Allergy and Allergic Diseases, 2 Volume Set

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Allergy and Allergic Diseases

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

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To our wives: Rosemary, Lee, Hélène and Barbara

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

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Allergy and Allergic Diseases Editor-in-Chief

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

Editors

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

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

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

SECOND EDITION

FOREWORD BY K. FRANK AUSTEN, MD

In two volumes Volume 1

A John Wiley and Sons, Ltd., Publication

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

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Contents

List of Contributors, ix

Part 2 Inflammatory Cells and Mediators

Foreword, xvii

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

Preface, xviii

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

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

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

41 Airway Smooth Muscle, 874 Stuart J. Hirst

Part 3 Pharmacology

Part 5 Allergens

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

42 Biochemistry of Allergens and Recombinant Allergens, 895 Rudolf Valenta

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

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

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

Part 6 Animal Models of Asthma

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

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

40 Biology of Vascular Permeability, 857 Peter Clark

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

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

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

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

Index

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

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

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

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

Part 10 Asthma and its Treatment

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

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

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

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

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

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

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

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

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Contents

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

Part 12 Allergy and the Skin

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

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

Part 14 Prevention of Allergic Disease

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

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

90 Urticaria and Angioedema, 1853 Allen P. Kaplan

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

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

Index

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

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

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List of Contributors

Cezmi A. Akdis

Tony R. Bai

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

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

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

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

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

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

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

Andrew J. Beavil

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

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

Pedro C. Avila

Rebecca L. Beavil

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

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

Mark V. Avdalovic

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

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

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

Bengt Björkstén The National Institute of Environmental Medicine/IMM

Division of Physiology Karolinska Institutet Stockholm, Sweden

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

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

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

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

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

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

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

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List of Contributors

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

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

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

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

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

Glenn Cruse

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

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

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

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

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

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

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

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

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

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

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

Anuk M. Das Immunobiology Centocor Radnor, Pennsylvania, USA

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

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

Alexander C. Drew Andrew S. Cowburn

Edwin R. Chilvers

Peter Clark

Pascal Demoly

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

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

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

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

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

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

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

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

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

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

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List of Contributors

David J. Fear

Arturo Genovese

Trevor T. Hansel

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

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

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

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

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

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

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

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

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

Howard Fox Novartis Horsham Research Centre Horsham West Sussex, UK

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

Cathryn C. Hassett

William G. Glass Immunobiology Centocor Radnor, Pennsylvania, USA

Catherine Hawrylowicz

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

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

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

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

Nicholas J. Gross

C. Garren Hester

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

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

Avinash Gurbaxani

Stuart J. Hirst

Moorfields Eye Hospital London, UK

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

Belinda J. Hales Michael M. Frank

Institute of Environment and Health Cranfield University Cranfield, UK

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

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

Paul Harrison

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

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

Arthur N. Freed

Qutayba Hamid

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

Meakins-Christie Laboratories McGill University Montreal, Quebec, Canada

Anthony J. Frew

Hamida Hammad

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

Postdoctoral Fellow University of Ghent Laboratory of Immunoregulation Ghent, Belgium

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

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

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List of Contributors

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

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

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

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

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

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

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

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

Royal Brompton Hospital London, UK

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

John Lacey Deceased

Paige Lacy

Department of Respiratory Medicine Ghent University Hospital Ghent, Belgium

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

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

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

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

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

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

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

Onn Min Kon

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

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

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

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

Guy F. Joos

Debbie L. Jarvis

Peter K. Jeffery

Kevin Kwong

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

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

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

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

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

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

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

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

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List of Contributors

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

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

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

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

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

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

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

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

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

University of California Davis, California, USA

St George’s Hospital London, UK

Miriam F. Moffatt

Robyn E. O’Hehir

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

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

Rafael I. Monsalve

Thomas G. O’Riordan

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

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

Redwan Moqbel

David I. Orton

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

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

Ulrich R. Müller

Reinhard Pabst

Allergy Division Medical Department Spital Bern Ziegler Bern, Switzerland

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

Michelle J. Muessel

Giovanni Passalacqua

Research Associate Glenfield Hospital Department of Respiratory Medicine Leicester, UK

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

Lynne A. Murray

Ian D. Pavord

Manager of Pharmacology Promedior Inc Malvern, Pennsylvania, USA

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

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

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

Dean D. Metcalfe

Wolfgang A. Nockher

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

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

Lisa A. Miller

Yee-Ean Ong

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

Consultant Physician and Honorary Senior Lecturer

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

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

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

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List of Contributors

Emily M. Pieczarka

Jamie A. Robyn

Edward S. Schelegle

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

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

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

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

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

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

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

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

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

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

Anna Pomés INDOOR Biotechnologies Inc. Charlottesville, Virginia, USA

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Rebecca Slack

Wayne R. Thomas

Laura S. Van Winkle

Institute of Environment and Health Cranfield University Cranfield, UK

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

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

Catherine Thornton

Ulrich Wahn

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

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

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

Suzette M. Smiley-Jewel

Stephen J. Till

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

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

Sanchaita Sonar

Isabelle Tillie-Leblond

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

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

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

Taniawati Supali Department of Parasitology University of Indonesia Jakarta, Indonesia

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

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

Department of Pneumology and Immunoallergology University Hospital Lille, France

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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List of Contributors

Reen Wu

Maria Yazdanbakhsh

Michael C. Zacharisen

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

Department of Parasitology Leiden University Medical Center Leiden, The Netherlands

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

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Foreword

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

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

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Preface

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

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

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

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

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

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Immunology of the Allergic Response

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

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Allergy and Hypersensitivity: History and Concepts A. Barry Kay

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Allergy and Hypersensitivity: History and Concepts

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

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

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

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

From reagin to IgE

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

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

6

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

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

(a)

Allergy and Hypersensitivity: History and Concepts

(b)

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

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

Mediator cells and mediators

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

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

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

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

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

8

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

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

(a)

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

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

Allergy and Hypersensitivity: History and Concepts

(b)

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

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

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(b)

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

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

10

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

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

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

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

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

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

Allergy and Hypersensitivity: History and Concepts

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

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

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

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

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

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

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

(a)

12

(b)

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

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

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

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

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

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

(a) (b)

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

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

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

14

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

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

Shen Nung

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

2698 BC 2641 BC

Huang Ti Menes

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

460–365 BC

Hippocrates

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

25 BC–AD 40

Aulus Celsus

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

AD

c. 40

Marcus Terrentius Varro

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

AD

b.41

Britannicus

Reported to be afflicted by acute allergic reactions to horses

AD

c. 60

Pedanius

Remedies for asthma in classical pharmacology treatise

Aretaeus of Cappadocia

First detailed description and coining of the word asthma

120–AD 180 AD

b.131

Galen

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

AD

b.865

Rhazes

Description of seasonal catarrh due to roses in Persia

1135–1204

Moses Maimonides

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

b.1306

John of Arderne

Prescription of a “syrup” for asthma

1530

Thomas Moore

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

1552

Girolamo Cardano

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

1565

Leonardhus Botallus

Description of “rose cold” in Pavia

1570

Pietro Mattioli

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

c. 1584

Johann Schenk

Coins the term “idiosyncrasy”

b.1603

Kenelm Digby

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

1603

Felix Platter

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

c. 1630

Sanctorius

Description of asthma to cat hair

1656

Pierre Borel

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

1662

K.V. Schneider

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

1665

Philipp Jacob Sachs

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

1673

Johannes Binneringus

Description of seasonal rose coryza in Basle

1675

Theophile Bonet

Idiosyncrasies to bread, strawberries and wine

1680

Thomas Willis

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

c. 1680

Nehemiah Green

First microscopic studies of pollen grains

1682

Joan van Helmont

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

1691

Jacob de Rebecque

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

1698

John Floyer

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

1713

Bernardino Ramazzini

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

1765

Debrest

Description of sudden death by bee sting in Montpellier

1775

William Cullen

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

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Table 1.1 (Cont’d ) 1776

Johann Murray

Ipecac root (emetine), causes asthma attacks in pharmacists

1778

Stolpertus

Description of acute angioneurotic oedema following ingestion of eggs

1783

Friedrich Schademantal

Allergic urticaria due to ingestion of fresh pork meat

1783

Philipp Phoebus

Comprehensive monograph on hayfever. First epidemiological enquiry

1802

William Heberden

Description of summer catarrh and asthma; differentiation from common cold

1816

Henri Laennec

Invents stethoscope. Identifies bronchospasm as important component of asthma

1819

John Bostock

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

1839

François Magendie

Description of sudden death of dogs repeatedly injected with egg albumin

1853 1886

Jean Martin Charcot and Ernst van Leyden

Description of Charcot–Leyden crystals in sputum of asthmatics

1868

Henry Hide Salter um

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

1872

Heinrich Quincke

First description of angioneurotic oedema

1872

Morrill Wyman

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

1873–80

Charles Blackley

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

1877

Paul Ehrlich

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

1894

Samuel Flexner

Experimental “toxic death” whilst injecting dog serum into rabbits

1895

Josef Jadassohn

Establishment of patch tests in contact dermatitis

1895

Josef Jadassohn

Description of various types of drug reactions in the skin

1900

Solomon Solis-Cohen

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

1902

Charles Richet Paul Portier

Discovery of experimental anaphylaxis in dogs

1903

Theobald Smith

Observations of anaphylactic reactions of guinea pigs to horse serum

1903

Maurice Arthus

Experimental localized acute necrotizing vasculitis, first described as local anaphylaxis

1905

Bela Schick

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

1905

Clemens von Pirquet

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

1906

A. Wolff-Eisner

Relationship of human hayfever and urticaria to experimental anaphylaxis

1909

William Schultz

Detection of anaphylaxis by contraction of isolated smooth muscle in vitro

1909

William B. Osler

Asthma associated with neurotic disease

1910

S. Meltzer

Bronchial asthma as a phenomenon of anaphylaxis

1910

William Dunbar

Methodology for pollen extraction; first approaches to pollen immunotherapy

1911

Leonard Noon John Freeman

Wide use of immunotherapy with pollen extracts in hayfever patients

1911

Henry Dale

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

1911

Tomaso Casoni

Skin test in patient infected with Echinococcus

1912

Oscar M. Schloss

Use of scratch test in allergy to foods

1913

William Dunbar

Methodology for pollen extraction, identification of allergenic protein

1914

A.T. Waterhouse

Anaphylactic reactions of beekeepers to stings

16

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Table 1.1 (Cont’d ) 1915

Warfield T. Longcope

Experimental lesions in organs from animals repeatedly injected with foreign proteins

1916

Robert A. Cooke

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

1918

Francis Rackemann

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

1919

M.A. Ramirez

Horse dander asthma following blood transfusion

1921

Carl Prausnitz Heinz Küstner

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

1921

Arent de Besche

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

1922

Fernand Widal Pierre Abrami J. Lermoyez

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

1923

Arthur Coca

Reagent for allergen extraction

1923

Arthur Coca

Coined the term “atopy”

1924

F.T. Codham

First description of mould allergy

1924

Ko Kuei Shen Carl F. Schmidt

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

1927

Thomas Lewis

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

1927

Bret Ratner

Experiments on allergic sensitization in utero

1928

Storm van Leeuwen

Inhalation allergy to house dust

1928

Edward Dienes

Induction of cellular delayed hypersensitivity by protein antigens

1934

Mataso Masugi

Experimental glomerulonephritis with anti-kidney antiserum

1935

Ulf von Euler

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

1937

Daniel Bovet

First synthesis of antihistaminic drugs

1939

Harry H. Donally

Transmission of food allergens in breast milk

1940

Charles H. Kellaway Everton T. Trethewie

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

1940

Mary Loveless

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

1941

Louis B. Jaques

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

1941

Joseph Harkavy

Bronchial asthma with recurrent pulmonary eosinophilic infiltration and polyserositis

1942

Merrill W. Chase

Transfer of tuberculin sensitivity by cells from immunized animals

1945

Merrill W. Chase

Transfer of contact dermatitis to simple chemicals by sensitized leukocytes

1945

Robin A. Coombs

Described the antiglobulin (“Coombs”) test

1949

Philip S. Hench Edward C. Kendall

Isolation of cortisone from adrenals for therapy of rheumatoid arthritis

1952

Zoltan Ovary

Development of passive cutaneous anaphylaxis (PCA) for quantification

1953

James F. Riley Geoffrey B. West

Mast cell granules as major source of histamine in tissues

1954

William Frankland Rosa Augustin

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

Continued p. 18

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Table 1.1 (Cont’d ) 1957

Ernest Witebsky

Experimental autoallergic thyroiditis: pathology and criteria of immune diseases

1958–60

Zoltan Ovary Herman N. Eisen

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

1960

Bernard. B. Levine Alain L. de Weck

Identification of major and minor antigenic determinants in penicillin allergy

1962

Alain L. de Weck Charles W. Parker

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

1963

Robin A. Coombs Philip Gell

Classification of the hypersensitivity reactions

1963

K. Frank Austen

Biochemistry of mast cell mediator release

1963

Jack Pepys

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

1964

Lawrence M. Lichtenstein Abraham G. Osler

Development of allergen-specific histamine release test

1964

T.E. King

Identification of major allergen in ragweed

1966

Barry R. Bloom John R. David

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

1966

David Marsh

Identification of major grass allergens

1966

J.J. Curry

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

1967

Kimishige Ishizaka Teruko Ishizaka

Characterization of reagins as IgE immunoglobulins

1967

Hans Bennich Gunnar Johansson

Identification of myeloma ND as IgE immunoglobulin

1967

L. Wide Gunnar Johannsson

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

1967

Reindert Voorhorst Frederick Spieksma

Identification of Dermatophagoides mites as major allergen source in house dust

1967

Bernard Halpern

Lymphocyte stimulation test in drug allergy

1967

Roger Altounyan

Discovery of sodium cromoglycate as anti-asthmatic drug

1981

K. Frank Austen

Biological properties of leukotrienes

1983

Bengt I. Samuelson

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

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

18

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

Soluble

Antigen

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

b

Antagonist

Neutrophil-rich inflammatory response

APC

MHC Class II

TCR

Macrophage-rich inflammatory response

Type 1 cytokines

AF

CD4+ type 1

T Lymphocyte

Soluble

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

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

Agonist

IgG

Microvasculature

Soluble

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

• • • •

MHC Class II

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

Eosinophil- and basophil-rich inflammatory response

Type 2 cytokines

APC

CD4+ type 2

T Lymphocyte

Soluble

MHC Class I

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

Cytotoxicity (apoptosis)

Target cell

CD8+ cytotoxic

T Lymphocyte

Cell-associated

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

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

Tissue injury by cytotoxic T lymphocytes

Th2

Th1

Type IV cytotoxic

Type IV

Type IV

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

Type III

Receptor Antigen-antibody complexes + ligand complement

Target cell Receptor

Cell-associated

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

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

Type II

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

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

Complement lysis or removal by the RE system

+/– complement

IgG

Cell surface antigen

Target cell

Cell-associated

IgG antibody interacting with cell surface antigen

Cytolytic, or cytotoxic, reactions

a

CHAPTER 1

Examples in humans

Allergen

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

IgE

Mast cell/basophil

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

Initiating event

Simplified scheme of the proposed mechanism

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

Descriptive term

Type I

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

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

20

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

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

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

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

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

Allergy and Hypersensitivity: History and Concepts

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

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

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

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

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

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

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

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

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

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

ia ter

ve

iti pos

Gram negative bacteria (including endotoxin) RSV F protein

?

bac

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

Flagellated bacteria

m Gra gi u F n

TLR10

TLR4

TLR6

TLR5

TLR2

Plasma membrane

TLR1 Viral ssRNA Viral/bacterial DNA Viral dsRNA

TLR7 TLR8 TLR3

24

TLR9

Intracellular vesicle membrane

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

OLD MODEL Th1

NEW MODEL Th2

Th1

Th2

Treg

Dendritic cell

Dendritic cell

Microbial stimulation

Microbial stimulation

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

ENDOTOXIN

Dendritic cell

Th1

Dendritic cell

Allergen

Th2

Th1

Allergen

Th2 Allergic inflammation Tissue remodeling

No allergic disease

Epithelium

ENDOTOXIN Allergic disease exacerbation

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

mCD14+ cells

Membrane CD14-negative cells

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

Signal

LPS

LBP

CD14

TLR4

MD2

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

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ALLERGIC DISEASE IgE sensitization

Environment

Tissue-specific factors

Development

Genetics

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

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

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

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T Cells and Cytokines in Asthma and Allergic Inflammation Chris Corrigan

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

Immature T cell

Thymic cortical epithelium Primary positive selection

CD4+

CD4+

Intermediate anti-self affinity

High anti-self affinity

HC

TSLPactivated DC

TSLP

Secondary positive selection

AIRE

Non-self reactive T cells eliminated

CD4+

Immature DC

Thymic medullary epithelium CD4+

CD4+ CD25+ FoxP3+

Low-affinity anti-self T cells

Negative selection

Naive T cells in the periphery

Death

Treg cell in the periphery

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

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

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

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Th2

Th1

Signature cytokines

IFN-g

IL-4, IL-5, IL-13

IL-12 receptor b2-chain

+++

+/−

Chemokine receptors

CXCR3 CCR5

CCR3 CCR4 CXCR4 CCR8

Chandra protein

+

−

Costimulatory molecule ICOS

−

+

CRTH2

−

+

TIM (mice)

TIM-3

TIM-2

Signaling

STAT4 T-bet

STAT6 GATA3 c-Maf

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

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

Table 3.1 The IL-17 cytokine family. Name

Alternative names

Cellular sources

Receptor

IL-17A

IL-17, CTLA-8

IL-17RA (IL-17R)

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

CXI, NERF CX2 IL-27 IL-25

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

IL-17F

ML-1

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

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

52

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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Table 3.2 T-cell costimulatory molecules and their receptors.

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

Naive T cells

Activated Th2 cells

Memory T cells

Other cells*

+++ ++ +++ +++ − − − − + − −

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

+++ +++ +++ ++ + − − − − − −

Yes Yes Yes Yes No No Yes Yes No No No

Counterreceptor(s)

Action

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

+ + +? − + + + + + − −?

CD80, CD86 PD-L1, PD-L2

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

56

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

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

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Pre-Th1 TCM

CXCR3

Pre-Th2 TCM

CCR4

Th2 TCM

CRTH2

Th1 TEM

CXCR3

Th2 TEM

CCR4

Th2 TEM

CRTH2

CCR7

TCM CD62L +

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

?

Antigen reexposure

TM CD45RO

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

TEM

?

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

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Immunology of the Allergic Response Allergen Mucosal surface DC

TSLP

Other sources?

TSLP DC

MHC/peptide/TCR DC

OX40/OX40L

Effector Th2

Lymph node

Th2 CRTH2

Th2 TEM CRTH2

Effector memory Th2

Naïve T cell

Trafficking to target organ

Th

Central memory Th2

Th2 TCM CRTH2

Inflammatory Th2 Th2 CRTH2

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

60

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

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

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

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

Th2

Th2 IL-10

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

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

TNF-a

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

Th1

Th1

Regulatory IFN-g

Inflammatory IFN-g

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

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

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

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

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Histamine Bronchospasm

Prostaglandin D2 Leukotrienes Mast cell

IL-4, IL-13, TGF-b

IgE

ACUTE CHRONIC

Allergen Other antigens?

B

Epi Fib ASM

B cell

TSLP IL-4, IL-13

TSLP IL-4

IL-13

AHR Remodeling Narrowing TGF-b

IL-5 Major basic proteins Leukotrienes

CD4+ DC Th2 T cell

Eosinophil

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

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

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

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

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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66

Th1

Th1/Th2

Th2

Th2

Th1/Th2

IL-2

IL-3

IL-4

IL-5

IL-6

Fibroblasts, macrophages, endothelial cells, epithelial cells

AHR, eosinophilia, mucus hypersecretion, airway remodeling

IL-16

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

Increased

Increased

Increased

CD8+, MC, eosinophils

T-cell growth factor; expands Treg

Th1

IL-15

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

Decreased: exogenous IL-12 decreased eosinophils but not AHR

Increased

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

Increased further by viral infection

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

Increased (in severe asthma)

Increased: antibody decreased eosinophils but not AHR

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

Increased

Increased

Human asthma

Decreased

Th2 cells, eosinophils, MC, basophils

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

Th2

IL-13

Exogenous IL-12 blocked eosinophils and AHR

Transgene had AHR and changes of remodeling

Adenoviral transfer decreased inflammation

Transgene had eosinophils, AHR and mucus; knockout no effect

IL-8 receptor knockout decreased AHR and neutrophils

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

Increased: knockout or blocking decreases eosinophils and AHR

Increased: blocking decreases AHR (but some residual)

Increased: blocking decreases eosinophilia

Decreased

Animal models of airway inflammation

Many non-T cells

DC, macrophages, B cells

Favors Th1 differentiation, inhibits IgE synthesis

Th1

IL-12

IL-11

T cells, macrophage

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

Neutrophil activation and chemotaxis; weak eosinophil chemotaxis

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

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

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

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

Th2 cells, eosinophils, MC, basophils

Eosinophil and basophil differentiation, maturation, and activation

IL-10

Th2 cells, eosinophils, MC, basophils

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

Th2 cells, eosinophils, neutrophils, MC, basophils

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

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

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

Th1 cells, eosinophils

T-cell growth factor

Th2

Producer cell in human patients

Actions

IL-9

IL-8

Th1/Th2/Th17/ Treg associated

PART 1

Cytokine

Table 3.3 Cytokines implicated in asthma.

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

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

“Regulatory” Th1/Th2, Treg

Th1

TGF-b

IFN-g

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

Th1 cells, NK cells

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

Macrophages, NK cells, T cells

Transgene develops atopic dermatitislike phenotype and airways inflammation

Decreased

Increased

Increased

Increased: transgene has airway inflammation

Not studied

Not studied

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

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

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

Wide variety of targets increasing cytokines, chemokines, and metalloproteinases

Increased in the bronchial mucosa; correlates with CC chemokine expression

Increased in viral infection and exacerbations

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

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

Increased

Not studied

Not studied

Elevated in serum

Reduced

Not yet measured

Increased

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

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

Activation of endothelial cells and epithelial cells

“Inflammatory” Th1/Th2

TNF-a

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

Macrophages, DC

Macrophages, DC

T cells

Macrophages, epithelial cells, DC

Th2 central memory cells

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

CHAPTER 3

TSLP

Differentiation and activation of eosinophils, neutrophils, basophils, MC

Th1/Th2

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

IL-27

GM-CSF

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

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

IL-23

Th1

IL-18

Maintains Th2-type T-cell activation and central memory

T-cell growth factor

Th2

IL-17E (IL-25)

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

IL-19

Th17

IL-17A/F

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

Microbial fragments

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

Th1-type

Th2-type

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

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

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

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

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

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

TLR Blockade

OX40L NF-AT TSLP

DC

AHR

IL-12

DC

IL-4, IL-13

IL-4

Mucus hypersecretion IgE switching

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

GATA-3 STAT6 C-maf

TSLPR

Mast cell activation Smooth muscle activation, remodeling

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

IL-25R

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

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

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TNF-a

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

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Addressing all these challenges provides exciting prospects for future studies.

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

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

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

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

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

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

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Induction of IgG4, IgA suppression of IgE

Suppression of Th2 cell homing to tissues Endothelial cells

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

B cell

IL-

4,

IL-

13

Decreased Th2 cytokines

Epithelial apoptosis

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

Th2

13

L,I

9 IL-

IL-10 TGF-b TReg

IL-

3,

IL-

4,

IL-10 TGF-b

IL-

5,

IL-

9

Mucus production

Suppression of Th0/Th1/Th2 effector cells

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

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

T regulatory cells

Suppressor mechanism(s)

Reference

Tr1, IL-10-Treg

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

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

Th3

TGF-b, IL-10

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

CD4+CD25+ Treg

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

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

CD8+CD25+CD28− Treg

Same as CD4+CD25+

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

CD4−CD8− Treg

Induction of apoptosis

Thomson et al. (2006)

TCRgd Treg

IL-10, TGF-b

Strober et al. (1996)

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Th0 Th1 IFN-g TNF-a

Smooth muscle cell activation and apoptosis

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

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

See text for definition of abbreviations.

IFN-g TNF-a

Basophil Eosinophil mast cell

Suppression of mucus production

FasL

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

IL-10

TGF-b

Dendritic cells

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

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

T cells

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

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

B cells and immunoglobulin

Enhances survival Promotes immunoglobulin production, including IgG4

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

IgE

Suppresses allergen-specific IgE

Suppresses allergen-specific IgE

CD25+

Treg

Upregulates Foxp3 Promotes generation in the periphery Potential effects on homeostasis

IL-10-Treg

Promotes IL-10-Treg induction

Can promote IL-10 synthesis

Monocytes/macrophages

Inhibits proinflammatory cytokine production and antigen presentation

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

Eosinophils

Inhibits survival and cytokine production

Chemoattractant

Mast cells

Inhibits mast cell activation, including cytokine production

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

Neutrophils

Inhibits chemokine and proinflammatory cytokine production

Potent chemoattractant

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

B cells

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

Dendritic cells

Suppressed IgE-facilitated antigen presentation

Eosinophils

Reduction of tissue numbers Decrease in mediator release

Mast cells

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

Basophils

Decrease in mediator release Decrease in proinflammatory cytokine production

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Evidence

Reference

Reconstitution of an extract of five recombinant allergens

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

Jutel et al. (2005)

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

Single dose prevents mice from developing IgE against native allergen

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

Trimer and fragments of one major allergen

IgG4 is induced

Gafvelin et al. (2005)

Peptide immunotherapy

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

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

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

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

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

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

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

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

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

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

Summary

Introduction

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

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

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

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

VH

VH

VL

Fab

1

Ce 1

C

L

CL

Ce2

Ce2

Ce3

Ce3

Fc

Ce4

Ce4

104

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

VL

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

Ce

PART 1

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

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CHAPTER 5

Ve

Vl

nm

Ce1

6 .9

Cl

Ce2

≥10 nm Ce4

Ce3

5.3 nm

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

D2

D1

a

ITAM b

g

g

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

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

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a(1)

a(2) CC′ peptide a(1) Ce3

Ce3

Ce3 N

N

CD23 binding site

(a)

Ce4

Ce4

Ce4

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

B

Cys 241 Ce2 Cys 328 Ce3

Ce4

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

106

To membrane

Ce4 (c)

(b)

A

a(2)

Ce3

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

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

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

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

FceRI

IgE and IgE Receptors

Residues which interact with FceRI a1 a2

CC’ loop

Ce2 Ce3 Ce2 Ce4 (a)

Fab

Fab

Conformational change

Fab Fab

(b) Membrane

Membrane

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

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

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

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

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

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

IgE and IgE Receptors

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

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

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C-type lectin head D

RG

Ca2 IgE binding site

DG

R

C-terminal tail a-helical coiled-coil stalk

avb5 integrin binding site

N

CD21 binding site C

N N N (a)

CD23

(b)

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

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

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

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

IgE and IgE Receptors

Allergen IgE

IgE mCD23

mCD23

mCD23

CD21

(a)

(b)

CD19

sCD23

sCD23

EMPD CD21

CD21 mIgE

(c)

CD19

CD19

(d)

CD21

CD19

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

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

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

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

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

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

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

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

IgE and IgE Receptors

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

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

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

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IgE and IgE Receptors

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

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

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

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

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

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

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

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

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

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

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

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Plasma cell

Antigen receptor signaling

BLIMP1

BCL6

PAX5 XBP1 Regulates: Ig CD19, AID expression

Regulates: ER production Ig secretion

Responses to cytokines and AgR signals

Cessation of cell cycle

Cell cycle progression SHM

High-level Ig secretion

CSR

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

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

IgG

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

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

IgD

IgA

IgE

Fab L Fc H

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

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

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FceRI

Mast cell

APC Allergen MHC II CD23

lgE

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

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

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

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

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

L2 Vl2

L Vl-30

Jl1

Cl1

Jl2

Cl2

Jl4

Cl4

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

L3 Vk-3

L Vk-40

Jk1–5

Ck

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

L3 VH-3

LH VH-50

DH1–25

JH1–6

Cm

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

D J genes genes

Cm Cd

3’E (Ca1) enhancer Cg3 Cg1

Ye

Ca1

Yg

3’E (Ca2) enhancer Cg2 Cg4 Ce

Ca2

(d)

Germline Ie promoter

Se

Ce1–4 M1,2

Mouse IgH locus Ei (Em) enhancer V genes

D genes

J genes

Cm Cd

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

Ca

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CACAGTG Heptamer GTGTCAC

Vk

VH

GGTTTTTGT CACTGTG Nonamer 12 Heptamer CCAAAAACA GTGACAC

ACAAAAACC Nonamer TGTTTTTGG

23

12

23

23

23

D

12

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

l chain

Jk

k chain

JH

H chain

12

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

L1 V1

Jl

Ln Vn

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

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

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

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

RAG1/RAG2 binds to the 12 bp spacer

DNA–RAG complex recruits the 23 bp spacer

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

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

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

Coding joint

Signal joint

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

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

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

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

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

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

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Transitions at C-G T

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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IeGLT

B cell

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IL-4 CD4 T cell

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Se

IL-4

P P

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AP1

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NK-kB ID2+PAX5

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

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

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

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

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

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

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

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

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IgD+

IgD– BcI6+ Tfh IL-21

+

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IgG1 BcI6

Th2 IL-4

IgG1– IgE+ IgG1+ PB IgG1+ MC

IgE+ PB

IgE+ PC

IgG1+ PC

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

Local IgE regulation in the target organs of allergy

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

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

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

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

Positive feedback

Allergen

CD23

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IgE synthesis

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IgE synthesis

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

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

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

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

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7

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

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

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

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

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

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

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

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

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

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

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

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

Environmental Factors in IgE Production

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

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

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

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

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

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

Air particulate matter Effect of particle size Ambient PM consists of very heterogeneous groups of components, arbitrarily divided into different size fractions: coarse (PM ≤ 10 μm), fine (PM ≤ 2.5 μm), and ultrafine (PM ≤ 0.1 μm). Particle size is very important as size determines, to a great extent, inhalation, deposition, and elimination of particles. Ultrafine particles have substantially higher deposition efficiency. Moreover, ultrafine particles may penetrate through the epithelium and vascular walls and be transported to the blood to distal organs where proinflammatory events may occur (Elder & Oberdorster 2006).

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Studies on ultrafine particles have shown that the allergic pulmonary effects were related to the total surface area and number of particles rather than to the instilled mass (Nygaard et al. 2004). Mouse footpad injections of coarse and fine PM sampled from different European cities showed that the fine particles had a stronger adjuvant effect on ovalbumin (OVA)-induced IgE (Dybing et al. 2004). When comparing fine with ultrafine PM, only the ultrafine PM was able to increase Th2 cytokine and allergen-specific IgE production to administration of the antigen in sensitized mice (Alessandrini 2006; De Haar et al. 2006). Short-term exposure to PM 2.5 has also been associated with increased allergic inflammation in nasal lavage from asthmatics, but not healthy controls (Nikasinovic et al. 2006).

Effect of age The age of the subjects is also an important consideration in PM exposure. Children have greater physical activities, spend more time outdoors, and are likely to be more exposed than adults. In exposure studies, childrens’ exposure was twice that of adults exposed to the same outdoor concentrations of PM 10 (Janssen et al. 1997, 1998). Children are also more vulnerable to the effects of pollution, because their lungs are not fully developed when exposure begins. In a prospective 8-year study, adverse effect on lung development, assessed by decreased flow expiratory volume, was associated with pollution by PM 2.5 and NO2 (Gauderman et al. 2004).

Effect of particle-associated metals Metals have been scrutinized as important constituents of PM. In the Utah valley, closure of a steel mill factory releasing metal-rich PM in the 1980s was associated with decreased mortality and morbidity (Ghio 2004). Metal-rich particles have been linked to allergic diseases. Evidence was provided from regional differences in industrial air pollution that were shown to account for differences in prevalence rates of allergic sensitization in children living in different cities in eastern Germany (Heinrich et al. 1999). These differences correlated with the presence in samples of PM 2.5 of severalfold higher levels of zinc, magnesium, lead, copper, and cadmium (Gavett et al. 2003). There is evidence that metals induce IgE production (Prouvost-Danon et al. 1981; Murdoch et al. 1986) and stimulate Th2-driven immune responses in different species (Heo et al. 1997). A correlation between in vitro biological effects of residual oil fly ash (ROFA), a fine PM sample, and its content in transition metals has been reported (Dreher et al. 1997), leading to its use as a surrogate of metals. In animals, ROFA and metal-rich particles can enhance allergic responses to OVA and HDM in models of asthma (Lambert et al. 1999, 2000; Gavett et al. 2003), including increased release of pro-Th2 cytokines, increase in allergenspecific IgE levels, eosinophil recruitment, and airways hyperresponsiveness (Gavett et al. 1999). Moreover, metal ions such as aluminum, cadmium, nickel, and strontium can directly

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Exacerbation of inflammation Many studies have shown that both particulate and gaseous pollutants can initiate and exacerbate cellular inflammation, by interacting with the immune system. Ozone, nitrogen dioxide (NO2), and DEPs can all initiate the recruitment of inflammatory cells such as neutrophils, lymphocytes, and macrophages (Sandstrom et al. 1990; Schelegle et al. 1991; Salvi et al. 1999), but also, through their direct interaction with the airway epithelium, can lead to the generation of increased amounts of proinflammatory cytokines, chemokines, and adhesion molecules. DEPs can induce release of soluble intercellular adhesion molecule (ICAM)-1, and cytokines like granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-8 from bronchial epithelial cells (Bayram et al. 1998; Takizawa et al. 2000). DEP extracts can also directly stimulate macrophages leading to the production

Pioneering studies in Japan, where diesel vehicles are most common, showed that respiratory allergy was more prevalent in subjects living near busy highways than in subjects living in less busy areas with equivalent atmospheric concentrations of cedar pollen allergens (Ishizaki et al. 1987). As discussed below, it is now appreciated that DEPs are able to potentiate the effect of the allergen on the immune response at several steps (Fig. 7.3). Macrophage

Airway epithelium IL-10 CD80

CD28

TH1

–

Proinflammatory IL-B, Rantes GM-CSF, others

TH0 APC

TCR

IL-4 IL-13

B cell Antigen DEP

YY

Y

YY

DEP + ROS

Y Specific lgE

YY

TH2

Y

MHC-II

Y

Diesel exhaust consists of a complex mixture of DEPs and gases. Diesel engines emit 30–100 times more PM than gasoline engines, making DEPs one of the major components of airborne PM in urban and industrialized areas, and the best-studied particulate pollutant. The 3.3 million diesel trucks and buses in the USA contribute 58% of respirable particulates, although they comprise less than 2% of the total vehicles (National Air Pollutant Emission Trends 1997). The same proportion is observed in France (55.6% of PM) (Kunzli et al. 2000), whereas in other countries such as Japan, diesel vehicles contribute almost 100% of PM emissions (Kagawa 2002). After combustion of diesel fuel, the exhaust components tend to aggregate into discrete spherical respirable particles approximately 0.1– 0.5 μm in diameter, which consist of an elemental carbon core with a large surface area to which hundreds of chemicals and transition metals are attached. The major particle-associated chemical compounds are PAHs, which contain three to five benzene rings that can easily diffuse through cell membranes. Of the gaseous pollutants, the main focus has been on ozone. The principal source of lower atmosphere ozone is automobile exhaust as a consequence of photochemical reactions of nitric oxides, volatile organic compounds, and ultraviolet light.

Interactions with allergen Diesel exhaust

Y

Diesel exhaust and gases

of chemokines able to recruit neutrophils and eosinophils (Fahy et al. 1999). Beside these interactions with the innate immune system leading to bronchial inflammation, interesting results have shown that pollutants are able to modify the IgE response by interacting with allergens.

YY

affect mast cells by enhancing their IL-4 release and degranulation (Walczak-Drzewiecka et al. 2003). Recently, aerosolized ROFA in pregnant mice was shown to favor IgE-dependent asthma susceptibility in their offspring (Hamada et al. 2007). Taken together, these studies suggest that soluble metal from PM has an influence on subsequent allergic responses. Identifying the specific causative agents in PM is difficult because of its multiple constituents. Beside metals, biological contaminants such as endotoxin (see Endotoxin section) and polyaromatic hydrocarbons (PAH), contained in diesel exhaust particles (DEPs) and cigarette smoke, have been shown to contribute to their adjuvant effect on IgE production.

Environmental Factors in IgE Production

Y

Y Mast cell

Plasma cell

Fig. 7.3 How diesel exhaust particles (DEPs) modify the immune response to allergen. DEPs impact on, and are internalized by, the airway epithelium and macrophages. The ensuing generation of oxidative stress causes the release of proinflammatory cytokines from both cell types. One outcome is increased antigen presentation because of decreased IL-10 plus upregulation of costimulatory molecules such as CD80 on antigenpresenting cells (APC). In this setting, APCs seem to favor the development of a Th2 cytokine milieu, which has a positive feedback loop on IL-4 production. DEPs have indirect effects on B-cell development and isotype switching through the secretion of IL-4 and IL-13 from Th2 cells. DEPs also have a direct effect on B cells that can drive enhanced IgE isotype switch and production. The IgE produced by the resulting antigen-specific plasma cells leads to the priming of mast cells that after antigen exposure degranulate and release cytokines such as IL-4 that produce positive feedback on both B and T cells. Finally, DEPs themselves can enhance mast cell and basophil degranulation, and cytokine release. MHC II, major histocompatibility class II; TCR, T-cell receptor. See text for definition of other abbreviations. (From Saxon & Diaz-Sanchez 2005, with permission.) (See CD-ROM for color version.)

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Effect on allergen presentation One level of action of pollutants is the modification of the immune system’s handling of the allergen. DEPs and their derived chemicals affect allergen presentation by upregulating costimulatory molecules such as CD80 and CD86 (Nel et al. 1998), and HLA-DR major histocompatibility complex class II molecule on human macrophages (Don Porto Carero et al. 2002) and nasal epithelial cells (Takizawa et al. 2007). In particular, pollutant-induced increases in allergen-induced macrophage-derived chemokine (MDC) production from human peripheral blood mononuclear cells (PBMCs) can be completely blocked by inhibition of the B7–CD28 pathway (Fahy et al. 2002). Furthermore, because DEPs and their resident chemicals may inhibit the tolerogenic cytokines IL-10 and transforming growth factor (TGF)-β while increasing IL-1 and soluble CD23 in lipopolysaccharide (LPS)stimulated PBMC/macrophages, exposure to DEPs has been suggested to be important in increasing allergenicity during the early phase of allergen presentation (Nel et al. 1998; Pacheco et al. 2001). In vivo human data support this concept by showing that primary human mucosal sensitization (IgE), as well as increased IL-4 levels, can be driven by a neoantigen (keyhole limpet hemocyanin, KLH) administered with DEPs, whereas the antigen alone leads only to a protective IgG response in allergic subjects (Diaz-Sanchez et al. 1999). Indirect effects on IgE production DEPs also augment the effect of the allergen on the immune system, by both inducing and exacerbating in vivo allergic responses in the human upper respiratory tract. In subjects challenged intranasally with DEPs, increased IgE isotype switching results in increased total IgE levels (Diaz-Sanchez et al. 1994, 1997; Fujieda et al. 1998). In atopic patients, in contrast to the two- to three-fold increase in allergen-specific IgE produced with allergen alone, combined challenge with DEPs plus allergen enhances local specific IgE production 16-fold (Diaz-Sanchez et al. 1997). Similar deviations of the IgE response have been seen in mice and rats repeatedly challenged with DEPs and ovalbumin (Muranaka et al. 1986; Fujimaki et al. 1994; Al-Humadi et al. 2002) and with PAH, in particular phenantrene, anthracene, and quinone derivatives (Heo et al. 2001; Hiyoshi et al. 2005). This potentiation of the IgE response is accompanied by an increased IL-4 and IL-13 Th2 cytokine profile evidenced both in mice (Takano et al. 1997; Yanagisawa et al. 2006; Inoue et al. 2007a) and humans, as well as a decrease in IFN-γ Th1 cytokine (Diaz-Sanchez et al. 1997). Along the same lines, in allergic subjects, diesel extracts also induce increased production of Th2-attracting chemokines (such as MDC and I-309) (Fahy et al. 2002; Senechal et al. 2003) and eosinophil-attracting chemokines (such as RANTES) (Diaz-Sanchez et al. 2000a; Fahy et al. 2000), and decreased production of Th1-attracting chemokines (such as IP-10) (Fahy et al. 2002). It is of interest that parts of these effects on the Th1/Th2 polarization of the immune

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response have also been shown in nonatopic subjects (Pourazar et al. 2004; Ohtani et al. 2005; Chang et al. 2006) or nonsensitized mice (Finkelman et al. 2004; Chan et al. 2006; Inoue et al. 2007b), suggesting that DEPs might also have a role in the genesis of allergic reactions. Moreover, DEPs and PAHs, such as benzo(a)pyrene and quinines, enhance IgEdependent histamine release and IL-4 production from human mast cells and basophils (Wang et al. 1999; Diaz-Sanchez et al. 2000b; Devouassoux et al. 2002; Kepley et al. 2003). Direct effects on B cells and IgE production In vitro studies have shown that human B cells and cell lines cultured with IL-4 and CD40 monoclonal antibodies in the presence of DEP-derived chemicals demonstrate up to a 360% increase in IgE production (Takenaka et al. 1995; Tsien et al. 1997). Moreover, DEP exposure in humans leads to the attraction of B cells in airway lavage, showing that a direct interaction can occur in the lung (Salvi et al. 1999). Thus, DEPs appear to have direct effects on B cells as well.

Gases Although not as well studied, gaseous pollutants may also modify responses to allergen. In animal models, ozone, SO2, and NO2 can all augment allergic antibody production and pulmonary inflammation after allergen challenge (Gilmour 1995; Park et al. 2001; Gershwin 2003). In particular for ozone, increases in Th2 cytokines and in IgE production have been observed after antigen challenge in mice (NeuhausSteinmetz et al. 2000), as well as in nonhuman primates (Schelegle et al. 2003). However, results of controlled human exposure studies have been more equivocal. Whereas some studies have reported enhanced airway responses to inhaled allergen after exposure to ozone (Molfino et al. 1991; Jorres et al. 1996), the combination of NO2 and SO2 (Devalia et al. 1994), or NO2 (Tunnicliffe et al. 1994; Strand et al. 1997), others have found enhanced late inflammatory or early bronchoconstrictor responses to inhaled antigen in only a subgroup of allergic asthmatic patients (Vagaggini et al. 2002; Chen et al. 2004). Overall, these data suggest that many airborne pollutants function as mucosal adjuvants and, in interacting with both innate and adaptive immune cells, skew the immune response to inhaled antigens toward a Th2-like phenotype (Saxon & Diaz-Sanchez 2005).

Mechanisms Despite our knowledge that air pollutants alter immunophysiologic outcomes, the mechanisms that underlie these outcomes are only now being elucidated. The most plausible model to explain these effects involves hierarchical oxidative stress (Xiao et al. 2003). This model postulates that with low exposure the formation of reactive oxygen species (ROS) leads to the activation of antioxidant response elements, followed by transcription of enzymes important in detoxifica-

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tion, cytoprotective, and antioxidant responses, such as phase II enzymes. At higher exposure, the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 response elements are activated, leading to NF-κB and mitogenactivated protein kinase (MAPK) signaling, and resulting in increased expression of proinflammatory and allergy-related genes. Enhanced inflammation leads to additional generation of ROS, which are normally curtailed by antioxidant defenses. With greater exposure, defenses are overwhelmed and inflammation and cytotoxic effects may occur. Experimental evidence in support of this model come from increases in markers of oxidative stress reported in both human and animal models after exposure to DEPs (Lim et al. 1998; Hiura et al. 1999; Li et al. 2002). Ultrafine particles, because of their greater relative surface area, contain significantly more organic carbons and PAHs than coarse or fine particles, and are more active in their redox cycling capacity (Li et al. 2003a), and may well have greater proinflammatory effects than larger particles. It is believed that particleassociated transition metals, such as iron and copper, generate formation of ROS through the Fenton reaction and also synergize with organic PM components in this generation (Gilmour et al. 1996; Barchowsky & O’Hara 2003). DEPs associated with PAHs and their quinone oxygenated derivatives directly induce oxidative stress. In animal models, antioxidant treatment can inhibit DEP-enhanced IgE production and its proinflammatory effects (Whitekus et al. 2002). PAHs and quinone derivatives have been shown to modify IL-10, RANTES, and macrophage inflammatory protein (MIP)-1α chemokine production (Shi et al. 1996; Nel et al. 1998), all able to modulate IgE production (Kimata et al. 1996; Jeannin et al. 1998). Moreover, DEPs and their resident chemicals can activate the redox-sensitive transcription factors N-terminal Jun kinase and p38 MAPK (Ng et al. 1998), and NF-κB pathways, leading to expression of proinflammatory cytokines, chemokines, and adhesion molecules (Ng et al. 1998; Takizawa et al. 1999; Fahy et al. 2000; Wang et al. 2005). DEPs and their associated PAHs can also activate phase II drug metabolizing enzymes (Li et al. 2000, 2004). Recently, it has been shown that B cells can make a cytoprotective response to diesel extracts by increasing such an enzyme, the NADPH quinone oxidoreductase (NQO1), and that when this response was sufficiently elevated it could block the IgE potentiation effect of DEP extracts (Wan & Diaz-Sanchez 2006). Therefore, oxidative stress can also be partly protective (Fig. 7.4). This hierarchical oxidative stress model can probably be extended to gaseous pollutants and thus underlies the adjuvant effects of air pollution in general. Ozone is a potent oxidant that produces free radicals and ROS. The epithelial surface of the respiratory tract is rich in antioxidants, such as glutathione and ascorbate. In controlled exposure experiments, ozone induces considerable depletion of this antioxidant storage (Mudway et al. 1999). Similarly, SO2 inhalation

Environmental Factors in IgE Production

Reactive oxygen species/ oxidative stress

Sensor/detector ASK1?

Keap 1 ?

MAPKs, e.g. JNK

NK-kB kinases

Nrf2

AP-1 RE

NK-kB RE

ARE

Transcription of: (a) Cytokine (b) Chemokines (c) Adhesion molecules

Transcription of: (a) Heme oxygenase-1 (b) Phase II enzymes, e.g. GST, NQO1

Fig. 7.4 Cellular sensors and signaling pathways involved in oxidative stress. Reactive oxygen species (ROS) generation and/or oxidative stress is detected by cellular sensors. The possible candidates for the sensors include ASK1 for the AP-1 pathway and Keap1 for the ARE pathway. These afferent components activate the mitogen-activated protein kinase (MAPK) cascade and, in the case of Keap1, lead to the release of the transcription factor, Nrf2, to the nucleus. The sensor for the NF-kB cascade is unknown, but ultimately leads to the phosphorylation and degradation of IkBa, thereby releasing the attached Rel protein to the nucleus. Binding of AP-1 and/or NF-kB transcription factors to their respective DNA-binding sites eventually leads to the production of cytokines, chemokines, and adhesion molecules. These products exert proinflammatory effects. The binding of Nrf2 to ARE results in the expression of heme oxygenase 1 (antioxidative) and phase II (detoxifying) enzymes. These products are cytoprotective. GST, glutathione S-transferase; NQO1, NADPH quinone oxidoreductase. (From Li et al. 2003b, with permission.) (See CD-ROM for color version.)

affects the intracellular glutathione redox state in airway epithelial cells (Todokoro et al. 2004), as well as NO2 (Kelly et al. 1996). In conclusion, there is growing evidence that pollutantinduced oxidative stress may be responsible for generating IgE production. Moreover, there is the realization that not all oxidative stresses are injurious; there is also a protective level of oxidative stress that could form the basis of disease susceptibility. However, the complex gene–environment interactions involved need to be further deciphered to understand the subtleties involved in pollution effects.

Genetics The results discussed above showing the underlying role of oxidative stress in the proinflammatory and proallergic effects of pollutants suggest that the key to protection from such pollutants is to mount an effective cytoprotective

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response. It follows that people with diminished ability to detoxify xenobiotics and metabolize ROS are at increased risk for adverse outcomes from pollutant exposure. Controlled human exposure studies have shown a large interindividual variation in responses to pollutants such as SO2, ozone, and DEPs (Horstman et al. 1986; Holz et al. 1999; Bastain et al. 2003), leading to the idea of pollutant “susceptibility genes.” Members of the glutathione S-transferase (GST) superfamily of phase II xenobiotic metabolizing enzymes, GSTP1 and GSTM1 are proving to be ideal candidate genes. Their products are present in the respiratory tract and they have common variant alleles with decreased or abolished enzyme function, which are associated with asthma (Fryer et al. 2000; Gilliland et al. 2002). The frequency of the homozygous GSTM1-null and of the GSTP1-Ile/Ile genotype is very high, around 40% (London et al. 1995). Taken together, these data suggest that a decreased ability to mount an effective cytoprotective response to pollutants in relation with a GST polymorphism may increase the allergic airway response. This hypothesis was confirmed in allergen-sensitive patients by nasal provocation challenges with allergen alone, or plus DEPs. Individuals with low responsive genotypes (GSTM1null or GSTP1-Ile/Ile) showed enhanced susceptibility to the adjuvant effect of DEPs, but not to allergen alone (Gilliland et al. 2004). These studies have been extended by showing that these genes are also involved in susceptibility to ozone in children who live in high-ozone areas in Mexico (Romieu et al. 2004, 2006). Asthmatic children with the nonfunctional variant GSTM1-null had greater ozone-related decreases in forced expiratory flow than children who received antioxidant supplementation with vitamins C and E. Interestingly, this population of GSTM1-null children with high ozone exposure were at significantly reduced risk of asthma when they also carried the protective NQO1 Pro-Ser polymorphism, whereas children with GSTM1 carrying the serine allele did not show modified risk of asthma (David et al. 2003). These data suggest a hierarchy of genes determining susceptibility, but also the complexity of the gene–environment interactions. In mice, other susceptibility genes involved in lung hyperpermeability to ozone have been located on chromosome 4, which contains the TLR4 gene, a regulator of innate immune responses (Kleeberger et al. 2000). It is of note that TLR4-deficient mice do not exhibit airway hyperreactivity after subchronic ozone exposure as do wild-type mice (Hollingsworth et al. 2004). Susceptibility to ROFA may similarly be associated with TLR4 (Cho et al. 2005). Tumor necrosis factor (TNF)-α has also been identified as a candidate gene in ozone susceptibility in mice (Kleeberger et al. 1997); however, in humans it is not clear if the –308G polymorphism of this gene protects (Li et al. 2006) or not (Yang et al. 2005) from ozone exposure.

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Tobacco Particulates are also a significant problem in indoor environments, with environmental tobacco smoke (ETS) being perhaps the most remediable source of indoor PM. Tobacco smoke is a complex mixture of more than 5000 components including SO2, NO2, particles, and PAH (Leikauf et al. 1995). An epidemiologic survey in the 1990s in the USA showed that 87.9% of nontobacco users had detectable levels of cotinine, a surrogate of ETS exposure (Pirkle et al. 1996). Epidemiologic studies have shown a link between tobacco and asthma, in particular in children. Metaanalyses found a dose-dependent increase in children’s rates of asthma related to increasing number of household smokers (Cook & Strachan 1997). Recent studies have addressed the role of prenatal versus postnatal exposure. In utero exposure to maternal smoking without subsequent ETS exposure was associated with asthma in school-age children (Gilliland et al. 2001), and the risk of developing asthma among children 7 years of age was increased with the mother’s smoking rates in pregnancy (Jaakkola & Gissler 2004). The finding that maternal smoking during pregnancy has a stronger relationship to asthma onset than later ETS exposures is supported by other studies that separated postnatal exposures from maternal in utero exposures (Cunningham et al. 1996). Recently, an increased risk of adult-onset asthma has been reported in relation to ETS exposure (Jaakkola et al. 2003; Piipari et al. 2004).

Effect on allergic primary and secondary responses Tobacco smoke is considered a cofactor in promoting IgE production in humans. Serum IgE, IL-4, and IL-13 are higher in smokers than in nonsmokers (Byron et al. 1994; Sherrill et al. 1994; Oryszczyn et al. 2000; Cozen et al. 2004), and are possibly higher in ETS-exposed subjects (Ronchetti et al. 1990; Oryszczyn et al. 2000; Feleszko et al. 2006). A recent experimental study has definitely shown that in humans ETS exacerbates allergic reactions by evidencing, in nasal lavages from allergic patients challenged with both allergen and ETS, enhanced allergen-specific IgE and Th2 cytokine milieu as compared with allergen alone (Diaz-Sanchez et al. 2006). However, few animal studies have addressed the mechanistic effects of tobacco on allergic responses. These few studies have shown that ETS exposure enhances secondary allergic responses to inhaled allergen in mice, in promoting IgE antibody, Th2 cytokines, peripheral eosinophilia (Seymour et al. 1997), and airway hyperresponsiveness (Moerloose et al. 2005). These results may be explained in part by PAHs contained in ETS and the generation of oxidative stress (see Pollutant section). Indeed, cigarette smoke is an incredibly potent oxidant mixture (Church & Pryor 1985), which is expected to induce ROS leading to the activation of a Th2

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pattern response, although this hypothesis awaits experimental evidence. The question arises whether environmental noxious agents, such as cigarette smoke, could be one of the mechanisms responsible for the suppression of the normal tolerogenic status to harmless allergens (Holt & Mcmenamin 1989). Animal studies have evaluated the effect of ETS on primary allergic sensitization and found increased IgE responses to harmless allergens in rats (Zetterstrom et al. 1985) and in mice, and induced airway eosinophilia on restimulation with allergen in mice (Rumold et al. 2001). More recently, ETS effect on primary sensitization in mice was found to be associated with increases in lung tissue dendritic cells, suggesting that the mechanism involved might involve altered antigen presentation (Moerloose et al. 2006).

Genetics Asthma gene linkage analysis has been performed, including ETS as a potential risk factor for asthma. Genes were found in three chromosomal regions (1p, 5q, 9q) that might interact with ETS to confer risk of asthma (Colilla et al. 2003). Chromosome 1p contains the GSTM1 gene. In the GST gene family, GSTP1 and GSTM1 variant alleles have been associated with asthma in smokers. In utero exposure to secondhand smoke has been associated with increased prevalence of early-onset asthma in GSTM1-null children (Gilliland et al. 2002), as well as passive smoking (≥ 20 cigarettes per day) (Kabesch et al. 2004). In ETS preexposed allergic subjects challenged with allergen, GSTM1-null individuals had a larger increase in allergen-specific IgE than nonaffected individuals (Gilliland et al. 2006). The 5q region was confirmed to interact with ETS exposure in infancy and asthma development (Meyers et al. 2005). This region contains genes coding for IL-13 and CD14, both of which have been linked to ETS and serum IgE levels by candidate gene approaches. Maternal smoking was found to increase the effects of –1055C→T and Arg130Gln IL-13 polymorphisms on total serum IgE level (Liu et al. 2003). Cigarette smoke also contains endotoxin, and passive smoking results in exposure levels of endotoxin that are 120 times higher than in smoke-free air. The CD14 gene encodes part of the endotoxin receptor and the CD14 −159 polymorphism has been associated with decreased IgE levels in ETS-exposed subjects (Choudhry et al. 2005). It is of interest that opposite effects of this polymorphism have been observed on IgE levels in children recruited from the same population but raised in environments with different levels of microbial exposure (see discussion on endotoxin above), suggesting that the environment may elicit different, and even opposite, phenotypes. One possible explanation is the involvement of epigenetic mechanisms able to affect gene expression, as exemplified by the effect of different amounts of folic acid in diet (required for DNA

Environmental Factors in IgE Production

methylation) in early life which change the coat color of agouti mice (Vercelli 2004). The extent to which environmental factors may provoke epigenetic responses represents a very exciting area of future research.

Endotoxin The “hygiene hypothesis” (see Chapter 2) proposes an association between the decreased exposure to microbes in early life and the increased incidence of atopic diseases in the recent decades. Exposure to bacteria and their relationship with atopy has thus attracted much scientific interest. Special attention has been focused on endotoxin, which comprises the outer LPS component of the cell wall of Gram-negative bacteria. Although the word “endotoxin” is used in epidemiologic literature, the effect of “LPS” is analyzed in experimental studies. Epidemiologic studies in humans have found that exposure to endotoxin can protect against, have no effect on, or exacerbate asthma. Similarly, LPS has variable effects on allergic pulmonary inflammation in the mouse depending on exposure time points, doses, and administration procedures of LPS, as well as the model of allergic sensitization. Clearly, this is a complex issue.

Endotoxin: beneficial or detrimental? Children raised on farms have been shown to be at lower risk for allergic sensitization to inhalant allergens. Studies analyzing rural communities in Switzerland and Austria found that farmers’ children who had regular contact with livestock were significantly less likely to demonstrate an elevated level of allergen-specific IgE (Braun-Fahrlander et al. 1999; Riedler et al. 2000). In a search for the protective factor in livestock exposure, endotoxin levels were measured in households and found to be associated with farming and nonfarming lifestyle (Von Mutius et al. 2000). A cross-sectional study in rural areas of Austria, Germany, and Switzerland showed that exposure to stables and farm milk in the first year of life was protectively associated with serum allergenspecific IgE compared with children with no such exposure in their first year. This suggests that the protective factor mediated its effect early in life (Riedler et al. 2001; Stern et al. 2007). Protection from allergic sensitization by early exposure to endotoxin was also found in nonfarming children (Gereda et al. 2000a; Gehring et al. 2002). However, no relationship was found between high endotoxin levels and sensitization to food allergens nor atopic dermatitis, but stratification by parental atopy showed a positive association of endotoxin exposure with sensitization to inhalant allergens in infants with parental atopy (Bolte et al. 2003). Finally, many studies have shown that endotoxin increases asthma severity in adults, but did not report their IgE levels (Michel et al. 1991).

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The dual effect of endotoxin was further analyzed using animal models. Timing of LPS exposure determines the outcome on IgE production, early exposure being associated with protection. Prenatal and neonatal LPS exposure diminished OVA-specific IgE, and suppressed IL-5 and IL-13 production while strongly enhancing IL-10 and IFN-γ production by spleen cells of OVA-sensitized mice (Gerhold et al. 2006). In a rat model, timing of LPS exposure modifies its effect on IgE production: early aerosol LPS inhibited the increase in OVAspecific IgE, while late exposure did not alter the serum antibody levels in sensitized animals (Tulic et al. 2000). The site of LPS injection differentially affects Th1 cytokine secretion, as both systemic and local LPS administration before OVA sensitization reduced serum OVA-specific IgE, and diminished Th2 cytokine production by splenic mononuclear cells, but only local LPS administration induced IFN-γ production by peribronchial lymph nodes (Gerhold et al. 2002). Finally, the effect of LPS exposure is also dependent on the dose of endotoxin. Indeed, mice sensitized by intranasal exposure to OVA plus a high or low dose of LPS showed a bimodal immune response. In contrast to low levels of LPS, which induce Th2 responses, inhalation of high levels of LPS with antigen induces Th1 responses (Eisenbarth et al. 2002). However, both doses of LPS increased serum IgE levels (Kim et al. 2007).

Association with expression of receptors for endotoxin The expression of, and polymorphisms in, the genes encoding endotoxin receptors have been linked to allergic diseases. Two TLRs were found to be differentially involved in endotoxin recognition: many LPS species (from Gram-negative bacteria) signal through TLR4 (Poltorak et al. 1998), while a few LPS species signal through TLR2 (Table 7.1) (Werts et al. 2001; Girard et al. 2003). CD14 is an important cofactor in binding endotoxin and initiating the immune response (Wright et al. 1990). Expression of mRNA encoding TLR2, TLR4, and CD14 was significantly higher among farm children than reference children, and was associated with endotoxin maternal exposure during pregnancy, which was found to be protective on atopic sensitization of the child (Ege et al. 2006). Similarly, prenatal and postnatal LPS exposure, which prevents allergic sensitization, inflammation, and hyperresponsiveness, increased soluble CD14 (sCD14) serum levels and TLR2 and TLR4 mRNA levels in lung tissues from OVA-sensitized mice (Gerhold et al. 2006). In contrast, low sCD14 levels in amniotic fluid or breast milk were found to be associated with an increased risk of developing atopic sensitization (Jones et al. 2002).

Table 7.1 Toll-like receptors (TLR) involved in the recognition of bacteria, viruses, and helminths. (From Duez et al. 2006, with permission.) Receptors

Microbial ligands

TLR2/TLR1 or TLR6

Diacyl lipopeptide (Pam2CSK4) (TLR2/TLR6), triacyl lipopeptide (Pam3CSK4) (TLR1/TLR2) Lipoteichoic acid (TLR2/TLR6) Lipoarabinomannan (Mycobacterium tuberculosis) (TLR2/TLR6) Porins (Neisseria meningitidis) (TLR2/TLR6) Macrophage-activating lipopeptide 2 (MALP2) (TLR2/TLR6) Glycophosphatidylinositol (Trypanosoma cruzi) Viral protein (herpes simplex virus) Phospholipomannan (Candida albicans)

TLR3

Double-stranded RNA from viruses or helminths (Schistosoma mansoni)

TLR4

Lipopolysaccharide (also involves CD14, MD2 molecules) F protein (from the respiratory syncytial virus) Phosphatidylinositol mannosides (Mycobacterium tuberculosis) Glycoinositol phospholipid ceramides (Trypanosoma cruzi)

TLR5

Flagellin

TLR7, TLR8

Guanosine and uridine-rich single-stranded RNA

TLR9

Bacterial and viral DNA

TLR11

Profilin (Toxoplasma gondii )

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Functional mutations in the genes encoding these receptors were found to be associated with risk of atopy. The CD14 gene was identified as the nearest gene to the loci most closely associated with total serum IgE (D5S399/D5S393 on chromosome 5q31–33) (Gao et al. 1999). A C→T transition at position –159 in the promoter of the gene encoding CD14 was associated with increased levels of serum circulating sCD14, which were positively and negatively related to IFN-γ and IL-4, respectively (Baldini et al. 1999). Several studies have found consistent associations between the CD14 –159TT genotype and lower levels of total IgE (Baldini et al. 1999; Koppelman et al. 2001; Buckova et al. 2003; Leung et al. 2003; O’Donnell et al. 2004), whereas others have not (Sengler et al. 2003; Kedda et al. 2005). Environment (endotoxin or allergen exposure) may greatly differ between the populations considered in these studies and gene–environment interactions might explain these discrepancies (Eder et al. 2005). Other studies suggest that the −159C→T CD14 promoter polymorphism might have a differential effect according to the dose and the timing of LPS exposure, and might influence the prevalence of atopy at different ages, as well as the degree of atopy among those already sensitized (Leynaert et al. 2006; Simpson et al. 2006). Results obtained with TLR4 polymorphisms are just as complex: 29 single-nucleotide polymorphisms identified in the TLR4 locus were analyzed in two different cohorts, but none were found to be associated with asthma or total serum IgE (Raby et al. 2002; Werner et al. 2003), whereas asthmatic people with the D299G polymorphism exhibit an increased severity of atopy (Yang et al. 2004). Analysis of polymorphisms in the TLR2 gene has shown that adult patients with atopic dermatitis with the TLR2 R753Q polymorphism had significantly higher total IgE levels (Ahmad-Nejad et al. 2004). However, in children with asthma, no association was found between polymorphisms in TLR2 and total IgE levels (Eder et al. 2004; Noguchi et al. 2004). In conclusion, the protective response to endotoxin exposure appears to be dependent in part on the time of exposure and on the receptors involved in endotoxin recognition. However, one might still elucidate why, paradoxically, a mutation that is thought to diminish responsiveness to LPS also appears to decrease the risk for atopy in highly exposed individuals. More studies are needed to clarify the gene– environment interaction.

Mechanisms of endotoxin effects Endotoxin exposure clearly modulates the profile of the immune response. Increased house-dust endotoxin concentration correlated with increased proportion of IFN-γproducing CD4 T cells, suggesting that indoor endotoxin exposure early in life may protect against allergen sensitization by enhancing type 1 immunity (Gereda et al. 2000b). In mice, an inhibitory effect of LPS was found to be dependent

Environmental Factors in IgE Production

on IL-12 (Gerhold et al. 2002) or nitric oxide synthase 2 activity (Rodriguez et al. 2003). Costimulation of explanted nasal mucosa from atopic children with allergen and LPS abrogated Th2 cytokine expression and increased Th1 cytokine production, as well as the number of IL-10+TLR4+ cells and CD4+CD25+ cells, when compared with allergen stimulation alone. LPS-induced Th1 cytokine production was dependent on IL-10, IL-12, and IFN-γ. LPS effect appeared diminished in adults compared with children (Tulic et al. 2004). In a mouse model, protection by LPS administration paralleled the generation of CD25+IL-10+ T cells (Wang & McCusker, 2006). Finally, a marked downregulation of immune responses was found in exposed children: the production of TNF-α, IFN-γ, IL-10, and IL-12 by peripheral blood leukocytes stimulated with LPS was shown to be inversely related to the endotoxin level in the bedding (Braun-Fahrlander et al. 2002). These findings suggest that LPS may downregulate allergic response through Th1 skewing and/or the expansion of regulatory T cells, but that these effects were more prominent in children, whose developing immune system is still susceptive. Different cytokines have been found to be involved in the IgE-enhancing effects of LPS, depending on the LPS dose. Thus, OVA-specific IgE and IL-4 production enhanced by low-dose LPS was dependent on TNF-α, whereas production enhanced by high-dose LPS was dependent on IL-4 and IFN-γ. IL-12p40 levels in bronchoalveolar lavage fluid were significantly higher in mice sensitized with OVA plus highdose LPS than in those sensitized with OVA alone or OVA plus low-dose LPS (Feleszko et al. 2007). TLR4 and dendritic cells play crucial roles in LPS effects. tlr4-deficient mice subjected to sensitization and pulmonary challenge with OVA had reduction in allergen-specific IgE levels and Th2 cytokine production compared with wildtype mice. The reduced response was attributable, at least in part, to decreased dendritic cell function, as dendritic cells from tlr4-deficient mice expressed lower levels of CD86 and induced less Th2 cytokine production by naive CD4 T cells in vitro (Dabbagh et al. 2002). Similarly, Th2 response induced by low-level inhaled LPS was shown to be mediated by signaling through TLR4, and activation of dendritic cells (Eisenbarth et al. 2002). Inhibition of Th2 responses by LPS was also found to be dendritic cell dependent, but IL-12 independent (Kuipers et al. 2003). Moreover, stimulation of TLR4 on dendritic cells was described to remove the suppressive effect of CD4+CD25+ regulatory T cells on effector T cells, suggesting that ineffective TLR4 signaling may result in unopposed inhibition of both Th1 and Th2 expression by regulatory T cells (Pasare & Medzhitov 2003). MyD88 is a critical common adaptor molecule shared by TLR. The MyD88-independent LPS signaling pathway induces Th2 cell development and is responsive to LPS at low concentration, whereas the MyD88-dependent pathway dominates at

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LPS

Viruses TLR4 EC Mast cell

NKT

Treg CD4+CD25+

BEC

Cell recruitment

mDC

Low High dose of LPS Th1

IL-13

IL-4 (early)

Th2

Increased suppressive function

B cell/plasmacyte Increased IgE Fig. 7.5 Effect of lipopolysaccharide (LPS) on IgE production. Differences in timing, dose, and compartment targeted (local/systemic) by LPS result in the stimulation of different cell types through TLR4, which leads to activation or suppression of IgE production. Plain lines indicate activation, dotted lines inhibition EC: Endothelial cells, BEC: bronchial epithelial cells, MDC: myeloid dendritic cells. (See CD-ROM for color version.)

high LPS concentration and favors Th1 cell development (Kaisho et al. 2002; Piggott et al. 2005). Finally, long-term allergen exposures of tlr4-deficient mice dramatically increased serum IgE and Th2 cytokines compared with similarly challenged wild-type mice, suggesting that tlr4 functions in an antiinflammatory pathway that limits the extent of allergic responses to continued allergen challenge (Hollingsworth et al. 2006). The effect of LPS on allergic sensitization is likely to be the result of the activation of multiple cells expressing LPS receptors (Fig. 7.5). Mast cells, which were found to be involved in Th2 cytokine production induced by low-dose LPS, express TLR4 and on LPS stimulation produced IL-5, IL-13, TNF-α, and IL-6 (the later cytokines being involved in dendritic cell maturation and participating in Th2 polarization, respectively) (Nigo et al. 2006). TLR4 is expressed on CD4+CD25+ T regulatory cells, and LPS activation of CD4+CD25+ cells increases cell survival/proliferation and suppressor efficiency (Caramalho et al. 2003). Functional TLR4 is expressed on invariant Vα14+Jα18+ natural killer (NK)T cells, and in vivo and in vitro LPS stimulation induced rapid production of IL-4 in hepatic iNKT (Askenase et al. 2005). Endothelium and epithelium also express TLR4 and, on LPS stimulation, upregulate the expression of adhesion molecules and increase their production of chemokines and proinflammatory cytokines (Monick et al. 2003; Dauphinee & Karsan 2006). All these mechanisms may contribute to the regulation of the IgE production and explain the apparently conflicting data of the literature.

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Clinical observations and experimental data clearly suggest that viruses may inhibit or exacerbate atopic disorders depending on the virus and the conditions of infection. Seropositivity for hepatitis A was associated with a lower prevalence of atopy (Matricardi et al. 1997; Linneberg et al. 2003), and this has now been linked to the TIM1 gene. TIM-1 functions as a receptor for hepatitis A and is expressed on Th2 cells (McIntire et al. 2001, 2004). Recent studies have characterized several TIM1 polymorphisms in human subjects and also demonstrated that hepatitis A seropositivity protects against atopy, but only in individuals with a specific insertional mutation in the sequence corresponding to amino acid position 157 of TIM1. In contrast, measles vaccination or infection was associated with a higher frequency of atopy (Shaheen et al. 1996; Paunio et al. 2000). Respiratory viruses (respiratory syncytial virus or RSV, rhinoviruses, influenza, and parainfluenza viruses) can also exacerbate atopy and the symptoms of asthma (Sigurs et al. 1995, 2005; Mallia & Johnston, 2002; Umetsu 2004). The role of RSV has been extensively studied and found to have either no effect or accelerate the rate of sensitization depending on the severity of infection and the timing of atopy assessment (Murray et al. 1992; Sigurs et al. 1995; Noble et al. 1997; Schauer et al.2002).

Regulation of IgE production by viruses Viral infections generally induce a Th1 cytokine pattern. It is therefore not surprising that children who had hepatitis A or B virus infection had lower levels of allergen-specific IgE than controls (Kocabas et al. 2006). However, increases in systemic IgE levels have been found following infection with several other viruses like the Epstein–Barr virus, cytomegalovirus, and the measles virus (Griffin et al. 1985), and following vaccination with influenza in nonallergic subjects (Davidsson et al. 2005). In allergic rhinitis subjects, total serum IgE levels have been noted to be increased following experimental rhinovirus infection (Skoner et al. 1995). Increases in IgE levels could be viral- or allergen-specific or could simply represent a polyclonal upregulation of IgE production. In some patients viral infections seem to induce allergic reactions directly. The most prominent example is the induction of acute urticaria shortly after viral infections, especially in children, suggesting the presence of virus-specific IgE (Mortureux et al. 1998). RSV-specific IgE has been detected after infection, the magnitude of the response correlating with degree of wheezing, and children with an atopic predisposition developed an IgE response more readily (Welliver et al. 1981). RSV-specific IgE was also detected in some studies in nasopharyngeal secretions and was significantly higher in infants with wheezing (Welliver et al. 1981), whereas others failed to measure RSV-IgE antibodies in nasal washes

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from children with wheezing (Toms et al. 1996; De Alarcon et al. 2001). Higher virus load associated with more severe illness may be needed to detect the presence of RSV-IgE (Russi et al. 1993; Rabatic et al. 1997). Specific IgE antibodies to parainfluenza virus have also been demonstrated (Welliver et al. 1982). Virus-specific IgE has been reproduced in mouse models, as mice infected with influenza A virus produce virus-specific IgE antibodies and develop active and passive cutaneous anaphylaxis with the influenza A virus antigen (Grunewald et al. 2002). Similarly, infection with RSV induces serum RSV-IgE (Dakhama et al. 2004). The role of IgE in the course of viral infection is unknown and has still to be elucidated. Viruses have also been shown to increase allergen-specific IgE. In a small study of 12 children with asthma attacks precipitated by influenza, the levels of HDM-specific IgE increased during the acute phase, although total serum IgE levels remained unchanged (Lin et al. 1988). Similarly, increased allergen-specific IgE has also been observed after infection of OVA-sensitized mice with influenza virus (Suzuki et al. 1998). Prospective study of a cohort of children hospitalized with RSV bronchiolitis in infancy found that serum IgE antibodies to common allergens were higher in this group compared with the control group, up to age 13 (Sigurs et al. 2000, 2005). The timing of RSV infection is critical for RSV effects on IgE production. Indeed, prior inoculation with RSV decreased the concentration of both total and OVA-specific IgE in serum of OVA/RSV-treated mice, compared with OVA/RSV-treated mice that were not previously inoculated with RSV (Barends et al. 2004). However, RSV infection before OVA sensitization did not enhance the production of OVA-specific IgE (Schwarze et al. 1997). Finally, in vitro, a human B-cell line treated with IL-4 and infected with measles virus increased its IgE class switching in comparison with control cells treated only with IL-4, suggesting that measles virus may increase the production of IgE during infection in allergic patients (Imani et al. 1999). RSV activation of the antiviral protein kinase in B cells was also shown to induce isotype switching to IgE in vitro (Rager et al. 1998). Therefore, virus effect on IgE production appears to depend on the virus, the severity of infection, and the timing of exposure and assessment. Moreover, it is likely that exposure to other environmental factors (as was shown with allergen) and genetic predisposition might affect the outcome of virus infection on allergic diseases. This latter is suggested by a study comparing the effect of influenza A viral infection on OVA-induced responses in C57BL/6 mice and BALB/c mice. Indeed, prior influenza A viral infection of C57BL/6 mice, which have a Th1 bias compared with BALB/c mice, prevented Th2-type allergic responses (Wohlleben et al. 2003).

Virus-induced mechanisms Most studies devoted to understanding the effect of viral infection on IgE production have focused on respiratory

Environmental Factors in IgE Production

viruses and their regulation of the production of Th1- and/or Th2-type cytokines. Virus infection alone was found to lead to Th1 cytokine production, both in respiratory secretions and PBMCs (Van Schaik et al. 1999; Gern et al. 2006). In mice, influenza A respiratory infection leads to high local IFN-γ production by CD4+ and CD8+ T cells, and strong Th1 responses in lymph nodes, while a low to undetectable amount of ex vivo Th2 cytokine expression is detected (Doyle et al. 1999; Grunewald et al. 2002; Roman et al. 2002). However, the initial induction of RSV-IgE antibody production is likely to be dependent on the Th2 response. Consistent data from clinical studies described a significant local and systemic Th2 response that was associated with RSV bronchiolitis, influenza, and parainfluenza infections in infants (Roman et al. 1997; Bendelja et al. 2000; Pala et al. 2002; Tripp et al. 2002; Legg et al. 2003; Kristjansson et al. 2005; Murai et al. 2007). RSV protein G was found to induce a Th2-like immune response both in humans and mice (Alwan et al. 1993; Jackson & Scott 1996). In mice, lung infection with RSV resulted in significant increases in mRNA expression for IL-4, IL-13, CD40, and IgE, and for both of the high- and low-affinity receptors in the lungs (Johnson & Graham 1999; Varga et al. 2000; Dakhama et al. 2004). IL-13 production was elicited upon reinfection only if RSV first infection occurred early in life with live viruses, emphasizing the requirement for active lung infection (Dakhama et al. 2005). Both these Th1 and Th2 responses induced by respiratory viruses might play a role in exacerbating asthma. KLH sensitization of BALB/c mice after influenza A viral infection and clearance promoted dual allergen-specific Th1 and Th2 responses and enhanced later allergen-specific asthma. This effect was dependent on IFN-γ secreted during acute viral infection and on lung dendritic cells (Dahl et al. 2004). Viruses have been found to upregulate some chemokines involved in IgE production, as well as Th1 and Th2 cytokines. Mononuclear cells from infants with severe respiratory tract illness stimulated in vitro with RSV produce RANTES and MIP-1α (Tripp et al. 2002). Significantly higher levels of RANTES and MIP-1α have been detected in nasopharyngeal secretions of RSV-, influenza virus- or parainfluenza virusinfected infants (Kristjansson et al. 2005; Murai et al. 2007). Moreover, RSV alone, or in combination with IFN-γ, increases the secretion of RANTES from respiratory epithelial cells (Saito et al. 1997; Olszewska-Pazdrak et al. 1998). All this may contribute to the exacerbation of allergic diseases. Molecular events involved in the regulation of Th1 and Th2 cytokine production induced by viruses have still to be identified, but some TLRs will certainly play a role. Indeed, rhinovirus and RSV are partly recognized by TLR3 and TLR4 (associated with CD14) (see Table 7.1), and were also found to increase the expression of these TLRs on airway epithelial cells (Kurt-Jones et al. 2000; Hewson et al. 2005; Groskreutz et al. 2006).

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Therefore, respiratory viruses may exacerbate asthma and IgE production not only by increasing the production of Th1 and Th2 cytokines and several chemokines, but also by modifying the response to subsequent exposure of other environmental factors.

Parasites Underdeveloped nations are relatively protected from the allergy epidemic, but are more chronically infected with various parasite worms than industrialized nations. A hallmark of a helminth infection is the large amount of IgE detected in the serum of infected animals and humans (Yazdanbakhsh et al. 2001). Helminths induce the production of large amounts of polyclonal IgE and very low levels of helminth antigen-specific IgE, the latter providing an important component of the immune protection (Lynch et al. 1993; Capron & Capron 1994; Allen & Maizels 1996).

Parasites and allergen-specific IgE Despite this enhanced total IgE production associated with helminths, a consistent negative relation between helminth infections and the prevalence of atopic responses has been seen in various developing countries (Van Den Biggelaar et al. 2000; Nyan et al. 2001; Yazdanbakhsh et al. 2001, 2002; Huang et al. 2002; Cooper et al. 2003). Along the same lines, long-term antihelminthic chemotherapy increased HDM reactivity in chronically infected children (Van Den Biggelaar et al. 2004). Experimental animal models also support a dual effect of helminths on allergen-specific and total IgE production. In mice, infection with Strongyloides stercoralis or Nippostrongylus brasiliensis leads to a reduction of allergenspecific IgE in the bronchoalveolar lavage fluid, whereas serum total IgE is increased (Wang et al. 2001; Wohlleben et al. 2004). In a model of food allergy, infection with the enteric helminth Heligmosomoides polygyrus resulted in a decrease of allergen-specific IgE production in the serum (Bashir et al. 2002). However, the role of helminths in protecting against allergies is not universally accepted, as several studies do not show a protective role (Cooper et al. 2006; Karadag et al. 2006). One explanation for contradictory observations may be provided by a paradigm in which lowlevel or acute helminth infections enhance allergic reactivity and high-level or chronic infections suppress allergic inflammation (Cooper 2002).

Mechanisms involved in IgE regulation by parasites The role of Th2 responses in protection against helminth infection has been extensively documented (Lynch et al. 1993; Capron & Capron 1994; Allen & Maizels 1996). An absolute requirement for IL-4 receptor-dependent mechanisms in the clearance of worms has been demonstrated

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(Urban et al. 1998), and thus it is not surprising that such infections lead toward a Th2 polarization. Indeed, Ascaris lumbricoides infections are associated with a highly polarized type 2 cytokine response as indicated by increased IL-4 and IL-5 production by mononuclear cells from infected patients (Cooper et al. 2000). Schistosoma mansoni egg-laying male and female worms and, more particularly, schistosome eggs are potent inducers of Th2 immune responses when injected into mice (Vella & Pearce 1992; Mangan et al. 2006). Mechanisms may partly involve recognition of schistosome N-glycans by CD1d, which induces Th2 polarization in vitro (Faveeuw et al. 2002, 2003). Helminth infections have also been associated with suppressing the development of allergic responses and appear to be, at least in part, associated with the induction of T regulatory responses (Kamradt et al. 2005; Maizels 2005). The regulatory cytokines IL-10 and TGF-β are produced after helminth infection (Mahanty & Nutman 1995; Doetze et al. 2000; Van Den Biggelaar et al. 2000; Cooper 2002). Protection against S. mansoni worms was suggested to involve compartmentalization of immunity, with a helminth-modified type 2 pulmonary immune response characterized by elevated IL10, specific IgG4 antibodies, and reduced IL-5 in the lungs (Mangan et al. 2006). Heligmosomoides polygyrus, S. mansoni and N. brasiliensis have been shown to prevent allergen-induced responses in an IL-10-dependent mechanism (Bashir et al. 2002; Mangan et al. 2004; Wohlleben et al. 2004; Kitagaki et al. 2006). Finally, increased natural regulatory CD4+CD25+Foxp3+ cells were found in thoracic lymph nodes of H. polygyrusinfected/OVA-exposed mice (Kitagaki et al. 2006). In contrast, the suppressive effect of N. brasiliensis excretory/secretory products on the development of allergic responses was found to be independent of the presence of TLR2 and TLR4, IFN-γ, and IL-10 (Trujillo-Vargas et al. 2007). However, these TLRs and others may potentially participate in the regulation of the T-cell response and IgE production, as other helminthderived products, such as helminth RNA, glycolipids, and profilin signal through TLR3, TLR2 and TLR4, and TLR11, respectively (see Table 7.1) (Campos et al. 2001; Oliveira et al. 2004; Aksoy et al. 2005; Yarovinsky et al. 2005). Another mechanism for reduced IgE in certain helminth infections may involve chemokine inhibition, as S. mansoni, S. haematobium and S. japonicum eggs were found to produce a chemokine-binding protein able to block the interaction of MIP-1α and RANTES with host chemokine receptors and their biological activity (Smith et al. 2005). Recent data suggest an evolutionary balance between allergy and helminth infection. Human polymorphisms originally described as predisposing toward asthma have now been linked to resistance to helminth infection. A polymorphism in the 3′ UTR of STAT6 was linked to differential resistance to Ascaris in a Chinese population, as well as to asthma in Japan (Peisong et al. 2004). Similarly, an IL-13

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promoter allele (1055T) first identified as increasing gene transcription, and thereby asthma risk, has subsequently been shown to confer greater resistance to schistosome infection in Mali (Kouriba et al. 2005). These findings suggest that one mechanism for increased susceptibility to allergy may be human evolutionary adaptation to ubiquitous helminth infection.

Conclusion Our expanding knowledge of the effects of environmental factors on IgE production (Fig. 7.6) has broad implications for unraveling the complex factors leading to both allergic disease pathogenesis and the ongoing marked increase in the prevalence of IgE-mediated disease. It is anticipated that an understanding of the genes and gene products that control inflammatory responses to environmental factors, and conversely of environmental factors that may regulate gene function, will clarify the molecular/cellular mechanisms of host response to the exposure, and may lead to new prevention and treatment approaches.

LPS Viruses

DEP LPS Viruses ETS

DEP DEP Metals Ozone ETS Viruses Parasites

–

+

+ CD80 + CD28 Th0

Th1 +

–

LPS Parasites

– IL-4 IL-13

IL-10

YY

Y

Y

Y

YY

LPS

+

YY

–

+

B cell

Th2

MHC-II + TCR Treg

Y

APC

YY

Y

Y +

+

Mast cell

Plasma cell Antigen Y Specific lgE

DEP Viruses

DEP Metals LPS Viruses

Fig. 7.6 Environmental factors and their sites of interaction with IgE production. Environmental factors can act at different stages of the immune allergic reaction, by affecting antigen-presenting cells (APC), the polarized T-cell subsets Th1, Th2, and regulatory T cells (Treg), B-cell switch, and mast cell activation. See text for definition of abbreviations. (See CD-ROM for color version.)

Environmental Factors in IgE Production

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Environmental Factors in IgE Production

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Antigen-presenting Dendritic Cells and Macrophages Bart N. Lambrecht and Hamida Hammad

Summary

Introduction

The lung contains many subsets of dendritic cells (DCs) that are distributed in various anatomic compartments. These cells possess all the machinery to take up and process antigen into the major histocompatibility complex class I and II presentation pathways. In homeostatic conditions, a fine-tuned balance exists between plasmacytoid and myeloid DCs necessary for maintaining tolerance to inhaled antigen and avoiding overt inflammation. Tolerance occurs when immature DCs present antigen to T cells. Several lung protective pathways therefore suppress the function of DCs in homeostatic conditions. One such pathway involves alveolar macrophages that actively suppress T cell and DC activation in the lung. The same subsets of DCs are also implicated in the process of allergic sensitization. Allergens can directly or indirectly activate the myeloid DC network of the lungs and this can lead to Th2 immunity. Wellknown adjuvant factors like endotoxin, cigarette smoke, diesel particles, ozone, and viral infection also lead to Th2 immunity by altering DC function. It is increasingly clear that DCs also play important roles in establishment of eosinophilic airway inflammation seen in ongoing asthma. DCs are important for local activation of memory Th2 cells, for attracting lymphocytes and inflammatory cells to the lungs, and for regulating the function of antiinflammatory Treg cells. In the future, it will be important to study how DCs communicate with the various structural cells of the airways, how they are recruited to the lungs and draining lymph nodes, and how their removal might affect antimicrobial defense mechanisms in the lungs. Critical cytokines produced by epithelial cells could perpetuate allergic inflammation through a DC intermediate mechanism. Based on these new insights on airway DC biology, several approaches that interfere with DC function show potential as new intervention strategies for these ever-increasing diseases.

The prevalence of sensitization to allergens and allergic diseases has reached epidemic proportions in Western societies. Allergic sensitization is the presence of IgE to common environmental allergens, and is controlled by Th2 cells that provide help for IgE synthesis by B cells. In addition, many of the inflammatory cell types found within sites of allergic inflammation, such as eosinophils and mast cells, depend on Th2 cells for their development and function. Th2 cells will only react to allergen when it is presented in the context of major histocompatibility complex (MHC) molecules by professional antigen-presenting cells such as dendritic cells, macrophages, and B cells. DCs are the most important antigen-presenting cells found throughout the body and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to (aero)allergens. Increasingly, these cells are also recognized for their potential to maintain ongoing effector responses and therefore they might be crucial in maintaining allergic inflammation. B cells present allergen to T cells mainly in the context of immunoglobulin synthesis, for which they need T-cell help. Macrophages are seen as scavenger cells that can also regulate the function of DCs. They are equally important in controlling pathogen clearance and tissue remodeling.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Dendritic cell terminology and heterogeneity DCs were originally described by their capacity to efficiently process and present antigens and to prime naive T cells (Steinman & Cohn 1973). Over the last three decades, multiple DC subtypes have been defined, differing in phenotype, localization, and immune function (Shortman & Liu 2002). Myeloid DCs, Langerhans cells (LC) as well as natural type I interferon (IFN)-producing cells (IPCs, also called plasmacytoid DCs, pDCs) are part of the hematopoietic system and have a relatively short half-life in tissues. To maintain DC numbers in the tissues, there is continuous renewal of DCs from hematopoietic precursors residing in the bone marrow or within the skin (for LC in steady-state conditions; Merad

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CD34+

GM-CSF TNF-a

CD14+ CD1a–

M-CSF IL-6

CD14– CD1a+

Monocyte

TGF-b IL-15

Interstitial Langerhans cell “dermal” DCs Fig. 8.1 MHCII-positive mucosal dendritic cell network visualized by MHCII staining on a murine tracheal wholemount. Trachea was taken from a naive unimmunized mouse. (See CD-ROM for color version.)

et al. 2002). A universal feature of DCs in tissues is their typical morphology with long dendrite-like extensions (hence their name) that can be demonstrated by staining for MHC class II (Fig. 8.1). Myeloid DCs in humans express markers shared with monocytes/macrophages such as CD33, CD4, and CD11c, whereas in the mouse they typically express CD11c and CD11b. In humans, pDCs were described in the bloodstream, lungs, and lymph nodes as lineageneg CD11clo CD123+ BDCA2+ cells (Shortman & Liu 2002). In the mouse, pDCs express specific markers (120G8, PDCA-1) as well as cell markers shared with myeloid DCs (MHC class I and II, CD11c) but also with granulocytes (Gr1) and B cells (B220) (Shortman & Liu 2002). LC express CD1a, langerin, and intracellularly demonstrate so-called Birbeck granules (BG), “tennis racket”shaped organelles. Recently, another DC subset sharing marker expression with natural killer (NK) cells was identified as “natural killer DC.” These DCs originate as cells with NK function, which are capable of taking up killed material for presentation to T cells (Taieb et al. 2006). DCs originate in the bone marrow from a CD34+ precursor and circulate in the bloodstream as a monocyte-like precursor before entering peripheral tissues (Geissmann et al. 2003; Bonasio & von Andrian 2006). The exact nature of the precursor DC cell type is currently unknown (Fig. 8.2), and could vary for myeloid versus plasmacytoid DC, inflammation versus steady state, or for lymphoid organ versus peripheral tissues (del Hoyo et al. 2002; Naik et al. 2006).

Antigen uptake There are various ways by which antigen-presenting cells can acquire foreign antigen. A first mechanism is via receptor-

CD11c+ CD1a– CD9 CD68 CD2 FXIIIa

CD11c+ CD1a+ Birbeck E-cadherin Langerin

GM-CSF IL-4

FIt3L

CD11c– IL-3Ra IL-3 CD40L

Monocyte-derived DC Plasmacytoid DC CD11c+ CD1a+/– CD83 DC-SIGN

CD11c– CD1a– CD4+ IL-3Ra (CD123) BDCA2/4

Fig. 8.2 Different origins and fate of dendritic cell (DC) subsets. All DCs originate from a CD34+ precursor in the bone marrow. These cells then further differentiate under the influence of various cytokines into Langerhans cells of the skin, interstitial DCs of tissues, monocyte-derived DCs, or plasmacytoid DCs, each expressing specific markers. Certain cytokines like IL-6 and M-CSF inhibit DC development when precursors are continuously exposed to them. (See CD-ROM for color version.)

mediated endocytosis involving clathrin-coated pits. Immature DCs express a plethora of specialized cell receptors for patterns associated with foreign antigens, such as the C-type lectin carbohydrate receptors (langerin, DC-SIGN, dectin, BDCA-2, macrophage mannose receptor, and the unique carbohydrate receptor DEC-205) (Figdor et al. 2002). Lectin-receptor mediated uptake by DCs results in about 100-fold more efficient presentation to T cells as compared with antigens internalized via fluid phase (Mahnke et al. 2000). Interestingly, langerin is a C-type lectin displaying mannose-binding specificity and is exclusively expressed by DCs that display BG, such as lung DCs and skin LC, but seems to be functionally irrelevant (Kissenpfennig et al. 2005). Pollen starch granules were shown to bind to C-type lectin receptors on alveolar macrophages (AMs) and DCs, although internalization occurred only in macrophages (Currie et al. 2000). Also, Pestel demonstrated that Der p1 uptake into cultured DCs involves mannose receptor-mediated endocytosis, and that process is more efficient in DCs obtained from allergic donors (Deslee Gt et al. 2002). In allergic individuals, DCs are furthermore armed with allergen-specific IgE bound to the high-affinity IgE receptor (FcεRI), thus enabling efficient receptor-mediated endocytosis of the allergen (Novak et al. 2003). A second mechanism of antigen uptake is constitutive macropinocytosis that involves the actin skeleton-driven engulfment of large amounts of fluid and solutes (about

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one cell volume/hour) by the ruffling membrane of the DC followed by concentration of soluble antigen in the endocytic compartment (de Baey & Lanzavecchia 2000). Macropinocytosis seems to be a dominant mechanism involved in the uptake of recombinant Bet v1 and Phl p1 pollen allergens by LCs and of Der p1 by cultured DCs, and can be inhibited by cytochalasin D and amiloride (Noirey et al. 2000). Thirdly, immature LCs, cultured DCs, pDCs, and macrophages have been shown to phagocytose particulate antigens such as latex beads and even whole bacteria, as well as apoptotic cells, and this could be the dominant mechanism of uptake of particulate allergens (Inaba et al. 1993; Ochando et al. 2006). The extracellular antigens that are taken up by any of these mechanisms accumulate in the endocytic compartment, where they are loaded on newly synthesized and recycling MHC class II molecules but may also be transported into the cytosol, where they become accessible to the class I antigen presentation pathway, a process called “cross-presentation.”

Antigen presentation

Foreign antigen

MHCI peptide

Allergens are extracellular antigens, and like most extracellular antigens they are processed for presentation onto MHC class II molecules. The T-cell receptor of CD4 T lymphocytes will respond only to processed antigen in the context of MHC class II, a process called “MHC restriction.” In contrast to MHC class I, which is expressed on all nucleated cells types, MHC class II is mainly expressed by professional antigenpresenting cells, but also to a lower extent by epithelial cells, mast cells, and eosinophils. MHC class II molecules, which consist of an αβ heterodimer, assemble in the endoplasmic reticulum with the chaperone invariant chain (Ii) (Fig. 8.3) (Chapman 1998). The cytoplasmic tail of Ii contains a motif that targets the Ii-MHC class II complexes to the endosomal pathway. After entry into endosomal/lysosomal compartments, Ii undergoes stepwise degradation by lysosomal proteases, leaving a small fragment of Ii known as the class II-associated invariant chain peptide (CLIP) associated with the MHC class II peptidebinding groove (Riese et al. 1996). This event stabilizes and protects the peptide-binding site from interacting with other polypeptides. To bind the antigenic peptides that have been generated in acidic endosomal and lysosomal compartments, the binding site of the MHC class II αβ dimers must be free of its previous occupant, CLIP. Displacement of CLIP is facilitated by several factors: (i) low pH, which favors an “open” conformation in the MHC class II molecule and peptide exchange; (ii) the activity of the chaperone H-2DM (HLADM), which stabilizes the open conformation (Denzin & Cresswell 1995); and (iii) by proteolytic elimination of the regions of Ii that flank the CLIP peptide (Kropshofer et al.

168

Recycling

Endocytosis Endosome

MIIC Cathepsin S MHC-CLIP

HLA-DM CLIP

TGN

ER

Cytosolic antigen

MHCI-b2

Proteasome

Presentation of exogenous antigens on MHC class II to CD4+ T cells

MHCII peptide

TAP

MHCII-Ii

ab heterodimer

hsp

Fig. 8.3 Antigen processing in DCs. The classical pathway of MHCI presentation is initiated by the cytosolic degradation of protein antigen by the proteasome, a complex multicatalytic protease. This ubiquitindependent protein degradation generates peptide fragments of defined length (“molecular ruler” function). These fragments bind to heat-shock proteins and target an ATPase-dependent TAP (transporter associated with antigen presentation) on the endoplasmic reticulum (ER) membrane. TAP transports peptides (8–12 amino acids) into the lumen of the ER. Inside the ER, peptides are loaded onto newly synthesized MHCI (a chain and b2-microglobulin) molecules. Chaperone molecules (tapasin, calnexin) prevent immature MHCI molecules from being loaded with irrelevant endogenous ER proteins and disengage after high-affinity binding of cytosolic peptides. Peptide-loaded complexes are transported to the cell membrane after passing through the Golgi and trans-Golgi (TGN) network. For successful expression of MHCII–peptide complexes on the cell surface, endocytosed and partly digested antigen needs to be efficiently mixed with newly synthesized or recycling MHCII molecules in a specialized subcellular compartment called MIIC. The highly polymorphic MHCII molecules consist of an ab heterodimer which assembles in the ER with a third molecule, the invariant chain (Ii). After transport to the TGN, the MHCII–Ii complex is targeted to the endocytic MIIC pathway, via a signal sequence on the Ii chain. In addition, some abIi complexes are directly targeted to the cell membrane, followed by recycling to the endocytic compartment. In the endocytic pathway, invariant chain proteolysis by cathepsin S generates the CLIP (class II-associated invariant chain peptide) fragment, which binds to the peptide-loading groove of the ab heterodimer. Binding of CLIP protects the peptide-binding groove from interacting with irrelevant ER peptides (generated for MHCI loading). The CLIP fragment is then exchanged for immunogenic peptides, generated in the endocytic/lysosomal pathway by proteolysis of intact protein antigen into peptides 12–20 amino acids long. The exchange is catalyzed by HLA-DM. The binding of high-affinity antigenic peptide stabilizes the ab heterodimer, HLA-DM binds the released CLIP fragment and physically disengages. Due to the loss of association with the intact invariant chain, stabilized ab–peptide complexes are targeted to the cell membrane and are transiently expressed before being recycled via the endocytic pathway.

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1995). CLIP removal and binding of the antigenic peptides direct the MHC class II/peptide complexes to the cell surface for presentation to CD4+ T cells. Within the endocytic compartment, antigen is cleaved into short immunogenic peptides by proteolytic enzymes of the cathepsin family. Antigen loading on MHC class II molecules occurs in an acidic cellular compartment rich in newly synthesized MHC class II molecules, called the “MIIC compartment” (Nijman et al. 1995). This multivesicular complex is located at the intersection of the biosynthetic [endoplasmic reticulum (ER), Golgi complex, secretory granules] and endocytic pathway of vesicle transport within the cell and contains the MHC II-related HLA-DM peptide exchanger, which is essential for loading high-affinity antigenic peptides on MHC II (Denzin & Cresswell 1995). Alternatively, there is a pathway of peptide loading onto preformed MHC class II molecules that have been internalized into mildly acidic endosomal vesicles after being expressed on the cell surface (Koppelman et al. 1997). Surprisingly, proteolysis of antigen by immature DCs can also occur extracellularly through secreted proteases, generating peptides that can be loaded onto empty cell surface-expressed MHC class II (De Bruijn et al. 1992). It is at present unclear how allergens are loaded onto MHC class II molecules by DCs. In sensitized individuals, internalization of allergens via receptor-mediated endocytosis by multivalent cross-linking of the high-affinity IgE receptor (FcεRI) on immature DCs targets the antigen to the MIIC compartment (Maurer et al. 1998; Holloway et al. 2001; Novak et al. 2003). In contrast, the generation of peptide–MHC complexes derived from macropinocytosis of Bet v1 and Phl p1 pollen allergens was shown to be inhibited only partly when the pH of the endosomes was altered, suggesting that parts of the molecules were not metabolized in the lysosomal MIIC compartment (Riese et al. 1996).

Presentation of endogenous antigen on MHC class I to CD8+ T cells After a virus enters a host and infects cells, the major adaptive immune response that clears the infection is mediated by CD8+ cytotoxic T lymphocytes (CTLs). These cells also provide the major defense against cancers. CD8+ lymphocytes recognize infected cells that display on their surface MHC class I molecules presenting antigenic peptides derived from viral proteins or tumor antigens expressed in the cytoplasm. All nucleated cells have the capacity to present peptides derived from the cytoplasm onto MHC class I molecules. After recognition of peptide–MHCI, the CTL kills the infected cells and thereby eliminates the source of viral replication or the abnormal/cancerous cells. In the classical view, bystander cells that have endocytosed viral debris cannot process such antigens to form MHC class I-restricted complexes and are therefore not targeted. Recently, however, this view has been challenged (see next section).

Antigen-presenting Dendritic Cells and Macrophages

The classical pathway of MHC class I presentation is initiated by the cytosolic degradation of the protein antigen by the proteasome, a complex multicatalytic protease (Fig. 8.3). This ubiquitin-dependent protein degradation generates peptides of a defined length. A fraction of these peptides (> seven residues) is transported into the ER through the transporter associated with antigen processing (TAP). Inside the ER, peptides are loaded onto newly synthesized MHCI, and the accompanying chaperone proteins that keep MHCI stabilized in the absence of peptide are released. Peptide-loaded complexes are transported to the cell surface for display.

Presentation of exogenous antigen on MHC class I to CD8+ T cells A second, less well-defined, approach to load peptides on MHCI molecules is for the DCs to capture extracellular antigens and to process these captured exogenous antigens into the MHC class I pathway. This form of presentation is referred to as “cross-presentation.” In the field of experimental allergy, evidence is present for this cross-priming. Aerosolization of ovalbumin (OVA) in OVA-sensitized mice leads to the generation of MHC class I-restricted CD8+ T cells that can regulate the magnitude and duration of IgE responses (MacAry et al. 1997) and suppress airway inflammation (Wells et al. 2007). The mechanisms underlying cross-presentation are not very well defined. Particulate exogenous antigens are phagocytosed and there is gradual accumulation of ER proteins in a form of fused ER/phagosome organelle where MHCI can be loaded (TAP-independent cross-presentation). Under some conditions, the phagocytosed antigen is transferred into the cytosol (Rodriguez et al. 1999), in a process involving Sec61, a transporter normally involved in ER import (Desjardins 2003). The protein can then be degraded by closely membraneassociated proteasome and subsequently transported back into the phagosome/ER by TAP in order to bind and be presented by MHC class I molecules (TAP-dependent crosspresentation) (York & Rock 1996). DCs are particularly efficient in generating antiviral or antitumoral MHCI-restricted CD8+ T cell responses in vitro and in vivo. Indeed, when DCs were purified from mice injected with protein antigens or viruses, they cross-presented the antigen on their MHC class I molecules. Moreover, in many of these experiments, antigen cross-presentation was found only in DCs (Heath & Carbone 2001). Consistent with this finding, Jung et al. (2002) generated transgenic mice expressing diphtheria toxin receptor under the control of CD11c promoter, and found that when CD11c+ DCs were depleted by treatment with diphtheria toxin, mice failed to generate a CD8+ T cell response to cell-associated antigen and intracellular pathogens. DCs are heterogeneous and can be divided into pDCs and conventional myeloid DCs (mDCs). The latter subset can be subdivided further according to their expression of CD8α.

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Which subsets are able to cross-present antigens to CD8+ T cells is not fully understood. In experiments in which mice were immunized with cell-associated antigen (den Haan et al. 2000) or infected with virus (Allan et al. 2003, 2006) and DC subsets isolated, the CD8α+ DC was identified as the primary APC that stimulated naive CD8+ T-cell responses. In viral infection models, however, it is less clear whether crosspriming is an exclusive function of CD8α+ DCs. The nature of the antigen also influences what type of DC participates in cross-priming. For example, cell-associated OVA is crosspresented by CD8α+ DCs, while OVA/anti-OVA immune complexes are cross-presented by both CD8α+ and CD8α− DCs. It is generally believed that pDCs do not cross-prime exogenous antigens. However, type I IFN has been suggested to promote cross-priming (Le Bon et al. 2003). As pDCs produce type I IFN during viral infections, they may enhance the cross-priming process to viral antigen during viral infections (Liu 2005).

Table 8.1 Expression of chemokine receptors by DC subsets.

mDCs

Integrated function of dendritic cells in the immune response Dendritic cell activation DCs originate in the bone marrow and circulate in the bloodstream as monocyte-like precursors before entering peripheral tissues (Geissmann et al. 2003; Bonasio & von Andrian 2006). DC migration is a tightly regulated process in which many chemokines and other factors are involved (Table 8.1 and Fig. 8.4) mDCs are attracted to peripheral tissues by a specific set of chemokines such as macrophage inflammatory protein (MIP)-3α (CCL20) and epithelial β defensins acting on CCR6 (Yang et al. 1999; Cook et al. 2000; Biragyn et al. 2002; Bonasio & von Andrian 2006). pDCs respond preferentially to SDF1 (CXCL12) and CXCL9-11 and the newly described chemerin, a ligand for ChemR23 (Vermi et al. 2005). Once DCs extravasate, they form a network in the upper layers of the epithelium and lamina propria of the airways (see Fig. 8.1), gut, and skin. Here, DCs are said to be in an immature state, specialized for internalizing foreign antigens but not yet able to activate naive T cells (Banchereau & Steinman 1998; Vermaelen et al. 2001). The DC network serves a patrolling function, continuously scanning the environment for foreign antigens. The DC is endowed with numerous ancient receptors for foreign antigenic signature molecules such as bacterial cell walls, viral and bacterial DNA, and foreign sugar molecules (Fig. 8.5). These so-called pathogen-associated molecular patterns (PAMPs) are recognized by Toll-like receptors (TLR1–10) and C-type lectin receptors, which are abundantly expressed on the surface of DCs (Figdor et al. 2002). The expression of various TLRs varies between DC subsets, particularly in human DCs. In humans and mice, pDCs preferentially express TLR7 and TLR9, and thus respond to the corresponding ligands (imidazoquinolines and single-stranded

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pDCs

Chemokine receptors/ molecules controlling migration

Ligands

CCR1 CCR2 CCR4 CCR5 CCR6 CCR7 CCR8 CXCR3 CXCR4

CCL5, CCL3, CCL7 CCL2, CCL7, CCL8, CCL13 CCL17, CCL22 CCL3, CCL4, CCL5, CCL8 CCL20 CCL19, CCL21 CCL1 CXCL9, CXCL10, CXCL11 CXCL12

PAFR S1PR EP4 CD38 CystLT1 ChemR23

PAF Sphingosine 1-phosphate Prostaglandin E2 CD31 Cysteinyl leukotrienes Chemerin

DP1 IP ChemR23 FPRL1

Prostaglandin D2 Prostaglandin I2 Resolvin E1 Lipoxin A4

CCR7 CXCR3 CXCR4 ChemR23

CCL19, CCL21 CXCL9, CXCL10, CXCL11 CXCL12 Chemerin

RNA versus CpG motif bacterial DNA) but not to ligands for TLR2, TLR3, TLR4, or TLR5. In contrast, in vitro generated conventional monocyte-derived DCs or ex vivo isolated mDCs express all TLRs except TLR9. In addition to the direct molecular recognition of foreign antigenic structures, exposure to foreign antigens or necrotic cell death leads to tissue damage and this by itself can activate the DC system. DCs express a plethora of receptors for these so-called damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1) protein, heatshock proteins, uric acid, adenosine triphosphate (ATP), complement cascade fragments, neuropeptides, prostaglandins, etc. (Fig. 8.5). Many of these compounds not only activate the already residing DCs but also attract new waves of cells to the periphery (Lambrecht 2001, 2006a; Lotze & Tracey 2005). DC activation and maturation in the periphery can occur directly by ligation of DAMP or PAMP receptors and can occur indirectly through activation of the same receptors on the surrounding structural cells such as keratinocytes, epithelial cells, or fibroblasts (Lambrecht & Hammad 2003a). Keratinocytes and lung epithelial cells make granulocyte–

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Epithelium

Allergen

Antigen-presenting Dendritic Cells and Macrophages

Microbes

4. “Danger” LPS, peptidoglycan Mannan CpG DNA, ds RNA PAMPs

3. MIP3a/CCL20 b-defensin 1.

2. TNF-a GMCSF O2 radicals DAMPs

DC

CCR+

CCR7+

5.

Migration via afferent lymph Immature DC High Ag uptake Low signal 2

MIP3b/CCL19

T cell area

Mature DC Low Ag uptake High signal 2

DC

HEV IL2 7. Selection Activation

IL2R CD8

CD4

8.

CXCR5 9.

Clonal expansion

CD44+ L-sel–

Th1/2

Efferent lymph

L-sel+ CD44–

6. Naive T cell

IL-4 Th2

B cell

CD40L germinal centre Bone marrow plasma cell

CD8

Recirculation to tissues

Fig. 8.4 Induction of the primary immune response by dendritic cells (DCs). 1 Under baseline conditions and on exposure to foreign antigens, epithelia produce macrophage inflammatory protein (MIP)-3a (CCL20) and b-defensin to attract CCR6+ immature DCs from the bloodstream. 2 Resident cell types produce inflammatory mediators and growth factors that attract and activate the recently recruited DC. 3 DCs capture allergens and other foreign antigens such as bacteria and viruses. 4 DCs can discriminate between “dangerous” antigens, and nonpathogenic antigens such as self antigens and probably most allergens, by recognizing certain viral and bacterial patterns. 5 The recognition of infection and tissue damage upregulates the CCR7 and CXCR4 and DCs migrate to the T-cell area of draining lymph nodes where the ligand MIP-3b and SDF-1 is constitutively expressed. During this migration, DCs lose the capacity to take up antigen, but become strong stimulators of naive T cells by their strong expression of costimulatory molecules (signal 2). 6 In the T-cell area, DCs produce chemokines to attract naive T cells that continuously leave the

bloodstream via the high endothelial venules (HEV). 7 Naive T cells are first arrested and then selected for antigen specificity. The recognition of the correct peptide–MHC induces the activation of naive T cells, which will lead to further terminal differentiation of DC function. 8 The activation of T cells leads to autocrine production of IL-2 and to clonal expansion of antigenspecific CD4+ and CD8+ T cells. These cells differentiate into effector cells that leave the lymph node via the efferent lymphatic. These effector cells are poised to migrate to peripheral tissues, especially to inflamed areas. 9 On contact with DCs, some antigen-specific CD4+ T cells upregulate CXCR5 receptor and migrate to the B-cell follicles of the draining lymph node. Here they further interact with germinal center DCs to induce CD40L-dependent B-cell immunoglobulin switching and affinity maturation (germinal center reaction). Most high-affinity B cells go to the bone marrow to become immunoglobulin-producing plasma cells. See text for definition of other abbbreviations. (See CD-ROM for color version.)

macrophage colony-stimulating factor (GM-CSF) and thymic stromal lymphopoietin (TSLP) that activate the underlying DC network. These cytokines are regarded as the principal maturation-inducing factors that can also be used to mature DCs in vitro.

Dendritic cell migration to the draining lymph nodes The recognition of danger (PAMPs or DAMPs) by peripheral dendritic cells dramatically alters the migration behavior of DCs and thus induces the surface expression of CCR7 on

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Toll-like receptors 1–10

Protease-activated receptors Complement receptors

Intracellular receptors NOD1/2 TLR7,9 PKB C-type lectin receptors Dectin Macrophage mannose receptor DEC205 BDCA-2

Prostanoid receptors DP1, EP4, IP Neuropeptide receptors NK1, CGRPR Purinergic receptors P2X, P2Y Receptors for Uric acid, HMGB1 Heat shock proteins

Fig. 8.5 Expression of “danger” receptors by dendritic cells. Dendritic cells express the ancient receptors of the innate immune system also expressed by macrophages, such as the Toll-like receptors (TLRs) and C-type lectin receptors. These receptors react to foreign pathogen-associated molecular patterns (PAMPs). In addition, DCs express numerous receptors for inflammatory mediators and necrotic cell debris, the so-called damageassociated molecular patterns (DAMPs). The exact receptors for uric acid, high mobility group box 1 (HMGB1) protein, and heat-shock proteins are not yet known. (See CD-ROM for color version.)

peripheral DCs (Fig. 8.4) (Bonasio & von Andrian 2006). The ligands for CCR7 are secondary lymphoid chemokine (SLC, now known as CCL21) and MIP-3β (CCL19), which are expressed at the luminal side of afferent lymph vessels and by the T-cell area of draining lymph nodes (Sallusto et al. 1998). Another factor attracting DCs to the lymph node is the lipid mediator sphingosine-1-phosphate (S1P). Blocking the S1P-type receptor dramatically reduces the migration of lung DCs to the mediastinal lymph nodes (Idzko et al. 2006). The responsiveness of CCR7 to CCL19 and CCL21 and the consequent lymph node migration of DCs are controlled by lipid mediators such as the leukotrienes and prostaglandins. Prostaglandin (PG)D2 acts on the DP1 receptor expressed by lung DCs and suppresses the migration of lung DCs (Hammad et al. 2003a). The downstream metabolite 15-deoxy(Δ)1214PGJ2 could also slow down the migration of DCs, by acting on the nuclear PPARγ receptor (Hammad et al. 2004a). Leukotriene (LT)B4 is exported from the cytoplasm of the DC by the multidrug resistance (MDR) protein, where it is metabolized into LTC4, which regulates the CCR7 responsiveness (Robbiani et al. 2000). In contrast to skin DCs, it was recently shown that lung DC migration is less dependent on the export of LTB4 by MDR (Jakubzick et al. 2006). For emigration of DCs from the skin, the CCR8 receptor for the chemokine CCL-1 (also known as I-309 in humans and TCA-3 in mice) acts in concert with CCR7 (Qu et al. 2004). Whether this is also true for lung DC migration remains to be shown. It is clear that the regulation of DC migration by arachidonic acid metabolites is very amenable to modification by various drugs already being developed for allergy treatment.

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Just as in the gut, airway DCs extend long dendrites to the lumen of the airways, forming bud-like extensions at the border of the air interface (Brokaw et al. 1998). Within a few hours after inhalation, airway mDCs and pDCs have taken up fluorescently labeled antigen within the draining mediastinal lymph nodes (Vermaelen et al. 2001; Hammad et al. 2003a; De Heer et al. 2004). After 24 hours, both mDCs and pDCs in the mediastinal lymph nodes contain antigen inside vesicles of the cytoplasm. What is unclear at present is whether pDCs take up antigen in the periphery of the lung and subsequently migrate to the nodes, or whether antigen is being transported to them by migratory mDCs or even a specific subset of CD8α− CD11b− migratory DCs recently described by Belz et al. (2004). Transport of immunogenic material from one nonmigratory DC to another is certainly a possibility, as CD8α+ DCs injected into the lung induce an immune response in the mediastinal node without migrating into it (Hammad et al. 2004b). Under steady-state conditions, mDCs continuously migrate to draining lymph nodes and present either (self-)autoantigens or harmless antigen in a tolerogenic form (Steinman & Nussenzweig 2002). Once they have reached the draining lymph nodes, mDCs express intermediate levels of costimulatory molecules and MHC class II.

T-cell activation by dendritic cells By upregulating the lymph node-homing chemokine receptors, DCs that have seen foreign antigen thus direct their interest to the regional draining lymph node T-cell area where they interact with recirculating T cells and B cells (Fig. 8.6) (Stoll et al. 2002). DCs that have arrived in the lymph node undergo short-lived interactions with T cells in the paracortical region and, during this initial antigen independent event, individual T cells are scanned for specificity for antigen. When antigen is being recognized, there is formation of a more long-term immunologic synapse, leading to full-blown T-cell activation, after which the T cell detaches, divides, and differentiates into an effector and possibly memory T cell. DCs also transport antigen without degrading it and thus offer intact protein to B cells at the interface between the paracortex and B-cell follicle (Castellino et al. 2006; Qi et al. 2006). DCs that have reached the T-cell area have lost the capacity to take up antigen, and now express a plethora of cell adhesion and surface molecules interacting with T cells not previously expressed on peripheral-based DCs. This phenotype is called “mature dendritic cell” implying that functionally these cells are now fully adapted to induce naive T-cell responses. DCs express the antigen on MHC molecules, and provide so-called costimulatory molecules [CD80/CD86 family; tumor necrosis factor (TNF)/TNFR family] (Fig. 8.7) together with cytokines to optimally expand and differentiate T cells for the particular task that needs to be carried out to clear the foreign antigen. Initially, T cells are stimulated in the draining lymph node, but after a

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Antigen-presenting Dendritic Cells and Macrophages

Uptake of Ag in mucosa Local presentation to Teff

DC network takes up antigen

Teff

Lung effector site Day 4 87654 0

Tcm DC migration DC maturation

Draining node Day 0 Tcm

Naive T

naive

Tcm

Other nodes/spleen Day 4 87654 0

Day 4 876543210

KJ1-26

Clonal selection Proliferation Differentiation (Tcm or Teff) CFSE Ovalbumin

T cell

Epithelial cell

Dendritic cell

Eosinophil

Goblet cell

Teff Tcm Tnaive

Fig. 8.6 Integrated overview of DCs and CD4+ T-cell migration during primary and secondary immune responses. Antigen (Ag) is taken up by DCs across the mucosal impermeable barrier. Mucosal DCs continuously migrate from the lungs to the T-cell area of mediastinal lymph nodes (MLNs). In the presence of inflammation, this process is amplified, increasing the possibility that pathogenic substances will be presented to recirculating naive T cells (Tnaive) or central memory T cells (Tcm). At the same time, DC maturation will be fully induced. When mature DCs arrive in the MLNs, they select specific T cells from the polyclonal repertoire of cells that migrates through the high endothelial venules and T-cell area. Within 4 days, this will lead to clonal expansion of antigen-specific T cells. This is illustrated in the FACS plot where antigen-specific T cells, identified by staining with a specific KJ1-26 antibody for the ovalbumin T-cell receptor, dilute the CFSE signal. When a T cell has acquired a certain threshold number of divisions (usually four or more), it will leave the MLN to become either a Tcm cell or an effector

T cell (Teff). This is where migration pathways separate and consequently the anatomic requirements for reactivation diverge. The Tcm cells will extravasate in other nondraining nodes and spleen, and will eventually accumulate in the spleen over time (see FACS plot for nonlung-draining lymphoid tissue where both divided Tcm and naive T cells can be found). Reactivation of these cells will therefore only occur in central lymphoid organs. In contrast, Teff cells will extravasate in peripheral sites of inflammation (see FACS plot for lungs, where only divided cells can be found), including the lung when the original inflammation is still present. In contrast to naive T cells, which are excluded from lung tissues, these Teff cells can be stimulated by local airway DCs to mediate their effector function. In this scenario, alternative antigen-presenting cells might be eosinophils or even epithelial cells, expressing MHC molecules. (See CD-ROM for color version.)

few cell divisions they acquire effector potential (Lambrecht et al. 2000a), start expressing chemokine receptors for inflammatory chemokines expressed at sites of pathogen entry, and lose the expression of CD69, thus rendering them insensitive to the lymph node-retention signal S1P (Shiow et al. 2006).

Th polarization by dendritic cells DCs are crucial in regulating the immune response by bridging innate and adaptive immunity. Signals from the type of antigen and the response of the innate immune system are translated by DCs into a signal that can be read by the cells of the adaptive immune response, leading to an optimal

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Surface ligand

MHC/peptide-TCR Signal 1

Ag uptake

Ag processing

Signal 2 DCSIGN-ICAM-3 CD80/86-CD28 B7RP-ICOS PDL1/PDL2-PD1 CD40-CD40L OX40L-OX40

T cell

T cell response Activation Differentiation

Fig. 8.7 Formation of the immunologic synapse. Dendritic cells internalize antigen and present it into the groove of MHCI and MHCII molecules to, respectively, CD8 and CD4 T lymphocytes (signal 1). In the process of recognizing foreign antigen, they are induced to express some costimulatory molecules for naive T cells (signal 2). The T cell bearing a specific receptor upregulates CD40L, which induces the terminal differentiation of dendritic cells (DCs) through CD40 signaling, inducing the full expression of all costimulatory molecules like CD80, CD86, ICOSL, etc., that further polarize the immune response. In addition, DCs produce cytokines to expand and differentiate the T-cell response. TCR, T-cell receptor. (See CD-ROM for color version.)

response for a particular insult (Fig. 8.8). Together, these signals consist of provision of a particular density of peptide– MHC, the expression of costimulatory or Th-polarizing cell surface molecules, and the expression of soluble cytokines and chemokines that polarize T cells or enhance their survival. At the same time, DCs also control the function and expansion of regulatory T (Treg) cells that tightly control overzealous inflammatory T-cell responses. Although controversial, it has been suggested over recent years that particular functions of DCs such as tolerance or immunity or Th1/Th2 differentiation might be a specialized function of defined subtypes of DCs (Maldonado-Lopez et al. 1999; Rissoan et al. 1999). Others have refuted this idea and have claimed that DCs are very versatile cells, and can virtually induce any type of response depending on the need of the moment (Kapsenberg 2003). Recent studies have suggested that lung mDCs mediate protective immunity to inhaled antigens only when properly activated by innate immune system-activating immune signals, acting through TLRs or other recognition receptors. Under inflammatory conditions such as those provided by lipopolysacharide (LPS) or virus infection, the expansion of T cells induced by mDCs leads to the generation of Th1 or Th2 effector cells in the mediastinal nodes (Eisenbarth et al. 2002; Brimnes et al. 2003). The signals that determine the type of response after encountering a pathogen in the lung are delivered by DCs in the lymph node. Sporri and Reis e

174

High IL-6 Low IL-12 T1/ST2L/IL-33 CCL2?

OX40L CD86 Jagged

DC

+

Secreted

ICOSL dim CD86 dim CD80 Jagged?

Th0

Low IL-6 Low IL-12 IL-10 TGFb PGD2

IL-4 IL-5 IL-13 TNF-a

Treg

IL-10 TGF-b

Th17

IL-17

Th1

IFN-g TNF-a

IL-2

High IL-6 High TGF-b CD80 Delta ICAM-1

Th2

IL-23 High IL-6 High IL-12 High IL-18 CCL3 High IFNg?

Fig. 8.8 T-helper cell polarization by dendritic cells (DCs). Depending on the type of antigen, the dose, the genetic background, and the tissue environment where antigen is first introduced, DCs can induce various types of Th response, tailormade to protect the host, while avoiding autoimmunity. Often the response is extremely well balanced, to avoid tissue damage, while allowing clearance of the threat. The various cytokines and costimulatory molecules that favor a particular direction are indicated. See text for further explanation and for definition of abbreviations. (See CD-ROM for color version.)

Sousa (2005) recently suggested that DC maturation and provision of peptide–MHC to T cells is not sufficient to generate effector cells. Cytokines are dominant signals that determine the quality and quantity of an effector immune response. During generation of an efficient effector immune response, DCs also have to overcome suppression by Treg cells, and the dominant way by which they seem to do this is by production of interleukin (IL)-6 that releases the suppression by naturally occurring Tregs (Pasare & Medzhitov 2003). Certain pathogens or pathogen-derived products induce the direct secretion of Th1-polarizing cytokines by DCs and thus instruct the type of immune response generated. Alternatively, it was shown that tissue environment can also determine Th differentiation. Stumbles et al. (1998) and Dodge et al. (2003) have shown that resting respiratory tract DCs mainly induced Th2 responses. As a direct proof that mDCs can induce Th2 sensitization in the lung, it was shown that intratracheal injection of bone marrow-derived mDCs pulsed with OVA induced a Th2 response to OVA and subsequently led to severe features of asthma when mice were rechallenged with OVA aerosol (Lambrecht et al. 2000b). Recently, much information has been gathered on how exactly Th2 polarization is controlled by DCs. Mice that

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conditionally overexpress TSLP in the lungs mount vigorous Th2 responses in the airways, in a process driven by DCs (AlShami et al. 2005; Zhou et al. 2005). TSLP is increased in the airways of asthmatic patients (Ying et al. 2005) and it can activate mDCs to prime naive CD4+ T cells to differentiate into proinflammatory Th2 cells (Watanabe et al. 2005). The Th2 skewing effect induced by TSLP-activated DCs was found to be dependent on OX40L, a costimulatory molecule shown to play a critical role in the development of allergic lung inflammation (Ito et al. 2005). As TSLP is such an important factor in the sensitization process, it will be very important to study how its release by epithelial cells and other inflammatory cells is regulated in response to natural allergen exposure. The type of immune response induced by mDCs also depends on the strength of the activating innate immune system stimulus. Elegant studies by Eisenbarth et al. (2002) showed that low-level TLR4 agonists prime mDCs to induce a Th2 response, by inducing their full maturation, but not their production of IL-12. High-level LPS administration induced high level IL-12. These findings might help to explain the effects of environmental exposure to LPS on the reduced incidence of allergic sensitization. IL-12 seems to be a dominant cytokine for Th1 responses in the lung, yet the LPS-induced Th1 response induced by mDCs in the lung was not dependent on IL-12 (Kuipers et al. 2003). Although IL-12 may be redundant for some Th1-inducing stimuli, it is certainly sufficient as retroviral overexpression of this cytokine in mDCs in the lung induced strongly polarized Th1 responses (Kuipers et al. 2004). The transcription factor Tbet is a master controller of Th1 development and was recently found to be expressed in DCs in addition to T cells. Tbet –/– DCs were less potent at inducing Th1 responses and produced less proinflammatory cytokines (Wang et al. 2006). The exact role of IL-23 and IL-27, as well as surface expression of the Notch ligands Delta/Jagged, in DC-driven Th1 development in the lung remains to be studied (Amsen et al. 2004). Cells of the innate immune system, such as NK cells, are recruited to the draining nodes by DCs and could also be an early source of Th1-polarizing cytokines (Martin-Fontecha et al. 2004). Recently, Th17 cells producing IL-17 that regulate autoimmune inflammation have been identified. They are induced by a cytokine cocktail of transforming growth factor (TGF)-β, IL-6 and their numbers are expanded by IL-23 (Veldhoen et al. 2006). DCs can produce all these factors, and have been shown to induce Th17 cells in a model of experimental allergic encephalomyelitis (Veldhoen et al. 2006). In view of the fact that IL-17 also plays a crucial role in regulating allergic airway inflammation, the involvement of the DC– Th17 axis in allergy will have to be ascertained (SchnyderCandrian et al. 2006).

Tolerance induction by dendritic cells Immature DCs are distributed throughout the lung and are at the focal control point determining the induction of

Antigen-presenting Dendritic Cells and Macrophages

pulmonary immunity or tolerance (Akbari et al. 2001, 2002; Lambrecht & Hammad 2003b). Airway DCs form a dense network in the lung, ideally placed to sample inhaled antigens, and these cells migrate to draining lymph nodes to stimulate naive T cells (Banchereau & Steinman 1998; Lambrecht et al. 1998; Vermaelen et al. 2001). As most allergens are immunologically inert proteins, the usual outcome of their inhalation is tolerance and inflammation does not develop on chronic exposure (De Heer et al. 2004; Ostroukhova et al. 2004). This is shown best for the model antigen OVA. When given to the airways of naive mice via aerosolization, nasal droplet aspiration, or intratracheal injection, it renders mice tolerant to a subsequent immunization with OVA in adjuvant, and effectively inhibits the development of airway inflammation, a feature of true immunologic tolerance (De Heer et al. 2004; Ostroukhova et al. 2004). It was, therefore, long enigmatic how sensitization to natural allergens occurred. An important discovery was the fact that most clinically important allergens, such as the major Der p1 allergen from house-dust mite (HDM), are proteolytic enzymes that can directly activate DCs or epithelial cells to break the process of tolerance and promote Th2 responses (Hammad et al. 2001; Kheradmand et al. 2002). However, other allergens, such as the experimental allergen OVA, do not have any intrinsic activating properties. For these antigens, contaminating molecules or environmental exposures (respiratory viruses, air pollution) might pull the trigger on DC activation (Dahl et al. 2004). Eisenbarth et al. (2004) showed that low-level TLR4 agonists admixed with harmless OVA prime DCs to induce a Th2 response, by inducing their full maturation, yet not their production of IL-12. This process has been recently described as being dependent on the activation of the adaptor molecule MyD88 in pulmonary DCs (Piggott et al. 2005). This is clinically important information as most natural allergens such as HDM, cockroach, and animal dander contain endotoxin, and undoubtedly other TLR agonists (Braun-Fahrlander et al. 2002). From the above, it seems that the decision between tolerance or immunity (in the lungs) is controlled by the degree of maturity of mDCs interacting with naive T cells, a process driven by signals from the innate immune system (Herrick & Bottomly 2003; De Heer et al. 2005). It has indeed been shown that immature mDCs induce abortive T-cell proliferation in responding T cells and induce Tregs (Akbari et al. 2001; Brimnes et al. 2003; Ostroukhova et al. 2004). Another level of complexity arose when it was shown that (respiratory) tolerance might be a function of a subset of pDCs (De Heer et al. 2004; Oriss et al. 2005). Removal of pDCs from mice using depleting antibodies led to a break in inhalational tolerance to OVA and to development of asthmatic inflammation (De Heer et al. 2004). The precise mechanisms by which pDCs promote tolerance are unknown but, in the absence of pDCs, mDCs become more immunogenic and induce the formation of effector cytokines from dividing T cells (De Heer

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et al. 2005). The negative signal that is delivered by pDCs has not been elucidated, but could be the high-level expression of programmed death ligand (PDL)-1, delivering a negative signal to T cells or to mDCs directly (De Heer et al. 2004; Kohl et al. 2006). Additionally, pDCs can produce the tryptophanmetabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which has strong inhibitory activity on T-cell proliferation (Fallarino et al. 2004) and inhibits inflammatory airway disease (Hayashi et al. 2004). Interestingly, IDO expression has been demonstrated recently in pulmonary CD11c+ cells (Swanson et al. 2004), although the exact cell type involved has not clearly been identified. Another explanation to the tolerogenic properties of pDCs is related to their immature phenotype, as it has been demonstrated that immature DCs can induce Tregs (Dhodapkar & Steinman 2002). Ex vivo at least, lung-derived pDCs promoted formation of Treg cells specific for OVA (De Heer et al. 2004). If pDCs promote tolerance and mDCs immunity, it is logical to assume that the balance between both subsets is tightly controlled. In support of this, the administration of Flt-3 ligand, a cytokine that induces the differentiation of pDCs, to sensitized mice reduced all the features of asthma (Edwan et al. 2004), whereas administration of GM-CSF expanded mDCs and strongly enhanced sensitization and inflammation (Stampfli et al. 1998). Kohl and colleagues recently demonstrated that blockade of the anaphylatoxin C5a receptor during priming reactions to HDMs or harmless OVA was able to strongly enhance Th2 priming, through effects on the selective recruitment of immunogenic mDCs over tolerogenic pDCs (Lambrecht 2006a; Kohl et al. 2006).

in the number of alveolar mDCs, displaying a mature phenotype with increased expression of MHCII, OX40L, and CCR7 (Fainaru et al. 2005), and demonstrating an increased immunostimulatory capacity. Moreover, RunX3–/– DCs are able to mount inflammatory responses to otherwise harmless inhaled antigens, possibly through their lack of responsiveness to locally secreted TGF-β (Fainaru et al. 2004). In mice normally resistant to HDM-induced asthma and airway hyperresponsiveness (AHR) (C3H mice), Treg cell depletion using the CD25-depleting antibody similarly led to increased numbers of pulmonary mDCs with elevated expression of MHCII, CD80, and CD86, and an increased capacity to stimulate T-cell proliferation and Th2 cytokine production. In normally susceptible A/J mice, Tregs did not suppress inflammation and AHR. These data suggest, therefore, that resistance to allergen-driven AHR is mediated in part by CD4+CD25+ Treg cell suppression of DC activation and that the absence of this regulatory pathway contributes to susceptibility (Lewkowich et al. 2005). In the rat, it was shown that Tregs also control the level of CD86 expression on lung DCs and are responsible for the tolerance to inhaled allergen that occurs upon repeated exposure to allergens (Strickland et al. 2006). In humans with allergy, there is a reduction in the number and possibly function of Tregs (Kuipers & Lambrecht 2004), but it is unclear at present whether this would also lead to altered function of DCs in these patients.

Control of lung dendritic cell function by regulatory T cells

Although it has not been proven directly in humans that DCs are responsible for the Th2 sensitization process, some in vitro findings strongly imply these cells. The way in which allergens are handled by DCs is fundamentally different between atopic and nonatopic individuals (Bellinghausen et al. 2000; De Wit et al. 2000; Hammad et al. 2002; Lambrecht & Hammad 2002). When DCs obtained from HDM-sensitive asthmatics were exposed to the endotoxin-free major allergen component Der p1 in vitro, they mainly produced IL-10, but little IL-12. They expressed the costimulatory molecules CD86 and PDL-1 (Hammad et al. 2001, 2003b). When monocytederived DCs from non-HDM-allergic donors or nonallergic donors were exposed to Der p1, they mainly produced IL-12, expressed CD80, and produced the Th1-cell-specific chemokine CXCL10. Not surprisingly, monocyte-derived DCs from allergic patients induced Th2 cell responses of naive alloreactive T cells in vitro, whereas those DCs from nonallergic individuals induced Th1 responses. Therefore, the way HDM is handled by DCs is crucial to the generation of Th2 cell sensitization, and is clearly different in patients with allergy to HDM. The cysteine protease activity of Der p1 induced these changes in the DCs of allergic individuals, indicating that the activation of a protease-activated receptor on DCs leads to

The signals that determine the type of response after encountering a pathogen in the lung are delivered by DCs in the lymph node. Induction of DC maturation and provision of peptide–MHC to T cells is not sufficient to generate effector cells (Sporri & Reis e Sousa 2005). During generation of an efficient effector immune response DCs have to overcome suppression by Tregs, and the dominant way by which they seem to do this is by producing the cytokine IL-6, which counteracts the suppression by naturally occurring CD4+CD25+ Tregs (Doganci et al. 2005). Established airway inflammation seems to be regulated by Tregs expressing membrane TGF-β or secreting bioactive TGF-β and possibly IL-10 (Kearley et al. 2005). This is a pleiotropic cytokine with significant antiinflammatory and immunosuppressive properties in the lungs, as reduced expression of this cytokine exacerbates airway pathology in an asthma model (Ostroukhova et al. 2004). Several papers now support the concept that Tregs alter airway DC function. Mice lacking the transcription factor RunX3, involved in downstream TGF-β signaling, spontaneously develop asthma features (Fainaru et al. 2004). In the lungs of these mice, there is a strong increase

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aberrant cellular activation in patients with asthma (Hammad et al. 2001). The enzyme activity of Der p1 could also indirectly facilitate antigen presentation by DCs in vivo, by allowing access to intraepithelial DCs through cleavage of epithelial tight junctions and by locally activating the release of epithelial GM-CSF (Wan et al. 1999; Lordan et al. 2002). In this way, the epithelial response to allergens might also determine the type of adaptive immune response induced by DCs. Supporting this idea, DCs treated with lipase (an industrial allergen displaying an enzymatic activity) have been reported to induce a strong recall CD4+ T-cell response associated with a high production of IL-4 and IL-13, and a low production of IFN-γ (Lindstedt et al. 2005). However, allergens without enzymatic activity can also directly activate DCs to induce Th2 priming. For instance, phytoprostane lipids contained in pollen allergens can induce DC maturation and inhibit IL-12 production by LPS-activated DCs. When cocultured with allogeneic naive T cells, pollen-treated DCs polarized the immune response toward Th2 (Traidl-Hoffmann et al. 2005).

Dendritic cells in allergic asthma Not only do DCs play a role in the primary immune response to inhaled allergens, but they are also crucial for the outcome of the effector phase in asthma. Indeed, the number of mDCs is increased in the airways of sensitized and challenged mice during the acute phase of the response (van Rijt et al. 2002). However, during the chronic phase of the pulmonary response, induced by prolonged exposure to a large number of aerosols, respiratory tolerance develops through unclear mechanisms. During this regulatory phase, the number of mDCs in the lungs steadily decreased, and this was associated with a reduction of bronchial hyperreactivity. Inflammation, however, reappeared when mDCs were given (Koya et al. 2006). The role of mDCs in the secondary immune response was further supported by the fact that their depletion at the time of allergen challenge abrogated all the features of asthma, including airway inflammation, goblet cell hyperplasia, and bronchial hyperresponsiveness (Lambrecht et al. 1998; van Rijt et al. 2005). Again, the defect was restored by intratracheal injection of mDCs. It therefore seems that mDCs are both necessary and sufficient for secondary immune responses to allergen. Costimulatory molecules expressed by DCs could play a crucial role in established asthma. Pulmonary DCs upregulate the expression of CD40, CD80, CD86, ICOS-L, PD-L1, and PD-L2 during eosinophilic airway inflammation, particularly on contact with Th2 cells (De Heer et al. 2004; van Rijt et al. 2004, 2005). Costimulatory molecules might be involved in activation of effector T cells in the tissues. In allergen-challenged mice, mDCs might also be a prominent source of the chemokines CCL17 and CCL22, involved in attracting CCR4+ Th2 cells to the airways (Vermaelen et al.

Antigen-presenting Dendritic Cells and Macrophages

2003; Kohl et al. 2006). The proallergic cytokine TSLP induces the production of large amounts of CCL17 by mDCs, thus contributing to the recruitment of a large number of Th2 cells to the airways, explaining how it may act to enhance inflammation (Zhou et al. 2005). In humans, allergen challenge leads to an accumulation of myeloid, but not plasmacytoid, DCs in the airways of asthmatics, concomitantly with a reduction in circulating CD11c+ cells, showing that these cells are recruited from the bloodstream in response to allergen challenge (Jahnsen et al. 2001; Upham et al. 2002). In stable asthma, the number of CD1a+ DCs is increased in the airway epithelium and lamina propria, and these numbers are reduced by treatment with inhaled corticosteroids (Moller et al. 1996). Based on the above data from mouse studies of asthma, it is very likely that part of the efficacy of inhaled steroids might be due to their effects in dampening airway DC function. According to current thinking, epithelial dysfunction, either intrinsic to asthma or caused by persistent inflammation, leads to epithelial release of profibrotic cytokines, such as epidermal growth factor and TGF-β, acting on fibroblasts and smooth muscle cells, disturbing the equilibrium between epithelial destruction, and growth and repair. Moreover, asthmatic epithelium might release factors such as GM-CSF, TSLP or chemokines that profoundly influence DC survival and/or function (Fig. 8.9). The exact consequences of this epithelial remodeling on the functioning of the airway DCs are currently unknown. Finally, many inflammatory cell types such as mast cells, basophils, and eosinophils are recruited to the airways in chronic asthma. These cells release many mediators such as cytokines, neuropeptides, enzymes, and lipid mediators that may also profoundly influence DC function and, in this way, might perpetuate ongoing inflammation (Lambrecht & Hammad 2003b). As only one example, it is known that histamine and PGD2, both released by mast cells upon crosslinking, reduce the potential of DCs to produce bioactive IL-12, and in this way contribute to Th2 polarization (Idzko et al. 2002; Hammad et al. 2003b). The exact role of pDCs in ongoing allergen-specific responses in asthma is currently unknown. It was shown that pDCs accumulate in the nose, but not lungs, of allergenchallenged atopics (Jahnsen et al. 2000). This is not strictly correct: the same author has shown that deliberate challenge of HDM-sensitized atopics with HDM leads to rapid recruitment of mDCs (but not pDCs) to the airway mucosa within the time-frame of the LPR and, moreover, that this response is attenuated by steroid treatment (Jahnsen et al. 2001). When pDCs were pulsed with pollen allergens, they were as efficient as mDCs in inducing Th2 proliferation and effector function (Farkas et al. 2004). Others have suggested that pDCs might also confer protection against allergic responses, as in the mouse. In children at high risk of developing atopic disease, the number of circulating pDCs was reduced (Hagendorens et al. 2003).

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Allergens

Allergens break down epithelial tight juntions

TSLP PGE2 IL-10 CCL5, attracts Eos, Th2, DCs

GM-CSF enhances DC survival

CCL20, attracts DCs

Th2 DC

CCL17/CCL22 chemokines attract Th2 cells

IL-4 IL-13

Th2

DC

CCR4 CCR8

Th2 Local costimulation to Th2 cells

New effector cells migrate to lung

Ag loaded DCs migrate to draining lymph nodes

CCR7 Th0

Th2 IL4

Stimulation of recirculating memory Th2 cells and naive T cells

IgE synthesis

Dendritic cells in atopic dermatitis Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by eczematous lesions and is associated with elevated serum IgE levels, and tissue and blood eosinophilia. AD is characterized by the infiltration of Th2 cells and the increased secretion of Th2-related cytokines (IL-4 and IL-5) and chemokines (TARC) in early lesions. However, Th1 cells also emerge during the chronic phase of the disease (Fiset et al. 2006). Recent evidence suggests that DCs in the skin and the blood of patients with AD play a pivotal role in the generation and/or control of inflammation. In patients with AD, DCs highly express FcεRI, the highaffinity receptor for IgE (Stary et al. 2005). Two FcεRI + subsets of mDCs have been identified in skin lesions of AD patients: (i) LC expressing CD1a and BG found in the epidermis; and (ii) inflammatory dendritic epidermal cells (IDEC) only found in inflamed skin (Wollenberg et al. 1995, 1996). In AD, FcεRI+ LCs bearing the antigen migrate from the skin to the draining lymph nodes where they activate FcεRI-mediated

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Fig. 8.9 Interaction between epithelial cells and dendritic cells (DCs) during established inflammation. Allergens stimulate epithelial cells to release chemokines and growth factors for DCs, Th2 cells, and eosinophils. Thymic stromal lymphopoietin (TSLP) and granulocyte–macrophage colony-stimulating factor (GM-CSF) are instrumental in inducing a Th2 prone phenotype in lung DCs. Epithelial cell tight junctions are opened up by protease activity of certain allergens, such as Der p1 from house-dust mite. In this way, allergens gain access to the DC extensions. The recruited DCs are also stimulated directly by allergen and produce even more chemokines for Th2 cells (TARC and MDC). Locally attracted Th2 cells interact with DCs in the airways, leading to local DC maturation and T-cell costimulation of effector cytokine production. These activated Th2 cells eventually control the inflammatory process by activating eosinophils and mast cells, and by feeding back on the epithelium and DCs. At the same time, DCs also migrate to the draining lymph nodes where they restimulate recirculating memory Th2 cells to become effector cells, and they recruit new cells into the response. In this way, effector cells are continuously replenished. DCs are also crucial for maintaining IgE synthesis, through their stimulation of IL-4-producing Th2 cells. See text for definition of abbreviations. (See CD-ROM for color version.)

Th2 immune responses. At the same time, LCs also present allergen-derived peptides locally to transiting T cells and induce a classic secondary immune response. Moreover, the aggregation of FcεRI on LCs stimulates them to release chemokines such as IL-16, TARC, MDC, and monocyte-attracting chemokines (Novak et al. 2004a). All these molecules contribute to the recruitment of FcεRI hi IDEC into the skin. IDECs are only found under inflammatory conditions, display high stimulatory capacities toward T cells, and serve as amplifiers of the allergic-inflammatory immune response. The stimulation of FcεRI on IDEC induces the release of IL-12 and IL-18 leading to the priming of Th1 cells, probably contributing to the Th1 response observed in the chronic phase of AD. In the mouse, overexpression of TSLP under the control of a keratinocytespecific promoter led to an AD-like phenotype. In these mice, skin DCs were likely activated to induce Th2 responses to some self or environmental antigen (Yoo et al. 2005). In addition to mDCs, pDCs have been found in increased numbers in the blood of AD patients and express FcεRI (Novak et al. 2004b; Stary et al. 2005). pDCs can process allergens by FcεRI-IgE and promote Th2-type immune responses.

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However, in contrast to LCs or IDEC, pDCs fail to accumulate in skin lesions of AD patients and seem to be retained in the bloodstream. Whether this is due to a lack of recruitment from the blood to the skin or to the high sensitivity of pDCs to proapoptotic signals present in AD skin, remains unclear.

Role of dendritic cells in allergic rhinitis In allergic rhinitis (AR), CD4+ Th2 cells control inflammation by secreting Th2 cytokines, but little is known about how these cells are activated to cause disease. Elevated numbers of CD1a+ LC are present in the nasal mucosa of symptomatic grass pollen-sensitive AR patients, and these numbers further increase upon relevant allergen challenge to the nose (Fokkens et al. 1989; Godthelp et al. 1996; Fokkens 1999). In symptomatic AR patients, DCs bearing allergenspecific IgE in the nasal mucosa are present (Kleinjan et al. 1997). In HDM-allergic perennial AR patients, the number of CD1a+ and CD11c+ MHCII+ DCs was higher in the epithelium and lamina propria of the nasal mucosa compared with healthy control subjects. In AR, DCs had a more mature phenotype and were found in close approximation with T cells. Similarly, in a mouse model of OVA-induced AR, CD11c+ DCs accumulated in areas of nasal eosinophilic inflammation and clustered with CD4+ T cells. To address the functional role of DCs in maintaining inflammation, CD11c+ DCs were conditionally depleted during allergen challenge by systemic administration of diphtheria toxin (DT) to CD11cDT-Receptor-Tg mice. In the absence of CD11c+ DCs, nasal OVA challenge in OVA-sensitized mice did not induce nasal eosinophilia, and did not boost OVA-specific IgE levels or Th2 cytokine production in the cervical lymph nodes. Conversely, when OVA pulsed DCs were administered intranasally to sensitized mice, they strongly enhanced OVAinduced nasal eosinophilia and Th2 cytokine production. These data in humans and mice suggest an essential role for nasal DCs in activation of effector Th2 function leading to allergic rhinitis, and identify DCs as a novel target for therapeutic intervention (Kleinjan et al. 2006). In support, treatment of AR patients with intranasal corticosteroid therapy reduced dramatically the numbers of DCs in the nasal mucosa (Holm et al. 1999).

Dendritic cells as drug targets in allergic diseases If DCs are so crucial in mounting immune responses during ongoing inflammation in the lung, nose, and skin, then interfering with their function could constitute a novel form of treatment for allergic diseases. Additionally, pharmacologic modification of DCs might fundamentally reset the balance

Antigen-presenting Dendritic Cells and Macrophages

of the allergic immune response in favor of regulatory T cells, and thus lead to a more long-lasting effect on the natural course of allergic disease. Steroids are currently the cornerstone of antiinflammatory treatment in allergic disease. Inhaled steroids reduce the number of lung and nose DCs in patients with atopic asthma (AA) and AD, whereas local application of steroids to the skin of AD patients reduces the influx of IDECs (Holm et al. 1995; Moller et al. 1996). The immunosuppressant drug tacrolimus is currently in use for topical treatment of AD. It suppresses the expression of MHCII and costimulatory molecules and FcεRI on LC from AD patients in vitro, and reduces the number of IDECs in lesional skin. Recently, several other new molecules have surfaced that may alter DC function in allergic inflammation and thus treat disease. In one report, administration of CpG-containing immunostimulatory DNA sequences to the lungs of allergenchallenged mice inhibited the upregulation of these costimulatory molecules, suggesting that this is one mechanism by which they suppress inflammation (Hessel et al. 2005). The S1P analog FTY720 is currently used in clinical trials for multiple sclerosis and transplant rejection. When given to the lungs of mice with established inflammation, it strongly reduced inflammation by suppressing the T-cell stimulatory capacity and migratory behavior of lung DCs (Idzko et al. 2006). Also, selective agonists of particular prostaglandin series receptors might suppress DC function. The DP1 agonist BW245C strongly suppressed airway inflammation and bronchial hyperreactivity when given to allergic mice by inhibiting the maturation of lung DCs. DCs thus exposed to DP1 agonists induced the formation of Foxp3+ Treg cells that suppressed inflammation upon adoptive transfer (Hammad et al. 2007). A very similar mechanism was described for inhaled iloprost, a prostacyclin analog acting on the IP receptor expressed by lung DCs (Idzko et al. 2007). A specific small-molecule compound (VAF347) that blocks the function of B cells and DCs was also shown to be effective in suppressing allergic airway inflammation in a mouse model of asthma (Ettmayer et al. 2006). Finally, specific inhibitors of syk kinase were shown to suppress DC function and cure established inflammation (Matsubara et al. 2006).

Origin and function of macrophages The lung contains a large variety of macrophages, of which the phenotype and function varies considerably in baseline and inflammatory conditions. The vast majority in the lung are, however, alveolar and interstitial macrophages. AMs originate from a CD34+ hematopoietic stem cell and differentiate along the myeloid pathway under the influence of MCSF and IL-6. These cells have therefore a lineage relationship with DCs. The immediate precursors of lung macrophages are blood monocytes, which have the potential to differentiate into macrophages upon arrival in the lung tissues and

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alveolar compartment. Apart from a bone marrow supply, it has been demonstrated by bone marrow irradiation experiments that alveolar macrophages derive also from a local proliferating pool of precursor macrophages that respond to M-CSF. The predominant function of AMs is to phagocytose inhaled particulate antigens, and to effectively sequester these antigens from the immune system. Therefore, AMs are endowed with many receptors, such as the CR1 and CR3 complement receptors and Fc receptors for opsonized antigens, and the macrophage mannose receptor, as well as scavenger receptors. A very efficient system of phagolysosomes fuses with endocytosed particles to neutralize the ingested material. Kradin has very elegantly demonstrated that AMs sequester inhaled particulate antigens to shield them off from the specific immune system induced by DCs. A pulmonary cellular immune response is generated to an inhaled particulate antigen when the protective phagocytic capacities of the lung are exceeded and antigen is able to interact directly with interstitial DCs. The diversion of particulate antigens by pulmonary phagocytes may help to limit undesirable pulmonary inflammation while allowing the generation of antigen-specific immune lymphocytes in vivo (MacLean et al. 1996).

Homeostasis in the alveolar compartment is maintained by alveolar macrophages Macrophages are specialized hematopoietic cells, distributed throughout different tissues of the body where they play a central role in homeostasis, tissue remodeling, host defense, and the response to foreign materials, including particulates. One of the key functional characteristics of macrophages is that, depending on their state of differentiation and the microenvironmental factors that they encounter in a particular tissue, they can be modulated to express whatever functions are required to deal most effectively with a given kind of inciting stimulus. In the lung, resident AMs are continuously encountering inhaled substances due to their exposed position in the alveolar lumen. To avoid collateral damage to type I and type II alveolar epithelia cells in response to harmless antigens, they are kept in a quiescent state, producing few inflammatory cytokines, and displaying poor phagocytic activity, as evidenced by downregulated expression of the phagocytic receptor CD11b (Holt 1978). In addition, AMs actively suppress the induction of adaptive immunity, through effects on alveolar and interstitial DCs and T cells. Elegant studies have demonstrated that in vivo elimination of AMs using clodronate-filled liposomes leads to overt inflammatory reactions to otherwise harmless particulate and soluble antigens (Thepen et al. 1989). AMs adhere closely to airway epithelial cells (AECs) at the alveolar wall and are separated by only 0.2–0.5 μm from interstitial DCs. In macrophage-depleted

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mice, the DCs have a clearly enhanced antigen-presenting function (Holt et al. 1993). When mixed with DCs in vitro, AMs suppress T-cell activation through release of NO (mainly in rodents), prostaglandins, IL-10, and TGF-β. The T cells that have been exposed to human or rodent AMs have a remarkable phenotype as they are locked in G0/G1 arrest, unable to proliferate, yet after T-cell receptor stimulation demonstrate normal calcium fluxes, produce IL-2 and IFN-γ, and express the IL-2 receptor CD25. However, this phenotype is completely reversible, and T cells resume dividing when AMs are removed. Under homeostatic conditions, AMs closely adhere to AECs and this in turn induces the expression of the integrin αvβ6 on AECs, in a TGF-β-dependent manner. It was previously shown that αvβ6 integrin-deficient mice have activated AMs. This integrin has the potential to activate latent TGF-β by binding to the latency-associated peptide (LAP), an N-terminal inactivating fragment of TGF-β. Binding of activated TGF-β to its receptors expressed on macrophages induces phosphorylation of SMAD2 and SMAD3, and to suppression of macrophage phagocytosis and cytokine production. The inhibition of macrophage function by αvβ6–TGF-β complex is unique to the lung, illustrating the microenvironmental specializations of macrophages to meet the needs of the tissue. AEC expression of the αvβ6–TGF-β complex could also suppress the function of alveolar DCs and adaptive immunity. The mechanism of immune homeostasis and tonic inhibition of macrophage function in the lung is so robust that it was long enigmatic how infection might lead to macrophage activation triggering of TLRs or, via non-TLR-mediated stimulation of innate immune receptors on macrophages, open a window of opportunity for macrophage activation. TLR stimulation of macrophages leads to a rapid loss of contact with AECs, in turn inducing a rapid loss of expression of αvβ6 integrin expression on AECs. Under these conditions, TGF-β is no longer activated, releasing the brakes over macrophage activation and innate immune function, and macrophages become primed to secrete proinflammatory cytokines (TNF-α, IL-6) and to phagocytose particulate matter. Once activated, AMs can clear the infectious threat on their own, at the same time avoiding collateral damage to the alveolus. In addition, many infectious agents lead to recruitment of CCR2+ inflammatory monocytes to the alveolar space. These freshly recruited monocytes are clearly proinflammatory and display phagocytosis and killing, and promote rather than suppress T cell and DC activation. It takes a few days before these monocytes acquire the suppressive phenotype of alveolar macrophages, allowing for another ‘window of opportunity’ for initiation of innate and adaptive responses in the lung. To avoid collateral damage and to restore gas exchange as quickly as possible, there needs to be a mechanism keeping macrophage activation in check. Activated lymphocytes secreting IFN-γ stimulate the production of matrix metalloproteinase (MMP)-9. This particular MMP has the potential to activate latent TGF-β and, in this way, tonic inhibition of macrophage

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function is restored, macrophages again adhere to AECs, and αvβ6 integrin expression is restored. It has been described previously by Holt and colleagues that freshly recruited monocytes gradually acquire the phenotype of resident suppressive AMs over a period of days (Bilyk & Holt 1993). Whether this would be a predefined process or an instruction by the lung TGF-β-rich environment remains to be shown. Certainly, the prolonged presence of activating cytokines such as GM-CSF keeps inflammatory monocytes from acquiring suppressive activities (Bilyk & Holt 1993). An additional advantage of enhanced TGF-β production would be the stimulation collagen synthesis in interstitial fibroblasts, necessary for restoring alveolar wall architecture (Lambrecht 2006b).

Function of alveolar macrophages in inflammatory conditions and asthma Under inflammatory conditions, fresh monocytes and DCs are recruited to the airways and these cells “dilute” the immunosuppressive AMs, allowing a window phase in which T-cell responses can be induced. Freshly recruited monocytes and AMs produce a variety of inflammatory mediators such as cytokines, cytokine antagonists, coagulation products, lipid mediators, and growth factors. The local production of GM-CSF in these conditions can also switch the immunosuppressive AMs into a stimulatory cell (Bilyk & Holt 1993). In humans, the recently recruited AMs can be discriminated from the resident AMs by means of staining with the combination of RFD1/RFD7, initially described by Spiteri et al. (1992). Using these antibodies, it was shown that RFD1+/RFD7 − AMs have a stimulating function, whereas the RFD1+/RFD7+ and RFD1−/RFD7+ subset has a suppressive function on T cells. Strikingly, the RFD1 marker is also expressed on lung DCs (Spiteri et al. 1992). The airways of AA patients and of sarcoidosis patients contain increased amounts of RFD1+ AMs. Numerous studies have now demonstrated that the phenotype and function of AMs in patients with asthma are fundamentally different from those of healthy controls. Most notably has been the increased expression of CD80 and/or CD86, CD1a, ICAM-1, and LFA-1 and of the low-affinity receptor for IgE on AMs from patients with AA (Aubas et al. 1984; Gosset et al. 1991; Chanez et al. 1993; Tang et al. 1998). Conversely, the expression of CD40 is lower in the AMs of asthma patients. When compared with healthy control subjects, the AMs of patients with AA produce more IL-1, IL-6, TNF-α, and IL-10, and less IL-12, explaining their preferential induction of Th2 responses. When AMs of AA patients were cocultured with syngeneic CD4+ T cells, they induced the production of IL-5 (Tang et al. 2001). In the same experiments, the AMs of healthy controls suppressed IL-5 production. In some experiments, the production of IL-5 could be inhibited by blocking antibodies against CD80 and/or CD86, IL-1, IL-6, or TNF-α (Balbo et al. 2001). Conversely, antibodies to CD40

Antigen-presenting Dendritic Cells and Macrophages

enhanced the AM-induced IL-5 production by T cells (Tang et al. 2001). These studies suggest that AMs from AA patients can activate effector function in Th2 cells, at least in vitro.

Conclusion DCs and macrophages are crucial in determining the functional outcome of allergen encounter in the lung, nose, and skin, and antigen presentation by mDCs leads to Th2 sensitization typical of allergic disease, whereas pDCs appear likely to play a more subtle regulatory role. It is increasingly clear that DCs have an antigen-presenting function beyond sensitization. DCs therefore constitute a novel target for the development of antiallergic therapy aimed at the origin of the inflammatory cascade.

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Innate Immunity in Allergic Disease Ian Sabroe

Summary The innate immune system comprises a complex network of overlapping defenses against infection, ranging from humoral to cellular components, and from structural barriers such as the epithelium to mobile response elements such as phagocytic leukocytes. Recent rapid expansion in our knowledge of many key pathways regulating innate immune activation (Toll-like receptors, Nod-like receptors, RIG-I-like helicases) has stimulated a resurgent interest in the mechanisms of innate immunity. This immune network regulates our every interaction with the pathogenic world around us, and has important capability in the sensing of tissue damage. Lifelong engagement of the innate immune system feeds into control and modulation of the function of the adaptive immune system. The timing and nature of activation of the innate immune system is an important determinant of our risks of developing allergic disease in childhood; continual engagement of innate immunity throughout our lives directly influences the severity of allergic disease once established. The most promising new treatments for allergic disease directly engage with innate immunity to rebalance the activity of the adaptive immune system. Allergic disease cannot be understood without reference to the central role of the innate immune system. This chapter describes both the basic science of innate immunity, and its dialogue with disease, highlighting the potential for targeting of these systems to dramatically influence the future management of allergic disease.

What constitutes the innate immune system? Present long before birth, our innate immune system provides intrinsic protection against a broad range of pathogens without the need for preexposure to the pathogen or formation of immunologic memory. This lack of targeted memory leads authors to describe the innate immune system as Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

“nonspecific,” though a series of proteins, receptors, and cells provide tailored responses to microbes, leading to effective responses to many different pathogens. Thus, the innate immune system exhibits some specificity, encoded through a large germline family of genes that are not rearranged as part of a memory-invoking process. The absolute requirement of the innate system for generation of effective memory further complicates the arbitrary division of immunity into innate and adaptive systems.

Humoral factors Innate immune responses are seen in a very broad range of tissues. Indeed, the Toll-like receptors (TLRs, one of the most important series of innate immune response proteins, described in detail below) are probably represented at some level in every cell in the body. Even before such systems are engaged, however, other levels of defense have important roles in mediating successful immunity. Barrier functions served by intact epithelia are fundamental to the prevention of infection. Such barriers are augmented by secreted milieu such as airway mucus, with its ability to bind and neutralize pathogens resulting in their clearance via the mucociliary escalator. Epithelial lining fluids often contain potent microbicidal proteins such as cathepsins, collectins, lysozyme, and defensins (Bals 2000; Ganz 2004; Hickling et al. 2004), with roles in pathogen neutralization, killing, and opsonization. A key part of any inflammatory response is the induction of capillary permeability, resulting in the delivery of further humoral factors such as complement to the site of tissue damage/infection (Walport 2001a,b). In addition to its roles in bacterial killing, complement is important in the regulation of leukocyte trafficking and inflammation. Mice deficient in the complement protein C3 show reduced production of Th2 cytokines in asthma (Drouin et al. 2001), though interestingly if allergen sensitization is performed epicutaneously, C3aR knockout mice show the opposite phenotype with enhanced Th2 responses perhaps mediated by alterations in antigen-presenting cell responses (Kawamoto et al. 2004). This interaction of innate and adaptive immunity is clearly complicated, and can involve multiple components of the complement system, since C5-deficient mice also show enhanced Th2-type allergic inflammation (Drouin et al. 2006). The role

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Contact of pathogens with the innate immune system will most frequently occur at epithelia, and the biology of the airway epithelium is of considerable importance in asthma (Davies et al. 2003). Airway epithelia express a range of innate immune receptors, allowing them to function as a line of first response to pathogens: their ability to detect and respond to pathogens must clearly be substantial, given that they form the main target for most respiratory viruses. There are also potentially close relationships between epithelial cells and other cells of the innate immune system such as DCs and macrophages (Rescigno et al. 2001; Morris et al. 2005, 2006; Sabroe et al. 2006). Cooperative networks that regulate airway inflammation are discussed in more detail below. Interestingly, defective responses to respiratory viruses are evident in epithelial cells from asthmatics (Wark et al. 2005), which may be relevant in the pathology of asthma exacerbations, and phenotypic differences in epithelia between asthmatics and normal subjects have been demonstrated (Kicic et al. 2006). Effective barrier functions result from a combination of humoral and cellular mechanisms, as classically illustrated in the respiratory tract, where mucus and lining fluid serve to neutralize and clear pathogens, and epithelial cells act to impede pathogen access to the body, as well as providing a first line of response to pathogens.

2006), and alveolar macrophages can inhibit DC maturation in the lung (Holt et al. 1993). These are long-lived cells derived from monocytes, but in chronic inflammation repeated rounds of cell recruitment may result in replacement of macrophages by cells with a more monocytic phenotype (Maus et al. 2006). Data from the gut and lung has suggested that DCs may sample the local microenvironment by extruding processes through epithelial cell tight junctions (Rescigno et al. 2001; Jahnsen et al. 2006): such mechanisms might also be theoretically important in the lung. Clearly the lung is a major site of antigen exposure, and resident DCs have vital roles to play in the regulation of the subsequent inflammatory and immunologic response, acting as a bridge between innate and adaptive immunity (discussed in more detail below). Again, subsets of DCs in the lungs show selective patterns of expression of innate immune receptors such as the TLRs, and differential ability to support T-cell proliferation (Demedts et al. 2006). Other cells which act as long-term resident early-warning cells include the lung mast cell. Mast cells respond to pathogenic stimuli in species- and tissue-specific patterns (Supajatura et al. 2002; Ikeda & Funaba 2003; McCurdy et al. 2003; Okumura et al. 2003; Kulka et al. 2004; Matsushima et al. 2004; Orinska et al. 2005; Nigo et al. 2006), and through dual signaling via pathogen-response systems and IgE-mediated mechanisms have the potential to integrate innate and adaptive responses into a regulation of Th2 immunity. Resident T-cell populations that form part of the innate immune system, such as natural killer (NK) and NKT cells, may also exert important roles in allergic inflammation. These cells again respond to agonists stimulating pathogen response systems such as TLRs, though some of these responses may be mediated by indirect networks (Herzyk et al. 1992; Korsgren et al. 1999; Hart et al. 2005; Akbari et al. 2006; Gorski et al. 2006; Kay 2006).

Resident leukocyte populations

Recruited leukocyte populations

Innate immunity depends on both resident and recruited leukocytes. The macrophage without doubt plays an important role in the detection of pulmonary infections. Low inocula of pneumococci are cleared by macrophages (Dockrell et al. 2003). Alveolar macrophages also have an important role in the initiation of responses to inhaled lipopolysaccharide (LPS) (Hollingsworth et al. 2005), and their function is altered by exposure to irritants such as cigarette smoke (McCrea et al. 1994; Medvedev et al. 2006). Alveolar macrophages may also have a role to play in the regulation of airway hyperresponsiveness. These cells can exhibit strain-dependent bias toward the support of Th1 or Th2 phenotypes (Mills et al. 2000; Careau & Bissonnette 2004; Peters-Golden 2004) and be rendered tolerogenic, resulting in downregulation of allergic airways disease (Korf et al. 2006). There is also evidence that macrophages have additional roles in the maintenance of normal peripheral tolerance in vivo (Hoves et al.

Once local defenses are activated, rapid recruitment of additional leukocytes is the rule, aiming for fast and effective pathogen clearance with rapid restoration of normal tissue architecture. Neutrophils are the most numerous early phagocyte recruited to inflammatory sites. Their principal role is the neutralization of bacterial and fungal pathogens, but they also interact with many other cell types to influence the development of the inflammatory lesion (discussed below). Their role in asthma is becoming increasingly discussed (Kamath et al. 2005), potentially contributing to specific disease phenotypes evident at a pathologic (Wenzel et al. 1999) or clinically relevant (Green et al. 2002) level. Clinical phenotypes associated with airways neutrophilia include exacerbations induced by viruses (Grissell et al. 2005), severe (Jatakanon et al. 1999) or steroid-resistant disease (Wenzel et al. 1999), and potentially fatal disease (Lamblin et al. 1998). These cells are not just there in their capacity as phagocytes,

of complement fragments is thus context dependent, and recent work suggests that, for example, C5a may reduce the establishment of Th2 responses, but in models in which allergic sensitization has already occurred, inhibition of C5a might reduce disease severity (Kohl et al. 2006; Lambrecht 2006). Again, these effects may involve, but are not exclusively dependent on, alteration in dendritic cell (DC) trafficking and function (Kohl et al. 2006; Lambrecht 2006).

Barrier cells, innate immunity, and allergic inflammation

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since they can contribute to the generation of delayed-type hypersensitivity immune responses (Molesworth-Kenyon et al. 2005), modify cytokine production from macrophages (Daley et al. 2005), activate epithelia (van Wetering et al. 2005), induce the death of smooth muscle cells (Oltmanns et al. 2005), and even in death influence the subsequent healing or proinflammatory response (Haslett 1997; Zheng et al. 2004; Serhan & Savill 2005). Thus, the subsequent influx of both innate immune cells (eosinophils, monocytes, mast cells, NK cells) and adaptive immune cells (T cells, B cells) is heavily influenced by the early innate immune response. Additional recruitment of monocytes rapidly follows the neutrophil, changing the monocyte/macrophage balance of the lung (Maus et al. 2006) and providing cells that engage in phagocytosis of pathogen and apoptotic neutrophil alike, regulating the balance between ongoing inflammation and injury resolution. Eosinophil recruitment, a hallmark of allergic inflammation, provides a further phagocytic cell whose biological role in immunity and disease still remains an uncertain area (Flood-Page et al. 2003a,b). The presence of a diverse and responsive resident leukocyte population thus enables a sophisticated early response to pathogens. Many day-to-day routine microbial encounters may never “make it through” to interaction with the adaptive immune system, effectively being targeted by the highly efficient innate response. Activation of the innate response is an inescapable component of inflammatory reactions pertaining to epithelial and mucosal surfaces, and a potentially important contributor to the pathology of allergic disease.

How does the innate immune system recognize pathogens? Many molecules contribute to pathogen recognition, from well-described families of receptors such as the TLRs (Basu & Fenton 2004; Chaudhuri et al. 2005), Nod family/Nod-like receptors (NLRs) (Meylan et al. 2006), and scavenger receptors, through to integrins and sometimes apparently surprising moieties such as the platelet-activating factor receptor (Fillon et al. 2006). These signaling systems are often studied in the context of investigation of responses to their purified putative ligands or agonists. While this gives a picture of the contribution of individual signaling systems to a given response, it is important to remember that pathogens or pathogen remnants will typically interact with multiple receptor systems, sometimes across a membrane surface and sometimes within a phagolysosome. The processes of phagocytosis, and the interaction of pathogen surfaces and soluble components presented simultaneously potentially across a measurable portion of an innate immune cell’s surface, are likely to generate summated signaling and responses driven by a range of mechanisms.

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Bacterium Virus

Lipoproteins

Flagellin ssRNA

LPS Proteins

1/2

3/3

4/4

5/5

2/6

dsRNA

7/7

8/8

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Fig. 9.1 Toll-like receptor (TLR) agonists. TLRs divide into two broad groups: those whose predominant role appears to be in the mediation of antibacterial responses, and those whose role appears more tied to meeting responses to viruses. TLRs colored dark blue appear more linked to antibacterial immunity. TLR1/2 heterodimers and TLR2/6 heterodimers mediate responses to a range of bacterial lipoproteins. Other TLRs function as homodimers. TLR4 responds to lipopolysaccharide (LPS), TLR5 to flagellin, and TLR9 to bacterial and viral DNA. TLRs colored pale blue are more central to responses to viruses. TLR3 mediates responses to double-stranded RNA, whereas TLR7 and TLR8 have been linked to responses to single-stranded RNA. As noted, TLR9 responds to some viral DNAs. The roles of TLR10 (white) have yet to be clarified. (See CD-ROM for color version.)

Toll-like receptors (Figs 9.1 & 9.2) The discovery of a germline-encoded family of pattern recognition receptors mediating responses to pathogens across species as evolutionarily distinct as fly and human has transformed our understanding of innate immunity. The story began when knockout of a gene in Drosophila led to a failure of fly embryos to undergo normal polarization (Anderson et al. 1985); in German, the language in which the protein underlying these abnormalities was named, toll has many meanings, including “great,” “weird” and “crazy.” A role for this molecule in fly development was later complemented by identification of a role for this protein in fly immunity (Lemaitre et al. 1996). The link to mammals came with the discovery of constitutive activation of human homologs of Toll-activated immune responses (Medzhitov et al. 1997) and that a human family of these proteins existed (Rock et al. 1998). Natural mouse strains unable to respond to LPS were subsequently shown to be lacking in a functional TLR4 (Poltorak et al. 1998; Qureshi et al. 1999), providing the key proof of the role of this family of proteins in innate immune signaling. In a remarkably short time we have come a long way in our understanding of TLRs. We now know that there are at least 10 TLRs in the human, with an eleventh that is

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Environmental microbial products

Allergens

Infections

Spectrum of contiguous immunity

Th1/innate immunity-type

Stimuli

Smoking, pollution

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Initiation signals Antigen presentation

Neutrophil recruitment

Eosinophil recruitment

Monocyte recruitment

Th2-type T-cell activation Processes

Mast cell recruitment

NK/NK T-cell stimulation Tissue cell activation

Bronchial hyperreactivity

functional in the mouse. Putative agonists or ligands have been assigned for each of these, except TLR10, whose function is still somewhat enigmatic, though its genetics appear to contribute to asthma risk (Lazarus et al. 2004). While not the only receptors employed for the detection of pathogens, they are clearly extremely important. In common with most pathogen-response elements, TLRs recognize not single specific ligands but molecules that have in common some molecular pattern. For example, TLR4 recognizes a range of LPS species, whereas TLR2 responds to lipoproteins, TLR5 to flagellins, and TLR9 to bacterial DNA. There are only limited data describing the molecular interaction of putative TLR ligands with their receptors, and further complications arise from the use of accessory proteins

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Fig. 9.2 Contiguous immunity in allergic disease. The pathology of chronic inflammatory diseases such as asthma are to some extent poorly described by traditional descriptors such as innate or adaptive disease, or Th1 vs. Th2 immunological phenotypes. These chronic diseases are perpetuated by repeated exposure to a variety of stimuli, activating a large number of inflammatory subsystems involving innate and adaptive, Th1 and Th2, type processes. Continual evolution of the inflammatory state occurs through dialogue between these inflammatory nodes, generating cooperative and regulatory networks that determine the disease phenotype and its progression or resolution. With time, as pathology evolves and the nature of the tissue changes in response, the components of the network alter. Such alterations occur both with respect to numbers and types of cells, and activation or differentiation status. Inflammation is regulated by networks that are physically adjacent with the potential for constant communication and dialogue (spatially contiguous), which may evolve in complex sequences over time (temporally contiguous). The relative contribution of each component is flexible, and understanding their different contribution to separate phases of the disease is essential to facilitate effective therapeutic targeting. (From Sabroe et al. 2007, with permission.) (See CD-ROM for color version.)

to enable signaling. For example, LPS signaling appears to require monomerization of LPS by LPS-binding protein, presentation to CD14 (a transmembrane or soluble nonsignaling protein), and transfer to a complex of MD-2 (a secreted protein) and TLR4 (Shimazu et al. 1999; Visintin et al. 2001a; Nagai et al. 2002; Gioannini et al. 2004; Kennedy et al. 2004). Under some circumstances, integrins such as CD11b/CD18 may play roles in LPS responses (Perera et al. 2001), and TLRs may aggregate in lipid rafts with other molecules that could facilitate signaling (Triantafilou & Triantafilou 2002; Triantafilou et al. 2004, 2006). Other TLRs broaden their range of agonists by heterodimerization: this is particularly seen for TLR2, which dimerizes with TLR1 or TLR6 (Ozinsky et al. 2000). The mechanism of interaction that enables broad

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detection of pathogens, while maintaining discrete nonrecognition of self, is still poorly understood with relatively little available pharmacologic data on the behavior of the TLRs. These receptors also show some ability to detect host damage, since self molecules including hyaluronan, fibrinogen, fibronectin, heat-shock proteins, and transcription factors can all potentially activate TLR4 (Ohashi et al. 2000; Okamura et al. 2001; Smiley et al. 2001; Guillot et al. 2002; Park et al. 2004; Tsan & Gao 2004; Jiang et al. 2005; Ohya et al. 2006). This literature has been bedevilled by the potential contamination of putative TLR4 agonists by LPS (Tsan & Gao 2004), an area fraught with difficulty (Wakelin et al. 2006). It appears true that TLR4 in particular can act as a sensor of tissue damage in the absence of pathogen exposure, but this is an area in which the evolution of our understanding remains hard to predict. TLRs are expressed widely, though some curiosities to these expression patterns are seen. In general, innate immune cells likely to come into contact with bacteria respond well to agonists of TLR2 (principally bacterial lipoproteins) and TLR4 (LPS), as well as to flagellin (TLR5), and often to bacterial DNA (TLR9). Many barrier cells such as airway epithelial cells also show similar responses and express similar patterns of TLRs (Hertz et al. 2003; Adamo et al. 2004; Armstrong et al. 2004; Guillot et al. 2004; Sha et al. 2004; Guillot et al. 2005). DCs show select patterns of TLR expression, with monocytederived DCs showing a monocyte-like expression pattern including TLR2 and TLR4, whereas plasmacytoid DCs tend to favor expression of TLR7 and TLR9 (Demedts et al. 2006). A wide variety of tissue cells, including epithelia, smooth muscle, and endothelial cells, respond to synthetic analogs of double-stranded RNA. Often generated as an intermediate in viral replication, double-stranded RNA is sensed by a variety of mechanisms, including protein kinase R (PKR), TLR3, and also the cytosolic receptors of the RIG-I/mda5 pathway (see below). TLR3 expression seems relatively widespread, although it is only found in rare leukocyte populations in the blood, including immature DCs (Muzio et al. 2000; Visintin et al. 2001b; Matsumoto et al. 2003). In the lung, monocytederived DCs express TLR3 (Demedts et al. 2006), as do alveolar macrophages, though unusually human alveolar macrophages do not show the classical induction of type I interferons (Punturieri et al. 2004) that is a hallmark of activation of the antiviral TLRs (TLR3, TLR7, TLR8). TLR3 also has a putative endogenous agonist (mRNA) (Kariko et al. 2004), and thus its common tissue expression may also indicate other roles in host defense or responses to tissue damage (Brentano et al. 2005) as yet relatively unexplored. In contrast to the “antiviral” receptor TLR3, TLR7 and TLR8 mediate responses to single-stranded viral RNA (Heil et al. 2004) and seem to show expression in selected leukocyte populations, including mast cells, neutrophils (Hayashi et al. 2003) and eosinophils (Nagase et al. 2003) but most notably plasmacytoid DCs, rather than in tissue cells (Hemmi et al.

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2002; Mohty et al. 2003; Nagase et al. 2003; Diebold et al. 2004; Matsushima et al. 2004; Berkeredjian-Ding et al. 2005; Hart et al. 2005; Demedts et al. 2006; Morris et al. 2006). These receptors seem to be dependent on acidification of their intracellular phagosomes for effective signaling (de Bouteiller et al. 2005; Gibbard et al. 2006). TLRs are also important in T-cell function, providing another point of interaction between the innate and adaptive immune systems. The role of TLRs in T-cell function is discussed in more detail below. TLRs signal through a family of adapter proteins. Currently five adapters have been identified. MyD88 is the prototypic adapter, shared with the interleukin (IL)-1 receptor. Utilized by all TLRs except TLR3, it forms a signaling complex with a series of other proteins to activate mitogen-activated protein kinase (MAPK) and NF-κB cascades. A further adapter, MAL/TIRAP, shares in this signaling cascade for the receptors TLR2 and TLR4, but MAL/TIRAP is not involved in the signaling of TLR5, TLR7, and TLR9. TLR3 uses the adapter TRIF to activate NF-κB and the generation of type I interferons, coordinating antiviral responses. TLR4 also uses TRIF, but couples to it via the adapter TRAM. The exact pattern of signaling, and potentially of adapters used, may vary from cell to cell. A fifth adapter, SARM, appears to be a negative regulator of TRIF signaling (Carty et al. 2006). Other pathways initiate signaling via tyrosine kinases (Jefferies et al. 2003; Doyle et al. 2005; Horwood et al. 2006), small GTPases, and phosphatidylinositol 3-kinases: such pathways are still relatively poorly understood. Signaling is very carefully regulated by some extraordinarily complex pathways. Adapter function and localization is regulated (Gray et al. 2006; Mansell et al. 2006; Rowe et al. 2006). TLR8 (Wang et al. 2006) and the IL-1R superfamily member T1/ST2 (Brint et al. 2004) can negatively regulate TLR signaling. A variety of further negative regulators of TLR signaling, including members of the IRAK family and SOCS families, control responses, and TLR signaling can be rapidly tolerized (Medvedev et al. 2006). Presumably these very complex patterns of signaling and regulation thereof all enable tailored and controlled responses to specific pathogens. The evolving map of TLR signaling is complex indeed (Oda & Kitano 2006), and remains to be fully elucidated. Many of these pathways are actively targeted by viruses, in order, presumably, to promote their survival in host tissues (Bowie et al. 2000; DiPerna et al. 2004).

Nod-like receptors The NLRs form another family of less well understood intracellular pattern-recognition receptors (Inohara et al. 2005), with the potential to cooperate with TLRs in the induction of effective immune responses (van Heel et al. 2005). NOD1 and NOD2 recognize components of Gram-positive bacterial peptidoglycans. The best characterized of these interactions is the ability of NOD2 to respond to muramyl dipeptide, a

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peptidoglycan fragment generated during bacterial replication and host degradation of phagocytosed bacteria (Meylan et al. 2006).

Other sensors of viral infection Recently, a further family of cytoplasmic receptors have been identified that respond to viral infection. RIG-I and mda5 sense viral infection, presumably through the detection of double-stranded RNA, and activate IRF and NF-κB signaling pathways through another, mitochondrial-bound, adapter, Cardif (also known as VISA, MAVS, and IPS-1, having been discovered almost simultaneously by several groups). Early data indicate potentially specific roles for mda5 and RIG-I in responses to individual viruses, but this remains a very new field with much work to be done in defining their contribution to antiviral responses in a range of settings (Meylan et al. 2006).

Other pattern-recognition receptors While TLRs, NLRs, and the helicase-like proteins RIG-I and mda5 are currently major targets of research, it is important to recall that the family of pattern-recognition molecules is broad. Humoral factors such as complement are important, scavenger receptors contribute to recognition of bacteria such as Neisseria meningitidis and the pneumococcus (Hampton et al. 1991; Haworth et al. 1997; van der Laan et al. 1999; Fitzgerald et al. 2000; Peiser et al. 2000; Thomas et al. 2000; Gordon 2002; Peiser et al. 2002; Arredouani et al. 2004; Mukhopadhyay et al. 2004), and integrins may contribute to TLR signaling (Perera et al. 2001). Responses to viruses also involve PKR (Saunders & Barber 2003), and foreign DNA may also involve complex sensing pathways beyond TLR9 (Alvarez et al. 2006; Wagner & Bauer 2006), which may be cell type dependent.

How does the innate immune system become activated? The first thing to note is that very often the innate immune system will not become activated on pathogen exposure, because barrier components of the innate immune system will control the stimulus effectively without recourse to activation of cell signaling and responses. Mucociliary clearance, interaction with neutralizing proteins in skin or lung lining fluid, and impermeable barriers all conspire to prevent stimuli ever reaching a cellular surface in a form that requires an active response. However, it should be apparent from the description of the innate immune system above that innate immunity is far from a simple mechanism that provides basic barrier functions, or one whose role has been apparently surpassed by the more complex adaptive immune response. Indeed, the innate immune system functions to maintain our health on an hour-by-hour basis, continually regulating interactions with both pathogens and commensals in our

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environment, with no need of recourse to immunologic memory. It is also becoming apparent that effective immunity is not the function of a single cell, protein, or pathway, but rather depends on the coordinated action of a complex interdependent system whose evolutionary roots probably go back to the first unicellular organisms. In the context of single exposures to specific insults, it is possible to begin to define the networks involved in the generation of effective innate immunity. Signaling from sentinel cells such as the macrophage appears to be important in the initiation of responses to bacteria and LPS. These signals cooperate with responses from tissue cells, generating rapid amplification of signals favoring an efficient innate response. Interestingly, signaling from tissue cells and leukocytes in response to molecules such as LPS may play separate roles, since there is evidence that signaling initiated by macrophages is important for leukocyte recruitment, whereas signaling from lung tissue cells is needed for LPS to induce bronchoconstriction (Noulin et al. 2005). Activation of macrophages leads, via secretion of molecules such as IL-1, to rapid activation of tissue cells, switching epithelia and smooth muscle into prosynthetic phenotypes, and driving rapid amplification of cytokine production. Rapid recruitment of granulocytes, monocytes, and lymphocytes ensues. Each cell type in turn may modify the immune response, through the release of cytokines, tissue-damaging proteases, and reactive oxygen species, and ultimately through their death. This appears to be particularly true for neutrophils, whose death by apoptosis is a crucial injury-resolving stimulus (Haslett et al. 1994; Haslett 1997; Bianchi et al. 2006). The epithelial cell provides another immunocompetent sensing cell with considerable potential to initiate responses to bacterial infection, through its expression of molecules such as TLR4. While it can undoubtedly respond to LPS, there is now evidence that the magnitude of this response is dwarfed by that which may be achieved when sentinel leukocytes such as monocytes use the epithelial cell to amplify their response to LPS (Tsutsumi-Ishii & Nagaoka 2003; Morris et al. 2005, 2006). In chronic inflammation, the repopulation of the lung with relatively young monocytes/macrophages (Maus et al. 2006) amplifies the potential for potent responses to microbial pathogen exposure. The epithelium, and other TLR-responsive tissue cells, perhaps play a much greater role in the initiation of responses to viruses. Expression of a variety of viral sensors by this cell type allows it respond rapidly to viral infection, though again there is potential for cooperation with other innate immune cells such as monocytes/ macrophages in the initiation of effective antiviral immunity, through expression of TLR7/8 on some leukocyte populations. In reality, exposure to single TLR agonists probably never occurs, since bacteria and viruses will interact with components of the innate immune system through multiple molecular recognition systems. In these settings, it is becoming increasingly apparent that cooperativity between cells

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and receptors is essential for rapid and efficacious signaling and activation of innate immunity (Morris et al. 2006).

How do the innate and adaptive immune systems interact? Interactions with T and B cells Activation of innate immunity is key to the generation of effective adaptive immunity. TLR signals are among the most potent inducers of DC maturation and trafficking, making their signaling central to new treatments for allergic disease (Creticos et al. 2006; Hayashi & Raz 2006; Krieg 2006). Specific patterns of TLR expression between monocyte-derived DCs and plasmacytoid DCs suggest that these cells may serve different functions in innate immunity, and there is some evidence to support this contention in vivo (De Heer et al. 2004; Demedts et al. 2006). Some TLRs may be more naturally biased to a specific cytokine phenotype: there is evidence, for example, that TLR2 signaling may favor a Th2 response (Redecke et al. 2004), though this is not a unanimous finding (Sieling et al. 2003; Komai-Koma et al. 2004). TLR4 signaling has been associated with prevention of airway inflammation (Rodríguez et al. 2003), but TLR4 knockouts may show reduced Th2 responses, which appears contrary to these data (Dabbagh et al. 2002). Both TLR2 and TLR4 agonists may reduce risks of allergic sensitization (Velasco et al. 2005). The key to understanding this complicated area lies in appreciating the timing, duration, and intensity of the DC-stimulating signal. Low amounts of LPS, acting via TLR4, may actually favor Th2 responses during sensitization, whereas higher doses drive Th1-type inflammation (Eisenbarth et al. 2002, 2003). Once disease is established, the picture may be different again, since low amounts of endotoxin administered with allergen may downregulate Th2 inflammation whereas high doses enhance it (Hollingsworth et al. 2006). Variations between mouse strains, amounts of endotoxin administered, sensitization protocols (which might have a profound impact depending on route of administration), and levels of endotoxin contamination of classical model allergens such as ovalbumin all conspire to generate a field that is still confusing and unclear. Innate immune signals also directly regulate the function of cells of the adaptive immune system. LPS is a stimulator of T-cell proliferation (Vogel et al. 1983) and TLR2 can function as a costimulatory molecule on memory T cells (Komai-Koma et al. 2004). TLR2 signaling can also temporarily reduce the inhibitory potential of CD25+ T regulatory (Treg) cells, as well as rendering effector T cells less susceptible to Treg suppression of function (Liu et al. 2006). Again, contradictory data in this rapidly emerging area also suggest a potential for TLR2 signaling to enhance the suppressive ability of CD4+CD25+ Treg cells (Zanin-Zhorov et al. 2006). LPS is, additionally, an effective stimulus of B-cell proliferation, via TLR4 and the

Innate Immunity in Allergic Disease

related receptor RP105 (Ogata et al. 2000). It is again important to understand that TLR-mediated signaling into B- and T-cell function may depend on direct stimulation, but also on indirect signaling. Inflammatory sites to which T cells are recruited will contain many cytokine signals from activated leukocytes and tissue cells that directly modulate T-cell function and proliferation, and in the context of antigen presentation the TLR-driven activation status of the antigenpresenting cell may have profound consequences for T-cell function.

Interactions at the level of the mast cell The mast cell probably represents an important interface between innate and adaptive immunity. Mast cells are clearly capable of expressing a range of TLRs, though there are likely to be differences in TLR expression between tissues and species (Applequist et al. 2002; Supajatura et al. 2002; Ikeda & Funaba 2003; McCurdy et al. 2003; Okumura et al. 2003; Rodríguez et al. 2003; Kulka et al. 2004; Matsushima et al. 2004; Orinska et al. 2005; Nigo et al. 2006). TLR4 signaling may be able to significantly enhance Th2 cytokine production in response to IgE stimulation (Nigo et al. 2006), which may also explain the ability of LPS to increase allergic inflammation in some settings. Mast cell recruitment, a hallmark of allergic asthma (Brightling et al. 2002), may also be driven by TLR-mediated responses to viral infection (Morris et al. 2006), through cytokine generation from airway smooth muscle cells, potentially in cooperation with signals from activated leukocytes.

How do we translate this information into an understanding of human allergic disease? It should now be apparent that the innate immune system is crucial to health, and has immense potential to play important roles in human allergic disease by the regulation of inflammation, the control of sensitization, and close liaison with the function of the adaptive immune system. Despite the huge contribution of the innate immune response to allergic disease, the subject has to some degree, until the excitement of the TLR field, been something of a Cinderella speciality. There are currently substantial problems in translating our understanding of innate immunity in vitro and in vivo into a clear view of the pathology of allergic diseases. Most models of allergen challenge employed in vivo are relatively biased to an understanding of the mechanisms of Th2-driven inflammation. It has only recently become apparent that levels of endotoxin in the commonest allergen used in these studies, ovalbumin, is of considerable significance in understanding these processes (Eisenbarth et al. 2002; Hollingsworth et al. 2006). While these models have made a very great contribution to our understanding of allergic inflammation, they have

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not typically been geared to investigating the contribution of the innate immune response to these processes; moreover, the stimulation of exacerbations of allergic diseases such as asthma by viral infections, driving neutrophilic inflammation, is harder to model. Likewise, in vitro studies of human or mouse cells are often limited in their scope, and hard to use to make general inferences. The complexities involved are complicated by important differences between the innate immune response in mouse and human, as illustrated by studies of IRAK-4, a key signaling protein in the TLR cascade. While knockout of this protein in humans is associated with profound susceptibility to bacterial infections (Medvedev et al. 2003; Picard et al. 2003), in IRAK-4–/– mice there is marked susceptibility to an even greater range of pathogens (Suzuki et al. 2002). It is also likely that species-specific evolution of innate immune systems has occurred to tailor the responses of individual species, such as mouse and human, to their principal pathogens and commensals. That this is true is borne out by the chaos that ensues when a pathogen jumps species, as illustrated by the current fears over avian influenza and the havoc wreaked by bovine spongiform encephalopathy. Nonetheless, we can begin to discern how innate immunity will modify allergic disease.

Disease phenotypes It is important to note that no one process explains all disease. Within the field of asthma, it is clear that there are several pathologic phenotypes, as yet poorly characterized. These may include phenotypes in which certain innate immune systems, such as neutrophilic inflammation, are more represented (Lamblin et al. 1998; Wenzel et al. 1999; Green et al. 2002; Kamath et al. 2005), potentially requiring individualized therapy (Green et al. 2002). We are yet to distinguish these phenotypes reliably, which hampers their modeling in vitro and in vivo. In some situations, such as asthma induced by LPS exposure in certain occupational groups, the contribution of the innate immune system is easy to discern. Similarly, the dominant role of infections in the initiation of asthma exacerbations, and their role in acute exacerbations of eczema, should point to an important role for innate immunity in the expression of established disease. The evidence of the hygiene hypothesis suggests that innate immunity plays an important role in disease initiation as well. Equally, it is feasible that some sufferers of allergic disease, e.g., those with asthma induced by exposure to small domestic or research animals, have a disease that is almost entirely dominated by purely allergic triggers with less of a role for innate immune responses. The role of the innate immune system in disease may therefore be broken down into specific areas.

The innate immune system in disease initiation The data above demonstrate that the actions of the innate immune system are crucial in determining patterns of sensitization. Although the relative contribution of differing

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amounts of TLR agonists to the severity of allergic disease is still a complicated area of much research (Eisenbarth et al. 2002, 2003; Hollingsworth et al. 2006), it is absolutely clear that exposure to microbial stimuli modulate sensitization. This has been well illustrated in humans, as evidenced by epidemiologic data and expressed in the hygiene hypothesis (von Mutius 2001; Braun-Fahrlander et al. 2002). Exposure to endotoxin and other microbial compounds, at appropriate doses and in early life, appears to be highly protective against development of atopy (Braun-Fahrlander et al. 2002; Gehring et al. 2002; Matricardi et al. 2002; Weiss 2002). To what degree this is by induction of Th1 cell function, or upregulation of Treg cell function, remains an active area of investigation. Importantly, polymorphisms in genes associated with detection of pathogens modulate the risks of developing atopic disease. In farming children, polymorphisms in TLR2 predict risk of developing allergic disease (Eder et al. 2004). An interesting association has been seen between polymorphisms in the TLR4 coreceptor, CD14: those with a specific CD14 genotype show a negative association between environmental endotoxin and risk of allergic disease but a positive association with nonatopic wheeze (Simpson et al. 2006), and provide support for the notion that low levels of endotoxin may favor a Th2 pattern of immunity (Simpson et al. 2006). Interestingly, activation of the epithelium can modify local DC responses, potentially affecting risks of sensitization or amplification of established disease (Pichavant et al. 2006). Finally, although a controversial area, childhood viral infections are associated with, or modify later risks of, allergic disease (Singh et al. 2006).

The innate immune system in disease perpetuation Animal studies have demonstrated that high doses of LPS may exacerbate airway inflammation (Hollingsworth et al. 2006). These studies are consistent with observations that endotoxin levels in house dust are correlated with disease severity (Michel et al. 1996), and the link between viral infections and exacerbations of allergic airways disease is well established. In most patients with asthma, exacerbation triggers are predominantly infective or nonspecific, and allergens are rarely inhaled in the absence of TLR agonists such as endotoxin (Michel et al. 1996). Repeated rounds of inflammation, most often triggered by pathogens and perhaps endotoxin, will drive activation of the innate immune response, triggering recruitment of innate and adaptive immune cells. Chronic inflammation will cause the lung to be repopulated with relatively young and biologically active monocytes (Maus et al. 2006). Acute and chronic exposure to even relatively low-level innate immune stimuli can initiate cooperative signaling between leukocytes and tissue cells that can result in the potent induction and perpetuation of inflammation (Morris et al. 2005, 2006; Sabroe et al. 2006). These inflammatory networks are likely to evolve over time, as the local tissue environment changes. For example,

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airway remodeling and smooth muscle hypertrophy in the airway results in a greater potential for airway smooth muscle to contribute to the inflammatory response and, given its huge cytokine-secreting potential in cooperation with monocyte activation (Morris et al. 2005, 2006; Sabroe et al. 2006), this is potentially very significant. Alterations or differences in smooth muscle and epithelial phenotypes between asthmatics and normal subjects may also underpin disease and affect disease phenotype as processes evolve over time. Chronic recruitment of neutrophils may exert multiple effects on disease, through their ability to secrete chemokines and cytokines, contribute to Th1-type cell recruitment (Molesworth-Kenyon et al. 2005), modulate the cytokine response of macrophages (Daley et al. 2005), and directly induce tissue cell activation and damage (Oltmanns et al. 2005; van Wetering et al. 2005). Thus it can be seen that during chronic disease, multiple interrelated networks will perpetuate inflammation. These networks involve continual dialogue between innate and adaptive immune systems, and are modulated with time as the nature of the tissue changes with remodeling and chronic inflammation. Repeated rounds of acute pathogen stimulation will interact with chronic inflammation that has often become self-perpetuating. Importantly, components

Innate Immunity in Allergic Disease

of this inflammatory network may play different roles at different points in the disease, influencing acute inflammation, airway remodeling, leukocyte survival, and chronic inflammatory responses. In the setting of chronic allergic diseases such as asthma, simple terms of innate and adaptive immunity describe the pathologic processes poorly. We have therefore recently proposed that such pathologies represent a process we have named “contiguous immunity,” where multiple networks are both physically adjacent and cooperating (physically contiguous) and evolving over time (temporally contiguous) (Sabroe et al. 2006) (Fig. 9.2). These networks also allow modeling of other environmental stimuli with the chronic inflammation of allergic disease. For example, ozone-induced hyperresponsiveness is partly dependent on TLR4 signaling (Hollingsworth et al. 2004). Significant single environmental exposures, such as seen in emergency workers at the World Trade Center collapse, can also result in dramatic effects on lung function (Banauch et al. 2006). Whether any of these effects are mediated by activation of the innate immune system is, of course, not clear, but it is tempting to speculate that cell networks incorporating elements of innate immune responses to inhaled stimuli and local tissue damage are likely to have participated in these significant episodes of lung damage.

INNATE IMMUNE RESPONSE Inflammation

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Fig. 9.3 Immune pathways activated by TLR signaling. TLR signaling exerts its effects on multiple cell types to regulate innate immune inflammation. Direct effects on individual cell types are indicated by solid lines, indirect effects by actions exerted by intermediary cells. EO, eosinophil; MNC, mononuclear cell; PMN, neutrophil. See text for definition of other abbreviations. (From Parker et al. 2006, with permission.) (See CD-ROM for color version.)

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Choosing targets

Impact of disease on innate immunity While our primary concern is the impact that the innate immune system has on allergic disease, it is essential to recognize that allergic disease also impacts on the functioning of the innate immune system. In part this is through treatment, since immunosuppressive therapies inevitably contribute to impairment of immunity at the levels of the innate and adaptive systems. Induction of inflammation might be thought to generate a local environment that is hostile to pathogens, and allergen provocation of the nose can, for example, enhance subsequent responses to endotoxin (Eldridge & Peden 2000). Beyond this, asthma itself is a risk factor for invasive pneumococcal disease (Talbot et al. 2005). Macrophages are crucial in defense against pneumococccus (Dockrell et al. 2003). It is interesting to note, therefore, that the phenotype of allergic disease and the response to infection may rest in part in macrophage phenotypes that may be better or worse at supporting Th2 and Th1 responses (Mills et al. 2000). Susceptibility to pneumococcal disease in different mouse strains is potentially associated with their ability to recruit and activate neutrophils (Gingles et al. 2001). Allergic inflammation further degrades the function of innate immunity, through suppression of the generation of antimicrobial peptides such as defensins from the epithelium, resulting in increased susceptibility to Pseudomonas aeruginosa infections in mouse models (Beisswenger et al. 2006). Abnormalities in the bronchial epithelium also appear to underlie risk factors for viral infection, since asthmatic airway epithelium shows an impaired interferon response to viral infection (Wark et al. 2005; Contoli et al. 2006). It is interesting to note that local immunity may be impaired even in the presence of a potentially vigorous chronic inflammatory response. Indeed, chronic inflammation may for other reasons provide an environment with potentially advantageous components from the point of view of the respiratory pathogen. Barrier disruption by epithelial damage may favor pathogen access, and upregulation of tissue cell adhesion molecule expression may make viral attachment and infection easier.

The complex immunopathology of asthma and other allergic diseases remains poorly understood. Making the link between the innate immune system and allergic disease has now allowed us to consider targets that had previously been overlooked. Each component of the innate immune system may be a feasible drug target, but we need to consider carefully how such targets should be handled. For example, if the innate immune system is activated repeatedly over time, generating repeated acute waves of high levels of activation (potentially on a background of chronic activation), it may be appropriate to target these separately from the underlying chronic disease. This might be achieved, for example, by neutralizing IL-1 or other important upstream cytokines during acute exacerbations. The long-lived nature of the immune response also requires serious thought when designing interventional therapies and drug trials. This is well illustrated by a cursory consideration of the biology of the macrophage. It is clear that monocytes and macrophages perform important functions as immune surveillance cells, with great potential to amplify inflammatory responses through cooperative signaling via tissue cells such as epithelium and smooth muscle (Morris et al. 2005, 2006). Macrophages are very long-lived cells, and a single inflammatory stimulus can result in the gradual repopulation of the lung by young monocytes that may have a different activation phenotype, and will persist for months or years in the lung tissue (Maus et al. 2006). Thus, preventing monocyte trafficking to the lung with drugs such as chemokine receptor antagonists is a potentially very useful therapy, but (i) the effects of these drugs may take months of administration to become apparent and (ii) their efficacy may be different on background chronic disease compared with the disease of acute exacerbations. However, targeting the processes of innate immunity is clearly effective and feasible. Considerable interest and excitement has been generated by the use of anti-tumor necrosis factor (TNF)-α strategies in the treatment of asthma (Howarth et al. 2005; Berry et al. 2006). Neutralization of IL-1 may also represent a useful treatment (Sabroe et al. 2006). Importantly, not all therapeutic options will involve the neutralization of cytokines: targeting innate immune deficiencies that lead to impairment of viral responses in asthmatics by replacement of absent cytokines is also theoretically feasible (Wark et al. 2005).

Therapeutic exploitation of the biology of the innate immune system

Taming the adaptive response

Activation of the innate immune system is an integral part of the pathology of allergic diseases such as asthma, with a dual role that has different emphases in disease initiation and disease perpetuation. Sadly underappreciated in the past, the resurgence in interest in innate immunobiology has been spearheaded by the identification of the TLR system and its huge contribution to health and disease.

One of the most exciting areas in the treatment of allergic disease is the prospect of using the power of the innate immune response to ameliorate allergic responses. Conventional immunotherapy aims to downregulate Th2 responses, while upregulating Th1 or Treg responses, by judicious administration of allergen. Although effective, it is timeconsuming and requires considerable clinical skill, being most effective in cases where single allergens are clearly dominant

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(Durham et al. 1999; Robinson et al. 2004). An understanding of TLR biology now allows the development of potentially more efficacious immunotherapy strategies. While much TLR signaling induces a Th1-type response, interest has focused on the biology of TLR9. This receptor responds to sequences in DNA that are more common in bacterial and viral DNA than human DNA, named “CpG motifs” (Hemmi et al. 2000; Bauer, S. et al. 2001; Lund et al. 2003). Short oligonucleotides containing CpG motifs are remarkably potent activators of TLR9-positive cells, including B cells (Bourke et al. 2003) and DCs, particularly the plasmacytoid DC subset (Bauer, M. et al. 2001; Kadowaki et al. 2001; Boonstra et al. 2003; Demedts et al. 2006). Other cells, such as the epithelium, also express TLR9 (Li et al. 2004; Sha et al. 2004). There is some variation between species and cell type in the nature of the optimal form of the CpG motif (Ballas et al. 2001; Roberts et al. 2005), but some form of secondary structure to the short oligonucleotide, such as the formation of hairpin loops, appears to facilitate immune responses. This activation induces a very strong Th1 response and can be used to induce Treg activity (Moseman et al. 2004). Add to these facts the potential to couple such short oligonucleotides directly to allergen (Marshall et al. 2001), minimizing the CpG dose and reducing the risk of nonspecific immune priming, and the potential to generate a very effective immunomodulatory therapy arises. Such strategies have been shown to be promising in animals (Tighe et al. 2000): there is now early human data showing that immunotherapy with CpG-conjugated allergen may be a very simple, safe, and effective treatment to reduce human allergic disease (Creticos et al. 2006). It is also feasible that administration of CpG motifs alone (also known as immunostimulatory DNA motifs) to the whole organism or to DCs ex vivo may be a useful strategy (Chiang et al. 2003; Ikeda et al. 2003; Fanucchi et al. 2004), but the potential for TLR9 signaling to contribute to disease pathologies such as systemic lupus erythematosus suggest that caution is needed with TLR-based therapies (Boulé et al. 2004; Barrat et al. 2005; Means et al. 2005), and dose limitation by allergen conjugation may be wise, though experience in humans has been generally reassuring to date (Krieg 2006). These data are obviously extremely exciting, but a note of caution needs to be sounded. Immunotherapy is traditionally most effective in individuals with a single dominant allergic disorder, such as grass pollen-induced hay fever. In chronic disease, allergen avoidance has been a disappointing treatment, and while the reasons for this may be legion, chronic allergen-independent disease mechanisms, which may in significant part be dependent on the innate immune system, may be dominant. Nonetheless, for hay fever, asthma related to single dominant allergens (e.g., animal danders), or venom allergies and anaphylaxis, TLR9-directed therapies are an extremely exciting area and several drugs are in clinical trials (Krieg 2006).

Innate Immunity in Allergic Disease

Conclusion The innate immune system is a multilayered, complex, germline-encoded system that interacts with the adaptive immune system at multiple levels. Its activation may underpin allergic disease, while serving different roles in disease initiation and disease persistence. Targeting innate immunity is feasible and already underway, with the potential to generate new and effective therapies for allergic disease.

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dendritic cells with potent functional activities. J Immunol 171, 3385– 93. Molesworth-Kenyon, S.J., Oakes, J.E. & Lausch, R.N. (2005) A novel role for neutrophils as a source of T cell-recruiting chemokines IP-10 and Mig during the DTH response to HSV-1 antigen. J Leukoc Biol 77, 552– 9. Morris, G.E., Whyte, M.K.B., Martin, G.F. et al. (2005) Agonists of Toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes. Am J Respir Crit Care Med 171, 814–22. Morris, G.E., Parker, L.C., Ward, J.R. et al. (2006) Cooperative molecular and cellular networks regulate Toll-like receptordependent inflammatory responses. FASEB J 20, 2153–5. Moseman, E.A., Liang, X., Dawson, A.J. et al. (2004) Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J Immunol 173, 4433– 42. Mukhopadhyay, S., Peiser, L. & Gordon, S. (2004) Activation of murine macrophages by Neisseria meningitidis and IFN-gamma in vitro: distinct roles of class A scavenger and Toll-like pattern recognition receptors in selective modulation of surface phenotype. J Leukoc Biol 76, 577– 84. Muzio, M., Bosisio, D., Polentarutti, N. et al. (2000) Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164, 5998– 6004. Nagai, Y., Akashi, S., Nagafuku, M. et al. (2002) Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 3, 667–72. Nagase, H., Okugawa, S., Ota, Y. et al. (2003) Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand. J Immunol 171, 3977– 82. Nigo, Y.I., Yamashita, M., Hirahara, K. et al. (2006) Regulation of allergic airway inflammation through Toll-like receptor 4-mediated modification of mast cell function. Proc Natl Acad Sci USA 103, 2286–9. Noulin, N., Quesniaux, V.F., Schnyder-Candrian, S. et al. (2005) Both hemopoietic and resident cells are required for MyD88dependent pulmonary inflammatory response to inhaled endotoxin. J Immunol 175, 6861– 9. Oda, K. & Kitano, H. (2006) A comprehensive map of the toll-like receptor signaling network. Mol Syst Biol 2, 2006.0015 (Epub). Ogata, H., Su, I., Miyake, K. et al. (2000) The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells. J Exp Med 192, 23– 9. Ohashi, K., Burkart, V., Flohe, S. & Kolb, H. (2000) Cutting edge: heat shock protein 60 is a putative endogenous ligand of the tolllike receptor-4 complex. J Immunol 164, 558–61. Ohya, M., Nishitani, C., Sano, H. et al. (2006) Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide. Biochemistry 45, 8657–64. Okamura, Y., Watari, M., Jerud, E.S. et al. (2001) The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 276, 10229–33. Okumura, S., Kashiwakura, J.I., Tomita, H. et al. (2003) Identification of specific gene expression profiles in human mast cells mediated by Toll-like receptor 4 and FceRI. Blood 102, 2547–54. Oltmanns, U., Sukkar, M.B., Xie, S., John, M. & Chung, K.F. (2005) Induction of human airway smooth muscle apoptosis by neutrophils and neutrophil elastase. Am J Respir Cell Mol Biol 32, 334–41.

Innate Immunity in Allergic Disease

Orinska, Z., Bulanova, E., Budagian, V. et al. (2005) TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment. Blood 106, 978–87. Ozinsky, A., Underhill, D.M., Fontenot, J.D. et al. (2000) The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc Natl Acad Sci USA 97, 13766–71. Park, J.S., Svetkauskaite, D., He, Q. et al. (2004) Involvement of tolllike receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J Biol Chem 279, 7370–7. Parker, L.C., Prince, L.R. & Sabroe, I. (2007) Networks regulated by Toll-like receptors mediate innate and adaptive immunity. Clin Exp Immunol 147, 199–207. Peiser, L., Gough, P.J., Kodama, T. & Gordon, S. (2000) Macrophage class A scavenger receptor-mediated phagocytosis of Escherichia coli: role of cell heterogeneity, microbial strain, and culture conditions in vitro. Infect Immun 68, 1953–63. Peiser, L., De Winther, M.P., Makepeace, K. et al. (2002) The class A macrophage scavenger receptor is a major pattern recognition receptor for Neisseria meningitidis which is independent of lipopolysaccharide and not required for secretory responses. Infect Immun 70, 5346–54. Perera, P.Y., Mayadas, T.N., Takeuchi, O. et al. (2001) CD11b/CD 18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxolinducible gene expression. J Immunol 166, 574–81. Peters-Golden, M. (2004) The alveolar macrophage: the forgotten cell in asthma. Am J Respir Cell Mol Biol 31, 3–7. Picard, C., Puel, A., Bonnet, M. et al. (2003) Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 299, 2076– 9. Pichavant, M., Taront, S., Jeannin, P. et al. (2006) Impact of bronchial epithelium on dendritic cell migration and function: modulation by the bacterial motif KpOmpA. J Immunol 177, 5912–19. Poltorak, A., He, X., Smirnova, I. et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 282, 2085–8. Punturieri, A., Alviani, R.S., Polak, T. et al. (2004) Specific engagement of TLR4 or TLR3 does not lead to IFN-beta-mediated innate signal amplification and STAT1 phosphorylation in resident murine alveolar macrophages. J Immunol 173, 1033–42. Qureshi, S.T., Lariviere, L., Leveque, G. et al. (1999) Endotoxintolerant mice have mutations in Toll-like receptor 4 (TLR4). J Exp Med 189, 615–25. Redecke, V., Hacker, H., Datta, S.K. et al. (2004) Cutting edge: activation of toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J Immunol 172, 2739–43. Rescigno, M., Urbano, M., Valzasina, B. et al. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2, 361–7. Roberts, T.L., Sweet, M.J., Hume, D.A. & Stacey, K.J. (2005) Cutting edge: species-specific TLR9-mediated recognition of CpG and nonCpG phosphorothioate-modified oligonucleotides. J Immunol 174, 605– 8. Robinson, D.S., Larche, M. & Durham, S.R. (2004) Tregs and allergic disease. J Clin Invest 114, 1389–97. Rock, F.L., Hardiman, G., Timans, J.C., Kastelein, R.A. & Bazan, J.F. (1998) A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 95, 588–93.

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Signal Transduction in Allergic and Inflammatory Cells Rafeul Alam

Summary Signal transduction is a fundamental cellular process that is essential for interpreting environmental cues and mounting an appropriate cellular response. Signal transduction is initiated by cell-surface receptors and involves a variety of enzymes, adapter molecules, chaperones, nucleotides, nucleotidebinding molecules, cytoskeleton, motor proteins, and ions. The generation of initial signal requires the activation of a kinase or a GTP-binding protein. The initial signal is then amplified through a variety of mechanisms including the recruitment of adapters and downstream targets. Many signaling pathways converge on a few final common pathways, including the NF-κB, Ca2+-calcineurin, mitogen-activated protein kinase (MAPK), Jak–STAT, and phosphatidylinositol 3-kinase (PI3K)–AKT pathways. Allergic inflammation is characterized by the presence of Th2 cells, eosinophils, and mast cells. Studies have identified important signaling molecules and master regulators of these inflammatory cells. Many cell-surface receptors, instead of stimulating, actually inhibit the cell through the generation of an inhibitory signal. Specific molecules have been identified that mediate this inhibitory signal. Therapeutic modalities are now being designed that either inhibit the activating signal or augment the inhibitory signal in order to control inflammatory diseases.

Table 10.1 Types of protein modification and the structural domains that recognize the modification. Amino acid

Modification

Signaling domain

Serine

Phosphorylation

14-3-3, KIX, MH2, WW, WD40, LRR, arrestin domain, PBD, BRCT, CID

Threonine

Phosphorylation

14-3-3, WW, FHA, PBD, BRCT

Tyrosine

Phosphorylation Nitration Sulfation

SH2, PTB, C2 ? ?

Lysine

Acetylation

Bromodomain

Arginine

Methylation Ubiquitination Sumoylation Neddylation

Chromodomain UIM, UBA, CUE, UEV, NZF, PAZ SIM, SHD ?

Proline

Isomerization

?

Aspartate

ADP-ribosylation

?

Glutamate

Methylation

?

Signal generation Introduction Many immune cells as well as resident tissue cells participate in allergic diseases. There are significant differences in signaling pathways leading to their activation. It is not the objective of this chapter to describe signaling mechanism of all allergic cells. Instead it provides a broad outline of general signaling mechanisms and then focuses on the signal transduction mechanisms of select cells of the immune system.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Most proteins have an innate ability to function as environmental sensors. They sense subtle changes in the surrounding microenvironment (at the cell membrane as receptors or inside the cell) and in response undergo a conformational change. This conformational change lies at the heart of signal generation. As a result of this conformational change the protein may become catalytically active (if the protein is an enzyme) and modify another protein or acquire affinity to interact with a new partner. This change(s) is sensed by other proteins, which creates a ripple effect and leads to signal transduction. The ultimate goal of this process is to read, generate/amplify, and convey information. Proteins are amenable to secondary modification on many residues (Yang 2005). A list of amino acid residues that undergo secondary modification is shown in Table 10.1. The secondary

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modification of proteins is sensed by an appropriate interactive domain (e.g., SH2 domain recognizing phosphorylation of tyrosine residue) leading to protein–protein interaction and exchange of information. A major focus of signaling research is to understand how a cell receives signals from the environment and conveys this signal to intracellular destinations for an appropriate biological response. Cell-surface receptors are primarily responsible for generating intracellular signals in response to extracellular cues. Intracellular molecules that generate and transduce signals can be classified into the categories shown in Table 10.2. These molecules utilize a variety of mechanisms to generate signals. Examples include association with GTP or GDP, phosphorylation by kinases and dephosphorylation by phosphatases, ubiquitylation (mono or poly), sumolation, and

Table 10.2 Intracellular molecules that generate and transduce signals. Enzymes Kinases Tyrosine Serine/threonine Leucine Threonine/tyrosine kinases Phosphatases Proteases Ligases (e.g., ubiquitin ligase) GTPases Heterotrimeric G proteins (a, b, g) p21 G proteins Ras (oncoprotein Ras) Rho (Ras homolog) Rab (Ras-related genes expressed in rat brain) ARF (ADP ribosylation factor) Adapter/scaffolding proteins Grb2 (growth factor receptor-bound protein 2) MP1 (MEK1 partner 1) MyD88 (myeloid differentiation factor 88) TIRAP (TIR domain-containing adapter protein) Regulatory/inhibitory proteins SOCS (soluble inhibitor of cytokine signaling) RGS (regulator of G-protein signaling) Lipids Inositol trisphosphate Nucleotides Cyclic AMP Cyclic GMP Ions Ca2+ Transcription factors

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acetylation/deacetylation. These modifications significantly alter the physiochemical behavior of the protein. A frequent outcome of this process is dimerization/oligomerization of signaling molecules, association with other signaling proteins and chaperones, and translocation to specific destinations. The secondary modification of amino acid residues of a protein or its conformational changes are recognized by specific structural domains that play a critical role in signal transduction. The PFAM (protein family database alignment and hidden Markov models) database (www.sanger.ac.uk/Software/Pfam/) lists 84 different modular domains. A few examples of these modular domains and their target amino acid residues are shown in Table 10.1. A typical example of this domain-based interaction is phosphotyrosine. When a protein is tyrosyl phosphorylated, it is recognized by three or more distinct modular domains: SH2, C2, and phosphotyrosine-binding domain. Which of these domains interacts with phosphotyrosine depends on conserved amino acid residues adjacent to the phosphotyrosine residue and availability of domaincontaining proteins in the surrounding milieu.

G protein-coupled signaling Heterotrimeric G proteins Activation of specific proteins through GTP binding represents a major form of signal transduction (reviewed by Hubbard & Hepler 2006; Pierce et al. 2002). There are two families of G proteins: heterotrimeric (big) G proteins and small G proteins. Heterotrimeric G proteins transduce signals from G protein-coupled receptors (GPCR). GPCR represent the biggest family of cell membrane receptors. The Swiss-Prot database (www.expasy.org/sprot) has 2841 entries, encompassing all species. GPCR relevant to allergic diseases include receptors for histamine, leukotrienes, prostacyclins, kinins, adenosine, neurokinins, chemokines, and most hormones and neurotransmitters (including epinephrine and acetylcholine). The heterotrimeric G proteins comprise three subunits: α, β, and γ. The Gα subunit has 25 family members (examples include Gi, Gs, Gq, and G12), Gβ has five members, and Gγ has 10 members. In unstimulated cells GPCR are associated with the GDP-bound form of Gα (Fig. 10.1). On ligand binding GDP is exchanged for GTP, which triggers dissociation of Gα from the β and γ subunits. Activated Gα then associates with and stimulates adenyl cyclase, phospholipase C (PLC)-β, Src family kinases, and Bruton’s tyrosine kinase (Btk), among others. The β and γ subunits activate PI3K-γ. The activation of these effectors transduce signals downstream. The GTP bound to Gα is rapidly hydrolyzed to GDP. As a result the GDP-bound Gα regains affinity for the β and γ subunits and homeostasis is restored. The G proteins represent a major mechanism of cAMP- and Ca2+-dependent signaling in the cell. PLC-β hydrolyzes phosphatidylinositol bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG), leading to the elevation of calcium and activation of protein kinase C (PKC). In addition, G protein activation is linked to

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H

Receptor

IP3

PLCb,g Ga

Ga family (23 members) • Gs • Gi • Gq • G12 Gb (5 members) Gg (10 members)

Ga

b g

c

b g

GTP GDP PI3Kg Src

GDP-a

b g

GTP-a

Effector Phospholipase Cb Adenyl Cyclase Bruton’s tyrosine kinase (Btk) Src-family

Fig. 10.1 Signal transduction mechanism of G protein-coupled receptors. Ligand binding to the receptor induces a conformational change in Ga and an increase in its affinity for GTP. The GTP-bound Ga dissociates from the b and g subunits. The dissociated subunits activate downstream signaling molecules. The GTPase activity of Ga hydrolyzes GTP into GDP. The GDPassociated Ga regains affinity for b and g subunits and homeostasis is restored. The ribbon diagram in the upper right corner shows the crystallographic structure of rhodopsin, a prototypic G protein-coupled receptor. (See CD-ROM for color version.)

the MAPK pathway (Kampen et al. 2000). Inhibition of Gi with pertussis toxin blocks the eotaxin-induced activation of ERK and p38 in eosinophils. There are also reports suggesting that the βγ dimer of G proteins can bind and activate PI3K-γ. Elevated intracellular Ca2+ activates a variety of downstream effectors including calcineurin, calmodulin, and myosine light chain kinase. Calcineurin is a phosphatase that dephosphorylates NFAT, a transcription factor. Phosphorylated NFAT is sequestered in the cytosol. Dephosphorylation allows its translocation to the nucleus, association with other transcription factors (e.g., AP1), and induction of new gene transcription (Fig. 10.2). Myosin light chain kinase activates myosin, an important motor protein involved in cell motility, contraction, degranulation, and cytoskeletal changes.

Small G proteins Small G proteins represent a large family of signaling molecules. Unlike the big G proteins, small G proteins do not associate with β and γ subunits. However, like big G proteins their activation status is determined by the association with GTP. There are multiple subfamilies including Ras (rat sarcoma), Rho (ras homology), Rab, Ran, ARF, and non-Ras GTPases (reviewed by Colicelli 2004). The Ras family has 35 members, Rho 23 members, Rab and Ran 71 members, and ARF and SARA 30 members. Ras and Rac play a major role in activation of the MAPK pathway. Rac, Rho, and cdc42 are critical regulators of the cytoskeleton (Etienne-Manneville & Hall 2002). Rac controls actin polymerization, membrane ruffles forma-

Ca2+ Calcineurin NFAT-p

Nucleus NFAT

NFAT

Fig. 10.2 Calcium and calcineurin signaling pathway. Receptor stimulation leads to activation of phospholipase C (PLC) b or g isoform, which then acts on membrane lipids and generates inositol trisphosphate (IP3). IP3 releases Ca2+ from intracellular stores. Increased Ca2+ activates calcineurin, which is a phosphatase. Calcineurin dephosphorylates NFAT (nuclear factor of activated T cells), which then migrates to the nucleus. (See CD-ROM for color version.)

tion, and cell movement, whereas Rho is essential for focal adhesion formation. Cdc42 is a master regulator of cell polarity and directional cell movement. Broadly, this family of G proteins determines cell shape, polarity, movement, phagocytosis, and secretion. Proteins of the Rab (reviewed by Jordens et al. 2005) and ARF (Randazzo et al. 2000) families are involved in endomembrane movement and vesicular transport and as such play a vital role in endocytosis and exocytosis.

Tyrosine kinases Tyrosine kinases catalyze the transfer of the γ phosphate of ATP to the hydroxyl group of tyrosine on proteins. This family of kinases consists of nearly 500 members. Most kinases, including tyrosine kinases, are maintained in an inactive conformation through intramolecular interactions. Activation of kinases requires disruption of these intramolecular bonds by interaction with other molecules/ligands. In the case of receptor tyrosine kinases (RTKs) extracellular ligands induce this conformational change and induce their catalytic activity. Examples of RTKs include receptors for growth factors such as epidermal growth factor (EGF), platelet-derived growth factor, and nerve growth factor. The structural mechanism of activation of some of these kinases has been elucidated. The EGF receptor undergoes a dramatic conformational change in its extracellular domain (Zhang et al. 2006), which is transmitted to the intracellular domain. This conformational change in the intracellular domain leads to head-to-tail positioning of the kinase domain of two adjacent receptors (Fig. 10.3). This head-to-tail positioning disrupts their intramolecular inhibitory binding and activates the kinase. Nonreceptor tyrosine kinases (NRTKs) bind to receptors in a noncovalent fashion. Some NRTKs do not bind cell

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EGFR ErbB2 ErbB4

EGFR ErbB2 ErbB3 ErbB4

membrane receptors and their activation occurs in the later phase of the signaling cascade. NRTKs include the following families: ACK, Csk, FAK, Fes, FRK, JAK, Src, Syk, and Tec. The Src family is one of the larger tyrosine kinase families. There are 10 family members: Blk, Brk, Fgr, Fyn, Hck, Lck, Lyn, Src, Srm, and Yes. Lck and Fyn play an essential role in signal generation by the T-cell receptor (TCR) (Palacios & Weiss 2004). Lck and Fyn associate, respectively, with CD4 and CD3 of the TCR complex and are critical for activation of T cells (Fig. 10.4). Activation of these kinases leads to phosphorylation of ZAP70 and propagation of downstream signals via the MAPK, PLC-γ and Ca2+ signaling pathways. Lyn, Fyn, and Syk kinases play a similar signal-generating role in B-cell receptor (BCR) and FcεRI signaling. Lyn and Syk are important for growth factor-mediated signal generation in eosinophils. Src family kinases also play an essential role in signaling processes mediated by cell–cell adhesions. The Janus family represents an important family of NRTKs. Most cytokine and many growth factor receptors utilize this family of tyrosine kinases to transduce signals. There are four members: Jak1, Jak2, Jak3, and Tyk2 (Aaronson & Horvath 2002). These kinases are especially important for hematopoiesis and development of the immune system. Jak kinases phosphorylate the STAT family of transcription factors and regulate cellular response to cytokine and growth factor stimulation (Kisseleva et al. 2002).

Clinical relevance The deficiency of many tyrosine kinases leads to severe immunodeficiency. Jak3 deficiency causes severe combined immunodeficiency (SCID) (Macchi et al. 1995). Tyk2 mutation has been linked to a patient with hyper-IgE syndrome (Minegishi et al. 2006). Lck deficiency causes T-cell deficiency (Goldman et al. 1998). ZAP70 deficiency/mutation has been linked to SCID and CD8 T-cell deficiency (Chan et al. 1994). Btk deficiency or mutation causes X-linked agammaglobulinemia (Rawlings et al. 1993). Mutation of EGF receptor family members is the cause of a variety of cancers.

Mechanism of activation of Src family kinases Most kinases are inactive in their native conformation. This inactive state is maintained through multiple intramolecular

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Fig. 10.3 Schematic presentation of activation of the epidermal growth factor (EGF) receptors: EGFR, ErbB2, ErbB3, and ErbB4 (ErbB is homologous to the viral erythroblastoma oncogene v-erbB). Following the binding of the ligand EGF, the intracellular domains of the dimerized receptor position themselves in a head-to-tail position. This conformational change opens up the activation loop, which results in activation of the kinase. (See CD-ROM for color version.)

CD4

Unc Lck

TCR ab d d zz

Fyn

T-cell signaling LAT

I ZAP70 PKC8

Grb2

P

PI4,5P2

P PLCg Ras

P P GADS SOS P SLP76

NFkB

IP3 MAPKKK

MAPK

Ca++ NFAT

IL-2 transcription Proliferation

Fig. 10.4 Simplified schematic presentation of signal transduction mechanism of the T-cell receptor (TCR). Antigen presentation leads to the formation of a multimolecular complex involving CD3g (not shown), d, e, two z subunits and CD4. The CD4-associated adapter protein Unc119 activates the tyrosine kinases Lck and Fyn, which then phosphorylate ITAM (I) residues on z subunits. Phosphorylated ITAMs recruit ZAP70. Activated ZAP70 then phosphorylates the transmembrane adapter protein LAT. Phosphorylated LAT recruits a number of signaling molecules including PLC-g, Grb2, GADS, SLP76, and SOS. The latter activates Ras and transduces signals via the MAPK pathway. The action of PLC-g leads to the generation of Ca2+ signals and activation of the transcription factor NFAT. TCR also activates PKC-q, which transduces signals via the NF-kB pathway. GADS, Grb2-like adaptor downstream of Shc; Grb2, growth factor receptor-bound 2; ITAM, immunoreceptor tyrosine-based activating motif; LAT, linker of activated T cells; NFAT, nuclear factor of activated T cell; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; SLP76, SH2-containing leukocyte-specific protein of 76 kDa; SOS, son of sevenless; ZAP70, z chain-associated protein of 70 kDa. (See CD-ROM for color version.)

bonds that impose a constrained conformation. An essential step in kinase activation is the disruption of these inhibitory intramolecular bonds, allowing a relaxed conformation and catalytic activation. The crystal structure of Src kinases has provided a basic understanding of kinase activation (Boggon & Eck 2004). Src kinases are composed of five structural components: a unique region, SH2 (SH, Src homology) and SH3 domains, a linker region, and the kinase domain. There are two inhibitory intramolecular bonds that keep the kinase in a closed “off” conformation. The first is a tyrosine residue

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at the C-terminus, which when phosphorylated binds to the SH2 domain. The linker region contains a proline-rich SH3-binding motif, which binds to the SH3 domain and creates a second intramolecular bond. Activation of the kinase requires a stepwise approach. The engagement of a tyrosine phosphatase (e.g., CD45) dephosphorylates the C-terminal phosphotyrosine and disrupts the first bond. The second step is disruption of the SH3–linker interaction. This involves interaction with a high-affinity SH3 motif-containing ligand, which disrupts the internal low-affinity binding and moves the SH3 domain away from the activation loop. This allows a relaxed conformation leading to kinase activation. The identity of the endogenous ligand was until now unknown. We recently reported the cloning and characterization of the new SH3 ligand called Unc119 (Cen et al. 2003; Gorska et al. 2004). Unc119 is associated with many membrane receptors including CD3, CD4, and interleukin (IL)-5 receptor α chain (see Fig. 10.4). On receptor stimulation Unc119 binds to the SH3 domain of Src kinases (e.g., Lck, Fyn, Lyn) and activates the kinase, which results in its autophosphorylation.

Signal amplification Adapter proteins The initial activation of receptor-associated signaling molecules is usually transient and weak in nature. In order to generate a conducive signal this initial receptor-associated signal is amplified through a variety of mechanisms. Adapter proteins are one class of molecules that function as amplifier of signals (Wonerow & Watson 2001; Lindquist et al. 2003). Adapter proteins are signaling molecules that do not have catalytic domains, but instead contain modular protein–protein and protein–lipid interaction domains or motifs. Adapter proteins are involved in the proper organization and activation of protein complexes. Following activation many receptors are phosphorylated. This phosphorylation causes recruitment of adapter proteins. The recruited adapters amplify and relay signals to a next set of signaling molecules. Examples of adapters include LAT, Shc, Grb2, GAD, Nck, SLP76, and MyD88. In the case of T cells or mast cells, initial tyrosine kinase activation leads to phosphorylation of LAT on up to nine different tyrosine residues (see Fig. 10.4). These phosphotyrosine residues then serve as docking sites for additional signaling molecules such as GAD, SLP76, and others. The recruited GAD and SLP76 also undergo phosphorylation and relay the signal to another set of signaling molecules.

Clinical relevance BLNK (B-cell linker protein, also known as BASH and SLP65) is an adapter protein that is expressed in B cells and is important for B-cell signaling. Deficiency of BLNK is a cause of hypogammaglobulinemia (Minegishi et al. 1999). Deficiency of another adapter protein IL-1 receptor-associated kinase

Signal Transduction in Allergic and Inflammatory Cells

(IRAK)-4 causes pyogenic bacterial infections (Picard et al. 2003). The mutation of SLAM-associated protein (SAP), also known as SH2 domain-containing adapter protein (SH2D1A), which is expressed in T cells and natural killer cells, causes X-linked lymphoproliferative disorder (Morra et al. 2001).

Lipid rafts Another mechanism of signal amplification is signaling through the lipid raft. Lipid rafts are freely diffusing, stable, lateral assemblies of sphingolipids and cholesterol. They constitute an important organizing principle for the plasma membrane. The basic concept is that lipid rafts facilitate selective protein–protein interactions by selectively excluding or including proteins. An example of this lipid raftfacilitated signaling is the concentration of Lck, CD2, CD28, and Cbp in T cells (Rodgers et al. 2005).

ITAM, ITIM, and ITSM Some transmembrane receptors have two tyrosine residues that are spaced at an appropriate distance and placed in the context of conserved residues: D/E-X7-D/E-X2-Y-X2-L-X7Y-X2-L (where X indicates any amino acid residue). These tyrosine residues, when phosphorylated, are able to recruit and activate downstream signaling molecules that contain phosphotyrosine-binding domains (e.g., SH2, C2, and the phosphotyrosine binding domain). This conserved sequence is known as ITAM (imunoreceptor tyrosine-based activation motif) (Flaswinkel et al. 1995). The following receptors contain ITAM in their cytosolic fragment: TCR ζ, γ, δ and ε; BCR α and β; FcεRI β and γ; and FcγRIIa. Another conserved tyrosine residue-based motif recruits signaling inhibitors (e.g., tyrosine and lipid phosphatases: SHP-1, SHP-2, SHIP) instead of signaling activators and thereby terminates signal transduction (Isakov 1997). This motif has the following sequence: I/L/V-X-Y-XX-L/V. This motif is preceded and followed by hydrophobic residues unlike the tyrosine residues in ITAM, which is preceded by acidic residues. ITIM (imunoreceptor tyrosine-based inhibitory motif) is present in the following receptors: FcγRII, KIR, NKG2A/B, mLy-49A, gp49B1, SIRPα, Siglec-3, -5, and -8, PIR-B, and MAFA. An example of ITAMbased signal amplification is signaling by the TCR. The CD3 complex has two CD3ζ subunits and the cytosolic fragment of each CD3ζ has three ITAMs. Since each ITAM binds two SH2 domain-containing molecules or a single molecule with two SH2 domains, one CD3 complex can bind a total of 12 SH2 domain-containing molecules. This results in substantial amplification of the initial signal at the receptor complex. In the case of T cells and CD3ζ (see Fig. 10.4), phosphorylation of ITAM results in the recruitment of multiple ZAP70 molecules and subsequent downstream signal transduction. It should be pointed out that although in the vast majority of cases ITAM generates an activation signal (as the name implies), there are examples where ITAM actually delivers an inhibitory signal and ITIM generates activation signals

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(Barrow & Trowsdale 2006). A variation of ITIM has recently been coined, the immunoreceptor tyrosine-based switch motif (ITSM), which has the following consensus sequence: T-X-Y-X-X-V/I (Shlapatska et al. 2001). This motif is present in CD150, 2B4, CD33, Siglec-9, and PD1. The presence of this motif allows binding of the adepter protein SH2D1A to the receptor and switches its interaction from a tyrosine phosphatase (SHP-1, SHP-2) to a lipid phosphatase (e.g., SHIP).

Signaling integration MAPK pathway An example of signaling integration is the activation of MAPKs (Roux & Blenis 2004). Various extracellular and intracellular signaling pathways converge on the MAPK signaling pathway. MAPKs are activated in a cascade involving two upstream protein kinases known as MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK). There are at least 10 different members of the MAPK family. Four pathways have been well characterized and extensively studied, including ERK1/2, JNK, p38, and ERK5 (Fig. 10.5). MAPK signaling regulates major cellular functions including proliferation, differentiation, survival, apoptosis, adhesion, migration, and degranulation. In a typical growth factor-initiated signaling pathway the phosphorylated receptor serves as the docking site for the SH2 domain of the adapter protein Shc. Subsequently, Shc undergoes tyrosine phosphorylation and recruits the adapter protein Grb2. Grb2, in turn, binds to Sos (son of sevenless), a Ras GTP/GDP exchange factor. Therefore, interaction of Shc

Raf-1, A & B-Raf, Cot/Tpl2

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Fig. 10.5 Mitogen-activated protein kinase (MAPK) signaling pathway. The generic MAPK cascade starts with a MAPK kinase kinase (MAPKKK), which phosphorylates the MAPK kinase (MAPKK), which in turn phosphorylates MAPK (left panel). Four major MAPK signaling pathways, ERK1/2, JNK1–3, p38 and ERK5 and their upstream activators, are shown. ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase. (See CD-ROM for color version.)

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with the growth factor receptor results in recruitment of the Grb2–Sos complex to the membrane, where the Sos effector Ras is localized. Sos induces GDP to GTP exchange in Ras, which leads to its activation. Ras stimulates the activity of the serine/threonine kinase Raf-1, which is a MAPKKK. Raf-1, in turn, induces the activation of MEK1, a MAPKK. MEK1 phosphorylates ERK on threonine and tyrosine residues on the consensus T-E-Y motif of ERK1 and ERK2. Activated ERK1/2 kinases then phosphorylate a multitude of downstream cytosolic and nuclear effectors in the consensus motif S/T-P. The ERK1/2 module (Raf-1–MEK1/2–ERK1/2) can be activated by a variety of upstream pathways including various small G proteins (Ras, Rap), Gα and Gβγ, Ca2+ signaling and adapter proteins (e.g., TAB). Like ERK1/2, the other members of this family (JNK and p38) are activated by multiple upstream mechanisms.

Clinical relevance Ras is one of the most frequently mutated genes in human cancer (Rodriguez-Viciana et al. 2005). For this reason various strategies are being developed to interfere with this signaling pathway. Genetic, molecular, and animal studies implicate ERK1/2, p38, and ERK5 in various inflammatory illnesses including rheumatoid arthritis, chronic obstructive pulmonary disease, and asthma (Duan & Wong 2006). Low-molecularweight inhibitors of these pathways are now undergoing human trials.

NF-kB pathway This pathway represents one of the most important signaling pathways for cell activation, survival, differentiation, and oncogenesis. It demonstrates signaling integration at key signaling hubs. Various signaling pathways converge on the IKK (inhibitor of κB kinase) complex (Fig. 10.6) (Li & Verma 2002). For example, the TCR complex activates PKC-θ, which transduces signals through the complex of BCL10, MAGUK, and MALT1 to the IKK complex. The tumor necrosis factor receptor superfamily engages the adapter proteins TRADD and TRAF2 to activate RIP and MKK3, which then converge on the IKK complex. IRAK binds to the cytosolic fragment of Toll-like receptors (e.g., TLR4). Activation of TLR leads to the recruitment of TRAF6 through IRAK. TRAF6 propagates signals via TAK1 and TAB, which leads to IKK activation. The IKK complex is composed of IKKα, IKKβ, and IKKγ (NEMO). IKKγ (NEMO) is a scaffold for IKKα and IKKβ and is important for proper signal transduction. Activation of IKKα, especially IKKβ, leads to phosphorylation of IκB. Phosphorylation of IκB is a signal for its ubiquitylation and proteosomal (β-TRCP ubiquitin proteasome) degradation. IκB binds NF-κB (p65, p50) and retains it in the cytosol. Its degradation allows NF-κB to migrate to nucleus. NF-κB is frequently phosphorylated and acetylated, which facilitates its DNA-binding activity. In the nucleus NF-κB binds to its

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TNFR

TCR

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TLR

Cytokine Cytokine receptor

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Proteosomal degradation Fig. 10.6 NF-kB signaling pathway. Activation of various receptors such as those for tumor necrosis factor (TNFR), T-cell receptor (TCR), or Toll-like receptor (TLR) leads to the generation of signals that converge on the inhibitor of kB (IKK) complex. IKK phosphorylates (P) IkB, which is then ubiquitylated (ub) and degraded. The IkB-bound NF-kB is then released and translocated to the nucleus for transcriptional activity. See text for definition of other abbreviations. (See CD-ROM for color version.)

Nucleus P Y PIAS

STAT PTP

Y P

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Transcription

consensus sequence on the promoter of more than 200 genes and induces gene transcription.

Clinical relevance Mutation of IKKγ (NEMO) causes X-linked hypohidrotic ectodermal dysplasia with variable immunodeficiency (Zonana et al. 2000). NEMO deficiency may manifest as severe profound immunodeficiency or as hyper-IgM syndrome.

Jak–STAT pathway The Janus kinases (Jak1– 3 and Tyk2) phosphorylate a family of transcription factors called STAT (signal transducer and activator of transcription). There are six members of the STAT family: STAT1–STAT6 (Kisseleva et al. 2002). Following tyrosine phosphorylation, the STATs undergo homodimerization and heterodimerization, which allows them to translocate to nucleus (Fig. 10.7). Frequently the STATs are additionally phosphorylated by MAPK, which enhances their transcriptional activity. Most cytokines signal through the Jak–STAT pathway. The receptor for IL-12 and interferon (IFN)-γ activates STAT1 and STAT4, respectively (O’Garra & Robinson 2004). Activation of STAT1 and STAT4 is important for the induction of the transcription factor T-bet and for a Th1 response (Fig. 10.8). The IL-4 receptor transduces signals through STAT6. In addition to STAT6, STAT5 is also important for a Th2 response. Both these molecules induce GATA-3 transcription, which is a master regulator for Th2 differentiation (Murphy & Reiner 2002).

Clinical relevance STAT1 mutation predisposes patients to extrapulmonary mycobacterial infections (Dupuis et al. 2001).

Fig. 10.7 Jak–STAT signaling pathway. The binding of cytokines to their respective receptors causes activation of receptor-associated Jak (Janus kinase) kinases: Jak1, Jak2, Jak3, and Tyk2. Activated Jak kinases phosphorylate the receptors and the recruited STATs. Phosphorylated STATs dimerize and translocate to nucleus. Phosphorylated Jaks are inhibited by protein tyrosine phosphatase (PTP) and SOCS (suppressor of cytokine signaling). The transcriptional activity of STAT is antagonized by PIAS (protein inhibitor of activated STATs) and PTP. (See CD-ROM for color version.)

STAT1 T-Bet

STAT6 STAT5

STAT4

GATA-3 C-Maf

RORgT

FoxP3

Th1

Th2

Th17

Treg

Fig. 10.8 Transcription factors and T-cell differentiation. Major transcription factors involved in Th1, Th2, Th17, and Treg are shown. The initial transcription factors that lead to the induction of RORgT (retinoic acid-related orphan receptor g expressed in T cells) and FoxP3 (forkhead box protein 3) are currently unknown.

PI3K–AKT pathway PI3K phosphorylates phosphatidylinositol lipids at the D3 position of the inositol ring and produces phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Cantley 2002). The reaction is reversed by the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10). Cellular membranes are the sources of PI3K substrates. PIP3 creates a membrane

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docking site for pleckstrin homology (PH) domain-containing proteins, e.g., the serine/threonine kinase AKT, tyrosine kinases belonging to Tec family, PLC-γ, and the GTP/GDP exchange factor Vav. The binding of PIP3 at the cell membrane is important for activation of these molecules. The activation of AKT (also known as protein kinase B) depends on additional kinases: phosphoinositide-dependent kinase 1 and 2 (PDK1 and PDK2). AKT, in conjunction with the ERK1/2 pathway, activates S6 kinase and facilitates activation of mTOR (mammalian target of rapamycin) (Shaw & Cantley 2006). Activation of S6 kinase and mTOR promotes cell survival and proliferation. AKT regulates a number of important signaling pathways including GSK3β, Bcl2 family members, and the transcription factors p53, FOXO and β-catenin. AKT regulates cell survival through its phosphorylation of Bcl family members and inhibition of GSK3β.

Signal termination The duration and amplitude of signals are under stringent control. Short-lasting or weak signals may not elicit any cellular response. On the other hand long-lasting or very strong signals may lead to pathologic changes, e.g., cell death or excessive proliferation. Signal termination is essential for cellular homeostasis. Cells employ a number of mechanisms to terminate signaling processes. Receptor endocytosis is one such mechanism. Receptors may also undergo degradation. Activation of many receptors leads to the recruitment of a ubiquitin ligase, e.g., Cbl. IL-5 induces ubiquitination and proteosomal cleavage of the cytoplasmic portion of β chain (Martinez-Moczygemba & Huston 2001). Enzymatic reversal of modified signaling molecules is another mechanism. Targeting of phosphorylated tyrosine, serine, threonine, and lipid residues by phosphatases is an efficient mechanism of restoration of homeostasis. The tyrosine phosphatase SHP-1 associates with the phosphorylated βc chain of the IL-3 receptor on stimulation of myeloid cells and negatively regulates IL-3-induced proliferation (Paling & Welham 2002). SHIP is an SH domain phosphatidylinositol phosphatase, which inactivates PIP3 and inhibits signal transduction. SHP and SHIP are preferentially recruited to the ITIM of inhibitory receptors such as FcRγII and PIR-B, and blocks signaling through activating receptors such as FcεRI on mast cells (Uehara et al. 2001). Signal termination also involves physical blocking of active sites. SOCS (suppressor of cytokine signaling) family members bind to phosphorylated tyrosine residues of other molecules, such as receptors or Jaks and block access of downstream molecules (e.g., STATs) (Alexander & Hilton 2004). CIS1 binds to the tyrosine-phosphorylated βc chain and prevents STAT5 recruitment (Matsumoto et al. 1997). IL-5 and granulocyte–macrophage colony-stimulating factor (GM-CSF) both upregulate mRNA for CIS in eosinophils, which is likely to function as a negative feedback mechanism.

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SOCS1 has been proposed to bind to the tyrosine residue in the activation loop of Jaks (including Jak2) and block access of Jak substrates (Ilangumaran & Rottapel 2003). Recently, a negative regulator of the ERK pathway has been identified in eosinophils (Inoue et al. 2005). Spred1 is a member of the Sprouty-related family of signaling molecules. It binds to Raf-1 and inhibits Raf-1 and ERK activation through a not-so-well characterized mechanism. IL-5-stimulated eosinophil differentiation is enhanced in Spred1-deficient mice.

Signal transduction in eosinophils The eosinophil is a major effector cell in allergic inflammation. Its growth and differentiation are regulated by hematopoietins such as IL-3, IL-5, and GM-CSF. The function of mature eosinophils is regulated by chemokines. The hematopoietins have their own α receptor and share a common β receptor. The common β receptor generates and transduces the majority of the intracellular signals. Tyrosine kinases of the Src and Janus family associate with the hematopoietin receptor (Adachi & Alam 1998). For example, IL-5 receptor associates with Jak1, Jak2, and Lyn kinase (Torigoe et al. 1992; Pazdrak et al. 1995; Van der Bruggen et al. 1995). Receptor dimerization leads to the interaction of the adapter protein Unc119 with Lyn (Fig. 10.9). As a result Lyn is activated. Lyn and

IL-5 receptor a bc Unc Jak1/2 STAT5

Lyn P P

P Syk P Shc

Ras c-raf

AKT

P ERK PLA2

p38 BclxL

GSK3b

Differentiation Survival Degranulation Cytokine Fig. 10.9 Eosinophil signaling through the interleukin (IL)-5 receptor. Engagement of IL-5 to the receptor induces interaction of Unc119 with Lyn kinase, which results in activation of the kinase. Lyn and Jak kinases phosphorylate the common b receptor (bc). Phosphorylated bc recruits Syk and Shc, which indirectly activate Ras, the MAPK signaling pathways and AKT. Both ERK and p38 pathways activate phospholipase (PL)A2 to generate eicosanoids. AKT regulates eosinophil survival by activation of the Bcl family of molecules and inhibition of GSK3b. Jak kinases phosphorylate STATs, including STAT1, STAT3 and STAT5. STATs regulate eosinophil differentiation. AKT, viral oncogene v-AKT; Bcl, B-cell lymphoma-associated gene product; ERK, extracellular signal-regulated kinase; GSK, glycogen synthase kinase; STAT, signal transducer and activator of transcription; Unc119, uncoordinated gene 119. (See CD-ROM for color version.)

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other tyrosine kinases contribute to the activation of Ras, PI3K, and MAPK signaling pathways, especially that of ERK1/2 and p38 (51). Jak kinases phosphorylate and activate STAT1, STAT3, and STAT5, which then translocate to the nucleus and induce new gene transcription. STAT5, ERK1/2, and p38 MAPK play an important role in eosinophil differentiation. ERK1/2 and p38 MAPK are involved in eosinophil chemotaxis, degranulation, and cytokine production (Pazdrak et al. 1995; Kagami et al. 2000; Kampen et al. 2000). The PI3K– AKT pathway is involved in eosinophil survival. Chemokines such as eotaxin activates CCR3 and transduces signals via the Gi protein. This pathway leads to activation of PI3K and MAPK signaling pathways (Kampen et al. 2000). There are important differences in signaling between the IL-5 receptor and CCR3 in regard to the amplitude, duration, and intracellular localization of PI3K and MAPK activation. The activation of PI3K, AKT, ERK1/2, and p38 by IL-5 is of higher amplitude and longer duration than that by eotaxin. This difference translates into a qualitatively different biological response.

Signal transduction in mast cells The high-affinity IgE receptor (FcεRI) is composed of four subunits: α, β, two γ subunits. The β subunit is associated with Lyn kinase, which is an important activator and regulator of signal generation by FcεRI (Nadler et al. 2000). On IgE binding to the receptor, Lyn is activated by an unknown mechanism, which leads to the phosphorylation of ITAM on γ subunits (Fig. 10.10). Syk is then recruited to these phosphorylated ITAMs, which then phosphorylates the membrane-associated adapter protein LAT (linker of activated T cells). Phosphorylated LAT then functions as a docking site for many downstream signaling molecules including PLC-γ, GADS, SLP76, and the Grb2–Shc complex. The action of PLC-γ leads to the generation of IP3 and mobilization of intracellular calcium. Further, it releases DAG, which activates PKC. The multimolecular complexes of Grb2–Shc, GADS, and SLP76 transduce signals via ERK1/2 and p38 MAPK signaling pathways (Gilfillan & Tkaczyk 2006). Both ERK1/2 and p38 MAPK phosphorylate phospholipase A2, leading to eicosanoid synthesis via the cyclooxygenase and lipoxygenase pathways. Calcium, PKC, and MAPK signaling are all important for mast cell degranulation. MAPK signaling seems to contribute to cytokine synthesis. In addition to Lyn, recent studies point to an important contribution by the Fyn kinase. Unlike Lyn, Fyn transduces signals by engaging NTAL, an adapter protein that is similar to LAT in its signaling role (Rivera 2005). Mast cell signaling is regulated by the input from the activating receptor such as FcεRI and c-kit as well as by inhibitory receptors such as FcγRIIb (receptor for IgG) (Ono et al. 1996), gp49B1 (Daheshia et al. 2001), MAFA (Ortega

Signal Transduction in Allergic and Inflammatory Cells

FceRI a b gg

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Fig. 10.10 Mast cell signaling through the high-affinity IgE receptor. FceRI is composed of one a, one b and two g subunits. The a and b subunits are associated with Lyn kinase. Binding of IgE to the receptor leads to activation of Lyn, which then phosphorylates the immunoreceptor tyrosine-based activating motif (ITAM) (I) residue on the g subunit. Syk is recruited to the phosphorylated ITAM and is then phosphorylated by Lyn. Activated Syk phosphorylates tyrosine residues on LAT (linker of activated T cells). Phosphorylated LAT serves as the docking site for many signaling molecules such as Grb2, phospholipase (PL)C-g, GADS, SLP76, and phosphatidylinositol-3 kinase (PI3K). PLC-g generates inositol trisphosphate (IP3) and diacylglycerol (DAG) from membrane lipids. IP3 releases Ca2+ whereas DAG activates protein kinase (PKC). GADS, Grb2, and SLP76 lead to the activation of various mitogen-activated protein kinase (MAPK) signaling pathways. ERK1/2 as well as p38 MAPK phosphorylate and activate PLA2. MAPK, PLA2, Ca2+, and PKC all play an important role in mast cell degranulation, cytokine secretion, and eicosanoid generation. Simultaneous cross-linking of FceRI and FcgRIIb leads to recruitment of SHIP and SHP-1 to the ITIM (immunoreceptor tyrosine-based inhibitory motif ) residue of FcgRIIb. SHP-1 and SHIP block signal generation by dephosphorylating protein and lipid kinases. GADS, Grb2-like adaptor downstream of Shc; Grb2, growth factor receptor-bound 2; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; SHIP, SH2 domain-containing inositol phosphatase; SHP, SH2 domain-containing phosphatase; SLP76, SH2-containing leukocyte-specific protein of 76 kDa. (See CD-ROM for color version.)

et al. 1991), and PIR-B (Uehara et al. 2001). The ligand for gp49B1 is integrin αvβ3. The ligands for MAFA and PIR-B are unknown. As mentioned previously, FcγRIIb, gp49B1, MAFA, and PIR-B contain ITIM sequences in their cytosolic domain, which recruit phosphatases such as SHP-1, SHP-2, SHIP, and the adapter protein DOK. The phosphatases dephosphorylate receptors and kinases such as Lyn, Syk, and PI3K. Simultaneous activation of FcεRI and the inhibitory receptors FcγRIIb, gp49B1, MAFA or PIR-B leads to attenuation and/or termination of the activation signal.

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Signal Transduction in Allergic and Inflammatory Cells

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Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Mast Cells: Biological Properties and Role in Health and Allergic Diseases Peter Bradding and Glenn Cruse

Summary Mast cells are derived from progenitor cells in the bone marrow, which circulate as undifferentiated CD34+ mononuclear cells in the peripheral circulation, and subsequently mature following migration into tissue. Stem cell factor (SCF) is the critical growth and differentiation factor for human mast cells. In health, mast cells are widely distributed throughout the body in both connective tissue and at mucosal surfaces, and form a heterogeneous population of cells with differences apparent in their mediator content, ultrastructure, and functional behavior. Human mast cells can be divided into two clear phenotypes based on their content of the neutral serine proteases tryptase and chymase. Mast cells have been implicated in many diverse biological processes in both health and disease, with an important role in host defense against invading pathogens. It is likely that their primary role is to sense the external environment, ready to respond to a variety of diverse tissue insults with an early and appropriate program of gene expression and mediator release aimed at initiating inflammation and then repair. When the insult becomes chronic, then it is our view that their continuing activation contributes to tissue dysfunction and remodeling. In addition, through the misguided generation of allergen-specific IgE by host B cells, they have the potential to induce acute, sometimes life-threatening symptoms on exposure to allergen. There is overwhelming evidence that mast cells play a central role in the pathophysiology of asthma, allergic rhinoconjunctivitis, urticaria, and anaphylaxis through their activation by both allergen and other nonimmunologic stimuli. Histamine, prostaglandin (PG)D2 and leukotriene (LT)C4 contribute to mucosal symptoms through the induction of mucus secretion and mucosal edema, and in the case of asthma bronchoconstriction. Mast cells also synthesize and secrete a number of proinflammatory chemokines and cytokines including interleukin (IL)-4, IL-5, and IL-13, which regulate both IgE synthesis and the development of eosinophilic inflammation. In addition, the mast cell proteases tryptase and chymase have the potential to cause tissue damage as well as contributing to the inflammatory response. Importantly, mast

cells are present in an activated secretory state in these diseases, with evidence of ongoing mediator release, degranulation visible with electron microscopy, and cytokine mRNA synthesis. Mast cells also relocate to specific structures within diseased tissues. This is particularly evident in asthma where they migrate into the airway epithelium, mucous glands and airway smooth muscle, placing them in direct contact with these dysfunctional airway elements. Developing a better understanding of the mechanisms driving chronic mast cell activation and migration may uncover truly novel therapies for the treatment of mast cell-mediated disease.

Introduction Since their discovery over a century ago, the role of mast cells in human pathophysiology has been the subject of much debate. Mast cells are ubiquitous throughout connective tissues and mucosal surfaces, particularly at the interface with the external environment such as the skin, respiratory tract, and gastrointestinal tract. At these sites they are well placed and well equipped to deal with a multitude of tissue insults. Mast cells contribute to the maintenance of tissue homeostasis, with examples including roles in wound repair (Iba et al. 2004; Weller et al. 2006) and revascularization (Heissig et al. 2005), and protective roles in acquired and innate immune responses to bacterial infection (Echtenacher et al. 1996). They are also implicated in many diverse diseases such as asthma and related allergies, pulmonary fibrosis, connective tissue disease, multiple sclerosis, and atherosclerosis. Mast cells can therefore be considered to represent a double-edged sword. This chapter focuses on the biological properties of mast cells, and how these fascinating cells contribute to both health and allergen-related disease with a fine balance between protection and destruction.

Mast cell development Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Mast cells derive from pluripotent hematopoietic stem cells in the bone marrow (Fig. 11.1). Mast cell-committed progenitors

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Bone Marrow

Pluripotent stem cell Myelocyte precursor

Mast cell precursor

Blood Vessel

Proliferation

Rolling

Adhesion

Lamina Propria

Chemotaxis Cell to cell contact with exchange of growth factors

Transendothelial migration (diapedesis)

Maturation

SCF

Epithelium

are released into the systemic circulation as agranular, undifferentiated, CD34+ mononuclear cells that then migrate into their destination tissue where they terminally differentiate (mature) under the influence of the local cytokine milieu (for review of mast cell ontogeny see Gurish & Boyce 2006). In addition, interactions with the cell matrix and resident cells such as fibroblasts profoundly alters their phenotype (LeviSchaffer et al. 1986; Rothenberg & Austen 1989; Swieter et al. 1993; Rubinchik & Levi-Schaffer 1994; Ogasawara et al. 1997; Hsieh et al. 2005). The vital growth factor for mast cells is the stromal cell and fibroblast-derived cytokine, SCF (Valent et al. 1992). Previously known as steel factor, SCF is the ligand for the receptor tyrosine kinase CD117, encoded by the protooncogene c-kit (Williams et al. 1990). SCF exists as both a cell membrane-bound protein and a soluble protein that can be detected in the blood (Langley et al. 1993). The activation of

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Fig. 11.1 Ontogeny of mast cells. Mast cells originate from pluripotent hematopoietic stem cells in the bone marrow. They enter the bloodstream as mast cell-committed CD34+ progenitor cells. Mast cells differentiate after tissue recruitment within the tissue itself, which accounts for much of their heterogeneity. For more details, see text. (See CD-ROM for color version.)

c-kit by SCF is vital for the growth and survival of mast cells, which undergo apoptosis on SCF withdrawal (Iemura et al. 1994). Mast cells can also undergo apoptosis in the presence of SCF since they have been found to have tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptors that initiate apoptosis (Berent-Maoz et al. 2006). Activation of c-kit on mast cells regulates expression levels of c-kit (Tsujimura et al. 1996), induces immature cell proliferation, promotes chemotaxis, and suppresses apoptosis, thus enhancing survival and promoting recruitment and growth (Li & Krilis 1999). The activation of c-kit also has a regulatory role in mast cell activation. At 10 ng/mL it potentiates IgE-dependent mast cell mediator and cytokine release, while at 100 ng/mL it directly activates the cells (Columbo et al. 1992; Takaishi et al. 1994; Taylor et al. 1995; Lin et al. 1996; Lukacs et al. 1996; Petersen et al. 1996; Frenz et al. 1997; Cruse et al. 2005).

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SCF is the critical growth factor responsible for mast cell growth and differentiation in humans. Mast cells will grow in vitro with SCF as the only exogenous factor, although the cells remain immature and are predominantly of the tryptase-only (MCT) phenotype (Mierke et al. 2000). However, cells that are grown in suspension culture in a medium supplemented with serum, SCF, and IL-6 are more mature in terms of their nuclear morphology and granular structure but retain the MCT phenotype (Saito et al. 1995; Yanagida et al. 1995; Saito et al. 1996; Bradding & Holgate 1999). Cells grown on a fibroblast or endothelial cell monolayer have a tryptase- and chymase-positive (MCTC) phenotype and resemble skin mast cells (Levi-Schaffer et al. 1987; Furitsu et al. 1989; Bradding & Holgate 1999; Mierke et al. 2000). In addition, bone marrow-derived mast cells (BMMC) and umbilical cord blood-derived mast cells (CBMC) grown in conditioned medium from a cell strain derived from a patient with systemic mastocytosis together with SCF yielded fully mature cells containing chymase only (MCC phenotype) (Li et al. 1996). These observations suggest that factors other than SCF are required for complete mast cell maturation. The phenotypic differences could be partly due to the presence of IL-4, which dramatically increases chymase expression in

CBMC (Toru et al. 1998). It is also possible that the expression of chymase is part of a maturation pathway, with MCT phenotypes maturing into MCTC phenotypes (Li & Krilis 1999). There are cofactors that can enhance or inhibit the effects of SCF, which appear to be dependent on the origin of the mast cells (Okayama 2000). Nerve growth factor (Kanbe et al. 2000), IL-3, IL-6, IL-9, IL-10 (Yanagida et al. 1995; Norrby 2002), and thrombopoietin enhance SCF-dependent mast cell growth. Conversely, granulocyte–macrophage colonystimulating factor (GM-CSF) (Welker et al. 2001), retinoids, interferon (IFN)-γ, and transforming growth factor (TGF)-β inhibit the growth and differentiation of mast cells (Ishida et al. 2003).

Mast cell heterogeneity Tissue-dependent influences result in marked heterogeneity of mast cells across species, between different organs, and even within the same organ. This heterogeneity is evident in terms of their structure, mediator content, immunologic and nonimmunologic activation, and pharmacologic responsiveness (Table 11.1) (reviewed by Church et al. 1994). Rodent mast

Table 11.1 Mast cell subtype characteristics. Rodent mast cells

Human mast cells

MMC

CTMC

MC T

MC TC

Protease content

Rat mast cell protease 2

Rat mast cell protease 1

Tryptase

Tryptase, chymase, carboxypeptidase, cathepsin G

Proteoglycan content

Chondroitin sulfate

Heparin

Heparin

Heparin

Scroll

Lattice

Predominant granule patterning evident with electron microscopy Common location

Mucosa

Submucosal tissues

Epithelium

Lamina propria, connective tissue and skin

Staining characteristics

Alcian Blue +

Safranin +

Safranin +

Safranin +

Suggested primary role

Host defense

Tissue repair

Host defense

Tissue repair

Relative histamine content

Low

High

Low

High

Relative LTC4 release

High

Absent

High

Skin low

Relative PGD2 release

Low

Low

High

Skin high

IL-4 (low) IL-5 (high) IL-6 (high) IL-13 (low)

IL-4 (high) IL-13 (high)

Cytokine profile

Activated by antigen

Yes

Yes

Yes

Yes

Activated by substance P

No

Yes

No

Yes

No

Yes

Yes (weak effect)

No

Responds to C5a Inhibited by sodium cromoglycate

No

Yes

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Chymase

Chymase

IL-4

IL-6

cells were originally classified according to the histochemical staining properties of their granules and their most common location (Enerback 1966). Using this nomenclature, safraninpositive, alcian blue-negative rodent mast cells are mostly associated with the submucosal tissues and are thus called connective tissue mast cells (CTMC); safranin-negative, alcian blue-positive rodent mast cells are predominantly mucosal and termed mucosal mast cells (MMC) (Table 11.1). This differential staining is due to the proteoglycan content of the granules. Rodent CTMC produce the highly sulfated glycosaminoglycan heparin, whereas rodent MMC produce chondroitin sulfate (Gurish et al. 1992). This classification does not work in humans because all human mast cells contain heparin (Craig et al. 1993), which is essential for the stabilization of the β-tryptase tetramer (Pereira et al. 1998). Thus human mast cells are classified according to their protease content. Mast cells that contain only tryptase (MCT) are usually situated in mucosal tissue, and may be functionally associated with the immune system and host defense (Irani et al. 1987). There are also mast cells containing tryptase, chymase, carboxypeptidase A, and cathepsin G (MCTC) (Table

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Tryptase

Tryptase

Fig. 11.2 Mast cell heterogeneity in terms of cytokine content. Three sequential 2-mm sections of nasal mucosa from a patient with allergic rhinitis stained for (a) tryptase, IL-4 and chymase, demonstrating that nearly all chymase-positive mast cells contain IL-4, and (b) tryptase, IL-6 and chymase, demonstrating colocalization of IL-6 to a tryptase-positive, chymase-negative mast cell (arrows). A chymase-positive mast cell does not contain IL-6 (arrowheads). (From Bradding et al. 1995a, with permission.) (See CD-ROM for color version.)

11.1), which are normally situated in the skin and submucosal connective tissue, and which are proposed to be involved in tissue repair, fibrotic reactions, and angiogenesis (Artuc et al. 1999; Trautmann et al. 2000). There are also reports of mast cells containing chymase and carboxypeptidase (MCC) without tryptase (Bradding et al. 1995a; Yamada et al. 2001; Horny et al. 2003) which vary in location and whose function has yet to be elucidated. The heterogeneity of mast cells also extends to their cytokine content (Fig. 11.2). MCTC predominantly express IL-4 and IL-13, whereas MCT express IL-5 and IL-6 almost exclusively (Bradding et al. 1995a; Anderson et al. 2001), again suggesting distinct roles for these phenotypes.

General morphology and biology Despite the heterogeneity of mast cells, their general morphology is similar regardless of the tissue site they reside in. Ultrastructurally, the cell membrane contains finger-like projections, and while immature mast cells may have a multilobed nucleus, mature cells have a monolobed nucleus

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with no apparent nucleoli and little condensed chromatin. They have few mitochondria and ribosomes, as well as an inconspicuous Golgi apparatus, scant rough endoplasmic reticulum (RER) and, in contrast to basophils, a lack of cytoplasmic glycogen aggregates (Fig. 11.3) (reviewed by Dvorak 2005). In fact, the only prominent cytoplasmic structures are the electron-dense granules (Fig. 11.3). Unusually, mature human mast cells have ribosomes closely associated with these secretory granules, and little association between ribosomes and RER (Dvorak & Morgan 2001). This suggests that the secretory granules play a significant role in RNA metabolism in human mast cells. The granules are membrane bound and contain preformed mediators, while dense lipid bodies are a store of arachidonic acid. The membrane-bound secretory granules contain crystalline structures that resemble scrolls, lattices, crystals, or whorls (Dvorak 2005). These

(a)

(b) Fig. 11.3 Scanning electron micrographs of mast cells. (a) Normal mast cell at rest showing electron-dense granules and a monolobed nucleus with little condensed chromatin. (b) Mast cell showing evidence of piecemeal degranulation with variable, selective loss of granule contents (arrows).

structures are more visible in MCT subtypes as the sheer volume of protease in the MCTC subtype masks their appearance. Despite this, electron microscopy shows that MCT granules contain predominantly scroll patterns and MCTC granules contain predominantly lattice patterns (Table 11.1) (Craig et al. 1988). Mast cells have basophilic cytoplasm that stains pink with Wright’s or May–Grünwald’s Giemsa, with a purple/blue nucleus and blackish granules. Mast cells can have an irregular shape in tissues but can be identified by selective staining using cationic dyes (such as aniline dyes) that bind to sulfated glycosaminoglycans specific to the mast cell granules. Using stains that utilize the anionic property of the mast cell granules to identify their presence is a useful tool. However, in humans the most effective way to identify the location and subtype of mast cells histologically is to use immunohistochemistry with antibodies raised against the mast cellspecific enzymes tryptase and chymase (Walls et al. 1990). The proteoglycans of the granules are the backbone of the granule matrix. They are a long single peptide with glycosaminoglycans attached. In human mast cells, the proteoglycan content of the granules is mainly heparin and chondroitin E. Neutral proteases, acid hydrolases, and histamine molecules are attached to the proteoglycans by ionic linkage to the sulfate groups on the glycosaminoglycans (Aborg et al. 1967). The sulfate groups generate an acidic environment within the granules that maintains the mediators in an inactive state (Humphries et al. 1999). IgE-dependent activation of the mast cell induces granule swelling, crystal dissolution, and granule fusion, both with surrounding granules as well as the cell membrane, followed by exocytosis with release of mediators into the extracellular space (Caulfield et al. 1980). This process is termed “compound exocytosis” or “anaphylactic degranulation.” Once in the extracellular space, the neutral pH activates the mediators (Caulfield et al. 1980, 1990). However, in many diseased tissues including asthmatic bronchial mucosa, the ultrastructural appearance of mast cells typically demonstrates piecemeal degranulation (Djukanovic et al. 1992a; Aoki & Kawana 1999; Begueret et al. 2007), in which there is variable loss of granule contents although the granules and their membranes remain intact (Fig. 11.3). The mechanisms leading to piecemeal degranulation in mast cells are poorly understood and require further research. The effects of preformed mediators often remain localized as histamine is short-lived in vivo, being broken down by histamine N-methyltransferase, and the active tetramer of tryptase rapidly dissociates into inactive monomers in the absence of heparin (Schwartz & Bradford 1986). In addition to stored granule-derived mediators, newly formed metabolites of arachidonic acid are also released from the cell following IgE-dependent activation (Table 11.2). This phospholipid is liberated from the cell membrane, nuclear envelope, or lipid bodies during immunologic activation and is rapidly oxidized by either the cyclooxygenase (COX) or

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lipoxygenase pathways to form the eicosanoids PGD2 and LTC4 respectively (Bradding et al. 1995b; Boyce 2003). In addition, the mast cell is capable of synthesizing and secreting numerous cytokines and chemokines depending on the stimulus (see below). The principal biological properties of mast cell autacoids (histamine, PGD2, LTC4), proteases, and cytokines are summarized in Tables 11.2–11.4.

Mechanisms of mast cell activation IgE-dependent activation The best-studied mechanism of mast cell activation, and that considered most relevant to allergic disease is activation through the high-affinity IgE receptor FcεRI (reviewed by Gilfillan & Tkaczyk 2006; Rivera & Gilfillan 2006). Most of the downstream signaling events identified following FcεRI engagement have been defined in rodent models, with relatively little known about events in human mast cells. Where human cells have been investigated, some important differences in signaling have been observed (Duffy et al. 2001a). FcεRI is a tetrameric structure that belongs to the multichain immune recognition receptor (MIRR) family. It comprises an α chain (FcεRIα) that binds IgE, a β-chain signaling subunit (FcεRIβ), and two γ subunits that exist as a homodimer signaling subunit (FcεRIγ) (Fig. 11.4). A detailed description of the multiple signaling cascades activated following receptor activation is beyond the scope of this chapter but is summarized in Fig. 11.4 (for reviews see Gilfillan & Tkaczyk 2006; Rivera & Gilfillan 2006). In terms of the proximal signaling pathways, the γ signaling subunits contain an immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995) within their cytoplasmic C-terminal domains, which bind to Syk tyrosine kinases initiating phosphorylation. The β chain of FcεRI also contains an ITAM. However, the FcεRIβ ITAM contains a noncanonical tyrosine residue that prevents binding of Syk kinase. Instead, FcεRIβ signals through activation of the lipid raft-associated Lyn tyrosine kinase, which in turn activates Syk kinase (Fig. 11.4) (Rivera & Gilfillan 2006). Signaling in vivo is initiated when multivalent allergen binds to allergen-specific IgE bound to the FcεRIα chain. This promotes FcεRI aggregation and can be mimicked in vitro by the use of anti-IgE antibodies. Receptor aggregation initiates association of the receptor with lipid rafts containing Lyn, a membrane-anchored member of the Src family of protein tyrosine kinases (reviewed by Dykstra et al. 2003). Lyn kinase transphosphorylates tyrosine residues in the ITAMs before binding strongly to the phosphorylated FcεRIβ ITAM through the SH2 (Src homology 2) domain (reviewed by Siraganian 2003). Syk protein tyrosine kinases are recruited to the rafts and bind the doubly phosphorylated ITAMs. They are themselves phosphorylated by recruited Lyn and Syk kinases, promoting the Syk activation loop which results in a fully activated Syk with adjacent tyrosine phosphorylation that

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Table 11.2 Classical human mast cell mediators and their biological effects. Preformed Histamine Bronchoconstriction Tissue edema Mucus secretion Fibroblast proliferation Collagen synthesis Endothelial proliferation, dendritic cell activation Heparin Anticoagulant Storage matrix for mast cell mediators Fibroblast activation Protects growth factors from degradation and potentiates their action Endothelial cell migration Tryptase Generates C3a and bradykinin Degrades neuropeptides Increases bronchial hyperresponsiveness Indirectly activates collagenase Fibroblast proliferation and collagen synthesis Bone remodeling and epithelial activation Potentiates mast cell histamine release Chymase Mucus secretion Extracellular matrix degradation Type I procollagen processing Converts angiotensin I to angiotensin II Inhibits T-cell adhesion to airway smooth muscle Activates IL-1b, degrades IL-4 and releases membrane-bound SCF Newly generated PGD2 Bronchoconstriction Tissue edema Mucus secretion Dendritic cell activation Chemotaxis of eosinophils, Th2 T cells and basophils via the CRTH2 receptor LTC4 /LTD4 Bronchoconstriction Tissue edema Mucus secretion Enhances IL-13-dependent airway smooth muscle proliferation Dendritic cell maturation and migration Eosinophil IL-4 secretion and mast cell IL-5, IL-8 and TNF-a secretion Tissue fibrosis

begins a cascade of events leading to the activation of inositol trisphosphate (IP3). The generation of IP3 induces calcium mobilization from intracellular RER stores, which initiates the influx of extracellular calcium via store-operated calcium

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Table 11.3 In vitro biological effects of human mast cell-derived cytokines. Cytokine

Target cells

Biological effects

IL-4

B cells

Eosinophils Fibroblasts Mast cells

IgE production; proliferation; MHC class II, CD40 and CD25 expression; IL-6 production Proliferation; induction of Th2 phenotype ↑ VCAM-1 and ↓ ICAM-1 expression; proliferation ↓ generation of H2O2 and O2−, ↓ parasite killing and ↓ tumoricidal activity; ↓ IL-1, IL-6, IL-8, and TNF-a production ↑ MHC class II and CD23 expression; monocyte/macrophage differentiation; 15-lipoxygenase expression Transendothelial migration Proliferation, chemotaxis and matrix protein secretion; ↑ ICAM-1 expression ↑ FceRI expression, ↑ ICAM-1 expression

IL-3, IL-5

Eosinophils and prolonged survival

Growth, adhesion, transendothelial migration, chemotaxis, activation, GM-CSF

IL-6

B cells T cells Airway glandular cells Mast cells

Immunoglobulin secretion including ↑ IgE synthesis Growth, differentiation and activation Mucus secretion Survival

IL-13

B cells Monocyte/macrophages Eosinophils Vascular endothelial cells

IgE synthesis As for IL-4 Activation, ↑ survival ↑ VCAM-1

IL-16

T cells

Chemotaxis

TNF-a

Monocyte/macrophages T cells Neutrophils Eosinophils Mast cells Vascular endothelial cells

Airway glandular cells

Enhanced cytotoxicity; chemotaxis and prolonged survival Class II antigen and IL-2R expression; proliferation Chemotaxis, enhanced cytotoxicity Enhanced cytotoxicity and oxidant production Histamine and tryptase secretion E-selectin, ICAM-1 and VCAM-1 expression. Adhesion and transendothelial migration of most leukocytes Growth and chemotaxis; ↓ collagen synthesis but ↑ collagenase production; IL-6 and IL-8 synthesis Mucus secretion

SCF

Mast cells

Growth, differentiation, survival, chemotaxis

NGF

Mast cells B cells T cells Eosinophils Basophils Neutrophils Monocyte/macrophages Fibroblasts Smooth muscle cells

Differentiation, survival, activation, mediator release Differentiation, proliferation, immunoglobulin synthesis Differentiation, proliferation Proliferation Activation, mediator release Chemotaxis, survival, mediator release Proliferation, mediator release Migration, contraction Migration, contraction, proliferation

TGF-b

Smooth muscle cells Epithelial cells Endothelial cells

Differentiation, activation Inhibition of proliferation Induces angiogenesis

bFGF

Fibroblasts Vascular endothelial

Proliferation Stimulates angiogenesis

IFN-a

NK cells Macrophages Dendritic cells T cells

Increased cytotoxicity Development, maturation Activation and maturation, upregulation of IFN-g production Promotes survival of activated T cells, induction of Th1 phenotype

T cells Vascular endothelial cells Monocyte/macrophages

Fibroblasts

NK, natural killer; VCAM, vascular cell adhesion molecule. See text for definition of other abbreviations.

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Table 11.4 In vitro biological effects of human mast cell-derived chemokines. Chemokine

Target cells

Biological effects

CCL1

T cells

Chemotaxis (selective for Th2), survival?

CCL2

T cells Mast cells Eosinophils Monocytes Fibrocytes Epithelial cells Basophils

T-cell polarization toward Th2 phenotype Chemotaxis Chemotaxis Chemotaxis Chemotaxis Proliferation, chemotaxis Activation, mediator release

CCL3

Macrophages Neutrophils Eosinophils Monocytes Mast cells Basophils T cells

Differentiation Chemotaxis (in vivo) and cytotoxicity Chemotaxis Chemotaxis Activation, mediator release Activation, mediator release Chemotaxis (selective for Th1), polarization toward Th1 phenotype

CCL4

Eosinophils Neutrophils T cells

Chemotaxis Chemotaxis (in vivo) Chemotaxis (selective for Th1), polarization toward Th1 phenotype

CCL5

Mast cells Eosinophils Monocytes T cells

Chemotaxis? Chemotaxis Chemotaxis Chemotaxis (selective for Th1), polarization toward Th1 phenotype

CCL7

Eosinophils Monocytes Basophils

Chemotaxis Chemotaxis Activation, mediator release

CCL12

Fibrocytes Monocytes Eosinophils Lymphocytes

Chemotaxis Chemotaxis Chemotaxis Chemotaxis

CCL17

T cells

Chemotaxis (selective for Th2)

CCL19

Airway smooth muscle

Chemotaxis

CCL20

Dendritic cells T cells

Chemotaxis Chemotaxis

CCL22

T cells

Chemotaxis (selective for Th2)

CXCL5

Neutrophils

Chemotaxis

CXCL8

Neutrophils Mast cells Endothelial cells Eosinophils

Chemotaxis Inhibition of chemotaxis, inhibition of mediator release Proliferation, survival, chemotaxis, angiogenesis Chemotaxis after priming with IL-3, IL-5 or GM-CSF

channels (Fig. 11.4) (Faeder et al. 2003). In terms of IgEdependent mediator release in both rodents and humans, influx of extracellular Ca2+ is a critical requirement for the release of both preformed and newly generated mediators (Church et al. 1982; Kim et al. 1997).

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The process of Ca2+ entry through the plasma membrane as a result of the depletion of Ca2+ from internal stores is termed capacitive Ca2+ entry. The Ca2+ current passing through the plasma membrane is known as the Ca2+ release activated Ca2+ current (ICRAC), and is believed to play a central role in

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Allergen

g

b

a

ICRAC

LAT

Syk

Syk Lyn

Lyn

PLC-g

Syk

Syk

DAG

PKC

Ca2+

RER

Ca2+

Sos

Ras

Grb2

+ KCa3.1

+

K+

Raf-1

IP3

IP3R

K+

Ca2+

IgE

Mediator release Cytokine production

MEK

Ca2+

ERK PLA2

Arachidonic acid metabolism

Mediator release Cytokine production Arachidonic acid metabolism

Fig. 11.4 Simplified schematic of mast cell signaling events leading to degranulation and mediator production. Cross-linking of IgE molecules bound to FceRI initiates ITAM phosphorylation at the cytoplasmic termini of the dimeric g signaling subunits of the receptor (depicted in green). Syk kinases are recruited to the activated ITAMs, which autophosphorylate and recruit more Syk kinases (along with other kinases) leading to a cascade of signaling events. The b chain of the receptor (depicted in blue) also contains an ITAM, which binds to the lipid raft-associated Lyn kinase which in turn recruits, to the lipid rafts, and activates Syk kinases, thus amplifying receptor signaling. Syk kinases activate the membrane-associated LAT, which activates phospholipase C (PLC)-g leading to the release of intracellular calcium stores from the rough endoplasmic reticulum via inositol trisphosphate (IP3) and its receptor. PLC-g also leads to activation of protein

kinase C (PKC), which induces mediator release and cytokine production. In addition, PLC-g initiates the Grb2/Sos/Ras pathway, which leads to extracellular regulated kinase (ERK) activation that initiates arachidonic acid metabolism via activation of phospholipase A2 (PLA2) and subsequent eicosanoid production and release. Mast cell degranulation is dependent on influx of extracellular calcium through store-operated calcium channels (SOCC) such as ICRAC, which is initiated by the release of calcium from internal stores (depicted by red dashed line). This influx of calcium activates the intermediate conductance Ca2+-activated K+ channel, KCa3.1 (depicted by blue arrow), leading to an efflux of K+ which counteracts the membrane depolarization induced by Ca2+ influx, thus increasing the driving force for Ca2+ entry. (See CD-ROM for color version.)

many physiologic processes such as gene transcription, proliferation and cytokine release (Lewis 2003). ICRAC has been well characterized electrophysiologically in several cells including rodent mast cells (Hoth & Penner 1992), but the channel carrying it has only recently been identified. Studies from separate groups using an RNAi screen in Drosophila cells have identified the gene product of FLJ 14466 as an essential component of ICRAC (Feske et al. 2006; Vig et al. 2006), and two further mutagenesis studies suggest this is indeed the poreforming protein (Prakriya et al. 2006; Yeromin et al. 2006). The gene product has been given different names (Orai1, CRACM1). We will refer to it as CRACM1. Although it is generally accepted that Ca2+ influx following store release is required for mediator release from human and rodent mast cells following immunologic activation, whether CRACM1 channels alone control granule exocytosis is not clear, and has not been investigated in human mast cells. One study in rat basophil leukemia (RBL) cells, which combined measurements of ICRAC with membrane capacitance measurements to monitor exocytosis, found that ICRAC did not provide suf-

ficient Ca2+ to support granule fusion (Artalejo et al. 1998), although it may well contribute to the production of lipid mediators in these cells (Chang & Parekh 2004). Human mast cells also express several members of the transient receptor potential family of ion channels that also have the potential to contribute to Ca2+ influx following immunologic activation (Bradding et al. 2003). K+ channels have the potential to modulate Ca2+ influx and hence mediator release due to their profound effects on the cell membrane potential. K+ channels hyperpolarize the plasma membrane when open and thus increase the electrical driving force for Ca2+ entry (Panyi et al. 2004), but perhaps more importantly, CRACM1 conducts larger currents at negative membrane potentials (Hoth & Penner 1992). In both RBL cells and rat IL-3-dependent BMMC, an inwardlyrectifying K+ channel (Kir2.1) is open when the cells are at rest (Lindau & Fernandez 1986; McCloskey & Qian 1994). However, the K+ channels present in human mast cells differ to those in rodents, highlighting an example of speciesdependent heterogeneity. Of note we have never seen a Kir

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indirectly enhances Ca2+ influx (Fig. 11.5c) and histamine release (Fig. 11.5d), but is not critical for secretion, and can thus be considered as increasing the gain of an immunologic stimulus (Duffy et al. 2004). This channel is closed by compounds that inhibit mast cell secretion such as β2-adrenoceptor agonists, providing a mechanism for the coupling of receptor activation and impaired secretion (Fig. 11.5e). Interestingly, this effect appears to act through a cAMP-independent mechanism (Duffy et al. 2005).

current in any human mast cell (Duffy et al. 2001a,b, 2003, 2004, 2005; Cruse et al. 2006). Thus the majority of human lung mast cells (HLMC) and human peripheral blood-derived mast cells are electrically “silent” at rest, with a resting membrane potential of around 0 mV (Fig. 11.5a,b). Following IgE-dependent activation, human mast cells rapidly open the intermediate conductance Ca2+-activated K+ channel KCa3.1 (Fig. 11.5a) (Duffy et al. 2001a; Kaur et al. 2005), which has not been described in rodent mast cells. The KCa3.1 channel

80 100

Membrane voltage (mV)

Anti-IgE

Current (pA)

80 60 40 20

Baseline

0 –20 –40 –150 –100 –50

0

50

0

–80

100 150

Command potential (mV)

(a)

Anti-IgE added

(b)

1:30000 Anti-IgE

500 300 100

1-EBIO 0

12

200 300 Time (sec)

Anti-IgE 1:30000 n=6

*

1:30000 300 Anti-IgE 200 100

1-EBIO 200 300 Time (sec)

500

1:30000 Anti-IgE

300 100

400

1-EBIO 0

100

200 300 Time (sec)

1-EBIO 100 mM

300

8 6 4

Wash

100 0

Salbutamol 1 mM Salbutamol 3 mM Baseline

–100 –200

2 SO .1% mM mM mM mM DM O 0 10 30 100 300 S IO IO IO O DM 1-EB 1-EB -EB -EBI 1 1

–300 –150 (e)

Fig. 11.5 (a) Whole-cell patch-clamp recording of a human peripheral blood-derived mast cell at baseline and following addition of anti-IgE. Current–voltage curve demonstrating a characteristic KCa3.1 current. (b) Current clamp trace demonstrating acute change in membrane potential as KCa3.1 opens. (c) Enhancement of Ca2+ influx by the KCa3.1 opener 1-EBIO following submaximal IgE-dependent activation of human

226

100

700

200

10

No

400

0

400

Current (pA)

Net IgE-dependent histamine release (% of cell total)

14

100

500

Cytosolic free Ca2+ (nM)

700

(c)

(d)

Cytosolic free Ca2+ (nM)

Cytosolic free Ca2+ (nM)

100 sec

–100

–50 0 50 Command potential (mV)

100

150

lung mast cells. (d) Enchancement of histamine release by the KCa3.1 opener 1-EBIO following submaximal IgE-dependent activation of human lung mast cells. (e) Reversible closure of KCa3.1 by the b2adrenoceptor agonist salbutamol. (a) and (b) from Duffy et al. 2001a, (c) and (d) from Duffy et al. 2004 and (e) from Duffy et al. 2005, with permission.

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Monomeric IgE

Allergen Tetrameric FceRI

Y

Y Stem cell factor

a Ig free light chains

c-kit +

gb

Protease-activated receptors

↓ SHIP (?) Chemokines

Protein tyrosine kinases Serine/threonine kinases

TLRs (viruses bacteria)

Second messengers IP3 DAG Cytokines (TNF-a)

Ca2+ release from stores

Adenosine Neuropeptides C5a

PKC

Further protein kinases

Hyperosmolality

Cell–cell signals Fig. 11.6 IgE-dependent and -independent mechanisms of mast cell activation. See text for definition of abbreviations. (See CD-ROM for color version.)

Cl− Ca2+

Monomeric IgE-dependent mast cell activation In addition to the cross-linking of FcεRI by allergen, the binding of monomeric IgE alone to FcεRI can initiate intracellular signaling events and Ca2+ influx (Fig. 11.6) (Kalesnikoff et al. 2001; Huber et al. 2002; Kitaura et al. 2003; Oka et al. 2004; Pandey et al. 2004; Cruse et al. 2005; Kitaura et al. 2005; Liu et al. 2005; Matsuda et al. 2005; Nunomura et al. 2005; Tanaka et al. 2005). In rodents this results in the release of granulederived mediators and the secretion of cytokines including IL-6 (Kitaura et al. 2003; Oka et al. 2004; Tanaka et al. 2005). This IL-6 acts in an autocrine manner and prolongs mast cell survival following growth factor withdrawal (Kitaura et al. 2003). In human CBMC, monomeric IgE alone induces the release of the cytokines I-309, GM-CSF, and MIP-1α without histamine release (Gilchrest et al. 2003). However, in HLMC that have been maintained in culture, IgE induces the secretion of histamine, LTC4, and IL-8, which is markedly enhanced in the presence of SCF (Cruse et al. 2005). Interestingly, in both rodent mast cells and HLMC, ongoing signaling is dependent on the presence of “free” IgE, and this ceases immediately when free IgE is removed, suggesting that these findings are physiologically relevant (Pandey et al. 2004; Cruse et al. 2005). The mechanisms behind this are uncertain but in part are thought to involve FcεRI aggregation. These observations

K+

are of great interest because SCF and free IgE concentrations are elevated in asthmatic airways, and there is a robust correlation between total serum IgE and the presence of asthma and bronchial hyperresponsiveness (Burrows et al. 1989; Sears et al. 1991; Sunyer et al. 1995, 1996). This provides a mechanism for the ongoing activation of mast cells through FcεRI in the absence of allergen, and could partly explain the efficacy of anti-IgE therapy in chronic allergic disease (D’Amato et al. 2004; Djukanovic et al. 2004; Holgate et al. 2005a; Ong et al. 2005).

Nonimmunologic stimuli Mast cells may also be activated through a plethora of non-IgE-dependent mechanisms (Fig. 11.6). These include proteases (including tryptase) (He et al. 2004; Moormann et al. 2006), cytokines (e.g., SCF, TNF-α, IFN-γ) (Columbo et al. 1992; Sperr et al. 1993; Yanagida et al. 1996; BrzezinskaBlaszczyk & Pietrzak 1997), complement (Nilsson et al. 1996; Ahamed et al. 2004), adenosine (Forsythe et al. 1999), Tolllike receptor ligands (Varadaradjalou et al. 2003; Kulka et al. 2004), neuropeptides (particularly skin mast cells) (Columbo et al. 1992; Heaney et al. 1995), and hyperosmolality (Eggleston et al. 1987; Genovese et al. 1996a,b; Peachell & Morcos 1998). For example, the C5a receptor CD88 was not thought to be

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expressed on HLMC, but recent work demonstrates that it is in fact expressed on the MCTC subset of HLMC (Oskeritzian et al. 2005). Elevated C5a concentrations have been identified in the induced sputum of asthmatic subjects (Marc et al. 2004), thus providing an alternative means of mast cell activation, and of particular relevance to those mast cells (MCTC) within the airway smooth muscle bundles (see below). Human progenitor-derived mast cells and mouse mast cells express Toll-like receptor (TLR)1–7 and TLR9 (Applequist et al. 2002; Matsushima et al. 2004). These play an important role in the innate host response to pathogens, activating diverse programs of gene expression depending on the stimulus. For example, in mouse mast cells functional responses to TLR2 (the receptor for bacterial peptidoglycan) results in production of TNF-α, IL-4, IL-5, IL-6, IL-13, and IL-1β, while activation of TLR4 (the receptor for lipopolysaccharide, LPS) induces production of TNF-α, IL-1β, IL-6, and IL-13 but not IL-4 or IL-5. In addition, activation of TLR2 but not TLR4 induces Ca2+ mobilization, degranulation, and LTC4 production (Supajatura et al. 2001, 2002; Ikeda & Funaba 2003). Examination of the gene expression profile from human CBMC using high-density oligonucleotide probe arrays following activation with LPS compared with anti-IgE demonstrates that both induce a core response, plus an LPS or anti-IgE specific program of gene expression (Okumura et al. 2003). Perhaps of more relevance to asthma is mast cell activation via TLR3, the ligand for which is double-stranded viral RNA (Kulka et al. 2004). Poly:IC, a synthetic activator of TLR3, induces the specific release of IFN-α as does exposure to respiratory syncytial virus (RSV) and influenza virus. Since viruses are a common cause for asthma exacerbations, the mast cell antiviral response may be an important contributor to the deteriorating airway physiology. A further interesting area of study is the role of immunoglobulin free light chains. These are present in serum in normal subjects and their production is augmented in inflammatory diseases such as rheumatoid disease. In mice, immunoglobulin free light chains can confer mast cell-dependent hypersensitivity through an unknown mechanism, and antigen-specific light chains can mediate mast cell-dependent bronchoconstriction following antigen challenge (Kraneveld et al. 2005). Concentrations of immunoglobulin free light chains are elevated in the sera of asthmatic compared with normal subjects, suggesting they may be relevant to the pathophysiology of human asthma (Kraneveld et al. 2005). Finally, there are likely to be genetic factors that lower the mast cell threshold for activation in asthma. For example, an important negative regulator of mast cell activation is SH2containing inositol phosphatase (SHIP) (Huber et al. 2002). SHIP-deficient rodent mast cells exhibit markedly enhanced secretory responses and, with respect to human basophils at least, cells that are “hyperreleasable” demonstrate a relative deficiency of this molecule (MacDonald & Vonakis 2002).

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Role of mast cells in health Interest in the function of mast cells in disease often takes precedence over their role in health. However, mast cells may play a significant role in the healing of wounds and defense against bacterial and parasitic infection, participating in both innate and adaptive immunity. They are also major effector cells in inflammatory processes, attracting leukocytes to the area of insult, which contributes to host defense and repair. It is likely therefore that their primary role is to sense the external environment, ready to respond to a variety of diverse tissue insults with an early and appropriate program of gene expression and mediator release aimed at initiating inflammation and then repair. When the insult becomes chronic, then it is our view that their continuing activation contributes to tissue dysfunction and remodeling. In addition, through the misguided generation of allergen-specific IgE by host B cells, they have the potential to induce acute, sometimes life-threatening symptoms on exposure to allergen.

Mast cells in wound repair and angiogenesis Because of the biological actions of their mediators (Tables 11.2–11.4), mast cells have been thought to be involved in the healing of wounds. Early studies using metachromatic staining showed that mast cells “disappear” at the wound edge in the first few days, possibly due to degranulation, then increase twofold over baseline by 10 days before returning to normal (Persinger et al. 1983). However, studies using mast cell-deficient mice have provided conflicting results. Egozi et al. (2003) found that mast cells modulated the early inflammatory response to wound healing and angiogenesis, but were not required for the late-phase proliferative response to injury and had no effect on collagen deposition and reepithelialization. In contrast, Iba et al. (2004) showed that mast cells contributed to the late-phase remodeling in wound healing and that collagen fibrils were more interwoven in wild-type mice than mast cell-deficient mice (Kit w/kit w-v). The differing results between these studies may be due to the experimental models used. However, neither study showed any real convincing evidence of a major role for mast cells in wound repair. In contrast, a study by Weller et al. (2006) suggested that mast cell deficient (Kit w/kit w-v) mice have significant retardation of wound closure compared with wild type, and that mast cells are required for normal wound healing. Mast cells may potentially inhibit thrombosis within damaged tissues through the release of heparin, tryptase, chymase, and tissue plasminogen activator, allowing perfusion and nutrition to the site of injury (Thomas et al. 1998; Valent et al. 2002). In support of this, mast cell-deficient mice are more susceptible to lethal thrombogenic stimuli than wild-type mice. Neovascularization occurs in a number of physiologic and pathologic situations including wound healing and tumor

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growth. Mast cells are usually found at sites of neovascularization such as around the periphery of solid tumors (Westphal 1891; Hartveit 1981; Welsh et al. 2005). Rodent mast cells and the human mast cell line HMC-1 induce proliferation of microvessels (Rizzo & DeFouw 1996). Several cytokines identified in human mast cells have potential roles in angiogenesis, including TNF-α, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). Supernatants from unstimulated HMC-1 cells induce proliferation of human microvascular endothelial cells, which is largely mediated by VEGF (Feoktistov et al. 2003). In a model of vascular tube formation, human dermal microvascular endothelial cells exposed to HMC-1 cell supernatants or cocultured with HMC-1 cells rapidly differentiate and mature into vascular tubes (Blair et al. 1997). This effect is

Mast cells and inflammation The mechanism by which leukocytes are recruited to sites of inflammation includes a series of steps, namely leukocyte– endothelial cell adherence, diapedesis or transendothelial migration, chemotaxis and, at various stages during these processes, cell activation for mediator secretion. A number of mast cell mediators and cytokines have the potential to orchestrate each of these events (Fig. 11.7; Tables 11.3 & 11.4), and robust evidence suggests that this is indeed the case (reviewed by Bradding & Holgate 1999).

Capillary leakage

Histamine LTC4 PGD2

Rolling adhesion Firm adhesion Transendothelial migration PSGL-1 E-selectin

PSGL-1 P-selectin

Histamine LTC4

TNF-a

VLA-4 VCAM-1

TNF-a IL-4 IL-13

CD11/18 ICAM-1

TNF-a

TNF-a IL-4 Chemotaxis

MC

IL-5 IL-8 IL-16 GM-CSF CCL2

Mast cell activation

Complement Antigen Proteases Bacteria

LPS

IL-4 TNF-a IL-5 SCF IL-6 CCL3 IL-13 Tryptase GM-CSF

Activation and mediator release

Neuropeptides

Fig. 11.7 The contribution of mast cell mediators to inflammatory cell recruitment. See text for definition of abbreviations. (See CD-ROM for color version.)

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Parasite infection Mast cells have been considered to represent the primary defense against parasitic infections and are thought to be important for the expulsion of parasites (reviewed by Finkelman et al. 1997). However, inoculation of mice with neutralizing antibodies to both IL-3 and IL-4 abrogates the mast cell hyperplasia that occurs in the gut of Nippostrongylus brasiliensis-infected mice and the associated IgE synthesis, but does not alter the clinical course (Madden et al. 1991). Experiments using parasite-infected mast cell-deficient mice have been contradictory, and so the role of mast cells in immunologic responses to parasites seems far from clear. It has been suggested that the inappropriate activation of mast cells by otherwise innocuous antigens, which is fundamental in allergic reactions, is a maladaption of their antiparasitic role. This “maladaption hypothesis” stems from the observation that the immunopathologic response to parasitic infection and strong allergens is remarkably similar (Finkelman et al. 1997).

Defense against bacterial and viral infection In mice, mast cells have a critical role in the host response to acute bacterial infection. A series of experiments in different laboratories have shown that the release of TNF-α from resident mast cells at various tissue sites is an essential prerequisite for the recruitment and activation of neutrophils required to control the infection. This has been demonstrated with a diverse range of bacteria and experimental models (Malaviya et al. 1996; Supajatura et al. 2001; Jippo et al. 2003). Mast cells can be activated through a variety of mechanisms, including bacterial peptidoglycan (binds to TLR2), LPS (binds to TLR4) and the type 1 fimbrial subunit (binds to CD48) (Malaviya et al. 1994, 1999). Mast cells can also ingest opsonized bacteria and potentially kill them following oxidative burst (Malaviya et al. 1999), and are activated following ligation of TLR3, indicating a likely role in the host response to viral infection (Kulka et al. 2004). These studies provide a clear example of where mast cells, in mice at least, provide an important protective role for the host. This role in the defense against local infection perhaps explains why mast cells are so widely distributed throughout the human body, particularly at mucosal sites and within the skin, which provide a ready portal of entry for foreign organisms.

Mast cell interactions with the specific immune system Mast cells, antigen presentation, and differentiation of Th2 T cells In addition to the innate mechanisms of mast cell activation, both rodent and human mast cells express class II major histocompatibility complex (MHC) antigens; additionally, in both mice and rats, bone marrow-derived and peritoneal mast cells, respectively, are capable of presenting soluble antigens to T cells with subsequent T-cell proliferation (Fox et al. 1994; Frandji et al. 1995). This antigen-presenting activ-

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ity of rat mast cells is enhanced by IL-4 and GM-CSF, and inhibited by IFN-γ (Frandji et al. 1995). IL-4 is required for the differentiation of the Th2 subset of T cells, which themselves produce IL-4 and IL-5. It has been hypothesized that the release of IL-4 from mast cells could provide the right microenvironment for such T-cell polarization (Bradding et al. 1992). One study has reported that rat mast cells can indeed shift the differentiation of T cells toward the Th2 phenotype (Huels et al. 1995). It is therefore plausible that antigen presentation by mast cells in concert with mast cell IL-4 secretion could contribute to Th2 skewing at the onset of an immune response. Mast cells also influence dendritic cell development and their ability to activate T cells. For example, histamine increases IL-10 and decreases IL-12 production by mature dendritic cells, with the result that naive T cells become polarized toward a Th2 phenotype (Idzko et al. 2002). Similar effects have been observed with PGD2 (Theiner et al. 2006), and mast cell dependence for the generation of Th2-promoting dendritic cells is evident in mice in vivo (Mazzoni et al. 2006). Mast cell exosomes induce immature dendritic cells to upregulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent antigen-presenting capacity to T cells (Skokos et al. 2003). Mast cell TNF-α is important for dendritic cell migration during immune responses (Suto et al. 2006), and of particular relevance to allergy, a population of FcεRI+ dendritic-like cells also emerge in parallel with developing mast cells from human hip bone marrow cultures (Kaur et al. 2005).

Mast cells as initiators of allergic inflammatory responses in the absence of IgE A number of diverse allergens, including bee venom phospholipase (PL)A2, Der p I and III, and schistosomal protease, induce the release of histamine and IL-4 from HLMC in the absence of cell-bound IgE (Dudler et al. 1995; Machado et al. 1996). These allergens are all enzymes, and the response to house-dust mite antigens requires the presence of catalytic activity. A rat basophilic leukemia cell line (RBL-2/2/C) also degranulates and secretes IL-4 in response to trypsin and Aspergillus protease. PLA2, PLC, thrombin, trypsin, and papain also induce mediator release from RBL cells, and all depend on the presence of an active catalytic site. Phospholipases, proteases, or both are associated with many allergens including those originating from house-dust mites, cockroaches, fungal spores, grain dust, plant pollens, cats, and insect venoms. The molecular mechanism by which proteases release mediators from mast cells independently of IgE cross-linking involves in part the protease-activated receptor PAR2 (He et al. 2004; Moormann et al. 2006). It is therefore plausible that activation of mast cells by allergens through an IgE-independent mechanism provides an initial stimulus in the immune response to these molecules and may promote the generation of Th2 cells. The reason why this does not occur in everyone can still be explained by

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environmental factors (e.g., level of allergen exposure) and genetic factors (e.g., mast cell releasability, epithelial integrity/ permeability, local antiprotease activity, regulation of cytokine production).

Role of mast cells in allergic diseases Despite the potential physiologic roles that mast cells perform, they are still synonymous with allergy. IgE-mediated allergic diseases include asthma, allergic rhinitis and conjunctivitis, eczema, urticaria, and systemic anaphylaxis. The incidence of these is increasing (Lack et al. 2002; Madsen 2005; Burr et al. 2006; Galassi et al. 2006), but the cause for this remains uncertain. It is evident from Tables 11.2 and 11.3 and the above discussion that mast cells secrete a plethora of autacoids, proteases and cytokines relevant to the pathophysiology of allergic diseases, and many of these are released via both IgE-dependent and -nondependent mechanisms. Depending on the site of mediator release, symptoms manifest clinically as rhinitis, conjunctivitis, urticaria, angioedema, erythema, bronchospasm, diarrhoea, vomiting, and hypotension, which can be fatal in severe reactions (such as anaphylactic shock). In some of these diseases, such as seasonal allergic rhinoconjunctivitis and anaphylaxis, the role of IgE and allergen are relatively clear-cut, while in chronic asthma and atopic eczema the involvement of IgE and mast cells is probably one of many factors contributing to disease expression. We initially focus in detail on the immunopathology of asthma: many of the principles discussed are also applicable to the immunopathology of related allergic diseases.

Mast cells in asthma Asthma is a complex disease characterized by the presence of airway obstruction. This obstruction is potentially reversible, at least in part, either spontaneously or with pharmacologic intervention, and is characterized by the symptoms of wheeze, dyspnea, cough, and chest tightness. Exacerbations may be triggered by a number of different stimuli, one or more of which may predominate in any individual. The major pathologic processes by which airflow obstruction occurs are smooth muscle contraction, mucosal edema due to increased vascular permeability, excessive mucus secretion, airway inflammation, and various structural changes referred to as airway wall remodeling. In this section we concentrate on the evidence that places mast cells as central effector cells in asthma pathophysiology as determined by the profile of mediators they release, their ongoing release of these mediators, and their relocation to key structures within the airways.

Evidence of mast cell activation in asthma Experimental allergen-induced asthma Approximately 90% of subjects with asthma under the age of 30 are atopic (Smith 1974), most frequently with reactions to the house-dust mite Dermatophagoides pteronyssinus or farinae, whereas in subjects developing asthma for the first time over the age of 40, the prevalence of atopy is no greater than in the general population. Allergen exposure clearly plays a role in asthma pathophysiology but it is not the whole story. The level of dust mite exposure in early life is a strong risk factor for the development of asthma, and it is clear that certain pollenallergic individuals only develop symptoms during the pollen season. Clusters of acute asthma exacerbations occur following the acute release of pollen particles into the atmosphere after thunderstorms (Newson et al. 1997, 1998). In addition, the clear clinical efficacy of the anti-human IgE monoclonal antibody omalizumab in severe allergic asthma and its ability to markedly reduce airway inflammation (Djukanovic et al. 2004) points to the importance of IgE in ongoing disease. However, omalizumab does not cure the disease, and symptoms often persist albeit at a lower level. Furthermore, allergen avoidance usually has only a minor effect on the state of established disease, which therefore appears to become “self-perpetuating.” This is typified in cases of occupational asthma, where asthma will persist on removal of the sensitizing agent if exposure is not prevented early in the course of the disease. Because of the evidence that asthma is at least partly an IgE-dependent disease, the method of acute bronchial challenge with a relatively large dose of allergen in the laboratory has provided a useful model for studying asthma pathophysiology. Following bronchial allergen challenge in nearly all atopic asthmatics and many atopic nonasthmatics, there is a rapid fall in pulmonary function (e.g., forced expiratory volume in 1 s or FEV1) at 10–20 min that gradually recovers over the following 2 hours, defined as the early asthmatic reaction (EAR). In about 50% of subjects, between 4 and 6 hours there is a further fall in FEV1, the late asthmatic reaction (LAR). This may last up to 12 hours and in some individuals may be followed by recurring airway obstruction for several days or even weeks (Booij-Noord et al. 1972).

Early asthmatic reaction During the EAR several vasoactive and spasmogenic mediators are released, most of which originate from mast cells resident in the airway mucosa (see Table 11.2). The relative rate of mediator release from HLMC in vitro is histamine > PGD2 > LTC4, with half-maximal release occurring at 2, 5, and 10 min respectively (Schleimer et al. 1986). This is reflected in vivo by the recovery of these mediators in bronchoalveolar lavage (BAL) fluid within 5–10 min following local bronchial allergen challenge (Fig. 11.8) (Murray et al. 1986; Casale et al. 1987a,b; Wenzel et al. 1988, 1990, 1991; Liu et al. 1991; Sedgwick et al. 1991).

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15

4

10

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PART 2

0

0 Pre

Post Histamine

Pre

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Tryptase

Fig. 11.8 Concentrations of histamine and tryptase in bronchoalveolar lavage fluid before and 5 min after local bronchial allergen challenge in a group of mild allergic asthmatic subjects. Medians are denoted by horizontal bars. (Data from Wenzel et al. 1988.)

The ability of histamine, PGD2 and LTC4 to produce bronchoconstriction, mucosal edema, and mucus secretion suggests that they are responsible for the acute airway narrowing following allergen challenge (Fig. 11.9). Calculations indicate that the concentrations of mediators generated are similar to those required to produce bronchoconstriction in vitro (Liu et al. 1991) or when delivered by nebulizer (Wenzel et al. 1990). Better evidence for their role is provided by studies using potent and selective receptor antagonists, which demonstrate that the EAR is significantly attenuated by antagonists of histamine (Curzen et al. 1987; Rafferty et al. 1987), LTC4 (Taylor et al. 1991; Findlay et al. 1992), and to a lesser extent PGD2 (Beasley et al. 1989a). The mast cell as a source of these mediators is supported by three lines of evidence. Firstly, the kinetics of IgE-dependent mediator release in vivo parallels that of purified mast cells in vitro. Secondly, the presence of mast cell activation during the EAR is confirmed by the rapid increase in concentrations of the preformed mast cell-specific protease tryptase, recovered by BAL within minutes following local bronchial allergen challenge (Wenzel et al. 1988; Sedgwick et al. 1991). Thirdly, β-agonists such as salbutamol, known inhibitors of mast cell degranulation when applied acutely in vitro (Church & Hiroi 1987), completely abolish the early reaction and the associated increase in plasma histamine levels (Pepys et al. 1968; Howarth et al. 1985). In contrast, corticosteroids administered just prior to allergen challenge do not inhibit the EAR (Booij-Noord et al. 1971) and do not inhibit mast cell degranulation (Schleimer et al. 1983), although repeated treatment for several days may lead to attenuation of the EAR (Burge et al. 1982), possibly as a result of a reduction in the number of mast cells in the bronchial mucosa (Djukanovic et al. 1992b). The EAR is markedly attenuated after pretreatment with anti-IgE (Boulet et al. 1997; Fahy et al. 1997), confirming that IgE-dependent signaling is critical.

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Late asthmatic reaction In contrast to the acute mediator-induced bronchoconstriction and mucosal edema characteristic of the EAR, the LAR is associated with inflammatory cell accumulation and activation. Subsequent mediator release and tissue damage following these events is thought to account for the ensuing airway obstruction and associated increase in bronchial hyperresponsiveness that accompanies the LAR. This situation has been considered to be analogous to that seen in chronic airway inflammation, although some caution is needed in extrapolating the results of applying a single large dose of allergen to the airways, in the presence of natural disease, to the natural disease itself. The role of the mast cell as a source of bronchospastic mediators during the LAR is more difficult to define than during the EAR due to the recruitment and activation of many cell types, including eosinophils (de Monchy et al. 1985; Metzger et al. 1987; Bentley et al. 1993; Montefort et al. 1994), activated CD4+ T cells (Bentley et al. 1993), activated macrophages (Tonnel et al. 1983; Diaz et al. 1989), and small numbers of basophils (Liu et al. 1991; Braunstahl et al. 2001), which are not usually present in stable asthma (Wardlaw et al. 1988; Beasley et al. 1989b). Increased concentrations of histamine, PGD2 and LTC4 are present in the LAR, but in different ratios than during the EAR, raising the possibility that a contribution comes from sources other than mast cells, such as macrophages and eosinophils (Liu et al. 1991; Sedgwick et al. 1991). Tryptase levels fall during the LAR (Sedgwick et al. 1991), which might indicate an absence of mast cell activity. However, mast cell degranulation is not “all or nothing,” with clear examples available of differential mediator release under various conditions (Theoharides et al. 1982; Benyon et al. 1989; Stellato et al. 1991; Leal-Berumen et al. 1994). Interestingly GM-CSF, which is released following allergen provocation (Broide & Firestein 1991), inhibits expression of tryptase in the immature mast cell line HMC-1 but does not attenuate histamine release (Finotto et al. 1996), and in HLMC may in fact potentiate IgE-dependent histamine release (Louis et al. 1995). Furthermore, in rodent mast cells, protease and proteoglycan peptide core mRNA transcripts remain low after the activation/ secretion response, suggesting that regranulation of mast cells is a slow process (Gurish et al. 1991). Thus plausible mechanisms exist to explain the disparity between tryptase and histamine levels during the LAR. Mast cell-derived mediators may therefore contribute directly to the symptomatology of the LAR, although their precise role is uncertain. However, the LAR is also attenuated markedly by anti-IgE therapy (Fahy et al. 1997), indicating that mast cell activation during the EAR initiates events leading to the LAR. It is therefore likely that the secretion of mast cell mediators and cytokines orchestrates the development of the late inflammatory response (Fig. 11.9).

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Mast Cells: Biological Properties and Role in Health and Allergic Diseases Epithelial denudation, activation, permeability

Tryptase, Chymase, IL-4, IL-13

Airway Lumen

Epithelium MC

MC

MC

Histamine, Tryptase, PGD2, LTC4, IL-4, IL-6, IL-13, TNF-a and amphiregulin

ASM

CXCL8 CCL11 CCL5

Submucosal gland

Subepithelial fibrosis Mucus hypersecretion

MC

MC

TGF-b bFGF Tryptase

CXCL8 CXCL10 CXCL12 Histamine, Tryptase, PGD2, LTC4, IL-4, IL-13

ASM hypertrophy and hyperplasia

Bronchoconstriction Airway hyperresponsiveness

ASM

ASM mast cell recruitment CXCL10 SCF

MC Mast cell differentiation, survival and activation

MC

Differentiation

SCF MC

MC

Rolling and adhesion

Blood Vessel MC

Fig. 11.9 Mast cell interactions with structural airway cells in the pathogenesis of asthma. See text for definition of abbreviations. (See CD-ROM for color version.)

Mast cell progenitors circulate in the blood

Chronic allergic asthma If mast cell activation is relevant to chronic everyday asthma, it would be expected that bronchial mucosal mast cells in asthmatic subjects would be in an activated state, which is indeed the case. Morphologic assessment using electron microscopy indicates that in “stable” atopic asthma there is continuous ongoing degranulation in both the airway epithelium and submucosa (Beasley et al. 1989b; Djukanovic et al. 1992a) (see Fig. 11.3). Several studies have also shown

MC

Precursor recruitment (CXCL12?)

increased numbers of mast cells in BAL fluid from stable asthmatics compared with normal controls (Flint et al. 1985; Casale et al. 1987b; Kirby et al. 1987), together with increased concentrations of histamine and tryptase, providing further evidence of ongoing mast cell degranulation (Casale et al. 1987b; Kirby et al. 1987; Wenzel et al. 1988) (Fig. 11.10). Mast cells within the bronchial mucosa in asthma express several cytokines, including IL-4, IL-5, IL-6, TNF-α, and IL-13 (Bradding et al. 1992, 1993, 1994; Ying et al. 1997; Berry et al.

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5 Mediator concentration

** 4

*

1800 **

6

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5 3

4

2

3

1200 900 600

2 1

300

1

0

0

0 Histamine (ng/mL)

Tryptase (ng/mL)

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Fig. 11.10 Concentrations of mast cell-derived histamine, tryptase, and PGD2 in bronchoalveolar lavage fluid recovered from airways of individuals with (solid bars) and without (shaded bars) asthma. The bars represent 1 standard deviation from the mean of 12 observations. **, P < 0.01; *, P < 0.05.

2004). When asthmatic airways are compared with normal airways, there is evidence of increased expression of IL-4 and IL-5 mRNA in mast cells (Ying et al. 1995), and increased expression of mast cell-associated IL-4 and TNF-α protein (Bradding et al. 1994). Strong correlations have been described between numbers of eosinophils and IL-4-, IL-5- and TNFα-positive mast cell densities, although correlations do not necessarily imply cause and effect. However it seems likely that mast cell-derived cytokines make a major contribution to the pathophysiology of asthma. BAL mast cells from symptomatic asthmatic subjects probably differ from normal BAL mast cells by exhibiting greater spontaneous histamine release (Flint et al. 1985; Broide et al. 1991). In addition, the secretory response of asthmatic BAL mast cells to IgE-dependent activation is altered. Broide et al. (1991) found that IgE-dependent activation of mast cells from symptomatic asthmatics did not produce any significant increase in histamine release compared with the already high spontaneous release, in contrast to the findings in asymptomatic asthmatic subjects, perhaps supporting the idea that high spontaneous release in the presence of symptoms is related to IgE-dependent activation in vivo. Both Flint et al. (1985) and Casolaro et al. (1989) reported increased IgEdependent histamine release from atopic asthmatic BAL mast cells compared with nonatopic normal controls. This may have resulted from greater mast cell sensitization by IgE in the atopic subjects, which is known to augment IgEdependent histamine release in a dose-dependent manner (Tunon de Lara et al. 1995). Increased FcεRI occupation by IgE not only enhances receptor aggregation by anti-IgE/ allergen, but also increases surface FcεRI expression (Saito et al. 1997; Welker et al. 1997; Yamaguchi et al. 1997, 1999), and activates HLMC in the absence of allergen (Cruse et al. 2005). Enhanced mediator releasability in asthma may also

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stem from in vivo activation by other inflammatory stimuli described above, but could also represent a fundamental functional difference predisposing to asthma, perhaps as a result of genetic factors. It is often suggested that mast cells cannot be important in asthma because so-called mast cell “stabilizing” drugs are not effective at preventing symptoms or attenuating the underlying inflammation. However, although several commonly used drugs are able to inhibit mast cell secretion, they either lack potency or are ineffective at inhibiting mast cell activation in chronic asthma. For example, sodium cromoglycate is only a weak inhibitor of IgE-dependent HLMC secretion, with maximal inhibition of histamine release in vitro of 10 –20% when used in the high micromolar range (Church & Hiroi 1987), and it also exhibits rapid tachyphylaxis. The β2-adrenoceptor agonists such as salbutamol are more potent inhibitors of HLMC mediator release in vitro (Church & Hiroi 1987), but again there is rapid tachyphylaxis so that with chronic administration the clinical evidence is that they do not attenuate mast cell secretion in the asthmatic airway and may even enhance it (Chong et al. 1995, 2003; Swystun et al. 2000; Scola et al. 2004). Furthermore, the morphology of mast cell degranulation in chronic asthma is predominantly piecemeal (Djukanovic et al. 1992a; Begueret et al. 2007), the mechanism of which is unknown, and this may simply not be susceptible to inhibition by drugs that attenuate classical IgE-dependent anaphylactic degranulation. Mast cells are therefore in a state of continuous activation in chronic allergic asthma, with some evidence to suggest that they may exhibit increased mediator releasability, both spontaneously and in response to IgE-dependent activation. When this information is coupled with the epidemiologic data implicating allergen exposure in the development of asthma and the efficacy of omalizumab, it becomes attractive to hypothesize that the atopic asthmatic phenotype results in part from the everyday interaction between allergens, IgE, and hyperreactive mast cells.

Nonallergic (“intrinsic”) asthma A subgroup of asthmatic patients have traditionally been described as having so-called “intrinsic” or nonallergic asthma, where no sensitivity to environmental allergens or occupational agents can be identified. This form of the disease is often of later onset, more severe and persistent, and more often associated with nasal polyposis and aspirin sensitivity. However, in spite of the different clinical picture, a pattern of inflammation more or less identical to that found in extrinsic asthma is present in the bronchial mucosa of intrinsic asthmatics (Bentley et al. 1992a). This suggests that a common mechanism may in fact be operating in the development of both phenotypes of asthma. Although the serum concentration of total IgE falls within the “normal range” in nonallergic asthma, it is still higher than in nonatopic nonasthmatic subjects (Bentley et al.

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1992a). Furthermore, it has been demonstrated in epidemiologic studies that total serum concentrations of IgE correlate with indices of asthma at all age groups, irrespective of atopic status, raising the possibility that IgE-mediated mechanisms are also relevant to intrinsic asthma (Burrows et al. 1989). This hypothesis is supported by several pieces of evidence that also implicate a role for mast cells. Firstly, FcεRI+ cells are present in increased numbers in the bronchial mucosa of both atopic and nonatopic asthmatic subjects compared with normal controls (Humbert et al. 1996a). Although total mast cell counts are the same in these three groups, the majority of the FcεRI+ cells are mast cells, indicating that FcεRI density is greatly increased on mast cells in both intrinsic and extrinsic asthma. Secondly, and of equal interest, there is increased expression of the Th2 cytokines IL-4 and IL-5 at both the mRNA and protein level in intrinsic asthma (Humbert et al. 1996b). As in atopic asthma, mast cells account for a significant proportion of the cells expressing mRNA and protein for both these cytokines (Ying et al. 1997), suggesting there is ongoing mast cell activation and cytokine secretion in intrinsic asthma. Both IL-4 and IgE are powerful inducers of FcεRI expression on mast cells (Saito et al. 1997; Toru et al. 1997; Welker et al. 1997; Xia et al. 1997; Yamaguchi et al. 1999), and IL-4 is of course a key cytokine involved in the promotion of IgE synthesis. There are increases in ε germline gene (Iε) and mature ε heavy chain (Cε) RNA-positive B cells in the bronchial mucosa in both atopic and nonatopic asthma, suggesting that there is local IgE synthesis (Ying et al. 2001). This is supported by the observation that the concentration of IgE in BAL fluid is increased 24 hours after bronchial allergen challenge (Wilson et al. 2002). Thus it is plausible that IgE production occurs locally within the bronchial mucosa in intrinsic asthma, promoting the increased expression of FcεRI, and perhaps explaining the absence of high serum concentrations. Since nasal mast cells can induce IgE synthesis by B cells in an IL-4 and IL-13 dependent manner (Pawankar et al. 1997), lung mast cells might contribute to local bronchial IgE production.

Virus-induced asthma Viral infections induce exacerbations of asthma in many individuals, particularly those caused by the rhinovirus (RV) (Minor et al. 1974; Johnston et al. 1995). Several mechanisms may be active including direct airway damage with loss of epithelial structure and function, and the presence of virus-specific IgE. The loss of epithelium might allow greater penetration of allergens into the bronchial mucosa, so it is interesting that experimental nasal infection with live RV16 increases the frequency of bronchial LAR after inhaled allergen provocation (Lemanske et al. 1989). Nasal RV16 infection also enhances both the immediate release of histamine into BAL fluid (presumably mast cell-derived) and the later recruitment of eosinophils into the airway after local bronchial allergen challenge (Calhoun et al. 1994).

Whether this is actually due to increased allergen availability or other mechanisms that amplify the inflammatory response is uncertain. Viral infection can also result in the production of virusspecific IgE, and the intensity of this antibody response has been correlated with changes in airway function during the acute infection (Welliver et al. 1981, 1986a). In addition, children who develop higher levels of virus-specific IgE during infection with RSV are more prone to later wheezing than children who do not (Welliver et al. 1986b). An in vivo animal model of asthma and RSV infection has shown that RSV can induce mast cell degranulation through the crosslinking of virus-specific IgE on mast cells, with associated increases in bronchial hyperresponsiveness (BHR) (Dakhama et al. 2004). In addition it is apparent that human mast cells can be activated via TLR3, a receptor for double-stranded viral RNA, secreting both IFN-α and IFN-β (Kulka et al. 2004). This provides a further mechanism through which viruses might directly interact with mast cells.

Occupational asthma Occupational asthma is defined as asthma that develops, or is exacerbated, following specific exposure in the workplace and should exclude nonspecific stimuli that will produce bronchoconstriction in any asthmatic subject. The prevalence of occupational asthma varies, but in some areas may account for 15–20% of adult asthma (Blanc 1987). Over 300 agents are recognized as inducers of occupational asthma, and common to all these compounds is that they are inhaled (which probably informs us about the induction of atopic and intrinsic asthma). They fall into three main groups, the first of which is associated with the synthesis of specific IgE antibodies. Compounds in this IgE-dependent group are either directly immunogenic proteins, such as high-molecularweight enzymes, or low-molecular-weight compounds that bind to body proteins and act as haptens, for example the acid anhydrides and complex halogenated platinum salts. The second group is thought to produce sensitization through as yet undefined immunologic mechanisms, with specific IgE antibodies usually absent. Examples from this group include the low-molecular-weight chemicals plicatic acid (present in the dust of western red cedar) and the isocyanates. The third group consists of agents that are mostly irritant gases, fumes, or chemicals, and which are capable of producing asthma after a single large exposure (reactive airways dysfunction syndrome or irritant-induced asthma). Interestingly, the pathology of occupational asthma (with the exception of irritant-induced asthma) is virtually identical to that seen in atopic and intrinsic asthma, including those cases where IgE-independent mechanisms have been advocated such as with western red cedar asthma (WRCA) and toluene diisocyanate (TDI) asthma (Frew et al. 1995; Saetta et al. 1992). Thus in TDI asthma, numbers of mast cells are increased in the bronchial epithelium compared with

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Histamine

LTE4

*

*

Mediator concentration (mg/mL)

7 6 5 4 3 2 1 0 Pre Post WRCA

Pre Post NOA

Pre Post WRCA

Pre

Post NOA

Fig. 11.11 Histamine and LTE4 concentrations in bronchoalveolar lavage fluid before and 10 min after bronchial provocation with plicatic acid in patients with western red cedar asthma (WRCA) and nonoccupational asthma (NOA). Medians denoted by horizontal bars. *, P < 0.01 (Mann–Whitney U test) for comparison of change between the two groups. (Data from Chan-Yeung et al. 1989.)

normal controls, and electron microscopy demonstrates that the majority of mast cells are degranulated (Saetta et al. 1992). Subjects who develop occupational asthma after a short period of exposure to TDI (2 years) have more mast cells in their airway mucosa than subjects who develop asthma after a long period of exposure (22 years) (Di Stefano et al. 1993). Whether this is a predisposing factor for the early onset of TDI asthma or a secondary effect due to different levels of exposure awaits clarification. Bronchial provocation with plicatic acid results in the rapid release of histamine into the BAL fluid of patients with WRCA but not normal subjects (Fig. 11.11) (Chan-Yeung et al. 1989). Similarly, in vitro, plicatic acid releases histamine from mast cells in both BAL fluid and bronchial mucosal biopsies obtained from patients with WRCA but not those with atopic asthma (Frew et al. 1993). This occurs via an undefined IgE-independent mechanism, which is in keeping with the observation that patients with WRCA do not usually have specific IgE to plicatic acid (Frew et al. 1993).

Exercise-induced asthma Of asthmatic subjects, 80% develop airway narrowing with exercise or hyperventilation of cold dry air. Typically bronchoconstriction occurs 5–10 min after exercise and usually recovers within 30 min. Approximately half these patients exhibit a refractory period for about 1 hour following exercise, during which additional exercise does not induce further bronchoconstriction. The mechanisms behind exercise-induced asthma probably relate to the effects of airway cooling and water loss during exercise and may be mimicked by hyperventilation of cold dry air (reviewed by Anderson 2006).

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Some but not all studies have identified increased concentrations of circulating histamine in the serum of asthmatic subjects following exercise, which has been taken to indicate a possible role for mast cell degranulation (Anderson et al. 1981; Barnes & Brown 1981; Lee et al. 1982). Both mast cells and basophils release histamine in a hyperosmolar environment (Eggleston et al. 1984, 1987), and this release is attenuated by sodium cromoglycate, providing evidence that could link changes in airway osmolarity to bronchoconstriction. Analysis of induced sputum following exercise has demonstrated increased concentrations of histamine, cysteinyl leukotrienes, and tryptase indicative of mast cell activation (Hallstrand et al. 2005). In support of this, terfenadine and clemastine (histamine H1 receptor antagonists), flurbiprofen (potent COX inhibitor), and the LTD4 receptor antagonists MK 571 and ICI 204219 all significantly attenuate exercise-induced bronchoconstriction (Hartley & Nogrady 1980; Finnerty & Holgate 1990; Manning et al. 1990; Finnerty et al. 1992). The refractory period following exercise remains unexplained. Interestingly, challenge of the airways with adenosine 5-monophosphate (AMP), which induces bronchoconstriction indirectly via activation of mast cells, exhibits crossrefractoriness with exercise, implicating a similar mechanism (Finnerty et al. 1990). Depletion of mast cell mediators has also been put forward as an explanation, but this seems unlikely. To summarize then, there is strong evidence implicating mast cell activation as an important component of the bronchoconstrictor response to exercise.

Aspirin-induced asthma Asthma is exacerbated by aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) in about 10% of all asthmatics. Classically these are nonatopic female patients with late-onset asthma who have associated nasal polyps, vasomotor rhinitis, and moderate blood eosinophilia. Asthmatic symptoms usually start 1–2 hours after drug ingestion and may be severe. The observation that this occurs with all NSAIDs suggests that manipulation of arachidonic acid metabolism is the cause. One hypothesis is that a conformational change in COX-2 induced by nonselective NSAIDS favors the formation of hydroxyeicosatetraenoic acids and leukotrienes via the lipooxygenase pathway, resulting in bronchospasm, increased mucus production, and airway edema (Mitchell & Belvisi 1997). This is supported by the finding of increased LTC4 in nasal secretions (Ferreri et al. 1988), increased LTE4 in the urine (Christie et al. 1992), and the protection afforded by LTC4 receptor antagonists (Dahlen et al. 1993) following ingestion of aspirin in aspirin-sensitive asthmatics. This could therefore involve mast cell leukotriene generation. In support of this, a recent study by Machado et al. (1996) reported that aspirin induces mediator release from RBL cells, a cell line with phenotypic features typical of rodent mucosal mast cells. A further clue that mast cells may be dysfunctional in

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aspirin-sensitive asthma comes from observations that there are increased numbers of mast cells in the airways of these patients, and that an increased proportion of these mast cells express COX-2 (Sousa et al. 1997). In addition, mast cells are the predominant cells expressing LTC4 synthase in the airways of aspirin-sensitive asthmatics (Cai et al. 2003).

Mast cell microlocalization in asthmatic airways Mast cells are present in normal airways adjacent to blood vessels, and scattered throughout the lamina propria (Pesci et al. 1993; Carroll et al. 2002a; de Magalhaes et al. 2005). The number of mast cells in the lamina propria is not increased in asthmatic compared with normal airways, but in asthma mast cells infiltrate three key sites: the airway epithelium (Pesci et al. 1993; Bradding et al. 1994; Amin et al. 2005), airway mucosal glands (Brightling et al. 2002a; Carroll et al. 2002b; Chen et al. 2004), and airway smooth muscle (Brightling et al. 2002a, 2005a; Berger et al. 2003; Chen et al. 2004; Amin et al. 2005; El-Shazly et al. 2006; Begueret et al. 2007). This anatomic relocation places the mast cell at the site of several dysfunctional airway elements and, as shown below and alluded to above, the local delivery of their mediators is likely to be central to the disordered airway physiology.

100 Mast cells/mm2 smooth muscle

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P < 0.0001

30 10 3 1

0 Normal (a)

Asthma

Eosinophilic bronchitis

Mast cell infiltration of airway smooth muscle as a key determinant of the asthmatic phenotype It is often stated that the disordered airway physiology and airway wall remodeling characteristic of asthma are consequences of Th2 lymphocyte-driven eosinophilic inflammation. However, there are many examples where the relationship between airflow obstruction and inflammation is weak. This is particularly evident in patients with eosinophilic bronchitis (EB). This disease is responsible for approximately 15% of cases of cough referred to respiratory specialists (Brightling et al. 1999). It is characterized by the presence of sputum eosinophilia occurring in the absence of variable airflow obstruction or BHR (Brightling et al. 1999), and usually responds to treatment with inhaled corticosteroids. An immunopathologic comparison of asthma and EB has revealed that these two conditions are identical in terms of the mucosal inflammatory cell infiltrate, mucosal IL-4 and IL-5 expression, and the degree of subbasement membrane collagen deposition (Brightling et al. 2000, 2002b, 2003a; Berry et al. 2004). Furthermore in BAL fluid and/or induced sputum, mediator concentrations including histamine and PGD2, and numbers of IL-4-expressing T cells are almost identical (Brightling et al. 2000, 2002b). This raises the possibility that many of the Th2-related changes in asthmatic airways considered fundamental in disease pathogenesis may not be so important for the development of airflow obstruction and BHR after all. The really striking difference between the pathology of asthma and EB lies within the airway smooth muscle (ASM). Although ASM function is a disease-defining feature of asthma, it has been largely ignored in previous immunopathologic

(b) Fig. 11.12 (a) Mast cell numbers in the airway smooth muscle of patients with asthma, eosinophilic bronchitis, and normal control subjects. (From Brightling et al. 2002a, with permission.) (b) Light micrograph of tryptasepositive mast cells within the airway smooth muscle bundle (arrows) of a patient with asthma. (See CD-ROM for color version.)

studies of the bronchial mucosa. In asthmatic bronchial biopsies, there are numerous mast cells present within the ASM bundles, but these are virtually absent in the ASM of normal subjects and patients with EB (Fig. 11.12) (Brightling et al. 2002a). In contrast, there are no T cells or eosinophils in the ASM in any of these subject groups. This suggests that mast cell infiltration of the ASM in asthma may be important for the development of BHR and variable airflow obstruction (see Fig. 11.9) (Brightling et al. 2002a). This hypothesis is supported by the finding of a significant correlation between the number of ASM mast cells and the severity of BHR (Brightling et al. 2002a). This increase in ASM mast cells in asthmatic compared with normal control subjects has been confirmed in several further studies (Berger et al. 2003; Amin et al. 2005; Brightling et al. 2005a; El-Shazly et al. 2006; Begueret et al. 2007). It is not linked to atopic status, and so to some extent can be said to extend across asthma phenotypes

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although further work is needed. The majority of mast cells in the ASM are of the MCTC subset (i.e., containing both tryptase and chymase) and express both IL-4 and IL-13 but not IL-5 (Brightling et al. 2002a, 2003b). Furthermore, the mast cells within the ASM demonstrate ultrastructural evidence of activation (Begueret et al. 2007). Taken together these observations suggest that ASM infiltration by mast cells is a key determinant of the asthmatic phenotype. There are also further implications. For example, not only might it clarify why many atopic patients do not have asthma but it could also explain why the presence of asthma is such a strong risk factor for death from anaphylaxis and allergen desensitization (Bock et al. 2001). A second pathologic difference between asthma and EB is that the concentration of IL-13 is elevated in the induced sputum supernatant of asthmatic compared with normal and EB subjects (Berry et al. 2004). Associated with this, the number of IL-13-positive cells in the airway mucosa in asthma is elevated when compared with EB, although nevertheless relatively low (Berry et al. 2004). Interestingly, most of the IL-13-positive cells in asthmatic biopsies in this study are in fact eosinophils, suggesting that there may be dysregulation of IL-13 expression in asthmatic as opposed to EB eosinophils.

Putative mast cell–airway smooth muscle interactions The localization of mast cells within the ASM in asthma is likely to facilitate specific interactions between these two cell types through the specific targeting of both soluble mediators and signals delivered via direct cell–cell contact. We would therefore predict that the microlocalization of mast cells within the ASM will contribute to the development of ASM hypertrophy and hyperplasia, and smooth muscle dysfunction expressed as BHR and variable airflow obstruction. In turn, we would predict that the ASM provides a suitable environment for mast cell growth and survival.

Mechanisms of mast cell recruitment by asthmatic airway smooth muscle The mechanism by which mast cells are recruited to the ASM is important because inhibiting this might offer a novel approach to the treatment of asthma. A likely source for the primary stimulus for mast cell recruitment is the ASM itself, and might involve the recruitment of mast cell progenitors or mature tissue-resident cells (see Fig. 11.9). The ASM secretes many chemokines and growth factors known to exhibit mast cell chemotactic activity, including CCL11, CXCL8, CXCL12, SCF, and TGF-β (John et al. 1998; McKay et al. 1998; Ghaffar et al. 1999; Kassel et al. 1999; Berger et al. 2003; Brightling et al. 2005a; Sutcliffe et al. 2006). A detailed characterization of the chemokine receptor profile of HLMC revealed that they express the relevant receptors for the above chemokines, namely CCR3, CXCR1, and CXCR4 respectively (Fig. 11.13a) (Brightling et al. 2005b). Of great interest, however, was the

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observation that they also express CXCR3 and that this was the most highly expressed receptor (Fig. 11.13a) (Brightling et al. 2005b). Furthermore, almost 100% of mast cells within asthmatic ASM bundles express CXCR3 compared with about 50% in the surrounding mucosa, suggesting that there is selective recruitment of CXCR3-positive mast cells to the ASM in asthma (Brightling et al. 2005a). ASM secretes the three CXCR3 ligands CXCL9, CXCL10, and CXCL11 (Hardaker et al. 2004; Brightling et al. 2005a) and, interestingly, cultured human ASM from asthmatic subjects preferentially secretes CXCL10 after cytokine activation. This CXCL10 accounts for greatly enhanced HLMC chemotaxis mediated by conditioned medium from asthmatic compared with normal cultured ASM (Fig. 11.13b) (Brightling et al. 2005a). The relevance of this to mast cell recruitment by the asthmatic ASM in vivo is further demonstrated by the increased expression of CXCL10 by the ASM in bronchial biopsies from asthmatic compared with normal subjects (Fig. 11.13c) (Brightling et al. 2005a). Other chemoattractants may also contribute to the ASM mast cell myositis. SCF (c-kit ligand) is produced by both ASM and mast cells and is both a chemoattractant and an essential survival factor for mast cells (Iemura et al. 1994; Nilsson et al. 1994; Kassel et al. 1999). In addition, TGF-β is another mast cell chemoattractant released by ASM following exposure to tryptase, providing a mechanism by which mast cells might contribute to further mast cell recruitment via autocrine pathways (Berger et al. 2003). However, there appears to be an added level of complexity in the regulation of mast cell migration by ASM in that normal ASM in vitro also appears to secrete an inhibitor of HLMC migration (Sutcliffe et al. 2006).

Mast cell adhesion to airway smooth muscle Cell–cell adhesion is a fundamental mechanism by which cells communicate, facilitating the targeting of specific signals. It is also important for the retention of cells at particular locations. Resting nonactivated HLMC adhere avidly to resting ASM cells in culture (Yang et al. 2006a). This alone is interesting because T cell and eosinophils, which are not found in the ASM bundles in asthma, adhere poorly unless activated (Lazaar et al. 1994; Hughes et al. 2000). This suggests that adhesion may be important for retaining HLMC within the ASM bundles. This adhesive process is mediated partly via an undefined Ca2+-dependent pathway, and partly through a Ca2+-independent pathway that utilizes a molecule known as TSLC-1 (tumor suppressor in lung cancer 1, also known as SgIGSF, IGSF4, RA175, Necl2, SynCAM). TSLC-1 is highly expressed by HLMC and mediates HLMC adhesion to ASM through a heterophilic mechanism (Yang et al. 2006a). TSLC-1 also mediates the adhesion of mouse mast cells to fibroblasts and nerves (Ito et al. 2003; Furuno et al. 2005; Koma et al. 2005), and may therefore represent a particularly important molecule facilitating mast cell interactions with various cell types.

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% of human lung mast cells expressing chemokine receptors

50 P < 0.05

40

30

20

10

CC C C R1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CC R9 R1 0 CX C CX R1 CXCR2 CXCR3 CXCR4 CXCR5 CX CR 6 3C R1

0 (a)

Fig. 11.13 (a) Chemokine receptor expression on ex vivo human lung mast cells. (b) Mast cell chemotaxis to conditioned media from cytokine-activated normal and asthmatic airway smooth muscle (ASM) cultures and its inhibition by chemokine receptor blockade. (c) CXCL10 protein expression in asthmatic but normal airway smooth muscle in situ. (a) from Brightling et al. 2005b, (b) and (c) from Brightling et al. 2005a, with permission. (See CD-ROM for color version.)

Fold difference in migration compared with control

6

*P < 0.05 compared with control ***P < 0.01 compared with control **P < 0.05 compared with asthmatic ASM alone

5

Normal

***

4

3

2

**

**

** *

1

(b)

CXCR3 CCR3 CXCR1 CXCR4 Normal ASM blocking antibody Supernatant Asthmatic ASM Supernatant

Biological effects of mast cell mediators on airway smooth muscle Immunohistochemical and electron microscopical analyses suggest that mast cells in the asthmatic ASM form intimate contact with ASM cells and are present in an activated state, with evidence of ongoing mediator release (Begueret et al. 2007). The effects of mast cell mediators on ASM are therefore likely to be profound. The classical mast cell autacoid mediators histamine, PGD2 and LTC4 are all potent agonists for ASM contraction. Exogenously administered tryptase induces bronchoconstriction and the development of BHR in dogs and sheep (Sekizawa et al. 1989), and tryptase can potentiate the contractile response of sensitized bronchi to histamine in vitro (Berger et al. 1999). In addition to its ability to stimulate cytokine release from ASM, tryptase can also act as a potent ASM mitogen in vitro (Berger et al. 2001; Brown et al. 2002). The precise mechanism whereby tryptase may interact with these cells is unclear because although several studies have suggested the need for an intact catalytic site

(c)

Asthma

(Brown et al. 1995; Berger et al. 2001, 2003), there is a report that nonproteolytic actions may be involved in mitogenesis (Brown et al. 2002). The recently identified transmembrane form of tryptase, but not the secreted form of tryptase (which is about 50% identical), also induces BHR in mice, an effect dependent on expression of the IL-4Rα subunit and STAT6 (Wong et al. 2002). Transmembrane tryptase increases expression of IL-13 in the airways of these mice and also stimulates IL-13 release from T cells, so it would be of great interest to know if it has a similar effect on mast cells, leading to autocrine amplification of mast cell cytokine release and the subsequent development of BHR. Chymase is a mast cell neutral protease that has been less extensively studied than tryptase, but is expressed by those mast cells infiltrating the ASM in asthma (Brightling et al. 2002a). Interestingly, chymase degrades human ASM pericellular matrix and inhibits T-cell adhesion to ASM, which might explain the paucity of T cells within this structure in asthma (Lazaar et al. 2002). However, unlike tryptase,

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chymase inhibits human ASM proliferation. The effects of the joint expression of these proteases and other mast cell mediators in vivo therefore remain unclear, but it is noteworthy that ASM proliferation in asthma in situ has never been observed (Benayoun et al. 2003; Woodruff et al. 2004). It has therefore been proposed that the ASM hyperplasia in asthma may be due to the recruitment of ASM precursors known as fibrocytes from the peripheral circulation (Kaur et al. 2006). In support of this, both ASM and myofibroblasts in tissue express the chemokine receptor CCR7, while one of the relevant ligands, CCL19, is expressed both by the ASM and by mast cells within the ASM bundles (Kaur et al. 2006). This suggests that mast cells and ASM could recruit ASM progenitors to the ASM bundle, a supposition supported by the ability of these cells to induce CCL19-dependent ASM migration in vitro (Kaur et al. 2006). Potential interactions between ASM and infiltrating mast cells are summarized in Fig. 11.9. IL-4 and IL-13 are believed to be key in the development of BHR. This is supported by an in vivo study in mice where instillation of Th2 cell-conditioned medium to the airways of naive mice induced BHR within 6 hours. This required expression of the IL-4Rα subunit and STAT6, suggesting a critical role for IL-4 and/or IL-13, and both of these cytokines produced similar effects when administered individually (Venkayya et al. 2002). IL-4 and IL-13 also enhance the magnitiude of agonist-induced intracellular Ca2+ responses in cultured human ASM. Since mast cells within the asthmatic ASM express both IL-4 and IL-13 (Brightling et al. 2003b), this may represent a further important pathway by which mast cells could contribute to the development of BHR.

Mast cells, airway epithelial dysfunction, and subepithelial fibrosis The epithelium is fragile and denuded even in mild asthmatics. Often the basal cell layer is left intact, suggesting the point of weakness is between this layer and the surface epithelium (Montefort et al. 1992). Whether this abnormality is primary or secondary is unclear, although evidence suggests the latter may be the case. Mast cells infiltrate the bronchial epithelium in asthma (Pesci et al. 1993; Bradding et al. 1994; Amin et al. 2005). This is of potential importance because it places mast cells at the portal of entry of noxious stimuli such as aeroallergens and viruses, which could facilitate an effector role in the ongoing immunologic response (antigen presentation, Th2 cell differentiation, IgE synthesis). Furthermore, there are likely to be important consequences of mast cell degranulation on epithelial function. For example, mast cells adhere avidly to bronchial epithelial cells (Sanmugalingam et al. 2000; Yang et al. 2006b) and tryptase stimulates airway epithelial cell IL-8 release and upregulates intercellular adhesion molecule (ICAM)-1 expression (Cairns & Walls 1996), a receptor for RV. That mast cell activation can actually damage epithelium is supported by a study investigating the

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mechanisms of ozone-induced epithelial damage in mast cell-deficient mice. This demonstrated that ozone-induced epithelial sloughing is entirely mast cell dependent (Longphre et al. 1996). The mechanisms behind this and the effects of mast cell activation on human airway epithelial function deserve further investigation. A characteristic histologic feature of asthma is thickening of the subbasement membrane due to deposition of type III and V collagen in the lamina reticularis (Roche et al. 1989). The most likely origin for this collagen is proliferating myofibroblasts whose number correlates with collagen thickness (Brewster et al. 1990). Mast cells in or adjacent to the bronchial epithelium also have the potential to activate subepithelial myofibroblasts as there is longstanding evidence that mast cells and fibroblasts interact intimately through several mechanisms. For example, cultured rat embryonic skin fibroblasts phagocytose rat mast cell granules, and this is followed by secretion of collagenase and β-hexosaminidase (Subba Rao et al. 1983). Histamine, TNF-α, bFGF, and IL-4 promote fibroblast proliferation in humans (Boucek & Noble 1973; Sugarman et al. 1985; Jordana et al. 1988; Feghali et al. 1992). IL-4 is a chemoattractant for human fibroblasts (Postlethwaite & Seyer 1991) and also induces human fibroblasts to secrete collagen types I and III and fibronectin (Postlethwaite et al. 1992). bFGF is a potent fibrogenic cytokine (Qu et al. 1995; Reed et al. 1995), and heparin stabilizes bFGF structurally and preserves its bioactivity by protecting it from degradation (Gospodarowicz & Cheng 1986). Furthermore, heparin and/or heparan sulfate are required for binding of bFGF to its receptors (Yayon et al. 1991), and also release it from basement membranes where it is stored (Folkman et al. 1988). Heparin may thus potentiate fibroblast activation and proliferation indirectly through the regulation of bFGF bioactivity. In coculture, human mast cells adhere avidly to human fibroblasts through an as yet undefined mechanism, which does not involve known integrin or cadherin receptors (Trautmann et al. 1997). Both HMC-1 cells and human skin mast cells augment proliferation of human skin fibroblasts, which is dependent on this heterotypic cell–cell contact (Trautmann et al. 1998). This proliferative response was dependent on the expression of mast cell-associated IL-4, although IL-4 could not be detected in the supernatant of the coculture system (but was present in lysates of HMC-1 cells), suggesting that IL-4 was secreted by mast cells in low amounts and strictly limited to cell–cell contacts with fibroblasts. Interestingly, we had suggested previously that mast cells may present IL-4 on their surface in order to confer local cytokine specificity (Bradding et al. 1993). This was based on the observation that in mucosal biopsies from patients with asthma and allergic rhinitis (where mast cells are chronically activated), but not normal subjects, we observed a ring-staining pattern of IL-4 immunoreactivity around mast cells (Bradding et al. 1992, 1993, 1994).

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Mast cell microlocalization within airway submucosal glands Severe mucus plugging is an established feature of severe fatal asthma but is also present in milder disease (Carroll et al. 2002b; Kuyper et al. 2003). This results from mucus hypersecretion by hyperplastic submucosal glands and epithelial goblet cells. Carroll et al. (1993) performed a detailed analysis of cartilaginous airways in postmortem lung specimens from patients with fatal asthma, patients with asthma who died from other causes (nonfatal asthma), and subjects without asthma who died of nonpulmonary causes. Immunohistochemistry for mast cell tryptase revealed a significant increase in the number of mast cells within the mucosal gland stroma in nonfatal asthma, and a marked increase in the number of degranulated mast cells in both fatal and nonfatal asthma compared with normal controls. There were significant correlations between the density of both intact and degranulated mast cells within the mucous glands and the degree of mucus obstruction in the airways. Numerous products released by these activated mast cells have the potential to contribute to both mucous gland hyperplasia and mucus secretion. In terms of the autacoids, their potency for stimulating mucus secretion is in the order LTD4 > LTC4 > prostanoids > histamine (Shelhamer et al. 1980; Marom et al. 1981, 1982). Canine mast cell chymase is also a potent mucus secretagogue when added to cultures of bovine airway glands (Sommerhoff et al. 1989). IL-6 and TNF-α induce mucous glycoprotein secretion and MUC-2 gene expression by both human bronchial organ explant cultures and airway epithelial cells obtained by bronchial brushing (Levine et al. 1994, 1995). IL-6 also induces expression of MUC5B and MUC5AC (Chen et al. 2003). This is relevant because mast cells within the bronchial mucous glands in asthma express IL-6 (Bradding et al. 1994). Animal models also indicate roles for tryptase and IL-13 in mucus hypersecretion (Zhu et al. 1999; Oh et al. 2002). A further molecule of interest and of relevance to both the airway epithelium and mucosal glands is amphiregulin, a member of the epidermal growth factor family. Amphiregullin expression is induced in human progenitor-derived mast cells following activation through FcεRI (Okumura et al. 2005; Wang et al. 2005), an effect which is not suppressed by dexamethasone. Amphiregulin expression by mast cells in the asthmatic bronchial mucosa is increased compared with normal controls, and mast-cell derived amphiregulin increases mucin gene expression in the NCI-H292 epithelial cell line in vitro (Okumura et al. 2005). Thus mast cell-derived amphiregulin may contribute to epithelial goblet cell metaplasia and mucus hypersecretion in asthma, a process refractory to corticosteroids clinically. In addition, recombinant amphiregulin induces the proliferation of human airway fibroblasts but not ASM cells, suggesting a further mechanism whereby mast cells might contribute to subepithelial fibrosis.

Mast cells in animal models of asthma Several models have been developed that aim to induce the airway features of asthma. The most widely reported is the mouse model using intraperitoneal antigen sensitization followed by antigen challenge of the airways. This most closely resembles the model of acute allergen challenge in the airways, although the route of sensitization is obviously different. The dependence on mast cells with regard to the development of airway hyperresponsiveness and inflammatory changes in the airways is highly contingent on the model studied and the mode of antigen sensitization. Thus sensitization without adjuvant generates a mast cell-dependent model, while sensitization with adjuvant creates a mast cell-independent model (Williams & Galli 2000). An alternative model uses airway sensitization without adjuvant from the outset and to some extent is more physiologic. In this setting, mast cells are again an essential component required for the development of airway hyperresponsiveness, inflammation, and remodeling (collagen deposition, goblet cell hyperplasia) (Taube et al. 2004; Yu et al. 2006). However, there are inevitably a number of problems in relating these models to the human disease. For example, mouse airways contain very few mast cells at baseline, so the changes seen following antigen challenge rely heavily on the recruitment of mast cell progenitors rather than the activity of resident cells, and it is perhaps not surprising that subsequent short-term studies using intraperitoneal sensitization did not find a role for mast cells in the outcomes commonly measured. In addition, mice have relatively little smooth muscle in their airways, and so there is no model described to date in mice that has recapitulated the infiltration of ASM by mast cells, a feature that may be key to the development of asthma in humans. So while mouse models are useful for generating hypotheses regarding the pathogenesis of asthma, their findings may also be potentially misleading.

Possible targets for novel therapies in asthma Asthma remains a considerable cause of morbidity and occasionally death, and constitutes a major economic burden: in consequence novel approaches to treatment are urgently required. It is self-evident from the information provided above that successfully inhibiting the release of various mediators from mast cells in asthma could be particularly effective for its treatment. A possible target for treatment has already been tested clinically. The nonanaphylactogenic humanized monomeric antibody omalizumab, which prevents IgE from binding to its high-affinity (FcεRI) and lowaffinity (CD23) receptors by binding to an epitope on the CH3 domain of IgE (Holgate et al. 1998), is partially effective and now licensed for the treatment of asthma in many countries. There are obviously many possible targets, but we discuss two in further detail.

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α as a pivotal Mast cell-derived TNF-α cytokine in asthma

Inhibiting mast cell migration as novel approach to asthma therapy

Several cytokines expressed by mast cells are attractive targets, but targeting IL-4 with soluble receptors or IL-5 with neutralizing monoclonal antibodies has met with little success (Leckie et al. 2000; O’Byrne 2006). In contrast, preliminary evidence suggests that neutralizing TNF-α in severe asthma may be more effective. TNF-α is a proinflammatory cytokine strongly implicated in the pathogenesis of asthma (Bradding et al. 1994). When administered by inhalation to animals and humans it induces both BHR and sputum neutrophilia and exacerbates BHR in patients with asthma (Thomas et al. 1995; Thomas & Heywood 2002). TNF-α immunoreactivity is increased in the airways of mild asthmatics, largely due to increased expression by mucosal mast cells (Bradding et al. 1994). Two studies have demonstrated that TNF-α expression is increased markedly in severe asthma, as shown by increased TNF-α protein in BAL fluid, increased protein expression on peripheral blood mononuclear cells, and both increased protein and a 30-fold increase in mRNA in the bronchial mucosa (Howarth et al. 2005; Berry et al. 2006). This is remarkable because these patients were receiving high-dose inhaled and oral corticosteroid therapy. Interestingly, the increased protein expression in endobronchial biopsies was accounted for by increased numbers of TNF-α-positive mast cells (Howarth et al. 2005). An uncontrolled proof-of-concept study demonstrated that administration of the soluble TNF-α P75 receptor–IgG1Fc fusion protein etanercept (Enbrel) for 12 weeks significantly improved quality of life, lung function, and BHR by 2.5 doubling dilutions of methacholine (Howarth et al. 2005). Interestingly, there was no change in sputum inflammatory cells. A further small, double-blind, placebo-controlled, crossover study administered etanercept for 10 weeks to patients with severe refractory asthma (Berry et al. 2006), leading to marked improvements in quality of life, lung function, and methacholine-induced BHR (3.5 doubling dilution improvement). Again there were no significant changes in sputum inflammatory cells but there was a marked reduction in sputum histamine concentration. This latter observation is compatible with the inhibition of lung mast cell activation. In vitro, TNF-α not only induces skin mast cell histamine and tryptase release (van Overveld et al. 1991) but, following IgE-dependent activation of HLMC, the release of preformed mast cell-associated TNF-α has been shown to serve as a positive autocrine feedback signal to augment NF-κB activation and further production of TNF-α and other cytokines, including GM-CSF and IL-8 (Coward et al. 2002). Taken together, these studies provide strong evidence that mast cell-derived TNF-α plays a major role in the pathophysiology of severe asthma, and that antagonizing TNF-α is useful in this group of patients who are otherwise largely resistant to treatment.

The reason for studying the mechanisms of mast cell migration is that if the relocalization of mast cells in asthmatic airways can be prevented, this may offer a truly novel means of treating the disease. As discussed above, the CXCL10/CXCR3 axis appears important for the recruitment of mast cells by the asthmatic ASM. This therefore represents an interesting target, but other chemoattractants may be involved under various conditions, and may be involved in the recruitment of mast cells to other sites such as the airway mucosal glands. A more generalized approach to inhibiting migration may therefore be more effective than targeting individual chemoattractants. Ion channels are emerging as potential therapeutic targets in inflammatory and structural cells. The intermediate conductance calcium-activated potassium channel KCa3.1 (also known as IKCa1) is expressed by human mast cells and is particularly interesting (Duffy et al. 2001a, 2004; Kaur et al. 2005; Cruse et al. 2006). We have recently demonstrated that blockade of KCa3.1 using the highly specific small-molecule blocker TRAM-34, and the peptide charybdotoxin (derived from scorpion venom), not only markedly attenuated HLMC migration toward CXCL10 and SCF but also stimulated asthmatic ASM supernatants (Fig. 11.14) (Cruse et al. 2006). Furthermore, this channel potentiates human mast cell and T-cell mediator release (Ghanshani et al. 2000; Fanger et al. 2001; Duffy et al. 2004), and may contribute to the pathologic function of other cell types in the airways. Since KCa3.1 can be blocked effectively in both animals and humans without causing undue toxicity (Kohler et al. 2003), blockers of this channel such as TRAM-34 represent a real

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100 90 ASM-induced migration (%)

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10 0 Control

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ChTX

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Fig. 11.14 Inhibition of human lung mast cell migration toward TNF-a-activated airway smooth muscle (ASM) supernatants following KCa3.1 blockade with TRAM-34 and charybdotoxin. *P < 0.05; ** P < 0.01. (From Cruse et al. 2006, with permission.)

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therapeutic possibility for mast cell-mediated inflammatory disease.

Mast cells in allergic rhinitis Allergic rhinitis is characterized by the symptoms of nasal and palatal itch, rhinorrhea, sneezing, nasal blockage and, in severe cases, anosmia. Symptoms may be either seasonal (SAR) due to sensitivity to seasonal allergens such as grass, tree or shrub pollens, or perennial (PAR), often due to sensitivity to house-dust mite. No firm definition of allergic rhinitis exists, and patients often present with symptoms when no identifiable allergy is present (nonallergic rhinitis).

Seasonal and perennial allergic rhinitis Allergic rhinitis is characterized by the presence of a mucosal inflammatory response, similar in many respects to that seen in asthma. In both SAR and PAR there are increased numbers of eosinophils in the lamina propria and epithelium, increased mast cell numbers in the epithelium, and increased expression of Th2 cytokines in mast cells, eosinophils and T lymphocytes (Bentley et al. 1992b; Durham et al. 1992; Bradding et al. 1993, 1995c; Ying et al. 1993, 1994). Increased numbers of CD34+ tryptase-negative cells have been seen in the nasal epithelium, suggesting that there is recruitment of mast cell progenitors to this site (Kawabori et al. 1997). These intraepithelial mast cells are predominantly of the MCT phenotype, whereas those in the lamina propria are about 60% MCTC (Bentley et al. 1992a; Otsuka et al. 1995). Mast cells in the airway epithelium and lamina propria are activated in that there is ultrastructural evidence of mast cell degranulation (Howarth et al. 1991), and increased concentrations of LTC4 but not histamine are present in nasal lavage (Volovitz et al. 1988). In addition, mast cells express IL-4, IL-5, IL-6, and TNF-α in the nasal mucosa and demonstrate increased expression of IL-4, a feature reversed by the application of topical corticosteroids (Bradding et al. 1995c). Although histamine concentrations are not elevated, in contrast to asthma, antihistamine therapy is highly effective at ameliorating symptoms (Bachert et al. 2004). Topical corticosteroids are very effective at reversing the inflammatory changes (Bradding et al. 1995c; Foresi et al. 1996; Holm & Fokkens 2001). Anti-IgE therapy is also effective at treating both SAR and PAR when serum IgE levels are suppressed effectively, supporting the view that this is an IgE-driven disease (Casale et al. 2001; Holgate et al. 2005a,b). Mast cells recovered from the nasal mucosa of patients with allergic rhinitis have been studied in vitro. Interestingly these cells are able to induce B-cell IgE synthesis through the IgE-dependent release of IL-4 and IL-13 and the interaction of CD40 (expressed by B cells) with its ligand (CD40L expressed by mast cells) suggesting that mast cells could

contribute to the local production of IgE in the nasal mucosa (Pawankar et al. 1997). This is relevant because increased numbers of IgE-secreting B cells are present in the allergic nasal mucosa (Coker et al. 2005). It is therefore evident from biological effects of mast cell products (see Tables 11.2 and 11.3) that ongoing mast cell activation in the allergic nasal mucosa can explain much of the symptomatology and pathology of allergic rhinitis.

Experimental allergen-induced rhinitis Nasal allergen challenge has been used extensively to explore the immunopathology of allergic rhinitis and provides further evidence that mast cells are important effectors and orchestrators of the disease. Nearly all patients with a history of allergic rhinitis develop an early-phase response (EPR) following appropriate allergen challenge, characterized by the acute development of rhinitic symptoms. In about 40% of subjects, a late-phase response (LPR) follows after about 6 hours with recurrence of symptoms. During the EPR there is the release of a spectrum of inflammatory mediators, including histamine (Juliusson et al. 1991; Proud et al. 1992), tryptase (Juliusson et al. 1991; Proud et al. 1992), and LTC4 (Wang et al. 1997; Zweiman et al. 1997), supportive of mast cell activation. These symptoms are attenuated by antihistamines (Klementsson et al. 1990) and markedly inhibited by anti-IgE therapy (Hanf et al. 2004). During the LPR there is tissue infiltration by eosinophils and CD4+ T cells. Again this phase is markedly attenuated by anti-IgE therapy (Djukanovic et al. 2004), suggesting that mast cell-driven events at the time of challenge, such as adhesion molecule upregulation, chemoattractant release and enhanced vascular permeability, are largely responsible. A model using low-dose repeat allergen challenge more closely mimics events during the pollen season (Pipkorn et al. 1989). After repeated daily challenges, mast cell numbers start to increase in the nasal epithelium at day 6, indicating that if this involves recruitment and differentiation of progenitors, then this occurs relatively quickly.

Mast cells in allergic conjunctivitis Allergic conjunctivitis commonly accompanies allergic rhinitis and the term “rhinoconjunctivitis” is commonly used. In mild allergic eye disease, patients complain of variable itch, tearing, and swelling that is uncomfortable but does not threaten sight. Chronic forms of the disease can give rise to more severe symptoms including pain, corneal scarring, cataract or glaucoma with the potential to threaten sight. The most common form is seasonal allergic conjunctivitis (SAC), with perennial allergic conjunctivitis (PAC), atopic keratoconjunctivitis (AKC), atopic blepharoconjunctivitis (ABC), and vernal conjunctivitis (VC) less common (for detailed

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review see McGill et al. 1998). Mast cells are recognized as central effector cells in all types of allergic eye disease, demonstrating increased numbers and morphologic evidence of degranulation (Cook et al. 2001; Graziano et al. 2001; Leonardi 2002). Following conjunctival allergen challenge, elevated levels of the mast cell products histamine, tryptase, and LTC4 have also been detected in tears (Margrini et al. 1996; Bacon et al. 2000). The MCTC phenotype predominates in normal conjunctiva but in PAC, SAC, and VC the number of MCT cells increases in both the conjunctival epithelium and subepithelial layers (Irani et al. 1990; Baddeley et al. 1995). In contrast, in AKC and ABC, the numbers of MCTC increase (Yao et al. 2003). It has been proposed that MCTC contribute to tissue fibrosis in various diseases (Gotis-Graham & McNeil 1997; Mitani et al. 1999; Yamada et al. 2001; Andoh et al. 2006), and their increased numbers may therefore contribute to the excess fibrosis evident in AKC and ABC. As in the nose and bronchi (Bradding et al. 1995a), the mast cells in the allergic conjunctiva in SAC demonstrate heterogeneity in terms of cytokine expression, with the MCTC subset expressing predominantly IL-4 and IL-13, while the MCT subset expresses IL-5 and IL-6 (Anderson et al. 2001). Allergen challenge again produces early and late-phase responses, suggesting similar mechanisms to those in the nose and lower airway. Antihistamines are useful for the treatment of symptoms, suggesting that mast cell-derived histamine contributes to the symptomatology (Yanni et al. 1994; Cook et al. 2002; Abelson 2004; Abelson et al. 2004).

assumed to relate to the systemic spread of allergen, but it also needs to be considered that amplification mechanisms might exist, for example neurologic reflexes. The best evidence that there is systemic mast cell activation in anaphylaxis is from studies demonstrating increased concentrations of tryptase, particularly β-tryptase, in the peripheral circulation (Schwartz et al. 1987; Schwartz 2006). Unlike α-tryptase, which is released by mast cells constitutively, β-tryptase is stored in mast cell granules and released following IgE-dependent activation and therefore represents a more specific marker of activation than total tryptase. Histamine and β-tryptase are released from mast cells together, but while histamine concentrations in the plasma peak within 5 min, the peak of tryptase is delayed due to its slower diffusion from the tissue. This is because it is a larger molecule than histamine but also remains bound to heparin longer. Thus serum histamine concentrations peak at about 5 min while those of β-tryptase levels are maximal 15–120 min after the onset of symptoms (Schwartz et al. 1989). Tryptase is therefore not only a more specific marker for mast cell activation than histamine, which is also expressed by basophils, but also more convenient to measure after a suspected anaphylactic event. Interestingly, patients with systemic mastocytosis are at increased risk of anaphylactic and anaphylactoid reactions (Biedermann et al. 1999; Ludolph-Hauser et al. 2001), and often demonstrate increased baseline concentrations of plasma tryptase (predominantly α-tryptase) (Schwartz et al. 1995).

Mast cells in anaphylaxis

Mast cells in atopic dermatitis (eczema) and urticaria

The most striking and immediately life-threatening IgEdependent reaction is that manifesting as anaphylaxis. Food allergies are the most common cause of anaphylaxis, with 1–2% of adults having a food allergy (Jansen et al. 1994; Young et al. 1994) and as many as 8% of children (Bock 1987; Lack et al. 2002). Peanut allergy is the most common cause of fatal food-induced anaphylaxis (Bock et al. 2001), although these statistics vary between countries (Shimamoto & Bock 2002). Anaphylaxis can also result from drug allergies, in particular to penicillin, and insect venom such as bee stings. Reactions clinically indistinguishable from anaphylactic reactions but which are not IgE-dependent are termed “anaphylactoid reactions.” It is generally agreed that anaphylaxis is a syndrome with varied mechanisms and clinical presentations, mediated predominantly by mast cells and basophils. The key difference between anaphylaxis and other mast cell-associated disease is that anaphylaxis involves the systemic activation of mast cells and basophils leading to cardiovascular collapse and respiratory embarrassment due to either bronchospasm or laryngeal edema. This is often fatal if left untreated. The reason for the systemic spread of mast cell activation is often

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While atopic dermatitis is strongly associated with atopy, as indicated by its name, the role of IgE in its pathogenesis is not well defined (Williams & Flohr 2006). In addition, there are no studies examining the effects of omalizumab in atopic dermatitis that might support or refute a pathogenic role for IgE. Unlike the other “allergic” diseases described above, evidence for a role for mast cells in atopic dermatitis is relatively sparse. However, bearing in mind their wide distribution in the skin and the multiple modes they exhibit for sensing the surrounding environment, it seems unlikely that they are not involved in the immunopathology. Studies have shown that the number of the MCT phenotype of mast cells increases in the skin of patients with atopic dermatitis (Irani et al. 1989; Jarvikallio et al. 1997). In addition, skin mast cells demonstrate increased expression of IL-4 in atopic dermatitis, suggesting this may have a pathogenic role. It is evident from the other sections in this chapter that a number of mast cell mediators have the potential to contribute to disease pathogenesis, but evidence for this is currently lacking. Urticaria occurs in acute and chronic forms, with a complex classification and multiple etiologies. This is covered in

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more detail in Chapter 93. In various forms of acute urticaria, antihistamines provide a useful means of treatment and support the view that the skin lesions occur predominantly as a result of mast cell activation. In chronic urticaria, mast cell activation is also a factor, although some patients require treatment with oral steroids to control the disease, indicating a more complex immunopathology. In about 30% of patients with chronic uritcaria, circulating autoantibodies to FcεRI or IgE have been identified, suggesting a pathogenic role involving mast cells (Hide et al. 1993; Niimi et al. 1996; Sabroe et al. 1999, 2002; Sabroe & Greaves 2006). However, these patients do not usually have evidence of mast cell activation elsewhere in the body, which is unexplained, although it has been suggested that these autoantibodies might actually activate skin mast cells through complement-dependent receptors such as the C5a receptor in addition to FcεRI-dependent mechanisms (Ferrer et al. 1999).

Concluding remarks In summary, mast cells are multifaceted tissue-resident cells capable of responding to a variety of noxious stimuli with the secretion of numerous multifunctional autacoids, proteases, and cytokines. Current evidence indicates roles in host defense and repair, as well as many diverse diseases. As evident from this chapter, they play a central role in many aspects of allergic disease, although it needs to be appreciated that their activity in these and other disorders will occur through complex interactions with other immunologic and structural cells. Developing drugs that inhibit pathologic mast cell secretion when administered regularly should improve the treatment of many patients with asthma and related allergic diseases.

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Eosinophils: Biological Properties and Role in Health and Disease Simon P. Hogan, Helene F. Rosenberg, Redwan Moqbel, Simon Phipps, Paul S. Foster, Paige Lacy, A. Barry Kay and Marc E. Rothenberg

Summary Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the emerging properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil-mediated mucosal diseases are conceptualized.

Introduction Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory processes including parasitic helminth, bacterial and viral infections, tissue injury, tumor immunity, and allergic diseases (Gleich & Loegering 1984; Weller 1994; Rothenberg 1998). In response to diverse stimuli, eosinophils are recruited from the circulation into inflammatory foci where they modulate immune responses through an array of mechanisms. Triggering of eosinophils by engagement of receptors for cytokines, immunoglobulins, and complement can lead to the secretion of an array of proinflammatory cytokines, such as interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-16, IL-18, and transforming growth factor (TGF)-α/β, chemokines such as CCL5/RANTES and

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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CCL11/eotaxin-1, and lipid mediators such as plateletactivating factor (PAF) and leukotriene (LT)C4 (Kita 1996). These molecules have proinflammatory effects that include upregulation of adhesion systems, modulation of cellular trafficking and activation and regulation of vascular permeability, mucus secretion, and smooth muscle constriction. Eosinophils can initiate antigen-specific immune responses by acting as antigen-presenting cells. Furthermore, eosinophils can serve as major effector cells inducing tissue damage and dysfunction by releasing toxic granule proteins and lipid mediators (Gleich & Adolphson 1986). In this chapter, we summarize eosinophil surface marker expression and the growing number of properties defined for eosinophil degranulation. We review the molecular mechanisms involved in eosinophil development and trafficking, including the role of the transcription factors GATA-1, PU.1, and c/EBP members and the eosinophil selective cytokine IL-5 and the eotaxin subfamily of chemokines. Furthermore, we discuss the views on the role of eosinophils in homeostatic function, including developmental biology and innate and adaptive immunity, and in disease processes including infections, asthma, and gastrointestinal disorders.

Eosinophil granule proteins Eosinophils contain up to four different populations of secretory organelles: crystalloid granules, primary granules, small granules, and secretory vesicles. The largest of the secretory organelles are the crystalloid granules (0.5–0.8 μm in diameter), which store the majority of granule proteins in eosinophils. The unique crystalloid granules are so called because they contain an intensely staining electron-dense crystalline core surrounded by an electron-lucent matrix when cells are stained and imaged by electron microscopy. Most of the granule proteins packaged into the crystalloid granules are composed of four highly basic proteins. Major basic protein (MBP) is crystallized in the core of the crystalloid granule, where it accounts for virtually all the protein (Gleich et al. 1973; Lewis et al. 1978). Eosinophil peroxidase (EPO),

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eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) reside in the granule matrix (Egesten et al. 1986; Peters et al. 1986). The primary granules appear during the promyelocytic stage of eosinophil development and are enriched in Charcot–Leyden crystal (CLC) protein. Small secretory vesicles have also been identified that overlap in their contents with those of small granules, and are packed densely in the cytoplasm of eosinophils. The biology of the major cationic proteins in eosinophils has been reviewed in

detail (Gleich & Adolphson 1986; Walsh 2001); their functions are summarized in Table 12.1.

Table 12.1 Functions of eosinophil cationic granule proteins. MBP Toxicity toward helminthic parasites such as schistosomulae of Schistosoma mansoni (Butterworth 1984; Ackerman et al. 1985; Gleich & Adolphson 1986) Cytotoxicity toward airway epithelium (Frigas et al. 1980; Hastie et al. 1987; Hisamatsu et al. 1990; Furuta et al. 2005) Bronchoconstriction and hyperresponsiveness on aerosolization in rats and monkeys (Gundel et al. 1991; Coyle et al. 1993; Uchida et al. 1993) Platelet agonist (Rohrbach et al. 1990) Activation of complement via classical and alternative pathways (Weiler et al. 1992, 1995) Antibacterial properties (Lehrer et al. 1989) Activation of remodeling factors from epithelial cells (Pégorier et al. 2006) Increased cutaneous vasopermeability (Davis et al. 2003) Stimulation of signaling pathways and mediator release from mast cells, neutrophils and basophils (Zheutlin et al. 1984; Haskell et al. 1995; Page et al. 1999; Shenoy et al. 2003) ECP Ribonuclease activity (100 times less potent than EDN) (Slifman et al. 1986) Toxicity toward helminthic parasites and mammalian epithelial cells (McLaren et al. 1981; Ackerman et al. 1985) Bactericidal properties (Lehrer et al. 1989) Induction of Gordon phenomenon (Durack et al. 1979; Fredens et al. 1982) Promotion of mast cell degranulation (Zheutlin et al. 1984) EDN Weakly toxic for parasites and mammalian cells (Ackerman et al. 1985) Induction of Gordon phenomenon (Durack et al. 1979) Antiviral activity in respiratory infection (Rosenberg & Domachowske 2001) EPO Toxic for mammalian cells and degradative toward connective tissue via ability to form hypohalous acids (Slungaard & Mahoney 1991; Wang & Slungaard 2006) Cytotoxicity toward airway epithelium (Brottman et al. 1996; Pégorier et al. 2006) Bactericidal, membrane lysis, and signaling pathway effects (Wang & Slungaard 2006) Induction of oxidative damage and mutagenesis of DNA and RNA (Shen et al. 2000; Henderson et al. 2001)

Major basic protein As one of the most highly cationic proteins synthesized by eosinophils, MBP is expressed as two different homologs (MBP1 and MBP2). MBP is a small protein that consists of a single polypeptide chain of 117 amino acids, with a molecular mass of 13.8 kDa and a high isoelectric point (>11), which cannot be measured accurately due to its extremely basic nature (Hamann et al. 1991). Its basicity is due to the presence of 17 arginine residues, and it also contains nine cysteine residues that enable it to form disulfide bonds. The cDNA for MBP encodes a pre-prosequence that includes a putative signal peptide and an acidic 90-amino acid prosequence that may serve to neutralize MBP’s highly basic charge as it is processed through the Golgi and transported to the granule, where the prosequence is cleaved (Barker et al. 1988; McGrogan et al. 1988; Popken-Harris et al. 1998). MBP is among the most abundant proteins in eosinophils, with as much as 250 pg/cell detected in guinea-pig eosinophils, while comparatively less is found in human eosinophils (5–10 pg/cell). MBP1 can also be detected in basophil granules, although there is considerably less expressed than in eosinophils (Ackerman et al. 1983). Mature eosinophils lose the ability to transcribe mRNA encoding MBP, indicating that all the MBP stored in crystalloid granules is synthesized during early eosinophil development prior to maturation (Popken-Harris et al. 1998). Plasma concentrations of MBP are elevated in the sera of pregnant women, with a peak 2–3 weeks before parturition. Placental eosinophils are few in numbers, and MBP1 has been shown to be synthesized by placental X cells and placentalsite giant cells (Maddox et al. 1984). MBP2 is exclusively expressed by eosinophils, and may be a more specific marker for elevated eosinophils in patients with eosinophilia than MBP1 (Plager et al. 2006). The classical role of eosinophils in protection against parasitic infections has been supported by the toxicity of MBP against helminthic worms (O’Donnell et al. 1983; Butterworth 1984; Ackerman et al. 1985; Gleich & Adolphson 1986). MBP has also been shown to be cytotoxic to airways and may be at least partly responsible for tissue damage associated with eosinophil infiltration in bronchial mucosa in asthma (Frigas et al. 1980; Hisamatsu et al. 1990; Furuta et al. 2005). The toxic effect of MBP is thought to result from increased membrane permeability through surface charge interactions leading to perturbation of the cell-surface lipid bilayer (Wasmoen et al. 1988).

Eosinophil cationic protein ECP is a member of a subfamily of ribonuclease (RNase)A multigenes expressed in eosinophils, with approximately 15–25 pg synthesized per cell in human eosinophils. Similarly to MBP, ECP is a single-chain cationic polypeptide with

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a pI > 11. On molecular sizing, ECP displays marked heterogeneity, as a result of differential glycosylation, with a molecular mass ranging between 16 and 21.4 kDa. Two isoforms, ECP-1 and ECP-2, have been identified using heparin Sepharose chromatography (Gleich et al. 1986). The cDNA for ECP encodes a leader sequence of 27 amino acids and a mature protein of 133 amino acids with a calculated molecular mass of 15.6 kDa (Barker et al. 1989; Rosenberg et al. 1989). The amino acid sequence is 66% homologous to EDN and 31% homologous to human pancreatic ribonuclease. ECP does have ribonuclease activity but is 100 times less potent than EDN (Slifman et al. 1986). ECP is bactericidal, promotes degranulation from mast cells, and is toxic to helminthic parasites on its own (Gleich et al. 1986; Lehrer et al. 1989). The mechanism of action of ECP is thought to involve pore formation in target membranes, which is apparently not dependent on its RNase activity (Young et al. 1986). ECP was originally characterized for its ability to elicit the Gordon phenomenon (neurotoxicity causing stiffness, ataxia and paralysis) when injected into the cranial ventricles of rabbits (Durack et al. 1979).

Eosinophil-derived neurotoxin A second member of the RNase A multigene family, EDN, also called EPX (Slifman et al. 1986), is expressed in eosinophils and is less basic than ECP or MBP, with a pI of 8.9 due to a smaller number of arginine residues in its sequence. EDN is a single-chain polypeptide with an observed molecular mass of 18.6 kDa. EDN expression is not restricted to eosinophils, as it is also detected in mononuclear cells and possibly neutrophils. ECP and EDN share high sequence homology of 70% at the amino acid level for the pre-form of both proteins, suggesting that these proteins derived from the same gene during evolutionary development (Hamann et al. 1990). Eosinophils express approximately 10 pg of EDN per cell, with marked variation between individuals. EDN similarly induces the Gordon phenomenon when injected intracranially in laboratory animals (Durack et al. 1979). EDN is also implicated in antiviral activity against respiratory infections (Rosenberg & Domachowske 2001). The gene family expressing ECP and EDN has one of the highest rates of mutation in the primate genome, ranking with those of immunoglobulins, T-cell receptors, and major histocompatibility complex (MHC) classes (Rosenberg et al. 1995). This extreme rate of mutation suggests that evolutionary constraints acting on the ECP/EDN subfamily have promoted the acquisition of a specialized antiviral activity, inferred by the high mutation rates of other genes commonly associated with host protection against viral infection.

neutrophil myeloperoxidase, suggesting that a peroxidase multigene family may have developed through gene duplication (Ten et al. 1989; Hamann et al. 1991). Eosinophils store approximately 15 pg/cell of EPO. The functional role of EPO is associated with bacterial killing. EPO catalyzes the peroxidative oxidation of halides (such as bromide, chloride, and iodide) and pseudohalides (thiocyanate) present in the plasma together with hydrogen peroxide generated by dismutation of superoxide produced during respiratory burst (Weiss et al. 1986; Mayeno et al. 1989; Thomas et al. 1995). This reaction leads to the formation of bactericidal hypohalous acids, particularly hypobromous acid, under physiologic conditions. Eosinophils are robust producers of extracellular superoxide due to expression of high levels of the enzyme complex that generates superoxide (NADPH oxidase) (Someya et al. 1997) and preferential assembly of the enzyme complex at the cell surface (Lacy et al. 2003).

Cytokines Eosinophils can synthesize and secrete at least 35 important inflammatory and regulatory cytokines, chemokines, and growth factors (Table 12.2). Many of these cytokines are potent inducers of immune responses in asthma, eczema, rhinitis, and other inflammatory diseases. Those eosinophilderived cytokines that have been quantified generally appear to be generated in relatively small amounts, suggesting an autocrine, paracrine, or juxtacrine role in regulating the function of the microenvironment. However, in some circumstances, eosinophils are the chief producers of cytokines such as TGF-β, which is linked with tissue remodeling in a variety of eosinophil-associated diseases, such as asthma (Kay et al. 2004). A major distinction in cytokine production between eosinophils and T cells, which generate much larger quantities of cytokines, is that eosinophils store their cytokines intracellularly as preformed mediators. Several eosinophil cytokines have been shown to be stored in crystalloid granules and small secretory vesicles, and possess bioactivity on their release (Lacy & Moqbel 2000). This allows the immediate release of cytokines on eosinophil activation, instead of the several hours or days required to generate cytokines from T cells. For example, release of the chemokine RANTES was shown to occur within 60–120 min of eosinophil stimulation by interferon (IFN)-γ. This was related to rapid mobilization (within 10 min) of RANTES in small secretory vesicles that translocated this chemokine to the cell membrane prior to its release (Fig. 12.1). Cytokines generated by eosinophils are discussed in more detail in several comprehensive reviews (Lacy & Moqbel 1997, 2000; Moqbel & Lacy 1998).

Other eosinophil-derived mediators Eosinophil peroxidase EPO is a heme-containing haloperoxidase with a high pI (> 11) composed of two subunits: a heavy chain of 50–57 kDa and a light chain of 11–15 kDa. EPO has 68% sequence identity to

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A major constituent of the human eosinophil is CLC protein, also known as galectin-10 (Ackerman et al. 2002). CLC is a hydrophobic protein of molecular mass 17.4 kDa that was thought to possess a weak lysophospholipase activity,

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Table 12.2 Cytokine generation by eosinophils.

Cytokine

Products

Stored protein in resting cells (/106 cells)

Interleukins Interleukin-1a Interleukin-2 Interleukin-3 Interleukin-4 Interleukin-5

mRNA, protein mRNA, protein mRNA, protein mRNA, protein mRNA, protein

– 6 ± 2 pg – ∼75 ± 20 pg –

mRNA, protein mRNA, protein mRNA, protein mRNA mRNA, protein mRNA, protein mRNA, protein

25 ± 6 pg – ∼25 pg – – – 1.6 ± 0.8 ng

– Crystalloid granules (core) – Crystalloid granules (core) Crystalloid granules (core/matrix?) Crystalloid granules (matrix) – – – – – –

mRNA, protein

–

–

mRNA, protein mRNA, protein mRNA, protein

– – 15.1 ± 0.3 pg

– Crystalloid granules (matrix) Crystalloid granules (core)

mRNA, protein

12 ± 2 pg

–

mRNA, protein mRNA, protein mRNA, protein mRNA, protein mRNA

19 ± 4 pg – 140 pg – –

Crystalloid granules – Cytoplasmic – –

mRNA, protein Protein

– –

– –

mRNA, protein mRNA mRNA mRNA, protein

– – – 72 ± 15 pg

– – – Crystalloid granules (matrix) and small secretory vesicles

mRNA

–

–

mRNA, protein mRNA mRNA, protein mRNA, protein

4 ± 2 pg – – 22 ± 6 pg

mRNA, protein

–

– – Membrane, cytoplasm Crystalloid granules (matrix) and small secretory vesicles –

Interleukin-6 Interleukin-9 Interleukin-10 Interleukin-11 Interleukin-12 Interleukin-13 Interleukin-16 Interleukin-17 Leukemia inhibitory factor Interferons and others Interferon (IFN)-g Tumor necrosis factor a Granulocyte–macrophage colony-stimulating factor Chemokines Epithelial cell-derived neutrophil activating peptide (ENA-78/CXCL5) Eotaxin (CCL11) Growth-related oncogene (GROa/CXCL1) Interleukin-8 (CXCL8) IFN-g-inducible protein (IP-10/CXCL10) IFN-inducible T-cell alpha chemoattractant (I-TAC/CXCL11) Macrophage inflammatory protein 1a Monocyte chemoattractant protein 1 (MCP-1/CCL3) Monokine induced by IFN-g (MIG/CXCL9) MCP-3 (CCL7) MCP-4 (CCL13) RANTES (CCL5)

Growth factors Heparin-binding epidermal growth factor-like binding protein (HB-EGF-LBP) Nerve growth factor Platelet-derived growth factor, B chain Stem cell factor Transforming growth factor a Transforming growth factor b1

Intracellular site of storage

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Fig. 12.1 Human eosinophils stimulated with interferon (IFN)-g to induce rapid release of RANTES. Cells were stained with antibody to major basic protein (MBP) (red) and antibody to RANTES (green) in a time course study of RANTES mobilization. Yellow indicates colocalization of MBP and RANTES to similar granule compartments, which become distinct as early as 10 min following stimulation with IFN-g (500 U/mL). Unstimulated cells (a) were compared with cells stimulated for (b) 5 min, (c) 10 min, (d) 30 min, (e) 60 min, and (f) 16 hours. Original magnification ×1000. (See CD-ROM for color version.)

but instead modulates this by interacting with eosinophil lysophospholipases. It is synthesized at very high levels by eosinophils, and is produced at lesser levels in basophils. CLC possesses strong sequence homology to the carbohydratebinding galectin family of proteins, hence its designation as galectin-10. CLC was first characterized by Charcot and Robin in 1853 for its abundance in sputum and fecal samples from patients with severe respiratory and gastrointestinal eosinophilia. Its release results in the formation of distinct, needle-shaped structures that are colorless, measuring 20– 40 μm in length and 2– 4 μm across. However, the function of CLC remains obscure. In addition, the eosinophil contains a number of other granule-stored enzymes whose exact role in eosinophil function has not been defined (Spry 1988). They include acid phosphatase (large amounts of which have been isolated from eosinophils), collagenase, arylsulfatase B, histaminase, phospholipase D, catalase, nonspecific esterases, and vitamin B12-binding protein. Eosinophils are also a source of matrix metalloproteases, which have an important role in cell transmigration and inflammation (Ohno et al. 1997; Okada et al. 1997; Schwingshackl et al. 1999; Gauthier et al. 2003; Wiehler et al. 2004), although much less is produced than from monocytes, macrophages, and neutrophils. The intracellular location of matrix metalloprotease-9 has been localized to perinuclear regions and not the crystalloid granules (Ohno et al. 1997).

Eosinophils express SNARE isoforms Secretory cells from diverse biological systems express components of a fusion complex of membrane-bound proteins known as the SNARE (SNAP Receptor) complex, which is essential for vesicular docking and fusion (Sollner et al. 1993; Sutton et al. 1998). This complex, originally characterized in

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neuronal cells, is composed of VAMP-1 (vesicle-associated membrane protein, also known as synaptobrevin-1), syntaxin1, and SNAP-25 (synaptosome-associated protein of 25 kDa). These molecules are categorized into two groups, namely vesicular SNAREs (v-SNAREs), which bind to plasma membrane target SNAREs (t-SNAREs). The SNARE molecules form a coiled-coil structure with four separate α-helices contributed by three different molecules during vesicle docking with the plasma membrane. The binding region associated with the four α-helices is known as the SNARE motif. Fusion of the granule membrane with the plasma membrane is dependent on cytosolic NSF (N-ethylmaleimide-sensitive factor) and α, β, or γ-SNAP (soluble NSF-attachment protein)mediated disassembly of the SNARE complex (Sollner et al. 1993). Cleavage of SNAREs can occur via clostridial neurotoxins containing zinc endopeptidase activity, particularly tetanus toxin and botulinum toxin serotypes (BoNT/A, B, C, D, E, F, and G). These toxins have been used to characterize the dependency of secretion on SNARE complex formation. Many SNARE proteins have also been identified in nonneuronal secretory cells including syntaxin-4 and SNAP-23 (Ravichandran et al. 1996), while VAMP-2 expression is distributed between neuronal and nonneuronal tissues (Rossetto et al. 1996). In addition, VAMP-4 (Steegmaier et al. 1999), VAMP-5 (Zeng et al. 1998), the tetanus toxin-insensitive proteins VAMP-7 (formerly known as tetanus toxin-insensitive VAMP or TI-VAMP) (Galli et al. 1998; Advani et al. 1999; Hibi et al. 2000; Ward et al. 2000) and VAMP-8 have been characterized in nonneuronal tissues (Mullock et al. 2000; Paumet et al. 2000; Polgar et al. 2002). Eosinophils express VAMP-2, VAMP-7, VAMP-8, syntaxin4, and SNAP-23 (Feng et al. 2001; Lacy et al. 2001; Logan et al. 2002, 2003, 2006), whereas they do not contain detectable levels of the classical neuronal SNARE proteins (syntaxin-1,

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(b)

Fig. 12.2 Translocation of VAMP-2 during interferon (IFN)-g stimulation of human eosinophils. Immunolabeling for VAMP-2 (green) showed moderate colocalization with RANTES (red) in (a) unstimulated eosinophils, but these were strongly colocalized at the cell membrane (arrow) following 5 min of stimulation with IFN-g 500 U/mL (b). Original magnification ×1000. (See CD-ROM for color version.)

SNAP-25, and VAMP-1) (Lacy et al. 1995). Eosinophil VAMP-2 is expressed in a population of small secretory vesicles that store the chemokine RANTES, which translocates to the cell membrane during IFN-γ stimulation (Fig. 12.2) (Lacy et al. 1999, 2001). Immunofluorescence staining showed that syntaxin-4 and SNAP-23 were localized to the cell membrane in eosinophils, where they may function as cognate intracellular receptors for VAMP-2 (Fig. 12.3) (Logan et al. 2002). Inhibition of VAMP-2 binding led to the loss of IgE-induced ECP release in permeabilized eosinophils (Hoffmann et al. 2001). A novel isoform of VAMP, known as tetanus-insensitive VAMP (TI-VAMP, also known as VAMP-7), is a putative vesicular SNARE isoform for regulation of lysosomal fusion (Advani et al. 1999; Ward et al. 2000; Rao et al. 2004). VAMP7 and VAMP-8 are abundantly expressed in eosinophil crystalloid granules. VAMP-7 has been shown to be required for

Fig. 12.3 Localization of SNAP-23 and syntaxin-4 in eosinophils. (Left panel ) Distribution of SNAP-23 at cell membranes (arrow) as well as at an intracellular site that colocalizes with the Golgi apparatus (arrowhead). (Right panel) Antibody to syntaxin-4 shows expression in the cell membrane (arrow) and endoplasmic reticulum (arrowhead). Lower panels indicate differential interference contrast images. Original magnification ×630. (See CD-ROM for color version.)

exocytosis of crystalloid granule as well as small secretory vesicles (Logan et al. 2006). This finding suggests that VAMP2 and VAMP-7 may play overlapping roles in the release of small secretory vesicles from eosinophils, although their release may be more dependent on VAMP-7 (Fig. 12.4). In

Resting

Stimulated

Plasma membrane VAMP-7

Fig. 12.4 Scheme showing SNARE-dependent exocytotic pathways for crystalloid granules and secretory vesicles in eosinophils. (See CD-ROM for color version.)

Syntaxin-4

SNAP-23 VAMP-2 or VAMP-7

SNARE complex

EPO, EDN, RANTES

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summary, SNARE isoforms may play a crucial role in the regulation of granule fusion in eosinophils.

Eosinophil surface markers The first characterization of eosinophil surface molecules demonstrated that eosinophils express a large number of cell-surface markers including adhesion molecules, apoptotic signaling molecules, chemokine, complement and chemotactic factor receptors, cytokine receptors, and immunoglobulin receptors (Gupta et al. 1976; Ebisawa et al. 1995). Since these studies and the discovery of new immune receptors (Toll-like receptors, inhibitory receptors and Siglecs) and development of new reagents, the list has been extended revealing that eosinophils express an array of surface structures that were previously thought to be exclusively expressed by other cell types (Tachimoto & Bochner 2000; Rothenberg & Hogan 2006).

Adhesion molecules Transmigration of the eosinophil through the vascular endothelium is a multistep process involving rolling, tethering, firm adhesion, and transendothelial migration (Wardlaw et al. 1994; Wardlaw 2000). The initial steps of eosinophil rolling and tethering are regulated by selectins and their counterligands expressed on the endothelium (Ebnet et al. 1996; Wardlaw 1999). Eosinophils have been shown to constitutively express L-selectin, which regulates eosinophil rolling on the endothelium in vivo (Georas et al. 1992; Sriramarao et al. 1994). Ligands for L-selectin include CD34 and MAdCAM-1 which are expressed endothelium (Berg et al. 1993). Eosinophils also express CD162 (P-selectin glycoprotein ligand-1 or PSGL-1) and sialyl-Lewis X (CD15s), which interact with Eselectin and P-selectins and regulate eosinophil tethering to endothelium (Symon et al. 1996). The firm adhesion of the eosinophil and transmigration across the vascular epithelium into tissues is regulated by coordinated interaction between networks involving chemokine and cytokine signaling, eosinophil adhesion molecules (e.g., selectins and integrins), and integrin receptors such as vascular cell adhesion molecule (VCAM)-1, mucosal addressin cell adhesion molecule (MAdCAM)-1 and intercellular adhesion molecule (ICAM)-1 expressed on vascular endothelial cells (Kunkel & Butcher 2002; Hogan et al. 2004). Integrins are heterodimeric surface molecules consisting of an α and β chain and eosinophils express members of the β1 (α4β1 and α6β1), β2 (αLβ2, αMβ2, αXβ2, and αDβ2) and β7 (α4β7) integrin families (Georas et al. 1993; Grayson et al. 1998; Tachimoto & Bochner 2000; Bochner & Schleimer 2001; Tachimoto et al. 2002). These various integrin molecules selectively interact with adhesion receptors (VCAM-1, MAdCAM-1, ICAM-1, -2 and -3, and fibrinogen) expressed on the vascular endothelium. The specific interaction of cell-surface integrins with ad-

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hesion receptors (VCAM-1, MAdCAM-1, ICAM-1, ICAM-2, ICAM-3, and fibrinogen) facilitates eosinophil migration into various tissue compartments during inflammation. For example, eosinophil recruitment to the site of allergic inflammation in the lung and skin is regulated by VLA-4 (α4β1 integrin)/VCAM-1-dependent processes (Weg et al. 1993; Abraham et al. 1994; Nakajima et al. 1994; Pretolani et al. 1994; Gonzalo et al. 1996). Pretreatment of mice with neutralizing monoclonal antibodies against α4 or β1 integrin or genetic deletion of VCAM-1 attenuates eosinophil accumulation in the lung during allergic airways disease (Weg et al. 1993; Abraham et al. 1994; Nakajima et al. 1994; Pretolani et al. 1994; Gonzalo et al. 1996). Recent experimental studies have demonstrated that eosinophil recruitment into different tissue compartments (gastrointestinal tract) is regulated by differential adhesion pathways. For example, eotaxin-1dependent eosinophil recruitment to the small intestine is MAdCAM-1/α4β7 integrin dependent (Mishra et al. 2002), whereas eosinophil accumulation in the colon is regulated by a β2 integrin pathway (ICAM-1) and can occur independently of α4 and β7 integrin pathways (Forbes et al. 2006).

Chemokine, complement and other chemotactic factor receptors Experimental investigations have shown eosinophils to constitutively express the chemokine receptors CCR3 and CCR1 (Ponath et al. 1996; Phillips et al. 2003; Elsner et al. 2005). Consistent with this observation, eosinophils respond to CCR1 and CCR3 ligands including macrophage inflammatory protein (MIP)-1α/CCL3, RANTES/CCL5, macrophage chemotactic protein (MCP)-2/CCL8, MCP-3/CCL7 and MCP-4/CCL-13, eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26, and mucosa-associated epithelial chemokine (MEC)/CCL28. Eosinophils have also been shown to express a number of other chemokine receptors including CXCR3, CXCR4, CCR5, CCR6, and CCR8 following activation by IL-5 (Sullivan et al. 1999; Nagase et al. 2000; Oliveira et al. 2002). While chemokines are thought primarily to regulate the migration pattern of eosinophils, they have also been shown to promote eosinophil activation and function (Zimmermann et al. 2003). For example, RANTES/CCL5 and eotaxin-1/CCL11 have been shown to promote cellular activation and modulate respiratory burst in eosinophils (Elsner et al. 1997, 1999).

Cytokine receptors Three cytokines, IL-3, IL-5, and granulocyte–macrophage colony-stimulating factor (GM-CSF), are particularly important in regulating eosinophil development, and eosinophils have been shown to express the specific cytokine receptor subunit for IL-3 (IL-3Rα, CD123), IL-5 (IL-5Rα, CD125) and GM-CSF (GM-CSFRα, CD116) as well as the shared β chain (CD131) (Lopez et al. 1986, 1988; Rothenberg et al. 1988; Takatsu et al. 1994). Functional studies have demonstrated that cytokines including stem cell factor (SCF), IFN-γ, tumor

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necrosis factor (TNF)-α, IL-4, and IL-9 activate eosinophil functions, suggesting that eosinophils express the c-kit receptor (CD117), IFN-γR α-chain (CDw119), TNF-α receptor types 1 and 2 (CD120a, CD120b), type 1 IL-4 receptor [IL-4R α-chain (CD124) and the common γ chain (CD132)], and the IL-9 receptor [IL-9R α-chain (CD129)/CD132] (Wallen et al. 1991; Yuan et al. 1997; Dubois et al. 1998; Nutku et al. 1999; Hauber et al. 2004). Consistent with these obervations, both the type 1 (CD120a) and type 2 (CD120b) TNF receptors have been identified on human eosinophils by fluorescence-activated cells sorting analysis and immune electron microscopy. Activation of these receptors are thought to promote eosinophil apoptosis (Zeck-Kapp et al. 1994; Zeck-Kapp & Kapp 1995). While eosinophils have been shown to express a number of IFN receptor superfamily members, including receptors for IFN-α, IFN-β, IFN-γ, and IL-10 (Giembycz & Lindsay 1999), only the receptor for IFN-γ (type 2 IFN) has been convincingly identified (Aldebert et al. 1996; Ishihara et al. 1997; Matsuyama et al. 1998; Ochiai et al. 1999). The IL-2 receptor is composed of three polypeptide chains, an α chain (p55) (CD25), β chain (p75) (CD122), and a γ chain (CD132) that is common to several other cytokine receptors. Experimental investigations have demonstrated that IL-2 induces eosinophil chemotaxis, suggesting that the cognate receptor for the cytokine was expressed on the eosinophils. This was confirmed by the observation that the IL-2-mediated effects could be blocked by antibodies against IL-2R α chain (p55) and β chain (p75) (Rand et al. 1991).

phils and immature eosinophil progenitors express CysLT1R, whereas CysLT2R has only been identified on mature eosinophils. Expression of these receptors has been shown to be upregulated on eosinophils from asthmatics during excerbations (Fujii et al. 2005). Notably, CysLT2R expression on eosinophils was selectively greater in nonatopic asthmatics (Fujii et al. 2005). The function of these receptors on eosinophils has not yet been fully defined. However, leukotrienes (LTB4, LTD4, LTE4), PAF, and 5-oxo-6,8,11,14-eicosatetraenoic acid induce eosinophil recruitment, suggesting that they may regulate eosinophil transmigration (Powell et al. 1995; Bandeira-Melo et al. 2000; Ohshima et al. 2002; Shiraishi et al. 2005). Previous reports have also demonstrated that in vitro suppression of cysteinyl leukotriene activity by a CysLT1R antagonist blocks eosinophil differentiation and/or maturation, suggesting that cysteinyl leukotrienes may play a role in eosinophil lineage commitment and maturation (Thivierge et al. 2000). Cysteinyl leukotrienes have also been shown to promote eosinophil release of cytokines including IL-4 (Bandeira-Melo et al. 2002a,b). Eosinophils have also been shown to express high levels of the histamine H4 receptor that mediate eosinophil chemoattraction and activation in vitro (O’Reilly et al. 2002).

Complement receptors Initial studies suggested that eosinophils express complement receptors for C3a and C5a (Daffern et al. 1995; DiScipio et al. 1999); however, more recent studies have revealed that eosinophils also express CR1 (CD35), CR3 (CD11b/CD18), CR4 (CD11c), CD103, and receptors for C1q (Walsh et al. 1990; Giembycz & Lindsay 1999). CR1 is recognized by the complement fragments C3b, C4b, iC3b, and C1q. The expression of CR1 on eosinophils is regulated by certain stimuli, including LTB4, 5-HETE, and 5-HPETE (Fischer et al. 1986). CR3 has also been shown to be expressed on eosinophils: CR3 interacts with a number of ligands including iC3b and ICAM-1, all of which could activate eosinophils resulting in eosinophil priming and degranulation (Koenderman et al. 1991).

Prostaglandin and leukotriene receptors Clinical and experimental studies have demonstrated that eosinophils express both cysteinyl leukotriene receptors (CysLT1R and CysLT2R), high-affinity prostaglandin (PG)D2 type 2 receptor, and the PAF receptor (Wang et al. 1999; Fujii et al. 2005; Zinchuk et al. 2005). Interestingly, the PGE2 receptor is also expressed by basophils and Th2 cells (and is now designated “chemoattractant receptor Th2 cells” or CRTH2) and appears to co-mediate Th2 cell and eosinophil/ basophil recruitment (Hirai et al. 2001). Both mature eosino-

Immunoglobulin receptors Eosinophils express Fc receptors for IgA, IgD, IgG, and IgM (Giembycz & Lindsay 1999). CD32 (FcγRII) is constitutively expressed on resting human eosinophils (Hartnell et al. 1990) and is upregulated by IFN-γ (Hartnell et al. 1992). These receptors not only function as IgG receptors but also appear to have a role in stimulating eosinophil survival, degranulation, and generation of leukotrienes (Cromwell et al. 1988, 1990; Kita et al. 1991; Kim et al. 1999). Eosinophils do not constitutively express FcγRI (CD64) or low-affinity FcγRIII (CD16), although expression can be upregulated by cytokines, IFN-γ, complement (C5a), and PAF (Hartnell et al. 1992). Eosinophils do appear to express IgA receptors (CD89) (Monteiro et al. 1993) and exvivo studies have demonstrated that eosinophil degranulation can be induced by IgA-coated particles, suggesting that IgA–receptor interaction induces eosinophil degranulation (Abu Ghazaleh et al. 1989). The expression or presence of the low-affinity IgE receptor (CD23) or the high-affinity IgE receptor on eosinophils remains controversial (Kita & Gleich 1997). Some studies suggest that eosinophils bind to IgE (Capron et al. 1985, 1995), although more recent investigations suggest that eosinophils express little if any α or β chains for the high-affinity receptor or the low-affinity CD23 IgE receptor (Ying et al. 1998; Kita et al. 1999; Seminario et al. 1999).

Other cell-surface structures Eosinophil apoptosis has been shown to be induced by two surface structures, CD95 (first) and CD69 (Matsumoto et al. 1995; Walsh et al. 1996). Eosinophils also express CD9,

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CD37, CD52, CD63, CD81, CD82, and CD151 (Ebisawa et al. 1995). Experimental investigations have demonstrated that eosinophils can express antigen to naive CD4+ T cells and promote T-cell proliferation and polarization (Shi et al. 2000, 2004; Shi 2004). Consistent with this observation, eosinophils have also been shown to express MHC class II and the necessary costimulatory signals for T-cell activation and proliferation, including the type 1 interval surface membrane glycoprotein CD40 as well as CD80 and CD86 (Ohkawara et al. 1996; Woerly et al. 1999). The expression of CD80 and CD86 appears to be independently regulated by IL-3 and GM-CSF (Tamura et al. 1996). Human eosinophils have also been shown to express the MHC class II protein human leukocyte antigen (HLA)-DR (Shi 2004). Interestingly, peripheral eosinophils of most normal eosinophilic donors do not express HLA-DR proteins, although sputum eosinophils and bronchoalveolar lavage (BAL) eosinophils from asthmatics have been shown to express HLA-DR (Hansel et al. 1991; Sedgwick et al. 1992). Notably, levels of HLA-DR expression on BAL eosinophils from allergic subjects following segmental challenge were elevated compared with peripheral blood eosinophils, suggesting that recruitment and activation of eosinophils following allergen challenge promotes HLA-DR expression (Sedgwick et al. 1992). HLA-DR expression on eosinophils has been shown to be regulated by IL-3, IL-4, GM-CSF, and IFN-γ (Lucey et al. 1989; Weller et al. 1993).

Inhibitory receptors The CD2 subfamily of the IgE superfamily includes CD2, CD48 (BLAST1) and BTM1 (CD58), LFA3, CD84, IL-9b, CD150, CD229, and 2B4 (CD244). Recent studies have demonstrated that eosinophils express the CD2 subfamily of receptors, namely CD48 and 2B4 (CD244) (Munitz et al. 2005). CD48 is a glycosylphosphatidylinositol (GPI)-anchored protein involved in cellular activation, costimulation, and adhesion. CD48 expression is elevated on human eosinophils from atopic asthmatics and is upregulated by IL-3 (Munitz et al. 2006). Cross-linking of CD48 on eosinophils triggers eosinophil degranulation (Munitz et al. 2005, 2006). Notably, CD48 is the high-affinity ligand for 2B4. Eosinophils also express inhibitory receptor IRp60/CD300a, p140, Siglec-8, Siglec-10, ILT5/LIR3, CD33, and p75/adhesion inhibitory receptor molecules (Munitz & Levi-Schaffer 2007). IRp60 activation has been shown to be involved in the suppression of eosinophil activation (Munitz et al. 2005). Interestingly, CCR3 has been shown to induce negative signaling in murine eosinophils following receptor engagement with the Th1 chemokine CXCL9 (MIg) (Fulkerson et al. 2005).

Toll-like receptors Eosinophils express mRNA for a number of Toll-like receptors (TLR) including TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 (Plotz et al. 2001; Sabroe et al. 2002; Nagase et al. 2003). The level of TLR expression on eosinophils is low

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relative to other granulocytes such as neutrophils, except for relatively elevated levels of TLR7/TLR8 (Nagase et al. 2003). The natural ligands for TLR7/TLR8 are currently not clear, although significant evidence suggests that guanosine- and uridine-rch ssRNA are physiologic ligands for these TLRs (Heil et al. 2004). Functional analysis using TLR-specific ligands reveals that TLR7/TLR8 ligands (R-848) induced eosinophil activation (superoxide production) and prolonged eosinophil survival. The expression of TLR7/TLR8 has been shown to be regulated by cytokines including IFN-γ (Nagase et al. 2003).

Eosinophils and immune regulation In recent years, eosinophils have been shown to possess the ability to perform numerous immune functions, including antigen presentation (Shi et al. 2000; MacKenzie et al. 2001) and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators (Gleich & Adolphson 1986; Weller 1994).

Thymic eosinophils Eosinophils transmigrate into the thymus during the neonatal period, reaching maximum levels by 2 weeks of age. Interestingly, their absolute levels are approximately equivalent to that of thymic dendritic cells (Throsby et al. 2000). Eosinophils primarily localize to the corticomedullary region of the thymus and reach basal levels by 28 days of age. Subsequently, an increase in thymic eosinophil levels at 16 weeks of age corresponds to the commencement of thymic involution. Notably, eosinophils at this stage localize to the medullary region. Thymic eosinophils express high levels of MHC class II molecules and moderate levels of MHC class I and the costimulatory molecules CD86 (B7.2) and CD30L (CD153) (Fig. 12.5). Furthermore, thymic eosinophils are CD11b/CD11c double-positive and appear to be activated, as they lose expression of GL-1 and CD62L and upregulate CD25 and CD69 surface expression. Analysis of thymic eosinophil cytokine production reveals that eosinophils express mRNA for the proinflammatory cytokines TNF-α, TGF-β, IL-1α and IL-6 and the Th2 cytokines IL-4 and IL-13 (Throsby et al. 2000). Notably, the recruitment of eosinophils into the thymus is regulated by eotaxin-1, which is constitutively expressed in the thymus (Matthews et al. 1998). It has been postulated that eosinophils are associated with MHC class I-restricted thymocyte deletion. Consistent with this notion, the biphasic recruitment of eosinophils and their anatomic localization within discrete compartments of the thymus coincides with negative selection of double-positive thymocytes (Throsby et al. 2000). Employing an experimental model of acute negative selection, increased thymic eosinophil levels have been demonstrated in MHC class I-restricted female H-Y T-cell receptor transgenic mice

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Immunoglobulin receptors and members of the immunoglobulin superfamily CD4 CD16 CD32 CD33 CD47 CD48 CD50 CD54

CD58 CD66 CD89 CD100 CD101 HLA class I HLA-DR FceRI

Chemokine, complement and other chemotactic factor receptors CD35 CD88 CD182 CD183 CD191 CD192 CD193 CD196

Enzymes

Cytokine receptors CD25 CD116 CD117 CD119 CD120 CD123 CD124

PAFR LTB4R C3aR CystLT1R CystLT2R fMLPR CRTH2 Histamine 4R

CD13 CD45 CD45RB CD45RO CD46

CD125 CD131 CD213 IL-9R IL-13Ra1 TGF-bR

CD55 CD59 CD87 PAR-2

Apoptosis, signaling and others Adhesion molecules Fig. 12.5 Schematic diagram showing surface molecules expressed by human eosinophils. Molecules have been listed based on convincing evidence for their expression as assessed by flow cytometry or inferred by cellular responsiveness to specific stimuli. Cluster designation (CD) for particular molecules is indicated based on the most recent classification (www.ncbi.nlm.nih.gov/prow/). (See CD-ROM for color version.)

CD11a CD11b CD11c CD15 CD18 CD29 CD44 CD49d

following cognate peptide injection. In addition, eosinophils are associated with clusters of apoptotic bodies, suggesting eosinophil-mediated MHC class I-restricted thymocyte deletion. Thymic eosinophils have the capacity to promote thymocyte apoptosis as they express costimulatory molecules involved in clonal deletions, such as CD30L (CD153) and CD66 (Throsby et al. 2000). Additionally, eosinophils may induce thymocyte apoptosis via free radicals, as thymic eosinophils express high levels of NADPH oxidase activity; notably, developing thymocytes have increased sensitivity to free radicals due to downregulation of Cu2+/Zn2+ superoxide dismutase.

Antigen presentation Recent clinical and experimental investigations have shown that eosinophils can function as antigen-presenting cells (Fig. 12.6). Eosinophils are capable of processing and presenting a variety of microbial, viral, and parasitic antigens, as well as superantigens (Shi 2004). GM-CSF-treated eosinophils promote T-cell proliferation in response to staphylococcal superantigen (Staphylococcus enterotoxins A, B and E) stimulation (Mawhorter et al. 1994). Furthermore, eosinophils incubated with human rhinovirus-16 promote rhinovirus-16-specific

CD49f CD62L CD156 CD162 CD174 ad integrin b7 integrin

CD9 CD17 CD24 CD28 CD37 CD39 CD43 CD48 CD52 CD53 CD63 CD65 CD69

CD71 CD76 CD81 CD82 CD86 CD92 CD95 CD97 CD97 CD98 CD99 CD137 CD139

CD148 CD149 CD151 CD153 CD161 CD165 Siglec-8 Siglec-10 LIR1 LIR2 LIR3 LIR7 TLR7 TLR8

T-cell proliferation and IFN-γ secretion (Handzel et al. 1998). Eosinophils can also effectively present soluble antigens to CD4+ T cells, thereby promoting T-cell proliferation and polarization. Adoptive transfer of antigen-pulsed eosinophils results in eosinophil-dependent T-cell proliferation (MacKenzie et al. 2001). Furthermore, addition of antigen to eosinophil and T-cell cocultures promotes heightened T-cell proliferative responses (Shi et al. 2000). The capacity of eosinophils to present antigen has been debated in some publications. It is interesting to note that the failure of eosinophils to present antigen may be related to the methods used for isolating eosinophils. For example, lysis of erythrocytes with ammonium chloride, an inhibitor of lysosome acidification (needed for antigen presentation), negatively correlates with eosinophil antigen presentation activity (Shi et al. 2000; van Rijt et al. 2003). Eosinophils secrete an array of cytokines capable of promoting T-cell proliferation, and activation of Th1 or Th2 polarization (IL-2, IL-4, IL-6, IL-10 and IL-12) (Kita 1996; Lacy & Moqbel 2000; Shi et al. 2000; MacKenzie et al. 2001) (Fig. 12.6 and Table 12.2). Recent attention has been drawn to the ability of murine eosinophils to produce IL-4. Employing mice with enhanced green fluorescent protein (GFP) in the

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Inflammatory Cells and Mediators Cytokines IL-2, IL-3, IL-4, IL-5 IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, TGF-a/b GM-CSF, TNF-a/b, IFN-g and IL-12

Stimuli Tissue injury Viral and bacterial infections Allergens Allografts Tumors

Growth Factors Heparin-binding epidermal growth factor-like binding protein (HB-EGF-LBP) NGF PDGF VEGF SCF

TLR FcR/Ig

Lipid Mediators Leukotrienes-LTD4, LTE4 'Prostaglandins-PGE1, PGE2 15-HETE Platelet-activating factor

IDO KYN IL-4

Neuromediators Substance P NGF VIP Chemokines CCL3/MIP-1a, CCL5/RANTES CCL7/MCP-3, CCL8/MCP-2 CCL11/eotaxin-1, CCL13/MCP-4 IL-8

Tc

ell

MBP Activation

Antigen Presentation MHC-II CD80/CD86

IL-5

Cytotoxic secretory products EPO MBP ECP Ribonucleases EDN Others MMP-9

Mast cell

Fig. 12.6 Schematic diagram of an eosinophil and its multifunctional effects. Eosinophils are bilobed granulocytes with eosinophilic staining secondary granules. The secondary granules contain four primary cationic proteins designated eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil derived neurotoxin (EDN). All four proteins are cytotoxic molecules; in addition, ECP and EDN are ribonucleases. Eosinophils respond to diverse stimuli including nonspecific tissue injury, viral infections, allografts, allergens, and tumors. In addition to releasing their preformed cationic proteins, eosinophils can also release a variety of cytokines, chemokines, lipid mediators, and neuromodulators. Eosinophils directly communicate with T cells and mast cells in a bidirectional manner. Eosinophils activate T cells by serving as

antigen-presenting cells and eosinophil-derived MBP is a mast cell secretagogue. Eosinophils can also regulate T-cell polarization through synthesis of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in oxidative metabolism of tryptophan, catalyzing the conversion of tryptophan to kynurenines (KYN), a regulator of Th1/Th2 balance. 15-HETE, 15-hydroxyeicosatetraenoic acid; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NGF, nerve growth factor; PDGF, platelet-derived growth factor; RANTES, regulated on activation normal T-cell expressed and secreted; SCF, stem cell factor; VEGF, vascular endothelial growth factor; VIP, vasoactive intestinal polypeptide. See text for definition of other abbreviations. (Adapted from Rothenberg & Hogan 2006, with permission.) (See CD-ROM for color version.)

IL-4 gene locus (4get mice), eosinophils appear to be a primary source of IL-4 following parasitic infection or anti-IgD treatment (a strong Th2 stimulator). Notably, while the IL-4 gene locus is transcriptionally active in eosinophils, the amount of IL-4 protein production appears to be lower than in T cells and basophils (Shinkai et al. 2002; Khodoun et al. 2004; Voehringer et al. 2004). Furthermore, murine eosinophils promote IL-4, IL-5, and IL-13 secretion by CD4+ T cells (MacKenzie et al. 2001). Eosinophils can also regulate T-cell polarization through their synthesis of indoleamine 2,3-dioxygenase, an enzyme involved in oxidative metabolism of tryptophan, converting tryptophan to kynurenines. Kynurenines regulate Th1 and Th2 imbalance by promoting Th1 cell apoptosis (Odemuyiwa

et al. 2004). The eosinophil-mediated T-cell proliferative and cytokine secretion responses are dependent on costimulation. Indeed, blockade of CD80, CD86, and CTLA-4 by neutralizing antibodies inhibits eosinophil-elicited T-cell proliferation and cytokine secretion (Shi 2004). Fluorescent labeling studies have revealed that eosinophils instilled into the trachea of mice traffic into the draining peritracheal lymph nodes and localize to the T-cell-rich paracortical regions (B-cell zones) within 24 hours (Shi et al. 2000). Employing models of allergic airway disease and gastrointestinal allergy, investigators have demonstrated that inhalation of antigen promotes eosinophil homing to the draining endotracheal lymph nodes and Peyer’s patches (Korsgren

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et al. 1997; Mishra et al. 2000; Hogan et al. 2001; MacKenzie et al. 2001). Interestingly, a recent investigation suggests that eosinophils can only promote proliferation of effector T cells but not naive T cells (van Rijt et al. 2003). Moreover, eosinophils pulsed with ovalbumin peptide and cocultured with ovalbuminspecific T-cell receptor transgenic T cells (D011.10 T cells) induced effector T-cell proliferation; however, when cocultured with naive CD4+ T cells, no T-cell proliferation was observed. It is tempting to speculate that eosinophils traffic to draining lymph nodes in order to recruit activated effector T cells and promote proliferation of effector T cells.

with shared properties of basophils and eosinophils, and then into a separate eosinophil lineage (Boyce et al. 1995). Initial studies examining the the eos 47 gene (encoding EOS47, the avian ortholog of the mammalian melanotransferrin gene), a gene specifically expressed by bone marrow eosinophils, revealed that a 309-bp promoter region consisting of binding sites for Myb-, Ets-, c/EBP-, and GATA-type transcription factors were responsible for governing lineage-specific expression (McNagny et al. 1998). More recent investigations have supported these initial observations, demonstrating that eosinophil lineage specification is dictated by the interplay of at least three classes of transcription factors including GATA-1 (a zinc finger family member), PU.1 (an Ets family member), and c/EBP members (CCAAT/enhancer-binding protein family) (Nerlov & Graf 1998; Nerlov et al. 1998; McNagny & Graf 2002). Interestingly, these three transcription factors are expressed in a variety of hematopoietic lineages, although their synergistic mechanism of action in eosinophils promotes lineage specificity. The expression level of PU.1 specifies distinct cell lineage fates, with low levels specifying lymphocytic and high levels myeloid differentiation (De Koter & Singh 2000; Du et al. 2002). In most cell types, GATA-1 and PU.1 antagonize each other, but have synergistic activity in regulating eosinophil lineage specification (and eosinophil granule protein transcription) (Du et al. 2002). The specificity of these factors for eosinophils is conserved across species, for example c/EBP factors and GATA-1 drive differentiation of chicken progenitor cells into eosinophils (McNagny & Graf 2002). Of these transcription factors, GATA-1 is clearly the most important for eosinophil lineage specification as revealed by loss of the eosinophil lineage in mice harboring a targeted deletion of the high-affinity GATA-binding site in the GATA-1 promoter (Yu et al. 2002), and based on eosinophil differentiation experiments in vitro (Hirasawa et al. 2002). In particular, the specific activity of GATA-1 in eosinophils but not other GATA-1-positive lineages (mast cells, megakaryocytes, erythroid cells) appears to be mediated by a high-affinity palindromic (or “double”) GATA site (Du et al. 2002). This double GATA site is present in the downstream GATA-1 promoter and also in the regulatory regions of eosinophil-specific genes, including the eotaxin receptor CCR3, MBP, and the IL-5Rα gene, and accounts for eosinophil-specific gene expression (Zimmermann et al. 2000a; Du et al. 2002; Yu et al. 2002). For example, the tandem double GATA site in the human MBP-P2 promoter is required for both promoter activity in human eosinophil cell lines and for synergistic transactivation by GATA-1 and PU.1 (Du et al. 2002). Previous studies have identified cis-acting sequences (cis elements) as important regulators of GATA-1 expression, particularly a 3-cis-acting sequence known as upstream enhancer HS1/G1HE (HS1) as a major enhancer of GATA-1 expression (McDevitt et al. 1997; Onodera et al. 1997). However, studies have demonstrated that HS1 deletion in mice does not affect eosinophil

Mast cell regulation A substantial body of literature has emerged demonstrating that eosinophils have the capacity to regulate mast cell function (see Fig. 12.6). Notably, human umbilical cord bloodderived mast cells can be activated by MBP to release histamine, PGD2, GM-CSF, TNF-α, and IL-8 (Piliponsky et al. 2002). The activation of mast cells by MBP elicits not only exocytosis but also eicosanoid generation and cytokine production, both of which are prominent responses following FcεRI-dependent activation of mast cells (Piliponsky et al. 2002). Incubation of rat peritoneal mast cells with native MBP, EPO, and ECP (but not EDN) results in concentrationdependent histamine release (Zheutlin et al. 1984). Several studies have shown that MBP induces mast cell activation via a similar pathway to that observed with other polybasic compounds such as substance P, compound 48/80, and bradykinin (Piliponsky et al. 2001). Freshly isolated human lung mast cells are resistant to IgE-independent activation; however, highly purified lung mast cells cocultured with human lung fibroblasts are sensitive to IgE-independent activation by MBP (Piliponsky et al. 2002). Interestingly, activation of eosinophils with the mast cell protease chymase promotes production of eosinophil-derived stem cell factor, a critical mast cell growth factor. Eosinophils also produce nerve growth factor (NGF) (Solomon et al. 1998), a cytokine not only involved in survival and functional maintenance of sympathetic neurons but also in immune regulation. For example, NGF promotes mast cell survival and activation (Horigome et al. 1994; Bullock & Johnson 1996). NGF is preformed in eosinophils and acts in an autocrine fashion by activating release of EPO (Solomon et al. 1998). EPO activates rat peritoneal muscles to release histamine, suggesting a role of eosinophil-derived NGF in mast cell-eosinophil interactions. Thus, eosinophils and mast cells communicate in a bidirectional fashion.

Eosinophil development Eosinophils are produced in the bone marrow from pluripotent stem cells, which first differentiate into a hybrid precursor

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GATA-1 mRNA expression and eosinophil differentiation (Guyot et al. 2004). A number of the c/EBP members have also been implicated in the regulation of eosinophil lineage commitment (Nerlov et al. 1998). Phenotypic characterization of c/EBPα-deficient mice revealed that these mice are devoid of eosinophils (Zhang et al. 1997). This is consistent with studies demonstrating eosinophil formation from cord blood progenitors by enforced expression of c/EBPα (Iwama et al. 2002). Collectively these studies suggest that coexpression of GATA-1 and c/EBPα are required for efficient eosinophil formation (Hirasawa et al. 2002; Iwama et al. 2002; McNagny & Graf 2002). More recent investigations have demontrated that expression of eosinophil granule proteins is also regulated by c/EBPε and PU.1. Eosinophils from c/EBPε–/– mice have an abnormal phenotype (Yamanaka et al. 1997). Notably, in these mice neutrophil secondary granule gene expression is severely impaired, suggesting that c/EBPε may be involved in granule gene expression (Gombart et al. 2003). c/EBPε and GATA-1 proteins have been shown to weakly induce MBP expression; however, addition of PU.1 dramatically upregulates endogenous MBP expression, suggesting that PU.1 may regulate eosinophil-specific gene expression (Gombart et al. 2003). Consistent with these observations, MBP and EPX gene expression was attenuated in myeloid cell lines derived from PU.1–/– mice. Furthermore, PU.1 has been shown to be involved in the expression of other eosinophil-specific genes including EDN (van Dijk et al. 1998). Granulocytes are generated from a small number of hematopoietic stem cells and this process is highly regulated and maintained at a constant level under steady-state conditions (Metcalf 1991; Tenen et al. 1997; Zhu & Emerson 2002). However, under conditions of inflammation or cytokine stimulation, termed “emergency granulopoiesis,” the hematopoietic system greatly amplifies granulocyte formation. This process has been shown to be regulated by many cytokines (G-CSF, GM-CSF and IL-3). A recent study suggests that c/EBP factors (c/EBPα and c/EBPβ) also play an important role in the regulation of different aspects of steady-state and inflammatory stimuli-induced (emergency) granulopoiesis (Kincade 2006). Employing c/EBPβ-deficient mice, investigators have demonstrated normal steady-state granulopoiesis in the absence of c/EBPβ, suggesting that c/EBPα is sufficient for steady-state granulopoiesis. This has been confirmed by the demonstration of complete loss of granulocytes in c/EBPα-deficient mice (Zhang et al. 1997). Notably, emergency granulopoiesis induced by cytokine stimulation was ablated in c/EBPβ-deficient mice (Hirai et al. 2006), suggesting that c/EBPβ selectively regulates emergency granulopoiesis. In support of this hypothesis, cytokine treatment induced c/EBPβ but not c/EBPα or c/EBPε transcripts in granulocyte progenitors. Furthermore, granulocytes can be generated from c/EBPα–/– progenitors following cytokine stimulation in vivo (Hirai et al. 2006). These studies suggest

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that c/EBPα regulates steady-state and c/EBPβ emergency granulopoiesis (Hirai et al. 2006; Kincade 2006). Three cytokines, IL-3, IL-5, and GM-CSF, are particularly important in regulating eosinophil development (Lopez et al. 1986, 1988; Rothenberg et al. 1988; Takatsu et al. 1994). These eosinophilopoietins likely provide permissive proliferative and differentiation signals following the instructive signals specified by the transcription factors GATA-1, PU.1, and c/EBPs. These cytokines are encoded by closely linked genes on chromosome 5q31. They bind to receptors that share a common β chain and have unique α chains (Vadas et al. 1994). Of these three cytokines, IL-5 is the most specific to the eosinophil lineage and is responsible for selective differentiation of eosinophils (Sanderson 1992). IL-5 also stimulates the release of eosinophils from the bone marrow into the peripheral circulation (Collins et al. 1995). The critical role of IL-5 in the production of eosinophils is best demonstrated by genetic manipulation of mice. Overproduction of IL-5 in transgenic mice results in profound eosinophilia (Dent et al. 1990; Tominaga et al. 1991; Lee et al. 1997; Mishra et al. 2002) and deletion of the IL-5 gene causes a marked reduction of eosinophils in the blood and lungs after allergen challenge (Foster et al. 1996; Kopf et al. 1996). The overproduction of one or a combination of these three cytokines occurs in humans with eosinophilia, and diseases with selective eosinophilia are often accompanied by overproduction of IL-5 (Owen et al. 1989). The critical role of IL-5 in regulating eosinophils in humans has been demonstrated by several clinical trials with humanized anti-IL-5 antibody; this currently unapproved drug dramatically lowers eosinophil levels in the blood and, to a lesser extent, in the inflamed lung (Leckie et al. 2000; Flood-Page et al. 2003a; Kips et al. 2003).

Eosinophil trafficking Chemokine regulation of eosinophil and CD4+ T-cell trafficking Under baseline conditions, most eosinophils traffic into the gastrointestinal tract where they normally reside within the lamina propria of all segments except the esophagus (Mishra et al. 1999). The gastrointestinal eosinophil is the predominant population of eosinophils. Under baseline conditions, eosinophil levels in the gastrointestinal tract occur independent of lymphocytes and enteric flora, indicating unique regulation compared with other leukocytes (Mishra et al. 1999). Indeed, the recruitment of gastrointestinal eosinophils is regulated by the constitutive expression of eotaxin-1, as demonstrated by the marked decrease of this population of eosinophils in eotaxin-1-deficient mice. The importance of eotaxin-1 in regulating the baseline level of eosinophils is reinforced by the observation that mice with targeted deletion of CCR3 (but not eotaxin-2-deficient mice) also have a deficiency in gastrointestinal eosinophils (Humbles et al.

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2002; Pope et al. 2005a). In addition eosinophils homing into the gastrointestinal tract, thymus, mammary gland and uterus, under homeostatic conditions has been shown to be dependent on eotaxin-1 (Gouon-Evans et al. 2000; Rothenberg et al. 2001a). Notably, trafficking into the uterus is regulated by estrogen, as eosinophil and eotaxin-1 levels cycle with estrus (Gouon-Evans & Pollard 2001). The trafficking of eosinophils into inflammatory sites has been shown to involve a number of cytokines (most notably the Th2 cell products IL-4, IL-5, and IL-13) (Sher et al. 1990a; Moser et al. 1992; Horie et al. 1997), adhesion molecules (e.g., β1, β2, and β7 integrins) (Bochner & Schleimer 1994), chemokines such as RANTES and the eotaxins (Zimmermann et al. 2003), and other recently identified molecules (e.g., acidic mammalian chitinase) (Zhu et al. 2004) (Fig. 12.7). Of the cytokines implicated in modulating leukocyte recruitment, only IL-5 and the eotaxins selectively regulate eosinophil trafficking (Rankin et al. 2000). IL-5 regulates growth, differentiation, activation, and survival of eosinophils and has been shown to provide an essential signal for the expansion and mobilization of eosinophils from the bone marrow into the lung following allergen exposure (Collins et al. 1995). However, antigen-induced tissue eosinophilia can occur independent of IL-5, as demonstrated by residual tissue eosinophils in trials using anti-IL-5 in patients with asthma (Flood-Page et al.

2003a), and in IL-5-deficient mice (Foster et al. 1996; Hogan et al. 1997). Recent studies have demonstrated an important role for the eotaxin subfamily of chemokines in eosinophil recruitment to the lung (Zimmermann et al. 2003). Eotaxin was initially discovered using a biological assay in guinea pigs designed to identify the molecules responsible for allergen-induced eosinophil accumulation in the lungs (Jose et al. 1994; Rothenberg et al. 1995; Rankin et al. 2000). Subsequently, utilizing genomic analyses, two additional chemokine genes have been identified in the human genome that encode CC chemokines with eosinophil-selective chemoattractant activity, and have hence been designated eotaxin-2 and eotaxin-3 (Zimmermann et al. 2003). Eotaxin2 and eotaxin-3 are only distantly related to eotaxin-1 since they are only about 30% identical in sequence and are located in a different chromosomal position (Shinkai et al. 1999; Zimmermann et al. 2000b). The specific activity of all eotaxins is mediated by selective expression of the seventransmembrane spanning, G-protein-coupled receptor CCR3, primarily expressed on eosinophils (Murphy 1994; Daugherty et al. 1996; Ponath et al. 1996). Notably, the eotaxin chemokines cooperate with IL-5 in the induction of tissue eosinophilia. IL-5 increases the pool of eotaxin-responsive cells and primes eosinophils to respond to CCR3 ligands (Zimmermann et al. 2003). Furthermore, when given exogenously, eotaxins

Eosinophil differentiation Transcription factors: GATA-1, PU.1 and c/EBP members (a, b and e) Cytokines: IL-5, IL-3 and GM-CSF Bone marrow

IL-5

Blood Selectins L-selectin PSGL-1 Sialyl-Lewis X Selectins P-selectin E-selectin

Eosinophil adhesion Integrins b1-a4, a6 b2-aL, aM, aD, aX b7-a4 Adhesion receptors ICAM-1 VCAM-1 MAdCAM-1

Eosinophil chemotaxis Chemokine receptors CCR1, CCR3, CCR5, CCR6, CCR8, CXCR3 and CXCR4

Chemokines CCL3, CCL5, CCL8, CCL7, CCL-11, CCL13, CCL24, CCL26 and CCL28

Tissue

Fig. 12.7 Schematic representation of eosinophil trafficking. Eosinophils develop in the bone marrow where they differentiate from hematopoietic progenitor cells into mature eosinophils under the control of the critical transcription factors GATA-1, PU.1 and c/EBP members. The eosinophilopoietins IL-3, IL-5, and GM-CSF regulate eosinophil expansion, especially in conditions of hypereosinophilia. Eosinophil migration out of the bone marrow into the circulation is primarily regulated by IL-5.

Circulating eosinophils subsequently interact with the endothelium by processes involving rolling, adhesion, and diapedesis. Depending on the target organ, eosinophils cross the endothelium into tissues by a regulated process involving coordinated interaction between networks involving the chemokines, eosinophil adhesion molecules, and adhesion receptors on the endothelium. (Adapted from Rothenberg & Hogan 2006, with permission.) (See CD-ROM for color version.)

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cooperate with IL-5 to induce substantial production of IL-13 in the lung (Zimmermann et al. 2003). The finding that IL-4 and IL-13 are potent inducers of the eotaxin chemokines by a STAT6-dependent pathway provides an integrated mechanism to explain the eosinophilia associated with Th2 responses (Zimmermann et al. 2003). Recent studies have identified that eosinophil recruitment to the lung is dependent on STAT6 and a bone marrow-derived lung tissue resident nonT or B cell (Voehringer et al. 2004); in particular, eotaxin-2 production by airway macrophages likely accounts for this (Pope et al. 2005a,b). Of further interest, recently CCR3 has been shown to also deliver a powerful negative signal in eosinophils, depending on the ligand engaged. For example, pretreatment with the chemokine Mig inhibits eosinophil responses by a CCR3- and Rac2-dependent mechanism (Fulkerson et al. 2005). Utilizing eotaxin-1 and eotaxin-2 single and double genedeficient mice or neutralizing antibodies, both chemokines have been shown to have nonoverlapping roles in regulating the temporal and regional distribution of eosinophils in an allergic inflammatory site (Rothenberg et al. 1997; Gonzalo et al. 1998; Pope et al. 2005a). Utilizing a standard experimental asthma model induced by systemic sensitization with ovalbumin/alum followed by respiratory ovalbumin challenge, only a modest reduction in lung eosinophils was found in CCR3-deficient mice (Humbles et al. 2002). However, when the same CCR3-deficient mouse line was subjected to experimental asthma induction by epicutaneous ovalbumin sensitization, there was a marked deficiency of lung and BAL eosinophils (Ma et al. 2002). It was proposed that these apparently conflicting results may be related to the sensitization protocol (Ma et al. 2002), but the reason for this apparent discrepancy remains unclear. Notably, another CCR3-deficient mouse strain has recently been shown to have a profound reduction in eosinophil recruitment to the lung in the standard ovalbumin/alum systemic sensitization model (Pope et al. 2005b). There is now substantial preclinical evidence supporting a role for the eotaxin chemokines in human allergic disease (Zimmermann et al. 2003). Experimental induction of cutaneous and pulmonary late-phase responses in humans has revealed that the eotaxin chemokines are produced by tissue-resident cells (e.g., respiratory epithelial cells and skin fibroblasts) and allergen-induced infiltrative cells (e.g., macrophages and eosinophils). Following allergen challenge in the human lung, eotaxin-1 is induced early (6 hours) and correlates with early eosinophil recruitment; in contrast, eotaxin-2 correlates with eosinophil accumulation at 24 hours (Zimmermann et al. 2003). In another study, eotaxin-1 and eotaxin-2 mRNA were increased in patients with asthma compared with normal controls; however, there was no further increase following allergen challenge (Zimmermann et al. 2003). In contrast, eotaxin-3 mRNA was dramatically enhanced 24 hours after allergen challenge (Zimmermann et al.

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2003). The chemoattractant activity of BAL fluid from patients with asthma is inhibited by antibodies against RANTES, MCP-3, MCP-4, and eotaxin-1 (Zimmermann et al. 2003). Further support for an important role of eotaxin-1 in human asthma is derived from analysis of a single nucleotide polymorphism (SNP) in the eotaxin-1 gene. A naturally occurring mutation encoding a change in the last amino acid in the signal peptide (alanine→threonine) results in less effective cellular secretion of eotaxin-1 in vitro and in vivo (Nakamura et al. 2001). Notably, this SNP is associated with reduced levels of circulating eotaxin-1 and eosinophils and improved lung function (e.g., forced expiratory volume in 1 s or FEV1) (Nakamura et al. 2001). Furthermore, an SNP in the eotaxin-3 gene is associated with atopy in a Korean population and eosinophilic esophagitis in a white population (Chae et al. 2005; Blanchard et al. 2006). Recently, the activity of eotaxin-1 and eotaxin-2 in humans has been investigated by injection of these chemokines into the skin; both eotaxin-1 and eotaxin-2 induce an immediate wheal and flare response associated with mast cell degranulation and subsequent infiltrations by eosinophils, basophils, and neutrophils (MenziesGow et al. 2002). The infiltration by neutrophils is likely to be mediated indirectly by mast cell degranulation. These results provide substantial evidence that the biological activities attributed to eotaxins in animals are conserved in humans.

Role of eosinophils in disease Eosinophils are closely associated with infection by parasitic helminths, as production of Th2 cytokines, specifically IL-5, within cells in infected tissue promotes expansion of progenitor populations in the bone marrow, leading to blood and tissue eosinophilia (Pearce et al. 2004; Wynn et al. 2004; Jankovic et al. 2006). Although in vitro studies suggest that eosinophils can destroy these organisms via secretion of cytotoxic proteins and reactive oxygen species, the results from infection studies carried out in vivo remain unclear and controversial. Eosinophils are also closely associated with the pathogenesis of allergy, specifically in the respiratory tract, with the development of allergic asthma (Bochner & Busse 2005). Symptomatic wheezing and bronchoconstriction associated with asthma exacerbations can result from a superimposed respiratory virus infection, with common inciting agents including the paramyxovirus pathogen respiratory syncytial virus (hRSV) (Singh et al. 2007). At the same time, primary hRSV infection has intriguing associations with the development of childhood asthma (Everard 2006; Martin et al. 2006; Schaller et al. 2006). In connection with these observations, eosinophil recruitment and degranulation has also been observed in response to primary infection with hRSV, an observation that has been explored in human tissues, in culture systems, and in mouse models.

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Infection Eosinophils and parasitic helminth infection Profound blood and tissue eosinophilia are among the hallmark features of parasitic helminth infection, observed in response to activation of CD4+ Th2 lymphocytes at specific stages of the parasite life cycle. As noted earlier, although it seems logical to conclude that eosinophils serve as a means of host defense, there are no “errors of nature” (i.e., human conditions or syndromes characterized by a unique eosinophil deficiency) that might provide direct insight into eosinophil function in helminth-related disease. The only eosinophil-specific condition is hereditary EPO deficiency (Romano et al. 1994), which is detected by laboratory analysis and determined to be unrelated to an increased susceptibility to helminth infection in human studies. Interestingly, most of the human data available suggest a role for eosinophils in preventing reinfection (Hagan et al. 1985; Sturrock et al. 1996), a subject that might be addressed further in rodent infection models (Knopf et al. 1977). At the same time, controlled mouse model studies of primary infection have yielded results that are equivocal and, as such, no specific conclusions regarding the role of eosinophils in promoting host defense can be reached. There are several recent reviews that discuss these data in great depth, and provide significant insight into the ongoing controversy (Behm & Ovington 2000; Meeusen & Balic 2000; Klion & Nutman 2004). The primary points are summarized here. The initial paradigm, that eosinophils might provide host defense against parasitic helminths, came from studies in which activated human eosinophils in the presence of antibody and/or complement, as well as specific eosinophil secretory components alone (e.g., MBP, ECP, EPO), reduced the viability of various helminths in vitro (Butterworth & Franks 1975; Glauert & Butterworth 1977; Hamann et al. 1987). The availability of a monoclonal antibody directed against the eosinophilopoietic cytokine IL-5 permitted studies of the role of eosinophils in host defense against helminth infection to be performed in vivo. Although this reagent resulted in a large-scale reduction in circulating and tissue eosinophilia, there was no evidence for any change in the nature or extent of helminth infection in mouse model studies (Sher et al. 1990a; Herndon & Kayes 1992). Similar conclusions were reached in several studies performed in genetically altered IL-5 transgenic hypereosinophilic mice and in IL-5 and IL-5Rα gene-deleted, eosinophil-deficient mice (Hokibara et al. 1997; Takamoto et al. 1997; Le Goff et al. 2000), as well as more recently in studies performed in the Δdbl-GATA and TgPHIL eosinophil-ablated strains of mice (Swartz et al. 2006). However, among the notable exceptions, eosinophils did appear to play a role in reducing the parasite burden in several studies performed with nematode Strongyloides and Angiostrongylus species. Among these experiments, Korenaga et al. (1991) demonstrated increased recovery of S. venezuelensis worms from lungs of eosinophil-depleted mice treated with anti-IL-5

Eosinophils: Biological Properties and Role in Health and Disease

Table 12.3 Eosinophils and host defense against helminth parasites: results from mouse model studies. (Adapted from Klion & Nutman 2004, with permission.) Organism

Results

Cestodes Mesocestoides corti

−

Trematode Fasciola sp. Schistosoma sp.

− −

Nematodes Angiostrongylus sp. Heligmosomoides polygyrus Brugia sp. Nippostrongylus sp. Onchocerca sp. Strongyloides sp. Toxocara sp. Trichinella sp. Trichuris sp.

+ − + ± + + − ± −

−, Eosinophils shown to play no role; +, eosinophils shown to provide host defense; ±, conflicting information in the literature.

monoclonal antibody, and Sasaki et al. (1993) and Yoshida et al. (1996) demonstrated prolonged survival and increased recovery of A. cantonensis worms from anti-IL-5-treated and IL-5Rα-deficient mice, respectively. Given the unique tissuemigratory phase of Strongyloides and related nematode species, a role for eosinophils in host defense against specific tissueinvading helminths has been suggested (Klion & Nutman 2004). A compilation of the reports documenting the results of mouse model studies that have addressed the role of eosinophils in host defense against helminth pathogens is shown in Table 12.3. There are a number of publications that have focused on the role of eosinophils in promoting tissue pathology. A specific role for eosinophils in promoting pathology has been defined in mouse models of corneal inflammation characteristic onchocercal keratitis (Pearlman et al. 1998). However, in other models, such as those exploring the pathogenesis of Schistosoma mansoni and Nippostrongylus brasiliensis infection in eosinophil-deficient mice, the characteristic liver and lung lesions, respectively, are eosinophil depleted, but pathology remains otherwise unchanged (Coffman et al. 1989; Sher et al. 1990b; Swartz et al. 2006) (Fig. 12.8). There are many reasons why it may be difficult to discern a role for eosinophils in vivo in the mouse experimental system. Among the possibilities, many of these experiments are performed with human pathogens that do not naturally infect rodent species, and thus there is no assurance that one is

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

engaging innate host defense in an evolutionarily meaningful fashion. Related to this point, mouse eosinophils and human eosinophils are not necessarily functionally equivalent (Lee & Lee 2005). While EPO is highly conserved between mouse and human, the eosinophil ribonucleases and MBP are highly divergent. Likewise, mouse and human eosinophils display significant differences in morphology, surface protein expression, and propensity to degranulate in response to physiologic stimuli (Denzler et al. 2001; Clark et al. 2004; Lee & Lee 2005). The inability to observe eosinophils degranulating in response to infectious stimuli in vivo would appear to be a major factor hindering the identification of the role of this cell in host immunity against helminth infection. Different responses observed among various inbred strains of mice likewise add to the overall complexity (Dehlawi & Goyal 2003). Furthermore, it may be simply that we are asking the wrong questions. It remains possible that eosinophils do contribute to host defense, not necessarily by reducing the number of pathogens or eliminating immediate pathologic responses, but instead in a more subtle fashion by contributing to tissue remodeling (De Jesus et al. 2004; Reiman et al. 2006), propensity for reinfection (Knopf et al. 1977; Sturrock et al. 1983), and/or other long-term immunomodulatory sequelae.

Eosinophils and respiratory syncytial virus infection hRSV is a single-stranded negative-sense RNA virus pathogen of the family Paramyxoviridae, subfamily Pneumovirinae that causes respiratory tract infection, primarily among infants and toddlers. The severity of infection can extend from mild upper respiratory symptoms to full-blown bronchiolitis and pneumonia, and may progress to acute respiratory distress syndrome and death, particularly among highly susceptible populations (reviewed in Tripp 2004; DeVincenzo 2005).

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Fig. 12.8 Microscopic pathology of hepatic granulomas of Schistosoma mansoni-infected wild-type and eosinophil-ablated Ddbl-GATA and TgPHIL mice. Giemsa-stained liver tissue sections featuring granulomas from S. mansoni-infected BALB/c (a, b), eosinophilablated Ddbl-GATA (c, d), C57BL/6 (e, f), and eosinophil-ablated TgPHIL (g, h) mice, all at 12 weeks of infection. Arrows indicate examples of eosinophils. Original magnifications: ×10 (a, b, e, f); ×40 (c, d, g, h). (From Swartz et al. 2006, with permission.) (See CD-ROM for color version.)

There is no specific treatment for this infection other than primary support, as neither the antiviral agent ribavirin nor antiinflammatory glucocorticoids have proven impact on the course of disease (Randolph & Wang 1996). A safe and effective human vaccine is not available, although humanized monoclonal antibody directed against the virus fusion (F) protein is approved for treatment of high-risk infants (Cardenas et al. 2005). Several independent groups have detected eosinophils and/or their degranulation products in BAL washings taken from infants undergoing mechanical ventilation secondary to severe hRSV disease (Garofalo et al. 1992; Harrison et al. 1999; Dimova-Yaneva et al. 2004; Kim et al. 2006). The signals promoting eosinophil recruitment and degranulation have not been defined, although several potential eosinophil chemoattractants, including RANTES and MIP-1α have been detected in BAL from hRSV-infected infants (Harrison et al. 1999; Garofalo et al. 2001) (Fig. 12.9). Likewise uncertain is the role of eosinophils in primary hRSV infection, as there is no clear evidence from any human studies as to whether they promote host defense or serve to enhance immunopathology. Among these immunopathologies, there is a clear association between severe hRSV infection, particularly among the youngest infants, and the development of postinfection wheezing and asthma (Everard 2006; Martin et al. 2006; Schaller et al. 2006; Singh et al. 2007), the latter related, among other things, to age-dependent Th2 cytokine-mediated recruitment of proinflammatory eosinophils (Zhao et al. 2002; Kristjansson et al. 2005). Specific observations made in vitro provide potential insight into the role of eosinophils in primary hRSV disease. In correlation with their presence in human BAL washings, hRSVinfected respiratory epithelial cells in culture synthesize both RANTES (CCL5) and MIP-1α (CCL3) (Saito et al. 1997;

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+C 1

2 3

4 5

6 7

8

Eosinophils: Biological Properties and Role in Health and Disease 9 10 11 12 13 14 15 16 17 18 19 20

EDN

ECP

MIP-1a (CCL3; pg/mL/mg protein)

RNase activity (pg/mL/mg protein)

(a)

30 25 20 15 10 5 0

1

3

5

7

9

11

13

15

17

19

(b)

1200 1000 800 600 400 200 0

1

3

5

7

9

11

13

15

17

19

(c)

Fig. 12.9 Immunoreactive eosinophil granule proteins detected in lower airway secretions. (a) Western blots from lower airway secretions obtained from patients with RSV bronchiolitis (lanes 1–10) or unrelated diagnoses (lanes 11–20) were probed with polyclonal anti-EDN or anti-ECP antisera; +C, control: human eosinophil lysate (∼ 1 mg loaded). Detection of (b) ribonuclease activity (units/mL per mg protein) and (c) the proinflammatory chemokine MIP-1a (pg/mL per mg protein) in the same samples. (Adapted from Harrison et al. 1999, with permission.)

Harrison et al. 1999; Miller et al. 2004). In support of a role in host defense, Domachowske et al. (1998) have shown that human eosinophils mediate a dose-dependent reduction in hRSV infectivity, an effect directly dependent on degranulation of its unique secretory ribonucleases. Likewise, Adamko et al. (2001) demonstrated that EPO inhibited replication of the related rodent RNA virus pathogen parainfluenza type I in a similar in vitro assay system. The role of eosinophils in primary infection has also been addressed in the hRSV challenge model in mice. There are some differences between the human and mouse responses to the hRSV pathogen. As hRSV is a human pathogen, it undergoes little or no overt replication in mouse lung tissue and disease does not progress beyond a limited inflammatory state even in response to relatively large viral inocula. It is also important to recognize that most of the information relating to eosinophil recruitment in mice is based on studies of “enhanced disease,” which is an IL-5-mediated allergic response to formalin-fixed virus and virion components (Piedra 2003; Openshaw & Tregoning 2005) and not directly related to primary hRSV infection per se. However, in a recent study, Phipps et al. (2007) demonstrated accelerated clearance of hRSV after primary challenge in IL-5 transgenic hypereosinophilic mice, an effect that is directly dependent on signaling through TLR7. Interestingly, eosinophil recruitment in the primary hRSV challenge model appears to be a function

of age, with increasing numbers of eosinophils observed on infection of younger (neonatal) mice (Culley et al. 2002); neonatal challenge is also accompanied by enhanced recruitment of Th2 cells and progression to an allergen-responsive phenotype (You et al. 2006; Barends et al. 2004), similar to what has been observed in hRSV-infected human neonates. The role of eosinophils in promoting host defense against natural rodent paramyxovirus pathogens has been explored in some detail. In a study directed toward understanding airway hyperresponsiveness, Adamko et al. (1999) identified an eosinophil-dependent reduction in titers of rodent parainfluenza I virus in lungs of mice subjected to ovalbumin sensitization and challenge. Likewise, eosinophil recruitment has been observed as an early response to infection with pneumonia virus of mice (PVM), the cognate rodent pathogen most closely related to hRSV (Domachowske et al. 2000a; Easton et al. 2007). Eosinophil recruitment in response to PVM is not dependent on IL-5, but is blunted (along with recruitment of neutrophils) in mice devoid of the chemokine MIP-1α or its receptor CCR1 (Domachowske et al. 2000b). Preliminary studies suggest that, similar to studies perfomed with hRSV (Phipps et al. 2007), higher titers of virus are detected in lung tissue of the eosinophil-ablated Δdbl-GATA mice (Foster et al. 2007), although protection from the characteristic severe disease state is known to be related to factors other than absolute virus titer (Rosenberg et al. 2005).

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In summary, eosinophils are recruited and degranulate in lung tissue in response to hRSV infection. Experiments performed in vitro and in several different mouse models suggest mechanisms underlying eosinophil recruitment, and provide evidence consistent with a role for these cells in promoting virus clearance.

Asthma Asthma phenotypes Asthma is a heterogeneous disease with several clinical subtypes and a wide spectrum, ranging from mild, episodic, wheezy breathlessness to chronic, intractable, corticosteroiddependent chronic airway narrowing (Bel 2004). The classical IgE-associated allergic asthma phenotype starting in childhood is the most widely studied, not least because this form of the disease can be provoked in the clinical laboratory under controlled conditions by inhalation of allergen or allergenderived T-cell peptides. In these patients, airway cells have a predominant Th2 cytokine profile (i.e., IL-4+, IL-5+, IL-9+ and IL-13+ mucosal cells). Some asthmatics have late-onset nonallergic (so-called “intrinsic”) asthma in which sensitivity to allergens cannot be identified but in which airway eosinophilia and Th2 cells are also prominent (Humbert et al. 1999). The characteristic features of most asthma phenotypes, including allergic asthma, are airway inflammation, airway hyperresponsiveness (AHR), excessive airway mucus production due to goblet cell hyperplasia, and thickness of the airway wall. This airway thickness, often referred to as remodeling, is consequent to excessive repair processes following repeated airway injury and involves an increase in airway smooth muscle mass, deposition of collagen and other matrix proteins, and new blood vessel formation (Bousquet et al. 1990).

Association with eosinophils Increases in eosinophils in the tissues, blood, and bone marrow are a hallmark of most asthma phenotypes and, in general, elevated numbers correlate with disease severity (although “noneosinophilic/nonneutrophilic” asthma is characteristic of bacterial, viral, and pollutant triggers (Douwes et al. 2002)). This has led to the hypothesis that the eosinophil is the central effector cell responsible for ongoing airway inflammation. Thus, the cell has the potential to cause damage to the airway mucosa and associated nerves through the release of granule-associated basic proteins (which damage nerves and epithelial cells), lipid mediators (which cause bronchoconstriction and mucus hypersecretion), and reactive oxygen species (which generally injure mucosal cells). The inflammatory milieu promotes the survival of eosinophils by the elaboration of agents that delay apoptosis. These include epithelial-derived GM-CSF and neurotrophins (e.g., NGF and brain-derived neurotrophic factor) (Hahn et al. 2006). Eosinophils are also highly sensitive to Fas-mediated apoptosis. In a mouse model of asthma, Fas-positive T cells

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were found to regulate the resolution of airway inflammation since Fas deficiency on T cells produced long-term allergic airways disease (Tong et al. 2006). Blood eosinophils from patients with asthma have a number of phenotypic alterations, particularly in relation to their adhesive properties. Thus airway eosinophils recovered after antigen challenge have enhanced adhesion to VCAM-1 (CD106) and other ligands including albumin, ICAM-1 (CD54), fibrinogen, and vitronectin. These hyperadhesive properties seem to be mediated by upregulated and activated αMβ2 (CD11b/18) (Barthel et al. 2006). Asthmatic eosinophils also have increased expression of collagen receptors α1β1 and α2β1 integrins (Bazan-Socha et al. 2006). More attention is now given to a possible role for the eosinophil in repair and remodeling processes since there is a well-documented association of tissue eosinophilia and eosinophil degranulation with certain fibrotic syndromes and the cell is the source of several fibrogenic and growth factors, including TGF-α, TGF-β, fibroblast growth factor (FGF)-2, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-9, IL-1β, IL-13, and IL-17 (see Fig. 12.6).

Eosinophils and animal models of asthma Over the years animal models of asthma have often given conflicting results, especially those involving short-term sensitization (models of repeat allergen inhalation appear to be more reproducible). For example, two groups used experimental models of airway inflammation in mice genetargeted for complete and selective ablation of the eosinophil lineage. In one model eosinophils were targeted through transgenic expression of the diphtheria toxin A chain under control of the eosinophil peroxidase promoter (PHIL) (Lee et al. 2004). The authors of the study concluded that eosinophils were required for both AHR and mucus accumulation. Other workers have ablated the eosinophil lineage by deleting the high-affinity GATA-1 binding site on the palindromic GATA-1 promoter (Δdbl) but found that these same features of experimental asthma were unaffected by eosinophil depletion, although the cell did appear to play a critical role in airway remodeling (Humbles et al. 2004). Such anomalies have previously been explained by differences in the strains of animals used (Shinagawa & Kojima 2003) and different experimental protocols. Animal studies have demonstrated a role for eosinophils in airway remodeling. The Δdbl-GATA animals were clearly protected from peribronchiolar collagen deposition and increases in airway smooth muscle (Humbles et al. 2004). Furthermore, in a chronic repetitive allergen challenge model, Cho et al. (2004) found that IL-5 gene deletion suppressed lung eosinophilia, peribronchial fibrosis, collagen III, collagen V and total lung collagen content in parallel. These changes were associated with decreased TGF-β1 content of lung tissue, with evidence that eosinophils were the major source. Interestingly, epithelial cell expression of αvβ6, an integrin that

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Early Asthmatic Reaction

Late Asthmatic Reaction

• • • •

• • • • •

Mast cells IgE Histamine Cysteinyl leukotrienes

Airway hyperresponsiveness • • • •

Eosinophils: Biological Properties and Role in Health and Disease

Wheezy breathlessness

Neural pathways ASM T cells Mast cells

T cells Th2 cytokines (e.g., IL-13) DCs/IgE T cell/ASM interactions Mast cells (cysteinyl leukotrienes) Airway remodeling • Fibrogenic factors (e.g. TGF-b) • Growth factors (e.g. VEGF) • EOSINOPHILS

Natural Exacerbations • Viral/nonviral • Chronic inflammation • EOSINOPHILS Fig. 12.10 Pathways to wheezy breathlessness in asthma. Airway narrowing, the cause of wheezy breathlessness, can result from several mechanisms, many of which overlap. Early and late reactions are clinical models. The early asthmatic reaction can be largely blocked by antihistamines and anti-cysteinyl leukotrienes and is largely mast cell/IgE-mediated. The late asthmatic reaction is partially dependent on the early response and is “blunted” by leukotriene receptor antagonists. The late-phase reaction also has a T-cell component and may involve antigen trapping and focusing by IgE-bound dendritic cells prior to presentation

to, and activation of, Th2 cells. In airway hyperresponsiveness (AHR), nonspecific triggers such as smoke, dust, and fumes induce wheezing on a background of airway inflammation. The mechanisms underlying AHR include enhanced neural pathways, alterations in airway smooth muscle (ASM), and T cell- and mast cell-dependent pathways. Airway remodeling probably has a major eosinophil component as shown in both animals and human. Eosinophils are also prominent in natural exacerbations of the disease triggered by viral and nonviral agents. DC, dendritic cell; Th, T helper cell. (See CD-ROM for color version.)

activates latent TGF-β1, was also suppressed. Peribronchial smooth muscle thickness and epithelial mucus expression were also reduced. Others have observed reduced airway eosinophilia, TGF-β production, and remodeling in IL-5 knockout animals, and increased airway fibrosis in IL-5 transgenic animals after repeated allergen inhalation (Tanaka et al. 2004). It was surprising therefore that the reduced subepithelial cell fibrosis observed in the Δdbl-GATA line were apparently independent of TGF-β expression. However there are other potential fibrogenic pathway activators as shown diagrammatically in Fig. 12.10.

sites of inflammation. It stimulates the expansion and differentiation of eosinophil precursors and upregulates expression of its own specific receptor α chain during human eosinophil development. Anti-IL-5 has a good safety profile and anxieties regarding increased susceptibility to helminths and tumors appear, so far, unfounded. When asthmatics were given three infusions of anti-IL-5 (mepolizumab) this produced about a 90% reduction in blood and bronchial lavage eosinophils but only 55% reduction in bronchial mucosal eosinophils (Flood-Page et al. 2003b). However, even this modest effect produced significant reduction in tenascin, lumican, and procollagen III compared with placebo (Flood-Page et al. 2003c). There was also a significant reduction in the numbers and percentage of tissue eosinophils expressing mRNA for TGF-β1 as well as the concentration of TGF-β1 in BAL fluid. Although there were no appreciable improvements in clinical outcomes, the study was not powered to detect changes in lung function or AHR. Nevertheless, the results do provide strong evidence that there is a causal relation between eosinophils and matrix deposition in the extracellular matrix. The clinical significance of these findings is unclear, especially as fibroblast accumulation and airway smooth muscle cell hypertrophy in proximal airways seem to be more selective determinants of severe persistent asthma than matrix deposition beneath the basement membrane (Benayoun et al. 2003). A further complicating factor is the role of atopy. For example, airway eosinophilia and angiogenesis were observed in bronchial biopsies from atopic children without asthma (Barbato et al. 2006) and eosinophilic inflammation and

Remodeling in clinical asthma The precise clinical significance of airway remodeling is debated. One view is that the thickness of the wall, the overall consequence of remodeling, leads to a decrease in baseline caliber (i.e., the radius of the airway lumen), resulting in a disproportionate increase in airway resistance, which in turn enhances AHR. Others point out that corticosteroids, the mainstay antiinflammatory treatment in the disease, reverse some but not all the features of remodeling. Nevertheless there is a hard core of asthmatics who are steroid resistant and airway remodeling is often quite marked in asthma deaths. Studies in humans using anti-IL-5 antibodies also support a role for eosinophils in events surrounding deposition of certain matrix proteins within the reticular basement membrane (Kay et al. 2004). IL-5 is a key cytokine in eosinophil differentiation, maturation, recruitment, and activation at

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increased AHR have been observed in adult patients with allergic rhinitis (Tatar et al. 2005). On the other hand, intense bronchial mucosal eosinophilia is a feature of nonatopic (“intrinsic”) asthma. One interpretation of these findings is that eosinophil-mediated damage precedes the development of overt asthma irrespective of the atopic status. In order to provide definitive evidence that eosinophils are key cells in airway remodeling, more effective strategies are required to deplete tissue eosinophils. Even in animal models of asthma there was residual tissue eosinophilia in the airways after anti-IL-5 administration (Foster et al. 2001). In fact depletion of both IL-5 and eotaxin are required to abolish tissue eosinophils and AHR in mice, suggesting that IL-5 blockade alone is insufficient. Combination therapy with, for example, anti-IL-5 and a CCR3 antagonist may be more useful than IL-5 blockade alone, since this would have the theoretical advantage of inhibiting both bone marrow maturation (mainly an IL-5 effect) and tissue accumulation (predominantly a CCR3-dependent effect). In a mouse model, ablation of eotaxin chemokines prevented antigeninduced pulmonary eosinophilia (Pope et al. 2005b) and antagonism of CCR3 reduced eosinophil numbers and this was accompanied by a diminution in asthma pathology (Weigmann et al. 2007). Eosinophils are also known to localize to cholinergic nerves in a variety of inflammatory conditions including asthma. This effect appears to be the result of enhanced eotaxin production by neurons, possibly as a result of IL-4 and IL-13 upregulation (Fryer et al. 2006). These events result in damage of inhibitory M2 receptors by eosinophil MBP (Evans et al. 1997) and reduced catabolism of acetylcholine (Durcan et al. 2006).

Eosinophils and the late asthmatic reaction When clinical asthma is provoked experimentally by inhalation of allergen, there are two general patterns of airway narrowing, termed the early asthmatic reaction (EAR) and late asthmatic reaction (LAR). The EAR (as measured by changes in FEV1 as a test of airway narrowing) peaks within 15–30 min after allergen challenge and returns nearly to baseline by 1 hour. In this sense it is a “bronchospastic” reaction and involves the IgE-dependent release from mast cells of histamine and other mediators, including leukotrienes, prostaglandins, and tryptase. The LAR, on the other hand, is characterized by a delayed course and slow decrease in FEV1 (which peaks between 3 and 9 hours), and tends to resolve by 24 hours. The mechanism(s) of the LAR is controversial, although there is good evidence to suggest that there is a significant T-cell component because isolated LAR (i.e., without the EAR component) can be provoked by inhalation of allergen-derived T-cell peptides (which do not cross-link IgE and cause mast cell activation) (Haselden et al. 1999). Furthermore, the immunosuppressant cyclosporin A blocks the late, but not the early, asthmatic reaction (Sihra et al.

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1997). Thus the LAR may involve direct interaction between activated T cells (presumed Th2 cells) and airway smooth muscle subsequent to IgE-dependent trapping and focusing by airway dendritic cells. The role of the eosinophil in the LAR and in AHR remains uncertain. The original observation of Cockcroft et al. (1977) was that allergen inhalation increased AHR in dual asthmatic responders which was sustained for at least 7 days. The temporal association between increased inflammatory cell infiltration and increased AHR at 24 hours after allergen challenge in dual responders (Brusasco et al. 1990; Flood-Page et al. 2003b; Dorman et al. 2004a) has led to the suggestion of a causal relationship between the two. However, Kariyawasam et al. (2007) showed that allergen-induced increases in eosinophilic airway inflammation, while marked at 24 hours in dual asthmatic responders, were virtually resolved by 7 days. On the other hand, increases in AHR and expression of collagen markers of airway remodeling persisted. Other less direct evidence also supports the view that cellular inflammation, particularly eosinophil infiltration (Dorman et al. 2004b), does not necessarily directly relate to AHR (Djukanovic et al. 1990; Ollerenshaw & Woolcock 1992; Iredale et al. 1994; Crimi et al. 1998; Dorman et al. 2004b). In an allergen-induced study by Gauvreau et al. (1999) on the cellular kinetics of cells in induced sputum, eosinophilia remained elevated 7 days after allergen inhalation challenge, albeit at levels considerably less than at 24 hours. This is in contrast to the finding of others who observed resolution of mucosal eosinophils to baseline levels by this time point (Kariyawasam et al. 2007). The reason for this difference between measures of airway luminal eosinophils and tissue cells is not clear but may reflect eosinophils that have been cleared from the submucosa but can still be detected in the sputum at a time point when tissue infiltration has resolved. Attempts to deplete eosinophils selectively in humans have been largely unsuccessful and as such we have been unable to fully resolve the role of this cell in the asthma process. The first study in humans showed that a single infusion of anti-IL-5 produced a significant reduction in both blood and induced sputum eosinophils, but no appreciable changes in either the LAR or AHR (Leckie et al. 2000). However, a subsequent study (Flood-Page et al. 2003c) involving three infusions indicated that mepolizumab was unable to deplete tissue eosinophils, i.e., from the bronchial mucosa, making interpretation of the single infusion study problematic (O’Byrne et al. 2001; Flood-Page et al. 2002, 2003b; Leckie 2003). Nevertheless, further evidence against a role for eosinophils as causative of the late reaction comes from studies in human skin in which it has been shown that the kinetics of eosinophil accumulation can be dissociated from the time course of the late-phase allergic reaction and that profound reduction of eosinophils (again by anti-IL-5) does not affect the magnitude of the allergen-induced cutaneous swelling and edema (Phipps et al. 2004a).

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Eosinophils and airway hyperresponsiveness There is no firm evidence that eosinophils or their products are directly causative in AHR in clinical asthma. The correlation between blood and tissue eosinophils and the degree of AHR is generally weak or nonexistent. Following a single infusion of anti-IL-5 there was no change in AHR, even when patients were followed up for several weeks, although this study may have been flawed for the reasons already stated above (Leckie et al. 2000). Further evidence casting doubt on a role for eosinophils in AHR comes from studies of eosinophilic bronchitis (Brightling et al. 2002, 2003). In this condition there is a similar distribution of eosinophils in the airways to that found in asthma, although there is no wheezy breathlessness or AHR (Brightling et al. 2003). The only histopathologic feature distinguishing asthma from eosinophilic bronchitis was mast cells associated with airway smooth muscle cells.

Eosinophils and natural exacerbations of asthma Severe persistent asthma is characterized by viral- and nonviral-induced natural exacerbations on a background of chronic inflammation. An increase in blood or sputum eosinophils often predates deterioration in symptoms and lung function. In fact a management strategy directed at normalizing the sputum eosinophil count was more effective than traditional management strategy (based on lung function, assessment of symptoms, and use of rescue β2 agonists) in reducing the number of asthma exacerbations (Green et al. 2002). It is interesting to speculate that eosinophil-derived fibrogenic/growth factors amplify airway remodeling and associated mucus production relatively rapidly and that this in turn leads to deterioration in symptoms. In a recent study it was shown that even a single allergen inhalation could induce acute airway remodeling in mild atopic asthmatics (Phipps et al. 2004b). Endobronchial mucosal biopsies obtained 24 hours after challenge showed significant increases in Hsp47, a chaperone of collagen synthesis as well as STAT6 and phospho-Smad2 as evidence of IL-4/IL-13 and TGF-β activated cells respectively. There were also increases in the thickness of tenascin within the reticular basement membrane. Therefore (“eosinophilic”) airway remodeling in asthma may partly result from repeated acute activation of the epithelial mesenchymal trophic unit by allergen exposure.

Conclusions There are many areas of remodeling that require further investigation. The precise relationship between chronic inflammation and remodeling is still unclear. Some studies suggest that remodeling might even predate the first signs of inflammation and be an independent event that is amplified rather than caused by Th2 inflammation. The genetic predisposition to airway remodeling, including gene expression by resident cells from normal and diseased airways, is an important area for future research as are the precise effects of the various com-

Eosinophils: Biological Properties and Role in Health and Disease ponents of remodeling on airway function. For example, the consequence of subepithial fibrosis to chronic airway obstruction is still unknown and the importance of angiogenesis, one of the few components of remodeling reversed by corticosteroids, in inflammation and edema is poorly understood. Animal and human studies point to an important function of eosinophils in airway remodeling in asthma. The cell probably also plays a critical role in natural exacerbations of the disease. The significance of eosinophils in the LAR and in AHR, at least in the clinical situation, is far less certain. Studies on the role of the cell in AHR and mucus production, using eosinophil-lineage depletion in mice, has given diametrically opposite results, emphasizing the difficulties of animal models in mimicking the natural disease in humans.

Eosinophil-associated gastrointestinal diseases Eosinophil accumulation in the gastrointestinal tract is a common feature of numerous gastrointestinal disorders, including classic IgE-mediated food allergy (Saavedra-Delgado & Metcalfe 1985; Moon & Kleinman 1995), eosinophilic gastroenteritis (Keshavarzian et al. 1985; Torpier et al. 1988), allergic colitis (Sherman & Cox 1982; Hill & Milla 1990; Odze et al. 1995), eosinophilic esophagitis (Furuta 1998; Rothenberg et al. 2001b; Fox et al. 2002), inflammatory bowel disease (IBD) (Sarin et al. 1978; Dvorak 1980; Walsh & Gaginella 1991), and gastroesophageal reflux disease (Winter et al. 1982; Brown et al. 1984; Liacouras et al. 1998). In IBD, eosinophils usually represent only a small percentage of the infiltrating leukocytes (Walsh & Gaginella 1991; Desreumaux et al. 1999), but their level has been proposed to be a negative prognostic indicator (Nishitani et al. 1998; Desreumaux et al. 1999). Primary eosinophil-associated gastrointestinal diseases (EGIDs) such as eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, and eosinophilic colitis are defined as disorders that primarily affect the gastrointestinal tract with eosinophil-rich inflammation in the absence of known causes of eosinophilia (e.g., drug reactions, parasitic infections, malignancy). Patients with EGIDs suffer a variety of problems, including failure to thrive, abdominal pain, irritability, gastric dysmotility, vomiting, diarrhea, and dysphagia (Guajardo et al. 2002; Khan & Orenstein 2002). Evidence is accumulating to support the concept that EGIDs arise secondary to the interplay of genetic and environmental factors. Notably, a large percentage (∼ 10%) of patients suffering from EGID have an immediate family member with EGID (Guajardo et al. 2002). Additionally, several lines of evidence support an allergic etiology: (i) about 75% of patients with EGID are atopic (Caldwell et al. 1975; Cello 1979; Scudamore et al. 1982; Furuta et al. 1995; Iacono et al. 1996; Sampson 1997; Walsh et al. 1999; Spergel et al. 2002); (ii) the severity of disease can sometimes be reversed by institution of an allergen-free diet (Kelly et al. 1995; Walsh et al. 1999; Spergel et al. 2002); and (iii) the common finding of mast cell degranulation in tissue specimens (Oyaizu et al.

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1985; Bischoff 1996). Importantly, our recent models of EGID support a potential allergic etiology for these disorders (Rothenberg et al. 2001c). Interestingly, despite the common finding of food-specific IgE in patients with EGID, foodinduced anaphylactic responses only occur in a minority of patients (Sampson 1999). Thus, EGIDs have properties that fall between pure IgE-mediated food allergy and cell-mediated hypersensitivity disorders (e.g., celiac disease) (Sampson 1999).

Hypereosinophilic syndromes The term “hypereosinophilic syndrome” (HES) was introduced by Anderson and Hardy (1968) to designate patients with marked eosinophilia. They reported three patients, all males, between the ages of 34 and 47 who suffered from cardiopulmonary symptoms, fever, sweats, weight loss, and marked eosinophilia. Two of the patients died, and at autopsy their hearts were enlarged and showed mural thrombi. The treatment for HES is similar to that used for patients with chronic myelogenous leukemia, including tyrosine kinase inhibitors (e.g., Imatinib) prednisone, hydroxyurea (hydroxycarbamide), and interferon (IFN)-α and humanized anti-IL-5 antibodies. Chusid et al. (1975) formulated the diagnostic criteria for HES to include (i) persistent eosinophilia, with an eosinophil cell count of at least 1.5 × 109/L for a minimum of 6 months; (ii) lack of known causes for eosinophilia (e.g., parasitic or allergic triggers); and (iii) symptoms and signs of organ system involvement. Based on these diagnostic criteria, patients with EGID and blood eosinophil counts in excess of 1.5 × 109/L meet the diagnostic criteria. However, patients with EGID generally do not have the high risk of lifethreatening complications associated with classic HES (i.e., cardiomyopathy or central nervous system involvement). Notably, considerable heterogeneity among patients with HES has been recognized. For example, T-cell clones producing the characteristic Th2 cytokines IL-4 and IL-5 have been found in patients satisfying the diagnostic criteria for HES (Simon et al. 1999; Roufosse et al. 2003). However, perhaps the most striking advance in our understanding of HES has come about following treatment of HES patients with the tyrosine kinase inhibitor imatinib mesylate (Schaller & Burkland 2001; Ault et al. 2002; Gleich et al. 2002; Cools et al. 2003; Cortes et al. 2003). Imatinib was introduced for the treatment of chronic myelogenous leukemia and has had a remarkable effect in that disease. Treatment of many HES patients with imatinib mesylate causes a dramatic reduction of peripheral blood and bone marrow eosinophils, suggesting that certain HES patients express a novel kinase sensitive to imatinib mesylate. Further investigation of the ability of imatinib mesylate to treat HES patients revealed the existence of an 800-kb deletion in chromosome 4 bringing together an upstream DNA sequence homologous to a yeast protein, referred to as FIP1, and designated as like FIP1 or FIP1-L1, and the gene for the cytoplasmic domain of the platelet-derived growth factor α (PDGFRA) receptor (Cools

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et al. 2003; Griffin et al. 2003). This fusion gene is transcribed and translated to yield a novel kinase referred to as FIP-L1PDGFRA; FIP-L1-PDGFRA is exquisitely sensitive to imatinib in vitro, thus explaining the remarkable sensitivity of HES patients to this drug. The FIP-L1-PDGFRA fusion gene cooperates with IL-5 overexpression in a murine model of HES, suggesting that both pathogenic events cooperate in disease etiology (Yamada et al. 2006). The patients responsive to imatinib are those most characteristic of “classic” HES, namely males between the ages of 20 and 50 who present clinically with marked peripheral blood eosinophilia. Recently, these patients have been shown to meet minor criteria for systemic mastocytosis, having elevated levels of serum mast cell tryptase and high numbers of dysplastic mast cells in the bone marrow (Klion et al. 2003, 2004a). These patients go on to develop eosinophilic endomyocardial disease with embolization to peripheral organs including the extremities and the brain, and they strikingly resemble the patients originally designated by Hardy and Anderson. However, it appears that any disease that results in prolonged and marked eosinophilia can be associated with endomyocardial disease. For example, endomyocardial disease has occurred during the course of helminth infections and also in various malignancies associated with marked eosinophilia (Hussain et al. 1994; Yoshida et al. 1995; Andy et al. 1998). Thus patients with marked eosinophilia are at risk of developing cardiac disease regardless of the underlying etiology of the eosinophilia. Accordingly, routine surveillance of the cardiorespiratory system (e.g., echocardiography and plethysmography) in patients with EGID and peripheral blood eosinophilia is warranted. Based on these concerns, the diagnosis of HES in patients with EGID should always be considered, especially in those who develop extragastrointestinal manifestations (e.g., splenomegaly, or cutaneous, cardiac or respiratory). As such, additional diagnostic testing for HES should be considered including bone marrow analysis (searching for evidence of myelodysplasia), serum mast cell tryptase and vitamin B12 levels (both moderately elevated in classic HES), and genetic analysis for the presence of the FIP1-L1PDGFRA fusion event (Klion et al. 2003).

Anti-eosinophil therapeutics Numerous drugs inhibit eosinophil production or eosinophilderived products, including glucocorticoids, myelosuppressive drugs, leukotriene synthesis or receptor antagonists, tyrosine kinase inhibitors, IFN-α, and humanized anti-IL-5 antibodies. The etiology of the primary disease often specifies the best therapeutic strategy. For example, a subset of patients with HES have an 800-kb interstitial deletion on chromosome 4 (4q12) that results in the fusion of an unknown gene FIP1-L1 with PDGFRA (Cools et al. 2003, 2004a,b). This fusion gene produces a constitutively active tyrosine kinase

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(PDGFRA) that is exquisitively sensitive to the inhibitor imatinib mesylate, which is now approved for the treatment of HES (Gleevec). Although PDGFRA is not normally active in hematopoietic cells, the activated kinase renders cells growth factor independent, perhaps by activating STAT5 signal transduction. Thus, eosinophilic patients with FIP1L1-PDGFRA-positive disease are now treated with Gleevec as first-line therapy (Gleich et al. 2002). In addition, a variety of other activated tyrosine kinases have just been associated with HES, including PDGFR-β, Janus kinase-2, and FGF receptor 1. In most other individuals, glucocorticoids are the most effective agents for reducing eosinophilia (Rothenberg 1998). They suppress the transcription of a number of genes for inflammatory mediators, including the genes for IL-3, IL-4, IL-5, GM-CSF, and various chemokines including the eotaxins. Recently, the main action of glucocorticoids on eosinophil active cytokines has been shown to involve mRNA destabilization, thus reducing the half-life of cytokines such as eotaxins (Stellato et al. 1999). In addition, glucocorticoids inhibit the cytokine-dependent survival of eosinophils (Schleimer & Bochner 1994). Systemic or topical (inhaled or intranasal) glucocorticoid treatment typically causes a rapid reduction in eosinophils, but a few patients are glucocorticoid resistant and maintain eosinophilia despite high doses (Barnes & Adcock 1995). The mechanism of glucocorticoid resistance is unclear, but a reduced level of glucocorticoid receptors and alterations in transcription factor AP-1 (activator protein 1) appear to be at least partially responsible in some of them (Barnes & Adcock 1995). Glucocorticoid-resistant patients sometimes require other therapy such as myelosuppressive drugs (hydroxyurea, vincristine) or IFN-α (Rothenberg 1998). IFN-α can be especially helpful because it inhibits eosinophil degranulation and effector function (Aldebert et al. 1996). Notably, patients with myeloproliferative variants of HES can often go into remission with IFN-α therapy. Cyclophilins (e.g., cyclosporin A) have also been used because they block the transcription of numerous eosinophil-active cytokines (e.g., IL-5, GM-CSF) (Rothenberg 1998). Recently, lidocaine has been shown to shorten eosinophil survival, and its effects mimic those of glucocorticoids and are noncytotoxic (Bankers-Fulbright et al. 1998). Indeed, an early clinical trial has shown that nebulized lidocaine is safe and effective in subjects with asthma (Hunt et al. 2004). Drugs that interfere with eosinophil chemotactic signals include recently approved leukotriene antagonists and inhibitors. Inhibition of 5-lipoxygenase (e.g., zileuton) blocks the rate-limiting step in leukotriene synthesis and inhibits the generation of the eosinophil chemoattractant LTB4 and the cysteinyl leukotrienes (Kane et al. 1996). Cysteinyl leukotriene receptor antagonists block the muscle contraction and increased vascular permeability mediated by leukocyte-derived leukotrienes (Gaddy et al. 1992). Some of

the third-generation antihistamines inhibit the vacuolization (Snyman et al. 1992) and accumulation (Redier et al. 1992) of eosinophils after allergen challenge and directly inhibit eosinophils in vitro (Rand et al. 1988; Snyman et al. 1992). Cromoglycate and nedocromil inhibit the effector function of eosinophils such as antibody-dependent cellular cytotoxicity (Rand et al. 1988). The identification of molecules that specifically regulate eosinophil function and/or production offers new therapeutic strategies in the pipeline. Agents that interrupt eosinophil adhesion to the endothelium through the interaction of CD18/ICAM-1 (Wegner et al. 1990) or VLA-4/VCAM-1 may be useful (Kuijpers et al. 1993; Weg et al. 1993). Indeed, antibodies that block these pathways have recently been approved for other indications, but their anti-eosinophil activity has yet to be determined (von Andrian & Engelhardt 2003). Antibodies against IL-5, now humanized by two different pharmaceutical companies, are under active clinical investigation (Egan et al. 1995; Mauser et al. 1995) and look particularly promising for the treatment of HES (Garrett et al. 2004; Klion et al. 2004b, 2006) and eosinophilic esophagitis (Stein et al. 2006). While its utility for asthma may be limited due to redundant pathways, anti-IL-5 is particularly promising for HES. Numerous inhibitors of the eotaxin/CCR3 pathway, including small-molecule inhibitors of CCR3 and a human anti-eotaxin-1 antibody, are being developed (Zimmermann et al. 2003). Early results with a phase I trial of human antieotaxin-1 antibody in patients with allergic rhinitis have demonstrated the ability of this apparently safe drug to lower levels of eosinophils in nasal washes and nasal biopsies, and to improve nasal patency (Zimmermann et al. 2003). Antihuman IL-13 antibody is now in early clinical trials (Blanchard et al. 2005) and looks promising for lowering tissue eosinophil levels and improving features of asthma. Finally, a recently identified eosinophil surface molecule, Siglec-8, may offer a therapeutic opportunity (Nutku et al. 2003). Siglec-8 is a member of the sialic acid-binding lectin family and contains ITIMs (immunoreceptor tyrosine-based inhibitory motifs) that can induce efficient eosinophil apoptosis when engaged by anti-Siglec-8 cross-linking antibodies. It is interesting to note that Siglec-8, as well as CCR-3 and CRTH2, are coexpressed by other cells involved in Th2 responses including Th2 cells, mast cells, and basophils. Thus, agents that block these receptors may be particularly useful for allergic disorders.

Conclusions Historically, eosinophils have been considered end-stage cells involved in host protection against parasites. However, numerous lines of evidence have now changed this perspective by showing that eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of

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adaptive immunity by directly activating T cells. As normal constituents of the mucosal immune system, particularly in the gastrointestinal tract, eosinophils are likely to have a physiologic function. Indeed, eosinophils have been implicated in innate immunity by being an early and possibly instrumental source of cytokines (e.g., IL-4) and have been shown to have a role in developmental processes such as mammary gland development. Analysis of recently generated genetically engineered eosinophil-deficient mice will soon answer critical questions concerning the true involvement of this cell type in a variety of processes. Breakthroughs in identifying key eosinophil regulatory cytokines such as IL-5 and the eotaxin subfamily of chemokines have uncovered mechanisms that selectively regulate eosinophil production and localization at baseline and during inflammatory responses. In particular, an integrated mechanism involving Th2 cell-derived IL-5 regulating eosinophil expansion in the bone marrow and blood and Th2 cell-derived IL-13 regulating eotaxin production now explains the means by which T cells regulate eosinophils. Based on these findings, it is predicted that targeted therapy against key eosinophil regulators (e.g., humanized anti-IL-5 and CCR3 antagonists) will likely transform medical management of eosinophilic patients.

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Neutrophils: Biological Properties and Role in Health and Allergic Diseases Alison M. Condliffe, Andrew S. Cowburn and Edwin R. Chilvers

Summary The polymorphonuclear leukocyte or neutrophil is the most abundant yet shortest lived of all myeloid cells. It develops and matures in the bone marrow under the influence of interleukin (IL)-3, granulocyte colony-stimulating factor (GCSF), and granulocyte–macrophage colony-stimulating factor (GM-CSF) and is released into the circulating pool to fulfil its normal and vital functions of bacterial and fungal surveillance and phagocytic removal. In recent years, molecular steps underlying bone marrow release, neutrophil migration, pathogen recognition, phagocytosis, degranulation, activation of NADPH oxidase, and the precise mechanisms underlying pathogen destruction have been elucidated. Neutrophils are remarkably well adapted to survive and function in highly acidic and hypoxic environments such as an abscess cavity or infected joint space, in part through their capacity for anaerobic glycolytic metabolism. The numerical dominance, migratory potential, and highly destructive capacity of these cells results in their frequent and pathogenic role in many inflammatory diseases, the most notable being acute respiratory distress syndrome (ARDS), inflammatory bowel disease, and rheumatoid arthritis. In addition, a more prominent role has recently been attributed to the neutrophil in allergic diseases, in particular in the development of asthma exacerbations and in steroid-resistant asthma. New studies have also highlighted additional functions for the neutrophil, including the production of neutrophil extracellular traps comprising granule proteins and chromatin designed to trap and neutralize extracellular bacteria, a role in early antigen processing and transfer to dendritic cells, and a role in facilitating the migration of other inflammatory cells such as the eosinophil. In vivo trafficking studies using 111In- and 99mTclabeled autologous neutrophils have now defined the major role of the spleen, liver, and bone marrow in human neutrophil margination, and complementary studies in mice have shown the potential for a minor fraction of neutrophils to migrate from an inflamed site back into the circulatory pool. The capacity for neutrophils to injure tissues demands tight regulatory control

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

via the processes of priming (which dictates the speed and magnitude of NADPH oxidase or respiratory burst response) and apoptosis (which regulates the functional longevity and clearance route for these cells). This latter pathway is now established as a major mechanism underlying the removal of neutrophils from the inflamed site, a process that involves recognition and uptake by inflammatory and tissue resident macrophages. The monitoring of neutrophilic inflammation and the therapeutic targeting of these cells, both to inhibit activation and to facilitate apoptotic clearance, remain important research targets.

Introduction Neutrophil granulocytes are the most abundant white blood cells and originate from the myeloid series in the bone marrow. They have an average volume of 330 fL and a mean diameter of 10 μm (Schmid-Schonbein et al. 1980). Together with the eosinophil and basophil they form the class of polymorphonuclear cells (PMN), so called because their nuclei have a characteristic multilobulated shape (Fig. 13.1) in contrast to the mononuclear white blood cells (monocytes and lymphocytes) whose nuclei are unsegmented. In health the normal peripheral blood neutrophil count is 2.5–7.5 × 109/L. Circulating neutrophils are short-lived cells with a half-life of just 6–8 hours. As a consequence, neutrophils must be continually replenished from hematopoietic stem cells within the bone marrow by the process of granulopoiesis. The bone marrow manufactures approximately 1011 neutrophils per day, with the capacity to increase production still further when required. The initial stages of neutrophil development involve the commitment and differentiation of stem cells through pluripotent common myeloid progenitors and bipotent granulocyte–macrophage progenitors toward unipotent progenitors committed to granulocyte lineage (Fig. 13.2). The early stages of neutrophil development are governed by a transcriptional program that progressively downregulates the genes associated with multiple lineages (Akashi et al. 2003); terminal differentiation into bone marrow neutrophils is directed by a transcriptional program that promotes cell

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Inflammatory Cells and Mediators cycle exit and acquisition of the neutrophil-specific proteins that enable these cells to fulfil their role within the framework of the overall immune system (Theilgaard-Monch et al. 2005).

Neutrophil structure

Fig. 13.1 Electron micrograph of an activated human neutrophil granulocyte showing the multilobed nucleus and cytoplasmic granules.

Antimicrobial and proteolytical granule proteins

Bone marrow Promyelocytes Cell cycle Metamyelocytes PM Exit MY

Human neutrophils are defined by the segmentation of their nucleus into three to five lobes connected by heterochromatin filaments. The physiologic purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation, although a role for the centrosome and associated microtubules has been suggested in the latter process (Olins & Olins 2005). Nuclear segmentation occurs during granulocyte development and maturation. In myeloid leukemias, the normal differentiation process is arrested and hence immature cells with large rounded nuclei may be seen in the circulation. During severe sepsis, neutrophils may be released from the bone marrow before completing nuclear maturation; such ‘band’ cells have indented rather than segmented nuclei. When maturation is delayed, as in megaloblastic anemia, hypersegmentation may occur. Finally, during apoptosis, dramatic chromatin condensation occurs to produce the characteristic dense and pyknotic nucleus of programmed cell death.

Blood PMN

az–GP3 Azurophil-granule proteins SPe GPs Specific gel GPs

Cytokines and chemokine

Receptors

Apoptosis Cell cycle

Gelatinase CDK2, 4, 6 E2F target genes

Death Rec. pathway TLR-2 apoptosis pathway

P53 pathway

Rec. for multiple lineage HGF

(Hematopoietic growth factors)

Rec. for proinflammatory cytokines/chemokines

Proinflammatory cytokines and chemokines

Fig. 13.2 Neutrophil origins and ontogeny. GP, granule protein. (Adapted from Theilgaard-Monch et al. 2006, with permission.)

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Neutrophils have been called the secretory organs of the innate immune response and contain heterogeneous and numerous populations of secretory granules. The manufacture of these granules and their contents is regulated by cytokines and transcription factors, many of which are shared with the neutrophil differentiation program; this ensures that granules develop at the correct stage of cell maturation (Gullberg et al. 1999). Neutrophil differentiation is characterized by the sequential formation of azurophil (primary), specific (secondary), and gelatinase (tertiary) granules, and this is synchronized with the synthesis and packaging of their specific granule proteins (Fig. 13.2). This has given rise to the “targeting-by-timing” hypothesis, which states that the packaging of proteins into distinct granule subtypes is determined principally by the time of their biosynthesis. Therefore granule proteins are characterized both by gene expression during neutrophil development and by subcellular localization (Faurschou & Borregaard 2003). Azurophil granules contain proteins that are released into the phagocytic vacuole at the end of the engulfment process or are secreted onto opsonized surfaces, including proteinases, hydrolases, and microbicidal proteins such as defensins and bactericidal/ permeability-inducing protein. Specific granules incorporate enzymes such as lysozyme and collagenase together with lactoferrin and vitamin B12-binding protein, and also provide an important intracellular reserve of membrane components. There appear to be several subtypes of tertiary granules, containing enzymes such as gelatinase and lysozyme, plus membrane components including constituents of the NADPH oxidase, in a form that can be rapidly mobilized to the cell surface (Faurschou & Borregaard 2003). It is likely that most granule proteins have evolved to aid the neutrophil’s rapid migration through tissues and to promote effective and targeted bacterial killing; it can be readily appreciated, however, that many of these agents can be highly toxic to normal tissue if released in an uncontrolled manner. The plasma membrane is of great importance to this highly motile and responsive cell, since it contains the receptors used to detect inflammatory events – such as adhesion molecules and ligands enabling interactions with the activated endothelial cell surface, pathogen-recognition receptors including Toll-like receptors (TLRs), and inflammatory cytokine receptors – and the molecules involved in the transduction of external signals into cellular responses, including locomotion, phagocytosis, secretion, and the oxidative burst. Therefore intimate communication arrangements must exist between the membrane and the granule populations that replenish these membrane components. The neutrophil also possesses a highly developed cytoskeletal framework responsible for coordinating its complex motility functions (see below). It was previously believed that neutrophils possess few or even no mitochondria: neutrophils utilize glycolysis rather than oxidative phosporylation to generate energy, mitochondrial poisons such as cyanide have little effect on neutrophil

function, and few intact mitochondria are evident on electron microscopy of neutrophil specimens. However, the use of mitochondrial-specific dyes has confirmed that mitochondria do exist in neutrophils, organized as a tubular network (Fossati et al. 2003). Functionally their role seems to be largely restricted to the regulation of apoptosis (Maianski et al. 2004). The endoplasmic reticulum (ER) is the largest endomembrane system within eukaryotic cells, and subserves a variety of functions, including calcium uptake and release, protein and lipid synthesis, posttranslational protein modification and export to the Golgi apparatus. Terminal differentiation of myeloid cells is associated with a decrease in ER content (Clark et al. 2002), perhaps reflecting decreased protein synthetic activity. Despite this reduction, mature neutrophils retain a plentiful ER network, participating in the calcium signaling pathways that are of fundamental importance to these rapidly responsive cells; it is also sufficient to induce the ER stress or unfolded protein response in patients with mutations in neutrophil elastase, with the resultant activation of pro-apoptotic signaling pathways producing congenital and cyclical neutropenia syndromes (Kollner et al. 2006). Likewise, while the Golgi apparatus is more prominent in immature neutrophils consistent with its importance in granule packaging and transport, it still has a role in maintaining homeostasis in the terminally differentiated cell; for example, monomeric lipopolysaccharide (LPS) is delivered to the neutrophil Golgi apparatus for processing, and defective function of the glycosylation/fucosylation enzymes located within the Golgi membrane results in leukocyte adhesion deficiency (LAD) type II, in which abnormal processing of selectin ligands leads to a profound defect in leukocyte rolling and hence an immunodeficient state (Etzioni & Tonetti 2000). Thus neutrophil development is tailored to produce a terminally differentiated cell, uniquely adapted to its role. Its highly developed and rapidly responsive cytoskeletal network allows rapid recruitment to sites of infection and the efficient engulfment of bacteria, the lack of dependence on mitochondrial respiration permits optimal function even in profoundly hypoxic environments, and the rapid mobilization of preformed granules and assembly of the NADPH oxidase complex allows rapid and efficient bacterial killing. The neutrophil is truly “fit for purpose.”

Neutrophil origin, maturation, and bone marrow release The production of neutrophils accounts for more than half of the cellular output of the bone marrow. The development of mature neutrophils from CD34+ stem cells typically takes 14 days and is dependent on growth factors such as IL-3, GM-CSF, and G-CSF, the latter being available for use as a therapeutic agent to enhance neutrophil production in vivo (Bai et al. 2005). Stromal cells in the marrow appear to be a

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particularly important source of these factors (Takagi 2005). In addition to this constitutive role, stromal cells are of great importance in the control of inflammation, as their release of growth factors is exquisitely sensitive to cytokines such as IL-1 and tumor necrosis factor (TNF)-α. Thus granulopoiesis can be increased several fold as part of the systemic response to infection and inflammation (Burman et al. 2005). Granulopoiesis is also subject to negative regulatory feedback. Neutrophils that have migrated into tissue compartments undergo apoptosis and are phagocytosed by resident macrophages and dendritic cells, with resultant suppression of IL-23, IL-17 and hence G-CSF production; neutrophils from mice lacking β2 integrins exhibit reduced tissue migration, hence this regulatory loop is lost, granulopoiesis is enhanced, and the mice have a persistent neutrophilia unrelated to infection (Stark et al. 2005). The events underlying neutrophil release from the bone marrow are also becoming clearer, with opposing roles for CD18 and CD49d (VLA4) in mediating retention and release respectively demonstrated in a rat model (Burdon et al. 2005). Neutrophil responses are regulated in large part by cytokines, chemokines, and microorganisms signaling through specific cell-surface receptors. The ontogeny of receptor expression during the different stages of neutrophil maturation has recently been analyzed by cDNA microarray (Theilgaard-Monch et al. 2006). Receptor expression for many inflammatory stimuli is very low in neutrophil progenitors but increases progressively as the cell matures, with terminally differentiated bone marrow neutrophils expressing a wide repertoire of cytokine, chemokine, growth factor, and pathogen-recognition receptors. Among these are the interferon receptors (αR-1 and -2, γR-1 and -2), interleukin receptors (IL-1, -4, -6, -10, -13, -17, -18), TNF receptors (TNF-R1 and -R2), and CXC and CC chemokine receptors (CXCR-1, -2, -4 and CCR-1, -2 , -3 respectively). In addition, the expression of a number of proinflammatory cytokines and chemokines is also increased in mature neutrophils, including IL-1, TNF-α, IL-8, and GRO-α. Thus overall the expression pattern for receptors and inflammatory cytokines suggests a marked increase in responsiveness toward inflammatory stimuli during terminal differentiation (Theilgaard-Monch et al. 2005).

Neutrophil kinetics The proportion of neutrophils circulating freely comprises only a small fraction of the total neutrophil cell pool. The bone marrow contains a large number of mature and nearmature neutrophils available for mobilization, and neutrophils within the vascular compartment also reside in the so-called “marginated” pools of the spleen, liver and bone marrow as well as in the active circulation. Margination represents prolonged physiologic transit of neutrophils through these organs with, for example, mean transit times in the

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spleen of 10 min. The mechanisms regulating the migration, capture, and release of neutrophils from these organs are poorly understood. Likewise, the function of the marginated neutrophil pool is unclear, although the rapid release of cells back into the active circulation from these sites through the shedding of CD62L may provide a mechanism for elevating neutrophil numbers acutely during stress or injury. Splenic transit may also afford a mechanism for sensing and removing damaged cells and/or marking them for subsequent removal. The presence and true size of a marginated pool within the lung is debated; early experiments showing significant neutrophil retention in the pulmonary circulation may have been influenced by inadvertent neutrophil priming during cell isolation (Peters 1998). Radiolabeled neutrophils leave the blood in an exponential and apparently random manner with a half-life of approximately 7 hours. This corresponds to a mean intravascular lifespan of 10 hours and a physiologic disposal rate of approximately 108 cells/min. In health the sites of neutrophil disposal are restricted to the reticuloendothelial system (RES), in particular the liver and bone marrow. Hematologic analysis of healthy subjects has failed to identify any evidence of circulating apoptotic cells, which suggests either a very efficient clearance mechanism for such cells or, more likely, that this program of cell death is not activated within the circulation. Once neutrophils undergo apoptosis in the RES they are recognized and removed by tissue resident macrophagelike cells. A study by Shi et al. (2001) has indicated that P-selectin expression on hepatic endothelial cells is functionally important in the clearance of neutrophils by Kupffer cells, which have been shown to contain apoptotic bodies. Neutrophil expression of CXCR4 and the chemotactic response to its cognate ligand SDF-1α have been shown to influence the release of mature neutrophils from the bone marrow and their subsequent return. Newly released neutrophils are low expressers of CXCR4, and in mice it has been shown that as neutrophils age they upregulate this receptor and acquire the ability to migrate toward SDF-1α, which is constitutively expressed by bone marrow stromal cells. Senescent CXCR4high neutrophils therefore appear to migrate back to the bone marrow and allow efficient clearance of effete cells from the circulation (Martin et al. 2003). Whether this mechanism exists in humans has not yet been determined.

Physiologic role of the neutrophil Enormous numbers of short-lived neutrophils are produced by the bone marrow, and are only rarely called upon to protect against significant microbial invasion. This high-turnover system allows a very rapid increase in neutrophil numbers following insult or injury to the host. Neutrophils are highly chemotactic and hence are usually the first immune cells to

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reach an inflamed site where, using their oxidative responses and array of granule proteins, they are rapidly effective in ingesting and killing fungal and bacterial pathogens. The ability of the neutrophil to employ glycolytic metabolism and thus to migrate into, and function and survive within, the highly hypoxic and acidic environment characteristic of an inflamed site is likewise an important aspect of their adaptable nature. Until quite recently the neutrophil was widely perceived as being an immutable end-stage cell with the sole capacity of detecting and removing bacteria, before finally disintegrating. However, it is now clear that the neutrophil is a key player in several aspects of the evolving inflammatory response. Moreover, each stage of its behavior at the inflamed site, including its final removal, has the potential to be finely controlled in order to limit incidental injury to normal tissues and to facilitate the resolution of inflammation and tissue repair, so that tissues can be restored to their normal homeostatic function once the bacterial invasion has been wiped out. For example, (i) the neutrophil can be involved in the initial generation of inflammatory edema (see below), (ii) the migration of eosinophils and monocytes appears to depend on the initial migration of neutrophils (Kikuchi et al. 2006), (iii) neutrophils play an important role in antigen digestion and processing (reviewed by Appelberg 2007), and (iv) it is now recognized that the neutrophil can generate a variety of important cytokines and chemokines that may amplify inflammation and attract additional cells to the inflamed site. It is also clear that different aspects of neutrophil behavior during the inflammatory response, including sequestration, endothelial adhesion, capillary transmigration, bacterial phagocytosis, oxidative burst, degranulation and removal, are interlinked. Exposure of the neutrophil to physiologic concentrations of inflammatory cytokines such as LPS, GM-CSF, or TNF-α engenders a “primed” state in which the functional responses [phagocytosis, degranulation, and generation of reactive oxygen species (ROS)] to subsequent stimuli are greatly upregulated (reviewed in Downey et al. 1995 and Condliffe et al. 1998) and also profoundly influences neutrophil longevity (Fig. 13.3). This dual effect may augment the ability of the innate immune response to clear a bacterial challenge, but may likewise underlie the tissue-damaging potential of the neutrophil in inflammatory disease.

Neutrophil recruitment to inflamed sites Neutrophil recruitment to an inflammatory site is a complex multistep process involving coordinated and sequential interactions of neutrophil adhesion molecules and ligands with those of vascular endothelium. Failure of recruitment, as exemplified by LAD [due to deletion of the gene coding for the β2 integrins (LAD type I) or failure to process the glycoprotein ligands recognized by the selectin adhesion

Priming/activation

Apoptosis

Injury

Resolution

Fig. 13.3 Regulation of neutrophil function and longevity. Mature neutrophils are poorly responsive to secretagogue agonists unless first exposed to a priming agent, such as lipopolysaccharide, granulocyte–macrophage colony-stimulating factor or platelet-activating factor; these agents on their own cause minimal activation but serve to induce cell polarization and substantially upregulate the cell’s secretory response to a second agent. Apoptosis, in contrast, is a constitutive process designed to limit the functional longevity of cells and aid macrophage clearance and the resolution of neutrophilic inflammation. (See CD-ROM for color version.)

molecules (LAD type II)], leads to recurrent bacterial infections, impaired wound healing, and early death. In response to an inflammatory (allergic or infective) stimulus, resident cells such as alveolar macrophages and mast cells release cytokines and chemokines including TNF-α and IL-8, which modify the local microcirculatory environment and lead to upregulation of vascular endothelial adhesion molecules. In addition, neutrophils exposed to circulating inflammatory mediators or such molecules presented on the endothelial surface themselves become “stickier” and less deformable. These two processes result in neutrophil sequestration and transmigration through the endothelial barrier. Neutrophils subsequently migrate toward the inflammatory focus by detecting and moving along concentration gradients of chemoattractant molecules (chemokines, complement components and bacterial products), a process known as chemotaxis. When the migrating neutrophil is exposed to higher concentrations of chemoattractants it becomes immobilized and an array of “activation” responses are progressively engaged, preparing the cell to phagocytose and kill invading bacteria.

Neutrophil sequestration and adhesion Egress of neutrophils from the circulation depends on the sequential interactions of cellular adhesion molecules (CAMs)

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(Fig. 13.4) and, in some circumstances, on factors influencing neutrophil deformability. The relative contribution of individual components of the recruitment cascade may vary according to the stimulus and site involved. In the systemic circulation following insults such as ischemia–reperfusion (Zahler et al. 2002), ventilator-induced lung injury (Miyao et al. 2006), and mast cell degranulation (Kubes & Kanwar 1994), neutrophils roll along the wall of the postcapillary venule. Rolling is principally mediated by the interaction of the selectin family of CAMs with their glycoprotein ligands (Fig. 13.4b); stored endothelial P-selectin is mobilized to the endothelial cell surface by inflammatory stimuli (McEver et al. 1989), including thrombin, histamine and complement, and binds to P-selectin glycoprotein ligand (PSGL)-1, which

is constitutively expressed on the cell surface of neutrophils, eosinophils, monocytes, and lymphocytes. The interaction of endothelial E-selectin and neutrophil L-selectin with their respective ligands also contributes to rolling (Jung & Ley 1999) (Fig. 13.4b), as may certain integrins such as the α4 integrins (Barringhaus et al. 2004). During severe sepsis, rolling of neutrophils may be compromised (Swartz et al. 2000), perhaps due to shedding of L-selectin from the neutrophil surface induced by inflammatory cytokines (Condliffe et al. 1996). Rolling neutrophils “taste” the endothelial surface and integrate a range of signals to determine whether they will detach or whether their rolling velocity will decrease, enabling initiation of firm adhesion and transendothelial migration (reviewed by Ley 2002). Factors that promote adhesion

G protein-coupled receptor Neutrophil L-selectin PSGL-1 (P-selectin ligand) VE-cadherin (a)

Endothelial L-selectin ligand P-selectin b2 integrin (inactive conformation) b2 integrin (active conformation) Chemoattractant Cytokine (b)

Cytokine receptor

ICAM-1 (c)

↑[Ca2+]

retraction

Junctional adhesion molecule (JAM) PECAM-1

(d)

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Fig. 13.4 Neutrophil adhesion molecules and recruitment (rolling, firm adhesion, and transmigration). (a) Circulating neutrophils do not interact with quiescent endothelial cells. (b) Cytokines such as TNF-a (here shown derived from inflammatory macrophages) activate the vascular endothelium to express P-selectin, which interacts with neutrophil P-selectin glycoprotein ligand (PSGL)-1 to induce rolling. Interaction of other selectins with their ligands may contribute to rolling. Chemokines (such as IL-8) may be bound by the endothelium and with circulating inflammatory mediators lead to activation of b2 integrins. (c) The activated endothelium expresses intercellular adhesion molecule (ICAM)-1, which binds activated b2 integrins on the neutrophil surface, leading to arrest and firm adhesion. (d) Endothelial cells adjacent to adherent neutrophils may experience a rise in intracellular calcium, leading to endothelial retraction. The interaction of neutrophils with endothelial junctional adhesion molecules (JAMs), of neutrophils with endothelial PECAM-1, and of integrins with their ligands may all encourage transmigration, which preferentially occurs at sites in the basement membrane with low protein expression. (See CD-ROM for color version.)

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include circulating inflammatory cytokines, interaction of selectins with their corresponding glycoprotein ligands, and interaction of neutrophil receptors with so-called “arrest chemokines” such as IL-8 and platelet-activating factor (PAF) tethered to the endothelial surface (Lorant et al. 1991; DiVietro et al. 2001; Laudanna & Alon 2006) (Fig. 13.4b). These varied inputs summate to induce a conformational change in the neutrophil β2 integrins (principally LFA-1 and Mac-1). In their active confirmation, these integrins bind to endothelial intercellular adhesion molecule (ICAM)-1, whose expression is substantially upregulated by inflammatory cytokines such as TNF-α and IL-1. Binding of neutrophil β2 integrins to ICAM-1 leads to firm adhesion to the endothelial surface (Lawrence & Springer 1991; Phillipson et al. 2006) (Fig. 13.4c), and induces further intracellular signaling events to prepare the neutrophil for transmigration. In the lung, diseases such as asthma and bronchiectasis affect the proximal airways, which are fed by the bronchial circulation, while the distal bronchi, bronchioles and alveoli (relevant to processes such as acute lung injury and bacterial pneumonia) derive their blood supply from the pulmonary circulation (Doerschuk 2000). Recruitment of neutrophils from the bronchial circulation is likely to follow the rolling– tethering–transmigration paradigm outlined above, and increased expression of relevant CAMs (including selectins and ICAM-1) has been demonstrated in the bronchial microvasculature in disease processes, for example in asthmatic versus nonasthmatic lungs (Feuerhake et al. 1998; Hirata et al. 1998). In contrast, in the pulmonary circulation, neutrophils emigrate from the pulmonary capillary bed, where spatial constraints preclude rolling, and even under normal flow conditions neutrophils must deform to squeeze through the narrow pulmonary capillaries (Doerschuk et al. 1993). This leads to a modest prolongation in neutrophil transit time in the lung under physiologic conditions (see above); however, inflammatory challenge has a major effect on the biomechanical properties of neutrophils and results in a significant increase in the sequestration of these cells in the pulmonary microvasculature. In response to inflammatory cytokines, and in a model of bacterial pneumonia (Yoshida et al. 2006), neutrophils become stiffer and less deformable due to changes in the actin cytoskeleton and such stiffened cells are preferentially sequestered in pulmonary capillaries. Once the neutrophils have arrested they may subsequently exit the capillary, and the dependence of this process on CAMs is highly variable according to the causative inflammatory insult (Doerschuk 2000; Reutershan & Ley 2004). Many studies on neutrophil recruitment have been performed in vitro using isolated cell populations. More recently, the role of other cellular components of the immune system in this process has been investigated in vivo, revealing a new layer of complexity. Neutrophils may be recruited to the lungs by the products of alveolar macrophages or mast cells, but they are themselves a potent source of cytokines and chemo-

kines, thus initiating a possible feed-forward inflammatory loop. Neutrophil–platelet aggregates have been detected in many inflammatory conditions and such aggregates may be more prone to sequestration due to their biomechanical properties; additionally, platelets adherent to the endothelium may interact with circulating leukocytes to promote their recruitment. Additionally, platelets and neutrophils may activate each other and there are intriguing suggestions that such interactions may enhance neutrophil-mediated tissue injury (Singbartl et al. 2001). Finally, as noted above, neutrophils may also promote the recruitment of eosinophils under certain conditions, thereby aggravating allergic inflammatory states (Kikuchi et al. 2006).

Neutrophil transmigration To exit the circulation, adherent neutrophils squeeze between endothelial cells and through the basement membrane and pericyte layer (diapedesis), usually with minimal disruption of vascular integrity. Engagement of leukocyte–endothelial cell adhesion molecule interactions leads to “loosening” of the interendothelial cell junctions with loss of VE-cadherin (Shaw et al. 2001) to allow the leukocyte to transmigrate (Fig. 13.4d). Endothelial cell-selective adhesion molecule (ESAM) has likewise been implicated in triggering the opening of endothelial cell contacts (Wegmann et al. 2006). Sequential homophilic interactions between PECAM-1 and CD99 expressed on both leukocyte and endothelial cells, plus interactions of endothelial cell junctional adhesion molecules (JAMs) with neutrophil JAMs and additionally neutrophil integrins, also seem to be integral to this process (reviewed by Muller 2003) (Fig. 13.4d). Endothelial cells adjacent to transmigrating neutrophils experience a rise in intracellular free calcium, which leads to endothelial retraction and is essential for diapedesis to occur. The second key barrier of the venular wall that the neutrophil must traverse is the perivascular basement membrane (BM). Transmigrated neutrophils express the protease elastase on their surface (Wang et al. 2005) and proteolytic cleavage of BM constituents such as laminin is a possible (though not universally accepted) mechanism by which leukocytes are thought to penetrate this membrane. In addition, neutrophils seem to utilize areas of the BM with reduced expression of matrix proteins as specific exit points (Wang et al. 2006).

Neutrophil chemotactic factors Interaction of chemoattractant molecules with specific neutrophil cell-surface receptors engages signaling mechanisms that result in the development of polarity and directional movement. The principal groups of neutrophil chemoattractants are (i) products of the complement (C5a) and eicosanoid (LTB4 and PAF) cascades, (ii) bacterial-derived peptides (classically N-formyl-methionyl-leucyl-phenylalanine or fMLP) and (iii) members of the chemokine family.

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Allergen–antibody complexes stimulate mast cells and macrophages to release LTB4, a potent chemoattractant for both neutrophils and eosinophils. Neutrophils also release LTB4 on stimulation, conferring the potential for positive feedback. Studies in mice lacking the LTB4 receptor BLT1 (Medoff et al. 2006) demonstrate that it is required for the early recruitment of both eosinophils and neutrophils to the airways following allergen challenge, but that the later phases of granulocyte recruitment and the development of airway hyperresponsiveness were not affected. Mechanisms leading to airway inflammation in asthma are likely to be complex and redundant, and it is possible that the LTB4 pathway may play a more important role in those asthmatics with neutrophil-predominant inflammation. fMLP and several other bacterial-derived and synthetic peptides bind the high-affinity formyl peptide receptor (FPR) (Selvatici et al. 2006) and mice lacking this receptor exhibit impaired antibacterial host defense (Gao et al. 1999). More recently, annexin 1 (lipocortin 1) has been identified as an endogenous FPR agonist (Walther et al. 2000). The loweraffinity FPR homolog FPR1 has also now been found to interact with other host-derived chemotactic agonists, including serum amyloid A (Su et al. 1999), peptides derived from prion protein (Le et al. 2001), and the neutrophil-granule protein cathelicidin (Yang et al. 2000). The significance of these endogenous agonists for FPRs is as yet uncertain, but they suggest a much broader role in inflammatory processes than has been hitherto appreciated. Chemokines are an extensive family of small heparinbinding proteins that direct the movement of leukocytes in both physiologic and pathologic situations (Baggiolini & Loetscher 2000; Charo & Ransohoff 2006). There are approximately 50 chemokines organized into four families: the CC chemokines (where the first two of four cysteine residues are adjacent to each other) act on T cells, monocytes, eosinophils, basophils, mast cells, and dendritic cells, while some CXC chemokines (where a single amino acid is interposed between the first two cysteine residues) are powerful neutrophil chemoattractants. Neutrophils express the chemokine receptors CXCR1 and CXCR2, enabling them to bind CXCL8 (IL-8, the prototypical neutrophil chemokine), CXCL1 (Gro-α), CXCL2 (Gro-β), CXCL5 (ENA-78), CXCL3, and CXCL6. Classically, IL-8 has been implicated in the pathogenesis of ARDS (Donnelly et al. 1993), but elevated IL-8 in sputum or bronchoalveolar lavage (BAL) fluid has also been detected in adults suffering acute severe asthma, where it correlates with the presence of a predominantly neutrophilic inflammatory response (Fahy et al. 1995; Lamblin et al. 1998; Tonnel et al. 2001). Elevated IL-8 in sputum is also associated with markers of epithelial damage and with neutrophil elastase in children with asthma exacerbations (Yoshihara et al. 2006). Finally, IL-8 has been found to be increased in allergic bronchopulmonary aspergillosis and to be correlated with airway neutrophilia and airway obstruction (Gibson et al. 2003). Whether these signs

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of neutrophil mobilization in the airway lumen reflect an increased number of neutrophils in the surrounding airway tissue has not yet been determined. Nonetheless it seems that chemokines, and in particular IL-8, have an important role in mediating the recruitment of neutrophils in several inflammatory and allergic disease states, and have hence been identified as potential therapeutic targets (see below).

Neutrophil chemotaxis Neutrophils exiting the circulation may be exposed to gradients of the chemoattractant molecules described above, in which case they develop polarity (i.e., form distinct front and back ends) and migrate in amoeboid fashion by extension of pseudopods at the leading edge, enabling directed movement toward the source of the stimulus (chemotaxis). The development of polarity is fundamental to chemotaxis and is associated with a dramatic redistribution of cytoskeletal proteins, with enrichment of F-actin at the leading edge and assembly of myosin posteriorly (reviewed by Parent 2004 and Niggli 2003). Chemoattractants generate intracellular signals by binding transmembrane receptors coupled to heterotrimeric G proteins. This binding induces dissociation of the G protein into α and βγ subunits, which activate an array of downstream effectors. While both receptors and G proteins are uniformly or near-uniformly distributed over the cellular surface (Servant et al. 1999; Jin et al. 2000), the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), the product of class 1 phosphatidylinositol 3-kinase (PI3K), accumulates at the leading edge (Fig. 13.5), and this seems to underpin subsequent responses (Servant et al. 2000). Studies using knockout mice have suggested a critical role for the PI3K-γ isoform in this process (Hirsch et al. 2000; Li et al. 2000; Sasaki et al. 2000) and PI3K-δ has also been implicated by studies employing isoform-specific PI3K inhibitors (Sadhu et al. 2003); both PI3K-γ and PI3K-δ are highly expressed in neutrophils. Precisely how the internal PIP3 gradient is generated remains unclear, but it may involve selective recruitment of PI3Ks to the leading edge together with a concentration of phosphatases that metabolize PIP3 (PTEN in Dictyostelium, SHIP-1 in mammalian neutrophils) at the trailing edge (Li et al. 2003; Nishio et al. 2007). PIP3 activates the small GTPases Rac and CDC42, which relay signals to the actin cytoskeleton resulting in actin polymerization and pseudopod protrusion (Srinivasan et al. 2003) (Fig. 13.5); activated Rac may additionally promote PIP3 formation in a feed-forward loop, reinforcing cellular polarity (Weiner et al. 2002). At the rear of the cell, in the absence of predominant PIP3-directed signaling, activation of the small GTPase RhoA seems to be important in organizing myosin-based contraction and retraction of the cell posterior (Xu et al. 2003) (Fig. 13.5). The net result of this spatially and temporally coordinated signaling process is the highly purposeful movement of the neutrophil to the source of the inflammatory cascade at speeds of up to 20 μm/min.

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Formation of protrusions at leading edge; directional locomotion

PIP3

PIP2

Ga/Gbg Ga/Gbg

PI3Kg/d Gbg

F-actin polymerization

GTP-Rac GTP-Cdc42

Direction of movement

Myosin activation

SHIP-1 PtdIns (3,4)P2

GTP-RhoA

PIP3

Tail retraction Rear release Allows directional locomotion

Fig. 13.5 Chemotaxis signaling in neutrophils. Stimulation of G protein-coupled receptors by chemoattractants leads to stimulation of phosphatidylinositol 3-kinase (PI3K)-g or PI3K-d, with the generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), activation of small GTPases (Rac and CDC42), F-actin polymerization, and hence the development of a leading edge. At the rear of the cell, PIP3 is metabolized by phosphatases such as SH2-containing inositol phosphatase (SHIP)-1, and activation of myosin leads to the development of a rear end or uropod. The net result of these diverse signaling events is the development of polarity and directional migration toward the concentration gradient. PIP2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate. (See CD-ROM for color version.)

Neutrophil activation Neutrophil phagocytosis Neutrophils and macrophages are professional phagocytes, highly evolved to recognize, engulf, and eliminate invading pathogens (Lee et al. 2003). Pathogen recognition occurs via TLRs, which initiate proinflammatory signaling pathways to trigger the innate immune response (reviewed in Wagner & Bauer 2006 and Pinheiro & Ellar 2006). TLRs identify molecular motifs of infectious agents (pathogen-associated molecular patterns); so far, expression of 10 TLRs is known in humans. The best characterized is TLR4, which recognizes LPS. Bacterial lipopeptides are recognized by TLR-1, TLR-2, and TLR-6; foreign (viral and bacterial) DNA is sensed by

TLR-3, TLR-7, TLR-8, and TLR-9; and various bacterial, protozoal, and yeast proteins are sensed by TLR2 and TLR5. The adapter proteins, myeloid differentiation factor 88 and Toll IL-1 receptor (TIR) domain-containing adapter inducing interferon (IFN)-β, are the key players in the TLR signaling cascade leading to the activation of NF-κB and interferon regulatory factor-3. Ingestion by the phagocyte is usually greatly facilitated if the target is coated (opsonized) by specific immunoglobulins (Fig. 13.6a) or by complement components (C3b), with immunoglobulins binding to neutrophil Fc receptors, principally FcγRIIa (CD32) and FcγRIIIb (CD16), and complement components binding to the β2 integrin CD11b/CD18. Receptor clustering initiates signaling events (unique to the receptor engaged), resulting in particle engulfment. In the case of Fcγ receptors, receptor phosphorylation by src-family kinases leads to recruitment and activation of the tyrosine kinase Syk (Kiefer et al. 1998), with subsequent stimulation of PI3K and local production of PIP3 at the evolving phagocytic cup. As during chemotaxis, PIP3-directed activation of Rac1 and CDC42 are thought to be required for actin assembly and hence pseudopodial extension; PIP3 additionally leads to local activation of phospholipase C (PLC) and protein kinase C (PKC), which have also been implicated in the uptake process. Since professional phagocytes can engulf multiple particles, large volumes of surface membrane may be internalized by a process that is compensated for by the delivery of cellular endomembranes to the vicinity of the nascent phagosome (Bajno et al. 2003). The newly formed and sealed phagosome is not initially antimicrobial, but must undergo maturation to acquire microbicidal enzymes and components of the NADPH oxidase (see below) that enable killing and disposal of the ingested microorganisms. In addition to their “classical” role in phagocytosis and pathogen destruction, neutrophils have also been shown to express molecules associated with antigen presentation, including major histocompatibility complex class II (DR) antigen (Sandilands et al. 2003); moreover, these molecules are translocated to the plasma membrane on treatment of neutrophils by a variety of stimuli. In the case of some pathogens, including Listeria monocytogenes, neutrophils have been shown to provide nonsecreted bacterial antigens for crosspresentation to the adaptive immune system (Tvinnereim et al. 2004) in addition to their role in innate defense against infection.

Neutrophil degranulation Neutrophil granules and secretory vesicles constitute an important reservoir of antimicrobial proteins, proteases, components of the NADPH oxidase, and a wide range of receptors for adhesion molecules, matrix proteins and bacterial products (Faurschou & Borregaard 2003). Regulated granule exocytosis is intimately involved in all aspects of neutrophil function, including recruitment, phagocytosis and

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SG

p22

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AG GG p91

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G protein-coupled receptor

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SG

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p91 p47 P p40 p22 p67 P GTP-Rac2

ROS release P p91 p40 p67 P p47 p22 P GTP-Rac2

(b)

killing, while dysregulated extracellular release of granule contents contributes to host cell damage and organ dysfunction in disease processes. All granule subsets are defined by a phospholipid bilayer that encompasses an intragranular matrix, but the proteins within the matrix differ between the granule subsets (Table 13.1). As introduced earlier, primary (azurophil) granules bud off from the Golgi apparatus in promyelocytes, are characterized by the presence of myeloperoxidase (MPO), and contain the structurally related serine proteases neutrophil elastase, cathepsin G, and proteinase-3. Secondary (specific) granules and tertiary (gelatinase) granules are formed as a continuum in myelocytes, metamyelocytes, band cells, and segmented neutrophils, but differ in protein content and secretory properties. Specific granules are rich in antibacterial proteins

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O2–

Fig. 13.6 Phagocytosis, degranulation, and assembly of the neutrophil’s NADPH oxidase. (a) In a resting neutrophil, the components of the NADPH oxidase reside in the cytoplasm (p47phox, p67phox, p40phox, and GDP-Rac2) or on secretory vesicle (SV) membranes (gp91phox, p22phox). Granule populations (azurophil granules, AG; gelatinase granules, GG; specific granules, SG) are distributed throughout the cytoplasm. (b) On phagocytosis of a bacterium (B), components of the oxidase become activated (phosphorylation of p47phox and p67phox, conversion of GDP-Rac2 to GTP-Rac2) and both the cytoplasmic and membraneassociated proteins are assembled at the phagosomal membrane and reactive oxygen species (ROS) are generated. Granules, particularly protease-containing AGs, are discharged into the phagosome, and ROS promote their liberation from proteoglycan matrix to digest the engulfed bacterium. If the neutrophil is instead stimulated by soluble agonists, these events may occur at the plasma membrane, leading to degranulation and external ROS release. (See CD-ROM for color version.)

such as lactoferrin, transcobalamin, and lysozyme, plus the matrix metalloproteinases (MMPs) gelatinase, collagenase and leukolysin and also a number of membrane receptors and proteins including CD11b/CD18 and cytochrome b558 (Gallin 1985). Tertiary granules contain abundant gelatinase (MMP-9) but no lactoferrin. Secretory vesicles are endocytic vesicles that form a reservoir to replenish plasma membrane lost during phagocytosis, and are also an important source of membrane receptors, which can be mobilized to allow the neutrophil to respond more readily to environmental signals. Such is the functional diversity of these granules that neutrophils can attack bacteria in a number of ways. For example, constituents such as lysozyme exert antimicrobial activity directly by disrupting bacterial membranes, lactoferrin binds and sequesters iron and copper to interfere with the

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Table 13.1 Neutrophil granule constituents. Azurophil

Specific

Gelatinase

Secretory

Membrane proteins

CD63, CD66

CD11b/CD18 (Mac1), CD18, CD66, CD67, Rap, receptors (TNF, fMLP, laminin, fibronectin, vitronectin), cytochrome b558

CD11b/CD18, cytochrome b558, LAMP-2, NRAMP-1, SNAPs, receptors for fMLP IgE, laminin

CD10, CD11b/CD18, CD13, CD14, CD16, CD35, CD45, alkaline phosphatase, cytochrome b558, decay accelerating factor, fMLP receptor, CR1

Proteinases

Elastase, cathepsin G, proteinase-3,

Collagenase, gelatinase, leukolysin

Gelatinase

Enzymes

MPO, lysozyme, azurocidain, neuraminidase

Lysozyme, neuraminidase

MPO, lysozyme

Hydrolases

Cathepsins B and D, bgalactosidase, b-glucuronidase, a-mannosidase, N-acetyl-bglucosaminidase

Lactoferrin, apolactoferrin, vitamin

Inhibitors

a1-antitrypsin, heparin-binding protein

B12-binding protein, transcobalamin-1, histaminase, heparinase

Others

Defensins, BPI, ubiquitin

Lipocalin, b2-microglobulin

Acetyltransferase

Plasma proteins

BPI, bactericidal/permeability-inducing protein; LAMP, lysosome-associated membrane protein; NRAMP, Natural resistance-associated protein; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein. See text for definition of other abbreviations.

metabolic pathways in bacteria, and MPO is both directly toxic and participates in the formation of oxygen radicals (see below). Several of the constituents, for example defensins and azurocidin, are also chemotactic toward CD4 and CD8 T cells, thus amplifying and diversifying the inflammatory response. The intracellular granule populations also differ in their availability for exocytosis and are thus mobilized in a hierarchical fashion (Sengelov et al. 1995). Secretory vesicles have the highest propensity for extracellular release, and are recruited to the cell surface by low concentrations of soluble agonists such as TNF-α, LPS, fMLP, and C5a, enabling the rapid deployment of additional membrane receptors to enhance cellular responses. Tertiary, secondary, and primary granules are progressively less prone to mobilize to the cell surface (a control mechanism reducing the likelihood of extracellular release of the potentially destructive serine proteases). The hierarchical mobilization of neutrophil granule populations can be reproduced in vitro by progressive elevations of intracellular calcium (Sengelov et al. 1993), suggesting that activation of PLC may be critical in controlling this response. Primary granule exocytosis is preferentially targeted to the emerging phagosome, a process that seems dependent on the microtubule network (Tapper et al. 2002) and on the GTPase Rac2 (Abdel-Latif et al. 2004). The precise molecular mechanisms providing this fine control of granule deployment are not

fully understood, but are likely to involve differential granule expression of SNARE (soluble N-ethylmaleimide-sensitive attachment protein receptor) proteins and differences in SNARE complex formation (Mollinedo et al. 2006). In addition to the classical degranulation response, neutrophils have also been shown to release a combination of primary and secondary granule proteins together with chromatin, forming an extracellular fiber network (so-called neutrophil extracellular traps). These neutrophil extracellular traps have been shown to be formed both in vitro and in vivo, to trap bacteria, and to kill both bacteria and fungi (Brinkmann et al. 2004; Urban et al. 2006). While the importance of this recently described phenomenon is as yet unclear, it provides another weapon in the neutrophil armamentarium, and likewise may contribute to the injurious potential of these cells. The essential role of neutrophil proteases in bacterial killing has been confirmed by gene targeting. Mice deficient in neutrophil elastase exhibit profound susceptibility to Gramnegative organisms and to some fungi, but not Gram-positive bacteria (Belaaouaj et al. 1998). While the deletion of cathepsin G alone had little effect on bacterial killing, mice lacking both cathepsin G and elastase are susceptible to both Grampositive and Gram-negative organisms (Tkalcevic et al. 2000), conferring a phenotype similar to that seen in chronic granulomatous disease (CGD), in which there is defective NADPH oxidase function (see below). Thus a combination of a

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functional respiratory burst and an intact degranulation response is required for optimal bacterial killing (see below).

Neutrophil NADPH oxidase The neutrophil NADPH oxidase is a multicomponent enzyme with a redox core that allows electron transfer from cytoplasmic NADPH to intraphagosomal or extracellular molecular oxygen, generating superoxide anions (O2–). The oxidase is composed of membrane-associated (p22phox, gp91phox) and cytosolic (p67phox, p47phox, p40phox, and Rac2) components (see Fig. 13.6a) and is assembled when the neutrophil becomes activated, by translocation of the cytosolic elements to dock with the membrane proteins. In resting cells, the membraneassociated cytochrome b558 (a heterodimer comprising one molecule of p22phox and one molecule of gp91phox) is predominantly located within the intracellular granule membranes (Fig. 13.6a), with only 15% present at the plasma membrane (Calafat et al. 1993); much of the initial oxidase assembly occurs on the intracellular granules, with subsequent movement to the phagosomes or to the plasma membrane (Kobayashi et al. 1998). The molecular interactions between the oxidase components and the mechanisms by which the multimeric oxidase is assembled have been extensively reviewed elsewhere (Roos et al. 2003; Sheppard et al. 2005). Neutrophil priming and/or activation results in phosphorylation of p47phox at several serine residues. The precise residues and the kinase responsible may vary according to the stimulus, but isoforms of PKC activated by PI3K have been heavily implicated. p47phox phosphorylation, and to a lesser extent phosphorylation of p67phox, induces conformational changes in the cytosolic complex of p47phox/p67phox/p40phox, thereby exposing new binding domains (SH3, proline-rich and PX domains). This results in translocation of the cytosolic phox proteins to the membrane and their association with cytochrome b558 (see Fig. 13.6b). Membrane recruitment may be further enhanced by the local activation of PI3Ks with consequent generation of 3-phosphorylated lipids (particularly phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4bisphosphate), which interact with the PX domains of p40phox and p47phox (Ellson et al. 2001; Kanai et al. 2001). Neutrophil activation also results in conversion of inactive GDP-Rac2 to active GTP-Rac2, which migrates to the membrane and binds p67phox. The assembled NADPH oxidase enables electron transfer that generates O2−, a short-lived radical that can be converted to other cytotoxic products (ROS) such as hydrogen peroxide and hypochlorous acid, or which can react with nitric oxide to form reactive nitrogen species. The importance of the NADPH oxidase in bacterial killing is exemplified by the rare genetic condition CGD. In this disease, mutations in genes coding for the oxidase components (gp91phox, p22phox, p47phox, or p67phox) lead to a failure of ROS generation and a profound immunodeficiency state characterized by infections with Staphylococcus aureus, Aspergillus, enteric

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Gram-negative bacteria, and Burkholderia cepacia. While no cases of CGD have been found to be due to mutations in p40phox, recent evidence from knockout mice (Ellson et al. 2006) and studies employing cellular reconstitution (Suh et al. 2006) have supported the critical role this protein plays in the generation of ROS at the phagosome. The mechanism by which such phagosomal ROS kill bacteria was assumed to be direct toxicity, but elegant experiments (Reeves et al. 2002) have suggested that an indirect mechanism is important. The influx of electrons consequent on NADPH oxidase activity results in influx of potassium ions (charge compensation), which are instrumental in liberating the serine proteases elastase, cathepsin, and proteinase-3 from the proteoglycan matrix within the granules, and it is suggested that these liberated proteases are the predominant microbicidal factor (Segal 2005). Generation of ROS in response to phagocytosed particles is tightly restricted to the phagosomal membrane, and soluble stimuli such as C5a and formylated peptides liberate minimal extracellular ROS release from quiescent neutrophils. However, cells that have been primed by prior engagement of adhesion receptors, by cytokines such as TNF-α, or by bacterial LPS exhibit massive upregulation (10–20 fold) of NADPH oxidase activity at the plasma membrane (see Fig. 13.6b), potentially contributing to tissue injury in a range of disease states (Condliffe et al. 1998). Degranulation responses are similarly augmented. The molecular basis for priming is complex and poorly understood, but enhanced activation of PI3-kinase, phosphorylation of p47phox and recruitment of tyrosine kinases may all contribute to the establishment of the primed state.

Neutrophil deactivation and removal The intense histotoxic potential of the neutrophil dictates that powerful control mechanisms need to be in place to regulate the activation status of these cells. As indicated, the normal short half-life of these cells and the presence of a marginated pool and significant bone marrow reserve of mature cells support a system that can respond rapidly to any demand on neutrophil numbers. In the same manner, healthy circulating cells fail to generate a large respiratory burst or extracellular degranulation response when challenged unless first primed (see above). Moreover, in contrast to unprimed cells, fully primed/activated neutrophils tend to be poorly motile, which may trap these cells within the immediate inflamed site. Priming of circulating neutrophils is a feature of several systemic inflammatory conditions, including vasculitis, graft-versus-host disease, and inflammatory bowel disease, and these cells can be identified by their polarized (“shape-change”) morphology, the upregulation of their cell-surface β2 integrins, and shedding of CD62L (Condliffe et al. 1996). As a consequence these cells are held up within the pulmonary capillary bed, but do not cause lung injury unless there is a secondary trigger, for example local

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infection, causing tissue migration and activation. It is also apparent that, at least in vitro, neutrophils can recover from this primed state and return to their previous quiescent form (Kitchen et al. 1996). Unfortunately, very little is known about the capacity for neutrophils to de-prime in vivo or the role of the lung and the spleen in this process. As described below, there is also a major interaction between neutrophil priming and survival, with the majority of priming agents having the further function of delaying apoptosis, the second vital control mechanism regulating the function and fate of these cells.

Neutrophil apoptosis Despite the importance of tissue residence time and the rate of removal of neutrophils in the control of neutrophil tissue “load,” there has been little formal study of the mechanisms controlling these events. It had been widely assumed that neutrophils inevitably undergo necrosis and disintegrate in situ before removal of their fragments by local macrophages (Hurley 1983). However, if this was the case, healthy tissues would be continuously exposed to injurious neutrophil contents. Although a number of pathologic descriptions have favored neutrophil necrosis as a major mechanism operating in inflammation, most of these examples have been derived from histologic observation of diseased tissues rather than “beneficial” inflammatory responses, such as the response to infection. Furthermore, there has been evidence from the classical observations of Metchnikoff (1886) more than a century ago of an alternative fate for neutrophils, whereby the intact cell is removed by local macrophages. More recently, it has been shown that a major mechanism available for the removal of intact neutrophils and other granulocytes at the inflamed site is the process of apoptosis or programmed cell death, which has major implications for the control of inflammation (Haslett 1992, 1999). There is now clear evidence of a role for apoptosis in the clearance of neutrophils from tissues in a variety of acute inflammatory disorders (Grigg et al. 1991; Cox et al. 1995; Brazil et al. 2005; Rossi et al. 2006). Moreover, it appears that apoptosis represents a pivotal control point that determines the functional longevity of granulocytes and which can be modulated by a variety of inflammatory mediators. In mice, a distinct subpopulation of circulating neutrophils has recently been identified that are CD54high and CXCR1low and appear to represent cells that have undergone reverse endothelial cell transmigration, from an inflamed site back into the circulation (Buckley et al. 2006). These cells appear to have a delayed rate of apoptosis and can account for up to 2% of the circulating neutrophil population. Thus neutrophil exit strategies include apoptosis, transepithelial migration into the airways and bowel, transendothelial migration back into the circulation, and necrosis; clearly the balance is dictated by the nature, site, and stage of the inflammatory response. Unlike necrosis, apoptosis is a process that occurs where death is predictable or physiologic, such as thymic involution,

or where cell turnover is physiologically rapid, for example crypt cells in the gut epithelium (Wyllie et al. 1980). During apoptosis, ultrastructural studies show that cells shrink yet the plasma membrane remains intact, the ability to exclude vital dyes is preserved, and the cell retains organelles including granules (Fig. 13.7). Apoptotic cells are ingested very rapidly by phagocytes (particularly macrophages) in vivo; for example, in tissue sections of the remodeling embryo, apoptotic cells are rarely seen outside phagocytes (Koenig & Yoder 2004). Furthermore, in these and other examples of programmed cell death there is no evidence of local tissue injury or the induction of an inflammatory response, suggesting that apoptosis may represent a tissue injury-limiting mechanism for the removal of neutrophils. Human neutrophils undergo spontaneous apoptosis both in vitro and in vivo and this process has been proposed as a key mechanism underlying the resolution of granulocytic inflammation. Following the emigration of neutrophils into tissues, a switch from prostaglandin and leukotriene production to lipoxin formation occurs and this, together with the local production of resolvins and protectins from docosahexaenoic acid, serves to limit further neutrophil recruitment and initiate apoptosis (Serhan & Savill 2005). The process of apoptosis involves the promotion of controlled cell death while maintaining cellular integrity; with the possible exception that apoptotic neutrophils may still be able to release macrophage migration inhibitory factor (MIF) (Daryadel et al. 2006), this process prevents the release of hazardous proinflammatory mediators and promotes the recognition and

Fig. 13.7 Electron micrograph of an apoptotic human neutrophil. Note the intact cell membrane, the retention of granules, and the nuclear chromatin condensation and cytoplasmic vacuolation that characterize apoptosis.

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Regulation of neutrophil apoptosis: LPS, IL-1b, TNF-a, IL-6, IFN-g G-CSF, GM-CSF, C5a, IL-8, NGF calcium, cAMP, corticosteroids Normal cell

INHIBIT

Apoptotic cell

ACCELERATE E. coli ingestion Oil red ingestion Peritoneal dialysate UV irradiation HIV infection

TNF-a/Fas-L CD11b/CD18 cross-linking Protein synthesis inhibitors Sodium arsenite, ROS, NO IL-10, hyperosmolarity

Fig. 13.8 Factors that influence rates of neutrophil apoptosis in vitro. cAMP, cyclic AMP; IFN, interferon; NGF, nerve growth factor; NO, nitric oxide; UV, ultraviolet. See text for definition of other abbreviations. (See CD-ROM for color version.)

phagocytic elimination of effete cells from the site of inflammation. Thus neutrophil apoptosis modulates the duration and intensity of the inflammatory granulocytic response and hence the extent of neutrophil-mediated tissue damage. A wide range of proinflammatory cytokines and mediators that serve as neutrophil priming or activating agonists also inhibit the progression of apoptosis in vitro and these two processes are thought to be important in the establishment of chronic inflammatory diseases. GM-CSF, IL-8, LPS, C5a, LTB4, insulinlike growth factor 1, respiratory syncytial virus infection, and hypoxia have all been documented to delay neutrophil apoptosis, whereas TNF-α, Fas-L, TRAIL, and ultraviolet radiation promote apoptosis (Ward et al. 1999a; Scheel-Toellner et al. 2004; Walmsley et al. 2005; Lindemans et al. 2006) (Fig. 13.8). In addition to physiologic and pathologic stimuli, several pharmacologic agents have been reported to modify rates of apoptosis. For example, corticosteroids have been documented to promote neutrophil survival, an effect in contrast to their major pro-apoptotic effect in eosinophils (Cox 1995; Meagher et al. 1996), whereas NF-κB and cyclin-dependent kinase inhibitors accelerate this process (Ward et al. 1999b; Rossi et al. 2006).

Signaling pathways regulating neutrophil apoptosis (Fig. 13.9) Caspases are essential proteases for the initiation and execution of apoptosis. The caspase family is subdivided into three groups, initiators (caspase-8, -9, and -10) and executioners (caspase-3, -6, and -7) of apoptosis, and cyokine-processing (caspase-1, -4, and -5). The initiators sense death signals and activate downstream executioner caspases, which cleave cellular substrates mediating the changes associated with apoptosis. Neutrophil apoptosis can be initiated by activation of either the extrinsic or the intrinsic cell death pathways (Akgul & Edwards 2003). The extrinsic pathway is initiated by death receptor–ligand interaction (e.g., Fas/FasL, TNF-α/

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TNF-R1 and -R2), leading to the formation of the deathinducing signaling complex and the cleavage of caspase-8. The intrinsic (or stress) pathway, typically initiated by ultraviolet radiation, rapid temperature shifts, or oxidant damage, leads to increased mitochondrial membrane permeability and a reduction in membrane potential; as a result cytochrome c and apoptosis-inducing factor are liberated from the mitochondria, activating caspase-9. Activation of the initiator complexes (Apaf-1, caspase-8) by either of these routes leads to cleavage of the effector caspases caspase-3 and caspase-7, the main proteolytic executioners of cell death. The intrinsic and extrinsic pathways can also interact to amplify the death initiation signal since the pro-apoptotic Bcl-2 protein Bid is cleaved by caspase-8 and translocates to the mitochondria, resulting in activation of Bak/Bax (Thornberry & Lazebnik 1998). Caspase-10, in contrast, is activated in spontaneous but not death receptor-mediated neutrophil apoptosis (Thornberry, Lazebnik & Goepel et al. 1998). The differential expression and phosphorylation status of Bcl-2 and Bcl-2-like proteins, which serve as a common checkpoint for several distinct death signaling pathways, appear to be important in controlling the apoptotic threshold of neutrophils. Pro- and anti-apoptotic members of the Bcl-2 family regulate death signaling through their ability to form complex homodimers and heterodimers, which ultimately influence the insertion of Bax and Bax-like proteins into the outer mitochondrial membrane. This event plays a key role in triggering neutrophil apoptosis and results in the release of cytochrome c and activation of the caspase-9/Apaf-1 apoptosome (Dibbert et al. 1999; Pryde et al. 2000). High levels of pro-apoptotic Bcl-2 family members have been identified in normal neutrophils and the presence of these may account for the short half-life of the neutrophil. Both Bax and Bak have been identified at the mRNA and protein level in mature neutrophils (Weinmann et al. 1999; Santos-Beneit & Mollinedo 2000); while the selective deletion of either Bax or Bak in mice does not influence neutrophil longevity, in dual Bax–/–/Bak–/– animals there is a significant increase in neutrophil accumulation suggesting a degree of functional crossover between these proteins (Strasser 2005). Neutrophils also express “BH3-only” members of the Bcl-2 family, such as Bad and Bim; deletion and mutagenesis analysis of the BH3 domain have shown this to be the minimal death sequence required for heterodimerization and the promotion of apoptosis (Ottilie et al. 1997). These BH3-only proteins show no intrinsic or independent cell destructive properties; instead they appear to function as dominant inhibitors of the anti-apoptotic Bcl-2-like proteins. Bad has been reported to be phosphorylated at Ser 112 and Ser 136 by GM-CSF, which was associated with its translocation into the cytosol (Cowburn et al. 2002). Phosphorylated Bad is unable to bind to Bcl-XL, and consequently Bcl-XL can block Bax-like proteins and delay apoptosis. Genetic deletion of Bim in mice again promotes the accumulation of neutrophils

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Cytokine withdrawal

Death receptor CD9SL

TNF-a

CD9SL Plasma membrane

TRAFF

TRADD

PUMA

BIM

BAD

HRK

Bcl-XL A1 Mcl-1 Pro-survival BCl-2-family

NOXA PUMA BIM P53

NOXA PUMA

DNA damage

Bid

FADD Pre-caspase-8

Caspase-8

Effector caspase (caspase 3)

BAX/BAK-like protein

Mitochondria

Cell death

Cytochrome c

Effector caspase (caspase 3) APAF1

Caspase-9 Caspase-9

Fig.13.9 Signaling pathways in neutrophil apoptosis.

in vivo and prolongs neutrophil survival in vitro (Strasser et al. 2000; Strasser 2005). Neutrophils appear to express a rather limited and individual array of anti-apoptotic Bcl-2 family members. Hence, mature circulating neutrophils do not express Bcl-2 itself but rather Mcl-1 and A1 with Bcl-XL also reported at mRNA

level. The expression of Mcl-1 has been described as transient, and in the absence of survival stimuli the protein is rapidly degraded below a protective threshold (Moulding et al. 1998). Notably, GM-CSF increases or maintains Mcl-1 and A1 expression in neutrophils in vitro (Derouet et al. 2004).

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Several intracellular signaling pathways have been proposed as involved in cytokine-mediated inhibition of neutrophil apoptosis. For GM-CSF (the growth factor most clearly implicated in the aberrant survival of neutrophils in ARDS), tyrosine phosphorylation of Lyn and subsequent activation of the JAK/STAT pathway together with activation of the PI3K and p42/44 mitogen-activated protein kinase (MAPK) pathways appear to be the dominant survival routes. Vlahos et al. (1995) first identified the contribution of the PI3K/Akt pathway in GM-CSF-stimulated neutrophil survival, with possible downstream targets including phosphorylation of the pro-apoptotic BH3-only Bcl-2 family member protein Bad, suppression of BAX translocation to the mitochondria, and phosphorylation of the inhibitor of NF-κB, IκB (Ward et al. 1999a; Cowburn et al. 2005; Walmsley et al. 2005; see below). The role of MAPK in neutrophil survival is more controversial, with some groups reporting only a marginal effect of specific inhibitors (Cowburn et al. 2002), whereas others report significant attenuation of GM-CSF-induced survival (Klein et al. 2000). TNF-α is a complex pluripotent cytokine, first reported to inhibit neutrophil apoptosis through activation of NF-κB (Colotta et al. 1992), but subsequently demonstrated to also stimulate apoptosis at early times via a caspase-dependent mechanism requiring the coligation of both TNF-R1 and TNF-R2 (Murray et al. 1997). NF-κB is a key transcription activator of several anti-apoptotic proteins including Bcl-2, Bcl-X, Bcl-2A1, and IL-8 (Chen et al. 2000; Glasgow et al. 2001). The activation of of NF-κB is regulated by a complex family of proteins termed IκBα/β/ε that sequester the transcription factor in an inactivated state in the cytosol. Phosphorylation of the IκB complex leads to its polyubiquitination and subsequent degradation, enabling NF-κB to translocate to the nucleus. NF-κB may also be important in the regulation of constitutive rates of neutrophil apoptosis; hence, under in vitro culture conditions both IκB and NF-κB expression decreases in these cells in a time-dependent manner and this may represent an essential trigger for the onset of apoptosis (Walmsley et al. 2005). The central role of NF-κB in neutrophil survival is reinforced by studies demonstrating that the introduction of a NEMO (NF-κB essential modulator)-binding peptide into the neutrophil using an HIV-TAT transduction shuttle causes selective inhibition of IκKγ (IκB-kinase-γ; NEMO)/IκKβ interaction and NF-κB activation, and a major increase in neutrophil apoptosis (Choi et al. 2003).

Phagocytic removal of apoptotic neutrophils The presence of neutrophils in tissues is predominantly observed at injured or inflamed sites, from which their effective elimination is essential for resolution of the inflammatory response. Intact apoptotic neutrophils can be cleared from tissues by phagocytosis, principally by inflammatory and tissue resident macrophages. Neutrophil apoptosis triggers this recognition and clearance event, and the process

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of phagocytosis initiates the migration of engorged macrophages from the inflamed site to the draining lymphatic system. Neutrophil apoptosis is associated with downregulation of several cell-surface proteins including CD16 and CD62L, as well as exposure of phoshatidylserine (PS) and “enabled” FcγRII that supports the binding of immune complexes (Dransfield & Rossi 2004). Bridging molecules including thrombospondin-1 appear to enhance the binding of apoptotic cells to phagocytes either directly or indirectly. The interaction of oxidized PS and these other bridging molecules with phagocyte receptors, including αvβ3 integrin and CD36, engages apoptotic cells (Savill et al. 1991, 1992; Greenberg et al. 2006). The involvement of the macrophage PS receptor in the process of apoptotic cell uptake is currently uncertain (Fadok et al. 1992; Devitt et al. 2003). The uptake of apoptotic neutrophils by inflammatory macrophages stimulates the release and activation of transforming growth factor (TGF)β1 and other antiinflammatory factors as a further mechanism to facilitate the resolution of the inflammatory response (Fadok et al. 1998). Finally, semiprofessional phagocytes, such as fibroblasts and mesangial cells, have the capacity to recognize and ingest apoptotic neutrophils but not with the same facility or capacity as inflammatory macrophages. The in vivo significance of this latter observation is uncertain but it could represent a “back-up” mechanism for the macrophage system. Thus apoptosis not only determines the functional longevity of granulocytes at the inflamed site but can be modulated and controlled by external mediators of relevance to the control of inflammation. In contrast with necrosis, it provides a neutrophil removal mechanism that again is influenced by inflammatory mediators and which, by a variety of mechanisms, would tend to limit rather than promote bystander injury to healthy tissues.

Neutrophils in disease Role of neutrophils in inflammatory disease Depletion of neutrophils by cytotoxic drugs confers an increased risk of bacterial and fungal sepsis, proportional to the decrease in the neutrophil count; rare genetic diseases that compromise neutrophil function likewise lead to recurrent infections. To evade killing by the innate immune system, many bacteria have evolved virulence factors to circumvent specific neutrophil functions. For example, certain Salmonella species can interfere with the assembly of the NADPH oxidase (Vazquez-Torres et al. 2000; Gallois et al. 2001), certain pathogenic streptococci inhibit azurophil granule fusion with phagosomes (Staali et al. 2006), and Anaplasma phagocytophila (the causative agent of tick-borne ehrlichiosis) downregulates the expression of oxidase components (Carlyon et al. 2002; Thomas et al. 2005). Thus pathogens and the host immune response are in a perpetual arms race, and neutrophils

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comprise the frontline defense. Not surprisingly, given the potency of the neutrophil’s bactericidal armamentarium, host damage from “friendly fire” may occur, and in some cases have serious consequences. Neutrophil-mediated vascular endothelial injury, both direct and indirect, has been implicated in the genesis of inflammatory edema in several animal models and human disease states. Neutrophil numbers correlate spatially and temporally with edema formation in simple models of inflammation, for example carrageenan-induced paw edema in rodents (Houle et al. 2005; Rocha et al. 2006). Similar correlations can be seen in more complex animal models, e.g., adjuvant-mediated arthritis (Barsante et al. 2005) and corneal inflammation (Sonoda et al. 2005), and in human inflammatory conditions such as wound healing in pressure ulcers (Diegelmann 2003). Multiple effectors may summate to induce vascular leakage; for example, both natural killer cells and neutrophils are required for IL-2-induced pulmonary vascular leak syndrome (Assier et al. 2004). In models of cerebral ischemia, the presence of neutrophils and the expression of MMP-9 have been associated with blood–brain barrier leakage (Fujimura et al. 1999; Rosell et al. 2006), but neutrophil depletion did not influence several indicators of ischemic brain damage including cerebral edema and infarct size (Harris et al. 2005). Thus the importance of neutrophilic inflammation in edema formation varies according to the stimulus or disease state. Neutrophils can migrate to tissues, perform their phagocytic and microbicidal functions, and be cleared from the inflammatory site without causing tissue injury, as occurs in lobar pneumonia (Haslett 1999). The activation status of the cell and the precise anatomic location at the time of activation seem to be crucial in determining whether a beneficial or a harmful response is elicited. Priming of neutrophils has been shown to be a requirement for neutrophil-induced endothelial injury in vitro (Smedly et al. 1986; Jacobi et al. 2006) and primed neutrophils have been demonstrated either in the circulation or at the sites of tissue injury in a variety of disease states (El Benna et al. 2002; Bhatia et al. 2006; Chen et al. 2006). Close apposition of the neutrophil with a host cell is likewise thought to facilitate injury, enabling labile ROS to reach and react with target proteins and excluding tissue antiproteases from the interacting surfaces (Campbell et al. 1982; Rice & Weiss 1990). Catalytically active neutrophil elastase has been demonstrated at the neutrophil plasma membrane; indeed, in vitro studies have shown that up to 12% of stored human neutrophil elastase is present at the cell surface after priming with TNF-α or activation with IL-8 (Owen et al. 1997). The protease/antiprotease theory of emphysema postulates that an imbalance between neutrophilderived proteases (principally elastase) and locally produced antiproteases (such as α1-antitrypsin) leads to the destruction of matrix proteins such as elastin and hence to the airspace enlargement typical of this condition. Factors such as cigarette smoking and antiprotease deficiency (α1-antitrypsin deficiency)

are thought to tip the balance in favor of protease-mediated tissue injury. While these mechanisms are still thought to have an important role in some forms of tissue injury, neutrophils are no longer believed to be the only source of elastolytic enzymes within the lung, and the role of MMPs produced by many cell types including macrophages is increasingly felt to be of significance (Parks & Shapiro 2001). The deleterious role of the neutrophil in inflammatory disease has been studied in greatest depth in ARDS, a state characterized by high-permeability pulmonary edema and refractory hypoxemia occurring in the setting of the systemic inflammatory response syndrome. This condition serves as a paradigm for other disease states in which the neutrophil is a key player. Neutrophil depletion is protective in a range of animal models of acute lung injury (Flick et al. 1981; Heflin & Brigham 1981; Shasby et al. 1982; Looney et al. 2006), and in patients with ARDS the degree of neutrophilic inflammation correlates with the severity of lung injury (Fowler et al. 1987). BAL fluid concentrations of the neutrophil chemokine IL-8 predict the development of ARDS in an at-risk trauma group (Donnelly et al. 1993) and an excess of neutrophil proteases (Lee et al. 1981; Christner et al. 1985) have likewise been identified in BAL fluid from patients with ARDS. Zimmerman et al. (1983) found evidence of activated neutrophils in the pulmonary circulation of patients with ARDS, and the extent of NF-κB activity in peripheral blood neutrophils predicts the outcome of ARDS (Yang et al. 2003). Thus neutrophil recruitment and activation status appear to correlate with the development and clinical course of ARDS, and strategies that prevent neutrophil recruitment are protective in experimental models. Neutrophils are also implicated to varying degrees in the pathogenesis of a wide range of other inflammatory conditions with various targets (Table 13.2). Such neutrophilic injury may be localized, for example to an acutely inflamed joint in crystal-induced arthropathy, or occur in the setting of a multisystem disorder such as Wegener’s granulomatosis. A characteristic feature of Wegener’s granulomatosis is the presence of pathogenic autoantibodies directed against neutrophil proteins (MPO and proteinase-3); interaction of these antibodies with their target antigens results in intense perivascular and systemic activation of neutrophils, contributing to widespread vasculitic injury. Thus in the future, either local targeting of neutrophils at the disease site or inhibition of specific neutrophil activation pathways may provide treatments for inflammatory diseases where current treatments are ineffective or rely on generalized immunosuppression (see below). Finally, recent research has suggested that failure of the innate immune response to clear pathogens may induce chronic inflammatory disease states, not just chronic infections. In patients with Crohn disease, trauma to the gut mucosa or skin was shown to induce defective neutrophil accumulation, and the acute inflammatory reaction to subcutaneous

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Total tissue neutrophil load

Table 13.2 Noninfectious diseases in which neutrophils are implicated. Respiratory system Acute respiratory distress syndrome Bronchiectasis (including cystic fibrosis) Chronic obstructive pulmonary disease Asthma (especially corticosteroid-resistant asthma) Silicosis Hypersensitivity pneumonitis Idiopathic pulmonary fibrosis

Disposal Apoptosis

Lifespan

Disintegration

Activation/secretion

+

+ – Neutrophil products

+

Antiproteases, antioxidants +

Tissue injury

Tissue protection

Cardiovascular system Acute coronary syndromes Gastrointestinal system Crohn disease Ulcerative colitis Acute pancreatitis Musculoskeletal system Rheumatoid arthritis Gout Chondrocalcinosis Multisystem disorders Vasculitides (ANCA positive and negative) Ischemia–reperfusion (including post transplantation and post infarct) Multiorgan failure complicating SIRS Genetic disorders Familial Mediterranean fever ANCA, antineutrophil cytoplasmic antibody; SIRS, systemic inflammatory response syndrome.

Escherichia coli was greatly attenuated, responses that were shown to be at least partly due to reduced production of IL-8 by macrophages (Marks et al. 2006). Such delayed or incomplete removal of bacteria could initiate the granulomatous inflammation seen in Crohn disease, a hypothesis strengthened by the fact that granulomatous inflammation is the hallmark of CGD. A further mechanism of ineffective neutrophil pathogen clearance leading to chronic inflammatory disease was suggested by van Zandebergen et al. (2004), who demonstrated that under certain conditions neutrophils can serve as Trojan vectors by conveying Leishmania parasites into macrophages (which phagocytose infected and apoptotic neutrophils). Thus the innate immune response must be exquisitely fine-tuned, not only to avoid tissue injury secondary to overexuberant responses, but also to achieve effective pathogen clearance so that overwhelming infection or the establishment of chronic infection/inflammation does not occur (Fig. 13.10).

Role of neutrophils in allergic disease Eosinophilic inflammation is widely regarded as the hallmark of allergic disease, and until recently the neutrophil

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Fig. 13.10 The “balance” of injurious and protective influences in the determination of inflammatory tissue injury.

has been dismissed as having no role in this spectrum of disorders. However, recent studies have suggested that in clinically relevant subsets of patients, the neutrophil may be a key effector of “allergic” responses. Neutrophils have been demonstrated in induced sputum and in bronchial biopsies in the allergen-induced late asthmatic response (Lopuhaä et al. 2002; Kariyawasam et al. 2007). Allergen provocation was likewise associated with a prominent neutrophil influx in both the early and late phase responses in allergic rhinitis (Fransson et al. 2004). Although the role of the eosinophil in mediating mild and moderate atopic asthma is well established, eosinophilic inflammation is present in the airway lumen of only 50% of asthmatic subjects (Douwes et al. 2002), intense eosiniphilic inflammation in the setting of eosinophilic bronchitis does not induce asthma (Brightling et al. 2003), and certain antieosinophilic strategies are poorly efficacious in asthma (Bryan et al. 2000; Leckie et al. 2000; Kips et al. 2003); thus the eosinophil appears to be neither necessary nor sufficient to induce asthma alone (reviewed in Kamath et al. 2005). However, a strong association has been established between airway neutrophilia and severe asthma (Wenzel et al. 1997; Jatakanon et al. 1999; Little et al. 2002), corticosteroid-resistant asthma (Wenzel et al. 1999; Green et al. 2002), asthma exacerbations (Fahy et al. 1995; Lamblin et al. 1998; Tonnel et al. 2001), and acute fatal asthma (Sur et al. 1993). Where measured, neutrophil numbers correlate with markers of neutrophil degranulation, implying that these cells are also activated. Airway neutrophilia may be triggered by physical agents such as bacteria and viruses (Stenfors et al. 2002; Wark et al. 2002), and IL-8 may be critically involved in the recruitment of these cells to the asthmatic airway (Ordonez et al. 2000). While in severe asthma eosinophils and neutrophils are often found together, neutrophils may gradually replace eosinophils in proportion to the severity and/or duration of the disease, perhaps reflecting the ability of corticosteroids to induce eosinophil apoptosis while inhibiting this process in neutrophils (Tanizaki et al. 1993; Meagher et al.

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1996; Hauber et al. 2003). In addition to the direct effects of proteases and ROS on airway epithelia, neutrophil elastase has been shown to stimulate TGF-β release from airway smooth muscle cells (Lee et al. 2006) and to convert latent TGF-β into the biologically active form (Taipale et al. 1995; Hyytiäinen et al. 1998), suggesting a potential role in the airway remodeling that occurs in chronic asthma. Bronchoscopic studies (Martin et al. 1991) have suggested that neutrophils are in a rapid state of flux in asthma, with a short half-life (< 8 hours) in the asthmatic airway. This plasticity of airway neutrophilia is supported by an equine model of asthma, where complete resolution of the neutrophilic airway response occurred over a few days following removal of allergen challenge (Brazil et al. 2005). Thus strategies aimed at blockade of neutrophil recruitment may be efficacious in this condition, which may be of particular importance given the disproportionate healthcare resources required to manage severe and corticosteroid-resistant asthma (Serra-Batlles et al. 1998).

Antineutrophil strategies IL-8 is a major neutrophil chemoattractant released by macrophages and epithelial cells and in many lung inflammatory situations appears to account for the majority of the neutrophil chemotactic signal (Kunkel et al. 1991; Beeh & Beier 2006). Fully human anti-IL-8 antibodies and CXC-chemokine receptor antagonists have now been developed (Yang et al. 1999) and early clinical trials in conditions such as chronic obstructive pulmonary disease are beginning to be published (Mahler et al. 2004). Targeting LTB4 is also possible, either through inhibition of its synthesis via the 5-lipoxygenase pathway or by using LTB4 receptor antagonists and these agents appear to show efficacy in reducing neutrophilic inflammation at least in antigen-challenged animal models (Silbaugh et al. 2000). The final current strategy to prevent neutrophil recruitment involves the use of selectin antagonists such as TBC1269, which targets P-, L- and E-selectin and following inhalation has been shown to cause potent inhibition of allergic airway responses and neutrophilic inflammation in a sheep model of allergy (Abraham et al. 1999). The most promising targets for reducing neutrophil activation and mediator release include adenosine A2A receptor agonists (which inhibit NADPH oxidase activation, degranulation, and adherence), elastase and metalloproteinase inhibitors (especially MMP-9), anti-TNF-α strategies, phosphodiesterase IV inhibitors, macrolide antibiotics, and various antioxidants. Of these, neutralizing monoclonal antibodies against TNF-α appear to be showing most promise (Howarth et al. 2005; Berry et al. 2006). These agents are reviewed extensively elsewhere (Beeh & Beier 2006; Yamagata & Ichinose 2006). Lastly, recent insights into the molecular mechanisms that regulate neutrophil apoptosis have offered valid new targets aimed at either blocking survival signaling or driving apop-

tosis itself. Such targets include inhibition of NF-κB, PI3K-γ, a Gβγ and p101-regulated PI3K isoform with restricted expression in the neutrophil, cyclin-dependent kinases, and Bad (Ward et al. 1999b; Rossi et al. 2006). Overcoming the redundancy in neutrophil signaling and testing of these agents in animal and human models and disease settings are the immediate challenges ahead.

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Basophils: Biological Properties and Role in Allergic Diseases Gianni Marone, Giuseppe Spadaro and Arturo Genovese

Summary Basophils and mast cells are the only cells expressing the tetrameric (αβγ2) structure of the high-affinity receptor for IgE (FcεRI) and synthesizing histamine in humans. These cells differ immunologically, biochemically and pharmacologically, which suggests that they play distinct roles in the appearance and fluctuation of the allergic phenotype. Monoclonal antibodies are now available to identify specific basophil epitopes (e.g., basogranulin), representing a breakthrough in clarifying the role(s) of these cells in allergic disorders. Recent data indicate the complexity of the involvement of human basophils in allergic diseases and have shed light on their control of recruitment and activation in different human tissues. Basophils may play a role in inflammatory angiogenesis through the expression of several forms of vascular endothelial growth factor (VEGF) and their receptors. Preliminary evidence suggests that these cells are not always harmful but, in some circumstances, they might exert a protective effect by influencing certain aspects of innate and acquired immunity and allergic inflammation.

Introduction Human basophils and mast cells, described by Paul Ehrlich in 1879, are unique in that they are the only cells that both express the tetrameric (αβγ2) high-affinity receptor for IgE (FcεRI) and synthesize histamine (Ehrlich 1879). Despite these similarities, however, they differ in various aspects. Mast cells are tissue-resident cells, while basophils are normally found only in peripheral blood. Both cell types are highly mobile and readily infiltrate tissues at sites of inflammation (Ying et al. 1999; de Paulis et al. 2004a). However, mast cells and basophils have different strategic microlocalizations in the human

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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compartments and synthesize different sets of proinflammatory mediators, cytokines and chemokines. For instance, basophils, not found in normal lung tissue, infiltrate the sites of allergic airway inflammation (Ying et al. 1999; KleinJan et al. 2000; de Paulis et al. 2006). A wide range of newly identified chemotactic receptors (e.g., CC and CXC chemokine receptors, c-kit, CRTH2, FPR, FPRL1, FPRL2, C3aR, C5aR), selectively displayed on basophils and mast cells, are responsible for their recruitment to different compartments of skin and lung tissues. The mast cell’s role in the pathogenesis of allergic disorders has been extensively investigated, whereas the basophil’s role is less well defined, mainly because of limitations in the experimental models available at present. There are four canonical mechanisms whereby cross-linking IgE high-affinity receptors for the IgE (FcεRI) network can induce the release of mediators from human basophils in vitro and, presumably, in vivo (Fig. 14.1). In the classical model, a multivalent antigen cross-links at least two specific IgE bound to these cells and can serve as a stimulus for histamine and cytokine release from basophils. There is also evidence that lectins such as concanavalin A induce histamine release from basophils and mast cells through interaction with the carbohydrate moiety of IgE (Magro, 1974; Siraganian & Siraganian 1974). In addition, anti-human IgE antibodies cross-link two binding sites on the Fcε region of IgE. Similarly, antibodies directed against an epitope of the α chain of human FcεRI can trigger the release of mediators from FcεRI+ cells. Finally, immune complexes containing IgG against human IgE can activate human basophils (Marone et al. 2004). A fifth mechanism was based on the observation that IgE-mediated activation of human basophils and mast cells can also be induced by endogenous (e.g., protein Fv), bacterial (e.g., protein A and protein L) and viral superallergens (e.g., gp120) (Marone et al. 2004; Marone 2007) (Fig. 14.2). The binding of certain types of monomeric IgE to FcεRI can influence murine mast cell survival, apoptosis (Asai et al. 2001) and cytokine production (Kalesnikoff et al. 2001; Lam et al. 2003; Pandey et al. 2004). Exposure of mouse bone marrow-derived mast cells to monomeric IgE induces histidine decarboxylase expression and consequently affects levels of stored histamine (Tanaka et al. 2002). Donald MacGlashan reported that three human monoclonal IgE

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Fig. 14.1 The four canonical mechanisms by which cross-linking of the IgE–FceRI network can induce the release of mediators from human basophils and mast cells. (a) A multivalent antigen can cross-link at least two specific IgE molecules bound to FceRI+ cells to release mediators. (b) Anti-human IgE (anti-IgE) antibodies possess two binding sites for the Fce region of human IgE and activate mediator secretion from these cells. (c) Antibodies directed against an epitope of the a chain of FceRI (anti-FceRIa) can also trigger the release of mediators. (d) Immune complexes containing IgG anti-IgE and anti-IgG can activate human basophils in vitro. (From Marone et al. 2004, with permission.)

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Fig. 14.2 A fifth potential mechanism by which endogenous (protein Fv), bacterial (protein A and protein L) and viral proteins (gp120) can activate human FceRI+ cells, acting as superallergens. (a) Protein Fv, synthesized in small amounts in normal liver, is released in biological fluids of patients with acute and chronic viral hepatitis. Protein Fv has six binding sites for the VH3 region of human immunoglobulin and is a potent stimulator of histamine and cytokine release from FceRI+ cells through the interaction with VH3+ IgE. (b) Protein A, a cell-wall protein of Staphylococcus aureus, has a classical binding site for Fcg and an alternative site that binds the Fab portion of 15–50% of human polyclonal IgM, IgA, IgG and IgE. Protein A

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induces mediator release from human basophils and mast cells through interaction with the VH3 domain of IgE. (c) Peptostreptococcus magnus is a bacterium expressing a cell-wall protein L that binds human Ig regardless of the heavy-chain class, through high-affinity interaction with k light chains, and is thus an immunoglobulin superantigen. Protein L is a potent stimulator of the activation of human FceRI+ cells through interaction with the k light chain of membrane-bound IgE. (d) HIV-1 gp120 from divergent HIV-1 isolates from different viral clades of various geographical regions activate human FceRI+ cells through interaction with membrane-bound VH3+ IgE.

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antibodies did not induce signaling or mediator release from human basophils (MacGlashan 2003). Clarification of the underlying molecular mechanism(s) and their relevance in vivo and in human in vitro models will shed further light on these observations.

Origin of human basophils Basophils circulate in human peripheral blood where they amount to less than 1% of total leukocytes. Peripheral blood also contains basophil precursors (Denburg et al. 1983). Interleukin (IL)-3 is the principal cytokine responsible for human basophil growth and differentiation (Valent et al. 1989; Valent et al. 1990) from CD34+ pluripotent progenitor cells (Kirshenbaum et al. 1992). Other cytokines, such as granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-4, IL-5, and nerve growth factor (NGF), are also important for basophil growth, differentiation, and mediator production (Denburg 1995).

Morphologic and ultrastructural characteristics of human basophils Human basophils have polylobed nuclei with condensed chromatin (Fig. 14.3). They are commonly identified by metachromatic staining with basic dyes, such as toluidine

Fig. 14.4 A basophil in the ileum of a patient with Crohn disease shows piecemeal degranulation in vivo. Empty granules (arrows), full granules and partially depleted granules are present in the cytoplasm (× 10 700). (From Dvorak 1988, with permission.)

blue. On the surface there are irregular, broad processes. The cytoplasm contains membrane-bound secretory granules, mitochondria, and small Golgi structures. The secretory granules of basophils are of two types: the most numerous are large, round, and filled with electron-dense particles and/or finely granular material. A small, paranuclear granule with homogeneous content has also been observed, but much less frequently (Dvorak 1995, 2005). Human basophils extrude secretory granules to the external microenvironment when stimulated with a variety of triggering agents. Bridging the IgE–FcεRI network in vitro triggers a series of biochemical and ultrastructural changes, termed “anaphylactic degranulation,” which culminate in fusion of cytoplasmic granule membranes with the plasma membrane (Dvorak 2000). Ann Dvorak showed that in certain immunologic responses in man the basophils progressively lost their cytoplasmic granule contents, with no evidence of anaphylactic degranulation. This vesicle-mediated release of granule content is termed “piecemeal degranulation” (Fig. 14.4). Until a few years ago, technical limitations hampered the identification of basophils infiltrating the sites of inflammation. In fact, it is hard to identify partially degranulated basophils with conventional morphologic techniques. The availability of monoclonal antibodies (BB1 and 2D7) that identify specific basophil epitopes (e.g., basogranulin) (Irani et al. 1998; McEuen et al. 2001; de Paulis et al. 2006) led to a technological breakthrough in these studies.

Mediators of human basophils

Fig. 14.3 The human peripheral blood basophil has a polylobed nucleus, irregular, short broad cell surface processes, cytoplasmic glycogen and large secretory granules filled with particles (× 18 000). (From Dvorak 1992, with permission.)

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Human basophils contain about 1 pg/cell of histamine (Fig. 14.5). Basogranulin, a basophil-specific granule protein recognized by monoclonal antibody BB1, used to identify basophils in tissues (McEuen et al. 2001; de Paulis et al. 2006), is secreted together with histamine in response to IgE-dependent

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Fig. 14.5 Human peripheral blood basophils synthesize histamine, which is stored in secretory granules as a preformed mediator (∼1 pg/cell). Basophils contain basogranulin in their secretory granules, a specific granule protein recognized by the monoclonal antibody BB1, and mature and enzymatically active tryptase at levels of less than 1% of those of mast cells. Immunologic activation of basophils leads to the production of a restricted profile of Th2-like cytokines (IL-4 and IL-13). Activated basophils also produce the chemokines IL-8/CXCL8 and MIP-1a/CCL3 and several isoforms of VEGF-A. Immunologically activated basophils produce only LTC4 (∼30 ng/106 cells); no cyclooxygenase metabolite has been conclusively demonstrated in these cells. Activated basophils also synthesize PAF and AAGPC in a ratio of 1 : 2. (From Marone et al. 2005c, with permission.) (See CD-ROM for color version.)

activation (Mochizuki et al. 2003). Basophils express mature and enzymatically active tryptase at levels less than 1% of those of mast cells (Foster et al. 2002; Jogie-Brahim et al. 2004). Immunologically activated human basophils produce leukotriene (LT)C4 (∼30 ng/106 cells). Arachidonic acid for conversion to LTC4 in basophils is supplied almost exclusively by the cytosolic group IV phospholipase (PL)A2 (Triggiani et al. 2004), even though these cells contain one or more secretory PLA2 isoforms (Hundley et al. 1998). No cyclooxygenase metabolite has been conclusively demonstrated in these cells. However, PGD2, unlike other prostaglandins, enhances histamine release from immunologically activated basophils. It also reverses the inhibition of release by drugs and hormones that activate adenylate cyclase to increase cellular cyclic AMP (cAMP) (Peters et al. 1984). Immunologically activated basophils produce two acetylated phospholipids, AAGPC and plateletactivating factor (PAF), in a ratio of 2 : 1 (Triggiani et al. 1991, Triggiani et al. 1995) which, interestingly, have different effects on basophils primed with cytokines (Brunner et al. 1991; Columbo et al. 1993a). Immunologic activation of human basophils leads to an increase in IL-4 mRNA transcription and IL-4 secretion (MacGlashan et al. 1994; Mueller et al. 1994; Gibbs et al. 1996; Ochensberger et al. 1996; Redrup et al. 1998; Genovese et al. 2003). Basophils secrete an average of 30 pg/106 cells of IL-4 (range 10– 80 pg/106 cells); IL-4 secretion starts 1 hour after stimulation and peaks at 6 hours. The kinetics of production

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of IL-13 are different: there is a lag period of 4 hours and a plateau is reached 18–24 hours after immunologic stimulation (Fig. 14.6). Under these conditions there was no evidence of interferon (IFN)-γ mRNA. Therefore it appears that human basophils are unique because they express large amounts of IL-4 and IL-13 without any Th1-like cytokine (e.g., IFN-γ) and without most of the proinflammatory cytokines [tumor necrosis factor (TNF)-α, IL-1, IL-6] (Dahinden 2000). Thus, basophils are not simply inflammation effector cells, but also play a key immunoregulatory role by skewing immune responses towards the Th2 type. Differently from mast cells, basophils express a restricted cytokine profile. The different profiles of cytokines synthesized and released by basophils and mast cells provide indirect evidence that these cells have distinct roles in the appearance and fluctuation (exacerbations and remissions) of the allergic phenotype. Basophils also express the chemokines IL-8/CXCL8 and MIP-1α/CCL3 on IgE receptor cross-linking (Li et al. 1996). Vascular endothelial growth factor (VEGF)-A has also been recently identified in the supernatants of activated basophils (de Paulis et al. 2006; Marone et al. 2007a).

Surface markers of human basophils Basophils express an impressive array of cell–cell signalling molecules and this might explain why these cells can be attracted and/or activated by a very wide variety of inflammatory and immune stimuli (Fig. 14.7). In addition to FcεRI, human basophils express the inhibitory receptor FcγRIIb (Kepley et al. 2000). Coligation of FcγRIIb and FcεRI inhibits mediator release from basophils. Of particular importance for the response to agonists is the priming effect of certain cytokines that enhance effector functions, such as mediator release and cytokine expression. IL-3, IL-5, GM-CSF, and NGF prime basophils with different potencies (IL-3 > NGF ≥ IL-5 ≥ GM-CSF) (Bischoff et al. 1990; Bischoff & Dahinden

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Fig. 14.7 Selective display of membrane receptors on human basophils. Basophils express the tetrameric high-affinity receptor for IgE (FceRI). They also express FcgRIIb, whose cross-linking inhibits mediator release, and Tolllike receptor 2 (TLR2), TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9. Basophils express various receptors for chemokines (CCR1, CCR2, CCR3, CXCR1, and CXCR4), and cytokines (IL-3Ra, IL-5Ra, GM-CSFRa, and TRAK-A), whose activation induces chemotaxis and/or mediator release. These cells express the seven-transmembrane receptor CRTH2, whose activation by prostaglandin D2 (PGD2) induces chemotaxis, and a receptor for PAF. They also express at least two receptors for cysteinyl leukotrienes (CysLTR1 and CysLTR2) and three formyl peptide receptors (FPR, FPRL1 and FPRL2). They express receptors for anaphylatoxins (C3a and C5a), for VEGF (VEGFR-2/KDR) and for IGF (IGF-1R and IGF-2R) and the urokinase plasminogen (uPA) receptor (uPAR). Human basophils express the histamine H2 receptor, whose activation inhibits the release of mediators. Basophils express low levels of LIR3 and LIR7; cross-linking of LIR7 induces the secretion of mediators, while coligation of LIR3 and FceRI inhibits mediator release. (From Marone et al. 2005c, with permission.) (See CD-ROM for color version.)

1992; Brunner et al. 1993). The priming effect of IL-3, IL-5, and GM-CSF depends on the expression of the common β chain in their receptors (Ochensberger et al. 1999). The priming and modulatory effects of NGF are due to the expression of the high-affinity receptor TRK-A (Burgi et al. 1996), and highlights the link between the nervous system and allergic inflammation. An important function of basophils is their ability to migrate from peripheral blood to sites of allergic inflammation, thanks to a complex interplay of different chemotactic factors of various origins that act on a wide spectrum of surface receptors. Basophils express various receptors for chemokines (CCR1, CCR2, CCR3, CXCR1, and CXCR4), whose activation induces chemotaxis and/or mediator release. The eotaxin receptor CCR3 is expressed on the majority (∼ 80%) of human basophils (Uguccioni et al. 1997; de Paulis et al. 2000, 2001), and also on human lung mast cells (∼ 20%) (Romagnani et al. 1999, 2000; de Paulis et al. 2000). CCR3

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is activated by eotaxin/CCL11, eotaxin-2/CCL24, eotaxin-3/ CCL26 and RANTES/CCL5 and governs mostly chemotaxis to sites of allergic inflammation. Basophils also express high levels of CCR2, which can be activated by MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, and MCP-4/CCL13. Activation of CCR2 seems mainly to cause mediator release from basophils with only weak migratory responses (Bischoff et al. 1992; Uguccioni et al. 1997). PGD2, a major mast cell mediator released during the allergic response (Schleimer et al. 1985; Genovese et al. 2000), is chemotactic for basophils, eosinophils, mast cells, and Th2 cells through activation of the CRTH2 receptor (Hirai et al. 2001). The anaphylatoxins C3a and C5a are potent chemoattractants for basophils and induce mediator release, particularly when these cells are primed with certain cytokines (IL-3, IL-5, GM-CSF) (Bischoff et al. 1990; Ochensberger et al. 1995, 1996). The effects of anaphylatoxins are due to the specific seven-transmembrane (STM) G protein-coupled receptors C3aR and C5aR. In this family of STM, de Paulis and collaborators have identified at least three receptors in human basophils that bind several natural N-formyl peptides, including the prototype N-formyl-methionyl-leucyl-phenylalanine (FMLP) (de Paulis et al. 2004a,b). Basophils express the high-affinity receptor FPR and its homologs FPR-like-1 (FPRL1) and FPR-like-2 (FPRL2). Both receptors serve as chemotactic receptors for endogenous or viral products. For example, two HIV-1 gp41 peptides act as chemoattractants for basophils by interacting with FPRL1 (de Paulis et al. 2002). It has also been found that urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for FPRL1 and FPRL2 (de Paulis et al. 2004b). These results indicate that the uPA/ uPAR system is involved in allergic disorders. Activated FcεRI+ cells express the ligand for CD40 (CD40L), which can provide the cell contact signal required for IgE synthesis by human B cells (Gauchat et al. 1993). Basophils may therefore play a key role in allergic disorders not only by producing inflammatory and fibrogenic mediators but also by directly (CD40L) and indirectly (IL-4 and IL-13) regulating IgE synthesis independently of T cells. Recent evidence indicates that human basophils contribute to various aspects of angiogenesis. They synthesize and release such potent proangiogenic factors as (VEGF). Furthermore, VEGF and placental growth factor (PlGF) are both chemotactic for basophils through the activation of VEGF receptors (Marone et al. 2005a; de Paulis et al. 2006). The leukocyte immunoglobulin-like receptors (LIRs) are a family of cell-surface receptors that include both activating and inhibitory receptors (Arm 2004). Human basophils express low levels of LIR3 and LIR7 (Sloane et al. 2004). Cross-linking of LIR7 induces the secretion of proinflammatory mediators and IL-4, whereas coligation of LIR3 and FcεRI inhibits mediator release.

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CD203c (ecto-nucleotide pyrophosphatase/phosphodiesterase 3), a transmembrane protein, has been described as being selectively expressed on basophils, mast cells and their CD34+ progenitors. As CD203c is rapidly upregulated after allergen challenge in sensitized patients, it has been proposed as a new tool for allergy diagnosis (Boumiza et al. 2003).

Evidence of basophil involvement in bronchial asthma On the basis of some apparent similarities to mast cells, basophils have often been considered (and neglected) as minor and possibly redundant “circulating mast cells.” It is now evident, however, that they differ morphologically, ultrastructurally, and biochemically and produce a different set of preformed and de novo synthesized mediators. More importantly, basophils express a restricted profile of Th2-like cytokines (IL-4 and IL-13), whereas mast cells synthesize a broad array of proinflammatory and immunoregulatory molecules (Marone et al. 2005b). These marked differences suggest that the two cells play different roles in the pathogenesis of allergic disorders. Basophils, usually confined to the circulation, are not found in normal tissues. However, they infiltrate the sites of allergic inflammation (Irani et al. 1998; Ying et al. 1999; KleinJan et al. 2000). In addition, they have been found in the airways of asthmatics (Liu et al. 1991; Gauvreau et al. 2000; Macfarlane et al. 2000), in postmortem cases of fatal asthma (Koshino et al. 1993; Kepley et al. 2001), after antigen challenge of airway mucosa (KleinJan et al. 2000; Nouri-Aria et al. 2001), and in nasal polyps (de Paulis et al. 2006). From a functional viewpoint, IgE-mediated basophil releasability (i.e., the ability of a basophil/mast cell to release a certain percentage of histamine in response to a given immunologic stimulus) is increased in asthma and allergic rhinitis (Casolaro et al. 1990). More importantly, allergen-induced asthmatic responses are accompanied by infiltration of basophils that express IL-4 mRNA (Nouri-Aria et al. 2001). Thus, basophils might be an important source of Th2-like cytokines (IL-4 and IL-13) in the lung microenvironment. Techniques for the isolation and purification of basophils from peripheral blood, and monoclonal antibodies (BB1 and 2D7) that recognize specific epitopes in these cells (Irani et al. 1998; McEuen et al. 2001), should permit further studies of the function of basophils in allergic diseases (Falcone et al. 2000; Marone et al. 2005b).

Basophil recruitment to allergic airways Human basophils constitutively express several chemokine receptors (CCR1, CCR2, CCR3, CXCR1, CXCR3, CXCR4) and about 80% express CCR3, which can be activated by eotaxin/

Basophils: Biological Properties and Role in Allergic Diseases CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, MCP-3/CCL7 and MCP-4/CCL13 (Uguccioni et al. 1997; Romagnani et al. 1999). Eotaxin increases FcεRI-dependent IL-4 and IL-13 generation, respectively, by basophils (Devouassoux et al. 1999) and mast cells (Price et al. 2003). It is likely that the production of RANTES/CCL5, eotaxin/ CCL11, and eotaxin-2/CCL24 by human airway epithelial cells (Stellato et al. 1997) and smooth muscle (Hirst et al. 2002) partly accounts for the recruitment of basophils and mast cells to the lung during allergic inflammation. Two cytokines, IL-4 and TNF-α, produced by basophils and mast cells, respectively, increase eotaxin mRNA stability in airway epithelial cells, further amplifying the recruitment of inflammatory cells (Atasoy et al. 2003). Insulin-like growth factor (IGF)-1 and IGF-2 have been identified as selective basophil chemoattractants in human nasal polyps (Hartnell et al. 2004). These factors induce chemotaxis of basophils, but not eosinophils or neutrophils, and may therefore help explain the preferential infiltration of basophils in certain allergic disorders, although in vivo studies are required to confirm this. Nasal polyps involve hyperplasia of the mucosal epithelium and submucosal mucous glands with underlying areas of infiltrating inflammatory cells and proliferating blood vessels (Kakoi & Hiraide 1987). Vascular endothelial growth factor and its receptors have been implicated in nasal polyposis (Wittekindt et al. 2002). Nasal polyp tissues from patients undergoing polypectomy were examined by immunohistochemistry using the monoclonal antibody BB1, which specifically recognizes human basophils in tissue (McEuen et al. 1999; Ying et al. 1999), and a rabbit polyclonal anti-VEGF-A. Figure 14.8 shows BB1+ basophils in the perivascular area stained positive for VEGF-A as well. Figure 14.8 also shows the colocalization of BB1 and VEGF-A. Thus, VEGF-A appears to be present not only in peripheral blood but also more importantly at sites of chronic inflammation. This suggests that VEGF-A synthesized and released from basophils plays a dual role in inflammatory angiogenesis. First, VEGF-A released from circulating basophils might activate VEGF receptors on circulating endothelial cell precursors and immune cells; second, VEGF-A released from basophils infiltrating the sites of chronic inflammation might serve as a local source of an important angiogenic and chemotactic factor. A variety of other receptors are implicated in basophil chemotaxis. Human basophils express specific receptors for C3a and C5a (Füreder et al. 1995). A recent intriguing example is how the FMLP receptors FPRL1 and FPRL2 mediate the activation and chemotaxis of basophils in response to viral (gp41 of HIV-1) and bacterial [Hp (2–20)] peptides (de Paulis et al. 2002, 2004a). Consequent to these observations, it became evident that basophils express several receptors involved in the innate pattern recognition of microbes (FPR, FPRL1, FPRL2, and complement receptors) and of danger signals (VEGFR-2/KDR,

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uPAR, IGF-1/2/R). Thus the concept emerged that basophils receive danger signals in infection and injury and release mediators that initiate a protective leukocyte response that facilitates repair and healing. Amplification of these processes, however, could lead to persistent inflammation, angiogenesis, and tissue remodeling.

Fig. 14.8 Confocal micrographs of nasal polyps stained for basophils (green) and VEGFA (red). (a) A cluster of BB1-immunoreactive basophils. (b) VEGF-A-positive cells. (c) Colocalization of basogranulin and VEGF-A in basophils in nasal polyps. (From de Paulis et al. 2006, with permission.) (See CD-ROM for color version.)

classical mechanisms of FcεRI+ cell activation. We have demonstrated that immunoglobulin superantigens of various origins (endogenous, bacterial, and viral) can activate FcεRI+ cells to release proinflammatory mediators and cytokines (Marone et al. 2007b).

Endogenous and viral immunoglobulin superallergens

Superallergens in allergic disorders A mechanism by which human FcεRI+ cells can be activated by immunoglobulin superantigens has recently been identified (Marone et al. 2004, 2007b; Marone 2007). This might play a physiologic protective role in certain bacterial and viral infections (see Fig. 14.2). Amplification of this mechanism could lead to superantigen/superallergen activation of FcεRI+ cells, enabling certain viruses and bacteria to cause exacerbations in allergic disorders. A conventional antigen can usually stimulate less than 0.001% of the naive lymphocyte pool, whereas a superantigen can stimulate more than 5% (Silverman 1998). This immunologic property derives from the superantigen’s unique ability to interact with most lymphocytes that express antigen receptors from a particular variable (V) region gene family (Silverman 1997). Classical superantigens are T-cell superantigens (staphylococcal enterotoxins and toxic shock syndrome toxin-1). Some naturally occurring proteins are B-cell superantigens with unconventional immunoglobulin-binding capacities. The best-characterized immunoglobulin superantigen is Staphylococcus aureus protein A (Inganäs et al. 1980; Graille et al. 2000). Other B-cell superantigens are the gp120 envelope glycoprotein of HIV-1 (Townsley-Fuchs et al. 1997), a human gut-associated sialoprotein known as “protein Fv” (Guihard et al. 1997), and protein L from Peptostreptococcus magnus (Björck 1988). The concept of immunoglobulin superantigens applied to the pathophysiology of allergic disorders could be translated as “superallergens” to indicate proteins of various origin able to activate FcεRI+ cells by interacting with membranebound IgE. A significant proportion of allergic diseases (e.g., certain cases of intrinsic asthma) cannot be explained by the four

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About 5% of patients with viral hepatitis suffer urticarial reactions (Segool et al. 1975; Popp et al. 1981; Vaida et al. 1983). Protein Fv is a sialoprotein produced in the human liver and released in biological fluids during hepatitis A, B, C, and E (Bouvet et al. 1990). A single protein Fv molecule can bind six F(ab′)2 fragments (Bouvet et al. 1991a) of human IgM, IgG, and IgE (Bouvet et al. 1990, 1991a,b). Protein Fv binds to the VH3 region of immunoglobulins in a domain outside the conventional antigen-binding pocket (Bouvet et al. 1991a,b). We found that protein Fv is the most potent IgE-mediated stimulus for the activation of human basophils and lung mast cells (Patella et al. 1998) by interacting with IgE VH3+. This is the largest immunoglobulin family in the human repertoire (∼ 50%) (Karray & Zouali 1997; Silverman 1997; Karray et al. 1998). Therefore, protein Fv can function as an endogenous immunoglobulin superantigen frequently interacting with IgE VH3+ bound to FcεRI+ cells (Patella et al. 1993, 1998). Low concentrations of protein Fv induce IL-4 secretion from basophils through interaction with IgE VH3+ (Patella et al. 1998). IL-4 is a critical cytokine in the regulation of IgE synthesis by B lymphocytes and it is intriguing that some patients with viral hepatitis have high serum IgE levels (Van Epps et al. 1976). HIV-1-infected patients have higher than normal prevalence and/or severity of allergic reactions (Kaplan et al. 1987; Coopman et al. 1993). Serum IgE levels are high in HIV-1infected children (Viganò et al. 1995; Koutsonikolis et al. 1996; Onorato et al. 1999) and adults (Wright et al. 1990; Paganelli et al. 1995; Rancinan et al. 1998). Immunoglobulin VH3+ is a ligand for gp120 (Berberian et al. 1993; Karray & Zouali 1997). We found that four recombinant gp120 from different HIV-1 isolates from viral clades of varying geographic origin stimulated the release of cytokines (IL-4

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Bacterial immunoglobulin superallergens Staphylococcus aureus can exacerbate atopic dermatitis (Leung et al. 1998) and certain forms of asthma (Suh et al. 2004). Most clinical isolates of S. aureus synthesize protein A, a cell wall protein that has unique immunoglobulin-binding properties. Protein A has a classical site that binds the Fcγ of IgG, and an alternative site that binds the Fab portion of 15–50% of human polyclonal IgG, IgM, IgA, and IgE (Inganäs 1981). Staphylococcus aureus Cowan 1, which synthesizes protein A and soluble protein A, gradually increased histamine release from basophils (Marone et al. 1982) and lung mast cells (Genovese et al. 2000). Protein A activated basophils through interaction with the alternative binding site of IgE VH3+. These observations raise the possibility that exacerbations of atopic dermatitis (Leung et al. 1998) and certain forms of asthma (Suh et al. 2004) associated with S. aureus infection might be caused through this mechanism. Protein L, a cell-wall protein synthesized by the bacterium Peptostreptococcus magnus (Björck 1988), consists of up to five repeated immunoglobulin-binding domains (B1–B5) (Kastern et al. 1992) and is a determinant of virulence (Ricci et al. 2001). Each homologous domain binds with high affinity to the variable domain of the VKI, VKIII and VKIV subgroups (Nilson et al. 1992). Protein L binds human Ig regardless of the H chain class, is mitogenic for B cells (Axcrona et al. 1995), and is an immunoglobulin superantigen (Silverman 1997). We found that protein L induced the release of proinflammatory mediators and cytokines (IL-4 and IL-13) from basophils by interacting with the κ light chains of IgE (Patella et al. 1990; Genovese et al. 2000; Genovese et al. 2003). The relation between certain viral and bacterial infections and the induction and/or exacerbation of allergic reactions is well established (Johnston 1997; Leung et al. 1998; Gern & Busse 2000; Holtzman et al. 2002; Suh et al. 2004). Although there is evidence that certain viruses may exert an inhibitor effect on human basophils (Shiratori et al. 2005), our results point to a novel mechanism by which these infections might be involved (Marone 2007; Marone et al. 2007b). The in vivo implications of IgE-mediated activation of human FcεRI+ cells by these immunoglobulin superallergens remain to be defined.

Role of basophils in allergic angiogenesis The formation of new blood vessels (angiogenesis) is vital for numerous inflammatory and immune disorders including asthma, atopic dermatitis, polyposis, and rheumatic diseases (Carmeliet 2003). It is a prerequisite for airway remodeling. Several growth factors may be important in allergic inflammation and angiogenesis. VEGF and (PlGF) are among the most potent proangiogenic factors (De Falco et al. 2002; Nagy et al. 2003). FcεRI+ cells, which are closely associated with blood vessels and are increased at angiogenic sites, contribute to various aspects of angiogenesis (Ribatti et al. 2002; Hiromatsu & Toda 2003). Mast cells synthesize and release proangiogenic factors (histamine, tryptase, transforming growth factor β, IL-8, VEGF, I-309/CCL1) (Boesiger et al. 1998; Grützkau et al. 1998; Gilchrest et al. 2003). PGE2 and other cAMP-elevating agents increase the production of VEGF by mast cells (Abdel-Majid & Marshall 2004). We have explored the expression of VEGF and its receptors, and their functional interactions in human basophils (Marone et al. 2005a). Basophils constitutively express several isoforms of VEGF-A (VEGF-A121, VEGF-A165, and VEGF-A189) and their immunologic activation induces the release of VEGF-A (Fig. 14.9). This factor is chemotactic for basophils, presumably through interaction with the VEGFR-2/KDR, a receptor phenotypically expressed on the vast majority of these cells. Basophils also constitutively express mRNA for the soluble VEGFR-1 (sVEGFR-1), which is biologically active in blocking endogenously expressed VEGF activity (Eubank et al. 2004).

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and IL-13), parallel to histamine secretion from basophils (Florio et al. 2000; Patella et al. 2000). The viral superantigen gp120 activates basophils through an interaction with IgE VH3+ (Fig. 14.8). The mechanism of FcεRI+ cell activation by protein Fv and gp120 represents a new pathogenic cascade consisting of viral infection, immunoglobulin superantigen production, activation of FcεRI+ cells and tissue injury (see Fig. 14.2). This sequence of events raises the possibility that further immunoglobulin superantigens induced by viruses cause tissue injury in allergic inflammation through this mechanism involving FcεRI+ cell activation.

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Time (hours) Fig. 14.9 Kinetics of VEGF-A and histamine release from human basophils. Basophils were incubated with buffer (spontaneous release) or with anti-IgE (0.3 μg/mL). At each time point, supernatants were collected and centrifuged (1000 × g, 4°C, 5 min). VEGF-A release from basophils induced by anti-IgE is indicated by filled circles and histamine release by open circles. VEGF-A and histamine release in the supernatants was determined by ELISA and fluorometric techniques, respectively. The values are mean of duplicate determinations from a typical experiment.

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Most basophils express neuropilin 1 (NRP1) (de Paulis et al. 2006), a coreceptor for VEGF-A 165 (Soker et al. 1998; Mamluk et al. 2002), which enhances VEGFR-2/KDR-induced responses (Yamada et al. 2003). Basophils also express NRP2 mRNA and protein. Neuropilin 1 has no known enzymatic activity and therefore participates in signal transduction events by forming a complex with tyrosine kinase receptors. However, it appears to support the autocrine functions of VEGF in cells lacking VEGFR-2 expression (Wang et al. 2003). This raises the possibility that in certain cells NRP1 functions either alone or with other tyrosine kinase-linked receptors to transduce VEGF signaling. Neuropilin 1 on cells other than endothelial cells can induce angiogenesis too (Yamada et al. 2003). Therefore, NRP1 highly expressed on basophils might enhance angiogenesis even when VEGF is not abundantly expressed. The urokinase plasminogen activator (uPA) and its highaffinity receptor (uPAR) are involved in tissue remodeling and vessel sprouting (Carmeliet 2003). The uPAR is expressed by human basophils (de Paulis et al. 2004b) and mast cells (Sillaber et al. 1997). uPA is a potent chemoattractant for basophils through exposure of a chemotactic uPAR epitope, which is a ligand for FPRL1 and FPRL2 (de Paulis et al. 2004b). The expression and functions of angiopoietins and their receptors in human basophils remain to be elucidated. These findings illustrate the complex roles of basophils in fine regulation of the homeostatic control of angiogenesis during chronic inflammation. The VEGF/VEGFR system is emerging as an important regulator of angiogenesis and tissue remodeling in inflammatory disorders. We believe that this VEGF/VEGFR network warrants consideration as a target for future therapeutic intervention in allergic diseases.

Pharmacologic modulation of human basophils Basophils, mast cells and their mediators play a pivotal role in most allergic diseases in humans. This has prompted a race within the pharmaceutical industry to achieve optimal therapeutic targeting of FcεRI+ cells in these disorders. It is now clear that this ambitious target is more elusive than expected, on account of the complexity of the human FcεRI+–IgE system. First, mast cells differ immunologically, biochemically, and pharmacologically from human basophils. Second, basophils and mast cells synthesize different sets of proinflammatory mediators, cytokines, and chemokines. Moreover, different subpopulations of mast cells and basophils might have different roles – even, in some cases, protective – in the appearance and disappearance of the allergic phenotype. In addition, under some circumstances, FcεRI+ cells are important in homeostasis (Marone et al. 2005b,c). Finally, human FcεRI+ cells can be activated by a variety of immunologic and nonimmunologic stimuli, besides IgE-cross-linking.

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Blockade of activating receptors There are four main areas for therapeutic targeting of basophils and mast cells in allergic disorders. The first is interfering with activating receptors (FcεRI, C3aR, C5aR, cytokine and chemokine receptors, etc.) on the cells. Using a monoclonal antibody anti-IgE this strategy has been partially successful in certain forms of allergic asthma (Milgrom et al. 1999). The partial efficacy might be because other receptors besides FcεRI trigger the release of mediators from human FcεRI+ cells. Chemokine receptor antagonists are another area with pharmacologic potential for the prevention or treatment of allergic disorders. Several chemokine receptors are selectively displayed on human basophils and mast cells. Their antagonism can interfere with chemotaxis and/or secretion of FcεRI+ cells (Lukacs 2001). The redundancy of chemokines and their receptors is a major obstacle to achieving specific inhibitory effects. VEGF and its receptors are expressed in human basophils (Marone et al. 2005a; de Paulis et al. 2006). Small-molecularweight antagonists of VEGF receptors are candidates for the prevention/treatment of certain aspects of tissue remodeling in asthma (Wood et al. 2000). Alternative approaches to VEGF-induced angiogenesis include the use of humanized monoclonal antibodies against VEGF (Presta et al. 1997) or its receptor Flk-1/KDR (Prewett et al. 1999), or a decoy of this receptor (Wood et al. 2000).

Inhibition of signal transduction A second therapeutic strategy for allergic diseases is to inhibit FcεRI+ cell activation by interfering with one or more biochemical events essential for signal transduction. Excellent reviews have focused on the molecular consequences of basophil (MacGlashan et al. 2000a; Lusková & Dráber 2004) and mast cell (MacGlashan et al. 2000b; Bastan et al. 2001; Siraganian 2003; Wymann et al. 2003; Blank & Rivera 2004) activation and their pharmacologic manipulation. Several steps in the signal transduction process appear to be promising targets for the action on mediator release, cell growth, proliferation, and survival of FcεRI+ cells. In particular, tyrosine kinases (Syk, Btk, Lyn, Fyn, etc.), protein kinase C, phosphatidylinositol 3-kinase, and adapter molecules (Gab1, Gab2, Cbl, LAT, Grb2, Vav, etc.) might qualify as drug targets for the treatment of allergic disorders. The pharmacologic effects of most of these compounds have been so far evaluated in vitro using rodent and/or mast cell lines. Their pharmacologic properties must be confirmed using human mast cells and basophils before they can be considered for clinical trials.

Adenylate cyclase activators and phosphodiesterase inhibitors Catecholamines inhibit IgE-mediated histamine release from basophils and mast cells by binding β2-adrenergic receptors

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(Lichtenstein & Margolis 1968; Marone et al. 1984). Phosphodiesterases (PDEs) are responsible for cAMP hydrolysis. At least 11 different classes of cAMP PDE isoenzymes have been identified; cAMP PDE3 and PDE4 have both been found in basophils and mast cells (Essayan 2001). PDE4 has attracted particular attention and four variants have been identified (PDE4A, PDE4B, PDE4C, PDE4D) (Giembycz 2000). The four gene products of PDE4 show sensitivity to inhibition by rolipram, which inhibits PAF- and anti-IgE-induced mediator release from basophils too (Peachell et al. 1992; Columbo et al. 1993b). PDE4 inhibitors also regulate mast cell functions (Torphy et al. 1992). In contrast, PDE3 and PDE5 inhibitors do not inhibit mediator release. Thus, it appears that PDE4 is the isoenzyme mainly responsible for action on FcεRI+ cells.

Glucocorticoids Glucocorticoids are one of the therapeutic mainstays in allergic disorders. In humans, intravenous glucocorticoids cause rapid basopenia, while skin tissue histamine remains unchanged (Dunsky et al. 1979). In contrast, prolonged treatment with topical glucocorticoids reduces skin mast cell density and inhibits the allergen-induced wheal-and-flare response (Lavker & Schechter 1985). High doses of inhaled glucocorticoids in mild-to-moderate asthma reduce mast cell density and the number of bronchial vessels (Chetta et al. 2003). Prolonged (12–24 hours) incubation with glucocorticoids inhibits IgE-mediated histamine release from basophils (Schleimer et al. 1981). In contrast, up to 24-hour incubation of mast cells isolated from lung parenchyma with glucocorticoids does not alter their immunologic release of histamine, PGD2, or LTC4 (Schleimer et al. 1983). These examples highlight the pharmacologic differences between human basophils and mast cells. Short preincubation (∼1 hour) of human basophils with glucocorticoids inhibits IL-4 release (Schroeder et al. 1997), without any inhibitory effect on histamine. These results suggest that the mechanisms controlling the release of cytokines differ from those controlling the release of histamine from basophils.

Immunophilin ligands Cyclophilin (CyP) is a protein with high affinity for cyclosporin A (CsA) (Hait et al. 1986) that belongs to a family of intracellular proteins, the immunophilins, which includes the FK-binding proteins (FKBPs) (Standaert et al. 1990). FK-506 (tacrolimus) binds with high affinity to FKBPs (Standaert et al. 1990). CsA–CyP and FK-506–FKBP complexes bind to calcineurin (Cn) (Stellato et al. 1992), which has a catalytic A subunit (CnA) and a regulatory B subunit (CnB). The CnA subunit has a binding site for calmodulin (CaM) and for the CnB subunit. Complexes of CsA–CyP or FK-506–FKBP inhibit the CaM-dependent protein phosphatase 2B, which is essential in the signal transduction pathway for basophils

Basophils: Biological Properties and Role in Allergic Diseases (Cirillo et al. 1990; de Paulis et al. 1991) and mast cells (Cirillo et al. 1990; de Paulis et al. 1992; Stellato et al. 1992). Low concentrations of CsA prevent histamine and LTC4 release from basophils and mast cells challenged with IgEmediated stimuli (Cirillo et al. 1990; de Paulis et al. 1991, 1992; Stellato et al. 1992), by interacting with CyP. A single oral dose of CsA rapidly inhibited histamine release from basophils ex vivo, providing a rare example of how a drug administered in vivo can affect basophil releasability ex vivo (Casolaro et al. 1993). CsA and tacrolimus are also potent inhibitors of the de novo synthesis of IL-4 and IL-13 from immunologically activated basophils (Patella et al. 1998; Florio et al. 2000; Genovese et al. 2003). Tacrolimus ointment is rapidly effective in patients with atopic dermatitis (Ruzicka et al. 1997). Skin mast cells and infiltrating basophils are important in atopic dermatitis (Mitchell et al. 1982). Tacrolimus exerts its potent antiinflammatory effects by inhibiting the release of histamine and eicosanoids from skin mast cells (de Paulis et al. 1991, 1992). These findings, together with its efficacy in atopic dermatitis, suggest that the beneficial effects of this compound in vivo are largely due to its antiinflammatory properties (Marone 1998).

Receptor antagonists of Fc εRI+ cell-derived mediators Antagonists of receptors activated by mediators synthesized by human FcεRI+ cells are widely used in the treatment of allergic disorders. The main property of H1 receptor antihistamines is to antagonize the effects of this mediator at the H1 receptor in different organs (Marone 1997). Certain antihistamines prevent the release not only of histamine, but also of other proinflammatory mediators, such as LTC4, PAF, and PGD2 (Church & Gradidge 1980; Patella et al. 1996; Genovese et al. 1997; Marone et al. 1999). Loratadine and desloratadine inhibit histamine release and cytokine production from human basophils induced by IgE-dependent mechanisms (Genovese et al. 1997; Schroeder et al. 2001); in human lung mast cells, desloratadine inhibits preformed (histamine and tryptase) and de novo synthesized mediators (LTC4 and PGD2) (Genovese et al. 1997). Not all H1 antagonists have antiinflammatory activity in vitro. For instance, mizolastine inhibits the de novo synthesis of LTC4 from basophils, but potentiates the secretion of histamine (Triggiani et al. 2004). Thus, the inhibitory effect of certain H1 receptor antagonists on mediator release from human FcεRI+ cells is not class-specific and is not a general property of all antihistamines. Selective cysteinyl (Cys)LT1 receptor antagonists, such as montelukast, have been employed in certain allergic disorders (Barnes et al. 2005). CysLT1 and CysLT2 receptors are also expressed on mast cells (Mellor et al. 2001) and leukocytes (Figueroa et al. 2001). It is possible that cysteinyl leukotriene antagonists influence certain aspects of their activation.

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PGD2 activates two important receptors: the DP receptor, whose activation elicits bronchoconstriction in asthmatics (Hardy et al. 1984), and the CRTH2 receptor present on human basophils and mast cells (Hirai et al. 2001). Specific antagonists of these receptors are being evaluated for use in allergic diseases. β Tryptase and chymase, stored and released by human mast cells, exert several biochemical and proinflammatory effects (Caughey 2004; Stevens et al. 2004). Specific smallmolecular-weight inhibitors of β tryptase and chymase are under investigation for the treatment of allergic disorders. Several VEGF-A isoforms have been identified in human basophils (Marone et al. 2005a) and mast cells (Boesiger et al. 1998; Grützkau et al. 1998). In addition, immunologic activation of human basophils induces the release of VEGF-A (de Paulis et al. 2006), which is chemotactic for human basophils (de Paulis et al. 2006). Therefore, the antagonism of VEGF and their receptors by different strategic approaches may serve to interrupt an autocrine loop affecting tissue remodeling and angiogenesis.

Conclusions and implications The ability of basophils to migrate to sites of chronic inflammation, their powerful effector repertoire, their different activating ligands, and their plasticity in response to various signals suggest that these cells have a central role in several allergic disorders. Mast cells differ from basophils immunologically, biochemically, and pharmacologically and therefore have distinct roles in the orchestration of inflammation. It is quite possible that different subpopulations of basophils have different, in some cases even protective, roles in the appearance of the allergic phenotype. We have to continue working to define the cytokines and chemokines and their receptors acting on or released by human basophils. We must also elucidate the complex biochemical steps in human basophils activated by immunologic and nonimmunologic stimuli. Another area to explore is the paracrine and autocrine interactions of basophils on other immune cells (e.g., eosinophils, macrophages, Th2 cells, B cells). Finally, it remains to be elucidated whether basophils have role(s), in some cases even protective, in angiogenesis and tissue remodeling. Until a few years ago, partially degranulated basophils at sites of inflammation were hard to identify with conventional morphologic techniques. The availability of monoclonal antibodies that identify specific basophil epitopes (e.g., basogranulin) marks a breakthrough in clarifying the roles of these cells in allergic diseases. However, we still need more knowledge of the biological properties of human basophils to modify their “bad” behavior without compromising their homeostatic and protective roles in innate and acquired immunity.

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Acknowledgments This work was supported by grants from the Ministero dell’Università e Ricerca, the Istituto Superiore di Sanità (AIDS Project 40G.38), Ministero della Salute “Alzheimer Project” (Rome, Italy), and Regione Campania (Naples, Italy). We thank Giorgio Giannattasio for a critical reading of the manuscript and Francesco Granata for the artwork. We also wish to acknowledge the important contribution of colleagues whose work has not been included due to space constraints.

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Leukocyte Adhesion in Allergic Inflammation Michelle J. Muessel and Andrew J. Wardlaw

Summary

Introduction

The pattern of inflammation in chronic allergic disease is controlled by selective expression of adhesion molecules and chemoattractants that cause the preferential migration, tissue localization and activation of eosinophils, mast cells, and Th2 cells. The adhesion receptors predominantly involved are the selectins and their ligands, in particular P-selectin glycoprotein ligand (PSGL)-1 and the leukocyte integrins and their ligands intercellular adhesion molecule (ICAM)-1–3 and vascular cell adhesion molecule (VCAM)-1. The function of the leukocyte integrins is controlled mainly by conformational changes in the receptor via a process called “inside-out” signaling, whereas expression of endothelial adhesion receptors is controlled largely by increased expression as a result of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-4. One of the most striking features of allergic disease is the increased numbers of tissue eosinophils. This results from a combination of increased eosinophilpoiesis, increased migration through the endothelium controlled by a combination of P-selectin/PSGL-1 and VCAM-1/VLA-4, and prolonged survival in tissue under the influence of IL-5 and granulocyte– macrophage colony-stimulating factor (GM-CSF). Mast cells migrate into tissue as precursor cells and their activation and localization in tissue is modulated by their integrin-mediated interaction with matrix proteins such as collagen and fibronectin as well as their ability to adhere avidly to structural cells through novel adhesion pathways. There does not appear to be any specific adhesion pathway for Th2 cells, with selective Th1 versus Th2 migration controlled principally by chemokines and their receptors. Our understanding of the adhesion receptors controlling leukocyte migration into tissue has yet to be translated into new therapies. Potent yet safe adhesion receptor antagonists have been difficult to develop and in the case of VLA-4 antagonists have been disappointing in allergen challenge studies. Nonetheless this remains a fruitful area for future drug development.

In the last decade, dramatic progress has been made in our understanding of the structural and functional properties of the molecules involved in leukocyte adhesion. The migration of leukocytes from the vasculature across the endothelial barrier to sites of allergic inflammation, known as transendothelial migration (TEM) or diapedesis, remains central to the immune response. This multistep sequential process involves (i) leukocytes rolling along the endothelium, (ii) arrest and firm adhesion, and (iii) subsequent transmigration through the endothelial barrier (Springer 1994). The adhesion molecules utilized during this process also work in a sequential fashion. Selectins and their counterstructures slow the velocity of leukocytes leading to rolling along the endothelial wall (Luscinskas et al. 1994; Alon et al. 1995). Firm adhesion is mediated by leukocyte integrins such as αLβ2 (LFA-1) or α4β1 (VLA-4) binding to members of the immunoglobulin superfamily such as ICAM-1 on endothelium (Lawrence & Springer 1991; Carlos & Harlan 1994). Subsequently, cells transmigrate through the endothelium either via transcytosis of the endothelial cell or via the predominant paracellular route between adjacent cells (Feng et al. 1998; Muller 2003; Zen et al. 2005; Dejana 2006). Transmigration is an active process engaging other members of the immunoglobulin superfamily, including junctional adhesion molecules (JAMs), platelet endothelial cell adhesion molecule (PECAM)-1, and CD99 (Muller 2003). Orchestrating these events is a variety of signaling molecules unique to each of these steps. Proinflammatory mediators such as TNF-α and IL-1 prime the endothelial cells, increasing the surface expression of various adhesion molecules that mediate rolling. As leukocytes roll along the endothelial surface, they encounter chemotactic factors such as chemokines that activate integrins, thus mediating firm adhesion (von Andrian & Mackay 2000). The selective recruitment of eosinophils and T cells to sites of allergic inflammation attest to the exquisite regulation of the immune response. The diversity of cytokines, adhesion molecules, and chemokines utilized in specific combinations allows a high level of specificity in recruitment of selective subpopulations of leukocytes. The selectivity can be modulated at each step in the process, thus offering the possibility of

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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inhibiting leukocyte migration at each step. Indeed, blockade of β2 or α4 integrins has been shown to be a promising route of therapeutic intervention for a variety of inflammatory and autoimmune disorders (Gottlieb et al. 2000; Jackson et al. 2002).

Structure and function of leukocyte adhesion receptors Selectins and their counterstructures Selectins Selectins and their counterstructures regulate the first step in the process of TEM, the slow rolling of leukocytes along the surface of endothelial cells (Luscinskas et al. 1994; Alon et al. 1995; Berlin et al. 1995). There are three members of the selectin family, L-, E- and P-selectin, which mediate cell contact by binding weakly to carbohydrate structures typified by sialyl Lewis x (sLex)-like glycans and with high affinity to specific glycoprotein counterstructures (covered later in the chapter) (Somers et al. 2000). Recent studies have demonstrated that selectins form “catch bonds” where dissociation rates decrease with increasing applied force (Marshall et al. 2003; Sarangapani et al. 2004; Yago et al. 2004; Hammer 2005; Pereverzev et al. 2005). The rolling/tethering step is a prerequisite for firm adhesion (Lawrence & Springer 1991; Milstone et al. 1998), allowing leukocytes to sense their local environment for proinflammatory stimuli such as cytokines and chemokines presented on the endothelial surface. L-selectin is constitutively expressed on most types of leukocytes, implying that the specificity of extravasation into tissue must be regulated by the inducibility of its endothelial ligands. L-selectin was first identified by the monoclonal antibody MEL 14, which blocks the binding of lymphocytes to lymph node high endothelial venules (HEV) (Gallatin et al. 1983). E-selectin is specific to endothelial cells. E-selectin was also found using the monoclonal antibody technique while searching for cytokine-inducible endothelial surface molecules (Bevilacqua et al. 1987). Proinflammatory stimuli such as TNF-α or IL-1β induce de novo gene expression and protein synthesis of E-selectin on endothelium through NF-κB and JNK/p38 mitogen-activated protein kinase (MAPK) pathways (Bevilacqua et al. 1987; Read et al. 1997). Surface expression of E-selectin is maximal at 4– 6 hours after stimulation and rapidly downregulated to baseline levels after 12–16 hours (Weller et al. 1992; Hahne et al. 1993). P-selectin was identified in platelet storage granules as a protein of unknown function and was later detected in endothelial cells (Hsu-Lin et al. 1984; McEver & Martin 1984). P-selectin is found on endothelium as well as platelets, where its expression is regulated by proinflammatory mediators such as TNF-α and lipopolysaccharide (LPS) (Weller et al. 1992; Hahne et al. 1993). However, the mechanism of regulation for P-selectin is different from other selectins. P-selectin protein is stored in

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cytosolic storage granules called Weibel–Palade bodies (Birch et al. 1992; Hannah et al. 2002). These intracellular vesicles then fuse with plasma membrane to release P-selectin in response to various stimuli such as histamine and thrombin. P-selectin is rapidly downregulated by internalization (Green et al. 1994) but can be recycled from endosomes to storage granules (Subramaniam et al. 1993). The extracellular domains of all three lectins are very similar. The N-terminus is a C-type lectin, followed by an epidermal growth factor-like domain, then a series of short consensus repeats that are variable in number, with L-selectin having two repeat units, E-selectin six, and P-selectin nine (Fig. 15.1). Soluble forms of all three selectins exist due to shedding or proteolytic cleavage (Kishimoto et al. 1990; Gearing & Newman 1993). The soluble form correlates with membrane expression of selectins (Leeuwenberg et al. 1992) and is increased in asthma and atopic dermatitis (Kobayashi et al. 1994; Wuthrich et al. 1995). Blockade of L-selectin shedding decreases leukocyte rolling in vivo, leading to the hypothesis that shedding may be important to prevent an excessively strong interaction mediated by L-selectin (HafeziMoghadam & Ley 1999). The C-type lectin domain binds to the carbohydrate family of sialylated fucosylated glycosaminoglycans typified by the sugar moiety sLex (Springer & Lasky 1991) (Fig. 15.2). The importance of fucosylation is Endothelium

Leukocyte

GlyCAM-1 L-selectin

CD34 MadCAM-1

E-selectin

ESL-1

P-selectin

PSGL-1

Lectin domain Transmembrane region EGF domain Cytoplasmic domain Short consensus repeats

Ig domain N-linked glycosylation O-linked glycosylation

Fig. 15.1 Schematic representation of the structure of selectins and their ligands. EGF, epidermal growth factor. (From Vestweber & Blanks 1999, with permission). (See CD-ROM for color version.)

NeuAca2

3Galb1

Sialylated Lewis x

4GlcNAcb1 3 Fuca1

3Galb1

4GlcNAcb1 3 Fuca1

Fig. 15.2 Structure of the carbohydrate moieties recognized by selectins including the structure of sialyl Lewis x. NeuAc, sialyl group; Gal, galactose; Fuc, fucose; GlcNac, N-acetylglucosamine.

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demonstrated by the genetic disorder leukocyte adhesion deficiency (LAD) II, in which patients are immunodeficient due to the inability of leukocytes to bind through selectins (Etzioni et al. 1993). LAD II has recently been shown to involve a genetic defect that results in a malfunctioning nucleotide sugar transporter (Luhn et al. 2001). All three selectins contain a single transmembrane region followed by a short cytoplasmic tail that consists of 17 amino acids in L-selecin, 32 in E-selectin, and 35 in P-selectin (Huang 1997). Selectin structure is most diverse in the transmembrane and cytosolic tail regions, implying that they are either differentially regulated or transduce different signals. Some intracellular binding partners have been identified for selectins, which give us some clues as to function. The cytoplasmic tail of selectins has been shown to bind the cellular cytoskeleton (Pavalko et al. 1995; Yoshida et al. 1996; Ivetic et al. 2002) and leukocytes show reduced tethering and rolling in vitro on deletion of this region in L-selectin (Dwir et al. 2001). Calmodulin binds to the L-selectin cytoplasmic tail and may negatively regulate shedding (Kahn et al. 1998; Diaz-Rodriguez et al. 2000). The cytoplasmic tail of selectins is also important for signaling, which may depend on selectin clustering. The intracellular signaling molecule, Rho A, a member of the small GTPase family, regulates E-selectin localization and clustering (WojciakStothard et al. 1999) and ligand-induced activation of phospholipase C-γ is dependent on clustering in lipid rafts (Kiely et al. 2003). Clustering also induces increases in intracellular free Ca2+, cytoskeletal shape changes, and F-actin distribution (Kaplanski et al. 1994; Lorenzon et al. 1998). The increase in F-actin filament polymerization has been shown to be dependent on the small GTPase Rac2 (Brenner et al. 1997). The signaling pathway has been further studied in L-selectin that activates p56lck, Grb2/sos, Ras, Rac2, and MAPK to increase synthesis of reactive oxygen species (Waddell et al. 1995; Brenner et al. 1996). In addition, E-selectin has also been shown to stimulate MAPK and Erk1/2 (Hu et al. 2000, 2001). Most importantly, L-selectin ligation has been shown to upregulate the surface expression of the αMβ2 integrin (Mac-1) (Crockett-Torabi et al. 1995), providing an important ingredient for the next step in the process of leukocyte extravasation into tissue.

Leukocyte Adhesion in Allergic Inflammation

1 (Berg et al. 1993), Sgp200 (Hemmerich et al. 1994), and podocalyxin-like protein (Sassetti et al. 1998). GlyCAM-1 and CD34 are both sialomucins displaying large sialic acidrich O-linked carbohydrate side chains that are necessary for binding to L-selectin. CD34 is a heavily O- and N-linked glycosylated transmembrane receptor expressed on most endothelial cells and hematopoietic progenitor cells (Greaves et al. 1992). GlyCAM-1 does not have a transmembrane region and is a secreted protein (Brustein et al. 1992) found in cytoplasmic granules (Kikuta & Rosen 1994). Sulfation is essential for GlyCAM-1 binding to L-selectin (Imai et al. 1993). Vascular MAdCAM-1 also contains a sialomucin domain and carries O-linked carbohydrate modifications recognized by L-selectin (Berg et al. 1993). Interestingly, MAdCAM-1 is unusual in that it also serves as a ligand for the integrin α4β7 and contains two immunoglobulin domains that are important for this interaction. The P-selectin counterstructure PSGL-1 is also a sialomucin, identified through expression cloning using a P-selectin– immunoglobulin fusion protein as bait (Sako et al. 1995). A 250-kDa protein identified earlier by affinity isolation using P-selectin an affinity probe was described on neutrophils (Moore et al. 1992) and was subsequently demonstrated to be identical to PSGL-1 (Moore et al. 1994). The carbohydrate modifications of sialylation and fucosylation are required for PSGL-1 binding (Larsen et al. 1992; Lenter et al. 1994) as are branched carbohydrate side chains generated by the core-2 enzyme (Wilkins et al. 1995; Kumar et al. 1996). Furthermore, sulfation at one of the three tyrosine residues at its N-terminus is necessary for binding to P-selectin (Sako et al. 1995; Wilkins et al. 1995) and probably also to L-selectin (Spertini et al. 1996) (Fig. 15.3). The recently elucidated co-crystal structure of PSGL-1 with P-selectin demonstrated that tyrosine sulfation and sialylated and fucosylated sugars within the terminal 19 amino acids of PSGL-1 bind separately to P-selectin adjacent to the calcium atom (Somers et al. 2000). PSGL-1 was found to be the major ligand for P-selectin on both neutrophils (Moore et al. 1995) and T cells (Vachino et al. 1995), although PSGL-1 is also recognized by E- and L-selectins (Li et al. 1996; Goetz et al. 1997). PSGL-1-deficient mice exhibit deficits in P-selectin-mediated rolling and neutrophil recruitment,

Selectin counterstructures The ligands for selectins are composed of a scaffolding protein modified by various carbohydrates and other posttranslational modifications such as sulfation. The protein core is important for increased specificity and affinity of binding while the cellular background itself is important for providing the necessary repertoire of glycosylation enzymes to confer the selectivity of selectin binding. Five ligands for L-selectin have been identified using soluble recombinant protein as affinity probes: glycosylated cell adhesion molecule (GlyCAM)-1 (Lasky et al. 1992), CD34 (Baumhueter et al. 1994), mucosal addressin cell adhesion molecule (MAdCAM)-

15/16 Decamer repeats NH2

Y

N-glycosylation sites

COOH

Ser/Thr/Pro rich region

Transmembrane region

Y Three sulfated tyrosine amino acids essential for ligand binding Fig. 15.3 Schematic representation of the structure of PSGL-1 showing the position of the sulfated tyrosines essential for high-affinity binding to P-selectin. (See CD-ROM for color version.)

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Inflammatory Cells and Mediators cell-surface counterreceptors and other extracellular ligands (Plow et al. 2000; Van der Flier & Sonnenberg 2001). They transmit signals both into cells regarding their environment (“outside-in”) and out of cells (“inside-out”) (Hynes 1992, 2002). Originally termed integrins due to the fact that they are integral membrane proteins linking extracellular contacts to the intracellular cytoskeleton, they regulate and modulate actin filaments through a complex array of submembrane linker proteins (Hynes 1992; Van der Flier & Sonnenberg 2001; Zamir & Geiger 2001). Integrins are heterodimers of two subunits, α and β. The combination of different α and β subunit partners makes each integrin unique, with each subunit contributing to ligand specificity. Most of each polypeptide subunit, greater than 1600 amino acids, resides in the extracellular space. The two short cytoplasmic domains usually comprise 20–50 amino acids. In mammals, there are known to be eight β and 18 α subunits comprising a total of 24 distinct integrins (Fig. 15.4). The uniqueness of each integrin is displayed by the specificity of its ligand binding partner, an arrangement reinforced by the specific defects in leukocyte function present in the phenotype of knockout mice: knockout of αL leads to impaired leukocyte recruitment (Schmits et al. 1996); αM deficient mice are defective in phagocytosis and apoptosis of neutrophils and mast cells (Coxon et al. 1996; Dong et al. 1997; Tang et al. 1997); knockout of αE leads to reduced numbers of intraepithelial lymphocytes (Schon et al. 1999); mice deficient in β2 have leukocytosis, impaired inflammatory responses, skin infections, and T-cell proliferation defects (Scharffetter-Kochanek et al. 1998); mice deficient in β6 display inflammation in skin and airways as well as impaired lung fibrosis, probably due to the failure to activate transforming growth factor (TGF)-β (Huang et al. 1996; Munger et al. 1999); and, finally, mice deficient in

demonstrating its in vivo relevance (Yang et al. 1999). PSGL-1 binding to E-selectin is also dependent on the same carbohydrate modifications as required for binding to P-selectin, although sulfation of tyrosine residues is unnecessary (Somers et al. 2000). Studies on PSGL-1 provide insights into two general points for selectin counterstructures. Firstly, while only 15% of lymphocytes bind P-selectin, the majority of lymphocytes express PSGL-1, emphasizing the importance of carbohydrate modification to binding specificity. Secondly, despite the fact that PSGL-1 represents only 1% of the sLex on the cell surface, it is a requirement for leukocyte binding to P-selectin (Norgard et al. 1993; Li et al. 1996), emphasizing the importance of a single glycoprotein to high-affinity binding and specificity. ESL-1 is a selective E-selectin ligand found on leukocytes that requires N-linked carbohydrate modifications for binding. The 150-kDa glycoprotein ESL-1 was identified using affinity isolation using an E-selectin–immunoglobulin fusion protein as bait (Levinovitz et al. 1993). ESL-1 is not a sialomucin and furthermore has been identified as a variant of the fibroblast growth factor receptor (Steegmaler et al. 1995). In contrast to these other structures, which are concentrated at the tips of microvilli, ESL-1 is expressed on the surfaces of microvilli and may have a function after the tethering step. Finally, another high-affinity ligand for E-selectin is L-selectin itself (Kishimoto et al. 1991; Lawrence et al. 1994). Affinity isolation experiments using E-selectin–immunoglobulin fusion protein provided direct evidence of this interaction (Jones et al. 1997; Zollner et al. 1997).

Integrins Integrins are major leukocyte receptors for mediating adhesion to a wide array of extracellular matrix proteins as well as

Alpha Chain

Beta Chain b1

a1 a2 a3 a4 a5 a6 a7 a8 a9

b2

b3

b4

b5

VLA-1 (L:C) VLA-2 (Fn:L:C) VLA-3 (L:C) VLA-4 (VCAM-1:Fn) VLA-5 (Fn) VLA-6 (L) a7b1(L) a8b1 a9b1

b6

b7

a4b6

a4b7:VCAM-1 MAdCAM-1:Fn

aEb7 E-cadherin

aE aL aM aX aD aIIb aV

340

b8

aVb1(Vn)

LFA-1 (ICAM-1-3) Mac-1 C3bi:ICAM-1:Fb p150,95 (Fb,ICAM-1, VCAM-1) aDb2 (VCAM-1) aIIbb3 (Fn:Fb) aVb3(Vn:Fn:Ln)

aVb5(Fn)

aVb6(Vn)

aVb8

Fig. 15.4 Structure of the integrin family showing the pairing of the a and b chains and, where known, the main ligands recognized by the integrin. Fn, fibronectin; Fb, fibrinogen; Vn, vitronectin; L, laminin; C, collagen.

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Leukocyte integrins Each leukocyte subtype expresses one or more members of the β2 (CD18) family of integrins; further, β2 and β7 are restricted in their expression to leukocytes. β2 is a common subunit of four different binding partners (αL, αM, αX, and αD), while β7 has just one binding partner, αE, making a total of five integrins unique to leukocytes (Fig. 15.5). The first leukocyte integrin, αMβ2 (Mac-1/Mo-1/CR3/ CD11b), was identified by using a monoclonal antibody as a marker for myloid cells (Springer et al. 1979). The expression of αMβ2 is restricted to myloid cells such as monocytes and neutrophils, large granular lymphocytes, and a subset of B and T cells (Arnaout & Colten 1984). αMβ2 has a wide array of binding partners including ICAM-1, -2, -3, -4 and -5 (Diamond et al. 1990; Xie et al. 1995; Ihanus et al. 2003), JAM-C (Santoso et al. 2002; Chavakis et al. 2004; Aurrand-Lions et al. 2005), “inactivated” opsonic C3b (iC3b) component (Beller et al. 1982), fibrinogen (Wright et al. 1988), vitronectin (Kanse et al. 2004), Leishmania gp63 (Russell & Wright 1988), the LDL receptor-related protein (Spijkers et al. 2005), matrix metalloproteinase (MMP)-9 (Stefanidakis et al. 2003), and Thy-1 (Wetzel et al. 2004). In addition, αMβ2 binds to yeast, zymosan,

Endothelium

MAdCAM-1 VCAM-1

a4b7 a4b1 aDb2

ICAM-1 ICAM-2

aXb2 Mac-1

Leukocyte

β7 show defects in gut-associated lymphocytes and reduced intraepithelial lymphocytes (Wagner et al. 1996). Leukocyte-specific α subunits have an inserted I/A domain. The crystal structure of this domain was elucidated by Lee et al. (1995) from αMβ2 (CD11b/CD18). There is a metal ion coordination site at the “top” of the I/A domain of αM, which explains the necessity of divalent cations for ligand binding. Additional structural and biochemical experiments show that ligand binding is regulated through coordination of the metal ion site in the I/A domain which causes confirmational changes allowing the integrin head to adopt either an open highaffinity “active” form or a closed low-affinity “inactive” form (Shimaoka et al. 2000; Xiong et al. 2000). However, there is some evidence to show that this “active” form may not be generally true of all leukocyte integrin α subunits (Walters et al. 2005). The recent elucidation of the crystal structure of one integrin, αVβ3, has led to advances in our understanding of integrin structure and function (Xiong et al. 2001, 2002). In agreement with a large body of evidence from other techniques, it was confirmed that the β subunit also contains an I/A domain (or β I-like domain) and together with the propeller domain from the α subunit forms the ligand-binding head of the integrin, which is attached to two legs, one from each subunit. However, the configuration of unliganded crystal structure yielded a surprise: an integrin bent over at an angle of 135°, which was presumed to be inactive. Crystal structure of the ligand-bound integrin confirmed that, on activation, presumably either through ligand binding or via “inside-out” signaling, straightening and separation of the legs allows the integrin to become fully functional.

Leukocyte Adhesion in Allergic Inflammation

LFA-1

Fig. 15.5 Schematic structure of the leukocyte integrins and the endothelial adhesion receptors they recognize.

plastic, and glass (Anderson et al. 1986; Wallis et al. 1986). Phosphorylation of the cytoplasmic tail of αMβ2 is important for activation (Fagerholm et al. 2006) as is selective recruitment of the src family kinase Hck (Tang et al. 2006). αLβ2 (LFA-1/CD11a) was identified as a surface antigen involved in cytotoxic T cell-mediated killing (Davignon et al. 1981). αLβ2 is expressed by virtually all leukocytes (Krensky et al. 1983). Binding partners for αLβ2 include ICAM-1, -2, -3 and -4 as well as JAM-A (Luscinskas et al. 1991; Ostermann et al. 2002; Ihanus et al. 2003; Fraemohs et al. 2004; Yang et al. 2005). Phosphorylation of the cytoplasmic tail of αLβ2 is important for adhesion (Fagerholm et al. 2005; Nurmi et al. 2007). Α third member of the family, αXβ2 (P150,95/CR4/CD11c), was identified shortly thereafter (Lanier et al. 1985; Springer et al. 1986). Expression of αXβ2 is restricted to monocytes, macrophages, and CD8− dendritic cells with only weak expression on neutrophils (Springer et al. 1986; Shortman & Liu 2002). Ligands for αXβ2 include collagen type I, iC3b, fibrinogen, LPS, ICAM-1 and -4, as well as Thy-1 (Loike et al. 1991; Diamond et al. 1993; Bilsland et al. 1994; Choi et al. 2005; Ihanus et al. 2006). Additionally, αXβ2 has been identified as a marker for hairy cell leukemia (Schwarting et al. 1985), as well as interacting with the rotavirus protein VP7 during viral cell entry (Graham et al. 2004). αXβ2 is more resistant to activation than other leukocyte integrins and recent evidence shows that this is due to activation restraints imposed by structural changes in the cytoplasmic domain (Zang & Springer 2001). αDβ2 (CD11d) is expressed on monocytes, macrophages, natural killer (NK) cells, neutrophils, eosinophils, dendritic cells, and a subset of T and B cells (Danilenko et al. 1995; Grayson et al. 1998; Mabon et al. 2000). The principal ligand for αDβ2 is VCAM-1 (Van der Vieren et al. 1999; Mabon et al. 2000). Administration of antibodies directed against αDβ2 significantly inhibited macrophage as well as neutrophil recruitment in vivo (Mabon et al. 2000). Phorbol esters increase αDβ2 surface expression as well as eosinophil αDβ2-mediated adhesion to VCAM-1 (Kikuchi et al. 2003). αEβ7 is expressed by dendritic cells (Pribila et al. 2004), mast cells (Tegoshi et al. 2005), and T cells in the lung and gut including regulatory T cells (Lehmann et al. 2002; Uss et al. 2006). Expression

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of αEβ7 is induced by TGF-β (Kilshaw 1999; Robinson et al. 2001). αEβ7 mediates lymphocyte adhesion to epithelial cells through its only known ligand, E-cadherin (Cepek et al. 1994; Kilshaw 1999). Other members of the integrin family are variably expressed on leukocytes depending on the activation state. αIIbβ3 is only expressed on platelets and megakaryocytes (Wee & Jackson 2005). αVβ3 is expressed by tissue macrophages and lymphocytes (Savill et al. 1990; Legler et al. 2004). Most members of the β1 family bind to extracellular matrix proteins such that α4β1 (VLA-4) binds to fibronectin, although it also recognizes immunoglobulin superfamily cell-surface counterparts such as VCAM-1 (Luscinskas et al. 1991) as well as JAM-B (Cunningham et al. 2002). α4β1 is expressed by T lymphoblastoid cell lines and mononuclear leukocytes (Hemler et al. 1984). The integrin α4β7 is expressed by lymphocytes, mast cells, eosinophils, and NK cells and recognizes MAdCAM-1 and VCAM-1 (Erle et al. 1994; Gurish et al. 2001). Although it is well documented that integrins mediate firm adhesion, it is becoming increasingly clear that, like selectins, several integrins also participate in the rolling and arrest of leukocytes under flow conditions including α4β1, α4β7, and αLβ2 (Berlin et al. 1995; Dunne et al. 2003; Rosenthal-Allieri et al. 2005; Pendu et al. 2006).

Regulation of integrin function Spatial and temporal regulation of leukocyte adhesion is an absolute necessity in many leukocyte functions including migration. It is important that integrins are inactive on resting leukocytes in order to avoid inflammation. In contrast, it is important that they are able to rapidly activate to facilitate leukocyte function. Elucidation of the crystal structure of αVβ3 integrin, as mentioned above, has allowed integration of the functional data with a large body of experimental work involving the regulation of integrin function. It has been known for many years that integrins on resting leukocytes are unable to bind ligand and do not signal. For example, resting T cells and granulocytes bind to purified ICAM-1 very weakly. It had been proposed that the changes in integrin function are due to a conformational change in the ligand-binding domain. It has now been shown that the I/A domains of αM can adopt either an open or closed position (Xiong et al. 2000). The open state is a high-affinity or “active” state while the closed position is low affinity or “inactive.” Ligand binding or stimulation by other known activating agents such as antibodies or divalent cations induces a conformational switch beween the two states. Integrins are also capable of regulating each other, either inhibiting or activating function. For instance, the major platelet integrin αIIbβ3 can be activated by collagen signaling through its receptors GPVI and the integrin α2β1. Conversely, it has been shown that antibodies to αVβ3 can inhibit the role of α5β1 in cell migration and phagocytosis (Simon et al. 1997; Scharffetter-Kochanek et al. 1998; Blystone et al. 1999).

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Another important aspect of integrin regulation is the coordination of adhesion molecule signaling with more traditional signaling pathways such as growth factor receptors. Integrin signaling that regulates many aspects of cell behavior such as proliferation, apoptosis, differentiation, migration, and gene expression is intimately coupled with pathways triggered by growth factor receptors to yield coordinated functional responses. Similarly, integrin attachment to substrate is necessary for many cellular responses to soluble growth factors such as thrombin, platelet-derived growth factor, and lysophosphatidic acid (LPA). Again, dysregulation of leukocyte integrins in both mice and humans attest to the importance of their tight regulation. For instance, as discussed earlier, knockout of β2 integrins leads to defects in leukocyte function (Scharffetter-Kochanek et al. 1998). The importance of β2 integrins is also demonstrated by the human disease LAD I, a genetic mutation in the gene for β2. These patients suffer from leukocytosis and failure to recruit leukocytes to sites of infection, leading to early death (Etzioni et al. 1999). A more recently described disease, LAD III, also demonstrates activation defects in integrin function possibly through signaling defects in the small GTPase Rap1, a key component in inside-out signaling (Etzioni & Alon 2004; Kinashi et al. 2004). These patients suffer from defects in platelet aggregation, recurrent bacterial infections, and a severe bleeding tendency.

Immunoglobulin family members ICAM-1 ICAM-1/CD54 is a cell-surface glycoprotein of the immunoglobulin superfamily that is critical for trafficking of leukocytes through leukocyte–leukocyte, leukocyte–endothelial, and leukocyte–epithelial interactions (Rothlein et al. 1986; Springer 1990). ICAMs are cellular binding partners for β2 integrins on leukocytes, for example ICAM-1 binds αLβ2 on lymphocytes or αMβ2 on neutrophils to facilitate TEM (Springer 1990; Staunton et al. 1990; Vonderheide et al. 1994). There are five members of the ICAM family, which show variable expression patterns. The most extensively studied member of this family, ICAM-1, is expressed constitutively at low levels on endothelial and epithelial cells as well as on some leukocytes, dendritic cells, and fibroblasts (Rothlein et al. 1986; Shappell et al. 1990; Kelly et al. 1992; Dippold et al. 1993). ICAM-2 is constitutively expressed on endothelial and mononuclear leukocytes, but not on neutrophils (de Fougerolles et al. 1991), ICAM-3 is restricted to leukocytes where it is constitutively expressed (de Fougerolles & Springer 1992), ICAM-4 is restricted to erythrocytes (Bailly et al. 1994), and ICAM-5 is strongly expressed in the brain (Mizuno et al. 1997). While ICAM-1 and ICAM-2 are both constitutively expressed on endothelium, surface expression of ICAM-1 but not ICAM-2 (Staunton et al. 1989) is upregulated in response to the inflammatory cytokines IL-1, interferon (IFN)-γ, and TNF-α (Rothlein et al. 1986; Rice et al. 1990; Min

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et al. 2005). ICAM-1 is a variably glycosylated, 76–114 kDa glycoprotein with a 55-kDa peptide backbone (Dustin et al. 1986). The extracellular portion of all five ICAMs possesses variable numbers of immunoglobulin-like domains. ICAM-1 consists of five immunoglobulin-like extracellular domains, with the first domain responsible for binding to αLβ2 (LFA-1) and the third domain responsible for binding to αMβ2 (MAC-1) (Staunton et al. 1990; Vonderheide et al. 1994). In addition to binding β2 integrins, ICAM-1 is also a receptor for Plasmodium falciparum (Berendt et al. 1989) and the major group of rhinovirus (Greve et al. 1989; Tomassini et al. 1989). Alternative splicing of ICAM-1 produces multiple isoforms (King et al. 1995) including a soluble form (Rothlein et al. 1991) that binds the human rhinovirus, inhibiting infection (Marlin et al. 1990). The soluble extracellular domain may inhibit leukocyte–endothelial interactions by acting as a decoy receptor (Kusterer et al. 1998). ICAMs possess one transmembrane segment and a short cytoplasmic tail, which interacts with the cytoskeletal protein vinculin and is important for intracellular signaling events as well as diapedesis (Carpen et al. 1992; Lyck et al. 2003; Yang et al. 2005; Nieminen et al. 2006). ICAM-1 mediates granulocyte extravasation and antibodies to ICAM-1 inhibit leukocyte binding to endothelium as well as extravasation (Smith et al. 1988, 1989; Barton et al. 1989). In light of this, it is unsurprising that ICAM-1deficient mice display impaired neutrophil trafficking (Sligh et al. 1993). Studies on how ICAM-1 mediates firm adhesion have recently demonstrated that ICAM-1 (and to a lesser extent VCAM-1) concentrates in an actin-rich, cup-like docking structure that not only binds leukocytes to the endothelium but is important for TEM (Barreiro et al. 2002; Carman & Springer 2004; Yang et al. 2005). Additionally, a recent study has proposed a more specific role for ICAM-1 in mediating TEM via the transcellular route (through single endothelial cells). Millan et al. (2006) have demonstrated that leukocyte adhesion induces clustering of ICAM-1 and caveolin which may form a channel through which the leukocyte migrates across individual endothelial cells.

VCAM-1 VCAM-1 (CD106) is a type I transmembrane glycoprotein member of the immunoglobulin superfamily that mediates TEM by binding to α4β1 (VLA-1) on leukocytes (Dustin & Springer 1988; Rice & Bevilacqua 1989; Elices et al. 1990; Cybulsky & Gimbrone 1991). It is expressed by a variety of cell types including endothelium, epithelium, fibroblasts, dendritic cells, and bone marrow stromal cells (Koopman et al. 1991; Miyake et al. 1991; Osborn et al. 1992; Salomon et al. 1997). Expression of VCAM-1 is upregulated by TNF-α, IL-1 and IL-4, as well as by nucleotides released by endothelial cells and detected by the P2Y2 receptor (Iademarco et al. 1992; Seye et al. 2004). Two alternatively spliced forms of VCAM-1 exist in humans (Cybulsky et al. 1991; Osborn et al. 1992; Chuluyan et al. 1995). The predominant form expressed

Leukocyte Adhesion in Allergic Inflammation

on the cell surface (7D VCAM-1) contains seven extracellular immunoglobulin-like domains, while the alternatively spliced form (6D VCAM-1) lacks immunoglobulin-like domain 4 (Hession et al. 1991). There is increasing evidence that the soluble form of 6D VCAM-1 augments leukocyte migration (Rose et al. 2001) possibly by binding α4β1 with higher affinity than 7D VCAM-1 (Woodside et al. 2006).

PECAM-1 PECAM-1/CD31 contains intracytoplasmic immunoreceptor tyrosine inhibitory motifs (ITIMs) that mediate an inhibitory function through recruitment of protein-tyrosine phosphatases (Jackson et al. 1997; Hua et al. 1998; Henshall et al. 2001; Newman et al. 2001). This has led to its subclassification into the immunoglobulin-ITIM superfamily, a subset of the conventional immunoglobulin superfamily (Hua et al. 1998; Newman 1999; Henshall et al. 2001). It is a 150-kDa glycoprotein consisting of six extracellular immunoglobulin domains, a transmembrane region, and a cytoplasmic domain. It has a number of O- and N-linked glycosylation sites as well as splice variants expressed in different cell types (Baldwin et al. 1994). PECAM-1 also has a soluble form that is generated by splicing out exon 9, which encodes the transmembrane region (Goldberger et al. 1994). PECAM-1 is expressed at high density on the lateral junctions of endothelial cells and on the surface of most leukocytes (Newman 1997; Mamdouh et al. 2003). Surface expression of PECAM-1 is regulated by shedding due to matrix metalloprotease and caspase activity (Ilan et al. 2001). The ligands for PECAM-1 include homophilic binding with itself on other cells, αVβ3, and CD38 as well as proposed interactions with proteoglycans (Piali et al. 1995; Sun et al. 1996; Deaglio et al. 1998). In addition, phosphorylation at tyrosine residue Y686F on the cytoplasmic tail results in ligand specificity switching from heterophilic to homophilic binding (Famiglietti et al. 1997). PECAM-1 has several important functions in both endothelial cells and leukocytes that contribute to leukocyte migration and TEM. PECAM-1 has been shown to be important for regulation of endothelial barrier function (Ferrero et al. 1995). This importance was reinforced by recent studies demonstrating that endothelial cells from PECAM-1-deficient mice show enhanced permeability in response to histamine (Graesser et al. 2002). PECAM-1 is required for leukocyte migration and TEM both in vitro and in vivo (Muller et al. 1993; Bogen et al. 1994; Nakada et al. 2000; Wang et al. 2005). Anti-PECAM-1 antibodies arrest leukocyte migration at the apical surface of endothelium prior to proceeding through the junction, demonstrating that PECAM-1 mediates the initial phase of diapedesis (Schenkel et al. 2002). There are several studies indicating that PECAM-1 may regulate these functions by regulating integrin activation (Piali et al. 1995; Chiba et al. 1999) and PECAM-1-deficient mice have impaired “outsidein” signaling by the platelet integrin αIIbβ3 (Wee & Jackson 2005).

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CD99 CD99 is a small, heavily O-glycosylated transmembrane glycoprotein expressed on most leukocytes, erythrocytes, and at endothelial cell junctions (Aubrit et al. 1989; Muller 2002; Schenkel et al. 2002). It shows no resemblance structurally or at amino acid level to any known protein family and its functions are poorly understood, although along with PECAM-1 and JAMs it is involved in diapedesis. The gene for CD99 encodes two alternatively spiced products that appear to have opposite effects on lymphocyte adhesion as well as on the expression of LFA-1 and ICAM-1 (Hahn et al. 1997; Byun et al. 2006). Anti-CD99 antibodies have demonstrated lymphocyte adhesion mediated by homotypic interactions (Kasinrerk et al. 2000; Imbert et al. 2006). In addition, ligation of CD99 on lymphocytes has been shown to induce adhesion by regulating expression of α4β1 binding to VCAM-1 (Bernard et al. 2000). Antibodies directed against CD99 block T-cell recruitment in vivo (Bixel et al. 2004). Recent work indicates that antibodies against CD99 arrest monocyte migration but, importantly, only when the cells are located deep within the paracellular pathway and close to completion of transmigration into tissue, suggesting that PECAM-1 and CD99 work in a sequential fashion to mediate paracellular diapedesis (Aurrand-Lions et al. 2002; Schenkel et al. 2002).

JAMs JAMs are recently discovered members of the immunoglobulin superfamily. JAM-A was first reported as the ligand for platelet activating antibody and thus was referred to as F11 receptor (Kornecki et al. 1990). Sequencing of JAM-A from epithelial and endothelial cells led to its initial description in the formation of tight junctions (Martin-Padura et al. 1998; Palmeri et al. 2000). JAM proteins are type I transmembrane receptors consisting of an extracellular domain containing two immunoglobulin-like domains, a transmembrane domain, and a short cytoplasmic tail (Malergue et al. 1998; Martin-Padura et al. 1998; Sobocka et al. 2000; Arrate et al. 2001). Two related JAMs were subsequently found, JAM-B and JAM-C, which were also localized primarily at cell–cell junctions on endothelial cells (Cunningham et al. 2000; Arrate et al. 2001; Aurrand-Lions et al. 2005). In addition, two more JAMs have been recently identified: JAML in humans (Moog-Lutz et al. 2003) and JAM4 in mice, although it is unknown in the latter whether a human homolog exits (Hirabayashi et al. 2003). In addition, JAM4 and JAML appear to be less closely related to JAM-A, JAM-B or JAM-C (Mandell & Parkos 2005). Subsequently, the expression pattern of JAM proteins has been found to be much wider than previously thought and these proteins have been implicated in a variety of events important to leukocyte function including tight junction assembly (Martin-Padura et al. 1998; Liang et al. 2000; Itoh et al. 2001; Ebnet et al. 2003; Mandicourt et al. 2007), platelet activation (Kornecki et al. 1990; Ozaki et al. 2000; Sobocka et al. 2000), and leukocyte transmigra-

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tion (Martin-Padura et al. 1998; Johnson-Leger et al. 2002; Ostermann et al. 2002). Indeed, antibodies directed against JAM-A and JAM-C inhibit leukocyte migration both in vitro and in vivo (Martin-Padura et al. 1998; Del Maschio et al. 1999; Chavakis et al. 2004). JAM-A is expressed on the surface of lymphocytes, monocytes, neutrophils, platelets, and erythrocytes (Martin-Padura et al. 1998; Williams et al. 1999; AurrandLions et al. 2001). JAM-A binds itself (Bazzoni et al. 2000) as well as reovirus protein (Barton et al. 2001). In addition, JAM-A on endothelial cells binds to αVβ3 (Naik et al. 2003; Naik & Naik 2006), as well as αLβ2 on neutrophils and T cells, facilitating TEM (Ostermann et al. 2002; Fraemohs et al. 2004). Furthermore, soluble JAM-A has been used to inhibit TEM of T cells under flow conditions (Ostermann et al. 2005). Inflammatory mediators such as TNF-α and IFN-γ cause junctional disassembly of JAM-A and relocalization to the apical surface of endothelial cells (Ozaki et al. 1999; Shaw et al. 2001). The JAM-A–αLβ2 interaction mediates tight adhesion or transmigration depending on the apical or junctional localization of JAM-A on endothelial cells (Ostermann et al. 2002). JAM-B has restricted expression to endothelial cells where it mediates leukocyte adhesion by binding to α4β1 (VLA-4) as well as JAM-C (Cunningham et al. 2002; Liang et al. 2002) and promotes lymphocyte TEM (Johnson-Leger et al. 2002). JAM-C is expressed on endothelial cells, leukocytes, and fibroblasts (Cunningham et al. 2000; Palmeri et al. 2000; Aurrand-Lions et al. 2001; Morris et al. 2006). JAM-C binds to JAM-B (Arrate et al. 2001) as well as αMβ2 (Mac-1/ CD11b/CD18) on neutrophils and mediates TEM both in vitro and in vivo (Santoso et al. 2002; Chavakis et al. 2004; AurrandLions et al. 2005; Zen et al. 2005; Mandicourt et al. 2007). JAML is found on human granulocytes (Moog-Lutz et al. 2003), and coxsackie and adenovirus receptor (CAR) has been found to be an epithelial counterreceptor that mediates neutrophil migration across tight junctions (Zen et al. 2005). Consequently, JAMs appear to mediate leukocyte adhesion through both homophilic interactions as well as heterophilic interactions with integrins and other JAMs (Bazzoni et al. 2000; Arrate et al. 2001; Cunningham et al. 2002; Santoso et al. 2002; Lamagna et al. 2005; Mandell et al. 2005). Like other leukocyte adhesion molecules, cytokines seem to be important for JAM regulation. However, to date, these studies demonstrate that, rather than regulating expression levels, cytokines regulate JAM localization from intercellular junctions to the apical membrane (Ozaki et al. 1999; Shaw et al. 2001) and internalization of JAMs (Bruewer et al. 2003, 2005; Utech et al. 2005). Phosphorylation of the cytoplasmic tail appears to be a molecular mechanism for regulation of intracellular binding partners, adhesive function, and localization (Ozaki et al. 2000; Ebnet et al. 2003; Dejana 2006; Mandicourt et al. 2007).

MAdCAM-1 MAdCAM-1 is an endothelial cell glycoprotein that specifies lymphocyte homing to mucosal sites (Sampaio et al. 1995).

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MAdCAM-1 is expressed at high density on HEV of lymphoid tissues such as Peyer’s patches and mesenteric lymph nodes and to a lesser extent in lamina propria (Picker & Butcher 1992; Briskin et al. 1997). Recent evidence indicates that MAdCAM-1 may be more widely expressed than previously thought, with evidence of expression on fibroblasts and melanoma cells (Leung et al. 2003) and in the brain (Kanwar et al. 2004). Cloning of human MAdCAM-1 revealed a multifunctional type I transmembrane adhesion molecule comprising two extracellular immunoglobulin-like domains, an extracellular mucin-like domain, one transmembrane region, and a short cytoplasmic tail (Shyjan et al. 1996; Leung et al. 1997, 2003). Its unique structure allows it to bind to lymphocytes and mediate homing through both α4β7 and L-selectin (Berg et al. 1993; Berlin et al. 1993; Shimizu & Shaw 1993; Hamann et al. 1994). Thus, MAdCAM-1 is uniquely able to participate in two phases of diapedesis: lymphocyte rolling with L-selectin and the firm adhesion step by binding with the integrin α4β7. The importance of MAdCAM-1 leukocyte trafficking is shown by studies demonstrating enhanced expression of MAdCAM-1 in patients with inflammatory disease (Souza et al. 1999; Arihiro et al. 2002; Ohara et al. 2003). Blocking antibodies to MAdCAM-1 have been shown to reduce leukocyte extravasation in vivo (Matsuzaki et al. 2005; Farkas et al. 2006). Numerous studies indicate that MAdCAM-1 expression can be induced by TNF-α and IL-1β (Sikorski et al. 1993; Takeuchi & Baichwal 1995; Ando et al. 2005). The cytoplasmic tail is involved in signaling via phosphorylation of cytoskeletal proteins (Murata et al. 2002).

Adhesion molecules in allergic disease Adhesion molecule expression Endothelial adhesion molecule expression Adhesion molecule function is regulated in a number of ways, including increased expression as with E- and P-selectin, ICAM-1 and VCAM-1, shedding as with L-selectin, and conformational changes in the binding affinity of the receptor as seen with many integrins. A number of groups have studied expression of E-selectin, ICAM-1, and VCAM-1 in asthma and other allergic inflammatory conditions. P-selectin expression has been less widely studied, partly because of the difficulty in distinguishing between intracellular and luminal staining. Studies of adhesion receptors expressed following allergen challenge have been generally consistent with observations in vitro with cytokine-stimulated human umbilical vein endothelial cells (HUVECs). In the skin, low background expression of ICAM-1 is seen with absent expression of E-selectin and VCAM-1. After allergen challenge, increased endothelial expression of all three receptors has been reported (KyanAung et al. 1991; Leung et al. 1991). In the airway, Montefort et al. (1994a) found increased expression of ICAM-1 and E-selectin 6 hours after local allergen challenge with no

Leukocyte Adhesion in Allergic Inflammation

increase in VCAM-1 expression. Bentley et al. (1993) reported a trend toward increased VCAM-1 expression (significance was lost through one outlier), with a good correlation between VCAM-1 expression and eosinophil infiltration 24 hours after aerosol allergen challenge. In sensitized lung explants, allergen challenge increased ICAM-1, E-selectin, and VCAM-1 expression in a manner similar to that seen in HUVECs. Upregulation was mediated by a combination IL-4 and TNF-α (Hirata et al. 1998). In clinical asthma, findings have been more variable, perhaps reflecting the inherent problems in accurately quantifying small changes in expression using immunohistochemistry. Montefort et al. (1992b) were unable to detect changes in adhesion receptor expression in atopic asthma compared with normal subjects. In a study of atopic and nonatopic asthma, Bentley et al. (1993) could only detect a modest increase in ICAM-1 and E-selectin expression in nonatopic asthmatics compared with normal subjects, with relatively high expression in normals. However, the E-selectin antibody cross-reacted with P-selectin, which makes the data on E-selectin difficult to interpret. In contrast, Gosset et al. (1995) found low expression in normal subjects and could detect increases in adhesion molecule expression in atopic but not nonatopic asthmatics. Ohkawara et al. (1995) agreed with these findings in six atopic asthmatics but Fukuda et al. (1996) detected no increase in ICAM-1 or E-selectin staining over controls. However, the E-selectin antibody they used also cross-reacted with P-selectin. Nevertheless, this group did find an increase in VCAM-1 expression that correlated with eosinophil counts but only in those subjects with detectable IL-4 in bronchoalveolar lavage (BAL) fluid. In nasal endothelium, generally weak expression of VCAM-1 has been observed, although increased over normal controls, in both perennial rhinitis and nasal polyps (Montefort et al. 1992a). Increased expression of ICAM-1, VCAM-1, and E-selectin was also observed after allergen challenge (Braunstahl et al. 2001). We have found E-selectin expression to be weak in nasal polyps, lung resection tissue, and perennial rhinitis. We have found that P-selectin is widely expressed in both nasal and lung tissue (Symon et al. 1994; Ainslie et al. 2002). Strong expression was seen in nasal biopsies from both normal controls and patients with perennial rhinitis, with little difference between the two groups after fixation with either acetone or paraformaldehyde (both of which favor surface as opposed to intracellular staining). Similarly, in endobronchial biopsies good expression was observed in sections from both asthmatic and control subjects. Consistent with this observation, P-selectin was well expressed in lung resection tissue from patients with lung cancer. Expression was seen on both bronchial and pulmonary venules but not the pulmonary capillaries. As mentioned above it is difficult to distinguish conclusively between intracellular and luminal staining using standard immunohistochemistry. However, the strong expression in the airway does

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suggest P-selectin rather than E-selectin is the major selectin involved in leukocyte migration into the lung in humans. ICAM-1 expression on epithelial cells is consistently increased on bronchial epithelium in asthma (Vignola et al. 1993). The role of ICAM-1 as a receptor for the major group of rhinoviruses means that the epithelium in asthmatics may be more vulnerable to viral infection. Expression of CD44, a receptor for the matrix protein hyaluronate, is increased on the bronchial epithelium in asthma, although it is also found on normal epithelium (Lackie et al. 1997).

Soluble adhesion molecules Several adhesion molecules can be detected in soluble form circulating in the plasma. Montefort et al. (1994b) found that concentrations of E-selectin, ICAM-1, and VCAM-1 were not elevated in stable asthma, but there was a significant increase compared with normal controls in concentrations of sE-selectin and sICAM-1 in patients with acute severe asthma. However, concentrations of these molecules did not correlate with disease severity and were therefore not thought useful in clinical management. In another study of 45 atopic and nonatopic asthmatics serum concentrations of sICAM-1, sE-selectin, and sVCAM-1 were increased during “asthma attacks” compared with stable periods (Kobayashi et al. 1994; Koizumi et al. 1995). Modest increases in concentrations of sICAM-1 and sE-selectin have also been detected in BAL fluid after segmental allergen challenge (Georas et al. 1992; Takahashi et al. 1994). Zangrilli et al. (1995) measured sVCAM-1 concentrations in BAL fluid 24 hours after segmental allergen challenge in 27 ragweed allergic asthmatics and 18 atopic nonasthmatics. A marked increase in sVCAM-1 concentrations was observed in BAL fluid that correlated with increased numbers of eosinophils and concentrations of IL-4 and IL-5. Most of the increase occurred in the late responders. In atopic dermatitis in children, plasma E-selectin was raised and correlated with disease severity during exacerbations but did not fall after treatment (Wolkerstorfer et al. 2003). Therefore as yet no clear-cut correlations between disease severity and concentration of soluble adhesion receptors have been found that would allow these molecules to be used as biomarkers of diagnosis or disease activity.

Leukocyte migration and adhesion Eosinophil migration and adhesion The mechanisms controlling eosinophil migration into tissue have been the subject of intensive study for over four decades and there is a wealth of literature on the subject summarized in several recent reviews (Weller 1997; Wardlaw 1999; Bochner 2000; Rothenberg & Hogan 2006). The eosinophil expresses a number of adhesion receptors that are involved in cell trafficking and effector function (Table 15.1). In the normal individual eosinophils make up only 1–5% of the white cell count and in the normal lung there are 50 neutrophils for every eosinophil. In severe asthma there is an average of four eosinophils for every neutrophil in the bronchial sub-

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Table 15.1 Eosinophil adhesion receptors and the ligands they recognize. Ligand Receptor Integrins a4b1(VLA-4) a4b6 a4b7 LFA-1(aLb2) Mac-1(aMb2) P150,95(axb2) adb2 Selectins and ligands PSGL-1 L-selectin Other CD44 ICAM-3 PECAM Siglec-8

Endothelial

Matrix protein

VCAM-1

Fibronectin Laminin Fibronectin

MAdCAM-1 ICAM-1-3 ICAM-1 VCAM-1(ICAM-3?)

P-selectin(E-selectin) GlyCAM-1,CD34,Podocalyxin

Hyaluronate PECAM Sialic acid

mucosa, representing a 200-fold enrichment of eosinophils over neutrophils in the process of accumulation. Even in mild asthma there is an up to 30-fold enrichment (Azzawi et al. 1992; Brightling et al. 2003). Historically this was thought to be due to a selective chemoattractant. A factor termed “eosinophil chemotactic factor of anaphylaxis” (ECF-A) was detected in supernatants from anaphylactically challenged guinea-pig lung that appeared to be selectively chemotactic for eosinophils (Kay et al. 1971). This was subsequently found to consist of a combination of leukotriene (LT)B4, which is active on guinea-pig eosinophils but less so on human eosinophils, and 15-hydroxyeicosatetraenoic acid (Sehmi et al. 1991). ECF-A from human lung was later identified and characterized as two tetrapeptides, Val-Gly-Ser-Glu and AlaGly-Ser-Glu (Goetzl & Austen 1975). However, in comparison with platelet-activating factor, these peptides were found to have negligible activity (Wardlaw et al. 1986). More recently it has become clear that eosinophil migration is the result of an interaction between selective expression of adhesion molecules that bias toward eosinophil recruitment and eosinophil-selective chemoattractants, particularly chemokines. Accumulation of eosinophils is not therefore the result of any single event but occurs because of selective pressure at every stage in the life cycle of the eosinophil, including eosinophilopoiesis and egress from the bone marrow, adhesion mechanisms, chemotaxis and prolonged survival under the influence of locally generated growth factors (Fig. 15.6).

Eosinophilopoiesis and egress from the bone marrow Eosinophils differentiate from bone marrow precursors under the influence of growth factors, especially IL-5. Increased

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IL-5 eotaxin 1

IL-4/ IL-13

PSGL-1/P-selectin VLA-4/VCAM-1 Chemotaxis CCL26/CCR3

Survival IL-5/GM-CSF IL-4 IL-5 IL-13

4

2

Leukocyte Adhesion in Allergic Inflammation

Tether/roll Flow

Activation Firm arrest

3

In situ Diff/IL-5 5 CCL26/CCR3

Th2

6 Apoptosis

Fig. 15.6 Selective accumulation of eosinophils is a multistep process directed by Th2-associated cytokines. Step 1 involves selective eosinophilopoiesis under the influence of IL-5 and egress from the bone marrow promoted by chemoattractants such as eotaxin 1. Step 2 involves selective migration through venular endothelium that is promoted by a4b1/VCAM-1, PSGL-1/P-selectin, and (CCL26) eotaxin 3. After transmigration (step 3), tissue accumulation is controlled by a balance between prolonged survival and retention in tissues as a result of adhesion to matrix proteins such as fibronectin (step 4), local differentiation from precursors within tissue (step 5), and signals for egress into the lumen provided by epithelial-derived chemoattractants such as CCL26 (step 6). All this is orchestrated by IL-4, IL-13 and IL-5 released by Th2 lymphocytes. (See CD-ROM for color version.)

amounts of IL-5 generated at sites of allergic inflammation act hormonally to increase eosinophil production. There is on average about a fourfold increase compared with normal subjects in the number of circulating eosinophils in allergic individuals. There is also evidence for increased numbers of circulating eosinophil precursors in the peripheral blood of allergic patients. This increase is the consequence of both increased production and egress from the bone marrow. In guinea pigs IL-5 selectively promoted the egress of eosinophils from the marrow (Collins et al. 1995). This was enhanced by eotaxin, an eosinophil-selective chemokine (Palframan et al. 1998a), and controlled by the adhesion molecules VLA-4, inhibition of which accelerates egress, and Mac-1, whose inhibition prevents egress (Palframan et al. 1998b). In this contex IL-5 is probably acting as a chemokinetic factor as well as priming eosinophils for chemotactic responsiveness.

Migration through vascular endothelium Adhesion to and migration through vascular endothelium is an important control point in the migration of all leukocytes including eosinophils. As for other leukocytes, migration in most situations occurs through the postcapillary venular endothelium and follows the well-established multistep paradigm of a capture step, often followed by rolling along the surface of the vessel until the leukocyte becomes activated, which results in arrest of the cell and transmigration (Fig. 15.7). An exception to this is migration into the peripheral lung

a4/b1/VCAM-1 PSGL-1/P-selectin L-selectin

Gai, PT sensitive 7TM receptors

CD18/ICAM-1 a4b1/VCAM-1

Fig. 15.7 Schematic illustration of the multistep paradigm of leukocyte migration through endothelium in the high-flow systemic circulation, which involves a capture step mediated by selectins and VCAM-1, an arrest step mediated by activating signals such as chemoattractants expressed on the endothelial surface, and a transmigration step mediated largely by b2 integrins and ICAM-1. (See CD-ROM for color version.)

compartment through the capillary bed and venules of the low-pressure pulmonary circulation, which is less dependent on adhesion receptors, being predominantly controlled by chemoattractant stimuli (Fig. 15.8). Eosinophils adhere in much greater numbers than neutrophils to vascular endothelium stimulated with the Th2 cytokines IL-4 and IL-13 as a result of selective adhesion to VCAM-1 and P-selectin, which work in a synergistic manner to capture and then arrest these cells on vascular endothelium (Patel 1998; Woltmann et al. 1999). Human endothelial cells stimulated for over 24 hours with IL-4 or IL-13, but not IL-1β or TNF-α, induced low levels of expression of both VCAM-1 and P-selectin (Yao et al. 1996). Eosinophils can adhere to VCAM-1 through VLA-4 whereas neutrophils that do not express this receptor are unable to do so (Walsh et al. 1991). Eosinophils adhere more avidly to low levels of P-selectin than neutrophils as a result of increased expression of the P-selectin receptor PSGL-1 and possibly because of differences in the pattern of O-glycosylation on the eosinophil PSGL-1 receptor (Symon et al. 1996; Edwards et al. 2000). P-selectin is well expressed on airway vascular endothelium and in an ex vivo assay of eosinophil adhesion to vascular endothelium in a nasal polyp model of eosinophilic inflammation P-selectin/PSGL-1 was the primary adhesion pathway involved (Symon et al. 1994). Eosinophil migration to the lung was reduced in a P-selectin gene-deleted mouse (Broide et al. 1998). In contrast, neutrophils bind more avidly to E-selectin, which is not well expressed on the airway vascular endothelium in asthma (Sriramarao et al. 1996; Kitayama et al. 1997a). VLA-4/VCAM-1 can also support selective transmigration through HUVECs and a number of animal models have shown that eosinophil migration into the lung is inhibited by anti-VLA-4 (Nakajima et al. 1994; Lobb et al. 1996; Cortijo et al. 2006). However, VCAM-1 expression is not particularly marked on the airway mucosal endothelium in asthma or nasal polyps; in both the nasal polyp model and in adhesion to IL-4-stimulated

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Bronchial artery and vein

Pulmonary vein Systemic circulation

Pulmonary artery

endothelium, inhibition of VLA-4/VCAM-1 on its own had a relatively minor effect (Bentley et al. 1993; McNulty et al. 1999; Woltmann et al. 2000). Antagonists of VLA-4 only partially blocked eosinophil adhesion to TNF-α- and IL-4-stimulated endothelium (Sedgwick et al. 2005). In addition VLA-4 antagonists had only a minor effect at best on eosinophil accumulation in the airways after allergen challenge, although it is possible that the inhaled route used in these studies was not optimal (Diamant et al. 2005; Norris et al. 2005). In summary, therefore, in vitro studies of adhesion to endothelium under conditions of Th2 cytokine generation have shown that selective adhesion is directed by the combined actions of VCAM-1/VLA-4 and P-selectin/PSGL-1, with the latter playing a dominant role in the capture of the cells. Under more Th1-like conditions where cytokines such as TNF-α and IL-1 are generated, adhesion will be through E-selectin, VCAM-1/ VLA-4, and ICAM-1/β2 integrins, but will not have a selective component (Bochner et al. 1991). Eosinophils express all four members of the β2 integrin family (Hartnell et al. 1990; Grayson et al. 1998). As is the case for neutrophils, αΜβ2 (Mac-1) and αLβ2 (LFA-1) have been shown to mediate eosinophil transmigration through TNFα- and IL-1β-stimulated endothelium mainly by binding to ICAM-1 (Yamamoto et al. 1998), although both αDβ2 and Mac-1 as well as VLA-4 have been shown to bind to VCAM-1 (Barthel et al. 2006a). Eosinophils bind VCAM-1 through podosomes (Johansson et al. 2004). Following the established paradigm for leukocyte adhesion to endothelium, after the capture and rolling step the eosinophil requires activation to mediate arrest and engagement of β2 integrins in particular. This then allows transmigration. Eosinophil activation in this context is complex and still relatively poorly understood. Although CCR3-binding chemokines can participate in this process, they have only a relatively minor effect on adhesion to cytokine-stimulated endothelium under flow conditions (Kitayama et al. 1998). Avidity for β1 and β2 integrins is differentially regulated depending on the timing and type of stimulus and eosinophils have possibly three distinct signal transduction pathways that regulate responses to chemo-

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Fig. 15.8 Migration into the lung differs between the large airways and the peripheral compartment. The bronchi are supplied by bronchial arteries that are part of the systemic circulation and therefore migration follows the multistep paradigm. Migration into the peripheral lung is through the capillary bed of the low-pressure pulmonary circulation and is relatively adhesion receptor independent. (See CD-ROM for color version.)

attractants (Weber et al. 1996; Kitayama et al. 1997b). More work is required in this area to understand the precise signals that control the activation step in eosinophil adhesion to endothelium. As well as VLA-4, eosinophils also express α4β7 and there is evidence that this receptor is involved in normal homing of eosinophils to the small intestine through binding to MAdCAM-1, although ICAM is the main receptor involved in migration into the large intestine (Mishra et al. 1999; Forbes et al. 2006). Transmigration of eosinophils is shear dependent and under flow conditions occurs on IL-4- but not TNF-α-stimulated endothelium. Under these conditions the CCR3-binding chemokine eotaxin 3 (CCL26) appeared to the main chemoattractant involved (Cuvelier & Patel 2001). Eosinophil adhesion to endothelium under shear flow resulted in endothelial activation of Rho-associated proteins and calpain, which are necessary for transmigration of eosinophils (Cuvelier et al. 2005).

Eosinophil migration within tissue Once eosinophils have migrated into tissue, as is the case with all leukocytes, they need to be able to integrate the various adhesive and chemotactic signals derived from structural cells and the extracellular matrix in order to direct their microlocalization within the tissue environment. This includes maintaining residence within the tissue or migrating through epithelium into the gut or airway lumen (Fig. 15.9). Eosinophils express the β1 integrins α4β1 and α6β1, which have been shown to play a role in binding to fibronectin and laminin respectively. These matrix proteins support eosinophil survival by triggering release of GM-CSF, an event inhibited by glucocorticoids (Walsh & Wardlaw 1997). With other aspects of eosinophil activation more variable effects with fibronectin have been observed, with some authors reporting enhancement and others inhibition of eosinophil degranulation (Neeley et al. 1994; Kita et al. 1996). Tissue eosinophils express a more activated phenotype than their blood counterparts, with increased expression of CD69, ICAM-1, and a number of other receptors (Hartnell et al. 1993). They also express an activated form of Mac-1 that results in a hyperadhesive phenotype to a

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Lymphatics

Airway lumen Fig. 15.9 Once through the endothelium, leukocytes undergo a process of microlocalization within the mucosa. This is controlled by integration of various adhesive and chemoattactant signals provided by the extracellular matrix, structural cells, and other leukocytes. Microlocalization is likely to be important in the effector role of leukocytes, which probably exert their effects across only a few micrometers. In particular mast cells colocalize with structural cells including airway smooth muscle in asthma, whereas T cells migrate into the epithelium or into the lymphatics on their way back to the blood. Eosinophils migrate into the lumen where they appear in induced sputum. (See CD-ROM for color version.)

number of matrix proteins, as well as VCAM-1 and ICAM-1 (Barthel et al. 2006b). The tissue phenotype can be mimicked by culturing blood eosinophils in IL-5 or GM-CSF for 24 hours. For eosinophils to migrate through epithelium to the lumen they need to migrate to and through the epithelium. Eosinophils adhere relatively weakly to cultured airway epithelial cells, which presumably allows them to escape into the lumen. Such adhesion is mediated by CD18/ICAM-1 interactions and is stimulated by IL-5 (Sanmugalingham et al. 2000). Migration through IL-4-stimulated epithelium, as is the case for endothelium, is primarily mediated by cell-bound eotaxin 3 (Yuan et al. 2006). Thus although there are a large number of chemoattractants involved in eosinophil migration under conditions of Th2 cytokine stimulation, the in vitro evidence points to eotaxin 3 binding to CCR3 as playing a central role. In summary, therefore, eosinophil trafficking into tissue involves a multistep process, with selective pressure on eosinophil recruitment occurring through selective expression of eosinophil-biased adhesion receptors and chemokines promoted by the Th2 cytokines IL-4 and IL-13. Once in tissue, adhesion to matrix proteins and structural cells plays an important role in survival, microlocalization, and eosinophilmediated tissue damage.

Basophil migration and adhesion Basophils, like eosinophils, are generated in the bone marrow and circulate in blood as mature granulocytes before migrating into tissue. Basophils express a similar but not identical pattern of adhesion and chemokine receptors to eosinophils.

Leukocyte Adhesion in Allergic Inflammation

They express PSGL-1 but at lower levels than eosinophils, and like neutrophils (but not eosinophils) express high levels of sLex, which suggests they preferentially bind E-selectin compared with P-selectin. They express L-selectin (which is shed on activation), all four β2 integrins, and α4β1 and α4β7. In addition they express α5β1 and αVβ3, but not α6β1 (Bochner & Schleimer 2001). Blocking antibodies against α4β1 have been shown to inhibit basophil binding to endothelium under static conditions (Bochner et al. 1996) as well as TEM through IL-3-stimulated endothelium, although β2 integrins binding to ICAM-1 was more important in TEM through IL-1-stimulated endothelium (Iikura et al. 2004). Under flow conditions basophil adhesion to IL-4-stimulated endothelium was mediated by both E-selectin and P-selectin, with α4β1 only playing a role if the basophils were activated, possibly through an Src-dependent pathway (Andrews et al. 2001; Kepley et al. 2002). Again under flow conditions, IL-3stimulated endothelium was able to bind basophils (as well as eosinophils but not neutrophils) through a PSGL-1/VLA-4 dependent pathway in a manner similar to IL-4- and IL-13treated endothelium. Of particular interest is the observation that the basophil arrest step appeared to be pertussis toxin sensitive and mediated by CCR7, whereas eosinophil arrest was not pertussis toxin sensitive as was also the case in the ex vivo nasal polyp model (McNulty et al. 1999; Lim et al. 2006).

Mast cell migration and adhesion Mast cells are released from the bone marrow and circulate in the blood as precursors (pMC) before fully differentiating in tissue. In blood they are found among a subset of mononuclear cells that express CD34, CD13, and the stem cell factor (SCF) receptor c-kit. Immature peripheral blood mast cells, or cells derived from human cord blood as a model of pMC, express α4β1, α4β7, αMβ2, αVβ2, and PSGL-1 (Inamura et al. 2001; Tachimoto et al. 2001; Boyce et al. 2002). They can therefore bind VCAM-1, MAdCAM-1, ICAM-1/2, E-selectin, and P-selectin. They do not express L-selectin. The patterning of binding of pMC to vascular endothelium is similar in some respects to basophils rather than eosinophils, with a relatively low affinity for P-selectin and therefore an inability to bind to IL-4-stimulated endothelium under flow conditions. They utilize predominantly E-selectin and VLA-4/VCAM-1 for binding to TNF-α-stimulated endothelium (Boyce et al. 2002). Interestingly, the β2 integrins and ICAM-1 do not play a major role in adhesion under these conditions despite these receptors being involved in IL-4-mediated homotypic adhesion (Toru et al. 1997). The chemoattractants involved in migration of pMC across the endothelium have not been well defined. pMC express CCR3 and may therefore be able to respond to eotaxin 3, which is secreted by endothelial cells (Romagnani et al. 1999; Juremalm & Nilsson 2005). They also express CXCR4 and transmigrate across endothelium in response to CXCL14 (SDF-1) (Juremalm et al. 2000; Lin et al. 2000).

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When pMC migrate into tissue they differentiate into tryptase-expressing mast cells (MCT) or chymase/tryptaseexpressing mast cells (MCTC) depending on local developmental signals. During this process they change their pattern of chemokine receptors, for example increasing expression of CXCR3 and reducing expression of CCR3 (Brightling et al. 2005a), a change that may promote their migration toward airway smooth muscle (ASM) (Brightling et al. 2005b). Mature mast cells can also respond to fractalkine, another chemokine released by ASM (Papadopoulos et al. 2000; El-Shazly et al. 2006). An important feature of mast cell biology is their anatomic proximity to the structural cells within the tissue, presumably in order to modulate the function of these structures. This includes vascular and neural tissue, mucous glands, epithelium, and ASM where colocalization has been shown to be related to presence of airway hyperresponsiveness (Costello et al. 1997; Brightling et al. 2002). Whether it is the pMC, mature mast cells, or both that migrate is not known but there is evidence that mature cells can move from the submucosa to the epithelium during allergen challenge (Viegas et al. 1987). Adhesion to matrix proteins also plays an important role in mast cell activation, acting as a priming stimulus for mediator release. Mast cells are able to adhere to fibrinogen and von Willebrand factor via αIIbβ3 after stimulation with SCF and cross-linking of FcεRI. Binding to fibrinogen has a marked effect on function, with enhanced proliferation, cytokine production, and migration (Oki et al. 2006). Similarly, binding to fibronectin and vitronectin primes the mast cell line HMC-1 for mediator release stimulated by phorbol myristate acetate and ionomycin (KrugerKrasagakes et al. 1999). Human intestinal mast cells are able to adhere to fibronectin, laminin and collagens I, III, VI, and XIV. Adhesion was enhanced fivefold by SCF and was mediated by a combination of α2β1–α5β1 and αVβ3. The effects of SCF were reduced by cotreatment with IL-4 and by blockade of phosphatidylinositol 3-kinase and MAPK (Lorentz et al. 2002). Adhesion to fibronectin and vitronectin was also inhibited by an antibody against CD63, a member of the tetraspanin family that forms multimolecular complexes with a variety of membrane proteins including β integrins. In addition, anti-CD63 inhibited FcεRI-mediated degranulation of adherent but not nonadherent mast cells (Kraft et al. 2005). Activation of Toll-like receptor (TLR)3 on mast cells had a similar effect to blockade of CD63. Mast cells express TLR1–7 and TLR9 and when stimulated through TLR3 mast cell adherence to fibronectin and vitronectin as well as FcεRI-mediated degranulation was reduced as a result of inactivation of CD29 (β1) (Kulka & Metcalfe 2006). The close proximity of mast cells to structural cells may also be the result of avid cell–cell adhesion. A striking feature of the cell differential from bronchial washings and induced sputum is the paucity of mast cells (and T cells) compared with neutrophils and (in asthma) eosinophils, despite these cells being present in similar numbers in the submucosa. This

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is likely to be due to the firmer anchoring of mast cells in the tissue as a result of binding to matrix proteins and structural cells including epithelium. In support of this hypothesis, mast cells bind much more firmly to bronchial epithelial cells than eosinophils (60% vs. 10%) (Sanmugalingam et al. 2000). Similar levels of mast cell adhesion have been observed to ASM and fibroblasts (Trautmann et al. 1997; Yang et al. 2006a). Interestingly this adhesion pathway, although partly calcium dependent, is not mediated by any of the classical adhesion receptors including the β1 or β2 integrins and appears to involve in part novel pathways, with tumor suppressor in lung cancer-1 mediating adhesion to ASM and a galactosebearing carbohydrate pathway adhesion to epithelial cells. Unlike matrix, adherence to epithelial cells attenuates mast cell degranulation stimulated by FcεRI (Yang et al. 2006b). Mast cell adherence to matrix proteins and structural cells is therefore of central importance to the function of these cells in allergic disease and dissection of the receptors involved and the consequences of their inhibition it likely to be a fruitful area of future research.

T-cell migration T cells, especially those of the Th2 subset, are thought to play a central part in allergic disease. The role of adhesion in T-cell migration and function is considerably more varied and complex than granulocytes and this topic can only be touched on in this section. The reader is referred to recent reviews of the subject for a detailed background (Campbell et al. 2003; Mora & von Andrian 2006). Unlike other leukocytes T cells migrate from blood into tissue and then back to the blood in a directed manner depending on their state of antigenic stimulation. Naive cells migrate only into lymph nodes whereas memory cells can migrate into both lymphatic tissue and organs which make up a tertiary lymphoid structure. T cells preferentially migrate back to the organ where they first encountered their cognate antigen, a process called lymphocyte homing. T cells expressing high levels of α4β7, which therefore bind MAdCAM-1, are thought to be preferentially gut homing whereas T cells that express CLA, a glycoform of PSGL-1 which preferentially binds E-selectin, are thought to be skin homing (Campbell & Butcher 2000). Lymphocyte homing may be important in directing allergen-sensitized T cells to the airway or skin. For example, caesin-reactive T cells from patients with milk-induced eczema had higher expression of the CLA antigen than Candida albicans-reactive T cells from the same patients or caesin-reactive T cells from nonatopic controls (Abernathy-Carver et al. 1995) . When house-dust mite-sensitive patients with asthma and atopic dermatitis were compared, the house-dust mite-responsive T cells from the eczema patients, but not from the asthma group, were in the CLA-positive T-cell subset (Santamaria Babi et al. 1995). Lung T cells are distinct from those of the gut and skin, being CLA negative and α4β7low (Campbell et al. 2001). CXCR6 is potentially involved in lung homing (Wardlaw et al. 2005)

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CLA/E-selectin CCR4/CCL17 CCR10/CCL27 CCR8/CCL1 Fig. 15.10 Lymphocytes migrate in a controlled manner, with naive lymphocytes only able to enter the secondary lymphoid tissue and memory lymphocytes preferentially returning to the organ where they encountered their cognate antigen. This process of lymphocyte homing is controlled by differential expression of a combination of chemoattractant and adhesion receptors and their ligands. (See CD-ROM for color version.)

Leukocyte Adhesion in Allergic Inflammation

P-selectin/PSGL-1 CXCR6/CXCL16

Skin

Lung

L-selectin/PNAd CCR7/CCL21 CCR7/CCL19 Lymph node

(Fig. 15.10). Migration during inflammatory states is superimposed on this homeostatic process. The number of T cells in tissue at any point in the normal state varies considerably. T cells are numerous in the gut and lung submucosa where many are likely to be long-term residents. However in the normal skin and in nonmucosal organs such as the heart and kidney, T cells are scanty. As with other leukocytes, T cells can be found in different compartments within tissue. For example in the lung there is the bronchial submucosa, epithelium, airway lumen, alveolar compartment, and interstitium. It is likely that the migration pathway into each compartment is different. As discussed above, in the lung there is a major difference between migration into the alveolar compartment through the low-pressure capillary bed, which is likely to be largely chemoattractant dependent, and migration into the bronchial mucosa via the systemic highpressure bronchial circulation, which involves the established multistep paradigm (Wardlaw 2002). There is a general assumption that Th2 cells are present in greater numbers in the tissue in allergic disease and there has therefore been considerable interest in the mechanisms driving this process, largely focused on the role of chemokines and their receptors. Most studies, the majority of which examined those with mild asthma, have not shown any increase in the numbers of T cells in the submucosa between asthmatics and normal subjects. There is also no consistent increase in T-cell numbers in BAL fluid or bronchial mucosa after allergen challenge (Aalbers et al. 1993; Frew et al. 1996). Again, at least in mild asthma, there is only very limited evidence for a major increase in IL-4-secreting T cells in the asthmatic lung (Morgan et al. 2005). Surprisingly, therefore, there is no strong signal for T-cell recruitment into the lung in asthma, although this picture may differ in more severe disease. T-cell capture by endothelium is mediated by a combination of P-, E- and L-selectin depending on the organ involved. L-selectin mediates adhesion to HEV in lymph nodes, and

a4b7/MAdCAM-1 CCR9/CCL25

Small intestine

L-selectin-negative T cells cannot migrate though HEV. As histamine stimulates endothelial expression of P-selectin in the short term and IL-4 and IL-13 in the long term, it might be considered that Th2 cells would preferentially bind to this receptor. In support of this, T-cell adhesion to nasal polyp endothelium was P-selectin/PSGL-1 dependent (Symon et al. 1999). However in vitro polarized Th1 cells preferentially bound P- and E-selectin compared with Th2 cells as a result of IL-4 inhibition of expression of FucTVII enzyme, which is necessary for the function of PSGL-1, although whether this is relevant in vivo is less clear (Wagers et al. 1998; Bonder et al. 2005a). T cells express both α4β1 and αLβ2 (LFA-1) and arrest and migration through endothelium is mediated by these receptors binding to VCAM-1 and ICAM-1 and -2 respectively, with no obvious differences between Th1 and Th2 cells despite the association of VCAM-1 expression with Th2 cytokines. Indeed in liver sinusoids, α4β1 mediated Th1 recruitment whereas vascular adhesion protein (VAP)-1 mediated Th2 recruitment (Bonder et al. 2005b). Once in the tissue T cells will integrate adhesive and chemoattractant signals in order to orientate themselves within various compartments. How this occurs is poorly understood. Unlike mast cells, T cells do not associate with ASM cells at least in mild asthma, but a subset do migrate into the epithelium where they remain anchored, appearing in the lumen in only small numbers as evidenced by the relatively small numbers of T cells in induced sputum in both asthmatics and normal controls (Brightling et al. 2002). Lung T cells express a variable pattern of activation receptors that probably directs their localization. About 30% of cells coexpress αEβ7, CD69, VLA-1, CXCR3, CCR5, and CXCR6 and it is likely that these cells are retained within the mucosa for prolonged periods. αEβ7-expressing cells (a receptor for E-cadherin) may become anchored within the epithelium, whereas VLA-1-expressing cells adhere to collagen within the airway mucosa. In contrast, cells that do not express any of these receptors but which

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tend to have increased expression of CCR7 are able to migrate into the lymphatics and recirculate back into the blood (Debes et al. 2005). ICAM-1 and VCAM-1 are expressed on ASM cells after stimulation with TNF-α and these receptors together with CD44 have been shown to mediate adhesion of activated T cells to ASM cells (Lazaar et al. 1994; Brightling et al. 2002). However, the lack of any association between T cells and ASM cells in bronchial biopsies from asthmatics raises questions about the physiologic relevance of this interaction.

In vivo studies of adhesion receptor function in allergic inflammation In vivo models of allergen challenge in a variety of animal species have been used extensively to investigate the role of adhesion receptors, mainly in terms of cell recruitment and airway hyperresponsiveness, using either monoclonal antibodies or other adhesion receptor antagonists or gene-deleted mice. These studies are detailed in Table 15.2. Inhibitory effects have been demonstrated using antibodies against a

Table 15.2 In vivo models of adhesion receptors in allergen challenge. Receptor

Species

Findings

Reference

E-selectin

Monkey

In this neutrophil-dependent model of late-phase response to Ascaris challenge, both cell migration and bronchoconstriction were inhibited

Gundel et al. (1991)

ICAM-1

Monkey

In this multiple allergen (Ascaris) challenge model of asthma, both airway eosinophilia and development of BHR were inhibited

Wegner et al. (1990)

Mac-1

Monkey

In the same multiple antigen challenge model, anti-Mac-1 inhibited development of BHR and ECP concentrations in BAL but not eosinophil counts

Wegner et al. (1993)

a4

Sheep

Late response to Ascaris challenge inhibited when antibody given both intravenously and by inhalation but no effect on BAL eosinophils

Abraham et al. (1994)

a4/Mac-1/LFA-1

Rat

Antibodies against all three receptors inhibited early and late response to ovalbumin challenge without any effect on cell counts in BAL at 8 hours

Rabb et al. (1994)

a4

Rat

Antibody against VLA-4 inhibited eosinophil and T lymphocyte infiltration at 24 hours

Richards et al. (1996)

a4

Guinea pig

Inhibition of eosinophil infiltration into the skin in both PCA reaction and chemoattractant induced

Weg et al. (1993)

a4

Guinea pig

Inhibition of BHR, EPO release, and eosinophil infiltration into the airway of ovalbumin-challenged animals

Pretolani et al. (1994)

a4/VCAM-1/ ICAM-1/LFA-1

Mouse

Eosinophil and T-cell infiltration inhibited by anti-a4 and anti-VCAM-1 but not by anti-ICAM-1 or anti-LFA-1

Nakajima et al. (1994)

a4

Mouse

Inhibition of eosinophil recruitment after ovalbumin challenge with antibody given either by inhaled or peritoneal route but inhibition of BHR, mucus and cytokine production only when inhaled

Henderson et al. (1997)

ICAM-1

Mouse

Eosinophil and T-cell influx inhibited by anti-ICAM-1 monoclonal antibody

Chin et al. (1998)

P-selectin KO

Mouse

Eosinophil and T-cell recruitment reduced in gene-deleted mouse after ovalbumin challenge

De Sanctis et al. (1997)

P-selectin/ICAM-1 KO

Mouse

Reduced migration of eosinophils into the lung after ovalbumin challenge in both P-selectin and ICAM-1 deficient animals

Broide et al. (1998)

Mac-1 KO

Mouse

Complex effect with increased eosinophilia in Mac-1 KO (ovalbumin challenge) and inhibitory effect with anti-Mac-1 monoclonal antibody

Kanwar et al. (2001)

ICAM-2 KO

Mouse

Increased eosinophil adhesion after allergen challenge

Gerwin et al. (1999)

ICAM-1/L-selectin KO

Mouse

Reduced inflammation and AHR in ICAM-1 KO. Reduced AHR in L-selectin KO

Tang & Fiscus (2001)

ICAM-1 KO

Mouse

Inhibition of inflammation in a TDI model of asthma with ICAM-1 gene deletion

Furusho et al. (2006)

AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BHR, bronchial hyperresponsiveness; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; KO, knockout; PCA, passive cutaneous anaphylactic; TDI, toluene diisocyanate.

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number of receptors including α4β7, Mac-1, LFA-1, ICAM-1, VCAM-1, and E-selectin, although differences in degree and pattern of inhibition have been observed depending on the species used and the exact conditions. Most support has been gained for an important role of α4β1 and VCAM-1 in both eosinophil and T-lymphocyte migration into the airways after allergen challenge and this has given impetus to the development of α4β1 antagonists in clinical trials. However, several studies have shown a clear dissociation between effects on leukocyte migration and effects on airway physiology. This mirrors the picture in asthma where there is only a weak relationship between the severity of airway inflammation and the severity of bronchial hyperresponsiveness (Wardlaw et al. 2002). Some studies with gene-deleted mice have given unexpected results, for example in an ICAM-2 knockout mouse there was increased eosinophilia after allergen challenge. With Mac-1 contrasting results were seen with an antibody and with gene deletion, which emphasizes the complexity of these models.

Adhesion receptor antagonists as treatments for allergic disease Chronic allergic disease is caused by persistent inflammation characterized by infiltration of tissues with eosinophils and activation of mast cells, basophils, and Th2 lymphocytes. As detailed above, recruitment of these cells into tissue is dependent on adhesion receptor interactions and an impetus for research in this area has been the hope that adhesion receptor antagonists will be effective forms of antiinflammatory treatment. From the above review of the literature it can be seen that both VLA-4 and P-selectin antagonists might be expected to selectively and effectively inhibit eosinophil migration. However, despite potential adhesion receptor targets having been identified for well over a decade, no such treatments have yet reached the clinic. The reasons for this include difficulty in designing potent low-molecular-weight antagonists, concern about redundancy on the one hand and immunosuppression on the other, and potential side effects of treatment. Most effort appears to have gone into the development of α4β1 antagonists, which have also been used for a number of other diseases such as multiple sclerosis and Crohn disease. Natalizumab is a humanized antibody against α4β1 that has recently been licensed for the treatment of multiple sclerosis despite being associated with the occurrence in three patients of progressive multifocal leukoencephalopathy, an opportunistic infection of the brain with polyoma virus JC (Khalili et al. 2007). A number of low-molecular-weight antagonists of α4β1 have been shown to have activity in animal models of asthma (Huryn et al. 2004; Singh et al. 2004; Lawson et al. 2006; Okigami et al. 2007). However, the early results of both inhaled and oral treatment with these compounds in humans in allergen challenge models has so far proved disappointing, with no effect on lung physiology and minimal effect on eosinophilic inflammation (Diamant et al. 2005; Norris

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et al. 2005; Ravensberg et al. 2006). Potent selectin antagonists have been difficult to develop because of the low affinity/highavidity nature of selectin-based carbohydrate interactions. The development of selectin antagonists has recently been reviewed (Kneuer et al. 2006). The only candidate that has been explored in asthma is the pan-selectin antagonist TBC1269 (bimosiamose) originally developed by Texas Biotechnology Corporation (Kogan et al. 1998). Intravenous TBC1269 had no effect on the early and late asthmatic response after allergen challenge, although inhaled TBC1269 did inhibit the late response to allergen challenge by about 50% without any effect on the early response, airway hyperresponsiveness, or exhaled nitric oxide (Avila et al. 2004; Beeh et al. 2006). This compound was unable to inhibit eosinophil adhesion to vascular endothelium under flow conditions (Davenpeck et al. 2000), and is probably not sufficiently potent to be an effective treatment, but may be a model for more potent compounds (Kranich et al. 2007). It is perhaps surprising that a humanized monoclonal antibody against P-selectin has not been investigated in severe asthma. The only other adhesion receptor that has been targeted in allergic disease is LFA-1. An antibody against LFA-1 significantly inhibited sputum eosinophilia and there was a trend toward inhibition of the late response after allergen challenge (Gauvreau et al. 2003). However, there was a high rate of adverse effects, particularly with flu-like symptoms, and this approach has not so far been pursued.

Conclusion There is a wealth of data concerning both the biology of adhesion receptors and their role in the pathogenesis of allergic disease, particularly in the context of leukocyte trafficking. So far this information has not been translated into promising new therapies with a frustrating lack of potent and safe compounds for human use. However, this remains a potentially fruitful approach to the treatment of allergic disease and further studies are eagerly anticipated.

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Airway Epithelium Pedro C. Avila and Robert P. Schleimer

Summary The airways form conduits to allow ambient air to reach alveoli in terminal airways, where gas exchange occurs, followed by elimination of exchanged air. Although apparently simple, this function requires the airways to condition inspired air, to maintain patent conduits, and to protect the lungs against airborne threats. Airway epithelium performs all these functions: it conditions and filters air in the upper airways, provides an important barrier, is intimately involved in gas exchange, and plays a key role in host defense because of its position at the boundary between the environment and the internal tissues. The epithelium responds to microbes and noxious stimuli that overcome the mucociliary barrier by releasing inflammatory mediators that recruit immune cells, triggering immune responses. This chapter reviews the anatomy, physiology, and pathophysiology of the airway epithelium as they relate to allergic diseases and asthma. Additional information on innate immunity (Chapter 9), the mucociliary system (Chapter 39), airway remodeling (Chapter 55), and airway pathology (Chapters 62, 68, 78 and 79) is reviewed elsewhere in this book.

Anatomy and physiology The upper airway consists of the nasal cavities, paranasal sinuses, pharynx, and larynx, namely all airways above the vocal cords. The nasal cavities filter, warm and humidify inhaled air. Airflow is made turbulent in the nasal airways and is ultimately diverted 90° as it flows into the nasal cavity (Fig. 16.1), causing impact of airborne particles against the mucus layer overlaying the epithelium. The 10–15 μm deep mucus layer traps particles, filtering the air. Larger particles are more efficiently filtered than smaller ones. Particles with

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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aerodynamic equivalent diameters of 30, 10, and 2.5 μm are 50%, 70%, and 90% respirable (able to reach lower airways) respectively (Eccles 2003). Once trapped in the mucus layer, particles are moved by the mucociliary escalator toward the pharynx and ultimately eliminated into the digestive tract. The abundant blood vessels in the nasal submucosa warm inhaled air to about 30–32ºC so that it reaches 37ºC after passing through a few bronchial branches (McFadden et al. 1985). Likewise, in the proximal airways, water vapor from the mucus layer humidifies the inhaled air before it reaches the distal airways in the lungs. The lower airway (below the vocal cords) consists of the trachea, right and left main bronchi, and the remainder of the bronchial tree, which undergoes a total of 18 branching generations before reaching bronchioles, all kept patent by their cartilaginous support (McFadden 1998). After about 18 generations, bronchial branches become bronchioles, which are 1 mm or less in diameter and remain patent because of the elastic fiber recoil of the lung parenchyma (McFadden 1998). The proximal bronchioles are called terminal bronchioles and they branch five additional generations before becoming respiratory bronchioles, at which point gas exchange begins to take place. Altogether, after a total of about 23 generations from the trachea, the respiratory bronchioles finally branch into alveolar ducts, which then end in the alveoli (Fig. 16.1). Diseases of the airways that affect epithelium include, among others, rhinitis (upper airways), sinusitis (sinuses), asthma (mostly medium-size bronchi), bronchitis (larger airways), and emphysema (small airways and alveoli). There are more than 50 cell types in the airways, of which 12 are considered to be epithelial cells (Harkema et al. 1991; Albertine et al. 2000). The epithelium in the conducting airways, which does not exchange air, is ciliated pseudostratified and columnar, except in the anterior tip of the lower turbinate, pharynx, and larynx, where it is squamous. In the bronchioles, the ciliated epithelium becomes thinner and cuboid (Fig. 16.2). The main epithelial cell types in the bronchial airways are the ciliated, goblet, and basal cells. Clara cells are present in the terminal bronchioles among ciliated cuboid cells. At the bronchioles the epithelium suddenly changes from ciliated pseudostratified to the alveolar epithelium, demarcating the transition from terminal to respiratory

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FS

ST SS

MT

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LT 1 AIR FLOW

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Uvula Oropharynx Epiglottis Larynx Glottis

Vocal cords Trachea

Esophagus

2 3 Bronchial tree

Respiratory bronchioles

4

Terminal bronchioles

I II

Alveoli

Alveolar ducts

Fig. 16.1 Anatomy of the airways. Stratified squamous epithelium (insert 1) lines pharynx and larynx. Pseudostratified ciliated columnar epithelium (insert 2) lines nasal and sinus cavities as well as lower airways where it becomes thinner and cuboid in the small peripheral airways (insert 3). Cartilaginous support maintains patency of trachea and bronchial tree and ends before terminal bronchioles. Bronchioles and alveoli are kept patent by parenchymal elastic recoil. The sudden transition from ciliated to alveolar

epithelium marks the change from terminal to respiratory bronchioles (insert 3). Alveolar epithelium (insert 4) carries out gas exchange and is composed of alveolar type I (flat cells in insert 4, marked I) and round type II (gray cell, marked II) cells. Pores of Kohn (not shown) present between adjacent alveoli allow further air circulation other than that via airways. FS, frontal sinus; SS, sphenoid sinus; LT, MT, ST, lower, middle and superior turbinates respectively. See text for further details. (See CD-ROM for color version.)

bronchioles. Gas exchange occurs from respiratory bronchioles to alveoli, the so-called respiratory airways, all lined by the single layer of alveolar epithelium, which is composed of alveolar type I and II cells (or type I and II pneumocytes) and form the lung parenchyma. Finally, mucous and serous cells form submucosal glands, which are present from the upper airways to distal bronchial branches.

Ciliated epithelial cells The ciliated epithelial cells move mucus toward the pharynx, conduct vectorial ion transport conferring electrical properties to the epithelium, form the epithelial permeability barrier through tight junctions, and play an important role in innate immunity and inflammation. Ciliated cells are rectangular, measuring up to 20–25 μm in length, but become short and

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Large airways Mc Ne

Eo

Mucus Cilia Small airways

Go

Ci

De Cla

PB Ly

PN

Ba

Ms

Basement membrane

Pore Vagus nerve

Sensory nerve

cuboid in the terminal bronchioles (Albertine et al. 2000; Shebani et al. 2005) (Fig. 16.2). Each cell has 200–300 cilia on its apical (luminal) surface. Cilia are 5– 6 μm high and 0.2– 0.3 μm wide. Each cilium contains a microtubule arrangement (axoneme) composed of a central pair and nine peripheral doublets of microtubules (9 + 2 axoneme) (Satir & Christensen 2007; Zariwala et al. 2007). The peripheral doublets are connected by dynein arms and nexin links, whereas radial spokes connect the peripheral with the central microtubules (Fig. 16.3, insert top right). At baseline, cilia beat at about 10 Hz in the sol phase of the mucus layer but can beat as fast as 100 Hz, moving the entire mucus layer at 2–20 mm/min (Harkema et al. 1991; Widdicombe 1991) (see Chapter 41). Ciliated cells are able to perform vectorial ion transport, polarizing the epithelium (Widdicombe 1991). Airway epithelium actively secretes Cl− into the lumen, mainly via cystic fibrosis transmembrane conductance regulator (CFTR) located in the apical membrane, which is followed by water diffusion, hydrating the mucus. On the other hand, active absorption of Na+ from the lumen, via the epithelial sodium channel (a Na+/K+-ATPase) located in the basolateral membrane, dehydrates the mucus layer. Thus, epithelial ion transport plays a role in the homeostasis of mucus hydration, maintaining the depth of the mucus sol layer similar to the height of the cilia so that cilia can beat freely and their tips can push the mucus gel layer. Ion transport electrically polarizes the epithelium, which is negatively charged at its luminal surface and is about –5 to –10 mV compared with the skin (Knowles et al. 1995; Chung et al. 2003; Schuler et al. 2004). This polarization is also possible because of the permeability barrier of the tight junctions (zona occludens, see below), which seal

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Fig. 16.2 Pseudostratified columnar epithelium lines upper and lower airways from trachea to terminal bronchioles. Epithelial cells include basal (Ba), parabasal (PB), ciliated (Ci), goblet (Go), and Clara (Cla) cells. Several types of immune cells traffic through the airway epithelium in normal conditions. Although overly represented in the figure for normal conditions, they may dramatically increase in numbers in disease processes. These cells include lymphocytes (Ly), dendritic cells (De), neutrophils (Ne), eosinophils (Eo), mast cells (Ms), macrophages (Mc), and monocytes (not shown). In addition, there are sensory nerve fibers and pulmonary neuroendocrine cells (PN). See text for further details. (See CD-ROM for color version.)

the apicolateral regions between superficial epithelial cells, restricting passive diffusion of water and ions between cells (paracellular space). This barrier also creates a transepithelial electrical resistance of more than 100 Ω cm2.

Apical intercellular adhesion complex Adhesion among epithelial cells is maintained by complex structures. Superficial epithelial cells adhere to each other via the apical intercellular adhesion complex (Fig. 16.3), which comprises three zonulae: the zonula occludens (or tight junctions), zonula adherens (or intermediate junctions), and the macula adherens (or desmosomes). The tight junctions are located in the apicolateral membrane closest to the lumen in the superficial epithelial cells (Shin et al. 2006; Godfrey 1997). They consist of interconnections of transmembrane proteins including occludins, claudins, junctional adhesion molecules, and crumbs 3. Claudins, a family of more than 20 members, form the calcium-independent tight interconnecting strands between cell membranes seen in freeze-fracture electron micrographs. They seal the epithelial cells at its apical–lateral areas and regulate paracellular permeability (Van Itallie & Anderson 2006). The transmembrane proteins interact with intracellular scaffold proteins such as zonula occludens protein (ZO)-1, ZO-2, ZO-3, and cingulin, which in turn interact with the actin cytoskeleton. In addition, tight junction proteins interact with signaling molecules so that disruption of the junction signals epithelial cells to undergo cell proliferation and initiate repair processes (Matter et al. 2005). The zonula adherens consists of epithelial cadherins (Ecadherin or cadherin 1, one of more than 20 members of the

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Cilium

Airway Epithelium

Plasma membrane Peripheral microtubule doublet Outer dynein arm Inner dynein arm Radial spoke Nexin link Central microtubule pair

Cilia Tight Junction

Occludin

Z0-1 Cing Z0-2 Z0-3 Actin

Claudin

Z0-1 Cing Z0-2 Z0-3

JAM

Cing

CRB3

Z0-3 Go Intermediate Junction

E-Cadherin

Ci Fig. 16.3 Ciliated epithelium: structure of cilium and apical intercellular adhesion complexes. Adhesion complex comprises three zonulae: the zonula occludens (or tight junctions), zonula adherens (or intermediate junctions), and macula adherens (or desmosomes). In addition, hemidesmosomes adhere basal cells to the basement membrane. See text for details and Fig. 18.2 for cell types. Actin, actin cytoskeleton; Cat, catenin; Cing, cingulin; CRB3, crumbs 3; DPK, desmoplakin; DSC, desmocollins; DSG, desmogleins; JAM, junctional adhesion molecules; KIF, keratin intermediate filaments; P120, catenin d; PKG, plakoglobin; PKP, plakophilin; ZO, zonula occludens protein. See text for further details. (See CD-ROM for color version.)

a-cat PB

b-cat p120

Actin PB

Ba

Desmosome PKG KIF DPK

PKP

DSG

Basement membrane

cadherin gene family), which form transmembrane homodimers that mediate calcium-dependent adhesion between epithelial cells throughout the epithelial layer (Lilien et al. 2002; McGuire et al. 2003; Lilien & Balsamo 2005). The intracellular domain of E-cadherin homodimers interact with β-catenin, catenin δ1 (p120ctn), and α-catenin. The latter catenin interacts with the actin cytoskeleton. Disruption of E-cadherin adhesion between cells augments epithelial permeability. Catenins also interact with protein tyrosine kinases and phosphatases mediating intracellular signaling (e.g., proliferation). Cadherins also serve as a substrate for adhesion of T lymphocytes or mast cells to the epithelium via the adhesion molecule αEβ7 (Taraszka et al. 2000). The macula adherens or desmosomes, although present in airway epithelium (Shebani et al. 2005), are best studied in the skin (Kottke et al. 2006). Desmosomes also consist of

Hemidesmosome

DSC

transmembrane proteins that connect with the cytoskeleton through scaffold proteins. In this case, the transmembrane proteins desmogleins and desmocollins mediate calciumdependent adhesion between cells throughout the epithelial layer. Their intracytoplasmic domains interact with desmoplakin, plakophilin, and plakoglobin, which form the characteristic paramembranous plaques of desmosomes seen in electron micrographs. These protein plaques interact with a dense bundle of filamentous proteins (actin and keratin intermediate filaments) of the cytoskeleton. Hemidesmosomes adhere basal epithelial cells to the extracellular matrix of the epithelial basement membrane. Integrins on the epithelial cells also contribute to the adhesion of basal epithelial cells to the basement membrane. Integrins are transmembrane heterodimeric adhesion molecules. The integrins α6β4, present in hemidesmosomes, and α3β1,

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present throughout the epithelium, are receptors for laminin 5 (Sheppard 2003), present in the basement membrane of conducting airways (Coraux et al. 2002). These integrins also bind to laminins 10 and 11. Integrin α2β1 is expressed on basal cells, binds to collagens I and IV, and tenascin C (expressed in injury), and functions as the receptor for echoviruses. In asthma, there seems to be an enlargement of the intervening space between basal cells and between basal cells and basement membrane, suggesting a defect in the integrity of desmosomes and hemidesmosomes (Shebani et al. 2005). Epithelial barrier function is regulated by a host of factors intrinsic to the epithelium (see above) as well as external factors such as proteases and inflammatory mediators. Although most studies have been performed with keratinocytes, recent data suggest that a family of cytokines related to interleukin (IL)-10 influences barrier function. These cytokines, which include IL-19, IL-20, IL-22, IL-24, and IL-26, modulate the expression of a host of molecules involved in regulating the protease–antiprotease balance and the expression of structural genes involved in maintaining epithelial barrier function (Hor et al. 2004; Boniface et al. 2005; Sa et al. 2007). Exogenous proteases from microorganisms or allergens can alter this balance. For example, Der p1, a major allergen of Dermatophagoides pteronyssinus and a cysteine proteinase, cleaves occludin and claudin 1, disrupting tight junction integrity and increasing airway epithelial permeability (Wan et al. 1999).

Goblet cells Goblet cells are nonciliated mucus-secreting cells interspersed between ciliated cells in the superficial layer of the epithelium (Albertine et al. 2000). They decrease in number toward the peripheral airways, disappearing in the terminal bronchioles. Their characteristic numerous, mucous, electron-lucent granules at the apical surface are loaded with acidic mucin, particularly Muc5AC, but also Muc1 and Muc5B (Ordonez et al. 2001). Together with mucins from submucosal glands, goblet cell mucins form the gel phase of airway mucus. Goblet cell exposure to airborne insults such as chemical irritants, pollutants (ozone, sulfur dioxide), allergens, tobacco smoke, and microbes can stimulate mucin secretion (Harkema et al. 1991). These insults can also induce goblet cell hyperplasia with repeated exposures. In airways of individuals with mildto-moderate asthma, there are twice as many goblet cells and twice the stored mucus content than in airways of healthy individuals (Ordonez et al. 2001) (see Chapter 41). An increased number or function of goblet cells is also observed in chronic obstructive pulmonary disease (COPD) and rhinitis.

Clara cells Although Clara cells resemble goblet cells, they are present in the terminal and respiratory bronchioles (Harkema et al. 1991; Albertine et al. 2000) and have less abundant apical granules. In contrast to goblet cell granules, Clara cell granules are electron-dense and have little if any mucin (glycoproteins).

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Clara cell granules contain proteins such as surfactant protein D, antileukoproteases, and uteroglobin (also called Clara cell secretory protein), among others. In addition, Clara cells have abundant agranular endoplasmic reticulum, glycogen stores, and mitochondria occupying 40% of the cytoplasm. Clara cells also possess cytochrome P450 monooxygenases for metabolism of xenobiotic substances. In animals, Clara cells are also precursors of ciliated cells (Harkema et al. 1991) and goblet cells (Hayashi et al. 2004). In mice, ciliated epithelial cells can also transdifferentiate into goblet cells via epidermal growth factor receptor and IL-13 signaling (Tyner et al. 2006).

Basal epithelial cells Basal epithelial cells are very small and closely attached to the basement membrane via hemidesmosomes. This attachment prevents loss of basal cells during processes that cause epithelial damage and desquamation. Basal cells are precursors for all epithelial cells, including parabasal cells, ciliated cells, and goblet cells (see Fig. 16.2). In normal airways, about 0.8% of the epithelial cells are proliferating at baseline, and among the proliferating cells are basal cells, parabasal cells, and Clara cells (Harkema et al. 1991; Boers et al. 1998, 1999). Proliferation rate can increase 10–20 fold, as shown in airways of rats exposed to nitrogen dioxide (Harkema et al. 1991). Basal cells are also affected by airborne insults and produce inflammatory mediators. Serous and mucous epithelial cells (Wine & Joo 2004) line submucosal glands, although occasional serous cells can be seen in the mucosal epithelium as well. Glands provide an estimated 95% or more of the mucus content of the upper airways. There is on average one gland every square millimeter of mucosa in the trachea down to the 10th generation of bronchial branches (airway diameter 1–2 mm), where they disappear. From the airway lumen, the gland duct (containing ciliated epithelium) penetrates into the submucosa and branches into numerous tubules, where they become lined with mucous cells proximally and with serous cells in distal acini. Normal glands are composed of 60% serous and 40% mucous cells by volume. Serous cells secrete water, ions, antimicrobials (e.g., lysozyme, lactoferrin, sIgA), and antiinflammatory and antioxidant agents, whereas mucous cells secrete sialomucins and sulfomucins. Glandular secretion is stimulated by acetylcholine released from the parasympathetic innervation, which does not affect goblet cell secretion.

Alveolar epithelium Alveolar epithelium lines the respiratory epithelium, from the respiratory bronchioles to the alveoli, and consists of alveolar type I and II cells. Type I cells are “fried egg”-like cells that line respiratory airways as a thin single-cell layer. They are adjacent to endothelial cells, with a very thin interstitium separating them so that the distance between the alveolar lumen and the capillary lumen is less than 1 μm, particularly when the alveoli are stretched at total lung

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capacity. Type I cells express aquaporin 5 and the epithelial sodium channel and may play an important role in the transport of water and ions across the alveolar epithelium (Albertine et al. 2000). Alveolar type II cells are cuboidal cells attached to type I cells via tight junctions. Type II cells have electron-dense lamellar cytoplasmic inclusions, or lamellar bodies. These are granules containing several proteins including surfactant proteins A, B, C and D, lysosomal enzymes, and α-glucosidase. One of the most important functions of type II cells is the production of surfactant lipids, which are required for the proper assembly and function of surfactant proteins (Ridsdale & Post 2004). Type II cells can also synthesize chemokines, cytokines, matrix proteins, and transporters for water and ions. They also participate in epithelial regeneration and act as stem cells, differentiating into type I cells. Although type II cells outnumber type I cells by 2 : 1, type I cells cover over 90% of the alveolar surface due to their attenuated morphology.

Pulmonary neuroendocrine cells Pulmonary neuroendocrine cells (PNECs) are scattered along the airways from trachea to bronchioles (Johnson 1991). They occur just above the basement membrane, typically in contact with afferent nerve fibers. They are more numerous in proximal than distal airways, and in airways of newborns, where they occur in clusters (neuroepithelial bodies). In adults, they occur as single cells, accounting for 0.41% of all epithelial cells (Boers et al. 1996). They have characteristic small dense-cored cytoplasmic vesicles and cytoplasmic projections that extend between epithelial cells toward the lumen and in parallel to the basement membrane (Johnson 1991). These vesicles contain amine hormones (serotonin, dopamine, norepinephrine) and peptide hormones (calcitonin, leu-enkephalin, bombesin, somatostatin, endothelin) (Johnson 1991). The adjacent sensory nerve fibers contain substance P and vasoactive intestinal peptide. One of the functions of the PNEC is believed to be as a chemoreceptor for sensing irritants (e.g., tobacco smoke), pollutants (e.g., ozone), hypoxia, and hypercapnia, all stimuli that increase the number of PNECs in the airways. Hyperplasia of PNECs also occurs in many lung diseases including, but not limited to, asthma, cystic fibrosis, COPD, and bronchiectasis (Johnson 1991; Joad et al. 2006). PNEC secretory products are believed to play a role in bronchospasm, vasospasm, inflammation, airway hyperreactivity, and airway remodeling (Johnson 1991).

Olfactory epithelium In the upper airways, the olfactory epithelium consists of specialized epithelial cells that interact with olfactory receptor neurons and two types of glial cells (sustentacular cells and olfactory ensheathing cells) that collectively mediate olfaction (Beites et al. 2005). Anosmia, in which the olfactory epithelium is depleted or fails, is not an uncommon occurrence in diseases of the upper airways, particularly nasal polyposis.

Airway Epithelium

Basement membrane The basement membrane separates epithelium from submucosa. It provides support and orientation for epithelial cell growth and repair, and serves as a semipermeable barrier. It has two layers, the more superficial lamina densa, and the deeper and thicker lamina reticularis. The lamina densa is 0.1 μm thick and is mainly composed of collagen type IV and laminin V, which are produced by epithelial cells. The lamina reticularis is 2–6 μm thick and is mostly composed of fibronectin and collagen type III and V, which are secreted by subepithelial fibroblasts and myofibroblasts (Paulsson 1992). In asthma, the lamina reticularis is thickened 40–50% (Shahana et al. 2005), and its thickness correlates with the number of subepithelial myofibroblasts (Brewster et al. 1990). Although some researchers have detected a 30% thinning of the reticularis membrane after inhaled corticosteroid therapy (Laitinen et al. 1997; Hoshino et al. 1998; Sont et al. 1999), others have failed to confirm this effect (Jeffery et al. 1992; Barnes et al. 2000; Boulet et al. 2000). Pores are also randomly scattered throughout the basement membrane, possibly from immune cell migration (Howat et al. 2002). In healthy airways the pores number about 740/mm2, are ovoid in shape, 2–4 μm long, and 0.4–2.7 μm in diameter with a corresponding cross-sectional area of 1–30 μm2.

Other cell types In addition to epithelial cells, the airway epithelium contains numerous other cell types (see Fig. 16.2). Sensory nerve fibers can penetrate far into the epithelium in close proximity to the airway lumen. All immune cells, including lymphocytes, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and macrophages, can traffic through the airway epithelium and are present in histologic sections of normal airways and in luminal secretions. The biology of these cells is discussed elsewhere in this book. Most importantly, epithelial cells can chemoattract and interact with these cells via contact through surface membrane molecules and/or via soluble molecules to alter the function of one another. Underneath the basement membrane, fibroblasts and myofibroblasts can interact with epithelial cells in health and airway diseases such as asthma (Holgate et al. 2004).

Models used to study epithelial cells The function of epithelial cells can be studied in several different models (Table 16.1). In vitro, cancer or virustransformed epithelial cell lines and primary culture of freshly isolated airway epithelial cells have been used to study their function. These cells are often used in their undifferentiated state, as a monolayer of flattened cells resembling the basal epithelial cells, which lack epithelial polarization. Some transformed cell lines and primary airway epithelial cells can be grown on a porous membrane bathed by medium underneath and

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Table 16.1 Common models used to study human airway epithelial cells. Cell line or model

Characteristics

Model of

A549 cells

Lung carcinoma cell line from a 58-year-old white male. Chromosomal abnormalities include two X and two Y chromosomes, and about 20% of cells with 64, 65 or 67 chromosomes

Alveolar type II cells

BEAS-2B

Normal bronchial epithelial cells transformed by infection with adenovirus 12 and simian virus 40

Bronchial epithelial cells

CALU-3

Lung adenocarcinoma cell line grown from pleural effusion of a 25 year-old white male. Abnormal chromosomes 1, 13, 15, 17 and Y

Bronchial gland epithelial cells

NCI-H292

Lung mucoepidermoid carcinoma isolated from a lymph node metastasis in a 32 year-old African female. About 36% of cells have 47 chromosomes

Mucus/mucin-producing bronchial epithelial cells

16HBE14o–

Normal tracheal epithelial cells transformed by infection with simian virus 40 that can differentiate in air–liquid interface cultures forming tight junctions and microvilli

Somewhat differentiated bronchial epithelial cells

Primary epithelial cells

Normal nasal, tracheal, and bronchial cells can be grown for a few passages. Early passages (P0 to about P2) can be differentiated in air–liquid interface cultures to form ciliated and polarized epithelium with vectorial ion transport and adhesion complex structures that resemble native pseudostratified ciliated epithelium

Normal differentiated pseudostratified ciliated epithelium to study mucociliary function, ion transport, and electrical properties

Tissue engineering

Airway fibroblasts are grown in a gel matrix and airway epithelial cells grown in air–liquid interface overlaying the gel. Leukocytes may be added to the system

Interactions between epithelium, fibroblasts, and leukocytes

Tissue explants

Segments of airways (e.g., trachea) or mucosal biopsies are directly cultured ex vivo to study their responses to different stimuli

Response of minimally manipulated tissues to stimuli

Human xenograft

Human bronchial epithelial cells grown inside rat trachea denuded of its own epithelium and implanted in the subcutaneous tissue of T-cell-immunodeficient mice (prevents rejection). Epithelium remains exposed to air

Normal human airway epithelium to study innate response and regeneration

Source: American Type Culture Collection (www.atcc.org).

exposed to air above, so-called air–liquid interface culture, which in combination with several hormones induces differentiation (Van Scott et al. 1991). Differentiated epithelial cultures resemble the natural ciliated pseudostratified columnar epithelium, forming three- to five-cell layers, a mucociliary system, polarization (tight junctions), vectorial ion transport, and normal electrical properties (Wu et al. 1985; Yamaya et al. 1992, 1993; Gruenert et al. 1995; Lopez-Souza et al. 2003; Widdicombe et al. 2003, 2005). It has also been possible to produce an engineered human bronchial mucosa in which bronchial fibroblasts are grown in a collagen gel on top of which primary epithelial cells are grown at air–liquid interface. This system allows study of the complex interactions between epithelial cells, leukocytes, and fibroblasts (Chakir et al. 2001). In addition, tracheal segments and biopsies from airway mucosa, so-called tissue explants, have been placed in culture medium ex vivo to study their response to stimuli (Warner & Azen 1984; Lundgren et al. 1991; Roat et al. 1993; Roca-Ferrer et al. 2000; Morin et al. 2005). In vivo models of the airway epithelium include studies

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in animal models, in humans, and human xenograft transplants in immunodeficient mice. In the latter model, human bronchial epithelial cells are grown inside rat trachea denuded of its own epithelium and implanted in the subcutaneous tissue of immunodeficient mice (e.g., nude mice, severe combined immunodeficient mice). The ends of the trachea are connected to ambient air via sterile polyethylene tubing (Puchelle & Peault 2000). The immunodeficient mice do not reject the rat trachea or human airway epithelial cells, allowing studies of innate immunity and regeneration processes of human cells in vivo.

Pathophysiology In asthma, the airway epithelium undergoes several pathologic changes even in mild disease (see also Chapters 56, 63, 69 and 80). As mentioned above, the airway epithelium of asthmatic individuals manifests goblet cell hyperplasia, thickening of the reticular basement membrane, and loosen-

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ing of epithelial intercellular adhesion. In addition, there are areas of desquamation, i.e., loss of superficial epithelial cells exposing basal cells to the lumen, and areas of epithelial denudation, in which all epithelial cells slough off and basement membrane is exposed to the lumen. These changes are more often seen in severe disease (Laitinen et al. 1991; O’Byrne 1991), and may not occur in mild asthma (Ordonez et al. 2000a). The lost airway epithelium can form Creola bodies, which are round clusters of epithelial cells seen in sputum cytology in asthma exacerbations (Naylor 1962; O’Byrne 1991). Also seen in sputum are corkscrew-shaped strings, so-called Curschmann’s spirals, caused by mucus hypersecretion and drying of mucus in the lumen of small airways. Tenacious mucus plugs occlude the airways of patients who die of asthma (Jeffery 1992). The epithelial damage is a consequence of inflammation. For example, major basic protein (MBP) derived from eosinophil degranulation can desquamate epithelium in guinea pigs (Motojima et al. 1989) and epithelial denudation is observed in areas containing large numbers of eosinophils in human asthma (Gleich et al. 1987). Other inflammatory cell products are also likely involved in epithelial damage, such as those from eosinophils (Hirata et al. 1996), the main inflammatory cell infiltrating the asthmatic airway lumen in asthma (see Chapters 67, 79 and 80), and products from neutrophils, the main infiltrating cells in asthma exacerbations (Fahy et al. 1995; Ordonez et al. 2000b).

Role of epithelium in innate immunity The first line of defense provided by the epithelium is the mucociliary system, present from the upper airways to the terminal bronchioles (see Chapter 41). As mentioned above, sudden changes in airflow direction in the upper airways impacts air against the mucosa, where particles are trapped in the mucus. Ciliary beating then moves mucus toward the pharynx where it is eliminated by the digestive system. Mucus is composed of long chains of glycoproteins that determine its viscosity, elasticity, and adhesive properties. There is population diversity in the carbohydrate composition of mucus glycoproteins. For example, different sugar residues decorate the H antigen, giving rise to different ABO blood group antigens. These antigens are also synthesized by epithelial cells. People who have functional fucosyltransferase 2 (FUT2) can add fucose to the H antigen enabling secretion of ABO blood group antigens in body fluids (so called secretors). Individuals with loss-of-function mutations in FUT2 can not secrete ABO antigens in their secretions (nonsecretors) (D’Adamo & Kelly 2001). Among several features of these phenotypes is the fact that secretors are more susceptible to respiratory virus infections (Raza et al. 1991), whereas nonsecretors are more susceptible to Helicobacter pylori infections and peptic ulcer disease (D’Adamo & Kelly 2001). The susceptibility mechanism seems related either to the ability of different sugars in mucus glycoproteins to bind and coat microbes in the lumen

Airway Epithelium

or to the ability of different sugars on cell-surface glycoproteins (glycocalyx) to allow adhesion of microbes. Therefore, the types of sugars in mucus influences susceptibility to infections, including to viral respiratory infections (Raza et al. 1991); which are known to play a role in the development of asthma (Friedlander et al. 2005) and precipitate asthma exacerbations (Johnston et al. 1995). Mucus composition can change as a result of several processes. The mucus is derived from products of airwayresident and immune cells, which can alter the content or volume of their secretions on stimulation by microbes, irritants, and inflammatory mediators. Airway-resident cells that contribute to mucus production include ciliated cells, goblet cells, Clara cells, and serous and mucous cells. Mucus also has components derived from plasma transudation and from immune cells such as mast cells, dendritic cells, neutrophils, eosinophils, monocytes, macrophages, and lymphocytes, which are normally present in the airways in small numbers. In addition, during inflammation, newly formed mediators and infiltrating cells alter mucus production and constituents (see Chapter 41). The important influence of epithelial cells on the ionic content of mucus is illustrated by abnormalities in cystic fibrosis, in which several antimicrobial substances fail to kill microorganisms as a result of alterations of the ionic properties of airway secretions (Travis et al. 1999). That mucus possesses antimicrobial properties was first recognized by Sir Alexander Fleming, who discovered the ability of lysozyme in saliva and nasal secretions to kill Micrococcus lysodeikticus (Fleming 1922). Mucus contains numerous other antimicrobial products secreted by epithelial cells and immune cells. The following text, and Table 16.2, list several antimicrobial products found in the airways, most produced by airway epithelial cells.

Secreted antimicrobials Lysozyme Lysozyme is a 14-kDa enzyme that cleaves the 1–4 β-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid residues in peptidoglycan, a cell wall component of Gram-positive bacteria (Ganz 2004). It can also kill bacteria by a nonenzymatic mechanism. It is ineffective against Gramnegative bacteria unless the outer envelope is damaged (e.g., as a result of the action of lactoferrin) and allows penetration of lysozyme. Lysozyme is decreased in patients with recurrent sinusitis and perennial allergic rhinitis (Kalfa et al. 2004).

Lactoferrin Lactoferrin is an 80-kDa glycoprotein related to transferrin that acts by chelating iron, an essential nutrient for respiration in microbes (Ganz 2004). This action also prevents biofilm formation (Singh et al. 2002). The cationic N-terminal of lactoferrin can also directly kill bacteria.

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Table 16.2 Antimicrobials produced in the airways. Antimicrobial

Action

Affected microbes

Sources

Lysozyme

Cleavage of peptidoglycan

Gram-positive bacteria

Serous epithelial cells, monocytes, macrophages

Lactoferrin

Chelation of iron, prevention of biofilm formation

Bacteria

Serous epithelial cells, neutrophils

Secretory leukocyte proteinase inhibitor

Direct antimicrobial activity

Bacteria, fungi

Serous epithelial and Clara cells, alveolar type II cells, monocytes, macrophages, neutrophils

Elafin

Direct antimicrobial activity

Bacteria, fungi

Serous epithelial and Clara cells, alveolar type II cells, macrophages

Phospholipase A2

Direct antimicrobial activity

Bacteria

Serous epithelial cells

Cathelicidins

Direct antimicrobial activity

Bacteria, fungi, enveloped viruses

Mucosal, serous and mucous epithelial cells, neutrophils, monocytes

b Defensins

Permeabilization of microbial membranes

Bacteria, fungi, enveloped viruses

Mucosal epithelial cells, monocytes, macrophages, dendritic cells

Collectins (SP-A, SP-D)

Opsonization, neutralization

Bacteria, fungi, viruses

Clara cells, alveolar type II cells

Collectin (MBL)

Opsonization, complement activation, neutralization

Bacteria, fungi, viruses, protozoa

Hepatocytes (leaked from plasma into airways)

Pentraxin-3

Opsonization, complement (C1q) activation

Bacteria, fungi

Mucosal epithelial cells, alveolar type II cells, dendritic cells, monocytes, macrophages, fibroblasts, endothelial cells

C-reactive protein

Opsonization, complement (C1q) activation

Phosphorylcholineexpressing bacteria

Hepatocytes and nasal epithelial cells

Serum amyloid A

Opsonization, complement (C1q) activation

Bacteria

Naso-sinusal epithelial cells

Complement components

Opsonization, complement activation, chemoattraction

Bacteria, fungi, viruses, parasites

Mucosal epithelial cells, liver, mononuclear phagocytes (monocytes, macrophages)

Reactive oxygen species

Direct antimicrobial activity

Bacteria, fungi, parasites, viruses

Mucosal epithelial cells, mononuclear and polymorphonuclear (eosinophils, neutrophils) phagocytes

Reactive nitrogen species

Direct antimicrobial activity

Bacteria, fungi, parasites, viruses

Mucosal epithelial cells, alveolar type II cells, macrophages, neutrophils, mast cells, endothelial cells among others (see text)

SP, surfactant protein; MBL, mannose-binding lectin, also known as mannan-binding protein (MBP).

Secretory leukocyte proteinase inhibitor Secretory leukocyte proteinase inhibitor (SLPI) is a 12-kDa polypeptide that contains two whey acid protein (WAP)/fourdisulfide core domains. Its N-terminal domain has modest antibacterial activity, whereas its C-terminal domain inhibits neutrophil elastase (Ganz 2004) as well as cathepsin G, trypsin, chymotrypsin, and chymase (Sallenave 2002). It also inhibits nuclear factor (NF)-κB activation (Hiemstra et al. 2004) and attenuates tumor necrosis factor (TNF)-α secretion by lipopolysaccharide (LPS)-stimulated monocytes (Sallenave 2002). These antiinflammatory effects may provide SLPI with the

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ability to inhibit the allergic late response to allergen in animal models of asthma (Wright et al. 1999). Elafin (6 kDa), or elastase-specific inhibitor, is another serine protease inhibitor that is structurally similar to the WAP/four-disulfide core domains of SLPI. Its precursor, pre-elafin (9.9 kDa), contains an N-terminal domain (cementoin) that is a substrate for the enzyme transglutaminase and is a member of the trappin family; “trappin” is an acronym for TRansglutaminase substrate and wAP domain containing ProteIN, and refers to its functional property of “getting trapped” in tissues by covalent cross-linking (Williams et al. 2006). Like SLPI, it is upregulated

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by LPS, IL-1, TNF-α, neutrophil elastase, and neutrophil αdefensins. Elafin exerts antibacterial effects on Gram-positive and Gram-negative bacteria and fungi (Simpson et al. 1999). Like SLPI, elafin also inhibits NF-κB activation and is chemotactic for neutrophils. The N-terminus of elafin forms polymers that bind to extracellular matrix proteins, making it a tissue-bound elastase inhibitor (Sallenave 2002).

Phospholipase A2 Phospholipase (PL)A2 has been described in nasal lavage (Stadel et al. 1994; Aho et al. 1997). It increases after nasal challenge with methacholine, histamine, and allergen. It has direct calcium-dependent antimicrobial activity against Gram-positive and Gram-negative bacteria (Cole et al. 1999). The PLA2 superfamily is large, and only selected members are antibacterial (Schaloske & Dennis 2006). Cathelicidins Cathelicidins comprise a family of antimicrobial polypeptides of 110–214 amino acids containing a variable C-terminal cationic antimicrobial domain (12–100 residues) that becomes active after being enzymatically freed in the extracellular space from the N-terminal conserved cathelin domain (98–114 residues) of the holoprotein (Zanetti 2004). Both domains have antimicrobial activity. The most studied human cathelicidin is the human cationic antimicrobial protein (hCAP)-18, which can be cleaved into LL-37 and cathelin by neutrophil elastase, proteinase 3, and prostate fluid pepsin C. hCAP-18 is produced by mucosal epithelial cells and submucosal glands and kills Gram-negative and Gram-positive bacteria by itself and in synergism with lactoferrin and lysozyme (Bals et al. 1998). LL-37 is a chemoattractant for neutrophils, monocytes, CD4 T cells, and mast cells. It also activates macrophages and epithelial cells, degranulates mast cells, and stimulates angiogenesis (Hiemstra et al. 2004). The mRNA for cathelicidin LL-37 is increased in nasal polyps from patients with chronic rhinosinusitis (CRS) (Chen & Fang 2004). Defensins Defensins are small antimicrobial polypeptides (3–5 kDa) of 30– 45 amino acids characterized by a three-dimensional β-sheet conformation due to three disulfide bonds (six cysteine residues) (Ganz 2004; Hiemstra et al. 2004). Defensins are classified into two groups, α and β defensins. The α defensins are present in the azurophil granules of neutrophils [human neutrophil defensins (DEFA)1–4] and in the secretory granules of Paneth cells (human α defensins DEFA5 and DEFA6). Paneth cells are involved in host defense and are found at the bottom of small intestinal epithelial crypts. DEFA5 is also produced by airway epithelium (Frye et al. 2000), and DEFA1 induces bronchial epithelial secretion of IL-1β and IL-8 via activation of NF-κB (Van Wetering et al. 1997; Sakamoto et al. 2005). The β defensins differ from their α counterparts by the position of the disulfide bonds

Airway Epithelium

and cysteine residues. Four human β defensins (DEFB1–4) are well described, although a whole-genome search reveals up to 28 genes possibly encoding additional DEFBs (Hiemstra et al. 2004). DEFB1–4 are present in mucosal epithelial cells of various organs and in skin. DEFB1 is constitutively expressed, whereas DEFB2 (also known as DEFB4) is inducible (Hiemstra et al. 2004). In the lungs, the conducting airway superficial and submucosal gland epithelial cells produce DEFB2 on stimulation via Toll-like receptor (TLR)4 with LPS and/or cytokines (e.g., IL-1) (Ganz 2004). Defensins kill Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses by permeabilizing membranes rich in anionic phospholipids and sparing the neutral and cholesterol-rich membranes of human cells. Defensins also stimulate mitogenesis of fibroblasts and bronchial epithelial secretion of IL-8 and SLPI, and they also inhibit fibrinolysis (Ganz 2004). DEFB2 is a chemoattractant for dendritic cells and memory T cells via CCR6, providing a link between innate and adaptive immunity.

Collectins Collectins are a small family of glycoproteins containing collagenous regions and C-type lectin domains (Hickling et al. 2004). The lectin domain (“head”) binds to a variety of monossacharides, conferring binding specificity to targets, whereas the collagen portion (“stalk”) mediates effector function. They trimerize to form a subunit, which in turn polymerizes via disulfide and noncovalent bonds into a cruciate or sertiform structure with up to six subunits. The human collectins include surfactant proteins (SP-A and SP-D), mannose-binding lectin (MBL) or mannan-binding protein, CL-L1 (liver, collectin 10), and CL-P1 (placenta, collectin 12). All four surfactant proteins are produced by alveolar type II cells (Albertine et al. 2000) and Clara cells (Hickling et al. 2004; Kishore et al. 2006), of which SP-A and SP-D are major collectin constituents of surfactant. SP-B and SP-C function as true surfactants, helping maintain the patency of terminal airways by their action on surface tension, whereas SP-A and SP-D participate in host defense by agglutinating and opsonizing microbes, facilitating their clearance by phagocytosis. Most collectins can activate the complement protein C1q and mediate their opsonizing effects partly through complement receptors on phagocytes. They can also bind to allergens and impair allergic response in animal models of allergy to fungal and dust mite allergens, presumably by inhibiting activation of basophils and mast cells and altering allergen presentation to T cells (Hickling et al. 2004; Kishore et al. 2006). The amount of SP-A is reduced in lungs of asthmatic individuals. SP-A decreases lymphocyte proliferation to allergens and attenuates allergic airway inflammation in mouse models of asthma. MBL is produced in the liver and found mainly in blood, but is also present in upper airways (Hickling et al. 2004; Dommett et al. 2006). It binds to several microbes, attenuating infections through neutralization, opsonization, and activation

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of complement. About 5% of the population can be deficient in MBL, as defined by low serum levels (< 50 ng/mL), a condition that is most often asymptomatic but which may predispose to recurrent infections in infants (Hickling et al. 2004; Dommett et al. 2006). Deficiency of MBL may also predispose adults to infections caused by mycoplasma, severe acute respiratory syndrome (SARS)-coronavirus, and to more severe forms of hepatitis B and C virus infections. MBL also facilitates macrophage clearance of apoptotic cells (Hickling et al. 2004).

Pentraxins Pentraxins are a superfamily of interspecies-conserved acutephase proteins that also facilitate opsonization and complement activation (Bottazzi et al. 2006). They are produced early during inflammation on stimulation by TLRs (Doni et al. 2006), IL-1β, or TNF-α (Bottazzi et al. 2006). They form multimers with 5–10 units. Structurally, they share a 200amino acid residue C-terminal pentraxin domain and have an N-terminal domain that varies in length between the short and long forms (Garlanda et al. 2005). For example, C-reactive protein is a short pentraxin produced in the liver and nasal epithelial cells (Gould & Weiser 2001), whereas pentraxin-3 (PTX-3) is a long pentraxin secreted by mononuclear phagocytes, alveolar type II cells, and mucosal airway epithelial cells (Han et al. 2005). PTX-3, a 45-kDa glycoprotein, binds to C1q to activate complement, binds to the extracellular matrix component TNF-α-induced protein 6 to facilitate assembly of hyaluronic acid-rich structures, and binds to some microbes such as Aspergillus fumigatus and Gram-negative bacteria including Pseudomonas aeruginosa (Garlanda et al. 2005; Bottazzi et al. 2006). PTX-3-deficient mice are more susceptible to invasive pulmonary aspergillosis (Garlanda et al. 2002). Serum amyloid A Serum amyloid A (SAA) binds directly to Gram-positive bacteria and is probably an important opsonin. mRNA for SAA is expressed in sinus mucosa and is increased in patients with recalcitrant CRS with polyps as compared to those with treatment-responsive CRS (Lane et al. 2006a). Experiments in bronchial epithelial cell lines indicate that SAA and complement components are expressed as part of a local epithelial acute-phase response reminiscent of the hepatic acute-phase response (Sha et al. 2004). Complement components Several complement components play roles in airway diseases. Anaphylatoxin receptors C3aR and C5aR are expressed on airway epithelial cells, smooth muscle cells (Drouin et al. 2001), endothelial cells, and leukocytes, more so in fatal asthma (Fregonese et al. 2005). In animal models of asthma, C3a and C5a mediate several features of Th2 inflammation in airways of mice sensitized and challenged to allergens

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(Sarma et al. 2006; Kohl & Wills-Karp 2007). However, during the sensitization phase of animals, C5a protects against generation of Th2 effector adaptive response (Drouin et al. 2006), probably via C5aR on dendritic cells and induction of tolerance by T regulatory cells (Kohl et al. 2006; Kohl & Wills-Karp 2007). Sinonasal mucosa can also produce C3, which is observed in inflamed mucosa of CRS (Vandermeer et al. 2004; Lane et al. 2006b). Cleavage products of C3 are potent chemoattractants for granulocytes and act to opsonize particulates for removal by phagocytes. Sinus mucosal epithelial cells also produce alternative complement proteins such as factors B, H and I and properdin (Vandermeer et al. 2004). In the airways, these proteins may thus be expected to derive from plasma exudation as well as local production by epithelium.

Reactive oxygen species Enzymes involved in the production of reactive oxygen species (ROS) participate in several biological processes. Production of ROS starts with formation of superoxide (O2–) from oxygen (O2) catalyzed by NADPH oxidase. Next, superoxide dismutase (SOD) transforms 2H+ and 2O2– molecules into one hydrogen peroxide (H2O2) molecule. Then peroxidases metabolize H2O2 and Cl– into H2O and hypochlorate (OCl–). These ROS damage nucleic acids, lipids and proteins (Valko et al. 2007). Similarly to phagocytes, airway epithelial cells also express these enzymes. The NADPH oxidase activity in epithelial cells is mediated by dual oxidases 1 and 2 (Duox 1 and 2), high-molecular-weight enzymes that also have peroxidase domains (Forteza et al. 2005). Epithelial Duox activity is important for epithelial production of mucin (Shao & Nadel 2005), epithelial repair (Wesley et al. 2007), epithelial production of acid (Schwarzer et al. 2004), innate immunity (Forteza et al. 2005), and for epithelial responses to ozone (Salmon et al. 1998), LPS (Koff et al. 2006), and cigarette smoke (Lavigne & Eppihimer 2005). Regarding innate immunity, epithelial Duox and SOD produce H2O2, which in the lumen is used by airway lactoperoxidase to oxidize thiocyanate anions (SCN–) to hypothiocyanite (OHSN), a potent antimicrobial (Wijkstrom-Frei et al. 2003; Forteza et al. 2005). Eosinophil peroxidase also converts SCN– into OHSN. Hypothiocyanite is an important antimicrobial that selectively kills microorganisms and spares host cells, unlike hypohalous acids that are often generated in the absence of a source of thiocyanate. Epithelial transport of thiocyanate is defective in cystic fibrosis (Moskwa et al. 2007). Upregulation of epithelial Duox activity in airway epithelial cells occurs in response to ATP and increased intracellular calcium (Forteza et al. 2005; Wesley et al. 2007). In alveolar type II cell lines, Duox activity increases as a result of mechanical stretching (Chapman et al. 2005). On the other hand, NADPH oxidase activity in neutrophils plays a role in transepithelial migration on chemoattraction by leukotriene (LT)B4 (Woo et al. 2003), and NADPH oxidase activity intrinsic

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to pollens (e.g., ragweed, plantain, birch, oak, grass pollen) can amplify Th2 sensitization and the allergic response to challenge in animal models of asthma (Boldogh et al. 2005; Ritsick & Lambeth 2005). Nonhematopoietic NADPH oxidase (e.g., in endothelial cells) regulates transendothelial migration of eosinophils into the lung (Abdala-Valencia et al. 2007). Inhalation of pollutants such as ozone (O3), sulfur dioxide (SO2), nitrogen dioxide (NO2), and particulate air pollutants (e.g., diesel exhaust particles) induce oxidative stress in the airways (Li et al. 2003). Airways resist endogenous and exogenous ROS damage through several mechanisms. The epithelial lining fluid contains antioxidants such as vitamins C and E, urate, and reduced glutathione. Intracellular ROS scavenger enzymes and antioxidants in airway cells include heme oxygenase I, glutathione-S-transferase, glutathione peroxidase, SOD, catalase, and thioredoxin reductase, many of which use gluthatione and thyoredoxin as substrates. If the intensity of oxidative stress overcomes these protective mechanisms, bronchial epithelial cells initiate inflammation by secreting cytokines and chemokines via activation of the transcription factors activator protein (AP)-1 and NF-κB. At high intensity of oxidative stress, however, the epithelium undergoes apoptosis via caspase activation or necrosis due to ROS cytotoxicity (Li et al. 2003).

Reactive nitrogen species Like ROS, reactive nitrogen species (RNS) participate in several biological processes. Indeed, ROS and RNS interact in disease, combining oxidative stress and nitrosative stress to cause tissue damage (Ricciardolo et al. 2006; Valko et al. 2007). Increased nitric oxide in exhaled air is a hallmark of asthma (Kharitonov & Barnes 2006). Nitric oxide (NO, or NO· because it has one unpaired electron) is produced in all organ systems by nitric oxide synthase (NOS), which use NADPH to transform the semi-essential amino acid L-arginine and oxygen into NO– and L-citrulline via a five-electron oxidative reaction (Ricciardolo et al. 2006). NOS has two domains, a C-terminal reductase domain with binding sites for NADPH, FAD and FMN (electron donors), and the N-terminal oxygenase domain with binding sites for L-arginine. Between these two domains is a consensus sequence that binds to calmodulin and which transfers electrons between the two domains. There are three members of the NOS family: constitutive neural NOS (NOS1 or nNOS, inducible NOS (NOS2 or iNOS), and constitutive endothelial NOS (NOS3 or eNOS). Airway epithelial cells express eNOS and iNOS. nNOS is expressed in airway nerves, where NO acts as a neurotransmitter and mediates airway smooth muscle relaxation via the inhibitory nonadrenergic, noncholinergic system. eNOS produces small amounts of NO and is constitutively expressed in airway epithelial cells, type II alveolar epithelial cells, and pulmonary vessel endothelial cells. iNOS is expressed in airway epithelial cells, endothelial cells, macrophages, airway and vascular smooth muscle cells,

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lung fibroblasts, mast cells, neutrophils, and chondrocytes. On stimulation, the iNOS gene is induced and large quantities of iNOS produced within hours; levels remain high for hours to days. Stimuli include, but are not limited to, TNF-α, IL-1β, interferon (IFN)-γ, chemokines, bacterial toxins, virus infections, allergens, pollutants (e.g., ozone, oxidative stress, silica), hypoxia, and the presence of tumors. Induction of iNOS expression is inhibited by glucocorticosteroids. iNOS produces large amounts of NO, which has a half-life of < 5 s in an aqueous environment because NO combines with oxygen donors to react with most biomolecules by nitration, nitrosation, and nitrosylation (addition of NO2, NO+, and NO, respectively). These reactions mediate the nitrosative stress that can result in inflammatory, antimicrobial, or antitumor effects, and in regulation of apoptosis. Physiologically, NO produced by constitutive NOS functions as an intracellular second messenger and neurotransmitter, inducing vasodilation and bronchodilation (Ricciardolo et al. 2006). As an antimicrobial (Mannick 2006), NO reacts with superoxide from the oxidative burst to form peroxynitrite (ONOO–), a potent oxidant. RNS deaminate DNA, inhibit DNA synthesis, and damage enzymes, which suppresses growth of, or kills, viruses, fungi, bacteria, and parasites. NO can inhibit apoptosis in low concentrations or cause apoptosis and necrosis in high concentrations by nitrosative stress in the context of inflammation and tumor suppression. NO inhibits apoptosis via a cGMP-induced increase in Bcl-2, via an increase in heat-shock protein 70 (which inhibits release of cytochrome c), via inhibition of caspase 3 by S-nitrosylation, and via an increase in the anti-apoptotic thioredoxin. In high concentrations, NO-induced nitrosylation leads to apoptosis by inhibition of NF-κB, reduction in Bcl-2, increase in p53, inhibition of the proteasome, and DNA damage (Mannick 2006). As with ROS induced-signaling and damage, the effects of RNS can be reversed by incompletely understood mechanisms. For example, the balance between nitrosylation and denitrosylation regulates apoptosis (Mannick et al. 1999; Mannick 2007) and enzyme activities such as that of tissue transglutaminase (Lai et al. 2001). In the airways of asthmatic individuals, increased oxidative and nitrosative stress (Misso & Thompson 2005) directly inhibits airway epithelial SOD, leading to increased epithelial apoptosis to an extent directly proportional to clinical severity of disease (Comhair et al. 2005). To counteract these stresses, the airway lumen of asthmatic individuals contains large amounts of glutathione peroxidase, a critical first-line antioxidant defense against ROS and RNS (Comhair et al. 2001). Inhibition of NOS by NG-monomethyl-L-arginine increases bradykinin-induced bronchospasm in asthmatic individuals, suggesting a bronchoprotective role for constitutive eNOS in asthma. On allergen challenge, however, eNOS is suppressed and iNOS upregulated, amplifying Th2 inflammation as suggested by asthma models in iNOS-deficient mice, which have attenuated eosinophil influx, vascular

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leakage, and airway obstruction compared with wild-type mice (Ricciardolo et al. 2006).

Epithelial receptors for sensing microbes Epithelial cells can detect the presence of microbes through several receptors, which recognize molecular patterns common to microbes and are designated pattern recognition receptors (PRRs). These receptors are located in the plasma membrane and in the intracellular compartments and can detect both extracellular and intracellular microbes. Among PRRs, there are 10 TLRs that recognize pathogen-associated molecular patterns (PAMPs) from viruses, bacteria, fungi, protozoa, and multicellular parasites (Table 16.3). TLRs were named for their homology to the Toll molecule in Drosophila melanogaster, which protects the fly against Aspergillus infection (Trinchieri & Sher 2007).

Toll-like receptors TLRs detect microbial products and transduce intracellular signals via adapter molecules (e.g., MyD88), leading to activation of the transcription factors NF-κB and interferonregulatory factor (IRF). These transcription factors activate innate immune responses such as the interferon response, expression of costimulatory molecules, and secretion of cytokines, chemokines and host defense molecules. Structurally, TLRs consist of three domains (Uematsu & Akira 2006). The extracellular domain is a leucine-rich repeat domain that accounts for specificity to PAMPs. The transmembrane domain anchors the molecule to the plasma membrane or intracellular membranes (e.g., endosomes). The cytoplasmic domain is similar to that of the IL-1 receptor, and is called the Toll/IL-1 receptor (TIR) domain. TIRs bind to adapter molecules (see Table 16.3) to transduce signal to cytosolic molecules including NF-κB, IRF, mitogen-activated protein kinases (MAPK), AP-1, and others (Hasan et al. 2005; West et al. 2006; Barton 2007; Trinchieri & Sher 2007). All 10 TLRs likely play roles in innate defense of the airways since they are expressed on epithelial and immune cells. mRNA for all 10 TLRs is present in sinonasal mucosa (Dong et al. 2005; Lane et al. 2006a). TLR2 mRNA is expressed at higher levels in the mucosa of subjects with CRS (Lane et al. 2006b), and TLR9 mRNA levels are higher in mucosal tissue from those with nasal polyps compared with mucosa from control subjects (Ramanathan et al. 2007). Bronchial epithelial cells express functional TLR1–6 and TLR9 (Sha et al. 2004; Guillot et al. 2005; Hewson et al. 2005; Ritter et al. 2005; Zhang et al. 2005; Groskreutz et al. 2006; Kato, A. et al. 2006; Mayer et al. 2007). Primary alveolar type II cells isolated from normal lungs express functional TLR2 and TLR4 (Armstrong et al. 2004). In BEAS-2B bronchial epithelial cells, IFN-γ increases expression of TLR3 and TLR4, whereas TNF-α increases expression of TLR2 (Homma et al. 2004; Ritter et al. 2005). Histamine increases TLR3 expression in the epithelial cell lines NCI-H292 and A549 (Hou et al. 2006).

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The glucocorticoid dexamethasone increases expression of TLR2 in epithelial cells (Shuto et al. 2002; Homma et al. 2004). Indeed, glucocorticoids seem to spare or increase expression of molecules related to innate immunity while inhibiting those involved in inflammation and/or adaptive immunity (Schleimer 2004). In addition to responding to microbial PAMPs, TLR2 can be involved in the response to particulate matter pollutants (Becker et al. 2005) and TLR2, TLR4, and TLR9 can also be involved in the response to dust mites (Boasen et al. 2005), although these environmental stimuli may also contain microbial components. TLR2 and TLR4 can also be activated by intrinsic ligands such as hyaluronan degradation products from extracellular matrix breakdown, resulting in inflammation and epithelial cell apoptosis (Jiang et al. 2005). Taken together, these studies demonstrate not only the presence of functional TLRs in airway epithelial cells, but also that their expression is modulated by mediators and hormones, which can modulate the ability of epithelial cells to sense and to respond to respiratory microbes and particles. In the context of an infection, more than one TLR can be simultaneously stimulated by different products of a single microbial species (Monick et al. 2003; Adamo et al. 2004; Morris et al. 2006; Trinchieri & Sher 2007). These TLR responses in epithelial and antigen-presenting cells (e.g., dendritic cells) result in increased expression of costimulatory molecules and secretion of cytokines and chemokines that can modulate the adaptive immune response. For example, stimulation of bronchial epithelial cells with ligands for TLR2, TLR8, and TLR9 (Lee & Ziegler 2007) and TLR3 (Kato et al. 2007) causes secretion of thymic stromal lymphopoietin (TSLP), an epithelial-derived cytokine that induces expression of OX40L on dendritic cells, which in turn induces differentiation of naive CD4 (Th0) cells into Th2 cells (Liu, Y.J. et al. 2007). Interestingly, rhinovirus synergized with IL-4 to induce TSLP in epithelial cells (Kato et al. 2007), providing a possible explanation for the enhanced allergic response in asthmatics undergoing rhinovirus infections (Lemanske et al. 2005).

Other microbe-sensing molecules In addition to TLRs, other recently recognized PAMP receptors are present in the cytosolic compartment and signal when microbes reach the intracellular compartment. Some of these intracellular receptors belong to two main families: the nucleotide-binding oligomerization domain (NOD)-like receptor (or NLR) family, and the helicase family (Trinchieri & Sher 2007). NLR family The NLR family consists of more than 20 members that are either NOD receptors, or NALPs (an acronym for NACHT-, leucine-rich repeat- and pyrin domain-containing proteins). NACHT is a common domain present in neuronal apoptosis inhibitory protein (NAIP), HLA class 2 transactivator (CIITA),

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Table 16.3 Toll-like receptors (TLRs) on human airway epithelial cells*. TLR response in airway epithelial cells

TLR

Ligands

Adapter molecules

TLR1

TLR1/TLR2 binds to triacyl lipopeptides

See TLR2

TLR1 heterodimerizes with TLR2, which activates adapter molecules. See TLR2

See TLR2

TLR2

TLR2 binds to peptidoglycan, atypical LPS, modulin (Staphylococcus epidermidis), porin (Neisseria). Trypanosoma cruzi GPI anchors. Measles, CMV, HSV-1. See also TLR1 and TLR6

MyD88. TIRAP/Mal

TIRAP/Mal → MyD88. Myd88 → IRAK4, IRAK1 → TRAF6 → TAK1 → IKK (→ IkB → NF-kB,p65p50) and JNK,p38 (→ AP-1). Induce gene transcription of chemokines, cytokines, and surface adhesion and costimulatory molecules

IL-6, TNF-a, IL-8, b-defensin 2 (DEFB2 also known as DEFB4), MIP-3a, GROa, IP10, TSLP

TLR3

Double-stranded RNA formed during replication of RNA viruses such as respiratory viruses

TRIF

TRIF → TRAF6 (see MyD88), RIP1 (→ TAK1, see MyD88), and TBK1. TBK1 → IRF3 → interferon (IFN)-induced genes

IL-6, TNF-a, IL-8, IFN pathway (IFN-b1), MIP-3a, GROa, IP10, I-TAC, RANTES, MMP13, TSLP, BAFF

TLR4

Endotoxin/LPS, RSV F protein, fungal mannan, protozoal GIP

TRAM. TIRAP/Mal

TLR4 associates with CD14 and other proteins to form the LPS receptor. TRAM → TRIF (see TLR3). TIRAP/Mal → MyD88 (see TLR2)

IL-6, IL-8, MIP-3a, GM-CSF

TLR5

Flagellin (Pseudomonas)

MyD88

Myd88 → See TLR2

MIP-3a, GM-CSF, GROa, MMP13

TLR6

TLR6/TLR2 binds to diacyl lipopeptides, lipoteichoic acid. Fungal zymosan. T. cruzi glycolipids

See TLR2

TLR6 heterodimerizes with TLR2, which activates adapter molecules. See TLR2. CD36 associates with TLR2/6

See TLR2

TLR7

Viral single-stranded RNA

MyD88

Myd88 → . . . → IRF7 instead of IRF3 (see TLR9). IRF7 → IFN-induced genes

TLR8

Viral single-stranded RNA

MyD88

Myd88 → . . . → IRF7 instead of IRF3 (see TLR9). IRF7 → IFN-induced genes

TSLP (weak response)

TLR9

Bacterial unmethylated CpG DNA. T. cruzi DNA. Hemozoin from Plasmodium falciparum. HSV-1 and HSV-2 DNA

MyD88

In plasmacytoid dendritic cells: TLR7 and TLR9 → Myd88 → IRAK4, IRAK1 → TRAF6 → TAK1 → IKK (→ IkB → NF-kB, p65p50) and IRF7 instead of IRF3 → IFN-induced genes

TSLP (weak response)

TLR10

Homodimerizes and also heterodimerizes with TLR1 or TLR6. TLR10 ligands are unknown

MyD88

Myd88 → See TLR2

Intracellular signaling pathways

* TLRs are expressed on the plasma membrane, except for TLR3, TLR7, TLR8 and TLR9, which respond to nucleic acids and are mostly expressed in endosomal membranes inside the cells. Mice also have a TLR11 that binds to protein-like proteins from uropathogenic. Escherichia coli and from Toxoplasma gondii. Depicted signaling pathways relate to studies in several cellular systems, whereas the last column only focuses on reports of TLR stimulation effects on human airway epithelial cells. Different ligands for the same TLR can induce mildly different responses, which may result from binding of ligand to different TLR heterodimers, to proteins associated with TLRs (e.g., CD14, CD36), or from differential activation of adapter molecules. Microarray studies show that expression of a large number of genes is altered by TLR ligand stimulation, particularly TLR3 stimulation with synthetic dsRNA poly deoxyinosinic:deoxycytidylic acid (poly I:C). GPI, glycosylphosphatidylinositol; LPS, lipopolysaccharide; GIP, glycoinositolphospholipids; CMV, cytomegalovirus; HSV, herpes simplex virus; RSV F, respiratory syncytial virus fusion; TIRAP/Mal, Toll/IL-1R (TIR)-domain-containing adapter protein; MyD88, myeloid differentiation primary-response gene 88; IRAK, IL-1R-associated kinase; TRAF, tumor necrosis factor receptor associated factor; TAK, transforming growth factor b (TGF-b)-associated kinase; IKK, IkB kinase; IkB, inhibitor of NF-kB; NF-kB, nuclear factor kB; JNK, c-Jun N-terminal kinase (p38 is a mitogen-activated protein kinase); TRIF, TIR-domain-containing adapter protein inducing IFN-b; TRAM, TRIF-related adapter molecule; RIP, kinases receptor interacting protein; TBK, tumor necrosis factor receptor (TNFR)-associated NF-kB kinase (TANK)-binding kinase; IRF, interferon-regulatory factor; MIP, macrophage inflammatory protein; GM-CSF, granulocyte–macrophage colony-stimulating factor; GRO1/CXCL1, growth-regulated oncogene 1; IP10/CXCL10, IFN-g-inducible protein 10; I-TAC/IP9/CXCL11, interferon-inducible T-cell a-chemoattractant; MMP, matrix metalloproteinase; RANTES/CCL5, regulated upon activation, normally T-expressed, and presumably secreted; TSLP, thymic stromal lymphopoietin; BAFF, B-cell-activating factor of TNF family.

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heterokaryon formation gene locus E (HET-E), and transition protein (TP)-1 (Trinchieri & Sher 2007). NLRs have three domains: 1 a carboxy-terminal series of leucine-rich repeats that bind to PAMPs; 2 an NOD that mediates self-oligomerization; 3 an amino-terminal effector domain that can be either a pyrin domain or a caspase-recruitment domain (CARD). Signaling occurs either via NOD activation of receptorinteracting serine/threonine kinase followed by NF-κB activation, or via CARD activation of caspase-1. Both signaling pathways lead to transcription of inflammatory cytokines. Most of the NLR molecules have only recently been discovered and relatively little information is available for epithelial cells. Thus far, the NLR member NOD1 has been found in airway epithelial cells (Slevogt et al. 2007). NOD1 recognizes bacterial wall components. Together with TLR2, NOD1 recognizes Moraxella catarrhalis infection of airway epithelial cells and induces production of IL-8 (Slevogt et al. 2007). It is likely that other NLRs and NALPs will be found in airway epithelial cells because they are found in intestinal epithelial cells (Rosenstiel et al. 2006). Helicase family The helicase or RLR (RIG-I-like receptor) family consists of two members: retinoic acid-inducible gene (RIG)-I and melanoma differentiation-associated gene (MDA)5 (Uematsu & Akira 2006). They recognize intracellular double-stranded RNA (dsRNA) produced on replication of RNA viruses, such as all respiratory viruses except adenovirus (which is a DNA virus). Helicases have an amino-terminal helicase domain that binds to and unwinds dsRNA via an ATPase-dependent mechanism and activates the two tandem CARD domains in the carboxy-terminus (Uematsu & Akira 2006), which binds the CARD domain of IFN-β promoter stimulator (IPS)-1 and activates two signaling pathways involving: 1 FAS-associated via death domain (FADD), receptor interacting protein (RIP)-1, inhibitor of NF-κB kinase (IKK)α, IKKβ and IKKγ, inhibitor of NF-κB (IκB), and finally NF-κB; 2 IKKi, TANK-binding kinase (TBK)1, and activation of IRF7 and/or IRF-3, leading to type I IFN production (Uematsu & Akira 2006). Helicases therefore provide a TLR-independent mechanism for recognizing RNA virus infections and induce type I IFN and NF-κB responses. RIG-I in the alveolar type II cell line A549 recognizes dsRNA formed by respiratory syncytial virus (RSV), activates NF-κB, and induces production of IFN-β, interferon-inducible protein 10 (IP-10/CXCL10), CCL5/ RANTES, and TLR3 (Liu, P. et al. 2007). RIG-I expression is increased by IFN-γ in the bronchial epithelial cell lines BEAS2B and NCI-H292 cells (Imaizumi et al. 2005). Animal studies suggest that RIG-I is important in response to parainfluenza and influenza viruses, whereas MDA5 is critical for response to picornaviruses (e.g., rhinoviruses) (Kato, H. et al. 2006).

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Another well-known receptor for dsRNA is the dsRNAdependent protein kinase (PKR) (Garcia et al. 2006). PKR is a cytosolic serine/threonine kinase that dimerizes when it binds to dsRNA, leading to autophosphorylation and subsequent phosphorylation of the α subunit of eukaryotic translation initiation factor (eIF)-2α. Once phosphorylated, eIF-2α is unable to deliver Met-tRNAi to the 40S ribosome, preventing initiation of protein synthesis, which impairs virus replication and cell proliferation and induces apoptosis. PKR is upregulated by IFN and signals through the MAPKs JNK and p38, and through NF-κB, STAT1, and IRF-1. In airway epithelial cells, PKR participates in the response to rhinovirus by mediating production of CCL5/RANTES (Gern et al. 2003). In addition, PKR mediates IgE class-switching in the human Burkitt’s lymphoma B-cell line Ramos on rhinovirus infection (Rager et al. 1998). Together, TLR3, PKR, and helicases detect and initiate mucosal innate responses to RNA viruses such as most respiratory viruses. Recent evidence suggests that bronchial epithelial cells from asthmatic individuals are deficient in their production of antiviral IFNs. These cells secrete less type I IFN (IFN-β1) and have impaired induction of apoptosis on rhinovirus infection compared with cells from healthy individuals, which allows replication of the virus (Wark et al. 2005). Likewise, bronchial epithelial cells from asthmatic individuals are also deficient in production of type III IFNs such as IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B) on rhinovirus infection (Contoli et al. 2006). In addition, production of lymphocyte-derived type II IFN (IFN-γ) is also impaired in asthmatic individuals (Gern et al. 2000). These deficiencies in type I, II, and III IFNs correlate with severity of rhinovirus colds. Taken together, asthmatic individuals probably have IFN deficiencies that enhance susceptibility to respiratory infections. Whether this deficiency is primary (genetic susceptibility) or secondary to allergen-driven inflammation is not known.

Role of epithelium in inflammation In the 1980s, the airway epithelium was first noticed to produce inflammatory mediators, including arachidonic acid metabolites, NO and cytokines such as granulocyte– macrophage colony-stimulating factor (GM-CSF), IL-8, and IL-6 (Henke et al. 1988; Vanhoutte 1988; Denburg et al. 1991; Holtzman 1991; Marini et al. 1991; Churchill et al. 1992). Until then, the airway epithelium was mostly known for its mucociliary system, ion transport, and electrical properties. In the last two decades, numerous other inflammatory mediators have been found to be generated in airway epithelial cells in response to environmental agents and also on stimulation by immune cells.

Lipid mediators In airway epithelial cells, PLA2 metabolizes membrane phospholipids and releases arachidonic acid (AA), which can be

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further metabolized into prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids (HETE) and lipoxins (Henke et al. 1988; Holtzman 1991, 1992; Goetzl 2003; Schaloske & Dennis 2006). Cyclooxygenase (COX)-1 and COX-2 (Balzary & Cocks 2006) both transform AA into prostaglandin (PG)E2, one of the first inflammatory mediators found to be secreted by airway epithelial cells (Holtzman 1992). Other epithelial COX minor products are PGF2α and thromboxane (TX)B2. Exogenous PGE2, administered by nebulization immediately prior to bronchial allergen challenge, attenuates early and late airway bronchoconstriction responses, airway hyperresponsiveness, and influx of eosinophils (Gauvreau et al. 1999). PGE2 relaxes airway smooth muscle and also has several anti-inflammatory actions (Holtzman 1992; Gauvreau et al. 1999). 5-Lipoxygenase (5-LO) together with 5-lipoxygenaseactivating protein (FLAP) transforms AA into 5-HETE and leukotriene (LT)A4 (Holtzman 1991, 1992), which is unstable and is quickly transformed by LTA4 hydrolase (Bigby et al. 1989) into LTB4, a potent chemoattractant for neutrophil and cytotoxic CD8 lymphocytes (Goodarzi et al. 2003; Ott et al. 2003). LTA4 can also be transformed into LTC4 by LTC4 synthase, but this occurs in leukocytes. Epithelial cells also express 15-lipoxygenase (15-LO), which transforms AA into 15-HETE and other minor products such as 12-HETE and 8-HETE (Henke et al. 1988; Widdicombe 1991), whose fuctions are not completely understood. Epithelial 15-LO has been shown to be induced by IL-13 and has been implicated in both asthma and COPD (Chu et al. 2002; Kuperman et al. 2005). Recent studies indicate that 15-LO may inhibit epithelial transport of IgA into the airways (Kuperman et al. 2007). Products from lipoxygenases interact to form lipoxins (Bonnans et al. 2004). Interactions between LTA4 and 15HETE form lipoxin (LX)A4, while those between LXB4 and 5-epi-lipoxin form 15epi-LXA4 (Bonnans et al. 2004). These lipoxins have antiinflammatory and epithelium repair actions via inhibition of a number of processes, such as LTB4-mediated chemotaxis of neutrophils, release of IL-8 and elastase by neutrophils, platelet-activating factor-induced chemotaxis of eosinophils, and LTC4-induced bronchospasm (Bonnans et al. 2004).

Adhesion molecules Epithelial cells express adhesion molecules that not only maintain structural integrity of the normal epithelium but also interact with leukocytes, initiate intracellular signaling in inflammation and repair, and participate in lung morphogenesis (Sheppard 2003). Airway epithelial cells express integrins, intercellular adhesion molecule (ICAM)-1, and CD44 (Peroni et al. 1996) (Table 16.4). Integrins are heterodimeric transmembrane glycoproteins composed of α and β subunits. There are at least 18 α subunits and 8 β subunits that can form 24 integrin heterodimers.

Airway Epithelium

Virtually all cells in the body express integrins. There is redundancy because several integrins bind to the same ligand, and the same integrin–ligand pair can result in different responses in different tissues. On interaction, the conformational changes in the integrin–ligand complex can result in downstream intracellular signaling in both interacting cells. In epithelial cells, integrins regulate wound repair, establishment of polarity, differentiation into secretory cells, and inflammation. After binding to ligand in extracellular matrix or on the surface of adjacent cells, integrins change their conformation, initiating or repressing intracellular signaling through interactions between the short intracytoplasmic domains of their subunits and a multiprotein signaling complex. In this complex, focal adhesion kinase (FAK) has binding sites to interact with several signaling molecules, including ras-associated protein Grb2, the SH2 domains of src, phosphatidylinositol 3-kinase and PLC-γ, and adapter proteins (Sheppard 2003). In addition, some integrins (e.g., α2β1 and αVβ3) use another transmembrane protein called integrin-associated protein (IAP) for signaling. These proteins allow integrins to activate a broad number of downstream signaling pathways (see Table 16.4). Bronchial and nasal epithelial cells also express ICAM-1, a member of the immunoglobulin superfamily. ICAM-1 is increased in bronchial epithelial cells of asthmatic patients (Wegner et al. 1990) and its blockade in a primate asthma model attenuates airway eosinophilia and hyperresponsiveness (Wegner et al. 1990). Bronchial epithelial expression of ICAM-1 increases 24 hours after allergen challenge in asthmatic patients (Bentley et al. 1993), and also on stimulation with TNF-α, IFN-γ, IL-4, and IL-13 (Bianco et al. 1998; Striz et al. 1999). It reduces on treatment with corticosteroids (Papi et al. 2000) and histamine H1 receptor blockers (Ciprandi et al. 2003). Epithelial ICAM-1 mediates adhesion to eosinophils (Burke-Gaffney & Hellewell 1998) and neutrophils via Mac-1 (CD11b/CD18) and to lymphocytes via LFA-1 (CD11a/ CD18). Interaction between goblet cells and neutrophils via ICAM-1 mediates degranulation of goblet cells induced by neutrophil elastase (Nadel et al. 1999). Besides being expressed on the plasma membrane (mICAM-1), a soluble form of ICAM-1 (sICAM-1) can derive from either mRNA splicing or from protease-mediated cleavage of mICAM-1. The function of sICAM-1 is not fully established, but it could be a decoy inhibitory molecule interfering with ICAM-1-mediated adhesion. sICAM-1 is elevated in the serum of patients affected by many chronic inflammatory diseases including asthma (Witkowska & Borawska 2004). It increases further in acute asthma. ICAM-1 is also the receptor for major group rhinoviruses, which precipitate 30–50% of all asthma exacerbations. Rhinovirus further increases ICAM-1 expression by epithelial cells. The minor group rhinoviruses represent 10% of the serotypes and utilize the LDL receptor for cell entry (Edwards et al. 2006a).

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Table 16.4 Adhesion molecules on human airway epithelial cells. Molecule

Ligands

Localization

Function

Integrins a2b1

Collagen I and IV, tenascin C, echovirus

All epithelium, particularly basal cells

Adhesion of basal cells to basement membrane

a3b1

Laminins 5, 10, 11

All epithelium, particularly basal surface of basal cells

Adhesion of basal cells to basement membrane

a5b1

Fibronectin

Expressed after injury on all epithelium

Binding to matrix and repair

a6b4

Laminins 5, 10, 11

Basal surface of basal cells

Adhesion of basal cells to basement membrane

a9b1

Tenascin C, osteopontin, VCAM-1, L1-CAM, vWF, factor XIII, tissue transglutaminase, fibronectin, angiostatin, ADAMS 1, 2, 3, 9 and 15

All epithelium, particularly basal cells

Adhesion to basement membrane and leukocytes, coagulation, and repair

aVb5

Vitronectin, osteopontin, adenovirus

All epithelium, particularly on basal cells

Binding to matrix and repair

aVb6

LAP of TGF-b1 and -b3, fibronectin, tenascin C, osteopontin, vitronectin, foot and mouth virus

Highly expressed after injury on all epithelium

Binding to matrix, initiation of fibrosis and repair

aVb8

LAP of TGF-b1 and -b3, vitronectin

Adhesion of basal cells to basement membrane

Binding to matrix, initiation of fibrosis and repair

Immunoglobulin superfamily molecule Eosinophils, neutrophils, lymphocytes, and ICAM-1 rhinovirus

Other adhesion molecule Hyaluronic acid, collagen and fibronectin CD44

Expressed on scattered epithelial cells. Increased in asthma

Mediates neutrophil-induced mucus secretion and adhesion to leukocytes

All epithelium, particularly on basal cells. Increased in asthma

Mediates neutrophil-induced mucus secretion

ICAM, intercellular adhesion molecule; LAP, latency-associated peptide; TGF, transforming growth factor; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor.

Cytokines, chemokines and neuropeptides In the past 20 years, airway epithelial cells have been found to secrete a vast number of cytokines and chemokines that recruit all types of leukocytes and regulate their functions. In this section, we focus on cytokines and chemokines secreted by the pseudostratified epithelium in the conducting airways as identified by airway biopsy studies using immunohistochemistry and in situ hybridization techniques and by cultures of bronchial or nasal epithelial cells (Tables 16.5 and 16.6). These tables do not include the biology of mediators secreted by other epithelial cell types such as alveolar epithelial cells, glandular epithelial cells, and squamous cells.

Cytokines Cytokines are secreted polypeptides that regulate growth, differentiation, and functions of leukocytes and structural cells. Epithelial cells can secrete pleiotropic cytokines such as TNF-α, IL-1β, IL-6, and IL-11 on respiratory virus infection

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and other stimuli. These cytokines initiate inflammation and the engagement of adaptive immune responses (Becker et al. 1991; Elias et al. 1994; Khair et al. 1995; Bitko et al. 1997; Terajima et al. 1997; Takizawa 1998; Nam et al. 2006). Epithelial cells can also secrete regulatory cytokines that affect monocytes (IL-10, IL-12p80, IL-19) (Bonfield et al. 1995; Walter et al. 2001; Bosson et al. 2003; Pathmanathan et al. 2003; Zhong et al. 2006), T cells (IL-15, IL16, TSLP) (Arima et al. 1999; Little et al. 2003; Ying et al. 2005; Kato et al. 2007; Regamey et al. 2007), and B cells (IL-6, IL-10, BAFF, APRIL) (Kato, A. et al. 2006). In addition, as part of the defense against respiratory viruses, epithelial cells release IFNs (Friedlander et al. 2005; Wark et al. 2005; Contoli et al. 2006; Edwards et al. 2006a). Epithelial cells also secrete growth factors that participate in injury repair, but can also be stimulated by inflammation and environmental agents (see Table 16.5) (Marini et al. 1991; Bedard et al. 1993; Coste et al. 1996; Hirst et al. 1996; Nakamura et al. 1996; Yi et al.

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Table 16.5 Cytokines and other mediators produced by nasal and/or bronchial airway epithelial cells*. Molecule

Function

Stimuli for production

Interleukins IL-1b IL-6 IL-10 IL-11 IL12p80 IL-15 IL-16 IL-19 IL-28A/B, IL-29

Lymphocyte activation, endothelial expression of ICAM-1 and VCAM-1 Induces acute-phase reactants, stimulates T and B cells Inhibition of activated T cells and mononuclear phagocytes. Induction of Treg Stimulates hematopoiesis, myelofibroblast and collagen deposition IL12p40 homodimer attracts macrophages Stimulates T and NK cells, and differentiates monocytes into DCs Chemoattracts CD4+ T cells, eosinophils and monocytes Induces monocytes to secrete TNF-a Type III (lambda) interferons. Induce antiviral response similar to IFN-b

Virus infections, asbestos, TDI, HDM, DEP, CSE Virus and bacterial infections, O3, IL-17 TNF-a, IFN-l RSV, IL-1b, TGF-b1, and histamine Virus infections, allograft rejection IFN-l Histamine, TNF-a, TGF-b1, IL-9 Adenosine via A2B receptor Virus infections

Interferon type I. Induces epithelial antivirus defense Airway hyperresponsiveness, stimulates myofibroblast, mediates eosinophilia, production of SP and growth factors Promotes differentiation of Th0 to Th2 via dendritic cells and OX40 ligand Vasoconstriction, bronchospasm, mucus secretion, fibroblast proliferation, vascular leakage, production of PGD2 and LTD4 CGRP is a neuropeptide that induces potent arterial and venous vasodilatation

Virus infections IL-1a, TNF-a, TGF-b1

Other mediators IFN-b1 NGF TSLP Endothelin 1 CGRP

TNF superfamily† Endothelial expression of ICAM-1, VCAM-1 TNF-a Stimulates B-cell proliferation and differentiation into plasma cells including IgE BAFF switching Stimulates B-cell proliferation and differentiation into plasma cells including IgA APRIL switching Growth factors GM-CSF/CSF2 G-CSF/CSF-3 FGF-1, FGF-2 FGF-7 or KGF TGF-a EGFR pathway TGF-b1 TGF-b2 PDGF-b SCF Heregulin-a

Stimulates hematopoiesis for granulocytes and monocytes. Differentiation of DCs Stimulates hematopoiesis for granulocytes, particularly neutrophils Fibroblast proliferation and angiogenic Proliferation of keratinocytes and airway epithelial cells Surface bound. Released by TACE and induces mucin synthesis via EGFR Mucin synthesis, mucous cell metaplasia and fibroblast proliferation, differentiation, and extracellular matrix production Stimulates myofibroblast proliferation. Induces epithelial–mesenchymal transition. Induces IgA class switch Stimulates myofibroblasts to secrete extracellular matrix proteins Epithelial, fibroblast and smooth muscle cell proliferation and collagen deposition Mast cell proliferation and differentiation On injury, apically secreted heregulin can reach and interact with erbB-2, erbB-3 and erbB-4 receptors on the basolateral membrane to induce epithelial proliferation and wound repair

IL-1b, TNF-a, rhinovirus, stimulation of TLRs EPO, MBP Produced in epithelial and T cells during late-phase response to allergens

Virus and bacterial infections TLR3 stimulation via IFN-b1 TLR3 stimulation via IFN-b1

TNF-a, IL-1, IL-4, IL-13, virus infection, DEP, NO2, O3 TNF-a, IL-1, IL-17A, IL-17F, virus infection Increased in COPD. Virus infection Unknown EPO, MBP CSE, virus and bacterial infections, Th2 cytokines, neutrophil elastase via TGF-a, EGF EPO, MBP IL-4, IL-13 EPO, MBP, PAR-1 agonist Allergen exposure in sensitized individuals Mechanical disruption of epithelium integrity

* For more details on biology of cytokines, see Chapter 23. † Surface molecules that bind to their respective receptors during cell–cell contact and/or are cleaved by surface enzymes (i.e., TACE) to become soluble factors. APRIL, a proliferation-inducing ligand (also TNFSF13); BAFF, B-cell-activating factor (also TNFSF13B); CGRP, calcitonin-gene related peptide; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; DCs, dendritic cells; DEP, diesel exhaust particles; EGFR, epidermal growth factor receptor; EPO, eosinophil peroxidase; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colonystimulating factor; HDM, house-dust mite; ICAM, intercellular adhesion molecule; KGF, keratinocyte growth factor; MBP, major basic protein; NGF, nerve growth factor; NK, natural killer; PAR, protease-activated receptor; PDGF, platelet-derived growth factor; RSV, respiratory syncytial virus; SCF, stem cell factor; SP, substance P; TACE, TNF-a converting enzyme; TDI, toluene diisocyanate; TLR, Toll-like receptor; Treg, CD4+ T regulatory cells; TSLP, thymic stromal lymphopoietin; VCAM, vascular cell adhesion molecule.

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Table 16.6 Chemokines secreted by nasal and/or bronchial airway epithelial cells*. Molecule

Cells attracted

Stimuli for production

CC chemokines CCL1/I-309 CCL2/MCP-1 CCL3/MIP-1a CCL5/RANTES CCL11/eotaxin CCL13/MCP-4 CCL17/TARC CCL20/MIP-3a CCL22/MDC CCL24/eotaxin 2 CCL26/eotaxin 3

Eosinophils, monocytes Basophils, monocytes, activated T, NK, immature DC Eosinophils, monocytes, activated T, NK, immature DC Eosinophils, monocytes, activated T, NK, immature DC Eosinophils, basophils, activated T, NK, immature DC Eosinophils, basophils, activated T, NK, immature DC Th2, eosinophils Immature DC Eosinophils, basophils, activated T, immature DC Eosinophils, basophils, activated T, immature DC Eosinophils, basophils, resting T

Unknown Virus infections, a defensin 1, IL-4, IL-13, Der p1, TNF-a, IL-1b, IFN-l Virus infections IL-1b, virus infections, Pseudomonas, Der p1, O3, TNF-a, DEP, IL-4 Virus infections, IL-4, IL-13, TNF-a, Staphylococcus aureus TNF-a, IL-1b, IFN-l Der p1, IL-4, TGF-b1 Der p1, extracellular nucleotides, IL-17 Virus infection, IL-4, IL-13 Virus infection, IL-4, IL-13 Virus infection, IL-4, IL-13

CXC chemokines CXCL1/GROa CXCL2/GROb CXCL3/GROl CXCL5/ENA78 CXCL6/GCP2 CXCL8/IL-8

Neutrophils Neutrophils Neutrophils Neutrophils Neutrophils Neutrophils, DC

IL-17A, IL-17F, virus infections, IL-4, IL-13, DEP

CXCL9 /MIG CXCL10/IP-10 CXCL11/I-TAC

Activated Th1, NK Activated Th1, NK Activated Th1, NK

CX3C chemokines CX3CL1/fractalkine

DC–epithelial interaction

TNF-a Virus infection, O3 IL-17 Virus and bacterial infections, HDM, DEP, CSE, O3, IL-17, a defensin 1, TNF-a, IL-1b, IL-4 Virus infection, IFN-l Virus infection, IFN-l, Der p1 Virus infection, IFN-l

* For more details on names and biology of chemokines, see Chapter 24. T, CD3+ lymphocytes; Th, T helper CD3+CD4+ cells; Th1, CD3+CD4+ lymphocytes secreting IFN-l; Th2, CD3+CD4+ lymphocytes secreting IL-4, IL-13; NK, natural killer cells; DC, dendritic cells; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIG, monokine induced by gamma interferon; MIP, macrophage inflammatory protein; TARC, thymus and activation-regulated chemokine; ENA, epithelial cell-derived neutrophilactivating peptide; GCP, granulocyte chemotactic protein; TECK/CCL25 and MEC/CCL28 have been described in mucosa of airways in animals. For definition of other abbreviations, see footnote of Table 16.3.

1996; Aoshiba et al. 1997; Ishibashi et al. 1998; Michelson et al. 1999; Gern et al. 2000; Shimizu et al. 2000; Nadel & Burgel 2001; Bosson et al. 2003; Pathmanathan et al. 2003; Sheppard 2003; Vermeer et al. 2003a; Al-Muhsen et al. 2004; Burgel & Nadel 2004; Holgate et al. 2004; Kowalski et al. 2005; Kranenburg et al. 2005; Shao & Nadel 2005; Takizawa 2005; Ingram & Bonner 2006; Koff et al. 2006; Pegorier et al. 2006; Tyner et al. 2006; Volonaki et al. 2006). In bronchial biopsies, epithelial cells from asthmatic subjects have increased expression of IL-12p80 (Walter et al. 2001), IL-16 (Laberge et al. 1997), eotaxins (Komiya et al. 2003), endothelin (Howarth et al. 1995), IL-11, IL-17, transforming growth factor (TGF)-β, (Chakir et al. 2003), TSLP (Ying et al. 2005), stem cell factor (Al-Muhsen et al. 2004), and nerve growth factor (Frossard et al. 2004). Epithelial cells are also a target for action of cytokines released by inflammatory cells and their own mediators

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(autocrine action). Most notably, in asthma, epithelial cells undergo mucous metaplasia driven particularly by IL-13 (Kuperman et al. 2002), but also by IL-4 (Iwashita et al. 2003), IL-9 (Vermeer et al. 2003b), IL-11, IL-25, and IL-33 (Tyner et al. 2006; Nakajima & Takatsu 2007). Indeed, in mice, tracheal administration of IL-13 or IL-4 causes airway hyperactivity within 6 hours without eosinophilic inflammation (Venkayya et al. 2002).

Chemokines As mentioned above, airway epithelial cells also secrete a large variety of chemokines that enable recruitment of all types of leukocytes on exposure to diverse stimuli including environmental agents and cytokines (see Table 16.5). Chemokines are small proteins of 8–12 kDa that act through chemokine receptors, with overlapping specificity constituting a redundant system. They are classified based on the

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number of amino acids between the closest cysteine residues forming disulfide bonds. The CC, CXC, and CX3C chemokine families have none, one or three intervening amino acids, respectively, between the cysteine residues forming the bonds. The C chemokine family has only one member, lymphotactin, not expressed by epithelial cells. Secreted chemokines are designated ligands, indicated by the letter L, and receptors, indicated by the letter R. For example, the chemokine RANTES is designated CCL5 and binds to three receptors, CCR1, CCR3, and CCR5. In general, CCL chemokines recruit lymphocytes, monocytes, eosinophils, basophils, and dendritic cells, whereas CXC chemokines recruit neutrophils. There is only one member of the CX3C chemokine category. There are 28 CCLs and 16 CXCs. Epithelial cells can secrete the CCL chemokines (see Table 16.6) CCL1/I-309 (Montes-Vizuet et al. 2006), CCL2/ monocyte chemoattractant protein (MCP)-1 (van der Velden et al. 1998; Ip et al. 2006; Liu, C.Y. et al. 2007), CCL5/RANTES (Teran 2000; Bayram et al. 2001), CCL11/eotaxin (Matsukura et al. 2001; Takamura et al. 2004; Escotte et al. 2006), CCL13/ MCP-4 (Stellato et al. 1997; Lamkhioued et al. 2000), CCL17 (Heijink et al. 2007), CCL20/macrophage inflammatory protein (MIP)-3α (Pichavant et al. 2005 and 2006; Marcet et al. 2007), CCL22/MDC (Lezcano-Meza et al. 2003), CCL24/ eotaxin 2 (Papadopoulos et al. 2001; van Wetering et al. 2007), and CCL26/eotaxin 3 (Komiya et al. 2003). CCL25/TECK (thymus-expressed chemokine) and CCL28/MEC (mucosaeassociated epithelial chemokine), B-cell attracting chemokines, have been described in mucosa of airways in mice (Meurens et al. 2006), where CCL28 was also involved in recruitment of eosinophils (John et al. 2005). Of the CXCL chemokines, airway epithelium can secrete CXCL1/GROα (Salvi et al. 2000; McAllister et al. 2005), CXCL5/ENA78 (Bosson et al. 2003; Edwards et al. 2006b), CXCL6/GCP2 (Prause et al. 2003; Rudack et al. 2006), and CXCL9–11 (MIG, IP10, I-TAC) (Sauty et al. 1999). In airways of asthmatics, the following chemokines are increased compared with nonasthmatic control subjects: CCL1/I309 (Montes-Vizuet et al. 2006), CCL2/MCP-1 (Sousa et al. 1994), CCL3/MIP-1α, CXCL8/IL-8 (Hamilton et al. 2003), CCL11/eotaxin (Pepe et al. 2005), CCL13/MCP-4 (Lamkhioued et al. 2000), and CX3CL1/fractalkine (Rimaniol et al. 2003).

Neuropeptides Neuropeptides are peptides released by nerve fibers that regulate function of nonneuronal tissue. The main neuropeptides secreted by nociceptive (pain sensation) nonmyelinated sensory nerve fibers (type C) in the airways are substance P (SP), neurokinin A (NKA), and calcitonin generelated peptide (CGRP). These neuropeptides induce bronchoconstriction (NKA > SP > CGRP) and vasodilatation (SP, CGRP), increase blood vessel permeability (SP), and induce glandular secretion (SP) (Casale & Baraniuk 1998). Among these neuropeptides, only CGRP increased in bronchial secre-

Airway Epithelium

tions and tissue in the late asthmatic response, i.e., 5 hours after inhalation challenge with cat allergen peptide (Kay et al. 2007). Immunostaining of bronchial biopsies revealed presence of CGRP in airway epithelium, smooth muscle, T cells, and macrophages. In this model, there is no early asthmatic response because peptides are too small to cross-link IgE and activate mast cells. Therefore, this model indicates that allergen stimulation of antigen-presenting cells and T cells leads to CGRP production, a potent arterial and venous vasodilator. This may be an important pathogenic event in late asthmatic responses to allergens because in the cat allergen peptide inhalation model these reactions are not mediated by histamine, leukotrienes, prostaglandins, or by influx of polymorphonuclear leukocytes.

Role of epithelium in adaptive immune responses Accumulating evidence indicates that epithelial cells play important roles in the initiation, maintenance, and regulation of adaptive immune responses in the airways. Epithelial cells can trigger and modify the activation and differentiation of dendritic cells (DCs), B cells, and T cells. They also play an important role in the formation of lymphoid structures in the airways. When the innate immune functions of epithelium discussed above fail, adaptive immune responses to potential pathogens are necessary and life-saving. In addition, restraint of inflammatory immune responses is essential to prevent excessive or unnecessary damage to the airways. While responses of cells armed with antigen-specific immunoglobulins (e.g., mast cells, basophils, eosinophils, neutrophils) are part of the adaptive response, they are discussed elsewhere in this volume and are not considered here. There is a highly meshed network of DCs within the respiratory epithelium. A subset of these DCs, referred to as intraepithelial dendritic cells, has a distinct phenotype, expressing αEβ7, Fc receptors, langerin, and tight junction proteins (claudin-1, claudin-7, and ZO-2) (Holt et al. 1989; Gong et al. 1992; Sung et al. 2006) Intraepithelial DCs extend processes into the airway lumen between epithelial cells, presumably to collect antigenic material from the mucosal surface. These cellular processes may interact with epithelial cells through the unusual membrane-associated chemokine CX3CL1/fractalkine, which is primarily expressed by epithelial cells (Lucas et al. 2001). Fractalkine and its receptor CX3CR1 have been shown to mediate DC–epithelial interactions in the gut (Rimaniol et al. 2003; Niess et al. 2005) and are elevated in asthmatic airways (Rimaniol et al. 2003). Mucosal M cells are a type of specialized epithelial cell found in the intestine that have high permeability and permit antigen tissue entry for access to subepithelial DCs. Analogous cells do not appear to be found in normal human airway or in bronchialassociated lymphoid tissue (BALT), which is generally only found in inflamed human airways (Richmond et al. 1993). There is growing evidence that epithelial cells play a role in the recruitment and local survival of DCs, as they produce

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the chemokine CCL20/MIP-3α and the cytokine GM-CSF, respectively, which promote these processes (Reibman et al. 2003; Sha et al. 2004). Epithelial IL-15 has been shown to transform monocytes into plasmacytoid DCs (Regamey et al. 2007). The nature of the immune response that occurs after DC exposure to antigen (e.g., Th1, Th2, Treg, Th17) is determined by the state of activation of DCs and the context in which they present antigen to T cells (level and type of costimulatory molecules expressed by the DC, production of IL-10, IL12, etc.). While factors associated with antigen, such as the presence of TLR ligands, can have a profound outcome on the nature of the DC response, it is now clear that epithelial cells can also influence subsequent DC activation status. Recent studies have focused on the epithelial-derived factor TSLP, which has been shown to skew DCs so that they activate formation of Th2 cells. TSLP is a four-helix bundle cytokine related to IL-7 and binds to a specific receptor composed of the IL-7R α chain and the TSLP receptor (Liu, Y.J. et al. 2007). Production of TSLP occurs primarily in keratinocytes in the skin and epithelial cells in the airways. Besides activating DCs, epithelial TSLP has been shown recently to activate cytokine secretion by mast cells (Allakhverdi et al. 2007). Elevated TSLP expression has been demonstrated in both atopic dermatitis and in asthma (Soumelis et al. 2002; Ying et al. 2005). A feature of its effects on DCs is that it activates costimulation processes without triggering DC generation of IL-12, a cytokine with potent Th1-skewing activity. Epithelial expression of TSLP is triggered by the TLR3 ligand dsRNA, by rhinovirus, and by Th2 cytokines and the process involves activation of NF-κB and IRF-3 in the case of dsRNA and STAT6 in the case of IL-4 (Kato et al. 2007). Other epithelial-derived factors that are likely to influence the Tcell-skewing characteristics of DCs are the type I interferons IFN-α and IFN-β (that skew toward Th1) and IFN-λ (IL-28/ IL-29) (skew toward Treg) (Rogge et al. 1998; Mennechet & Uze 2006). Epithelial cells can shape the tissue response during adaptive immune effector responses to conform to the nature of the T-cell response and the leukocytes required (neutrophils for Th1 and Th17 and eosinophils and basophils for Th2). For example, epithelial cells release Th1-related chemokines (CXCL10/IP-10 and CXCL9/MIG to attract Th1 cells and neutrophil chemoattractant chemokines) in response to IFN-γ produced by Th1 cells, and produce Th2-related chemokines (CCL22/MDC and CCL17/TARC to attract Th2 cells and eotaxins to attract eosinophils and basophils) in response to IL-4 and IL-13 produced by Th2 cells (Nickel et al. 1999). Epithelial cells produce CXCL8/IL-8 and CXCL1–3/GROα–γ in response to Th17 to recruit neutrophils (Laan et al. 1999; McAllister et al. 2005). The chemokines responsible for Th17 recruitment have not been evaluated, but it is reasonable to expect that they will be partly epithelial cell-derived. It is not clear whether epithelial cells also shape the T-cell response

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during initial sensitization, although it is tempting to speculate that epithelial cells may influence the phenotype of outgoing DCs to modify their T-cell differentiation tendencies in draining lymph nodes. Epithelial cells do express receptors for some factors that drive the T-cell differentiation process, such as IL-31, which may regulate both Th1 and Th2, and TGF-β, which skews toward Th17 (Chattopadhyay et al. 2007). It has not been tested whether epithelial cells have receptors for other such factors that influence the nature of the T-cell response, such as IL-25, that skews toward Th2, IL-28B/IL-29 that skew toward Treg, TSLP that skews to Th2 (see above), or IL-23 that skews to Th17 (see above). However, IL-22 appears to be a prominent Th17-produced cytokine and epithelial cells (keratinocytes) display a robust response to this cytokine (Zheng et al. 2007). Although epithelial cells express HLA-I, HLA-II and costimulatory molecules such as CD80, CD86 (Oei et al. 2004), B7-H1–B7-H4, B7-DC (Kurosawa et al. 2003; Kim et al. 2005; Tsuda et al. 2005), and CD40 (Propst et al. 2000), the roles of these molecules in antigen presentation to T cells and in modulation of T-cell responses are not completely understood. Studies in the 1970s demonstrated that IgA- and IgEexpressing B cells are found in the airways and that these cells produce IgE and IgA specific to known inhaled antigens (Huggins & Brostoff 1975; Nakajima et al. 1975). Several studies have shown that levels of aeroallergen-specific IgE are much higher in the airways than in the serum when normalized to total IgE or albumin. It is not uncommon for individuals with allergic rhinitis or CRS to manifest antigen-specific IgE in nasal secretions or nasal tissue with no apparent specific IgE in the serum for the same antigen, and documented cases exist in which nasal antigen challenge responses are elicited in individuals who lack skin-test sensitivity. A survey study found that 19% of patients with rhinitis and polyposis had specific IgE in the nose but not the serum (Shatkin et al. 1994). Recent studies that used timeresolved fluorescence immunosorbent assays to determine the relative proportions of total and specific immunoglobulins in the airways and circulation concluded that the majority of the total body aeroallergen-specific antibodies of the IgE and IgA isotypes are produced in the airways, and that systemic sensitization largely reflects spillover of immunoglobulins from the mucosal site of their production into the circulation (Yoshida et al. 2005). It has been suggested that IgE is tightly regulated in this way to avoid the danger of anaphylaxis that accompanies the presence of high concentrations of circulating IgE (Geha et al. 2003). It is now clear that antigen-specific B cells are activated and undergo class-switch recombination in the gut and airway submucosae. It thus becomes important to consider the role that local factors in the mucosae play in the recruitment, differentiation, activation, and survival of B cells. Epithelial cells, especially in the gut, have been shown to release chemokines that attract B cells in general, and IgA-secreting B cells

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and plasma cells in particular. These chemokines include CCL25/TECK, CCL28/MEC, CXCL13/BLC (B-lymphocyte chemoattractant), and CXCL12/stromal-cell derived factor (SDF)-1α. In many cases, studies have been restricted to mucosal epithelium of the intestine (Hieshima et al. 2004). In the airways, epithelial production of CCL28/MEC has been largely of interest since it attracts eosinophils and Th2 cells via CCR3 and CCR10 (John et al. 2005). However, this chemokine is also likely to be an important epithelial-derived chemokine that attracts B cells in both the gut and the airways (Lazarus et al. 2003; English et al. 2006). CXCL12/ SDF-1α is another known B cell-attracting chemokine that has nonetheless been of interest in airway disease for another reason; it has recently also been shown to play a role in recruitment of epithelial stem cells to injured trachea as part of the repair process mediated by keratinocyte growth factor (Gomperts et al. 2006, 2007). More information is needed to better clarify the role of epithelium in B-cell recruitment. Recent studies indicate that epithelial cells produce several factors that can modify the differentiation of B cells, much in the same way that has been described above for DCs and T cells. Epithelial cells have long been known to be a rich source of IL-6 and TGF-β, cytokines that have profound B cell-activating properties (see above). In addition, recent studies indicate that epithelial cells produce B-cell activating factor (BAFF)/BLyS (see Table 16.5) or TNFSF13B, a member of the TNF superfamily that is essential for B-cell development via the BAFF receptor and which mediates B-cell class-switch recombination through another receptor, TACI [transmembrane activator and CAML (cyclophilin ligand protein) interactor] (Mackay et al. 2003; Kato, A. et al. 2006). More studies are needed to determine the relative importance of epithelial-derived BAFF versus other sources of BAFF on local class-switch recombination and plasma cell differentiation of B cells in the airways. In the intestine, it has been concluded that epithelial BAFF is the major trigger for regulating immunoglobulin class switching and that this process is promoted further by epithelial-derived TSLP and regulated by the protease inhibitor SLPI (Xu et al. 2007). Epithelial cells perform a well-known role in the transport of IgA and IgM across the epithelium into mucosal secretions (Kaetzel 2005; Brandtzaeg et al. 2006). This process is likely to be of importance both in innate and adaptive immunity, as natural IgA antibodies, i.e., those not generated by somatic hypermutation, can be produced locally in mucosal tissues, in some cases without the participation of T cells. Mucosal B cells produce dimeric IgA or pentameric IgM with monomers joined by the J chain. These multimers bind to the polymeric immunoglobulin receptor (pIgR), which transports them across epithelial cells into the airway lumen. This process occurs to a significant extent in airway mucosal glands as well as in the lamina propria of intestine and conducting airways (Fagarasan & Honjo 2004). During the process of

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transport of IgA (or IgM) by pIgR, the transported antibodies are covalently linked to a portion of pIgR that becomes the secretory component (SC) to produce the secretory forms sIgA or sIgM. This process is quite important in mucosal immunity as well as in the neutralization of potential antigens in the gut and airways (immune exclusion). pIgR and SC have important immunologic roles beyond immunoglobulin transport. It has been established that epithelial pIgR expression and function is regulated by numerous cytokines, hormones and PAMPs (Kaetzel 2005). There are reports suggesting that defective epithelial transport of IgA may play a role in mucosal airway diseases such as COPD, CRS, and asthma (Buckley 1975; Daele 1997; Chee et al. 2001; Pilette et al. 2004). More information is needed to determine the role of local B-cell responses in both inflammatory disease and protective inmmunity in the airways. It should be clear from this section that awareness that epithelial cells drive adaptive immune responses in the airways at the level of DCs, T cells and B cells has clearly risen significantly.

Enzymes and enzyme inhibitors Several epithelial-derived enzymes and their inhibitors participate in airway inflammation and are briefly mentioned here. Protease-activated receptors (PARs) are seven-transmembrane G protein-coupled receptors that are activated by protease cleavage (Sokolova & Reiser 2007). The proteolytic cleavage of the extracellular N-terminal domain of PAR by proteases present in inflammation such as tryptase, cathepsin G, elastase, and allergen proteases (i.e., dust mite proteases) exposes a new N-terminus that is the PAR’s own ligand. This new N-terminal ligand folds onto the extracellular loops of the PAR, activating the receptor. G-protein signaling then induces diverse responses. Airway epithelium expresses all four members of the PAR family, particularly PAR-1 and PAR-3. PAR-1 and PAR-2 transactivate epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), resulting in cell proliferation or migration (wound repair). PAR-2 is increased in airways of asthmatic individuals and seems to mediate allergen-induced airway hyperresponsiveness and eosinophil influx, although this is controversial. PAR-2 activation by serine proteases Der p3 and Der p9 from house-dust mite induces epithelial secretion of IL-6, IL-8, and CCL17/TARC. Matrix metalloproteinases (MMPs) depend on zinc to degrade matrix proteins (Elliott et al. 2007). There are 24 MMPs that function as collagenases, gelatinases, or stromeolysins. In homeostatic conditions they are inhibited by tissue inhibitors of MMP (TIMP) that maintain the balance of formation and destruction of the extracellular matrix. In asthma, the elevated ratio between MMP-9 (gelatinase B) and TIMP-1 correlates with airflow obstruction, although these products may originate from other cell types other than epithelial cells. MMP-9 is increased in epithelium of nasal polyps (Chen et al. 2007) and is secreted by epithelial cells

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infected with RSV or exposed to eosinophil peroxidase and MBP (Pegorier et al. 2006). Epithelial cells also express surface-bound proteinases of the ADAM family (disintegrin and metalloproteinase domain), which are membrane proteins containing a signal peptide followed by proprotein, zinc metalloprotease, disintegrin, transmembrane, and cytoplasmic regions. Most noteworthy in airway epithelial cells is ADAM17, or TNF-α-converting enzyme, which cleaves membrane-bound TNF-α and TGF-α, which in turn become soluble and mediate inflammation and mucin production, respectively. Genotype variations of ADAM33 have been associated with asthma (Holgate et al. 2006) and its airway expression increases as disease severity worsens (Foley et al. 2007), although its function is not completely understood. Besides TIMP, epithelial cells produce another enzyme inhibitor, neutral endopeptidase, which degrades endothelins (Goldie & Henry 1999), SP, and other peptides (Widdicombe 1991). Chitin is the second most abundant biopolymer in nature after cellulose (Elias et al. 2005). It is not produced in humans but is a component of the walls of fungi; the exoskeleton of crabs, shrimp and insects; the microfilarial sheath of parasitic nematodes; and the lining of the digestive tracts of many insects. When mammalians are infected or infested with these organisms, they produce acidic mammalial chitinase (AMC). Neutralization of AMC in an asthma mouse model attenuated Th2 inflammation and airway hyperresponsiveness by inhibiting chemokine production and the IL-13 pathway (Zhu et al. 2004).

Conclusion We hope that this review illustrates the diverse and essential roles played by epithelium in homeostasis as well as in host responses to the environment and in disease. The older concepts of epithelium as a relatively simple hydrated barrier have been supplanted by the recognition that epithelial cells are centrally involved in immunity and inflammation, airway physiology, and repair. Inasmuch as numerous severe diseases of the airways are characterized by abnormal or excessive activation of one or more of these processes in the epithelium, future investigations to better understand the function of epithelium will certainly pay dividends in the form of improved understanding of disease and new therapeutic strategies to alleviate airways disease.

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Airway Vascularity in Asthma John W. Wilson

Summary

Role in asthmatic responses

The airway circulation has many potential roles in asthma. The vasculature has a major influence on upper airway patency in nasal infection and atopic responses. Given its responsiveness to provocative stimuli in asthma and vasoconstrictive and antiproliferative response to corticosteroids, the bronchial circulation is likely to be a future target for novel asthma therapies.

The classical early asthmatic response to allergen is associated with the release of numerous preformed and newly generated inflammatory mediators capable of causing vasodilatation and capillary leakage. Edema formation is an important component of this response, and may be life-threatening when critical upper airway structures are involved. The nasal obstruction seen in susceptible individuals after aeroallergen exposure is a manifestation of highly reactive airway vessels responding with dilation and leakage, though difficult to detect in the stable state between episodes (Braunstahl et al. 2003). In addition, the airway vascular response accompanying viral infection may be significantly upregulated by vascular endothelial growth factor (VEGF) released by fibroblasts (De Silva et al. 2006). The cellular infiltration that is characteristic of the late asthmatic response is a complex process that involves mobilization of cells from bone marrow and vessel margination sites, adhesion to vessels in the local inflammatory environment, cell rolling and transmigration between endothelial cells (von Andrian & Mackay 2000). The cell infiltrate includes eosinophils, mast cells and activated Th2 lymphocytes (Djukanovic et al. 1990; Wilson et al. 1992; Jeffery 1999). The central role of the eosinophil in the pathogenesis of asthma is well described (Kay 2005). Regulation of eosinophil numbers in the airway may be achieved through a number of therapeutic strategies including antagonism of the trophic factor interleukin (IL)-5 (Menzies-Gow et al. 2003), the use of corticosteroids acting through multiple pathways (Djukanovic et al. 1992), as well as via effective inhibition of adhesion glycoproteins in the bronchial circulation (Wegner et al. 1990; Sedgwick et al. 2005). Vessels in asthma are therefore capable of acting through a range of mechanisms to promote the inflammatory response and airway remodeling typical of asthma (Fig. 17.1).

Introduction Increased vascularity of the bronchial wall is now recognized as a key feature of the tissue remodeling that is characteristic of asthma, (Li & Wilson 1997b; Bousquet et al. 2000). Originally described in the era of Leonardo da Vinci, the bronchial circulation has until recently been considered to be primarily a source of oxygenation and tissue nutrients for the airway wall (Mitzner & Wagner 1992; Widdicombe 1993), as well as thermoregulation and humidification of inspired gas. It is also recognized as a primary pathway for elimination of drugs. The bronchial circulation has specific functions of relevance to asthma through regulation of fluid shift into the submucosa, by attracting and controlling the migration of inflammatory cells into the airway wall, as well as being a source of microenvironmental factors for the regulation of tissue remodeling. It is reactive to multiple stimuli and is able to increase blood flow dramatically to provide effective thermoregulation in response to heat stress. Its responsiveness to chemical mediators of inflammation, capacity to participate in immunologic responses and ability to remodel in response to trophic stimuli make the airway circulatory bed both an important determinant of airway wall thickness and the acute changes in airflow characteristic of asthma.

Descriptions of airway vessels Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Modern descriptions of airway vessels have been qualitative comparisons of findings in postmortem specimens. In these

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Airway vessels

Fig. 17.1 Role of bronchial vessels in asthma.

Angiogenesis

studies, vessel congestion, edema and airway fluid were characteristic findings (Dunnill 1960; Dunnill et al. 1969). Using rubber injection-digestion preparations, the rich network of vessels deep to the surface mucosa appears as a superficial plexus, which is joined to a deeper system of capacitance vessels through connecting perforator vessels (Widdicombe 1992, 1993). This is exemplified by the typical findings seen in animal models of the bronchial circulation in proximal airways (Fig. 17.2). Flow in the bronchial circulation can increase dramatically in heat stress, with unit flow of over 7 mL/min (Baile et al. 1985), compared with 4 mL/min in cardiac muscle and kidney or 1 mL/min in resting skeletal muscle (Schindt & Thews 1987). The combination of constriction of efferent vessels and increased flow causes congestion of the submucosal plexus, evidenced by vasodilatation and vessel leakage. The net effect of these changes is thickening of the airway wall (Corfield et al. 1991a). Confined by remodeled components of the bronchial wall, including intramural scar-type collagen (Li & Wilson 1997a), swelling in the submucosa further contributes to luminal narrowing in bronchoconstriction and loss of distensibility during inspiration, typical of asthma (Wilson et al. 1993). Direct evidence for hypervascularity and angiogenesis in asthma has come from bronchial biopsy studies in volunteers using anticollagen IV to detect vessels (Li & Wilson 1997b). Earlier studies describing inflammation used nonspecific

Dilatation

Permeability

Cell infiltrate

Mediators

stains that were not vessel specific and found no increase in vessel numbers in asthma (Beasley et al. 1989). The use of factor VIII antigen to detect vessels in postmortem and resected lung specimens found that the vessel contribution to the thickness of the airway submucosa in sections was 3.3% in asthma and 0.6% in controls (Kuwano et al. 1993). This marginal increase was assessed as being unlikely to be a significant contributor to thickening of the airway wall. A later study in postmortem specimens from fatal asthma also using factor VIII Ag, in which vessel congestion was maximal, found dilatation of larger vessels and vascularity of 10.2% compared with 6.6% in controls (Carroll et al. 1997). This study also concluded that the increase in vascularity was not contributory to bronchial wall thickening and, indeed, no increase in vascularity was seen in nonfatal asthma. Possibly, vessel markers such as factor VIII Ag and CD31 are indicators of vessel activation or maturity (Tanaka et al. 2001; Kinouchi et al. 2003). The method of measurement in histological material appears to be crucial to discern differences in airway vascularity. It is now apparent that collagen IV is likely to be a component of almost all airway vessels and does not define any specific subset (Baluk et al. 2003). Virtually all bronchial vessels have collagen IV as a basement membrane (Baluk et al. 2003). In mild asthmatics, it has been established that 17% of the submucosal section is covered in vessels with a density of 740/mm2 (Li & Wilson 1997b). These are both significantly greater than the 10% of submucosal area and density of 555/mm2 seen in control subjects (Fig. 17.3).

Airway blood flow

Fig. 17.2 Bronchial capillary bed in the proximal sheep airway. A, afferent arteriole; C, capillary; V, venule. (See CD-ROM for color version.)

The total volume of blood flow through a tissue bed is determined by resistance (vessel caliber) and perfusing pressure (blood pressure). Variations in these determinants can result in marked changes in tissue blood flow. Physiologic examples include skeletal muscle during exercise and erectile tissue after stimulation. Flow in the tracheal capillaries in heatchallenged dogs may be as high as 7 mL/min per unit volume, compared with maximal flow in cardiac muscle of 4 mL/min or skeletal muscle of 1 mL/min (Baile et al. 1985, 1987). The increase in flow in specific capillary beds occurs because of diversion in blood flow from deep capacitance vessels into more superficial capillaries (Widdicombe 1992). It is likely that the combination of increased flow and vasodilatation in these vessels leads to mucosal thickening (Corfield

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Inflammatory Cells and Mediators 1994). Although bronchopulmonary anastamoses are well described (Jindal et al. 1985; Charan et al. 1986), there do not appear to be significant changes in bronchial flow after lung parenchymal injury (Behera et al. 1995); however, the addition of positive end-expiratory pressure (PEEP) does reduce flow (Behera et al. 1995). Bronchial flow is both highly tissuedependent and reactive to provocative stimuli. It provides both a mechanism to regulate humidification and heat exchange as well as acting as a means to increase the thickness of the bronchial submucosa. The homeostatic role of the bronchial circulation in maintaining thermoregulation was initially made evident from studies in animal models (Baile et al. 1985; Solway et al. 1986; Manohar 1990). Saturation with water vapor increases as inhaled gas is heated to body temperature and hence thermoregulation is associated with net water loss (Hanna & Scherer 1986). This has been the subject of controversy in the study of exercise-induced asthma, where both heat and water loss may occur (Gilbert et al. 1987; McFadden 1992). One view is that exercise-induced asthma may be almost solely a vascular phenomenon (McFadden 1990). Heat loss occurs in association with the hyperventilation of exercise and water loss may cause mucosal hyperosmolarity, leading to secondary mast cell and eosinophil activation with spasmogenic mediator release (McFadden 1990; Anderson & Daviskas 1992; McFadden & Hejal 1995; Moloney et al. 2003; Anderson 2006). Of importance is the finding that heat loss from the airway in asthma is increased (Paredi et al. 2002), although reduced in chronic obstructive pulmonary disease where vascularity is not increased (Paredi et al. 2003). It is likely that the vitally important role of the airway vasculature in maintaining thermoregulation in smaller animals is less significant in humans, but that during hyperventilation heat and water losses are substantial. Local mast cell activation produces inflammatory mediators that cause bronchoconstriction, but may be inhibited by mast cell stabilizing agents (Spooner et al. 2003). The role of the vasculature appears to be crucial in maintaining airway surface conditions, thereby avoiding destabilizing of mast cells and promoting the removal of released mediators from the local microenvironment.

(a)

(b)

Impact of increased airway vascularity on airway caliber (c) Fig. 17.3 Photomicrograph of vessels: (a) asthma. (b) control. (c) cystic fibrosis (× 400). (See CD-ROM for color version.)

et al. 1991a). In addition, blood flow in the human trachea at rest has been estimated to be 7.2 mL/min, falling to 3.3 mL/min during Valsalva maneuver and increasing to 17.1 mL/min with the Muller maneuver (Breitenbucher et al.

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Early studies in severe and fatal asthma showed evidence of tissue swelling and vasodilatation (De Burgh Daly 1935; Renault et al. 1943; Vallery-Radot et al. 1950; Dunnill 1960; Dunnill et al. 1969; Li & Wilson 1997b). The association of increased airway vascularity with asthma has led to speculation regarding a causal relationship (Baier et al. 1985; Lockhart et al. 1992; Li & Wilson 1997b). There is compelling evidence for a causal relationship based on detailed

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Poor distensibility of airway lumen on inspiration

Submucosal swelling internal to rigid zone with edema Remodeled airway Limited response to bronchodilator or antiinflammatory therapy

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Sheep and dog models do, however, differ from the human airway in having significantly denser subepithelial capillary networks (Widdicombe 1996). Additionally, airway smooth muscle constriction may itself cause increased local tissue pressure, causing impaired bronchial venous drainage and vessel dilatation. The site of airway vascular engorgement is important in relation to any potential effect on airflow obstruction. Proximal airways are larger with deep and superficial plexuses of vessels (Widdicombe 1992). Maximal flow regulation appears to occur in the postcapillary venules, resulting in proximal congestion. Additional flow limitation may occur in the arterioles during acute events (Salvato 2001). Together, this evidence demonstrates the response of the airway to fluid loading and vascular expansion, as well as providing a view of how submucosal swelling may add to factors that reduce airflow in asthma.

Fig. 17.4 Potential limitations on airway function caused by submucosal swelling. (See CD-ROM for color version.)

Vasodilatation and angiogenesis morphologic analysis of bronchial biopsies in asthma of varying severity (Vrugt et al. 2000) and extent of inflammatory response (Salvato 2001). Theoretical models taking into account the site and degree of mucosal swelling have contributed significantly to the understanding of the role of minimal increases in submucosal edema (Hogg et al. 1987). Moderate submucosal swelling in conjunction with smooth muscle shortening of 20– 40% can lead to profound airway narrowing in this model (Wiggs et al. 1990, 1992). Mucosal vascular congestion and edema may: (i) reactively contribute to airway wall thickening, (ii) impinge on the airway lumen through swelling internal to submucosal, scar-type collagen (Wilson & Li 1997) and smooth muscle hyperplasia (Ebina et al. 1993), (iii) cause loss of distensibility of the airway wall (Wilson et al. 1993), (iv) potentially reduce responsiveness to bronchodilators (Li & Wilson 1997), and (v) reduce clearance of locally active inflammatory mediators (Cabanes et al. 1989; Lockhart et al. 1992) (Fig. 17.4). In a series of studies in sheep, it has been shown that fluid loading, resulting in elevated left atrial pressure, caused airway wall thickening and luminal narrowing (Wagner & Mitzner 1990; Blosser et al. 1994). The effect of fluid loading with saline appeared to cause greater wall thickening than loading with blood, possibly because of transudation associated with lower oncotic pressure. The important observation of increased reactivity to methacholine in left ventricular failure further supports the view that bronchial vascular engorgement will alter the mechanics of airway narrowing to stimuli. It also gives credence to the concept of cardiac asthma being potentially responsive to diuretic therapy through fluid unloading. Earlier use of the sheep model has shown a significant increase in bronchial blood flow after allergen challenge in animals sensitized to Ascaris suum antigen (Long et al. 1988).

The expansion of the bronchial vasculature may occur through either neovascularization or vasodilatation, or potentially both mechanisms. Vasodilatation may occur following exposure to a wide variety of inflammatory mediators and pharmacologic agents (Table 17.1). Much evidence for the potential role of these factors is circumstantial, as they have in many cases been shown to be active in animal models, as well as having been shown to be present or relevant to human asthma. Early studies in bronchial biopsies from mild asthma suggested that larger vessels over 300 mm in diameter were increased in number (Li & Wilson 1997). Whether this was

Table 17.1 Vasodilating factors acting on airway vessels. Angiogenin (Delvigne & Rozenberg 2002; Distler et al. 2003) bFGF (Tiefenbacher & Chilian 1997) Bradykinin (Laitinen et al. 1986) Histamine (Alving 1991) Heparin (Compton et al. 2002) Hypoxia (Wagner & Mitzner 1988) Hyperosmolar stimuli (Widdicombe 1996) LTD4 (Bisgaard 1987) Methacholine (Laitinen et al. 1986) Nitric oxide (NO) (Barnes 1996) Platelet-activating factor (PAF) (Corfield et al. 1991b) PGD2 (Alving et al. 1991) Salbutamol (Laitinen et al.1987b) Substance P (Laitinen et al. 1986) Transforming growth factor (TGF)-b (Armstead et al. 1993) Tumor necrosis factor (TNF)-a (Patel et al. 2002) Tryptase (Dumitrascu 1996) VEGF, via NO and PGI2 (He et al. 1999) Vasoactive intestinal polypeptide (VIP) (Laitinen et al. 1986)

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due to stretch vasodilatation or nonsprouting proliferation of endothelial cells was unclear. Intrinsically, bronchial endothelial cells have little replicative activity. Currently, evidence suggests that angioblasts and hemopoietic progenitors may be derived from a common lineage, sharing surface markers including CD31, CD34, and a receptor for vascular endothelial growth factor (VEGFR-2) (Shalaby et al. 1995). Endothelial progenitors may be either local in origin, or migrate to sites of injury from the circulation. This process is dependent on attraction to local sites (chemotactic) and specific adhesion to activated vessel walls (von Andrian & Mackay 2000). Factors likely to be responsible include VEGF, fibroblast growth factor (FGF)-2, angiopoietin, stromal cell-derived factor-1, and insulin-like growth factor (IGF) (Hattori et al. 2001; Moore et al. 2001). The active production of angiogenic factors by local endothelial cells and their precursors appears to be a potent stimulus for remodeling of airway vessels and accompanies intense vascular remodeling such as that seen in cystic fibrosis (Solic et al. 2005). In the latter study, the role of storage sites of IL-8 was emphasized, when bound to glycosaminoglycan in airway cells. The process of angiogenesis proceeds along a sequence of regulated steps (Conway et al. 2006). In sprouting angiogenesis, breakdown of the extracellular matrix (ECM) occurs initially, followed by elaboration of chemotactic factors and extracellular proliferation. Lumen formation is accompanied by collagen deposition and vessel maturation. Matrix metalloproteinases (MMPs) perform multiple roles in this environment including the degradation of matrix (Visse & Nagase 2003; Chung et al. 2004; Conant et al. 2004; Jozic et al. 2005; Nagase et al. 2006) and release of growth factors from known ECM storage sites (Visse & Nagase 2003; Solic et al. 2005; Nagase et al. 2006). The leakage of plasma proteins then contributes to the migration and differentiation of angioblasts in extravascular sites. This process is tightly regulated by local tissue inhibitors of metalloproteinases (TIMPs), that act to counterregulate ECM breakdown as well as performing multiple immunologic roles (Visse & Nagase 2003).

Table 17.2 Factors and states known to stimulate angiogenesis. aFGF (Rogala et al. 2001) avb3 integrin (Drake et al. 1995) Angiogenins (Hoshino et al. 2001b) Angiopoietin-1 (Koblizek et al. 1998) bFGF (Folkman et al. 1988) Calcitonin gene-related peptide (CGRP) (Alving 1991) ELR + CXC chemokines IL-8 (Koch et al. 1992; Norrby 1997) ENA-78 (Gillitzer et al. 1996) GCP-2 (Addison et al. 2000; Keane et al. 2002) ECM (Ingber et al. 1987) ENA-78 (Donninger et al. 2003) Ephrin-B1 (Sawai et al. 2003) Ephrin-B2 (Murohara et al. 1998) Estrogens (Johns et al. 1996) GRO-a (Shibata et al. 2003) Hepatocyte growth factor (HGF) (Bevan et al. 2004) Histamine (Sorbo et al. 1994; Norrby 1997) Hypoxia and HGF-1 (Bevan et al. 2004) IGF-1 (Warburton et al. 2000) IL-1 (Li et al. 1995) IL-4 (Fukushi et al. 2000) IL-13 (Fukushi et al. 2000) IL-6 (Huang et al. 2004) Lipopolysaccharide (Mattsby Baltzer et al. 1994) LTC4 (Kanayasu et al. 1989) MMP (Haas et al. 1998; Foda et al. 1999) Neurokinin (NK)A (Martling et al. 1990) NO (Morbidelli et al. 1996) PAF (Murohara et al. 1998; Russo et al. 2003) Prostaglandins (Ruegg et al. 2004) Pulmonary arterial occlusion (Mitzner et al. 2000) Stromal cell-derived factor-1 (Hoshino et al. 2003) Substance P (McDonald et al. 1996) TGF-a (Norrby 1997) TGF-b (Roberts et al. 1986) TNF-a (Norrby 1997) Vascular cell adhesion molecule (VCAM)-1 (Fukushi et al. 2000) VEGF (Leung et al. 1989; Jakeman et al. 1993; Hoshino et al. 2001a) VIP (Laitinen et al. 1987b)

Drivers of angiogenesis The phenotypic expression of angiogenic factors and their receptors is counterregulated to allow appropriate tissue homeostasis without hypervascularity (Hoshino et al. 2001b; Favre et al. 2003; Russo et al. 2003; Thurston et al. 2005). However, the genetic polymorphisms in angiogenic factors or their receptors may also contribute to the wide variation in response seen in human disease (Thurston et al. 1998). A range of growth factors and local conditions play defined roles in inflammation and angiogenesis through effects on local cell activity and the endothelium (Ribatti et al. 2004; Khurana et al. 2005) (Table 17.2). Neovascularization and the remodeling of existing vessels are likely to be induced by

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multiple growth factors, including VEGF (Hoshino et al. 2001a), angiopoietin 1 (McDonald 2001), and stromal cell-derived factor-1 (Hoshino et al. 2003). Many counterregulatory factors inhibit angiogenesis. Amongst these, corticosteroids, angiostatins (O’Reilly et al. 1997), and TIMPs (Johnson et al. 1994) are significant in asthma (Table 17.3). VEGF exists in a number of well characterized isoforms, including vascular permeability factor (VPF) (Tammela et al. 2005; Voelkel et al. 2006). Evidence of increased levels of VEGF has been found in the bronchial submucosa in asthma (Hoshino et al. 2001a), and sputum levels have been found to correlate with disease activity (Abdel-Rahman et al. 2006). Airway vascularity was found to be dependent on gene

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Table 17.3 Antiangiogenic factors relevant to asthma. Angiopoietin-2 (Maisonpierre et al. 1997; Holash et al. 1999; Kim et al. 2000) Angiostatin (O’Reilly et al. 1994) Anti-VCAM-1 (Fukushi et al. 2000) Anti-IL-8 antisera (Smith et al. 1994) Cancer therapy (Gourley & Williamson 2000) Corticosteroids (Smink et al. 2003) Endostatin (O’Reilly et al. 1997) IL-2 with histamine (Johansson et al. 2000) IL-12 (Voest et al. 1995; Yao et al. 2000) Immunization to CM101 (Fu et al. 2001) Monoclonal antibody blockade of VEGFR2 (Witte et al. 1998) MMP inhibition (marimistat) (Wada et al. 2003) Platelet factor 4 inhibits VEGF (Gengrinovitch et al. 1995) Prolactin (anti-VEGF) (D’Angelo et al. 1999) Suramin (Danesi et al. 1993) TIMP-1 (Johnson et al. 1994) TSP-1 (DiPietro et al. 1994) Thalidomide (Folkman 2001) Thrombospondin (Iruela-Arispe & Dvorak 1997; Tokunaga et al. 1999) Vasostatin (Yao et al. 2000) VEGFR-2 antibodies (Menrad et al. 1997) VEGFR2 blockade using VEGF-165 peptides (Siemeister et al. 1998; Piossek et al. 1999) TSP, thrombospondin.

expression of VEGF and its receptors flt-1 (VEGFR-1) and flk-1 (VEGFR-2) (Hoshino et al. 2001a). Given the potentially important role of VEGF in determining the extent of airway vascularity, identification of cellular origins becomes pivotal to understanding the regulation of production of this factor. A range of cells have been identified as sources in the airway, including smooth muscle (Alagappan et al. 2007), epithelium (Verhaeghe et al. 2007), mast cells (Chetta et al. 2005), macrophages, eosinophils, and CD34+ cells (Hoshino et al. 2001a) (Fig. 17.5).

Epithelium Mast cell

VEGF Poly

Eos

Vessel

EC Mf

Smooth muscle Fig. 17.5 Local sources of VEGF in asthma. EC, endothelial cell, Eos, eosinophil, Mf, macrophage; poly, polymorphonuclear leukocyte. (See CD-ROM for color version.)

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VEGF, through the isoform VPF, increases vascular permeability and vessel fenestration (Roberts & Palade 1997); however, angiopoietin-1 has the opposite effect (McDonald 2001). Opposing functions of VEGF and angiopoietin-1 may signify complementary and coordinated roles in regulation of the bronchial vasculature. Fundamentally, regulation of vascular permeability by VEGF/VPF and angiopoietin-1 may be their most significant action in asthma, as nongenomic actions are more rapid than their more protracted effects on angiogenesis and vessel remodeling. The relative contributions of growth factors and inflammatory, vasoactive mediators remain undetermined. Identification of the inhibitory effect of angiopoietin and other factors on vascular leakiness raises the potential for new strategies for reducing airway obstruction in asthma and chronic bronchitis (McDonald 2001).

Bronchial vessels and the immune response Local microenvironmental factors are crucial in determining both susceptibility to vascular remodeling and the extent of angiogenesis. Major exogenous triggers of airway inflammation in asthma include viruses and inhaled aeroallergens, both of which are known to be associated with the production of angiogenic factors (Ribatti et al. 2004; Ghildyal et al. 2005). These stimuli elicit reciprocal immune responses, through elaboration of Th1 and Th2 cytokines. While there may be a genetic predisposition to bronchial hyperreactivity (Van Eerdewegh et al. 2002), the link to exaggerated Th2 responses characteristic of atopic asthma (Robinson et al. 1992) is currently less well defined. A link between airway inflammation and vessel remodeling may be understood by examining nonvascular actions of VEGF. Vascular endothelial growth factor levels are associated with increased severity of clinical asthma (Lee & Lee 2001), and may enhance Th2 lung inflammation murine asthma models (Lee et al. 2004). Together with the finding of VEGF production by mast cells (Ribatti et al. 2004), it is clear that there is a role in addition to its action on vessels in the effector arm of the early and late asthmatic responses. Regulation of the inflammatory response in asthma occurs through intermediary transcription factors such as NF-κB, that also signal gene transcription for angiogenic factors including IL-8, ENA-78, and GRO-α (Shibata et al. 2003). Indeed, endothelial cell cytokine production of angiogenic factors such as IL-8 is mediated by NF-κB (Tanner 2004). Treatment of asthma with inhaled corticosteroids may suppress NF-κB, and, coincidentally, local inflammation with angiogenesis (Orsida et al. 1999, 2001; Wilson et al. 2001). Cell infiltration, angiogenesis, and vessel leakage may also occur through the activation of structural cells, including fibroblasts, by viral infection that induces production of IL-8 and ENA-78 (Donninger et al. 2003; Ghildyal et al. 2005). Nasal obstruction during viral upper respiratory tract infections indicates the

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significance of this mechanism to cause luminal occlusion, independent of atopic status. With the stimuli of either infection or atopic inflammation, increased vascular surface area and endothelial fenestrations lead to increased plasma protein leakage. As well as elevated baseline leakage, remodeled vessels in the submucosa are abnormally sensitive to substance P, but not to plateletactivating factor or serotonin, implying that at least infection is associated with a selective upregulation of NK1 receptors on the vasculature (McDonald 2001). Inflammation and angiogenesis are codependent phenomena (McColley et al. 2000). The preparation of vessels with upregulated adhesion glycoprotein expression in an appropriate field, allows angiogenesis to arise from resident tissue progenitors, or alternatively from migrating angioblasts with endothelial lineage capacity.

Angioblasts and stem cells It has been known for some time that hemopoietic progenitors can be mobilized from bone marrow by inflammatory responses (Metcalf & Wilson 1976). Stem cells from an adult are generally classified as hemopoietic stem cells (HSCs) or mesenchymal stem cells based on their differentiation capacity. There are no specific markers for stem cells in the postnatal period. Nonetheless, HSCs used in bone marrow transplantation for hematologic malignancies and bone marrow failure are distinguishable by the presence of CD34 and CD45 surface markers (Reyes et al. 2002; Dao et al. 2003). These cells have been shown to include a subpopulation of progenitors that can differentiate from angioblasts into endothelial cells (Reyes et al. 2002; Young 2004), migrate to sites of ischemic injury in the heart, induce angiogenesis, and lead to adaptive tissue remodeling with improved cardiac function (Wollert et al. 2004). Mesenchymal stem cells may also act in a similar capacity (Vulliet et al. 2004); however, these CD34– cells may be preangioblasts capable of bidirectional CD34 expression (Dao et al. 2003). There are at least two lineages of cells with potential to contribute to angiogenesis, detectable in the peripheral circulation. Other components of airway remodeling have been shown to arise from mobilized, homing progenitors (Grove et al. 2004; Albera et al. 2005). Hemopoiesis and progenitor cell production in the bone marrow is stimulated by circulating factors elaborated as a byproduct of airway inflammation (Denburg 1998; Denburg et al. 2000). These mobilized cells may play an important role in determining chronic inflammatory responses in the airway. Typically this may be in infection, either as suppressive regulators of the immune response (Le Blanc et al. 2004), or as initiators of remodeling of different tissue components, including epithelium (Delplanque et al. 2000), collagen (Hashimoto et al. 2004), blood vessels (Kamihata et al. 2002), and smooth muscle (Yeh et al. 2003). It would seem likely

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Angiogenesis Growth factors

CD34+ EC progenitor migration

EC proliferation

Homing to vessel

Vessel expansion/ sprouting

Angioblast proliferation

Angiogenesis

Angiogenesis

Fig. 17.6 Pathways to bronchial angiogenesis. (See CD-ROM for color version.)

that homing of angioblast progenitors to “prepared fields” is a mechanism of angiogenesis and a component of airway wall remodeling. Augmented mobilization of angioblasts through the use of granulocyte–macrophage colony-stimulating factor may improve graft function after pancreatic islet cell transplantation (Contreras et al. 2003), possibly because of the inflammatory microenvironment associated with the transplant, resulting in enhanced homing of angioblasts. In the absence of a “prepared field,” there is little possibility of homing and transmigration of circulating progenitors (Davies et al. 2002). Abrogation of homing through the use of integrin antagonists has been found to reduce airway hypervascularity and airway remodeling (Simper et al. 2002), and airway hyperresponsiveness in a primate model (Wegner et al. 1990). The relative contribution of migratory versus endogenous endothelial cell progenitors to angiogenesis is unknown at present (Fig. 17.6). The resolution of this issue may be key to novel strategies aimed at addressing remodeling associated with chronic inflammatory diseases.

Vessel leakage The morphologic description of edema in severe and fatal asthma is consistent with the role of the bronchial vasculature and the known actions of angiogenic factors, inflammatory mediators, and their counterregulators (Table 17.4). Observations in fatal asthma identified tissue expansion secondary to classical vasodilatation and plasma exudation (Dunnill 1960; Dunnill et al. 1969). Recognition of the importance of plasma extravasation has led to interpretations of the role of current asthma therapies in limiting airway edema based on animal studies (Barnes et al. 1990; Chung et al. 1990; Boschetto et al. 1991). Accurate quantification of plasma leakage in human asthma has been difficult to achieve; however, indirect assessment using plasma protein detection methods (Persson et al. 1986) has enabled quantitative assessments to be performed (Chu et al. 2001; Wilson & Wilson 2001). The action of corticosteroid therapy to limit extravasation has been inferred from levels of α2-macroglobulin in bronchoalveolar

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Table 17.4 Factors known to regulate microvascular leakage. Bradykinin (Laitinen et al. 1987b; Murohara et al. 1998) CGRP (Madeddu et al. 2001) Endotoxin (Grimminger et al. 1997) ET-1 (Eibl et al. 2002) Formoterol (Baluk & McDonald 1994) Histamine (Murohara et al. 1998) LTB4 (Bannenberg et al. 2004) LTC4 (Bureau et al. 1997) LTE4 (Joris et al. 1987) Mycoplasma (Chu et al. 2001) NKA (Baluk et al. 1999) NO (Murohara et al. 1998) O 2– (Dauber et al. 1991) PAF (Murohara et al. 1998) (Bjork & Smedegard 1983) PGI2 (Murohara et al. 1998) Salmeterol (Bolton et al. 1997) Substance P (Baluk et al. 1997; Van Rensen et al. 2002) Terbutaline (Svensjo et al. 1977) TNF-a (Koizumi et al. 2003) VEGF (Esser et al. 1998; Hippenstiel et al. 1998) VPF (Connolly et al. 1989)

lavage fluid from asthmatics (Nocker et al. 1999), and estimates have been made in noninvasive studies of induced sputum (Van Rensen et al. 2002; Kanazawa et al. 2007). Plasma leakage from vessels may be inhibited by βadrenergic agonists (Tokuyama et al. 1991; Bolton et al. 1997), corticosteroids (Bowden et al. 1994), and angiopoietins through regulation of the role of VEGF/VPF (Gamble et al. 2000; Koh et al. 2002; Makinde et al. 2006). Vessel dilatation cannot be easily separated from microvascular leakage, as they frequently increase under similar conditions. Leakage is an important component of the vascular response that enhances thickening of the airway wall.

Pharmacologic responsiveness Early information on the response of bronchial vessels to pharmacologic agents is available from canine models (Laitinen et al. 1986, 1987a). The β2-adrenergic agonists including salbutamol (Laitinen et al. 1987b) dilate vessels, while both corticosteroids (fluticasone propionate) and leukotriene receptor antagonists (monteleukast) are vasoconstrictors in the airway (Mendes et al. 2004). Using a soluble, inert gas uptake method, Wanner and colleagues have shown relative potencies of inhaled corticosteroids to cause bronchial vasoconstriction and found budesonide to have the greatest relative effect compared with beclomethasone dipropionate and fluticasone propionate in asthma (Mendes et al. 2003). The actions of known asthma treatments to modify airway blood flow

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are relatively minor compared with their other known properties as antiinflammatory and bronchodilating agents.

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Airway Vascularity in Asthma

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Fibroblasts and the Extracellular Matrix Lynne A. Murray, William G. Glass, Anuk M. Das and Geoffrey J. Laurent

Summary Chronic respiratory diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and asthma, collectively affecting more than 300 million people worldwide, place a huge burden on our health systems. A feature of these diseases, which currently remains poorly treated, is the destruction of normal airway and parenchymal structures and replacement with scar tissue, or fibrosis. We propose that the aforementioned changes not amenable to current therapies are integral to the pathophysiology of these diseases. The nature of this fibrotic response, characterized by proliferation of fibroblasts and myofibroblasts, is being challenged recently as it becomes clear that these cells may be derived from blood-borne precursors, or fibrocytes, as well as from transdifferentiation of resident cells, including epithelial cells. This chapter proposes that remodeling and fibrosis are central to normal host defense and are driven by multiple pathways including the coagulation and inflammation cascade. In disease settings, these pathways may proceed unabated, resulting in aberrant tissue function. For example, the pathways that give rise to key growth factors and cytokines including transforming growth factor (TGF)-β, thrombin and interleukin (IL)-13 are discussed. Furthermore, we describe the activation of resident cells including alveolar macrophages and epithelial cells as well as the processes that cause the cells to adopt a pathogenic phenotype. Finally, we review the opportunities arising out of research for better therapeutic intervention strategies that will either halt or potentially reverse fibrosis.

Introduction Fibrosis as a component of asthma Fibrosis with excessive accumulation of extracellular matrix (ECM) represents an element of airway remodeling and as such is part of the wider structural changes to lung archiAllergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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tecture. The features of airway remodeling associated with asthma that contribute directly to an increase in airway wall thickness are subepithelial fibrosis, angiogenesis, and smooth muscle and mucous gland hypertrophy (reviewed by Cohn et al. 2004). The consequences of this airway wall remodeling include airway narrowing leading to lung function impairment (Paganin et al. 1996; Chetta et al. 1997; Bousquet et al. 2000; Cohn et al. 2004). Furthermore, inflammation and increased wall thickness is more pronounced in fatal asthma compared with mild/moderate asthma, suggesting an association between the degree of remodeling and disease severity (Carroll et al. 1993, 2002). Fibrosis is defined as the excessive accumulation of ECM proteins. Although the fibrosis observed in many pulmonary diseases may be an important part of host defense akin to collagen deposition during acute wound healing, we propose that the increased deposition of ECM proteins leads to pathophysiologic symptoms of chronic asthma, particularly those seen in chronic severe asthma. Significant differences in collagen type and deposition have been observed in the lung of asthma subjects. Types I and III are the predominant collagen types in the normal lung, with type I collagen the most abundant (Laurent 1986). The subepithelial tissues of asthmatics have been shown to contain significantly higher levels of collagen types I and III, contributing to reduced elasticity and compliance of the tissue (Wilson & Li 1997; Hoshino et al. 1998; Ward et al. 2001). Increased levels of collagen type V, fibronectin, and tenascin have also been reported to occur in the airways of chronic asthmatics (Laitinen et al. 1997; Hoshino et al. 1998; Cohn et al. 2002). In another study, although no changes in the extent of collagen I was observed, asthma subjects exhibited an abundance of collagens III and V and fibronectin in the thickened basement membrane compared with nonasthmatic subjects (Roche et al. 1989). Finally, although the total amount of elastic fiber appears to be unchanged in asthmatics (Godfrey et al. 1995), subepithelial elastin fibers of the airways are fragmented and fibers in the deeper layer are often patchy, tangled, and thickened indicating impaired ECM formation (Bousquet et al. 1996). These data suggest that the precise ratios of ECM components may not be identical

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between all asthma subgroups. Further, diversity is observed between patient groups depending on which area of the lung is evaluated.

Fibrosis and altered ECM turnover is seen in diverse lung disease pathologies Fibrosis associated with exuberant matrix turnover is also a feature in interstitial lung disease (ILD). The most common ILD is idiopathic pulmonary fibrosis (IPF), with an incidence of 3–29 per 100 000 (Coultas et al. 1994). IPF is a fibroproliferative disease characterized by excessive collagen accumulation in the lung, which ultimately leads to impaired gas exchange. Characteristic computed tomography (CT) findings of IPF include the presence of patchy heterogeneous lung fibrosis, which as the disease progresses often results in alveolar collapse, bronchiolectasis, and honeycombing (Dacic & Yousem 2003). As the underlying cause of IPF is unknown, many hypotheses have arisen as to the mechanisms of disease exacerbation and perpetuation. There is current debate as to whether IPF is due to repeated injury to the epithelium driving the fibrotic response. Another hypothesis suggests that chronic inflammation drives IPF. Both are likely important and further research is needed to dissect which of multiple pathways predominates in individual patients. Airway wall remodeling, characterized by emphysema (alveolar wall breakdown and airspace enlargement) and small airways fibrosis, is seen in the small airways of patients with chronic obstructive pulmonary disease (COPD). This raises an interesting phenomenon where excessive ECM breakdown and ECM deposition are occurring in adjacent sites in the same disease state. It is thought that the main pathologic alterations in the lungs of patients with COPD are due to impaired protease–antiprotease balance, resulting in dysregulated matrix degradation and generation (Shapiro 2003; Shapiro & Ingenito 2005). The extent of peribronchial fibrosis (which, like asthma, also occurs in the lamina reticularis of the basement membrane) increases with disease severity (Hogg et al. 2004; Chung 2005) and increased collagen and elastin deposition have also been observed in smokers with emphysema (Lang et al. 1994). COPD is hypothesized to be due to exposure to particulate matter such as occupational exposure or cigarette smoke. The increase in ECM found in the lungs of these patients may be due to pronounced activation of cells by the foreign particles, resulting in downstream inflammatory events mediating disease pathology. However, as has been hypothesized for IPF, the remodeling events may be an aberrant homeostatic process serving to protect the lung. This chapter explores the mechanisms involved in the generation and maintenance of the asthma-specific subepithelial fibrosis in the lamina reticularis of the basement membrane. The diversity in ECM components and how, when organized, their ultimate structure is central to tissue function

Fibroblasts and the Extracellular Matrix

is described. The main area of discussion with regard to the ECM focuses on the alteration and potential role the ECM may play during airway disease. The mechanisms by which the ECM signals to cells to regulate cellular function are also described. Further, the numerous cell types that directly and indirectly contribute to ECM generation, as well as how these cells often have altered phenotype and function during fibrotic disease states, is discussed. Other pulmonary diseases associated with fibrotic remodeling such as IPF and COPD are highlighted to better elucidate underlying mechanisms. Although the fibrosis associated with these diseases occur in distinct anatomic sites, we propose that common final pathways are likely in these apparently diverse diseases.

ECM and the basement membrane The ECM comprises a diverse group of proteins and glycoproteins that provide structural integrity and mechanical support for tissues and provides a meshwork for cell adhesion and motility. Cells are tethered to components of the ECM through cell–ECM adhesions mediated by a variety of cellsurface receptors such as integrins. The ECM can also bind growth factors and cytokines. This provides a readily available reservoir of mediators that may be released by either enzymatic activity or changes in the chemistry of the environment. Due to the diversity of the ECM components, this section initially focuses on each major component individually: collagen, elastin, proteoglycans (and glycosaminoglycans), laminin, and tenascin. It then describes how specific components of the ECM come together to mediate tissue integrity and function in the basement membrane.

Components of the ECM Collagen There are currently 28 types of collagen and they can be divided into fibrillar and nonfibrillar collagen subtypes. Fibrillar collagen, such as types I, II and III, forms banded fibrils and is the main constituent of connective tissue, cartilage, and bone. The basic unit of collagen is the triple helix, which consists of three polypeptide chains containing Gly-X-Y repeated amino acid sequences. Approximately every third X is proline and every third Y is hydroxyproline. Given the high concentration of hydroxyproline, this analyte is often used to quantitate the extent of collagen deposition in tissues. Collagen generation, with respect to amount and relative quantities of the different types, is site specific and is regulated at both the transcriptional and translational level, as well as being subject to posttranslational control. Ultimately, the physical properties of collagen depend on final structural assembly (Khoshnoodi et al. 2006). For example, tissues that are required to withstand high tensile strength such as tendons have a higher fibrillar to nonfibrillar collagen ratio.

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Inflammatory Cells and Mediators

Collagen forms a scaffold for supporting cells and connects to cells directly through interactions with collagen receptors such as integrins α1β1 and α2β1 or the discoidin domain receptors, and indirectly via fibronectin–α5β1 and αVβ3 integrin bonds (Vogel et al. 1997; Vogel 1999). Other functions of collagen, besides providing tissue integrity, include activating platelets at sites of wound healing, thus triggering the formation of a hemostatic plug (Sixma & Wester 1977; Laurent 1986; Stassen et al. 2004). Furthermore, collagen can inhibit cell apoptosis in adherent cells thus promoting cell survival. This may be potentially either deleterious if the cell is contributing to aberrant pathogenesis such as fibrosis or be protective if the cell is maintaining homeostasis (Laurent 1986; Bijian et al. 2004; Sturm et al. 2004).

Elastin Elastic fibers are made up of a core of insoluble elastin and microfibrils predominantly consisting of fibrillin. Elastin fibers provide the required flexibility of lung tissue (Mithieux & Weiss 2005) and are essential for lung development, as elastin gene-deficient mice fail to develop adequately branched terminal bronchi and die 3 days postnatally (Dietz & Mecham 2000; Wendel et al. 2000). Fibrillin I-deficient mice also have severe airway malformation. In contrast, tight-skin mice, which have a duplication in the fibrillin I gene resulting in overexpression of fibrillin I, develop emphysema (Foster & Curtiss 1990). This suggests that both the elastin core and microfibril components of elastic fibers are required for normal lung development. Within the lung, elastin is produced by myofibroblasts and fibroblasts, endothelial cells, and smooth muscle cells (Foster & Curtiss 1990; Starcher 2000). Mechanical stress has been shown to upregulate elastin expression. Furthermore, mediators such as TGF-β1 and tumor necrosis factor (TNF)-α also increase elastin. Interestingly, although elastin expression declines after lung development, aberrant disorganized elastin production has been observed during IPF (Laurent & Tetley 1984).

Proteoglycans Proteoglycans are hydrophilic molecules consisting of a core protein with at least one of a diverse group of covalently attached glycosaminoglycan (GAG) chains that confer hydrophobicity by binding to water and cations (Hardingham & Fosang 1992). More than 20 genetically different species of core proteins have been identified (reviewed by Iozzo & Murdoch 1996). Proteoglycans are also found in cell membranes and thus may exert pleiotropic biological activities (Yanagishita & Hascall 1992; Iozzo & Murdoch 1996). The proteoglycans versican, biglycan, and decorin predominantly localize to the subepithelial layer, whereas perlecan is associated with the basal lamina (Huang et al. 1999; Johnson et al. 2004; de Medeiros Matsushita et al. 2005). Versican levels have been shown to be increased in a number of lung pathologies associated with fibrosis including

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IPF (Bensadoun et al. 1996). Interestingly, altered mass and composition of proteoglycans have been observed in the asthmatic lung (Huang et al. 1999; de Medeiros Matsushita et al. 2005). Patients with severe asthma also have increased versican and biglycan, which has been shown to correlate with disease severity (de Medeiros Matsushita et al. 2005). The GAGs most commonly found in the lung include heparan sulfate, hyaluronan (HA), chondroitin/dermatan sulfate, and heparin. GAGs can also act as docking stations for growth factors and cytokines, allowing retention of mediators locally and protection from proteolytic degradation and/or cleavage (Li et al. 2002). In disease models, alterations in GAG constituents have been observed, with the rank order of predominance being shown to shift from heparan sulfate to chondroitin/dermatan sulfate (Cantor et al. 1983). One of the GAGs widely distributed in the body is HA, which binds to various molecules including other matrix proteins such as versican (Laurent & Fraser 1986; LeBaron et al. 1992). Relatively low levels of HA are distributed ubiquitously in most tissues, although the amount of HA increases during wound healing responses (Hamann et al. 1995; Laurent et al. 1996). HA has been shown to promote cell migration and proliferation through binding to the cell-surface receptor CD44, which is expressed on many cell types including hematopoietic cells (Jalkanen 1987; Miyake & Kincade 1990; Hamann et al. 1995; Laurent et al. 1996; Teder et al. 2002). Increased levels of HA have been seen in the bronchoalveolar lavage (BAL) fluid of asthmatics and the level of HA correlates with disease severity (Bousquet et al. 1991).

Laminin Laminins are the predominant noncollagenous component of the basal lamina (Aumailley & Smyth 1998). They contribute to cell attachment, differentiation and cell motility, and have anti-apoptotic effects on cells through interactions with other ECM components such as collagen type IV (Wondimu et al. 2004). Furthermore, laminin expressed on epithelial cells has recently been shown to be essential in lung development and in epithelial cell differentiation, a process that may contribute to lung fibrosis and which is discussed further in the section on epithelial cells (Nguyen et al. 2005).

Tenascin Tenascin is another ECM glycoprotein which is upregulated during morphogenesis and tissue repair (Mackie et al. 1988; Sechler et al. 1998). Increased levels of tenascin have been observed in the bronchial lamina reticularis in various asthma subgroups such as patients with chronic asthma or seasonal asthma, in comparison with control patients (Laitinen et al. 1997). No correlation was reported between the number of eosinophils or lymphocytes and the level of tenascin expression, indicating that tenascin levels may correlate with the extent of tissue remodeling as opposed to inflammatory events or exacerbations (Laitinen et al. 1997). The potential

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contribution of eosinophils and lymphocytes to fibrosis is discussed later in this chapter (see section Indirect mediators of fibrosis).

The basement membrane During airway remodeling in asthma, it is collagen accumulation in the lamina reticularis layer of the basement membrane that leads to the characteristic subepithelial fibrosis associated with the disease (Roche et al. 1989; Brewster et al. 1990; Evans et al. 1993). The structure of the basement membrane is maintained through interactions with ECM components, for example homeotypic cross-linking between collagen types or proteoglycans. Specific cell–matrix interactions can regulate cell functions such as proliferation, differentiation, and survival and these are discussed further in this chapter. The true basement-membrane zone appears as three component layers: the lamina lucida, lamina densa, and lamina reticularis (Fig. 18.1). Together, the lamina lucida and lamina densa make up the basal lamina. The lamina lucida functions as the region of attachment between the epithelium and lamina densa and contains cell adhesion molecules and anchoring filaments of laminin. In contrast, the lamina densa is predominantly composed of collagen type IV, laminin, entactin, and heparan sulfate proteoglycans. On the ECM side of the lamina densa, anchoring fibrils of type VII collagen loop through strands of collagen in the lamina reticularis (Merker 1994). The lamina reticularis is variable in distribution, thickness, and composition. It forms a complex structure within the tracheal basement-membrane zone, under the respiratory epithelium (Evans et al. 2000). The lamina reticularis is especially pronounced under the respiratory epithelium of large conducting airways, where it may be several micrometers thick and becomes thicker as the airway increases in diameter. Immunohistochemical studies have shown that the collagen fibrils of the lamina reticularis consist of types I, III, V, VI, and VII collagen. Within and around these collagen fibrils are fibronectin, tenascin, and proteoglycans (Merker 1994).

Fibroblasts and the Extracellular Matrix

ECM-producing cells Most cells in the body have the ability to produce matrix proteins (Fig. 18.2). The major ECM-producing cells are fibroblasts and myofibroblasts, although smooth muscle cells also generate large quantities of ECM (Dabbagh et al. 1998). The derivation of both fibroblasts and myofibroblasts is currently under examination and reports indicate that there may be multiple pathways through which fibroblasts and myofibroblasts are derived. For example, bone marrow-derived circulating collagen I-positive cells, or fibrocytes, have been shown to traffic to sites of active lung fibrosis (Quan et al. 2006). These cells have been hypothesized to differentiate into fibroblasts or myofibroblasts (Quan et al. 2004). In addition, studies in IPF patients have also highlighted the airway epithelium as being a source of fibroblast-like and myofibroblastlike cells during remodeling (Willis et al. 2005). The magnitude of these pathways during fibrosis is currently uncertain. These cell differentiation pathways are discussed below.

Fibroblasts Fibroblasts produce numerous matrix proteins including collagen, proteoglycans, and glycoproteins (Wahl et al. 1978; Hibbs et al. 1983; Derdak et al. 1992; Sheppard & Harrison 1992). In the lung they are associated with the ECM in the subepithelial layer, conducting airways, and also in the interstitial space of the lung parenchyma. They also interact with airway epithelial cells; this interaction may be important in disease settings and is discussed later in this chapter. Fibroblasts play a key role in ECM homeostasis. For example, they generate matrix metalloproteinases (MMPs), which break down collagen thereby maintaining homeostasis of the subepithelial basement membrane. However, the pleiotropic functions of fibroblasts, including the production of many cytokines and growth factors, may contribute to the perpetuation and maintenance of the aberrant fibrotic environment (Bergeron et al. 2003).

Lamina lucida

Basal lamina

Lamina densa

Basement membrane zone

Lamina reticularis Fig. 18.1 Schematic of the anatomic distribution within the basement membrane zone. The basement membrane consists of three layers: the lamina reticularis, lamina densa, and lamina lucida. Each layer contains multiple different extracellular matrix components that provide tissue architecture and strength, as well as influencing cellular function. (See CD-ROM for color version.)

ECM components of the lamina densa: Collagen type IV, heparan sulfate, laminin, entactin Collagen type VII ECM components of the lamina reticularis: Collagen types I, III, V, VI, VII; Fibronectin; Tenascin; Proteogyclans Epithelial layer

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Inflammatory Cells and Mediators Fibrocyte Recruitment and Accumulation Chemokines (e.g. MCP-1/CCL2, SDF1a/CXCL12)

Fibrocyte Differentiation Growth factors (e.g. TGF-b1) Fibroblast Activation Proliferation and Survival

Myofibroblast Activation and Survival Differentiation (e.g. TGF-b1, thrombin, IL-13)

Epithelial to Mesenchymal Transition Growth factors (e.g. TGF-b1)

Epithelial Cell Injury

Fibroblasts are activated by numerous signals: mechanical forces imposed during bronchoconstriction, matrix interactions, hypoxia, growth factors such as TGF-β1 and platelet-derived growth factor (PDGF), and cytokines including the type 2 cytokines IL-4 and IL-13 (Desmouliere et al. 1993; Doucet et al. 1998). Fibroblasts are capable of generating chemokines, including macrophage chemotactic factor (MCP)-1/CCL2, RANTES/CCL5, and eotaxin/CCL11 (Hogaboam et al. 1999; Teran et al. 1999; Wenzel et al. 2002; Chibana et al. 2003). Indeed, the Th2-associated cytokines IL-4 or IL-13 synergize with TGF-β to further enhance the secretion of proinflammatory mediators such as eotaxin/CCL11 from human airway fibroblasts in vitro (Doucet et al. 1998; Wenzel et al. 2002), demonstrating the interactions between the proinflammatory and profibrotic processes in the lung. Further, lung eosinophilia has been demonstrated to correlate with the extent of subepithelial fibrosis (Wenzel et al. 1999), again emphasizing the potential links between inflammation and fibrosis. The aberrant fibrotic response associated with diseases may be due to phenotypic alterations in fibroblasts (Scaffidi et al. 2002; Moodley et al. 2003). These studies have been performed with fibroblasts isolated from the lungs of patients with IPF. IPF-derived fibroblasts are more resistant to apoptosis, which is partly mediated through a TGF-β-independent pathway (Scaffidi et al. 2002). These cells also have low levels of cyclooxygenase (COX)-2 activity and an inability to upregulate COX-2 that may impact the proliferative capacity of fibroblasts (Moore et al. 2003). Further COX-2-deficient mice are more susceptible to bleomycin-induced lung fibrosis due to loss of the regulatory functions mediated by this component of the arachidonic acid cascade (Keerthisingam et al.

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Fig. 18.2 Derivation and potential differentiation of matrix-producing cells. Fibroblasts and myofibroblasts are the major sources of collagen within the lung. During fibrotic pathologies such as asthma and idiopathic pulmonary fibrosis, the extent of collagen production increases. This is due to heightened fibroblast and myofibroblast activation and number, as well as the recruitment of bone marrow-derived fibrocytes and differentiation of epithelial cells into a collagen-producing phenotype. See text for definition of abbreviations. (See CD-ROM for color version.)

2001; Hodges et al. 2004). Fibroblasts from a profibrotic environment exhibit altered responsiveness to growth factors, express enhanced receptor levels for chemokine receptors such as CCR7 and cytokines such as IL-4 and IL-13, and secrete a different array of mediators including MCP-3/CCL7 (Hogaboam et al. 1999; Choi et al. 2004, 2006; Jakubzick et al. 2004a,b; Renzoni et al. 2004). Another functional difference in fibroblasts derived from the lungs of IPF patients is the extent to which this cell type can modulate angiogenesis. Lung tissue sections from IPF patients express increased levels of the angiogenic chemokine IL-8/CXCL8 and decreased levels of the angiostatic chemokine IP10/CXCL10, thereby suggesting an imbalance in net angiogenesis in the lungs of IPF patients. Interestingly, IPF patientderived fibroblasts express increased levels of IL-8/CXCL8, suggesting that the fibroblast is the main effector cell causing the angiogenic imbalance (Keane et al. 1997). Reports dating back to 1952 have indicated that fatal asthma is associated with dilated, congested blood vessels (Walzer & Frost 1952; Dunnill 1960). Vascular changes often occur early in the disease process (Orsida et al. 1999) and studies have suggested that the extent of angiogenesis parallels the severity of asthma (Vrugt et al. 2000). Due to the other pathologies associated with severe asthma, such as thickening of the airways, an increase in vascularization is required to support the increased tissue mass. However, the extent and function of angiogenesis and whether angiogenesis is a major contributing factor to disease severity remains to be elucidated. Increased angiogenesis in the bronchial vasculature in asthma may contribute to the increased interstitial edema due to microvasculature leakage. Also, an increase in vessel number may

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increase leukocyte infiltration into the lung, thus perpetuating the inflammatory cascade in the lung. Interestingly, phenotypic differences have also been observed between fibroblasts isolated from mice with Th1-biased or Th2-biased experimentally induced granulomas (Hogaboam et al. 1999). Pulmonary fibroblasts isolated from the Th2 fibrotic environment expressed increased levels of MCP-1/ CCL2 and its receptor CCR2. These fibroblasts also expressed increased levels of procollagen mRNA when stimulated with MCP-1/CCL2, in comparison with Th1 environment-derived fibroblasts (Hogaboam et al. 1999). A better understanding of the phenotypes of disease-associated fibroblasts may highlight pathways specific to disease pathologies giving rise to targeted therapeutics.

Myofibroblasts Myofibroblasts were first described by Gabbiani and colleagues as cells central to wound healing (Gabbiani & Badonnel 1976). As has already been alluded to, the presence of myofibroblasts in the lung is characteristic of aberrant fibrotic remodeling and IPF (Desmouliere & Gabbiani 1995; Phan 2002). They are “smooth muscle-like” cells as myofibroblasts are morphologically similar to fibroblasts; however, they also express α-smooth muscle actin (α-SMA) fibers (Ohta et al. 1995). The actin filaments result in myofibroblasts having a contractile phenotype, which at sites of wound healing serves to close the wound. Myofibroblasts are rarely seen in the normal lung, although the presence of myofibroblasts is characteristic of fibrotic remodeling and IPF (Adler et al. 1989; Mitchell et al. 1989; Kuhn & McDonald 1991; Pache et al. 1998). However, the presence of contractile myofibroblasts in the interstitium of the lung may cause either retraction of parenchymal tissue, resulting in alveolar collapse and creating the characteristic honeycombing observed in the lungs of IPF patients, or add to the increase in alveolar size, which is characteristic of COPD (Dacic & Yousem 2003). There is also a significant increase in myofibroblast numbers in the lungs of asthmatic patients following allergen challenge (Gizycki et al. 1997). An increase in the number of myofibroblasts in the mucosa of asthmatics has been shown to correlate with subepithelial collagen deposition (Brewster et al. 1990). Indeed, during fibrotic pathologies, myofibroblasts have been shown to be the predominant contributors to ECM deposition and structural remodeling (Phan 2002). For example, colocalization of myofibroblast α-SMA and procollagen I at the mRNA level has been reported by in situ hybridization, indicating that these cell types are a significant source of collagen during pulmonary fibrosis (Zhang et al. 1994). Additionally, myofibroblasts also produce elastin, fibronectin, and laminin and they are significant sources of chemokines and growth factors (Baur & Parks 1983; Singer et al. 1984; Zhang et al. 1994; Weill et al. 1997; Thannickal et al. 2003). Given the importance of myofibroblasts in the fibrotic state, defining both the origin and mechanisms leading to the

Fibroblasts and the Extracellular Matrix

clearance of these cells will greatly add to our understanding of the role of this cell in driving fibrosis. Myofibroblasts express a panel of markers and these markers have been correlated with site of derivation. For example, myofibroblasts found in the peripheral and subpleural regions of fibrosis express α-SMA, vimentin, and desmin, whereas cells found in other regions of the lung do not express desmin (Zhang et al. 1994). This suggests that there may be differential sources of myofibroblasts and indeed multiple potential processes have been described. Fibroblast-to-myofibroblast transdifferentiation can be induced in vitro by TGF-β1 stimulation of fibroblasts and it has been hypothesized that TGF-β1 found locally at sites of fibrosis will differentiate resident fibroblasts into myofibroblasts (Desmouliere et al. 1993; Chambers et al. 2003). The environment at sites of fibrotic remodeling also contains a variety of other soluble factors, such as type 2 cytokines which have also been shown to potentiate fibroblast differentiation (Mattey et al. 1997). At sites of normal wound healing, once sufficient matrix has been deposited, fibroblasts and myofibroblasts undergo apoptosis (Darby et al. 1990; Desmouliere et al. 1995). This serves to limit the excessive deposition of ECM and also dampen the proinflammatory and profibrotic milieu. However, myofibroblasts persist in the IPF lung and this may be due to the profibrotic environment as TGF-β1 promotes IPF lung-derived myofibroblast survival (Kuhn & McDonald 1991; Zhang & Phan 1999). These cells generate mediators such as TNF-α and IL-1α that may drive epithelial cell apoptosis yet prevent eosinophil apoptosis (Zhang et al. 1996). Thus, myofibroblasts appear to have an integral role in perpetuating the inflammatory processes involved in asthma and fibrosis. Another possible source of myofibroblasts in the fibrotic lung is the epithelium, through a process of epithelial-tomesenchymal transition (EMT) (Kalluri & Neilson 2003). Interestingly, this process was also alluded to in 1976, with epithelial cells being described as differentiating into spindlelike myofibroblasts during reepithelialization (Gabbiani & Badonnel 1976). Overall, EMT describes a process where resident epithelial cells can be induced, through stimulation with TGF-β1, to transition into a mesenchymal phenotype, thus changing their properties such as the extent of ECM generation and enhanced motility (Okada et al. 1997). The hypothesis that EMT may be driving fibrosis was first described in vivo at sites of renal fibrosis and is discussed later in this chapter (Okada et al. 1997). Another mechanism by which myofibroblasts may arise is due to differentiation of recruited bone marrow-derived collagen I-positive cells, commonly referred to as “fibrocytes” (Epperly et al. 2003; Direkze et al. 2004; Forbes et al. 2004; Hashimoto et al. 2004; Deb et al. 2005; Yamaguchi et al. 2005; Quan et al. 2006).

Fibrocytes Circulating type I collagen-positive (collagen I+) cells have been identified as having a role in collagen deposition during

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remodeling pathologies (Quan et al. 2006). A variety of extracellular and intracellular markers have been used to identify fibrocytes. Fibrocytes express CD45, indicating hematopoietic origin. They also express stem cell markers, for example CD34 or CD13, as well as intracellular procollagen I. Furthermore, fibrocytes have been shown to express multiple chemokine receptors including CCR2, CCR7, and CXCR4 (Hashimoto et al. 2004; Phillips et al. 2004; Moore et al. 2006). Inhibition of the respective chemokine ligands in mouse models of fibrosis inhibits fibrocyte recruitment and ultimately reduces collagen deposition (Hashimoto et al. 2004; Phillips et al. 2004; Moore et al. 2005, 2006). Interestingly, fibrocytes also express major histocompatibility complex (MHC) class II molecules and have been shown to be able to present antigen to naive T cells, indicating that these cells may function as antigen-presenting cells at sites of fibrosis (Chesney et al. 1997, 1998). Fibrocytes have also been hypothesized to be a circulating progenitor cell in that they have pleuripotent potential to differentiate into other cell lineages, as has been demonstrated with fibrocyte-derived adipocytes (Hong et al. 2005). These circulating collagen I+ cells, or “fibrocytes,” were initially described at sites of wound repair (Bucala et al. 1994). However, fibrocytes have been demonstrated to generate a variety of ECM components and cytokines both in vitro and at sites of tissue remodeling (Chesney et al. 1998; Yoshida et al. 1999; Abe et al. 2001; Phillips et al. 2004; Hong et al. 2005; Moore et al. 2006). In particular, these cells have been demonstrated to secrete other matrix proteins, including type I and type III collagen as well as fibronectin (Bucala et al. 1994; Quan et al. 2006; Varcoe et al. 2006). Fibrocytes may also play a role in tissue remodeling through the production of MMPs (Hartlapp et al. 2001). Moreover, it has recently been postulated that an overexuberant recruitment of these cells to sites of pulmonary injury contributes to the aberrant deposition of collagen, which ultimately induces pathologic fibrosis (Bucala et al. 1994; Chesney et al. 1998; Chesney & Bucala 2000; Abe et al. 2001; Schmidt et al. 2003). It has been suggested that fibrocytes have the ability to differentiate into myofibroblasts, although this is not consistently reproducible (Jester et al 1987; Delanian et al. 1998; Schmidt et al. 2003). In vitro stimulation of fibrocytes with TGF-β1 results in the cells transitioning into a myofibroblast phenotype and producing fibronectin and type III collagen (Schmidt et al. 2003). Using an adoptive transfer model of bone marrow cells from green fluorescent protein (GFP) transgenic mice into mice challenged with intratracheal bleomycin (to initiate lung injury), it was demonstrated that recruited GFPpositive fibrocytes differentiated into fibroblasts and resident lung fibroblasts differentiated into myofibroblasts (Hashimoto et al. 2004). Further studies are necessary to delineate the fate of fibrocytes and the total impact of this pathway to fibrogenesis. There is increasing evidence that fibrocytes may also have a pathogenic role in asthma (Schmidt et al. 2003; Phillips et al.

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2004). A correlation in the number of fibrocytes in the basement membrane of asthmatics and the extent of subepithelial fibrosis has been described (Nihlberg et al. 2006). Increased numbers of bone marrow-derived fibrocytes have been reported in the airways of asthmatics, and allergen challenge further induced CD34+ cell recruitment to the subepithelial region (Schmidt et al. 2003). Interestingly, CD34 colocalized with α-SMA, suggesting that CD34+ fibrocytes traffic to sites of active remodeling and differentiate into myofibroblast-like cells (Schmidt et al. 2003). The mobilization of fibrocytes in repair during other diseases has also been demonstrated with burn patients (Yang et al. 2002). Adherent cells isolated from the peripheral blood of patients with severe burns could be rapidly induced to a fibrocyte phenotype in vitro and the peak number of these potential “pre-fibrocyte” cells occurred 3 weeks after injury, which was also when peak circulating TGF-β1 levels were detected (Yang et al. 2002).

Epithelial cells Alterations in epithelial cell phenotype to a more mesenchymal phenotype, i.e., the process of EMT, is a concept that is emerging for IPF and pulmonary disorders associated with fibrotic remodeling. One of the causative factors hypothesized to be driving IPF is repeated injury to the epithelium, which cannot regenerate adequately to cover denuded surfaces and leads to epithelial cell hyperplasia (Hyde et al. 1992; Gross & Hunninghake 2001). IPF epithelial cells have altered synthetic capacity and produce increased levels of profibrotic factors such as TGF-β (Khalil et al. 1996). This finding has also been correlated with disease progression in that advanced IPF lesions have increased TGF-β1 expression compared with epithelial cells from early lesions (Khalil et al. 1996). EMT describes a process by which epithelial cells undergo phenotypic transition to fully differentiated mesenchymal cells, such as fibroblasts and myofibroblasts (Zavadil et al. 2004; Zavadil & Bottinger 2005). The differentiation of airway epithelial cells of one type to another, for example type I pneumocytes transitioning into goblet cells, has been previously described (Danto et al. 1995; Borok et al. 1998; Torday et al. 2003; Kim et al. 2006). However, the switching of an epithelial cell into a phenotype that moves beyond the original cell’s embryonic lineage has only recently been hypothesized as a driving factor in fibrosis (Kalluri & Neilson 2003; Selgas et al. 2004; Valcourt et al. 2005). EMT is a dynamic process in which the epithelial cells must lose polarity, downregulate adhesion molecule expression, and upregulate the machinery necessary for motility (Zavadil et al. 2004; Zavadil & Bottinger 2005). It is a wellestablished phenomenon in tumor metastases, in that primary tumor cells change phenotype to become more motile and mesenchymal in phenotype and then engraft at sites distal to the initial primary tumor (Kang & Massague 2004). Recent publications indicate that epithelial cells at sites of

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fibrosis also transition into a mesenchymal phenotype following injury and this can facilitate the fibrotic state in various organs (Strutz et al. 1995; Ng et al. 1998; Iwano et al. 2002). This has been shown using cells in vitro and also immunohistochemical colocalization of epithelial and mesenchymal cell markers on the same cells (Willis et al. 2005). Epithelial cells that undergo EMT downregulate epithelialspecific markers such as E-cadherin and concomitantly acquire some of the fibroblast and/or myofibroblast-associated markers such as expression of fibroblast-specific protein (FSP)-1 or α-SMA (Okada et al. 1997; Jechlinger et al. 2002; Grunert et al. 2003). The idea of EMT promoting the fibrosis observed in asthma and IPF is rapidly evolving. Induction of EMT requires the presence of profibrotic growth factors such as TGF-β1 (Citterio & Gaillard 1994; Pagan et al. 1999; Grande et al. 2002; Xu et al. 2003; Zeisberg et al. 2003; Liu 2004; Strizzi et al. 2004; Aguilera et al. 2005; Demir et al. 2005; Han et al. 2005; Zavadil & Bottinger 2005; Ahmed et al. 2006; Gotzmann et al. 2006; Lee & Kay 2006; Sam et al. 2006; Yang et al. 2006). Epithelial cells exposed to TGF-β1, alone or in combination with other growth factors such as epidermal growth factor, begin the process of EMT through a loss of polarity and the increased expression of MMPs, which enable basement membrane degradation. The cells also undergo cytoskeletal changes as well as altered expression of surface molecules. For example, downregulation of E-cadherin and zona occludens (ZO)-1 with a concomitant upregulation of vimentin is needed for migration and transition to a mesenchymal phenotype (Iwano et al. 2002; Grunert et al. 2003). The majority of the work evaluating EMT has been performed in vitro, and the full extent of this pathogenic pathway in vivo is currently being evaluated. It is still not known whether EMT contributes to the excess ECM deposition. Future work correlating the time-course of EMT induction with disease staging will also be very insightful. This pathogenic process may provide novel therapeutic targets such that inhibiting or reversing EMT may provide clinical benefit to patients with lung fibrosis-associated diseases. In asthma there is also evidence of altered epithelial cell phenotype and function (Fahy 2001). Not only is there an increased goblet cell number in asthma (Fahy 2002), which may occur through differentiation of type I cells into mucusproducing type II cells (Hogg 1993), but epithelial cell shedding (sloughing) is also a characteristic feature of asthma (Chanez et al. 1999). This phenomenon may be due to shearing of the epithelium following bronchoconstriction or airway hyperresponsiveness, or may be due to a rapid proliferative rate of the epithelium in asthmatics (Montefort et al. 1993). Interestingly, the epithelium has been demonstrated to regulate fibroblast proliferation through the generation of mediators including prostaglandins and chemokines (Moore et al. 2003). Therefore, any alteration in epithelial phenotype may deleteriously affect fibroblast function.

Fibroblasts and the Extracellular Matrix

There is emerging data indicating that bone marrow-derived progenitor epithelial cells may play a role in re-modeling. Resident progenitor epithelial cells in the lung have been hypothesized to maintain a homeostatic epithelial layer. In contrast, circulating progenitor epithelial cells are recruited to promote repair following excessive injury. Evidence for this has come from sex-mismatched human allograft transplants where recipient epithelial cells contribute toward the regeneration of donor lung epithelium (Kleeberger et al. 2003; Spencer et al. 2005). Further, using an animal model of tracheal transplantation, recipient bone marrow-derived epithelial cells repopulated the transplanted tracheas (Gomperts et al. 2006). Once recruited, the progenitor epithelial cells localized to the basement membrane and then migrated through the basement membrane to the apical epithelial layer where the cells further differentiated (Gomperts et al. 2006). Therefore, as with the literature surrounding fibrocytes, this body of work demonstrates the plasticity of bone marrowderived cells in reconstituting damaged ECM. Although this field is still emerging, this work does highlight a role for the epithelium in remodeling through epithelial–fibroblast communication as well as transition into a fibroblast-like phenotype via EMT.

Indirect mediators of fibrosis The lung contains numerous other structural and inflammatory cells that have been shown to be involved in the fibrotic response. These cell types are not responsible for directly generating collagen, although they have been shown to be indirectly contributing to fibrosis through the generation of other mediators such as growth factors and cytokines. This section outlines some of the cell types involved indirectly in fibrosis and the current understanding of how these cells drive fibrosis.

Airway smooth muscle Airway smooth muscle (ASM) cells have the ability to secrete large amounts of cytokines and chemokines such as RANTES/CCL5, MCP-1/CCL2, eotaxin/CLL11, and MCP-3/ CCL7 (Baraldo et al. 2003; Wuyts et al. 2003a,b; John et al. 2004; Peng et al. 2004). These chemokines and cytokines likely play a strong role in both the initiation and maintenance of chronic airway inflammation by recruitment and activation of immune cells (Murray et al. 2006). ASM cells have been shown to produce MMPs which, as discussed later in the chapter, can also act on the local ECM environment (Xie et al. 2005). Increased procollagen I gene expression has been demonstrated in ASM cells, although their ability to produce other ECM components is currently undefined (Dabbagh et al. 1998). There is also evidence indicating that ECM components themselves induce chemokine production by ASM cells and that this synthesis is increased in asthma (Johnson 2001; Black et al. 2003; Elshaw et al. 2004).

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Alveolar macrophages Alveolar macrophages have been widely examined in the pathogenesis of asthma. Several studies have demonstrated a significant positive correlation between macrophage activation and the severity of asthma (Duddridge et al. 1993; Woolley et al. 1995; Virchow et al. 1996). Further, allergen challenge has been demonstrated to induce the activation of these cells (Carroll et al. 1985; Viksman et al. 2002; Ferreira 2003; Lensmar et al. 2006). It is long established that alveolar macrophages have a role in lung fibrosis. One role for macrophages in tissue remodeling may be through the production of MMPs, other proteolytic enzymes and mediators that degrade local tissue but which also exacerbate inflammation and enhance mucus production (Page & Morley 1986; Page & Coyle 1989; Henderson et al. 2000; Maisi et al. 2002). Crucially, several reports have shown that macrophages are sources of profibrotic growth factors such as PDGF, connective tissue growth factor (CTGF), and TGF-β1 (Chanez et al. 1995; Lee & Lee 2001; Burgess 2005; Chetta et al. 2005; Howell & McAnulty 2006). Further, the extent of growth factor production is enhanced in alveolar macrophages isolated form the lungs of patients with ILD, as is the levels of chemokine production (Bitterman et al. 1982; Nagaoka et al. 1990; Zhang & Phan 1996; Hasegawa et al. 1999). Alveolar macrophages from asthmatic patients also secrete significantly more fibronectin and TGF-β compared with macrophages from nonasthmatic patients (Vignola et al. 1996). Interestingly, stimulating macrophages with the type 2 cytokines IL-13 or IL-4, which are commonly associated with atopic diseases, results in an “alternatively activated macrophage” (aaAM) phenotype that may be additive to the fibrotic environment (Fig. 18.3) (Gordon 2003). These

Classically Activated Alveolar Macrophage

Predominant Response

Nitric oxide, IL-6, TNF-a

TGF-b1, PDGF, TARC, IL-10, arginase, fibronectin Alternatively Activated Fig. 18.3 Predominant responses of classically activated and alternatively activated alveolar macrophages. Classical activation of macrophages by, for example, interferon (IFN)-g or microbial pathogens induces microbicidal activity such as increased generation of nitric oxide and TNF-a. In contrast, stimulation of alveolar macrophages with type 2 cytokines such as IL-4 or IL-13 results in an alternatively activated phenotype. This results in the production of profibrotic growth factors and extracellular matrix components. See text for definition of abbreviations. (See CD-ROM for color version.)

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aaAMs have been demonstrated to generate fibronectin, promote proliferation, and augment ECM generation by fibroblasts in vitro (Song et al. 2000). Several reports have indicated altered cytokine and growth factor production by aaAMs, including increased TGF-β1, Th2-associated chemokines TARC/CCL17 and MDC/CCL22 (Andrew et al. 1998; Bonecchi et al. 1998; Imai et al. 1999; Song et al. 2000; Lee, C.G. et al. 2001; Fichtner-Feigl et al. 2006). Another chemokine associated with aaAMs is PARC/CCL18 and interestingly the majority of alveolar macrophages isolated from the BAL fluid of IPF patients spontaneously release PARC/CCL18 (Prasse et al. 2006). Moreover, aaAM-derived PARC/CCL18 has been shown to feed back to fibroblasts to promote collagen generation (Prasse et al. 2006). Currently the receptor for PARC/CCL18 is unknown. Another characteristic marker for the aaAM phenotype is the presence of arginase. Arginase inhibits nitric oxide production in an autocrine manner, potentially inhibiting the nitric oxide host defense role of these cells and has also been reported to further promote ECM generation (Hesse et al. 2001). This early work suggests that alveolar macrophages may be central to the pathogenesis of IPF and fibrosis in general. Emerging evidence suggests a role for aaAM in fibrotic disease pathologies. Further studies establishing the role of aaAM in diseases associated with fibrosis are warranted to identify novel pathways amenable to therapeutic intervention.

T cells T cells have the ability to orchestrate a number of features of remodeling (reviewed in Kay 1997). The airways of patients with moderate, severe, and fatal asthma have increased numbers of T lymphocytes including both CD4+ and CD8+ T cells (Kline & Hunninghake 1994; Mazzarella et al. 2000; Redington et al. 2000; Tsoumakidou et al. 2004; van Oosterhout et al. 2004). T cell-derived cytokines, in particular IL-4 and IL-13, can mediate various pathologies associated with the initiation, stabilization, and progression of fibrosis (Fig. 18.4). The animal models of asthma and fibrosis show a strong presence of CD4+ cells in the lung and these cells are thought to participate in the fibrotic process through the secretion of Th2 cytokines such as IL-4, IL-5, and IL-13 (Wise et al. 1999; van Rijt et al. 2005). CD4+ T cells are required for eosinophil recruitment since CD4 gene-deficient mice did not exhibit pulmonary eosinophilia (Gonzalo et al. 1996). Although the extent of eosinophilia does not correlate with disease severity in this model, it is interesting to note that the eosinophilia observed in asthmatic patients correlates with certain features of airway remodeling such as the extent of ECM deposition and subepithelial membrane thickness (Wenzel et al. 1999).

Eosinophils The role of eosinophils in fibrosis may be twofold: through the release of soluble mediators and through cell–cell

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Monocyte

IL-13 Fig. 18.4 Effects of IL-4 and IL-13 on key features of fibrotic remodeling in the lung. IL-4 and IL-13, generated by Th2 cells, exert pleiotropic functions on a variety of cells within the lung. Reports indicate that these two cytokines have overlapping roles yet each exert the pathologic changes to different extents and this is represented with the shaded gradients. For example, IL-13 is more efficient at promoting the generation of alternatively activated macrophages, whereas IL-4 is more efficient at inducing isotype switching in B cells. These cellular responses are hallmarks of asthma and idiopathic pulmonary fibrosis and have been associated with disease progression and severity. AHR, airway hyperresponsiveness; EMT, epithelial to mesenchymal transition. See text for definition of other abbreviations. (See CD-ROM for color version.)

Fibroblasts and the Extracellular Matrix

Alternatively Activated Macrophage TGF-b production

Fibroblasts

ECM generation Proliferation Differentiation in myofibroblasts

Smooth Muscle Th2 Cell

Hyperreactivity Hypertrophy

Eosinophil Activation Survival Lung infiltration Epithelium

Mucos production Goblet cell hyperplasia EMT

B Cell

Mast Cell IgE

IL-4

interactions. For example, the eosinophil granule proteins major basic protein (MBP) and eosinophil peroxidase (EPO) can directly damage host tissue, and eosinophil cationic protein (ECP) directly induces fibroblast ECM production (Heyneman et al. 1975; Dechatelet et al. 1978; Bozeman et al. 1990; Hernnas et al. 1992; Romano et al. 2000). Further, eosinophils are sources of TGF-β1 during fibrosis (Wong et al. 1993; Elovic et al. 1994; Minshall et al. 1997). In fact, increased numbers of eosinophils as well as their products are often found in patients with IPF (Reimert et al. 1994; Fujimoto et al. 1995; Kroegel et al. 1998) and this cell type has been demonstrated to directly activate fibroblasts and promote fibroblast proliferation through cell–cell interactions (Shock et al. 1991). Further, fibroblasts generate granulocyte–macrophage colony-stimulating factor (GM-CSF), which has been shown to promote eosinophil survival (Vancheri et al. 1989). IL-5, another Th2-associated cytokine, has been shown to be central to eosinophil recruitment, activation, maturation, and survival (Yamaguchi et al. 1988; Clutterbuck et al. 1989). Treatment of asthmatics with a neutralizing monoclonal antibody to IL-5 reduced the number of eosinophils in the airways and also impacted subepithelial fibrosis (Leckie et al. 2000). Critically, however, lung function in asthmatic patients was not improved following a single dose of the anti-IL-5 antibody (Leckie et al. 2000; Flood-Page et al. 2003; Menzies-Gow et al. 2003). However it is important to note that the investigators in the anti-IL-5 study reported that there was not complete inhibition of eosinophil numbers in the lungs (Flood-Page et al. 2003). Overall, the role of the eosinophil in fibrosis requires further evaluation (Kay et al. 2004).

Degranulation Histamine release AHR

Mast cells Mast cells are strongly implicated in the pathogenesis of asthma, because these cells are found activated in the lungs of asthmatics (Synek et al. 1996; Boyce 2003). Although the major role of the mast cell appears to be limited to degranulation following engagement of cell-surface IgE receptors, evidence suggests that the mast cell plays a larger role in asthma and fibrosis. For example, mast cells produce the proteolytic enzymes tryptase and chymase, which may promote aberrant tissue degradation (Boyce 2003). Mast cell-derived chymase can have numerous downstream activities including the cleavage of latent TGF-β1 into active TGF-β1 (Rothe & Kerdel 1991; Howard et al. 2004). Chymase has also been demonstrated to generate type I collagen peptides which, by the nature of these peptides, may exacerbate the proinflammatory milieu (Kofford et al. 1997; Wang et al. 2001). Use of mast cell chymase inhibitors has shown efficacy in animal models of lung fibrosis, thus highlighting the potential deleterious role of these cells in fibrosis (Tomimori et al. 2003).

Neutrophils Neutrophils appear to be an indicator of the severity of asthma as they are found in the airways of patients with severe or chronic asthma (Martin et al. 1991; Sur et al. 1993; Carroll et al. 1996). One potential role of neutrophils in driving fibrotic disease is through the degradation of ECM by proteolytic neutrophil-derived mediators. Neutrophil elastasedeficient mice are protected from bleomycin-induced lung injury that is partly mediated through decreased TGF-β generation (Dunsmore et al. 2001). Further, during disease exacerbations in asthma, IPF and COPD, the pulmonary localization

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of neutrophils may excessively cleave ECM components into profibrotic and proinflammatory peptides. Increased levels of neutrophil-recruiting CXCR1- and CXCR2-binding chemokines such as ENA78/CXCL5 and IL-8/CXCL8 have been described in the IPF lung (Keane et al. 1997, 2001). However, the consequences of elevated levels of these chemokines has been correlated with an increase in angiogenesis, which may worsen fibrotic disease manifestations as described earlier (Folkman 1995). The potential consequences of matrix degradation are discussed subsequently in the section Matrix metalloproteinases and matrix degradation.

Mediators of fibrosis There are a number of mediators of fibrosis that have been shown to activate fibroblasts, including growth factors and cytokines. One of the most potent and well-described growth factors is TGF-β. This cytokine is found upregulated in numerous fibrotic diseases and not only contributes directly to fibrosis by acting on fibroblasts but also induces the secretion of other profibrotic mediators such as PDGF and CTGF. Of the classic Th2 cytokines, both IL-4 and, to a greater extent, IL-13 have been shown to be profibrotic. IL-4 is profibrotic predominantly via an indirect mechanism, whereas IL-13 acts directly on fibroblasts and virtually every other cell type involved in asthma and fibrosis. We also discuss thrombin as a soluble mediator of fibrosis that mediates its fibrotic activities via two distinct mechanisms: initiation of coagulation and activation of its cell-surface protease-activated receptors. Finally, we highlight the importance of ECM homeostasis, which is mediated by various proteases and protease inhibitors.

TGF-b Three TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, have been described (Coker et al. 1997; Chakravarthy et al. 1999). TGF-β1 appears to be the predominant isoform found in fibrotic lung tissue. The extent and expression pattern of TGF-β2 and TGF-β3 are comparable between nonfibrotic and fibrotic tissue (Khalil et al. 1996). TGF-β1 is upregulated in bronchial epithelial cells and alveolar macrophages in the IPF lung (Khalil et al. 1991, 1996; Coker et al. 1996). Numerous cell types generate and secrete TGF-β1 including lymphocytes, macrophages, eosinophils, epithelial cells, fibroblasts, and endothelial cells (Yoshida & Gage 1992; Okumura et al. 1997). While not thought to be the predominant source of TGF-β1 in the local tissue environment, platelets store and can secrete large amounts of this potent growth factor (LangRollin et al. 2001). TGF-β1 regulates numerous biological activities such as proliferation, apoptosis, and differentiation (Pinkas & Teicher 2006; Sanders et al. 2006). This protein also acts on cells to induce secretion of the profibrotic growth factors such as CTGF and PDGF (Kothapalli et al. 1998).

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Interestingly, TGF-β1 is a bimodal protein: it induces the proliferation of smooth muscle cells and fibroblasts at low concentrations but inhibits cell growth at higher concentrations through the induction of PGE2 (Battegay et al. 1990; McAnulty et al. 1997). TGF-β1 expression is increased in the lungs of asthma patients (Vignola et al. 1997; reviewed in Howell & McAnulty 2006). It has been further demonstrated that TGF-β1 is nearly absent in bronchial epithelial cells but is highly expressed in inflammatory cells beneath the basement membrane where subepithelial fibrosis predominates (Magnan et al. 1997). Eosinophils in the lungs of asthmatic patients have also been shown to have increased expression of TGF-β1 compared with control patients (Nomura et al. 2002; Kay et al. 2004). Moreover, TGF-β1 expression in the patients correlated with the observed increase in fibroblast number, as well as with thickness of the basement membrane (Minshall et al. 1997; Kay et al. 2004; Balzar et al. 2005). Transient overexpression of TGF-β1 or pulmonary delivery of this cytokine to mouse lungs induces a pronounced interstitial fibrosis mediated by aberrant ECM generation and deposition, as well as the presence of myofibroblasts (Sime et al. 1997). The in vitro profibrotic functionality of TGF-β1 and the association of TGF-β1 with fibrotic pathologies suggest that TGF-β1 is a central mediator of fibrosis (Bergeron et al. 2003). A polymorphism in TGF-β1 conferring greater production has been associated with disease progression but not with predisposition to developing IPF (Xaubet et al. 2003). Interestingly, this same polymorphism has also been associated with COPD (Wu et al. 2004). The polymorphism occurs at a lower frequency in COPD patients, suggesting that this particular genetic alteration may be protective (Wu et al. 2004). The emergence of therapeutics directed against the TGF-β pathway will shed light on the role of this growth factor in disease (see Table 18.1).

IL-13 and IL-4 IL-13 and IL-4 are two pleiotropic Th2-associated cytokines, with numerous distinct and overlapping functions (see Fig. 18.4). IL-13 is elevated in the lungs of IPF patients and is associated with fibrotic pathologies and aberrant remodeling at various tissue sites (Hancock et al. 1998; Wynn 2004). IL-13 is produced predominantly by T cells and exerts the majority of its biological effects via a heterodimeric receptor composed of IL-4Rα and IL-13Rα1 (Schnyder et al. 1996; Akbari et al. 2003; Wynn 2003). The profibrotic role for IL-13 in asthma and pulmonary fibrosis has been reviewed (Finkelman et al. 2004; Wills-Karp 2004). IL-4 is a potent cytokine that shares many functions with IL-13, which is not surprising given that IL-4 and IL-13 share the common IL-4Rα receptor subunit (Wynn 2003; Finkelman et al. 2004; Wills-Karp 2004). IL-4 is also secreted predominantly by Th2

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cells and is a characteristic feature defining CD4+ T cells as Th2 cells (Mosmann & Coffman 1989). There are differences in the downstream events following IL-4 or IL-13 signaling as these two cytokines have overlapping but not redundant function. For example, IL-4 more potently induces B-cell proliferation as well as immunoglobulin production and class switching to expression of IgE, which in turn activates mast cells by cross-linking the Fcε receptor (Cocks et al. 1993). In contrast, IL-13 activates epithelial cells and goblet cells, causing mucus production, goblet cell hyperplasia, and EMT (Atherton et al. 2003; Yasuo et al. 2006). Moreover, IL-13, to a greater extent than IL-4, stimulates epithelial cells to generate and secrete eotaxin/ CCL11 and MCP-1/CCL2, chemoattractants for eosinophils and monocytes respectively (Pope et al. 2001; Moore et al. 2002; Ip et al. 2006). IL-13 also induces eosinophil activation and promotes survival, a function not yet attributed to IL-4 (Luttmann et al. 1996). Various animal models of fibrosis have indicated a more profibrotic role for IL-13 than IL-4. One study using pulmonary-specific IL-13 transgenic overexpression demonstrated many hallmarks of asthma and IPF (Zhu et al. 1999). There was an increase in subepithelial fibrosis, excess mucus production, and pronounced pulmonary inflammation (Zhu et al. 1999). IL-13 has also been shown to be involved in the maintenance of lung fibrosis. Selective depletion of IL-13Rα1-positive cells following establishment of experimental fibrosis improved disease pathology (Jakubzick et al. 2003). In the fluorescein isothiocyanate (FITC) mouse model of fibrosis, IL-4 gene-deleted mice were not protected from fibrosis while both IL-13 gene-deleted and IL-4/IL-13 dual gene-deleted mice were both significantly protected. This protection correlated with reduced eosinophilia but appeared to be independent of the TGF-β pathway (Kolodsick et al. 2004). Similarly, in the bleomycin-induced mouse model of pulmonary fibrosis, neutralization of IL-13, but not IL-4, attenuated the pulmonary fibrosis as assessed by collagen deposition (Belperio et al. 2002). Further, IL-13 has been demonstrated to play a significant role in maintenance of the subepithelial fibrosis seen in the ovalbumin-induced mouse model of asthma, as anti-IL-13 monoclonal antibody treatment halted development of subepithelial fibrosis and reversed mucous hyperplasia (Yang et al. 2005). Both IL-13 and IL-4 induce profibrotic responses in fibroblasts, responses comparable to those induced by TGF-β1 (Oriente et al. 2000; Hashimoto et al. 2001). Both IL-4 and IL-13 induce fibroblast proliferation (Saito et al. 2003). Interestingly, IL-4 and IL-13 induced greater proliferation of fibroblasts derived from patients with mild asthma compared with responses elicited on fibroblasts derived from subjects with severe asthma (Kraft et al. 2001). This suggests an altered fibroblast phenotype that is dependent on disease staging. IL-13 and IL-4 have also been demonstrated to play

Fibroblasts and the Extracellular Matrix

profibrotic roles on airway epithelial cells by promoting epithelial cell proliferation and also inducing mitogenic TGF-β2 production from these cells (Richter et al. 2001). Interestingly, IL-13 but not IL-4 promotes the differentiation of fibroblasts to myofibroblasts (Kraft et al. 2001; Saito et al. 2003). The other mechanism by which IL-13 can mediate fibrosis is via the second IL-13 receptor, IL-13Rα2 (Fichtner-Feigl et al. 2006). This receptor was, until very recently, thought to be a decoy receptor as it has a very short cytoplasmic tail and is frequently shed from the surface of cells (Gauchat et al. 1997; Orchansky et al. 1997). However IL-13 binds to IL13Rα2 with a much higher affinity than IL-13Rα1 (Rahaman et al. 2002; Wu & Low 2003). Recent data suggest that IL13Rα2 has signaling capabilities resulting in TGF-β1 secretion from macrophages (Fichtner-Feigl et al. 2006). Overall the vast majority of literature suggests that IL-4 plays a role in asthma, likely at the acute stage. In contrast, IL-13 appears to play a more central role through the maintenance and progression of fibrosis. This is further highlighted by the recent evidence demonstrating a functional role for the IL-13-specific receptor IL-13Rα2.

Thrombin and coagulation pathway-associated proteases Thrombin is the final serine protease generated during blood coagulation and fibrin formation (Overduin & de Beer 2000). The extrinsic coagulation cascade is initiated following tissue injury, with tissue factor (TF) interacting with factor VII. This then initiates the conversion of factor VII to VIIa. Factor VIIa converts factor X to Xa, which acts in concert with factor V to convert prothrombin to thrombin (Chambers & Laurent 2002). Thrombin catalyzes the conversion of soluble fibrinogen to insoluble fibrin monomers. Thrombin can generate a positive feedback loop to achieve sustained coagulation by activating the intrinsic pathway, starting with the conversion of factor XI to XIa. Factor XIa converts factor IX to IXa. Factor IXa converts factor X to Xa, which, as with the extrinsic pathway, converts prothrombin to thrombin (Schenone et al. 2004). Thrombin has been implicated in pulmonary fibrotic diseases such as acute lung injury (Schmidt et al. 1996; Kipnis et al. 2004), acute respiratory distress syndrome (ARDS) (Burchardi et al. 1984), ILD (Fujimoto et al. 2003; Kimura et al. 2005), and IPF (Hernandez-Rodriguez et al. 1995; Howell et al. 2002; Ludwicka-Bradley et al. 2004). Indeed, BAL fluid from ARDS patients was shown to have complexes of TF– factor VII–VIIa capable of triggering the extrinsic coagulation cascade. Active thrombin has also been found elevated in the BAL fluid of scleroderma patients (Ohba et al. 1994). Active thrombin is also found at increased levels in patients with IPF and eosinophilic pneumonia (Kimura et al. 2005). Further, thrombin also represents a large proportion of the fibroblast proliferation capacity of BAL fluid (Hernandez-Rodriguez et al. 1995). Animal models of fibrosis have strengthened the

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connection between thrombin and fibrosis. Increased thrombin is found in the lungs of mice challenged with bleomycin and pharmacologic inhibition of thrombin significantly reduced collagen deposition (Howell et al. 2002). At the cellular level, thrombin has numerous biological effects that are divergent from its role as a coagulation pathway proteinase. It has been demonstrated to be a chemoattractant for leukocytes and can initiate the release of cytokines and chemokines such as MCP-1/CCL2, IL-6, and IL-8/CXCL8 from numerous cells (Sower et al. 1995; Gordon et al. 2000; Brandes et al. 2001; Liu et al. 2006). Specific to fibrosis, thrombin can act directly on smooth muscle cells and fibroblasts to induce matrix production by promoting the release of PDGF and TGF-β1 (Ohba et al. 1994; Chambers et al. 2000; Howell et al. 2002; Vesey et al. 2005). The cell-based activities of thrombin are mediated through a family of receptors termed proteinase-activated receptors (PARs). PARs are G protein-coupled receptors (GPCRs) that, unlike other GPCRs, are activated by proteolytic unmasking of a ligand attached to the receptor. Thrombin, trypsin, and tryptases are the most well-characterized proteinases capable of this unmasking (Molino et al. 1997; Fan et al. 2005). Four PARs have been identified in humans with differing functions and tissue expression patterns. Thrombin activates PAR-1, PAR-3, and PAR-4, whereas PAR-2 is activated by trypsin, mast cell tryptase, and coagulation factors VIIa and Xa (Molino et al. 1997; Camerer et al. 2000; Kawabata 2002; Ossovskaya & Bunnett 2004). As with thrombin, a profibrotic role for PARs has also been described. PAR-1, which is activated by thrombin, is expressed on T cells, monocytes, and dendritic cells, although the functional significance of this interaction is not completely understood (Steinhoff et al. 1999; Fields et al. 2003). PAR-2 is expressed in the skin, specifically keratinocytes, endothelial cells, and dermal dendritic cells (Steinhoff et al. 1999). However, PAR-2 expression has also been shown on vascular smooth muscle cells as well as on fibroblasts (Molino et al. 1998; Gruber et al. 2004). PAR-3, which is not as well characterized as PAR-1 or PAR-2, has been found on endothelial cells and platelets, although the function of this PAR is still being determined (Schmidt et al. 1998). PAR-4, which is cleaved by thrombin, is also found predominantly on platelets (Henriksen & Hanks 2002). Given the tissue localization and activation of PARs, it is interesting to note that both PAR-1 and PAR-2 have been implicated in fibrosis. Indeed, PAR-1-deficient mice are protected from bleomycininduced lung fibrosis (Howell et al. 2005). Further, PAR-2deficient mice had decreased eotaxin/CCL11 and reduced eosinophilia in the lungs following antigen challenge in an allergen sensitization and challenge model of asthma (Schmidlin et al. 2002; Takizawa et al. 2005).

Matrix metalloproteinases and matrix degradation The ECM is a dynamic structure that is continuously turning over and it is the fine balance between matrix generation and

424

breakdown that maintains the functional tissue environment (Laurent 1987; McAnulty & Laurent 1987; Mosher et al. 1992). Disruption of this homeostatic environment leads to excessive degradation or aberrant accumulation of the ECM. It has been hypothesized that breakdown of the matrix and its degradation products significantly contributes to the fibrotic process, potentially through the generation of proinflammatory and profibrotic ECM peptides (Laurent & Tetley 1984; Crouch 1990). Major elements associated with matrix degradation are proteases and protease inhibitors, which act on ECM components and also further enhance their own activity through activation of local protease cascades (Matrisian 1992; O’Connor & FitzGerald 1994). MMPs are central to ECM formation (Demedts et al. 2005). As well as directly altering ECM components, MMPs have been shown to cleave mediators such as chemokines from GAGs (Li et al. 2002). This cleavage increases the local concentration of these factors and may potentially increase their biological activity (Van den Steen et al. 2000). Tissue inhibitors of metalloproteinases (TIMPs) inhibit MMPs by binding to the catalytic site on these proteinases (Visse & Nagase 2003). A role for MMPs in disease activity has been reported for asthma and COPD (reviewed in Demedts et al. 2005). In asthma, it has been suggested that MMP-9 is central to disease activity and severity, with eosinophils and macrophages being the major source of this protease (Mautino et al. 1997; Ohno et al. 1997). Increased MMP-2 and TIMP-1 have also been detected in the lungs of asthmatics (Cataldo et al. 2000). However, only MMP-9 has been reportedly elevated in both BAL fluid and plasma of asthmatics during disease exacerbations, while MMP-2 and TIMP-1 levels were unaltered (Lee, Y.C. et al. 2001; Mattos et al. 2002; Cundall et al. 2003; Oshita et al. 2003). In contrast, increased MMP-8, MMP-9, and MMP-12 have been found to be elevated in the lungs of COPD patients (Finlay et al. 1997). In IPF, MMP-1, MMP-2, and MMP-9 were colocalized to the epithelium surrounding fibrotic lesions (Fukuda et al. 1998). There was also increased TIMP-2 in the myofibroblasts, which suggests that MMP activity may be inhibited and that the fibrotic region is not degraded (Fukuda et al. 1998). Another protease known to degrade matrix is the serine protease neutrophil elastase (NE). NE is released from azurophil granules in neutrophils and has microbicidal properties but it can also damage local tissue when expression is prolonged and thus promote aberrant remodeling (Travis 1988; Chua & Laurent 2006). Furthermore, as with MMPs, NE can cleave other proteolytic enzymes, rendering them more biologically active (Ferry et al. 1997). In disease, elevated levels of elastase have been detected in the sputum of asthmatic patients in comparison with control subjects, and elastase concentrations highly correlated with lung function as measured by FEV1 (Vignola et al. 1998). In addition, mice deficient in NE are resistant to bleomycin-induced pulmonary

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Table 18.1 Current therapeutics in development for disease indications where targeting these pathways may have profibrotic potential and thus may be suitable therapeutic candidates for diseases associated with pulmonary remodeling and fibrosis. Target

Compound

Company

Therapeutic strategy

Current reported indications

Status

CTGF

FG-3019

Fibrogen

Anti-CTGF mAb

Diabetic nephropathy, idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis

Phase I

PDGF

ARC-127 CR-002

Archemix Curagen

Anti-PDGF-B aptamer Anti-PDGF-D mAb

Age-related macular degeneration Diabetic nephropathy, IgA nephropathy, lupus nephritis

Preclinical Phase I

TGF-b

GC-1008

Cambridge Antibody Technology/Genzyme Archemix

Anti-TGF-b mAb

Idiopathic pulmonary fibrosis, renal cell carcinoma, malignant melanoma

Phase I

ABX10-0122 DOM 1000P IMA-638 TNX-650

Abgenix/Amgen Domantis Millenium/Wyeth Tanox

Anti-IL-13 mAb Anti-IL-13 dAB Anti-IL-13 mAb Anti-IL-13 mAb

CAT-354

Cambridge Antibody Technology

DOM-0910 AMG-317

Domantis Amgen

IL-13

IL-13/IL-4

TGF-b2 aptamer

Preclinical Preclinical Preclinical Phase I Phase I

Anti-IL-13 mAb

Asthma Asthma Asthma Hodgkin’s lymphoma, moderate to severe asthma Severe asthma

Anti-IL-4/IL-13 dAb Anti-IL-4 receptor mAb

Asthma Asthma

Preclinical Phase I

Phase I/II

mAb, monoclonal antibody; dAb, domain antibody.

fibrosis possibly because NE plays a role in TGF-β1 activation (Dunsmore et al. 2001). Thus it might exert important profibrotic effects.

Clinical targeting of fibrosis in lung pathologies Until recently, analysis of the fibrotic component of pulmonary disease processes required the examination of resected tissue. However, advances in CT and other imaging modalities now permit more accurate and insightful analysis of the structure of the lung parenchyma and airway wall in a noninvasive manner (Coxson & Rogers 2005). These techniques will prove to be extremely important in the evaluation of disease staging and progression. For example, in COPD, accurate diagnosis of patients regarding emphysematous disease or a disease with more airway wall remodeling will allow patient stratification for therapeutic intervention, which will depend on the specific pathologic manifestation of the disease. Currently there are no therapeutic molecules in use in clinical respiratory medicine that specifically attenuate fibrotic remodeling in the lung. However, Table 18.1 shows examples of some of the approaches currently being applied within the pharmaceutical industry that may modulate some of the key pathways associated with fibrotic remodeling

described in this chapter. The table focuses on agents in development that are specific inhibitors of numerous molecular targets. The table also highlights the large number of ongoing trials with these potential therapeutics. Data from the clinical trials will provide some very important insights in the next few years into the role of these molecular targets in different diseases. Some of the therapeutics in development are targeting diseases associated with significant fibrotic pathologies. Outcomes from these studies will provide invaluable information regarding the importance of the molecular target in ECM accumulation. Further, information from ongoing trials in IPF may also give us some important insights into the potential contribution of aberrant ECM deposition to asthma physiology.

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Immune Complexes and Complement: Their Role in Host Defense and in Disease Michael M. Frank and C. Garren Hester

Summary The formation of circulating immune complexes is thought to play an etiologic role in many autoimmune and infectious diseases. These complexes activate a variety of effector systems that contribute to tissue pathology. One important, highly complex, effector system is comprised of the proteins of the complement system. Our current understanding of immune complexes, their formation, and their contribution to disease manifestations is reviewed. Similarly, we provide a brief review highlighting the chemistry, mechanisms of action, and function of the complement system proteins.

Overview During the course of ordinary life, normal individuals are exposed to microbes that have invaded the tissues or intravascular space as well as to circulating foreign proteins absorbed from the gut or transported across the respiratory mucosa. To deal with these kinds of threats even primitive animals and plants have evolved a variety of defense mechanisms that together are termed “innate immunity.” Innate immune mechanisms are “hardwired” in the genome. Animals more advanced than sharks have evolved adaptive immune responses as well. These responses allow for a greater degree of flexibility in that the profile of responding cells and proteins undergoes alteration to more precisely fit the antigenic target. The innate immune system has many overlapping systems for dealing with invaders. Toll-like receptors on a variety of cells, including all phagocytes, recognize repeating structures on microbes, as exist in the polysaccharide capsule of bacteria or microbial DNA and RNA, and activate the phagocytes to destroy the microbes (Hoebe et al. 2006). There are receptors on Kupffer cells that recognize the terminal

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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mannose on the sugar moiety of partially degraded glycoproteins and there are scavenger receptors on phagocytic cells that also bind and phagocytose these materials. Defensins and granzymes, microbicidal peptides, are present in a variety of cells and are released to aid in host defense. The complement proteins represent yet another sophisticated innate immune system designed to deal with invaders or foreign materials both in circulation and in tissues. The adaptive immune response developed to provide more precise control and more rapid elimination of microbes from the circulation or tissues, since infection is such an important factor disrupting normal homeostasis. All these systems are designed to clear abnormal cells, foreign proteins, and microbes from the body, but, like many sophisticated systems, there are problems that may arise from their presence as well as the benefits usually obtained. Adaptive immune clearance mechanisms often involve the interaction of specific antibody with the antigenic or microbial target, leading to the formation of immune complexes in the circulation or in tissues. This interaction in turn often activates the complement system leading to yet another series of downstream consequences. This chapter reviews aspects of immune complex formation and pathophysiology, and the role of complement in the immunologic and inflammatory response. Investigators over a century ago recognized that the immune system responds to a foreign protein with the formation of antibody. Shortly thereafter, horse anti-diphtheria toxin antiserum was found to be successful in treatment of children with diphtheria; tens of thousands of such children were treated and a new illness was noted that had not been recognized earlier. This illness was systemically studied by von Pirquet and Schick (1905). They noted that the administration of horse serum did not produce immediate toxicity. However, 5–8 days later, the children frequently developed urticaria, particularly at the site of subcutaneous injection of the horse serum, fever, lymphoadenopathy of the draining lymph nodes in the region of the injection, leukopenia, edema, and proteinuria. This toxic effect of the horse serum disappeared over the next 4 or 5 days. In their careful clinical investigation of these phenomena, they came to realize that the phenomena were due to formation of antibody in the recipient to the infused horse protein, the formation of

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antigen–antibody complexes in the circulation, and toxicity derived from these antigen–antibody complexes. They further appreciated the fact that if a child was reexposed to horse serum at a later time the symptom complex reappeared, often in more severe form and much more rapidly. This was the first demonstration in humans that products of the immune response could be damaging as well as beneficial. They termed this phenomena “serum sickness” as it was brought on by the administration of horse antiserum. Further understanding of serum sickness was limited until the 1950s when radiolabeled proteins became available for study. In a classic series of studies, radiolabeled bovine serum albumin (BSA) was given to rabbits intravenously and the effects of the infusion followed (Dixon 1963). It was noted that there was a period of equilibration of the BSA in the circulation, followed by the normal degradation of the protein with its slow disappearance from the circulation. In the rabbit, as in humans, at 5– 8 days there was a sudden acceleration of clearance of the injected protein, as the animal made antibody, and a depression in serum complement associated with the appearance of circulating immune complexes in the rabbit’s blood. When the immune complexes were cleared some days later, free antibody appeared in the rabbit’s circulation. These animals developed marked organ pathology during the period of circulating immune complexes that resolved after the complexes were cleared from the circulation. An important feature of this disease in the rabbit was that it was much more severe than the disease usually associated with injection of horse serum in children with diphtheria. In rabbits, serum sickness included arthritis, glomerulonephritis, and vasculitis. The glomerulonephritis was characterized by proteinuria and swelling of endothelial cells, but there was little hematuria and few red cell casts. Neutrophils did not seem to be very important in the development of this acute glomerulonephritis, and, in fact, it was not clear that complement was important either, although C3 was deposited along the glomerular basement membrane in a typical granular pattern. Arteritis in this acute serum sickness in rabbit was noted at branching points of the aorta. In this case, both neutrophils and complement appeared to be required for the development of the lesions. A chronic rabbit model of serum sickness was also developed in which antigen was given repeatedly over a period of weeks. It was found that to develop chronic glomerulonephritis in these animals, it was important the animals made an immune response with circulating antibody, and the amount of the antigen given had to be titrated such that it overwhelmed the antibody at the time of each injection. If insufficient antigen was given, it was removed from the circulation rapidly by the large amounts of circulating antibody, and glomerulonephritis was not seen. If antigen excess was achieved, proteinuria became so severe that it caused a nephrotic syndrome. The striking thing about this model was

that it mimicked many of the features seen in patients with systemic lupus erythematosus (SLE). Studies showed that patients with SLE have circulating immune complexes to a wide variety of antigens, particularly antinuclear antibodies and antibodies to double-stranded DNA, and may develop vasculitis and glomerulonephritis, similar to the rabbits with circulating immune complexes. Thus, it was concluded that lupus erythematosus is the prototype of an immune complex disease in humans and that the rabbit model was an excellent model for defining the pathology of lupus. As work proceeded and many studies were moved into murine models, a variety of immune complex-mediated lupus-like models were also developed in mice. Studies in mice were particularly useful as, with the advent of genetic homologous recombination technology, it was possible to explore systematically the factors that lead to immune complex formation and tissue destruction in animals missing various proteins important in disease. Nevertheless, it is fair to say that there are still many features of immune complex pathology that are not fully understood, and there is a considerable way to go before we have detailed insight into these immune complex models. With the advent of a convenient model in rabbits of immune complex pathology, many studies were performed to determine the mechanisms by which immune complexes cause disease. It was clear that when immune complexes are formed in the circulation, they are rapidly removed by the cells of the mononuclear phagocyte system or, as it was originally termed, the reticuloendothelial system (Aschoff 1924). The majority of these cells are in the liver, but the spleen makes an important contribution to the clearance of immune complexes from the bloodstream and even the lung has some contribution to make. It was shown that when one forms immune complexes in the test tube and injects them into rabbits, high-molecular-weight complexes (greater than 11S in sedimentation characteristics) are rapidly removed from the circulation and that these appear to be responsible for the development of disease (Mannik et al. 1971). However, studies showed that experimentally it was extremely difficult to cause preformed immune complexes to be deposited in tissues like the kidney and, in fact, in most situations, only very tiny amounts of such preformed immune complexes escape the circulation to be deposited in tissues (Mannik & Arend 1971). The role of complement in immune complex clearance was studied by depleting animals of C3, alternative pathway proteins, and the late-acting complement components, by the injection of cobra venom factor (Mannik & Arend 1971). This protein, known for a century and isolated from the venom of cobras, is a C3b analog that causes massive alternative complement pathway activation and, therefore, depletes the animal of complement. The nature of the C3b fragment and the mechanism of its action are discussed later in the chapter.

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The use of cobra venom factor in studies of clearance of immune complexes from the circulation suggested that complement plays no role in their clearance: the rates of clearance of immune complexes were exactly the same in cobra venom and treated and control animals. Injected complexes were removed rapidly from the circulation and it was found that to have complexes deposited in tissue sites, they had to circulate for a prolonged period. One way this could be accomplished was by reduction and alkylation of disulfide bonds in the antibody used to form the complexes (Haakenstad & Mannik 1976). This chemically altered antibody interacted poorly with complement and poorly with cellular receptors for complement and antibody. The complexes continued to circulate for a longer time and were more likely to be deposited in tissues (Haakenstad et al. 1976). The characteristics of antigens associated with immune complex disease pathology were examined systematically. It was found that charged antigens, such as cationically charged proteins, were more likely to cause glomerulonephritis than neutral or negatively charged proteins (Gauthier et al. 1982). Presumably, the negative charges on the basement membrane of the glomerulus bound these positively charged proteins and facilitated their deposition in the kidney. Other factors that were determined to be important were the size of the antigenic protein, the degree of “foreignness,” hydrophobicity, and the ability to form a lattice with antibody (Berzofsky & Berkower 1993). IgG, IgE, and monomer IgA are divalent. IgM has five effective binding sites. IgG deposition in tissues is characteristic of immune complex diseases and the interaction of IgG with antigens was studied in detail. In the presence of antigen excess, that is, far more antigen than specific antibody in a mixture, antigen molecules will bind to each of the IgG antibody binding sites. Theoretically two antigen molecules can bind to each divalent antibody molecule. The resulting product is soluble. Similarly, in vast antibody excess the resulting product is usually soluble. Here, the number of antibody molecules that bind to each antigen is determined by the size of the antigen, and the number of antigenic groupings on the antigen surface. Thus, if the antigen has ten antigenic groupings, ten molecules of antibody theoretically can combine with each antigen, each by one of its two binding sites. In general, this combination still leads to soluble products. In the zone of equivalence, where neither the antigen nor antibody is in excess, there tends to form a lattice in which antibody molecules crosslink antigens and in the test tube form a precipitate. It was in slight antigen excess or equivalence that maximum pathology was observed in the animal models. The characteristics of antibody also were found to be important in determining the nature of the immunopathology observed. In most of the diseases studied, IgG antibody appeared to be more responsible for clinical symptomatology than IgM, although clearly there are situations in which IgM immune complexes are present. For example, in some cases

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of cryoglobulinemia, IgM antibody complexed to hepatitis C virus antigen on the virus surface may be responsible for the cold precipitins found in serum (Gertz 2005). Nevertheless, in most diseases, it appeared that IgG deposition was responsible for pathology. It was recently noted that the degree of sialation of IgG increases during immunization and the degree of sialation can be correlated with biological effect (Kaneko et al. 2006). IgA complexes were found present in such diseases as Henoch–Schönlein purpura, but these situations appeared to be in the minority (Coppo et al. 2006). The ability of the antibody to activate complement proved to be important (Cochrane & Koffler 1973). Although this did not appear to be true for clearance of the complexes, it did seem to be required for maximum damage, and often complement proteins were found deposited in the tissues at sites of inflammation. It was found that certain IgG antibodies, as well as IgM, activate the classical complement pathway. This is discussed in greater detail later in the chapter. The proteins or components of the classical pathway are activated by antibody binding to its antigen and in turn binding C1, C4, C2, and C3 of the classical pathway. A second pathway of complement activation, the alternative pathway, was later discovered, and it was found that antigen–antibody complexes of many classes and isotypes can activate this pathway and bind C3 (Thurman & Holers 2006). In early investigations, there was a particular focus on the ability of IgA to activate the alternative pathway, as it was found that IgA could not activate the classical pathway and C3 was often found in the IgA containing lesions in Henoch–Schönlein purpura. The lectin pathway, found more recently, has a reaction mechanism similar to that of the classical pathway (Jensenius 2005). It differs in that the recognition element is not antibody and C1, but mannose-binding lectin (MBL), a serum protein that recognizes the repeating sugar structures on many pathogens. The classical pathway is activated by IgM and IgG subclasses 1, 2, and 3. Subclass 4, like the other subclasses, activates the alternative pathway, but it does not activate the classical pathway to induce complement-mediated inflammation (Walport 2001a,b). The affinity of the antibody for antigen also proved to be important; in general, high-affinity interactions activate more complement and cause more damage than low-affinity interactions (Fauci et al. 1970). Because it proved difficult to develop models in which preformed immune complexes leave the circulation to deposit in tissues, there was interest in the possible formation of the immune complexes within the tissue that was damaged. It was shown that in some cases an antigen could be planted or deposited in the kidney and that antibody diffusing through tissue could meet that antigen, form immune complex in situ, and cause the immunologic damage observed (Nangaku & Couser 2005). Although in situ complex formation clearly occurs, and it has been shown that a renal metalloproteinase is the antigen in one rare type of membranoproliferative glomerulonephritis, it is not clear exactly how important this phenomenon

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is, and people have turned their attention back to deposition of circulating immune complexes as a major cause of disease. Our group was able to perform the only reported prospective study of serum sickness and immune complex disease in humans and the results are quite instructive (Lawley et al. 1984). At the time of our study, there was great interest in the use of horse anti-thymic globulin for treating patients with aplastic anemia. It had been found that this treatment leads to remission of disease in about one-third of patients. The treatment had been instituted to prevent graft-versus-host disease in patients who were being prepared for bone marrow transplantation, but in this one-third of patients the bone marrow transplantation was not needed. All patients receiving horse antithymic globulin were treated with high-dose steroids but, even so, they developed serum sickness. As in the original studies of von Pirquet and Schick, the serum sickness started 5– 8 days after the injection of horse antithymic globulin. Unlike the patients studied by von Pirquet and Schick, these patients, who presumably had immunologic abnormalities leading to aplastic anemia to begin with, and who tended to be adults rather than children, became far more ill. It was shown that they had circulating immune complexes at the time that they became ill and a striking fall in serum complement. At the time that immune complexes were in the circulation, they developed urticaria, but also a variety of other skin lesions. They developed mild albuminuria, edema, hematuria and decreased creatinine clearance, severe arthralgias, which in some patients progressed to arthritis, leukopenia, an increase in blood detected in the stool, an increase in hepatic transaminases, and many of the features that we consider part of the lupus syndrome. All these symptoms cleared when the immune complexes disappeared from the patients’ circulation. Thus, it was possible in this prospective study to show that many of the symptoms that we associate with immune complex diseases like SLE are associated with circulating immune complexes patients with a propensity to develop autoimmune disease. More recently, attention has turned to the molecular factors that promote the pathologic effects of immune complexes. As mentioned, it is known that the removal of immune complexes from the circulation is the responsibility of phagocytes predominantly of the liver and spleen. These cells have a wide variety of receptors including complement receptors and receptors for the Fc fragment of IgG (FcγR), as well as receptors for IgE, etc. It is known that in the mouse there are four groups of IgG Fc receptors. It is assumed that there are four groups of FcγR in humans as well, but only three groups have been identified and studied thus far (Ravetch & Bolland 2001; Salmon & Pricop 2001). These groups of receptors have extracellular domains that recognize the Fc fragment of IgG. We focus on IgG receptors in this chapter as receptors for IgE are considered in detail elsewhere in this book. In general, these receptors are associated with transmembrane proteins

and, usually, these receptors are associated with signaling functions within cells. FcγR have been divided into “activating” and “inhibiting” groups of receptors. Activating receptors include FcγRI, FcγRIII, and FcγRIV in the mouse, and FcγRI, FcγRIIA, and FcγRIII in humans. These receptors do not signal directly, but in most cases have an associated membrane protein, the γ chain, with a large intracytoplasmic tail that transduces the activation signal. Activation of the receptor is associated with phosphorylation of the γ chain. The γ chain resides in the membrane as a dimer or is associated with another chain, the ζ chain. All the receptors are transmembrane proteins except FcγRIII which exists in two forms, FcγRIIIa and FcγRIIIb. FcγRIIIa is a typical transmembrane protein while FcγRIIIb is linked to the membrane by a phosphatidylinositol linkage. Receptor activation leads in turn to phosphorylation of a series of intracellular signaling molecules including src and syk protein kinases, ultimately leading to cellular activation. FcγRIIB is the only FcγRII receptor in the mouse. It and FcγRIIB in humans are inhibitory receptors. Inhibitory receptors differ from the activating receptors in that they do not transmit their information via phosphorylation of the γ chain, but via an ITIM (immunoreceptor tyrosine-based inhibition motif) or inhibition motif on the intracellular portion of the Fc receptor. This is a 13-amino acid sequence in the cytoplasmic domain that becomes tyrosine phosphorylated upon activation, creating a binding site for an inhibitory signal protein, SHIP (SH2-containing inositol phosphatase), that hydrolyzes activated membrane inositol phosphate, thereby disrupting immunoreceptor tyrosine-based activation motifs and B-cell receptor-mediated calcium influx. Also, other cellular activation pathways are inhibited by ligation of the ITIM-bearing protein. Many studies have assessed the function of FcγR in the development of autoimmunity and in the development of the immune response. FcγRI in both human and mouse is a high-affinity receptor (KA ∼ 1 × 108 − 1 × 109) (Nimmerjahn & Ravetch 2005). Under usual circumstances, it is expected to be complexed with serum IgG and, presumably, crossligation of receptors is important in its function. Its function is the most poorly understood of the receptor group. FcγRII and FcγRIII are relatively low-affinity receptors (KA ∼ 1 × 107 and 3 × 107, respectively), and when cells with these receptors are suspended in serum and then washed there is little IgG bound to their surface. These receptors are inefficient at binding ligand, except in the presence of large immune complexes. In that situation, multiple receptors can be occupied by the antigen–antibody lattice leading to cellular activation. In the mouse, FcγRIV is of somewhat higher affinity than the lowaffinity receptors and, presumably, is a typical activating receptor. As mentioned, FcγRII in humans includes both the activating receptor, FcγRIIA, and inhibiting receptor, FcγRIIB. These receptors are almost identical in their extracellular region and, therefore, bind ligand similarly but, as mentioned, the crosslinking of these receptors has exactly the opposite

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Table 19.1 In vivo phenotypes. Deleted gene: FcgRIIB Enhanced skin and lung Arthus reaction Enhanced IgG- and IgE-induced systemic anaphylaxis Enhanced anti-GBM (glomerular basement membrane) antibodyinduced glomerulonephritis Enhanced bleeding in anti-aIIBb3 antibody-induced ITP Enhanced collagen-induced arthritis Development of Goodpasture syndrome Spontaneous development of glomerulonephritis in B6 background Deleted gene: FcgRIII Attenuated skin and lung Arthus reaction Attenuated anti-GBM antibody-induced glomerulonephritis Attenuated cryoglobulin-induced vasculitis Reduced sensitivity to autoimmune hemolytic anemia Reduced sensitivity to hypothermia and bleeding in ITP Reduced sensitivity to anti-GPI antibody-induced arthritis

effect on cells. These, like FcγRIII, are low-affinity receptors and require crossligation to have their effect. Mice have been generated by targeted gene deletion that are missing each of the receptors or the gamma chain common to the activating receptors. In general, mice missing the activating receptors have a decreased immunopathologic response in all of the models of immunologic disease in mice (Table 19.1) (Schmidt & Gessner 2005). Thus, immune complex deposition in the skin, with resulting vasculitis, a phenomenon called the “Arthus reaction,” is decreased in these animals. Similarly, they have attenuated immune complex-induced glomerulonephritis. They have attenuated cryoglobulin-induced vasculitis, reduced sensitivity to antibody-induced autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (ITP), and reduced development of antibody-induced arthritis. As might be expected, mice deficient in the inhibitory receptor, FcγRIIB, on the other hand have an enhanced immunologic reaction in each of these areas, including Arthus reactions in the skin and lung, glomerulonephritis, ITP, collagen-induced arthritis, Goodpasture-like syndrome, and indeed they even have enhanced IgG- and IgE-induced systemic anaphylaxis. This large group of studies with similar findings suggests that Fc receptors are of critical importance in the development of autoimmunity, not only in animals, but also likely in humans. In the past several decades, there have been additional studies in animals and humans of the efficiency of the mononuclear phagocyte system in the removal of immune complexes from the circulation. In these studies, the definition of immune complexes has varied. In initial studies that we performed years ago, we radiolabeled animal or patient red cells with chromium-51, sensitized them with antibody, and followed their rate of clearance from the circulation after sensitization of the cells with a known amount of antibody (Frank et al. 1977, 1983). In animals and humans we studied

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the effect of IgG and IgM antibody. We had available guinea pigs deficient in the classical pathway protein C4. As the antibodies studied did not appear to activate the alternative pathway, we could study the effect of IgG and IgM Fc fragment-mediated clearance and complement-related clearance independently. It was found that IgM coating of red cells did not cause clearance of the red cells in C4-deficient animals; complement was responsible for the clearance of cells coated with IgM. In this case, the IgM-coated cells were cleared by the liver. IgM plus complement on a target cell is a weak stimulus for phagocytosis and these cleared cells were never phagocytosed efficiently, unless the phagocytes were activated by various inflammatory cytokines. In the normal resting state, the cleared cells remained stuck to the surface of Kupffer cells for a period of time, but then were released back into the circulation as the C3 on their surface became degraded to a nonopsonic form by mechanisms discussed later, and they circulated as Coombs-positive erythrocytes with normal survival. In striking contrast, IgG is a strong stimulus for phagocytosis, and in the presence of IgG antibody the cells were efficiently removed from the circulation and phagocytosed. Interestingly, about 100 or 200 complementactivating IgM antibody sites per red cell were required for removal of the red cells from the circulation, while only one or two complement-fixing sites were required for removal of the IgG-coated cells from the circulation. Perhaps the fact that they had less C3 on their surface is the reason the IgGcoated cells tended not to be cleared by the liver, but by the spleen. As 50% of the cardiac output goes to the liver, one must assume that the IgG- and complement-coated cells pass through the liver to ultimately be cleared by the spleen. Presumably, this occurs because there is insufficient complement on their surface to be recognized and bound by Kupffer cells, and the Fcγ receptors, partially inhibited in function by plasma concentrations of IgG, require an extensive array of IgG bound to antigen for effective binding and clearance. In humans, it was also possible to show that IgM-coated erythrocytes required complement for clearance, and the clearance mechanisms and survival of the cells seemed very similar to that in guinea pigs. For IgG-coated erythrocyte clearance, our studies used anti-Rh antibody. Rh antibody has the major advantage that it does not activate complement efficiently and, therefore, any removal of Rh IgG-coated cells from the circulation requires IgG Fc receptor activity. We showed that these Fc receptors cause progressive removal by the spleen of the red cells from the circulation. Moreover, we found a defect in such clearance in patients with lupus erythematosus and various other autoimmune diseases associated with the deposition of immune complexes in tissues. We hypothesized that the Fc receptor defect might delay the removal of the complexes from the circulation and thereby facilitate the ability of the complexes to escape from the circulation and be deposited in tissues, where they could cause inflammation and tissue damage.

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Later experiments in both animals and humans by others, studied the removal of antigen–antibody complexes formed with tetanus toxin antitoxin or hepatitis B surface antigen– antihepatitis B antibody (Davies et al. 2002). Unlike the antibody-sensitized red cells, these are true soluble complexes. In this case again, radiolabeled complexes were introduced into the circulation and their clearance was studied. It was found in these studies that patients with lupus erythematosus have more than the defect in splenic clearance that we observed in our studies of Rh-coated red cells. These additional studies showed that in humans the clearance from the circulation of these immune complexes by the liver was hyperfast. Thus, patients with lupus, as well as animals in lupus models, had an increased clearance of the complexes by the liver and a decreased clearance by the spleen. The reasons for this have never been fully explored, but we hypothesize that the difference has to do with the fact that the soluble immune complexes injected into patients or animals activate complement and can interact with both IgG Fc receptors and complement receptors simultaneously, providing much more efficient hepatic sequestration. In patients and animals with autoimmune diseases, this combination of antibody and complement combined in an immune lattice, allows for efficient hepatic sequestration by activated Kupffer cells. In summary, immune complexes are known to form regularly in healthy individuals and to be removed from the circulation without causing any toxicity. In patients with a number of autoimmune diseases, unlike in healthy individuals, immune complexes deposit in tissues to mediate various types of pathology for as yet unclear reasons. It is clear that receptors for antibody and complement are important in immune complex clearance, and it is believed that the receptors are not functioning normally in these patient populations. These various receptors are responsible for cellular activation and inhibition signals and it is thought that ultimately an abnormal signaling process contributes importantly to disease manifestations.

Introduction to complement Complement is a term originally introduced 100 years ago to designate factors in fresh serum that in the presence of specific

Lectin pathway 1990

Classical pathway 1890

antibody are able to kill microorganisms (Frank 1998). Later work showed the killing principle was heat-labile. Over time, it was found that this term is a collective one for a group of about 30 known proteins and protein regulators, some of which circulate in the blood and some of which are cell membrane-bound, which play a role in host defense and innate immunity. In recent years, it has become clear that complement is also important in the afferent limb as well as the efferent limb of the immune response. Phylogenetically, the complement proteins are ancient, being present in primitive animals where they serve a host-defense function, even in the absence of any immune system. The adaptive immune system appears in evolution at the level of the fish and, by this point in evolution, all the various complement proteins are arrayed to produce their regulatory and host-defense functions. Over the years, three pathways of complement activation have been identified (Fig. 19.1) (Walport 2001a,b). The first pathway was defined almost a century ago and for this reason is termed the “classical pathway.” It is this pathway that is usually activated by antibody and that is usually responsible for the killing of microorganisms and cells in the presence of specific antibody. A second pathway, first noted in the 1950s but studied in more detail in the 1970s and 1980s is termed the “alternative pathway” (Thurman & Holers 2006). The alternative pathway is thought to be phylogenetically older than the classical pathway; it does not require antibody to function and predates antibody in phylogenetic development. Although antibody is not required for function, the presence of antibody usually allows this pathway to function more efficiently. A third pathway described in the past two decades is still being defined in detail. This pathway, termed the “lectin pathway,” is also phylogenetically ancient and does not require antibody to function (Jensenius 2005). All pathways proceed through a series of proteins that will be discussed below, to the activation and binding of a plasma protein, C3, which is central to all three pathways. The pathways then proceed together through the binding of an additional series of proteins to the lytic steps in complement action. Complement is generally believed to have three major functions in the effector steps in host defense. The most striking and the first defined is its lytic function. On binding to an antibody-sensitized microorganism or cell, complement

MBL MASP 1,2,3

C1q, C1r, C1s

C4

C2

C4b,2a C3

C5 to C9 lysis

C3(H2O)Bb, P Fig. 19.1 The complement pathways. MBL, mannose-binding lectin; MASP, MBL-associated serine proteases.

Alternative pathway 1970

C3(H2O)

Factors B, D, Properdin (P)

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may cause direct lysis. Lysis is due to the formation by the late-acting complement proteins (C5, C6, C7, C8, and C9) of a doughnut- or cylinder-like structure with a hydrophobic exterior and a hydrophilic center, through which water and electrolytes can pass freely. The hydrophobic exterior allows the doughnut-like structure to insert into the lipid membrane of cells and microbes. It floats in the membrane and is oriented such that the hydrophilic center allows free flow between the interior of the cell or microbe and the exterior environment surrounding the cell. The cell cannot maintain its osmotic equilibrium; it swells and lyses. The second and biologically more important function of complement is the ability to opsonize particles, that is coat the particles with complement-derived protein fragments that allow them to be phagocytosed easily. Phagocytic cells have on their surface specific receptors for complement-derived peptides, often cleavage fragments of C3. When these fragments are deposited on microbes, they can link the microbe to the phagocyte receptors; the adherence facilitates the phagocytic process. The third important effector function of complement is generation of inflammatory peptides. Many of the plasma complement proteins circulate in an inactive form and on activation of the complement system are cleaved, with the formation of a large fragment and a small fragment. In most cases, the large fragment continues the lytic sequence and the small fragment has inflammatory activity. For example, the small fragment of C5, C5a, can cause mast cells to degranulate and release histamine, as if they were coated with IgE and antigen. It can cause migration of phagocytic cells toward the place where the peptide is generated, that is induce chemotaxis. These peptides have a variety of other inflammatory properties including the contraction of smooth muscle, and even may have direct bactericidal activity against a variety of bacteria and other microbes (Hugli 1986; Nordahl et al. 2004). Complement proteins are rarely completely absent because of genetic abnormalities and usually the system is acting appropriately, that is damaging cells and tissues that it recognizes as foreign (Frank 2000; Sjoholm et al. 2006). However, although it is extremely rare to have someone missing a complement protein, it is quite common to have complement causing immunologic damage under inappropriate circumstances. Thus, for example, if one produces an abnormal antibody to the basement membrane of the glomerulus, and the antibody binds to the glomerulus, activates complement, and causes inflammatory damage, the complement is acting normally. It is the antibody that is inappropriate. Complement is found in the kidneys, deposited in glomeruli in glomerulonephritis, and along the glomerular basement membrane in Goodpasture disease. It is found in vasculitic lesions, etc. In all these locations, it is presumed that complement is contributing to local tissue damage because of inappropriate activation. Because of these untoward effects of inappropriate activation, there are control proteins that downregulate

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activated complement proteins at each step in the various cascades of reaction. The importance of these proteins is that they prevent unwanted damage of one’s own tissues and cells. Although the absence of complement proteins is unusual, the absence of control proteins is more common, and patients who have absent control proteins may have ongoing “autoimmune” immunologic disease. Moreover, as mentioned in our discussion of the lectin pathway below, there are a sizable group of patients with allotypic variations of MBL that lead to very low levels of this circulating protein. To this point we have discussed effector functions of complement in host defense and it is this aspect of complement function that has received most attention over the years. It is clear that complement also functions in the afferent phase of the immune response, but here there has been far less study and less information is available (Carroll 2004). One reason that information on this important function of complement is coming to light slowly, is there are so few complementdeficient individuals to study. With the advent of knockout gene technology it has been possible to develop mouse strains missing one or more complement proteins or complement receptors, and it has been found that these animals have defects in development of many aspects of the normal murine immune response (Holers 2000). This aspect of complement physiology is discussed toward the end of this chapter.

The classical pathway The classical pathway is usually activated by antibody. IgM, and IgG subclasses IgG1, IgG2, and IgG3, bind the first component of complement to activate the classical pathway (Walport 2001a,b). C1, the first acting component, exists in serum as a three-part molecule (C1q, C1r, C1s) held together in the presence of ionic calcium. C1q has a central protein core and six radiating arms each ending in a pod-like protein domain that recognizes the Fc fragment of IgG or IgM. Each of the six arms is made up of three intertwined chains C1q A, B, and C, and has a triple helix structure like collagen, providing great flexibility. In the case of IgG, the binding of multiple IgG molecules to an antigenic surface allows binding of multiple arms of C1q, each to an Fc fragment with sufficient affinity of the C1q to allow C1 activation. In the case of IgM, a single molecule bound to an antigenic surface by multiple binding sites, with the availability of five Fc fragments within one molecule, is sufficient to bind C1q and activate the classical pathway. On binding of C1q to antibody, a distortion of the C1q molecule takes place, which in turn causes autoactivation of C1r, which then activates C1s. There are two single-chain molecules each of C1r and C1s associated with each C1q, and activation is associated with cleavage of each of the two molecules of C1r and C1s into a short fragment and a long fragment. In each case the short fragment formed has an active enzymatic site that continues the complement cascade. C1

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requires calcium for selfassociation and, therefore, the classical pathway requires calcium for initiation. One function of active C1 is to bind and cleave C4, the next protein in the activation sequence. As with cleavage of the C1 chains, C4 is cleaved into a large fragment, C4b, and a small fragment, C4a. The large fragment continues the complement cascade and the small fragment, like small fragments of C3 (C3a) and C5 (C5a) later acting proteins in the sequence, has anaphylatoxic activity. All of these fragments are able to cause mast cell degranulation with resulting histamine release. Upon activation of C4, a thioester-containing binding site is exposed on the C4b generated, which allows covalent attachment of the C4b to the target of covalent attack. Binding can be via an ester or an amide linkage. The nature of the binding site on C4 and C3 is similar and is discussed in further detail in the section on the alternative pathway. The C14 site, now bound to a target, allows C2 binding to the C4b portion of the site. On C2 binding to the C14b site, C1s cleaves the C2 also into a large and small fragment. Again the large fragment, C2a, remains bound to the assembling protein complex and the small fragment, C2b, is released. C2 binding to C4 requires the presence of ionic magnesium. The new site, consisting of C4b and C2a, no longer requires C1 for activity. Enzymatic activity resides in C2a. This site, termed the “C3 convertase” of the classical pathway, can bind the next complement protein in the sequence, C3, to continue the complement cascade. C3 is cleaved into a large fragment, C3b, and a small fragment, C3a, which again has inflammatory activity. Just as in the case of C4b, C3b can bind covalently to the target of attack. In many cases it binds directly to the C4b on the target. As mentioned earlier, C3 is the central component of all three complement pathways and is present at high concentration in serum, about 1.2 mg/mL. On the addition of C3b to the C4b, C2a site, a new binding site is created that can bind C5, the next protein in the sequence. Again, C5 is cleaved into a large fragment and a small fragment. The large fragment, C5b, continues the complement cascade, although it does not form a covalent bond with the target; it remains associated with the C3b. The small fragment released, C5a, is one of the most potent inflammatory peptides released by complement activation and has strong neutrophil aggregating activity, strong neutrophil chemotactic activity, and is an excellent anaphylatoxin. Injection of a large amount of C5a into an animal will cause anaphylaxis and death due to neutrophil aggregation in the circulation and massive histamine release (Hugli 1986; Shushakova et al. 2002). The complement cascade continues following C5b binding with the binding of C6, C7, C8, and C9. One molecule of C6 and C7 each bind to C5b on the target surface. If this binding takes place at the surface of a cell or microbe, the introduction of C7 to the binding site leads to an increase in hydrophobicity of the C5–C7 complex and insertion of the complex into the lipid cytoplasmic membrane of the cell. Under these circumstances, the cell is targeted for lysis. With the binding of one

molecule of C8 to the C5–C7 complex, a slow leak in cells such as erythrocytes appears, and with the binding of up to 16 molecules of C9, a cylinder or doughnut-like structure is formed, containing all the proteins C5b–C9, that penetrates the cell membrane leading to rapid lysis. Cells protect themselves from lysis in a variety of ways. The many complement-control proteins are discussed in greater detail below, but also the lytic C5b–C9 complex can be shed from the surface of some cells or internalized and destroyed, as the cell attempts to protect itself from damage. Cells such as erythrocytes with little intracellular protein synthetic machinery to help repair their membranes, rely on the control proteins for protection. Cells such as macrophages and endothelial cells have these extra mechanisms for clearing their membranes of deposited complement proteins.

The lectin pathway The lectin pathway unlike the classical pathway does not require antibody to function and appears to be developmentally more primitive than the classical pathway. Nevertheless, it is quite similar in function (Jensenius 2005). In the classical pathway, the recognition molecule that sees the foreign antigen and induces complement activation is antibody. The lectin pathway does not use antibody but has its own recognition molecule. The pathway is initiated by a plasma protein, MBL, or by related proteins, the ficolins. MBL has a structure rather similar to C1q, with a central core and a series of radiating arms composed of a flexible triple helix, each ending in a binding structure. Unlike C1q, in MBL the helix contains three copies of a single chain. In the case of C1q, the binding structure at the end of the arms recognizes immunoglobulin and it is the binding of the antibody that is the critically important feature of the activation sequence. In the case of MBL, there are three lectin-binding sites at the termination of each of the arms of the MBL. Each lectinbinding site has low affinity for sugars like mannose but with the binding of multiple arms of the MBL, each with three binding sites to, for example, the repeating polysaccharides on the surface of a bacterium, the association is stabilized and the complement pathway is activated. Therefore, the protein that recognizes the foreign structure is not specific antibody, but one of the complement proteins, MBL. MBL circulates as a series of multimers and may have two arms, four arms, or six arms. In general, it is thought that the four-arm structure predominates. Associated with MBL is another group of proteins called MASPs (MBL-associated serine proteases). The functional structure again resembles C1, as C1q, the subunit with collagen-like arms that binds to antibody, also has associated with it serine proteases, C1r, and C1s. In the case of MBL, the associated serine proteases are MASP1, MASP2, and MASP3, as well as some other related molecules. It is believed that

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MASP2 is the principal serine protease involved in continuation of the complement cascade, with MASP1 also active. The function of MASP3 is not known at this time and it is not clear why two MASPs, MASP1 and MASP2, exist with much the same function. Currently, it is thought that the main path of activation following MBL binding is via activation of C4 by MASP2. Thus, lectin pathway activation is very much like classical pathway activation. In the classical pathway, antibody is the recognition molecule followed by C1 activation. In the lectin pathway, the recognition molecule is MBL and MASP2 is the C1-like molecule that cleaves C4 into C4a and C4b. The C4b then binds C2, the C2 is cleaved by MASP2, and the pathway continues to C9, just as in the classical pathway.

The alternative pathway The alternative pathway, probably the oldest of the complement pathways in phylogenetic terms, is more difficult to understand in that it operates by a mechanism which is fundamentally different and more primitive than that of the classical and lectin pathways. In these latter two cases, the pathway is specifically activated by a recognition molecule that binds to the target of attack and activates a serine protease; the protease then continues the complement sequence. In the alternative pathway, C3 is itself the recognition molecule and activation of the pathway is inefficient. C3 is a two-chain molecule, α and β, with an internal thioester joining a cystine at position 988 and a glutamine at position 991 in the α-chain backbone. The tertiary configuration of the molecule protects the internal thioester from cleavage through nucleophilic attack by water; however, even so, it undergoes slow hydrolysis in the circulation. When water penetrates to the thioester bond, the bond undergoes hydrolysis with cleavage of the thioester, leaving a free sulfhydryl at position 988 and a hydrated carboxyl ion at position 991. This is associated with a marked change in tertiary structure and the molecule comes to resemble C3b. Hydrolyzed C3, like C3b itself, is capable of binding factor B, a protein of the alternative pathway very much like C2 of the classical pathway. On binding to hydrated C3 or C3b, factor B can be cleaved by a serine protease, very much like C1s of the classical pathway, termed factor D. Thus, a protein complex is formed consisting of hydrated C3 or C3b and the large fragment of cleaved factor B, termed Bb, with the release of the small fragment Ba. This complex is the C3 convertase of the alternative pathway. It may bind a new molecule of C3 and cleave it into C3a and C3b. The major difference between the C3 convertase (C3 cleaving enzyme) of the alternative pathway and the C3 convertase of the classical and lectin pathways, is that there is no C4b in the convertase of the alternative pathway. C3b itself takes the place of the C4b with factor B acting like C2 and factor D acting like C1. A sec-

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ond difference is that factor D, the molecule that resembles C1s of the classical pathway and MASP2 of the lectin pathway, is not physically bound to the active site, but acts as a fluid phase enzyme. The initial alternative pathway C3 convertase (C3(H2O)Bb) binds and cleaves another molecule of C3. When C3a is cleaved from C3b, the thioester becomes immediately available. If this cleavage occurs close to the surface of a cell or microbe, the carboxyl on the C3b generated can form an ester or amide bond with the surface of the cell or microbe. This target-bound C3b can accept another factor B molecule and in the presence of factor D can cleave more C3 into C3a and C3b, with more C3b becoming target bound. In the case of the classical and lectin pathways, the C42 complex is unstable and slowly decays. In the case of the alternative pathway, the C3bBb complex is also unstable. It rapidly decays and is stabilized in the circulation by yet another protein termed “properdin.” Like the classical pathway convertase, the alternative pathway convertase requires magnesium ions to function. Presumably, the first pathway to develop in the complement system, in terms of phylogenetic development, was the alternative pathway. Because initiation is not directed and requires, by chance, hydrolysis of C3 close to the target of destruction with binding of additional C3 to the target, it is inefficient. It is believed that the lectin pathway evolved to recognize the target more directly, and with the appearance of antibody the target could be even more specifically identified. C3b deposited on a target by the lectin or classical pathway can also engage proteins of the alternative pathway to further amplify C3 deposition. C3b undergoes a complex sequence of degradation steps with each degradation product having different biological activity. Because these steps are regulated by control molecules they are considered in the sections below.

Complement receptors and complement control molecules By definition, complement receptors recognize and bind various complement proteins and fragments. As with other receptors, this may cause cellular activation. However, unlike most cellular receptors, some of the complement receptors also act as control molecules. They may interact with the molecules they bind to allow for further degradation of the bound fragment. In performing this function, the receptors act like the many complement control molecules that also regulate the activity of complement proteins to control their biological function. As has been mentioned, C3 has many functions in the complement system and many of the control molecules regulate C3. Four of the major complement receptors (CR1, CR2, CR3, and CR4 in one terminology) bind fragments of C3. Nevertheless, for clarity we will take each step in the complement cascade of reactions in turn. At virtually each step of the complement cascade, control points are established to downregulate the possibility of untoward complement activation.

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Control of activity of C1 and MASPs

Control of the activity of C4 and C2

In the classical pathway, the activation of C1 with cleavage of C4 is downregulated by C1 inhibitor (Han Lee et al. 2003). This single chain molecule is a serpin, (serine protease inhibitor). Inhibitors of this class present a bait sequence to the enzyme to be inhibited. When an enzyme cleaves the inhibitor at the site of the bait sequence (amino acid 444 of the C1 inhibitor), the inhibitor springs apart uncovering a highly reactive site that forms a covalent bond with the active site on the enzyme. The C1 inhibitor has been termed a “suicide” inhibitor, as it is used up during the inhibition process. It functions to inhibit both activated C1r and C1s. Two molecules of inhibitor interact with each of the two C1r active sites and two molecules of interact with each of the two C1s active sites in C1 as part of the inhibition process. Therefore, four molecules of C1 inhibitor are used up, with each molecule of active C1 inhibited. During the process of C1 inhibition, the C1 molecule is taken apart, the Clr and Cls dissociated, and the C1q left bound to the antibody site. As discussed elsewhere in this volume, C1 inhibitor inhibits enzymes in a number of other mediator pathways in plasma, including the kinin-generating pathway, and patients with abnormalities in even one of the genes for normal Cl inhibitor have hereditary angioedema, a swelling disorder. C1 inhibitor is capable of inhibiting MASPs in exactly the same way as it inhibits C1. Receptors also exist for the C1q fragment of C1. Here there has been considerably less research and there is less understanding of how the several C1q receptors that have been described function in normal physiology and in disease.

The next steps in the classical complement pathway, the interaction of C4 and C2, are also under the control of a circulating protein, C4 binding protein. This protein binds to C4b, preventing its interaction with C2 and accelerating the decay of the C4b,C2a site once formed. It also is capable of binding to C3b when these reactants are present at high concentration. As discussed, the C4b,C2a site is further controlled because it is subject to spontaneous degradation over time, losing its activity. Loss of activity is accompanied by the release of C2a from the C4b site. The C4b site can accept another C2 and, in the presence of the C1, can regenerate the C4b/2a site.

Control molecules and cellular receptors that interact with C3 As an essential component in the lytic pathway, C3 functions in the classical, lectin, and alternative pathways. C3b on the cell surface is also a potent opsonin, aiding the phagocytic process. As C3b, if deposited on tissue cells, can become a focus of tissue damage, its formation and degradation are under tight regulation. It is simplest to describe the steps in degradation and then the receptors (Fig. 19.2). There are a great many circulating proteins and cellsurface receptors that can interact with this protein and the results of the interaction may differ depending on the set of control proteins with which it interacts (Barilla-LaBarca et al. 2002; Kim 2006). Virtually all normal cells have these control molecules. Some of the plasma and cellular membrane-bound =

O

S-C

22.5 kDa Fig. 19.2 The two-chain molecule C3 is shown first. There are no receptors that recognize this molecule. The C3 convertase of the classical or alternative pathway cleaves off C3a, an anaphylatoxin. The remainder of the molecule C3b undergoes a marked molecular rearrangement and now is recognized by CD35 (CR1), as well as by the recently recognized receptor on Kupffer cells, C3Ig. C3b binds factor H and now can be cleaved by factor I to iC3b. iC3b is recognized by CD11b/CD18 and CD11c/CD18. These two-chain receptors are on all phagocytes and on dendritic cells. They aid the processing of antigen. In serum, the cleavage of C3 stops at this point, but when an immune complex is bound to cellular CD35 or when C3 is deposited on a cell with CD35, it is cleaved further by factor I to C3c and C3d. The latter fragment is recognized by CD21 found on B cells and dendritic cells. Antigen with multiple bound C3d molecules can interact with both CD21 and the B-cell receptor, which can augment the immune response. (See CD-ROM for color version.)

C3

39.5 kDa

Receptors

35 kDa

S S

S

S 75 kDa

None

R H O S C C3a+C3b

CD35 (C3bR) C3aR

S S

S

S

H S S S

iC3b

R O C

S

Factors H & I CD11b,c; CD18 (iC3bR) S

H R S O C

CD35 Factor I C3c+C3dg

CD21 (C3dR) S S

S

S

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proteins also act as complement receptors, aiding the phagocytic process or providing signals for cellular activation. Two plasma proteins are critical regulators of C3b, factors H and I. C3b when generated will bind factor H, and the complex of C3b and H can be attacked by the circulating complement enzyme, factor I, which can then cleave the C3b α chain leading to the formation of iC3b or inactivated C3b. Cleaved C3b will no longer function as a C3 or C5 convertase, but it remains cell bound and remains a potent opsonin. The rare patients missing factor I have low C3 in the circulation, because the alternative pathway stays active and cleaves C3, and have urticaria, presumably because they continuously generate the anaphylatoxin C3a. It is interesting that, at this step, there is the possibility of separating self from nonself, one of the cardinal features of the adaptive immune response. During an immune response it is essential to separate self from nonself, to prevent the development of autoimmune disease and damage to one’s own tissues. In the case of the complement system, the system does this, albeit in a fairly primitive way. C3b deposited on one’s own tissues or cells is often close to a sialic acid, present in relatively large amounts on normal tissue and cellular membrane carbohydrates. Factor H activity is facilitated by sialic acid. Any C3b deposited on one’s own cells, therefore, tends to be cleaved by factor I, preventing further complement activation. The microorganism surface usually does not have sialic acid. Factor H function is not facilitated. C3b remains on the organism surface and the C3 convertase of the alternative pathway continues to deposit additional C3b on the microbe. Many pathogens have evolved mechanisms to incorporate sialic acid into surface structures, to protect themselves in part from complement attack. For example, Esherichia coli K1 has developed sialic acid-containing capsules to mimic the surface of the normal cell and thus protect the bacterium from destruction. Four different cellular receptors appear to be important in the phagocytosis of C3-coated particles. CD35 (also termed CR1) recognizes C3b, as does a recently described receptor, present on Kupffer cells, termed CRIg. The β2 integrins (CD11b/CD18 also termed CR3, and CD11c/CD18 also termed CR4) principally recognize iC3b, the product formed by the action of factors H and I, and mediate phagocytosis. Receptors for iC3b are present on all phagocytes and dendritic cells, although they are not present on lymphocytes. The β2 integrins are two-chain molecules (α and β chains) (Staunton et al. 2006). The α chain (CD11b or CD11c) provides the ligand recognition and the β chain (CD18) is required for transport of the two-chain complex to the cell surface. Patients with leukocyte adhesion deficiency have a defect leading to their inability to express these molecules on the cell surface and are highly susceptible to infection. Phagocytes bind iC3b-coated particles by virtue of their β2 integrin receptors, which markedly facilitates the phagocytic process. CD11c/CD18 is the signature receptor used in identification of monocytic

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dendritic cells. Presumably this receptor, acting through complement bound to antigen, is of critical importance in processing of antigen for presentation to the immune system. CD35 (CR1) is present on erythrocytes, phagocytes, dendritic cells, and all B cells. As mentioned, binding of a particle to a phagocyte surface by CD35 aids in the phagocytic process. However, if an immune complex forms in the circulation and binds C3b, most often it will bind not to the surface of a phagocyte, but to the surface of an erythrocyte by erythrocyte CD35, because of the high density of erythrocytes in the circulation. The immune complex, bound to the surface of the red cell is effectively out of the circulation and cannot easily leave the intravascular space to be deposited in tissues like the kidneys. As the erythrocyte circulates through the liver and spleen, the immune complex comes in contact with the fixed phagocytes in the sinusoids of these organs and is removed from the red cell surface and phagocytosed. The red cell exits the liver or spleen free of the complex and continues to have normal survival. During this process, some of the CD35 is removed from the red cell as the immune complex is removed. The infusion of normal erythrocytes into patients with active systemic lupus erythematosus is followed by those erythrocytes gradually losing their CD35, as the CD35 on the infused erythrocytes binds circulating immune complexes and transports them to the liver and spleen. CD35 itself may act as a cofactor protein for degradation of C3, but its function is different from the proteins listed above. Like C3b that has bound factor H, C3b bound to CD35 can be cleaved by factor I, but the cleavage leads to a different fragmentation pattern. Cleavage of the α chain leads first to the formation of iC3b, but the process does not stop at this step. Further cleavage of the α chain leads to the release from the target bound C3b of the largest part of the iC3b, C3c, with retention of a 40-kDa fragment of the α chain of iC3b, C3dg, bound to the target. This fragment can be further trimmed by proteases to C3d. C3dg and C3d do not bind to CD35 or to the β2 integrins, but do bind to CD21 (CR2), present on all B cells, a T cell subset, and on follicular dendritic cells. As β2 integrins are not on B cells and CD21 is not on most phagocytes, the fragmentation pattern of C3 mediated by the various cofactor proteins can direct targets of attack or antigens to phagocytes, antigen-presenting cells, or B cells. A group of other complement control molecules on the membrane of normal cells also acts to dampen the activity of C3 when it is deposited. Thus, MCP (CD46, membrane cofactor protein) acts as a cofactor for the cleavage of C3b by factor I, just as factor H does. Another molecule present on most cells, bound to the cells by a phosphatidylinositol linkage, DAF (decay accelerating factor, CD55), interacts with both the classical and alternative pathway C3 convertase to increase the rate of degradation of the convertase, destroying its activity. It is interesting that these two molecules, widely distributed on cells of the body, together have much of the activity of CD35 on immune cells and phagocytes. CD35 has

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both decay-accelerating and cofactor activity in the same receptor molecule, and these activities are separated and slightly changed in CD46 and CD55. As discussed, the complement system has been present over much of mammalian evolution and microorganisms have evolved mechanisms for using these proteins as docking sites for entry into cells. Thus, MCP has been shown to be a docking site for measles virus, for certain adenoviruses, and for some Neisseria; CD21 is a docking site for Epstein–Barr virus. Each year the list of control molecules that have been found to be docking sites for various viruses or bacteria grows. Several of the complement receptors are thought to aid directly in cellular activation or inhibition. CD35 has been discussed above as a facilitator of phagocytosis. Particles with bound CD35 will attach to the surface of phagocytic cells. This provides the first signal for phagocytosis to the cell. A second signal must be provided to start the phagocytic process. This can be accomplished by IgG interacting with an IgG Fc-activating receptor, like FcγRIII, or by any of a large number of cellactivating cytokines. The β2 integrins CD11b,c/CD18 are the principal iC3b receptors and like CD35 provide a first signal for the phagocytic process. These receptors are present on all phagocytes and natural killer cells. As mentioned, CD11c/ CD18 is used as an identifying marker of dendritic cells. Follicular dendritic cells, and some T cells have CD21 (CR2) on their surface. This receptor recognizes C3d, C3dg, and polymerized iC3b. It is believed that antigens with C3d on their surface can cross-link CD21 with the B-cell receptor. Cross-linking these two receptors can augment the ability of antigen to activate B cells by as much as a 1000-fold. For the purposes of this chapter, it is important to note that factor H abnormalities have been reported in two important medical situations. Lack of factor H activity has been shown to be important in patients with familial atypical hemolytic– uremic syndrome, namely hemolytic–uremic syndrome usually not associated with bacterial infection and diarrhea (Zipfel et al. 2006). Three molecules, each of which play a role in C3 degradation, have been reported to be abnormal in various subgroups of these patients. The three proteins are factor H, factor I, and MCP. The defects in the proteins can be present in either the heterozygous or homozygous state, probably reflecting the fact that half the normal number of C3 control molecules is not sufficient to protect from untoward immunologic activation. One way of thinking about the pathogenesis of this syndrome is that Shiga toxin from bacteria or other toxins enter the circulation and are deposited on endothelial cells, particularly in the kidney, and on erythrocytes. As the individual makes an immune response to the toxin, in the absence of sufficient control molecules, antibody binds to the toxin and cells with toxin and antibody are destroyed by complement activation. It has also recently been reported that the largest risk factor in the development of macular degeneration in the elderly is an alteration of the amino acid at position 402 in factor

H (Tyr→His) (Haines et al. 2005). It is believed from statistical studies of DNA sequences from pedigrees of families with inherited macular degeneration that approximately 50% of cases are due to this alteration in this one amino acid, although the factor H allele with histidine in position 402 is fairly common in the population and other factors must be involved.

Receptors for the anaphylatoxins C3a, C4a and C5a Of the anaphylatoxins, C5a has been studied in greatest detail (Hugli 1986). It is a potent chemotactic factor, causing the directed migration of phagocytes. It contracts smooth muscle cells and causes mast cells to degranulate in the absence of IgE antibody. It caused neutrophils to adhere to one another and to endothelium in vessels. It clearly plays a part in the damage observed during the course of immunologic lung disease. Mice with a defect in the C5a receptor do not develop all of the manifestations of immunologic or allergic lung disease. It is likely that far more information will become available about this important receptor in the development of asthma. There is less information available on C4a and C3a binding. The membrane receptor for C3a is clearly different from that of C5a and can be triggered to cause mucus secretion in the airways, but its role in immunologic airways disease is still speculative.

Control of the late steps in the complement cascade The later steps in the complement cascade are also under tight control. The site composed of the C3 convertase with bound C5 will decay if it does not bind C6 rapidly, and there are a series of molecules that downregulate the late-acting proteins both in serum and on cells. S protein, a plasma protein, interacts with C7 as the C5–C7 complex forms and becomes hydrophobic (Liszewski & Atkinson 1998). On binding S protein, this complex is neutralized and can no longer bind to cell surfaces. Similarly, clusterin, another plasma protein, binds to the forming C5–C9 complex and prevents its activation and completion. Most cells in the body have membrane-bound CD59, which interacts with the C5b–8 site decreasing the binding of C9 and preventing polymerization of C9. It protects the cell by preventing effective pore formation. All of these control molecules are important in maintaining homeostasis and loss of the control molecules often leads to disease. CD59, like CD55, is linked to cell membranes by a phosphatidylinositol linkage. By not having a transmembrane domain, the protein is free to move rapidly in the fatty hydrophobic plane of the cell membrane to intercept forming C5b–9 and prevent cell lysis. No patient has been described with a genetic defect leading to absence of CD59. However, patients with the disease paroxysmal nocturnal hemoglobinuria (PNH) have an acquired bone marrow defect in which they develop a mutation in bone marrow stem cells of the gene PIG A (phosphatidylinositol glycan enzyme A), the first enzyme in the development of phosphatidylinositol bonds (Luzzatto 2006). This is a gene

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present on the X chromosome and a single gene defect in a bone marrow stem cell leads to an inability to synthesize the first intermediate in the phosphatidylinositol linkage pathway, and therefore the failure to have phosphatidylinositollinked proteins on the cell membrane. A failure to generate hematopoietic cells with CD59 leads to all hematopoietic cells of bone marrow origin derived from the abnormal clone being easily lysed by complement. As mentioned, alternative pathway proteins in the circulation undergo slow activation; CD59 is critical for neutralizing complement proteins when they bind to the body’s own cells. In patients with PNH, this mechanism is defective and patients have a hemolytic anemia, often thrombocytopenia, and often low neutrophil count. As this chapter is written, papers have appeared promoting the use of monoclonal anti-C5 to turn off the late components of complement, thereby preventing lysis while allowing opsonization to proceed. This is the first medication that improves cell survival in this patient group with a disease that has a generally grim prognosis. The role of complement in the generation of immunologic lung disease is of particular interest. For many years, it was taken as gospel that complement plays no role in IgE-mediated lung disease or in asthma. Recent work has suggested that this may not be the case. Complement may play a number of interesting functions in the generation of lung pathology. First, it has been suggested that complement functions importantly in directing immune responses toward Th1 or Th2 type immunity. Th1 immunity is generally considered most important in prevention of infection and Th2 immunity is associated with asthma and other allergic diseases. It is believed that the activation of C5 and the generation of C5a are important in directing the immune response toward a Th1 phenotype and lack of C5 therefore skews the system toward the generation of Th2 immunity (Peng et al. 2005; Kohl et al. 2006). On the other hand, once immunity or allergy is established, it is believed that C5a may be generated during immunologic responses in the lung and, acting as an anaphylatoxin, may cause mast cell degranulation, smooth muscle contraction, etc., contributing to the asthmatic response.

Complement in the afferent limb of the immune response In recent years, there has been considerable interest in the role of complement in the development of immunity (Carroll 1998; Carroll & Holers 2005). The discussion up to this point has focused on the efferent limb of the response, how tissue damage is caused or controlled by complement. As mentioned early in the chapter, the complement system is phylogenetically older than the adaptive immune system, and many of the complement proteins existed as the afferent immune system evolved (Nonaka 1998). It is not surprising, therefore,

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that elements of the complement system were incorporated into the afferent immune system and these elements are only now being slowly identified. As mentioned in an earlier section, the binding of complement to an antigen allowing cross-linking of CD21 and the B cell receptor increases antigenicity by up to 1000-fold. In this case, complement augments the immune response. It is also known that patients deficient in complement, although rare, often have major defects in adaptive immunity. Patients deficient in C1, C4 and, to a lesser extent, C2, have a high propensity to develop SLE (Walport et al. 1998; Manderson et al. 2004). In fact, of the relatively few C1q-deficient patients who have been described, 96% have had systemic lupus. Of the relatively few C4-deficient individuals who have been described, 75% have had lupus. Even heterozygosity of the genes for C4 predisposes individuals to the development of lupus. This propensity to cause SLE seems to be independent of the genetic localization of C4, C3 and factor B in the major histocompatibility locus as class III genes, and therefore their linkage to the MHC. It is known that animals deficient in C1q, C4, C3, and CR1/2 make a poor immune response, particularly to T-dependent antigens, have poor germinal center formation, and have poor immunologic memory. It is known that complement aids in the localization and retention of antigens within the germinal center and it is believed that this localization of antigen to the germinal center facilitates an ongoing immune response. In addition to the above, animals, particularly those deficient in C1q and C4, fail to develop normal tolerance as well, although animals deficient in C3 and CR1/2 do not appear to have this defect. Although these are intriguing findings and have been repeated in many laboratories, it is still not completely clear how complement functions in the afferent limb of the immune response. It is quite likely that this question will be clarified over the next few years.

Complement deficiencies and clinical illness Patients and animals with defects in specific complement proteins have similar phenotypes. Both patients and animals deficient in the classical pathway factors and C3 have an increased propensity to infection, particularly with high-grade pathogenic bacteria like pneumococci, as opposed to viruses (Figueroa & Densen 1991; Sjoholm et al. 2006). Patients with late component defects, that is of C5–9, have a propensity to develop systemic Neisseria infections with Neisseria gonorrhoeae or Neisseria meningitidis. Why opsonization, which only requires complement through C3, is not sufficient to protect against these two organisms is not clear, but repeated infection with either of these two organisms is often an excellent tip to the clinician that a late complement protein deficiency is present. Alternative pathway defects are rarer and, in fact, no factor B deficiency has ever been described (Thurman

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Table 19.2 Consequences of complement deficiency. C3 and factors that control C3 levels

Late-acting proteins: C5–9

Decreased C′ activation in the absence of antibody

Decreased opsonization: if control factors are abnormal, increased C′ effect

Inability to form lytic lesions C5 important in PMN chemotaxis

Increase in infection with high grade pathogens: some increase in Neisseria infections

Marked increase in infection with highgrade pathogens: failure to downregulate C3 associated with hemolytic–uremic syndrome and adultonset macular degeneration

Marked increase in Neisseria infection

Defect

Classical pathway

Lectin pathway

Alternative pathway

Functional consequences

Delayed C′ activation Decreased immune response Poor antibody activation of C′

Decreased activation in the absence of antibody

Clinical consequences

Increased incidence of autoimmune disease: infection with highgrade pathogens (Pneumococcus, etc.)

Infection in the newborn: question of increased rheumatic disease

& Holers 2006). The few patients with factor D deficiency also have a propensity to develop infection, but autoimmunity has not been seen in either animals or patients with defects in this pathway. Defects in the lectin pathway are being defined currently (Jensenius 2005). As discussed earlier in the chapter, MBL has a central core and a series of radiating arms ending in the lectin-binding sites. The radiating arms have the structure of collagen and, like collagen, are composed of three intertwined chains; however, unlike collagen, the chains are identical. It has been noted that single gene defects affecting these chains can lead to improper winding of the chains about one another during the formation of the protein, leading to low levels of MBL. This protein is present at very low levels to begin with (2 μg/mL) and patients, commonly with one of three genetic defects in the MBL gene, even when present in the heterozygous state, have inefficient chain matching, and as little as one-tenth of the normal level of MBL. It is reported from Europe that children with these defects have a high frequency of infection, although few studies have been done to confirm this finding. It is also reported that individuals with abnormalities of MBL often may die early during the course of cystic fibrosis. As patients with cystic fibrosis typically have high titer antibody to their organisms, it is not known why the MBL deficiency should lead to early death. It is also suggested that MBL deficiencies facilitate the pathogenesis of rheumatic disease. All these observations are intriguing and all require considerably more study before we understand both the observations and their meaning (Table 19.2). It should be clear from this brief review that antigen– antibody complexes and complement are capable of having important biological effects and can influence the expression of a wide variety of autoimmune and allergic diseases.

We believe that as we develop a clearer understanding of the complex interactions involved in pathogenesis, we will develop a far more insightful approach to the treatment of these important illnesses.

References Aschoff, L. (1924) Lectures on Pathology. Paul B. Hoeber, New York. Barilla-LaBarca, M.L., Liszewski, M.K. et al. (2002) Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. J Immunol 168, 6298–304. Berzofsky, J.A. & Berkower, I.J. (1993) Immunogenicity and Antigen Structure. Raven Press, New York. Carroll, M. (1998) The role of complement and complement receptors in induction and regulation of immunity. Ann Rev Immunol 16, 545– 68. Carroll, M. (2004) The complement system in regulation of adaptive immunity. Nat Immunol 5, 981–6. Carroll, M.C. & Holers, V.M. (2005) Innate autoimmunity. Adv Immunol 86, 137–57. Cochrane, C.G. & Koffler, D. (1973) Immune complex disease in experimental animals and man. Adv Immunol 16, 185–264. Coppo, R., Andrulli, S., Amore, A. et al. (2006) Predictors of outcome in Henoch–Schonlein nephritis in children and adults. Am J Kidney Dis 47, 993–1003. Davies, K.A., Robson, M.G., Peters, A.M. et al. (2002) Defective Fc-dependent processing of immune complexes in patients with systemic lupus erythematosus. Arthritis Rheum 46, 1028–38. Dixon, F. (1963) The role of antigen–antibody complexes in disease. Harvey Lectures 58, 21. Fauci, A.S., Frank, M.M. & Johnson, J.S. (1970) The relationship between antibody affinity and the efficiency of complement fixation J Immunol 105, 215–20. Figueroa, J. & Densen, P. (1991) Infectious diseases associated with complement deficiencies. Clin Microbiol Rev 4, 369–95.

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Frank, M.M. (1998) Complement, Introduction and Historical Notes. Marcel Decker, New York, Frank, M.M. (2000) Complement deficiencies. Pediatr Clin North Am 47, 1339–54. Frank, M.M., Lawley, T.J., Hamburger, M.I. et al. (1983) NIH Conference: Immunoglobulin G Fc receptor-mediated clearance in autoimmune diseases. Ann Intern Med 98, 206–18. Frank, M.M., Schreiber, A.D., Atkinson, J.P. & Jaffe, C.J. (1977) Pathophysiology of immune hemolytic anemia. Ann Intern Med 87, 210–22. Gauthier, V.J., Mannik, M. et al. (1982) Effect of cationized antibodies in performed immune complexes on deposition and persistence in renal glomeruli. J Exp Med 156, 766– 77. Gertz, M.A. (2005) Cold agglutinin disease and cryoglobulinemia. Clin Lymphoma 5, 290– 3. Haakenstad, A.O. & Mannik, M. (1976) The disappearance kinetics of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice. Lab Invest 35, 283–92. Haakenstad, A.O., Striker, G.E. et al. (1976) The glomerular deposition of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice. Lab Invest 35, 293– 301. Haines, J.L., Hauser, M.A., Schmidt, S., et al. (2005) Complement factor H variant increases the risk of age-related macular degeneration. Science 308, 419– 21. Han Lee, P.E., Scafidi, J. & Davis., A.E. III (2003) Approaches toward reversal of increased vascular permeability in C1 inhibitor deficient mice. Immunol Lett 89, 155– 60. Hoebe, K., Jiang, Z., Tabeta, K. et al. (2006) Genetic analysis of innate immunity. Adv Immunol 91, 175– 226. Holers, V.M. (2000) Phenotypes of complement knockouts. Immunopharmacology 49, 125– 31. Hugli, T.E. (1986) Biochemistry and biology of anaphylatoxins. Complement 3, 111– 27. Jensenius, J.C. (2005) The mannan-binding lectin (MBL) pathway of complement activation: biochemistry, biology and clinical implications. Adv Exp Med Biol 564, 21–2. Kaneko, Y., Nimmerjahn, F., Madaio, M.P. et al. (2006) Antiinflammatory activity of immunoglobulin G resulting from Fc sialation. Science 313, 670– 3. Kim, D.D., Song, W. (2006) Membrane complement regulatory proteins. Clin Immunol 118, 127–36. Kohl, J., Baelder, R., Lewkowich, M. et al. (2006) A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma. J Clin Invest 116, 783– 96. Lawley, T.J., Bielory, L., Gascon, P., Yancey, K.B., Young, N.S. & Frank, M.M. (1984) A prospective clinical and immunologic analysis of patients with serum sickness. N Engl J Med 311, 1407–13. Liszewski, M.K. & Atkinson, J.P. (1998) Regulatory proteins of complement. In: Volanakis, J.A. & Frank, M.M., eds. The Human Complement System in Health and Disease. Marcel Dekker, New York, pp. 149–66.

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Luzzatto, L. (2006) Paroxysmal nocturnal hemoglobinuria: an acquired X-linked genetic disease with somatic-cell mosaicism. Curr Opin Gen Dev 16, 317–22. Manderson, A.P., Botto, M. & Walport, M.J. (2004) The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol 22, 431–56. Mannik, M., Arend, M.P., Hall, A.P. et al. (1971) Studies on antigenantibody complexes. I. Elimination of soluble complexes from rabbit circulation. J Exp Med 133, 713–39. Mannik, M. & Arend, W.P. (1971) Fate of preformed immune complexes in rabbits and rhesus monkeys. J Exp Med 134, 19s–31s. Nangaku, M. & Couser, W.G. (2005) Mechanisms of immune-deposit formation and the mediation of immune renal injury. Clin Exp Nephrol 9, 183– 91. Nimmerjahn, F. & Ravetch, J.V. (2005) Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science 310, 1510–12. Nonaka, M. (1998) Phylogeny of the complement system. In: Volanakis, J.A. & Frank, M.M., eds. The Human Complement System in Health and Disease. Marcel Dekker, New York, pp. 203–16. Nordahl, E.A., Rydengard, V., Nyberg, P., et al. (2004) Activation of the complement system generates antibacterial peptides. Proc Natl Acad Sci USA 101, 16879–84. Peng, T., Hao, L., Madri, J.A. et al. (2005) Role of C5 in the development of airway inflammation, airway hyperresponsiveness, and ongoing airway response. J Clin Invest 115, 1590–600. Ravetch, J.V. & Bolland, S. (2001) IgG Fc receptors. Annu Rev Immunol 19, 275–90. Salmon, J.E. & Pricop, L. (2001) Human receptors for immunoglobulin G: key elements in the pathogenesis of rheumatic disease. Arth Rheum 44, 739–50. Schmidt, R.E. & Gessner, J.E. (2005) Fc receptors and their interaction with complement in autoimmunity Immunol Lett 100, 56–67. Shushakova, N., Skokowa, J. et al. (2002) C5a anaphylatoxin is a major regulator of activating versus inhibitory FcγRs in immune complexinduced lung disease. J Clin Invest 110, 1823–30. Sjoholm, A.G., Jonsson, G., Braconier, J.H. et al. (2006) Complement deficiency and disease: an update. Mol Immunol 43 78–85. Staunton, D.E., Lupher, M.L., Liddington, R. et al. (2006) Targeting integrin structure and function in disease. Adv Immunol 91, 111–57. Thurman, J.M. & Holers, V.M. (2006) The central role of the alternative complement pathway in human disease. J Immunol 176, 1305–10. von Pirquet, C.F. & Schick, B. (1905) Die Serumkrankeit. Deuticke, Leipzig. Walport, M., Davies, K.A. & Botto, M. (1998) C1q and systemic lupus erythematosus. Immunobiology 199, 256–85. Walport, M.J. (2001a) Complement: first of two parts. N Engl J Med 344, 1058–66. Walport, M.J. (2001b) Complement: second of two parts. N Engl J Med 344 1140–4. Zipfel, P.F., Misselwitz, J., Licht, C. et al. (2006) The role of defective complement control in hemolytic uremic syndrome. Semin Thromb Hemost 32, 146–54.

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Bradykinin Pathways and Allergic Disease Allen P. Kaplan

Summary Bradykinin is generated by cleavage of low-molecular-weight kininogen (LK) by tissue kallikrein to release kallidin (lysylbradykinin) followed by removal of N-terminal lysine by an aminopeptidase and by activation of a plasma cascade consisting of coagulation factor XII, prekallikrein (PK), and high-molecular-weight kininogen (HK). PK and HK circulate in plasma as a complex. The plasma cascade is initiated by contact with negatively charged macromolecules, including the surface of endothelial cells, leading to factor XII activation, conversion of PK to kallikrein, and digestion of HK to liberate bradykinin. The HK–PK complex binds to endothelial cells by HK interaction with free gC1qR, as well as gC1qR complexed to cytokeratin 1, while factor XII binds to a complex of the urokinase plasminogen activator receptor (u-PAR) and cytokeratin 1. Bypass of factor XII to initiate the cascade can occur along the surface of endothelial cells by direct activation of the PK–HK complex by heat-shock protein 90 and a prolylcarboxypeptidase. Bradykinin is degraded by angiotensin-converting enzyme (ACE) and plasma carboxypeptidase N. Therapy with ACE inhibitors can lead to bradykinin accumulation due to a decreased rate of degradation, resulting in complications including cough and angioedema; the latter can be very severe, including airway obstruction reminiscent of C1 inhibitor deficiency. Hereditary angioedema or acquired C1 inhibitor deficiency can lead to peripheral angioedema, swelling of the face, lips, tongue, pharynx and larynx, and bowel wall edema causing abdominal attacks that can be particularly painful.

and mobilization of arachidonic acid. Kinins also stimulate sensory nerve endings to cause a burning dysesthesia. Thus the classical parameters of inflammation (i.e., redness, heat, swelling, and pain) can all result from kinin formation. Bradykinin is the best characterized of this group of vasoactive substances. There are two general pathways by which bradykinin is generated (Margolius 1998) (Fig. 20.1). The simpler of the two has only two components: an enzyme, tissue kallikrein, and a plasma substrate, low-molecular-weight kininogen (LK) (Jacobson & Kritz 1967; Muller-Esterl et al. 1985a). Tissue kallikrein is secreted by many cells throughout the body; however, certain tissues produce particularly large quantities. These include glandular tissues (salivary and sweat glands and pancreatic exocrine gland) and the lung, kidney, intestine, and brain. The enzyme is processed intracellularly from a precursor, prokallikrein, to produce tissue kallikrein; however, the enzyme responsible for this conversion has not been identified. Tissue kallikrein is secreted and digests LK to yield a 10-amino-acid peptide, lysyl-bradykinin (kallidin), with the sequence Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-PheArg. A plasma aminopeptidase cleaves the N-terminal Lys leaving the 9-amino-acid peptide bradykinin.

Surface factor XII, prekallikrein, HK

Tissue kallikrein LK

Lys-bradykinin

Plasma kallikrein

Aminopeptidase

Introduction Bradykinin

Kinins are low-molecular-weight peptides that participate in inflammatory processes by virtue of their ability to activate endothelial cells and, as a consequence, lead to vasodilatation, increased vascular permeability, production of nitric oxide,

Kininase I Des-arg9-bradykinin

HK Kininase II (ACE) Arg-Pro-Pro-Gly-Phe-Ser-Pro + phe arg

Kininase II (ACE) Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Arg-Pro-Pro-Gly-Phe + ser pro phe

ACE Arg-Pro-Pro-Gly-Phe + ser pro

Fig. 20.1 Pathways for bradykinin formation and degradation.

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Trace factor XIIa Factor XII

Surface

Prekallikrein Factor XIIa

HK Surface

HK

HK Kallikrein Bradykinin

Factor XI

Factor XIa

Factor XII Intrinsic coagulation

HK

Factor XIIa

Factor XIIf

Autodigestion kallikrein C1

CI

C4 and C2 digestion

The second pathway for bradykinin formation is far more complex and is part of the initiating mechanism by which the “intrinsic” coagulation pathway is activated (Kaplan et al. 1998) (Fig. 20.2). Factor XII is the initiating protein that binds to certain negatively charged macromolecular surfaces and autoactivates (autodigests) to form factor XIIa (Silverberg et al. 1980a; Tans & Griffin 1982). There are two plasma substrates of factor XIIa, namely prekallikrein (PK) (Mandle & Kaplan 1977) and factor XI (Bouma & Griffin 1977; Kurachi & Davie 1977), and each of these circulates as a complex with high-molecular-weight kininogen (HK) (Mandle et al. 1976; Thompson et al. 1977). These complexes also attach to initiating surfaces, and the major attachment sites are on two of the domains of HK, thereby placing both PK and factor XI in optimal conformation for cleavage to kallikrein (plasma kallikrein) and factor XIa, respectively. It is important to note that plasma kallikrein and tissue kallikrein are separate gene products and have little amino acid sequence homology, although they have related functions (i.e., cleavage of kininogens). The main substrate of tissue kallikrein is LK, but it is also capable of cleaving HK. Plasma kallikrein cleaves HK exclusively. The two kininogens have an identical amino acid sequence starting at the N-terminus and continuing to 12 amino acids beyond the bradykinin moiety but differ in C-terminal domains because of alternative exon splicing (Kitamura et al. 1985; Takagaki et al. 1985). This scheme for production and degradation of kinins is shown in Fig. 20.1, and further details of the plasma cascade are given in Fig. 20.2. The enzymes that destroy bradykinin consist of kininases I and II. Kininase I is also known as plasma carboxypeptidase N (Erdos & Sloane 1962), which removes the C-terminal Arg from bradykinin or kallidin to yield des-Arg9-bradykinin or des-Arg10-kallidin, respectively (Sheikh & Kaplan 1986a). It is the same enzyme that cleaves

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Fig. 20.2 Pathway for bradykinin formation indicating the autoactivation of factor XII, the positive feedback by which kallikrein activates factor XII, cleavage of high-molecular-weight kininogen (HK) to release bradykinin, formation of factor XII fragment, and enzymatic activation of C1. The steps inhibitable by C1 inhibitor are indicated by the black rectangles.

the C-terminal Arg from the complement anaphylatoxins C3a and C5a. Kininase II is identical to angiotensin-converting enzyme (ACE) (Yang & Erdos 1967). Bradykinin and kallidin stimulate constitutively produced B2 receptors (Vavrek & Stewart 1985), whereas des-Arg9-bradykinin or des-Arg10Lys-bradykinin both stimulate B1 receptors (Regoli & Barabe 1980), which are induced as a result of inflammation. Stimuli for B1 receptor transcription include interleukin (IL)-1 and tumor necrosis factor (TNF)-α (Davis & Perkins 1994; Marceau 1995). Kininase II is a dipeptidase that cleaves the C-terminal Phe-Arg from bradykinin to yield a heptopeptide, which is cleaved once again to remove Ser-Pro to leave the pentapeptide Arg-Pro-Pro-Gly-Phe (Sheikh & Kaplan 1986b, 1989a). If the C-terminal Arg of bradykinin is first removed with kininase I, then ACE functions as a tripeptidase to remove SerPro-Phe to leave the above pentapeptide (Sheikh & Kaplan 1986b).

Contact activation The concept of contact activation was originally developed because it was found that addition of blood to a glass tube leads to coagulation. Thus “contact” with the silicate surface appeared to initiate a proteolytic cascade culminating in the conversion of fibrinogen of fibrin. At the same time, bradykinin is generated. The reactions that occur during activation in this fashion are shown in Fig. 20.2. It has been shown that factor XII (Hageman factor) circulates as a β-globulin (molecular weight 80 000) that autoactivates on binding to surfaces bearing negative charges (Silverberg et al. 1980a). Because the zymogen has no detectable enzymatic activity (Silverberg & Kaplan 1982) it has been proposed that trace quantities of the active enzyme

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3

5 BK HMWK Transcription Poly-A site 5 5

3 3 Unprocessed mRNAs

Fig. 20.3 The gene for high-molecular-weight kininogen (HK). The 9 boxes shown in the mature preHMWK mRNA represent the exons coding for the heavy chain of both HK and lowmolecular-weight kininogen (LK). Exon 10 codes for the bradykinin sequence and the light chain of HK, whereas exon 11 codes for the light chain of LK. The mature mRNAs are assembled by alternative splicing events in which the light chain sequences are attached to the 3′ end of the 13-amino acid common sequence C-terminal to bradykinin.

RNA Splicing 5

3 Mature pre-HMWK mRNA

3

5

Poly A

Mature preLMWK mRNA

Poly A

Translation

Heavy chain

BK

Light chain

Heavy chain

Pre-HMWK

(factor XIIa) actually circulate but that digestion of factor XII by factor XIIa occurs only on binding of factor XII to the surface. Thus the surface renders factor XII a substrate (Griffin 1978) for traces of preformed factor XIIa. PK and coagulation factor XI each circulate as a complex with HK (Mandle et al. 1976; Thompson et al. 1977) with stoichiometry of 1 : 1 and 1 : 2 respectively (factor XI is a dimer). The binding sites for PK and factor XI on HK overlap (Tait & Fujikawa 1986, 1987) to such a degree that HK can bind only one molecule of each, but never both. However, HK is present in considerable molar excess. Thus there are separate complexes of PK–HK and factor XI–HK and the percentage bound to HK in each case is 85% and 95%, based on equilibrium considerations (Scott & Colman 1980). HK is a key factor regulating contact activation. It is also the link protein that allows assembly of the kinin-forming cascade along the surface of cells and we therefore consider its structural features in some detail. Human plasma has two kininogens designated HK and LK. They are assembled by alternative splicing of the terminal exons (Fig. 20.3) such that a large portion of their amino acid sequence is identical (Kitamura et al. 1985). HK consists of six domains (Fig. 20.4). At the N-terminus are three domains (encoded by exons 1–9) that are homologous to cystatins and stefans (Kellermann et al. 1986a), including sulfhydryl proteases such as cathepsins B, H, and L; domains 2 and 3 actually retain cysteine protease inhibitory activity (Gounaris et al. 1984; Muller-Esterl et al. 1985b; Higashiyama et al. 1986; Ishiguro et al. 1987). Domain 4 contains the bradykinin sequence plus the next 12 amino acids. Up to this point LK and HK have identical amino acid sequences. Then exon 10, which includes bradykinin plus domains 5 and 6, is added for HK (Fig. 20.3), while exon 11 is added for LK with the attachment at the C-terminus of domain 4. When HK is cleaved by plasma kallikrein to release

BK Light chain

Pre-LMWK

bradykinin (fast cleavage occurs at a C-terminal Arg–Ser bond, followed by cleavage at an N-terminal Lys–Arg bond) (Mori & Nagasawa 1981; Mori et al. 1981), and the kinin-free HK is reduced and alkylated, one can isolate a heavy chain (domains 1–3) and a light chain (the C-terminal 12 amino acids of domain 4 plus domains 5 and 6) (Thompson et al. 1979). Thus the light chain of HK and LK are quite different (Kellermann et al. 1986b) and this accounts for the difference in molecular weight and many of the functional properties of HK not shared by LK. As noted above, plasma kallikrein

HK Domains NH2

COOH

1 6

2 Cysteine protease inhibitor

3

5

Prekallikrein and factor XI binding

Bradykinin Fig. 20.4 The structure of high-molecular-weight kininogen (HK). The heavy-chain region consists of three homologous domains (1–3) of which the latter two are sulfhydryl protease inhibition sites. Domain 4 contains the bradykinin moiety. The light-chain region contains the surface-binding site (domain 5) and overlapping binding sites for prekallikrein and factor XI (domain 6).

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preferentially cleaves HK (Reddigari & Kaplan 1988, 1989) while tissue kallikrein (encoded by a separate gene from that of plasma kallikrein) cleaves both HK and LK, but with more favorable kinetics if LK is the substrate (Lottspeich et al. 1984; Muller-Esterl et al. 1985a). The functions of HK in contact activation, as depicted in Fig. 20.2, are multiple. First, it accelerates the conversion of PK and factor XI to kallikrein and factor XIa, respectively (Griffin & Cochrane 1976; Meier et al. 1977; Wiggins et al. 1977). This acceleration appears due to the ability of PK and factor XI to bind to HK; as a result, each is in a more favorable conformation for activation than when they are tested unbound. In addition, HK provides the attachment to initiating surfaces and brings both PK and factor XI to the surface as a complex. If PK and factor XI bind to the surface in the absence of HK, activation by factor XIIa is markedly inhibited, even when compared with activation in the fluid phase. Thus the conformational effects of binding of PK and factor XI to HK are even more evident when activation along the surface is compared with fluid-phase activation (since factor XII is activated along surfaces, this comparison is made by adding preformed factor XIIa to PK or PK–HK either in solution or bound to a surface). Figure 20.2 also depicts a positive feedback in which kallikrein activates surface-bound factor XII to form factor XIIa (Cochrane et al. 1973; Meier et al. 1977; Silverberg et al. 1980b; Dunn et al. 1982). In fact the bound factor XII undergoes a conformational change that renders it a substrate for factor XIIa (Dunn et al. 1982). Thus autoactivation of factor XII can initiate the cascade once sufficient factor XIIa forms to overcome plasma inhibitors (Silverberg et al. 1980b; Tankersley & Finlayson 1984) and only a few percent conversion to factor XIIa is required. Then the kallikrein formed activates the remaining surface-bound factor XII at a much more rapid rate. This positive feedback is also accelerated by the presence of HK (Griffin & Cochrane 1976; Meier et al. 1977; Silverberg et al. 1980b). The factor XIIa formed remains attached to the initiating surface; further digestion of factor XIIa by kallikrein (Fig. 20.2) yields a 32.5-kDa factor XII fragment (factor XIIf) (Kaplan & Austen 1970, 1971; Dunn & Kaplan 1982), which retains the active site of factor XIIa but lacks the binding site to the surface. It is a doublet on SDS gel, with bands at 30 and 28.5 kDa. Factor XIIf consists of the light chain of factor XIIa, containing the active site, disulfide linked to small C-terminal fragments of the heavy chain of 2000 or 500, corresponding to the two SDS bands. Factor XIIf lacks the binding site for the surface and is released into the fluid phase where it can continue to activate PK until it is ultimately inactivated by plasma protease inhibitors. Factor XI is activated by factor XIIa in the presence of HK, but factor XIIf possesses only 2– 4% of the coagulant activity of factor XIIa and HK does not augment its reaction rate. Thus factor XIIf can be important for bradykinin formation, but not for intrinsic coagulation.

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The mechanism by which HK catalyzes factor XII activation is multifactorial and indirect since HK does not increase the enzymatic activity of kallikrein, nor does it interact with factor XII to render it a better substrate (Silverberg et al. 1980b). Its main effect is to allow dissociation of kallikrein from its complex with HK so that it can enzymatically activate factor XII along the cell surface (Cochrane & Revak 1980). Kallikrein bound directly to the surface is much less effective and cannot disseminate the reaction (Silverberg et al. 1980b). Since HK is required for the formation of kallikrein, i.e., activation of PK, the amount of kallikrein is increased when HK is present. Thus the effective ratio of kallikrein/factor XII in this enzymatic reaction is significantly augmented when HK is present. The percentage augmentation of contact activation in the presence of a surface plus HK is estimated to be 3000–12 000 fold (Tankersley & Finlayson 1984; Rosing et al. 1985). If one considers the rate of factor XI activation, HKdeficient plasma is almost as abnormal as factor XII-deficient plasma in a coagulation assay. HK increases the rate of formation of kallikrein, facilitates factor XII conversion to factor XIIa by kallikrein, and facilitates factor XI activation by factor XIIa. For comparison, it is of interest to consider the rate of factor XI activation in PK-deficient plasma where the kallikrein feedback activation of factor XII is not possible, and the only role of HK is in conversion of factor XI to factor XIa. In this case contact activation of coagulation is very slow, but if the time of incubation of citrated plasma with the surface is increased prior to recalcification, the clotting time approaches normal (Wuepper 1973; Saito et al. 1974; Weiss et al. 1974). This is due to gradual conversion of factor XI to factor XIa as a result of factor XII autoactivation on the surface. The next step, conversion of factor IX to factor IXa by factor XIa, is dependent on calcium, and thus prolonged incubation allows the amount of factor XIa to increase toward normal. It should be evident from Fig. 20.2 that plasma deficient in factor XII, PK, or HK cannot generate bradykinin via contact activation. Any bradykinin formed is dependent on tissue kallikrein activation of LK, or for factor XII-deficient plasma a bypass that requires endothelial cells, PK, and HK (see below). A detailed discussion of the structure of each protein, transcriptional and translational events involved in the synthesis of each protein, and mechanistic details regarding activation of factor XII, PK, and factor XI has been presented in Kaplan et al. (1997) and the reader is referred to this publication for further details.

Assembly on cell surfaces Binding of HK to human umbilical vein endothelial cells The first studies of the interaction of proteins of the kininforming cascade with cells were performed initially with platelets (Greengard & Griffin 1984; Gustafson et al. 1986)

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and then human umbilical vein endothelial cells (HUVECs) (Schmaier et al. 1988; van Iwaarden et al. 1988). In each instance, HK was shown to bind to each cell type in a zincdependent fashion. The binding was saturable and reversible, although binding was found to be dependent on domains 3 and 5 (Reddigari et al. 1993a; Hasan et al. 1995; Herwald et al. 1995) so that both heavy chain and light chain were capable of similar ion-dependent interactions (Reddigari et al. 1993a). Factor XII interacts with HUVECs in a similar fashion to HK; the interaction requires zinc ion and factor XII and HK can compete for binding to the cell surface (Reddigari et al. 1993b). The latter observation suggests that they bind to very similar cell-surface proteins with comparable affinity (Fig. 20.5). This binding protein was purified and further characterized by Joseph et al. (1996). These results are summarized below,

120

% 125I-FXII bound

100 80 60 40 Factor XII HK Human lgG

20 0 0

10

(a)

100

1000

10000

1000

104

[Competitor], nM 120

% 125I-HK bound

100 80 60 40 20 0 1 (b)

10

100 Factor XII, nM

Fig. 20.5 High-molecular-weight kininogen (HK) competes with factor XII for the same binding sites on human umbilical vein endothelial cells (HUVECs). (a) HUVECs were incubated with 1 mg/mL [125I]-FXII in triplicate in the presence of incremental concentrations of unlabeled factor XII, HK, or normal human IgG for 120 min and bound ligand was determined. The percentage bound in the presence of a competitor is plotted against the concentration of the competitor. (b) HUVECs were incubated with 1 mg/mL [125I]-HK in triplicate in the presence of increasing concentration of unlabeled factor XII and bound ligand was determined.

Bradykinin Pathways and Allergic Disease

and correspond to a p33 endothelial cell protein isolated by Herwald et al. (1996) and identified to be gC1qR, the receptor for the globular heads of C1q discovered by Ghebrehiwet et al. (1994). A solubilized endothelial cell membrane preparation was passed over an HK affinity column in the presence or absence of zinc ion, eluted, fractions neutralized, and an immunoblot performed with biotinylated HK. A prominent increase in HK binding was observed after elution in the presence of zinc. These fractions were then pooled, concentrated, and analyzed by SDS-PAGE. The main feature was the appearance of a new prominent band at 33 kDa that was visible with a Coomassie Blue stain, and ligand blot with biotinylated HK demonstrated binding only to the 33-kDa band. Based on this information, the 33-kDa protein was subjected to N-terminal amino acid sequence analysis and the first 13 amino acids were found to be identical to the known NH2 terminus of gC1qR (Ghebrehiwet et al. 1994). A Western blot using anti-gC1qR monoclonal antibody 60.11 was employed to further assess the identity of these two proteins. Monoclonal antibody 60.11, which interacts with an epitope at the N-terminus of gC1qR, identified the 33-kDa HUVEC-derived membrane-binding protein. The possible binding of factor XII to gC1qR was studied next. HUVEC membrane-purified gC1qR or recombinant gC1qR at 1.0–2.0 μg were applied to nitrocellulose membranes and blotted with biotinylated HK or factor XII in the presence or absence of 50 μmol/L zinc. We found that both HK and factor XII bind to either purified or recombinant gC1qR in the presence of zinc. Addition of excess unlabeled HK reversed the ability of factor XII to bind to gC1qR by over 90% as quantitated by scanning, which suggests interaction with a common domain within the protein. Factor XII only partially (46%) reverses HK binding. This difference may be due to relative affinity of the two ligands for the gC1qR molecule. Nevertheless, these data completely paralleled those observed on binding of factor XII and HK to endothelial cells. Herwald et al. (1996) also demonstrated that gC1qR is a major endothelial cell binding protein for HK used a domain 5-derived peptide from the light chain rather than whole HK as the ligand. In aggregate these data indicate that gC1qR serves as a zinc-dependent binding protein for factor XII as well as for HK, and that binding to HK occurs via the light chain moiety. The specific location within HK for binding to endothelial cells is within domain 5 (Hasan et al. 1995; Schmaier 2003) and this also appears to be the site of interaction with gC1qR. It is also clear that the HK heavy chain also binds to endothelial cells. This interaction has been shown to require domain 3. The methods employed to identify the cell-surface protein that interacts with heavy chain are analogous to those described above for isolation of gC1qR. Later, a second HKbinding protein was identified in HUVECs by affinity chromatography employing HK as ligand (Hasan et al. 1998; Shariat-Madar et al. 1999) and was identified as cytokeratin 1.

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It seemed likely that this protein would contribute to heavy chain interaction with cells and an affinity column was prepared by covalently coupling peptide LDC27 sequence to the matrix; this is a 27-amino acid peptide derived from domain 3 that has been identified as an HK-binding site (Herwald et al. 1995). When cell membranes derived from HUVECs were applied to the column in the presence and absence of 50 μmol/L zinc and each eluted with 0.1 mol/L glycine-HCl, pH 2.5, a band was noted at 68 kDa in the zinc-containing eluate. A ligand blot with HK confirmed binding to this band. Attempts to sequence the protein failed because the Nterminus was blocked. It was therefore digested with cyanogen bromide and the mixture subjected to mass spectrometry. A major peptide at molecular weight 2721.7 was identified, its sequence determined, and shown to correspond to an internal peptide derived from cytokeratin 1 (see Fig. 20.4) (Joseph et al. 1999a,b). Thus HK binding to HUVECs appears to depend on interaction with two proteins, cytokeratin 1 and gC1qR, with binding to each by domains 3 and 5 of HK respectively (i.e., binding of heavy chain to cytokeratin 1 and light chain to gC1qR). We demonstrated that gC1qR cannot bind heavy chain at all, whereas cytokeratin 1, when tested as a purified protein, can bind both heavy and light chains, although binding to heavy chain clearly predominates. Factor XII is capable of binding to both proteins. To confirm that these proteins are important for binding to endothelial cells, we performed an inhibition experiment in which antibody to gC1qR and antibody to cytokeratin 1 were employed. Antibody to gC1qR inhibited zinc-dependent binding by 65%, antibody to cytokeratin 1 inhibited binding by 30%, while a combination of antisera inhibited binding by 85%. Since 15% binding corresponds to zinc-independent binding, our data suggest that we accounted for most if not all HK binding to endothelial cells by these two proteins. A third protein has been reported to be important for HK binding to HUVECs and this has been identified as the urokinase plasminogen activator receptor (u-PAR) by inhibition of HK binding with antisera to domain 2/3 of u-PAR (Colman et al. 1997). However, our laboratory has not been able to isolate u-PAR from cell membranes (confirmed to contain considerable u-PAR) by HK affinity chromatography. One difference in the experiments is that the studies by Colman et al. employed cleaved HK lacking the bradykinin moiety and it is possible that cleaved HK binds more avidly to u-PAR than does native HK, while native HK binds more avidly to gC1qR and cytokeratin 1 than it does to u-PAR.

Binding of factor XII to HUVECs Early studies demonstrated that factor XII binds to both gC1qR and cytokeratin 1 and that it competes for the same binding sites as HK (see Fig. 20.5). The first study to attempt to identify the binding site on HUVECs (rather than testing purified proteins shown to bind HK) was by Mahdi et al. (2002) in which antisera to u-PAR, cytokeratin 1, and gC1qR

456

Endothelial cell

Zn2+

B2 receptor 2+

Zn

HK FXII

BK

2+

Zn

2C-HK

HK PK

K

FXIIa Fig. 20.6 Generation of bradykinin (BK) along the endothelial cell surface via zinc-dependent interaction of high-molecular-weight kininogen (HK) and factor XII with endothelial cell surface receptors (representing gC1qR, cytokeratin 1, and u-PAR). PK, prekallikrein; K, kallikrein. (See CD-ROM for color version.)

were employed to inhibit cell binding of factor XII. A surprising result was that antibody to u-PAR inhibited best, although the other antisera were contributory. We therefore sought to corroborate the observation by isolation of factor XII-binding proteins directly from HUVEC-derived cell membrane preparations by affinity chromatography employing factor XII as ligand. The major zinc-dependent binding protein was clearly u-PAR; gC1qR was also isolated as well as small amounts of cytokeratin 1 (Joseph, K. and Allen P. Kaplan, unpublished data). A higher avidity of binding of factor XII to u-PAR than to either gC1qR or cytokeratin 1 was demonstrated by competitive displacement of factor XII bound to one protein employing increasing quantities of either of the other two. Thus a model for assembly of the kinin-forming cascade on endothelial cells was developed in which zinc-dependent binding of factor XII is associated predominantly with u-PAR, and HK binds to gC1qR as well as cytokeratin 1 while PK is bound to HK. A diagrammatic representation of such binding is shown in Fig. 20.6, as well as activation of the cascade along the endothelial cell surface to generate bradykinin. Table 20.1 summarizes the interactions of the proteins of the kinin-forming cascade with the endothelial cell-binding proteins for each. How that binding occurs, particularly for HK, depends on the way these proteins are distributed within the cell membrane of HUVECs.

Interaction of gC1qR, cytokeratin 1, and u-PAR within HUVEC cell membranes A dilemma posed by antibody inhibition studies in which all three antisera were employed was that the total inhibition obtained with all three antisera combined exceeded 100%. One possible explanation is that these proteins might interact in some fashion within the cell membrane and a trimolecular complex containing all three was proposed (Colman & Schmaier 1997).

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Table 20.1 Plasma inhibitors of enzymes of contact activation: relative percentage contributions to inhibition in normal human plasma. Enzyme Inhibitor

Factor XIIa

Factor XIIf

Kallikrein

Factor XIa*

C1 inhibitor Antithrombin III† a2-Macroglobulin a1-Protease inhibitor a2-Antiplasmin

91.3 1.5 4.3 – 3.0

93 4 – – 3

52 (84)† ND 35 (16)† ND ND

8 (47) 16 (5) – 68 (23.5) 8‡ (24.5)

* Data given are from kinetic studies and irreversible complexes formed in plasma are given in parentheses. † Data obtained from generation of kallikrein in situ. ‡ Data are for results obtained in the absence of added heparin. ND, not determined separately.

We (and others) have addressed each of these issues. Employing high-titer monospecific antisera to gC1qR, the protein was clearly demonstrated to be at the HUVEC cell surface (Joseph et al. 1999b). Mahdi et al. (2001, 2002) then demonstrated the presence of all these proteins within cell membranes by immunoelectron microscopy. Cytokeratin 1 and u-PAR appeared to be colocalized while gC1qR was present throughout the cell membrane. The interactions of these proteins with each other was then examined. First it was shown that gC1qR binds to cytokeratin 1 but not u-PAR, while u-PAR also binds to cytokeratin 1 but not gC1qR. Thus a trimolecular complex is not possible but two bimolecular complexes seemed feasible. gC1qR and u-PAR were then precipitated from cell membrane preparations and the composition of the precipitated materials was analyzed. Cytokeratin 1 was precipitated with both anti-gC1qR and anti-u-PAR; however, the gC1qR/cytokeratin 1 fraction had no u-PAR, whereas the cytokeratin 1/u-PAR fraction contained no gC1qR. A current view of the assembly of the proteins of the kinin-forming cascade on HUVECs envisions factor XII bound to a complex of u-PAR/cytokeratin 1 while HK binds to a complex of gC1qR/cytokeratin 1. It is not known whether HK binds to the complex by both domain 3 and domain 5 simultaneously or whether binding to one site affects binding to the other. Complicating this assessment is the fact that the number of gC1qR sites within the cell membrane is at least three times that of u-PAR or cytokeratin 1; thus gC1qR unassociated with either cytokeratin 1 or u-PAR is likely present and can bind HK or factor XII. Given the relative affinities of factor XII, HK heavy chain, and HK light chain for gC1qR, we would anticipate preferential binding of light chain (domain 5) of HK to gC1qR. Consistent with this is the very prominent inhibition of HK binding to the cell employing peptide HKH20 derived from domain 5 of the light chain (Nakazawa et al. 2002).

Binding to other cells The interaction of factor XII and HK with other cell types resembles that seen in HUVECs, although there are differences in number of binding sites, affinity of binding, and nature of the binding proteins. We recently reported studies of microvascular endothelial cells derived from skin and lung, and compared binding with that seen in HUVECs since these cells are more likely to be “physiologic” (Fernando et al. 2003). Whereas HUVECs had approximately 710 000 binding sites per cell, dermal microvascular cells had 53 000 per cell and lung microvascular cells 316 000 per cell. The binding affinity was 2.5- and 10-fold higher for dermal and lung endothelial cells, respectively. It should be noted that a very large number of HK-binding sites were demonstrated for HUVECs by two separate methods, including a fluid-phase based assay where 850 000 binding sites per cell were documented. It has been suggested that values in the 107 range (Motta et al. 1998) may have been due to ligand binding to the plates used rather than to the cells coating the plates, and thus the cell surface represented only a fraction of the total binding seen (Baird & Walsh 2002, 2003). However, the inhibition of such binding employing antisera to the cellsurface ligands suggests some other interpretation (Mahdi et al. 2003), and our values in the fluid phase (696 427 binding sites/cell) are in close agreement with binding studies within microtiter plates (771 666 binding sites/cell). Binding of HK and factor XII to neutrophils and platelets has also been studied. HK interacts with neutrophils in a zinc-dependent fashion analogous to that seen with other cell types, although the protein with which it interacts is MAC-1 (C3bi receptor; CD11b/CD18) (Wachtfogel et al. 1994). Zinc-dependent HK binding to platelets is dependent on interaction with glycoprotein 1b (Bradford et al. 1997; Joseph et al. 1999c). Since both PK and factor XI circulate as complexes with HK, HK–factor XI and HK–PK should bind to

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HUVECs in an analagous fashion (Shariat-Madar et al. 2001). However, Baird and Walsh (2003) reported that although preformed HK–PK binds to endothelial cells as a complex, HK–factor XI does not. This is in contrast to studies in which HK is bound and factor XI is added separately, in which case factor XI will bind to HK. In response, Mahdi et al. (2003) agree that HK–PK predominates, but that HK–factor XI can bind with lower affinity. Binding to platelets differs because factor XI can interact with platelets in the absence of HK, and there appear to be separate receptors for factor XI and factor XIa (Sinha et al. 1984). Platelets also possess an intrinsic protein with factor XI activity that cross-reacts with plasma factor XI immunologically but differs in molecular weight and isoelectric point (Hsu et al. 1998). This form of factor XI is present even in patients deficient in plasma factor XI (Tuszynski et al. 1982) and has been shown to be an alternatively spliced form of factor XI in which one exon is missing (Hsu et al. 1998). During blood coagulation by the extrinsic (tissue factor) pathway, these forms of factor XI are more likely activated by thrombin feedback (Gailani & Broze 1991; Naito & Fujikawa 1991; Baglia & Walsh 2000; Baglia et al. 2002) than by factor XIIa to augment clot formation. This has been shown to occur as a late event within a fibrin matrix (Rand et al. 1996; Bouma & Meijers 2000). Thus activation on platelets involves factor XII-dependent (Walsh & Griffin 1981; Brunnee et al. 1993) and factor XII-independent (thrombin) pathways (von dem Borne et al. 1994), and factor XI may be attached to the platelets via HK or by separate receptors. LK, which has a separate light chain from HK, interacts with platelets (as is true of endothelial cells) and, of necessity, does so solely via domain 3 (Jiang et al. 1992a,b; Herwald et al. 1995) since the light chains of the two kininogens are completely different.

Activation of the kinin cascade: the role of endothelial cells Activation by binding to the cell surface Factor XII has been shown to slowly autoactivate when bound to endothelial cells and addition of kallikrein can digest bound HK to liberate bradykinin at a rate proportional to the kallikrein concentration, with a final bradykinin level dependent on the amount of bound HK (Nishikawa et al. 1992). Thus, activation of the cascade along the endothelial cell surface is likely; bradykinin is liberated and then interacts with the B2 receptor to increase vascular permeability. Bradykinin can also stimulate cultured endothelial cells to secrete tissue plasminogen activator (Smith et al. 1985), prostaglandin I2 (prostacyclin), thromboxane A2 (Crutchley et al. 1983), and nitric oxide and can thereby modulate platelet function and stimulate local fibrinolysis. To determine whether factor XII binding to gC1qR is capable of initiating this cascade, purified factor XII was incubated with a wide dose

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range of gC1qR (0–100/μL) for 30 min and replicate samples were incubated in the absence of zinc ion. We found that the rate of PK conversion to kallikrein increased as the concentration of gC1qR increased (Joseph et al. 2001a,b) and there was no activation if zinc was eliminated from the reaction mixture. Purified cell membrane (native) gC1qR yields a response that is indistinguishable from a recombinant protein, indicating that gC1qR glycosylation does not affect its “surface” properties. If gC1qR is incubated directly with PK or with PK plus HK, there is no conversion of PK to kallikrein , again emphasizing the requirement for factor XII. This appears to be a physiologic phenomenon controlled by C1 inhibitor and α2-macroglobulin. This may be one source of the minute quantities of factor XIIa that escape inhibition that are requisite for contact activation in plasma or during pathologic processes and may be operative in patients with C1 inhibitor deficiency. Other data employing endothelial cells have corroborated the aforementioned effect of gC1qR when endothelial cells are incubated with normal plasma and the rate of kallikrein formation compared with that seen with plasma deficient in factor XII, PK, or HK. There was no detectable activation in any plasma except normal plasma (Fig. 20.7a) and the activation was inhibited by antisera to gC1qR and cytokeratin I (Fig. 20.7b). However, when the reaction proceeds beyond 2 hours, factor XII-deficient plasma activates, but HK-deficient plasma and PK-deficient plasma do not; thus a cell-dependent activation of PK in the presence of HK but absence of factor XII appears possible.

Factor XII-independent activation of the PK–HK complex Studies have demonstrated that binding of the PK–HK complex to endothelial cells leads to activation in the absence of factor XII (Rojkjaer et al. 1998; Rojkjaer & Schmaier 1999a,b) and that the kallikrein which forms can digest HK to liberate bradykinin and also initiate fibrinolysis (Lin et al. 1997). The latter reaction is dependent on kallikrein activating prourokinase (bound to cell membrane u-PAR) to urokinase, which in turn converts plasminogen to the fibrinolytic enzyme plasmin. Once such a reaction is set in motion, the addition of factor XII leads to a marked increase in reaction kinetics as a result of the conversion of factor XII to factor XIIa by kallikrein. These observations raise two important questions. First, what is the nature of the PK activator? Second, when factor XII is present (the normal circumstance), is the cascade initiated by factor XII autoactivation or is PK first activated by some cell-derived factor and kallikrein then activates factor XII? The cell-derived protein(s) responsible for PK activation in the absence of factor XII has been purified and characterized. The activity was present in the cell membrane fraction as well as the cytosol derived from endothelial cells and we chose to isolate it from the cytosol. The PK-activating moiety appeared to be inhibitable by corn trypsin inhibitor (CTI) (as

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Pooled normal plasma

Anti-gC1qR

PK-deficient plasma

Anti-cytokeratin 1

FXII-deficient plasma

0.20

Pooled normal plasma

0.25

HK-deficient plasma

Absorbance (OD 405 nm)

Absorbance (OD 405 nm)

0.25

Bradykinin Pathways and Allergic Disease

0.15

0.10

0.05

Anti-gC1qR

0.20

anti-cytokeratin 1

0.15

0.10

0.05 0

20

40

60

80

100

(b)

Incubation time (min)

(a)

0

120

20

40

60

80

100

120

Incubation time (min)

Fig. 20.7 Prekallikrein activation on endothelial cells. (a) Endothelial cells were incubated with normal, prekallikrein-deficient, factor XII-deficient or highmolecular-weight kininogen (HK)-deficient plasmas for 1 hour at 37°C. After incubation, the cells were washed with HEPES-buffered saline containing 50 mmol/L zinc chloride and prekallikrein activation was monitored by the cleavage of a kallikrein-specific substrate, S2302 (0.6 mmol/L) at 405 nm. (b) Endothelial cells were preincubated with antibodies to cytokeratin 1, gC1qR, or a combination of both for 30 min before addition of normal plasma.

is factor XIIa) and a CTI affinity column was shown to bind the activity and it was recoverable by eluting the column. A single-step purification followed by sequence analysis of suspect bands seen on SDS gel electrophoresis ultimately determined that heat-shock protein (HSP)-90 is responsible for the activity seen. Thus when cloned HSP-90 was incubated with PK and HK, the PK was converted to kallikrein and HK was cleaved to liberate bradykinin (Fig. 20.8) (Joseph et al. 2002a,b). This is also demonstrable by binding PK and HK to

0.9

HK+PK+HSP90+Zn HK+PK+HSP90

0.8

PK+HSP90+Zn

Absorbance (OD 405 nm)

0.7

PK+HSP90

0.6 0.5 0.4 0.3 0.2 0.1 0 0

20

40

60 Time (min)

80

100

Fig. 20.8 Prekallikrein (PK) activation on heat-shock protein (HSP)-90. Purified HSP-90 (2 mg) was incubated with PK (20 mmol/L), highmolecular-weight kininogen (HK) (20 mmol/L), zinc (50 mmol/L), and S2302 (0.6 mmol/L) and chromogenic activity was monitored. Controls were performed in the absence of either zinc or HK, or both.

120

endothelial cells and assessing the rate of conversion of PK to kallikrein. Both HK and zinc ion are requisite and the rate is fast. However, this is in contrast to the very slow and factor XII-dependent activation seen when whole plasma is employed (Fig. 20.7). The reaction is readily demonstrable in the fluid phase as well as by assembly of components along the cell surface, although it differs strikingly from that seen with factor XIIa. The most critical difference is that PK is not activated unless HK is present. Factor XIIa readily activates PK, although the presence of HK does augment the reaction rate. Second, the reaction is stoichiometric, i.e., the amount of PK activated has a 1 : 1 molar ratio to the amount of HSP90 present. When the structural features of HK required for binding were determined, the individual heavy and light chains were inactive, and cleaved HK, with bradykinin removed (i.e., two-chain rather than single-chain HK), lost about 70% of the activity (Fig. 20.9). Thus native HK is required. Addition of a peptide that prevents the interaction of PK with HK also completely inhibits the effect of adding HSP-90. HSP-90 is therefore a stoichiometric activator of the PK–HK complex and not a PK activator as is factor XIIa. One of the interesting questions we might consider is whether HSP-90 has enzymatic activity, with the PK–HK complex as substrate. HSP-90 does have ATPase activity but it is not known to be a proteolytic enzyme. The PK-activating activity can be inhibited by diisopropylfluorophosphate (DFP) but it has been difficult to characterize the active site. Although DFP inhibits the reaction, we have been unable to incubate DFP with individual components, dialyze it out, and inhibit the reaction. In fact, if DFP is added to a mixture of HSP-90, PK and HK, it inhibits conversion of PK to kallikrein; however, if DFP is then dialyzed out of the mixture, PK activation

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Activation when factor XII is present

0.6

Absorbance (OD 405 nm)

0.5

0.4

0.3

0.2

0.1

0 SC-HK

2C-HK

LK

HC-HK

LC-HK

Fig. 20.9 Effect of high-molecular-weight kininogen (HK) on prekallikrein activation. Cytosol (20 mg) was incubated with 20 nmol/L of HK, lowmolecular-weight kininogen (LK), cleaved HK (2C-HK), purified heavy chain of HK (HC-HK), or light chain of HK (LC-HK) in the presence of 20 nmol/L prekallikrein, 50 mmol/L zinc and 0.6 mmol/L S2302. After 2 hours the chromogenic activity was measured at 405 nm.

proceeds normally. Thus DFP behaves as a reversible inhibitor instead of as an irreversible inhibitor, and is not phosphorylating an active site serine as is its usual effect on serine proteases. Alternative possibilities were considered, for example autoactivation of PK within the PK–HK complex on addition of HSP-90, or even that HK becomes an enzyme that converts PK to kallikrein when HSP-90 binds, but the mechanism is thus far unknown. Other studies have isolated yet another protein with very similar functional capability. Shariat-Mader and colleagues isolated a membrane protein that converts PK to kallikrein within the PK–HK complex and identified it to be prolylcarboxypeptidase (Motta et al. 2001; Shariat-Madar et al. 2002). This is an exopeptidase which, if its enzymatic capability is relevant, is behaving as an endopeptidase. It is said to be active along the cell surface, but not in the fluid phase, which is different from HSP-90; otherwise, its mechanism of action is strikingly similar. Both require the presence of HK and zinc ion, the reaction in each case is stoichiometric, and each is inhibited by DFP. Although the polylcarboxypeptidase is assumed to be the enzyme that activates PK within the PK– HK complex, we suspect that some other mechanism may be operative, perhaps common to both. The prolylcarboxypeptidase provides an interesting link from the kinin-forming cascade to the biology of angiotensin since its function, when originally isolated, was to convert angiotensin II to angiotensin III, which inactivates it. Thus a molecule that can generate bradykinin, a vasodilator, inhibits another that is a vasoconstrictor. HSP-90 is also of particular interest since this is a protein that is constitutively present yet upregulated with tissue stress such as hypoxia or during an inflammatory response.

460

Since the endothelial cell participates in the activation of the bradykinin-forming cascade, when all three components are present, factor XII might be activated by autoactivation on gC1qR, requiring trace amounts of factor XIIa present in plasma, or factor XII might be activated by kallikrein. In the latter scenario, the source of kallikrein would be the stoichiometric interaction of PK–HK with HSP-90 and/or prolylcarboxypeptidase. Formation of factor XIIa then markedly accelerates activation of PK–HK since this reaction has typical Michaelis–Menten kinetics. Of course all these may be occurring simultaneously, but the evidence thus far suggests that the rate of activation of the PK–HK complex exceeds that of factor XII autoactivation. Thus it is possible that the initiation of the cascade on the surface is actually kallikrein, while factor XII has the role of an accelerator. Some favor this possibility, employing endothelial cells and purified protein constituents (Schmaier et al. 1987; Schmaier 1997, 1998; Rojkjaer et al. 1998). On a quantitative basis, the cascade remains factor XII-dependent. However, the data in Fig. 20.7 suggest that factor XII may truly initiate when whole plasma is studied, i.e., minimal dilution in the presence of all the plasma inhibitors.

Inhibition of contact activation Regulation of factor XII-dependent pathways occurs by both intrinsic and extrinsic controls. Cleavage of factor XIIa to XIIf (see Fig. 20.2) is one example of an intrinsic control. The factor XIIf produced is not surface bound and is a very poor activator of factor XI. At the same time, the heavy chain moiety, which has no enzymatic activity, retains the surfacebinding site and can compete with factor XII and HK for binding to the surface. Thus, the conversion of factor XIIa to factor XIIf will reduce the rate of the surface-dependent reactions of coagulation, whereas bradykinin generation via fluid-phase activation of PK continues. Similarly, digestion of kinin-free HK by factor XIa has been reported to limit its coagulant activity (Scott et al. 1985), although in this case the kinetics appear to be too slow to be of physiologic importance (Reddigari & Kaplan 1988). Extrinsic controls are provided by plasma inhibitors for each enzyme. Table 20.1 indicates the major inhibitors of each active enzyme and, where known, their relative contributions to the total inhibition in plasma. Inhibition of the contact activation proteases is clearly different from that of the rest of the coagulation pathways in that antithrombin III (ATIII) appears to play only a minor role. Instead, contact activation appears to be limited mainly by C1 inhibitor, which is not active against any of the other clotting factors except for inhibition of factor XI. C1 inhibitor is cleaved by the protease it inhibits and attaches to the active site in a covalent complex. It may remain in the stable form of the

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acyl enzyme intermediate that characterizes the normal serine protease mechanism (Travis & Salvesen 1983). Thus, after a protease has reacted with C1 inhibitor, it cannot digest protein substrates or hydrolyze small synthetic substrates, and the reaction of the active site serine with DFP is abolished. C1 inhibitor is the only major plasma inhibitor of factor XII and factor XIIf (de Agostini et al. 1984; Pixley et al. 1985). Although ATIII can inhibit activated factor XII (de Agostini et al. 1984; Cameron et al. 1989), its contribution to factor XIIa inhibition in plasma is apparently only a few percent of that due to C1 inhibitor (de Agostini et al. 1984; Pixley et al. 1985). Disagreement exists over the effect of heparin on the inhibition of activated factor XII by ATIII. Some investigators have observed little enhancement of the rate of factor XIIa inhibition (Pixley et al. 1991), whereas others have observed a significant increase (Cameron et al. 1989). Heparin can act as an activating surface for contact activation, and factor XII and factor XIIa can bind to it (Hojima et al. 1984; Silverberg & Diehl 1987). This binding is a factor in the inhibition by ATIII since inhibition of factor XIIf, which lacks the surfacebinding site, is not augmented in the presence of heparin as much as that of factor XIIa (Cameron et al. 1989). Curiously, α2-macroglobulin, although thought of as a “universal” protease inhibitor, does not significantly inhibit either form of activated factor XII. The two major inhibitors of plasma kallikrein are C1 inhibitor and α2-macroglobulin (Gigli et al. 1970; Harpel 1973; Harpel et al. 1985). Together they account for over 90% of the kallikrein inhibitory activity of plasma, with the remainder contributed by ATIII (Schapira et al. 1982a; van der Graaf et al. 1983a). When kallikrein is added to plasma, approximately half is bound to C1 inhibitor and half to α2macroglobulin (Harpel et al. 1985). α2-Macroglobulin does not bind to the active site of kallikrein but appears to trap the protease within its structure so as to sterically interfere with its ability to cleave large protein substrates. The degree of inhibition is greater than 95%, but the residual activity is detectable when assayed for lengthy incubation periods. In contrast, digestion of small synthetic substrates is much less affected, and approximately one-third of the starting activity is retained. When a surface such as kaolin is added to plasma so that kallikrein is generated in situ, close to 70 or 80% of it is bound to C1 inhibitor (Harpel et al. 1985). The reason for the difference between the patterns of inhibition of added kallikrein and of endogenously produced kallikrein is unknown. Interestingly, at low temperatures, most of the inhibition of added kallikrein is accounted for by α2-macroglobulin (Harpel et al. 1985); C1 inhibitor appears to be ineffective in the cold (Cameron et al. 1989) and this may underlie the phenomenon of “cold activation” of plasma. The inhibition of kallikrein by ATIII is also enhanced by heparin and may therefore become significant in heparinized plasma. The inhibition profile of factor XI is complicated by the

Bradykinin Pathways and Allergic Disease

involvement of several factors. In kinetic studies of purified components, α1-antiproteinase inhibitor (α1-antitrypsin) appears to be the most significant inhibitor of factor XIa (Heck & Kaplan 1974; Scott et al. 1982a), whereas α1-antitrypsin is not a major inhibitor of other coagulation factors. However, when the generation of irreversible enzyme inhibitor complexes was assessed in plasma, C1 inhibitor was found to be the key inhibitor (Wuillemin et al. 1995), with approximately equal contributions by α2-antiplasmin and α1-antiproteinase inhibitor. ATIII is also an inhibitor of factor XI with potential for augmentation by heparin, the magnitude of which is unclear (Scott et al. 1982b; Beeler et al. 1986). The predominant role of C1 inhibitor in the regulation of contact activation in human plasma is underscored by the fact that it alone is an efficient inhibitor of activated factor XII, kallikrein, and factor XIa. In plasma from patients with hereditary angioedema, in which C1 inhibitor is absent, the amount of dextran sulfate required to produce activation is reduced tenfold compared with normal plasma (Cameron et al. 1989); similar results are obtained in cold plasma. Because some surface was still required for activation under these conditions, we may surmise that the other inhibitors which are active against the contact factors do serve to limit their reactions, but that in normal plasma it is inhibition by C1 inhibitor that forms the barrier to the initiation of contact activation. The plasma concentration of C1 inhibitor is approximately 2 μmol/L, and it is remarkable that its inhibition is ever overcome. That surfaces are able to induce activation must reflect the protection of the proteases at the surface from inhibition. It has also been proposed that kallikrein bound to HK is protected from inactivation by C1 inhibitor (Schapira et al. 1981, 1982b) and α2-macroglobulin (Schapira et al. 1982b; van der Graaf et al. 1983a,b) and that factor XIa is similarly protected from α1-antiproteinase inhibitor (Scott et al. 1982a); however, this mechanism has been ruled out in the case of kallikrein and C1 inhibitor (van der Graaf et al. 1983a; Silverberg et al. 1986).

Inactivation of bradykinin Bradykinin is an exceedingly potent vasoactive peptide that can cause venular dilatation, activation of arterial endothelial cells, increased vascular permeability, hypotension, constriction of uterine and gastrointestinal smooth muscle, constriction of the coronary and pulmonary vasculature, bronchoconstriction, and activation of phospholipase A2 to augment arachidonic acid metabolism. Its regulation is of prime importance, and a variety of enzymes in plasma contribute to kinin degradation. Carboxypeptidase N (Erdos & Sloane 1962) removes the C-terminal Arg from bradykinin to leave an octapeptide, des-Arg9-bradykinin (Sheikh & Kaplan 1986a), which is then digested by ACE, acting as a tripeptidase, to separate the tripeptide Ser-Pro-Phe from the

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pentapeptide Arg-Pro-Pro-Gly-Phe (Sheikh & Kaplan 1986b). Enzymes that have not been characterized rapidly digest Ser-Pro-Phe to individual amino acids and more slowly convert the pentapeptide to Arg-Pro-Pro plus Gly and Phe. The final products of bradykinin degradation are the peptide Arg-Pro-Pro, plus 1 mol each of Gly, Ser, Pro, and Arg, and 2 mol of Phe (Sheikh & Kaplan 1989b,c). The change from bradykinin to des-Arg9-bradykinin formed by this initial cleavage retains some but not all the various activities of bradykinin (Marceau & Bachvarov 1998). It can, for example, interact with B1 receptors (Regoli & Barabe 1980) induced by inflammation (e.g., IL-1, TNF-α) in the vasculature and cause hypotension, but bradykinin’s activities on the skin and the contraction of other smooth muscles are abolished. Bradykinin interacts with constitutively expressed B2 receptors to mediate all its functions. Selective B2 and B1 receptor antagonists have been synthesized (Vavrek & Stewart 1985; Beierwaltes et al. 1987; Stewart et al. 1999). When blood is clotted and serum is studied, all the reactions for bradykinin degradation occur as described, but the rate of the initial Arg removal is accelerated fivefold compared with plasma (Sheikh & Kaplan 1989b). This is probably due to the action of a plasma carboxypeptidase that is distinct from carboxypeptidase N and is expressed (activated) as a result of blood coagulation. It has been designated “thrombin activatable fibrinolysis inhibitor” (TAFI) (Bajzar et al. 1995, 1996). It should also be noted that bradykinin degradation in vivo occurs largely along the pulmonary vasculature and that endothelial cells there have carboxypeptidase as well as ACE activities. In the pulmonary circulation, the initial cleavage may occur by ACE acting as a dipeptidase to first remove Phe-Arg and then Ser-Pro (each of which is next cleaved to free amino acids), leaving the pentapeptide Arg-Pro-Pro-GlyPhe. This is then metabolized further. The cough, wheeze, and angioedema sometimes associated with use of ACE inhibitors for treatment of hypertension or heart failure is likely due to inhibition of kinin metabolism leading to increased levels of bradykinin (Nussberger et al. 1998). Because bradykinin is a peripheral vasodilator, it has been considered to be a counterbalance to the vasopressor effects of angiotensin II. It is clear that the two peptides are also related in terms of metabolism, because ACE cleaves His-Leu from the C-terminal of angiotensin I, a decapeptide, to leave the octapeptide angiotensin II. Thus, ACE creates a vasoconstrictor and inactivates a vasodilator. The prolylcarboxypeptidase discussed above does the reverse: it can produce bradykinin, a vasodilator, and inactivate angiotensin II, a vasoconstrictor.

Considerations in human diseases Bradykinin mediates its effects by interaction with receptors termed B1 and B2. B2 receptors were actually discovered first

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and exhibit high affinity for bradykinin and Lys-bradykinins (kallidin) and mediate effects such as vasodilatation, increased vascular permeability, and contraction of smooth muscle. A B2 receptor antagonist (Icatibond or HOE 140) is being evaluated for possible clinical utility, but has been used in animal models for many years. Once a ligand interacts with B2 receptors, there is rapid internalization and desensitization (Marceau et al. 2002). The B1 receptor, in contrast, is not present constitutively but is induced during an ongoing inflammatory process by cytokines such as IL-1 and TNF-α. This receptor is selectively activated by des-Arg9-bradykinin and des-Arg10-Lys-bradykinin, the products of carboxypeptidase removal of C-terminal Arg from bradykinin and kallidin, respectively. At least one additional cleavage, typically by ACE, is required to inactivate the system. Both B1 and B2 receptors signal via G proteins compled to them. However, the B1 receptor remains at the cell surface, once activated, and continues to function as long as ligands are available.

C1 inhibitor deficiency Although C1 inhibitor was defined as an inhibitor of the activated first component of complement, it is clearly a key control protein of the plasma kinin-forming cascade. The pathogenesis of the swelling in C1 inhibitor deficiency is dependent on the plasma kinin-forming pathway rather than complement; however, it is germane to review the history of the complement data and to point out how it was first thought to be the key, and then to present the more recent data that suggest otherwise. Intracutaneous injection of C1 into normal individuals was reported to cause the formation of a small wheal reaction whereas injection into patients with hereditary angioedema yielded localized angioedema, i.e., an augmented response because of low C1 inhibitor (Klemperer et al. 1968). A kinin-like peptide was isolated from such patients and its formation appeared to be inhibited in C2deficient plasma. Thus C2 was considered to be the source of the pathogenic peptide (Donaldson 1968). However, direct demonstration of such a kinin-like peptide on interaction of activated C1 and C4 and C2 or with C2 alone is lacking. Although it was originally reported that cleavage of C2b by plasmin generates a kinin (Donaldson et al. 1977), attempts to confirm this experiment have all failed (Fields et al. 1983). The only identifiable kinin seen in subsequent studies was bradykinin (Fields et al. 1983). On the other hand, the amino acid sequence of C2b is known, and Strang et al. (1988) synthesized peptides of various lengths and tested each for kinin-like activity. One such peptide was shown to cause edema when injected intracutaneously, reminiscent of the C2 kinin originally described. However, this peptide has not been shown to be a cleavage product of C2b, nor has it been shown to be present during attacks of swelling in patients with hereditary angioedema. Thus, at this point it seems unlikely that a kinin-like molecule is derived from C2b as a result

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of enzymatic cleavage. On the other hand, the presence of bradykinin has been documented, as described below, and it is the likely cause of the swelling. In fact when one of the proponents of the C2 kinin reexamined kinin formation in the plasma of patients with hereditary angioedema, only bradykinin was found (Shoemaker et al. 1994). It should be noted that 24-hour urine histamine excretion may also be increased during attacks of angioedema, suggesting that C3a, C4a, or C5a is being generated. Although the plasma levels of C3 and C5 are normal in this disorder, C3 turnover is clearly enhanced. The lesions, however, are not pruritic, and antihistaminics have no effect on the clinical course of the disease. Thus, complement activation is undoubtedly occurring, perhaps even during quiescent periods, leading to a low level of C4, but the vasoactive consequences of the augmented complement activation that occurs during attacks of hereditary angioedema do not appear to be the cause of the swelling. C1 inhibitor inhibits all functions of factor XIIa and is one of the two major plasma kallikrein inhibitors (Gigli et al. 1970), the other being α2-macroblobulin (Harpel et al. 1985), and all functions of kallikrein are thereby inhibited including the feedback activation of factor XII, the cleavage of HK, and the activation of plasma pro-urokinase (Ichinose et al. 1986) leading to plasmin formation. C1 inhibitor also inhibits the fibrinolytic enzyme plasmin, although it is a relatively minor inhibitor compared with α2-antiplasmin or α2-macroglobulin. Patients with hereditary angioedema appear to be hyperresponsive to cutaneous injections of kallikrein, as they are to C1, and elevated levels of bradykinin and cleaved kininogen have been observed during attacks of swelling (Nussberger et al. 1998). There is also evidence that C1 activation observed in hereditary angioedema may also be factor XII dependent (Donaldson 1968). Thus, a factor XII-dependent enzyme may be initiating the classic complement cascade. Plasmin is capable of activating C1s and may represent one such enzyme (Ratnoff & Naff 1967). Ghebrehiwet et al. (1981, 1983) demonstrated that Hageman factor fragment (factor XIIf) can directly activate the classical complement cascade by activating C1r and to a lesser degree C1s. This may represent a critical link between the intrinsic coagulation–kinin cascade and complement activation (see Fig. 20.2). The presence of kallikrein-like activity in induced blisters of patients with hereditary angioedema supports this notion, as does the progressive generation of bradykinin on incubation of hereditary angioedema plasma in plastic noncontact-activated test tubes (Fields et al. 1983) and the low PK and HK levels seen during attacks (Schapira et al. 1983). More recent data support these indirect observations, favoring bradykinin as the critical pathogenic peptide for hereditary angioedema and, probably, acquired C1 inhibitor deficiency as well. One unique family has been described in which there is a point mutation in C1 inhibitor (Ala443→Val), leading to inability to inhibit the complement cascade but normal inhibition

Bradykinin Pathways and Allergic Disease

of factor XIIa and kallikrein (Zahedi et al. 1995, 1997). No family member of this type II mutation has had angioedema. In recent studies, plasma bradykinin levels have been shown to be elevated during attacks of swelling in patients with hereditary and acquired forms of C1 inhibitor deficiency (Cugno et al. 1996; Nussberger et al. 1998) and local bradykinin generation has been documented at the site of the swelling (Nussberger et al. 1999). The role of fibrinolysis also needs to be considered in the pathogenesis of the disease since antifibrinolytic agents such as ε-aminocaproic acid and tranexamic acid appear to be efficacious (Lundh et al. 1968; Frank et al. 1972; Sheffer et al. 1972) and plasmin is generated during active disease (Cugno et al. 1993). Although kallikrein, factor XIa, and even factor XIIa have some ability to activate plasminogen directly, the plasma pathway via the pro-urokinase intermediate appears to be the major factor XII-dependent fibrinolytic mechanism (Fig. 20.10). Among the functions of plasmin are activation of C1s, the ability to cleave and activate factor XII as kallikrein does (Kaplan & Austen 1971), and digestion of C1 inhibitor (Wallace et al. 1997). Each of these would serve to augment bradykinin formation and further deplete the levels of C1 inhibitor. Thus, the formation of plasmin may in this fashion contribute to the pathogenesis of the disease.

Regulation of blood pressure The possible relationship of the contact system to blood pressure regulation is an intriguing question. As already seen, ACE creates the hypertensive peptide angiotensin II, and plays a major role in inactivating the hypotensive product of contact activation, bradykinin. An unfortunate circumstance has dramatized the kinin-forming capacity of factor XIIf: trauma patients given plasma protein fractions as plasma expanders that were contaminated with factor XIIf showed profound hypotension (Alving et al. 1978, 1980). The mechanism by which infused factor XIIf causes hypotension has been demonstrated to be due to bradykinin formation. A more tenuous connection exists between blood pressure regulation and the contact system in the factor XII-dependent activation of plasma prorenin. Prorenin is activated to renin by cold treatment of plasma or acidification to pH 3.3. With acid treatment, most of the renin activity is produced after reneutralization; this alkaline-phase activation is mediated by kallikrein (Derkx et al. 1979; Sealey et al. 1979), as is the cold-induced activation (Brown & Osmond 1984). Kallikrein is able to activate purified prorenin (Yokosawa et al. 1979), but when added to plasma it does not cause prorenin activation in the absence of an acidification step. Although it has been supposed that acid treatment serves to destroy kallikrein inhibitors, prorenin is not activated in plasma deficient in C1 inhibitor or α2-macroglobulin (Purdon et al. 1985). Thus, some other unknown event must occur on acidification. The physiologic significance of this reaction is uncertain.

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Factor XII Prekallikrein HK Contact activation Activation along cell surface

Plasmin kallikrein Prorenin

Angiotensinogen

HK

Renin pH

HK Angiotensin I

ACE

Prekallikrein

Bradykinin

Prolylcarboxypeptidase

Angiotensin II

Angiotensin III ACE

Glucose

mRNA plasma prekallikrein

Plasma kallikrein

AT1 receptor B2 receptor upregulation

Kinins and ACE inhibition Severe angioedema often involving the face, tongue, or both is seen as a complication of the use of ACE inhibitors. It appears that this swelling is also a consequence of elevated levels of bradykinin (Nussberger et al. 1998); however, the accumulation of bradykinin is due to a defect in degradation rather than excessive production. ACE, being identical to kininase II, is the major enzyme responsible for bradykinin degradation (see Fig. 20.1) and although it is present in plasma, the vascular endothelium of the lung appears to be its major site of action (Alabaster & Bakhle 1992). The action of ACE always leads to the formation of degradation products with no activity, whereas kininase I alone yields the des-Arg products, which are capable of stimulating B1 receptors. The excessive accumulation of bradykinin implies that production is ongoing or that some event leads to activation of the plasma cascade or release of tissue kallikrein, and then faulty inactivation leads to swelling. Continuous turnover of the cascade is implied by data demonstrating activation along the surface of cells and cellular expression or secretion of a PK activator other than factor XII.

Kinins and allergic rhinitis Early studies suggested activation of the plasma bradykininforming cascade in allergic rhinitis on the basis of finding tosylarginine methyl ester esterase activity in the secretions, indicative of production of plasma kallikrein (Naclerio et al. 1983). Assessment of kinins by means of high-performance liquid chromatography demonstrated the presence of both lysyl-bradykinin and bradykinin during the immediate phase and the late phase of allergen-induced rhinitis (Proud et al.

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Arg-Pro-Pro-Phe + Ser-Pro + Phe-Arg

Fig. 20.10 Diagrammatic representation of the many interconnections between the renin–angiotensin pathway and the bradykinin-forming pathway. HK, highmolecular-weight kininogen.

1983; Naclerio et al. 1985). The presence of lysyl-bradykinin indicated release of tissue kallikrein, whereas bradykinin can be derived from lysyl-bradykinin by the action of plasma aminopeptidase or it can be produced directly by plasma kallikrein. Both HK and LK were present, and therefore the preferred substrate for each type of kallikrein was present (Baumgarten et al. 1985). Chromatographic assessment of the secretions demonstrated that both tissue kallikrein and plasma kallikrein are produced (Baumgarten et al. 1986a,b). Bradykinin can produce hyperemia, rhinorrhea, and nasal congestion; however, it has not been possible to assess its contribution to symptoms because a potent and selective bradykinin receptor antagonist that can be administered in vivo has not been available.

Bradykinin and asthma Bradykinin has many effects on the airway including both bronchoconstriction and bronchdilatation, stimulation of cholinergic and sensory nerves, mucus production, and edema (Barnes et al. 1998). Inhalation of bradykinin provokes prominent bronchoconstriction in asthmatics (Fuller et al. 1987; Polosa & Holgate 1990). Nevertheless, bradykinin causes bronchial relaxation in rodent models of asthma via prostaglandin E release if there is pretreatment of the muscle preparation by cholinergic agonists such as carbachol (Li et al. 1998). Studies of asthmatics have revealed the presence of kinins in bronchoalveolar lavage fluid. Early studies demonstrated tissue kallikrein in bronchoalveolar lavage fluid (Christiansen et al. 1992), but there has been no definitive assessment of the plasma bradykinin-forming cascade in bronchoalveolar

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lavage fluids or within lung parenchyma. Bradykinin challenge of asthmatic subjects leads to symptomatic and physiologic changes similar to those seen in the natural disease (Fuller et al. 1987), and allergen challenge of asthmatic subjects leads to an increase in kinin levels in conjunction with histamine release during both the early and late-phase time intervals (Liu et al. 1991; Christiansen et al. 1992). Of particular interest is that Icatibant (HOE 140), a B2 receptor antagonist, inhibited histamine-induced bronchial hyperresponsiveness induced by allergen in allergic rhinitis, and more prolonged treatment improved pulmonary function in asthmatic subjects followed for 1 month (Akbary et al. 1996; Turner et al. 2001). In rats a B1 receptor antagonist diminished airway hyperresponsiveness, suggesting induction of B1 receptors as a result of the inflammatory process (Huang et al. 1999). A role for kinins in general is suspected as mediators of bronchial hyperreactivity, but this need not be restricted to bradykinin or lysyl-bradykinin and may involve neurokinins secreted from type C sensory nerve fibers, such as substance P, neurokinin A, or vasoactive intestinal polypeptide. Bradykinin many be a stimulus for secretion of these neuropeptides (Geppetti 1993).

Bradykinin and anaphylaxis The hypotension of anaphylaxis is clearly multifactorial, including the effects of multiple vasoactive mediators leading to hypovolemia and edematous tissues. Bradykinin is certainly one of those capable of causing hypotension, and one study of insect-sting anaphylaxis demonstrated cleavage of HK concomitant with changes in blood coagulation and fibrinolytic parameters indicative of disseminated intravascular coagulation (Smith et al. 1980). Thus massive release of bradykinin seems likely, although kinin levels were not measured directly. It is of interest that in a rhesus monkey model of endotoxic shock, infusion of a monoclonal antibody to factor XII leads to a 50% improvement in blood pressure, although disseminated intravascular coagulation was unaffected.

Bradykinin formation The mechanism of bradykinin formation in any IgEmediated reaction is multifactorial, including not only tissue kallikrein secretion (see Fig. 20.1) but also direct activation of the plasma cascade by secreted heparin (Hojima et al. 1984; Brunnee et al. 1997), activation along the surface of endothelial cells, or exposure to connective tissue proteoglycans. Any local dilution of plasma constituents decreases the effect of protease inhibitors and leads to an increased rate of the enzymatic reactions to augment kinin formation. Plasma leakage can initially be the result of other permeability factors (e.g., histamine or leukotriene C4/D4), which is then augmented as bradykinin is produced. Bradykinin may be formed (and rapidly degraded) in virtually every type of inflammation. However, its contribution to disease manifestations can only

Bradykinin Pathways and Allergic Disease

be assessed when we have high-potency oral agents or parenteral monoclonal antibodies that inhibit bradykinin formation or block its effects at the receptor level.

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Chemokines James E. Pease and Timothy J. Williams

Summary

Introduction

A basic cell function is the ability to detect a chemotactic gradient and move along it. This function is especially important in leukocytes. Leukocyte chemoattractants, such as C5a and formyl-methionyl-leucyl-phenylalanine (fMLP), have been known for a long time, but their lack of specificity did not explain how specific leukocyte types are able to localize to distinct sites under basal conditions or how these cells are recruited to sites of inflammation. This was a particular issue in allergic inflammation, which is characterized by Th2 lymphocyte recruitment and activation, and the accumulation of large numbers of eosinophils. The discovery of a large family of small proteins, the chemokines, in the 1980s began to throw light on the mechanisms involved in leukocyte recruitment. In humans, around 40 chemokines have been identified, small proteins that signal through 18 different G protein-coupled receptors. There has been extensive research into the differential expression of these receptors on different leukocyte types, and on what chemokines are generated in the context of different types of inflammatory reaction. The mechanisms involved in this cell–cell signaling are complex, as cell types tend to express more than one chemokine receptor and a particular chemokine often can act on more than one receptor. Moreover, chemokine receptor expression is a dynamic process, different receptors being upregulated or downregulated during the life history of the cell. Despite these difficulties, we now have a comprehensive knowledge of chemokine biology. Complementing this, smallmolecule antagonists have been produced that can render a particular cell type insensitive to the attraction of a defined chemokine. All this information can be utilized for the design of new drugs aimed at blocking leukocyte recruitment and, thus, the pathology associated with the activation of this cell once in the inflamed tissue. Choice of the chemokine receptor target has proved to be a major challenge for those trying to produce new therapeutic agents.

The ability to sense a chemical gradient and respond accordingly by migration is crucial to the functioning of many cells, whether they be cells from higher order organisms such as humans, or unicellular organisms such as the slime-mould Dictyostelium discoideum. This property, chemotaxis, is critical for reproduction, the organization of cells during development and growth, and repair. In higher order organisms, it is chiefly recognized because of its importance in localization of immune cells under basal conditions, and in their recruitment at sites of inflammation. The study of this process in Dictyostelium has shed much light on the basic mechanisms involved (Van Haastert & Devreotes 2004). Dictyostelium detects cAMP in its immediate environment using a G protein-coupled receptor (GPCR). The cAMP is released by the organisms themselves during times of nutrient deprivation, and coordinates the directed migration of cells toward one another. There, they cluster to form an aggregate known as a “fruiting body,” with the sole aim of maintaining spores until conditions are appropriate for their germination. The sensing of the cAMP gradient requires that the organism is able to detect tiny differences in concentration between the front of the cell and its rear, which direct its subsequent migration, a process known as chemotaxis. The GPCR for cAMP sits in the cell membrane and acts as a transducer, with the external cAMP gradient reflected as the degree of G-protein activation inside the cell. Subsequent activation of the enzyme phosphatidylinositol 3-kinase (PI3K) results in the generation of an intracellular gradient of secondary messengers such as phosphatidylinositol 3,4,5-trisphosphate (PIP3), with its highest concentration at the region of the cell membrane associated with the greatest amount of G-protein activation. This is further polarized by the activity of the enzyme PTEN, which is displaced from this region to the rear of the cell membrane where it inhibits the activity of PI3K by breaking down PIP3 into phosphatidylinositol 4,5bisphosphate (PIP2). This results in the amplification of the external polarizing signal and is followed by locomotion of the organism along the cAMP concentration gradient, which requires both activation of adhesion molecules at the front of the cell and their inactivation at the rear.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Chemokines In mammals, the family of chemokines (chemotactic cytokines) has been recognized as being particularly important in guiding cellular migration. Their discovery was a result of investigations by several laboratories of the molecules responsible for mediating inflammation. These proteins have an essential role in host defense, organizing the localization of leukocytes to specialized tissues, such as the thymus, under basal conditions, and also coordinating their recruitment and activation in response to inflammatory stimuli. Until the discovery of chemokines almost 30 years ago, the mechanisms by which specific subpopulations of leukocytes were selectively recruited to distinct sites in the body was poorly understood. The so-called classical chemoattractants such as C5a and fMLP that were known at the time were able to recruit several different leukocyte subtypes and did not explain the selective recruitment observed in vivo, for example the recruitment of eosinophils to the allergic lung. The identification of chemokines and their specific leukocyte cell-surface receptors has greatly enriched our understanding of the process of leukocyte recruitment. Since the excessive production of chemokines has been linked with the pathogenesis of several clinically important diseases, including asthma, considerable efforts have been undertaken to understand how chemokines and their receptors function at the molecular level, with a view to selective blockade as a means of modifying undesirable immune responses such as those seen in allergy. As the chemokine

Relationship between chemokines, cytokines, and adhesion molecules Before we look in detail at the molecules themselves, it is worth taking an overall view of the links between the cytokines, chemokines (a subdivision of cytokines), and cellular adhesion molecules to gauge the context in which chemokines exert their effects. Figure 21.1 depicts the recruitment of effector cells (in this case eosinophils) to sites of allergic inflammation. Th2 lymphocytes typical of allergic inflammation are activated by antigen-presenting cells (APCs) and release the cytokines interleukin (IL)-4, IL-13, and IL-5, able to induce synthesis of the chemokine CCL11/eotaxin-1 in airway epithelial cells. Eosinophils, like other leukocytes, form loose interactions with the luminal surface of the venular endothelium, via low-affinity binding of selectin adhesion molecules so that the cells tend to roll along the endothelial surface. CCL11 acts on CCR3 receptors on eosinophils within venules, and induces the upregulation of integrins on the eosinophil cell surface such as integrin α4β1. The integrins bind to complementary receptors on the venule wall resulting initially in the arrest of eosinophils, subsequently followed

Allergen

Epithelium APC (Mo/DC) IL-5

IL-4 IL-13 Th2 cell

Priming, survival

CCL11/eotaxin-1

Adhesion

Rolling Selectins

a4b1

Emigration

VCAM Venule

Eosinophil

Endothelium

472

Fig. 21.1 An overview of chemokines, cytokines, and cells in allergic inflammation. This diagram illustrates the many links between the cytokine, chemokine, and cellular adhesion systems, which mediate the recruitment of effector cells at sites of allergic inflammation typified by eosinophils. The role of the chemokine CCL11/eotaxin-1 is highlighted as inducing firm adhesion to the endothelium via the activation of integrin a4b1, and also the subsequent emigration of the eosinophils to the tissues. DC, dendritic cell; Mo, monocyte; VCAM, vascular cell adhesion molecule. (See CD-ROM for color version.)

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by their chemotactic migration through the vessel wall and to the source of CCL11 production, in this case the airway epithelia. The cytokine IL-5 increases the survival of accumulated eosinophils and also primes them for enhanced responses to diverse chemoattractants such as leukotriene (LT)B4 (Sehmi et al. 1992), fMLP (Sehmi et al. 1992) and CCL11 (Shahabuddin et al. 2000). Similarly, chemokine-mediated signaling can influence the downstream signaling pathways of other cell surface receptors. For example, although the chemokine CCL11 alone is unable to induce IL-4 production by basophils, it can readily potentiate IL-4 production following their stimulation with an allergen such as cat dander (Devouassoux et al. 1999).

Classification of chemokines In the human, around 40 chemokines have been identified through a combination of proteomic and genomic efforts. Chemokines are typically small proteins of around 8–10 kDa and, despite often low sequence homology, share the same common protein fold as deduced by NMR and crystallographic studies. The tertiary structure has both common elements of secondary structure and contains a “Greek key” motif, in which three antiparallel β-pleated strands overlie a C-terminal α helix (Fig. 21.2). With a few rare exceptions, four conserved cysteine residues within chemokines form intermolecular disulfide bonds, which serve to stabilize the overall conformation, typically in the order Cys1–Cys3 and Cys2–Cys4. The cysteine residues also allow the convenient subdivision of chemokines into four families, based on the arrangement of two cysteine residues within the aminoterminal region of the protein (Fig. 21.3). The majority of chemokines fall into either the CC or CXC classes, where the

Fig. 21.2 Secondary structural elements of chemokines as typified by the CC chemokine CCL5/RANTES. The three antiparallel b-pleated sheets (the so-called “Greek key” motif) overlay a C-terminal, a-helical domain. The basic residues located in the region known as the “40s loop” and implicated in glycosaminoglycan binding are shown as ball-and-stick representations. (See CD-ROM for color version.)

two amino-terminal cysteines are either adjacent or have a single amino acid inserted between them (Zlotnik & Yoshie 2000). Two further classes of chemokine have also been described sharing only three members between them; namely the C class featuring a single amino-terminal cysteine residue and the CX3C class in which the two cysteines are separated by three residues. This latter class is quite intriguing as the sole member, a chemokine called CX3CL1 or fractalkine, is expressed as a membrane-bound form on the end of a

CC chemokines

C chemokines

NH2

NH2

C C C CC

C

C

COOH

Fig. 21.3 Subdivision of the chemokine family. Shown are the four classes of chemokines based upon their arrangement of amino-terminal cysteine residues. Conserved cysteine residues are shown as black spheres with disulfide bonds depicted as dashed lines. The sole CX3C ligand is presented as a bound form attached to a mucin-like stalk decorated with oligosaccharides (diamonds).

CXC chemokines

CX3C chemokines NH2

NH2 C

C

C C

COOH

C C C

C

COOH

COOH

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mucin-like stalk. This allows it to serve as an effective adhesion molecule, allowing leukocytes expressing the respective cognate receptor to bind. CX3CL1 can also be released from the cell surface as a soluble chemokine by the action of metalloproteinases such as ADAM17 (Garton et al. 2001). Likewise, the CXC chemokine CXCL16 is also expressed in both stalk and soluble forms. The majority of CXC and CC chemokines have been shown by NMR and crystallographic studies to form dimers and higher order oligomers, although the biological significance of this dimerization is unclear. The concentrations of chemokines employed for these studies are typically in the millimolar range, several orders of magnitude higher than those observed in vivo. Earlier studies of the chemokine IL-8 suggested that at physiologic concentrations, the dimer readily dissociates to the monomeric form and it is this form which activates its specific receptor (Burrows et al. 1994). In contrast, studies of an amino-terminal deletion variant of monocyte chemoattractant protein (MCP)-1, which is able to form heterodimers with wild-type MCP-1, was found to act as a dominant negative, inhibiting monocyte chemotaxis in response to MCP-1 (Zhang & Rollins 1995).

Nomenclature of chemokines Like many areas of molecular immunology, intensive research in the chemokine field has been undertaken by several different laboratories worldwide, with the result that many chemokines were discovered simultaneously and given quite different colloquial names, usually descriptive of their activity. An extreme example is the chemokine known as PARC (pulmonary and activation-regulated chemokine), MIP-4 (macrophage inflammatory protein-4), AMAC-1 (alternative macrophage activation-associated CC chemokine-1), and DC-CK1 (dendritic cell chemokine-1). This created a sense of confusion even among those in the field, and consequently a systematic nomenclature was devised and introduced (Zlotnik & Yoshie 2000). Chemokines are now given the prefixes CCL (CC ligand), CXCL (CXC ligand), CX3CL (CX3C ligand), and XCL (C ligand), together with an identifying number. Table 21.1 shows the chemokines identified to date in the human, together with their previous colloquial numbers where given. This nomenclature has been embraced by the research community and is used throughout the remainder of this chapter.

Interaction of chemokines with glycosaminoglycans Chemokines bind readily to proteoglycans, molecules expressed on the surface of many cells such as endothelial cells and

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which consist of a core protein and glycosaminoglycan (GAG) side chains. These GAG chains typically contain repeating disaccharide units and can often be up to 200 molecules in length. The presence of carboxyl groups and high levels of sulfation results in a high density of negative charge, which can interact electrostatically with the basic chemokine (Witt & Lander 1994; Hoogewerf et al. 1997; Kuschert et al. 1999). These interactions are typically of micromolar affinity, although some chemokines such as CXCL4/platelet factor 4 can bind to GAGs with nanomolar affinity (Loscalzo et al. 1985), indeed CXCL4 was discovered by virtue of its affinity for the GAG heparin (Deuel et al. 1977). The interaction of chemokines with endothelially expressed proteoglycans has been proposed to immobilize a high concentration of chemokine locally upon the luminal surface of the venular endothelium, maintaining chemokine concentrations under conditions of shear flow (Handel et al. 2005). The GAG binding domains of a few CC chemokines have been determined by a combination of structural studies and site-directed mutagenesis and they are typically constructed of a cluster of basic residues located within a region known as the “40s loop” (Fig. 21.2). A mutant CCL5 molecule in which thee basic residues within this cluster were mutated to alanine was observed to be unable to bind to GAGs yet still maintained its in vitro chemotactic activity (Proudfoot et al. 2001). In contrast, the same mutant chemokine was found to be unable to recruit cells when administered intraperitoneally to mice, suggesting that GAG binding is essential for in vivo activity of certain chemokines (Proudfoot et al. 2003).

Chemokine receptors Like many other cytokines, chemokines mediate their effects upon leukocytes by engagement with cell surface receptors. These belong to the superfamily of GPCRs, which is thought to make up almost 5% of the coding portion of the human genome and number over 1000 members (Fredriksson et al. 2003). Chemokine receptors are typically in the region of 350 amino acids long, and belong to the class A of rhodopsin-like receptors, with seven putative hydrophobic regions thought to take the form of α helices that span the cell membrane in a serpentine fashion, leaving the amino terminus extracellular and the carboxyl terminus intracellular (Fig. 21.4). To date, 19 human chemokine receptors have been formally identified, with 10 binding CC chemokines, seven binding CXC chemokines, and solitary receptors for the CX3C and C chemokines (Murphy et al. 2000; Murphy 2002). Nomenclature of the receptors is similar to that of the chemokines with CCR, CXCR, CX3CR used to depict the class of chemokine receptor and a number used to distinguish it from other members of the family (Table 21.2). XCR is used to describe

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Table 21.1 Human chemokines. The systematic names of chemokines, together with their most common colloquial names and receptor agonist activity are shown, although some human chemokines appear to be missing from the list, e.g., CCL6. In such instances, while a chemokine of that name has been identified in the mouse, no human ortholog has been documented. Systematic name

Colloquial name

Receptor usage

Systematic name

Colloquial name

Receptor usage

CCL1 CCL2 CCL3 CCL3L1 CCL4 CCL4L1 CCL5 CCL7 CCL8 CCL11 CCL13 CCL14 CCL15 CCL16 CCL17 CCL18

I-309 MCP-1/MCAF MIP-1a/LD78a LD78b MIP-1b LAG-1 RANTES MCP-3 MCP-2 Eotaxin MCP-4 HCC-1 HCC-2/Lkn-1/MIP-1d/MIP5 HCC-4/LEC TARC DC-CK-1/AMAC-1/ MIP5/PARC/MIP-4 MIP-3b/ELC/Exodus-3 MIP3a/LARC/Exodus-1 SLC/6Ckine/Exodus-2 MDC/STCP-1 MPIF-1 MPIF-2/eotaxin-2 TECK Eotaxin-3 CTACK/ALP/ILC/ESkine MEC

CCR8 CCR2 CCR1,5 CCR1,5 CCR5 CCR5 CCR1,3,5 CCR1,2,3 CCR3 CCR3 CCR2,3 CCR1 CCR1,3 CCR1 CCR4 Unknown

CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL16

GROa/MGSA-a GROb/MGSA-b GROg/MGSA-g PF-4 ENA-78 GCP-2 NAP-2 IL-8 Mig IP-10 I-TAC SDF-1a /b BLC/BCA-1 BRAK/Bolekine SR-PSOX

CXCR2 CXCR2 CXCR2 CXCR3B CXCR2 CXCR1,2 CXCR2 CXCR1,2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5 CXCR6

CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR10,3

XCL1 XCL2

Lymphotactin/SCM-1a/ATAC SCM-1b

XCR1 XCR1

CX3CL1

Fractalkine, neurotactin

CX3CR1

CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28

the sole receptor for the C class chemokines and is so named to distinguish it from the complement receptors which have the prefix CR. Ligand binding by chemokine receptors is typically of low nanomolar affinity and is class restricted, in that CXC receptors generally bind only chemokines of the CXC family and, likewise, CC receptors bind only chemokines of the CC family. Receptors are generally promiscuous and bind several different ligands, with a distinctive “pecking order” in terms of affinity for receptor, and potency and efficacy in in vitro assays such as chemotaxis and intracellular calcium flux.

Structural determinants of chemokine binding and receptor activation The amino termini of chemokine receptors are typically negatively charged, containing numerous acidic side chains. This is thought to play a part in tethering the basic chemokine to

the receptor with high affinity. This interaction may be facilitated further by glycosylation or sulfation of additional sidechain residues within the receptor amino terminus (Farzan et al. 1999; Bannert et al. 2001). Experiments in which the amino termini of chemokine receptors were exchanged to generate chimeric constructs suggest that activation occurs in two stages. Following initial binding of the chemokine to the amino terminus of the receptor, the chemokine is subsequently “delivered” to the remaining extracellular regions of the receptor (Monteclaro & Charo 1996; Pease et al. 1998). This second interaction most likely induces a conformational change within the intracellular regions of the receptor, increasing the affinity of the receptor for heterotrimeric G proteins and initiating intracellular signaling. Structural information regarding the activation of chemokine receptors is scant, as only one GPCR has had its conformation solved by

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N

N

C

b g PI3K

PLC

PIP2

a

DAG

MAPK

+ 2+ i[Ca ]

IP3

Akt

CHEMOTAXIS Fig. 21.4 Chemokine receptors and downstream signaling. A diagram depicting the binding of chemokines by their receptors and the subsequent activation of downstream signaling cascades. Chemokines (black) are thought to be tethered to the amino-terminus of the receptor, allowing the chemokine N-terminus to activate the receptor by disruption of interhelical interactions. This facilitates the recruitment of heterotrimeric G proteins to the receptor, which induce a series of intracellular signals resulting typically in chemotaxis. DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

crystallographic techniques, namely rhodopsin (Palczewski et al. 2000). As chemokine receptors belong to the same class A group of GPCRs as rhodopsin, homology modeling approaches using rhodopsin as a template allow structures to be generated that can be examined by site-directed mutagenesis. Conserved between the majority of class A GPCRs is an aspartate–arginine–tyrosine motif (Vincent et al. 1991) at the cytoplasmic end of the third transmembrane α helix, which is thought to act as an ionic lock, holding the receptor in an inactive state prior to its activation by ligand (Ballesteros et al. 2001). On ligand binding, a conformation change in the GPCR is believed to result in rearrangement of the intracellular loops, facilitating the recruitment of G protein. The importance of the DRY motif in chemokine receptor structure and function was shown in a collaborative study from

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our own group, where nonconservative mutagenesis of this domain in the receptor CCR3 led to a dramatic loss of function and greatly reduced cell-surface expression (Auger et al. 2002). Homology modeling and mutagenesis studies of the receptor CCR5 have given insights into chemokine receptor activation, highlighting an interaction between the N-terminus of the chemokine and transmembrane helices of the receptor (Blanpain et al. 2003; Govaerts et al. 2003). In the inactive receptor, the side chains of helix II and helix III are believed to make contact via hydrophobic interactions, the perturbation of which by the chemokine N-terminus, is thought to induce the conformational changes needed for receptor activation (Fig. 21.4). This fits nicely with experimental data describing the truncation of the chemokine N-terminus. Truncation of the chemokine CCL5/RANTES by eight residues to generate CCL5 (9–68) produces a receptor antagonist as deduced by several assays, blocking responses to ligands that use the same receptor (Gong et al. 1996). Likewise, extension of the CCL5 N-terminus with a single methionine residue (Met-CCL5/RANTES generates a potent receptor antagonist (Proudfoot et al. 1996).

G protein coupling of chemokine receptors and downstream signaling Like other members of the GPCR super family, chemokine receptors relay signals from the exterior to the interior of the cell, by interacting with heterotrimeric G proteins which comprise three subunits named α, β, and γ. While around 20 α subunits have been identified in mammals, experiments employing pertussis toxin suggested that Gαi proteins were primarily responsible for signaling downstream from chemokines, as physiologic responses such as chemotaxis are readily inhibited by preincubation of cells with the toxin (Thelen et al. 1988). The toxin ADP-ribosylates a cystine residue within the carboxyl-terminus of the Gαi subunit, rendering it nonfunctional. The crystal structure of a Gαi βγ heterotrimer has been solved and suggests that the carboxyl-terminal helix of the α subunit binds to the third transmembrane domain of the GPCR, and perhaps the receptor carboxy terminus (Wall et al. 1995). Once the heterotrimer is recruited to the activated GPCR, a guanosine disphosphate (GDP) bound within the α subunit is replaced by a molecule of GTP, resulting in the displacement of the βγ from the Gα subunit and the release of both subunits. These can then go on to activate various effectors and multiple downstream signaling events. Activation of the Gα subunit is terminated by an intrinsic GTPase activity and results in the reassociation of the α subunit with the βγ dimer. Activation of G proteins is thought to occur catalytically, with a single activated GPCR able to activate several G proteins (Janetopoulos et al. 2001). A rapid occurrence following binding of chemokine to its receptor is a rise in the intracellular calcium concentration

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Table 21.2 Chemokine receptors and their principal ligands. Listed are the principal ligands of chemokine receptors and their distribution over a range of cell types. Also listed are the chromosomal locations of each receptor. Although there are reports in the literature of chemokine receptors on nearly every type of cell, those shown above represent the commonly agreed attributions.

Receptor

Principal ligands

Chromosomal location

Receptor principally expressed by

CCR1

CCL3/MIP-1a CCL5/RANTES CCL7/MCP-3

3p21

Mo, DC, Eo, Bs, T, PMN, NK

CCR2

CCL2/MCP-1 CCL7/MCP-3 CCL8/MCP-2 CCL13/MCP-4

3p21

Mo, DC, T, Bs

CCR3

CCL11/eotaxin-1 CCL13/MCP-4 CCL24/eotaxin-2 CCL26/eotaxin-3

3p21

Eo, T, Bs, Mc

CCR4

CCL17/TARC CCL22/MDC

3p24

DC, T, Bs, NK

CCR5

CCL3/MIP-1a CCL4/MIP-1b CCL5/RANTES

3p21

Mo, DC, T

CCR6

CCL20/MIP-3a

6q27

DC, T

CCR7

CCL21/MIP-3b

17q12

DC, T, B, NK

CCR8

CCL1/I-309

3p22

Mo, T, NK

CCR9

CCL25/TECK

3p21.3

T

CCR10

CCL27/CTACK

17q21

T

CXCR1

CXCL1/Groa CXCL6/GCP-2 CXCL8/IL-8

2q35

N, Mo

CXCR2

CXCL1/Groa CXCL2/Grob CXCL3/Grog CXCL5/ENA-78 CXCL6/GCP-2 CXCL8/IL-8

2q35

N, Mo

CXCR3

CXCL9/Mig CXCL10/IP-10 CXCL11/I-TAC CXCL4/PF-4

Xq13

T, B

CXCR4

CXCL12/SDF-1a /b

2q21

T, B, DC, Mo

CXCR5

CXCL13/BCA-1

11q23

B, T

CXCR6

CXCL16

3p21

T, NK

XCR1

XCL1/lymphotactin XCL2

3p21

T, NK

CX3CR1

CX3CL1

3p21

T, NK, DC, Mo

B, B lymphocyte; Bs, basophil; DC, dendritic cell; Eo, eosinophil; Mc, mast cell; Mo, monocyte; N, neutrophil; NK, natural killer cell; PMN, polymorphonuclear leukocyte; T, T lymphocyte.

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within the cell ([Ca2+]i) and this has commonly been used in the laboratory as a measurement of receptor activation. This is mediated by the βγ subunits, which activate phospholipase Cβ leading to hydrolysis of phosphatidylinositol to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is able to induce the release of Ca2+ from intracellular stores and the elevated calcium levels subsequently induce protein kinase (PK)C activation. This activity is necessary for leukocyte responses such as the neutrophil respiratory burst (Li et al. 2000). The βγ subunit also activates PI3K. The use of PI3K inhibitors in vitro has demonstrated a significant role for PI3K in the process of leukocyte chemotaxis (Turner et al. 1995) and has been further corroborated by studies using mice deficient in various PI3K isoforms. Mice deficient in PI3K-γ show impaired neutrophil recruitment in a model of peritonitis and defective leukocyte chemotaxis in response to CCL3 (Li et al. 2000), while mice deficient in PI3K-δ exhibit attenuated allergic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge (Lee et al. 2006). In T lymphocytes at least, a more complicated signaling mechanism may be apparent as PI3K has been reported to be dispensable for the chemotaxis of Th2 cells in response to the CCR4 ligands CCL22 and CCL17 (Cronshaw et al. 2004). These studies identified a pathway involving small GTPases and, more specifically, Rho-associated coiled-coil forming protein kinase (ROCK), as being the critical PI3K independent pathway for T lymphocyte migration. ROCK is an effector of Rho, a small GTPase that is also activated by the βγ G protein subunit via the other GTPase Ras/Rac that has been previously shown to be associated with cytoskeletal rearrangements, effecting cellular responses such as shape change, adhesion, and chemotaxis (Bokoch 1995).

Regulation of chemokine receptor expression In order to respond to a particular chemokine, leukocytes must express the cognate receptor at the cell surface. To compound the issue of promiscuity, whereby receptors typically bind several chemokines, leukocytes also generally express several different chemokine receptors. These receptors have been broadly divided by others into two groups, those constitutively expressed by leukocytes and those whose expression is dynamic and induced by external stimuli, such as observed during inflammation (Proudfoot 2002). The former are especially involved in homeostasis for example the receptor CCR7 and its ligands CCL19 and CCL21 are vital for directing B and T lymphocytes to the secondary lymphoid organs, while the latter are important for the recruitment of leukocytes to inflamed tissues. A good example of the dynamic manner in which chemokine receptors can be expressed is the differential expression pattern observed at both the mRNA and protein level on Th1 and Th2 subsets of T lymphocytes following their polarization (Bonecchi et al. 1998; Sallusto et al. 1998). Th1 lymphocytes selectively express

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CCR1, CCR5, and CXCR3, whereas CCR3, CCR4, and CCR8 are found on Th2 lymphocytes. This fine tuning of receptor expression enables the cells to respond to a variety of different chemokines in vivo, for example, enabling them to leave one tissue compartment and migrate to another. It is likely that such flexibility is vital for a focused adaptive immune response. It should be noted that the division of chemokine receptors into either inducible and inflammatory subsets of receptors is not perfect, as some receptors can be involved in both the basal homing of leukocyte to tissues and also their recruitment in inflammation. For example, the eosinophil receptor CCR3 mediates both the recruitment of eosinophils to the gut under noninflammatory conditions and also their recruitment to the lung following allergen challenge (Gurish et al. 2002; Ma et al. 2002). The number of chemokine receptors on the leukocyte surface results from a balance between the rate of internalization and the rate of replacement (recycling and synthesis of nascent receptor). Following ligand binding, the carboxyltermini of chemokine receptors undergo phosphorylation by protein kinases at a series of serine and threonine residues, a process known as “desensitization.” This facilitates the recruitment of β arrestins to the carboxyl-terminus, rendering the receptor unable to interact with G proteins and thereby switching the receptor off. Desensitization can be described as homologous or heterologous, dependent on the ligand observed to induce the process. Homologous desensitization occurs following repeated exposure of leukocytes to the same ligand and is mediated by specific G protein-coupled receptor kinases (GRKs) following occupation of the receptor by ligand. In contrast, heterologous desensitization does not require direct activation of the chemokine receptor itself and can be mediated by protein kinases, such as PKA and PKC, following the activation of downstream signaling pathways by other receptors (Ali et al. 1999; Bohm et al. 1997). These can be either distinct chemokine receptors expressed on the same leukocyte, or other GPCRs such as those for fMLP and C5a which cross-desensitize signals via the chemokine receptor CXCR2 on human neutrophils (Sabroe et al. 1997). The recruitment of arrestin also initiates the endocytosis or internalization of receptors by binding to clathrin with the receptor–arrestin complex subsequently sequestered in clathrin-coated pits. This pathway is often considered a default system for the degradation and recycling of GPCRs (Pelchen-Matthews et al. 1999; Shenoy & Lefkowitz 2003). Although the rate of internalization of a receptor is an important factor in determining its level at the cell surface, the rate of recycling and the rate of synthesis of new receptors are also important. The concept of two different classes of receptor (as distinguished by their recycling) has been introduced recently, in which class A receptors traffic to recycling endosomes and are rapidly returned to the cell surface (Shenoy & Lefkowitz 2003). In contrast, class B receptors are dephosphorylated in endosomes, followed by slow recycling back to

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the plasma membrane. Sequentially, the receptors pass through late endosomes and the Golgi and, finally, are transported back to the cell surface. Quite unexpected was the finding from Fong and colleagues that T and B lymphocytes from mice deficient in β arrestin-2 had impaired chemotactic responses to the CXCR4 ligand CXCL12 (Fong et al. 2002). Until this publication, as their name implies, arrestins were thought to provide solely a stop signal to impede G protein activation and facilitate endocytosis. Following allergen-challenge, the same β arrestin-2-deficient mice also exhibit reduced T lymphocyte accumulation in the airways (Walker et al. 2003). In light of these findings, it is now believed that arrestins additionally function as an adapter scaffold, allowing the docking of kinases such as JNK-3 (McDonald et al. 2000), Hck, or c-Fgr (Barlic et al. 2000). These latter two molecules have been shown to associate with β arrestin to induce neutrophil degranulation following treatment with the chemokine CXCL8. The additional finding that arrestins can modulate histone acetylation suggests that these molecules may also play a role in the transcriptional profiles of leukocytes following their recruitment to inflamed tissues (Kang et al. 2005).

Modulation of chemokine function Proteolytic processing of chemokines As mentioned earlier in this chapter, the N-terminus of the chemokine is instrumental in inducing activation of the receptor and subsequent signaling. This permits a means of regulating signaling by means of proteolysis (reviewed in Struyf et al. 2003). The type II membrane protein CD26/ DPP IV has an aminopeptidase activity, which is known to cleave dipeptides selectively from the N-terminus of proteins with a proline or alanine residue at the second position of their sequence, an attribute common to chemokines. Indeed, several CC chemokines have greatly impaired responses at their receptors following CD26 processing, including CCL5 (Oravecz et al. 1997), CCL22 (Proost et al. 1999), and CCL11 (Struyf et al. 1999). This latter chemokine, a potent eosinophil attractant, has its chemotactic activity for eosinophils reduced 30-fold on truncation by CD26/DPP IV (Struyf et al. 1999). Several matrix metalloproteinases (MMPs) also have been implicated in the modulation of chemokine activity. For example, CCL7 is processed by MMP2/gelatinase A resulting in the removal of four N-terminal residues and the generation of a receptor antagonist with activity both in vitro and in vivo (McQuibban et al. 2000). In addition, MMPs are also able to process the chemokines CCL2, CCL8, and CCL13 (McQuibban et al. 2002; Overall et al. 2002). Such a strategy has not been lost on the hookworm Necator americanus, which secretes metalloproteases to degrade CCL11 and thereby inhibits eosinophil recruitment (Culley et al. 2000).

Chemokines

Chemokine scavenging by the silent receptors D6 and DARC The chemokine receptor D6 was originally identified by Nibbs and colleagues following RT-PCR analysis of murine spleen with the human ortholog being subsequently amplified from human genomic DNA (Nibbs et al. 1997a,b). D6 is an extremely promiscuous receptor, binding with high affinity a large number of inflammatory CC chemokines, including CCL2, CCL3L1, CCL4, CCL5, CCL7, CCL11, and CCL16. However, despite considerable efforts, no cell signals have been documented to be transduced following chemokine binding, leading the receptor to be described as “silent.” D6 is expressed at high levels on the surface of lymphatic endothelial cells and on syncytial trophoblasts of the placenta (Nibbs et al. 2001), and undergoes rapid constitutive internalization enabling it to rapidly remove chemokines from the endothelial cell surface (Fra et al. 2003; Bonecchi et al. 2004; Galliera et al. 2004) which suffer a proteolytic fate while the receptor is recycled back to the cell surface (Weber et al. 2004). This has led to the suggestion that D6 acts as a “gatekeeper” preserving the integrity of lymphoid tissue by the scavenging of inflammatory chemokines (Young et al. 1955; Fra et al. 2003; Nibbs et al. 2003). This has been supported by recent studies using mice deficient in D6. Exacerbated inflammation was observed following application of phorbol ester to the skin, with the histology of lesions reported to resemble those of human psoriasis (Jamieson et al. 2005; Martinez de la Torre et al. 2005). These results support the notion that D6 is involved in the resolution of the inflammatory response. Similarly, the Duffy antigen receptor complex (DARC) is thought to act as a scavenger of both CC and CXC chemokines and is expressed primarily on erythrocytes. Although DARC was originally defined serologically in the 1950s as a minor red blood cell antigen, it was not until 1990s that it was identified at the cDNA level. It was quickly noted that the molecule had considerable homology to chemokine receptors and it was subsequently shown that DARC could bind the chemokine CXCL8 with high affinity (Chaudhuri et al. 1993; Horuk et al. 1994; Neote et al. 1994; Peiper et al. 1995). In addition to its expression on erythrocytes, DARC has also been identified on the postcapillary venule endothelial cells of several organs (Horuk et al. 1997) and on subsets of neurons in the central nervous system (Dawson et al. 2000). Studies of mice in which the DARC gene has been deleted suggest that it functions as a biological “sink” for chemokines, with both an antiinflammatory role and antiangiogenic role (Neote et al. 1994). As is the case with D6, chemokine binding by DARC does not appear to result in signal transduction, either in erythrocytes or transfectant systems. This is thought to be primarily due to the lack of a DRY motif in the putative third transmembrane helix, present in the majority of signaling chemokine receptors, and thought to play a critical role in maintaining GPCR conformation (Ballesteros et al. 2001).

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Chemokines and their receptors in allergic inflammation Having described in some detail the biology of chemokines and their receptors, in the following sections we attempt to address which chemokines and which chemokine receptors are implicated in the pathogenesis of allergic disease. We will do this on a cell-by-cell basis, looking at the individual subsets of leukocytes implicated in the allergic response and in doing so, we will dissect relevant in vitro and in vivo data and, where applicable, the translation of this basic research into the clinic setting. It should be stated at this juncture that it would be erroneous to view chemokines in isolation of other in vivo chemoattractant systems, as leukocytes are also equipped with receptors for chemoattractants other than chemokines. These molecules also have a potentially important role in the pathogenesis of allergic reactions. Although non-chemokine chemoattractants typically exhibit low specificity for leukocytes, they may be particularly important in the early stages of an allergic response, to be replaced by the more specific chemokines as the response develops. For example, C5a, a fragment of the complement component C5, generated during complement activation, is a potent chemoattractant for neutrophils and other subsets of leukocytes. C5a has been shown to appear before CXCL8 in certain inflammatory models (Ivey et al. 1995). Likewise, the lipid mediators LTB4 and prostaglandin (PG)D2 are potent leukocyte chemoattractants and have been postulated to “grease the way” of T lymphocytes to allergic airways, cooperating with chemokines in a sequential manner (reviewed by Luster & Tager 2004). LTB4 is produced rapidly by activated mast cells and recent research by ourselves and others has highlighted its potential to recruit mast cell progenitors (Weller et al. 2005) and lymphocytes (Goodarzi et al. 2003).

DCs downregulate CCR6 expression and consequently lose responsiveness to CCL20. Instead, they upregulate expression of the receptor CCR7 (Yoshida et al. 1997), which makes them responsive to the chemokines CCL19/MIP-3β (Dieu et al. 1998; Sozzani et al. 1998) and CCL21/SLC (Saeki et al. 2001). This maturation is accompanied by the production of chemokines such as CCL22/MDC which will recruit activated T cells expressing the receptor CCR4 (Tang & Cyster 1999). Initial studies employing mice deficient in either CCL19 and CCL21 (Gunn et al. 1999) or CCR7 (Forster et al. 1999; Ohl et al. 2004) revealed an impaired capacity for the recruitment of DCs to draining lymph nodes. This was subsequently supported by studies in which a mouse with severe combined immunodeficiency (SCID) was reconstituted with peripheral blood mononuclear cells from allergic humans following the neutralization of CCL21 by antibodies (Hammad et al. 2002). In this model, impaired DC homing to the draining mediastinal lymph nodes was observed following antigen challenge, with a resultant decrease in both Th2 cytokine production and T cell recruitment. At the genomic level, regulation of this key chemokine receptor appears to be mediated by the transcription factor Runx3 (Fainaru et al. 2005). Mice deficient in this transcription factor exhibit enhanced expression of CCR7 on alveolar DCs, and subsequently display increased migration to the lymph nodes draining the lung. Interestingly, the resultant accumulation of activated DC within the lymph nodes is associated with features typical of asthma, including increased serum IgE levels and AHR to the nonselective muscarinic receptor agonist methacholine. Of note is a point made by the authors of that study, namely that the gene encoding Runx3 lies within a region of chromosome 1p36 previously linked by another group to asthma and atopy in a scan of the human genome for susceptibility genes (Haagerup et al. 2002).

Chemokines as recruiters of T lymphocytes Chemokines as recruiters of dendritic cells Dendritic cells (DCs) play a crucial part in the allergic response by presenting antigen to other leukocytes. In their role as a sentinel cell, they need to be able to traffic to tissues where they can survey the environment and following encounter with antigen, return to the lymph nodes as an activated APC. Chemokines carefully regulate these migratory steps via a selection of different CC chemokines and receptors. The recruitment of immature DCs to inflamed tissues is facilitated by the receptors CCR2, CCR5, and CXCR4, which recognize a variety of different ligands, although use of these receptors appears to be selective depending on whether the cells have myeloid or plasmacytoid origins (Penna et al. 2001). The chemokine receptor CCR6 was originally identified as being expressed by lung-derived DCs (Power et al. 1997), and mediates chemotaxis in response to CCL20/MIP-3α which is expressed by epithelial cells from a variety of tissues (Greaves et al. 1997). On maturation,

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T lymphocytes Naive T lymphocytes As is the case for DCs, T lymphocytes also have a critical requirement for directed migration to and from secondary lymphoid organs. Migration to the secondary lymphoid organs is also controlled by the CCR7 ligands CCL19 and CCL21, and plt mice deficient in both chemokines exhibit a failure in naïve T cells homing to lymph nodes (Gunn et al. 1999). The receptor for both ligands, CCR7, also appears to determine, at least in part, T cell exit from peripheral tissues, as T cells from CCR7-deficient mice are unable to leave the lung or skin following allergen challenge and enter the draining lymph nodes (Bromley et al. 2005; Debes et al. 2005). In addition to CCR7, naive T lymphocytes also express the receptor CXCR4 (Bleul et al. 1997). In a well-characterized murine model of allergic airway disease, neutralizing antibodies to both CXCR4 and its ligand CXCL12 were observed to reduce lung eosinophilia and AHR (Gonzalo et al. 2000),

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and treatment of mice with the small-molecule antagonist of CXCR4 resulted in a significant reduction in AHR, eosinophilia, and the production of Th2-associated cytokines following allergen challenge (Lukacs et al. 2002). Collectively these data suggest that the CXCR4–CXCL12 axis may play a role in the pathology of allergic disease.

Activated T lymphocytes As we have already mentioned, T cells polarized in vitro to either the Th1 or Th2 subset can dynamically regulate their cell surface levels of several different chemokine receptors, allowing them to respond to a variety of signals (Sallusto et al. 2000). Th2 cells are distinguished from Th1 cells by their expression of the chemokine receptors CCR3, CCR4, and CCR8. Such polarization has also been observed in vivo, with IL-4 producing cells recovered by bronchoalveolar lavage (BAL) shown to preferentially express CCR3 and CCR4 (Morgan et al. 2005). We and others have shown in vitro that CXCR3 ligands are natural antagonists of CCR3-mediated responses (Loetscher et al. 2001; Xanthou et al. 2003) and we postulated that this counterplay between Th1- and Th2associated chemokines and their receptors might result in the fine tuning of leukocyte recruitment in vivo. Subsequent studies have shown that CXCR3 ligands are upregulated following allergen challenge in both the human (Bochner et al. 2003) and murine lung (Fulkerson et al. 2004) and, in support of our postulate, intravenous administration of low doses of CXCL9 was demonstrated to inhibit allergen-induced eosinophil recruitment (Fulkerson et al. 2004). Consistent with a role for the CCR4 and CCR8 axes in T-cell recruitment to the allergic lung, CCL22 and CCL17 have also been observed to be upregulated in the human lung following allergen challenge (Bochner et al. 2003; Pilette et al. 2004). Neutralization of both CCL11 and CCL22 by specific monoclonal antibodies has been reported to block early stage recruitment of Th2 cells to the murine lung following allergen challenge with the long-term blockade of Th2 cell recruitment, following repeated antigen stimulation only inhibited by CCL22 blockade, suggesting roles for either chemokine in the initial and late stages of T cell recruitment, respectively (Lloyd et al. 2000). Coexpression of CCR4 and CCR8 on a significant percentage of T cells in the allergic lung as revealed by immunohistochemical study of bronchial biopsies was reported in an early study (Panina-Bordignon et al. 2001). A more recent study using flow cytometry found an increased percentage of CCR4+ CD4+ T cells recovered in the BAL following allergen challenge, although the percentage of CD4+ or CD8+ T cells expressing CCR4 was unchanged (Thomas et al. 2007). As the authors of this later study suggest, this may be due to differences in either the methodologies employed for staining or in cell isolation from different tissue compartments (BAL compared with bronchial biopsy). The lack of commercially available CCR8-specific antibodies that work efficiently in

Chemokines

immunostaining (Fox et al. 2006) has not assisted the elucidation of the role of CCR8 in T cell trafficking to the allergic lung, although Thomas et al. (2007) recently observed that CCR8 mRNA was found at similar levels in both CD4+ peripheral blood and BAL T cells following allergen challenge. This is not supportive of an active role for this receptor in cell recruitment, although elevated levels of the ligand have been recently reported to be significantly elevated in the BAL fluid of asthmatics compared with normal controls (MontesVizuet et al. 2006). Attempts to demonstrate an absolute requirement for CCR4 or CCR8 in rodent models of allergic inflammation have met with mixed results. Following challenge with ovalbumin, CCR4-deficient mice revealed little difference from their wild-type counterparts allergic airways inflammation (Chvatchko et al. 2000), although attenuation of chronic AHR has been reported in CCR4-deficient mice challenged with Aspergillus fumigatus spores (Schuh et al. 2002). Likewise, our own groups reported that monoclonal antibody neutralization of CCR4 in guinea pigs was ineffective at modulating the allergic response in the lung following challenge with ovalbumin (Conroy et al. 2003). Mice deficient in CCR8 exhibit similar airway inflammation to wild-type mice following ovalbumin challenge (Chung et al. 2003; Goya et al. 2003), and neutralization of the CCR8 ligand CCL1 reduced eosinophil migration to the murine lung, but had no effect on Th2 cell recruitment following allergen challenge (Bishop & Lloyd 2003). In contrast to both these reports, defects in Th2 responses have been reported in CCR8-deficient mice following both ovalbumin and cockroach antigen-induced airway inflammation (Chensue et al. 2001). Thus, like CCR4, the role of CCR8 in allergic inflammation remains less than clear-cut. The roles of the chemokine CCL2 and its receptor CCR2 in allergic disease appear to be quite complicated. Studies using CCL2-deficient mice suggest that the chemokine is critical for the in vitro polarization of T cells to the Th2 subclass as ovalbumin challenge of deficient mice led to reduced IL-4 and IL-5 production and an inability to undergo immunoglobulin class switching (Gu et al. 2000). Supportive of this, depletion of CCL2 in murine models of allergic airways disease has been demonstrated to reduce AHR (Gonzalo et al. 1998; Campbell et al. 1999). Likewise, mice deficient in CCR2 exhibit reduced pulmonary granuloma formation following the injection of Schistosoma egg antigen (Warmington et al. 1999). However, in contrast, studies by other groups have shown enhanced Th2 responses to ovalbumin (Kim et al. 2001) and Aspergillus (Blease et al. 2000a) in CCR2-deficient mice. A more recent study comparing both CCR2- and CCL2deficient mice has reported intact Th2-mediated responses and lung fibrosis in both animals following challenge with Aspergillus (Koth et al. 2004). In the context of the other studies, it thus appears that there is considerable variation in the importance of either chemokine or ligand in allergic

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pathology depending on the experimental model employed, making the relative importance of either CCL2 or CCR2 in human allergic disease difficult to gauge.

Regulatory T cells IL-10 is a potent antiinflammatory cytokine that works in part by the inhibition of APC function and cytokine production by DCs and macrophages. In the asthmatic setting, IL-10 can inhibit the production of cytokine by both Th2 cells and mast cells and also modulate eosinophil function (reviewed in Hawrylowicz & O’Garra 2005). CD4+CD25+ regulatory T cells (Tregs) are a major source of IL-10 and recent studies have focused on these cells as a means of suppressing allergic inflammation. CD4+CD25+ Tregs purified from peripheral blood make up around 10% of the CD4+ population and have been reported to express CCR4 and CCR8, which may facilitate their migration toward APCs and activated T cells allowing them to inhibit APC function and suppress responding T cells (Iellem et al. 2001). In vitro, CD25 + Tregs have been reported to suppress the differentiation of murine CD4+ T cells towards Th2 cells via a contact-dependent mechanism (Stassen et al. 2004) and defects in the ability of human Tregs to suppress allergen-driven T cell activation have been proposed to result in asthma (Bellinghausen et al. 2003; Ling et al. 2004). CD4+ Tregs differentiated from cell lines derived from the blood and skin of patients presenting with allergic contact dermatitis to nickel, have been shown to express CCR3, CCR4, CCR5, CXCR3, and CCR8 (Sebastiani et al. 2001). Curiously, functional responses to most ligands were lost on activation, with the exception of responses to the chemokines CCL17, CCL2, and CCL1. Similarly, in mice immunized with Schistosoma mansoni egg antigen-coated beads in order to elicit a Th2 response, CD4+CD25+ IL-10-producing cells were shown to selectively express CCR8, and their ability to infiltrate the resulting granulomas coincided with the production of CCL1, the main CCR8 ligand (Freeman et al. 2005). Natural killer T cells In contrast to Tregs, which regulate the adaptive arm of the immune system, natural killer T cells (NKT cells) regulate the innate immune system and are a subpopulation of αβ T cells, defined by the expression of a conserved T-cell receptor (V Vα24Jα 18-Vβ11 in humans) which recognizes the glycolipid α-galactosylceramide (α-GalCer) (Dellabona et al. 1994). Flow cytometric analysis suggests that the majority of NKT cells express the chemokine receptors CCR1, CCR2, CCR5, CXCR3, CXCR4, and CXCR6 (Kim et al. 2002; Motsinger et al. 2002). Unlike other T cells, NKT cells have low levels of CCR7, which is required for effective migration to secondary lymphoid organs. As a consequence, they are preferentially recruited to extra lymphoid tissues by the production of chemokines such as CCL2, CCL3, and CXCL10. As for CD4+ T cells, NKT subsets can be further divided based on their cytokine expression. A CD4+ IL-4/IL-2-producing subset, has

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been reported to express CCR4, while CD8– and CD4–CD8– low cytokine-producing subsets expressed the receptors CCR1, CCR6, and CXCR6 (Kim et al. 2002). As is the case for the differential expression of chemokine receptors on Th1 and Th2 lymphocytes, it is envisaged that such flexible programs of expression allow the fine tuning of NKT cell recruitment in vivo. Mice deficient in CD1 also lack NKT cells and in a ragweedinduced model of allergic airways disease, in vivo stimulation with α-GalCer has been reported to result in reduced CCL11, IL-4 production and subsequent eosinophilia, when compared with their wild-type counterparts. This suggests that NKT cells may play a proinflammatory role in allergic airways inflammation. This finding is in contrast with a reported suppressive role in bleomycin-induced models of pulmonary fibrosis, where administration of α-GalCer resulted in increased survival of mice (Kimura et al. 2004). Patients presenting to clinic with allergic asthma have been documented as having elevated levels of peripheral blood NKT cells compared with healthy volunteers (Sen et al. 2005). These NKT cells express significant levels of CCR9 and are subsequently responsive to CCL25 in vitro. Histochemical analysis identified NKT cells as having infiltrated the bronchi mucosa of asthmatics and, following purification, these cells were identified as having the potential to drive in vitro cocultures of CD3+ T cells into the expression of IL-4 and IL-13, unlike those isolated from healthy volunteers which drove interferon (IFN)-γ production. This ability to induce a Th2 bias was dependent on cross-talk between activated CCR9 and CD226, as shown by independent blockade of CD226 expression by shRNA and transfection of NKT cells from nonasthmatics with CCR9 cDNA, suggesting that blockade of CCR9 may prove to be fruitful in the treatment of asthma (Sen et al. 2005). Despite an initial study suggesting that approximately 60% of the pulmonary CD4+CD3+ cells in patients with moderate-to-severe persistent asthma were NKT cells (Akbari et al. 2006), two subsequent studies have shown comparatively few NKT cells in allergen-challenged lung compared with control (Thomas et al. 2006; Vijayanand et al. 2007), with the stringency of the flow cytometry parameters employed in the original study a source of much debate (Ho 2007). Thus, at the time of writing, despite strong evidence that NKT cells play a proinflammatory role in murine models of allergic airways inflammation, such findings have not been translated to humans.

Chemokines as recruiters of mast cells Mast cells are principally recognized for their effector functions in allergic reactions and express FcεR1 receptors that are capable of binding IgE with high affinity. Recognition of a polyvalent antigen triggers receptor cross-linking, resulting in the release of degranulation products such as histamine and the de novo synthesis of mediators with potent inflammatory activity such as prostaglandins and cytokines (Marshall

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2004). Mast cells characteristically express the receptor c-kit and, accordingly, its ligand SCF induces their chemotaxis (Boyce et al. 2002), in addition to its other important effects such as proliferation, differentiation, and inhibition of apoptosis. Several independent studies have described a repertoire of chemokine receptors expressed by mast cells or mast cell lines. CCR1 (Juremalm et al. 2002), CCR3 (Ochi et al. 1999; Romagnani et al. 1999), CCR4 (Juremalm et al. 2002), CCR5 (Ochi et al. 1999), CXCR2 (Ochi et al. 1999), CXCR3 (Brightling et al. 2005), and CXCR4 (Ochi et al. 1999; Juremalm et al. 2000) have all been described as being expressed by mast cells. In vivo studies employing blocking antibodies to chemokines or mice deficient in receptors have suggested a role for the CCR1–CCL3 signaling pathways leading to mast cell degranulation (Toda et al. 2004). Following costimulation of both FcεRI and CCR1, reduced chemotaxis was observed, which was postulated to focus the inflammatory response by maintaining cells at the site of allergen accumulation. Subsequent in vivo experimentation using a murine model of allergic conjunctivitis has expanded these findings and shown that costimulation of both receptors optimizes their capacity for degranulation, a phenomenon absent in CCR1deficient mice and which can be impeded in wild-type mice by neutralization of CCL3 (Miyazaki et al. 2005). Antagonism of CCR3 by small molecules in the same model is also reported to be beneficial with an impaired early phase reaction, again thought to be due to the inhibition of mast cell degranulation (Nakamura et al. 2005). CCR3 is expressed on both mature human mast cells and their progenitors (Ochi et al. 1999), although attempts to describe a definitive role for CCR3 in mast cell trafficking have proved difficult, with studies often throwing up apparently conflicting results. CCR3-deficient mice infected with the helminth Trichinella spiralis were reported to exhibit a normal jejunum and cecum mast cell hyperplasia and unaffected worm expulsion (Gurish et al. 2002), while intraperitoneal sensitization of the same mice, followed by aerosol challenge, resulted in increased numbers of tracheal intraepithelial mast cells and increased AHR compared with wild type (Humbles et al. 2002). Work from our own group has demonstrated that immature murine and human mast cells express the receptor BLT1, unlike their mature counterparts, which suggests that circulating mast cell progenitors can migrate along a gradient of LTB4 generated by the activation of mast cells in tissues (Weller et al. 2005). This provides a potential component of mast cell hyperplasia associated with allergy. CXCR2deficient mice have been reported to have reduced numbers of intestinal mast cell progenitors as determined by limiting dilution assays, suggesting that mediators such as CXCL1/KC and CXCL2/MIP-2 may also be involved in trafficking, in contrast to mice deficient in CCR2, CCR3, and CCR5, which show no reduction in the numbers of mast cell progenitors (Abonia et al. 2005).

Chemokines

Chemokines as recruiters of basophils Like mast cells, following their activation, basophils can also release histamine and are a source of leukotrienes and cytokines with the potential to amplify inflammation. Hence, the mechanism by which they are recruited to sites of allergic inflammation is of interest. The question of which chemokine receptors are expressed by basophils was originally addressed at the mRNA level with transcripts for CCR1, CCR2, CCR3, and CCR5 detected, although curiously migration was only observed in vitro to the chemokines CCL2 and CCL11 (Iikura et al. 2001). In a model of cutaneous inflammation in which humanized SCID mice received autologous human skin grafts, CCL11 was reported to be a potent inducer of basophil migration (Fahy et al. 2001). Likewise, CCL5, a ligand for the receptors CCR1, CCR3, and CCR5 has been reported to play a role in recruiting basophils into the nasal mucosa of allergic patients following allergen challenge (Kuna et al. 1998). When injected intradermally into rats, CCL5 has been reported to induce basophilic cell recruitment and induction of histidine decarboxylase mRNA transcripts, with the potential to further exacerbate inflammation by elevating histamine production (Conti et al. 1997). Transcripts for CXCR4 have also been reported in basophils coupled with functional data linking CXCL12-induced [Ca2+]i flux and chemotaxis, although there has been debate as to whether the receptor is present on resting cells (Jinquan et al. 2000; Iikura et al. 2001). Several chemokines have been shown to induce basophil degranulation, including CCL2 (Kuna et al. 1992; Iikura et al. 2001), CCL3 (Alam et al. 1992), CCL7 (Dahinden et al. 1994), and CCL13 (Garcia-Zepeda et al. 1996), although CCL11 is without effect in this respect. CCL11 can, however, potentiate the production of IL-4 production by basophils, mediating this effect via CCR3 (Devouassoux et al. 1999). CCL13 has been reported to induce a biphasic response in assays of basophil shape change, with the receptors CCR2 and CCR3 apparently cooperating to coordinate responses across the concentration range examined (Heinemann et al. 2000), which is reminiscent of the actions of CXCR1 and CXCR2 on neutrophils to coordinate responses to CXCL7 (Ludwig et al. 1997). In humans, intradermal injection of CCL11 has been reported to induce an acute wheal and flare reaction, most likely mediated by the degranulation of mast cells (MenziesGow et al. 2002). Supportive of this, numbers of detectable mast cells were also observed to decrease in the 24 hours following injection of CCL11, and the ability of the basophil to produce CCL3 following IgE cross-linking suggests that it may constitute a positive-feedback mechanism for amplification of leukocyte recruitment in allergy (Li et al. 1996).

Chemokines as recruiters of eosinophils Although eosinophils are characteristically found in large numbers at sites of allergic inflammation, defining a precise role for the cells in the pathogenesis of asthma has been difficult.

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Neutralization of IL-5 in vivo or the use of IL-5-deficient mice has been shown to suppress eosinophil production, suggestive of a link between the activation of lung eosinophils and the development of AHR (Eum et al. 1995; Foster et al. 1996). This postulate was dealt a blow with the finding that neutralization of IL-5 in asthmatic patients did not show any effects on airway function (Leckie et al. 2000), although a considerable number of eosinophils remained in the lung tissue (FloodPage et al. 2003a). However, subsequent studies in both mice (Humbles et al. 2004; Lee et al. 2004) and humans (FloodPage et al. 2003b) have suggested a critical role for eosinophils in airway remodeling with eosinophil-derived TGF-β thought to be a key driver of the remodeling process. Whilst CCL5/RANTES was shown to be chemotactic for eosinophils in an earlier study (Kameyoshi et al. 1994), the chemokine is capable of attracting numerous other leukocyte subtypes and it was not until the discovery of a chemokine named “eotaxin” that a highly potent, highly selective eosinophil chemoattractant was described (Jose et al. 1994). CCL11/ eotaxin-1 was discovered following protein purification of BAL fluid from allergen-sensitized guinea pigs. Following the identification of murine and human orthologs, the chemokine was subsequently found to signal via the receptor CCR3 (Ponath et al. 1996). This receptor is highly expressed on human eosinophils at around 50 000 receptors/cell and is very promiscuous, binding upwards of a dozen different chemokines with varying potency and efficacy. Two other chemokines CCL24/eotaxin-2 and CCL26/eotaxin-3 (Forssmann et al. 1997; Patel et al. 1997; White et al. 1997; Kitaura et al. 1999; Shinkai et al. 1999) were subsequently identified as signaling via CCR3 and are so-named because of their chemotactic activity for eosinophils, having little amino acid sequence similarity with CCL11. CCR3 is also expressed by basophils (Uguccioni et al. 1997), mast cells (Ochi et al. 1999), and a subpopulation of Th2 lymphocytes (Sallusto et al. 1997), making it a likely player in the pathogenesis of allergic disease. Several studies have demonstrated that CCL11, CCL24, and CCL26 are generated in allergic reactions and that their production correlates with eosinophil recruitment in both animals and humans (Rothenberg et al. 1995a,b; Humbles et al. 1997; Ying et al. 1997, 1999a,b; Berkman et al. 2001). There is also evidence to suggest that CCL11 can act in concert with IL-5 to prompt release of eosinophils from the bone marrow, leading to peripheral blood eosinophilia (Collins et al. 1995; Palframan et al. 1998a, 1998b). Use of mice deficient in CCL11 has led to conflicting reports in the literature, most likely due to genetic differences between the mouse strains used. Whilst BALB/c mice deficient in CCL11 were reported to show a reduction in ovalbumin-induced lung eosinophilia (Rothenberg et al. 1997), CCL11-deficient mice of the outbred ICR strain exhibited no difference in the numbers of BAL eosinophils following challenge with the same allergen (Yang et al. 1998). Similarly, the ability of mice deficient in the CCL11 receptor CCR3 to

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develop AHR to methacholine following antigen inhalation appears to be dependent on the route of sensitization, be it intraperitoneal (Humbles et al. 2002) or epicutaneous (Ma et al. 2002). A more recent study using an assortment of CCL11/CCL24 doubly deficient mice, CCR3-deficient mice, and eosinophil-deficient Δdbl-GATA mice, reported that airway eosinophil recruitment was ablated following allergen challenge (Fulkerson et al. 2006a). A subsequent study by the same group employed mice deficient in CCR3, which had been backcrossed onto an IL-13 transgenic background in which expression of the cytokine was targeted to the lung. In this model, a 98% reduction in lung eosinophil numbers was observed compared to littermate controls, which correlated with reduced collagen deposition in the airways (Fulkerson et al. 2006b). Expression of CXCR4 mRNA by freshly isolated eosinophils has previously been reported although detection of cell-surface receptor was only apparent following their culture for 24 hours in a medium containing IL-5 (Nagase et al. 2000). This upregulation of CXCR4 is reminiscent of a previously described mechanism for the clearance of senescent neutrophils, which preferentially home to the bone marrow where CXCL12 is produced (Martin et al. 2003). So, although the lack of CXCR4 on freshly isolated eosinophils suggests that the receptor is unlikely to mediate eosinophil recruitment in the context of allergy, its upregulation as eosinophils age may mark the cells for clearance from the circulation. Another receptor with variable expression on eosinophils is CCR1. While our own laboratory has been able to detect CCR3 on the surface of eosinophils from all the donors that we have examined, we found that in approximately 20% of individuals CCR1 was also expressed at high levels (Sabroe et al. 1999; Phillips et al. 2003). This renders the eosinophils responsive to the chemokine CCL3/MIP-1α and suggests that, in a significant proportion of the population, the CCR1– CCL3 axis has the potential to recruit eosinophils in allergic disorders. Indeed, expression of CCL3 in the human asthmatic lung has previously been reported by several other groups (Alam et al. 1996; Holgate et al. 1997; Tillie-Leblond et al. 2000; Zimmermann et al. 2000) with increased serum CCL3 levels also reported in atopic dermatitis patients (Kaburagi et al. 2001). In murine models of allergic airways disease employing challenge with cockroach allergen, administration of a blocking monoclonal antibody to CCL3 resulted in reduced AHR and reduced eosinophil recruitment to the lungs in the initial stages of disease (Campbell et al. 1998). As with the data on AHR in CCR3-deficient mice, the allergen/sensitization protocol used appears to be important, as similar neutralization of CCL3 in an ovalbumin-induced model resulted in only a partial reduction in AHR and eosinophil recruitment to the lung (Gonzalo et al. 1998). Likewise, a role for the CCR1–CCL3 axis in airways remodeling observed during chronic lung inflammation has been put forward (Blease et al. 2000b).

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Following challenge of CCR1-deficient mice with Aspergillus fumigatus, significantly lower levels of Th2 cytokines and fibrosis were observed in the lungs of the deficient mice compared with their wild-type counterparts.

Blockade of the chemokine system as a therapeutic strategy for the treatment of allergy As we have seen, the leukocytes implicated in allergic inflammation express a variety of chemokine receptors that appear to play distinct roles in the pathology of allergic disease. Consequently, blockade of the chemokine system has great appeal as a possible therapeutic strategy. An initial key question in this respect is how selective an approach to take. A broad approach might try to inhibit the activities of several chemokines simultaneously. Since the finding that chemokines defective in their ability to interact with GAGs are inactive in vivo (Proudfoot et al. 2003), this raises the possibility that antagonism of the chemokine–GAG interaction may be a useful therapeutic angle (Handel et al. 2005). Indeed, soluble GAGs have been used by our own group to inhibit the actions of chemokines on eosinophils in vitro by sequestering the chemokine such that it can no longer bind to the specific receptor (Culley et al. 2003). Heparin, produced in vivo exclusively by mast cells, has been shown to inhibit eosinophil recruitment in a guinea pig model of allergic lung inflammation (Seeds et al. 1995), and to attenuate the late asthmatic response to allergen challenge in patients with atopic asthma suggesting that it may represent a natural antiinflammatory agent able to attenuate allergic inflammation in the lung following mast cell degranulation. If a more selective approach is to be taken, the key question is surely which chemokine receptor should be blocked, and typically results from experimental observations about whether a particular cell type expressing a particular receptor plays a key role in the pathogenesis of disease (Wells et al. 2006). As CCR3 is expressed on the cell types implicated in the initiation and maintenance of the allergic phenotype, e.g. eosinophils (Ponath et al. 1996), basophils (Uguccioni et al. 1997), Th2 lymphocytes (Sallusto et al. 1997), and mast cells (de Paulis et al. 2001), the receptor has become a focus for the pharmaceutical industry, with the development of small molecule antagonists of CCR3 a goal of several companies. Initial in vitro and in vivo proof of principle studies suggested that CCR3 blockade was feasible (Sabroe et al. 2000; White et al. 2000; Dhanak et al. 2001a; Naya et al. 2001a; Varnes et al. 2004), and subsequent high-throughput screens identified several potent small-molecule antagonists of CCR3, typically with activities in the low nanomolar range. High-throughput screening of chemical libraries in biotechnology/pharmaceutical companies led to the discovery of a plethora of smallmolecule CCR3 antagonists with in vitro activities typically in the low nanomolar range (Sabroe et al. 2000; White et al. 2000; Dhanak et al. 2001a,b; Naya et al. 2001a,b, 2003; Saeki et al. 2001; Bryan et al. 2002; De Lucca et al. 2002; Wacker

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et al. 2002; Wan et al. 2002; Warrior et al. 2003; Hodgson et al. 2004; Varnes et al. 2004; Anderskewitz et al. 2005; De Lucca et al. 2005; Ting et al. 2005a,b; Fryer et al. 2006; Morokata et al. 2006; Suzuki et al. 2006). Enthusiasm for CCR3 antagonists appeared to wane with the report that IL-5 blockade had little effect on lung function, as recently reviewed by Wells et al. (2006). However, as mentioned in an earlier section, subsequent studies in both humans and animals have highlighted an additional role for eosinophils in the process of airways remodeling associated with asthma. Deletion of the murine IL-5 gene was observed to suppress both lung eosinophilia and tissue remodeling, with a decrease in the growth factor TGF-β1 (Cho et al. 2004) correlating with clinical data in which anti-IL-5 blockade resulted in a reduction in the numbers of airway eosinophils expressing mRNA for TGF-β1, and in the deposition of extracellular matrix proteins in the reticular basement membrane of bronchial biopsies (Flood-Page et al. 2003b). The recent generation of mice deficient in eosinophils has allowed the role of the cell to be probed further, with data derived from allergen challenge implicating the eosinophil in both airway remodeling (Humbles et al. 2004) and AHR (Lee et al. 2004). Consequently, interest in CCR3 as a therapeutic target has been rekindled, particularly as it is also expressed by basophils, Th2 cells, and mast cells, and blockade on these cells may also be therapeutically beneficial. An often insurmountable obstacle is that finding that limited homology between human and rodent orthologs of chemokine receptors means that potent, efficacious inhibitors of a human receptor often have little if any activity at their rodent ortholog. This makes target validation impossible and reports describing the in vivo efficacy of such compounds generally lag well behind in vitro studies. However, a flurry of recent papers have described activities of CCR3 antagonists in a variety of animal models. The Yamanouchi Pharmaceutical Company have recently described compounds with efficacy in an assay of eosinophil recruitment to the macaque lung following bronchoprovocation with CCL11 (Morokata et al. 2006), and in a murine model of cutaneous inflammation (Suzuki et al. 2006). Likewise, Abbot Laboratories have described the antagonist A-122058, which was effective in reducing the number of eosinophils following intraperitoneal injection of CCL11 into mice (Warrior et al. 2003). To date, the only small-molecule antagonist of CCR3 to have entered phase II trials has been the GSK766994 compound from GlaxoSmithKline and, following a lack of efficacy in a study of allergic rhinitis, there are no plans to assess the efficacy of this molecule in the asthmatic setting (Murdoch 2006). This follows on from the reports of efficacy of their GW701897B compound in a model of vagally mediated bronchoconstriction in antigen-challenged guinea-pigs (Fryer et al. 2006). One point worth making is the fact that chemokine antagonists, like many agonists and antagonists of GPCRs,

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exert their effects by binding to the transmembrane helices (de Mendonca et al. 2005), a region often highly conserved between different receptors. Thus, it is not surprising that some of these compounds have selectivity for more than one receptor, such as the compound UCB 35625 (a trans isomer of BANYU J113863) which has nanomolar activity at both CCR1 and CCR3 (Sabroe et al. 2000). It may prove beneficial to target one or more receptors with such compounds to provide a more broad-spectrum approach to chemokine receptor blockade.

Acknowledgments We are grateful to Asthma UK and the Wellcome Trust for their support of our research in this field.

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co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells. Eur J Immunol 27, 3507–16. Ying, S., Meng, Q., Zeibecoglou, K. et al. (1999a) Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (Intrinsic) asthmatics. J Immunol 163, 6321–9. Ying, S., Robinson, D.S., Meng, Q. et al. (1999b) C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES). J Immunol 163, 3976– 84.

Chemokines

Yoshida, R., Imai, T., Hieshima, K. et al. (1997) Molecular cloning of a novel human CC chemokine EBI1-ligand chemokine that is a specific functional ligand for EBI1, CCR7. J Biol Chem 272, 13803–9. Young, M.D., Eyles, D.E., Burgess, R.W. & Jeffery, G.M. (1955) Experimental testing of the immunity of Negroes to Plasmodium vivax. J Parasitol 41, 315–18. Zhang, Y. & Rollins, B.J. (1995) A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer. Mol Cell Biol 15, 4851–5. Zimmermann, N., Hogan, S.P., Mishra, A. et al. (2000) Murine eotaxin-2: a constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression. J Immunol 165, 5839–46. Zlotnik, A. & Yoshie, O. (2000) Chemokines: a new classification system and their role in immunity. Immunity 12, 121–7.

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22

Neurotrophins Wolfgang A. Nockher, Sanchaita Sonar and Harald Renz

Summary Neurotrophins, namely nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), neurotrophin (NT)-3, NT-4, and NT-5, were first discovered as target-derived growth factors responsible for the survival and maintenance of specific subsets of central and peripheral neurons. Additional neurotrophin-responsive cells are now known to include immune cells such as mast cells, lymphocytes, and eosinophils, as well as other ectodermal-derived cells such as keratinocytes and epithelial cells. Moreover, these cells are also shown to synthesize neurotrophins, especially after activation during an inflammatory response. Therefore, neurotrophins not only modulate various cell to cell signaling within an inflamed tissue, such as neuroimmune interactions, but also interactions with structural tissue cells. Initial observations have revealed elevated levels of systemic neurotrophin levels in allergic inflamed tissues. The pathophysiology of allergic diseases is characterized by an initial immune response as well as alterations of the local innervation and the integrity of tissue cells. Neurotrophins have been shown to modulate the sensory innervation of allergic inflamed tissues resulting in an enhanced neuronal activity. In allergic airway diseases such as asthma or rhinitis, neurotrophins account for major clinical symptoms such as bronchial hyperreactivity or rhinorrhea and sneezing reflex. Furthermore, in allergic skin diseases, neurotrophins have been shown to modulate cutaneous neuroimmune interactions resulting in sensory hyperreactivity and enhanced pruritus. More recent data indicate that neurotrophins not only account for changes in neuronal density and activity, but also influence the function of immune and structural cells during allergic inflammation. Neurotrophins promote the activation and survival of infiltrating and resident immune cells, e.g., eosinophils and mast cells, and studies of various allergic diseases reviewed over the last two decades indicate that neurotrophins behave as inflammatory cytokines delivering and activating survival signals to effector cells of the allergic response. In addition, it has also been shown

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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that neurotrophins may also modify diverse functional activities of structural tissue cells, e.g., fibroblasts and keratinocytes. While most knowledge comes from studying NGF, the most prominent member of the neurotrophin family, the contribution of other neurotrophins like BDNF, NT-3, and NT-4 remains to be more intensively elucidated. In various animal studies, local inhibition of neurotrophin signaling significantly abrogated development or magnitude of the allergic inflammation and airway hyperreactivity. Therefore, novel strategies for therapy of allergic diseases may result from a precise understanding of the molecular mechanisms of neurotrophin functions as well as unraveling differential activities of single members of the neurotrophin family.

Neurogenic inflammation Allergic diseases are characterized by an activation of the immune system resulting in an induction of cellular and humoral immune response followed by eosinophilia and synthesis of IgE antibodies. Recent studies indicate an extensive communication between cells of the nervous and the immune system during such an inflammatory process. The direct potentiation of local inflammation by directional pathways connecting neurons and immune cells has led to the concept of “neurogenic inflammation.” The classical concept of this hypothesis defined an alteration of the inflammatory response by the local nervous system (Barnes 1992). Stimulation of an afferent nerve results in the propagation of the action potential not only to the spinal cord but also to a retrograde activation of synaptic endings in the tissue resulting in a release of neuropeptides and neurotransmitters (axon reflex). These neurotransmitters then modulate functional activity of immune cells. However, this concept may also work in the opposite direction, namely that inflammatory cells influence the structure and activity of tissue nerves. Thereby, inflammation-induced alterations in the local neuronal function can lead within a vicious cycle to increased release of proinflammatory neuropeptides on stimulation (Fig. 22.1). Neuroimmune interactions have been well described in local allergic inflammation of the skin or the airways, such as atopic

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Neurotrophins

Stimulation of an afferent nerve

Epithelial barrier

Fig. 22.1 Axon reflex and neurogenic inflammation in allergic airway diseases. Neurotrophins potentiate release of neuropeptides resulting in enhanced vessel and immune cell reactivity. Eos, eosinophil. (See CD-ROM for color version.)

Vessel: Vasodilation Extravasation of plasma proteins Immune cells: Chemotaxis Activation

dermatitis or asthma (James & Nijkamp 1999; Darsow & Ring 2001). One important feature of this neuroimmune interaction is the alteration of the local innervation by growth and activation factors like interleukins and cytokines liberated during the allergic inflammation by immune cells. Vice versa, the inflammatory process represented by infiltrated immune cells, is modulated by neurotransmitters and neuropeptides that are released by the local nervous system. Neurogenic inflammation may also be initiated by activation of sensory nerves, inflammatory mediators or irritants invading the skin or the airways. The cross-talk between neurons and immune cells is then mediated via cytokines, neuropeptides and their receptors. Neuropeptides, the main neurotransmitters of sensory nerves, such as tachykinins (substance P and neurokinin A and B) or calcitonin gene-related peptide (CGRP), are transferred via axonal transport not only to presynaptic axons endings in the spinal cord but also to peripheral nerve endings. On stimulation with mechanical, thermal, chemical or inflammatory stimuli, tachykinins are released and act in a dual fashion, as afferent neurotransmitters to spinal cord motor neurons as well as efferent neurosecretory mediators diffusing into the peripheral tissue (Barnes 1996). Many studies indicated that tachykinins as well as other neuromediators released from sensory nerve endings participate in allergic airway and skin inflammation. Substance P, the most prominent member of the tachykinin family, modulates a broad range of functional responses of immune cells including lymphocytes, eosinophils, mast cells, macrophages, and nonimmune cells such as microvascular endothelial cells, fibroblasts, keratinocytes, and epithelial cells. These functional responses include cellular development, growth, differentiation, chemotaxis, vasoregulation, and wound healing. Neurogenic inflammation is mainly involved in allergic airway diseases, such as asthma or rhinitis, contributing to the inflammatory response to allergens, infections, and various irritants. Following irritation, activation of sensory airway nerves leads to an axonal response that acts as an immediate protective mucosal defense mechanism resulting in coughing and sneezing. Coughing, sneezing and other protective mechanisms clear the upper and lower airways from offend-

Neurotrophins

Eos

Tissue Vessel T cell

Retrograde propagation of the action potential

Neuropeptides

Propagation of the action potential to the spinal cord

ing agents. However, dysfunction of sensory nerves due to an enhanced and/or chronic activation results in an overexpression of neuropeptides. Increased concentrations of neuropeptides modify the functional activity of other nerves, structural cells, e.g., smooth muscle cells, as well as resident and invading immune cells. While the immunoregulation of neuropeptides has been intensively studied, an important question is related to the mechanism of neurotransmitter induction during the inflammatory process. It is well known that various interleukins like IL-1 mediate signals from the immune to the nervous system and activate tachykinin synthesis; however, communication between neurons and immune cells is not exclusively restricted to these “classical” cytokines. Recent studies have indicated that neurotrophins, a protein family of growth factors originally found in the nervous system, trigger neuroimmune interactions. Neurotrophins exert their signals through specific cell-surface receptors, which are found on various cell types within and outside the nervous system. Neurotrophins have been reported to influence the intensity and duration of a local immune response through the regulation of neuropeptide synthesis in the local nervous system. As neuropeptides are short-lived signaling molecules that are rapidly degraded at the site of synthesis, their action is therefore mainly restricted to the site of synthesis. Neurotrophins, however, are produced constitutively by various tissue and immune cells during the allergic inflammation. Therefore, acting as long-term modulators, neurotrophins potentiate interactions between the nervous and immune systems (Fig. 22.2). In addition to controlling neuropeptide synthesis, neurotrophins modulate the number and activity of peripheral nerves resulting in neuronal hyperplasia. Recent data suggest that neurotrophins exert direct effects via their receptors on immune cells and also modulate biological activities of structural tissue cells, such as fibroblasts, keratinocytes, epithelial, and smooth muscle cells. During allergic tissue inflammation, structural cells respond morphologically and functionally resulting in increased proliferation, cytokine, and mediator release, and synthesis of extracellular matrix components. Enhanced neurotrophin expression triggered

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PART 2

Inflammatory Cells and Mediators are well defined: BDNF, NT-3, and NT-4/5. NT-4 was first discovered in Xenopus laevis and it supports the survival of chick neurons, whereas NT-5, thought to be the mammalian equivalent of NT-4, does not. Whether or not NT-4 and NT-5 are different members of the neurotrophin family, or the same protein in different species is presently unclear. Today the affiliation of additional factors (e.g., NT-6 and NT-7) to this protein family is under discussion. Translated neurotrophins contain a signal sequence for secretion and are produced as precursor proteins (proneurotrophins) of 30–35 kDa. Following proteolytic C-terminal cleavage, a biologically active 12–14 kDa protein is released. The mature proteins are very well conserved and approximately 50% of the amino acids are common to all neurotrophins, indicating a functional homology. Within the cells, the neurotrophins are stored in vesicles and secreted as biologically active protein dimers associated by noncovalent bonds. However, the unprocessed 30–35 kDa proforms can also be released and there is growing evidence that the proneurotrophins are not only intracellular precursor molecules, but may also possess special functions in signal transduction outside the cell. There is also some evidence that NGF and pro-NGF differentially activate pro- and antiapoptotic cellular responses through differential activation of neurotrophin receptors, and the physiologic significance of proneurotrophin signaling is still under investigation. All members of the neurotrophin family use a common group of specific high-affinity cell-surface receptors: the tropomyosin-related tyrosine kinase (Trk) receptors (Fig. 22.4). Additionally, neurotrophins also signal through a common low-affinity neurotrophin receptor, the p75NTR (Pattarawarapan & Burges 2003). The p75NTR is a member of the tumor necrosis factor (TNF) receptor/Fas/CD40

Neurotrophins

Neuropeptides

Chronicity of the disease (t) Fig. 22.2 Potentiation of neurogenic inflammation by neurotrophins. (See CD-ROM for color version.)

by inflammatory cytokines integrates structural tissue cells into the neurotrophin signaling network during allergic inflammation (Fig. 22.3).

Biology of neurotrophins and neurotrophin receptors Neurotrophins are a family of homologous proteins that have similarities in structure, and receptor utility as well as physiologic activities. While the first member of the protein family, NGF, was discovered about half a century ago, in the following decades additional neurotrophins have been reported and the discovery of additional members remains open. Today, at least three additional members of this family

NEUROTROPHINS: NGF, BDNF, NT-3 Excitability Structural cells: Epithelia, Fibroblasts NGF induction CYTOKINES: IL-1, TNF, etc.

Synthesis

Biological effects

NEUROPEPTIDES: Substance P, Neurokinin Immune cells: Macrophages Lymphocytes Eosinophils Mast cells

Airway remodeling

Airway inflammation

• Subepithelial thickening • ECM deposition • Proliferation of ASMC

• Activation and survival of eosinophils in the lung • Accumulation and mediator release of lung mast cells

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Airway nerves

Activity

Airway hyperresponsiveness • Unspecific irritation • Low stimulation threshold • Enhanced contractility

Fig. 22.3 Neurotrophin signaling modulates the activity of neurons, and immune and structural cells during allergic inflammation of the airways. ASMC, airway smooth muscle cells; ECM, extracellular matrix. See text for definition of other abbreviations. (See CD-ROM for color version.)

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NGF BDNF NT-3 NT-4

p75NTR Low-affinity

NGF

trkA

BDNF NT-4

trkB

NT-3

trkC

High-affinity

Fig. 22.4 Signaling of neurotrophins by cell-surface receptors. See text for definition of other abbreviations. (See CD-ROM for color version.)

superfamily and all mature neurotrophins bind with low affinity to this receptor. In contrast, the Trk receptors bind neurotrophins specifically and with a higher affinity; however, there exists some crossreactivity between the Trk receptors and the neurotrophin ligands. NGF interacts preferentially with TrkA receptors and BDNF binds preferentially to TrkB. NT-3 interacts preferably with the TrkC receptor but can also bind to TrkA and TrkB. NT-4/5 overlaps with BDNF for TrkB receptor usage. Following specific binding of neurotrophins to their respective Trk receptors, they induce receptor homodimerization, which initiates kinase activation and subsequent transphosphorylation. The Trks are receptor tyrosine kinases that utilize a complex set of intracellular substrates and adaptor proteins to activate a defined secondary signaling cascade required for neurotrophin signal transduction. Classical signaling molecules such as the phosphatidylinositol 3-kinase pathway and the mitogen-activated protein (MAP) kinase cascade, have been identified as the primary downstream cellular events following Trk activation. However, the specificity of downstream Trk receptor-mediated signaling is controlled by the expression of intermediate molecules in these pathways as well as membrane trafficking that regulates the localization of different signaling components. As a consequence, in various cell types Trk receptor activation may result in different biological effects. While activation of the Trk receptor system generally induces cell differentiation, proliferation, and survival, signaling pathways activated by p75NTR appear to promote survival as well as apoptosis of cells. Thus, the signaling of neurotrophins via their complex receptor systems is not thoroughly and completely understood. In the absence of Trk receptors, p75NTR mostly induces apoptosis in conjunction with non-Trk receptor complexes such as sortilin. However, coexpression of p75NTR and TrkA may form high-affinity NGF binding sites by the interaction of transmembrane and cytoplasmic domains of both receptor molecules, and, thus, the p75NTR enhanced NGF-mediated TrkA receptor activity. In this way, it seems that the traditional view of an independent signaling of p75NTR and the Trk receptors may change to provide a more interactive cooperation during neurotrophin signaling.

Neurotrophins

Cellular sources and effects of neurotrophins The important role of neurotrophins as neuronal growth factors has been well known for about five decades, and the contribution of the peripheral nervous system to the pathogenesis of allergic diseases has also been well recognized. However, it is now well established that the expression as well as the signaling of neurotrophins via their specific receptors is not only restricted to the nervous system but also found in various other cell compartments (Fig. 22.5). These “other cell compartments” comprise also cells of the immune system that are able to express both neurotrophins and their corresponding receptors. These observations provide growing evidence that neurotrophins play a key role in influencing the developing allergic immune response. As the pathophysiology of allergic diseases depends on the progression of allergic inflammation, the biological functions of neurotrophins within the immune system are very important and will be discussed in detail. In addition, recent findings in asthma research lead to the suggestion that neurotrophins may also influence tissue remodeling processes, which are hallmark events that accompany chronic asthma. Moreover as discussed, in addition to cells of the nervous and immune system, neurotrophins are also expressed by structural tissue cells such as epithelia, fibroblast, and smooth muscle cells. Preliminary data indicate that neurotrophins may have autocrine and/or paracrine effects on functional activities of resident tissue cells. Based on all these observations, it is obvious that neurotrophins are not only growth factors modulating the peripheral nervous system, but also could play a more general role as cytokines mediating/delivering signals within and/or between various cell systems (Nockher & Renz 2006b). In this way, the definition of neurotrophins as “neurotrophic factors” seems to reflect a scientific relic rather than the biological tissue specificity of these signaling molecules (Fig. 22.5).

Peripheral nervous system Disturbances of neuronal activities within the allergic inflamed tissues are well known. For example, pruritus in atopic dermatitis and airway hyperresponsiveness in allergic asthma are essential features of these diseases and mainly contribute to the morbidity of these diseases. Therefore, unraveling the pathophysiology of neuronal changes in allergic inflammation is a key to the development of clinical strategies in the treatment of allergic diseases. The neurotrophins were originally identified as essential factors for the development of the vertebrate nervous system (Lewin & Barde 1996). They regulate the survival, death or the differentiation of neurons in the embryonic and postnatal stages, as well as neuronal maintenance later in life. While many sympathetic neurons depend on NGF for survival into adulthood, postnatal sensory neurons cease to rely on this

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Inflammatory Cells and Mediators

Sources

Targets

Effects Neuropeptide secretion Activity

Airway epithelium Neurons Keratinocytes

NGF BDNF NT-3 NT-4

Proliferation

ASM

Eos

Lym

MF MC

Activation Proliferation Mediator release

Inflammatory cells

factor. Somatic sensory neurons have their cell bodies within the dorsal root ganglia and their processes project to the spinal cord and to the periphery via the dorsal nerve root. Sensory ganglion cells are heterogeneous with regard to cell body size, fiber type and sensory receptor subtype. Moreover, they vary in neuropeptide content and signaling properties. In general, large-fiber sensory neurons are attached to muscle spindles and mediate proprioception. Neurons with mediumsized fibers subserve mechanoreceptors, which are located in joints as well as in the deep cutaneous layers of the skin, and mediate touch, pressure and vibratory sensations. In contrast, small-fiber neurons have terminals in the superficial skin, and mediate pain and temperature sensation. In general, larger axonal diameters are associated with larger cell bodies of the neurons and this relationship, although only approximate, has enabled researchers to correlate specific neurotrophin receptor expression with functional aspects of neurons within the dorsal root ganglion. The NGF receptor TrkA is expressed predominantly on small-diameter neurons, some of which are nociceptors. In contrast, TrkB, the high-affinity receptor for BDNF, is mainly expressed on medium-sized sensory neurons, which generally function as mechanoreceptors. The NT-3 receptor TrkC is found on large-diameter sensory neurons, which mediate proprioception. In chicken embryos, about 75% of large-diameter muscle afferents express TrkC, whereas only about 7% of large-diameter cutaneous afferents express this protein. The corresponding neurotrophin ligands NGF, BDNF and NT-3 were expressed in the appropriate target tissues that are innervated by these neurons, e.g., superficial epidermis, deep dermal layers, and muscle fibers. Thus, neurotrophins are target-derived neuronal growth factors that regulate neuronal outgrowth towards their place

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Fig. 22.5 Cellular sources and responders to neurotrophins in allergic inflammation. ASM, airway smooth muscle; Eos, eosinophil; Lym, lymphocyte; MC, mast cell; Mf, macrophage. See text for definition of other abbreviations. (See CD-ROM for color version.)

of synthesis (Ernfors 2001). Overexpression of neurotrophins in peripheral body tissues results in hyperinnervation as demonstrated in genetically modified animals. Transgenic mice with constitutive NGF overexpression in the clara cells of the lung exhibit an increased number of tachykinincontaining sensory as well as sympathetic nerve fibers in the airways. Vice versa, homozygous mice with disruption of the NGF gene showed extensive sensory and sympathetic neuronal cell loss. In addition, mice that lack expression of the p75NTR were shown to have a deficit in sensory innervations. On the other hand, mice lacking BDNF expression develop severe sensory deficits, while survival of sympathetic neurons is not affected. While visceral sensory neurons require neurotrophins for survival during development, these neurons do not further depend on neurotrophins for their survival throughout adulthood. Nevertheless, neurotrophins influence the function of peripheral sensory neurons after development, such as upregulation of neuropeptide synthesis (Lindsay & Harmer 1989), increase in excitability, establishment of functional synapses, or peripheral axonal branching. Therefore, enhanced tissue expression of neurotrophins results in functional changes and hyperactivity of the peripheral nervous system.

Immune system While the allergic inflammation is the initial trigger of the development and magnitude of all allergic diseases, the contribution of neurotrophin signaling to the local and systemic inflammatory response is of outstanding interest. The beststudied neurotrophin within the immune system is NGF, the first discovered and most prominent member of the neurotrophin family. Nevertheless, for many cells of the immune

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Differentiation

Mediators (IL-4, EPX) Cytotoxicity Chemotaxis

NT-3

Survival

Th2 Th1

Eos

NGF BDNF NT-3

? Survival

T-naive NGF

B cell

Survival Phagocytosis NO-synthesis

M NGF NT-3

MC

Priming Maturation Degranulation Survival

Fig. 22.6 Stimulatory effects of neurotrophins on various biological functions in the immune system. While the role of NGF has been most intensively studied, actions of other neurotrophins are less well characterized. Eos, eosinophil; EPX, eosinophil peroxidase; MC, mast cell; Mf, macrophage; NO, nitric oxide. See text for definition of other abbreviations. (See CD-ROM for color version.)

system the production of one or more neurotrophins and/or the expression of various neurotrophin receptors has been described. Consequently, neurotrophins mediate autocrine as well as paracrine signaling, which results in diverse biological effects. These effects besides cell survival, differentiation, and/or proliferation, also include other functions such as activation and cytokine or mediator release (Fig. 22.6).

Monocytes, macrophages, and dendritic cells Dendritic cells and tissue macrophages are important in the pathogenesis of local inflammatory processes. As antigenpresenting cells, they are mainly involved in the initiation and acceleration of the allergic immune response. While the involvement of dendritic cells and tissue macrophages within the cytokine signaling network of an inflammatory process is well established, their contribution to neurotrophin signaling is, so far, less well resolved. Whereas some data about neurotrophin expression are available for the monocytes/ macrophage system, detailed information about neurotrophin signaling in dendritic cells is absent. Within macrophage populations in various body tissues, lung macrophages are the best studied with regard to neurotrophin expression. In a murine model of allergic asthma our group has shown that macrophages found in bronchoalveolar lavage (BAL) fluid produce NGF and BDNF after allergen challenge (Nockher & Renz 2003), and others have shown a constitutive expression of NT-3 and NT-4 in murine alveolar macrophages. While mouse alveolar macrophages express NT-3 and NT-4/5 without their corresponding receptors, interstitial lung macrophages expressed BDNF and NT-4/5 as well as the receptors TrkB and TrkC. This implies that interstitial macrophages show a bidirectional mode of neurotrophin signaling both

Neurotrophins

via autocrine and paracrine mechanisms; however, alveolar macrophages are insensitive for neurotrophins due to receptor deficiency. In humans, macrophage populations are less studied. However, monocytes isolated from human peripheral blood show a constitutive expression of BDNF and NGF, and the release of NGF is significantly enhanced in monocytes from allergic patients compared with those from healthy donors (Rost et al. 2005). Monocyte secretion of BDNF has been shown to be increased by inflammatory cytokines such as IL-6 and TNF-α, which are released by activated cytokines (Schulte-Herbruggen et al. 2005). Neurotrophins function as survival factors for monocytes and macrophages especially after activation. Monocytes express the NGF receptor TrkA and ligand activation protects the cells from apoptosis induced by gliotoxin or UV-radiation. When monocyte-derived macrophages were stimulated with lipopolysaccharide (LPS), their NGF as well as TrkA expression was significantly enhanced while neutralization of endogenous NGF in LPS-activated macrophages induced apoptosis. Therefore, NGF exerts a survival effect on cells of the monocyte/macrophage lineage and activated cells especially required NGF for protection against apoptosis.

Lymphocytes T lymphocytes are important immune cells responsible for directing and maintaining a local allergic inflammatory response by production of Th2-type cytokines. Additionally, the synthesis of neurotrophins has been shown in T cells. Splenic T cells from mice express basal levels of NGF and NT-3, and activation with concanavalin-A upregulates their synthesis. BDNF release occurs after antigen stimulation of cultured splenic T cells from sensitized mice. Interestingly, tissue-specific BDNF production by T cells was observed in this study. T cells isolated from the inflamed lung showed high levels of BDNF synthesis without stimulation, whereas unstimulated T cells from the spleen did not produce detectable levels of BDNF. These differences may result from different T cell populations in spleen and lung, and/or a preactivation state of lung T cells at the site of local inflammation. In humans, CD4+ and CD8+ T cells, as well as CD19+ B cells purified from human peripheral blood lymphocytes, constitutively synthesize increased amounts of BDNF after stimulation. In contrast CD19-sorted B cells obtained from mouse spleen or lungs did not synthesize BDNF even under stimulation. Lymphocytes are not only able to synthesize neurotrophins, but also depend on these factors for maturation and maintenance. The expression of various neurotrophin receptors has been shown in human T cell clones, and in the mouse BDNF/TrkB signaling is necessary for the development of thymocyte precursors. Failure of Trk signaling results in mass apoptosis of lymphocytes, as has been shown in the thymus of TrkB-deficient mice. In the B cell system, NGF acts as an autocrine survival factor for surface γ+/α+ memory B

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lymphocytes but NGF had no effect on IgM→IgG or IgM→ IgA class switching (Torcia et al. 1996). Thus, NGF seems to be essential for the maintenance of specific secondary IgG- and IgA-mediated immune responses. On the other hand, divergent findings were reported with regard to IgE production. In human B lymphocytes, NGF inhibits the production of IgE after induction by IL-4, while in mononuclear cell preparations from spleens of allergic sensitized mice NGF favors IgE synthesis (Nockher & Renz 2003). While allergic inflammation is driven by a Th2-biased immune response, the possible role of neurotrophins in initiating or maintaining this process may be of interest. It has been found that Th1 but not Th2 or Th0 cell clones express the active BDNF receptor gp145trkB, and the Th1 or Th2 cytokine milieu favors expression of different members of the neurotrophin family. Both Th1 and Th2 CD4+ T cell clones produce low amounts of NGF in culture; however, upon mitogen stimulation, only Th2 cells upregulate NGF secretion, indicating a preferential Th2 source for this factor following T-cell activation (Lambiase et al. 1997). Additionally, when naive ovalbumin-specific CD4+CD45RB+ T cells from ovalbumin-specific D011.10 T cell receptor transgenic mice were directed to Th1/Th2 differentiation, the NT-3 receptor TrkC was expressed in Th2 cells but not in Th1 and naive CD4+ T cells (Sekimoto et al. 2003). Consequently, NT-3 synergistically enhanced anti-CD3-induced IL-4 production by Th2 cells but did not affect interferon (IFN)-γ synthesis by Th1 cells. Thus, at least in this mouse model, NT-3 seems to play a critical role in regulating Th1/Th2 balance, favoring a Th2-biased immunity. However, further studies are necessary to clarify if enhanced neurotrophin signaling directly influences the direction of T helper cell maturation during allergic inflammation in humans.

Eosinophils Eosinophils are the most important infiltrating cell population in allergic reactions, especially in allergic airway diseases. Eosinophils produce an array of cytokines and chemokines that contribute to the allergic inflammatory response, and also cell growth factors involved in tissue repair and fibrosis. During the last decade, it has been recognized that although human eosinophils produce NGF and NT-3 both factors are differentially regulated (Kobayashi et al. 2002). Whereas NT3 is constitutively expressed without further regulation, the amount of NGF synthesis depends on cellular activation. Synthesis of NGF is enhanced by Fc-receptor-mediated stimuli and eosinophil stimulation with IL-5 and soluble immune complexes results in an increased immunologically and biologically active NGF within the cells (Kobayashi et al. 2002). Although eosinophils are potentially able to express cellsurface receptors for neurotrophins, receptor expression may depend on the maturation or activation of these cells. In the bone marrow, eosinophils express the TrkB and TrkC receptors for BDNF and NT-3, respectively, but not the NGF

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receptor TrkA as well as the p75NTR. The expression profile of NT receptors in peripheral blood eosinophils is not clear. In one report, expression of all receptors was shown; however, the Trk expression varied both on mRNA as well as on the protein level between single patients. In contrast, in our experiments, circulating blood eosinophils from allergic patients did not show any Trk expression, but, more importantly, eosinophils obtained from BAL fluid after allergen provocation expressed all the neurotrophin receptors (Nassenstein et al. 2003). When BAL fluid eosinophils were cultured in the presence of neurotrophins, an antiapoptotic effect of neurotrophins was observed. Consequently, all members of the neurotrophin family are survival factors for eosinophils obtained from BAL fluid, but not for circulating blood eosinophils of patients with allergic asthma. Thus, the induction of neurotrophin receptors in activated eosinophils makes them susceptible to the antiapoptotic signals delivered by neurotrophins acting in an autocrine or paracrine manner. As cytotoxic activity and production of IL-4 is enhanced by NGF, one may speculate that an increased local neurotrophin synthesis supports maintenance as well as cellular activity of eosinophils within the inflamed tissue.

Mast cells Mast cells are important tissue-resident effector cells of the allergic immune response as they mainly contribute to immediate hypersensitivity reactions when activated through binding of IgE–antigen complexes. Numbers of mast cells are increased in allergic tissues, and recent studies have shown that NGF and NT-3, especially, play a pivotal role in mast cell biology as they mediate survival and differentiation. Reports on neurotrophin receptor expression in mast cells vary in a species- and tissue-dependent way. Mast cells obtained from different sources as well as from different species were all shown to respond to NGF through expression of the specific NGF receptor TrkA (Sawada et al. 2000); however, the expression pattern of other neurotrophin receptors is not unique. In mice, mast cells obtained from neonatal skin express TrkC; mast cells prepared from human lung express TrkB and TrkC but human umbilical cord blood-derived mast cells lack TrkB expression. While NGF alone does not support the survival of cultured mast cells, NGF acts as a cofactor together with stem cell factor (SCF) to prevent apoptosis. This effect is clearly antiapoptotic rather than proliferative as NGF does not stimulate changes in cell cycle progression. A similar finding has been recently observed in NT-3-overexpressing transgenic mice, where increased numbers of mast cells in the skin did not correlate with local mast cell proliferation (Metz et al. 2004). The authors speculate that NT-3 modulates skin mast cell numbers via differentiation of preexisting, immature mast cells and/or by enhancing the migration of circulating mast cells into the skin. At least, NGF has been shown to act as a chemoattractant for mast cells and induces chemotactic

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movement together with morphologic changes within the cytoskeleton (Sawada et al. 2000). Furthermore, NGF is involved in mast cell development such as differentiation from cord blood cell progenitors. NGF, and even more the combination of NGF and SCF, increased or induced expression of typical mast cell markers: IgE-receptor type I (FceRI), chymase, or mast-cell specific tryptase. Additionally, NGF modulates inflammatory cytokine expression such as induction of IL-6 expression and inhibition of TNF-α. NGF also regulates mast cell degranulation and subsequent cytokine and mediator release. In vitro, NGF stimulates the release of serotonin in cultured mast cells. Controversially, NT-3 failed to increase serotonin release in cultured mast cells but induced a significant increase in mast cell degranulation in vivo (Metz et al. 2004). While mast cells respond to NGF and NT-3, expression of NGF, NT-3, as well as NT-4/5, has also been shown. Therefore, neurotrophins are involved in vital functions of tissue mast cells, and mast cells have the capacity to produce these factors in an autocrine manner. Spontaneous NGF release is significantly increased in response to IgE and specific allergen (Xiang & Nilson 2000), and thus NGF could be specifically released by stimuli causing an allergic reaction and mast cells can thereby be a source of NGF in IgE-mediated inflammatory diseases.

Structural tissue cells The neuroimmune cross-talk involved in airway diseases also involves structural cells and their contribution to both neurotrophin production and responsiveness via autocrine and/or paracrine manner. Synthesis of neurotrophins like NGF and BDNF by structural cells such as the airway epithelia has been clearly demonstrated. Various in vitro studies with human lung cells in culture have identified bronchial epithelial cells, primary fibroblasts, and bronchial smooth muscle cells to express NGF (reviewed by Nockher & Renz 2003). Besides bronchial structural cells, human cutaneous fibroblast cell lines, intestinal epithelium, retinal epithelium, human vascular epithelium, and also vesical smooth muscle cells are other potential sources of NGF. Moreover, the synthesis of BDNF and NT-3 has been shown in structural cells like rat thoracic aortic smooth muscle cells, and human keratinocytes were identified to express NT-4/5 (Grewe et al. 2000). In vitro stimulation with IFN-γ, a marker cytokine for atopic eczema, was shown to induce keratinocyte NT-4 production (Grewe et al. 2000). In the same study, IFN-γ-injected skin and prurigo lesions of atopic dermatitis skin were characterized by intense epidermal staining for NT-4. Consistent with the in vitro studies, in vivo studies corroborate very well the expression of NGF by structural cells in the airways. Immunolabeling of bronchial biopsies from healthy patients without asthma show considerable staining of NGF in epithelial cells, bronchial smooth muscle cells, and fibroblasts (OlgartHögland et al. 2002). Immunohistochemistry performed in

Neurotrophins

mouse airways in our laboratory showed epithelial cells lining the airways as potential sources of neurotrophins like NGF, BDNF, and NT-3. The epithelial neurotrophin production was shown to be upregulated by IL-1β, TNF-α and Th2 cytokines. Inflammatory cells like eosinophils express the TrkA and TrkB receptors responsive to NGF and BDNF, respectively, and coculture with airway epithelial cells results in an enhanced epithelial neurotrophin production, which in turn enhances eosinophil survival (Hahn et al. 2006). Although most neurotrophins are clearly produced from structural cells, the relative expressions of each are different. This becomes more relevant in pathologic conditions as differential modulation of neurotrophins could shed more light on their individual relevance and functions, rather than all being referred to equally for the condition. Furthermore, in inflammatory conditions, the infiltrating inflammatory cells further supplement the increases in NT levels. Therefore, besides a physiologic function, their levels could be important in pathologic conditions and the role of each in such a scenario needs further investigation. The increases in neurotrophins could then have an autocrine or a paracrine effect on the structural cells lining the airway. One function for the responsiveness of structural cells to neurotrophins could be proliferation. Studies in human airway cells in culture show that NGF production depends on cell density. This has been shown in human fibroblasts and in bronchial smooth muscle cells (Olgart & Frossard 2001). It has been suggested that NGF is synthesized at higher rates by proliferating cells than by confluent cells. This could be an important factor in disorders like asthma where injury to airway epithelial cells triggers proliferation and therefore the increases in NGF levels. Other factors that lead to an induction in neurotrophin levels by structural cells are cytokines. Proinflammatory cytokines such as IL-1β and TNF-α or transforming growth factor (TGF)-β, increase the synthesis of NGF by airway structural cells. Human pulmonary fibroblasts, airway epithelial cell lines and bronchial smooth muscle cells, have all been shown to respond to proinflammatory cytokines by NGF production (reviewed by Nockher & Renz 2003). The increases in neurotrophin levels by structural cells observed in asthmatic airways and allergies seem to be due to a concerted outcome brought about by varied signals that are also upregulated in inflammatory conditions. Recent studies have also suggested that NGF is capable of directly stimulating structural cells. NGF has been shown to induce contraction and migration of human pulmonary fibroblasts (Micera et al. 2001) and vascular smooth muscle cells. The presence of specific receptors for neurotrophins in structural cells like TrkA and p75NTR further suggests an autocrine effect that could be useful for the cells in pathologic conditions. NGF, however, is also able to induce the proliferation of structural cells (Botchkarev et al. 2004) and can support the regeneration of injured tissue. Clinical studies as well as animal studies support an important role of NGF in wound healing.

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Topical treatment of corneal ulcers with NGF promotes restoring of corneal integrity (Lambiase et al. 1998). In a case report, NGF application reduced the size of pressure ulcers by favoring the increase of epithelial tissue (Bernabei et al. 1999). Moreover, topical administration of NGF into wounds increases the degree of reepithelialization and is able to accelerate the rate of wound healing in the skin (Matsuda et al. 1998).

Pathophysiology of neurotrophins in allergic diseases Initially, elevated neurotrophin synthesis was described in a study of patients with vernal keratoconjunctivitis, a disease with local mast cell activation. Later on, NGF serum levels were found to be increased in patients with various allergic diseases such as allergic asthma, urticaria, and atopic dermatitis (Bonini et al. 1996). Neurotrophin levels were also increased in local body fluids such as BAL fluids or nasal fluids of allergic patients, and local neurotrophin production resulted from enhanced neurotrophin expression of immune as well as structural cells. Consequently, an enhancement of local production of NGF and other neurotrophins has been shown in body fluids and tissue biopsy specimens obtained from allergic patients. However, the exact cellular sources of circulating neurotrophins are still not well defined. While a spill-over from enhanced local production into the bloodsream is noteworthy, direct proof is still lacking. One hint may come from asthmatic patients after immunosuppressive treatment with glucocorticoids, who show a significant decline in circulating neurotrophin levels (Noga et al. 2001). Inhaled glucocorticoid drugs were designed to exert their immunosuppressive effects mostly locally at the site of administration with only negligible systemic effects. Therefore, one may speculate that the reduction of blood neurotrophin levels originates from the suppression of an upregulated neurotrophin synthesis in the airways. Vice versa, increased levels of circulating neurotrophins may originate from enhanced local production within the inflamed tissue. It is well known that human platelets contain large amounts of BDNF, probably accumulated during vascular circulation and which could be released after platelet activation (Lommatzsch et al. 2005a). Recently, it has been shown in asthmatics that plasma and platelet levels of BDNF correlate with parameters of airway hyperresponsiveness (AHR) in steroid-naive but not corticosteroid-treated patients (Lommatzsch et al. 2005b). As corticosteroids were able to suppress BDNF secretion in blood mononuclear cells, this clinical study gives indirect evidence of a role of neurotrophins in the progression of inflammatorymediated airflow limitation in asthma.

Allergic asthma Allergic asthma is characterized by chronic airway inflammation, development of AHR, recurrent reversible airway

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obstruction, and subsequent airway remodeling. It is now clear that neurotrophins mainly contribute to the pathogenesis of inflammation and AHR, as well as tissue remodeling in allergic asthma.

Airway inflammation Allergic inflammation results in elevated serum IgE antibody titers, cellular infiltration of eosinophils and lymphocytes into lung tissue, and high levels of inflammatory cytokines such as IL-4 or IL-5 in BAL fluids. Asthmatic patients display increased levels of neurotrophins in serum and BAL fluid. Increased neurotrophin production has also been reported in response to segmental allergen provocation in the lung. Notably, this upregulation occurred at 18 hours after allergen challenge, when a marked infiltration of immune cells was also evident. This finding implies that inflammatory cells are a major source of neurotrophin synthesis in the allergic lung and eosinophils, lymphocytes, and macrophages have been shown to produce these factors (Nockher & Renz 2003). However, whether neurotrophin expression is upregulated even earlier after allergen challenge has not been investigated in humans. Animal models of allergic airway inflammation have also revealed that T lymphocytes and macrophages represent potential sources of neurotrophin production in the inflamed lung, and in vitro allergen stimulation of mononuclear cells from sensitized animals was shown to result in enhanced NGF synthesis. While immune cells are also able to express neurotrophin receptors, autocrine/paracrine effects of enhanced neurotrophin expression may modulate functional aspects of these cells. On the one hand, infiltrating immune cells such as eosinophils and lymphocytes, a hallmark of acute allergic inflammation, may also be a major source of NGF in the airways. On the other hand, in a murine model of asthma, our group has shown that NGF-overexpressing transgenic mice, which constitutively overexpress NGF in lung epithelial cells, recruited significantly more eosinophils after allergic sensitization compared with wild-type animals (Päth et al. 2002). Likewise, the p75NTR knockout mice exhibit a significant decrease in eosinophilic infiltration and similar effects were found after inactivation of NGF by intranasal application of an anti-NGF antibody during allergic sensitization (Kerzel et al. 2003) (Fig. 22.7). These data clearly show that allergic eosinophilia is associated with enhanced neurotrophin production in the inflamed lung. As has been shown previously, neurotrophins support survival of activated eosinophils and thus enhanced neurotrophin expression by either resident lung tissue cells or invading immune cells supports eosinophil homoeostasis in the airways (Nassenstein et al. 2003). However, whether neurotrophins in the lung besides supporting the survival of invading eosinophils also possesses chemoattractive activities, remains to be determined. Moreover, NGF amplifies the humoral allergic immune response such as stimulation of Th2 cytokine and IgE synthesis.

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Neurotrophins

Effects

Eos

Airway

T cell

Airway inflammation • Cells in the BALF

Fig. 22.7 Effects of NGF overexpression (NGF-tg mice) or depletion of p75NTR expression (p75NTR–/– mice) in a mouse model of allergic airway inflammation. Airway inflammation, characterized by eosinophil infiltration, and activity of sensory nerves, measured by response to capsaicin, are augmented in NGF-transgenic mice and diminished in p75NTR–/– mice. Bronchial smooth muscle activity, characterized by response to methacholine, is not affected by neurotrophin signaling. BALF, bronchoalveolar lavage fluid; Eos, eosinophil; wt, wild type. (See CD-ROM for color version.)

Tissue

Bronchial hyperreactivity • Capsaicin response (sensory nerves) • Methacholine response (smooth muscle)

Mouse:

wt

A Th2-biased immune response is critically important for the development of asthma-associated symptoms, and inhibiting the activity of NGF by anti-NGF treatment inhibits the local production of IL-4, the most important Th2-related cytokine. In addition to infiltrating eosinophils, macrophages, and T cells, tissue-resident mast cells are also involved in neurotrophin signaling. These cells secrete NGF after IgE-mediated stimulation and also depend on NGF as a maturation and survival factor. As mentioned above, NGF and BDNF levels in BALF of asthmatic patients are markedly increased 18 hours after segmental allergen provocation, whereas no change was observed directly (20 min) after the challenge (Virchow et al. 1998). These data may suggest that increased neurotrophin production is only associated with the allergic late phase reaction, which is characterized by a marked infiltration of immune cells. However, abrogation of NGF signaling by intranasal application of neutralizing antibodies inhibits allergen-induced early phase reactions in animal studies (de Vries et al. 2002). Vice versa, in transgenic mice that constitutively overexpress NGF in the lung, the early phase reaction, characterized by allergen-induced bronchoconstriction and serotonin release, was examined compared with wild-type animals. Similarly, other studies suggest a local upregulation of NGF expression in the bronchi as an early event in asthma, as increased transcripts of NGF were found in airway tissue from mild asthmatics following challenges of subclinical doses of allergen without inducing asthma symptoms (Kassel

NGF-tg

p75NTR–/–

et al. 2001). An increase in mast cells in the tissue parallels the upregulation of NGF expression in the airways and these cells were intensively immunostained for NGF. Hence, many immunocompetent cells involved in the allergic reaction, either tissue-resident and acutely reactive or infiltrating during the ongoing inflammation, may contribute to enhanced local neurotrophin expression (Fig. 22.8).

Airway hyperreactivity A key feature of chronic allergic asthma is the development of AHR. Target and effector cells responsible for AHR and airway obstruction include sensory and motor neurons as well as smooth muscle cells. Airway hyperreactivity is characterized by an increased susceptibility to a broad range of physiologic and nonphysiologic stimuli, including methacholine, histamine, hypertonic saline, cold air, and cigarette smoke. One possible mechanism for this is an inflammation-induced hypersensensitivity of sensory neurons that augments neuronal reflex circuits controlling lung function (Braun et al. 2002). During allergic asthma, qualitative and quantitative changes in the functional activity of lung sensory neurons were observed, such as an increase in mechanosensitivity resulting in an exaggerated neuronal excitability. There is growing evidence that changes in sensory airway innervation in the lung are under the control of inflammatory mediators released during allergic inflammation. The detailed mechanisms linking airway inflammation and AHR are not

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Airway Epithelium

Mast cell T cell Macrophage

Neurotrophins Eosinophil

Tissue

Vessel

completely defined, but neurotrophins represent candidate molecules regulating and controlling the cross-talk between the immune and peripheral nervous system (Nockher & Renz 2006a). NGF amplifies inflammatory AHR by upregulation of substance P production in lung sensory neurons and AHR has shown to be blocked by a substance P receptor antagonist (de Vries et al. 1999). In NGF-transgenic mice, airway inflammation is at least partly mediated by neuropeptides, as treatment of allergen-sensitized animals with a neurokinin receptor antagonist prevents eosinophil influx and IL-5 synthesis (Quarcoo et al. 2004). In a human study, the inflammatory cytokine IL-1β induced the release of NGF from isolated bronchi as well as the airway response to a tachykinin NK-1 receptor agonist, demonstrating that NGF mediates AHR induced by inflammation (Frossard et al. 2005). Allergen-challenged animals display an increase in substance P-immunoreactive nodose neurons, and a similar effect was shown following tracheal NGF application. Tracheal injection of NGF also changed substance P expression in mechanically sensitive Aδ-fibers, which do not express this neuropeptide under normal conditions (Hunter et al. 2000). Thus, NGF induced a switch in the phenotype of nodose neurons to become a component of the tachykinergic lung innervation. Recently, it has been shown in allergen-sensitized guinea pigs that inhibition of the NGF-receptor TrkA prevents the development of AHR as well as the increase in lung concentrations of substance P and substance P-positive neurons in nodose ganglia (de Vries et al. 2006). However, NGF also increases the release of stored substance P from sensory nerve endings without the need for neuropeptide synthesis in the neuronal cell body. This is obvious by the observation that NGF can increase AHR in the absence of sensory nerve cell bodies such as in isolated tracheal rings. The contribution of NGF expression on the development of neuronal AHR

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Fig. 22.8 Neurotrophins in the allergic inflamed lung. Immune cells as well as structural lung cells, e.g., epithelia, fibroblasts, and smooth muscle cells, produce neurotrophins constitutively or upregulate neurotrophin synthesis during inflammatory stimulation. Increased production of neurotrophins by inflammatory and structural lung cells results in elevated neurotrophin levels in the blood as well as in the bronchoalveolar fluid. (See CD-ROM for color version.)

is further demonstrated by sensory hyperreactivity in a mouse model of allergic airway inflammation using the NGF overexpressing transgenic mice (Päth et al. 2002). Sensory airway reactivity is measured via reactivity against capsaicin, which acts specifically via vaniloid receptors expressed on sensory neurons. Hyperreactivity caused by capsaicin-induced irritation of lung sensory neurons was markedly increased in allergen-sensitized NGF-transgenic mice compared with allergen-sensitized control animals. Consequently, in transgenic mice depleted for the expression of the pan-neurotrophin receptor p75NTR, the capsaicin-induced sensory hyperreactivity was almost abolished in allergen-challenged animals (Kerzel et al. 2003) (Fig. 22.7). Neurotrophins act via direct mechanisms through neurotrophin receptor signaling such as increasing the capsaicin receptor expression (Zhang et al. 2005) but also via indirect effects such as enhanced neuropeptide synthesis. The latter mechanism is supported by enhanced substance P-positive sensory innervation in the lungs of NGF-transgenic mice and a reduced amount of substance P in the lungs of p75NTR−/− mice (Kerzel et al. 2003). Nonspecific AHR in allergic asthma results in airway narrowing, which depends on an increased reactivity of sensory nerves and on the release of bronchoconstrictor stimuli that modulate airway smooth muscle cells. While reactivity to capsaicin reflects local activity of sensory nerves, methacholine directly induces smooth muscle contraction via muscarinic acetylcholine type 3 (M3) receptor expressed on airway smooth muscle cells. Animal studies have demonstrated that increased NGF expression or altered NGF signaling modulates sensory hyperreactivity; however, no effect of NGF on modulation of methacholine airway reactivity was shown in the same study (Nockher & Renz 2006a). After allergen challenge, no significant differences were found in the induction of an enhanced methacholine response between NGF-transgenic

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and wild-type mice, as well as in p75NTR−/− and wild-type animals (Fig. 22.7). Recently, comparable observations were demonstrated by modulation of BDNF signaling in the allergic lung. While treatment with anti-BDNF antibodies significantly reduced capsaicin-mediated sensory hypersensitivity, the methacholine response was unaltered (Quarcoo et al. 2004). However, using the p75NTR–/– mice, the upregulation of cholinergic AHR by acetylcholine was significantly diminished in these animals compared with wild-type mice (Tokuoka et al. 2001). In contrast to methacholine, which is relatively specific to M3 receptors on smooth muscle cells, the response to acetylcholine also includes reactivity of cholinergic airway neurons via neuronal muscarinic M2 receptors. Thus, the altered acetylcholine response in p75NTR–/– mice indicates that neurotrophins are able to modulate the activity of local cholinergic nerves. This is consistent with the recent observation of an enhanced cholinergic innervation as well as increased cholinergic activity in explanted mouse tracheal segments after culture with NGF (Bachar et al. 2004). Therefore, it seems that neurotrophin synthesis and signaling in the lung modulates local neuronal hyperreactivity of sensory as well as cholinergic neurons, but may not directly affect smooth muscle reactivity (Fig. 22.7).

Airway remodeling Accumulating evidence suggests the potential role of NGF in bronchial remodeling. The final phenotype of AHR and increased subepithelial thickening is a conglomeration of many events and mediators, and NGF seems to be playing a prominent role in mediating these events. Airway remodeling mainly consists of deposition of collagen beneath the epithelial basal lamina, which is associated with an increased number of myofibroblasts, hypertrophy of bronchial wall smooth muscle, and hyperplasia of mucus glands and goblet cells. Edema, vascular dilatation, and increased numbers of blood vessels also contribute to the thickening of the airway wall. The NGF-transgenic mouse (NGF-tg) overexpressing NGF in the clara cells of the lung shows an increased subepithelial thickening in the absence of any ongoing inflammation (Hoyle et al. 1998). NGF stimulates the contraction and migration of pulmonary fibroblasts as well as their differentiation into myofibroblasts (Micera et al. 2001). Isolated lung fibroblasts constitutively express TrkA. Although NGF has the modulatory effects mentioned above on fibroblasts, it does not influence lung fibroblast proliferation, collagen production or metalloproteinase production or activation (Micera et al. 2001). These effects of NGF could be due to the induction and release of profibrotic factors like TFG-β and fibroblast growth factor (FGF)-2, and of cytokines involved in the remodeling mechanisms like IL-4 and IL13. Experiments in human conjunctival fibroblasts have shown an enhanced TGF-β1 production after long-term NGF exposure, and following specific TGF-β1 neutralization all the NGF-induced effects were ceased (Micera et al. 2005).

Neurotrophins

In NGF-treated PC12 cells, the activation of the smad signal transduction pathway, typical of TGF-β1, has also been reported (Lutz et al. 2004). Remodeling events in tissue repair, consequential of any inflammatory reaction or injury, are a complex process that can result in pathologic outcomes like fibrosis. Fibroblasts have been clearly shown to mediate most of the wound healing processes; one of the prime ways is by extracellular matrix (ECM) production. NGF stimulates fibroblast production of type I collagen (Nithya et al. 2003) and bronchial biopsy sections from patients with interstitial fibrosis show strong NGF staining (Micera et al. 2001). NGF also stimulates the proliferation of bronchial smooth muscle cells and is, therefore, perhaps a prime candidate involved in smooth muscle cell hypertrophy observed in asthma. Matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), also play an important role in the development of tissue fibrosis. In addition to ECM proteolysis, MMPs also process growth factors including the immature precursors pro-NGF and proBDNF (Lee et al. 2001) as well as the receptors, including TrkA, TrkC, and p75NTR. It has also been reported that NGF-induced activation of TrkA receptor induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in smooth muscle cell lines genetically manipulated to express TrkA (Khan et al. 2002). This activation of MMP-9 by NGF was shown to occur by the Shc/mitogen-activated protein kinase pathway, indicating it to be a mediator in remodeling events occurring in the vascular wall in response to injury. After injury, MMP-9 mediates the migration of smooth muscle cells by releasing the attachment of the surrounding extracellular matrix. This allows the smooth muscle cells to migrate into the intimal space and is an essential step in formation of angiogenesis and increased vascularity. The microvasculature has been shown to contribute to airway wall remodeling in asthmatic patients and neurotrophin effects may support this process. TIMPs are secreted proteins that serve to inhibit MMPs and have relatively low selectivity. The relative proportion of the MMPs and their inhibitor TIMPs determines the proteolytic activity and is therefore critical for the turnover of extracellular matrix components within the tissues. Beside MMP expression, expression of TIMP-2 is also upregulated by NGF in various neuronal cell lines. However, so far the role of neurotrophins in regulation of extracellular matrix proteins, MMPs, and TIMPs is not well investigated in tissue remodeling in asthma. Further studies in this direction are required to better understand the airway remodeling observed in allergic asthma.

Allergic rhinitis Allergic rhinitis is a common chronic inflammatory disease of the upper airways with a pathophysiology similar to asthma, its counterpart of the lower airways. Asthma and allergic rhinitis are common comorbidities, suggesting the concept

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of “one airway, one disease.” Like asthma, allergic rhinitis is characterized by an inflammatory response including mucosal edema, increased vascular permeability, and leukocyte infiltration. The allergic responses are largely divided into two phases: systemic response in the induction phase and local allergic inflammation in the effector phase. The contribution of neurotrophins to the induction of the allergic response may be the same as in other allergic diseases, and the immunotrophic functions of neurotrophins for several immune cells involved in the initiation of the allergic Th2-response have been discussed above. The effector phase is characterized by sneezing, pruritus, rhinorrhea, and infiltration of the nasal mucosa with inflammatory cells, including eosinophils, neutrophils, lymphocytes, and mast cells. While direct studies of neurotrophins on these effector cells were not performed in the context of allergic rhinitis, the pathophysiologic events (e.g., epithelial–eosinophil or epithelial–mast cell interactions) are likely comparable to those in allergic asthma. Baseline levels of NGF protein are increased in the nasal fluid of rhinitis patients compared with control subjects (Sanico et al. 2000). Nasal provocation with allergen induces immediate symptoms of nasal irritation in allergic patients, together with a dose-dependent increase in NGF protein levels in nasal lavage fluid after allergen challenge. This increase in nasal fluid NGF concentrations is not attributable to plasma extravasation, as provocation with histamine increases concentrations of albumin but not NGF in nasal fluids. Therefore, enhanced NGF levels must originate from local sources of the allergic inflamed nose. Within the allergic inflamed nose tissue, NGF expression is found in infiltrated eosinophils but also in structural elements of the nasal mucosa such as the epithelium or the submucosal glands (Wu et al. 2006). The enhanced NGF expression in allergic rhinitis may provide a link between allergic inflammation and neural hyperresponsiveness to various inhalants. Allergic rhinitis is characterized by significantly increased reactivity against various inhalants, allergens, and environmental irritants such as tobacco smoke or cold dry air, resulting in sneezing and secretion of nasal fluid. Patients with allergic rhinitis show significantly greater nasal responses to stimulation with cold air compared with healthy volunteers (Sanico et al. 1999). In addition, rhinitis patients show a higher responsiveness of the neural apparatus of the nose as demonstrated by (i) significantly greater sneezing reflex after histamine provocation, (ii) significantly greater secretory responsiveness to capsaicin stimulation, and (iii) more intense plasma extravasation after capsaicin stimulation. These reflexes may be actively modulated by NT signaling, as has been shown in the lower respiratory tract. Stimulation of sensory neurons with capsaicin via activation of the vanilloid receptor-1 (TRPV1) results in an increased release of neuropeptides such as the tachykinins, which have multiple effects including the induction of vascular permeability, manifested by plasma protein extravasation in nasal fluids.

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Nasal epithelium Mucous glands RHINORRHEA

Blood vessels VASODILATATION PLASMA EXTRAVASTION

NGF Mast cell Histamine C5 Spinal cord

SNEEZING REFLEX Vidian nerve

Sensory nerve

CNS Fig. 22.9 In allergic rhinitis NGF modulates disease activity. Mast cells release histamine, which activates sensory nerves to produce pruritus and sneezing, as well as a nasal central reflex. This leads to activation of submucosal glands and rhinorrhea as well as vessel dilatation and plasma extravasation. Enhanced NGF expression by the nasal mucosa triggers mast cell and neuronal activity. (See CD-ROM for color version.)

Neurotrophins, in turn, increase membrane expression of TRPV1 nociceptors as well as modulating their signal transduction, resulting in an enhanced response to capsaicin (sensitization, hyperalgesia). Moreover, neurotrophins are target-derived growth factors and overexpression of NGF in the bronchial epithelium is associated with enhanced innervation (Hoyle et al. 1998). Therefore, increased NGF expression by the nasal mucosa may also alter the magnitude of innervation alongside elevated functional activity. However, enhanced sensory nerve activity results in increased pruritus and sneezing reflex as well as activation of submucosal glands leading to rhinorrhea (Fig. 22.9). Immunosuppressive therapy with local application of corticosteroids is a common treatment in allergic rhinitis. This is effective in inhibition of the allergic inflammation and also in the treatment of the clinical symptoms such as sneezing, pruritus, and rhinorrhea. The immunosuppressive action of corticosteroids results in a downmodulation of various biological functions of resident (e.g., mast cells) or infiltrating (e.g., eosinophils, immune cells). However, corticosteroids are also able to suppress neurotrophin synthesis in the airway epithelium (Hahn et al. 2006), and, thus, may block the described neuronal dysfunction triggered by enhanced local neurotrophin concentrations.

Atopic dermatitis Atopic dermatitis (AD) is a chronic multifactorial skin disease, which is the result of a complex network of genetic immunologic, and also environmental and psychologic factors. In atopic dermatitis various interactions between structural skin

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cells and the local nervous system as well as immune cells contribute to the pathology of the disease. The interplay between the local nervous system and infiltrating immune cells is particularly becoming a focus of clinical and basic research in AD. Elevated levels of NGF (Toyoda et al. 2002) and BDNF (Raap et al. 2005) were found in the plasma of AD patients and plasma NGF levels correlated positively with the disease activity. This finding indicates that neurotrophins are involved during the ongoing allergic inflammatory response in AD.

Neurotrophins

Mast cell Histamine

NGF Keratinocytes Substance P

Allergic skin inflammation Similar to other allergic diseases, e.g., those of the airways, neurotrophins are also produced in allergic skin disease by immune cells during the local inflammatory process. In AD many local as well as infiltrating immune cells (e.g., dendritic cells, Langerhans cells, mast cells, macrophages, lymphocytes, and eosinophils) are involved in the initiation as well as progression of the disease. Within the inflamed skin, a huge number of infiltrating cells show strong NGF expression while the normal skin areas of AD patients show weak NGF immunoreactivity (Dou et al. 2006). Moreover, the NGF content inside the granules of eosinophils obtained from AD patients was significantly elevated compared with cells from normal donors (Toyoda et al. 2003). Keratinocytes comprise an important source of neurotrophins in the diseased skin. Keratinocytes are recognized as a primary source of cutaneous neurotrophin production and keratinocyte neurotrophin release is enhanced during inflammatory conditions. Psoriatic keratinocytes release higher amounts of NGF than normal cells, and in AD skin an intense expression of NT-4 is found in pruritic lesions. IFN-γ is a strong inducer of keratinocyte NT4 as has been shown in culture as well as in INF-γ-injected skin. Recent findings indicate that NGF may also function as a signaling molecule between keratinocytes and mast cells. In the early phase of AD, mast cells are increased in the dermis and it is well known that NGF supports chemotaxis, maturation, and survival of mast cells in the body tissues. Thus, it is clear that enhanced NGF synthesis by keratinocytes may also promote dermal infiltration of mast cells as well as their activation, within the inflamed skin. Vice versa, mast cellderived histamine induces keratinocytes to upregulate NGF production (Kanda & Watanabe 2003), as do various other inflammatory cytokines. While NGF has been shown to degranulate mast cells in the skin, it stimulates its own synthesis via release of histamine (Fig. 22.10). Subsequently, plasma levels of histamine, as well as NGF, were significantly higher in patients with AD but also in serum and affected skin regions of DS-Nh mice, an animal model of AD. In vitro, the augmentation of keratinocyte NGF synthesis is inhibited by addition of loratadine, which is a strong histamine H1receptor antagonist (Yoshioka et al. 2006). The histamineinduced production of NGF, but also other cytokines such as GM-CSF, is mediated via H1 receptors on the keratinocyte

Sensory nerve Fig. 22.10 Nerve growth factor modulates interaction of keratinocytes with sensory nerves as well as mast cells in a vicious cycle within the allergic inflamed skin. (See CD-ROM for color version.)

cell surface and subsequent activation of intracellular protein kinase C. Thus, the NGF release after H1 histamine receptor activation contributes to a common inflammatory response of keratinocytes and is yet another example that neurotrophins play a role in the vicious cycle that amplifies allergic skin inflammation.

Pruritus In the epidermis of lesional skin of AD patients, cutaneous nerve fibers are present at higher densities. This increase in epidermal nerve fibers is at least partly responsible for the intense itch, one of the major clinical symptoms in AD. It is now accepted that NGF is one of the major mediators that determine skin innervation, corroborated in transgenic mice overexpressing NGF where hypertrophic sensory ganglia along with increased skin innervation is found. In a murine model of AD, treatment with a neutralizing anti-NGF antibody inhibited the proliferation of skin lesions and epidermal innervation, as well as intensity of itch–scratch cycles (Takano et al. 2005). However, the nature of relationship between NGF and pruritic conditions in AD is complex and involves multiple signaling between neurons, and structural and immune cells. Neuroimmune interactions are thought to be the most important trigger during the progression of this disease. In inflammatory skin disease, bidirectional pathways between the peripheral nervous system and immune system have been described. The interaction between cutaneous nerves and the immune cells is modulated by neuromediators such as neuropeptides and neurotrophins, which activate neurons and immune cells and also structural skin cells, such as keratinocytes, through specific receptors. Recent research indicates that the peripheral nervous system alters the course of AD through changes in cutaneous innervation and upregulated neuropeptide expression in lesional skin (Tobin et al.

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1995). AD lesions are characterized by high numbers of nerve fibers containing substance P, the most prominent member of the tachykinin neuropeptide family, while somatostatinimmunoreactive nerves were not found in the skin of AD patients in contrast with healthy subjects. Neuropeptides play an important role in immediate and delayed-type hypersensitivities and also mediate neurogenic inflammation in the skin. In particular, the release of substance P induces vascular responses, such as expression of adhesion molecules on endothelial cells, and also pruritus as a result of mast cell degranulation. Neuronal expression and release of tachykinins is mainly regulated by neurotrophins and the increased substance P may result from enhanced neurotrophin synthesis in AD skin. An increased epidermal expression of NGF and NT-4 in prurigo lesions of AD skin clearly supports this hypothesis (Grewe et al. 2000). Thus, neurotrophin expression in keratinocytes is upregulated in atopic dermatitis and may contribute to pruritus, which involves a complex signaling network between cutaneous nerves, mast cells, and also cells of the skin surface. While the primary function of keratinocytes is to provide the structural integrity of the skin and to protect against external injury, they are actively engaged in inflammatory skin responses by attracting immune cells and modulating their functions. However, these activities are also extended in chronic inflammatory skin disorders such as psoriasis or atopic dermatitis. Thereby, the expression of neurotrophins by activated keratinocytes is thought to regulate keratinocyte– nerve interactions during skin inflammation. Peptidergic sensory nerve fibers extending into the epidermis are in direct contact with keratinocytes. Enhanced synthesis of neurotrophins in the inflamed skin induces neuropeptide production and release in sensory skin innervation. Vice versa, substance P and neurokinin A are able to induce keratinocyte NGF (Burbach et al. 2001). While the direct effect of the neurosensory system on keratinocyte NGF production may be physiologically important for the maintenance and regeneration of cutaneous nerves in the normal skin, enhanced keratinocyte NGF synthesis may stimulate sensory innervation in a vicious cycle during allergic inflammation (Fig. 22.10).

Conclusions Studies of blood levels of neurotrophins revealed increased systemic concentrations of these factors during allergic diseases such as asthma or atopic dermatitis. While NGF is also elevated in autoimmune diseases such as systemic lupus erythematosus or systemic sclerosis, we might speculate whether upregulated neurotrophin expression is specifically linked to Th2-type allergic inflammation or more generally associated with an ongoing inflammatory response. There is growing evidence that NGF and also, potentially, the other members of the neurotrophin family are part of an integrated

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adaptive response to several offending stimuli, which connects cells of the immune and nervous system as well as structural cells. Initially discovered as growth factors supporting neuronal maintenance, it is now clear that their target cells are not limited to the nervous system. Cells of the immune system produce and respond to neurotrophins and studies of various allergic diseases reviewed over the last two decades indicate that neurotrophins behave as inflammatory cytokines delivering activating and survival signals to effector cells of the allergic response. In particular, the biology of mast cells and eosinophils is mainly affected by neurotrophins. However, a discussion on the potential mechanisms by which neurotrophins may participate in allergic inflammation will stray beyond what can be actually supported by experimental data. This also includes the functions of neurotrophins within structural tissue cells such as keratinocytes or epithelia which themselves contribute to the pathogenesis of allergic disease. On the other hand, it is indisputable from the results of many studies that enhanced local neurotrophin expression modulates activity of the peripheral nervous system and alterations in sensory innervation are frequently observed in allergic diseases. Moreover, they are responsible for typical clinical symptoms such as AHR in asthma or pruritus in atopic dermatitis. Based on these observations, a more intense study of the complex biological function of neurotrophins will become a potential tool for therapeutic intervention in the future.

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Neuropeptides David A. Groneberg and Axel Fischer

Central reflex

Summary Inflammatory events in allergic diseases such as allergic asthma or atopic dermatitis encompass the release of various neuropeptides from nerve fibers. This neurogenic response can be initiated not only by allergens but also by exogenous irritants, such as cigarette smoke or gases, and is characterized by a bidirectional linkage between neurons and inflammatory cells. The molecular mechanisms underlying neurogenic inflammation are orchestrated by a large number of neuropeptides including proinflammatory peptides such as tachykinins, neuropeptide tyrosine, or calcitonin gene-related peptide. Also, other biologically active molecules such as vasoactive intestinal polypeptide or pituitary adenylate cyclaseactivating peptide play a role in the modulation of inflammatory responses. Whereas numerous aspects of neuropeptide effects have been studied in laboratory animal models, little is known about precise neuroimmune events in human allergic diseases. However, different neuropeptide-modulating compounds may be used as targets for future therapeutic strategies.

Nodose ganglion

Vagus nerve

“Peripheral reflex“ Respiratory tract

Antidromic neuropeptide release Neurogenic inflammation

Allergen

Introduction In the event of allergic inflammation, neuropeptides such as the tachykinin substance P or calcitonin gene-related peptide (CGRP) are released into the periphery and can lead to inflammatory effects. The concepts of neurogenic inflammatory events in the airways and their potential importance for bronchial asthma have been established for more than a decade (Fig. 23.1). The initial activation of airway nerve fibers is based on the stimulation of receptors expressed on the terminal varicosities of airway nerves with exogenous irritants such as allergens, tobacco smoke or other air pollutants, or endogenous inflammatory mediators. This activation may

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Central nervous system

Airway lumen

Fig. 23.1 Concept of neurogenic inflammation. After peripheral noxious stimulation by airborne substances such as allergens or tobacco smoke, orthodromic activation of sensory nerve fiber endings takes place. The classical pathway then leads via orthodromic transmission into the brainstem. Here, the signal is modulated via interneurons and then transmitted via efferent parasympathetic nerve fibers to induce parasympathetic effects after synaptic transmission in local intrinsic airway ganglia. The neural events orchestrating neurogenic inflammation start in the sensory ganglia perikarya with the induction of proinflammatory neuropeptide gene expression. Then the neuropeptides are transported antidromically via the sensory nerve fibers back to the peripheral endings in the airways. Here, they are locally released and propagate the events of neurogenic inflammation. (From Groneberg et al. 2004e, with permission.)

then lead via orthodromic central and antidromic local reflex pathways to symptoms of allergy including bronchoconstriction or mucus secretion (Vignola et al. 2000). Numerous effects are exerted after nerve stimulation and it is accepted that there is broad communication between

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neurons and inflammatory cells. These interactions are orchestrated by a large number of neuromediators such as tachykinins (Harrison & Geppetti 2001), CGRP, vasoactive intestinal polypeptide (VIP), gaseous molecules such as nitric oxide or carbon monoxide (Eynott et al. 2002, 2003), or endogenous opioids (Groneberg & Fischer 2001). The role of the innervation for inflammatory events is well established for experimental models of airway inflammatory and obstructive diseases such as bronchial asthma. In this respect, numerous mediators of inflammation are known to influence sensory and cholinergic nerve activity under conditions of allergic inflammation. It is also accepted that the airway innervation, and especially sensory nerve fibers, regulates all major features of human respiratory function. However, findings from animal experiments on the significance of neurogenic inflammation have not yet been fully extrapolated to the human situation. Since the different pharmacologic properties of airway nerve mediators orchestrate many aspects of airway inflammation and may be useful as targets for future therapeutic strategies, neurogenic inflammation is an important area of current research. The present chapter aims to give a comprehensive summary of neuropeptide actions in allergic inflammation.

neurons can be variable in their myelination, caliber, and central nervous input (Solway & Leff 1991). The largest portion of mammalian airway-innervating sensory nerve fibers originates from the vagal ganglia (Kummer et al. 1992; Fischer et al. 1996). A smaller number of airway sensory nerves originate from the dorsal root ganglia (Dalsgaard et al. 1984; Saria et al. 1985; Springall et al. 1987) (Fig. 23.2).

Brainstem

Vagus nerve Parasympathetic

Nodose ganglion

Sensory

Sympathetic ganglia

Local intrinsic ganglia

Symp.

Airway innervation Sensory

Alongside the two classical components of the autonomic nervous system, the sympathetic and parasympathetic innervation (Table 23.1), a third system termed “sensory innervation” projects to the airways and innervates all major respiratory effector cells. The sensory innervation of the mammalian airways, which has also been called the noncholinergic nonadrenergic innervation, constitutes a heterogeneous population of neurons. In this respect, sensory

DRG Fig. 23.2 Anatomy of airway innervation: schematic illustration of human airway innervation. The vagus nerve supplies all parasympathetic preganglionic neurons and the majority of sensory nerve fibers. Sympathetic nerve fibers originate from sympathetic cervical and thoracic ganglia. A minority of sensory nerve fibers originate from dorsal root ganglia (DRG). (From Groneberg et al. 2004e, with permission.)

Table 23.1 Neuromediators. Mediator

Receptor

Major origin

Major effects

Acetylcholine

Nicotinergic and cholinergic receptors

Parasympathetic fibers

Bronchoconstriction

Catecholamines

Adrenergic receptors

Sympathetic fibers

Bronchodilation

CGRP

CGRP receptors

Sensory fibers

Vasodilation, bronchial tone

NPY

NPY receptors

Sympathetic fibers

Inflammation

PACAP

VPAC1, VPAC2, PAC1

Parasympathetic, sympathetic, and sensory fibers

Bronchodilation, vasodilation, immunomodulation

Tachykinins

Tachykinin receptors (NK1, NK2)

Sensory fibers

Inflammation, bronchoconstriction, mucus secretion, plasma exudation

VIP

VPAC1, VPAC2

Parasympathetic, sympathetic, and sensory fibers

Bronchodilation, vasodilation, immunomodulation

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The majority of neurons that innervate the airways end in the commissural, ventrolateral, and medial areas of the nucleus tractus solitarius in the brainstem (Haxhiu et al. 1993; Perez Fontan et al. 2000a,b). In these areas, they form synapses with interneurons communicating with medullary networks (Haxhiu & Loewy 1996; Hadziefendic & Haxhiu 1999; Perez Fontan 2002). Even now, little is known about the resulting multineuronal circles. It is generally assumed that the circles represent reflex loops that are activated by the peripheral stimulation of sensory airway neurons leading to the enhancement or inhibition of cholinergic nerve fibers, which project to numerous target cells in the respiratory tract (Fig. 23.1). Apart from central multineuronal reflex circuits there is also a second mode of operation present in sensory neurons: peripheral stimulation in the respiratory tract can also lead to a local neuronal mechanism that is orchestrated by sensory neurons (Coleridge & Coleridge 1984; Coleridge et al. 1989) expressing proinflammatory neuropeptides, such as CGRP or tachykinins. As nerve fiber terminals, and also the receptors for these neuropeptides, are localized in the epithelium, vessel walls, bronchial smooth muscles, and around mucus glands (Lundberg et al. 1984a), the local stimulation of sensory nerve fibers projecting to these target cells and the subsequent neuropeptide release can lead to features of allergic inflammation such as hyperemia (Salonen et al. 1988), edema (Lundberg et al. 1984b), mucus hypersecretion (Coles et al. 1984), and contraction of the bronchial smooth muscle. It is also known that sensory nerve fibers project to local intrinsic airway ganglia (Lundberg et al. 1984a; Myers et al. 1996), which may depolarize in response to the presence of tachykinins (Myers & Undem 1993; Belvisi et al. 1994). Therefore, peripheral activation of sensory nerve fibers with consecutive neuropeptide release may also lead to modulation of centrally mediated medullary reflexes (Tanaka & Grunstein 1990).

Human respiratory sensory innervation There are major species-specific differences between human and animal respiratory tract innervation, as many of the findings on airway innervation or promising effects of compounds targeting sensory nerves were not replicated with human tissues. While guinea-pig innervation is the closest approach to the human situation among all common laboratory species, there are still differences present: in general, sensory nerve fibers containing substance P and CGRP are of lower number in the human respiratory tract as compared with guinea-pig airways. Whereas these fibers constitute a large portion of total epithelial nerve fibers in the guinea-pig airways, they have been estimated in preliminary studies at only about 1% in humans (Bowden & Gibbins 1992). However, these preliminary data need further morphologic

Neuropeptides

studies to clarify this issue. In contrast to the potential anatomic and functional discrepancies, airway neuronal plasticity is generally accepted to occur in human pulmonary diseases. To assess if alterations in neuronal subpopulations are disease-specific or an epiphenomenon of the inflammation, different subtypes of human chronic upper airway diseases including hyperreflectoric rhinitis (Heppt et al. 2002), aspirin-sensitive rhinitis (Groneberg et al. 2003a) and toxic rhinitis (Groneberg et al. 2003b) were examined. Hyperreflectoric rhinitis is a chronic upper airway inflammatory disease related to nonspecific hyperreactivity. Expression profiling for substance P, CGRP, VIP, and neuropeptide tyrosine (NPY) revealed an abundant presence of nerve fibers expressing these peptides in the airways. Neuropeptide expression in mucosal nerves was also quantitatively assessed and significant increases were found for substance P (3.00 ± 0.37 vs. 1.64 ± 0.34 staining intensity in the control group) and VIP (2.33 ± 0.42 vs. 0.82 ± 0.33) (Heppt et al. 2002). These results demonstrate differences in levels of neuropeptides in the innervation of human nasal mucosa between nonrhinitic and hyperreflectoric rhinitic subjects and point to a modulatory role of neuropeptide-specific subpopulations of nerve fibers in hyperreflectoric rhinitis. Irritative toxic rhinitis is induced by chemical compounds such as tobacco smoke, ozone, solvents, formaldehyde, nickel, or chrome, which are also known to be associated with the development and progression of chronic obstructive pulmonary disease (COPD) and asthma. Semiquantitative immunohistochemistry for substance P, CGRP, VIP and NPY demonstrated significant differences between rhinitis patients and control subjects: toxic rhinitis patients had significantly increased expression scores for VIP (2.83 ± 0.31 vs. 1.27 ± 0.47 control group) and NPY (3.17 ± 0.31 vs. 0.91 ± 0.37 control group) (Groneberg et al. 2003b). These results indicated a differential participation of subclasses of mucosal nerves in the pathophysiology of toxic rhinitis and suggested that the changes in nerve profiles found in toxic rhinitis and hyperreflectoric rhinitis are disease-specific and not an epiphenomenon of inflammation. Next to toxic rhinitis and hyperreflectoric rhinitis, aspirinsensitive rhinitis was investigated which represents the manifestation of aspirin intolerance in the upper airways (Groneberg et al. 2003a). The disease is a pseudoallergy against aspirin and related nonsteroidal antiinflammatory drugs. Immunohistochemical analysis demonstrated that aspirinsensitive rhinitis patients also had a significant increase in VIP-immunoreactive nerve fibers (Groneberg et al. 2003a). These changes observed for the different forms of chronic upper airway inflammation may be partly regulated via neurotrophins. Nerve growth factor (NGF) significantly increases the transcription of the preprotachykinin-A gene, which encodes for substance P and neurokinin A in vitro (Lindsay & Harmar 1989). NGF expression is increased in asthma (Olgart Hoglund & Frossard 2002), and might therefore account for the induction of substance P in airway

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nerves under inflammatory conditions. In this respect, earlier studies reported an increase in the number of substance P-immunoreactive nerve fibers in the airways of patients with fatal asthma (Ollerenshaw et al. 1991). Also, increased concentrations of substance P were documented in the bronchoalveolar lavage fluids of patients with asthma, with a further rise following allergen challenges (Nieber et al. 1992). Substance P has also been found in the sputum of asthmatic patients after inhalation of hypertonic saline (Tomaki et al. 1995). However, it was difficult to replicate these findings in other populations and other studies did not reveal any increases (Howarth et al. 1991; Lilly et al. 1995). At the receptor level, it was reported that NK1 receptor gene expression is increased in the airways of asthmatic patients (Adcock et al. 1993). Also, abnormal expression of NK2 receptors was documented in asthma (Bai & Bramley 1993).

Neuropeptides involved in neurogenic inflammation Among the different neuropeptides stored in and secreted from airway nerves, predominantly those expressed in sensory nerves have been shown to contribute to the events of neurogenic inflammation. They are secreted during airway inflammation in reaction to a multitude of inflammatory mediators. More than 100 different nonneuronal mediators of airway inflammation have been described so far (Barnes et al. 1998), which may all propagate directly or indirectly neuropeptide release from nerve fibers in diseases such as bronchial asthma or COPD (Chung 2001). These mediators may not only induce neuropeptide expression and secretion but also increase the transcriptional and translational expression of neuropeptide receptors on either neuronal, inflammatory, or respiratory target cells. They may also influence the degradation of neuropeptides in the periphery. Although neuropeptides usually originate from airway nerves, recent studies indicate nonneuronal cellular sources such as inflammatory or epithelial cells, especially in states of airway inflammation as found in bronchial asthma (Germonpre et al. 1999).

Tachykinins Tachykinins such as substance P or neurokinin A encompass a family of peptides released from airway nerves after noxious stimulation (Fig. 23.3). They influence numerous respiratory functions under both normal and pathologic conditions, including the regulation of airway smooth muscle tone, vascular tone, mucus secretion, and immune functions (Maggi et al. 1995). Apart from neuronal cells as the source of tachykinins, inflammatory and immune cells may also synthesize and release tachykinins under certain conditions. This second cellular source of tachykinins may play a role in inflammatory airway diseases. Dual tachykinin NK1 and

514

Fig. 23.3 Tachykinin ligand and receptor expression in airways. Doublefluorescence immunohistochemistry illustrating the abundant presence of tachykinergic nerve fibers and NK2 receptors in the airways. Tachykinergic fibers are identified with an anti-substance P antibody in the smooth muscle layer and in the subepithelial layer. The presence of the NK2 receptors in smooth muscle cells is visualized with an NK2 antibody in guinea-pig airways. Original magnification ×200. (See CD-ROM for color version.)

NK2 receptor antagonism has been shown to exert major bronchoprotective effects and may be of special interest for the development of novel therapeutic approaches. Also, NK3 receptors may participate in the mediation of effects but their precise role in the human respiratory tract still needs to be investigated. The family of tachykinins has recently been extended by the discovery of a third tachykinin gene that encodes previously unknown NK1 receptor-selective tachykinins such as hemokinin 1 or endokinin A and B. Together with other novel tachykinin peptides, such as C14TKL-1 and virokinin, they may also play a role in the respiratory tract and further research is required to define their respiratory biological role in health and disease. The release of the major tachykinins from sensory nerves is affected by numerous other mediators including opioids, dopamine, or histamine (Fig. 23.4). The neuronal induction of tachykinin expression has been demonstrated in animal models of allergic inflammation, where tachykinins act as potent regulators of neurogenic inflammation due to their proinflammatory effects on many airway effector cells (Fischer et al. 1996). Both substance P and neurokinin A derive from the preprotachykinin A gene. Both peptides share a common carboxy-terminus amino acid sequence containing the biologically active domain (Escher et al. 1982). In the upper and lower respiratory tract, tachykinin immunoreactivity has been shown to be present in nerve fibers localized to submucosal glands, airway smooth muscle, and vasculature (Lundberg et al. 1984a; Heppt et al. 2002). Retrograde neuronal tracing studies in rats and guinea pigs have demonstrated that sensory nerve fibers, which innervate the trachea, mainly originate from the jugular and nodose vagal sensory ganglia (Springall et al. 1987; Kummer et al. 1992). To the same

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Inhibition

Activation Sensory nerve +

Activation

CB2 −

B2 GABA-B H1 Capsaicin LTB-4 12-HPETE Proton Heat

H3

Inhibition TRPV1 −

a2

+

OP3(m) D2

Tachykinins, CGRP

NK1

NK2

CGRP-R

Respiratory target cells

Fig. 23.4 Modulation of sensory nerve activity. Sensory nerve-mediated airway effects are induced via antidromic release of proinflammatory neuropeptides such as tachykinins or CGRP and their receptors NK1, NK2, and CGRP-R. Sensory nerve activity is regulated via stimulation of the vanilloid receptor TRPV1, bradykinin (B2), or histamine (H1) receptor, and inhibited via activation of numerous receptors including adrenergic (a2), opioid (OP3), cannabinoid (CB2), dopaminergic (D2), or histamine (H3) receptors. (From Groneberg et al. 2004e, with permission.)

extent, tachykinin-expressing nerve fibers mainly originate from neurons localized in the jugular and dorsal root ganglia (Saria et al. 1985; Kummer et al. 1992). It was recently shown that a furin-mediated cleavage of the bovine respiratory syncytial virus fusion protein leads to the release of a peptide converted into a biologically active tachykinin termed “virokinin” (Zimmer et al. 2003). The cellular enzymes involved in the C-terminus maturation of virokinin are present in many established cell lines and, indeed, virokinin is secreted by virus-infected cells. In vitro experiments revealed that virokinin by itself is capable of inducing smooth muscle contraction (Zimmer et al. 2003). Therefore, the viral tachykinin-like peptide virokinin may be considered a novel form of molecular mimicry, by which a virus may benefit by affecting host immune responses (Zimmer et al. 2003; Patacchini et al. 2004). Tachykinins bind to three different G-protein-coupled receptors. These receptors can be distinguished by their molecular properties and different pharmacologic affinities (Nakanishi

Neuropeptides

et al. 1993). While substance P principally activates NK1 receptors, neurokinin A mainly acts via NK2, and neurokinin B via NK3 receptors (Frossard & Advenier 1991; Devillier et al. 1992; Joos et al. 2000a). A differential pattern of tachykinin receptor distribution is known to be present in the respiratory tract: while NK1 receptors are predominantly localized in the airway epithelium and around submucosal glands and vessels, NK2 receptors are mainly expressed in the airway smooth muscle layer (Martling et al. 1987; Komatsu et al. 1991; Laitinen et al. 1992). The majority of human data available is on the regulation and function of tachykinin ligands and their receptors in allergic inflammatory diseases (Brunelleschi 1999; Honore et al. 2000; Joos et al. 2000b; Collins 2001). The interactions of tachykinins and their receptors in diseases such as COPD are largely unknown. However, it has been reported that tachykinin gene expression is increased after chronic exposure to cigarette smoke in animals (Kwong et al. 2001) and this mechanism may also exist in humans.

Pulmonary effects of tachykinins Tachykinins such as substance P and neurokinin A exert a multitude of effects on respiratory target cells. Tachykinins, particularly NKA, potently constrict human airway smooth muscles in vitro via NK2 receptors, with significantly greater potency in smaller airways (Frossard & Barnes 1991). This suggests that tachykinins can play a role in regulating constrictory effects in more peripheral airways, whereas cholinergic fiber effects may dominate in larger airways. In asthma, neurokinin A leads to bronchoconstriction after both inhaled and intravenous administration (Crimi et al. 1993). In vitro, the removal of the epithelial layer significantly increases bronchoconstrictor responses to tachykinins (Frossard et al. 1989). This effect points to an increased importance of tachykinins in diseases with a distorted airway epithelium such as bronchial asthma or COPD. Tachykinins are also known to stimulate secretion from human submucosal glands in vitro. This effect is mediated via NK1 receptors. The exact molecular pathways leading to the induction of mucin genes such as the primary gel-forming mucins expressed in the upper and lower respiratory tract, MUC5AC or MUC5B (Groneberg et al. 2002a,b, 2003c, 2004a), have not been elucidated so far. Further effects mediated via NK1 receptor signaling are plasma vasodilatation and exudation, as well as acetylcholine release facilitation at cholinergic nerve terminals, thereby enhancing cholinergic neurotransmission (Watson et al. 1993).

Metabolism of tachykinins The predominant mode of airway enzymatic tachykinin cleavage is via two enzymes, neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) (Di Maria et al. 1998). As NEP is expressed in the airway mucosa and submucosa (Fig. 23.5), it plays a major role in the degradation of

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Fig. 23.5 Neuropeptide hydrolysis. Neutral endopeptidase (NEP) activity is abundantly present in the airways as demonstrated using an NEP activity assay in guinea-pig airways. (From Groneberg et al. 2006, with permission.) (See CD-ROM for color version.)

tachykinins in the airways. In contrast, ACE is predominantly expressed in vascular cells and therefore predominantly regulates the cleavage of intravascular peptides. The activity of NEP can affect the responsiveness to tachykinins in the airways (Fig. 23.5). NEP inhibition has been demonstrated to significantly increase tachykinin-induced bronchoconstriction. Removing the epithelium also decreases the activity of NEP and it is known that the removal of the epithelial layer significantly increases bronchoconstrictory responses to tachykinins. Also, tobacco smoke or exposure to viral infections can decrease NEP activity and thereby may diminish the cleavage and degradation of proinflammatory tachykinins (Di Maria et al. 1998).

Tachykinin antagonists: clinical applications Antagonism of the selective neuropeptide receptors represents a useful tool for discriminating the precise role of neuropeptides. This approach was used to identify the effect of tachykinins: the NK1 receptor antagonist CP96345 significantly blocked plasma exudation in response to vagal stimulation and to cigarette smoke (Lei et al. 1992). This receptor-specific antagonism did not have any impact on bronchoconstrictor responses, but was blocked by selective NK2 antagonists (Advenier et al. 1992). Antagonism of NK1 receptors also led to a decrease in bradykinin- and hyperpnea-induced plasma exudation, but did not alter acute allergen-induced plasma exudation in sensitized guinea pigs (Sakamoto et al. 1993). Although the precise role and extent of tachykinin-related neurogenic events is not completely understood for bronchial asthma, tachykinin receptor antagonism represents a potential target for novel therapeutic approaches. Initial clinical studies with asthmatic patients have not led to very promising results. The nonselective tachykinin antagonist FK-224 was found to have inhibitory effects on bradykinin-induced bronchoconstriction in asthmatic patients (Ichinose et al.

516

1992). A more potent NK1 receptor antagonist also revealed a reduction in the duration of exercise-induced bronchial asthma. However, there was no consistent effect on the maximal bronchoconstriction (Ichinose et al. 1996). The potent NK1 receptor antagonist CP99994 did not have effects on either hypertonic saline-induced bronchoconstriction or on cough (Fahy et al. 1995). In contrast to these early studies that were characterized by the use of single receptor antagonists, in a recently published study a dual tachykinin NK1/NK2 antagonist (DNK333) was shown to inhibit neurokinin A-induced bronchoconstriction in patients with bronchial asthma patients (Joos et al. 2004). A total of 19 male adults with mild asthma participated in the randomized, double-blind, placebo-controlled, crossover study protocol in which increasing concentrations of NKA (3.3 × 10–9 to 1.0 × 10–6 mol/mL) were inhaled at 1- and 10hour intervals. Either a single oral dose of DNK333 (100 mg) or a placebo was given. It was shown that DNK333 did not affect baseline lung functions but it protected against NKAinduced bronchoconstriction. The mean log10 provocative concentration leading to a 20% fall in forced expiratory volume in 1 s (FEV1) for NKA was –5.6 log10 mol/mL at 1 hour (DNK333) and –6.8 log10 mol/mL (placebo). The ratio is equivalent to a difference of 4.08 doubling doses. Ten hours after treatment, the ratio decreased to a difference of 0.9 doubling doses (Joos et al. 2004). The findings from this study demonstrate that the dual receptor antagonist DNK333 can be used to efficiently block neurokinin A-induced bronchoconstriction in bronchial asthma. It was also suggested that although NK2 receptors predominate in mediation of neurokinin A smooth muscle contraction, NK1 receptors might also be involved in these events. In view of the bronchoprotective effects of DNK333, further clinical studies need to be performed on the efficacy and safety of dual tachykinin receptor antagonists with regard to bronchial asthma.

Calcitonin gene-related peptide CGRP is a further major neuropeptide that belongs to the class of proinflammatory sensory neuropeptides in the airways (Springer et al. 2003). CGRP consists of 37 amino acids and is a product of calcitonin pre-mRNA alternative splicing. The neuropeptide is expressed and stored together with tachykinins in sensory nerve fibers (Palmer & Barnes 1987; Palmer et al. 1987; Groneberg et al. 2003b). In addition to its expression in airway sensory neurons, CGRP has also been detected in pulmonary neuroendocrine cells where it is expressed in both the solitary type and cluster type (neuroepithelial body) (Keith et al. 1991; Buvry et al. 1999). The molecular identity of CGRP receptors has not been identified in detail. Numerous receptors have been suggested to be associated with CGRP or related peptides but had to be reclassified as orphan receptors (Kapas & Clark 1995; Kapas et al. 1995; Hanze et al. 2002). However, it was recently shown that CGRP receptors belong to the family of G-protein-

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ADM receptor

CGRP-A receptor N

N N

N

N or C

C C

C

RAMP2 or 3

CL-R

C

CL-R C

RAMP1

CGRP-B receptor

C

N

C C N

N

CL-R

N

RAMP2/3

RAMP1

C

N

C RCP ER

??? Fig. 23.6 CGRP receptors. Illustration of the currently known and predicted receptors for CGRP and related peptides. The CL protein (formerly CRLR) in coexpression with RAMP1 leads to a functional CGRP-A receptor, whereas the coexpression of CL with RAMP2 or RAMP3 leads to a receptor with higher affinity for adrenomedullin (ADM). The CT protein functions alone as a calcitonin receptor (CT-R), but in coexpression with RAMP1 or RAMP3 it serves as an amylin receptor. A further receptor (CGRP-A receptor), which is related to a receptor component protein (RCP), has been suggested. ER, endoplasmic reticulum; RAMP, receptor affinity modifying protein. (From Springer et al. 2003, with permission.)

N N

N

N

CT-R

RCP C

C

Plasma membrane

C RAMP1 or 3

CT-R

CT-R C

C

C

C or

N

coupled seven transmembrane receptors, as do many other neuropeptide receptors (Fischer et al. 2001, 2002; Groneberg et al. 2001a). In contrast to other neuropeptide receptors, the activity of CGRP receptors is regulated by a family of receptor activity-modifying proteins leading to different type A or type B CGRP receptors (McLatchie et al. 1998) (Fig. 23.6). Binding sites for CGRP in human airways were identified first using autoradiography (Mak & Barnes 1988) with a wide distribution throughout the respiratory tract. Immunohistochemistry to precisely identify CGRP type A receptor expression in human bronchial blood vessels revealed receptor immunoreactivity in the endothelium of venules but not in the endothelium of arterioles (Springer et al. 2003).

Pulmonary effects of CGRP Numerous studies have provided evidence for a modulatory role of CGRP in multiple pulmonary functions. These include the regulation of airway smooth muscle tones and vessel tones (Springer et al. 2003). However, the exact mode of

N Amylin receptor

N

N CT-receptor

CGRP effects on the airway smooth muscle tone is still unclear as controversial bronchoconstrictor or bronchodilator effects have been reported in the past years in different species and preparations (Springer et al. 2003). Recently it was shown for human bronchi in vitro that CGRP causes a concentration-dependent contraction of epithelium-denuded human bronchi whereas no significant effects are found in epithelium-intact bronchi (Springer et al. 2004), indicating a potential altered effect of CGRP on airway tone in respiratory diseases with a damaged epithelial layer such as asthma or COPD. In human pulmonary arteries, CGRP causes a concentration-dependent relaxation of both endothelium-intact anddenuded arteries. Pretreatment with indomethacin prevents the CGRP-induced relaxation in pulmonary arteries suggesting that prostaglandins are involved in the intracellular signal transduction pathway. In contrast, nitric oxide (NO) is not involved in this mechanism as pretreatment with the NO synthase inhibitor L-NAME does not affect CGRP-induced

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vascular relaxation. CGRP-induced effects on both bronchi and vessels are prevented by application of the CGRPantagonist CGRP (8-37) (Springer et al. 2004). There are no published data about the effects of CGRP on human airway mucus secretion. However, although glandular areas only display a very low density of CGRP binding sites (Mak & Barnes 1988), CGRP may affect mucus secretion indirectly by increasing the blood flow to submucosal glands.

Metabolism of CGRP CGRP is subjected to proteolytic inactivation by several enzymes expressed in the human respiratory tract. The exact cleavage pathways have not been revealed so far but NEP (EC 3.4.24.11) inhibitors have been shown to increase some of the CGRP effects in the airways (Katayama et al. 1991).

sent in the airways. In the respiratory tract (Groneberg et al. 2002c) and in other organs (Groneberg et al. 2002d,e; Doring et al. 2005), short-chain peptide fragments of NPY may be rescavenged by peptide transporters such as PEPT2 (Groneberg et al. 2004c).

NPY: clinical applications Whereas the peptide has been focused for therapeutic options in the central nervous system, a potential use in the treatment of pulmonary inflammatory disorders has not been revealed so far due to the complex pulmonary effects of NPY. However, as selective antagonists and agonists and gene-depleted animals for the different receptors are now available, NPY may be of value for future strategies in airway nerve modulation.

Vasoactive intestinal polypeptide CGRP: clinical applications The complex effects of CGRP on the airway and vascular tone and the immune system have so far prevented the development of any therapeutic strategy based on CGRP targets (Springer et al. 2003).

Neuropeptide tyrosine NPY has long been proposed to play a role in the pathogenesis of inflammatory diseases (Groneberg et al. 2004b). NPY is a 36 amino acid neuropeptide that participates in the regulation of a large number of physiologic and pathophysiologic processes in the cardiorespiratory system, immune system, nervous system, and endocrine system.

Pulmonary effects of NPY Serum levels of NPY are increased during exacerbations of asthma, whereas the number of NPY-immunoreactive nerves in the airways remained constant in patients with inflammatory airway diseases such asthma or rhinitis. In addition to a role in the regulation of glandular activity, NPY exerts a major influence on humoral and cellular immune functions (Groneberg et al. 2004b). In this respect, NPY is known to modulate potent immunologic effects, such as immune cell distribution, T helper cell differentiation, mediator release, or natural killer (NK) cell activation. In addition to these direct effects, NPY also acts as an immunomodulator by influencing the effects of a variety of other neurotransmitters (Groneberg et al. 2004b).

Metabolism of NPY The active form of NPY can be degraded by specific enzymes, leading to the generation of NPY2–36 and NPY3–36. It is suggested that these truncated fragments may be ligands of particular NPY receptors. Dipeptidyl peptidase IV (EC 3.4.14.5) is a likely candidate responsible for the cleavage of NPY as this exopeptidase is a membrane-bound protease, which cleaves proline in the penultimate position, and is abundantly pre-

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One of the most important bronchodilatory peptides found in the human body is VIP (Groneberg et al. 2001a). It is one of the most abundant neuropeptides present in both the upper and lower human respiratory tract (Ghatei et al. 1982; Baraniuk et al. 1990; Groneberg et al. 2003a) and a likely neurotransmitter of the inhibitory nonadrenergic noncholinergic airway nervous system. Together with pituitary adenylate cyclase-activating peptide (PACAP), VIP influences numerous pulmonary functions (Said 1982). It is expressed in human airway nerve fibers innervating the pulmonary and bronchial vessels, the tracheobronchial airway smooth muscle layer, and submucosal glands (Dey et al. 1981; Lundberg et al. 1984c). Also, receptors for VIP have been identified in the airway epithelium, glands, and in inflammatory cells (Fischer et al. 2001, 2002; Groneberg et al. 2001b, 2003d).

Metabolism of VIP Similar to proinflammatory peptides, VIP is subjected to degradation by airway enzymes such as NEP (Goetzl et al. 1989), mast cell chymase, and mast cell tryptase (Caughey 1989). NEP hydrolysis fragments of VIP are physiologically inactive (Lilly et al. 1993). Also, chymase and tryptase VIP degradation products fail to relax vascular or airway smooth muscle (Caughey et al. 1988).

Pulmonary effects of VIP VIP has been shown to be a potent relaxant of airway smooth muscle. As the bronchodilatory effect of VIP in human bronchi is almost 100 times more potent than adrenergic dilation by isoproterenol, VIP is the most potent endogenous bronchodilator described so far (Palmer et al. 1986a). The predominant site of VIP-induced pulmonary dilatation appears to be the central airways. Vasodilation of pulmonary and systemic vessels is the second principal effect of VIP. VIP potently relaxes the vessels supplying the upper airways (Lundberg et al. 1981; Lung & Widdicombe 1987), trachea, bronchi (Laitinen et al. 1987),

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and pulmonary vessels (Hamasaki et al. 1983; Nandiwada et al. 1985; Obara et al. 1989). VIP-induced vasodilation is more potent in the tracheal circulation than in the bronchial circulation (Matran et al. 1989). The vasodilatory effect of VIP is 50 times more potent than prostacyclin (Saga & Said 1984) and is independent of endothelial cells (Barnes et al. 1986; Greenberg et al. 1987). There is a dense network of VIP-immunoreactive nerve fibers around airway submucosal glands (Dey et al. 1981), and VIP-binding sites have been localized to human submucosal glands, suggesting a regulatory role of VIPergic nerves in human airway mucus secretion. The effects of VIP on mucus secretion are very complex: VIP stimulates the secretion of mucus from tracheal submucosal glands of ferrets in vitro, being significantly more potent than isoproterenol (Peatfield et al. 1983). Furthermore, mucus secretion stimulated by cholinergic agonists is inhibited by VIP in ferret trachea but stimulated in cat trachea (Webber & Widdicombe, 1987; Shimura et al. 1988). In contrast to ferret trachea, VIP exhibits an inhibitory effect on methacholine-stimulated release of glycoproteins and lysozyme in human trachea (Coles et al. 1981), which is surprising because VIP increases cyclic AMP formation in submucosal gland cells (Frandsen et al. 1978), and therefore it would be expected to stimulate secretion.

VIP: clinical applications The precise role of VIP in the pathogenesis of asthma and COPD is still uncertain. Although therapy using the strong bronchodilator effects of VIP would offer potential benefits, the rapid inactivation of the peptide by airway peptidases has prevented a widespread use of effective VIP-based drugs so far. The potent bronchodilator properties of VIP gave rise to studies addressing the effectiveness of VIP-based therapy in bronchial asthma. Contrary to early studies (Morice et al. 1983) that reported a significant bronchodilation and protection against histamine-induced bronchoconstriction in asthmatic subjects, later studies failed to show protective effects of the peptide. In this respect, systemic administration was reported to have no effect on the airway tone in normal subjects (Palmer et al. 1986b). At higher doses, VIP even caused blood pressure decreases and tachycardia. Aerosol administration of VIP was shown to reduce the bronchial reactivity to histamine in mild asthma but did not significantly increase airway conductance (Barnes & Dixon 1984). Also, a further study using inhaled VIP as pretreatment did not significantly prevent exercise-induced asthma (Diamond et al. 1983). The explanation for the inability of VIP to cause protective effects is the rapid inactivation of the peptide by airway peptidases. To bypass the limited clinical effectiveness of the native peptide, which is subject to degradation by many enzymes, peptidase-resistant VIP analogs were generated. After the characterization of amino acid residues required for VIP receptor binding and activation (O’Donnell et al. 1991),

Neuropeptides

various compounds were synthesized and examined. The identification of major cleavage sites led to the synthesis of metabolically more stable analogs, namely cyclic peptides incorporating disulfide and lactam ring structures (Bolin et al. 1995). The most potent synthetic VIP analogs that proved to have the best metabolic stability and the longest duration of activity are the cyclic peptides Ro 25-1553, developed as an antiinflammatory bronchorelaxant agent (O’Donnell et al. 1994), and Ro 25-1392 (Xia et al. 1997). Both compounds have been characterized to be highly selective agonists of the VPAC2 receptor (Gourlet et al. 1997; Xia et al. 1997; Schmidt et al. 2001). Due to the extremely promising profiles of the respiratory effects of VIP, a double-blind, randomized, placebo-controlled, crossover study was recently performed to study the effects of Ro 25-1553 (Linden et al. 2003). The compound was administered by inhalation to patients with asthma and compared with the long acting β2-adrenoceptor agonist formoterol. Twenty-four patients with moderate stable asthma were included in a crossover protocol with the primary variable being the bronchodilatory effect assessed as increase in FEV1 after inhalation of Ro 25-1553 (100 or 600 μg) or formoterol (4.5 μg) (Linden et al. 2003). Side effects were identified by assessing blood pressure, serum potassium, electrocardiography, and echocardiography. In contrast to earlier unsuccessful studies with the native peptide VIP, it was shown that the inhalation of 600 μg Ro 25-1553 caused a rapid bronchodilatory effect (geometric mean increase in FEV1 compared with placebo) within 3 min of 6% (95% CI 4–9). The corresponding maximum bronchodilatory effect during 24 hours was similar for 600 μg Ro 25-1553 (7%; 95% CI 4–10) and the reference bronchodilator formoterol (10%; 95% CI 7–12). However, for both doses of the synthetic VIP agonist, the bronchodilatory effect was attenuated 5 hours after inhalation whereas formoterol had a bronchodilatory effect 12 hours after inhalation (Linden et al. 2003). Safety profiling of Ro 25-1553 and formoterol did not reveal any clinically relevant adverse reactions and side effects. Given the advantages of the strong topical effects of VIP, newly developed VIP agonists with a longer duration of bronchodilatory effects will be very attractive for potential clinical use.

Neuropeptides and cough Cough is an important defensive reflex of the airways and is also a very common symptom of respiratory diseases. Cough following a viral infection of the upper respiratory tract is usually transient, while persistent cough is associated with a whole range of conditions, including bronchial asthma or rhinosinusitis. A variety of studies focused on the involvement of neuropeptides in the modulation of the cough reflex. Direct functional effects were not found in pharmacologic

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studies, i.e., the potent NK1 receptor antagonist CP 99994 did not have effects on cough (Fahy et al. 1995). However, it was established that the vanilloid receptor TRPV1 plays a crucial role in the pathophysiology of the cough reflex (Groneberg et al. 2004d; Geppetti et al. 2006). Clinical trials using TRPV1 antagonists have not been carried out on cough so far but this receptor seems to be a principal target for the development of more effective antitussives devoid of side effects. Also, novel molecular targets for the delivery of drugs have been identified (Groneberg et al. 2004c) that offer promising pathways to deliver compounds directly to the airways via proton-coupled drug transport mechanisms (Groneberg et al. 2003e).

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Sakamoto, T., Barnes, P.J. & Fan Chung, K. (1993) Effect of CP96,345, a non-peptide NK1 receptor antagonist, against substance P-, bradykinin- and allergen-induced airway microvascular leakage and bronchoconstriction in the guinea pig. Eur J Pharmacol 231, 31–8. Salonen, R.O., Webber, S.E. & Widdicombe, J.G. (1988) Effects of neuropeptides and capsaicin on the canine tracheal vasculature in vivo. Br J Pharmacol 95, 1262–70. Saria, A., Martling, C.R., Dalsgaard, C.J. & Lundberg, J.M. (1985) Evidence for substance P-immunoreactive spinal afferents that mediate bronchoconstriction. Acta Physiol Scand 125, 407–14. Schmidt, D.T., Ruhlmann, E., Waldeck, B. et al. (2001) The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery. Naunyn Schmiedebergs Arch Pharmacol 364, 314–20. Shimura, S., Sasaki, T., Ikeda, K., Sasaki, H. & Takishima, T. (1988) VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands. J Appl Physiol 65, 2537–44. Solway, J. & Leff, A.R. (1991) Sensory neuropeptides and airway function. J Appl Physiol 71, 2077–87. Springall, D.R., Cadieux, A., Oliveira, H., Su, H., Royston, D. & Polak, J.M. (1987) Retrograde tracing shows that CGRP-immunoreactive nerves of rat trachea and lung originate from vagal and dorsal root ganglia. J Auton Nerv Syst 20, 155–66. Springer, J., Geppetti, P., Fischer, A. & Groneberg, D.A. (2003) Calcitonin gene-related peptide as inflammatory mediator. Pulm Pharmacol Ther 16, 121–30. Springer, J., Amadesi, S., Trevisani, M. et al. (2004) Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery. Regul Pept 118, 127–34. Tanaka, D.T. & Grunstein, M.M. (1990) Maturation of neuromodulatory effect of substance P in rabbit airways. J Clin Invest 85, 345–50. Tomaki, M., Ichinose, M., Miura, M. et al. (1995) Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med 151, 613–17. Vignola, A.M., Kips, J. & Bousquet, J. (2000) Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 105, 1041–53. Watson, N., Maclagan, J. & Barnes, P.J. (1993) Endogenous tachykinins facilitate transmission through parasympathetic ganglia in guinea-pig trachea. Br J Pharmacol 109, 751–9. Webber, S.E. & Widdicombe, J.G. (1987) The effect of vasoactive intestinal peptide on smooth muscle tone and mucus secretion from the ferret trachea. Br J Pharmacol 91, 139–48. Xia, M., Sreedharan, S.P., Bolin, D.R., Gaufo, G.O. & Goetzl, E.J. (1997) Novel cyclic peptide agonist of high potency and selectivity for the type II vasoactive intestinal peptide receptor. J Pharmacol Exp Ther 281, 629–33. Zimmer, G., Rohn, M., McGregor, G.P., Schemann, M., Conzelmann, K.K. & Herrler, G. (2003) Virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus. J Biol Chem 278, 46854–61.

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Late-phase Allergic Reactions in Humans Yee-Ean Ong and A. Barry Kay

Summary The late-phase reaction (LPR) in humans is an experimental model of allergic inflammation elicited usually in the nose, lower airways, or skin of atopic subjects following challenge with specific allergen. The LPR is a delayed-in-time reaction peaking between 3 and 9 hours after challenge, and is preceded by an early immediate response. A common feature of LPRs is vascular leakage with tissue swelling and edema. A wide range of cell types, pharmacologic mediators, cytokines, and chemokines have been identified in fluids and tissue sampled from LPRs. So far, no single agent has been shown to account for all the features of these tissue responses. Late asthmatic reactions can be provoked in isolation (i.e., without an immediate response) by allergen-derived T-cell peptide epitopes. Peptide challenge, like whole allergen provocation, leads to increased airway hyperresponsiveness in those subjects who experience a late asthmatic reaction. In the peptide model, the potent vasodilator calcitonin gene-related peptide was highly expressed in epithelial cells, infiltrating T cells, and smooth muscle at the peak of the late response, suggesting one mechanism that explains, at least in part, the events surrounding the formation of tissue swelling. However, the precise mechanisms of vascular leakage and edema are still not fully elucidated, although it is likely that agents derived from mast cells and T cells are crucial for the full expression of LPRs.

Introduction When the skin, nose, or airways of atopic subjects are provoked with a single dose of allergen this produces, respectively, and within minutes, an immediate cutaneous wheal-and-flare reaction, sneezing and runny nose, or wheezing. Depending on the dose of allergen, these immediate-type hypersensitivity

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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responses are followed by a late-phase allergic reaction (LPR), which is slow to peak (6–9 hours) and slow to resolve. In the skin, LPRs are characterized by an edematous, red, and slightly indurated swelling, in the nose by sustained blockage, and in the lung by further wheezing. The LPR is extensively used as a model for studying the mechanisms of allergic inflammation and for screening drugs. Charles Blackley was the first to recognize, over 100 years ago, the association of allergen inhalation with symptoms of asthma several hours later, which often continued for several days (Blackley 1959). In 1934, Stevens noted that asthma symptoms could persist several days after allergen inhalation challenge (Stevens 1934). It was not until the 1950s that the response and time-course to allergen inhalation was more clearly described by Herxheimer (1952). Investigations into the pathogenesis of responses to inhaled allergens were well established by the 1970s (Booij-Noord et al. 1972; Pepys 1973). The development of a late asthmatic reaction (LAR) often results in increased airway responsiveness, which is reflected as an increase in morning dipping in peak expiratory flow rates for a few days following a single allergen challenge (Taylor et al. 1979). In 1977, Cockcroft made the classic observation that subjects who develop LARs following allergen provocation also develop an increase in airway responsiveness to histamine identified 24 hours after the challenge test, which persists for several days (Cockcroft et al. 1977a). The cutaneous late-phase reaction (CLPR) is characterized by a slowly developing, pink, edematous swelling at the challenge site. In large CLPR, there may be pruritus and systemic malaise, but these are unusual features. In most cases, the reaction is surprisingly indolent and the subject may be unaware of the reaction unless he or she is asked to look at the skin test site 4–6 hours after challenge. The reaction diameter can be measured accurately up to 8 hours after challenge, but becomes rather ill-defined thereafter. Following large CLPR, some diffuse swelling may persist for 48 hours, but this is not usually accompanied by erythema, and does not resemble the classical delayed-type hypersensitivity (tuberculin) reaction. The CLPR was originally considered to be an infrequent sequel to the immediate wheal-and-flare reaction. However, Frew and Kay (1988a) showed that all

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Methods of eliciting late-phase allergic reactions Airway challenge Late asthmatic responses can be achieved by experimental challenge in the laboratory. A number of methods have been employed, although perhaps the simplest and most reproducible is the tidal breathing method using a Wright nebulizer (Cockcroft et al. 1977b). Doubling or half-log incremental increasing concentrations of allergen are inhaled for 2 min from a Wright nebulizer at 15-min intervals. The starting allergen concentration is based on skin-prick tests of increasing concentrations of allergen extracts, with the starting concentration for inhalation being one half-log dilution lower than that which gave a 2-mm skin wheal. Baseline forced expiratory volume in 1 s (FEV1) (best of three attempts) is most commonly recorded initially after a 2-min inhalation of saline. Measurements are then repeated every 15 min after each allergen inhalation. If FEV1 has fallen by less than 10%, the next allergen concentration is inhaled. If FEV1 has fallen by 10–20%, it is repeated every 10 min until the maximal fall in FEV1 is achieved. If there is then a greater than 20% fall in FEV1, allergen inhalations are discontinued. If the fall in FEV1 is between 10 and 20%, the next concentration of allergen may be given. Once the end point has been achieved, the FEV1 is measured every 10 min

3.0

Normal saline control

Inhalational challenge 2.5 FEV1 (L)

atopic subjects are capable of developing a skin LPR if sufficient allergen is administered. The immediate response to nasal antigen challenge in sensitized patients comprises nasal congestion, rhinorrhea, sneezing, pruritus, and, often, conjunctival symptoms. These symptoms peak within a few minutes of the appropriate challenge and decrease over the next 1–2 hours (Naclerio et al. 1985). In 80% of subjects, the symptoms reach baseline levels within a couple of hours (Iliopoulos et al. 1990). Thereafter, in about 50% of these cases, recurrence of symptoms occurs 3–12 hours following the challenge. In some individuals, the decline in symptoms after the immediate reaction does not reach baseline levels; the clinical picture is that of continuing long-lasting reaction. Similarly, increased sneezing responsiveness to histamine has been observed after nasal allergen challenge, which dissipated after 12 days (Andersson et al. 1989a; Walden et al. 1991). In general, the molecular pathology of allergen-induced, late-phase lung, nasal, and skin reactions is similar. However, there are clear anatomic differences as regards permeability, content of smooth muscle, and autonomic nerve supply between the three tissues. The composition of mast cell granules between the different tissues (Irani et al. 1986) also varies, and may account for the different profile of response to various immunologic and nonimmunologic stimuli (Church et al. 1989).

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Late asthmatic reaction

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Time (h) Fig. 24.1 An example of an early and late asthmatic reaction provoked by inhalation of whole allergen (cat dander) in a cat-allergic subject. The early reaction is followed by a delayed-in-time late-phase response, which in this subject peaked at 8 hours. The response to allergen is compared with the control day in which the subject inhaled normal saline. (See CD-ROM for color version.)

until 1 hour and then hourly for up to 8 hours or longer depending on the protocol (Fig. 24.1). When performing repeated allergen inhalation challenges, identical conditions should be observed. These would include performing the challenges at the same time of day and adhering to the same technical and nontechnical factors. It is well recognized that an LAR may not be obtained in subjects challenged in the early morning, whereas this may be identified when challenged later in the day or evening. It has been postulated that this may be related to endogenous cortisol response, which might limit the development of a late response in some individuals. However, it has been shown that there are no changes in diurnal variation in serum cortisol concentrations in asthmatic subjects after allergen inhalation and no relationship to the size of the late response (Durham et al. 1989). Thus, the reason for the diurnal variation remains uncertain.

Nasal challenge Late nasal reactions can be produced by introduction of appropriate allergen into the nasal cavity either by means of a spray or presoaked disks. Allergen extracts are given in 10-fold increasing concentrations, with the starting concentration being the dose that caused a 2-mm wheal. Subjects are asked to inhale through the mouth to total lung capacity and hold their breath to minimize lower airway contamination by the test agent. Then, one squirt of the starting concentration is sprayed into one or both nostrils according to protocol from a metered-dose pump spray (Erin et al. 2005), or a disk is applied to the middle portion of the anterior nasal septum, posterior to the mucocutaneous junction for 1 min (Wagenmann et al. 2005). After 10–15 min, symptom scores derived from blockage, secretion, sneezes, itching eye, throat, conjunctivitis, cough, urticaria, or dyspnea are recorded

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Symptom score

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Time after challenge (hours) Fig. 24.2 Examples of immediate- and late-phase reactions in the nose assessed through symptom scores. (a) Example 1: the introduction of allergen in the nose leads to an immediate increase in nasal symptoms. These symptoms begin to dissipate soon after they peak and, within approximately 1 hour, they have almost returned to baseline. Later, 3– 4 hours after the allergen challenge, the symptoms return spontaneously and last for many hours and are still present 24 hours after allergen challenge. (b) Example 2: an identical immediate phase is followed by a partial reduction in symptoms, which remain elevated above baseline for many hours. A distinct late phase is not discernible but the magnitude of the symptomatology during the period that would be conventionally considered the “late phase” is similar to Example 1. (From Peebles & Togias 1997, with permission.)

(Fig. 24.2). The procedure is repeated until the highest concentration is reached or a positive response occurs (based on a predefined cumulative symptom score). The dose can be increased by increasing the number of squirts, until a response is obtained. Physiologic measurements can be made assessing resistance to air (which essentially reflects vascular engorgement), using rhinomanometry or measuring peak nasal inspiratory flow rate. Thickening of the paranasal sinus mucosa has been monitored (Pelikan & Pelikan-Filipek 1990). Nasal secretions can be collected, either spontaneously or by nasal lavage, and nasal biopsies taken. The glandular secretory apparatus can be assessed by local stimulation with a cholinergic agent such as methacholine (Raphael et al. 1988; Baroody et al. 1993). The activity of the neuronal apparatus can be monitored in the nose because stimulation of a mucosal irritant receptor inside one nasal cavity leads to the generation of a central reflex, the efferent arm of which produces a secretory response in the contralateral cavity (Konno & Togawa 1979; Baroody et al. 1993).

Cutaneous challenge Intradermal allergen challenge can be safely performed with predetermined concentrations of allergen using a 29G microfine syringe. The dose of allergen can be titrated according to the skin-prick test reaction (Charlesworth et al. 1989), or set using previous data (Frew & Kay 1988a). The immediate wheal

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(a)

(b)

Fig. 24.3 An early-phase and late-phase allergic skin reaction in an atopic subject challenged with house-dust mite extract by intradermal injection. (a) The early-phase reaction (15 min) is essentially a wheal-and-flare response, similar to that observed in a diagnostic skin-prick test. (b) The latephase reaction (6 h) is slightly raised, erythematous and with an edematous swelling, which is easy to palpate. (See CD-ROM for color version.)

and flare of the early reaction and the swollen erythematous skin characteristic of the CLPR is shown in Fig. 24.3. The late-phase skin reaction can be measured using a sharpened pencil point to detect the edge of the induration caused by the injections as previously described (Frew & Kay 1988a). An outline of the induration is drawn in ink and transferred using tape to form a permanent record. Alternatively, scanning laser Doppler imaging can be used to assess changes in skin blood flow (Clough et al. 1998). Samples can be obtained by means of skin biopsy to assess cellularity, while the skin chamber/skin window technique and cutaneous microdialysis are particularly useful to examine mediators. Recent alterations have been made to the traditional skin window technique in order to obtain sufficient cells to study by flow cytometry, while also collecting supernatant for study of mediators (Fernvik et al. 1999; Ong & Kay 2006).

Progression of early- and late-phase reactions Initially, atopic individuals were thought to be either single early responders or to have both early and late reactions (dual responders) to intradermal allergen challenge (Slavin et al. 1964). It has now been shown that there is a dose– response relationship between allergen dose and continuation of the skin reaction to the formation of an LPR (Frew & Kay 1988a). Formation of a CLPR after allergen challenge required a higher concentration threshold of allergen than the early-phase reaction (EPR), but all atopics were capable of mounting a LPR if challenged with sufficient allergen. It is

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possible that late asthmatic and late nasal reactions would also be ubiquitous if the airway effects of the EPR did not limit the dose of allergen that can safely be given. Isolated LARs have been shown to be induced by occupational agents (Paggiaro & Chan 1987), allergen-derived peptides (Haselden et al. 1999; Ali et al. 2004), and low-dose allergen (Ihre et al. 1988). Similarly, late nasal reactions have also been reported in the absence of early responses (Pelikan 1978). However, isolated late cutaneous reactions have not been reported, even when intradermal challenge with allergen-derived peptides has resulted in distant LARs (see below) (Haselden et al. 1999; Oldfield et al. 2001). The situation is more complex in patients with urticaria (see Chapter 90). Delayed-pressure urticaria is an example of a skin reaction that resembles an LPR but has no preceding immediate phase. In addition, a large immediate hive can be provoked with an ice cube in cold urticaria, but this is not followed by an LPR even with very large wheals. Thus, in this situation, the magnitude of the EPR does not seem related to an LPR.

Priming Following nasal or inhaled allergen challenge, the response to repeated local stimulation with the same allergen is increased. Priming was first described in the 1960s and its occurrence has since been confirmed by several investigators in both experimental and natural allergen exposure (Connell 1968, 1969; Bacon et al. 1981; Iliopoulos et al. 1990; Koh et al. 1997). In contrast, repeated cutaneous allergen challenges have shown both a reduction in the size of response (so-called refractory period) (Shaikh et al. 1977) or priming (Weller et al. 1996), depending on the time between challenges. The mechanism by which priming occurs is unclear. Although nonspecific target-organ responsiveness to inflammatory mediators released during allergen challenge can be implicated as a possible contributor to priming, there is evidence that the amount of inflammatory mediators recovered after allergen challenge in the primed state is significantly elevated as compared with baseline provocation (Pipkorn et al. 1987a; Wachs et al. 1989; Iliopoulos et al. 1990).

Late-phase Allergic Reactions in Humans

Mast cells There is incontrovertible evidence that the allergen-induced EPR is due to allergen cross-linking IgE antibodies on mast cells. The number of mast cells in bronchoalveolar lavage (BAL) (Diaz et al. 1989), skin (Macfarlane et al. 2000), and nasal epithelium (Fokkens et al. 1992) has been shown to decrease after allergen challenge. However, mast cell numbers were raised in bronchial mucosal biopsies 24 hours after allergen challenge (Crimi et al. 1991), and nasal lamina propria also shows increased levels of perivascular mast cells 10 hours after challenge (Juliusson et al. 1992). It is possible that this apparent discrepancy occurs as a result of continuous migration of mast cells into the epithelial surface fluid (Fokkens et al. 1992). The cells show an increased capacity to degranulate as they approach the airway surface (Djukanovic et al. 1990). Mapping techniques have shown that mast cells are present in normal skin with the greatest density occurring just below the dermal–epidermal junction (Cowen et al. 1979), particularly concentrated around blood vessels, nerves, and appendages (Eady et al. 1979). Application of inhalant allergen to the abraded skin of sensitive individuals with atopic dermatitis has been shown to induce an increase in the number of cutaneous mast cells (Mitchell et al. 1986). Studies using immunohistochemistry have shown a decrease in tryptase-containing mast cells after allergen provocation suggesting mast cell degranulation had occurred, and these remained reduced for up to 48 hours (Macfarlane et al. 2000). In nasal studies, some authors have found a correlation between mast cell numbers and the severity of early phase symptoms reported (Juliusson et al. 1992); however, others have not (Fokkens et al. 1992). In seasonal allergen exposure, a positive correlation has been reported between local mast cell density at the start of the allergy season and the severity of symptoms (Pipkorn et al. 1988). Circulating basophils infiltrate tissues at sites of allergen challenge in the skin (Solley et al. 1976; Mitchell et al. 1986; Charlesworth et al. 1989), nose (Iliopoulos et al. 1992), and lung (Guo et al. 1994). However, their numbers are small and influx after allergen challenge tends to occur later than the peak of the LPR itself, suggesting that this cell type is not directly responsible for tissue edema.

Immunology of late-phase reactions Eosinophils (see Chapter 12) Pepys, studying patients with allergic bronchopulmonary aspergillosis, initially proposed that the LAR was a type III reaction initiated by IgG antibodies (Pepys et al. 1968). However, later studies identified the IgE dependency of late responses occurring in the skin and the lung (Dolovich et al. 1973; Solley et al. 1976; Kirby et al. 1986). Mast cells, basophils, and dendritic cells possess high-affinity IgE receptors (FcεRI), and other cell types including monocytes, alveolar macrophages, and eosinophils recognize IgE through the low-affinity receptor (FcεRII, CD23).

In recent years, eosinophils have been regarded as proinflammatory cells in allergic disease that show selective enrichment in involved tissues (Wardlaw 1999). Allergen-induced LARs are accompanied by changes in blood eosinophils (Booij-Noord et al. 1972; Durham & Kay 1985; Cookson et al. 1989), which have both a temporal and quantitative relationship with the size of the late response and increase in airway responsiveness (Frick et al. 1989). Late-phase responses are associated with eosinophilia in the lung (de Monchy et al. 1985), nose (Bascom et al. 1988), and skin (Frew & Kay 1988b),

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and the appearance of hypodense eosinophils suggests they are in a state of activation (Frick et al. 1989). There is evidence of eosinophil degranulation in asthmatics (Wardlaw et al. 1988; Adelroth et al. 1990), after nasal allergen challenge (Andersson et al. 1989b) and in skin blister fluid after cutaneous challenge (Zweiman et al. 1991). Disease severity in asthmatics correlates with the number and state of activation of eosinophils in the bronchial mucosa and BAL fluid (Wardlaw et al. 1988). In contrast, there was no correlation between eosinophil numbers and the size of the allergen-induced LPR at 6 hours in the skin model (Frew & Kay 1988b). It seems unlikely that eosinophils are essential for the expression of LPRs. Anti-interleukin (IL)-5 had no effect on the LAR (Leckie et al. 2000) or the CLPR (Phipps et al. 2004). It is uncertain whether, in the former study, IL-5 depleted bronchial mucosal eosinophils; in the skin the eosinophildepleting effect was far more effective. Also, as described below, isolated LAR induced by inhalation of T-cell peptides was not associated with a significant increase in airway eosinophils (Ali et al. 2007).

Neutrophils (see Chapter 13) Neutrophils are residents of the airways in normal individuals as well as subjects with asthma. They accumulate in large numbers after allergen challenge in the lungs (Nocker et al. 1999; Macfarlane et al. 2000), nose (Bascom et al. 1988), and skin (Ying et al. 1999). The CXC chemokine IL-8, initially described as neutrophil chemotactic factor, was first noted after allergen challenge (Lee & Kay 1982). Although allergic asthma is traditionally thought to be due to eosinophilic inflammation, several studies have described the presence of neutrophils and IL-8 particularly in severe asthma (Jatakanon et al. 1999; Wenzel et al. 1999). In addition, peripheral blood neutrophils become activated in association with allergen-induced early and late-phase asthmatic reactions (Papageorgiou et al. 1983; Durham et al. 1984; Carroll et al. 1985). Although neutrophils are identified after allergen challenge in rhinitis (Lim et al. 1995) and asthma, their functional role in the development of late responses has yet to be determined. Neutrophils have the potential to release a mixture of lysozomal enzymes, oxygen metabolites, and leukotriene (LT)B4, which may be of relevance to the LPR. For example, vascular endothelial growth factor (VEGF) has been found in several cell types, including neutrophils and eosinophils, and is localized to the secretory vesicles (Gaudry et al. 1997). Tumor necrosis factor (TNF)-α stimulation has been shown to induce VEGF release by isolated human neutrophils (Gaudry et al. 1997). VEGF has an important role in angiogenesis but also induces endothelial hyperpermeability via the action of endothelial VEGF-R2 and thus may play a role the development of edema (Bates & Harper 2002; Byrne et al. 2005). The role of vascular permeability is considered later.

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T cells (see Chapter 3) Most of the work examining the role of T cells in allergic disease has been in the field of asthma. In the 1980s the concept arose that chronically activated T helper cells perpetuated the inflammatory response in allergic diseases and an association between T cells, their products, and asthma severity was noted (Walker et al. 1991). The T-cell hypothesis of asthma developed from studies of allergen-induced LARs (Gonzalez et al. 1987) and acute severe asthma (Corrigan et al. 1988), and was supported by the observation that there was a Th2 cytokine profile in asthma (Robinson et al. 1992; Bentley et al. 1993). Interest in the role of T cells in asthma arose from the concept that, in addition to participating in IgE synthesis, T-cell products may also have effects on the airways either through direct action or via the recruitment of inflammatory cells. Sampling of both peripheral blood of asthmatics during exacerbations as well as the airways of asthmatic patients revealed T cells with features of activation (Gonzalez et al. 1987; Azzawi et al. 1990; Robinson et al. 1993a). After description of the Th1/Th2 dichotomy, cells bearing mRNA for IL-4 and IL-5 (signature Th2 cytokines) were detected in the airways of atopic asthmatic patients (Ying et al. 1997). This linked Th2 cells with IgE synthesis through the actions of IL-4, and also eosinophil airway inflammation through IL-5, together with IL-3 and granulocyte–macrophage colonystimulating factor (GM-CSF). Over recent years it has been suggested that Th2 cytokines contribute to asthma pathology in a variety of ways. In addition to stimulating IgE production (IL-4, IL-13), the effects include maturation of eosinophils (IL-5, IL-9), upregulation of the eosinophil/basophil selective adhesion molecule vascular cell adhesion molecule (VCAM)-1 (IL-4, IL-13), mast cell development (IL-3, IL-9, stem-cell factor), and, in the context of asthma, airway hyperreactivity (IL-9, IL-13) and mucus hypersecretion (IL-4, IL-9, IL-13) (Kay 2001). It is also suggested that there may be direct interaction between T cells and airway smooth muscle, and IL-5 has been shown to increase smooth muscle contractility in vitro (Hakonarson et al. 1999). In humans, LARs were provoked by intradermal challenge with T-cell peptide epitopes. However, as mentioned above, the expected infiltration of eosinophils and Th2 cells into bronchial mucosa was not observed and there was no measurable release of histamine or eicosanoids in BAL, raising the possibility that there may be purely T cell-dependent pathways to airway narrowing in asthma (Haselden et al. 2001). Although some studies also showed the presence of Th1 cytokines in serum and BAL fluid from asthmatics (Krug et al. 1996), most studies confirm a Th2 predominance. An increase in nasal mucosal CD4+ T lymphocytes is also seen after nasal allergen challenge, and there is a correlation between expression of the IL-2 receptor (CD25+) and CD4+ cells, suggesting that CD4+ cells may be activated after allergen challenge (Varney et al. 1992a; Lim et al. 1995). Interest-

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ingly, in some studies, mononuclear cells (which presumably include lymphocytes) do not rise significantly, suggesting that the allergen-induced inflammatory response may vary between individuals (Lim et al. 1995). As with the lung, the majority of the lymphocytes in the nose (Durham et al. 1992) and skin (Barata et al. 1998) after allergen challenge were Th2 type.

Antigen-presenting cells (see Chapter 8) Increased numbers of Langerhans cells have been found in epithelium and lamina propria of the nose after sequential nasal allergen challenge (Godthelp et al. 1996). Similar results have been observed during grass pollen season in patients with seasonal allergic rhinitis and these increases were inhibited by the use of topical corticosteroids (Till et al. 2001).

Late-phase Allergic Reactions in Humans

It is not possible to determine the cellular sources of histamine, PGD2, and LTC4 recovered in these studies. Mast cells have been shown to produce all three substances, eosinophils the lipid-derived mediators, while basophils release only histamine and LTC4. Therefore, it is possible that mast cells are responsible for the early release of histamine and, in conjunction with eosinophils, for PGD2 and LTC4 levels in the first 6–8 hours. The absence of tryptase and PGD2 from the LPRs suggests that basophils rather than mast cells may be responsible for the second wave of mediators in the airways. There is also evidence that kinins may be involved in the late-phase skin response, as activation of the kallikrein–kinin cascade (see Chapter 20) paralleled the magnitude of the LPR (Atkins et al. 1987).

Cytokines (see Chapter 3) Pharmacologic mediators Early release of the mast cell products histamine, tryptase, prostaglandin (PG)D2, and LTC4 has been detected in nasal lavage fluid (Naclerio et al. 1985; Juliusson et al. 1991), and also in BAL (Sedgwick et al. 1991; Wenzel et al. 1991) after local allergen challenge. Skin blister studies have also shown histamine release within 5 min of allergen challenge (Ting et al. 1980), as well as tryptase and PGD2 (Shalit et al. 1988; Charlesworth et al. 1989; Reshef et al. 1989). Dermal microdialysis techniques have detected histamine release in the wheal area after allergen challenge, but not in the flare which is thought to occur by a neurogenic reflex mechanism (Petersen et al. 1997). The quantity of mediators produced by mast cells correlates significantly with the clinical intensity of the immediate-phase nasal reaction (Naclerio et al. 1985). There is a second increase in histamine, LTC4, and kinins in those patients who exhibit a late nasal response (Naclerio et al. 1985; Pipkorn et al. 1987b). Although the late increase in LTC4 is small compared with levels found in the early phase (Pipkorn et al. 1987b), a metabolite of LTC4, LTE4, is also detected in the late phase in higher quantities than in the early phase, perhaps as a result of metabolism. Interestingly, PGD2 and tryptase do not reappear in increased concentrations in the late-phase nasal response (Naclerio et al. 1985; Gosset et al. 1993). Similarly, increases have been seen in histamine and LTC4 levels in BAL 6 hours after challenge (Diaz et al. 1989), and the terminal metabolite LTE4 has been recovered in increased amounts in the urine (Taylor et al. 1989). In the skin after allergen challenge, histamine continues to be detected at a lower level for up to 12 hours, although some investigators have noted a second peak at 12 hours (Charlesworth et al. 1989). PGD2 levels peak 1–4 hours after allergen challenge before decreasing over the next 8 hours (Charlesworth et al. 1989; Reshef et al. 1989). In contrast, LTC4 is elevated for 7 hours with significantly raised levels only between 4 and 6 hours (Reshef et al. 1989).

After antigen challenge, elevated levels of several cytokines have been detected in nasal lavage, BAL, and skin blister fluid during the LPR. These include IL-1β (Bochner et al. 1990; Sim et al. 1994; Virchow et al. 1995) and IL-6 (Lee et al. 1992; Sim et al. 1994; Virchow et al. 1995). IL-4, IL-5, IL-13, GM-CSF, and TNF-α have all been detected in lavage fluid after nasal and bronchial challenges (Sim et al. 1994; Bradding et al. 1995; Huang et al. 1995; Virchow et al. 1995; Batra et al. 2004; Erin et al. 2005; Ong & Kay 2006). The source of cytokines that are found in lavage or skin blister fluid is yet to be fully determined. Bradding et al. (1993) found, in immunohistochemical staining of inferior turbinates of patients with perennial rhinitis, that 90% of the IL-4 and IL-6 immunoreactive cells were mast cells, while 50% of the IL-5 immunoreactive cells were mast cells. IL-5 immunoreactivity was also found in eosinophils. Studies have also shown that the majority of the cells expressing IL4, IL-5 in the nose and lungs after allergen challenge are CD3+ T cells, with the remainder being mast cells and some eosinophils (Ying et al. 1993, 1995, 1997). Although allergic disease has traditionally been viewed as a Th2-driven disease, there is some work that has suggested inbuilt regulatory mechanisms with Th1 or regulatory cytokines playing a role. Previous work using in situ hybridization to assess mRNA for Th1 and Th2 cytokines did not show a significant increase in interferon (IFN)-γ mRNA (Kay et al. 1991). However, some studies have shown elevated IFN-γ in acute and chronic asthma (Corrigan & Kay 1990; Cembrzynska-Nowak et al. 1993), and correlated this with disease activity in subjects with asthma (ten Hacken et al. 1998). Furthermore, T cells obtained from BAL are able to produce IFN-γ on stimulation (Cho et al. 2005). There is also growing evidence to suggest that Th1 cytokines counteract the activity of Th2 lymphocytes, with IFN-γ being the most potent in suppressing Th2-type allergic responses (Gavett et al. 1995; Lack et al. 1996; Tang et al. 2001). Therefore, Th1 cytokines within areas of allergic inflammation may be acting to “switch off” the allergic response.

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In addition, there were significant increases in the level of the regulatory cytokine IL-10 in allergen-challenged blisters. IL-10 is produced by a variety of cells, including regulatory T cells (Tr1) as well as macrophages (Moore et al. 1993; Groux et al. 1997). It is thought to play a possible role in the regulation of allergic responses through a number of different pathways, including its suppressive effects on Th1 and Th2 cells (Romagnani 1995), and cytokines (Fiorentino et al. 1991), as well as by inhibiting eosinophil survival (Takanaski et al. 1994). It is also a potent suppressor of IgE while it simultaneously increases IgG4 production (Akdis et al. 1998). Given its antiinflammatory effects, it has been suggested that a deficiency in this cytokine may result in a more intense inflammatory reaction in asthmatic subjects. Although some investigators have reported reduced levels of IL-10 in asthmatics (Borish et al. 1996), others have reported higher levels of both IL-10 mRNA (Robinson et al. 1996; Magnan et al. 1998), as well as IL-10 protein in the BAL of asthmatics compared with normal individuals (Colavita et al. 2000). Similarly, elevated levels of transforming growth factor (TGF)-β have been detected in the BAL of asthmatics (Redington et al. 1997). Previous work has suggested that TGF-β may have antiinflammatory properties (Haneda et al. 1997, 1999), as well as a role in the remodeling seen in chronic asthma (Kumar et al. 2004). It is possible that these regulatory cytokines attempt to act in conjunction with Th1 cytokines to downregulate the allergic inflammatory response that has been elicited.

Chemokines Following allergen challenge in asthmatics, both macrophagederived chemokine (MDC) and thymus and activationregulated chemokine (TARC) were strongly upregulated on airway epithelial cells, and Th2 cells in the bronchial mucosa expressed CCR4, and some of them CCR8, but no CCR3 (Panina-Bordignon et al. 2001). Macrophage inflammatory protein (MIP)-1β, the ligand for CCR5 and interferon-inducible protein (IP)-10, the CXCR3 ligand, preferentially attract Th1 cells in vitro. In sarcoidosis, a typical Th1 cell-mediated lung disease, the T cells isolated from BAL expressed high levels of CXCR3 (Agostini et al. 1998). However, Campbell et al. (2001) examined BAL T cells comparing asthmatic subjects and nonasthmatic control subjects and showed no difference in the expression of CCR5 and CXCR3, with only low expression of CCR4. Both Th1 (IP-10) and Th2 (MDC and TARC) chemokines have been recovered from BAL after allergen challenge (Liu et al. 2004). Furthermore, some work suggests that chemokines may induce tissuespecific (rather than disease-specific) migration of T cells. Memory for skin-homing circulating lymphocytes is found in a population of circulating lymphocytes expressing cutaneous lymphocyte antigen (CLA) (Berg et al. 1991). In addition, TARC (CCL17) has been shown to be expressed by cutaneous endothelium and binds to CCR4 expressed at high levels on CLA-positive skin memory lymphocytes (Campbell et al.

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1999). More recently, keratinocytes have been shown to express cutaneous T cell-attracting chemokine (CTACK, CCL27), which binds to the receptor CCR10, also specifically expressed on circulating skin-homing CLA-positive T lymphocytes (Morales et al. 1999). Increased levels of CCR4 have been found in the skin after allergen challenge (Nouri-Aria et al. 2002), and elevated levels of CCR4 and CCR10 have also been demonstrated in an animal model of delayed-type hypersensitivity (Reiss et al. 2001) suggesting that, in the skin, the TARC/CCR4 axis may act as a specific skin-homing mechanism rather than having a Th2 disease bias. Increased levels of RANTES and MIP-1α have been identified in nasal secretions after allergen challenge (Weido et al. 1996; Kramer et al. 2001). Similarly, increased levels of eotaxin, eotaxin-2, RANTES, MIP-1α, and MCP-4 have been found after intradermal allergen challenge (Zweiman et al. 1997; Ying et al. 1999, 2001).

Neural inflammation LPRs in the lungs, nose, and skin provide three different methods of assessing the role of neurogenic inflammation. The lower airways allow us to study the interaction of nerves, neuropeptides, and smooth muscle; the nose allows investigators to stimulate one nasal passage and study the effects of neurogenic inflammation in the other; and the skin model can be used to study the disruption of nerve supply whether by use of local anesthetics or interruption of nerve supply. Initial interest in the concept of neurogenic inflammation arose when stimulation of dorsal root ganglion (a sensory nerve) led to vasodilation via an antidromic (conduction opposite to normal) pathway (Bayliss 1901). The axon reflex model was proposed whereby local injury could spread by orthodromic and then antidromic conduction of impulses (Langley 1900). Bruce (1913) demonstrated that acute inflammation caused by the application of mustard oil to the skin required an intact sensory system. Lewis and Grant (1924) then showed that sensory innervation provides the pathway for a local reflex which is required for the flare of the wheal-and-flare response after allergen challenge.

Airways Substance P is the predominant neuropeptide found in BAL of asthmatics (Nieber et al. 1991, 1992). Substance P and neurokinin A increase in BAL after allergen challenge (Nieber et al. 1992; Tomaki et al. 1995; Heaney et al. 1998). It appears that substance P exerts its proinflammatory effects mainly by stimulation of the neurokinin (NK)1 receptors, whereas neurokinin A (NKA) mainly acts on NK2 receptors. Increased levels of NK1 and NK2 receptor mRNA expression have been found in asthmatics as compared with nonasthmatics (Adcock et al. 1993; Bai et al. 1995). On inhalation, substance P induces airway hyperresponsiveness (AHR)

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mainly characterized by microvascular leakage, mucus secretion, and inflammatory cell responses (Joos et al. 2000), whereas inhaled NKA mainly causes bronchoconstriction (Joos et al. 1987; Cheung et al. 1993). Calcitonin gene-related peptide (CGRP), which acts predominantly as a vasodilator, has also been localized in airway nerves (Palmer et al. 1987; Martling et al. 1988). Although neuropeptides were traditionally thought to derive from nerves, there is considerable evidence that there are cellular sources that produce neuropeptides and also bear NK receptors (Ho et al. 1997; Kay et al. 2007). Several studies have been conducted assessing tachykinin NK1/NK2 receptor antagonists. In one study, the NK1/NK2 receptor antagonist FK224 failed to protect against NKAinduced bronchoconstriction (Joos et al. 1996). In contrast, other tachykinin receptor antagonists, DNK333 and CS-003, have been shown to produce a significant rightward shift of the dose–response curve to NKA (Joos et al. 2004; Schelfhout et al. 2006). More recently, a dual NK1/NK2 receptor antagonist, AVE5883, conferred protection against NKA-induced bronchoconstriction but had no beneficial effects against allergen-induced early or late-phase asthmatic reactions, methacholine PC20, or sputum differential cell counts, raising a question as to the role of neurogenic inflammation in this model (Boot et al. 2007).

Late-phase Allergic Reactions in Humans

Skin Nerve fibers containing a variety of neuropeptides, including substance P and CGRP, have been demonstrated in human skin (Schulze et al. 1997). Intradermal injection of substance P results in an immediate wheal-and-flare reaction (Foreman et al. 1983), whereas the main effect of CGRP is a long-lasting (4–6 hours) intense erythema (Brain et al. 1985). Additionally, CGRP potentiates edema formation evoked by mediators such as substance P (Brain & Williams 1985). This work suggested that neuropeptides may have an important role to play in both the allergen-induced EPR and LPR. Previous work on two subjects with impaired sensory function (one with sensory denervation due to trauma, the second after administration of local anesthetic) showed that stimulation of mast cells with anaphylactogenic anti-IgE resulted in a wheal, but the complete absence of a flare, corroborating evidence that the flare in the EPR is due to a neurogenic reflex mechanism. However, in both of these subjects, there was no apparent reduction in the LPR on the sensorydeprived site as compared with a control site (Umemoto et al. 1976), suggesting that while neurogenic inflammation was responsible for the flare of the EPR, it had a limited role in the LPR.

Action of drugs Nose After nasal allergen challenge, substance P and CGRP are released into nasal secretions of atopic patients (Mosimann et al. 1993; Nieber et al. 1993). In allergic rhinitis patients, but not in normal control subjects, exogenous substance P induces nasal obstruction (Devillier et al. 1988). In contrast, CGRP instillation in the nasal cavity increased nasal blood flow and caused the sensation of nasal obstruction (Rangi et al. 1990), but had no immediate effects on albumin leakage or glandular secretion (Guarnaccia et al. 1994). Nerve growth factor has been found in nasal lavage fluid and also in cells, particularly mast cells, in allergic rhinitis (Sanico et al. 2000; Wu et al. 2006). There is evidence for the participation of nasal nerves in aspects of the LPR. Most studies have reported that the number of sneezes during the LPR, although much less than the EPR, is elevated compared with appropriate controls (Naclerio et al. 1985). When a localized, unilateral nasal allergen challenge is performed, ipsilateral and contralateral elevations in airway resistance, as well as in levels of histamine, IL-4, IL-8, and airways secretions are seen (Baroody et al. 1993; Wagenmann et al. 2005). Although it is not possible to refute the hypothesis that local allergen application may lead to systemic contralateral effects unrelated to neurogenic inflammation, there is evidence that stimulation of local sensory nerves with capsaicin can lead to dose-dependent leukocyte influx, albumin leakage, and glandular secretion (Sanico et al. 1997).

The b agonists The β agonists, when given prior to allergen, will generally inhibit the immediate asthmatic response but not the late response (Cockcroft & Murdock 1987). In nebulized form, salbutamol has been shown to partially inhibit the late response when given prior to allergen, and similarly inhaled β agonists may have a partial effect when given during the late response (Twentyman et al. 1991). Their effects may be more a reflection of prolonged functional antagonism rather than a specific effect on the underlying mechanism of the LAR (Twentyman et al. 1990). The cutaneous allergeninduced EPR can also be attenuated by the administration of β2 agonists by inhibition of allergen-induced mast cell degranulation both in vitro and in vivo. Intradermal terbutaline and salbutamol administration resulted in inhibition of the allergen-induced early and late-phase skin reactions (Petersen & Skov 1999) and also an immediate reduction in the release of histamine and PGD2 synthesis (Petersen & Skov 2003).

Antihistamines Antihistamines have been an integral part of the treatment of allergic rhinitis for many years and predominantly reduce symptoms of sneezing and pruritis, but have limited effects on nasal blockage (Hilberg 1995). Antihistamines have been shown to predominantly affect the early phase nasal reaction, with limited effect in the late-phase reaction (Baroody &

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Naclerio 2000; Saengpanich et al. 2002). Although antihistamines are not used in the treatment of asthma, they have been shown to reduce the size of both the early and late asthmatic reactions (Hamid et al. 1990; Wasserfallen et al. 1993), as well as reduce eosinophil accumulation 24 hours after allergen challenge (Redier et al. 1992). Similarly, histamine H1 receptor antagonists such as cetirizine have been shown to inhibit both the early and, to a lesser extent, late-phase allergen-induced skin reactions (Varney et al. 1992b; Nielsen et al. 2001). Although cetirizine has been shown to reduce early total protein extravasation, there was no evidence of a reduction in the release of inflammatory mediators, such as histamine, tryptase, or LTB4, or a reduction in the numbers of inflammatory cells after allergen challenge.

Mast cell stabilizers Sodium cromoglycate has been shown to inhibit both the early and late asthmatic response to allergen (Crimi et al. 1986; Cockcroft & Murdock 1987). Cromolyn has been shown to be effective in protecting the nasal mucosa from both the early and late nasal responses to allergen (Pelikan 1982); however, the therapeutic role of cromolyn in allergic rhinitis is rather limited (Naclerio 1991). There have been contradictory results as to the effect of mast cell stabilizers such as sodium cromoglycate on allergen-induced skin reactions. Locally applied cromolyn does not appear to cause a reduction in the allergeninduced EPR when administered at the same time as allergen or anaphylactogenic anti-IgE (Ting et al. 1983; Kimata & Igarashi 1990). However, when pretreatment with cromolyn was given, there was a reduction in the EPR (Kimata & Igarashi 1990). One other study has shown that although local cromolyn administered at the same time as anti-human IgE did not alter the EPR, there was a significant reduction in the LPR (Gronneberg & Zetterstrom 1985). Differences in dosage, as well as the time of administration, may have played a role in the differences between these studies.

Leukotriene receptor antagonists Early trials of leukotriene receptor antagonists (LTRAs) showed benefit only on the early asthmatic response (Fuller et al. 1989). However, later LTRAs have been shown to inhibit both the early and late asthmatic response to inhaled allergen as well as the development of AHR (Taylor et al. 1991; Hamilton et al. 1998). There is general agreement that LTRAs do not inhibit allergen-induced cutaneous EPRs (Hill & Krouse 2003; White et al. 2005). However, there has been contradictory work as regards their effects on the cutaneous LPR, with some authors finding a reduction in size of these reactions after administration of LTRAs while others have not (Simons et al. 2001; Sekerel & Akpinarli 2003).

inhaled corticosteroids inhibit the late response almost completely and some authors have also shown partial inhibition of the early response (Burge et al. 1982; Cockcroft & Murdock 1987). In addition, there is a reduction in bronchial hyperreactivity after inhaled allergen challenge (Kraan et al. 1985; Cockcroft & Murdock 1987). Even when inhaled beclomethasone dipropionate was administered after the early asthmatic response caused by inhaled allergen challenge, the LAR was attenuated (Cockcroft et al. 1993; Paggiaro et al. 1994). Inhaled beclomethasone dipropionate given for a week prior to allergen challenge was able to reduce the allergen-induced eosinophil accumulation and activation (Gauvreau et al. 1996; Kelly et al. 2000; Parameswaran et al. 2000), and certain inhaled steroid formulations have been shown to reduce sputum expression of IL-4 and IL-5 after allergen challenge (Hauber et al. 2006). Similarly, short-term local steroid application has a greater effect on the allergen-induced late nasal reaction as compared with the early reaction (Pelikan 1982). However, with pretreatment for 7 days, the early as well as the late nasal reaction is reduced, with a reduction in histamine and kinins in nasal lavage fluid (Pipkorn et al. 1987a). Six weeks treatment with topical steroids prior to allergen challenge showed a reduction in T cell as well as eosinophil accumulation (Rak et al. 1994). Cytokine and chemokine levels in nasal mucosa are also reduced after treatment with topical steroids (Kleinjan et al. 2000; Erin et al. 2005). Systemic treatment with steroids prior to allergen challenge inhibited eosinophil influx and the release of eosinophil granule proteins, but had no effect on T cells (Bascom et al. 1988, 1989). In contrast, a single dose of oral corticosteroid has been shown to significantly reduce the size of the allergen-induced CLPR, and also the infiltration of inflammatory cells such as T cells and eosinophils in the absence of any effect on the EPR (Charlesworth et al. 1991; Varney et al. 1992b). Prednisolone has also been found to ablate the secondary rise of histamine seen during allergen-induced late-phase reactions, and reduce levels of LTC4 (Charlesworth et al. 1989). Prolonged corticosteroid use led to a reduction in allergen-induced early phase skin reactions and also a reduction in the number of skin mast cells (Pipkorn et al. 1989).

Cyclosporin A Evidence for a T-cell component to the LAR was provided by Sihra et al. (1997), who showed that a single dose of cyclosporin A inhibited the late, but not the early, reaction in mild asthmatics after allergen challenge. This supported the view that T-cell products may be acting independently of mast cell mediators in at least some aspects of the late phase mechanism(s).

Corticosteroids

Anti-IgE

Inhaled corticosteroids are the mainstay of treatment in all but the mildest of asthmatics. Studies have shown that

Two studies have shown that anti-IgE (omalizumab) has a greater inhibiting effect on the LPR than the EPR. Fahy et al.

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Anti-IgE (Omalizumab)

100%

105 95 85 75 65 0

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(1997) showed that anti-IgE, given twice weekly for 3 weeks, suppressed the early and late asthmatic reaction, as well as the number of circulating eosinophils. Inhibition of the LAR was more marked than that of the early asthmatic response, suggesting either mast cell activation leading to downstream inflammatory effects, or nonmast-cell IgE-dependent events involving IgE (Fc) receptors on antigen-presenting cells (Fig. 24.4). Ong et al. (2005) studied 24 atopic allergic volunteers who received omalizumab or placebo for 12 weeks. Paired intradermal challenges of allergen (30 biological units) and diluent control were administered on nine occasions at 2-week intervals. Compared with placebo, omalizumab-treated patients had a progressive reduction in the LPR, which was significantly greater than the effect on the EPR. In addition, significant reduction of the LPR was reached within 2 weeks of commencing treatment, compared with 8 weeks for the EPR. The study raised the possibility that a major effect of IgE in asthma may be related to its role in antigen presentation to T cells.

Immunotherapy Allergen-specific immunotherapy has been shown to reduce LARs in children after 1 year of therapy, without a significant change in the EPR (Warner et al. 1978). Later studies have shown that immunotherapy reduced both the allergeninduced early and late asthmatic reactions, as well as airway hyperreactivity, and abrogated eosinophil influx and eosinophil cationic protein release after inhaled allergen challenge (Arvidsson et al. 2004). The majority of studies have been conducted to assess the effect of immunotherapy with respect to seasonal allergic rhinitis, rather than using models of allergic disease. Successful immunotherapy has been associated with a reduction of eosinophil accumulation into tissues after local allergen provocation. This has been demonstrated in the nose (Furin et al. 1991), together with a decrease in CD4+ T lymphocytes and an increase in IFN-γ (Durham et al. 1996). Allergen-specific immunotherapy has also been shown to cause a reduction in the size of cutaneous allergen-induced LPRs (Pienkowski et al. 1985; Varney et al. 1993). Although

3 4 Time (h)

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Per cent of baseline FEV1

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Fig. 24.4 Effect of a humanized murine monoclonal antibody directed to the FceR1binding domain of human IgE (omalizumab) on allergic airway responses. The effects of 56 days of treatment with anti-IgE in a parallel group, randomized, double-blind, placebocontrolled study of 19 allergic asthmatic subjects was studied. There was partial inhibition of the early reaction and almost total abrogation of the late asthmatic reaction. (From Fahy et al. 1997, with permission.) (See CD-ROM for color version.)

Late-phase Allergic Reactions in Humans

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the mechanisms by which immunotherapy exerts its beneficial effects have still not been fully elucidated, skin biopsies taken during the LPR have shown a reduction in the number of CD4+ T cells and eosinophils. In addition, although the Th2 pattern of cytokines persisted, there was an enhanced expression of Th1 cytokine mRNA (Varney et al. 1993). Similarly, studies that examined the effect of Fel d1-derived peptide immunotherapy have also shown a reduction in the size of the cutaneous and bronchial LPR after local allergen challenge (Oldfield et al. 2002; Alexander et al. 2005a), and this has been associated with an increase in cells with a Th1 cytokine profile (Alexander et al. 2005b), although other studies have shown an increase in serum IL-10 (Oldfield et al. 2002).

Late-phase reaction provoked by allergenderived T-cell peptides A novel method of antigen challenge has been developed that involves administration of allergen-derived T-cell peptide epitopes. It was originally shown that direct T-cell activation through intradermal injection of short peptides derived from the cat allergen Fel d1 could induce an isolated LAR in asthmatic patients with cat allergy (Haselden et al. 1999; Oldfield et al. 2001). These LPRs peaked between 3 and 9 hours after peptide inhalation and had a similar time-course of onset and resolution to LARs induced by whole allergen extract. They are termed “isolated” late reactions as there was no early (immediate) asthmatic reaction, presumably because the peptides were too short to cross-link IgE on mast cells (Fig. 24.5). Like whole antigen, peptides administered by either intradermal injection (Haselden et al. 1999) or by inhalation (Ali et al. 2004), induce LARs in a proportion of atopic asthmatics (responders) but not in others (nonresponders) (Fig. 24.6). Peptides were shown to bind to major histocompatibility complex (MHC) class II molecules, but did not cross-link IgE in a basophil histamine-release assay. The peptide-induced LAR was MHC-restricted in that it only occurred in those individuals with MHC class II able to bind the injected peptides, further supporting a role for T cells in the observed response. Thus, the model has the advantage of

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Early asthmatic reaction

Mast cell

FEV1 (% baseline)

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Fig. 24.6 Characteristics of T-cell peptide responders and nonresponders. Changes in FEV1 after peptide challenge (closed circles) in 12 responders (a) and 12 nonresponders (b), as well as the effect of diluent (open triangles).

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Fig. 24.5 Diagrammatic representation of mechanisms of late asthmatic reactions elicited by (a) whole allergen and (b) allergen-derived peptides. APC, antigen-presenting cell. (See CD-ROM for color version.)

providing information on the T-cell component of allergic airway inflammation, independently of initial mast cell activation. Examples of the different patterns of airway response after challenge with whole allergen or T-cell peptides are shown in Fig. 24.7. T-cell peptide challenge, either by the intradermal route or by inhalation, was not associated with increases in BAL, in a wide range of pharmacologic mediators including histamine, leukotrienes, prostaglandins, and neurotrophins. Also, there were no increases in the numbers of eosinophils, neutrophils, and basophils in the airway wall (Haselden et al. 2001). On the other hand, responders, but not nonresponders, had high expression of the potent vasodilator CGRP (but not neurokinin-A or substance P) in bronchial biopsies and BAL fluid recovered at 6 hours, i.e., when the LAR was well established.

Calcitonin gene-related peptide CGRP is a 37-amino acid peptide resulting from alternative splicing of mRNA from the calcitonin gene (Springer et al. 2003), and is part of the adrenomedullin, calcitonin, amylin family of polypeptides and a potent arterial and venous vasodilator (Brain & Grant 2004). Brain et al. (1985) showed, in experimental animals and humans, that CGRP had a pro-

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longed mode of action when injected into the skin (5–6 hours with as little as 15 pmol). CGRP does not induce permeability per se, but appears to act synergistically with several mediators of inflammation, including histamine, to produce marked and prolonged edema (Brain & Williams 1985). In the airways, in health, CGRP is contained within small sensory nerves, epithelial neuroendocrine cells (NEC), and aggregates of NEC, neuroendocrine bodies. Aoki-Nagase et al. (2002) showed CGRP immunoreactivity throughout the airway epithelium and in the submucosa of mice sensitized and challenged with specific antigen. Furthermore, sensitized CGRP gene-disrupted mice had significant attenuation of both airway hyperreactivity and CGRP expression after antigen challenge. CGRP has also been shown to be synthesized and secreted in vitro by the type II alveolar cell line A549 (Li et al. 2004). There are also many studies showing that the related neuropeptide, substance P, is expressed by several inflammatory cells, including neutrophils and eosinophils (O’Connor et al. 2004). There is controversy regarding the effects of CGRP on bronchial smooth muscle. Earlier claims that it is a potent bronchoconstrictor (Palmer et al. 1987) have not been confirmed and, in any event, this would have been surprising as CGRP increases levels of cAMP in airway smooth muscle cells indicating a relaxing effect. On the other hand, the peptide has been shown to have constrictor effects on damaged (epithelium-denuded) human airways (Springer et al. 2004). To date, there is no convincing evidence that CGRP is overtly expressed in asthma or allergic disease, although previous studies were performed on biopsies from asthmatics obtained at baseline (Howarth et al. 1995; Chanez et al. 1998). Dakhama et al. (2002) suggested that CGRP may have a regulatory role,

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as its administration to sensitized and challenged mice resulted in the normalization of AHR. Nevertheless, these clinical data indicated that changes in the vasculature may be critical in the pathogenesis of late-phase allergic reactions and, if confirmed, will have important implications for understanding mechanisms in bronchial, nasal, and cutaneous late-phase allergic reactions. CGRP immunoreactivity in bronchial biopsies from peptide-challenged asthmatics with LARs were similar to the findings in wild-type mice with positive cells being observed throughout the airway wall, i.e., in epithelial cells, infiltrating inflammatory cells, and airway smooth muscle (Fig. 24.8). Colocalization experiments showed that these were largely CD3+/CD4+ and CD68+ cells (Fig. 24.9). The concept that T cells and macrophages can be recruited in allergic airway disease for the release of the vasoactive peptide CGRP is novel and

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supports the view that vascular leakage may be a component of subacute (i.e., LAR) as well as acute airway narrowing.

T cells and airway hyperresponsiveness It has also been shown that, in asthmatic subjects, selective activation of allergen-specific T cells by inhalation of allergenderived peptides is sufficient to induce increases in nonspecific AHR, a cardinal feature of asthma, which in turn is accompanied by a predominantly T cell, bronchial mucosal inflammatory response. Thus, in responders but not nonresponders, inhalation of peptides produced increased AHR (measured 7 days after peptide provocation), as well as an LAR that peaked at 6 hours after challenge. The elevated numbers of CD3+, CD4+, and TARC+ cells in biopsies supports the view that increased AHR is linked to T-cell activation and was in keeping with previous animal studies. For example, adoptive transfer experiments in Brown Norway rats have shown that AHR can be transferred by allergen-specific CD4+ T cells (Haczku et al. 1977; Mishima et al. 1998). Moreover, in mice, using a combination of antiT-cell monoclonal antibody, T-cell transfer, and bone marrow transplantation, it was shown that T cells enhanced genetically determined AHR (De Sanctis et al. 1977). Also, depletion of murine CD4+ T lymphocytes prevented antigen-induced AHR and pulmonary eosinophilia (Gavett et al. 1995; Hogan et al. 1998).

Peptide-specific T cells

–60 (a)

Late-phase Allergic Reactions in Humans

Time (h)

It was also found that at baseline, i.e., before challenge, the responders had significantly raised serum cat-specific IgE compared with nonresponders. It was previously shown Fig. 24.7 (left) Different patterns of airway response after challenge with whole allergen or T-cell peptides. In approximately 50% of allergic asthmatic patients, inhalation of whole-allergen extract results in a biphasic reduction in FEV1. The early asthmatic reaction occurs within the first hour. The late asthmatic reaction (LAR) is initiated between 2 and 4 hours and peaks at 6–9 hours. Challenge of allergic asthmatic patients with T-cell peptides through either the intradermal route or the inhaled route results in an isolated LAR. (a) Whole cat dander. An asthmatic patient with cat allergy was challenged with either nebulized saline (open circles) or nebulized whole cat dander allergen extract (filled circles) through the inhaled route. In contrast to saline, challenge with allergen resulted in both early asthmatic response and LAR. A reduction of 20% in FEV1 was arbitrarily considered significant to allow for normal variation in airway caliber in an asthmatic subject (dotted line). (b) Intradermal peptides. An asthmatic patient with cat allergy was challenged with either saline or a mixture of overlapping peptides spanning the majority of the Fel d 1 molecule. Administration of saline or peptide was through intradermal injection in the volar aspect of the forearm. In contrast to the saline control, injection of peptides resulted in an isolated LAR. (c) Peptides by inhalation. An asthmatic patient with cat allergy was challenged with either saline or a mixture of overlapping peptides spanning the majority of the Fel d 1 molecule. Administration of saline or peptide was through inhalation of nebulized material (particle size approximately 5 mm). In contrast to challenge with saline, peptides induced an isolated LAR. (From Larche et al. 2003, with permission.)

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

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CD3

Late-phase Allergic Reactions in Humans

CD68

Fig. 24.9 Bronchial biopsies from T-cell peptide challenge showing colocalization of CGRP immunoreactivity to CD3+ and CD4+ T lymphocytes, neutrophils, eosinophils, and CD68+ macrophages. The left panel shows colocalization of CGRP to CD3 cells (indicated by arrows). Single CGRP cells are green (indicated by white arrows) and single CD3+ cells are red (indicated by red arrows). Double-positive (yellow) CGRP/CD68+ cells are shown in the right panel. (From Kay et al. 2007, with permission.) (See CD-ROM for color version.)

that dual responders, i.e., those who develop early and late asthmatic reactions and subsequent increased AHR after whole allergen challenge, tended to have raised allergenspecific IgE compared with single early responders (Cockcroft et al. 1979). This observation may help to predict why some cat-sensitive individuals, and not others, develop an LAR after the Fel d1 peptide challenge. Thus, elevated allergenspecific serum IgE may reflect high IgE density on airway dendritic cells and/or the frequency of allergen-specific T cells in the airway mucosa. This study supported the view that increased AHR occurs after an LAR, irrespective of whether this is induced by whole allergen (and therefore preceded by a mast cell-dependent early response) or is an isolated peptide-induced LAR as in the present study. In both situations, i.e., whole allergen- or peptide-challenge, the subjects who develop an LAR have elevated serum allergen-specific IgE. Thus, T-cell activation, rather than mast cell activation, may Fig. 24.8 (opposite) Photomicrographs of CGRP immunoreactivity in mucosal bronchial biopsies from peptide-induced late asthmatic reactions. (a) Responder challenged with peptides showing CGRP-immunoreactive cells in basal cells along a length of basement membrane. The epithelium is denuded. (b) Responder challenged with diluent showing basal cells and denuded epithelium but no immunoreactivity. (c) Responder challenged with peptide showing numerous immunoreactive cells within a disrupted epithelium together with positive infiltrating cells below the basement membrane, as well as in association with small blood vessels. (d) A nonresponder after peptide challenge. There are occasional CGRPimmunoreactive cells below the basement membrane. The epithelium

be more crucial for the development of increased AHR as previously observed in animal studies (De Sanctis et al. 1977; Haczku et al. 1977; Mishima et al. 1998).

Downstream events The following scenario is proposed to explain the events occurring after peptide challenge. Inhaled T-cell peptide epitopes bind to MHC class II molecules expressed in the airways, leading to activation of peptide-specific resident effector memory T cells. Activation of these cells enhances local production of TARC from antigen-presenting cells, structural cells, and accessory cells. Elevated TARC expression leads to recruitment and activation of CD3+/CD4+ T cells, production of inflammatory cytokines and, ultimately, increased AHR. As described above, the mechanism of the LPR itself may be related to elevated expression of CGRP in epithelial cells, infiltrating submucosal CD3+/CD4+ cells, and in smooth is intact. (e) Responder challenged with peptide showing numerous infiltrating CGRP-positive inflammatory cells below the basement membrane and also immunoreactivity in association with airway smooth muscle. (f) A responder challenged with diluent showing no immunoreactive submucosal inflammatory cells or CGRP staining in association with airway smooth muscle. (g) A confocal micrograph of a responder challenged with peptide showing CGRP-positive epithelial cells and immunoreactive smooth muscle. (h) A nonresponder challenged with peptide showing no smooth muscle or epithelial immunoreactivity. (See CD-ROM for color version.)

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muscle in responders (but not nonresponders) after peptide challenge.

Concluding comments The concept that mast cell products are largely responsible for EPRs in the lung, nose, and skin is uncontroversial. In addition, mast cell degranulation may contribute to the LPR by means of histamine-induced eotaxin upregulation contributing to eosinophil accumulation (Menzies-Gow et al. 2004). In the skin model, stimulation of mast cells with anaphylactogenic IgE is sufficient to cause both early- and late-phase skin reactions (Dolovich et al. 1973), suggesting that mast cells may cause the cutaneous LPR through as yet undetermined mediators. Antigen-presenting cells take up allergen, process it, and present peptides in the context of MHC class II to CD4+ Th2 cells. This is presumed to cause release of the Th2 cytokines IL-4, IL-5, and IL-13. In humans, IL-5 acts selectively on eosinophils and basophils. By associating with its receptor, IL-5 effects eosinophil growth and differentiation (in particular the terminal differentiation of committed eosinophil precursors) (Yamaguchi et al. 1988; Clutterbuck et al. 1989; Lopez et al. 1992), migration (Warringa et al. 1992), activation and effector function (Kita et al. 1992; Carlson et al. 1993), and survival (Yamaguchi et al. 1991). In allergic disease, eosinophils migrate to sites in a relatively select fashion with up to 100-fold enrichment of eosinophils over neutrophils in tissues. This is a multistep process directed by Th2-cytokine producing T cells (Wardlaw 1999). The first step involves increased production and release of eosinophils from the bone marrow under the influence of IL-5, acting synergistically with specific chemoattractants such as eotaxin (Clutterbuck et al. 1989; Palframan et al. 1998). Next, the target organ vasculature displays increased adhesiveness for eosinophils. The Th2 cytokines IL-4 and IL-13 induce expression of the adhesion molecule VCAM-1 on endothelium during allergic inflammation, which binds eosinophils through very late antigen (VLA)-4, a receptor not expressed on neutrophils, and P-selectin to which eosinophils bind with greater avidity than neutrophils (Symon et al. 1996; Edwards et al. 2000; Woltmann et al. 2000). In addition, IL-4 and IL-13 contribute towards perpetuating IgE production, working in conjunction with CD40 and CD40 ligand. IL-13, in addition, contributes to bronchial hyperresponsiveness. The association between eosinophils and allergy is well established. Local tissue eosinophilia has been described in asthma, atopic rhinitis, and atopic dermatitis. Leukotrienes, which can be produced by eosinophils as well as eosinophil granule products, are found during LPRs in all three tissues after allergen challenge, suggesting that eosinophils actively secrete mediators (Naclerio et al. 1985; Andersson et al. 1989b; Diaz et al. 1989; Reshef et al. 1989; Wardlaw et al. 1989;

538

Zweiman et al. 1991). Disease severity in asthmatics correlated with the number and state of activation of eosinophils in the bronchial mucosa and BAL fluid (Wardlaw et al. 1988). Thus, the general belief over the last 10–15 years has been that the LPR is largely due to the release of eosinophil products. However, as stated above, studies with mepolizumab, a humanized monoclonal anti-IL-5 antibody, produced no appreciable effects on the LAR, AHR, or other clinical outcomes including lung function (Leckie et al. 2000; Flood-Page et al. 2003a). Anti-IL-5 also had no effect on the late-phase skin reaction (Phipps et al. 2004). There is currently an interest in the concept that eosinophils play a role in repair and remodeling processes (Levi-Schaffer et al. 1999) and, indeed, in both the lung and skin models, reduction of eosinophils led to a decrease in markers of airway and skin remodeling (FloodPage et al. 2003b; Phipps et al. 2004). Moreover, Kariyawasam et al. (2007) found that in dual responders with asthma the 24-hour increase in airway wall cellular inflammation, including tissue eosinophilia, resolved by 7 days, whereas the increases in AHR and markers of remodeling persisted. Thus, persistent inflammation does not appear to be necessary for ongoing remodeling and AHR. A further difference between the tissues may lie in T-cell activation and function. Again, as described above, studies using allergen-derived peptides have shown that inhalation of Fel d1-derived peptides by cat allergic asthmatics resulted in isolated LARs (Ali et al. 2004). It has also been shown that intradermal administration of these peptides cause distant LARs in the absence of cutaneous LPRs (Haselden et al. 1999), suggesting that either the T cells in the skin are not able to respond to the peptides or that the mechanisms that cause the LAR are not able to manifest themselves in skin. For example, if the peptide-induced LAR was due to smooth muscle contraction, there would be a lack of response in the skin even if mediators responsible for the LAR were released in the skin. Furthermore, Frew and Kay (1988b) had previously suggested that the skin might lack “activated” T cells in contrast to the airways of asthmatics (Robinson et al. 1993b). It is possible that “inactive” T cells in the skin are unable to generate a response when exposed to the Fel d1-derived peptides. Furthermore, repeat allergen challenges result in increased T-cell accumulation without causing an increase in the size of the allergen-induced LPR (Ong et al. 2005). Taken together with previous Fel d1 peptide studies, this suggests that LARs may have a predominant T-cell component, whereas, in the skin, late reactions are largely mast cellmediated. The common denominator is vascular permeability and vascular leakage, which presumably involve several mediators. This hypothesis is diagrammatically represented in Fig. 24.10. These speculations are in keeping with a number of studies on the role of the vasculature in asthma. These include the demonstration of increases in angiogenic factors, such as VEGF and angiogenin (Hoshino et al. 2001), as well as stromal cell-derived factor 1 in asthmatic airway epithelium

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Ag

Early-phase reaction

MC

(Prominent in late skin reactions) Vascular permeability factors APC

Late-phase reaction

(Prominent in late asthmatic reactions) CD4+ Th2 Eos

Remodeling

IL-5 Bone marrow Eotaxin Fig. 24.10 Hypothesis for mechanisms of late-phase allergic reactions in the skin and airways. It is proposed that eosinophils (Eos), although mobilized by antigen, play a role in downstream events (i.e., remodeling and repair) rather than contribute directly to tissue swelling. Mast cell (MC) permeability factors are prominent in skin late-phase reactions, whereas T-cell permeability factors play a major role in late asthmatic reactions. The role of airway smooth muscle in late asthmatic reactions is unclear. Airway wall edema may be sufficient to account for narrowing. APC, antigen-presenting cell. (See CD-ROM for color version.)

(Hoshino et al. 2003). Furthermore, increases in subepithelial vessels of the airways were observed in newly diagnosed asthmatics, but not in COPD patients or normal control subjects using a high magnification bronchovideoscope (Tanaka et al. 2003). The role of airway smooth muscle in LARs is unclear. Airway wall edema may be sufficient to account for narrowing. Of interest was the observation that inhalation of the loop diuretic furosemide had a protective effect on the allergen-induced early and late asthmatic reaction (Bianco et al. 1989). Therefore, there may be differences in emphasis of the prominent cell type involved in the formation of the LPR in the airways as compared with the skin. Finally, it is important to emphasize that the common feature of late-phase allergic reactions, whatever the organ, is tissue swelling and edema. Increases in our knowledge of mechanisms of vascular permeability and leakage in the context of allergic tissue reactions may well hold the key to understanding the final common pathways involved in this useful model of allergic inflammation.

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Antihistamines F. Estelle R. Simons and Keith J. Simons

Summary In this chapter, the important role played by histamine in human health, in immunomodulation, and in allergic inflammation is reviewed. The four currently known types of histamine receptors are compared and contrasted. The main focus of the chapter is on H1-antihistamines, which are among the most commonly used medications in the world. The molecular basis for H1-antihistamine action as inverse agonists, rather than antagonists or blockers, is described. The first-generation, potentially sedating H1-antihistamines, which have never been optimally investigated in humans, are discussed briefly. The therapeutically more important and well-investigated secondgeneration, nonsedating H1-antihistamines are discussed more extensively. Clinically relevant differences in the pharmacologic profiles of H1-antihistamines are reviewed, with emphasis on their onset of action, potency, and duration of action. H1-antihistamines play an important role in the treatment of allergic rhinitis, allergic conjunctivitis, and urticaria, and their efficacy as well as their limitations in these disorders is described. In contrast to the first-generation H1-antihistamines, the second-generation H1-antihistamines are relatively free from adverse effects, including central nervous system adverse effects and cardiac toxicity, not only when administered in usual doses but also if taken in overdose. The second-generation H1-antihistamines are therefore medications of choice. Use of H1-antihistamines in special populations such as infants, young children, pregnant women, and the elderly is discussed. Clinically advantageous H1-antihistamines developed with the aid of molecular techniques might be available in the future.

Histamine and histamine receptors Histamine is an important natural body constituent that is expressed in central nervous system (CNS) neurons, gastric mucosa parietal cells, mast cells, and basophils, and exerts its

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

diverse biological effects through four or more types of histamine receptors. H1, H2, H3, and H4 receptors differ in their expression, signal transduction, and function (Table 25.1). All types of histamine receptors are heptahelical transmembrane molecules that transduce extracellular signals through G proteins to intracellular second-messenger systems. The four different types of histamine receptors have distinct intracellular signals. In addition, differences in the affinities of these receptors for histamine are highly decisive for the biological effects of histamine and for drugs that target histamine receptors (Leurs et al. 2002; de Esch et al. 2005; Jongejan et al. 2005; Bongers et al. 2007). All known types of histamine receptors have constitutive activity, which is defined as the ability to trigger downstream events in the absence of ligand binding (Fig. 25.1). The active and inactive states of the receptors exist in equilibrium. Agonists stimulate the active conformation, and inverse agonists (historically termed histamine antagonists) stimulate the inactive conformation. H1 and H2 receptors are widely expressed in the body, in contrast to H3 and H4 receptors. H1-receptor polymorphisms have been described, but not yet optimally studied. In humans, H1 receptors have approximately 45% homology with muscarinic receptors (Leurs et al. 2002). Through the H1 receptor, histamine is involved in cell proliferation and differentiation, hematopoiesis, embryonic development, regeneration, and wound healing. In the CNS, histamine is produced in neurons with cell bodies in the tuberomamillary nucleus of the posterior hypothalamus, from which axons project to the frontal and temporal cortices and other regions of the brain. In this phlyogenetically old neurotransmitter system, histamine is involved in the regulation of basic body functions such as energy and endocrine homeostasis, sleeping, waking, cognition, memory, and anticonvulsant activity (Schneider et al. 2002; Haas & Panula 2003). Targeted disruption of H1 receptors in a murine model results primarily in the impairment of neurologic function such as learning, memory, and locomotion, and in aggressive behavior, although some immunologic abnormalities, including impaired antigen-specific responses of T cells and B cells also occur (Toyota et al. 2002).

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Table 25.1(a) Histamine receptors.

H1 receptor

Year described/

Receptor

Chromosomal

year human

proteins,

localization,

gene cloned

humans

humans

Receptor expression

coupling

Activated intracellular signals

1966/1993

487 amino

3p25, 3p14–p21

Nerve cells, airway and vascular smooth

Gaq/11

Ca2+, protein kinase C, PLC, cGMP,

G protein

PLD, PLA2, NF-kB

muscle, endothelial cells, epithelial cells,

acids, 56 kDa

neutrophils, eosinophils, monocytes/ macrophages, DCs, T and B cells, hepatocytes, chondrocytes H2 receptor

1972/1991

359 amino

5q35.3

Gas

Nerve cells, airway and vascular smooth

Adenylate cyclase, cAMP, c-Fos, cJun, protein kinase C, P70S6K

muscle, endothelial cells, epithelial cells,

acids, 40 kDa

neutrophils, eosinophils, monocytes/ macrophages, DCs, T and B cells, hepatocytes, chondrocytes H3 receptor

1983/1999

445 amino

20q13.33

High expression in histaminergic neurons,

Gi/o

Inhibition of adenocyclase, activation

acids, 70 kDa;

eosinophils, DCs, monocytes, low expression

of PLA2, modulation of the MAPK

splice variants

in peripheral tissues

pathway, activation of the Akt/GSK3b axis, modulation of intracellular Ca2+, and inhibition of Na+/H+ exchanger activity

H4 receptor

1994/2000

390 amino

18q11.2

acids, kDa n/a

Gi/os

High expression on bone marrow and

Enhanced Ca2+, inhibition of cAMP

peripheral hematopoietic cells, neutrophils, eosinophils, DCs, T cells, basophils, mast cells

Table 25.1(b) Histamine receptors (contd). H1 receptor

H2 receptor

H3 receptor

H4 receptor

Histamine

↑ pruritus, ↑ pain, ↑ vasodilation

↑ gastric acid secretion

Prevents excessive

Differentiation of

function,

↑ vascular permeability

↑ vascular permeability

bronchoconstriction

myeloblasts and

general

↑ hypotension, flushing, headache, tachycardia,

↑ hypotension, flushing, headache,

Mediates pruritus (no mast

promyelocytes

bronchoconstriction, stimulation of airway vagal

tachycardia

cell involvement)

Mediates pruritus (no

afferent nerves and cough receptors

↑ chronotropic and ↑ inotropic activity,

Nasal congestion

mast cell involvement)

↓ atrioventricular node conduction time

bronchodilation

Nasal congestion

↑ mucus production (airway) Histamine

↑ release of histamine and other mediators

↓ eosinophil and neutrophil chemotaxis

Probably involved in control

↑ calcium flux in human

function

↑ cell adhesion molecule expression and

↓ IL-12 by dendritic cells

of neurogenic inflammation

eosinophils

in allergic

chemotaxis of eosinophils and neutrophils

↑ IL-10 and development of Th2 or

through local neuron–mast

↑ eosinophil chemotaxis

inflammation

↑ APC capacity, costimulatory activity on B cells

tolerance-inducing DCs

cell feedback loops

↑ IL-16 production

and immune

↑ cellular immunity (Th1)

↑ humoral immunity

↑ proinflammatory activity

(H2 receptor also involved)

modulation

↑ IFN-g, autoimmunity

↓ cellular immunity

↑ APC capacity

↓ humoral immunity and IgE production

suppresses Th2 cells and cytokines indirect role in allergy, autoimmunity, malignancy, graft rejection Presynaptic heteroreceptor

Histamine

Sleep/wakefulness, food intake, thermal

function

regulation

↓ histamine, dopamine,

in the CNS

Emotions/aggressive behavior, locomotion,

serotonin, norepinephrine,

memory, learning

and acetylcholine release

Neuroendocrine

To be defined

Inverse agonists

> 40, including cetirizine, desloratadine,

Cimetidine, famotidine, nizatidine, and

Medications in development

Medications in

(formerly called

fexofenadine, levocetirizine, and loratadine for

ranitidine for peptic ulcer and related

for treatment of narcolepsy,

development for allergic

antagonists)

allergic rhinoconjunctivitis and urticaria

disorders

dementia, schizophrenia,

rhinitis treatment

and other CNS disorders APC, antigen-presenting cell; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DCs, dendritic cells; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase; n/a, information not available; NF-kB, nuclear factor kB; NOS, nitric oxide synthase; PL, phospholipase. The information about histamine receptors and their inverse agonists is based on in vitro studies, studies in animal models (H1, H2, H3, and H4 receptor-deficient mice, H1/H2receptor-deficient mice and histidine decarboxylase-deficient mice), and on studies in humans. Peripheral and central H1 receptors do not differ, although isoforms of H1 receptors, including species isoforms, exist. Receptor binding varies depending on the system used to study it and the inverse agonist being investigated; for example, in the Chinese hamster ovary cell membrane model, the Ki (nmol/L) of second-generation H1-antihistamines in current use ranges from 0.87 (desloratadine) to 175 (fexofenadine).

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Extracellular

Extracellular

Extracellular

Effect of agonist (histamine)

Inactive

Effect of inverse agonist (antihistamine)

Inactive

Inactive

Active (a)

Intracellular

Active (b)

Antihistamines

Intracellular

Fig. 25.1 The histamine H1 receptor: a simplified two-state model. (a) The inactive state of the histamine H1 receptor is in equilibrium with the active state. (b) An agonist has a preferential affinity for the active state and stabilizes the H1 receptor in this conformation, consequently causing a shift in the equilibrium toward the active state. (c) An inverse agonist such as an H1-antihistamine has a preferential affinity for the inactive state,

Active (c)

stabilizes the receptor in this conformation, and causes a shift in the equilibrium toward the inactive state. All H1-antihistamines described to date function as inverse agonists. Intracellular and extracellular are defined in relation to the cell membrane. (From Leurs et al. 2002, with permission.) (See CD-ROM for color version.)

Histamine and immunomodulation Histamine affects chronic inflammation through at least four distinct receptor types on macrophages, dendritic cells, T lymphocytes, B lymphocytes, epithelial cells, endothelial cells, and other cells such as eosinophils, basophils, fibroblasts, and keratinocytes (Traidl-Hoffmann et al. 2006). In this complex system, the expression of the receptor types is influenced by the microenvironment, and differs according to the stage of cell differentiation (Triggiani et al. 2007). Histamine regulates a number of essential events in the immune response, including cell proliferation, cytokine production, and expression of cell adhesion molecules and major histocompatibility complex (MHC) class II antigens (Jutel et al. 2006). Through the H1 receptor and the H3 receptor, histamine induces proinflammatory activity and increased antigenpresenting cell capacity. Through the H2 receptor, it plays a suppressive role on monocytes and monocyte-derived dendritic cells. Th1 cells show predominant, but not exclusive, expression of the histamine H1 receptor, and histamine induces increased proliferation of Th1 cells, as well as interferon-γ production. On Th2 cells, the expression of the histamine H2 receptor predominates and, through it, histamine acts as a negative regulator of proliferation, and of interleukin (IL)-4 and IL-13 production. Histamine enhances Th1-type responses through the histamine H1 receptor, whereas both Th1- and Th2-type responses are negatively regulated through the histamine H2 receptor. These distinct effects suggest roles for the histamine H1 receptor and the histamine H2 receptor on T cells for autoimmunity and for peripheral tolerance, respectively (Akdis & Simons 2006) (Fig. 25.2).

Intracellular

HR2 Induction of IL-10 Suppression of IL-12 Th2 or tolerance Inducing DC

High HR2 Low HR1 Suppressed Th2 cytokines tolerance

Th2

HR2: induction of humoral immunity and suppression of cellular immunity. HR2-deficient mice show suppressed specific IgE

Monocyte dendritic cell HR1-4 DC

HR1/HR3 proinflammatory activity, increased APC capacity

Histamine

Th1

High HR1 Low HR2 Increased IFN-g autoimmunity

B cell

HR1: blocking of humoral immunity and induction of cellular immunity. HR1-deficient mice show increased specific IgE

Fig. 25.2 Histamine plays an important role in allergic inflammation. In lymphatic organs and subepithelial tissues, it regulates monocytes and dendritic cells that express all four known subtypes of histamine receptors, and T cells which express histamine H1 receptors and histamine H2 receptors, as well as B cells and other cells. APC, anti-presenting cell; DC, dendritic cell. (From Akdis & Simons 2006, with permission.) (See CD-ROM for color version.)

Histamine also modulates antibody production, as a costimulatory receptor on B cells. Histamine H1 receptors predominantly expressed on Th1 cells may block humoral immune responses by enhancing the Th1 cytokine response. In contrast, histamine enhances humoral immune responses through the histamine H2 receptor. Allergen-specific IgE production

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is differentially regulated in histamine H1 receptor and histamine H2 receptor-deficient mice. Histamine H1 receptordeleted mice show increased allergen-specific IgE production, in contrast to histamine H2 receptor-deleted mice, which show suppressed IgE production. Bphs, a non-MHC-linked gene involved in the susceptibility to many autoimmune diseases, has been identified as the histamine H1-receptor gene in mice (Jutel et al. 2006).

Histamine in allergic inflammation Histamine plays a key role in the intricate network of cellular events described as allergic inflammation (MacGlashan 2003). Along with preformed mediators of inflammation such as tryptase, as well as newly generated leukotrienes, prostaglandins, and other mediators, it is released from the granules of mast cells and basophils after cross-linking of surface high-affinity IgE receptors by allergen. Less commonly, it is released from these cells through mechanisms that are independent of IgE and the IgE receptor. Although histamine is less potent than some other mediators such as leukotrienes, after allergen challenge it is found locally in relatively large microgram quantities per one million cells. In contrast, leukotrienes are found in picogram quantities per one million cells. In the early allergic response, most of the effects of histamine occur through H1 receptors; these include itching and sneezing due to neural reflexes, edema due to vasodilation and increased permeability of the vascular endothelium, and bronchoconstriction due to smooth muscle contraction. In addition to its role in the early allergic response, histamine plays an important role in the late allergic response by activating various signal cascades, including NF-κB, followed by the production of proinflammatory cytokines such as IL-4 and IL-13, the expression of cell adhesion molecules and class II antigens, and chemotaxis of eosinophils and other cells. The clinical significance of the complex interrelationships of histamine, its receptors, and other G-protein coupled receptors remains to be elucidated. In anaphylaxis, some symptoms such as hypotension, tachycardia, flushing, and headache occur through both H1 and H2 receptors in the vasculature. Cutaneous itching in atopic dermatitis and nasal congestion in allergic rhinitis may occur through H3 or H4 receptors, as well as through H1 receptors. There are also some hints that H1-antihistamines subtly modulate the allergic response by affecting the balance between Th1, Th2, and Treg cells when suppressing the inflammatory cell accumulation. For example, in humans, H1-antihistamine premedication during the initial dose escalation phase of allergen-specific immunotherapy may improve the longterm efficacy of treatment (Muller et al. 2001; Golightly & Greos 2005; Bryce et al. 2006; Caproni et al. 2006; Petecchia et al. 2006).

554

Clinical pharmacology During the past decade, understanding of the molecular mechanisms by which H1-antihistamines interact with the histamine H1 receptor has increased greatly. It is now known that they do not “block” these receptors. Rather, all H1antihistamines in use function as inverse agonists that have a preferential affinity for the inactive state of histamine H1 receptors. They stabilize the receptors in this conformation, with a shift in equilibrium toward the inactive state and consequent downregulation of acute and chronic inflammation (Leurs et al. 2002). Traditionally, H1-antihistamines have been classified into six chemical groups: the ethanolamines, ethylenediamines, alkylamines, piperazines, piperidines, and phenothiazines. Now, the more commonly used classification is a functional one in which H1-antihistamines are divided into firstgeneration, potentially sedating agents and second-generation, relatively nonsedating H1-antihistamines (Simons 2003). The term third-generation has been used to market some newer H1-antihistamines; however, this designation should be reserved for clinically advantageous H1-antihistamines designed with the use of molecular techniques, and no such H1-antihistamines are currently available (Holgate et al. 2003). Many second-generation H1-antihistamines have been identified by screening and structural modification of preexisting medications in the class. For example, acrivastine is structurally similar to triprolidine; cetirizine is a metabolite of hydroxyzine; levocetirizine is the active R-enantiomer of cetirizine; desloratadine is a metabolite of loratadine; and fexofenadine is a metabolite of terfenadine (Simons 2003; Hair & Scott 2006). New H1-antihistamines continue to be developed and introduced for clinical use (Corcostegui et al. 2006; Keam & Plosker 2007).

Pharmacokinetics Pharmacokinetics is defined as the study of the absorption, distribution, metabolism, and elimination of medications. The pharmacokinetics of the first-generation H1-antihistamines have never been optimally investigated, even in healthy adults or adults with allergic diseases, let alone in special groups such as children, the elderly, or patients with hepatic or renal dysfunction. Clearance rates and terminal elimination half-life values are known for a few of these medications, but not for all of them. Although all the first-generation H1antihistamines are metabolized by the hepatic cytochrome P450 (CYP450) system, and interactions with other drugs, foods, or herbal products potentially occur, few interaction studies of these medications have been performed (Table 25.2). In contrast, the pharmacokinetics of most of the secondgeneration H1-antihistamines have been extensively studied. After oral administration, peak plasma concentrations of

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Table 25.2(a) Pharmacokinetics and pharmacodynamics of oral H1-antihistamines differ in healthy young adults.

H1-antihistamine (metabolite)

tmax (hours) after single dose§

Terminal elimination half-life (hours)

Elimination unchanged in the urine/ feces (%)

Clinically relevant drug/drug interactions*

Onset/ duration of action† (hours)

First-generation Chlorpheniramine‡

2.8 ± 0.8

27.9 ± 8.7

n/a

Possible

3/24

Diphenhydramine‡

1.7 ± 1.0

9.2 ± 2.5

n/a

Possible

2/12

Doxepin‡ Hydroxyzine‡

2 2.1 ± 0.4

13 20.0 ± 4.1

n/a n/a

Possible Possible

n/a 2/24

Second-generation Acrivastine Cetirizine Desloratadine Ebastine (carebastine) Fexofenadine*

1.4 ± 0.4 1.0 ± 0.5 1–3 (2.6–5.7) 2.6

1.4–3.1 6.5–10 27 (10.3–19.3) 14.4

59/0 60/10 0 (75–95)/0 12/80

Unlikely Unlikely Unlikely n/a Unlikely

1/8 1/≥ 24 2/≥ 24 2/≥ 24 2/24

Levocetirizine Loratadine (descarboethoxyloratadine) Mizolastine Rupatadine

0.8 ± 0.5 1.2 ± 0.3 (1.5 ± 0.7) 1.5 0.75–1.0

7 ± 1.5 7.8 ± 4.2 (24 ± 9.8) 12.9 6 (4.3–13.0)

86/13 trace

Unlikely Unlikely

0.5/0 n/a

n/a Proven

Usual adult dose

4 mg t.i.d.–q.i.d. 12 mg (SR) b.i.d.† 25–50 mg t.i.d.–q.i.d. or h.s.† 25–50 mg t.i.d. or h.s.† 25–50 mg t.i.d. or h.s.†

Population in which dose adjustment may be required

G G, H H H

n/a G, R, H R, H R, H R

1/> 24 2/24

8 mg t.i.d. 5–10 mg daily 5 mg daily 10–20 mg daily 60 mg b.i.d. or 120 mg or 180 mg daily 5 mg daily 10 mg daily

1/24 2/24

10 mg daily 10 mg daily

n/a G, R, H

R, H H

The legend for Table 25.2(a) and Table 25.2(b) appears on p. 556.

these medications are reached within 1–2 hours. Terminal elimination half-life values range from about 2 hours for acrivastine to 27 hours for desloratadine. Protein binding of the second-generation H1-antihistamines ranges from 60% (fexofenadine) to 99% (levocetirizine). Cetirizine and levocetirizine are eliminated largely unchanged in the urine, and fexofenadine is eliminated mostly unchanged in the feces (Simons 2003, 2004; Molimard et al. 2004; Golightly & Greos 2005; Hair & Scott 2006; Keam & Plosker 2007). The pharmacokinetics of many of the second-generation H1-antihistamines have been studied in special groups such as children, the elderly, and patients with hepatic or renal dysfunction. For example, in very young children, traditional pharmacokinetic studies and population pharmacokinetic studies have been performed for cetirizine, desloratadine, levocetirizine, and loratadine (Salmun et al. 2000; Simons 2003; Cranswick et al. 2005; Simons & Simons 2005; Simons et al. 2005; Gupta 2007; Gupta et al. 2007). Clinically relevant pharmacokinetic interactions between second-generation H1-antihistamines and other drugs, foods, or herbal products, are few (Zhou et al. 2004; Bressler 2006; Prenner et al. 2006). For example, although some individuals have decreased ability to convert desloratadine to 3-hydroxy-desloratadine, the major metabolite, clinically relevant drug accumulation does not occur. It is recommended, however, that fexofen-

adine not be administered within 15 min after the ingestion of antacids that contain aluminum and magnesium, which potentially decrease its absorption (Golightly & Greos 2005). Rupatadine is metabolized by the hepatic cytochrome P450 system, and significant drug interactions between rupatadine and agents such as ketoconazole, erythromycin, and grapefruit juice that inhibit CYP3A4 activity have been reported (Picado 2006; Keam & Plosker 2007).

Pharmacodynamics Pharmacodynamics involves the study of the onset, amount, and duration of action of medications in relationship to their plasma concentrations. The pharmacodynamics of H1-antihistamines are readily studied using inhibition of the histamine-induced wheal and flare (erythema), a standardized biological assay of peripheral H1-activity. H1-antihistamines decrease wheal size by decreasing the vascular permeability and leakage of plasma proteins. They decrease flare (erythema) size by decreasing vasodilation caused by the histamineinduced axon reflex. Less commonly, suppression of the allergen-, compound 48/80-, or codeine-induced wheals and flares (erythema) is used in this bioassay. There are few pharmacodynamic studies of the firstgeneration H1-antihistamines. In contrast, the pharmacodynamics of most of the second-generation H1-antihistamines

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Table 25.2(b) Pharmacokinetics and pharmacodynamics of H1-antihistamines for intranasal/ophthalmic use.

H1-antihistamine (metabolite)

tmax (hours) after single dose§

Topical intranasal/ophthalmic Azelastine** 5.3 ± 1.6 (desmethylazelastine) (20.5)

Terminal elimination half-life (hours)

Elimination unchanged in the urine/ feces (%)

Clinically relevant drug/drug interactions

Onset/ duration of action¶ (hours)

22–27.6 (54 ± 15)

2(3)/0

No

0.5/12

0.137 mg/nasal spray, 1–2 sprays b.i.d.; 0.5% ophthalmic solution, 1 drop b.i.d.

n/a

Usual adult dose§

Population in which dose adjustment may be required

Emedastine**

1.4 ± 0.5

7

6.7/n/a

No

0.25/12

0.05% ophthalmic solution, 1 drop q.i.d.

n/a

Epinastine**

2–3

6.5

25/70

No

0.1/12

0.05% ophthalmic solution, 1 drop b.i.d.

n/a

Ketotifen**

2–4

20–22

60–70/ 30–40

No

0.25/12

0.025% ophthalmic solution, 1 drop b.i.d. (q8–12h)

n/a

Levocabastine**

1–2

35–40

65–70/20

No

0.25/12

50 mg/nasal spray 2 sprays b.i.d.; or 0.05% ophthalmic solution, 1 drop t.i.d.–q.i.d.

n/a

Olopatadine**

0.5–2

7.1–9.4

59–73/n/a

No

0.25/12

0.1% ophthalmic solution, 1 drop b.i.d. (q6–8h); 0.2% ophthalmic solution, 1 drop once daily

n/a

Results are expressed as mean ± standard deviation, unless otherwise indicated. b.i.d. twice daily; G, geriatric; H, hepatic impairment; n/a, information not available or incomplete; q.i.d., four times daily; R, renal impairment; t.i.d., three times daily. * Drug interactions: fexofenadine should not be administered within 15 min of ingestion of aluminum- and magnesium-containing antacids, which decrease its absorption; although grapefruit juice and rifampin may also decrease its absorption, and erythromycin, ketoconazole, and verapamil may increase its absorption, fexofenadine has a very wide benefit-to-risk ratio and these effects are unlikely to have clinical significance; nevertheless, effectiveness should be monitored. † Onset/duration of action is based on wheal and flare studies. ‡ Five or six decades ago when many of the first-generation H1-antihistamines were introduced, pharmacokinetic and pharmacodynamic studies were not required by regulatory agencies. They have subsequently been performed for some of these drugs. Empirical dosage regimens persist; for example, the manufacturers’ recommended diphenhydramine dose for allergic rhinitis is 25–50 mg q4–6h and the diphenhydramine dose for insomnia is 25–50 mg at bedtime. The use of sustained-action formulations persists despite the long terminal elimination half-life values identified for medications such as chlorpheniramine. § Time from oral intake to peak plasma concentration. ¶ Intranasal and ophthalmic H1-antihistamines: onset and duration of action, and usual adult dose refers to topical application; for nasal use, the doses refer to sprays in each nostril, and for ophthalmic use the doses refer to one drop in each eye. ** Intranasal and ophthalmic H1-antihistamines: pharmacokinetic parameters after oral administration.

have been well studied (Murdoch et al. 2003; Simons 2003, 2004; Curran et al. 2004; Golightly & Greos 2005; Hair & Scott 2006; Keam & Plosker 2007). In healthy and allergic individuals, all H1-antihistamines inhibit the histamine-induced wheal and flare to some extent. The magnitude of the effect, the time to peak effect, and the duration of the effect are medication and dose-related (Popov et al. 2006). For a few

556

H1-antihistamines such as cetirizine and fexofenadine, wheal and flare (erythema) suppression has been shown to correlate with H1-antihistamine concentrations in the skin (Simons et al. 2002). For other H1 receptors such as levocetirizine, suppression has been shown to correlate with H1-receptor occupancy by free (unbound) H1-antihistamine (Simons KJ et al. 2007).

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The onset of action ranges from about 1 hour for cetirizine and levocetirizine to about 2 hours for fexofenadine, desloratadine, and loratadine. The duration of action for most of the orally administered second-generation H1-antihistamines is at least 24 hours, facilitating once-daily dosing. Tolerance to the suppressive effect on skin test reactivity to allergens does not occur, as documented in rigorously controlled, double-blind studies of several months duration. After discontinuing a second-generation H1-antihistamine that has been taken regularly for 1 week or longer, residual suppression of skin test reactivity to histamine and to allergens varies with the H1-antihistamine (Simons 2003, 2004). Available evidence suggests that residual effects of a short course of loratadine or desloratadine last for 1 day, those of fexofenadine last for 2 days, and those of cetirizine and levocetirizine last for 3– 4 days. Some systemic absorption of intranasal and ophthalmic H1-antihistamines such as azelastine, emedastine, epinastine, levocabastine, and olopatadine occurs, and may be associated with transient suppression of skin-test reactivity. Regardless of the terminal elimination half-life of these medications, which ranges from about 7 hours (emedastine, epinastine, olopatadine) to 40 (levocabastine) hours, they are administered at 6–12 hour intervals because of washout from the nasal mucosa or conjunctivae. No dose adjustments are required in special populations (Bielory et al. 2005). In individuals with allergic rhinoconjunctivitis, pharmacodynamic studies of orally or topically active H1-antihistamines, with a focus on onset of action, can be conducted using nasal or ophthalmic challenge tests. Challenges with histamine or allergen can be administered on an individual basis, or groups of individuals can be challenged with allergen, either in a purpose-built chamber or room (Day et al. 2006; Horak et al. 2006; Stuebner et al. 2006), or in an outdoor setting when ambient seasonal pollen counts are high. Doses and dose intervals for the second-generation H1antihistamines are therefore based on objective information obtained from pharmacokinetic and pharmacodynamic studies. Moreover, modified dose regimens have been defined, where required, in infants, children, elderly persons, patients with hepatic or renal dysfunction, or those taking other medications concurrently (Table 25.2) (Simons 2003; Golightly & Greos 2005; Hair & Scott 2006). For some H1-antihistamines, such as cetirizine and levocetirizine, wheal and flare inhibition correlates with relief of symptoms of allergic rhinitis, beginning at 1 hour and peaking at 5–7 hours after the dose, several hours after the peak plasma concentration. H1-antihistamine effects persist even when plasma concentrations become undetectable, probably due to high plasma/tissue concentration ratios, as determined by measurement of H1-antihistamine concentrations in skin biopsy specimens, and for some medications, the presence of active metabolites (Simons et al. 2002). For other H1antihistamines, such as loratadine and desloratadine, the

Antihistamines

pharmacodynamics of the wheal and flare (erythema) response do not seem to predict efficacy in allergic rhinitis. In this model, desloratadine 10 mg and levocetirizine 1.25 mg provide equivalent suppression 4 hours after dosing, although both of these medications are administered in a dose of 5 mg daily for allergic rhinoconjunctivitis or urticaria treatment (Popov et al. 2006).

Efficacy H1-antihistamines prevent and relieve symptoms in seasonal/ intermittent and perennial/persistent allergic rhinitis, allergic conjunctivitis, and acute and chronic urticaria. The rationale for their use in these and other allergic disorders is based on the following: (i) local challenge with histamine reproduces many of the acute symptoms of these diseases; (ii) challenge with allergen or other relevant stimulus can result in local or systemic increases in histamine concentrations; (iii) during disease activity, histamine concentrations may increase locally or even systemically; (iv) pretreatment with an H1antihistamine prevents or relieves symptoms after challenge with histamine or with allergen (Simons 2003). H1-antihistamines sometimes relieve symptoms incompletely in allergic disorders because, in addition to histamine, leukotrienes and other mast cell and basophil products also play a role in allergic inflammation. The dose–response curve for efficacy of H1-antihistamines in allergic rhinoconjunctivitis and urticaria is relatively flat, i.e., doubling or trebling of the manufacturers’ recommended dose does not necessarily result in a corresponding two- or three-fold increase in efficacy. In chronic/perennial/persistent allergic diseases, H1antihistamines are best taken on a regular basis rather than as needed, in order to prevent allergic inflammation and associated symptoms. Tolerance to clinical efficacy does not develop. First-generation H1-antihistamines have not been optimally studied in any allergic disease, and the evidence base for their use is largely empirical. In contrast, the use of second-generation H1-antihistamines for relief of symptoms in seasonal/intermittent, and perennial/persistent allergic rhinoconjunctivitis, and in chronic urticaria is supported by a strong evidence base consisting of randomized, doublemasked, placebo-controlled clinical trials lasting weeks or months. In these trials, there are adequate numbers of participants enrolled, and data on attrition and adherence are reported. Second-generation H1-antihistamines are therefore medications of choice in these diseases (Murdoch et al. 2003; Curran et al. 2004; Simons 2004; van Cauwenberge et al. 2004; Golightly & Greos 2005; Hair & Scott 2006; Keam & Plosker 2007).

Allergic rhinoconjunctivitis In allergic rhinoconjunctivitis, H1-antihistamines prevent and relieve the sneezing, nasal and conjunctival itching, rhinorrhea, tearing, and conjunctival erythema of the early response to allergen (Fig. 25.3). They also have a small beneficial effect

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Calcium-ion channels

H1-receptor Nuclear factor-kB Decreased allergic inflammation, itching, sneezing, rhinorrhea, and whealing

Decreased antigen presentation, expression of cell-adhesion molecules, chemotaxis, and proinflammatory cytokines

Decreased mediator release

Potential adverse effects of H1-antihistamines

H1-receptor

Muscarinic receptor

a-adrenergic receptor

Serotonin receptor

IKr and other cardiac-ion channels

Decreased neurotransmission in the central nervous system, increased sedation, decreased cognitive and psychomotor performance, and increased appetite

Increased dry mouth, urinary retention, and sinus tachycardia

Hypotension, dizziness, and reflex tachycardia

Increased appetite

Prolonged QT intervals, sometimes resulting in ventricular arrhythmias

Fig. 25.3 Benefits and potential adverse effects of H1-antihistamines. H1-antihistamines downregulate allergic inflammation mainly through the H1 receptor. First-generation H1-antihistamines potentially cause adverse effects not only through H1 receptors in the central nervous system, but also through the muscarinic, a-adrenergic, serotonin receptors, and cardiac ion channels. (From Simons 2004, with permission.) (See CD-ROM for color version.)

on the nasal blockage that characterizes the late allergic response (Plaut & Valentine 2005). They improve quality of life (Juniper et al. 2005). Second-generation H1-antihistamines such as cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mizolastine, and rupatadine have greater efficacy than placebo, as documented in well-designed clinical trials (Simons et al. 2003a; Golightly & Greos 2005; Pasquali et al. 2006; Canonica et al. 2007). There are relatively few published clinical trials in which their efficacy relative to each other, or to first-generation H1-antihistamines has been investigated; however, when such trials have been conducted, no clear overall superior efficacy of one H1antihistamine over another has been documented consistently (van Cauwenberge & Juniper 2000; Berger et al. 2006). When orally administered H1-antihistamines are compared with intranasal or ophthalmic H1-antihistamines, the latter are found to have a more rapid onset of action; for example, 15 min for topical azelastine versus 150 min for orally administered desloratadine (Horak et al. 2006); however, they require administration several times daily, in contrast to once-daily dosing for the orally administered second-generation H1antihistamines (Bielory et al. 2005). Few H1-antihistamines have been optimally studied in children with allergic rhinoconjunctivitis (Wahn et al. 2003).

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Selection of an H1-antihistamine for an individual should be based on his/her preference for a particular H1-antihistamine formulation, route of administration, or dose regimen, and above all, on considerations of benefits versus risk of adverse effects (Blaiss 2006; Raphael et al. 2006). More head-to-head comparisons among the H1-antihistamines are needed. In allergic rhinitis, second-generation H1-antihistamines have comparable efficacy to intranasal cromolyn or nedocromil, and to the leukotriene modifier montelukast, although in seasonal and persistent allergic rhinitis the combination of desloratadine or levocetirizine with montelukast may be more effective than monotherapy with any of these agents (Ciebiada et al. 2006). In order to provide increased relief of nasal congestion, H1-antihistamines are sometimes marketed in fixed-dose combinations with pseudoephedrine or other decongestants. H1-antihistamines are less effective than intranasal glucocorticoids, especially for relief of nasal congestion (Saengpanich et al. 2003; Barnes et al. 2006). In individuals with allergic rhinoconjunctivitis whose symptoms are primarily conjunctival, H1-antihistamines administered topically to the affected areas are the medications of choice, not only for their antihistaminic effects, but also for their antiallergic and antiinflammatory effects, and their rapid onset of action (3–15 min) (Bielory et al. 2005).

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H1-antihistamines, whether topically or orally administered, have a more favorable therapeutic index than any other class of medications used in the treatment of allergic conjunctivitis (Treatment Guidelines from The Medical Letter 2007).

Other airway disorders H1-antihistamines are widely used to relieve symptoms of upper respiratory tract infections, otitis media, sinusitis, and nonspecific cough; however, the published evidence does not support their use in these disorders (De Sutter et al. 2003; Simons 2003; Chang et al. 2006; Griffin et al. 2006). Pretreatment with an H1-antihistamine provides some protection against bronchospasm induced in the clinical laboratory by histamine, exercise, hyperventilation, cold, dry air, hypertonic or hypotonic saline, distilled water, adenosine-5 monophosphate, or allergen (Wilson 2006). Although H1antihistamines do no harm in asthma, they are not recommended, as such, for the treatment of asthma. While they are reported to decrease concurrent symptoms of allergic rhinitis and asthma significantly in individuals who have allergic inflammation throughout the upper and lower airways, their effect on allergic rhinitis is greater than their effect on asthma (Bousquet et al. 2001; Baena-Cagnani et al. 2003). In an 18month study in very young children with atopic dermatitis and house-dust mite or grass sensitization who were at risk for development of asthma, regular administration of cetirizine was reported to delay asthma onset (Warner et al. 2001); however, in a population of more highly atopic young children, this observation was not confirmed with levocetirizine.

Urticaria H1-antihistamines are efficacious in acute urticaria, defined as lasting < 6 weeks, and chronic urticaria, defined as lasting ≥ 6 weeks, including physical urticarias such as cholinergic, cold, and pressure-induced urticarias. They are not effective in urticarial vasculitis or non-allergic angioedema. They decrease itching and reduce the number, size, and duration of wheals and erythema (flares), and significantly improve quality of life. In acute urticaria, both first- and secondgeneration H1-antihistamines are widely used, although there is not much published evidence in support of their efficacy in this disorder (Zuberbier et al. 2006). In two different large, randomized, double-masked, placebo-controlled studies in young atopic children in which efficacy in acute urticaria was a planned secondary outcome, cetirizine and levocetirizine effectively prevented and treated the hives (Simons, F.E.R. et al. 2001, 2007a). The first-generation H1-antihistamines remain in widespread use for chronic urticaria, despite a paucity of efficacy studies, and concerns about potential adverse effects, which may or may not be clinically apparent. In contrast, the secondgeneration H1-antihistamines cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mizolastine, and rupatadine have been well studied in chronic urticaria

Antihistamines

(Simons 2003, 2004; Kozel & Sabroe 2004; Golightly & Greos 2005; Hair & Scott 2006; Kapp & Pichler 2006; Keam & Plosker 2007; Nettis et al. 2006; Zuberbier et al. 2006; Dubertret et al. 2007; Gimenez-Arnau et al. 2007; Ortonne et al. 2007). In chronic urticaria, although use of two different H1antihistamines on the same day (specifically, a nonsedating medication in the morning and a sedating medication at bedtime) has been recommended (Kaplan 2002), this strategy has not been prospectively tested in randomized, doublemasked, placebo-controlled trials. In individuals with chronic urticaria that is not relieved by an H1-antihistamine alone, an H2-antihistamine such as cimetidine administered concurrently with an H1-antihistamine may give added relief. This treatment strategy is worth a 3–4 week trial (Kaplan 2002). Simultaneous administration of the H1-antihistamine desloratadine with the leukotriene D4-antagonist montelukast does not appear to offer any consistent therapeutic advantage over desloratadine alone (Di Lorenzo et al. 2004). In some individuals, chronic urticaria and associated pruritus can be difficult to treat. If H1-antihistamine treatment fails, they may require an immunomodulator such as an oral corticosteroid, cyclosporin, sulfasalazine, hydroxychloroquine, mycophenolate, or tacrolimus; however, all these medications have a more limited evidence base for efficacy than H1-antihistamines in chronic urticaria, and most of them potentially have severe adverse effects (Kozel & Sabroe 2004; Treatment Guidelines from The Medical Letter 2007).

Atopic dermatitis and other skin disorders The evidence that H1-antihistamines relieve itch in atopic dermatitis, and provide a glucocorticoid-sparing effect in atopic dermatitis is not convincing (Klein & Clark 1999). In this disorder, histamine may act as a pruritogen through H4 receptors on peripheral neurons (Dunford et al. 2007); in addition, cytokines such as IL-31, and other agents, may play an important pruritogenic role (Boguniewicz et al. 2006). The use of H1-antihistamines to relieve symptoms in individuals with mastocytosis, or to prevent itchy local allergic reactions to bites of insects such as mosquitoes, is supported by small clinical trials (Karppinen et al. 2006).

Anaphylaxis A recent Cochrane collaboration review of 2070 studies of H1-antihistamines in anaphylaxis did not reveal even one study that provided high-quality evidence for or against the use of H1-antihistamines in this disease (Sheikh et al. 2007). Individuals who experience anaphylaxis in the community should not depend on an oral H1-antihistamine for first-aid treatment, because H1-antihistamines are not life-saving, and do not relieve respiratory, cardiovascular, or gastrointestinal symptoms, although they decrease itch and hives. After administration by mouth, absorption and onset of action of H1-antihistamines takes at least 1–2 hours. First-generation H1-antihistamines, such as diphenhydramine, potentially

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cause somnolence and impaired recognition of anaphylaxis symptoms. In many individuals, improvement in anaphylaxis attributed to H1-antihistamine treatment likely reflects spontaneous improvement due to endogenous epinephrine, endothelin, or angiotensin II (Simons 2006).

CNS and vestibular system disorders: the unfavorable therapeutic index of first-generation H1-antihistamines Although not medications of choice, the first-generation H1-antihistamines diphenhydramine, doxylamine, and pyrilamine are the most widely used medications in the world for insomnia relief. They are also still used for treatment of akathisia, serotonin syndrome, anxiety, and other conditions affecting the CNS. Diphenhydramine, hydroxyzine, cyproheptadine, and promethazine remain in use for perioperative sedation and for analgesia. Concerns have been raised about their use in conscious sedation, not only because of their potential CNS adverse effects, but also because they may cause respiratory depression, and fatality. Promethazine has received a black-box warning from the United States Food and Drug Administration regarding use in young children, due to its association with a high rate of occurrence of CNS adverse effects, respiratory depression, and death in this age group (Starke et al. 2005). Dimenhydrinate, diphenhydramine, meclizine, and promethazine block the histaminergic signal from the vestibular nucleus to the vomiting centre in the medulla, and are used for antiemetic effects, and for prevention and treatment of motion sickness, vertigo, and related disorders. The firstgeneration H1-antihistamines have an unfavorable benefitto-risk ratio in the treatment of CNS and vestibular disorders. Military and commercial aviation authorities do not permit pilots to use these medications. Other public transportation workers such as ships’ captains, bus, truck, and taxi drivers, and members of the general public, must decide for themselves whether or not they are willing to take the risk of using a first-generation H1-antihistamine while operating a vehicle.

Adverse effects First-generation H1-antihistamines First-generation H1-antihistamines, administered in usual doses, potentially cause a wide variety of adverse effects in many body systems (Simons 2003, 2004). In contrast to the flat dose–response curve for efficacy, the dose–response curve for adverse effects is steep; that is, the adverse effects potentially increase with increasing doses. Through muscarinic receptors, these older H1-antihistamines may cause pupillary dilation, dry eyes, dry mouth, urinary retention and hesitancy, decreased gastrointestinal motility and constipation. Through α-adrenergic receptors, they potentially

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cause peripheral vasodilation, postural hypotension, and dizziness. Through serotonin receptors, they potentially cause appetite stimulation and weight gain (Simons 2004) (Fig. 25.3). The main concern, however, is that all first-generation H1-antihistamines administered in usual doses interfere with histamine’s neurotransmitter effects through H1 receptors in the CNS, and potentially cause CNS adverse effects (Shamsi & Hindmarch 2000; Tashiro et al. 2005). These include drowsiness, sedation, somnolence, fatigue, and impairment of memory, cognitive function, and psychomotor performance, and, less commonly, headache, dystonia, dyskinesia, agitation, confusion, and hallucinations. Positron emission tomography (PET) with 11C-doxepin as the positron-emitting ligand reveals that these medications occupy 70–90% of the H1 receptors in the frontal cortex, temporal cortex, hippocampus, and pons (Tashiro et al. 2002, 2006). Blood–brain barrier penetration is related to their lipophilicity, relatively low molecular weights, and lack of substrate recognition by the P-glycoprotein efflux pump that is expressed on the luminal surfaces of nonfenestrated endothelial cells in the CNS vasculature (Chen et al. 2003). Impairment of CNS function by first-generation H1antihistamines in usual doses has been documented in the absence of CNS symptoms. After taking one of these older medications at bedtime, some individuals have residual CNS adverse effects the next morning, the so-called antihistamine “hangover.” Tolerance to the CNS effects of the firstgeneration H1-antihistamines does not necessarily occur. The CNS effects of a first-generation H1-antihistamine are similar to, and exacerbate, those produced by alcohol or by a major tranquilizer. After overdose with first-generation H1-antihistamines, CNS symptoms predominate. In adults, these symptoms may culminate in delirium and coma. In children, paradoxical excitation, irritability, hyperactivity, insomnia, hallucinations, and seizures may occur. After overdose, first-generation H1antihistamines, such as diphenhydramine and hydroxyzine, also potentially cause dose-related cardiac adverse effects, including sinus tachycardia, reflex tachycardia, supraventricular arrhythmias, and prolongation of the QT interval and ventricular arrhythmias. These effects occur through interaction with muscarinic receptors, α-adrenergic receptors, and blockade of cardiac ion currents, especially the rapid component of the delayed rectifier potassium current (Yap & Camm 2002; Liu et al. 2007). Deaths attributed to first-generation H1-antihistamines due to accidental overdose, suicide, and homicide (in infants) have been reported in the literature for more than half a century (Wyngaarden & Seevers 1951). The plasma concentrations associated with fatality have been identified for medications such as diphenhydramine and brompheniramine (Simons 2003; Nine & Rund 2006). Up to 30 000 diphenhy-

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dramine overdoses are reported to poison control centers in the USA every year. Triage guidelines have been published in order to help identify individuals with these overdoses who urgently require transportation to a hospital emergency department for supportive treatment, and those who can be safely managed in other healthcare facilities, or at home (Scharman et al. 2006).

Second-generation H1-antihistamines In contrast to all of the foregoing, the second-generation H1antihistamines are unlikely to cause CNS effects when taken in manufacturers’ recommended doses, although in higher doses some of them, such as cetirizine, may cause sedation (Simons 2004; Golightly & Greos 2005). They are therefore medications of choice for pilots and others in safety-critical jobs, elite and professional athletes, and indeed in anyone who needs to remain alert. Relative lack of penetration into the CNS (Chen et al. 2003) has been documented by PET scan studies, which demonstrate that 0 to ≤ 30% of H1 receptors in the CNS are occupied by these newer medications, and by electroencephalographic monitoring, sleep latency studies, and standardized performance tests ranging from simple reaction time tests to complex sensorimotor tasks such as computermonitored driving (Shamsi & Hindmarch 2000; Hindmarch et al. 2002; Nicholson et al. 2003; Tashiro et al. 2005). Lack of CNS effects, confirmed objectively for the most part in healthy volunteers, may be of even greater significance in individuals with allergic rhinoconjunctivitis or urticaria, in whom symptoms such as congestion and extreme itching may reduce the duration and quality of nocturnal sleep, and increase daytime somnolence. In “real world,” prescription-event monitoring studies conducted in thousands of individuals with allergic rhinitis during the first 30 days after introduction of a new H1antihistamine in the UK, a low risk of sedation has been reported for medications such as fexofenadine, cetirizine, and loratadine (Mann et al. 2000), and more recently, for desloratadine and levocetirizine (Layton et al. 2006). The second-generation H1-antihistamines do not exacerbate the CNS effects of alcohol, diazepam, or other CNS-active substances when coadministered with these substances (Weiler et al. 2000; Ridout et al. 2003; Barbanoj et al. 2006; Scharf & Berkowitz 2007). Since withdrawal of regulatory approval for astemizole and terfenadine in most countries more than a decade ago (Woosley 1996), the second-generation H1-antihistamines remaining in use are free from potential cardiac adverse effects (Yap & Camm 2002; Hove-Madsen et al. 2006; Liu et al. 2007). According to published reports, accidental massive (e.g., up to 20-fold) overdoses of second-generation H1antihistamines such as cetirizine, fexofenadine, and loratadine have not resulted in any serious CNS or cardiovascular adverse effects, or in any deaths (Golightly & Greos 2005).

Antihistamines

Individuals who suffer from one or more allergic diseases often use H1-antihistamines intermittently or regularly for months, years, or even decades, yet there are only a few published randomized, controlled studies documenting the long-term safety of these medications. These include 6–12 month-long studies of fexofenadine and of levocetirizine in adults with allergic rhinitis, and three studies in very young children: a 12-month study of loratadine, an 18-month study of cetirizine, and an 18-month study of levocetirizine (Simons et al. 1999; Bachert et al. 2004; Grimfeld et al. 2004; Simons, F.E.R. et al. 2007b).

Other considerations Uncommonly, after usual doses, euphoria and “getting high” are reported for diphenhydramine, dimenhydrinate, and other first-generation H1-antihistamines. Rare adverse effects of both first- and second-generation H1-antihistamines include contact dermatitis, fixed-drug eruptions, photosensitivity, fever, elevation of liver enzymes and hepatitis, cytopenias, urticaria, and even anaphylaxis. First-generation H1-antihistamines, particularly those in the phenothiazine class, have been associated with sudden infant death syndrome, although causality has never been proven. First-generation H1-antihistamines applied topically to excoriated or abraded skin may cause systemic and local adverse effects. Intranasal or ophthalmic H1-antihistamines may cause stinging or burning of the nasal mucosa or the conjunctivae, and some such as azelastine potentially cause dysgeusia (bitter taste). No H1antihistamines currently approved for use are considered to have carcinogenic or tumor-promoting effects in humans (Nadalin et al. 2003; Simons 2003; Golightly & Greos 2005; Vythoulka et al. 2006).

Use of H1-antihistamines in infants and very young children H1-antihistamines are commonly given to infants and young children, not only for treatment of allergic rhinoconjunctivitis and urticaria, but also for colds, otitis media, and other disorders in which their efficacy remains unproven. Firstgeneration H1-antihistamines are available without prescription and are extensively marketed in a wide variety of tasty liquid formulations, often in combination with other medications. There are no prospective safety studies of these older H1-antihistamines in infants or very young children. The second-generation H1-antihistamines cetirizine and desloratadine have been prospectively studied in infants aged 6–11 months (Simons et al. 2003b; Gupta 2007). The long-term safety of cetirizine, levocetirizine, and loratadine has been confirmed in children aged 12–36 months, as noted previously. Studies of all three medications involved monitoring of adverse event reports, body mass and height measurements, and blood hematology and chemistry tests; in addition, electrocardiograms were performed in the cetirizine

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and loratadine studies. The 18-month duration cetirizine and levocetirizine studies included documentation of developmental milestones, and testing behavior and intellectual performance (Stevenson et al. 2002). For these second-generation H1-antihistamines, safety profiles were similar to safety profiles of children being treated with placebo.

Use of H1-antihistamines in pregnancy and lactation With any medication, and any class of medications, potential teratogenicity is a concern. Few medications, and no H1antihistamines, have been designated as FDA Category A, denoting negative studies in animals and negative human data. Several H1-antihistamines, such as the first-generation medications chlorpheniramine and diphenhydramine, and the orally administered second-generation medications cetirizine, levocetirizine and loratadine, as well as emedastine for ophthalmic use, have been designated as FDA Category B, denoting that studies in animals have shown no adverse effects and data in humans are not available; or that studies in animals have shown adverse effects but studies in humans have not shown adverse effects. These six medications are therefore considered to be relatively safe for use if needed in pregnancy. All other H1-antihistamines are designated as Category C, denoting drugs for which studies in animals have shown adverse effects, and data in humans are not available, or, drugs for which neither studies in animals nor studies in humans have been performed (Simons 2004). H1-antihistamines that are not approved for use in the USA (ebastine, mizolastine, and rupatadine) have not been categorized by the FDA. H1-antihistamines are secreted into breast milk. Nursing infants receive approximately 0.1% of an orally administered maternal dose, and first-generation H1-antihistamines potentially cause sedation and other adverse effects in these infants.

Use of first-generation H1-antihistamines in the elderly The vulnerability of the elderly to adverse effects from any CNS-active chemical is an important issue, because 25% of individuals older than age 65 years have some baseline cognitive impairment, often with no obvious sign of dysfunction. Use of first-generation H1-antihistamines for allergic rhinoconjunctivitis and urticaria in elderly individuals is a concern because of potential drug–drug and drug–herbal product interactions, potential antimuscarinic effects, and potential CNS effects through the H1 receptor. Diphenhydramine administration for insomnia in institutionalized elderly individuals has been associated with an increased cognitive impairment, delirium, inattention, disorganized speech, altered consciousness, and need for urinary catheter placement (Simons 2003; Hansen et al. 2005).

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Future directions H1-antihistamines are first-line medications in the treatment of allergic rhinitis, allergic conjunctivitis, and urticaria. Secondgeneration H1-antihistamines, which are relatively free from adverse effects, should be used in preference to the firstgeneration medications in the treatment of these disorders. The latter remain in use, however, and, in many countries, regulatory agencies appear to be less concerned about the potential CNS toxicity of these older H1-antihistamines than they are about the potential cardiac toxicity of H1antihistamines. Most of the new H1-antihistamines developed in recent years are structurally related to existing medications in the class. To date, no second-generation H1-antihistamine appears to be superior to the others, and none of them appears to be a true third- or next- or new-generation H1-antihistamine (Holgate et al. 2003). Nevertheless, in the future, clinically advantageous H1-antihistamines may be designed with the use of molecular techniques and will merit the designation “third-generation.” Some of these medications may possess intrinsic H2, H3, or H4 antihistamine properties, or antileukotriene properties.

References Akdis, C.A. & Simons, F.E.R. (2006) Histamine receptors are hot in immunopharmacology. Eur J Pharmacol 533, 69–76. Bachert, C., Bousquet, J., Canonica, G.W. et al. (2004) Levocetirizine improves quality of life and reduces costs in long-term management of persistent allergic rhinitis. J Allergy Clin Immunol 114, 838–44. Baena-Cagnani, C.E., Berger, W.E., DuBuske, L.M. et al. (2003) Comparative effects of desloratadine versus montelukast on asthma symptoms and use of beta 2-agonists in patients with seasonal allergic rhinitis and asthma. Int Arch Allergy Immunol 130, 307–13. Barbanoj, M.J., Garcia-Gea, C., Antonijoan, R. et al. (2006) Evaluation of the cognitive, psychomotor and pharmacokinetic profiles of rupatadine, hydroxyzine and cetirizine, in combination with alcohol, in healthy volunteers. Human Psychopharmacol 21, 13–26. Barnes, M.L., Ward, J.H., Fardon, T.C. & Lipworth, B.J. (2006) Effects of levocetirizine as add-on therapy to fluticasone in seasonal allergic rhinitis. Clin Exp Allergy 36, 676–84. Berger, W.E., Lumry, W.R., Meltzer, E.O. & Pearlman, D.S. (2006) Efficacy of desloratadine, 5 mg, compared with fexofenadine, 180 mg, in patients with symptomatic seasonal allergic rhinitis. Allergy Asthma Proc 27, 214–23. Bielory, L., Lien, K.W. & Bigelsen, S. (2005) Efficacy and tolerability of newer antihistamines in the treatment of allergic conjunctivitis. Drugs 65, 215–28. Blaiss, M.S. (2006) Diphenhydramine vs desloratadine comparisons must consider risk-benefit ratio. Ann Allergy Asthma Immunol 97, 121–2.

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Boguniewicz, M., Schmid-Grendelmeier, P. & Leung, D.Y.M. (2006) Atopic dermatitis. J Allergy Clin Immunol 118, 40–3. Bongers, G., Bakker, R.A. & Leurs, R. (2007) Molecular aspects of the histamine H3 receptor. Biochem Pharmacol 73, 1195–204. Bousquet, J., van Cauwenberge, P.B., Khaltaev, N. in collaboration with the World Health Organization (2001) Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol 108, 1A–14A, S147–S334. Bressler, R. (2006) Grapefruit juice and drug interactions. Exploring mechanisms of this interaction and potential toxicity for certain drugs. Geriatrics 61, 12–18. Bryce, P.J., Mathias, C.B., Harrison, K.L., Watanabe, T., Geha, R.S. & Oettgen, H.C. (2006) The H1 histamine receptor regulates allergic lung responses. J Clin Invest 116, 1624–32. Canonica, G.W., Tarantini, F., Compalati, E. & Penagos, M. (2007) Efficacy of desloratadine in the treatment of allergic rhinitis: a meta-analysis of randomized, double-blind, controlled trials. Allergy 62, 359– 66. Caproni, M., Volpi, W., Giomi, B., Torchia, D., Del Bianco, E. & Fabbri, P. (2006) Cellular adhesion molecules in chronic urticaria: modulation of serum levels occurs during levocetirizine treatment. Br J Dermatol 155, 1270– 4. Chang, A.B., Peake, J. & McElrea, M.S. (2006) Antihistamines for prolonged non-specific cough in children. Cochrane Database Syst Rev 3, CD005604. Chen, C., Hanson, E., Watson, J.W. & Lee, J.S. (2003) P-glycoprotein limits the brain penetration of nonsedating but not sedating H1antagonists. Drug Metabol Dispos 31, 312–18. Ciebiada, M., Gorska-Ciebiada, M., DuBuske, L.M. & Gorski, P. (2006) Montelukast with desloratadine or levocetirizine for the treatment of persistent allergic rhinitis. Ann Allergy Asthma Immunol 97, 664–71. Corcostegui, R., Labeaga, L., Innerarity, A., Berisa, A. & Orjales, A. (2006) In vivo pharmacological characterisation of bilastine, a potent and selective histamine H1 receptor antagonist. Drugs Res Dev 7, 219–31. Cranswick, N., Turzikova, J., Fuchs, M. & Hulhoven, R. (2005) Levocetirizine in 1–2 year old children: pharmacokinetic and pharmacodynamic profile. Int J Clin Pharmacol Ther 43, 172–7. Curran, M.P., Scott, L.J. & Perry, C.M. (2004) Cetirizine: a review of its use in allergic disorders. Drugs 64, 523–61. Day, J.H., Ellis, A.K., Rafeiro, E., Ratz, J.D. & Briscoe, M.P. (2006) Experimental models for the evaluation of treatment of allergic rhinitis. Ann Allergy Asthma Immunol 96, 263–77. de Esch, I.J.P., Thurmond, R.L., Jongejan, A. & Leurs, R. (2005) The histamine H4 receptor as a new therapeutic target for inflammation. Trends Pharmacol Sci 26, 462–9. De Sutter, A.I.M., Lemiengre, M., Campbell, H. & Mackinnon, H.F. (2003) Antihistamines for the common cold. Cochrane Database Syst Rev 3, CD001267. Di Lorenzo, G., Pacor, M.L., Mansueto, P. et al. (2004) Randomized placebo-controlled trial comparing desloratadine and montelukast in monotherapy and desloratadine plus montelukast in combined therapy for chronic idiopathic urticaria. J Allergy Clin Immunol 114, 619–25. Dubertret, L., Zalupca, L., Cristodoulo, T. et al. (2007) Once-daily rupatadine improves the symptoms of chronic idiopathic urticaria: a randomised, double-blind, placebo-controlled study. Eur J Dermatol 17, 223– 8.

Antihistamines

Dunford, P.J., Williams, K.N., Desai, P.J., Karlsson, L., McQueen, D. & Thurmond, R.L. (2007) Histamine H4 receptor antagonists are superior to traditional antihistamines in the attenuation of experimental pruritus. J Allergy Clin Immunol 119, 176–83. Gimenez-Arnau, A., Pujol, R.M., Ianosi, S. et al. (2007) Rupatadine in the treatment of chronic idiopathic urticaria: a double-blind, randomized, placebo-controlled multicentre study. Allergy 62, 539– 46. Golightly, L.K. & Greos, L.S. (2005) Second-generation antihistamines: actions and efficacy in the management of allergic disorders. Drugs 65, 341–84. Griffin, G.H., Flynn, C., Bailey, R.E. & Schultz, J.K. (2006) Antihistamines and/or decongestants for otitis media with effusion (OME) in children. Cochrane Database Syst Rev 4, CD003423. Grimfeld, A., Holgate, S.T., Canonica, G.W. et al. (2004) Prophylactic management of children at risk for recurrent upper respiratory infections: the Preventia I Study. Clin Exp Allergy 34, 1665–72. Gupta, S., Khalilieh, S., Kantesaria, B. & Banfield, C. (2007) Pharmacokinetics of desloratadine in children between 2 and 11 years of age. Br J Clin Pharmacol 63, 534–40. Gupta S.K., Kantesaria, B., Banfield, C., Wang, Z. (2007) Desloratadine dose selection in children aged 6 months to 2 years: comparison of population pharmacokinetics between children and adults. Br J Clin Pharmacol 64, 174–84. Haas, H. & Panula, P. (2003) The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci 4, 121–30. Hair, P.I. & Scott, L.J. (2006) Levocetirizine: a review of its use in the management of allergic rhinitis and skin allergies. Drugs 66, 973–96. Hansen, J., Klimek, L. & Hormann, K. (2005) Pharmacological management of allergic rhinitis in the elderly: safety issues with oral antihistamines. Drugs Aging 22, 289–96. Hindmarch, I., Shamsi, Z. & Kimber, S. (2002) An evaluation of the effects of high-dose fexofenadine on the central nervous system: a double-blind, placebo-controlled study in healthy volunteers. Clin Exp Allergy 32, 133–9. Holgate, S.T., Canonica, G.W., Simons, F.E.R. et al. (2003) Consensus Group on New-Generation Antihistamines (CONGA): present status and recommendations. Clin Exp Allergy 33, 1305–24. Horak, F., Zieglmayer, U.P., Zieglmayer, R. et al. (2006) Azelastine nasal spray and desloratadine tablets in pollen-induced seasonal allergic rhinitis: a pharmacodynamic study of onset of action and efficacy. Curr Med Res Opin 22, 151–7. Hove-Madsen, L., Llach, A., Molina, C.E. et al. (2006) The proarrhythmic antihistaminic drug terfenadine increases spontaneous calcium release in human atrial myocytes. Eur J Pharmacol 553, 215–21. Jongejan, A., Bruysters, M., Ballesteros, J.A. et al. (2005) Linking agonist binding to histamine H1 receptor activation. Nat Chem Biol 1, 98–103. Juniper, E.F., Stahl, E., Doty, R.L., Simons, F.E.R., Allen, D.B. & Howarth, P.H. (2005) Clinical outcomes and adverse effect monitoring in allergic rhinitis. J Allergy Clin Immunol 115 (suppl. 1), S390–S413. Jutel, M., Blaser, K. & Akdis, C.A. (2006) Histamine receptors in immune regulation and allergen-specific immunotherapy. Immunol Allergy Clin North Am 26, 249–59.

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Pharmacology

Kaplan, A.P. (2002) Clinical practice. Chronic urticaria and angioedema. N Engl J Med 346, 175–9. Kapp, A. & Pichler, W.J. (2006) Levocetirizine is an effective treatment in patients suffering from chronic idiopathic urticaria: a randomized, double-blind, placebo-controlled, parallel, multicenter study. Int J Dermatol 45, 469–74. Karppinen, A., Brummer-Korvenkontio, H., Petman, L., Kautiainen, H., Herve, Jean-P. & Reunala, T. (2006) Levocetirizine for treatment of immediate and delayed mosquito bite reactions. Acta Dermato-Venereologica 86, 329–31. Keam, S.J. & Plosker, G.L. (2007) Rupatadine: a review of its use in the management of allergic disorders. Drugs 67, 457–74. Klein, P.A. & Clark, R.A.F. (1999) An evidence-based review of the efficacy of antihistamines in relieving pruritus in atopic dermatitis. Arch Dermatol 135, 1522–5. Kozel, M.M.A. & Sabroe, R.A. (2004) Chronic urticaria: aetiology, management and current and future treatment options. Drugs 64, 2515–36. Layton, D., Wilton, L., Boshier, A., Cornelius, V., Harris, S. & Shakir, S.A.W. (2006) Comparison of the risk of drowsiness and sedation between levocetirizine and desloratadine: a prescription-event monitoring study in England. Drug Safety 29, 897–909. Leurs, R., Church, M.K. & Taglialatela, M. (2002) H1-antihistamines: inverse agonism, antiinflammatory actions and cardiac effects. Clin Exp Allergy 32, 489–98. Liu, B., Jia, Z., Geng, X. et al. (2007) Selective inhibition of Kir currents by antihistamines. Eur J Pharmacol 558, 21–6. MacGlashan, D., Jr. (2003) Histamine: a mediator of inflammation. J Allergy Clin Immunol 112 (suppl.), S53–S59. Mann, R.D., Pearce, G.L., Dunn, N. & Shakir, S. (2000) Sedation with “non-sedating” antihistamines: four prescription-event monitoring studies in general practice. BMJ 320, 1184–7. Molimard, M., Diquet, B. & Benedetti, M.S. (2004) Comparison of pharmacokinetics and metabolism of desloratadine, fexofenadine, levocetirizine and mizolastine in humans. Fund Clin Pharmacol 18, 399–411. Muller, U., Hari, Y. & Berchtold, E. (2001) Premedication with antihistamines may enhance efficacy of specific-allergen immunotherapy. J Allergy Clin Immunol 107, 81–6. Murdoch, D., Goa, K.L. & Keam, S.J. (2003) Desloratadine: an update of its efficacy in the management of allergic disorders. Drugs 63, 2051–77. Nadalin, V., Cotterchio, M. & Kreiger, N. (2003) Antihistamine use and breast cancer risk. Int J Cancer 106, 566– 8. Nettis, E., Colanardi, M.C., Barra, L., Ferrannini, A., Vacca, A. & Tursi, A. (2006) Levocetirizine in the treatment of chronic idiopathic urticaria: a randomized, double-blind, placebo-controlled study. Br J Dermatol 154, 533– 8. Nicholson, A.N., Handford, A.D.F., Turner, C. & Stone, B.M. (2003) Studies on performance and sleepiness with the H1-antihistamine, desloratadine. Aviation Space Environ Med 74, 809–15. Nine, J.S. & Rund, C.R. (2006) Fatality from diphenhydramine monointoxication: a case report and review of the infant, pediatric, and adult literature. Am J Forensic Med Pathol 27, 36– 41. Ortonne, Jean-P, Grob, Jean-J, Auquier, P. & Dreyfus, I. (2007) Efficacy and safety of desloratadine in adults with chronic idiopathic urticaria: a randomized, double-blind, placebo-controlled, multicenter trial. Am J Clin Dermatol 8, 37– 42.

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Simons, F.E.R. (2004) Advances in H1-antihistamines. N Engl J Med 351, 2203–17. Simons, F.E.R. (2006) Anaphylaxis, killer allergy: long-term management in the community. J Allergy Clin Immunol 117, 367–77. Simons, F.E.R. & Simons, K.J. (2005) Levocetirizine: pharmacokinetics and pharmacodynamics in children age 6 to 11 years. J Allergy Clin Immunol 116, 355– 61. Simons, F.E.R., on behalf of the ETAC Study Group (1999) Prospective, long-term safety evaluation of the H1-receptor antagonist cetirizine in very young children with atopic dermatitis. J Allergy Clin Immunol 104, 433– 40. Simons, F.E.R., on behalf of the ETAC Study Group (2001) Prevention of acute urticaria in young children with atopic dermatitis. J Allergy Clin Immunol 107, 703– 6. Simons, F.E.R., Silver, N.A., Gu, X. & Simons, K.J. (2002) Clinical pharmacology of H1-antihistamines in the skin. J Allergy Clin Immunol 110, 777– 83. Simons, F.E.R., Prenner, B.M., Finn, A., for the Desloratadine Study Group (2003a) Efficacy and safety of desloratadine in the treatment of perennial allergic rhinitis. J Allergy Clin Immunol 111, 617–22. Simons, F.E.R., Silas, P., Portnoy, J.M., Catuogno, J., Chapman, D. & Olufade, A.O. (2003b) Safety of cetirizine in infants 6 to 11 months of age: a randomized, double-blind, placebo-controlled trial. J Allergy Clin Immunol 111, 1244– 8. Simons, F.E.R., on behalf of the ETAC study group (2005) Population pharmacokinetics of levocetirizine in very young children: the pediatricians’ perspective. Pediatr Allergy Immunol 16, 97–103. Simons, F.E.R., on behalf of the Early Prevention of Asthma in Atopic Children (EPAAC) Study Group (2007a) H1-antihistamine treatment in young atopic children: effect on urticaria. Ann Allergy, Asthma, Immunol 99, 261– 6. Simons, F.E.R., on behalf of the Early Prevention of Asthma in Atopic Children (EPAAC) Study Group (2007b) Safety of levocetirizine treatment in young atopic children: an 18-month study. Pediatr Allergy Immunol 18, 535– 42. Simons, K.J., Strolin-Benedetti, M., Simons, F.E.R., Gillard, M. & Baltes, E. (2007) Relevance of H1-receptor occupancy to antihistamine dosing in children. J Allergy Clin Immunol 119, 1551–4. Starke, P.R., Weaver, J. & Chowdhury, B.A. (2005) Boxed warning added to promethazine labeling for pediatric use. N Engl J Med 352, 2653. Stevenson, J., Cornah, D., Evrard, P. et al. (2002) Long-term evaluation of the impact of the H1-receptor antagonist cetirizine on the behavioral, cognitive and psychomotor development of very young children with atopic dermatitis. Pediatr Res 52, 251–7. Stuebner, P., Horak, F., Zieglmayer, R. et al. (2006) Effects of rupatadine vs placebo on allergen-induced symptoms in patients exposed to aeroallergens in the Vienna Challenge Chamber. Ann Allergy Asthma Immunol 96, 37– 44. Tashiro, M., Mochizuki, H., Iwabuchi, K. et al. (2002) Roles of histamine in regulation of arousal and cognition: functional neuroimaging of histamine H1 receptors in human brain. Life Sci 72, 409–14. Tashiro, M., Horikawa, E., Mochizuki, H. et al. (2005) Effects of fexofenadine and hydroxyzine on brake reaction time during cardriving with cellular phone use. Hum Psychopharmacol 20, 501–9.

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Lipid Mediators: Leukotrienes, Prostanoids, Lipoxins, and Platelet-activating Factor Sophie P. Farooque, Jonathan P. Arm and Tak H. Lee

Summary The eicosanoids are metabolites of the 20-carbon fatty acid arachidonic acid. The best-known eicosanoids are the prostanoids (prostaglandins and thromboxane) and leukotrienes, which are implicated in a large number of physiologic and pathologic processes and are synthesized during allergic and asthmatic reactions. Monocytes, alveolar macrophages, and peripheral blood neutrophils preferentially generate leukotriene (LT)B4, whereas eosinophils, mast cells, and basophils preferentially generate the cysteinyl leukotrienes (LTC4, LTD4 and LTE4). In addition to production by inflammatory leukocytes, structural cells within the lung can produce prostaglandins. Lipoxin are also eicosanoids, and are endogenous antiinflammatory mediators. Arachidonic acid is the parent compound from which leukotrienes and prostanoids are synthesized de novo upon cellular activation. It is cleaved from the cell membrane by one of the isoforms of the enzyme phospholipase A2 and if metabolized through the 5-lipoxygenase (5-LO) pathway from an unstable intermediary LTA4 yields LTB4 or the cysteinyl leukotrienes (CysLTs), whereas if metabolized through the cyclooxygenase (COX) pathway yields the prostanoids. 5-LO is the rate-limiting step in leukotriene formation but is dependent on 5-LO-activating protein, which serves as a necessary presentation molecule. Arachidonic acid release is the rate-limiting step for prostanoid formation.

orchestrate amplification of the allergic inflammatory response in the airway, and influencing neural transmission. Release of CysLTs has been found to be a final common response to mast cell activation regardless of the specific route of inflammation. Four leukotriene receptors have been cloned: BLT1 and BLT2 which both bind LTB4, and CysLT1 and CysLT2 which bind the cysteinyl leukotrienes. The expression of BLT1 is limited to leukocytes whilst BLT2 is believed to be more widely expressed, with BLT2 mRNA found in several tissue types. Biological activities of LTB4 include chemotaxis for mast cell progenitors and neutrophils, weak chemotactic activity for eosinophils, and a role in increasing leukocyte adherence to endothelium. The role of the LTB4 in the pathogenesis of airway inflammation in humans is less well elucidated compared with the CysLTs. Patients with aspirin-sensitive respiratory disease demonstrate particularly high levels of CysLTs and increased CysLT1 receptor expression compared with nonaspirin-sensitive cohorts. Most of the actions of the CysLTs are believed to be mediated through the CysLT1 receptor, which is highly expressed in human leukocytes and is upregulated in the airways of asthmatics. It has been hypothesized that CysLTs may act on more than two receptors, as several studies have recorded that the biological properties of the CysLTs cannot be satisfactorily explained on the basis of recognized pharmacologic properties of CysLT1 and CysLT2.

Prostanoids Leukotrienes Originally described as the slow-reacting substances of anaphylaxis (Brocklehurst et al., 1956), the CysLTs are strongly implicated in the pathogenesis of both allergic rhinitis and asthma. CysLTs were first recognized for their airway bronchoconstrictor activity and appear to play a central role in the airway smooth muscle hypertrophy and hyperplasia found in chronic severe asthma. Other biological properties include stimulation of airway mucus secretion, recruitment and activation of eosinophils, upregulation of the cytokines that

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The generation of prostanoids is a two-step process involving COX activity to convert arachidonic acid to PGG2 and a peroxidase reaction to produce PGH2. PGH2 serves as a substrate for the prostaglandin synthase enzymes, which are responsible for the production of the five physiologically bioactive prostaglandins generated in vivo, PGE2, PGF2α, PGD2, PGI2, and TXA2 (thromboxane). At least two isoforms of COX exist. COX-1 is constitutively expressed and is responsible for basal prostanoid production and hence is often referred to as the “housekeeping” COX isoform. COX-2 is expressed in leukocytes following cellular activation by inflammatory stimuli and is involved in inflammation. In the asthmatic human airway smooth muscle, eosinophils, bronchial epithelial cells, mast cells, and macrophages all exhibit COX-2 immunoreactivity. The expression of COX-1 and COX-2 in human lungs results in the production of two different classes of prostanoids. These are

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broadly divided into the bronchoconstrictive prostaglandins (PGD2 and PGF2α) and the bronchoprotective prostaglandins (PGE2 and PGI2). Although COX is present in most cells including the respiratory tract, airway smooth muscle and epithelial cells, the COX metabolites released from a particular cell are cell specific, reflecting the isomerase and synthase components of that cell. Prostanoid receptors are specific cell surface G-protein-coupled receptors and are classified according to the prostanoid causing selective activation.

Lipoxins Perhaps the lesser known of the eicosanoids, lipoxins are endogenous antiinflammatory mediators distinct from leukotrienes and prostaglandins in structure and function. They are double lipoxygenase products and may require separate cell sources for the two lipoxygenases and are synthesized via transcellular cooperation. Their synthesis has been strongly implicated in the resolution of inflammation and they are generated in normal and asthmatic subjects. Exogenous lipoxin A4 has been shown to block asthmatic responses in both human and animal models.

Introduction The eicosanoids are metabolites of the 20-carbon fatty acid arachidonic acid. The best-known eicosanoids are the leukotrienes and prostanoids (prostaglandins and thromboxane), which form part of a family of oxygenated fatty acids found in virtually every mammalian cell. Physiologically these products of arachidonic acid metabolism participate in many normal processes, which include body temperature regulation, the coagulation cascade, the control of parturition, blood pressure maintenance, and mediation of the immune system. Pathologically, these lipid mediators have been incriminated in a wide range of diseases, including asthma, psoriasis, rheumatoid arthritis, inflammatory bowel disease, and malignancy. Much of the early work on prostaglandins was carried out by three groups led by Sune Bergstrom, Bengt Samuelsson, and John Vane. Bergstrom determined the structure and biological role of the prostaglandins and discovered that they are synthesized in vivo from dietary polyunsaturated fatty acids; Samuelsson determined the metabolic fate and disposition of the prostaglandins; and Vane demonstrated the ability of antiinflammatory substances such as aspirin to inhibit prostaglandin synthesis. However, the original investigation of these compounds began in 1930, with the work of Raphael Kurzok and Charles Lieb, who studied the ability of human semen either to relax or to contract isolated strips of uterine tissue. Three years later, Goldblatt and von Euler independently observed a similar phenomenon using human seminal plasma and extracts from sheep seminal

vesical glands. Von Euler therefore coined the term “prostaglandin,” believing that he had isolated a product of the prostate gland. Leukotrienes were discovered during the elucidation of a mixture of compounds referred to as the slow-reacting substance of anaphylaxis or SRS-A. In 1938, Feldberg and Kellaway coined the term SRS to define the smooth muscle contracting activity found in the effluent of the perfused lungs of guinea pigs and cats following treatment with cobra venom. In 1940, Kellaway and Trethewie found that smooth muscle contractions in anaphylaxis were caused not only by histamine but also in part by a slow-reacting substance. Brocklehurst (1956) unmasked the SRS by demonstrating that the contraction it produced on the isolated guinea-pig ileum was not inhibited by an antihistamine added to the organ bath. He also showed that the pattern of contraction was different from bradykinin, substance P, and 5-hydroxytryptamine and added the suffix “A” to indicate that this was a particular slow-reacting substance associated with anaphylaxis. The 1960s and 1970s saw the purification and description of many biological and physicochemical properties of SRS-A by Orange, Austen, and Murphy (Orange & Austen 1969; Orange et al. 1973), Morris et al. (1980), Lewis et al. (1980a), and others. The covalent structure and total synthesis of SRS-A and its identification as a mixture of the sulfidopeptide leukotrienes, leukotriene (LT)C4, and its biologically active metabolites LTD4 and LTE4 was achieved by Samuelsson et al. (1980) and Corey et al. (1980). The leukotrienes and the prostanoids are not preformed, but upon cell activation are synthesized de novo. The primary substrate fatty acid which acts as a precursor for leukotrienes and prostanoids is 5,8,11,14-eicosatetraenoic acid (arachidonic acid). Leukocyte activation initiates the rapid generation of eicosanoids via the release of arachidonic acid from cell membrane phospholipids by one of the isoforms of phospholipase A2 (PLA2). Subsequent oxidative metabolism of this polyunsaturated fatty acid by the lipoxygenase pathway yields the leukotrienes, and by the cyclooxygenase (COX) pathway leads to prostanoid synthesis. Asthma exacerbations are characterized by increased leukotriene biosynthesis (Green et al. 2004). In 1984, Serhan, Samuelsson and their coworkers observed that labeled arachidonate was transformed by suspensions of mixed human leukocytes into polar compounds with physical properties distinct from prostaglandins, thromboxanes, and leukotrienes (Serhan et al. 1984). These compounds were named “lipoxins” (an abbreviation for “lipoxygenase interaction products”) as they are generated from arachidonic acid via sequential action of two or more lipoxygenases. Structurally distinct, the lipoxins carry a tetraene chromophorein and, in contrast to proinflammatory prostanoids and leukotrienes, have antiinflammatory actions and are known to have an important role in inhibiting the host response and promoting the resolution of asthma exacerbations.

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Phospholipase A2 PLA2 hydrolyzes the sn-2 ester bond of cell membrane glycerophospholipids to release free polyunsaturated fatty acids and generate lysophospholipids. The first mammalian PLA2 enzymes to be structurally characterized were those isolated from the pancreas and from the synovial fluid of rheumatoid arthritis (De Haas et al. 1968; Seilhamer et al. 1989). These were termed group IB and group IIA PLA2, respectively, based on their structural similarity to the already-characterized snake venom PLA2 enzymes. They were also termed secretory PLA2 due to the presence of a leader sequence predicting their intracellular targeting to the secretory pathway of the cell, which was validated by their presence in extracellular fluids (pancreatic secretions and synovial fluid, respectively). They are low-molecular-weight enzymes (∼ 14 kDa), are highly disulfide-linked with 14 cysteine residues, and use a catalytic histidine residue. They are distinguished from one another by the distribution of their cysteine residues, a carboxylterminal extension in the group IIA enzyme, and the “pancreatic loop” of the group IB enzyme (Schaloske & Dennis 2006) In addition, the group IB enzyme has an activation peptide that is cleaved by pancreatic trypsin. However, these enzymes are poor candidates for providing arachidonic acid as substrate for eicosanoid generation. They are secreted (not intracellular) enzymes, are active at high micromolar or millimolar concentrations of calcium, and have no specificity for arachidonic acid. Three major groups of PLA2 have been identified (Underwood et al. 1998; Pickard et al. 1999; Ohto et al. 2005): high-molecular-weight cytosolic PLA2 (cPLA2α), lowmolecular-weight secretory PLA2 (sPLA2), and calciumindependent PLA2 (iPLA2) isoforms.

cPLA2a When cytosolic PLA2 (cPLA2; group IV PLA2) was characterized it was considered a prime candidate for the enzyme initiating eicosanoid biosynthesis (Leslie et al. 1988; Clark et al. 1991). cPLA2α is an 85-kDa enzyme that uses a catalytic serine residue, is active at low micromolar concentrations of Ca2+, is present in the cytosol of cells that generate eicosanoids, and preferentially hydrolyzes phospholipids with arachidonic acid in the sn-2 position. cPLA2α translocates from the cytosol to the nuclear envelope, a prominent site of eicosanoid biosynthesis (Schievella et al. 1995). The activity of cPLA2α is regulated by phosphorylation at serine residues 505 and 727 by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 (BorschHaubold et al. 1998). The generation of mice in which the gene encoding cPLA2α was deleted by recombinant DNA technology confirmed its essential role in eicosanoid biosynthesis by leukocytes in response to a diverse array of stimuli (Bonventre et al. 1997; Uozumi et al. 1997). Mice lacking

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cPLA2α have attenuation of allergic pulmonary inflammation (Uozumi et al. 1997), acute lung injury in response to acid aspiration (Nagase et al. 2000), cerebral infarction following ischemia–reperfusion injury (Bonventre et al. 1997), and bleomycin-induced pulmonary fibrosis (Nagase et al. 2002a), as well as difficulties with fertilization and parturition (Uozumi et al. 1997). Five other isoforms of cPLA2α have been characterized whose biological roles have yet to be determined (Underwood et al. 1998; Pickard et al. 1999; Ohto et al. 2005).

sPLA2 The explosion of information on the human genome and the widespread use of in silico cloning in the 1990s led to the identification of multiple mammalian sPLA2 enzymes, each sharing a common catalytic motif (CCXXHDXC). The nomenclature built on what already existed (Schaloske & Dennis 2006), each new structure being assigned to a new group (Schaloske & Dennis 2006). Thus, the group III enzyme was so named due to the homology of its core to the group III hymenopteran PLA2, although the mammalian group III enzyme has long amino-terminal and carboxy-terminal extensions. Group V sPLA2 contains the six core disulfidelinked cysteine pairs of the group IB and IIA enzymes and no other cysteines (Chen, J. et al. 1994), whereas group X sPLA2 contains the six core cysteine pairs and both cysteine pairs characteristic of group IB and group IIA sPLA2, respectively (Cupillard et al. 1997). Group III sPLA2 and group XII sPLA2 are cysteine-rich but have a completely novel distribution of cysteine residues (Valentin et al. 2000; Ho et al. 2001). The diverse isoforms of sPLA2 enzymes do not merely serve redundant functions. They have widely differing tissue, cellular, and subcellular distributions (Bingham et al. 1999; Valentin et al. 1999). In contrast to cPLA2α most sPLA2 enzymes are released in the extracellular environment on appropriate cell activation. They also have different biochemical properties. PLA2 must be able to gain access to its substrate, which is an integral structural component of cell membranes. The group V and group X enzymes are the only isoforms of sPLA2 capable of directly binding to phosphatidylcholine, so-called interfacial binding, and can therefore release arachidonic acid from cell membrane phospholipids when applied exogenously or when secreted from the cell. Thus they have the potential for acting in a transcellular manner, being released from one cell and stimulating eicosanoid generation in a neighboring cell (Bezzine et al. 2000; Kim et al. 2002; Munoz et al. 2003). Three sPLA2 enzymes, groups IIA, IID, and V, are able to bind to cell surface proteoglycans. When transfected into transformed kidney cells, they are released from the cell on stimulation with A23187 or with interleukin (IL)-1β and serum, bind to glypican in caveolae, are internalized, and couple to COX for prostaglandin (PG)E2 biosynthesis (Murakami et al. 1998). The idea that sPLA2 enzymes can augment the essential function of cPLA2α in providing arachidonic acid for eicosanoid generation was indicated in transfection studies

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and by the actions of exogenous group V and group X sPLA2 (Murakami et al. 1998; Kim et al. 2002; Han et al. 2003; Munoz et al. 2003). Recent studies with transgenic mice lacking group V sPLA2 have demonstrated that it not only augments cPLA2αdependent eicosanoid generation in zymosan-stimulated macrophages (Satake et al. 2004) but it also regulates phagocytosis of zymosan (Balestrieri et al. 2006).

Nuclear membrane phospholipids Phospholipase A2 Arachidonic acid 5-lipoxygenase

5(S)-HPETE

Cyclooxygenase

iPLA2

Calcium-independent iPLA2 isoforms, like cPLA2α, use a catalytic serine residue but do not require calcium for activity (Wolf & Gross 1996; Balboa et al. 1997). They have been implicated in membrane remodeling (Balsinde et al. 1997), regulation of store-operated calcium channels (Smani et al. 2003) and the ion channel TRPM8 (Vanden Abeele et al. 2006), apoptosis (Zhang et al. 2006), regulation of insulin secretion (Ramanadham et al. 1999), and spermatogenesis (Bao et al. 2004), as well as in the release of arachidonic acid.

5(S)-HETE

Prostaglandins and thromboxanes

5-lipoxygenase Leukotriene A4 LTA4 hydrolase

LTC4 synthase

Leukotriene B4

Leukotriene C4

Platelet-activating factor acetylhydrolase The acetylhydrolases of platelet-activating factor (PAF) are PLA2 enzymes that cleave acetate from the sn-2 position of PAF. They also release F2-isoprostanes from phospholipids (Stafforini et al. 2006) and hydrolyze oxidized phospholipids (Marathe et al. 2003).

Gammaglutamyl transpeptidase Leukotriene D4

Slow reacting substance of anaphylaxis

Dipeptidases

Leukotrienes Biosynthesis Samuelsson conceived the term “leukotriene” to describe a family of compounds each containing three conjugated double bonds and derived from the conversion of arachidonic acid by 5-lipoxygenase (5-LO) in leukocytes. He subjected arachidonic acid to the actions of various lipoxygenases and found that rabbit polymorphonuclear leukocytes (PMNs) metabolized arachidonate to a family of dihydroxy acids that showed triple spectrophotometric absorption peaks at 259, 269 and 279 nm. These triple peaks suggested the existence of triple conjugated double bonds in these compounds. Arachidonic acid is a 20-carbon polyunsaturated fatty acid released from membrane phospholipids by the action of PLA2. The enzyme 5-LO catalyzes the first step in arachidonic acid metabolism (Fig. 26.1) by inserting oxygen at C-5 to produce the unstable intermediate 5S-hydroperoxyeicosatetraenoic acid (5-HPETE). This is either reduced to the alcohol 5Shydroxyeicosatetraenoic acid (5-HETE) or converted by 5-LO via a dehydrase step to a C-5,6-transepoxide with three conjugated (7,9-trans,11-cis) olefinic bonds, and a fourth, unconjugated double bond at C-14. This compound was the first leukotriene described and designated LTA4 (the subscript “4” refers to the number of carbon–carbon double bonds in the molecule). Subsequent metabolism of LTA4 takes place via two alternative enzymatic pathways: (i) via an epoxide

Leukotriene E4 Fig. 26.1 Leukotriene biosynthesis from nuclear membrane phospholipids and arachidonic acid via the 5-lipoxygenase pathway. 5(S)-HPETE, 5(S)-hydroperoxyeicosatetraenoic acid; 5(S)-HETE, 5(S)hydroxyeicosatetraenoic acid.

hydrolase to 5S,12R-dihydroxyeicosatetraenoic acid (LTB4) (S and R denote the chirality of the molecule at the 5 and 12 carbon atoms, respectively); or (ii) by opening the epoxide and conjugation of the tripeptide, glutathione, at C-6 by a glutathione S-transferase (S in this case refers to the sulfur moiety of glutathione) termed LTC4 synthase to LTC4. In the absence of either activated enzyme system, LTA4 degrades spontaneously to 6-trans-LTB4, which has significantly less bioactivity. LTC4 is exported from the cell by the multidrug resistance-associated protein (MRP)1 (Leier et al. 1994; Muller et al. 1994). After its export from the cell, LTC4 is subsequently cleaved to form the 6R-S-cysteinylglycine analog LTD4 by removal of glutamic acid from the peptide by γglutamyl-transpeptidase (Leier et al. 1994; Muller et al. 1994). LTD4 is further cleaved by a dipeptidase to remove glycine to form its 6R-S-cysteinyl analog (LTE4). These latter three compounds are known collectively as the cysteinyl leukotrienes, because of the presence of a cysteine linked to the eicosanoid backbone through a thiolether link at C-6. The chemical

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O COOH

Leukotriene A4 OH

OH COOH

Leukotriene B4 OH COOH S Leukotriene C4

Cys

Gly

Glu OH COOH S

Cys

Gly

Leukotriene D4 OH COOH S

Cys

Leukotriene E4 Fig. 26.2 Chemical structure of the leukotrienes.

structure of the leukotrienes is shown in Fig. 26.2. The cysteinyl leukotrienes comprise the activity previously recognized as SRS-A. Although the enzyme 5-LO is the rate-limiting step for leukotriene formation, it requires the association of an additional factor. Mammalian osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations but no leukotriene metabolites on stimulation with the calcium ionophore A23187 (Rouzer et al. 1988). In addition, a novel indole leukotriene biosynthesis inhibitor MK-886 was found to act only on intact PMNs but had no direct inhibitory effect on soluble 5-LO activity (Gillard et al. 1989). An 18-kDa membrane protein with high affinity for MK-886 was identified from rat and human leukocytes, and it was demonstrated that expression of both 5-LO and this MK-886 binding protein was necessary for leukotriene synthesis. This was confirmed by its specific labeling with a 125I-radiolabeled photoaffinity

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probe and by its retention on agarose gels to which analogs of MK-886 had been bound. This protein was termed “5lipoxygenase-activating protein” (FLAP) (Miller et al. 1990). In osteosarcoma cells transfected with 5-LO or FLAP alone and stimulated with the calcium ionophore A23187, no arachidonic acid metabolites were detected. In contrast, A23187 treatment of cell lines expressing both 5-LO and FLAP resulted in significant production of 5-LO products (Dixon et al. 1990). The human promyelocytic cell line U937 expresses FLAP but not 5-LO, and is unable to synthesize leukotrienes after A23187 stimulation but does so after transfection by a retroviral vector encoding 5-LO mRNA (Kargman et al. 1993). Human B-lymphocyte lines and normal human tonsillar B lymphocytes have 5-LO activity and can produce LTB4 on cell activation (Jakobsson et al. 1991), but five T-cell lines were found to express only FLAP and not 5-LO and therefore were unable to generate leukotrienes on cell activation (Jakobsson et al. 1992). The mechanism of cellular activation and subsequent leukotriene generation is believed to arise as a result of translocation of 5-LO from cytosolic to membrane compartments in order to bring the enzyme into close proximity with FLAP, which acts as an anchor for 5-LO and provides access to substrate and other components of leukotriene synthesis. In osteosarcoma cells expressing 5-LO but not FLAP, 5-LO is able to associate with cell membranes following A23187 stimulation and this is not inhibited by MK-886. This led the authors to conclude that 5-LO membrane association and activation can be divided into a two-stage process: (i) Ca2+dependent movement of 5-LO to membrane without product formation which can occur in the absence of FLAP; and (ii) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (Kargman et al. 1992). High levels of FLAP were expressed in SF9 insect cells transfected with recombinant baculovirus, and this system was used to demonstrate that FLAP specifically binds [125I]L739,059, a photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886 and suggests that FLAP may activate 5-LO by bringing enzyme and substrate together (Mancini et al. 1993). Ultracentrifugation (Peters-Golden & McNish 1993) and immunoelectron microscopy studies have demonstrated that on cellular activation 5-LO translocates to the nuclear envelope, which is also the location of FLAP (Woods et al. 1993). The situation in resting cells is still variable, and 5-LO may be located in the cytosol (Rouzer & Kargman 1988) or nuclear euchromatin (Woods et al. 1994).

Cellular sources Because of the requirement to express both 5-LO and FLAP, the range of cells known to synthesize leukotrienes is limited. Cells of myeloid lineage provide the major source of leukotriene production, although B lymphocytes have also been shown to be capable of generating small quantities of

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Table 26.1 Cellular source of human leukotriene generation. Predominant leukotriene product

Activating stimulus

Approximate amount generated/106 cells

Neutrophils

LTB4 LTC4

A23187 A23187

50 ng 10 ng

Eosinophils

LTB4 LTC4

A23187 A23187

10 ng 40 ng

Peripheral blood monocytes

LTB4 LTC4

A23187 A23187

70 ng 30 ng

Alveolar macrophages

LTB4

A23187

30 ng

Mast cells

LTC4

A23187/ or IgE antigen

20 ng

Basophils

LTC4

A23187/ or IgE antigen

10 ng

Cell

A23187, divalent calcium ionophore.

leukotrienes (Jakobsson et al. 1991). Cells can be divided by their ability to preferentially synthesize LTB4 or the cysteinyl leukotriene LTC4, depending on the intracellular predominance of either LTA4 epoxide hydrolase or LTC4 synthase. Monocytes, alveolar macrophages, and peripheral blood neutrophils preferentially generate LTB4, whereas eosinophils, mast cells, and basophils preferentially generate cysteinyl leukotriene products (Table 26.1). Monocytes and macrophages have the capacity to generate both LTB4 and LTC4. Human peripheral blood monocytes in adherent monolayers produce 68 ng LTB4 and 30 ng LTC4 per 106 cells on stimulation with the calcium ionophore A23187 (Williams et al. 1984). LTB4 is the major lipoxygenase product of human alveolar macrophages, which generate 17–30 ng per 106 cells in response to ionophore A23187 stimulation (Fels et al. 1982). Peripheral blood neutrophils from normal donors produce about 48 ng LTB4 per 106 cells, with only oneseventh as much LTC4 per 106 cells after A23187 stimulation. Eosinophils from the same subjects preferentially synthesize LTC4, producing 38 ng LTC4 per 106 cells and about 6 ng LTB4 per 106 cells in response to A23187 stimulation (Weller et al. 1983). Activation of leukocytes is accompanied by respiratory burst activity, with reduction of oxygen to form superoxide anion (O 2–) and hydrogen peroxide (H2O2) and the release of cytoplasmic contents, including eosinophil peroxidase (EPO) from eosinophils and myeloperoxidase (MPO) from neutrophils or monocytes. This leads to oxidative degradation of the cysteinyl leukotrienes and acts as a control on their biological effects. EPO or MPO catalyze the oxidation of halides by H2O2 to form hypohalous acids such a HOCl, HOBr, or HOI, which readily degrade cysteinyl leukotrienes to their inactive sulfoxide derivatives and to 6-trans-stereoisomers

of LTB4 (Henderson et al. 1982; Henderson & Klebanoff 1983). Human lung mast cells release 22 ng LTC4 per 106 cells, with similar amounts released in response to either IgE- or calcium ionophore A23187-mediated degranulation (Peters et al. 1984). Basophils obtained from the peripheral blood of patients with chronic myelogenous leukemia were found to release LTB4 (Rothenberg et al. 1987). Basophils also release LTC4 in response to cross-linking of FcεRI or engagement of leukocyte immunoglobulin-like receptor A2, but unlike mast cells do not produce PGD2 (MacGlashan et al. 1986; Sloane et al. 2004). Local instillation of specific allergen into the nasal mucosa of sensitive subjects leads to recovery of histamine, PGD2, and LTC4 from nasal secretions during the early-phase reaction, but only histamine and LTC4 during the late-phase reaction. These findings suggest that mast cell degranulation is important in the early-phase response to allergen and that basophils mediate the late reaction (Naclerio et al. 1983; Creticos et al. 1984). B lymphocytes produce only small amounts of LTB4 and 5-HETE on stimulation with A23187, in comparison with the amounts produced by the sonicates of these cells. However, preincubation with a glutathionedepleting agent prior to stimulation leads to similar amounts of LTB4 generated by the intact cells as are formed by the sonicated cells. T lymphocytes express the FLAP gene but not the 5-LO gene and therefore do not generate leukotrienes (Jakobsson et al. 1992). Guinea-pig lung parenchyma has been shown to convert LTA4 to LTB4, LTC4, LTD4, and LTE4 (Sirois et al. 1985) and human lung parenchyma converts LTC4 to LTD4 and LTE4 (Aharony et al. 1985; Conroy et al. 1989). Unlike 5-LO, LTC4 synthase and LTA4 hydrolase, which convert LTA4 to LTC4 and LTB4 respectively, are widely distributed. Thus, LTA4 released by a cell capable of generating

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15–LO

Endothelial cell

AA

AA LTB4

15–HPETE

5–HPETE

trans locat

ion

5–LO/FLAP complex (active)

15–LO

5–LO

AA

LTA4 hydrolase

Erythrocyte LTA4 LTA4

FLAP LTB4

Epithelial cell

LTC4 synthase

5–LO

Platelet

12–LO

Neutrophil

LXA4 LXB4

LTC4 LXA4

LXB4

Fig. 26.3 Transcellular biosynthesis of leukotrienes and lipoxins by cell–cell interaction between polymorphonuclear (PMN) leukocytes and platelets for cysteinyl leukotriene production or PMN leukocytes and either platelets, epithelial or endothelial cells for lipoxin generation. Human 5-hypoxygenase (5-LO) is located in the cytosol or nuclear euchromatin of the resting cell, and on activation is translocated to form a complex with 5-LO activating protein (FLAP) to express enzymatic activity. Endothelial and epithelial cells contain 15-LO and platelets contain both 12-LO and leukotriene C4 (LTC4) synthetase activity.

eicosatetraenoic acid (20-COOH-LTB4). 20-CHO-LTB4 can be converted via an alternative pathway by an aldehyde reductase in the microsomes back to 20-OH-LTB4, thereby allowing tightly regulated LTB4 degradation. LTC4 can be bioconverted by three major metabolic pathways. The first comprises conversion of LTC4 by peptide cleavage to LTD4 and then to LTE4 by the action of γ-glutamyl transpeptidase and one of a variety of dipeptidases, respectively. This does not require activation of the cells involved and may only result in some loss of biological activity. The second pathway involves oxidative metabolism of the cysteinyl leukorienes and depends on the production of hydrogen peroxide by the respiratory burst and secretion of cell-specific peroxidase, which occurs only in the extracellular microenvironment of activated neutrophils, eosinophils, and monocytes. A peroxidase–hypochlorous acid reaction then transforms LTC4, LTD4, and LTE4 to their respective Sdiastereoisomeric sulfoxides and to the 6-trans-diastereoisomers of LTB4. Each of the S-diastereomeric sulfoxides is then converted to its respective sulfone, which results in considerable loss of bioactivity. The 6-trans-LTB4 diastereoisomers are not immunoreactive in a sulfidopeptide leukotriene assay and are nonspasmogenic. The sulfoxides are fully immunoreactive but possess less than 5% spasmogenic activity. The third pathway involves β-oxidation and elimination leading to carboxylation, hydroxylation, and the gradual shortening of the ω-segment of the molecule. A fraction of infused LTE4 is excreted unchanged in urine and because this fraction remains constant, urinary concentrations of LTE4 have been used to reflect systemic synthesis of LTC4. It is likely that cellular activation leads not only to enhanced leukotriene release but also to increased capacity to degrade them.

Regulation of leukotriene generation 5-LO products may be utilized for further metabolism by other cells without the enzyme 5-LO. Such transcellular metabolism can lead to the generation of LTC4 by platelets (Edenius et al. 1988; Maclouf & Murphy 1988), mast cells (Dahinden et al. 1985), airway epithelium and vascular endothelial cells (Feinmark & Cannon 1986), or to generation of LTB4 by erythrocytes from LTA4 supplied by neutrophils (McGee & Fitzpatrick 1986) (Fig. 26.3).

Leukotriene metabolism and elimination Removal of leukotrienes from sites of inflammation requires rapid inactivation by specific and appropriately located enzymes. LTB4-20-hydroxylase (P450LTB) is located exclusively in neutrophil microsomes and converts LTB4 by ωoxygenation to 20-OH-LTB4, which has substantially less PMN chemotactic and activating activity than LTB4. Further metabolism takes place by oxidation to 5S,12R-dihydroxy20-aldehyde-6,14-cis-8,10-trans-eicosatetraenoic acid (20CHO-LTB4), which has no biological activity. This is followed by irreversible conversion to 20-carboxy-6,14-cis-8,10-trans-

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The generation of lipoxygenase products is tightly controlled and is subject to regulation at several levels. Firstly, the expression of individual lipoxygenase pathway enzymes is subject to developmental and cytokine regulation. This is illustrated by the requirement for IL-3 for the full expression of 5-LO, FLAP, and LTC4 synthase in developing mouse mast cells (Murakami et al. 1995). The generation of LTC4 in human culture-derived mast cells was upregulated by IL-4, which markedly induced the expression of LTC4 synthase (Hsieh et al. 2001). Leukotriene generation was further upregulated by IL-5, which induced the redistribution of 5-LO to the nucleus (Hsieh et al. 2001). 5-LO is a pivotal point of the cross-talk between the 5-LO and COX pathways. PGE2, through the induction of adenosine 3′,5′-cyclic monophosphate (cAMP) and phosphorylation of 5-LO, regulates both the activity of the enzyme and its subcellular location. Thus, phosphorylation of 5-LO by protein kinase A (PKA) at serine 523 leads to inhibition of 5-LO activity (Luo et al. 2004). Substitution of Ser with Ala abolished the effect of PKA, whereas substitution with glutamate

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to mimic the effects of phosphorylation led to inhibition of 5-LO activity and cellular leukotriene generation. In addition to reducing the intrinsic catalytic activity of the enzyme, PKA phosphorylation at Ser 523 leads to redistribution of 5-LO from the nucleus to the cytoplasm (Luo et al. 2005), an event associated with reduced cellular capacity for leukotriene biosynthesis (Luo et al. 2003). It may be of note that Ser 523 is located within a putative nuclear import sequence (Jones et al. 2003).

In addition to the perinuclear region, lipid bodies have been implicated as a site of eicosanoid biosynthesis. The evidence for this is based on the following observations: 1 Lipid body formation is induced in leukocytes in response to cis-unsaturated fatty acids (Bozza et al. 1996) and in eosinophils in response to PAF (Bozza et al. 1997) and the chemokines eotaxin and RANTES (Bandeira-Melo et al. 2001). IL-16 via release of endogenous chemokines (Bandeira-Melo et al. 2002a) or PGD2 (Mesquita-Santos et al. 2006) is associated with increased leukotriene generation. 2 The enzymes of eicosanoid biosynthesis (cPLA2α, 5-LO, LTC4 synthase, COX) can be detected in lipid bodies by immunofluorescence labeling (Bozza et al. 1997; Yu et al. 1998). 3 Immunostaining of carbodiimide-fixed cells reveals cysteinyl leukotriene formation in lipid bodies, for example in chemokine-primed eosinophils in response to A23187 (Bandeira-Melo et al. 2001). In addition, the upstream signaling molecules phosphoinositide 3-kinase, ERK, and p38 MAPK are present in lipid bodies and are required for lipid body-associated leukotriene generation (Yu et al. 1998; Bandeira-Melo et al. 2001).

Sites of leukotriene biosynthesis As noted above, the perinuclear region is well established as a prominent site of eicosanoid biosynthesis. cPLA2α translocates from its cytosolic location to the nuclear envelope or Golgi apparatus in response to a calcium flux in a manner that depends on its C2 (calcium-binding) domain. Likewise, 5-LO translocates from either the cytosol or the nucleosol to the nuclear envelope in response to intracellular calcium mobilization. FLAP and LTC4 synthase are integral perinuclear membrane proteins that are able to form heterotrimers (Mandal et al. 2004) (Fig. 26.4).

Plasma membrane

Cytosol

Nuclear envelope

Nucleus

ER

COX-1

export PGD2

PGH2 hPGDS

SCF

AA

c-kit AA cPLA2

Ca2+

IgE Ag

AA

PO4

FceRI

FLAP 5-LO

FceRI

2+

LTA4 GSH

IgE export

Fig. 26.4 Subcellular regulation of eicosanoid biosynthesis depicting the pathways of LTC4 and PGD2 in the mast cell. The calcium flux elicited by cross-linking of FceRI or c-kit elicits translocation of cytosolic phospholipase A2a (cPLA2a) to the nuclear envelope where it releases esterified arachidonic acid (AA) from the sn-2 position of membrane glycerophospholipids; the enzymatic activity of cPLA2a is augmented by phosphorylation by MAP kinases. 5-Lipoxygenase (5-LO) likewise translocates from either a nuclear or a cytoplasmic location to the nuclear envelope in response to Ca2+ flux. AA is presented to 5-LO by the integral

Ca LTC4 synthase

LTC4

perinuclear membrane protein 5-LO activating protein (FLAP), and metabolizes AA to the unstable epoxide intermediate leukotriene LTA4. LTC4 synthase, also an integral perinuclear membrane protein, conjugates glutathione (GSH) to LTA4 to generate LTC4 that is exported from the cell by the multidrug resistance transporter, MDR1. Constitutively expressed cyclooxygenase (COX)-1 and inducible COX-2 are present in the nuclear envelope and contiguous endoplasmic reticulum (ER); they metabolize AA to the unstable intermediate PGH2 which is metabolized to PGD2 by hematopoietic PGD synthase (hPGDS).

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The ability of different stimuli to elicit LTC4 formation at different subcellular location in the eosinophils is illustrated by the observation that CCL11 (eotaxin) elicits cysteinyl leukotriene formation in lipid bodies where stimulation via stimuli that elicit calcium flux, such as A23187 or leukocyte immunoglobulin-like receptor A2, elicits cysteinyl leukotriene formation in a perinuclear distribution (Tedla et al. 2003). The relevance of lipid body formation as a site of leukotriene biosynthesis is suggested by the observations that lipid bodies are induced in eosinophils within the lungs of allergenchallenged mice, and in the leukocytes of lipopolysaccharide (LPS)-challenged mice (Vieira-de-Abreu et al. 2005) and the leukocytes of patients with sepsis (Pacheco et al. 2002). The evidence supports the concept that lipid bodies are rapidly inducible, specialized cytoplasmic domains for eicosanoid generation at sites of inflammation. Recent reports reveal that cPLA2α, 5-LO, FLAP, LTC4 synthase, and COX-2 translocate to the phagosome of mouse resident peritoneal macrophages after ingestion of zymosan (Girotti et al. 2004; Balestrieri et al. 2006) and that LTC4 formation was revealed at the phagosome by immunostaining of cardbodiimide-fixed macrophages after ingestion of zymosan.

Biological activity LTB4 LTB4 acts at the level of the microvasculature by increasing leukocyte adherence to endothelium (Hoover et al. 1984) and enhancing cutaneous microvascular permeability (Bray et al. 1981; Issekutz 1981). Other activities include potent chemotaxis for neutrophils (Ford-Hutchinson et al. 1980), mediated by a subset of high-affinity receptors, and chemokinetic and weak chemotactic activity for eosinophils (Palmer et al. 1980; Nagy et al. 1982) (Table 26.2). Consistent with the

cheomotactic activity of LTB4 for PMNs, mice transgenic for the high-affinity LTB4 receptor (BLT1) exhibited increased PMN recruitment in sites of ischemia–reperfusion (Chiang et al. 1999), whereas PMN accumulation was attenuated in mice lacking 5-LO. Infusion of aspirin-triggered lipoxins attenuated PMN accumulation into the lungs following hindlimb ischemia–reperfusion in BLT1 transgenic mice, and topical lipoxin A4 attenuated PMN accumulation at the site of dermal inflammation elicited by LTB4 (Chiang et al. 1999). Recent studies have extended our understanding of the chemotactic capacity of LTB4 beyond granulocytes and cells of the monocyte lineage. Thus LTB4 shows chemotactic activity to progenitor, but not mature, mast cells (Weller et al. 2005). Mature mast cells are not normally found in the circulation. Rather, they circulate as immature progenitors (Arinobu et al. 2005) that differentiate further on recruitment to tissues (Gurish et al. 2001) where they undergo phenotypic maturation that is dependent on the tissue microenvironment (Friend et al. 1996). These studies suggest that LTB4 may regulate the trafficking of progenitor mast cells and reactive mastocytosis at sites of inflammation. BLT1 is expressed at low levels on naive circulating T cells, and is upregulated substantially in CD8+ effector T cells (TEFF) (Goodarzi et al. 2003) and on CD4+ T cells challenged with antigen in nonskewing conditions, or in conditions that favor the development of Th1 or Th2 cells (Tager et al. 2003). LTB4 is a potent chemotactic factor both in vitro and in vivo for effector but not memory CD8+ T cells (Goodarzi et al. 2003). LTB4 augmented adhesion, but not rolling, of both TEFF and central memory CD8+ T cells in the microvasculature of tissue inflammation induced by tumor necrosis factor (TNF)-α and of Th1 and Th2 cells on mouse lung endothelial cells. LTB4 is chemokinetic for monocytes (Palmer et al. 1980), enhances expression of CR1

Table 26.2 Biological activity of LTB4. Activity

Reference

Increase leukocyte adherence to endothelium Increase microvascular permeability (secondary action) Neutrophil chemotaxis and weak eosinophil chemotaxis Monocyte chemokinesis Enhanced expression of CR1 and CR3 receptors on neutrophils and eosinophils Neutrophil aggregation Lysosomal release and superoxide generation Bronchoconstriction via secondary release of TXA2 Enhanced NK cell cytotoxicity for virus-infected and tumor cells Induction of suppressor T lymphocytes Enhances IgE production by human B lymphocytes and augments IL-2 and IFN-g production by T lymphocytes

Hoover et al. (1984) Bray et al. (1981); Issekutz (1981) Palmer et al. (1980) Palmer et al. (1980) Nagy et al. (1982); Lee et al. (1988)

See text for definition of abbreviations.

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Ford-Hutchinson et al. (1980) Goldman & Goetzl (1984) Sirois et al. (1982) Rola-Pleszczynski (1985) Atluru & Goodwin (1986) Rola-Pleszczynski et al. (1986)

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and CR3 (CD11b/CD18) receptors on neutrophils and eosinophils (Nagy et al. 1982; Lee et al. 1988), aggregates neutrophils (Ford-Hutchinson et al. 1980), and releases lysosomal enzymes and generates superoxide via a subset of low-affinity receptors (Goldman & Goetzl 1984). LTB4 is bronchoconstricting, especially when administered intravenously, and this action is mediated indirectly through the action of thromboxane (TX)A2 synthesized locally in lung tissue (Sirois et al. 1982). LTB4 also has immunoregulatory functions. At concentrations of 10 −12 to 10 −8 mol/L, LTB4 significantly enhances natural killer cytotoxicity for virus-infected cells and the tumor cell line K-562 (Rola-Pleszczynski 1985), and induces increased numbers of suppressor T lymphocytes from precursors (Atluru & Goodwin 1986). It also directly increases IgG production by highly purified human tonsillar B lymphocytes and augments IL-2 and interferon (IFN)-γ production in human T lymphocytes (Rola-Pleszczynski et al. 1986). The importance of leukotrienes in PAF-induced shock was initially revealed in 5-LO knockout mice (Chen, X.S. et al. 1994). Interestingly, survival in response to PAF was increased in female but not male mice lacking BLT1 (Haribabu et al. 2000). The importance of autocrine LTB4 in regulating leukocyte responses to PAF was revealed in human PMNs in which the second phase of Ca2+ mobilization was inhibited by LTB4 pretreatment (that would desensitize LTB4 receptors) or a BLT1 receptor antagonist (Gaudreault et al. 2005). BLT1 receptor antagonists also inhibited PAF-induced PMN degranulation and chemotaxis. In human PMNs, LTB4 acts in an autocrine manner to elicit Ca2+ flux, phospholipase D activation, and translocation of the small GTPases ARF1 and RhoA (Grenier et al. 2003).

Cysteinyl leukotrienes The cysteinyl leukotrienes have numerous biological effects (Table 26.3). LTD4, and with lesser potency LTC4 and LTE4, augment postcapillary dermal venular permeability when

administered locally to guinea pigs, as shown by the leakage of intravenously administered dye (Drazen et al. 1980; Lewis et al. 1980b). The cysteinyl leukotrienes increase microvascular permeability influenced by the contraction of adjacent endothelial cells (Drazen et al. 1980; Joris et al. 1987) and augment leukocyte adhesion to endothelial cells (McIntyre et al. 1986). Local application of the cysteinyl leukotrienes to the buccal mucosa produces submucosal edema in hamsters (Dahlén et al. 1981) and subcutaneous injection in humans produces dermal edema sustained for 2–4 hours (Soter et al. 1983). As little as 1 nmol/L LTC4 and LTD4 have been postulated to stimulate mucus secretion from studies of bronchial mucosal explants, and this may contribute to the excess mucus secretion found in bronchial asthma (Marom et al. 1982). The cysteinyl leukotrienes are potent contractile agonists for bronchial smooth muscle in isolated human lobar and segmental bronchi, with LTC4 and LTD4 reported to be approximately 1000 times more potent than histamine in contracting human bronchi in vitro (Dahlén et al. 1980) (Table 26.3). Studies using inhaled cysteinyl leukotrienes have demonstrated the potency of these mediators, in both normal and asthmatic subjects, in causing bronchoconstriction, with LTC4 being the most potent and LTE4 the least active (Adelroth et al. 1986; Drazen 1988; Arm et al. 1990). LTC4 and, to a lesser extent, LTD4 have demonstrated vasoconstrictor properties in guinea-pig skin after intradermal injection (Drazen et al. 1980; Lewis et al. 1980b) and in normal human skin by blanching of an elicited weal at the injection site (Camp et al. 1983; Soter et al. 1983). Coronary vasoconstriction has been demonstrated in sheep after direct infusion into a coronary vessel in vivo (Michelassi et al. 1982). In the rat, intravenous infusion of LTC4 led to systemic vasoconstriction (Pfeffer et al. 1983) and renal vasoconstriction (Badr et al. 1984). LTE4 has been demonstrated to recruit granulocytes and particularly eosinophils into the lamina propria of asthmatic airways (Laitinen et al. 1993) (Fig. 26.5).

Table 26.3 Biological activity of cysteinyl leukotrienes. Activity

Reference

Augment postcapillary dermal vascular permeability in guinea pigs Increased microvascular permeability Increase leukocyte adhesion to endothelial cells Increase mucus secretion Potent contractile agonists for bronchial smooth muscle Vasoconstriction Coronary vasoconstriction in sheep Systemic vasoconstriction in rats Renal vasoconstriction in rats Recruit granulocytes into the lamina propria of asthmatic airways (LTE4)

Drazen et al. (1980); Lewis et al. (1980a) Drazen et al. (1980); Joris et al. (1987) McIntyre et al. (1986) Marom et al. (1982) Dahlén et al. (1980) Camp et al. (1983); Soter et al. (1983) Michelassi et al. (1982) Pfeffer et al. (1983) Badr et al. (1984) Laitinen et al. (1993)

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1000

Cell numbers/mm2

100

10

1

Pre

Post LTE4

Pre Post Methacholine

Fig. 26.5 Change in the number of eosinophils (closed circles) and neutrophils (open circles) in the lamina propria of airways mucosa before and after provocation with inhaled LTE4 or methacholine. (From Laitinen et al. 1993, with permission.)

Receptors The leukotriene receptors were first characterized based on their relative potencies in tissue and cellular assays and on the actions of novel pharmacologic antagonists. As in the case of the prostanoid receptors, that classification has been retained with the molecular characterization of the leukotriene receptors (Coleman et al. 1995).

LTB4 receptors The LTB4 receptor was initially identified in PMNs and later demonstrated in eosinophils, monocytes, and lymphocytes (Rola-Pleszczynski et al. 1986). Scatchard analysis of the binding of [3H]LTB4 to freshly isolated PMNs demonstrated the expression of two distinct subsets of receptors: a highaffinity (Kd 0.5– 5 nmol/L), low-density (20 × 103 sites per neutrophil) receptor and a low-affinity (Kd 15–500 nmol/L), high-density (40– 400 × 103 receptors per neutrophil) receptor (Goldman & Goetzl 1982). These two receptor classes for LTB4 also differ in their stereospecificity for stereoisomers of 5,12-diHETE. A decreased chemotactic response to subsequent LTB4, termed “deactivation,” was achieved by preincubating PMNs with 10 nmol/L LTB4 followed by washing and this was paralleled by a selective loss of high-affinity receptors, indicating that the high-affinity receptor mediates chemotactic migration and aggregation. The elicitation of degranulation

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BLT1 The high-affinity cell-surface receptor for LTB4, BLT1, was identified using a cDNA subtraction strategy (Yokomizo et al. 1997). HL-60 cells treated with retinoic acid differentiate into granulocytes that exhibit increased LTB4 binding. Therefore a subtraction cDNA library was generated using HL-60 cells cultured with and without retinoic acid. Of 66 clones that were sequenced, one encoded the 3′ untranslated region of an orphan G protein-coupled receptor (GPCR). Using this original cDNA as a probe, two identical full-length cDNA clones were isolated from a cDNA library of retinoic-acid differentiated HL-60 cells. Northern blotting revealed that mRNA for BLT1 was strongly expressed in peripheral blood leukocytes. The cDNA for BLT1 was most closely related to the type 3 and type 5 somatostatin receptors, the human IL-8 receptor, the formyl-peptide-related receptor/lipoxin A4 receptor, and the formyl peptide receptor. A variety of cell lines, including COS-7 cells and HEK-293 cells transfected with the BLT1 cDNA and retinoic acid-differentiated HL-60 cells, demonstrated high-affinity specific binding of LTB4 (Kd 0.1–0.25 nmol/L). Binding of [3H]LTB4 to membrane fractions of transfected COS-7 cells revealed competition by LTB4, 20OH-LTB4, 12-oxo-LTB4, 12-(R)-HETE, and 20-COOH-LTB4, with KI values of 0.38, 7.6, 7.6, 30, and 190 nmol/L, respectively. In CHO cells stably expressing BLT1, LTB4 inhibited forskolin-induced elevations in cAMP and this inhibition was blocked by pertussis toxin. LTB4 also elicited calcium flux and inositol 1,4,5-trisphosphate (IP3) accumulation in a manner that was partially sensitive to inhibition by pertussis toxin. In contrast, calcium fluxes induced in HL-60 cells by LTB4 are completely blocked by pertussis toxin, indicating that BLT1 can couple to different G proteins. LTB4 was both chemotactic and chemokinetic for transfected CHO cells and was inhibited by pertussis toxin and by the BLT1 receptor antagonists U-75302 and ONO-4057. The cDNA for BLT1 was identical to that for a putative purinergic receptor, P2Y7. However, C6-15 glioma cells expressing the BLT1 cDNA responded with intracellular calcium flux to LTB4 but not to ATP. The BLT1 gene lacks TATA and CAAT boxes in the 5′ flanking region. Rather, there is a GC element in the promoter

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region that binds SP1 (Kato et al. 2000). Expression of the gene is regulated by methylation. Thus there is methylation of the CpG sites in the promoter region in nonhematopoietic cells that lack expression of BLT1. Structure and function of BLT1 A low-resolution structural characterization of the structure of BLT1 expressed in Escherichia coli was compatible with seven transmembrane helices (Baneres et al. 2003), with evidence for conformational change on ligand binding. In addition to its seven transmembrane helices, BLT1 is predicted to have a C-terminal helix similar to that observed in the crystal structure of rhodopsin, but without the cysteine residue that is palmitoylated in the rhodopsin receptor to tether it to the plasma membrane. Absence of helix 8 (Okuno et al. 2003) increases the affinity of BLT1 for LTB4 and prevents ligandinduced lowering of the affinity of BLT1 for its ligand, leading to prolonged calcium flux in response to LTB4 (Okuno et al. 2003). Mutation of the di-leucine motif (Leu 304/305) in the C-terminal tail of BLT1 led to constitutive activation and failure of ligand-dependent internalization (Gaudreau et al. 2004). BLT1 is desensitized by protein kinase C (PKC) and G protein receptor-coupled kinases (GRKs). Thus internalization of BLT1 expressed in RBL-2H3 cells was blocked by dominant negative GRK2 and promoted in HEK-293 cells cotransfected with GRK2 (Chen et al. 2004). Deletion mutants revealed the importance of the C-terminal tail for association with GRK2 and internalization (Chen et al. 2004). Substitution of Thr 308 within helix 8 prevented GRK-6 mediated inhibition of LTB4-induced phospholipase C activation (Gaudreau et al. 2002). The importance of dimerization of BLT1 for its function was demonstrated using a mutant carrying C97S, which has 100-fold reduced affinity for LTB4 compared with the wild-type receptor, and a fluorescent 5-hydroxytryptophan probe at residue 234. The mutant receptor was expressed as a dimer with the wild-type receptor. Ligation of the wild-type receptor with low concentrations of LTB4 elicted a conformational change in the mutant, unliganded receptor, revealing cooperativity of the dimers. Reconstitution of the BLT1/Gprotein complex using partially purified cell preparations of each (Igarashi et al. 1999) or recombinant proteins expressed in E. coli or in insect cells (Baneres & Parello 2003; Masuda et al. 2003) demonstrated that the affinity of BLT1 is regulated by its association with heterotrimeric G proteins (Igarashi et al. 1999; Baneres & Parello 2003), specifically Gi and Go but not Gs (Masuda et al. 2003). Cross-desensitization of BLT1 by CXC chemokines was suggested in a study of BLT1 expression in patients with trauma. BLT1-dependent chemotaxis of human PMNs was reduced in patients after severe trauma, concomitant with increased circulating levels of CXCL8 (IL-8). Pretreatment of human PMNs with CXCL8 or CXCL1 (Gro-α) led to reduced Ca2+ flux in response to subsequent stimulation with subnanomolar concentrations of LTB4, suggesting cross-desensitization

of BLT1 by CXCR1 and CXCR2 (Tarlowe et al. 2003). LTB4 did not downregulate Ca2+ flux to either CXCL8 or CXCL1. The expression of BLT1 is limited to leukocytes. Expression of BLT1 is increased on human PMNs in response to dexamethasone and also, surprisingly, in response to LTB4 (Stankova et al. 2002). Both dexamethasone and LTB4 prolong the survival of PMNs in culture, and their actions are inhibited by a BLT1 antagonist. Expression on monocytes is reduced on CD14+/CD16+ cells (Pettersson et al. 2005), and is diminished on exposure to the proinflammatory cytokines IFN-γ, TNF-α, and LPS, and increased in response to IL-10 or dexamethasone (Pettersson et al. 2005). In contrast, expression of BLT1 in human umbilical vein endothelial cells (HUVECs) was increased more than 10-fold by LPS or IL-1β (Qiu et al. 2006) and LTB4 elicited the generation of NO and CCL2 (MCP-1) from HUVECs (Qiu et al. 2006).

BLT2 The gene encoding BLT2 was identified during characterization of the gene encoding BLT1 (Kamohara et al. 2000; Tryselius et al. 2000; Wang et al. 2000; Yokomizo et al. 2000). The gene encoding BLT2 was identified within the promoter region of the BLT1 gene. In contrast to the restricted distribution of BLT1 in leukocytes, Northern analyses indicate that BLT2 mRNA is more widely expressed, with prominent expression in spleen, ovary, and liver as well as leukocytes, with lower expression in most tissues examined. [3H]LTB4 bound specifically to membranes of HEK-293 cells transfected with BLT2 cDNA (Kd 23 nmol/L). Binding was inhibted by the BLT1 receptor antagonist ONO 4057, but not by U-75302. In CHO cells stably expressing BLT2, LTB4 elicited calcium flux. The maximal response of cells expressing BLT2 was approximately one-third that of cells expressing BLT1 and required about 100-fold higher concentration of LTB4. BLT2, in addition to its lower affinity for LTB4 compared with BLT1, also recognizes a wider range of ligands (Yokomizo et al. 2001). Thus, LTB4 binding was competed at BLT2 by 12-(S)-HETE, 12-(R)-HETE, 15-(S)-HETE, as well as 20-OH-LTB4 and 20-epi-LTB4. 20-OH-LTB4 and 12-HETE and 15-HETE at concentrations up to 1 μmol/L did not elicit calcium flux from CHO cells transfected with BLT2. LTB4induced chemotaxis of CHO cells transfected with BLT2 was completely inhibited by pertussis toxin, whereas calcium fluxes were only partially inhibited. As with BLT1, LTB4 did not elicit an increase in cAMP in BLT2-transfected CHO cells, but did inhibit the rise in cAMP elicted by forskolin that was pertussis toxin insensitive (in contrast to BLT1). Thus, BLT2 signals by coupling to Gi and Gq and Gz (Kamohara et al. 2000).

Cysteinyl leukotriene receptors The evidence for separate cysteinyl leukotriene receptors was initially supported by differences in biological activity of the individual leukotrienes, the effects of leukotriene receptor

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antagonists, and by radioligand binding studies. Much of this evidence came from work carried out in the guinea-pig lung, which supported the existence of distinct receptors for LTC4, LTD4, and LTE4. The evidence for human tissue was more limited but was supported by the demonstration in human lung of a ligand-specific and stereospecific LTD4 receptor regulated by GTP. In guinea-pig lung the existence of separate receptors for LTC4, LTD4, and LTE4 was demonstrated by physiologic and radioligand binding studies. The rank order of potency of the sulfidopeptide leukotrienes for contraction of tracheal spirals (LTE4 > LTD4 = LTC4) is different from that for contraction of parenchymal strips (LTD4 > LTE4 > LTC4), thereby suggesting three separate receptors (Drazen et al. 1983; Lee, T.H. et al. 1984). Furthermore, whereas LTC4 and LTE4 elicited monophasic contraction in peripheral airway strips, LTD4 evoked a biphasic response (Drazen et al. 1980). Evidence for a separate LTE4 receptor in guinea-pig lung is provided by the capacity of LTE4 to enhance histamine responsiveness in a time- and dose-dependent manner, an effect that could not be reproduced by LTC4 and LTD4 despite eliciting the same magnitude of contraction of tracheal smooth muscle as LTE4 (Lee, T.H. et al. 1984). Studies in guinea-pig lung tissue in the presence of L-serine borate, which blocks the conversion of LTC4 to LTD4, showed that the LTD4/LTE4 antagonist FPL 55712 was unable to antagonize the contractile activity of LTC4 (Snyder & Krell 1984). Other selective LTD4 antagonists were shown to antagonize LTD4-induced contraction of guinea-pig tracheal strips but had little or no effect on the contractile effects mediated by LTC4 (Fleisch et al. 1985; Snyder et al. 1987). In guinea-pig uterus, specific binding of [3H]LTD4 could not be detected but specific and saturable binding of [3H]LTC4 was observed and reversed with unlabeled LTC4 (Levinson 1984). The binding of [3H]LTD4 and [3H]LTE4, but not [3H]LTC4, in guinea-pig lung was enhanced by divalent ions and the rate of dissociation was accelerated by either NaCl or GTP, providing further evidence for a multireceptor theory (Pong & DeHaven 1983). Several studies provided evidence for the heterogeneity of LTD4 receptors, both in the guinea-pig lung and across other tissues and species (Fleisch et al. 1982; Krell et al. 1983; Hua et al. 1985). Radioligand receptor binding assays with guinea-pig trachea (Krell et al. 1983) and lung membranes (Aharony et al. 1989), using specific LTD4 receptor antagonists, supported the existence of at least two distinct receptors for LTD4, with LTE4 preferentially interacting with only a subset of LTD4 receptors. This contrasted with the evidence in studies of human airways. Responses elicited by LTC4 and LTD4 are both inhibited to a similar degree by agents that are selective LTD4 receptor antagonists, with the inference that all the biological effects attributed to LTC4 can be explained by the bioconversion of LTC4 to LTD4 (Drazen & Austen 1987). Using radiolabeled LTD4 binding displacement assays, it was demonstrated that LTE4 binds to the identical receptor as

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LTD4 (Cheng & Townley 1984). However, these studies may be flawed because the prevention of metabolic conversion of LTC4 to LTD4 was not demonstrated experimentally, and this could explain the discrepancy in the literature.

CysLT1 Identification of the receptor A number of expression cloning and biochemical strategies were employed unsuccessfully in the 1990s to attempt to clone a cDNA for the cysteinyl leukotriene receptor CysLT1. Rapid progress in cDNA cloning, including the generation of databases of expressed sequence tags, facilitated the molecular characterization of the cysteinyl leukotriene receptors. In 1999 a previously cloned orphan GPCR, HG55 (identical to the cDNA HMTMF81) was “de-orphanized” and shown to have the pharmacologic properties of CysLT1 (Lynch et al. 1999; Sarau et al. 1999). CysLT1 has 32% amino acid identity to a purinergic receptor, P2Y1, and 28% identity to BLT1. Xenopus laevis oocytes injected with the complementary RNA for CysLT1 responded to LTD4 with a brisk calcium-dependent chloride flux (EC50 3 nmol/L, threshold 0.01 nmol/L) that was blocked by the selective CysLT1 receptor antagonist MK-571. Xenopus laevis melanophores, which respond to GPCR signaling with either aggregation or dispersion of pigment, were used to further characterize agonist responses of CysLT1. In this system the order of potency of the cysteinyl leukotrienes was LTD4 > LTC4 > LTE4, with EC50 values of 0.4, 21, and 212 nmol/L, respectively. Similar values were obtained in transfected HEK-293 cells: 2.3, 24, and 240 nmol/L, respectively. Signaling through CysLT1 was insensitive to pertussis toxin, indicating that it is not linked to Gi. In saturation binding assays the rank order of affinities was LTD4 >> LTC4 = LTE4 >> LTB4. The CysLT1 receptor antagonists montelukast, pranlukast, and zafirlukast all demonstrated highaffinity binding to membranes transfected with CysLT1 and dose-dependently displaced [3H]LTD4 from HEK-293 cells transfected with the cDNA encoding CysLT1. Molecular characterization of CysLT1 facilitated studies of its regulated expression and function in cells of both hematopoietic and nonhematopoietic origin, allowed the characterization of its gene, and permitted the generation of transgenic mice lacking expression of its gene, Cysltr1, extending our understanding of the function of the cysteinyl leukotrienes and CysLT1 beyond contraction of airway and vascular smooth muscle. CysLT1 gene The gene encoding CysLT1 was mapped to the human X chromosome at Xq13–q21 (Lynch et al. 1999). It contains five exons, the fifth exon containing the open-reading frame and 5′ untranslated sequence (Woszczek et al. 2005). There are multiple transcription starts and variable splicing of the first four exons, providing transcripts with variable 3′ untranslated sequence. Various polymorphisms of the human CysLT1

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gene have been described in association with allergy. There is increased frequency of the −634T/− 475C/−336G haplotype in a Korean population with a history of aspirin-sensitive respiratory disease (Kim, S.H. et al. 2006) . The T927C polymorphism was reported to be increased in Spanish males with atopic asthma and atopic dermatitis (Arriba-Mendez et al. 2006) and to be associated with more severe atopy in females in the UK (Hao et al. 2006). Expression and function Northern blot analysis showed high expression of CysLT1 in spleen and leukocytes, with lower expression in several other tissues including lung and small intestine (Lynch et al. 1999; Sarau et al. 1999). In situ hybridization revealed CysLT1 not only in smooth muscle of lung tissue but also in lung macrophages (Lynch et al. 1999). The expression of CysLT1 has been demonstrated in human nasal polyps (Chao et al. 2006), with markedly increased expression in individuals with aspirin-exacerbated respiratory disease that was decreased following desensitization to aspirin (Sousa et al. 2002). CysLT1 expression is increased in the airways in asthma (Zhu et al. 2005) and is further increased in severe asthma, with immunohistochemical identification of its expression in eosinophils, PMNs, mast cells, macrophages, B cells and plasma cells, but not T cells (Figueroa et al. 2001; Zhu et al. 2005). The promoter region of the human CysLT1 gene contains an active STAT6 binding site and expression is regulated by IL-4 and IL-13 (Woszczek et al. 2005). Thus THP-1 cells primed with IL-4 demonstrated increased generation of CCL2 (MCP-1) in response to LTD4 and LTC4 (Woszczek et al. 2005). IL-4 primed human cord blood-derived mast cells (hCBMCs) for increased Ca2+ flux (Mellor et al. 2001) and release of IL-5, TNF-α, and CCL4 (MIP-1β) in response to LTC4, LTD4, and UDP without concomitant release of histamine or PGD2 (Mellor et al. 2002). Pretreatment of hCBMCs with the CysLT1 antagonist MK-571 inhibited the generation of IL-5 and TNF-α in response to cross-linking of FcεRI, implying an autocrine role for CysLTs in amplifying IgE-mediated cytokine generation by mast cells (Mellor et al. 2002). Expression of CysLT1 by human monocytes is upregulated by IL-13 and IL-4, with a concomitant increase in Ca2+ flux and chemotaxis to LTD4 (Thivierge et al. 2001). Treatment of human fetal lung fibroblasts with IL-13 increased the expression of CysLT1 (Chibana et al. 2003). The regulation of CysLT1 is not confined to IL-4 and IL-13. The expression and function of CysLT1 in human airway smooth muscle cells is augmented by IFN-γ (Amrani et al. 2001). Treatment of eosinophilic HL-60 cells with IL-5 increased the expression of CysLT1 (Thivierge et al. 2000). CysLT1 is expressed on human T cells and its expression is increased in response to T-cell receptor (TCR) engagement (Spinozzi et al. 2004). Montelukast inhibited T-cell proliferative responses to TCR engagement with concomitant increase in IFN-γ release and increased apoptosis (Spinozzi et al. 2004). T cells from mice carrying a mutant form of linker for activa-

tion of T cells have increased numbers of CysLT1 and exhibit increased Ca2+ flux and chemotaxis to LTD4 (Prinz et al. 2005). In addition to its ability to contract bronchial smooth muscle, LTD4 augmented the mitogenic response to human airway smooth muscle cells to epidermal growth factor (Panettieri et al. 1998). With respect to the function of CysLT1 in cells of hematopoietic origin, in addition to eliciting Ca2+ flux and release of cytokines, LTD4 upregulated the expression of CD11b/CD18 on human eosinophils leading to increased adhesion to intercellular adhesion molecule (ICAM)-1 (Nagata et al. 2002). The regulation of dendritic cell migration by CysLTs was revealed in mice carrying targeted disruption of multidrug resistance-associated protein 1 (MRP1), which is essential for export of LTC4 from cells (Leier et al. 1994; Muller et al. 1994). Thus there was impaired migration of dendritic cells lacking MRP1 from the skin to the lymphatics which was restored by exogenous cysteinyl leukotrienes (Robbiani et al. 2000). Maturation of human monocytes to dendritic cells in the presence of LPS led to decreased expression of CysLT1 and increased expression of CysLT2 in a COXdependent manner that was reproduced by TNF-α and PGE2 (Thivierge et al. 2006). In contrast, dendritic cells differentiated in the presence of the TLR3 agonist poly I:C exhibited chemotaxis to LTD4, which also enhanced migration to CCL19 (MIP-3) (Thivierge et al. 2006). CysLT1 undergoes agonist-induced internalization that depends on phosphorylation by PKC of critical serene residues in the carboxyl tail of the receptor (Naik et al. 2005). Deletion of residues 310–321, mutation of serines 313–316, or inhibition of PKC leads to reduced agonist-induced internalization, with reduced generation of inositol phosphates, and reduced Ca2+ flux to CysLT stimulation (Naik et al. 2005).

CysLT2 Identification of the receptor The screening of public databases for novel orphan GPCRs led to the identification of CysLT2 (Heise et al. 2000; Nothacker et al. 2000; Takasaki et al. 2000). When the cRNA encoding the full-length open reading frame of CysLT2 was injected into X. laevis oocytes the cells responded to LTC4 and LTD4 with Ca2+-dependent chloride flux that was ablated by pretreatment with either LTC4 or LTD4 and was resistant to pertussis toxin. The response was resistant to inhibition by MK-571, but was inhibited by the CysLT1/CysLT2 antagonist Bay u9773. The rank order of potency of the cysteinyl leukotrienes in eliciting Ca2+ flux in HEK-293T cells transfected with CysLT2 and in radioligand binding studies of membranes of COS-7 cells transfected with CysLT2 was LTC4 = LTD4 >> LTE4. EC50 values for LTC4, LTD4, and LTE4 in eliciting Ca2+ flux from transfected HEK-293 cells were 8.9, 4.4, and 293 nmol/L, respectively. IC50 values for displacing [3H]LTC4 from transfected COS-1 cells by LTC4, LTD4, and LTE4 were 1.5, 59, and 1890 nmol/L, respectively (Takasaki et al. 2000). Northern blotting revealed widespread expression of CysLT2 in human

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tissues, including leukocytes, heart, spleen, lymph nodes, and neural tissues. In situ hybridization revealed expression of mRNA for CysLT2 in lung macrophages and peripheral blood leukocytes including eosinophils, in pheochromocytes and ganglion cells of the adrenal gland, and in Purkinje cells of the heart. The gene encoding CysLT2 was localized to human chromosome 13q14. Transgenic mice At present there are no specific inhibitors of CysLT2, so transgenic mice lacking expression of CysLT2 may be informative as to the role of the receptor. As in mice lacking CysLT1, the vascular leak in response to passive cutaneous anaphylaxis (PCA) was attenuated in Cysltr2-null mice. Unlike mice lacking CysLT1, the vascular leak in response to intraperitoneal zymosan was intact in mice lacking CysLT2 (Beller et al. 2004a), suggesting tissue-specific roles for the two cysteinyl leukotriene receptors in regulating vascular permeability. Interestingly, the pulmonary fibrotic response to bleomycin was augmented in Cysltr2-null mice (Beller et al. 2004a), suggesting a protective role for cysteinyl leukotrienes acting at CysLT2, in contrast to the attenuated fibrotic response to bleomycin in Cysltr1-null mice (Beller et al. 2004b). The effects of CysLT2 on vascular permeability were confirmed in mice with transgenic overexpression of the receptor in endothelial cells under the control of the Tie 2 promoter (Hui et al. 2004). These mice showed increased vascular leak in response to PCA and LTC4, but a decreased pressor response to intravenous LTC4 associated with increased NO generation. Expression and function Compared with CysLT1, there is limited information on the regulation of the expression and function of CysLT2. CysLT2 is prominently expressed by endothelial cells. Gene profiling of HUVECs treated with LTD4 yielded 37 genes, most of which were also upregulated by thrombin (Uzonyi et al. 2006). Genes induced by LTD4 included EGR1, E-selectin, CXCL2, CXCL8 (IL-8), and COX-2. Transcript induction was not blocked by CysLT1 receptor antagonism. The combined results suggest that CysLT2 may act in synergy with protease-activated receptor 1 to elicit vascular injury. Polymorphisms of the gene encoding CysLT2 The –1220 A/C polymorphism of the human gene encoding CysLT2, CYSLTR2, has been associated with asthma in a Japanese population (Fukai et al. 2004). Four SNPs in the 5′ and 3′ flanking region of the CysLT2 gene and five haplotypes were identified in a Korean population (Park et al. 2005). The frequency of certain rare alleles (− 819 T/G, 2078 C/T, and 2534 A/G) was higher in individuals with a history of aspirin-exacerbated respiratory disease. The M202V SNP encodes a receptor that has attenuated Ca2+ flux to LTD4 and is associated with atopy in the population of Tristan da Cunha (Thompson et al. 2003).

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Is there another CysLT receptor? Several studies have reported biological properties of the cysteinyl leukotrienes that cannot be explained on the basis of the recognized pharmacologic properties of CysLT1 and CysLT2, whether encoded on separate genes or generated by heterodimerization of cysteinyl leukotriene and/or related receptors. The cysteinyl leukotrienes were first recognized for their ability to potently constrict smooth muscle. While bronchoconstriction elicited by LTD4 was inhibited by the CysLT1 antagonists pranlukast, pobilukast, and zafirlukast, the co-mitogenic activity of LTD4 with epidermal growth factor was inhibited by pranlukast and pobilukast but was resistant to zafirlukast, suggesting that it might be mediated by a receptor separate from CysLT1 (Panettieri et al. 1998). Studies suggesting that there is a receptor with selective sensitivity to LTE4 have been referred to above. Cysteinyl leukotrienes and the pyrimidinergic agonist UDP elicited concentration-dependent calcium fluxes in hCBMCs that were blocked by the CysLT1 receptor antagonist MK571 (Mellor et al. 2001). Priming of hCBMCs with IL-4 for 5 days enhanced their sensitivity to each agonist, but preferentially lowered the threshold for activation by LTC4 and UDP by approximately 3 logs without altering CysLT1 receptor mRNA or surface protein expression, implying the induction of a second cysteinyl leukotriene receptor. IL-16, eotaxin, and RANTES stimulate vesicular transportmediated release of preformed, granule-derived IL-4 and RANTES from eosinophils and the synthesis of LTC4 at intracellular lipid bodies (Bandeira-Melo et al. 2002b). 5-LO inhibitors blocked IL-16-, eotaxin-, and RANTES-induced IL-4 release. After membrane permeabilization, LTC4 and LTD4 stimulated the release of IL-4 but not RANTES. This was blocked by pertussis toxin, implying the involvement of a GPCR. Inhibitors of CysLT1 and CysLT2 did not block LTC4elicited IL-4 release. LTC4 was 10-fold more potent than LTD4 and at low concentrations (0.3–3 nmol/L) elicited, and at higher concentrations (> 3 nmol/L) inhibited, IL-4 release from permeabilized eosinophils. These results demonstrate that LTC4, well recognized as a paracrine mediator, may also dynamically govern inflammatory and immune responses as an intracrine mediator of eosinophil cytokine secretion mediated by an intracellular GPCR distinct from CysLT1 and CysLT2. Ciana et al. (2006) recently characterized GPR17, an orphan GPCR intermediate in phylogeny between the cysteinyl leukotriene receptors and the P2Y pyrimidinergic receptors, as a dual UDP/CysLT receptor. Using a [35S]GTP-γ-S binding assay, the authors demonstrated responses to LTC4, LTD4, UDP, UDP-glucose, and UDP-galactose in the 1321N1 astrocytoma cell line transfected with GPR17. There was no response to ATP, ADP, 2-methyl-thio-ADP, UTP, α,β-methylene ATP, and guanosine, indicating that GPR17 responds to a different array of agonists than the already cloned cysteinyl leukotriene and P2Y receptors. Similar responses were seen to

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cysteinyl leukotrienes in COS-7 cells and to nucleotides in CHO cells and HEK cells transfected with GPR17. Agonist responses were inhibited by both P2Y antagonists and CysLT1 antagonists. Gi linkage was demonstrated by inhibition of agonist responses by pertussis toxin and by the ability of UDP and LTD4 to inhibit forskolin-induced elevations of cAMP. Immunoreactive GPR17 was seen within and at the borders of rat ischemic cerebral cortex, and ischemic damage in rats was inhibited by the P2Y inhibitor cangrelor, by the CysLT1 inhibitor montelukast, and by antisense knockdown of GPR17 expression in the brain. Thus, GPR17 is a dual UDP/CysLT receptor.

Actions in bronchial asthma It was the lasting contractile property of SRS-A at very low concentrations that originally generated interest in these lipid compounds as putative mediators in bronchial asthma. Isolated tracheal, bronchial, or parenchymal tissues from guinea pigs (Piper & Samhoun 1981), dogs (Johnson et al. 1983) and rats (Szarek & Evans 1988) have been found to contract in response to LTC4 at nanomolar concentrations. In guinea-pig tissue, LTC4 and LTD4 are approximately equipotent as contractile agonists, with EC50 values of 0.1–1 nmol/L. LTE4 is less potent in the same model, with EC50 values of 30–100 nmol/L (Dahlén 1983; Drazen & Austen 1987). Similar effects of the cysteinyl leukotrienes have been demonstrated in studies in vitro of human bronchus (Dahlén et al. 1980) and tracheal smooth muscle (Jones et al. 1982). The contractile action of LTC4 is at least 1000 times more potent than histamine in causing muscle contraction, and LTD4 has a similar potency to LTC4. Hanna et al. (1981) found a lower contractile activity of LTC4 and LTD4 than that of histamine. This contrasts with the findings of Chagnon et al. (1985), who showed that LTA4, LTC4, and LTD4 are at least 200 times more potent than histamine in human lung parenchyma in vitro. The release of LTC4, LTD4, and LTE4 was demonstrated in vitro in isolated bronchi from two birch pollen-sensitive asthmatic subjects after antigen challenge, and the amount of leukotriene released was found to correlate with the contraction evoked by allergen (Dahlén et al. 1983). In the guinea pig, pretreatment with a COX inhibitor led to a diminished LTC4- and LTD4-induced contractile response in isolated perfused lung, suggesting that bronchoconstrictor prostanoids are released by leukotriene action and contribute significantly to their contractile activity (Piper & Samhoun 1981). In contrast, in isolated human lung tissue indomethacin pretreatment had no effect on the contractile response to antigen induced by leukotriene release, which could be abolished by a leukotriene biosynthesis inhibitor (Dahlén et al. 1983). In humans, in vivo studies of inhaled leukotrienes have demonstrated potent stimulation of contractile activity in the airways in both normal and asthmatic subjects. Inhalation of nebulized solutions of leukotrienes leads to airway obstruc-

tion measured by falls in specific airway conductance or flow as measured from full or partial expiratory flow–volume curves. In five normal subjects the potency of LTC4 was 600– 9500 times greater than that of histamine, with 20 μg/mL LTC4 and 2–10 mg/mL histamine concentrations required to produce a 30% fall in expiratory flow rate at 30% of baseline vital capacity above residual volume (Vmax30) (Weiss et al. 1982a). In the same study, LTD4 was 6000-fold more potent than histamine (Weiss et al. 1983). If data on normal subjects from different sources are combined (Holroyde et al. 1981; Weiss et al. 1982a, 1983; Barnes et al. 1984a; Smith et al. 1985; Adelroth et al. 1986; Kern et al. 1986; Bel et al. 1987), inhaled LTC4 and LTD4 in normal subjects are 2000 times more potent than histamine or methacholine in producing airway obstruction. LTE4, on the other hand, is only 40–60-fold more potent than histamine but produces longer-lasting bronchoconstriction (Davidson et al. 1987; O’Hickey et al. 1988). Despite the similar potencies for LTC4 and LTD4, the timecourse of their action is different. LTC4 has a slower onset of action (10–15 min) than LTD4 and LTE4 (4–6 min), but the response for LTC4 is more prolonged (20–40 min) (Weiss et al. 1983; Drazen 1988). The reason for this difference in timecourse is unclear, but may be related either to the action of the cysteinyl leukotrienes at different receptors or because LTC4 requires metabolism to LTD4 prior to action at a specific receptor site. The discrepancy in the literature on the potency of the cysteinyl leukotrienes using a variety of measures of bronchoconstriction is likely to be due to differences in these parameters as measures of constriction at different sites within the lung. Changes in Vmax30 are mediated by bronchoconstricting agonists considered to act at a peripheral site of action, whereas forced expiratory volume in 1 s (FEV1) and specific airway conductance are measures of a central site of action. Inhalation of LTC4 and LTD4 in normal subjects produced a fall in Vmax30 with little effect on FEV1 (Holroyde et al. 1981). Similarly, Weiss et al. (1982b) demonstrated that a 50-fold greater concentration of LTC4 was required to achieve a 20% fall in FEV1 compared with the concentration required to cause a 30% fall in Vmax30. These studies on normal subjects suggested a predominantly peripheral site of action for LTC4 and LTD4. In asthmatic subjects, inhalation of LTC4 and LTD4 showed similar effects on specific airway conductance and Vmax30 (Barnes et al. 1984b; Smith et al. 1985; Kern et al. 1986), indicating that the site of LTC4 and LTD4 bronchoconstrictor activity in asthma is likely to be in both central and peripheral airways (Barnes et al. 1984a; Pichurko et al. 1989; Molfino et al. 1992). Asthmatic airways also respond by bronchoconstriction to inhaled leukotrienes, but in contrast to normal airways they exhibit smaller responses when comparison to a reference agonist is made. LTC4 and LTD4 have been reported to be of the order of 40-fold (Adelroth et al. 1986) to 140-fold (Griffin et al. 1983) more potent than histamine or methacholine in their bronchoconstrictor effects on asthmatic airways, which

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compares with 600– 9500-fold for LTC4 (Weiss et al. 1982a) and 6000-fold for LTD4 (Weiss et al. 1983) in normal airways compared with histamine. A correlation between airway responsiveness to methacholine (Adelroth et al. 1986) and histamine (Barnes et al. 1984c) and airway responsiveness to LTC4 and LTD4 has been established, but these studies also confirmed the relative lack of airway responsiveness to LTC4 and LTD4 in asthmatic subjects compared with normal subjects (Barnes et al. 1984c). The data for LTE4 is more limited. Using Vmax30 as a measure of airway obstruction, LTE4 was 39-fold more potent than histamine in normal subjects and 14-fold more potent than histamine in asthmatic subjects (Davidson et al. 1987). However, using a 35% fall in specific airway conductance as a measure of bronchoconstriction, the relative potency of LTE4 was found to be two to three times greater in asthmatic than in normal individuals (O’Hickey et al. 1988). In view of the discordance in the literature, a more extensive study was performed to compare the relative potencies of LTC4, LTD4, and LTE4 and the reference agonists histamine and methacholine in the same normal and asthmatic individuals (Arm et al. 1990). The airways of the subjects with asthma were approximately 14-fold, 15fold, sixfold, ninefold, and 219-fold more responsive to histamine, methacholine, LTC4, LTD4, and LTE4, respectively, than for normal subjects. Furthermore, as airway hyperresponsiveness (AHR) to histamine and methacholine increased, so too did the potency of LTE4, in contrast to LTD4 and LTC4 which decreased. Therefore, the results of these studies taken together suggest that, compared with normal airways, asthmatic airways are relatively less responsive to LTC4 and LTD4 but have a disproportionate hyperresponsiveness to the bronchoconstricting effects of LTE4. This indicates an important role for LTE4 in bronchial asthma, which may be due to its relative stability among the cysteinyl leukotrienes and because it persists for the longest time at the site of release (Lam et al. 1988). A subset of asthmatic individuals develop bronchospasm on ingestion of aspirin and other COX inhibitors. These subjects are unusually sensitive to the bronchoconstrictor effects of inhaled LTE4, which is 1870 times more potent than histamine in aspirin-sensitive subjects and only 145 times more potent than histamine in asthmatics who are aspirin tolerant (Arm et al. 1989). Following aspirin desensitization, there was a mean 20-fold decrease in airway responsiveness to LTE4 but no change in histamine responsiveness. In aspirin-sensitive subjects, this selective hyperresponsiveness is exclusive to LTE4 and not found with LTC4 (Christie et al. 1993), suggesting an important role in this form of asthma.

Airway responsiveness Bronchial hyperresponsiveness to contractile agonists such as histamine and methacholine and to nonspecific irritants is a key pathophysiologic feature of asthma. Studies in vitro on guinea-pig ileum have demonstrated that SRS-A enhanced

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the contractile response to histamine (Brocklehurst 1962). Pretreatment of guinea-pig spirals with a contracting dose of LTE4 accentuated the subsequent contractile response to histamine in a time- and dose-dependent fashion (Lee, T.H. et al. 1984). LTC4 and LTD4 did not enhance histamine responsiveness in parenchymal strips. Indomethacin pretreatment abolished the hyperresponsiveness to histamine, despite a lack of effect on the contractile activity of LTE4. A further study in guinea-pig tracheal spirals demonstrated enhanced contractile activity to histamine following LTC4 only in the presence of 0.1 mmol/L Ca2+ ions and this effect was blocked by the leukotriene antagonist FPL 55712 (Creese & Bach 1983). This AHR found in guinea-pig tracheal spirals was specific for histamine and was not found with carbachol or substance P. Furthermore, the effect was blocked not only by indomethacin but also by a TXA2/PGH2 (TP) receptor antagonist (GR32191). Pretreatment with LTE4 induces a similar effect on isolated human bronchus, with a fourfold leftward displacement of the histamine dose–response curve. This effect is blocked by GR32191, suggesting that the observed LTC4/ LTE4-induced hyperresponsiveness to histamine found in human and guinea-pig trachea is mediated by the secondary generation of COX products (Jacques et al. 1991). In normal subjects, inhalation of either a subthreshold dose of LTD4 (Barnes et al. 1984c) or bronchoconstricting doses of LTC4, LTD4, or LTE4 (Arm et al. 1988; O’Hickey et al. 1991) did not significantly enhance airway responsiveness to subsequent histamine inhalation. Inhalation of a bronchoconstricting dose of LTD4 produced an approximately twofold increase in airway responsiveness to methacholine (Kern et al. 1986). In an earlier study, inhalation of a bronchoconstricting dose of LTD4 led to significantly increased airway methacholine responsiveness, which was maximal at 7 days in six of eight subjects and persisted for 2–3 weeks in five subjects (Kaye & Smith 1990). Interestingly, in the same study the degree and duration of changes in methacholine airway responses were similar to those found after PAF inhalation. In asthmatic subjects, pre-inhalation of LTE4 that caused a 41% fall in specific airway conductance produced a doseand time-dependent increase in histamine responsiveness, which reached a peak of 3.5-fold at 7 hours after LTE4 inhalation (Arm et al. 1988). A subsequent study by the same authors found that each of the cysteinyl leukotrienes, LTC4, LTD4 and LTE4, produced an approximately threefold to fourfold increase in histamine responsiveness at 4 hours after inhalation in seven asthmatic individuals (O’Hickey et al. 1991) (Fig. 26.6). The magnitude of this enhanced histamine responsiveness is similar to that observed after inhaled allergen challenge (Cockcroft et al. 1977; Cartier et al. 1982). Prior inhalation of LTC4 was found to have no effect on airways response to inhalation of distilled water in nine asthmatic subjects (Bianco et al. 1985). Predosing with indomethacin significantly inhibited the LTE4-induced hyperresponsiveness to histamine in

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0.5

increase the extent of maximal airway narrowing, two cardinal features of airway responses in bronchial asthma.

0.4

Airway secretions

PD35 histamine (mmol)

0.3

0.2

0.1

0.05 1

4 Time (hours)

7

Fig. 26.6 Time-course of changes in airway histamine responsiveness after inhalation of phosphate-buffered saline (closed triangles), methacholine (closed circles), LTC4 (open triangles), LTD4 (closed squares), and LTE4 (open circles). Each point represents the geometric mean for seven subjects with asthma. (From O’Hickey et al. 1991, with permission.)

eight asthmatic subjects (Christie et al. 1992a). Indomethacin was also found to inhibit the increase in airway responsiveness following inhaled allergen provocation in seven atopic subjects, without an effect on either the early or late asthmatic reaction (Kirby et al. 1989). This suggests that the cysteinyl leukotrienes make a significant contribution to the enhanced airways responsiveness that is so characteristic of asthma and they may exert part of this effect through the secondary generation of COX pathway-derived products. Asthmatic airways are more sensitive to bronchoconstricting agonists such as histamine and methacholine than normal airways, with a leftward shift in the dose–response curve and a greater absolute response. Normal airways are found to reach a plateau of maximum bronchoconstriction at mild degrees of airway narrowing. In a study of eight normal subjects, LTD4 was found to produce a maximal response plateau at a higher level than methacholine. The addition of methacholine at the top of the LTD4 plateau caused a further fall of 6.6% and 4.8% in FEV1 and expiratory flow at 40% vital capacity (V40P), respectively (Bel et al. 1987). This effect could be demonstrated for at least 3 days following LTD4 inhalation. A later study by the same group reported that prior administration of budesonide for 6 days could diminish the maximal response to LTD4 in eight nonasthmatic subjects by 7.9% and 8.4% for FEV1 and V40P, respectively (Bel et al. 1989). Thus, the cysteinyl leukotrienes increase nonspecific AHR and also

In bronchial asthma, abnormalities of airway mucociliary function are suggested by the clinical observation of excessive tracheobronchial secretions, which are difficult to clear and may contribute to bronchial obstruction. The cysteinyl leukotrienes can influence both mucus secretion and composition as well as mucociliary transport within the airways. Cultured human airway explants when exposed to LTC4 and LTD4 increased the rate of secretion of radiolabeled glucosamine as part of a high-molecular-weight glycoprotein by 15 and 26%, respectively (Marom et al. 1982; Coles et al. 1983). Repeated stimulation of the explants led to diminished radiolabeled product secretion with the ratio of bound radiolabel to protein remaining unchanged, suggesting that LTC4 and LTD4 stimulate secretion rather than de novo mucus synthesis (Marom et al. 1982). LTC4 was found to have 1000fold more potent secretagogue effects on mucus secretion than LTD4 when injected into the artery supplying the cervical canine trachea (Johnson & McNee 1983). In vitro studies on cultured airway goblet cells demonstrated enhanced mucin release by the action of physiologic concentrations of LTC4 and LTD4 (Kim et al. 1989). LTC4 and LTD4 also mediate an increase in chloride flux of isolated canine tracheal epithelium, which induces an increase in the short-circuit ionic current (Leikauf et al. 1986) and is accompanied by enhanced fluid secretion (Johnson et al. 1983). In sheep allergic to Ascaris suum, concentrations of LTD4 as low as 25 μg/mL produced significant decreases in tracheal mucus velocity, with the maximum effect observed at 3 hours after leukotriene challenge (Russi et al. 1985). The overall effect of the leukotrienes therefore leads to a thickening of the mucous gel layer and a decrease in tracheal mucus velocity. Cysteinyl leukotrienes were detected in the sputum of 16 of 25 patients with cystic fibrosis and one of five patients with chronic bronchitis (Cromwell et al. 1982). In a study of 30 children with cystic fibrosis, urinary LTE4 levels were found to correlate with sputum LTE4 values, and one-third of the children with cystic fibrosis had urinary LTE4 greater than 200 pmol per mmol creatinine compared with only 3% of normal children (Sampson et al. 1990). The same group reported that in a study of 13 children with cystic fibrosis, the logarithm of sputum LTE4 levels and total cysteinyl leukotriene levels correlated with the overall severity of pulmonary disease, as assessed by Chrispin–Norman chest radiograph scores (Spencer et al. 1992). These findings suggest that cysteinyl leukotrienes may have a role in the sputum abnormalities characteristic of cystic fibrosis and chronic bronchitis.

Leukotriene release in disease Leukotrienes have been detected in a variety of biological fluids by employing sensitive assay systems to detect picogram

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Table 26.4 Release of leukotrienes into biological fluids. Disease

Biological fluid

Leukotriene release

Asthma Clinical disease Symptomatic Acute

BAL BAL Urine

LTE4 detected in mild/severe disease LTB4/LTC4 LTE4: highest levels in patients responding

BAL Urine Urine

LTC4 increases ninefold LTE4 increases 2–4 hours after allergen challenge LTE4 sixfold higher at baseline and increases a further fourfold after aspirin challenge LTC4 levels increase after lysine–aspirin challenge LTB4 rises 12-fold and LTC4 rises fivefold after isocapnic hyperventilation LTE4 found to increase 1.7-fold in only one study of children with severe asthma

Allergen challenge Early-allergen response Aspirin sensitive Exercise induced

Nasal lavage BAL Urine

Cryptogenic fibrosing alveolitis

BAL

LTB4 detected

Persistent pulmonary hypertension

BAL

LTC4 and LTD4 detected in newborn infants

Adult respiratory distress syndrome

Pulmonary edema fluid

LTD4 increased fourfold

Rheumatoid arthritis

Synovial fluid

LTB4 detected in active disease

Gouty arthritis

Joint fluid

LTB4 levels significantly increased

Psoriasis

Skin chamber fluid

LTB4, LTC4 and LTD4 levels detected in increased quantities

Inflammatory bowel disease

Intestinal mucosal fluid

LTB4 detected in both ulcerative colitis and Crohn disease

Acute myocardial infarction

Urine

LTE4 levels raised and return to normal day 3

Hepatorenal syndrome

Urine

LTE4 levels elevated threefold

BAL, bronchoalveolar lavage.

quantities, such as high-performance liquid chromatography (HPLC), radioimmunoassay, and mass spectrometry (Table 26.4). Bronchoalveolar lavage (BAL) has been used as a tool to obtain fluid in pulmonary disease states. Because the ratio of the volume of lavage fluid instilled to that recovered is not always constant, BAL suffers from the drawback that it is not easy to interpret the absolute values given and the results are usually expressed in terms of amounts per volume recovered. Similar concerns have been expressed regarding measurements of mediators in exhaled breath condensates. Urine is a more reliable biological fluid and amounts of mediator are easily standardized by expressing quantities per unit of creatinine, which is excreted in a relatively constant fashion throughout a 24-hour period (Smith et al. 1992). Maltby et al. (1990) found that by infusing three subjects with three doses of radiolabeled LTC4, it was possible to recover a constant 4.1–6.3%, regardless of the amount infused, in the form of LTE4, the most stable of the cysteinyl leukotrienes. Radiolabeled LTC4 instilled into the airways of asthmatics, nonasthmatics, and asthmatics challenged with allergen was found to be excreted in a constant fashion in all three groups,

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with LTE4 as the major metabolite (Westcott et al. 1993). Christie et al. (1994) found a significant correlation between the dose of inhaled LTC4 and the amounts excreted as urinary LTE4. These studies provide strong evidence for the utility of urinary LTE4 as a marker of pulmonary cysteinyl leukotriene release.

Clinical asthma On BAL, 15 of 17 asthmatic subjects with mild to severe disease were found to have detectable LTE4 in BAL fluid, but there was no correlation between LTE4 levels and pulmonary function. None of the group of nine control subjects had detectable LTE4 (Lam et al. 1988). In a study by Wardlaw et al. (1989), eight symptomatic asthmatic subjects had significantly higher levels of LTB4 and LTC4 in BAL fluid than a control group of 14 without asthma. A comparison of 11 healthy with 11 atopic subjects with mild asthma found no difference in cysteinyl leukotriene, histamine, or PAF levels between the two groups, but there was a higher level of PGD2 and a lower level of LTB4 in the asthmatic group (Crea et al. 1992). These findings are in contrast to the two studies above and

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may be due to selection of very mild asthmatic subjects. In a study using urinary LTE4 as a marker for pulmonary cysteinyl leukotriene release, Smith, C.M. et al. (1992 found no difference in baseline urinary levels of LTE4 between 17 normal and 31 asthmatic subjects. In addition, there was no correlation between urinary LTE4 levels as measured in picograms per milligram of creatinine and baseline airway responsiveness to histamine or FEV1 as a percentage of predicted values (Crea et al. 1992). Drazen et al. (1992) examined 72 subjects presenting to accident and emergency departments and classified 22 patients with a doubling of peak expiratory flow rate (PEFR) following nebulized salbutamol as responders, and 19 patients with < 25% increase in PEFR as nonresponders. Urinary LTE4 levels were assayed by precolumn extraction, HPLC, and radioimmunoassay in these two groups and compared with 13 normal controls. Urinary LTE4 levels were significantly higher in responders compared with nonresponders or normal subjects. The authors concluded that the highest levels of LTE4 found in those with acute reversible airflow obstruction were consistent with a bronchospastic role for cysteinyl leukotrienes in spontaneous acute asthma.

Allergen-induced asthma Leukotriene release has been measured in studies employing various bronchial challenge procedures. Wenzel et al. (1990) reported that using HPLC the predominant leukotriene in BAL fluid in atopic asthmatic subjects after endobronchial allergen challenge was LTC4, with an approximate ninefold rise in levels compared with baseline. Measurable levels of LTC4 at baseline were found in 9 of 11 atopic asthmatic subjects but in only one of seven atopic nonasthmatic subjects and one of six nonatopic subjects. There was only a slight rise in BAL LTC4 levels after allergen in the atopic nonasthmatic group and no change in the nonatopic samples. In a further study by Wenzel et al. (1991), the levels of PGD2, TXB2, LTC4, and histamine were measured in BAL fluid before and 5 min after endobronchial allergen challenge in three groups of atopic subjects: seven nonasthmatic subjects, six asthmatic subjects without a late asthmatic response, and six asthmatic subjects with a late asthmatic response. LTC4 was detected in 9 of 12 asthmatic subjects but in only one of seven subjects without asthma. Significant increases in all mediator levels were observed in both groups with asthma post allergen challenge compared with the nonasthmatic group. Interestingly, the asthmatic group without a late asthmatic response recorded significantly higher levels of all four mediators post challenge than the groups with a late response and the nonasthmatic controls. In a study of 17 allergic asthmatic subjects undergoing allergen provocation, there was a fall in FEV1 within the first 2 hours of 25– 59%, and this was accompanied by a rise in urinary LTE4 levels from 46 to 92 ng over a 12-hour collection period. Methacholine challenge alone, which led to similar falls in FEV1, did not significantly change urinary LTE4 excretion. There was a significant correlation

between the decrease in FEV1 during the early asthmatic response, the excretion of urinary LTE4, and airways reactivity. No correlation was found between urinary LTE4 excretion and the severity of late response to allergen, but there was significant prolonged elevated urinary LTE4 excretion in those patients with the most severe late asthmatic responses (Westcott et al. 1991). Manning et al. (1990a) studied 18 asthmatic subjects who were divided into three groups: those with an isolated early asthmatic response (EAR), those with an isolated late asthmatic response (LAR), and those with a dual asthmatic response (DAR). Urinary LTE4 rose significantly from baseline values only in the two groups with an EAR, with a rise from 150 to 1816 pg/mg creatinine in the group with an isolated EAR and a rise from 66 to 174 pg/mg creatinine in the group with an EAR preceding an LAR. No increase in urinary LTE4 was found in the group with an isolated LAR. Furthermore, the degree of maximum bronchoconstriction during the EAR correlated with urinary LTE4 release, suggesting that the cysteinyl leukotrienes are only released during the EAR and that they contribute significantly to the bronchoconstriction found during this phase of the asthmatic response. Several subsequent studies have also documented urinary LTE4 release 2–4 hours following specific bronchial allergen challenge (Taylor et al. 1989; Hui & Barnes 1991; Kumlin et al. 1992a; Nasser et al. 1994b).

Exercise-induced asthma Pliss et al. (1990) found increases in BAL fluid levels of LTB4 (from 10 to 121 pg/mL) and immunoreactive cysteinyl leukotrienes (from 46 to 251 pg/mL) following isocapnic hyperventilation as a model for exercise-induced asthma. There were also increases in eosinophil and epithelial cell numbers, but no changes in prostaglandin or histamine levels were detected. A further study was unable to find evidence for mast cell-derived mediator release, with no increase in BAL fluid histamine, tryptase, PGD2 or LTC4 following treadmill exercise in seven atopic asthmatic subjects. However, the sensitivity of the LTC4 assay in this study may not have been sufficient to detect any LTC4 released (Broide et al. 1990). Urinary levels of LTE4 were not found to increase in a study of six asthmatic subjects after treadmill exercise, leading to a mean 22% fall in FEV1 (Smith, C.M. et al. 1991). However, small increases in urinary LTE4 (14.3 ng/mg creatinine before and 24.3 ng/mg creatinine after exercise) were found in 8 of 10 children with severe asthma following exercise, which produced a 60% fall in FEV1 but no increases in urinary LTE4 in seven children with moderate asthma who experienced only a 24% fall in FEV1 (Kikawa et al. 1991). Despite the conflicting evidence, pharmacologic studies have indicated an important role for cysteinyl leukotrienes in exercise-induced bronchospasm (Makker et al. 1993). It is likely that our inability to detect these mediators consistently in biological fluids may be because whole-body cysteinyl leukotriene release does not change significantly after exercise, which may be

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further modified by changes in bronchial or pulmonary blood flow in response to airway cooling and local changes in pH and osmolarity. Furthermore, the modest local increases in cysteinyl leukotriene release in exercise-induced asthma are likely to be difficult to detect with the assays employed.

Aspirin-sensitive asthma The cysteinyl leukotrienes have considerable importance in the pathogenesis of aspirin-sensitive asthma (ASA). Mean resting urinary LTE4 levels in ASA subjects are significantly higher than in normals or non-ASA subjects. Christie et al. (1991a) found sixfold higher urinary LTE4 levels in ASA subjects compared with non-ASA controls. Furthermore, oral aspirin challenge which led to a mean 21% fall in FEV1 in six ASA subjects resulted in a fourfold increase in urinary LTE4 values over baseline values. There was no such increase in urinary LTE4 levels in the control subjects and no fall in FEV1 on aspirin ingestion. Smith et al. (1992) reported that baseline urinary LTE4 levels in 10 ASA subjects were 101 pg/mg creatinine compared with 43 pg/mg creatinine in 31 nonASA subjects and 34 pg/mg creatinine in 17 normals. There was substantial overlap between the groups and no correlation was found between urinary LTE4 and histamine PD20 or baseline FEV1, and so measurement of LTE4 in a single sample of urine does not predict the degree of resting airflow obstruction, the degree of bronchial hyperresponsiveness, or diagnose aspirin sensitivity. Subsequent studies have affirmed these findings (Knapp et al. 1992; Kumlin et al. 1992a; Sladek & Szczeklik 1993). Studies have also confirmed increased urinary LTE4 levels in ASA subjects following lysine-aspirin bronchial challenge (Christie et al. 1992b; Sladek et al. 1994). Nasal lavage mediator levels have been studied in aspirinsensitive rhinosinusitis. Following lysine-aspirin challenge increased levels of both histamine and LTC4 were detected in nasal lavage samples in three of four ASA subjects with both naso-ocular symptoms and a bronchospastic reaction. No increases in these mediators were found in normals or nonaspirin-sensitive subjects or aspirin-sensitive subjects in whom lysine-aspirin did not provoke naso-ocular symptoms (Ferreri et al. 1988; Picado et al. 1992).

Biological roles of leukotrienes and their receptors in asthma revealed through gene disruption in mice Potent inhibitors of CysLT1 have been used extensively in humans with minimal toxicity and have unequivocally demonstrated a role for the cysteinyl leukotrienes acting at CysLT1 in bronchial asthma, rhinitis, and chronic idiopathic urticaria (see Chapter 90). However, there are no existing BLT or CysLT2 antagonists approved for use in humans, and information on their biological activities is imputed largely from models of disease in mice engineered to lack one or more receptor. These studies also allow for dissection of putative mechanisms that is not possible in humans.

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Various models have been developed to study pulmonary allergic inflammation (Boyce & Austen 2005), each revealing different aspects of the disease. The actions of LTB4 in recruiting effector CD8+ and CD4+ T cells has been noted above (Goodarzi et al. 2003; Tager et al. 2003). In a mouse model of allergic pulmonary inflammation, there was substantially reduced recruitment of CD3+, CD4+, and CD8+ T cells to the BAL fluid of ovalbumin-sensitized mice after the first two of three sequential challenges with inhaled ovalbumin (Tager et al. 2003). No differences were seen in recruitment of T cells to the lung of ovalbumin-sensitized and -challenged mice, nor to the BAL fluid after a third ovalbumin challenge (Tager et al. 2003). A role for CD8+ T cells has been demonstrated in allergen-induced AHR and pulmonary inflammation (Hamelmann et al. 1996). Both AHR and pulmonary inflammation were restored by the adoptive transfer of antigen-primed CD8+ T cells from BLT1-sufficient but not from BLT1-null mice (Miyahara et al. 2005a), suggesting an important function for LTB4 acting at BLT1 in the recruitment of CD8+ effector T cells in allergic pulmonary inflammation. Similar findings were reported in a passive sensitization and ovalbumin challenge model of allergic pulmonary inflammation that is mast cell dependent (Taube et al. 2006), suggesting that the mast cell may be the source of LTB4. Two different strains of BLT1-null mice, sensitized and challenged with ovalbumin, developed less AHR and less goblet cell hyperplasia than wild-type BLT1-sufficient control mice, with reduced IL-5 and IL-13 in the BAL fluid and reduced staining for these cytokines in both CD4+ and CD8+ T cells (Miyahara et al. 2005b; Terawaki et al. 2005). Antigen restimulation of peribronchial lymph node cells from sensitized mice elicited less Th2 cytokine generation in vitro (Terawaki et al. 2005). Transfer of antigen-primed but not naive BLT1-sufficient T cells restored antigen-induced AHR consistent with the increased expression of BLT1 on effector compared with naive T cells. In a separate model of ovalbumin sensitization and challenge, a role for LTB4 acting at BLT1 was revealed for the early recruitment of neutrophils and eosinophils into the airways of ovalbumin-challenged mice (Medoff et al. 2006). That the findings regarding BLT1 on effector T cells in mice may be relevant to human disease is indicated by the finding that in humans BLT1-positive T cells, which are normally few in number, are found in increased numbers in individuals following infection with Epstein–Barr virus and in the lungs of individuals with asthma compared with normal controls (Islam et al. 2006). In addition to BLT1, these cells express IFN-γ and IL-4 and the chemokine receptors CCR1, CCR2, CCR6, and CXCR1. A role for the cysteinyl leukotrienes acting at CysLT1 is conclusively proven in human asthma through the use of CysLT1 antagonists (see Chapter 30). Studies in mice lacking LTC4 synthase suggest a distinct role for the cysteinyl leukotrienes in the epithelial reactive mastocytosis that is seen in asthma (Kim, D.C. et al. 2006). These mice have markedly

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attenuated AHR, eosinophilic pulmonary inflammation, goblet cell hyperplasia, and mucus hypersecretion compared with wild-type control mice following ovalbumin sensitization and airway challenge. The expansion of activated mast cells in the epithelium, but not in the submucosa, of allergic mice was markedly attenuated in mice lacking LTC4 synthase. Antigen restimulation of peribronchial lymph node cells from sensitized LTC4 synthase-null mice elicited less Th2 cytokine generation in vitro compared with wild-type control mice. It is also of note that the induction of allergen-specific IgE was attenuated in mice lacking LTC4 synthase (Kim, D.C. et al. 2006), as has also been seen in mice lacking 5-LO (Hashimoto et al. 2005) and BLT1 (Terawaki et al. 2005). A role for the cysteinyl leukotrienes in airway remodeling is suggested in a mouse model of chronic allergen challenge. Administration of the CysLT1 receptor antagonist montelukast to mice sensitized with ovalbumin and then subjected to repetitive ovalbumin challenge over several weeks attenuated the induction of airway goblet cell metaplasia, smooth muscle hypertrophy, and subepithelial fibrosis (Henderson et al. 2002). The increase in mRNA for IL-4 and IL-13 in the lung induced by allergen challenge was also attenuated by montelukast (Henderson et al. 2002). Of note, montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition (Henderson et al. 2006). Thus studies in mice engineered to lack enzymes of leukotriene biosynthesis and specific leukotriene receptors suggest that the cysteinyl leukotrienes have potent inflammatory properties and are not merely constrictors of smooth muscle and mucus secretogogues. Mice models of allergic inflammatory disease address specific aspects of the asthma phenotype and can be manipulated to address selective aspects of the inflammatory process. Furthermore, mice lacking a specific protein from conception cannot address the role of leukotrienes in established disease. To our knowledge there have been no studies of leukotriene-modifying drugs in the prevention of asthma or allergic disease. Whether findings of allergic pulmonary inflammation in mice will be effectively extended to human asthma requires the development of specific inhibitors of downstream enzymes and receptors beyond the current generation of CysLT1 receptor antagonists.

Other diseases Leukotrienes have also been recovered in a number of other nonallergic disease states. Increased quantities of LTB4 were detected in lavage fluid of patients with cryptogenic fibrosing alveolitis and correlated with the recovery of PMNs in BAL fluid, suggesting that leukotriene release may be responsible for granulocyte recruitment in this disease (Wardlaw et al. 1989). LTC4 and LTD4 have been found in increased concentrations in the lung lavage fluids of newborn infants with persistent pulmonary hypertension requiring assisted ventilation (Stenmark et al. 1983). In the adult respiratory distress

syndrome (ARDS), LTD4 was detected in fourfold higher concentrations in pulmonary edema fluid compared with patients with cardiogenic pulmonary edema. Furthermore, the LTD4 levels correlated with the ratio of edema fluid to plasma concentrations of albumin, suggesting that cysteinyl leukotrienes contribute to the permeability defect that allows accumulation of protein-rich fluid in ARDS (Matthay et al. 1984). Small quantities of LTB4 have been detected in the synovial fluid of joints from patients with active rheumatoid arthritis (Davidson et al. 1983). In gouty joint effusions, significantly higher quantities of LTB4 are detected compared with those found in either normal or rheumatoid joints. This may be partly due to monosodium urate crystals, which have been shown to inhibit metabolism and biological deactivation of LTB4 by polymorphonuclear granulocytes (Rae et al. 1982). Skin chamber fluid from abraded psoriatic skin lesions contains significantly higher quantities of LTB4 (Brain et al. 1984) and immunoreactive LTC4 and LTD4 (Brain et al. 1985) than that from normal skin, and this may partly explain the characteristic intraepidermal neutrophil infiltration and local vasodilatation found in psoriasis. By measuring the incorporation of radiolabeled thymidine, picomolar to nanomolar concentrations of LTB4 were found to increase DNA synthesis in cultured epidermal keratinocytes in vitro. The peptide leukotrienes LTC4 and LTD4 were less potent than LTB4, but also stimulated keratinocyte DNA synthesis. This finding may relate to the excessive epidermal hyperplasia seen in psoriasis (Kragballe et al. 1985). Both LTB4 and the cysteinyl leukotrienes are generated and released in vitro in greater quantities from intestinal mucosa obtained from patients with ulcerative colitis and Crohn disease (Sharon & Stenson 1984; Peskar et al. 1986; Lauritsen et al. 1987). Intestinal mucosa from patients with inflammatory bowel disease was found to contain 50-fold higher quantities of LTB4 per gram of tissue than that from normal mucosa (Sharon & Stenson 1984). The use of rectal pouches has allowed the measurement of mediator levels in vivo in inflammatory bowel disease and has demonstrated significantly higher LTB4 levels in active ulcerative colitis (Lauritsen et al. 1987). Treatment with either prednisolone or aminosalicylic acid decreased the generation of LTB4 in vivo, which was associated with clinical improvement. Pretreatment levels of both PGE2 and LTB4 were significantly higher in patients not responding to 5-aminosalicylic acid or prednisolone treatment, and the authors of the study suggested that intraluminal levels of these mediators are more useful predictors of relapsing ulcerative colitis than clinical indices of disease severity (Lauritsen et al. 1986). Levels of urinary LTE4 were found to be raised in a study of 16 patients presenting with acute myocardial infarction, with a fall to normal levels by day 3. Similar elevated levels of urinary LTE4 were found in unstable angina, with a reduction to normal levels once the chest pain had resolved. Treatment with thrombolytic agents leading to early coronary

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COOH

Arachidonic acid

COOH O

COOH

O

O COOH O

O

Prostaglandin H synthase (cyclooxygenase)

TXA2

OH

OOH HO

PGI2

OH

HO O HO

COOH

COOH

O

COOH

O

HO OH

HO

O

PGH2

TXB2

OH

HO

OH 6-keto-PGF1a

HO COOH COOH

O

HO

HO

O OH

COOH

HO

HO OH

PGE2 COOH

HO

HO

COOH

COOH

O HO

OH

O

O

15-keto-PGF2a

COOH

HO

2,3-dinor TXB2

O

OH

PGD2

O OH 11-dehydro TXB2

15-keto-PGE2 HO COOH

HO

OH

9a, 11b-PDF2a

Fig. 26.7 Prostanoid biosynthetic pathway. Cyclooxygenase catalyzes the first two steps and converts arachidonic acid to the endoperoxides PGG2 and PGH2, which are then enzymatically transformed to the prostaglandins, prostacyclin and thromboxanes by the action of the respective synthesis.

reperfusion resulted in a faster decline in LTE4 levels compared with patients who did not receive such treatment, suggesting that cardiac ischemia and necrosis were responsible for cysteinyl leukotriene release (Carry et al. 1992). In hepatorenal syndrome and decompensated liver disease, the rate of urinary LTE4 excretion per hour is threefold higher than that found in compensated liver disease. The additional finding of reduced renal clearance of a radiolabeled LTC4 infusion in one such patient suggested that the increased excretion of urinary LTE4 is due to excessive production or reduced metabolism of LTE4 and not due to reduced renal clearance of LTE4 (Moore et al. 1990). The fact that LTC4 and LTD4 are potent renal vasoconstrictors suggests that these mediators may modulate glomerular function and be involved in the pathogenesis of this disease.

Prostaglandins and thromboxanes Biosynthesis and cellular source TXA2 and prostaglandins belong to the series 2 prostanoids

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since two double bonds are present in the side chains attached to the cyclopentane ring; they are formed by the metabolic pathway outlined in Fig. 26.7. Arachidonic acid, a 20-carbon polyunsaturated fatty acid, is hydrolyzed from phospholipid cell membranes by PLA2, in response to various stimuli. This is followed by the subsequent enzymatic conversion of arachidonic acid to PGH2 by the COX enzymes (also termed prostaglandin H2 synthases). This is a two-step process involving COX activity to oxidise arachidonic acid to PGG2 at the cyclooxygenase site of the COX and a peroxidase reaction to reduce PGH2 at the peroxidase site of COX (Garavito & deWitt, 1999) . These two reactions occur at distinct but structurally and functionally interconnected sites. The peroxidase reaction occurs at a heme-containing active site located near the protein surface. The cyclooxygenase reaction occurs in a hydrophobic channel in the core of the enzyme (Smith et al., 1999). PGH2 serves as a substrate for the prostaglandin synthase enzymes, which are responsible for the production of the five principal bioactive prostaglandins generated in vivo: PGE2, PGF2α, PGD2, PGI2 (prostacyclin), and TXA2 (thromboxane).

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Hence it is the release of arachidonic acid that is the ratelimiting step in prostanoid synthesis. It is now well recognized that there are at least two COX enzymes, COX-1 and COX-2, both are homodimeric, heme-containing, glycosylated, integral membrane associated enzymes found in the endoplasmic reticulum (ER) and the nuclear envelope (Smith et al., 1996). Immunostaining suggests that COX-2 is expressed both in the cytoplasm and at the nuclear membrane whilst COX-1 localises to the cytoplasm alone (Morita et al., 1995). Both forms are expressed in normal human respiratory epithelium. COX-1 is constitutively expressed in most tissues and in blood platelets and the metabolites derived from COX-1 are involved in cellular housekeeping functions. COX-2 is the predominant isoform expressed in injured tissue and a main source of prostanoids during inflammation and is expressed in leukocytes only following cellular activation by inflammatory stimuli (Sousa et al. 1997). In the asthmatic human airway: airway smooth muscle (Belvisi et al. 1997), eosinophils (Sousa et al. 1997), bronchial epithelial cells (Mitchell et al. 1994), mast cells (Sousa et al. 1997), and macrophages (Hempel et al. 1994) all exhibit COX-2 immunoreactivity. The expression of COX-1 and COX-2 in human lungs results in the production of two different classes of prostanoids. These are broadly divided into the bronchoconstrictive prostaglandins (PGD2 and PGF2α) and the bronchoprotective prostaglandins (PGE2 and PGI2) (Samuelsson et al. 1978; Uotila & Vapaatalo 1984). Although COX is present in most cells including the respiratory tract, airway smooth muscle and epithelial cells, the COX metabolites released from a particular cell are cell specific reflecting the isomerase and synthase components of that cell and the quantity and variety of prostanoids produced determined by the nature and the activation state of the cells present at the site of inflammation (Gilroy et al., 1999).

Role of cyclooxygenase COX-1 was originally characterized from bovine seminal vesicles (Miyamoto et al. 1976) and subsequently the inducible form called COX-2 was isolated in a number of species and tissues (Simmons et al. 1991). These isoforms are the products of distinct genes. The gene for COX-1 is situated on chromosome 9q32– 33.3 (Funk et al. 1991) whereas the gene for COX-2 is localized to chromosome 1q25.2–25.3 (Tay et al. 1994). The differences between these two isoforms of COX appear to involve their regulation and cell expression. COX-1 is known to be constitutively expressed at varying levels in virtually all mammalian tissues and is responsible for the basal production of prostanoids. In contrast, COX-2 is undetectable in most tissues but its expression increases substantially in situations associated with cell replication and differentiation and in response to inflammation and mitogenic stimuli, with typical increases of 10–80-fold (Kujubu et al. 1991,

1993). The potential role of COX-2 in the pathogenesis of inflammation and cancer is suggested by its upregulation by bacterial LPS, cytokines, growth factors, and tumor promoters (Kujubu et al. 1991; Lee et al. 1992; O’Banion et al. 1992; O’Sullivan et al. 1992). The differences in expression of both isoforms of COX is reflected in their gene structure. The human COX-2 gene is 8.3 kb whereas the COX-1 gene is much larger at 22 kb (Kraemer et al. 1992; Tazawa et al. 1994). COX-1 has no TATA or CAAT box in its promoter region and is GC-rich, features consistent with its role as a housekeeping gene providing a continuously transcribed stable message (Tanabe & Tohnai 2002). In contrast, the gene structure of COX-2 suggests that it is an immediate early gene product that can be switched on rapidly during inflammation. The COX-2 gene promoter contains a canonical TATA box 31 bases upstream of the transcriptional start site and several functionally important enhancer elements, including a cAMP-response element (CRE), E box and AP-1 regulatory element complex situated very close to TATA, a C/EBP site and two nuclear factor (NF)-κB sites (Appleby et al. 1994). The 72-hour kinetics of the expression of COX-isoform mRNA has been examined in ovalbumin-sensitized and -challenged guinea-pig lungs (Oguma et al. 2002). The sensitized animals demonstrated a robust transient induction of COX-2 mRNA expression within 1 hour after ovalbumin challenge, while their COX-1 mRNA levels remained unchanged. Upregulation of the level and activity of COX-2 protein followed the induction of COX-2 mRNA. Polymorphisms in COX-1 appear not to play a substantial role in genetic predisposition for asthma or asthma severity (Shi et al. 2005) and to date no COX-2 polymorphism has been shown to be associated with asthma or atopy. Despite the differences in gene structure between the two COX isoforms, the catalytic activities and tertiary structures of COX-1 and COX-2 are remarkably similar: there is 61% homology between their amino acid sequences (Hla & Neilson 1992; Filizola et al. 1997), but COX-2 has a broader specificity for substrates because the hydrophobic channel leading to the active site of the enzyme is more accommodating. More recently, more COX enzymes have been discovered: COX-3 and two smaller forms of COX-1. All these are derived from the COX-1 gene by alternative splicing of COX-1 mRNA. COX-3 is also constitutively expressed (Chandrasekharan et al. 2002); it remains unclear whether the splice variants of COX-1 are constitutively expressed or are inducible.

Cyclooxygenase enzymes and asthma In humans, COX-1 and COX-2 immunoreactivities have been reported in bronchial mucosa of normal and asthmatic lungs (Demoly et al. 1997; Sousa et al. 1997). The relative expression of COX-1 and COX-2 in the airways of patients with asthma remains unresolved. Demoly et al. found that neither COX-1 nor COX-2 is upregulated in stable asthma.

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Sousa et al. found enhanced expression of COX-2 but not COX-1 in asthmatic airways, whereas Taha et al. (2000) found increased immunoreactivity of both COX-1 and COX-2 in induced sputum cells from asthmatics. In normal and asthmatic human lung COX-2 is expressed in activated eosinophils, mast cells, and macrophages but not T cells (Sousa et al. 1997). It is thought that increased COX-2 expression in alveolar macrophages from asthmatic subjects may contribute to enhanced eosinophil survival through an increase in PGE2 production (Profita et al. 2003). In human bronchial smooth muscle cells, COX-2 expression is easily induced by proinflammatory IL-1β, leading to increased PGE2 formation (Bonazzi et al. 2000. In human airway epithelial cells, COX-2 expression is also upregulated by inflammatory stimuli via a mechanism that appears to involve ERK and possibly p38 MAPK (Lin et al. 2002) and JNK as well (Newton et al. 2000; Mizumura et al. 2003). In human neutrophils LTB4 is the sole eicosanoidable to up-regulate COX-2 expression (St-Onge et al. 2007). A further regulatory property of COX-2 is that its induced expression can be completely inhibited by antiinflammatory glucocorticoids such as dexamethasone (Masferrer et al. 1992). Inhibitors of transcription and translation such as cycloheximide and actinomycin D also inhibit COX-2 induction in response to either cytokines (Pang & Knox 1997a) or bradykinin (Pang & Knox 1997b) The most likely mechanism for the effect of dexamethasone is inhibition of the transcription factor NF-κB (Cembrzynska-Nowak et al. 1993). It has therefore been hypothesized that the constitutively expressed COX-1 is a housekeeping gene involved in the production of prostanoids that regulate normal cellular function, and that inducible COX-2 is involved in the production of prostanoids that mediate inflammatory and mitogenic responses. Data from mice expressing neither COX-1 nor COX-2 have suggested that COX-1 attenuates airway responsiveness under basal conditions and is important in lung protection, probably due to more efficient coupling with constitutive cytosolic isomerase producing PGE2 (Gavett et al. 1999; Zeldin et al. 2001). Under basal conditions, mice deficient in either COX-1 or COX-2 showed no difference compared with wild-type mice with respect to basal lung function and lung histology, although mice not expressing COX-1 had significantly lower PGE2 levels in BAL fluid (Zeldin et al. 2001). Following allergen sensitization and exposure, lung inflammatory indices (BAL cells, proteins, IgE, lung inflammation score) and airway hyperreactivity were significantly greater in COX-1-deficient compared with COX-2-deficient mice and both were far greater than in wild-type mice (Zeldin et al. 2001), suggesting that in allergic mouse lung COX-1 products have mainly bronchprotective effects. In contrast, much increased airway inflammation in mice not expressing COX-2 was not associated with bronchial hyperreactivity to methacholine, thus dissociating airway inflammation and methacholine responsiveness (Zeldin et al. 2001).

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Recently, further understanding of the relative contribution of the COX enzymes to allergic lung inflammation has been facilitated by the development of transgenic mice that overexpress human COX-1 on airway epithelium. No differences in basal respiratory or lung mechanical parameters were observed between COX-1 transgenic and wild-type mice. However, consistent with Zeldin’s data, COX-1 transgenic mice had almost three times increased PGE2 content in BAL fluid compared with wild-type littermates and exhibited decreased airway responsiveness to inhaled methacholine (Card et al. 2006). In response to ovalbumin challenge, comparable upregulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 transgenic mice. In order to eliminate the presumed confounding effects of COX-2 upregulation, COX-1 transgenic mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly reduced allergen-induced AHR, coincident with reduced airway leukotriene levels (Card et al. 2006). The role of COX-1 and COX-2 in the pulmonary response to bacterial LPS has also been examined. Bacterial LPS is a known risk factor for exacerbation of asthma and a cause of airway inflammation. Following exposure to LPS, all mice exhibited increased bronchoconstriction and methacholine responsiveness; however, these changes were much more pronounced in mice that did not express COX-1 or COX-2 relative to wild-type mice (Zeldin et al. 2001). The degree of airway inflammation did not differ between the genotypes, indicating that airway inflammation and bronchial hyperreactivity remain dissociated in COX-1- and COX-2-deficient mice exposed to LPS. Lung fibrotic responses following vanadium pentoxide exposure have also been investigated in COX-1- or COX-2-deficient mice (Bonner et al. 2002). Mice deficient in COX-2 but not COX-1 exhibited severe inflammatory responses by 3 days following exposure and developed pulmonary fibrosis 2 weeks after vanadium exposure. Exposure to vanadium pentoxide led to an increase in PGE2 levels in BAL fluid from wild-type and COX-1-deficient mice within 24 hours, whereas PGE2 was not upregulated in COX2-deficient mice. COX-2 appears therefore to be protective against pulmonary fibrogenesis. This finding is significant, as chronic asthma is characterized by airway remodeling, in part secondary to subepithelial fibrosis.

Cyclooxygenase and aspirin sensitivity Aspirin-sensitive respiratory disease (ASRD) refers to a particular phenotype of adult asthma that is characterized by chronically severe asthma, rhinosinusitis, and nasal polyposis (Widal et al. 1922; Samter & Beers 1968). This phenotype affects around 10% of asthmatics (Sturtevant 1999) and like other forms of adult asthma affects more women than men, with an average age of onset at around 34 years (BergesGimeno et al. 2002). It is characterized by a potentially lifethreatening exacerbation of asthma following the ingestion

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of nonsteroidal antiinflammatory drugs (NSAIDs). Often ASRD is aggressive and refractory to treatment and patients are overrepresented in the severe asthma group, with 50% of patients with aspirin hypersensitivity being steroid dependent (Stevenson & Simon 1998). The ability of NSAIDs to exacerbate symptoms in ASRD is directly related to their COX-1 inhibitory activity. COX-1 inhibitors induce acutely elevated local and systemic cysteinyl leukotriene release in aspirin-sensitive patients (Christie et al. 1992b; Nasser et al. 1995) and in the event of COX-1 inhibition, it has been hypothesized that there is insufficient upregulation of COX-2 to produce sufficient PGE2 to compensate for loss of production. With the advent of selective COX-2 inhibitors, it has been demonstrated that inhibition of COX-1 but not COX-2 precipitates asthma attacks in ASRD (Stevenson & Simon 2001; Szczeklik et al. 2001; Gyllfors et al. 2003; Senna et al. 2004). COX-2 expression is known to be diminished and its activity reduced in nasal polyps from patients with ASRD (Pujols et al. 2004). It has therefore been hypothesised that selective COX-2 inhibitors are well tolerated by patients with ASRD because COX-2 expression is diminished in the lung and does not contribute much to eicosanoid balance (Szczeklik & Sanak 2006).

Receptors Following intracellular synthesis, prostanoids leave the cell probably by facilitated diffusion (Smith 1986). Outside the cell prostanoids act either on the parent or neighboring cells in an autocrine or paracrine fashion through specific G proteinlinked prostanoid receptors to stimulate or inhibit changes in the levels of second messengers (Smith 1989). Pharmacologically, prostanoid receptors are classified according to the prostanoid that causes selective activation of GPCRs. In the early 1980s, five different receptors were described, designated DP, EP, FP, IP, and TP on the basis of preferential sensitivity to PGD2, PGE2, PGF2α, PGI2, and TXA2 (Coleman et al. 1984). Within each receptor type there may be distinct subtypes, many of which have been identified using radioligand binding studies and cloning. Individual prostanoid receptors share 20–30% sequence identity with each other and encode specific motifs common only to members of the subfamily (Hata & Breyer 2004). Recently, a ninth prostaglandin receptor has been identified, CRHT2 (chemoattractant receptor homologous molecule expressed on Th2 cells), which binds PGD2 (Hirai et al. 2001). Activation of a given prostaglandin receptor by its cognate ligand may elicit varying responses depending on the cell type and tissue in which it is expressed. Evidence is also emerging that prostanoid receptors may be expressed not only on the cell surface but also on the nuclear membrane (Gobeil et al. 2002). Increased understanding of prostaglandin receptor function has started to reveal a complicated picture in which prostaglandins serve to both promote and inhibit inflammation.

Pharmacologic properties of prostanoids in asthma PGD2 PGD2 has long been associated with inflammation and allergy. It is mainly produced by activated mast cells or T cells (Roberts et al. 1980). PGD2 is the most abundant eicosanoid generated during IgE-mediated allergic responses (Lewis et al. 1982) and by ionophore-stimulated (Holgate et al. 1984) human pulmonary mast cells. In a murine model of asthma, PGD2 was found to increase the recruitment of eosinophils and CD4+ Th2 cells into the lung to induce expression of macrophage-derived chemokine, a chemoattractant for Th2 cells in (Honda et al. 2003). In patients with exercise-induced asthma, dietary supplementation with fish oil has been demonstrated to improve pulmonary function and decrease symptoms and β2-agonist use (Mickleborough et al. 2006. These findings were associated with a reduction in induced sputum differential cell count percentage. Concentrations of cysteinyl leukotrienes, PGD2, IL-1β, and TNF-α were also significantly reduced before and following exercise on the fish-oil diet compared with normal and placebo diets. PGD2 is synthesized by PGD synthase, which catalyzes simple nonoxidative rearrangements (isomerization) of PGH2 (Urade et al. 1995). The neural form of PGD synthase is glutathione (GSH) independent and is a member of the lipocalin superfamily (Nagata 1991). In contrast, the hematopoietic form of PGD synthase is GSH dependent and is a member of the sigma class of glutathione-S-transferases (Kanaoka et al. 1997). In the united airways theory, both allergic rhinitis and asthma are viewed as manifestations of a single allergic airway. It is therefore of note that in both normal controls and patients with allergic rhinitis the hematopoietic form of PGD synthase, but not lipocalin-type PGD synthase, is the predominant form expressed in inflammatory cells (mast cells, eosinophils, macrophages, and lymphocytes) and structural cells (epithelial cells and fibroblasts). Higher levels of the hematopoietic form of PGD synthase are also found in patients with allergic rhinitis compared with normal controls (Okano et al. 2006). In murine asthma models, mice that overexpress lipocalin-type PGD synthase have elevated PGD2 levels and an increased allergic response in the ovalbumininduced model of airway hyperreactivity (Fujitani et al. 2002). PGD2 is able to bind with similar high affinity and to activate two distinct GPCRs: DP (Boie et al. 1995) and CRHT2 (Hirai et al. 2001; Nagata et al. 1999a,. The proinflammatory effects of PGD2 therefore appear to be mediated by its actions on both DP and CRHT2 receptors. PGD2 may also contribute to the nonimmune pathogenesis of asthma. Hyperplasia of airway epithelial goblet cells leading to mucus hypersecretion is a feature of airway remodeling. Mucus secretion by colonic goblet cells has been linked to the DP receptor (Wright et al. 2000). The DP receptor is also expressed on bronchiole epithelial cells in antigen-challenged mice (Matsuoka et al. 2000) and is found on nasal epithelial goblet cells in normal human volunteers (Nantel et al. 2004).

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Taken together, these observations suggest that the DP receptor may evoke mucus secretion in response to PGD2 in asthma and allergic rhinitis. PGD2 is a potent contractile agent and elicits a rapidly developing bronchoconstriction (Hardy et al. 1984; Beasley et al. 1987a,b). In asthmatic subjects it has a potency of approximately 3.5 and 10 times greater than PGF2α and histamine, respectively, with the maximal effect evident 3 min following inhalation. However, these effects are unlikely to be mediated by the DP receptor since an increase in intracellular cAMP is typically associated with smooth muscle relaxation (Hall 2000). Despite the fact that PGD2 has relatively low affinity for the TP receptor (Abramovitz et al. 2000), it has been suggested that TP receptors mediate the bronchoconstrictive response to PGD2 (Beasley et al. 1989; Francis et al. 1991; Johnston et al. 1992). The TP antagonist ramatroban is a CRHT2 antagonist (Sugimoto et al. 2003) and the TXA2 metabolite 11-dehydro-TXB2 has been demonstrated to be a CRHT2 agonist (Bohm et al. 2004). Exposure to respiratory RNA viruses often aggravates the airway inflammation and bronchial responsiveness, characteristic of asthma. In a rat model of asthma, dsRNA (a nucleotide synthesized during viral replication) was instilled into the trachea and an increase in airway eosinophilia and enhanced bronchial hyperresponsiveness to methacholine in ovalbumin challenge was observed. These changes were associated with induction of COX-2 expression and COX-2-dependent PGD2 synthesis in the lungs, particularly in alveolar macrophages – suggesting that COX-2-dependent production of PGD2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenetic factors responsible for the dsRNAinduced enhancement of airway inflammation and responsiveness (Shirashi et al. 2008). These observations raise the possibility that activation of the CRHT2 receptor may contribute to bronchoconstriction in asthma. The DP receptor is expressed on bronchial epithelium. Its activation leads to increased intracellular concentrations of cAMP and mediates physiologic and pathologic events, including the allergic response, by producing chemokines and cytokines that recruit inflammatory lymphocytes and eosinophils (Matsuoka et al. 2000) to the airway. BAL fluid analyses have demonstrated that PGD2 is released in the airways following antigen challenge during acute allergic response (Murray et al. 1986). In 2000, a series of landmark experiments by Matsuoka and colleagues with DP receptordeficient mice clearly demonstrated that PGD2 is a proinflammatory prostanoid in allergic asthma. Sensitization and aerosol challenge with ovalbumin induced increases in the serum concentration of IgE that was similar in both DP receptor-deficient and wild-type mice. However, the concentrations of Th2 cytokines and the extent of lymphocyte accumulation in the lung of ovalbumin-challenged DP receptor-deficient mice was greatly reduced. Furthermore, DP receptor-deficient mice showed only marginal infiltration of eosinophils and did not develop airway hyperreactivity.

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These findings were strongly suggestive that the DP receptor plays a key role in mediating the effects of PGD2 released by mast cells during an asthmatic response (Matsuoka et al. 2000). DP antagonists have also been shown to inhibit ovalbumin-induced airway hyperreactivity in mice (Arimura et al. 2001). Furthermore, PGD2 may inhibit eosinophil apoptosis via the DP receptor (Gervais et al. 2001). In humans, the CRHT2 receptor is expressed on Th2 lymphocytes, eosinophils, and basophils (Nagata et al. 1999a,b; Hirai et al. 2001), key cells in the allergic response. The CRHT2 receptor (also known as the DP2 receptor) is a GPCR but shares little sequence homology with prostanoid DP receptor and structurally is closely related to chemoattractant receptors. As with many chemoattractant receptors, CRHT2 couples to a Gi-type protein leading to inhibition of intracellular cAMP and to an increased intracellular concentration of calcium (Hirai et al. 2001; Sawyer et al. 2002), with the specific response being influenced by cell type (Nagata et al. 2003). The CRHT2 receptor binds an overlapping but distinct set of ligands compared with the DP receptor. For example, the CRHT2 receptor binds the PGD2 metabolite 15-deoxy-Δ12,14-PGJ2 with equally high affinity as PGD2 itself (Sawyer et al. 2002), raising the possibility that metabolites of PGD2 may differentially exert effects through CRHT2 but not the DP receptor. The TP antagonist ramatroban (BAY u4305) is also a CRHT2 antagonist (Sugimoto et al. 2003) and this may explain the reduction in eosinophilic infiltration observed following antigen challenge in ramatroban-treated animals (Nagai et al. 1995; Narita et al. 1996), since TP receptor activation has no effect on eosinophil migration (Monneret et al. 2001). In allergic rhinitis, expression of the CRHT2 but not the DP receptor has been found to strongly correlate with eosinophilic inflammation in the nasal mucosa (Okano et al. 2006). Activation of CRHT2 by PGD2 has been shown to induce a modulation of eosinophil morphology, and an increase in both eosinophil and Th2 cell motility (Nagata et al. 2003). Shiraishi et al. (2005) have also underlined the significant role of PGD2 in eosinophil trafficking from the bloodstream to the airways during bronchial inflammation. Therefore, PGD2 acting through both DP and CRHT2 receptors is likely to contribute to the eosinophilic infiltration that is a hallmark of allergic asthma and rhinitis. Recent studies using two in vivo models of allergic inflammation (atopic dermatitis and allergic asthma) showed that CRHT2 plays a critical role in allergic diseases and underlined the interest of CRHT2 antagonists in human therapy (Spik et al. 2005). Characterization of mice deficient in the CRHT2 receptor will begin to clarify the exact role that each receptor plays in mediating the effects of PGD2 in allergic inflammation.

PGF2αα Human lung parenchyma, and to a lesser extent bronchial tissue fragments, spontaneously release PGF2α under resting conditions, leading to the suggestion that this prostanoid,

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together with PGE2, may be important in maintaining bronchial tone (Schulman et al. 1982; Cuthbert & Gardiner 1983). PGF2α is synthesized by PGE 9-ketoreductase via three pathways from PGE2, PGD2, or PGH2, PGD 11-ketoreductase, or PGH 9,11-endoperoxide reductase (Watanabe 2002). It binds the FP receptor, which was originally cloned from human kidney, uterus, and placental cDNA libraries (Abramovitz et al. 1994) and has two isoforms, FP(A) and FP(B), which differ from each other in C-terminal tail length (Pierce et al. 1997). Recently, a six-transmembrane splice-variant FP receptor mRNA from human heart and placenta cDNA, named hFP(S), was described (Vielhauer et al. 2004). The FP receptor is the least selective of the prostanoid receptors in binding the principal endogenous prostaglandins and binds both PGD2 and PGE2 at nanomolar concentrations (Abramovitz et al. 2000). When activated the FP receptor signals via Gq-mediated IP3 generation and increases in intracellular calcium (Abramovitz et al. 1994; Sugimoto et al. 1994). FP receptor expression has not been observed on immune cell populations (Tilley et al. 2003. Therefore, unlike the other prostaglandins, there is very little evidence to support a role for PGF2α–FP receptor signaling in inflammatory and immunologic processes. Nonetheless the inhalation of PGF2α has contractile activity on isolated human airway tissue (Sweatman & Collier 1968; Orehek et al. 1973) and causes bronchoconstriction in vivo, with asthmatic subjects demonstrating greater sensitivity to the effects of inhaled PGF2α than normal control subjects. In a study of 10 asthmatic and 10 healthy controls, it was reported that asthmatic subjects were 10 times more sensitive to histamine but 8000 times more sensitive to the bronchoconstrictor action of inhaled PGF2α (Mathe et al. 1973). Furthermore, whereas the healthy controls had reproducible decreases in specific airway conductance in response to repeated PGF2α challenge, the asthmatic subjects demonstrated tachyphylaxis (Mathe & Hedqvist 1975). Other studies have also reported similar responses to PGF2α inhalation in asthmatic subjects but with wide interstudy variation in the degree of hyperreactivity, ranging from 150-fold (Smith et al. 1975) to 8000-fold (Mathe et al. 1973) greater sensitivity compared with nonasthmatic subjects. In studies of asthmatic individuals that examined the effect of PGF2α on airway responsiveness, inhalation of a nonbronchoconstricting dose of PGF2α led to a fourfold enhancement of bronchial responsiveness to histamine (Walters et al. 1981; Heaton et al. 1984), but had no effect on subsequent methacholine provocation (Heaton et al. 1984).

TXA2 The majority of TXA2 produced in vivo is generated from platelets (Hamberg et al. 1975) but it is also produced after activation of a number of inflammatory cells, including alveolar macrophages (Nusing et al. 1990) and eosinophils (Kroegel et al. 1993. Due to its prothrombotic and vasoconstrictor effects, TXA2 has been described as the physiologic

antagonist of prostacyclin (Rolin et al. 2006). In addition to its roles as a mediator of platelet function and its association with heart failure (Castellani et al. 1997) and ischemic heart disease (Oates et al. 1988), TXA2 is a potent bronchoconstrictor and is increased in the airways of asthmatic patients on antigen challenge (Wenzel et al. 1991). Its other pharmacologic actions include increased microvascular leakage, impairment of mucociliary clearance, (Kurashima et al. 1995), and induction of AHR (Fujimura et al. 1995). TXA2 can also stimulate proliferation of airway smooth muscle cells, suggesting a role in the pathogenesis of asthma (Devillier & Bessard 1997). Increased concentrations of TXA2 and its metabolites have been demonstrated in BAL fluid, urine, plasma, and exhaled breath condensate from asthmatic patients (Wenzel et al. 1989; Kumlin et al. 1992a; Huszar et al. 2005). In a murine asthma model, blockade of either thromboxane synthesis or TP receptor activation reduced ovalbumin-induced cytokine production, which in turn inhibited eosinophil infiltration into the murine airway, with splenic mononuclear cells from treated mice exhibiting impaired antigeninduced cytokine production (Shi et al. 1998). TXA2 also appears to facilitate allergen-induced cough in a guinea-pig asthma model (Xiang et al. 2002). These data suggest that TP receptor signaling may promote cytokine production and allergic inflammation, although the exact mechanism and target cell population(s) responsible are not clear. In humans, the administration of the TXA2 receptor antagonist seratrodast is associated with a decrease in the concentration of eosinophilic cationic protein in the sputum of asthmatic patients (Fukuoka et al. 2003). The enzymes responsible for catalyzing the conversion of PGH2 to TXA2 are called the TXA synthases and have now been cloned from human platelets (Yokoyama et al. 1991) and lung (Ohashi et al. 1992). A member of the cytochrome P450 family, TXA synthase localizes to the epithelial goblet cells and subtracheal gland in a guinea-pig model (Xiang et al. 2002). In vivo, the half-life of TXA2 is only about 30 s and therefore much of the information about its biological activity comes from studies employing synthetic compounds that are stable analogs of the TXA2 structure, such as the long-acting thromboxane mimetic U-46619. TXA2 is readily degraded to its more stable and relatively inactive metabolite TXB2 which can be measured in biological fluids such as BAL and urine. Prostanoid TP receptors are distributed in both plasma membrane and cytosolic compartments and are found in tissues rich in vasculature such as lung, heart, and kidney (Rolin et al. 2006). The human TXA2 receptor, termed TP, was the first eicosanoid receptor cloned (Hirata et al. 1991). Two G proteincoupled isoforms of this receptor have been subsequently described, TPα (placental/platelet) and TPβ (endothelial), and these two splice variants differ in the length and sequence of the carboxyl-terminal tail distal to Arg328 (Raychowdhury

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et al. 1994, 1995). Both TPα and TPβ were initially characterized as functionally coupling to a Gq protein (Shenker et al. 1991), leading to phospholipase C activation, calcium release, and activation of PKC. Subsequently, differential G-protein coupling (Gi, Gs, Gh) of the two isoforms has been noted in cultured cells, where TPα and TPβ show similar ligand binding characteristics and phospholipase C activation but oppositely regulate adenylyl cyclase activity. TPα activates adenylyl cyclase, whereas TPβ inhibits it (Hirata et al. 1996). However, the physiologic role for differences in coupling between the TP receptor splice variants remains to be determined. Intrareceptor differences in C-terminal tail sequence also allow for significant differences in their ability to internalize in response to agonist exposure. For example, in HEK-293 cells, TPβ but not TPα undergoes U46619-induced GRK phosphorylation and internalization (Parent et al. 1999), whereas the C-terminus of TPα is not capable of being phosphorylated by GRKs (Zhou et al. 2001). TXA2 is a potent stimulator of airway smooth muscle cell proliferation (Devillier & Bessard 1997), smooth muscle constriction, and bronchial hyperresponsiveness (Arimura et al. 1994). In human airway smooth muscle cells the TP receptor, predominantly coupled to Gi/Go proteins, activates the Ras/ERK pathway to induce mitogenesis, probably with the involvement of nonreceptor tyrosine kinases and PKC (Citro et al. 2005). In bronchiole smooth muscle cells stimulation by U46619 leads to an increase in mitogenesis that can be blocked with the TP antagonist SQ29548 (Capra et al. 2003). It is therefore not inconceivable that TP receptor signaling partly mediates the airway smooth muscle hyperplasia seen in the airway remodeling of asthma (Vignola et al. 2003). The mechanism by which TXA2 induces bronchoconstriction and an increase in lung resistance remains unclear. Activation of TP receptors expressed on bronchiole smooth muscle cells leads to intracellular calcium mobilization (Capra et al. 2003), the predominant pathway mediating constriction of airway smooth muscle cells (Hall 2000). However, although TXA2 appears to cause airway constriction by TP receptor activation, it has been demonstrated that this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor antagonists (Allen et al. 2006). TXA2 may also exacerbate AHR by potentiating airway smooth muscle contraction elicited by cholinergic parasympathetic neurotransmission (Chung et al. 1985; Munoz et al. 1986). Pretreatment with ipratropium bromide, a muscarinic receptor antagonist, attenuates U46619-induced bronchoconstriction in asthmatic subjects, suggesting that TXA2-mediated bronchoconstriction may itself be mediated by cholinergic neurons (Saroea et al. 1995). TXA2 receptor antagonists and thromboxane synthase inhibitors have been developed that inhibit the physiologic actions of TXA2. Double-blind, placebo-controlled clinical trials have shown limited clinical benefit of the thromboxane receptor antagonist seratrodast and the selective thromboxane

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synthase inhibitor ozagrel in the treatment of patients with asthma. In 1992, ozagrel was released onto the Japanese market (Nakazawa et al. 1994). In humans, oral and inhaled administration of ozagrel hydrochloride reduced bronchial hyperresponsiveness to acetylcholine and leukotriene D4 in asthmatic subjects (Fujimura et al. 1986, 1990a,b). Seratrodast (AA-2414) was the first thromboxane receptor antagonist to be developed as an antiasthmatic drug and has received marketing approval in Japan (1997). Since 1999, four relevant clinical studies have been published with seratrodast that confirm its effect in asthma. Seratrodast treatment has been shown to decrease daytime asthma symptoms, bronchial hyperresponsiveness to methacholine, and supplemental use of inhaled β2 agonists but did not change baseline pulmonary function (Fukuoka et al. 2003). These results support the concept that TXA2 may play a part in the pathogenesis of bronchial hyperresponsiveness in asthma. Furthermore, antagonsim of the TXA2 receptor, while having minimal effects on pulmonary function, has also been shown to decrease the amount of sputum produced by asthmatics along with alterations in its physicochemical properties (Tamaoki et al. 2000). The TP antagonist, BAY u3405 or ramatroban, is also active on a wide range of airway smooth muscles cells across species, including human bronchial smooth muscle (McKenniff et al. 1991). It is highly effective in reducing antigen-, LTC4- and LTD4-induced bronchoconstriction in guinea pigs (Francis et al. 1991; Iwamoto et al. 1995) and PGD2-, exercise-, bradykinin- and histamine-induced bronchoconstriction in humans (Magnussen et al. 1992; Rajakulasingam et al. 1996). Moreover, Aizawa et al. (1996) demonstrated that oral ramatroban (75 mg/day) attenuated bronchial hyperresponsiveness to methacholine in asthmatics. Ramatroban is currently under clinical evaluation as an antiasthmatic agent (Rolin et al. 2006) and has been shown to partially attenuate PGD2induced bronchial hyperresponsiveness in humans, probably through its actions as an antagonist at the CRHT2 receptor and as an inhibitor of PGD2-induced chemotaxis of eosinophils (Sugimoto et al. 2003). TP receptors can be activated by high levels of other eicosanoids, including PGD2 and 8-iso-PGF2α, which act as TP receptor agonists (Elmhurst et al. 1997; Audoly et al. 2000) and may play an important role in activating prostaglandin receptors in settings of oxidative stress. The isoprostane 8-iso-PGF2α, a chemically stable product of oxidative stress, is also known to activate TP receptors, albeit as a partial agonist (Kinsella 2001). This perhaps limits the clinical use of thromboxane synthase inhibitors.

PGE2 PGE2 and allergic inflammation Prostaglandin E synthase (PGES), which generates PGE2 from COX-derived PGH2, occurs in multiple forms, two of which are membrane bound and one of which is cytosolic. Microsomal PGES (mPGES)-1 is a member of the MAPEG (microsome-

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associated proteins of eicosanoid generation) family of proteins, as are FLAP and LTC4 synthase, and like these proteins it is inhibited by MK-886, raising caution about use of this drug, originally described as a FLAP inhibitor, in discerning the role of leukotrienes in any particular process. mPGES-1 is induced by proinflammatory stimuli and downregulated by antiinflammatory glucocorticoids (Murakami et al. 2000). mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to formation of a mature cytosolic enzyme, although its cellular function remains uncertain (Tanikawa et al. 2002; Murakami et al. 2003). Whereas mPGES-1 is an inducible enzyme, cytosolic PGES is constitutively expressed, and in vitro these enzymes have been shown to be functionally coupled to COX-2 (Murakami et al. 2000; Mancini et al. 2001; St Onge et al. 2007) and COX-1 (Tanioka et al. 2000), respectively. PGE2 itself is rapidly degraded by the enzyme 15prostaglandin dehydrogenase (15-PGDH) (Bergholte & Okita 1986). Innate factors such as IL-1β suppress 15-PGDH, suggestive of overall upregulation of PGE2 and its relevant enzymes in response to innate stimuli (Mitchell, J.M. et al. 2000). It is now appreciated that IL-13 significantly decreases the inducible mPGES-1, while markedly upregulating 15PGDH, the enzyme preferentially metabolizing PGE2 (Trudeau et al. 2006). Asthma is a chronic Th2-dominated inflammatory disease. Despite both proinflammatory and antiinflammatory actions in vitro, in vivo inhaled PGE2 has been shown to attenuate exercise-induced (Melillo et al. 1994), allergen-induced (Pavord et al. 1993), and aspirin-induced bronchoconstriction (Sestini et al. 1996), as well as protecting against bronchoconstrictor agents such as methacholine and histamine (Walters et al. 1982; Manning et al. 1989). Hence, PGE2 is widely regarded as the prostanoid with the most prominent immunomodulatory, antifibrotic, and bronchodilating properties. It is spontaneously released from both human epithelial cells (Churchill et al. 1989) and lung fibroblasts (Korn 1983). Further sources include macrophages (Macdermott et al. 1984), follicular dendritic cells (Heinen et al. 1986), and endothelial cells. There are many events leading to the development of allergic inflammation which PGE2 may modulate. The adherence of inflammatory cells to endothelium is one of the initial events necessary for migration of these cells through the vascular wall. It is likely that PGE2 can modulate cell recruitment indirectly through inhibition of TNF-α. PGE2 inhibits both TNFα-induced expression of ICAM-1 and vascular cell adhesion molecule (VCAM)-1 on airway smooth muscle and the adhesion of activated T cells to TNF-α-stimulated airway smooth muscle (Panettieri et al. 1995). It also blocks the synthesis of endothelial–leukocyte adhesion molecule (ELAM)-1 and VCAM-1 in endothelial cells, which contributes to the reduced adhesion of activated T cells (Pober et al. 1993). The mechanisms by which PGE2 may regulate AHR and

airway inflammation after allergen challenge are speculative. Both eosinophils and mast cells, which are present in greater numbers in the asthmatic lung (Pin et al. 1992), are rich sources of proinflammatory cysteinyl leukotrienes and are immunomodulated by PGE2. Acting via the EP2 receptor (Kay et al. 2006), PGE2 inhibits antigen-induced human mast cell degranulation (Peters et al. 1982). Conversely, in vitro it appears to potentiate IgE-mediated histamine release from human peripheral blood-derived cultured mast cells via its actions on EP3 and/or EP1 receptors. Endogenous PGE2 inhibits PAFinduced synthesis of the potent bronchoconstrictor LTC4 by eosinophils (Tenor et al. 1996). It has been hypothesized that the ability of inhaled PGE2 to inhibit the late response is secondary to its ability to inhibit allergen-induced synthesis of LTC4. PGE2 also inhibits human eosinophil degranulation in vitro (Kita et al. 1991) chemotaxis, and cytokine-stimulated survival (Alam et al. 1993) and reduces arachidonic acid and TXA2 release from bronchial biopsies. Raised intracellular levels of cAMP generated by PGE2 have been shown to suppress leukotriene and prostaglandin production by neutrophils and eosinophils (Keuhl et al. 1987). However, increased levels of PGE2 in sputum from asthmatic patients may prolong eosinophil survival (Profita et al. 2003). Profita et al. which obtained induced sputum from 14 control and 30 asthmatic subjects, and demonstrated a striking correlation between PGE2 concentration and eosinophils numbers (P < 0.0001) and eosinophil cationic protein concentration (P < 0.0001). Immunostaining for COX-2 showed enhanced expression in macrophages of asthmatic subjects compared with control subjects, and incubation of sputum with the highly-specific COX-2 inhibitor SC58236 led to marked suppression of PGE2 levels, providing evidence that PGE2 synthesis was the result of COX-2 enzymatic activity in asthma-induced sputum cells. Incubation of peripheral blood eosinophils with induced sputum supernatant of subjects with a high eosinophil count caused decreased apoptosis of peripheral blood eosinophils compared with control subjects, but immunoprecipitation of PGE2 significantly reverted this phenomenon. PGE2 enhancement of eosinophil survival may therefore also contribute to the development of the “classic” eosinophilic inflammation of the airways of asthmatic subjects. PGE2 also inhibits leukotriene biosynthesis both directly and indirectly in certain specific cell types. In 2002, using human polymorphonucleocytes stimulated with PAF and thapsigargin, it was demonstrated that a number of cAMP-elevating agents including PGE2 inhibited leukotriene biosynthesis and 5-LO translocation to the nucleus (Flamand et al. 2002). PGE2 inhibits the production of LTB4 from human bone marrow-derived dendritic cells. This is achieved indirectly by an IL-10-dependent mechanism that inhibited expression of FLAP (Harizi et al. 2003). The presence of PGE2 during T-cell activation and maturation following priming by antigen-presenting cells (APCs) also appears to modulate the nature of the cellular immune

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response. Th1 and Th2 cells are differentiated from naive T cells after antigenic stimulation by the influence of cytokines (IL-12, IFN-γ) in the case of Th1 and IL-4/IL-13 in the case of Th2 or costimulatory molecules (CD80/CD86) provided by APCs. In vivo, PGE2 is thought to polarize the balance in favor of a Th2 response partly by EP4 receptor-mediated inhibition of IL-12 production by monocytes and macrophages (Van der Pouw Kraan et al. 1995; Nataraj et al. 2001) and partly by acting directly on T cells to suppress production of IL-2 and IFN-γ (Hilkens et al. 1995; Kabashima et al. 2003b). However, some reports suggest that PGE2 can also inhibit Th2 cytokine secretion by EP2/EP4 receptor-driven cytokine-induced class switching and IgE production by B cells (Fedyk & Phipps 1996). Despite evidence suggesting that PGE2 influences CD4+ T-cell maturation in favor of a Th2-dominant immune response, in vivo studies suggest that PGE2 suppresses Th2mediated allergic inflammation. As well as inhibiting transendothelial migration of human T cells, PGE2 also upregulates expression of Th2 cytokines following allergen challenge. In a rat model of allergic asthma, pretreating rats with PGE2 prior to challenging them with ovalbumin reduced the increase in the number of cells expressing IL-4 and IL-5 mRNA and eosinophils in lavage fluid, to levels comparable to those seen in sham-challenged animals. This suggested that in part the antiinflammatory effects of PGE2 are mediated by reducing allergen-induced Th2 cell activation (Martin et al. 2002). Dendritic cells (DCs) are thought to play an important role in the pathogenesis of allergic disorders via their interactions with T cells to initiate and amplify Th2 immune responses. DCs are the most potent APCs and are highly responsive to inflammatory stimuli such as bacterial LPS and TNF-α (Steinman 1991). DCs express all four EP receptor subtypes (Harizi et al. 2003) and PGE2 has been shown to modulate DC function. PGE2 upregulates IL-10, which downregulates IL-12 production and the antigen-presenting capabilities of bone marrow cells (Harizi et al. 2002). When activated in the presence of PGE2, DCs lose their ability to secrete cytokines such as IL-12, CCL3, and CCL4, but upregulate expression of chemokine receptors necessary for migration to lymph nodes (Luft et al. 2002; Scandella et al. 2004 Jing et al. 2003). In human monocyte-derived dendritic cells (MoDCs), stimulation of EP2 and EP4 by PGE2 promotes MoDC maturation and inhibits LPS-induced cytokine production (Kubo et al. 2004). Coculture of naive T cells with matured MoDCs showed that EP2/EP4-stimulated MoDCs preferentially induced Th2 polarization, indicating the importance of EP2 and EP4 receptors in the determination of Th1/Th2 development of naive T cells (Kubo et al. 2004). In vitro, LPS-stimulated MoDCs from individuals with asthma exhibited increased PGE2 and IL-10 production compared with DCs from normal subjects. Increased PGE2 synthesis by DCs from subjects with asthma was associated with an increase in COX-2 mRNA expression (Long et al. 2004). Alveolar macrophages represent a major source of PGE2, in

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particular after expression of COX-2 (Hempel et al. 1994 ). The resolution of inflammation in asthma and tissue homeostasis is dependent on clearance of apoptotic cells by alveolar macrophages. In animal and in vitro studies, apoptotic cell clearance induces secretion of antiinflammatory mediators such as transforming growth factor (TGF) and PGE2, with associated suppression of proinflammatory cytokines, chemokines, and eicosanoids (Fadok et al. 1998). Clearance of apoptotic cells is crucial to the resolution of inflammation and development of fibrosis, but the process is not well understood in normal or diseased human lungs. Human BAL macrophages from normal control subjects and subjects with mild/moderate or severe asthma have been examined in vitro for phagocytosis of apoptotic human T-cell line Jurkats and secretion of inflammatory mediators (Huynh et al. 2005). Alveolar macrophages from normal subjects and patients with mild/ moderate asthma were able to phagocytose apoptotic cells in response to LPS, resulting in PGE2 and 15-HETE induction; in contrast, cells from patients with severe asthma had defective LPS-stimulated uptake of apoptotic cells, with associated failure to induce PGE2 and 15-HETE. A decrease in the responsiveness of alveolar macrophages to LPS in severe asthma is manifested by defective apoptotic cell uptake and reduced secretion of inflammatory mediators and this may contribute to the chronicity of inflammation and remodeling in lungs of patients with asthma (Huynh et al. 2005). Finally, PGE2 has a fundamental role in tissue repair and fibrosis. Airway remodeling in chronic asthma is characterized by specific structural changes in the airway wall including subepithelial fibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia and hypertrophy, mucous gland and goblet cell hyperplasia, and epithelial disruption. These changes are typically refractory to corticosteroids and make a major contribution to disease chronicity. Unstressed bronchial epithelium releases PGE2 and 15-HETE, which supress mesenchymal cell activation. In asthma, however, the epithelium has increased susceptibility to oxidant injury and imparied epithelial repair. PGE2 acts as a potent inhibitor of mitogenesis, collagen synthesis, and mesenchymal cell chemotaxis and can therefore suppress inflammation and fibroblast activation. In human lung explants PGE2 also reduces mucus glycoprotein secretion (Marom et al. 1981).

PGE2 receptors PGE2 activates four GPCRs (EP1–EP4) that are present in differing ratios depending on the cell system. Stimulation of EP1 receptors results in activation of phosphatidylinositol hydrolysis and elevation of the intracellular Ca2+ concentration. EP2 and EP4 ligation increases intracellular cAMP through activation of adenyl cyclase (Narumiya et al. 1999. However, direct comparison of the relative abilities of EP2 and EP4 to increase cAMP reveals much weaker Gs coupling by EP4 compared with the EP2 receptor (Fujino et al. 2002). The striking feature of the EP3 receptor is that it exists in

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multiple isoforms generated by alternative splicing of the C-terminal tail. In humans, at least eight EP3 receptor splice variants have been identified, and multiple splice variants exist for other species including mouse, rabbit, and cow (Hata & Breyer 2004). EP3 generally couples to Gi and its stimulation inhibits adenylate cyclase, leading to a reduction in intracellular cAMP (Narumiya et al. 1999) but individual splice variants can also couple to stimulation of cAMP and IP3 generation (Irie et al. 1993; Namba et al. 1993). Hence, the diverse effects of PGE2 may be accounted for in part by the existence of EP1, EP2, EP3, and EP4 receptors, and heterogeneity in the coupling of these receptors to intracellular signal transduction pathways. It is known that EP receptors are localized to the plasma membrane. However, radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver has revealed perinuclear localization of EP3 and EP4 (Bhattacharya et al. 1999) and, in humans, perinuclear EP3 receptors have been found in brain endothelial cells (Gobeil et al. 2002) and adult epidermis (Konger et al. 2005). The EP receptors in brain endothelial cells have been proven to be functional as they modulate transcription (Gobeil et al. 2002), including the gene encoding inducible nitric oxide synthase (iNOS). COX-1 and COX-2 have been shown to be present on the inner and outer nuclear membranes and in similar proportions (Spencer et al. 1998). As cyclooxygenases localize to the nuclear envelope and play a dominant role in prostaglandin production, it could be speculated that PGE2-induced regulation of genes in asthma might involve EP receptors localized intracellularly and specifically at the cell nucleus. Of the four EP receptors, the EP3 and EP4 receptors bind PGE2 with highest affinity (Kd < 1 nmol/L), whereas the EP1 and EP2 receptors bind with lower affinity (Kd > 10 nmol/L) (Abramovitz et al. 2000). To elucidate the differential effects of PGE2 on individual EP receptors, a number of EP receptor agonists and antagonists have been used. First-generation EP receptor agonists include butaprost (EP2), the mixed EP1 and EP3 agonist sulprostone, and PGE1-OH (EP4). More recently, second-generation EP receptor ligands with higher receptor selectivity have been developed and include the agonists ONO-DI-004 (EP1), ONO-AE1-259-01 (EP2), ONO-AE-248 (EP3), and ONO-AE1-329 (EP4) and antagonists ONO-8713 (EP1), ONO-AE3-240 (EP3), and ONO-AE208 (EP4) respectively (Suzawa et al. 2000; Watanabe et al. 2000; Kabashima et al. 2002; Amano et al. 2003). Analyses of expression patterns of the four EP receptors have revealed their presence on most major subsets of cells involved in the adaptive immune response including macrophages, neutrophils, CD3+ T lymphocytes, and eosinophils in asthmatic patients and normal controls (Ying et al. 2004), suggesting that PGE2 may play a role at multiple levels within the immune system. EP3-deficient mice, but not EP1-, EP2-, or EP4-deficient mice, exhibited enhanced allergic inflamma-

tion in an EP receptor knockout model of murine ovalbumininduced allergic inflammation (Kunikata et al. 2005) and the selective EP3 agonist AE-248 suppressed the increased inflammation. In humans, induced sputum from atopic asthmatic patients contained greater numbers of macrophages expressing all four EP receptors and elevated percentages of these cells expressing EP2 and EP4 (Ying et al. 2004). The elevated expression of the PGE2 receptors EP2 and EP4 on bronchial macrophages in asthma, demonstrated in this study, is particularly interesting because studies in knockout mice have shown that EP2 and EP4, but not EP1 and EP3, agonists reduce the production of proinflammatory cytokines such as TNF-α but increase that of antiinflammatory cytokines such as IL-10 by macrophages (Shinomiya et al. 2001). PGE2 also reduces chemokine production in human macrophages through action on the EP4 receptor (Takayama et al. 2002). Furthermore, EP2 and EP4 receptor agonists, including PGE2 itself, upregulates COX-2 expression in activated macrophages, which might form a mechanism for further stimulation (Hinz et al. 2004). Ying et al. (2006) have also examined the expression of EP1–EP4 in nasal biopsies from patients with ASRD. ASRD is characterized by asthma often poorly responsive to treatment, rhinosinusitis, and recurrent nasal polyps. Impaired braking of inflammatory cell cysteinyl leukotriene production by PGE2 has been implicated in the pathogenesis of aspirin-exacerbated airways disease, but the mechanism is obscure. To address the hypothesis that expression of one or more EP receptors on nasal mucosal inflammatory cells was deficient in patients with aspirin-sensitive compared with nonaspirin-sensitive polypoid rhinosinusitis, double-staining was used to phenotype inflammatory leukocytes expressing EP1–EP4. Global mucosal expression of EP1 and EP2, but not EP3 or EP4, immunoreactivity was significantly elevated in aspirin-sensitive and nonaspirin-sensitive rhinosinusitis compared with controls. In contrast, the percentages of neutrophils, mast cells, eosinophils, and T cells expressing EP2 but not EP1, EP3 or EP4 was significantly reduced (P ≤ 0.04) in aspirin-sensitive compared with nonaspirin-sensitive patients. These findings are suggestive of a possible role for PGE2 in mediating epithelial repair in rhinitis and asthma. Because PGE2 exerts a range of inhibitory actions on inflammatory leukocytes via the EP2 receptor, its reduced expression in aspirin-sensitive rhinosinusitis may be partly responsible for the increased inflammatory infiltrate and production of cysteinyl leukotrienes that characterize aspirin-sensitive disease. Further confirmation of the likely relevance of these findings in ASRD comes from a Japanese genetics study, which has shown that polymorphisms in the PGE2 receptor subtype 2 gene confer susceptibility to aspirin-intolerant asthma (Jinnai et al. 2004). To identify the receptors mediating the actions of PGE2 on bronchomotor tone, Tilley et al. (2003) developed a mouse model and examined the effects of PGE2 on the airways of

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wild-type and EP receptor-deficient mice. In conscious mice, they found that administration of PGE2 increased airway responsiveness primarily through the EP1 receptor. Pretreatment with either atropine or bupivacaine eliminated these effects and AHR was also undetectable in anesthetized mice or denervated tracheal rings and only EP2-mediated relaxation of airway smooth muscle was observed. PGE2/EP1/ EP3-induced airway constriction appeared to occur indirectly through activation of neural pathways, whereas PGE2induced bronchodilation resulted from direct activation of EP2 receptors on airway smooth muscle. These finding raised the possibility that selective EP receptor analogs may prove useful in the treatment of asthma. EP2 and EP3 alone are expressed on bronchial smooth muscle (Burgess et al. 2004). In humans the action of PGE2 at the EP2 receptor appears to mediate against proliferation of airway smooth muscle cells (Burgess et al. 2004). One of the cardinal features of chronic asthma is airway remodeling and in particular increased airway smooth muscle bulk. PGE2 has previously been reported to inhibit the proliferation of nonasthmatic airway smooth muscle cells. Burgess et al. (2004) hypothesized that the proliferative control exerted by PGE2 was altered in the airways smooth muscle of asthmatic patients. Human airway smooth muscle cells from 17 nonasthmatic and 18 asthmatic patients were cultured in the presence and absence of PGE2 and tritiated thymidine uptake used to measure cell proliferation. The asthmatic airway smooth muscle cells were significantly more sensitive to inhibition of proliferation by PGE2 than the nonasthmatic cells. EP2 and EP3 receptors were also significantly increased in the asthmatic cohort compared with control subjects. The asthmatic cells also had increased sensitivity to inhibition of proliferation by EP2-specific agonists (butaprost and CAY10399) but not by EP3-specific agonists. Characterization of the structure of the human gene encoding EP2 has identified a CCAAT enhancer binding protein (C/EBP) consensus binding site 66 bp upstream of the transcription initiation site and several other C/EBP consensus binding sites in the 5′ flanking region (Smock et al. 1999). Three consensus sequences for the C/EBPs have also been described in the 5′ flanking sequence of the mouse gene encoding the EP2 receptor (Katsuyama et al. 1998). It is possible that the increased numbers of EP2 receptors observed on asthmatic airway smooth muscle cells is because these cells lack C/EBPα (Borger et al. 2003; Roth et al. 2003), the negative regulatory C/EBP. The authors hypothesized that the absence of this transcription factor might lead to disruption of the regulation of transcription, translation, or both of the EP2 receptor mRNA and therefore could contribute to the increased numbers of EP2 receptors observed. Hence the increased growth observed in asthmatic airway smooth muscle cells is not the result of impaired responsiveness to PGE2, as unexpectedly these cells have increased sensitivity to the antiproliferative effects of PGE2.

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The role of the EP1 in modulating airway function is just beginning to be elucidated and it has been suggested that its activation may be associated with the β-agonist resistance found in asthma (McGraw et al. 2006). Using a mouse model, activation of EP1 did not elicit airway smooth muscle contraction in mouse trachea. However, EP1 activation markedly reduced the bronchodilatory function of β2-adrenoceptor agonists. Activation of EP1 reduced β2-adrenoceptor-stimulated cAMP in airway smooth muscle but did not promote or augment β2-adrenoceptor phosphorylation or alter β2adrenoceptor trafficking. Bioluminescence resonant energy transfer, indicative of receptor cross-talk, showed that EP1 and β2-adrenoceptors formed heterodimers and this ultimately affected β2-adrenoceptor-mediated bronchial relaxation (McGraw et al. 2006). It therefore appears that lone stimulation of EP1 does not directly modify airway tone but that EP1 acts as a modulator of the β2-adrenoceptor.

PGE2 in asthma Shortly after its discovery in the 1960s, PGE2 was observed to have potent relaxant effects on airway smooth muscle, making this endogenous lipid an attractive candidate drug for asthma (Main 1964; Mathe & Hedqvist 1975). Inhalation of 55 μg PGE1 and PGE2 in normal subjects increased specific airway conductance by 10 and 18%, respectively, and in asthmatic subjects by 41 and 39%, respectively (Smith et al. 1975). However, the effect of PGE2 on human smooth muscle is much more complex. Inhalation in normal subjects produces an initial bronchoconstriction within 5 min, followed by bronchodilatation peaking at 15 min and returning to baseline after 30 min (Walters & Davies 1982). In asthmatic subjects the response to inhaled PGE2 is more often biphasic, with the degree of initial bronchoconstriction inversely related to resting airway tone and enhanced following bronchodilatation with the anticholinergic agent ipratropium bromide (Walters & Davies 1982). Additionally, PGE2 has paradoxical effects against the bronchoconstrictor activity of histamine and methacholine, protecting against bronchoconstriction during the bronchodilator phase and enhancing bronchial responsiveness to these reference agents if administered after the end of this phase (Walters & Davies 1982). It is likely that some of these seemingly paradoxical effects are likely to be explained by the actions of PGE2 on four receptor subtypes. Ex vivo studies from humans and animals have also shown that PGE2 can both relax and constrict airway smooth muscle, a paradox explained when it was recognized that PGE2 could act through more than one receptor. A number of studies have examined the usefulness of PGE2 in various models of asthma. Inhaled PGE2 has also been shown to protect against the early- and late-phase bronchoconstriction and AHR induced by allergen (Pavord et al. 1993; Gauvreau et al. 1999), inhalation of ultrasonically nebulized distilled water (Pasargiklian et al. 1976), and sodium metabisulfite (Pavord et al. 1991). Exogenously administered

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PGE2 reduces exhaled nitric oxide in both asthmatic and normal subjects, an effect seen independently of PGE2-induced airway bronchoconstriction and thought to be secondary to iNOS inhibition (Kharitonov et al. 1998). Inhaled PGE2 also reduced Th2 cytokine and cysteinyl leukotriene production in a rat model of ovalbumin-induced allergic inflammation (Martin et al. 2002). Inhaled PGE2 has been shown to decrease the maximal fall in exercise-induced FEV1 by 63% and reduce the duration of exercise-induced asthma (Melillo et al. 1994). This effect is comparable to that found with leukotriene receptor antagonists (Manning et al. 1990b), sodium cromoglycate (Robuschi et al. 1992), and β2 agonists (Anderson et al. 1979). In a randomized, double-blind, crossover study, induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 min after exercise challenge in 25 individuals with asthma with exercise-induced symptoms (Hallstrand et al. 2005). The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of exerciseinduced asthma. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and PGE2 and TXB2 significantly decreased in the airways, and there was an increase in airway columnar epithelial cells. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. PGE2 is the predominant eicosanoid synthesized by the airway epithelium and injury to the airway epithelium is known to decrease the production of PGE2 (Holgate et al. 2003). The decreased levels of PGE2 after exercise and the increased numbers of columnar epithelial cells strongly suggests that the relative underproduction of epithelial PGE2 leads to an overproduction of cysteinyl leukotrienes by leukocytes such as bronchial macrophages. Alteration in this ratio leads to a predominance of the effects of cysteinyl leukotrienes, which are counteracted in part by PGE2 under normal circumstances. In a placebo-controlled, randomized, double-blind, crossover study, Hartert et al. (2000) noted that PGE2 reduced allergen-stimulated release of PGD2 in subjects with asthma. PGE2 has been shown to affect airway physiology, inhibiting both the early and late asthmatic response, without resulting in significant bronchodilation (Pavord et al. 1993). PGE2 may attenuate the early asthmatic response by decreasing allergen-induced release of PGD2 in the airways of asthmatic patients. This process, involving the actions of histamine, PGD2, and cysteinyl leukotrienes, is thought to be largely mast cell dependent (Holgate et al. 1991) and PGD2 is thought to be principally released from activated mast cells. It also seems likely that the mechanisms of action of PGE2 in attenuating the late asthmatic response is antiinflammatory rather than bronchodilatory. There is good evidence for PGE2 ameliorating aspirin sensitive respiratory disease (ASRD) and exogenous PGE2 has been shown to prevent aspirin-induced bronchoconstriction and urinary LTE4 excretion (Sestini et al. 1996) and the release of

cysteinyl leukotrienes from cultured peripheral blood leukocytes (Celik et al. 2001). Therefore a special role for PGE2 in the pathogenesis of ASRD has been suggested (Szczeklik et al. 1995). PGE2 has the potential to reverse at least three of the cardinal features of aspirin-sensitive asthma: enhanced cysteinyl leukotriene production, smooth muscle hyperplasia (smooth muscle cells from asthmatic patients over-express PGE2 receptors) and airways epithelial damage. It has been suggested that COX-1 inhibitor induced exacerbations of ASRD are secondary to a depletion of bronchoprotective PGE2, which is more vulnerable because of a functional deficiency in COX-2 (Pujols et al. 2004). Alternatively it has been hypothesized that decreased expression of the EP2 receptor in inflammatory cells isolated from nasal biopsies in aspirin sensitive patients, may impair PGE2 in its anti-inflammatory effects (Ying et al. 2006). If deficient PGE2 production does lie at the heart of ASRD conclusively proving this hypothesis has been difficult as in vivo and in vitro studies have yielded inconsistent results. In 2005, a paper measuring PGE2 production at baseline in nasal polyps from aspirin sensitive and aspirin tolerant patients was published (Perez-Novo et al. 2005). The authors homogenised nasal polyps, in 15% methanol and 0.1M sodium phosphate buffer and measured PGE2 in the supernatant. Polyps in patients with ASRD had significantly lower PGE2 production (30 000 pg/ml) compared to aspirin tolerant patients (50 000 pg/ml). Homogenisation can disrupt the cell membrane and it is possible that the authors were measuring intracellular as well as extracellular PGE2 levels, as the PGE2 levels measured are significantly higher than in other in vitro studies. The authors also noted that local down-regulation of PGE2 was specifically observed in both nasal polyp groups compared to samples from patients with chronic rhinosinusitis (but without nasal polyps) and normal controls. This led them to question whether diminished local production of PGE2 might not be a unique characteristic of aspirin sensitivity. Other, in vitro studies, addressing PGE2 production in aspirin-sensitive patients have involved stimulation of peripheral blood cells which are remote from the site of the disease or prolonged culture of tissues in vitro. A study published in 2000, measured PGE2 production in cell culture supernatants, from nasal polyp epithelial cell explants (Kowalski et al. 2000). The authors found diminished PGE2 production at baseline in aspirin sensitive compared to aspirin tolerant patients but on stimulation with aspirin, a similar relative inhibition of PGE2 production was seen in both groups and on stimulation with calcium ionophore a similar relative increase in PGE2 production. In contrast, a study a few years later, stimulated bronchial fibroblasts with cytomix (lipopolysaccharide, il-1α, and tumour necrosis factor-α) for 18 hours. They found that after stimulation, in aspirin sensitive patients the bronchial fibroblasts did produce less PGE2 but spontaneous production was equivalent in both groups (Pierzchalska et al. 2003).

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In vivo studies, have directly quantified PGE2 production by measuring PGE2 release in nasal lavage fluid, exhaled breath condensate and bronchealveolar lavage fluid in aspirinsensitive and aspirin-tolerant patients. The findings from these studies are inconsistent. The first study measuring eicosanoid levels in nasal lavage fluid from aspirin tolerant, aspirin sensitive, aspirin desensitised and normal patients; was published in 1988. The authors found no significant decreases in PGE2 levels after aspirin challenge in patients with ASRD but levels did decrease in all other groups (Ferreri et al. 1988). Four year later, Picado et al. published a double blind placebo controlled study where they measured CysLT and PG production in nasal lavage fluid in aspirin sensitive and tolerant patients after nasal lysine aspirin challenge. They found that there was inhibition of PGE2 production in both groups of patients (Picauo et al. 1992). In 1996 Szczeklik et al. published their findings in bronchealveolar lavage fluid after segmentally challenging, aspirin sensitive and aspirin tolerant patients with lysine aspirin. They found no variation at baseline in PG, CysLT, tryptase, IL-5 or ECP levels. But, 15 minutes after aspirin instillation, there was a statistically significant rise in CysLT, IL-5, and eosinophil number in the aspirin sensitive patients only. The administration of aspirin significantly depressed PGE2 levels in aspirin sensitive and tolerant patients. Exhaled breath condensates have also been measured in patients pre- and post-aspirin challenge and no change in measurable PGE2 was noted after aspirin challenge in either group (Sanak et al. 2004).

PGI2 (prostacyclin) Prostacyclin is generated by macrophages and constitutes a major product of endothelial cells with potent cAMPdependent smooth muscle relaxant activity for the maintenance of vascular patency. The hydrolysis product of PGI2, 6-keto-PGF1α, is released into biological fluids such as BAL and provides a reliable index of PGI2 generation and release. This metabolite has been detected as the major COX product released from both resting and allergen-stimulated fragments of human bronchi, although the cells responsible remain unidentified (Schulman et al. 1982). In studies of airway smooth muscle, PGI2 was found to cause relaxation of guinea-pig tracheal strips and of isolated precontracted human bronchus, although smooth muscle contraction was also sometimes seen (Gardiner & Collier 1980). In a study of 8 asthmatic and 10 normal controls, the effects of inhaled PGI2 and 6-oxo-PGF1α on airway caliber were examined. Inhalation of both COX products caused cough and retrosternal discomfort and no consistent effect was found in either group, except for reproducible bronchodilatation in two asthmatic subjects (Hardy et al. 1985). The same group went on to examine the effect of doubling doses of both prostanoids using different parameters of airway caliber in mild asthmatic subjects. With doses of PGI2 of up to 1 mg/mL there

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was no effect on specific airway conductance, but a concentration-dependent fall in FEV1 and Vmax30 was observed in all subjects. In two of the four subjects, there was an increase in residual volume and reduction in vital capacity without a change in total lung capacity. Furthermore, PGI2 but not 6-oxo-PGF1α protected against PGD2- and methacholineinduced bronchoconstriction, as measured using any parameter of airway caliber (Hardy et al. 1988). An explanation of these seemingly paradoxical results may be that the principal action of PGI2 in the airways is to cause vasodilatory mucosal engorgement and hence a reduction in small-airway caliber as measured by FEV1 and Vmax30, but to protect against the spasmogenic effects of other mediators by increasing their clearance from receptor sites by increasing blood flow. PGI2 is less potent than PGE2 in relaxing human airways in vitro (Tamaoki et al. 1993) and, in contrast to PGE2, does not protect against histamine-induced contraction (Knight et al. 1995). Prostacyclin binds the prostanoid IP receptor, which predominantly couples to a Gs-type G protein, leading to an increase in cAMP (Boie et al. 1994; Katsuyama et al. 1994; Nakagawa et al. 1994). C-terminal modification of the IP receptor allows the receptor to couple to other signal transduction pathways such as Gq-dependent phosphoinositide turnover (Katsuyama et al. 1994 ) and Gi-dependent inhibition of cAMP (Hebert et al. 1998). The human prostacyclin IP receptor was first cloned from lung and megakaryocyte cDNA libraries (Boie et al. 1994; Katsuyama et al. 1994; Nakagawa et al. 1994) and has been shown to be expressed in many tissues including the lung. Due to its unstable nature, PGI2 has limited experimental use. Therefore, synthetic agonists such as iloprost, cicaprost, and carbacyclin are commonly used to study IP receptor function. Studies examining signaling through the IP receptor support the hypothesis that IP receptor signaling suppresses Th2-mediated allergic inflammatory responses. IP receptor mRNA is upregulated in CD4+ Th2 cells and inhibition of PGI2 formation by the COX-2 inhibitor NS-398 during antigeninduced airway inflammation results in greater lung Th2mediated lung inflammation (Jaffar et al. 2002). Prostacyclin has been suggested to exert this effect in part by enhancing Th2 production of the antiinflammatory cytokine IL-10. An immunosuppressive role for the IP receptor in Th2-mediated inflammation is supported by the observation that in the ovalbumin-induced asthma model, IP receptor-deficient mice have increased antigen-induced leukocyte accumulation in BAL fluid and peribronchiolar and perivascular inflammatory infiltration (Takahashi et al. 2002). In a murine model of asthma, IP receptor-deficient mice were challenged with ovalbumin. They developed goblet cell hyperplasia, and subepithelial fibrosis compared to wild-type animals. These changes were suggestive of allergen induced airway remodeling and were probably due to the upregulation of Th2 cytokine production, IgE production, or airway eosinophilic inflammation

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(Nagao et al. 2003). Thus, PGI2 may shift the balance within the immune system away from a Th2 dominant response and inhibits allergic inflammation. One potential mechanism by which PGI2 inhibits allergic inflammation is by increasing T-cell production of IL-10, which suppresses Th2 immunity. PGI2 or its stable analog carbaprostacyclin augmented IL-10 production by Th2 cells.

Lipoxins Nicknamed the “good” lipids for asthma (Peters-Golden 2002), lipoxins were discovered just over 20 years ago following experiments where labeled arachidonate was subjected to stimulation with a mixed leukocyte suspension (Serhan et al. 1984). Two new polar compounds with distinct physical properties from prostaglandins, leukotrienes and thromboxanes were identified using mass spectrometry. The name “lipoxin” was conceived to describe them, an abbreviation for lipoxygenase interaction product. Since then, Serhan and others have identified lipoxins in biological fluids and made structurally stable lipoxin analogs permitting further elucidation of their functions. Four years after Serhan’s initial description, lipoxins were isolated from rat alveolar macrophages (Kim 1988) and guinea-pig lung parenchyma (Cristol & Sirois 1988). Lipoxins have since been measured in a wide range of biological fluids and human tissues using enzyme-linked immunosorbent assay (ELISA), reverse-phase HPLC, and radioimmunoassay. In 1990, Lee and colleagues were the first to demonstrate the presence of lipoxins in BAL fluid from patients with a variety of pulmonary pathologies including asthma (Lee et al. 1990). Lipoxins have now been identified in patients with a range of respiratory conditions including from bronchial tissue in patients undergoing lung resection (Edenius et al. 1990), nasal lavage fluid from aspirinsensitive asthmatics (Levy et al. 1993), induced sputum from asthmatic patients (Vachier et al. 2005), and nasal polyp homogenates from both aspirin- and nonaspirin-sensitive patients (Perez-Novo et al. 2005).

Lipoxin biosynthesis, metabolism, and elimination Lipoxins are now known to be trihydroxytetraene eicosanoids that modulate leukocyte trafficking and vascular tone and are generated via the sequential actions of two or more lipoxygenases. Interaction of leukocytes with epithelium, endothelium, or platelets results in the formation of lipoxin (LX)A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) and its positional isomer LXB4 (5S,14R,15S-trihydroxy6,10,12-trans-8-cis-eicosatetraenoic acid). In contrast to cysteinyl leukotrienes, lipoxins have potent antiinflammatory effects. They are formed at sites of inflammation by transcellular cooperation. There are three principal biosynthetic pathways for their formation. The first involves cooperation between 15-LO and 5-LO, the second involves acetylation of

COX-2 by aspirin to trigger 15-epi-LXA4 biosynthesis, and the third involves interactions between 12-LO and 5-LO. 15-LO is an enzyme that is abundant in human lung and which is upregulated in the asthmatic airway (Bradding et al. 1995). It is present in airway epithelial cells and eosinophils (Nadel et al. 1991; Bradding et al. 1995), macrophages (Profita et al. 2000), and other leukocytes. In the lung 5-LO from circulating eosinophils (Steinhilber & Roth 1989), macrophages (Chavis et al. 1992), neutrophils (Chavis et al. 1996), or monocytes (Chavis et al. 1998) uses 15(S)-hydroxyeicosatetraenoic acid (15S-HETE), which is released from airway epithelial cells by airway epithelial 15-LO, as a substrate to generate lipoxins. Aspirin also interacts with the lipoxin system. Aspirin-acetylated COX-2 no longer produces prostaglandins but remains catalytically active in cells and generates 15RHETE from arachidonic acid; this becomes a substrate for leukocyte 5-LO-mediated conversion to 15-epi-LXA4 and 15-epi-LXB4 (Clària & Serhan 1995). 15-epi-LXA4 and 15epi-LXB4 are also known as aspirin-triggered lipoxins (ATL). 15R-HETE is also generated via the cytochrome P450 metabolism of arachidonic acid in epithelial cells (Clària et al. 1996). The third major route of lipoxin biosynthesis in humans involves an LTA4-dependent step, with lipoxin generation within the vascular lumen during platelet–leukocyte interactions and at mucosal surfaces via leukocyte–epithelial cell interactions. Platelets adhere to neutrophils, LTA4 is exported from leukocytes and converted rapidly to bioactive LXA4 and LXB4 in a 1 : 1 ratio by platelets rich in 12-LO and 15-LO (Serhan & Sheppard 1990). The process is similar in leukocyte– epithelial cell interactions but 15-LO from macrophages and bronchial tissue acts as a lipoxin synthase instead of 12-LO. Lipoxins are rapidly generated, act locally, and then rapidly inactivated by metabolic enzymes. The major route of lipoxin inactivation is rapid dehydrogenation and inactivation by monocyte 15-hydroxyprostaglandin dehydrogenase (PGDH). This process occurs in several stages. Firstly, 15PGDH catalyzes a dehydrogenation step that converts LXA4 to biologically inactive 15-oxo-LXA4 (Serhan et al. 1995) or LXB4 to 15-oxo-LXB4 (Maddox et al. 1998). This is followed by specific reduction of the double bond adjacent to the ketone (Serhan et al. 1995) by eicosanoid oxidoreductase to give 13,12-dihydro-15-oxo-LXA4. Further metabolism of the 15oxo group by 15-PGDH yields 13,14-dihydro-LXA4 (Clish et al. 2000). In comparison with lipoxins, the metabolic inactivation of ATLs is less efficient, with slower conversion in vitro to their 15-oxo-metabolite. Hence when ATLs are generated or cytochrome 450 15-epi-lipoxins, their biological half-life is around twofold greater than LXA4, leading to an enhanced bioavailability and duration of action (Serhan et al. 1995). However, the rapid degradation of lipoxins was a barrier to further investigation of lipoxin function, and this led to the design and synthesis of first-generation PGDH-resistant LXA4, LXB4 and ATL stable analogs by Serhan, Maddox and colleagues between 1995 and 1998 (Serhan et al. 1995; Maddox

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et al. 1998). These are relatively stable analogs that resist inactivation and retain biological activity and, together with myeloid-specific ALX-R-expressing transgenic mice, have provided powerful tools for exploring lipoxin functions in vivo.

Receptors When administered to human cells in vitro or murine systems in vivo, lipoxins act on at least two classes of receptors. Lipoxins competitively antagonize cysteinyl leukotrienes for binding at their cognate receptor CysLT1 (Gronert et al. 2001) and LXA4 binds to a seven-transmembrane-spanning GPCR designated ALX (Takano et al. 1997). Signal transduction pathways activated on stimulation of the ALX receptor (ALXR) are still being identified. In 1993 using [3H]-LXA4, Fiore et al. (1993) described the presence of a high-affinity lipoxin receptor (kd ∼ 0.5 nmol/L) on differentiated HL-60 cells. They also noted a second lowaffinity receptor (kd ∼ 10 nmol/L) on endothelial cells. A year later the same group identified the cDNA of the highaffinity receptor as a homolog of the low-affinity formylmethyl-peptide receptor (Fiore et al. 1994). The binding of other eicosanoids including LXB4, LTD4, LTB4, and PGE2 against this receptor was also tested but only LTD4 showed weak competition, with a Ki of 80 nmol/L (Fiore et al. 1994). Hence the ALXR is also known as formyl peptide receptor like-1 and formyl peptide receptor-2. The mRNA for ALXR has been isolated from a number of mammalian tissuesand cell types including human neutrophils (Fiore et al. 1994) and monocytes (Yang et al. 2001) (Table 26.5).

Table 26.5 Mammalian tissues expressing ALX mRNA. Tissue/cell type

Reference

Human cells Neutrophil Enterocytes Monocytes Synovial fibroblasts Activated T cells Bronchial epithelial cells

Fiore et al. (1994) Gronert et al. (1998) Yang et al. (2001) Sodin-Semrl et al. (2000) Ariel et al. (2003) Bonnans et al. (2006)

Human tissues Lung, placenta Spleen, liver (lesser amounts), heart (lesser amounts)

Fiore et al. (1994) Takano et al. (1997)

Murine cells Neutrophil

Takano et al. (1997)

Murine tissues Spleen, lung

Takano et al. (1997)

Rat Leukocytes

Chiang et al. (2003)

602

Mouse and rat ALXR have been cloned from a spleen cDNA library (Takano et al. 1997) and from peripheral blood leukocytes, respectively (Chiang et al. 2003). Across species, the human, mouse, and rat ALXR share a minimum of 74% homology (Serhan 2002). A second mouse lipoxin GPCR has also been identified using a macrophage cDNA library and, like its previously described counterpart, activation of this receptor is coupled to antiinflammatory responses (Vaughn et al. 2002). Using a murine model of zymosan-induced peritonitis in transgenic mice with myeloid-specific expression of the human ALXR, it has been shown that increased ALX signaling considerably abrogates the inflammatory response in vivo (Devchand et al. 2003). Adding further to the complexity of ALXR signaling, this study also showed that a 30–130% increase in human ALXR compared with endogenous murine ALXR led to a 20-fold enhancement in levels of endogenous LXA4, with a concomitant decrease in LTB4. This has led to suggestions that ALXR is not only activated by LXA4 but also amplifies lipoxin-mediated signal transduction (Parkinson 2006). ALXR is increasingly known as a “promiscuous receptor” as a number of peptide ligands can activate it in addition to LXA4 (Migeotte et al. 2006). These include serum amyloid A, synthetic peptides derived from HIV glycoprotein 120, and annexin 1, all of which bind the receptor but at much lower affinity than LXA4 (Su et al. 1999; Shen et al. 2000; Perretti et al. 2002). Differential responses depending on the ligand bound to ALXR have been noted (Sodin-Semrl et al. 2004) and may reflect “agonist-dependent trafficking.” Of interest in asthma, from murine data it is known that glucocorticoids such as dexamethasone induce expression of annexin 1 which is enzymatically cleaved to produce peptides and that these peptides interact with ALX to induce antiinflammatory cascades (Perretti et al. 2002). It has recently been hypothesized that glucocorticoids exert their antiinflammatory actions in part via upregulation of the ALXR; furthermore, in human neutrophils isolated from blood, the administration of dexamethasone was shown to upregulate the expression of ALX in a dose- and time-dependent manner (Hashimoto et al. 2007). As well as binding ALXR, lipoxins competitively antagonize the binding of cysteinyl leukotrienes to the CysLT1 receptor. In a study using human vascular endothelial cells it was demonstrated that both aspirin-triggered 15-epi-LXA4 and LTD4 bind and compete with equal affinity at the CysLT1 receptor. The clinically used CysLT1 receptor antagonist montelukast showed a lower rank order for competition with a [3H]-ATL analog (Gronert et al. 2001). In contrast, LTD4 was an ineffective competitive ligand at the recombinant ALXR compared with the [3H]-ATL analog, suggesting that lipoxin can act at the CysLT1 receptor but that the reverse does not hold (Gronert et al. 2001). It is unknown whether lipoxins also bind to and antagonize CysLT2.

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LXA4 also binds the nuclear aryl hydrocarbon receptor (Ah), a ligand-activated transcription factor (Schaldach et al. 1999) In human neutrophils, subcellular fractionation showed that tritiated LXA4 binding sites were found in the plasma membrane and endoplasmic reticulum (42.1%) and granules (34.5%) as well as nuclear-enriched fractions (23.3%) (Fiore et al. 1992). The finding that LXA4 acts as a ligand for the Ah receptor might relate to these reports of specific binding of LXA4 to intracellular recognition sites after transport and uptake (Levy 2005). In murine DCs, it has been demonstrated that lipoxins activate both Ah and ALXR and that this activation triggers expression of suppressor of cytokine signaling (SOCS)-2 (Machado et al. 2006). It is not known whether LXB4 binds to a specific high-affinity receptor, although functional studies have indicated such a putative receptor may exist (Maddox et al. 1998). However, presently there is no known receptor for LXB4 and research in this area has been slow because of lack of a suitable LXB4 radiolabel with high specificity (Brink et al. 2003).

Physiologic actions of lipoxins The lipoxins and their bioactive structural analogs have numerous biological effects, with cell-type specific actions. It is believed that the immunomodulatory role of lipoxins

may be important in regulating inflammatory processes in a number of diseases including asthma (Nasser & Lee 2002). Hence while inhibiting neutrophil chemotaxis and eosinophil activation at nanomolar concentrations (Lee et al. 1989; Soyombo et al. 1994), lipoxins stimulate monocyte trafficking (Maddox et al. 1997) and macrophage phagocytosis of apoptotic PMNs (Godson et al. 2000). Table 26.6 summarizes some of the key immunomodulatory actions of lipoxins. Increasingly the role of structural cells in the orchestration of ongoing inflammatory processes has been highlighted and a number of nonmyeloid cell functional responses have also been shown to be potently regulated by lipoxins (Serhan 2002). Chronic asthma is associated with airway remodeling, where structural changes including subepithelial fibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia and hypertrophy, mucous gland and goblet cell hyperplasia, and epithelial disruption may be seen. Of particular interest in asthma, LXA4 has been shown to inhibit proliferation of human lung fibroblasts induced by connective tissue growth factor (Wu et al. 2006). Lipoxins have also been shown to stimulate cytosolic Ca2+ release in bronchial epithelium, with resultant Cl– secretion (Bonnans et al. 2002), indicating that in airway epithelial cells LXA4 is involved in ionic transport regulation. It has also been hypothesized that the

Table 26.6 Immunomodulatory activities of lipoxins/lipoxin analogs in leukocytes. Activity

Reference

Neutrophils Inhibit neutrophil transmigration across: Epithelial cells Endothelial cell monolayers Inhibition of LTB4-induced neutrophil migration Inhibition of neutrophil adhesion molecule expression Inhibit neutrophil degranulation Inhibit neutrophil superoxide anion generation Inhibit TNF-a-induced IL-1b release from neutrophils Inhibit LTB4 release from neutrophils

Colgan et al. (1990) Papayianni et al. (1996) Lee et al. (1991) Papayianni et al. (1996) Hachicha et al. (1999); Vachier et al. (2002) Hachicha et al. (1999) Hachicha et al. (1999) Vachier et al. (2002)

T lymphocytes Inhibit NK cell cytotoxicity Inhibit TNF-a secretion

Ramstedt et al. (1987) Ariel et al. (2003)

Dendritic cells Inhibit IL-12 production from murine dendritic cells

Aliberti et al. (2001)

Eosinophils Inhibit eosinophil chemotaxis and degranulation Inhibit allergen-induced trafficking

Soyombo et al. (1994) Bandeira-Melo et al. (2000)

Monocytes Stimulate monocyte adhesion Stimulate chemotaxis Stimulate phagocytosis of apoptotic neutrophils by macrophages

Maddox & Serhan (1996) Maddox et al. (1997) Godson et al. (2000)

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LXA4-induced [Ca2+]i increase might play a central role in autocrine production of LXA4 and constitute an amplification mechanism for the biosynthesis of LXA4 itself (Bonnans et al. 2002). More recently it has been demonstrated that injured bronchial epithelial cells upregulate ALX to help promote resolution of bronchial inflammation by LXA4 (Bonnans et al. 2006). In vitro, lipoxins have been shown to mediate relaxation of precontracted pulmonary arteries and bronchi (Dahlén et al. 1988). It is also worth noting that the role of LXA4 may not be solely confined to antiinflammatory actions and recently several studies have suggested that it may also have an antiproliferative role in various tissue types including human mesangial cells and HUVECs (Mitchell, D. et al. 2000; Fierro et al. 2002). Remodeling in chronic asthma is also characterized by angiogenesis, and using a murine chronic granulomatous air pouch model in vivo, ATL-1 has been shown to inhibit angiogenesis (Fierro et al. 2002), a reminder perhaps that the vascular biology of lipoxins should not be forgotten.

Lipoxin and asthma Since Lee and colleagues first measured lipoxins in BAL fluid from patients with different pulmonary pathologies including asthma, a number of groups have confirmed that lipoxins and ATLs are generated in vivo and in vitro by asthmatic subjects (Levy et al. 1993; Chavis et al. 2000; Bonnans et al. 2002), as well as normal subjects (Bonnans et al. 2002). However, the data regarding increased or decreased lipoxin production in asthmatic patients is limited and presents a contradictory picture. LXA4 production, as measured by ELISA in supernatants of induced sputum, was significantly decreased in moderate and severe asthmatic compared with mild asthmatic subjects (Vachier et al. 2005). In contrast, levels of 15-epi-LXA4 from the same cohort were significantly higher in the severe asthmatics compared with normal controls. A study later the same year, using a novel fluorescencebased system to measure lipoxin synthesis in whole blood, found that severe asthmatic patients underproduced both 15-HETE and LXA4 (Levy et al. 2005). As inhaled lipoxins are inactivated rapidly, there is only limited information about their actions in humans in vivo. LXA4 has been shown to attenuate LTC4-induced bronchoconstriction in human asthmatic volunteers (Christie et al. 1992c), an effect explained partly by its antiinflammatory actions and also by its competitive binding to the CysLT1 receptor. ASRD is an eosinophil-driven, leukotriene-dependent phenotype of asthma that is often aggressive and refractory to treatment. The ingestion of aspirin is associated with a fourfold increase in already raised cysteinyl leukotriene levels and an acute exacerbation of symptoms (Christie et al. 1991a). In this cohort of patients, lipoxin synthesis from stimulated whole blood is reduced compared with aspirintolerant subjects (Sanak et al. 2000; Kowalski et al. 2003). Furthermore, patients with ASRD demonstrate upregulation

604

of CysLT1 expression on inflammatory leukocytes (Sousa et al. 2002; Corrigan et al. 2005) but have decreased levels of lipoxins to compete with cysteinyl leukotrienes for binding at their cognate receptor. Therefore diminished capacity to generate lipoxins may contribute to the uncontrolled chronic inflammation that characterizes this phenotype of asthma. Although it has been more recently suggested that diminished LXA4 synthesis is a feature of severe asthma regardless of aspirin sensitivity (Celik et al., 2007). There is some evidence that patients with aspirin sensitivity have decreased COX-2 expression and its activity is reduced (Picado et al. 1999; Pujols et al. 2004). One of the biosynthetic pathways for lipoxin generation is the aspirin-induced acetylation of COX-2 (Clària & Serhan 1995). This generates 15R-HETE, a biosynthetic precursor of the ATLs (15-epi-LXA4 and 15-epi-LXB4). 15-epiLXA4 is more potent than native LXA4 as an inhibitor of PMN trafficking and PMN-mediated inflammation in vivo (Fierro et al. 2003). It is therefore conceivable that reduced generation of 15-epi-LXA4 may contribute to the acute worsening of ASRD on aspirin ingestion. In 2002, Levy, Serhan and colleagues used a murine model of asthma to demonstrate that ovalbumin challenge induced airway biosynthesis of LXA4 and its leukocyte receptor (Levy et al. 2002). In the same model, preadministration of an LXA4 analog (designed using the 15-epi-LXA4 structure as a template) was administered at least 60 min prior to ovalbumin challenge and its administration was associated with a significant attenuation in AHR. Levy (2005) noted that with an ED50 of less than 0.05 mg/kg, the lipoxin analog compared favorably with the CysLT1 receptor antagonist montelukast (0.03 mg/kg in rats) and dexamethasone (0.5–3 mg/kg in mice). Furthermore, the LXA4 analog also blocked many of the features of allergen-induced inflammation including recruitment and activation of eosinophils and neutrophils. Decreased measurement of proinflammatory lipid mediators in BAL fluid including IL-5, IL-13, eotaxin, prostanoids, and cysteinyl leukotrienes was also noted. Using transgenic mice expressing leukocyte restricted human ALX, the authors then went on to investigate the importance of lipoxin receptors and their ligands. In the lung they found that there was significant inhibition of pulmonary inflammation and eicosanoidinitiated eosinophil tissue infiltration but AHR was not attenuated. Peters-Golden (2002) has hypothesized that this may be because the effects of the LXA4 analog on bronchial hyperreactivity are mediated entirely by its competitive interaction with CysLT1 receptors or alternatively because the analog’s inhibitory effects on AHR may depend on its interactions with ALXRs on airway smooth muscle and bronchial epithelium rather than on leukocytes. Using an ovalbumin model of allergic sensitization in rats, Bandeira-Melo et al. (2000) have demonstrated that stable analogs of LXA4 and ATLs attenuated allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, hence inhibiting the earlier edema and neutrophilia associated with allergic reac-

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tion. This eosinophilic blockade was independent of effects on mast cell recruitment and degranulation but involved analog inhibition of both IL-5 and eotaxin generation, as well as PAF. More recently, ATL has been found to attenuate LPSinduced acute lung injury in a mouse model (Jin et al. 2007). It has been just over 20 years since lipoxins were discovered by Serhan and colleagues. Since then a body of literature has accumulated describing their vascular biology and immunomodulatory and antiproliferative effects. LXB4 remains the less understood of the two lipoxins and the location and structure of LXB4 receptors and the extent to which LXB4 pharmacology varies from LXA4 is unknown. Nonetheless the evidence collected thus far suggests a pivotal role for lipoxins in mediating airway homeostasis and a potential role as a new therapeutic strategy in the management of asthma.

Platelet-activating factor PAF is an ether-linked phospholipid (1-O-alkyl-sn-glycero-3phosphocholine) first described as a substance released from IgE-stimulated basophils that was found to aggregate rabbit platelets (Benveniste et al. 1972). In 1979, Demopoulos et al. described a semisynthetic phosphoacylglycerol, 1-O-alkyl2-acetyl-sn-glycero-3-phosphocholine (AGEPC), which was able to aggregate platelets and release serotonin and had the same physicochemical properties as PAF. In 1980, Hanahan et al. purified and characterized PAF from activated basophils and concluded that AGEPC and PAF are the same compound. PAF has long been implicated in the pathophysiologic mechanisms of asthma, because exogenous PAF closely mimics many of the clinical features of asthma, including AHR. Since its discovery over three decades ago, PAF has emerged as one of the most important lipid mediators known.

Biosynthesis PAF is not a single molecule and should be considered as a family of structurally related autacoid phopholipids synthesized by inflammatory cells that have platelet-stimulating and neutrophil-priming activity and whose main members include 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine. The most commonly recognized chemical structure of PAF is shown in Fig. 26.8. PAF is synthesized in a wide variety of inflammatory cells, including platelets, neutrophils, basophils, macrophages, and eosinophils (Barnes et al. 1989; Chung 1992). The synthesis of PAF in stimulated inflammatory cells is generally via a two-step enzymatic pathway. Cell stimulation leads to the release of alkylacylglycerophosphocholine from cell membranes, which can be converted to PAF via either the “remodeling” pathway or the de novo pathway (Fig. 26.9). In the remodeling pathway, ether-linked phospholipids that form part of the structure of membranes are structurally

H2C

O

(CH2)n

CH3

O CH3

C

O

C

CH CH3

O CH2

O

P

CH2

CH2

O–

N+

CH3 CH3

sn-1, alkyl group sn-2, acetyl group sn-3, O-phosphocholine n = 15:0 or 17:1 Fig. 26.8 Chemical structure of platelet-activating factor.

modified. The initial step is catalyzed by the action of PLA2 on 1-alkyl-2-acyl-glycerophosphocholine, a membrane phospholipid (Hanahan 1986; Sturk et al. 1989). This deacylation results in lyso-PAF formation and the release of arachidonic acid, which has PLA2 inhibitory activity, thereby reducing further PAF synthesis; alternatively, arachidonic acid may act as the substrate for subsequent eicosanoid synthesis. LysoPAF is then acetylated by a specific lyso-PAF acetyltransferase to form 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine-PAF. PAF can subsequently be remetabolized back to its precursor lyso-PAF by the action of a specific PAF acetylhydrolase. The other route for PAF biosynthesis is known as the de novo pathway and begins with an intermediate in the synthesis of ether-linked membrane phospholipids. This is acetylated by a specific acetyltransferase and the phosphate is removed from the sn-3 carbon by a phosphohydrolase to form alkylacetylglycerol, which is subsequently converted by choline phosphotransferase to PAF.

Biological activity Soon after the identification of this proinflammatory phospholipid mediator, PAF was found to have activity beyond the originally described platelet aggregation and was demonstrated to possess a unique profile of biological effects. Furthermore, it was found to be the most potent lipid mediator known, with biological responses detectable at concentrations as low as 10–14 mol/L and an ED50 value for guinea-pig platelet activation of about 3 × 10–10 mol/L. PAF causes potent airway obstruction in vivo but in vitro effects on smooth muscle preparations are negligible, suggesting that this effect may be mediated through increased vascular permeability and vasoconstrictor effects on airway smooth muscle (Denjean et al. 1983; Chung et al. 1986; Cuss et al. 1986). PAF produces a receptor-mediated increase in airway microvascular leakage with a 1000-fold greater potency than histamine (Evans et al. 1987). Further evidence supporting this dual mechanism of PAF-induced airway obstruction comes from studies demonstrating that as this effect is not totally reversed by the airway smooth muscle

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De novo pathway O

CH2

Remodeling pathway R

O

CH2

R

O O

H

P 1-O-alkyl-2-lyso-sn-glycerol-3-phosphate

O

C

R′

P

Choline

1-O-alkyl-2-acyl-sn-glycerol-3-phosphocholine

Acetyl CoA Phospholipase A2

Acetyltransferase

Arachidonate

CoASH O

CH2

R

O

CH2

R

CH3

O

H

P

Choline

O O

C

P 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphate

1-O-alkyl-2-lyso-sn-glycerol-3-phosphocholine Lyso-PAF Acetyl CoA Acetyltransferase

Phosphohydrolase

CoASH

P O

CH2

O

R

O

CH2

R

O Cholinephosphotransferase

O

C

CH3 CDP-choline

O

H

1-O-alkyl-2-acetyl-sn-glycerol

O

C

P

Choline

CMP

1-O-alkyl-2-acetyl-sn-glycerol-phosphocholine (PAF)

relaxant salbutamol (Diaz et al. 1997), it cannot be completely attributed to vasoconstriction.

Vascular permeability PAF has been shown to be one of the few endogenous mediators that can induce increased vascular permeability in the pulmonary circulation within minutes. This is partly through a direct effect on the vascular endothelium (Humphrey et al. 1984) and partly an indirect effect through leukocyte activation (Bjork & Smedegard 1983), particularly neutrophils.

606

CH3

Fig. 26.9 Biosynthetic pathways of plateletactivating factor (PAF) showing the de novo and “remodeling” routes.

PAF induces this increase in vascular permeability by simultaneous activation of two independent pathways: a COX-independent pathway requiring the sphingomyelinase metabolite ceramide (Goggel et al. 2004), whereas the other is dependent on PGE2 (Goggel et al. 2002). Agents that interfere with PAF-induced ceramide synthesis, such as glucococorticoids, attenuate pulmonary edema formation induced by PAF (Goggel et al. 2002). In a murine model, PAF-induced vascular permeability appeared to be mediated via the actions of PGE2 on EP3 receptors (Goggel et al. 2002). PGE2 has been

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shown to both increase (Goggel et al. 2002) and decrease (Gillespie et al. 1987) vascular permeability. Recently, using perfused lung isolated from rats it has been demonstrated that PAF-induced edema is mediated by activation of the IP3 pathway, influx of extracellular calcium, and subsequent activation of a myosin light chain kinase-dependent and Rhoassociated protein kinase-independent mechanism (Goggel & Uhlig 2005).

Activity on airway smooth muscle PAF inhalation induces bronchoconstriction within 2–3 min and this is followed by the rapid development of tachyphylaxis (Cuss et al. 1986). Increases in airway responsiveness to reference agonists such as histamine and methacholine were originally described in both the guinea pig (Mazzoni et al. 1985) and the dog (Chung et al. 1986). PAF produces acute bronchoconstriction when inhaled by patients with asthma (Barnes et al. 1989). The major mediator responsible for PAFinduced bronchoconstriction in animal models (both in vivo and in vitro) is TXA2 (Tokuyama et al. 1991; Olson et al. 1993) and to a lesser degree leukotrienes (Uhlig et al. 1994). In animal models, dual inhibition of COX and 5-LO has a pronounced inhibitory effect on PAF-induced vasoconstriction and bronchoconstriction but does not prevent the increase in vascular permeability (Uhlig et al. 1994). In humans, PAF has been shown to release TXA2 and leukotrienes but their roles are less defined (Taylor et al. 1991). In two small trials with eight healthy men, PAF-induced bronchoconstriction was partially attenuated by the leukotriene antagonists SKF 104353-Z (Spencer et al. 1991) and ICI 204,219 (Kidney et al. 1993). However, the COX inhibitor indomethacin did not inhibit PAF-induced bronchoconstriction in seven subjects (three controls and four with asthma).

Activity on secretions PAF stimulates fluid secretion from porcine isolated trachea via activation of PAF receptors and via a mechanism that does not depend on the release of acetylcholine, histamine, or cysteinyl leukotrienes (Steiger et al. 1987). In cultures of guinea-pig tracheal epithelial cells, PAF stimulates release of mucin-like glycoprotein. The mechanism involves binding of PAF to receptors on epithelial cell surfaces, intraepithelial stimulation of lipoxygenase metabolism of arachidonic acid to HETEs, and stimulation of secretion by these epithelialderived HETEs via an autocrine or paracrine mechanism (Adler et al. 1992). In isolated human airways, PAF enhances respiratory glycolipid and glycoprotein release in vitro; this may be partly dependent on cysteinyl leukotriene generation but occurs independently of acetylcholine release (Goswami et al. 1989).

Inflammatory cells and asthma PAF is a potent activator of inflammatory cells. The activation of inflammatory cells leads to the release of other inflam-

matory mediators, such as the products of both the COX (Chung et al. 1986) and the lipoxygenase (Voelkel et al. 1982) pathways and the release of oxygen free radicals (Rouis et al. 1988). Activated eosinophils (Cromwell et al. 1990) and mast cells (Schleimer et al. 1986) in the bronchial airway of asthmatics may be the cellular source of PAF as these cells produce PAF in vitro when stimulated. With the advent of the “united airway theory” and allergic inflammation in upper and lower airways being increasingly viewed as one disease, it is worth noting that PAF is thought to be a potent mediator of allergic rhinitis and has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation. Immunohistochemistry has revealed the presence of anti-PAF receptor antibody-labeled eosinophils, macrophages, neutrophils, mast cells, lymphocytes, vascular endothelial cells, epithelial cells, and submucosal glands in nasal mucosa (Shirasaki et al. 2005). PAF-induced degranulation of mast cells appears to be inhibited by stimulation of β2-adrenergic receptors. The effect of PAF on eicosanoid generation was investigated by measuring a wide range of lipoxygenase and COX pathway products and release in cultured feline tracheal epithelial cells. HPLC chromatograms revealed that PAF augmented the release of PGE2, PGF2α, 12-HETE, and arachidonic acid. Among these eicosanoids, PGE2 predominated under baseline conditions and following PAF exposure. Using radio-immunoassay, PAF was also demonstrated to increase the production of 6-keto-PGF1α, TXB2, PGD2, 5-HETE, and 15-HETE, as well as PGE2, PGF2α, and 12-HETE. The PAF-induced eicosanoid augmentation was dose-dependent and occurred within 1 hour, with a prompt decline following termination of PAF exposure. This stimulating effect of PAF on eicosanoid release was blocked by two PAF receptor antagonists, Ro 19-3704 and WEB 2086, suggesting that PAF stimulates the production of COX and lipoxygenase pathway products from airway epithelial cells via PAF receptors (Wu et al. 1995). In macrophages isolated from BAL fluid in asthmatic patients, stimulation with PAF led to enhanced 5-LO activity and increased LTB4 release compared with alveolar macrophages isolated from normal controls (Shindo et al. 1998). The in vitro actions of PAF on eosinophils include chemotaxis and adhesion (Kimani et al. 1988; Kroegel et al. 1991). Eosinophil chemotaxis appears to be inhibited by the phosphoinositide-3 inhibitors wortmannin and LY294002. However, in vitro, two phosphoinositide 3-kinase inhibitors inhibit PAF-induced eosinophil chemotaxis and PAF-induced respiratory burst but not PAF-induced LTC4 secretion (Mishra et al. 2005). PAF also acts as an eosinophil priming agent (Zoratti et al. 1992). PAF-mediated priming of eosinophils is mediated via different signaling pathways, compared with IL-5-induced priming, because it is not blocked by tyrosine kinase inhibitors (Van der Bruggen et al. 1998). Exposure to PAF induces cytoplasmic alkalinization and granule acidification in human eosinophils (Bankers-Fulbright et al. 2004). It

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has been proposed that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins (Bankers-Fulbright et al. 2004). After allergen challenge in asthmatic patients, PAF induces greater activation of circulating eosinophils, suggestive of cross-talk between PAF and other priming factors such as IL-5 and GM-CSF (Evans et al. 1996). PAF also has a greater activating effect on neutrophils from asthmatic patients compared with those from normal control subjects (Shindo et al. 1997). In vivo, PAF elicits marked eosinophilic infiltration into lung tissue after both intravenous and aerosol administration to guinea pigs (Lellouch Tubiana et al. 1988) and rabbits (Coyle et al. 1990). PAF-induced eosinophilia in guinea pigs is associated with increased bronchial responsiveness to inhaled histamine, but not methacholine. In guinea pigs, PAF-induced bronchoconstriction is abolished by treatment with the timedependent, reversible COX-1 inhibitor, indomethacin, suggesting that in vivo airway eosinophils may reduce nonspecific bronchial responsiveness through production of inhibitory or bronchoprotective prostanoids (Ishiura et al. 2005). PAF and its metabolite lyso-PAF have been demonstrated in BAL fluid from stable atopic asthmatic subjects (Stenton et al. 1990a) and in plasma from asthmatic subjects during both early (Chan-Yeung et al. 1991) and late (Nakamura et al. 1987) responses to inhaled allergen. Increased levels of PAF have also been observed following allergen challenge in asthmatic patients. In asthmatic patients, inhalation of PAF is associated with an increase in respiratory system resistance, increased urinary LTE4, decreased PaO2, and a transient decrease in blood neutrophils (within 5 min) followed by a rebound neutrophilia (15 min later), but no increase in total antioxidant capacity and levels of lipid peroxides (Echazarreta et al. 2005). PAF also elicits bronchoconstriction and airway microvascular leakage, resulting in disturbances of pulmonary gas exchange similar to those observed during naturally occurring acute asthma, and has been reported to induce bronchial hyperresponsiveness in humans (Kaye & Smith 1990), although this has not been universally shown (Lai et al. 1990, Spencer et al., 1990). Increased levels of PAF have been measured in plasma from asthmatic patients during an acute attack (Kurosawa et al. 1994) and this is associated with diminished levels of PAF acetylhydrolase (Tsukioka et al. 1996). PAF receptor mRNA has also been detected in airway smooth muscle in human peripheral lung (Shirasaki et al. 1994a). Transgenic mice that either overexpress or underexpress the PAF receptor have proven useful in further elucidating the role this mediator may play in the pathogenesis of asthma. Transgenic mice that overexpress the PAF receptor (PAFR) gene exhibit bronchial hyperreactivity and this appears to be mediated in part by a muscarinic pathway. Nagase et al. (2002b) examined airway responsiveness to methacholine

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and serotonin (5-hydroxytryptamine) in PAFR transgenic mice. The PAFR transgenic mice exhibited hyperresponsiveness to methacholine and PAF, but no significant differences in serotonin responsiveness were observed between control and PAFR transgenic mice. The administration of atropine significantly blocked PAF-induced responses in PAFR transgenic mice, suggesting that the muscarinic pathway may have a key role in AHR associated with PAFR gene overexpression. In a murine asthma model, subsequent to ovalbumin sensitization, mice that did not express PAFR had no alteration in the total number of cells or in the proportions of eosinophils, lymphocytes, macrophages/monocytes, and neutrophils in BAL fluid compared with mice expressing PAFR (Ishii et al. 2004). PAFR-negative and PAFR-positive mice did not exhibit any histologic differences in the degree of bronchial inflammation. Following antigen challenge, PAFR-negative mice developed significantly increased airway responsiveness compared with their saline-treated controls. Furthermore, their responsiveness proved to be significantly lower than that of PAFR-positive mice sensitized with ovalbumin. Thus, PAFR is critical for the development of AHR following repeated aeroallergen challenge in sensitized mice, and this develops via PAFR-dependent and -independent pathways.

Cellular release Resident pulmonary inflammatory cells such as mast cells, (Triggiani et al. 1991), eosinophils (Cromwell et al. 1990), and alveolar macrophages (Bratton et al. 1994) are capable of PAF synthesis via specific agonist–receptor interactions. Many phagocytic stimuli, such as bacteria and parasites opsonized by IgG or complement, immune complexes, complement chemotactic factors C5a and C3a, and eosinophil chemotactic factor A, can stimulate inflammatory cells to initiate PAF synthesis. Macrophages activated by opsonized zymosan and by immune complex in hypersensitivity reactions are capable of PAF synthesis. PAF is synthesized by peripheral blood monocytes in response to 4β-phorbol-12-myristate13-acetate (PMA), opsonized zymosan, and the calcium ionophore A23187, an effect regulated by protein kinase C (Elstad et al. 1991). Both unopsonized zymosan and A23187 induce a dose- and time-dependent increase in PAF synthesis by eosinophils (Burke et al. 1990). Neutrophils stimulated with opsonized zymosan, A23187, and formyl-methionyl-leucylphenylalanine generate PAF (Sisson et al. 1987). Cytokines such as IL-1 and TNF stimulate macrophages, neutrophils, and endothelial cells to generate PAF (Camussi et al. 1987; Bussolino et al. 1988). Endothelial cells also produce PAF in response to specific stimuli such as vasopressin, angiotensin II, thrombin, bradykinin, and histamine (Camussi et al. 1983; Prescott et al. 1984; McIntyre et al. 1985). Human basophils have also been shown to produce PAF in vitro after costimulation with a combination of IL-3 and thapsigargin, although this effect is blocked by inhibitors of cytoslic PLA2, suggesting this enzyme’s involvement (Lie et al. 2003). Lymphocytes

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are unable to synthesize PAF, although natural killer cells may produce PAF in response to IgG binding to Fc receptors (Malavasi et al. 1986). Antigen or IgE-stimulated mast cells and basophils generate PAF (Schulman et al. 1983), supporting a role for PAF in the pathogenesis of acute anaphylaxis. Vadas et al. have investigated this and measured serum PAF levels and PAF acetylhydrolase activity in 41 patients with anaphylaxis and in 23 control patients. Serum PAF acetylhydrolase activity was also measured in patients with peanut allergy who had fatal anaphylaxis and compared to several control groups including patients with nonfatal anaphylaxis, children who died of non-anaphylactic causes and children with lifethreatening asthma. The severity of anaphylaxis directly correlated with serum PAF levels whilst serum PAF acetylhydrolase activity was inversely correlated with the severity of anaphylaxis. PAF acetylhydrolase activity was significantly lower in patients with fatal anaphylactic reactions to peanuts than in patients in any of the control groups. These findings suggest that failure of PAF acetylhydrolase to inactivate PAF may contribute to the severity of anaphylaxis (Vadas et al. 2007).

Catabolism The major enzyme responsible for the catabolism of PAF is PAF acetylhydrolase (PAF-AH), which is unusually a secretory Ca2+-independent PLA2. PAF-AH was initially described as being abundant in human plasma but by 1987 it had been shown to be associated with low-density lipoproteins (Stafforini et al. 1987) and to a lesser extent high-density lipoproteins. PAF-AH not only degrades PAF but also degrades the oxidation products of phosphatidylcholine, some of which express PAF-like activity (Blank et al. 1981; Heery et al. 1995). Oxidization of the ample phosphatidylcholine-containing arachidonate at the sn-2 position of glycerol (found in lowdensity lipoproteins) gives rise to numerous oxidized products with short chains at the sn-2 position of glycerol. Acting as an esterase, PAF-AH cleaves the acetyl group at the sn-2 position of oxidized 1-palmitoyl-2-arachidonyl phosphatidylcholine (oxPAPC) and PAF, producing lysophosphatidylcholine and lyso-PAF and short-chain fatty acids (Subramanian et al. 1999). Several inflammatory cell types, including mast cells (Nakajima et al. 1997), macrophages (Elstad et al. 1989), and platelets (Korth et al. 1993), synthesize and excrete active PAF-AH. Furthermore, recent studies have identified an acetylhydrolase in BAL fluid that is distinct from either plasma acetylhydrolase or erythrocyte-derived acetylhydrolase (Triggiani et al. 1997). This enzyme was present in smaller amounts in BAL fluid obtained from patients with mild asthma (Triggiani et al. 1997).

PAF receptor The PAFR was the first lipid mediator receptor to be cloned. A guinea-pig lung cDNA library was screened using a phage

clone shown to induce PAF-dependent responses in Xenopus laevis oocytes (Honda et al. 1991). Confirmation of the identity of the receptor was carried out based on its pharmacologic properties when expressed in COS-7 cells or oocytes and using known PAFR agonists and antagonists. A 3020nucleotide sequence was reported and the 342-amino-acid sequence was deduced from the longest open reading frame of the cloned cDNA. Hydropathy profile analysis revealed the existence of seven hydrophobic transmembrane segments characteristic of GPCRs, suggesting that like previously described receptors such as the α1- and α2-adrenergic and muscarinic acetylcholine receptors, the PAFR belongs to the G protein-linked receptor superfamily. Human PAFR cDNA was isolated from a human leukocyte cDNA library using a 0.8-kb fragment of the guinea-pig PAFR cDNA as a probe. Both the guinea-pig and human PAFR were shown to possess seven putative transmembrane domains, contain 342 amino acid residues, and demonstrate 83% homology in amino acid sequence (Nakamura et al. 1991). Activation of the receptor yielded IP3 in transfected oocytes and COS-7 cells, and guanosine 5′-O-(2-thio)biphosphate injection into these cells inhibited a PAF-induced Cl–? current, providing evidence that PAF stimulates phosphoinositide turnover via G proteins. When expressed in CHO cells the PAFR couples with various second messenger systems, leading to phospholipase C activation, inhibition of adenylate cyclase, and activation of the MAPK cascade and arachidonate release. A subsequent study identified the PAFR cytoplasmic tail as not being required for forward signal transduction. The several phosphorylation sites on the cytoplasmic tail are postulated to play a critical role in the rapid agonist-induced desensitization so characteristic of PAF activity (Takano et al. 1994). It is now known that substitution of the Cys90, Cys95, or Cys173 residues in the PAFR with alanine or serine yields mutant receptors that do not bind PAF and are not expressed on the surface of cells but are found intracellularly (Le Gouill et al. 1997). PAF appears to act in an autocrine manner and may modulate the expression of its own receptor at a transcriptional level in human monocytes (Shirasaki et al. 1994b). The cell signaling pathways initiated by PAF interaction with its receptor are well characterized and include increases in [Ca2+]i (Mazer et al. 1991), increases in IP3 and diacylglycerol levels, and induction of cell cycle-active genes such as fos, jun, and egr-1 (Mazer et al. 1991; Schulam et al. 1991). Through its action on PAFR, PAF also activates the transcription factor AP-1 in airway epithelial cells (Le Van et al. 1998). More recently, increasing evidence is emerging that intracellular binding sites for PAF contribute to its proinflammatory actions. It has also been suggested that PAF may act via more than one receptor. Evidence from both human and animal studies suggests that there may be heterogeneity of PAF receptors (Kroegel et al. 1989; Hwang et al. 1990). For example, PF10040 can antagonize PAF-induced edema formation

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(Rossi et al. 1992) and PAF-induced bronchial hyperresponsiveness (Herd et al. 1994) but has no effect on PAF-induced bronchoconstriction (Herd et al. 1994). Furthermore, it has been demonstrated that only a small part of the total amount of PAF generated by cells is actually released, with intracellular PAF having been proposed as a signaling molecule itself (Stewart & Harris 1991). This observation raised the possiblity of a distinct intracellular PAFR. Almost 10 years later, receptors for PAF have been shown to be localized at the cell nucleus of Chinese hamster ovarian cells, cerebral microvascular endothelial cells of newborn pigs (Marrache et al. 2002), and rat hepatocytes (Miguel et al. 2001). The nucleus has been suggested as a putative organelle that both generates PAF and expresses its receptor (Marrache et al. 2002). Marrache et al., stimulated isolated nuclei in porcine brain endothelial cells with methylcarbamate-PAF and found this evoked the expression of the genes for proinflammatory iNOS and COX-2. Gene expression was associated with augmented ERK-1/2 phosphorylation and NF-κB binding to the DNA consensus sequence. COX-2 expression was inhibited by mitogen-activated protein kinase kinase/extracellular signalregulated kinase 1/2 and NF-kappaB inhibitors. These above observations have led to the suggestion that the immediate effects of PAF are mediated via cell-surface receptors whereas long-term responses are dependent on intracellular receptor effects (Zhu et al. 2006). It is now recognized that subsequent to PAF stimulation, cells become rapidly desensitized and this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization, and downregulation. PAFR degradation can occur via both the proteasome and lysosomal pathways and ligand-stimulated degradation is ubiquitin-dependent (Dupre et al. 2003).

PAFR antagonists Due to the association of PAF with diverse physiologic and pathologic processes, considerable efforts have been invested in the development of antagonists to PAFR. These have been used to characterize PAFRs on a wide variety of inflammatory cells and also to elucidate the part PAF may play in the pathogenesis of asthma. Despite considerable in vitro and in vivo data in animal and human studies suggesting that PAF is an important mediator of asthma, clinical studies with PAFR antagonists have been disappointing. • WEB 2086 is a hetrazepine compound that inhibits the allergen-induced late response in allergic sheep (Abraham et al. 1989). In human subjects, WEB 2086 prevents PAFinduced bronchoconstriction (Adamus et al. 1990) and prevents the histamine hyperresponsiveness induced by PAF in vitro (Johnson et al. 1990). In a study of eight mild atopic asthmatic subjects, 1 week’s treatment with WEB 2086 (300 mg daily) did not attenuate the allergen-induced early or late responses or the subsequent histamine bronchial hyperre-

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sponsiveness (Freitag et al. 1993). In a subsequently reported study of 65 asthmatic subjects, 6 weeks’ treatment with WEB 2086 (120 mg daily) did not significantly reduce the requirement for inhaled corticosteroids compared with placebo (Spence et al. 1994). • The dihydropyridine compound UK74,505 is a specific and potent PAFR antagonist that shows 10-fold greater inhibition of PAF-induced aggregation of washed rabbit platelets than WEB 2086 (Alabaster et al. 1991). In a study of 12 normal subjects, inhalation of PAF, induced broncoconstriction for 60 min, neutropenia at 5 min, rebound neutrophilia at 2 hours, and stimulated the production of urinary eicosanoids (O’Connor et al. 1994). Premedication with UK74505 completely abolished the bronchoconstrictor response, neutropenia, and rebound neutrophilia, and significantly reduced urinary levels. In asthmatic patients who received a 4 week course of modipafant (the (+)-enantiomer of UK-74505) for 4 weeks, no clinical benefit was observed (Kuitert et al. 1995). • A 1-week course of the potent, long-acting PAFR antagonist foropafant (SR27417A) produced a modest reduction in the magnitude of the allergen-induced late response, although there was no effect on the early response, allergen-induced airway responsiveness, or baseline lung function (Evans et al. 1997). • Another PAF antagonist, Y24180, has also been shown to reduce airway responsiveness to inhaled methacholine in asthmatics (Hozawa et al. 1995), although these data are at variance with findings from other studies (Evans et al. 1997). • In patients with acute asthma, where endogenous release of PAF may be enhanced, the response to a potent selective PAFR antagonist (SR27471A) on pulmonary gas exchange was studied with ventilation–perfusion (VA/Q) scans (Gomez et al. 1999). SR27471A inhibited platelet agregation tests, suggesting good bioactivity in vivo. However, no differences were observed between placebo and active groups with regard to baseline FEV1, respiratory system resistance, alveolar–arterial pressure difference for oxygen (5.2 ± 0.4 kPa), PaO2, or VA/Q distributions, as expressed by the dispersion of pulmonary blood flow. This led to the conclusion that SR27417A has limited value when added to the conventional treatment of acute asthma (Gomez et al. 1999). This disappointing clinical response may reflect the unimportance of PAF in acute or chronic asthma, or reflect an impaired ability of the antagonists used to block endogenously produced PAF, which acts locally in the airways with “paracrine” effects (Barnes et al. 1988). Alternatively, as a number of assumed PAFR antagonists (WEB 2086, SM 10661, alprazolam) have now been shown to be in fact acting as inverse agonists on the human PAFR and inducing effector cascades of LTE4 and 2,3-dinor-TXB2 (Dupre et al. 2001), this may provide another explanation for the poor clinical performance of these drugs.

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Ying, S., O’Connor, B.J., Meng, Q. et al. (2004) Expression of prostaglandin E(2) receptor subtypes on cells in sputum from patients with asthma and controls: effect of allergen inhalational challenge. J Allergy Clin Immunol 114, 1309–16. Ying, S., Meng, Q., Scadding, G., Parikh, A., Corrigan, C.J. & Lee, T.H. (2006) Aspirin-sensitive rhinosinusitis is associated with reduced E-prostanoid 2 receptor expression on nasal mucosal inflammatory cells. J Allergy Clin Immunol 117, 312–18. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. & Shimizu, T. (1997) A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387, 620–4. Yokomizo, T., Kato, K., Hagiya, H., Izumi, T. & Shimizu, T. (2001) Hydroxyeicosanoids bind to and activate the low affinity leukotriene B4 receptor, BLT2. J Biol Chem 276, 12454–9. Yokomizo, T., Kato, K., Terawaki, K., Izumi, T. & Shimizu, T. (2000) A second leukotriene B4 receptor, BLT2. A new therapeutic target in inflammation and immunological disorders. J Exp Med 192, 421–32. Yokoyama, C., Miyata, A., Ihara, H., Ullrich, V. & Tanabe, T. (1991) Molecular cloning of human platelet thromboxane A synthase. Biochem Biophys Res Commun 178, 1479– 84. Yu, W., Bozza, P.T., Tzizik, D.M. et al. (1998) Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies. Am J Pathol 152, 759–69. Zeldin, D.C., Wohlford-Lenane, C., Chulada, P. et al. (2001) Airway inflammation and responsiveness in prostaglandin H synthasedeficient mice exposed to bacterial lipopolysaccharide. Am J Respir Cell Mol Biol 25, 457–65. Zhang, X.H., Zhao, C., Seleznev, K., Song, K., Manfredi, J.J. & Ma, Z.A. (2006) Disruption of G1-phase phospholipid turnover by inhibition of Ca2+-independent phospholipase A2 induces a p53-dependent cell-cycle arrest in G1 phase. J Cell Sci 119, 1005– 15. Zhou, H., Yan, F. & Tai, H.H. (2001) Phosphorylation and desensitization of the human thromboxane receptor-alpha by G proteincoupled receptor kinases, J Pharmacol Exp Ther 298, 1243–51. Zhu, J., Qiu, Y.S., figueroa, D.J. et al. (2005) Localization and upregulation of cysteinyl leukotriene-1 receptor in asthmatic bronchial mucosa. Am J Respir Cell Mol Biol 33, 531–40. Zhu, T., Gobeil, F., Vazquez-Tello, A. et al. (2006) Intracrine signaling through lipid mediators and their cognate nuclear G-proteincoupled receptors: a paradigm based on PGE2, PAF, and LPA1 receptors. Can J Physiol Pharmacol 84, 377–91. Zoratti, E.M., Sedgwick, J.B., Bates, M.E., Vrtis, R.F., Geiger, K. & Busse, W.W. (1992) Platelet-activating factor primes human eosinophil generation of superoxide. Am J Respir Cell Mol Biol 6, 100– 6.

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Summary

Table 27.1 Allergic diseases where IgE-driven mechanisms are implicated.

Cyclic nucleotide phosphodiesterases (PDEs) currently account for at least 50 distinct proteins that are all able to inactivate the ubiquitous second messengers cAMP and/or cGMP. These enzymes have proven to be very “drugable” targets and compounds are on the market or in late stage clinical development for a variety of dissimilar diseases. This chapter describes the multiplicity of mammalian PDE isoenzymes, the tissue distribution of PDEs expressed in cells which participate in allergic inflammatory reactions, and the potential sites where PDE inhibitors could act to alleviate the acute and chronic manifestations of allergic disease including clinical trial data.

Asthma Atopic and contact dermatitis Rhinitis Eczema Sinusitis Hypersensitivity pneumonitis Extrinsic alveolitis Angioedema and anaphylaxis Certain forms of migrane and gastrointestinal disorders Urticaria

Introduction The term “allergy” is often used to describe all aspects of immunology. However, this definition of allergy has been refined to refer specifically to the tissue-damaging or irritant effects to the host of immunologic reactions. Allergic diseases, including all of those listed in Table 27.1, affect 20% of the population and represent a highly significant cause of morbidity and mortality. Taking allergic asthma as a specific example, a recent assessment indicates that the prevalence and severity of this disease is increasing (Braman 2006), although the number of reported cases of fatal asthma has declined from the peak reached in the mid 1980s (Sidebotham & Roche 2003). Although significant advances in our understanding of the pathogenesis of many allergic disorders have been made, the etiology of allergy is still incompletely understood. However, as described in Chapters 2, 5, 6, 7 and 8 the likely participation of IgE-driven mechanisms in many allergic diseases, including all of those cited in Table 27.1, has been identified and

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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recognized by both the World Allergy and World Health Organizations (Asher et al. 2004). Since the incidence of allergy has reached epidemic proportions, it is only too clear that drugs which can prevent the overt and covert manifestations of allergic reactions and, ideally, suppress or even prevent the process of host sensitization could have a profound clinical and economic impact in the control of these diseases. While glucocorticosteroids are currently considered the most effective antiallergic/antiinflammatory drugs currently available, they are nonselective in action and not without adverse effects. New drugs with enhanced selectivity and improved side-effect profiles are clearly required. One group of drugs which, from a theoretical perspective, may exhibit powerful antiinflammatory and immunomodulatory activity are inhibitors of certain of the cyclic nucleotide phosphodiesterase (PDE) isoenzymes that selectively degrade cyclic adenosine3′,5′-monophosphate (cAMP) and/or cyclic guanosine-3′,5′monophosphate (cGMP) (Torphy 1998; Giembycz 2005a,b; Houslay 2005; Houslay et al. 2005; Bender & Beavo 2006a; Lugnier 2006). The prototype PDE inhibitors that have been used in the treatment of asthma for many years are the alkylxanthines of which theophylline is the most widely prescribed. The main beneficial activity of theophylline was originally attributed to its weak bronchodilator action. However, evidence accumulated in the 1990s suggested that this molecule may have antiinflammatory activity at

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Table 27.2 Representative PDE4 inhibitors for inflammatory disorders.

Company

Drug

Isoenzyme selectivity

Indication

Development stage

Almirall Glenmark Pfizer ONO Otsuka Nycomed GlaxoSmithKline GlaxoSmithKline Celgene ICOS MediciNova

Arofylline Oglemilast (GRC 3886) Tofimilast (CP-325366) ONO 6126 Tetomilast (OPC 6535) Roflumilast Cilomilast (SB 207499) AWD-12-281 (GSK 842470) CC-10004 IC-485 Ibudilast

4 4 4 4 4 4 4 4 4 4 4

Bronchitis Asthma, COPD Asthma Asthma, COPD COPD Asthma, COPD COPD Asthma Asthma, psoriasis COPD Multiple sclerosis

Phase II/III Phase III Phase II Phase II Phase III Phase III Phase III Phase II Phase II Discontinued Phase II

COPD, chronic obstructive pulmonary disease. Data collated from Mealey & Bayes (2005).

sub-bronchodilator doses (Ward et al. 1993; Sullivan et al. 1994; Djukanovic et al. 1995) which fuelled the idea that theophylline and so-called “second generation” PDE inhibitors could act as bronchodilators and potential antiallergic and/or antiinflammatory agents (Banner & Page 1995; Dent & Giembycz 1996; Palfreyman & Souness 1996; Torphy 1998; Doherty 1999; Essayan 1999; Giembycz 2000). The rationale for developing new PDE inhibitors has stemmed primarily from the realization that PDEs represent a highly heterogeneous group of enzymes (11 families have thus far been identified) that are differentially expressed between different cell types and almost certainly regulate specific functional responses. Accordingly, it was rapidly appreciated that drugs which selectively suppress the activity of a particular PDE isoenzyme may result in a discrete functional alteration of cells which express that PDE variant and, theoretically, specific functional responses within the same cell. Given the prevalence of allergic diseases as a whole, it is only too apparent that the potential clinical reward of developing a class of steroid-sparing drugs of general utility in a variety of allergic disorders is enormous and explains why many of the world’s major pharmaceutical companies have an active PDE research program and have developed highly selective PDE inhibitors, many of which are currently undergoing clinical trials for asthma and other allergic and nonallergic inflammatory disorders (Table 27.2). It is the purpose of this chapter to describe the multiplicity of mammalian PDE isoenzymes, the tissue distribution of PDEs expressed in cells which participate in inflammatory reactions and the potential sites where selective PDE inhibitors could act to alleviate the acute and chronic manifestations of allergic disease including clinical trials data. For conciseness, only those PDEs that, for established or theoretical reasons, may be exploited to therapeutic advantage in inflammatory diseases are discussed in detail. Readers interested in the

more general aspects of PDE biology are directed to recent reviews on this subject (Bender & Beavo 2006a; Lugnier 2006).

Generic properties and characteristics of cyclic nucleotide phosphodiesterases Cyclic nucleotide PDEs (EC 3.1.4.17) comprise a large group of enzymes whose sole function is to hydrolyze and thereby inactivate the biologically active cyclic purines cAMP and cGMP (Fig. 27.1). PDEs which act on cyclic pyrimidine monophosphates have also been described (see Newton & Salih 1986), although investigators have tended to focus, almost exclusively, on the PDEs which hydrolyze cyclic purine nucleotides for which functionally important second messenger roles have unequivocally been established. A cyclic nucleotide PDE that hydrolyzed the 3′-ribose phosphate bond of cAMP to the catalytically inactive 5′-AMP was first identified in 1962 (Butcher & Sutherland 1962). Since then multiple families and, indeed, subfamilies of PDEs which selectively act on cAMP and/or cGMP have been identified (see Bender & Beavo 2006a; Lugnier 2006 for reviews). The PDEs that metabolize cyclic purine nucleotides comprise at least 11 distinct families and can be distinguished by a number of criteria including substrate specificity, kinetic properties, responsiveness to endogenous allosteric regulators, susceptibility to inhibition by various compounds and in primary amino acid sequence (Table 27.3). Molecular biological studies have discovered that many PDEs are separate gene products and express multivariant regulatory domains linked to highly conserved homologous catalytic sequences located near the C-terminus of the protein. Members of one family share 20–25% sequence homology with members of another family. Furthermore, at least 6 of the 11 gene families can be divided into subfamilies that are 70–90%

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Pharmacology NH2

NH2 N

N

N

N N

N

PDE

N

N

H2O; Mg2+

cAMP

O

O

OH

O O

O P

O

HO

HO

O O

O N

N H2N

H2N

N N H

cGMP

O

N

PDE O

H2O; Mg2+

OH

O

OH

5′-GMP

OH

HO

P HO

N N H

N

O

O Fig. 27.1 cAMP and cGMP hydrolysis by cyclic nucleotide phosphodiesterases.

P

O

HO

homologous and which are derived from similar but distinct genes or, in some cases, from the same gene through alternate mRNA splicing or from differences in the initiation start sites for the translation of the protein. At the end of 2006 in excess of 22 PDE genes were unequivocally identified which, in mammals, probably encode for more than 50 distinct PDE enzymes. For further information on the molecular diversity of PDE isoenzymes, interested readers are directed toward a number of comprehensive reviews (Conti & Jin 1999; Conti 2000; Soderling & Beavo 2000; Houslay 2001; Houslay & Adams 2003; Bender & Beavo 2006a; Lugnier 2006). A diagrammatic representation of the primary structure of cyclic purine PDEs is shown in Fig. 27.2. All mammalian PDEs share essentially the same structure: there is a central cAMP cGMP

5′′-AMP 5′-GMP

Allosteric/small molecule Binding site (e.g. cGMP, Ca2+/calmodulin) Phosphorylation sites (e.g. PKA at S54 in PDE4D3)

O

core located close the C-terminus of the protein that shares 25–35% homology between isoenzyme families and which features a highly conserved domain of some 270 amino acids. Studies performed in a number of laboratories indicate that this conserved region within this central core of all PDEs represents the catalytic site (Charbonneau et al. 1986). The central core of mammalian PDEs is linked via so-called hinge regions to carboxy- and amino-terminal extensions (Fig. 27.2) which show relatively little sequence homology between PDE families (Conti & Jin 1999). This finding has led to the view that the nonconserved domains subserve regulatory functions of the protein (Conti & Jin 1999). Indeed, it is now appreciated that the N-termini of PDEs can feature binding sites for calmodulin (PDE1) and cGMP (PDEs 2, 5, 6,

Phosphorylation sites (e.g. ERK-2 at S579 in PDE4D3 and S651 in PDE4D5)

Catalytic domain Dimerization domains

Targeting domains (e.g. TAPAS-1 in PDE4A1) NH 2

636

5′-AMP

OH

HO

P

OH

CO

OH

Fig. 27.2 Generic structure of mammalian cyclic purine PDEs. Limited proteolysis and protein sequencing suggest that all mammalian PDEs so far examined contain a conserved central core that features the catalytic site. This region of PDE proteins is flanked, through putative hinge regions, by highly heterologous C- and N-termini which are believed to express regulatory domains, dimerization motifs, phosphorylation sites and sequence information which determine the subcellular localization of PDEs. See text for further details. (See CD-ROM for color version.)

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Table 27.3 Properties and selective inhibitors of cyclic nucleotide phosphodiesterases. Km (mmol/L) PDE family

cAMP

cGMP

Selective inhibitors

References

1

Ca2+/calmodulin-stimulated

0.3–124

0.6–6

SCH 57801 KS-505a IC-224

Ahn et al. (1997) Ichimura et al. (1996) Snyder et al. (2005)

2

cGMP-stimulated

28–100

10–28

ENHA Oxindole BAY 60-7550 IC933 PDP

Podzuweit et al. (1995) Chambers et al. (2006) Boess et al. (2004) Snyder et al. (2005) Seybold et al. (2005)

3

cGMP-inhibited

0.02–0.3

0.1–0.5

Siguazodan SK&F 95654 Cilostamide Cilostazol

Murray et al. (1990) Murray et al. (1992) Hidaka et al. (1979) Tanaka et al. (1988)

4

cAMP-specific

1–10

> 50

Rolipram Ro 20-1724 Cilomilast Roflumilast Piclamilast

Reeves et al. (1987); Schwabe et al. (1976) Sheppard & Tsien (1975) Barnette et al. (1998) Hatzelmann & Schudt (2001) Souness et al. (1995)

5

cGMP-binding

280

3–7

Zaprinast Sildenafil Vardenafil Tadalafil

Lugnier et al. (1986) Boolell et al. (1996) Saenz et al. (2001) Padma-Nathan et al. (2001)

6

Photoreceptor

600–700

15–17

None reported

7

Rolipram-insensitive, cAMP-specific

0.01–0.5

NA

BRL 50481 PF 332040 IC242

8

Rolipram-insensitive, cAMP-specific

0.06

NA

None reported

9

Zaprinast-insensitive, cGMP-specific

228

0.7–0.17

BAY 73-6691

10

AMP-inhibited (?)

0.22–1.1

13–14

None reported

11

Dual-selective

1–2

2–3

None reported

Smith et al. (2004) Giembycz & Smith (2006a); Jones et al. (2007) Lee et al. (2002)

Wunder et al. (2005)

NA, reliable data not available.

10 and 11), so-called GAF domains (Martinez et al. 2002a); membrane-association domains (PDE3); subcellular targeting sequences (PDE4, PDE7); phosphorylation sites (most PDEs) that control catalytic activity; and PAS domains (PDE8), involved in ligand recognition and protein–protein interactions. Similarly, sequences at the C-terminus express dimerization motifs (most PDEs exist as dimers (Charbonneau et al. 1991; Kovala et al. 1997; Martinez et al. 2002b; Richter & Conti 2002; Zoraghi et al. 2005) and/or are substrates for phosphorylation (Hoffmann et al. 1998b; MacKenzie et al. 2000). Readers should note that GAF and PAS (domains) are acronyms of the names of the first three groups of proteins identified to contain them, i.e., cGMP-regulated cyclic nucleotide phosphodiesterase, cyanobacterial Adenylyl cyclase and E. coli transcription factor, Fh1A (formate-hydrogen lyase),

and Period circadian protein, Ah receptor nuclear translocator protein and Single minded protein, respectively.

Standardized nomenclature A standardized GenBank nomenclature for existing and newly discovered PDEs was introduced in 1994 (Beavo et al. 1994). This taxonomy is based on traditional biochemical criteria and on protein sequencing and analyses of partial and full length cDNA clones. Consider two hypothetical PDEs, CPPDE4D4B and HSPDE11A1A (Fig. 27.3). The first two letters refer to the species (CP for Cavia porcellus, HS for Homo sapiens). The next three letters plus an Arabic numeral correspond to the PDE gene family (i.e., PDE, PDE11) while the

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Species

Pharmacology

PDE family

Gene

Splice variant

Report

HSPDE4D4B CPPDE11A1A Green Purple Red Orange Yellow

Species (first 2 letters) PDE gene family (3 capital letters + 1 or 2 Arabic numerals) PDE gene within family (capital letter) Splice variant (Arabic numeral) Report (capital letter)

Fig. 27.3 GenBank nomenclature for the classification of cyclic purine PDEs. The first two letters (green) represent the species, the next three letters plus an arabic numeral denote the PDE family (purple), the next letter and numeral refer to the gene product within the PDE family (red) and the spliced variant (if appropriate; orange) respectively and the last letter indicates the report (yellow). See text for further details. (See CD-ROM for color version.)

next letter and Arabic numeral (i.e., D4, A1) denote the gene product and spliced variant (if appropriate). This nomenclature is used throughout this chapter.

Phosphodiesterase 1 PDE1 is a generic term that describes a family of hydrolytic enzymes that are active only in the presence of calmodulin and micromolar concentrations of cytosolic free Ca2+ (Conti & Jin 1999; Goraya & Cooper 2005). This Ca2+/calmodulin dependency, originally documented in the early 1970s (Cheung 1970, 1971; Kakiuchi & Yamazaki 1970), is a characteristic of all members of this enzyme family and results in a 5–20-fold increased in catalytic activity depending on the particular isoform studied (Sharma & Wang 1986a,b). Detailed kinetic analyses indicate that calmodulin modulates PDE1 activity allosterically by substantially increasing the Vmax of the reaction while reducing, relatively modestly, the affinity (Km) of the substrate for the enzyme (Sharma & Wang 1986a,b). Evidence for PDE1 multiplicity was originally provided by immunologic studies (Hansen and Beavo 1982), which effectively discriminated two bovine isoforms: a 61-kDa enzyme in brain and a 59-kDa variant in heart. Sequence analysis has revealed that the 59-kDa and 61-kDa isoenzymes (BTPDE1A1 and BTPDE1A2 respectively) are alternatively spliced variants of the same gene (Charbonneau et al. 1991; Novack et al. 1991). Similar studies, subsequently reported by Sharma et al. (1984) identified a 63-kDa isoenzyme in bovine brain that is immunologically distinct from BTPDE1A2 and represents a different gene product (BTPDE1B1). This enzyme was cloned in 1992 from bovine and rat brain cDNA libraries (Repaske et al. 1992). It is now known that PDE1 comprises a large family of closely related proteins that are encoded by at least three genes (PDE1A, PDE1B, PDE1C) with further diversity arising from mRNA splicing due to differential exon usage (Snyder et al. 1999; Michibata et al. 2001; Fidock et al. 2002; Goraya

638

& Cooper 2005). In humans 13 PDE1A cDNAs have been described (Loughney et al. 1996; Snyder et al. 1999; Michibata et al. 2001) together with two splice variants for both PDE1B (PDE1B1, PDE1B2) and PDE1C (PDE1C1, PDE1C3) (Loughney et al. 1996; Fidock et al. 2002). All PDE1A and PDE1B isoforms as well as PDE1C1 preferentially hydrolyze cGMP, whereas PDE1C3 degrades cAMP and cGMP with equal efficiency (Loughney et al. 1996; Snyder et al. 1999).

Tissue distribution and selective inhibitors Of those proinflammatory and immunocompetent cells implicated in human inflammatory and allergic diseases, only alveolar macrophages (Tenor et al. 1995a), epithelial cells (Fuhrmann et al. 1999; Rousseau et al. 1994; Wright et al. 1998) and airway smooth muscle (Torphy et al. 1993a; Souness & Giembycz 1994) express appreciable PDE1 constitutively. Indeed, depending on species, PDE1 can account in airways smooth muscle for ~90% of the cyclic nucleotide hydrolysing activity (Souness & Giembycz 1994). In addition, trace amounts of this isoenzyme are present in other cells including CD4+ and CD8+ T lymphocytes (Tenor et al. 1995b) where they may have a role in regulating mitogenesis (see below). Although PDE1 is ubiquitously expressed, establishing functional roles for these isoenzymes has been greatly hindered by a lack of selective pharmacologic tools. Vinpocetine and 8-methoxymethyl-3-isobutyl-1-methylxanthine are often used for this purpose but, in fact, these compounds are nonselective and, minimally, will inhibit PDE5 (Ahn et al. 1989) and block certain Na+ and K+ channels (Bonoczk et al. 2000) at concentrations similar to those required for PDE inhibition (Bukanova & Solntseva 1998). However, in 1997 chemists from Schering-Plough described a number of selective and potent PDE1 inhibitors based on the tetracyclic guanine template (Ahn et al. 1997). Two of these compounds (1 and 2 in Fig. 27.4) are at least 280 times more selective for PDE1 over the other major PDEs and are suitable for in vitro pharmacologic testing. In addition, it was reported in the early 1990s that a heterocyclic molecule isolated from the bacterium Streptomyces argenteolus is a potent and highly selective inhibitor of PDE1 (Nakanishi et al. 1992; Kase et al. 1993; Nagashima et al. 1993; Ichimura et al. 1996). This compound, designated KS505a, is somewhat unique as it selectively inhibits PDE1A2. Indeed, KS505a is approximately 80-fold more potent against PDE1A2 from bovine brain (IC50 ∼ 170 nmol/L) than of PDE1A1 from bovine heart (Nakanishi et al. 1992; Kase et al. 1993; Ichimura et al. 1996). These are fascinating findings. Not only do they identify KS505a as a selective inhibitor of PDE1, but demonstrate that compounds can discriminate between two highly homologous proteins that are derived from the same gene by alternate splicing. Perhaps the most exciting prospect that arises from these data is the likelihood that third generation PDE inhibitors can be synthesized which would allow the selective targeting of specific enzymes encoded by the same gene.

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O

O H 3C

N

Fig. 27.4 Chemical structures of two novel and selective inhibitors of PDE1. See text and Ahn et al. (1997) for further details.

H3C

N

N N

N H

N

N N

1

Therapeutic indications Mitogenesis The functional roles in mammals of PDE1 are largely unknown. However, evidence has emerged since the early 1990s that certain PDE1 isoforms may regulate cell division in both lymphocytes and smooth muscle. The human lymphoblastoid RPMI-8392 B-cell line, which was established from a patient with acute lymphocytic leukemia, expresses two major PDEs: PDE1 (probably PDE1B) and PDE4 (Epstein et al. 1987; Jiang et al. 1996). In contrast, normal quiescent human B lymphocytes lack PDE1 (Epstein et al. 1987; Gantner et al. 1998). However, following exposure of normal B lymphocytes to the mitogen phytohemagglutinin (PHA), significant PDE1 activity was detected together with mRNA transcripts for PDE1B (Hurwitz et al. 1990; Jiang et al. 1996). The induced enzyme eluted from anion-exchange columns as a 63-kDa protein that was indistinguishable from PDE1B purified from bovine brain (Hurwitz et al. 1990; Bentley et al. 1992). It is known that mitogens effect transcription of PDE1B (Spence et al. 1995, 1997) and it is possible that induction of this gene occurs when lymphocytes switch from a quiescent to a proliferative phenotype. As cGMP suppresses lymphocyte proliferation (Fischer et al. 2001), the induction of PDE1B by PHA could, by lowering the basal cGMP content, represent a physiologic response to permit B-cell mitogenesis. It is clear from these data that inhibition of PDE1 could prevent B-cell expansion and so compromise their ability to synthesize allergen-specific IgE. To date, the possibility that PDE1 may also regulate T-cell proliferation in an analogous manner to B lymphocytes has not been examined. Nevertheless, PDE1B1 mRNA levels are elevated in human T lymphocytes exposed to PHA and antiCD3/antiCD28 (Kanda & Watanabe 2001), which has been linked to the expression of interleukin (IL)-13, and so this possibility seems likely. Thus, PDE1 inhibition could have a profound effect on aspects of T-cell function upon which the induction and perpetuation of allergic reactions depend. Evidence has been published that the proliferation of human vascular smooth muscle cells is also controlled by PDE1 (Rybalkin et al. 1997, 2002). A discussion of these data

N H

2 SCH 57801

here is merited as one or more PDE1 isoenzymes could serve the same function in airway myocytes and so impact on the remodeling of the airways that is a characteristic of subjects with asthma (Huber & Koessler 1922; Dunnill et al. 1969; Heard & Hossain 1973; Ebina et al. 1993; Jeffery 2001; Benayoun et al. 2003; Hirst et al. 2004; Woodruff et al. 2004). The salient observation made by Rybalkin and colleagues was that PDE1C was markedly induced in proliferating but not quiescent smooth muscle cells derived from human aorta (Rybalkin et al. 1997). Subsequently, it was established that induction of PDE1C correlated with cell cycle progression and that inhibition of this enzyme with either 8-methoxymethyl3-isobutyl-1-methylxanthine (a modestly-selective PDE1 inhibitor) or antisense oligonucleotides directed against PDE1C, significantly reduced mitogenesis (Rybalkin et al. 2002). As inhibitors of PDE5 (which selectively hydrolyze cGMP) were not antimitogenic in the same system, it was concluded that proliferation was due to the induction of PDE1C and involved degradation of cAMP rather than cGMP (Rybalkin et al. 2002). Collectively, these data, obtained in B cells and vascular myocytes, provide compelling support for the hypothesis that the appearance of a PDE1 isoenzyme in dividing cells, that is normally absent or expressed at a relatively low level in the quiescent phenotype, represents a normal physiologic, mitogen-induced response that would otherwise be suppressed by the relatively high resting cyclic nucleotide levels.

Monocyte differentiation Human blood monocytes have the ability to differentiate into a variety of cell types that is governed by cytokines and other stimuli. With respect to the immune system, monocytes may differentiate into dendritic cells, which are professional antigen-presenting cells, or various types of tissue macrophages that have distinct characteristics. In all cases, the phenotypical changes are accompanied by a marked remodelling of the cell’s PDE profile. Thus, exposure of human peripheral blood monocytes (CD14+/CD1a−) to GMCSF and IL-4 (in serum) results, over a period of several days, in the formation of dendritic cells (CD14−/CD1a+) and this

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process is associated with a marked upregulation of PDEs 1 and 3 and a decline in PDE4 (Gantner et al. 1999). A similar pattern of PDE remodeling is seen when monocytes are aged for several days in serum alone, or granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence of IL-10, which results in the generation of cells with macrophagelike phenotypes (Gantner et al. 1997; Bender et al. 2004). Molecular studies have found that human blood monocytes express predominantly PDE1B2 mRNA and protein and that the abundance of this transcript and enzyme is markedly higher in GM-CSF-induced macrophage-like cells (Bender et al. 2004, 2005). PDE1B is also induced in monocytes treated with M-CSF, which again results in the formation of cells with a macrophage-like phenotype (Bender et al. 2004). The functional significance of PDE1B2 upregulation has been studied using gene silencing technologies using HL-60 cells differentiated into macrophage-like cells by phorbol esters (Bender & Beavo 2006b). The results from those studies suggest that PDE1B2 preferentially regulates the cGMP content in these cells but this does not affect many of the markers that are typical of differentiation such as adherence and the expression of CD71 and CD87. However, selective knockout of PDE1B2 mRNA alters certain characteristics of the final macrophage phenotype. In particular, CD11b expression, cell spreading and phagocytosis are all augmented (Bender & Beavo 2006b). The clinical impact of inhibiting PDE1 in cells of the monocyte/macrophage/dendritic cell lineage is unclear but enhanced phagocytic capacity could be of benefit.

Phosphodiesterase 2 The first description of, what is now termed, a PDE2 isoenzyme was reported in 1971 following the observation that cAMP hydrolysis in rat liver supernatant was stimulated by micromolar concentrations of cGMP (Beavo et al. 1971). Since then, many reports have documented the presence of cGMP-stimulated PDEs in a number of cells and tissues including those relevant to the pathogenesis of allergic reactions (Table 27.3). PDE2 isoenzymes do not discriminate between cAMP (Km 28–100 μmol/L) and cGMP (Km 10– 28 μmol/L) to any great extent, exhibit positive homotropic cooperative behavior with respect to both substrates and feature two high-affinity (Kd ∼ 0.1 μmol/L), noncatalytic dimerization/allosteric binding sites for cGMP (Moss et al. 1977; Martins et al. 1982; Yamamoto et al. 1983; Stroop & Beavo 1992; Martinez et al. 2002b). An interesting property of these enzymes is that low concentrations (1– 5 μmol/L) of cGMP enhance the degradation of cAMP approximately sixfold by a mechanism that requires occupancy of the noncatalytic cGMP-binding domain (Yamamoto et al. 1983; Stroop & Beavo 1992). In contrast, cAMP does not detectably stimulate PDE2-catalyzed cGMP hydrolysis (Stroop & Beavo 1992). A single gene has thus far been identified which encodes PDE2. However, transcription of this gene can produce at

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least three spliced variants (PDE2A1, PDE2A2 and PDE2A3) that occurs at the 5′-end of the sequence (Trong et al. 1990; Sonnenburg et al. 1991; Yang et al. 1994; Rosman et al. 1997). It is noteworthy that only a single human PDE2 splice variant has so far been identified, which is highly homologous to bovine PDE2A3. Hence it has been named HSPDE2A3 (Rosman et al. 1997). These proteins share identical C-termini but differ in their N-termini, which dictate whether these enzymes are soluble or membrane-associated. PDE2s can be cytosolic or located to specific membrane structures including the Golgi apparatus, plasma membrane, endoplasmic reticulum and nuclear envelop where they presumably subserve distinct functions (Lugnier et al. 1999a,b; Geoffroy et al. 1999). Like many cyclic nucleotide PDEs, native PDE2s are homodimeric with a molecular mass of about 210 kDa (Stroop & Beavo 1992).

Tissue distribution and selective inhibitors Northern blot analysis has identified PDE2A mRNA in trachea and lung (Sonnenburg et al. 1991) and abundant PDE2-like enzyme activity has also been reported in these tissues from several species including human (Torphy et al. 1993a; Souness & Giembycz 1994). PDE2 is found in platelets (Simpson et al. 1988) and epithelial cells (Rousseau et al. 1994), although expression may be species dependent as it is seemingly absent from human airway epithelia (Wright et al. 1998; Fuhrmann et al. 1999). Trace amounts of PDE2 have been identified in T lymphocytes and alveolar macrophages (Tenor et al. 1995a,b; Bender et al. 2004) (Table 27.4). A study published in 1999 reported the expression profile of mRNA and protein for PDE2A3 in endothelial cells lining a variety of human vessels (Sadhu et al. 1999). Interestingly, Western blot analysis identified PDE2A3 only in certain populations of vascular endothelial cells, in particular the vasa

Table 27.4 Established PDE isoenzyme profiles in human proinflammatory and immune cells implicated in allergic disorders. Immune/proinflammatory cell

PDE profile

Mast cell Alveolar macrophage T lymphocyte B lymphocyte Eosinophil Neutrophil Basophil Monocyte Platelet Epithelial cell Endothelial cell Dendritic cell Airway myocyte Vascular myocyte

3, 4, 5, 7 1, 3, 4, 5, 7 1, 2, 3, 4, 5, 7, 8(?) 3, 4, 7 4 4, 7 4, 7 1, 3, 4, 7 1, 2, 3, 5 1, 2, 3, 4, 5, 7, 8 2, 3, 4, 5, 7 1, 3, 4, 7 1, 2, 3, 4, 5, 7, 8 1, 2, 3, 4, 5, 7

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O N N

NH2

N N

N

3 EHNA

N

S

CH3

H 3C

O O

N

N

NH

OH

H3C

HN

O

N

N

CH3

H3C

OH O

N H3C

CH3 4 Oxindole

O 5 BAY 60-7550

Fig. 27.5 Chemical structure of three selective inhibitors of PDE2. See text for further details.

vasorum and those of microvessels, whereas it was absent or very lowly expressed in large vessels such as the pulmonary artery, aorta and renal arteries (Sadhu et al. 1999). The subcellular localization of PDE2 in human endothelial cells requires clarification although in most cells it appears, by immunocytochemistry, to be cytosolic or perinuclear (Sadhu et al. 1999). Erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) was the first small molecule to be described with selective PDE2 inhibitory activity (Podzuweit et al. 1993, 1995) (see 3 in Fig. 27.5). The compound has an IC50 value of 0.5–1 μmol/L against PDE2 and is approximately 100-fold less potent against the other PDEs (Podzuweit et al. 1995). However, one drawback of ENHA is its ability to potently inhibit adenosine deaminase, which can compromise its use in certain tissues (Schaeffer & Schwender 1974). Two new PDE2 inhibitors were described in 2004, an oxindole (4), reported by Pfizer (Chambers et al. 2006), and the Bayer compound BAY 607550 (5) (Boess et al. 2004) (Fig. 27.5). The oxindole has an IC50 against human recombinant PDE2 of 40 nmol/L, over an order of magnitude more potent than EHNA. Moreover, the compound is > 290-fold selective over representatives from all other enzyme families and, in the rat, has favorable ADME, physicochemical and pharmacokinetic properties (Chambers et al. 2006). Several comprehensive screens have found that the oxindole has negligible off-target activity being devoid of significant interaction with a large panel of receptors, ion channels and enzymes. The most potent PDE2 inhibitor reported to date is BAY 60-7550 (IC50 4.7 nmol/L against human PDE2A) (Boess et al. 2004). Its selectivity relative to other PDEs ranges from 50-fold (PDE1) to > 800-fold (PDEs 3B, 7B, 8A, 9A, 11A) (Boess et al. 2004). BAY 60-7550 is bioavailable in rats and mice (Boess et al. 2004).

Therapeutic indications Despite being PDE2 are still relatively rich and/or cGMP

discovered in 1971, the functional roles of largely undetermined. In endothelial cells, a source of this enzyme, an elevation of cAMP can improve barrier function (Suttorp et al.

1993, 1996a,b; Westendorp et al. 1994; Seeger et al. 1995; Lum et al. 1999), inhibit mitogenesis (D’Angelo et al. 1997) and modulate (usual reduce) the elicited expression of certain adhesion molecules (Pober et al. 1993; Ghersa et al. 1994; Morandini et al. 1996). However, the principle PDEs implicated in the regulation of these responses is only now being addressed. In 2005, direct evidence was provided that PDE2 can regulate the integrity of the endothelium. In an elegant study Seybold and colleagues (2005) found that exposure of human umbilical vein endothelial cells (HUVECs) to tumor necrosis factor (TNF)-α resulted in a marked, time-dependent induction of PDE2A3 mRNA and protein that was associated with a decline in intracellular cyclic nucleotide levels and an increase in endothelial cell permeability. A disruption of endothelial cell integrity was also evoked in the same cells transfected with an expression vector encoding HSPDE2A3. Of significance was the additional finding that a selective PDE2 inhibitor, PNP, blocked the increase in permeability induced by thrombin in HUVECs transfected with the PDE2A3 expression vector, and this effect was reproduced in a murine model of endothelial barrier dysfunction (Seybold et al. 2005). From an allergy perspective, destabilization of the microvascular endothelium would be predicted in diseases such as asthma where TNF-α is known to be elevated (Broide et al. 1992; Ying et al. 1991) and this may facilitate edema formation and pulmonary leukocyte recruitment. A detrimental action on barrier function may not be restricted to TNF-α as many cytokines relevant to the pathogenesis of airway inflammation activate protein kinase C (PKC) (Kontny et al. 2000), which can also upregulate PDE2 (Geoffroy et al. 1999). Moreover, certain growth factor such as vascular endothelial growth factor also promote PDE2 gene expression (Favot et al. 2004) and, like TNF-α, is elevated in the asthmatic airway (Lee & Lee 2001; Chetta et al. 2005). Thus, if PDE2 also negatively regulates adhesion molecule expression and endothelial cell proliferation (Favot et al. 2003), inhibition of this enzyme could exert marked therapeutic benefit by suppressing the chronic inflammation and, potentially, airway remodeling that characterize asthma.

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Phosphodiesterase 3 Three genetically distinct cAMP-specific PDE families have been described and characterized (Bender & Beavo 2006a; Lugnier 2006). One of these, PDE3, is selectively inhibited by micromolar concentrations of cGMP and was originally classified as the cGMP-inhibited, cAMP PDE. The first clear demonstration of a cGMP-inhibited cAMP PDE was reported in 1982 (Weber & Appleman 1982). Since then, a PDE3 isoenzyme has been highly purified and characterized from a number of tissues including human platelets (Grant & Colman 1984), bovine heart (Harrison et al. 1986), rat liver (Pyne et al. 1987) and rat adipocytes (Degerman et al. 1987). Generally, members of this isoenzyme family hydrolyze cAMP and cGMP with high affinity but the apparent Vmax when cGMP is substrate is only about 10% of that achieved with cAMP (Grant & Colman 1984; Manganiello et al. 1992). For this reason, cGMP behaves as a potent and competitive inhibitor of cAMP hydrolysis (KI ∼ 0.1 μmol/L). It is noteworthy, however, that not all PDE3 preparations hydrolyze cGMP with a low Vmax (Maurice & Haslam 1990; Torphy & Cieslinski 1990; Manganiello et al. 1992; Torphy et al. 1993a). This is especially true for the PDE3 in human trachealis where less than a twofold difference in Vmax has been reported (Torphy et al. 1993a). The potential inhibitory action of cGMP on PDE3 activity may have significant physiologic implications in cells implicated in the pathogenesis of allergic diseases. Indeed, in PDE3expressing cells stimulants of guanylyl cyclase may inhibit cAMP hydrolysis and so potentiate cAMP-mediated functional responses. Evidence to support this possibility is available from studies performed with human platelets (Levin et al. 1982; Maurice & Haslam 1990), vascular smooth muscle (Shimokawa et al. 1988; Maurice et al. 1991), thymocytes (Marcoz et al. 1993) and airways smooth muscle (Turner, N.C. et al. 1994). In the latter tissue, sodium nitroprusside (SNP) markedly potentiated relaxation of guinea-pig trachea by the PDE4 inhibitor, rolipram, a finding consistent with the ability of SNP to increase cAMP in this tissue. In mammals, PDE3 isoenzymes are encoded by two genes: PDE3A and PDE3B. However, for only the former isoenzyme has evidence of multiplicity been convincingly provided (three variants have been described to date). These proteins have been denoted PDE3A1, PDE3A2 and PDE3A3 and arise from different start codon usage (Choi et al. 2001; Wechsler et al. 2002). PDE3A and PDE3B have a high degree of identity at the amino acid level (> 80% for most of the catalytic region), display similar kinetic properties and are both upregulated by phosphorylation catalyzed by PKA and PKB (Shakur et al. 2001). Unlike other PDE families, PDE3s contain a 44-amino acid insert within their catalytic domain of uncertain function although this additional sequence may contribute to both catalytic activity and inhibitor recognition (Manganiello & Degerman 1999). Full-length PDE3A (i.e., PDE3A1) and PDE3B feature one

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large (193 amino acids) and one small (50 amino acids) Nterminal hydrophobic membrane association region, NHR1 and NHR2, that are important in determining the subcellular localization of the protein (Shakur et al. 2000). Indeed, heterologous expression of these enzymes in Sf9 cells yields proteins that are entirely particulate (i.e., membraneassociated) (Kenan et al. 2000). In contrast, PDE3A2 and PDE3A3 in which the first and second NHRs are truncated are enzymes that are 50% cytosolic and totally cytosolic respectively (Kenan et al. 2000; Wechsler et al. 2002; Maurice et al. 2003).

Tissue distribution and selective inhibitors PDE3 isoenzymes are ubiquitously expressed in immune and proinflammatory cells (Table 27.4) including the platelet (Hidaka & Endo 1984; Macphee et al. 1986), basophil (Peachell et al. 1992), mast cell (Bergstrand et al. 1978; Weston et al. 1997), alveolar macrophage (Tenor et al. 1995a), T lymphocyte (Tenor et al. 1995b; Giembycz et al. 1996) and epithelial and endothelial cell (Lugnier & Schini 1990; Souness et al. 1990; Rousseau et al. 1994; Wright et al. 1998; Fuhrmann et al. 1999). PDE3 is not apparently present in eosinophils (Dent et al. 1991, 1994; Hatzelmann et al. 1995) or neutrophils (Nielson et al. 1990; Wright et al. 1990; Schudt et al. 1991a). The coexpression of PDE3A and PDE3B across tissues has not been systematically studied. However, where coincident expression has been found, PDE3A usually predominates (Manganiello & Degerman 1999; Maurice et al. 2003). Selective PDE3 inhibitors were developed in the 1980s for the treatment of dilated cardiomyopathy with the hope that a greater therapeutic index could be achieved over cardiac glycosides (Movsesian 2003). Accordingly, many potent and structurally distinct inhibitors have been synthesized some of which are currently used in the clinic for cardiovascular disease. In addition, many of these compounds have also been studied in nonvascular settings including asthma.

Therapeutic indications PDE3 has been considered a target for the treatment of asthma since selective inhibitors promote bronchodilatation in humans (Leeman et al. 1987; Fujimura et al. 1995; Bardin et al. 1998; Myou et al. 1999). Compounds of this class may also have desirable effects on the function of certain proinflammatory and immune cells although this is likely to be of relevance only during concurrent PDE4 inhibition (see below for further details). Thus, the concept of hybrid inhibitors that block PDE3 and PDE4 has found favor with some pharmaceutical companies as such compounds are predicted to have both bronchodilator and antiinflammatory activity.

Phosphodiesterase 4 A cAMP-specific PDE that was inhibited by the alkoxybenzylsubstituted imidazolidone, Ro 20-1724, but largely unaffected by cGMP was originally purified from canine kidney

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(Thompson et al. 1979; Epstein et al. 1982). It is now appreciated that this was the first report of a member of a large family of similar proteins collectively known as PDE4 isoenzymes. Since that original description, PDE4s have been purified and partially characterized from a number of sources including human monocytes (Torphy et al. 1993b) and human leukocytes (Truong & Muller 1994). Human recombinant proteins have also been heterologously expressed and purified to facilitate the identification of selective inhibitors by high throughput screening (Salanova et al. 1998). Without apparent exception, PDE4 isoenzymes are acid proteins (pI 4–6) and preferentially or even exclusively hydrolyze cAMP (Table 27.3). In 1989 Davis et al. (1989) isolated and cloned the Drosophila melanogaster “dunce” gene and found that it encoded a PDE of the PDE4 family. In the same year four rat cDNA homologs (Colicelli et al. 1989; Swinnen et al. 1989a,b) of the “dunce” cAMP PDE were identified, establishing a molecular basis for the observed heterogeneity of gene products within the PDE4 family. These clones represent transcripts of four different genes and are now named RNPDE4A, RNPDE4B, RNPDE4C and RNPDE4D according to the nomenclature proposed by Beavo and colleagues (1994). Four human genes (A–D) have also been identified and many of their products studied in some detail (see Bender & Beavo 2006a; Lugnier 2006). An astonishing finding that emerged from the molecular cloning of PDE4 isoenzymes is the presence of mRNA transcripts of different sizes for each of the four variants (greater than 26 have been unequivocally identified so far) that are differentially expressed between tissues (Houslay & Adams 2003; Bender & Beavo 2006a; Lugnier 2006). The basis for this profound heterogeneity of PDE4 isoenzymes is attributable to both alternative mRNA splicing and to the fact that the PDE4 genes express multiple promoter regions and, therefore, provide several potential start codons for the translation of the protein. Proteins encoded by each PDE4 gene may be either of the “short” or “long” form (see Bender & Beavo 2006a). Structurally, long PDE4 isoforms have an N-terminal domain, two socalled upstream conserved regions (UCR1 and UCR2; thought to mediate dimerization of long PDE4s (Richter & Conti 2002)), a highly conserved catalytic center and a unique C-terminus; in contrast, short PDE4 variants are truncated proteins that lack UCR1 (Houslay et al. 1998).

Tissue distribution and selective inhibitors Given the ubiquitous expression of PDE4 in cells implicated in the pathogenesis of allergic disease (Table 27.4), it is perhaps not surprising that many of the major pharmaceutical companies have invested heavily in PDE research over the last two decades, which has produced a variety of potent and selective inhibitors with which probe the pharmacologic consequences in vitro and in vivo of PDE4 inhibition (6–14 in Fig. 27.6; Table 27.3).

Therapeutic indications PDE4 inhibitors have been tested for efficacy in a number of immunologic and immunodeficiency conditions including asthma, chronic obstructive pulmonary disease (COPD), Crohn disease, myasthenia gravis, atopic dermatitis, psoriasis, systemic lupus erythematosis, rheumatoid arthritis, diabetes and multiple sclerosis (Sommer et al. 1997; Dyke & Montana 2002; Banner & Trevethick 2004; Aricha et al. 2006; Moore et al. 2006). Asthma and COPD are indications where development of PDE4 inhibitors is most advanced (Table 27.2). The rationale for developing compounds that attenuate PDE4 activity is based on three critical findings: (i) PDE4 is abundant and the major regulator of cAMP metabolism in almost every pro-inflammatory and immune cell; (ii) PDE4 inhibitors, of varied structural classes, suppress a myriad of in vitro responses such as cytokine generation, NADPH oxidase activity, degranulation, IgE production, proliferation, lipid mediator and histamine generation, and chemotaxis; and (iii) PDE4 inhibitors are efficacious in animal models of inflammation (Torphy 1998). If these observations hold in humans then, conceptually, PDE4 inhibitors should show a pleiotropic inhibitor profile of activity on proinflammatory and immune cell function. A further prediction is that inhibition of PDE4 should potentiate the effects of endogenous antiinflammatory agents that increase cAMP such as catecholamines, prostaglandin E2 and prostacyclin. Taken together, the preclinical pharmacology of these compounds provided an exciting rational basis for the development of novel antiinflammatory pharmaceuticals that may display steroid-like activity without the associated side-effects.

Regulation of PDE4 PDE4 is subject to both short and long-term regulation by agents that elevate cAMP. This finding has led to the proposal (see Giembycz 1996) that upregulation of PDE4 in allergic asthma could contribute to the tolerance of airway smooth muscle cells to the bronchodilator and protective effects of β2-adrenoceptor agonists (see Cheung et al. 1992; O’Connor et al. 1992; Cockcroft et al. 1993; Bhagat et al. 1995; Hancox et al. 1999, 2002; Jones et al. 2001; Wraight et al. 2003; Haney & Hancox 2005). Indeed, a metaanalysis of 22 independent randomized, placebo-controlled studies conducted between 1989 and 2001 lead the authors to conclude that regular use of β2-adrenoceptor agonists for at least one week in patients with asthma results in tolerance to the drug’s bronchodilator activity and that this may be associated with poorer asthma control (Salpeter et al. 2004). It is pertinent to mention that the mechanism of tolerance is not resticted to β2-adrenoceptor desensitization. It is likely that in many subjects poor asthma control reduces the efficacy of β2-adrenoceptor agonists. For example, mucus impaction may limit the delivery of drug to the desired sites of action and so compromise efficacy by physical means. Upregulation of PDE4 can occur through either posttranslational modification (e.g., phosphorylation) of existing

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H 3C

O

OH H N

O

HN

N

N

O

N

N

N O

N

N

N

S

N

O H3C

Cl 6 Arofylline

CH3 7 Tofimilast

8 Cilomilast OH HN

N

O

O HO

O

Cl O

CH3

O

O

N

O NH

N

N

O

Cl

CH3

N

S

O F

9 Tetomilast

F

CH3

10 Roflumilast

11 ONO 6126

F

N

N

CH3

N

O

Cl

N CH3

NH

N

HO O

O

CH3 O

CH3

12 AWD-12-281 (GSK 842470)

O

O

O

H 3C

Cl

CH3

O CH3 13 Ibudilast

CH3 14 IC-485

Fig. 27.6 Chemical structure of selective inhibitors of PDE4 that have been evaluated in the clinic for allergic diseases. See text for further details.

enzyme or gene induction. Indeed, the catalytic activity of long PDE4 isoforms is upregulated following phosphorylation by PKA of S54 within UCR1 (Sette et al. 1994; Sette & Conti 1996; MacKenzie et al. 2002). Similarly, the induction of certain PDE4 variants can be induced in a PKA-dependent manner that involves the phosphorylation of CREB (D’Sa et al. 2002; Wang et al. 2003). With respect to β2-adrenoceptors in the lung, it is the PDE4 isoenzyme family that is a primary regulator of cAMP metabolism (Torphy 1998). In this paradigm, tolerance to β2-adrenoceptor agonists is directly related to an increase in PDE activity. This effect would theoretically compromise cell signaling through all Gs-coupled receptors leading to heterologous desensitization of susceptible cells to cAMP-dependent events. It is hypothesized that this could occur as a direct consequence of regular treatment of asthmatic subjects with β2-adrenoceptor agonists (Giembycz 1996).

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While generally ignored, the concept of increased cAMP PDE activity as a mechanism of reducing the sensitivity of cells to hormones and other agonists that interact with Gscoupled receptors is not new. In fact, evidence that this phenomenon accounts for much of the reduced responsiveness that cells exhibit to chronic hormone exposure was provided in 1978 (Barber et al. 1978) and has since been documented in vitro in many cells implicated in the pathogenesis of asthma such as T lymphocytes, neutrophils, monocytes, macrophages, platelets and airway smooth muscle (Barber et al. 1978; Conti et al. 1986; Ashby 1989; Torphy et al. 1992, 1995; BousquetMelou et al. 1995; Verghese et al. 1995; Seybold et al. 1998; Mehats et al. 1999, 2001; Dasi et al. 2000; Ortiz et al. 2000; Persani et al. 2000). An important issue that arises from the aforementioned discussion is whether induction and/or phosphorylation of PDE4 can be demonstrated in immune/proinflammatory cells

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and in vivo in response to β2-adrenoceptor agonists. Although limited data are available, the answer to both parts of this question is yes. Torphy and colleagues (1995) demonstrated that the β2-adrenoceptor agonist, salbutamol, and the selective PDE4 inhibitor, rolipram when given in combination to the human monocytic cell line, U-937, increased PDE4 activity a time-dependent manner. Significantly, this effect required new protein synthesis indicating that the increase in enzyme activity was attributable to the induction of one or more PDE4 isogenes. Reverse transcription-PCR and Western analyses performed by the same authors demonstrated subsequently that salbutamol and rolipram increased the expression of PDE4A and PDE4B at the mRNA and protein level. A similar investigation by Verghese et al. (1995) essentially confirmed these observations. Thus, exposure of human peripheral blood monocytes and Mono Mac 6 cells to cyclic AMP-elevating agents promoted the transcription of the PDE4A, B and D isogenes with the generation of at least three distinct mRNA transcripts and proteins. Engels et al. (1994) have also reported induction of PDE4 isogenes in U-937 and Jurkat T-cells in response to prolonged exposure to dibutyryl cAMP and, more recently, the same phenomenon was documented in guinea-pig macrophages (Kochetkova et al. 1995), human T lymphocytes (Seybold et al. 1998), human neutrophils (Ortiz et al. 2000) and human airway smooth muscle cells (Le Jeune et al. 2002). In the latter study, upregulation of the PDE4D5 splice variant was described and this may have particular significance given that this isoform interacts preferentially with β-arrestins and may play a role in β2-adrenoceptor desensitization (Perry et al. 2002; Bolger et al. 2003). A consistent and highly significant finding is that the responsiveness of many cells in which PDE4 is induced to cAMP-generating agonists is restored, at least in part, by the addition of a PDE inhibitor providing compelling evidence that upregulation of PDE is a significant contributing factor in the development of tolerance. In 2000, Finney et al. reported the upregulation of PDE4 in the lungs of rats treated chronically for 7 days with salbutamol. Thus, this phenomenon can be reproduced in vivo and may have clinical relevance in the development of tolerance following chronic use of β2-adrenoceptor agonists.

Phosphodiesterase 5 Three major cyclic nucleotide PDE families have been described which preferentially hydrolyze cGMP. Two of these, PDE6 (or photoreceptor) and PDE9, are not implicated in asthma or allergy and are not discussed here. The other family comprises a group of cGMP-binding, cGMP PDEs that have been isolated and studied primarily from peripheral tissues and are collectively known as PDE5 isoenzymes. PDE5 was first identified in lung (Hamet & Coquil 1978; Coquil et al. 1980; Francis et al. 1980) and has since been purified to homogeneity from a number of tissues (Francis & Corbin 1988). In humans and other species PDE5 is a cytosolic

enzyme that is insensitive to Ca2+ and calmodulin and is selectively inhibited by zaprinast (Lugnier et al. 1986). PDE5 exist as a homodimer, displays selectivity for cGMP over cAMP (Table 27.3) (Thomas et al. 1990; Corbin & Francis 1999) and features two N-terminal GAF domains. In contrast to PDE2, cGMP binds only to GAF-A in PDE5 (Zoraghi et al. 2005) stimulating enzyme activity about 10-fold; this implies that PDE5 is inactive when GAF-A is not occupied by cGMP (Rybalkin et al. 2003). PDE5 is phosphorylated by PKG at a serine residue close to the GAF-A domain and is believed to stabilize the cGMP–GAF-A interaction by increasing the affinity of cGMP for GAF-A and thereby provide a means for prolonged activation of PDE5 (Corbin et al. 2000; Francis et al. 2002; Shimizu-Albergine et al. 2003). Only a single PDE5 gene has thus far been identified that encodes PDE5; however at least three variants (PDE5A1, PDE5A2, PDE5A3) can be generated from differentially regulated promoters that differ at their N-termini (Kotera et al. 1999; Loughney et al. 1998; Stacey et al. 1998; Yanaka et al. 1998; Lin et al. 2000a,b). Presumably, these promoters allow for differential control of PDE5 gene expression.

Tissue distribution and selective inhibitors PDE5 isoenzymes are widely distributed. Significant amounts are present in vascular (Lin et al. 2006) and airway smooth muscle (see Souness & Giembycz 1994), T lymphocytes (Tenor et al. 1995b; Giembycz et al. 1996), platelets (Hidaka & Endo 1984) and airways epithelial cells (Rousseau et al. 1994; Wright et al. 1998; Fuhrmann et al. 1999). Traces of this enzyme have also been detected in alveolar macrophages (Tenor et al. 1995a), vascular endothelial cells (Souness et al. 1990; Suttorp et al. 1993) mast cells and basophils (Peachell et al. 1992). The serendipitous discovery of sildenafil for the treatment of erectile dysfunction has led to the description of several new and potent PDE5 inhibitors including vardenafil and tadalafil, some of which have, in humans, a significantly longer half-life than sildenafil (Gupta et al. 2005).

Therapeutic implications From a therapeutic standpoint, selective inhibitors of the PDE5 isoenzyme family are not obvious candidates for having a major impact on allergic inflammation, although a modest degree of bronchodilatation may be elicited and afford some benefit in disease states characterized by reversible airflow limitation such as allergic asthma (see Murray 1993; Souness & Giembycz 1994). However, recent data indicate that PDE5 inhibitors can indeed suppress pulmonary inflammation and airways hyperreactivity following allergen challenge, at least in animal models of asthma (Toward et al. 2004). Moreover, there is emerging evidence that PDE5 may also play a major role in the vasculature regulating mitogenesis and angiogenesis (Ladha et al. 2005). These processes are involved in the development of airways remodeling (Wilson

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& Hii 2006) and, accordingly, may provide a viable therapeutic target for selective PDE5 inhibitors or drugs that inhibit more than a single PDE isoenzyme family.

Phosphodiesterase 7 In 1993, Micheali et al. reported the development of a highly sensitive functional screen for the isolation of cDNAs that encode cAMP PDEs. Using this technology three groups of cDNAs were isolated from a human glioblastoma cDNA library. Two of those genes were closely related to the Drosophila “dunce” cAMP PDE (i.e., PDE4-like), while the third encoded an enzyme that readily degraded cAMP but with characteristics distinct from all other known PDEs. At that time, the new PDE was given the name high affinity cAMP-specific phosphodiesterase 1 (HCP-1). Primary sequence analysis established that HCP-1 shared significant homology with a stretch of 280 C-terminal amino acids that constitute the catalytic domain of other mammalian cAMP PDEs (Michaeli et al. 1993). Within this highly conserved region HCP-1 exhibited the highest and lowest degree of homology to PDE4 (35% identity; 51% similarity) and PDE2 (24% identity; 37% similarity) family members respectively. However, given that the homology in the catalytic core between PDE4 variants varies between 85 and 95% and that cAMP hydrolysis by HCP-1 was insensitive to the selective PDE4 inhibitors, Ro-20,1724 and rolipram, it was concluded that the novel enzyme represented the first member of a previously unknown cAMP PDE family and was designated PDE7 (Michaeli et al. 1993). It is noteworthy that the discovery of PDE7 coincided with a report published at the same time of an atypical cAMP hydrolytic activity (called JK-21) in several human T-cell lines including Jurkat, MOLT-4, HPB-ALL and HUT-78 (Ichimura & Kase 1993), which is now known to be a PDE7 family member (Bloom & Beavo 1996). Two genes (PDE7A, PDE7B) have been identified in the mouse, rat and human that encode PDE7 isoenzymes (Michaeli et al. 1993; Hoffmann et al. 1998a; Hetman et al. 2000; Sasaki et al. 2000, 2002). However, it is only products derived from PDE7A, which encodes three variants (PDE7A1, PDE7A2, PDE7A3), that are potential targets for antiallergic drugs. Translation in humans of PDE7A1, PDE7A2 and PDE7A3 mRNA transcripts yields proteins that are composed of 482, 456, and 424 amino acids respectively. On SDS polyacrylamide gels the three splice variants migrate as 55–57 kDa (7A1), 50–52 kDa (7A2) and 50 kDa (7A3) proteins, which is similar to the masses predicted from their amino acid composition. The N-terminus of PDE7A1 and PDE7A3 is rich in proline, serine and positively charged amino acids whereas the same region of PDE7A2 is hydrophobic (Bloom & Beavo 1996; Han et al. 1997; Glavas et al. 2001) and features potential myristoylation (G2) and palmitoylation (C8) sites that may dictate intracellular localization. Indeed, following subcellular fractionation of a variety of tissues PDE7A2 has been found

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only in the particulate fraction, consistent with the hydrophobicity of its N-terminus; in contrast PDE7A1 is predominantly cytosolic (Bloom & Beavo 1996; Han et al. 1997). The subcellular distribution of PDE7A3 has not been investigated but it shares the same N-terminus as PDE7A1 and could localize to soluble cellular structures.

Tissue distribution and selective inhibitors PDE7A is widely expressed in the lungs and across a variety of immune and proinflammatory cells (Table 27.5) (Miro et al. 2001; Smith et al. 2003; Barber et al. 2004). Accordingly, it seems likely that this enzyme family may play an important role in regulating many cAMP-dependent processes related to the immune system. Indeed, all human proinflammatory and immune cells (primary and cells lines) that have been studied express mRNA for PDE7A1 and PDE7A2 at an approximate ratio of 4 : 1. However, irrespective of the method used for detection (i.e., Western blotting, immunocytochemistry, confocal microscopy) PDE7A2 has never been found at the protein level despite unequivocal identification of PCR products corresponding to this transcript. This finding is not peculiar to PDE7A. In human mononuclear cells several of the PDE4D splice variants are expressed only at the mRNA level (Giembycz et al. 1996; Nemoz et al. 1996). Whether this is due to translational repression or a low translation rate of these particular mRNA transcripts, instability of the active enzymes or to the fact that the proteins are expressed at very low but nevertheless functional levels is unexplored. Of the immunocompetent/proinflammatory cells that have been studied, CD4+ and CD8+ T lymphocytes express relatively high levels of PDE7A1 that are readily detected by RT-PCR and Western blotting (Table 27.5) (Giembycz et al. 1996; Seybold et al. 1998; Secchiero et al. 2000; Smith et al. 2003). Human airway smooth muscle cells, blood monocytes and lung macrophages together with several cell lines including HUT-78 (T-cell) and BEAS-2B (epithelial) are also PDE7A1+ under the same experimental conditions (Table 27.5) (Smith et al. 2003). In contrast, Western blotting has failed to detect PDE7A1 in human neutrophils although the protein can clearly be labeled using immunoconfocal laser microscopy (Smith et al. 2003). This more sensitive technique has also been employed to determine the expression profile of PDE7A in cells such as macrophages present in sputum and bronchoalveolar lavage fluid. The inability to detectable PDE7A1 in neutrophils by western blotting suggests that the concentration of this enzyme is lower than that found in the other cell types examined. Although immunoconfocal laser microscopy cannot distinguish between the PDE7A splice variants, much of the staining is cytosolic indicating that PDE7A1 (and/or PDE7A3 see below) may be the most abundant isoform expressed. Very little is known about PDE7A3. Studies by (Glavas et al. 2001) identified this splice variant in human CD4+ T lymphocytes after costimulation with antiCD3/CD28 anti-

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Table 27.5 Expression of PDE7A1 and PDE7A2 in human proinflammatory and immune cells. mRNA

Protein

Cell type

HSPDE7A1

HSPDE7A2

HSPDE7A1

HSPDE7A2

Human primary cells CD4+ T lymphocyte CD8+ T lymphocyte B lymphocyte Neutrophil Alveolar macrophage Monocyte Eosinophil Bronchial epithelial cell Lung mast cell Lung basophil Airway smooth muscle cell Vascular smooth muscle cell Vascular endothelial cell

+ + +* + + + ND +* +* +* + + +

+ +

+ + + +* +* + + ND − − + + ND

− − ND

− − ND

Human cell lines HUT-78 (T cell) Jurkat (T cell) 16-HBE14 (epithelial cell) NCIH292 (epithelial cell) BEAS-2B (epithelial cell) U-937 (monocyte) MRC-5 (fibroblast) AML14.3D10 (eosinophil) Jiyoye (B cell)

+ + + +* + + + + +

+ + + ND + + + + −

− − − ND − − − − ND

+ + + ND

+ + + + + + + + + + ND

− −

+, enzyme detected; −, enzyme not detected; ND, not determined; *, isoenzyme not determined. Data taken from Smith et al. (2003).

bodies but its distribution in other cells is unknown. Given that PDE7A3 and PDE7A1 are probably regulated by the same promoter (Torras-Llort & Azorin 2003) it is possible that the expression pattern of both transcripts is similar. PDE7A encodes cAMP-specific PDEs that are insensitive (IC50 > 100 μmol/L) to cGMP and standard inhibitors of PDE2, PDE3, PDE4 and PDE5 (Michaeli et al. 1993; Hetman et al. 2000; Richter et al. 2002; Smith et al. 2004), although it is inhibited by the generally nonselective compound 3-isobutyl1-methylxanthine (IBMX) with an IC50 of 5–53 μmol/L adopted across experiments (Wang et al. 2000; Lee et al. 2002; Richter et al. 2002; Smith et al. 2004). Despite the discovery of PDE7A in 1993, there were until 2001 surprisingly few reports of selective inhibitors. However, examples of compounds with PDE7 inhibitory activity have emerged and some of these have acceptable selectivity for both in vitro and in vivo studies (reviewed in Vergne et al. 2005; Giembycz & Smith 2006a,b). For example, Smith et al. (2004) reported on a sulphonamide derivative, BRL 50481 (see 16 in Fig. 27.7) that is > 200-fold selective for hrPDE7A1 expressed in baculovirus-infected Sf9 cells over PDEs 1 to 5. BRL 50481 is

a purely competitive inhibitor (with respect to substrate) of the enzyme with a Ki of 180 nmol/L. Another PDE7 inhibitor based on the 1,3,4-thiadiazole template is the Pfizer compound PF 332040 (see 16 in Fig. 27.7), which is at least 600-fold selective over other PDE isoenzyme families. To date, none of the compounds described are reported to discriminate between PDE7A and PDE7B (Bernardelli et al. 2004; Lorthiois et al. 2004; Vergne et al. 2004a,b).

Me Me

O S

N Me

O H 2N

N N S

O

N+ O

N

OH

−

O

15 BRL 50481

16 PF332040

Fig. 27.7 Chemical structure of two novel and selective inhibitors of PDE7. See text and Giembycz & Smith (2006a) for further details.

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Therapeutic indications The advent of selective PDE7 inhibitors has allowed an assessment of the functional role of PDE7. In the context of allergy, excitement in this area initially was fuelled by a report published in 1999 that IL-2 production by, and the subsequent proliferation of, antiCD3/antiCD28-stimulated human T lymphocytes was associated with induction of PDE7A1. Significantly, delivery to these cells of antisense oligonucleotides directed against PDE7A mRNA transcripts prevented these responses in a PKA-sensitive manner (Li et al. 1999; Glavas et al. 2001). The results described by Li et al. (1999) are supported by studies conducted in mice deficient in the PDE7A gene (Michaeli 2004). Thus, the ability of antiCD3 antibodies to promote the proliferation of splenocytes and purified T lymphocytes was reduced in PDE7A–/– mice by 66% and 60% respectively relative to their wild-type counterparts; in contrast, cell proliferation evoked by a combination of ionomycin and phorbol ester was unaffected by PDE7A gene deletion (Michaeli 2004). Similarly, the number of IFNγ +/ CD3+ T cells harvested from mice lacking the PDE7A gene and their ability to release interferon (IFN)-γ in response to antiCD3 antibodies both in vitro and ex vivo was significantly lower when compared to wild-type mice. Taken together these data implicate PDE7A in the regulation of murine T-cell proliferation and Th1 cytokine generation. However, subsequent investigations with small molecule inhibitors that are selective for PDE7A have not corroborated these findings. Thus, BRL 50481 (15 in Fig. 27.7) had no effect on IL-15-induced proliferation of human CD8+ T lymphocytes (Smith et al. 2004). Similarly, in another study where PDE7A–/– mice were used, T-cell proliferation and Th1 (IL-2, IFN-γ, TNF-α) and Th2 (IL-4, IL-5, IL-13) cytokine production evoked by ligation of CD3/CD28 was preserved (Yang et al. 2003). The same result was found using wild type mice treated with BMS-586353, a highly potent PDE7 inhibitor (Yang et al. 2003). It is unclear why the results of Smith, Yang and their respective colleagues do not concur with data reported by Li et al. (1999) or Michaeli (2004) but they are

O2N

O

NH

unlikely to be species related or to a redundant mechanism in mice that compensates for the deficiency in PDE7A. Two additional possibilities may account for some of the discrepancies. In the study reported by Smith et al. (2004), CD8+ T lymphocytes were isolated from other leukocytes by negative immunoselection using a mixture of antibodies against CD11b, CD16, CD19, CD36, CD56 and CD4. Although the same methodology was used by Li et al. (1999) antibodies against CD25 and HLA-DR were also employed, which will remove all activated and proliferating T cells. Thus, it is possible that naive T cells are regulated differently by PDE7A when compared to their activated and proliferating counterparts. Alternatively, the use of naked antisense oligonucleotides, as used by Li et al. (1999), may not have targeted specifically the mRNA of interest or, alternatively, evoked toxic effects that were sequence nonspecific (Stein 2001). The divergence in data between the two studies conducted with PDE7 –/– mice currently is inexplicable. Of potential interest is the report by Smith and colleagues in 2004 that the culture of human monocytes in RPMI-1640 for 24 hours resulted in upregulation of PDE7A1 and conferred functional sensitivity to BRL 50481, i.e., lipopolysaccharide (LPS)-induced TNF-α release was significantly inhibited (Smith et al. 2004). Moreover, in monocytes in which PDE7A1 was upregulated, the inhibition of TNF-α release evoked by rolipram and other cAMP-elevating agents was enhanced in a purely additive manner. These data imply that PDE7A inhibitors alone may regulate the responsiveness of monocytes and possibility other proinflammatory and immune cells under circumstances when PDE7A is highly expressed such as in chronic inflammation. In this respect many chemokines and cytokines relevant to the pathogenesis of chronic inflammatory diseases signal, in part, through a PKC-dependent mechanism (Kontny et al. 2000; New & Wong 2003; Johnson et al. 2004) and it is known that the human PDE7A1 promoter is activated by phorbol esters (Torras-Llort & Azorin 2003). Although inhibition of PDE7A has no demonstrable antiinflammatory activity under normal conditions, PDE7A–/– mice respond to immunization (with keyhole limpet hemocyanin) with a significantly enhanced antibody response when compared to wild-type animals. Thus, PDE7A may play a central role in cAMP/PKA signaling processes that are unrelated to T-cell activation (Yang et al. 2003) such as B-cell function.

Phosphodiesterase 8 F N NH O 17 Fig. 27.8 Chemical structure of a selective inhibitor of PDE3B. See text and Edmondson et al. (2003) for further details.

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The PDE8 isoenzyme family is composed of two genes, PDE8A and PDE8B, that encode predominantly cytosolic proteins (Gamanuma et al. 2003) that hydrolyze, almost exclusively, cAMP (Fisher et al. 1998; Hayashi et al. 1998) (Table 27.3). Multiple isoforms can be derived from PDE8A and PDE8B (5 and 4 variants respectively) that arise through alternative splicing and/or start codon usage (Wang et al. 2001; Hayashi et al. 2002). Putative regulatory domains of unknown func-

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tion have been identified towards the N-terminus of these enzymes including a “receiver” and a PAS domain. In other proteins PAS domains represent sites for ligand binding and protein–protein interaction but, thus far, no ligands have been identified that interact with the PAS domain on PDE8. In the context of allergy, PDE8A1 has been identified in human primary T-lymphocytes and T-cell lines (Glavas et al. 2001) and evidence, albeit circumstantial in nature, suggests that inhibition of PDE8 can attenuate T-cell chemotaxis (Dong et al. 2006). However, no selective PDE8 inhibitors have been described with which to more formally address this possibility.

Theophylline, hybrid and nonselective PDE inhibitors The nonselective PDE inhibitor, theophylline, was originally identified by Kossel in Berlin in 1888 and was synthesized 12 years later by Boehringer. However, more than two decades elapsed before the bronchodilator activity of theophylline was realized (Schultze-Werninghaus & Meier-Sydow 1982) and a further 56 years before a possible mechanism of action was suggested (Butcher & Sutherland 1962). Since the early 1950s, theophylline and some related alkylxanthines have been used widely in the treatment of asthma (and latterly, COPD) although recent international guidelines recommend that these drugs be relegated to third-line therapy for both indications. The primary use of theophylline is as a bronchodilator and its mechanism of action is due primarily to inhibition of PDE3 in airways smooth muscle cells (Cortijo et al. 1993). Indeed, in vitro studies have found that the EC50 of theophylline for the relaxation of human isolated tracheal smooth muscle is ∼ 70 μmol/L (Finney et al. 1985; Goldie et al. 1986; Cortijo et al. 1993), which equates to a plasma concentration of 32 μg/mL assuming that 60% of the drug is bound to plasma proteins (Guillot et al. 1984). Theophylline has also been reported to suppress a number of inflammatory indices associated with respiratory diseases (Pauwels et al. 1985; Mapp et al. 1987; Ward et al. 1993; Sullivan et al. 1994; Jaffar et al. 1996; Kraft et al. 1996; Lim et al. 2001; Culpitt et al. 2002) at low doses (5–10 μg/mL) that produce only a modest but nevertheless physiologically significant inhibition of PDE (see Dent & Rabe 1996 and Clinical Pharmacology). Based on these data it is tempting to speculate that second generation, nonxanthine PDE inhibitors that are devoid of activity at adenosine receptors with broader PDE specificity may have superior therapeutic activity over theophylline. Improved activity may be realized for several reasons. First, it is well established that inhibitors of PDE4 can act synergistically in many proinflammatory and immune cells with compounds that block PDE3 (Robicsek et al. 1991; Schudt et al. 1995; Giembycz et al. 1996) or PDE7 (Smith et al. 2004).

Indeed, in the context of asthma, T lymphocytes, macrophages, monocytes, endothelial and endothelial cells are sources of PDE3, PDE4 and PDE7 (Torphy 1998; Smith et al. 2003). Functionally, in vitro studies have shown that while PDE3 inhibitors generally have little or no effect themselves on T-cell proliferation or on IL-2 generation, they significantly enhance the antimitogenic effect of PDE4 inhibitors (Robicsek et al. 1991; Giembycz et al. 1996). Similar results have been reported for TNF-α release from human alveolar macrophages (Schudt et al. 1995). Concurrent inhibition of PDE4 and PDE7 may also promote a superior antiinflammatory effect than a PDE4 inhibitor alone. Thus, while inhibition of PDE7A with BRL 50481 does not attenuate the proliferation of T cells per se, it significantly augments the antimitogenic and cAMP-elevating activity of rolipram (Smith et al. 2004). The suppression by PDE4 inhibitors of TNF-α release from LPS-stimulated human blood monocytes and lung macrophages is also significantly enhanced by BRL 50481 (Smith et al. 2004). Taken together, these in vitro data imply that a nonselective compound may be more efficacious than a PDE4 inhibitor administered as a monotherapy. Moreover, because of synergy, lower doses of hybrid inhibitors may be efficacious such that adverse events attributed to the inhibition of PDE4 in nontarget tissues are reduced (see section on Adverse Events). Second, regardless of synergy, concurrent inhibition of multiple PDEs would be expected to exert clinically relevant effects (not induced by PDE4 inhibition alone) on other processes that contribute to the pathogenesis of asthma including airways/vascular remodeling (PDE1 and PDE5 regulated), endothelial cell permeability (PDE2 regulated), mast cell stabilization (PDE3 mediated) and airways smooth muscle tone (PDE3 regulated).

Antiinflammatory effects of PDE inhibitors in vivo The actions of theophylline and isoenzyme-selective PDE inhibitors upon the acute (IgE-mediated) or chronic (proinflammatory/immunocompetent cell-mediated) consequences of allergen provocation in vivo have, in general, been documented in some detail and include studies of passive cutaneous anaphylaxis (PCA), cell infiltration into sites of inflammation and microvascular leakage.

IgE-mediated processes It seems unlikely that PDE inhibitors of any isoenzyme family reduce IgE levels in vivo. In humans, it is reported that ibudilast, which is an essentially nonselective PDE inhibitor (Souness et al. 1994) is without effect on IgE levels in asthmatic individuals (Kawasaki et al. 1992). Similarly, the elevated level of total serum IgE in a murine model of dermatitis caused by the repeated application of 2,4,6-trinitro-1chlorobenzene is unaffected by oral administration of the PDE4 inhibitor rolipram (Harada et al. 2006). In contrast, PDE3 and PDE4 inhibitors exhibit efficacy at reducing PCA reactions in rats, mice and guinea-pigs (Davies

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& Evans 1973; Broughton et al. 1975). Furthermore, rolipram, but neither zaprinast nor SK&F 94120, is effective at reducing the infiltration of indium-labeled eosinophils into the skin of guinea-pigs following a PCA reaction (Teixeira et al. 1994). Collectively, these data imply that PDE4 inhibitors can suppress the degranulation of IgE-bearing leukocytes and, therefore, allergen-induced mediator release. Further support for this proposal derives from studies in sensitized guineapigs where rolipram, administered intravenously, inhibits antigen- but not leukotriene (LT)D4-induced bronchoconstriction (Howell et al. 1993). Thus, rolipram preferentially exerts an inhibitory influence at the level of mast cells and basophils rather than exerting a direct antispasmogenic action at the level of airways smooth muscle. In contrast, PDE3 inhibitors including CI-928 inhibit both allergen- and LTD4induced bronchoconstriction under identical conditions indicating a direct smooth muscle effect of these compounds (Howell et al. 1993).

after chronic (6 days) dosing (Sanjar et al. 1990a,b). Studies conducted more recently with rolipram and second generation PDE4 inhibitors including cilomilast, roflumilast and AWD 12-281 have corroborated these findings both in the airways an in allergic cutaneous reactions (Underwood et al. 1993, 1994, 1998; Griswold et al. 1998; Bundschuh et al. 2001; Baumer et al. 2002, 2003; Kuss et al. 2003; Draheim et al. 2004; Hoppmann et al. 2005). Similarly, treatment of sensitized cynomolgus monkeys with rolipram abrogates the pulmonary neutrophilia and eosinophilia, and airways hyperresponsiveness after multiple exposures to the antigen but is without effect on the immediate increase in airways resistance that follows acute antigen provocation (Turner, C.R. et al. 1994). Thus, these data are consistent with the findings of that PDE4 inhibitors may be antiinflammatory and act primarily to prevent the activation of immune cells in the lung rather than by exerting a antispasmogenic or spasmolytic effect the level of airways smooth muscle (Howell et al. 1993).

Proinflammatory cell infiltration Intravenous injection of a low dose of theophylline in to a guinea-pig model of allergic asthma immediately prior to antigen prevents both the immediate bronchoconstriction and the late-phase reaction (LPR) (Andersson et al. 1985). Qualitatively identical results were obtained in an allergic sheep and rabbit model (Ali et al. 1992). Independent experiments conducted with sensitized guinea-pigs and rats have demonstrated that theophylline suppresses pulmonary eosinophil and, in the case of the rat, also neutrophil recruitment which is believed to be intimately associated with the development of the LPR (Sanjar et al. 1990b; Gristwood et al. 1991; Tarayre et al. 1991a,b, 1992; Lagente et al. 1994; Manzini et al. 1993). In many of these studies, however, theophylline was administered acutely as a single dose that is significantly higher than that used therapeutically indicating that the observed effects may not be relevant to the clinical situation. In contrast, chronic treatment of guinea-pigs for seven days with theophylline effectively prevents plateletactivating factor (PAF)- and allergen-induced eosinophil recruitment at therapeutically-relevant doses (Sanjar et al. 1990a,b). A large number of investigations (too many to describe individually herein) have evaluated the effect of PDE inhibitors upon the infiltration of proinflammatory cells into the airways lumen, skin and eye of guinea-pigs, rats and monkeys in responses to a variety of dissimilar stimuli including allergen. For example, pretreatment of sensitized guinea-pigs with zardaverine, a mixed PDE3/4 inhibitor, markedly suppressed allergen-induced infiltration of eosinophils, macrophages and neutrophils into the bronchoalveolar lavage (BAL) fluid to a level achieved with dexamethasone (Schudt et al. 1991b). Qualitatively identical data have been reported for the PDE3/4 inhibitor, benafentrine, on PAF- and allergeninduced pulmonary eosinophil recruitment in guinea-pigs

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Microvascular leakage and edema It is well recognized that the microcirculation plays an important role in inflammatory reactions. Under normal conditions, the endothelium lining the postcapillary microvenules in the skin and bronchial circulation (capillaries in the pulmonary vasculature) is largely impermeable to blood cells and to macromolecules but following a proinflammatory insult, localized arteriolar vasodilatation occurs with a consequent increase in blood flow. This effect is induced by the liberation of proinflammatory mediators such as histamine and tachykinins. An increase in capillary and microvenular pressure then ensues together with the liberation of other mediators such LTD4, which contract directly the microvenular endothelial cells. Together these effects, by increasing microvenular permeability, permit the loss of plasmaproteins from the vascular compartment. Furthermore, the resulting increase in osmotic pressure due to loss of solute from the circulation leads to marked fluid exudation and to oedema. In guinea-pigs, theophylline reduces plasma exudation in to the trachea, bronchi and BAL fluid but its efficacy may depend upon the nature and strength of the leak-evoking stimulus (Boschetto et al. 1989; Erjefalt & Persson 1991; Raeburn & Karlsson 1993; Raeburn & Woodman 1994). With respect to selective drugs intravenous, oral and intratracheal administration of representative inhibitors of the PDE4 and PDE5 isoenzyme families markedly attenuate PAF-induced microvascular leakage in both small and large airways, and into the BAL fluid of anesthetized guinea-pigs (Ortiz et al. 1992; Raeburn & Karlsson 1993; Raeburn et al. 1994). The finding that rolipram and zaprinast are active when given directly in to the airways indicates an important local action in the lung and highlights that systemic administration is not necessary for these compounds to exert an antiinflammatory influence. This is an important observation

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since the administration of PDE inhibitors by the inhaled route should reduce untoward side effects while maintaining efficacy. Drugs which inhibit PDE1 and PDE3 do not inhibit PAF-induced microvascular leakage in guinea-pig airways (Ortiz et al. 1992; Raeburn & Karlsson 1993). This latter finding is curious given that a PDE3 isoenzyme has been identified in endothelial cells, and that a selective PDE3 inhibitor, motapizone, blocks the increase in permeability elicited by H2O2 in vitro (Suttorp et al. 1993). It is likely, however, that this discrepancy is due to a difference in species, the leak-evoking stimulus or in vessel type. In keeping with this latter possibility, Teixeira et al. (1994) have reported that rolipram does not inhibit edema formation in guinea-pig skin in response to histamine, zymosan-activated plasma and PAF (cf. guinea-pig lung). It is tempting to speculate that the complement of PDE isoenzymes and/or the regulation of endothelial cell contractility by cyclic nucleotides varies significantly between vessels of the pulmonary and systemic vascular beds. Svensjo et al. (1993) reported that PDE4 and PDE3/4 inhibitors attenuate the increase in microvascular permeability evoked by bradykinin in the hamster cheek pouch. Qualitatively identical results are obtained for the effect of rolipram and denbufylline upon arachidonate-induced edema in rodents (Crummey et al. 1987).

Clinical pharmacology Theophylline Until relatively recently, the therapeutic efficacy of theophylline in asthma was attributed to its weak bronchodilator activity. However, while theophylline is an effective bronchodilator, there is now increasing evidence that this drug may also exert an immunomodulatory action at plasma concentrations that do not affect airway smooth muscle tone (reviewed in Spina 2003). Several lines of investigation have lead to this conclusion. In essentially all studies, theophylline protects against the LPR following allergen provocation implying that the emigration of proinflammatory and immunocompetent cells from the circulation in to the lung and/or their subsequent activation is suppressed. Indeed, suppression of the LPR at nonbronchodilator doses (< 10 mg/L) of theophylline is associated with a reduction in the typical increase in CD4+ and CD8+ T lymphocytes. Theophylline also suppresses the activity of other proinflammatory and immune cells ex vivo including neutrophils and macrophages that is positively correlated with the concentration of theophylline measured in the BAL fluid. Similar experiments have demonstrated that the number of activated eosinophils and CD4+ T cells are reduced in allergic subjects given low dose theophylline and that eosinophil accumulation in bronchial tissue in patients with atopic asthma is also reduced. All these changes are mirrored by improvements in lung function.

PDE4 inhibitors PDE4 inhibitors have been shown to be effective in patients with exercise-induced asthma. Nieman et al. (1998) have reported the results of a randomized, placebo-controlled, double blind crossover trial with cilomilast in 27 patients with exercise-induced asthma. Subjects were randomized to receive cilomilast or placebo for 7 days followed by a 7 day washout and then the alternative treatment for 7 days. The primary efficacy variable was the maximum percentage decrease (MPD) in forced expiratory volume in 1 s (FEV1) in response to exercise. In the placebo group the mean fall in FEV1 after exercise was 32.9%, which was significantly greater than the deterioration in lung function seen when the same subjects that received a single dose of cilomilast (23.6% reduction in FEV1). The improvement in lung function was incremental such that after 7 days of therapy the MPD in FEV1 was further reduced to 21.8% (Nieman et al. 1998). There were also improvements in the MPD in peak expiratory flow rate (PEFR), time to recovery after exercise, and percent protection against exercise-induced bronchoconstriction (Nieman et al. 1998). Similar data have been obtained with roflumilast. Sixteen patients with exercise-induced asthma were recruited in to a placebo-controlled, randomized, double-blind, two-period crossover study where placebo or roflumilast (500 μg o.d.) was administered in random order for 28 days (Timmer et al. 2002). FEV1 was measured before and repeatedly up to 12 min after the end of the exercise challenge. Blood was taken for determination of LPS-induced TNF-α released ex vivo as a surrogate marker of inflammatory cell activation. At the end of the study the mean fall in FEV1 in response to exercise was significantly reduced (by 41%) when compared to placebo (Timmer et al. 2002). Similarly, the median TNF-α concentration was reduced by 21% during roflumilast treatment whereas it remained constant under placebo (Timmer et al. 2002), indicating that an antiinflammatory plasma concentration of the drug had been achieved. Other trials of cilomilast in asthma have yielded equivocal results. In one of these, the results of a multicenter, placebocontrolled, double-blind, randomized parallel group study with cilomilast (5, 10 and 15 mg b.i.d. for 6 weeks) involving 283 patients taking inhaled corticosteroids concurrently was reported (Compton et al. 1999). All patients had an FEV1 of approximately 66% of predicted, expressed a 12% or greater responsiveness to salbutamol and had asthma that was inadequately controlled with inhaled corticosteroids (Compton et al. 1999). Two hundred and sixty six patients completed the study. At the highest tolerated dose (15 mg b.i.d.) cilomilast as well as placebo increased FEV1 from week 1 onwards and this effect was greater with the active treatment group (Compton et al. 1999). However, the improvement in lung function failed to reach statistical significance at any time except at week two when the mean difference in trough FEV1 was 210 mL greater than in the placebo treatment group

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(Compton et al. 1999). Improvements, relative to placebo, in forced expiratory flow at 25–75% of forced vital capacity (FEF25 –75) and domiciliary PEFR were also detected but, again, statistical significance was not achieved. However, in the physicians’ global assessment 59% of patients taking cilomilast (15 mg b.i.d.) were rated as “markedly improved” compared to 39% of patients given placebo, which was significant. Similarly, in the patients’ global assessment 69% of patients in the active treatment group (15 mg b.i.d.) indicated that they were “markedly improved” compared to 41% of patients that received placebo (Compton et al. 1999). An international (Germany, UK, France and South Africa), multicenter phase IIb, double-blind, parallel group 12 month efficacy, safety and tolerability study of cilomilast (10 and 15 mg b.i.d.) has also been evaluated in 211 asthmatic patients between 19 and 70 years of age, which was an extension of three double-blind randomized phase II studies of 4–6 weeks’ duration (Compton et al. 2000a,b). One hundred and fifty eight patients received cilomilast and the remainder were given placebo. Inclusion criteria included a history of episodic wheezing for at least 6 months, an FEV1 ≥ 45% and ≤ 90% of predicted for height, sex and weight, and responsiveness to salbutamol (≥ 12%) at time of screening. Clinically relevant and statistically significant improvements above placebo were seen in forced vital capacity (FVC) and PEFR, which were sustained from week one to the end of month 12. A consistent improvement in FEV1 was also noted but this did not reach statistical significance. Diary asthma symptom scores also indicated a reduction in cough, wheeze, and breathlessness/chest tightness in the active treatment group when compared to placebo (Compton et al. 2000a). More encouraging data have been obtained with roflumilast. In particular, a trial in 23 subjects with mild asthma showed that roflumilast (250 or 500 μg od) given for 7–10 days reduced the early and late asthmatic reactions evoked by allergen challenge (van Schalkwyk et al. 2005). These are potentially exciting data as the suppression of LPR (43% in the 500 μg treatment group) indicates that the drug may have exerted a clinically relevant antiinflammatory effect. Indeed, in many clinical trials of oral PDE4 inhibitors, a maximum level of lung function improvement is achieved within the first 2–4 weeks of treatment implicating a nonbronchodilator mode of action. Further studies have also found that roflumilast improved FEV1 in subjects with mild to moderate asthma (Bateman et al. 2006) and also reduced allergen-induced AHR to histamine (Louw et al. 2006). One study has also found that roflumilast is as effective as an inhaled corticosteroid at improving lung function (Bousquet et al. 2006). In this study 499 asthmatic patients were randomized to receive either roflumilast (500 μg od) or beclomethasone dipropionate (200 μg b.i.d.) through a metered-dose inhaler for 12 weeks. At the end of the study, both treatment groups had significant and comparable improvements in FEV1, FVC and PEFR; asthma symptoms and β2-agonist use were also reduced.

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Finally, roflumilast has been shown to be effective in the treatment of allergic rhinitis (Schmidt et al. 2001), which supports the idea that PDE4 inhibitors may have utility in treating a variety of allergic (and nonallergic) inflammatory disorders.

Theophylline and PDE4 inhibitors: a comparison of their safety, tolerability, drug metabolism and pharmacokinetics Table 27.6 shows a comparison of the drug metabolism, pharmacokinetics (DMPK) and clinical safety of theophylline and PDE4 inhibitors for which information is available. Perhaps the most striking difference is in the pharmacokinetics, which has implications for patient compliance and the extent to which the plasma concentration requires monitoring. At bronchodilator doses, intra- and inter-subject variability to theophylline together with a low therapeutic ratio poses a significant clinical problem requiring careful titration with routine plasma monitoring to avoid serious cardiac and CNS side effects (Vignola 2004). This is a particular problem with smokers as polycyclic aromatic hydrocarbons present in vapor phase of cigarette smoke are known to induce drug-metabolizing enzymes including CYP1A1 and CYP1A2 (Campbell et al. 1987; Shimada et al. 1989; Vistisen et al. 1992). As theophylline is principally metabolized by CYP1A2 (Fuhr et al. 1992; Sarkar et al. 1992), dose adjustments are often necessary to compensate for the increased clearance in cigarette smokers (Hunt et al. 1976; Jusko et al. 1978; Jusko 1979). Age is another factor that has a marked effect on the pharmacokinetics of theophylline. Indeed, the clearance of theophylline decreases 15–28% in the elderly when compared to young adults, which probably reflects a decrease in the elimination of theophylline by CYP1A2 (Antal et al. 1981; Shin et al. 1988; Ohnishi et al. 2003). In contrast, the pharmacokinetics of cilomilast and roflumilast are linear providing dose-proportional systemic exposure that is essentially unaffected by age and cigarette smoking status indicating that no dose adjustments will be necessary in elderly smoking subjects with COPD (see Giembycz 2001, 2002b; Vignola 2004). Despite producing bronchodilatation, theophylline is prone to cause adverse events. These are particularly pronounced when dosed to give plasma concentrations of 20 mg/L or greater, although these unwanted actions can be offset by gradually titrating the dose of theophylline until therapeutic levels are achieved (Barnes 2003). In contrast, a major benefit of the PDE4 inhibitors in clinical trials is their superior safety and tolerability profile over theophylline. Although nausea and vomiting are not uncommon with PDE4 inhibitors, this is usually of moderate severity and is reported to be selflimiting (Compton et al. 2001). Moreover, PDE4 inhibitors are generally well tolerated in both short- and long-term dosing trials with a low incidence of adverse events; they have no action at adenosine receptors and, with the exception

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Table 27.6 Comparison of the mechanism of action, DMPK, safety and tolerability of theophylline and PDE4 inhibitors. Theophylline

PDE4 inhibitors*

Unclear (inhibition of PI3K-d; histone acetylation status) PDE inhibition only at high (> 20 mg/L) doses

Selective inhibition of PDE4

Nonlinear Significant intersubject variability affected by age, smoking status, and concomitant medication necessitating plasma monitoring

Linear providing dose proportional systemic exposure Low intersubject variability: no plasma monitoring required

Absorption

Variable, depends on formulation

Oral formulations (o.d. or b.i.d) tmax ∼ 1–2 hours

Bioavailability

Variable, depends on formulation

> 80%, unaffected by food or antacids

Half-life

7–9 hours

7–16 hours depending on inhibitor

Volume of distribution

500 mL/kg Plasma protein binding ∼ 56%

Low High

Clearance

∼ 400 mL/kg per hour Affected by genetic factors, cigarette smoking, coexisting pathology, and drugs that affect hepatic metabolism

Low

Metabolism

∼ 90% metabolized by liver (CYP1A2)

Negligible first-pass hepatic metabolism

Drug interactions

High potential for drug interactions including propafenone, mexiletine, enoxacin, ciprofloxacin, cimetidine, propranolol, oral contraceptives, erythromycin, rifampicin, phenytoin, carbamazepine, phenobarbital, isoproterenol, tobacco smoke

Low potential for drug interactions Can be taken with other drugs prescribed for asthma and COPD

Excretion

10% excreted unchanged via the kidneys

Depends on inhibitor

Dosing adjustment

May be required in cigarette smokers, the elderly, individuals with liver disease, and subjects taking concomitant medication Contraindicated in individuals with heart disease, seizure disorders, and gastroesophageal reflux

None except in individuals with moderate hepatic and severe renal impairment Contraindicated in subjects with severe hepatic impairment

Clinical efficacy

Effective in a subset of patients with COPD Nonbronchodilator doses effective in asthma; steroid sparing

Phase III clinical trials ongoing

Safety and tolerability

Serious cardiovascular and CNS side effects Gastrointestinal irritation, nausea, insomnia in 10–15% of patients

No cardiovascular or CNS side effects Headache, nausea, vomiting, arteriopathy(?)

Mechanism of action

DMPK Pharmacokinetics

* Details refer to cilomilast and roflumilast, which are in late Phase III clinical trials. COPD, chronic obstructive pulmonary disease; PI3K, phosphatidylinositol 3-kinase.

of headache, are devoid of adverse cardiovascular activity (see Giembycz 2001, 2002b). Nevertheless, despite these improvements over theophylline, there are still major safety concerns with current second generations PDE4 inhibitors at the doses believed necessary to impart therapeutic benefit (see Adverse events, below). Another significant clinical problem is that theophylline has a high potential for drug interactions (see Giembycz 2001, 2002b). Thus, in addition, to CYP1A2, theophylline is

also metabolized, albeit to a lesser extent, by CYP2E1 and CYP3A4. Accordingly, many drug interactions may occur including all of those indicated in Table 27.6. In contrast, PDE4 inhibitors have in general a far reduced propensity for drug interactions. With respect to cilomilast, none of the metabolic pathways involve, to any great extent, cytochrome P450 enzymes (CYP1A2, CYP2D6, CYP3A4) most susceptible to competitive inhibition by other drugs. Indeed, the only P450 enzyme implicated (CYP2C8), has few other substrates

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or inhibitors. Moreover, cilomilast does not inhibit any important hepatic cytochrome P450 enzymes in vitro. These data are supported by the finding that, at steady state, cilomilast has no clinically meaningful effect on the pharmacokinetics of digoxin, theophylline or prednisolone. Conversely, neither theophylline nor Maalox Plus, an antacid commonly used in the elderly that contains salts of calcium, magnesium and/or aluminum that can alter the absorption or bioavailability of some drugs, had any significantly influence on the pharmacokinetics of cilomilast. Similar results have been reported for roflumilast. Thus, taken together these data demonstrate that the two most clinically advanced PDE4 inhibitors are not contraindicated with commonly prescribed medications for asthma and can be safely coadministered with these drugs.

Adverse events of theophylline and PDE inhibitors Theophylline Despite demonstrable therapeutic benefit in asthma, theophylline has a low therapeutic ratio. This undesirable property is a major cause for concern as adverse events tend to occur when the plasma concentration exceeds 20 μg/mL (110 μmol/L). The most common adverse events include nausea, vomiting, gastrointestinal irritation and headache (probably due to PDE4 inhibition, see below). Other, more serious side effects such as CNS stimulation, diuresis and cardiac arrhythmias occur at higher doses and may be due to the ability of theophylline at act as an adenosine A1 receptor antagonist (Fredholm 1980; Barnes 2003). Higher plasma concentrations still may precipitate convulsions and even death (Barnes 2003).

PDE4 inhibitors Despite some encouraging data from Phase III efficacy studies in asthma and the superior DMPK over theophylline, roflumilast and other PDE4 inhibitors are still hampered by a low therapeutic ratio. This limitation became clear early on in the development of these compounds with nausea, diarrhea, abdominal pain, vomiting and dyspepsia being the most common adverse events reported (reviewed in Giembycz 2001, 2002b) (see Fig. 27.2). For example, the number of subjects failing to complete all controlled trials of cilomilast conducted by GSK due to an adverse event was positively dose-related with gastrointestinal disturbances being the most prevalent (GlaxoSmithKline 2003a). Unfortunately, these unwanted actions, which are mediated both locally (i.e., in the gastrointestinal tract) and centrally, can be accounted for by the ubiquitous distribution of PDE4 isoforms across many tissues, and represent an extension of the pharmacology of PDE4 inhibitors that is typical of first generation compounds such as rolipram.

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Documentation of serious toxicities resulting from the administration of PDE4 inhibitors is relatively sparse (cf. PDE3 inhibitors: Hanton et al. 1995; Zhang et al. 2002). However, the most worrying potential toxicity generic to PDE4 inhibitors is arteritis. This condition is characterized by inflammation, hemorrhage and necrosis of blood vessels, and is believed to be irreversible in animals. Mechanistically, arteritis is thought to result from hemodynamic changes produced by excessive and prolonged vasodilatation of specific vascular beds, although the means by which PDE4 inhibitors cause certain vessels to become targets of inflammation is unknown. In nonhuman primates, studies with PDE4 inhibitors generally have not identified pathologies, including arteritis, similar to those reported in other species used for toxicology (Larson et al. 1996; Robertson et al. 2001), and this has lead to a view that arteriopathies maybe nonprimate-specific. Indeed, rats and dogs may have an increased susceptibility to drug-induced vascular lesions because of the common occurrence of arteriopathies in these species (Bishop 1989; Ruben et al. 1989). Consistent with this hypothesis, cilomilast is reported not to produce vascular lesions in primates unlike comparable studies performed in rodents where medial necrosis of mesenteric arteries is reproducibly precipitated (GlaxoSmithKline 2003c). However, a recent comprehensive toxicologic study found that a PDE4 inhibitor, SCH 351591, produced, in cynomolgus monkeys, acute to chronic inflammation of small to medium sized arteries in many tissues and organs (Losco et al. 2004). These findings of arteriopathy in primates, which were previously thought to be resistant to toxicity, have serious implications for human risk, and it is noteworthy that Merck in 2003 abandoned development of their lead PDE4 inhibitor (licensed from Celltech Group) due to an incidence of colitis, raising the possibility that it was secondary to arteritis (Data Monitor 2003). Moreover, as asthma is a chronic disease requiring, in many subjects with moderate to severe disease, long-term therapy, a wide margin of safety will be needed because toxicity cannot be adequately monitored. The major problem for the physician is that presentation of mesenteric ischemia is vague in humans and diagnostic tools are poor. Indeed, attempts by the pharmaceutical industry to develop biomarkers of arteritis to assist the development of PDE4 inhibitors have, to date, been unsuccessful. However, perhaps some comfort can be derived from the knowledge that no clinically relevant effects have been produced in patients treated for many years with bronchodilator doses of theophylline (which produce medial necrosis of mesenteric vessels in rats) (Collins et al. 1988; Nyska et al. 1998) as well as more selective PDE4 inhibitors including rolipram and denbufylline (GlaxoSmithKline 2003c). Other adverse events generic to PDE4 inhibitors and of potential concern based on margin of safety calculations are testicular toxicity, manifested as degeneration of the epithelium lining the seminiferous tubules, hypertrophy and

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hyperplasia of the adrenal cortex, focal myocardial necrosis, erosion of the gastrointestinal mucosa and squamous cell hyperplasia of the nonglandular stomach, which is indicative of an irritant action of cilomilast on the gut (GlaxoSmithKline 2003a–c). However, it is currently believed that these findings do not have clinical relevance. Indeed, there is no evidence from the Phase II or Phase III trials conducted to date that cilomilast at a dose 15 mg (b.i.d.) produces these adverse effects in humans.

PDE3 inhibitors Although the clinical rationale is clear for developing hybrid PDE3/PDE4 inhibitors, there are also major safety concerns with this approach, not least the potential of these compounds to cause arteriopathies. There is an extensive literature on PDE3 inhibitor-induced arteriopathy in laboratory animals with the splanchnic vessels and coronary arteries of rats and dogs respectively being the most susceptible to lesions (Joseph 2000). Whether clinically meaningful bronchodilatation can ultimately be achieved in humans with an acceptable degree of vasodilatation is largely unexplored but coincident headache is not uncommon with current PDE3 or PDE3/PDE4 inhibitors at bronchodilator doses (Wilsmhurst & Webb-Peploe 1983; Yamashita et al. 1990; Brunnee et al. 1992; Fujimura et al. 1995). Conceivably, airway-selective compounds could be realized through formulation, route of administration or by the exploiting the fact that PDE3 and PDE4 inhibitors may interact synergistically to reduce human airways smooth muscle tone (Torphy et al. 1993a). Selective targeting of either PDE3A or PDE3B could also offer a more novel approach to reduce cardiovascular toxicity. Indeed, when PDE3 heterogeneity was first appreciated, the terms cardiac-type and adipocyte-type PDE3 were used to describe PDE3A and PDE3B respectively. This crude taxonomy is based on the finding that PDE3A is expressed primarily in cardiac and vascular myocytes whereas adipocytes, among other cells, are rich in PDE3B. While differential expression of PDE3 gene expression might lead one to hope that the cardiovascular and bronchodilator actions of PDE3 inhibitors can be separated pharmacologically, human cardiac myocytes and airways smooth muscle cells express mRNA for both isoforms. This raises the question of whether PDE3A and PDE3B mediate nonoverlapping functions in the same tissue. While this issue is unexplored, studies in PDE4A-, PDE4B- and PDE4D-deficient mice have found there to be no functional redundancy suggesting that PDE3A and PDE3B likely mediated discrete responses. Thus, it is possible that the cardiovascular actions of PDE3 inhibitors can be dissociated from their bronchodilator and antiinflammatory activities. Fortuitously, subtype selective compounds are beginning to emerge such as vinylogous amide pyrazolones (17 in Fig. 27.8), which preferentially target PDE3B, that could be employed to assess this possibility (Edmondson et al. 2003).

PDE7 inhibition Although very little is known of the adverse effects that may be produced by inhibiting PDE7 in nontarget tissues, this enzyme family is expressed widely throughout the CNS and could therefore compromise the beneficial activity of selective inhibitors that are able to penetrate the blood–brain barrier (Hoffmann et al. 1998a; Miro et al. 2001; Perez-Torres et al. 2003). In addition, compounds that inhibit both PDE7A and PDE7B, which probably include all of those thus far described, may be of particular concern as high levels of PDE7B mRNA are expressed in the area postrema (and adjacent nucleus tractus solitarius) of the rat brain (Reyes-Irisarri et al. 2005), which lies outside the blood–brain barrier (Miro et al. 2001). If the same holds true in the human brain, then additional caution is warranted as these structures are intimately involved in regulating emesis (Miller & Leslie 1994; Miller 1999) and are targets for the vomiting-inducing effects of PDE4 inhibitors (Robichaud et al. 1999). Thus, PDE7 inhibitors that do not discriminate between the two gene families may evoke the same or similar adverse events that have plagued the development of PDE4 inhibitors. This may be a particular problem with dual PDE4/PDE7 inhibitors if simultaneous inhibition of these enzymes in the area postrema results in a synergistic activation of those mechanisms that promote vomiting.

Concluding remarks Many of the major pharmaceutical companies in the world have developed potent and novel “second generation” PDE4 inhibitors for the treatment of a number of allergic and nonallergic inflammatory disorders (see Table 27.2). There is abundant predictive evidence from preclinical studies that these new PDE4 inhibitors should demonstrate global antiinflammatory activity with an improved and acceptable therapeutic ratio over so-called first-generation compounds (Souness & Rao 1997; Torphy 1998; Torphy et al. 1999; Giembycz 2001). That said, one must be cautious of this interpretation since only the acute effects of PDE4 inhibitors in animals have been demonstrated and whether they translate into a useful therapy in chronic human allergic diseases remains to be determined. Indeed, despite initial optimism, the disappointing results of a number of Phase III studies indicate that dose-limiting adverse events are a major cause for concern and probably reflect an interaction of the compound with PDE4 expressed in “nontarget” tissues. An additional contributing factor may lie in the knowledge that all of the PDE4 inhibitors currently in development will inhibit PDE4D, which may promote emesis (Giembycz 2002a; Robichaud et al. 2002) but not suppress inflammation (Hansen et al. 2000). Nevertheless, by 2010 it is likely that results from clinical trials will reveal whether the optimism in the therapeutic potential of this class of compounds was justified.

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Adrenergic Agonists and Antagonists Tony R. Bai

Summary Adrenergic agonists and antagonists act as ligands at either α or β adrenoceptor subtypes to mimic or antagonize the action of circulating epinephrine or neuronally released norepinephrine. Most cells possess adrenoceptors of varying subtype; the effects of ligand binding can be diametrically opposite in different cell systems, specificity being determined by the second messenger pathway coupled to the adrenoceptor. Adrenergic agonists are usually administered by inhalation or parenterally, and antagonists typically orally. In the field of allergy and immunology, the key benefit of α-adrenoceptor ligands is vasoconstriction induced by the agonism of medications such as epinephrine, pseudoephedrine (acting on both α1 and α2 adrenoceptors) or xylometazoline (α1 selective). The primary role of β-adrenoceptor ligands is the β2-agonist action of agents such as isoprenaline or salbutamol, causing smooth muscle relaxation, and hence inducing bronchodilation, although β2 agonist-mediated inhibition of mast cell mediator release is one mechanism of action of epinephrine in reducing symptoms of anaphylaxis such as itch and edema. A similar mechanism is involved in the inhibition of the early airway response to inhaled allergen. The β2-selective agonists are preferred as bronchodilators. Both short-acting (1–4 hours) and long-acting β2-selective bronchodilators (8–12 hours) are widely available. These medications vary in both adrenoceptor selectively, rapidity of action, and efficacy (maximal response). They should generally not be used in asthma more than two to three times per week without concomitant antiinflammatory therapy. β2-Adrenoceptor desensitization (i.e., waning of the response in the face of continuous or repeated agonist exposure) can occur rapidly; the receptor becoming uncoupled from its stimulatory G protein that usually is responsible for initiating downstream intracellular effects. The β2 adrenoceptor can also be internalized (downregulation) as a response to prolonged agonist stimulation. Uncoupling can be enhanced by proinflammatory mediators and downregulation reversed to some extent by corticosteroids.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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The use of β-adrenoceptor antagonists predictably increases the severity of anaphylaxis, and nonselective β-adrenoceptor antagonists are contraindicated in asthma but generally tolerated in chronic obstructive pulmonary disease. Relatively selective β1 antagonists such as bisoprolol may be tolerated in some asthmatics as long as asthma is controlled as assessed by standard measures such as normal exhaled nitric oxide fractional concentrations and lack of induced sputum esosinophila.

Introduction Ligands acting at adrenergic receptors are among the most commonly used medications in clinical immunology and allergy. Long before Langley (1905) and Dale (1906) developed the concept that the specific biological effects of hormones, neurotransmitters and drugs result from high-affinity stereospecific interactions with tissues, the English physician Henry Salter reported in 1859 what is probably the first account in modern times of the therapeutic effects of activation of adrenergic receptors when he wrote that “asthma is immediately cured in situations of either sudden alarm or violent fleeting excitements.” Endogenous levels of circulating catecholamines, particularly epinephrine (adrenaline), influence airway caliber in asthmatic patients, and it is likely that Salter was describing sympathoadrenal release of epinephrine following emotional triggers. Around 1900, the vasodilator hypothesis of asthma had considerable support in both Germany and the USA. This hypothesis stated that airway obstruction was caused by swelling of the bronchial mucosa secondary to vasodilatation. The other major hypothesis at that time was that asthma was due to “the spasm of the circular muscles of the bronchi.” Thus, in 1900, Solis-Cohen, encouraged by reports that adrenal extracts caused vasoconstriction, gave large oral doses of desiccated adrenal glands to asthmatic subjects with success, which he interpreted to be consistent with his view that asthma was a “vasomotor ataxia of the relaxing variety” (Solis-Cohen, 1990). However, it is unlikely the epinephrine

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content of the adrenal could have survived the oral route as an active drug and, indeed, the slow onset of action of the extract treatment reported in his paper is now thought to be more likely the demonstration of the beneficial effects of glucocorticosteroids (Persson 1989). Soon after this, epinephrine became available as a pure substance and in 1903 Bullowa and Kaplan successfully gave injections of it to asthmatic patients. They too thought this success was consistent with the vascular hypothesis of asthma but in 1907 epinephrine was shown to relax airway smooth muscle (Kahn 1907). Although it is possible in some asthmatics that the α-adrenoceptor agonist (α-agonist) effect of epinephrine contributes to increased airway caliber, it is probable that the β-adrenoceptor agonist (β-agonist) effect dominates (see later). In 1924, ephedrine was introduced to Western medicine, although the plant from which it is derived has been used for more than 5000 years in China for respiratory and other allergic conditions. Ephedrine, an α agonist with a weak β-agonist activity, and epinephrine were widely used over the ensuing decades in the treatment of asthma, rhinitis and anaphylaxis. Konzett (1941) isolated isoproterenol (isoprenaline), the first β agonist devoid of α-adrenergic effect. Subsequently, Ahlquist (1948) used isoproterenol to partition sympathomimetic effects into α (mainly excitatory) and β (mainly inhibitory) based on physiologic responses in isolated tissues.

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Table 28.1 Tissue distribution of adrenoceptor subtypes. Airways Lung parenchyma Nose Heart Blood vessels Inflammatory cells Gut, kidney, liver, pancreas, spleen Uterus Adipose tissue Noradrenergic and cholinergic nerve terminals Brain

a1, b2 b1 a1, a2, b2 a1, a2, b1, b2 a1, b2 a2, b2 a1, a2, b2 b2 b3 a2, b2 a1, a2, b1, b2

erally, but not invariably, β1 responses appear to be initiated by the neurotransmitter norepinephrine in innervated tissues, whereas β2 responses are triggered by the circulating hormone epinephrine (O’Donnell 1991). Subsequently, a third subtype of β adrenocepter, β3, was defined (Emorine et al. 1989). The β adrenoceptors are, in general, a low abundance receptor (500–5000/cell), although on airway smooth muscle they number 30 000–40 000/cell. Tissue distributions of both α and β adrenoceptors are summarized in Table 28.1. Heart In the ventricle 40% and in the atrium up to 55% of β adrenoceptors are of the β2 subtype (Bristow & Ginsburg 1986). The remainder are β1 subtype.

Adrenoceptor localization The mammalian G protein-coupled receptors (GPCRs) can be divided in three main classes according to sequence homology: class I or rhodopsin-like (which is the largest subfamily), class II or secretin-like, and class III or glutamatemetabotropic-like. Within class I, the adrenoceptors mediate the functional effects of epinephrine and norepinephrine. The adrenoceptor family includes nine different gene products: three β (β1, β2, β3), three α2 (α2A, α2B, α2C) and three α1 (α1A, α1B, α1D) receptor subtypes. The receptor population mediating α effects is characterized by a rank order of potency of epinephrine > norepinephrine > isoproterenol and β effects isoproterenol > epinephrine > norepinephrine (Table 28.1).

The b adrenoceptors Evaluation of a large volume of data generated in the study of β-adrenergic pharmacology enabled Lands et al. (1967) to suggest a further division of the β-adrenoceptor response into subtypes termed β1 and β2. Again this distinction was based on the relative potency of the naturally occurring catecholamines, epinephrine and norepinephrine. The β1 responses are equally sensitive to these two agonists; β2 responses are more potently stimulated by epinephrine. Gen-

Vasculature β2 Adrenoceptors predominate on vascular smooth muscle. Nose A homogeneous population of β2 adrenoceptors has been noted in several studies (van Megen et al. 1991). Lung Organ bath and autoradiographic studies (Fig. 28.1) have demonstrated that the airway smooth muscle relaxant effect of β agonists is largely via β2 adrenoceptors directly on the muscle surface (Nadel & Barnes 1984; Carstairs et al. 1985; Bai et al. 1992). This is not unexpected, given that β1 adrenoceptors are found at sites of sympathetic innervation responding to norepinephrine release and there is no direct sympathetic innervation of human airway smooth muscle (Daniel et al. 1986). Similarly, the adrenoceptors on mucous and serous glands and inflammatory cells are largely of the β2 type (Basbaum et al. 1990). β2 Adrenoceptors also predominate on bronchial epithelium, type I and II pneumocytes, and pulmonary vascular smooth muscle so that they make up 70% of the β adrenoceptors in the human lung, the other 30% being β1 on alveolar walls. The density of β2 adrenoceptors increases from the large to small airways and is much greater

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(a)

(b)

(c)

(d) Fig. 28.1 Distribution of b adrenoceptors in human normal and asthmatic bronchi. (a, b) Dark-field photomicrographs of sections showing the distribution of autoradiographic grains after incubation with 25 pmol/L 125I-iodocyanopindolol. (c, d) Bright-field photomicrographs of adjacent sections showing the epithelium (Ep), smooth muscle (SM) and submucosal glands (G) after staining with 1% cresyl fast violet. The epithelium is partially shed in the asthmatic sections. (From Bai et al. 1992, with permission.)

on alveolar walls than other structures in the lung (Carstairs et al. 1985). Other sites Functional and gene expression studies suggest adipocytes contain β1 and β3 adrenoceptors.

of α1 and α2 subtypes are summarized in Table 28.2. In the nasal vasculature, both α1 and α2 adrenoceptors are present (Lacroix, 1989; van Megen et al. 1991). In the nasal capacitance vessels, α2 adrenoceptors dominate over α1 adrenoceptors.

Adrenoceptor biology The a adrenoceptors At least seven α adrenoceptor subtypes (α1A–D and α2A–C) were initially proposed based on pharmacologic experiments and gene cloning. However, as α1A and α1C subtypes have very similar pharmacologic properties (Hieble & Bond 1994), only six subtypes are now accepted (Cotecchia 2007). The α1 subtypes are located on smooth muscle membranes of most sympathetically innervated tissues, mediating contraction. α1 Adrenoceptors are found on arterioles in skin, mucosa, viscera, and resistance vessels in the kidney as well as in all veins. The α2 adrenoceptors are primarily presynaptic, inhibiting traffic through autonomic ganglia and other nerve terminals. The localization and pharmacologic properties

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Molecular structure Prior to 1974, the adrenergic receptors were known only indirectly as entities that responded to drugs in a selective manner to mediate a variety of physiologically important responses. Then a variety of high-affinity 125I-labeled radioligands selective for these receptors were developed that led to experiments utilizing direct binding assays to establish the biochemical properties of the receptor proteins. These techniques, when coupled with efficient methods for detergent solubilization, formed the basis of receptor purification using affinity chromatography, and when coupled with autoradiographic methods lead to the cellular localization and quanti-

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b2AR agonist

Table 28.2 a2 Receptor subtypes.

b2AR

Subtype

Localization

Pharmacologic properties

a1A

Brain, vas deferens, kidney, heart, spleen

Norepinephrine > epinephrine > phenylephrine

a1B

Lung, brain, heart, liver, kidney, spleen

Oxymetazoline > epinephrine > norepinephrine

a1D

Vascular smooth muscle

Norepinephrine > epinephrine > phenylephrine

a2A

Brain and platelets

p-Aminoclonidine > epinephrine

a2B

Kidney, neonatal lung

Clonidine > norephinephrine > oxymetazoline

a2C

Kidney

Oxymetazoline > norepinephrine

fication of adrenergic receptors on thin sections of tissues (Stadel & Lefkowitz 1991). The availability of substantial amounts of purified β2 adrenoceptor allowed determination of the molecular mass and amino acid sequence of part of the receptor. This was the first adrenergic receptor isolated. This new information in turn led to the production of polynucleotide probes and eventually to cloning of the receptor gene and determination of the complete primary sequence of the receptor protein (Strader et al. 1989; Fraser & Venter 1990). The β2 adrenoceptor gene maps to chromosome 5 and encodes a protein of 413 amino acids, only 54% of which are shared with β1 adrenoceptors. α Adrenoceptors were cloned using similar techniques and show many homologies to β adrenoceptors (Fraser & Venter 1990). Current models of the adrenoceptors show seven transmembrane segments connected by alternating intracellular and extracellular loops (Fig. 28.2). Homology among all the members of the seven transmembrane region (serpentine) receptor family is greatest in the transmembrane-spanning domains. Genetic and biochemical manipulation of the β2 adrenoceptor has identified that the ligand-binding domain is a pocket buried within the membrane bilayer, with agonists interacting with amino acid residues within transmembrane helices III, V and VI (Johnson 2006). Four residues are of critical importance, namely the aspartate residue 113, positioned on the third domain, two serine residues, 204 and 207, on the fifth domain, and asparagine 293 in the sixth domain. Aspartate binds to the nitrogen of the β2-agonist molecule while the two serine residues interact with the hydroxyl groups on the phenyl rings. Antagonists do not bind with the same amino acids. Thus, β antagonists bind to aspartate 113 and a residue in the seventh domain rather than with serine residues in the fifth domain (Tota et al. 1991; Liggett 2002). Most β2 adrenoceptors exist as homodimers for at least part of the time, homodimers being essential for transport to the cell surface from the endoplasmic reticulum (Salahpour et al.

G AC

ATP

cAMP

Bronchodilatation Fig. 28.2 Simplified model of b2-adrenoceptor (b2-AR) signaling. Following agonist binding the Gs protein couples with, and stimulates, adenylate cyclase (AC) to catalyze the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). (See CD-ROM for color version.)

2004), but the importance of cell-surface dimerization in determining high-affinity binding remains controversial. The receptors do oscillate between an activated and inactivated state, and are at equilibrium under resting conditions (Liggett 2002). In the past decade it has become apparent that some ligands act as “inverse agonists,” decreasing the number of spontaneously active receptors (Bond & Ijzerman 2006). Studies of the regulation of adrenergic receptor gene transcription are incomplete. In both cell culture and homogenized human lung (Mak et al. 1995) as well as in nasal mucosa in vivo (Baraniuk et al. 1997), glucocorticoids increase β2adrenoceptor mRNA levels and receptor protein by increasing the rate of gene transcription and isoproterenol decreases mRNA levels by decreasing stability of the mature mRNA. Hamid et al. (1991) have reported the distribution of β2adrenoceptor mRNA in human lung by in situ hybridization and correlated this with receptor autoradiographic distribution. They report qualitative differences between the densities of labeling with the two techniques in different cell types. Pulmonary vascular and airway smooth muscle showed a high intensity of mRNA hybridization but only a low density of adrenoceptors and the converse was reported for the alveolar epithelium. These differences may be due to either a rapid rate of β2-adrenoceptor synthesis or high stability of mRNA in the airways and may explain contribute to the difficulty in demonstrating desensitization in airway smooth muscle (see later). G proteins are membrane-associated heterotrimers composed of α, β and γ subunits. Interaction with a receptor causes the release of GDP from the α subunit of the G protein, allowing GTP to bind and leading to the dissociation of the activated α subunit from the receptor and from the βγ complex (Fig. 28.2). Various G proteins activate or inhibit different effector enzymes, modulating the levels of intracellular second messengers. In the case of the β2 adrenoceptor, which

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is coupled to the stimulatory guanine-nucleotide binding protein, Gs, binding of an agonist to the receptor catalyzes the release of GDP from the α subunit of the G protein (αs), allowing the binding of GTP; this in turn leads to the direct activation of adenylate cyclase by αs-GTP. Adenylate cyclase catalyses the formation of the classical second messenger cyclic AMP so that levels of cAMP up to 400-fold over basal can occur within minutes of agonist exposure (Johnson, 2006). Upon removal of agonist, the activation of adenylate cyclase persists until the intrinsic GTPase activity of αs hydrolyses the bound nucleotide. Sequencing of ADRB2 has identified at least 58 single nucleotide polymorphisms (SNPs) within the coding and promoter region (Weiss et al. 2006). To date, the most frequently studied ADRB2 coding SNPs are characterized by substitutions of glycine for arginine at position 16 (Arg16Gly) and glutamine to glutamic acid at position 27 (Gln27Glu), both of which occur commonly in the population (minor allele frequency approximately 0.4– 0.5 in whites, with some differences in allelic frequency in blacks).

Mechanisms of adrenoceptor agonist action The contractile state of smooth muscle, the primary therapeutic target of most adrenergic agonists, is determined by cytoplasmic calcium concentrations. The cell membrane is negative with respect to the extracellular space and more positive potentials (depolarization) opens voltage-gated calcium channels, causing calcium influx to increase cytoplasmic calcium and trigger contraction. The effect of adrenergic agonists on smooth muscle are complex and vary from one site to the next. For example, α agonists relax intestinal smooth muscle and contract vascular smooth muscle by different and now well understood mechanisms involving coupling of G proteins with different second messenger pathways. Thus, the contractile response to α1 agonists is determined by activation of G protein l-nked adrenoceptors that activate phospholipase C, releasing inositol 1,4,5-trisphosphate (IP3) and increasing intracellular calcium (Hague et al. 2006). α2 Agonists inhibit adenylate cyclase activity and decrease cAMP levels. A number of other mechanisms, apart from increases in cyclic AMP, have been shown to be involved in the smooth muscle relaxation induced by β agonists (Table 28.3). Relaxation is primarily determined by generation of cAMP and activation of cAMP-dependent kinases which have several actions including shifting myosin light chain kinase to a less active form. The rise in cAMP also leads to calcium reuptake into the sarcoplasmic reticulum and organelles and calcium extrusion from the cell. cAMP also causes suppression of IP3 formation. β Agonists also reduce acetylcholine release from smooth muscle cholinergic nerve terminals and thus inhibit contraction (Bai et al. 1989). Finally, activation of β2 adrenoceptors stimulates a calcium-activated potassium channel in the cell membrane which leads to hyperpolarization of the membrane and cell relaxation (Johnson 2006).

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Table 28.3 Mechanisms of smooth muscle relaxation by b2 agonists. Stimulation of cAMP and subsequent reduction of myosin light chain kinase activity cAMP inhibition of phospholipase C with reduction of IP3 formation Stimulation of calcium extrusion pumps Direct interaction of Gs with membrane potassium channels Inhibition of acetylcholine release from cholinergic nerve terminals

b-Adrenoceptor agonists Structure and metabolism The structures of commonly used agonists and the natural catecholamines from which they are derived are shown in Fig. 28.3. The term “catecholamine” refers to all compounds with a catechol nucleus (benzene plus two adjacent hydroxyl groups) and an amine group. The three principal naturally occurring catecholamines are dopamine (dihydroxyphenylethylamine) and the metabolic products of dopamine, norepinephrine, and epinephrine. Monoamine oxidase, predominantly an intraneuronal enzyme, and catechol-Omethyltransferase, predominantly an extraneuronal enzyme, are the two enzymes primarily responsible for degradation of catecholamines. Ligand receptor interactions are stereospecific. All the commonly used β agonists exist in racemic mixtures of optical isomers referred to as R and S enantiomers. The agonist activity lies predominantly in the R enantiomer. The in vitro effects of the R and S enantiomers of some but not all β2 agonists have been shown to be different (Volcheck et al. 2005). There has been speculation that the S enantiomers possess adverse effects in clinical usage but this remains unclear. There are now preparations of R enantiomers of salbutamol alone available for commercial usage. However the clinical significance of such differences remains uncertain. Isoproterenol is a catecholamine like epinephrine and this class of compounds is both chemically and metabolically unstable. The resorcinol analog metaproterenol (orciprenaline) is structurally closely related to isoproterenol, and thus shares the nonselective actions of isoproterenol but is more stable. Both salbutamol and terbutaline, the first of the current generation of “short-acting” relatively β2 adrenoceptor-specific agonists used in the treatment of asthma were synthesized and characterized before Lands subtyped β adrenoceptors. Since the 1960s, a number of other β2 agonists have been developed as therapeutic agents. The key substitutions to the β-phenylethylamine parent have been to the catechol ring, or related structure, to make the compounds resistant to metabolism by endogenous methyltransferases and monoamine oxidase, and addition of an ethanolamine side chain of varying length (Fig. 28.3). Such alterations prolong half-life and increase the selectivity of these agents for β2 adrenoceptors. A short-acting β2-selective compound still metabolized by endogenous enzymes is sometimes useful in clinical research;

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b-agonists

Catecholamines HO

HO

OH

HO

CH

NHCH3

CH2

HO

OH

HO

CH

CH3

CH

CH3

HO

CH3 CH3

OH

NHCH

Isoprenaline

NHC

CH2

Terbutaline

HO

CH3 CH2

CH3

CH

Epinephrine HO

OH

CH2

NHCH CH2

Fenoterol

OH

a-agonists CH

CH2

OH

CH3

NH

HOCH2

CH3

OH

HO

CH

Ephedrine

OCH3

HOCH2 CH2

OH

CH3

NHC

NH2

CH3 CH3

OH

HO

CH

CH2

NH

(CH2)6

O

(CH2)4

Salmeterol

Methoxamine O

HO CH

CH2

OH Fig. 28.3 Representative catecholamines and selective a- and b-adrenoceptor agonists.

CH2

Salbutamol

OCH3 CH

CH3

NH

CH3

HCNH HO

OH CH

Phenylephrine

rimiterol is one such compound, although only modestly β2 selective. The prototypic short-acting β2 agonists salbutamol and terbutaline, despite the subsequent development of many other compounds such as fenoterol, clenbuterol and procaterol (Table 28.4), remains the most widely prescribed of this class of drug. The major shortcoming of these medications is duration of action and much effort has been expended to prolong duration beyond 4 hours. Agents such as pirbuterol and clenbuterol have been reported to possess a significantly longer duration of action but this is marginal at best and, in the case of clenbuterol, only after oral administration. A more recently developed compound, bambuterol, does have a more extended duration of action but is a prodrug of terbutaline and is effective only after oral administration, the least preferred route of administration because of systemic side effects. Two compounds in common clinical use, formoterol and salmeterol, have been shown to have a therapeutically significant increased duration of action by inhalation. Even

CH3 CH2

NHCH

Formoterol

CH2

OCH3

Table 28.4 b Agonists in clinical use. Short-acting (b2 selective) Salbutamol Terbutaline Fenoterol Metaproterenol (orciprenaline) Clenbuterol Pirbuterol Bitolterol Procaterol Long-acting (b2 selective) Salmeterol Formoterol Catecholamines Epinephrine (adrenaline) Isoproterenol (isoprenaline) Isoetharine

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longer-acting agents such as indacaterol, which increase forced expiratory volume in 1 s (FEV1) > 15% 24 hours after delivery, are in phase III trials. Formoterol was developed by investigating β-agonist analogs with increasing affinity for the β2 adrenoceptor itself. In contrast, salmeterol was designed to introduce large lipophilic N-substituents into saligenin ethanolamines to facilitate binding in hydrophobic regions of the cell membrane or to nonpolar amino acid residues in the β2-adrenoceptor protein (Johnson 2006). These differences in design lead to important differences between these two long-acting β2-adrenoceptor agonists. Both are moderately lipophilic compared to salbutamol and terbutaline which are hydrophilic in nature; this property leads to greater persistence in the cell membrane and may explain some but not all of the prolonged duration of action. The duration of action of short-acting β2 agonists is determined in part by the rate of diffusion of these hydrophilic compounds away from the receptor site. In contrast, salmeterol seems bound to the cell membrane for prolonged periods and offset of action may be determined by internalization of the bound receptor–drug complex. All the commonly used β2 agonists are either excreted unchanged in the urine or excreted in a conjugated fashion. For example, salbutamol and terbutaline are susceptible to 4-O ′-sulfate conjugation in intestinal wall and liver (when administered by the oral route) (Morgan et al. 1986). Following aerosol administration, the significant proportion that impacts in the oropharynx and is swallowed is also conjugated in the intestinal wall. Following intravenous administration, more of the free drug is excreted compared with the oral and aerosolized route. Fenoterol is also susceptible to 5-O′sulfation. Salmeterol is extensively metabolized by hydroxylation and formoterol is excreted unchanged or subject to glucuronide conjugation.

Selectivity, affinity, and efficacy The basis of β1/β2 adrenoceptor selectivity may be in differ-

ences in the amino acid sequences of the two adrenoceptors (Tota et al. 1991). Selectivity is determined by comparing the potency of β agonists on tissue preparations containing primarily β1 adrenoceptors (e.g., atrial inotropic responses) versus preparations containing primarily β2 adrenoceptors (e.g., bronchial relaxant responses). In this situation, agents are ranked compared to the effect of a completely nonselective β-adrenoceptor agonist, usually isoproterenol (Table 28.5). The ratio of the relative potencies at β2 vs. β1 sites gives the selectivity ratio. Using these criteria, the long-acting agent, salmeterol, is the most β2-selective agent in common use and fenoterol is the least selective. Formoterol is not very selective using these approaches. The potency of a given agonist is usually measured as the concentration of the drug required to cause 50% of maximum response to that agonist. Potency is a function of both receptor affinity and receptor efficacy and of tissue-related factors such as receptor density and efficiency of G-protein coupling. Affinity describes the degree of attraction of a ligand for a receptor, as determined by binding studies. A radiolabeled version of agonist is used in increasing concentrations until the maximum is reached for bound label compared to labeling in the presence of a high concentration of unlabeled agonist. Efficacy describes the ability of an agonist to induce a response in a particular tissue. β Agonists and β antagonists may both share similar affinities for β adrenoceptors but have different efficacies; a full agonist will have a high efficacy while a pure antagonist will have low or zero efficacy. The majority of short-acting β2-selective agonists have intermittent efficacy and potency compared to isoproterenol (Table 28.5). β2 Agonist efficacy is usually assessed by examining relaxation responses of contracted preparations of airway smooth muscle. Again, one compares the maximum relaxant response with isoproterenol. This value is called the intrinsic activity and is a ratio of the maximum response of a given β agonist to the maximum response of isoproterenol. Using these criteria,

Table 28.5 Potency, selectivity, and intrinsic activity of commonly used b-adrenoceptor agonists.

Agonist

Potency b1

Potency b2

Selectivity ratio (b2 : b1)

Airway smooth muscle intrinsic activity

Isoproterenol Salbutamol Terbutaline Fenoterol Salmeterol Formoterol

1.0 0.0004 0.003 0.005 0.001 0.05

1.0 0.48 0.08 0.9 8.5 25

1.0 1375 267 180 85 000 100

1.0 0.91 0.83 0.99 0.70 0.94

Data are relative to response to isoproterenol. Intrinsic activity is a measure of efficacy (the ratio of the maximum response to a given b agonist to the response to isoproterenol). Data compiled from O’Donnell & Wanstead (1979); Decker et al. (1982); Bai et al. (1989); Johnson (1991).

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none of the synthetic β agonists have higher intrinsic activity than isoproterenol. Agents with equivalent efficacy are procaterol and formoterol whereas most saligenins and resorcinols are of moderate efficacy (65– 85% of isoproterenol). The efficacy of β agonists at extrapulmonary sites may be of clinical relevance. Fenoterol and formoterol have the same efficacy as isoproterenol at cardiac β1 adrenoceptors, despite being less potent, whereas salbutamol and salmeterol have very low efficacy. In contrast to efficacy, lung β2-adrenoceptor potency can be greater than isoproterenol. For example, salmeterol is five times more potent than isoproterenol, formoterol 25-fold more potent, and procaterol 24-fold more potent. Neither intermediate intrinsic activity (efficacy) nor intermediate potency negates the clinical value of a given β agonist as a bronchodilator drug. Rather, the adverse consequences of prolonged use of these drugs may be influenced by whether they are partial or full agonists. The rate of desensitization is one phenomenon that could be influenced by full versus partial agonist activity (see later).

Physiologic effects Lung cells Although in vivo the most obvious and therapeutic pulmonary effect of β-adrenergic stimulation is bronchodilation mediated by airway smooth muscle relaxation, a number of other effects also occur (Table 28.6). β Agonists promote secretion from serous cells and, to a lesser extent, mucous cells, in mucous glands. Serous cell stimulation produces antibacterial proteins such as lysosomes and lactoferrin. This effect has been demonstrated convincingly in vitro only, using human tracheal explants at relatively high concentrations of β agonists (Basbaum et al. 1990) However, theoretical calculations of luminal β2 agonist concentrations following inhalation indicate such levels may be achieved in vivo. Furthermore, β2 agonists increase chloride iron transport through apical membranes of epithelial cells via an increase in cyclic AMP. Sodium follows passively via paracellular channels and water by osmosis. The net effect is to increase periciliary fluid (Wanner 1988). The combined effects of stimulation Table 28.6 Physiologic effects of b2-adrenoceptor stimulation in human lung. Airway smooth muscle relaxation Prejunctional inhibition of acetylcholine release from parasympathetic neurons in airway smooth muscle Stimulation of mucus and serous cell secretion Stimulation of chloride ion secretion across the apical membrane of airway epithelial cells Increase in ciliary beat frequency Stimulation of surfactant secretion from alveolar type II cells Inhibition of mediator release from lung mast cells and neutrophils ? Reduction in microvascular permeability (animal models) ? Increase in bronchial blood flow (animal models)

Adrenergic Agonists and Antagonists

of mucous glands and chloride channels, together with an increase in ciliary beat frequency (Wanner 1988), is to increase mucocilary clearance. Increased clearance has been demonstrated in vivo using radio tracer methods although the clinical relevance of this enhancement in patients with asthma is unknown. β2 Agonists also stimulate the secretion of surfactant from alveolar type II cells in vitro, although the magnitude of the effect is modest (Mason & Williams 1991). In animal models, inflammatory mediators increase microvascular permeability by contracting postcapillary venular endothelial cells so that spaces form between the cells. In such models, β2 agonists relax endothelial cells and therefore reduce permeability (Baluk & McDonald 1994). However, β2 agonists also increase bronchial blood flow by acting as vasodilators of bronchial arterioles (Kelly et al. 1992). The net effect of these two opposing effects on exudation or transudation of fluid into the lumen and wall of inflamed human airways is unclear. A report that nebulized epinephrine was no more effective in producing bronchodilatation in acute asthma than a nebulized β agonist, which lacks an α-adrenergic vasoconstrictor effect, suggests that potential alterations in bronchial blood flow induced by β2 agonists do not adversely affect fluid shifts across the lumen wall (Coupe et al. 1987). Moreover, the lack of additional benefit by epinephrine suggests that the potential decrease in lumen area produced by mucosal bronchodilation induced by β agonists is not an important component of airflow resistance in asthma. β Adrenoceptors are present in peribronchial parasympathetic ganglia, which receive direct sympathetic innervation (Widdicombe 2003). β2 Adrenoceptors are also present on cholinergic nerve terminals in airway smooth muscle and act here to inhibit acetylcholine release (prejunctional inhibition), thereby reducing the cholinergic component of bronchoconstriction. It is possible that β antagonists such as propranolol induce asthma exacerbations not only by reducing the tonic bronchodilator effect of circulating epinephrine on airway smooth muscle in maintaining airway patency, but also by blocking the effect of epinephrine on cholinergic nerve terminals leading to the exuberant release of acetylcholine. The observation by Grieco and Pierson (1971) that cholinergic antagonists partially reverse propranolol-induced bronchoconstriction provides some support for this hypothesis.

Inflammatory cells β2 Adrenoceptors are present on a variety of inflammatory cells including lymphocytes, granulocytes, mast cells, and macrophages. Stimulation of β receptors on immunocompetent cells primarily results in antiinflammatory effects. Circulating lymphocytes and neutrophils have low numbers of adrenoceptors which appear to be relatively poorly coupled to second messenger pathways in that they are easily downregulated (Insel 1991). Human neutrophils possess approximately 900–1800 β2 adrenoceptors per intact cell and mediator

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Cardiovascular β Agonists increase the force and rate of cardiac contraction and thus cause an increase in systolic blood pressure. Increases in inotropic reponses are predominantly mediated via β1 adrenoceptors, although β2 adrenoceptors also contribute. In contrast, chronotropic responses are predominantly β2 mediated (Fig. 28.4). β2 Agonists are also vasodilators, via β2 adrenoceptors on vascular smooth muscle, which leads to a slight fall in diastolic blood pressure. The role of baroreceptormediated reflex withdrawl of cardiac vagal tone in response to peripheral vasodilatation, in determining heart rate increases seems less important than direct cardiac effects of β agonists. β Agonists cause a dose-dependent increase in QTc interval, which has been reported to be correlated with

676

the degree of hypokalemia induced by these agents (Fig. 28.4) (Crane et al. 1989). However, isoproterenol, which causes minimal hypokalemia, prolongs QTc interval to a similar degree as salbutamol, suggesting a direct cardiac β adrenoceptor-mediated effect (Lipworth & McDevitt 1992).

DHR (beats/min)

30 25 20 15 10 5 0 100

200

500

1000

2000

4000

Dose salbutamol (mg)

ΔK (mmol/L)

release is inhibited in a dose-dependent manner by isoproterenol. Triggering of β2-receptors on neutrophils also results in inhibition of oxygen release and a reduction of the adhesion of neutrophils to the vascular endothelium and airway epithelial cell (Maris et al. 2005). Studies employing circulating lymphocytes or neutrophils as a marker of pulmonary β-adrenoceptor function can therefore be misleading (see below). However, one report showed a strong relationship between β2-adrenoceptor densities on circulating mononuclear leukocytes and in lung tissue obtained at thoracotomy (Liggett et al. 1988). β Agonists modulate lymphocyte cytokine production. Type 2 T cells freshly isolated from peripheral blood mononuclear cells directly respond to β agonists to activate protein kinase A and inhibit CD3-stimulated interleukin (IL)-13 production (Loza et al. 2006). Human alveolar macrophages contain 5000 β2 adrenoceptors per cell (Liggett 1988). There is controversy as to whether β2 agonists prevent mediator release from activated human alveolar macrophages; short-acting agents may not (Fuller et al. 1988) as opposed to longer-acting agents (Maris et al. 2005). There is evidence that β agonists reduce the release of histamine from mast cells. β Agonist-mediated inhibition of mast cell mediator release is probably one mechanism of action of epinephrine in reducing symptoms of anaphylaxis such as itch and edema. In addition, inhibition of mast cell mediator release is part of the mechanism of action of β agonists in abating the early response to allergic bronchial challenge. β Agonists are also functional antagonists of the airway smooth muscle contraction induced by release of mediators. β Agonists may also inhibit mediator release from basophils (Weston & Peachell 1998). Mediator release from the human eosinophil, although possessing a greater density and affinity of β2 adrenoceptors (5000 sites per cell) than neutrophils, was not shown to be inhibited by isoproterenol (Yukawa et al. 1990), but more recent studies show inhibitory effects on IL-5 induced superoxide production, an effect of the R but not the S enatiomer of salbutamol (Volcheck et al. 2005).

0.10 0.00 –0.10 –0.20 –0.30 –0.40 –0.50 –0.60 –0.70 –0.80 –0.90 –0.00 100

200

500 1000 Dose salbutamol (mg)

2000

4000

100

200

500 1000 Dose salbutamol (mg)

2000

4000

0.80

0.60 DFEV1 (L)

PART 3

0.40

0.20

0.00

Fig. 28.4 Changes in heart rate (upper panel), plasma potassium (middle panel), and FEV1 (lower panel) in response to cumulative doubling doses of inhaled salbutamol from pressurized metered-dose inhalers in 12 mild asthmatic patients. Subjects were studied after 2 weeks’ pretreatment with placebo (circles), low-dose regular salbutamol (800 mg/day) (triangles) or high-dose regular salbutamol (4000 mg/day) (squares). Randomized crossover design. Note that significant tolerance to the systemic but not bronchodilator effects of the b agonist develop with regular use. (Redrawn from Lipworth et al. 1989, with permission.)

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Pharmacokinetics of b-adrenoceptor agonists

Salbutamol 200 mg

Oral administration

Parenteral administration Subcutaneous, intramuscular, or intravenous injection of terbutaline, salbutamol or epinephrine provides almost immediate action and assured delivery. Following subcutaneous terbutaline 0.5 mg, significant levels are present within a few minutes and peak at 20 min (van den Berg et al. 1984). Epinephrine has a slightly shorter duration of action although a slow-release form is available in some countries. In an intensive care setting, intravenous infusion is sometimes used when patients are moribund or responding poorly to intermittent administration of β agonists by other routes. Both intravenous terbutaline and salbutamol have been quite widely used in this context, with a loading dose followed by a continuous infusion of 10– 20% of the loading dose. Intravenous isoproterenol has little advantage apart from quicker offset of action, and has greater side effects.

Aerosol administration This is the preferred method of administration of all β agonists as there is an effect on airway caliber within seconds, with the effect of short-acting β agonists such as salbutamol reaching 80% of maximum in 5 min (Figs 28.4 and 28.5). Compared to parenteral or oral administration, following aerosolization a given degree of bronchodilatation is achieved with significantly less adverse effects such as tremor or palpitations. Shortacting β2 agonists achieve peak effects within 30–60 min and bronchodilatation slowly reduces over a variable time after this, in part dependent on the severity of asthma, but airway caliber is back to baseline within 4–5 hours. There are no important clinical differences between commonly available short-acting β2 agonists in terms of bronchodilatation or duration of action. The effects of catecholamine aerosols such as rimiterol or epinephrine peak earlier and bronchodilatation persists for only 30 min to 2 hours. Serum levels are very low after inhaled administration and do not correlate

Salmeterol 50 mg Salmeterol 100 mg Salmeterol 200 mg

100

Per cent of max PEF1

After administration of an oral or parenteral β agonist, the bronchodilator effect is closely dependent on serum levels (Morgan et al. 1986). Orally administered β agonists are incompletely absorbed and the greater proportion metabolized by sulfate conjugation in the gut epithelium and liver. For example, oral terbutaline absorption varies from 30 to 65%, and of this only 25% remains unconjugated in the urine. Net biovavailability is thus 10–15%. Protein binding of terbutaline varies from 14 to 25%. Salbutamol is more completely absorbed with peak levels within 1 hour of administration when the subject is fasting; 60% of the absorbed dose is conjugated. Sustained release preparations of salbutamol and terbutaline are available in various formations in some countries, and may be useful in asthmatics with marked nocturnal symptoms, although long-acting inhaled β2 agonists have superseded such preparations.

90

80

70 0

120

240

360

480

600

720

Time (hours) Fig. 28.5 Time-course of bronchodilator response to standard shortand long-acting b agonists. Peak expiratory flow (PEF) after inhalation of salbutamol (200 mg) or salmeterol (50, 100 or 200 mg). (From Ullman & Svedmyr 1988, with permision.)

with bronchodilatation. The effects of different aerosol delivery devices on intrapulmonary β agonist deposition is discussed in a subsequent chapter. Systemic effects of inhaled β2 agonists are in part dependent on airway caliber, which influences peripheral deposition such that normal individuals have more systemic effects than asthmatics (Mortimer et al. 2006). Formoterol is long acting only by inhalation, reflecting unusually prolonged retention in the airway wall. Systemic effects are less prolonged (Lotvall 2002). In contrast to salmeterol, which is “dose loaded” (i.e., available only as a dose that has a maximal effect), formoterol is available in a range of doses and shows dose-dependent increases in both maximal effect and duration of action in clinical use.

Adverse effects Desensitization β-Adrenoceptor desensitization, i.e., waning of the response in the face of continuous or repeated agonist exposure, can occur by several mechanisms (McGraw et al. 2003; Johnson, 2006). Rapid desensitization is mediated by an alteration in the function of the β adrenoceptor in that it becomes uncoupled from the stimulatory G protein, Gs. This uncoupling phenomenon involves phosphorylation of receptor proteins in the third transmembrane intracellular loop and the terminal intracellular segment by at least two different kinases, protein kinase A and a β adrenoceptor-associated kinase (βARK) or related kinases, which are activated under different desensitization conditions. Phosphorylation leads to binding of β-arrestin and thus decreased efficiency of

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coupling of the β adrenoceptor to Gs, leading to decreased adenylyl cyclase activity and hence decreased cAMP levels. Desensitization can also occur by intracellular sequestration of the receptor complex or by downregulation, which refers to agonist-induced decrease in receptor number. Downregulation occurs on prolonged exposure to agonists and results in degradation of the receptor, presumably via a lysozymal pathway. Both uncoupling and sequestration (internalization) occur within minutes of exposure to micromolar concentration of β-adrenoceptor agonists and the process is essentially complete within 30 min. Downregulation is evident after only several hours of exposure. It has been proposed that the rapid desensitization mechanisms involving phosphorylation of the β adrenoceptor (uncoupling) may be operative mainly for nonneural β2 adrenoceptors that respond to circulating concentrations of epinephrine which are in the nanomolar range (Stadel & Lefkowitz 1991). Downregulation is also mediated by a decrease in β-adrenoceptor mRNA, caused by a decrease in mRNA stability rather than a decreased rate of transcription. Airway smooth muscle contains much less βARK and related kinase activity compared with mast cells for example, and this may explain discrepancies in the ability to induce desensitisation in inflammatory cells versus smooth muscle preparations. Relatively large numbers of β adrenoceptors on human airway smooth muscle or rapid turnover of adrenoceptors may explain why this tissue is relatively resistant to desensitization (McGraw & Liggett 1997). Phosphorylation and therefore uncoupling of the β adrenoceptor can also be induced by stimulation of adjacent GPCRs (receptor cross-talk) such as cholinergic muscarinic M3 receptors (Grandordy et al. 1994) and prostaglandin EP1 receptors (McGraw et al. 2006). Activation of muscarinic receptors leads to stimulation of phosphatidylinositol pathways with secondary activation of protein kinase C by diacylglycerol which, in turn, can phosphorylate and uncouple the β adrenoceptor (Malbon 1989). Prostaglandin EP1 receptors modulate β2 adrenoceptors directly by receptor heterodimerization, and thus PGE2, which has variable effects on smooth muscle, reduces the smooth muscle relaxation effect of β agonist via uncoupling of β2 adrenoceptors from Gs (McGraw et al. 2006). As discussed above, glucorticosteroids have been demonstrated in vitro to reverse desensitization and this is probably due to increased β2-adrenoceptor gene transcription (Mak et al. 1995), and possibly increased coupling, and may be an important mechanism of action of glucocorticosteroids in the treatment of asthma. There have been many clinical studies of desensitization following regular short-acting β-agonist use (reviewed by Broadley 2006). There is evidence in some studies of a small decrease in peak bronchodilator effect and duration of action in stable mild asthma but not in peak bronchodilator effect in more severe asthmatic patients (Lipworth et al. 1989) (see Fig. 28.4). Genetic polymorphisms in the β2

678

adrenoceptor influence this effect (see later). Oral β agonists are more likely to induce tolerance. In contrast to studies in asthmatics, normal subjects readily demonstrate desensitization both in the lung and in nonpulmonary β-adrenergic systems. Small increases in airway responsiveness have been detected following cessation of regular short-acting β agonists. One explanation of these findings is desensitization of airway smooth muscle β adrenoceptors (Vathenen et al. 1988). There is no evidence that long-term use of long-acting β2 agonists such as salmeterol or formoterol leads to clinically relevant tolerance to their bronchodilating effects. However, regular treatment of patients with asthma with long-acting β2 agonists leads to tolerance to the protective effect against exercise induced bronchoconstriction (Storms et al. 2004). Differences in the effects of regular long- versus short-acting β2 agonists, even when used with corticosteroids, are evident, supporting the as-needed rather than regular use of shortacting β2 agonists (Frey et al. 2005). In this study, despite improved basal airflow rates with either short- or long-acting β agonist, regular treatment with salmeterol reduced risk of asthma attacks compared with regular salbutamol. Overall, the importance of desensitization as a clinically relevant effect of β-agonist treatment remains unclear.

Tremor Tremor, due to activation of β2 adrenoceptors in skeletal muscle, occurs in up to 20% of patients at initiation of β2agonist therapy. Tremor usually abates with regular use due to the development of desensitization.

Cardiac effects Palpitations are reported by up to 5% of asthmatics at initiation of therapy, more so with agents which are full rather than partial agonists at β2 adrenoceptors. Desensitization develops with regular therapy (see Fig. 28.4). Despite concern that tachyarrhythmias could develop secondary to effects of β agonists on QTc interval and potassium in hypoxemic patients, serious cardiovascular events are extremely rare. However, caution should be exercised in individuals with unstable ischemic heart disease receiving high doses of nebulized β agonists as angina has been precipitated in this situation.

Hypokalemia and other metabolic effects Hypokalemia is seen following both inhaled or systemic administration of β agonists due to stimulation of Na+/K+ATPase activity and stimulation of insulin secretion (see Fig. 28.4). When high cumulative inhaled doses of salbutamol and fenoterol (1200 μg) were given to normal subjects, decreases in K+ of 0.67 and 1.13 mmol/L, respectively, were observed. Again desensitization is observed to this effect with chronic use of β agonists (see Fig. 28.4). Glycolysis also occurs secondary to β2-adrenoreceptor activation; the changes

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induced are small and of uncertain significance in patients with diabetes mellitus. Lipolysis is activated by β agonists via β1, β2, and possibly β3 adrenoceptors and results in the mobilization of free fatty acids from adipose tissue.

Hypoxemia All β agonists, including epinephrine, can reduce arterial oxygen tension. These changes are apparent 5 min after administration of inhaled β agonists and return to normal values by 30 min. These changes are secondary to an increase in pulmonary blood flow in poorly ventilated regions of the lung, hence worsening ventilation–perfusion inequality. The increase in blood flow may be secondary to pulmonary vasodilatation via stimulation of β2 adrenoceptors on vascular smooth muscle in the lung and also to increases in cardiac output following cardiac adrenoceptor stimulation. The reduction in arterial oxygen tension is small and unlikely to be clinically significant.

Pharmacogenetics Associations between β2-adrenoceptor SNPs and therapeutic outcomes suggest that genetic variation can influence the response to a variety of β2 agonists; however, the clinical significance of these observations is not yet clear (Tattersfield & Harrison 2006). Carefully designed studies have shown individual β2adrenergic receptor genotype can predict lung function in response to β2 agonists. With daily salbutamol therapy, morning peak expiratory flow progressively decreases in patients homozygous for an arginine SNP at amino acid position 16 of the coding region of the β2 adrenoceptor, but not in Gly16 homozygotes (Israel et al. 2004). Other work has shown an increased number of asthma exacerbations in individuals homozygous for the Arg16 allele who were given long-term salbutamol treatment, but not in those homozygous for the Gly16 variant (Taylor et al. 2000). A potentially critical factor in unravelling pharmacogenetic associations centers on the pharmacologic properties of the β2 agonists administered. The predominance of literature has centered on the short-acting β2 agonist, salbutamol, but because pharmacologic properties of β2 agonists vary, conclusions drawn from clinical studies that investigate associations between lung function and ADRB2 pharmacogenetics may not be comparable between short- and long-acting β2 agonists. In a single 24-week crossover study comparing use of short-acting β2 agonists with salmeterol evaluating patients by Arg16Gly genotype, lower exacerbation rates were reported for Arg/Arg patients receiving salmeterol compared with regular or as-needed albuterol use (Taylor et al. 2000). In this study the authors also demonstrated that while there was reduced peak flow over time with albuterol in the Arg/Arg genotype, there was no diminution of pulmonary function with the Arg/Arg genotype in patients receiving salmeterol. In another study, Arg16Gly effects were associated with lower

Adrenergic Agonists and Antagonists

morning peak expiratory flow in response to salmeterol compared with placebo, regardless of baseline inhaled corticosteroids (ICS) therapy (Wechsler et al. 2006), but there was no difference in exacerbation rates based on genotype. Only limited pharmacogenetic information is available for β2 agonists when coadministered with ICS. No pharmacogenetic associations predictive of clinical response to salmeterol, salmeterol coadministered with fluticasone proprionate (FP), or formoterol coadministered with budesonide have been identified in exploratory retrospective analyses performed to date. In one such study, the ADRB2 Arg16Gly SNP was evaluated in subjects with persistent asthma (N = 183). Following 12 weeks of chronic dosing with salmeterol administered with FP in a single device, no differences in morning peak expiratory flow were observed across Arg16Gly genotypes (Dorinsky et al. 2004). Studies evaluating the ADRB2 Arg16Gly SNP and asthma deaths in whites (who are using overusing β2 agonists) have not detected a risk of this allele (Weir et al. 1998). It is clear that the predictability of β2-agonist pharmacogenetics is low, due to incomplete understanding of the haplotypic structure of the gene and incomplete understanding of the genetic variants in other genes likely to be important determinants of β2-agonist responses (Weiss et al. 2006).

b-Adrenoceptor antagonists Propranolol was the first β antagonist developed for clinical use and remains the prototypic agent (van Zwieten 1988). It is a nonselective competitive antagonist without agonist activity. Nadolol, timolol, pindolol, and sotalol are other examples of this group. Although selectivity is not absolute, metoprolol, atenolol, acebutolol, bisoprolol, and esmolol are relatively β1 adrenoceptor selective. Labetalol represents a class of drugs that act as competitive antagonists at both α1 and β adrenoceptors, with greater affinity for the latter as well as some partial agonist activity at β2 adrenoceptors. Pindolol and acebutolol also have partial agonist activity (intrinsic sympathomimetic activity) at β1 adrenoceptors. Esmolol is a very short-acting β antagonist useful intravenously when short-duration β blockade is desired. Some, indeed most, antagonists, for example metoprolol, exhibit inverse agonism, as defined above, and this may be part of their therapeutic benefit in diseases such as heart failure (Bond & Ijzerman 2006) versus “neutral antagonists” such as bucindolol. None of these antagonists can be safely used in asthmatics, even by topical application, although most patients with chronic obstructive pulmonary disease (COPD) do tolerate these agents (Salpeter et al. 2005), reflecting the differences in the pathogenesis of the two conditions. It is likely, although unproven, that if asthma control is assured with antiinflammatory treatment and careful follow-up, that β1-selective antagonists are tolerated in asthma. The use of β antagonists,

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predictably, also increases the severity and resistance to treatment of anaphylaxis (Schellenberg et al. 1991).

ergic and cholinergic neurons. They inhibit norepinephrine release and they also inhibit, in some circumstances, acetylcholine release.

a-Adrenoceptor agonists a-Adrenoceptor antagonists Most of these compounds are drugs with mixed effects, i.e., they both displace norepinephrine from storage sites within the neuron and have direct α-adrenoceptor-stimulating effects. As for β agonists, the receptor subtype specificity and potency is determined by chemical structure (see Fig. 28.1). Ephedrine is the classic compound in this category. Ephedrine is an alkaloid derived from ma-huang (Ephedra equisetina) and exists in four enantiomers: (+) and (−) ephedrine and (+) and (−) pseudoephedrine. The most potent form in relation to sympathomimetic activity is (−)-ephedrine; this compound is used clinically, as is (−)-pseudoephedrine. Ephedrine produces a prompt rise in blood pressure, causes coronary vessel vasodilatation, and increases heart rate. The other commonly used oral α agonist is phenylpropanolamine. Ephedrine and phenylpropanolamine possess both α1- and α2-agonist activity and are both commonly used as nasal and sinus decongestants. In the nasal vasculature, stimulation of the α1 and α2 adrenoceptors on resistance blood vessels regulates blood flow. In the capacitance vessels, regulating blood volume changes (Lacroix & Lundberg 1989), the α2-adrenoceptor response predominates over α1 adrenoceptors. Phenylpropanolamine and pseudoephedrine are well absorbed with a half-life of 4 hours. Slow-release preparations enable twice daily dosage. Direct effects of ephedrine on airway β2 adrenoceptors result in bronchodilation. Both α1 and α2 agonists reduce vascular engorgement and therefore improve nasal patency (Bende & Loth 1986). There may be advantages to using selective α1 agonists as α2 agonists reduce mucosal blood flow by 30– 40%, which may impair long-term mucosal (i.e., nasal epithelium) health. Phenylephrine is a selective α1 agonist used clinically that is chemically closely related to epinephrine but is less potent and has a longer duration of action. It has little effect on α2 adrenoceptors. It is a useful agent as a topical nasal decongestant but is poorly absorbed orally. Methoxamine is also an effective α1 agonist, used sometimes to reduce airway narrowing when the latter is due to the dilatation of bronchial mucosal vessels secondary to left heart failure (“cardiac asthma”). Selective α1 agonists have a short duration of action and the predominantly α2-agonist imidazole derivatives oxymetazoline and xylometazoline are useful sympathomimetic agents with a larger duration of action. Use of α agonists causes rebound vasodilatation in the nose following offset of action, and prolonged use may lead to rhinitis medicamentosa. Other selective α2 agonists include clonidine and αmethyldopa which are widely used in hypertension and in conditions of “sympathetic overactivity.” These compounds stimulate presynaptic α2 adrenoceptors present both on adren-

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Currently there are no specific indications for α-adrenergic antagonists in the management of allergic diseases. Dale (1906) reported that the pressor effects of large doses of epinephrine could be reversed to cause a fall in blood pressure by certain ergot preparations now known to have αadrenoceptor blocking properties. Large numbers of drugs with the ability to antagonize the effects of α-adrenoceptor stimulation have been synthesized. They are classified into reversible noncompetitive antagonists, for example phenoxybenzamine, and reversible competitive antagonists such as phentolamine and prazosin. Phentolamine and phenoxybenzamine are nonselective antagonists whereas prazosin selectively blocks α1 adrenoceptors. Yohimbine is a plant alkaloid with selective α2-antagonist action. Interest has continued in the possibility of using α-adrenergic antagonists in the treatment of asthma as, in some patients, the administration of an α agonist such as methoxamine provokes airway narrowing (Black et al. 1984). There is also the longstanding observation of increased α-adrenergic responsiveness in patients with allergic disorders. Overall, the response to αadrenergic antagonists in clinical trials has been disappointing (Barnes 1986), although the possibility of individual responses to α-adrenergic antagonists still exists.

Acknowledgment Supported by the Canadian Institutes of Health Research.

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Cholinergic Antagonists Nicholas J. Gross

Summary Anticholinergic agents have important uses as bronchodilators, particularly in chronic obstructive pulmonary disease (COPD). They dilate the central airways by relaxing peribronchial smooth muscle using biochemical pathways that are distinct from other bronchodilators. Currently approved agents of this class are synthetic quaternary ammonium compounds and thus very poorly absorbed following inhalation. Ipratropium and oxitropium are relatively short-acting (4–8 hours). Tiotropium was introduced in the early 2000s and is unique in having a duration of action in excess of 24 hours, making it ideally suited for once-a-day routine use. Clinical studies show that, with daily routine use, tiotropium improves both airway function and lung hyperinflation. It also reduces symptoms and improves exercise tolerance and quality of life by clinically meaningful amounts. In patients with moderate or severe COPD, it also reduces the frequency of acute exacerbations and hospitalization by about 20–25%. All agents of this class have a wide therapeutic margin and are safe and well tolerated.

this was due to the discovery of adrenergic agents and methylxanthines, but the fact that atropine and its congeners have a narrow therapeutic margin and their use resulted in many systemic side effects. Interest in the use of anticholinergic agents as bronchodilators was revived by physiologic studies which showed that the airways, both of animals and humans, were largely under the control of the cholinergic parasympathetic nervous system. Moreover, many bronchospastic stimuli acted through cholinergic reflexes (Widdicombe 1979; Nadel 1980). Inhibition of these reflexes by anticholinergic agents prevented or relieved bronchospasm. The resulting need for anticholinergic agents that could be effective when taken by inhalation and that lacked widespread systemic effects led to the development of ipratropium and its approval specifically as a bronchodilator in the 1980s. Subsequently, longer-acting agents such as oxitropium and tiotropium have been developed and several other anticholinergic bronchodilators are in development.

Rationale for use of anticholinergic agents Introduction Cholinergic antagonists form an important class of agents in the treatment of respiratory disorders. Their use goes back millennia in several Asian countries where many alkaloids with anticholinergic properties are found and available in indigenous plants (Gandevia 1975). The leaves of stramonium and belladonna, for example, were eaten or smoked releasing atropine and related agents. These traditional herbal remedies were imported and used in Western medicine until the early part of the 20th century, together with moreor-less purified extracts of their bronchoactive components. However, their use declined in the 1920s and 1930s. Partly

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Autonomic control of airways The human airways of large and intermediate size are surrounded by smooth muscle whose tone, a low level of contraction, controls the caliber of the airways. The smooth muscle is supplied by branches of the efferent autonomic system, almost all of which derive from the vagus nerve, a branch of the parasympathetic nervous system (Richardson 1982). These preganglionic cholinergic nerves enter the lung at the hila and divide with the airways, ending as varicosities in peribronchial ganglia where they synapse with short postganglionic fibers that supply cholinergic drive to the peribronchial smooth muscle. They also supply other airway structures such as mucosal cells and possible the ciliated epithelial cells although the latter is not certain. So-called cholinergic drive is limited to central and intermediate airways where cholinergic nerve endings and the relevant receptors are found and results in contraction of peribronchial smooth muscle, increased release of mucus from mucosal cells in the epithelium, and possibly increased bronchial ciliary activity.

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The low level of cholinergic bronchomotor tone that is present at rest (and whose role in health is poorly understood) can be augmented in a phasic manner via neural reflex pathways by a number of factors. Stimulation of irritant receptors and C fibers in a wide territory of the lower and upper airways, and possibly the esophagus and carotid bodies, results in the rapid onset (within seconds) of bronchoconstriction and increased mucus production in the central airways. Stimuli which can initiate these effects include mechanical irritation, a variety of irritant gases, aerosols and particles, rapid temperature changes, allergens, and several mediators such as histamine and some eicosanoids (Widdicombe 1979; Nadel 1980; reviewed by Gross & Skorodin 1984a). The bronchoconstriction resulting from these stimuli can be shown to be inhibitable or reversed at least in part by administration of atropine, supporting the notion that cholinergic mechanisms are responsible. This provides the rationale for the use of anticholinergic agents in diseases of airways obstruction. In addition there is indirect evidence that resting bronchomotor tone is increased in both asthma (Shah et al. 1990) and chronic obstructive pulmonary disease (COPD) (Gross et al. 1989a). Indeed, as discussed below (section Protection against bronchospastic stimuli), inhibiting bronchomotor tone may be the only means of producing bronchodilatation in COPD.

Muscarinic receptor subtypes Muscarinic receptors are a family of typical transmembrane protein receptors, three of which (M1, M2, and M3) are expressed in the human lung (Fig. 29.1). Our understanding of their interrelated functions is not complete (Gross & Barnes 1988; Fryer & Jacoby 1991, 1993; Coulson & Fryer 2003). The current concept is that M1 receptors are located in the peribronchial ganglia where they facilitate transmission of cholinergic traffic or possibly expand cholinergic activity to adjacent cells. M3 receptors are located on the effector cells, namely smooth muscle cells, bronchial mucosal cells, and possibly bronchial epithelial cells at which sites they mediate bronchoconstriction and other cholinergic actions. M2 recep-

Pharmacology and available agents The naturally occurring atropine-like alkaloids are all tertiary ammonium compounds, a designation which refers to the

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tors are located on postganglionic nerves themselves and, in contrast, limit release of acetylcholine from those nerve terminals when stimulated by acetylcholine. Thus they have an autoreceptor action, braking or modulating cholinergic activity. The difference in activity of M1 and M3 receptors that mediate and promote bronchoconstriction on the one hand, and M2 receptors which tend to limit it on the other, has bearing on the development and clinical properties of selective anticholinergic agents as discussed in the next section. The important role of M2 receptors in limiting cholinergic activity may also be said to represent a potential vulnerability. Fryer and Jacoby have shown that M2 receptors are selectively damaged by parainfluenza virus infections (Fryer & Jacoby 1991) and by exposure to eosinophil products (Fryer & Jacoby 1993), providing an explanation for the bronchospasm commonly seen in association with these clinical situations. The distribution of muscarinic receptors in airways has been mapped by autoradiographic methods (Mak & Barnes 1990). Consistent with the location of vagal nerve endings, all three muscarinic receptor subtypes are located predominantly in the central airways. There are also some M1 receptors in the alveolar regions; their physiologic function is unknown. It should be stated that the rationale for the use of anticholinergic agents in diseases of airways is limited to their bronchodilator action by the inhibition of smooth muscle contraction. They have little or no effect on the numerous other mechanisms of airways obstruction. Although they have been shown to have some antiinflammatory actions in vitro (Morr 1979; Sato et al. 1998), the relevance of these to clinical practice is uncertain. In similar vein, experimental evidence from guinea-pig airways suggests that regular anticholinergic administration may decrease airway remodeling (Gosens et al. 2005).

SMOOTH MUSCLE

3

2

3

2

3

Fig. 29.1 Muscarinic receptor subtypes in airways. M1 receptors are localized to parasympathetic ganglia, M2 receptors (autoreceptors) are found on postganglionic cholinergic nerves, and M3 receptors on airway smooth muscle and mucus-producing cells.

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N

CH3 + CH3 N

CH3

O O Fig. 29.2 Structures of atropine sulfate, a tertiary ammonium anticholinergic, and tiotropium bromide, a quaternary ammonium anticholinergic.

Cholinergic Antagonists

C

CH2OH O

CH

ATROPINE

valence of the nitrogen atom on the tropane ring (Fig. 29.2). Consequently, atropine, scopolamine and other members of that family of alkaloids are freely soluble in aqueous media and lipids and cross mucosal surfaces and the blood–brain barrier rapidly and quantitatively. Following either inhalation or oral ingestion they are thus very widely distributed throughout the body and counteract multiple homeostatic actions of the parasympathetic system, producing dose-related systemic effects. This limits their clinical utility. Atropine inhalation, for example, causes bronchodilation in adult doses of about 1.0– 2.0 mg. At the same dose, patients are quite likely to experience dryness of the mouth, skin flushing, and mild tachycardia. At only slightly larger doses effects such as urinary retention, blurred vision, and mental changes can occur. Because of their very narrow therapeutic margin, tertiary ammonium anticholinergic agents are no longer recommended for purposes of bronchodilation. On the other hand, quaternary ammonium compounds, all of which are synthetic, have a five-valent nitrogen atom in the tropane ring which carries a charge. They are only moderately soluble, and cross lipid barriers with difficulty. They have local anticholinergic activity and will, for example, dilate the airways if inhaled or dilate the pupil when instilled in the eye. However, they are not significantly absorbed into the systemic circulation from either site even when delivered in large doses (Gross & Skorodin 1985). They can thus be regarded as topical anticholinergic agents and have a very wide therapeutic margin. This makes them well suited for use as bronchodilators. The class of quaternary anticholinergic agents includes ipratropium bromide (Atrovent), oxitropium bromide (Oxivent) and, most recently, tiotropium bromide (Spiriva). Other related quaternary agents that have been studied but not approved for routine clinical use as bronchodilators include atropine methonitrate, glycopyrrolate bromide (Robinul), and several long-acting agents in various stages of development that have only been identified by numbers as yet. Pharmacokinetic studies on the quaternary ammonium agents in humans show that, following oral or inhaled doses, the serum levels are relatively low, with peak blood levels being obtained at about 1–2 hours and half-lives of about 4 hours. However, blood level values do not reflect the bio-

O TIOTROPIUM

O

OH

C

C

S Br+ · H2O

S

Table 29.1 Dissociation half-lives (hours) of ipratropium and tiotropium on muscarinic receptor subtypes in Chinese hamster ovary cells. (From Disse et al. 1993, with permission.)

Ipratropium Tiotropium

M1

M2

M3

0.11 14.6

0.035 3.6

0.26 34.7

logical half-lives of the agents. The half-life of the agent on the muscarinic receptor is a more important determinant of the duration of activity of the agent. Table 29.1 shows the dissociation half-lifes of ipratropium at each of the relevant muscarinic receptors in comparison with those of tiotropium (Disse et al. 1993). The notable difference between the agents is the very much longer half-life of tiotropium than ipratropium on the M3 receptor. Consistent with this, the duration of bronchodilator effect of ipratropium is approximately 4–6 hours, and that of tiotropium is longer than 24 hours. (The corresponding value for oxitropium 6–8 hours, while that of glycopyrrolate is quite long, possibly also 24 hours.) Another notable feature of the data in Table 29.1 is the relatively short half-lifes of ipratropium and tiotropium on the M2 receptor. The significance of this is that these agents are functionally selective antagonists of M1 and M3 receptors. This is seen as an advantage over nonselective antimuscarinic agents such as atropine in that M3 antagonism is desirable from a therapeutic viewpoint and retention of M2 activity is also desirable. Concerning the pharmacodynamics of current antimuscarinic agents, as relatively small amounts are absorbed into the systemic circulation following inhalation or oral ingestion, exposure of bodily systems to quaternary agents is minimal, again in contrast to atropine and other tertiary ammonium compounds which are rapidly absorbed and widely distributed. Effects on the eye such as pupillary dilatation, leading to blurred vision and possibly acute glaucoma in susceptible individuals, or urinary retention in men, are very unlikely and rarely seen in practice. Furthermore, for the same reasons, very little crosses the blood–brain barrier to reach the central nervous system.

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The dose–response of anticholinergic agents given by various inhalational methods was provided in a previous review (Gross & Skorodin 1987) and is summarized only briefly here. For ipratropium by metered-dose inhaler (MDI), the optimal dose in young adults with asthma is 40–80 μg, but in older patients with COPD the optimal dose is much higher, possibly 160 μg, particularly when airways obstruction is severe (Gross et al. 1989b). That for oxitropium MDI is similar. By nebulized solution, the optimal dose of ipratropium is 500 μg in adults and 125– 250 μg in children (Gross et al. 1989b). With inhalers that employ a dry powder form without propellants the optimal dose may be a little lower; thus 10 μg of ipratropium delivered by Turbohaler was equipotent to 20 μg delivered by MDI (Bollert et al. 1997). The optimal dose of glycopyrrolate is 0.02 mg/kg. Dose-ranging studies of tiotropium dry powder administered by the Handihaler device showed little dose–response between 9 and 80 μg (Maesen et al. 1995; Littner et al. 2000). The approved dose of 18 μg once daily is also the optimal dose. Combinations of bronchodilators often result in greater bronchodilation than do single agents with less potential for adverse effects. Fixed-dose combinations of ipratropium and a short-acting β agonist, Combivent, Berodual and DuoVent, have been in wide use for both COPD and asthma since the l980s. Use of a fixed combination of ipratropium and albuterol for COPD, either in a metered-dose formulation (Petty 1994; Ikeda et al. 1995; Combivent Study Group 1997) or in a nebulized formulation, DuoNeb (Gross et al. 1998), shows that the combination results in greater bronchodilation than each of its components, although the duration of action is not significantly prolonged. The greater bronchodilation achieved by the combination was achieved without increasing the risk of side effects. Recently, combinations of tiotropium and the long-acting β agonist formoterol have been explored (Cazzola et al. 2005; Richter et al. 2006; van Noord et al. 2006). No such fixed combination is currently commercially available.

Clinical uses Protection against bronchospastic stimuli This topic is relevant mainly in asthmatic subjects. The protection afforded by anticholinergic agents against specific bronchospastic stimuli in a research setting has been reviewed (Gross & Skorodin 1984a). The degree of protection varies, being more or less complete against cholinergic agonists such as methacholine, but somewhat less against histamine, prostaglandins, nonspecific dusts and irritant aerosols, exercise, and hyperventilation due to cold dry air in asthmatic subjects (Ayala & Ahmed 1989; Azevedo et al. 1990). Ipratropium has no prophylactic effect against leukotriene-induced bronchoconstriction (Ayala et al. 1988). Ipratropium can prevent bronchospasm induced by beta-blocking agents, where

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β-adrenergic agents tend to be ineffective, and by psychogenic factors.

In stable COPD Stable COPD is the condition for which anticholinergic agents offer the most benefit and the prime indication for their use. Although patients with COPD usually do not exhibit as much response to any bronchodilator as do patients with asthma, most are capable of a clinically meaningful bronchodilator response (Gross 1986). A large number of studies have compared anticholinergic agents with other bronchodilators in patients with COPD (Thiessen & Pedersen 1982; Passamonte & Martinez 1984; Gross 1988). Most show that the anticholinergic agent is a more potent bronchodilator than other agents in COPD (Bleecker & Britt 1991; Braun et al. 1989; Tashkin et al. 1986). After large cumulative doses, an anticholinergic agent alone achieved all the available bronchodilatation in subjects with predominantly emphysema (Gross & Skorodin 1984b). In studies where bronchodilator responsiveness was compared between patients with asthma and COPD who had similar baseline airflows, patients with bronchitis had a better response to ipratropium than to adrenergic agents, the reverse being true for patients with asthma (Lefcoe et al. 1982). The possible explanation for the difference in responsiveness to an anticholinergic agent may be that in asthma, airflow obstruction has multiple mechanisms including mucosal inflammation and swelling besides smooth muscle contraction. Many of these mechanisms are at least partially modified by adrenergic agents but not by anticholinergics. In COPD, the major reversible component, and perhaps the only one, is bronchomotor tone which, being cholinergic in origin, is best reversed by an anticholinergic agent. Increases in forced expiratory volume in 1 s (FEV1) of 100–250 mL following optimal dosage of an anticholinergic agent can be seen in typical COPD patients who are in stable condition; and these changes are also typically associated with reductions in hyperinflation and increases in inspiratory capacity (Celli et al. 2003). The role of ipratropium for symptomatic relief and bronchodilation in COPD is now well established and ipratropium is currently recommended as a first-line treatment option for stable COPD in most current guidelines (Celli & MacNee 2004; GOLD 2006). The newer anticholinergic agent, tiotropium bromide, has somewhat different properties that merit separate consideration, as reviewed by Gross (2004). The prolonged half-life of tiotropium on the M3 receptor has been referred to previously. This confers the very long duration of action, more than 24 hours, on the agent. Following once-daily use, the trough FEV1, namely the FEV1 shortly before each daily administration, is consistently 100–200 mL greater than at the original baseline before the first dose (Casaburi et al. 2002; Vincken et al. 2002). The elevation in trough and postdose FEV1 seen after 6 weeks of regular once-

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Day 1

1.6

Cholinergic Antagonists

Day 8 Tiotropium day 8

Fig. 29.3 FEV1 profiles of tiotropium and ipratropium on day 1 and day 8 of a 1-year trial showing the increase in trough level seen with tiotropium. (From Vincken et al. 2002, with permission from the European Respiratory Journal.) (See CD-ROM for color version.)

Tiotropium day 1 Ipratropium day 1 Ipratropium day 8

1.4

1.3

Tiotropium (n = 329)

1.2

Ipratropium (n = 161) 1.1 –60

daily tiotropium were similar to those of twice-daily salmeterol plus fluticasone in a pilot study involving 107 patients with COPD (Bateman et al. 2006). The elevation in trough FEV1 is due to persistence of bronchodilatation from the previous doses, or carry-over of effect (Fig. 29.3). Not only is the trough FEV1 increased but so is the plateau level seen at about 3 hours after administration, albeit by a relatively small amount. This round-the-clock persistence of bronchodilatation is thought to provide better symptom control than the saw-tooth rise and fall seen in the 4–6 hours following repeated administrations of short-acting bronchodilators. This change in the FEV1 profile occurs over the first 2–4 days of regular once-daily administration of the drug (van Noord et al. 2002) and is not seen with other currently available long-acting bronchodilators. Trough forced vital capacity (FVC) rises in the same way but continues over the first 8 days of tiotropium administration. In the same way that airway function improves day-by-day following treatment initiation, it takes approximately 3 weeks to return to its original baseline following discontinuation (Littner et al. 2000). Another unique feature of tiotropium is that regular daily use has not been associated with any loss of efficacy or tachyphylaxis over a 1-year period of continuous treatment; there is even the as yet unconfirmed impression that the baseline FEV1 does not fall as much as would be expected from the usual age-related rate of FEV1 decline (Casaburi et al. 2002; Vincken et al. 2002). The latter impression is the subject of a 4-year ongoing trial (UPLIFT). (A similar long-term study with ipratropium failed to show any decrease in the agerelated decline of lung function (Anthonisen et al. 1994).) Along with the improvement in airflow, Celli et al. (2003) reported a decrease in static lung volumes consistent with a reduction in the static hyperinflation typically associated with moderate to severe COPD (Fig. 29.4). This would be expected to provide symptomatic relief from breathlessness and indeed the transition dyspnea index (TDI) of Mahler et al. (1996) was shown, in each of the 1-year studies cited above,

–5

30

60 Time (min)

120

Tiotropium

600

180

Placebo

400 200 0 mL

FEV1 (L)

1.5

–200 –400 –600 –800

FEV1

FVC

IC

FRC

SVC

Fig. 29.4 Changes in spirometry and static lung volumes following 4 weeks’ treatment of COPD patients with tiotropium or placebo. All differences between treatments were statistically significant (P < 0.01). (From Celli et al. 2003, with permission from Chest.)

to show a clinically meaningful decline in those patients who received tiotropium. Other studies show that regular tiotropium use also decreases the dynamic hyperinflation associated with moderate and severe COPD (O’Donnell et al. 2004; Maltais et al. 2005), dynamic hyperinflation being the augmentation of static hyperinflation that develops within minutes of the onset of an increase in ventilation. O’Donnell et al. (2004), for example, in a double-blind study, found that endurance time on a cycle ergometer improved approximately 21% in the COPD patients who had received tiotropium for 6 weeks as compared to the group that received placebo. Associated with this effect were reductions in residual volume and functional residual capacity, and increases in FVC and inspiratory

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Placebo: Exercise

Maximal 10

Recovery

Tiotropium: Exercise

Very, very severe 9

Recovery

8 Very severe 7

* †

6 Severe 5

†

Somewhat severe 4 *

Moderate 3 Slight 2 Very slight 1 †

Nothing at all 0 0

2

4

6

8

10

12

Exercise time (min)

capacity. Maltais et al. (2005) performed a similar doubleblind study with 261 COPD patients who received tiotropium or placebo. After 6 weeks, patients performed constant work rate cycle ergometry 2.25 and 8 hours after receiving their morning treatments. Patients in the tiotropium group were able to continue almost 3 min longer (> 30% increase in endurance time) with less dyspnea (as measured on a Borg scale) at each time point, the differences being statistically significant (Fig. 29.5). In the same study, lung volumes were measured before and during the ergometry protocol; these showed that operating lung volumes were significantly lower at baseline and at the end of exercise in the tiotropium group. Thus, after 6 weeks of regular tiotropium treatment, both static and dynamic hyperinflation were significantly reduced as was the intensity of exercise-induced dyspnea. One other clinically important outcome that has been extensively studied with regular tiotropium administration is quality of life (health status). Quality of life has been measured in each of the long-term tiotropium studies and almost uniformly shows an improvement in health status that is not only statistically significant but also clinically meaningful. The improvement may be detected at first measurement, e.g., 8 weeks into regular therapy, and persists throughout the months of study (Donohue et al. 2002). In the latter study, tiotropium was statistically better than placebo, but not better than salmeterol. Sleep disturbance is common in patients with COPD. A randomized double-blinded study involving 36 patients with moderate to severe COPD showed that ipratropium increased total sleep time, decreased the severity of nocturnal desaturation, and improved the patient’s perceptions of sleep quality (Martin et al. 1999).

Acute exacerbations of COPD Two questions are considered: whether an anticholinergic agent should be used for the management of an acute exacer-

688

14

16

18

20

Fig. 29.5 Intensity of dyspnea (Borg scale) 8 hours after tiotropium or placebo during constant work rate cycle ergometry to symptom limitation at 75% Wmax and the first 5 min of recovery on day 42. *, P = 0.05; †, P < 0.01 difference between groups. (From Maltais et al. 2005, with permission from Chest.)

bation, and whether regular use of an anticholinergic agent reduces the frequency of acute exacerbations. Regarding the first question, four studies failed to discern a difference among adrenergic agents, anticholinergic agents, or their combination (Rebuck et al. 1987; Karpel et al. 1990; Patrick et al. 1990; Koutsogiannis & Kelly 2000). Current guidelines (Celli & MacNee 2004; GOLD 2006) recommend the use of a short-acting β agonist initially, to which an anticholinergic agent may be added if the episode is severe or if the response to the adrenergic agent alone is unsatisfactory. Whether maintenance use of an anticholinergic agent reduces the frequency of acute exacerbations of COPD has been a secondary outcome in many ipratropium trials and in all the long-term tiotropium trials previously mentioned. However, it has been definitively studied (as a primary outcome) in two large trials. Niewoehner et al. (2005) performed a prospective study of 1829 US Veterans with moderateto-severe COPD. Patients were randomly assigned to either tiotropium in the approved dose or placebo once daily and were followed for 6 months. Both co-primary outcomes, the percentage of patients with an acute exacerbation and the percentage of patients requiring a COPD-related hospitalization, were reduced by about 25%. The numbers needed to treat to prevent one of these events were 23 and 40 respectively. A very similar study in Europe on 1010 patients with moderate to severe COPD has since been reported by Dusser et al. (2005) with almost identical results. In a metaanalysis that included 15 276 patients in 22 selected trials, Salpeter et al. (2006) found that anticholinergics but not β agonists significantly reduced the frequency of a severe exacerbation or death. The relative risk of a severe exacerbation with an anticholinergic agent compared with placebo was 0.67 (95% CI 0.53–0.81); that for “death related to a lower respiratory event” was 0.27 (95% CI 0.09–0.81). Thus tiotropium not only improves lung physiology in COPD, but improves a wide variety of clinically relevant

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outcomes. Accordingly, tiotropium is recommended by guidelines as an appropriate long-acting bronchodilator treatment option in patients with moderate or worse COPD. Some practical consideration concerning tiotropium use in the clinic should be mentioned. Because of its slow onset of action, tiotropium is not appropriate for use as a rescue bronchodilator but rather, if it is to be used, should be used as a regular daily maintenance treatment. The recommended dose (18 μg q.a.m.) is optimal and should not be exceeded. Tiotropium has no unfavorable interactions with other bronchoactive agents and can be used concomitantly with any other respiratory drugs, e.g., formoterol as recently described by van Noord et al. (2006). However, it is not appropriate to use tiotropium with other anticholinergic bronchodilators. There are theoretical concerns that its access to muscarinic receptors may be blocked if it is coadministered with another anticholinergic. While this has never been shown in a clinical study, nor is there evidence of any safety concern, as a practical matter ipratropium has almost no bronchodilator potency when the patient is receiving regular tiotropium (Kerstjens et al. 2004). So the use of ipratropium would be a waste. If and when patients on regular tiotropium experience “breakthrough” dyspnea, they should take a short-acting β-adrenergic agent for relief. A bronchodilator reversibility test before starting regular tiotropium administration is not predictive of a beneficial response to tiotropium and is unnecessary (Tashkin & Kesten 2003). Tiotropium is not recommended for the management of acute exacerbations of COPD; however, if the patient is already receiving tiotropium on a maintenance basis, it can be continued through the exacerbation.

Adult asthma When the first quaternary ammonium anticholinergic agent, ipratropium bromide, was being developed, it was assumed that it would be used exclusively to treat asthma. Consequently, the literature contains a substantial number of studies comparing the bronchodilator potential of anticholinergic agents with that of other bronchodilators in patients with asthma. These studies mostly date to the 1970s and 1980s. Typically, they show that ipratropium had a relatively slow onset of action by comparison with a short-acting β agonist, and that, on average, the peak improvement in airflow was not as great as that seen with the β agonist but the duration of action tended to be perhaps an hour or two longer than that of the β agonist (Ruffin et al. 1977). Thus, anticholinergic agents tended not to find much use in the treatment of stable asthma except possibly as adjunctive treatment. Nor have they received an asthma indication in the USA and some other countries. Nevertheless, it has been the clinical experience of many physicians that occasional asthma patients respond quite well to ipratropium. The reasons for this are unclear and attempts to identify subgroups of asthmatics likely to benefit from a response to anticholinergic therapy

Cholinergic Antagonists

have not been very successful. The bronchodilating effect of ipratropium may increase with age, in contrast to the decline in response to albuterol (Ullah et al. 1981). Patients with intrinsic asthma and those with longer duration of asthma may also respond better than individuals with extrinsic asthma (Jolobe 1984). However, both of these factors appear to be poor predictors of a response. An individual trial has been suggested as the best way to identify responsiveness (Brown et al. 1984). There are at present no definitive publications of the effects of tiotropium in asthma, nor is this agent approved in the USA for the treatment of asthma. Ipratropium is probably most widely used for asthmatic bronchospasm as a component of a number of combinations with β-adrenergic agents, examples being with salbutamol, as Combivent MDI, or in combination with fenoterol as Berodual MDI or DuoVent MDI. However, there are two instances where ipratropium may have a uniquely valuable role in asthma. Ipratropium is capable of achieving bronchodilatation in situations where a β-adrenergic blocking agent has been administered to an asthmatic patient who then experiences bronchospasm (Grieco & Pierson 1971). The β agonists are of course of little use in this situation. The second instance is in the treatment of psychogenic bronchospasm. McFadden et al. (1969) showed that prior administration of atropine prevented the bronchospasm induced by suggestion in susceptible subjects, ipratropium not being clinically available at that time. Additionally, Rebuck and Marcus (1979) showed that subjects whose asthma was believed to be psychogenic in origin experienced better and more prolonged bronchodilatation to ipratropium than did subjects whose asthma was believed not to be of psychogenic origin. Mention has been made above to the possible antiinflammatory effects of anticholinergic agents (Morr 1979; Sato et al. 1998; Gosens et al. 2005). A recent review of this field (Kanazawa 2006) provides further laboratory evidence pointing to possible future exploitation of anticholinergic agents in the long-term control of asthma. In acute severe asthma (status asthmaticus) there is a clearer picture of the role of anticholinergic agents, particularly ipratropium. The β-adrenergic agonists are more effective bronchodilators in the setting of acute severe asthma, and an anticholinergic agent should not be the sole initial bronchodilator. However, an anticholinergic agent may add to the bronchodilatation achieved by adrenergic monotherapy. Rebuck et al. (1987) found that the combination of 500 μg nebulized ipratropium with 1.25 mg nebulized fenoterol resulted in significantly more bronchodilatation during the first 90 min of treatment than either agent alone. Moreover, patients with more severe airway obstruction obtained the greatest benefit from the combination. Other studies have addressed this same question and a metaanalysis of 10 such studies (total 1377 patients) concluded that the addition of ipratropium reduced hospital admissions (relative risk 0.73) and increased FEV1 by 7.5% (average 100 mL, 95% CI

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50–149 mL) more than groups that received a beta adrenergic agent alone (Stoodley et al. 1999). These benefits were both statistically and clinically significant. It seems appropriate to recommend that both classes of bronchodilators be given in acute severe asthma, especially in the early hours of treatment (Brophy et al. 1998), and particularly in patients with more severe airflow obstruction. They can be given separately, or in a fixed combination (e.g., Combivent by MDI with spacer, or DuoNeb by nebulization). Conventionally, two to three doses should be given in the first hour of treatment.

Pediatric asthma Evidence in support of the use of anticholinergic agents in stable childhood asthma is sparse by comparison with that in adults, and is mostly confined to relatively small short-term clinical studies. Two consensus reports reviewed the published evidence and concluded that although ipratropium was safe for the pediatric population, its benefit as monotherapy compared with adrenergic monotherapy was slight at best (Warner et al. 1989; Hargreave et al. 1990). As in adult asthma, there is evidence that the addition of an anticholinergic may augment the bronchodilation achieved by albuterol alone in children aged 10–18 years with stable asthma (Vichyanond et al. 1990). There are no systematic studies of long-acting anticholinergic agents in childhood asthma to my knowledge. There are also scattered reports of ipratropium use in other pediatric conditions such as cystic fibrosis, viral bronchiolitis, exercise-induced bronchospasm, and bronchopulmonary dysplasia, but these do not provide strong and consistent evidence for the benefit of ipratropium over alternative bronchodilators. For acute severe asthma in children, two well-conducted trials in the 1980s showed that the addition of ipratropium accelerated the rate of improvement in airflow over albuterol alone (Beck et al. 1985; Reisman et al. 1988). More recent studies have yielded conflicting results regarding the efficacy of combination therapy over monotherapy (Schuh et al. 1995; Ducharme & Davis 1998; Qureshi et al. 1998; Zorc et al. 1999). A systematic review of this question that included 10 selected studies (Plotnick & Ducharme 1998) concluded that combination therapy which included ipratropium was safe, improved lung function, and reduced hospitalization rates, especially in children with severe asthma. Therefore, as in adults, an anticholinergic alone is not recommended in acute severe asthma but the combination of ipratropium with an adrenergic agent is safe and may be more effective than adrenergic monotherapy, particularly in severe exacerbations.

Adverse effects Atropine and its natural congeners, being well absorbed and widely distributed in the body produce numerous systemic

690

side effects, which is the principal reason they are no longer used as bronchodilators. The quaternary agents that are currently used for respiratory purposes are poorly absorbed. Ipratropium bromide was carefully monitored for atropinelike adverse effects, particularly for effects on the eye (narrowangle glaucoma), urinary tract (urinary retention in males), and respiratory mucus transport. Ipratropium was found to be essentially free of such atropine-like effects after extensive investigation (Gross 1988). It can, for example, be given to patients with glaucoma without affecting intraocular tension, provided it is not sprayed directly into the eye (Watson et al. 1994). It has been found not to affect urinary flow characteristics in older men. Nor has it been found to alter the viscosity and elasticity of respiratory mucus, or mucociliary clearance, as does atropine (Pavia et al. 1979). It has negligible effects on hemodynamics, minute ventilation (Tobin et al. 1984), and the pulmonary circulation (Chapman et al. 1985). Consequently, administering ipratropium or tiotropium does not carry the risk of worsening hypoxemia (Gross & Bankwala 1987; Gross et al. 2003) as do adrenergic agents (Ashutosh et al. 1995), a theoretical consideration in exacerbations of asthma and COPD. Even massive, inadvertent overdosage of one such agent resulted in trivial effects (Gross & Skorodin 1985). In normal clinical use, the only side effects of ipratropium and tiotropium are dryness of the mouth, a typical local effect of anticholinergic agents. Sometimes a brief coughing spell occurs. Paradoxical bronchoconstriction occurs in perhaps 0.3% of patients that receive ipratropium. Paradoxical bronchoconstriction, which may also occur with other anticholinergic agents, warrants withdrawing the drug from that patient. Concerns about the high incidence of cardiovascular events in patients with COPD have led to studies of the cardiac effects of long-term bronchodilator use. Covelli et al. (2005) reported a study of 225 patients with moderately severe COPD who received tiotropium or placebo for 12 weeks. Serial cardiograms and Holter monitoring were performed. The investigators found no evidence of significant ECG changes, or changes in heart rate, rhythm, or QT intervals during the study. The frequency of cardiac events of any kind was low, serious adverse events occurring in one patient in the tiotropium group (onset of atrial fibrillation) and two patients in the placebo group. For similar purposes, a retrospective review of several long-term tiotropium trials was performed by Morganroth et al. (2004). In all, 1167 patients received tiotropium, 405 patients received salmeterol, and 846 patients received placebo for periods from 1 to 12 months of observation. In each study, serial ECGs and Holter monitoring were performed. No significant differences in any of the ECG or Holter outcomes were detected. Specifically, there were no clinically relevant arrhythmias or changes in heart rate or conduction patterns. Thus, extensive investigation and the worldwide use of ipratropium for over two decades and tiotropium for 3 years demonstrate a low incidence of untoward reactions.

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Antileukotriene Agents Graeme P. Currie and Brian J. Lipworth

Summary A variety of proinflammatory cells, mediators, and cytokines orchestrate the development of airway hyperresponsiveness, which results in the episodic airflow obstruction characteristic of asthma. As a consequence, modulation of the underlying disease process with antiinflammatory agents is firmly established as being the cornerstone of successful management. Inhaled corticosteroids are the most potent antiinflammatory agents available and satisfactorily suppress underlying airway inflammation in most individuals. However, despite regular treatment with inhaled corticosteroids, patients can experience persistent symptoms and exacerbations due to unchecked airway inflammation and hyperresponsiveness. Moreover, children and the elderly may have a dislike of inhaled treatment and demonstrate difficulties in coordinating some hand-held devices, while some individuals may also express a preference for oral medication. Cysteinyl leukotrienes are potent proinflammatory and bronchoconstrictor lipid mediators that perpetuate the asthmatic inflammatory response. Accumulating data suggest that inhaled corticosteroids fail to have a significant impact on cysteinyl leukotriene levels in the airway, which in turn has prompted the development and introduction of drugs (antileukotrienes) that attenuate their effects in the airway. For example, drugs such as 5-lipoxygenase inhibitors prevent the synthesis of cysteinyl leukotrienes and leukotriene receptor antagonists interfere with the binding of cysteinyl leukotrienes to their cellular receptor. Leukotriene receptor antagonists are used across the world and demonstrate a safe adverse-effect profile, although 5-lipoxygenase inhibitors are less widely used mainly due to multiple daily dosing regimes and concerns relating to hepatotoxicity. Over the past decade, leukotriene receptor antagonists have emerged as useful oral nonsteroidal antiinflammatory adjuncts both as monotherapy and in combination with other classes of drugs across the full spectrum of asthma severities. Moreover, they may have a particularly beneficial role in exercise-induced

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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asthma, aspirin-sensitive asthma, and individuals with concomitant allergic rhinitis. As more data emerges, it is becoming increasingly noticeable that monitoring the effects of leukotriene receptor antagonists in terms of lung function alone may result in clinicians missing potentially beneficial effects on inflammatory biomarkers, airway hyperresponsiveness, and subsequent exacerbations. Recent studies have also suggested that some individuals with asthma may be predisposed to exhibit a preferential response to antileukotrienes according to genotype, which may well tailor the decision to initiate such treatment in the future.

Introduction A variety of proinflammatory cells, mediators and cytokines orchestrate the development of airway hyperresponsiveness, which results in the episodic airflow obstruction characteristic of asthma. As a consequence, suppression of inflammation using inhaled corticosteroids forms the gold standard treatment and are advocated in all but the mildest of disease (British guideline on the management of asthma 2003; GINA Workshop Report 2004). Once bound to cytoplasmic receptors, inhaled corticosteroids increase and decrease the gene transcription of antiinflammatory and proinflammatory mediators respectively. They also exert a direct inhibitory effect on a number of cells such as eosinophils, T lymphocytes, and epithelial cells implicated in the asthmatic inflammatory process. As a result, they attenuate airway hyperresponsiveness over several weeks, although the maximal effect may not be achieved until after several months of regular use (Barnes 1990; Barnes et al. 1998). It is important to note that inhaled corticosteroids do not attenuate all the effects of inflammatory cells and mediators involved in the pathogenesis of asthma (Schleimer et al. 1989; Cox 1995; Gyllfors et al. 2006). Moreover, their effects on the synthesis and release of cysteinyl leukotrienes, which are potent bronchoconstrictor and proinflammatory mediators, are limited (Booth et al. 1995; Wenzel et al. 1997; Pavord et al. 1999). After being established on inhaled corticosteroids, additional second-line preventative therapy is advocated in cur-

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Phospholipid bilayer Phospholipase A2 Cyclooxygenase

Arachidonic acid

5-Lipoxygenase/ FLAP

Prostaglandins Thromboxanes

5-HPETE Leukotriene A4 hydrolase Leukotriene B4

5-Lipoxygenase/ FLAP Leukotriene A4 Leukotriene C4 synthase Leukotriene C4 g-Glutamyl transpeptidase

Fig. 30.1 Synthesis pathway of cysteinyl leukotrienes and their effects in the airway. FLAP, 5-lipoxygenase-activating protein; HPETE, 5-hydroxyperoxyeicosatetraenoic acid.

rent guidelines for patients with persistent symptoms (British guideline on the management of asthma 2003; GINA Workshop Report 2004). The use of concomitant second-line therapy has become increasingly important as the dose–response effect (in terms of lung function) for inhaled corticosteroids such as fluticasone propionate becomes relatively flat at daily doses greater than 500 μg (Holt et al. 2001). Moreover, there is a greater propensity to develop systemic adverse effects with increasing doses (Lipworth 1999), while patients may express a preference for treatment that avoids an excessive corticosteroid burden. This all suggests that alternative nonsteroidal antiinflammatory therapy may play an increasingly prominent role in the successful management of asthma. Over the past few decades, a substantive amount of data has emerged indicating that cysteinyl leukotrienes, a family of bioactive fatty acids, play an important part in mediating crucial aspects of the asthmatic inflammatory cascade (Drazen 1998). In this chapter we provide a summary of the pharmacologic and clinical effects of drugs which block the actions of cysteinyl leukotrienes in the airway (antileukotrienes). We also highlight the role of antileukotriene drugs across a variety of asthma phenotypes and severities, and of their effects when used as both monotherapy and concomitant second-line treatment.

Pathophysiology Cysteinyl leukotriene biosynthesis The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) are lipid mediators produced from an arachidonic acid precursor fol-

Cysteinyl leukotriene receptor

Leukotriene D4 Dipeptidase Leukotriene E4

lowing a series of enzymatic steps. Arachidonic acid is firstly released from the phospholipid bilayer by phospholipase A2 and may be metabolized by either the cyclooxygenase (COX) or 5-lipoxygenase pathway (Fig. 30.1) (Lam et al. 1990; Horwitz et al. 1998). Once the unstable precursor LTA4 has been produced, it may be converted in neutrophils or monocytes to the noncysteinyl LTB4 by LTA4 hydrolase. In mast cells, eosinophils, macrophages, and basophils, LTA4 may alternatively be converted into LTC4 by LTC4 synthase and subsequently into LTD4 and LTE4. The cysteinyl leukotrienes then exert their effects following activation of specific receptors located on cell membranes of airway smooth muscle and macrophages. LTB4 activates the BLT receptor, while LTC4, LTD4 and LTE4 activate cysteinyl leukotriene receptor subtypes 1 and 2.

Effects of cysteinyl leukotrienes The cysteinyl leukotrienes exhibit a variety of effects associated with perpetuating the asthmatic inflammatory process (Table 30.1). LTC4, LTD4 and LTE4 are also potent bronchoconstrictor stimuli and have been shown to increase the

Table 30.1 Effects of cysteinyl leukotrienes in asthma leading to airway hyperresponsiveness and airflow obstruction. Mucus hypersecretion Inhibition of mucociliary clearance Hypertrophy and proliferation of smooth muscle Increased pulmonary vascular permeability Recruitment of inflammatory cells Release of acetylcholine from nerve fibers

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extent of airway hyperresponsiveness following exposure to histamine and methacholine (O’Hickey et al. 1991). Cysteinyl leukotrienes are also found in sputum, urine, nasal secretions, plasma, and bronchoalveolar lavage fluid following bronchial challenge tests and in individuals with spontaneous exacerbations of asthma (Lam et al. 1988; Wenzel et al. 1990; Wenzel et al. 1995). LTB4 is a neutrophil chemoattractant and is involved in the production of interleukin (IL)-5 (Yamaoka & Kolb 1993), although not thought to be closely implicated in the pathophysiology of asthma. The cysteinyl leukotrienes therefore appear to be potentially important mediators intrinsically associated with the asthmatic inflammatory response, and this knowledge has prompted the development and subsequent introduction of drugs which attenuate their effects in the airway.

Antileukotriene drugs Two main strategies have been developed to block the effects of cysteinyl leukotrienes in the airway. One method is to use drugs that prevent their synthesis (using a 5-lipoxygenase inhibitor) and the other involves interfering with the binding of cysteinyl leukotrienes to their cellular receptor using a leukotriene receptor antagonist (LTRA).

Leukotriene receptor antagonists LTRAs such as montelukast (the most commonly used), zafirlukast, and pranlukast selectively antagonize the cysteinyl leukotriene 1 receptor. These drugs have been shown to reduce airway hyperresponsiveness following exposure to both direct and indirect bronchoconstrictor stimuli. For example, in a metaanalysis of 12 trials (N = 353 subjects with asthma) (Currie & Lipworth 2002), significant effects on attenuating airway hyperresponsiveness was observed amounting to a 0.85 doubling dose/dilution shift (95% CI 0.69–1.02) from baseline. LTRAs have also been shown to reduce a variety of surrogate inflammatory biomarkers such as airway and blood eosinophils, eosinophil cationic protein, and exhaled nitric oxide (Yoshida et al. 2000; Wilson et al. 2001a; Minoguchi et al. 2002; Bjermer et al. 2003; Currie et al. 2003a; Ilowite et al. 2004). Moreover, LTRAs are effective following single oral doses (Dempsey et al. 2000), and unlike long acting β2 agonists (Grove & Lipworth 1995), tolerance to their bronchoprotective effects has not been demonstrated (Wilson et al. 2001a; Sims et al. 2003). There are also animal data which suggest that LTRAs may significantly reduce parameters of airway remodeling (Henderson et al. 2002).

it is generally no longer widely prescribed mainly due to concerns regarding hepatotoxicity. Far fewer data have been published regarding the putative effect of 5-lipoxygenase inhibitors and effects on surrogate inflammatory biomarkers and airway hyperresponsiveness. However, there is evidence in both animals and asthmatics that zileuton does have inhibitory effects on eosinophils (Munoz et al. 1997; Dahlen et al. 1998). Single doses of zileuton have been shown to significantly attenuate airway hyperresponsiveness to direct bronchoconstrictor stimuli (Dekhuijzen et al. 1997). For example, in a randomized, double-blind, placebo study in mild asthmatics receiving inhaled corticosteroids, a single dose of zileuton 400 mg increased the provocative dose of histamine causing a 20% fall in forced expiratory volume in 1 s (FEV1) by 2.1 doubling doses compared with placebo (P < 0.03). Chronic dosing with zileuton has also been shown to attenuate airway hyperresponsiveness to a significant degree (Fischer et al. 1995).

Prescribing antileukotrienes In the UK, Europe and the USA, montelukast is licensed for once-daily oral administration in adults and is also available as a cherry flavored pink tablet or as granules for use in children over the age of 6 months. Zafirlukast is licensed for use in individuals over 12 years of age (Fig. 30.2). In some countries such as Japan, another LTRA, pranlukast, is available for use. In the USA, zileuton is licensed for use in those over 12 years of age (Table 30.2).

CH3

Zafirlukast

N

O N H

O

CH3 O N H O

O

S O CH3

COONa

Montelukast

OH S

5-Lipoxygenase inhibitors These drugs are divided into those which bind and deactivate 5-lipoxygenase-activating protein or directly inhibit 5lipoxygenase. Zileuton is the only 5-lipoxygenase inhibitor available for use in some countries such as the USA. However,

696

N CI

Fig. 30.2 The chemical structures of zafirlukast and montelukast.

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CHAPTER 30

Antileukotriene Agents

Table 30.2 Adult prescribing information and pharmacokinetics relating to antileukotrienes.

Use in pregnancy Protein binding Half-life Time to peak levels Bioavailability

Montelukast Singulair Inhibitor of leukotriene receptor 10 mg daily Yes No dose adjustment No dose adjustment in mild-tomoderate dysfunction Limited data > 99% 2.7–5.5 hours 3–4 hours 64%

Special instructions Interaction with warfarin

Can be taken with food Not known to interact

Generic name Brand name Mode of action Adult dose Pediatric licence Prescribing in renal impairment Prescribing in hepatic impairment

Advantages of oral treatment over inhaled treatment Patients with asthma notoriously find difficulty in using inhalers. For example, children and the elderly frequently have problems coordinating some hand-held devices, while spacers (which are designed to help overcome this problem) tend to be cumbersome, less portable, and pose difficulties in optimal use and care. This in turn may lead to suboptimal adherence to inhaled treatment regimens. Patients started on inhaled drugs for asthma require initial education and subsequent reinforcement in terms of correct use of a particular device. As a consequence, it is conceivable that oral asthma medication (such as with antileukotrienes) is advantageous in the “real world” and may confer some benefit over and above inhaled antiinflammatory therapy. Indeed, previous data have shown that patient compliance and acceptance of treatment are significantly superior for oral compared with inhaled regimens (Kelloway et al. 1994; Bukstein et al. 2003).

Adverse effects LTRAs are generally well tolerated (Storms et al. 2001), even in children (Price 2001). However, adverse effects such as hypersensitivity reactions, arthralgia, pulmonary eosinophilia, gastrointestinal disturbances, sleep disorders, respiratory tract infections, hallucinations, seizures, and raised transaminases have been reported (www.bnf.org). Due to lack of data, their use in pregnancy is not advised. Concerns have been raised regarding the development of Churg–Strauss syndrome and administration of LTRAs (Hosker & Tuggey 2001). Many, although not all, of the documented cases of Churg–Strauss syndrome have been in patients in whom concomitant LTRA treatment has permitted a reduction in dose of inhaled corticosteroid. This in turn suggests that latent Churg–Strauss syndrome may have been unmasked by a reduction in anti-

Zafirlukast Accolate Inhibitor of leukotriene receptor 20 mg twice daily No No dose adjustment Reduced clearance

Zileuton Zyflo 5-lipoxygenase inhibitor 600 mg four times daily No No dose adjustment Contraindicated

Limited data > 99% 10 hours 3 hours Unknown, but reduced by 40% with food Avoid with food Increases prothrombin time

Limited data 93% 2.5 hours 1.7 hours Unknown Can be taken with food Increases prothrombin time

inflammatory therapy delivered to the lungs (Lipworth & Wilson 2001). Liver function derangement has been reported with zileuton (elevated transaminase levels) and may be a greater problem in females over 65 years of age and in those with preexisting elevated transaminases. As a consequence, liver function tests should be monitored and the drug discontinued if signs or symptoms of liver dysfunction were to develop. Other reported adverse effects include headache, dyspepsia, nausea, abdominal pain, leukopenia, asthenia, insomnia, rashes, and myalgia.

Current position of antileukotrienes Mild persistent asthmatics should preferably be treated with a low to medium dose (400–800 μg/day of beclomethasone dipropionate or equivalent) of inhaled corticosteroid, although guidelines suggest that an alternative option, especially in individuals with prominent exercise-related symptoms, is monotherapy with an antileukotriene. When symptoms persist, despite good compliance with inhaled corticosteroids, patients should preferentially be started on a long-acting β2 agonist. An antileukotriene should then be considered if symptoms persist or the therapeutic trial of long-acting β2 agonist is unsuccessful (British guideline on the management of asthma 2003; GINA Workshop Report 2004).

Antileukotrienes as monotherapy in asthma Patients frequently express a preference for oral rather than inhaled treatments, which can translate into greater acceptance and better compliance with oral formulations of antiasthma drugs (Kelloway et al. 1994). It therefore appears logical to consider whether antileukotrienes might be a satisfactory

697

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Pharmacology

nonsteroidal antiinflammatory alternative to inhaled corticosteroids as first-line controller therapy in mild persistent asthmatics. In a double-blind, multicenter, placebo-controlled trial, 146 mild-to-moderate asthmatics not using inhaled corticosteroids were randomized to receive zafirlukast 20 mg twice daily or placebo for 13 weeks (Suissa et al. 1997). The zafirlukast group (mean FEV1 74% predicted) had 89% more days without symptoms (P = 0.03), 89% more days without reliever use (P = 0.001), and 98% more days without episodes of asthma (P = 0.003) compared to the placebo group (mean FEV1 84% predicted). Individuals randomized to receive zafirlukast also experienced 55% fewer healthcare contacts (P = 0.007) and 55% fewer days of absence from work or school (P = 0.04). Thus, the clinical and putative economic benefits associated with zafirlukast in this study indicate a possible role for LTRAs as monotherapy in patients with mild to moderate asthma. Another study collected data from five randomized double-blind trials where the effects of zafirlukast 20 mg twice daily was compared to placebo in corticosteroid-naive asthmatics in terms of effects on exacerbations (Barnes & Miller 2000). Overall, the risk of an asthma exacerbation requiring withdrawal from zafirlukast-treated groups was approximately half that of placebo (odds ratio 0.45, 95% CI 0.26– 0.76; P = 0.003). Similar beneficial effects with zafirlukast versus placebo were also observed for exacerbations requiring additional medication (odds ratio 0.47, 95% CI 0.30–0.74; P = 0.001) and oral corticosteroid use (odds ratio 0.53, 95% CI 0.32– 0.86; P = 0.010). Studies with montelukast have also shown that benefits occur when LTRAs are used as monotherapy (Laviolette et al. 1999; Malmstrom et al. 1999). For example, 895 asthmatics (mean FEV1 65% predicted) were randomized to receive montelukast 10 mg/day, inhaled beclomethasone 400 μg/day or placebo (Malmstrom et al. 1999). Over the 12-week treatment period, the average improvement from baseline in FEV1 was 13.1% with beclomethasone, 7.4% with montelukast, and 0.7% with placebo (P < 0.001 for each active treatment vs. placebo and P < 0.01 for beclomethasone vs. montelukast). The average reduction from baseline in daytime symptom score was 0.62 for beclomethasone, 0.41 for montelukast, and 0.17 for placebo (P < 0.001 for each active treatment vs. placebo and P < 0.01 for beclomethasone vs. montelukast). Both active treatments significantly improved peak expiratory flow (PEF) and quality of life, reduced nocturnal awakenings, and decreased the number of days with asthma exacerbations (P < 0.001 for each active treatment vs. placebo and P < 0.01 for beclomethasone vs. montelukast for each end point). Treatment with montelukast and inhaled beclomethasone also resulted in similar significant reductions in blood eosinophils (P < 0.05 vs. placebo). Thus, beclomethasone demonstrated superior clinical benefits versus montelukast, although montelukast conferred significant superiority over placebo. In the same study, montelukast had a faster onset

698

*

8 * Fold difference from placebo at 4 weeks

PART 3

4 *

*

2

1

0.5 Methacholine

AMP

Fig. 30.3 Effects on airway hyperresponsiveness with methacholine and adenosine monophosphate (AMP). Geometric fold differences (95% CI) from placebo for inhaled corticosteroid (open bars) and leukotriene receptor antagonist (blue bars) are shown. *, P < 0.05 vs. placebo. (From Dempsey et al. 2002, with permission.)

of action and a greater initial effect than beclomethasone. In another study comparing once-daily hydrofluoroalkane triamcinolone 450 μg/day versus montelukast 10 mg/day as monotherapy in mild persistent asthma, significant and similar improvements were observed on the primary outcome of airway hyperresponsiveness to methacholine and adenosine monophosphate after 4 weeks (Fig. 30.3) (Dempsey et al. 2002). However, triamcinolone was superior to montelukast in attenuating other surrogate inflammatory biomarkers such as exhaled nitric oxide, blood eosinophils, and eosinophil cationic protein. Other head-to-head comparisons of inhaled corticosteroids with LTRAs have tended to favor the former across a variety of end points. For example, a metaanalysis of 13 trials demonstrated that inhaled corticosteroids equivalent to beclomethasone 400 μg/day were more effective than LTRAs in adults with mild or moderate asthma (Ducharme 2003). Individuals treated with LTRAs were 60% more likely to experience an exacerbation requiring oral corticosteroids (relative risk 1.6, 95% CI 1.2–2.2), while a 130-mL greater improvement (95% CI 80–170 mL) in FEV1 and a 19 L/min greater increase (95% CI 14–24 L) in morning PEF was found when inhaled corticosteroids were compared to LTRAs. However, in one large study over 6 weeks evaluating mild asthmatics (mean FEV1 67% predicted, N = 782), montelukast was as effective as 400 μg/day of inhaled beclomethasone in preventing asthma exacerbations (Israel et al. 2002), despite the latter being significantly superior in improving the FEV1. This in turn leads to the notion that monitoring lung function alone may be a poor indicator of overall asthma control (Teeter & Bleecker 1998), and doing so may miss potentially beneficial effects. A disconnection between effects of antiinflammatory

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CHAPTER 30

treatment on lung function and exacerbations has also been observed in more severe asthmatics (Pauwels et al. 1997). For example, a fourfold increase from 200 to 800 μg/day of budesonide conferred no further improvement in lung function but did result in a further significant reduction in exacerbations (P < 0.001) (Pauwels et al. 1997). Thus, when optimizing antiinflammatory treatment in asthma, lung function is relatively distant from the underlying inflammatory process, and despite little change further beneficial clinical effects may actually occur. Thus, while end points such as lung function are of undoubted value, clinicians must not lose sight of the basic pathophysiologic hallmarks of the asthma syndrome (i.e., airway hyperresponsiveness and inflammation) along with the impact of asthma pharmacotherapy on exacerbations. Indeed, since LTRAs principally exhibit antiinflammatory actions and attenuate airway hyperresponsiveness, and are only weak bronchodilators, monitoring lung function alone may miss beneficial effects occurring in both “real-life” and in randomized trials (Currie et al. 2003a; Currie & Lee 2005). The current literature presently suggests that when used as first-line therapy, LTRAs confer significant improvements in parameters of asthma control, although they tend to be clinically inferior to low to moderate doses of inhaled corticosteroids. Asthmatics with mild disease, especially those with exercise-induced symptoms (Leff et al. 1998), who are disinclined to use regular inhaled corticosteroids, LTRAs appear to be suitable asthma controller therapy. As several inhaled corticosteroids are now licensed for once daily use, including easy to use breath-actuated devices (e.g., budesonide dry powder, mometasone dry powder, ciclesonide pressurized metereddose inhalers), there would appear to be little rationale for choosing an antileukotriene as first-line antiinflammatory therapy in persistent asthmatics, unless patients are unable or unwilling to use inhaler devices. However, one potential advantage of using montelukast in concomitant atopic asthma and rhinitis is its efficacy in allergic rhinitis which would not occur with orally inhaled corticosteroids alone.

Antileukotrienes as add-on therapy to inhaled corticosteroids in asthma Despite optimum drug delivery and good compliance with inhaled corticosteroids, many patients experience symptoms and exacerbations. Dose–response studies using inhaled corticosteroids have generally been unable to demonstrate any significant difference between individual doses of inhaled corticosteroids (Holt et al. 2001; Masoli et al. 2005). For example, a metaanalysis evaluated eight studies (2324 asthmatics) where the effects of at least two doses of fluticasone were measured (Holt et al. 2001). Most therapeutic gain (in terms of airway caliber, symptoms, and effects on exacerbations) was achieved at daily doses of fluticasone between 100 and 250 μg. Indeed, it is generally accepted that at daily doses

Antileukotriene Agents

greater than 800 μg of beclomethasone or equivalent in adults, the dose–response curve for desired effects becomes flat, while that for systemic adverse effects becomes steep (Lipworth & Wilson 1998). Since inhaled corticosteroids have only limited impact on the synthesis or release of cysteinyl leukotrienes (O’Shaughnessy et al. 1993; Dworski et al. 1994; Booth et al. 1995; Wenzel et al. 1997; Pavord et al. 1999), concomitant inhaled corticosteroid plus antileukotriene therapy may facilitate more complete attenuation of the inflammatory pathway. Moreover, even a high dose of inhaled fluticasone propionate has been shown to confer no significant effect on airway hyperresponsiveness to LTD4 or urine cysteinyl leukotriene concentrations (Gyllfors et al. 2006). Numerous studies, including a Cochrane metaanalysis, have suggested that LTRAs are useful as add-on therapy to inhaled corticosteroids across a range of outcome parameters (Laviolette et al. 1999; Lofdahl et al. 1999; Virchow et al. 2000; Ducharme 2002; Vaquerizo et al. 2003). For example, in a 16-week, multicenter, double-blind study, the efficacy of adding montelukast to a constant dose of inhaled budesonide (400 –1600 μg/day) was evaluated (Vaquerizo et al. 2003). A total of 639 symptomatic asthmatics (mean FEV1 81% predicted) were randomized to receive add-on montelukast 10 mg/day (N = 326) or placebo (N = 313). The frequency of asthma exacerbation days was 35% lower (P = 0.03) and asthma-free days was 56% higher (P = 0.001) in the montelukast versus placebo group. Individuals receiving montelukast also benefited from significant reductions in nighttime symptoms and reliever use (P < 0.05), and a significant improvement in PEF (P < 0.05) compared to placebo. In the same study, improvements in asthma control occurred despite no significant improvement in FEV1. This may well be because the mean FEV1 at baseline was within the lower limits of normal (81%) thus allowing little further room for improvement. Similar to the findings by Israel et al. (2002), this provides further evidence that FEV1 may not be the most sensitive parameter by which to detect improvements in asthma control. In other words, significant reductions in exacerbation frequency can occur (due to reduction in inflammation and airway hyperresponsiveness) without altering airway caliber to any great extent. Similar effects have also been observed with zafirlukast as add-on therapy to inhaled corticosteroids. In a double-blind parallel group study, 368 symptomatic asthmatics receiving high-dose inhaled corticosteroids (1000–4000 μg/day) were randomized to receive add-on zafirlukast 80 mg twice daily over 6 weeks (Virchow et al. 2000). Compared with placebo, zafirlukast produced a significant improvement over baseline in the primary end point of morning PEF (18.7 vs. 1.5 L/min, P < 0.001), in addition to evening PEF (P < 0.01), FEV1 (P < 0.05), daytime symptom scores (P < 0.001), and reliever use (P < 0.001). Furthermore, zafirlukast significantly reduced the risk of an exacerbation of asthma (odds ratio 0.61, 95% CI 0.38–0.99) and the risk of patients requiring a further

699

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Pharmacology

increase in asthma controller therapy (odds ratio 0.4, 95% CI 0.2–0.8). In another study, the inhaled corticosteroid-sparing effects of montelukast was evaluated in a double-blind, randomized, placebo-controlled, parallel group design (Lofdahl et al. 1999). Following a single blind placebo run-in period, during which at least two decreases in inhaled corticosteroid dose occurred, patients (N = 226) were randomized to receive montelukast 10 mg/day or matching placebo for up to 12 weeks. Every 2 weeks, the inhaled corticosteroid dose was tapered, maintained, or increased based on a standardized clinical score. Compared with placebo, montelukast allowed a significant (47% vs. 30%, P = 0.046) reduction in the inhaled corticosteroid dose. Although LTRAs confer additive effects when used as second-line therapy, few studies have directly examined the effects of LTRAs plus inhaled corticosteroid versus a higher dose of the latter. However, in a double-blind, randomized, parallel group, multicenter 16-week study, the effects of adding montelukast to budesonide, compared to doubling the budesonide dose in adult asthmatics was evaluated (Price et al. 2003). Following a 4-week run-in period, patients inadequately controlled on 800 μg/day of inhaled budesonide were randomized to receive add-on montelukast 10 mg (N = 448) or double the dose of budesonide (N = 441) for 12 weeks. Both groups showed progressive improvement in several measures of asthma control compared with baseline. For example, the mean PEF improved to a similar extent in the last 10 weeks of treatment compared with baseline in both the LTRA and higher inhaled corticosteroid dose group (34 vs. 30 L/min respectively). Moreover, patients in the former group experienced a quicker improvement in PEF. Both randomized treatments showed similar improvements in terms of reliever requirement, daytime symptom score, nocturnal awakenings, exacerbations, asthma-free days, blood eosinophils, and asthma-specific quality of life. The implication from this study is that adding montelukast conferred a steroid-sparing effect equivalent to 800 μg of budesonide, although no intermediate dose of budesonide (such as 1200 μg) was evaluated. In another study, adding twice-daily zafirlukast 20 mg to extra-fine hydrofluoroalkane beclomethasone dipropionate 100 μg/day produced the same effect as increasing the dose of beclomethasone alone to 400 μg/day in terms of attenuation of methacholine hyperresponsiveness and reducing exhaled breath nitric oxide (Fig. 30.4) (Dempsey et al. 2002).

Antileukotriene or long-acting b2 agonist as add-on therapy to inhaled corticosteroids in persistent asthmatics? Current internationally recognized guidelines indicate that symptomatic asthmatics using a low to medium inhaled

700

60 Per cent fall NO from baseline

PART 3

+ ‡

+ ‡

BDP 400 mg

BDP 100 mg + Zafirlukast

40

20

0 BDP 100 mg

BDP 100 mg + Theophylline

Fig. 30.4 Effects of beclomethasone (BDP) 100 mg/day, beclomethasone 400 mg/day, beclomethasone 100 mg/day plus zafirlukast, and beclomethasone 100 mg/day plus theophylline on exhaled nitric oxide (NO). Values are shown as percentage fall from pretreatment baseline. *, P < 0.05 vs. baseline; +, P < 0.05 vs. beclomethasone 100 mg/day alone. (From Dempsey et al. 2002, with permission.)

corticosteroid dose (400–800 μg/day of beclomethasone or equivalent) alone should preferentially be commenced on a long-acting β2 agonist prior to an LTRA (British guideline on the management of asthma 2003; GINA Workshop Report 2004). However, two recent large trials have performed headto-head comparisons of add-on long-acting β2 agonist versus LTRA as therapeutic adjuncts to inhaled corticosteroids, using exacerbation frequency (rather than lung function and symptoms) as the primary end point (Bjermer et al. 2003; Ilowite et al. 2004). Indeed, studies comparing the effects of either long-acting β2 agonist or LTRAs on measures of airway caliber, reliever use, and symptoms will tend to favor the former since they relax airway smooth muscle more completely and thereby exhibit superior bronchodilator effects. Moreover, since many trials evaluating the effects of asthma treatment require reversibility of at least 12% to short-acting β2 agonist to be demonstrated as a prerequisite to entry, long-acting β2 agonists will frequently appear superior to antiinflammatory therapy. In a study involving 1490 persistent asthmatics, the effects of montelukast 10 mg/day versus salmeterol was evaluated in patients whose symptoms were inadequately controlled with fluticasone 100 μg twice-daily alone (Bjermer et al. 2003). Following 52 weeks of treatment, 20.1% of patients receiving add-on montelukast had an asthma exacerbation compared with 19.1% in the group receiving add-on salmeterol, with no significant difference (95% CI −3.1 to 5%) between randomized treatments (Fig. 30.5). As expected, treatment with add-on salmeterol resulted in a significantly greater improvement in lung function, while treatment with montelukast resulted in a significantly greater reduction in peripheral blood eosinophils. A similar nonsignificant difference in exacerbation frequency was observed in another 52-week study evaluating effects of add-on montelukast 10 mg/day

9781405157209_4_030.qxd 4/1/08 17:33 Page 701

Per cent of patients with an asthma exacerbation

CHAPTER 30

25

Montelukast plus fluticasone Salmeterol plus fluticasone

20

15

10

5

0 0

12

24 36 Weeks since randomization

48

Fig. 30.5 Effects of fluticasone plus salmeterol or montelukast in terms of cumulative percentage of patients with asthma exacerbations (P = 0.599 by log rank test). (From Bjermer et al. 2003, with permission.) (See CD-ROM for color version.)

versus salmeterol in 1473 randomized asthmatics using inhaled corticosteroids (Ilowite et al. 2004); 80% of patients receiving montelukast and 83.3% of patients receiving salmeterol remained free of exacerbations during the treatment period (95% CI 0.96–1.49 for the difference between randomized

Antileukotriene Agents

treatments). In the same study, montelukast significantly reduced blood eosinophils compared with salmeterol, whereas salmeterol significantly increased FEV1, asthma-specific quality of life and morning PEF and decreased nocturnal awakenings compared with montelukast. It is pertinent to point out that most studies (Nelson et al. 2000; Fish et al. 2001; Nelson et al. 2001; Wilson et al. 2001a; Bjermer et al. 2003; Currie et al. 2003a; Ringdal et al. 2003; Ilowite et al. 2004; Storms et al. 2004) making head-tohead comparisons have demonstrated that the addition of an LTRA is generally as effective at reducing exacerbations as adding a long-acting β2 agonist to an inhaled corticosteroid (Table 30.3) (Currie et al. 2005a). However, add-on long-acting β2 agonist tends to be superior to add-on LTRA in improving lung function, while the latter treatment confers superior antiinflammatory activity and attenuates airway hyperresponsiveness to a greater extent (Currie et al. 2005a). Moreover, it could be argued that some of these studies were biased in favor of long-acting β2 agonists, since individuals were required to exhibit a high degree of reversibility to shortacting β2 agonist (salbutamol) as an entry requirement. In other words, adding a long-acting β2 agonist would always be destined to be more effective in terms of lung function in study participants who had been selected a priori to respond favorably.

Table 30.3 Studies comparing effects of long-acting b2 agonists with leukotriene receptor antagonists as add-on therapy to inhaled corticosteroids. Duration (weeks)

Treatment

ICS (mg)

FEV1

PEF

Inflammatory biomarker

AHR

Exacerbations

QOL or symptoms

13–14

SM vs. ML

606

NA

*LABA+

NA

NA

↔

LABA+

1490

52

SM vs. ML

200

LABA+

LABA+

LRTA+

NA

*↔

↔

PG

1473

52

SM vs. ML

220

LABA+

LABA+

LRTA+

NA

*↔

LABA+

XO

22

8

SM vs. ML

466

↔

LABA+

LRTA+

*LRTA+

NA

↔

PG

447

15

SM vs. ML

200

LABA+

*LABA+

NA

NA

LABA+

↔

XO

20

6

SM vs. ML

↔

↔

LRTA+

*↔

NA

↔

PG

725

16

SM vs. ML

800 (median) 200

LABA+

*LABA+

NA

NA

LABA+

LABA+

PG

122

6

SM vs. ML

200

*LABA+

NA

NA

NA

NA

↔

PG

429

4–6

SM vs. ZL

NA

LABA+

*LABA+

NA

NA

↔

LABA+

Reference

Design

N

Fish et al. (2001) Bjermer et al. (2003) Ilowite et al. (2004) Currie et al. (2003a) Nelson et al. (2000) Wilson et al. (2001a) Ringdal et al. (2003) Storms et al. (2004) Nelson et al. (2001)

PG

948

PG

PEF, peak expiratory flow; FEV1, forced expiratory volume in 1 s; QOL, quality of life; N, number of randomized subjects; ICS, mean daily inhaled corticosteroid dose at study entry (unless specified); SM, salmeterol; ML, montelukast; PG, parallel group; XO, crossover; AHR, airway hyperresponsiveness; LABA, long-acting b2 agonist; LTRA, leukotriene receptor antagonist. LABA+, significant superiority vs. LTRA; LTRA+, significant superiority vs. LABA; ↔, no significant difference between randomized treatments; *, primary end point; NA, not documented or measured.

701

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Pharmacology

PART 3

Adenosine monophosphate PC20 doubling dose difference vs placebo

2.5

2.0

1.5

*+

* *

1.0

NS 0.5

0.0 Montelukast 1 day

Salmeterol 2 wks

1 day

2 wks

Fig. 30.6 Means (SE) for the doubling dilution difference from placebo for the adenosine monophosphate threshold concentration after the first and last doses of montelukast and salmeterol. *, P < 0.05 difference from placebo; +, P < 0.05 between the first and last doses of each drug. (From Wilson et al. 2001a, with permission.)

A further issue encountered when prescribing long-acting β2 agonists (but not with antileukotrienes) is the development of tolerance to their bronchoprotective effects (Grove & Lipworth 1995). Regular use of long-acting β2 agonists results in β2-adrenoceptor downregulation, receptor internalization, and uncoupling of the G protein–adenyl cyclase unit with subsequent subsensitivity (or tachyphylaxis) of response to effects on airway smooth muscle and inflammatory cells. As

a result, an attenuated bronchoprotective response can be observed following exposure to different types of inhaled stimuli (Lipworth 1997). For instance, in a randomized, placebo-controlled, crossover study, 20 asthmatics not controlled on inhaled corticosteroids received either add-on montelukast 10 mg/day or salmeterol for 2 weeks each (Wilson et al. 2001a). For the primary end point of adenosine monophosphate threshold concentration (a marker of mast cell-mediated activity), compared to placebo there were significant differences with montelukast for first and last doses, but only after the first dose of salmeterol (Fig. 30.6). Similar results were also observed in another study where montelukast conferred sustained bronchoprotection against inhaled adenosine monophosphate over 2 weeks of treatment, whereas formoterol only conferred significant protection following first but not last dose (Sims et al. 2003). Whether these observations lead to future guidelines in asthma management acknowledging that addition of an LTRA is as effective in reducing exacerbations as adding a long-acting β2 agonist to inhaled corticosteroids remains to be seen. However, it appears reasonable to consider that in persistent asthmatics using inhaled corticosteroids who have impaired FEV1 (e.g., FEV1 < 80% predicted) and who exhibit a high degree of reversibility to short-acting β2 agonist, should proceed to have a therapeutic trial with a long acting β2-agonist as add-on therapy. However, perhaps those patients with preserved lung function (e.g., FEV1 > 80%), especially those with symptomatic allergic rhinitis or prominent exercise-induced symptoms, and who are therefore less likely to benefit from the bronchodilator effects of a longacting β2 agonist, should be started on an LTRA (Fig. 30.7).

Persistent asthmatic using 400–800 µg/day of inhaled corticosteroid (beclomethasone equivalent)

• • • •

Assess inhaler technique Check compliance Exclude avoidable trigger factors Exclude concomitant diseases

Persistent asthmatic with preserved FEV1, symptomatic allergic rhinitis or exerciseinduced symptoms

Persistent asthmatic with impaired FEV1 and with reversibility to short-acting b2 agonist > 12%

Add a LTRA

Add a LABA

Symptoms controlled?

Yes

No

No Add a LABA

702

Symptoms controlled?

Arrange further review

Add a LTRA

Fig. 30.7 Suggested algorithm for guiding clinicians as to whether a long-acting b2 agonist (LABA) or leukotriene receptor antagonist (LTRA) should be used as additional second-line therapy in persistent asthmatics using a low to moderate dose of inhaled corticosteroid. (From Currie et al. 2005a, with permission.)

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CHAPTER 30

Antileukotriene Agents

asthma severities, montelukast was observed to be an effective and well-tolerated treatment in everyday life in as many as 66% of individuals, including symptomatic individuals already using inhaled corticosteroids plus long-acting β2 agonists (Barnes et al. 2005). In a randomized placebo-controlled trial evaluating 72 moderate-to-severe asthmatics maintained on inhaled corticosteroids and mostly taking long-acting β2 agonists, the addition of montelukast 10 mg/day for 2 weeks conferred no significant improvement in terms of PEF and symptom scores (Robinson et al. 2001). A major limitation of the study was failure to evaluate any antiinflammatory biomarkers or airway hyperresponsiveness, while the relative short treatment duration would have precluded identification of any reduction in exacerbation frequency (Green & Pavord 2001). The effects of “triple therapy” with inhaled corticosteroid, long-acting β2 agonist plus LTRA was evaluated in 22 mild-to-moderate persistent asthmatics (mean FEV1 80% predicted) in a randomized, double-blind, placebo-controlled, crossover study (Currie et al. 2003a). After a 2-week run-in using twice-daily fluticasone propionate 250 μg with salmeterol, patients entered a randomized crossover period where they received additional montelukast 10 mg daily or identical placebo for 3 weeks each. Treatment with montelukast was significantly (P < 0.05) better than placebo in reducing airway hyperresponsiveness to adenosine monophosphate (Fig. 30.8a) and surrogate biomarkers of inflammation including exhaled nitric oxide (Fig. 30.8b) and blood eosinophils (Fig. 30.8c), despite conferring no further improvement in lung function. This in turn implies that serial monitoring of airway caliber does not necessarily reflect any potential benefit of nonsteroidal antiinflammatory treatment in patents already receiving inhaled corticosteroids and long-acting β2 agonists. Longer studies with greater numbers of patients

Antileukotrienes as add-on therapy to inhaled corticosteroids plus long-acting b2 agonists in persistent asthma Over and above inflammation of underlying endobronchial mucosa, intermittent bronchial smooth muscle dysfunction (generally responsive to β2 agonists) is a fundamental component in the overall asthma syndrome. This knowledge has led to increased use of inhalers using fixed combinations of corticosteroids and long-acting β2 agonists (Currie et al. 2005b), which facilitate patient compliance by reducing the numbers of inhalations and inhalers required. Current guidelines (British guideline on the management of asthma 2003; GINA Workshop Report 2004) advocate that in individuals with persistent symptoms despite regular use of fixed combination inhalers, the main options consist of a therapeutic trial of LTRA, oral theophylline, or higher dose of inhaled corticosteroid. In these symptomatic patients, the widespread use of theophylline is often limited due to unwanted drug interactions, adverse effects, and need to monitor plasma levels. Moreover, some patients are reluctant to use higher inhaled corticosteroid doses in view of greater risk of unwanted local and systemic sequeluae, while dose–response studies have suggested that little further therapeutic benefit may be gained from doing so (Holt et al. 2001; Masoli et al. 2005). It is important to point our that few studies have actually examined whether additional treatment with a LTRA in conjunction with an inhaled corticosteroid plus long-acting β2 agonist (“triple therapy”) does in fact confer further clinical benefit. However, in a large retrospective study (1351 patients) evaluating the effects of LTRAs across a range of

P < 0.05 1.5

P < 0.05

2

P < 0.05

P < 0.05

P < 0.05

0.5

FP/SM +ML

FP +ML

FP/SM +PL

FP +PL

0

–2

FP/SM +ML

FP +ML FP/SM +PL

FP +ML

FP/SM +PL

FP +PL

–100

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Fig. 30.8 Change from fluticasone/salmeterol (FP/SM) 250 mg b.i.d. run-in with add-on montelukast 10 mg (ML) or placebo (PL) for (a) AMP PC20 threshold doubling dilution shift, (b) exhaled nitric oxide, and (c) blood eosinophils. Montelukast or placebo was given with fluticasone (FP) alone or fluticasone plus salmeterol in combination (FP/SM). All changes are referred to baseline after run-in on FP/SM. (From Currie et al. 2003a, with permission.)

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Antileukotrienes in acute asthma Leukotrienes can be found in the airway and urine following both spontaneous exacerbations of asthma and acute exposure to bronchoconstrictor stimuli in the laboratory (Hui et al. 1991; Wenzel et al. 1995). This in turn indicates that they may have a role in the pathogenesis of acute episodes of bronchoconstriction. Although antileukotrienes are not currently advocated in the management of acute asthma, there are data to suggest that they might be of some potential benefit. Prior treatment with montelukast has been shown in several studies to significantly shorten the time taken to recover (in terms of FEV1) following exposure to a bronchoconstrictor stimulus (Brannan et al. 2001; Currie et al. 2003a,b). For example, in a study evaluating mild atopic asthmatics, single doses of montelukast 10 mg given 14 hours earlier, either alone or in combination with an antihistamine, resulted in a significantly (P < 0.05) shortened recovery time back to baseline FEV1 compared with placebo following indirect bronchial challenge with adenosine monophosphate (Fig. 30.9) (Currie et al. 2003b). This amounted to approximately 30 min of a difference compared to placebo. These data suggest that cysteinyl leukotrienes are important mediators in prolonging the bronchoconstrictor response following an acute episode of airway narrowing. The “real-life” implication of this was observed in a study by Camargo et al. (2003). In a doubleblind, parallel-group controlled trial, 210 patients with acute asthma were randomized to receive either intravenous montelukast (7 or 14 mg) or placebo along with standard treatment. Montelukast improved FEV1 over the first 20 min after intravenous administration (mean percentage change from baseline, 14.8% vs. 3.6% for pooled montelukast and placebo groups respectively; P = 0.007). Moreover, patients given active drug had a significantly quicker improvement in FEV1 over the entire 2-hour period and received less inhaled β2 agonist compared with placebo. In another study, the effects of zafirlukast was evaluated in patients with acute asthma over a 28-day follow-up period (Silverman et al. 2004). In a double-blind multicenter study, 641 acute asthmatics presenting to the emergency room were randomized to receive single doses of either 160 mg zafirlukast, 20 mg zafirlukast, or placebo as an adjunct to standard care. Patients who were discharged after 4 hours continued randomized treatment over 28 days and received either zafirlukast 20 mg twice daily (276 patients) or placebo (270 patients) in addition to prednisolone and their previous asthma medications. At the end of the emergency room treatment period, 16 of 162 patients (10%) treated initially

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100 Per cent subjects with FEV1 > 5% from baseline

are required to establish whether these preliminary findings extrapolate into long-term clinical advantages in asthma control such as reductions in exacerbation frequency and airway remodeling.

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Fig. 30.9 Percentage of patients during recovery with a fall in FEV1 > 5% from baseline following (a) adenosine monophosphate challenge and (b) mannitol challenge. ML, montelukast alone; ML/DL, montelukast plus desloratadine in combination. (From Currie et al. 2003b, with permission.)

with a single dose of zafirlukast 160 mg, 26 of 158 patients (17%) treated with a single dose of zafirlukast 20 mg, and 48 of 321 patients (15%) treated with placebo required extended care (P = 0.052 for the difference between zafirlukast 160 mg and placebo). Moreover, at the end of the 28-day treatment period, 65 of 276 patients (24%) treated with twice-daily zafirlukast 20 mg and 78 of 270 patients (29%) treated with placebo relapsed (P = 0.047 for the difference). These findings were supported by a significant improvement in lung function and reduction in breathlessness in the emergency room with zafirlukast 160 mg and with similar improvements during the 28-day treatment period for patients receiving twice-daily zafirlukast. Some benefit with LTRAs was also observed in a randomized, double-blind, placebo-controlled

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study evaluating 51 preschool children with a mild to moderate exacerbation of asthma (Harmanci et al. 2006). At presentation, subjects received a single dose of montelukast 4 mg or placebo in addition to inhaled salbutamol and were followed up over a 4-hour period. Active treatment was associated with a significantly lowered respiratory rate at all time points, while 38.5% in the placebo group versus 20.8% in the montelukast group required oral steroid (P = 0.22). Whether LTRAs prove to be useful adjuncts in the management of acute asthma (in conjunction with oral corticosteroids and nebulized bronchodilators) remains to be seen. Further studies are required to confirm whether administration of a LTRA along with conventional treatment confers any advantage over addition of intravenous magnesium or aminophylline in the setting of an acute episode of asthma. Moreover, whether treatment with an LTRA at the onset of an exacerbation of asthma confers clinically relevant and cost-effective advantages in the short and medium term needs prospective evaluation.

Antileukotrienes in allergic rhinitis Allergic rhinitis is a common inflammatory condition of the upper airway characterized by sneezing, nasal pruritus, rhinorrhea, and nasal obstruction (Haberal & Corey 2003). Current management strategies are centered around the use of allergen avoidance, immunotherapy, corticosteroids, and antihistamines. Since the upper and lower airways have a direct anatomic connection, share similar epithelial lining, and release similar inflammatory mediators (Busse 1996; Lipworth & White 2000), it has been suggested that asthma and allergic rhinitis represent a continuation of the same inflammatory disease process. Indeed, 20–40% of individuals with allergic rhinitis are thought to have concomitant asthma, while 30– 90% of asthmatics are thought to have allergic rhinitis (Simons 1999; Leynaert et al. 2000). Uncontrolled allergic rhinitis is known to precipitate and exacerbate asthma, with the inference that clinicians should positively search for typical nasal and ocular symptoms (Rachelefsky 1999). Moreover, successful treatment of allergic rhinitis can confer benefits in overall asthma control (Henriksen & Wenzel 1984; Reed et al. 1988; Ragab et al. 2006). Nasal allergen challenge has been shown to result in dose-related increased levels of cysteinyl leukotrienes in nasal lavage fluid (Naclerio et al. 1983; Creticos et al. 1984; Pipkorn et al. 1987), while other studies have demonstrated the importance of these mediators in individuals with allergic rhinitis following exposure to natural allergens (Skoner et al. 1990; Knani et al. 1992). Moreover, activated eosinophils and mast cells, both of which release cysteinyl leukotrienes, play a prominent role in the pathophysiology of allergic rhinitis (Knani et al. 1992). This knowledge has raised interest and prompted considerable research into the potential therapeutic role of antileu-

Antileukotriene Agents

kotrienes in individuals with allergic rhinitis both with and without concomitant asthma. It is important to note that montelukast has gained approval from the US Food and Drug Administration for the management of allergic rhinitis alone, although LTRAs are not currently licensed for this use in the UK. They can however be prescribed for patients with uncontrolled asthma who may have active symptoms of allergic rhinitis. Montelukast as monotherapy has been shown to be effective in the treatment of allergic rhinitis. For example, a study of 1302 patients with allergic rhinitis demonstrated that montelukast 10 mg/day improved daytime and nighttime nasal symptoms plus quality-of-life parameters (Philip et al. 2002). Moreover, the adverse-effect profile with montelukast was comparable to placebo. In two other large studies (van Adelsberg et al. 2003a,b), montelukast 10 mg/day was significantly superior in reducing symptoms of allergic rhinitis compared to placebo, and was similar to treatment with loratadine. A study of 1862 symptomatic patients with allergic rhinitis showed superiority of montelukast over placebo in improving nasal symptoms, and also demonstrated a superior response in individuals who were exposed to a higher pollen counts (Chervinsky et al. 2004). Similarly, zafirlukast has been shown to significantly reduce nasal symptoms, nasal resistance, and lavage eosinophil counts in patients with allergic rhinitis (Piatti et al. 2003). A recent systematic review and metaanalysis examined the efficacy of LTRAs in patients with allergic rhinitis incorporating 11 randomized controlled trials (Wilson et al. 2004). Treatment with LTRA reduced mean daily rhinitis symptom scores 5% (95% CI 3–7%) more than placebo. In the same study, antihistamines improved nasal symptom scores 2% (95% CI 0–4%) more than LTRAs, and nasal corticosteroids improved scores 12% (95% CI 5–18%) more than LTRAs. This all suggests that LTRAs appear to be superior to placebo, generally as effective as antihistamines, but less effective than topical corticosteroids in improving symptoms and quality of life in patients with allergic rhinitis. Similar findings were also obtained in an another metaanalysis evaluating the effects of LTRAs as monotherapy or when combined with other drugs in patients with allergic rhinitis (Rodrigo & Yanez 2006). Seventeen randomized controlled trials (N = 6231 subjects) were eligible for inclusion, with 16 of the studies using montelukast and one zafirlukast. LTRAs significantly reduced daytime and nighttime nasal symptoms and eye symptoms, and also significantly improved quality of life compared to placebo. Moreover, no significant differences were observed between LTRAs and antihistamines, although nasal corticosteroids were significantly superior to LTRAs. Data relating to the combined effects of LTRAs plus antihistamines in allergic rhinitis have shown conflicting results. One study of 460 patients with seasonal allergic rhinitis demonstrated that the combination of montelukast plus loratadine improved daytime nasal symptoms, while less benefit

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was observed with either drug as monotherapy (Meltzer et al. 2000). In contrast, in another larger study of 907 patients with seasonal allergic rhinitis, both drugs alone improved daytime nasal symptoms with no further additional benefit from the combination (Nayak et al. 2002). A study of 62 patients with seasonal allergic rhinitis found that the combination of montelukast and loratadine was no more effective than montelukast alone on daytime or nighttime nasal symptoms (Pullerits et al. 2002), while a study of 60 patients with seasonal allergic rhinitis found that the combination of montelukast and cetirizine was more effective than cetirizine alone (Kurowski et al. 2004). When compared to intranasal corticosteroids, the combination of LTRA plus antihistamine has been shown to be similar in some studies (Wilson et al. 2001b,c) and less effective in others (Pullerits et al. 2002; Saengpanich et al. 2003; Di Lorenzo et al. 2004) in terms of effects on nasal symptoms in allergic rhinitis.

Antileukotrienes in concomitant asthma and allergic rhinitis In a multicenter trial, the efficacy of antileukotrienes in individuals (N = 831) with both symptomatic allergic rhinitis and active asthma was evaluated (Philip et al. 2004). Subjects were randomized to receive daily montelukast 10 mg (N = 415) or placebo (N = 416) over a 2-week, double-blind treatment period. Montelukast significantly (P = 0.001) reduced rhinitis symptoms scores compared to placebo, with improvements also being observed in nasal and eye symptoms. Moreover, active treatment provided greater benefit in overall asthma control and reduced the requirement for reliever use. A post hoc analysis from the COMPACT study (Price et al. 2003) examined whether those asthmatics with concomitant allergic rhinitis responded differently depending on whether they were randomized to receive add-on montelukast (N = 216) or double the dose of inhaled corticosteroid (N = 184) (Price et al. 2006). In terms of the primary outcome, there was a 9.2% increase in morning PEF from baseline in the montelukast group compared with a 6% increase in the higher inhaled corticosteroid group (P = 0.028 for the difference), although secondary end points were not significantly different between randomized groups. Thus, in the subgroup of asthmatic patients with allergic rhinitis, a combined treatment approach that included montelukast and budesonide provided significantly greater efficacy in improving lung function compared with doubling the dose of budesonide. This in turn supports the notion that a unified approach aimed at treating airway inflammation common to both diseases is beneficial for the large proportion of asthmatics who have concomitant allergic rhinitis. In a double-dummy crossover study, patients with both asthma and concomitant allergic rhinitis were randomized to

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receive 14 days of 400 μg/day of orally inhaled budesonide plus 200 μg/day intranasal budesonide or once-daily 10 mg montelukast plus 10 mg oral cetirizine (Wilson et al. 2000). Both treatments were significantly (P < 0.05) better than placebo, but of similar efficacy in terms of improving lung function, symptoms and inflammatory biomarkers. In another study of patients with asthma and allergic rhinitis, montelukast was as effective as orally inhaled and intranasal budesonide on lower airway parameters, with both treatments conferring benefit on symptoms of allergic rhinitis (Wilson et al. 2001d). Whether in the future a therapeutic trial of LTRA becomes standard practice in individuals symptomatic of both upper and lower airways inflammation remains to be seen. Indeed, it could be argued that asthmatics using inhaled corticosteroids with concomitant allergic rhinitis requiring additional second-line therapy should preferentially be commenced on an LTRA (instead of a long-acting β2 agonist), which in turn may confer a positive effect on both the upper and lower airways (see Fig. 30.7).

Other uses of antileukotrienes Aspirin-induced asthma The prevalence of aspirin-sensitive asthma is uncertain although it may exist in up to 20% of all asthmatics (Jenkins et al. 2004). The characteristic features include profound bronchoconstriction following aspirin ingestion, rhinosinusitis, nasal polyps, and abdominal cramps. Aspirin and nonsteroidal antiinflammatory drugs selectively inhibit COX-1, which in turn shunts arachidonic acid down the 5-lipoxygenaseactivating protein pathway, causing overproduction of cysteinyl leukotrienes. As a consequence, elevated levels of cysteinyl leukotrienes can be found in bronchial and nasal aspirates, and in urine following aspirin challenge (Christie et al. 1991; Knapp et al. 1992). Moreover, the rate-limiting enzyme, LTC4 synthase, which converts LTA4 to LTC4, is overexpressed in eosinophils and mast cells in patients with aspirin-sensitive asthma (Cowburn et al. 1998). Since cysteinyl leukotrienes appear therefore to be important mediators in the pathogenesis of aspirin-sensitive asthma, it is logical to consider that antileukotrienes may prevent development of symptoms. In one study, 80 aspirin-sensitive asthmatics receiving moderate to high doses of inhaled corticosteroid were randomized to receive placebo or montelukast 10 mg/day for 4 weeks (Dahlen et al. 2002). Compared to placebo, significant improvements in FEV1 and morning PEF (P < 0.001) were observed along with reductions in symptoms and reliever use (P < 0.05). Other studies have specifically evaluated the airway response following exposure to aspirin (Christie et al. 1991; Dahlen et al. 1993; Israel et al. 1993; Lee et al. 2004). In one randomized study of 12 patients with aspirin-sensitive

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asthma, nasal lysine aspirin challenge was performed after single doses of montelukast 10 mg, montelukast 40 mg, or placebo taken 12 hours earlier (Lee et al. 2004). The former treatment partially protected against the local effects (in terms of peak nasal inspiratory flow) of nasal lysine aspirin challenge compared to placebo, with no further benefit being observed with the higher dose of montelukast. In another randomized crossover study, bronchial provocation with lysine aspirin was performed at baseline and 1 hour after placebo or single dose of another LTRA (MK-0679) (Dahlen et al. 1993). Following active treatment, a rightward shift in the dose–response relationship for all eight subjects was observed (median shift 4.4-fold), with three individuals failing to produce a 20% decrease in FEV1 despite inhalation of the highest dose of lysine aspirin. In aspirin-sensitive asthmatics, zileuton decreased baseline urinary LTE4 excretion (P < 0.02) and blunted the maximum increase in urinary LTE4 after ingestion of aspirin (P < 0.01) (Israel et al. 1993). In the same randomized crossover study in contrast to placebo, zileuton prevented any significant fall in FEV1 following ingestion of aspirin (P < 0.014). However, in a further small study evaluating aspirin-sensitive asthmatics, montelukast was only partially effective in preventing bronchoconstriction following exposure to aspirin (Stevenson et al. 2000). Similarly in another study, 3 weeks of montelukast 10 mg/day conferred an overall improvement in asthma control, although no differences in the clinical response between aspirin-sensitive and aspirin-tolerant individuals were observed (Mastalerz et al. 2002). Antileukotrienes therefore appear to be of some value in preventing the airway response following exposure to aspirin in predisposed asthmatics, although they do not appear to attenuate the effects completely. Further large-scale prospective studies are required to further delineate the place of chronic dosing with antileukotrienes, perhaps in conjunction with antagonists of other mediators such as histamine, in aspirin-sensitive asthmatics.

Exercise-induced asthma Many patients with asthma develop symptoms as a response to exercise. This is thought to be due to drying and cooling effects occurring in the airway, with the subsequent release of proinflammatory mediators such as cysteinyl leukotrienes and histamine (Anderson & Brannan 2002). Indeed, cysteinyl leukotrienes can be found in urine following exerciseinduced bronchoconstriction (Kikawa et al. 1992; Reiss et al. 1997). LTRAs and other inhibitors of the cysteinyl leukotriene pathway have been shown to protect against exerciseinduced bronchoconstriction in a number of studies in both adults and children (Meltzer et al. 1996; Bronsky et al. 1997; Reiss et al. 1997; Kemp et al. 1998; Leff et al. 1998; de Benedictis et al. 2006). For example, in 100 corticosteroid-naive asthmatics with a mean FEV1 of 83% predicted, the effects of montelukast 10 mg/day was evaluated over a 12-week period

Antileukotriene Agents

(Leff et al. 1998). Compared to placebo, montelukast was significantly superior in protecting against exercise-induced bronchoconstriction, while patients also experienced better asthma control during active treatment. In the same study, tolerance to its effects were not observed, which is often a problem encountered with long-acting β2 agonists (Ramage et al. 1994). In a double-blind multicenter trial, asthmatics (N = 122) with a history of exercise-induced symptoms uncontrolled on low-dose fluticasone were randomized to receive montelukast 10 mg/day, salmeterol, or placebo for 4 weeks (Storms et al. 2004). The aim of this study was to compare the effects of chronic dosing on airway responses following exercise challenge and rescue short β2 agonist. The maximum FEV1 after short-acting β2 agonist improved in the montelukast (1.5%) and placebo (1.2%) groups at 4 weeks, but diminished in the salmeterol group by 3.9% (P < 0.001 for salmeterol vs. placebo and montelukast). Although the pre-exercise FEV1 was greatest with salmeterol (P = 0.10), individuals taking montelukast had significantly greater protection from an exercise-induced decrease in FEV1 than those taking salmeterol (P < 0.001). Both the magnitude and rate of rescue bronchodilation were greater with montelukast compared with salmeterol (P < 0.001). Moreover, 5 min after rescue short-acting β2 agonist, 92% of patients taking montelukast and 68% of those taking placebo had recovered to preexercise levels, while only 50% of those taking salmeterol had recovered. In other words, add-on therapy with montelukast facilitated a greater and more rapid degree of rescue bronchodilation with short-acting β2 agonist than addition of salmeterol, and provided superior protection against exerciseinduced bronchoconstriction. In another randomized doubleblind study, the effects of 8 weeks of montelukast 10 mg/day versus salmeterol was evaluated in individuals with exerciseinduced asthma (N = 191, mean FEV1 88% predicted) who were not using inhaled corticosteroids. Montelukast provided significantly greater protection against exercise-induced bronchoconstriction versus salmeterol after both 4 weeks (P = 0.015 for the difference) and 8 weeks (P = 0.002 for the difference) (Edelman et al. 2000). Similar beneficial effects on attenuation of exerciseinduced bronchoconstriction have also been observed with zafirlukast (Dessanges et al. 1999). In a randomized, doubleblind, three-way, crossover trial, 20 mg or 80 mg twice daily of zafirlukast or placebo was given for 14 days with a 7-day washout period between treatments. Exercise challenges were performed at 2 and 8 hours after the morning dose on day 14. Compared to placebo, both doses of zafirlukast significantly reduced exercise-induced bronchoconstriction as measured by the area under the FEV1 time curve after the 2-hour (P < 0.001) and 8-hour (P < 0.001) exercise challenges and maximum fall in FEV1 at the 2-hour challenge (P < 0.001). Mannitol is a bronchoconstrictor stimulus which can be used to asses the extent of airway hyperresponsiveness in

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asthmatics (Anderson et al. 1997). When given as a dry powder, inhaled mannitol increases the surface osmolarity of the bronchial mucosa resulting in the release of inflammatory and bronchoconstrictor mediators from a variety of cells. Bronchoprovocation with mannitol has been shown to demonstrate a high degree of sensitivity and specificity to eucapnic voluntary hyperpnea, and as a result suggested to be an alternative and more convenient means by which to identify exercise-induced bronchoconstriction (Holzer et al. 2003). Several studies evaluating the effects of prior dosing with montelukast (Brannan et al. 2001; Currie et al. 2003b) have demonstrated a significantly quicker rate of recovery following bronchoprovocation with mannitol, in turn suggesting that such drugs may be advantageous in individuals with exercise-induced symptoms. These data all indicate that drugs which antagonize the effects of cysteinyl leukotriene pathway should be considered in patients who require prevention of symptoms related to activity. Moreover, LTRAs may facilitate a simple and effective treatment option in patients with exerciseinduced symptoms, when either used alone or in combination with inhaled corticosteroids. The fact that they are effective even following single doses (Currie et al. 2003b) suggests that they may be a particularly attractive pharmacologic option in individuals with only mild, intermittent exercise-induced asthma who do not require daily inhaled corticosteroids.

Pharmacogenetic determinants of responsiveness to antileukotrienes An interesting property of antileukotrienes in “real life” is their apparent ability to demonstrate preferential therapeutic effects in subgroups of asthmatics. This in turn has prompted research into possible pharmacogenetic determinants that may govern the response to antileukotriene agents. LTC4 synthase is the terminal and rate-limiting enzyme involved in the production of cysteinyl leukotrienes. Polymorphisms of this critical enzyme, characterized by adenine (A) to cytosine (C) translocations at the −444 nucleotide, have been identified and prompted investigation into whether they might be related to asthma severity or phenotype, or in determining a preferential response to antileukotrienes. In aspirin-sensitive asthmatics, the variant C allele has been reported to be present in as many as 76% of cases, compared with around 40% in normal individuals and asthmatics without aspirin sensitivity (Sanak et al. 1997). However, the association between polymorphisms of LTC4 synthase and asthma phenotype and severity is controversial, and has generally revealed conflicting results. One large study of mild, moderate, and severe asthmatics failed to demonstrate a link between the variant C allele and disease severity or sensitivity to aspirin (Kedda et al. 2004). Similarly

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in another study, no significant association was observed between the presence of the C allele and asthma severity or presence of aspirin intolerance (Isidoro-Garcia et al. 2005). Conversely, a study in children did show that, compared to controls, those possessing the C allele had a lower mean percent predicted FEV1 (97.4% vs. 92.7%, P = 0.005) (Sayers et al. 2003). Only a few studies have examined the effects of polymorphisms of LTC4 synthase and clinical response to antileukotriene treatment. In a study by Sampson et al. (2000), the effects of zafirlukast 20 mg twice daily for 2 weeks in 23 asthmatics was evaluated according to genotype. In those with the variant LTC4 synthase genotype (AC and CC, N = 13), FEV1 and forced vital capacity increased by 9% and 15% respectively with zafirlukast compared to baseline levels, whereas individuals with the wild type (AA, N = 10) had a falls of 12% and 18% respectively. Although the differences were nonsignificant in this small sample (P = 0.1), these findings did raise the possibility that individuals with the variant C allele might represent those with a preferential response to antileukotrienes. Furthermore, Asano et al. (2002) evaluated the effects of pranlukast 225 mg twice daily for 4 weeks in 48 patients with moderate-to-severe persistent asthma according to genotype. Those with genotypes AC or CC experienced a greater improvement in FEV1 (P = 0.01) following LTRA treatment compared to those with the wild type. In a retrospective analysis (Currie et al. 2003c), polymorphisms of LTC4 synthase were not associated with clinical response to LTRAs in terms of surrogate inflammatory biomarkers and lung function. However, it may not be surprising to discover that a single allelic variation does not determine the response to LTRAs, since cysteinyl leukotrienes are synthesized via a cascade of enzymes. Indeed, in patients using montelukast over a 6-month period, a variety of single nucleotide polymorphisms in different genes were found to be implicated in determining preferential response to montelukast in terms of improving FEV1 and reducing exacerbations (Lima et al. 2006). Further large prospective studies specifically designed to evaluate whether interindividual variability in response to antileukotrienes in asthmatics is determined according to genotype are required.

Conclusion Due to greater understanding of the underlying inflammatory process implicated in the pathogenesis of asthma, antileukotrienes, particularly LTRAs, have emerged as useful oral nonsteroidal antiinflammatory agents that are effective in patients with symptomatic allergic airways disease (Currie et al. 2005c). Accumulating evidence indicates that there is a role for antileukotrienes across a broad spectrum of asthma severities, both as monotherapy and in conjunction with other pharmacologic adjuncts. Indeed, although corticosteroids

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are the most potent antiinflammatory drugs available, they fail to completely attenuate all aspects of the inflammatory disease process and many patients remain symptomatic despite their use. This in turn suggests that when used in combination with inhaled corticosteroids, antileukotrienes may facilitate more complete attenuation of the inflammatory cascade. Moreover, they also appear to confer some benefit in asthmatics with aspirin sensitivity, exercise-related symptoms, and concomitant allergic rhinitis. There is little doubt that inhaled corticosteroids remain the cornerstone of asthma management in all but the mildest of disease. However, antileukotrienes do allow an alternative therapeutic option in mild asthmatics perhaps unwilling or unable to use low to medium doses of inhaled corticosteroids. Indeed, this approach may well be best suited in those with predominant exercise-related symptoms. In the “real-world” setting, patient preference with oral treatment and a superior adverse-effect profile compared to inhaled corticosteroids may in turn lead to better compliance rates and superior long-term effect on airway inflammation. Current guidelines (British guideline on the management of asthma 2003; GINA Workshop Report 2004) suggest that antileukotrienes should be considered when the combination of a low to medium inhaled corticosteroid dose plus long-acting β2 agonist fails to adequately control symptoms. Alternatively, LTRAs may be tried in patients maintained on inhaled corticosteroids after a failed therapeutic trial of long-acting β2 agonist. However, randomized controlled trials suggest that when evaluating parameters such as exacerbation frequency, LTRAs may in fact have similar efficacy as long-acting β2 agonists. Indeed, it is important to note that many patients already using inhaled corticosteroids have relatively preserved lung function, with the inference that underlying inflammation and airway hyperresponsiveness are the driving forces behind episodic airflow obstruction and persistent symptoms. In these patients, add-on therapy with an LTRA would therefore appear to be a reasonable therapeutic option in view of its dual actions of attenuating airway hyperresponsiveness and suppressing inflammation. Indeed, the use of a long-acting β2 agonist, which as a class lacks intrinsic antiinflammatory properties (Gardiner et al. 1994; Roberts et al. 1999), would do little to deal with these underlying problems. This option may also be particularly attractive since concerns have been raised regarding the long-term safety of long-acting β2 agonists and their associated risks of increased asthma exacerbations and deaths (Nelson et al. 2006; Salpeter et al. 2006). However, in individuals with persistent symptoms and impaired lung function (e.g., FEV1 < 80% predicted), adding in a long-acting β2 agonist, while keeping the inhaled corticosteroid dose the same, would appear to be a logical step. Indeed, further increases in antiinflammatory therapy (either with an LTRA or increased inhaled corticosteroid dose) would be unlikely to improve lung function (Holt et al. 2001), and a combined inhaled cor-

Antileukotriene Agents

ticosteroid plus long-acting β2 agonist inhaler would ensure maximal bronchodilation. While not universally licensed for use in patients with allergic rhinitis alone, LTRAs have demonstrated efficacy in asthmatics with concomitant upper airway inflammation. The treatment of “one airway” therefore appears a logical step by which to attenuate both lower and upper airway inflammation and symptoms. Moreover, this rationale would confer a more favorable adverse-effect profile when compared to patients using both inhaled and nasal corticosteroids. In conclusion, antileukotrienes are one of the first asthma drugs developed as a consequence of exploring the possibility of antagonizing the effects of a specific inflammatory mediator. They not only provide a further therapeutic tool with which to attenuate inflammation and airway hyperresponsiveness, but facilitate an orally active means to target the clinical burden of asthma in both primary and secondary care settings. Indeed, the challenge facing clinicians must surely be to decide where this class of drug sits most comfortably in stepwise treatment algorithms favored by guidelines.

Conflicts of interest G.P.C. has received funding from MSD (who make montelukast) and AstraZeneca (who make zafirlukast) for attending postgraduate international conferences, and for giving talks. B.J.L. has received funding from MSD for giving talks and grant support for clinical trials.

References Anderson, S.D., Brannan, J., Spring, J. et al. (1997) A new method for bronchial-provocation testing in asthmatic subjects using a dry powder of mannitol. Am J Respir Crit Care Med 156, 758–65. Anderson, S.D. & Brannan, J.D. (2002) Exercise-induced asthma: is there still a case for histamine? J Allergy Clin Immunol 109, 771– 3. Asano, K., Shiomi, T., Hasegawa, N. et al. (2002) Leukotriene C4 synthase gene A(−444)C polymorphism and clinical response to a CYS-LT(1) antagonist, pranlukast, in Japanese patients with moderate asthma. Pharmacogenetics 12, 565–70. British guideline on the management of asthma (2003) Thorax 58 (suppl. 1), i1–i94. Barnes, P.J. (1990) Effect of corticosteroids on airway hyperresponsiveness. Am Rev Respir Dis 141, S70–S76. Barnes, N.C. & Miller, C.J. (2000) Effect of leukotriene receptor antagonist therapy on the risk of asthma exacerbations in patients with mild to moderate asthma: an integrated analysis of zafirlukast trials. Thorax 55, 478–83. Barnes, P.J., Pedersen, S. & Busse, W.W. (1998) Efficacy and safety of inhaled corticosteroids. New developments. Am J Respir Crit Care Med 157, S1–S53. Barnes, N., Thomas, M., Price, D. & Tate, H. (2005) The national montelukast survey. J Allergy Clin Immunol 115, 47–54.

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Stevenson, D.D., Simon, R.A., Mathison, D.A. & Christiansen, S.C. (2000) Montelukast is only partially effective in inhibiting aspirin responses in aspirin-sensitive asthmatics. Ann Allergy Asthma Immunol 85, 477–82. Storms, W., Michele, T.M., Knorr, B. et al. (2001) Clinical safety and tolerability of montelukast, a leukotriene receptor antagonist, in controlled clinical trials in patients aged ≥ 6 years. Clin Exp Allergy 31, 77–87. Storms, W., Chervinsky, P., Ghannam, A.F., Bird, S., Hustad, C.M. & Edelman, J.M. (2004) A comparison of the effects of oral montelukast and inhaled salmeterol on response to rescue bronchodilation after challenge. Respir Med 98, 1051–62. Suissa, S., Dennis, R., Ernst, P., Sheehy, O. & Wood-Dauphinee, S. (1997) Effectiveness of the leukotriene receptor antagonist zafirlukast for mild-to-moderate asthma. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 126, 177–83. Teeter, J.G. & Bleecker, E.R. (1998) Relationship between airway obstruction and respiratory symptoms in adult asthmatics. Chest 113, 272–7. van Adelsberg, J., Philip, G., LaForce, C.F. et al. (2003a) Randomized controlled trial evaluating the clinical benefit of montelukast for treating spring seasonal allergic rhinitis. Ann Allergy Asthma Immunol 90, 214–22. van Adelsberg, J., Philip, G., Pedinoff, A.J. et al. (2003b) Montelukast improves symptoms of seasonal allergic rhinitis over a 4-week treatment period. Allergy 58, 1268–76. Vaquerizo, M.J., Casan, P., Castillo, J. et al. (2003) Effect of montelukast added to inhaled budesonide on control of mild to moderate asthma. Thorax 58, 204–10. Virchow, J.C. Jr, Prasse, A., Naya, I., Summerton, L. & Harris, A. (2000) Zafirlukast improves asthma control in patients receiving highdose inhaled corticosteroids. Am J Respir Crit Care Med 162, 578–85. Wenzel, S.E., Larsen, G.L., Johnston, K., Voelkel, N.F. & Westcott, J.Y. (1990) Elevated levels of leukotriene C4 in bronchoalveolar lavage fluid from atopic asthmatics after endobronchial allergen challenge. Am Rev Respir Dis 142, 112–19. Wenzel, S.E., Trudeau, J.B., Kaminsky, D.A., Cohn, J., Martin, R.J. & Westcott, J.Y. (1995) Effect of 5-lipoxygenase inhibition on bronchoconstriction and airway inflammation in nocturnal asthma. Am J Respir Crit Care Med 152, 897–905. Wenzel, S.E., Szefler, S.J., Leung, D.Y., Sloan, S.I., Rex, M.D. & Martin, R.J. (1997) Bronchoscopic evaluation of severe asthma. Persistent inflammation associated with high dose glucocorticoids. Am J Respir Crit Care Med 156, 737–43. Wilson, A.M., Orr, L.C., Sims, E.J., Dempsey, O.J. & Lipworth, B.J. (2000) Antiasthmatic effects of mediator blockade versus topical corticosteroids in allergic rhinitis and asthma. Am J Respir Crit Care Med 162, 1297–301. Wilson, A.M., Dempsey, O.J., Sims, E.J. & Lipworth, B.J. (2001a) Evaluation of salmeterol or montelukast as second-line therapy for asthma not controlled with inhaled corticosteroids. Chest 119, 1021–6. Wilson, A.M., Sims, E.J., Orr, L.C. et al. (2001b) Effects of topical corticosteroid and combined mediator blockade on domiciliary and laboratory measurements of nasal function in seasonal allergic rhinitis. Ann Allergy Asthma Immunol 87, 344–9. Wilson, A.M., Orr, L.C., Sims, E.J. & Lipworth, B.J. (2001c) Effects of monotherapy with intra-nasal corticosteroid or combined oral

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histamine and leukotriene receptor antagonists in seasonal allergic rhinitis. Clin Exp Allergy 31, 61– 8. Wilson, A.M., Dempsey, O.J., Sims, E.J. & Lipworth, B.J. (2001d) A comparison of topical budesonide and oral montelukast in seasonal allergic rhinitis and asthma. Clin Exp Allergy 31, 616–24. Wilson, A.M., O’Byrne, P.M. & Parameswaran, K. (2004) Leukotriene receptor antagonists for allergic rhinitis: a systematic review and meta-analysis. Am J Med 116, 338– 44.

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Glucocorticosteroids Peter J. Barnes

Summary

Table 31.1 Effect of glucocorticoids on gene transcription.

Glucocorticoids are the most effective controllers of asthma and other allergic diseases. They suppress inflammation by switching off multiple activated inflammatory genes through reversing histone acetylation via the recruitment of histone deacetylase 2. Through suppression of airway inflammation inhaled glucocorticoids reduce airway hyperresponsiveness and control asthma symptoms. Inhaled glucocorticoids are now first-line therapy for all patients with persistent asthma, controlling asthma symptoms and preventing exacerbations. Inhaled long-acting β2 agonists added to inhaled glucocorticoids further improve asthma control and are commonly given as combination inhalers, which improve compliance and control asthma at lower doses of glucocorticoid. Inhaled glucocorticoids, which are absorbed from the lungs into the systemic circulation, have negligible systemic side effects at the doses most patients require. Systemic glucocorticoids are used in the treatment of acute exacerbations of asthma and as maintenance treatment in patients with severe asthma not controlled by maximum inhaled therapy. Oral steroids have numerous metabolic and endocrine side effects and the lowest dose needed to control the disease should be used. A few patients with asthma are resistant to the antiinflammatory effects of glucocorticoids and several molecular mechanisms for this have recently been described.

Increased transcription Lipocortin 1 b2-Adrenoceptor Secretory leukocyte inhibitory protein IkB-a (inhibitor of NF-kB) Antiinflammatory or inhibitory cytokines: IL-10, IL-12, IL-1 receptor antagonist Mitogen-activated protein kinase phosphatase 1 (inhibits MAP kinase pathways) Decreased transcription Inflammatory cytokines: IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, IL-13, IL-15, TNF-a, GM-CSF, SCF Chemokines: IL-8, RANTES, MIP-1a, eotaxin Inducible nitric oxide synthase (iNOS) Inducible cyclooxygenase (COX-2) Inducible phospholipase A2 (cPLA2) Endothelin 1 NK1 receptors Adhesion molecules (ICAM-1,VCAM-1) ICAM, intercellular adhesion molecule; SCF, stem cell factor; VCAM, vascular cell adhesion molecule. See text for definition of other abbreviations.

Introduction

Mechanisms of action

Glucocorticosteroids (also known as glucocorticoids, corticosteroids, steroids) are by far the most effective controllers used in the treatment of asthma and the only drugs that can effectively suppress the characteristic inflammation in asthmatic airways. They are also highly effective in the treatment of rhinitis and other allergic diseases. In this chapter, the mechanism of action and pharmacology of glucocorticoids and their use in the treatment of asthma will be discussed.

There have been major advances in understanding the molecular mechanisms whereby glucocorticoids suppress inflammation, based on recent developments in understanding the fundamental mechanisms of gene transcription (Rhen & Cidlowski 2005; Barnes 2006a). Glucocorticoids activate and suppress many genes relevant to understanding their action in asthma and other allergic diseases (Table 31.1).

Cellular effects Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

At a cellular level glucocorticoids reduce the numbers of inflammatory cells in the airways, including eosinophils, T lymphocytes, mast cells, and dendritic cells (Fig. 31.1). These effects of glucocorticoids are produced through inhibiting the

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Inflammatory cells

Structural cells

Eosinophil ↓ Numbers (apoptosis)

Epithelial cell ↓ Cytokines mediators

T-lymphocyte ↓ Cytokines Mast cell

Endothelial cell ↓ Leak

CORTICOSTEROIDS

↓ Numbers

Airway smooth muscle ↑ b2-Receptors ↓ Cytokines

Macrophage ↓ Cytokines

Mucus gland Dendritic cell

Mucus ↓ secretion

↓ Numbers

Fig. 31.1 Cellular effect of glucocorticoids. (See CD-ROM for color version.)

recruitment of inflammatory cells into the airway by suppressing the production of chemotactic mediators and adhesion molecules and by inhibiting the survival in the airways of inflammatory cells, such as eosinophils, T lymphocytes, and mast cells. Epithelial cells may be the major cellular target for inhaled glucocorticoids, which are the mainstay of modern asthma management. Inhaled glucocorticoids suppress many activated inflammatory genes in airway epithelial cells (Fig. 31.2). Epithelial integrity is restored by regular inhaled glucocorticoids. The suppression of mucosal inflammation is relatively rapid, with a significant reduction in eosinophils detectable within 6 hours and associated with reduced airway hyperresponsiveness (AHR) (Gibson et al. 2001; Ketchell et al. 2002). Reversal of AHR may take several

Epithelial cells

Enzymes iNOS COX-2 cPLA2

Peptides ET-1

Adhesion molecules ICAM-1

↓ INFLAMMATION

Fig. 31.2 Inhaled glucocorticoids may inhibit the transcription of several “inflammatory” genes in airway epithelial cells and thus reduce inflammation in the airway wall. GM-CSF, granulocyte–macrophage colony-stimulating factor; IL, interleukin; iNOS, inducible nitric oxide synthase; COX-2, inducible cyclooxygenase; cPLA2, cytoplasmic phospholipase A2; ET, endothelin; ICAM, intercellular adhesion molecule.

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Glucocorticoid receptors Glucocorticoids diffuse across the cell membrane and bind to glucocorticoid receptor (GR) in the cytoplasm (Rhen & Cidlowski 2005). There is only one form of GR that binds glucocorticoids termed GRα. GRβ is an alternatively spliced form of GR that interacts with DNA but not with glucocorticoids, so may act as a dominant negative inhibitor of glucocorticoid action by interfering with the binding of GR to DNA (Lewis-Tuffin & Cidlowski 2006). Whether GRβ is involved in steroid resistance in asthma is controversial. Activated GRs rapidly translocate to the nucleus where they produce their molecular effects. A pair of GRs (GR dimer) bind to glucocorticoid response elements (GREs) in the promoter region of steroid-responsive genes and this interaction switches on (and sometimes switches off) gene transcription (Fig. 31.3). Examples of genes that are activated by glucocorticoids include genes encoding β2-adrenergic receptors and the antiinflammatory proteins secretory leukoprotease inhibitor and mitogenactivated protein kinase phosphatase-1 (MKP-1) which inhibits mitogen-activated protein (MAP) kinase pathways. These effects may contribute to the antiinflammatory actions Glucocorticoid

Glucocorticoid receptor trans-activation

GRE Antiinflammatory Annexin-1 SLPI MKP-1 IkB-a

Inhaled glucocorticoids

Cytokines IL-1b IL-6 GM-CSF RANTES Eotaxin MIP-1a

months to reach a plateau, probably reflecting recovery of structural changes in the airway (Juniper et al. 1990a).

cis-repression

Negative GRE Side effects POMC CRF-1 Osteocalcin Keratin

trans-repression NF-kB CBP Inflammatory Cytokines Chemokines Adhesion molecules Inflammatory enzymes Inflammatory receptors Inflammatory proteins

Fig. 31.3 Glucocorticoids may regulate gene expression in several ways. Glucocorticoids enter the cell to bind to glucocorticoid receptors (GR) in the cytoplasm that translocate to the nucleus. GR homodimers bind to glucocorticoid-response elements (GRE) in the promoter region of steroid-sensitive genes, which may encode antiinflammatory proteins. Less commonly, GR homodimers interact with negative GREs to suppress genes, particularly those linked to side effects of corticosteroids. Nuclear GR also interact with coactivator molecules, such as CREB-binding protein (CBP), which is activated by proinflammatory transcription factors such as nuclear factor (NF)-kB, thus switching off the inflammatory genes activated by these transcription factors. SLPI, secretory leukoprotease inhibitor; MKP-1, mitogen-activated kinase phosphatase-1; IkB-a, inhibitor of NF-kB; GILZ, glucocorticoid-induced leucine zipper protein; POMC, proopiomelanocortin; CRF, corticotrophin-releasing factor. (See CD-ROM for color version.)

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of glucocorticoids (Clark 2003; Barnes 2006b). GR interaction with negative GREs may suppress gene transcription and it is thought that this may be important in mediating many the side effects of glucocorticoids. For example, glucocorticoids inhibit the expression of osteocalcin involved in bone synthesis (Dostert & Heinzel 2004).

Switching off inflammation The major action of glucocorticoids is to switch off multiple activated inflammatory genes that encode for cytokines, chemokines, adhesion molecules, inflammatory enzymes, and receptors (Barnes & Adcock 2003). These genes are switched on in the airways by proinflammatory transcription factors, such as nuclear factor (NF)-κB and activator protein (AP)-1, both of which are activated in asthmatic airways and switch on inflammatory genes by interacting with coactivator molecules, such as CREB-binding protein, that have intrinsic histone acetyltransferase activity, resulting in acetylation of core histones, which opens up the chromatin structure so that gene transcription is facilitated (Barnes et al. 2005). In artificial overexpression systems, activated GR may directly interact with NF-κB and AP-1 to inhibit their activity, but this does not appear to occur in asthmatic patients treated with inhaled glucocorticoids (Hart et al. 2000). Glucocorticoidactivated GR also interact with coactivator molecules and this inhibits the interaction of NF-κB with coactivators, thus reducing histone acetylation (Ito et al. 2000; Barnes 2006b). Reduction of histone acetylation also occurs through the recruitment of histone deacetylase 2 (HDAC2) to the activated inflammatory gene complex by activated GR, thereby resulting in effective suppression of all activated inflammatory genes within the nucleus (Fig. 31.4). This accounts for why glucocorticoids are so effective in the control of asthmatic inflammation, but also why they are safe, since other activated genes are not affected. There may be additional mechanisms that are also important in the antiinflammatory actions of glucocorticoids. Glucocorticoids have potent inhibitory effects on MAP kinase signaling pathways through the induction of MKP-1 and this may inhibit the expression of multiple inflammatory genes (Clark 2003; Barnes 2006a). Some inflammatory genes, for example granulocyte–macrophage colony-stimulating factor (GM-CSF), have an unstable mRNA that is rapidly degraded by certain RNAses but which is stabilized when cells are stimulated by inflammatory mediators. Glucocorticoids reverse this effect, resulting in rapid degradation of mRNA and reduced inflammatory protein secretion (Bergmann et al. 2004). This may be through the inhibition of proteins that stabilize mRNAs of inflammatory proteins, such as tristretraprolin (Brook et al. 2006).

Interaction with b2-adrenergic receptors

Inhaled β2 agonists and glucocorticoids are frequently used together in the control of asthma and it is now recognized

Glucocorticosteroids

Inflammatory stimuli e.g. IL-1b, TNF-a

Glucocorticoid (low dose)

IKK-2 NF-kB

p65 p50

GR

AF

pC kB Inflammatory genes, cytokines, chemokines, adhesion molecules, inflammatory receptors, enzymes, proteins

CBP p65 HAT p50 Acetylation –

↑ Gene transcription

GR

HDAC2 Deacetylation

Gene repression

Fig. 31.4 Glucocorticoid suppression of activated inflammatory genes. Inflammatory genes are activated by inflammatory stimuli, such as interleukin (IL)-1b or tumor necrosis factor (TNF)-a, resulting in activation of IKK2 (inhibitor of IkB kinase-2) which activates the transcription factor nuclear factor (NF)-kB. A dimer of p50 and p65 NF-kB proteins translocates to the nucleus and binds to specific kB recognition sites and also to coactivators, such as CREB-binding protein (CBP) or p300/CBP-activating factor (pCAF), which have intrinsic histone acetyltransferase (HAT) activity. This results in acetylation of core histone H4, resulting in increased expression of genes encoding multiple inflammatory proteins. Glucocorticoid receptors (GR) after activation by glucocorticoids translocate to the nucleus and bind to coactivators to inhibit HAT activity directly and recruiting histone deacetylase 2 (HDAC2), which reverses histone acetylation leading to suppression of these activated inflammatory genes. (See CD-ROM for color version.)

that there are important molecular interactions between these two classes of drug (Barnes 2002). As discussed above, glucocorticoids increase the gene transcription of β2-adrenergic receptors, resulting in increased expression of cell-surface receptors. This has been demonstrated in human lung in vitro (Mak et al. 1995a) and nasal mucosa in vivo after topical application of a glucocorticoid (Baraniuk et al. 1997). In this way glucocorticoids protect against the downregulation of β2 receptors after long-term administration (Mak et al. 1995b). This may be important for the nonbronchodilator effects of β2 agonists, such as mast cell stabilization. Glucocorticoids may also enhance the coupling of β2 receptors to G proteins, this enhancing β2-agonist effects and reversing the uncoupling of β2 receptors that may occur in response to inflammatory mediators, such as interleukin (IL)-1β through a stimulatory effect on a G protein-coupled receptor kinase (Mak et al. 2002). There is also evidence that β2 agonists may affect GR and thus enhance the antiinflammatory effects of glucocorticoids. β2 Agonists increase the translocation of GR from cytoplasm to the nucleus after activation by glucocorticoids (Roth et al.

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Pharmacology picin, phenobarbital or phenytoin, which induce cytochrome 450 enzymes, lower the plasma half-life of prednisolone (Gambertoglio et al. 1980). The plasma half-life is 2–3 hours, although its biological half-life is approximately 24 hours, so that it is suitable for daily dosing. There is no evidence that previous exposure to steroids changes their subsequent metabolism. Prednisolone is approximately 92% protein bound, the majority to a specific protein transcortin and the remainder to albumin; it is the unbound fraction that is biologically active. Some patients, usually with severe asthma, apparently fail to respond to glucocorticoids. “Steroid-resistant” asthma is not due to impaired absorption or metabolism of steroids, but is due to reduced antiinflammatory actions of glucocorticoids. Measurement of plasma concentrations of prednisolone are useful in monitoring compliance with inhaled glucocorticoids and in assessing whether a poor therapeutic response to glucocorticoids is due to poor absorption or increased metabolism.

2002). This effect has now been demonstrated in sputum macrophages of asthmatic patients after an inhaled glucocorticoid and inhaled long-acting β2 agonist (Usmani et al. 2005). This suggests that β2 agonists and glucocorticoid enhance each other’s beneficial effects in asthma therapy.

Chemical structures The adrenal cortex secretes cortisol (hydrocortisone) and, by modification of its structure, it was possible to develop derivatives, such as prednisolone and dexamethasone, with enhanced corticosteroid effects but with reduced mineralocorticoid activity. These derivatives with potent glucocorticoid actions were effective in asthma when given systemically but had no antiasthmatic activity when given by inhalation. Further substitution in the 17α ester position resulted in steroids with high topical activity, such as beclomethasone dipropionate (BDP), triamcinolone, flunisolide, budesonide, and fluticasone propionate (FP), which are potent in the skin (dermal blanching test) and were later found to have significant antiasthma effects when given by inhalation (Fig. 31.5).

Inhaled steroids The pharmacokinetics of inhaled glucocorticoids is important in relation to systemic effects (Barnes et al. 1998; Derendorf et al. 1998; Lipworth 1999). The fraction of steroid inhaled into the lungs acts locally on the airway mucosa, but may be absorbed from the airway and alveolar surface. This fraction therefore reaches the systemic circulation (Fig. 31.6). The fraction of inhaled steroid deposited in the oropharynx is swallowed and absorbed from the gut. The absorbed fraction may be metabolized in the liver before reaching the systemic circulation (first-pass metabolism). Budesonide and FP have a greater first-pass metabolism than BDP and are therefore less likely to produce systemic effects at high inhaled

Pharmacokinetics Prednisolone is readily and consistently absorbed after oral administration with little interindividual variation. Prednisone is converted in the liver to the active prednisolone. Enteric coatings to reduce the incidence of dyspepsia delay absorption but not the total amount of drug absorbed. Prednisolone is metabolized in the liver and drugs such as rifam-

CH2

HO

CH3

OH

CH2OCOC2H5 C=O

C=O 17

CH2

OH

C=O OCOC2H5 CH3

HO

HO

OH O O

C

H CH2CH2CH3

CH3 Cl O

O

O Hydrocortisone

Beclomethasone dipropionate

SCH2F

CH2 C=O

C=O HO

OCOC2H5 CH3

HO

Budesonide

OH O O

CH2 C

C=O

CH3

HO

CH3

O F Fluticasone propionate

Fig. 31.5 Chemical structures of inhaled glucocorticoids.

718

O O

F

F O

OH

O F Flunisolide

Triamcinolone acetonide

C

CH3 CH3

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MDI ~ 10–20% inhaled Mouth and pharynx ~ 80–90% swallowed (↓ spacer/mouth-wash)

Lungs

Systemic circulation

Absorption from GI tract Liver

GI tract

Inactivation in liver “first pass”

Systemic side effects

Fig. 31.6 Pharmacokinetics of inhaled glucocorticoids. GI, gastrointestinal. (See CD-ROM for color version.)

doses. The use of a large-volume spacer chamber reduces oropharyngeal deposition and therefore reduces systemic absorption of glucocorticoids, although this effect is minimal in glucocorticoids with a high first-pass metabolism (Brown et al. 1993). Mouth rinsing and discarding the rinse has a similar effect and this procedure should be used with highdose dry powder steroid inhalers, since spacer chambers cannot be used with these devices. The ideal inhaled corticosteroid with optimal therapeutic index should have high lung bioavailability, negligible oral bioavailability, low systemic absorption, high systemic clearance, and high protein binding (Derendorf 2005). A recently introduced glucocorticoid, ciclesonide, is an inactive prodrug that is activated by esterases in the lung to the active metabolite des-ciclesonide (Reynolds & Scott 2004). This may reduce oropharyngeal side effects as esterases appear to be less active in this site than in the lower airways. Ciclesonide is also claimed to be effective as a once-daily therapy.

Systemic steroids Hydrocortisone is given intravenously in acute severe asthma. While the value of glucocorticoids in acute severe asthma has been questioned, others have found that they speed the resolution of attacks. There is no apparent advantage in giving very high doses of intravenous steroids (such as methylprednisolone 1 g) as this only increases the risk of side effects, such as hyperglycemia and increased susceptibility to infections. Intravenous steroids are indicated in acute asthma if lung function is < 30% predicted and there is no significant improvement with a nebulized β2 agonist. Intravenous therapy is usually given until a satisfactory response is obtained and then oral prednisolone may be substituted. Oral prednisolone (40–60 mg) has a similar effect to intravenous hydrocortisone and is easier to administer (Harrison et al. 1986; Storr et al. 1987). High doses of inhaled glucocorticoids may also substitute for a course of oral steroids in controlling acute exacerbations of asthma. High-dose FP (2000 μg daily) was as effective as a course of oral prednisolone in controlling

Glucocorticosteroids

acute exacerbations of asthma in a family practice setting and in children in an emergency room setting, although this route of delivery is more expensive (Levy et al. 1996; Manjra et al. 2000). Although doubling the dose of inhaled glucocorticoids has been recommended for mild exacerbations of asthma, this does not appear to be useful (FitzGerald et al. 2004; Harrison et al. 2004), but a fourfold increase in dose does appear effective (Foresi et al. 2000). Inhaled steroids have no proven effect in the management of severe acute asthma in a hospital setting (Edmonds et al. 2003), but trials with nebulized steroids, which can deliver large doses, are underway. Maintenance treatment with oral steroids are reserved for patients who cannot be controlled on maximum doses of other therapy, the dose being titrated to the lowest which provides acceptable control of symptoms. For any patient taking regular oral steroids, objective evidence of steroid responsiveness should be obtained before maintenance therapy is instituted. Short courses of oral steroids (30–40 mg prednisolone daily for 1–2 weeks) are indicated for exacerbations of asthma, and the dose may be tailed off over 1 week once the exacerbation is resolved (although the tail-off period is not strictly necessary, patients often find it reassuring).

Inhaled glucocorticoids There is no doubt that the early use of inhaled glucocorticoids has revolutionized the management of asthma, with marked reductions in asthma morbidity and improvement in health status. Inhaled steroids are now recommended as first-line therapy for all patients with persistent asthma (British Thoracic Society 2003; Global Initiative for Asthma 2006). Inhaled glucocorticoids are very effective in controlling asthma symptoms in asthmatic patients of all ages and severity (Kamada et al. 1996; Barnes 1999). Inhaled glucocorticoids improve the quality of life of patients with asthma and allow many patients to lead normal lives, improve lung function, reduce the frequency of exacerbations, and may prevent irreversible airway changes. They were first introduced to reduce the requirement for oral glucocorticoids in patients with severe asthma and many studies have confirmed that the majority of patients can be weaned off oral glucocorticoids (Barnes et al. 1998).

Studies in adults As experience has been gained with inhaled glucocorticoids, they have been introduced in patients with milder asthma, with the recognition that inflammation is present even in patients with mild asthma. Inhaled antiinflammatory drugs have now become first-line therapy in any patient who needs to use a β2-agonist inhaler more than two to three times a week and this is reflected in national and international guidelines for the management of chronic asthma. In patients with newly diagnosed asthma an inhaled corticosteroid (budesonide

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600 μg twice daily) reduced symptoms and β2-agonist inhaler usage and improved lung function. These effects persisted over the 2 years of the study, whereas in a parallel group treated with inhaled β2 agonists alone there was no significant change in symptoms or lung function (Haahtela et al. 1991). In another study patients with mild asthma treated with a low dose of inhaled corticosteroid (budesonide 400 μg daily) showed less symptoms and a progressive improvement in lung function over several months and many patients became completely asymptomatic (Juniper et al. 1990b). There was also a significant reduction in the number of exacerbations; in patients with mild asthma a low dose of glucocorticoids (budesonide 400 μg daily) significantly reduced exacerbation by around 40% over a 3-year period (Pauwels et al. 2003). Although the effects of inhaled glucocorticoids on AHR may take several months to reach a plateau, the reduction in asthma symptoms occurs much more rapidly (Vathenen et al. 1991) and reduced inflammation is seen within hours (Gibson et al. 2001; Ketchell et al. 2002). High-dose inhaled glucocorticoids may be used for the control of more severe asthma. This markedly reduces the need for maintenance oral glucocorticoids (Mash et al. 2000). With the use of add-on therapies, particularly long-acting β2 agonists, most patients can now be controlled on much lower doses of inhaled glucocorticoids so that high doses are needed in only a few patients with severe disease. Inhaled glucocorticoids are the treatment of choice in nocturnal asthma, which is a manifestation of inflamed airways, reducing nocturnal awakening and the diurnal variation in airway function. Inhaled glucocorticoids effectively control asthmatic inflammation but must be taken regularly. When inhaled glucocorticoids are discontinued there is usually a gradual increase in symptoms and airway responsiveness back to pretreatment values (Vathenen et al. 1991), although in patients with mild asthma who have been treated with inhaled glucocorticoids for a long time symptoms may not recur in some patients (Juniper et al. 1991). Reduction in the dose of inhaled glucocorticoids is associated with an increase in symptoms and this is preceded by an increase in exhaled nitric oxide (NO) and sputum eosinophils (Jatakanon et al. 2000; Leuppi et al. 2001).

Studies in children Inhaled glucocorticoids are equally effective in children. In an extensive study of children aged 7–17 years there was a significant improvement in symptoms, peak flow variability, and lung function compared to a regular inhaled β2 agonist which was maintained over the 22 months of the study (van Essen-Zandvliet et al. 1992), but asthma deteriorated when the inhaled glucocorticoids were withdrawn (Waalkens et al. 1993). There was a high proportion of drop-outs (45%) in the group treated with inhaled β2 agonist alone. Inhaled glucocorticoids are more effective than a long-acting β2 agonist in

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controlling asthma in children (Simons 1997). Inhaled glucocorticoids are also effective in younger children. Nebulized budesonide reduces the need for oral glucocorticoids and also improved lung function in children under the age of 3 years (Ilangovan et al. 1993; Berger 2005). Inhaled glucocorticoids given via a large-volume spacer improve asthma symptoms and reduce the number of exacerbations in preschool children and in infants.

Dose–response studies Surprisingly, the dose–response curve for the clinical efficacy of inhaled glucocorticoids is relatively flat and while all studies have demonstrated a clinical benefit of inhaled glucocorticoids, it has been difficult to demonstrate differences between doses, with most benefit obtained at the lowest doses used (Kamada et al. 1996; Barnes et al. 1998; Busse et al. 1998; Adams et al. 2002; Adams & Jones 2006). This is in contrast to the steeper dose–response for systemic effects, implying that while there is little clinical benefit from increasing doses of inhaled glucocorticoids, the risk of adverse effects is increased. However, the dose–response effect of inhaled glucocorticoids may depend on the parameters measured and, while it is difficult to discern a dose–response when traditional lung function parameters are measured, there may be a dose–response effect in prevention of asthma exacerbations. Thus, there is a significantly greater effect of budesonide 800 μg daily compared to 200 μg daily in preventing severe and mild asthma exacerbations (Pauwels et al. 1997). Normally, a fourfold or greater difference in dose has been required to detect a statistically significant (but often small) difference in effect on commonly measured outcomes such as symptoms, peak expiratory flow (PEF), use of rescue β2 agonist, and lung function, and even such large differences in dose are not always associated with significant differences in response. These findings suggest that pulmonary function tests or symptoms may have a rather low sensitivity in the assessment of the effects of inhaled glucocorticoids. This is obviously important for the interpretation of clinical comparisons between different inhaled glucocorticoids or inhalers. It is also important to consider the type of patient included in clinical studies. Patients with relatively mild asthma may have relatively little room for improvement with inhaled glucocorticoids, so that maximal improvement is obtained with relatively low doses. Patients with more severe asthma or with unstable asthma may have more room for improvement and may therefore show a greater response to increasing doses, but it is often difficult to include such patients in controlled clinical trials. More studies are needed to assess whether other outcome measures such as AHR or more direct measurements of inflammation, such as sputum eosinophils or exhaled NO, may be more sensitive than traditional outcome measures such as symptoms or lung function tests (Jatakanon et al. 1998, 1999; Lim et al. 1999; Kharitonov et al. 2002). Higher

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doses of inhaled glucocorticoids are needed to control AHR than to improve symptoms and lung function, and this may have a better long-term outcome in terms of reduction in structural changes of the airways (Sont et al. 1999). Measurement of sputum eosinophils to adjust the dose of inhaled glucocorticoids may reduce the overall dose requirement for inhaled glucocorticoids and exacerbations (Green et al. 2002; Jayaram et al. 2006). Monitoring of exhaled NO also reduces the requirement for glucocorticoids but is not yet practical in clinical practice (Smith et al. 2005).

Prevention of irreversible airway changes Some patients with asthma develop an element of irreversible airflow obstruction, but the pathophysiologic basis of these changes is not yet understood. It is likely that they are the result of chronic airway inflammation and that they may be prevented by treatment with inhaled glucocorticoids. There is some evidence that the annual decline in lung function may be slowed by the introduction of inhaled glucocorticoids (Dompeling et al. 1992) and this is supported by a 5-year study of low-dose budesonide in patients with mild asthma (Lange et al. 2006; O’Byrne et al. 2006). Increasing evidence also suggests that delay in starting inhaled glucocorticoids may result in less overall improvement in lung function in both adults and children (Agertoft & Pedersen 1994; Haahtela et al. 1994; Selroos et al. 1995). These studies suggest that introduction of inhaled glucocorticoids at the time of diagnosis is likely to have the greatest impact (Agertoft & Pedersen 1994; Selroos et al. 1995). So far there is no evidence that early use of inhaled glucocorticoids is curative and even when inhaled glucocorticoids are introduced at the time of diagnosis, symptoms and lung function revert to pretreatment levels when glucocorticoids are withdrawn (Haahtela et al. 1994).

Reduction in mortality Inhaled glucocorticoids may reduce the mortality from asthma but prospective studies are almost impossible to conduct. In a retrospective review of the risk of mortality and prescribed antiasthma medication, there was significant protection provided by regular inhaled corticosteroid therapy (Suissa et al. 2000). By contrast, asthma mortality appears to increase with increasing usage of short-acting β2 agonists, reflecting the fact that increased rescue therapy is a marker of poor asthma control (Ernst et al. 1993). The increase in use of rescue therapy should result in an increase in the maintenance dose of inhaled glucocorticoids. The long-acting inhaled β2 agonist salmeterol is associated with a small increase in asthma mortality, but the excess deaths appear to be related to underuse of inhaled glucocorticoids (Nelson et al. 2006).

Comparison between inhaled glucocorticoids Several inhaled glucocorticoids are currently prescribable in asthma, although their availability varies between countries.

Glucocorticosteroids

There have been relatively few studies comparing efficacy of the different inhaled glucocorticoids, and it is important to take into account the delivery system and the type of patient under investigation when such comparisons are made. Because of the relatively flat dose–response curve for the clinical parameters normally used in comparing doses of inhaled glucocorticoids, it may be difficult to see differences in efficacy of inhaled glucocorticoids. Most comparisons have concentrated in differences in systemic effects at equally efficacious doses, although it has often proved difficult to establish dose equivalence. There are few studies comparing different doses of inhaled glucocorticoids in asthmatic patients. Budesonide has been compared with BDP and in adults and children it appears to have comparable antiasthma effects at equal doses, whereas FP appears to be approximately twice as potent as BDP and budesonide (Adams & Jones 2006). Studies have consistently shown that FP and budesonide have less systemic effects than BDP, triamcinolone, and flunisolide (Lipworth 1999). The new inhaled glucocorticoids mometasone and ciclesonide are claimed to have less systemic effects (Nathan et al. 2001; Reynolds & Scott 2004).

Clinical use in asthma Inhaled glucocorticoids are now recommended as first-line therapy for all patients with persistent symptoms. Inhaled glucocorticoids should be started in any patient who needs to use a β2-agonist inhaler for symptom control more than three times weekly. It is conventional to start with a low dose of inhaled corticosteroid and to increase the dose until asthma control is achieved. However, this may take time and a preferable approach is to start with a dose of glucocorticoids in the middle of the dose range (400 μg twice daily) to establish control of asthma more rapidly (Barnes 1996). Once control is achieved (defined as normal or best possible lung function and infrequent need to use an inhaled β2 agonist), the dose of inhaled corticosteroid should be reduced in a stepwise manner to the lowest dose needed for optimal control. It may take as long as 3 months to reach a plateau in response and any changes in dose should be made at intervals of 3 months or more. When daily doses of ≥ 800 μg daily are needed, a large-volume spacer device should be used with a metered-dose inhaler (MDI) and mouth-washing with a dry powder inhaler in order to reduce local and systemic side effects. Inhaled glucocorticoids are usually given as a twice-daily dose in order to increase compliance. When asthma is unstable four times daily dosage is preferable (Malo et al. 1989). For patients who require ≤ 400 μg daily, oncedaily dosing appears to be as effective as twice-daily dosing, at least for budesonide (Jones et al. 1994). The dose of inhaled corticosteroid should be increased to 2000 μg daily if necessary, but higher doses may result in systemic effects. It may be preferable to add a low dose of oral corticosteroid, since higher doses of inhaled glucocorticoids are expensive and have a high incidence of local side effects.

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Nebulized budesonide has been advocated in order to give an increased dose of inhaled corticosteroid and to reduce the requirement for oral glucocorticoids (Otulana et al. 1992), but this treatment is expensive and may achieve its effects largely via systemic absorption. The dose of inhaled corticosteroid should be the minimal dose that controls asthma and once control is achieved the dose should be slowly reduced (Hawkins et al. 2003).

Add-on therapy Previously it was recommended that the dose of inhaled glucocorticoids be increased if asthma was not controlled, on the assumption that there was residual inflammation of the airways. However, the dose–response effect of inhaled glucocorticoids is relatively flat, so that there is little improvement in lung function after increasing the dose of inhaled glucocorticoids. An alternative strategy is to add some other class of controller drug and this is more effective than increasing the dose of inhaled glucocorticoids for most patients (Kankaanranta et al. 2004). In patients in general practice who are not controlled on BDP 200 μg twice daily, addition of salmeterol 50 μg twice daily was more effective than increasing the dose of inhaled corticosteroid to 500 μg twice daily, in terms of lung function improvement, use of rescue β2-agonist use, and symptom control (Greening et al. 1994). This has been confirmed in several other studies (Shrewsbury et al. 2000). Similar results have been found with another long-acting inhaled β2agonist, formoterol, which in addition reduced the frequency of mild and severe asthma exacerbations in patients with mild, moderate, and severe persistent asthma (Pauwels et al. 1997; O’Byrne et al. 2001). These studies showing the great efficacy of combined glucocorticoids and long-acting β2 agonist compared with increased doses of long-acting β2 agonist have led to the development of fixed combinations of glucocorticoids and long-acting β2 agonists, such as FP/ salmeterol and budesonide/formoterol, which may be more convenient for patients (Chapman et al. 1999; Shapiro et al. 2000). These fixed combination inhalers also ensure that patients do not discontinue their inhaled glucocorticoids when a long-acting bronchodilator is used. For patients with mild persistent asthma, combination inhalers are no more effective than the inhaled glucocorticoids alone in controlled trials (Ni et al. 2005), but may have an advantage in the real world where adherence to regular inhaled glucocorticoids is very low. Recently studies have demonstrated that when formoterol combined with budesonide is used as a reliever therapy this gives better control of asthma compared with the normally used short-acting β2 agonist as rescue therapy with either the same dose of combination inhaler or a high dose of inhaled glucocorticoids as maintenance treatment (O’Byrne et al. 2005; Rabe et al. 2006a). This advantage is particularly striking in terms of reducing the number of severe exacerbations. When formoterol was used as reliever therapy, exacerbations

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were reduced to a greater extent than with the short-acting β2 agonist terbutaline, but the combination was even more effective (Rabe et al. 2006b). This suggests that the “as-required” use of inhaled glucocorticoids contributes to the marked reduction in acute exacerbations. The mechanisms by which as-required glucocorticoids improve asthma control and reduce exacerbations are not completely understood, but exacerbations of asthma evolve over several days when patients take increasing amounts of rescue medication (Tattersfield et al. 1999). During this time there is increasing inflammation of the airways, as may be measured by exhaled NO and sputum eosinophils (Jatakanon et al. 2000). Taking the inhaled corticosteroid at the same time as the formoterol to relieve symptoms may suppress this evolving inflammation, particularly since glucocorticoids appear to have a relatively rapid onset of effect in suppressing airway inflammation (Barnes 2006c). Addition of low doses of theophylline (plasma concentration < 10 mg/L) is more effective than doubling the dose of inhaled budesonide, either in mild or severe asthma (Evans et al. 1997; Ukena et al. 1997; Lim et al. 2000). However this is less effective than using a long-acting inhaled β2 agonist as add-on therapy (Wilson et al. 2000). Antileukotrienes have also been used as an add-on therapy (Laviolette et al. 1999; Price et al. 2003), although this is less effective than addition of long-acting β2 agonists (Nelson et al. 2000; Ducharme et al. 2004).

Side effects The efficacy of inhaled glucocorticoids is now established in short- and long-term studies in adults and children, but there are still concerns about side effects, particularly in children and when high inhaled doses are used. Several side effects have been recognized (Table 31.2).

Local side effects Side effects due to the local deposition of the inhaled corticosteroid in the oropharynx may occur with inhaled glucocorticoids, but the frequency of complaints depends on Table 31.2 Side effects of inhaled glucocorticoids. Local side effects Dysphonia Oropharyngeal candidiasis Cough Systemic side effects Adrenal suppression Growth suppression Bruising Osteoporosis Cataracts Glaucoma Metabolic abnormalities (glucose, insulin, triglycerides) Psychiatric disturbances

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the dose and frequency of administration and on the delivery system used. The commonest complaint is of hoarseness of the voice (dysphonia) and may occur in over 50% of patients using an MDI. Dysphonia is not appreciably reduced by using spacers, but may be less with dry powder devices. Dysphonia may be due to myopathy of laryngeal muscles and is reversible when treatment is withdrawn (Williamson et al. 1995). For most patients it is not troublesome but may be disabling in singers and lecturers. Oropharyngeal candidiasis (thrush) may be a problem in some patients, particularly in the elderly, with concomitant oral glucocorticoids and more than twice-daily administration (Toogood et al. 1980). Large-volume spacer devices protect against this local side effect by reducing the dose of inhaled corticosteroid that deposits in the oropharynx. There is no evidence that inhaled corticosteroid, even in high doses, increase the frequency of infections, including tuberculosis, in the lower respiratory tract. There is no evidence for atrophy of the airway epithelium and even after 10 years of treatment with inhaled glucocorticoids there is no evidence for any structural changes in the epithelium. Cough and throat irritation, sometimes accompanied by reflex bronchoconstriction, may occur when inhaled glucocorticoids are given via an MDI. These symptoms are likely to be due to surfactants in pressurized aerosols as they disappear after switching to a dry powder corticosteroid inhaler device.

The systemic effect of an inhaled corticosteroid is dependent on the amount of drug absorbed into the systemic circulation (Fig. 31.6). Approximately 90% of the inhaled dose from an MDI deposits in the oropharynx and is swallowed and subsequently absorbed from the gastrointestinal tract. Use of a large-volume spacer device markedly reduces oropharyngeal deposition and therefore the systemic effects of inhaled glucocorticoids, although this is less important when oral bioavailability is minimal, as with FP. For dry powder inhalers similar reductions in systemic effects may be achieved with mouth-washing and discarding the fluid. All patients using a daily dose of ≥ 800 μg of an inhaled corticosteroid should therefore use either a spacer or mouth-washing to reduce systemic absorption. Approximately 10% of an MDI enters the lung and this fraction (which presumably exerts the therapeutic effect) may be absorbed into the systemic circulation. As the fraction of inhaled corticosteroid deposited in the oropharynx is reduced, the proportion of the inhaled dose entering the lungs is increased. More efficient delivery to the lungs is therefore accompanied by increased systemic absorption, but this is offset by a reduction in the dose needed for optimal control of airway inflammation. For example, a multiple dry powder delivery system, the Turbohaler, delivers approximately twice as much corticosteroid to the lungs as other devices, and therefore has increased systemic effects. However this is compensated for by the fact that only half the dose is required.

Systemic side effects

Adrenal suppression Glucocorticoids may cause hypothalamic–pituitary–adrenal (HPA) axis suppression by reducing corticotrophin (ACTH) production, which reduces cortisol secretion by the adrenal gland. The degree of HPA suppression is dependent on dose, duration, frequency, and timing of corticosteroid administration. There is no evidence that cortisol responses to the stress of an asthma exacerbation or insulin-induced hypoglycemia are impaired, even with high doses of inhaled glucocorticoids. However, measurement of HPA axis function provides evidence for systemic effects of an inhaled corticosteroid. Basal adrenal cortisol secretion may be measured by a morning plasma cortisol, 24-hour urinary cortisol, or plasma cortisol profile over 24 hours. Other tests measure the HPA response following stimulation with tetracosactrin (which measures adrenal reserve) or stimulation with metyrapone and insulin (which measure the response to stress). There are many studies of HPA axis function in asthmatic patients with inhaled glucocorticoids, but the results are inconsistent as they have often been uncontrolled and patients have also been taking courses of oral glucocorticoids (which may affect the HPA axis for weeks) (Barnes et al. 1998). BDP, budesonide, and FP at high doses by conventional MDI (> 1600 μg daily) give a dose-related decrease in morning serum cortisol levels and 24-hour urinary cortisol, although values still lie well within the normal range. However, when a large-volume

The efficacy of inhaled glucocorticoids in the control of asthma is undisputed, but there are concerns about systemic effects of inhaled glucocorticoids, particularly as they are likely to be used over long periods and in children of all ages (Kamada et al. 1996; Lipworth 1999). The safety of inhaled glucocorticoids has been extensively investigated since their introduction 30 years ago (Barnes et al. 1998). One of the major problems is to decide whether a measurable systemic effect has any significant clinical consequence and this necessitates careful long-term follow-up studies. As biochemical markers of systemic corticosteroid effects become more sensitive, then systemic effects may be seen more often, but this does not mean that these effects are clinically relevant. There are several case reports of adverse systemic effects of inhaled glucocorticoids, and these may be idiosyncratic reactions, which may be due to abnormal pharmacokinetic handing of the inhaled corticosteroid. The systemic effect of an inhaled corticosteroid will depend on several factors, including the dose delivered to the patient, the site of delivery (gastrointestinal tract and lung), the delivery system used and individual differences in the patient’s response to the corticosteroid. Recent studies suggest that systemic effects of inhaled corticosteroid are less in patients with more severe asthma, presumably as less drug reaches the lung periphery (Brutsche et al. 2000; Harrison et al. 2001).

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spacer is used, doses of 2000 μg daily of BDP or budesonide have little effect on 24-hour urinary cortisol excretion. Stimulation tests of HPA axis function similarly show no consistent effects of doses of 1500 μg or less of inhaled corticosteroid. At high doses (> 1500 μg daily) budesonide and FP have less effect than BDP on HPA axis function. In children no suppression of urinary cortisol is seen with doses of BDP of 800 μg or less. In studies where plasma cortisol has been measured at frequent intervals there was a significant reduction in cortisol peaks with doses of inhaled BDP as low as 400 μg daily, although this does not appear to be dose related in the range 400–1000 μg. The clinical significance of these effects is not certain.

Bone metabolism Glucocorticoids lead to a reduction in bone mass by direct effects on bone formation and resorption and indirectly by suppression of the pituitary–gonadal and HPA axes, effects on intestinal calcium absorption, renal tubular calcium reabsorption, and secondary hyperparathyroidism (Efthimou & Barnes 1998). The effects of oral glucocorticoids on osteoporosis and increased risk of vertebral and rib fractures are well known, but there are no reports suggesting that longterm treatment with inhaled glucocorticoids is associated with an increased risk of fractures. Bone densitometry has been used to assess the effect of inhaled glucocorticoids on bone mass. Although there is evidence that bone density is less in patients taking high-dose inhaled glucocorticoids, interpretation is confounded by the fact that these patients are also taking intermittent courses of oral glucocorticoids. Changes in bone mass occur very slowly and several biochemical indices have been used to assess the short-term effects of inhaled glucocorticoids on bone metabolism. Bone formation has been measured by plasma concentrations of bone-specific alkaline phosphatase, serum osteocalcin, or procollagen peptides. Bone resorption may be assessed by urinary hydroxyproline after a 12-hour fast, urinary calcium excretion, and pyridinium cross-link excretion. It is important to consider the age, diet, time of day, and physical activity of the patient in interpreting any abnormalities. It is also necessary to choose appropriate control groups as asthma itself may have an effect on some of the measurements, such as osteocalcin. Inhaled glucocorticoids, even at doses up to 2000 μg daily, have no significant effect on calcium excretion, but acute and reversible dose-related suppression of serum osteocalcin has been reported with BDP and budesonide when given by conventional MDI in several studies. Budesonide consistently has less effect than BDP at equivalent doses and only BDP increases urinary hydroxyproline at high doses. However, with a large-volume spacer, even doses of 2000 μg daily of either BDP or budesonide are without effect on plasma osteocalcin concentrations. Urinary pyridinium and deoxypyridinoline cross-links, which are a more

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accurate and stable measurement of bone and collagen degradation, are not increased with inhaled glucocorticoids (BDP > 1000 μg daily), even with intermittent courses of oral glucocorticoids. It is important to monitor changes in markers of bone formation as well as bone degradation, as the net effect on bone turnover is important. There is no evidence that inhaled glucocorticoids increase the frequency of fractures. Long-term treatment with high-dose inhaled glucocorticoids has not been associated with any consistent change in bone density. Indeed, in elderly patients there may be an increase in bone density due to increased mobility.

Connective tissue effects Oral and topical glucocorticoids cause thinning of the skin, telangiectasiae, and easy bruising, probably as a result of loss of extracellular ground substance within the dermis, due to an inhibitory effect on dermal fibroblasts. There are reports of increased skin bruising and purpura in patients using high doses of inhaled BDP, but the amount of intermittent oral glucocorticoids in these patients is not known. Easy bruising in association with inhaled glucocorticoids is more frequent in elderly patients (Roy et al. 1996) and there are no reports of this problem in children. Long-term prospective studies with objective measurements of skin thickness are needed with different inhaled glucocorticoids. Cataracts Long-term treatment with oral glucocorticoids increase the risk of posterior subcapsular cataracts and there are several case reports describing cataracts in individual patients taking inhaled glucocorticoids (Barnes et al. 1998). In a recent crosssectional study in patients aged 5–25 years taking either inhaled BDP or budesonide no cataracts were found on slitlamp examination, even in patients taking 2000 μg daily for over 10 years (Simons et al. 1993). However, epidemiologic studies have identified an increased risk of cataracts in patients taking high-dose inhaled steroids over prolonged periods (Cumming et al. 1997). A slight increase in the risk of glaucoma in patients taking very high doses of inhaled glucocorticoids has also been identified (Garbe et al. 1997). Growth There has been particular concern that inhaled glucocorticoids may cause stunting of growth and several studies have addressed this issue. Asthma itself (as with other chronic diseases) may have an effect on the growth pattern and has been associated with delayed onset of puberty and deceleration of growth velocity that is more pronounced with more severe disease (Pedersen 2001). However, asthmatic children appear to grow for longer, so that their final height is normal. The effect of asthma on growth makes it difficult to assess the effects of inhaled glucocorticoids on growth in cross-sectional

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studies, particularly as courses of oral glucocorticoids is a confounding factor. Longitudinal studies have demonstrated that there is no significant effect of inhaled glucocorticoids on statural growth in doses of up to 800 μg daily and for up to 5 years of treatment (Barnes et al. 1998). A metaanalysis of 21 studies, including over 800 children, showed no effect of inhaled BDP on statural height, even with higher doses and long duration of therapy (Allen et al. 1994) and in a large study of asthmatics treated with inhaled glucocorticoids during childhood there was no difference in statural height compared to normal children (Silverstein et al. 1997). Another long-term follow-up study showed no effect of glucocorticoids on final height in children treated over several years (Agertoft & Pedersen 2000). Short-term growth measurements (knemometry) have demonstrated that even a low dose of an oral corticosteroid (prednisolone 2.5 mg) is sufficient to give complete suppression of lower leg growth. However, inhaled budesonide up to 400 μg is without effect, although some suppression is seen with 800 μg and with 400 μg BDP. The relationship between knemometry measurements and final height is uncertain since low doses of oral corticosteroid that have no effect on final height cause profound suppression.

Metabolic effects Several metabolic effects have been reported after inhaled glucocorticoids, but there is no evidence that these are clinically relevant at therapeutic doses. In adults fasting glucose and insulin are unchanged after doses of BDP up to 2000 μg daily and in children with inhaled budesonide up to 800 μg daily. In normal individuals high-dose inhaled BDP may slightly increase resistance to insulin. However, in patients with poorly controlled asthma high doses of BDP and budesonide paradoxically decrease insulin resistance and improve glucose tolerance, suggesting that the disease itself may lead to abnormalities in carbohydrate metabolism. Neither BDP 2000 μg daily in adults nor budesonide 800 μg daily in children have any effect on plasma cholesterol or triglycerides. Psychiatric effects There are various reports of psychiatric disturbance, including emotional lability, euphoria, depression, aggressiveness, and insomnia, after inhaled glucocorticoids. Only eight such patients have so far been reported, suggesting that this is very infrequent and a causal link with inhaled glucocorticoids has usually not been established. Pregnancy Based on extensive clinical experience inhaled glucocorticoids appear to be safe in pregnancy, although no controlled studies have been performed. There is no evidence for any adverse effects of inhaled glucocorticoids on the pregnancy, the delivery, or the fetus (Schatz 1999). It is important to

Glucocorticosteroids

recognize that poorly controlled asthma may increase the incidence of perinatal mortality and retard intrauterine growth, so that more effective control of asthma with inhaled glucocorticoids may reduce these problems.

Systemic glucocorticoids Oral or intravenous glucocorticoids may be indicated in several situations. Prednisone is converted in the liver to the active prednisolone. In pregnant patients prednisone is preferable as it is not converted to prednisolone in the fetal liver, thus diminishing exposure of the fetus to glucocorticoids. Enteric-coated preparations of prednisolone are used to reduce side effects (particularly gastric side effects) and give delayed and reduced peak plasma concentrations, although the bioavailability and therapeutic efficacy of these preparations is similar to uncoated tablets. Prednisolone and prednisone are preferable to dexamethasone, betamethasone or triamcinolone, which have longer plasma half-lives and therefore an increased frequency of adverse effects.

Short courses Short courses of oral glucocorticoids (30–40 mg prednisolone daily for 1–2 weeks or until the peak flow values return to best attainable) are indicated for exacerbations of asthma, and the dose may be tailed off over 1 week once the exacerbation is resolved. The tail-off period is not strictly necessary, but some patients find it reassuring.

Maintenance glucocorticoids Maintenance oral glucocorticoids are only needed in a small proportion of asthmatic patients (approximately 1%) with the most severe asthma that cannot be controlled with maximal doses of inhaled glucocorticoids (2000 μg daily) and additional bronchodilators. The minimal dose of oral corticosteroid needed for control should be used and reductions in the dose should be made slowly in patients who have been on oral glucocorticoids for long periods (e.g., by 2.5 mg per month for doses down to 10 mg daily and thereafter by 1 mg per month). Oral glucocorticoids are usually given as a single morning dose, as this reduces the risk of adverse effects since it coincides with the peak diurnal concentrations. There is some evidence that administration in the afternoon may be optimal for some patients who have severe nocturnal asthma (Beam et al. 1992). Alternate-day administration may also reduce adverse effects, but control of asthma may not be as good on the day when the oral dose is omitted in some patients. Intramuscular triamcinolone acetonide (80 mg monthly) has been advocated in patients with severe asthma as an alternative to oral glucocorticoids (McLeod et al. 1985; Ogirala et al. 1991). This may be considered in patients in whom

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compliance is a particular problem, but the major concern is the high frequency of proximal myopathy associated with this fluorinated corticosteroid. Some patients who do not respond well to prednisolone are reported to respond to oral betamethasone, presumably because of pharmacokinetic handling problems with prednisolone.

Acute severe asthma Intravenous hydrocortisone is given in acute severe asthma, with a recommended dose of 200 mg i.v. While the value of glucocorticoids in acute severe asthma has been questioned, others have found that they speed the resolution of attacks (Engel & Heinig 1991). There is no apparent advantage in giving very high doses of intravenous glucocorticoids (such as methylprednisolone 1 g). Indeed, intravenous glucocorticoids have occasionally been associated with an acute severe myopathy (Decramer et al. 1995). There was no difference in recovery from acute severe asthma when intravenous hydrocortisone in doses of 50, 200 or 500 mg 6-hourly was used (Bowler et al. 1992) and another placebo-controlled study showed no beneficial effect of intravenous glucocorticoids (Morell et al. 1992). Intravenous glucocorticoids are indicated in acute asthma if lung function is < 30% predicted and there is no significant improvement with nebulized β2 agonist. Intravenous therapy is usually given until a satisfactory response is obtained and then oral prednisolone may be substituted. Oral prednisolone (40– 60 mg) has a similar effect to intravenous hydrocortisone and is easier to administer (Harrison et al. 1986; Engel & Heinig 1991). Oral prednisolone is the preferred treatment for acute severe asthma, providing there are no contraindications to oral therapy (British Thoracic Society 1997). There is some evidence that high doses of nebulized glucocorticoids may also be effective in acute exacerbations of asthma, with a more rapid onset of action (Devidayal et al. 1999).

Corticosteroid resistance Although glucocorticoids are highly effective in the control of asthma and other chronic inflammatory or immune diseases, a small proportion of patients with asthma fail to respond even to high doses of oral glucocorticoids (Adcock & Lane 2003; Leung & Bloom 2003; Barnes 2004). Resistance to the therapeutic effects of glucocorticoids is also recognized in other inflammatory and immune diseases, including rheumatoid arthritis and inflammatory bowel disease. Steroidresistant patients, although uncommon, present considerable management problems. Steroid-resistant asthma is defined as a failure to improve FEV1 by > 15% after treatment with oral prednisolone 30– 40 mg daily for 2 weeks, providing the oral steroid is taken (verified by plasma prednisolone level or reduction in early-morning cortisol level). These patients are not addisonian and they do not suffer from the abnorm-

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alities in sex hormones described in the very rare familial glucocorticoid resistance. Plasma cortisol and adrenal suppression in response to exogenous cortisol is normal in these patients, so they suffer from side effects of glucocorticoids. Complete glucocorticoid resistance in asthma is very rare, with a prevalence of less than 1 in 1000 asthmatic patients. Much more common is reduced responsiveness to glucocorticoids, so that large inhaled or oral doses are needed to control asthma adequately (steroid-dependent asthma). It is likely that there is a range of responsiveness to glucocorticoids and that steroid resistance is at one extreme of this range. It is important to establish that the patient has asthma rather than chronic obstructive pulmonary disease (COPD), “pseudoasthma” (a hysterical conversion syndrome involving vocal cord dysfunction), left ventricular failure, or cystic fibrosis that do not respond to glucocorticoids (Thomas et al. 1999). Asthmatic patients are characterized by variability in PEF and, in particular, diurnal variability of > 15% and episodic symptoms. Biopsy studies have demonstrated the typical eosinophilic inflammation of asthma in these patients.

Molecular mechanisms The molecular mechanisms of steroid resistance are still not understood, but there are likely to be several mechanisms (Adcock & Lane 2003; Leung & Bloom 2003). GRs appear to function normally, but there are defects in the interaction of GR with other transcription factors. Certain cytokines are able to reduce steroid responsiveness by enhancing the activity of transcription factors, such as AP-1, which then combine with and divert GR (Adcock et al. 1995). Other patients appear to have a defect in nuclear localization of GR as a result of phosphorylation of the receptor by p38 MAP kinase (Irusen et al. 2002; Matthews et al. 2004). In other patients there is a defect in corticosteroid-induced histone acetylation, with defective acetylation of the lysine-5 residue on histone-4, presumably resulting in reduced transcription of important antiinflammatory genes (Matthews et al. 2004). It has been proposed that increased expression of GRβ interferes with the antiinflammatory action of corticosteroids by competing with GRα for binding to GREs or coactivator molecules (Leung et al. 1997), but this is unlikely as the amount of GRβ expressed in inflammatory cells is insufficient to interfere by competition with the effects of corticosteroids on GRα (Pujols et al. 2002). As discussed above, HDAC2 plays a critical role in mediating the antiinflammatory effects of corticosteroids. In COPD, corticosteroid resistance is explained by a marked defect in activity and expression of HDAC2 (Barnes 2006d; Ito et al. 2005, 2006). This defect in HDAC2 appears to be due to increased oxidative stress. There is also a defect in HDAC2 in patients with severe asthma who have a poor response to glucocorticoids (Hew et al. 2006). Asthmatic patients who smoke are also glucocorticoid insensitive (Thomson & Spears 2005) and this is likely to be explained by impairment in

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Steroid-resistant asthma Smoking asthma Severe asthma

Normal asthma

Cigarette smoke Stimuli Alveolar macrophage

Corticosteroids

a beneficial effect in some patients with steroid-dependent asthma (Kidney et al. 1995), and identification of the molecular mechanisms of theophylline-induced HDAC activation may lead to a new approach to the treatment of glucocorticoid resistance in some patients.

Oxidative stress Peroxynitrite

References

GR NF-kB

NF-kB ↑ HDAC2

↓ HDAC2

Histone acetylation ↑ TNF-a ↑ IL-8 ↑ GM-CSF

Glucocorticosteroids

↓ Histone acetylation

Histone acetylation TNF-a IL-8 GM-CSF

Fig. 31.7 Proposed mechanism of glucocorticoid resistance in severe asthma and smoking asthma. Stimulation of normal and asthmatic alveolar macrophages activates nuclear factor (NF)-kB and other transcription factors to switch on histone acetyltransferase leading to histone acetylation and subsequently to transcription of genes encoding inflammatory proteins, such as tumor necrosis factor (TNF)-a, interleukin (IL)-8, and granulocyte–macrophage colony-stimulating factor (GM-CSF). Corticosteroids reverse this by binding to glucocorticoid receptors (GR) and recruiting histone deacetylase 2 (HDAC2). This reverses the histone acetylation induced by NF-kB and switches off the activated inflammatory genes. In smoking asthmatic patients, cigarette smoke generates oxidative stress (acting via the formation of peroxynitrite) and in severe asthma intense inflammation generates oxidative stress to impair the activity of HDAC2. This amplifies the inflammatory response to NF-kB activation, but also reduces the antiinflammatory effect of corticosteroids, as HDAC2 is now unable to reverse histone acetylation.

HDAC2 in airway cells as a result of oxidative stress (Barnes et al. 2004) (Fig. 31.7).

Therapy Patients with glucocorticoid-resistant asthma may be difficult to manage, as they do not benefit from corticosteroids or require high doses of oral steroids that are associated with unacceptable side effects. Steroid-sparing therapies, including methotrexate, cyclosporin A, and oral gold have proved to be disappointing. Alternative antiinflammatory therapies, including phosphodiesterase-4 inhibitors, p38 MAP kinase inhibitors and NF-κB inhibitors, may prove useful in the future but there is a concern about potential side effects with these agents. Omalizumab is effective in a few allergic patients with steroid-dependent asthma. Anti-tumor necrosis factor therapies have been reported to be beneficial in patients with refractory asthma in small trials (Berry et al. 2006), but this has not been confirmed in larger controlled trials. Theophylline is able to reverse the glucocorticoid resistance in COPD by restoring HDAC activity (Cosio et al. 2004) and has

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Otulana, B.A., Varma, N., Bullock, A. & Higenbottam, T. (1992) High dose nebulized steroid in the treatment of chronic steroid-dependent asthma. Respir Med 86, 105–8. Pauwels, R.A., Lofdahl, C.-G., Postma, D.S. et al. (1997) Effect of inhaled formoterol and budesonide on exacerbations of asthma. N Engl J Med 337, 1412–18. Pauwels, R.A., Pedersen, S., Busse, W.W. et al. (2003) Early intervention with budesonide in mild persistent asthma: a randomised, double-blind trial. Lancet 361, 1071–6. Pedersen, S. (2001) Do inhaled corticosteroids inhibit growth in children? Am J Respir Crit Care Med 164, 521–35. Price, D.B., Hernandez, D., Magyar, P. et al. (2003) Randomised controlled trial of montelukast plus inhaled budesonide versus double dose inhaled budesonide in adult patients with asthma. Thorax 58, 211–16. Pujols, L., Mullol, J., Roca-Ferrer, J. et al. (2002) Expression of glucocorticoid receptor alpha- and beta-isoforms in human cells and tissues. Am J Physiol 283, C1324–C1331. Rabe, K.F., Pizzichini, E., Stallberg, B. et al. (2006a) Budesonide/ formoterol in a single inhaler for maintenance and relief in mildto-moderate asthma: a randomized, double-blind trial. Chest 129, 246–56. Rabe, K.F., Atienza, T., Magyar, P., Larsson, P., Jorup, C. & Lalloo, U.G. (2006b) Effect of budesonide in combination with formoterol for reliever therapy in asthma exacerbations: a randomised controlled, double-blind study. Lancet 368, 744–53. Reynolds, N.A. & Scott, L.J. (2004) Ciclesonide. Drugs 64, 511–19. Rhen, T. & Cidlowski, J.A. (2005) Antiinflammatory action of glucocorticoids: new mechanisms for old drugs. N Engl J Med 353, 1711–23. Roth, M., Johnson, P.R., Rudiger, J.J. et al. (2002) Interaction between glucocorticoids and β2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling. Lancet 360, 1293–9. Roy, A., Leblanc, C., Paquette, L. et al. (1996) Skin bruising in asthmatic subjects treated with high does of inhaled steroids: frequency and association with adrenal function. Eur Respir J 9, 226– 31. Schatz, M. (1999) Asthma and pregnancy. Lancet 353, 1202–4. Selroos, O., Pietinalcho, A., Lofroos, A.-B. & Riska, A. (1995) Effect of early and late intervention with inhaled corticosteroids in asthma. Chest 108, 1228–34. Shapiro, G., Lumry, W., Wolfe, J. et al. (2000) Combined salmeterol 50 mg and fluticasone propionate 250 mg in the Diskus device for the treatment of asthma. Am J Respir Crit Care Med 161, 527–34. Shrewsbury, S., Pyke, S. & Britton, M. (2000) Meta-analysis of increased dose of inhaled steroid or addition of salmeterol in symptomatic asthma (MIASMA). BMJ 320, 1368–73. Silverstein, M.D., Yunginger, J.W., Reed, C.E. et al. (1997) Attained adult height after childhood asthma: effect of glucocorticoid therapy. J Allergy Clin Immunol 99, 466–74. Simons, F.E. (1997) A comparison of beclomethasone, salmeterol, and placebo in children with asthma. N Engl J Med 337, 1659–65. Simons, F.E.R., Persaud, M.P., Gillespie, C.A., Cheang, M. & Shuckett, E.P. (1993) Absence of posterior subcapsular cataracts in young patients treated with inhaled glucocorticoids. Lancet 342, 736– 8. Smith, A.D., Cowan, J.O., Brassett, K.P., Herbison, G.P. & Taylor, D.R. (2005) Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 352, 2163–73.

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Sont, J.K., Willems, L.N., Bel, E.H., van Krieken, J.H., Vandenbroucke, J.P. & Sterk, P.J. (1999) Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. The AMPUL Study Group. Am J Respir Crit Care Med 159, 1043– 51. Storr, J., Barrell, E., Barry, W., Lenney, W. & Hatcher, G. (1987) Effect of a single oral dose of prednisolone in acute childhood asthma. Lancet i, 879– 82. Suissa, S., Ernst, P., Benayoun, S., Baltzan, M. & Cai, B. (2000) Lowdose inhaled corticosteroids and the prevention of death from asthma. N Engl J Med 343, 332– 6. Tattersfield, A.E., Postma, D.S., Barnes, P.J. et al. (1999) Exacerbations of asthma. A descriptive study of 425 severe exacerbations. Am J Respir Crit Care Med 160, 594– 9. Thomas, P.S., Geddes, D.M. & Barnes, P.J. (1999) Pseudo-steroid resistant asthma. Thorax 54, 352– 6. Thomson, N.C. & Spears, M. (2005) The influence of smoking on the treatment response in patients with asthma. Curr Opin Allergy Clin Immunol 5, 57– 63. Toogood, J.A., Jennings, B., Greenway, R.W. & Chung, L. (1980) Candidiasis and dysphonia complicating beclomethasone treatment of asthma. J Allergy Clin Immunol 65, 145–53. Ukena, D., Harnest, U., Sakalauskas, R. et al. (1997) Comparison of addition of theophylline to inhaled steroid with doubling of the dose of inhaled steroid in asthma. Eur Respir J 10, 2754–60.

Glucocorticosteroids

Usmani, O.S., Ito, K., Maneechotesuwan, K. et al. (2005) Glucocorticoid receptor nuclear translocation in airway cells following inhaled combination therapy. Am J Respir Crit Care Med 172, 704– 12. van Essen-Zandvliet, E.E., Hughes, M.D., Waalkens, H.J., Duiverman, E.J., Pocock, S.J. & Kerrebijn, K.F. (1992) Effects of 22 months of treatment with inhaled corticosteroids and/or b2agonists on lung function, airway responsiveness and symptoms in children with asthma. Am Rev Respir Dis 146, 547–54. Vathenen, A.S., Knox, A.J., Wisniewski, A. & Tattersfield, A.E. (1991) Time course of change in bronchial reactivity with an inhaled corticosteroid in asthma. Am Rev Respir Dis 143, 1317–21. Waalkens, H.J., van Essen-Zandvliet, E.E., Hughes, M.D. et al. (1993) Cessation of long-term treatment with inhaled corticosteroids (budesonide) in children with asthma results in deterioration. Am Rev Respir Dis 148, 1252–7. Williamson, I.J., Matusiewicz, S.P., Brown, P.H., Greening, A.P. & Crompton, G.K. (1995) Frequency of voice problems and cough in patients using pressurised aersosol inhaled steroid preparations. Eur Respir J 8, 590– 2. Wilson, A.J., Gibson, P.G. & Coughlan, J. (2000) Long acting betaagonists versus theophylline for maintenance treatment of asthma. Cochrane Database Syst Rev CD001281.

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32

Immunomodulating Drugs Iain A.M. MacPhee

Summary

Overview

The immune response causing tissue-damaging inflammation can be modulated at a number of points. Drugs with different modes of action are often used in synergistic combinations in an attempt to maximize efficacy with reduced drug toxicity. The most established of the immunosuppressive drugs act by inhibiting lymphocyte proliferation, the most widely used of which (azathioprine, mycophenolate and cyclophosphamide) are described here. These agents have effects on both T and B lymphocytes but are relatively nonspecific in their action, resulting in problems with toxicity. The calcineurin inhibitors ciclosporin A and tacrolimus block the production of interleukin (IL)-2 by T lymphocytes and their introduction resulted in significant improvements in the outcome for solid organ transplant recipients with more recent application to a number of autoimmune diseases. Nephrotoxicity is a major problem with calcineurin inhibitor use and this has led to a continued search for equally effective but less toxic agents. The mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus inhibit the response of lymphocytes to cytokines. They also inhibit wound healing and angiogenesis, with some potential benefits at the expense of side effects. The development of monoclonal antibodies has offered the potential for more specifically targeted drugs. Antibodies with specificity for a broad range of lymphocytes such as antithymocyte globulin and alemtuzumab are potently immunosuppressive but probably increase the risk of infectious complications. Antibodies with specificity for the IL-2 receptor, expressed on activated T lymphocytes, have been used to good effect in solid organ transplantation but have not been applied to other conditions. Rituximab, an antibody specific for the CD20 molecule expressed on B lymphocytes has been used successfully in the treatment of a number of antibody-mediated autoimmune diseases. These are all potent drugs with a narrow therapeutic index requiring a high degree of caution in prescribing.

T lymphocytes and their products are known to play a critical role in the pathogenesis of asthma and atopic allergic diseases (see Chapters 2 and 3) and “anti-T lymphocytes” agents have been evaluated in treatment of these conditions for many years. For example, the calcineurin inhibitors pimecrolimus and tacrolimus have an established place as topical immunosuppressants in atopic dermatitis (Alomar et al. 2004; Iskedjian et al. 2004; Breuer et al. 2005) with antiinflammatory potency similar to corticosteroids of moderate potency. Immunomodulatory drugs have been used for many years in the treatment of asthma. A metaanalysis of the effects of concomitant methotrexate therapy in oral glucocorticoid-dependent asthmatics suggested an oral glucocorticoid-sparing effect (Marin 1997). Two blinded placebo-controlled trials of cyclosporin A in severe oral glucocorticoid-dependent asthmatics showed improved lung function while reducing oral glucocorticoid requirements (Alexander et al. 1992; Lock et al. 1996). In a small controlled study a single injection of an anti-CD4 antibody improved lung function in steroid-dependent asthmatics (Kon et al. 1998). The currently available range of immunosuppressive drugs target a number of distinct steps in the generation of the immune response (Table 32.1). The synergistic effect of drugs acting at different points in the response is often used to good effect, allowing reduced drug doses with minimization of drug-specific side effects. Many of these drugs have a narrow therapeutic index with wide variation between individuals in the blood concentration achieved by a given dose. Therapeutic drug monitoring is often employed in an attempt to maximize efficacy and safety. In general, the immunosuppressive effect is proportional to the area under the concentration–time curve (AUC). Clearly, measurement of the AUC is impractical in clinical practice and limited sampling strategies, usually based on a single time point that correlates best with the AUC, are employed routinely. This chapter provides an overview of the immunosuppressive drugs used in current practice, with the exception of steroids (see Chapter 31).

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Table 32.1 Targets of the immunosuppressive drugs. Site of action

Drugs

Inhibition of IL-2 production by T lymphocytes Inhibition of T-lymphocyte response to IL-2 Inhibition of proliferation of T and B lymphocytes Depletion of T and B lymphocytes Depletion of T lymphocytes Depletion of B lymphocytes Inhibition of costimulatory signals

Ciclosporin A, tacrolimus Anti-CD25 antibodies, sirolimus, everolimus Azathioprine, mycophenolate, cyclophosphamide, methotrexate, leflunomide Polyclonal antilymphocyte antibodies, alemtuzumab Muromonab CD3 Rituximab Azathioprine, abatacept, belatacept

Generic complications of immunosuppression Inhibition of the host defense mechanisms to prevent tissuedamaging inflammation has the inevitable consequence of increased risk of infection and malignant disease, primarily due to defective control of intracellular pathogens. The commonest opportunistic infections in immunosuppressed patients are caused by latent viruses, in particular those in the herpesvirus family that reactivate. Individuals with a history of exposure to tuberculosis are at risk of recurrence following immunosuppression and prophylactic antituberculous therapy should be considered. Fungal infections are a rare but serious complication. Malignancy caused by oncogenic viruses is a potentially life-threatening complication, particularly skin cancer and lymphoma (Opelz & Dohler 2004).

The immunosuppressive drugs Antiproliferative agents Suppression of lymphocyte proliferation has proven to be a very effective way of modulating the immune response. The choice of antiproliferative agent for a given indication is often rooted in history rather than the drug having specific benefits for a given condition. The most widely used antiproliferative agents are discussed here.

Azathioprine/6-mercaptopurine Azathioprine is the best-established immunosuppressive drug and remains a useful and effective agent. Azathioprine was initially introduced in the early 1960s for solid organ transplantation and was subsequently adopted as therapy for a wide range of immunologically mediated diseases.

Structure Azathioprine is a purine analog (an imidazolyl derivative of 6-mercaptopurine; Fig. 32.1). Essentially, the features of

N H3C

N NO2 S

H N

N N

N

Fig. 32.1 Azathioprine: chemical structure.

azathioprine discussed below also apply to 6-mercaptopurine, an alternative orally active agent.

Pharmacokinetics Azathioprine is well absorbed orally and is rapidly metabolized nonenzymatically through reduction by glutathione and other sulfhydryl-containing compounds to 6-mercaptopurine (approximately 90%). Subsequently, 6-mercaptopurine is metabolized along three competing pathways (summarized in Fig. 32.2). The pathway leading to the active metabolite is initially catalyzed by hypoxanthine phosphoribosyltransferase via several steps to 6-thioguanine nucleotides. Competing pathways leading to the generation of inactive metabolites are catalyzed by xanthine oxidase and thiopurine-S-methyltransferase (TPMT). The plasma half-life of 6-mercaptopurine is short (38–114 min) but persistence of 6-thioguanine nucleotides in the tissues provides a basis for the once-daily dosing that is conventional (Chan et al. 1990). A typical initial daily dose would be 1–5 mg/kg body weight given either orally or intravenously (no adjustment required for parenteral administration), with dose adjusted according to clinical response and toxicity. Pharmacogenetics Variable activity of TPMT is the best-characterized example of a pharmacogenetic effect on an immunosuppressive drug (reviewed in Evans 2004). Approximately 90% of the

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Azathioprine Xanthine oxidase

Glutathione

6-Thiouracil

6-Methylmercaptopurine

6-Mercaptopurine

Hypoxanthine phosphoribosyltransferase

Thiopurine-S-methyltransferase

6-Thioinosine-monophosphate

6-Methylmercaptopurine ribonucleotides

Inosine monophosphate dehydrogenase 6-Thioxanthine-monophosphate Guanosine monophosphate pathways

6-Thioguanine nucleotides Active metabolite

population are homozygous for normal alleles with normal enzyme activity, 10% are heterozygotes with reduced enzyme activity, and 0.3% are homozygotes for nonfunctional alleles with functional enzyme deficiency (Yates et al. 1997). At least 11 variant alleles have been associated with reduced TPMT activity, three of which (TPMT*3C, *3A and *2) account for in excess of 95% of inherited TPMT deficiency. TPMT deficiency has been linked to myelotoxicity but not hepatotoxicity. Patients with TPMT deficiency require 5–10% standard dose of thiopurines, while heterozygotes can be treated with standard doses but are more likely to require dose reduction. The most widely applied assay for TPMT deficiency is measurement of enzyme activity in erythrocytes. As with all erythrocyte-based assays, it is important to avoid the assay within 30– 60 days of blood transfusion. The guidelines for the management of a number of autoimmune diseases now recommend screening for TPMT deficiency prior to treatment, although this may not be cost-effective in the presence of regular pharmacodynamic monitoring in the form of testing for myelosuppression.

Mechanism of action Azathioprine is a purine analog and inhibits the de novo and salvage pathways of purine synthesis resulting in the inhibition of DNA synthesis and the proliferation of both T and B lymphocytes. An additional effect, described recently, is inhibition of the transduction of the costimulatory signal delivered to T lymphocytes by ligation of CD28 (Tiede et al. 2003). Method of monitoring Plasma concentrations of azathioprine or 6-mercaptopurine are not predictive of efficacy or toxicity, although some recent publications suggest that measurement of 6-thioguanine

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Fig. 32.2 Simplified metabolic pathway for azathioprine. Enzymes-catalyzing steps on the pathway to the active drug are shown in black boxes and enzymes involved in the generation of inactive metabolites are shown in blue boxes. (See CD-ROM for color version.)

nucleotide concentrations in erythrocytes may be helpful in planning drug doses (Gearry & Barclay 2005). Generally, a pharmacodynamic approach is employed. The principle dose-limiting toxicities are myelotoxicity and hepatotoxicity. Routine monitoring of full blood count and liver blood tests are essential, weekly for at least the first 4 weeks after initiation (the summary of product characteristics recommends 8 weeks but this may be overcautious), followed by a reduced frequency but not less than every 3 months. Dose reduction should be considered when the white blood cell count falls below 4 × 109/L or platelets below 100 × 109/L or there is evidence of hepatic injury.

Established indications Use of azathioprine is well established for solid organ transplantation and a number of autoimmune disorders, including systemic lupus erythematosus (SLE), inflammatory bowel disease, rheumatoid arthritis, systemic vasculitis, dermatomyositis and polymyositis, autoimmune chronic active hepatitis, pemphigus vulgaris, autoimmune hemolytic anaemia, and chronic refractory idiopathic thrombocytopenic purpura. Safety profile/side effects (including drug interactions) The most important side effect of azathioprine is dose-related myelosuppression, as noted above, and macrocytosis is an expected observation. Hepatic problems including cholestasis and hepatic venoocclusive disease are much less common. Immunosuppression per se increases the risk of skin cancer but azathioprine may also have an added specific effect through the potentiation of oxidative damage to DNA (O’Donovan et al. 2005). The epidemiology for this issue is difficult to interpret as the cohort of patients with the longest duration of exposure to immunosuppression were mostly treated with azathioprine. The single most important drug interaction for

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Pharmacokinetics MPA is well absorbed orally with a peak concentration in blood at around 2 hours and a plasma half-life of around 17 hours (Weber et al. 1998). The principle difference in pharmacokinetics between MMF and enteric-coated MPA is the time of peak blood concentrations (Tmax), which occurs later and is more variable with enteric-coated MPA. The principal metabolite is the pharmacologically inactive phenolic

CH3 O O

O O N O OH Mycophenolate

25 20 15 10 5 0 4

8

12

16

20

24

Hours after oral dose

The active drug, mycophenolic acid (MPA), was initially identified in the 1940s as a product of Penicillium brevicompactum with antifungal activity. Mycophenolate is an alternative purine synthesis inhibitor to azathioprine. It is a more potent agent and has largely replaced azathioprine in solid organ transplantation, with increasing use for autoimmune indications (Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group 1996). Two preparations are available that deliver MPA, the morpholinoethyl ester mycophenolate mofetil (MMF, Cellcept) and enteric-coated mycophenolate sodium (Myfortic) (Fig. 32.3). Comparative studies suggest that these formulations have equivalent efficacy and safety (Salvadori et al. 2004). With orally administered MMF, the ester group is cleaved by first-pass metabolism in the enterocyte and liver resulting in undetectable concentrations of the parent drug in blood. Mycophenolate was initially introduced in solid organ transplantation but data are now accumulating for efficacy in the treatment of a number of autoimmune diseases, in particular SLE (Chan et al. 2000; Ginzler et al. 2005).

O

30

0

Mycophenolate

CH3

35

MPA (mg/mL)

azathioprine is that with allopurinol. Allopurinol is an inhibitor of xanthine oxidase, a key enzyme in purine metabolism, and inhibits the metabolism of azathioprine leading to severe myelotoxicity with potentially fatal consequences. Some would advocate the use of reduced-dose azathioprine with allopurinol but the only genuinely safe approach is to use an alternative antiproliferative agent such as mycophenolate or to avoid allopurinol. Caution must be exercised in the coprescription of other drugs that inhibit TPMT, including the 5-aminosalicylic acid derivatives. Azathioprine should not be coadministered with mycophenolate.

Immunomodulating Drugs

CH3 Mofetil

Fig. 32.3 Chemical structure of mycophenolate. Mycophenolic acid is available either as the morpholinoethyl ester (mofetil) or as a sodium salt.

Fig. 32.4 Pharmacokinetics of mycophenolate mofetil (MMF): a typical 24-hour pharmacokinetic profile for a patient given a single oral dose of 1 g MMF. Coprescription of drugs that inhibit ABCC2 (MRP2), e.g., cyclosporin A, inhibit enterohepatic recirculation resulting in loss of the second peak of absorption, which typically comprises 30–50% of the AUC.

glucuronide (MPAG) generated by uridine glucuronyltransferase but a small proportion is metabolized to the active acyl glucuronide that may be responsible for some of the side effects (Oellerich et al. 2000). These glucuronides are primarily cleared by renal excretion but a proportion are secreted in bile by the drug efflux pump ABCC2 (MRP2). Subsequently, a proportion is deconjugated by bacterial flora in the gut with a second peak of absorption at around 6–8 hours due to enterohepatic recirculation. This second peak accounts for 30–50% of the total AUC (Fig. 32.4). When coprescribed with drugs that inhibit ABCC2 (e.g., ciclosporin A, but not tacrolimus or sirolimus) the AUC is significantly reduced (Hesselink et al. 2005). It must be noted that the registration studies where the recommended dose of mycophenolate was defined were conducted in patients coprescribed ciclosporin A (Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group 1996). It is often stated that patients genetically of sub-Saharan African origin require higher doses of mycophenolate but this is not supported by the available pharmacokinetic data (Shaw et al. 2000). Taking mycophenolate along with food delays absorption but has no effect on total exposure. MPA is highly protein bound (97–99%) and the free fraction is increased by conditions that result in displacement of MPA from its protein binding sites or reduced plasma protein concentration (Weber et al. 1998; Kuypers et al. 2003). In renal impairment, accumulation of MPAG displaces MPA and uremic plasma and acidemia have been shown to have an independent effect (Nowak & Shaw 1995; Kaplan et al. 1999; MacPhee et al. 2000). An increased free fraction results in increased conjugation to MPAG with reduced plasma MPA concentrations but an increase in the degree of enterohepatic recirculation, which correlates with an increased incidence of diarrhoea. Conditions that increase the free fraction of MPA

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may result in poor tolerance due to gastrointestinal toxicity in the face of subtherapeutic MPA concentrations. The current recommended initial oral dose in renal transplant recipients is 1 g MMF twice daily, and 720 mg twice daily as the bioequivalent dose of enteric-coated MPA (Johnston et al. 2006). There is no dose adjustment for intravenous administration.

Pharmacogenetics Individuals with the g275T > A and g2152C > T single nucleotide polymorphisms (SNPs) in the UGT1A9 promoter region, mutations associated with reduced in vitro enzyme activity, have significantly reduced exposure to the active drug MPA, of a magnitude likely to be significant clinically (Kuypers et al. 2005). The ABCC2 g24C > T and g3972C > T polymorphisms have been found to prevent the reduced exposure to MPA found in renal transplant recipients with mildly altered liver dysfunction (Naesens et al. 2006). These observations do not yet provide a basis for the application of pharmacogenetics in planning mycophenolate dosing. Mechanism of action MPA is an inhibitor of inosine monophosphate dehydrogenase (particularly the type II isoform), a key rate-limiting enzyme for the de novo pathway of purine synthesis. Both T and B lymphocytes are particularly dependent on this pathway (Allison & Eugui 1993). There is some experimental evidence to suggest the inhibition of smooth muscle cell proliferation and pathologic scarring processes (Romero et al. 1999). Method of monitoring The role of therapeutic drug monitoring for MPA is an area of current controversy. The available data apply to MMF only. Enteric-coated MPA has an unpredictable Tmax and no abbreviated sampling strategies have been shown to correlate with AUC. Two methods are available for the assay of MPA. The high performance liquid chromatography (HPLC)-based approach measures only MPA while the enzyme-multiplied immunoassay technique (EMIT) measures both MPA and the acyl glucuronide (Shipkova et al. 1999). There is no single time point where measurement of plasma MPA correlates sufficiently well with the AUC to be of practical value. The best clinically applicable sampling strategies are based on three samples collected predose and 30 and 120 min after drug dosing, with the use of established algorithms to estimate the AUC (Pawinski et al. 2002). In renal transplantation the established therapeutic range for the estimated AUC is 45–60 mg/L per hour (van Gelder et al. 1999). There are no published data on the therapeutic range for other indications. Some experimental work has correlated the degree of suppression of inosine monophosphate dehydrogenase with efficacy but this has yet to translate to use in clinical practice (Glander et al. 2004).

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Full blood count should be monitored weekly during the first month of treatment, twice monthly during the second and third months, and then monthly throughout the first year. Dose reduction should be considered when the white blood cell count falls below 4 × 109/L or platelets below 100 × 109/L.

Established indications Established indications include solid organ transplantation and a number of autoimmune diseases including SLE. Safety profile/side effects (including drug interactions) The principal side effects of MPA are gastrointestinal toxicity, which is less severe when the drug is coprescribed with inhibitors of ABCC2, and myelosuppression. A common practice when faced with severe diarrhoea is to increase the dosing frequency to three or four times daily with the same total daily dose. However, there are no data to support equivalent efficacy with this approach. Drugs that inhibit enterohepatic recirculation including cholestyramine may reduce exposure to MPA. The absorption of MPA is reduced when coadministered with antacids.

Cyclophosphamide Cyclophosphamide is a nitrogen mustard and was originally derived from mustard gas used as a weapon during the First World War. It was noted to have myelosuppressive effects in the 1940s. It is the most potent and most toxic of the antiproliferative agents used for immunosuppression, being more widely used as a chemotherapeutic agent for malignant disease. It is reserved for the treatment of life- or organthreatening disease, primarily in systemic vasculitis and SLE. Protocols have evolved, for example in the treatment of systemic vasculitis, employing cyclophosphamide to induce remission, with switch to a less toxic agent such as azathioprine for maintenance treatment (Jayne et al. 2003).

Structure The chemical structure of cyclophosphamide is shown in Fig. 32.5. Pharmacokinetics The oral bioavailability of cyclophosphamide is approximately 75%, with a plasma half-life of 3.5–12 hours (Colvin 1999). Cyclophosphamide is a prodrug that is metabolized Cl

Cl

N O

P

NH

O Fig. 32.5 Cyclophosphamide: chemical structure.

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by cytochrome P450 (CYP), primarily CYP2C9, CYP3A4 and CYP3A5, to a number of active metabolites. The most important of these is 4-hydroxycyclophosphamide, a circulating metabolite that enters cells and is cleaved nonenzymatically to phosphoramide mustard, the principle alkylating substance, and acrolein (Ren et al. 1998). Clearance with urinary excretion follows further metabolism by cytochrome P450, with 30% excreted unmodified in urine. For the treatment of systemic vasculitis, typical daily doses of oral cyclophosphamide are 2 mg/kg (maximum 200 mg/day), with a 25% dose reduction for patients aged greater than 60 years and 50% reduction for those aged greater than 75 years (Savage et al. 2000). It has been demonstrated clearly in the treatment of lupus nephritis that the complication rate of pulsed intravenous cyclophosphamide is significantly lower than that for regular oral cyclophosphamide. This is particularly true for gonadal toxicity and malignant disease (Austin et al. 1986). Typical pulsed intravenous regimens comprise 500– 750 mg/m2 every 2– 4 weeks (Boumpas et al. 1992; Houssiau et al. 2002).

Immunomodulating Drugs

reduced by provoking high urine flow (> 100 mL/hour) for 24–48 hours after intravenous infusion and administration of the sulfhydryl-containing compound mesna. Mesna is given intravenously at a total dose of 60% that of cyclophosphamide at 0, 4 and 8 hours or is available orally. There is an increased risk of malignancy, particularly bladder cancer. Gonadotoxicity is a particular problem that is minimized, though not eliminated, by the use of pulsed intravenous therapy. Patients should be counselled on this and given the opportunity for sperm or egg storage prior to treatment. Hair loss is more common than with the other antiproliferative drugs.

Other antimetabolites Several other antiproliferative agents are used for immunomodulation. Methotrexate inhibits the enzyme dihydrofolate reductase with consequent inhibition of purine and pyrimidine synthesis. Leflunomide is a pyrimidine synthesis inhibitor. Chlorambucil has been used with some success in the treatment of membranous nephropathy (Ponticelli et al. 1995).

Calcineurin inhibitors Pharmacogenetics There are some published data to suggest that mutations in the genes encoding CYP3A5, CYP2B6, and CYP2C19 had a significant influence on efficacy failure and toxicity in patients treated with pulsed intravenous cyclophosphamide for lupus nephritis (Takada et al. 2004). Mechanism of action Phosphoramide mustard is a bifunctional alkylating agent that cross-links DNA, preventing cell division. Method of monitoring Regular monitoring of full blood count is required and cyclophosphamide should be withheld when the white blood cell count falls below 4 × 109/L or platelets below 100 × 109/L until the counts recover. With pulsed intravenous cyclophosphamide the nadir of blood cell counts occurs at around 10 days after dosing and should be monitored at this point, with reduction in subsequent doses in the event of excessive myelosuppression. Established indications Cyclophosphamide is indicated for a number of autoimmune conditions including, lupus nephritis and systemic vasculitis. Safety profile/side effects (including drug interactions) Parenteral administration is often followed by nausea or vomiting after 24 hours and very high doses can cause diarrhoea. As with the other antiproliferative agents, myelosuppression is a major problem, cell counts reaching a nadir 10–12 days after administration of a single dose, with return to baseline within 18–21 days. Hemorrhagic cystitis is thought to be caused by the metabolite acrolein and risk can be

There are two licensed drugs in this class, ciclosporin A and tacrolimus, both of which are fungal products. Ciclosporin was initially introduced as Sandimmun in the early 1980s (European Multicentre Trial Group 1983), with the introduction of a microemulsion formulation with more predictable absorption (Neoral) (Kovarik et al. 1994) in the early 1990s. Generic forms of ciclosporin A are now available but must used with caution as they have different pharmacokinetic properties and cannot be used interchangeably with Neoral (Kees et al. 2006). Tacrolimus was known for many years by its experimental name FK506 and is available as Prograf given twice daily and the recently introduced delayed release preparation (Pirsch et al. 1997). The primary use of these drugs has been in solid organ transplantation but use for autoimmune indications is increasing. Prior to the introduction of ciclosporin A, when azathioprine and prednisolone were the primary drugs used in immunosuppression for renal transplantation, 40% of transplants were lost to acute rejection during the first year. With the introduction of ciclosporin A, 1-year graft survival improved to greater than 80% and it is now unusual to lose a transplanted kidney through acute rejection.

Mode of action These drugs bind to intracellular binding proteins, cyclophilin A in the case of ciclosporin A or FK-binding protein 12 (FKBP12) in the case of tacrolimus, and inhibit the phosphatase calcineurin (Colgan et al. 2005; Fruman et al. 1992). Calcineurin is essential for activating the transcription factor NF-AT (nuclear factor of activated T lymphocytes) that is required for transcription of the IL-2 gene. The most important toxicity of the calcineurin inhibitors is nephrotoxicity. There are two different processes involved.

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(a)

(b) Fig. 32.6 Chronic calcineurin inhibitor toxicity. Sections are from a renal transplant biopsy performed in a tacrolimus-treated patient in order to investigate chronic renal transplant dysfunction. (a) Typical change of stripe fibrosis; (b) typical arterial changes with a hyaline deposit (arrow). (See CD-ROM for color version.)

The first is an acute reversible vasoconstriction of the renal vasculature, possibly mediated by endothelin or sympathetic nervous stimulation (Murray et al. 1985; Curtis et al. 1986; Kon et al. 1990). The vasoconstriction can, to some extent, be reversed by the administration of calcium channel blockers (Ruggenenti et al. 1993). Acute tubular toxicity is also manifest by tubular vacuolation. A more chronic effect is marked by hypertrophy of the arterial media and striped fibrosis (Fig. 32.6). Clear evidence on the long-term nephrotoxic effects comes from recipients of transplants other than kidneys, where the 5-year risk of developing chronic renal failure with glomerular filtration rate < 30 mL/min is 7– 21%, depending on the type of transplant (Ojo et al. 2003). Serial renal transplant biopsies in kidney/pancreas transplant recipients demonstrated changes of acute calcineurin inhibitor toxicity peaking at around 20% by month 6 after transplantation but, more ominously, over 75% of patients had evidence of chronic calcineurin inhibitor toxicity by 4 years after transplantation and almost all patients by 10 years (Nankivell et al. 2004). There is probably little difference between calcineurin inhibitors in the degree of nephrotoxicity (Solez et al. 1998). In a recent metaanalysis of the calcineurin inhibitors in renal transplantation, tacrolimus was shown to be more efficacious than ciclosporin A but at the cost of a higher rate of new-onset diabetes after transplantation (Webster et al. 2005). Both calcineurin inhibitors cause hypertension, but this is probably less common with tacrolimus. The key side effects of ciclosporin A and tacrolimus are compared in Table 32.2. Thrombotic microangiopathy is a rare but serious complication common to both calcineurin inhibitors and should be managed by drug withdrawal (Ponticelli & Banfi 2006).

Oral bioavailability Ciclosporin A and tacrolimus are both metabolized by the oxidative enzymes CYP3A4 and 3A5 and are substrates for

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Table 32.2 Calcineurin inhibitor side effects. Side effect

Ciclosporin A

Tacrolimus

Nephrotoxicity Hypertension Hyperkalemia Diabetes mellitus Gum hyperplasia Hypertrichosis Hair loss

++ +++ + + + + −

++ ++ ++ ++ − − +

the drug efflux pump P-glycoprotein (P-gp). Both drugs have oral bioavailability of 25–30%, in part due to an active barrier to drug absorption mediated by a combination of P-gp in the enterocyte, and first pass metabolism by CYP in the enterocyte and liver. Drugs that inhibit CYP and P-gp significantly enhance the absorption of the CNIs (Hebert et al. 1992; Tuteja et al. 2001) and vice versa for inhibitors (Gupta et al. 1989). Patients on treatment with CNIs should be advised against the consumption of grapefruit juice which can result in significant inhibition of CYP3A with increased blood drug concentrations and subsequent toxicity. Unfortunately, there is batch to batch variability in this effect preventing the use of regular grapefruit consumption as a strategy for reducing drug dose requirements (Lown et al. 1997).

Ciclosporin A Structure The chemical structure of ciclosporin A is shown in Fig. 32.7.

Pharmacokinetics The barriers to the oral absorption of ciclosporin A have been described above and the recommended intravenous dose is

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H HO

H3C

H3CO H H3C

H (CH3)2CHCH2

HO

CH3

H

O H3C

N

N O O

(CH3)2CHCH2 N H3C

N H

O N

N O

CH3

CH3

H N

H O

O N

HO

H

N

CH3

CH2CH3 O

N O

H

CH2CH(CH3)2

H

O

CH3

O

CH3 O

O

N

N CH3 CH(CH3)2

CH3

H

CH3 OH

H3C

CH3

H

O O

· H2O

CH3

O

H

H CH2CH(CH3)2 H

O CH(CH3)2

H3CO

Ciclosporin A

H H

OCH3

Tacrolimus

Fig. 32.7 Calcineurin inhibitors: chemical structure.

30% of the standard oral dose. The peak concentration in blood (Cmax) occurs at around 2 hours with a highly variable half-life, generally between 5 and 10 hours (Kees et al. 2006). The CYP3A metabolites are primarily excreted in bile.

Pharmacogenetics Unlike tacrolimus (see below) no clear genetic influences on ciclosporin A pharmacokinetics have been described. Specifically, SNPs in the genes encoding CYP3A5 and ABCB1 (MDR-1) have been shown to have no significant impact (MacPhee et al. 2005a; Fredericks et al. 2007). Method of monitoring Therapeutic drug monitoring is employed routinely for ciclosporin A. In view of uptake by erythrocytes, whole-blood concentrations are assayed. Initially this was by sampling at 12 hours after the previous dose (trough or C0 concentration). However, most of the heterogeneity in ciclosporin pharmacokinetics is in the absorption phase and blood concentration measured at 2 hours after the last dose (C2) correlates better with the AUC (Canadian Neoral Renal Transplantation Study Group 2001; Clase et al. 2002). C2 monitoring requires more accurate timing of the blood samples than C0 sampling, with a tolerance of only ± 10 min, which has generated logistical problems in the introduction of this sampling strategy. The therapeutic range for cyclosporin A in solid organ transplantation is 75–300 μg/L for C0 or 800– 1700 μg/L for C2 (Clase et al. 2002; Cole et al. 2003). Established indications Established indications for ciclosporin A are solid organ and bone marrow transplantation and a number of autoimmune diseases, including, psoriasis, atopic dermatitis, rheumatoid arthritis, and immunologically mediated nephrotic syndrome.

Safety profile/side effects (including drug interactions) Apart from the general comments on calcineurin inhibitor toxicity above, ciclosporin A has some adverse cosmetic effects with hypertrichosis and gum hypertrophy. Ciclosporin A increases plasma uric acid concentrations with increased incidence of gout.

Tacrolimus Tacrolimus is a macrolide with structure shown in Fig. 32.7. Occasionally there is cross-reaction with allergy to the chemically related antibiotic erythromycin.

Pharmacokinetics The barriers to the oral absorption of tacrolimus have been described above and the recommended intravenous dose is 20% of the standard oral dose. The peak blood concentration occurs at 1–2 hours after an oral dose, with a half-life of around 40 hours (Bekersky et al. 1999). When taken along with food, absorption is significantly inhibited and increased absorption is seen in fasting patients. The increased oral bioavailability of tacrolimus in patients with diarrhoea is intriguing and has been ascribed to reduced efficiency in the active barrier to drug absorption (Lemahieu et al. 2005). Tacrolimus is metabolized by CYP3A4 and CYP3A5 (Kamdem et al. 2005), with eight known metabolites labeled M-I to M-VIII. M-II has significant immunosuppressive activity and cross-reacts with tacrolimus in some immunoassays (Iwasaki et al. 1995). The metabolites are eliminated by biliary excretion with essentially all clearance in stool. Pharmacogenetics There are important differences between ethnic groups in the blood concentration achieved by a given dose of tacrolimus. Individuals genetically of sub-Saharan African origin require

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twofold higher doses of tacrolimus to achieve target blood concentrations compared to individuals from other ethnic groups (MacPhee et al. 2002). This difference was demonstrated to be due to reduced absorption rather than more rapid clearance (Mancinelli et al. 2001) and is possibly due to the high prevalence of CYP3A5 expression in black individuals. There are several SNPs in the CYP3A5 gene that result in greatly reduced levels of expression in homozygotes, the most prevalent of which is CYP3A5*3. CYP3A5*3 homozygotes require twofold lower doses of tacrolimus to achieve target blood concentrations than individuals with at least one CYP3A5*1 allele (CYP3A5 expressers). CYP3A5 expressers experienced a significant delay in achieving target blood concentrations after renal transplantation (MacPhee et al. 2005a,b). The ABCB1 (MDR-1) gene codes for P-gp. SNPs in this gene have been associated with high and low levels of expression of P-gp. Individuals with the wild type at these SNPs have lower dose-normalized blood concentrations of tacrolimus, although this is a minor effect (MacPhee et al. 2002). However, the ABCB1 genotype may influence pharmacodynamics by controlling distribution of the drug between cellular compartments. This has been hypothesized to be the mechanism underlying the augmentation of calcineurin inhibitor nephrotoxicity by sirolimus (Anglicheau et al. 2006).

Method of monitoring Therapeutic drug monitoring is employed routinely with tacrolimus, with 12-hour postdose trough whole blood concentrations being used as standard. Tacrolimus is taken up by erythrocytes and results should be interpreted with caution in the presence of anaemia. Some older publications report plasma concentrations which are significantly lower. Dogma suggests that sampling at this time-point correlates well with AUC but this is controversial (Wallemacq et al. 1998). However, while more precise limited sampling strategies including measurement at 2 and 4 hours after dosing have been suggested, none have been adopted widely in routine clinical practice (Wong et al. 2000). The therapeutic range for solid organ transplantation lies between 5 and 15 μg/L (Bottiger et al. 1999; Undre et al. 1999; Staatz et al. 2001; Maes et al. 2001). Established indications The primary indication for tacrolimus is solid organ transplantation. A topical formulation is also available for use in atopic eczema. Safety profile/side effects (including drug interactions) Apart from the general comments on calcineurin inhibitor toxicity above, the main concern with tacrolimus is that it is diabetogenic, more so than ciclosporin A. Neurotoxicity, manifest most commonly by tremor or paresthesia, is more common with tacrolimus than ciclosporin A.

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OH OMe

O

OH

N O

O

HO O

O

O

MeO

O

OMe

Fig. 32.8 Sirolimus: chemical structure.

Mammalian target of rapamycin inhibitors Sirolimus and everolimus History of discovery Sirolimus (previously known as rapamycin) is available as Rapamune, a product of the fungus Streptomyces hygroscopicus and was initially isolated in soil samples from Easter Island. Everolimus (Certican) is a derivative of sirolimus with a 2hydroxyethyl chain substitution at position 40, resulting in more efficient absorption following oral administration and a shorter half-life.

Structure Sirolimus is a macrolide antibiotic with some similar structural features to tacrolimus (Fig. 32.8) and binds to the same intracellular protein, FKBP12. Despite initial concerns that this would result in mutual antagonism of tacrolimus and sirolimus used together, this has been shown not to be a problem. The sirolimus–FKBP12 complex does not inhibit calcineurin. Pharmacokinetics Sirolimus is conventionally administered as a once-daily oral dose. Oral bioavailability is around 14%, with peak absorption (Tmax) at 1–2 hours and a very long half-life of around 60 hours allowing once-daily dosing (MacDonald et al. 2000). Everolimus has a half-life of 16–19 hours requiring twicedaily dosing (Kahan et al. 1999). Metabolism is primarily by CYP3A4 and CYP3A5 with biliary excretion of metabolites. Pharmacogenetics Given that sirolimus is metabolized by CYP3A4 and CYP3A5 and is a P-gp substrate, it might be predicted to be susceptible to the same pharmacogenetic influences as tacrolimus. However, this issue remains somewhat unclear with two studies reporting lower dose-normalized sirolimus blood concentrations in CYP3A5 expressers (Anglicheau et al. 2005; Le Meur et al. 2006) and one negative study (Mourad et al. 2005). A caveat is that the association was lost in the event of coadministration of a calcineurin inhibitor. No influence of

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the MDR-1 SNP was identified in these studies. The use of a pharmacogenetic strategy would be particularly attractive for sirolimus given the long half-life, with a resultant long response loop for therapeutic drug monitoring.

Mechanism of action The sirolimus–FKBP12 complex binds to mammalian target of rapamycin (mTOR), a serine/threonine kinase involved in activating the phosphatidylinositol 3-kinase/AKT pathway. This pathway plays a key role in controlling the cellular response to cytokines, primarily by preventing cell cycle progression from G1 to S phase. Sirolimus inhibits the downregulation of IκB-α, a key step in the protection of cells from apoptosis mediated by ligation of CD28 (Hay & Sonenberg 2004). The effects of fibroblast growth factors and vascular endothelial growth factor (VEGF) are also inhibited, resulting in beneficial effects in diseases mediated by endothelial proliferation such as cardiac allograft vasculopathy (Guba et al. 2002; Eisen et al. 2003) but at the expense of a delay in wound healing (Dean et al. 2004). The rate of restenosis of vascular stents due to endothelial proliferation has been reduced by the use of sirolimus-eluting stents (Eisenberg & Konnyu 2006). These factors may underlie the observed inhibition of the growth of malignant cells in vitro and suppression of the growth of experimental tumors in mice (Luan et al. 2002). There are epidemiologic data suggesting a lower rate of new malignancies in immunosuppressed patients treated with sirolimus (Kauffman et al. 2005), in particular cutaneous lesions (Stallone et al. 2005; Euvrard et al. 2006). Method of monitoring There is wide variability between individuals in the blood concentrations achieved by a given dose of sirolimus. Therapeutic drug monitoring has been applied from the outset, initially by methods based on HPLC with mass spectrophotometric detection on whole blood. An immunoassay has now been introduced which produces results that are on average 24% higher than measurements with HPLC due to crossreactivity of the antibody with some metabolites. In interpreting blood concentrations it is essential to know which assay methodology was used. The usual sample is whole blood collected immediately before the next dose. The target concentration required in renal transplantation is now believed to be lower than when the drug was first introduced, typically 10–15 μg/L in the early period and 5–10 μg/L for stable maintenance patients (Kahan et al. 2000). Established indications The main indication for sirolimus is solid organ transplantation but potential as an anticancer drug is under investigation. Safety profile/side effects (including drug interactions) On initial introduction, it was hoped that the mTOR inhibitors would offer an alternative to the calcineurin inhibitors

Immunomodulating Drugs

by being nonnephrotoxic, nonhypertensive, and nondiabetogenic. However, a number of toxicities make the mTOR inhibitors difficult drugs to use (Kahan 2000). Hyperlipidemia is a common complication and it remains uncertain whether this will translate into an increased risk of atherosclerotic complications. Myelosuppression, particularly anemia and thrombocytopenia, are seen, especially when used with one of the antiproliferative agents. A range of other complications are likely to be related to the effects on wound healing or VEGF, including oral ulcers, skin rashes, ankle odema, and proteinuria. Interstitial pneumonitis is a rare but serious complication that may resolve on drug withdrawal (Haydar et al. 2004). Initially, the mTOR inhibitors were thought to be nondiabetogenic, but evidence for toxicity to pancreatic B cells and inhibition of intracellular signalling downstream from the insulin receptor is accumulating (Di Paolo et al. 2006). Several important drug interactions have come to light through the use of sirolimus in multiple drug combinations. Dose-normalized blood concentrations of sirolimus were significantly higher in patients coprescribed ciclosporin A than in those treated with MMF (Cattaneo et al. 2004). Sirolimus appears to increase the nephrotoxic effect of the calcineurin inhibitors, possibly through inhibition of P-gp, allowing increased entry of the calcineurin inhibitors to renal tubular epithelial cells (Anglicheau et al. 2006).

Antibodies A major problem with all the drugs described above is their lack of fine specificity for the immune response, resulting in a narrow therapeutic index and a significant burden of toxicity. The use of antibodies, initially polyclonal and more recently monoclonal, has offered the prospect of more specifically targeted therapy.

Polyclonal antibodies A number of different polyclonal preparations raised by the immunization of rabbits or horses with human thymocytes or T lymphocytes are available. These agents are conventionally known as antithymocyte globulin (ATG) or antilymphocyte globulin (ALG). They contain a broad range of antibody specificities including T and B lymphocytes and a number of more broadly expressed cell-surface molecules including adhesion molecules (Bourdage & Hamlin 1995). These agents are potent immunosuppressants, causing severe lymphopenia, and as a consequence are associated with a high rate of infectious complications. Allergic reactions to the foreign protein are a common problem, with the added potential for neutralization of the therapeutic antibody.

Monoclonal antibodies The development of monoclonal antibodies has allowed the possibility of drugs with a “designed” specificity. The use of genetic engineering to molecularly graft the antibody-binding domain of the primary mouse antibody onto the constant

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Mouse monoclonal antibody Muromonab . . . . .

Chimaeric antibody . . . . . . ximab

Humanized antibody . . . . . zumab

Fig. 32.9 Monoclonal antibodies showing the species origin of the immunoglobulin domains in genetically engineered antibodies. The murine component is shown in blue and the human component in black. The DNA encoding the variable domains of a murine monoclonal antibody can be grafted onto the genes for a human IgG molecule to form a chimaeric antibody, or for the hypervariable regions of the antibody-binding pocket to form a humanized antibody. The taxonomy used in naming these antibodies is indicated. (See CD-ROM for color version.)

region of a human IgG molecule has helped to address the problems associated with the administration of antigenic foreign protein. The taxonomy conventionally used for these antibodies is outlined in Fig. 32.9. The variable domains of a murine monoclonal antibody can be grafted onto the genes for a human IgG molecule to form a chimaeric antibody, or for the hypervariable regions of the antibody-binding pocket to form a humanized antibody. While the development of anti-murine immunoglobulin antibodies is minimized with chimeric antibodies, there can still be a human antichimaeric antibody (HACA) response resulting in either an allergic or neutralizing response. Typically these agents have the halflife of human IgG, around 20 days.

Muromonab CD3 Muromonab CD3 (OKT3) was the first therapeutic monoclonal antibody to be used for immunosuppression (Cosimi et al. 1981). This is a murine monoclonal antibody to the CD3 molecule in the T lymphocyte receptor complex. One major drawback of OKT3 is that it activates T lymphocytes before killing them, causing a cytokine release syndrome. It is potently immunosuppressive and as a consequence is associated with a high complication rate, including significantly increased risk of lymphoma (Opelz & Dohler 2004). The primary use of OKT3 was in renal transplantation but it is now rarely used due to concerns about toxicity. Antibodies to the IL-2 receptor (CD25) Basiliximab (Simulect) and daclizumab (Zenapax) were the first widely used genetically engineered antibodies. They bind the α-chain of the IL-2 receptor, which is only expressed on activated T lymphocytes. Basiliximab is a chimaeric antibody licensed for use in a regimen comprising two intravenous infusions on day 0 and on day 4 following renal

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transplantation (Nashan et al. 1997). Daclizumab is a humanized antibody licensed for use as an intravenous infusion at the time of transplantation followed by four further infusions at 2-weekly intervals (Vincenti et al. 1998). Both agents were effective in reducing the rate of acute rejection. Almost uniquely in the evolution of immunosuppression for solid organ transplantation, the introduction of the CD25 antibodies resulted in reduced rates of acute rejection of renal transplants without an increase in the infectious complication rate (Adu et al. 2003). While these antibodies are very effective in reducing the rate of rejection when present from the start of the evolution of the immune response, there is no evidence for their efficacy in reversing acute rejection, which may limit their application in autoimmunity where the tissue-damaging immune response is already established.

Alemtuzumab (Campath-1H) This humanized monoclonal antibody is specific for CD52 a cell-surface molecule expressed on a number of circulating mononuclear cells, including T and B lymphocytes and natural killer cells. Having been developed originally for the treatment of lymphoma, it is acquiring widespread use in solid organ transplantation (Magliocca & Knechtle 2006) and has been used in the treatment of systemic vasculitis (Lockwood et al. 1993). Despite profound immunosuppression, safety up to 5 years in renal transplant recipients appears to be acceptable (Watson et al. 2005). Rituximab Rituximab is a chimaeric monoclonal antibody specific for CD20, a molecule expressed on B lymphocytes but not plasma cells. It was initially developed for the treatment of B-cell lymphoma but has been used successfully in a number of autoimmune diseases including rheumatoid arthritis (Edwards et al. 2004), systemic vasculitis, and SLE (Smith et al. 2006). It has demonstrated efficacy in the prevention of rejection in solid organ transplant recipients in the presence of preformed anti-donor antibody and may have a role in the treatment of humorally mediated acute rejection (Salama & Pusey 2006).

Fusion proteins T lymphocytes require at least two signals for activation, the first delivered by the specific receptor for antigen and a second delivered through a number of costimulatory pathways. The most important of these in early T-lymphocyte activation is the cross-linking of CD28 on the T lymphocyte through interaction with the ligands CD80 and CD86. CTLA4 (CD152) also binds to these ligands but with higher affinity than CD28. Abatacept is a genetically engineered protein that combines a CTLA4 molecule with a human immunoglobulin domain. Abatacept has been demonstrated to be effective in the treatment of rheumatoid arthriris (Kremer et al. 2003). In contrast, abatacept was ineffective in transplantation, leading

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to the development of belatacept a molecule derived from abatacept but with several amino acid substitutions resulting in increased avidity for CD80 and CD86 and evidence of efficacy in renal transplantation (Vincenti et al. 2005).

Immunosuppression and pregnancy All the immunosuppressive drugs are either contraindicated in pregnancy or come with advice to avoid. However, there are circumstances where immunosuppression cannot be withdrawn during pregnancy. There are reasonable data from the solid organ transplant literature supporting the safe use of azathioprine, ciclosporin A, and tacrolimus in pregnancy. Cyclophosphamide, sirolimus, and mycophenolate carry a high risk of teratogenicity and should be avoided. These drugs are all present in breast milk and conventional advice is to avoid breast-feeding. While blood concentrations in the baby are likely to be small from the given dose, the immature cytochrome P450 system may result in drug accumulation.

References Adu, D., Cockwell, P., Ives, N.J., Shaw, J. & Wheatley, K. (2003) Interleukin-2 receptor monoclonal antibodies in renal transplantation: meta-analysis of randomised trials. BMJ 326, 789. Alexander, A.G., Barnes, N.C. & Kay, A.B. (1992) Trial of cyclosporin in corticosteroid-dependent chronic severe asthma. Lancet 339, 324– 8. Allison, A.C. & Eugui, E.M. (1993) Immunosuppressive and other effects of mycophenolic acid and an ester prodrug, mycophenolate mofetil. Immunol Rev 136, 5– 28. Alomar, A., Berth-Jones, J., Bos, J.D. et al. (2004) The role of topical calcineurin inhibitors in atopic dermatitis. Br J Dermatol 151 (suppl. 70), 3– 27. Anglicheau, D., Le Corre, D., Lechaton, S. et al. (2005) Consequences of genetic polymorphisms for sirolimus requirements after renal transplant in patients on primary sirolimus therapy. Am J Transplant 5, 595– 603. Anglicheau, D., Pallet, N., Rabant, M. et al. (2006) Role of Pglycoprotein in cyclosporine cytotoxicity in the cyclosporinesirolimus interaction. Kidney Int 70, 1019–25. Austin, H.A., Klippel, J.H., Barlow, J.E. et al. (1986) Therapy of lupus nephritis: controlled trial of prednisone and cytotoxic drugs. N Engl J Med 314, 614–19. Bekersky, I., Dressler, D. & Mekki, Q.A. (1999) Dose linearity after oral administration of tacrolimus 1-mg capsules at doses of 3, 7, and 10 mg. Clin Ther 21, 2058– 64. Bottiger, Y., Brattstrom, C., Tyden, G., Sawe, J. & Groth, C.-C. (1999) Tacrolimus whole blood concentrations correlate closely to sideeffects in renal transplant recipients. Br J Clin Pharmacol 48, 445–8. Boumpas, D.T., Austin, H.A., Vaughan, E.M. et al. (1992) Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis. Lancet 340, 741–5. Bourdage, J.S. & Hamlin, D.M. (1995) Comparative polyclonal antithymocyte globulin and antilymphocyte/antilymphoblast globulin

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Nashan, B., Moore, R., Amlot, P., Schmidt, A.G., Abeywickrama, K. & Soulillou, J.P. (1997) Randomised trial of basiliximab versus placebo for control of acute cellular rejection in renal allograft recipients. CHIB 201 International Study Group. Lancet 350, 1193– 8. Nowak, I. & Shaw, L.M. (1995) Mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics. Clin Chem 41, 1011–17. O’Donovan, P., Perrett, C.M., Zhang, X. et al. (2005) Azathioprine and UVA light generate mutagenic oxidative DNA damage. Science 309, 1871–4. Oellerich, M., Shipkova, M., Schutz, E. et al. (2000) Pharmacokinetic and metabolic investigations of mycophenolic acid in pediatric patients after renal transplantation: implications for therapeutic drug monitoring. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients. Ther Drug Monit 22, 20–6. Ojo, A.O., Held, P.J., Port, F.K. et al. (2003) Chronic renal failure after transplantation of a nonrenal organ. N Engl J Med 349, 931– 40. Opelz, G. & Dohler, B. (2004) Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant 4, 222– 30. Pawinski, T., Hale, M., Korecka, M., Fitzsimmons, W.E. & Shaw, L.M. (2002) Limited sampling strategy for the estimation of mycophenolic acid area under the curve in adult renal transplant patients treated with concomitant tacrolimus. Clin Chem 48, 1497–504. Pirsch, J.D., Miller, J., Deierhoi, M.H., Vincenti, F. & Filo, R.S. (1997) A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group. Transplantation 63, 977–83. Ponticelli, C. & Banfi, G. (2006) Thrombotic microangiopathy after kidney transplantation. Transpl Int 19, 789–94. Ponticelli, C., Zucchelli, P., Passerini, P. et al. (1995) A 10-year followup of a randomized study with methylprednisolone and chlorambucil in membranous nephropathy. Kidney Int 48, 1600–4. Ren, S., Kalhorn, T.F., McDonald, G.B., Anasetti, C., Appelbaum, F.R. & Slattery, J.T. (1998) Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients. Clin Pharmacol Ther 64, 289–301. Romero, F., Rodriguez-Iturbe, B., Parra, G., Gonzalez, L., HerreraAcosta, J. & Tapia, E. (1999) Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats Kidney Int 55, 945–55. Ruggenenti, P., Perico, N., Mosconi, L. et al. (1993) Calcium channel blockers protect transplant patients from cyclosporine-induced daily renal hypoperfusion. Kidney Int 43, 706–11. Salama, A.D. & Pusey, C.D. (2006) Drug insight: rituximab in renal disease and transplantation. Nat Clin Pract Nephrol 2, 221–30. Salvadori, M., Holzer, H., de Mattos, A. et al. (2004) Enteric-coated mycophenolate sodium is therapeutically equivalent to mycophenolate mofetil in de novo renal transplant patients. Am J Transplant 4, 231–6. Savage, C.O., Harper, L., Cockwell, P., Adu, D. & Howie, A.J. (2000) ABC of arterial and vascular disease: vasculitis. BMJ 320, 1325–8. Shaw, L.M., Korecka, M., Aradhye, S. et al. (2000) Mycophenolic acid area under the curve values in African American and Caucasian renal transplant patients are comparable. J Clin Pharmacol 40, 624–33.

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Physiologic Aspects of Asthma Philip W. Ind and Neil B. Pride

Summary Physiologic measurement of variable narrowing of intrathoracic airways remains essential to the diagnosis of asthma. Methods of determination of airway caliber and their practical use in asthma management are covered with emphasis on the primacy of forced expiratory volume in 1 s (FEV1) but usefulness of peak expiratory flow (PEF) in clinical practice. Airways resistance (Raw), usually measured by body plethysmography, gives an estimate of the dimensions of the total airway tree but is only available in lung function laboratories. Two other simpler methods of measuring resistance, airflow interruption and the forced oscillation technique, are becoming more widely used. Forced oscillation has usually been used to determine resistance of the total respiratory system but can potentially enable estimation of the relative contributions of central and peripheral airways to increased resistance. Measures from the latter part of maximum expiratory flow–volume (MEFV) curve (e.g., MEF25) have been proposed as a sensitive test of small airway function. Increase in maximum expiratory flow and reduction in pulmonary resistance breathing helium (less dense than air, preferentially affecting central rather than laminar peripheral airflow) has not been widely adopted. Endobronchial catheter techniques provide the best physiologic method of defining contributions of large and small airway narrowing but are invasive and unsuitable for wider application. Three-dimensional imaging techniques have increasingly been used to study airways in asthma despite limitations of resolution. Computed tomography (CT) techniques demonstrate increased airway wall thickness. Air trapping, regional hyperinflation, particularly on expiration, and lung attenuation reflect changes in small airways. Closed airway volume has been measured using single photon emission computed tomography (SPECT) using Technegas. Positron emission tomography (PET) using 13N injection quantifies regional hypoventilation and the accompanying hypoperfusion. Magnetic resonance imaging (MRI) using 3He demonstrates ventilatory defects more marked in more severe asthma. All these techniques confirm previous

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Kr radioisotopic scan demonstrations of gross ventilatory inhomogeneity as predicted from the classic multiple inert gas elimination technique (MIGET). PET modeling of 13N infusion has defined regions of severe hypoventilation and heterogeneity “clusters of self-organized patchiness” below the physical limits of PET resolution. Deep inspiration (DI) typically produces transient airway widening after induced bronchoconstriction in normal subjects while it leads to subsequent airway narrowing in spontaneous asthma. Prohibition of DI produces marked hyperresponsiveness in normal subjects and augments bronchoconstriction in asthma. DI-induced bronchoprotection appears to be absent to inhaled allergen in asthma and allergic rhinitis. Inhaled steroid therapy improves airway responses to DI. The potential importance of airway wall thickening in exaggerating the influence of airway smooth muscle shortening contributing to airway hyperresponsiveness is highlighted. The interaction of airway smooth muscle and lung volume and the possibility of increased shortening due to reduced alveolar wall coupling to intrapulmonary airways secondary to inflammation may contribute to “remodeling.” The use of MEFV and maximum inspiratory flow volume (MIFV) curves for clinically diagnosing extrathoracic airflow obstruction is emphasized. Vocal cord dysfunction can pose major clinical challenges as it often coexists with asthma.

Introduction Asthma is conventionally regarded as a syndrome involving narrowing of airways of all sizes. The magnitude of the narrowing varies over time, either spontaneously or following treatment, but can become persistent. In addition to variable airway narrowing, airway hyperresponsiveness (AHR) and airway inflammation are the most important characteristics of asthma. This chapter focuses on practical measurements of simple lung function and their clinical relevance (see Box 33.1) but includes sections on the site of airway narrowing, the small airways, ventilation inhomogeneity, extrathoracic airways with brief discussion of the nose and larynx, and some specific current areas of interest. Space does not allow a comprehensive description of applied physiology in asthma.

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Box 33.1 Clinical role of lung function testing in asthma.

Box 33.2 Methods for detecting peripheral airways obstruction.

Diagnosis Demonstration of airflow obstruction and reversibility (FEV1) Assessment of PEF variability/reversibility at home (PEF) Bronchial responsiveness testing (PC20 FEV1) Exercise-induced fall in FEV1 Assessment of occupational asthma (PEF, PC20 FEV1, challenge)

Reduced MEF25 Increased residual volume Nitrogen washout detection of increased closing volume Reduced response to breathing helium/oxygen mixture Forced oscillation technique Endobronchial catheter measurement of peripheral airway resistance Tests showing uneven distribution of ventilation Increased alveolar–arterial PO2 difference Single breath N2 test Multiple inert gas elimination technique Frequency dependence of lung compliance or respiratory resistance Radioisotope gas gamma camera imaging SPECT Technegas studies HRCT imaging MRI imaging 13 N2 PET studies

Assessment Demonstration of maximal attainable lung function (spirometry, lung volumes) Assessment for medicolegal purposes (e.g., scuba diving) Demonstration of parenchymal function (gas transfer) Assessment of comorbid lung disease (lung volumes, gas transfer) Assessment of breathlessness (lung volumes, gas transfer, exercise testing) Effect of therapy adjustment (home PEF, FEV1, lung volumes) Home monitoring (PEF) Acute severe asthma (PEF) Clinical research (FEV1, FOT, etc.) Pharmacologic assessment FEV1, PEF, MEFV, PEFV curve, VC, SGaw, PC20 FEV1, lung volumes

at home by the patient. These spirometry tests include forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), or peak expiratory flow (PEF). Measurement of airways resistance in the tidal range gives a direct indication of airway dimensions but is only available in lung function laboratories.

Standard lung function tests Airway function and assessment of airflow obstruction (Box 33.1)

Tests of forced expiration

The primary abnormality in asthma is variable narrowing of the intrathoracic airways. In clinical practice this is most commonly assessed by tests of forced expiration because these tests are simple and can be performed at the bedside or even

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Fig. 33.1 (a) Normal forced expiratory spirogram: change in expired volume (L) against time (s). FEV1 3.2 L, FEV1/FVC ratio 3.2/3.7 (86%). (b) Maximum expiratory flow (L/s) vs. expired volume (L) (MEFV) curve derived from the same spirogram. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; PEF, peak expiratory flow; MEF50, maximum expiratory flow when 50% of FVC remains to be expired; MEF25, maximum expiratory flow when 25% of FVC remains to be expired. Note that MEF50 and MEF25 may also be termed “forced expiratory flow” and abbreviated as FEF50 and FEF25.

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residual volume (RV). The same forced expiration can also be plotted as rate of change in volume (expiratory flow) versus expired volume to obtain a maximum expiratory flow–volume (MEFV) curve (Fig. 33.1b).

FEV1 and FVC In healthy subjects FEV1, which is reproducible in an individual to 5% or better, and FVC both depend on height, age, and gender. At a given height and age, FEV1 is about 20% greater in men than women. Maximum FEV1 is reached in the early twenties and begins to decrease after age 25 years; the rate of decline may slightly accelerate with increasing age but in practice reference values assume an unchanged rate of decline. When vital capacity (VC) is reduced by restrictive disease (pulmonary fibrosis, heart failure, etc.), FEV1 is also reduced, so to diagnose airflow obstruction both FEV1 and the FEV1/FVC ratio have to be reduced (a crude boundary for FEV1/FVC ratio is < 70%). Because of the wide range of values of FEV1 according to height and age, a reduced FEV1/FVC ratio is often used to diagnose airflow obstruction. Once this is established, FEV1 is used to establish reversibility for the diagnosis of asthma. An increase of 12% over baseline and > 200 mL absolute increase are considered significant, while > 400 mL is clinically important. FEV1 remains the gold standard in clinical research. Instruments for measuring FEV1 at home are available but so far have not come into widespread use (Reddel et al. 1998). Because the complete forced expiratory maneuver often causes coughing as RV is approached, the FVC is often less than the VC obtained with a slow expiratory maneuver, so the best practice is to express FEV1 as a ratio of a separate slow VC maneuver. In routine clinical practice in a series of measurements (such as during measurement of airway responsiveness) the forced expiration is often discontinued once the 1-s volume has been obtained. However, it is always useful to know change in (F)VC as well as change in FEV1. As severity of asthma increases, RV increases due to air trapping with reciprocal decreases in FVC and VC. This volume reduction inevitably itself reduces FEV1 and has led to the suggestion that the two components to the spirometric change in asthma, the volume change reflected by the decrease in (F)VC and the obstructive change reflected by the fall in FEV1/(F)VC ratio, should be distinguished (Gibbons et al. 1996). The extent of the volume change as asthma severity increases is quite variable and a disproportionately large increase in air trapping may be an important determinant of symptoms and an unfavorable clinical course. The underlying reason for the usefulness of tests of forced expiration is that when strongly positive pleural pressures are generated, the rate of volume change (flow) at any lung volume reaches a maximum level which cannot be increased by further increases in expiratory effort and pleural pressures (expiratory flow limitation). This occurs because although progressively increasing pleural pressure increases the driving

Physiologic Aspects of Asthma

pressure for expiratory flow, it also increasingly compresses the larger intrathoracic airways, increasing their resistance and opposing the increased driving pressure. The result is that submaximal expiratory efforts are sufficient to generate maximum expiratory flow (MEF). Furthermore, when asthma is active, the required expiratory pressure to achieve MEF is less than when the subject is in remission. Although airway dimensions are crucial in determining the absolute level of MEF, other factors such as lung volume (including reduced VC due to air trapping) and lung recoil pressures (a measure of elasticity of the lungs) are important.

PEF The measurement of PEF is simpler and easier to apply to routine monitoring but is less robust, less reproducible, and more effort dependent than FEV1. Because peak flow meters/gauges measure the highest expiratory flow developed during a forced expiration, the PEF value depends on rapidly developing a positive expiratory pressure early in the expiratory maneuver and close to TLC (in healthy subjects PEF is usually achieved within the first 100 ms after the start of the maneuver when only about 10–15% of the VC has been expired). When measured off a spirometric trace it is usually expressed in liters per second BTPS, whereas it is usually expressed as liters per minute from a PEF meter or gauge. True expiratory flow limitation is present even at volumes very close to TLC, but the precise lung volume at which PEF is measured depends on how rapidly a positive expiratory pressure can be generated at the start of forced expiration. This makes the value obtained more susceptible to technical errors than measuring the FEV1. Normal values of PEF depend on height, age, and gender and are reduced by restrictive lung diseases that reduce lung volumes and VC. Hence PEF should not be used to establish the presence of airflow obstruction (this requires full spirometry). Once obstruction is known to be the cause of a reduction in PEF, it is particularly useful for monitoring progress because of the number of measurements that can be recorded over time, at home, as part of a therapeutic trial or personal management plan. PEF gauges are plastic, portable, and inexpensive and most patients can produce reproducible measurements (see Box 33.3). It is important for patients to use the same meter when assessing trends over time. PEF can be expressed as percent of personal best as well as percent predicted. It is useful to record it first thing, on waking, before therapy, in the morning (PEFAM) when it is usually lower than later in the day and this value can then be compared with an evening reading (PEFPM); the difference between PEFPM and PEFAM constitutes diurnal variation, which has been found to correlate well with other direct measures of bronchial responsiveness. It is also useful to measure PEF at the time of increased symptoms, e.g., if the patient wakes at night and to assess reversibility after administration of a β2-agonist bronchodilator. The amplitude of variation in PEF has more formally been

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Box 33.3 Comparison of methods for monitoring airway obstruction. FEV1 Spirometer Relatively cheap Relatively simple Portable Accurate Less effort dependent Gives other information (FVC) Future telemedicine

PEF Meter or gauge Cheap Simple Hand-held Fairly accurate Effort dependent No other information Computerized with diary

Changes in PEF do not precisely equate with changes in FEV1; typically percent-predicted PEF is higher than percentpredicted FEV1. PEF will tend to underestimate airflow limitation as this worsens and gas trapping occurs. FEV1 and PEF are sometimes described as tests of large intrathoracic airways which are insensitive to change in more peripheral airways function. This view is based on the serial distribution of resistance in normal subjects where the peripheral airways (so-called “silent zone”) make little contribution to total resistance. In mild airflow obstruction this may continue to be the case, but in symptomatic asthma, obstruction of peripheral airways usually makes a larger contribution to the total resistance, which is reflected in reduction in FEV1 and PEF. Hence these tests reflect total resistance of the airways (and so are useful for following the severity of airway narrowing) but are not useful in determining where in the intrathoracic airways most narrowing is situated.

Box 33.4 Assessment of diurnal variation in PEF. Airways resistance Morning dip PEFPM – PEFAM Amplitude % mean (Maximum PEF – minimum PEF)/average daily PEF (over 1–2 weeks) × 100 Min%Max Minimum PEFAM pre bronchodilator/recent maximum PEF × 100 Diagnosis of asthma > 20% diurnal variation ≥ 3 days in a week for 2 weeks on PEF diary

suggested as maximum minus minimum PEF over the average daily PEF as a percentage over 1– 2 weeks (Aggarwal et al. 2002). An alternative method (see Box 33.4) is minimum morning PEF before bronchodilator over 1 week expressed as a percentage of recent maximum (Min%Max). Home PEF recording can be used for the diagnosis of asthma. Maximum daily variation in normal subjects is 5– 8%. A 60 L/min or ≥ 20% improvement post bronchodilator or diurnal variation of > 20% on ≥ 3 days in a week for 2 weeks suggests the diagnosis of asthma (Reddel et al. 1995; Aggarwal et al. 2002). Asthma outcomes have been shown to be improved with self-monitoring of symptoms and/or PEF. This may be particularly in “poor perceivers” (Killian et al. 2000) and in preventing exacerbations (Gibson & Powell 2004). Repeated PEF measurement throughout the day in the workplace (every 2 hours waking to sleeping for 4 weeks) is useful in identifying occupational asthma, with a reported sensitivity of 80% and a specificity of around 90% (Leroyer et al. 1998; Baldwin et al. 2002). The objective measurement of PEF has been repeatedly shown to aid clinical assessment of the severity of an acute exacerbation of asthma, improving outcome.

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The resistance of the airways (Raw) is the pressure generating flow (the difference between mouth and alveolar pressures) divided by the instantaneous flow. It is usually measured at the volumes used during tidal breathing, but can be measured at other lung volumes. Raw gives an estimate of the dimensions of the total airway tree but again gives no indication of the location of any airway narrowing. The main established method for measuring airflow resistance is body plethysmography. When there is airflow obstruction, expiratory resistance can be much greater than inspiratory resistance due to dynamic narrowing of the airways during expiration. If Raw is measured during tidal breathing, usually an average of inspiratory and expiratory resistance, which includes glottal changes, is measured. If the primary interest is in dimensions of the intrathoracic airways, it is preferable to measure Raw on inspiration during a shallow panting maneuver which widens the vocal cords and minimizes resistance of the extrathoracic airway. An advantage of the plethysmographic method is that usually absolute lung volume (VL), which is an important determinant of resistance, is also measured in the same maneuver. This strong dependence on lung size is included in the measurement of “specific” Raw (SRaw = Raw × VL). More commonly adjustment for VL is applied to the reciprocal of Raw, airway conductance (Gaw) yielding “specific” airway conductance (SGaw = Gaw/VL). SGaw, measured at functional residual capacity (FRC) in an individual, is remarkable in that its normal range of values does not change between childhood and old age and is similar in men and women. For example, values of Raw decrease as the lungs grow in childhood but SGaw, which corrects for the growth of the lung, stays constant. Women have higher Raw (and lower Gaw) than men due to the larger VL of men but again SGaw and SRaw are similar in men and women. The requirement for expensive and bulky instrumentation makes the method most suitable for elective full laboratory

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MEFV curve and other flow–volume tests The MEFV curve is a different method of displaying the forced expiratory maneuver used in spirometry in which flow is measured at the mouth with a pneumotachograph and plotted against expired volume (Fig. 33.1b). The MEFV curve shows impressive repeatability when performed properly, so that visual inspection provides excellent quality control of the forced expiratory maneuver, including how rapidly PEF was developed and whether expiration was continued until expiratory flow ceased. In asthma the MEFV curve is often curvilinear, with MEF being particularly reduced in the lower half of the FVC (Fig. 33.2). This qualitative evidence of airflow obstruction can be quantified by measuring MEF at specific points on the MEFV curve, usually when 50% or 25% of the FVC remains to be expired. In mild obstruction changes are characteristically in the last part of the expiration (in part due to the closure of some airways, see below) so MEF25 has been proposed as a sensitive test; its main weakness is that the normal range of values is very wide and not much reduced by adjusting for the factors influencing spirometry, such as age, height and gender. PEF can also be measured from the MEFV curve, but there is little point in doing this when a complete forced expiratory maneuver is obtained. In addition values may differ from those obtained from a peak flow gauge. Maximum inspiratory flow–volume (MIFV) curves can be obtained from a forced inspiratory maneuver started after a slow full expiration to RV and continuing to inspire until TLC is reached. In contrast to MEF, maximum inspiratory flow

100% Maximum expiratory flow (% of baseline)

assessments or the study of pharmacologic responses. Two other simpler methods of measuring resistance, airflow interruption and the forced oscillation technique (FOT), are less demanding experimentally and becoming more widely available. These methods are applied during tidal breathing and may be particularly useful in young children (Delacourt et al. 2001). The interruption technique simply estimates airflow resistance in either inspiration or expiration. The FOT technique is most commonly used to measure resistance of the total respiratory system, averaged over several breaths, but it can also be used to obtain a much fuller analysis of the mechanical properties of the lungs (Oostveen et al. 2003). This analysis depends on subdividing the primary measurement obtained, the instantaneous relation between the applied pressure and the resultant flow oscillation (impedance), into its in-phase (resistance) and out-of-phase (reactance) components. Furthermore, impedance can be measured over a wide range of frequencies (typically 4– 26 Hz) so that its frequency dependence can be determined. There are suggestions that by studying the frequency dependence of resistance and the values of reactance, the relative contributions of central and peripheral airways to increased resistance can be estimated. If these claims are confirmed, FOT may gain a significant role in assessment of airway function in asthma.

Physiologic Aspects of Asthma

PEF

MEF50

MEF25

100% Forced vital capacity (% of baseline) Fig. 33.2 Characteristic changes in maximum expiratory flow–volume (MEFV) curve as asthma becomes more severe. Curves were obtained in five patients admitted with severe asthma who were studied sequentially until normal airway function was obtained. The curves were normalized to the values obtained in full remission of asthma (top curve, 100% function). Note progressive convexity of curve to volume axis as airflow limitation worsens. Because of reduction in forced vital capacity and increase in residual volume, MEF50 and MEF25 are measured at successively larger volumes as airflow limitation worsens. With mild airflow limitation, the largest proportional changes are in MEF25 and MEF50. See Fig. 33.1 for definition of other abbreviations.

(MIF) depends on the driving pressure generated and so requires more trial efforts to achieve repeatable results. MIF is reduced in active asthma but to a lesser extent than MEF and has not found a useful place in routine assessment of asthma. Only in very severe asthma in children or the very elderly will MIF be reduced enough to compromise effective use of some of the dry powder inhalers. The main clinical relevance of the MIFV curve is that, when studied in conjunction with the MEFV curve, it helps distinguish extrathoracic obstruction, for instance in the larynx, from asthma. The shape of the MEFV curve may be distinctive (Fig. 33.3), with reduced MEF at high lung volume but a normal MEF as RV is approached (quite different from asthma) and MIF may be reduced throughout the inspiratory VC. Sometimes obstruction of the extrathoracic airways may only reduce MIF, the MEF curve remaining normal. Several other techniques using flow–volume curves have been developed. A full inspiration to TLC in healthy subjects leads to short-lived bronchodilatation which may increase flow during a subsequent complete forced expiratory maneuver; in contrast in many asthmatic subjects, a full inspiration causes transient bronchoconstriction. To study these effects, maximum expiratory flow and volume during a forced expiration started from the end of a tidal inspiration (partial expiratory

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flow–volume PEFV curve) may be compared with that during a conventional MEFV curve started from TLC. MEFV curves breathing air have been compared with MEFV curves after breathing helium/oxygen mixtures to investigate the predominant site of airway narrowing. Flow– volume curves during tidal breathing have been studied at baseline and after a small negative expiratory pressure (NEP) has been applied at the mouth during expiration (Koulouris et al. 1997); usually NEP results in an increase in tidal expiratory flow but in severe airflow obstruction there may be no increase, indicating the presence of expiratory flow limitation during tidal breathing.

Measurement of lung volumes: assessment of hyperinflation and air trapping The VC and FVC are frequently reduced in symptomatic asthma. They are usually measured in an expiratory maneuver and provide no information on absolute lung volumes (TLC or RV) or on the presence of hyperinflation. Absolute lung volumes can be measured in asthma by body plethysmography or by a multiple breath equilibration technique in which the subject re-breathes a helium mixture until a steady concentration is reached. Both methods have potential problems particularly when obstruction is severe. Plethysmography may overestimate lung volume because during the panting maneuver used mouth pressure may not equilibrate with alveolar pressure (Stanescu et al. 1982). In contrast, the helium equilibration method tends to underestimate volume because equilibration is incomplete. With both techniques the volume directly measured is usually the expiratory end-tidal volume, i.e., FRC; the volumes at the extremes of the VC (TLC and RV) are obtained by an additional two-part VC maneuver (full inspiration to TLC, full expiration to RV) started from FRC. Sometimes the volume directly measured using body plethysmography is termed “thoracic gas volume.” In healthy subjects FRC is the volume where the inward (expiratory) recoil of the lungs is exactly balanced by the outward (inspiratory) recoil of the relaxed chest wall (relaxation

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Fig. 33.3 Maximum expiratory and inspiratory flow–volume curves in extrathoracic airway obstruction. (a) Fixed narrowing of the glottis. Reduced maximum expiratory flow (MEF) over the first 75% of the forced vital capacity (FVC) with normal decline in MEF over the last 25% of the FVC. Maximum inspiratory FEV1/FVC remains normal flow (MIF) reduced at all volumes. (b) Variable narrowing in this patient due to subglottic polyp which occludes the glottis during a forced inspiration. Maximum expiratory flow–volume curve virtually normal but MIF reduced at all lung volumes.

volume). At rest the later part of a tidal expiration is passive so that there is no activation of respiratory muscles and FRC corresponds to the relaxation volume. Typically at rest FRC is about 50% TLC in young healthy subjects, rising to 55– 60% TLC in elderly subjects. During exercise there is activation of expiratory muscles and end-tidal volume is lower than the resting FRC. To reach TLC requires full activation of inspiratory muscles; conversely RV requires full activation of expiratory muscles to reduce the volume of the chest wall. In healthy young adults the increasing stiffness of the chest wall limits deflation but after age 40 years airway closure, probably in small bronchioles, determines RV. RV rises more than FRC with increasing age, and because TLC does not change, the rise in RV accounts for the reduction in VC with age. The difference between FRC and TLC is termed “inspiratory capacity”; this falls with increasing age but to a smaller extent than VC. Fewer measurements are made of closing volume (CV), which requires a separate single breath technique, usually based on the change in nitrogen concentration during a full expiration after a full inspiration of 100% oxygen from RV. CV is recognized by the beginning of a steeper rise in expired nitrogen concentration, which corresponds to the beginning of airway closure on expiration. CV is usually expressed as milliliters above RV or as percent VC but the absolute volume at which closure begins, closing capacity (CC), can be obtained as the sum of CV + RV. In young healthy subjects closure begins well below FRC, but CV (and CC) rise with age and may exceed FRC by 70 years of age. In active asthma RV is characteristically elevated due to enhanced airway closure (air trapping). It is often assumed that closure occurs in the terminal bronchioles (and so is an indicator of small airways disease), but this is not necessarily the case in asthma where airway smooth muscle contraction and mucoinflammatory plugs may result in closure of more proximal airways. Even in asthma remission, when VC and RV (and FEV1) are normal, CV is often increased (McCarthy & Milic-Emili 1973). Imaging with scintigraphy, high-resolution

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CT (HRCT), or MRI shows that in healthy subjects airway closure commences in the most dependent parts of the lungs and progresses up the lungs as full expiration continues (see below). In asthma this pattern of closure is much more disorderly, presumably reflecting difference in the extent of pathologic changes between adjacent regions of lung. FRC also increases as asthma worsens but to a lesser extent than RV. Some of this increase may be due to an increase in relaxation volume but usually FRC is greater than relaxation volume and is maintained by glottal “braking” of expiration, which is terminated by the next inspiration before relaxation volume is reached. The increase in FRC widens airways but at the cost of increased work by the inspiratory muscles to overcome the greater elastic load offered by lungs and chest wall as lung volume is increased. When spontaneous or bronchodilator-induced improvement occurs there is usually some reduction in FRC which removes part of the improvement gained in airway dimensions, but most often both airway resistance and FRC are reduced. TLC in stable asthma is usually normal or may be slightly increased, especially in patients who had asthma during growth (Greaves & Colebatch 1985). Whether TLC increases in acute asthma has been controversial since first proposed more than 40 years ago (Woolcock & Read 1966; Woolcock et al. 1971). Some of the early increases reported using body plethysmography were artifactual (Stanescu et al. 1982). Subsequently the consensus for many years was that TLC did not increase acutely when airway narrowing was induced in asthma (Kirby et al. 1986). Recently this controversy has been revived by an analysis suggesting that an acute increase in TLC is an important adaptation to a large increase in RV (Brown et al. 2006; and see commentary by Irvin 2006). It remains unclear where an increase in TLC could be accommodated within the thoracic cavity. Perhaps the controversy will be resolved by the sequential use of an imaging technique to estimate volume at full inflation during an acute episode of asthma. Woolcock and Read (1966) also first suggested that spontaneous or treatment-induced improvement in symptoms could be due to a fall in FRC without accompanying improvement in spirometry or PEF. Probably most such examples occur when overall physiologic changes are small; with large responses, there is usually both an improvement in spirometry and a fall in FRC. Because measurement of FRC cannot be routinely assessed in acute asthma, the true prevalence of such isolated volume responses remains uncertain. Measurement of any of these absolute volumes is important because FEV1 and PEF only assess airflow obstruction, while hyperinflation (breathing at large operating volumes) is an important cause of discomfort in acute asthma. “Hyperinflation” is probably best used to refer to increased FRC because this indicates tidal breathing is occurring at larger than normal operating volumes. It is sometimes used to indicate a large RV, RV/TLC ratio, or even TLC, none of which are

Physiologic Aspects of Asthma

directly related to tidal breathing difficulty. The most practical method for monitoring hyperinflation in acute severe asthma is to measure inspiratory capacity (difference between FRC and TLC). This can be done with any spirometer which allows measurement of inspiratory volumes. As discussed above, a reduction in (F)VC indicates a reciprocal rise in RV.

Assessment of alveolar function Conventionally, asthma is regarded as a disease of the airways without significant changes in the lung tissue or airspaces. Although pulmonary gas exchange is inefficient and arterial oxygen pressure is reduced in attacks of asthma, this is caused by uneven distribution of ventilation (and blood flow) not by any alveolar abnormality. Carbon monoxide transfer (CO diffusing capacity) is usually normal or near normal in fully reversible asthma (Keens et al. 1979; Collard et al. 1994), but there is often a small loss of lung recoil pressure, especially at large volumes (Kraft et al. 2006) while in persistent asthma these losses may be larger (see below). Histologically human alveolar ducts contain smooth muscle and in animals increase in the resistance of lung tissue accounts for a considerable proportion of the response to bronchoconstrictor aerosols (Ludwig et al. 1989). In human asthma it is difficult to measure lung tissue resistance directly but modeling suggests that any contribution from lung tissue is certainly much smaller and is not found in all subjects (Kaczka et al. 1999).

Chronic asthma with persistent airflow obstruction In middle-aged and older patients with asthma, optimized airway function is often below predicted values (Brown et al. 1984). This confuses the distinction from, and results in genuine overlap with, smoking-related chronic obstructive pulmonary disease (COPD) (Boulet et al. 1998; Fabbri et al. 2003), although the prognosis in chronic asthma is much better. Airways resistance is increased in both central and peripheral airways (Yanai et al. 1992) but little is known about the pathophysiologic basis of these changes. Airway wall thickening is the most obvious and consistent finding on HRCT (Boulet et al. 1995; Awadh et al. 1998; Ketai et al. 2001; Nimi et al. 2003). Conventionally chronic asthma, in the absence of smoking, is not associated with the development of emphysema, although HRCT scans do show limited changes in a minority of patients (Boulet et al. 1995; Gelb et al. 2002). Carbon monoxide transfer may be modestly reduced but not to the extent commonly found in COPD (Boulet et al. 1998; Gelb et al. 2002; Fabbri et al. 2003). More striking reductions in lung recoil pressure, which would decrease MEF significantly, have been reported by Gelb and colleagues in a few patients with little or no emphysema on HRCT and normal carbon monoxide transfer (Gelb & Zamel 2000; Gelb et al. 2002); these findings need confirmation by further studies. Two factors that may contribute to loss of lung recoil are stress relaxation of alveolar tissue due to chronic hyperinflation

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(Gelb et al. 2002) and alveolar inflammation (Kraft et al. 2001). Peribronchial inflammation may damage alveolar walls (Mauad et al. 2004) and lead to loss of their recoil, while expansion of the airway outer perimeter may reduce coupling between the small airways and alveolar attachments. Hence even in the absence of overt destructive emphysema it is possible that more subtle alveolar disease contributes to the persistent airway obstruction of chronic asthma.

Changes in clinical remission Because of the enormous expansion of the cross-sectional area of the peripheral airways (their great increase in number far outweighing their much smaller diameter compared to more central airways) considerable disease can be present in small airways without obvious effect on spirometric function or total airways resistance. Many studies have shown that patients in clinical remission frequently show persisting abnormalities of RV, CV, abnormal distribution of ventilation, and peripheral lung resistance, changes all compatible with persistent obstruction in peripheral airways.

Site of increased airway resistance: large versus small airways Airway narrowing in episodic asthma is due to varying combinations of airway smooth muscle (ASM) contraction, mucosal swelling, and luminal secretions. These factors all vary during and between attacks. Pathologic studies have shown that inflammation of the airway mucosa and increased ASM mass extend throughout the tracheobronchial tree, with inflammation into the alveoli (Carroll et al. 1993; Kraft et al. 1996, 1999) indicating the potential for narrowing to develop in airways of all sizes. Studies of regional ventilation (see below) have consistently shown that airway narrowing varies greatly between parallel airways, despite the likely presence of inflammatory changes in all airways. Presumably there can also be longitudinal nonuniformity. Given such heterogeneity and the apparently invariable involvement of small airways, identifying precisely which airways contribute most to airflow obstruction is very difficult and may not even be clinically useful. Despite considerable research efforts, a simple robust measure of the predominant site of airflow obstruction is still needed (see Box 33.2).

receptors, and induce reflex bronchoconstriction and mucus production. The assumption is made that the peripheral lung beyond the catheter tip is “typical” of all other regions of peripheral lung (it has not been practical to place catheters in multiple regions of the lung) in an individual. Two different wedged-catheter techniques have been developed that measure pressure at a peripheral site; in one, a 3-mm catheter is wedged in a right lower-lobe bronchus and measuring lateral pressure just proximally to the wedged site allows the peripheral airways resistance of the adjacent ventilated lung to be determined (Yanai et al. 1992). Total lung resistance in normal subjects with this technique appears about 50% higher than without the presence of a catheter. In asthma in remission, inspiratory (but not expiratory) peripheral airway resistance was slightly increased. In chronic asthma with persistent airflow obstruction in middle-aged patients, both central and peripheral resistances were increased. Allowing for the contribution of the extrathoracic airway to total lung resistance, peripheral resistance accounted for about onethird of intrathoracic resistance in normal subjects and about 50% in chronic persistent asthma (Yanai et al. 1992). Another less physiologic technique measures pressure– flow relations within the occluded lung beyond a bronchoscope wedged in a segmental bronchus. Gas is introduced via the bronchoscope and slowly inflates the occluded lung while the pressure at the end of the bronchoscope is measured (Wagner et al. 1990). In asthmatic subjects in remission, with normal FEV1 and total airway resistance, a considerable increase in this peripheral lung resistance was found. This resistance not only reflects the resistance of peripheral airways, but also of collateral channels and lung tissue. These results directly confirm earlier suggestions from pathologic and physiologic studies that there are residual changes in the peripheral airways even in asthma in remission. This technique has also been used to show a rise in peripheral lung resistance at night in nocturnal asthma (Kraft et al. 2001) and to measure increases in the mean level and frequency dependence of this resistance after methacholine (Kaminsky et al. 2004). Although these results imply an increase in resistance of collateral channels in asthma, gas exchange studies suggest that these channels continue to allow some collateral ventilation except in the most severe asthma attacks.

Airway response to breathing a helium–oxygen mixture

Endobronchial catheter studies

MEFV curves

Endobronchial pressure measurements define the airway distribution of airflow resistance directly, and provide the best physiologic evidence for the respective contributions of large and small airways, but their invasive nature has restricted their use to stable patients. There are also important physiologic limitations. Endobronchial catheters inevitably reduce the area available for gas flow, may stimulate mucosal

The density of 80% helium/20% oxygen is approximately one-third that of air. In some (but not all) asthmatic subjects, MEF does not show the normal increase when breathing helium/oxygen (Despas et al. 1972) suggesting that the major site of flow limitation is no longer in central airways, as in normal subjects, but has moved to more peripheral airways, where flow is presumed to be laminar and independent of

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density (Ingram & McFadden 1977). This change is usually attributed to increased frictional pressure losses in narrowed peripheral airways. Some asthmatic subjects consistently lose or consistently retain density dependence of maximum flow with repeated attacks. In general, loss of density dependence becomes more common as expiratory airflow limitation increases in severity (Fairshter & Wilson 1980; Partridge & Saunders 1981) and is particularly observed in asthmatic subjects who smoke (Antic & Macklem 1976). Reduction in density dependence of MEF should not be interpreted as indicating that only peripheral airways are narrowed, even if they are the site of flow limitation. Considerable variation in size of the helium response in the normal population and in disease has been noted; changes in helium response in an individual before and after an acute intervention may be more reliable.

Pulmonary resistance An alternative method is to study the reduction in pulmonary resistance (RL) (measured by the esophageal balloon technique) when tidal breathing is switched from breathing air to a helium/oxygen mixture (Lisboa et al. 1980). A large decrease in RL suggests a large component is in the central conducting airways while a small response indicates that much of the resistance breathing air is in the peripheral airways. This tidal breathing technique is probably more relevant to the use of helium/oxygen in acute severe asthma to reduce resistive work of breathing. There are practical problems in clinical use because Heliox density rises once helium concentration falls below 70% due to increased inspired oxygen or entrainment of room air. A recent Cochrane metaanalysis of 544 patients in 10 studies concluded that there was no clinical role of Heliox, although FEV1 improved more in the subgroup with the most severe asthma (Rodrigo et al. 2006).

Physiologic Aspects of Asthma

explains the variable changes in airway function after DI (Ingram 1987, 1990). When airway hysteresis exceeds that of parenchyma, DI results in temporary airway widening when tidal breathing is resumed. When parenchymal hysteresis exceeds that of the airways, DI results in airway narrowing. Equal degrees of hysteresis result in no effect of DI on resting airway caliber, as found in most normal subjects. A major interest of this idea is that it might localize the site of disease within the lung. Contraction of smooth muscle in conducting airways would be expected to increase airway hysteresis without affecting parenchymal hysteresis. This is the typical pattern of induced bronchoconstriction. Increased tone or other causes of airway narrowing in the extreme periphery of the lung (respiratory bronchioles and alveolar ducts) would be expected to increase parenchymal hysteresis with only a small increase in airway hysteresis. This is the typical pattern in spontaneous asthma.

Other evidence of small airway involvement in asthma Histologic and morphometric data have long suggested that important structural changes occur in the airway walls and ASM of the peripheral as well as central airways (Ebina et al. 1990; Carroll et al. 1993; Hamid 1997). The bronchoscopic catheter studies directly, and the frequency dependence of dynamic resistance and elastance indirectly, provide strong physiologic evidence of peripheral airway involvement. Multibreath nitrogen (Verbanck et al. 1999) and single-breath SF6 and helium washout studies have provided evidence of airway closure at acinar level contributing to peripheral airway response (Gustafsson et al. 2003). Mathematical modeling approaches (Lutchen et al. 2001; Kaminsky et al. 2004) relate inflammation, airway thickening, and amplification of heterogeneous constriction to determine mechanical response.

Airway response to a deep inspiration Deep inspiration (DI) produces airway widening transiently when tidal breathing is resumed in normal subjects after narrowing is induced by inhaled histamine or methacholine. In contrast, DI can lead to subsequent airway narrowing in subjects with acute spontaneous asthma (Simonsson et al. 1967). These differences between normal and asthmatic subjects have practical effects exaggerating differences in airway responsiveness as assessed by conventional tests of forced expiration. Partial expiratory flow–volume curves surmount some of these difficulties, with measurements usually made at volumes between 30 and 40% VC, thus balancing the conflicting demands of avoiding undue lung inflation and artifactual flow at the start of expiration, while still ensuring an adequate flow signal. Initially, changes after DI were attributed to a change in bronchial muscle activity (direct or reflex) produced by stretch (see further discussion in section Airway smooth muscle). An alternative hypothesis is that the relation between parenchymal and airway hysteresis

Imaging of the airways and ventilation (Box 33.5)

Visualization of the airways Direct visualization of airways, either at bronchoscopy or by external imaging (plain radiographs, bronchography, CT), is confined to the larger conducting airways. Narrowing of the large airways has occasionally been reported when acute bronchoconstriction has developed during bronchoscopy (or, in the past, at bronchography) or on segmental airway challenge. Airway lumens as small as 0.5 mm in diameter have been reportedly measured using CT but large errors are likely (De Jong et al. 2005). HRCT can interrogate small airways indirectly by demonstrating air trapping and regional hypoventilation (exaggerated by expiration), assumed to be due to enhanced closure of peripheral airways (reflecting increase in CV and RV on standard lung function testing).

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Computed tomography Airway size HRCT and multidetector row CT (see De Jong et al. 2005 for methodology) allow determination of airway luminal area, which has been reported to be both decreased (Lynch et al. 1993) but normalized after salbutamol in some patients (Beigelman-Aubry et al. 2002) but not in others with more severe asthma (Park et al. 1997; Nimi et al. 2000). However, all studies have demonstrated increased airway wall thickness in asthma of all severities in children as well as adults. Degree of wall thickening is related to duration and severity and level of airway obstruction (De Jong et al. 2005). Many authors have measured bronchoconstriction of larger airways following methacholine. However, the relationship between airway wall thickness and AHR is complex. Thicker airways may be less compliant and therefore less responsive: increase in wall thickness in larger airways or decrease in luminal diameter of medium airways (5–10 mm diameter) and dynamic hyperinflation with critical narrowing of large airways are important determinants of responsiveness (Brown et al. 2006).

Gas trapping Air trapping, and regional hyperinflation, particularly on expiration, allow indirect assessment of small airways qualitatively (Park et al. 1997) and semiquantitatively (Laurent et al. 2000). The distribution of Hounsfield units represents a frequency distribution of lung attenuation (LA) values and dynamic analysis of the distribution of LA yields reproducible quantitative data. Reduction of gas trapping by hydrofluoroalkane small-particle beclomethasone dipropionate (BDP) more than conventional chlorofluorohydrocarbon (CFC)-BDP (Goldin et al. 1999) and other inhaled corticosteroids (Aubier et al. 2001) and montelukast has been shown (Zeidler et al. 2006).

Single photon emission computed tomography SPECT measurements of closed airway volume (inaccessible to a Technegas bolus) correlated with CV and CC and did not differ in mild asthmatics with normal and abnormal spirometry from normal subjects (King et al. 1998), although the distribution differed in asthma. Regional expiratory flow limitation was demonstrated using Technegas with hotspots thought to represent exaggerated impaction of Technegas on severely narrowed airways (Pellegrino et al. 2001).

Positron emission tomography Early studies (Rhodes et al. 1989a,b) used inhaled 19Ne and intravenous infusion of 13N2 to measure regional ventilation– perfusion ratios in asthma. There was a greater variation in regional ventilation and ventilation–perfusion ratio than in normal subjects. Modeling of a 13N2 bolus injection followed by 30–60 s apnea enabled quantitation of “fast” (normal ventilation) and “slow” (hypoventilation) compartments (Musch & Venegas 2005) occurring below the limited resolution of PET

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(approx 1 cm). Regional hypoventilation and heterogeneity in asthma was quantified as injected tracer is retained locally after resumption of breathing (Venegas et al. 2005a). The relative contributions of large and small airways have been analyzed using an image functional modeling approach mapping three-dimensional ventilation images onto an anatomically explicit computational model using forced oscillometry at 0.15–8 Hz to determine dynamic lung resistance and elastance pre and post methacholine (Tgavalekos et al. 2005). Closure of airways < 2.39 mm and mainly < 0.44 mm in diameter best mimicked the experimental data. Patterns of constriction involving larger airways could mimic the results only in the presence of small airway closure.

MRI studies Gaseous MRI contrast agents such as 129Xe and 3He have led to new imaging of ventilation without the use of ionizing radiation (Middleton et al. 1995; de Lange et al. 1999). Single breaths of hyperpolarized 3He, which provides a large MRI signal of ventilated airspaces because of its low water and lipid solubility, have demonstrated wedge-shaped ventilatory defects, mostly < 3 cm in size, more consistently and more extensively in more severe asthma correlating with percentpredicted FEV1 (Altes et al. 2001; Samee et al. 2003). Ventilation defects per slice were significantly greater in moderate and severe persistent asthma than in mild intermittent or mild persistent asthma and correlated best with FEV1/FVC (r = −0.51, P < 0.002), MEF25–75 (r = −0.5, P = 0.001) and FEV1 (r = −0.40, P = 0.002) (de Lange et al. 2006). Many defects persisted when MRI was repeated. Induced bronchoconstriction resulted in the appearance of new defects; these are detected more sensitively by 3He MRI than change in spirometric indices including MEF25–75 (de Lange et al. 2007). Furthermore, defects often recurred in the same site when methacholine challenge was repeated. Dynamic imaging using ultra-fast pulse sequences to image the respiratory cycle has the potential to produce quantitative regional measurements of airflow (Salerno et al. 2001). Other MRI techniques including measurement of ventilation volumes, diffusion imaging (measurement of apparent diffusion coefficient), and regional oxygen tension mapping have not been applied specifically to asthma (van Beek et al. 2004).

Ventilation inhomogeneity Inhomogeneity of ventilation is well recognized clinically in asthma; breath sounds may be absent to auscultation regionally and segmental atelectasis may sometimes be visualized radiologically. The multiple inert gas elimination technique (MIGET) demonstrated a clear bimodal distribution of ventilation–perfusion ratios (Wagner et al. 1978) and different airways were shown to respond differently to inhaled histamine using the wedged bronchoscope technique (Wagner et al. 1998). Heterogeneity of conducting airways (Scond) using multiple breath nitrogen washout was recently suggested as

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Box 33.5 Imaging of airways and ventilation. Imaging of airways Chest radiography Bronchography CT fine section and reconstruction, multidetector row Imaging of ventilation Scintigraphy: 81Kr, 133Xe, 127Xe, 99mTc aerosols SPECT: xenon, Technegas MRI: hyperpolarized 3He, 129Xe PET: inhaled or intravenous 13N, inhaled 19Ne

a major determinant of responsiveness uncoupled from airway inflammation (using inhaled nitric oxide as a surrogate by treatment with BDP (uncontrolled) (Downie et al. 2007). A variety of three-dimensional imaging techniques demonstrate ventilatory inhomogeneity dramatically. Patchy defects in 81mKr ventilation scans have long been recognized (Fig. 33.4), particularly on induced bronchoconstriction and in severe asthma (Sovijarvi et al. 1982; Vernon et al. 1986; Orphanidou et al. 1986). HRCT has been used to demonstrate heterogeneous airway narrowing particularly of larger airways to inhaled methacholine in dogs, normals and asthmatics (Brown & Mitzner 2003; King et al. 2004; Brown 2007). Spotty deposition of Technegas was greater in the lower zones (and less in the upper zones) in asthma (King et al. 1998). Defects were larger, more patchy, and often peripheral and wedge-shaped (Fig. 33.5). Complete absence of Technegas in an entire lobe or part of it, mainly in dependent lung regions, occurring at high doses of methacholine were thought to suggest full airway closure (Pellegrino et al. 2001). Patchy ventilatory defects were also noted in early PET studies using infused 13N2 (Rhodes et al. 1989b; Rhodes & Hughes 1995). Quantification of regional hypoventilation and heterogeneity in asthma was possible using PET modeling of a 13N2 bolus injection (Venegas et al. 2005a). Methacholineinduced bronchoconstriction is associated with regions with severe hypoventilation or “clusters of self-organized patchiness” (Venegas et al. 2005b). Bronchoconstriction is characterized by patchy distribution of regional ventilation with bimodal V/Q distributions in line with classic physiologic studies in asthma using MIGET (Wagner et al. 1978; RodriguezRoisin et al. 1991). In mild asthma, ventilation defects (varying from subsegmental to lobar) induced by methacholine were associated with a systematic reduction in regional perfusion of the order of 30% (Harris et al. 2006). This would operate to reduce regional ventilation–perfusion mismatch, though it is not clear to what extent local regional increase in gas volume leads directly to reduced blood flow and to what extent active pulmonary vasoconstrictor mechanisms are responsible.

Physiologic Aspects of Asthma

Airway smooth muscle and change in lung volume Both alveoli and airways are elastic structures, changing size when exposed to alterations in distending forces, as occurs every few seconds with tidal breathing, and to a greater extent with an occasional sigh or DI. However, whereas alveoli have rather fixed and predictable elastic properties, contraction of ASM changes airway dimensions and compliance drastically in a few seconds, putting ASM at the heart of the asthmatic process. The physiology of ASM is extensively discussed in Chapter 42. This brief section outlines the important interaction of ASM with lung volume and other determinants of its function in vivo.

Normal airways The area of a normal intrathoracic airway increases by 45–50% when a DI is made from FRC to TLC. This increase occurs due to increased distending forces by the lungs themselves as lung volume increases or, in the case of extrapulmonary intrathoracic airways, directly from the inspiratory reduction in pleural and mediastinal pressure. Normal airways have a baseline “tone” due to activation of ASM by vagal stimulation. This slightly reduces their dimensions from the relaxed state and accounts for the small response in airway function found with bronchodilators. Variations in this vagal “tone” determine the circadian rhythm in airway function found in normal subjects, which has the same timing but smaller amplitude compared with the circadian rhythm in asthma.

Asthma AHR to bronchoconstrictor challenge (see Chapter 38) is a central abnormality in asthma. For many years it has been known that the bulk of ASM is increased in asthma (Carroll et al. 1993; Woodruff et al. 2004) but there has been uncertainty whether it has increased contractility. Historically, AHR has been explained in terms of enhanced “twitchiness” and contraction of ASM. This could be due to external influences such as altered adrenergic receptors, altered neural control, inflammatory or other mediators, or the proximity of mast cells. Alternatively, it could be intrinsic to ASM itself: its increased mass or enhanced contractility. In recent years it has been emphasized that enhanced bronchoconstriction could occur without postulating any abnormality of ASM response because (i) a normal amount of shortening of ASM can give a disproportionate reduction in luminal caliber in the presence of relatively subtle wall thickening; or (ii) greater ASM shortening is produced for a given stimulus because the restraining force applied by surrounding alveolar walls attached to the external perimeter of intrapulmonary airways is reduced. Airway wall–parenchyma coupling might be reduced by peri-airway inflammation and damage to

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Mild asthma

Visit 1 FEV1 104%

Visit 2 FEV1 101%

Visit 1 FEV1 66%

Visit 2 FEV1 68%

Male Age 48 Grade 0

(a)

Male Age 42 Grade 1

(b) Severe asthma

Visit 1 FEV1 48%

Posterior ventilation

Visit 2 FEV1 52%

Male Age 66 Grade 2

(c) Visit 1 FEV1 41%

Visit 2 FEV1 43%

Male Age 53 Grade 3

(d)

alveolar walls and/or expansion of the airway perimeter. Although airway wall thickening (remodeling) might be induced by inflammation, if it does not completely regress with suppression of inflammation, AHR could be sustained solely by airway wall thickening.

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Fig. 33.4 Krypton-81m ventilation scans demonstrating heterogeneity, with defects visible in more severe asthma graded 0, 1, 2, and 3 in four patients studied on two occasions, when stable, 2 weeks apart. (a) Posterior scans in a 48-year-old mild asthmatic: FEV1 104% and 101% predicted on the two occasions, both graded 0 (normal). (b) Posterior scans in a 42-year-old asthmatic: FEV1 66% and 68% predicted on the two visits, both graded 1 (minor defects). (c) Posterior scans in a 66-year-old asthmatic: FEV1 48% and 52% predicted on the two visits, both graded 2. (d) Posterior scans in a 53-year-old asthmatic: FEV1 41% and 43% predicted on the two visits, graded 3 (multiple large defects). Note how ventilation has improved in the right lower lobe and deteriorated in the left between the two visits.

Effects of DI and its prohibition Further interest in the effects of DI and stretch on ASM was stimulated by the observation that prohibition of DI could produce striking hyperresponsiveness to methacholine in normal subjects (Skloot et al. 1995). This suggested that

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Physiologic Aspects of Asthma

Fig. 33.5 Use of three-dimensional imaging to show heterogeneity of airway closure (at residual volume) and inhomogeneity of ventilation. (Top) Single coronal slices from three normal subjects aged 27, 32 and 43 years. Unventilated regions are relatively uniform and contiguous. (Bottom) Slices show typical large peripheral triangular defects in asthma suggesting airway closure. Superimposed emission and transmission images; ventilated lung is white, unventilated lung gray, and soft tissue black. (From King et al. 1998.) (See CD-ROM for color version.)

failure to periodically inflate the lung to large volumes results in loss of a bronchoprotective effect, which was subsequently shown to be considerably stronger than the bronchodilator effect of DI in normal subjects (Schichilone et al. 2000). However, this bronchoprotective effect was lacking in asthmatics and even in patients with rhinitis alone (Kapsali et al. 2000; Schichilone et al. 2001). However, using a different protocol administering a PC15 dose of methacholine every 5 min and measuring FEV1 at the beginning and then after five challenges, King et al. (2001) found that inhibition of DI during challenges had an augmenting effect on bronchoconstriction in asthma, suggesting that DI may limit induced bronchoconstriction in asthma as well. Pyrgos et al. (2003) showed no effect of DI-induced bronchoprotection against inhaled allergen in subjects with allergic rhinitis, who nevertheless demonstrated a protective effect of DI against methacholine-induced bronchoconstriction. Inhaled steroids (Corsico et al. 2000) and oral in addition to inhaled steroids (Slats et al. 2006) have been shown to improve airway responses to deep inspiration, while Schichilone et al. (2005) suggested that inhaled steroid treatment can normal-

ize the protective effect of DI in mild but not more severe hyperresponsiveness. Recently, inflammation in ASM and the submucosa on bronchial biopsies has been shown to be associated with an impaired response to DI measured by the FOT in asthma (Slats et al. 2007). In the last decade these clinical observations have stimulated many investigations into the effect of stretch and its absence on ASM both ex vivo and in intact animals (see extensive review by An et al. 2007). These experiments suggest that in the absence of repeated stretch ASM can stiffen and increase its bronchoconstrictor potential. Even quite small stretches, such as occur in normal tidal breathing, may be required to maintain ASM in its optimal compliant state. It is suspected that stretching of ASM during breathing might be reduced in asthma. Understanding of the dynamics of ASM has greatly advanced, but dissecting out which of several proposed abnormalities of ASM are important in vivo in asthma remains a formidable task, which is not assisted by great uncertainty about normal physiologic function (if any) of ASM in health (Mitzner 2004). Development of ablation of ASM by bronchial thermoplasty as a treatment for asthma

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may provide insights into its physiologic role (Solway & Irvin 2007).

Involvement of the extrathoracic airway

Bronchial thermoplasty

Nose

Radiofrequency ablation by controlled heating of the airway wall using a catheter system at bronchoscopy (bronchial thermoplasty) produced wide ablation of smooth muscle affecting the airway wall and immediate peribronchial tissue in airways over 3 mm diameter in dogs, lasting at least 3 years (Danek et al. 2004). Thermoplasty of segmental airways down to 3 mm diameter, in both lower lobes and both upper lobes, in three sessions of 30 min, under either general or local anesthesia, was shown to be feasible and safe in mild stable asthma (Cox et al. 2006). Modest increases in FEV1 were accompanied by geometric mean PC20 methacholine improvements of 2.4, 3.0 and 2.3 doublings at 12 weeks, 1 year, and 2 years. A recent randomized study (Cox et al. 2007) reported 12-month follow-up after thermoplasty in 55 asthma patients compared with a control group of 54 patients taking inhaled steroid and long-acting inhaled β2-agonist (LABA) therapy. Mild exacerbations were significantly reduced over 1 year in the thermoplasty group, with significant improvement in morning PEF, symptom scores, and quality of life with reduced rescue β2-agonist use. These effects were seen at 3 months, when all patients were taking LABA, and at 12 months in the treatment group when all patients had attempted to stop LABA (achieved in about half of all patients in both groups). Evaluations were carried out after 2 weeks off LABA. Bronchial thermoplasty improved asthma control in moderate to severe asthma. A placebo-controlled study is required to assess true clinical benefit and the contribution of more central airways to AHR.

Nasal obstruction due to rhinitis and/or polyps is the most obvious site of extrathoracic airway involvement. Rhinitis is common in the general population, affecting up to 30–40% at some age. However, in severe asthma several studies have suggested a prevalence approaching 100% (Bresciani et al. 2001). In normal subjects, during tidal breathing, the nose provides a resistance of about 2 cmH2O/L per s, about 50% of the total airflow resistance. Nasal resistance may be considerably higher in the presence of rhinitis, particularly when supine (Table 33.1) (Duggan et al. 2004). Presumably, if nasal resistance is greatly increased, tidal breathing is divided between the oral and nasal routes, with reduction of the protective and humidifying effects of the nose. Reflexes between the nose and intrapulmonary airways have been investigated extensively in experimental animals, but their importance in humans remains uncertain. The better-established interaction is between acute nasal stimulation and transient narrowing of intrapulmonary airways, but the reverse relationship, nasal congestion following the provocation of intrapulmonary airway narrowing, has been described (Yap & Pride 1994). Whether these interactions can lead to sustained airway narrowing or nasal congestion and whether they are exaggerated by chronic nasal or bronchial disease is unknown.

Pharynx, glottis and trachea Detailed studies of resistance during air and helium/ oxygen breathing suggest involvement of the glottis and/or

Table 33.1 Contributions of nasal resistance to total respiratory resistance during tidal breathing. (Data from Duggan et al. 2004.)

Total respiratory resistance (cmH2O/L per s) Mouth breathing (m)

Nose breathing (n)

Nasal airway resistance (n-m) (cmH2O/L per s)

Normal subjects (N = 10) Sitting 2.2 (0.1) Supine 3.0 (0.1)

4.2 (0.4) 5.7 (0.4)

2.1 2.7

Asthma alone (N = 7) Sitting 3.8 (0.5) Supine 4.6 (0.6)

6.7 (0.6) 8.2 (0.3)

2.9 3.7

7.6 (1.4) 11.2 (1.9)

3.9 5.8

Asthma with nasal symptoms (N = 10) Sitting 3.7 (0.2) Supine 5.4 (0.3)

Values are mean (SE). Resistance was measured by forced oscillation at 6 Hz at the airway opening.

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extrathoracic trachea is present in a significant proportion of asthmatic attacks (Lisboa et al. 1980). Expiratory narrowing of the glottis and supraglottic airway can be visualized during provoked bronchoconstriction in asthmatic subjects and may play a physiologic role in “braking” expiration and increasing FRC. The airway obstruction of asthma has to be distinguished from structural or functional obstruction of the extrathoracic airways which may cause similar symptoms (Bucca et al. 1995). Structural narrowing results in distinctive changes in maximum expiratory and inspiratory flow–volume curves (Miller & Hyatt 1973) (see Fig. 33.3). Functional obstruction may mimic acute asthma and because of its variability be more difficult to diagnose.

Vocal cord dysfunction Vocal cord dysfunction (VCD) is defined as paradoxical adduction of the vocal cords during inspiration or expiration or both. This causes airflow obstruction, obvious wheezing, and breathlessness. VCD frequently coexists with, and may be clinically indistinguishable from, asthma, with a spectrum from mild breathlessness to respiratory distress leading to intubation or tracheostomy (Newman et al. 1995). There may be accompanying hyperventilation, which could itself act as a bronchoconstrictor. There is often considerable shortterm variability which makes it difficult to obtain consistent measurements of lung function but this may assist the diagnosis. Typically, tidal flow–volume curves show that breathing is at very small lung volumes close to RV, while MEFV and MIFV curves when wheezing is present show reduction in MIF, with an MEF50/MIF50 ratio > 2.0. Inspiratory Raw may be greatly increased during panting in the body plethysmograph (Vlahakis et al. 2002). Bronchial challenge may be negative; if there is accompanying asthma, the level of responsiveness may be mild in relation to the prominence of symptoms. When VCD is the sole cause of wheeze, evidence of ventilatory inhomogeneity, so typical of even mild asthma, is absent. The wheeze characteristically disappears when the patient sleeps or is anesthetized but its pathogenesis, and whether psychopathologic factors are involved, is uncertain. An excessive response to such recognized stimuli as gastroesophageal reflux, postnasal drip, or inhaled irritants or autonomic imbalance (Ayres & Gabbott 2002) have all been proposed.

Bronchial blood flow Increase in bronchial blood flow in asthma has received relatively little attention because of the invasive methods required for measurement. Angiogenesis and vascular remodeling with increased vascularity (a hallmark of inflammation) have increasingly been recognized in asthma (Salvato 2001; Vignola et al. 2003). Recently a more rapid exponential rise in exhaled breath temperature (Δ°C/s) during a flow-

Physiologic Aspects of Asthma

and pressure-controlled single exhalation in asthmatics compared to normal subjects (Paredi et al. 2002) was correlated with exhaled nitric oxide (NO). This was confirmed in a follow-up study (Paredi et al. 2005) with a faster rise of exhaled breath temperature (7.27 ± 0.6 in asthmatics vs. 4.23 ± 0.4 Δ°C/s in normal subjects). Furthermore, bronchial blood flow (Qaw) using the Fick principle and measuring the soluble gas acetylene with online mass spectrometry was elevated in mild persistent asthma (46.0 ± 51 vs. 31.6 ± 1.6 μL/mL per min in normal subjects). Qaw was weakly correlated with Δ°C/s (r = 0.52) and exhaled NO (r = 0.57). As measured, Qaw includes tracheal blood flow and dead space effects but is similar to other techniques that have previously been validated (Scuri et al. 1995). The effect of asthma drugs on Qaw has been studied: the inhaled steroid budesonide produced an approximate 60% reduction from 30–60 min in asthmatics while Qaw was increased by salbutamol about 67% in normal controls but not in asthmatics. Nonsignificant changes in Δ°C/s were seen after budesonide in asthmatics and normals, while in five normal subjects (but not in asthmatics) salbutamol significantly increased Δ°C/s. These findings are in line with older work in sheep and the established vasoconstrictor and vasodilator pharmacology of these agents. Furthermore, these recent results confirm others in asthma (Brieva et al. 2000; Horvath et al. 2007) suggesting that they may be of clinical significance. Increased bronchial blood flow in asthma probably contributes to increased heat transfer across the airway walls, accounting for the faster rise in breath temperature on exhalation. These may represent novel noninvasive markers of airway inflammation, potentially linked to the elevated levels of the potent endogenous vasodilator NO, found in asthma.

References Aggarwal, A.N., Gupta, D., Kumar, V. & Jindal, S.K. (2002) Assessment of diurnal variability of peak expiratory flow in stable asthmatics. J Asthma 39, 487–91. Altes, T.A., Powers, P.L., Knight-Scott, J. et al. (2001) Hyperpolarized 3 He MR lung ventilation imaging in patients with asthma: preliminary findings. J Magn Reson Imaging 13, 378–84. An, S.S., Bai, T.R., Bates, J.H.T. et al. (2007) Airway smooth muscle dynamics: a final common pathway of airway obstruction in asthma. Eur Respir J 29, 834–60. Antic, R. & Macklem, P.T. (1976) The influence of clinical factors on site of airway obstruction in asthma. Am Rev Respir Dis 114, 851–9. Aubier, M., Wettenger, R. & Gans, S.J. (2001) Efficacy of HFAbeclomethasone dipropionate extra-fine aerosol (800 μg/day) versus HFA-fluticasone propionate (1000 μg/day) in patients with asthma. Respir Med 95, 212–20. Awadh, N., Muller, N.L., Park, C.S., Abboud, R.T. & FitzGerald, J.M. (1998) Airway wall thickness in patients with near fatal asthma with or without fixed airflow obstruction. Thorax 53, 248–253.

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Aerosol Delivery Systems Thomas G. O’Riordan and Gerald C. Smaldone

Summary

Introduction

The inhaled route of administration is used for bronchodilator therapy for asthma as well as for delivery of antiinflammatory medications for allergic rhinitis and asthma. For most patients with asthma, pressurized metered-dose inhalers (pMDIs) and dry powder inhaler (DPI) devices are used for maintenance therapy but nebulizers are useful in young children and critically ill patients. Chlorofluorocarbons (CFCs) have been used as propellants in MDIs for 50 years but are now being replaced by hydrofluoralkane propellants in pMDIs because of concerns about the impact of CFCs on the ozone layer. Dry powder inhaler technology has been enhanced in recent years and modern DPIs produce aerosols of comparable quality and reproducibility as pMDIs. Aerosol quality control is important if the aerosolized medications are to be delivered in predictable quantities to the lower airways, as small changes in aerosol particle size or velocity can significantly alter lung deposition. In addition, breathing pattern and changes in airway anatomy due to disease can affect not only the quantity of medication delivered to the lung, but also its regional distribution within the lung (e.g., airway vs. alveolar deposition). Most aerosol delivery systems in current use cause significant extrapulmonary deposition of medication in the pharynx and can result in local irritation. Once extrapulmonary-deposited medication has been swallowed, it has the potential to be absorbed and cause systemic adverse events. Inhaled formulations of corticosteroids (ICS) have been developed to reduce this oral bioavailability and some preparations of ICS have negligible oral bioavailability. However it should be remembered that systemic exposure to ICS can also occur when medication that deposits in the lungs is absorbed. The delivery of aerosolized medications to children poses additional challenges. Masks used to facilitate the use of nebulizers or MDIs can also alter patterns of drug delivery. Nasal formulations of corticosteroids using aqueous suspensions or solutions and larger particles are effective at treating rhinitis.

Most medications used to treat asthma are delivered by the aerosol route. The rationale for therapeutic preference is that delivering a drug to the target organ will increase medication levels at the airway while reducing reduce systemic exposure. This assumption is on the whole valid but there are complicating factors that are the focus of this chapter. The human respiratory tract has evolved in a manner that makes it difficult for inhaled particles to enter the distal airspaces, thus protecting these delicate structures from the many noxious and infectious particles present in the atmosphere. The design of inhaled therapies must take these functional barriers to aerosol penetration into account. However, the principles of aerosol deposition and clearance apply not only to therapeutic aerosols but also to studies of how inhaled allergens, irritants, and infections also enter the lung.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Definition and description of an aerosol An aerosol can be defined as a system of solid particles or liquid droplets that can remain dispersed in a gas, usually air (Agnew 1984). Because the aerodynamic behavior of an aerosolized particle is critically influenced by its mass, it is important to be able to precisely describe the size distribution of aerosolized particles. In clinical studies, the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (σg) are often used to characterize the dimensions of an aerosol. The MMAD represents the point in the distribution above which 50% of the mass resides, expressed as the diameter of a unit density (1 g/mL) sphere having the same terminal settling velocity as the aerosol particle in question, regardless of its shape and density. The σg is the ratio of the size at 84% (or 16%) to the MMAD and is an indicator of the variability in particle diameters (one geometric standard deviation, if the distribution is log-normal). If the particle size varies over a wide range (σg > 1.2), it is described as having a polydisperse particle distribution. Pharmaceutical aerosols are polydisperse. If all the

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particles in an aerosol are of similar size (σg < 1.2), the particle distribution is described as monodisperse. Monodisperse aerosols are usually only encountered in research studies where specialized generators are used to create such aerosols (Stahlhofen et al. 1980; Smaldone et al. 1983)

Aerodynamic behavior of aerosols Mechanisms of deposition in human subjects Therapeutic aerosols, with diameters of 0.5–10 μm, deposit in the lung by inertial impaction and gravitational sedimentation. Inertial impaction occurs when an inhaled particle is not able to follow the air stream and impacts into the airway wall usually at airway bifurcations. Larger particles traveling at high velocities are more likely to impact in the upper airway than smaller particles at slower velocities. Particles less than 5 μm can also settle by gravitational sedimentation in more distal airways. Deposition by sedimentation is critically time dependent, with breath-holding for example significantly increasing the number of particles deposited (Bennett 1991). In healthy subjects, both the regional distribution as well as the absolute number of inhaled particles that deposit in the lung are primarily determined by the size of the particles and by the breathing pattern. Conversely, in disease states, the geometry of the airways and changes in regional ventilation may be the dominant influences (Smaldone & Messina 1985a & b).

Strategies to optimize deposition of therapeutic aerosols

combined with particle inertial effects and the geometry of the oropharynx result in upper airway deposition ranging from 30 to 90% of the total deposition in the patient for typical commercial delivery systems. Only nebulizers producing particles with particularly small MMAD (e.g., AeroTech II, MMAD 1.0 μm) bypass the upper airways (5% oropharyngeal deposition in adults) (Smaldone et al. 1998a). In children, the smaller oropharyngeal airways make the task more difficult (Diot et al. 1997). Thus, the demands placed on traditional devices can be overwhelming if the drug must efficiently bypass the upper airways.

Control of breathing pattern and aerosol deposition In normal subjects, the pattern of breathing is the most important factor affecting aerosol delivery and deposition. How the patient breathes affects the device (Smaldone et al. 1991), the penetration of particles past the oropharynx, and deposition within the parenchyma. Earlier studies suggested that much of the variation in parenchymal deposition was related to differences in airway geometry between subjects. However, variability in deposition between subjects appears well controlled if the pattern of breathing is controlled (Bennett & Smaldone 1987). Figure 34.1 depicts data from 11 subjects inhaling 2.6–μm monodisperse particles. The fraction of inhaled particles depositing in the lung (DF) can be closely related to the period of breathing. In simplified form, points near the origin of the horizontal axis represent normal tidal volumes and frequencies. As tidal volume increases and breathing frequency decreases, the time of inspiration is prolonged

The determinants of aerosol deposition apply to both therapeutic and environmental inhaled particles. To optimize deposition of therapeutic aerosols, a number of strategies have been developed based on these principles.

0.6

Getting particles past the oropharynx

0.4

In clinical studies, predicting penetration of aerosol beyond the oropharynx and subsequent lung deposition is a major criterion for device selection. Deposition estimations are often based on particle size measurements defined by in vitro characterization of the aerosol produced by a given device. During tidal breathing, most investigators would expect aerosols below 5 μm to be deposited primarily in the lungs (the fine particle fraction or FPF) (Smaldone & Messina 1985a & b; Smaldone et al. 1998a). However, in a recent clinical study, we found that the so-called FPF for wet nebulizers might be closer to 2.5 μm (Sangwan et al. 2003). Our data for wet nebulizers was similar to that for other traditional devices when data defining FPF were critically analyzed. Most of the reported data from previous studies as well as our data were obtained in patients receiving their aerosol during tidal breathing, rapid inhalation, or metered-dose inhaler (MDI) aerosol delivery. In all cases particle size effects

Aerosol Delivery Systems

DF

0.5 SLOW & DEEP

0.3 TIDAL BREATHING 0.2 Controlled breathing measurements in one subject Spontaneous breathing measurements in each of 10 patients

0.1 0.0 0

5

10

15

VT/f ′2 20

Fig. 34.1 Deposition (defined as deposition fraction or DF, the fraction of particles inhaled that are deposited) of 2.6-mm monodisperse particles versus breathing pattern. Filled circles, data from one subject during controlled breathing; open circles, data from 10 patients during spontaneous breathing. Breathing pattern was defined by a relationship that represents a measure of the period of breathing (tidal volume divided by breathing frequency squared or VT/f ′2). Typical “tidal breathing” parameters found near origin, “slow and deep” inspirations found away from the origin. (From Bennett & Smaldone 1987, with permission.)

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10 L/s L 2 (a)

GD

WM

RH

KS

ON

5 L/s L 1 (b)

SS

AT

RP

BH

HM

HS

Fig. 34.2 Tracings of maximal flow and tidal flow vs. volume. (a) Normal subjects. (b) Patients with severe obstructive lung disease. Note the change in the sensitivity of the axes (ordinate: flow range from 0 to 10 or 0–5 L/s; abscissa: volume range from 0 to 2 or 0–1 L respectively). The patients’ tidal loops are superimposed on maximal flow–volume curves. (From Smaldone et al. 1993, with permission.)

(e.g., slow and deep inspiration). The curve depicted in Fig. 34.1 represents maximum deposition with a slow and deep inspiration for particles of 2.6 μm. The curve would be shifted upwards with deposition approaching 100% for larger particles.

Expiration and problems with aerosol deposition In normal subjects, those particles that do not deposit during inspiration are largely exhaled completely. Particles that pass through the oropharynx during inspiration enter the central airways and traverse them without difficulty because lobar and segmental bronchi are generally widely patent during inspiration. Then the particles enter alveoli with a few depositing by sedimentation and, like cigarette smoke, the bulk of the aerosol begins to be exhaled. Deposition is controlled by sedimentation in small airways, influenced by local geo-

(a)

770

(b)

metry and to a strong degree by the residence time (period of breathing). In obstructive lung disease, maximal expiratory flows are diminished. With moderate disease, maximal flows can be superimposed on tidal breathing, and as the disease progresses maximal flows can be reduced even further (Smaldone 1997) Therefore, it is common to observe that patients are often breathing on their maximal expiratory flow–volume curves even during quiet tidal breathing (Fig. 34.2) (Smaldone et al. 1993). In these patients, flow-limiting segments exist in the same airways found in normal subjects during forced expiration but, for these patients, they form during every tidal breath. In patients with obstructive lung disease the promotion of deposition in the peripheral lung poses a significant challenge as deposition of aerosol is enhanced during expiration at sites of flow limitation (Fig. 34.3) (Smaldone & Messina 1985b; Smaldone 2001). Based on these physiologic considerations peripheral deposition of aerosol in these subjects would be favored using a system that provides a slow prolonged inspiration (to promote deposition by settling) with a breath long enough to minimize the particles available to the airways during expiration.

Slow and deep breathing For many aerosol applications inhaling particles slowly and deeply solves the problems outlined above. A slow inhalation will minimize oropharyngeal deposition. “Slow” inspiration reduces particle inertia and allows inhalation of particles larger than the 2.5–μm cutoff for the FPF that defines deposition in the oropharynx during tidal breathing. This concept has been exploited in physiologic studies of mucociliary clearance in the distal airways utilizing particles as large as 6 μm (Anderson et al. 1995). More recently commercial applications of these principles have combined slow and deep inspiration with direct mechanical feedback to the patient for particles of relatively large MMAD. This is discussed in more detail in a later section.

Fig. 34.3 (a) Sites of flow-limiting segments in humans. (b) Corresponding deposition image in patient with severe chronic obstructive pulmonary disease (posterior view) with maximal expiratory flow–volume curve superimposed on tidal loop (as shown for patients in lower part of Fig. 34.2). Particle deposition occurs in sites of flow-limiting segments. (From Smaldone 2001, with permission.)

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Interface between device and patient and drug delivery In spontaneously breathing adult patients, during quiet tidal breathing, aerosol is inhaled via a mouthpiece. In young children and adults with coordination problems, the situation is more complex as the patient is often interfaced to the device via a facemask and the impact of a facemask on deposition is discussed in a later section.

Measurements of particle size There are two principal approaches to the measurement of particle size: cascade impaction and the use of methods (Baron 2001) based on light scattering. We briefly discuss both of these methods and the limitations of in vitro measurements when extrapolated to the in vivo setting.

Cascade impaction A cascade impactor consists of a series of stages with differentsized orifices that fix local linear velocities of the carrier gas of the aerosol as it passes through the device. After particles pass through the orifice of a given stage, they must turn at 90° around an obstruction or “baffle” or impact on the stage. The ability of a particle to negotiate the baffles downstream from each orifice is a function of the aerodynamic particle diameter (Baron 2001).

Light scattering The scattering of light can be used to measure particle size and has been reviewed elsewhere (Baron 2001). Originally developed to measure monodisperse aerosols, newer devices can measure polydisperse aerosols. Small particles produced by a pressurized MDI (pMDI), whose dimensions are measured by a light-scattering device, may have high velocity and can thus have the same inertial properties as larger particles at slower velocities.

deposition within the lungs. The latter measurement is performed in vivo and is time-consuming, costly, and involves some degree of risk and uncertainty to the patient. The other components of the aerosol delivery process can be well characterized and studied in vitro. The field of aerosol delivery has advanced significantly in the last 10 years such that device characteristics and aerosol behavior can be significantly optimized on the bench before exposure to patients (O’Riordan et al. 1994)

Inhaled mass Figure 34.4 depicts a simple in vitro set-up for measuring the quantity of aerosol produced by a nebulizer (Smaldone 1991). This system does not require an understanding of nebulizer function from first principles. An absolute filter (i.e., a filter that captures all the particles in an aerosol) has replaced the mouthpiece. Because the nebulizer is attached to a breathing device (Harvard pump, Harvard Apparatus, South Natick, MA), the conditions of delivery such as routine tidal breathing can be duplicated. The quantity of drug captured on the filter represents the amount that passes the lips of the patient. To distinguish this quantity from a “dose” or deposited drug the term “inhaled mass” has been coined and thus the filter can be called the inhaled mass filter. The inhaled mass represents “delivery” of drug to the patient, constrained by conditions that should mimic actual clinical delivery.

Aerosol A cascade impactor can be inserted into the circuit depicted in Fig. 34.4 and provide information regarding the aerodynamic distribution of a given aerosol (MMAD and σg). Depending on circumstances, knowledge of MMAD and σg can predict the behavior of particles in the lungs. Figure 34.5

Inhaled mass filter

Expiration

Application of in vitro measurements of particle size to clinical studies Particle size data obtained with different techniques are not necessarily interchangeable. Meaningful comparisons of the sizes of clinical aerosols, should be compared only if obtained with identical techniques. Nevertheless, despite the technical difficulties encountered in measuring the size of polydisperse clinical aerosols, some investigators have established that when used with appropriate caution, in vitro measurement of particle size does provide useful predictive data for subsequent clinical studies (Newman 1996).

Principles of assessment of aerosol delivery systems Assessing effects of an aerosolized drug requires the understanding of three major factors: the aerosol delivery system, the quality of the aerosol produced, and the quantification of

Inspiration

1 2 3 4 5 6

Compressed air

7 8

Harvard pump 10-stage cascade impactor

9 10

Fig. 34.4 Technique for bench measurements of inhaled mass and particle distribution: breathing pattern defined by settings on Harvard pump. Particles presented to “patient” are captured on the inhaled mass filter. In separate experiments, the cascade impactor measures inspired aerosol. (From Smaldone 1991, with permission.)

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Fig. 34.5 Deposition scans of MistyNeb (top) and AeroEclipse (bottom) for three subjects 1, 2 and 3 (left to right) receiving aerosolized interferon-g for tuberculosis. Subject 2 has only the right lung. The lung outlines (133Xe equilibrium scan, central, peripheral regions and horizontal line separates upper from lower lung regions) and stomach outlines for patients 1 and 2 are shown. Deposition in the oropharynx is reduced with AeroEclipse. (From Sangwan et al. 2003, with permission.)

depicts deposition images for three subjects following inhalation of interferon (IFN)-γ aerosols generation by MistyNeb (Allegiance, McGraw Park, IL) and AeroEclipse (Trudell Medical International, Canada) nebulizers. All three patients show an increased deposition in the lungs for the AeroEclipse nebulizer as evidenced by increased activity in the lung fields with reduced deposition in the oropharynx (reduced stomach activity). For MistyNeb, lung deposition varied from 28 to 32% (mean ± SD, 30.9 ± 0.03%) of the total aerosol deposited in the patients. AeroEclipse deposited 59– 73% (68.1 ± 0.08%) of total deposition (Sangwan et al. 2003). In Table 34.1 the distribution of particles between the oropharynx or upper airways and the deep lung are shown in bold for both devices. The aerodynamic distributions from cascade impaction measurements for the different nebulizers are also listed. By

Table 34.1 In vitro assessment of interferon-g aerosol. Aerosol distribution determined by cascade impaction listed for the test nebulizers. All data in this table are from the in vitro cascade studies but bold numbers represent the cascade stages representing mean lung deposition (see text) measured from deposition images in vivo for the three subjects. (From Sangwan et al. 2003, with permission.) Nebulizer

Misty-Neb

AeroEclipse

MMAD (mm) Particles < 6 mm Particles < 3 mm Particles < 2 mm

3.10 55% 49% 30%

2.20 77% 73% 53%

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inspection, one can see that a cutoff of approximately 2.5 μm defines particles that pass the oropharynx and deposit in the lung. For the Misty-Neb approximately 30% of the particles are 2.5 μm or less and this corresponds to the lung depositions seen in the images for the three patients studied. For the AeroEclipse, which produces a smaller particle distribution, the 2.5–μm cutoff predicts that approximately 70% of the particles will deposit in the lung versus the upper airways. Our results are a strong function of the design and control of the experimental set-up. However, for certain devices, knowledge of the MMAD measured on the bench can assist in the design of appropriate aerosol delivery systems prior to in vivo testing.

Deposition The term “deposition” begins to imply a “dose” to the patient. The term deposition needs to be further refined in a given situation, for example oropharyngeal versus parenchymal deposition, or central versus peripheral deposition within the lung. Each of these terms may be important depending on the disease entity to be treated. Obviously the measurement of deposition requires an in vivo experiment. However, deposition can be related to parameters that are measured in vitro as shown in equation (1): Deposition = aerosol inhaled − aerosol exhaled

(1)

Because the term “aerosol” is a little vague with respect to drug activity, equation (1) can be rewritten as: Deposition = inhaled mass − exhaled mass

(2)

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(a)

Fig. 34.6 In vitro set-up: (a) constant flow experiment and (b) breathing simulator for each inhalation device (nebulizer, valved holding chamber). The connection with the flow-generating apparatus was made with a flat plate (sealed configuration) or a face (face configuration). (From Smaldone et al. 2005, with permission.)

Aerosol Delivery Systems

Plate (sealed) or face (not-sealed)

Gast vacuum pump

VHC + MDI or nebulizer + compressor

Inhaled mass filter

Computer with control card (b)

MIMIC breathing emulator

Many experiments can be performed on the bench to define the parameters that define the inhaled mass for different devices and experimental conditions (Smaldone 1991).

Measurement of inhaled mass Pediatric in vitro models Figure 34.6 represents a more complex situation than that of Fig. 34.4. An MDI and a valved holding chamber (VHC) are connected in series. For many patients the VHC utilizes a mouthpiece, but younger patients require a facemask. Measurement of inhaled mass requires a strategically placed filter. Reported studies to date place the inhaled mass filter on the VHC and capture particles using a suction device or a breathing machine (Fig. 34.6a). However, the principles illustrated above require the inhaled mass filter to mimic par-

ticles that actually pass the lips. To complete the in vitro model, therefore, the facemask must be placed on a face. Finally, the face must “breathe” with a breathing pattern that is representative of the patient population to be treated. As depicted in Fig. 34.6b, the correct filter location is shown between the breathing emulator and the face facsimile. Thus, all the components of the aerosol delivery system including the MDI, VHC, facemask, and pattern of breathing will be reflected in the measured inhaled mass. Table 34.2 illustrates summary data from a recent study comparing MDIs and nebulizers using patterns of breathing appropriate for pediatric patients using facemasks (Smaldone et al. 2005). The effects of the breathing pattern, the VHC employed, the facemask and different nebulizers were tested. VHCs were also washed with detergent to eliminate static charge. The influence of VHC

Table 34.2 Inhaled mass (percent of label dose) (mean ± SD) as a function of breathing pattern, VHC condition, and presence of facemask for budesonide (nebulizers) and fluticasone propionate (pMDI VHC). (From Smaldone et al. 2005, with permission.) VT Nebulizer (drug) Hudson Updraft II (budesonide) Pari LC plus (budesonide)

VHC (drug) pMDI AeroChamber (fluticasone) pMDI OptiChamber (fluticasone)

Sealed configuration

24.3 ± 3.1 9.6 ± 0.7 18.7 ± 1.9 10.0 ± 1.1

207 75 207 75

207 75 207 75

Face configuraton

Unwashed 7.2 ± 2.0a 2.9 ± 1.5a 7.7 ± 1.6a 0.7 ± 0.5a

19.3 ± 2.3 4.1 ± 0.8 11.7 ± 1.1 9.2 ± 1.9 Washed 53.3 ± 6.2b 30.5 ± 3.2b 50.2 ± 1.2b 27.2 ± 1.4b

Unwashed 2.4 ± 0.7a 3.1 ± 2.4a 2.9 ± 0.3a 1.0 ± 0.2a

Washed 13.6 ± 2.7b 4.7 ± 0.7b 28.6 ± 2.5b 4.0 ± 1.6b

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(a)

(b)

conditioning combined with effects of breathing pattern resulted in inhaled mass ranging from 0.7 ± 0.5 to 53.3 ± 6.2% (mean ± SD). Nebulizers were less variable (9.6 ± 0.7 to 24.3 ± 3.1%). Detergent coating of VHC markedly increased inhaled mass and reproducibility of drug delivery (27.2 ± 1.4 to 53.3 ± 6.2%) for pMDI/VHC combinations but these effects were lost in the presence of facemasks. Using pediatric patterns of breathing, nebulizer/facemask combinations delivered 4.1 ± 0.8 to 19.3 ± 2.3% of the labeled dose, while pMDI and detergent-coated VHC delivered 4.0 ± 1.6 to 28.6 ± 2.5%. Facemask seal was a key factor in drug delivery. Leaks around the facemask reduced drug delivery and for pMDI/VHCs negated effects of detergent coating (Smaldone et al. 2005).

Fig. 34.7 (a) Preferential eye deposition with Laerdal and Pari LC Plus nebulizer (b) Diffuse facial pattern with Salter mask and AeroTech II nebulizer. (From Sangwan et al. 2004, with permission.)

leaks near the bridge of the nose is reduced. An example is shown in Fig. 34.8, which consists of a deposition image created under the same conditions as for Fig. 34.7a using a modified mask design. Deposition on the eyes and face was markedly reduced, while drug delivery to the patient (inhaled mass) was preserved (Smaldone 2004).

Facemasks and facial deposition For pMDI/VHC, leaks around the facemask limit the exchange of tidal air with air in the VHC, reducing the inhalation of aerosol by the patient. For nebulizers operated with compressors the facemask can be kept filled with particles in spite of leaks because the compressor flow can exceed the minute ventilation of the child. However, the very leaks that may preserve delivery to nebulizers result in deposition of drug on the face and in the eyes. Figure 34.7 demonstrates characteristic deposition patterns following nebulizer therapy on the face using a pediatric model of aerosol delivery with a tightly fitted facemask (left) and a straight-in nebulizer and a commercially fitted mask with a straight-up nebulizer (right) (Sangwan et al. 2004). Recent experiments have indicated that deposition on the face and particularly in the eyes can be minimized if masks are designed such that linear velocity in the region of the

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Fig. 34.8 Reduced facial and eye deposition with Pari nebulizer and prototype facemask designed to reduce particle acceleration in the region of the eyes. (From Smaldone 2004, with permission.)

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Fig. 34.9 Lung images from seven patients following inhalation of 99mTc-HSA-labeled cyclosporin A aerosol. n indicates native lungs (see text). There is considerable variation in regional particle deposition. (From Smaldone 2005, with permission.)

Deposition and dose versus response

50 40 ΔFEV1% Day 0

For conventional bronchodilators and steroids, the dose and response are not critical for clinical efficacy because most delivery systems provide drug to the patient on the flat portion of the dose–response curve. Safety of most preparations is enhanced because of the high potency of most drugs and a high threshold for toxicity. The situation for bronchodilators and steroids may not carry over to newer drugs. For example, aerosolized cyclosporin A used in the treatment of lung transplant rejection has recently been shown to significantly reduce mortality. In vivo measurements of deposition have been related to clinical effects (dose vs. response). For example, Fig. 34.9 shows gamma camera images of individual patients from an early study assessing the effects of inhalation of aerosolized cyclosporin A aerosol (Iacono et al. 1997). Analysis of cyclosporin A deposition was related to clinical effects as shown in Fig. 34.10. After institution of aerosolized cyclosporin A therapy in patients with persistent acute rejection, pulmonary function improved (after 3 months of therapy) as illustrated by increases in forced expiratory volume in 1 s (FEV1). However, there was a suggestion of a dose–response relationship, with those patients receiving less than 20 mg of drug per allograft having minimal response. A similar situation will exist for systemically absorbed drugs such as insulin where the dose to the lung parenchyma will be critical in

30 20 10 0 –10 0

10

20

30

40

CSA deposition in transplanted lung(s) Fig. 34.10 Changes in spirometry assessed by change in FEV1 (ΔFEV1 Day 0) from the initiation of aerosol cyclosporin A (CSA) to approximately 200 days of therapy, as a function of cyclosporin A deposited in transplanted lung(s). (From Iacono et al. 1997, with permission.)

patient management. While clinical studies have shown that aerosolized cyclosporin A appears to be effective overall in a population of patients, data from the individual patients shown in Fig. 34.10 suggest that conventional aerosol delivery systems leave some patients at risk for inadequate dosing, while others may be overdosed and exposed to potential toxicity.

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Generation of aerosols

Oropharynx

Oropharynx Rondo

To generate an aerosol, energy must be applied to solid or liquid material in order to disperse the material and cause it to be suspended in a carrier gas which, for the purpose of this chapter, will nearly always be air. Lungs

Pressurized metered-dose inhalers pMDIs consist of a pressurized metal can that contains a mixture of propellants (freon) which, when in equilibrium, are both in liquid and gaseous form at room temperature (Morâen 1993). Suspended in the liquid, the agonist usually consists of solid particles that have been milled into an aerodynamically respirable distribution of diameters. In addition to the medication and propellant, many preparations contain a surfactant or dispersal agent such as lecithin or oleic acid because surface tension affects aerosol behavior. In addition, ethanol is used to enhance the solubility of some formulations. This two-phase suspension is evenly distributed by shaking and then a “metered dose” is released into the atmosphere via the valving system when the canister is compressed and triggered. The metering valve carefully controls the volume of liquid released into the atmosphere. At that point, the freon instantly evaporates, imparting a relatively high kinetic energy to the solid particles. Thus, the metering valve provides precise control of the triggered amount of drug. The volume of drug released per actuation is a function of the size of the metering valve. As the metering volume is increased, a greater amount of each spray will be deposited on the actuator mouthpiece. In addition, however, the proportion of particles depositing in the central airways relative to the lung periphery increases proportionate to metering volume. In contrast, warming of the MDI canister has been reported to increase drug deposition in the distal parts of the lung. An increase in concentration of propellant loaded into the canister elicits a finer aerosol at a higher ejection velocity (as discussed earlier, a high ejection velocity will increase the inertia of the particle with important clinical consequences). The milled size of the drug powder will be another limiting factor determining the particle size of the MDI aerosol: the coarser the drug powder, the larger the aerosol. If the pMDI is actuated within 60 s of a prior actuation, the dose of drug released may decrease significantly. Concern about the effects of subtle changes in the formulation of pMDI medications on aerosol delivery has led regulatory agencies to demand strict requirements that the manufacturers of generic formulations document clinical “equivalence” with existing products (Food & Drug Administration 1998).

Use of accessory devices with pMDIs There are practical difficulties encountered when administering drugs by pMDI. There is a need to coordinate actuation

776

Stomach

Fig. 34.11 Influence of intrinsic particle inertia on deposition pattern. (Left) A patient inhaling particles generated by a metered-dose inhaler without a spacer. (Right) The same patient inhaling the same aerosol but after modification by a Rondo spacer device. (From Newman et al. 1991, with permission.)

with respiration. Even with the use of optimal inhaler technique, significant extrapulmonary deposition occurs because while the particles are of respirable diameters, there is high inertia. Recognition of these problems has led to important attempts to modify the behavior of the aerosol as it is generated. Spacer devices have had a significant influence on drug delivery. As shown in the deposition study illustrated in Fig. 34.11, the spacer device absorbs those particles that have high inertia and which would, in the absence of the spacer, be likely to deposit on the pharynx and larynx (Newman et al. 1991). Significant losses occur in the spacer as particles impact on the walls but the available data in human studies indicate that the parenchymal deposition of drug within the lung is similar with and without the spacer, resulting in decreased exposure of the larynx and pharynx to the inhaled agonist. In addition some spacer devices have been designed to provide coordination control for the patient. Rather than relying on the patient to inhale at the instant of MDI triggering, the spacer serves as a reservoir from which particles remaining in the gas phase can be inhaled more conveniently. The presence of one-way valves within some spacer devices facilitates this maneuver (hence the term “valved holding chamber”; see below). In summary, the use of spacer devices reduces extrapulmonary deposition. This is especially important in preventing toxicity when doses of inhaled medications in excess of the usual maintenance doses are used. Pharyngeal deposition of high doses of inhaled corticosteroid agents, which are designed to be poorly absorbed, can cause local toxicity such as pharyngitis and Candida infection. Pharyngeal deposition of high doses of inhaled β-sympathetic agonists can cause systemic toxicity because these agents are swallowed

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and absorbed through the gastrointestinal tract. Controversy exists as to whether the use of spacer devices improves airway deposition of inhaled medications in situations where optimum inhaler technique is used. However, because patients’ pMDI techniques in “real world” clinical practice are frequently suboptimal, the use of spacers most likely does result in increased airway drug deposition for many patients (Moren 1982). Commercial holding chambers vary in design, size, and construction materials. Some are designed for use with only one drug formulation but most are marketed to be used with most MDIs. The simplest chambers are simple tubes, which should be at least 10 cm in length and 3 cm in diameter (Nelson & Loffert 1994; Newman & Newhouse 1996). These devices decrease oropharyngeal deposition and compensate in part for delay in inhalation after actuation of the device. If the patient exhales during actuation, simple tube spacers are not effective. While chambers of 150 mL volume are efficient, larger chambers up to 750 mL may provide enhanced drug delivery, albeit at a cost of reduced portability and convenience. The effects of poor patient coordination can be reduced by the addition of one-way valves (VHC) that prevent patients from exhaling into the device. A variety of valve designs have been patented. For portability, spacers and VHC may be collapsible. In addition, some devices provide sonic feedback if the patient’s inhalation flow rate is too rapid. It has also been noted that electrostatic interactions between aerosol and plastic chambers may reduce drug delivery (Wildhaber et al. 1996). As a result some manufacturers now include more detailed cleaning instructions (i.e., wash plastic chamber with detergent and leave to dry without wiping) while others have produced chambers that have less electrostatic interactions.

(Goldin et al. 1999): the development of an aerosol with both different aerosol properties and clinical effects (Busse et al. 1999). Compared with the CFC formulation, HFA BDP particles are significantly smaller in size, resulting in reduced extrapulmonary deposition in the oropharynx (which is universally accepted to be an advantage). On the other hand, penetration of the aerosol into the peripheral regions of the lung in enhanced. The benefits of the latter are more controversial and are discussed below. The pace of introduction of new HFA products and dry powder inhalers (DPIs) has been slower in the USA than in Europe, due in part to stricter regulatory requirements in the USA. The US Food and Drug Administration has been reluctant to approve products that do not meet the quality standards that apply to existing CFC formulations in terms of fine particle mass (dose in particles < 5 μm in diameter), dose-to-dose variability, and stability of the formulation over a 12–month storage time (Food & Drug Administration 1998). In addition, extensive clinical programs have been required for regulatory approval of these products because in vitro aerosol measurements are not viewed as acceptable surrogates for clinical data. The changeover from CFC to HFA has therefore been costly. Producing new HFA formulations equivalent in clinical efficacy to CFC formulations is difficult on a cost-of-goods basis when compared with the manufacture of generic CFC formulations. Governments will therefore consider banning the sale of generic CFC products when reliable supplies of the HFA versions of these products become available. Therefore one effect of the initiative to eliminate CFCs will be increased costs to consumers and insurers because of the extension of patent protection to these new HFA formulations.

Reformulation pMDIs without chlorofluorocarbons

Dry powder inhaler devices

Environmental pressures have led to the development of aerosol dispensers free of chlorofluorocarbons (CFCs), the production of which ceased in 1996 (although manufacture of therapeutic aerosols with CFC continues from reserved supplies). Such research has centered on dry powder systems (see below) and pMDIs free of CFCs. For example, the hydrofluoralkane (HFA) 1,1,1,2–tetrafluorethane (HFA 134a) is a propellant that does not affect the ozone layer and is found in the majority of reformulated pMDIs. All existing CFC MDIs will be replaced by HFA and/or dry powder formulations. As a result of this ruling clinical trials for the purpose of registering new products no longer involve CFC formulations. In replacing CFC formulations with HFA formulations, the pharmaceutical industry has adopted two strategies. The first replaces existing CFC formulations with HFA products designed to be clinically equivalent to each other. For example, Proventil (albuterol) HFA and Proventil CFC are very similar in terms of their in vitro aerosol characteristics and clinical effects. The reformulation of beclomethasone dipropionate (BDP) from CFC to HFA has involved a different strategy

Dry powder devices store the drug as either premeasured quantities of powder in capsules or blistered foil strips, from which a single dose of powdered drug is released into an inhalation chamber by opening or puncturing the capsule/ foil blister (Newman 1991; Nerbrink et al. 1994; Thorsson et al. 1994; Fuller 1995; Bisgaard et al. 1998). Alternatively, the powdered drug is stored in a multidose reservoir from which single doses are forced into the inhalation chamber (e.g., Turbuhaler, Astra-Zeneca, Lund, Sweden). To suspend the powder in air, the required energy is provided by the patient. This is usually accomplished via a rapid inhalation through the device and turbulence, created by a series of baffles, blends the powder into a respirable distribution, which is inhaled from the device. By modifying the design features, there is precise control of the released dose, but these systems are flow dependent to a varying degree and, in a manner similar to the MDI, the inhaled material of necessity leaves the device at relatively high velocity. Some DPIs contain only drug particles (e.g., Pulmicort Turbuhaler) whereas other DPIs (e.g., Salmeterol Diskus)

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also contain large particles of an inert dispersal agent such as lactose. Because they require the patient’s own rapid inhalation to generate the aerosol, DPIs are by definition coordinated with the patient’s breathing. Nevertheless, significant extrapulmonary deposition occurs because of the high inspiratory flow rates needed to disperse the powder. Therefore, while dry powder devices may be preferable to a pMDI in patients with suboptimal coordination who are unwilling to use a spacer, the high extrapulmonary deposition raises safety considerations in using higher than usual doses of corticosteroids or bronchodilators. A further reservation concerning these devices is that a threshold inspiratory flow rate is required to actuate these devices and patients with acute exacerbations of asthma may not be able to reach the required flow rates. Because of this concern, manufacturers are endeavoring to reduce the required threshold flow rates of their respective devices. An alternative to lowering the required flow rate is to lower the resistance of the device, so that an equivalent flow could be generated with less respiratory effort.

Nebulizers Nebulizers generate aerosols usually from liquids. Energy can be transferred to the liquid surface by either a jet as the liquid phase is forced through a narrow orifice at high velocity (Newman 1991; Nerbrink et al. 1994) or by sheer forces at the surface generated by ultrasonic waves. Usually, the agonist is dissolved in the solvent, but it is also possible to nebulize suspensions, although the efficiency of some nebulizers may be less with suspensions than with solvents. In terms of the basics of aerosol generation, the major difference between nebulizers and the metered systems (MDIs and DPIs) is that the velocity of particles leaving the generator is really a function of the patient’s method of breathing. Commonly, a patient quietly breathing through a nebulizer will inhale particles whose inertia is a function of the aerodynamic diameter and the local convective flows of the physiologic situation defined by the breathing pattern rather than the rapid evaporation of freon in the MDI or the high flow rates necessary via rapid inhalation in a dry powder device. Therefore, even though particles may have the same aerodynamic characteristics as described by cascade impaction, the nature of the aerosol generator significantly influences the actual aerosol inhaled (aerosol being defined as the distribution of particles and the carrier gas, the carrier gas imparting its own velocity to the particles) and the subsequent deposition patterns are, as described above, significantly affected. While MDIs and DPIs are approved for use by regulatory agencies in concert with a specific medication and are therefore highly regulated, there is much more variability and nonuniformity in the manufacture of nebulizers. Aerosols emitted from nebulizers are modified primarily by baffling systems that are either internal or external to the device.

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Nebulizer delivery systems must be assessed with the tubing and mouthpieces with which they are marketed. In addition, the efficiency of the nebulizer itself will affect the quantity of aerosol delivered in concert with the patient’s breathing pattern (Devadason et al. 1997).

New developments in aerosol delivery systems Significant enhancements in drug delivery by nebulizers are possible by coordinating nebulization with inspiration (e.g., breath actuation) that essentially turns the nebulizer off during expiration. Another improvement in efficiency is called “breath enhancement,” which utilizes the patient’s inspiratory flow through the nebulizer to increase drug delivery (e.g., LC Star Pari, Germany; Ventstream, Medicaid, Bognor Regis, UK) (Devadason et al. 1997). Recently the design of aerosol delivery systems has combined slow and deep inspiration with direct mechanical feedback to the patient for particles of relatively large MMAD. As discussed earlier, a “slow” inspiration reduces particle inertia and allows inhalation of particles larger than the 2.5–μm cutoff for the FPF that defines deposition in the oropharynx during tidal breathing and will thus minimize oropharyngeal deposition Adaptive aerosol delivery (AAD) technology analyzes parameters of inspiration and provides feedback to guide the patient’s inspiratory maneuvers. The I-neb AAD system (Respironics Inc., Cedar Grove, NJ), a hand-held device, combines the latest AAD technology with an optimized form of vibrating mesh technology. The I-neb system can deliver aerosol via two modes of inspiration: (i) the tidal breathing mode (TBM), which sets the device to deliver aerosol in the first 50% of the patient’s inspiration (Fig. 34.12); and (ii) a new algorithm that guides the patient to a slow and deep inspiration, the target inhalation mode (TIM) (Fig. 34.13). Testing of I-neb prototypes has demonstrated that TIM is capable of delivering between 19 and 20 times as much drug per breath as simple tidal (TBM) breathing. In addition, the slow and deep breath provided by the TIM pattern of breathing enhances deposition as much as 2.5–fold. Therefore, when compared with tidal breathing, in vivo measurements of deposition have indicated that TIM can be up to 51 times more efficient per breath in depositing particles in the lungs (Smaldone & Nikander 2004).

Application of principles in aerosol technology to treatment strategies for allergic disease Asthma Inhaled corticosteroids are the mainstay of maintenance therapy of asthma (Brutsche et al. 2000; Selman et al. 2001). One of the most important features of commercial formulations of inhaled corticosteroids (ICS) is reduced oral bioavailability (Derendorf et al. 1998). With many delivery systems

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Pulses aerosol into subsequent breaths

Exhalation flow

Inhalation flow

Monitors the 1st three breaths

Aerosol Delivery Systems

Time

Fig. 34.12 The I-neb adaptive aerosol delivery system can be set to two patterns of breathing (Figs 34.4 and 34.5). Tidal breathing mode (TBM), or tidal breathing, sets the device to deliver aerosol in the first 50% of the patient’s inspiration. The patient inhales spontaneously during tidal breathing. The device measures “flow” (vertical axis) and time on the horizontal axis. After the device determines the maxima and minima of three breaths, the software defines the “start” of the next

breath (zero flow) and then injects aerosol into the inhaled gas as a series of pulses or boluses over 50% of the time of inhalation. For each succeeding breath the aerosol pulse is adjusted for the average of the three preceding breaths. The inhaled mass is determined by the sum of the aerosol pulses. (Copyright of Profile Therapeutics, a Respironics Company.) (From Smaldone 2005, with permission.)

for ICS having high levels or oropharyngeal deposition (up to 80% with pMDIs and DPIs), it is crucial that when this oropharyngeally deposited drug is subsequently swallowed that its systemic exposure be kept as low as possible. The oral bioavailability of BDP, one of the oldest ICS, is about 20%, fluticasone is about 1%, and momethasone less than 1% (Derendorf et al. 1998). A newer drug, ciclesanide, does not become activated until it deposits in lung tissue (Humbert 2004). However even if there is no systemic bioavailability, systemic exposure can still occur because of ICS being absorbed through lung deposition. It appears that alveolar deposition may give rise to more systemic exposure because particles depositing in ciliated airways are subject to mucociliary clearance and because the barrier to diffusion may be more permeable in the alveoli than the airway. There is some pharmacodynamic data suggesting that when fluticasone is administered to normal subjects and asthmatics, the normal subjects are more susceptible to hypothalamic–pituitary– adrenal axis suppression (Brutsche et al. 2000). This finding is most likely due to the more proximal deposition in the asthmatic subjects because of reduced airway caliber (Fuller 1995). Most pharmaceutical manufacturers have therefore tried to target airway deposition over alveolar deposition. This appears reasonable because asthma is thought to be an airways disease. However some investigators have suggested that there may be an alveolar inflammatory component in asthma, thus suggesting that alveolar deposition may be beneficial, although this remains a minority viewpoint (Kraft et al. 2001). Targeting of the airways in asthma is complicated by the polydisperse nature of therapeutic aerosols. Making average

aerosol diameter smaller reduces oropharyngeal deposition but increases the amount of alveolar deposition. A low oral bioavailability is more important for polydisperse aerosols with larger MMADs so as to minimize systemic exposure from swallowed drug. While the search continues for the ideal method delivering inhaled steroids to patients with asthma, advances in drug design, formulation, and delivery systems now provide a wider array of options for the clinician. However, no delivery system can be considered to be intrinsically superior to all others. The delivery system should be judged instead by its ability to optimize the pharmacokinetic properties of the drug, most notably oral bioavailability, and by its suitability for the target subpopulation of asthmatics.

Aerosolized drug delivery in infants and young children For children less than 4 years old, inhaled medications can be delivered by a nebulizer and facemask or using a pMDI with holding chamber and mask. DPIs can be used from age 4 years and up. The breath-actuated feature of these devices makes them preferable to pMDIs (without a holding chamber) in children aged 4–6 years. After age 6 years children can be taught how to use a pMDI without a spacer, although the use of spacers should continue to be encouraged (Everard 1996). For young children with asthma, nebulized treatment with a suspension of budesonide is an effective and well-tolerated alternative (Szefler 1999). It should be noted that nebulizers can be less effective at aerosolizing suspensions than solutions

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TRAINING BREATH 20 Inhalation flow (L/min) 0

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Fig. 34.13 Target inhalation mode (TIM), or slow and deep breathing, a new algorithm for the I-neb that guides the patient to a slow and deep inspiration. Typical inspiratory flows are reduced to approximately 20 L/min via a high-resistance mouthpiece (inhalation is upward). With each breath the system trains the patient to lengthen inspiration via a vibration feedback. As shown in the top panel of the figure, the patient inhales the first training breath and after 3 s the system vibrates, signaling the patient to exhale. A bolus of aerosol was delivered for the first second of the breath. Because inspiration exceeded the so-called “target inhalation time” (vertical mark on horizontal axis), the device automatically extends the target inhalation time in subsequent training breaths (middle panel) and the time of inspiration is gradually lengthened. After approximately 30 breaths the process is complete and the time of inspiration is set at approximately 9 s (the maximum set by the device, lower panel). In this mode the aerosol pulse (darkly shaded area) begins with inspiration and lasts for 7 s. The device stops extending the target inhalation time at this point because the length of the patient’s inspiration is now approximately the same as the target inhalation time. The device “remembers” this pattern and gives the vibratory feedback at the same point in each succeeding breath and for all future treatments. (Copyright of Profile Therapeutics, a Respironics Company.) (From Smaldone 2005, with permission.)

and clinicians are advised to prescribe budesonide only with the brand of nebulizers used in the clinical trials of this product (Smaldone et al. 1998b). Masks are used with holding chambers and nebulizers because young children breathe nasally and because they

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Aerosol delivery during mechanical ventilation Aerosolized β-sympathomimetic agents are an essential component of the treatment of status asthmaticus. Such delivery is complicated if the patient is undergoing mechanical ventilation. However, recent studies have identified ways in which such delivery can be optimized. The use of a pMDI is feasible provided certain conditions are met (Dhand & Tobin 1997). For example, it is necessary to use a spacer/holding chamber when using a pMDI in this setting. Not all holding chambers are equivalent in efficiency and different brands are not necessarily interchangeable. In contrast to the treatment of spontaneously breathing patients, delivery during mechanical ventilation must be synchronized with respiration. It is essential that a dose escalation protocol be employed because doses far in excess of those used for maintenance therapy may be needed. Hence, efforts to obtain objective evidence on response to treatment (e.g., peak airway pressure, dynamic compliance) and toxicity (tachycardia, arrhythmias) should be sought. In endeavoring to maximize delivered doses, however, it needs to be remembered that the efficiency of pMDI delivery decreases significantly if the interval between serial actuations is less than 1 min and if the synchronization with the respiratory cycle is suboptimal. The efficiency of jet nebulizers in delivering aerosols in the setting of mechanical ventilation is very variable and affected by several factors (O’Riordan et al., 1994). Different brands of commercial nebulizers vary in terms of their output per minute and the duration of treatment. Breath actuation by mechanical ventilators (nebulization takes place only during inspiration) may prolong treatment times. Humidification may cause “rain out” of aerosol in tubing and reduce aerosol delivery by 50%. The use of helium/oxygen mixtures has been shown to affect aerosol delivery from MDIs and jet nebulizers in a model of mechanical ventilation, and could result in either enhanced or impaired delivery, depending on how the system was configured (O’Riordan et al. 1992, 1994). In conclusion, once technical factors have been identified and optimized, efficient delivery of aerosolized medications to patients undergoing mechanical ventilation is readily attainable.

Delivery of therapeutic aerosols to the nasal mucosa Among an increasing list of aerosolized medications being used to treat diseases of the nasal mucosa are corticosteroids, cromolyn, anticholinergics (ipratroprium bromide), saline, and decongestants. The particle size of nasal aerosols tends to be larger than pulmonary inhalers. Manual pumps that produce large, relatively low velocity particles are being used as an alternative to high-velocity freon-based pMDIs. There

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are isolated reports of nasal perforation occurring with highvelocity inhalers (Soderberg-Warner 1984) and patients should be advised to direct the spray in the direction of the ipsilateral ear and away from the nasal septum. In severe allergic sinusitis, the mucosa may be so congested that a short course of systemic corticosteroids may be needed to allow penetration of aerosolized therapy. Prolonged use of topical decongestant sprays may lead to rebound hyperemia and intractable nasal congestion and systemic administration of decongestants may be preferable. Intranasal corticosteroids are an effective therapy for seasonal and perennial rhinitis. While regular use of such an agent has been shown to lead to short-term growth suppression in children (Skoner et al. 2000), it appears that this complication can be prevented by the use of a formulation with low oral bioavailability (e.g., mometasone) (Schenkel et al. 2000).

References Agnew, J. (1984) Physical properties and mechanisms of deposition of aerosols (2 ed.). London; Boston: Butterworths. Anderson, M., Philipson, K., Svartengren, M. & Camner, P. (1995) Human deposition and clearance of 6-micron particles inhaled with an extremely low flow rate. Exp Lung Res, 21(1), 187–195. Baron, P.A. (2001) Measurement of airborne fibers: a review. Ind Health, 39(2), 39–50. Bennett, W.D. (1991) Aerosolized drug delivery: fractional deposition of inhaled particles. J Aerosol Med, 4(3), 223–227. Bennett, W.D. & Smaldone, G.C. (1987) Human variation in the peripheral air-space deposition of inhaled particles. J Appl Physiol, 62(4), 1603–1610. Bisgaard, H., Klug, B., Sumby, B.S. & Burnell, P.K. (1998) Fine particle mass from the Diskus inhaler and Turbuhaler inhaler in children with asthma. Eur Respir J, 11(5), 1111–1115. Brutsche, M.H., Brutsche, I.C., Munawar, M., Langley, S.J., Masterson, C.M., Daley-Yates, P.T. et al. (2000) Comparison of pharmacokinetics and systemic effects of inhaled fluticasone propionate in patients with asthma and healthy volunteers: a randomised crossover study. Lancet, 356(9229), 556–561. Busse, W.W., Brazinsky, S., Jacobson, K., Strieker, W., Schmitt, K., Vanden Burgt, J. et al. (1999) Efficacy response of inhaled beclomethasone dipropionate in asthma is proportional to dose and is improved by formulation with a new propellant. J Allergy Clin Immunol, 104(6), 1215–1222. Derendorf, H., Hochhaus, G., Meibohm, B., Mollmann, H. & Barth, J. (1998) Pharmacokinetics and pharmacodynamics of inhaled corticosteroids. J Allergy Clin Immunol, 101(4 Pt 2), S440–446. Devadason, S.G., Everard, M.L., Linto, J.M. & Le Souef, P.N. (1997) Comparison of drug delivery from conventional versus “Venturi” nebulizers. Eur Respir J, 10(11), 2479–2483. Dhand, R. & Tobin, M.J. (1997) Inhaled bronchodilator therapy in mechanically ventilated patients. Am J Respir Crit Care Med, 156(1), 3–10. Diot, P., Palmer, L.B., Smaldone, A., DeCelie-Germana, J., Grimson, R. & Smaldone, G.C. (1997) RhDNase I aerosol deposition and

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related factors in cystic fibrosis. Am J Respir Crit Care Med, 156(5), 1662–1668. Everard, M.L. (1996) Aerosol delivery in infants and young children. J Aerosol Med, 9(1), 71–77. Food and Drug Administration. (1998) Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug Products Chemistry, Manufacturing, and Controls Documentation. Center for Drug Evaluation and Research (CDER), http://www. fda.gov/cder/guidance/2180dft.htm. Fuller, R. (1995) The Diskus: a new multi-dose powder device– efficacy and comparison with Turbuhaler. J Aerosol Med, 8 Suppl 2, S11–17. Goldin, J.G., Tashkin, D.P., Kleerup, E.C., Greaser, L.E., Haywood, U.M., Sayre, J.W. et al. (1999) Comparative effects of hydrofluoroalkane and chlorofluorocarbon beclomethasone dipropionate inhalation on small airways: assessment with functional helical thin-section computed tomography. J Allergy Clin Immunol, 104(6), S258–267. Humbert, M. (2004) Ciclesonide: a novel inhaled corticosteroid. Expert Opin Investig Drugs, 13(10), 1349–1360. Iacono, A.T., Smaldone, G.C., Keenan, R.J., Diot, P., Dauber, J.H., Zeevi, A. et al. (1997) Dose-related reversal of acute lung rejection by aerosolized cyclosporine. Am J Respir Crit Care Med, 155(5), 1690–1698. Kraft, M., Pak, J., Martin, R.J., Kaminsky, D. & Irvin, C.G. (2001) Distal lung dysfunction at night in nocturnal asthma. Am J Respir Crit Care Med, 163(7), 1551–1556. Morâen, F. (1993) Aerosol dosage forms and formulations (2nd, rev. ed.). Amsterdam; New York: Elsevier. Moren, F. (1982) Drug deposition of pressurized inhalation aerosols. Eur J Respir Dis Suppl, 119, 51–55. Nelson, H.S. & Loffert, D.T. (1994) Comparison of the bronchodilator response to albuterol administered by the OptiHaler, the AeroChamber, or by metered dose inhaler alone. Ann Allergy, 72(4), 337–340. Nerbrink, O., Dahlback, M. & Hansson, H.C. (1994) Why do medical nebulizers differ in their output and particle size characteristics? J Aerosol Med, 7(3), 259–276. Newman, S.P. (1991) Aerosol generators and delivery systems. Respir Care, 36(9), 939–951. Newman, S.P. (1996) Characteristics of radiolabelled versus unlabelled inhaler formulations. J Aerosol Med, 9 Suppl 1, S37–47. Newman, S.P. & Newhouse, M.T. (1996) Effect of add-on devices for aerosol drug delivery: deposition studies and clinical aspects. J Aerosol Med, 9(1), 55–70. Newman, S.P., Talae, N. & Clarke, S.W. (1991) Pressurized aerosol in man with the Rondo spacer. Acta Therapeutica, 17, 49–58. O’Riordan, T.G., Greco, M.J., Perry, R.J. & Smaldone, G.C. (1992) Nebulizer function during mechanical ventilation. Am Rev Respir Dis, 145(5), 1117–1122. O’Riordan, T.G., Palmer, L.B. & Smaldone, G.C. (1994) Aerosol deposition in mechanically ventilated patients. Optimizing nebulizer delivery. Am J Respir Crit Care Med, 149(1), 214–219. Sangwan, S., Condos, R. & Smaldone, G.C. (2003) Lung deposition and respirable mass during wet nebulization. J Aerosol Med, 16(4), 379–386. Sangwan, S., Gurses, B.K. & Smaldone, G.C. (2004) Facemasks and facial deposition of aerosols. Pediatr Pulmonol, 37(5), 447–452. Schenkel, E.J., Skoner, D.P., Bronsky, E.A., Miller, S.D., Pearlman, D.S., Rooklin, A. et al. (2000) Absence of growth retardation in

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children with perennial allergic rhinitis after one year of treatment with mometasone furoate aqueous nasal spray. Pediatrics, 105(2), E22. Selman, M., King, T.E. & Pardo, A. (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med, 134(2), 136–151. Skoner, D.P., Rachelefsky, G.S., Meltzer, E.O., Chervinsky, P., Morris, R.M., Seltzer, J.M. et al. (2000) Detection of growth suppression in children during treatment with intranasal beclomethasone dipropionate. Pediatrics, 105(2), E23. Smaldone, G. (1997) Respiratory physiology in asthma: pulmonary function testing bronchoprovocation, and mucociliary clearance (2nd ed.). Philadelphia: Saunders. Smaldone, G.C. (1991) Drug delivery via aerosol systems: concept of “aerosol inhaled”. J Aerosol Med, 4(3), 229–235. Smaldone, G.C. (2001) Deposition and clearance: unique problems in the proximal airways and oral cavity in the young and elderly. Respir Physiol, 128(1), 33–38. Smaldone, G.C. (2004) Respiratory physiology and disease effects on aerosol deposition. Respir Drug Delivery IX, 1, 179–186. Smaldone, G.C. (2005) Assessing new technologies: patient-device interactions and deposition. Respir Care, 50(9), 1151–1160. Smaldone, G.C., Berg, E. & Nikander, K. (2005) Variation in pediatric aerosol delivery: importance of facemask. J Aerosol Med, 18(3), 354–363. Smaldone, G.C., Cruz-Rivera, M. & Nikander, K. (1998) In vitro determination of inhaled mass and particle distribution for Budesonide nebulizing suspension. J Aerosol Med 11, 113–125. Smaldone, G.C., Diot, P., Groth, M. & llowite, J. (1998) Respirable mass: vague and indefinable in disease. J Aerosol Med, 11 Suppl 1, S105–111. Smaldone, G.C., Foster, W.M., O’Riordan, T.G., Messina, M.S., Perry, R.J. & Langenback, E.G. (1993) Regional impairment of mucociliary clearance in chronic obstructive pulmonary disease. Chest, 103(5), 1390–1396.

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Smaldone, G.C., Fuhrer, J., Steigbigel, R.T. & McPeck, M. (1991) Factors determining pulmonary deposition of aerosolized pentamidine in patients with human immunodeficiency virus infection. Am Rev Respir Dis, 143(4 Pt 1), 727–737. Smaldone, G.C., Itoh, H., Swift, D.L., Kaplan, J., Florek, R., Wells, W. et al. (1983) Production of pharmacologic monodisperse aerosols. J Appl Physiol, 54(2), 393–399. Smaldone, G.C. & Messina, M.S. (1985a) Enhancement of particle deposition by flow-limiting segments in humans. J Appl Physiol, 59(2), 509–514. Smaldone, G.C. & Messina, M.S. (1985b) Flow limitation, cough, and patterns of aerosol deposition in humans. J Appl Physiol, 59(2), 515–520. Smaldone, G.C. & Nikander, K. (2004) Bench performance and in vivo deposition efficiency of the I-neb adaptive aerosol delivery system (AAD) during tidal versus slow and deep breathing. Proceedings of the Aerosol Society, DDL15, 17–20. Soderberg-Warner, M.L. (1984) Nasal septal perforation associated with topical corticosteroid therapy. J Pediatr, 105(5), 840–841. Stahlhofen, W., Gebhart, J. & Heyder, J. (1980) Experimental determination of the regional deposition of aerosol particles in the human respiratory tract. Am Ind Hyg Assoc J, 41(6), 385–398a. Szefler, S.J. (1999) Pharmacodynamics and pharmacokinetics of budesonide: a new nebulized corticosteroid. J Allergy Clin Immunol, 104(4 Pt 2), 175–183. Thorsson, L., Edsbacker, S. & Conradson, T.B. (1994) Lung deposition of budesonide from Turbuhaler is twice that from a pressurized metered-dose inhaler P-MDI. Eur Respir J, 7(10), 1839–1844. Wildhaber, J.H., Devadason, S.G., Eber, E., Hayden, M.J., Everard, M.L., Summers, Q.A. et al. (1996) Effect of electrostatic charge, flow, delay and multiple actuations on the in vitro delivery of salbutamol from different small volume spacers for infants. Thorax, 51(10), 985–988.

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Bronchial Hyperresponsiveness Guy F. Joos

Environmental risk factors (causes)

Summary Measures of bronchial responsiveness are widely used for diagnosis and monitoring of asthma. A wide array of nonspecific bronchoconstrictor stimuli is available. Methacholine and histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Indirect challenges (adenosine, exercise, hypertonic saline) induce airflow limitation by an action on cells other than smooth muscle cells. A variety of cells, mediators, and receptors are involved in the airway narrowing caused by indirect airway challenges. Whereas bronchial responsiveness to a direct stimulus is only weakly related to airway inflammation, indirect airway challenges might better reflect active airway inflammation. Both direct and indirect airway challenges are useful outcome parameters in clinical studies of asthma. For example, an indirect challenge responds within hours to days to treatment with inhaled steroids, while improvement in direct responsiveness may take months to years. Bronchial challenges are also an essential step in the development of some of the potential new antiasthma treatments, such as adenosine or tachykinin receptor antagonists.

Introduction Bronchial hyperresponsiveness (BHR) is one of the hallmarks of bronchial asthma. Other important characteristics include variable airflow obstruction, airway inflammation, and airway remodeling (Bousquet et al. 2000; Global Initiative for Asthma 2006) (Fig. 35.1). Measures of bronchial responsiveness are widely used for diagnosis and monitoring of asthma, in epidemiologic studies, and in clinical trials. They are important outcome parameters in studies evaluating treatment of asthma and are nowadays often combined with noninvasive

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

INFLAMMATION

Airflow limitation

Airway hyperresponsiveness Triggers

Symptoms Fig. 35.1 Relationship between airway inflammation, bronchial hyperresponsiveness and airflow limitation. (Redrawn from GINA 2006, with permission.) (See CD-ROM for color version.)

measures of airway inflammation such as induction of sputum or measurement of exhaled nitric oxide (NO) (American Thoracic Society 2000; Jayaram et al. 2000; Kharitonov & Barnes 2000; Sterk et al. 1993). Bronchial responsiveness is measured as the change in airway caliber occurring after inhalation of a bronchoconstrictor agent (Lötvall et al. 2000). The development of methods to demonstrate and quantify BHR has greatly contributed to the understanding of the pathogenesis of asthma and its diagnosis and treatment. Studies on BHR have also highlighted the complexity of mechanisms involved in the increased airway responses in asthma (Joos & O’Connor 2003). BHR is also observed in diseases other than asthma and in some apparently healthy subjects. For instance, BHR to methacholine is present in a majority of patients with mild to moderate chronic obstructive pulmonary disease (COPD) (Tashkin et al. 1992). Moreover, the severity of BHR predicts the response to inhaled corticosteroids in patients with asthma (Juniper et al. 1981) and the progression of airflow limitation in patients with COPD (Tashkin et al. 1992). An array of different pharmacologic and physical stimuli is available to measure bronchial responsiveness (Table 35.1). They are called nonspecific bronchoconstrictor stimuli, and differ from specific stimuli, such as allergen or aspirin, which cause bronchoconstriction only in a specific subset of asthmatic patients.

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Table 35.1 Nonspecific bronchoconstrictor stimuli. (Modified from Joos & O’Connor 2003.) Direct stimuli Cholinergic agonists: pilocarpine, acetylcholine, methacholine, carbachol Histamine Prostaglandin D2 Cysteinyl leukotrienes: LTC4, LTD4, LTE4 Indirect stimuli Physical stimuli Exercise Nonisotonic aerosols (hypertonic and hypotonic aerosols, distilled water, mannitol) Eucapnic voluntary hyperpnea of dry air Pharmacologic stimuli Adenosine Tachykinins (substance P, neurokinin A) Bradykinin Metabisulfite/SO2 Propranolol Endotoxin (lipopolysaccharide) Platelet-activating factor Ozone

Historical background In 1921, Alexander and Paddock found that asthmatic subjects developed asthma-like symptoms following subcutaneous administration of pilocarpine in doses that had no effect on normal subjects. In 1932, Weiss et al. showed that asthmatics were hyperresponsive to intravenous histamine. Curry (1947) reported that inhalation of small quantities of inhaled histamine induced bronchoconstriction. Bronchoconstriction induced in asthmatics by acetylcholine and histamine was also described by Tiffeneau & Beauvallet (1945), Herxheimer (1951), and De Vries et al. (1962). In the 1970s and 1980s, methods for bronchial provocation testing with nonspecific stimuli were standardized (Chai et al. 1975; Cockcroft et al. 1977). The number of stimuli that were demonstrated to cause an increased bronchial response in asthma compared with healthy subjects gradually enlarged, as well the understanding that mechanisms involved in BHR to these different stimuli were multiple and complex (Pauwels et al. 1988).

refers to a comparison with the airway response to the same agonist, using the same method to measure the airflow limitation, in a group of healthy subjects. The wording “airflow limitation” is chosen because it encompasses the different mechanisms that can lead to a decrease in the parameters of airflow used in studies on BHR, such as forced expiratory volume in 1 s (FEV1) or airway resistance (Raw). A decrease in FEV1 or an increase in Raw following exposure to a stimulus can reflect constriction of airway smooth muscle, edema of the airway wall, increased amounts of fluid or sputum in the airway wall, or a decrease in the elastic recoil pressure on the airways. The phenomenon of BHR is sometimes referred to as “nonspecific,” i.e., that most patients with active asthma will react to these stimuli. The stimuli used to reveal it, however, act by highly specific mechanisms. As multiple pathophysiologic pathways are involved, it is therefore no surprise that the results of the different challenge tests are only weakly correlated and not mutually interchangeable. Bronchoconstrictor stimuli may be classified into direct and indirect, according to the main mechanism through which they induce airflow limitation (Table 35.1). Combinations of mechanisms are possible. Direct stimuli induce airflow limitation through direct action on the effector cells involved in the airflow limitation. These comprise in the first place airway smooth muscle cells, but bronchial vascular endothelial cells and/or mucus-producing cells may also be involved. Indirect stimuli, on the other hand, act on intermediary cells such as inflammatory cells, bronchial epithelial cells and/or neuronal cells; the proinflammatory mediators and/or neurotransmitters liberated by these cells will then interact with the effector cells to cause airflow limitation (Fig. 35.2) (Van Schoor et al. 2000). It has become clear that several agonists can be used to study bronchial responsiveness. The most widely used pharmacologic agents to asses bronchial responsiveness are methacholine and histamine. Methacholine and histamine cause airflow limitation predominantly through a direct effect on airway smooth muscle and are therefore called direct broncoconstrictor agents.

Direct stimulus

Indirect stimulus

Effector cells:

Intermediary cells:

– Airway smooth muscle cells – Bronchial endothelial cells – Mucus-producing cells

– Inflammatory cells – Neuronal cells

Definition Airflow limitation

Bronchial or airway hyperresponsiveness can be defined as an abnormal increase in airflow limitation following the exposure to a stimulus. In this definition the word “abnormal”

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Fig. 35.2 Cellular pathways involved in the bronchoconstriction induced by direct and indirect stimuli. (After Van Schoor et al. 2000, with permission.) (See CD-ROM for color version.)

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60

FEV1 (% fall) Methacholine Histamine

50 ASTHMA

40 30 20

COPD SEVERE

MODERATE

MILD

SLIGHT

NORMAL 10 0 0.01

0.1

1.0 10 Dose (µmol)

100

Fig. 35.3 Dose–response curves for methacholine and histamine in patients with asthma, chronic obstructive pulmonary disease (COPD), and normal airways. (From Woolcock et al. 1991, with permission.) (See CD-ROM for color version.)

Methods for measuring bronchial responsiveness Bronchial responsiveness is measured as the change in airway caliber occurring after inhalation of a bronchoconstrictor agent. Typically, a hyperresponsive pattern is observed in patients with asthma: the dose –response curve to a bronchoconstrictor agent displays a leftward shift, an increased slope, and an enhanced maximal response (Fig. 35.3). Thus, the optimal way to measure bronchial responsiveness to one or another agonist is to perform a dose–response curve to the selected agonist and to quantify the response.

Methods for delivering the stimulus Various routes of delivery of stimuli have been used to demonstrate BHR in patients with asthma, including intravenous, subcutaneous, and oral administration, but most stimuli used in studies on BHR have been given via inhalation for reasons of safety and specificity. By use of aerosol, lower doses of the bronchoconstrictor substances are administered and unpleasant systemic side effects are avoided. Aerosols can be produced from solutions either by atomization with compressed air (jet nebulizers) or by the vibration of piezoelectric crystals at ultrasonic frequencies (ultrasonic nebulizers). Three types of nebulizer delivering methods are in use: the tidal breathing, the reservoir, and the dosimeter methods. With the tidal breathing method the aerosol is generated continuously by a nebulizer and inhaled by the subject via a facemask or a mouthpiece, with a nose-clip occluding the nose. The aerosol is inhaled for a set time during tidal breathing (e.g., 2 min) (Cockcroft et al. 1977). This system has the advantage that it is a well-tried method and does not need sophisticated equipment. However, although the output of the

Bronchial Hyperresponsiveness

nebulizer can be measured, the dose of the aerosol inhaled is not exactly known and depends on the minute volume of the subjects (Eiser et al. 1983). The reservoir and dosimeter methods deliver standardized doses of the aerosol during inspiration only. In the reservoir method a storage bell is filled with aerosol, freshly generated from a nebulizer. The subject then inhales a fixed volume from the bell via the mouthpiece and breath-holds for 4 s before exhaling in a balloon. In the dosimeter method, the dose of the inhaled aerosol is standardized via a breath-actuated dosimeter (e.g., Rosenthal-French dosimeter). In comparative studies the results obtained with the tidal breathing method compared well with those obtained with the dosimeter method (Beaupré & Malo 1979; Ryan et al. 1981). In the past few years, a novel osmotic bronchial challenge using mannitol dry powder has been developed as an alternative for the wet aerosol of hypertonic saline. From a practical point of view, it does not require a nebulizer and can be performed outside the laboratory setting using a simple hand-held inhaler device, the mannitol being contained in gelatin capsules (Anderson et al. 1997). The output of the nebulizer is a major determinant of the airway response to bronchial challenge. For instance, a threefold increase in the output of the nebulizer resulted in a threefold lower PC20 (provocative concentration causing a 20% decrease in FEV1) for methacholine (Ryan et al. 1981). Standardization of the droplet size and the flow characteristics and output of the nebulizers are all relevant to this issue, but a discussion of these important parameters of administration via nebulizers is outside the scope of this chapter and readers are referred to other sources for more technical details (Sterk et al. 1993; American Thoracic Society 2000; Anderson & Brannan 2003; Joos & O’Connor 2003). The important message is that the nebulizer should be well characterized so that results of bronchial challenges with the same stimulus can be compared within the same laboratory or lung function department and between investigations. This interinvestigator reproducibility is especially important in the settings of epidemiologic studies and multicenter therapeutic studies.

Methods for measuring the airway response The changes in airway caliber occurring after inhalation of the bronchoconstrictor stimulus can be estimated by measuring changes in airflow during a forced expiratory maneuver or by measuring changes in airways resistance (Raw) and conductance (Gaw). The tests of forced expiration are FEV1, peak expiratory flow rate (PEFR), and flow rates at different levels of the maximal or partial expiratory flow–volume curve (Table 35.2). The choice of which parameter to use is almost always a trade-off between the reproducibilty and the sensitivity of the parameter. Measurement of FEV1 is simple to perform, reproducible (intrasubject coefficient of variation 0–8%) (Eiser et al. 1983), and can be achieved with simple equipment. The FEV1 is relative insensitive at detecting changes in

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Table 35.2 Bronchial provocation testing: measurement of the airway response. Peak expiratory flow rate Spirometry Forced inspiratory maneuvers (if upper airway involvement is suspected) Body plethysmography Transcutaneous oxygen saturation Forced oscillometry

airway caliber, in comparison to Raw and specific (s)Gaw. The measurement of FEV1 involves a deep inspiration. In normal subjects, a deep inspiration may overcome a small induced increase in airway resistance. In asthmatics a deep inspiration may induce bronchoconstriction (Fish & Kelly 1979; Orehek et al. 1981). The measurement of Raw and sGaw more accurately reflect airway patency, but is more affected by changes in large than in smaller airways. It is more sensitive to airway caliber changes, but its reproducibility is less good than for FEV1 (intrasubject coefficient of variation for Raw 10–20%, for sGaw 9%; see Eiser et al. 1983). It does not involve a full inspiratory maneuver and hence does not affect airway tone.

Quantification of BHR Bronchial responsiveness is usually quantified by performing a dose–response study with the agonist, and expressing the result as a dose or quantity of the agonist that causes a predetermined change in the measured airway parameter (Fig. 35.3). The most frequently used measures are the PC20 FEV1 or the PD20 FEV1, respectively the concentration and dose of the agonist that cause a fall in FEV1 of 20% from baseline. To determine the PC20 or PD20, the dose–response curve is plotted on a semilogarithmic scale and the PC20 or PD20 calculated by interpolation. Similar procedures can be used for other parameters, such as the PC35 or PD35 sGaw (concentration or dose that decreases the sGaw by 35% from baseline) or the PC100 or PD100 Raw (concentration or dose that doubles the airway resistance). A dose–response curve to histamine or methacholine is characterized by the threshold concentration, the slope, and the maximal bronchoconstriction that can be reached. The threshold concentration, also called the sensitivity, is the dose at which the curve changes from baseline. The threshold can be defined as the concentration where a change of lung function of twice the coefficient of variation of the baseline measurement has occurred. The slope, also called the reactivity, is obtained by linear regression of the data points from threshold, using the formula y = mx + b (m = slope). Normal individuals have limited bronchoconstriction even if the amount of histamine or methacholine administered is further increased (Sterk et al. 1985). This plateau level is not observed in patients with moderate to severe asthma.

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Mechanisms and receptors involved in BHR Direct stimuli Histamine causes bronchoconstriction by binding to the histamine H1 receptor on airway smooth muscle cells. The cholinergic agonists cause bronchoconstriction by directly acting on the airway smooth muscle cell via the M3 muscarinic receptor. The leukotrienes (LT)C4, LTD4 or LTE4 cause airway narrowing by interaction with the CysLT1 receptor.

Indirect stimuli Several cells are involved in the airway narrowing caused by indirect stimuli. These include epithelial cells, inflammatory cells (including mast cells), nerve cells, and vascular smooth muscle cells. Upon application of a constrictor agent, a wide array of mediators are released: histamine, cysteinyl leukotrienes, prostaglandins, adenosine, acetylcholine, substance P (SP), neurokinin A (NKA), and nitric oxide (NO). Moreover, the nature and amount of mediator released can vary from one indirect stimulus to another (Van Schoor et al. 2000). Adenosine causes bronchoconstriction in asthma by interaction with mast cells; histamine has been shown to contribute to this reaction (Polosa et al. 2002). Using the experimental 5-lipoxygenase inhibitor ABT-761, a role for leukotrienes in the bronchoconstriction induced by inhaled adenosine was suggested (Van Schoor et al. 1997). In addition, Rorke et al. (2002) have produced direct evidence for involvement of cysteinyl leukotrienes in adenosine-induced bronchoconstriction in asthma by using the CysLT1 receptor antagonist montelukast. Adenosine exerts its effect on human cells through interaction with specific adenosine (P1) receptors, of which four subtypes (A1, A2A, A2B and A3) have been described. The A1, A2B and A3 receptors have been shown to be involved in various animal and human models of inflammation (Joos & Pauwels 1996). In particular, the potential role of A2B receptors is being increasingly recognized (Polosa 2002). Airway effects of the tachykinins SP and NKA are mediated through tachykinin NK1 and NK2 receptors (Joos et al. 2001). In vitro, tachykinins constrict human airway smooth muscle through NK2 receptors; in small- and medium-sized bronchi, NK1 receptors are also involved. In vivo inhaled NKA causes bronchoconstriction by indirect mechanisms (via interaction with cholinergic nerves and inflammatory cells); both NK1 and NK2 receptors are involved in this bronchoconstrictor effect (Joos et al. 2004). Exercise causes airway narrowing by the loss of water through evaporation from the airway surface. The mechanism whereby the loss of water causes airways to narrow is thought to relate to thermal (cooling and rewarming) and osmotic (increase in airway osmolarity) effects of dehydration (Anderson & Daviskas 2000). Mediators involved in exercise-induced bronchoconstriction include histamine,

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prostaglandins, leukotrienes, and tachykinins. In addition, an increase in plasma adenosine concentration during exercise was reported: in patients with asthma, a relationship was observed between the increase in adenosine concentration and the level of exercise-induced bronchoconstriction (Vizi et al. 2002). The mechanisms involved in the bronchoconstriction induced by other physical stimuli are similar to those described for exercise. The reader is referred to reviews on this topic (Joos & O’Connor 2003; Van Schoor et al. 2005).

BHR in asthma: relation to airway inflammation and airway remodeling Bronchial responsiveness to a direct stimulus is only weakly related to airway inflammation It is frequently assumed that measures of bronchial responsiveness and airway inflammation can be used interchangeably. However, this does not seem to be the case. Correlations between markers of airway inflammation (bronchoalveolar lavage, bronchial biopsy and/or sputum) and BHR at baseline (i.e., before any intervention) have been examined in a large number of studies. In some instances weak correlations have been found between one or more inflammatory markers and the degree of BHR. However, an almost equal number of studies did not find significant correlations between airway inflammation and BHR (reviewed in Brusasco et al. 1998; Rosi & Scano 2000). When asthmatic patients are examined during the course of their disease, bronchial responsiveness and markers of airway inflammation cannot be considered as overlapping dimensions. This has been demonstrated by the use of factor analysis. The statistical method of factor analysis allows the many parameters that characterize the disease to be reduced to a few independent factors, each factor grouping associated parameters. In a study on 99 stable asthmatics using factor analysis it was concluded that baseline BHR (measured by histamine challenge), airway function, and airway inflammation are to be considered separate dimensions in the description of chronic asthma, thus supporting the utility of routine measurement of all these dimensions (Rosi et al. 1999). A subsequent study, which performed factor analysis on 66 stable asthmatics, assessed whether the relationship between BHR (measured by methacholine challenge), noninvasive markers of airway inflammation, and baseline lung function depended on the duration of the disease; the results of the analysis suggested that with shorter duration of the disease, BHR is associated with airway inflammation, whereas with a longer duration it is associated with impaired lung function, suggesting that in longstanding asthma ongoing alterations become the primary determinant of functional characteristics (Gronke et al. 2002). The distribution of sputum eosinophil count and its relationship with BHR to methacholine has been studied in mild

Bronchial Hyperresponsiveness

to moderate steroid-naive asthmatic people. In this study, 118 patients with asthma and 44 healthy people were examined; 69% of the asthma group had a significantly raised sputum eosinophil count (i.e., > 2%). Although the sputum eosinophil count was significantly associated with bronchial responsiveness to methacholine, the relationship was found to be weak (Louis et al. 2002a). The cross-sectional relationships between parameters of airway physiology, inflammation, and remodeling was studied in a group of patients with mild symptomatic asthma; 40% of the variability in BHR could be explained by multiple regression containing remodeling and inflammatory components (reticular basement membrane thickness accounting for 20%, epithelial cells for 11%, and eosinophils for 8%). Thus, while inflammation was an important predictor for BHR, remodeling represented a stronger predictor (Ward et al. 2002). The relation with remodeling might even be stronger in patients with asthma of longer duration (Gronke et al. 2002).

Caliber of the airways and bronchial responsiveness Airway caliber in vivo is the result of a balance between the force generated by the airway smooth muscle and a number of opposing factors. The latter are mainly presented by autonomic mechanisms that tend to limit the airway smooth muscle tone and by mechanical factors that oppose airway smooth muscle shortening (Brusasco et al. 1998). Patients with nonasthmatic fixed airflow limitation also demonstrate BHR to histamine and methacholine. The characteristics are somewhat different in that there is a strong linear relationship between BHR and the reduction in FEV1 in subjects with chronic airflow limitation (Joos & O’Connor 2003). It is difficult to interprete the meaning of a low PC20 in a patient with baseline airway obstruction. For instance, most patients with smoking-related COPD and mild to moderate baseline airway obstruction have BHR, but most have no acute or chronic bronchodilator response or symptoms of asthma (Mullen et al. 1986; American Thoracic Society 2000).

Indirect airway challenges might better reflect active airway inflammation It has been suggested that indirect markers of airway responsiveness are a better reflection of active airway inflammation. One of the best-studied indirect-acting bronchoconstrictors is adenosine monophosphate (AMP) (Holgate 2002a). Indirect challenges are thought to better reflect ongoing inflammation than direct challenges; this hypothesis is based on data obtained from studies in which indirect and direct challenges have been compared head-to-head. This has been most extensively studied in the case of adenosine challenge (van den Berge et al. 2002). Allergen avoidance at high altitude led to a significant diminution of eosinophils and eosinophil cationic protein in blood; this was accompanied by a significant

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decrease in BHR to AMP (2.1 doubling concentrations), BHR to methacholine remaining unchanged (van Velzen et al. 1996). In a group of 120 patients with atopic asthma, the concentrations of AMP causing the FEV1 to drop by more than 20% (PC20 AMP) was found to be more closely associated with inflammatory parameters than PC20 methacholine. In this study PC20 methacholine was predominantly predicted by baseline lung function (FEV1 in percent of predicted value), while PC20 AMP was predominantly predicted by the percentage of eosinophils in sputum. So, PC20 AMP reflects more closely the extent of airway inflammation due to asthma than PC20 methacholine (van den Berge et al. 2001a). Even in patients with clinical remission of asthma, elevated levels of exhaled NO and increased bronchial responsiveness to AMP and methacholine are found. It is interesting to note that in these patients a significant correlation between exhaled NO and responsiveness to AMP, but not methacholine, has been reported (van Den Toorn et al. 2001).

Asymptomatic BHR BHR can be shown in virtually all patients who experience symptoms of asthma. Moreover, a significant proportion of individuals with no past or present history of asthma or of other respiratory diseases, no current symptoms, and no respiratory medication show an increased physiologic airway response (fall in expiratory flows) to agents such as histamine or methacholine or to stimuli such as exercise (reviewed in Boulet 2003). Most studies in adults and children report a prevalence of asymptomatic BHR under 15% (Salome et al. 1987; Rasmussen et al. 1999; Boulet 2003). Subjects with asymptomatic BHR may show a more rapid decline in lung function, and this might be associated with an increased risk of developing asthma (American Thoracic Society 2000; Boulet 2003). Some patients with asthma can evolve toward asymptomatic bronchial hyperreponsiveness or in some cases even normalize bronchial responsiveness (Boulet 2003). Adolescents in clinical remission of atopic asthma are less responsive to the bronchial stimuli adenosine monophosphate and methacholine, in comparison to subjects with current asthma; however they are still more responsive than healthy controls (Van den Toorn et al. 2000, Van den Toorn et al. 2001).

BHR and antiasthma drugs Use of bronchial challenges to evaluate new antiasthma drugs Challenges with specific neurotransmitter (e.g., a cholinergic antagonist) or a specific mediator (e.g., LTD4) have proved useful in the development of new neurotransmitter antagon-

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ists (such as the long-acting anticholinergic agent tiotropium bromide) or mediator antagonists (such as the CysLT1 receptor antagonists) (Pauwels et al. 1995; O’Connor et al. 1996). The tachykinins SP and NKA are able to mimic various pathophysiologic features of asthma and COPD. There is an interest in developing tachykinin receptor antagonists as possible treatment for obstructive airway diseases (Joos & Pauwels 2001). Inhaled NKA induces bronchoconstriction in patients with asthma. A shift in the dose–response curve to NKA suggests airway activity of the compound. Using this approach the potent tachykinin NK2 receptor antagonist SR 48968 was found to have an only limited protective activity in patients with asthma (Van Schoor et al. 1998). Using a similar approach a dual tachykinin NK1/NK2 receptor antagonist, DNK333, and a triple tachykinin NK1/NK2/NK3 antagonist were able to prevent NKA- induced bronchoconstriction (Joos et al. 2004; Schelfhout et al. 2006). Similar approaches are being considered for adenosine receptor antagonists, where challenge with AMP is a logical first step in testing potential airway activity of an adenosine receptor antagonist (Holgate 2002b).

Direct and indirect bronchial responsiveness are useful outcome measures in clinical studies and provide different information The monitoring of symptoms, airflow obstruction, and exacerbations is essential to asthma management. Regular monitoring by physicians improves health outcomes provided it includes monitoring of control of asthma, medication and skills at regular intervals (Gibson 2000). Measures of airway responsiveness and airway inflammation (e.g., sputum induction and exhaled air) can be assessed at regular clinic visits and are increasingly included in studies evaluating control of asthma (Sont et al. 1999; Joos 2001; Green et al. 2002; Smith et al. 2005). It is well known that treatment with inhaled steroids slowly improves the bronchial responsiveness to a direct stimulus (Woolcock et al. 1988). In a prospective longitudinal study on the effect of an inhaled steroid, fluticasone, various markers of airway physiology, inflammation, and remodeling were incorporated. A 1-year treatment with an inhaled steroid was shown to improve airway caliber, improve direct bronchial responsiveness, and decrease inflammatory cells in bronchoalveolar lavage (BAL) fluid, as well as reduce thickness of the reticular basement membrane (a measure of airway remodeling). Whereas the improvement in airway caliber and the decrease in inflammatory cells recovered from BAL reached a plateau after 3 months of treatment, BHR to methacholine (PD20) continued to improve over the same time period as changes in airway remodeling, with improvements in both reticular basement membrane thickness and PD20 occurring with 12 months of treatment. It is of interest to note that in this study one-third of the improvement in BHR with fluticasone propionate was associated

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with early changes in inflammation, but the more progressive and larger improvement was associated with the later improvement in airway remodeling (Ward et al. 2002). Recent findings indicate that using direct BHR as a parameter might be of practical clinical relevance in the optimization of the long-term maintenance treatment of asthma. Commonly used clinical indices of asthma severity, such as symptoms, β2-agonist use, FEV1, and peak flow variabilility, were shown not to be significantly correlated with inflammatory cell counts in the lamina propria of the airways in asthmatics not treated with inhaled corticosteroids (ICS) (Djukanovic et al. 1992) as well as in those receiving ICS (Sont et al. 1996). In addition, infiltration with inflammatory cells in the lamina propria seems to persist despite regular treatment with ICS; the number of infiltrating leukocytes appears to be correlated with direct BHR, but not with symptoms or lung function. In many patients whose disease is considered clinically controlled, it appears that BHR and airways inflammation persist (Sont et al. 1996). According to international guidelines, the level and adjustment of antiinflammatory treatment is based solely on symptoms and lung function, and in the light of the above one could question whether such an approach really leads to the optimal long-term outcome of asthma. Researchers from the Leiden group have postulated that, in addition to the current guidelines, asthma treatment should also be directed toward reducing BHR. They have shown in a subsequent 2-year prospective parallel-group trial (AMPUL study) that a treatment strategy with stepwise adjustments of ICS doses, aimed at reducing BHR on top of optimizing symptoms and lung function, indeed leads to more effective asthma control (significantly greater improvements in FEV1, significant 1.8fold decrease in mild exacerbation rate) and greater improvement of chronic airways remodeling (significantly greater reduction in subepithelial reticular layer thickness), when compared with a “reference” strategy based on current treatment guidelines (Sont et al. 1999). Whereas the steroid-induced improvement in direct bronchial responsiveness may take months or even years, indirect challenges may respond more rapidly (Van Schoor et al. 2002; Reynolds et al. 2002). In a study on the effects of two doses of the inhaled steroid fluticasone propionate, the severity of exercise-induced bronchoconstriction decreased significantly as compared to placebo within 3 weeks. In contrast, responsiveness to methacholine improved during the first 6 weeks of the treatment with fluticasone propionate, and steadily increased during the 24 weeks of treatment (Hofstra et al. 2000). In a study on the effect of 2 weeks’ treatment with oral or inhaled steroids in adult asthmatic patients, PC20 AMP was found to be more sensitive to changes in acute airway inflammation compared with PC20 methacholine (van den Berge et al. 2001b). So, BHR to inhaled AMP is an early and sensitive indicator of the beneficial antiinflammatory effects of topical steroids (Prosperini et al. 2002).

Bronchial Hyperresponsiveness

Specific uses of direct and indirect bronchial challenges We now have at our disposal a broad and heterogeneous array of stimuli that can be used for bronchial challenge testing in asthmatic patients. How we should best measure BHR is as yet uncertain. The choice of stimulus depends at least in part on the reason for performing the challenge and on the type of information one wants to obtain. Moreover, one should be aware of the fact that various stimuli can temporarily increase BHR (Table 35.3). In general, BHR to direct stimuli is extremely sensitive for clinically current asthma, and a negative test permits one to exclude with reasonable certainty the diagnosis of asthma in symptomatic patients. However, these tests lack specificity both in differentiating asthma from normal and asthma from COPD (Tables 35.4 and 35.5); moreover, they perform less well in the epidemiologic setting. Parameters of direct BHR appear to be better correlated with the long-term structural alterations of remodeling than with indices of inflammation. In contrast, tests of indirect BHR are both more specific (but less sensitive) for asthma and correlate better with the airway inflammation of the disease. Bronchial responsiveness to indirect stimuli may be preferred for confirming a diagnosis of asthma and for monitoring disease activity and the antiinflammatory effects of antiasthma therapy in the short and medium term, while direct stimuli may be preferred for

Table 35.3 Factors that increase bronchial responsiveness. (From American Thoracic Society 2000, with permission.) Factor

Duration of effect

Exposure to environmental antigens Occupational sensitizers Respiratory infection Air pollutants Cigarette smoke Chemical irritants

1–3 weeks Months 3–6 weeks 1 week Few hours Days to months

Table 35.4 Diseases manifesting bronchial hyperresponsiveness on testing with histamine or methacholine. Asthma COPD Congestive heart failure Cystic fibrosis Bronchiectasis Viral respiratory infections Allergic rhinitis Sarcoidosis

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Table 35.5 Value of direct and indirect bronchial challenges in the diagnosis and monitoring of asthma. (From Anderson & Brannan 2003; American Thoracic Society 2000.) Direct challenge

Indirect challenge

Diagnosis

Not specific for asthma High negative predictive value (approaching 100% for clinically active asthma)

Specific for asthma Less sensitive than direct challenge

Monitoring

Progressive improvement on treatment with inhaled steroid (months to years)

Rapid response to antiinflammatory treatment (days to weeks)

excluding asthma and optimizing antiinflammatory treatment in the long term (Cockcroft 2001; Joos & O’Connor 2003). Both types of challenges thus appear to have their value in the assessment of the different pathophysiologic components of asthma.

Pharmacologic stimuli Adenosine occupies a unique place in the group of indirect stimuli, as it is the challenge which, to date, is the best reflection of Th2-mediated inflammation, dominated by activated mast cells and eosinophils (Holgate 2002a). This means that BHR to AMP can also be present in other conditions characterized by mast cell priming, such as allergic rhinitis and a subgroup of patients with COPD. Adenosine challenge may be a useful tool in making the differential diagnosis of asthma and COPD in cases where the diagnosis is uncertain. This is especially the case in nonsmokers, as smokers with COPD may show AMP responsiveness as well (Polosa et al. 2002; van den Berge et al. 2002). The role of the mast cell in COPD is currently poorly defined. It has been shown that the number of epithelial mast cells in the bronchioli of smokers with COPD is increased (Grashoff et al. 1997). Interestingly, a subset of COPD patients has been described characterized by sputum eosinophilia and mast cell activation; the hypothesis has been raised that responders to steroids have both eosinophil- and mast-cell driven disease, regardless of whether they have asthma or COPD (Louis et al. 2002b). Concerning the association of BHR to AMP with upper airway allergic inflammation, an increasing dose–response slope along the symptom axis from asymptomatic controls to allergic rhinitis/conjunctivitis to allergic asthma has been demonstrated, which was in addition stronger than for methacholine (De Meer et al. 2002). In summary, the specificity of AMP challenge for asthma, together with its high repeatability, could be useful for epidemiologic purposes. AMP challenge testing will also become increasingly used for pharmacologic testing, once specific adenosine receptor antagonists become available for use in humans. Such investigations will allow determination of whether adenosine is a clinically important mediator of asthma,

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as well as identifying which adenosine receptor subtypes play a main role in asthma. Adenosine is inexpensive, and the challenge is technically easy to perform (Joos & O’Connor 2003). Tachykinins are of potential importance as mediators of asthma. Performing bronchial challenges with tachykinins is mainly of pathophysiologic importance, in order to elucidate the actions of the different tachykinins and to study the role of airway tachykinin receptors. Challenge tests with SP and NKA are currently employed to evaluate newly developed tachykinin receptor antagonists. However, the high cost of these peptides will probably limit their use to fundamental and clinical research purposes (Joos & Pauwels 2000). The other pharmacologic stimuli have been much less studied. To date, specific clinical uses have not been identified and these challenges remain essentially of pathophysiologic interest in the laboratory setting .

Physical and physicochemical stimuli This group of challenges better mimics the stimuli that asthmatic patients are likely to encounter in their everyday life. In this respect, exercise challenge is considered to be the most physiologic of all stimuli used for challenge testing. Several international guidelines have described the indications for exercise testing, and these include (i) making a diagnosis of exercise-induced bronchoconstriction (EIB) in asthmatic patients with a history of breathlessness during or after exertion; (ii) evaluating the ability to perform demanding or lifesaving work (e.g., military, police, fire fighting) in persons with a history suggesting asthma; and (iii) determining the effectiveness and optimal dosing of medications prescribed to prevent EIB and evaluating the effects of antiinflammatory treatment (Sterk et al. 1993; American Thoracic Society 2000). Eucapnic voluntary hyperpnea of dry air mimics exercise and has the same clinical significance. In contrast to exercise challenge, dose–response curves can be constructed. Hypertonic saline challenge is easy to perform and allows construction of a dose–response curve. The principal indications for this challenge are to identify BHR consistent with active asthma and to evaluate bronchial responsiveness that will respond to treatment with antiinflammatory drugs. A

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hypertonic saline challenge is an alternative to exercise or hyperventilation for identifying patients with EIB (Anderson & Brannan 2003). It can also be used in the assessment of patients with a history of asthma who wish to scuba-dive (British Thoracic Society 2003). Hypertonic saline challenge has gained widespread use for inducing sputum, and this procedure has become an important research tool for assessing acute and chronic airway inflammation in a noninvasive fashion. The mechanisms by which sputum is induced are not known, but different mechanisms are thought to be involved, such as increased vascular permeability, stimulation of mucus production, humidification of airway mucus, and stimulation of mucociliary clearance and of the cough reflex (Djukanovic et al. 2002). So, hypertonic saline challenge is unique as a challenge tool in that it permits documentation of BHR at the same time as collecting sputum. Recently, hypertonic saline challenge has found a novel application in the noninvasive assessment of microvascular leakage in the monitoring of airway inflammation, via the model of “dual induction” (first leakage, then sputum). In this model, pharmacologic agents that induce leakage (e.g., tachykinins) are inhaled in a first step; this is then followed by sputum induction through a hypertonic saline challenge, allowing quantification of macromolecules which serve as markers of leakage (Van Rensen et al. 2002). Another hypertonic challenge is the recently developed inhalation of hypertonic mannitol dry powder, the results of which show good correlation with the other physical challenges exercise, hypertonic saline, and eucapnic voluntary hyperpnea. A major advantage of this procedure is its technical simplicity as there is no need for an energy source, nor for sophisticated machinery to aerosolize the molecule; the system is portable, which could make it very useful for epidemiologic purposes. The efficacy (in comparison to hypertonic saline) and the safety of this challenge has been demonstrated in a large population of asthmatic and nonasthmatic subjects (Brannan et al. 2005).

Conclusions BHR is a major characteristic of asthma. The methods to measure it have evolved greatly and have revealed the complexity of the airway response in patients with asthma. Measures of bronchial responsiveness are widely used for diagnosis and monitoring of asthma (Table 35.5). A wide array of nonspecific bronchoconstrictor stimuli is available. Methacholine and histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Indirect challenges (adenosine, exercise, hypertonic saline, etc.) induce airflow limitation by an action on cells other than smooth muscle cells. Whereas bronchial responsiveness to a direct stimulus is only weakly related to airway inflammation, indirect airway challenges might better reflect active

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airway inflammation. Both direct and indirect airway challenges are useful outcome parameters in clinical studies of asthma. Bronchial challenges are also an essential step in the development of some of the potential new antiasthma treatments, such as adenosine or tachykinin receptor antagonists.

References Alexander, H.L. & Paddock, R. (1921) Bronchial asthma: response to pilocarpine and epineprine. Arch Intern Med 27, 184–91. American Thoracic Society (2000) Guidelines for methacholine and exercise challenge testing: 1999. Am J Respir Crit Care Med 161, 309– 29. Anderson, S.D. & Brannan, J.D. (2003) Methods for “indirect” challenge tests including exercise, eucapnic voluntary hyperpnea, and hypertonic aerosols. Clin Rev Allergy Immunol 24, 27–54. Anderson, S.D. & Daviskas, E. (2000) The mechanism of exerciseinduced asthma is . . . J Allergy Clin Immunol 106, 453–9. Anderson, S.D., Brannan, J., Spring, J. et al. (1997) A new method for bronchial-provocation testing in asthmatic subjects using a dry powder of mannitol. Am J Respir Crit Care Med 156, 758–65. Beaupré, A. & Malo, J.L. (1979) Comparison of histamine bronchial challenges with the Wright nebulizer and the dosimeter. Clin Allergy 9, 575– 83. Boulet, L.P. (2003) Asymptomatic airway hyperresponsiveness. A curiosity or an opportunity to prevent asthma? Am J Respir Crit Care Med 167, 371–8. Bousquet, J., Jeffery, P.K., Busse, W.W., Johnson, M. & Vignola, A.M. (2000) Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 161, 1720–45. Brannan, J.D., Anderson, S.D., Perry, C.P., Freed-Martens, R., Lassig, A.R. & Charlton, B. (2005) The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline. Respir Res 6, 144. British Thoracic Society (2003) British Thoracic Society guidelines on respiratory aspects of fitness for diving. Thorax 58, 3–13. Brusasco, V., Crimi, E. & Pellegrino, R. (1998) Airway hyperresponsiveness in asthma: not just a matter of airway inflammation. Thorax 53, 992–8. Chai, H., Farr, R.S., Froehlich, L.A. et al. (1975) Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol 56, 323–7. Cockcroft, D.W. (2001) How best to measure airway responsiveness. Am J Respir Crit Care Med 163, 1514–15. Cockcroft, D.W., Killian, D.N., Mellon, J.J. & Hargreave, F.E. (1977) Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 7, 235– 43. Curry, J.J. (1947) Comparative action of acetyl-beta-methyl choline and histamine on the respiratory tract in normals, patients with hay fever, and subjects with bronchial asthma. J Clin Invest 26, 430–8. De Meer, G., Heederik, D. & Postma, D.S. (2002) Bronchial responsiveness to adenosine 5′-monophosphate (AMP) and methacholine differ in their relationship with airway allergy and baseline FEV(1). Am J Respir Crit Care Med 165, 327–31. De Vries, K., Goei, J., Booy-Noord, H. & Orie, N. (1962) Changes during 24 hours in the lung function and histamine hyperreactivity of

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induced bronchoconstriction in asthma patients. Eur Respir J 23, 76– 81. Juniper, E.F., Frith, P.A. & Hargreave, F.E. (1981) Airway responsiveness to histamine and methacholine – relationship to minimum treatment to control symptoms of asthma. Thorax 36, 575–9. Kharitonov, S.A. & Barnes, P.J. (2000) Clinical aspects of exhaled nitric oxide. Eur Respir J 16, 781– 92. Lötvall, J., Inman, M. & O’Byrne, P. (2000) Measurement of airway hyperresponsiveness: new considerations. Thorax 53, 419–24. Louis, R., Sele, J., Henket, M. et al. (2002a) Sputum eosinophil count in a large population of patients with mild to moderate steroidnaive asthma: distribution and relationship with methacholine bronchial hyperresponsiveness. Allergy 57, 907–12. Louis, R.E., Cataldo, D., Buckley, M.G. et al. (2002b) Evidence of mast-cell activation in a subset of patients with eosinophilic chronic obstructive pulmonary disease. Eur Respir J 20, 325–31. Mullen, J.B., Wiggs, B.R., Wright, J.L., Hogg, J.C. & Pare, P.D. (1986) Nonspecific airway reactivity in cigarette smokers. Relationship to airway pathology and baseline lung function. Am Rev Respir Dis 133, 120–5. O’Connor, B.J., Towse, L.J. & Barnes, P.J. (1996) Prolonged effect of tiotropium bromide on methacholine-induced bronchoconstriction in asthma. Am J Respir Crit Care Med 154, 876– 80. Orehek, J., Nicoli, M.M., Delpierre, S. & Beaupre, A. (1981) Influence of the previous deep inspiration on the spirometric measurement of provoked bronchoconstriction in asthma. Am Rev Respir Dis 123, 269–72. Pauwels, R., Joos, G. & Van der Straeten, M. (1988) Bronchial hyperresponsiveness is not bronchial hyperresponsiveness is not bronchial asthma. Clin Allergy 18, 317–21. Pauwels, R.A., Joos, G.F. & Kips, J.C. (1995) Leukotrienes as therapeutic target in asthma. Allergy 50, 615–22. Polosa, R. (2002) Adenosine-receptor subtypes: their relevance to adenosine-mediated responses in asthma and chronic obstructive pulmonary disease. Eur Respir J 20, 488–96. Polosa, R., Rorke, S. & Holgate, S.T. (2002) Evolving concepts on the value of adenosine hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Thorax 57, 649–54. Prosperini, G., Rajakulasingam, K., Cacciola, R.R. et al. (2002) Changes in sputum counts and airway hyperresponsiveness after budesonide: monitoring anti-inflammatory response on the basis of surrogate markers of airway inflammation. J Allergy Clin Immunol 110, 855–61. Rasmussen, F., Lambrechtsen, J., Siersted, H.C., Hansen, H.S. & Hansen, N.C. (1999) Asymptomatic bronchial hyperresponsiveness to exercise in childhood and the development of asthma related symptoms in young adulthood: the Odense Schoolchild Study. Thorax 54, 587–9. Reynolds, C.J., Togias, A. & Proud, D. (2002) Airways hyper-responsiveness to bradykinin and methacholine: effects of inhaled fluticasone. Clin Exp Allergy 32, 1174–9. Rorke, S., Jennison, S., Jeffs, J.A., Sampson, A.P., Arshad, H. & Holgate, S.T. (2002) Role of cysteinyl leukotrienes in adenosine 5′monophosphate induced bronchoconstriction in asthma. Thorax 57, 323–7. Rosi, E. & Scano, G, (2000) Association of sputum parameters with clinical and functional measurements in asthma Thorax 55, 235–8. Rosi, E., Ronchi, M.C., Grazzini, M., Duranti, R. & Scano, G. (1999) Sputum analysis, bronchial hyperresponsiveness, and airway

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function in asthma: results of a factor analysis. J Allergy Clin Immunol 103, 232– 7. Ryan, G., Dolovich, M.B., Obminski, G. et al. (1981) Standardization of inhalation provocation tests: influence of nebulizer output, particle size, and method of inhalation. J Allergy Clin Immunol 67, 156– 61. Salome, C.M., Peat, J.K., Britton, W.J. & Woolcock, A.J. (1987). Bronchial hyperresponsiveness in two populations of Australian schoolchildren. I. Relation to respiratory symptoms and diagnosed asthma. Clin Allergy 17, 271– 81. Schelfhout, V., Louis, R., Lenz, W., Heyrman, R., Pauwels, R. & Joos, G. (2006) The triple neurokinin-receptor antagonist CS-003 inhibits neurokinin A-induced bronchoconstriction in patients with asthma. Pulm Pharmacol Ther 19, 413–18. Smith, A.D., Cowan, J.O., Brassett, K.P., Herbison, G.P. & Taylor, D.R. (2005) Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 352, 2163–73. Sont, J.K., Han, J., van Krieken, J.M. et al. (1996) Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids. Thorax 51, 496– 502. Sont, J.K., Willems, L.N., Bel, E.H., van Krieken, J.H., Vandenbroucke, J.P. & Sterk, P.J. (1999) Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. The AMPUL Study Group. Am J Respir Crit Care Med 159, 1043– 51. Sterk, P.J., Daniel, E.E., Zamel, N. & Hargreave, F.E. (1985) Limited maximal airway narrowing in nonasthmatic subjects. Role of neural control and prostaglandin release. Am Rev Respir Dis 132, 865– 70. Sterk, P.J., Fabbri, L.M., Quanjer, Ph.H. et al. (1993) Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Report Working Party Standardization of Lung Function Tests. Eur Respir J 6 (suppl. 16), 53–83. Tashkin, D.P., Altose, M.D., Bleecker, E.R. et al. (1992) The lung health study: airway responsiveness to inhaled methacholine in smokers with mild to moderate airflow limitation. The Lung Health Study Research Group. Am Rev Respir Dis 145, 301–10. Tiffeneau, R. & Beauvallet, M. (1945) Epreuve de bronchoconstriction et de bronchodilation par aerosols. Bull Acad Med 129, 165–8. van den Berge, M., Meijer, R.J., Kerstjens, H.A. et al. (2001a) Pc(20) adenosine 5’-monophosphate is more closely associated with airway inflammation in asthma than pc(20) methacholine. Am J Respir Crit Care Med 163, 1546– 50. van den Berge M., Kerstjens, H.A., Meijer, R.J. et al. (2001b) Corticosteroid-induced improvement in the PC20 of adenosine monophosphate is more closely associated with reduction in airway inflammation than improvement in the PC20 of methacholine. Am J Respir Crit Care Med 164, 1127–32. van den Berge M., Kerstjens, H.A. & Postma, D.S. (2002) Provocation with adenosine 5′-monophosphate as a marker of inflammation

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in asthma, allergic rhinitis and chronic obstructive pulmonary disease. Clin Exp Allergy 32, 824–30. van den Toorn, L.M., Prins, J.B., Overbeek, S.E., Hoogsteden, H.C. & de Jongste, J.C. (2000) Adolescents in clinical remission of atopic asthma have elevated exhaled nitric oxide levels and bronchial hyperresponsiveness. Am J Respir Crit Care Med 162, 953–7. van den Toorn, L.M., Overbeek, S.E., de Jongste, J.C., Leman, K., Hoogsteden, H.C. & Prins, J.B. (2001) Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Crit Care Med 164, 2107–13. Van Rensen, E.L., Hiemstra, P.S., Rabe, K.F. & Sterk, P.J. (2002) Assessment of microvascular leakage via sputum induction: the role of substance P and neurokinin A in patients with asthma. Am J Respir Crit Care Med 165, 1275–9. Van Schoor, J., Joos, G.F., Kips, J.C., Drajesk, J.F., Carpentier, P.J. & Pauwels, R.A. (1997) The effect of ABT-761, a novel 5–lipoxygenase inhibitor, on exercise- and adenosine-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med 155, 875–80. Van Schoor, J., Joos, G.F., Chasson, B.L., Brouard, R.J. & Pauwels, R.A. (1998) The effect of the NK2 tachykinin receptor antagonist SR 48968 (saredutant) on neurokinin A-induced bronchoconstriction in asthmatics. Eur Respir J 12, 17–23. Van Schoor, J., Joos, G.F. & Pauwels, R.A. (2000) Indirect bronchial hyperresponsiveness in asthma: mechanisms, pharmacology and implications for clinical research. Eur Respir J 16, 514–33. Van Schoor, J., Joos, G. & Pauwels, R.A. (2002) Effect of inhaled fluticasone propionate on bronchial responsiveness to neurokinin A in asthma. Eur Respir J 18, 997–1002. Van Schoor, J., Pauwels, R. & Joos, G. (2005) Indirect bronchial hyperresponsiveness: the coming of age of a specific group of bronchial challenges. Clin Exp Allergy 35, 250–61. van Velzen, E., van den Bos, J.W., Benckhuijsen, J.A., van Essel, T., de Bruijn, R. & Aalbers, R. (1996) Effect of allergen avoidance at high altitude on direct and indirect bronchial hyperresponsiveness and markers of inflammation in children with allergic asthma. Thorax 51, 582–4. Vizi, E., Huszar, E., Csoma, Z. et al. (2002) Plasma adenosine concentration increases during exercise: a possible contributing factor in exercise-induced bronchoconstriction in asthma. J Allergy Clin Immunol 109, 446–8. Ward, C., Pais, M., Bish, R. et al. (2002) Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma. Thorax 57, 309–16. Weiss, S., Robb, G.P. & Ellis, L.B. (1932) The systemic effects of histamine in man with special reference to the cardiovascular system. Arch Intern Med 49, 360–9. Woolcock, A.J., Yan, K. & Salome, C.M. (1988) Effect of therapy on bronchial hyperresponsiveness in the long-term management of asthma. Clin Allergy 18, 165–76. Woolcock, A.J., Anderson, S.D., Peat, J.K. et al. (1991) Characteristics of bronchial hyperresponsiveness in chronic obstructive pulmonary disease and in asthma. Am Rev Respir Dis 143, 1438–43.

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Exercise-induced Bronchoconstriction: Animal Models Arthur N. Freed and Sandra D. Anderson

Summary Hyperventilation with cool dry air damages bronchial mucosa and increases airway resistance in all mammalian species examined to date. Although hyperventilation-induced bronchoconstriction (HIB) appears to be qualitatively similar in most animal models and closely mimics exercise-induced asthma in human subjects, species-specific differences do exist. These differences must be taken into account when extrapolating model-derived data to the human condition. Data from animal models confirm that hyperventilation with dry air stimulates the production and release of numerous bronchoactive mediators including leukotrienes, prostanoids, and tachykinins. These mediators clearly modulate the development of HIB. Airway cooling may counterbalance the constrictor effect of airway drying by inhibiting either mediator release or the neuronal activity that accompanies HIB. Various animal models have shown that HIB and bronchovascular hyperpermeability develop simultaneously, making it likely that they also co-occur in humans. However, animal and human studies indicate that bronchovascular leakage and HIB result from independent mechanisms. Although the role of airway edema formation in the development of HIB remains unclear, the former may amplify the effects of smooth muscle contraction. Animal model data confirm human studies implicating the existence of a hyperventilation-induced late-phase response. Delayed airway obstruction develops as a direct result of acute airway injury and mediator release. Both airway hyperosmolarity and airway cooling appear necessary for all aspects of this late response to develop. Canine model data also reveal that an acute hyperventilation-induced mucosal injury is likely to heal quickly, whereas repeated hyperventilation with cold dry air impairs the repair process, and results in persistent inflammation. Chronic inflammation promotes airways remodeling in this model, and results in an asthma-like condition. Thus, repetitive exposure to cold dry air may be one of many environmental factors that contribute to the development of asthma in normal human subjects.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Animal models of exercise-induced bronchoconstriction Isocapnic hyperventilation-induced bronchoconstriction (HIB) occurs in all mammalian species examined to date (Jammes et al. 1983; Chapman & Danko 1985; Freed et al. 1985; Biagini et al. 1991; Koyama et al. 1992; Yang et al. 1999a), reflecting a conserved physiologic mechanism for limiting access of environmental irritants to the distal lung. Smooth muscle contraction enhances airway barrier function by reducing transepithelial conductance (Freed & Croxton 1993). This mechanism may provide additional protection to subepithelial tissues from environmental insult. Hyperventilation with cold dry air elicits qualitatively similar responses from normal and asthmatic subjects (O’Cain et al. 1980; Therminarias et al. 1998) (Fig. 36.1a). This suggests that the mechanisms directly contributing to the development of HIB are present in the normal population. Although HIB in normal individuals involves very small changes in forced expiratory volume in 1 s (FEV1) (4% on average), the exaggerated response measured in asthmatic individuals probably reflects smooth muscle sensitivity to inflammatory mediators released in response to hyperpnea. Whole-lung animal models are those in which dry air or a dry gas mixture bypasses the upper airway and directly enters the lower trachea via a tube. Guinea pigs are the most commonly used species (Chapman & Danko 1985; Ray et al. 1988; Nagase et al. 1996; Lai & Lee 2000), although rats (Yang et al. 1999a), rabbits (Koyama et al. 1992; Yuan & Nail 1995; Högman et al. 1997), cats (Jammes et al. 1983), and monkeys (Biagini et al. 1991) have also been studied with varying levels of success. The main criticisms of these models are that (i) they are anesthetized and mechanically ventilated during hyperventilation, (ii) the condition of the air as it passes from the tracheal tube to the lung is not the same as if it passed over the upper airway mucosa, and (iii) the tidal volumes and frequencies used during hyperventilation do not truly mimic natural breathing conditions. In addition to whole-lung models (Fig. 36.1b,c), a sublobar airway model is used in which canine peripheral airways are isolated and hyperventilated via a bronchoscope (Freed et al.

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same magnitude of respiratory heat and water loss as smaller increases in ventilation with cold air. Finally, racehorses and sled dogs are two natural models that can be used to study HIB. Racehorses routinely increase minute ventilation 20-fold or greater during strenuous exercise, often under subfreezing conditions (Davis et al. 2002a). Racing sled dogs (Davis et al. 2002b), which can sustain speeds as high as 25 km/hour (Van Citters & Franklin 1969), are capable of running great distances in extremely cold and desiccating environments. Both species exhibit hyperventilationinduced mucosal injury and inflammation similar to that seen in asthmatic subjects.

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Fig. 36.1 Hyperventilation-induced bronchoconstriction in mammals. (a) Asthmatic (closed circles) (Finnerty et al. 1992) and normal nonatopic (O’Cain et al. 1980) humans (open circles). Asthmatic subjects hyperventilated with room temperature air. Normal subjects hyperventilated with either saturated room temperature air at 123 L/min (1), dry subfreezing air at 61 L/min (2), or dry subfreezing air at 123 L/min (3). (b) Guinea pigs: hyperventilated with dry gas containing 95% O2 (closed circles) (data from Ray et al. 1988); hyperventilated with dry gas containing 21% O2 (open circles) (data from Chapman & Danko 1985). (c) Rabbits: sensitized to ovalbumin (closed circles); non-sensitized (open circles) (data from Koyama et al. 1992). (d) Dogs: hyperventilated with 2000 mL/min for 5 min (closed circles) (data from Freed & Adkinson 1990); hyperventilated with 1500 mL/min for 2 min (open circles) (data from Freed et al. 1994b). RL, pulmonary resistance; Rp, peripheral airway resistance; FEV1, forced expiratory volume in 1 s; Vmax 30% VC, maximum flow at 30% of vital capacity. Unless stated, isocapnic hyperventilation was done with either dry room air or 5% CO2 and dry air.

1985) (Fig. 36.1d). In this model, peripheral airway resistance typically increases between 50 and 200% above baseline, and the severity of HIB increases with increasing rates of ventilation (Freed et al. 1987a; Freed 1989). A major criticism of this model is that airflow is unidirectional, eliminating a primary heat and water recovery mechanism that functions under normal conditions in all mammals including humans. However, unidirectional hyperventilation simply increases the evaporative cooling and water loss at any given location in the airway when compared to normal ventilation (Daviskas et al. 1991). This is analogous to increasing tidal volume in whole-lung models to increase the penetration of dry air into the distal lung (Ray et al. 1990). In fact, air (or airway) temperatures in human airways recorded during and after normal hyperventilation with subfreezing air are similar to those recorded in similar-sized canine bronchi exposed to unidirectional hyperventilation with room temperature gas (McFadden et al. 1985; Freed et al. 1987a). Thus local heat and water fluxes created by these two modes of hyperventilation can be similar. Equivalent losses can be generated by brief periods of hyperventilation with cold air and prolonged periods of hyperventilation with warm air. Similarly, big increases in ventilation with warm air can approximate the

Early response to hyperventilation with dry air HIB usually develops and resolves within 30–60 min. Maximal narrowing typically occurs 2–10 min after hyperpnea ends in rats (Yang et al. 1999a), guinea pigs (Ray et al. 1991), rabbits (Koyama et al. 1992), cats (Jammes et al. 1983), dogs (Freed et al. 1989), and human (Blackie et al. 1990) (see Fig. 36.1). This delay may result from either thermal (Freed et al. 1987b, 1991) or mechanical (Blackie et al. 1990; Ray et al. 1991) mechanisms that modulate mediator release during hyperventilation (Freed et al. 1999). Studies in humans and dogs show that HIB is attenuated when airway cooling is sustained into the recovery period. Bronchoconstriction develops slowly and mediator release is delayed during exposure to cool dry air, as opposed to warm humid air, suggesting that cooling affects mediator release during hyperpnea (Freed et al. 1999). These events can account for the slow rise in airway resistance that characterizes this early response. Thus, exercise-induced bronchoconstriction (EIB) may result from an imbalance between two opposing stimuli: evaporative water loss that stimulates and evaporative cooling which inhibits the development of airway obstruction (Freed et al. 1987b). The time over which HIB develops may be species- or even strain-dependent. Of all the animal models examined to date, the rabbit appears to be less sensitive to hyperpnea than guinea pigs, dogs, and asthmatic humans (Fig. 36.1), whereas recovery appears to occur more rapidly in cats (Jammes et al. 1983). The one study examining the effect of cold dry air in monkeys suggests that obstruction develops slowly, with maximal changes in pulmonary resistance appearing about 25 min after the challenge (Biagini et al. 1991). Brown Norway rats appear to require even longer periods for obstruction to develop, whereas August-Copenhagen Irish (ACI) rats exhibit an unusual biphasic peak in airway obstruction (Yang et al. 1999a). However, full recovery of normal lung function was not reported for rat or monkey models, and the unusually long recovery times may result from mechanisms unrelated to HIB. HIB in normal human subjects is associated with mild central airway narrowing (O’Cain et al. 1980), whereas EIB is associated with severe peripheral airway narrowing (Wessling & Wouters 1992). Mild hyperpnea in guinea pigs elicits central

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airway narrowing, whereas more severe levels produce peripheral airway narrowing (Ray et al. 1990; Nagase et al. 1994). Thus, penetration of dry air into the distal lung is a key determinant of HIB in all mammals, regardless of disease state. Studies in dogs (Freed et al. 1985) and in human subjects (Kaminsky et al. 1995, 2000) confirm the direct involvement of small airways in HIB. Both smooth muscle contraction and airway edema have been proposed as the mechanism for the airway narrowing of HIB. β2-Adrenoreceptor agonists inhibit or abolish HIB in guinea pigs (Chapman & Danko 1985), rabbits (Horie et al. 1992), dogs (Teeter & Freed 1991; Wang et al. 1992), and humans (Anderson & Schoeffel 1982). They do so by inhibiting mediator release and reducing smooth muscle responsiveness, and by enhancing water replacement to the airway and its surface during hyperpnea in guinea pigs and dogs (Wang et al. 1992; Yang et al. 1999b). The efficacy of β2 agonists in preventing HIB and the speed with which the airway obstruction develops and subsides (Freed et al. 1985; Blackie et al. 1990; Ray et al. 1991; Koyama et al. 1992) (Fig. 36.1) favors mediator-induced smooth muscle contraction over airway edema as the primary cause of HIB. The parasympathetic nervous system modulates HIB in some but not all asthmatic humans (Finnerty & Holgate 1993). Although parasympathetic activity plays no role in the guinea-pig model (Chapman & Danko 1985; Ray et al. 1988), it does so to varying degrees in rabbits (Horie et al. 1992; Yuan & Nail 1995), cats (Jammes et al. 1983), and dogs (Freed et al. 1987a, 2000; Tang & Freed 1992). Blocking parasympathetic activity reduces HIB in dogs and abolishes it in rabbits and cats, making the latter two models ideal for studying the vagal component of this phenomenon. Engorgement and leakage of bronchial vessels are also likely to contribute to HIB (McFadden 1990). Hyperpnea with dry air causes vasodilation in dogs (Omori et al. 1995a) and sheep (Parsons et al. 1989); vascular hyperpermeability in rats (Yang et al. 1999a), guinea pigs (Fig. 36.2a,b), and dogs (Fig. 36.2c,d); and airway edema in rabbits (Högman et al. 1997). These phenomena are also likely to occur in human subjects. Although animal data reveal that airway edema and not bronchovascular engorgement causes airway obstruction in response to volume loading (Tang & Freed 1994), neither directly contributes to the development of HIB. HIB and bronchovascular hyperpermeability develop simultaneously (Garland et al. 1991; Freed et al. 1994a; Yang et al. 1997), and the latter persists for at least 24 hours in the canine model (Omori et al. 1995b). It is unlikely that this prolonged vascular response is responsible for HIB. Bronchial hyperpermeability induced by hyperpnea is unaffected by eicosanoid and tachykinin antagonists in guinea pigs, or by β2-receptor agonists in dogs despite their ability to inhibit HIB (Garland et al. 1993; Solway et al. 1993; Omori et al. 1995a; Yang et al. 1997; Lai & Lee 1999). Leukotriene and tachykinin antagonists do inhibit bronchial hyperpermeability

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Fig. 36.2 Bronchovascular leakage in unchallenged control animals (Cont, open bar) and animals hyperventilated with either warm moist air (WAC, light blue bar) or cool dry air (DAC, dark blue bar). (a) Guinea pig main bronchi; (b) guinea pig lower bronchi (data from Garland et al. 1991). (c) Canine peripheral airways (regardless of size); (d) canine peripheral airways (as a function of airway diameter) (data from Freed et al. 1994a). Bronchovascular leakage is expressed as extravasation of either Evans blue dye, monastral blue, or carbon black. The latter two colloids are “vascular labels.” **, P < 0.01 compared to control.

in rats, but this effect is likely to be strain dependent (Yang et al. 1999a). Morphometric analysis of guinea-pig airways confirmed that smooth muscle contraction and not airway edema was responsible for HIB (Nagase et al. 1994). Finally, ligation of the bronchial artery prior to hyperpnea abolishes microvascular leakage in the canine model without affecting bronchoconstriction (Freed et al. 1995), demonstrating that the bronchial circulation plays little if any role in HIB. Bronchovascular leakage may protect airway mucosa from desiccation injury (Persson et al. 1991), the latter resulting in mucus secretion, goblet cell degranulation, microvascular hyperpermeability, and smooth muscle contraction in canine airways (Freed et al. 1994a; Omori et al. 1995b) (Fig. 36.3). Smooth muscle contraction may provide protection by narrowing the bronchial lumen, inhibiting cold air penetration, and decreasing the mucosal surface exposed to desiccation. Microvascular leakage may account for the enhanced mucociliary clearance reported in normal and asthmatic subjects (Daviskas et al. 1995, 1996), and in turn may enhance the removal of bronchoactive mediators (Kelly et al. 1986; Wagner & Mitzner 1990). Finally, the fact that furosemide, norepinephrine, and methoxamine each enhance bronchovascular

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Normal epithelium

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IL-2 IL-4 IL-6 IL-8 IL-16 NO LTB4 EO GM-CSF RANTES

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Exercise-induced Bronchoconstriction: Animal Models

a-agonists

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Fig. 36.3 Schematic of proposed cascade of events that produce hyperventilation-induced bronchoconstriction, late-phase airway obstruction, and airway remodeling. Ach, acetylcholine; ASL, airway surface liquid; CAP, CAP37/azurocidin; CGRP, calcitonin gene-related peptide; Ciliated, ciliated epithelium; CNS, central nervous system; Def-1 and Def-2, defensins HNP-1 and HNP-2; Endo, endothelin; EO, eotaxin; Eos, eosinophil; Goblet, goblet cell; GM-CSF, granulocyte–macrophage colonystimulating factor; Heat/H2O, loss of heat and water from airway surface

and vasculature; IL, interleukin; LTs, leukotrienes; Mast, mast cell; Macro, macrophage; MMP-9, matrix metalloproteinase-9; NO, nitric oxide; PGs, prostaglandins; PMN, neutrophil; RANTES, a chemokine “Regulated by Activation, Normal T-cell Expressed and Secreted”; NANC, nonadrenergic noncholinergic nervous system; NKA, neurokinin A; SP, substance P; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a; Tx, thromboxane; VIP, vasoactive intestinal polypeptide. (See CD-ROM for color version.)

leakage induced by hyperpnea in dogs (Freed et al. 1996, 1997) suggests that they may serve to counterbalance the effects of evaporative water loss. Although unknown, even small changes in microvascular permeability may be sufficient to compensate for water loss during exercise in cold environments.

ments in dogs revealed that ASL osmolarity of the peripheral airways does increase during hyperventilation with dry air (Freed & Davis 1999). This increase in osmolarity is thought to initiate the synthesis and release of bronchoactive mediators (Fig. 36.4). The extent of bronchoconstriction produced by hyperventilation correlates with that produced by hypertonic saline in asthmatic humans (Belcher et al. 1989; Smith & Anderson 1989) and dogs (Freed et al. 1989), and supports the hypothesis that ASL hyperosmolarity initiates HIB (Anderson et al. 1982). Hypertonicity triggers mediator release in vitro from mast cells (Eggleston et al. 1987) and tachykinin release from cultured C-fiber neurons (Garland et al. 1995). Hyperventilation increases in vivo eicosanoid metabolism in guinea pigs, dogs, and asthmatic subjects (Fig. 36.4). Dietary salt loading in guinea pigs (Mickleborough et al. 2001) and asthmatic subjects (Mickleborough et al. 2005) also elevates mediator activity and enhances HIB, confirming the modulatory role of hypertonicity in the development of this response.

Role of airway surface liquid osmolarity Indirect estimates of hyperventilation- or exercise-induced airway water flux and airway surface liquid (ASL) osmolarity have provided contradictory results (Anderson et al. 1989; McFadden et al. 1999). Although subfreezing air penetrates to at least the level of fifth-generation bronchi during hyperventilation (McFadden et al. 1985), direct measurement of ASL osmolarity failed to detect any postchallenge changes in either normal (Kotaru et al. 2002) or asthmatic (Kotaru et al. 2003) human subjects. Unfortunately, ASL samples were obtained from the trachea, and as such are inappropriate to detect desiccation of the peripheral airways. However experi-

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* *

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Fig. 36.4 Mediators in bronchoalveolar lavage fluid recovered from unchallenged control animals (open bar) and from animals hyperventilated with either warm moist air (light blue bar) or cool dry air (dark blue bar). (a) Guinea pigs (data from Ingenito et al. 1990, 1992). (b) Guinea pigs (data from Lai & Lee 1999). (c) Canine peripheral airways (data from Freed et al. 1999). (d) Asthmatic human subjects (data from Pliss et al. 1990). LTB4, leukotriene B4; LTE4, leukotriene E4; LTC4-E4, leukotrienes C4, D4, and E4; PGF2a, prostaglandin F2a; SP, substance P; TxB2, thromboxane B2. *, P < 0.05; **, P < 0.01 compared to control.

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Mucosal injury following hyperventilation was originally documented using bronchoalveolar lavage fluid cell analyses in guinea pigs, dogs, horses, and asthmatic subjects (Fig. 36.5), and more recently using similar analyses of sputum induced after exercise challenge in human subjects with EIB (Hallstrand et al. 2005). Morphometric analyses of hyperventilated canine airways confirmed that these sampling techniques accurately reflect injury of the bronchial mucosa (Freed et al. 1994a; Omori et al. 1995b). Warming and humidifying the air reduces bronchoconstriction in rats (Yang et al. 1999a), guinea pigs (Ray et al. 1988; Yang et al. 1997), rabbits (Ohtsuka et al. 1993), dogs (Freed et al. 1985, 1987a), and human (Anderson et al. 1982; Stensrud et al. 2006), and nearly eliminates all evidence of airway mucosal injury (Freed et al. 1987c, 1994a, 1999). β2 agonists also protect the bronchial mucosa from injury (Omori et al. 1995a). In contrast to fully or partially conditioned air that reduces the strength of the stimulus, β2 agonists probably decrease desiccation and injury by increasing tramucosal fluid flux (Davis et al. 1979; Wang et al. 1992). Mucosal injury appears to initiate airway narrowing by the release of mediators, and the magnitude of mediator release is directly related to the severity of mucosal injury (Omori et al. 1995a,b; Freed et al. 1999). In canine airways the injury occurs before the release of eicosanoids. This release occurs during or soon after hyperventilation ends (Freed et al. 1999). Thus endogenous release of mediators is unlikely to cause mucosal injury during hyperventilation, although it is possible that mediators do contribute to the mucosal injury. Because hyperventilation with fully conditioned air precludes mucosal injury and inhibits bronchoconstriction (Freed et al.

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Fig. 36.5 Epithelial cells in bronchoalveolar lavage fluid recovered from unchallenged control animals (Cont, open bar) and animals hyperventilated with either warm moist air (WAC, light blue bar) or cool dry air (DAC, dark blue bar). (a) Guinea pig (data from Ingenito et al. 1990). (b) Canine peripheral airways (data from Freed et al. 1999). (c) Horse (data from Davis et al. 2002a). (d) Asthmatic human (data from Pliss et al. 1990). *, P < 0.05; **, P < 0.01 compared to control.

1985, 1994a, 1999), it is unlikely that the mechanical stress developed during hyperventilation causes mucosal injury. Finally, hypertonicity per se does not cause mucosal injury in either dogs or asthmatic subjects (Freed et al. 1994b; Makker

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et al. 1994). Thus the mechanism that results in mucosal injury remains unknown.

Hyperventilation-induced mediator production and release Hyperventilation in dogs (Omori et al. 1995b) and exercise in humans (Crimi et al. 1992) causes mast cell degranulation, and represents direct evidence that mediators contribute to the development of HIB (Fig. 36.3). Mediator release typically occurs 2–5 min after hyperventilation stops (Freed et al. 1999). Although this delay may simply reflect de novo biosynthesis, the temperature change that occurs during hyperventilation may retard mediator release. While hyperventilation with warm humid air inhibits HIB in dogs, the residual obstruction develops earlier, a finding consistent with the reduced amount of cooling that occurs during that challenge (Freed et al. 1999). The fact that hypertonic saline aerosol produces bronchoconstriction more rapidly than either HIB in dogs (Freed et al. 1991) or HIB and EIB in asthmatic subjects (Boulet & Turcotte 1989; Smith & Anderson 1989) supports the hypothesis that cooling per se retards mediator release. Eicosanoids appear to play a significant role in the development of EIB. Numerous studies have shown that leukotrienes modulate HIB in human subjects (van Schoor et al. 1997; Kemp et al. 1998; Lehnigk et al. 1998) (Fig. 36.4d) and in animal models including rats (Yang et al. 1999a), guinea pigs (Garland et al. 1993; Yang et al. 1997; Lai & Lee 2000; Mickleborough et al. 2001) (Fig. 36.4a,b), and dogs (Omori et al. 1996; Freed et al. 2000) (Fig. 36.4c). By comparison, the role of prostanoids in HIB has received far less attention. Hyperpnea-induced prostanoid activity reported in guinea pig (Garland et al. 1993; Yang et al. 1997; Suman et al. 2000) (Fig. 36.4a) and canine models (Freed et al. 1987a,c, 2000; Omori et al. 1996) (Fig. 36.4c) mirror results from similar human studies (Shimizu et al. 1997; O’Sullivan et al. 1998) (Fig. 36.4d), implicating products of cyclooxygenase activity in the development of HIB. Pharmacologic studies indicate that approximately half of the airway narrowing that occurs in response to hyperventilation can be independently attributed to prostanoid and leukotriene activity in the canine model (Freed et al. 1987a,c; Omori et al. 1996) and in asthmatic human subjects (Adelroth et al. 1997; Reiss et al. 1997; Shimizu et al. 1997; van Schoor et al. 1997). However, inhibition of cyclooxygenase and 5lipoxygenase simultaneously provides no more protection than when blocking either pathway alone (Freed et al. 2000). This suggests that these eicosanoids act in series (i.e., either leukotrienes stimulate prostaglandin activity or vice versa) and not in parallel to produce HIB. Eicosanoid mediators released in response to hyperpnea may act directly or indirectly on airway smooth muscle to initiate airway narrowing. Neurokinin (NK) receptor activity modulates HIB in guinea pigs (Ray et al. 1989; Higashide et al. 1997; Anthes et al. 2002) and dogs (Freed et al. 2003), which in turn stimulates afferent C-fibers to locally release tachykinins

Exercise-induced Bronchoconstriction: Animal Models

(Fang & Lai 1993). Initially it was proposed that leukotrienes directly mediated HIB via the secondary release of neuropeptides (Garland et al. 1993; Lai & Lee 1999). Later it was suggested that dry gas directly stimulated tachykinin release, which in turn stimulated leukotriene activity (Yang et al. 1997). The fact that leukotriene antagonists inhibit tachykinin release induced by hyperpnea in guinea pigs (Lai & Lee 1999, 2000) and NK1 and NK2 antagonists fail to inhibit eicosanoid production in dogs (Freed et al. 2003) strongly supports the initial hypothesis. However, the use of hyperoxic gas during hyperventilation (Ray et al. 1988; Yang et al. 1999a; Lai & Lee 2000) may exaggerate the role of tachykinins in guinea pig and rat models because reactive oxygen species produced during hyperventilation enhance tachykinin release and bronchoconstriction (Fang & Lai 1993; Lai et al. 2002). This, and the fact that NK receptor antagonists do not abolish HIB in either guinea pigs (Solway et al. 1993) or dogs (Freed et al. 2003), suggests that other pathways are activated during hyperventilation (Fig. 36.3). Nitric oxide (NO) has received considerable attention for its potential role in HIB. NO was reported to have little effect on HIB in guinea pigs (Nogami et al. 1998) and asthmatic subjects during and after exercise (Suman & Beck 2002). Other studies concluded that endogenous NO did not inhibit EIB (De Gouw et al. 2001) and markedly augmented HIB (Kotaru et al. 2001). In contrast to these findings, hyperpnea-induced ASL hyperosmolarity inhibits NO-induced relaxation of airway smooth muscle in rabbits (Hogman et al. 1998), and endogenous NO inhibits HIB in dogs (Suzuki & Freed 2000a) and in normal human subjects (Therminarias et al. 1998). Although further study is clearly necessary before any conclusions can be made, endogenous NO production is likely to modulate hyperventilation-induced responses in animal and human subjects. Finally, other inhibitory mediators appear to be activated in response to hyperventilation (Fig. 36.3). PGE2 is inversely correlated with the magnitude of HIB (Omori et al. 1996), supporting the hypothesis that inhibitory mediators counterbalance the development of HIB. Generation of inhibitory prostanoids in asthmatic subjects during hyperpnea supports this scenario (Pavord et al. 1992; Manning et al. 1993). In addition, hyperpnea stimulates release of vasoactive intestinal peptide (VIP) (Tang & Freed 1992) in dogs and calcitonin gene-related peptide (CGRP) in guinea pigs (Nohr et al. 1995; Nagase et al. 1996), and both may modulate HIB in concert with inhibitory prostanoids.

Late-phase airway obstruction in animals Unlike human subjects (Speelberg et al. 1991; Koh et al. 1994; Chhabra & Ojha 1998), the late response to hyperventilation (which is characterized by leukocyte infiltration, mediator release, airway obstruction, and airway remodeling) is readily

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elicited in the canine model. Isocapnic hyperventilation produces airway obstruction in normal canine bronchi within 5 hours after challenge (Freed & Adkinson 1990) that persists for as long as 24 hours after hyperventilation (Davis & Freed 1999, 2001). The magnitude of this late-phase airway obstruction is approximately the same as the initial early response in dogs (Freed & Adkinson 1990). Hyperventilation with dry air transiently alters airway morphology (Omori et al. 1995b), and the extent of these changes probably determines if late-phase airway obstruction develops. The mucosal injury induced by hyperventilation persists throughout the late phase. Injury of the airway mucosa may result in airway obstruction due to impaired production of PGE2 and NO. Although impaired PGE2 production during the early part of the late phase is not evident (Davis et al. 2002c), endogenous NO clearly modulates latephase airway obstruction and inflammation in dogs (Suzuki & Freed 2000a). The bronchovascular leakage induced by hyperventilation (Fig. 36.2) persists throughout the late phase (Omori et al. 1995b), and may amplify the effects of smooth muscle contraction via edematous thickening of the airway wall (Tang & Freed 1994) (Figs 36.3 & 36.6b,c). Although mucosal injury could cause impaired mucociliary clearance and accumulation of intraluminal debris, data discussed below make it unlikely that impaired mucociliary clearance can account for the late increase in peripheral airway resistance seen in the canine model. Pretreatment with drugs that block either cyclooxygenase or lipoxygenase will inhibit the development of latephase obstruction (Davis et al. 2002c), indicating a role for eicosanoids in the late-phase response. The fact that neither leukotrienes nor prostaglandins increase during the late phase (Freed & Adkinson 1990; Davis et al. 2002c; Suzuki & Freed 2002) suggests that early-phase mediator release initiates late-phase airway obstruction in the canine model.

50 mm

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Late-phase airway inflammation in dogs Late-phase airway obstruction in dogs is accompanied by an influx of leukocytes (Freed & Adkinson 1990; Omori et al. 1995b; Davis et al. 2002c) (Figs 36.3, 36.6 and 36.7). Leukocyte infiltration is not dependent on eicosanoids released during or after hyperventilation, as blockade of either cyclooxygenase or lipoxygenase immediately prior to the challenge has no effect on this inflammatory response (Davis et al. 2002c). Furthermore, although hypertonic aerosol challenge causes late-phase production of eicosanoids in canine peripheral airways, it does not stimulate inflammatory cell infiltration (Suzuki & Freed 2000b), suggesting that mucosal injury is necessary to initiate this event (Fig. 36.3). Bronchial epithelial cells can produce a wide variety of proinflammatory eicosanoids and cytokines (Knobil & Jacoby 1998; Holgate et al. 2000) (Fig. 36.3), leukotriene (LT)B4 and interleukin (IL)-8 act as chemoattractants for neutrophils, and the latter stimulates neutrophils to release other chemoattractants that

800

(c) Fig. 36.6 Photomicrographs of formalin-fixed, light microscopy sections of canine bronchi. (a) Normal bronchial epithelium and lamina propria (vertical bar, i.e., distance between basement membrane and smooth muscle) from a 3.4-mm diameter airway that received repeated bronchoscopy but no challenge. (b, c) Squamous metaplasia in a 3.2-mm airway 24 hours after repetitive hyperventilation with dry air. Note thickening of the lamina propria in (b) and (c) compared with (a). Note the eosinophilic influx in (c). (Adapted from Davis et al. 2003.) (See CD-ROM for color version.)

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70 1.6

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Fig. 36.7 (a) Baseline resistance in airways repeatedly wedged but not challenged (open circles) and airways repetitively hyperventilated with dry air (closed circles). Data are expressed as mean (± SEM). *, P < 0.05 compared to day 1. (b) Bronchoalveolar lavage fluid (BALF) cell concentrations from control (open bar), repeated wedge (light blue bar), and repeatedly hyperventilated (dark blue bar) airways. Mac, macrophage; Lym, lymphocytes; PMN, neutrophils; Eos, eosinophils; Epi, epithelial cells. *, significantly different from control, P < 0.05; **, significantly different from repeated wedge, P < 0.05. (Data from Davis & Freed 1999.)

Exercise-induced Bronchoconstriction: Animal Models

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mediate T-lymphocyte infiltration (Taub et al. 1996) (Fig. 36.3). The release of IL-16 from epithelial cells and mast cells (Laberge et al. 1997; Rumsaeng et al. 1997) can selectively recruit CD4+ T cells into the airway wall, although without antigen presentation it seems unlikely that these cells would play any role in the late response. Mast cells degranulate in response to hyperventilation (Freed et al. 1994a; Omori et al. 1995b) and secrete IL-4 (Bradding et al. 1994) and IL-5 (Oliveira et al. 1996; Rumsaeng et al. 1997), the latter being a potent chemoattractant for eosinophils in asthmatic airways (Shi et al. 1997). Given the mediator-producing capability of bronchial epithelial and mast cells, a proinflammatory type-2 cytokine environment could be produced even in the absence of T-cell infiltration. The fact that Th2 cytokine expression is upregulated in horses 5 hours after breathing subfreezing air during exercise supports this hypothesis (Davis et al. 2005). The release of these mediators provides the link between the mucosal injury induced by hyperventilation and the eosinophilic inflammation (Becker & Soukup 1999) that slowly develops in human asthmatics (Crimi et al. 1992) and the canine model (Freed & Adkinson 1990; Suzuki & Freed 2002) (Fig. 36.3).

Late-phase airway hyperreactivity in dogs In dogs, but not humans, isocapnic hyperventilation with dry air and inhalation of hypertonic saline result in airway hyperresponsiveness 5– 24 hours after challenge (Davis et al. 2002c; Suzuki & Freed 2002). This increase in airway responsiveness is accompanied by increased production of eicosanoids (Suzuki & Freed 2000b, 2002; Davis et al. 2002c), which are known to contribute to the development of airway hyperreactivity in mice, dogs, and humans (Becker et al. 1995; Fischer et al. 1995; Kleeberger & Freed 1995; Nagase et al. 1997). Thus, increased eicosanoid metabolism may be responsible for the late-phase increase in airway reactivity that develops after inhalation of hypertonic saline in dogs. In contrast to hyperventilation (Freed et al. 1999; Freed & Adkinson 1990; Davis et al. 2002c), hypertonic saline challenge (Suzuki & Freed 2000b) does not cause either leukocyte infiltration or

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late-phase airway obstruction in dogs. The fact that hypertonic saline does not cause mucosal injury in either dogs or asthmatic subjects (Freed et al. 1994b; Makker et al. 1994) may account for these differences. If the main difference between a hypertonic aerosol and hyperpnea challenge is the lack of temperature change with the former stimulus, and only the latter stimulus produces a late-phase response, then unlike the early response, both airway cooling and hyperosmolarity appear to be necessary for late-phase development. Finally, late-phase airway obstruction after exercise or isocapnic hyperpnea in humans is so rare that most investigators dismiss it as experimental artifact. However, canine model data reveal that the dose–response relationship between challenge severity and HIB is distinctly different from the dose–response relationship between challenge severity and the late-phase response (Davis et al. 2002d). The early response increases at a decreasing rate with higher levels of ventilation (i.e., stimulus strength), whereas late-phase bronchoconstriction only occurs after a minimum threshold stimulus is exceeded. This observation implies that the level of ventilation attained in most human HIB and EIB studies is probably below the threshold stimulus that initiates late-phase airway obstruction. Many late-phase studies limited the maximum level of hyperpnea attained during a trial (Zawadski et al. 1988; Crimi et al. 1992; Hofstra et al. 1996), while others used only room temperature air as a stimulus (Hofstra et al. 1996). The use of subthreshold stimuli to initiate the early response may preclude the development of late-phase airway obstruction. Thus, late-phase airway obstruction may be produced if either (i) asthmatic subjects capable of achieving very high minute ventilations were selected for study and/or (ii) frigid inspired air was used for the challenge. Because the development of late-phase airway obstruction is likely to require a much stronger stimulus than the early response, it still may not be demonstrable in many human subjects. Even though cross-country skiers exercise at up to 200 L/min there are no reports of late responses in this group despite many having diagnosed asthma (Anderson et al. 2003). However, the high prevalence of chronic inflammation reported in winter

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athletes (see below) suggests that hyperventilation-induced airway inflammation is an epiphenomenon that can be readily induced, providing a more reliable indicator for late-phase study. Finally, the recognition of a threshold effect reconciles the apparent discrepancies between human and animal studies regarding the existence of the late-phase response to exercise or isocapnic hyperventilation.

Repetitive airway exposure to cold air in humans Elite athletes who exercise in cold environments exhibit an increased incidence of airway hyperreactivity and asthma (Larsson et al. 1993; Leuppi et al. 1998; Sue-Chu et al. 1999; Karjalainen et al. 2000), without a significant increase in atopy (Larsson et al. 1993; Leuppi et al. 1998; Sue-Chu et al. 1999; Karjalainen et al. 2000). These athletes typically exhibit airway inflammation, characterized by eosinophil, mast cell, and T-leukocyte infiltration, and airway remodeling (SueChu et al. 1999; Karjalainen et al. 2000). The fact that elite cold-weather athletes appear to disproportionately suffer from airway inflammation and airway hyperresponsiveness supports the concept that repeated exposure of the lung periphery to unconditioned air could result in chronic airways disease and asthma (Larsson et al. 1993; Leuppi et al. 1998; Sue-Chu et al. 1999; Karjalainen et al. 2000).

Repetitive hyperventilation-induced airway obstruction in dogs Repetitive hyperventilation over a period of days or weeks results in chronic lower airways disease. Peripheral airways in dogs anesthetized and hyperventilated every other day for 2 weeks exhibited significant obstruction after four challenges, and then stabilized at about 150% of their original airway resistance (Davis & Freed 1999) (Fig. 36.7a). Airway obstruction developed more rapidly when canine peripheral airways were challenged daily. Airway resistance was increased within 24 hours after the first challenge, and then stabilized at approximately 200% of the original value (Davis & Freed 2001).

Repetitive hyperventilation-induced airway inflammation Repeated injury of canine peripheral airways with unconditioned air results in an airway phenotype similar to asthma. Repetitive hyperventilation causes airway inflammation in dogs characterized by mast cell infiltration and the progressive development of neutrophilia and eosinophilia (Davis & Freed 1999, 2001; Davis et al. 2003) (Figs 36.6c and 36.7b). Whether granulocytic inflammation and airway obstruction reflect cause and effect or independent events initiated by a common stimulus remains unknown. Although neutrophil infiltration was correlated with late-phase airway obstruc-

802

tion in dogs (Davis & Freed 2001) and has been associated with airway obstruction in response to a variety of noxious stimuli (Fabbri et al. 1984; Seltzer et al. 1984; Cormier et al. 1993), other studies suggest that neutrophils are neither necessary nor sufficient to cause obstruction (O’Byrne et al. 1984; Imai et al. 1990). Thus, neutrophil influx may not be directly related to the development of airways obstruction. However, eosinophil influx was also correlated with the severity of peripheral airway obstruction in dogs (Davis et al. 2003). These granulocytes secrete cationic proteins which can block prejunctional M2 muscarinic receptors, leading to increased tonic secretion of acetylcholine and increased airway resistance (Fryer & Jacoby 1992). They can also damage airway mucosa and connective tissue (Coyle et al. 1994). The former effect could reduce inhibitory prostanoid production and impair mucociliary clearance; the latter would promote edema formation via decreased tissue pressure and increased bronchovascular permeability. Both mucosal injury and lamina propria thickening are present in airways after repeated hyperventilation with unconditioned air (Davis et al. 2003), providing additional evidence for the role of eosinophils in this canine model of EIB.

Repetitive hyperventilation-induced airway remodeling Submucosal edema is believed to contribute to airway obstruction in asthmatic human subjects (Hogg et al. 1987). Data demonstrating a thickened lamina propria in dogs repeatedly hyperventilated with unconditioned air supports this belief (Davis et al. 2003) (Fig. 36.6a vs. Fig. 36.6b,c). Airway wall thickening is in part due to edema that may result from increased bronchovascular permeability, and/or decreased tissue hydrostatic pressure, and/or increased tissue oncotic pressure. A single bout of hyperventilation will produce bronchovascular hyperpermeability that persists for at least 24 hours after acute bronchoconstriction occurs (Omori et al. 1995b). The contribution of other factors, such as increased oncotic pressure (due to leakage and retention of plasma proteins) or increased compliance of the interstitial space (due to the breakdown of interstitial matrix by granulocyte-derived proteases) (Koller et al. 1993) is unknown. However, given the magnitude of granulocyte influx, these cells likely contribute to the development of repetitive hyperventilationinduced airway wall thickening. Repeated hyperventilation also causes the remodeling of normal ciliated epithelium into stratified squamous epithelium. Under normal circumstances, damaged epithelium is quickly repaired by the migration and expansion of adjacent basal cells (Rennard 1996). Undifferentiated cell stratification occurs within 2–3 days, followed by transformation into normal ciliated columnar epithelium. Thus, at the normal rate of repair, airways that were injured one day prior to harvesting would be expected to have only a single layer of cells covering the basement membrane. The appearance of

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multiple layers of squamous epithelium in repetitively injured canine airways (Figs 36.3 and 36.6b,c) suggests that either repair mechanisms are upregulated or the areas of stratification represent a more advanced stage of repair resulting from previous exposures. Repetitive hyperventilation-induced airway mucosal remodeling may reflect functional adaptations that protect against further desiccation-induced injury. The appearance of stratified squamous epithelia is normally associated with protection from desiccation and mechanical injury (Banks 1986). This raises the possibility that squamous metaplasia of the airway mucosa protects the underlying tissues from desiccation better than normal ciliated columnar epithelium. However, it may also impede movement of extravasated fluid into the lumen, resulting in further edema formation and thickening of the airway wall. The airway remodeling observed after repetitive hyperventilation may have other adverse consequences on airway function. Bronchial epithelium is an important source of PGE2, which can inhibit cholinergic neurotransmission (Ito et al. 1990), mast cell degranulation (Kleeberger & Freed 1995), and smooth muscle contraction (Spannhake et al. 1978). Thus, repetitively injured and remodeled airway mucosa may produce less PGE2. In addition, mucociliary clearance is likely to be impaired in repetitively hyperventilated airways due to a decrease in ciliated cell density. Indeed, the deleterious effects of airway remodeling may outweigh any protective benefits the remodeling may provide.

Summary and conclusion Hyperventilation with cool dry air causes airway mucosal injury and increases resistance in all mammals examined to date. Although the functional response to hyperventilation in most mammalian models appears qualitatively similar, species-specific differences exist. Thus caution is required in the extrapolation of data from any given animal model to the human condition. Data from animal models indicate that hyperventilation with dry air increases bronchoactive mediator metabolism. Both human and animal studies suggest that airway cooling counterbalances the constrictor effect of airway drying, and cooling may inhibit either the neuronal or mediator activity that contributes to HIB. Although eicosanoids and neurokinins contribute to the development of airway obstruction, the exact pathways leading to acute airway narrowing are unknown, and the hierarchy of mediator activity may be species-dependent. Less clear is the role for other types of mediators such as NO and VIP, and the contribution of airway edema to airway narrowing. All animal models examined to date exhibit bronchovascular hyperpermeability, suggesting that it also occurs in human. HIB and bronchovascular leakage are products of

Exercise-induced Bronchoconstriction: Animal Models

independent mechanisms. The former response probably reflects a protective mechanism that minimizes distal lung desiccation and injury, with asthmatic individuals exhibiting an exaggerated version of this normal mammalian response. The latter response appears to further modulate the bronchoconstriction, possibly protecting the airway mucosa from desiccation injury. Although HIB is primarily the result of airway smooth muscle contraction, edema may amplify its effect. Late-phase airway obstruction begins during the early phase and both ASL hyperosmolarity and airway cooling appear to be necessary to produce all aspects of the response. Bronchial epithelial cells and mast cells may generate proinflammatory type-2 cytokines even in the absence of T-cell infiltration, providing a link between acute mucosal injury and the delayed eosinophilic inflammation that develops in response to acute hyperventilation in canine and asthmatic human subjects. Finally, although acute mucosal injury induced by hyperventilation is likely to be quickly repaired in normal individuals, repetitive hyperventilation with cold dry air appears to interfere with mucosal repair and results in chronic inflammation. This persistent inflammation promotes airway remodeling, and may ultimately result in asthma. Thus, repeatedly overwhelming mechanisms that normally protect peripheral airways from desiccation may account for the high incidence of asthma-like symptoms and inflammation reported in winter athletes (Larsson et al. 1993; Karjalainen et al. 2000). In fact, repetitive exposure to dry air may be one of many environmental factors that can contribute to the development of asthma.

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Ray, D.W., Hernandez, C., Munoz, N., Leff, A.R. & Solway, J. (1988) Bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs. J Appl Physiol 65, 934– 9. Ray, D.W., Hernandez, C., Leff, A.R., Drazen, J.M. & Solway, J. (1989) Tachykinins mediate bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs. J Appl Physiol 66, 1108–12. Ray, D.W., Eappen, S., Hernandez, C. et al. (1990) Distribution of airway narrowing during hyperpnea-induced bronchoconstriction in guinea pigs. J Appl Physiol 69, 1323– 9. Ray, D.W., Garland, A., Hernandez, C., Eappen, S., Alger, L. & Solway, J. (1991) Time course of bronchoconstriction induced by dry gas hyperpnea in guinea pigs. J Appl Physiol 70, 504–10. Reiss, T.F., Hill, J.B., Harman, E. et al. (1997) Increased urinary excretion of LTE4 after exercise and attenuation of exercise-induced bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax 52, 1030– 5. Rennard, S.I. (1996) Repair mechanisms in asthma. J Allergy Clin Immunol 98, S278–S286. Rumsaeng, V., Cruikshank, W.W., Foster, B. et al. (1997) Human mast cells produce the CD4+ T lymphocyte chemoattractant factor, IL-16. J Immunol 159, 2904–10. Seltzer, J., Scanlon, P.D., Drazen, J.M., Ingram, R.H. Jr & Reid, L. (1984) Morphologic correlation of physiologic changes caused by SO2-induced bronchitis in dogs. The role of inflammation. Am Rev Respir Dis 129, 790–7. Shi, H., Qin, S., Huang, G. et al. (1997) Infiltration of eosinophils into the asthmatic airways caused by interleukin 5. Am J Respir Cell Mol Biol 16, 220– 4. Shimizu, T., Mochizuki, H., Shigeta, M. & Morikawa, A. (1997) Effect of inhaled indomethacin on exercise-induced bronchoconstriction in children with asthma. Am J Respir Crit Care Med 155, 170–3. Smith, C.M. & Anderson, S.D. (1989) A comparison between the airway response to isocapnic hyperventilation and hypertonic saline in subjects with asthma. Eur Respir J 2, 36– 43. Solway, J., Kao, B.M., Jordan, J.E. et al. (1993) Tachykinin receptor antagonists inhibit hyperpnea-induced bronchoconstriction in guinea pigs. J Clin Invest 92, 315– 23. Spannhake, E.W., Lemen, R.J., Wegmann, M.J., Hyman, A.L. & Kadowitz, P.J. (1978) Effects of arachidonic acid and prostaglandins on lung function in the intact dog. J Appl Physiol 44, 397–405. Speelberg, B., Panis, E.A., Bijl, D., van Herwaarden, C.L. & Bruynzeel, P.L. (1991) Late asthmatic responses after exercise challenge are reproducible. J Allergy Clin Immunol 87, 1128–37. Stensrud, T., Berntsen, S. & Carlsen, K.H. (2006) Humidity influences exercise capacity in subjects with exercise-induced bronchoconstriction (EIB). Respir Med 100, 1633– 41. Sue-Chu, M., Larsson, L., Moen, T., Rennard, S.I. & Bjermer, L. (1999) Bronchoscopy and bronchoalveolar lavage findings in cross-country skiers with and without “ski asthma”. Eur Respir J 13, 626– 32. Suman, O.E. & Beck, K.C. (2002) Role of airway endogenous nitric oxide on lung function during and after exercise in mild asthma. J Appl Physiol 93, 1932– 8. Suman, O.E., Morrow, J.D., O’Malley, K.A. & Beck, K.C. (2000) Airway function after cyclooxygenase inhibition during hyperpneainduced bronchoconstriction in guinea pigs. J Appl Physiol 89, 1971– 8.

Exercise-induced Bronchoconstriction: Animal Models

Suzuki, R. & Freed, A.N. (2000a) Endogenous nitric oxide modulates acute and late phase response to hyperventilation with dry air in dogs. Eur Respir J 16, A189. Suzuki, R. & Freed, A.N. (2000b) Hypertonic saline aerosol increases airway reactivity in the canine lung periphery. J Appl Physiol 89, 2139–46. Suzuki, R. & Freed, A.N. (2002) Heparin inhibits hyperventilationinduced late-phase hyperreactivity in dogs. Am J Respir Crit Care Med 165, 27–33. Tang, G.J. & Freed, A.N. (1992) The autonomic nervous system modulates dry air-induced constriction in the canine lung periphery. Am Rev Respir Dis 145, 1301–5. Tang, G.J. & Freed, A.N. (1994) The role of submucosal oedema in increased peripheral airway resistance by intravenous volume loading in dogs. Eur Respir J 7, 311–17. Taub, D.D., Anver, M., Oppenheim, J.J., Longo, D.L. & Murphy, W.J. (1996) T lymphocyte recruitment by interleukin-8 (IL-8). IL8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J Clin Invest 97, 1931–41. Teeter, J.G. & Freed, A.N. (1991) Effect of salbutamol on dry air- and acetylcholine-induced bronchoconstriction in the canine lung periphery. Eur Respir J 4, 972– 8. Therminarias, A., Oddou, M.F., Favre-Juvin, A., Flore, P. & Delaire, M. (1998) Bronchial obstruction and exhaled nitric oxide response during exercise in cold air. Eur Respir J 12, 1040–5. Van Citters, R.L. & Franklin, D.L. (1969) Cardiovascular performance of Alaska sled dogs during exercise. Circ Res 24, 33–42. van Schoor, J., Joos, G.F., Kips, J.C., Drajesk, J.F., Carpentier, P.J. & Pauwels, R.A. (1997) The effect of ABT-761, a novel 5-lipoxygenase inhibitor, on exercise- and adenosine-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med 155, 875–80. Wagner, E.M. & Mitzner, W.A. (1990) Bronchial circulatory reversal of methacholine-induced airway constriction. J Appl Physiol 69, 1220–4. Wang, D., Chen, H.I., Chou, C.L., Hsu, K. & Freed, A.N. (1992) Terbutaline acts at multiple sites to inhibit bronchoconstriction induced by dry air in canine peripheral airways. Am Rev Respir Dis 145, 1295–300. Wessling, G.J. & Wouters, E.F.M. (1992) Respiratory impedance measurements in a dose-response study of isocapnic hyperventilation with cold air. Respiration 59, 259–64. Yang, X.X., Powell, W.S., Hojo, M. & Martin, J.G. (1997) Hyperpneainduced bronchoconstriction is dependent on tachykinin-induced cysteinyl leukotriene synthesis. J Appl Physiol 82, 538–44. Yang, X.X., Powell, W.S., Xu, L.J. & Martin, J.G. (1999a) Strain dependence of the airway response to dry-gas hyperpnea challenge in the rat. J Appl Physiol 86, 152–8. Yang, X.X., Ho, G., Xu, L.J., Powell, W.S. & Martin, J.G. (1999b) The beta(2)-agonist salbutamol inhibits bronchoconstriction and leukotriene D(4) synthesis after dry gas hyperpnea in the guinea-pig. Pulm Pharmacol Ther 12, 325–9. Yuan, L. & Nail, B.S. (1995) A differential bronchomotor response to cooling and drying the upper airway. Respir Physiol 101, 121–8. Zawadski, D.K., Lenner, K.A. & McFadden, E.R. Jr (1988) Reexamination of the late asthmatic response to exercise. Am Rev Respir Dis 137, 837–41.

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Exercise-induced Bronchoconstriction: Human Models Arthur N. Freed and Sandra D. Anderson

Summary

5

4 FEV1 in litres

Airway narrowing following exercise, usually known as exercise-induced bronchoconstriction (EIB), is common in clinically recognized asthmatics and becoming increasingly reported in asymptomatic healthy subjects, particularly athletes. EIB appears to be an early manifestation of asthma in children and studies are required to determine if this is also the case when it develops in young adults. The stimulus for EIB is the evaporative loss of water in conditioning air during exercise. The mechanism relates to the osmotic and thermal consequences of the evaporation of water. The osmotic consequences include the release of inflammatory mediators that act on bronchial and possibly vascular smooth muscle to cause the airways to narrow. In asthmatics, EIB is associated with airway inflammation and EIB severity may be an indirect marker of disease activity. The severity of EIB is reduced in all asthmatics in response to chronic treatment with inhaled corticosteroids. Less is known about the pathophysiology, repeatability, or response to drugs of EIB that occurs in otherwise asymptomatic healthy subjects. An objective test with hyperpnea of dry air or a hyperosmolar aerosol should still be regarded as important for patients suspected of having EIB and who complain of breathlessness on or after exertion. By documenting the hyperresponsiveness to hyperpnea or a hyperosmolar aerosol, a diagnosis of EIB, and even asthma itself, can be confirmed and the appropriate therapy prescribed.

A diagnosis of EIB is made by measuring a fall in forced expiratory volume in 1 s (FEV1), an indirect measure of airways resistance, after exercise of 10% or more of the value measured immediately before exercise (Sterk et al. 1993; Roca et al. 1997; Task Force on Recognizing and Diagnosing ExerciseRelated Asthma Respiratory and Allergic Disorders in Sports 2005) (Fig. 37.1). Data in children and young adults indicate that a fall in FEV1 of 13% has 94% specificity for identifying those with a clinical diagnosis of asthma (Godfrey et al. 1999). A provisional diagnosis of EIB can be made on history if a person complains of breathlessness that is worse 5–10 min after ceasing exercise compared with during exercise. Vocal cord dysfunction (VCD) (McFadden & Zawadski 1996; Morris et al. 1999), or inspiratory stridor, a symptom of VCD (Rundell & Spiering 2003) or exercise hyperventilation syndrome (Hammo & Weinberger 1999), can be incorrectly diagnosed as EIB. For these disorders the symptoms occur during rather than after exercise.

Introduction Exercise-induced bronchoconstriction (EIB) and exerciseinduced asthma (EIA) are the terms used to describe the transient increase in airways resistance that follows vigorous exercise. While these terms are used interchangeably, EIA is best used only for those with a clinical diagnosis of asthma (Anderson & Henriksen 1999). For the purposes of this chapter the term EIB is used.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN 978-1-4051-5720-9.

808

< 10% Normal

< 25% Mild

3

> 25% Moderate 2

> 50% Severe Exercise

1 0

8

14

20

Time (min) Fig. 37.1 Pattern of change in forced expiratory volume in 1 s (FEV1) after 8 min of vigorous exercise inspiring dry air at a ventilation exceeding 50% of maximum in a normal healthy subject without exercise-induced bronchoconstriction (EIB) and in subjects with mild, moderate or severe EIB. The severity of the response is based on the fall in FEV1 after exercise expressed as a percentage of the baseline value. If a subject is taking inhaled corticosteroids on a daily basis, a postexercise fall in FEV1 of 30% or more would be considered as severe.

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Exercise-induced Bronchoconstriction: Human Models Arthur N. Freed and Sandra D. Anderson

Summary

5

4 FEV1 in litres

Airway narrowing following exercise, usually known as exercise-induced bronchoconstriction (EIB), is common in clinically recognized asthmatics and becoming increasingly reported in asymptomatic healthy subjects, particularly athletes. EIB appears to be an early manifestation of asthma in children and studies are required to determine if this is also the case when it develops in young adults. The stimulus for EIB is the evaporative loss of water in conditioning air during exercise. The mechanism relates to the osmotic and thermal consequences of the evaporation of water. The osmotic consequences include the release of inflammatory mediators that act on bronchial and possibly vascular smooth muscle to cause the airways to narrow. In asthmatics, EIB is associated with airway inflammation and EIB severity may be an indirect marker of disease activity. The severity of EIB is reduced in all asthmatics in response to chronic treatment with inhaled corticosteroids. Less is known about the pathophysiology, repeatability, or response to drugs of EIB that occurs in otherwise asymptomatic healthy subjects. An objective test with hyperpnea of dry air or a hyperosmolar aerosol should still be regarded as important for patients suspected of having EIB and who complain of breathlessness on or after exertion. By documenting the hyperresponsiveness to hyperpnea or a hyperosmolar aerosol, a diagnosis of EIB, and even asthma itself, can be confirmed and the appropriate therapy prescribed.

A diagnosis of EIB is made by measuring a fall in forced expiratory volume in 1 s (FEV1), an indirect measure of airways resistance, after exercise of 10% or more of the value measured immediately before exercise (Sterk et al. 1993; Roca et al. 1997; Task Force on Recognizing and Diagnosing ExerciseRelated Asthma Respiratory and Allergic Disorders in Sports 2005) (Fig. 37.1). Data in children and young adults indicate that a fall in FEV1 of 13% has 94% specificity for identifying those with a clinical diagnosis of asthma (Godfrey et al. 1999). A provisional diagnosis of EIB can be made on history if a person complains of breathlessness that is worse 5–10 min after ceasing exercise compared with during exercise. Vocal cord dysfunction (VCD) (McFadden & Zawadski 1996; Morris et al. 1999), or inspiratory stridor, a symptom of VCD (Rundell & Spiering 2003) or exercise hyperventilation syndrome (Hammo & Weinberger 1999), can be incorrectly diagnosed as EIB. For these disorders the symptoms occur during rather than after exercise.

Introduction Exercise-induced bronchoconstriction (EIB) and exerciseinduced asthma (EIA) are the terms used to describe the transient increase in airways resistance that follows vigorous exercise. While these terms are used interchangeably, EIA is best used only for those with a clinical diagnosis of asthma (Anderson & Henriksen 1999). For the purposes of this chapter the term EIB is used.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

808

< 10% Normal

< 25% Mild

3

> 25% Moderate 2

> 50% Severe Exercise

1 0

8

14

20

Time (min) Fig. 37.1 Pattern of change in forced expiratory volume in 1 s (FEV1) after 8 min of vigorous exercise inspiring dry air at a ventilation exceeding 50% of maximum in a normal healthy subject without exercise-induced bronchoconstriction (EIB) and in subjects with mild, moderate or severe EIB. The severity of the response is based on the fall in FEV1 after exercise expressed as a percentage of the baseline value. If a subject is taking inhaled corticosteroids on a daily basis, a postexercise fall in FEV1 of 30% or more would be considered as severe.

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RESPIRATORY WATER LOSS Mucosal cooling

Mucosal dehydration Increase [Na+] [Cl–] [Ca2+] [K+]

Vasoconstriction

Cough

Increased osmolarity

Mucus

Rapid rewarming of airways Cells shrink and mediators released Vascular engorgement ± edema

Smooth muscle contraction ± edema Exercise-induced bronchoconstriction

Fig. 37.2 Schematic diagram showing the two hypotheses (Anderson & Daviskas 2000; McFadden et al. 1996) that explain the events leading to exercise-induced bronchoconstriction. Respiratory water loss can cause airway cooling and mucosal dehydration. Airway cooling is associated with vasoconstriction, vascular engorgement, and edema. Airway dehydration is associated with an increase in osmolarity of the airway surface, with a consequent release of mediators that act on smooth muscle causing it to contract. An increase in osmolarity also causes cough and mucus independently of airway narrowing.

The stimulus for EIB is the loss of water by evaporation from the airway surface that is the consequence of humidifying large volumes of ambient air in a very short time (Anderson & Daviskas 2000). Thus EIB is most likely to occur in a dry environment in response to an exercise intensity that raises the minute ventilation to a value that is 50% or more of predicted maximum achievable. The mechanism for dehydration causing the airways to narrow relates to the osmotic and thermal effects of evaporation (McFadden et al. 1996; Anderson & Daviskas 2000) (Fig. 37.2). The thermal effect, i.e., airway cooling, is thought to cause vasoconstriction of the bronchial vasculature, an event followed by a reactive hyperemia at the end of exercise when the airways rapidly rewarm (Gilbert & McFadden 1992). These thermal events may be accompanied by an increase in vascular permeability, exudation of plasma, and airway edema that all contribute to airway narrowing (Kanazawa et al. 2002). This vascular hypothesis does not implicate the bronchial smooth muscle in EIB. The osmotic effect, i.e., hyperosmolarity of the airway surface liquid (ASL), is thought to provoke release of mediators that act on specific receptors on bronchial smooth muscle causing it to contract and the airways to narrow (Reiss et al. 1997; O’Sullivan et al. 1998; Hallstrand et al. 2005a). Increased vascular permeability and airway edema may be caused by the same mediators and plasma exudation may also occur in response to an osmotic stimulus (Greiff et al. 2003a,b). The increase in ASL osmolarity may be the signal for the release of nitric oxide (NO) (Smith et al. 1993; Anderson & Daviskas 1999; Högman et al. 2001) and could account for some of the vascular events (Kanazawa et al.

Exercise-induced Bronchoconstriction: Human Models

2002). In asthmatics exercising under conditions of breathing cold dry air, both thermal and osmotic effects are likely to contribute to EIB. Under temperate or hot dry environmental conditions, when significant airway cooling is unlikely to occur, it is the osmotic effects that are the most important (Aitken & Marini 1985; Anderson & Daviskas 1992, 2000; Argyros et al. 1993; Anderson & Holzer 2000; Evans et al. 2005). In elite athletes without a clinical diagnosis of asthma, EIB may be a consequence of epithelial injury and subsequent changes to the contractile properties of bronchial smooth muscle that make it more responsive (Anderson & Kippelen 2005). The injury may lead to an imbalance between the bronchodilator mediator prostaglandin (PG)E2 and the bronchoconstricting cysteinyl leukotrienes, making the muscle appear more hyperresponsive (Hallstrand et al. 2005b). Excessive exposure to cold air, airborne allergens, pollutants or particulate matter, and viral infections may also amplify this problem in athletes (Heir & Larsen 1995; Heir et al. 1995; Rundell et al. 2003; Bjermer & Anderson 2005).

EIB and airway inflammation In asthmatics, EIB is associated with active airway inflammation, and the severity of EIB serves as an indirect marker of the severity of inflammation. Eosinophils, a well-known marker of asthmatic inflammation, are higher in sputum from asthmatic children and adults with EIB relative to those without EIB (Yoshikawa et al. 1998; Hallstrand et al. 2005b). The severity of EIB is also related to airway injury, as reflected by the number of columnar epithelial cells collected in sputum before exercise (Hallstrand et al. 2005b). The severity of EIB is also related to the concentration of NO derivatives in sputum (Kanazawa et al. 2000), and to expired levels of NO (eNO) (Terada et al. 2001; Buchvald et al. 2005). The relationship between eNO and EIB is only significant in those who are atopic (Rouhos et al. 2005). EIB is markedly reduced by drugs that exert an antiinflammatory effect, such as inhaled corticosteroids (ICS) (Hofstra et al. 2000; Jonasson et al. 2000; Subbarao et al. 2006) (Fig. 37.3), and drugs that inhibit the products of 5-lipoxygenase (LTB4, LTC4, LTD4, LTE4) (Meltzer et al. 1996; Lehnigk et al. 1998) or which antagonize the effects of leukotrienes at the receptor.

Mediators and EIB The potential for inflammatory mediators such as histamine, prostaglandins, and leukotrienes to cause EIB has been acknowledged for many years, based on the finding that specific receptor antagonists provide some protection. The precise role of the preformed mediator histamine in EIB remains unclear. Histamine is probably important in contributing to the early fall in FEV1, whereas the prostaglandins

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70

70

60

60

50

50 % Fall in FEV1

% Fall in FEV1

PART 4

40 30 20 10

40 30 20 10

10/14 (71%)

0 Pre

9/14 (64%)

0

Post

Pre

Post

100 mg/day for 12 weeks

200 mg/day for 12 weeks

Baseline: FEV1 100 ± 12% Predicted

FEV1 101 ± 10% Predicted

and leukotrienes appear to be more important for sustaining the fall in FEV1. The intensity of the exercise stimulus may be the determining factor for histamine to contribute significantly to the maximal fall in FEV1 (Anderson & Brannan 2002). Histamine receptor antagonists were found effective in inhibiting EIB in some studies (Patel 1984; Baki & Orhan 2002 ) but not others (Dahlén et al. 2002; Peroni et al. 2002). The same histamine antagonist that was reported as effective in one study (Baki & Orhan 2002) was ineffective in another (Dahlén et al. 2002). It is not known if the presence of IgE is important in determining the efficacy of a histamine antagonist. The leukotriene antagonists montelukast and zafirlukast both inhibit and enhance recovery from EIB (Kemp et al. 1998; Pearlman et al. 1999; Villaran et al. 1999; Dahlén et al. 2002) (Fig. 37.4).

Fig. 37.3 Maximum percentage fall in FEV1 from baseline after exercise in two groups of children who received 100 mg or 200 mg of budesonide daily for 2 weeks. The percentage of children who had a response within the normal range after treatment is shown. (Individual data kindly provided by Jonasson et al. 2000.)

There is now direct evidence to implicate mediator release in EIB, mainly due to the availability of sensitive assays for detecting leukotrienes, prostaglandins, and histamine, and the use of induced sputum and urine. Urinary excretion of LTE4 and the metabolite of PGD2 (9α,11β-PGF2) are increased after exercise in both asthmatic (Fig. 37.5) and healthy subjects (Caillaud et al. 2003; Mickleborough et al. 2003). The same findings have been reported following inhalation of the hyperosmolar aerosol mannitol (Brannan et al. 2003). In sputum, histamine, tryptase, and cysteinyl leukotrienes (Hallstrand et al.

100

P < 0.05

P < 0.05

30 min

90 min

5

% Change FEV1 (mean ± SEM)

0 –5 –10 Placebo % fall 26 ± 14 Montelukast % fall 18 ± 13 = 25

–15 –20

9a,11b-PGF2 ng/mmol creatinine

80

60

40

20

–25 0 –30 0

5

10

15

30

45

60

Time after exercise (min) Fig. 37.4 Mean percentage change in FEV1 in children in response to 6 min of running exercise 20–24 hours after 5 mg of montelukast once daily in the evening for 2 days. The maximum fall in FEV1 is also given. Note the rapid return of FEV1 to baseline following exercise in the presence of the montelukast. (Data from Kemp et al. 1998.)

810

Before

Fig. 37.5 Mean (SEM) values for urinary excretion of the mast cell mediator prostaglandin D2 metabolite 9a,11b-PGF2 measured before and after exercise in asthmatic patients who responded or did not respond to challenge by exercise. The values at 30 and 90 min after exercise were significantly higher in the seven responders than in the five nonresponders and the values at 90 min in the responders were significantly increased above baseline. (From O’Sullivan et al. 1998, with permission.)

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Exercise-induced Bronchoconstriction: Human Models

40

10

500

0

Baseline Post-exercise

5

Baseline Post-exercise

2000

4

10

1500 CysLT pg/mL

Tryptase ng/mL

20

3 2

Treatment

0

1000

500

1

Placebo

1000

treatment with oral formulations of histamine and leukotriene antagonists, significantly lower levels of histamine and cysteinyl leukotrienes were found in sputum after exercise (Hallstrand et al. 2005a) (Fig. 37.7). It is not known if this is due to an antagonist action reducing vascular permeability or a pharmacologic effect on mast cells. Enhanced transcription of two genes coding for 5-lipoxygenase (ALOX5) and ALOX5activating protein, together with enhanced levels of LTB4 and LTC4 in plasma, were reported following cycling exercise in healthy subjects (Hilberg et al. 2005). These data provide evidence to connect the putative mediators to the stimulus of exercise. The beneficial effects of the 5-lipoxygenase inhibitor zileuton (Meltzer et al. 1996) and leukotriene antagonists on EIB in asthmatics (Reiss et al. 1997; Kemp et al. 1998) provide the evidence for connecting these mediators with the bronchoconstrictor response. In elite athletes, the events that accompany airway injury may lead to the bronchial smooth muscle becoming responsive to leukotrienes and prostaglandins, particularly in athletes with allergies. EIB is followed by a period of refractoriness, and approximately 50% of asthmatics will be protected from the effects of repeated exercise for 2–3 hours. The mechanism for refractoriness probably involves both prostaglandins and leukotrienes, mediators that are released in response to the initial exercise (Wilson et al. 1994; Dessanges et al. 1999). The ability to become refractory is lost by taking nonsteroidal

30 Histamine ng/mL

2

0

Baseline Post-exercise

40

0

3

1

2005b) are increased after exercise (Fig. 37.6). Underproduction of PGE2 in response to exercise may also play a significant role in determining EIB (Hallstrand et al. 2005a). This underproduction may relate to the airway epithelial cell injury. Mast cells are likely the primary source of PGD2. While macrophages also produce PGD2, the concentration is only one-ten thousandth of that produced by mast cells. The source of leukotrienes is probably multicellular, with mast cells and eosinophils and macrophages all contributing. Eosinophils release leukotrienes (Moloney et al. 2003) and mast cells release histamine in response to a hyperosmotic stimulus (Flint et al. 1985; Eggleston et al. 1990). Nasal epithelial cells in the nose release 15-hydroxyeicosatetraenoic acid in those with allergic rhinitis in response to a hyperosmolar stimulus. Neuropeptides (Ichinose et al. 1996) and adenosine (Csoma et al. 2005) may also play an important role in EIB and both are stimulated by hyperosmolarity (Garland et al. 1995; Tarran et al. 2005). Evidence to support a cause and effect role for PGD2 in EIB comes from studies using the hyperosmotic stimulus mannitol. Premedication by inhaling an aerosol of either sodium cromoglycate or eformoterol inhibited both the increase in urinary excretion of 9α,11β-PGF2 and the airway response (Brannan et al. 2006). Since both drugs were inhaled, it is likely that the effect was superficial in the airways as sodium cromoglycate is not readily absorbed. Further, following preFig. 37.7 Effect of treatment with montelukast and loratadine on the level of mast cell mediators and eisosanoids in induced sputum after exercise in individuals with asthma and exercise-induced bronchoconstriction. Comparisons were made between treatment with montelukast and loratadine as compared with administration of two matched placebos on average 6.8 days apart (range 4–14 days). The level of histamine (P < 0.001) and cysteinyl leukotrienes (CysLT) (P < 0.001) were significantly decreased. (Original individual data from Hallstrand et al. 2005a.)

1500 CysLT pg/mL

Tryptase ng/mL

20

0

2000

4

30 Histamine ng/mL

Fig. 37.6 Effect of exercise on the level of mast cell mediators in induced sputum of asthmatic subjects with exercise-induced bronchoconstriction. Comparisons were made between baseline and 30 min after exercise on separate days on average 9.9 days apart (range 4–18 days). The levels of histamine (P < 0.002), tryptase (P < 0.02), and cysteinyl leukotrienes (CysLT) (P < 0.03) were significantly increased. (Original individual data from Hallstrand et al. 2005a.)

5

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Treatment

0

Placebo

Treatment

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antiinflammatory agents and is well described following treatment with indomethacin for 3 days (O’Byrne 1986; Margolskee 1988; Wilson et al. 1994).

Prevalence The treatment of asthma with antiinflammatory agents over the last 10 years means that most asthmatics have received these drugs in one form or other. This therapeutic approach has had a beneficial impact on both the prevalence and severity of EIB measured in the asthmatic population at any one time. Under standardized exercise conditions, EIB occurs in the majority of steroid-naive asthmatic subjects and about 55% of steroid-treated asthmatics referred to hospital laboratories (Waalkans et al. 1993; Anderson et al. 2001c). Similarly, 50% of adults taking ICS remain hyperresponsive to challenge with hyperosmolar aerosols of 4.5% saline or mannitol, both stimuli used as surrogate challenge tests for identifying EIB (du Toit et al. 1997; Brannan et al. 2002; Koskela et al. 2003). EIB appears to be an early sign of asthma in asymptomatic children (Jones & Bowen 1994; Lombardi et al. 1997; Ernst et al. 2002; Porsbjerg et al. 2005), so its recognition and treatment may reduce the risk of developing symptomatic asthma at a later age. EIB is one of the last signs of asthma to respond to treatment with aerosol corticosteroids (Pedersen & Hansen 1995; Subbarao et al. 2006), making it important to investigate before, or at least during. treatment. EIB is equally common in asthmatic adults, although many adults do not give a history of EIB as they avoid vigorous exercise. Further, in the laboratory, older adults are less responsive to challenge with cold dry air than younger adults (Koskela et al. 1997, 2003). This may be due to the short duration of the cold air challenge and the lower values for FEV1 in older people that would limit maximum ventilation and thus the stimulus, i.e., water loss. Neither the presence nor the severity of EIB can be predicted from the resting level of FEV1 (Cabral et al. 1999; Anderson et al. 2001a). EIB occurs more frequently in asthmatics whose lung function is abnormal but it also occurs in asthmatics with normal lung function (Hofstra et al. 2000; Jonasson et al. 2000; Subbarao et al. 2006). The resting values for FEV1 in elite athletes are commonly higher than predicted normal yet they often record moderate to severe EIB (Rundell et al. 2000, 2004a; Holzer et al. 2003; Anderson et al. 2006a). Treatment with antiinflammatory agents is the likely reason that the prevalence of EIB in known asthmatic populations is lower than the previously reported 70–80% (West et al. 1996; Sano et al. 1998; Cabral et al. 1999; Anderson et al. 2001a; Hallstrand et al. 2002). The prevalence of EIB is higher in the presence of atopy (Koh et al. 2002), exercise-induced wheeze (Ponsonby et al. 1996), and a past history of asthma (Sinclair et al. 1995), relative to those without those features.

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There are some advantages in using exercise in the field to measure the airway hyperresponsiveness of asthma. First, exercise is a common noninvasive stimulus for provoking an attack of asthma, and many asthmatics have EIB. Second, using an objective test overcomes the problems with questionnaires and using a “one dose fits all stimulus” gives insight into who has excessive airway narrowing, a feature that distinguishes the asthmatic. However, a low prevalence of EIB in known asthmatics has been reported in some field studies. These findings may relate to the diagnosis of asthma having been made (i) on a questionnaire that covers 12 months, (ii) that treatment for asthma was not withheld before testing, (iii) that only a single measurement of lung function was made after exercise (Terblanche & Stewart 1990; Bardagi et al. 1993; Jones 1994; Riedler et al. 1994), or (iv) that an exercise or hyperpnea stimulus was not long or intense enough (Nicolai et al. 1993). In a field study in schoolchildren that did optimize all these factors, 19% had EIB defined as a fall in FEV1 of 15% or more (Riedler et al. 1994; Haby et al. 1995) and 40% of these children did not have a clinical diagnosis of asthma. The prevalence of EIB is higher in city children compared with country children (Keeley et al. 1991; Ng’ang’a et al. 1998). It is of interest that the prevalence of EIB increased in schoolchildren in Wales between 1974 and 1989 (Burr et al. 1989). Over the last decade there has been increasing recognition of EIB in athletes. The prevalence has been higher in those studies that have used questionnaires to identify EIB (Weiler et al. 1986; Helenius et al. 1996, 1997; Weiler et al. 1998), relative to those studies where exercise or surrogates of exercise have been used to identify EIB (Rundell et al. 2001; Dickinson et al. 2005). The prevalence of EIB was 21–35% in figure skaters (Mannix et al. 1996; Provost-Craig et al. 1996; Wilber et al. 2000), 43% in short track speed skaters (Wilber et al. 2000), 21% in ice hockey players (Lumme et al. 2003; Rundell et al. 2004b), 50% in cross-country skiers (Wilber et al. 2000), and 17% in long-distance athletes (Helenius et al. 1997). There are now excellent data on prevalence of asthma and/or EIB in elite athletes because the International Olympic Committee Medical Commission (IOC-MC) has required objective evidence for approval to inhale a β2 agonist during competition. There is a much higher prevalence in those athletes performing endurance sports. In the Winter Olympics of 2002, 70% of the applicants approved to use a β2 agonist were skaters or skiers (Anderson et al. 2003). For the Summer Olympics of 2004, 76% of the submissions came from canoeing, rowing, swimming, modern pentathlon, cycling and triathlon, sports that represented only 43% of the participating athletes (Anderson et al. 2006a). In sensitized asthmatics, EIB is more severe during the pollen season and, for some, this may be the only time it occurs (Karjalainen et al. 1989). Exposure to allergens can increase the severity of EIB (Mussaffi et al. 1986). However, a single exposure to an allergen in a laboratory failed to demonstrate

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enhancement of EIB 48 hours later (Boulet et al. 1992). Allergen avoidance reduces severity of EIB (Benckhuijsen et al. 1996). Exposure to environmental pollutants (nitrogen dioxide, ozone or tobacco smoke) in the short term (1 hour) has little or no enhancing effect on EIB severity. Low levels of sulfur dioxide can have an effect though it is variable (Roger et al. 1985). Breathing cold air can reveal EIB or make it more severe. However, the most important determinant of severity of EIB in an asthmatic is not the temperature but the water content of the air inspired, and the ventilation reached and sustained during exercise. Cold air always has a lower water content as the capacity of the air to hold water increases with increasing temperature.

Diagnosing EIB A laboratory evaluation of the response to exercise, or a surrogate for the stimulus of exercise, is useful for revealing the potential for EIB and for predicting and monitoring the benefits of treatment. Unfortunately the exercise protocols recommended for provoking EIB do not always have a high success rate, and this is particularly true when evaluating athletes (Sterk et al. 1993; Carlsen et al. 2000; Crapo et al. 2000; Anderson et al. 2001a). For this reason, testing with surrogates of the exercise stimulus, such as hyperpnea of dry air or inhalation of hyperosmolar aerosols, has become increasingly popular. The eucapnic voluntary hyperpnea (EVH) test has the advantage of being a test where it is easy to simulate the high minute ventilation of exercise, such as rowing and crosscountry skiing, that would otherwise be difficult using a laboratory ergometer. The inhalation of hyperosmolar aerosols requires even less expensive equipment than EVH. It is important that, whatever the stimulus used, the subject should withhold medications for the required time before challenge by exercise EVH or hyperosmolar aerosols. Furthermore, to avoid the possibility of refractoriness, more than 3 hours should have passed since exercise was last performed. A baseline spirometry value of 75% of predicted FEV1 or more is recommended for challenge to proceed. However, for most people, lung function is preserved or improved during exercise, probably as a result of the high tidal volume exerting a relaxing effect on bronchial smooth muscle (Suman et al. 1995). If exercise is used it is important that it is of sufficient duration and intensity to raise the ventilation to > 17.5 and preferably > 21 times the FEV1, values that represent 50 and 60% of the predicted maximum respectively. The duration needs to be at least 6 min and preferably 8 min in adults. The intentisty needs to be sufficient to generate the required ventilation and have it sustained for at least 4 and preferably 6 min. The inspired air needs to be is dry (water less than <10 mg/L, i.e., 50% relative humidity at 20°C). Compressed dry air is suitable and for most assessments it is not necessary to cool the inspired air (Carlsen et al. 2000; Evans et al. 2005). Some

Exercise-induced Bronchoconstriction: Human Models

protocols use heart rate to monitor exercise intensity but it should be noted that ventilation and not heart rate is the stimulus to EIB. Before excluding a diagnosis of EIB, a measure of ventilation during exercise is recommended in order to ensure that the appropriate level was reached and sustained. Bicycle exercise is commonly used in the laboratory as it is safe and it is easy to measure ventilation and heart rate while cycling. Running at high speeds on a treadmill is not always practical or safe and it is difficult to make measurements of exercise intensity. As cycling exercise increases minute ventilation more slowly than running, intensity or duration of the exercise may need to be increased to achieve the target ventilation. The major issue in laboratories nowadays is the referral of athletes who perform a sport other than cycling or running. This has accelerated the move to testing with EVH (Holzer & Brukner 2004).

Expression of the bronchoconstrictor response EIB is expressed as the percent fall index. This is calculated by subtracting the lowest FEV1 measured after exercise from the pre-exercise FEV1 and expressing it as a percentage of the pre-exercise FEV1. The times recommended for measuring FEV1 after exercise are usually 1, 3, 5, 7, 10, or 15 min. The measurements of FEV1 at 1 and 3 min are recommended when there is a need to monitor a severe response and permit early reversal of bronchoconstriction if required. The maximum fall in FEV1 usually occurs 5–10 min after exercise and EVH. It is recommended that two time points after exercise or EVH are positive for the response as a single low reading may reflect poor expiratory effort. EIB can also be expressed by the area above the FEV1 time curve (Kemp et al. 1998), but this index is usually used for assessing effects of treatment rather than for diagnosis. Late responses following EIB, while reported (Iikura et al. 1985; Speelberg et al. 1989), are relatively uncommon (Karjalainen 1991). Thus there seems to be no need to follow subjects for longer than recovery to baseline lung function. EIB is considered to be mild if FEV1 falls by 10–25%, moderate if it falls by 25–50%, and severe if it falls by > 50% (Fig. 37.1). If the person is taking inhaled corticosteroids, a fall in FEV1 of 30% or more from baseline is regarded as severe. EIB can be variable over time particularly if it is mild (Dahlén et al. 2001). For inclusion in clinical studies evaluating the effect of treatment, a repeatable fall in FEV1 of 20% or more is recommended (Anderson et al. 2001a). EIB in known asthmatics is frequently associated with significant arterial hypoxemia, ventilation–perfusion inequality, and hyperinflation, and for these reasons medical intervention is aimed at preventing EIB (Haverkamp et al. 2005). Fortunately spontaneous recovery from EIB occurs (Hofstra et al. 1995; Brannan et al. 1998), with oxygen tension and lung function returning to baseline usually within 45–90 min depending on severity of the response (Anderson et al. 1972; Bye et al. 1980; Munoz 2006). For moderate to severe EIB,

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rescue medication with a β2-adrenoceptor agonist is often used to reduce recovery time to 15–30 min. In addition to the percent fall index, the pre-exercise FEV1 and the lowest FEV1 recorded after exercise should be reported and expressed as a percentage of the predicted normal value. The measurement of peak expiratory flow (PEF) and flow rates through the mid-portion of the vital capacity (FEF25–75 or FEF50) have proved to be much more variable than FEV1 (Nicolai et al. 1993), and are not recommended for diagnosing EIB in the laboratory. Contrary to expectation, the inclusion of FEF50 in addition to FEV1 in the diagnosis of EIB reduces the sensitivity and does not enhance the sensitivity or specificity of the diagnosis. Further the use of FEF50 alone is insufficiently sensitive to diagnose EIB in elite athletes (Dickinson et al. 2006). In patients suspected of having VCD, an inspiratory flow– volume curve should be performed and checked for abnormality. Exercise-induced hyperventilation can be confirmed or excluded by monitoring end-tidal carbon dioxide.

Surrogate challenges for identifying EIB Eucapnic voluntary hyperpnea with dry air The EVH of dry air challenge (Phillips et al. 1985; Argyros et al. 1996; Anderson et al. 2001b) has become widely used to identify EIB in potential recruits and elite athletes (Mannix et al. 1999; Holzer et al. 2003; Rundell et al. 2004a; Dickinson et al. 2005), children (Zach et al. 1984; Zach & Polgar 1987), and even infants (Nielsen & Bisgaard 2000). The major advantage of EVH is the ease with which the subject can achieve and sustain the required target minute ventilation of exercise. This test is easy to perform in a laboratory setting and the equipment needed is less costly than that required for exercise. The test requires the person to voluntarily hyperventilate a dry gas containing 4.9% CO2, 21% O2, and N2. The level of ventilation can be either increased progressively (10, 21, 32 times FEV1) or performed as a single-stage test (target between 21 and 30 times FEV1). The single-stage protocol of 6 min at a target ventilation of 30 times FEV1 should be used with some caution, particularly in those with poorly controlled asthma or moderate to severe asthma, as severe airway narrowing can occur. For the progressive protocol the person hyperventilates for 3 min at each stage followed by a measurement of FEV1 3 min later, and the next stage performed if the fall in FEV1 does not exceed 10% of the baseline value. The protocol for a progressive increase in the ventilatory stimulus is performed for reasons of safety in people with a strong history of EIB. However, this multistage protocol has been associated with the induction of a state of refractoriness (Argyros et al. 1995) and is not recommended for assessing elite athletes. For athletes with normal lung function, a single challenge for 6 min with a target minute ventilation of 30 times FEV1 is recommended (Spiering et al. 2002).

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The EVH test for identifying EIB in a nonathlete is valid if the ventilation is greater than 21 times FEV1 and is sustained for 6 min because this ventilation is not usually exceeded during exercise in an untrained person. However, for an elite athlete the minute ventilation achieved during exercise can be 28 or more times FEV1 so voluntary ventilation needs to exceed this in order to identify or exclude a diagnosis of EIB. The EVH test is also useful for determining the minute ventilation below which a subject may exercise in order not to provoke EIB. The duration of exercise, the intensity of ventilation, and the temperature of the inspired air can all be matched, if necessary, to reproduce the exercise task that provokes the athlete’s symptoms. A 10% or more fall in FEV1 in response to EVH is abnormal (Hurwitz et al. 1995) and this value has been adopted by various sporting bodies as the cutoff point to permit pre-exercise medication with a β2 agonist (Anderson et al. 2006a; Task Force on Recognizing and Diagnosing Exercise-Related Asthma Respiratory and Allergic Disorders in Sports 2005). For the single-stage EVH test the grades of severity of the response are the same as those described above for EIB. However, for the multistage test the ventilation rate required to provoke a 10, 15, and 20% fall in FEV1 (PVE10, PVE15, PVE20) is used for assessing severity and the effect of treatment. The EVH is a potent challenge that may or may not be the best test for assessing the protective effects of drugs. The high cardiac output and sympathetic drive of exercise are absent during EVH and this may be a disadvantage. Yet another disadvantage is the cost of the prepared gas mixtures. While this cost can partly be avoided by using dry air and adding carbon dioxide, this approach necessitates monitoring of end-tidal CO2 and this requires a rapid gas analyzer.

Challenge with hyperosmolar aerosols At the time investigators were trying to identify the mechanism whereby the evaporative water loss of hyperpnea provoked airway narrowing, it was found that asthmatics sensitive to the effects of dry air hyperpnea were also sensitive to hyperosmolar aerosols. This was initially shown for saline (Schoeffel et al. 1981; Smith & Anderson 1986; Belcher et al. 1989; Boulet & Turcotte 1989) and later for mannitol (Brannan et al. 1998) (Fig. 37.8). The development of standardized protocols for administering these hyperosmolar aerosols (Riedler 1997; Anderson & Brannan 2003; Anderson et al. 2005; Brannan et al. 2005) has further reduced the need for expensive laboratory equipment for identifying EIB. A dry powder mannitol challenge test kit is commercially available, increasing the opportunity for a common operating procedure to be widely used both within and outside a laboratory environment (Aridol, Pharmaxis Ltd, French’s Forest, NSW, Australia). It has been shown that people who have a 15% or more reduction in FEV1 after inhaling 4.5% saline or mannitol are likely to have EIB (Smith & Anderson 1990; Brannan et al. 1998; Holzer et al. 2003). The response to 4.5% saline (in mL)

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PD15 mannitol (mg)

1000

100

rp = –0.68 < 0.01 = 13

10 0

10

20

30

40

50

60

70

% fall in FEV1 to exercise Fig. 37.8 Provoking dose inducing a 15% fall in forced expiratory volume in 1 s (PD15 FEV1) in response to the inhalation of mannitol in relation to the maximum percentage fall in FEV1 from baseline following bicycle exercise in a group of 13 clinically recognized adult asthmatic subjects who were not taking inhaled corticosteroids. (From Brannan et al. 1998, with permission.)

or mannitol (in mg) is said to be mild when the PD15 is greater than 6 mL or 155 mg; moderate when 2–6 mL or 36–155 mg; and severe when < 2 mL or < 36 mg. The test protocols are likely to be faster to perform than exercise when the response is positive. The hyperosmolar aerosol challenge tests take about 25 min when the response is negative (Brannan et al. 2005).

Exercise-induced Bronchoconstriction: Human Models

In a study of 100 defense force recruits with a past history of asthma, but free of symptoms and off all treatment for at least 4 years, 29% were found to have EIB (Sinclair et al. 1995). Further, 18% of people with a past history of asthma found medically fit to scuba dive were shown to have positive responses to hyperosmolar saline, suggesting that they still had active airway inflammation and could have been at increased risk for problems while diving (Anderson et al. 1995, 2006b). The hyperresponsiveness to exercise, hyperpnea of dry air, and hyperosmolar aerosols is markedly reduced and, in 50% of cases, abolished in patients with mild to moderate asthma who are given 8 weeks treatment with steroids (Waalkans et al. 1993; Anderson et al. 1994; du Toit et al. 1997; Brannan et al. 2002, 2005; Koskela et al. 2003). Thus these provoking stimuli have been used to monitor response to treatment and asthma control. A negative response to a hyperosmolar aerosol challenge after treatment is indicative of control of EIB and it may also provide an opportunity to start back-titration of steroid dose (Leuppi et al. 2001). A negative response on treatment with ICS may permit a person to enter an occupation previously not open to people with asthma. If a known asthmatic tests positive while on treatment with inhaled steroids, the physician should check that the patient is adhering to the prescribed dose. If so, consideration should be given to increasing the dose of inhaled steroid and to repeating the challenge at a later time. A positive response to these tests indicates currently active asthma consistent with potential for EIB and potential to respond to treatment with ICS. It is also reasonable to consider that negative responses to the surrogate challenges for EIB as being consistent with normality or mild asthma of insufficient severity to require any treatment.

Who to challenge

Challenge with pharmacologic agents to identify EIB

Exercise is used to make a diagnosis of EIB in asthmatic patients and those suspected of having asthma with a history of breathlessness during or after exertion. Many people, particularly those with a past history of asthma, may have avoided vigorous exercise or they do not consider the breathlessness of vigorous exercise to be a sign or symptom of EIB. These reasons provide a good argument for objective testing to detect EIB, particularly in those for whom it may have relevance to an occupational setting. While symptoms such as breathlessness, wheeze, cough, and mucus production may be indicative of EIB in a clinically recognized asthmatic, they are poor predictors in athletes (Holzer et al. 2002), in whom it has been reported that 50% with symptoms do not have EIB (Rundell et al. 2001; Thole et al. 2001; De Baets et al. 2005). Many healthy subjects, particularly regular runners, may have exercise-induced cough that is not associated with airway narrowing (Rundell et al. 2001), and cough when related to exercise should not be thought synonymous with EIB.

EIB can neither be excluded by a negative response to challenge with methacholine or histamine, nor confirmed by a positive response. The exclusion aspect has been noted particularly in those with normal lung function such as schoolchildren (Backer et al. 1991; Haby et al. 1994), adult athletes (Holzer et al. 2002), and defense force recruits (Nish & Schwietz 1992). Finding a person with good lung function negative to pharmacologic challenge but positive to exercise, or surrogate challenge for exercise, would not be an expectation of most respiratory physicians. The findings may be due to the mediators associated with EIB. Thus leukotrienes and prostaglandins are 1000 and 100 times, respectively, more potent in provoking airway narrowing than histamine or methacholine (O’Byrne 1997). This may also explain why EIB may be the first sign of asthma. Ernst et al. (2002) found that airway hyperresponsiveness to methacholine at 14 years was predicted by the presence of EIB at 6 years, and similar implications were made by others (Lombardi et al. 1997; Porsbjerg et al. 2005).

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For these reasons pharmacologic challenges are not recommended for excluding EIB in recruits for the defense forces, in clearance for scuba diving, or in other professions and sports where EIB would be potentially hazardous on the job (Anderson et al. 2006b).

Prevention of EIB In asthmatic children with good lung function, chronic treatment with ICS as single therapy (i.e., not in combination with a β2 agonist) can markedly reduce the fall in FEV1 after exercise (Fig. 37.3) (Pedersen & Hansen 1995; Hofstra et al. 2000; Jonasson et al. 2000; Subbarao et al. 2006). Similar benefits have been documented in adults taking ICS as single therapy (Weiler et al. 2005). This beneficial response to ICS appears to be universal in the asthmatic population. Chronic treatment with a leukotriene receptor antagonist (LTRA) in children (Kemp et al. 1998) and adults (Villaran et al. 1999; Edelman et al. 2000) reduces the severity of EIB and enhances recovery from any residual EIB (Storms et al. 2004). A major advantage in using leukotriene receptor antagonists for control of EIB is that tolerance does not occur with daily treatment and, in the event of breakthrough EIB, the recovery of lung function is rapid and usually within 10 min (de Benedictis et al. 2006). For subjects who do not have normal lung function at rest, adding a long-acting β2 agonist (LABA) in combination with ICS is very effective in preventing EIB (Weiler et al. 2005). LABAs alone have also been shown to be effective against EIB in single doses (Bronsky et al. 2002; Richter et al. 2002; Shapiro et al. 2002), and there is little or no difference between salmeterol and formoterol in the onset of the protective effect (Richter et al. 2002). β2 Agonists probably prevent EIB by inhibiting mast cell release of mediators in addition to antagonizing the bronchoconstricting effects of these mediators at the smooth muscle (Church & Hiroi 1987; Anderson & Brannan 2004; Gebhardt et al. 2005). Unfortunately, tolerance to β2 agonists is common. This tolerance manifests itself as a reduction in duration of protection from EIB (Nelson et al. 1998), and this is not prevented by using LABAs in combination with ICS (Kalra et al. 1996; Simons et al. 1997). Because of the tolerance issue (Anderson & Brannan 2004), LABAs alone are recommended only when the need to control EIB is intermittent, and preferably three or fewer times per week (Davis et al. 2003). The tolerance issue appears to have been overlooked by those willing to prescribe extra doses of β2 agonist before exercise. Prescribing extra doses contributes further to the problem of tolerance, a consequence of which may be more symptoms and a perceived need to increase the dose of steroids. Tolerance to β2 agonists is most evident on the mast cell release of mediators (Scola et al. 2004; Tsuji et al. 2004; McGraw & Liggett 2005; Peachell 2006). It is well described that tolerance to the acute bronchodilating effect of

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short-acting β2 agonists (SABAs) also occurs with chronic use of either a SABA (Jones et al. 2001) or a LABA (van der Woude et al. 2001; Haney & Hancox 2005). The more severe the bronchoconstriction, the more evident the tolerance to the bronchodilator effects (Wraight et al. 2003) and this could be of some concern for those with EIB. In people with good lung function, SABAs are commonly recommended as first-line treatment to be taken immediately before exercise. When used intermittently these drugs are very effective in preventing EIB. As with LABAs, SABAs also induce tolerance at the mast cell (Chong et al. 2003; Peachell 2006), and patients should be made aware that frequent use may result in dose escalation and worsening of EIB (Inman & O’Byrne 1996; Hancox et al. 2002; Anderson et al. 2006c) or asthma provoked by an allergen (Swystun et al. 2000). Sodium cromoglycate (2, 10 or 20 mg) and nedocromil sodium (usually 4 or 8 mg) provide about 50–80% protection from EIB (Schoeffel et al. 1983; Tullett et al. 1985; Albazzaz et al. 1989; Pfleger et al. 2002) and they enhance recovery from EIB (Spooner et al. 2000). These drugs are instantaneously effective and can be taken immediately before exercise. For more details the reader is referred to reviews on treatment of EIB in asthmatics (Massie 2002; Tan & Spector 2002; Anderson 2004; Moraes & Selvadurai 2004). In the presence of sodium cromoglycate, the increase in 9α,11β-PGF2 release is inhibited in response to a hyperosmolar aerosol, and blocking either the production or release of PGD2 may be the mode of action whereby sodium cromoglycate prevents EIB. It is not known if nedocromil sodium has the same effect. The LABA formoterol also inhibits PGD2 release in response to an osmotic stimulus; however, neither sodium cromoglycate nor formoterol have a significant inhibitory effect on the release of leukotrienes in response to the same stimulus (Brannan et al. 2006).

Nonpharmacologic methods for preventing EIB There are a number of techniques that can be used to prevent EIB without recourse to the use of pharmacologic agents. Any intervention that reduces respiratory water loss or increases delivery of water to the airways may be expected to reduce the severity of EIB. For example, EIB can be prevented or markedly reduced by inhaling air of alveolar conditions (34– 37°C, 100% relative humidity) (Chen & Horton 1977; Strauss et al. 1978; Anderson et al. 1982) but this is impractical and not recommended. Breathing through the nose also reduces severity of EIB by reducing the air-conditioning burden on the lower airways. However, the resistance of the upper airways limits the nasal flow to about 35 L/min, and breathing by mouth is usual above this moderate ventilation rate. Masks and mouthpieces that capture expired water and permit some degree of rebreathing (Nisar et al. 1992; Millqvist et al. 1995; Beuther & Martin 2006) have been developed and used successfully. For example, one mask with low resistance at high flow rates will recover 42% of water at 16°C

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(Nisar et al. 1992), and some of this water will be available for rebreathing. One important nonpharmacologic approach for preventing EIB is to recognize if the person becomes refractory to the exercise stimulus. Refractoriness can be identified by repeating exactly the same exercise task after spontaneous recovery from EIB; 50% of people will have less than half the severity of the response when the task is repeated within 1 hour. Many who participate in competitive sport have recognized that they can become refractory to the effects of exercise and choose to exercise and have their EIB 30– 60 min before the “real game” begins.

Physical training and EIB It has been suggested that low physical fitness is associated with developing asthma in young adults (Rasmussen et al. 2000), but it is possible that these people have avoided exercise unconsciously because of EIB. Thus attention has been given to improving physical fitness and the role it plays in controlling EIB (Emtner et al. 1996; Hallstrand et al. 2000; Ram et al. 2005). With appropriate medication to prevent EIB, fitness can be improved in most asthmatics by exercising regularly. Physical training is associated with an increase in cardiopulmonary fitness as reflected by an increase in oxygen consumption, a decrease in heart rate, and a decrease in ventilatory equivalent (Hallstrand et al. 2000; Ram et al. 2005). As ventilation rate is a major determinant of the severity of EIB, a general increase in fitness will increase the threshold for EIB. The benefits of training on well-being are well described, although there is no improvement in lung function with training (Ram et al. 2005). Further, EIB severity is not affected when the person is tested at the same ventilation after physical training (Fitch et al. 1986). This is not surprising as the stimulus for EIB, i.e., respiratory water loss, will still be the same. Sprints or warm-ups are likely to increase the rate of delivery of water to the airway mucosa by increasing bronchial blood flow. Thus after warm-up the airways are able to cope better with the dehydrating effects of hyperpnea.

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cold-air hyperventilation in normal and in asthmatic children in a survey of 5,697 schoolchildren in southern Bavaria. Am Rev Respir Dis 147, 565–72. Nielsen, K.G. & Bisgaard, H. (2000) Lung function response to cold air challenge in asthmatic and healthy children of 2–5 years of age. Am J Respir Crit Care Med 161, 1805– 9. Nisar, M., Spence, D.P.S., West, D. et al. (1992) A mask to modify inspired air temperature and humidity and its effect on exercise Induced asthma. Thorax 47, 446– 50. Nish, W.A. & Schwietz, L.A. (1992) Underdiagnosis of asthma in young adults presenting for USAF basic training. Ann Allergy 69, 239– 42. O’Byrne, P.M. (1997) Leukotrienes in the pathogenesis of asthma. Chest 111 (suppl. 2), 27S–34S. O’Byrne, P.M., Jones, G.L. (1986) The effect of indomethacin on exercise-induced bronchoconstruction and refractoriness after exercise. Am Rev Respir Dis 134, 169–72. O’Sullivan, S., Roquet, A., Dahlén, B. et al. (1998) Evidence for mast cell activation during exercise-induced bronchoconstriction. Eur Respir J 12, 345– 50. Patel, K.R. (1984) Terfenadine in exercise-induced asthma. BMJ 85, 1496–7. Peachell, P. (2006) Regulation of mast cells by β2-agonists. Clin Rev Allergy Immunol 31, 131– 42. Pearlman, D.S., Ostrom, N.K., Bronsky, E.A., Bonuccelli, C.M. & Hanby, L.A. (1999) The leukotriene D4 receptor antagonist zafirlukast attenuates exercise-induced bronchoconstriction in children. J Pediatr 134, 273– 9. Pedersen, S. & Hansen, O.R. (1995) Budesonide treatment of moderate and severe asthma in children: a dose–response study. J Allergy Clin Immunol 95, 29– 33. Peroni, D.G., Piacentini, G.L., Pietrobelli, A. et al. (2002) The combination of single-dose montelukast and loratadine on exerciseinduced bronchospasm in children. Eur Respir J 20, 104–7. Pfleger, A., Eber, E., Weinhandl, E. & Zach, M.S. (2002) Effects of nedocromil and salbutamol on airway reactivity in children with asthma. Eur Respir J 20, 624– 9. Phillips, Y.Y., Jaeger, J.J., Laube, B.L. & Rosenthal, R.R. (1985) Eucapnic voluntary hyperventilation of compressed gas mixture. A simple system for bronchial challenge by respiratory heat loss. Am Rev Respir Dis 131, 31– 5. Ponsonby, A.-L., Couper, D., Dwyer, T., Carmichael, A. & WoodBaker, R. (1996) Exercise-induced bronchial hyperresponsiveness and parental ISAAC questionnaire responses. Eur Respir J 9, 1356– 62. Porsbjerg, C., von Listow, M.L., Ulrik, C.S., Nepper-Christensen, S.C. & Backer, V. (2005) Outcome in adulthood of asymptomatic airway hyperresponsivenss to histamine and exercise-induced bronchospasm in childhood. Ann Allergy Asthma Immunol 95, 137– 42. Provost-Craig, M.A., Arbour, K.S., Sestili, D.C., Chabalko, J.J. & Ekinci, E. (1996) The incidence of exercise-induced bronchospasm in competitive figure skaters. J Asthma 33, 67–71. Ram, F.S.F., Robinson, S.M., Black, P.N. & Picot, J. (2005) Physical training for asthma. Cochrane Database Syst Rev 4, CD001116. Rasmussen, F., Lambrechtsen, J., Siersted, H.C., Hansen, H.S. & Hansen, N.C. (2000) Low physical fitness in childhood is associated with the development of asthma in young adulthood: the Odense schoolchild study. Eur Respir J 16, 866–70.

Exercise-induced Bronchoconstriction: Human Models

Reiss, T.F., Hill, J.B., Harman, E. et al. (1997) Increased urinary excretion of LTE4 after exercise and attenuation of exerciseinduced bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax 52, 1030–5. Richter, K., Janicki, S., Jorres, R.A. & Magnussen, H. (2002) Acute protection against exercise-induced bronchoconstriction by formoterol, salmeterol and terbutaline. Eur Respir J 19, 865–71. Riedler, J. (1997) Nonpharmacological challenges in the assessment of bronchial responsiveness. Eur Respir Monogr 5, 115–35. Riedler, J., Reade, T., Dalton, M., Holst, D.I. & Robertson, C.F. (1994) Hypertonic saline challenge in an epidemiological survey of asthma in children. Am J Respir Crit Care Med 150, 1632–9. Roca, J., Whipp, B.J., Agustí, A.G.N. et al. (1997) Clinical exercise testing with reference to lung diseases: indications, standardization and interpretation strategies. Position document of the European Respiratory Society. Eur Respir J 10, 2662–89. Roger, L.J., Kehrl, H.R., Hazucha, M. & Horstman, D.H. (1985) Bronchoconstriction in asthmatics exposed to sulfur dioxide during repeated exercise. J Appl Physiol 59, 784–91. Rouhos, A., Ekroos, H., Karjalainen, J., Sarna, S. & Sovijarvi, A.R. (2005) Exhaled nitric oxide and exercise-induced bronchoconstriction in young male conscripts: association only in atopics. Allergy 60, 1493–8. Rundell, K. & Spiering, B.A. (2003) Inspiratory stridor in elite athletes. Chest 123, 468–74. Rundell, K.W., Wilber, R.L., Szmedra, L., Jenkinson, D.M., Mayers, L.B. & Im, J. (2000) Exercise-induced asthma screening of elite athletes: field vs laboratory exercise challenge. Med Sci Sports Exerc 32, 309–16. Rundell, K.W., Im, J., Mayers, L.B., Wilber, R.L., Szmedra, L. & Schmitz, H.R. (2001) Self-reported symptoms and exercise-induced asthma in the elite athlete. Med Sci Sports Exerc 33, 208–13. Rundell, K.W., Spiering, B.A., Judelson, D.A. & Wilson, M.H. (2003) Bronchoconstriction during cross-country skiing: is there really a refractory period? Med Sci Sports Exerc 35, 18–26. Rundell, K.W., Anderson, S.D., Spiering, B.A. & Judelson, D.A. (2004a) Field exercise vs laboratory eucapnic voluntary hyperventilation to identify airway hyperresponsiveness in elite cold weather athletes. Chest 125, 909–15. Rundell, K.W., Spiering, B.A., Evans, T.M. & Baumann, J.M. (2004b) Baseline lung function, exercise-induced bronchoconstriction, and asthma-like symptoms in elite women ice hockey players. Med Sci Sports & Exerc 36, 405–10. Sano, F., Sole, D. & Naspitz, C.K. (1998) Prevalence and characteristics of exercise-induced asthma in children. Pediatr Allergy Immunol 9, 181–5. Schoeffel, R.E., Anderson, S.D. & Altounyan, R.E. (1981) Bronchial hyperreactivity in response to inhalation of ultrasonically nebulised solutions of distilled water and saline. BMJ 283, 1285–7. Schoeffel, R.E., Anderson, S.D. & Lindsay, D.A. (1983) Sodium cromoglycate as a pressurized aerosol (Vicrom) in exercise-induced asthma. Aust NZ J Med 13, 157–61. Scola, A.M., Chong, L.K., Suvarna, S.K., Chess-Williams, R. & Peachell, P.T. (2004) Desensitisation of mast cell β2-adrenoceptormediated responses by salmeterol and formoterol. Br J Pharmacol 141, 163–71. Shapiro, G.S., Yegen, Ü., Xiang, J., Kottakis, J. & Della Cioppa, G. (2002) A randomized, double-blind, single-dose, crossover clinical trial of the onset and duration of protection from exercise-induced

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Sensory and Autonomic Nervous System in Asthma and Rhinitis Bradley J. Undem and Kevin Kwong

Summary Studies of nerve–immune interactions often focus on mechanisms by which the nervous system can modulate immunity. With respect to inflammatory disease there has been a longstanding interest in nerve-mediated (neurogenic) inflammation. In allergic airway disease, however, it may be more useful to reverse the arrow of this interaction and evaluate the neuronal consequence of IgE-mediated inflammation. This chapter, in large part, focuses on this issue. Many of the signs and symptoms of allergic airway disease can be attributable to neural dysregulation. These include the self-evident neuronal symptoms of itchy sensations, constant sneezing, dry nonproductive coughing, inappropriate sensations of air hunger, as well as less conscious corruptions in autonomic reflexes including excessive secretions and inappropriate reflex bronchoconstriction. Moreover, the perceived triggers of symptoms in allergic rhinitis and asthma are often not only allergens but also emotions and inhalation of nonallergenic irritants. To explain this, one might logically hypothesize that the airway nervous system itself is a target of the allergic inflammatory reaction. In fact, in the laboratory, allergic inflammation has been shown to modulate all aspects of airway innervation, including modulation of the excitability of sensory nerve terminals, synaptic modulation in the central nervous system (CNS), and changes in activity of the autonomic airway parasympathetic nerves. This modulation may involve acute and short-lasting mechanisms, or long-lasting phenotypic changes in the nerves. This chapter provides an overview of airway innervation, how airway neurophysiology may change in the face of allergic inflammation, and how this may contribute to allergic airway diseases.

Introduction Nerves innervating the upper and lower airways play important roles in both health and disease. The reflexes initiated on activation of the airway afferent (sensory) nerves help estabAllergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

lish physiologic homeostasis and provide for defense against the inhalation of potentially damaging substances (e.g., productive cough, bronchoconstriction, increase in mucus production). However, reflex functions of the very same nerves may become exaggerated or corrupted such that what was once an appropriate physiologic response is perverted into a pathophysiologic one. In addition, sensations arising from stimulation of respiratory afferent nerve fibers (e.g., urge to cough, urge to sneeze, dyspnea) can also become exaggerated to such an extent as to become inappropriate so that they also contribute to airway pathology (e.g., incessant urge to cough even when there is nothing to “cough-up” as in the dry nonproductive cough). In this chapter we provide a brief overview of the anatomy and physiology of upper and lower airway innervation. This is followed by two additional sections, one on how the nervous system may change in the face of allergic inflammation, and one that draws on experimental and clinical observations to discuss the potential mechanisms by which the nervous system contributes to asthma and rhinitis.

Basic neurophysiology of the airways Upper airways Sensory (afferent) innervation The neural pathways of nasal innervation are illustrated in Fig. 38.1. The lining of the nasal cavity is divided into the olfactory mucosa, which is situated at the roof of the nasal cavity and whose main function is odor detection, and the respiratory mucosa, which is responsible in part for conditioning inhaled air. Although the olfactory nerves innervating the olfactory mucosa play an obvious role in avoidance behaviors in response to potentially noxious or offending odors, no known evidence exists which indicate that they participate in the respiratory functions of the normal nose or in the etiology of rhinitis. The sensory nonolfactory nerves of the nasal respiratory mucosa arise from cell bodies situated in the trigeminal ganglion. Central projections of neurons innervating the nasal mucosa have been mapped to the subnucleus caudalis and subnucleus interpolaris of the medulla, areas considered to

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Blood vessel

Trigeminal ganglion

Gland Sphenopalatine (parasympathetic) ganglion

Sympathetic ganglion

be involved in sensations of pain and temperature (Anton & Peppel 1991; Wallois et al. 1995). The primary afferent nerves in the nasal respiratory mucosa comprise both myelinated (A) and unmyelinated (C) fibers. Functional evidence (e.g., conduction velocity) on the exact types of fibers innervating the nasal mucosa is sparse (Lucier & Egizii 1989). However, based on studies in experimental animals and on analogies from studies on the lower airways and other visceral systems, it would seem likely that the A-fibers are low-threshold mechanosensors that can respond to light touch or airflow, whereas many of the Cfibers are nociceptors responding to noxious inputs that are potentially tissue damaging (e.g., excessive temperatures, large osmolarity shifts) and to events associated with tissue inflammation (Lucier & Egizii 1989).

Autonomic (efferent) innervation Parasympathetic The parasympathetic innervation of the nasal airways originates from the facial nucleus of the brainstem and the superior salivatory nucleus. Cholinergic preganglionic fibers follow the greater superficial petrosal nerve and the vidian nerve to synapse on neurons clustered in the sphenopalatine ganglion (also known as the pterygopalatine ganglion) (Cauna 1982). The postganglionic fibers are distributed to the nasal mucosa via the branches of the posterior nasal nerve. Postganglionic fibers innervate serous and mucous glands, arteries, veins, and arteriovenous anastomoses (Cauna et al. 1969, 1972; Cauna 1970a,b; Ishii 1970; Nomura & Matsuura 1972; Cauna & Cauna 1975). Postganglionic fibers release acetylcholine that acts on muscarinic receptors in the membranes of various effector cells. There are five muscarinic receptor subtypes, M1–M5 (Wess 1993). The most extensively distributed of all the muscarinic receptor subtypes is the M3 receptor, which is found on arteries and veins, and is responsible for cholinergic glandular secretions (Nakaya et al. 2002). In addition to

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Fig. 38.1 The extrinsic (sensory and autonomic) innervation of the nasal respiratory mucosa.

acetylcholine, postganglionic fibers innervating the nose may contain neuropeptides such as vasoactive intestinal peptide (VIP) and express nitric oxide synthase, the enzyme that catalyzes nitric oxide (NO) production. Two major sources of nasal secretions are serous/mucous glands and plasma extravasation from blood vessels in the mucosa. Based on the efficacy of anticholinergic drugs, it appears that most of the secretory effects of parasympathetic nerve activation are dependent on cholinergic muscarinic receptor activation (Bates et al. 1971). Stimulation of the parasympathetic nerves causes glandular secretions rich in mucous glycoproteins, lactoferrin, lysozyme, secretory leukoprotease inhibitor, neutral endopeptidase, and secretory IgA (Raphael et al. 1991; Kaliner 1992). Direct stimulation of parasympathetic nasal nerves may also elicit vasodilation of vessels in the nasal mucosa through both cholinergic and noncholinergic mechanisms (Änggård 1974, 1977). The noncholinergic component (atropine resistant) may be due to either neuropeptide and/or NO release from the nerves as both of these transmitters are effective vasodilators. Although immunohistochemical evidence indicates that there are nitric oxide synthase-immunopositive nerves innervating the human nasal mucosa (Riederer et al. 1999), a nonadrenergic, noncholinergic (NANC) neural control of the nasal blood flow has not been thoroughly explored (Hanazawa et al. 1997; Lacroix et al. 1998; Okita & Ichimura 1998).

Sympathetic Sympathetic neural output to the human nose originates from preganglionic fibers in the thoracolumbar region of the spinal cord that synapse on neurons in the superior cervical ganglion (Dahlstroem & Fuxe 1965; Baroody & Canning 2003). The postganglionic fibers form the petrosal nerve, which joins the greater superficial nerve to form the vidian nerve. Thus the vidian nerve contains both the parasympathetic and sympathetic innervation to the nasal mucosa. The postganglionic sympathetic nerves contain catecholamines,

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but may also contain neuropeptides, most notably neuropeptide Y (NPY). Sympathetic nerve stimulation induces vasoconstriction and increases nasal airway patency (Rooker & Jackson 1969; Malm 1977; Eccles 1983; Lacroix 1989; Revington et al. 1997; Kawarai & Koss 2001). There is also some evidence that sympathetic activity can induce airway glandular secretion through stimulation of serous cells (Baraniuk 1992). The effects of sympathetic nerves are mediated mainly through adrenoreceptors. Stimulation of α1- and α2-adrenoceptors on the smooth muscle of resistance vessels (which control blood flow) and of venous sinusoids (which are responsible for blood pooling leading to mucosal engorgement and modulation of nasal airway resistance) leads to vasoconstriction and consequently to reduced blood flow and reduced blood pooling. Activation of β1- and β2-adrenoceptors, also present on the vasculature of the nasal mucosa, can lead to vasodilation of resistance vessels and increased blood flow (Malm 1977; Wang & Lung 2003). However, the effects of βreceptor stimulation are less pronounced than those induced by α-receptor stimulation. NPY, the peptide often colocalized with norepinephrine in the nasal mucosa, also causes vasoconstriction and decongestion (Fischer et al. 1993; Malmström et al. 1996; Cervin et al. 1999; Tai & Baraniuk 2002).

Sensory neuropeptides and neurogenic inflammation Generally speaking, stimulation of airway sensory nerves regulates end-organ activity by increasing or decreasing autonomic reflex tone. However, there is substantial evidence for the existence of local reflex regulation of end-organ activity that occurs independently of centrally mediated autonomic mechanisms. An interesting characteristic of a subset of Cfibers is that neuropeptides such as substance P, neurokinin A, and calcitonin gene-related peptide are stored in the peripheral (and central) terminals where they can be released on activation. The concept is that action potential discharge initiated at a terminal site of the axon reaches a branch in the nerves where it then antidromically (away from the CNS) travels back down a collateral branch of the same nerve resulting in the local release of bioactive peptides. This process is referred to as the “axon reflex,” and was originally evaluated in the skin as contributing the flare component of the triple response (see Chapman 1977). Release of neuropeptides from sensory nerve endings can lead not only to vasodilatation but also to increase leukocyte infiltration and increased vascular permeability. These events are collectively referred to as neurogenic inflammation (Lundblad et al. 1984). Additional effects of these neuropeptides in human nose include glandular activation (Baraniuk et al. 1991, 1999) and modulation of ciliary beat frequency (Schuil et al. 1995a,b; Smith et al. 1996). The neurogenic inflammation evoked via sensory C-fiber activation appears to be well developed in guinea-pig and rodent airways, but its role in human airways is less obvious. Some afferent nerves in the human nasal mucosa store sensory neuropeptides in their terminals. Moreover, the neurokinin (NK)1 receptor, which is activated by substance P and neurokinin A, has been identified on human nasal glands and epithelium (Shirasaki et al. 1998) and the NK2 receptor, which is preferentially activated by neurokinin A, has been identified on nasal mucosal blood vessels (Shirasaki et al. 2004). Concentrations of the C-fiber stimulant capsaicin that are sufficient to cause pain also cause plasma extravasation and inflammatory cell influx, suggesting that neurogenic inflammation may indeed take place in the human nose (Sanico et al. 1997). Baraniuk and colleagues used hypertonic saline to activate afferent nerves nonselectively and noticed a nasal secretory response, but they failed to generate evidence of plasma extravasation despite the demonstration of substance P release (Baraniuk et al. 1999). As discussed below the extent to which neurogenic inflammation is evoked upon nasal afferent activation may be heightened when studied in the face of ongoing allergic inflammation (Sanico et al. 1998).

Nasal reflex physiology Activation of afferent nerves in the respiratory mucosa can lead to changes in autonomic outflow to the nose, sneezing reflexes, and reflex changes in breathing patterns. Stimulation of nasal afferent nerves can also lead to tactile sensations, sensations of cooling and burning, and pain. Selective stimulation of C-fibers with capsaicin leads to sensations of burning pain and increases in autonomic reflex nasal secretion, but does not consistently cause sneezing (Philip et al. 1994). Based on analogies with itch, it has been hypothesized that a subset of histamine-sensitive C-fibers may underlie the sneezing reflex, but the urge to sneeze sensation may be masked (overwhelmed by painful sensations) when capsaicin is used to stimulate all C-fibers (Taylor-Clark et al. 2005). Activation of nasal afferent nerves can also alter autonomic outflow to the lower respiratory tract (Togias 2004). This provides one mechanism by which changes in the nose can lead to increases in airflow resistance in the lower airways. The nasal cycle reveals the potential for the autonomic nervous system to cause substantial nasal congestion (Stoksted 1952; Eccles 1978). In a majority of humans, there is a cycling of resistance to nasal airflow between the two nostrils. This so-called nasal cycle appears to be due to unilateral cyclical changes in parasympathetic and sympathetic drive to the nasal vasculature. The congestion occurs concurrently with a decrease in sympathetic drive, and perhaps an increase in parasympathetic drive. The nasal cycle is driven by central mechanisms (Eccles & Lee 1981; Bamford & Eccles 1982) and does not appear to involve nasal afferent nerves.

Lower airways The lower airways of all mammals, including humans, are densely innervated with sensory (afferent) and autonomic (efferent) nerves. Histologic studies reveal a dense network of nerve fibers forming a plexus near the smooth muscle and just beneath the epithelium. Whole-mount preparations nicely

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Fig. 38.2 Nerves revealed with PGP 9.5 staining in whole mounts of rat trachea (left), guinea-pig trachea (middle), and human bronchus (right). The whole mounts are situated such that the subepithelial plexus is seen. (Courtesy of Drs Stuart Mazzone and Brendan Canning.)

illustrate this subepithelial plexus (Fig. 38.2). The general scheme of lower airway innervation is illustrated in Fig. 38.3.

Sensory (afferent) innervation Afferent (sensory) nerves represent the most abundant nerve type innervating the lower airways (Agostoni et al. 1957). There are three sources of afferent innervation to the lower airways. Most of the afferent fibers are vagal afferents with their cell bodies in the nodose vagal ganglia. In addition, neurons in the jugular vagal (or supranodose) ganglia also project vagal afferent fibers to the lower airways (Kummer et al. 1992). The third source comprises spinal afferent nerves with cell bodies residing in the dorsal root ganglion that also innervate the lungs (Plato et al. 2006). The primary afferent fibers innervating the lower airways are populated by myelinated A-fibers and unmyelinated Cfibers. In addition these fibers are further classified based on functional properties into three major groups: bronchopulmonary C-fibers, slowly adapting receptors, and rapidly adapting receptors (Widdicombe 1982; Coleridge & Coleridge 1984). By convention, small afferent nerves with unmyelinated axons that conduct action potentials at relatively slow

velocities (0.1–2 m/s) are referred to as C-fibers. Afferent nerves with larger myelinated axons that conduct action potentials at relatively high velocities (3–50 m/s) are referred to as A-fibers. The A-fibers comprise slowly adapting receptors (SAR) and rapidly adapting receptors (RAR). While SARs possess slightly higher conduction velocities than RARs, the main factor differentiating the two is their pattern of action potential discharge in the face of prolonged lung inflation (Sant’Ambrogio 1982). SARs are highly mechanosensitive neurons that maintain action potential discharge on stimulation with little or no attenuation. RARs are so named because of their quick attenuation in action potential discharge (on the order of a few seconds) in response to a prolonged mechanical stimulus (Knowlton & Larrabee 1946). The SARs are the most mechanically sensitive afferent nerves in the lungs and are thought to be involved in the mechanoception of breathing (Coleridge & Coleridge 1984). SARs are known as “stretch receptors” because they function to regulate breathing pattern: as lung volume increases and the airway wall is stretched, fiber SAR activity increases, eliciting an inhibition of inspiratory activity and lengthening of expiratory time (Bartlett & St John 1979; Coleridge &

Blood vessel Airway smooth muscle

Jugular ganglion

A i r w a y

Nodose ganglion

Vagus nerve Gland Parasympathetic ganglion

Sympathetic ganglion

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e p i t h e l i u m Fig. 38.3 The basic wiring diagram of the sensory and autonomic innervation of the lower airways.

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Coleridge 1984). The RAR and SAR fibers are generally insensitive to chemical stimuli, except when the stimulus indirectly results in mechanical changes in the tissues (e.g., bronchoconstriction, vascular edema) (Dawes et al. 1951; Coleridge & Coleridge 1984). One exception to this may be ATP that can act on P2X receptors to directly stimulate lung stretch sensors (Canning et al. 2004). In contrast to RARs and SARs, many inflammatory mediators elicit action potential discharge in bronchopulmonary C-fibers independently of mechanical events (Carr & Undem 2003). Many of the stimuli that activate C-fibers are associated with inflammation or potential tissue injury (excessive heat or stretch). These fibers are therefore thought to provide the tissue with a sense of its own potential injury; accordingly they are often referred to as nociceptors. The C-fiber endings can be directly activated by a variety of stimuli, e.g., capsaicin, noxious heat, bradykinin, ATP, adenosine, 5hydroytryptamine, hypertonic saline, mechanical stimuli, and acid (Coleridge & Coleridge 1984; Lee & Pisarri 2001; Carr & Undem 2003). The C-fibers constitute about 75% of pulmonary afferents and provide the primary chemosensory input from lungs and airways (Evans & Murray 1954; Agostoni et al. 1957). Central terminations for each broadly defined type of sensory neuron (SAR, RAR and bronchopulmonary C-fibers) occur at distinct largely nonoverlapping loci in the middle and caudal portions of the nucleus tractus solitarii (NTS) (Kubin et al. 2006).

major parasympathetic NANC neurotransmitters identified thus far in human airways are NO and VIP (Laitinen et al. 1985; Ward et al. 1995). When the action potential threshold is reached, the impulses travel down the postganglionic axon triggering the release of acetylcholine or NANC neurotransmitters at the effectors cells (smooth muscle, glands, and vasculature). The parasympathetic nervous system is the dominant regulator of airway smooth muscle tone in all mammals including human (Canning & Fischer 2001). These nerves control the smooth muscle in the trachea to the terminal bronchioles. In humans, the only autonomic contractile innervation is cholinergic. Although bronchial smooth muscle expresses both muscarinic M2 and M3 receptors, the latter are responsible for cholinergic contractions. There are muscarinic M2 receptors on the cholinergic terminals that serve to inhibit acetylcholine release. Thus, selectively blocking muscarinic M2 receptors in human bronchi can actually enhance nerve-evoked cholinergic contractions (ten Berge et al. 1996). The parasympathetic nerves (NANC) also provide the only relaxant innervation to human bronchial smooth muscle. The parasympathetically mediated relaxations are inhibited by drugs that block the production of NO (Belvisi et al. 1992; Ellis & Undem 1992). Tracheobronchial submucosal glands and goblet cells receive both parasympathetic and sympathetic efferent innervation, though they are dominated by parasympathetic tone. The neurogenic increase in mucus secretion in human airways is largely cholinergic via muscarinic M3 receptor stimulation (Barnes 1993). The parasympathetic system may play a role in mucociliary clearance in the upper airways. Administration of atropine, a muscarinic receptor antagonist, decreases ciliary beat frequency, suggesting that the role is stimulatory (Wanner 1988). Increases in parasympathetic outflow to the airways results in bronchial and pulmonary vasodilation. In experimental animals, this neurogenic relaxation of the bronchial vasculature is mediated through both cholinergic and NANC mechanisms (Widdicombe 1990).

Autonomic (efferent) innervation Parasympathetic The majority of autonomic nerves innervating the human lower airways are parasympathetic in nature. The preganglionic parasympathetic neurons are situated in the brainstem in and around the compact formation of the nucleus ambiguus, the dorsal motor nucleus of the vagus, and in the reticular formation located between these nuclei. These neurons are rhythmically active during eupnic breathing and send volleys of action potentials in excess of 25 impulses per second down the preganglionic axons. The preganglionic parasympathetic fibers arise at the airway via the vagus nerves, where they synapse with principal ganglion neurons (postganglionic neurons) located in small ganglia within or near the airway wall. Much of the preganglionic input is filtered at the parasympathetic ganglion because the excitatory postsynaptic potentials fail to reach action potential threshold (Myers 2001). Parasympathetic ganglia are located mainly in the larger airways, but the postganglionic fibers innervate the conducting airways to the bronchioles (Canning & Fischer 2001). Thus, the ganglia neurons function to filter, integrate, and distribute parasympathetic input to the airways. Neurons in the bronchial parasympathetic ganglia are cholinergic (release acetylcholine) and NANC, or both. The

Sympathetic Human bronchial smooth muscle receives little, if any, sympathetic innervation. As mentioned above, both the contractile and relaxant innervation is derived from postganglionic parasympathetic nerve fibers. However, sympathetic β2-adrenoceptor mediated bronchial smooth muscle relaxation can occur via circulating epinephrine derived from the adrenal medulla. As in other organs the sympathetic nervous system innervates the blood vessels in the airways and lungs. The pulmonary and bronchial circulations are under adrenergic control, and perhaps also under nonadrenergic (NPY) sympathetic control (Widdicombe 1990). Sympathetic transmitters can constrict and relax vascular smooth muscle, but studies in

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Physiology vagi are cut, or if large doses of anticholinergic drugs are administered, the baseline smooth muscle tone is essentially abolished and the airways maximally dilate (Fig. 38.4, bottom). Stimulation of SARs leads to decreases in tonic parasympathetic outflow to the lungs, whereas RAR and bronchopulmonary C-fiber activation is thought to increase airway parasympathetic tone and, as a consequence, bronchoconstriction and vasodilation (Canning 2006).

Control

Control

Albuterol

Ipratropium (low dose)

Ipratropium (large dose)

Fig. 38.4 (Top) Matched high-resolution computed tomography (CT) images of three airways (arrows) from one patient at baseline and after albuterol treatment. (Bottom) Matched high-resolution CT images of airways of approximately 4 mm in diameter (arrows) from one dog at control (left), after 10 mg/mL ipratropium aerosol (center), and after 1000 mg/mL ipratropium aerosol. (From Groeben & Brown 1996, with permission.)

experimental animals would indicate that the net effect of increasing sympathetic outflow to the lungs is one of vasoconstriction.

Reflexes in lower airways Autonomic reflexes Mammalian airways are under tonic parasympathetic tone (Canning 2006). Both cholinergic and NANC parasympathetic fibers are active in airways, but the net effect is one of cholinergic constriction. In a study in which computed tomography was used to visualize human airway dimensions, functionally antagonizing this baseline tone resulted in a 10–15% increase in the mean diameter of small and midsized bronchi (Fig. 38.4, top) (Brown et al. 2006). Studies on experimental animals indicate that most if not all of the baseline cholinergic tone is due to vagal reflexes initiated on activation of vagal mechanosensors during the course of breathing. When the

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Breathing pattern Activation of afferent nerves in the lower airways can influence respiration (Kubin et al. 2006). SAR stimulation regulates breathing pattern by evoking the Hering–Breuer reflex. Stimulation of RAR fibers and nociceptive C-fibers can lead to apnea and rapid shallow breathing (Coleridge & Coleridge 1984). Cough The cough reflex is initiated by vagal afferent nerves. There appears to be at least two afferent neural pathways initiating cough (Canning et al. 2006). The rapid cough evoked on mechanical perturbation of the larynx or trachea (e.g., cough of aspiration) is most likely initiated by a mechanosensitive vagal A-fiber. This cough can be evoked under light anesthesia, and can be initiated by spritzing acid or water onto the larynx or trachea (Addington et al. 2003; Ishikawa et al. 2005). This convulsive cough is not under voluntary control. In guinea pigs, a specialized nodose vagal Aδ “cough fiber” that is likely involved in the initiation of aspiration-induced cough has been described in the larynx, trachea, and main bronchi (Canning et al. 2004). In contrast to the rapid convulsive cough experienced on aspiration or mechanical perturbation of the tracheal mucosa, the C-fiber-mediated cough is difficult to observe in anesthetized animals (or humans), and more likely underlies the irritating “urge to cough” sensations associated with respiratory tract infections and experienced by many asthmatic subjects before and during their attacks (Canning et al. 2006). C-fiber-mediated cough is likely involved in the cough reflex evoked experimentally by inhalation of C-fiber stimulants such as capsaicin or bradykinin. Neurogenic inflammation The effects of local release of sensory neuropeptides due to axon reflexes (discussed above) have been well documented in the lower airways of guinea pigs and rodents. In these species, antidromic stimulation of vagal C-fibers can lead to bronchoconstriction, vasodilation, and extensive plasma extravasation (McDonald 1988; McDonald et al. 1988). However, the extent to which human airways are innervated by neuropeptide-laden C-fibers is much less than in rodents, and consequently there is likely to be less neurogenic inflammatory responses in the lower respiratory tract of humans. In human bronchi, neuropeptide-containing C-fibers are found preferentially surrounding local parasympathetic gan-

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glia (Lundberg et al. 1984). Axon reflexes in these fibers may result in release of neurokinins at the parasympathetic synapse where they can increase the efficacy of parasympathetic transmission (Myers et al. 1996, 2005).

for these triggers, each of them is known to be associated with sensory and autonomic nerve stimulation.

Neuromodulation in airway inflammation As discussed elsewhere in this book, a large and impressive literature has accumulated over the past few decades that details the characteristics of allergic airway inflammation. Much less progress has been made on the key question as to how this inflammation associated with asthma perverts the nervous system such that many of the symptoms of asthma are expressed. Results mainly from studies on experimental animals reveal that allergic inflammation can quantitatively and qualitatively modulate airway neurophysiology at virtually all points along the classical reflex arc, from the primary afferent nerves, to synaptic events in the CNS, to synaptic events within autonomic ganglia, and to events at the autonomic neuro-effector junction in the airway wall. One might predict that if allergic inflammation is adept at increasing the “reactivity” of the sensory–CNS–autonomic arc, then it may predispose individuals to triggers of asthma that would otherwise be innocuous. This indeed would appear to be the case. For example, a recent study of about 250 asthmatic individuals revealed that over 50% of the subjects identified irritants in the air, changes in climate, and physical exercise as major triggers of their asthma (Fig. 38.5) (Ritz et al. 2006). Although other explanations may be found

Modulation of primary afferent nerves A hallmark of asthma is inflammation of the airway mucosa. Considering the location of nerve terminals within the airway mucosa, it would seem likely that airway inflammation will lead to situations in which the terminals of afferent nerves are bathed in and ultimately altered by inflammatory mediators. It is also likely that these alterations in afferent nerves will be reflected in quantitative or qualitative changes in the information (action potentials) received by CNS neurons from the airways. Allergic inflammation is associated with several mediators known to be capable of directly evoking action potential discharge in vagal sensory fibers, notably bronchopulmonary C-fibers, in the airways (Table 38.1). Other mediators are known to sensitize or “prime” the nociceptive nerve terminals such that their threshold to activation is reduced (Table 38.1). Similarly, allergen-induced activation of mast cells can lead to increases in the sensitivity of lowthreshold Aδ mechanosensors, including those involved with cough reflexes (Riccio et al. 1996). In addition, the activity in low-threshold mechanosensors is likely to be increased during allergic asthma attacks indirectly via bronchoconstriction, vasodilation, or edema. Finally, evidence exists supporting the notion of a “phenotypic switch” wherein an inflammatory mediator(s) induces changes in gene expression in neurons. In particular, nerve growth factor or allergen has been shown to induce substance P production in A-fibers, which would not normally express it (Hunter et al. 2000; Chuaychoo et al. 2005b).

Medication Mold Alcohol Sleep Food Respiration Psychology Pollen allergy Animal allergy House dust Infection Air pollution Fig. 38.5 Frequency of main categories of asthma triggers perceived by patients as most relevant to their condition (based on N = 247 subject). (From Ritz et al. 2006, with permission.)

Physical Climate 0

10

20

30

40

50

60

% of patients

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Table 38.1 Activation and sensitization of vagal C-fibers by mediators associated with allergic inflammation. Receptors

Reference

Mediators that activate C-fibers* Bradykinin B2 via TRPV1 Adenosine A1, A2a ATP P2X Acid TRPV1/ASIC Serotonin 5HT3 Thromboxane ?

Fox et al. (1993); Carr et al. (2003); Lee et al. (2005) Hong et al. (1998); Chuaychoo et al. (2006) Pelleg & Hurt (1996); Undem et al. (2004) Fox et al. (1995); Kollarik & Undem (2002) Coleridge & Coleridge (1984); Chuaychoo et al. (2005a) Karla et al. (1992)

Mediators that sensitize C-fibers† Histamine H1 CysLT1 LTD4 Tryptase PAR2 via TRPV1 ? PGD2 EP2 PGE2 MBP ?

Lee & Morton (1993) McAlexander et al. (1998) Gu & Lee (2006) Fowler et al. (1985) Kwong & Lee (2005) Lee et al. (2001)

* Activating mediators are those that overtly evoke action potential discharge. † Sensitizing mediators are those that may not evoke action potential discharge alone, but increase the sensitivity of the nerve such that activating stimuli evoke either discharge at a higher frequency or at a lower threshold. MBP, major basic protein.

Modulation in the CNS Too little attention has been given to how airway inflammation affects synaptic transmission between the primary afferent nerve and second-order neurons in the brainstem (NTS). One mechanism by which integration of afferent information in the CNS can be qualitatively altered is by changing the neurochemistry of afferent fibers (i.e., changes in neurotransmitter released from the central terminals of the afferent nerves). The low-threshold mechanosensors in the airway use excitatory amino acids (EAAs) as their principal central neurotransmitter (Kubin et al. 2006). When EAAs, such as glutamate, are released onto second-order neurons in the brainstem they cause fast excitatory postsynaptic potentials. The efficacy by which an EAA results in synaptic transmission in the CNS may be increased by the activation of converging C-fibers due to the actions of sensory neuropeptides released from the central terminals (Mutoh et al. 2000). This enhancement of synaptic transmission by converging inputs in the CNS has been termed “central sensitization.” Central sensitization likely contributes, for example, to the augmentation of reflex parasympathetic drive and cough seen after stimulation of bronchopulmonary C-fibers (Mazzone & Canning 2002; Mazzone et al. 2005). The concept of central sensitization has been studied extensively in the somatosensory system where it appears to be a major mechanism underlying certain types of hyperalgesia and allodynia (Ji & Woolf 2001). Central sensitization may be increased during allergic inflammation, when the inflammation leads to neuropeptide expression in lowthreshold mechanosensors. This, in theory, could lead to

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central sensitization during respiration, independent of Cfiber nerve stimulation (Chuaychoo et al. 2005b). There have been few direct studies that have evaluated the influence of allergic airway inflammation on CNS integration of primary afferent signals. In one such study, nonhuman primates were sensitized and repeatedly exposed to housedust mite allergen. The airways of these animals become inflamed in a fashion similar to that seen in human asthma. Interestingly, the second-order neurons in the CNS (NTS) were “sensitized” relative to those in nonallergic monkeys (Chen et al. 2001). This was revealed by a substantial increase in their electrical excitability.

Modulation of autonomic nerves The neuromodulation associated with airway inflammation is not limited to primary afferent nerve activity and CNS integration. Airway inflammation also directly affects autonomic neuronal activity. Allergen challenge in vitro has been associated with long-lasting increases in electrical excitability of both sympathetic, enteric, and bronchial parasympathetic ganglion neurons (Weinreich & Undem 1987; Kajekar et al. 2003; Liu et al. 2003). This increase in electrical excitability leads to an increase in the amplitude (efficacy) of the postsynaptic excitatory synaptic potentials, and consequently a decrease in the capacity of the ganglia to filter preganglionic input. Airway inflammation can also lead to an increase in the amount of acetylcholine released per action potential from the postganglionic fibers at the level of the neuroeffector cells. This has been explained by mediators inhibiting certain

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potassium channels on postganglionic nerves, or by causing a decrease in the presynaptic inhibitory influence of cholinergic muscarinic M2 receptors (Fryer & Wills-Karp 1991).

Concept of critical period (speculation) The lack of tight correlations between neuronal symptoms and various markers of inflammation suggests that there is a differential sensitivity of the nervous system to airway inflammation among different patients. In this regard, it may be useful to think about early life events. It is well established that development of sensory neural systems often requires use-dependent activity early in life (Berardi et al. 2000). During postnatal sensory nerve development there is a defined window of time during which the nerves are susceptible to this experience-dependent plasticity. For example, if a young animal is deprived of vision by eyelid closure (or various other techniques), changes occur in the neural circuitry of the visual cortex leading to severe and permanent loss in visual acuity. This only occurs if the vision deprivation occurs during a critical period of time. Even prolonged vision deprivation after the critical period is without effect on visual acuity. Since these pioneering studies, critical periods have been defined in audio and somatosensory systems, and have been noted in virtually all species from humans to songbirds to Drosophila (Berardi et al. 2000). Two recent reports support the concept that inflammationdependent sensory nerve activity during a critical period can lead to persistent changes in somatosensory and vagalsensory neural circuits. In these studies, when the animals were subjected to inflammation during an early life “critical period” there were persistent abnormalities in pain regulation (Ruda et al. 2000) and, more relevant to the present discussion, in a model of visceral hyperreflexivity (Al-Chaer et al. 2000). The results in these studies support the hypothesis that airway inflammation early in life may have long-lasting consequences with respect to sensory hyperirritability.

Role of nerves in allergic airway diseases Asthma One is hard pressed to find a more scholarly and lucid defense of the hypothesis that bronchial asthma is partly a disorder of the nervous system than that penned in 1860 by Henry Salter (Salter 1882). In the second chapter, titled “The pathology of asthma: its absolute nature,” he discusses eight lines of evidence favoring his conclusion that asthma is: A morbid proclivity of the musculo-nervous system of his bronchial tubes to be thrown into a state of activity; the stimulus may be either immediately or remotely applied, but in either case would not normally be attended by any such result. There is no peculiarity in the stimulus, the air breathed is the same to the asthmatic and non-asthmatic; . . . nor probably is there any peculiarity in the irritability of the bronchial muscle; the pecu-

Sensory and Autonomic Nervous System in Asthma and Rhinitis liarity is confined to the link that connects these two – the nervous system – and consists in its perverted sensibility in its receiving and transmitting on to the muscle, as a stimulus to contraction, that which it should take no cognizance.

“On Asthma” predated carefully designed clinical trials, and as such depends heavily on anecdotal evidence. The clinical observations, though, were weighed against an accurate if unsophisticated physiology. This is not the place to review Salter’s eight lines of evidence, but before the skeptic dismisses a major role for the nervous system in asthma, he or she is encouraged to read at least the first two chapters of his monograph. The basic neuroanatomy of the human respiratory system was understood in Salter’s time, but was reflned in the ensuing 50 years. By the 1920s, enough was understood that surgeons had confidence to treat severe asthma by nerve resection. In 1929, Phillips and Scott wrote what was in essence a metaanalysis of the burgeoning literature in this area that sprouted up during the first 20 years or so of the 20th century. They reviewed some 300 surgical cases, but considered informative only those cases in which asthma was accurately diagnosed and the subject was followed and evaluated for a minimum of 6 months. With this filter in mind, they found 29 of the 300 cases of scientific value. They summarize by stating that “there are a few brilliant cures in extremely severe forms of asthma. Roughly on half the patients definitely improved, while frequently the other half, after temporary improvement, are in no better condition than before the operation. The patients who were cured have been followed on average almost two years.” (Phillips & Scott 1929) Among these 29 cases there was little consistency in the operational procedure, and some of the surgeries described can be questioned based on present knowledge of the physiology and extrinsic innervation of the airways. Over the next decade the surgical technique was further reflned by Rienhoff at Johns Hopkins Hospital. Rienhoff made a bilateral resection of the posterior pulmonary nerve plexus, thereby selectively denervating the nerve supply to the lungs. He teamed up with Leslie Gay a noted allergist and asthmologist and reported their findings in the Archives of Surgery in 1938 (Rienhoff & Gay 1938). The patients were selected on the basis of an unquestionable diagnosis of bronchial asthma, and on severity of disease with only those totally incapacitated by their disease being included. The report covers 11 patients economically, physically, and socially incapacitated by their disease. All the subjects were then followed for 1.75–2.75 years postoperatively. In their thorough account of the operations they concluded that approximately 2 years post surgery: Of the 10 patients discharged from the hospital 1 was entirely unimproved; 1 improved for 3 months, finally succumbing to what seemed to be cardiac failure; 4 are completely well at the time of writing, having been free of attacks since the operation or a short time later, and are able to resume their former work;

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4 have occasional mild attacks of asthma, all of which are amenable to control by means of small doses of ephedrine.

No lung function data are reported in these studies, so it is unclear just how impressive the “cures” were. Fortunately at about this time adrenocorticotropic hormone (ACTH) was synthesized and found to be effective in the treatment of arthritis. By 1950, it was evident that severe cases of asthma could effectively be treated with systemic cortisone (Carey et al. 1950; Randolph & Rollins 1950). This, fortunately, put an end to the surgical treatment of asthma. Nevertheless, looking back on these surgical reports, even with a skeptic’s eye, one finds additional experimental support for Salter’s hypothesis. Asthma is defined largely by symptoms of episodic wheezing, breathlessness, chest tightness, and cough. Each of these major symptoms can be explained in part or in toto by alteration in the nervous system.

Wheezing The reversible episodic wheezing and chest tightness of asthma is a consequence of a narrowing of the bronchi due to the constriction of the bronchial smooth muscle. As described above, the major regulator of bronchial smooth muscle is the parasympathetic nervous system. Even in healthy individuals there is a baseline cholinergic tone on the muscle due to reflexes initiated by mechanosensitive vagal afferent nerves responding to the mechanical forces of respiration. In asthmatic lungs with compromised lung function, studies have revealed that the increased airflow evoked by functional antagonism of smooth muscle contractions with a maximum concentration of β-adrenoceptor agonist can be nearly mimicked by simply antagonizing cholinergic muscarinic receptors (Gross & Skorodin 1984; Grandordy et al. 1988). These data lead to the conclusion that although inflammatory mediators may be present in the airways of asthmatics, the substance responsible for the vast majority of smooth muscle contraction is acetylcholine, and by inference cholinergic nerve activation. This is not unexpected; as predicted from studies on animals, with each breath the airways are subjected to an endogenous “acetylcholine challenge” as a consequence of parasympathetic reflex drive. A diagnostic feature of asthmatic airways is “airway hyperreactivity” to bronchoconstricting stimuli. This is most often quantified by measuring the concentration of an inhaled stimulus required to decrease forced expiratory volume in 1 s (FEV1) by 20%. The difference in this parameter between non-asthmatic and asthmatic airways is often quite staggering. It typically takes concentrations of histamine or methacholine in excess of 20 mg/mL to cause an appreciable drop in FEV1 in nonasthmatic subjects, whereas it is not uncommon for concentrations of these agonists to influence FEV1 in asthmatic subjects at concentrations far below 1 mg/mL (Cockcroft 1997).

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Surprisingly little is known about the mechanisms that underlie asthma-associated airway hyperreactivity. The hyperreactivity appears to be associated with airway inflammation, but a cause–effect relationship between inflammation and hyperreactivity remains obscure. Several studies have documented that inhibiting airway inflammation with inhaled corticosteroids has either no effect or very modest effects on the chronic hyperreactivity in asthma (Cockcroft & Davis 2006). A large study on childhood asthma revealed that long-term treatment with inhaled corticosteroids decreased asthma-related hospitalizations in children, but only trivially influenced airway hyperreactivity (Childhood Asthma Management Program Research 2000). Moreover, some allergic rhinitic subjects without asthma have lung inflammation similar to that seen in asthmatic subjects, yet do not have airways hyperreactivity (Braunstahl et al. 2003). There is indirect evidence in support of the concept that abnormal neuronal reflex activity may contribute at least in part to hyperreactive airways, but again specific cause–effect relationships are unknown. Typically, the component of a bronchial provocation challenge with stimuli other than muscarinic agonists, which is inhibited by antimuscarinic drugs such as ipratroprium, is suggested to be due to cholinergic reflex activity. There is large variability among these types of studies with respect to the inhibitory efficacy of ipratroprium (or atropine) (Van Schoor et al. 2000). This may be due to the fact that these drugs are relatively short-acting and are reversible competitive antagonists, making timing and dosage of critical importance. As seen in Fig. 38.4, it takes a relatively large dose of ipratroprium to even reverse baseline parasympathetic tone. It is easy to envisage a scenario where a dose of ipratroprium that effectively antagonizes the response to a PD20 dose of inhaled methacholine is completely surmounted by the very large local concentrations of acetylcholine likely found at certain cholinergic nerveeffector junctions during cholinergic reflex contractions. Studies with relatively large doses of a more irreversible antagonist such as tiotropium would likely yield more consistent results in this area (Barnes 2000). Nevertheless, many stimuli to which asthmatics have been found to be hyperreactive modulate airflow resistance in part or entirely by mechanisms that can be inhibited by ipratroprium or atropine. These experimental provocations include hypertonic solutions, adenosine, substance P, bradykinin, histamine, propranolol, sulfur dioxide, sodium metabisulfite, and isocapnic hyperventilation (Van Schoor et al. 2000; Canning & Fischer 2001). Even the so-called called direct-acting smooth muscle agonist methacholine may have a mixed direct and reflex component in its action, depending on concentration (Wagner & Jacoby 1999). Some mechanistic insights on airway hyperreactivity may be derived from those studies that induce the phenomenon in nonasthmatic subjects. In one such study, upper respiratory tract viral infection was shown to lead to airway hyperreact-

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ivity to histamine, and this hyperreactivity persisted well beyond the infection (much like postviral cough syndromes) (Empey et al. 1976). The second method by which increased airway reactivity can be observed in nonasthmatic subjects is by preventing them from taking deep inspirations or “sighs” for a short period of time (e.g., 20 min) prior to the provocation. When nonasthmatic subjects avoid deep inspiration they respond to concentrations of methacholine or histamine aerosol that under normal conditions have no effect (Skloot et al. 1995). Specifically relevant to the present chapter are the findings that the heightened sensitivity to histamine induced by upper respiratory tract infection or by deep breath avoidance is prevented by pretreatment with an anticholinergic drug (Empey et al. 1976; Kesler et al. 2001). These studies circumstantially support the hypothesis that the induced airway “hyperreactivity” was due to a “hyperreflexivity.”

striction, but also directly via activation of relevant vagal sensory nerves. In support of this hypothesis, ATP, via P2X receptor activation, is a strong stimulus of pulmonary C-fibers (Pelleg & Hurt 1996; Undem et al. 2004). Considered together, these findings suggest that discharge of action potentials in certain types of airway sensory nerves can lead to dyspnea or augment dyspnea, and that this is not strictly dependent on bronchoconstriction. This can lead to a situation where the sense of breathlessness is not matched to the physiologic condition; i.e., when lung function is near normal. In asthma there may also be a situation where dyspnea is “desensitized” such that the sense of breathlessness per unit fall in FEV1 is actually dampened. The mechanisms underlying these mismatches in sensation versus physiology are unknown.

Breathlessness/dyspnea The episodic bronchospasm associated with asthma certainly contributes to sensations of dyspnea (“chest tightness”) that typifies the asthmatic condition. There are multiple neuronal pathways capable of underlying sensations of dyspnea, several of which do not depend on intact vagal pathways (Kimoff et al. 1990). Accumulating data, however, support the hypothesis that increases in vagal afferent activity caused by inflammation, independent of bronchospasm, may also play a role in causing or augmenting the dyspnea. The evidence for such a hypothesis is derived from several disparate studies: 1 Electrical stimulation of the vagus nerves, using stimulation paradigms that do not cause bronchoconstriction or changes in heart rate, can lead to dyspnea in some humans (Handforth et al. 1998). 2 Inhalation of histamine, adenosine, or sodium metabisulfite, chemicals associated with activation of vagal afferent nerves, leads to a more profound sensation of discomfort for a given fall in FEV1 than methacholine inhalation (Marks et al. 1996; Tetzlaff et al. 1999). 3 Dyspnea associated with histamine inhalation can be relieved by lidocaine inhalation (Taguchi et al. 1991). 4 Prostaglandin E2, a mediator that increases vagal sensory nerve excitability, exacerbates the dyspnea associated with exercise, despite being a bronchodilator (Taguchi et al. 1992). 5 Inhalation of furosemide, a drug with known vagal sensory neuromodulatory activity, alleviates experimentally induced dyspnea (Nishino et al. 2000). 6 On inhalation of ATP there is a positive correlation between the change in FEV1 and the intensity of dyspnea in asthmatics, although there is also a very tight negative correlation between the intensity of dyspnea and the concentration of ATP required to cause a 20% fall in FEV1 (Basoglu et al. 2005). The interpretation of this may not be immediately apparent, but the results would be consistent with the hypothesis that ATP evokes dyspnea indirectly via bronchocon-

Cough Cough is a common symptom of asthma (Chang 1999). Cough-inducing sensations in asthma may be caused by the presence of mucus in the airways. Mucus secretion, like bronchospasm, can be increased as a consequence of altered autonomic reflex activity. Often, however, patients describe an indefinable and persistent itch or irritation in the airway that provokes a dry unproductive cough reflex. This type of cough is unlikely related to either bronchospasm or mucus secretion, but is most likely related to vagal nociceptive Cfiber activation. Decreasing FEV1 with methacholine is not an effective tussive stimulus in most asthmatic subjects (Chausow & Banner 1983). It is therefore informative that asthmatic subjects are not only “hyperreactive” to adenosine and ATP in the classical sense when the outcome is a fall in FEV1, but are also hyperreactive to these nociceptor stimulants when the outcome is cough (Basoglu et al. 2005). The problem in asthma may be interpreted as an exaggeration of normal reflex behavior. Analogies to this type of process may be found in other systems. In the study of pain it has long been recognized that inflammation can lead to a state of hyperalgesia such that the threshold for painful stimuli is decreased (Treede et al. 1992). Hyperalgesia thus has similarities to airway hyperreactivity. Inflammation can also lead to painful sensations in response to normally nonpainful stimuli such as gentle brushing of the skin or hair. The term given to this inappropriate pain sensation is “allodynia” (Cervero & Laird 1996). A similar phenomenon occurs with respect to inappropriate itch sensations, termed “alloknesis.” In some cases of asthma, one might argue that when a severe shortness of breath is experienced, despite only a mild compromise in lung function, there is an inappropriate hunger for air, i.e., an “allodyspneic” sensation. Similarly asthmatics may experience irritating itch sensations in their airways leading to an urge to cough despite the lack of physical objects in the airway provoking the irritation. This “allotussive” effect may be analogous to alloknesis.

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Nocturnal asthma The CNS regulation of the autonomic nervous system follows a circadian cycle. Indirect evidence for a role of the nervous system in asthma is therefore found in the circadian rhythm of variations in lung function. Fluctuations in FEV1 of a few percent are seen in healthy individuals such that the best lung function occurs around 4 p.m. and reaches a nadir 12 hours later. In asthmatic individuals, however, fluctuations of 20% or greater can be observed. It has been estimated that as many as 30– 40% of asthmatics complain of awakening nightly due to asthma symptoms (Sutherland 2005). There are many potential explanations for this (e.g., circadian changes in levels of epinephrine, oxygen radicals, or cortisol) (Barnes et al. 1980; Jarjour et al. 1992), but it should not go unnoticed that this may coincide with the times at which parasympathetic activity is reaching its peak. Indeed, clinical studies have shown that administration of the anticholinergic drugs oxitropium bromide or atropine has been able to ameliorate the nominal fall in lung function in subjects with nocturnal asthma (Coe & Barnes 1986; Morrison et al. 1988).

Placebos and nocebos A perusal of the literature pertaining to phase III drug trials in asthma or rhinitis reveals that, depending on the symptom, there can be rather large “responses” in the placebo limb of the study. This would suggest that the CNS can, in the absence of active drug, result in the alleviation of asthma symptoms in certain individuals. Perhaps more relevant to the present chapter is the so-called nocebo responses. In this context, a nocebo response is the opposite of the placebo response in that the “dummy challenge” causes rather than alleviates a symptom. There have been a large number of studies showing that a subset of individuals with asthma respond to the suggestion of a bronchoconstrictive challenge (Luparello et al. 1968; McFadden et al. 1969; Horton et al. 1978; Isenberg et al. 1992; Leigh et al. 2003). Typically, in these studies, the subjects are asked to inhale normal saline, but are told that they are taking a large dose of methacholine or a dose of allergen that, based on previous experience, is expected to cause bronchconstriction. In about 30–40% of individuals with asthma, the suggestion leads to a substantive decline in FEV1. Perhaps not surprisingly, the suggestioninduced bronchoconstriction is also associated with sensations of dyspnea (Leigh et al. 2003). In fact, some subjects who do not show a decline in lung function still experience dyspnea on inhalation of the “dummy bronchoconstrictor.” As always, these data need to be cautiously interpreted; in some cases the suggestion-induced decline in FEV1 may be due to actual physical properties of the “dummy” saline (pH, temperature, etc.) (Lewis et al. 1984). Tellingly, however, most studies have also shown that the subject’s FEV1 is actually improved if the same “dummy” saline is inhaled with the suggestion that it is a potent bronchodilator. The mechanism of psychogenic changes in measured lung function has not been

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completely resolved. The fact that FEV1 itself is a voluntary act makes it possible that a decrease in FEV1 could occur independently of changes in airway caliber. Several studies, however, have gone on to show that the suggestion-induced decrease in FEV1 is prevented by pretreatment with an anticholinergic drug (McFadden et al. 1969; Isenberg et al. 1992). Horton et al. (1978) also showed that the suggestion-induced decrease in FEV1 may be positively correlated with airway hyperreactivity. Considered together, the data are most consistent with a “suggestion-induced” increase in parasympathetic cholinergic drive to the airways. These experimental studies on psychogenic changes in lung function would not surprise Salter, who reported several cases where asthma attacks were triggered or relieved by “sudden or violent mental emotion.” The CNS control over airway neurophysiology may also provide some causal insights into the concept of stress and psychogenic triggers of asthma (Ritz et al. 2006).

Rhinitis The symptoms of rhinitis (nasal irritation, runny nose, sneezing and congestion) can each be evoked by stimulating or inhibiting certain components of the nervous system. At a conceptual level, the role of nerves in rhinitis more or less parallels that described above in asthma, and will therefore be discussed rather briefly here. For a recent extensive review of the experimental literature that addresses the role of the nervous system in allergic rhinitis see Sarin et al. (2006). Although electrophysiologic studies are lacking, one might predict that, as in the lower airways, allergic inflammation in the nose can lead to the production and release of inflammatory mediators that result in action potential discharge in sensory nerve terminals in the nasal mucosa. This could then lead to certain sensations, sneezing, and autonomic reflexes described above. Also analogous to lower airway disease, allergic rhinitis may also lead to a situation whereby the nasal nervous system itself is “hyperreactive” to a given amount of stimulus. This is evidenced by several clinical studies which show that the same concentration of sensory nerve stimulant (e.g., bradykinin, histamine, endothelin) leads to much larger reflex secretory and sneezing responses when studied in the context of allergic nasal inflammation (Baraniuk et al. 1994; Riccio et al. 1995; Riccio & Proud 1996; Sanico et al. 1999; Sheahan et al. 2005). This “hyperreflexia” may help to explain why subjects with allergic rhinitis, as with asthma, often associate their symptoms with nonallergen triggers. For example, in one study of 350 individuals with allergic rhinitis, approximately half listed smoke, cold air, or other airborne irritants as triggers to their nasal symptoms (Diemer et al. 1999). Clear evidence of the role of nerves in allergic mediatorinduced nasal secretion has come from elegant studies in which the vidian nerve was unilaterally sectioned in human subjects (Konno & Togawa 1979). As discussed above, this nerve carries both sympathetic and parasympathetic post-

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Histamine in right side

Histamine-induced secretion (mL/10 min)

0.8 Right nasal cavity Left nasal cavity

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

Before vidian neurectomy

After right vidian neurectomy

After right vidian neurectomy and right nasal anesthesia

Fig. 38.6 Nasal secretion from the right and left nostril after unilateral histamine application to right nostril, and before and after vidian neurectomy in a subject with rhinitis. Before neurectomy, histamine caused secretions in both the ipsilateral and contralateral nostril. After the vidian nerve in the right side was severed, ipsilateral secretion was abolished, suggesting even the ipsilateral secretion was dependent on nerve stimulation. As expected, the right-side vidian neurectomy did not block the contralateral reflex secretion, because the vidian nerve does not carry sensory afferent nerves. When these nerves in the right nostril were anesthetized, however, contralateral secretion was abolished. Considered together, these data suggest that nearly all histamine-induced secretions in the human nose are due to neuronal reflexes.

ganglionic fibers. These studies show that the nasal secretion evoked by histamine or allergen challenge depends nearly entirely on an intact vidian nerve. By contrast, histamineinduced nasal congestion depends only modestly on the nervous system (Fig. 38.6).

Conclusion The upper and lower airways are innervated by sensory and autonomic nerves that, in addition to participating in the maintenance of physiologic homeostasis, serve to defend the airspaces against foreign substances. In allergic airway disease

Sensory and Autonomic Nervous System in Asthma and Rhinitis the experimental data support the hypothesis that, as with the immune system, this process is corruptible in a manner such that the responses become inappropriately matched to the stimulus (excessive autonomic bronchospasm, mucus secretion, inappropriate urge to cough, sensations leading to dry and unproductive cough, and perhaps mismatched sensations of dyspnea). The altered neuronal activity in allergic airway disease may also help explain the prevalence of disparate nonallergic triggers in both asthma and rhinitis. The mechanisms by which allergic inflammation alters airway neurophysiology include changes in excitability of the afferent nerve terminals within the airways, changes in gene expression in the sensory cell soma, changes in synaptic transmission with the CNS, increases in synaptic efficacy within autonomic ganglia, and increases in transmitter secretion at the neuroeffector junction. Other than anticholinergic drugs, there are no therapies specifically aimed at normalizing the altered neuronal state in airway disease. This is likely to change in the future as pharmaceutical scientists recognize the potential of such drugs to alleviate symptoms of not only asthma and rhinitis but also of other visceral disorders, such as inflammatory diseases of the gastrointestinal tract and bladder, typified by inappropriate sensations and abnormal reflex physiology.

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Mucus and Mucociliary Clearance in Asthma and Allergic Rhinitis Duncan F. Rogers

Summary

Introduction

Coughing up phlegm or battling a relentlessly runny nose are signs of airway mucus hypersecretion in patients with asthma or allergic rhinitis. The increased luminal liquid is due to increased mucus secretion and plasma exudation from postcapillary venules in the airway mucosa (bronchial or nasal). The increased mucin is associated with goblet cell hyperplasia and submucosal gland hypertrophy. However, although there are many similarities in the mucus hypersecretory phenotype between asthma and allergic rhinitis, there are a number of important differences. For example, goblet cell hyperplasia is a characteristic feature of asthma but is less consistent in rhinitis. In asthma, the hypertrophied bronchial glands maintain the normal ratio of serous cells to mucous cells (about 50 : 50), whereas in rhinitis there is complete replacement of mucous by serous cells in the nasal glands. Biological targets for suppression of hypersecretion range from the inflammatory cells that initiate airway inflammation and which are associated with induction of the mucus hypersecretory phenotype to specific cellular elements such as antiapoptotic factors (e.g., Bcl-2), calciumactivated chloride (CACL) channels, epidermal growth factor receptor tyrosine kinase and exocytosis regulators (e.g., Munc18B). Identification of these targets is driving development of novel pharmacotherapeutic compounds. Apart from specific instances where a single mediator has a major impact on hypersecretion, for example histamine in rhinitis, it is likely that compounds with broad-spectrum antiinflammatory activity will be more effective than compounds with restricted activity. However, certain highly specific cellular moieties, such as CACL channels, which appear to be intimately associated with development of a hypersecretory phenotype, or molecules involved specifically with mucin exocytosis, such as MARCKS or SNARE complex proteins, are tantalizing targets. Data from clinical trials with blockers of these targets are awaited with great interest, not only for disease management but also to determine the clinical benefit of selective inhibition of airway hypersecretion in asthma and allergic rhinitis.

Asthma and allergic rhinitis share airway hypersecretion as a prominent feature (Rogers 2003, 2004; Mahr & Sheth 2005; Plaut & Valentine 2005). In asthma, viscous mucus accumulates in the airways, initiating cough and sputum production, and contributes to difficulty in breathing. In rhinitis, watery liquid pours uncontrollably out of the nose. This contributes to difficulty in nasal breathing, and is uncomfortable and embarrassing for patients. Whether thick, as in asthma, or thin, as in rhinitis, the excessive mucus is difficult to clear from the airways. In severe cases of asthma, mucociliary clearance can be so compromised that mucus blocks the airways and may be a cause of death. Consequently, treatment of airway hypersecretion is clinically important (Table 39.1). However, it should be noted that although often perceived as detrimental, because of its association with abnormal physiology and airflow obstruction, airway hypersecretion is a protective homeostatic mechanism. It is only when the increased secretion becomes excessive and longstanding that hypersecretion changes from being protective to contributing to pathophysiology and clinical symptoms. Consequently, effective treatment would entail return to a normal homeostatic phenotype, rather than mere inhibition of secretion. It should also be noted that the extent of the contribution of mucus to pathophysiology and clinical symptoms in asthma, and to a lesser extent in allergic rhinitis, has not been specifically quantified. In addition, the association of mucus with asthma or rhinitis does not necessarily imply causation; mucus may be a result of the pathophysiologic processes causing the disease. Treating the airway hypersecretion will not inevitably treat the disease, especially if the changes that have led to increased mucus production are irreversible. The present chapter considers new therapies for airway hypersecretion in asthma and allergic rhinitis. These two conditions are currently considered to be different manifestations of a larger systemic inflammatory syndrome involving both the upper and lower airways (Passalacqua et al. 2004). Consequently, they share similar current pharmacotherapeutic management, for example treatment with topical glucocorticosteroids. As an extension of this, it may be that new

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Table 39.1 Neuronal and humoral mediators of mucus secretion, goblet cell hyperplasia and MUC gene expression/synthesis, and plasma exudation in the airways.

Stimulus

Secretion

Hyperplasia

MUC

Plasma exudation

b-Adrenoceptor agonists Bradykinin Cholinergic nerves Cholinoceptor agonists EGF (+ TNF-a) Endothelin Endotoxin Histamine IL-1b IL-4/IL-13 IL-6 IL-9 Leukotrienes (cysteinyl) Neurokinin A Nicotine Nitric oxide Phosphodiesterase-4 inhibitors Platelet-activating factor Prostaglandins Proteinases Purine nucleotides Reactive oxygen species Sensitization followed by challenge Substance P Tachykininergic nerves TNF-a

+ + ++ ++ NP 0/+ + 0/+ + + + NP 0/+ + ++ –/+ + 0/+ 0/+ +++ + 0/+ + ++ 0/++ ++

Yes NP NP Yes Yes NP Yes NP NP Yes NP NP NP NP Yes NP NP Yes* NP Yes NP NP Yes NP NP Yes1

NP NP NP NP Yes NP Yes NP NP Yes Yes Yes NP NP NP NP NP Yes* NP NP NP NP Yes NP NP Yes

Inhibit ++ 0 0 NP + ++ ++ NP NP NP NP ++ + ++ + ?Inhibition +++ 0/+ NP NP + ++ +++ ++ NP

* Effect observed only in combination with platelet-activating factor. Scoring: +++, highly potent; ++, marked effect; +, lesser effect; 0, minimal effect. MUC, mucin; NP, effect not published; EGF, epidermal growth factor; TNF, tumor necrosis factor.

therapeutic compounds being developed for one disease will be effective in the other. A range of novel pharmacotherapeutic targets and compounds is presented in this chapter. As a background to these therapies, the chapter begins with considerations of airway mucus and the pathophysiology of hypersecretion in asthma and allergic rhinitis.

Airway mucus Airway mucus is a complex dilute aqueous solution of salts, enzymes and antienzymes, oxidants and antioxidants, exogenous bacterial products, endogenous antibacterial agents, cell-derived regulatory mediators and other proteins, plasma-derived mediators and proteins, and cell debris such as DNA. The mucus forms an upper gel layer and a lower sol layer (Knowles & Boucher 2002) (Fig. 39.1). Inhaled par-

ticles are trapped in the gel layer and, by movement on the tips of beating cilia, are removed from the airways, a process termed “mucociliary clearance.” Airway mucus requires an optimal combination of viscosity and elasticity for efficient ciliary interaction. Viscoelasticity is conferred on the mucus primarily by high-molecular-weight mucous glycoproteins termed “mucins” that, in the absence of respiratory disease, comprise up to 2% by weight of the mucus (Davies et al. 2002). Airway mucins are produced by goblet cells in the epithelium (Rogers 2002a) and seromucous glands in the submucosa (Finkbeiner 1999) (Fig. 39.1). Mucins are threadlike molecules comprising a highly glycosylated linear peptide sequence termed “apomucin” that is encoded by specific mucin (MUC) genes (Fig. 39.2). To date, 18 human MUC genes are reported, namely MUC1, 2, 3A, 3B, 4, 5AC, 5B, 6–9, 11–13 and 16–19 (Lapensee et al 1997; Wu et al. 2001; Gum et al. 2002; Chen et al. 2004). Ten of these genes are

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Physiology respiratory secretions from normal subjects (Fig. 39.2). Very small amounts of MUC2 mucin may also be present. The mucin content of airway secretions from patients with asthma or allergic rhinitis appears to differ from normal (see below).

Trapped particles Gel Mucus Sol

Mucociliary transport

Epithelium

Ciliated cell Goblet cell

PP

Salts water

Venule

Pathophysiology of airway hypersecretion in asthma and allergic rhinitis

Serous cell Mucous cell

Nerve

The pathophysiology of airway hypersecretion in asthma and allergic rhinitis shows many similarities, but also significant differences, as discussed in the following sections (Fig. 39.3).

Submucosal gland Mediators Drugs

Asthma Asthma is a chronic inflammatory condition of the lower airways (Eapen & Busse 2002). It is characterized clinically by variable airflow limitation that is at least partially reversible, either spontaneously or with treatment (National Institutes of Health 2002). Asthma is invariably an allergic disease and shares the general feature with allergic rhinitis of a Th2 lymphocyte-driven airway eosinophilia. Tissue inflammatory cells and “structural” cells (e.g., epithelial cells, airway smooth muscle cells, bronchial endothelial cells) release a range of inflammatory mediators, chemokines and cytokines, many of which, either directly or indirectly (e.g., by triggering neuronal pathways), have the capacity to increase airway mucus output (Table 39.1). Airway mucus hypersecretion and plasma exudation are characteristic pathophysiologic features of asthma (Figs 39.3 and 39.4) (Greiff et al. 2003; Rogers 2004). The plasma exudation is due to inflammatory-mediator induced bulk flow of plasma from bronchial postcapillary venules in the airway submucosa and thence into the airway lumen (Figs 39.3 and

Fig. 39.1 Production of airway mucus. Airway mucus forms an upper gel phase, in which inhaled particles are trapped and removed by mucociliary clearance, and a lower sol phase in which the cilia beat. Mucins, secreted by surface epithelial goblet cells and mucous cells in the submucosal glands, play a vital role in conferring the correct viscoelasticity on the mucus for optimal mucociliary transport. Plasma proteins (PP), exuded from postcapillary venules in the bronchial microvasculature, also contribute to production of the airway surface liquid. Hydration of mucus is controlled by active transepithelial transport of electrolytes and water, both across the epithelium and secreted by the glands. The processes which contribute to the production of airway mucus are under neural and humoral control, and may also be influenced by pharmaceutical compounds. (See CD-ROM for color version.)

expressed in the respiratory tract, namely MUC1, 2, 4, 5AC, 5B, 7, 8, 11, 13 and 19 (Davies et al 2002; Chen et al 2004). However, of the 10 MUC genes expressed in the airways, it is only the MUC5AC and MUC5B gene products (mucin proteins) that comprise the major gel-forming mucins in MUC5AC MUC5B

SH SH

SH

C

Mucin subunit (monomer)

C

MUC2

SH

Folded region

Disulfide bond -S–S

-

C

C

Oligosacchariderich region

C

C

-

-S–S

Mature mucin

S-

S-

C

C

-S–S-

SH

C C -S–S-

C

-S–S-

C

C

C

C

842

-S– C

-S–

C

-S–S-

C

C

-S–S-

SH

Fig. 39.2 Schematic representation of a gel-forming mucin molecule. The two main mucin species in respiratory tract secretions are MUC5AC and MUC5B, with very small amounts of MUC2 sometimes present. The mucin subunit (~ 500 nm in length) comprises an amino acid backbone (longer for MUC5B than for MUC5AC or MUC2) with extensive highly glycosylated areas and intermittent folded regions, stabilized via disulfide bonds, with little or no glycosylation. Glycosylation is via O-linkages and is highly diverse. In secretions, the subunits are joined end-to-end by disulfide bonds (S-S) into long thread-like mature mucin molecules. (See CD-ROM for color version.)

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Fig. 39.3 Putative differences in pathophysiology of airway hypersecretion in allergic rhinitis and asthma. Compared with normal, in rhinitis there is nasal inflammation, increased luminal secretions comprising plasma exudate and mucins (more MUC5AC than MUC1 or MUC2), goblet cell hyperplasia, and submucosal gland hypertrophy with a switch to a serous phenotype (mucous cells absent). In asthma, there is airway inflammation, increased luminal plasma exudate and mucins (an increased ratio of MUC5B to MUC5AC and the appearance of MUC2 mucin), epithelial “fragility” with loss of ciliated cells, marked goblet cell hyperplasia, submucosal gland hypertrophy (although without a change in mucous to serous ratio), and “tethering” of mucus to goblet cells. Many of these features require confirmation (or refutation) by data from greater numbers of subjects. (See CD-ROM for color version.)

Mucus and Mucociliary Clearance in Asthma and Allergic Rhinitis

Mucus

Epithelium

Goblet cell

Allergic rhinitis MUC5B ≥ MUC5AC >> MUC2

Inflammation

BAL total protein/serum TP

Immediately post-allergen 2

Pre-allergen

a1Pi

Alb Trans

Cer

MUC5AC >> MUC1, MUC2

Plasma exudation

3

0

Mucous cell Submucosal gland Serous cell

Ciliated cell

Asthma

4

1

MUC5AC + MUC5B

Normal

Fib

a2M

Relative molecular weight (increasing →) Fig. 39.4 Airway plasma exudation in asthma. Bronchoalveolar lavage (BAL) fluid of allergic asthmatics, taken before and immediately after antigen challenge, was analyzed for protein markers of plasma exudation (low relative molecular weight) and secretion (high relative molecular weight). Data were expressed as a ratio of BAL total protein to serum total protein (TP). Antigen challenge was associated with an overall increase in BAL proteins, but with a shift in the curve toward a disproportionate increase in plasma proteins compared with secreted proteins, indicating increased plasma exudation in allergic asthmatics when challenged. a1Pi, a1-protease inhibitor; Alb, albumin; Trans, tranferrin; Cer, ceruloplasmin; fib, fibrinogen; a2M, a2-macroglubulin. (Redrawn after data in Fick et al. 1987.) (See CD-ROM for color version.)

39.5). The mucin hypersecretion and plasma exudation not only increase mucus volume but also have synergistic interactions that further contribute to airway obstruction (Fig. 39.6). The latter is particularly evident in a proportion of patients who die in status asthmaticus, where many airways are occluded by gelatinous, “glairy”-appearing plugs (Rogers 2004). The plugs are highly viscid and comprise

Inflammation

mucins, plasma proteins, DNA, cells, and proteoglycans (Bhaskar et al. 1988; Sheehan et al. 1995). Incomplete plugs are often found encrusting the airway lumina of asthmatics who have died from causes other than asthma. There is more mucus in the central and peripheral airways of both chronic and severe asthmatics compared with control subjects, and the mucus comprises mucins, DNA, lactoferrin, eosinophil cationic protein, and plasma proteins such as albumin and fibrinogen (Fahy et al. 1993a,b; Ordonez et al. 2001) (Fig. 39.4). Correspondingly, the sputum coughed up by asthmatic patients is more viscous than that from patients with chronic bronchitis or bronchiectasis (Charman & Reid 1972; Shimura et al. 1988). The increased amount of luminal mucus is associated with goblet cell hyperplasia (Ordonez et al. 2001) and submucosal gland hypertrophy, although the latter is not characteristic of all patients with asthma (Jeffery & Zhu 2002). Hypertrophied glands are otherwise morphologically normal, with an equal ratio of mucous to serous cells. The latter is in contrast to allergic rhinitis where the glands become exclusively serous (no mucous acini) (Mir-Salim et al. 1998) (Fig. 39.3). More is known about the biochemical and biophysical properties of airway mucus in asthma than in allergic rhinitis (Rogers 2004). Firstly, the airway mucus in asthma may have an intrinsic biochemical abnormality. For example, the mucus plugs in asthma differ from the mucus found obstructing the airways in chronic bronchitis or cystic fibrosis by being stabilized by noncovalent interactions between abnormally large mucins assembled from conventional-sized subunits (Sheehan et al 1995). This indicates a defect in assembly of the mucin molecules, and could account for the increased viscosity of the mucus plugs in asthma. Plug formation may also be due, at least in part, to increased airway plasma exudation (Greiff et al. 2003). In addition, exocytosed mucins in asthma are not released fully from the goblet cells, leading to “tethering” of

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Epithelium Plasma Endothelial cell

SP, NKA

NK1-R, CGRP-R SP, NKA, CGRP

Inflammatory mediators

NK1-R Inflammatory mediators (e.g., leukotrienes, bradykinin)

Sensory nerve

Blood flow Arteriole

Capillary

Venule

Fig. 39.5 Mechanism of plasma exudation in the airways. Contraction of endothelial cells in postcapillary venules forms gaps between the cells and allows outflow of plasma into the submucosal tissue, to form edema, and into the airway lumen, to contribute to mucus formation. Humoral mediators and sensory neuropeptides released from “sensoryefferent” nerves cause venular endothelial cell contraction, but may also enhance plasma exudation by inducing arteriolar contraction and increasing blood flow to postcapillary sites of leakage. SP, substance P; NKA, neurokinin A; CGRP, calcitonin gene-related peptide. (See CD-ROM for color version.)

Luminal liquid Mucus Mucus viscosity

M Plasma exudation Postcapillary venule

Luminal liquid Mucus secretion

Fig. 39.6 Airway mucus–plasma interactions in asthma and allergic rhinitis. Increased mucus secretion and plasma exudation will both increase the amount of liquid in the airways (bronchial and nasal). Plasma has also been shown to induce mucus secretion in experimental systems (Williams et al. 1983), thereby further increasing the amount of luminal liquid. In addition, in vitro, albumin synergistically enhances the viscosity of mucus (List et al. 1978), thereby leading to the possibility of thickening of the already increased luminal liquid. (See CD-ROM for color version.)

luminal mucins to the airway epithelium (Shimura et al. 1996) (Fig. 39.7). This tethering may also contribute to plug formation. MUC5AC and a low-charge glycoform of MUC5B are the major mucin species in airway secretions from patients with asthma (Thornton et al. 1997; Sheehan et al. 1999), and both mucins are increased above the levels found in secretions from control subjects (Hovenberg et al. 1996; Wickstrom et al. 1998; Sheehan et al. 1999). There is significantly more of the low-charge glycoform of MUC5B in asthmatic than in normal control secretions (Kirkham et al. 2002) (Fig. 39.3). Interestingly, MUC5AC and MUC5B show a similar histologic distribution to that in normal controls, with MUC5AC found primarily in the goblet cells and MUC5B primarily in the submucosal glands (Groneberg et al. 2002). This distribu-

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GC

Fig. 39.7 Airway mucus “tethering” in asthma. Light microscopy of a peripheral airway from a patient with severe asthma showing incomplete release, or tethering (arrows), of secreted mucus (M) from goblet cells (GC). (See CD-ROM for color version.)

tion is similar to the localization of MUC gene expression in nasal turbinates (Aust et al. 1997). MUC2 mucin is not found in airway secretions from normal subjects (Davies et al. 2002) but, in a similar manner to the low expression of MUC2 mRNA in nasal epithelial cells (Voynow et al. 1998), MUC2 mucin protein is found in small amounts in secretions from asthmatic patients (Kirkham et al 2002).

Allergic rhinitis Allergic rhinitis is a heterogeneous disorder characterized clinically by one or more of the symptoms of sneezing, itching, congestion, and rhinorrhea (Ferguson 1997), which give rise to difficulty in breathing through the nose. From the viewpoint of the present chapter, namely nasal hypersecretion, the rhinorrhea is due to excessive secretion of mucins

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and liquid (water efflux), and to plasma exudation from the nasal microvasculature (Fig. 39.3). Thus, excessive mucus secretion and plasma exudation contribute to the excess nasal and airway liquid, respectively, in allergic rhinitis and asthma (Fig. 39.6). In addition, similarly to asthma, allergic rhinitis is currently considered a chronic inflammatory condition of the airways, albeit of the upper respiratory tract (Skoner 2001). However, it should be noted that the majority of the immediate symptoms of allergic rhinitis are caused by histamine, namely itching, nasal swelling, and watery secretion. This has prompted the suggestion that the role of chronic inflammation is overestimated in the pathophysiology and symptoms of rhinitis (Mygind 2001). In general terms, the chronic inflammation in allergic rhinitis comprises a Th2 lymphocytic orchestration of nasal eosinophilia (Holgate & Broide 2003). Tissue mast cells, basophils, Langerhans cells, epithelial cells, and endothelial cells, and their respective chemokines and cytokines, are also involved. Subsequent release of inflammatory mediators, including mast cellderived histamine, leukotrienes, tryptase, and prostaglandin (PG)D2 (Table 39.1), contribute to development of symptoms, in part via triggering of nasal neurogenic mechanisms (Tai & Baraniuk 2002). From the viewpoint of hypersecretion, these mediators combine to induce mucin output and plasma exudation, both of which contribute to the nasal discharge associated with rhinitis. The increase in nasal mucin secretion is invariably associated with an increased amount of mucin-secreting tissue, namely submucosal glands and goblet cells (Fig. 39.3). However, it should be noted that changes in number of the latter are equivocal. A transitory goblet cell hyperplasia has been noted in patients with allergic rhinitis after nasal allergen challenge (Pelikan & Pelikan-Filipek 1995a,b). Similarly, nasal goblet cell hyperplasia is noted in patients during the grass pollen season (Gluck & Gebbers 2000), and also in allergic patients with bronchial hyperreactivity (Bavbek et al. 1996). In contrast, nasal goblet cell hyperplasia was not detected in other studies of patients with allergic rhinitis, including the influence of natural allergen exposure (Karlsson & Pipkorn 1989; Berger et al. 1997). The reason(s) for these discrepant observations is unclear, but presumably relates to time of sampling (in or out of the allergen season), and to difficulties in quantification of goblet cell number associated with marked variability, not only between patients but also to the heterogeneous distribution of goblet cells in the nasal epithelium (Berger et al. 1997). Whether hyperplastic or not, nasal goblet cells from patients with allergic rhinitis discharge more mucus than cells from patients with nonallergic rhinitis or controls (Berger et al. 1999). Compared with the goblet cells, there is less information on the submucosal glands in allergic rhinitis, presumably because their deeper location limits sampling by nasal biopsy. Two studies in a limited number of patients demonstrate gland hypertrophy, characterized by an increase in serous

acini and an absence of mucous acini (Toppozada & Gaafar 1973; Mir-Salim et al. 1998); “normal” nasal glands have a relatively even distribution of mucous and serous cells (Toppozada & Gaafar 1973), as do the hypertrophied glands in the airways of asthmatics (Jeffery & Zhu 2002) (Fig. 39.3). The identities of the nasal mucins in allergic rhinitis are beginning to be elucidated. MUC1, 2 and 5AC mucin genes are expressed in the nasal mucosa of patients with allergic rhinitis (Takeuchi et al. 1995; Voynow et al. 1998). In epithelial cells, the MUC5AC gene is expressed up to 10-fold above that of MUC1 or MUC2. MUC5AC protein is also expressed in allergic rhinitis (Wood et al. 1997). Information is now needed on the relative amounts of the MUC1 and MUC2 proteins, and whether or not there are differences in MUC gene and product expression between allergic rhinitis and controls. Plasma exudation is a major contributor to nasal discharge, possibly greater than in the airways in asthma (Greiff et al. 2003). Interestingly, in patients with allergic rhinitis, the nasal venules, the primary site of plasma exudation, exhibit “gaping” of the interendothelial junctions (Toppozada & Talaat 1976). There are similar changes in the capillaries. These observations are consistent with an increased potential for plasma exudation from the nasal microvasculature and subsequent transepithelial transudation into nasal secretions in allergic rhinitis.

Mucociliary clearance in asthma and allergic rhinitis Clearance of mucus from the airways is impaired in patients with a variety of respiratory diseases, including asthma and rhinitis (Wanner et al. 1996). However, there are often discrepancies in results between studies that are invariably due to differences in methodology (Clarke & Pavia 1980; Pavia et al. 1983a), but may also be due to observations made at different stages of disease.

Asthma Airway mucociliary clearance is well documented as being impaired in asthma (Del Donno et al. 2000). Clearance is impaired even in patients in remission (Pavia et al. 1985) and in those with mild stable disease (Bateman et al. 1983). Mucus clearance is proportionally reduced in symptomatic asthmatics (Foster et al. 1982) and during exacerbations (Messina et al. 1991). In addition, the normal slowing of mucus clearance during sleep is more pronounced in asthmatic patients (Bateman et al. 1978; Pavia et al. 1987), and this could be a contributory factor in nocturnal asthma. The mechanisms underlying the reduced mucus clearance in asthma are not clearly defined, although airway inflammation is considered to be a major contributor (Del Donno et al. 2000).

Allergic rhinitis Although rhinorrhea is uncontrolled outpouring of liquid from the nose, there is nevertheless reduced nasal mucociliary

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clearance in rhinitis (Stanley et al. 1985; Prior et al. 1999; Sun et al. 2002). The reduction in clearance is predominantly due to an increase in viscosity of the mucus (Passali et al. 1995; Schuhl 1995).

Considerations for effective treatment of airway hypersecretion The prevalence of cough and sputum production at presentation of asthma patients, and the impact on quality of life of a perpetually runny nose in patients with allergic rhinitis, indicates an important role for airway mucus (mucin secretion and plasma exudation) in the pathophysiology of these conditions. Consequently, treatments are being developed to treat airway hypersecretion (Willsie 2002; Rogers & Barnes 2006). Numerous naturally occurring and synthetic compounds are available worldwide that are perceived to have potentially beneficial actions on some aspect of mucus or its secretion (Rogers 2002b). There are two overall objectives for treatment of airway hypersecretion, namely short-term relief of symptoms and long-term benefit (Table 39.2). In rhinitis, short-term benefit would be achieved by inhibiting the rhinorrhea by reducing mucin secretion and plasma exudation. In contrast, in asthma, short-term benefit would be to facilitate clearance of excessive airway mucus. The latter

would entail inhibiting secretion and exudation, reducing the viscosity of the mucus to an optimal level (and possibly increasing elasticity), increasing ciliary function and, possibly, encouraging cough. In addition, in asthmatic patients, facilitating release of the tethered goblet cell mucin should improve airflow. Long-term benefit in asthma and allergic rhinitis entails reversal of the hypersecretory phenotype. For both conditions, treatment of the airway inflammation would be expected to be associated with treatment of hypersecretion. Specific interventions would be similar for both conditions and include reducing the number of goblet cells (particularly in asthma) and the size of the submucosal glands and, in rhinitis, reversing the switch to a predominant serous phenotype (Table 39.2). Additional interventions to reverse changes in airway mucins and MUC gene expression may also be required for comprehensive treatment, although information on this aspect is currently scarce. The following section considers the mediators involved in pathophysiology of allergic rhinitis and asthma (Table 39.1) and the variety of pharmacotherapeutic interventions (with emphasis on new/novel targets and drugs) aimed at alleviating airway hypersecretion. However, it should be noted that with the possible exception of glucocorticosteroids, it is unlikely that any one class of compound will provide comprehensive treatment of the pathophysiology of excessive production of airway secretions.

Table 39.2 Objectives for effective pharmacotherapy of airway hypersecretory pathophysiology in asthma and allergic rhinitis. Overall objectives Asthma Facilitate mucus clearance (short-term relief of symptoms)

Reverse hypersecretory phenotype (long-term benefit)

Allergic rhinitis Inhibit nasal discharge (short-term relief of symptoms)

Reverse hypersecretory phenotype (long-term benefit)

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Specific objectives

Inhibit mucin secretion Inhibit plasma exudation Reduce mucus viscosity (?increase elasticity) Increase ciliary function Induce cough Facilitate release of “tethered” goblet cell mucin Treat airway inflammation Reduce goblet cell number Reduce submucosal gland size Reverse increased ratio of MUC5B (low-charge glycoform) to MUC5AC Inhibit production/secretion of MUC2 (albeit minor proportion of secretions)

Inhibit mucin secretion Inhibit plasma exudation Treat nasal inflammation Reverse widening of gaps between microvascular endothelial cells Reduce goblet cell number Reduce submucosal gland size Reduce disproportionate increase in gland serous cells ?Increase gland mucous cells to normal numbers

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Allergen

Glucocorticosteroids

Allergen

Th2 cell

Suplatast tosilate

Bcl-2

EGFR

CLCA

Munc

Th2 cell Soluble receptors Monoclonal antibodies

Goblet cell

IL-9, IL-4/IL-13

Ciliated cell

Cytokines

Small molecule inhibitors

Goblet cell hyperplasia/mucin synthesis

Fig. 39.8 Relationship between Th2 lymphocyte-mediated airway inflammation and generation of a mucus hypersecretory phenotype. Allergic inflammation leads to generation of a variety of inflammatory cytokines of which interleukin (IL)-9 and IL-4/IL-13 have been shown to induce mucin production and/or goblet cell hyperplasia in experimental systems. Dampening of the inflammation at different “levels” with antiinflammatory drugs or cytokine inhibitors suppresses the induction of the mucus hypersecretory phenotype. (See CD-ROM for color version.)

Goblet cell

Goblet cell hyperplasia/mucin synthesis

Ciliated cell

Fig. 39.9 Upregulation of epithelial cell factors during generation of airway goblet cell hyperplasia. Cytokines (e.g., IL-4/IL-13) upregulate mucin production and/or induce goblet cell hyperplasia in experimental systems, with associated increases in expression of the anti-apoptotic factor Bcl-2, calcium-activated chloride channels (CLCA), epidermal growth factor receptors (EGFR), and the exocytosis regulator Munc18B. (See CD-ROM for color version.)

Glucocorticosteroids

New drugs for airway hypersecretion The recent upsurge in the perceived importance of mucus in the pathophysiology and clinical presentation of asthma and rhinitis has fuelled interest in development of novel compounds to treat airway hypersecretion (Figs 39.8–39.10 and Table 39.3). The following sections consider these compounds, including mention of improvements in more established drugs such as glucocorticosteroids, antihistamines, and anticholinergics.

Inhaled corticosteroids are highly effective in clinical management of asthma (Barnes 2006) and nasal glucocorticosteroids are equally effective in treatment of allergic rhinitis (Holm & Fokkens 2001; Naclerio et al. 2003; Meltzer 2005). Corticosteroids effectively inhibit plasma exudation (Rogers & Evans 1992) and although it is unclear whether they have direct inhibitory effects on mucin secretion, corticosteroids inhibit MUC5AC gene expression (Tanaka et al. 2001) and experimentally induced airway goblet cell hyperplasia

Allergen

Fig. 39.10 Schematic diagram of the pathophysiology of mucus hypersecretion in asthma and allergic rhinitis, and putative sites of action of pharmacotherapeutic compounds. Note that some drugs may act at more than one site, and the precise site of action of some compounds is unclear. CLCA, calcium-activated chloride channel; COX, cyclooxygenase; EGFR, epidermal growth factor receptor; GCs, glucocorticosteroids; MARCKS, myristoylated alanine-rich C-kinase substrate; MEK, mitogenactivated protein kinase kinase; MUC, mucin; PDE, phosphodiesterase. See text for definition of other abbreviations. (See CD-ROM for color version.)

GCs COX-2 inhibitors Suplatast tosilate PDE4 inhibitors

Inflammation

Mediator antagonists Cytokine antagonists Protease inhibitors Anti-MARCKS Anti-Munc18 BoNT endopeptidases Pro-apoptotic drugs Anti-anti-apoptotic drugs P2Y2 agonists/antagonists Macrolide antibiotics

Nerve activation

Secretagogues Plasma “exuders” Mucus secretion

Neural inhibitors/neurotransmitter release inhibitors

M3 anticholinergics Tachykinin NK1 antagonists EGFR tyrosine kinase inhibitors MEK/MAPK inhibitors hCLCA1 inhibitors MUC antisense oligomers

MUC expression

Gland, goblet cell hyperplasia

RAR-a antagonists

Mucus hypersecretion

Mucolytic/mucoactive/antioxidant drugs

Pathophysiology and clinical symptoms

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Table 39.3 Novel therapeutic targets and inhibitors of airway mucus hypersecretion. Target

Inhibitor

Bcl-2 Bradykinin (B2 receptor) Calcium-activated chloride channels (hCLCA1) EGFR tyrosine kinase Endothelin-1 (ETA receptor) ERK Histamine (H1 receptors) Leukotriene (CysLT1 receptors) p38 MAPK Oxidants Platelet-activating factor Prostaglandins (EP, FP receptors) PGD2 (?CRTH2 receptor) MARCKS Mast cell tryptase MUC gene upregulation Neutrophil elastase

Antisense oligonucleotides (e.g., ODN64, ODN83) Icatibant Lomucin (MSI-1995) Iressa (ZD1839) Bosentan ERK inhibitors (e.g., PD 098059), MEK inhibitors (e.g., U0126) Desloratadine, fexofenadine, ebastine Montelukast, zafirlukast (pralukast) p38 MAPK inhibitors (e.g., SB 203580) Superoxide dismutase mimetics (e.g., AEOL 10150C) Apafant, modipafant Indomethacin, selective COX-2 inhibitors PGD2 antagonists (e.g., S-5751) MARCKS inhibitors (antisense oligomer) APC, BABIM Antisense oligonucleotides (18-mer MUC antisense oligomer) Elastase inhibitors (e.g., batimastat, suramin and ?macrolide antibiotics such as erythromycin and flurithromycin) Antioxidants (e.g., N-acetylcysteine) No selective antagonists. Selective agonist (INS37217)

Oxidative stress Purine nucleotides (ATP, UTP: P2Y2 receptors)

EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MARCKS, myristoylated alanine-rich C-kinase substrate; MEK, MAPK kinase; MUC, mucin.

(Rogers 1994). In both asthma and rhinitis, the increased airway secretion is associated with the activity of multiple inflammatory mediators, including cysteinyl leukotrienes and cytokines such as interleukin (IL)-4, IL-9, and IL-13 (Howarth et al. 2000; Rogers 2004; Ciprandi & Passalacqua 2005). Corticosteroids effectively suppress expression of inflammatory genes, including those coding for cytokines (Barnes 2006). Consequently, inhibition by corticosteroids of mucus hypersecretion in asthma and rhinitis is likely to be via a combination of direct effects on plasma and mucus and indirect effects on airway inflammation (Figs 39.8 and 39.10).

Phosphodiesterase 4 inhibitors Phosphodiesterase (PDE)4 is the major PDE isoform in a variety of inflammatory cells implicated in pathophysiology of allergic rhinitis and asthma. Maintenance of intracellular levels of cyclic AMP by inhibition of PDE4 is considered to be of potential therapeutic benefit. For example, rolipram, a selective PDE4 inhibitor, inhibited airway inflammation and goblet cell hyperplasia in a mouse model of allergic asthma (Kanehiro et al. 2001). In a small study in patients with allergic rhinitis, another selective PDE4 inhibitor, roflumilast, produced a transitory reduction in sensations of rhinorrhea (Schmidt et al. 2001). Larger studies, including measurements of volume of secretion, are warranted.

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Anticholinergics Anticholinergics are recommended in clinical management of asthma (Gross 2006) and may also have some benefit in rhinitis (Baraniuk 1998; Jacoby & Fryer 2001). The cholinergic nervous pathway represents the dominant neural stimulus to mucin secretion in the nose (Tai & Baraniuk 2002) and the lower airways (Rogers 2002c). The mucus secretory response of submucosal glands to cholinergic stimulation is largely via muscarinic M3 receptors, with water secretion mediated via M1 receptors (Mullol et al. 1992; Ramnarine et al. 1996; Nakaya et al. 2002). Anticholinergics should reduce mucus and liquid secretion by blocking vagal cholinergic tone (Fig. 39.10). However, it is unclear whether inhaled nonselective anticholinergics decrease mucus secretion or alter mucus viscosity (Pavia et al. 1983b). Tiotropium bromide is a new long-acting anticholinergic that has kinetic selectivity for M1 and M3 receptors over autoinhibitory M2 receptors (Gross 2004). It may therefore have greater efficacy than first-generation anticholinergics. Although its effects on airway secretion have not been reported, 3 weeks’ treatment of chronic obstructive pulmonary disease (COPD) patients with tiotropium did not alter mucociliary clearance (Hasani et al. 2004). The latter observation is difficult to interpret in terms of the effect of tiotropium on airway secretion because of the contribution of both mucus secretion and ciliary activity to clearance.

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Inhibition of nerve activity (“sensoryefferent” nerves) In the airways of a number of animal species, including humans, neuronal C-fibers containing the neurotransmitters substance P (SP), neurokinin A (NKA), and calcitonin generelated peptide (CGRP) form a sensory neural system with a motor function (termed “sensory-efferent” nerves) (Rogers 2002c). The sensory neuropeptides have a variety of biological effects, including on secretion (Khawaja & Rogers 1996). The tachykinins SP and NKA increase plasma exudation, an effect potentiated by the potent vasodilator activity of CGRP, and also increase mucin secretion. The secretory effects are mediated via interaction with tachykinin NK1 receptors. Activation of sensory nerves might therefore contribute to the pathophysiology of airway hypersecretion in allergic rhinitis (Howarth et al. 2000; Tai & Baraniuk 2002) and asthma (Rogers 2002c), although it has so far been difficult to demonstrate for the latter condition. The secretory effects of sensory nerve activation can be inhibited in three ways, namely inhibition of nerve activation, inhibition of release of sensory neuropeptides, or inhibition of neurokinin activity by use of tachykinin receptor antagonists.

Inhibition of sensory nerve activation A variety of irritants activate the vanilloid VR-1 receptor on sensory nerves, for which selective antagonists, such as capsazepine, are in development (Caterina & Julius 2001). Cannabinoids inhibit sensory nerve activation, including the endogenous cannabinoid anandamide, which also inhibits VR-1 (Pertwee 2001). To my knowledge, inhibition by cannabinoids on neurogenic airway mucus secretion has not been explored.

Inhibition of neurotransmitter release Neurotransmitter release, from both sensory and cholinergic nerves, can be inhibited by activation of several types of prejunctional receptor (Rogers 2002c). Opioids are the most effective neuroregulators, and inhibit mucus secretion induced by a variety of stimuli. Inhibition is via both OP1 (previously known as δ) and OP3 (μ) opioid receptors, which have peripheral activity. Peripherally acting opioids such as BW443c have been developed, but have not been tested in patients with airway mucus hypersecretion. Interestingly, cannabinoids inhibit acetylcholine release (Spicuzza et al. 2000) via interaction with prejunctional CB2 receptors. It would be of interest to determine whether they also inhibit sensory neuropeptide release. CB2-selective agonists, such as AM1241 and SR144528, might therefore be effective in reducing neurogenic airway hypersecretion. Many of the prejunctional inhibitory receptors on airway sensory nerves act via a common molecular mechanism that involves opening of large-conductance calcium-activated potassium (BKCa) channels (Rogers 2002c). Drugs that open BKCa channels, such as NS1619, are therefore potential treat-

ments for neurogenic hypersecretion, but to date no clinical studies have been reported.

Tachykinin receptor antagonists Numerous tachykinin receptor antagonists are in development (Rogers 2001). These include peptide and nonpeptide antagonists selective for the NK1, NK2, or NK3 receptor, as well as dual antagonists at NK1 and NK2 receptors. Few of these are in clinical trials for airway diseases, and the results from a limited number of trials in asthma have for the most part been inconclusive. Trials with the newer antagonists, including evaluation in allergic rhinitis, are warranted. However, it may be that, for example, in rhinitis, inhibition of both the secretory effects of tachykinins and the vasodilator effects of CGRP are required for effective control of hypersecretion. Consequently, more than one antagonist would be required. This issue could be resolved by use of a single inhibitor of nerve activation or neurotransmitter release (see above). It should also be noted that inhibition of neurotransmitter release might be expected to extend to inhibition of acetylcholine release, thereby giving additional benefit by inhibiting cholinergic secretion.

Inflammatory mediator antagonists: antihistamines and antileukotrienes Many of the inflammatory mediators that are increased in asthma and rhinitis stimulate mucin secretion and/or plasma exudation, for example bradykinin and endothelin (Table 39.1) (Rogers 2004; Rogers & Evans 1992). However, it could be argued that individual mediators are unlikely to contribute significantly to pathophysiology of these complex disease conditions. Consequently, single-mediator antagonists might not be expected to be particularly effective in disease management, including reduction of mucus secretion. Conversely, single-mediator antagonism would be effective if a particular mediator contributed markedly to pathophysiology and clinical symptoms (Fig. 39.10). Histamine and cysteinyl leukotrienes (LTC4 and LTD4) are two such mediators with a particular involvement in rhinitis. Histamine H1-receptor antagonists are not recommended in the management of asthma (National Institutes of Health 2002) but are extremely effective in rhinitis (Mygind 2001; Meltzer 2005). Part of this effectiveness is likely to be due to inhibition of endogenous histamine-induced nasal secretion (Greiff et al. 2002). Cysteinyl leukotriene receptor (CysLT1) antagonists have also been shown to be effective, to a greater or lesser extent, in clinical management of asthma and rhinitis (Borish 2002). Part of their efficacy may be due to inhibition of mucus secretion (Liu et al. 1998) and plasma exudation (Rogers & Evans 1992). The latter effect may be more pertinent to asthma than rhinitis because the CysLT1 receptor shows minimal localization to the nasal epithelium or glands (Shirasaki et al. 2002). In contrast, the receptor is markedly expressed on

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blood vessels. This localization indicates predominant vascular activity and is consistent with a previous suggestion that, in terms of nasal discharge, leukotrienes are primarily responsible for plasma exudation rather than mucus secretion (Howarth et al. 2000).

Cytokine antagonists A variety of inflammatory cytokines are implicated in the pathophysiology of asthma and allergic rhinitis (Howarth et al. 2000; Simon 2006) (Table 39.1). This suggests that inhibition of these cytokines has therapeutic potential (Fig. 39.10). Certain cytokines have been particularly linked to airway mucus hypersecretion. Tumor necrosis factor (TNF)-α causes a prolonged increase in mucus secretion and upregulation of MUC genes (Levine et al. 1995; Busse et al. 2005), which suggests that inhibitors of TNF-α would be beneficial. However, unless a “pivotal” cytokine is discovered, it seems unlikely that a single cytokine will account for mucus hypersecretion in inflammatory airway disease, and antagonizing a single cytokine is therefore unlikely to have a significant therapeutic impact. The Th2 lymphocyte-derived cytokines IL-4, IL-9, and IL-13 all cause mucus hypersecretion in experimental asthma models (Wills-Karp 2000), and may also be relevant to hypersecretion in allergic rhinitis (Howarth et al. 2000). One therapeutic approach, therefore, would be to inhibit these individual cytokines using soluble receptors or antibodies (Fig. 39.8). Another approach would be to use selective immunomodulators to inhibit the Th2 cells that secrete these cytokines. For example, suplatast tosilate is a Th2-cell inhibitor that suppresses goblet cell metaplasia in mice (Figs 39.8 and 39.10) (Shim et al. 2000).

Protease inhibitors Proteases are potent stimulants of secretion, and a variety of these enzymes are generated in asthma and allergic rhinitis. Consequently, blocking their activity may suppress airway hypersecretion (Fig. 39.10).

Mast cell tryptase inhibitors Mast cells are important inflammatory cells in allergic rhinitis and asthma, and mast cell tryptase induces marked increases in mucus secretion (Sommerhoff et al. 1989). Tryptase inhibitors are in clinical development and two of these, APC 366 and BABIM, suppress airway inflammation in allergic sheep, although effects on mucus were not evaluated (Clark et al. 1995).

Neutrophil protease inhibitors Although neutrophils are possibly not as significant in the pathophysiology of asthma or allergic rhinitis as they are, for example, in COPD, they are implicated at certain stages of disease, for example in acute severe asthma (Woodruff & Fahy 2002). Neutrophil elastase, cathepsin G, and proteinase-3 are

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all potent stimulants of airway secretion (Sommerhoff et al. 1990; Nadel et al. 1999; Witko-Sarsat et al. 1999). This links neutrophilic inflammation in the airways with mucus hypersecretion and suggests that inhibitors of neutrophil proteases would be effective in reducing mucus hypersecretion. Several neutrophil elastase inhibitors are in clinical development (Donnelly & Rogers 2003). Since these proteases are derived from neutrophils, another strategy would be to inhibit neutrophil influx to chemotactic factors such as LTB4, IL-8, and GRO-α. Small-molecule inhibitors of LTB4 receptors (BLT1 receptors) and of CXCR2, the chemokine receptor mediating the neutrophil chemotactic effects of CXC chemokines, are now in clinical trial. PDE4 inhibitors (see above) also inhibit neutrophil infiltration and might therefore inhibit proteaseinduced mucus hypersecretion.

Inhibition of mucus secretion The cellular and molecular mechanisms underlying the process of mucin secretion (exocytosis) are being elucidated, with the possibility of developing specific antisecretory drugs for asthma and rhinitis (Fig. 39.10).

MARCKS inhibitors The myristoylated alanine-rich C-kinase substrate (MARCKS) is a key signaling molecule in the intracellular pathways involved in mucus exocytosis in response to a variety of stimuli (Li et al. 2001), and may therefore represent a novel therapeutic target for inhibiting mucus hypersecretion from multiple causes. An antisense oligonucleotide directed against MARCKS attenuates mucus secretion in vitro, and a MARCKrelated compound (MANS peptide) inhibits hypersecretion in vivo in a mouse model of asthma (Singer et al. 2004).

Munc18B The Sec1/Munc18 family are critical to exocytosis in airway goblet cells, and experimental induction of Munc18B (mammalian homolog of the nematode unc-18 gene product, also known as syntaxin-binding protein 1) induces a marked experimental airway hypersecretory phenotype (Evans et al. 2002). However, to my knowledge, no anti-MUC18B drugs have been reported.

Retargeted clostridial endopeptidases Clostridium botulinum neurotoxins (BoNT) inhibit neurotransmitter (e.g., acetylcholine) release by cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, for example syntaxin, in the nerve terminal (Foster 2005). Conversely, BoNTs could be retargeted to airway secretory cells via a fusion ligand to selectively inhibit mucin exocytosis, and thereby reduce mucus output. For example, we have shown that the LHN fragment (representing the light chain and the N-terminal domain of the heavy chain coupled via a single disulfide bond) of BoNT-C fused to epidermal growth factor (EGF), termed EGF–LHN-C, as

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targeting ligand for EGF receptors on airway epithelial cells markedly inhibits stimulated mucin output from a human respiratory epithelial cell line (Foster et al. 2006).

clinical trial for hypersecretory airway diseases. The results of these trials are awaited with great interest.

MUC gene suppression Epidermal growth factor receptor tyrosine kinase inhibitors The epidermal growth factor receptor (EGFR) and its tyrosine kinase appear to be a fundamental signaling pathway involved in upregulation of MUC gene expression and mucus synthesis, with associated goblet cell hyperplasia in both the nasal mucosa and lower airways (Fig. 39.9) (Lee et al. 2001; Nadel & Burgel 2001). EGFRs may be involved in the increased mucus secretory response to Th2 cytokines and oxidative stress. Several inhibitors of EGFR tyrosine kinase activity, such as AG1571 and ZD1839 (Iressa), are in clinical development for cancer and may prove useful in airway hypersecretory diseases if shown to be safe.

MAP kinase inhibitors

Inhibition of MUC gene expression, with presumed associated inhibition of mucin synthesis and goblet cell hyperplasia, is a future possibility (Fig. 39.10). For example, an 18-mer mucin antisense oligonucleotide not only suppressed MUC gene expression induced by wood smoke in rabbit airway epithelial cells, but also inhibited mucous metaplasia of these cells (Bhattacharyya et al. 1998). It may be possible to develop small-molecule inhibitors of MUC gene expression.

Induction of goblet cell apoptosis Airway goblet cells in asthma models with an associated hypersecretory phenotype express apoptotic factors. For example, in rat airways, Bcl-2, an inhibitor of apoptosis, is expressed in hyperplastic goblet cells, and the proportion of Bcl-2-positive cells is reduced prior to resolution of the hyperplasia (Tesfaigzi et al. 2000). Thus, Bcl-2 appears to be involved in maintenance of metaplastic goblet cells (Fig. 39.9). Targeted reduction of Bcl-2 expression by antisense oligonucleotides (ODN64 or ODN83) causes a dose-dependent resolution of the hyperplasia (Tesfaigzi 2002).

Upregulation of MUC genes is associated with increased airway mucin synthesis and goblet cell hyperplasia. This is presumably due to increased gene expression in response to inflammatory mediators. This in turn activates specific signal transduction pathways, for example EGFR tyrosine kinases and mitogen-activated protein (MAP) kinases in mucussecreting cells, that initiate differentiation of nonmucussecreting cells (primarily basal cells) into goblet cells (Nadel & Burgel 2001). There is emerging evidence that p38 MAP kinase and ERK MAP kinase pathways are central to these signaling pathways (Wang et al. 2002), and small-molecule inhibitors are in development, for example SB 203580 and SB 239063.

Retinoic acid (vitamin A) acting on the RAR-α receptor appears to be involved in increasing mucin expression (Apfel et al. 1992; Yoon et al. 1997; Koo et al. 1999) and in the development and maintenance of a hypersecretory phenotype (Apfel et al. 1992). RAR-α antagonists such as RO-41-5253 inhibit a number of these activities (Bhattacharyya et al. 1998).

Calcium-activated chloride channel inhibitors

P2Y2 receptors: agonist or antagonist?

The human calcium-activated chloride channel (originally reported as Gob-5; now termed mCLCA3 in mice and hCLCA1 in humans) is upregulated in goblet cells in the airways of asthmatic patients (Toda et al. 2002). Similarly, oxidative stress activates these channels in nonciliated nasal epithelial cells (Jeulin et al. 2005). The latter observations, and early data in mice showing that suppression of mCLCA expression inhibited goblet cell hyperplasia whereas overexpression increased goblet cell number (Nakanishi et al. 2001), led to the supposition that these channels are critical in the development of the airway mucus hypersecretory phenotype (Fig. 39.9). However, more recent data in knockout mice either implicate mCACL in airway mucus hypersecretion (Long et al. 2006) or fail to demonstrate such an effect (Robichaud et al. 2005). However, nonselective inhibitors of CACL such as niflumic acid inhibit mucin production and goblet cell hyperplasia in in vitro and in vivo models of asthma (Fig. 39.10) (Hauber et al. 2005; Nakano et al. 2006; Yasuo et al. 2006). Lomucin (MSI 1956, talniflumate) is a smallmolecule putative inhibitor of hCLCA1 that is currently in

The purine nucleotides, adenosine 5′-triphosphate (ATP) and uridine triphosphate (UTP), are stimulants of airway mucin and water secretion (Roger et al. 2000). These effects appear to be mediated by P2Y2 receptors (Fig. 39.10). Consequently, it may be that P2Y2 antagonists would be effective in inhibiting airway hypersecretion. However, mucus hydration, with associated improvements in mucociliary clearance, induced by stimulation of water secretion is currently perceived to be of greater therapeutic potential than inhibition of P2Y2-mediated secretion (Knowles & Boucher 2002). Consequently, there is considerable interest in development of P2Y2 agonists. In phase I clinical trial, a second-generation P2Y2 agonist, INS365, was safe, well tolerated, and significantly enhanced sputum expectoration (Kellerman 2002). The compound is now in phase II trial.

Retinoic acid receptor antagonists

Macrolide antibiotics The macrolide antibiotic erythromycin is reported anecdotally to reduce airway liquid in patients with bronchial hypersecretion (Marom & Goswami 1991), while clarithromycin

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inhibits nasal secretion in patients with purulent rhinitis (Rubin et al. 1997) (Fig. 39.10). In vitro, erythromycin inhibits mucus secretion in human airway explants (Goswami et al. 1990), while azithromycin inhibits MUC5AC production in a human respiratory epithelial cell line (Imamura et al. 2004). These direct effects on mucus secretion extend the growing awareness that macrolide antibiotics have antiinflammatory and immunomodulatory activities additional to their antibacterial properties (Tamaoki et al. 2004).

Mucoactive agents: mucolytics and antioxidants “Mucoactive” agents have some biochemical/biophysical property that is perceived to have an effect on mucus such that administration of these compounds is of benefit to patients (Fig. 39.10). These drugs can be divided into mucolytics, peptide mucolytics, nondestructive mucolytics, expectorants, mucokinetic agents, abhesives, and mucoregulators (Rubin 2002). At least 15 drugs with mucoactive properties are listed in international pharmacopeias for treatment of respiratory conditions associated with airway mucus hypersecretion (Rogers 2002b). However, the results of clinical trials on the effectiveness of these compounds are equivocal. Nevertheless, mucoactive drugs such N-acetylcysteine that are also, or possibly primarily, antioxidants have some beneficial effects in inflammatory airway disease (Poole & Black 2001). Reactive oxygen and nitrogen species are potent stimulants of mucus secretion (Wright et al. 1996). Consequently, antioxidants and inhibitors of inducible nitric oxide synthase may have clinical benefit for airway mucus hypersecretion.

Conclusions From the above, it may be seen that mucus hypersecretion and plasma exudation are features of asthma and allergic rhinitis. Although there are many similarities in the pathophysiology of the mucus hypersecretory phenotype between the two diseases, there are also important differences. For example, in allergic rhinitis, the hypertrophied nasal submucosal glands demonstrate a markedly disproportionate increase in serous cells over mucous cells compared with the more even distribution of these two cell types in the hypertrophied bronchial glands in asthma. The clinical significance of these differences is unclear and warrants investigation. There are numerous biological targets linked to pathophysiology of airway mucus hypersecretion in asthma and allergic rhinitis. These range from the inflammatory cells involved in initiation of the inflammatory response that generates the hypersecretory phenotype to highly specific cellular elements such as CACL channels and EGFR tyrosine kinases. Numerous other targets lurk in the wings awaiting recognition and investigation, for example trefoil factors and aquaporins. In concert with identification of these factors is development of different classes of pharmacotherapeutic molecules directed

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at these targets. Within each class, pharmaceutical companies vie with each other to develop a more marketable compound. Apart from specific instances where a single mediator has a major impact on airway hypersecretory pathophysiology, for example histamine in rhinitis, compounds with a broader spectrum of antiinflammatory activity, for example the effectiveness of corticosteroids in allergic rhinitis and asthma, would be expected to be more effective than compounds with a restricted activity. However, certain highly specific targets, such as hCACL1, appear to be intimately associated with development of an airway hypersecretory phenotype. Data from clinical trials with blockers of these targets are awaited with great interest, not only for potential patient benefit but also to address the tantalizing concept that these specific, almost “innocuous,” targets play a significant role in airway hypersecretory pathophysiology.

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Ramnarine, S.I., Haddad, E.B., Khawaja, A.M., Mak, J.C. & Rogers, D.F. (1996) On muscarinic control of neurogenic mucus secretion in ferret trachea. J Physiol 494, 577– 86. Robichaud, A., Tuck, S.A., Kargman, S. et al. (2005) Gob-5 is not essential for mucus overproduction in preclinical murine models of allergic asthma. Am J Respir Cell Mol Biol 33, 303–14. Roger, P., Gascard, J.P., Bara, J., de Montpreville, V.T., Yeadon, M. & Brink, C. (2000) ATP induced MUC5AC release from human airways in vitro. Mediators Inflamm 9, 277– 84. Rogers, D.F. (1994) Airway goblet cells: responsive and adaptable front-line defenders. Eur Respir J 7, 1690–706. Rogers, D.F. (2001) Tachykinin receptor antagonists for asthma and COPD. Expert Opin Ther Patents 11, 1097–121. Rogers, D.F. (2002a) The airway goblet cell. Int J Biochem Cell Biol 35, 1–6. Rogers, D.F. (2002b) Mucoactive drugs for asthma and COPD: any place in therapy? Expert Opin Investig Drugs 11, 15–35. Rogers, D.F. (2002c) Pharmacological regulation of the neuronal control of airway mucus secretion. Curr Opin Pharmacol 2, 249– 255. Rogers, D.F. (2003) Pulmonary mucus: pediatric perspective. Pediatr Pulmonol 36, 178– 88. Rogers, D.F. (2004) Airway mucus hypersecretion in asthma: an undervalued pathology? Curr Opin Pharmacol 4, 241–50. Rogers, D.F. & Barnes, P.J. (2006) Treatment of airway mucus hypersecretion. Ann Med 38, 116– 25. Rogers, D.F. & Evans, T.W. (1992) Plasma exudation and oedema in asthma. Br Med Bull 48, 120– 34. Rubin, B.K. (2002) The pharmacologic approach to airway clearance: mucoactive agents. Respir Care 47, 818– 22. Rubin, B.K., Druce, H., Ramirez, O.E. & Palmer, R. (1997) Effect of clarithromycin on nasal mucus properties in healthy subjects and in patients with purulent rhinitis. Am J Respir Crit Care Med 155, 2018– 23. Schmidt, B.M., Kusma, M., Feuring, M. et al. (2001) The phosphodiesterase 4 inhibitor roflumilast is effective in the treatment of allergic rhinitis. J Allergy Clin Immunol 108, 530–6. Schuhl, J.F. (1995) Nasal mucociliary clearance in perennial rhinitis. J Invest Allergol Clin Immunol 5, 333– 6. Sheehan, J.K., Richardson, P.S., Fung, D.C., Howard, M. & Thornton, D.J. (1995) Analysis of respiratory mucus glycoproteins in asthma: a detailed study from a patient who died in status asthmaticus. Am J Respir Cell Mol Biol 13, 748–56. Sheehan, J.K., Howard, M., Richardson, P.S., Longwill, T. & Thornton, D.J. (1999) Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug. Biochem J 338, 507–13. Shim, J.J., Dabbagh, K., Takeyama, K. et al. (2000) Suplatast tosilate inhibits goblet-cell metaplasia of airway epithelium in sensitized mice. J Allergy Clin Immunol 105, 739– 45. Shimura, S., Sasaki, T., Sasaki, H., Takishima, T. & Umeya, K. (1988) Viscoelastic properties of bronchorrhoea sputum in bronchial asthmatics. Biorheology 25, 173– 9. Shimura, S., Andoh, Y., Haraguchi, M. & Shirato, K. (1996) Continuity of airway goblet cells and intraluminal mucus in the airways of patients with bronchial asthma. Eur Respir J 9, 1395– 401. Shirasaki, H., Kanaizumi, E., Watanabe, K. et al. (2002) Expression and localization of the cysteinyl leukotriene 1 receptor in human nasal mucosa. Clin Exp Allergy 32, 1007–12.

Simon, H.U. (2006) Cytokine and anti-cytokine therapy for asthma. Curr Allergy Asthma Rep 6, 117– 21. Singer, M., Martin, L.D., Vargaftig, B.B. et al. (2004) A MARCKSrelated peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med 10, 193–6. Skoner, D.P. (2001) Allergic rhinitis: definition, epidemiology, pathophysiology, detection, and diagnosis. J Allergy Clin Immunol 108 (suppl.), S2–S8. Sommerhoff, C.P., Caughey, G.H., Finkbeiner, W.E., Lazarus, S.C., Basbaum, C.B. & Nadel, J.A. (1989) Mast cell chymase. A potent secretagogue for airway gland serous cells. J Immunol 142, 2450–6. Sommerhoff, C.P., Nadel, J.A., Basbaum, C.B. & Caughey, G.H. (1990) Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 85, 682–9. Spicuzza, L., Haddad, E.B., Birrell, M. et al. (2000) Characterization of the effects of cannabinoids on guinea-pig tracheal smooth muscle tone: role in the modulation of acetylcholine release from parasympathetic nerves. Br J Pharmacol 130, 1720–6. Stanley, P.J., Wilson, R., Greenstone, M.A., Mackay, I.S. & Cole, P.J. (1985) Abnormal nasal mucociliary clearance in patients with rhinitis and its relationship to concomitant chest disease. Br J Dis Chest 79, 77–82. Sun, S.S., Hsieh, J.F., Tsai, S.C., Ho, Y.J. & Kao, C.H. (2002) Evaluation of nasal mucociliary clearance function in allergic rhinitis patients with technetium 99m-labeled macroaggregated albumin rhinoscintigraphy. Ann Otol Rhinol Laryngol 111, 77–9. Tai, C.F. & Baraniuk, J.N. (2002) Upper airway neurogenic mechanisms. Curr Opin Allergy Clin Immunol 2, 11–19. Takeuchi, K., Yuta, A. & Sakakura, Y. (1995) MUC2 mucin gene expression in the nose and maxillary sinus. Am J Otolaryngol 16, 391–5. Tamaoki, J., Kadota, J. & Takizawa, H. (2004) Clinical implications of the immunomodulatory effects of macrolides. Am J Med 117 (suppl. 9A), 5S–11S. Tanaka, T., Kobayashi, T., Sunaga, K. & Tani, S. (2001) Effect of glucocorticoid on expression of rat MUC5AC mRNA in rat gastric mucosa in vivo and in vitro. Biol Pharm Bull 24, 634–7. Tesfaigzi, Y. (2002) The role of apoptotic regulators in metaplastic mucous cells. Novartis Found Symp 248, 221–30. Tesfaigzi, Y., Fischer, M.J., Martin, A.J. & Seagrave, J. (2000) Bcl-2 in LPS- and allergen-induced hyperplastic mucous cells in airway epithelia of Brown Norway rats. Am J Physiol 279, L1210–L1217. Thornton, D.J., Howard, M., Khan, N. & Sheehan, J.K. (1997) Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule. J Biol Chem 272, 9561–6. Toda, M., Tulic, M.K., Levitt, R.C. & Hamid, Q. (2002) A calciumactivated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma. J Allergy Clin Immunol 109, 246–50. Toppozada, H.H. & Gaafar, H.A. (1973) Electron microscopy of the human nasal mixed glands. J Laryngol Otol 87, 639–45. Toppozada, H.H. & Talaat, M.A. (1976) Tunica propria in chronic allergic rhinitis. Electron-microscopic study. I. The nasal blood vessels. ORL J Otorhinolaryngol Relat Spec 38, 86–92. Voynow, J.A., Selby, D.M. & Rose, M.C. (1998) Mucin gene expression (MUC(1) MUC(2) and MUC5/5AC) in nasal epithelial cells of cystic fibrosis, allergic rhinitis, and normal individuals. Lung 176, 345– 54.

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Wang, B., Lim, D.J., Han, J., Kim, Y.S., Basbaum, C.B. & Li, J.D. (2002) Novel cytoplasmic proteins of nontypeable Haemophilus influenzae up-regulate human MUC5AC mucin transcription via a positive p38 mitogen-activated protein kinase pathway and a negative phosphoinositide 3-kinase-Akt pathway. J Biol Chem 277, 949–57. Wanner, A., Salathe, M. & O’Riordan, T.G. (1996) Mucociliary clearance in the airways. Am J Respir Crit Care Med 154, 1868–902. Wickstrom, C., Davies, J.R., Eriksen, G.V., Veerman, E.C. & Carlstedt, I. (1998) MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem J 334, 685–93. Williams, I.P., Rich, B. & Richardson, P.S. (1983) Action of serum on the output of secretory glycoproteins from human bronchi in vitro. Thorax 38, 682– 5. Willsie, S.K. (2002) Improved strategies and new treatment options for allergic rhinitis. J Am Osteopath Assoc 102 (suppl. 2), S7–S14. Wills-Karp, M. (2000) Trophic slime, allergic slime. Am J Respir Cell Mol Biol 22, 637– 9. Witko-Sarsat, V., Halbwachs-Mecarelli, L., Schuster, A. et al. (1999) Proteinase 3, a potent secretagogue in airways, is present in cystic fibrosis sputum. Am J Respir Cell Mol Biol 20, 729– 36.

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Wood, S.J., Birchall, M.A., Carlstedt, I. & Corfield, A.P. (1997) The expression of MUC5AC in allergic rhinitis. Biochem Soc Trans 25, 504S. Woodruff, P.G. & Fahy, J.V. (2002) A role for neutrophils in asthma? Am J Med 112, 498–500. Wright, D.T., Fischer, B.M., Li, C., Rochelle, L.G., Akley, N.J. & Adler, K.B. (1996) Oxidant stress stimulates mucin secretion and PLC in airway epithelium via a nitric oxide-dependent mechanism. Am J Physiol 271, L854–L861. Wu, G.J., Wu, M.W., Wang, S.W. et al. (2001) Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression. Gene 279, 17–31. Yasuo, M., Fujimoto, K., Tanabe, T. et al. (2006) Relationship between calcium-activated chloride channel 1 and MUC5AC in goblet cell hyperplasia induced by interleukin-13 in human bronchial epithelial cells. Respiration 73, 347–59. Yoon, J.H., Gray, T., Guzman, K., Koo, J.S. & Nettesheim, P. (1997) Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix. Am J Respir Cell Mol Biol 16, 724–31.

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Biology of Vascular Permeability Peter Clark

Summary The leakage of plasma into tissues, leading to edema formation, is one of the hallmarks of inflammation, and it is becoming increasingly apparent that allergic edema can have profound effects on the pathophysiology and clinical manifestations of allergic disease. This chapter examines the basic biology of vascular permeability and reviews the major molecular and cellular mechanisms involved in controlling endothelial barrier function. In particular, recent findings in the signaling mechanisms leading to the loss, or enhancement and maintenance, of vascular barrier properties are considered. The relative contributions of actomyosin-mediated endothelial cell contractility and the reorganization of interendothelial junctions are considered to be key to understanding the modulation of vascular barrier function. Recent findings on the mechanisms of leukocyte-mediated edema, and on endogenous and pharmacologic agents enhancing barrier function, provide insights into potential therapeutic targets in allergy.

Introduction Increased vascular permeability, leading to edema formation, is one of the defining responses to inflammatory stimuli. In allergic inflammation, it is well established that edema significantly contributes to clinical symptoms. For example, edematous airways are narrowed, they exhibit increased airflow resistance and altered mechanical properties, and it has been suggested that their hyperreactivity is enhanced (Bucca & Rolla 1989; Chung et al. 1990). Also, swelling of the deeper layers of the skin is the hallmark of angioedema, a disease which often has an allergic basis. This review considers the basic biology of vascular permeability and examines our current understanding of the mechanisms of its control.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Although transport of solutes and fluid can occur to some degree across any vessel wall, most exchange between the blood and the tissues takes place in the microvessels, particularly the capillaries and venules (Michel & Curry 1999; Curry 2005; Mehta & Malik 2006). While hydrophobic molecules and respiratory gases can freely exchange via cellular membranes, the walls of exchange vessels act as restrictive filters to water, hydrophilic solutes, and blood cells. Vessels are lined with endothelial cells that are commonly assumed to constitute the vessel barrier, although a variety of studies have estimated that the extracellular matrix (ECM), and nonendothelial cells associated with the vessel wall, could contribute up to 50% of the barrier (Michel & Curry 1999). Changes to ECM organization that may affect vessel permeability are not well understood, but they could be important in relation to chronic pathologic remodeling of the interstitium. In resting (unstimulated) conditions, the endothelial cells lining vessels form an antithrombogenic surface poised to react to a wide variety of stimuli that alter vessel permeability and promote the recruitment of leukocytes (Galley & Webster 2004). It is these properties of the endothelial lining of vessels that have received most attention in terms of the control of vessel permeability.

Basal permeability of microvessels The fluid and solute fluxes across a resting vessel wall are described in biophysical terms as a balance between two pressure differences: the filtration pressure (i.e., the difference between intraluminal hydrostatic pressure and the pressure in the surrounding tissue), and the oncotic pressure resulting from the difference in the osmotic pressure exerted by macromolecules inside and outside of a vessel (Michel & Curry 1999; Curry 2005; Mehta & Malik 2006). This model was first proposed by Starling more than 100 years ago, but has been generally confirmed and refined by subsequent studies, and is still considered to be a realistic description of the biophysical properties of microvessels. The full modern expression of Starling’s principle is: Jv/S = Lp[(Pc – Pi) – σ(πc – πi)]

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N (b)

Caveolae

VVO forming transcellular channel

(d) Fenestrae with diaphragms 0.5 µm (e) (c) (a)

where Jv/S is the volume of fluid flux per unit surface area of vessel wall, Lp the hydraulic conductivity (i.e., permeability to water), Pc and Pi vessel and interstitial hydrostatic pressures, πc and πi the vessel and interstitial osmotic pressures of macromolecules (oncotic pressures), and σ the oncotic reflection coefficient of macromolecules (a measure of permeability to plasma proteins, where 0 = fully permeable, 1 = impermeable). In essence, this equation predicts that when the vessel luminal hydrostatic pressure is greater than that in the interstitium, fluid flow out of the vessel is promoted. However, this is opposed by the oncotic pressure, where the concentration of macromolecules (i.e., plasma proteins) in the vessel lumen is usually greater than in the interstitium. If there is no net flow across a vessel’s wall, then the opposing Starling forces are balanced. Where a concentration difference of a small solute (e.g., glucose) between the vessel lumen and the interstitium exists, the solute can pass into the tissues relatively easily in most vascular beds, and vice versa. Further biophysical studies have shown that microvessels behave as filters containing two populations of cylindrical or slit-like pores: relatively numerous small pores of radius or width 4–5 nm (close to the effective radius of albumin), and rare large pores of radius or width 20–30 nm. The frequency and proportion of these two populations of pores can account for the variability in the basal permeability in different microvascular beds. The structural basis of the pathways across small and large pores has proved difficult to determine, although the differences in basal permeability between different vessels have some basis in obvious structural differences: continuous endothelia (lacking fenestrae, e.g., dermal, lung, skeletal and heart muscle capillaries) are generally less permeable than fenestrated endothelia (e.g., small intestinal capillaries, renal glomerular capillaries). The impermeable blood–brain barrier endothelia also provide strong clues as to the nature of the pores. Blood–brain barrier endothelia form interendothelial junctions that are considerably tighter than in peripheral

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Fig. 40.1 Endothelial cell structure. (a) Lowpower transmission electron micrograph (TEM) of a rat cardiac capillary endothelial cell showing the thin capillary wall, except in the region of the nucleus (N). (b) Detail of the box in (a) showing an intercellular cleft (arrow). (c) Detail of the capillary wall showing caveolae and intercellular vesicles. (d) Diagram showing the organization and relationships of caveolae and vesiculovacuolar organelles (VVO). (e) Diagram of the thin wall of a fenestrated endothelial cell. (a, b and c courtesy of Matthew Glyn, Imperial College London.) (See CD-ROM for color version.)

vessels (Wolburg & Lippoldt 2002). Passive diffusion of small hydrophilic solutes across the blood–brain barrier vessels is severely restricted, but the endothelia express transporters (e.g., glucose transporter-1) to allow controlled transcellular exchange (Abbott 2005). They also lack caveolae, this being an indication of reduced transcellular vesicular transport. The obvious pathway for the passage of small solutes is via the paracellular pathway, i.e., through the narrow spaces between adjacent endothelial cells, the interendothelial clefts (Fig. 40.1). The organization of the interendothelial clefts is known to be crucial to the formation of coherent endothelial layers and involves adhesive junction structures similar to those formed between epithelial cells. The two main types of junction between endothelial cells are adherens junctions and tight junctions (Fig. 40.2) (Aurrand-Lions et al. 2002; Bazzoni & Dejana 2004; Dejana 2004).

Adherens junctions Adhesion at adherens junctions (AJs) is mediated by cadherin cell–cell adhesion molecules (Fig. 40.2). Cadherins are calcium ion-dependent adhesion molecules that normally associate with identical cadherins on adjacent cells, i.e., homophilic adhesion (Takeichi et al. 2000). Endothelial cells express a specific cadherin, vascular endothelial cadherin (VE-cadherin, also designated as cadherin-5) (Lampugnani et al. 1992; Leach et al. 1993), which is essential for the formation and maintenance of endothelial barriers (Corada et al. 1999). One exception is in blood–brain barrier vessels where VE-cadherin is downregulated and cadherin-10 is expressed (Breier et al. 1996; Williams et al. 2005). Classical cadherins, including VE-cadherin, span the plasma membrane and their cytoplasmic domains are associated with catenins (Fig. 40.2), molecules that link them to the actin cytoskeleton and control their turnover (Yagi & Takeichi 2000). A specific sequence in the cadherin cytoplasmic domain binds a molecule of either β-catenin or γ-catenin (plakoglobin), which binds to α-catenin. α-Catenin in turn associates this

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Occludin, claudins

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IgCAMs (PECAM-1, JAMs, ESAM, ICAM-2, nectins)

TJ

ZO-1,2,3

Actin

VE-cadherin b/g

a

AJ

FA Integrins

p120

Fig. 40.2 Basic molecular organization of endothelial adhesions. The two main types of interendothelial junctions are the tight junction (TJ) and the adherens junction (AJ); TJ integral membrane proteins, occludin and claudins, link to the actin cytoskeleton via zonula occludens (ZO)-1, ZO-2 and ZO-3, as well as other components not shown. Vascular endothelial (VE)-cadherin links to the actin cytoskeleton via either b-catenin (b) or g-catenin (g). p120 catenin (p120) binds to a juxtamembrane site of the cytoplasmic domain of VE-cadherin. Immunoglobulin-like cell adhesion

molecules (IgCAMs) present at interendothelial junctions include platelet endothelial cell adhesion molecule (PECAM)-1, junction adhesion molecules (JAMs), endothelial-selective adhesion molecule (ESAM), intercellular adhesion molecule (ICAM)-2, and members of the nectin family. Adhesion to the basement membrane is mediated by integrin complexes that cluster to form focal adhesions (FA). Integrins link to the actin cytoskeleton via talin, paxillin, vinculin, a-actinin, and others (not shown). (See CD-ROM for color version.)

complex with the filamentous actin (F-actin) cytoskeleton. Disrupting these cadherin–cytoskeleton associations, or directly disrupting the organization of F-actin, leads to the breakdown of cell–cell contacts and a loss of barrier function (Yuan 2002; Bazzoni & Dejana 2004; Verin 2005). Desmosomes are not present in endothelia, although plakoglobin associated with VE-cadherin can recruit desmosomal plaque proteins (Dejana 2004). In epithelia, desmosomal plaque proteins associate with cytokeratin intermediate filaments. In endothelial cells, the “hybrid” AJ/desmosome junctional structures associate with vimentin intermediate filaments, this having been best described as the complexus adhaerentes in lymphatic vessels, although there is some evidence of these structures in blood vessel endothelium, particularly in culture (Lampugnani et al. 1995; Andriopoulou et al. 1999; Venkiteswaran et al. 2002). The functional significance of these different AJ components is not clear. It has been suggested that vimentin-coupled adhesions are required for normal endothelial organization in vessel formation. Plakoglobin recruitment to AJs in cultured human umbilical vein endothelial cells (HUVECs) was shown to be associated with the formation of mature junctions, while β-catenin predominates in nascent and immature junctions (Lampugnani et al. 1995). A different catenin, p120ctn, binds to a juxtamembrane region of the cytoplasmic tail of cadherins distinct from the β-catenin/γ-catenin binding site. p120 is known to regulate cadherin trafficking and also signaling involved in actin organization (Iyer et al. 2004). Lack of association of p120 with VE-cadherin can lead to cadherin internalization and loss of cell–cell adhesion, and to

altered signaling from AJ (Iyer et al. 2004). The cadherin/ catenin/actin complex forms the core of AJ, but a number of other molecules, including cell adhesion molecules (e.g., nectins) and signaling molecules (e.g., growth factor receptors, small GTPases, protein kinases, and phosphatases), are also present and play important roles in AJ organization (Bazzoni & Dejana 2004). AJs are required for the formation of other intercellular junctions; tight junctions will not normally form in the absence of AJ formation (Gonzalez-Mariscal et al. 2003).

Tight junctions Tight junctions (TJs, zonula occludens) are made up of networks of strands of integral membrane proteins that associate with complementary strands to mediate close contact between the plasma membranes of adjacent cells (Fig. 40.2). When these networks of strands are sufficiently extensive, they can allow the effective sealing of the paracellular pathway (Gonzalez-Mariscal et al. 2003). The paracellular passage of water and solutes is severely restricted in cell layers with well-formed TJs. In most endothelia, TJs are not well developed. The TJ strands are generally sparse and discontinuous, offering only a partial restriction of the paracelluar pathway. The major exception to this is again in the blood–brain barrier, where interendothelial TJs are more extensive, contributing to the impermeability of these vessels (Wolburg & Lippoldt 2002). TJ strands are made up of transmembrane proteins, occludin and members of the claudin family. These integralmembrane TJ proteins associate with the actin cytoskeleton

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via ZO-1, ZO-2, and ZO-3 and other plaque proteins. Occludin is present in epithelial tight junctions and blood–brain barrier endothelial junctions, but is only weakly and patchily expressed in other endothelia. The claudin family members are differentially expressed, some with wide expression (e.g., claudin-1 in many epithelia), with others having more limited expression patterns. Claudin-5 is expressed mainly in endothelial cells, although the more extensive TJs of the blood–brain barrier have been shown to also contain claudins 1, 3, and 12 (Gonzalez-Mariscal et al. 2003; Wolburg et al. 2003). Some claudins (e.g., claudin-16) have been shown to form ion-selective pores through the “sealed” TJ strands, although it is not clear if this is a feature in endothelial TJs. In addition to restricting the paracellular passage of water and solutes, TJs have an important role in forming and maintaining the apical/basolateral membrane polarity of epithelial and endothelial monolayers, by virtue of their role in restricting the diffusion of membrane components between the apical and basolateral plasma membrane domains (GonzalezMariscal et al. 2003). As with AJs, other cell-adhesion and signaling molecules are associated with TJs. Junctional adhesion molecules (JAMs) and endothelial selective adhesion molecule (ESAM) are immunoglobulin superfamily members associated with tight junctions, and are known to play a role in the development and organization of TJs by recruiting cytoplasmic binding partners such as ZO-1 and the mammalian homologs of the nematode Caenorhabditis elegans, partitioning defective gene products, the Par proteins (Ebnet et al. 2003; Ilan & Madri 2003; Wegmann et al. 2006). Such recruitment is required for the establishment of polarity in endothelial and epithelial cells. Other immunoglobulin superfamily molecules, e.g., PECAM-1 (CD31) and ICAM-2, are also associated with interendothelial clefts; there appears to be a large degree of functional redundancy, since knockout of individual junction-associated immunoglobulin superfamily molecules shows that they are not essential for endothelial junction organization. Many of these immunoglobulin superfamily molecules have ligands on leukocytes, and thus play a role in leukocyte–endothelial adhesion and signaling during diapedesis and extravasation (Nourshargh et al. 2006). An important difference in the arrangement of intercellular junctions exists between endothelial and epithelia. In simple epithelia, AJs and TJs are spatially and biochemically distinct, typically found as an apical TJ belt, with AJs as a separate belt basal to them. In endothelial cells, immunoelectron microscopy and biochemical fractionation have shown that TJs and AJs are intermingled, i.e., TJ strands are “embedded” in the AJ belt (Schulze & Firth 1993; Bazzoni & Dejana 2004; Ruffer et al. 2004).

Pores in the resting endothelium The most compelling model that accounts for the small pore of endothelial barriers involves both TJs and AJs. The extent of the discontinuities in TJ strands were quantified by ultra-

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thin serial-sectioning of endothelia, but these proved to be too large (20 nm width) and too abundant to account solely for the small pore component of the endothelial filter (theoretically 4 nm in width) (Michel & Curry 1999). However, recent models have proposed that the glycocalyx in the interendothelial cleft, together with the partial restriction of the discontinuous TJs, provides the biophysically predicted filtration. This “fiber matrix model” proposes that water and solutes are funneled through the breaks in the TJ strands, with a second, finer level of filtration being the glycocalyx at the luminal entrance and in the 20-nm wide AJ regions (Firth 2002; Curry 2005). For many years, the nature of the large pore has been the subject of considerable controversy. However in recent years, morphologic studies and molecular biology have provided strong evidence that transcellular transport via the caveolar system in endothelial cells could effectively function as the large pore, providing the mechanism for the passage of macromolecules across resting endothelial cells (Tuma & Hubbard 2003; Gratton et al. 2004). Caveolae (Fig. 40.1) are small (50–100 nm with ∼ 25-nm cell surface openings), uncoated, flask-shaped pits or invaginations of the plasma membrane. Caveolae at the cell surface can possess a diaphragm at the extracellular “opening.” Large numbers of individual uncoated vesicles are observed in endothelia by transmission electron microscopy. Three-dimensional reconstruction of serial images from ultrathin sections failed to conclusively show the presence of individual caveolar vesicles, suggesting that these structures are part of larger interconnected structures that might directly connect the luminal and abluminal spaces, i.e., forming a transcellular channel (Dvorak et al. 1996). However, the thickness of ultrathin sections (50–100 nm) is similar to the dimensions of vesicles, and makes such reconstructions difficult to interpret. Larger interconnected groups of “vesicles” are observed in a single ultrathin section, and have been described as vesiculovacuolar organelles (VVOs) (Fig. 40.1) (Dvorak et al. 1996). These caveolar and VVO membranes contain members of the caveolin family of proteins, caveolin-1 and -2 (Williams & Lisanti 2004). Caveolincontaining membranes are also membrane microdomains (i.e., lipid rafts) enriched with signaling molecules such as protein kinase C (PKC) isoforms, src family tyrosine kinases, and endothelial nitric oxide synthase (eNOS). In unstimulated endothelia, the constitutive association of eNOS with caveolin holds the enzyme in the inactive state. Receptors involved in macromolecular transport, e.g., the albumin receptor gp60, are also present in caveolar membranes. Recent studies have provided evidence that the baseline passage of macromolecules, particularly albumin, is transcellular, via caveolae. It is now known that caveolae possess the molecular repertoire of proteins required for docking and scission during vesicular trafficking, and that downregulation or inhibition of these proteins leads to loss of albumin transport (Tuma & Hubbard 2003; Mehta & Malik 2006).

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Caveolin knockout mice have provided some of the strongest evidence for the role of caveolae in macromolecular transport (Predescu et al. 2004; Lin et al. 2007). In wild-type mice, labeled albumin is ultrastructurally localized to caveolae and uncoated vesicles, but is not detected in interendothelial clefts. In caveolin-1 knockout mice, endothelial cells lack caveolae, and the transcellular passage of albumin is not observed. However, albumin is detected in the intercellular clefts. This is likely to be due to the fact that in the absence of caveolin-1, eNOS is deregulated (i.e., loss of caveolinmediated eNOS inhibition) and nitric oxide (NO) is produced at higher levels than in wild type, which leads to enhanced paracellular permeability (Predescu et al. 2005) (see below). In this model, the loss of transcellular transport of albumin is compensated for by the NO-induced increase in paracellular permeability. A recent study has shown that cross-linked intercellular adhesion molecule (ICAM)-1 is internalized from apical caveolae and is transported to the basal plasma membrane in caveolin-containing vesicular and VVO structures (Millan et al. 2006). Although such vesicular transport

(ai)

does not fulfil the strict morphologic or biophysical criteria of a “pore”, there is little doubt that the constitutive transcellular transport of macromolecules accounts for, at least partly, the large pore function.

Hyperpermeability in microvessels Although capillaries are induced to be leaky in extreme situations, such as thermal shock and systemic capillary leak syndrome (Fishel et al. 2003), agents that increase the permeability of vessels act primarily on postcapillary venules (Fig. 40.3). The seminal work by Palade and his colleagues showed that histamine and serotonin each induced the formation of gaps in the endothelial linings of postcapillary venules where circulating colloidal tracer leaked into the interstitium (Majno & Palade 1961; Majno et al. 1961). They showed that these gaps were associated with focal openings of the interendothelial clefts, and little or no tracer could be seen in intracellular vesicles in the endothelium. Many

a-smooth muscle actin

Vessel endothelium (aii)

Biology of Vascular Permeability

Tracer leakage (aiii)

No stimulus (bi)

(bii)

(biii)

Bradykinin Fig. 40.3 Sites of leakage in stimulated vessels. Confocal projections of mouse dermis after intravascular injection of fluorescent submicrometer particles, followed by intradermal injection of (a) control buffer or (b) bradykinin. Skin biopsies were stained to show endothelium (ai and bi) and a-smooth muscle actin (aii and bii). Little accumulation of the fluorescent particles is seen with the control intradermal injection (aiii), while particle accumulation is observed in venules, but not capillaries, after intradermal injection of bradykinin (biii). (Images courtesy of Adam Al-Kashi, Imperial College London.)

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subsequent studies have confirmed and extended these findings for a wide variety of agents in vivo (Thurston et al. 1996; Baluk et al. 1997, 1998; McDonald et al. 1999; Michel & Curry 1999; Bates & Harper 2002; Thurston et al. 2000a), and using cultured endothelial cell monolayers (Andriopoulou et al. 1999; Wojciak-Stothard et al. 2001; Wojciak-Stothard & Ridley 2002; Sun et al. 2006). It is generally believed that gap formation results from a combination of increased endothelial actomyosin-dependent cell tension and reduced intercellular adhesion. The nature and origin of at least a proportion of the induced gaps has been called into question. Michel and his colleagues used serial ultrathin sectioning and electron microscopy of frog and rat mesenteric vessels to show that for some stimuli, such as calcium ionophore, heating, and vascular endothelial growth factor (VEGF), the induced gaps were predominantly transcellular, while gaps induced by other mediators (e.g., histamine) were mainly intercellular (Michel & Curry 1999). It is now well established that, in addition to passage via the interendothelial clefts, transcellular openings are an important pathway for the extravasation of leukocytes from the blood to the tissues (Feng et al. 1996), and it has recently been shown that these openings are lined by caveolin-rich membranes (Millan et al. 2006). It has also been proposed that transcellular gaps induced by inflammatory agents at, or close to, interendothelial clefts involve the fusion of caveolae and VVO, although there is a suggestion that this may only apply in the presence of particulate tracers (Curry 2005). A study examining the kinetics of agonist-induced gap formation and leakage of circulating tracer found that after intravenous injection of substance P, gaps formed in rat tracheal venules persisted after the fall in circulating tracer leakage (Baluk et al. 1997). This suggests that the restoration of the endothelial barrier, at least to colloidal tracers, may be more complex than simply closing the gaps, and could involve rearrangements of the glycocalyx of the interendothelial cleft.

i.e., stress fibers. At the same time, cortical F-actin at cell–cell junctions is reduced, as are various other components of AJs and TJs. The signaling mechanisms leading to these changes have been studied in detail in a variety of conditions. The main pathways involved in the induction of contractility could be summarized as those leading to increased intracellular Ca2+ concentration ([Ca2+]i) and activation of the small GTPase, Rho; and those leading to disruption of cell–cell adhesion involving phosphorylation of junction components and inhibition of the small GTPase, Rac (Wojciak-Stothard & Ridley 2002; Millan & Ridley 2005; Mehta & Malik 2006). Increased [Ca2+]i has been shown to be necessary for hyperpermeability induced by most agents (Michel & Curry 1999; Tiruppathi et al. 2002; Curry 2005). Agonist/receptor-induced activation of phospholipases (PLC and/or PLD isoforms) leads to the production of diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 binds to its receptors on endoplasmic reticulum (ER) membranes to stimulate the release of ERstored Ca2+. This increase in [Ca2+]i stimulates the opening of nonselective divalent cation transient receptor potential channels (TRPC) in the plasma membrane (Ahmmed & Malik 2005). These “store-operated channels” (SOC), e.g., TRPC4, allow extracellular Ca2+ to enter the cell to enhance and sustain the increase in [Ca2+]i. Some TRCP channels are activated by the binding of DAG, independently of stored Ca2+. These receptor-operated channels (ROC) (e.g., TRPC6) have been shown to act in VEGF-induced permeability in isolated, single, perfused venules and in cultured cells (Pocock et al. 2004). Endothelial cells express multiple TPRCs, and both SOC and ROC could contribute to [Ca2+]i. A number of signaling pathways are influenced by increased [Ca2+]i. In particular, Ca2+/calmodulin-dependent myosin light chain

Ca2+/Calmodulin MLCK

MLCK

Rho-GTP

Inactive

Active

Rho kinase

Cell signaling and vessel permeability The signaling pathways involved in the control of vessel permeability have recently been comprehensively reviewed (Mehta & Malik 2006). Here, the major issues and some recent findings relating to potential therapeutic targets are considered.

Stress fibers Contractility

MLC-p

MLC

High affinity

Low affinity MLCptase

MLCptase-p

Active

Inactive

Calcium ions, NO, and endothelial contractility As mentioned above, increased permeability coincides with the formation of intercellular gaps, and is believed to be the result of a breakdown of the balance between endothelial centripetal cell tension produced by actomyosin-mediated contraction and interendothelial adhesion. Studies in cultured endothelial cells consistently show that agents which increase permeability induce the formation of contractile actomyosin bundles associated with focal adhesions (cell–ECM adhesions),

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Permeability Fig. 40.4 Cell signaling leading to actomyosin contractility. Endothelial cell contractility is increased by activation of the regulatory myosin light chain (MLC) kinase and Rho. MLC is phosphorylated by myosin light chain kinase (MLCK) and Rho-activated Rho kinase to increase its affinity for binding to actin. Myosin light chain phosphatase (MLCptase) is deactivated by Rho kinase-mediated phosphorylation, which also promotes MLC phosphorylation. (See CD-ROM for color version.)

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kinase (MLCK) is activated by [Ca2+]i (Fig. 40.4). Activated MLCK phospholylates myosin light chain (MLC) to promote myosin binding to F-actin and stress fiber formation, resulting in actomyosin contraction and gap formation. At odds with this, there are examples (discussed below) of agents that induce the phosphorylation of MLCK but which enhance endothelial barrier function. Endothelial cells express a nonmuscle isoform of MLCK, EC-MLCK, which has a unique N-terminal domain containing phosphorylation sites for cAMP-dependent kinase (PKA) and Src (Garcia et al. 1997; Bogatcheva et al. 2002). PKA-mediated serine/threonine phosphorylation reduces EC-MLCK catalytic activity. The consequences of Src-induced tyrosine phosphorylation of EC-MLCK are not clear. However, selective knockout of ECMLCK in mice has emphasized its role in vascular permeability, since knockout mice are resistant to lipopolysaccharide (LPS)- and thrombin-induced hyperpermeability in lung microvessels (Mehta & Malik 2006). The production of NO by eNOS increases the permeability of exchange microvessels, but can enhance endothelial barrier function in arteries. The inhibition of eNOS reduces agonist-induced hyperpermeability for a number of agents (e.g., histamine, bradykinin, thrombin, VEGF), but has no observable effect on the induced increase in [Ca2+]i, suggesting that eNOS is a major target for increased [Ca2+]i (Yuan 2002; Predescu et al. 2005; Yuan 2006). Cytoplasmic Ca2+ activates calcium/calmodulin-dependent eNOS (NOS III) leading to the production of NO (Fig. 40.5). NO binds to the heme group of soluble guanylate cyclase to activate it and increase cyclic GMP (cGMP) levels. cGMP activates cGMP-dependent kinase (PKG) which may act to promote actomyosin contractility. In addition, cGMP can either activate or inhibit phosphosdiesterases (PDE), depending on the isoform. When PDE isoforms are activated by cGMP (e.g., PDE4 in microvascular endothelial cells), levels of cAMP fall. Since cAMP enhances and maintains endothelial barrier function (see below), the action of PDE might lead to increased permeability by destabilizing interendothelial junctions (Yuan 2006). The vasodilatory effects of NO will increase blood flow through an increase in the number of microvessels being perfused, and will increase intraluminal pressure in these microvessels (Bates & Harper 2002). The Starling principle predicts that both of these hemodynamic effects will lead to increased plasma leakage, even if endothelial barrier function is not compromised.

Rho and endothelial contractility In recent years, the role of small GTPases in vascular permeability has been subject to considerable study. Members of the Rho subfamily, Rho, Rac and Cdc42, have been most extensively studied in this context (Wojciak-Stothard & Ridley 2002; Millan & Ridley 2005; Minshall & Malik 2006). These small GTPases act as molecular switches under the control of a number of regulatory proteins (Fig. 40.6). Rho

Biology of Vascular Permeability

Ca2+ eNOS

eNOS

Inactive

Active NO

G-cyclase

G-cyclase

Inactive

Active

cGMP

GMP

PDE4

PKG

PKG

Inactive

Active

cAMP

Junction disassembly

MAP kinases, cytoskeletal targets? Permeability

Fig. 40.5 Nitric oxide signaling. Calcium ions (Ca2+) activate endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) which activates guanylate cyclase (G-cyclase) leading to an increase in cyclic GMP (cGMP). cGMP activates cGMP-dependent protein kinase (PKG), and can also activate some isoforms of phosphodiesterase (PDE), such as PDE4. Active PDE4 will reduce cyclic AMP levels (cAMP), which will lead to disassembly of interendothelial junctions. Activated PKG may increase cell contractility by acting on cytoskeletal targets, though the details of PKG activity are unclear. (See CD-ROM for color version.)

Rho-GDP (inactive)

GDI

Rho-GDP (inactive)

GAP

GEF

Rho-GTP (active)

Binds and activates effectors e.g., ROK; LIM kinase Fig. 40.6 Regulation of Rho GTPases. Rho GTPases are inactive in the GDP-bound state and active when binding GTP. Guanine nucleotide exchange factors (GEFs) promote the exchange of GTP for GDP, promoting the active state. GTPase-activating proteins (GAPs) stimulate Rho proteins to hydrolyze GTP to GDP, promoting the inactive state. Guanine dissociation inhibitors (GDIs) prevent GDP/GTP exchange and hold Rho proteins in the inactive state. (See CD-ROM for color version.)

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proteins are active (i.e., they bind and activate their effector proteins) in the GTP-bound state, and are inactive when GDP-bound. Guanine nucleotide exchange factors (GEFs) enhance the exchange of GDP for GTP, promoting the active state. GTPase-activating proteins (GAPs) activate intrinsic GTPase activity of Rho proteins, stimulating the conversion of bound GTP to GDP, promoting the inactive state. In addition, guanine nucleotide dissociation inhibitors (GDIs) bind to Rho proteins and prevent their conversion from the GDPto GTP-binding form, thereby maintaining the inactive state. GEFs, GAPs, and GDIs control many other classes of small GTPases, including the Ras-related Rap1, whose role in the control of interendothelial junction assembly is discussed later. Rho was originally described as a factor that increases actomyosin stress fiber formation in stimulated fibroblasts, and activation of Rho by a variety of agents is associated with endothelial contractility and increased permeability (Braga et al. 1999; Wojciak-Stothard & Ridley 2002). Inhibition of Rho reduces agonist-induced hyperpermeability (WojciakStothard & Ridley 2002). Events both upstream and downstream of Rho activation have been elucidated. Receptors of permeability-increasing agents can activate Rho directly by activating Rho GEFs, or via the activation of other signaling molecules such as PKC and phosphatidylinositol 3-kinase (PI3K) (Rossman et al. 2005). A major effector of Rho is Rho kinase (ROK). Rho-activated ROK promotes cell contractility by increasing the phophorylation of MLC in two ways (Fig. 40.4): ROK phosphorylates MLC directly, but also promotes MLC phosphorylation by phosphorylating myosin light chain phosphatase (MLCP), reducing its ability to dephosphorylate MLC (Bogatcheva et al. 2003a; Breslin et al. 2006; Mehta & Malik 2006; Shen et al. 2006; Sun et al. 2006). Ischemia and reperfusion, which is known to increase permeability, causes a ROK-dependent constriction of rat cardiac capillary endothelium in situ (Glyn et al. 2003).

Loss of intercellular adhesion and vessel permeability In addition to stimulating cell contractility, agents inducing hyperpermeability of intact vessels and cultured endothelial cell monolayers also affect the organization of interendothelial junctions. A reduction in interendothelial adhesion usually leads to an increase in permeability. For example, blocking intercellular cadherin-mediated adhesion (e.g., using adhesionblocking VE-cadherin antibodies) increases vascular permeability in culture and in vivo (Corada et al. 1999), although interendothelial junctions may not be grossly altered, and the distribution of other junction components (e.g., PECAM-1) may be unaffected. It is well established, in both endothelial and epithelial cells, that interfering with cadherin/catenin/Factin associations leads to the redistribution (both dispersion and internalization) and loss of intercellular junction components of both AJs and TJs, and a consequent loss of barrier

864

function (Yuan 2002; Bazzoni & Dejana 2004). A number of mechanisms may contribute to this. Histamine, VEGF, reactive oxygen species (ROS), and other permeability-inducing agents lead to the phosphorylation of VE-cadherin and its cytoplasmic partners, β-catenin, plakoglobin and p120, with a similar time-course to the development of increased permeability (Rabiet et al. 1996; Esser et al. 1998; Andriopoulou et al. 1999; Yuan 2002; Lilien & Balsamo 2005; Alema & Salvatore 2006; Ali et al. 2006). In general, tyrosine phosphorylation of cadherin and/or the catenins leads to the loss of binding between them, and loss of association with the actin cytoskeleton. Directly disrupting the actin cytoskeleton also leads to loss of interendothelial adhesion and increased permeability. Kinases present at AJs (e.g., Src) promote the phosphorylation of junction components. The nonreceptor tyrosine kinase, Src, associates with VEGFR2 and VE-cadherin at AJs, and with cell–matrix focal adhesions. Src activity is upregulated by permeabilityinducing agents, and inhibition of Src inhibits agonist-induced permeability. By phosphorylating β-catenin and VE-cadherin, Src destabilizes interendothelial adhesions. In addition, Src may phosphorylate MLCK to act on the contractile apparatus. Src may be directly activated by associating with agonistactivated receptors (e.g., VEGFR2), or by second messengermediated mechanisms such as Ca2+ and DAG-activated PKC. A recent study provides evidence that agents which promote an increase in the phosphorylation of AJ components (phosphatase inhibitors or VEGF) can cause a rapid transient increase in Rac function in cultured HUVECs, correlating with transiently enhanced barrier function (Seebach et al. 2005). The authors suggest that the pattern of phosphorylation may be important in determining the effect on permeability. Basal levels of junction-molecule phosphorylation are normally repressed by phosphatases present at AJs, e.g., PTP-μ and the VE-cadherin-specific phosphatase VE-PTP (Nawroth et al. 2002; Lilien & Balsamo 2005). Inhibition of phosphatases increases phosphorylation of junction components and increases vascular permeability. AJ phosphorylation may also be potentiated by a stimulus-mediated loss or inhibition of phosphatases from AJs (Nawroth et al. 2002; Bazzoni & Dejana 2004). TJ integrity is closely linked with that of AJs. Disruption and disassembly of AJs typically leads to loss of TJ function (Gonzalez-Mariscal et al. 2003). However, agents inducing hyperpermeability of endothelial barriers may also act directly on TJs. The phosphorylation of TJ components is associated with junction disassembly and loss of barrier function. Phosphorylation of occludin, claudins, and TJ cytoplasmic plaque proteins, such as ZO-1, have been shown to be associated with altered TJ organization and increased permeability of endothelial monolayers and vessels (Borbiev et al. 2001; Hirase et al. 2001; Smales et al. 2003; D’Souza et al. 2005; Harhaj et al. 2006). The signaling mechanisms involved are not as well characterized as for AJs, but some recent studies

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have suggested that PKC, PKA, and casein kinase 2 (CK2) are involved in the phosphorylation of TJ components (Yuan 2002; Smales et al. 2003).

Mitogen-activated protein kinases in endothelial hyperpermeability A number of studies have found that inhibition of mitogenactivated protein (MAP) kinases can reduce hyperpermeability, and that Raf/MEK/Erk, JNK and p38 MAP kinase signaling cascades are involved in agonist-induced hyperpermeability, but the details of these mechanisms are only beginning to be understood (Bogatcheva et al. 2003b). In particular, VEGFstimulated hyperpermeability is sensitive to inhibition of the Raf/MEK/Erk pathway downstream of NO-activated PKG, and to inhibition of p38 MAP kinase (Pocock & Bates 2001; Bogatcheva et al. 2003b; Issbrucker et al. 2003; Usatyuk & Natarajan 2004; Fischer et al. 2005; Wu et al. 2005). It is still unclear whether MAP kinase activity is a general requirement for agonist-induced permeability. p38 MAP kinase inhibitors failed to inhibit bradykinin-induced leakage in mouse dermis (Adam Al-Kashi, Tim Williams & Peter Clark, unpublished observations). Further studies of the range of stimuli sensitive to MAP kinase inhibition, and the identification of the cellular targets of these kinases, will provide significant insights into the mechanisms controlling endothelial barrier function. Some recent studies have shown that p21-activated kinase (PAK) is a key signaling intermediate that is activated by permeability-inducing agents and promotes MLCK phosphorylation via Erk-MAP kinase activation (Orr et al. 2007; Stockton et al. 2007).

Agents enhancing endothelial barrier function It has been known for some time that agonists that elevate the level of cAMP in endothelial cells can reduce the basal permeability of endothelial cells in culture and in vivo, and can oppose the hyperpermeability response induced by a number of agents (Michel & Curry 1999; Mehta & Malik 2006). Adenosine and β-adrenergic agonists increase intracellular levels of cAMP, via their G protein-coupled receptors (GPRC), by activation of membrane-associated adenylate cyclase (Fukuhara et al. 2005; Jacobson et al. 2006). Because of potential therapeutic benefits, a greater understanding of the signaling events leading to cAMP-induced endothelial barrier enhancement is of considerable interest. It has been known for some time that cAMP-activated PKA is involved in reducing the phosphorylation of MLC, thereby suppressing the contractility of endothelial cells (Patterson et al. 2000). Recent studies in bovine arterial endothelial cells have now provided new insights into cAMP activity. Activated PKA phosphorylates Rho, reducing its activity, and that of its downstream effectors, ROK and LIM kinase (Goeckeler & Wysolmerski 2005). As discussed earlier, active ROK and MLCK promote MLC phosphorylation to increase actomyosinmediated contractility (see Fig. 40.4), and therefore inhibition

Biology of Vascular Permeability

of ROK will result in reduced contractility. However, direct pharmacologic inhibition of MLCK, while blocking MLC phosphorylation, only moderately inhibited thrombin-induced contractility in this model. The Rho effector, LIM kinase, phosphorylates and deactivates the actin-severing protein cofilin. MLC phosphatase, which is deactivated by ROK, also dephosphorylates cofilin. Inhibition of Rho can therefore promote the dephosphorlylation (activation) of cofilin in two ways: (i) lack of activation of ROK will promote the activation of MLC phosphatase, increasing cofilin dephosphorylation; and (ii) lack of activation of LIM kinase will reduce cofilin phosphorylation. Cofilin-mediated actin severing will reduce the amount of F-actin, and may be responsible for the observed loss of stress fibers and cell contractility in some models of endothelial barrier enhancement. Details of how cAMP can enhance interendothelial junction stability, independently of any effects on the Rho/MLC/ contractility pathway have recently been shown. In general, activation of the small GTPase Rac is associated with interendothelial junction assembly and a reduction in vascular permeability (Lampugnani et al. 2002; Wojciak-Stothard & Ridley 2002; Goeckeler & Wysolmerski 2005; WojciakStothard et al. 2005; Waschke et al. 2006), and cAMP prevents agonist-induced deactivation of Rac (Waschke et al. 2004a). Recent studies have provided details linking cAMP to Rac activation (Fig. 40.7). Rap1 is a closely-related homolog of Ras which has recently been placed at the center of the control of interendothelial and interepithelial junction assembly (Bos 2005; Wittchen et al. 2005). In endothelial cells, the Rap1 GEF, Epac1, is directly activated by cAMP, leading to Rap1 activation (Fukuhara et al. 2005). Two important effectors of Rap1 are Vav2 and Tiam, both of which are GEFs cAMP

EPAC

EPAC (Rap1 GEF)

Inactive

Active

Rap1-GDP

Inactive

Rap1-GTP

Active

Tiam (Rac GEF)

Tiam

Active

Inactive

Rac-GTP

Rac-GDP

Active

Inactive

Rac effectors e.g., IQGAP PAK?

Junction assembly

Fig. 40.7 Mechanism of cyclic AMP endothelial barrier enhancement (see Fig. 40.6 for the mechanisms regulating small GTPases). Cyclic AMP (cAMP) activates the Rap1 GEF, Epac, which activates Rap1. Some effectors of Rap1 are Rac GEFs (e.g., Tiam) and will activate Rac, which in turn promotes interendothelial junction assembly and reduced permeability. (See CD-ROM for color version.)

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for Rac, and therefore cAMP-mediated Rap1 activation will lead to Rac activation. Recently, two members of the JAM family of adhesion molecules, and their influence on Rap1 activity, have been shown to have opposite effects on both endothelial and epithelial junction integrity. Blocking or knockdown of JAM-A (JAM-1) results in increased permeability, while the same treatment of JAM-C (JAM-3) leads to junction stabilization, decrease in basal permeability, and prevention of histamine- or VEGF-induced hyperpermeability (Mandell et al. 2005, 2006; Orlova et al. 2006). The different effects of these two JAMs correlates with their regulation of Rap1 activity: JAM-A expression is associated with Rap1 activation and JAM-C with the downregulation of Rap1 activity. The role of Rac in the formation and maintenance of interendothelial junctions is well established, while the role of the related Rho GTPase, Cdc42, is less well defined (Wojciak-Stothard et al. 2001), although some reports do implicate Cdc42 in barrier maintenance and recovery after stimulation (Kouklis et al. 2004; Broman et al. 2006; Waschke et al. 2006). Inhibition of Rac inhibits junction-assembly in cultured endothelia, and increases basal permeability and prolongs agonist-induced hyperpermeability in intact vessels (Braga et al. 1999; Lampugnani et al. 2002; Wojciak-Stothard & Ridley 2002; Wojciak-Stothard et al. 2005). Consistent with this, activation of Rac enhances basal endothelial barrier function and opposes agonist-induced hyperpermeability in cultured cells, but the effect on basal permeability is less apparent in intact vessels, where basal barrier function may already be maximal (Waschke et al. 2006). Rac promotes the formation of peripheral or cortical F-actin in many cell types, and is believed to induce and strengthen interendothelial junctions by enhancing the association of VE-cadherin with the actin cytoskeleton (Liu et al. 2001; Lampugnani et al. 2002; Wojciak-Stothard & Ridley 2002; Waschke et al. 2004a,b, 2005; Jacobson et al. 2006). However, some studies implicate the Rac effector, PAK, in increased endothelial permeability by acting on both cell contractility and the disruption of AJs (Stockton et al. 2004; Fryer et al. 2006; Gavard and Gutkind 2006). PAK activity, elicited by integrin–matrix adhesion, has also recently been shown to promote the hyperpermeability of atherogenic regions in a mouse model (Orr et al. 2007). These contradictory findings might be explained by the suggestion that the effect of Rac activation can be determined by GEF-specific subcellular localization and activation of Rac, i.e., different signals may activate different Rho GEFs that can act to promote either the assembly or breakdown of junctions. While activated PAKs may lead to increased permeability, another Rac effector, IQGAP, sequesters β-catenin and will release this bound β-catenin upon Rac activation and binding, to promote junction assembly and barrier function (Briggs & Sacks 2003; Yamaoka-Tojo et al. 2006). The importance of intracellular localization on the effects of a given signal has been highlighted in a recent report showing

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that cAMP produced by a plasma membrane-targeted adenylate cyclase promoted enhanced endothelial barrier function, while cytoplasmic adenylate cyclase activity, as can be observed in some bacterial infections, led to barrier disruption (Sayner et al. 2006). Rac activation is also known to lead to the inhibition of Rho activity, via Rac stimulation of ROS production which results in the oxidation and deactivation of low-molecular-weight protein tyrosine phosphatase (LMWPTP). This reduction in phosphatase activity results in enhanced Src-mediated phosphorylation and activation of p190Rho-GAP, leading to stimulation of Rho’s intrinsic GTPase activity and the formation of the inactive Rho-GDP (Nimnual et al. 2003; Wildenberg et al. 2006). Clearly, Rac’s ability to enhance interendothelial adhesion, while inhibiting the Rho-mediated contractile phenotype, will efficiently increase barrier function. However, it must be emphasized that the source of Rac stimulation, and therefore the subcellular localization of activated Rac, can substantially affect the outcome (Wildenberg et al. 2006). Endogenous agents that enhance endothelial barrier function are thought to be important in the maintenance of basal endothelial barrier function in vivo (McVerry & Garcia 2005; Brinkmann & Baumruker 2006). Sphingosine 1-phosphate (S1P) is a vasoactive phospholipid produced primarily by activated platelets. In endothelial cells, S1P activates Rac in a pertussis toxin-sensitive manner via members of the Edg family of receptors, Edg-1 and Edg-3. This activation of Rac promotes the translocation of the actin-binding proteins, cortactin and VASP, together with MLCK, to the cortical cytoskeleton, and this rearrangement is required for the robust barrier-enhancing response to S1P (Garcia et al. 2001). However, it should be noted that while S1P promoted barrier function in the vessels, it has the opposite effect on pulmonary alveolar epithelia, inducing a breakdown of barrier function and fluid loss (Brinkmann & Baumruker 2006). An important angiogenic factor, angiopoietin-1, is also an endogenous endothelial barrier stabilizer that can oppose agonist-induced vessel hyperpermeability, e.g., by preventing bradykinin-induced gap formation in rat tracheal vessels in vivo (Thurston et al. 2000b). Angiopoietin-1 can be produced by pericytes and vascular smooth muscle cells and, acting though its receptor Tie2, it stimulates a number of signaling pathways involved in endothelial proliferation, survival, and motility (Gamble et al. 2000; Thurston et al. 2000b; Brindle et al. 2006; Eklund & Olsen 2006). The signals involved in barrier enhancement are not clear, although PI3K activation of Rac is likely to be involved (Eklund & Olsen 2006).

Differences between cultured endothelia and intact microvessels Some studies have questioned the precise roles of endothelial cell contractility and gap formation in hyperpermeability. In HUVECs, histamine-induced gap formation was observed

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in recently confluent cells, but not in cells that had been confluent for longer periods, though in both culture conditions a similar increase in monolayer permeability was observed (Andriopoulou et al. 1999). Collaborative studies from the laboratories of Curry and Drenckhahn have shown that extrapolation of findings in endothelial cell culture models should be done with care. They showed that inhibition of Rho or MLCK reduces agonist-induced hyperpermeability in culture but may have no effect in intact mesenteric vessels, although basal permeability may be reduced (Waschke et al. 2004a, 2006; Curry 2005). However, neutrophil-induced permeability in isolated cardiac venules is sensitive to MLCK inhibition (Yuan et al. 2002). Most cultured endothelia exhibit a robust contractile and permeability response to thrombin; however, thrombin fails to induce hyperpermeability in intact vessels unless they are prestimulated with a proinflammatory agent such as LPS (Curry et al. 2003). Activation of Rac enhances basal barrier function in cultured endothelial monolayers, but not in resting intact vessels, suggesting that Rac-mediated junction formation/stabilization is maximal in intact vessels (Waschke et al. 2004a,b, 2005, 2006). Interestingly, elevated cAMP could enhance basal barrier function in intact vessels, indicating additional, Rac-independent activities (Waschke et al. 2004c). Together, these findings, and those discussed earlier, suggest that cultured endothelial monolayers may represent a partially activated state compared with intact vessels. They also indicate that in resting vessels there is a degree of contractility that is opposed by Rac-mediated maintenance of intercellular adhesion, and this tension may contribute to the rapid responsiveness of vessels to permeability-inducing stimuli.

Fig. 40.8 Possible mechanisms of neutrophilmediated edema. Circulating neutrophils (PMN), if activated or if encountering activated endothelial cells, will form firm adhesions with endothelial cells. This adhesion is by b2 integrins on neutrophils binding to intercellular adhesion molecule (ICAM)-1 on endothelial cells. These adhesion molecules cluster and induce downstream signaling in both the neutrophil and the endothelial cell. Therefore, neutrophil/endothelial adhesion can stimulate signals (i) directly by clustering endothelial ICAM-1, or (ii) indirectly by clustered b2 integrin-stimulated neutrophil secretion, a secreted product or products, then signaling after binding to the endothelium. Signals downstream of direct or indirect stimulation may act on the actin cytoskeleton, stimulating cell contractility, and/or act directly on interendothelial junctions to promote junction disassembly. (See CD-ROM for color version.)

Biology of Vascular Permeability

Leukocyte-mediated endothelial hyperpermeability It is well established that early vascular permeability responses to allergen are mediated by secretion of vasoactive agents by tissue-resident inflammatory cells, and that the delayed late-phase reactions are associated with the recruitment of circulating leukocytes (see relevant chapters in this book). In animal models, neutrophils are the predominant cell type whose peak of infiltration coincides with late-phase edema formation, while other cell types, such as eosinophils, have peak infiltration considerably later. Indeed, depletion studies have shown that late-phase responses are neutrophildependent (discussed in Baluk et al. 1998). The situation in humans is less clear, although neutrophils are strongly implicated in severe asthma and in many types of skin allergy (Tillie-Leblond et al. 2005; Beeh & Beier 2006). Neutrophil-mediated edema is clearly important in both allergic and nonallergic inflammation. This phenomenon was first characterized in rabbit skin, where plasma leakage, induced by intradermal injection of neutrophil chemoattractants, was abolished in neutrophil-depleted animals (Wedmore & Williams 1981; Williams et al. 1984). Since this study, considerable progress has been made in understanding the mechanisms involved, but key details are still outstanding. Studies in cultured cells and in vivo have found that neutrophil αMβ2 integrin (MAC1, CD11b/CD18) engagement with endothelial ICAM-1 (Fig. 40.8) causes an increase in endothelial [Ca2+]i, production of ROS, activation of PKC, src, Rho, MLCK, and p38 MAP kinase, and induces the formation of stress fibers and disassembly of interendothelial junctions (Gautam et al. 1998, 2000; Edens & Parkos 2003; Millan &

PMN

2 Vessel lumen

1

b2 integrin

F-actin Intercellular junction adhesion molecule

ICAM-1

Unknown receptor(s) for PMN-secreted product(s)

Extravascular space

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Ridley 2005). Interendothelial junctions become disorganized, with a loss of AJ and TJ components and the formation of intercellular gaps (Del Maschio et al. 1996; Millan & Ridley 2005). In isolated cardiac venules, activated neutrophils induced the activation of MLCK and focal adhesion kinase (FAK), both of which were shown to be required for increased permeability (Yuan et al. 2002; Guo et al. 2005; Breslin et al. 2006). Therefore, neutrophils are capable of stimulating the major signaling pathways involved in inducing endothelial hyperpermeability. When neutrophils form αMβ2 integrin-mediated firm adhesions with endothelial ICAM-1, two routes of signaling to the endothelium may operate (Fig. 40.8). The clustering of endothelial ICAM-1 is known to be capable of eliciting signals in endothelium (Etienne et al. 1998; Greenwood et al. 2002; Millan & Ridley 2005), though this appears not to be sufficient to induce hyperpermeability, since T lymphocytes, which also adhere to endothelial ICAM-1, did not alter AJ organization in cultured HUVECs nor did antibody crosslinking of ICAM-1 (Del Maschio et al. 1996). The other event that occurs with neutrophil/endothelial firm adhesion is that clustering of β2 integrins on the neutrophil stimulates them to secrete. Cell-free supernatant from anti-β2 antibody crosslinked neutrophils had been shown to stimulate a similar hyperpermeability response (decreased electrical resistance) in bovine aortic endothelial cell monolayers, and could induce leakage of a macromolecular tracer in hamster cheekpouch vessels (Gautam et al. 2000). Polycations (e.g., poly-Llysine) induce vascular hyperpermeability (Needham et al. 1988; Jones et al. 2001, 2002), while dextran sulfate (a polyanion) could prevent LTB4-induced hyperpermeability but not neutrophil extravasation (Rosengren et al. 1989). These findings suggested that a positively charged agent secreted by neutrophils was responsible for inducing the permeability increase. A subsequent study found that azurocidin (heparin-binding protein, CAP37), a 28-kDa protein with an asymmetric cluster of positively charged amino acid residues, is the neutrophil-secreted agent responsible (Gautam et al. 2001). Azurocidin is present in cell-free supernatant from anti-β2 antibody cross-linked neutrophils. Depleting the supernatant of azurocidin (but not elastase or cathepsin G) or neutralizing with dextran sulfate ablated its hyperpermeability-inducing activity. It is not known to which receptor(s) on endothelial cells azurocidin binds to activate the hyperpermeability response, though it is known to bind to heparansulfated proteins. This study also examined the effect of the protease inhibitor aprotinin, finding that it binds to azurocidin and neutralizes azurocidin’s activity, possibly by masking the positive charge of the molecule. Aprotinin is used clinically after thoracic surgery to minimize bleeding by inhibiting fibrinolytic enzymes, although additional antiinflammatory properties, possibly due to its interaction with azurocidin, are being recognized (Clark 2006; Sodha et al. 2006). Because cell-mediated vascular hyperpermeability may be of particu-

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lar importance in acute severe allergy, the details of how neutrophils stimulate endothelial cells, particularly determining the mechanism of azurocidin’s activity, could prove fruitful for pointing to new therapeutic intervention strategies. However, there are reports that the sites of vessel leakage and neutrophil/endothelial adhesion do not necessarily coincide. One experimental study of rat airway responses to allergen showed that the majority of leakage sites were upstream of leukocyte adhesion (Baluk et al. 1998). A recent report suggests that fMLP-induced permeability in rat mesenteric venules does not require neutrophil adhesion to endothelia, but is dependent on ROS production by the neutrophils (Zhu et al. 2005), although a previous study in cultured HUVECs showed that inhibition or scavenging of ROS did not affect neutrophil-mediated interendothelial junction disassembly or hyperpermeability (Del Maschio et al. 1996). Clearly, there is still uncertainty as to the mechanisms involved in neutrophil-mediated vascular hyperpermeability. As mentioned earlier, systemic capillary leak syndrome (Clarkson disease) is a relatively rare life-threatening problem. Clarkson disease is episodic and occurs in the absence of sepsis or other obvious causes (Teelucksingh et al. 1990). Reversible increased permeability of the normally unresponsive capillaries is also known to occur in bacterial and viral septic shock conditions (Fishel et al. 2003; Rechner et al. 2003). In these conditions, there is massive reduction of blood volume, blood cell concentration, and loss of plasma proteins, which can ultimately lead to multiple organ failure. The details of how capillaries are rendered leaky are not fully understood, but systemic neutrophil activation and recruitment has been implicated in at least some instances. In this context, it is worth noting that there has been a recent increase in the incidence of capillary leak syndrome with the clinical use of granulocyte colony-stimulating factor (G-CSF), and other cytokines, after bone marrow stem cell transplantation, where G-CSF will mobilize and prime neutrophils (Deeren et al. 2005). Capillary leak syndrome might therefore be an extreme example of neutrophil-mediated edema.

Conclusions Although the mechanisms leading to increased vascular permeability may vary with different stimuli in different vessels and vascular beds, a number of consistent findings are emerging from the body of research in this area. Reorganization of the actin cytoskeleton in relation to changes in the integrity of interendothelial junctions appears to be the core mechanism, leading to the breakdown of endothelial barrier function. Our increased understanding of the mechanisms involved in maintaining or disrupting vascular permeability is allowing the development of new therapeutic approaches to inflammatory disorders. Current drug therapies may act to reduce stimulated vascular permeability, although this is

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often not their primary, or intended, action. The prevention of vascular hyperpermeability can be approached at a variety of levels, from receptor antagonists (e.g., established drugs such as antihistamines), to inhibition of second messengers (e.g., prevention of [Ca2+]i using Ca2+ blockers), to promoting barrier function by β-adrenergic elevation of cAMP. The endogenous junction-stabilizing agents are providing physiologic and biochemical clues as to how, in the future, clinicians could intervene to control edema. One potentially important area will be to develop the means to activate the small Rho GTPase, Rac, in a specific subcellular compartment. Elevating cAMP by PDE inhibition could prove to be too indiscriminate to be clinically useful, but an indication of how this might be avoided is seen in work showing the cAMP analog, 8CPT-2′-O-Me-cAMP (also known as 007), is a specific activator of Epac, and not PKA, that can oppose agonistinduced hyperpermeability in vivo (Fukuhara et al. 2005; Kooistra et al. 2005). Intervention at the level of circulating leukocyte-mediated effects of allergen exposure will clearly be an important area of future research. Another important consequence of endothelial barrier enhancement is that leukocyte extravasation is also inhibited, adding a further antiinflammatory dimension to the manipulation of interendothelial junctions (Gamble et al. 2000; van Nieuw Amerongen & van Hinsbergh 2002; Millan & Ridley 2005; Wittchen et al. 2005). Preventing leukocyte recruitment is an approach that is currently receiving a great deal of attention in inflammation research, and further developments in this area, and in how leukocytes induce edema, will significantly improve our ability to control vascular permeability.

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Airway Smooth Muscle Stuart J. Hirst

Summary

Introduction

Below the level of the main bronchus, airway smooth muscle encircles the entire airway in a roughly circular orientation and is the primary effector cell regulating bronchomotor tone. In the healthy adult airway its physiologic function has been questioned. In contrast, in lung disease its role seems unequivocal, particularly in allergic asthma. Contemporary concepts center on its remarkable mechanical and functional plasticity. The traditional view that contraction was the sole function of airway smooth muscle has given way to a new paradigm in which both its contractile and noncontractile properties contribute to the development and perpetuation of airway hyperresponsiveness. Ideas of contraction have advanced from classical sliding filament theory to models that reflect the oscillatory nature of dynamic loads imposed on the smooth muscle in the airways. Cyclical interactions of myosin crossbridges with actin filaments underpin the initial shortening, while dynamic reorganization and polymerization of these filaments ensure maintenance of contraction during nonstatic conditions. Aside from contraction, additional pathologic functions of airway smooth muscle include its newly described allergic and immunomodulatory properties in which the muscle contributes to known changes in the composition of the extracellular matrix in asthma, as well as to mucosal inflammatory processes by expression of cellular adhesion molecules and release of multiple chemokines and cytokines. Furthermore, airway smooth muscle content is pathologically increased in allergic asthma. Mechanisms underlying its accumulation remain elusive but likely involve multiple pathways, including activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinases, and the generation of reactive oxygen species. Thus, an integrated paradigm for plasticity of function has emerged that encompasses both acute and long-term regulation of contraction, as well as immunomodulation and proliferation as smooth muscle effector mechanisms in lung disease. Targeting airway smooth muscle by thermoablation offers a potential new treatment that limits both its contractile and synthetic functions in asthma.

Airway smooth muscle is the major contractile tissue of the airways in the lung. Its localization and arrangement within the airway wall appears optimized to effect changes in airway caliber through active force generation and cell shortening. However, like smooth muscle present in other hollow organs, airway smooth muscle retains a capacity for other functions including growth, migration, and synthesis of a vast array of regulatory molecules, which are manifest during disease. Coordination of these varied functions allows airway caliber to be regulated both transiently via reversible muscle shortening and in the longer term by alterations in airway smooth muscle content and synthetic activity, perhaps as part of the normal contractile and repair programming of airway smooth muscle. Available data suggest that in disorders of the lung involving airway narrowing, exemplified by asthma, this programming is disrupted and the function of airway smooth muscle becomes aberrant, involving enhanced synthetic activity and impaired responses to antiasthma therapy.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Normal anatomy and location Airway smooth muscle was first described in 1804 by the German anatomist Franz Reisseisen who, using a hand-held magnifier, reported its arrangement in bundles in the bronchial tree to the level of the membranous bronchioles, from where it was inferred to continue to the lung periphery (see Otis 1983). Later works established that airway smooth muscle extends from the cartilaginous airways of the trachea and bronchi to the terminal bronchioles where the smooth muscle fibers of the smallest bronchi are referred to as Reisseisen’s muscles, although the functional properties of these fibers were not considered until almost a century later by Einthoven (1892) and Dixon and Brodie (1903). The arrangement of airway smooth muscle varies according to its location in the airway tree. In the posterior aspect of the adult trachea it forms the trachealis, a unified layer of thick unbranched bundles from the larynx to the carina in which the parallel smooth muscle fibers are oriented perpendicular to the long axis of the trachea to span the gap between the ends of incomplete cartilaginous rings (Fig. 41.1).

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(a)

n

n

(b) Actin filament

Nucleus

Myosin filament Plasmalemma

Dense plaque

Dense body

(c) Fig. 41.1 Morphologic and ultrastructural features of airway smooth muscle showing (a) peribronchial view of a-actin-labeled bundles in developing airways and (b) general orientation of contractile filaments and dense bodies (arrowheads) along the long axes of individual cells. Intercellular coupling via dense plaques appears within the oval. Inset shows thick myosin filaments running toward and past the nucleus (n). Bar represents 100 mm in (a) and 1 mm in (b). (c) Schematic of (b) showing overall contractile filament architecture in an airway smooth muscle bundle. ((a) From Sparrow & Lamb 2003, with permission; (b) and (c) from Kuo & Seow 2004, with permission.)

The trachealis comprises more than 75% muscle, with connective tissue, fibroblasts, and neuronal structures comprising the major portion of the balance. Airway smooth muscle cells in the large segmental or lobar bronchi are similar to those in trachealis. Muscle bundles encase the bronchi in a roughly circular orientation but appear devoid of a specific anatomic arrangement where they branch and interdigitate with plates of cartilage and other

Airway Smooth Muscle

connective tissue structures within the airway wall. More distally, in the peripheral noncartilaginous airways the smooth muscle bundles surround the airway in a helix–antihelix pattern with extensive criss-crossing of fibers between bundles, giving an overall geodesic arrangement (Mitchell & Halayko 1997; Sparrow & Lamb 2003). The helical pitch of these bundles varies with lung inflation and airway level but is in the range ±13–30° in noncartilaginous airways (Ebina et al. 1990). However, because of the regularity of crossover of fibers from one bundle to another, the magnitude of this obliquity may not result in physiologically important changes in airway length during bronchoconstriction (Sparrow & Lamb 2003). While absolute amounts of airway smooth muscle are less in peripheral airways compared with proximal airways, the proportion of airway wall structure occupied by muscle is higher in the periphery (15–25%) than the proximal airways (2–4%) (Mitchell & Halayko 1997; Sparrow & Lamb 2003). Classically, smooth muscle is categorized into single unit, multiunit, or intermediate types based on the extent of innervation to individual muscle cells and on its capacity to generate spontaneous contractile responses and myogenic tone via propagation of membrane depolarizing pulses. Resting airway smooth muscle from the large central airways including the trachealis display multiunit or intermediate properties, having few gap junctions and being devoid of spontaneous action potential generation or myogenic tone, although individual smooth muscle cells are relatively sparsely innervated (Mitchell & Halayko 1997). However, under some experimental situations the trachealis exhibits spontaneous action potentials and myogenic contractile activity, reminiscent of single-unit smooth muscle. Likewise, smooth muscle in the smaller bronchioles appears to be single-unit, typified by spontaneous action potential generation and myogenic tone, and is less innervated and has more gap junctions than smooth muscle in the larger central airways. This is discussed in more detail by Mitchell and Halayko (1997).

Ultrastructural features: contractile and cytoskeletal elements Airway smooth muscle bundles interconnect with numerous collagen-rich elastic fibers of the extracellular matrix to provide mechanical coupling between the muscle and surrounding tissues. Individual airway smooth muscle cells are fusiform (spindle-shaped) and typically are 3–5 μm wide and can exceed 1 mm in length (Kuo & Seow 2004). The detection and identification of many ultrastructural features within these cells is dependent on orientation. Mechanical coupling between cells occurs via dense plaques and gap junctions as well as frequent small invaginations of membrane called caveolae. Each smooth muscle cell may contain more than 100 000 of these invaginated structures, occupying as much as 30% of the plasma membrane (Kuo et al. 2003). Characteristic of smooth muscle, the nucleus is elongated and

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centrally located within the cytoplasm and often appears crenulated at higher power (Fig. 41.1b). Mitochondrial clusters and Golgi apparatus are perinuclear, most often appearing adjacent to each pole of the nucleus. The sarcoplasmic reticulum accounts for 2– 6% of the cytoplasm. In mature smooth muscle it is in intimate contact with pinocytic vesicles and caveolae at the plasma membrane. Recent evidence suggests two types of sarcoplasmic reticulum are found in airway smooth muscle: superficial and deep. In three dimensions it is likened to a hollow pancake with its flat face adjacent to the plasma membrane in the case of superficial sarcoplasmic reticulum or perpendicular for the deep sarcoplasmic reticulum, where the opposite edge extends to the mitochondria (Kuo et al. 2003). The superficial sarcoplasmic reticulum and its proximity to the plasma membrane may govern local calcium gradients or oscillations, whereas the deep sarcoplasmic reticulum may provide a direct conduit for calcium transport within the cell and perhaps ultimately regulation of mitochondrial calcium concentration (Lee et al. 2002). The major feature of any mature smooth muscle cell under the transmission electron microscope is the dense appearance of numerous parallel myofilaments in the cytoplasm running along the longitudinal axis (Fig. 41.1). These comprise mainly smooth muscle myosin heavy-chain thick filaments and smooth muscle α-actin thin filaments that form the backbone of the force-generating machinery, as well as desminand vimentin-containing intermediate filaments (Kuo et al. 2003). Frequent cytoplasmic and membrane-associated dense bodies and dense plaques form anchor points for attachment of thin and intermediate filaments (Fig. 41.1c). Dense plaques are synonymous with focal adhesions, sites of linkage between actin filament polymerization and membrane-spanning β subunits of collagen-ligating integrins. Other actin filaments appear to attach to the nuclear envelope, making it a forcetransmitting structure (Fig. 41.1b). The resultant mesh of thin and intermediate filaments connected through dense plaques also includes long hollow tubular structures at regular intervals called microtubules, composed of tubulin proteins. Together, these structures form much of the cytoskeleton scaffold that give the cell its shape. The free ends of α-actin filaments interdigitate with myosin filaments and the crossbridge interactions formed allow the actin and myosin filaments to slide over one another in opposite directions during cross-bridge cycling, pulling the dense plaques closer and shortening the cell during contraction (Fig. 41.1c). The parallel arrangement of actomyosin filaments via dense plaques is aligned with similar sites in adjacent cells to create a transcellular parallel alignment of myofilaments that may allow airway smooth muscle to shorten as a syncytium (Fig. 41.1c) (Ali et al. 2005). Recent studies indicate the presence of sarcomeric structures, as in striated muscle, which allow the sliding of contractile filaments to be translated into cell shortening. However, distinct from striated muscle it is believed that

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a malleable sarcomeric structure exists that is composed of contractile units assembled in series and in parallel. In this model, the geometric organization of the basic building blocks (contractile units) within the assembly and the dimension of individual contractile units can be altered when the muscle cells adapt to different lengths (Herrera et al. 2005). A similar remodeling is proposed for the actin cytoskeleton during contraction (Gunst & Fredberg 2003; Gunst et al. 2003). Thus, the ultrastructural arrangement and density of thick (myosin) and thin (actin) filaments in airway smooth muscle appears highly dynamic, involving actin and myosin polymerization and elongation of both actin and myosin filaments (Gunst et al. 2003; Herrera et al. 2005; Seow 2005). Such structural alterations may account for the different length–force relationships of the muscle obtained at different adapted cell lengths (Bai et al. 2004). Moreover, the structural malleability necessary for length adaptation also precludes a requirement for a permanent filament lattice and explains the lack of aligned filament arrays seen in striated muscle and probably explains why smooth muscle is “smooth” (Herrera et al. 2005).

Ontogeny and myogenesis Although substantial morphologic similarities exist between smooth muscle of the airways and that residing in other hollow organs, its pattern of development in the airways differs from other smooth muscle tissues (Sparrow & Lamb 2003). During embryogenesis airway smooth muscle appears ahead of vascular smooth muscle and originates from mesenchymal cells during branching morphogenesis, a period of prolific branching during the pseudoglandular stage of lung development that establishes the bronchial tree. Initiation of airway smooth muscle differentiation and its arrangement into bundles begins by induction of a thin ring of mesenchyme cells around the base of the epithelial bud immediately under the epithelium at sites of laminin basement membrane assembly (Tollet et al. 2001). The basement membrane plays a critical role in airway smooth muscle development by facilitating mesenchymal cell spreading/elongation and maturation (Tran et al. 2006). When laminin polymerization in the basement membrane is inhibited, spreading of mesenchymal cells is prevented and these cells are negative for differentiation marker proteins such as smooth muscle α-actin, sm-22α, and calponin. Serum response factor is the transcription factor that induces expression of these smooth muscle-specific proteins (Yang et al. 2000). It is assumed, as with myogenesis elsewhere, that maturation involves transition of mesenchyme cells to smooth muscle myoblasts and on to immature smooth muscle cells followed by maturation. In addition to laminin, soluble factors including transforming growth factor (TGF)-β released by the epithelium are also important in progressing maturation (Yang et al. 1998). The maturation ring steadily widens as new bundles appear distally such that maturation progresses from the terminal tubules to the trachea

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as the developing epithelial bud extends. Initially, bundles encircle the developing trachea but this is progressively restricted to the dorsal wall to form the trachealis as the incomplete cartilage rings develop in the pseudoglandular stage. The first detected maturation and differentiation marker proteins are smooth muscle α-actin and calponin (Yang et al. 1998). Other markers follow, also in the early pseudoglandular stage, including smooth muscle myosin heavy chain, sm-22α, and desmin. The appearance of mature airway smooth muscle cells in developing airway walls is temporally linked to the generation of regular waves of spontaneous action potentials and rhythmic contractions and relaxations, similar to that in the developing gut. These propel liquid toward the periphery and cause marked pulsatile distensions of the epithelial buds and elongation of the mesenchyme cells therein. The signals driving airway smooth muscle myogenesis and differentiation are poorly defined but recent observations suggest that contact with polymerized laminin in basement membranes and rhythmic mechanical stretch during branching morphogenesis are required via induction of serum response factor and its cofactor, myocardin, as well as other transcription factors (Yang et al. 2000; Sparrow & Lamb 2003; Tran et al. 2006). Mechanical forces exerted on airway smooth muscle by tethering of the lung parenchyma may also modulate airway smooth muscle differentiation via induction of transcription of smooth muscle contractile protein genes (Yang et al. 2000). Postnatally, airway smooth muscle undergoes slow graded shortenings where action potentials are no longer required to support rhythmic contractions.

Is airway smooth muscle important in the healthy lung? Airway smooth muscle is exposed continuously to changing mechanical conditions during normal breathing and is periodically subjected to larger forces of expansion by intermittent deep breaths. Identification of a normal physiologic role of airway smooth muscle continues to be elusive (Otis 1983). This contrasts with smooth muscles in other hollow organs such as the vasculature or gut whose primary functional roles are self-evident. Thus, smooth muscle in the airways has been described as a “frustrated” cell or the “appendix of the lung” because its role in normal lung physiology has been questioned (Seow & Fredberg 2001; Mitzner 2004). Proponents of this argument suggest that much of the physiology of ventilation and its regional distribution can be explained by regarding the airways as passive conducting tubes or conduits, with the respiratory skeletal muscles providing the major power stroke for ventilation and the smooth muscle being superfluous in these events. Indeed, relaxation of airway smooth muscle in healthy subjects by inhalation of the β2-adrenoceptor agonist salbutamol rarely results in improved

Airway Smooth Muscle

lung spirometry or ventilation. Speculation on possible functions has centered on its contractile role and includes peristalsis to assist exhalation, optimization of epithelial ciliary function and mucus propulsion, and the regional distribution of airflow during ventilation and optimization of anatomic dead space volume for matching of alveolar ventilation with blood perfusion to reduce the work of breathing (Widdicombe 1963). Other possibilities include stabilization of the airway wall to enhance the effectiveness of cough or ejection of foreign material or to stiffen the airway sufficiently to prevent extreme airway collapse during forced expiration. Recently, it has been suggested that airway smooth muscle serves to match the mechanical hysteresis of small airways and alveolated ducts to the mechanical hysteresis of the alveolar surface film in a way that allows for synchronous and uniform alveolar expansion (Mitzner 2004; Fredberg 2007; Mead 2007). Finally, based on the capacity of airway smooth muscle in fetal but not in postnatal airways to generate spontaneous and rhythmic contractions and relaxations (Solmann & Gilbert 1937), akin to its embryologic counterpart in the gut, it has been proposed that these peristaltic contractions are important in fetal lung to generate fluid pressure for fetal airway differentiation and branching (see above) (Schittny et al. 2000). Most explanations for the presence of airway smooth muscle in healthy adults appear plausible but have received little experimental validation. It might appear therefore that airway smooth muscle in healthy adult airways is vestigial, having no identifiable function, but as with other vestigial structures such as wisdom teeth or the appendix, when diseased they are the cause of major medical complications. Proponents of its vestigial nature have suggested its removal by pharmacologic targeting with a directed toxin or physicochemical disruption to provide a somewhat drastic if not effective future treatment for lung disease. The latter is under current evaluation in the form of bronchial thermoplasty, where delivery of radiofrequency energy as heat to the airway wall results in localized and apparently selective ablation of airway smooth muscle (see later sections; Mitzner 2006). Perhaps this novel nonpharmacologic intervention will provide the ultimate clue in establishing the importance of airway smooth muscle in maintaining normal lung physiology as well as its role in the diseased lung.

Airway smooth muscle in lung disease There is no known pathologic condition or appreciable physiologic defect that results in loss of airway smooth muscle function. Instead, in stark contrast to its subtle importance in the healthy airway, its amplified function is only too apparent in lung disease. Understanding these processes, in particular pathways that regulate cell differentiation and intracellular signaling, has relied largely on the development of cell culture-based methods. Accumulating evidence from

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these and intact tissue systems from human or animal airways suggests that airway smooth muscle function is remarkably diverse (Hirst et al. 2000). It can respond to external stimulation in a variety of ways: by contraction and relaxation, by increased secretion, and by altered growth. Such studies must be seen in the context of model systems informing the future design and development of improved methodologies in order to test the importance of airway smooth muscle in the cause or progression of lung disease. Inappropriate airway smooth muscle responses are hypothesized to contribute to various lung pathologies, including asthma, chronic obstructive pulmonary disease, chronic lung disease of immaturity (bronchopulmonary dysplasia), and the rare disorder lymphangioleiomyomatosis, which affects young women (Black et al. 2005; Chung 2005; Borger et al. 2006). Limited evidence also supports alterations in airway smooth muscle in sudden infant death syndrome. However, most research has centered on understanding amplified airway smooth muscle responses as a basis for the airway hyperresponsiveness in allergic asthma and to a lesser extent in chronic obstructive pulmonary disease (Chung 2005).

Contractile role and mechanical plasticity in asthma The underlying abnormality causing airway smooth muscle contraction in response to such diverse stimuli as allergens, cold air and occupational triggers in an asthmatic but not in a healthy individual remains unknown. Nevertheless, moderate or excessive airway smooth muscle shortening is the unequivocal basis of asthmatic symptoms and disability, particularly during acute allergen-triggered exacerbations. Attempts to show that airway smooth muscle in asthma is hypercontractile have met with mixed findings, as the few studies available have demonstrated increased contractility, decreased relaxation, decreased contractility, or no clear difference (Goldie et al. 1986; Whicker et al. 1988; Bai 1991; Seow et al. 1998). Theories of airway smooth muscle contraction and its dysfunction in asthma are currently under radical review (Gunst & Fredberg 2003). The observation that a prolonged bronchodilatation to deep inspiration seen in healthy subjects is diminished in asthmatics has provided a major impetus for renewed research (Cockcroft & Davis 2006). Previously, airway smooth muscle shortening leading to airway lumen narrowing in asthma has been considered strictly within the confines of a static equilibrium of forces. New concepts have displaced much of the classical understanding of contraction to account for the continuously changing oscillatory stresses and strains exerted on airway smooth muscle in the fundamentally dynamic, nonequilibrium environment created by tidal loading during spontaneous breathing and intermittent deep breaths (Fredberg 2001). Typically, force generation and cell shortening depends critically on levels of cytosolic calcium and the interaction of smooth muscle α-actin with myosin myofilaments via cross-bridge cycling. A prerequisite for the latter is phospho-

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rylation of 20-kDa myosin light chains by smooth muscle myosin light chain kinase by the binding of free calcium to calmodulin (Fig. 41.2). Airway spasmogens such as acetylcholine, leukotriene (LT)D4, endothelin-1, thrombin, or histamine released from either airway nerves or inflammatory cells activate cognate cell-surface Gα-type G protein-coupled receptors via phospholipase C to cause a rise in cytosolic

Histamine, acetylcholine, leukotrienes, endothelin-1 Ca2+ channels GPCR

Ca2+

Ga SR IP3 RyR Ca2+ Ca2+ Ca2+ CaM Ca2+ Ca2+

RhoGEF

DAG

RhoA

PKC

Ca2+ sensitization

CPI-17

P

Rho-kinase

MLCK MLCK

↑Contraction

PIP2 PLC

MLC20 MLC20P

MLC phosphatase

Contraction↓ Airway smooth muscle cell Fig. 41.2 Major pathways leading to contraction in airway smooth muscle. Activation of cell surface G protein coupled-receptors (GPCR) via phospholipase C (PLC) results in generation of two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), from membrane lipid phosphatidylinositpl 4,5-bisphosphate (PIP2). Release of stored calcium (Ca2+) from the sarcoplasmic reticulum (SR) occurs via IP3- and ryanodine (RyR)-activated channels. Agonist stimulation also activates receptoroperated Ca2+ channels in the plasma membrane and allows cytoslic Ca2+ influx. Binding of Ca2+ to calmodulin (CaM) activates myosin light chain kinase (MCLK) to phosphorylate (P) 20-kDa myosin light chains (MLC) for the initiation of actomyosin cross-bridge cycling and active force generation. Additionally, Ca2+ sensitization, resulting from inhibition of MLC phosphatase, is initiated at the same time. The nature of the activation of RhoA by GPCR agonists is unclear but involves guanine nucleotide exchange factors (RhoGEFs), which activate RhoA by exchanging GDPwith GTP-bound form of RhoA. Activated GTP-bound RhoA increases Rho-kinase activity, leading to inhibition of MLC phosphatase to promote the contractile state. In addition to RhoA/Rho-kinase-mediated inhibition of MLC phosphatase, CPI-17, which is activated by protein kinase C (PKC)mediated phosphorylation, also inhibits MLC phosphatase. Relaxation arising from reduced cross-bridge formation and force generation results from MLC20 dephosphorylation by MLC phosphatase. (See CD-ROM for color version.)

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calcium concentration and propagation of calcium oscillations (reviewed in Janssen & Killian 2006). Calcium influx derives from the extracellular space and from release via second messengers of intracellular calcium stores, principally the sarcoplasmic reticulum. Two major types of calcium-release channels are postulated in airway smooth muscle sarcoplasmic reticulum: one activated by inositol 1,4,5-trisphosphate (IP3) and the other termed the ryanodine receptor (RyR). The initial phase of the biphasic calcium response derives primarily from IP3-dependent calcium release, while more sustained elevations are more dependent on calcium-induced calcium release via RyR channels. The frequency of calcium oscillations occurring via repetitive release from RyR channels increases with agonist concentration, resulting in an overall higher mean calcium concentration (Chiba & Misawa 2004; Janssen & Killian 2006). Airway smooth muscle cells also contain myosin light chain phosphatase, which dephosphorylates myosin and so counters myosin light chain kinase to limit or reverse airway contraction (Fig. 41.2). If myosin light chain phosphatase activity is reduced, net myosin phosphorylation in response to a given level of cytosolic calcium is enhanced or prolonged (i.e., calcium sensitization), resulting in greater contraction. At least two different signaling pathways in airway smooth muscle have been found to mediate this increased calcium sensitivity of the contractile apparatus (Fig. 41.2). The first involves activation of RhoA by G protein-coupled receptor agonists and guanine nucleotide exchange factors (RhoGEFs), which activate RhoA by exchanging the GDP- for GTP-bound form of RhoA. Activated GTP-bound RhoA increases Rho kinase activity, leading to inhibition of myosin light chain phosphatase. The second mechanism involves diacylglycerol (second messenger) and protein kinase C, via CPI-17 phosphorylation, which inhibits myosin light chain phosphatase leading to calcium sensitization. These pathways are shown in Fig. 41.2 and reviewed in detail by Chiba and Misawa (2004). Additionally, several proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and Th2 cytokines (IL-4, IL-5, IL-13) in the asthmatic inflammatory environment may induce airway hyperresponsiveness by acting directly on airway smooth muscle to enhance agonistevoked calcium signals and contractile responses (Amrani & Bronner 1993; Amrani et al. 1996; Tliba et al. 2003) and by uncoupling β2 adrenoceptors, thereby diminishing relaxations evoked by salbutamol and other β-adrenoceptor agonists (Moore et al. 2001). Enhanced responsiveness of airway smooth muscle after cytokine treatment appears dependent on RyR channel activation, and components of the cyclic ADP ribose pathway including transcriptional activation of CD38 are implicated in the amplification and this may be a third mechanism by which calcium sensitization occurs in airway smooth muscle (Deshpande et al. 2005). Perhaps the most significant development and radical departure from the classical sliding filament hypothesis is the

Airway Smooth Muscle

recent emergence of mechanical plastic theory for the understanding of sustained airway smooth muscle contraction under nonequilibrium conditions (Fabry & Fredberg 2003; Gunst et al. 2003; Seow 2005). Its tenet is that while the rate and extent to which airway smooth muscle shortens in response to a spasmogen are linear functions of smooth muscle length, active force generation is independent of length (Bai et al. 2004; Fredberg 2004). Plastic restructuring is envisaged when dynamic length changes occur between sequential contractile events or during a single contraction. On a molecular level several dynamic models have been proposed to explain airway smooth muscle mechanical adaptation during single or multiple stimulations. One of these is referred to as the series-to-parallel filament transition theory and involves the net addition in series of thick myosincontaining filaments to increase the number of parallel contractile elements when optimal force generation is exceeded (Gunst & Fredberg 2003; Fredberg 2004). At shorter lengths, depolymerization of contractile elements occurs. A major attraction of this model is that myosin filament lengthening alone can explain the observed slowing of muscle shortening velocity occurring when approaching the sustained phase of a single contraction without any need to postulate a change in the rate of cross-bridge cycling, previously known as the “latch” state. Other theories of mechanical plasticity operating during sustained length adaptation between contractions involve polymerization and elongation of thin smooth muscle α-actin-containing filaments following the activation and clustering of β1 integrin focal adhesions (Halayko & Solway 2001; Gunst et al. 2003). In a refinement of both these possibilities, there is the perturbed equilibrium model of myosin binding in which the oscillatory strain on airway smooth muscle occurring with each lung inflation during breathing is transmitted directly to the myosin head to cause unending perturbations of optimal actin and myosin binding (Fredberg 2000; Mijailovich et al. 2000; Fredberg 2004). The result is that the contractile machinery is normally kept off-balance and unable to achieve its full force-generating potential. Each of these possibilities may operate coincidentally and is integrated completely by considering airway smooth muscle as a glassy material in which the cytoskeletal and contractile filament deformation and reorganization and gross mechanical properties of muscle are regulated from a state of relative fluidity (“hot”) to the rigidity (“cold”) of a solid (Fig. 41.3). This is now referred to as the “glass hypothesis” (Fabry & Fredberg 2003) because glasses are substances with the structural disorder of a liquid but the stiffness of a solid. In the relaxed inactivated state, airway smooth muscle cell can be thought of as “cold,” but with the onset of contractile stimulation it rapidly becomes “hot,” during which there is rapid rearrangement of the cytoskeleton and rapid cross-bridge cycling. Following this hot initial transient, the cell cools if the contractile stimulation is sustained, becoming gradually cooler until it approaches a steady state that approximates

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Cytoskeletal signaling proteins undergo mechanosensitive phosphorylation Mechanosensitive activation of contractile/cytoskeletal proteins Intracellular Ca2+, MLC phosphorylation increase

Contraction/ length change

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Integrin proteins sense mechanical strain on ECM Contractile stimulus onset

HSP27 Elongation/reorganization of actomyosin filaments

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RhoA

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a “frozen” or rigid state not only mechanically but also biochemically and metabolically when the contracted muscle cell is in “latch” (Fig. 41.3). The underlying signaling pathways responsible for mechanical plasticity or soft glassy behavior are only just being investigated and available information relates to airway smooth muscle from nonasthmatics. Initial findings implicate agonist-induced activation of Rho GTPbinding proteins such as RhoA and its downstream intermediates in polymerization of myosin thick filaments; while RhoA, p38 mitogen-activated protein (MAP) kinase, focal adhesion components, and small heat-shock proteins such as HSP27 appear to be effectors of actin thin fiber polymerization (Halayko & Solway 2001; An et al. 2004) (Fig. 41.4). It remains uncertain whether a definite intrinsic abnormality exists in the contractile machinery of airway smooth muscle from asthmatics to account for the enhanced bronchoconstriction in asthma. A seminal study showed that the shortening ability of unloaded bronchial smooth muscle cells from human subjects with asthma is increased (Ma et al. 2002). The degree to which myosin light chain is phosphorylated partly determines the velocity of shortening and maximum contraction via increased actomyosin ATPase activity, and expression of myosin light chain kinase, the rate-limiting enzyme in this sequence, is increased by the coexistence of allergy or sensitization (Ammit et al. 2000). In keeping with this possibility, Ma et al. (2002) reported that mRNA transcripts for smooth myosin light chain kinase were more abundant in smooth muscle from asthmatics. Abnormalities

P

P

P

Myosin monomers

Time

Fig. 41.3 Summary of major events during airway smooth muscle stimulus–response contraction coupling from the resting state to tension maintenance. Mechanical plasticity events are shown in the context of the “glass hypothesis” by considering airway smooth muscle as a soft glassy material in which the cytoskeletal and contractile filament deformation and reorganization and gross mechanical properties are regulated from a state of relative fluidity (“hot”) to the rigidity (“cold”) of a solid. MLC, myosin light chain. (From Gunst & Fredberg 2003, with permission.) (See CD-ROM for color version.)

880

P

COLD

Tension maintenance

P

Cytoskeletal lattice Crossbridge cycling stabilizes and becomes rigid slows, latch

Rho kinase

P

P

P

P

Myosin polymerization

G-actin Actin polymerization

Fig. 41.4 Mechanism of mechanical plasticity in airway smooth muscle. Polymerization of globular (G) to filamentous (F) actin is regulated by pathways requiring RhoA, p38 mitogen-activated protein (MAP) kinase, and heat-shock protein (HSP)27. Polymerization of myosin monomers is regulated by RhoA and by Rho kinase. (Adapted from Halayko & Solway 2001, with permission.)

may also be present in the mechanisms that limit bronchoconstriction. In particular, bronchoconstriction in asthmatics induced by spasmogens such as methacholine is not reversed by deep inspiration unlike the bronchoconstriction in healthy individuals. Two possibilities need to be tested to explain these differences. One is that airway smooth muscle in asthmatics shortens faster and thus recontracts more quickly after deep inspiration than in healthy airways, with the result that any bronchodilating effect of deep inhalation does not persist in the asthmatic. The second relates to differences in the plasticity (stretch without regaining original configuration)–elasticity balance of airway smooth muscle from healthy or asthmatic individuals. This may result from loss of interdependence between airway smooth muscle and lung parenchyma (due to airway wall structural changes in asthma) or, at the level of contractile filament function, from excessive elasticity of asthmatic muscle thus limiting the effects of deep inspiration, perhaps because the soft glassy properties of healthy and asthmatic airway smooth muscle cells differ (Fredberg 2001, 2004).

Allergic role in asthma The link between asthma and allergy is well established but a direct role of the airway smooth muscle cell in allergic events is contentious. Incubation of airway smooth muscle with serum from atopic individuals (so-called passive sensitization), which contained high levels of IgE, has long been known to induce hyperreactivity in isolated airway preparations (Black

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et al. 1989; Villanove et al. 1993; Mitchell et al. 1997; Watson et al. 1997). Treatment of airway smooth muscle preparations with atopic serum increases the number of IgE-bearing cells, which have been identified mainly as mast cells (Berger et al. 1998). While these early findings imply a role for IgE in the regulation of contractile and relaxant properties of passively sensitized airway smooth muscle, IgE depletion from atopic serum did not prevent passive sensitization (Watson et al. 1998) and IgE is reported to induce airway smooth muscle cell membrane hyperpolarization (Souhrada & Souhrada 1984). However, recent findings suggest that human airway smooth muscle constitutively expresses both the high-affinity (FcεRI) and low-affinity (FcεRII/CD23) receptor for IgE (Gounni et al. 2005) as well as Fc receptors for IgG, FcRI (CD64), FcRII (CD32), and FcRIII (CD16) (Hakonarson et al. 1999a). More is known of the expression of FcRII/CD23 by airway smooth muscle than FcεRI, where it is linked to induction of proasthmatic contractile changes as well as their enhanced synthetic function. Hakonarson and Grunstein (1998) showed that treatment with atopic asthmatic serum (containing high levels of IgE) or exogenously administered IgE immune complexes upregulated FcRII/CD23 expression, and subsequent activation provoked proasthmatic changes in airway smooth muscle responsiveness including increased agonist-induced contractility and attenuated relaxation. These changes were abrogated by preincubation with a monoclonal anti-CD23 neutralizing antibody. Additionally, FcRII/CD23 expression in human airway smooth muscle was induced by either IL-4 or granulocyte–macrophage colony-stimulating factor (GMCSF), which was accompanied by changes in cell morphology (Belleau et al. 2005). In another study, incubation of airway smooth muscle cells with IgE induced expression of low levels of the proasthmatic Th2 cytokines IL-13 and IL-5. Neutralization of these cytokines reduced IgE-induced agonistmediated constrictor responsiveness and restored impaired relaxations to β2-adrenoceptor agonists (Hakonarson et al. 1999a; Grunstein et al. 2002). The same authors report that rhinovirus inoculation induces expression of FcεRII/CD23 protein in nonsensitized airway smooth muscle cells, and that this is potentiated in airway smooth muscle cells already passively sensitized with atopic asthmatic serum (Grunstein et al. 2001). FcεRI immunoreactivity has recently been detected in vivo within airway smooth muscle cells in bronchial biopsy specimens from mild atopic asthmatic subjects (Gounni et al. 2005), although a contribution from FcεRI-positive mast cells cannot be fully excluded because both smooth muscle cells and mast cells are intimately located in the airways (Brightling et al. 2002). In the same unconfirmed report, cross-linking of FcεRI on airway smooth muscle induced calcium release from intracellular stores and caused low levels of release of the eosinophil-mobilizing and chemoattractant factors IL-5, IL-13, and eotaxin-1/CCL11 (Gounni et al. 2005). Together, there is tentative support for FcεRI activation on

Airway Smooth Muscle

airway smooth muscle modulating both calcium signaling and cytokine secretion to promote a hyperresponsive phenotype. It is of interest that the anti-human IgE antibody omalizumab has beneficial effects as adjunctive therapy in asthma but it is uncertain whether these can be related to effects on the airway smooth muscle cell. Airway smooth muscle cells have been suggested as participating in antigen presentation, a function usually restricted to the professional antigen-presenting cells (dendritic cells, macrophages, B lymphocytes) of the immune system. Although, airway smooth muscle is reported to express major histocompatibility complex (MHC) class II (Lazaar et al. 1987) and the costimulatory molecules CD80 and CD86 (Hakonarson et al. 2001), it is unable to present antigen to CD4 T cells. However, as pointed out by Oliver and Black (2006), alveolar macrophages from asthmatic but not healthy subjects can function as antigen-presenting cells (Balbo et al. 2001) and so it remains to be determined if the same is true of asthmatic muscle cells.

Immunomodulatory role in asthma In addition to a possible direct role in allergy through expression of IgE receptors (Gounni et al. 2005), a growing body of evidence suggests that airway smooth muscle expresses a vast array of immunomodulatory factors and can therefore participate in the orchestration and/or perpetuation of inflammation at multiple points in the inflammation cascade. Examples of some of the factors expressed by airway smooth muscle include the following: • Inflammatory cytokines: IL-1β, IL-5, IL-6, GM-CSF, G-CSF. • Inflammatory chemokines: IL-8, RANTES, eotaxin, monocyte chemotactic protein (MCP)-1, MCP-2, MCP-3, thymus and activation-regulated chemokine (TARC). • Polypeptide growth factors: platelet-derived growth factor (PDGF), TGF-β, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), c-kit ligand/stemcell factor. • Interstitial extracellular matrix proteins: collagen, fibronectin, perlecan, laminin, elastin. • Cell adhesion molecules: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CD44, integrins. • Cytokine (TNFR1/2, IL-1, IL-4Rα, IL-13Rα1/2) and chemokine (CCR3) receptors. • Costimulatory molecules: CD80/CD86, CD40, OX40L (reviewed in Hirst 2001; Howarth et al. 2004). Additionally, polymorphisms in the IL-4 receptor on airway smooth muscle influence chemokine release (Faffe et al. 2003) and many of the mediators secreted by airway smooth muscle occur in response to stimulation with proasthmatic cytokines such as IL-1β, TNF-α, and IL-13. These are elevated in bronchoalveolar lavage fluid and lung tissue of patients with asthma and thus in vitro stimulation likely mimics components of the inflammatory cascade occurring within

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Mast cell IgE/FceRI IgE/FceRI T cell

CXCR3

CD44 IgE/FceRI

IL-4/IL-13 IP-10 SCF

Hyaluronan CD44 OX40 /

CD40 /

OX40 L

CD40 L T cell

Eotaxin IL-5 GM-CSF

TSLC1

ICAM-1/VCAM-1

Eosinophil

Fig. 41.5 Putative interactions of airway smooth muscle cells with inflammatory cells. The possibility exists for many inflammatory cells to interact with smooth muscle cells in the allergic airway. These are mediated via soluble cytokine interactions as well as direct adherence of multiple inflammatory cells to airway smooth muscle via adhesion molecules such as intercellular adhesion molecule (ICAM)-1, vascular endothelial adhesion molecule (VCAM)-1, and tumor suppressor in lung cancer (TSLC)-1, as well as components of the extracellular matrix such as hyaluronan, and costimulatory molecules including CD40 and OX40. (Adapted from Oliver & Black 2006, with permission.)

the asthmatic airway. Furthermore, many cytokine and chemokine receptors are present on the surface of these cells, suggesting that both autocrine and paracrine signaling pathways regulate airway smooth muscle function in vivo. Airway smooth muscle may also participate in innate immune responses since it expresses and responds to ligation of multiple Toll-like receptors (TLRs) including TLR2, TLR3, and TLR4 (Morris et al. 2005; Sukkar et al. 2006). Assimilation of this emerging information suggests that airway smooth muscle has the capacity to recruit, activate and retain inflammatory cells (eosinophils, mast cells and leukocytes) in asthmatic airways (Fig. 41.5). Thus, airway smooth muscle can be activated to induce both the recruitment and survival of eosinophils through secretion of eotaxin/CCL11, GM-CSF, and IL-5 (Saunders et al. 1997; Hallsworth et al. 1998; Hakonarson et al. 1999b; Hirst et al. 2002) and constitutively it produces stem-cell factor, the c-kit ligand, which acts to recruit and retain mast cells within the smooth muscle layer (Kassel et al. 1999). Other pathways that may contribute to the selective mast cell accumulation in airway smooth muscle bundles observed in asthma (Brightling et al. 2002) include preferential secretion of the chemokine CXCL10 (IP-10) by airway smooth muscle from asthmatic patients (Brightling et al. 2005) and direct adherence of human lung mast cells to airway smooth muscle cells via tumor suppressor in lung cancer (TSLC)-1 located on the mast cell surface (Liu et al. 2006; Yang et al. 2006) (Fig. 41.5). Mast cells located within the airway bundles of asthmatic subjects also express Th2 cytokines, such as IL-4 and IL-13

882

(Brightling et al. 2003), and coculture of mast cells with airway smooth muscle enhances mast cell degranulation via a mechanism that requires cell contact between mast cells and airway smooth muscle (Thangam et al. 2005). Leukocyte retention by airway smooth muscle involves expression and activation of cell adhesion molecules. Airway smooth muscle cells express ICAM-1 and VCAM-1, which are inducible by inflammatory mediators, and constitutively express CD44 (Lazaar et al. 1994). ICAM-1 is a cellular receptor for many rhinoviruses and like VCAM-1 can be used by activated T cells to mediate adhesion (Fig. 41.5). CD44, the cellular receptor for hyaluronan, is also constitutively expressed on the surface of both airway smooth muscle cells and T cells (Lazaar et al. 1994). Significantly, coculture of activated T cells with airway smooth muscle induces DNA synthesis in airway smooth muscle cells (Lazaar et al. 1994) and adhesion of activated T cells increases smooth muscle cell contractility (Hakonarson et al. 2001). Further evidence of possible interactions between airway smooth muscle cells and T cells in immune responses is suggested by expression of the costimulatory molecule OX40L by airway smooth muscle (Fig. 41.5). OX40L is involved in T-cell activation and OX40, the receptor, is expressed on the surface of T cells activated through T-cell receptor stimulation. OX40L engagement on airway smooth muscle cells by recombinant human OX40 induces IL-6 secretion (Burgess et al. 2004) and levels of OX40L are greater on airway smooth muscle cells grown from asthmatics (Burgess et al. 2005). In addition to cellular adhesion and costimulatory molecules, airway smooth muscle cells express variable levels of integrin subunits, with the α2, α5, αv, and β1 subunits predominating (Freyer et al. 2001; Nguyen et al. 2005). Integrins are the major receptors for many extracellular matrix proteins. In asthma there are increases in airway wall hyaluronan, fibronectin, tenascin, versican, laminin, and collagen types I, III, and V. Human airway smooth muscle cells secrete multiple extracellular matrix proteins in response to asthmatic sera (Johnson et al. 2000), identifying them as a putative cellular source for extracellular matrix deposition in airways and implicating a novel mechanism by which these cells might upregulate autocrine responses (Freyer et al. 2001; Parameswaran et al. 2004; Nguyen et al. 2005; Peng et al. 2005). Airway smooth muscle cells from asthmatic subjects secrete higher levels of extracellular matrix proteins (Johnson et al. 2004; Chan et al. 2006) and this partly mediates both the hypersecretory (Chan et al. 2006) and putative hyperproliferative (Johnson et al. 2001, 2004) function of these cells. The increase in extracellular matrix deposition in asthma likely results from mesenchymal cells as well as an imbalance between matrix metalloproteinase (MMP) enzymes and endogenous inhibitors of these matrix-degrading proteases. Airway smooth muscle cells express multiple MMPs including pro-MMP-2, pro-MMP-3, and active MMP-3 (Elshaw et al. 2004). MMP-2 (progelatinase A) is constitutively released by

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airway smooth muscle cells and required for proliferation (Johnson & Knox 1999). The composition of the extracellular matrix affects airway smooth muscle cell function. Proliferation is enhanced by monomeric collagen I, fibronectin, and vitronectin, whereas other extracellular matrix components such as fibrillar collagen I, collagen III, and tenascin-C are without effect (Hirst et al. 2001; Nguyen et al. 2005). Likewise, collagen type I and fibronectin amplify other responses of airway smooth muscle including cell survival (Freyer et al. 2001), cellular migration (Parameswaran et al. 2004), and chemokine production (Peng et al. 2005). Cells plated on extracellular matrix derived from asthmatic airway smooth muscle also show enhanced proliferation and chemokine secretion (Johnson et al. 2004; Chan et al. 2006). Finally, extracellular matrix composition may determine the responses of airway smooth muscle to front-line antiasthma therapies. Freyer et al. (2004) demonstrated that fibronectin increased, whereas collagen V and laminin decreased, accumulation of the second messenger cyclic AMP following β2-adrenoceptor activation compared with collagens I or IV. Likewise, collagen type I renders airway smooth muscle cells refractory to the antiproliferative effects of glucocorticoids (Bonacci et al. 2003). Putative bidirectional intregrin-dependent interactions between the extracellular matrix and airway smooth muscle are shown in Fig. 41.6. Thus the available data support airway smooth muscle cells having an immunomodulatory function by contributing and responding to the known alterations within the extracellular matrix in asthma and to actively perpetuate airway mucosal inflammatory processes, including mast cell, eosinophil, and leukocyte activation and recruitment (Figs 41.5 and 41.6).

Airway Smooth Muscle

Orchestration of these events by airway smooth muscle may be especially relevant in the diseased lung where its content is increased, and initial findings with cultured airway smooth muscle cells derived from asthmatics suggest that these cells respond differently, both qualitatively and quantitatively. Moreover, the production of antiinflammatory mediators by airway smooth muscle that exert a braking effect on local inflammation, such as prostaglandin E2, may be impaired in airway smooth muscle from asthmatics (Chambers et al. 2003).

Proliferative role in asthma Increased airway smooth muscle content in small and large airways is a characteristic finding in fatal and nonfatal severe asthma. Airway smooth muscle accumulation also occurs in severe chronic obstructive pulmonary disease but only in those patients where there is persistent airflow obstruction and not to the extent found in asthma (Chung 2005). As with other functions of airway smooth muscle, such as contraction and immunomodulation, most of the information for airway smooth muscle accumulation relates specifically to asthma where the increase correlates directly with both the severity and duration of asthma. Although not proven directly, it is generally agreed from mathematical modeling of thickened large and small airways that accumulation of airway smooth muscle explains the major component of the development of airway hyperresponsiveness in severe asthma (Bai & Knight 2005). Recent bronchial biopsy studies reveal increased airway smooth muscle content even in mild asthmatics. In these, there is evidence for hypertrophy (Ebina et al. 1993; Benayoun et al. 2003), although detailed stereologic studies emphasize the importance of hyperplasia (Woodruff

EXTRACELLULAR SPACE Proteoglycan ril Collagen fib

Fibronectin Integrin b1 a2 Collagen fragment

CLEAVAGE a5

b1

a3 a6

Laminin b1 b4

Fig. 41.6 Putative bidirectional integrindependent interactions between the extracellular matrix and airway smooth muscle as well as other airway mesenchymal cells that result in altered cellular function responses or antiasthma therapy. (Modified from Fernandes et al. 2006, with permission.)

Airway smooth muscle cell

INTEGRIN-MEDIATED REGULATION OF: Adhesion, proliferation, migration Mediator release, survival Response to growth factors Response to anti-asthma drugs

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et al. 2004). Thus the precise cellular mechanisms that account for accumulation of airway smooth muscle in asthma are undefined. While the in vitro proliferative capacity of human airway smooth muscle from asthmatics is reported to be greater (Johnson et al. 2001), other mechanisms must also be considered. Airway smooth muscle accumulation could also result from reduced apoptosis of resident smooth muscle cells, or from migration of interstitial mesenchymal cells or circulating stem cells that then differentiate into fibroblasts or muscle precursor cells to form the additional muscle bulk. The varying occurrence of these processes in asthma may also explain why so few studies have detected increased markers of proliferation in intact airway smooth muscle bundles from asthmatics (Madison 2003; Stewart 2004). The mitogens responsible for increased airway smooth muscle growth in vivo are unknown, although several candidate mediators and mitogenic intracellular signaling pathways have emerged as leading candidates using in vitro airway smooth muscle cell culture-based studies (Hirst et al. 2004; Lazaar & Panettieri 2005). Some, but not all, airway smooth muscle mitogens also promote migration of airway smooth muscle in vitro, although whether airway smooth muscle cell migration occurs in the intact airway wall is uncertain (Stewart 2004). The majority of mitogens identified fall into two broad categories: polypeptide growth factors that activate receptors with intrinsic protein tyrosine kinase activity and those that act through receptors coupled to heterotrimeric G proteins, which includes the thromboxanes, acetylcholine, endothelin-1, LTD4, and inflammatory cell-derived proteases such as thrombin. Many of these are comitogens that synergize with polypeptide growth factors to promote accelerated growth in vitro (Hirst et al. 2004). Molecular mechanisms accounting for airway smooth muscle growth have been reviewed extensively elsewhere (Hirst et al. 2001, 2004; Zhou & Hershenson 2003) and are summarized in Fig. 41.7. Major effector signaling pathways include activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signalregulated kinase (ERK) as well as NADPH oxidase pathways (Fig. 41.7). p21ras, a 21-kDa guanosine triphosphatase (GTPase), may act as a point of convergence for the mitogenic signal activated by varying receptor-operated mechanisms. In its active GTP-bound state, p21ras binds downstream effectors such as PI3K and the 74-kDa cytoplasmic serine/ threonine kinase Raf-1. Recruitment of Raf-1 to the plasma membrane activates the ERK pathway. PI3K activation is critical for cell-cycle progression in airway smooth muscle (Walker et al. 1998; Krymskaya et al. 1999, 2001), although additional signaling events are necessary for maximal airway smooth muscle growth responses including sustained ERK activity (Karpova et al. 1997; Fernandes et al. 1999; Orsini et al. 1999). Enhanced proliferation of airway smooth muscle cells from patients with asthma (Johnson et al. 2001) has been linked to lack of expression of the transcription factor

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P Rb

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Fig. 41.7 Putative mitogens and key signal transduction mechanisms that regulate airway smooth muscle cell division. Mitogens acting predominantly through receptor tyrosine kinase-linked receptors (RTK) or G protein-coupled receptors (GPCR) activate small GTPase p21ras proteins, which interact with downstream effectors. These include phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK). PI3K, via 3-phosphorylated phosphoinositides and other intermediates, activates the NADPH oxidase complex to induce cell cycle progression and cell proliferation. Cdk, cell cycle-dependent kinase; p70S6K, ribosomal S6 kinase; R, cell cycle restriction point; P, phosphorylation; Rb, retinoblastoma protein; ROS, reactive oxygen species. (See CD-ROM for color version.)

C/EBPα in asthmatic airway smooth muscle cells. Reduced C/EBPα protein expression is implicated also in resistance to the antiproliferative effects of glucocorticoids (Roth et al. 2002, 2004). Reactive oxygen species also regulate airway smooth muscle proliferation. Airway smooth muscle cells express several, but not all, components of the phagocyte NADPH oxidase, and reactive oxygen species can modulate mitogen-induced airway smooth muscle cell proliferation (Page et al. 1999; Brar et al. 2002; Pandya et al. 2002). Additionally, activation of Janus kinase (JAK)-2 and signal transducer and activator of transcription (STAT)-3 is required for PDGF-dependent proliferation of human airway smooth muscle cells. Overexpression of catalase attenuates STAT3 phosphorylation, suggesting that JAK/STAT activation is redox-dependent and

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that JAK/STAT-dependent signaling constitutes an alternative downstream target of reactive oxygen species in regulating airway smooth muscle expansion (Simon et al. 2002).

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points therefore exist for the suppression of airway smooth muscle synthetic responses by β2-adrenoceptor activation. These are reviewed in detail in Howarth et al. (2004).

Glucocorticoids

Actions of existing front-line antiasthma drugs on airway smooth muscle Impaired relaxation of airway smooth muscle and incomplete bronchodilatation are characteristic features of asthma. Although currently available therapies such as corticosteroids and β2-adrenoceptor agonists are varyingly effective in suppressing in vitro responses of airway smooth muscle such as contraction, proliferation, migration, and secretion (reviewed in Hirst et al. 2004; Howarth et al. 2004), their efficacy in reducing key responses of airway smooth muscle in the intact asthmatic airway is uncertain or unknown. Cell culture-based studies suggest that β2-adrenoceptor agonists decrease activation of PI3K and ERK, whereas glucocorticoids interfere with pathways leading to an increase in cyclin D1 message levels and prevent progression through the cell cycle. Emerging in vitro evidence suggests that contact with the extracellular matrix modulates the efficacy of these drugs (Fig. 41.6).

b2-Adrenoceptor agonists To date, there is no evidence that the effects of long-acting β2-adrenoceptor agonists are different from those of shortacting β2-adrenoceptor agonists, but it appears likely that persistent β2-adrenoceptor stimulation would be important in the inflamed airway in contributing to suppression of airway smooth muscle responses, because stimuli for contractile and synthetic responses may be active for periods longer than the periods of perceptible airway obstruction. β2-Adrenoceptor agonists attenuate DNA synthesis and increases in airway smooth muscle cell number in response to a diverse range of mitogenic stimuli (Hirst et al. 2004). Similarly, they reduce spontaneous migratory responses in these cells as well as PDGF-induced migration enhanced by urokinase (Carlin et al. 2003). All β2-adrenoceptor agonists exert their biological and therapeutic effects through cellsurface β2 adrenoceptors. After ligand binding to the active site of the receptor, the α-component of the associated Gs protein dissociates and activates adenylate cyclase, leading to the production of intracellular cyclic AMP and subsequent activation of protein kinase A (PKA), which then phosphorylates several intracellular regulatory proteins. β2-Adrenoceptor agonists may also influence gene transcription through a cyclic AMP mechanism whereby there is increased translocation of the catalytic subunit of PKA to the nucleus and phosphorylation of cyclic AMP response element (CRE)-binding protein, enhancing its DNA binding and transactivation effects. It has been suggested that β2-adrenoceptor agonists activate C/EBPα as do glucocorticoids (Roth et al. 2002). Multiple

The target receptor for corticosteroids is the intracellular glucocorticoid receptor (GR). Under resting conditions, inactive GR is found mainly in the cell cytosol, associated with multichaperone proteins. Binding of glucocorticoids to the GR through the corticosteroid-binding domain induces a conformational change in the receptor protein, dissociation of the chaperone proteins, and formation of an active corticosteroid–GR complex. The complex translocates from the cytosol to the nucleus, where it binds to specific DNA sequences called glucocorticoid response elements (GRE) in the promoter region of target genes, leading to cofactor activation and either an increase or decrease in gene transcription. Alternatively, the active corticosteroid–GR complex can interact directly with intracellular transcription factors such as AP-1 or NF-κB through protein–protein interaction to attenuate transcription factor-mediated proinflammatory processes. This transrepression process involves recruitment of histone deacetylases and modulation of chromatin structure. Glucocorticoids, like β2-adrenoceptor agonists, suppress the synthetic behavior of airway smooth muscle against a broad spectrum of mitogens, migratory stimuli, and proinflammatory cytokines (reviewed in Hirst 2001; Howarth et al. 2004). Antiproliferative activity occurs through binding to the GR, because it is attenuated by the transactivation antagonist RU486 and is mimicked by glucocorticoids but not mineralocorticoids. Glucocorticoids decrease the transcription and the translation of cyclin D1 and consequently arrest progression through the cell cycle (Fig. 41.7). Unlike β2-adrenoceptor agonists, glucocorticoids have no effect on ERK activity, but they share a stimulatory effect on C/EBPα (Roth et al. 2004).

Factors limiting the actions of b2-adrenoceptor agonists and glucocorticoids Different mitogens are differentially sensitive to the action of the β2-adrenoceptor agonists and glucocorticoids. In general, thrombin and other G protein-coupled receptor stimuli are more sensitive to the antiproliferative actions of either glucocorticoids or β2-adrenoceptor agonists (Vlahos et al. 2003). Moreover, the antiproliferative action of glucocorticoids in vitro is suppressed in airway smooth muscle cells grown from asthmatics, which lack normal expression of C/EBPα. When overexpressed by transient transfection, C/EPBα restores the antiproliferaitve action of glucocorticoids (Roth et al. 2004). Such a mechanism could explain the accumulation of airway smooth muscle occurring in the airways of asthmatics, which otherwise appears unchecked by therapy. Perhaps related to this observation, the extracellular matrix may regulate the antiproliferative actions of glucocorticoids

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in airway smooth muscle. Thus, the antiproliferative effects of glucocorticoids are also lost when nonasthmatic airway smooth muscle cells are plated on monomeric collagen I, but not when seeded on plastic or a laminin substrate (Bonacci et al. 2003). Given that asthmatic airway smooth muscle cells synthesize more collagen I relative to nonasthmatic cells (Johnson et al. 2004), it is possible that the insensitivity to glucocorticoids might be mediated in part by an altered extracellular matrix environment. Consistent with this notion, Johnson et al. (2000) reported that the glucocorticoid beclomethasone enhanced rather than decreased the synthesis of several extracellular matrix substrates induced by atopic asthmatic serum. The extracellular matrix environment may also regulate responses to β2-adrenoceptor agonists. Freyer et al. (2004) noted that a fibronectin-dominated environment increased responses to β2-adrenoceptor activation, while exposure to collagen type V or laminin reduced β2-adrenoceptor signaling compared with collagens types I or IV. This effect was accompanied by a reduction in intracellular cyclic AMP levels that was associated with modulation of Giα. More recently, it was reported that neither corticosteroids nor long-acting β2adreonoceptor agonists reduced TGF-β-induced collagen type I or fibronectin in airway smooth muscle from individuals with or without asthma (Burgess et al. 2006). In contrast, corticosteroids alone induced the expression of collagen type I and fibronectin. However, the phosphodiesterase (PDE)4 inhibitor roflumilast prevented upregulation of induced collagen type I, fibronectin, or CTGF by TGF-β in intact bronchial rings from nonasthmatics. In contrast, the PDE inhibitor was less effective against TGF-β-induced extracellular matrix production from airway smooth muscle cells cultured from healthy or asthmatics donors (Burgess et al. 2006). Thus the therapeutic actions of the major classes of current antiasthma agents on airway smooth muscle are modified or impaired by contact with an altered extracellular matrix environment (Fig. 41.6).

Novel approach to decreasing airway wall smooth muscle content in asthma While accumulation of airway smooth muscle is considered quantitatively important and a target for antiasthma therapy, available data from in vitro studies and those performed with intact tissue sections have not revealed the predominant cellular or molecular events by which airway smooth muscle accumulates in asthma. Cell culture studies have not revealed a predominant signaling mechanism for cell proliferation and doubt now exists as to whether smooth muscle proliferation per se can explain the increase in smooth muscle cell number in asthma. With alternative explanations emerging, including the possibility that cells migrate either from the interstitial compartment or from a circulating precursor stem-cell popu-

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lation, targeting of airway smooth muscle accumulation via current pharmacologic approaches remains an uncertain way forward, especially when little change has occurred in asthma morbidity and mortality over the last 15–20 years (Mannino et al. 2002). Thus, an alternate strategy has emerged to target airway smooth muscle accumulation in subjects with moderately severe asthma. An exciting and radical new development is to ablate the airway smooth muscle itself through the controlled delivery of radiofrequency thermal energy to the airways. To achieve this, an expandable heating filament in circumferential contact with the airway wall is inserted through the channel of a bronchoscope and the airway wall heated to 65°C or more, a procedure now referred to as bronchial thermoplasty. The technique was originally intended to serve as a treatment for chronic obstructive pulmonary disease, in which collapse of the airways and gas trapping is a major problem. The rationale was that induction of scarring of the airways by heat stress would make them stiffer and thus less susceptible to collapse. Treatment appears to cause an acute blanching at the site of treatment, and histology shows epithelial disruption at treated sites. Subsequent regrowth occurs after as little as 12–20 days in the epithelium, blood vessels, mucosa, and nerves, with the unexpected finding that fibrosis or scarring is not evident (Miller et al. 2005). Airway smooth muscle, however, seems to have almost no capacity for regeneration. Thus, treated airways appear normal except for the absence of airway smooth muscle in those areas where heat was applied (Fig. 41.8) (Mitzner 2006). Optimization studies, performed in dogs, determined the intensity and duration of delivery of thermal energy required to achieve a 50% reduction in airway smooth muscle content (Danek et al. 2004). Following this treatment, a degree of airway responsiveness to locally applied methacholine persisted in the airways in which airway smooth muscle was replaced by loose connective tissue. Airway smooth muscle between treatment sites appeared unaffected, the muscle being sufficiently contractile to cause some airway narrowing on challenge (Danek et al. 2004; Brown et al. 2005). In humans, the first studies using bronchial thermoplasty were carried out in nonasthmatic subjects with preoperative lung cancer (Cox et al. 2004; Miller et al. 2005). This allowed an opportunity for the ablation procedure to be performed in airways that would later be available for histologic examination. This safety study confirmed that bronchial thermoplasty was well tolerated in human airways, leading to a marked and localized reduction of airway smooth muscle content. A longer-term evaluation was recently reported in patients with mild or moderate asthma (Cox et al. 2006). Patients were followed initially for 12 months without any evidence of chronic or progressive airway injury. All accessible airways greater than 3 mm in diameter were treated in the lower and upper lobes in three bronchoscopy sessions, each at least 3 weeks apart. These subjects have now been monitored for

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Seromucosal gland Airway smooth muscle Ciliated epithelium

Phenotypic plasticity

Maturation Modulation

Airway Smooth Muscle

Mechanical plasticity

Contractile filament Reorganization

• Length change • Contraction “Synthetic” airway smooth muscle • Cell division & hypertrophy • Migration • Extracellular matrix production • Immunomodulation

Parenchyma (a)

Airway smooth muscle absent

Seromucosal gland Ciliated epithelium

“Contractile” airway smooth muscle • Contraction • Do not migrate • Immunomodulation? • Extracellular matrix production

Fig. 41.9 Integrated paradigm for functional and phenotypic plasticity of airway smooth muscle. This schematic diagram shows the association between phenotypic and mechanical plasticity in smooth muscle. Phenotypic plasticity results from reversible modulation and maturation of smooth muscle cells between a functionally synthetic and contractile state. This diversity is chiefly controlled through regulated expression of specific genes in response to a number of factors, which can include proinflammatory stimuli, changes in extracellular matrix, and cell–cell interactions. Mechanical plasticity occurs in contractile smooth muscle cells as a result of rapid subcellular reorganization of the contractile thick and thin filaments in response to acute or chronic muscle length change during contraction or due to stretch. (Modified from Halayko & Amrani 2003, with permission.)

Parenchyma

2 years following treatment. Results to date show marked consistent improvements in lung function, symptom-free days, and decreased airway responsiveness to methacholine challenge. The success of this approach extends the validity of targeting airway smooth muscle for the effective treatment of asthma.

tone in response to various neurotransmitters, proinflammatory mediators, and exogenous substances is outmoded. In its place, an integrated paradigm for plasticity of airway smooth muscle function in lung disease has emerged that encompasses both acute and long-term modulation of cell contraction, proliferation, and synthesis/secretion of inflammatory mediators (Fig. 41.9). Growing evidence supports a principal role for airway smooth muscle as an effector in airway hyperresponsiveness not only through its increased contractile responses to bronchoconstrictors and attenuated responses to bronchodilators, but also as a result of its increased content in the airway wall, its production of extracellular matrix proteins in the muscle layer, submucosa and adventitia, and by its capacity to perpetuate local inflammation (Halayko & Amrani 2003). Added to this, selective ablation or reduction in airway smooth muscle content in asthma underscores the overall importance of this cell type in the pathogenesis of asthma and provides a convincing argument for targeting this cell type for development of new effective treatments of asthma.

Concluding remarks

Acknowledgments

The traditional passive-partner view in which airway smooth muscle is the major effector cell regulating bronchomotor

The author thanks Dr Jane E. Ward (University of Melbourne, Australia) and Dr Andrew J. Halayko (University of Manitoba,

(b) Fig. 41.8 Histopathologic features of (a) untreated control canine airway and (b) an airway treated with radiofrequency thermal energy at 65°C, 12 weeks post treatment (trichrome stain, original magnification ×100). Airway smooth muscle in the untreated airway is normal, but is absent in the treated airway. Airway smooth muscle is reduced throughout the circumference of the treated airway (b) and present in normal amounts throughout the circumference of the untreated airway (a). The parenchyma, epithelium, and mucous glands are normal in the treated airway. (From Danek et al. 2004, with permission.) (See CD-ROM for color version.)

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Winnipeg, Canada) for helpful comments and the reading of this manuscript during its preparation.

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Biochemistry of Allergens and Recombinant Allergens Rudolf Valenta

Summary

Introduction

This chapter attempts to give a comprehensive overview of biochemical, biological, and clinical aspects of allergens involved in IgE-mediated allergy. With the application of recombinant DNA technology to the field of allergen characterization, our knowledge regarding the structure, biological, and immunologic properties of allergens has tremendously increased. DNA and amino acid sequences of the most common allergens have become available and recombinant allergens mimicking the characteristics of the natural allergens have been produced. The detailed knowledge of allergen structures has influenced nomenclature, standardization, basic research, diagnosis, prevention, and therapy in allergology. The availability of allergen sequences and their three-dimensional structures has facilitated experimental studies regarding the recognition of allergens by T cells and antibodies and has allowed allergens to be grouped into families of related proteins based on sequence similarity and immunologic cross-reactivity. It is expected that the progress made in the field of molecular allergen characterization will lead to considerable changes in allergen nomenclature. According to the DNA sequences of allergens it has become possible to produce large amounts of pure allergens by recombinant DNA technology, which may bring about numerous advantages for allergy diagnosis and treatment. New diagnostic tests based on recombinant allergens allow a more precise diagnosis of patients’ reactivity profiles and represent better tools for prescription of therapies and preventive measures. Furthermore, they allow monitoring of the allergic immune response during the course of disease and changes of allergen-specific immune responses during treatment. Finally, several new approaches for allergy treatment based on recombinant allergens and allergen-derived peptides have shown good results in clinical trials and thus hold promise that better forms of allergen-specific therapy and perhaps even prophylaxis will become available soon. The chapter provides basic definitions and a summary regarding general properties of allergens. Furthermore, it attempts to guide the reader through the rapidly expanding field of allergen biology and the numerous clinical applications of recombinant allergens.

The term “allergy,” coined by Clemens von Pirquet in 1906, intended to discriminate hypersensitivity from normal immunity as a faster and more intense response that gives rise to clinical reactions (von Pirquet 1906; Kay 2006). The foreign substance which induces the organism to react in such a “changed manner” to its single or repeated introduction into the body was defined as an “allergen.” The classification of Coombs and Gell, and a modified version of this classification, groups allergic reactions according to fundamental pathomechanisms into several types of hypersensitivities (Coombs & Gell 1963; Kay 1997). Type I allergy (i.e., immediate-type hypersensitivity) defines the reaction induced by allergens when they combine with specific IgE antibodies to form allergen–IgE immune complexes that activate immune cells, in particular mast cells and basophils, via Fcε receptors. A more recent classification has suggested the term “IgEmediated allergy” for an IgE-mediated hypersensitivity reaction, although such reactions do not necessarily need to be limited to atopic subjects (Johansson et al. 2001). Atopy as such has been defined as a personal or familial tendency to produce IgE antibodies in response to low doses of allergens. In this scenario allergens involved in IgEmediated allergy are therefore defined by their ability to recombine via their epitopes with the variable regions (i.e., paratopes) of specific IgE antibodies. Allergens thus represent antigens that are specifically recognized by IgE antibodies and need to be discriminated from substances which bind to IgE in a nonspecific manner such as certain lectins or other ligands for IgE. Allergen recognition by IgE antibodies is hence the result of a specific immune response. In the case of IgE-mediated allergies, almost all clinically relevant allergens are protein antigens that induce IgE responses in a T cell-dependent manner (reviewed in Valenta & Kraft 2001; Blumenthal & Rosenberg 2004). Carbohydrate moieties can also induce IgE responses. In this case IgE production may receive T-cell help via peptide epitopes derived from the protein backbone of various glycoproteins (Mari 2002; van Ree 2002; Altmann 2007). Similar mechanisms may be operative in certain forms

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of IgE-mediated drug allergies (Gerber & Pichler 2004; Pichler 2004). There are also a few reports that nucleic acids, lipids, and other substances may behave as allergens (Nagpal et al. 1987; Corinti et al. 1997; Agea et al. 2005). The initiation of an allergic immune response, commonly termed “allergic sensitization,” is the primary immune response which may lead to the development of an IgEmediated allergy (reviewed in Valenta 2002; Valenta et al. 2004). It occurs in early childhood and often has a characteristic pattern of a first phase of food allergy which is then followed by the development of respiratory allergy, commonly termed the “allergic march” (Kulig et al. 1999). Also, IgE autosensitization may occur early in childhood (Mothes et al. 2005). Allergic sensitization is a T cell-dependent process which leads to the production of allergen-specific IgE antibodies. The process of class switching to IgE requires typical Th2 cytokines and there is evidence that this process does not necessarily evolve in a sequential form (Niederberger et al. 2002a; Geha et al. 2003). Whether a given antigen induces tolerance or a specific IgE response, or a normal IgG response, may be determined by a large variety of host and environmental factors, of which some are listed in Table 42.1. Allergic sensitization leads to the establishment of an allergen-specific IgE and T-cell memory response. Allergenspecific IgE memory responses are strongly boosted by repeated allergen contact and results from animal studies suggest that this secondary, established IgE response can potentially be maintained by T cell-independent mechanisms (Linhart et al. 2007; Niederberger et al. 2007b). Several studies performed in patients demonstrate that respiratory, particularly nasal, allergen contact strongly boosts systemic IgE production but much less is known about whether allergen exposure via the skin or the upper and lower gastrointestinal tract can boost systemic IgE responses in allergic patients (Reininger et al. 2003; Niederberger et al. 2007b).

Table 42.1 Host and environmental factors influencing the development of an allergen-specific IgE response. Host factors Route/site of contact Mechanisms of uptake and processing Status of immune system (childhood vs. senescence) Genetic predisposition Antigen-specific (e.g., MHC) General atopic vs. nonatopic (e.g., IL-4 promoter) Environmental factors Allergen structure (folded, unfolded) Allergen immunogenicity Allergen exposure and dose Adjuvants and immunemodulating factors Pollution, endotoxins, infections

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The ability of an allergen to induce an allergic immune response may be defined by the term “allergenicity.” Allergenicity is similar to “immunogenicity,” which defines the ability of an antigen to induce a specific immune response. Several reports suggest that certain allergens may have an intrinsic property to induce IgE responses due to certain biological functions (e.g., protease activity) but these properties are not common to all the important allergens (Chua et al. 1988; Hewitt et al. 1995; Gough et al. 1999, 2001; Wan et al. 1999; Valenta & Kraft 2001; Shreffler et al. 2006). In addition, in nonallergic persons allergens induce “normal” IgG responses, suggesting that the allergenicity of a given antigen is primarily determined by its immunogenicity in combination with certain host and environmental conditions that favor the development of an IgE response (Akdis et al. 2004; Stern et al. 2007). In principle, it is possible to induce allergic sensitization in experimental animal models with any antigen that exhibits sufficient immunogenicity provided that adjuvants or experimental conditions are chosen which favor the development of IgE responses (Epstein 2004). In fact, in a murine model comparing different allergens it has been found that the overall allergenicity of allergens is related to their immunogenicity (Vrtala et al. 1998). However, the term “allergen” implies that the antigen, besides specifically inducing an IgE response and binding to IgE antibodies, can also induce a clinically relevant allergic reaction. Whether a clinically relevant allergic reaction occurs on allergen contact depends, for the most common forms of allergic manifestations, on the ability of an allergen to crosslink mast cell- and basophil-bound IgE antibodies. IgE antibodies bind with high affinity to FcεRI on mast cells and basophils, and on cross-linking by allergens induce a signaling cascade that leads to the release of stored mediators, proteases, and cytokines as well as to de novo synthesis of mediators and proinflammatory substances. Mast cell and basophil degranulation is one key mechanism of allergic inflammation but there is evidence that allergens can also induce allergic inflammation via T-cell activation through IgE-facilitated antigen presentation (reviewed in Stingl & Maurer 1997; Novak et al. 2003; van Neerven et al. 2006) and via non-IgEmediated mechanisms (Haselden et al. 1999; Larché et al. 2001). In order to induce mast cell or basophil degranulation, an allergen needs to have at least two epitopes for IgE binding. The extent of mast cell and basophil degranulation depends on the number of IgE epitopes and the levels of allergen-specific IgE (Gieras et al. 2007). For many important allergens it has been demonstrated that they contain several different IgE epitopes (Chua et al. 1991; Greene & Thomas 1992; Ball et al. 1994, 1999b; Bufe et al. 1994; Fedorov et al. 1997; Schramm et al. 1997; Flicker et al. 2000; Schramm et al. 2001). There is also evidence that the structural topology of epitopes may have an influence on the extent of the degranulation reaction. For example, clustering of epitopes on certain parts of an allergen may facilitate receptor aggregation

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(Flicker et al. 2006). The weak or even nonexistent ability of carbohydrates to activate mast cells and basophils and hence to induce immediate-type allergic reactions may be explained by the fact that they contain only a few IgE epitopes or even behave as IgE-reactive haptens. Nonallergenic IgE-reactive haptens can be prepared from potent allergens by proteolytic cleavage or by recombinant DNA technology (Malley & Perlman 1970; Chakrabarty et al. 1980; Ball et al. 1994). Allergen-derived IgE-reactive haptens, IgE-reactive carbohydrates, and some of the recently discovered IgE-reactive autoantigens bind specifically IgE but are weak inducers of mast cell or basophil degranulation (Ball et al. 1994; Mari 2002; Aichberger et al. 2005). In principle such structures may be defined as “IgE-reactive antigens” as compared with “allergens.” Yet the question is open whether such structures can contribute to allergic inflammation. In principle it is possible that IgE-reactive antigens without the ability to cross-link mast cells or basophils may contribute to allergic inflammation by inducing T-cell activation. For example, certain IgE-reactive autoantigens were found to induce the secretion of interferon (IFN)-γ, a cytokine that leads to epithelial cell damage (Aichberger et al. 2005; Reisinger et al. 2005a). Furthermore, it has been demonstrated that allergenderived peptides without IgE reactivity can induce late-phase allergic reactions via major histocompatibility complex (MHC) class II-dependent T-cell activation (Haselden et al. 1999). Finally, it has been observed that allergen derivatives engineered to reduce their ability to induce IgE-mediated mast cell activation more than 100-fold compared with the wildtype allergen could induce late-phase reactions in allergic patients by patch testing and in the course of allergen-specific immunotherapy (Campana et al., Purohit et al. submitted). All the latter results indicate that it may be necessary to investigate in more detail the mechanisms of “allergic inflammation” that are independent from IgE-mediated mast cell and basophil degranulation. Such studies would ideally identify clinically relevant criteria for defining the allergenic activity of allergens. Furthermore, they may additionally allow estimates to be made of the importance of such mechanisms to overall allergic inflammation, and in doing so provide new information relevant to reducing side effects in the course of allergen-specific therapies.

Allergen sources, allergen extracts, and purified natural and recombinant allergen molecules From historical and clinical perspectives it has proven useful to identify and define allergen sources for seasonal, perennial, indoor, outdoor, respiratory, food, skin, and systemically acting allergens. The abundance of allergen sources as well as the degree to which they contain and liberate allergens determine their importance, among other parameters. For

Biochemistry of Allergens and Recombinant Allergens

example, grasses are very important allergen sources because they occur worldwide and produce large amounts of airborne pollen (Esch 2004). In contrast, other plants (e.g., oilseed rape) have a more restricted distribution and their pollen is carried mainly by insects. Therefore they have lower importance as allergen sources. Local climate conditions as well as other geographic factors have implications for the occurrence of certain allergen sources in certain areas and their importance. For example, birch pollen is a very important allergen source in northern and central Europe but has little relevance in Mediterranean countries (Mothes et al. 2004). Similar rules apply for other allergen sources, such as certain species of mites (e.g., house-dust mites versus tropical mites), and it is hence possible to identify characteristic profiles of sensitization to individual allergens that can be explained by the local prevalence of the allergen sources. For example, the different sensitization profiles found among patients from southern and northern parts of Europe reflect the local flora, and there are also differences regarding the profiles of indoor allergens in different continents (Moverare et al. 2002b; Westritschnig et al. 2003). Allergen sources may contain varying numbers of allergens (Fig. 42.1). There are certain allergen sources (e.g., birch pollen, cat, fish) which contain primarily one major allergen that is recognized by the majority of patients and accounts for most of the allergenic activity, whereas other allergen sources contain several important allergens. Besides factors related to the allergen itself, several other factors related to the characteristics of allergen sources may influence whether a certain protein becomes an allergen. For example, it has been shown that potent pollen allergens elute very quickly and in large amounts from pollen grains when they come into contact with the mucosa and there are different mechanisms (e.g., pollen rupture after hydration) favoring the release of respirable allergen-containing particles (Suphioglu et al. 1992; Vrtala et al. 1993; Schappi et al. 1997; Grote et al. 2000, 2003; Taylor et al. 2002; Motta et al. 2006). Allergens may also be released from the primary allergen source and may then become bound to other materials (e.g., diesel exhaust particles) which may act not only as allergen carriers but may also have adjuvant activity and push the immune response along a Th2 pathway (Diaz-Sanchez et al. 1994, 1997; Knox et al. 1997). Rather recent data even indicate that allergen sources, besides containing the disease-eliciting allergens, may also contain substances that can favor the development of allergic immune responses (Traidl-Hoffmann et al. 2005). The contents of individual allergens in allergen sources is regulated by a variety of biological processes. For example, it has been shown that the expression of certain allergens is highly upregulated during pollen maturation; there is evidence that the expression of allergens depends on certain growth factors, hormones, and environmental conditions; and that strain and gender aspects may have an influence

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Allergen source

Allergen extract

Allergen

MC

Mixture of allergenic and non-allergenic components Disease-eliciting molecule

on the amounts of allergens in certain allergen sources (Savolainen et al. 1989; Hjelmroos et al. 1995; Hsieh et al. 1995; Mittermann et al. 1995; Jalil-Colome et al. 1996; Midoro-Horiuti et al. 2001; Tashpulatov et al. 2004; Martínez et al. 2006). From what has been said it is clear that there can be huge variability regarding the presence of certain allergens in a particular allergen source and there are also numerous, as yet only partly characterized, other substances in allergen sources. These substances may have an influence on immune responses or represent a matrix that may carry and protect allergens or enhance their intrusion to varying degrees, for example in the respiratory tract, through the skin and intracellular matrix, or during gastrointestinal processing. The variability of individual allergens and the presence of undefined nonallergenic materials in allergen sources also has great impact on the daily practice of allergy diagnosis and treatment. At present, allergen extracts prepared from allergen sources by various protocols are used for diagnostic testing and treatment. Manufacturers of allergen extracts attempt to control the quality of allergen extracts using immunochemical and biological standardization techniques, but it is becoming ever clearer that there are numerous problems with allergen extracts that cannot be overcome (Akkerdaas et al. 2003). It is possible to determine the concentrations of some but not all important allergens in such extracts but it is not possible to influence the concentrations of the individual allergens except by the addition of purified allergens. Contamination of allergen extracts with allergens from other allergen sources have been reported and unwanted materials such as endotoxins and bacterial components have been found in commercial allergen extracts (van der Veen et al. 1996; Trivedi et al. 2003; Valerio et al. 2005). While it must be appreciated that standardization of methods has improved the quality of allergen extracts from 1930 onwards, one must

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Fig. 42.1 Allergen source, allergen extract and allergen molecule. (See CD-ROM for color version.)

also accept that certain fundamental problems related to natural allergen extracts cannot be overcome. Instead, new technologies that allow the preparation of extremely pure and defined allergens have to be applied. The need for such pure and defined allergens for diagnosis and treatment can in fact be satisfied by the application of recombinant allergens (reviewed in Valenta & Kraft 2002). Several research groups have been isolating cDNAs that code for allergens and producing the corresponding allergens by recombinant expression since 1988. As fruit of this work and its explosive expansion in the last two decades, recombinant allergens for the most common allergen sources have become available and have been used for research purposes, diagnosis, treatment, and prevention of allergy (reviewed in Valenta & Kraft 2002).

Recombinant allergens Recombinant allergens are allergens which are produced by transcription and translation of the corresponding DNA in an expression system. Allergen-encoding DNAs can be obtained in principle by functional or DNA-based screening assays. Figure 42.2 illustrates one of the most common and successful strategies for the isolation of recombinant allergens. In a first step, mRNA is isolated from the allergen-expressing source and converted into cDNA by reverse transcription. The cDNA molecules can then be inserted into phage DNA, which on in vitro packaging gives rise to a phage library containing a repertoire of cDNA molecules that represent the pool of mRNA molecules which have been transcribed in the allergen source and hence also contain allergen-encoding mRNA. It has proven very useful to use phage systems such as λgt11 phages, which on infection of bacteria are capable of producing recombinant proteins from the allergen-encoding

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Allergen source (e.g., pollen, animal dander, house dust mite. . . . )

Isolation of the mRNA Synthesis of cDNA Phage library Infection of E.coli

Serum IgE IgE immunoscreening

Isolation of phage DNA Fig. 42.2 Definition and production of recombinant allergens.

cDNA template. Using this system it is possible to select phage clones that express allergen-encoding DNAs with IgE antibodies from patients and to obtain the allergen-encoding DNA after a few cycles of recloning. The technology of IgE immunoscreening of phage libraries has allowed access to a large number of allergen-encoding cDNAs in a fast and convenient form. Several other technologies, such as DNA-based screening approaches, have been used for the isolation of allergen-encoding cDNAs. They are based on the creation of synthetic DNA probes, which can be obtained by deduction from allergen-derived amino acid sequences. Such probes can then be used for DNA-based screening of libraries or for polymerase chain reaction (PCR)-based cloning of allergenencoding sequences. PCR-based allergen cloning may be a fast method but can also deliver nonauthentic DNA sequences or DNA sequences coding for allergen isoforms with low or no IgE reactivity (Breiteneder et al. 1993; Ferreira et al. 1996). The availability of allergen-encoding cDNAs opens numerous possibilities for allergen characterization. Further, it facilitates understanding of the structure and biological functions of allergens and of the immune responses to allergens (e.g., mapping of T-cell and B-cell epitopes). Finally, it is possible to produce recombinant allergens that exactly mimic the properties of the corresponding natural allergens for clinical applications (reviewed in Valenta & Kraft 2002). Figure 42.3 is a summary of the many opportunities presented by the availability of recombinant allergens. From the DNA sequence of allergens, it is immediately possible to deduce the corresponding amino acid sequence of the allergen. Allergen sequences can be used to search the databases of known sequences for similar proteins or for certain sequence motifs that allow the possible biological function of the allergen to be deciphered. Furthermore, it has become

Cloning into a vector

DNA sequence analysis Expression of the recombinant allergen

Panel of recombinant allergens

possible to produce recombinant allergens for use as research tools in the study of allergen-specific immune responses as well as for diagnostic and therapeutic purposes.

From traditional allergen nomenclature to new forms of allergen classification The explosive increase in knowledge regarding allergen structure and biology through allergen characterization by recombinant DNA technology will dramatically change the way in which we designate and characterize allergens, the allergen nomenclature system. Allergen nomenclature has evolved out of early attempts to purify allergens from natural allergen sources using a variety of classical biochemical and immunochemical techniques (reviewed in Chapman et al. 2006). Initially these techniques were used to enrich certain allergenic fractions from allergen extracts and allowed the purification of several important allergens from pollens, cat, fish, and other allergen sources between 1960 and 1970. In 1986, the allergen nomenclature subcommittee, formed under the auspices of the World Health Organization (WHO) and the International Union of Immunological Societies (IUIS), first published a list of highly purified allergens (Marsh et al. 1986). According to the allergen nomenclature system, allergens are designated as follows: the genus is indicated by three letters and the species by one letter and an Arabic numeral that normally indicates the chronologic order of identification. For example, Bet v 1 is the major allergen of white birch, Betula verrucosa. The designation “major allergen” is reserved for allergens that react with IgE antibodies from more than 50% of patients allergic to a given allergen source, whereas the term “minor allergen” indicates that this allergen is

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Standardization • Pure recombinant allergens in defined mass units available • Establishment of assays for measuring allergen contents in diagnostic and therapeutic extracts

Basic science • Defined molecules and epitopes for basic in vitro and in vivo experiments

Allergen structure

Therapy • Recombinant allergens • T cell epitope-containing peptides • DNA vaccines • CpG-coupled allergens • B cell epitope-derived peptides • Mimotopes • Modified recombinant allergens

Prevention • Establishment of assays for measuring allergen exposure • ldentification of hypoallergenic products • Generation of hypoallergenic plants and food

Diagnosis • Component-resolved diagnosis with defined molecules • Marker allergens as gatekeeping tests • Microarrayed recombinant allergens for chip-based allergy diagnosis

Fig. 42.3 Impact of recombinant allergens on research, diagnosis, treatment and prevention. (From Valenta & Kraft 2002, with permission from Elsevier.)

recognized by IgE of less than 20% of patients. Within a given allergen source, several different genes may code for isoforms of a given allergen that differ in sequence from each other, often only in a few amino acids. Isoforms can be denoted by the addition of numeral suffixes to the allergen name. There are isoforms that are almost indistinguishable from each other with regard to IgE reactivity and allergenic activity, whereas others show greatly varying IgE reactivity and allergenic activity despite only a marginal sequence variation (Breiteneder et al. 1993; Ferreira et al. 1996). Allergens purified from natural allergen sources may therefore consist of several isoforms. They are indicated as natural allergens by the addition of the letter “n” for natural, whereas the letter “r” indicates that an allergen has been produced as recombinant protein in a foreign host system according to one defined cDNA template. The traditional nomenclature system has been extremely useful in the past for compiling lists of known allergens for each allergen source, but does not take into account the biological functions of allergens and the fact that allergens with similar structure, function, immunologic, and allergenic activity are present in different unrelated allergen sources. The family of profilins, a group of actin-binding proteins distributed throughout eukaryotic organisms, is a good example of a family of highly cross-reactive allergens not restricted to certain taxonomically related allergen sources (Valenta et al. 1991, 1992). It has therefore been suggested that allergens can be grouped according to sequence similarity and immunologic cross-reactivity rather than by the traditional con-

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cept of grouping by allergen sources (Valenta et al. 1996). This concept of grouping allergens by sequence, functional characteristics and, most importantly, immunologic cross-reactivity rather than by the traditional Linnaean taxonomy continues to gain support and appears likely to change the traditional allergen nomenclature system (Radauer & Breiteneder 2006). One strong argument for grouping allergens according to cross-reactivity is that such a system provides a better basis for determining the conditions of original sensitization in a patient, i.e., differentiation between co- and cross-sensitization, and is extremely useful for diagnosis and the prescription of allergen-specific forms of treatment (Pauli 2000; KazemiShirazi et al. 2002). As exemplified in Fig. 42.4, allergen sources (e.g., A, B, C) contain allergens that are only present in certain sources or which exist as cross-reactive allergens in different allergen sources. Those allergens present only in certain allergen sources can be considered as marker allergens for a genuine sensitization against these sources, whereas cross-reactive marker allergens indicate that patients may exhibit cross-reactivity between different unrelated allergen sources (Fig. 42.5). For example, IgE reactivity and clinical sensitivity to marker allergens that occur as cross-reactive structures in two (McAC), three (McABC), or more different allergen sources indicate that the patient can in principle be allergic against each of the allergen sources in which the cross-reactive structure is present (Fig. 42.4). On the other hand, marker allergens that occur only in certain allergen sources (e.g., only in A, B or C) can be considered as marker allergens (MgA, MgB, MgC) for genuine

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MgA

MgB

A

Biochemistry of Allergens and Recombinant Allergens

Table 42.2 Prevalence of IgE reactivity to birch pollen allergens in population. Population

Bet v 1 (%)

Bet v 2 (%)

Bet v 4 (%)

Finland Sweden Austria France Switzerland Italy Zimbabwe

100 98 98 90 65 62 0

2 12 30 20 43 33 100

5 8 11 6 7 27 0

B

C

McABC

McAC MgC

Fig. 42.4 Cross-reactive and source-specific allergens. Different allergen sources contain overlapping and nonoverlapping allergen repertoires.

“Species-specific” marker allergens

Cross-reactive marker allergens

Genuine sensitization against a certain allergen source

Cross-sensitization

Fig. 42.5 Marker allergens.

sensitization against these sources (Fig. 42.4). For example, IgE reactivity to MgA indicates genuine sensitization to the allergen source A because MgA does not occur in other sources. The usefulness of using marker allergens instead of allergen extracts for diagnosis becomes apparent from population studies conducted with allergen extracts and marker allergens. For example, the increase in false-positive diagnostic test results with birch pollen extracts in southern populations can be revealed with the marker allergen for birch sensitization, rBet v 1. “Birch pollen-allergic” patients from Zimbabwe, a country with virtually no birch pollen exposure, are all negative for rBet v 1 but positive for cross-reactive grass or weed pollen allergens that cross-react with homologous allergens in birch pollen extract (e.g., Bet v 2) (Table 42.2) (Moverare et al. 2002b; Westritschnig et al. 2003). The rate of truly birch pollen sensitized, i.e., rBet v 1-positive, patients is highest in Nordic countries with high birch pollen exposure (Moverare et al. 2002b). As for birch pollen, marker allergens that can be used as diagnostic markers for genuine sensitization to grass, tree, and weed pollens as well as to house-dust mites, cat, and fish have been identified (Kazemi-Shirazi et al. 2002; Grönlund et al. 2003; Stumvoll et al. 2003; Swoboda et al. 2002, 2004; Mothes et al. 2004; Pittner et al. 2004; Palomares et al. 2006). Such marker allergens can be used to diagnose genuine

sensitization against a certain allergen source and consequently are more precise diagnostic tools for the prescription of allergen-specific immunotherapy than allergen extracts (Fig. 42.5). Marker allergens are also useful for establishing the hierarchy of sensitization in allergic patients. For example, patients suffering from birch pollen allergy also frequently suffer from allergies to pollens of botanically related trees such as alder and hazel and to certain plant foods, e.g., apple. The clinically relevant question arising is whether birch pollen or another tree pollen or plant food has been the original sensitizing agent. One possibility for addressing such a question is to compare the levels of IgE antibodies directed against the marker allergens from each of the allergen sources suspected of being the origin of sensitization. This can be achieved by quantitative IgE measurements and by serologic competition experiments in which sera are preadsorbed with each of the suspected allergens and the remaining (i.e., unbound) IgE is allowed to bind to the individual allergens after preadsorption (Niederberger et al. 1998a,b; Kazemi-Shirazi et al. 2000). It may be reasonable to assume from such experiments that the allergen which binds most of the IgE and depletes most of the IgE directed against the other related allergens has been the primary sensitizing agent, particularly if such experiments have been performed in representative populations of allergic patients. The establishment of hierarchies of sensitization among allergens may be considered of particular relevance to the choice of allergens for allergen-specific immunotherapy. Using such an approach evidence has been provided that birch pollen, and in particular the major birch pollen allergen rBet v 1, contains the majority of IgE epitopes present in pollens of related trees and plant food (Fig. 42.6). It is thus not surprising that several studies indicate that immunotherapy with birch pollen or rBet v 1 derivatives is effective and sufficient not only for treating birch pollen allergy but also for treating allergies to pollens from related trees and plant food (Petersen et al. 1988; Asero 1998; Bolhaar et al. 2004; Bucher et al. 2004; Niederberger et al. 2007a).

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lgE

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rBet v 1

rAIn g 1

rCor a 1

rMal d 1

Fig. 42.6 Hierarchy of IgE cross-reactivity according to quantitative IgE measurements and cross-inhibition tests.

The spectrum of respiratory, food, and insect allergens The application of recombinant DNA technology to the field of allergen characterization has led to an explosive increase in our knowledge of the structures and characteristics of allergen molecules. In order to obtain an overview of important allergens, the reader is advised to consult Table 42.3,

where useful references, mostly review articles and book chapters, provide summaries and descriptions of the allergens in the listed allergen sources. The table contains information regarding the allergens from the following allergen sources: pollens (trees, grasses, weeds), mites (house-dust mites, storage mites, tropical mites), cockroaches, fungi (Aspergillus, Alternaria, Cladosporium, Penicillium, Trichophyton, Candida, Malassezia), animals (cat, dog, horse, cow, mouse, rat, guinea pig, rabbit, human), food (cows’ milk, chicken egg, peanut, soybean, fruits, vegetables, spices, wheat, fish, shrimp), insect (Hymenoptera: bees, fire ants, wasps, hornets; Biting insects: mosquitoes, flies, bugs, fleas, ticks), and latex allergens. In addition to the review articles and book chapters the reader may want to consult, allergen databases containing information about individual allergen molecules. An update of currently available allergen databases is given in Mari (2005). The website of the WHO/IUIS Allergen Nomenclature subcommittee (www.allergen.org) lists allergens that have been submitted to the subcommittee by researchers for approval and registration. The Structural Database of Allergenic Molecules (SDAP) from the University of Texas Medical Branch offers structural data. Information particularly regarding food allergens may be obtained from the Food

Table 42.3 Important allergen sources and their allergens. Allergen sources

References

Tree pollen (e.g., birch, alder, hazel, plane, olive, ash, cypress, cedar, juniper, etc.)

Mothes et al. (2004), Rodriguez et al. (2001), Niederberger et al. (2002b)

Grass pollen

Andersson & Lidholm (2003), Esch (2004), Suphioglu (2000)

Weed pollen (e.g., ragweed, mugwort, Parietaria, etc.)

Colombo et al. (2003), Wopfner et al. (2005), Mohapatra et al. (2004), Gadermaier et al. (2004)

Mites (e.g., house-dust mites, storage mites, tropical mites)

Thomas et al. (2002), Fernandez-Caldas et al. (2004)

Cockroach

Helm & Pomes (2004), Arruda (2005)

Fungi (e.g., Aspergillus, Alternaria, Cladosporium, Penicillium, Trichophyton, Candida, Malassezia, etc.)

Kurup et al. (2002), Vijay & Kurup (2004)

Animals (e.g., cat, dog, horse, cow, mouse, rat, guinea pig, rabbit, human)

Virtanen & Mäntyjärvi (2004), Spitzauer (1999)

Food allergens (e.g., cows’ milk, chicken egg, peanut, soybean, fruits, vegetables, spices, wheat, fish, shrimp, etc.)

Roux et al. (2003), Burks (2004), Wal (2001), Schöll & Jensen-Jarolim (2004)

Insects (Hymenoptera: bees, fire ants, wasps, hornets, etc. Biting insects: mosquitos, flies, bugs, fleas, ticks, etc.)

Peng & Simons (2004), King & Guralnick (2004), Hoffman (2004), King & Spangfort (2000)

Latex allergens

Wagner & Breiteneder (2005), Slater (2004)

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Allergy Research and Resource Program (Farrp) (www. allergenonline.com) and the Protall database (www.ifr.bbsrc. ac.uk/protall). Allergome (www.allergome.org) represents a frequently updated and well-kept allergen database based on published allergen sequences and studies using allergen molecules. Besides specialized allergen databases, DNA and protein sequences as well as three-dimensional structures of allergens are part of large public databases, such as that at the National Center for Biotechnology Information (www.ncbi. nlm.nih.gov/).

Biological functions and structural characterization of allergens The isolation and sequencing of allergen-encoding cDNAs has allowed the amino acid sequences of many important allergens to be deduced. Once the cDNA and deduced amino acid sequence of an allergen are available, it possible to compare the sequences with sequences of known proteins deposited in databases. Such sequence comparisons have revealed the biological functions of many allergens and attempts have been made to relate the allergenic activity of a given allergen to certain biological functions. There are indeed interesting data showing that the major house-dust mite allergen Der p 1 may act as a protease and through cleavage of immune cell-surface structures or damage to respiratory epithelial cell layers promote its own allergenicity and perhaps that of other allergens present in house-dust mite extracts (Chua et al. 1988; Hewitt et al. 1995; Gough et al. 1999, 2001; Wan et al. 1999; Kikuchi et al. 2006). However, if one reviews the list of characterized allergens for which biological functions are known or suspected, it becomes apparent that allergens have numerous biological functions for which no immediate connection to allergenicity can be established. For example, the highly cross-reactive profilin allergens belongs to a family of actin-binding cytoskeletal proteins (Valenta et al. 1991, 1992). Muscle proteins such as parvalbumin or tropomyosin, serum proteins such as albumin, calcium-binding proteins, pathogenesis-related plant proteins or storage proteins are also important allergens (reviewed in Valenta & Kraft 2001). In light of these data it is therefore not possible to entertain the idea that allergens fall exclusively into a group of proteins with predefined biological functions (e.g., enzymatic activity) that would favor allergenicity. Other attempts to explain and predict allergenic activity on the basis of sequence or structural motifs have also been quite unsuccessful (reviewed in Spoek et al. 2005). The possibility of producing large quantities of recombinant allergens by NMR and X-ray crystallography has rapidly increased our knowledge of the three-dimensional structures of allergens. However, if one reviews the current list of available allergen structures, it becomes obvious that there is no common conformation that would predetermine the aller-

Biochemistry of Allergens and Recombinant Allergens

genicity of a given protein. Allergens with almost exclusive β-sheet structure, mixed β-sheet/α-helical structures, and exclusively α-helical structures have been reported (reviewed in Valenta & Kraft 2001). As mentioned above, it is possible to group allergens according to sequence and structural similarities (Valenta et al. 1996; Radauer & Breiteneder 2006). In fact, allergens exhibiting sequence and/or structural similarities very often contain cross-reactive IgE and T-cell epitopes, but there may be exceptions to this rule and it is therefore not possible to determine the degree of sequence or structural similarity that allows prediction of the presence and extent of immunologic cross-reactivity with certainty. The extent and hierarchy of cross-reactivity therefore always needs to be established by representative experiments, such as immunologic crossreactivity studies at the antibody and T-cell level. There are also several good examples contradicting the assumption that high sequence similarity predicts allergenic cross-reactivity with certainty, among them the fact that certain allergen sources such as birch pollen contain nonallergenic or low-allergenic isoforms which differ from the major allergen Bet v1 in only a few amino acids (Ferreira et al. 1996). It should be mentioned at this point that attempts to predict the allergenic activity of proteins exclusively on the basis of sequence comparisons requires particular caution when it comes to allergenicity assessment of genetically modified organisms (Spoek et al. 2005). Instead experimental approaches based on in vitro and in vivo experiments have been suggested as more reliable methods for allergenicity assessment (Spoek et al. 2005). Nevertheless, knowledge of allergen structures has had great impact on several areas of allergology (Fig. 42.3). For basic science, recombinant allergens represent pure tools for studying the immune mechanisms underlying allergy. Based on the availability of allergen sequences and structure, it has become possible to study in detail epitopes recognized by T cells and IgE antibodies (reviewed in Valenta & Kraft 2002). Epitopes defined by IgE antibodies are crucially involved in IgE-mediated allergic inflammation (e.g., mast cell degranulation). Our knowledge of IgE epitopes is continuously increasing. Interestingly, most respiratory allergens seem to contain primarily conformational IgE epitopes that depend on the intact three-dimensional structure of these allergens (reviewed in Valenta & Kraft 2001). Accordingly, several research groups have engineered recombinant allergen derivatives with reduced IgE reactivity and allergenic activity by disrupting conformational IgE epitopes or by removing amino acids critically involved in combining with the IgE paratopes (reviewed in Linhart & Valenta 2005). Although there seem to be exceptions to the rule (e.g., parvalbumin) (Bugajska-Schretter et al. 1998), “true” food allergens that can elicit systemic IgE-mediated reactions often contain sequential IgE epitopes (Helm et al. 2000; Jarvinen et al. 2001; Shreffler et al. 2004). Whether a given food allergen can

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induce systemic IgE-mediated inflammation and thus anaphylactic reactions seems to be associated with the preservation of IgE epitopes after passing through the gastrointestinal tract. Allergens with high resistance against destruction of IgE epitopes, for example high stability or presence of sequential IgE epitopes less affected than conformational epitopes by digestion, more likely induce systemic IgE-mediated reactions (Sanch et al. 2005; Vassilopoulou et al. 2006). Allergens with conformational and/or labile IgE epitopes trigger more local reactions in the mouth (e.g., oral allergy syndrome) or purely T cell-mediated reactions (e.g., exacerbation of atopic dermatitis after food ingestion) because small allergen-derived peptides recognized by T cells can survive the digestion process and, once resorbed from the gastrointestinal tract, can induce T cell-mediated symptoms in other organs (Schimek et al. 2005; Bohle et al. 2006). The binding sites for IgE antibodies on allergens have been mapped for many important allergens and demonstrate that allergens contain several of these binding sites. IgE epitopes belong to the conformational or sequential type and include several different surface structures of an allergen. The number of IgE epitopes on a given allergen, together with the level of IgE antibodies specific for these epitopes, determine the capacity of an allergen to induce mast cell and basophil degranulation (Gieras et al. 2007). There is also evidence that structural constraints, i.e., orientation of IgE epitopes, may affect the allergenic activity of an allergen, specifically when a particular clustering of epitopes on an allergen may foster the aggregation of mast cell-bound IgE (Flicker et al. 2006). Recently, the structural biology of allergens has become extremely important for the design of new types of allergy vaccines. Technologies are being applied and developed which allow dissection of IgE and T-cell epitopes for engineering of hypoallergenic allergen derivatives, and it has become possible to even alter the immunologic properties of allergens (e.g., immunogenicity, tolerogenic activity, Th1/Th2 activity) by means of allergen engineering (reviewed in Linhart & Valenta 2005).

Biochemical, immunologic, and clinical characterization of allergens Natural allergens are often difficult to purify from allergen sources because they may occur only in small amounts or they may be difficult to separate from other allergens or nonallergenic materials. For example, several important allergens (e.g., house-dust mite allergens) have been discovered only by cDNA cloning techniques because they exist in trace amounts in natural allergen extracts (Lin et al. 1994). Obviously one may have doubts that an allergen which is basically absent from natural allergen extracts may indeed be clinically important, but many of the allergens that have been only discovered by molecular cloning show IgE reactiv-

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ity and allergenic activity in a considerable percentage of allergic patients, leaving no doubt regarding their clinical relevance (Lynch et al. 1997). In this context it should be noted that it has become a quite common habit to improve the quality of natural allergen extracts by the addition of recombinant allergens (Lundberg et al. 2001). Another problem related to purified natural allergens is the fact that many allergen sources contain several isoforms of a given allergen, which may differ substantially regarding allergenic activity. Even if one applies the most sophisticated purification protocols, a “purified natural allergen” may consist of several isoallergens with different sequences and immunologic and clinical properties. From what has been said it becomes clear that it may be impossible to obtain the complete allergen repertoire from an allergen source in the form of purified natural allergens. Furthermore, it is almost impossible to obtain pure and homogeneous natural allergen preparations for comparison with recombinant allergens. The discussion about natural allergens has been initiated to point out that the traditional strategy of comparing the biochemical, immunologic, and clinical properties of natural versus recombinant allergens may not always be applicable. Instead it may be more straightforward to go directly for the production of biochemically well-characterized recombinant allergens and evaluate the immunologic and clinical properties of these molecules. The availability of different strategies for the expression of high levels of properly folded recombinant proteins using prokaryotic and eukaryotic systems allows production of large amounts of defined recombinant allergens (reviewed in Valenta & Kraft 2004). The proteins are then subjected to a variety of protein chemical techniques such as mass spectrometry and circular dichroism, which allow determination of the precise mass and measure the folding of the protein. Whether the allergen is aggregated can be determined by gel filtration or small-angle X-ray scattering (SAXS) (Ferreira et al. 2005). The purified recombinant allergens are then usually assessed for IgE reactivity and their ability to stimulate allergenspecific T cells (e.g., proliferation, cytokine release) and to induce IgE-dependent activation of basophils and mast cells in vitro. The basophil activation tests can be performed using either basophils from allergic patients or rat basophil leukemia cells, which can be loaded with serum IgE from allergic patients, by measuring the release of biological mediators after allergen contact or by recording the upregulation of certain activation markers (e.g., CD63, CD203c) by flow cytometry (Valenta et al. 1993; Hauswirth et al. 2002; Valent et al. 2004; Kleine-Tebbe et al. 2006; Kaul et al. 2007). The latter assays seem to be particularly useful for estimating the allergenic activity of allergens because it has become apparent that the levels of allergen-specific IgE and the extent of in vivo allergenic activity as measured by skin or nasal provocation testing are often not well correlated (Heiss et al. 1999; Niederberger et al. 2001; Purohit et al. 2005).

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Evaluation of the clinical relevance of a given allergen molecule should therefore also include studies of its in vivo allergenic activity in representative populations of patients. Such evaluation will have impact on the selection of allergens that should be included in therapeutic allergy vaccines. Clearly it will not be necessary to include allergens which fail to induce relevant clinical reactions, whereas frequently recognized allergens with high allergenic activity need to be covered by the vaccine. These considerations will be particularly important for the design of new recombinant allergenbased vaccines, which can be tailored according to patient needs (reviewed in Valenta & Niederberger 2007).

Clinical use of recombinant allergens The clinical uses of recombinant allergens comprise basically three areas, diagnosis, prevention, and treatment. Numerous studies have demonstrated that recombinant allergens can be used to replace traditional natural allergen extracts for in vitro (antibody measurements, cell activation tests) and in vivo diagnosis (reviewed in Valenta & Kraft 2002). In principle it is possible to mix recombinant allergens of a given allergen source to obtain diagnostic tests that can replace extracts or to add recombinant allergens to allergen extracts to include components present in insufficient amounts (reviewed in Valenta et al. 1999). The improvement or replacement of allergen extracts is especially important for those natural extracts that are difficult to prepare such as food extracts (Lidholm et al. 2006). Diagnostic tests based on allergen extracts or allergen mixtures only indicate whether a patient reacts with an unspecified component in an extract but fail to identify the disease-eliciting allergens. Testing with the individual recombinant allergens, termed “component-resolved diagnosis” (CRD), reveals the patient’s reactivity profile to each of the allergens (Valenta et al. 1999). Since CRD performed in vivo with many allergens may be cumbersome, diagnostic tests have been developed based on microarrayed recombinant allergens (Hiller et al. 2002; Harwanegg et al. 2003). Such allergen arrays allow determination of the antibody reactivity profile against a larger number of molecules in tiny blood samples using a single test. Subsequent clinical testing, e.g., by skin testing, nasal or bronchial provocation testing, or oral challenge, allow confirmation of the clinical relevance of IgE reactivities (reviewed in Schmid-Grendelmeier & Crameri 2001; van Hage-Hamsten & Pauli 2004). As mentioned above, recombinant marker allergens can be used to differentiate the clinical conditions of co- and cross-sensitization (Pauli 2000; Kazemi-Shirazi et al. 2000). Such molecules have been identified for many important allergen sources, such as birch pollen, grass pollen, house-dust mites, cat, and weed and olive pollens (Kazemi-Shirazi et al. 2002; Grönlund et al. 2003; Stumvoll et al. 2003; Swoboda et al. 2002, 2004; Mothes et al. 2004; Pittner et al. 2004; Palomares et al. 2006). Diagnostic tests based on

Biochemistry of Allergens and Recombinant Allergens

allergen source-specific marker allergens allow diagnosis of genuine sensitization against certain allergen sources and are therefore useful tools for accurate prescription of allergenspecific immunotherapy. Recombinant allergens have been used to decipher sensitization profiles in children, monitor reactivity profiles, and investigate the mechanisms underlying boosting of the allergic immune response in allergic patients and the development of IgE and IgG reactivities during allergen-specific immunotherapy (Ball et al. 1999a; Niederberger et al. 2002a, 2004, 2007b; Mothes et al. 2003; Reisinger et al. 2005b; Rossi et al. 2007). Measurements of IgE reactivity profiles during allergenspecific immunotherapy with allergen extracts have revealed the induction of new IgE specificities during treatment (van Ree et al. 1996; Ball et al. 1999a; Moverare et al. 2002a). It has been shown that specific immunotherapy with crude allergen extracts may fail to induce IgG responses against important allergens and false-positive IgG responses against nonallergenic material may develop (Birkner et al. 1990; Mothes et al. 2003). Only recombinant allergen-based tests allow detection of true allergen-specific IgG responses (reviewed in Flicker & Valenta 2003). Such tests are useful for monitoring allergenspecific immunotherapy because they allow determination of whether an allergy vaccine can induce allergen-specific immune responses or if there is nonresponsiveness in a patient. Several studies also emphasize the importance of the development of allergen-specific IgG antibodies in the course of immunotherapy (reviewed in Larché et al. 2006). It has been shown that therapy-induced allergen-specific IgG antibodies compete with the IgE recognition of allergens and that this mechanism reduces IgE-mediated activation of mast cells/basophils and thus immediate-type symptoms (Ball et al. 1999a,b; Mothes et al. 2003; Niederberger et al. 2004). Furthermore, it has been shown that blocking IgG can inhibit IgE-facilitated allergen presentation to T cells and thus suppresses T-cell activation (van Neerven et al. 1999, 2004, 2006; Wachholz et al. 2003). Finally, there is evidence that allergenspecific IgG can prevent the boosting of IgE production caused by natural allergen exposure and may thus contribute to the downregulation of allergen-specific IgE responses during immunotherapy (Mothes et al. 2003; Niederberger et al. 2004; Creticos et al. 2006). Recombinant DNA technology and recombinant allergens have also impacted on the field of allergy prevention. Recombinant marker allergens can be used to quantify allergen-specific IgE levels, which reflect whether a patient has been exposed to the allergen (Niederberger et al. 2007b). Such measurements are therefore useful for monitoring whether preventive measures have been effective or not. Using recombinant allergens it is possible to obtain allergenspecific antibody probes that allow detection of nanogram amounts of allergens in our environment and food and thus contribute to measures of allergen avoidance (Valenta et al. 1997; Marth et al. 2004; Earle et al. 2007). Furthermore,

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Allergen sources Grasses Weeds

Trees

Animals

Mites

Food

Insects

Allergen-encoding cDNAs

Recombinant allergens • Research tools • Diagnosis • Vaccines • Allergen detection for prevention

Genetically modified allergens • Vaccines

• Research tools Peptides T cell peptides • Diagnosis • Vaccines B cell

DNA vaccines

attempts have been made to identify hypoallergenic organisms or breeds as well as to reduce the allergenic activity of allergen sources by genetic engineering (Tada et al. 1996; Bhalla et al. 1999; Gilissen et al. 2005; Le et al. 2006). Probably the most important clinical application of recombinant allergens is therapy and prophylaxis of allergy. Based

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Fig. 42.7 From allergen sources to recombinant allergens and new allergy vaccines. (See CD-ROM for color version.)

on allergen-encoding cDNAs, various strategies for prophylaxis and treatment have been developed (Fig. 42.7) (reviewed in Linhart & Valenta 2005). These include allergy vaccines based on recombinant allergens that have been engineered to reduce their allergenic activity and thus therapy-induced side effects, highly specific allergy vaccines based on recombinant wild-type

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allergens, T-cell as well as B-cell peptide-based vaccine approaches, DNA vaccination and other forms allergen-specific vaccination or tolerance induction. An overview of recombinant allergen-based treatment and prophylaxis strategies can be found in Valenta (2002), Valenta et al. (2004), Linhart and Valenta (2005), and Valenta and Niederberger (2007). Several successful clinical immunotherapy trials have been performed with recombinant allergens and a state of the art overview of can found in Valenta and Niederberger (2007). It is expected that we will see the development, clinical evaluation, and application of recombinant allergen-based therapeutic vaccines for the most common allergen sources in the next few years. Furthermore, it is possible that recombinant DNA technology will allow the development of prophylactic approaches to reduce the rapidly growing prevalence of allergic diseases.

Acknowledgments This chapter is dedicated to my mentor Professor Dietrich Kraft on the occasion of his 70th birthday. The study has been supported by a grant from the Christian Doppler Research Association Austria and by the Austrian Science Fund. The secretarial help of Michelle Mueller and Irmgard Lubenik with the preparation of the manuscript and the figures is greatly appreciated. The author thanks Dr Verena Niederberger for help with illustrations.

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of dendritic cell-specific ICAM-grabbing nonintegrin and acts as a Th2 adjuvant in vitro. J Immunol 177, 3677–85. Shreffler, W.G., Beyer, K., Chu, T.H. et al. (2004) Microarray immunoassay: association of clinical history, in vitro IgE function, and heterogeneity of allergenic peanut epitopes. J Allergy Cli Immunol 113, 776– 82. Slater, J.E. (2004) Latex allergens. In: Lockey, R.F., Bukantz, S.C. & Bousquet, J., eds. Allergens and Allergen Immunotherapy. Marcel Dekker, New York, pp. 369– 86. Spitzauer, S. (1999) Allergy to mammalian proteins: at the borderline between foreign and self? Int Arch Allergy Immunol 120, 259–69. Spoek, A., Gaugitsch, H., Laffer, S. et al. (2005) Suggestions fort he assessment of the allegenic potential of genetically modified organisms. Int Arch Allergy Immunol 137, 151– 2. Stern, D.A., Riedler, J., Nowak, D. et al. (2007) Exposure to a farming environment has allergen-specific protective effects on T(H)2dependent isotype switching in response to common inhalants. J Allergy Clin Immunol 119, 351– 8. Stingl, G. & Maurer, D. (1997) IgE-mediated allergen presentation via Fc epsilon RI on antigen-presenting cells. Int Arch Allergy Immunol 113, 24– 9. Stumvoll, S., Westritschnig, K., Lidholm, J. et al. (2003) Identification of cross-reactive and genuine Parietaria judaica pollen allergens. J Allergy Clin Immunol 111, 974– 9. Swoboda, I., Bugajska-Schretter, A., Verdino, P. et al. (2002) Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy. J Immunol 168, 4576– 84. Swoboda, I., Grote, M., Verdino, P. et al. (2004) Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles. J Immunol 172, 6490– 500. Tada, Y., Nakase, M., Adachi, T. et al. (1996) Reduction of 14–16 kDa allergenic proteins in transgenic rice plants by antisense gene. FEBS Lett 391, 341– 5. Suphioglu, C. (2000) What are the important allergens in grass pollen that are linked to human allergic disease? Clin Exp Allergy 30, 1335– 41. Suphioglu, C., Singh, M.B., Taylor, P. et al. (1992) Mechanism of grass-pollen-induced asthma. Lancet 339, 569–72. Tashpulatov, A.S., Clement, P., Akimcheva, S.A. et al. (2004) A model system to study the environment-dependent expression of the Bet v 1a gene encoding the major birch pollen allergen. Int Arch Allergy Immunol. 134, 1– 9. Taylor, P.E., Flagan, R.C., Valenta, R. et al. (2002) Release of allergens as respirable aerosols: a link between grass pollen and asthma. J Allergy Clin Immunol 109, 51– 6. Thomas, W.R., Smith W.A., Hales, B.J. et al. (2002) Characterization and immunobilogy of house dust mite allergen. Int Arch Allergy Immunol 129, 1–18. Traidl-Hoffmann, C., Mariani, V., Hochrein, H. et al. (2005) Pollenassociated phytoprotanes inhibit dendritic cell interleukin 12 production and augment T helper 2 cell polarization. J Exp Med 201, 627–36. Trivedi, B., Valerio, C. & Slater, J.E. (2003) Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 111, 777–83. Valent, P., Hauswirth, A.W., Natter, S. et al. (2004) Assays for measuring in vitro basophil activation induced by recombinant allergens. Methods 32, 265– 70.

Biochemistry of Allergens and Recombinant Allergens

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Host Responses to Allergens Wayne R. Thomas and Belinda J. Hales

Summary This chapter describes and compares the immune responses induced by the common allergens of pollen, house-dust mites, cockroaches, and cats along with the common food allergens of milk, eggs, and peanut and the venom allergens of the honeybee. The IgE, IgG, IgA, and T-cell effector and regulatory responses are described with express reference to responses measured to major allergens and knowledge of the responses produced to the different specificities of each allergen source. IgE binding is usually directed to one or two major allergens. Good examples of this are birch, olive and grass pollen, house-dust mite, milk and cat. Major allergens of cockroach, ragweed, peanut, bee venom, and egg have also been defined but are less dominant or universally recognized. Sera typically contain an average of around 50 ng/mL of IgE antibody to major allergens, with Fel d 1 being an exception with many patients showing low titers. For all allergens, however, many asthmatics only produce a few nanograms per milliliter of antibody. IgG antibody responses to pollen, food, and mite allergens are restricted to allergic subjects. For house-dust mite it has been shown that the IgG binding activity of individual allergens correlates with their allergenicity. As well as IgG4, the Th1-type IgG1 subclass is produced in titers similar to those to bacterial antigens. The IgG responses to pollens are low. IgG binding to individual cockroach allergens has less concordance with IgE, and cat and mouse allergens induce IgG antibody in the absence of IgE. This has been associated with the high levels of cat allergen in homes but comparable exposure levels are not found for the mouse allergen. The precursor frequency of allergen-specific T cells is higher in allergic than nonallergic subjects, but even nonallergic subjects show a sizeable T-cell expansion in response to allergic exposure. T cells from allergic subjects produce Th2 cytokines, and when stimulated directly ex vivo a similar Th1 response to cells from nonallergic subjects, and in similar amounts to that induced by bacterial antigens. The expression of T-cell chemokines and chemokine receptors shows the same pattern.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

The regulatory cytokine IL-10 and CD4+CD25+ T cells have both been shown to be able to inhibit responses of T cells to allergens in in vitro cultures. However, their relative levels in allergic and nonallergic people have been the subject of conflicting reports and they appear to have a regulatory role in the responses of both healthy and allergic subjects.

Allergens Most common sources of allergen such as pollen grains, house-dust mites, cockroaches, pets, foods, and venom produce a large number of allergens. For example the current International Union of Immunological Societies (IUIS) allergen database lists over 20 house-dust mite allergens and a similar number of grass pollen allergens. However, this is only based on the ability to detect IgE binding to the allergen in 5% of people with an allergy to a particular source. It does not depend on the quantitative measurement of IgE or the ability of the allergen to elicit allergic reactions in vivo. The responses of people to most sources of allergen are biased to small number of specificities. These are called the major allergens (Table 43.1). Their current definition as an allergen that binds IgE from the sera of 50% of allergic subjects was made at a time when few proteins had been purified in sufficient quantity for quantitative measurements of IgE binding. It is now known that many allergens bind IgE at high frequency but they often only bind small amounts. The term “major” will be used here in a dictionary sense meaning the main allergens. Bet v 1 from birch pollen is the best-defined major allergen. About 80% of birch-allergic people in regions with high exposure to birch pollen produce IgE antibody in quantities that account for 90% of the response. The remaining responses are directed to at least six other pollen proteins (Moverare et al. 2002). The Ole e 1 allergen of olive pollen is also quantitatively very dominant (Lombardero et al. 1992) even though there are other allergens that bind at high frequency (Rodriguez et al. 2002). The group 1 and group 5 major allergens of grass pollen collectively bind IgE in 95% of sera from grass-allergic subjects, accounting for 80% of the IgE binding to pollen extracts in most cases (Andersson &

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Table 43.1 Common major allergens.

Allergen source

Allergens

Dermatophagoides pteronyssinus (house-dust mite) Timothy grass (grass pollen)

Der p 1 Der p 2 Phl p 1 Phl p 5 Bet v 1 Ole e 1 Cry j 1 Cry j 2 Cup a 1 Cup a 2 Jun a 1 Jun a 2 Art v 1 Amb a 1 Bla g 2 Bla g 5 Fel d 1 Mus m 1 Api m 1 Bos d 8 Gal d 1 Ara h 2

Birch pollen Olive pollen Sugi, cypress, mountain cedar (conifer pollens)

Mugwort pollen Ragweed pollen Cockroach (Blatella germanica) Cat Mouse Bee venom Milk Egg Peanut

Percent IgE binding (of extract)

Biochemical names

50%

Cysteine protease, ML-domain lipid-binding protein

90%

Expansin-like protein and unknown

90% 95% Not documented

PR-10 pathogenesis protein “Inactive” homolog of trypsin inhibitor Pectate lyase, polymethylgalacturonase

Not documented 25–85% 50%*

Defensin Pectate lyase “Inactive” aspartic protease homolog, glutathione-S-transferase Uteroglobin Urinary lipocalin Phospholipase A Caseins Ovomucoid Conglutinin

50% Not documented 30–40%* 90% 30–50%* 30–40%*

* Estimates made from published average IgE binding values of individual allergens.

Lidholm 2003). Most other major allergens account for about 50% of the IgE-binding activity of extracts made from their respective sources. These include the combination of the group 1 and 2 allergens of house-dust mites (Trombone et al. 2002; Hales et al. 2006) and Fel d 1 from the cat. The major group 2 and 5 allergens of the cockroach bind IgE with prevalences of 54 and 37% but the contribution increases to 70% in patients with high levels of anti-cockroach antibody (Satinover et al. 2005). It appears that for patients the contribution of IgE binding is about 50% of the combined and cockroach specificities. Amb a 1 from ragweed also accounts for an average of 50% of IgE binding to the pollen extract but unlike birch, grass and mite allergens, the percentage contribution of the anti-Amb a 1 IgE binding to total binding of the pollen extract varies within a continuum, ranging from 25 to 85% (Zeiss et al. 1978). Food allergy can also be directed to major allergens, with much of the response of milk being to the casein family (Shek et al. 2005). The major allergens of egg and peanut are not so dominant. Ovomucoid is the major allergen of egg especially with respect to the induction of allergic reactions (Bernhisel-Broadbent et al. 1994) but the allergens ovalbumin, ovotransferrin, and lysozyme induce significant IgE titers and can induce food allergy (Urisu et al. 1997). The major allergen of peanut is Ara h 2. It reacts with the IgE from nearly all peanut-allergic people (Astier et al.

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2006) but Ara h 1, Ara h 3, and related Ara h 4 as well as Ara h 2-related conglutinins like Ara h 6 (Koppelman et al. 2005) also have high IgE binding activity. The major allergen of bee venom, phospholipase A2 (PLA2), induces skin-test reactions and binds IgE in nearly all venom-allergic subjects (Muller et al. 1997), but from a quantitative perspective patients have substantial levels of IgE to other allergens (Muller et al. 1997) especially hyaluronidase and acid phosphatase (Kemeny et al. 1983). The amount of IgE antibody binding to the major allergens of birch, grass, and mite is about 50 ng/mL, while Amb a 1 has been reported as 20 ng/mL and the cockroach Bla g 2 and Bla g 5 a combined average of 10 ng/mL. Many people have low levels of IgE to cat so average anti-Fel d1 levels are about 4 ng/mL (Erwin et al. 2005a). However, some people have very high levels, 1–200 ng/mL being not uncommon (van Ree et al. 1999). Food allergens induce levels of IgE antibody similar to the inhalants as shown for the quantitative measures of anti-Ara h 2 from peanut (Hales et al. 2004) and from measurements with egg and milk preparations (Sampson & Ho 1997; Celik-Bilgili et al. 2005). The IgE directed to PLA2 in bee venom is found at about 5 ng/m compared with an average of 10 ng/mL IgE measured to unfractionated venom by Phadebas CAP (Muller et al. 1997). Bee sting thus induces lower amounts of IgE and systemic reactions can occur with very

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low, including undetectable, levels of IgE antibody (Golden et al. 2001). It should be noted that the CAP assay system is calibrated by anti-IgE coupled in saturating quantities to the CAP matrix. For valid measurements of anti-allergen antibody, the allergen must be conjugated to the CAP matrix in sufficient quantities to allow near-complete binding of the test IgE (Yman 2001). Valid assays can be conducted when allergens are provided in excess and the chemical coupling does not cause undue denaturation. When extracts are coupled to the matrix, it is unlikely that antigen excess is achieved for any except the most abundant allergens.

IgE and allergenicity IgE antibody titers provide an accurate and reproducible measure of allergic sensitization. However, their relationship with the ability of the allergen to induce a hypersensitivity reaction is not straightforward. The analysis of Witteman et al. (1996) with a panel of major allergens showed that people with the same skin-test reactivity to an allergen had a 10-fold range of IgE binding titers to the allergen, and Purohit et al. (2005) showed no correlation with basophil degranulation tests and IgE titer. As shown for grass pollen, the relationship between IgE binding and allergic responses can depend on the allergen. Nasal provocation and skin tests with grass pollen allergens showed that the major allergen Phl p 1 induced low in vivo responses despite its high IgE binding activity (Niederberger et al. 2001). The minor allergen Phl p 2, as defined by IgE binding, was far more allergenic in vivo. Recent studies indicate that the lower ability of Phl p 1 to induce tissue responses may be due to the fact that its IgE binding is restricted to a small region of the molecule and this could make cross-linking of IgE on mast cells difficult (Flicker et al. 2006). However, restricted binding is not a common feature of allergens. Studies using monoclonal antibodies to block IgE binding to Bet v 1 suggested that the P-loop region had high binding activity but the binding was also found in other regions (Holm et al. 2004). Similar analyses of Der p 1 (Chapman et al. 1987; Horn & Lind 1987) and Der p 2 (Mueller et al. 2001) show multiple binding regions. Immunoglobulin VH gene usage of IgE antibodies shows no evidence of restriction or lack of mutation (Eibensteiner et al. 2000), despite early reports of this with a small number of atopic dermatitis patients. An association between the severity of peanut allergy and the ability of IgE from the patients to bind to multiple peptides representing different sequences spanning the molecule has been noted (Shreffler et al. 2005). Since antibody binding to peptides typically only accounts for a small proportion of the IgE that can bind to a whole allergen, the immunologic significance is uncertain. It may be a surrogate measure of affinity or it is possible that the recognition of peptides by antibodies could be special for food allergy with respect to resistance to digestive enzymes. The ability of Ara

Host Responses to Allergens

h 1 and Ara h 2 peanut allergens to induce histamine release has been reported to differ, with Ara h 2 being able to induce 100-fold more basophil degranulation (Palmer et al. 2005). Both are multimeric proteins but Ara h 2 has some repeating sequences that could be important for cross-linking. However, the dangers of simplistic explanations are illustrated by the finding that Ara h 6, a conglutinin homolog of Ara h 2, induces less IgE antibody than Ara h 2 despite being produced in similar amounts in the peanut, but has the same ability to induce degranulation (Lehmann et al. 2006). IgE antibody can be produced to the carbohydrate determinants of allergens. This provides a diagnostic problem because IgE binding can cross-react across disparate species. As particularly noted for bee venom and peanut allergy, the IgE reactivity to carbohydrate is not associated with allergic disease (Altmann 2006). An explanation for this is that the reactivity is directed to N-linked glycans and most allergens are monovalent for these substitutions. Cross-linking of IgE receptors would therefore not be expected. A circumstantial case has nonetheless been made for possible importance in food allergy and it has been shown that Phl p 13, a grass pollen allergen with multiple glycans, can induce mediator release by binding to anti-carbohydrate antibodies (Wicklein et al. 2004).

Studies on the spectrum of IgE antiallergen responses Quantitative studies of IgE reactivity to panels of recombinant or purified pollen, cockroach, and mite allergens have been conducted. The use of panels of purified allergens instead of allergen extracts to measure IgE has shown that extracts underestimate the concentration of IgE in sera, presumably because of under-representation of many of the allergens. This was a striking eightfold increase for grass allergy even using a panel of allergens that did not include the important polygalacturonase group 13 allergen (Rossi et al. 2001; Mari 2003). Panels of cockroach and mite allergens have shown significant but less spectacular increases (Satinover et al. 2005; Hales et al. 2006). All the sera examined with the panel of mite allergens had IgE to several of the allergens even though the panel did not include the potentially important paramyosin, apolipophorin, and chitinase allergens (Thomas et al. 2002; Thomas & Hales 2007). In contrast to mite, not all the sera examined for IgE binding to the grass and cockroach panels bound one of the purified allergens. However, the titers of the nonbinding sera were low so it is possible that the binding detected by the extract was due to cross-reactivity and even possibly to carbohydrate determinants. Analyses with grass allergens showed that the proportion of people with IgE binding increased from 86 to 93% when the number of allergens examined was progressively increased from the two major allergens Phl p 1 and Phl

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p 5, to a panel of eight allergens. While it is possible that IgE binding in the absence of binding to the major allergens was cross-reactivity, the patients were recruited because they had seasonal grass pollen allergy. The fallibility of clinical assessments for the determination of the source of allergen in allergic disease is well documented (Williams et al. 2003) and so is the reproducibility of skin tests (Bodtger et al. 2003). The overall IgE antibody-binding pattern to arrays of grass allergens confirmed the high IgE binding to Phl p 1 and Phl p 5, with IgE binding to the nonmajor allergens in 10–70% of sera depending on the allergen, with no particular pattern or hierarchy in terms of the degree of response. Responses to the cross-reacting EF-hand calcium-binding proteins (Phl p 7 for grass and Bet v 4 for birch pollen) and to profilin (Phl p 12 for grass and Bet v 2 for birch) have the potential to mediate polysensitization to different pollens. About 25% of pollenallergic people make IgE to profilin but the titers are generally low (< 5 ng/mL) while the EF calcium binding induces IgE in fewer subjects but with high titers (Rossi et al. 2001). Multiple pollen sensitivity was not just associated with responses to the cross-reacting allergens. People with multiple sensitivities had higher IgE titers to all the grass pollen allergens compared with monosensitized and oligosensitized patients but they did not have more severe disease (Mari 2003). A comparative analysis of the IgE binding to a panel of 5 cockroach allergens showed a broader of range of IgE binding but with Bla g 2 and Bla g 5 binding IgE in 54 and 37% of the sera from cockroach allergic patients. The prevalence increased to 70 and 60% when the high titer sera were considered. The prevalence of binding to the other allergens ranged from 12– 26%. The less frequently recognized allergen could induce high titer IgE responses and there was no particular pattern of recognition. Indeed the study emphasized the individual patterns for each patient. 36% of sera did not bind any of the allergens tested but 75% of these had low titers. The study also used recombinant allergens that had not been validated for structure so this or the reactivity of the patients examined to other allergens could explain the lack of reactivity. A panel of nine house-dust mite allergens, comprising natural allergens or recombinant allergens with natural IgE binding activity, was found to detect IgE binding in all 182 mite-allergic subjects. All except 4% bound either Der p 1 or Der p 2 and those that did not only had low titers to other allergens. This differs from other investigations which reported that 20% of mite-allergic people did not have IgE to either of these allergens (Meyer et al. 1994; Trombone et al. 2002). While many of the non-Der p 1–Der p 2 binders had low titers, some were high (Trombone et al. 2002) and given the high prevalence of mite allergy this could represent a substantial number of people. The discrepancies may be the result of the geographic location of the studies. One was performed in Brazil where there is a rich acarofauna, including Dermatophagoides farinae, Blomia tropicalis and other nonDermatophagoides mites (Rizzo et al. 1997). Another was from

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Massachusetts in the USA where the mites are mainly D. farinae (Huss et al. 2001). Cross-reactivity of IgE binding to the major allergens of D. pteronyssinus and D. farinae is common but it can vary by 100-fold (Horn & Lind 1987). The majority of people studied by Hales and colleagues in Perth would have only been sensitized to D. pteronyssinus (Colloff et al. 1991). The pattern of IgE binding to the mite allergens differed from the binding pattern to grass and cockroach allergens by showing a distinct hierarchy (Hales et al. 2006). IgE to Der p 1 and Der p 2 constituted about 50% of the binding, while mid-potency allergens Der p 4, Der p 5, and Der p 7 each accounted for about 10%. The same hierarchy was found regardless of whether the total response to the mite allergens were high or low. IgE titers to the mid-potency allergens of 15 ng/mL were still high compared to allergens from other sources so they could be important specificities for hypersensitivity reactions. The responses to Der p 3, Der p 8, Der p 10, and Der p 20 were low despite a high prevalence of reactivity for some of them. IgE binding to Bet v 1, Bet v 2 and Bet v 4 of birch pollen has been systematically compared (Moverare et al. 2002; Rossi et al. 2003). As presented above for grass, the profilin and EF-hand calcium-binding protein allergens, Bet v 2 and Bet v 7, were of interest because of their cross-reactivity with related pollen allergens from disparate species. The very high reactivity, almost monoreactivity, of the IgE of patients in Scandinavian countries to Bet v 1 was confirmed, but sera from other countries had varying degrees of reactivity to the Bet v 2 and Bet v 4 allergens. In Italy, 11% of patients did not have IgE binding to any of the allergens, showing a potential importance of other specificities (Rossi et al. 2003). There are no quantitative comparisons of IgE binding to different cat allergens. It has long been known that there are several cat allergens that bind IgE at high frequency (Lowenstein et al. 1985; Mosimann et al. 1994) but they have not been investigated. Although absolute quantitation has not been used it is clear that cat albumin (Fel d 2) is a minor specificity that binds IgE in 15% of sera and is the dominant allergen in only 2% (van Ree et al. 1999). The comparative IgE binding of Fel d 3 is unknown. A study on the salivary lipocalin allergen Fel d 4 showed that half of the 70% of people with IgE to this allergen had higher titers than to Fel d 1 (Smith et al. 2004). The IgE titers were typically low but this needs to be viewed in the knowledge that IgE titers to Fel d 1 are low (Erwin et al. 2005a). It is possible that the specificity of cat allergy could be viewed differently when comparative studies are performed especially the patients that only have low titers of IgE to the cat dander extracts. There has not been a quantitative study of IgE binding to a panel of peanut allergens but it is clear that Ara h 1, Ara h 2, and Ara h 3 (and Ara h 4 which is almost identical to Ara h 3) are important. Ara h 2 binds IgE in nearly all peanut-allergic people and, depending on the study, 40% of people produce IgE to Ara h 1 and Ara h 3 with no particular pattern of

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reactivity (Astier et al. 2006). Ara h 1 binds a similar titer of IgE to Ara h 2 but is less potent in skin tests and degranulation (Palmer et al. 2005). Patients allergic only to Ara h 3 have been reported (Restani et al. 2005). Allergy to milk is dominated by IgE responses to α- and β-casein compared with κcasein and whey proteins α-lactalbumin and β-lactoglobulin. All allergic patients produce IgE to casein and only about 10% to α-lactalbumin and β-lactoglobulin (Shek et al. 2005). Ovomucoid, called Gad c 1, has been proposed as the dominant allergen of egg. Although accurate quantitative studies have not been done, IgE responses to the other allergens, ovalbumin and particularly ovotransferrin, are (as judged by RAST and ELISA binding) very similar to ovomucoid, and while lysozyme appears less reactive IgE can be readily detectable (Yamada et al. 2000). Egg allergy has a heterogeneous clinical presentation and it has been proposed that this could reflect different allergen recognition patterns (Walsh et al. 2005). It is clear that allergic reactions to ingested heatdenatured egg are due to ovomucoid, but half the allergic population do not make allergic responses to oral challenge with heat-treated egg, indicating they must respond predominantly to another allergen (Urisu et al. 1997). It might be expected that the diversity of the response to different allergens from the same source would have some relationship with the severity of disease. For example it has been reported that people with reactivity to more sources of allergen have more disease (Pallasaho et al. 2006; Tepas et al. 2006). The development of allergic disease is also correlated with the sum of IgE antibody measured to all sources of allergen (Simpson et al. 2005; Wickman et al. 2005). The same relationship does not appear to exist for the recognition of allergens within a source. Mari (2003) found no relationship with the diversity of IgE binding to grass allergens, including for subjects who recognize cross-reacting allergens for multiple pollen allergy. Studies with mites concurred with this (Pittner et al. 2004; Hales et al. 2006). Pittner et al. (2004) identified patients with complex allergen IgE binding pattern by Western blotting and showed that they also had a broader reactivity with recombinant allergens and that this extended to nonmite allergens and allergens of disparate mite species. Despite the broader pattern and increased IgE immunoglobulin, as also found by Mari in grass pollen allergy, there was no indication of poorer health. Hales et al. (2006) compared patients attending a hospital emergency department for asthma with those detected in a community sample. Not only was the spectrum of allergen recognized the same in each group but the responses of the acute asthma patients had a trend to be more directed to the major group 1 and 2 allergens. The study by Satinover et al. (2005) also noted that although asthma was more prevalent in patients with high titers to the cockroach allergen, it was not associated with the number of specificities recognized. Peanut allergy appears to be different because correlations with the number of specificities recognized in Western blotting assays (Lewis

Host Responses to Allergens

et al. 2005) and peanut allergy has been reported while IgE binding to extract has not been associated. However, more quantitative assays found that people monosensitized to Ara h 2 had less allergy than the polysensitized but their IgE titers were also much lower (Astier et al. 2006).

IgG antibody IgG antibodies to allergens are, with some notable exceptions, predominantly produced by people allergic to the allergens. This has been found for grass (Platts-Mills 1979), ragweed (Platts-Mills et al. 1976), mite (Miyamoto et al. 1981; Hales et al. 2006), and birch pollen (Harfast et al. 1998; Benson et al. 2003). The exceptions are the mammalian cat (Platts-Mills et al. 2001) and mouse (Matsui et al. 2006) allergens that induce IgG antibodies in most exposed people. It has been proposed for cat that this could be related to a high dose of allergen exposure compared with mite and pollen and that the high doses could tolerize for IgE and induce IgG antibody by an immune deviation phenomenon (Platts-Mills et al. 2001). Cat allergens are made airborne on balloon-like sebum particles from the sebaceous and anal glands. The amounts found in inhalable air in homes with cats has been measured as 50–100-fold more than that found for mite and pollen and even homes without cats have 10-fold more cat than mite or pollen allergen (Custis et al. 2003). However, mouse allergen exposure that also induces IgG is 50 times less than cat (Matsui et al. 2006) and the known major allergen is urinary protein that is not released in sebum particles. Other factors could be involved, such as an effect of the sebum particle on delivery through the mucosa or adjuvant molecules contained within the particle. The association of IgE and IgG antibody is also shown for the food allergens from peanut (Kolopp-Sarda et al. 2001), milk (Shek et al. 2005), and egg (Lilja et al. 1990). Bee venom is another allergen where IgG is produced in the absence of IgE (Garcia-Robaina et al. 1997). Allergic and nonallergic people, especially beekeepers, make high titers that increase with the number of stings and length of exposure. The lack of IgG antibody to pollen and mite allergens shows that people who do not become allergic either do not respond to the allergens or have responses that do not lead to significant antibody production. The results of investigations of IgG responses are not without a degree of between-studies variation. To take mite allergen as an example, some studies show a very marked difference between the sera from allergic and nonallergic subjects for all IgG subclasses measured (Hales et al. 2006), others only show an increase in the Th2dependent IgG4 isotype (Erwin et al. 2005b), and a minority have shown differences in only IgG1 or none at all. Some of the best discrimination has been obtained with solid-phase assays (Hales et al. 2006) so it is not a matter of measuring lower affinities by multipoint binding. IgG antibody is far

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more difficult to measure than IgE antibody, particularly IgG1 where the specific antibody response only constitutes about 0.1% of the 10 mg/mL of this isotype in serum. Protocols that minimize nonspecific binding while maintaining the signal need to optimized. It is essential that the assays titrate the antibody against standards, and also essential to use assays with a suitable and known dynamic range. Absolute quantitation is recommended because it shows how significant the responses are. As measured for bee and mite, IgG antibody titers are in the region of 10 μg/mL (Garcia-Robaina et al. 1997; Hales et al. 2006). This is of similar magnitude to antibody responses to immunization with pneumococcal conjugate vaccine (Edwards 2001) and tetanus toxoid (Vance et al. 1998) so they are biologically relevant titers. IgG titers to pollen are lower, at less than 1 μg/mL (Rossi & Monasterolo 2004; Jutel et al. 2005). Not all allergic people produce detectable IgG. The study of Hales et al. (2006) found IgG1 and IgG4 in the sera of 70% of mite-allergic children but only in the significantly less 40% of sera from adults. Further, only 25% of children admitted to an emergency department had IgG, suggesting a relationship with susceptibility to exacerbation. The spectrum of IgG binding specificities has been studied for mite, cockroach, and pollen allergens. For mites the major Der p 1 and Der p 2 allergens bound high titers of IgG1 and IgG4 antibody and the mid-strength allergens Der p 4, Der p 5, and Der p 7 bound IgG but in lower frequency and titer (Hales et al. 2006). The weak allergens Der p 3, Der p 8, Der p 10, and Der p 20 rarely bound IgG. This not only shows the relationship between IgE and IgG but also that the allergens which only induce low IgE responses do not induce Th1-skewed responses associated with IgG1. Cockroach allergen showed a slightly different relationship between IgE and IgG binding specificities (Satinover et al. 2005). The major allergen Bla g 5 bound the most IgG antibody but the minor Bla g 4 and Bla g 7 allergens also had high IgG titers including in sera that did not have IgE antibody. Sera with IgG binding to the Bla g 7 tropomyosin allergen also had IgG binding to the Der p 10 tropomyosin allergen of the house-dust mite. The same Der p 10 allergen rarely bound IgG from the sera of mite-allergic subjects so it appears that cockroach but not mite exposure can induce IgG to tropomyosin. IgG binding to Timothy grass allergens, which is low, was highest to Phl p 5 with anti-Phl p 1 responses being low (Rossi & Monasterolo 2004). The responses increased after immunotherapy mainly to Phl p 5 and to the allergens that had induced IgE in the sensitized individuals. However, Jutel et al. (2005) reported that immunotherapy induced anti-Phl p 5 IgG in people without IgE to this allergen. The IgG1 and IgG4 responses to Phl p 1 remained low even after immunotherapy with optimized doses of recombinant allergen. In a study of IgG subclass antibodies before and after pollen immunotherapy, Heiss et al. (1999) reported that IgG antibody binding to Phl p 1, Phl p 2 and Phl p 5, Bet v 1 and Bet v 2 was very variable and not correlated with IgE but the assays were not titrated.

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IgA antibody IgA antibodies are found in mucosal secretions of people with inhalant and food allergies but not nonallergic people. Allergen specificity has been demonstrated by measured IgA binding with purified major birch (Benson et al. 2003), cat (Bottcher et al. 2002), mite (Chapman & Platts-Mills 1978), and milk (Shek et al. 2005) allergens. Although IgA antibodies to purified peanut allergens have not been described, IgA antibodies reacting with peanut extract have been correlated with the presence of anti-peanut IgE (Kolopp-Sarda et al. 2001). An association between high levels of IgA in bronchoalveolar lavage and late-phase reactions has been described for ragweed extract (Peebles et al. 2001). Increased production of IgA antibody has been described for ragweed (Reed et al. 1991) and the Bet v 1 allergen (Keen et al. 2005) but this has not been universally found (Benson et al. 2003). An important fact about IgA is that like IgE much is produced locally (Shimoda et al. 2001), as shown by the presence of allergen or antigen-driven class-switch recombination and local somatic mutation in the mucosa (Fujieda et al. 1998). The specificity of the locally produced antibody appears to be a significant factor in rhinoconjunctivitis to grass and pollen allergens. While the IgE in tears was very similar to serum IgE, IgA antibodies are directed to different specificities and thus may not have a direct blocking effect (HoffmannSommergruber et al. 1996). Perhaps related to local production, IgA antibodies have been found to inhalant antibodies in milk regardless of allergic status (Casas et al. 2000) so this appears to be different to other secretions.

T-cell effector responses T-cell responses to allergens have been studied in vivo by examining biopsies and lavages after bronchial or skin-test challenge with allergen, and in vitro by tissue explants, T-cell cloning, and allergen-stimulated responses of peripheral blood mononuclear cells (PBMC). Much of the work in this field has been conducted with allergen extracts and not allergens. This not only ignores the important effects of allergen doses on T-cell responses but also that allergen extracts contain mitogenic and inflammatory substances. It is possible that pollen extracts may resemble the pollen grain dissolved in mucosal fluids but people do not inhale house-dust mite, cockroach, and cat dander extracts. T cells are not only required for antibody responses but also appear to have a more direct role in the pathophysiology of allergic reactions (Larche et al. 2003). However, the extent to which this occurs is unknown. Late-phase reactions in asthma, which are accompanied or caused by a mixed cellular infiltrate including eosinophils and T cells, are produced by people with higher T-cell reactivity as shown by in vitro

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responses of PBMC to house-dust mite extracts (Burastero et al. 1993; van Neerven et al. 1999). However, they can at least for skin reactions, be induced by the injection of antiIgE antibodies, indicating that T cells are not absolutely required. Anti-IgE therapy also removes late-phase reactions, although this could act via IgE antibody-facilitated antigen presentation to T cells. The studies of Haselden et al. (2001) provide direct evidence for T-cell involvement by showing that allergic patients injected with peptides representing the T-cell epitopes of Fel d 1 developed late-phase bronchoconstriction. The reactions were not associated with any increase in inflammation or T-cell infiltration into the airways. The histopathology of lung biopsies of asthmatics, in contrast to that of healthy individuals, shows the presence of T cells producing Th2 cytokines in the airways walls. The same difference is not found for interferon (IFN)-γ-producing cells, which are low for both normal and asthmatic people (Larche et al. 2003). Natural exposure to allergen, as examined by biopsies taken at the start of the pollen season, increases the infiltration of memory and activated T cells shown by interleukin (IL)-2R-bearing cells, the release of soluble IL-2R, and human leukocyte antigen (HLA)-DR staining. Increased numbers of eosinophils in the airway walls and IL-5-producing cells can also be demonstrated. There are many studies on the inflammatory responses induced by bronchial challenge with allergen extracts. Challenge induces a rapid infiltration of dendritic cells into the bronchial mucosa and a decrease in number of allergen-reactive T cells in the peripheral blood (Borgonovo et al. 1997). The infiltration of T cells producing Th2 cytokines and the Th2 master transcription factor GATA3 can then be demonstrated (Erpenbeck et al. 2006; Larche et al. 2003), paralleled by an eosinophilic infiltrate (Teran et al. 1999). The same events occur in the upper airways with nasal provocation of allergic rhinitis patients (Nakamura et al. 2000). Not all bronchial challenge studies have used allergen extracts. T-cell responses following bronchial challenge with major allergens have been demonstrated for the Der p 1 and Der p 2 house-dust mite allergens (Van Der Veen et al. 2001). They induced early asthmatic reactions, increased serum IL-5, and late-phase reactions. The IL-5 and late responses induced by the major allergens were less than those produced with a dose of house-dust mite extract that induced the same immediate reactions. This possibly indicated that other allergens or other substances in the extract were important, although it may result from different dose–response characteristics of early and late reactions. PBMC provide a convenient source of T cells to study in vitro responses. The fact that bronchial challenge depletes the allergen-responsive cells (Burastero et al. 1993) indicates that they are the cells that infiltrate the lungs. T-cell cloning from this source has definitively established that both allergic and nonallergic people have allergen-specific T cells. The precursor frequencies of T cells responding to defined allergens have not been examined but pollen (Assing et al. 2006) and house-

Host Responses to Allergens

dust mite (Burastero et al. 1993; Richards et al. 1997) extracts have been studied. Frequencies of 0.05–0.1% for allergic subjects and 0.01–0.02% for nonallergic subjects were reported. Analyses of responses of unimmunized people to other antigens show frequencies in the region of 0.001% (Ford & Burger 1983; Gabaglia et al. 2000; Avanzini et al. 2001), which rise to about 0.02% after vaccinations (Ford & Burger 1983; Avanzini et al. 2001). The precursor frequencies found in nonallergic subjects are thus indicative of a considerable amount of expansion in the absence of allergic sensitization. The allergen-responsive T cells of allergic subjects are mainly in the memory CD45RO+ population compartment (Richards et al. 1997), while nonallergic subjects show both CD45RO+ and CD45RA+ cells (Richards et al. 1997). The induction of the Th2 transcription factor GATA-3 by house-dust mite extract has also been shown to be stronger in CD45RO+ cells (Macaubas et al. 2002). T-cell responses to the major allergens from common sources such as grass (van Neerven et al. 1999; Burton et al. 2002), birch (Ebner et al. 1995), weeds (Eisenbrey et al. 1985; Jahn-Schmid et al. 2005), mite (O’Brien et al. 1992), cat (Reefer et al. 2004), milk (Ruiter et al. 2006), egg (Cooke & Sampson 1997), and peanut (Bannon et al. 2001; Turcanu et al. 2003; Glaspole et al. 2005) have been studied. In general the proliferative responses of PBMC from allergic people are better than those of cells from nonallergic people, although there is considerable overlap. The induction of the Th2 cytokines IL-5 and IL-13 can be readily detected. The measurement of IL-4 from primary cultures must use ultrasensitive assays (Simons et al. 2004) or inferred from measuring RNA transcripts. Some investigators induce higher levels of cytokine release by multiple stimulation, either with repeated doses of allergen or by allergen and then polyclonal stimulators such as phorbol myristate acetate (Akdis et al. 2004), but the results are open to question with respect to the specificity and accumulation of tissue-culture artifacts. The discovery that T-cell lines or clones cultured from allergic people produced Th2-biased cytokines and that the clones from nonallergic subjects were Th1-biased was an important observation, showing that Th1 and Th2 polarization could occur in human cells (Parronchi et al. 1991; Wierenga et al. 1991). It is likely that much of the effect is due to the strong in vitro polarizing activity of IL-4. When the responses of T cells are measured without extended culture, most studies with mite and pollen allergen stimulation show that cells from allergic and nonallergic subjects produce similar amounts of IFN-γ (Akdis et al. 2004) or there is considerable overlap (Li et al. 1996). Other studies even show increased IFN-γ production by cells from allergic subjects (O’Brien et al. 2000; Smart & Kemp 2002; Heaton et al. 2005). The level of IFN-γ induced by allergen is similar to that induced by classical Th1-type bacterial antigens and vaccines (Holt et al. 2000; Epton et al. 2002; Laaksonen et al. 2003). Similar increased IFN-γ production, as well as Th2 cytokine production, has been reported

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for PBMC of egg-allergic people stimulated with the food allergen ovalbumin (Smart & Kemp 2002) or with milk allergens from people with persistent cows’ milk allergy (Tiemessen et al. 2004). However, decreased IFN-γ production has been reported for egg, milk, and peanut allergens in children with multiple allergies (Scott-Taylor et al. 2005). A study of primary responses to peanut extract by Thottingal et al. (2006) found that PBMC from both allergic and non-allergic people released Th2 responses and that the extract rarely induced IFN-γ. There was only a tendency for the PBMC from allergic people to make more Th2 cytokines but the results imply that peanut extract had a special Th2-inducing activity regardless of the allergic status of the donor. However, a previous study with multiple stimulations with peanut extracts and Ara h 2 had reported the induction of Th2-biased responses induced from PBMC of allergic but not nonallergic subjects (Turcanu et al. 2003). The cytokine bias of allergen-responsive T cells is also indicated by their chemokine receptors and chemokines found in vivo. Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their respective ligands, eotaxin (CCL11), monocyte-derived chemokine (MDC) (CCL22), and thymus- and activation-regulated chemokine (TARC) (CCL17). Bronchial lavages of asthmatics taken without allergen challenge show the presence of CCR4+CD4+ cells and their ligands TARC and MDC (Hartl et al. 2005). When endobronchial biopsies were taken after allergen challenge, virtually all T cells expressed IL-4 and CCR4 with some coexpression of CCR8 and epithelial cells produced MDC and TARC, suggesting a role in lymphocyte recruitment (PaninaBordignon et al. 2001). Panina-Bordignon contrasted the findings in asthma to the T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and sarcoidosis, which produce IFN-γ and express high levels of CXCR3, while lacking CCR4 and CCR8. Nevertheless, it does appear from other studies that bronchial challenge with allergen can induce the Th1-associated chemokines as well as the Th2 chemokines and that they may be important. Segmental lung challenge with ragweed, house-dust mite, and cat extracts has been shown to induce the Th1-type chemokine IP-10 (Bochner et al. 2003; Liu et al. 2004) and demonstrated that patients with late-phase reactions to allergen challenge produce more of both the Th1 and Th2 chemokine (Liu et al. 2004). PBMC also show responses that reflect chemokine bias. Notably, PBMC stimulated with grass extract demonstrate proliferation of CCR4+ T cells, but only in cultures from allergic and not nonallergic subjects, in keeping with the Th2 phenotype of allergy (Assing et al. 2006). CCR4 could be detected on 40% of the responding T cells. T cells from PBMC of allergic subjects also produce the Th2 chemoattractants TARC and MDC (Simons et al. 2004; Thottingal et al. 2006) in greater quantity than PBMC from nonallergic people. CD4+ cells are the major source of Th2 cytokines in PBMC (Till et al. 1997) and predominate in the cellular infiltrates

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of allergic hypersensitivity reactions (Frew & Kay 1988). However, the Der p 1 allergen has also been shown to stimulate the release of IFN-γ from CD8+ cells, although in in vitro cultures this required IL-2 supplementation (O’Brien et al. 2000). The amounts released were similar to the release from unfractionated cultures. Th2 cytokine production could not be detected. The Der p 1-specific CD8 cells have also been detected by isolating T cells with a major histocompatibility complex (MHC) class I-binding peptide tetramer (Seneviratne et al. 2002).

T-cell recognition The T-cell epitopes of many major allergens have been studied, including Bet v 1, Phl p 1 and Phl p 5, Lol p 1 and Lol p 5, Der p 1 and Der p 2, Der f 1, Amb a 1, Art v 1, Fel d 1, and PLA2 (van Neerven et al. 1996; Thomas & Hales 2007). For the most part, both allergic and nonallergic people respond to an array of epitopes with no special pattern. Early studies on Amb a 5 had pointed to HLA-DR gene control of responses (Marsh et al. 1982). However, Amb a 5 is a minor allergen with a sequence of only 45 amino acids so it is a good candidate for a limited pattern of MHC restriction. There are many reports in the literature about HLA class II associations with either asthma or immune responses to highly purified allergens but they are not consistent from study to study (Thomas & Hales 2007). Whole-genome analysis also does not show an association of allergic disease with the HLA region, although there have been reports of an effect of tumor necrosis factor (TNF)α polymorphism, a gene also found in this region, on asthma (Blumenthal et al. 2006). A feature of most of the common major allergens is that they show enormous sequence variation by being members of gene families or being products of highly polymorphic alleles or both, and by the coexistence of allergens from related species. Bet v 1, for example, is part of the PR-10 multigene family, which is produced in pollen as products from at least seven different genes (Schenk et al. 2006). On top of this the allergens from other Fagales trees including different birch species, alder, hazel, and chestnut show about 80–85% identity, with varying degrees of antibody and T-cell cross-reactivity. Plants have a great deal of heteroploidy, up to octoploidy, so even within the one tree there can be great sequence variation. The weed allergen Amb a 1 consists of four different isoallergens with about 70% identity to each other and to Amb a 2, which also belongs in this family (Wopfner et al. 2005). The group 5 grass allergens have two to four different genes with 70–80% sequence identity, and while the group 1 allergens show less variation, the group 2 and 3 allergens have related sequences (Andersson & Lidholm 2003). Mite allergens are single gene products but different cDNA clones typically have 5% sequence variation (Smith et al. 2001; Piboonpocanun et al. 2006). The mites D. farinae and D. pteronyssinus coexist in many countries, although

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D. pteronyssinus can be very dominant in some regions such as England and Australia (Colloff et al. 1991). With this variation it would seem unlikely that a restriction in the number of MHC-binding peptides would be a limiting factor for immune responses. Some special cases of T-cell recognition have been identified. Firstly, responses to the major mugwort allergen Art v 1 are strongly linked to the HLA-DRB1*01 allele and the T cells from the patients recognize the same immunodominant peptide presented by this allele (Jahn-Schmid et al. 2005). Such a clear-cut MHC restriction is the exception and it is possible that the heavily O-glycosylated nature of Art v 1 limits processing of the allergen by antigen-presenting cells and restricts presentation. Another preferential recognition has been found for Der p 1. Peptides in the central domainconnecting loop preferentially stimulate T-cell responses (Higgins et al. 1994; Hales & Thomas 1997; Kircher et al. 2002) and the responding T cells have a biased use of T-cell receptor Vβ18.1 (Wedderburn et al. 1993; Kircher et al. 2002). The responses in the region are not restricted to one epitope, and allergen presentation by DR, DP, and DQ alleles have been demonstrated (Higgins et al. 1994). The T-cell responses of wasp-allergic subjects to the Ves v 5 allergen has been shown to be strongly directed to a particular epitope in over 50% of patients and this allergen was not recognized by T cells from nonallergic subjects (Bohle et al. 2005). The responders all had the HLA-DR3*0202 allele but this allele was also found in nonresponders, showing that MHC restriction was not the only determinant of the response. Woodfolk and colleagues found concordance between modified Th2 responses to the cat allergen Fel d 1 and the HLA-DRB1*0701 allele. T cells from people with this allele made strong IL-10 responses to a peptides representing the residues 1–24 of chain 2 (Reefer et al. 2004). Patients with atopic dermatitis did not produce T-cell responses to this epitope, indicating a possible lack of epitope-specific immunoregulation (Carneiro et al. 2004). The same authors found that patients with Th1 or Th2 responses to the Trichophyton rubrum antigen Tri r 2 recognized different epitopes (Woodfolk et al. 1998). These epitope-specific cytokine responses are interesting but remain uncorroborated. Previous studies have reported the absence of epitope-specific cytokine bias (van Neerven et al. 1994) but were based on T-cell clones that would have had their cytokine profile changed by nonphysiologic in vitro polarization.

T-cell regulatory responses The induction of regulatory responses by allergens is a likely mechanism for controlling allergic responses. However, the knowledge of such responses is in its infancy. The ability of IL-10 to inhibit Th1 and Th2 immune responses in experimental animals and in vitro responses of human cells is well documented (Taylor et al. 2006a). Several investigations have

Host Responses to Allergens

also shown that T cells from PBMC produce more IL-10 after immunotherapy (Taylor et al. 2006a). While it is reasonable to propose that a similar mechanism might operate in the healthy nonallergic response, IL-10 production in standard in vitro cultures of allergen-stimulated PBMC from such subjects has frequently been found to be decreased compared with atopics. This has been shown for patients allergic to house-dust mite allergens (Macaubas et al. 1999; Hales et al. 2002; Heaton et al. 2005) as well as cat and pollen allergens (Matsumoto et al. 2002). More IL-10 can also be found in bronchial and skin challenge sites of house-dust mite-allergic people, as indicated by mRNA transcription (Robinson et al. 1996). However, it has been reported that PBMC from nonallergic children produce more IL-10 than those of allergic children when stimulated with cat or birch allergen. Bullens and colleagues found no difference in the responses of allergic and nonallergic subjects with either birch (Bullens et al. 2004) or Der p 2 (Bullens et al. 2005) but found that the addition of IL-4 to Bet v 1-stimulated cultures increased the IL-10 response of PBMC from nonallergic but not allergic subjects. This raises the possibility that T cells of allergic subjects produce regulatory IL-10 if they attempt to mount a Th2 response. There are indications that although IL-10 production can be increased in allergy, it inhibits hypersensitivity reactions and disease. There are several examples. T cells from people with cows’ milk allergy produce much larger quantities of IL-10 than cells from nonallergic people, but people who do not develop disease produce the highest amounts (Tiemessen et al. 2004). A similar indication for a regulatory role for IL-10 was also evident from two independent studies of house-dust mite allergy. Although allergen-induced IL-10 production by T cells was increased in allergic subjects, there was a negative correlation between IL-10 and the size of the wheal induced by skin-prick testing the donors (Macaubas et al. 1999; Heaton et al. 2005). The possibility that IL-10 might also regulate the responses of cells from healthy individuals has been demonstrated by experiments where the addition of anti-IL-10 receptor antibodies to cultures of PBMC from nonallergic subjects enhanced proliferative responses to Der p 1 (Jutel et al. 2003). A follow-up showed that unlike previous reports allergenstimulated cultures from nonallergic people had more IL10-producing T cells than cultures from allergic subjects. Perhaps importantly, this was measured 12 hours after stimulation with allergen (Akdis et al. 2004). The IL-10producing cells had poor proliferative activity unless one of the cytokines IL-2, IL-4, IL-7 or IL-15 was added. Nonallergic people produce less of these cytokines so the IL-10producing cells may not be observable in the 5–6 day culture periods used in the standard cytokine-producing cell assays. The regulatory activity of IL-10 was first noted by its ability to inhibit IFN-γ production. In nickel atopic dermatitis, increasing doses of IL-10 have been shown to first inhibit the Th1 and then the Th2 response to PBMC cultures (Minang

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et al. 2006). From these observations, the effect of IL-10 remains uncertain because it is likely to be critically dependent on the dose and timing and the presence of other cytokines and regulatory elements. Suppressive effects of CD4+CD25+ T cells have been demonstrated in in vitro cultures of PBMC stimulated with cat and pollen allergens, although no easily interpretable difference has been demonstrated in the activity of cells from allergic and nonallergic subjects (Bellinghausen et al. 2003; Ling et al. 2004). Several early investigations observed increased numbers of CD4+CD25+ cells in allergen-stimulated culture of PBMC from allergic people or after allergen challenge of bronchial tissue (Chang et al. 1996). These were interpreted as a marker of effector T-cell activation but these conclusions could be revisited in the light of the more recent knowledge of regulatory cells. Similarly, studies of atopic dermatitis showed that house-dust mite extract stimulated more of the regulatory cell transcription factor FOXP3 from PBMC from house-dust mite-allergic subjects than PBMC from nonallergic subjects (Taylor et al. 2006b). It is possible these effects are linked to increased IL-2 production by the higher responses of allergic subjects. The studies on induction of FOXP3 by allergen have only examined allergen extracts so it not known if allergens themselves induce the regulatory effects and how this relates to allergenicity.

Conclusions The hallmark of an allergic response is the production of IgE antibody. While most of the common sources of allergen contain many specificities, the majority of the IgE binding in sera is typically directed to one or two major allergens. This is particularly true for birch and grass pollen, house-dust mite, and milk and probably for cat. The major Bla g 2 and Bla g 5 allergens of the cockroach show a similar dominance for most patients. IgE binding to ragweed, peanut, bee venom, and egg is reportedly less focused but major specificities have been identified. The major allergens induce an average of around 20–50 ng/mL of IgE antibody, a known exception being the cat. Fel d 1 can induce very high titers of antibody but the average level for cat-allergic people is about 4 ng/mL. Even for other allergens the lower limit of IgE antibody associated with allergic disease is small, with many asthmatics showing less than a few nanograms per milliliter of antibody. People with undetectable levels of IgE antibody make anaphylactic reactions to bee sting. The production of IgG antibody to pollen, food, mite and most cockroach allergens is associated with IgE antibody. The lack of allergic responses in nonallergic people to these is therefore not the result of immune deviation to IgG production. In contrast, cat and mouse allergens induce IgG antibody in the absence of IgE in a phenomenon that has been proposed to be a high-dose immune deviation. The high dose certainly applies for the

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Fel d 1 cat allergen made airborne on sebum particles but the mouse allergen is a urinary protein released in smaller amounts. Allergens within the one source can also differ in their IgG-inducing ability, with the minor cockroach allergen Bla g 7 binding IgG in high frequency in sera without IgE antibodies. As shown for mites, the IgG responses can be as high as those induced by strong bacterial antigens. Where measured, the precursor frequency of allergen-responsive T cells in the blood is also similar to that found after vaccination with protein antigens. The frequencies are higher for allergic than nonallergic subjects but even nonallergic subjects show evidence of considerable T-cell expansion in response to in vitro allergen challenge. The signature response of T cells from allergic subjects to allergen is the production of Th2 cytokines. The strong and rapid polarizing effect of exposure to IL-4 in lymphocyte culture often results in low IFN-γ production from even short-term T-cell lines and has given the impression of low Th1 responses to allergens. When measured without extended culture, PBMC from allergic subjects produce similar, or even greater, levels of IFN-γ than nonallergic subjects and the levels are high compared with those induced by bacterial antigens in similar assays. Chemokine production can be measured from T cells of allergenstimulated PBMC, and these studies have also shown that allergic but not nonallergic subjects produce the Th2 chemokines TARC and MDC; allergic subjects can make similar Th1 chemokine responses as shown by IP-10 production. The allergen-responsive T cells of allergic subjects display the Th2-regulated CCR4 chemokine receptor indicative of ongoing Th2 responses to inhaled allergen. The regulatory cytokine IL-10 is produced in responses of T cells to allergen and, in standard in vitro allergen stimulation assays of PBMC, it is reportedly released in higher amounts by cells from allergic subjects. However, some studies have demonstrated the T cells from healthy people produce a higher early IL-10 response with possible regulatory functions. In particular, studies with mite and milk allergens suggest that allergic subjects produce more IL-10 but that the level of production is inversely correlated with hypersensitivity responses to natural exposure or skin test. Evidence for allergen-induced CD4+CD25+ T regulatory cells has been revealed but, like IL10, there is evidence for higher activity in allergic responses, and evidence for the regulation of responses of cells from both allergic and nonallergic subjects.

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fluid are associated with stronger antigen-induced late phase reactions. Clin Exp Allergy 31, 239– 48. Piboonpocanun, S., Malainual, N., Jirapongsananuruk, O., Vichyanond, P. & Thomas, W.R. (2006) Genetic polymorphisms of major house dust mite allergens. Clin Exp Allergy 36, 510–16. Pittner, G., Vrtala, S., Thomas, W.R. et al. (2004) Component-resolved diagnosis of house-dust mite allergy with purified natural and recombinant mite allergens. Clin Exp Allergy 34, 597–603. Platts-Mills, T.A. (1979) Local production of IgG, IgA and IgE antibodies in grass pollen hay fever. J Immunol 122, 2218–25. Platts-Mills, T.A., von Maur, R.K., Ishizaka, K., Norman, P.S. & Lichtenstein, L.M. (1976) IgA and IgG anti-ragweed antibodies in nasal secretions. Quantitative measurements of antibodies and correlation with inhibition of histamine release. J Clin Invest 57, 1041–50. Platts-Mills, T., Vaughan, J., Squillace, S., Woodfolk, J. & Sporik, R. (2001) Sensitisation, asthma, and a modified Th2 response in children exposed to cat allergen: a population-based cross-sectional study. Lancet 357, 752– 6. Purohit, A., Laffer, S., Metz-Favre, C. et al. (2005) Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to Fc epsilon RI. Clin Exp Allergy 35, 186– 92. Reed, C.E., Bubak, M., Dunnette, S. et al. (1991) Ragweed-specific IgA in nasal lavage fluid of ragweed-sensitive allergic rhinitis patients: increase during the pollen season. Int Arch Allergy Appl Immunol 94, 275– 7. Reefer, A.J., Carneiro, R.M., Custis, N.J. et al. (2004) A role for IL-10–mediated HLA-DR7-restricted T cell-dependent events in development of the modified Th2 response to cat allergen. J Immunol 172, 2763– 72. Restani, P., Ballabio, C., Corsini, E. et al. (2005) Identification of the basic subunit of Ara h 3 as the major allergen in a group of children allergic to peanuts. Ann Allergy Asthma Immunol 94, 262–6. Richards, D., Chapman, M.D., Sasama, J., Lee, T.H. & Kemeny, D.M. (1997) Immune memory in CD4+ CD45RA+ T cells. Immunology 91, 331–9. Rizzo, M.C., Fernandez-Caldas, E., Sole, D. & Naspitz, C.K. (1997) IgE antibodies to aeroallergens in allergic children in Sao Paulo, Brazil. J Invest Allergol Clin Immunol 7, 242– 8. Robinson, D.S., Tsicopoulos, A., Meng, Q., Durham, S., Kay, A.B. & Hamid, Q. (1996) Increased interleukin-10 messenger RNA expression in atopic allergy and asthma. Am J Respir Cell Mol Biol 14, 113–17. Rodriguez, R., Villalba, M., Batanero, E. et al. (2002) Allergenic diversity of the olive pollen. Allergy 57 (suppl. 71), 6–16. Rossi, R.E. & Monasterolo, G. (2004) Evaluation of recombinant and native timothy pollen (rPhl p 1, 2, 5, 6, 7, 11, 12 and nPhl p 4)specific IgG4 antibodies induced by subcutaneous immunotherapy with timothy pollen extract in allergic patients. Int Arch Allergy Immunol 135, 44– 53. Rossi, R.E., Monasterolo, G. & Monasterolo, S. (2001) Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen. Allergy 56, 1180– 5. Rossi, R.E., Monasterolo, G. & Monasterolo, S. (2003) Detection of specific IgE antibodies in the sera of patients allergic to birch pollen

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using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles. Allergy 58, 929–32. Ruiter, B., Tregoat, V., M’Rabet, L. et al. (2006) Characterization of T cell epitopes in alphas1-casein in cow’s milk allergic, atopic and non-atopic children. Clin Exp Allergy 36, 303–10. Sampson, H.A. & Ho, D.G. (1997) Relationship between foodspecific IgE concentrations and the risk of positive food challenges in children and adolescents. J Allergy Clin Immunol 100, 444–51. Satinover, S.M., Reefer, A.J., Pomes, A., Chapman, M.D., PlattsMills, T.A. & Woodfolk, J.A. (2005) Specific IgE and IgG antibodybinding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 115, 803–9. Schenk, M.F., Gilissen, L.J., Esselink, G.D. & Smulders, M.J. (2006) Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen. BMC Genomics 7, 168. Scott-Taylor, T.H., Hourihane, J.B., Harper, J. & Strobel, S. (2005) Patterns of food allergen-specific cytokine production by T lymphocytes of children with multiple allergies. Clin Exp Allergy 35, 1473–80. Seneviratne, S.L., Jones, L., King, A.S. et al. (2002) Allergen-specific CD8(+) T cells and atopic disease. J Clin Invest 110, 1283–91. Shek, L.P., Bardina, L., Castro, R., Sampson, H.A. & Beyer, K. (2005) Humoral and cellular responses to cow milk proteins in patients with milk-induced IgE-mediated and non-IgE-mediated disorders. Allergy 60, 912–19. Shimoda, M., Nakamura, T., Takahashi, Y. et al. (2001) Isotypespecific selection of high affinity memory B cells in nasalassociated lymphoid tissue. J Exp Med 194, 1597–607. Shreffler, W.G., Lencer, D.A., Bardina, L. & Sampson, H.A. (2005) IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2. J Allergy Clin Immunol 116, 893–9. Simons, F.E., Shikishima, Y., Van Nest, G., Eiden, J.J. & HayGlass, K.T. (2004) Selective immune redirection in humans with ragweed allergy by injecting Amb a 1 linked to immunostimulatory DNA. J Allergy Clin Immunol, 113, 1144–51. Simpson, A., Soderstrom, L., Ahlstedt, S., Murray, C.S., Woodcock, A. & Custovic, A. (2005) IgE antibody quantification and the probability of wheeze in preschool children. J Allergy Clin Immunol. 116, 744– 9. Smart, J.M. & Kemp, A.S. (2002) Increased Th1 and Th2 allergeninduced cytokine responses in children with atopic disease. Clin Exp Allergy 32, 796–802. Smith, W., Butler, A.J., Hazell, L.A. et al. (2004) Fel d 4, a cat lipocalin allergen. Clin Exp Allergy 34, 1732–8. Smith, W.A., Hales, B.J., Jarnicki, A.G. & Thomas, W.R. (2001) Allergens of wild house dust mites: environmental Der p 1 and Der p 2 sequence polymorphisms. J Allergy Clin Immunol 107, 985–92. Taylor, A., Verhagen, J., Blaser, K., Akdis, M. & Akdis, C.A. (2006) Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells. Immunology 117, 433–42. Taylor, A.L., Hale, J., Hales, B.J., Dunstan, J.A., Thomas, W.R. & Prescott, S.L. (2007) FOXP3 mRNA expression at 6 months of age is not affected by giving probiotics from birth, but is higher in infants who develop atopic dermatitis. Pediatr Allergy Immunol 18, 10–19. Tepas, E.C., Litonjua, A.A., Celedon, J.C., Sredl, D. & Gold, D.R. (2006) Sensitization to aeroallergens and airway hyperresponsiveness at 7 years of age. Chest 129, 1500–8.

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Teran, L.M., Carroll, M.P., Shute, J.K. & Holgate, S.T. (1999) Interleukin 5 release into asthmatic airways 4 and 24 hours after endobronchial allergen challenge: its relationship with eosinophil recruitment. Cytokine 11, 518– 22. Thomas, W.R. & Hales, B.J. (2007) T and B-cell responses to HDM allergens and antigens. Immunol Res 37, 187–99. Thomas, W.R., Smith, W.A., Hales, B.J., Mills, K.L. & O’Brien, R.M. (2002) Characterization and immunobiology of house dust mite allergens. Int Arch Allergy Immunol 129, 1–18. Thottingal, T.B., Stefura, B.P., Simons, F.E., Bannon, G.A., Burks, W. & HayGlass, K.T. (2006) Human subjects without peanut allergy demonstrate T cell-dependent, TH2-biased, peanut-specific cytokine and chemokine responses independent of TH1 expression. J Allergy Clin Immunol 118, 905–14. Tiemessen, M.M., Van Ieperen-Van Dijk, A.G., Bruijnzeel-Koomen, C.A., Garssen, J., Knol, E.F. & Van Hoffen, E. (2004) Cow’s milkspecific T-cell reactivity of children with and without persistent cow’s milk allergy: key role for IL-10. J Allergy Clin Immunol 113, 932– 9. Till, S., Durham, S., Dickason, R. et al. (1997) IL-13 production by allergen-stimulated T cells is increased in allergic disease and associated with IL-5 but not IFN-gamma expression. Immunology 91, 53–7. Trombone, A.P., Tobias, K.R., Ferriani, V.P. et al. (2002) Use of a chimeric ELISA to investigate immunoglobulin E antibody responses to Der p 1 and Der p 2 in mite-allergic patients with asthma, wheezing and/or rhinitis. Clin Exp Allergy 32, 1323–8. Turcanu, V., Maleki, S.J. & Lack, G. (2003) Characterization of lymphocyte responses to peanuts in normal children, peanut-allergic children, and allergic children who acquired tolerance to peanuts. J Clin Invest 111, 1065– 72. Urisu, A., Ando, H., Morita, Y. et al. (1997) Allergenic activity of heated and ovomucoid-depleted egg white. J Allergy Clin Immunol 100, 171– 6. Vance, E., George, S., Guinan, E.C. et al. (1998) Comparison of multiple immunization schedules for Haemophilus influenzae type b-conjugate and tetanus toxoid vaccines following bone marrow transplantation. Bone Marrow Transplant 22, 735–41. Van Der Veen, M.J., Jansen, H.M., Aalberse, R.C. & van der Zee, J.S. (2001) Der p 1 and Der p 2 induce less severe late asthmatic responses than native Dermatophagoides pteronyssinus extract after a similar early asthmatic response. Clin Exp Allergy 31, 705–14. van Neerven, R.J., van de Pol, M.M., Wierenga, E.A., Aalberse, R.C., Jansen, H.M. & Kapsenberg, M.L. (1994) Peptide specificity and HLA restriction do not dictate lymphokine production by allergenspecific T-lymphocyte clones. Immunology 82, 351–6. van Neerven, R.J., Ebner, C., Yssel, H., Kapsenberg, M.L. & Lamb, J.R. (1996) T-cell responses to allergens: epitope-specificity and clinical relevance. Immunol Today 17, 526–32. van Neerven, R., Wissenbach, M., Ipsen, H., Bufe, A., Arnved, J. &

Host Responses to Allergens

Wurtzen, P.A. (1999) Differential recognition of recombinant Phl p 5 isoallergens by Phl p 5-specific T cells. Int Arch Allergy Immunol 118(2–4), 125–8. van Ree, R., van Leeuwen, W.A., Bulder, I., Bond, J. & Aalberse, R.C. (1999) Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 104, 1223–30. Walsh, B.J., Hill, D.J., Macoun, P., Cairns, D. & Howden, M.E. (2005) Detection of four distinct groups of hen egg allergens binding IgE in the sera of children with egg allergy. Allergol Immunopathol (Madr) 33, 183–91. Wedderburn, L.R., O’Hehir, R.E., Hewitt, C.R., Lamb, J.R. & Owen, M.J. (1993) In vivo clonal dominance and limited T-cell receptor usage in human CD4+ T-cell recognition of house dust mite allergens. Proc Natl Acad Sci USA 90, 8214–18. Wicklein, D., Lindner, B., Moll, H. et al. (2004) Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens. Biol Chem 385, 397–407. Wickman, M., Lilja, G., Soderstrom, L., van Hage-Hamsten, M. & Ahlstedt, S. (2005) Quantitative analysis of IgE antibodies to food and inhalant allergens in 4-year-old children reflects their likelihood of allergic disease. Allergy 60, 650–7. Wierenga, E.A., Snoek, M., Jansen, H.M., Bos, J.D., van Lier, R.A. & Kapsenberg, M.L. (1991) Human atopen-specific types 1 and 2 T helper cell clones. J Immunol 147, 2942–9. Williams, P.B., Ahlstedt, S., Barnes, J.H., Soderstrom, L. & Portnoy, J. (2003) Are our impressions of allergy test performances correct? Ann Allergy Asthma Immunol 91, 26– 33. Witteman, A.M., Stapel, S.O., Perdok, G.J. et al. (1996) The relationship between RAST and skin test results in patients with asthma or rhinitis: a quantitative study with purified major allergens. J Allergy Clin Immunol 97, 16–25. Woodfolk, J.A., Wheatley, L.M., Piyasena, R.V., Benjamin, D.C. & Platts-Mills, T.A. (1998) Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity. Sequence homology to two families of serine proteinases. J Biol Chem 273, 29489– 96. Wopfner, N., Gadermaier, G., Egger, M. et al. (2005) The spectrum of allergens in ragweed and mugwort pollen. Int Arch Allergy Immunol 138, 337–46. Yamada, K., Urisu, A., Kakami, M. et al. (2000) IgE-binding activity to enzyme-digested ovomucoid distinguishes between patients with contact urticaria to egg with and without overt symptoms on ingestion. Allergy 55, 565–9. Yman, L. (2001) Standardization of in vitro methods. Allergy 56 (suppl. 67), 70–4. Zeiss, C.R., Levitz, D. & Suszko, I.M. (1978) Quantitation of IgE antibody specific for ragweed and grass allergens: binding of radiolabeled allergens by solid-phase bound IgE. J Allergy Clin Immunol 62, 83–90.

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Allergen Extracts and Standardization Ronald van Ree

Summary Since the discovery of IgE antibodies, standardization of allergen extracts has been dominated by measurement of IgE-binding potencies (biological activity). This focus has in the first place been driven by the desire to control the safety of immunotherapy. The identification of major allergens and the production of specific antibody reagents has facilitated the introduction of major allergen measurements into standardization protocols. Measurement of major allergens has revealed that their quantities and ratios can differ considerably between allergen products. This of course has relevance for safety, but also for efficacy. Effective immunotherapy minimally requires administration of quantities of major allergen in the order of 5–20 μg per maintenance dose. For optimal control of safety and efficacy, standardization protocols should therefore include both assessment of biological activity and measurement of major allergens. Immunoassays can only reliably quantify major allergens if they do not distinguish between isoforms. Purified recombinant allergens are ideal candidates as reference preparations, provided that they are good mimics of their natural counterparts. Antibody-independent techniques combining high-performance liquid chromatography or capillary electrophoresis with mass spectrometry hold promise for the future of standardization of complex allergen extracts. Replacement of these extracts by (cocktails of) recombinant allergens or peptides will simplify the need for standardization to the level of simple protein determinations. This is therefore the ultimate solution for the problems posed by standardization of allergen extracts.

A short history of allergen standardization Allergen extracts have been used for diagnosis (skin testing) and immunotherapy since the early 20th century. It started with the work of Robert A. Cooke and Mary Hewitt-Loveless

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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(Cohen 2003), almost 50 years before the discovery of IgE by the Ishizakas and Johansson (Bennich et al. 1968a,b). Before this discovery, no serious attempts had been made to standardize allergen extracts. A major step was the development of an assay to measure allergen-specific IgE antibodies, the radioallergosorbent test (RAST) (Johansson et al. 1973) Standardization was (and is) largely driven by the safety aspect of immunotherapy, i.e., determining the potency of an extract to induce IgE-mediated reactions (van Ree 2006). From this it is easily understood why the development of RAST sparked off initiatives to standardize allergen extracts (de Weck 2003). These initiatives were embedded in developments toward closer international collaboration between immunologists, leading to the establishment in 1969 of an umbrella organization for national immunology societies, the International Union of Immunological Societies (IUIS). One of the first activities of IUIS was the creation of several committees, of which one was the Committee on Standardization, aiming at standardization of immunologic reagents. A subcommittee was created that dealt with standardization of allergen extracts, the IUIS Allergen Standardization Subcommittee. In the early days of its existence, close links were established to the World Health Organization (WHO), which was the only organization legitimized to establish certified international references. Toward the end of the 1970s the initiative was taken to organize 3-yearly meetings entirely devoted to allergen standardization, the Paul Ehrlich Seminars. The first meeting was held in 1979 and 10 meetings have followed since. In 1981 the IUIS International Allergen Standards Program (IASP) commenced, aiming at the establishment of international allergen standards to be recognized by the Committee on Biological Standardization of the WHO. Originally, the program included 16 allergen sources: Dermatophagoides pteronyssinus and D. farinae, Phleum pratense, Cynodon dactylon and Lolium perenne pollen, birch, ragweed, plane, mugwort, oak, olive and Parietaria pollen, cat and dog, and Alternaria and Cladosporium. It delivered five certified international standards (D. pteronyssinus, P. pratense, ragweed, birch and dog) between 1981 and 1987 (Helm et al. 1984; Ford et al. 1985a,b; Gjesing et al. 1985; Larsen et al. 1988; Arntzen et al. 1989) The duration (and cost) of the program, together with increasing pressure from regulatory authorities to implement standardization protocols, prompted

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allergen manufacturers to develop their own in-house references (IHR) to be able to comply with the rapidly changing regulatory requirements. As a consequence, the momentum for the IASP was lost, and by the end of the 1980s the unfinished reference program was stopped. In the end, the international references were never really implemented by the allergen manufacturers. The original idea of comparability of products from different companies by the use of a uniform international unit (IU) never materialized. This was not only because manufacturers had started using their own IHR preparations with company-specific units, but also because a system based on RAST inhibition and allergen extracts does not easily facilitate comparability (van Ree 1997, 1999, 2006). Serum pools are variable in composition between companies and in time, and so are allergen extracts. With the identification, purification, and cloning of most major allergens from the important allergen sources in the 1980s and 1990s and the production of specific monoclonal antibodies, a new strategy emerged to give allergen standardization its desired comparability: mass units of major allergen (van Ree 2004). Despite the attractiveness of this idea, to date it has not yet been implemented. Why?

Allergen Extracts and Standardization

Table 44.1 Correlation between major allergen (MA) and RAST inhibition (RI) assessed by Spearman rank correlation.*

Mite Cat Grass Olive Alternaria

Major allergen

R (Spearman) MA vs. RI

P (significance)

Der p 1 Der p 2 Fel d 1 Lol p 1 Lol p 5 Ole e 1 Alt a 1

0.785 0.876 0.658 0.849 0.875 0.423 0.058

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.001 > 0.5

* Correlations are based on measurement of 42, 44, 34, 39, and 32 extracts for mite, cat, grass, olive, and Alternaria, respectively.

2.5 Mite

Cat

Grass

Olive

2.0

Biological standardization versus major allergen measurements

1.5

The traditional focus of allergen standardization has been on safety of allergen-specific immunotherapy, i.e., on IgEbinding potencies of allergen extracts. It is this property of allergen extracts that is most directly linked to the risk of adverse reactions. How well do IgE-independent measurements of major allergens correlate with IgE-based standardization? A limited number of studies has addressed this issue, reporting good correlations between major allergen content and IgE-binding potency (Dreborg & Einarsson 1992; Ramirez et al. 1997a,b; Duffort et al. 2004). Indeed, when analyzing extracts from a single company by RAST inhibition and major allergen tests, in most cases acceptable to good correlations were found (Table 44.1). Only in the case of Alternaria no significant correlation was observed. For extracts with multiple major allergens like house-dust mite and grass pollen, this analysis revealed that Der p 2 and Lol p 5, respectively, gave the closest correlation (Spearman R 0.88) to the IgE-binding potency of the extracts. Ole e 1 levels correlated relatively poorly to IgE-binding potencies, indicating that one or more additional important allergens are involved in IgE binding. For Alternaria no significant correlation was found at all between Alt a1 levels and IgE-binding potencies, and other allergens are likely to be more relevant to measurement. Strong correlations between IgE-binding potencies and major allergen levels should not be overinterpreted. The ratio between both parameters roughly varies fourfold to eightfold (Fig. 44.1). In other words, two house-dust mite extracts

1.0

0.5

0.0 Der p 1

Der p 2

Fel d 1

Lol p 1

Lol p 5

Ole e 1

Fig. 44.1 Variability in relation between biological potency and major allergen content. Ratios of results obtained by RAST inhibition (percentage relative to in-house references) and major allergen test (weight/volume) were calculated. The median ratios were determined, and then given a value 1 by multiplication with 1/median. All other values were multiplied by the same factor to allow easier comparison between extracts from different allergen sources in a single graph. Box plots indicate the factorized medians (i.e., all are 1), and the 10, 25, 75, and 90 percentiles.

designated to have identical potency (biological units) can have up to eightfold different Der p 1 content and up to fourfold different Der p 2 content. Conversely, cat extracts with similar Fel d 1 content can demonstrate up to eightfold differences in biological potencies. From a safety perspective, such a difference is relevant and argues for continuation of biological standardization. Why then should major allergen measurements be introduced in standardization protocols, apart from the fact that significant differences in major allergen content are relevant for safety?

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Allergens

Major allergens, the active ingredients of immunotherapy Although this has never really been demonstrated, major allergens are thought to be the active ingredients of allergen extracts used for allergen-specific immunotherapy. The strongest support for a decisive role of major allergens comes from recent clinical trials in which (cocktails of) recombinant major allergens were demonstrated to be an effective replacement of such extracts (Jutel et al. 2005; Creticos et al. 2006). From the perspective of efficacy, major allergen measurements on extract-based immunotherapy products are therefore extremely relevant. The 1998 WHO position paper on allergen immunotherapy reported on optimal doses for injection immunotherapy that should be the target maintenance dose for all patients (Bousquet et al. 1998). The optimal dose was defined as “the dose of an allergen vaccine inducing a clinically relevant effect in the majority of patients without causing unacceptable side effects.” The designation “optimal” is perhaps somewhat misleading and easily interpreted as optimal with respect to efficacy. Safety considerations (side effects) have most likely limited the possibility to reach optimally effective allergen doses. From a limited number of studies it was concluded that doses of 5– 20 μg of major allergen are optimal (Van Metre et al. 1988; Creticos et al. 1989, 1996; Haugaard et al. 1993; Alvarez-Cuesta et al. 1994; Olaguibel et al. 1997). Only a few of these studies actually included dosefinding protocols (Haugaard et al. 1993; Olaguibel et al. 1997). However, the question asked in these studies was not really at what concentration optimal efficacy was reached but what the lowest allergen concentration was that still gave a significant improvement over placebo. True dose-finding studies aiming at maximum efficacy cannot easily be performed, and will require strategies to reduce side effects, i.e., to achieve hypoallergenicity. Generally speaking the so-called “optimal dose” has been estimated by applying major allergen measurements to existing safe dosing schemes of conventional treatments, and subsequently designating these doses as optimal. Despite these limitations, the range between 5 and 20 μg of major allergen has, since the publication of the position paper, been promoted as the target for efficient immunotherapy. From that date, many additional clinical trials have been performed that largely report very similar effective concentrations of major allergen in therapeutic extracts, although in most cases again without dose-finding protocols (Garcia-Villalmanzo et al. 1999; Nelson 2000; Bodtger et al. 2002; Gonzalez et al. 2002; Nettis et al. 2002; Ewbank et al. 2003; Mirone et al. 2004; Nanda et al. 2004; Rossi & Monasterolo 2004; Tabar et al. 2005; Casanovas et al. 2006; Frew et al. 2006; Lent et al. 2006). More recently, trials for sublingual immunotherapy (SLIT) were published that also reported major allergen content (Bousquet et al. 1999; La Rosa et al. 1999; Pradalier et al. 1999; Guez et al. 2000; Grosclaude et al. 2002; Andre et al.

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2003; Khinchi et al. 2004; Rolinck-Werninghaus et al. 2004; Smith et al. 2004; Tonnel et al. 2004; Dahl et al. 2006a,b; Durham et al. 2006; Larsen et al. 2006). In most cases the dose was significantly higher than administered during injection immunotherapy (10- to 100-fold). On the basis of the available data for injection and sublingual immunotherapy, it has to be concluded that an optimal dose with respect to efficacy is actually still unknown. To improve insight into the dose– response relation between active ingredient and efficacy, measurement of major allergens should be routinely included in standardization protocols next to biological potency testing. Is it worth the trouble? In other words, are there significant differences between major allergen contents of allergen products? Several examples have been reported, both for inhalant allergen extracts (Carreira et al. 1994) and food allergen extracts (Akkerdaas et al. 2003). In the latter case, important allergens were even undetectable in commercially available hazelnut skin prick test (SPT) reagents. For example, it was demonstrated that some diagnostic extracts did not contain any detectable Cor a 8, the hazelnut lipid transfer protein (LTP). LTP has been convincingly shown to be a highly relevant food allergen in fruits and nuts (van Ree 2002). Sensitization to LTP is a risk factor for severe systemic reactions (Schocker et al. 2004; Fernandez-Rivas et al. 2006). Standardization of SPT reagents on the presence of LTP is therefore highly relevant. Also for immunotherapy products significant differences in major allergen content exist. In a recent analysis of commercially available SLIT products, Bet v 1 concentrations were shown to vary up to more than tenfold in birch pollen extract (van Ree 2006). Similar variation was observed for Der p 1 and Der p 2 concentrations in house-dust mite extracts (van Ree 2006). For Der f 1 and Der f 2, differences between the extremes were even 40- and 375-fold, respectively (Fig. 44.2). Ratios of Der f 1 over Der f 2 ranged from 0.6 to > 60. High group 1 to group 2 ratios point toward the use of mites plus spent growth medium as source material instead of purified mite bodies, because fecal particles contain high concentrations of group 1 allergen. The observed differences emphasize the importance of major allergen tests in standardization protocols.

Immunoassays for measuring allergen molecules There are various immunoassay formats for measuring major allergens, all having the use of allergen-specific antibody reagents in common. Monoclonal antibodies are most commonly used. In a review on allergen standardization published in 1997, allergen-specific monoclonal antibodies available up to that year were listed (van Ree 1997). At that time around 80 allergens were covered. Ten years later, at least 30 new allergens have been added to the list (Table 44.2) (Arilla et al. 1997, 2004, 2005, 2006; Banerjee et al. 1997; Sander et al. 1997;

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90

Table 44.2 New allergens against which monoclonal antibodies were raised and reported between 1997 and 2006.

80

Der f 1 (mg/mL)

70

Pollen Grasses Group 2 (Marth et al. 2004) Group 4 (Fahlbusch et al. 1998)

60 50 40

Trees Ole e 9 (Duffort et al. 2006) Pla a 1 (Arilla et al. 2005) Cup a 1 (Arilla et al. 2004) Hazel 70 kDa (Gruehn et al. 2003) Bet v 5 (Karamloo et al. 1999)

30 20 10 0 1

2

3

4

5

6

7

SLIT extract 12

Der f 2 (mg/mL)

Weeds Par j 2 (Arilla et al. 2006) Art v 1 (Jimeno et al. 2004) Pla l 1 (Calabozo et al. 2001) Mites B. tropicalis Blo t 1 (Ramos et al. 2004a) Blo t 3 (Yang et al. 2003) Blo t 5 (Yi et al. 2005) Blo t 11 (Ramos et al. 2003, 2004b) Blo t 13 (Labrada et al. 2002)

10 8 6 4

D. farinae Der f 7 (Shen et al. 1997) Der f 15 (McCall et al. 2001) Der f 98 kDa (Tsai et al. 1998)

2 0 1

2

3

4 SLIT extract

5

6

7

D. simoney Der s 1 (Sewer et al. 2000) Epithelia Horse Equ c 1 (Lascombe et al. 2000) Equ c ?? (Emenius et al. 2001)

70 60 Ratio Der f 1/Der f 2

Allergen Extracts and Standardization

Insects Vespid venoms Ves v 5 (Suck et al. 2000)

50 40

Molds Aspergillus a-Amylase (Sander et al. 1997) Serine protease (Lin et al. 2000) Asp f 2 (Banerjee et al. 1997)

30 20 10 0 1

2

3

4

5

6

7

SLIT extract Fig. 44.2 Variability of major allergen concentrations in sublingual immunotherapy (SLIT) products. Dermatophagoides farinae extracts of seven companies were compared with respect to Der f 1 and Der f 2 content. Concentrations are given in mg/mL. In the bottom graph, the ratio Der f 1/Der f 2 is given.

Penicillium Serine protease (Lin et al. 2000) Latex Hev b 1 (Raulf-Heimsoth et al. 2000) Food Apple Mal d 3 (Zuidmeer et al. 2005) Peach Pru p 3 (Duffort et al. 2002) Continued p. 932

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Allergens assay formats, of which the sandwich ELISA is the most commonly used. Competitive immunoassays and quantitative immunoblotting are however also used frequently.

Table 44.2 (Cont’d) Abalone Hal d 1 (Lu et al. 2004)

Sandwich ELISA

Scampi Nep n 97 kDa (Griffin et al. 2001) Wheat Tri a bd 17 kDa (Yamashita et al. 2001) a-Amylase inhibitor (Wiley et al. 1997) Soy Gly m 1 (Gonzalez et al. 2000) Sunflower Hel a 2 (Arilla et al. 1997; Asturias et al. 1999)

Shen et al. 1997; Wiley et al. 1997; Fahlbusch et al. 1998; Tsai et al. 1998; Asturias et al. 1999; Karamloo et al. 1999; Gonzalez et al. 2000; Lascombe et al. 2000; Lin et al. 2000; RaulfHeimsoth et al. 2000; Sewer et al. 2000; Suck et al. 2000; Calabozo et al. 2001; Emenius et al. 2001; Griffin et al. 2001; McCall et al. 2001; Yamashita et al. 2001; Duffort et al. 2002, 2006; Labrada et al. 2002; Gruehn et al. 2003; Ramos et al. 2003, 2004a,b; Yang et al. 2003; Jimeno et al. 2004; Lu et al. 2004; Marth et al. 2004; Yi et al. 2005; Zuidmeer et al. 2005) and additional monoclonal antibodies were reported for allergens that were already covered (Ramirez et al. 1997a; Portnoy et al. 1998; Aden et al. 1999; Yong et al. 1999; Batard et al. 2000; Chien et al. 2000; van Ree et al. 2000; Arilla et al. 2001, 2002, 2006; Jeong et al. 2002; Parvaneh et al. 2002; Asturias et al. 2003; Pomes et al. 2003; Duffort et al. 2004; Abebe et al. 2006). The number of allergens for which monoclonal antibodies come available is constantly increasing. This process has at least partly been driven by the need to develop immunoassays for measurement of (major) allergens. Monoclonal antibodies have found their way into different

Strep biot

Strep biot

or

In a sandwich enzyme-linked immunosorbent assay (ELISA) (Fig. 44.3), a minimum of two antibodies with specificity for the allergen are needed. Usually, a combination of two mouse monoclonal antibodies or one monoclonal and one (monospecific) polyclonal antibody is used. Polyclonal antibodies are mostly produced in rabbits. The capturing (monoclonal) antibody is coated to a microtiter plate in sodium bicarbonate buffer at a concentration of around 1 μg per well. After coating, plates are blocked using reagents like bovine serum albumin, gelatin, or skimmed milk. Between each step in the ELISA procedure, plates are washed with a buffer containing a detergent like Tween-20. Allergen extracts are incubated with the coated plates to allow the major allergen to bind to the monoclonal antibody. Unbound material is washed out prior to addition of the second antibody, the so-called detecting antibody. In the case of a monoclonal antibody, the detecting antibody is usually conjugated with an enzyme (horseradish peroxidase or alkaline phosphatase) or with biotin. In the latter case, the enzyme of choice is conjugated to (strept)avidin. When a polyclonal rabbit antiserum is used as detecting antibody, a third (enzyme-conjugated) antibody with specificity for rabbit IgG is usually introduced. In an ELISA the quantity of enzyme bound to the plate determines the degree of consumption of a substrate, resulting in a measurable color development. Of course, for calculation purposes a standard curve with known major allergen content is needed. Such a calibration curve can be composed of dilutions of a calibrated extract, a purified major allergen, or a recombinant version thereof. The sensitivity of this type of assay is typically in the range 1–10 ng of major allergen.

Strep biot Detecting antibody Capturing antibody

(a) nd

2 detecting antibody 1st detecting antibody Capturing antibody (b)

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Allergen

Enzyme

Fig. 44.3 Sandwich ELISA. Schematic representation of sandwich ELISA with two monoclonal antibodies (a) or with monoclonal and polyclona antibodies (b). In (a), the enzyme is either directly conjugated to the detecting antibody or, in combination with biotinylated detecting antibodies, to streptavidin. When polyclonal rabbit antisera are used for detection (b), a second antibody against rabbit IgG conjugated with enzyme is usually included. (See CD-ROM for color version.)

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Coated allergen

Allergen Other proteins

Enzyme

Inhibiting extract

Enzyme-conjugated monoclonal Bound Unbound

Fig. 44.4 Competitive ELISA. Schematic representation of the process of inhibition in a competitive ELISA. Enzyme-conjugated monoclonal antibody is partly prevented from binding to immobilized allergen by soluble allergen in allergen extract. (See CD-ROM for color version.)

Allergen Extracts and Standardization

extract. The antibody reagent can be a mouse monoclonal antibody, polyclonal rabbit antibodies or even IgE antibodies from human serum (pool). For the sake of consistency over a period of time, of course a monoclonal antibody has preference. Compared with a sandwich ELISA, where purified major allergen is only used for the calibration curve, much more of this reagent is needed. From this perspective, the second format for competitive assays with (radio)labeled purified allergen in the liquid phase is a good alternative (Fig. 44.5). In this case, only around 1–10 ng of purified (radio)labeled allergen are needed per test. In such assays, allergen-specific antibodies are bound to an affinity matrix for (rabbit) IgG (e.g., protein A or protein G Sepharose) or for mouse monoclonal antibodies (e.g., Sepharose-coupled goat antibodies against mouse IgG). Binding of these antibodies to the labeled major allergen is inhibited with the allergen extract. The label can be radioactive iodine (125I) or biotin. In the latter case a streptavidin–enzyme conjugate is needed to facilitate a color reaction. For broad application, monoclonal antibodybased sandwich ELISA is usually the technique of choice.

Competitive allergen assays Competitive assays for allergen measurement exist in two formats, one with purified allergen immobilized to the solid phase of the assay, the other with an antibody-capturing solid phase and labeled allergen in the liquid phase. Examples of the former are ELISA inhibition (Fig. 44.4), RAST inhibition, and CAP inhibition. In an inhibition ELISA, major allergen is coated to the microtiter plate in the order of 100 ng to 1 μg per well. In RAST or CAP inhibition, allergen is covalently coupled to a solid phase (paper disks, Sepharose, etc.) at very similar concentrations per test. Binding of allergen-specific antibodies to the coated allergen is inhibited with allergen

125

Quantitative immunoblotting An immunoassay occasionally used for measuring allergen levels is immunoblotting, either as a simple dot blot or after separation of samples by SDS-PAGE. In the former case, extract is spotted onto nitrocellulose or polyvinyldifluoride (PVDF) membrane and probed with a specific antibody for the allergen. Spots with dilutions of a solution with known concentrations of the allergen serve as standard curve. Bound allergen-specific antibodies are detected with secondary antibodies (e.g., antimouse IgG or anti-rabbbit IgG) that are radioactively labeled or conjugated with an enzyme or chemiluminescent probe.

I-allergen

Bound

Unbound

Allergen Other proteins

Inhibiting extract

Protein A sepharose

Fig. 44.5 Schematic representation of a competitive radioimmunoassay. IgG antibodies from a monospecific polyclonal rabbit antiserum are immobilized using protein A or protein A sepharose. Binding of radiolabeled allergen is inhibited by nonlabeled allergen in allergen extract. (See CD-ROM for color version.)

Protein A sepharose

Uninhibited

Inhibited

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Allergens Slot for sample loading −

W

gh

−

−

−

−

−

−

M

Hi

Standard curve

W

w

M

Lo

+

Extracts +

+

+

+

+

+

MW markers

SDS-PAGE separation of proteins +

Nitrocellulose

+ −

−

ingredients of diagnostic and therapeutic extracts individually. At best, IgE-binding potency tests evaluate the sum of major allergen activities without revealing differences in ratios between them (van Ree 1997, 1999, 2006). Another major strength of most allergen tests is that they are based on highly reproducible antibody reagents that are readily available in unlimited quantity and can be used all over the world. In contrast, IgE-binding potency tests are based on limited volumes of pooled sera. This implies variability in time and place. Technically, validated major allergen tests are accurate and reproducible. However, this does not mean that there are no weaknesses associated with major allergen tests like the sandwich ELISA. Problems do exist related to the interplay between the fine specificity of the antibodies in the assay, the composition of allergen extracts, and the allergen preparation used as standard curve, i.e., the reference preparation.

Fine specificity of monoclonal antibodies Transfer of SDS-PAGE separated proteins to nitrocellulose

Allergen-specific antibody

Secondary radiolabeled (or conjugated) antibody

Measured allergen

Resulting autoradiogram Fig. 44.6 Quantitative immunoblot. Schematic representation of a (semi)quantitative immunoblot. Allergens (extract and purified standard) are separated by SDS-PAGE followed by electroblotting to nitrocellulose (or PVDF) membrane. After detection with a specific antibody, bands of standard curve and samples are quantified by densitometry and the allergen content of samples is calculated. (See CD-ROM for color version.)

The intensity of bands can be measured on a densitometer and provides a measure of the quantity of allergen. Prior to immunodetection, extracts and standards can first be separated by SDS-PAGE (Fig. 44.6). This provides additional certainty with respect to the identity of the band recognized because the apparent molecular weight is revealed.

Major allergen tests: strength and weaknesses Immunoassays measuring individual allergens have some important advantages compared with those assessing overall IgE-binding potencies. Major allergen tests measure the active

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For the production of monoclonal antibodies against major allergens, allergen extracts, purified natural allergens, or purified recombinant allergens are used as immunogen. The choice can significantly influence the fine specificity of the resulting monoclonal antibodiy. With respect to isoform composition, an extract as immunogen is most complex. Purified natural allergen is likely to be enriched for one or more isoforms and a recombinant allergen represents a single isoform. Therefore, the chance of isoform specificity of laboratory antibodies increases from extract to recombinant. For measuring the major allergen content of allergen extracts, isoform specificity is undesirable because one or more isoforms may remain undetected. Examples of assays missing isoforms have been reported, e.g., measurement of the major house-dust mite allergen Der p 2 (Hakkaart et al. 1998; Smith et al. 2001). Extraction, recombinant production, and purification steps can each in their own way influence the molecular appearance of major allergens with respect to denaturation, degradation, and aggregation. The particular state of an immunogen is potentially reflected in the fine specificity of resulting monoclonal antibodies, e.g., an incorrectly folded aggregated recombinant protein or an allergen fragment will induce different antibodies than a well-folded intact natural allergen.

Composition of allergen products Major allergens in allergen extracts (can) have different appearances, ranging from a (variable) mixture of well-folded monomeric isoforms to a (variable) mixture of well-folded protein and denatured protein, fragments and/or aggregates. A purified (natural or recombinant) standard used for reference purposes will have a different molecular composition. It will depend on the fine specificity of the assay’s antibodies as to how effectively each molecular form of a major allergen will be detected. The situation becomes even more complex in the case where allergen products are composed of mixtures of different related species, e.g., mixtures of four

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to ten different grass pollen species or of multiple house-dust mite species. Accurate measurement of the total grass pollen group 1 content of a mixture is impossible by immunoassay. Group 1 allergen from Lolium perenne will be recognized differently than that from Secale cereale. A suitable standard curve cannot be designed. Should it be a single group 1 allergen or a mixture? And if a mixture of purified group 1 allergens, what should be their proportion? The only solution for assessing the major allergen content of mixtures is to measure each component of allergen mixtures individually using species-specific standards. Production protocols of several companies do not allow individual assessment because source materials are mixed prior to extraction. The best chance for obtaining reliable data is by using assays that are maximally cross-reactive between isoforms, fragments, aggregates, and species.

Natural or recombinant standards As already mentioned above, the choice of a reference preparation for application as a calibrator has a major impact as well. It is tempting to choose a recombinant allergen because such a reference preparation can easily be produced in a highly homogeneous and reproducible way (van Ree 2004). However, the choice of the isoform and the folding and aggregation state of the recombinant molecule will influence the performance of the standard. Is a natural reference preparation better? Not necessarily. Denaturation, degradation, and aggregation can also occur on extraction and purification

Allergen Extracts and Standardization

and the isoform composition of a standard is unlikely to be identical to that of sample extracts.

Major allergen immunoassays: complex interpretation The interplay between the fine specificity of antibodies, the allergenic composition of (mixtures of) extracts, and the source and characteristics of reference preparations makes a straightforward interpretation of assay results complex. There are many scenarios that vary from (close to) accurate quantification to overestimation or underestimation. Detailed knowledge about the fine specificity of antibodies and molecular characteristics of the standard is an absolute requirement to be able to judge whether the outcome of a standardization assay is reliable enough or not. To illustrate this we take the following example. We simplify the situation by assuming that in a given allergen extract only two isoforms, A and B, exist, and that the antibodies used are directed to correctly folded allergen. Then already at least 18 different scenarios can be worked out, depending on the isoform specificity of the antibodies and the characteristics of the standard used (Table 44.3). Again following a simplified scheme, the antibodies in the assay used are assumed to be either selective for isoform A or B, or completely cross-reactive between the two. The standard is either a mixture of natural isoform A and B, enriched for either one of them, or a recombinant version of isoform A or B in a correctly folded or inadequately folded version. Incorrectly folded standard will not (effectively) be

Table 44.3 Representation of the accuracy of possible outcomes of major allergen assays.*

Standard

Antibody specificity Folded only/isoform A

Folded only/isoform B

Folded only/cross-reactive

Natural Enriched for isoform A

Low degree of overestimation of isoform A; isoform B not detected → overall unclear

High degree of overestimation of isoform B; isoform A undetected → overall unclear

~ Accurate

Natural Enriched for isoform B

High degree of overestimation of A; isoform B not detected → overall unclear

Low degree of overestimation of isoform B; isoform A undetected → overall unclear

~ Accurate

Recombinant Isoform A/folded

Underestimation: isoform A correctly measured; isoform B not detected

Standard cannot be used

~ Accurate

Recombinant Isoform B/folded

Standard cannot be used

Underestimation: isoform B correctly measured; isoform A not detected

~ Accurate

Recombinant Isoform A/partly folded

Isoform A overestimated; isoform B not detected → overall unclear

Standard cannot be used

Overestimation

Recombinant Isoform B/partly folded

Standard cannot be used

Isoform B overestimated; isoform A not detected → overall unclear

Overestimation

* Antibodies were assumed to be specific for well-folded allergen. Three antibody variants were considered: specific for isoform A, specific for isoform B, or reactive with both isoforms (cross-reactive). The standard used as reference was either natural or recombinant. The natural was assumed to be well-folded and a mixture of isoform A and B (A > B or A < B). The recombinant was either isoform A or B in well-folded or partly unfolded molecular appearance.

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Table 44.4 Fictitious numerical example to illustrate the effect of difference in isoform composition between standard and extract.*

Extract (allergen content: 10)

Standard (allergen content: 10) Composition (detected/undetected)

1/9

5/5

9/1

1/9

10

2↓

1.1 ↓

5/5

50 ↑

10

5.6 ↓

9/1

90 ↑

18 ↑

10

Calculated allergen content * Antibodies are assumed to recognize one of two isoforms selectively and not respond to the second isoform. Three different ratios between detected isoform and undetected isoform were used: 10% vs. 90%, 50% vs. 50%, and 90% vs. 10%. Arrows indicate overestimation (↑) and underestimation (↓).

detected by antibodies that are exclusively or preferentially reactive with well-folded allergen. The absorbance/protein ratio will be lower than for a standard that is (partly) incorrectly folded. Assuming that extracts contain predominantly well-folded allergen, a standard curve of inadequately folded allergen will lead to overestimation of the allergen content because the absorbance/protein ratio is higher for allergen in the extract than for the standard. When isoform specificity comes into play, things get even more complicated. In general one can say that, similar to preference of antibodies for folded allergen, preference for an isoform will result in overestimation of that particular isoform in an extract if the standard contains a mixture of isoforms. At the same time any other isoform will not be detected or will be less efficiently detected. The higher the share of poorly detected isoform in a standard, the larger the overestimation of the detected isoform in a test sample. In particular, if the isoform composition of standard and extract is different but unknown (the real situation), it is virtually impossible to predict whether allergen content is overestimated or underestimated. This is illustrated again schematically in Table 44.4. Only when standard and extract have a similar isoform composition is the calculated content close to accurate. This example is purposely oversimplified by the assumption that the antibodies react to one isoform and not at all to the other. In reality, it is far more likely that the situation is not as black and white and that antibodies have preference for one isoform compared with another, resulting in different sensitivities and nonparallel curves. Is the situation hopeless for major allergen tests? Not really. If assays are based on antibodies raised against well-folded allergen (natural or recombinant) that do not distinguish between the major isoforms of an allergen (cross-reactive), and if the standard is composed of well-folded natural or recombinant allergen, accurate measurements are realistic. For many allergens, good-quality recombinant allergens are available and so are assays in which standard curves made of purified natural

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and purified recombinant allergen overlap (i.e., isoform crossreactive antibodies). In that case, major allergen content can reliably be determined.

The future: standardization without antibodies? The first methods used to standardize allergen extracts were independent of the use of antibodies. These included weight per volume and measurement of total protein, total nitrogen content, or protein nitrogen units (PNU) (Pepys et al. 1975; Arbesman et al. 1977; Brighton et al. 1979; May et al. 1979, 1981; Richman & Cissel 1988). In particular PNU have sometimes been used in more recent publications to express potencies (Fitzsimons et al. 1986; Darsow et al. 1995; Bhagat et al. 1996; Trimmer et al. 2005), despite the fact that allergenspecific immunoassays are now widely available. Knowing that the major allergen composition of allergenic protein extracts can differ significantly, such general methods can no longer play a dominant role in allergen standardization. Does this mean that we have to disqualify all methods that measure total protein-related parameters? If used in conjunction with potency tests and allergen-specific assays, they certainly have added value for characterization of allergen products. A method that falls in the same category is SDS-PAGE, providing useful qualitative and semiquantitative information on the (batchto-batch consistency of the) composition of extracts. More recently, other antibody-independent techniques have been introduced into the area of allergen standardization (Natale et al. 2004; Batard et al. 2006). These include combinations of state-of-the-art techniques for separating complex protein mixtures like high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) and mass spectrometric techniques for identifying proteins like matrix-assisted laser desorption/ionization time of flight

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(MALDI-TOF) and electrospray ionization (ESI)-TOF mass spectrometry (Natale et al. 2004; Batard et al. 2006; Canas et al. 2006). Mass spectrometry is rapidly developing from a qualitative tool for identification of molecules into a technique that can also be applied for quantitative purposes (Ohnesorge et al. 2005; Ong & Mann 2005; Fenselau 2006). This will facilitate standardization of complex allergen extracts on the basis of major allergen content without allergen-specific antibodies.

The future: recombinants, hypoallergens, and peptides Recombinant allergens Allergen extracts have been used in immunotherapy for almost 100 years now. The level of standardization and characterization has certainly improved enormously, especially over the last three decades. These improvements were largely made on the basis of the identification, purification, and characterization of the most important major allergens and subsequent generation of specific antibody reagents. The first major allergen was cloned and expressed by the end of the 1980s by Breiteneder et al. (1989), i.e., the major birch pollen allergen Bet v 1. In the years following this milestone, the major allergens of the most frequently used other allergen sources for immunotherapy (grass pollen, olive pollen, ragweed pollen, house-dust mite, cat and dog epithelium) have successfully been cloned and expressed as well (www.allergome.org). Several research groups and companies are now pursuing the replacement of allergen extracts by (cocktails of) recombinant (or natural) major allergens. The first clinical trials have been quite promising (Jutel et al. 2005; Creticos et al. 2006), and it is therefore not unlikely that extracts will slowly make place for such biotechnology products. Of course this will have great impact on allergen standardization, because products can simply be labeled in mass units of major allergen(s). A straightforward protein measurement or quantification of protein on the basis of an amino acid determination is sufficient. The level of complexity of immunotherapy products and consequently the requirements for reliable standardization will significantly decrease.

Hypoallergenic approaches Recombinant allergens that display all characteristics of their natural counterparts can of course also be standardized on the basis of IgE-binding (biological) potency. With the advent of recombinant allergens, new strategies to decrease side effects have also come in reach. Recombinant allergens can be mutated by site-directed mutagenesis to knock out major IgE-binding epitopes (Ferreira et al. 2002). For similar reasons, synthetic peptides mimicking major T-cell epitopes have been developed for the treatment of (cat) allergy (Kay & Larche 2004). Hypoallergens and peptides cannot be standardized by biological potency measurements, as is now required for

Allergen Extracts and Standardization

current extract-based products. New regulations will need to be developed to accommodate this biotechnology revolution. Mass units of protein or peptide will become the major potency designation. Of course, IgE potency tests will still be needed to check levels of hypoallergenicity.

Concluding remarks Standardization of allergen extracts is moving from biological standardization to a more labor-intensive combination of biological testing and (multiple) major allergen measurements. In the (near) future, replacement of extracts by biotechnology products will offer the possibility to reverse this trend and simplify the needs for allergen standardization.

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Emenius, G., Larsson, P.H., Wickman, M. & Harfast, B. (2001) Dispersion of horse allergen in the ambient air, detected with sandwich ELISA. Allergy 56, 771– 4. Ewbank, P.A., Murray, J., Sanders, K., Curran-Everett, D., Dreskin, S. & Nelson, H.S. (2003) A double-blind, placebo-controlled immunotherapy dose-response study with standardized cat extract. J Allergy Clin Immunol 111, 155– 61. Fahlbusch, B., Muller, W.D., Rudeschko, O., Jager, L., Cromwell, O. & Fiebig, H. (1998) Detection and quantification of group 4 allergens in grass pollen extracts using monoclonal antibodies. Clin Exp Allergy 28, 799– 807. Fenselau, C. (2007) A review of quantitative methods for proteomic studies. J Chromatogr B Analyt Technol Biomed Life Sci 885, 14–20. Fernandez-Rivas, M., Bolhaar, S., Gonzalez-Mancebo, E. et al. (2006) Apple allergy across Europe: how allergen sensitization profiles determine the clinical expression of allergies to plant foods. J Allergy Clin Immunol 118, 481– 8. Ferreira, F., Wallner, M., Breiteneder, H., Hartl, A., Thalhamer, J. & Ebner, C. (2002) Genetic engineering of allergens: future therapeutic products. Int Arch Allergy Immunol 128, 171–8. Fitzsimons, R., Grammer, L.C., Shaughnessy, M.A. & Patterson, R. (1986) A comparison of the immune response to immunotherapy with polymerized grass allergen and monomeric grass allergen. Ann Allergy 57, 291– 4. Ford, A., Seagroatt, V., Platts-Mills, T.A. & Lowenstein, H. (1985a) A collaborative study on the first international standard of Dermatophagoides pteronyssinus (house dust mite) extract. J Allergy Clin Immunol 75, 676– 86. Ford, A.W., Rawle, F.C., Lind, P., Spieksma, F.T., Lowenstein, H. & Platts-Mills, T.A. (1985b) Standardization of Dermatophagoides pteronyssinus: assessment of potency and allergen content in ten coded extracts. Int Arch Allergy Appl Immunol 76, 58–67. Frew, A.J., Powell, R.J., Corrigan, C.J. & Durham, S.R. (2006) Efficacy and safety of specific immunotherapy with SQ allergen extract in treatment-resistant seasonal allergic rhinoconjunctivitis. J Allergy Clin Immunol 117, 319– 25. Garcia-Villalmanzo, I., Hernandez, M.D., Campos, A. et al. (1999) Immunotherapy with a mass unit Parietaria judaica extract: a tolerance study with evidence of immunological changes to the major allergen Par j 1. J Investig Allergol Clin Immunol 9, 321–9. Gjesing, B., Jager, L., Marsh, D.G. & Lowenstein, H. (1985) The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract. J Allergy Clin Immunol 75, 258– 67. Gonzalez, P., Florido, F., Saenz de San, P.B. de la T.F., Rico, P. & Martin, S. (2002) Immunotherapy with an extract of Olea europaea quantified in mass units. Evaluation of the safety and efficacy after one year of treatment. J Invest Allergol Clin Immunol 12, 263–71. Gonzalez, R., Duffort, O., Calabozo, B., Barber, D., Carreira, J. & Polo, F. (2000) Monoclonal antibody-based method to quantify Gly m 1. Its application to assess environmental exposure to soybean dust. Allergy 55, 59– 64. Griffin, P., Allan, L., Gibson, M., Elms, J., Wiley, K. & Curran, A.D. (2001) Measurement of personal exposure to aerosols of Nephrops norvegicus (scampi) using a monoclonal-based assay. Clin Exp Allergy 31, 928– 33. Grosclaude, M., Bouillot, P., Alt, R. et al. (2002) Safety of various dosage regimens during induction of sublingual immunotherapy. A preliminary study. Int Arch Allergy Immunol 129, 248–53.

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Gruehn, S., Suphioglu, C., O’Hehir, R.E. & Volkmann, D. (2003) Molecular cloning and characterization of hazel pollen protein (70 kD) as a luminal binding protein (BiP): a novel cross-reactive plant allergen. Int Arch Allergy Immunol 131, 91–100. Guez, S., Vatrinet, C., Fadel, R. & Andre, C. (2000) House-dust-mite sublingual-swallow immunotherapy (SLIT) in perennial rhinitis: a double-blind, placebo-controlled study. Allergy 55, 369–75. Hakkaart, G.A., Chapman, M.D., Aalberse, R.C. & van Ree, R. (1998) Immune-reactivity of recombinant isoforms of the major house dust mite allergen Der p 2. Clin Exp Allergy 28, 169–74. Haugaard, L., Dahl, R. & Jacobsen, L. (1993) A controlled dose– response study of immunotherapy with standardized, partially purified extract of house dust mite: clinical efficacy and side effects. J Allergy Clin Immunol 91, 709–22. Helm, R.M., Gauerke, M.B., Baer, H. et al. (1984) Production and testing of an international reference standard of short ragweed pollen extract. J Allergy Clin Immunol 73, 790–800. Jeong, K.Y., Jin, H.S., Oh, S.H. et al. (2002) Monoclonal antibodies to recombinant Der f 2 and development of a two-site ELISA sensitive to major Der f 2 isoallergen in Korea. Allergy 57, 29– 34. Jimeno, L., Duffort, O., Serrano, C., Barber, D. & Polo, F. (2004) Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1. Allergy 59, 995–1001. Johansson, S.G., Bennich, H. & Foucard, T. (1973) Quantitation of IgE antibodies and allergens by the radioallergosorbent test, RAST. Int Arch Allergy Appl Immunol 45, 55–56. Jutel, M., Jaeger, L., Suck, R., Meyer, H., Fiebig, H. & Cromwell, O. (2005) Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 116, 608–13. Karamloo, F., Schmitz, N., Scheurer, S. et al. (1999) Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins. J Allergy Clin Immunol 104, 991–9. Kay, A.B. & Larche, M. (2004) Allergen immunotherapy with cat allergen peptides. Springer Semin Immunopathol 25, 391–9. Khinchi, M.S., Poulsen, L.K., Carat, F., Andre, C., Hansen, A.B. & Malling, H.J. (2004) Clinical efficacy of sublingual and subcutaneous birch pollen allergen-specific immunotherapy: a randomized, placebo-controlled, double-blind, double-dummy study. Allergy 59, 45– 53. Labrada, M., Uyema, K., Sewer, M. et al. (2002) Monoclonal antibodies against Blo t 13, a recombinant allergen from Blomia tropicalis. Int Arch Allergy Immunol 129, 212–18. La Rosa M., Ranno, C., Andre, C., Carat, F., Tosca, M.A. & Canonica, G.W. (1999) Double-blind placebo-controlled evaluation of sublingual-swallow immunotherapy with standardized Parietaria judaica extract in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 104, 425–32. Larsen, J.N., Ford, A., Gjesing, B. et al. (1988) The collaborative study of the international standard of dog, Canis domesticus, hair/dander extract. J Allergy Clin Immunol 82, 318–30. Larsen, T.H., Poulsen, L.K., Melac, M., Combebias, A., Andre, C. & Malling, H.J. (2006) Safety and tolerability of grass pollen tablets in sublingual immunotherapy: a phase-1 study. Allergy 61, 1173– 6. Lascombe, M.B., Gregoire, C., Poncet, P. et al. (2000) Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgEreactive epitopes. J Biol Chem 275, 21572–7.

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Allergens

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Grass, Tree, and Weed Pollen Jean Emberlin

Summary Pollen from wind-pollinated (anemophilous) plants is the main problem in pollinosis because vast amounts are released to the air in an evolutionary adaptation to overcome wastage. In contrast, pollen from insect-pollinated (entomophilous) plants does not typically become airborne in large amounts and is normally a problem only in occupation exposures such as horticulture or floristry. It is very difficult to avoid contact with pollen from anemophilous plants as some is in the air during most months of the year in the majority of climates and pollen allergens persist in house dust for months after the period of pollen release. Most pollen grains are 25–50 μm in diameter and so nearly all are removed from the airflow in the nose but plant allergen is also present in the air on other particles. These can be paucimicronic or submicronic and can include pollution particulates, aerosolized sap, or mist droplets. This small particle allergenic fraction can be important in asthma. Interactions between pollen and air pollutants can enhance the allergenicty of pollen grains. Similarly, environmental stresses resulting from climate changes can increase the allergen load of the grains. Climate changes also exert an influence on the aeroallergen load by affecting plant distributions, for example enabling ragweed to spread further northwards in Europe. The timing and severity of pollen seasons differs notably between biogeographic areas due to variations in climate, vegetation, and land use. On a regional basis the pollen seasons differ annually due to weather factors and some trees have biennial or longer-term patterns in severity because of inherent biological rhythms. Quality-controlled information on pollen seasons and forecasts is available with regional coverage for Europe and the USA but elsewhere coverage is limited or nonexistent. Pollen count data are usually supplied as daily average counts per cubic meter of air but these can mask short-term peaks, which can be important triggers of symptoms. Also the counts are taken at rooftops to sample from background airflows so the amounts may not correspond closely to personal exposure. Grass pollen is ranked among the top plant aeroallergens, especially in temperate areas, including most of Europe and

North America. In many parts of the world it is the allergen most implicated in allergic rhinoconjunctivitis and for asthma it is second only to house-dust mite. Trees differ in importance for allergy geographically, for example birch is most important in northwestern Europe whereas olive is most important in Mediterranean areas. Pollen from weeds also differs in significance in various areas, for instance ragweed is very important in the USA and parts of central and southern Europe, and Parietaria in the Mediterranean. Considerable cross-reactivity exists between pollen from both related and different taxa. Also cross-reactivities exist between pollen allergens and foods, for example between Bet v1 and apples.

Introduction The timing and severity of pollen seasons differs notably between bigeographic regions due to variations in climate, vegetation, and land use. These contrasts are enhanced by the influence of air pollutants and other environmental stresses on the allergenicity of pollen, to give complex temporal and spatial mosaics of aeroallergen load. In addition, evidence shows that pollen seasons are changing due to global warming, not only in the timing of pollen release but more fundamentally through changing the distributions of plants. Information about the concentration and periodicity of pollen types is useful for interpreting symptoms of intermittent allergic rhinitis and seasonal asthma. It also provides a useful prophylactic and avoidance tool for allergic people. This chapter focuses on the most important pollen types in temperate and Mediterranean climates and provides background information on pollen seasons and data sources.

Main features of pollen Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Pollen transmits the male genetic material in sexual reproduction in the seed plants (Spermatophyta), which include the flowering plants and the gymnosperms or cone-bearing

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Fig. 45.1 Scanning electron micrograph of pollen grain of Solidago spp. (goldenrod). Magnification ×3500. (See CD-ROM for color version.)

plants. Most pollen grains are 10–100 μm in diameter, with the majority being in the range 25–50 μm (Linder 2000). Typically, pollen grains can be identified to the level of genus fairly easily by examining them under ×400 magnification, but only a few species have pollen that is sufficiently distinct to allow identification by visual inspection. In some families, all the genera have very similar pollen and it is impossible to distinguish them. The majority of flowering plants are insect-pollinated (entomophilous) and are insignificant as causes of pollinosis, except in occupational exposures, such as horticulture or floristry (Emberlin et al. 2004). Typically, the transfer of pollen from the anther to the stigma is achieved efficiently, with little release to the air, so relatively small quantities are produced. The grains tend to be large, heavy, sticky, and highly sculptured (Fig. 45.1) to help them adhere to the insect or other vector. The pollen may have antigens but exposures are so limited that sensitizations do not normally occur. It is the windpollinated (anemophilous) plants that are most important in pollinosis. Wind pollination is typical of many temperate zone plants but it is also abundant in many tropical ecosystems. Plants that have evolved wind pollination have a number of features in common. The pollen grains have characteristics to facilitate wind dispersal. They tend to be small, dry, and smooth, typically 20– 45 μm in diameter (Fig. 45.2), with slow terminal velocities, normally in the range of 2–6 m/s. If breathed in, pollen is deposited mostly in the nasal cavities and turbinates. Inevitably, vast amounts of pollen from anemophilous plants do not reach their targets so to compensate for this wastage, enormous numbers of pollen grains are produced, resulting

Grass, Tree, and Weed Pollen

Fig. 45.2 Scanning electron micrograph of grass pollen (Dactylis glomerata). Magnification ×3500.

in very high concentrations in the air. Deviations from the normal pattern feature among some wind-pollinated plants. Certain types produce only small quantities of pollen or release grains that do not travel very far. For instance, Zea mays (maize), a member of the grass family, is not often a cause of hay fever although it is wind pollinated and has allergenic proteins. This is probably because the large heavy grains are deposited close to the plant or are filtered from the wind efficiently by the large tassels of the stigmas. Certain anemophilous plants produce pollen grains with sculptured exine, such as Ambrosia (the ragweeds). There are also many examples of species that are not totally wind or insect pollinated, such as oilseed rape (Brassica napus). Since wind-pollinated plants produce such vast quantities of pollen, it is highly likely that pollen from several different species will land on one stigma. If all these grains started to germinate, the style would be occupied and rendered useless by pollen tubes of the wrong type. A pollen grain will grow on a stigma only if the chemical signatures are compatible. These signatures are enzymes composed of highly soluble substances, mostly proteins or polypeptides but also polysaccharides, glycoproteins and lipoproteins, held in micropores on the surface and deeper in the wall. The speed of release depends on several factors including pH. In the mature dormant pollen grain, most of the enzyme activity is associated with the walls, with only a relatively minor amount in the protoplast of the vegetative cell. For example, the immunodominant component of Lolium perenne (ryegrass), Lol p 1, is located mainly on the micropores of the exine but some allergen, Lol p 1X, is present within the pollen cell in starch granules (Knox 1993). When the pollen grain comes into contact with the moist surface of the stigma, the proteins to be discharged

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first are those held in the tiny crevices on the surface. These are lightly sealed in by a fatty layer, ready for quick release. The first-release proteins can be liberated within seconds and act as chemical passwords. If this is correct, the enzymes produced from deeper in the pollen cell react with the stigma to make a hole through the style. This sequence is mimicked when pollen grains land in the nose. The quick-release recognition proteins are discharged rapidly when the pollen grain comes into contact with the mucous membranes, whereas the slower dissolving enzymes inside the grains are liberated gradually through the pores and furrows. These compounds, as well as those held in the micropores on the exine, are the pollen allergens, which may elicit IgE production. In most conifers, the pollen grains germinate very slowly after transfer to the female cones. Fertilization may not take place until several months after pollination. This slower germination of the pollen tube is associated with a thick layer of lipid that seals in the recognition proteins on the pollen wall. The proteins are unloaded much more slowly than in the flowering plants. This is probably one of the reasons why conifer pollen is usually less allergenic but there are some exceptions, such as cypress and Japanese red cedar.

Patterns of abundance and dispersal The amount of pollen in the air not only differs with location and time of year but also depends on a large number of factors, including those which effect its production, release, and dispersal. Some of the controlling factors are shared in common between species, especially those relating to the mechanical aspects of pollen dispersal in the wind. Other variables, such as those which influence pollen abundance on the plants and its liberation into the atmosphere, differ between types of plants or even species. Tree pollen seasons can differ a great deal annually. Many temperate-zone trees form pollen in the late summer or early autumn preceding the spring of release. In these cases, the weather during the late growing season exerts an influence on pollen production. Many trees have inherent reproductive rhythms of high and low years for the abundance of pollen and subsequent seed because of the interactions of hormone production. These rhythms may be biennial, as in the cases of Betula (birch), Olea (olive), and many fruit trees, or they may occur over longer periods, as in Fagus (beech) and Quercus (oaks), which tend to have high-production years followed by four or five low-yield years. The alternating patterns may be disrupted by extreme winter temperatures for example, or modified or even obscured by the influence of weather during the times of pollen dispersal. Pollen seasons for most herbaceous plants, including grass, do not differ annually as much as those for trees. Herbaceous plants tend to form pollen in the anthers immediately before

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the flowering season, so pollen production is largely determined by the weather in the few weeks or months before pollination. Ideal conditions for pollen dispersal in the air are a steady wind and no precipitation, as even a slight drizzle will wash particles from the atmosphere. Plants have evolved a combination of physiologic and mechanical ways to maximize the chance of pollen release coinciding with this type of weather. For example, birch catkins need to be shaken to release pollen otherwise their physical structure of overlapping plates keeps them closed. For most plants, low relative humidity is essential for pollen release because the grains must be dry on the anther before they can be detached by the wind. However, drought may lead to pollen retention. Pollen will spread furthest downwind in steady nonturbulent airflows. If mixing depths are limited, pollen may transported for hundreds of kilometers in dry weather, but in most usual situations pollen would be deposited within about a 50-km radius of its source. Pollen concentrations will be diluted most rapidly in highspeed winds, with turbulent flows and unstable synoptic situations. Conversely, pollen will accumulate in slowly moving air, giving rise to high concentrations locally. The dispersal patterns of pollen are essentially the same as for any particles. Their distribution in the airflow tends to be heterogeneous, due to local eddying, shearing of wind directions, and so on. The concentrations can vary greatly over short distances, both vertically and horizontally, depending on the nature of the airflow, local topography, and position of the sources. The amount of pollen released by a species in a region will vary through its pollen season. In some species, pollination starts very suddenly, with a close synchronization of flower maturation and a sharp rise in the amounts of pollen released. In other genera, there is a more gradual start to pollination, with a staggered maturation of flowers through the season. Many plants have a pattern of pollen release between these two extremes. Different weather patterns from year to year, especially in temperate areas, are responsible for marked variation in start dates and duration of most pollen seasons. In some perennials, including both woody and herbaceous plants, overwintering dormancy may have to be broken by vernalization (exposure to cold). If insufficient cold occurs, more warmth will be needed in the spring to start growth again. In many species, for example many temperate grasses, reproduction starts in response to changes in day length (photoperiod). For these plants, responses to changes in weather, for example exceptionally warm springs, can be made only within the framework of their photoperiodic controls. Most plant taxa will have some capacity to respond to broad-scale changes in weather patterns, such as those associated with the North Atlantic oscillation or with global warming. Trends in weather patterns will result in alterations to plant distributions and pollen seasons. Both of these aspects will exert important influences

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on the aeroallergen load. Examples are given later in this chapter in the sections on individual plant types.

Plant allergen on particles other than pollen Allergens from plants are present in the air on a wide range of particle sizes other than pollen and can be important in allergic diseases. These particles may be fairly large, such as fragments of plant hairs and parts of decaying leaves, or they may be very small particles, either derived from plant material or formed through the transfer of allergen to soil particles or air pollution particulates. Allergenic particles and aerosols have been detected as small as 0.1 μm in diameter. As these are of respirable dimensions, they may penetrate deep into the lung and could be involved in the provocation of asthma attacks. Allergens on the particles are also important as agents causing pollinosis. The existence of these allergenic paucimicronic and submicronic particles has been demonstrated since the 1980s for several plant groups including Ambrosia (ragweed) (Solomon et al. 1983), Gramineae (grasses) (Stewart & Holt 1985; Spieksma et al. 1990), and Betula (birch) (RantioLehtimaki 1991). They can be detected before the pollen season and can persist long after pollen is no longer present in the air. They can also make a notable contribution to the total plant allergen load in the air during pollen seasons. For example, early work on this topic showed that during the peak ragweed season, almost 50% of the total allergen can be associated with particles of less than pollen-grain dimensions (Reed et al. 1986). Transfer of allergens can occur by leaching of proteins from pollen grains onto amorphous particles, including suspended particulate matter (SPM) and diesel exhaust particulates (DEP), e.g., Lol p 1 was found to bind with DEP in vitro (Knox et al. 1997). This may increase the potential of the allergens to sensitize individuals. Other allergenic paucimicronic and submicronic particles emanate from orbicules (spherical bodies c. 0.02 μm, formed in the anthers) which may become airborne during the release of pollen (Vinckier et al. 2005). Some allergens may become airborne as aerosols from buds or sap, as for example when lawns are cut (Rowe et al. 1986; Morrow-Brown 1989; Fernandez-Caldas et al. 1992). Allergen can also be present in microdroplets formed in mists and fog. Mature pollen grains of some species can rupture through osmotic shock. As they burst, the starch granules inside the grain are released into the airflow. A single mature pollen grain from Lolium perenne (ryegrass) contains about 700 starch granules in the size range 0.6– 2.5 μm, each containing allergen (Fig. 45.3). Interestingly, this phenomenon of granule release was first observed by Charles Blackley (1873), but its significance was not recognized until more recently in the context of thunderstorm asthma (Davidson et al. 1996; Newson et al. 1997; Venables et al. 1997). It does not in itself explain the phenomenon of thunderstorm asthma as grains

Fig. 45.3 Grass pollen rupturing under osmotic shock and releasing allergenic granules (0.6–2.5 mm). Magnification ×400. (See CD-ROM for color version.)

burst in ordinary rainfall episodes but these are not associated with excess asthma.

Pollen and air pollution Pollen counts are typically much lower in cities than in the surrounding rural areas but prevalence rates of hay fever and pollen-related asthma are frequently as high or higher (D’Amato et al. 2002). The abundance of airborne allergens and certain air pollutants (e.g., vehicle particulates, photochemical gases) is influenced by the same factors, such as temperature and rainfall, so their peak concentrations often coincide. Air pollution exerts both direct and indirect effects on the amount of pollen in the air and its allergenicity. Indirect effects result from growth stress on the plants, reducing productivity so that fewer and smaller pollen grains are produced. However, evidence indicates that in these cases the pollen grains have enhanced allergen content per weight. For example, Bet v 1, one of the main allergens of birch, is similar to pathogenesisrelated proteins, and is present in increased amounts in trees which are environmentally stressed (Ahlholm et al. 1998). Direct effects can occur through exposure of pollen on the plant or during flight in air. The surface of the grains is slightly sticky, which results in particles collecting on the surface. Pollen allergens can interact with air pollution particles and can modify their antigenic potency. Diesel exhaust emissions are thought to stimulate IgE synthesis, facilitating allergic sensitization in predisposed subjects (D’Amato 2000). The proteins on pollen may be modified by exposure to gaseous pollutants. This is important, for example, in the case of leukotriene (LT)B4-like pollen-associated lipid mediators (PALMLTB4), which can activate and stimulate both eosinophil and neutrophil leucocytes (Thiel et al. 2005). In a comparison of birch pollen from western and southern Germany,

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Kashe et al. (2005) found significant differences in proinflammatory substances. Urban pollen had greater chemotactic activity on human neutrophils than rural extracts and the authors concluded that air pollution seems to have enhancing effects on release of PALMLTB4 from birch pollen. Evidence indicates that nitrous oxides and ozone can cause nitrate to be added to protein on pollen grains. For instance, birch pollen has seven tyrosine components and is readily nitrated by traffic smog. Of birch pollen exposed by a busy road for a few days, 10% exhibited nitrate addition, and it was found that nitrated proteins bind more strongly to the antibodies involved in allergic reactions (Franze et al. 2005). The mode of action leading to enhanced allergenicity is unclear but the authors suggest that possibly the body uses tyrosine nitration as a marker to attract antibodies to inflamed tissue, although this is unlikely to be the only factor. Increased CO2 concentrations and amounts of ultraviolet light lead to stronger pollen allergenicity exacerbated by air pollution (Frenguelli 2002). Research indicates that either protein modification occurring following cell stress can lead to increases in cross-linker transglutaminase in/on pollen, which has a role in allergy-associated inflammation, or increased allergenictiy occurs via activation of secretory phospholipase A2 responsible for inflammation responses in humans following pollen contact (Brunner et al. 2002; Del Duca & Serafini-Fracassini 2005).

Sources of information

Fig. 45.4 Burkard volumetric traps at the national network pollen monitoring site at the National Pollen and Aerobiology Research Unit, University of Worcester, UK. (See CD-ROM for color version.)

Pollen counting and other traditional methods Information about the pollen and spore load of the air comes mainly from volumetric trapping but other simpler methods such as gravitational sampling may be used to produce basic data in some parts of the world. Two volumetric techniques are applied most frequently. One is the Rotorod system, which was widely used in the USA until recently for routine counting. It consists of a rotating impact sampler that collects particles on the surfaces of two upright metal arms (3-cm long) mounted on a small motor. The arms rotate at about 2500 rpm, so theoretically the volume of air passing over the surfaces can be calculated. The Rotorod system provides information on average concentrations over the duration of sampling time. It is relatively cheap and simple to operate but is generally considered to be unsuitable for work related to allergy, although it is widely used for pollen dispersal surveys for agricultural and horticulture. The other main technique is suction trapping. In this category, the most widely used system is based on the Hirst (1952) spore trap. These volumetric traps are made for continuous isokinetic sampling, although this parameter may not be fulfilled in all wind conditions. Air is sucked into the trap at a rate of 10 L/min through a narrow slit, which is oriented toward the direction of the prevailing wind by a vane (Fig. 45.4). The

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air flows over a rotating drum coated in adhesive, which moves past the orifice at 2 mm/hour to give a time-related sample. The internal dimensions of the trap provide a system that is aerodynamically efficient at capturing particles in the size range of pollen and many fungal spores. The volumetric spore trap is ideal for routine pollen survey work and is a useful compromise of many conflicting requirements. Occasionally, aerobiological reports are published which include general pollen surveys from other sampling methods. For instance, depositional or gravitational samplers were used extensively for many years and are still utilized in some studies. These inert, passive collectors have been favored for their cheapness and simplicity, but the information they yield is very limited. The Durham or gravity slide does not measure actual deposition or give a reasonable indication of airborne concentrations, because the catch is a function of wind speed and turbulence at the site. At best, these samplers give an idea of what pollen types are present in the atmosphere so that crude pollen calendars may be drawn.

Using pollen counts Pollen counts from volumetric traps are usually given as the daily average number of grains of a particular pollen taxon per

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cubic meter of air sampled over the 24-hour period. Several aspects need to be noted here, especially that pollen concentrations can vary greatly during the day even when the weather does not change. On showery days, the average pollen count may be low, but short-term peaks during dry intervals may trigger allergic reactions. In these cases short-term peak counts may give better correlations with symptoms. The pollen traps are usually situated on rooftops in exposed areas in order to sample from a mixed airflow. The resultant figures are essentially background readings and do not relate directly to the concentrations of exposure for individuals. If attempts are being made to relate symptoms to daily pollen counts or other environmental data, care needs to be taken to coordinate the time periods. In the UK, pollen count days run from 0900 hours local time but in other countries they may run from 1200 or 2400 hours. It is well known that pollen concentrations vary greatly over short distances, both vertically and horizontally (Emberlin et al. 1990). The counts should be considered as a guide to the conditions in the surrounding areas and not the specific level of exposure. In most pollenmonitoring networks, the sites are located considerable distances apart to represent various climatic and vegetation types. In each case, details of the surroundings of individual sites, such as topography, vegetation and altitude, are used to aid interpretation of the results and extrapolation to a wider area. The onset and the severity of pollinosis symptoms do not always relate closely to the magnitude of daily pollen counts or indeed the course of pollen seasons. The factors mentioned here account in part for this, but it should also be remembered that symptoms may have a delayed response. If lag times are incorporated into the analysis and peak 2hourly pollen concentrations are used instead of daily averages, then a closer correspondence may be revealed. Another source of potential difficulty is that the pollen count is for intact grains so the technique does not take into account submicronic allergenic particles. Also, proteins may be leached from grains and pollen may be resuspended after deposition, especially in urban environments. In these cases, the grains may not evoke allergic reactions.

Monitoring plant aeroallergens using immunologic techniques Attention is currently focused on developing reliable methods of assessing the aeroallergen content of the air rather than counting pollen grains. Until recently most researchers in this field used high-volume samplers (HVS), mainly because of the need to collect sufficient allergen for quantification. These are usually combined with cascade impactors to fractionate particles by size classes, which are then collected on filters. The capture efficiency for particles over 0.3 μm diameter typically reaches 96– 99%. If the approach is used with radioallergosorbent assay (RAST), it can be very sensitive but there are several major disadvantages. One of the main problems is the use of large filters, which need to be

Grass, Tree, and Weed Pollen

subsampled, require long elution times, and yield samples subject to considerable dilution. Also, the sampling is nonisokinetic and usually nondirectional. The airflow rates range up to 1.2 m3/min. This fast flow may rupture pollen grains, producing bias in the samples. Some of these problems have been overcome by using low-volume samplers (LVS). The results give a better representation of the allergen challenge and may be compared with the usual volumetric pollen counts more easily. This aspect is important because volumetric pollen counts are likely to remain the standard source of information for some time. LVS have been used in conjunction with cascade impactors and RAST or enzyme-linked immunsorbent assay (ELISA) techniques to produce quantification of allergens. Like the HVS, LVS systems mainly use filters for particle collection. The main problem with these is the time taken for elution and the large volumes of solvent needed. LVS are less likely to damage pollen grains and so generally produce a better representation of the size ranges of particles in the atmosphere. The Finnish Bioaerosol sampler (Rantio-Lehtimaki et al. 1987) was a major advance in LVS, particularly because it has a bifurcated flow system to overcome some of the fractionation problems inherent in other samplers. A nonfractionating directional cyclone sampler was developed to collect particles directly into a dry Eppendorf tube for immunologic analysis or for particle counting (Emberlin & Baboonian 1995). This is an LVS (16 L/min) providing a quick and relatively cheap method for routine assessments of the aeroallergen content of the atmosphere. Another cyclonebased system has been developed which has a larger volume of flow (200 L/min) and samples directly into eluting liquid but is nondirectional (Thibaudon et al. 2006).

Pollen monitoring networks The USA and most countries in Europe have well-developed pollen monitoring networks that provide quality-controlled data and forecasts, but other regions have either no service or very limited coverage. In some cases only one part of a country has a service, e.g., eastern Canada, or there are very few sites, e.g., Australia has only about six. There are virtually no pollen monitoring sites in Africa, and very few in Asia, the Far East, and South America. For these areas some general information about pollen seasons is available based on vegetation type, phenology, and climate (Emberlin 2002a,b).

European Pollen Information European Pollen Information (EPI) links the national pollen monitoring networks of 29 countries to provide a coordinated quality-controlled service. It was originally formed as the European Aeroallergen Network (EAN) in 1986 with 12 countries and started its central database of European pollen counts in 1988. It has grown steadily to its present coverage of 32 countries and a total of 339 sites; all use volumetric traps based on the Hirst design (Fig. 45.5) and must adhere to

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Active stations 1985

©2006 EAN

Active stations 2000

©2006 EAN

Active stations 1995

©2006 EAN

Active stations 2006

©2006 EAN

Fig. 45.5 Growth of the European Pollen Information network. (Maps by permission of European Pollen Information Ltd.) (See CD-ROM for color version.)

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certain standards of procedure. The database, which is held in Vienna, is a unique resource of daily pollen counts for all allergenic taxa, in some cases extending for several decades. Data are used for a variety of purposes including producing generalized maps of pollen seasons across Europe, analysis of trends in relation to climate change, and provision of international pollen datasets for clinical trials. Further information is available on www.polleninfo.org which also gives links to national pollen network sites. Most of the member countries of the EPI issue regional pollen forecasts and a few include fungal spores, but the coverage differs considerably. Some countries, such as the UK, France and Italy, have numerous sites whereas others, such as Greece, have very few.

The USA network The USA network is run by the National Allergy Bureau (NAB) under the auspices of the American Academy of Allergy Asthma and Immunology. There are about 86 stations, most using Hirst-type volumetric traps. Data on spores are not accepted from the few sites that still use Rotorods. Sites can select which pollen types to count from an approved list (Table 45.1) based on the vegetation at each location. Not all stations count spores. All sites must have certification, which initially includes taking an approved aerobiology course, an online written examination, and a practical test in pollen and spore identification. Recertification entails maintaining active status as a network station, taking the online examination every other year, attending a pollen/spore course at least once every 5 years, or passing the pollen and spore slide tests again. Pollen and spore data are sent to a central database maintained by Data Harbor and are used for providing information on the NAB website (www.aaaai.org/nab/index.cfm).

Table 45.1 USA National Allergy Bureau: list of recommended pollen and spore types for monitoring sites. Pollen Acer Alnus Ambrosia Arecaceae Artemisia Asteraceae (excluding Ambrosia and Artesimia) Betula Carpinus/Ostrya Carya Celtis Chenopodiaceae/Amaranthaceae Corylus Cupressaceae Cyperaceae Eupatorium

Grass, Tree, and Weed Pollen

Fagus Fraxinus Gramineae/Poaceae Juglans Ligustrum Liquidambar Morus Myrica Olea Pinaceae Plantago Platanus Populus Prosopsis Pseudotsuga Quercus Rumex Salix Tilia Tsuga Typha Ulmus Urticaceae Other tree pollen Other weed pollen Unidentified pollen Fungal spores Agrocybe-type basidiospore Alternaria Botrytis Cercospora Chaetomium ascospore Cladosporium Coprinus-type basidiospore Curvularia Diatrypaceae ascospore Dreshslera/Helminthosporium Epicoccum Fusarium Ganoderma basidiospore Leptosphaeria-type ascospores Nigrospora Oidium/Erysiphe Penicillium/Aspergillus Periconia Peronospora Pithomyces Pleospora ascospore Polythrincium Rusts Smuts/Myxomycetes Stemphylium Torula Undifferentiated ascospores Undifferentiated basidiospores Other fungi Unidentified fungi

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Grass

Table 45.2 The main subfamilies and tribes of the grass family. Examples of genera are given for each tribe.

Grass pollen is ranked among the top plant aeroallergens, especially in temperate areas, including most of Europe and North America. In many parts of the world it is the allergen most implicated in allergic rhinoconjunctivitis and for asthma it is second only to house-dust mite (Burr et al. 2003). Grasses belong to the class Liliopsida (monocotyledons) in the family Poaceae, also known as Gramineae. This is extremely large, consisting of about 620 genera and 10 000 species, of which over 1000 occur in North America and over 420 in Europe. Grasses are highly successful ecologically and are present in every type of terrestrial habitat. It is estimated that grasslands cover about 20% of the world’s surface. The majority of species have cosmopolitan distributions; for instance, many have ranges that extend throughout the northern hemisphere. Grass pollen is extremely difficult to avoid. It can even persist indoors in dust well after the pollen season (Fahlbusch et al. 2000). Typically grasses are specialized for wind pollination. In most cases vast amounts of pollen are produced, but the quantities per anther differ considerably between species. Generally, grasses of lowlands and meadows tend to produce more pollen than those of acidic moorlands and uplands. Some grasses are regularly self-pollinating, including many cultivars, such as species of Avena (oats), Oryza (rice), and Triticum (wheat).

Subfamily Bambusoideae Tribe Oryzae Oryza (rice) Zizania (wild rice)

Subgroups and cross-reactivity

Tribe Andropogoneae Andropogon (beard grass) Zea (corn or maize)

In evolutionary terms, the grass family is relatively young as recent fossil evidence places their origins at about 65 million years ago. Although speciation has been rapid, the family has not diverged very widely in its physiology. Some crossreactivity is seen between species that are closely related, such as between members of the same tribe, subfamily, or genus (Table 45.2). For instance, a high grade of immunologic similarity exists among the Pooideae subfamily, which includes many widespread allergenic genera, such as Dactylis, Festuca, Lolium, Poa, and Holcus. The antigens from species in other subfamilies differ from those of the pooids and also from each other. This can be illustrated by the arundinoids, chloridoids and panicoids, which show relatively little crossreactivity outside their taxonomic groups but some exceptions to this general pattern do appear (Muller et al. 1996). Potential cross-reactivity exists between the pollen of a grass species and its edible grain or even related ones, for example between rye pollen and rye flour and also between rye pollen and wheat flour.

Pollen grains Grass pollen grains are spherical to ovoid and range from about 8 to 122 μm, but most are between 30 and 60 μm. They have one circular or ovoid pore, usually 2– 8 μm, surrounded

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Subfamily Arundinoideae Tribe Arundineae Arundo (giant reed) Phragmites (common reed) Subfamily Chloridoideae Tribe Eragrotideae Eragrostis (lovegrass) Tribe Chloroideae Bouteloua (grama) Buchloe (buffalo grass) Cynodon (Bermuda grass) Tribe Muhlenbergia Muhly Tribe Zoysieacae Hilaria (galleta) Subfamily Panicoideae Tribe Paniceae Digitaria (crabgrass) Panicum (switch grass, millet) Pennisetum (elephant grass)

Subfamily Pooideae Tribe Poeae Briza (quaking grass) Cynosurus (dogtail) Dactylis (cocksfoot or orchard grass) Festuca (fescues) Lolium (ryegrass) Poa (meadow or June grass) Anthoxanthum (vernal grass) Holcus (velvet grass) Tribe Agostideae Agrostis (bent grass) Alopecurus (foxtail) Phleum (Timothy grass) Tribe Bromeae Bromus (brome grass) Tribe Triticeae Agropyron (couch grass) Hordeum (barley) Secale (rye) Triticum (wheat)

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by a thickened protruding ring (an annulus) and covered by an operculum (Fig. 45.2). The allergens are complex. For example, Lolium perrene (ryegrass) pollen contains at least 32 antigens, of which 13 could be bound by IgE sera from 11 patients with well-established allergic rhinitis (Ruden & Steinman 2004). Molecular masses of characterized allergens range from 11 to 57 kDa. Most grass pollen grains appear very similar under the light microscope, so it is difficult to distinguish those from different species. In routine counting, grass pollen grains from all species are grouped together. This is generally satisfactory due to the high degree of cross-reactivity between them. The latest immunologic techniques for assessing aeroallergen load also group the grasses together.

Pollen seasons Timing In northern hemisphere temperate climates most species of grass flower between mid-May and the end of July, with the greatest number flowering in June. However, some species will flower much earlier while others may flower throughout August. Certain species, such as Poa annua, will flower throughout the year, except during very cold winters. During mild winters, up to 20 species with fully developed flowerheads have been observed in December and January at Kew Gardens in London (Hubbard 1992). In Mediterranean climates, even more grasses are able to flower during the winter, but the majority will flower in the spring and early summer. In any one region, a progression of flowering of different species occurs through the growing season. In Europe, grass pollen seasons usually start first in the southern Mediterranean where the main season extends from late April to the end of May. This peak travels northwards through France and Germany in May and June, reaching Scandinavia in June and July (Fig. 45.6). The timing of seasons can vary considerably annually, depending mainly on cumulated temperatures over 5.5°C and precipitation in the months proceeding the flowering period. For example, the start date of the grass pollen season in London differed by 30 days over the 31 years 1971–2002 (Emberlin et al. 1993; Smith 2003). The progress of start dates over large areas also differs from year to year, as a function of seasonal weather patterns rather than average climate. For instance, data from the UK national pollen count database shows that the lag time between start dates in the Isle of Wight, in southern England, and Edinburgh, in Scotland, a difference of 5.5° latitude, was 25 days in 2005 but only 13 days in 2006. The duration of grass pollen seasons in many areas of Europe are becoming slightly longer due to changing weather patterns.

Severity of seasons The severity of the grass pollen season in any location depends on the climate, the species spectrum of the vegetation, the amount of pollen produced, and the weather during flowering. The last three aspects can result in considerable differ-

Grass, Tree, and Weed Pollen

ences, even within the same broad climatic zone (Fig. 45.7). On a continental scale, seasons are shorter and tend to be less severe with increasing latitude. Pollen production is governed by many factors, including growth conditions during the pollen formation period. Warm wet weather at this stage is conducive to high pollen productivity. When the flowers are mature, the release and dispersal of pollen depend on having dry weather, ideally with warm conditions and a light wind. Grass pollen seasons in both temperate and Mediterranean areas tend to vary annually, reflecting fluctuations in precipitation and temperature. For example, the total of daily grass pollen counts in London was 4424 in 2004, when only 21 days had counts over 50 grains/m3, but was 10 791 in 2006 with 45 days on which counts exceeded 50 grains/m3. Contrasts of a similar magnitude can occur in Mediterranean climates. For instance, in Cordoba, in central southern Spain, the total daily grass pollen count was 7543 in 2003, with 25 days over 50 grains/m3, but was 1689 in 2005, with 7 days over 50 grains/m3 (data kindly supplied by Profesor Carmen Galan, University of Cordoba). The pollen record from north London (Emberlin et al. 1993; Smith 2003) is one of the longest continuous datasets for pollen counts in the world. Analysis of this dataset reveals some interesting trends in the light of reports that the prevalence of pollen allergy has increased notably over the last 30 years (Law et al. 2005). Decreases in the severity of the grass pollen seasons in London have occurred, particularly between the 1960s and the early 1970s but also over 20 years to 1990. This decrease seems to be related directly to changes in land use and agricultural practices. Similar patterns of decline in grass pollen counts are evident for these periods at Derby (Corden & Millington 1991), but land-use statistics do show increases in grassland areas for some parts of the UK, such as South Wales, and it cannot be assumed that the pollen counts declined in parallel over the whole country. During the last few years grass pollen counts in the London area have increased again (Fig. 45.8). This trend seems mainly due to recent changes in weather patterns, which are very conducive to grass growth and pollen release (Emberlin & Adams-Groom 2004).

Daily and diurnal variation The main controls on daily variations in grass pollen counts are precipitation, temperature, wind speed, and relative humidity. The operation of these factors is broadly similar in different climates, but interrelationships vary. For instance, in Mediterranean climates high temperatures are negatively related to high pollen counts after the peak of the season, whereas they are positively related through the whole season in northwestern temperate climates (Galan et al. 1995). Pollen counts can differ markedly from day to day, especially in temperate areas with varied weather (Fig. 45.9). Grass species have fairly regular daily flowering patterns, although the florets will remain shut on dull or wet days. In

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Fig. 45.6 Monthly course of the grass pollen season over Europe in 2006. (Maps by permission of European Pollen Information Ltd.) (See CD-ROM for color version.)

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the early evenings because pollen will take some time to be transported from rural areas. On dry sunny days in summer the pollen will be kept aloft and the air mixed due to convection currents until solar heating decreases. For example, in central London major source areas of grass pollen are at least 20 km away. On dry summer days peak concentrations of grass pollen feature in the evening from about 1600 to 2100 hours (Norris-Hill & Emberlin 1991). Occasionally, pollen concentrations have been observed to increase to short-term peaks with the sudden onset of heavy convectional showers. Strong downdrafts associated with the start of precipitation may transport particles, including pollen, to ground level (Norris-Hill & Emberlin 1993).

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Trees

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Fig. 45.6 (Cont‘d )

most species, flowering occurs in the early morning, typically between 0400 and 0900 hours, with a few about noon and some in the afternoon and evening from 1500 to 1900 hours. These periods of pollen release produce distinct patterns of diurnal variation in pollen concentrations. On dry days in rural areas, peak grass pollen concentrations will usually occur from early to mid morning with a second smaller peak during the late afternoon and evening. In large cities the peak grass pollen concentrations on dry days tend to occur in

The allergens of temperate-zone trees are acidic proteins with molecular masses of about 20 kDa and are mostly structurally and immunologically similar resulting in marked crossreactivity between different taxonomic groups. There are also numerous cross-reactions with foods, e.g., Bet v 1 (the major allergen of birch) with apples and other fruits of the Rosaceae. Tree pollen seasons differ more in severity than those of grass for the reasons explained earlier in this chapter. Consequently symptoms may differ greatly in severity annually and may be absent in some years. The timing of pollen seasons for trees depends more directly on weather factors as typically trees are not dependent on photoperiod. In many temperate areas the start of the tree pollen season is becoming earlier due to changing weather patterns, especially milder winters and warm springs, while the grass and weed pollen seasons are extending longer into autumn. This means that overall the hay-fever season is getting longer. The impact of warming climates on the start of tree pollen seasons differs geographically, with most effect occurring in northern and central Europe where sufficient cold still occurs to break dormancy. Examples are given in the following sections on selected tree types. It is not possible to include details

Daily average count grains/m3

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on all allergenic tree types in this chapter. Further information is available on pollen websites.

Alder Alder (Alnus) is in the Betulaceae (birch family), which also includes Betula (birch), Corylus (hazel), and Carpinus (hornbeam). The group contains some 100 species distributed across the northern temperate regions. The genus Alnus has 30 species distributed across the northern hemisphere, Peru, and western China. Within this distribution range, alders are common deciduous trees. In North America, alders are fairly frequent in all regions except the southern and central areas. In Europe, alders are found in all temperate climates, east to Siberia and south to North Africa, but the majority of species thrive on wet soils so they are most frequent in the lowlands. Alders are typically wind pollinated and shed large amounts of pollen in early spring. The grains range from 19 to 30 μm in diameter, with a smooth surface containing five pores (occasionally three or seven) connected by raised ridges. In oceanic temperate climates Alnus is the first airborne pollen of the growing season and may occur from mid-December onwards in mild years in the southern and central parts of its range, and from March in the northern parts. Peak daily average concentrations in northern Europe usually occur between late February and mid-April. Over the last decade the start date for pollination of Alnus has become noticeably

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Fig. 45.9 Daily grass pollen counts for 2005 in London and Worcester, UK. (See CD-ROM for color version.)

earlier over much of northern Europe, in response to milder winters and warmer springs (Emberlin et al. 2006). Sensitization to Alnus pollen extract has been recorded frequently (Smith et al. 2006), but this is not matched in the expression of symptoms. The importance of the Alnus pollen season lies mainly in the cross-reactivity with birch pollen. The major allergens of alder (Aln g 1), birch (Bet v 1), hazel (Cor a 1), and hornbeam (Car b 1) have many features in common (Ipsen & Hansen 1991), although some of the allergenic properties of Alnus pollen have been described as species specific. Alnus pollen acts as a primer, making allergic people more sensitive to other pollens later in the season.

Birch The genus Betula (birch) is by far the most important of the Betulaceae as a cause of symptoms of pollinosis and asthma. Betula pollen is well known as a significant aeroallergen, especially in northern European countries, where it appears among the top-ranked allergenic pollens. Estimates suggest that between 10 and 20% of the population of northern and central Europe are allergic to birch pollen and that prevalence rates are increasing (Spieksma et al. 1990; Moverare et al. 2006). The genus Betula includes some 40 species distributed throughout the northern hemisphere. For example, Betula pendula (silver birch) grows wild throughout the whole of Europe, east to Siberia, and closely related species extend

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through North America. They are also planted extensively as ornamental trees. Birch trees shed large amounts of wind-borne pollen. The grains range between 19 and 22 μm in diameter and have a distinct appearance, with three collared pores. The antigenic and allergenic material is located mainly inside the pollen grain within the cytoplasm; however, some is also present in various parts in the pollen wall and in the aperture regions (Grote et al. 1988). The surface allergens can diffuse from the grain within seconds of being deposited on the mucous membranes, while the capsule antigens can diffuse through the apertures from the cytoplasm within minutes and most of the antigen diffuses out of the grain within 45 min. Cross-reactions, with both other Betulaceae and other tree pollens, contribute to a prolonged season of symptoms for many allergic people. For example, cross-reactivity occurs between Fra e 1 from ash (Fraxinus excelsior) pollen and the major allergen of olive, Ole e 1, and also with Bet v 1 and with group 11 grass pollen allergens (Niederberger et al. 2002).

Start dates of seasons The birch pollen season starts typically in the southern Mediterranean in early March and progresses northwards, reaching France, Germany and Austria in early to mid April and Scandinavia in late April to mid-May. The factors that exert the most influence on trends in start dates and fluctuations are mean day temperatures in the period preceding pollination or anthesis (Emberlin et al. 2002). The earliest starts of the Betula pollen season occur with a combination of a warm autumn, a cold snap in the winter to achieve vernalization, followed by a warm spring with dry weather in March and April. Evidence from the records of the pollen monitoring sites in northwestern Europe shows a trend toward earlier start dates (Emberlin et al. 2002) due to warmer spring temperatures. For example, in London the start dates for birch pollen seasons have become earlier by about 5 days per decade over the last 30 years (Fig. 45.10).

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In southern Scandinavia, birch pollen may be present in the air before the start of local flowering because of longrange transport of pollen from areas further south where the season is more advanced (Ranta et al. 2006). Birch-sensitive patients have been found to experience symptoms about 2 weeks before the birch pollen was released locally. Little work has been done on dose–response relationships between concentrations of Betula pollen and clinical symptoms, apart from the research of Viander and Koivikko (1978) and Koivikko et al. (1986). These workers reported that 90% of patients with Betula allergy had mild symptoms at pollen counts of 80 grains/m3 or more in the early season and that 80% of patients remained symptomatic at a level of about 30 grains/ m3 during the late season. The prevalence of Betula pollinosis is greatest in northern Europe, especially Scandinavia.

Annual variations in severity Season severity differs markedly, partly because of the weather during dispersal but also because of preseason factors and inherent rhythms of productivity. Long-term records indicate that birch trees often have alternating high and low years of pollen production in northwestern Europe. Results suggest that this pattern may become established for a run of years and then may fade or switch (Spieksma et al. 1995). There is some evidence of synchronization across regions and it is likely that these rhythms are similar to those exhibited by apple trees, in which the developing fruits produce hormones that reduce flower-bud development the following year. Environmental factors, such as severe frost or drought, can induce this cycle. This can result in a series of years with alternating high and low Birch pollen seasons after a period of years during which there have been no alternating rhythms (Lavee 1989).

Chestnut Chestnuts belong to the Fagaceae family, together with beeches and oaks. The 10 species of the chestnut genus (Castanea)

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have a natural distribution ranging through southern parts of north temperate regions. Sweet chestnut (Castanea sativa) is endemic to southern Europe across to west Asia and south to North Africa but its distribution has been extended deliberately through history. Several authors (e.g., Laurent et al. 1993) suspect that chestnut pollinosis has been overlooked in many places, possibly because chestnut pollen is released later than that of most other trees. For example, in France and southern Britain it usually features during the first half of July (Emberlin et al. 1990), coinciding with the second half of the grass pollen season. In the USA the season extends from May to July. In some French cities, the quantity of pollen released by sweet chestnut exceeds that produced by either birch or oak (Sutra & Peltre 1990), but the grains are smaller in size, ranging from 10.8 to 13.8 μm in diameter. The start of pollination is usually sudden and intense (Sutra et al. 1987). The resulting clinical manifestations are severe, with adverse effects on asthmatic patients. Cross-reactivity is common among the Betulaceae and the Fagaceae, as demonstrated by Bessot et al. (1981) with RAST inhibition and by Sutra and Peltre (1990) with immunoblotting techniques.

Cypress The cypress family (Cupressaceae) has a worldwide distribution. It is divided into three subfamilies, Thujoidaeae, Cupressoidaeae and Juniperoidaeae, which together comprise 16 genera and 140 species. The main genera in the cypress family are Cupressus, Juniperus, Callitris, Tetraclinis, Libocedrus, and Chamaecyparis. The genus Cupressus itself includes 12 species, which differ in their geographic ranges. For example, Cupressus sempervirens and Cupressus arizonica are common in southern France and in Italy. Cupressus alba extends through most of Italy, and cultivars of Cupressus lawsoniana are planted frequently in the UK as a quick-growing evergreen. In North America Cupressus is common in the southwest. The majority of pollinosis cases in the USA attributable to cypress allergy have been ascribed to Juniperus (red cedar, mountain juniper), but other genera may be involved (Steinman & Ruden 2005). Cypress pollen was not considered to be allergenic until the mid 1970s, although a number of cases of allergy to C. sempervirens had been reported from both France (Panzani 1962) and Italy (Tas 1965). Also allergic reactions to the pollen of the related mountain cedar (Juniperus sabinoides) had been described in the USA (Black 1929). Since 1975, an increasing number of cases have been noted in southern Europe, especially in the south of France, Italy and Spain, and in Israel (Bousquet et al. 1984; Charpin et al. 1993). This allergy may have been underestimated because the symptoms may have been attributed to other allergens present at the same time of year. Apparent increases in prevalence in some regions may be due to more extensive planting of cypress trees. All the cypress subfamilies are wind pollinated. The grains are mostly sphericoidal, ranging in size from fairly small (about 25 μm diameter) to rather large (about 44 μm diameter).

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Throughout most of southern Europe, the cypress pollen season begins in January and continues until the end of March or April. The peak season in France, Spain and Italy occurs between mid-February and the end of March, varying with latitude and local climate. In northern regions, such as the UK, the peak season occurs in April or May. The quantity of pollen present in the air differs greatly daily and annually. Average daily pollen counts can be relatively high, often reaching 1500 grains/m3 locally in the main season. Cypress pollen may travel a long way on the airflow, affecting regions downwind at considerable distances from the sources.

Cross-reactivity Pham et al. (1994) demonstrated frequent cross-reactivity between members of the Cupressaceae. For example, in 80% of cases, skin tests to Juniperus communis correlated with skin tests to C. sempervirens, but with Thuja orientalis the correlation was lower (60%). Soman et al. (2000) characterized IgEbinding epitopes of mountain cedar allergen. The results indicate that pollen allergenic cross-reactivity is much more wide-ranging than is generally appreciated. White cypress pine pollen (Cupressus glaucophylla), which has been implicated as an important seasonal allergen in parts of rural Australia, has shown cross-reactions with birch, grass and olive. These results could not be explained on the basis of taxonomic relationships. Sugi pollen (Cryptomeria japonica), one of the most important aeroallergens in Japan (Saito 1993), belongs to the Taxodiaceae family, which also includes swamp cypress and sequoia and is closely related to the Cupressaceae. Crossreactivity between the pollen of C. sempervirens (Italian cypress) and Cryptomeria japonica has been demonstrated by Panzani et al. (1986).

Olive In Mediterranean regions, pollen from olive trees (Olea europaea) is a major cause of pollinosis and can make asthma worse (Wheeler 1992). Olive pollinosis has been reported throughout most of the olive-producing areas bordering the Mediterranean, particularly in Italy, Spain, France and Greece. In favorable locations, such as Bari in Italy and Cordoba in Spain, over 55% of entire areas are devoted to olive production. In Spain the percentage prevalence of Olea pollinosis has increased in recent years (Hidalgo et al. 2002) and since 1988 it has ranked more highly than grass pollinosis. In the Bari region, the prevalence of olive pollinosis is estimated to be 30–40% of all pollen-sensitized subjects. Olive flowers from January through to June depending on location, with the peak season from mid-April to the end of June. The trees are insect pollinated, but large amounts of pollen become airborne. The pollen grains are relatively small (17–20 μm in diameter). Typically, the pollen season is quite short, usually lasting about 40 days, but it can be very intensive, with daily average pollen counts often exceeding thousands of grains per cubic meter. Flowering commences

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suddenly, giving a sharp start to the season, which is reflected in the abrupt onset of symptoms in sensitive people. In contrast, symptoms persist for several weeks at the end of the season when the pollen count has already decreased.

Severity of seasons Olive pollen seasons alternate between severe and moderate in a fairly predictable pattern. This phenomenon depends on competition between ripening fruits and developing buds (Orlandi et al. 2005). The pattern is reflected strongly in the severity of olive pollinosis each year. It is useful in forecasting and makes the short-term planning of therapeutic approaches possible.

Cross-reactions Many olive tree varieties have been developed and not all allergens are found in every olive cultivar. In Italy, evidence suggests that groups of patients from the north and the south, where different varieties of olive are grown, may have different IgE specificity (Wheeler 1992). However, work by Obispo et al. (1993) demonstrated the presence of a shared major allergen in the Oleaceae (olive family). This explains the high degree of cross-reactivity that has been demonstrated between O. europaea and other Oleaceae species, particularly Fraxinus excelsior (ash), Ligustrum vulgare (privet), and Syringa vulgaris (lilac) (Steinman & Ruden 2005). The existence of a common allergen in the Oleaceae family has clinical implications, due to the different geographic distributions of these plants. Fraxinus excelsior and Ligustrum vulgare are very common in northern Europe and North America, and sensitization to these may be more important than previously thought. Pollen from Fraxinus can be abundant in the air in northern and central Europe during April and May, about 3– 4 weeks later than in Mediterranean areas. Pollen concentrations of Ligustrum are low in all the regions and is likely to be significant only if there is direct exposure, such as in hedge-cutting.

Grass, Tree, and Weed Pollen

Chenopodiaceae and Amaranthaceae, within the order Caryophyllales. Both families contain numerous genera and species. Pollination methods differ, even within genera, with both anemophilous and entomophilous species occurring in closely related plants. The allergenic status of the species is largely dependent on the amount of pollen produced. The Chenopodiaceae, the goosefoot family, has 100 genera and about 1500 species. They are predominantly perennial herbs, adapted to soils with a high percentage of inorganic salts. Many are halophytes, often in arid areas. For example, of the 22 genera found in North America, 18 are in western regions on salt-rich soils. Atriplex, the genus of saltbushes, grows in arid soils and sheds large amounts of pollen. Fortunately, the areas are mainly of low population density. Kochia scoparia (burning bush) is significant in some areas, such as the upper great plains of the USA, where it has become the most prolific source of chenopod pollen. Within Europe, the family is represented by a number of species that are recognized as being allergenic, such as Chenopodium album, C. ambrosioides, C. murale, Atriplex hastate, Atriplese patula, and Beta vulgaris. Many occur throughout Europe, but the chenopods tend to be particularly important in certain areas. In Mediterranean regions, some chenopods are used for hedges (e.g., Atriplex halimus) or as garden plants. Salsola pestifera or S. kali (Russian thistle) is a dominant weed, mainly in dry or saline soils and sandy shores. It produces large amounts of pollen and is probably the most important allergenic chenopod in Europe. The amaranth family consists of 65 genera with about 900 species. These are mainly herbs, with a few shrubs and include some noxious weeds. The most important allergenic plants in the group are predominantly anemophilous, including species in the genus Amaranthus such as pigweed and water hemp. Amaranthus is common throughout the USA and southern Canada. In Europe, they are particularly important in southern regions, often coincident with the chenopods.

Chenopod pollen

Weeds Weed pollen is almost ubiquitous in the air during growing seasons and is often present locally at high concentrations near ground level. This abundance is typically not reflected in the readings from rooftop pollen traps. Many of the weed pollen seasons coincide with those of other allergenic plants, such as the grasses, and their importance for allergy may be underestimated. There are some exceptions, such as ragweed and Parietaria, which are well recognized as notable allergenic weeds.

Chenopods The chenopods are widespread weeds and shrubs of temperate areas, consisting of two closely related families, the

Most chenopod pollen grains are spheroidal and between 18 and 30 μm in diameter, with sizes differing between species. The pollen grains of chenopods are very similar, apart from the numbers of pores, and are usually described together in aerobiological studies. Flowering peaks from July to October, although some pollen may be in the air from the end of April through to December in warmer climates. Reports of daily variations from several locations indicate a diurnal peak from 1000 to 1500 hours, reaching a maximum at noon (Galan et al. 1989). In North America, the contribution from chenopod pollen differs greatly regionally. In certain areas, such as the western states, anemophilous chenopods shed large amounts of pollen and can make an important contribution to the pollen spectrum. Very little is known about the detailed characteristics of the allergens from chenopods. Studies have

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shown a high degree of cross-reactivity among the different chenopods and amaranths and have concluded that many of the species share common allergens, although some are unique.

Parietaria The nettle family, Urticaceae, contains two genera that are markedly different in their allergenicity but have pollen grains that are not easily distinguished. Parietaria (pellitory of the wall) is an important cause of pollinosis in Mediterranean Europe, especially in some parts of southern Italy and the southern coast of Spain, where it ranks as the most important allergenic plant (Steinman & Ruden 2006). In contrast, Urtica species (nettles) are generally regarded as being insignificant as allergens. There are six species of Parietaria, of which Parietaria officinalis and P. judaica are the most common. These two are separate ecologically, in that P. officinalis grows on hilly and mountain areas up to 1000 m altitude in many southern and central European countries, whereas P. judaica is more strictly Mediterranean, being warmth and light-loving, growing in coastal areas and typically colonizing walls. The species was introduced to the south coast of Britain by monks in the Middle Ages and has spread to London and the Midlands. Extensive cross-reactivity of the pollen extracts of P. officinalis and P. judaica had been reported (Corbia et al. 1985) but wider cross-reactivity seems more limited (Asero et al. 2004). In Mediterranean areas, Parietaria has two cycles of flowering. The first runs from March to late summer, with a peak between April and June, and then a shorter second period extends from August through to October (Guardia & Belmonte 2004). Parietaria produce vast amounts of light small pollen grains (approximately 12–16 μm diameter) that are wind dispersed and which can remain airborne for a long time. The genus has evolved a “spring-release mechanism,” whereby pollen leaves the plant even without air movement. In still air, pollen can accumulate to very high concentrations near ground level, which induce severe symptoms in sensitive people. The threshold of response after the priming effect is estimated to be about 30 grains/m3 (D’Amato & Lobefalo 1989). This is exceeded through much of the flowering periods, resulting in a long duration of symptoms in susceptible people.

Ragweed Ragweeds (Ambrosia species) are probably the most notorious hay-fever plants in North America but are less well known in Europe. Most of the 18 species are indigenous to the New World, but a few are cosmopolitan weeds. In the USA allergy to ragweed has a prevalence of about 26% (White & Bernstein 2003; Arbes et al. 2005). Ambrosia artemisiifolia and A. trifida alone are thought to cause over half of all hay-fever cases. The allergens of the various species are not identical but have sufficient similarity to invoke broad cross-reactions. Ambrosia occurs throughout most of the USA, but the abundance and the number of species growing vary regionally.

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In Europe, there is one native species, sea ragweed (A. maritima), which grows mainly on coasts round the Mediterranean. Four other species were introduced accidentally to Europe from North America with grain shipments during the 19th century and have become well established. For instance, silver ragweed (A. tenuifolia) has been naturalized in France and Spain, and the perennial ragweed (A. psilostachya) had spread to 11 countries by 1994 (Rich 1994). Ambrosia elatior spread to Hungary from the south in the 1970s and had colonized most of the country by 1990. It spread further through Europe in the 1990s and is now present extensively even as far north as the Netherlands (Fig. 45.11). In all these regions, the main pollen season runs from August to late September. During this time, ragweeds produce vast quantities of pollen. For instance, giant ragweed (A. trifida) can produce 8000 million pollen grains in just 5 hours. The grains are fairly small (16–21 μm in diameter), and can be carried for hundreds of kilometers in the wind. An average plant produces approximately 3000 viable seeds in the autumn, which retain their ability to germinate for up to 30 years. In suitable climates, ragweeds have an enormous potential to spread rapidly. Release of pollen starts about 0800 hours, when temperature is increasing and humidity is decreasing, and ends about noon. This pattern causes a morning peak in ragweed concentrations in the local area. In Austria, ragweed occurs sparsely in the country but large amounts of pollen are imported by southeasterly winds from sources 200 km distant in Hungary. This long-range transport of pollen takes about 10 hours so that concentrations reach a peak at midnight in Vienna and reduce to almost zero during late morning (Jaeger 1998). The threshold of response seems to be in the region of 20 grains/m3 (Makra et al. 2005). Several allergens have been isolated from Ambrosia, of which the most important are Amb a 1 (378 kDa) and Amb a 2 (382 kDa), representing about 6% and 3% of the pollen protein respectively. Amb a 3, Amb a 5, and Amb a 6 are also important but have lower molecular weights. The northward distribution of ragweed species in Europe is limited by climate, because seed maturation requires a longlasting autumn. If annual average temperatures in Europe continue to increase at the current rate, then ragweed could spread to be persistent as far north as central England by the year 2050.

Other weeds Less is known about the importance of pollen from most other weed groups. For example, pollens from mugwort (Artemisia), plantain (Plantago), and sorrel (Rumex) are generally regarded as only minor contributors to pollinosis symptoms, although they may be significant in some areas. In most of these cases, the clinical relevance of the weeds is not easily defined. Often, they are widespread but do not contribute much to the pollen spectrum. For instance, the Rumex genus (sorrels and docks) includes 18 different species spread

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Ambrosia 1990

Ambrosia 1995

none

none

very low

very low

low

low

medium

medium

high

high

very high

very high

Ambrosia 2000

Ambrosia 2006

none

none

very low

very low

low

low

medium

medium

high

high

very high

very high

Fig. 45.11 Changing distribution of Ambrosia spp. pollen in Europe. (Maps by permission of European Pollen Information Ltd.) (See CD-ROM for color version.)

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through a variety of different habitats in Europe. Species of Rumex occur throughout North America but are especially common in the western half of the USA, from southern Wyoming to eastern Arizona and New Mexico. All the species are wind pollinated, with most seasons starting about May and peaking between June and August.

Artemisia The genus Artemisia (wormwoods and mugworts) belongs to the Compositae family, which also includes the ragweeds (Ambrosia) and the feverfews (Parthenium). Several species of Artemisia occur as widespread weeds of disturbed ground in cities and rural wasteland in temperate areas. The abundance of the plants and the various species differ locally. The small inconspicuous flowers produce relatively modest quantities of pollen, which results in low airborne concentrations. In northern Europe, the flowering season runs from about late July to the end of August and in the south it occurs about 1 month later. The allergenic status of Artemisia is unclear.

Parthenium hysterophorus Sometimes allergenic weeds are spread to new areas accidentally. For instance, the South American weed Parthenium hysterophorus was introduced into India with cereal grains in the 1960s. It has extended its range over most parts of India and has resulted in the sensitization of a considerable proportion of the population. Similarly, cases of rhinitis due to exposure to Parthenium pollen have been reported from other regions where the weed has spread, including the USA and Australia (Sriramaro et al. 1991; Stewart 1993).

Mercurialis annua In some cases, the roles of long-established or indigenous species in provoking allergic disease may not have been recognized. Mercurialis annua (annual mercury), a member of the spurge family, is a long-lasting flowering weed, widespread in Europe and North America. In the 1990s, pollen from this species was found to cause sensitization and allergic disease in atopic patients (Garcia-Ortega et al. 1992). Sensitization was also found to develop in healthy nonatopic people if close contact was maintained through occupational exposure as, for example, in handling flowering plants daily (GarciaOrtega et al. 1992).

Acknowledgments The author is grateful to Professor Dr Siegfried Jaeger and Christoph Jaeger for supplying the pollen maps from the European Information Service; to Dr Estelle Levetin for help with information about pollen monitoring in the USA; to Professor Dr Carmen Galan for information about Spain; and to Rachael Marks and Sally Wall for help in preparing the manuscript.

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Grass, Tree, and Weed Pollen

Ipsen, H. & Hansen, O.C. (1991) The NH2-terminal amino acid sequence of the immunochemically partial identical major allergens of alder (Alnus glutinosa) Aln gl, birch (Betula verrucosa) Bet v I, hornbeam (Carpinus alba) Car b I and oak (Quercus alba) Que a I pollens. Mol Immunol 28, 1279–88. Jaeger, S. (1998) Global aspects of ragweed in Europe. Presented at 6th International Congress on Aerobiology. Sattelite symposium Procedings. Ragweed in Europe ALK Abello. Kashe, A., Klaus, S., Thiel, M. et al. (2005) Release of LTB4-like pollen associated lipid mediators (PALMLTB4) from birch pollen in different air polluted areas in West and Southern Germany. Allergy Clin Immunol Int 394 (1), p. 394. Knox, R.B. (1993) Grass pollen, thunderstorms and asthma. Clin Exp Allergy 23, 354–9. Knox, R.B., Suphioglu, C., Taylor, P. et al. (1997) Major grass pollen allergen Lolp1 binds to diesel exhaust particles: implications for asthma and air pollution. Clin Exp Allergy 27, 246–52. Koivikko, A., Kupius, R., Makinen, Y. & Pohjala, N. (1986) Pollen seasons: forecasts of the most important allergenic plants in Finland. Allergy 41, 233–42. Laurent, J.M., Lattanzi, B., Le Gall, C. & Sauvaget, J. (1993) Evidence for chestnut pollinosis in Paris. Clin Exp Allergy 23, 39–43. Lavee, S. (1989) Involvement of plant growth regulators and endogenous growth substances in the control of alternate bearing: growth regulators in fruit production. Acta Hortic 239, 311–22. Law, M., Morris, J.K., Wald, N., Luczynska, C. & Burney, P. (2005) Changes in atopy over a quarter of a century based on cross sectional data at three time periods BMJ 330, 1187–8. Linder, H.P. (2000) Pollen morphology and wind pollination in angiosperms. In: Harley, M.M. Morton, C.M. & Blackmore, S., eds. Pollen Morphology and Biology. Royal Botanic Gardens, London, pp. 73–88. Makra, L., Juhasz, M., Beczi, R. & Borsos, E. (2005) The history and impacts of airborne Ambrosia (Asteraceae) pollen in Hungary. Grana 44, 57–64. Morrow-Brown, H. (1989) Grass juice is an allergen as well as grass pollen. Abstracts of XIVth Congress of EACCI, Berlin, 17– 22 September. Moverare, R., Kosunen, T.U. & Haahtela, T. (2006) Change in the pattern of IgE reactivity to timothy grass and birch pollen allergens over a 20 year period. J Invest Allergol Clin Immunol 16, 274–8. Muller, W.D., Karanfilov, T., Bufe, A., Fahlbush, B., Wolf, I. & Jager, L. (1996) Group 5 allergens of timothy grass (Phl p 5) bear cross reacting T cell epitopes with group 1 allergens of rye grass (Lol p 1). Int Arch Allergy Immunol 109, 352–5. Newson, R., Strachan, D., Archibald, E., Emberlin, J., Hardaker, P. & Collier, C. (1997) Effect of thunderstorms and airborne grass pollen on the incidence of acute asthma in England, 1990–94. Thorax 52, 680– 5. Niederberger, V., Purohit, A., Oster, J.P., Spitzauer, S., Valenta, R. & Pauli, G. (2002) The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species. Clin Exp Allergy 32, 933–41. Norris-Hill, J. & Emberlin, J. (1991) Diurnal variation of pollen concentration in the air of London. Grana 30, 229–34. Norris-Hill, J. & Emberlin, J. (1993) The incidence of increased pollen concentrations during rainfall in the air of London. Aeroboligica 9, 27– 32. Obispo, T.M., Melero, J., Carpizo, J., Carreira, J. & Lombarero, M. (1993) The main allergen of Olea europaea (Ole e I) is also present

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in other species of the Oleaceae family. Clin Exp Allergy 23, 311– 16. Orlandi, F., Romano, B. & Fornaciari, M. (2005) Relationship between pollen emission and fruit production in olive (Olea europaea L.). Grana 44, 98–104. Panzani, R. (1962) L’ Allergie respiratoire aux pollens de coniferes. Rev Fr Allergol 2, 164– 8. Panzani, R., Yasueda, H., Shimizu, T. & Shida, T. (1986) Cross reactivity between the pollen of Cupressus sempervirens (common cypress) and of Cryptomeria japonica (Japanese cedar). Ann Allergy 57, 26–30. Pham, N.H., Baldo, B.A. & Bass, D.J. (1994) Cypress pollen allergy: identification of allergens and cross reactivity between divergent species. Clin Exp Allergy 24, 558– 65. Ranta, H., Oksanen, A., Bergmann, K.C. et al. (2006) Spatio-temporal patterns of annual loads of airborne birch pollen in Europe. Presented at 8th International Congress in Aerobiology, Neuchatel, Switzerland, August 2006. Rantio-Lehtimaki, A. (1991) Aerobiology in Finland: Information Service, Methodology and Allergological Applications. Reports from the University of Turku No. 28. Turku, Finland. Rantio-Lehtimaki, A., Kauppinen, E. & Koivikko, A. (1987) Efficiency of a new bioaerosol sampler in sampling Betula pollen for antigen analysis. Adv Aerobiol Exp 51, 383– 90. Reed, C.E., Swanson, M.C. & Yunginger, J.W. (1986) Measurement of allergen concentration in the air as an aid to controlling exposure to aeroallergens. J Allergy Clin Immunol 78, 1028–30. Rich, T. (1994) Ragweeds (Ambrosia L.) in Britain 1994. Grana 33, 38–44. Rowe, M., Bailey, J. & Ownby, D. (1986) Evaluation of the cause of nasal and ocular symptoms associated with lawn mowing. J Allergy Clin Immunol 77, 714–17. Ruden, S. & Steinman, H. (2004) Allergy-Which Allergens: grass pollens. Aller Res Int South Africa, p. 80. Saito, Y. (1993) Regional character of pollinosis in Kanto area. Allergy Pract 13, 28–32. Smith, M. (2003) Developing medium and long range forecast models for allergenic pollen in the United Kingdom. PhD Thesis, University of Worcester in conjunction with University of Coventry. Smith, M., Emberlin, J., Stach, A., Czarnecka-Operacz, M., Jenerowicz, D. & Silny, W. (2007) The regional importance of Alnus pollen as an aeroallergen: a comparative study of Alnus pollen counts from Worcester (UK) and Poznan´ (Poland). Ann Agric Environ Med 14, 123–8. Solomon, W.R., Burge, H.A. & Muilenberg, M.L. (1983) Allergen carriage by atmospheric aerosol I. Ragweed pollen determinants in smaller micronic fraction. J Allergy Clin Immunol 72, 443–7. Soman, K.V., Midoro-Hoiuti, T., Ferreon, J.C. et al. (2000) Homology modelling and characterisation of IgE binding epitopes of mountain cedar allergen Jun a3. Biophys J 79, 1601– 9.

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Fungi as Allergens Cathryn C. Hassett, W. Elliott Horner, Estelle Levetin, Laurianne G. Wild, W. Edward Davis, Samuel B. Lehrer and John Lacey Dedicated to the memory of John Lacey

Summary Fungal spores and particles occur widely as normal components in both indoor and outdoor atmospheres. Fungi in either environment are potential respiratory allergens, implicated in allergic rhinitis, asthma, and occupational asthma. Fungi are eukaryotic organisms that colonize various organic materials (subtrates), and thus most derive nutrients from digesting wood, paper, soil, dust, and so on, or infecting plants or less commonly animals. Substrates must contain adequate moisture to support fungal growth. Airborne spore loads indoors typically resemble the outdoor load but lower concentrations are found indoors, unless indoor materials are damp enough to support mold colonization. Fungi vary in size and morphology from single spores to large fruiting bodies. Fungal spores or fragments contain allergens and usually provide the initial encounter between fungi and lung surface. Species from any of the major fungal groups can be causes of respiratory allergy if their spores become airborne. Fungal allergens are generally proteins and glycoproteins. Methods to identify and characterize different fungal allergens have improved over the past several years, although fungal allergen extracts are still difficult to produce and in contrast to other aeroallergens most fungal extracts are not well characterized nor standardized. This, in addition to exposure and sensitization to multiple fungi, makes diagnosis of fungal allergy more difficult than other respiratory allergies. Occurrence of allergic symptoms is affected by the amount of fungal exposure. Fungal spores are almost always present in the air, but their numbers and types change with time of day, weather, season, and geographic location. Airborne spore loads may also be affected by local sources of spores, among which agricultural crops play a prominent role. Fungal growth may also produce metabolites like mycotoxins and microbial volatile organic compounds that may affect the lung but not via an IgE-mediated process. A recent major natural disaster in the USA, Hurricane Katrina, left thousands of homes in New Orleans, Louisiana flooded for

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

weeks with extensive mold growth in buildings. Air samples showed elevated levels of various fungi, especially Penicillium and Aspergillus species. Although most studies focused on indoor spore levels, the outdoor spora also showed significant increases of Penicillium/Aspergillus-type spores compared with pre-Katrina levels. The massive mold contamination following Hurricane Katrina illustrates the potential for massive and continual domestic as well as occupational exposure to molds yet also provides an excellent opportunity to study the health effects of such mold exposure.

Introduction Spores and other particles of fungi and bacteria, including actinomycetes, occur widely in the air. They are normal components of the outdoor and indoor aerial environment but can be especially numerous in indoor environments with microbial growth, ranging from residential buildings through offices and industrial structures to farms, often as a consequence of human activities. Although neglected as potential causes of disease, fungi and actinomycetes in these environments have often been implicated in respiratory allergies, including seasonal rhinitis, asthma, and occupational asthma. In retrospect, fungi are among the earliest recognized aeroallergens. Wheezing in damp weather, described by Moses Maimonides in the 12th century, probably resulted from fungal allergy. Later, Floyer (1726) noted the development in a patient of violent asthma after visiting a wine cellar; almost 150 years later in 1873, Blackley in his classic researches into the causes of hay fever reported bronchial catarrh and severe chest tightness after inhaling spores from Penicillium colonies and further suggested Chaetomium as another possible cause. In 1924 Cadham recognized the fungi that induced rust disease on cereals as causes of asthma and Prince et al. (1934), Feinberg (1935) and Van der Werff (1958) frequently recorded allergy to molds among their patients, from both outdoor and indoor exposure. Fungal spores are among the most numerous airborne biological particles in outdoor air. They are far more numerous

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than pollen but, because they are much smaller, total volumes of fungal spores in the air approximately equal those of pollen. Especially large concentrations of fungal spores can occur outdoors during harvesting and similar activities, and indoors when handling stored products. Actinomycetes and other bacteria are also numerous outdoors but have rarely been associated with allergy. However, like fungal spores, they can occur in extremely large numbers when colonized stored products are handled. This chapter briefly reviews the occurrence of microorganisms in the air, their role in allergy, and the nature of fungal allergens.

c f h e d a b

k

g j

The agents Currently the classification of life recognizes three domains (or superkingdoms), the Archaea, Eubacteria and Eukaryota. Fungi are eukaryotic organisms, with a nucleus contained within a nuclear membrane. They lack chlorophyll and are dependent on external sources of energy. Most are saprotrophs, obtaining nutrients and energy from decaying organic matter (heterotrophic), but some are parasitic, causing infections of plants, animals and humans. As saprotrophs, they can gather nutrients from wood, paper, paints, glues, soils, dust, or food chips. They can even grow on inorganic surfaces like glass or fiberglass covered with dust. Dust trapped by fiberglass is hygroscopic and prone to absorb nutrients that enable the growth of mold (Van Loo et al. 2004). More detailed discussions of basic mycology, including the various means of spore production and dispersal, are available in general or medical mycology texts (Rippon 1988; Alexopoulis et al. 1996). In morphology, they range from single cells, through threadlike mycelial colonies to the large complex fruiting bodies of the mushrooms and toadstools. Individual cells range in size from about 1 to 300 μm and their colonies may appear dark colored, colorless, or brightly colored from pigmentation of both mycelium and spores. Some species are pleomorphic and produce different morphologic forms in different environments, often yeast-like in one and mycelial in another. Fungi reproduce by spores which may be one-, two-, or multi-celled, from about 2.5 to 100 μm in size (Gregory 1973; Lacey & West 2006). The majority of fungal spores are dispersed by the atmosphere; to aid their dispersal, spores sometimes have specialized liberation mechanisms. Some fungal spores may be dispersed in rain splashes and never truly become airborne, while others may be dispersed by insects or larger animals but these are unlikely to be important in allergy. The cell and spore walls of fungi control release of allergens in culture filtrates and the availability of allergen to the lung when they are inhaled (Fig. 46.1). The cell wall of true fungi is complex, with successive layers of mixed polysaccharides (glucans), an inner reticulum of glucans plus proteins, then a protein layer and, on the inner side, chitin ((1→4)-β-Nacetylglucosamine) fibrils embedded in protein, as discussed

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10 mm

u

Fig. 46.1 Airborne fungal and actinomycete spores showing relative sizes and shapes. (a) Mastigomycotina: sporangium of Phytophthora infestans. (b–f) Ascomycotina: ascospores of (b) Claviceps purpurea; (c) Pyronema omphalodes; (d) Sordaria fimicola; (e) Xylaria polymorpha; (f) Didymella exitialis. (g) Actinomycete spores: Thermoactinomyces vulgaris. (h–n) Deuteromycotina and anamorphs of Ascomycotina: conidia of (h) Botrytis sp.; (i) Penicillium chrysogenum; ( j) Aspergillus fumigatus; (k) Cladosporium herbarum; (l) Erysiphe graminis; (m) Alternaria sp.; (n) Drechslera sp. (o–u) Basidiomycotina: ballistospores of (o) Armillaria mellea; (p) Serpula lacrimans; (q) Ganoderma applanatum; (r) Sporobolomyces sp.; (s) teliospore of Ustilago avenae; (t) urediniospore of Puccinia graminis; (u) teleutospore of Puccinia graminis. (From Carlile & Watkinson 1994, with permission.)

in Griffin (1994). Another wall component, (1→3)-β-Dglucans, can initiate a wide range of biological responses in vertebrates. Since spores contain glucans as well as allergens and other components, spore deposition simultaneously coexposes respiratory mucosa to several biologically active materials. Although speculative, a principal effect of glucans may be the modulation of responses to other components. The polysaccharides of the cell walls may be immunogenic themselves, but they also may allow the passage of antigens from within the cell into culture media or into contact with the lung surface.

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Spores characteristically provide the initial contact between fungus and the lung surface, and differ structurally from the mycelium. Spores include the ascospores and basidiospores produced by the sexual process of ascomycetes and basidiomycetes, and include the asexual externally produced spores of (mostly) ascomycetes that are termed “conidia.” Spore wall structure determines whether allergens are already available on the surface or how easily they may diffuse through it, especially important for immediate reactions. However, although there is much information on the structure of yeasts and some on that of conidia, there is little on the structure of ascospores and basidiospores. Empirical evidence for differences in allergen release from spores was presented by Horner et al. (1993); these patterns correlated generally with spore wall structure, with slow release from either thick or pigmented walls and rapid release from thin-walled colorless spores, although only four spore types were assessed.

Fungi as causes of allergy Although fungi are chiefly known as respiratory allergens, superficial contact allergy has also been described (Austwick 1967). Skin irritation was caused in a farmer by maize heavily infected with Ustilago maydis (Preininger 1937–38); recurrent eczematous dermatitis in 13 of 50 patients, distinguished from atopic dermatitis, was attributed to Alternaria, Aspergillus or Penicillium (Fujisawa et al. 1966); and skin lesions on the shoulders of workers who carry bundles of reeds colonized by Apiospora montagnei are characteristic of mal des cannes de Provence (Duché 1944). Although, allergic eczema in dogs and horses has also been attributed to Alternaria species, little is known of the processes of sensitization and skin penetration by the antigen or how the host reaction is mediated. It is estimated that 10% of the population have IgE antibodies to commonly inhaled mold spores (Bush et al. 2006). There is sufficient evidence of a cause and effect relationship between fungal exposure and asthma symptoms in sensitized patients (Institute of Medicine Report 2004; Richardson et al. 2005). Fungi implicated in respiratory allergy are represented in all the major fungal groups, including Rhizopus, Mucor and Absidia in the Zygomycota, Chaetomium, Didymella, Erysiphe and Eurotium in the Ascomycota, Agaricus, Coprinus, Pleurotus, Serpula, Ganoderma, Puccinia and Ustilago in the Basidiomycota, and Cladosporium, Alternaria, Aspergillus, Botrytis, Penicillium, Phoma, Stemphylium, Trichothecium and many other anamorphic fungi. However, not all fungi implicated as allergens have formed a significant proportion of the air spora in any study or had their status as allergens confirmed by inhalation tests, passive transfer or a corresponding history. Many of the species listed by Hyde (1972) are seldom, if ever, abundant in outdoor air and some have no ready means for becoming airborne. There is still little information on doses necessary for sensitization. The best-known allergens are indeed those

Fungi as Allergens

found most abundantly in the air, e.g., Cladosporium, Alternaria, Didymella, Sporobolomyces, although it is uncertain whether numbers or volumes are more important. To be dispersed, fungal spores must escape from the layer of still air that surrounds the surface on which they grow into the more turbulent air above. This is often achieved through specialized dispersal mechanisms and may be aided by growth high on plants. Many spores are trapped within the vegetation in which they are formed, but perhaps 5–10% of the total escape into the atmosphere to be dispersed for long distances. Some fungi reported to be allergens produce spores in slimy masses, which can only be dispersed by rain. However, they may also produce ascospores, which can readily become airborne. Thus, allergy attributed to Phoma is probably caused by inhalation of Leptosphaeria ascospores, to Ascochyta by Didymella ascospores, and to Fusarium by Nectria ascospores. However, fungal allergen extracts are difficult to produce because of a lack of source material availability, lack of characterized allergenic extracts compared with other bettercharacterized aeroallergens such as pollen, and the apparent cross-reactivity among many species. In contrast to other aeroallergens, fungal extracts are not standardized, and sensitized patients may respond to a number of fungi (Horner et al. 1992). Sensitization to fungi, particularly Alternaria alternata, has been linked to the presence, persistence, and severity of asthma (Bush et al. 2006). Diaries recording when symptoms occur can be instructive in indicating the cause of allergy. Both time of day and weather should be noted and compared with spore-trap results, although late asthmatic responses sometimes occur several hours after exposure. Didymella exitialis was implicated as a cause of late-summer asthma in the UK by correlating patients’ records with concentrations of airborne spores (Harries et al. 1985). Symptoms were worst after rain, when D. exitialis spores were numerous in the air. The sporetrapping method used when making such correlations must catch the spores of interest volumetrically and allow time discrimination. Automatic volumetric spore traps revolutionized our concept of the air spora, especially in indicating the existence and importance of the night-time damp-air spora of ballistospores of Sporobolomycetaceae, basidiospores and ascospores. These were sampled inefficiently by gravity slide traps, while settle plates were rarely exposed at an appropriate time and often grew colonies that were either sterile or produced an alternative spore type.

Occurrence of allergenic fungi outdoors Fungal spores are almost always present in the air, but their numbers and types change with time of day, weather, season, and geographic location and may be affected by local sources of spores, among which agricultural crops occupy a prominent position (Gregory 1973). These differences result from the

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different environmental cropping practices. Together, they affect the occurrence and time of presentation of allergic symptoms (Lacey 1981, 1991).

(a) 75 50

Weather and spore release mechanisms

Circadian periodicity Characteristic diurnal changes in the concentrations of different spore types result from the different requirements of the liberation mechanisms described above. Mechanisms activated by drying give greatest airborne spore concentrations from about 0700 to 1000 hours as dew dries from crop canopies (post-dawn pattern). Mechanical disturbance gives maxima from 1000 to 1600 hours (midday pattern), when wind speeds, turbulence, and convection are greatest, or double maxima from 0800 to 1000 hours and 1400 to 1800 hours (double-peak pattern), probably when, close to midday, spores are carried away faster than they are released. Finally, mechanisms requiring water give maxima from 2000 to 2200 hours (post-dusk pattern) or from 0200 to 0400 hours (Levetin & Horner 2002; Horner et al. 2004) after wetting by dew (night pattern). However, some ascospores, especially of discomycetes, are released after dawn, perhaps because some drying is necessary to cause shrinkage of the fruiting body, and exert additional pressure on the asci. These circadian periodicities may be modified by rain.

966

25

(b) 75 50 25 Per cent of maximum spore count

Fungal spores are released from their fruiting structures in many different ways. Some have active spore release mechanisms activated by drying, catapulting spores into the air. Other mechanisms require water for the sudden rounding (Conidiobolus coronatus, Epicoccum nigrum, Arthrinium cuspidatum) or bursting (Nigrospora sphaerica, ascomycetes) of turgid cells. Many spore types are released by mechanical disturbance, especially by movement of leaves and litter in wind. Handling or removal of colonized building material is adequate mechanical disturbance to release spore levels comparable to the high levels seen when handling colonized hay or grain. Numbers of spores released increase with increasing wind speed and turbulence or vigor of handling and with decreasing humidity. Falling raindrops also vibrate vegetation when they impact and, as they spread out, push a fast-moving cushion of air before them, which disturbs the still air surrounding the surface and disperses spores into the air. Rain has other effects on the occurrence of airborne spores. Droplets falling on some fruiting bodies (puffballs) expel puffs of spores, while they disperse spores from others in splashes. The largest droplets follow ballistic trajectories and hardly become airborne, while the smallest can evaporate and allow any spores they carry to be dispersed by wind. Besides dispersing spores, continued rain removes them from the atmosphere by impaction on raindrops. Finally, rain provides water for ascospore release, resulting in greatly increased numbers in the air soon after the rain has ceased (Harries et al. 1985), perhaps causing epidemic outbreaks of acute asthma (Packe & Ayres 1985).

(c) 75 50 25

75

(d)

50 25

(e) 75 50 25

06

Noon

18

Fig. 46.2 Diurnal periodicities exhibited by different fungal spore types: (a) post-dawn pattern, e.g., Phytophthora infestans; (b) midday pattern, e.g., Cladosporium, Alternaria; (c) double-peak pattern, e.g., Cladosporium; (d) post-dusk pattern, e.g., Ustilaginoidea virens; (e) night pattern, e.g., ascospores, basidiospores. (From Lacey 1981, with permission.)

Season There is no clear season for mold spores; however, seasonal trends are often related to crop growth cycles or climatic factors. In the UK, a temperate region, airborne spores are usually fewest during winter and spring and most abundant in summer, when Cladosporium usually predominates by day and Sporobolomyces by night (Fig. 46.2). In tropical areas, basidiospores are most abundant during the wet season and Cladosporium during the following cool, dry season (Fig. 46.3). Few fungi are abundant during the hot, dry season. Plantpathogenic fungi, in particular, have seasonal trends linked to crop growth cycles. For instance, in the UK (Fig. 46.4a), Erysiphe (powdery mildew) spores are most abundant in June–July, when the disease is most abundant on cereals, and Ustilago (smuts) during the flowering periods of their grass

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Per cent of maximum spore concentration

100 80 60 40 20 0 12

18

06

4

100 80

3

60 2 40 1

20

0

0 18

12 (b)

Rain (mm)

Per cent of maximum spore concentration

24 Time

(a)

31 July

24

06 1 Aug

Fig. 46.3 Effect of rain on the diurnal periodicity of ascospores of Didymella exitialis: (a) periodicity in the absence of rain; (b) release of spores following rain. (From Harries et al. 1985, with permission.)

hosts. Alternaria and Didymella both grow on ripening cereal straw and are most numerous close to harvest, when a second peak of Cladosporium may also occur. Basidiospores become abundant during the autumn, when heavy dews favor fruiting of many mushrooms and toadstools. Conversely, Penicillium and Aspergillus are often most abundant in cities in winter (Hamilton 1959). In tropical regions, e.g., India (Fig. 46.4b), the incidence of Cladosporium and Nigrospora is correlated with similar growth stages of each of the two rice crops, but Dieghtoniella torulosa occurs on rice only in the rainy season. Sphacelotheca sorghi is associated chiefly with the earlier of the two sorghum crops, Cercospora with the later, and Drechslera turcica with both. Aspergillus is present throughout the year and Alternaria chiefly at the end of the hot season as the rains start. However, the occurrence of spores identified as Fusarium correlates only with rainfall.

Geographic location The predominant spore types are remarkably widespread in their occurrence. Regional differences are mainly between minor components of the air spora, which tend to increase in number and variety from cooler to warmer climatic zones. Cladosporium is the most abundant spore type over the whole year in temperate and most tropical regions, even though it is exceeded by other spore types in some regions and seasons

Fungi as Allergens

of the year. Spore concentrations of Cladosporium may reach 240 000/m3 and form 93% of the total air spora, but mean daily concentrations are usually of the order of 5000/m3. Alternaria spores are the second most abundant overall and can exceed concentrations of Cladosporium in warm dry regions. The mean daily spore concentration is usually only about 150/m3, although maxima up to 6000/m3 have been found over short periods. Curvularia and Nigrospora sometimes make large contributions to the air spora of tropical regions, with maxima of 4000–9000/m3, although mean daily concentrations over the year are only 50/m3 or fewer. Aspergillus species are particularly characteristic of humid tropical regions. In Mysore, 37 Aspergillus species accounted for 5.6% of the colonies isolated and Penicillium for only 1.7%, while in the UK Penicillium accounts for 2.5–13% of catches and Aspergillus only 0.9–3%. In sub-Arctic and Arctic zones, where seasons are short, maximum spore concentrations are less than 10 000/m3.

Local spore sources Although the air spora of different regions is generally similar, locally nearby sources of spores and differences in microenvironment and levels of human activity can have considerable effects (Levetin and Horner 2002). Lacey (1962) found 2.6 times more spores in a valley, close to a stream, than on an exposed hill nearby. These included five times more ascospores and three times more basidiospores, but only 1.4 times more conidia. Crops and natural vegetation can form vast sources of spores, especially agricultural crops close to harvest (e.g., Cladosporium, Alternaria, Didymella species). In forested areas, ascospores and basidiospores are among the most common types. Mowing, haymaking, and harvesting can put vast numbers of spores into the air, giving concentrations locally up to 109/m3. Differences over a wide area may be similar to those found around Derby, where concentrations of Cladosporium at eight sites within a 60-km radius were 83–125%, and Alternaria 54–102%, of the catch in the city (Broun & Jackson 1978). However, catches of some other spore types were only 25% of those found in the city. Catches in London were less than 50% of those 40 km north at Rothamsted (Hamilton 1959). Similarly, catches in Cardiff were smaller than at a nearby rural site and were especially small when winds blew from the sea (Harvey 1967).

Fungal particles from colonized building materials serve as allergen sources Recent descriptions of the mix of particles shed from moldcolonized surfaces suggest that current views of exposure assessment may be grossly inaccurate (Gorny 2004; Green et al. 2006). Although the studies were directed at mold particles from the indoor environment of water-damaged buildings, the conclusions likely pertain to outdoor bioaerosols as well. A new particle size sampler for emissions from surfaces indicates that much of the airborne material shed from moldy surfaces is not in the form of intact spores (Sivasubramani

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20 000

Cladosporium

10 000 0 10 000 0 3000

Basidiospores

Aspergillus

2000 30 000

Sporobolomyces

1000 0 2000

10 000

1000

0 20 000

Spores m–3 air

20 000

Cladosporium

10 000

Spores per m3 air

0

Nigrospora

400 200 0

Erysiphe

1000

600

Ascospores

Curvularia 200

0 1000 0 1000 0 1000 0 100 0

100

Ustilago

0 200 Alternaria

Tilletia type

100 0 100

Basidiospores Uredospores

0 20

Pyricularia oryzae Uredospores

10 40

Phytophthora 0

0 (a)

Jun

Jul

Aug

Sep

Oct

1960

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Season (b)

Cool, dry

Hot, dry

Wet, monsoon

Cool, dry

Fig. 46.4 Seasonal periodicities of some fungal spores in (a) the UK and (b) India. (From Lacey 1988, with permission.)

et al. 2004). Studies of these particles also show that the particles are of respirable sizes and are immunologically reactive. Analogous to the recognition in the 1990s that starch grains from pollen carry allergen (Spieksma et al. 1990), recent data confirm that broken spores, hyphal fragments, and digested substrate particles carry fungal allergens as well as intact fungal spores (Fig. 46.5). Although empirical evidence is currently sparse, presumably other metabolites such as proteases, glucans or any toxins present would also occur on these particles. Gliotoxin has been recovered from building material substrates colonized by Aspergillus fumigatus, and Stachybotrys chartarum toxicity was putatively associated with submicronic airborne particles (Nieminen et al. 2002; Brasel et al. 2005). Intact spores are typically released from mold-colonized building materials that occur in water-damaged buildings. The broken spores, hyphal fragments, and particles of degraded substrate that are also released may substantially outnumber

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intact spores (Gorny et al. 2002). In the limited laboratory studies reported to date, intact spores may represent only a few percent of the total fungal particles shed from moldy surfaces. The presence of fungal antigens on nonspore particles was demonstrated with studies that labeled mold antigens with monoclonal antibodies (Gorny et al. 2002). These studies were corroborated with double-labeling procedures that demonstrated the presence of fungal antigens and IgE binding on the same particles (Green et al. 2005a). This procedure, in conjunction with traditional light microscopy, was able to confirm allergenicity in spores from multiple new genera of fungi (Fig. 46.6). Spores of all fungi have long been considered potentially allergenic, and these studies strongly reinforce this notion. The recent development of new samplers for fungal particles is yielding new information about nonspore fungal

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Fungi as Allergens

particles released from mold-colonized building materials. New applications of immunoassays have demonstrated that metabolites of interest (allergens) are present on nonspore particles. Based on these recent findings, it is feasible to postulate that much of the exposure to fungal metabolites, at least allergens, may correlate poorly with spore exposure. This in turn indicates (i) the need for rethinking our assumptions about mold exposure and (ii) an opportunity to apply these new techniques for more productive work on fungal exposures than was possible with the more traditional techniques.

Occupational exposure to spores outdoors

Fig. 46.5 Immunostaining of allergen release from fragment of fungal hyphae. (From Green et al. 2005, with permission from the American Academy of Allergy, Asthma, and Immunology.) (See CD-ROM for color version.)

Occupational allergy outdoors is difficult to define, because the boundary between everyday exposure and occupational exposure is indistinct. Recorded examples refer only to agriculture (Table 46.1). However, the incidence of allergy and asthma in farmers is similar to that in the general population (Heinonen et al. 1987). Many of the species normally present in air grow abundantly on agricultural crops, but their numbers locally can be greatly enhanced by agricultural operations, e.g., haymaking and harvesting. Harvesting can lead to spore concentrations up to 2 × 108/m3 near combine harvesters. Spore concentrations near the driver are smaller, reaching about 2 × 107/m3. Cladosporium accounted for 46– 75% of spores, Alternaria for 9–28%, and Verticillium for 1–9%. Rust diseases caused by Puccinia species within the crop contributed 3–4% and smuts (Ustilago species) up to 10% of spores. Bacteria contributed less than 10% of the total. About 20% of a small group of British farm workers complained that they were affected by harvester dust. Symptoms ranged from irritation, through rhinitis and asthma, to allergic alveolitis and positive skin tests, and precipitins were found to some fungi (Darke et al. 1976). Exposure of workers can be decreased by using helmets or cabs with filtered air suppliers that remove up to 98% of spores from the breathing zone. Dust produced during haymaking is similar to that produced during cereal harvest, but yeasts, particularly Sporobolomyces, contributed 43%, bacteria 29%, and Cladosporium 24% to the total spore content.

Table 46.1 Fungi implicated in occupational asthma in outdoor environments.

Fig. 46.6 Immunostaining of allergen release from conidium of Spegazzinia. Note that as with all fungi, Spegazzinia was previously considered potentially allergenic but is clearly demonstrated here as releasing IgE-binding material. Note also the extensive area surrounding the spore that allergen is spread through. Unrelated particulate matter in the vicinity of a wetted spore could thus become a “carrier” of fungal allergens without being recognizable as a fungal spore. (From Green et al. 2005b, with permission from the American Academy of Allergy, Asthma, and Immunology.) (See CD-ROM for color version.)

Source of allergen

Fungus implicated

Cereal grains and straw

Puccinia species (Cadham 1924) Ustilago species (Harris 1939) Tilletia caries (Jiminez-Diaz et al. 1947) Verticillium lecanii (Darke et al. 1976) Aphanocladium album Paecilomyces farinosus

Reeds

Apiospora montagnei (Duché 1944) (Arthrinium arundinis)

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Table 46.2 Fungi implicated in occupational asthma in indoor environments. Source of allergen

Species implicated

Tomato growing

Verticillium albo-atrum (Davies et al. 1988)

Mushroom culture

Aspergillus fumigatus (Sakula 1967) Lentinus edulis (Kondo 1969) Pleurotus ostreatus (Zadrazil 1974; Schulz et al. 1974) Oidiodendron species (Olivier et al. 1975)

Soup manufacture

Agaricus bisporus (Symington et al. 1981) Boletus edulis

Flour

Alternaria species (Klaustermeyer et al. 1977) Aspergillus species

Cheese dairy

Penicillium camembertii (Gari et al. 1983)

Tobacco

Scopulariopsis brevicaulis (Lander et al. 1985)

Enzyme production (surface culture)

Aspergillus flavus (Izrailet & Feoktistova 1970) Aspergillus awamori

Food-protein culture

Candida tropicalis (Cornillon et al. 1975)

Chiropody

Trichophyton rubrum (Pepys 1986)

(a)

Fungi in indoor environments (b)

Spore types in the air of indoor and outdoor environments often differ markedly. In the absence of indoor sources, airborne spore types will be similar to those outdoors, but concentrations will be smaller. Fungi need a source of organic material and moisture to grow (Portnoy et al. 2005). Differences in either levels or types of fungi may indicate that excess moisture and resultant fungal growth may be problematic (Petronella et al. 2005). Fungi may grow on structural materials and furnishings where humidity is high enough or there is condensation, on foods and stored products, and in dust deposits. Stored products can form vast sources of fungal and actinomycete spores, which overwhelm the small numbers from outdoor sources. However, their numbers and types change with storage conditions, degree of disturbance, and ventilation. Many fungi from these sources have been implicated in occupational asthma (Table 46.2). Dry-spored fungi producing abundant spores smaller than 5 μm, especially Aspergillus and Penicillium species, are chiefly implicated. Apart from their direct allergenic effect, active fungal growth may produce various metabolites including mycotoxins and microbial volatile organic compounds (Flannigan et al. 2002). Inhalation of mycotoxins produced especially by Aspergillus, Penicillium and Fusarium species, including aflatoxins, secalonic acid D, zearalenone and trichothecenes, may affect the immunologic response of the lung or present other hazards to health (Gerberick et al. 1984). However, the health effects of volatile metabolites of fungi are not resolved. In some cases,

970

Fig. 46.7 Immunostaining of ungerminated spores (a) and of germinated Alternaria spores (b). Note the increased staining surrounding the germinated spores and the hyphae (thread-like filaments) compared with the ungerminated spores. This indicates an increase of IgE binding components associated with germination, and the release of these components (i.e., the allergens are no longer bound to fungal particles) into the environment. (From Mitakakis et al. 2001, with permission from the American Academy of Allergy, Asthma, and Immunology.) (See CD-ROM for color version.)

there appears to be correlation of symptoms and mold exposure with mechanisms not suggestive of an IgE-mediated process. Mycotoxin-mediated mechanisms may account for these symptoms, although there are currently no means to test this. The etiology of these symptoms therefore remains unclear and warrants further investigation (Edmondson et al. 2005). Similarly, other products related to mold colonization, such as glucans or proteases, or combinations thereof may account for these symptoms, although effective exposure assessment tools are not currently available for these either. One difficulty with previous exposure tools is their reliance on countable or culturable propagules for quantitation of exposures. Mitakakis et al. (2001) using an immunostaining technique demonstrated the dramatic release of allergens from spores and hyphae (Fig. 46.7). Indoor air may also carry endotoxins, derived from the cell walls of Gram-negative bacteria, causing febrile and other reactions and perhaps enhancing immunologic effects (Michel et al. 1990). The effects

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of interactions of endotoxin with mold products have yet to be fully considered experimentally, although coexposure to endotoxin and mold products in damp indoor environments is very plausible. Exposure to fungal spores in indoor environments is seldom constant but changes both quantitatively and qualitatively with time and place. The way in which the air spora and consequent allergenic risk can change in different environments is described below.

Nonindustrial and residential environments Concentrations of airborne allergens in residential and office buildings are affected by similar factors, but the scale is generally much larger in commercial buildings. Airborne spores may enter buildings from outdoors with ventilation, especially in summer, or come from wood-rotting fungi, from molds growing in condensate on walls and windows, on food scraps and other organic material in house dust, or in humidifiers or air-conditioning systems. However, in contrast with dwellings, air in modern nonindustrial buildings is less likely to enter by natural ventilation, less timber is used in construction, and condensation may be controlled by central airconditioning systems. However, these systems can also rapidly disperse microorganisms or their products throughout the building and, as in houses, humidifiers can provide sources of contamination. Where timber is affected by the dry-rot fungus Serpula lacrymans, basidiospores may be produced prolifically and can be dispersed throughout the house. Thus, extensive fruiting bodies, yielding nearly 80 000 spores/m3 in the cellar of a house, yielded 16 000–26 000 spores/m3 in other parts of the house, with no fruiting bodies (Gregory et al. 1953). Similarly, there were 360 000 spores/m3 in the cellar of a country mansion, and 1600 spores/m3 in a draughty corridor above. Other wood-decaying basidiomycetes can attack wooden window frames or other wooden materials in houses and release high levels of spores (Hunter et al. 1988). Concentrations of outdoor spore types, e.g., Cladosporium, Alternaria, Botrytis and Epicoccum, in houses are generally smaller than those outdoors. However, in winter, when outdoor spore levels are low and there is less ventilation, indoor spore concentrations are likely to exceed those outdoors, especially if the house is damp and there is visible fungal growth. Total concentrations may range from less than 12 to 450 000 colony-forming units (cfu)/m3. Water for fungal growth is chiefly provided by rising damp, water penetration, high relative humidity from insufficient ventilation, air infiltration due to building depressurization in hot humid climates, and condensation on cold surfaces. Xerophilic fungi commence growth at a relative humidity greater than about 70% and, as humidity increases, more species are able to grow. Nutrient may be provided by paint or wallpaper, cotton and other materials containing amorphous (pulped) cellulose or food scraps and other organic matter in house dust. The

Fungi as Allergens

predominant types will be determined by their source but are generally species of Cladosporium, Penicillium and Aspergillus, although many other genera have occasionally been recorded, including abundant Alternaria in winter, when Penicillium may form more than half the colonies isolated. Fungi isolated from walls include Cladosporium herbarum, Aspergillus versicolor, Penicillium brevicompactum, and P. glabrum (P. frequentans), with P. chrysogenum and P. aurantiogriseum (P. cyclopium) common in bathrooms and other wet rooms. Aureobasidium can grow actively, with Penicillium, Phoma, Trichoderma, Sporobolomyces and Cryptococcus, on the frames of visibly moldy windows, with Penicillium particularly numerous in the air in older wooden houses in Finland (Käpyla 1985). Fungi colonizing painted surfaces include Cladosporium sphaerospermum, C. cladosporioides, Penicillium species, Aspergillus flavus, Acremonium species, Gliomastix murorum, Phoma herbarum, P. nebulosa, Phialophora fastigiata, Alternaria alternata, Fusarium species, Rhodotorula glutinis, and Ulocladium atrum (Springle 1990). Food scraps and other organic materials are commonly colonized by Aspergillus, Eurotium and Penicillium species, which produce large numbers of spores that can easily become airborne when disturbed. Eurotium species and Wallemia sebi are xerophilic fungi, which can grow at low relative humidity and are often important components of the indoor air spora. However, they have been inadequately studied because suitable low-water-activity media, e.g., DG18, have seldom been used until the late 1990s. Aspergillus versicolor has often been isolated from house dust and exposed surfaces but is replaced by Penicillium species, especially Penicillium viridicatum, P. fellutanum and P. decumbens, in Canada. Carpeting can provide a reservoir of dust and spores that can become airborne. Penicillium, Cladosporium and Aspergillus were more numerous in schools and offices where floors were carpeted, especially if these had been wetted frequently. However, presence of fungi in carpet dust, e.g., Fusarium, did not necessarily indicate their presence in the air. Potted plants may be sources of Aspergillus species, including A. fumigatus, although few spores are said to become airborne from these sources. Exceptionally, slimy-spored species, such as Phoma violacea, can become airborne in aerosols created by showers. Stachybotrys chartarum, well known as a producer of immunotoxic macrocyclic trichothecene mycotoxins, has been isolated from ducts, insulation and structural materials of a Chicago house (Croft et al. 1986). It was also frequent in the air and surface mold in Scottish houses, with spores numbering up to 1.8 × 104/m3 (Hunter et al. 1988). This species grows readily on wet cellulose-based building materials such as acoustic tiles and the backing of gypsum board. The exact effect of mycotoxins from indoor air exposures on human health remains controversial. Fungi in dwellings generally have no specialized sporeliberation mechanisms and largely depend on disturbance. Thus, spore concentrations often reflect the level of activity

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in a building. This underscores the danger of characterizing the airborne spore load in a building by relying on only a few air samples taken under quiet conditions. High concentrations often occur during construction, repairs and cleaning, especially if surface mold on walls is disturbed, and low concentrations occur when there is little activity. Vacuum cleaning can cause up to a 17-fold increase in numbers immediately and leave numbers elevated for at least 1 hour afterwards, while disturbance of surface mold can increase airborne fungi within 0.3 m of the source 3300 times. The effect of disturbance on airborne spore loads is the basis for strict dust control precautions that are recommended in North America for remediation of mold-colonized building materials. Wood chips used as heating fuel can be sources of abundant airborne spores that can spread throughout a building (Kotimaa 1990). Heating, ventilation and air-conditioning (HVAC) systems can be important sources of allergens, as well as dispersing them throughout buildings. These systems may decrease spore levels of air due to filtration, but since water often accumulates in HVAC systems from condensation on cooling coils or other surface or accumulated dusts and this may permit colonization within the air supply system. Starting the fan coil unit or agitating the drain pan can increase concentrations of airborne fungi from 7000/m3 in undisturbed conditions to almost 107/m3, while operating cold-mist vaporizers increased airborne cell/spore loads 100to 240-fold, sometimes to > 14 000/m3. Humidifier fever has been attributed variously to allergy and alveolitis to different airborne microorganisms from contaminated humidifiers and to endotoxin. However, sources of antigenicity from humidifiers have often been difficult to determine (Hodges et al. 1974); in some outbreaks, symptoms have been attributed to bacteria (Flavobacterium, Cytophaga), actinomycetes (Saccharopolyspora rectivirgula, Thermoactinomyces species), or even amoebae.

Agricultural environments The microflora of harvested crops changes during storage, depending on environmental conditions, especially water content. Preharvest fungi survive only in materials stored dry (water activity < 0.70; about 12–13% water content in starchy cereal grains). Other species, especially Aspergillus and Penicillium, grow as water content increases, and increasing metabolic activity causes spontaneous heating. Above 35% water content, temperatures of 65°C may occur, allowing growth of thermophilic and thermotolerant fungi and actinomycetes. These include species that can cause infection (A. fumigatus, Absidia corymbifera), asthma (A. fumigatus), and allergic alveolitis (thermophilic actinomycetes, Aspergillus species). Most species produce abundant spores that easily become airborne when the substrate is disturbed. Molding and heating may be decreased if the air supply is restricted and carbon dioxide concentrations increase to inhibitory

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levels. The numbers and types of fungi and actinomycetes depend on the microflora of the substrate, the degree of disturbance, and the amount of ventilation. Removal of moldy grain from silos and elevators can also generate concentrations of actinomycete and fungal spores of up to 1010/m3, although Penicillium species may be more numerous in grain than in hay. In Canadian grain elevators, Aspergillus and Penicillium species predominate in settled dust and have been implicated in respiratory allergy and allergic alveolitis (Lacey 1980). However, endotoxins have been implicated in the bronchitis and chronic obstructive lung disease often found in grain handlers. Additionally, mites may be attracted by molding of stored products and can also be potent allergens (Revsbech & Andersen 1987).

Mushroom farms Fungi and actinomycetes present two different types of hazard on mushroom farms: allergic alveolitis (mushroomworker’s lung) during the preparation and spawning of composts, and occupational asthma among pickers in the growing houses. Exposure in the growing houses is more likely to be to fungi than to actinomycetes. Few spores are released when mushrooms are picked as “buttons,” but up to 106 spores/m3 can become airborne if the caps are allowed to open. Pickers probably disperse molds growing on the compost and on the moist timber of the compost trays. These include Cephalotrichum (Doratomyces) stemonitis (up to 2.1 × 105 spores/m3), A. fumigatus (up to 9 × 104 cfu/m3), and Penicillium species (up to 4 × 104 cfu/m3). The oyster mushroom Pleurotus ostreatus, and other oriental mushrooms such as Lentinus edodes (Shiitake) and Pholiota nameko, produce spores prolifically from an early stage in their development, giving spore concentrations up to 4 × 107/m3. These can cause asthma and allergic alveolitis (Gandy 1955; Cox et al. 1988; Sastre et al. 1990). Pleurotus ostreatus was shown to produce two proteinaceous cytolytic toxins, which could contribute to its pathogenic effects (Anderson et al. 1988). Cases of hypersensitivity pneumonitis have been associated with chronic inhalation of the spores of Hypsizygus marmoreus, also known as Bunashimeji mushroom (Tsushima et al. 2005).

Food processing Although allergy to spores of Agaricus bisporus has been found in a soup-processing factory; exposure to fungal spores during food processing usually results from the presence of contaminating mold. For instance, coffee beans prior to roasting usually carry fungal spores, which can give up to 2.4 × 104 cfu/ m3 in containers when unloaded at the warehouse. They include Eurotium species, A. fumigatus, A. flavus, A. niger, and Wallemia sebi. In bakeries, fungi from flour and dried fruit, especially Aspergillus, Penicillium and Mucor species, are frequent in the air. Up to 1.2 × 103 cfu/m3, mostly Penicillium species, were found in a British bakery where flour was weighed, and up to

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3.7 × 103 cfu/m3, mostly W. sebi and yeasts, where dry fruit was handled. Alternaria and Aspergillus species, fungal amylase, flour protein and mites have been suggested as possible causes of baker’s asthma (Klaustermeyer et al. 1977; Baur et al. 1986; Revsbech & Dueholm 1990). Penicillium is associated with occupational asthma at food processing plants. Alternaria and Aspergillus are associated with occupational asthma in flour mills, bakeries and food processing plants (Orman et al. 2005). Penicillium species are important in the maturation of some cheeses and sausages and grow abundantly on their surfaces. When these products are cleaned before sale, spores are dispersed and cause asthma and allergic alveolitis in exposed workers (Table 46.2). However, exposure has rarely been quantified aerobiologically.

Fungal allergens Fungal allergens are generally proteins and glycoproteins with molecular masses of 5–70 kDa. The number and strength of antigenic components, which depend on the nutritional composition of the growth medium and period of incubation, are notoriously variable in the fungi. In the past, there has been little standardization of fungal antigenic preparations and those used in clinical allergy generally consists of aqueous extracts of variable stability and quality, often prepared from ill-defined raw materials, lacking well-defined allergens and thus yielding unreliable results (Aukrust & Aas 1978; Aas et al. 1980; Al-Doory & Domson 1984; Malling et al. 1985). Although previous methods of assay have sometimes been insensitive (Nüsslein et al. 1987), the advent of Western blotting and allergen cloning has substantially aided fungal allergen identification and characterization (Britenbach et al. 2002; Vijay & Kurup 2004). Fungi are intrinsically variable and batches of allergenic material produced successively, even from the same strain, can differ widely. Such variability makes fungal allergen extracts difficult to produce because of a lack of consistent source material and apparent cross-reactivity among many species; thus there has been a lack of standardized fungal allergenic extracts compared with other better-characterized aeroallergens such as pollen. For this reason diagnosis of mold allergy is more complex, especially since sensitized patients may respond to a number of fungi (Horner et al. 1992). Sensitization to fungi, particularly Alternaria alternata, has been linked to the presence, persistence, and severity of asthma (Bush et al. 2006). Recent rapid accumulation of allergen information in online databases renders catalogs of allergens somewhat obsolete (http://www.allergome.org). This is especially true for fungal allergens for which information is still rapidly emerging. At present characterized allergens are reported for some 81 species of fungi. The number of characterized allergens from these fungi varies from only a few, such as with Psilocybe cubensis and Epicoccum nigrum, to some two dozen

Fungi as Allergens

allergens characterized to date for Aspergillus fumigatus. The following sections briefly address general patterns of emerging information, with Alternaria discussed in more detail as an example.

Alternaria Throughout much of the world, Alternaria is the second most numerous spore type in air on dry days, although in some seasons and climates it may be more numerous than Cladosporium (Lacey 1981). In temperate climates, numbers of Alternaria are often only 2–4% of those of Cladosporium, but on a volume basis the discrepancy is less, as its spores are 20–40 times larger. Alternaria alternata is very common worldwide as an indoor allergen. It grows well at 20–30°C in humid places, such as kitchens, bathrooms, and garages in all seasons of the year but especially in summer. The major allergen Alt a 1 has been well characterized as a 29-kDa dimer that binds IgE from up to 80% of Alternaria-sensitized subjects (Aden et al. 1999). The release of Alt a 1 from spores is greatly enhanced by germination of the spores (Mitakakis et al. 2001) and the role of Alt a 1 homolog in the related species Alternaria brassicae was putatively implicated in the pathogenesis on the leaves of plants (Cramer & Lawrence 2003). Environmental measures of both Alt a 1 and of Alternaria antigen in settled dust have been made, and although more specific have not proved useful (O’Connor et al. 2004; Salo et al. 2006). The clinical relevance of Alternaria antigen measurements is just beginning to emerge but it has been suggested that levels of Alternaria antigens (or antigens crossreactive with the sera used) are a significant risk factor for current asthma. Other minor A. alternata allergens, such as Alt a 11, Alt a 3, Alt a 4, Alt a 6, Alt a 7, Alt a 10, Alt a 12, and nuclear transport factor 2, have been reported (Asturias et al. 2005).

Cladosporium Cladosporium is the most numerous airborne spore type over much of the world, and is a major source of fungal allergen in cooler climates (Vijay & Kurup 2004). Cladosporium spore count has been shown to be correlated with asthma severity, peak flow decrease, and use of antihistamine medication by patients, although only the last correlation was statistically significant (Malling 1986). The most abundant species are C. herbarum and C. cladosporioides, which together predominate on dry summer days in the UK. Current database records include 24 entries for nine designated allergens from C. herbarum. These include ribosomal protein P2 (Cla h 4), enolase (Cla h 6), mannitol dehydrogenase (Cla h 8), vacuolar serine protease (Cla h 9), aldehyde dehydrogenase (Cla h 10), and ribosomal protein P1 (Cla h 12). Heatshock proteins and glutathione-S-transferase (GST) have also been characterized as allergens from C. herbarum, and incompletely characterized allergens have been identified from both C. sphaerospermum and C. cladosporioides.

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Aspergillus fumigatus and other Aspergillus species Human allergic diseases involving Aspergillus include asthma, allergic bronchpulomnary aspergillosis, allergic fungal sinusitis, and hypersensitivity pneumonitis (Kurup 2005). Aspergillus fumigatus can cause several of these and is one of the most frequently implicated molds in human disease. Other species of Aspergillus that are recognized human pathogens are Emericella (Aspergillus) nidulans, A. oryzae, A. terreus, A. flavus, and A. niger. These are all species that occur widely in nature. The allergens of A. fumigatus have received as much attention as those of any other fungal species. Mitogillin (Asp f 1) is a potent cytotoxin that may have a role in pathogenesis. Other allergens are constitutive enzymes, a pattern seen already in the discussion of Alternaria and Cladosporium. Examples of these include peroxisomal protein (Asp f 3), metalloprotease (Asp f 5), manganese superoxide dismutase (Asp f 6), and cyclophilin (Asp f 11). For many other characterized allergens, the biological function remains unknown.

Penicillium Species of Penicillium are among the most prevalent airborne mold types in all regions. Penicillium species are common soil molds, so exposure to airborne Penicillium spores is certainly typical of all outdoor environments. Since damp indoor materials are commonly colonized by Penicillium, exposure is also typical of damp indoor environments. Allergens have been characterized from at least four species: Penicillium brevicompactum, P. chrysogenum, P. citrinum, and P. oxalicum. Evidence has been presented that among these species, and four species of Aspergillus, the serine proteinases represent a major source of allergenic activity (Shen et al. 1999). Significantly, cross-reactivity was also detected among the allergens of these eight common allergenic molds. Further evidence of general patterns of allergenicity has been implied by comparison of sequence orthologs (Bowyer et al. 2006). Consistent with earlier observations based on blotting experiments, orthologs of some allergens occurred among distantly related species of fungi, but other relatively unique orthologs occurred in a very restricted pattern on a few or even a single species. A specific example of the former situation is the 94% identity reported for the deduced amino acid sequence of the P. citrinum and the A. fumigatus enolases (Lai et al. 2002). Enolases of Alternaria, Candida, Cladosporium, Curvularia, Rhodotorula and Saccharomyces have also been described as allergens.

Basidiomycetes Basidiospores are characteristic of the night-time dampair spora, with daily mean spore concentrations in the UK between June and September seldom less than 1000/m3. However, their abundance was not totally appreciated before the advent of the volumetric spore trap (Gregory & Hirst

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1952). Although Herxheimer et al. (1969) showed basidiospores to give positive skin tests and to cause asthma in inhalation challenge tests, their importance as allergens has been established only during the last 30 years. Airborne basidiospores were then found in large concentrations coincident with late summer and autumn epidemics of New Orleans asthma, while atopic patients gave positive skin tests and radioallergosorbent test (RAST) assays to mycelial extracts of different basidiomycete species (Lopez et al. 1976; Lehrer et al. 1986). Positive skin tests for at least one mycelial extract from 10–15 basidiomycetes were found in 27% of a group of 150 patients with respiratory allergy and to basidiospores in 32%. Sensitivity to individual species ranged from 5% to Cantharellus cibarius to 17% to Scleroderma spp. Recently, 25% of 700 patients living in the USA or in western Europe who had symptoms of asthma, rhinitis or both were shown to react to at least one basidiospore extract in skin-prick tests. More than half of the patients reacting to basidiospore extracts had both asthma and rhinitis, while there was no statistically significant association between the occurrence of rhinitis alone and basidiospore reactivity. There were no significant differences in incidence between the USA and Europe, and for both sets of patients Psilocybe cubensis (12.3% positive skin tests in the USA, 16.0% in Europe) was the most potent allergen source, followed by Pleurotus ostreatus (10.7%) and Geastrum saccatum (8.4%) in the USA and by Ganoderma meredithiae (11.4%) and P. ostreatus (10.3%) in Europe. Overall, Pisolithus tinctorius and Coprinus quadrifidus showed least reactivity (Lehrer et al. 1994). Ganoderma extracts contain complex mixtures of allergens, but differences between G. meredithiae and Ganoderma applanatum, common in air in Europe, were few (Horner et al. 1993). Basidiospores may also form spore concentrations up to 100 000/m3 in New Zealand, with many, including Ganoderma species, giving positive skin tests in up to 22% of allergic patients (Hasnain et al. 1984, 1985). Santilli et al. (1985) also found an average of 4.4 immediate skin-test reactions to dialyzed spore extracts from different basidiomycetes in 100 American patients with asthma with symptoms October– November. Another group, with seasonal rhinitis in the same season, gave only 1.3 immediate reactions per patient but more late reactions than in the asthmatic group. Patients showed significant differences in their skin-test reactions to extracts of spores and mycelia of P. ostreatus (Lopez et al. 1985). Some reacted only to mycelial extracts, some only to spores, and only one to all extracts. Although spore extracts contained antigens common to the mycelium, they also contained unique antigens. In crossed immunoelectrophoresis, P. ostreatus spores yielded at least 27 precipitating antigens (Weissman et al. 1987). Crossed-line immunoelectrophoresis comparing extracts of spores, caps and mycelia of P. ostreatus confirmed the presence of both common and spore-specific antigens in these materials, while RAST inhibition suggested

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that they contained common allergenic components. There was little correlation in skin-test reactivity between caps and spores, and spores were recommended as the best source of antigens for clinical studies. Spores of 12 basidiomycete species gave positive skin tests in 28.6% (P. tinctorius) to 55.6% (G. saccatum) of 42 patients with perennial rhinitis and/or asthma, but not in skin testnegative atopic or in nonatopic subjects (Butcher et al. 1987). Positive RAST significantly correlated with skin-test results in all but three species, confirming the development of IgE antibodies to spores in skin test-positive individuals. Positive reactions by both skin test and RAST to some species were sometimes closely associated. Pooled fractions of spore extracts from four puffballs (Calvatia cyathiformis, G. saccatum, P. tinctorius and Scleroderma areolatum), analyzed by skin testing and RAST, all contained allergenic components with molecular masses from 10.5 to 20 kDa, except that the most reactive fraction from S. areolatum was > 70 kDa (Ibanez et al. 1988). Further fractionation of the Sephadex G-75 allergenic pool of C. cyathiformis produced at least 15 Coomassie blue-staining bands after isoelectric focusing, of which seven bound IgE from allergy patients. A band with pI 9.3, reacting with 63% of RAST-positive sera, was common to three species (Horner et al. 1991). After purification, it produced a single band by isoelectric focusing and had a molecular mass of 16 kDa but still cross-reacted with similar and other bands in other species of basidiomycete. The protein was rich in glycine, serine and glutamic acid/glutamate, which together formed 47 mmol/L% of the protein. Cross-reactivity occurs among basidiomycetes, including among some distantly related species. De Zubiria et al. (1990) showed specific inhibition of Western-blotted allergens by extracts of species from different taxonomic families and even orders. This implies that sensitization to these fungal allergens should be broadly interpreted as regards exposure source(s).

Other fungi and current allergen recognition approaches Using traditional methods, allergens from various fungi have been characterized. As discussed, the advent of blotting, peptide sequencing, and DNA sequencing techniques dramatically changed the approach to fungal allergen identification. Identification of allergen genes permits more rapid detection of homologous allergens in different fungi, facilitates direct comparison of allergens from different fungi, and often provides functional identifications as well. Although most allergens were initially identified with traditional methods, allergen gene sequences have now been identified in Epicoccum nigrum, Ulocladium species, Stachybotrys chartarum, Coprinus comatus, Psilocybe cubensis, Embillisia species, Curvularia lunata, Fusarium culmorum (and F. solani), Stemphylium species, Trichophyton species, and the yeasts Saccahromyces cerevisiae, Candida albicans (and C. boidinii), Rhodotorula

Fungi as Allergens

mucilaginosa, and Malassezia furfur (and M. sympodialis) (list compiled from www.allergome.org; accessed March 2007). The rate of description of new allergens and the availability of online databases now greatly facilitate identification of allergenic fungi. The long-established doctrine that all fungi may be allergenic is supported by the application of these molecular techniques since allergens tend to be usually found wherever known allergen sequences are sought. Many fungal allergens are now recognized from comparison of peptide sequences (determined in vitro or in silico) rather than from protein purification and IgE binding. Comparison from sequence databases has revealed distinct allergen distribution patterns; some allergens are highly conserved across many taxa, whereas other allergens occur only among limited groups of fungi (Bowyer et al. 2006). A range of over 30 species of Alternaria and fungi from five other genera were surveyed for the gene for Alt a 1 (Hong et al. 2005). Homologs were identified throughout the group of fungi, all of which were from the order Pleosporales. Predicted structures were all similar, which implies a conserved function. A large portion of the airborne pigmented spores are from fungi within the Pleosporales, so that establishing distribution allergen patterns within this group could yield significant insights into overall allergen exposure and sources. Antibodies to GST identified it as an allergen in extracts from Epicoccum, Aspergillus and Curvularia, which are rather distantly related fungi (Shankar et al. 2005). The GST in these extracts was shown to be both enzymatically active and able to bind serum IgE, and were cross-reactive. A major allergen of Stachybotrys was identified with a combination of IgE blotting and amino acid sequencing (Karkkainen et al. 2004). Based on sequence homology, this allergen is a cellulose, or component of the cellulase complex. A key aspect of this is that cellulases are not cytosolic enzymes, but rather are secreted into the environment. This has important implications for indoor air quality issues in water-damaged/damp buildings since mold growth is almost always on cellulosic building materials, and thus cellulases from a variety of fungi may accumulate in this situation. The recognition of these features would be far more challenging with traditional methods of allergen characterization. Allergens have been identified and characterized from other yeasts and plant pathogenic fungi (beyond those discussed above) and from dermatophyte fungi as well. As current techniques are applied to the detection, identification and characterization of fungal allergens, gaps in the knowledge of allergens from these other groups will be filled. These current techniques generate results with great speed relative to the traditional protein purification methods. Obviously, this will permit delineation of the patterns of occurrence, sensitization and exposure to allergens from all groups of fungi.

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Nonallergic factors associated with exposure to fungi

PM2.5 alone should be explored, but possible interactions with biologically active materials (allergens, glucans, etc.) on the particles should not be ignored.

Glucans (1→3)-β-Glucans (with 1→6 branches) are important structural carbohydrates in the cell walls of higher fungi, which include the majority of fungi that become airborne (Williams 1994). Although fungi are not the only source of β-glucans, it would be expected that all particles of fungi (spores, fragments of spores or mycelium) would contain β-glucans. Although currently there are mixed results on the health effects (Douwes 2005), since β-glucans have long been known to have significant immunomodulatory effects (DiLuzio 1985) possible interactions with allergens should not be disregarded.

Mycotoxins Controversy persists about the role that mycotoxins may play in inhalational exposures, particularly in moldy indoor environments. As with glucans, however, mycotoxins on fungal particles will be copresented to the respiratory mucosa along with allergens and also with glucans. Research on mycotoxin exposures should thus consider interactions with these other factors.

PM2.5 As discussed in the section on particles, building materials colonized by fungi are significant sources of fine particulate matter (PM2.5). PM2.5 has potent respiratory effects and levels are even regulated in outdoor air. The effect of fungal

Fungal studies in the aftermath of Hurricane Katrina Hurricane Katrina was a tropical cyclone that struck the Gulf Coast of the USA in late August 2005. It was the deadliest hurricane to strike the USA since 1928, with over 1200 hurricane-related deaths (CDC 2006a). On the morning of August 29, 2005, Hurricane Katrina came ashore in southeastern Louisiana. At this time the storm intensity was rated as a strong category 3, although it had been category 5 the previous day. In addition to the direct effects of the wind and rain, levees protecting greater New Orleans were breached in several places, and water poured into the city from Lake Pontchartrain whose levels were elevated by the storm surge from the Gulf of Mexico. Approximately 80% of the city was flooded (Fig. 46.8), and in some areas flood waters were over 6 m deep (Reid 2006). In various parts of the city the flooding lasted several weeks, such as South Lakeview (Fig. 46.9). Although the destruction of Hurricane Katrina is generally associated with New Orleans, the storm created havoc along the Gulf Coast from Louisiana to Florida. About 1 month later Hurricane Rita also struck the Gulf Coast, reflooding some areas that were just beginning to dry out. Overall more than 230 000 km2 of the Gulf Coast were affected by the storms, 1.3 million people displaced, and over 200 000 homes destroyed,

Fig. 46.8 Map of New Orleans area showing flooding. (See CD-ROM for color version.)

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Fig. 46.9 Photograph of flooded New Orleans neighborhood. This picture was taken from a South Lakeview area home. (See CD-ROM for color version.)

Fig. 46.10 Photographs of mold from a flooded house from the South Lakeview area shown in Fig. 46.9. (See CD-ROM for color version.)

the majority in New Orleans (Reid 2006). The extensive mold growth in flooded buildings raised concerns about possible health effects as residents began returning to the city (CDC 2006b). Several studies examined air quality issues in the New Orleans area 7–8 weeks following Hurricane Katrina. A preliminary survey of 112 homes conducted by representatives of the Centers for Disease Control and Prevention during October 22–28, 2005 found visible mold growth in 46% of the homes (CDC 2006b) (Fig. 46.10). Air samples were collected in 20 of these homes by filtration and analyzed for culturable fungi, β-glucans, and endotoxin. Outdoor samples were collected at 11 of these locations. Species of Penicillium and Aspergillus were the predominant fungi isolated indoors as well as outdoors; however, concentrations were not reported. Levels of β-glucans and endotoxin were higher indoors compared to outdoors but the differences were not significant (CDC 2006b). Solomon et al. (2006) examined the potential heath risk to returning residents and remediation workers from exposure to airborne fungal spores and endotoxin. These researchers conducted air sampling on four days in October and four days in November 2005 in New Orleans and surrounding towns. Samples were collected in a variety of indoor and outdoor locations using spore traps for total fungal spores and filter sampling for endotoxin. Outdoor locations included flooded and nonflooded areas as well as regions remote from flooding. Indoor samples were collected from two homes with less than 4 cm flood water and six homes with over 1 m of flooding. Outdoor fungal spore levels ranged from around 21 000

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to 102 000/m3, with concentrations in flooded areas higher than nonflooded areas and those areas remote from flooding. Spore concentrations found in indoor samples ranged from 11 000 to 645 000/m3, with samples from flooded homes significantly higher than outdoor samples. In contrast, the homes with minimal flooding had lower fungal spore levels than the corresponding outdoor samples. Cladosporium and Aspergillus/Penicillium-type spores were the dominant fungi identified from the outdoor air samples and accounted for 60–90% of the total spores. In nonflooded areas, Cladosporium spores were dominant with 70–75% of the total spores. In flooded homes Penicillium/Aspergillus-type spores accounted for over 70% of the fungal spores registered in the samples. In one flooded home the concentration of airborne Stachybotrys spores was over 300 000/m3. There were no significant differences in the endotoxin levels measured in the flooded compared with the nonflooded area or between indoor and outdoor measurements. Chew et al. (2006) examined airborne fungi and endotoxin levels in three flooded homes in order to determine the efficacy of remediation procedures. Indoor and outdoor samples were collected before, during, and after remediation using Teflon filters which were assayed for culturable fungi and endotoxin. Polymerase chain reaction (PCR) analyses for specific fungi were also performed on aliquots of the filter eluate. In addition spore trap samples were collected from indoor and outdoor locations; however, post-remediation samples were not collected at two of the houses. Prior to remediation, the concentration of indoor fungi showed considerable range in the three homes using three methods of analysis. Culturable fungi ranged from 22 000 to 515 000 cfu/m3, total spore levels from over 82 000 to over 634 000/m3, and PCR levels ranged from over 80 000 to over 1 million spore equivalents/m3. For all methods, indoor levels were higher than outdoor concentrations. During the remediation, all fungal concentrations increased but decreased again post remediation, often by two orders of magnitude. Penicillium, Aspergillus, and Paecilomyces were the most frequently recovered fungi by all sampling methods. The percent occurrence of other fungi varied with the sampling method. Endotoxin levels were higher indoors prior to and during remediation when compared witho outdoor levels. Following remediation, one house still had elevated endotoxin levels (Chew et al. 2006). Ochsner Clinic Foundation, a large medical center in New Orleans, is located in an area of the city that was not flooded, although flood waters came within 1–2 km of the facility. Air sampling with a Burkard spore trap has been ongoing at Ochsner Clinic since August 2003, although the sampling record is not complete for 2004. The sampler is located on the roof of a five-story building at the clinic. Following Hurricane Katrina, sampler operations resumed on October 8, 2005. To determine the effects of the storm on the outdoor air spora, samples collected during October 8–31, 2005 were compared to samples available from 2003 and 2004 during the same

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time period (Levetin et al. 2007). There was no significant difference in mean total spore levels during the period October 8–31 in 2003 and 2005. The mean for this period in 2003 was 6935 spores/m3 and the mean for 2005 was 6828 spores/m3 (t = 0.41, P > 0.05). The mean for October 2004 was 4690 spores/m3; however, data were only available for 7 days. The Cladosporium concentration was slightly higher in October 2005 (mean 3232 spores/m3) than 2003 (mean 3009 spores/ m3) but the difference was not significant. In contrast, the post-Katrina air samples did show significant changes in the composition of the air spora for other taxa. There were significant increases in the Penicillium/Aspergillus and Chaetomium concentrations in October 2005 compared with October 2003. In contrast, basidiospore and ascospore concentrations were significantly lower in October 2005. Also Eurotium ascospores, Stachybotrys conidia, and Myrothecium conidia were registered in the post-Katrina air samples. Short-term grab samples collected with a Burkard personal sampler during the fall of 2005 at several locations in the flooded areas of the city showed similar increases of Penicillium/Aspergillus-type spores (Levetin et al. 2007). These results from the Burkard at Ochsner Clinic are also in line with the samples collected by other investigators in fall 2005 (CDC 2006b; Chew et al. 2006; Solomon et al. 2006). Although the concentrations at Ochsner Clinic were lower than those reported by other investigators, the Burkard samples were obtained in an area that was not flooded and were collected at rooftop level not at ground level. Limited data are available on any lasting effects caused by flooding on indoor mold growth. Pearce and Huelman (1995) conducted long-term monitoring of fungal growth in eight homes that were flooded during the spring or summer of 1993 in Minnesota. Sampling was undertaken following remediation and continued for up to 1 year for two homes and 17 months for six homes. Results indicated that mold spore levels in the homes remained elevated long after remediation. Although concentrations generally returned to normal after 1 year, some contamination remained at the end of the study. Few studies exist relating adverse health effects to elevated mold spore exposure following flooding. Ross et al. (2000) examined the relationship between asthma severity and indoor environmental factors in homes that had been flooded the previous year and in nonflooded homes. Repeated measures of fungal spores and other environmental factors showed that increasing levels of airborne mold spores were associated with increased risk of emergency room visits for asthma; however, there was no association between asthma severity and flooding status of the home. Dalan et al. (2007) reported increases in patients’ visits to an allergist for allergic rhinitis complaints following the Red River flood in Fargo, North Dakota during the spring of 1997. Skin-test records for 1628 patients from 1995 to 2005 were examined to determine if any changes in reactivity followed the flooding. Cephalosporium acremonium sensitivity showed a significant increase 3 years

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after the flood. Reactions showed increased rates among patients living in areas adjoining the river and in flooded areas. Yearly fluctuations in sensitivity to other fungi, several weeds, and grasses were found to correlate with airborne levels of pollen and mold spores. The massive mold contamination following Hurricane Katrina provides an excellent opportunity to study the health effects of mold exposure (Hampton 2006). At Ochsner Clinic in New Orleans, a random review of patient charts showed no increase in mold sensitivity from Hurricane Katrina detected in skin-prick testing through June 2006 (Levetin et al. 2007). However, the report by Dalan et al. (2007) suggests that changes could occur several years following flooding. Clearly, longterm monitoring of the health effects of mold exposure and measurement of the mold levels in indoor and outdoor environments of mold exposure and measurement of the mold levels in indoor and outdoor environments assessment of any mold-related effects.

Conclusion Fungi are abundant and widespread allergens and their importance in causing seasonal allergy is probably underestimated because of the variable, poor-quality, unstandardized allergens used for diagnosis and, in the past, the use of inadequate sampling methods. Fungi are the most numerous particles in outdoor air and can occur in even larger numbers, often with actinomycetes, in working environments, leading to occupational asthma and allergic alveolitis in workers. These diseases have a long history. In the early 18th century, Ramazzini (1713) described how threshers, sifters and measurers of grain were plagued by the dust, especially from heated and crumbling grain, with symptoms closely resembling allergic alveolitis. Such occupational exposure occurs widely in agriculture and is now recognized in many other industries, especially where stored products are handled. The growth of biotechnology-related industry will require, in the future, an awareness of the potential dangers of exposure and of occupational respiratory disease. The use of adequate sampling equipment is important for determining exposure to spore allergens in both indoor and outdoor situations. The Durham slide and settle plates produced much useful information but are subject to many errors of interpretation, resulting from their bias toward large spores and pollens, their greater settling speeds, and the erratic effects of wind speed and turbulence on deposition. As a consequence, the importance of ascospores and basidiospores was long neglected. Even some newer samplers, favored for their convenience, are subject to errors because sampling rates cannot be accurately determined and irregular patterns of deposition, particularly of larger particles, make interpretation difficult. The suitability of different microbiologic sampling methods has been reviewed by Cox and Wathes (1995).

Fungi as Allergens

It should be noted that exposure to airborne fungal material, particularly where mold growth occurs indoors, includes exposure to allergens, fine particulate matter, glucans, and possibly toxins. These components all are particle-associated and hence will impact on respiratory mucosa together. Several non-IgE-mediated factors are involved in exposure to fungi, including glucans, mycotoxins and PM2.5. The importance of any of these factors alone is poorly understood, and the occurrence of interactions or synergistic effects of coexposures of these factors with allergens is entirely unexplored and remains an important area of future studies. Good diagnostic antigens are urgently required if the importance of fungal allergy is to be fully established, and methods of standardization need to be developed. The major allergens in all the most common airborne fungi need to be identified and standardized and sensitive and rapid assays developed in order to diagnose disease in patients and detect the presence of allergen in the air. The development of immunologic assay methods could even allow the detection in the atmosphere of unknown allergens relevant to seasonal and occupational lung disease, as has been done for mite, insect and domestic and laboratory animal allergens (Vijay & Kurup 2004). Immunoassays are being developed for airborne phytopathogenic Alternaria, Botrytis and Sclerotinia species trapped in enzyme-linked immunosorbent assay (ELISA) strip wells mounted on a whirling arm (Rotorod) sampler, which could perhaps be extended to detect potential allergens if sensitivity can be improved (Schmechel et al. 1994). Despite many attempts, no threshold values have yet been applied to residential or occupational exposure to fungal spores. If these are to be applied, they will require adequate and reproducible sampling and assay methods to monitor and control concentrations, greater understanding of the dose–response relationships in occupational respiratory diseases than at present, and knowledge of how constitutional factors affect such relationships in the exposed population. However, they also require a detailed understanding of outdoor and indoor air spora to identify the changes that might be significant in relation to allergy. Numbers and types of different fungi and constitutional factors in the exposed population are all important in determining whether a given exposure is likely to be hazardous.

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Dust Mites and Asthma Thomas A.E. Platts-Mills and Judith A. Woodfolk

Summary In prospective and cross-sectional studies, dust mites are the single most important allergen associated with asthma. This is despite the fact that exposure is generally low even in countries where mites flourish. Thus, there is increasing interest in understanding why exposure to mite feces is such a potent cause of IgE antibody production. The possible factors influencing this response include the enzymatic activity of Der p 1, the effects of endotoxin on Toll-like receptor (TLR)4 or of DNA-derived from mites or bacteria on TLR9. More recently it has been proposed that the immunogenicity of mite allergens also reflects the “foreignness” of their amino acid sequence, which is largely a function of evolutionary distance. Exposure to dust mite allergens is not only a cause of IgE antibody production, but is also considered to be an important cause of bronchial hyperreactivity. However, because of the form of exposure, this effect is delayed in onset and the inflammation can last for weeks or months. The result is that even if patients are aware of being allergic to dust they generally do not appreciate the relevance of exposure to their lung symptoms. In keeping with the high prevalence of mite sensitization, these patients have been the focus of a large number of studies on allergen avoidance and immunotherapy as treatment for asthma. Evaluation of the evidence strongly favors a central role for allergen-specific treatment in asthma.

Introduction and history The realization that asthma is an inflammatory disease came very rapidly and was in large part dependent on biopsy studies of patients with mild to moderate asthma. Of course, it had been known for many years that these patients have eosinophilia, as well as eosinophils and Charcot–Leyden crystals in their sputum. Furthermore, Altounyan had already shown in 1970 that bronchial hyperreactivity (BHR) was reversible, and increased during periods of exposure to pollen. It was also

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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clear that removing mite-allergic patients from their houses could result in a decrease in BHR (reviewed in Platts-Mills & Chapman 1987). Nonetheless, until about 1985 there was still a majority opinion that asthma was primarily a physiologic property of the lungs. In this model, allergen exposure could never be more than a “trigger factor” for lungs that were already hyperreactive. In addition, if allergens were a trigger factor, it was to be expected that the response would be rapid and obvious to the patient. Thus, many textbooks included phrases such as “allergens should be considered as a factor [only] when there is a history of increasing symptoms following exposure.” The epidemiology of dust mite sensitivity and the detailed study of dust mite allergens has played a major role in reshaping our understanding of both the role of allergen exposure in asthma and the relevance of allergenspecific treatment in the management of asthma. As early as 1921 it was reported that many asthmatics had strongly positive skin tests to house dust (Kern 1921; Spain & Cooke 1924). Although some of the allergenicity of dust could be explained by animal dander, it was obvious that there was a major allergen content in house dust that was not explained. The breakthrough came in the mid-1960s, when Spieksma and Voorhorst established that there was a strong relationship between the number of dust mites (actually Dermatophagoides pteronyssinus) and the allergenicity of house dust (Voorhorst et al. 1967). Dekker had suggested that mites played an important role in 1928, but his evidence was unconvincing and it was not possible to grow mites in culture at that time (Dekker 1928). Spieksma and colleagues succeeded in culturing dust mites and this made it possible to produce extracts for routine use. In turn, this led to a series of studies demonstrating the association between immediate hypersensitivity to dust mites and asthma (Miyamoto et al. 1968; Smith et al. 1969; Clarke & Aldons 1979). The association was reported from Holland, England, Australia, Japan, and many other countries. In some studies, the association was so strong that it could be taken to imply a causal relationship. In addition, the ability to identify mites in dust samples made it possible to study the distribution of dust mites in houses and in different climatic regions (Voorhorst et al. 1967). Thus, it was demonstrated that dust from houses in Holland close to canals (i.e., most houses) often had ≥ 500 mites/g. In contrast, the sanatoria in Davos

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(Thomas Mann’s Magic Mountain) contained very few mites. Indeed, the Dutch group ascribed the improvement in asthma symptoms in Switzerland to decreased exposure to dust mites. Despite (or perhaps because of) the strength of the evidence from Europe, the importance of dust mites as a source of indoor allergens was not initially recognized in the USA (Kawai et al. 1972). However, the situation in the USA is complex because of the wide range of climatic and living conditions (Sporik et al. 1995).

Mite allergens and mite fecal particles The next step forward came when Bencard in England developed large-scale culture techniques so that it was possible to

Dust Mites and Asthma

introduce widespread use of immunotherapy for asthma. As a byproduct of those cultures, sufficient material (i.e., ∼ 400 g) was available from D. pteronyssinus cultures to purify a major allergen, Der p 1 (initially antigen p1) (Chapman & Platts-Mills 1980). Der p 1 is a glycoprotein of molecular weight 24 000 which is freely water soluble, labile to heat or pH, and highly immunogenic in rabbits and mice (Table 47.1) (Chapman et al. 2007). Radioimmunoassays for mite allergens were used to prove the fecal origin of Der p 1, to measure the concentrations in dust samples, and to measure the quantities of mite allergen that become airborne (Tovey et al. 1981a,b). By purifying sufficient Der p 1 to weigh, it was possible to standardize the assays in absolute units, i.e., micrograms, and thus express airborne allergen in ng/m3. From the first experiments, it was obvious that mite allergen was not airborne in

Table 47.1 Nomenclature and characteristics of indoor allergens. Source

Allergen

MW (kDa)

Function

Sequence†

25

Cysteine protease

cDNA

14

Unknown

cDNA

~ 30

Serine protease

cDNA/protein

Euroglyphus maynei Blomia tropicalis (Arruda et al. 1995a) Lepidoglyphus destructor

Group 1 (Der p 1, Der f 1) Group 2 (Der p 2, Der f 2) Group 3 (Der p 3, Der f 3) Der p 4 Der p 5 Der p 6 Der p 7 Der p 8 Eur m 1 Blo t 5 Lep d 1

~ 60 14 25 22–28 26 25 14 14

Amylase Unknown Chymotrypsin Unknown Glutathione transferase Cysteine protease Unknown Unknown

Protein cDNA Protein cDNA cDNA PCR cDNA cDNA

Mammals Felis domesticus Canis familiaris Mus musculus

Fel d 1 Can f 1 Mus m 1

36 25 19

(Uteroglobin) Unknown Calycins, pheromonebinding proteins

PCR cDNA cDNA

Rattus norvegicus

Rat n 1

19

Bla g 1 Bla g 2 Bla g 4 Bla g 5 Bla g 6 Per a 1 Per a 3

20–25 36 21 23 18 20–25 72–78

Unknown Aspartic protease Calycin Glutathione transferase Troponin C Unknown Unknown

cDNA cDNA/protein cDNA cDNA/protein cDNA cDNA cDNA

Asp f 1

18

Cytotoxin (mitogillin)

cDNA

House-dust mite Dermatophagoides spp. (Thomas 1996)

Cockroach Blattella germanica (Arruda et al. 1995b; Pomes et al. submitted; Arruda et al. submitted)

Periplaneta americana

Fungi Aspergillus fumigatus (Arruda et al. 1992)

cDNA

† Method given for full sequence determination, where available. However, protein sequences are incomplete; usually N-terminal or internal peptide sequences have been determined.

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an undisturbed room and also that the allergen that became airborne during disturbance fell within 20 min after the end of a disturbance, such as vacuum cleaning (Tovey et al. 1981b; Custis et al. 2003). These observations fitted with the evidence that Der p 1 became airborne almost exclusively on mite fecal particles (Tovey et al. 1981b). The properties of mite fecal particles are important both to the biology of the mites and to understanding the ways in which allergen exposure can contribute to asthma. The fecal particles range in size from 15 to 30 μm diameter and consist of an amorphous center encased in a chitinous peritrophic membrane. Several of the main allergens, including Der p 1, Der p 3 and Der p 4, are digestive enzymes. It seems likely that the center of the particle continues to digest after it is released. Coprophagia is normal for some species of mites, and this practice has been observed in cultures of D. pteronyssinus (Stewart et al. 1989). Thus, the particle might be designed to allow easy recognition by the mite of its own feces. Alternatively, the peritrophic membrane may be important in protecting the hindgut from mechanical abrasion by the gut contents or helping to reduce water loss in the feces. A wide range of studies confirmed that dust mite sensitivity was very strongly associated with asthma in those countries where the climate favored mite growth, i.e., humid for 6 months or more (Platts-Mills & De Weck 1989). For many years we assumed that the fecal particle was simply an efficient method of delivering mite allergens, e.g., Der p 1 and Der p 2 to the nose. However, calculation of the quantities of mite allergen inhaled are very low, i.e., 2–10 ng/day. Thus, there are real questions about how the human immune system succeeds in identifying and responding to these proteins. At the same time it has become clear that both endotoxin and the DNA of nonvertebrate animals can act as a potent adjuvant by binding to Toll-like receptors (TLR)4 and TLR9, respectively (Eisenbarth et al. 2002; Muller et al. 2008). Perhaps equally important, it has become clear that TLR ligands can enhance responses to proteins that are presented in the same phagosome (Blander & Medzhitov 2006; Blander 2007). Thus, we now see the fecal particle as a method of delivering mite allergens in proximity to potent TLR ligands, including endotoxin, mite DNA, and bacterial DNA. If mite allergens become airborne on fecal particles, then we assume that this is the normal form in which the allergens are inhaled. From the airborne data, it seems likely that mite particles are inhaled during domestic disturbance or when the patient’s head is close to pillows or furniture. It has proved very difficult to make accurate measurements of natural exposure; however, measurement during disturbance suggests that inhalation of 5–20 ng is likely. This represents ∼ 25–100 fecal particles; however, from experimental studies one would only expect 5–10% of these particles to get beyond the larynx (Task Group on Lung Dynamics 1966; Svartengren et al. 1987). Thus, our best estimates suggest that natural exposure represents a relatively small number of “large” particles,

986

Sinusitis (Bacteria?) Fungi?

Colonization of the lungs Aspergillus

Inhalants Dust mites# Cat, dog Cockroach ?Other mammals

* T cells?

* Sensitization – lgE ab

*

*

or

* Tolerance Th2 + lgG4 (Modified TH2 response)

Bronchial inflammation‡ Mast cells, eosinophils, T cells Rhinovirus

* Airway obstruction

* Possible sites for genetic control ‡ Bronchial inflammation is related to but not the same as bronchial hyperreactivity # Prolonged exposure to nonmammalian allergens generally at a very low dose Fig. 47.1 Etiology of bronchial reactivity in adults.

each of which contains a high concentration of allergen and would be expected to produce a small focus of inflammation in the lung. The contrast with bronchial provocation is dramatic; nebulization of an extract inhaled over 2 min could equal as many as 107 or 108 droplets inhaled with a very small quantity of allergen in each particle. Thus, although bronchial provocation appears to represent a higher total quantity of allergen inhaled, the concentration at the site of impact is probably much lower than when exposed to the naturally occurring particles. Daily exposure to a relatively small number of mite particles may be the best way to gradually increase inflammation in the bronchi and the related BHR (Fig. 47.1). As envisaged, this process would not be apparent to patients, who would have no reason to connect a progressive increase in nocturnal coughing, or wheezing on exercise, with chronic exposure to dust on the sofa or in bed. Remember that cat-allergic patients are often much more aware of the relationship between exposure and development of symptoms; this appears to reflect the fact that cat allergen remains airborne and is associated with smaller particles (Luczynska et al. 1990; Custis et al. 2003).

Measurement of exposure Mites, like any animal, produce many different proteins, any of which would be expected to be highly immunogenic in humans if injected with an appropriate adjuvant. Thus, the factors influencing the importance of different mite proteins as allergens may be largely physical, i.e., quantity arriving on the nasal mucosa and solubility; however, there may be an added role for enzymatic activity as an adjuvant (Stewart et al. 1989). Measurement of exposure could be designed either to detect all the important allergens or to use one protein as a marker. For practical reasons, it is impossible to measure all

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allergens separately. Radioallergosorbent test (RAST) inhibition, which theoretically measures all the important allergens, is extremely difficult to standardize, cannot give results in absolute units, and is too technically demanding for routine use. Thus, the practical approach is to use assay of a single protein, and the easiest proteins to use are the group 1 proteins Der p 1 and Der f 1 (Chapman & Platts-Mills 1980; Chapman et al. 1985; Luczynska et al. 1989). The important factor is that these proteins represent a significant proportion of the protein in mite extracts; as digestive enzymes, their production is likely to be essential for the survival of mites, and thus they are almost certainly a good marker for the presence of mites (Platts-Mills et al. 1997). However, the relative proportions of the mite allergens in mite extracts produced in different ways or in different parts of the mite are not the same. The ratio of group 1 to group 2 proteins in an extract can vary from 20 : 1 to 1 : 2. Thus, the measurement of group 1 proteins in dust must be judged as an index of exposure, i.e., as a marker, not as a measurement of total exposure. Many different assays for the group 1 antigens have been developed, including fluid-phase radioimmunoassays and solid-phase assays using monoclonal antibodies or polyclonal antibodies and radioactive or enzyme markers. While there are major attractions to using monoclonal antibodies, there are no absolute reasons for using one assay rather than another (Van Ree 2007). On the other hand, there are overwhelming reasons for using absolute units referred to national or international standards (Ford et al. 1985; Chapman & Platts-Mills 1988). The current standard [National Institute of Biological Sciences and Control 82/518] is a D. pteronyssinus culture extract that has been considered to contain 12.5 μg Der p 1/ampoule (Ford et al. 1985). All values referred to in this chapter are dependent on that estimate. The primary question about collection of samples is whether to attempt to measure the allergen being inhaled. As we have already discussed, there is no significant airborne mite allergen in an undisturbed room. The measurements of airborne allergen reported in houses without artificial disturbance have been either very low (i.e., < 1 ng/m3) or have had a poorly defined degree of disturbance (Tovey et al. 1981b; Price et al. 1990; Sakaguchi et al. 1990; Woodfolk et al. 1995). In our view, it is still not possible to standardize disturbance and therefore not appropriate to use airborne measurements in epidemiologic studies. There has also been extensive discussion about techniques for collecting dust samples from reservoirs within the house. However, there are two important factors to consider: firstly, if the sample is too small, i.e., < 20 mg of sieved dust, measurements become increasingly imprecise; and, secondly, most homeowners will vacuum-clean prior to the visit. Because of this, we find that estimates of total allergen per m2 of carpet are difficult to obtain and that values relating to the concentration of allergen in dust are more reliable. The three international workshops on mite allergens and asthma concluded that the concentration of group 1 mite allergen per gram of dust should be the primary index of

Dust Mites and Asthma

exposure (Third International Workshop 1997). This was not meant to discourage the use of other methods of measuring or expressing results. It was strongly suggested that this measurement should be given and that other measurements, such as airborne or total allergen recovered, should be related to or compared with the concentration of allergen found in reservoir dust.

Epidemiology Relationship of dust mite sensitization to asthma Following the discovery of dust mites and the development of skin-test reagents, it rapidly became obvious that there was a strong association between positive wheal and flare skin tests to dust mites and asthma (Miyamoto et al. 1968; Smith et al. 1969; Sears et al. 1989). This association is present in studies from many different parts of the world and is clearest in children and young adults (Peat et al. 1987; Pollart et al. 1989; Sporik et al. 1990; Charpin et al. 1991; Gelber et al. 1993, Illi et al. 2006; Sporik et al. 1999; Sporik & Platts-Mills 2001). In some areas, the association is so strong, e.g., 80 – 90% of young people with asthma in Japan or New Zealand, that it can be taken to imply a causal relationship between mite allergen sensitivity and asthma (Pollart et al. 1989; Sporik et al. 1990; Gelber et al. 1993; Squillace et al. 1997; Sporik et al. 1999). In addition, in these countries the prevalence of sensitization is so high that any increase in asthma prevalence must have included mite-allergic children. It is very important to recognize that such an increase in asthma could be predominantly affecting mite-allergic children and yet not be caused by an actual increase in mite allergens (Platts-Mills 2005). On the other hand, in some countries much of the increase in asthma symptoms could be explained simply by an increase in mite allergen exposure resulting from changes in the furnishing, ventilation, and temperature of houses, coupled with increased time indoors. In all studies, mite sensitization is also found in a proportion of the asymptomatic or nonasthmatic controls. This proportion ranges from 5 to nearly 20%; thus, it is clear that there must be factors other than sensitization that dictate the phenotype of active disease. Finally, it is clear that the association between mite sensitization and asthma is restricted to areas of the world where mites can grow in houses; hence, there is little or no association between mite allergens and asthma in northern Sweden, central Australia, the French Alps, i.e., Briançon, or the mountain regions of the USA (Charpin et al. 1991; Wickman et al. 1993; Peat et al. 1994; Ingram et al. 1995; Sporik et al. 1995; Perzanowski et al. 2002; Erwin et al. 2007). Furthermore, recent evidence from some northern cities in the USA suggests that mite sensitization is not very common among inner-city asthmatics (Rosenstreich et al. 1997; Eggleston et al. 1998); this may well reflect the long dry winters, which would be expected to reduce mite populations to levels that are unable to recover during the humid summers (Arlian et al. 1982; Korsgaard 1983a).

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Relationship between exposure to dust mites and sensitization Specific immune responses in humans, as in all other species, require immunization or natural exposure. However, dust mites are present in the majority of houses at concentrations ranging from < 10 000 to 24 000/g, and it is clearly important to know what concentration of mites and mite allergens creates a risk of sensitization. Early studies suggested that 100 mites/g was a level that could increase the risk of asthma (Voorhorst et al. 1967; Korsgaard 1983a). In 1987, we proposed that a value of group 1 allergen of 2 μg/g should be considered a threshold for sensitization (Platts-Mills et al. 1987). Subsequently, evidence for this value was obtained from several studies and considered by three international workshops (Lau et al. 1989; Sporik et al. 1990; Charpin et al. 1991; Third International Workshop 1997). By 1994, it was clear that this concentration was a useful value for comparing studies, but it is not a threshold in the sense that this word is used for toxicology. It is apparent that in towns where the majority of houses contain > 2 μg/g dust, mite sensitization will be common among asthmatic children. However, some children can become sensitized on exposure to lower levels. Furthermore, children who are not genetically predisposed to become allergic are only likely to become sensitized on exposure at much higher levels. Kueher et al. (1994) demonstrated a clear difference in “threshold” between atopic and nonatopic children in Germany. The real distinction from exposure thresholds for gases, such as SO2 or ozone, or chemicals, such as lead, is that mite allergens are not inherently toxic, so that for much of the population even very high levels do not present any danger. Equally important, the threshold is not truly a value below which no harmful effects will occur, because a few children will become sensitized to dust mites even though they live in houses where group 1 allergen is below 2 μg/g dust, and some highly allergic individuals appear to have ongoing symptoms even though they are only exposed to low levels in the house. Nonetheless, we believe that the evidence now shows that in an area where most or all houses contain less than 2 μg/g, very few children, i.e., < 5% of children, will become sensitized to mites and mite sensitization will not be significantly associated with symptomatic asthma (Martinez et al. 1995; Sporik et al. 1995; Erwin et al. 2007).

Relationship between exposure to dust mites (and other indoor allergens) and asthma When the original thresholds were presented, it was also suggested that a group 1 allergen concentration of 10 μg/g dust was one that would produce increased symptoms, including acute attacks of asthma. However, since that time there has been only marginal support for such a view. Indeed, it has proved difficult to obtain evidence supporting a simple dose– response relationship between exposure and symptoms (Sporik et al. 1990; Charpin et al. 1991; Call et al. 1992; Gelber et al. 1993; Marks et al. 1995; Lau et al. 2001). What is absolutely clear is

988

that some individuals who have IgE antibodies to dust mites do not experience any symptoms of asthma and do not develop BHR, even though they have high levels of mite allergen in their houses. On the other hand, there are individuals who become so sensitive that even levels as low as 1 μg/g will give rise to symptoms. If we assume that a group of patients has lived in an area where mite allergen levels are stable, then the fact that there are more individuals who are sensitized to dust mites than there are individuals with asthma means that the average level of allergen necessary to induce BHR and/or symptoms is higher than the level necessary to sensitize. However, there is a wide range of sensitivity, such that some patients only have symptoms with high levels of exposure while others are so sensitive that it would be difficult to reduce their exposure below the level at which they get symptoms.

Allergen-specific treatment Allergen avoidance If it is accepted that mite allergens entering the lung predominantly are an important cause of the inflammation that is a common feature of patients with asthma, then reducing exposure is the logical first line of treatment. There is abundant evidence to show that radical reduction, i.e., > 90%, in mite allergen exposure can reduce symptoms and reverse bronchial reactivity (Kerrebijn 1970; Platts-Mills et al. 1982; Vervloet et al. 1982; Murray & Ferguson 1983; Ehnert et al. 1992). However, many of these studies have involved moving the patients into a sanatorium or hospital, where mite group 1 allergen levels are typically < 0.4 μg/g dust. Six well-conducted controlled trials have demonstrated success of avoidance measures in the patient’s home (Murray & Ferguson 1983; Ehnert et al. 1992; van der Heide et al. 1997), and it is these studies that provide the best evidence about effective avoidance measures (Table 47.2). The assumption is that effective avoidance measures will reduce inflammation in the lungs; indeed, it seems inevitable that there must be a decrease in cellular influx and in mediator release in order to observe the major decreases in BHR that have been observed. Boner and his colleagues in Verona have reported progressive decreases in eosinophils in induced sputum occurring in parallel with decreases in BHR among children staying in the sanatorium at Misurina in the Dolomites (Boner et al. 1985; Peroni et al. 2002). Thus, it seems reasonable to conclude that allergen avoidance can be an “antiinflammatory” treatment. The next question is whether avoidance measures are practical and effective when included in a normal treatment plan. Some results have suggested that simple advice given in a clinic is ineffective (Korsgaard 1983b; Woodcock et al. 2003). Two different conclusions can be drawn from this: either that avoidance is too difficult for routine use, or that it needs to be taken seriously (Platts-Mills 2003). Given the protocol set out in Table 47.2, there are several factors that influence success. The patient must be convinced that he/she is allergic, e.g., by seeing the skin-test response. The patient must also

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Table 47.2 University of Virginia Allergy Clinic instructions for reducing exposure to house-dust mites. House-dust mites require humidity (> 50% relative humidity) and warmth (> 21°C) to grow. Because mites avoid the light and because surfaces dry out rapidly, mites flourish in mattresses, bedding, upholstered furniture, carpets, pillows, and quilts. Under very humid conditions, mites will also grow in clothing, curtains, and any material. Procedures to reduce dust mites should focus first on the bedroom because more time is spent there than any other room and it is generally easiest to change. However, in the long run, it is best to modify much of the house and this should certainly be considered when moving. Priority objectives 1 Mattresses and pillows should be enclosed in a zippered, plastic cover or a special vapor-permeable, allergen-proof fabric. Damp-wipe the mattress cover every 2 weeks. 2 Wash all bedding, including mattress pad, pillowcases, and blankets, in hot cycle (~ 54°C) weekly. Comforters (or duvets) should be replaced with Dacron or Orlon, which can be washed with the bedding or covered with vapor-permeable covers. 3 Small objects that accumulate dust, such as knick-knacks, books, stuffed animals, and records, should be placed in drawers or closed cabinets. Clothing should be stored in drawers enclosed in plastic or in a closed closet. Unused clothing should be stored away from the bedroom. 4 Carpets should be vacuum-cleaned weekly, using a vacuum cleaner with an effective filter (Woodfolk et al. 1993). The patient should either avoid vacuum cleaning or wear a mask during cleaning. In general, dust mite allergens will take about 20 min to fall after cleaning. Medium-term objectives 1 Removing carpets from bedrooms makes it much easier to control mites. This is because carpets are very difficult to clean and will tend not only to grow mites (in humid seasons) but also to act as a source to reinfest bedding, clothing, etc. 2 Replace curtains with washable cotton curtains or venetian/slat blinds. 3 Control humidity in the house; this can be achieved by increasing ventilation if the outdoor conditions are cold and/or dry; alternatively, reducing humidity can be achieved with central air-conditioning. Dehumidifiers are helpful in basements. The objective is to keep relative humidity below 50%. 4 Treat carpets with acaricides (e.g., Acarosan) or 3% tannic acid (Woodfolk et al. 1994). Choice of houses/apartments 1 Basements are not recommended for any allergic patients (in some cases moving out of basement may be urgent because it is so difficult to control mite and/or fungal growth in the basement). Bedrooms should be upstairs. 2 Carpets fitted to a concrete slab, either in a basement or on the ground floor, tend to become damp and remain damp. We recommend that all floors should have a primary polished floor (vinyl or wood) and carpets should be movable. 3 Upholstered sofas and chairs should be avoided. 4 Air filters on central air-conditioning should be cleaned regularly. High-quality (e.g., electrostatic) filters may be helpful, but are no substitutes for reducing available mite nests in the house.

understand the biology of mites and be convinced about their presence in his or her house. In some cases, simple explanation is sufficient, but written or video presentations are generally more effective. Interactive computer education programs have been shown to produce better results (Huss et al. 1992). Assay of mite allergens in dust samples from the patient’s house may be helpful. Obviously, any measurement can only be useful to the patient if it comes with an interpretation. Thus, establishing the relevance of different concentrations is essential for developing avoidance advice. Many upstairs carpets have low or very low concentrations, i.e., < 2 μg or < 0.4 μg/g dust, and removal is not a priority. In contrast, carpets in basements or on concrete-slab foundations will become very heavily infested in humid climates and replacement should be a priority (Platts-Mills et al. 1987). Many societies historically have engaged in practices in carpet maintenance that would be expected to be effective at killing mites and/or removing accumulated allergen, e.g., washing carpets in the sea in Finland; airing carpets in the sun in the Middle East and India; beating carpets in England and the USA. Furthermore, some religious groups in the USA still have specific rules against fitting carpets in houses. The

importance of a comprehensive program of avoidance is illustrated by the failure of a large study using mattress covers only without education or a comprehensive program. That study did not help the patients and did not produce a significant decrease in mite allergen exposure. (Woodcock et al. 2003). An important aspect of avoidance protocols is to establish priorities, starting with covering pillows and mattresses, continuing with the bedding and the rest of the bedroom, and finally addressing the rest of the house. All avoidance advice should be adapted to the circumstances of the patient. One approach is to develop a list of priorities and persuade the patient to make decisions about a series of specific actions with target dates. These can then be checked during a return visit to the clinic. The following are key decisions: • The type of covers to use, cost, and availability. • Changes in bedding and feasibility of hot-water washing. • Changes in the ventilation rate of the building and how to determine whether this is necessary. • Replacement of carpets if needed and possibility of removal. • Concurrent decisions about other allergen sources, such as animal dander and/or cockroaches.

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The important conclusion is that avoidance advice needs to be taken as seriously as advice about medicines for asthma, and that all members of the medical staff should reinforce the message (NAEPP Guidelines for Asthma Management 2007).

MHC/ peptide

Immunotherapy Immunotherapy with injected allergen extracts is fully established as a treatment for seasonal allergic rhinitis and for seasonal asthma (Reid et al. 1986; Creticos et al. 2006). Although not all studies on perennial asthma have been successful, the evidence regarding immunotherapy with mite extracts is compelling (see metaanalysis by Abramson et al. 2003). Severe and fatal reactions have occurred with mite extract injections. This may reflect the fact that in many cases the buildup to full dose has to be given to patients who are exposed year-round. On the other hand, mite extracts may have been the first potent extract used for immunotherapy among patients with high levels of bronchial reactivity. There is no doubt that immunotherapy should be given with great care in patients who have unstable asthma. This should include peak flow measurements before and after treatment and minimum waiting periods of 30 min (Reid et al. 1993). Because of progressive increases in the evidence for the efficacy of immunotherapy in allergic asthma, the national guidelines in the USA now recognize a significant role for this mode of treatment (NAEPP Guidelines for Asthma Management 2007). By contrast, the UK guidelines do not recognize chronic asthma as an indication for immunotherapy. Thus, there is clearly a place for improvement in immunotherapy both in terms of efficiency and safety.

Allergic response to mite antigens and the relevance to future developments in immunotherapy IgE antibodies to dust mites can be measured either against crude extract or against specific purified antigens (Chapman & Platts-Mills 1980; Erwin et al. 2005). These antibodies correlate with skin-test reactivity and Prausnitz–Küstner activity of the serum, but do not define symptoms. Patients with IgE antibodies also have serum IgG antibodies and secretory IgA antibodies to mite allergens. Furthermore, the serum IgG response includes a significant proportion of IgG4 antibodies (Aalberse et al. 1983; Rowntree et al. 1987; Platts-Mills et al. 2001; Aalberse & Platts-Mills 2004). It was inevitable that these responses required T-cell help and it is well established that mite-allergic patients have T cells that proliferate in vitro; therefore, the details of T-cell control are highly relevant to the future design of immunotherapy (Fig. 47.2). The actual mechanism of conventional immunotherapy is not clear; however, there is good evidence for increases in IgG antibodies to mite and a marked change in the in vitro behavior of T cells (Varney et al. 1993; Reefer et al. 2004; Akdis & Akdis 2007; Chapman et al. 1980). Recent evidence has suggested that there is at least some change in the cytokine profile of

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High-dose antigen e.g., DPT, viral or bacterial infection

Macrophage

+

Th

Delayed hypersensitivity

Th2

IgG1

Th1

IL-12 CD4+ helper T cell

Th1

IFN-g

IL-2 IFN-g

IL-4 MHC/ peptide

Th2

Dendritic cell

Th2

IL-5

Eos

IL-4, IL-13 IgE, IgG4

B IgE Low-dose antigen e.g., allergens

IL-5

Eos

Mast cell* * Activated mast cells produce histamine, tryptase, leukotrienes, and also IL-3, GM-CSF, TNF-a, IL-4, IL-5, RANTES Fig. 47.2 T-cell differentiation during human immune responses (immune deviation).

T cells during immunotherapy. In Fig. 47.2, we show two pathways of eosinophil recruitment: either through direct T-cell production of interleukin (IL)-5 (or other cytokines) or mast cell production of cytokines (including IL-3, IL-4, IL5, granulocyte–macrophage colony-stimulating factor, and RANTES). At present, it is clear that allergic and asthmatic patients who have IgE antibodies to dust mite proteins generally have mite-specific CD4+ T cells which are of the T helper 2 (Th2) phenotype, i.e., they produce IL-4 and IL-5 (Wierenga et al. 1991; Woodfolk 2007). Some nonatopic individuals also have T cells responsive to mite antigens, and these appear to be of the Th1 type, but other groups find that the majority of nonallergic individuals have no T-cell response (Rawle et al. 1984; Upham et al. 1995). Antigenic peptides or epitopes that are specifically recognized by T cells have been defined for both Der p 1 and Der p 2; however, the use of peptides for immunotherapy has only been clearly demonstrated using peptides derived from Fel d 1 (Haselden et al. 1999; Larché et al. 2003). Experiments are planned using injections of peptides to treat mite allergy; however, it is not clear what aspect of T-cell behavior will be changed and there are many possible targets. The simple view of changing T-cell responses from that of a Th2 to a Th1 phenotype is attractive, but is only one of many possible outcomes. Furthermore, there are other possible

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approaches, including site-directed mutagenesis of the whole molecule, which aims to reduce or abolish reactivity with IgE antibodies while preserving all T cell-responsive elements (Smith et al. 1998). A completely different approach would be to immunize at-risk children with a mite antigen linked to an adjuvant that would induce responses of the Th1 type. This would require bacterial products or modified proteins that would induce IL-12 production by macrophages (Afonso et al. 1994; Creticos et al. 2006). Analysis of the T cell response to peptides of Fel d1 in individuals who are tolerant to cat allergen suggest that IL-10 production may be critical (Reefer et al. 2004). However, it is not clear how we should modify mite proteins or peptides in order to enhance IL-10 production.

Discussion Role of mite exposure in disease Dust mites are but one of many sources of foreign proteins in the human ecosystem. However, they play a central role both in understanding the mechanisms by which allergens contribute to asthma and in evaluating the role of allergenspecific treatment for asthma. On a world scale, sensitization to dust mite allergens is associated with more cases of asthma than any other causal factor. In addition, dust mites are the cardinal example of an allergen source of which the patients are not aware; this is not only because mites cannot be seen (and do not smell) but also, perhaps more importantly, because the relationship between exposure and symptoms is delayed, prolonged and, in some senses, indirect. Thus, the inflammatory effects of leukotrienes and eosinophil influx may take hours to develop and last for days or weeks. Furthermore, if exposure represents multiple small inflammatory foci that occur over a period of weeks, there would not be a temporal relationship that the patient could appreciate. This is supported by the fact that cockroach-allergic patients also generally have no awareness of the role that cockroaches or cockroach debris (which they can see) play in their asthma. Assuming that inflammation produced by allergen exposure is an important cause of increased BHR, it is tempting to conclude that there will be a direct relationship between increased exposure and increased symptoms. However, simple cross-sectional studies do not always demonstrate such a relationship. The implication is that the two steps, i.e., exposure causing inflammation and inflammation giving rise to increased symptoms, are too complicated and too variable to allow the demonstration of a simple dose–response relationship between exposure and the severity of asthma. Obviously, there are many different factors that are known to trigger episodes of wheezing among patients who have reactive airways. These include exercise, psychologic stress, allergen exposure (most obviously to cats), gaseous air pollution (particularly passive smoke and ozone), viral infections, and cold air. At this point, it is important to distinguish those “triggers” that can induce a reversible episode of bron-

Dust Mites and Asthma

chospasm without any evidence of a persistent effect and those “triggers” which have been reported to produce longerlasting effects, in most cases by interacting with allergen exposure. The primary examples of the former category are histamine, methacholine, cold air, and exercise. It is certainly clear that repeated histamine or cold air challenges do not increase BHR, do not induce an eosinophil influx and do not prevent reversal of BHR in mite-allergic individuals (Kerrebijn 1970; Platts-Mills et al. 1982).

Interactions between inflammation caused by allergen exposure and enhancers such as rhinovirus There is now strong evidence both from observations and challenge studies that infection with the common cold virus can upregulate inflammatory events in the lung. This has been demonstrated both in experimental studies, where rhinovirus infection followed by allergen challenge leads to an increased eosinophil response (Lemanske et al. 1989; Calhoun et al. 1994), and in studies on children and adults, where admission to hospital or emergency rooms is associated with rhinovirus infection (Duff et al. 1993; Johnston et al. 1995; Heymann et al. 2004). For ozone, the evidence has come from experimental studies showing a positive interaction between exposure and the subsequent response to an allergen challenge (Molfino et al. 1991; Cockcroft et al. 1993; Peden et al. 1995; Peden 2005). It is possible that exercise can induce a prolonged effect, but most investigators have not observed late reactions after exercise challenge. The clearest evidence is now for the interaction of picornaviruses (including the rhinoviruses) and allergen-induced bronchial inflammation. In the centers where this interaction has been documented, the primary allergen associated with asthma is the dust mite (Duff et al. 1993; Johnston et al. 1995; Heymann et al. 2004). Thus, the model for dust mites and asthma is of prolonged exposure to a particulate allergen giving multiple foci of inflammation, which collectively add to BHR. On this background of chronic inflammation caused by allergens, increased episodes of asthma can be caused by intercurrent viral infection, increased exposure to allergens, air pollution, passive smoke, or other nonspecific trigger factors.

Dose–response: contrast between mite (or cockroach) and the mammalian allergens Since the first edition of this text there has been a major change in our understanding of the dose–response relationships for different allergens. The most obvious contrast is between dust mite and cat (Fig. 47.3). The quantity of cat allergen inhaled is much higher than for mites, and many children with the highest exposure become tolerant (Hesselmar et al. 1999; Platts-Mills et al. 2001, 2002; Erwin et al. 2005; Kurosaka et al. 2006; Lau et al. 2005). Recently it has become clear that this form of tolerance, which includes IgG4 antibody production, is also common with high exposure to rat, mouse, and dog allergens (Jeal et al. 2006; Matsui et al. 2006). In our own studies,

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m

Allergens

CD25+ TReg

Th2

most nonmammalian allergen sources, e.g., mites, cockroach, pollens, and fungi.

Bone marrow

e

IL-4

e

lgE

(a) Germinal center g1

g1

g4

g4

e

IL10

m

Lymph node

IL-4

*

g1

*

g4

lgG1 lgG4

IL-4

T cell

(b)

e

lgE

Tr1 * Circulating memory cells

Apoptosis of lgE-switched B cells

Fig. 47.3 Differences in the response to (a) low dose (i.e. Mite) and (b) high dose (i.e. Cat) allergen exposure. Source (evolutionary distance+) Particle Mites Fecal particles 600+ 20 mm #

Contents

“Dose”

Der p 1 – cystein protease Der p 2 – not an enzyme Endotoxin – TLR Mite-DNA – TLR-9 Bacterial–DNA – TLR-9

~ 10 ng/day

The challenge of avoidance as a treatment Precisely because the patients are not aware of the presence of dust mites in their houses, allergen-specific treatment presents a special challenge. In order to persuade patients to carry out avoidance measures, it is necessary to convince them that they are allergic, that dust mites exist, and that the measures proposed are practical and effective. This is a challenge that requires good educational material, repeating the message several times and detailed support for decisionmaking. The real question may be whether avoidance measures are practical without a house visit, to reinforce the message, install covers, collect dust, and help the family make decisions. At present, there is overwhelming evidence that full avoidance of mite allergens can produce major decreases in symptoms and decrease BHR. Although it is obvious that simple avoidance measures should be advised for mite-allergic patients, it is not clear whether full avoidance in the patient’s house is practical. Furthermore, although there were a series of changes in our houses that could have increased the concentration of mite allergens, it is likely that some of this has now reversed, and it is not clear that this is a major cause of increased asthma prevalence.

Cat 60+

Dander 2–10 mm

Fel d 1 Endotoxin Cat-DNA

CCSP* TLR-4 methylated

~ 500 ng/day

# Chitin in the peritrophic membrane may also be a Toll-like receptor (TLR) ligand * CCSP = homologous to clara cell secretory protein which has welldefined immunomodulatory effects in mice + Evolutionary distance in millions of years Fig. 47.4 Relevance of evolutionary distance to differences between mite and cat allergens. (See CD-ROM for color version.)

we have observed “tolerance” to cat allergens among miteallergic children with asthma who are living in a house with a cat (Erwin et al. 2005, 2007). While the mechanisms of this tolerance are not clear, it is likely that it includes: (i) a T-cell response dominated by IL-10 production; (ii) IgG4 antibody production without IgE antibody production; and (iii) controlled titers of IgE antibodies even in those who make an IgE antibody response (Platts-Mills et al. 2001; Reefer et al. 2004; Matsui et al. 2006; Erwin et al. 2007)(Fig. 47.3). The interesting possibility is that the allergens that induce tolerance are characterized by limited amino acid sequence diversity in keeping with their “evolutionary distance” (Platts-Mills 2007) (Fig. 47.4). Certainly, the allergen sources that have been shown to give rise to tolerance are all mammalian in origin and therefore only have limited difference from primate proteins (Dawkins 2004; Jenkins et al. 2007). However, what is important from the point of view of the present chapter is that this form of tolerance appears to be very unusual with

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Conclusions In the UK, New Zealand, and coastal Australia, it is likely that increased carpeting and higher temperatures increased the quantity of dust mite allergens over the period 1955–1985, which coincided with increases in asthma prevalence and severity. However, asthma has also increased in countries where mites are not a problem (Haahtela et al. 1990; Lundback 1990; Peat et al. 1994; Sporik et al. 1995). This suggests that there must have been some change in “lifestyle” contributing to the asthma increase that was common to all “western” countries. Possible candidates for the lifestyle changes include both those seen as increased hygiene, i.e., clean water, decreased infection, and increased immunization, as well as those changes related to the indoor lifestyle, i.e., decreased physical activity, obesity, and dietary changes (Platts-Mills 2005). However, more recent analyses of the epidemiology have identified major differences in prevalence between different countries and communities. In several studies, it is clear that the highest prevalence of asthma symptoms or diagnosis (i.e., > 20%) was observed in communities characterized by high concentrations of mites in the homes, e.g., Australia, New Zealand, the UK, and Japan. By contrast, prevalence in some countries in continental Europe has been as low as 6% (Eder et al. 2006). Our recent analysis suggests that much of this difference can be attributed to the effects of sensitization to dust mites or cockroach allergens (Erwin

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et al. 2007). Thus, even though there must have been a major contribution of lifestyle to the increase in asthma, the large differences in prevalence are best explained by the remarkable ability of dust mite allergens to induce high-titer IgE antibodies.

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schoolchildren. III. Effect of exposure to environmental allergens. Clin Allergy 17, 297–300. Peat, J.K., Tovey, E., Gray, E.J., Mellis, C.M. & Woolcock, A.J. (1994) Asthma severity and morbidity in a population sample of Sydney schoolchildren: Part II. Importance of house dust mite allergens. Aust NZ J Med 24, 270–5. Peden, D.B. (2005) The epidemiology and genetics of asthma risk associated with air pollution. J Allergy Clin Immunol 115, 345–50. Peden, D.B., Setzer, R.W. Jr & Devlin, R.B. (1995) Ozone exposure has both a priming effect on allergen induced responses as well as an intrinsic inflammatory action in the nasal airways of perennially allergic asthmatics. Am J Respir Crit Care Med 151, 1336–45. Peroni, D.G., Piacentini, G.L., Costella, S. & Pietrobelli, A. (2002) Mite avoidance can reduce air trapping and airway inflammation in allergic asthmatic children. Clin Exp Allergy 32, 850–5. Perzanowski, M.S., Ronmark, E., Platts-Mills, T.A.E. & Lundback, B. (2002) Effect of cat and dog ownership on sensitization and development of asthma among preteenage children. Am J Respir Crit Care Med 166, 696– 702. Platts-Mills, T.A.E. (2003) Allergen avoidance in the treatment of asthma and rhinitis. N Engl J Med 349, 207–8. Platts-Mills, T.A.E. (2005) Asthma severity and prevalence: an ongoing interaction between exposure, hygiene, and lifestyle. PLoS Med 2(2), e34; Epub [review]. Platts-Mills, T.A.E. (2007) The role of indoor allergens in chronic allergic disease. J Allergy Clin Immunol 119, 297–302. Platts-Mills, T.A.E. & Chapman, M.E. (1987) Dust mites: immunology, allergic disease, and environmental control. J Allergy Clin Immunol 80, 755– 75. Platts-Mills, T.A.E. & De Weck, A. (1989) Dust mite allergens and asthma: a world wide problem. J Allergy Clin Immunol 83, 416–27. Platts-Mills, T.A.E., Tovey, E.R., Mitchell, E.B., Moszoro, H., Nock, P. & Wilkins, S.R. (1982) Reduction of bronchial hyperreactivity during prolonged allergen avoidance. Lancet ii, 675– 8. Platts-Mills, T.A.E., Hayden, M.L., Chapman, M.D. & Wilkins, S.R. (1987) Seasonal variation in dust mite and grass-pollen allergens in dust from the houses of patients with asthma. J Allergy Clin Immunol 79, 781– 91. Platts-Mills, T.A.E., Vervloet, D., Thmoas, W.R. et al. (1997) Indoor allergens and asthma: report of the Third International Workshop. J Allergy Clin Immunol 100, S2– 24. Platts-Mills, T.A.E., Vaughan, J., Squillace, S. & Woodfolk, J.A. (2001) Sensitisation, asthma, and a modified Th2 response in children exposed to cat allergen: a population-based cross-sectional study. Lancet 357, 752– 6. Platts-Mills, T.A.E., Perzanowski, M., Woodfolk, J.A. & Lundback, B. (2002) Relevance of early or current pet ownership to the prevalence of allergic disease. Clin Exp Allergy 32, 335–8. Pollart, S.M., Chapman, M.D., Fiocco, G.P., Rose, G. & Platts-Mills, T.A.E. (1989) Epidemiology of acute asthma: IgE antibodies to common inhalant allergens as a risk factor for emergency room visits. J Allergy Clin Immunol 83, 875– 82. Pomes, A., Melen, E., Vailes, L.D., Retief, J.D., Arruda, L.K., Chapman, M.D. (1998) Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem 273, 30801–7. Price, J.A., Pollock, J., Little, S.A., Longbottom, J.L. & Warner, J.O. (1990) Measurements of airborne mite allergen in houses of asthmatic children. Lancet 336, 895– 7.

Dust Mites and Asthma

Rawle, F.C., Mitchell, E.B. & Platts-Mills, T.A.E. (1984) T cell responses to the major allergen from the house dust mite Dermatophagoides pteronyssinus, Antigen P1: comparison of patients with asthma, atopic dermatitis, and perennial rhinitis. J Immunol 133, 195–201. Reefer, A.J., Carneiro, R.M., Custis, N.J. & Platts-Mills, T.A.E. (2004) A role for IL-10-mediated HLA-DR7-restricted T cell-dependent events in development of the modified Th2 response to cat allergen. J Immunol 172, 2763–72. Reid, M.J., Moss, R.B., Hsu, Y.P. et al. (1986) Seasonal asthma in northern California: allergic causes and efficacy of immunotherapy. J Allergy Clin Immunol 78, 590–600. Reid, M.J., Lockey, R.F., Turkeltaub, P.C. & Platts-Mills, T.A.E. (1993) Surveys of fatalities from skin testing and immunotherapy 1985–90. J Allergy Clin Immunol 92, 6–15. Rosenstreich, D.L., Eggleston, P., Kattan, M. & Baker, D. (1997) The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. N Engl J Med 336, 1356–63. Rowntree, S., Platts-Mills, T.A.E., Cogswell, J.J. & Mitchell, E.B. (1987) A subclass IgG4-specific antigen-binding radioimmunoassay (RIA). J Allergy Clin Immunol 80, 622–30. Sakaguchi, M., Inouye, S., Yasueda, H., Irie, T., Yoshizawa, S. & Shida, T. (1990) Measurements of allergens associated with dust mite allergy. II. Concentrations of airborne mite allergens (Der I and Der II) in the house. Int Arch Allergy Appl Immunol 90, 190–3. Sears, M.R., Hervison, G.P., Holdaway, M.D., Hewitt, C.J., Flannery, E.M. & Silva, P.A. (1989) The relative risks of sensitivity to grass pollen, house dust mite, and cat dander in the development of childhood asthma. Clin Exp Allergy 19, 419–24. Smith, A.M., Chapman, M.D., Taketomi, E.A. et al. (1998) Recombinant allergens for immunotherapy: a Der p 2 variant with reduced IgE reactivity retains T-cell epitopes. J Allergy Clin Immunol 101, 423–5. Smith, J.M., Disney, M.E., Williams, J.D. & Goels, Z.A. (1969) Clinical significance of skin reactions to mite extracts in children with asthma. BMJ 1, 723– 6. Spain, W.C. & Cooke, R.A. (1924) Studies in specific hypersensitivity: II. The familial incidence of hay fever and bronchial asthma. J Immunol 9, 521–5. Sporik, R. & Platts-Mills, T.A.E. (2001) Allergen exposure and the development of asthma. Thorax 56 (suppl. 2), ii58–ii63. Sporik, R., Holgate, S.T., Platts-Mills, T.A.E. & Cogswell, J. (1990) Exposure to house dust mite allergen (Der p 1) and the development of asthma in childhood: a prospective study. N Engl J Med 323, 502–7. Sporik, R., Ingram, J.M., Price, W., Sussman, J.H., Honsinger, R.W. & Platts-Mills, T.A.E. (1995) Association of asthma with serum IgE and skin-test reactivity to allergens among children living at high altitude: tickling the dragon’s breath. Am J Respir Crit Care Med 151, 1388–92. Sporik, R., Squillace, S.P., Ingram, J.M. & Rakes, G. (1999) Mite, cat, and cockroach exposure, allergen sensitization, and asthma in children: a case-control study of three schools. Thorax 54, 675–80. Squillace, S.P., Sporik, R.B., Rakes, G. & Couture, N. (1997) Sensitization to dust mites as a dominant risk factor for asthma among adolescents living in central Virginia. Multiple regression analysis of a population-base study. Am J Respir Crit Care Med 156, 1760–4. Stewart, G.A., Thompson, P.J. & Simpson, R.J. (1989) Protease antigens from house dust mite [letter]. Lancet ii, 154–5.

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Svartengren, M., Falk, R., Linnman, L., Philipson, K. & Camner, P. (1987) Deposition of large particles in human lung. Exp Lung Res 12, 75–88. Task Group on Lung Dynamics (1966) Deposition and retention models for internal dosimetry of the human respiratory tract. Health Phys 12, 173– 207. Thomas, W.R. (1996) Molecular analysis of house dust mite allergens. In: M.R. Walker & A.M. Roberts, eds. Molecular Analysis of IgEMediated Hypersensitivity and Strategies for Immunological Intervention. John Wylie & Sons, Chichester, UK. Tovey, E.R., Chapman, M.D. & Platts-Mills, T.A.E. (1981a) Mite faeces are a major source of house dust allergens. Nature 289, 592–3. Tovey, E.R., Chapman, M.D., Wells, C.W. & Platts-Mills, T.A.E. (1981b) The distribution of dust mite allergen in the houses of patients with asthma. Am Rev Respir Dis 124, 630– 5. Upham, J.W., Holt, B.J., Baron-Hay, M.J. et al. (1995) Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals. Clin Exp Allergy 25, 634–42. van der Heide S., Kauffman, HF, Dubois, AE. (1997) Allergen reduction measures in houses of allergic asthmatic patients: effects of air cleaners and allergen-impermeable mattress covers. Eur Respir J 10, 1217–23. Van Ree, R. (2007) Indoor allergens: relevance of major allergen measurements and standardization. J Allergy Clin Immunol 119, 270–7. Varney, V.A., Hamid, Q.A., Gaga, M. et al. (1993) Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergen-induced late phase cutaneous reactions. J Clin Invest 92, 644– 51.

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Vervloet, D., Penaud, A., Razzouk, H. et al. (1982) Altitude and house dust mites. J Allergy Clin Immunol 69, 290–6. Voorhorst, R., Spieksma, F.Th.M., Varekamp, H., Leupen, M.J. & Lyklema, A.W. (1967) The house dust mite (Dermatophagoides pteronyssinus) and the allergens it produces: identity with the house dust allergen. J Allergy 39, 325–39. Wickman, M., Nordvall, L., Pershagen, G., Korsgaard, J. & Johannsen, N. (1993) Sensitization to domestic mites in a cold temperate region. Am Rev Respir Dis 148, 58–62. Wierenga, E.A., Snoek, M., Jansen, H.M., Bos, J.D., Van Lieu, R.A.W. & Kapsenberg, M.L. (1991) Human atopen-specific types 1 and 2 helper T cell clones. J Immunol 147, 2942–9. Woodcock, A., Forster, L., Matthew, E. et al. (2003) Control of exposure to mite allergen and allergen-impermeable bed covers for adults with asthma. N Engl J Med 349, 225–36. Woodfolk, J.A. (2007) T-cell responses to allergens. J Allergy Clin Immunol 119, 280–94. Woodfolk, J.A., Luczynska, C.M., de Blay, F., Chapman, M.D. & Platts-Mills, T.A.E. (1993) The effect of vacuum cleaners on the concentration and particle size distribution of airborne cat allergen. J Allergy Clin Immunol 91, 829–37. Woodfolk, J.A., Hayden, M.L., Miller, J.D., Rose, G., Chapman, M.D. & Platts-Mills, T.A.E. (1994) Chemical treatment of carpets to reduce allergen: a detailed study of the effects of tannic acid on indoor allergens. J Allergy Clin Immunol 94, 19–26. Woodfolk, J.A., Hayden, M.L., Couture, N. & Platts-Mills, T.A.E. (1995) Chemical treatment of carpets to reduce allergen: comparison of the effects of tannic acid and other treatments on proteins derived from dust mites and cats. J Allergy Clin Immunol 96, 325–33.

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Animal Allergens Adnan Custovic and Angela Simpson

Summary Cat and dog allergens are ubiquitous in the regions with high proportion of pet ownership; in such areas, they are detectable in air and dust in all sorts of places (from homes without pets to hospitals). They are carried on small respirable particles that remain airborne for long periods and which are very difficult to remove from the domestic environment, even after the permanent removal of the pet. Sensitization to domestic pets is a risk factor for asthma. Individuals may become sensitized to cats and dogs even though they have never lived with pets and have very low levels of pet allergens in the home. Patients with established asthma who are sensitized to pets and exposed either to the pet or to high levels of pet allergen tend to have more severe asthma than those who are not exposed. The clear advice to pet-sensitized pet owners who experience symptoms upon exposure is to remove the pet from the home. The questions that remain unclear from the literature include the following. How and why do some people become sensitized to pets? Is cat and dog ownership a risk factor for sensitization or is it protective? Is the effect of direct pet exposure different in subjects with different relative risks based on personal or parental allergy or asthma, and is the effect independent of age? Do cats and dogs behave in the same way? While numerous studies addressed these issues, there are still no unequivocal answers to these questions. Clinical outcomes reported from different observational studies appear inconsistent and often confusing. For allergic sensitization, the overall trend is for pet ownership to be associated with a decrease in the risk, and this is seen more commonly for dogs than for cats. It does appear that for allergic sensitization, dogs may be protective or at worst have no effect. The discrepancies between different studies are at least partly due to the fact that exposure to domestic pets may have different, or even opposite effect on the development of sensitization and allergic disease in individuals with different genetic polymorphisms. Thus, the future advice on pet ownership is unlikely to be blanket advice aimed at and

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

applicable to everybody, but rather tailor-made, individualized advice targeting individuals with specific susceptibilities who will benefit from a particular intervention, i.e., it may well be that some individuals may benefit from having a cat or a dog, while in others pet ownership may increase the risk of allergic disease.

Introduction Numerous studies have been published investigating the association between exposure to pets, sensitization and asthma, with confusing and often conflicting results. Most of the published work is heterogeneous not only in results but also design: for example, some studies include exposure to birds and rodents as well as cats and dogs, some are conducted in high-risk children only, some look at early life exposure, others at exposures later in life. All of this makes it difficult to draw out common themes. However, let us first ask the question of why people keep cats and dogs. They are kept for different reasons in different countries: in Africa, cats are outside the house controlling rodents, whereas in European cities they stay indoors depositing allergen on the bed and the sofa and this is reflected in widely differing allergen levels between countries. In a strictly nonscientific poll of friends and family, when asked why they keep pets, many say “we have always had cats/dogs” and for some families there seems to be a familial component to pet ownership. No one answered “to reduce the risk of me/my children developing allergy.” So why do some of us not keep pets? The decision for most is not a random one and factors that determine pet ownership would be interesting subjects in their own right, but are beyond the scope of this chapter. For some individuals, it is clearly because exposure to pets causes an exacerbation of symptoms of allergic disease; there is evidence to suggest that pet-sensitized pet owners have more severe asthma than petsensitized nonpet owners (Tunnicliffe et al. 1999). Indeed, a recent report suggests that children with early-onset asthma symptoms are less likely to keep a cat for the rest of childhood, although this did not influence cat-owning behavior in adulthood unless the person had symptoms in adulthood

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(Svanes et al. 2006). Others may add that they are afraid that their children will develop allergy or asthma if they keep pets. Some feel that pets are dirty and should not be in homes with babies and young children. Are pets relevant in asthma and allergy? Undoubtedly, a considerable body of evidence suggests that sensitization to cats is a major risk factor for asthma, with some data suggesting that early sensitization is associated with more severe disease. There are less data for dogs, and it cannot be assumed that the effects of dogs are the same as for cats. However, before discussing all these issues in greater detail, it is important to understand the allergen sources and aerodynamics of allergen-carrying particles if we are to begin unraveling the mechanisms of sensitization and the clinical presentation of allergic disease associated with pet allergy. In addition, this knowledge is critical for the design of successful strategies to reduce personal exposure.

Sources of allergens Cats Allergenic activity has been demonstrated in dander and pelt extracts, as well as in saliva, serum, and urine. Although several molecules in cat extracts are allergenic, the most important one from a clinical point of view is Fel d 1 (Felis domesticus allergen I) (Ohman 1978). Fel d 1 is responsible for approximately 90% of the cat-specific IgE in individuals allergic to cats, and it is the only cat antigen that fulfils the criteria to be considered a “major allergen.”. Fel d 1 was first described by Ohman et al. (1973) and has subsequently been better defined with monoclonal antibodies and specific immunoassays (Chapman et al. 1988). Fel d 1 is a heterodimer comprising chain I (70 amino acids) and chain II (90/92 amino acids). The sequences were obtained by polymerase chain reaction (PCR), and the protein sequencing and the genomic DNA sequence of Fel d 1 have been determined (Morgenstern et al. 1991; Griffith et al. 1992). The molecule comprises two polypeptide chains and N-linked carbohydrate. One of these chains lacks homology with other proteins, while the other chain shows homology to two wellcharacterized proteins (human Clara-cell phospholipid-binding protein and the progesterone-binding rabbit uteroglobin). Fel d 1 is present in several tissues, e.g., sebaceous glands (Brown et al. 1984) and salivary glands (Bartholome et al. 1985), and its concentration is 10 times greater at the root than at the tip of the hair (Charpin et al. 1991). Its production is under hormonal control, and a single cat can produce 3–7 μg daily (Dabrowski et al. 1990). Castration of male cats results in a threefold to fivefold reduction of Fel d 1 concentration in skin washing, while testosterone treatment of the castrated cats restores the Fel d 1 levels to precastration values (Zielonka et al. 1994). The commonly asked question is whether one breed of cat (or dog) can produce more allergens, or different allergens than another. As all cats belong to the same species, it is not likely

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that different breeds should exhibit breed-specific allergen molecules (Schou 1993), but it is possible that variations in the relative concentration of allergen between different breeds (e.g., short hair and long hair) do occur (Blands et al. 1977).

Dogs It is estimated that there are 7.3 million domestic dogs in the UK (RSPCA, personal communication). Although dog allergy has been recognized as a clinical problem, it has received less scientific interest than cat allergy. Dander, pelt hair, and saliva are the most important sources of dog allergens, while urine does not exhibit significant allergenic activity (Uhlin et al. 1984). A major dog allergen, Can f 1, has been purified from house dust extracts by monoclonal antibody affinity chromatography (de Groot et al. 1991). Can f 1 is a protein produced in the canine Von Ebner’s glands. The ducts of these small lingual salivary glands open in the lingual epithelium, suggesting a possible role of Can f 1 in taste reception (Schmale et al. 1990). Can f 1 has been shown to account for at least half of the allergenic activity in extracts of dog hair and dander and to induce positive skin tests in 92% of dog allergic patients (Schou et al. 1991). De Groot et al. (1991) reported varying degrees of Can f 1 among different dog breeds and among individual dogs. The existence of breed-specific dog allergens has been suggested, as about 15% of patients with hypersensitivity to dogs have different skin test responses to different dog breeds (Lindgren et al. 1988). This is, however, still a controversial issue.

Allergen distribution in homes Levels in the dust reservoirs In the areas of the world with high proportions of pet ownership, the overwhelming majority of homes without pets contain quantifiable levels of pet allergens in at least one dust reservoir (Bollinger et al. 1996; Custovic et al. 1997, 1998a). However, it is worth emphasizing that the levels in the homes of pet owners are approximately 250-fold higher (Fig. 48.1). The distribution of pet allergen in the dust differs between the homes with or without an animal. The highest allergen concentrations in the homes without a pet are found in the upholstered furniture from the lounge, supporting the view that allergen can be passively transferred into houses without pets, probably on the pet owners’ clothing. Not surprisingly, in homes with pets, the distribution of allergen reflects that of the animal: the highest levels are usually found in living room carpets, and the lowest in beds (Custovic et al. 1998a; Addo-Yobo et al. 2001).

Aerodynamics of cat and dog allergen Physical properties of the airborne particles including size, shape, and density are important determinants of the site of deposition within the human respiratory tract (Findlay et al.

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10000

0 g Can f 1/g dust

1000

100

10

1

Fig. 48.1 Distribution of Can f 1 in the settled dust from four sampling sites in homes with and without dogs. The lower level of sensitivity of the assay for Can f 1 was 0.2 mg/g. (From Addo-Yobo et al. 2001, with permission.)

0.1 Mattress

Bedroom carpet

Living room carpet

Homes with dogs

Upholstered furniture

Homes without dogs

50

40

%

30

20

10 Fig. 48.2 Particle size distribution of airborne Fel d 1. (From Custovic et al. 1998a, with permission.)

0

>9

1983). The intrathoracic deposition of relatively large particles (> 10 μm) is still controversial, while particles 2–5 μm in diameter can more readily penetrate into the lower airways. The majority of both Fel d 1 and Can f 1 (∼ 50%) is carried on large particles > 10 μm in diameter (Luczynska et al. 1990; De Blay et al. 1991a; Custovic et al. 1997, 1998a). However, about 20% is carried on particles < 4.7 μm in diameter (Fig. 48.2). Although small particles may be more relevant in terms of acute symptoms, by their virtue of lung deposition, the relative role of particles of different sizes and shapes is as yet undetermined. Large particles are likely to be effective in perpetuating the IgE response, thus possibly contributing to the chronic inflammation. There are considerable differences in total airborne allergen levels between different homes. However, whatever the absolute quantity of the allergen in the air, approximately 20% is associated with particles < 5 μm. These particles would be expected to remain airborne for several hours and, when inhaled, to penetrate into the lung.

5.8–9

4.7–5.8

3.3–4.7

2.1–3.3

1.1–2.1

0.65–1.1 0.43–0.65

mm

Even in the absence of disturbance, airborne cat and dog allergens can be detected in virtually all homes with an indoor pet, but the levels vary greatly between homes. There are several possible explanations for this finding (e.g., differences in the air exchange rate between the houses, variability in the amount of allergen shed by different animals). More surprisingly, airborne pet allergens can be found in the absence of disturbance in homes that have never housed a pet, albeit in comparatively low concentrations (Custovic et al. 1998a) (Fig. 48.3). Consequently, individuals living in homes without an animal can be exposed to low levels of pet allergens in their homes. The animal is the major source of allergen in homes with pets. For example, with the cat in the room, the level of Fel d 1 in the air is more than fivefold higher than when the cat is not in the room. Excluding cats from the living areas of the home results in a dramatic fall in the total airborne Fel d 1, but mostly due to a decrease in larger particles (Custovic et al. 1998a).

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Fel d 1 & Can f 1 ng/m3

100

Fel d 1 (ng/m3)

10

1

10

1

0.08

0.1

0.01 0.1

Can f 1

Fel d 1

Fig. 48.5 Airborne levels of cat and dog allergen in the outpatient area. (Derived from data published in Custovic et al. 1998b.) Without cats

With cats

Fig. 48.3 Airborne Fel d 1 in 50 homes with cats and 75 homes without a cat. (From Custovic et al. 1998a, with permission.)

Pet allergens in public buildings and public transport High levels of cat and dog allergen can be found in public buildings and public transport (Munir et al. 1993, 1995; Custovic et al. 1994, 1996, 1998b; de Andrade et al. 1995; Al Mousawi et al. 2001). For example, very high levels of both cat and dog allergen were found in virtually all upholstered seats in hotels, cinemas, pubs and public transport (Fig. 48.4). It has been suggested that cat allergen is transported into houses without cats on the clothes of cat owners who come to visit or on the clothes of the inhabitants when allergen has been picked up on the clothing while out of the home.

Community prevalence of cat ownership has recently been shown to correlate with cat allergen levels in those not owning cats (Heinrich et al. 2006). Higher levels of Fel d 1 were found on chairs than on floors in Swedish schools, probably as a result of children and teachers carrying it on their clothing (Munir et al. 1993). It is likely that some very sensitive patients may have symptoms when exposed to very low doses of allergen, while in others the required dose for the same effect will be much higher. It is of interest that both cat and dog airborne allergen were readily detectable in the outpatient waiting areas in hospitals (Custovic et al. 1998b) (Fig. 48.5).

Conclusions on distribution and aerodynamics Allergens from cats and dogs have different aerodynamic properties from those of mites and cockroaches (Custovic et al.

Can f 1 (mg/g)

100

10

1

0.1 Carpets

Seats

Public houses

1000

Carpets

Seats

Cinema

Carpet

Seats Hotel

Mattress

Carpets

Seats

School

Public transport

Fig. 48.4 Concentration of Can f 1 in the dust from different sampling sites. (Derived from data published in Custovic et al. 1996.)

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Table 48.1 Differences in the aerodynamic properties between house dust mite and cockroach and pet allergens. Allergen

Particle size

Airborne level

Mite: Group 1, Group 2 Cockroach: Bla g 1, Bla g 2

Large particles > 10 mm

Undisturbed: undetectable with conventional assays (< 0.2 ng/m3 for mite allergens, < 0.02 ng/m3 for cockroach)

Disturbed: detectable after vigorous disturbance

Cat: Fel d 1 Dog: Can f 1

Large particles > 5 mm (∼ 75%) Small particles < 5 mm (∼ 25%)

Homes with animal: detectable in all homes. Levels four to five times higher with animal in the room

Homes without animal: detectable in about one-third of the homes without artificial disturbance

1999a,b) (Table 48.1). Mite and cockroach allergens can be detected in the air in significant amounts only after vigorous disturbance, and are contained within relatively large particles (> 10 μm diameter). In contrast, airborne cat and dog allergen are readily measured in houses with pets (and in one-quarter of the homes without pets), and about 25% of airborne Fel d 1 and Can f 1 is associated with small particles (< 5 μm diameter). These differences underlie the difference in the clinical presentation of the disease. Mite- and cockroach-sensitive asthmatics are usually unaware of the relationship between allergen exposure at home and asthma symptoms (exposure is low grade and chronic). The large particles, however, may contain a large quantity of allergen, and even small numbers may cause a significant inflammatory response when impacted in the airways. In contrast, cat- or dog-allergic patients often develop symptoms within minutes of entering a home with a pet due to the inhalation of large amounts of airborne allergen on small particles, which can penetrate deep into the respiratory tract inducing acute asthma. Application of this information is important, implying, for example, that air filtration units have no place in mite or cockroach avoidance, but may be useful in removing cat and dog allergen from the air. The aerodynamic differences between mite and pet allergens have to be taken into account in assessing exposure. While levels in settled dust are the best available index for mite allergens, airborne levels might be more suitable for defining exposure to Can f 1 and Fel d 1. It is likely that the majority of exposure to allergens of domestic pets occurs in the living room area, and this must be taken into account when planning avoidance strategies. Passive exposure in homes without pets and in public places (schools, restaurants, cinemas, public transport, and even hospitals) may be important, and we should be thinking in terms of community exposure for pet allergens, rather than just domestic exposure. Studies from Sweden suggest that such exposure is sufficient to cause sensitization in susceptible children who have never been pet owners (Munir et al. 1997). As yet, there are no data on the dose of airborne allergen needed to cause sensitization, and this is likely to be different in individuals with different genetic predispositions.

Pet sensitization, ownership and asthma severity The severity of asthma symptoms has a major impact on patients’ lives and the quantity of medical care they require. One of the numerous factors associated with asthma severity is exposure to allergens. However, showing the direct relationship between a particular allergen exposure and symptoms has always been difficult, in part due to a number of confounding factors (e.g., patients are usually sensitized and exposed to more than one allergen, and viral infection and medication may obscure the relationship). Nevertheless, a case–control study from Birmingham, UK has shown that patients with very severe, brittle asthma are significantly more often both sensitized and exposed to high levels of dog allergen compared with patients with mild disease (Tunnicliffe et al. 1999). The combination of sensitization and high exposure to pet allergens has been associated with more severe asthma in a large sample of patients (Langley et al. 2003), although this finding could not be confirmed in a subsequent study that assessed exposure by the measurement of personal, inhaled allergen load (Gore et al. 2006a). Furthermore, high exposure to dog allergen has been associated with more severe asthma among nonsensitized asthmatics (Langley et al. 2005). This raises the question as to whether reducing exposure to pet allergen may improve asthma severity.

Pet allergen avoidance Pet allergen avoidance measures The only way to effectively reduce exposure to cat or dog allergen is not to have one in the home. It is worth emphasizing that even after permanent removal of an animal from a home it can take many months for the allergen reservoir levels to fall (Wood et al. 1989). Unfortunately, despite the advice by health professionals, a large number of pet-sensitized individuals will continue to live with their animal and an attempt to control allergen levels will be needed. High-efficiency particulate air (HEPA) filters can reduce the

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Allergens

airborne concentrations of cat and dog allergens in homes with pets (Green et al. 1999a). The high flow rate (250 m3/ hour) of the HEPA air cleaner enables the air within the average room to circulate through the device about 10 times each hour. The absolute allergen levels in rooms containing a pet during air filtration are comparable with the baseline levels in the same rooms without active air filtration, but with the dog elsewhere in the house. While measurements of the effectiveness of HEPA filters under experimental conditions suggest marked reduction in airborne allergen levels (Green et al. 1999a), field studies using measurements of personal exposure are far less convincing (Gore et al. 2003a). Several studies have investigated the effect of cat washing on levels of Fel d 1. Klucka et al. (1995) compared the relative efficacy of washing with water, Allerpet-C spray, and acepromazine in decreasing cat allergen shedding in 24 cats, and found no beneficial effect. De Blay et al. (1991b) reported a reduction in airborne Fel d 1 following washing one cat weekly over a 4-week period, and similar, but short-lived, reduction was confirmed in a later study (Perzanowski et al. 1997). Washing dogs with shampoo significantly reduces the levels of dog allergen in fur and dander samples, but the levels increase to the starting values within 3– 4 days (Hodson et al. 1999). This would suggest that pet washing needs to be done a minimum of twice a week to be effective. Twice-weekly washing could decrease the buildup of allergen in the dust reservoirs within the home. In addition, washing the pet regularly may reduce the level of airborne allergen in homes with pets (De Blay et al. 1991b; Hodson et al. 1999). However, it is unlikely that the short-term and modest reduction in the airborne allergen achieved by washing would significantly improve asthma control in sensitized individuals. In the experimental chamber, vacuum cleaners with builtin HEPA filters and double-thickness bags do not leak Can f 1 (Green et al. 1999b). However, in a real-life study using intranasal air samplers, we have recently demonstrated a threefold to fivefold increase in personal cat allergen exposure while using new high-efficiency vacuum cleaners, despite the fact that these vacuum cleaners clearly performed well in the experimental chamber (Gore et al. 2003b). This is likely consequent to the beating bar action, the air disturbance from the exhaust flow, and the back and forth motion of the cleaning head. The disparity between the experimental chamber and allergen exposure in real life means that the use of the experimental chamber alone is insufficient to justify the current recommendations of high-efficiency vacuum cleaners to allergy sufferers (Gore et al. 2006b). Pet removal remains the best advice to patients with pet allergy who experience symptoms on exposure and who wish to reduce their exposure.

Clinical effectiveness of pet allergen avoidance Although it is accepted that there is a significant clinical improvement associated with the absence of contact with the sensitizing pet in cat- or dog-allergic patients, based on clinical

1002

experience and observational studies (Shirai et al. 2005), it is also known that these allergens are very difficult to eradicate from the homes, and even then exposure may be maintained as cat and dog allergens are ubiquitous (Almqvist et al. 1999). Three studies have addressed the effects of pet allergen control measures in pet-sensitized pet owners. Two showed small improvements in asthma-related outcomes (Van Der Heide et al. 1999; Francis et al. 2003), but one did not (Wood et al. 1998). However, the number of subjects was small and larger studies would be needed before public health recommendations can be made. A Cochrane review reported no beneficial effect of pet allergen control measures for allergic asthma in children and adults; however, only two small studies met the inclusion criteria (Kilburn et al. 2003).

Pets and development of allergic sensitization and asthma Unequivocal facts about pets, allergies, and asthma Pet allergens are ubiquitous, detectable in air and dust in all sorts of places (from homes without pets to hospitals). They are carried on small respirable particles that remain airborne for long periods, and are very difficult to remove from the domestic environment, even after the pet has gone. Community exposure is directly related to the community proportion of pet ownership (Woodcock et al. 2001). Sensitization to pets is a major risk factor for asthma in the areas of the world with high proportion of pet ownership (Simpson et al. 2001), and even in some (Al-Mousawi et al. 2004) but not all (Addo-Yobo et al. 2001) areas with low proportion of pet ownership. Individuals may become sensitized to pets even though they have never lived with pets and have very low levels of pet allergens in the home (Munir et al. 1997). Patients with established asthma who are sensitized to pets and exposed either to the pet or to high levels of pet allergen tend to have more severe asthma than those who are not exposed (Tunnicliffe et al. 1999).

Equivocal findings about pets, allergies, and asthma What is unclear from the literature is how and why some people become sensitized to pets, and this has not become materially clearer since we last reviewed the literature (Simpson & Custovic 2003, 2005). Is pet ownership a risk factor for sensitization or is it protective? Is the effect of direct pet exposure different in subjects with different relative risks based on personal or parental allergy or asthma, and is the effect independent of age? In most areas, the data remain confusing and contradictory. One cannot prevent allergic sensitization in children by avoiding pets in childhood, which is true at least in areas with high community prevalence of pet ownership (Munir et al. 1997; Perzanowski et al. 2002). It is also interesting to note that across Europe the community prevalence of sensitization to cat among those not owning cats correlates positively with community cat ownership rates, i.e., more

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CHAPTER 48

cats in the community means that more people who are not cat owners will be sensitized (Roost et al. 1999). One may choose any relationship between cat and dog ownership and sensitization/asthma and find a study to support a favored theory (i.e., pets are good, pets are bad, pets do not matter), and study design may contribute to this heterogeneity. Overall, considerably more studies have reported on the association between cat ownership and sensitization to cat than on the association between dog ownership and sensitization to dog. This may in part account for the increased heterogeneity of results seen for cat compared with dog. The list of studies reviewed in this chapter is not exhaustive, but focuses on relevant studies to illustrate various important points. The studies reviewed are summarized in Table 48.2, sorted by study design, presenting results first for studies in children and then for those in adults.

Birth cohort studies Different types of birth cohort studies (population-based, enriched with high-risk children, and high-risk only) are considered separately, as the difference in design is likely to be reflected in the results.

Population-based Of the eight population-based birth cohort studies with published data reporting associations between pet ownership, allergies, and allergic disease, one presents results on only high- and low-risk children from this cohort, so is considered in the section below (Custovic et al. 2001b). Of the remaining seven, only four looked for an association between cat ownership and sensitization to cat. Three reported no association (Arshad et al. 2001; Remes et al. 2001; Ownby et al. 2002), and one reported that there was an increase in sensitization to cat with increasing Fel d 1 exposures, which was still seen in the multivariate model (Torrent et al. 2006). The authors found a steep increase in risk at low levels, with the association flattening out above 1 μg/g Fel d 1, without any reduction in risk at very high levels (Torrent et al. 2006). Only two of these studies reported on the relationship between dog ownership and sensitization to dog, one finding no association (Arshad et al. 2001) and the other reporting a decrease in sensitization to dog among children who owned a dog in infancy (Ownby et al. 2002). Three studies reported on the association between pet ownership and sensitization to other allergens: two found no association (Remes et al. 2001; Hagendorens et al. 2005), the other a reduced risk of sensitization in children who owned two or more dogs or cats (Ownby et al. 2002). For wheeze/ asthma, four reported no association (Horwood et al. 1985; Nafstad et al. 2001; Ownby et al. 2002; Hagendorens et al. 2005), one reported that early-onset persistent wheeze was reduced in cat owners (Kurukulaaratchy et al. 2005), with no effect on transient early or late-onset wheeze, and the Tucson Children’s Respiratory Study reported a decrease in frequent wheeze for those who had a dog in infancy in the absence of

Animal Allergens

a family history of asthma, but no association for cat (Remes et al. 2001).

Enriched with high-risk children Findings appear different in birth cohort studies using populations enriched with high-risk children. All three studies suggest an increase in sensitization to cat with cat exposure (Lau et al. 2000; Custovic et al. 2001b; Brussee et al. 2005), although the effect of family history differed between studies (Custovic et al. 2001b; Brussee et al. 2005). No association was seen for dog ownership and sensitization to dog (Custovic et al. 2001b; Brussee et al. 2005). High-risk children Of the five high-risk birth cohorts, only one (from Sweden, with a surprisingly low rate of cat ownership of 8%) has reported on specific sensitization to cats and dogs. This study reported no association between cat ownership or cat allergen levels and sensitization to cat when children were aged 5 years (Munir et al. 1997). Sensitization to cat had occurred in some high-risk children with very low levels of cat allergen in the home, but too few were sensitized to dog to make any conclusion. Two studies reported on allergic sensitization. The UK study found a decrease in the risk of allergic sensitization using IgE (AlaTOP multiallergen screen) among subjects with a cat or dog in infancy; the authors commented that the effect appeared greater for dogs than cats. However, when allergic sensitization was defined as one or more positive skin tests, there was no association between sensitization and pet ownership (Burr et al. 1997). In the smaller Childhood Origins of Asthma Study, dog ownership was associated with a reduced risk of allergic sensitization on skin test at age 1 year (Gern et al. 2004), but there was no association between cat ownership and sensitization outcomes. For asthma, two of the high-risk birth cohorts have not reported (Munir et al. 1997; Gern et al. 2004), two found no association (Burr et al. 1997; Rhodes et al. 2001) and one found asthma decreased among cat owners if the mother did not have asthma, but increased if she did, with no association for dog (Celedon et al. 2002). In summary, for population-based and high-risk cohorts there is little evidence of any association between pet ownership, allergic sensitization, and symptoms, with a suggestion that dog ownership is protective. However, the three enriched studies suggest increasing risk of sensitization to cat with cat ownership, with inconsistent effects of family history.

Cross-sectional and cohort studies Of the 21 different studies listed in Table 48.2, mostly from Europe with a large preponderance from Sweden, 10 have reported on the relationship between skin tests or IgE data and allergy. In three populations studied in Sweden, cat ownership was associated with a decrease in the risk of sensitization to cat (Hesselmar et al. 1999; Braback et al. 2001; Perzanowski

1003

1004 Definition of exposure (pets and time groups, allergens) C, D indoors Infancy

C, D Current

C, D, ownership

C, D, current infancy, Fel d 1

C, D, any pet, at birth Fel d 1, Can f 1

C, D During first year of life

Design, age, numbers UBC, 13 years, N = 1076

UBC, 4 years, N = 1218

UBC, 1 year, N = 517

UBC, 6–7 years, N = 474

UBC, 4 years, N = 2531

UBC, 1 year, N = 810

Study

Remes et al. (2001), USA

Arshad et al. (2001), UK; Kurukulaaratchy et al. (2005)

Custovic et al. (2001b), UK

Ownby et al. (2002), USA

Nafstad et al. (2001), Norway

Hagendorens et al. (2005), Belgium

Q, IgE

Q

Spt, IgE, BHR

Q, Spt, IgE

Spt, Q

Spt (but not to D), Q

Outcome measures

Sens to C: NR Sens to D: NR AS: NA

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NA Sens to D: decreased with D in infancy AS: decreased with 2 or more D or C

Sens to C: increased with C if positive FH Sens to D: NA AS: NR

Sens to C: NA Sens to D: NA AS: NR

Sens to C: NA Sens to D: NR AS: NA

Association between exposure and specific sensitization to C or D and between exposure and allergic sensitization (AS)

A: NA for wheeze E: postnatal exposure to C protective R: NR

A: NA for Fel d 1 and Can f 1, NA with C or D at 4 years (increased bronchial obstruction with D at 2 years) E: decreased with C or D if FH R: decreased with any P, NA for C or D individually

A: NA with asthma diagnosis, but less BHR for boys with increasing C and D exposure in infancy. NA for current C or D E: NR R: NR

A: NA for respiratory symptoms E: NR R: NR NR (but NA for respiratory symptoms, too young to diagnose asthma)

A: early onset persistent wheeze reduced in cat owners E: NR R: NR

A: decreased frequent wheeze with D in infancy if no history of parental asthma; NA for C E: NR R: NR

Association between exposure to C or D and asthma, eczema, and rhinitis

Table 48.2 Summary of studies of the effect of exposure to cats and dogs on the development of allergies and asthma in adults and in children.

C: 53% D: 35% 13% sensitized in total, mostly to food PIPO

C: 8% D: 9%

C: 32% in infancy D: 45% in infancy No difference in parents of pet owners and nonpet owners

C: 15% D: 14% 1-year-old children Presented data on high- and low-risk children only in this publication MAAS

C: 39% D: 30% No association with transient early or late onset wheeze Isle of Wight Study

C: 22% D: 30% Lowest risk of frequent wheeze at 13 was in persistent D owners, highest risk in never D owners Tucson CRS

C/D/pet ownership rates ( if given); general comments; study acronym

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UBC, 6 years, N = 1056

UBC, 4 years, N = 1019

SBC, 7 years, N = 939

SBC (high and low risk only), N = 1127

HRBC, 5 years, N = 86

HRBC, 7 years, N = 440

HRBC, 22 years, N = 63

HRBC, 1 year, N = 285

HRBC, 5 years, N = 448

Horwood et al. (1985), NZ

Torrent et al. (2006), UK and Spain

Lau et al. (2000), Germany

Brussee et al. (2005), The Netherlands

Munir et al. (1997), Sweden

Burr et al. (1997), UK

Rhodes et al. (2001), UK

Gern et al. (2004), USA

Celedon et al. (2002); Tepas et al. (2006), USA C, D, current Fel d 1, Can f 1

C, D ownership

C, D Before 5 years

C, D Current

C, D Current Peak Fel d 1, Can f 1

Fel d 1, Can f 1 at 3 months

C Current Fel d 1

Fel d 1, Can f 1 at 3 months

C, D Current

Q

Q, IgE

Spt, IgE, Q, BHR

Spt, IgE, Q

Spt

IgE to cat, dog Q

IgE to cat, BHR, Q

IgE Q

Q

A: NA E: NR R: NR

A: NA E: NR R: NR A: NR E: decreased with D or D + C; NA for C alone R: NR A: for C, risk of wheezing decreased if mother did not have asthma, increased if mother had asthma. NA for D E: NR R: NR

Sens to C: NR Sens to D: NR AS: NR Sens to C: NR Sens to D: NR AS: Reduced with D; NA for C

Sens to C: NR Sens to D: NR AS: NR

A: NR E: NR R: NR

A: increased transient early wheeze with increasing Can f 1 if mother nonatopic. NA with DD asthma E: NR R: NR

A: NA at 7 years for ownership or Fel d 1 levels E: NR R: NR

A: NR E: NR R: NR

A: NA for C or D ownership E: NR R: NR

Sens to C: NR Sens to D: NR AS: Reduced (IgE) in C or D in infancy NA for spt

Sens to C: NA with Fel d 1 levels or C ownership Sens to D: Too few sens to D to comment AS: NR

Sens to C: increased with increasing Fel d 1 exposure if mother nonatopic Sens to D: NA AS: NR

Sens to C: increased with higher Fel d 1 levels at 3 and 7 years. NR for C Sens to D: NR AS: NR

Sens to C: increased with increasing Fel d 1 exposure Sens to D: NR AS: NR

Sens to C: NR Sens to D: NR AS: NR

C: 16% D: 21%

Continued p. 1006

C: 29% D: 35% Effect of dogs was confined to one CD14 genotype group (TT)

C: 21% D: 29% 24% had asthma

C: NR D: NR

C: 8% D: 9% Sens to C or D can occur at low allergen levels

C: NR D: 16% 68% of homes had no detectable dog allergen, compared with 13% for cat allergen PIAMA

C: NR D: NR Levels of Fel d 1 generally low MAS-90

C: 16.7% in first year D: NR AMICS

C: NR D: NR 82% had C or D in home

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1005

1006 HRC (sibs of above), 1–5 years, N = 226

CS, prevalence at 7–8 years, N = 3525

Cohort (above population followed up), incidence at 10–11 years, N = 3525

Cohort, 7– 8 years, followup 12–13 years, N=?

Cohort, (4-year incidence), 7–17 years, N = 1347

Ronmark et al. (1999), Sweden

Perzanowski et al. (2002), Sweden

Hesselmar et al. (1999), Sweden

Smedje & Norback (2001), Sweden

Design, age, numbers

Litonjua et al. (2002), USA

Study

Table 48.2 (Cont’d )

C, D Current Fel d 1, Can f 1 in school

C, D inside in infancy

C, D Ever/never

C, D Ever/never

C, D, Fel d 1 (Can f 1 incomplete)

Definition of exposure (pets and time groups, allergens)

Q

Spt, Q

Q, Spt 2/3

Q, Spt on 2/3

Q

Outcome measures

Sens to C: NR Sens to D: NR AS: NR

Sens to C: decreased at 12 years with C in infancy (but effect lost when avoiders excluded) Sens to D: NA AS: NA

Sens to C: decreased if C ever and positive FH Sens to D: decreased if D ever and positive FH AS: NR

Sens to C: decreased if C ever (no stats) Sens to D: decreased if dog (no stats) AS: NR

Sens to C: NR Sens to D: NR AS: NR

Association between exposure and specific sensitization to C or D and between exposure and allergic sensitization (AS)

A: 4-year incident asthma less likely in current C owners, but more common in schools with higher Fel d 1; NA for D E: NR R: NR

C: 34% D: 25% Self-reported pet allergy results were not included in this table

C: NR D: NR P: 23% had C or D in infancy Excluded those who avoided pets because of symptoms (avoiders)

C: NR D: NR

A: decreased incident asthma with C; trend for D. Reanalyzed prevalence data and found protective effect of C was in those with positive FH ( not significant for D) E: NR R: NR A: decreased risk of asthma at 12 years with C or D in infancy, even when avoiders excluded E: NA R: lower at 7–8 years with C or D in infancy (not at 12–13 years)

C: NR D: NR P: 72% had animals at home

C/D/pet ownership rates ( if given); general comments; study acronym

A: decreased atopic asthma if C or D ever E: NR R: NR

A: repeated wheezing decreased with D; trend towards decreased repeated wheeze with higher Fel d 1 E: NR R : NR

Association between exposure to C or D and asthma, eczema, and rhinitis

9781405157209_4_048.qxd 4/1/08 17:12 Page 1006

CS, 1–6 years, N = 10 851

CS, 10–11 years, N = 2108

CS, 7–16 years, N = 367

CS, 7–16 years, N = 446

CS, 6 –12 years, N = 3344

Cohort, 14–16 years, N = 2289

CS, 7–12 years, N = 2729

CS, 12–14 years, N = 25 393

Bornehag et al. (2003), Sweden

Braback et al. (2001), Sweden

Von Hertzen et al. (2006), Finland

Von Hertzen et al. (2006), Russia

Brunekreef et al. (1992), Holland

Withers et al. (1998), UK

Anyo et al. (2002), Holland

Burr et al. (1997), UK

Pet ownership (furry or other)

Pet (C, D, rodents, birds), owner early C/D

Current furry pets

Pets (C, D, birds, rodents)

Indoor C, D current, previous

Indoor C, D current, previous

C, D current, previous

Pets (cat, dog, hamster/rabbit, bird, fish) Current Birth

Q

Spt and/or IgE, BHR, Q

Q

Q

Q, Spt

Q, Spt

Q, Spt

Q

A: increased in previous P owners, NA with early or current P owners E: NA R: decreased in current P owners

A: furry pets associated with increased risk of wheeze and wheeze with colds (small effect) E: NR R: NR

Sens to C: NR Sens to D: NR AS: NR

A: furry pets in household currently associated with decreased current wheeze in multivariate analysis E: NR R: NR

A: decreased asthma in current P owners, trend towards increase with previous C owners E: NR R: NR

A: NR E: NR R: NR

A: NR E: NR R: NR

A: NR E: NR R: decreased in current C owners

A: increased wheeze and diagnosed asthma with furred pets at birth in multivariate E: NA in multivariate R: increased with furred pet at birth in multivariate

Sens to C: decreased in current P owners Sens to D: decreased in current P owners AS: decreased in current P owners

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NR Sens to D: NR AS: C in first year protective

Sens to C: NR Sens to D: NR AS: D in first year protective

Sens to C: decreased in C owners (past or present) with positive FH Sens to D: NA AS: decreased in C owners (past or present). NA for D

Sens to C: NR Sens to D: NR AS: NR

C: NR D: NR P: 66% had a furry pet

Continued p. 1008

C: NR D: NR 36% had C/D in infancy. Conclude effect due in part to avoidance of P in allergic families

C: NR D: NR

C: 14% D: 31% 14% avoided or removed pets because of allergy

C: 35% D: 22% In first year of life, increases thereafter

C: 18% D: 24% In first year of life, increases thereafter

C: 29–41% D: 23–41% Higher community ownership of C and D associated with higher rates of sensitization to C and D

C: 25.7% D: 18.2% P: 42.3% current 8.5% got rid of pets because of allergy (asked why avoided), i.e., pet avoidance skews pet ownership distribution in the population

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1007

1008 Design, age, numbers CS, 12–14 years, N = 1537

CS, 5–14 years. N = 1893

CS, 5–7 years, N = 8216

CS, 3–5 years, N = 1402 CS, 7–17 years, N = 1002

CS, 13–14 years, N = 5035

CS, 5–14 years, N = 7448

Study

Austin & Russell (1997), UK

Ritz et al. (2002), Germany

Oberle et al. (2003), Germany (Bavaria)

Oberle et al. (2003), Italy

Cooper et al. (2004), Ecuador

Yarnell et al. (2003), Ireland

Holscher et al. (2002), Germany

Table 48.2 (Cont’d )

C, D, rodents, birds (contact with)

D, C, other furry pets, birds in household

Animals inside and outside house

Furred pets at home

C, D, infancy, now (hamster/rabbit, bird, fish)

Regular contact with C

Current furry pet ownership

Definition of exposure (pets and time groups, allergens)

Q, IgE (not D)

Q

Q, Spt (not D)

Q, Spt

Q

IgE, Q

Q

Outcome measures

A: NR E: NR R: NR

Sens to C: NR Sens to D: NR AS: NA between C or D (inside or out) and atopy

Sens to C: increased with current C contact (NA with C in first year of life); decreased in current D contacts Sens to D: NR AS: decreased with current D contact, NA for C

A: decreased with current D contacts, NA for C E: decreased with D in first year of life, NA for C R: decreased with D ever, NA for C

A: NA with wheeze for C or D. Other furry pets were independent associates of wheeze E: NR R: NR

A: NR E: NR R: increased if P at home

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NR Sens to D: NR AS: NR

A: reduced with continuous exposure to C, NA for D E: NR R: increased if P in first year of life only, decreased if C continuously, NA for D

A: NA E: NR R: NA

A: NA with C or D E: increased if mammal other than C or D R: NA

Association between exposure to C or D and asthma, eczema, and rhinitis

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NA with C contact, increased in non-C owners if many C owners at school Sens to D: NA AS: NR

Sens to C: NR Sens to D: NR AS: NR

Association between exposure and specific sensitization to C or D and between exposure and allergic sensitization (AS)

C: 34% current, 18% first year D: 38% 21% first year D contact in first year more protective if positive FH. A, E, R were doctor diagnosed

C: 28% D: 52% 10% other furry pet Asthma and on treatment in > 20%. NA with reported contact with farm animals

C: 35% mostly outdoors D: 71% mostly outdoors Pigs associated with increased atopy 0.2% sens to C, atopy 20%

C: NR D: NR 18.6% reported rhinitis ever

C: NR D: NR P: 36.4% ever, 4.5% in first year. Excluded farmers and those who gave up cats because of allergies. Most protection seen if C allowed in bedroom

C: NR D: NR 36% had regular C contact

C: 37% D: 44%

C/D/pet ownership rates ( if given); general comments; study acronym

9781405157209_4_048.qxd 4/1/08 17:12 Page 1008

Current C, D ownership Fel d 1, Can f 1 levels

C,D current, birth or both, never

Pets in the house

C, D, before or after 2 years

C, Fel d 1 in dust and air

CC, 12–14 years, N = 332

CC, 11–16 years, N = 961

CC, 6–10 years, N = 1209 (1 : 2)

CC (A or AR), 13–19 years, N = 431

AC, 2–14 years, N = 68

Ingram et al. (1995); Sporik et al. (1999); Platts-Mills et al. (2001), USA

Strachan & Carey (1995), UK

Zheng et al. (2002), China

Henriksen et al. (2001), Norway

Warner & Warner (1991), UK

Current C, D owners Fel d 1, Can f 1

CC, 8–17 years, N = 100

Addo-Yobo et al. (2001), Ghana

C, D in house

CS, 6 years, N = 35 552

Kurosaka et al. (2006), Japan

Spt, IgE

Q, PFT

Q

Q

Spt and IgE, BHR, Q

Spt, IgE, Q

Q

Sens to C: increased if C at birth, or if detectable Fel d 1 in air Sens to D: NR AS: NR

Sens to C: decreased if ever had C Sens to D: NA AS: NR

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NR Sens to D: NR AS: NR

Sens to C: decreased IgE to C with increasing Fel d 1 exposure, NR for C ownership Sens to D: NA with Can f 1, NR for D ownership AS: NA with allergen levels, NR for P ownership

Sens to C: NR Sens to D: NR AS: NR

Sens to C: NR Sens to D: NR AS: NR

A: NR E: NR R: NR

A: eNO higher in those sensitized and exposed to C or D; no other associations E: NR R: NR

A: increased if both D + C. NA for individual pets E: NR R: NR

A: increased severe wheeze with furry pet at birth or current, when pet avoiders excluded. Current furry pets associated with increased risk of speech-limiting wheeze E: NR R: NR

A: NA for allergen levels. NR for C or D ownership E: NR R: NR

A: decreased with D in univariate but NA in multivariate analysis; NA for C E: NR R: NR

A: NA E: lower prevalence among current C owners. NA for D R: lower prevalence among current C owners and current C and D owners. NA for D alone

C: 31% D: NR

Continued p. 1010

C: 40% ever D: 38% ever > 30% of cases had parted with C

C: 18% D: 46% 12% both

C: NR D: NR P: 39% of cases and 37% of controls kept furred pets. 30% of cases and 10% of controls avoided pets. Feather pillows were protective

C: NR D: NR Wide range of allergen levels

C: 36 D: 35% Poor correlation between Spt and IgE

C: 3.9% D: 2.2% Current C also associated with lower Japanese cedar pollinosis

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1009

1010 Asthma cohort, 2–4 years, N = 181

AC, 6–13 years, N = 271

AC, 1–34 years, N = 504

RC, 2–14 years, N = 202

Farmers vs. nonfarmers, 6–13 years, N = 765 CS, adults, N = 1112 Incident over 8 years

CS, 19–29 years, N = 525

Almqvist et al. (1999), Sweden

Ichikawa et al. (1999), Japan

Kidon et al. (2004), Singapore

Remes et al. (2002), Finland

Linneberg et al. (2001, 2003), Denmark

Von Linstow et al. (2002), Denmark

Design, age, numbers

Melen et al. (2001), Sweden

Study

Table 48.2 (Cont’d )

Pets in home in childhood

C, D never, past, current

Livestock, pets (C or D)

Current furry pet ownership

C, D ownership

C contact, C ownership rate in school class ( 18%)

C, D, no, low, high exposure Fel d 1

Definition of exposure (pets and time groups, allergens)

Sens to C: NR Sens to D: NR AS: NR

Sens to C: increased in C owners (twofold but no statistics given) Sens to D: NA AS: NR Sens to C: NA Sens to D: NA AS: NR Sens to C: NR Sens to D: NR AS: decreased with livestock or pets in infancy or current Sens to C: increased with previous C. Incident sens higher with current C Sens to D: NA AS: decreased with previous D (Spt); NA for C

Sens to C: NR Sens to D: NR AS: decreased atopy in C owners (effect lost in multivariate analysis). NA for D

IgE

Spt

Q, Spt

Spt, IgE, Q

Q, Spt, PFT

Sens to C: increased with exposure to C before age 2; increased at high Fel d 1 levels Sens to D: NA AS: NR

PEF, b2 agonist use

IgE, Spt, Q

Outcome measures

Association between exposure and specific sensitization to C or D and between exposure and allergic sensitization (AS)

A: NR E: NR R: NR

A: current C associated with incident “allergic asthma to animals” E: NR R: previous C associated with increased “AR to animals”, previous D associated with decreased “AR to animals”

A: NR E: NR R: NR

A: NR E: NR R: NR

A: NR E: NR R: NR

A: poorer control in children without direct C contact with high C ownership rates in class E: NR R: NR

A: NA for C or D exposure. High Fel d 1 associated with increased risk of severe asthma E: NR R: NR

Association between exposure to C or D and asthma, eczema, and rhinitis

C: 39.7% in childhood D: NR AS less common in those exposed to tobacco smoke in childhood

C: 42% ever D: 66% ever Associations still seen when those who got rid of pets because of symptoms were excluded

C: 47% D: 48% AS less common in farmers’ children, livestock more important than P

19% kept pets. 20% sensitized to pets

C: 10% D: 13%

C: NR D: NR

C: 14% at 2 years, 7% at 4 years D: 8% at 2 years, 4% at 4 years

C/D/pet ownership rates ( if given); general comments; study acronym

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Animals at home in first 5 years of life

P (C or D) before or after age 2 years

CS, 18–58 years, N = 2254

CS, 24–48 years, N = 485

CS, 17–66 years, N = 516

AC, N = 187 cases

AC, adults, N = 200

Custovic et al. (2001a), UK

Dharmage et al. (2001), Australia

Raukas-Kivioja et al. (2003), Estonia

Oryszczyn et al. (2003), France

Desjardins et al. (1993), Canada Spt, Q

Q, Spt (not D)

Q, Spt, BHR

IgE, Spt, Q

Spt

Q Spt on 504 to C

Q, IgE (not D)

Sens to C: NR Sens to D: NR AS: NA

Sens to C: NA with current P, decreased if P before 2 years Sens to D: NR AS: NA with current P, decreased if P before 2 years

Sens to C: increased if animals at home before 5 years Sens to D: NA AS: NA

Sens to C: increased with high Fel d 1 levels Sens to D: NR AS: NR

Sens to C: decreased in lowest and highest Fel d 1 exposure groups Sens to D: NR AS: NR

Sens to C: NA Sens to D: NR AS: NR

Sens to C: increased in asymptomatics with C indoors currently. Decreased in childhood P/C owners with FH atopy Sens to D: NR AS: decreased if D in childhood. NA for C

A: NR E: NR R: NR

A: NR E: NR R: NR

A: NR E: NR R: NR

A: current asthma assoc with Fel d 1 in bed in highest quartile E: NR R: NR

A: NR E: NR R: NR

A: NA for C or D in childhood, increased with current C E: NR R: NR

A: increased nonatopic wheeze if D in childhood or adulthood. Increased atopic wheeze if C in childhood E: NR R: decreased if D in childhood

C: NR D: NR 91% had owned a furry pet at some stage; 53% parted with P because of symptoms or Spt

C: NR, D: NR P: 54.6% currently, 39.7% before 2 years Protective effect on AS seen if P ownership predated onset of A

C: NR D: NR AS in 35% 9% sens to D, 7% sens to C

C: NR D: NR

C: 20% D: NR MAAS parents

C: 21% D: 10%

C: NR D: NR P: current 41%, childhood 65% Among noncat owners community prevalence of C correlated with sens to C and respiratory symptoms ECRHS

A, asthma; AC, asthma cases; AR, allergic rhinitis; AS, allergic sensitization (one or more allergens); BC, birth cohort; BHR, bronchial hyperresponsiveness; C, cat; CC, case–control study; CS, crosssectional study; D, dog; DD, doctor diagnosed; FH, family history; HRBC, high-risk birth cohort; HR, high risk; IgE, specific IgE; NA, no association; NR, not reported; PO, pet owners; RC, rhinitis cases; Sens, sensitivity; S, selected; Spt, skin-prick test; U, unselected.

C, D, current previous, never

Fel d 1 in mattress and floor

Fel d 1 in mattress

C, D, pets,current, previous, childhood

CS, 20–44 years, N = 2999

Noertjojo et al. (1999), Canada

C, D current, childhood

CS, 20–44 years, N = 18 097 (blood on 13 509)

Svanes et al. (1999); Roost et al. (1999); Svanes et al. (2003)

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et al. 2002), but this effect was only seen among high-risk children in two (Braback et al. 2001; Perzanowski et al. 2002). The effect appeared the same in relation to the timing of cat ownership (i.e., whether the cat was present in infancy or currently). In the third study, the apparently protective effect of cat ownership on sensitization was lost when families who had deliberately avoided cat ownership were excluded from the analysis (Hesselmar et al. 1999). It is noteworthy that in one study, of the 287 children who were sensitized to cat, 237 had never lived with a cat (Perzanowski et al. 2002). Two studies from Germany, however, suggest that cats can be a risk, either among noncat owners with high levels of cat ownership at school (Ritz et al. 2002), or in terms of current cat contact (Holscher et al. 2002). In the Netherlands, sensitization to cats was reduced in pet owners (Anyo et al. 2002). Of the five studies reporting, three found no association between dog ownership and sensitization to dog (Hesselmar et al. 1999; Braback et al. 2001; Ritz et al. 2002) and two found dogs to be protective (Anyo et al. 2002; Perzanowski et al. 2002). With respect to the relationship between pet ownership and sensitization to other allergens, results suggested either no association (Hesselmar et al. 1999; Cooper et al. 2004), or that cat (Braback et al. 2001; Anyo et al. 2002; von Hertzen et al. 2006) or dog (Anyo et al. 2002; Holscher et al. 2002; Von Hertzen et al. 2006) ownership was protective. For asthma, three studies (which together included almost 40 000 children) found that pet ownership was associated with an increased risk of asthma; for one of these the effect was seen with pet ownership at birth (Bornehag et al. 2003), one was seen in previous pet owners (Anyo et al. 2002), and one commented that the effect was small (Burr et al. 1997). One large Japanese study of > 35 000 children found no association, (Kurosaka et al. 2006), as did three smaller European studies (Austin & Russell 1997; Ritz et al. 2002; Yarnell et al. 2003). Pets in general were protective in three studies, each numbering about 3000 participants (Brunekreef et al. 1992; Withers et al. 1998; Perzanowski et al. 2002). Others found a specific protective effect of dogs (Holscher et al. 2002; Litonjua et al. 2002) and cats (Smedje & Norback 2001; Oberle et al. 2003). The one study that measured Fel d 1 as well as cat ownership commented that although cat ownership appeared to be protective, higher exposure to Fel d 1 at school appeared to be a risk (Smedje & Norback 2001). Again, it is difficult to draw themes. Passive exposure to cats among children not owning cats is clearly a risk for sensitization to cats, and dogs appear to be protective in general.

Asthma case–control and asthma clinic studies The remaining studies of this topic conducted in childhood comprise mostly asthma case–control studies or asthma clinic populations. For dog sensitization, there was no association between ownership and sensitization to dog whenever this relationship was reported (Ingram et al. 1995; Ichikawa et al.

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1999; Henriksen et al. 2001; Melen et al. 2001; Kidon et al. 2004). For cat sensitization, case–control studies suggest protection for cat owners (Henriksen et al. 2001; Platts-Mills et al. 2001), whereas asthma cohort studies suggest an increase in risk for cat owners. For asthma, the results were so individualized it is not possible to summarize them. Again, it is important to note that those not owning cats exposed to high levels in schools were at increased risk of symptoms (Almqvist et al. 1999). In summary, these studies have shown inconsistent results for cat ownership, with fewer results, but more consistency, for dog ownership.

Adult studies There are fewer published studies in adults; seven crosssectional studies with results on large numbers of young adults from Europe, Canada, Australia, and Estonia, and two asthma case studies. All relied upon recall for the information on pet ownership and pet contact. The results for the relationship between cat ownership and cat sensitization were mixed, with almost every conceivable relationship being reported by one or another study. For dog, the two studies that reported found no association (Linneberg et al. 2003; Raukas-Kivioja et al. 2003). For allergic sensitization, pets were either not associated (Desjardins et al. 1993; Raukas-Kivioja et al. 2003) or were protective (Svanes et al. 1999; Von Linstow et al. 2002; Linneberg et al. 2003; Oryszczyn et al. 2003). For asthma, cats appeared to increase the risk of symptoms (Noertjojo et al. 1999; Dharmage et al. 2001; Linneberg et al. 2003; Svanes et al. 2003), with fewer reports for dog. In conclusion, the results of studies in adults are inconsistent, particularly for the relationship between cat ownership and sensitization to cat. For sensitization to other allergens, the trend is for a decrease in risk among those with pets in childhood, particularly dogs, with no suggestion of an increase in risk in any studies. It remains unclear how exposure to domestic pets could provide a degree of protection against the development of allergic sensitization and/or asthma. There is a possibility that the explanation lies within the context of the hygiene hypothesis, i.e., pet owners may be exposed to a broader range of microorganisms via their pets. The other explanation may be that the effect is due to the exposure to very high levels of pet allergens, i.e., a form of “natural specific immunotherapy.” It has been hypothesized that children who are exposed to high levels of cat allergen may make a modified Th2 response characterized by the presence of IgG4 antibody to cat proteins without IgE response (Platts-Mills et al. 2001). If true, this would suggest that the shape of the dose–response curve between pet-allergen exposure and the risk of sensitization may not be linear, but bell-shaped, with very low and very high exposure providing protection. If this is the case, the results of many of the studies (e.g., intervention studies) would be influenced by where the “start” point was for exposure.

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Conclusions Cat and dog allergens are ubiquitous in areas with high proportion of pet ownership. They are carried on small particles that remain airborne for long periods, and are very difficult to remove from the domestic environment, even after the permanent removal of the pet. Sensitization to pets is a risk factor for asthma, but pet ownership is most certainly not a prerequisite for sensitization. Patients with established asthma who are sensitized to pets and exposed to the pet tend to have more severe asthma than those who are not exposed. The clear advice to pet-sensitized pet owners who experience symptoms on exposure is to remove their pet from the home. What is as yet unclear is how and why some people become sensitized to pets, whether pet ownership is a risk factor for sensitization or is protective, and whether the effect of direct pet exposure differs in subjects with different genetic predispositions. While numerous studies have addressed these issues, there are still no unequivocal answers to these questions, and clinical outcomes reported from different observational studies appear inconsistent and often confusing. These discrepancies between different studies are at least partly due to the fact that exposure to domestic pets may have different, or even opposite effects on the development of sensitization and allergic disease in individuals with different genetic polymorphisms (Karjalainen et al. 2005; Simpson et al. 2006). Thus, the future advice on pet ownership is unlikely to be blanket advice aimed at and applicable to everybody, but rather tailor-made, individualized advice targeting individuals with specific susceptibilities who will benefit from a particular intervention (Custovic & Simpson 2004), i.e., it may well be that some individuals may benefit from having a cat or a dog, while in others pet ownership may increase the risk of allergic disease.

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Animal Allergens

(Can f I) allergens in dust from Swedish schools is high enough to probably cause perennial symptoms in most children with asthma who are sensitized to cat and dog. J Allergy Clin Immunol 91, 1067–74. Munir, A.K., Einarsson, R. & Dreborg, S.K. (1995) Mite (Der p I, Der f I), cat (Fel d I) and dog (Can f I) allergens in dust from Swedish day-care centres. Clin Exp Allergy 25, 119–26. Munir, A.K., Kjellman, N.I. & Bjorksten, B. (1997) Exposure to indoor allergens in early infancy and sensitization. J Allergy Clin Immunol 100, 177–81. Nafstad, P., Magnus, P., Gaarder, P.I. & Jaakkola, J.J. (2001) Exposure to pets and atopy-related diseases in the first 4 years of life. Allergy 56, 307–12. Noertjojo, K., Dimich-Ward, H., Obata, H., Manfreda, J. & ChanYeung, M. (1999) Exposure and sensitization to cat dander: asthma and asthma-like symptoms among adults. J Allergy Clin Immunol 103, 60–5. Oberle, D., Von Mutius, E. & Von Kries, R. (2003) Childhood asthma and continuous exposure to cats since the first year of life with cats allowed in the child’s bedroom. Allergy 58, 1033–6. Ohman, J.L. (1978) Allergy in man caused by exposure to mammals. J Am Vet Med Assoc 172, 1403–6. Ohman, J.L., Lowell, F.C. & Bloch, K.J. (1973) Allergens of mammalian origin: characterization of allergen extracted from cat pelts. J Allergy Clin Immunol 52, 231–41. Oryszczyn, M.P., Annesi-Maesano, I., Charpin, D. & Kauffmann, F. (2003) Allergy markers in adults in relation to the timing of pet exposure: the EGEA Study. Allergy 58, 1136–43. Ownby, D.R., Johnson, C.C. & Peterson, E.L. (2002) Exposure to dogs and cats in the first year of life and risk of allergic sensitization at 6 to 7 years of age. JAMA 288, 963–72. Perzanowski, M.S., Wheatley, L.M., Avner, D.B., Woodfolk, J.A. & Platts-Mills, T.A. (1997) The effectiveness of Allerpet/C in reducing the cat allergen Fel d 1. J Allergy Clin Immunol 100, 428–30. Perzanowski, M.S., Ronmark, E., Platts-Mills, T.A. & Lundback, B. (2002) Effect of cat and dog ownership on sensitization and development of asthma among preteenage children. Am J Respir Crit Care Med 166, 696–702. Platts-Mills, T., Vaughan, J., Squillace, S., Woodfolk, J. & Sporik, R. (2001) Sensitisation, asthma, and a modified Th2 response in children exposed to cat allergen: a population-based cross-sectional study. Lancet 357, 752–6. Raukas-Kivioja, A., Raukas, E., Loit, H.M. et al. (2003) Allergic sensitization among adults in Tallinn, Estonia. Clin Exp Allergy 33, 1342– 8. Remes, S.T., Castro-Rodriguez, J.A., Holberg, C. ., Martinez, F.D. & Wright, A.L. (2001) Dog exposure in infancy decreases the subsequent risk of frequent wheeze but not of atopy. J Allergy Clin Immunol 108, 509–15. Remes, S.T., Pekkanen, J., Soininen, L., Kajosaari, M., Husman, T. & Koivikko, A. (2002) Does heredity modify the association between farming and allergy in children? Acta Paediatr 91, 1163–9. Rhodes, H.L., Sporik, R., Thomas, P., Holgate, S.T. & Cogswell, J.J. (2001) Early life risk factors for adult asthma: a birth cohort study of subjects at risk. J Allergy Clin Immunol 108, 720–5. Ritz, B.R., Hoelscher, B., Frye, C., Meyer, I. & Heinrich, J. (2002) Allergic sensitization owing to “second-hand” cat exposure in schools. Allergy 57, 357–61. Ronmark, E., Jonsson, E., Platts-Mills, T. & Lundback, B. (1999) Different pattern of risk factors for atopic and nonatopic asthma

1015

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Allergens

among children: report from the Obstructive Lung Disease in Northern Sweden Study. Allergy 54, 926– 35. Roost, H.P., Kunzli, N., Schindler, C. et al. (1999) Role of current and childhood exposure to cat and atopic sensitization. European Community Respiratory Health Survey. J Allergy Clin Immunol 104, 941–7. Schmale, H., Holtgreve-Grez, H. & Christiansen, H. (1990) Possible role for salivary gland protein in taste reception indicated by homology to lipophilic-ligand carrier proteins. Nature 343, 366–9. Schou, C. (1993) Defining allergens of mammalian origin. Clin Exp Allergy 23, 7–14. Schou, C., Svendsen, U.G. & Lowenstein, H. (1991) Purification and characterization of the major dog allergen, Can f I. Clin Exp Allergy 21, 321–8. Shirai, T., Matsui, T., Suzuki, K. & Chida, K. (2005) Effect of pet removal on pet allergic asthma. Chest 127, 1565–71. Simpson, A. & Custovic, A. (2003) Early pet exposure: friend or foe? Curr Opin Allergy Clin Immunol 3, 7–14. Simpson, A. & Custovic, A. (2005) Pets and the development of allergic sensitization. Curr Allergy Asthma Rep 5, 212– 20. Simpson, A., John, S.L., Jury, F. et al. (2006) Endotoxin exposure, CD14, and allergic disease: an interaction between genes and the environment. Am J Respir Crit Care Med 174, 386– 92. Simpson, B.M., Custovic, A., Simpson, A. et al. (2001) Nac Manchester Asthma and Allergy Study (Nacmaas): risk factors for asthma and allergic disorders in adults. Clin Exp Allergy 31, 391–9. Smedje, G. & Norback, D. (2001) Incidence of asthma diagnosis and self-reported allergy in relation to the school environment: a four-year follow-up study in schoolchildren. Int J Tuberc Lung Dis 5, 1059–66. Sporik, R., Squillace, S.P., Ingram, J.M., Rakes, G., Honsinger, R.W. & Platts-Mills, T.A. (1999) Mite, cat, and cockroach exposure, allergen sensitisation, and asthma in children: a case-control study of three schools. Thorax 54, 675– 80. Strachan, D.P. & Carey, I.M. (1995) Home environment and severe asthma in adolescence: a population based case-control study. BMJ 311, 1053– 6. Svanes, C., Jarvis, D., Chinn, S. & Burney, P. (1999) Childhood environment and adult atopy: results from the European Community Respiratory Health Survey. J Allergy Clin Immunol 103, 415–20. Svanes, C., Heinrich, J., Jarvis, D. et al. (2003) Pet-keeping in childhood and adult asthma and hay fever: European Community Respiratory Health Survey. J Allergy Clin Immunol 112, 289–300. Svanes, C., Zock, J.P., Anto, J. et al. (2006) Do asthma and allergy influence subsequent pet keeping? An analysis of childhood and adulthood. J Allergy Clin Immunol 118, 691– 8.

1016

Tepas, E.C., Litonjua, A.A., Celedon, J.C., Sredl, D. & Gold, D.R. (2006) Sensitization to aeroallergens and airway hyperresponsiveness at 7 years of age. Chest 129, 1500–8. Torrent, M., Sunyer, J., Munoz, L. et al. (2006) Early-life domestic aeroallergen exposure and IgE sensitization at age 4 years. J Allergy Clin Immunol 118, 742–8. Tunnicliffe, W.S., Fletcher, T.J., Hammond, K. et al. (1999) Sensitivity and exposure to indoor allergens in adults with differing asthma severity. Eur Respir J 13, 654–9. Uhlin, T., Reuterby, J. & Einarsson, R. (1984) Antigenic/allergenic composition of poodle/alsatian dandruff extract. Allergy 39, 125–33. Van Der Heide, S., Van Aalderen, W.M., Kauffman, H.F., Dubois, A.E. & De Monchy, J.G. (1999) Clinical effects of air cleaners in homes of asthmatic children sensitized to pet allergens. J Allergy Clin Immunol 104, 447–51. Von Hertzen, L., Makela, M.J., Petays, T. et al. (2006) Growing disparities in atopy between the finns and the russians: a comparison of 2 generations. J Allergy Clin Immunol 117, 151–7. Von Linstow, M.L., Porsbjerg, C., Ulrik, C.S., Nepper-Christensen, S. & Backer, V. (2002) Prevalence and predictors of atopy among young Danish adults. Clin Exp Allergy 32, 520–5. Warner, J.A. & Warner, J.O. (1991) Allergen avoidance in childhood asthma. Respir Med 85, 101–5. Withers, N.J., Low, L., Holgate, S.T. & Clough, J.B. (1998) The natural history of respiratory symptoms in a cohort of adolescents. Am J Respir Crit Care Med 158, 352–7. Wood, R.A., Chapman, M.D., Adkinson, N.F., Jr. & Eggleston, P.A. (1989) The effect of cat removal on allergen content in householddust samples. J Allergy Clin Immunol 83, 730–4. Wood, R.A., Johnson, E.F., Van Natta, M.L., Chen, P.H. & Eggleston, P.A. (1998) A placebo-controlled trial of a hepa air cleaner in the treatment of cat allergy. Am J Respir Crit Care Med 158, 115–20. Woodcock, A., Addo-Yobo, E.O., Taggart, S.C., Craven, M. & Custovic, A. (2001) Pet allergen levels in homes in Ghana and the United Kingdom. J Allergy Clin Immunol 108, 463–5. Yarnell, J.W., Stevenson, M.R., Macmahon, J. et al. (2003) Smoking, atopy and certain furry pets are major determinants of respiratory symptoms in children: The International Study of Asthma and Allergies in Childhood Study (Ireland). Clin Exp Allergy 33, 96–100. Zheng, T., Niu, S., Lu, B. et al. (2002) Childhood asthma in Beijing, China: a population-based case-control study. Am J Epidemiol 156, 977– 83. Zielonka, T.M., Charpin, D., Berbis, P., Luciani, P., Casanova, D. & Vervloet, D. (1994) Effects of castration and testosterone on Fel d I production by sebaceous glands of male cats: I. Immunological assessment. Clin Exp Allergy 24, 1169–73.

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49

Airborne Allergens and Irritants in the Workplace Xaver Baur

Summary This chapter is intended to help identify agents causing occupational asthma in individual workplaces and initiate appropriate and effective primary as well as secondary preventive measures. It is based on a literature search and lists more than 400 occupational agents described in the medical literature as causing asthma. Approximately two-thirds are allergens, one-third irritants. Allergens are predominantly high-molecular-weight components, such as proteins of plants, microorganisms, or animals. Low-molecular-weight chemicals mainly represent irritants but some of them also behave as allergens. Newly identified causes include environmental tobacco smoke, fire smoke (e.g., during the World Trade Center disaster), and endotoxins. Several occupational confinement facilities with a distinctly increased prevalence or incidence of asthma where the causative agents have not been (fully) identified are also mentioned. According to occupational disease statistics, irritant asthma attributed mainly to isocyanates, welding fumes, chlorine, and formaldehyde is too rarely diagnosed. In addition to the listed medical literature, more than 600 occupational agents are defined as “irritating the respiratory system” (by phrase R37) and “may cause sensitization by inhalation” (by phrase R42) (European Union directives; ILO/CIS 2002) and/or agents causing respiratory effects by the American Conference of Governmental Industrial Hygienists (ACGIH). A separate disorder is occupational rhinitis, a frequent precursor of occupational asthma. Contrary to irritant asthma, occupational irritant rhinitis is a relatively unknown disorder that has not been systematically investigated by epidemiologic or other scientific studies.

Introduction Approximately 10% of asthma cases are caused by agents in the workplace. Investigations in recent years have identi-

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

fied approximately 400 causative occupational allergens and nearly 200 causative irritants. Their number is steadily increasing. Most of the allergens are high-molecular-weight components, mainly proteins of plants, microorganisms, or animal origin. However, several low-molecular-weight allergens also exist. Some of them, e.g., isocyanates and acid anhydrides, also have irritative effects on the airways. The objective of this chapter is to provide an overview of known occupational airway sensitizers and irritants eliciting allergic or nonallergic asthma, in order to support occupational physicians and primary caregivers in identifying causative agents in affected employees, as well as in initiating effective preventive measures. This list includes neither substances mentioned in publications and reports not found on the Internet nor occupational agents recently introduced into industry that have not yet been clinically or epidemiologically studied. Therefore, it is not complete and has to be updated continuously.

Results Listed regulatory airway sensitizers and regulatory irritants Listed regulatory airway sensitizers (ACGIH 2007; European Union 1967, 2001, 2004 with regard to Phrase R42) and listed regulatory irritants (ACGIH 2007; European Union 1967, 2001, 2004 with regard to Phrase R37) are given in the Appendix Table available at www.uke.uni-hamburg. de/institute/arbeitsmedizin.* The medical literature, as summarized in Tables 49.1 and 49.2, frequently mentions clearly defined asthma-inducing agents, e.g., acids, alkali, and chlorine. However, many reports deal with less-defined causative substances and materials, such as resins, adhesives, flaxes, metals, drugs, biocides, dust, and fumes. Furthermore, industrial mixtures or occupational activities like “cleaning” or “farming” are also mentioned in the literature. They are included in Table 49.2 if there is no specific causative agent that employees are known to be exposed to.

* Click on navigation term “Publikationen”

1017

Veld et al. 2002

(Amblyseius

119/725 16.4

25.7

129/725

78/109*

17.8

71.6

%

nd

nd

n/n

%

nd

nd

n/n

BHR %

nd

nd

n/n

PFT %

nd

nd

n/n

SIC % i(n)

l(n)

SIC reaction d(n)

nd

63/109

n/n

57.8

%

spider mite; sign. increased

*SPT+ with at least one

Apple-cultivating farmers.

not listed

rhinitis, individual results

employees without WR

higher responses than

WR-rhinitis had sign.

employees: those with

nasal Ch in 23 sensitized

for sensitized subjects only;

(62/472 SPT+). *WRS listed

Tyrophagus putrescentiae

plant (80/109 SPT+) and

bell pepper pollen and/or

sensitized to both sweet

co-exposed and co-

bell pepper horticulture,

Greenhouse employees in

Remarks

putrescentiae)

(Tyrophagus

Mould mite

98/465

21.1

asthmatics

urticae)

37/118 SPT+ were

16.6

23.2

25.4

23.1

%

Spec. IgE

(Tetranychus

77/465

108/465

465*

118/

109/472

n/n

SPT

spider mite

32

48

37 (5.1)

28/109*

n/n

LFT

among SPT+ subjects;

725

28 (25.7)

%

Rhinitis

prevalence of asthma

Survey

472

n/n

Asthma

Work-related symptoms

Two-spotted

1999

Kim, Lee et al.

sectional

Cross-

%)

ence,

(preval-

cases, n

Evidence (pathological results)

(Panonychus ulmi)

European red mite

Spider mites

Spider mites (Tetranychidae)

cucumeris)

Groenewoud,

Thirps mite

(Phytoseiidae)

Predatory mites

Mites (Acarina)

(ARACHNIDA)

ARACHNIDS

(ARTHROPODS)

JOINT-LEGS

(ANIMALIA)

ANIMALS

type

CAS no. studied, n

subjects

Study

[synonyms]

Reference

ally exposed

Occupation-

Agents

asthma

Allergic

Table 49.1 Allergenic agents reported to cause occupational asthma.

9781405157209_4_049.qxd 4/1/08 17:12 Page 1018

series

Case series Case series

1999

Michel, Guin

et al. 1977

Carbonnelle,

Lavaud

(Panonychus citri)

European red mite

(P. ulmi)

MacDaniel spider

mite (T. macdanieli)

series

Franzese

series

Enrique et al.

2000

Case

Cisteró-Bahima,

et al. 1994

Case

Astarita,

1

46

246

7

5

16

control

Ruoppi et al.

2006

Flour mite

(Acarus siro),

68.8

24/

20.7

nd

nd

1/1

46*

19/

nd

41.3

1/1*

nd

nd

nd

nd

1

1

32/

6/12*

1/1

36/46

61/241

7/7

5/5*

16/16

24.6

78.3

25.3

100

30/

0/12

1/1

36/46

29/110

nd

nd

16/16

23.4

78.3

store. *6/12 SPT+ with T.

moisture-damaged grocery

The entire personnel of a

carnation

simultaneous OA to

Flower cultivator with

also sensitized

asthmatics; 16 asthmatics

workers. *PEFR+ all

workers, 16 greenhouse

Farm workers: 30 fields

asthmatics not listed

among exposed; sensitized

sensitization sign. increased

(n = 46); specific

of sensitized symptomatics

of exposure and number

correlation between time

Greenhouse workers. Sign.

Wine growers

Apple growers. *IC

with normal LFT

fruit. *BHR in all subjects

Farmers cultivating citrus

116

20/130

130** 15.4

9/128

128** 7

at least one storage mite;

**SPT and IgE results with

with storage mite mix;

Grain-store workers. *SIC

Acarus farris

(T. putrescentiae) 4/130

21/130

3.1

16.2

15/128

21/128

11.7

16.4

least one

9.4

13/130 SPT+ with at

12/128

Mold mite

9.2

(G. domesticus)

12/130

and sensitization; also

9.4

association between WRS

12/128

House itch mite

10

(G. destructor)

13/130

15 asthmatics IgE+; sign.

116

***21 asthmatics SPT+ and

(15.8)***

Fodder mite

1989

Topping et al.

Blainey,

(A. siro)

Flour mite

Storage mites

(T. putrescentiae)

not listed

11/

nd

1/1

nd

nd

nd

nd

1/1

Ch+; sensitized asthmatics

–

nd

0/1

nd

nd

nd

nd

nd

Mold mite

100

17.5

nd

nd

7/7*

reactive; 4/6 SPT+ nasal

32.3

12/12

–

46/46

43/246

nd

nd

9/16

destructor),

43/133

8.3

41.3

6.9

4/7

4/5

15/16

putrescentiae as most

21

1/12

1/1

19/46

17/246

4/7

2/5

16/16

(Lepidoglyphus

133

12

1

16

4

2

16 (100)

Fodder mite

Survey

Case

Koistinen,

Storage mites:

Storage mites (Acaridae, Glycyphagidae) (see also bakery confinement)

“

“

sectional

Delgado et al.

mite (T. urticae)

2000

Cross-

Navarro,

Two-spotted spider

et al. 1986

Case

Kim, Son et al.

Citrus red mite

9781405157209_4_049.qxd 4/1/08 17:12 Page 1019

1979

(A. siro)

1

nd

1/1

nd

nd

n/n

SIC %

1

i(n)

l(n)

SIC reaction d(n)

10/16

37/38

34/38

37/38

34/38

38/38

74/290

103/290

69/290

108/290

n/n

SPT

62.5

97.4

89.5

97.4

89.5

100

37.2

%

13/14

6/38

20/38

4/38

42/219

n/n

Spec. IgE

92.9

15.8

52.6

10.5

19.2

%

Poultry workers. 14/16

listed

of farmers not separately

18 kids, 1 father); results

family members (6 wives,

13 farmers and their 25

asthmatics SPT+

Farm workers. 27/36

Remarks

Stevenson,

Mathews et al.

1967

Cimarra,

Martínez-

Cócera et al.

1999

Bee moth larvae

(Galleria mellonella),

wax worm, wax moth

Champignon

(mushroom) flies;

family: Phoridae

and Sciaridae

INSECTS (INSECTA)

report

Case

report

Case

control

1

1

1

1/1

1/1

1/1

1/1

1/1

0/1

nd

nd

1/1*

nd

nd

1/1*

1

1/1

1/1**

1/1

nd

1/1 conj. Ch+

and off-work for 15 days;

Sciaridae); *PEFR at work

Phoridae and 2%

mix of both flies (98%

Clinical tests with extract-

Champignon cultivator.

adult bee moth; HR+; PK+

extract; **scratch test with

company. *SIC with wing

Employee in a fishbait

asthmatics not listed

serum); sensitized

SPT+ with at least one feathers, litter, feed and

nd

%

poultry allergen (NFM,

nd

nd

nd

n/n

PFT

et al. 1982

12.5

nd

%

Lutsky, Bar-Sela

2/16

42.9

nd

n/n

BHR

sylviarum)

87.5

12/28

nd

%

et al. 1984;

14/16

28.9

24.8

n/n

LFT

Lutsky, Teichtahl

16

11/38

12.4

36/290

%

(Ornithonyssus

Case

Survey

n/n

n/n 72/290

Rhinitis %

Asthma

Work-related symptoms

Evidence (pathological results)

Northern fowl mite

Poultry mites (Macronyssidae)

(T. putrescentiae)

Mould mite

(G. domesticus)

House itch mite

(L. destructor)

Fodder mite

Brostoff et al.

Flour mite

Storage mites

(T. longior)

Seed mite

(G. destructor)

11 (28.9)

1984

(A. siro)

38

Jeffrey et al.

Flour mite

Cuthbert,

27 (9.3)

290

Cross-

Cuthbert,

Storage mites

Fodder mite

%)

studied, n

type

sectional

ence,

subjects

Study

Reference

CAS no.

(preval-

ally exposed

[synonyms]

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1020

1

2 (20)

1/1

2/10

15.4

20

1/1

3/10

15.4

30

nd

0/10

nd

1/1

nd

1/1

nd

2/2

21.4

1

1

3

1

1/1

3/10

38.4

30

1/1

1/10

30.8

10

factory and 1 former

9 workers of a natural dye

report

Roggenbuck

report Case report

Case reports with

et al. 2003

Alanko, Tuomi

et al. 2000

Bagenstose,

Mathews

et al. 1980

Confused flour beetle

(Tribolium confusum)

Cricket (Acheta

domestica)

report

2002

beetle (order

sectional

Vooren et al.

melanogaster)

2/2

18.2

1/2

1/1

7/22

1/1

2/13

1/1

1/1

31.8

nd

1/1

nd

nd

2/2

nd

nd

1/1

1/1

nd

0/1

nd

nd

nd

nd

1/1*

nd

1/1*

nd

nd

nd

2/2

nd

14*

3/

1/1

2/2

1/1*

nd

1

1

2

1

2/2

1/1

9/22**

1/1

5/13

1/1

1/1

40.9

2/2

1/1

10/22

1/1

4/13

1/1

1/1

45.5

water. *Serial PEFT at work

Employee bottling mineral

**IC

Ch, 9/14 nasal reaction;

laboratory. *nasobronchial

Workers in a scientific

Ch+

daily for 3 months; conj.

Wool worker. *PEFR twice

asymptomatic coworkers

facility workers and 11

2 symptomatic amphibian

storage mites

and SPT− with flour and

contaminated flour; IgE−

*SIC+ with T. confusum

crispbread factory.

Employee in a rye

Farmer

reports

Lombardero

(Anisakis simplex)

report

Swanson

(Apis mellifera)

report

Lombardero

(Bruchus lentis)

et al. 2003

Case

Armentia,

Lentil pest

et al. 1986

Case

Ostrom,

Honeybee

et al. 1998

Case

Armentia,

Herring worm

albofasciato)

Microplax

1

1

1

1

1/1

1/1

1/1

–

nd

0/1

nd

nd

nd

nd

1/1*

1/1

1

1/1

1/1

1/1**

1/1

and SIC– with lentil

infested lentil; SPT–, IgE–

type not listed; **IgE+ with

Agronomist. *SIC reaction

employee. IgE− with pollen

Honey-processing plant

monger. 1/1 SPT– with fish

Chicken breeder and fish

conj. Ch+

2

1/1

4/22

1/1

2/13

1/1

1/1

and off-work for 6 weeks;

2

1

6 (27.3)

1

2 (15)

1

1

ditomoides et

report

et al. 1997

(family Lygaeidae:

1

22

1

13

1

1

Flavourer in a food factory

Metopoplax

Case

Lázaro, Muela

Ground bugs

1986

Cross-

Spieksma,

Fruit fly (Drosophila

Coleoptera)

Case

Brito, Mur et al.

Dermestidae spp.

survey

Case

Focke, Hemmer

(Musca domestica)

et al. 1996

Case

Stücker,

Common housefly

“

E 120

due to carmine

1

10

employee with previous OA

Survey

coccus), carmine red

et al. 1994

(dried bodies of

female Dactylopius

Quirce, Cuevas

Cochineal

9781405157209_4_049.qxd 4/1/08 17:12 Page 1021

14.4

22.2

nd

nd

l(n)

d(n)

(5/7)

19/76

24/76

3/3

n/n

SPT

33.3

31.6

%

18.8

%

workers. Sensitization to

8 retailers, 18 laboratory

50 workers in 8 LFB farms,

Research entomologists

Remarks

et al. 1980a

gregaria and Locusta

migratoria), cicada

Burge, Edge

Locust (Schistocerca

Crosssectional

90

11 (12.2)

13/90 20/90

x*

nd

nd

nd

29/87

x**

partellus)

SPT+) to moth (Chilo

co-sensitization (11/87

co-exposure and

asthmatics had SPT+;

WR asthma; 11/12

degree of exposure and

corelated sign. with

mean FEV1; **IgE+

*asthmatics had a reduced

7/28 admin. staff SPT+;

staff) in a research centre;

non-exposed (admin.

90 exposed and 28

larvael extracts detected

no cross-reactivity to

mollitor)

3/13

12 asthmatics sensitized; 3/13

(Tenebrio

(2 immediate, 3 late);

Mealworm

12/13

and non-exposure day 12/13

farm and 11 anglers.

3 workers of a fish bait

(Lucilia caesar)

4/13

13/14

*PEFR at exposure day

2/13

13/14

Greenbottle

series

(1/7)

2/64

brackets

results for symptomatics in

(G. mellonella)

Beemoth

Live fish bait (LFB)

et al. 1994

(2/7)

(Tenebrio

mollitor)

8/76

Mealworm

moorei)

(2/7)

5/64

(3/7)

5/64

(3/7)

6/64

12/64

3/3

n/n

Spec. IgE

(3/7)

5/7*

nd

i(n)

SIC reaction

(Cilecomadia

7/13

nd

%

8/76

nd

nd

nd

n/n

SIC

Gusano rojo

14/14

6.6

%

associated; IgE and SPT

13/14

5/76

nd

n/n

PFT

(4/7)

12

3.9

%

(G. mellonella)

14

3/76

nd

n/n

BHR

LFB and WRS were strongly

Case

3 (3.9)

%

13/76

Siracusa, Bettini

76

nd

n/n

LFT

Beemoth

2003

(Calliphora

sectional

Cross-

%

vomitoria)

Marcucci et al.

Siracusa,

Live fish bait (LFB)

Bluebottle

et al.1988

diaperinus (Panzer))

reports

2/3

2/3

2

3

Meier-Davis

(Alphitobius

Schroeckenstein, Case

n/n

Lesser mealworm

Rhinitis

n/n

%) %

Asthma

ence,

studied, n

type

subjects

Study

Reference

CAS no.

(preval-

[synonyms]

cases, n

ally exposed

Work-related symptoms

Evidence (pathological results)

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1022

sectional

Case series

et al. 1988

Bernstein,

Gallagher

et al. 1983

Wittich 1940

Mealworm (larva

of beetle Tenibrio

molitor)

Mexican bean

et al. 1965

(Cochliomyia

hominivorax)

Gibbons, Dille

Screwworm fly

Survey

182

46 (25.3)

+* +* nd

nd

nd

nd

nd

nd

nd

nd

10/11**

51/94

90.9

nd

76/225

33.8

symptomatics

listed; **SPT+ in all

individual figures not

exposed 68.8–71%,

5.6–7.1%, among heavily

and shortness of breath)

(primarily cough, wheezing

*Prevalence of symptoms

fly eradication programme.

Workers of the screwworm

exposure

symptoms and degree of

also association between

fish-food factory. 34/37

Aquarists and workers in a

sign. associated with WRS;

54/225

asthmatics had IgE+; IgE

37/225

spective)

main allergen Chi t I

34 (15.1)

(retro-

225

et al. 1993

54.3

Aquarium keeper

Ch+ and 1/1 conj. Ch+

Bean sorters. *IC; 1/1 nasal

sensitized

symptomatics were

asthmatics only; only

Fish bait handlers. *SIC in

mixture of S.g. and L.m.

antigen; **IgE+ with a

*SPT+ with at least one

centre with past exposure.

20 employees in a research

SPT and exposure

association between

sensitized; sign.

symptomatics were

of S.g. and L.m.; all

**IgE+ with a mixture

with at least one antigen;

a research centre. *SPT+

15 currently exposed in

thummi thummi ),

24

35

73.3

sectional

1/1

nd

2/5

7/20**

11/15**

Cross-

1/1

2/2*

4/5

10

66.7

Hoernstein

1

2

2/20*

10/15*

Liebers,

1/1

nd

*

2/2

nd

nd

(Chironomus

nd

nd

nd

nd

nd

Non-biting midges

nd

nd

nd

nd

nd

1982

nd

nd

nd

nd

nd

plamosus) 16.4

60

report

1/1

2/2

4/5

–

9/15

Case

1/1

33.3

Barbaro et al.

1

2/2

2/5

–

5/15

Resta, Foschino-

1

2

2

5 (33.3)

(Echinodorus

reports

2

5

20

15

Mosquito larvae

subfasciatus boh.)

weevil (Zabrotes

Case

Cross-

Tee, Gordon

“

9781405157209_4_049.qxd 4/1/08 17:12 Page 1023

Crosssectional

et al. 1985

Kaufman,

Gandevia et al.

1989

Charpin and

Blanc 1967

Uragoda and

Wijekoon 1991

alternata)

Sheep blowfly,

Australian

(Lucilia cuprina)

Silkworm, silk,

sericin

“

23.1

34

1/1

6/13

2/2

–

46.2

24.1

0/1

nd

1/1

nd

nd

nd

%

1/1

nd

nd

nd

nd

nd

nd

n/n

%

nd

nd

nd

18*

4/

nd

nd

nd

n/n

PFT %

1/1

nd

1/1

nd

nd

nd

1/1

n/n

SIC %

1

1

1

i(n)

l(n)

SIC reaction d(n)

1/1

nd

2/2*

nd

1/1

nd

1/1

n/n

SPT %

Lobster report

Desjardins

et al. 1996

Case

Lemière,

CRUSTACIANS (CRUSTACEA)

Ostrinia nubilalis

decemlineata

Leptinotarsa

carnea

1/1

4/7

4/7

3/7

5/7

8/13*

nd

nd

nd

19/52*

1/1

n/n

Spec. IgE

61.5

36.5

%

of beneficial arthropods.

Employees in a production

SPT+

store. *1/1 IC+ and 1/1

Workers in the fish food

*PEFR in all asthmatics

Silk processing workers.

Hairdressers

not listed

sensitized asthmatics

symptomatics IgE+;

programme. *10/14

blowfly breeding

Workers in a sheep

cockroach; HR+; PK+

black fly, mosquito and

with wax moth, deer fly,

treatment plant. Also SPT+

Worker at a sewage

Remarks

with shrimp (immediate)

crab, and crawfish; SIC+

clam; IgE+ with shrimp,

Also SPT+ with shrimp and

co-sensitized to shrimp.

shop, co-exposed and

Worker in a fishmonger

*All symptomatics IgE+

1/1

3/13

2/2

18/53

2/2

13/54

0/1

n/n

BHR

with at least one insect

1

3 (23.1)

2

4 (7.5)

2/2

11.1

%

LFT

Chrysoperla

1

13

2

53

2

6/54

1/1

n/n

n/n 1/1

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

kuehniella

sectional

Cross-

et al. 1994

Lugo, Cipolla

Various insects

Ephestia

reports

Case

reports

Case

sectional

Cross-

(Daphnia)

Meister 1978

1

1

Case

Gold, Mathews

Sewer fly (Psychoda

Water-flea

%)

studied, n

type

Reference

CAS no.

2

ence,

subjects

Study

[synonyms]

54

(preval-

ally exposed

Agents

report

cases, n

asthma

Allergic Occupation-

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1024

Case report

Carino, Elia

et al. 1985

Malo et al. 1995

Desjardins, Survey

report

Whisman et al.

2000

Case

Goetz &

(Penaeus shrimp,

Crosssectional Case

Jyo, Kohmoto

et al. 1980

Zadda 1967

Hoya (sea-squirt)

report

sectional

et al. 1984a

(Chinoecetes opilis)

Nacre dust

Cross-

Cartier, Malo

Snow crab

Artemia salina)

brine shrimp

5.3

1/1

18

12/

4.4

66.7

1/1

nd

54.4

nd

nd

1/1

2/2

33.3

1

1

23

1

1

1/1

13/50

15.8

26

1/1

8/50

14.5

16

Employee in a fish-food

sensitized

asthmatics were not

IgE+, the other 11

asthmatics had SPT+ and

Prawn processors. 7/18

1

1413

303

1

57

1

410 (29)

27 (8.9)

1

1 (1.8)

1

511/1413

64/303

1/1

2/57

1/1

36.2

21.1

+*

1/1

3/57

–

298

13/

0/1

nd

nd

114

62/

1/1

4/8

1/1

**

14

12/

nd

nd

1/1

1/1

nd

46

33/

1/1

1/3

1/1

71.7

1

1

1

9

1/1

511

*419/

65/298

1/1

9/57

1/1

82

21.8

nd

160/180

nd

1/1

8/55

1/1

89

Nacre buttons

*SPT in asthmatics only

Workers of oyster farm.

SPT+

46 subjects with OA had

to work (n = 13); 27 out of

and/or FEV1 after return

changes in PEFR, PC20

2 of the following: sign.

as +SIC (n = 33) or at least

only; 46 had OA, defined

PFT, BHR in asthmatics

after return to work; SIC,

**Serial PEFR before and

rhinitis and/or conj.;

*55/303 (18.2%) WR

Snow crab processors.

aquaculture

Technician for experimental

shrimp; see also clam

SPT+, IgE+ and SIC+ to

production period had

asthmatics during shrimp-

SIC in sensitized only; 1/2

exposed to clam. BHR and

including index case, co-

Food company workers

handler. See scallop

Restaurant seafood

Chi t 1–9 of C. thummi

3.5

22

factory. Also IgE+ with

–

11/50

t

1/1

36

repor

1

18/50

et al. 2000

1

7 (14.0)

Case

Shrimp meal

“

Shrimp

Shrimp, Gammarus

50

Baur, Huber

sectional

et al. 1980

norwegicus),

Norway lobster

Cross-

Gaddie, Legge

Prawn (Nephrops

9781405157209_4_049.qxd 4/1/08 17:12 Page 1025

Orriols, Aliage

“

sectional

et al. 1986b

Scallop

Cuttle-fish

Clam

series

Weclawik

report

Whisman

et al. 2000

Case

Goetz,

et al. 1988

Case

Tomaszunas,

Malo et al. 1995

Desjardins,

Survey

Cross-

Cartier, Malo

et al. 1997

Malo, Chretien

2001

Kamiya et al.

Onizuka,

sectional

et al. 1990

MOLLUSKS (MOLLUSCA)

“

Snow crab

“

Cross-

Onizuka, Inoue

1

66

57

303

1

61

2 (3.5)

9

%)

studied, n

type

Reference

CAS no.

74

ence,

subjects

Study

[synonyms]

et al. 1977

(preval-

ally exposed

Red soft coral

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

1/1

61/66

3.5

–

–

4/57

n/n

n/n

2/57

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

7.0

%

nd

nd

nd

nd

n/n

LFT %

1/1

nd

4/8

n/n

BHR %

1/1

nd

nd

n/n

PFT %

1/1

nd

2/2

nd

nd

n/n

SIC

100

%

1

1

i(n)

l(n)

SIC reaction

1

d(n)

1/1

nd

4/57

65/119

2/2

n/n

SPT

7.0

54.6

%

1/1

nd

4/55

62/115

nd

n/n

Spec. IgE

7.3

53.9

%

and shrimp

reactivity between scallop

shrimp; significant cross-

handler. Co-exposure to

Restaurant seafood

survey)

reactions (5 year

incidence of allergic

*Estimated annual

Deep-sea fishermen.

also shrimp

and SIC+ to clam; see

period had SPT+, IgE+

during clam-production

only; both asthmatics

in sensitized subjects

shrimp. BHR and SIC

case, co-exposed to

including the index

Food company workers

about symptoms

years; no information

Data collection over several

Fishermen

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1026

et al. 1982

powdered (Dysidea report

Case

McSharry

(Salmo salar)

51

1 (2.0)

2

1/51

2/2

70/791

2.0

24.1

–

1/2

+*

–

37.5

17/

nd

nd

1/1

33.3

nd

1/1

nd

nd

2/2

23.5

2/2

25/291

100

8.6

Employees of a salmon-

HR+ to D. herbacea

and 2 soft coral species;

IgE+ with 7 other sponge

Laboratory worker. Also

nd

2/2*

2/51

1

12/51*

nd

work and away from work;

*Serial PEFR for 2 weeks at

Fish-processing workers.

salmon

9 PFT+ not sensitized to

asthmatic WRS, the other

were sensitized and had

PEFR levels, of whom 15

changes in daily mean

subjects showed marked

over several weeks: 24

not listed; *serial PEFR

*Prevalence of indiv. WRS

2

nd

1/1

**

2/2

nd

nd

processing plant.

66.7

8.2

nd

291

24/

nd

5

6/8

75

3/8

1/8

7/8

4/6*

nd

**

nd

8/8**

species; **nasal Ch

*IC with a mix of 10 fish

Fish meal factory workers.

Trout, rainbow

shrimp, mussel

sectional

Hansen et al.

1989

Cross-

Sherson,

endotoxin/mL

water containing 1 µg

trout-contaminated

+BHR; **IgE done with

done in 1 asthmatic with

*PEFR at work, PFT was not

trout-processing factory.

Production workers of

FEV1 in asthmatic subject

8

51

induced a 20% decrease in

et al. 1981

fish, plaice, eel,

sectional

(tuna), salmon,

Kowalski

herring, cod, shell

SIC+ with raw salmon

sardine, tunny-fish

Droszcz,

Fishmeal: pickling,

trout, anchovy, salmon;

Atlantic pomfret; IgE+ with

sardine, trout, salmon, sole,

and cooked anchovy,

subject 2: SPT+ with raw

hake, salmon, plaice, tuna;

salmon; SIC+ with raw

hake, tuna; IgE+ with

and cooked plaice, salmon,

Cross-

2

15 (5.2)

1/1

subject 1: SPT+ with raw

1997

hake, plaice,

reports

Case

291

1

sole, trout, tuna

Reaño et al.

Atlantic pomfret,

sectional

Cross-

1

salmon, sardine,

Rodríguez,

Fish: anchovy,

et al. 1995

Douglas,

Atlantic salmon

FISH (PISCES)

SPINAL CORDS (CHORDATA), VERTEBRATA

herbacea)

Baldo, Krilis

Marine sponge,

SPONGES (PORIFERA)

9781405157209_4_049.qxd 4/1/08 17:12 Page 1027

Fruhmann

parrot, canary)

reports

et al. 1997

Bat (Chiroptera)

Bat

Gordon et al.

[chaerephon] major)

report

Johansson

et al. 1996

Case

report

et al. 2000

Spiewak,

Case

series

Case

Senti, Lundberg

1987

El-Ansary,

Black bat (Tandarida

MAMMALS (MAMMALIA)

Case

Perfetti, Cartier

“

report

Schwartz 1994

Case

series

Chicken and turkey

et al. 1994

Tauer-Reich,

Birds (budgerigar,

1

1

7

4

1

5

1

1

7

4

1

5

%)

studied, n

type

CAS no.

Case

ence,

subjects

Study

[synonyms]

BIRDS (AVES)

(preval-

ally exposed

Reference

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

1/1

1/1

7/7

4/4

1/1

–

1/1

6/7

3/4

1/1

2/5

n/n

n/n

5/5

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

%

1/1

0/1

nd

nd

0/1

2/5

n/n

LFT %

nd

0/1

nd

nd

nd

5/5

n/n

BHR %

nd

nd

nd

4/4*

nd

nd

n/n

PFT %

nd

nd

nd

nd

nd

nd

n/n

SIC % i(n)

l(n)

Reaction d(n)

1/1

1/1

7/7*

4/4**

1/1*

nd

n/n

SPT

100

100

%

1/1

1/1

7/7*

nd

nd

5/5*

n/n

Spec. IgE

100

%

with bat hair

Bat scientist. Clinical tests

(bat feed)

to Tenebrio molitor

and co-sensitization

bat guano; co-exposure

Zoologist. Clinical test with

guano/droppings

*Clinical tests with bat

cracked ceiling in Sudan.

bat droppings from

housewife exposed to

6 employees and 1

and off-work

feathers; *FEV1 at work

workers. **SPT with

Poultry-slaughterhouse

turkey

with raw chicken and raw

Food processing. *SPT+

of diff. bird species

serum and feather allergen

cross-reactivity between

and feathers; SDS-PAGE:

Bird fanciers. *IgE with sera

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1028

41

1

1

x**

Dairy farmers with

1/1 1/1

41/100

nd

nd

1/1

x*

nd

nd 100*

11/

1/1*

7/41**

1/1

4/41**

38 OA cases was SPT+ with

Farmers. Sign. risk factor of

anti-BEA IgE

report

Case report

et al. 1996

Armentia,

Martin-Santos

report

Anton et al.

Case reports

Newman Taylor,

Longbottom

Mouse

Brennan, 1985 report

Case

reports

1985

et al. 1977

Case

Petry, Voss et al.

1996

Case

Jimenez Gomez,

et al. 1988

Case

Nahm, Park

1

4

2

1

1

1

4

2

1

1

1/1

4/4

2/2

1/1

1/1

–

4/4

1/2

1/1

1/1

1/1

nd

nd

1/1

nd

nd

nd

nd

1/1

nd

1/1*

nd

nd

nd

nd

nd

0/4

4/4

4/4*

nd

1/1*

nd

1/1

3

3

1

1

urine, pig skin

weeks. **IgE+ with pig

work and off work for 2

Butcher. *Serial PEFR at

Mouse hair 1/1**

nd 4/4 nd

Mouse serum

urine; see rats

to rats. *Clinical tests with

exposed and co-sensitized

Laboratory workers co-

dander

*Clinical tests with monkey

professor and his assistant.

Research psychology

urine

*Clinical tests with mink

Worker at a mink farm.

extract

*Clinical tests with venom

diagn. of seasonal asthma.

Frog catcher with prev.

cow, deer

3/4

4/4*

2/2*

0/1*

1/1*

3/4

4/4*

2/2*

1/1*

1/1*

deer, goat, sheep, camel,

*SPT+ with dander of

Farmer raising red deer.

allergens in symptomatics

**SPT and IgE with animal

*SIC or nasal Ch with cow;

1

38/100

storage mites and cereals.

1

100

antigen); sign. higher

BUA (bovine urinary

allergen), 26/51 IgE+ with

BEA (bovine epithelial

diagnosed bovine asthma.

2004

control

Krawczyk-

tamarin)

Pig

38

x**

Adamus et al.

Case

Walusiak,

49*

or SPT+; 30/51 IgE+ with

49/

listed; **49/49 either IgE+

nd

study

nd

*Type of reaction not

nd

parative

–

com-

49/49

et al. 1992

49

series,

Mäntyjärvi

49

Case

Ylönen,

Monkey (Cotton top

Mink (Mustela vison)

Frog (Rana esculenta)

Deer

“

Cow

9781405157209_4_049.qxd 4/1/08 17:12 Page 1029

series

Thompson

reports

Longbottom

Infante-Rivard

et al. 2001

and mouse proteins

and rabbit dander

Gautrin,

animals, urinary rat

case

et al. 1999

203

53/

nd

nd

nd

nd

%

2

d(n)

3/5

3/5*

14/29*

20/32*

n/n

nd

129/373

54/342*

1

l(n)

nd

1

3

i(n)

4/5

%

0/2

2/5

5/5*

nd

nd

n/n

*

34.6

**

15.8

**

48.3

%

nd

nd

nd

2/5

5/5*

16/148

17/32*

n/n

Spec. IgE

**

10.8

%

apprentices. Peak incidents

Animal laboratory

**incidence in 3 years

first 2 years of exposure;

symptoms within the

sensitisation and chest

response relationship with

SPT+; sign. exposure-

urine; 46% of asthmatics

within the cohort; *with

case-referent analysis

Cohort study of 7 years and

Laboratory animal workers.

Rat hair

Rat serum

*with urine; see mice

co-sensitization to mice;

Co-exposure and

Laboratory workers.

in 1 year of employment

sensitized; **incidence

SPT; 2/3 asthmatics

symptomatics underwent

*Only IgE+ subjects and

in their 1 year of work.

Laboratory animal workers

had SPT+ and IgE+

urine; 12/13 asthmatics

allergic symptoms. *With

who previously reported

Laboratory animal workers

Remarks

*incidence in 3– 4 years

cases had asthmatic WRS;

in PC20, only 8/28 OA

SPT+ and 3.2 fold decrease

OA cases: subjects with

througout the first 3 yrs;

nd

nd

26.1

nd

nd

n/n

SPT

beginning, and that of OA

*

26.5

**

24.6

nd

%

Reaction

4 years

99/373

84/342

nd

nd

nd

n/n

SIC

1–2 years after exposure

5.9*

**

10.5

5/5

nd

%

PFT

over 3–

22/373

36/342

5/5

–

3/29

n/n

BHR

of skin reactions are max.

28 (7.5)*

(5.0)**

17/342

5

**

2.0

%

LFT

study

373

342

5

3/148

21/32

n/n

n/n 13/32

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

cohort

spective

Pro-

control

Nested

Cullinan, Cook

et al. 1977

Case

study

tive

Newman Taylor,

Various laboratory

“

“

12 (37.5)

32

Case

Davies,

Rat

2 (1.4)**

%)

studied, n

type

Reference

CAS no.

148

ence,

subjects

Study

[synonyms]

Prospec-

(preval-

ally exposed

Agents

et al. 1983

cases, n

asthma

Allergic Occupation-

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1030

1

1

1

pharmaceutical company.

Employees in a

1/1 1/1

0/1 nd

nd

1/1

0/1*

nd

pharmaceutical company.

Employees in a

report

report

Hernandez

Case report

Vargiu, Vargiu

et al. 1994

Milk proteins (casein,

lactoglobulin)

et al. 1990

Case

Olaguibel,

protein)

et al. 1994

Case

Echechipia

Casein (main milk

Alpha-lactalbumin

Bernaola,

report

et al. 1988

Milk proteins

Case

Armstrong, Neill

africana)

1999

report

Ivory (Loxadonta

Honey

origin Case

1989

pancreas, adrenal

Dittrick et al.

report

Leneutre et al.

(ovaries, testes,

Johnson,

Case

Breton,

Endocrine glands

glands) of bovine

report

Case

Weber 1991

(BSA) powder

Bovine serum albumin Joliat and

ANIMAL PRODUCTS

1

1

1

1

1

1

1

1

1

1

1

1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

–

–

1/1

nd

nd

0/1

nd

0/1

1/1

nd

1/1

1/1

nd

1/1

nd

nd

nd

nd

1/1*

1/1

nd

nd

1/1

1/1

1/1

1/1

1/1*

**

1

1

1

1/1

1/1

1/1

0/1

1/1

1/1

1/1

1/1

1/1

nd

1/1

nd

Cattle farmer

Tannery worker

Conj Ch+

Chocolate candy maker.

and off work for 2 weeks

shop. *Serial FEV1 at work

Worker in an ivory-carving

producing company

Worker in a cereal-

and adrenal glands

powdered bovine testes

Pharmacist. *SIC with

BSA

BSA; SIC+ with aqueous

to lab animals. *IC+ with

exposed and co-sensitized

Laboratory technician co-

sensitized

only; 13/15 asthmatics

dander in symptomatics

**SPT with animal

FEV1/FVC in asthmatics;

1

nd

*Sign. decrease in

1

22/48**

pig, mouse, rat etc.

sectional

1981

animals: dander

extracts of guinea

Cross-

Slovak and Hill

Various laboratory

12/15 asthmatics IgE+

37.7

7/15 asthmatics SPT+ and

49/130*

24/130 45.8

12.8

13/133

17/133*

Rat nd

nd

least one urine extract;

nd

nd

20/130

nd

nd

6/133

x*

nd

Rabbit

32.9

37

*SPT+ and IgE+ with at

48/146

51/138

40/130

10.3

10.9

7/133

15/146

15/138

Mouse

13 (8.9)

12 (8.7) 23/130

146

138 10/133

sectional

et al. 1988

animals (urine)

Guinea pig

Cross-

Venables, Tee

Various laboratory

9781405157209_4_049.qxd 4/1/08 17:12 Page 1031

et al. 1990

ovalbumin,

– nd

1/4

1/1

4/4

nd

n/n

BHR %

1/1*

nd

86

19/

n/n

PFT

22

%

nd

n/n

SIC

report

et al. 1993

1

1

1

1/1

1/1

1/1

1/1

0/1

1/1

nd

1/1

nd

1/1*

1994

Schroeckenstein, Case

paniculata)

“

et al. 1990

Meier-Davis

Jáuregui et al.

(Gypsophila

report

report

Antépara,

Baby’s breath

1

1

1/1

1/1

0/1

nd

nd

1/1

1/1

**

1/1

1

1

d(n)

1/1

1/1***

1/1

1/4

2/4

4/4

2/4

4/4

29/86

n/n

SPT

33.7

%

1/1

1/1***

1/1

2/4

3/4

2/4

2/4

4/4

34/86

n/n

Spec. IgE

39.5

%

of work days and off work;

Greenhouse worker. *PEFR

ovomucoid, ovoalbumin

IgE+ with conalbumin,

at work. Also SPT+ and

worker. *PEFR for 2 weeks

Lysozyme production

and barley

sensitized to wheat, rye

Bakery workers, all

with ≥ 2 egg proteins

asthma plus WRS plus SPT+

physician diagnosis of

concordance between the

defined by dual

SPT, IgE, PFT; asthma cases

asymptomatics underwent

symptomatics and 44

production plant. 44/58

Employees of egg products

Remarks

1

1/1

1/1

Florist

Florist

pollen, stem, petal

***SPT+ and IgE+ with

Case

1

l(n)

**SIC+ with pollen;

report

1

1

2

4

i(n)

SIC reaction

of hippeastrum

et al. 1996

hippeastrum),

1/1

%

hybrid cultivate

Jansen, Visser

Amaryllis (Amaryllis

Case

Case

Bernstein, Kraut

Flowers, ornamental plants

PLANTS (PLANTAE)

Egg lysozyme

1/1 1/1

4/4

nd

%

2/2

1

4/4

–

n/n

LFT

Ovomucoid (Gal d 1) 1

4

30.9

%

Ovalbumin (Gal d 2)

2003

Egg yolk

reports

4

58/188

n/n

n/n

4/4

Quirce et al.

Egg white

Case

14 (7.4)

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

Lysozyme (Gal d 4)

Escudero,

Egg proteins

ovomucoid, egg yolk

Smith, Bernstein

Egg white, lysozyme,

188

%)

studied, n

type

CAS no.

Survey

ence,

subjects

Study

[synonyms]

Egg proteins

(preval-

ally exposed

Reference

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1032

report

Yunginger

report

Case report

Case report

1994

Miesen, van der

Heide et al.

2003

Kiistala,

Mäkinen-

Kiljunen et al.

Bells of Ireland,

pollen of (Molucella

laevis)

Bishop’s weed

(Ammi majus),

Queen Anne’s lace

1/1

7.7

–

48.1

0/1

nd

1/1

nd

1/1

1

1

1

1

1

1

1/1

15/16

1/1

1/1

1/1

1/1

1/1

20.2

1/1

11/14

1/1

1/1

1/1

0/1

1/1

10.6

carnation cultivation.

Employees in indoor

(P. dactylifera) pollen

pollen and date palm

reactivity btw. P. c.

Gardener. Sign. cross-

Florist. Nasal Ch+

*serial PEFR for 1 week

during seasonal exposure;

18% decline of FEV1

Greenhouse employee.

see Sanyak

co-sensitization to Sanyak;

Co-exposure and

medicine pharmacy.

Employee of herbal

Florist

1/1 nd

1/1

nd

1/1

1/1

1/1

report

Crosssectional

Groenewoud,

Jong et al.

Chrysanthemum

(Chrysanthemum)

Case report Case reports

Piirilä, Keskinen

et al. 1994

Bolhaar and van

Ginkel 2000

2002b

Case

et al. 2003

2004

Gutiérrez et al.

González-

2

1

104

1

1

1

8 (7.7)

1

1/2

1/1

8/104

1/1

2/2

–

50/104

1/1

nd

1/1

nd

nd

nd

1/1

nd

nd

nd

1/1*

nd

nd

nd

1/1

nd

nd

2/2

1/1

21/104

1/1

2/2

nd

11/104

nd

Floriculturists

at home and at work

Florist. *Serial PEFR or FEV1

mugwort

Chrysanthemum and

Cross-reaction between

Greenhouse employees.

flower. *Nasal Ch+

and co-sensitized to lime

Cosmetician co-exposed

Flowershop worker

1/1

report

exposed and co-sensitized

Flower cultivator co-

work

Case 1

nd

14*

13/

nd

nd

1/1

1/1

1/1

Fernández,

1

1/1

16

15/

1/1

nd

1/1*

nd

nd

Sáncher-

Rudzki, Rapiejko

Cyclamen pollen

1

15/16

0/1

0/1

1/1

0/1

nd

to Tetranychus urticae

report

Enrique et al.

chamomilla)

“

1

15/16

1/1

0/1

0/1

nd

nd

2000

Case

Cisteró-Bahima,

Camomile (Matricaria

“

“

13

1/1

1/1

1/1

1/1

1/1

at work and 2 weeks off

series

Guerrero,

caryophillus)

16

1

1/1

1/1

1/1

1/1

*Serial PEFR for 2 weeks

Case

Sancher-

Carnation (Dianthus

1

1

1

1

1

et al. 1999

report

et al. 1995

(Phoenix canariensis)

1

1

1

1

Escudero

Case

Blanco, Carrillo

Canari palm pollen

1999

Case

Park, Kim et al.

ternata)

et al. 1982

Case

Twiggs,

Banha (Pinellia

“

9781405157209_4_049.qxd 4/1/08 17:12 Page 1033

van and Dieges

1984

Toorenenbergen

hybride)

Case report

nd

nd

nd

nd

1/1

9/39

Scrophulariaceae

Freesia (Freesia

5/39

Ranunculacea

7/39 7/39

et al. 1998

Gentianaceae

36/39

Liliaceae

Confino-Cohen

Asteraceae

–

12/14

1/1

5/14

Solidago

1

3/13

Saintpaulia

1

3/13

Pelargonium

39/75

11/14

Narcissus

nd

0/13

Matricaria

nd

6/13

Limonium

sectional

4/13

Helianthus

Flowers

6/14

6/14

7/14

1/1

0/1*

n/n

SPT

Gerbera

1

d(n)

7/14

l(n)

Freesia

i(n)

Reaction

6/13

%

Eustoma

nd

nd

1/1

nd

n/n

SIC

Euphorbia

%

5/14

nd

nd

1/1

nd

n/n

PFT

8/14

+*

nd

%

Dianthus

+*

nd

1/1

nd

n/n

BHR

2/14

75

14/14

0/1

%

Chrysanthemum

Cross-

10/14

1/1

nd

n/n

LFT

4/14

Goldberg,

14

1/1

%

Aster

et al. 1998

series

Case

up

follow-

with

report

Case

1/1

n/n

n/n 1/1

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

Asclepias

Alstroemeria

Jong de,

Flowers

Vermeulen

et al. 1999

longiflorum)

Ageratum

Piirilä, Kanerva

Easter Lily (Lilium

1

et al. 1987

pseudonarcissus),

1

1

1

Case

Gonçalo, Freitas

Daffodil (Narcissus

Trumpet narcissus

%)

studied, n

type

report

ence,

subjects

Study

Reference

CAS no.

(preval-

ally exposed

[synonyms]

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

23

13

18

18

92

52

%

1/1

nd

11/11

10/11

1/1

nd

n/n

Spec. IgE %

and stem

Clinical tests with flower

Greenhouse worker.

symptomatics only

individual flowers in

listed; SPT results with

*Individual symptoms not

Rural flower growers.

Flower industry workers

IgE+ and SPT+ with tulip

Floral shop worker. Also

leaf of N.p.

test+ with flower and

with N.p. juice; patch

Flower grower. *SPT

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1034

report Case report Case

Case report

Survey

et al. 1994

Piirilä, Hannu

et al. 1998

Lahti 1986

Rudzki, Rapiejko

et al. 2003

Zee van der, de

Jager et al. 1999

Hyacinth (Hyacinthus

orientalis)

Lily (L. longiforum)

Lime flower

Madagascar jasmine

(Stephanotis

series

Rowe et al.

(Rosa rugosa)

1

1/1

12/70

7/13

6/28

4/8

17.1

21.4

1/1

8/41

7/13

6/28

9/34***

19.5

21.4

26.5

nd

0/1

nd

1/1

1

1/1

1/1

1985

1/1

annuus)

1/1

Employee in the

1

report

1

agricultural department

*Serial PEFR at work and

Floral industry worker.

Dhivert et al.

1/1

(Helianthemum

1/1

at home for 4 weeks

1/1

Case

1/1*

Bousquet,

1/1

Sunflower pollen

0/1

1993

1/1

lavender

1/1

report

Figueroa et al.

tataricum), see

1

Case

Quirce, García-

Statice (Limonium

exposed

exposed and co-sensitized

Caretaker of plants, co-

asthma

18/290 (6.2%) new-onset

aggravated symptoms,

*New-onset and

Rose cultivators.

asthmatics

asthma. *SIC in sensitized

1 with work-aggrav.

of vitamin manufact. plant,

13 symptomatic employees

asthmatics

sensitized; *SIC only in

factory. 6/6 asthmatics

Workers of pharmaceutical

had IgE+

***only symptomatics

cases with asthma;

symptoms” and 4 index

with “airway and skin

4 index cases. **5 subjects

of 5 nurseries including

Greenhouse employees

camomile. *Nasal Ch+

and co-sensitized to

Cosmetician co-exposed

co-sensitized to tulips

Florist co-exposed and

Gardener

home and at work

*Subject 8 months not

0/1*

nd

2

4

1

nd

nd

nd

Gardener. *PEFR or FEV1 at

to Dracaena fragrans.

nd

nd

2/4*

4/4*

1/1

1/1

1/1

0/1

nd

1995

0/1

nd

nd

nd

2/2

nd

nd

1

0/1

Kiljunen et al.

0/1

x

nd

nd

1/3

nd

nd

1/1

0/1

wallisii)

13.1

nd

nd

0/3

nd

nd

1/1

1/1*

report

1/1

38/290*

5/13

–

–

nd

nd

0/1

1/1

Case

23.8

21.4

11.8

1/1

1/1

0/1

0/1

Mäkinen-

1/1

69/290*

9/13

6/28

4/34

1/1

1/1

1/1

–

Kanerva,

1

18 (6.2)

2

6 (21.4)

4

1

1

1/1

1/1

(Spathiphyllum

1

290

13

28

34

1

1

1

1

Spathe flower

et al. 2002

Demir, Karakaya Survey

Case

Kwaselow,

Rose hips

Rose (R. rugosa)

sectional

et al. 1993

somniferum)

1990

Cross-

Moneo, Alday

Poppy (Papaver

floribunda)

report

Case

Piirilä, Keskinen

“

9781405157209_4_049.qxd 4/1/08 17:12 Page 1035

reports

21.4

%

Florist co-exposed and

with Tuliposide-A

work and at home; SPT−

*Serial PEFR or FEV1 at

Gardener and florist.

asthmatics not listed

group; sensitized

workers than in unexposed

lower in highly exposed

LFT parameters significantly

skin) 29/102 (28.4%); **

symptoms (rhinitis, conj.,

factory. *Allergic

Sunflower-processing

Remarks

report

et al. 1998

et al. 1996

Quirce, Tabar

et al. 1996

Lopez-Rubio,

Rodriguez et al.

(Pimpinella anisum)

Artichoke, globe

(Cynara scolymus)

Asparagus

(Asparagus officinalis)

1998

Fraj, Lezaun

Aniseed dust

report

Case

report

Case

report

Case

Case

Grob, Wüthrich

Vegetables, legumes, other plants

“

Survey

reports

Case

1

1

1

1

84

2

1

1

1

6

1

1/1

1/1

1/1

1/1

6/84

1/2

1/1

1/1

–

1/1

17/84

2/2

1/1

0/1

0/1

–

nd

nd

nd

nd

1/1

0/1

1/1

nd

6/9

1/1

nd

nd

nd

1/1*

nd

nd

1/1

nd

1/1

0/1

1/1

nd

6/6*

1/1

nd

1

1

4

1/1

1/1

1/1

1/1

18/84

2/2

1/1

1/1

1/1

1/1

1/1

18//84

2/2

1/1

Ch– with raw asparagus

Harvesting worker. Oral

processing plant

Worker in a vegetable-

2 weeks

work and off work for

Butcher. *Serial PEFR at

umbrella tree

Indoor gardener. See

Ch+

Plant keepers, greenhouse

Vineyard farmers

benjamina.

co-sensitization to Ficus

Co-exposure and

Indoor gardener.

report

1/1

co-sensitized to lily 1

Case 1

report

asthmatics; 9/9 rhinoconj.

21.4

nd

1/1

nd

n/n

Spec. IgE

workers. *SIC in sensitized

2

23.5

%

1987

100

1/1

1/2

24/102

n/n

SPT

Johansson et al.

20.2

1

d(n)

Axelsson,

l(n)

benjamina)

1

1

i(n)

SIC reaction

Weeping fig (Ficus 7.1

%

2001

nd

1/1

nd

n/n

SIC

Brito, Mur et al.

%

(Diplotaxis erucoides)

nd

1/2*

nd

n/n

PFT

Wall rocket pollen

nd

1/1

%

et al. 1998

nd

1/2

nd

n/n

BHR

(Schefflera)

1/1

2/2

%

Grob, Wüthrich

1/1

2/2

x**

n/n

LFT

Umbrella tree

1

2

+*

%

Lahti 1986

1

2

16.6

n/n

n/n 17/102

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

“

Case

Case

sectional

et al. 2002

et al. 1994

102

Cross-

Atis, Tutluoglu

“

Piirilä, Keskinen

%)

studied, n

type

Tulip (Tulipa)

ence,

subjects

Study

Reference

CAS no.

(preval-

ally exposed

[synonyms]

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1036

report Case report

Case report

et al. 1991

Perfetti, Lehrer

et al. 1997

Brempt van der,

Ledent et al.

(Pfaffia paniculata)

Cacao beans

(Theobroma cacao)

Carob bean flour

(Ceratonia siliqua)

report

Case series

et al. 1994a

Panzani,

Johannson et al.

(Ricinus communis)

“

et al. 1982

4u1kin, Vali´c

(Cafe arabica), green

“

“

Survey

Jones, Hughes

Coffee bean dust

series

Kanceljak

et al. 1985

Case

Zuskin,

Survey

report

et al. 1996

intybus)

et al. 1981

Case

Cadot, Kochuyt

Chicory (Cichorium

1986

Case

Merget, Heger

Castor beans

1992

Case

Subiza, Subiza

Brazil ginseng root

9

45

372

1

15

1

1

1

1

4

2 (4.4)

7 (1.9)

1

1

1

1

1

6/9

4/45

7/372*

1/1

+*

1/1

1/1

1/1

1/1

8.9

1.9

9/9

+*

–

1/1

+*

1/1

1/1

1/1

1/1

4/9

nd

x**

nd

nd

0/1

1/1

0/1

nd

nd

nd

nd

nd

nd

1/1

nd

1/1

1/1

nd

x**

x***

nd

nd

nd

1/1

1/1*

nd

4/9

nd

nd

nd

nd

1/1*

1/1

1**

1/

1/1

4

1

1

1

1

6/9*

5/45***

****

93/362

1/1

0/15

1/1

nd

1/1

1/1

12

25.7

nd

nd

39/331

1/1

2/15

1/1

1/1

1/1

1/1

11.8

all SIC+ had SPT+

SPT+ with roasted coffee;

Coffee workers.* IC; 1/9

coffee bag dust

coffee and 18/45 IC with

also 4/5 IC with roasted

over Monday shift; ***IC;

mean decrease in FEF50

rhinitis or conj.; **sign.

Coffee workers. *17/45

collector dust

−0.024 L; ****SPT with

all 66 tested subjects was

mean change in FEV1 for

before and after shift: The

and FEF25–75; ***PFT

declines of FEV1, FEV1/FVC

IgE+ subjects had sign.

roasted coffee exposure,

as compared to those with

exposure and FEV1 decline

between length of

asthma; **sign. association

workers. *New-onset

Coffee-processing plant

Vegetable wholesaler

allergic rhinitis

15 farmers. *Asthma or

fertilizer

extract of castor bean

merchant. *SIC with

Agricultural products

guar gum

and co-sensitized to

factory co-exposed

Employee in a jam

**SIC with cocoa powder

*Serial PEFR and FEV1;

Confectionery worker.

Laboratory worker

9781405157209_4_049.qxd 4/1/08 17:12 Page 1037

et al. 1997

Falleroni, Zeiss

et al. 1981

Subiza, Subiza

et al. 1995

Igea, Fernandez

et al. 1994

(Foeniculum vulgare)

Garlic dust

(Allium sativa)

Grass juice

(Lolium perenne)

Green bean

(Phaseolus

multiflorus)

Schwartz, Jones

Fennel seed

report

Case

report

Case

report

Case

report

Case

report

et al. 1999

tenacissima)

Case

Case

Gil, Hogendjik

Eggplant pollen

report

Baz, Hinojosa

et al. 2000

pepo)

Case

Esparto grass (Stipa

Miralles, Negro

Courgette (Cucurbita

report

Case

report

et al. 1996

(Solanum melongena) et al. 2002

Sastre, Olmo

report

et al. 1996

(Coriandrum sativum)

Case

Lemiere, Malo

Coriander

“, roasted

series

Johansson et al.

1985

Case

Osterman,

1

1

1

1

1

1

1

1

1

22

1

1

1

1

1

1

1

1

1

8

%)

“

ence,

studied, n

type

subjects

Study

Reference

CAS no.

(preval-

ally exposed

[synonyms]

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

20/22

n/n

n/n 11/22

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

%

0/1

0/1

1/1

1/1

0/1

1/1

nd

nd

1/1

nd

n/n

LFT %

1/1

1/1

nd

nd

1/1

nd

nd

1/1

nd

22

14/

n/n

BHR %

nd

nd

nd

nd

nd

nd

nd

nd

1/1*

x*

n/n

PFT %

1/1

1/1

1/1

nd

1/1

nd

nd

1/1

1/1

*

22*

8/

n/n

SIC %

1

1

1

1

6

i(n) 2

l(n)

SIC reaction

1

1

d(n)

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

18/22

n/n

SPT %

1/1

1/1

1/1

1/1

1/1

nd

1/1

1/1

1/1

11/22

n/n

Spec. IgE %

green beans

beans. SPT– with cooked

vapor of boiling green

Homemaker exposed to

Gardener

co-sensitization to onion

Co-exposure and

firm packaging spices.

aggravated asthma in a

Employee with work-

worker

Sausage-processing

SIC+ with A. fumigatus

Aspergillus fumigatus;

and co-sensitized to

Stucco maker, co-exposed

Ch+ with pollen

Agricultural worker. Conj.

Nasal Ch+

vegetable warehouse.

Worker in the fruit and

mace and paprika

mace and paprika. See

Butcher co-exposed to

IgE+ with green coffee

off work; also SPT+ and

*Serial PEFR at work and

Coffee production worker.

sensitized asthmatics

declined; **all SIC+

in subjects with SPT+ sign.

off-work for 1 week: PEFR

*Serial PEFR at work and

Coffee roastery workers.

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1038

Case report

Crosssectional

Parra, Lázaro

et al. 1993

Malo, Cartier

et al. 1990b

Guar gum

(Cypamopsis

22.8

1/1

59/162

1/1

36.4

1/1

3/40

0/1

7.5

Case report

Pepys,

Hutchcroft

Henna (roots of

Lawsonia lnermis)

et al. 1982

sectional

Marshall et al.

1984

(Cephaelis

ipecacuanha and/or

report

1994

pentandra Gaertner)

up

follow-

with

Case

Kern and Kohn

Kapok (Ceiba

Cephaelis acuminata)

Cross-

Luczynska,

report

Case

Ipecacuanha

lupulus)

Hops (Humulus

Newmark 1978

report

et al. 1996

bay leaf, garlic)

Case

Lemière, Cartier

report

Case

(thyme, rosemary,

et al. 1986

Herbs, aromatic

clover, mint etc.)

chaparral, red

reports

Starr, Yunginger Case

Herbal tea (containing Blanc, Trainor

“

report

et al. 1997

(Indigofera argentea)

et al. 1976

Case

Scibilia, Galdi

Henna, black

1

42

1

1

1

2

1

1

1

1

1

1/1

nd

1/1

1/1

1/1

2/2

1/1

–

nd

1/1

1/1

–

2/2

1/1

1/1

nd

nd

0/1

1/1

0/2

0/1

nd

nd

nd

1/1

nd

nd

nd

1/1

40*

11/

1/1

27.5

nd*

nd

nd

1/1

nd

nd

nd

1/1

**

0/5

nd

1/1

nd

nd

1/1

1/1

1/2

0/1

nd

***

2/4

1/1

50

1

2

1

1

1/1

8/162

1/1

30.8

4.9

1/1

11/133

1/1

37.5

8.3

manufact. plant. *BHR in

Employees of a carpet

chard

and co-sensitized to Swiss

of boiling GB, co-exposed

Housewife inhaling vapors

1

1

0/1

12/39

1/1*

1/1

nd

2/2

1/1

0/1

12/32**

nd

1/1

nd

2/2

nd

co-workers

Sewer. *0/9 PFT+

symptomatic

**all IgE+ subjects were

further differentiated);

and chest tightness, not

(rhinitis, conjunctivitis

*Allergic symptoms

Ipecacuanha tablets.

Workers packing

hops flowers

*Scratch test+ with

Brewery worker.

garlic, bay leaf

thyme, bayleaf. IgE+ with

with garlic, rosemary,

Butcher. SIC+ and SPT+

herbal tea

Worker processing

Beauticians

persulfate salts (late)

persulphates. SIC+ with

Hairdresser, co-exposed to

Herbal shop worker

and BHR+

in asthmatics with SPT+

BHR+ subjects; ***SIC only

weeks at work in SPT– and

only; **Serial PEFR for 2

1/1

37/162

1/1

symptomatics or sensitized

1

3 (1.9)

1

9000-30-0

1

162

1

tetragonolobus)

“

9781405157209_4_049.qxd 4/1/08 17:12 Page 1039

%)

studied, n

type

review of cross-

Reference

Bousquet,

Flahault et al.

2006

CAS no.

Latex (Hevea

braziliensis)

“

“

“

“

sectional

Ownby

sectional

Delwiche

series

Binard-van-

et al. 2001

Cangh

Case

Vandenplas,

et al. 1990

Survey

sectional

et al. 1995

Tarlo, Wong

Cross-

Baur, Chen

et al. 1995

Cross-

Vandenplas,

et al. 1996

Cross-

Grzybowski,

studies

sectional

45

69

111

273

741

31

3

3 (2.7)

7 (2.6)

ence,

subjects

Study

[synonyms]

“

(preval-

ally exposed

Systematic 9056

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

36/45

42/69

4/111

5/273

27/741

60.9

3.6

1.8

3.6

43/45

52/68

12/111

25/273

99/741

76.5

10.8

9.2

13.4

x

1/1

nd

12*

1/

nd

45

44/

6/12

nd

12*

12/

nd

%

nd

6/51

nd

nd

nd

n/n

%

45

31/

nd

nd

12*

7/

nd

n/n

%

4

i(n)

l(n)

3

d(n)

42/45

8/65

nd

13/273

nd

n/n

SPT %

10.9

4.8

7.09

n/n

SIC reaction

CP:

%

SIC

2.73

n/n

PFT

OR:

%

BHR

1.55

n/n

n/n

LFT

OR:

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

nd

nd

17/111

nd

65/741

n/n

Spec. IgE

15.3

8.8

6.30

CP:

%

All SIC+ were also SPT+

45 latex-exposed subjects.

clinical tests

asthmatics underwent

BHR+, and PFT+; not all

asthmatic WRS, SPT+,

case; 3 subjects had

Results include 1 index

glove manufacturing plant.

Employees of a surgical

sensitized

IgE+; 3/4 asthmatics

sign. associated with

Hospital staff. WRS

BHR+ and SPT+

2 other subjects had SIC+,

in addition to 5 asthmatics

underwent clinical tests;

*Only sensitized subjects

Hospital employees.

with IgE+

listed; WRS sign. associated

sensitized asthmatics not

symptomatics IgE+;

Registered nurses. 56

with asthma (OR 3.95)

Sensitization sign. assoc.

Health care workers.

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1040

report

Crosssectional

et al. 2002

Crespo,

Rodríguez et al.

(Glycyrrhiza glabra)

Lupine seed flour

(Lupinus albus)

report

Case report Case reports

et al. 1996

Suh, Park et al.

1998

Valdivieso,

Subiza et al.

fragans), nutmeg

Oilseed rape (Brassica

napus spp., oleifera)

Onion

Crosssectional

Groenewoud,

de Jong et al.

Paprika (Capsicum

annum)

1

1/1

1/1

167/472

1/1

4/4

1/1

1/1

3/7

35.4

42.9

1/1

1/1

88/472

1/1

3/4

1/1

1/1

2/7

18.6

28.6

Clinical tests with flower,

Greenhouse worker.

asthmatics

42/472 sensitized

asthmatic symptoms;

MEAN in those with

showed sign. lower PEF

2 weeks in 436 subjects

stem; *PEFR twice/day for

stamen, juice, leaf and/or

Clinical test with pollen,

Greenhouse employees.

seed; IgE– with onion

white and violet onion

seeds. SPT+ with Italian,

Worker packing red onion

3 homemakers and 1 cook

feed industry

Employee in the animal

also paprika and coriander

paprika and coriander. See

Butcher, co-exposed to

laboratories

Employees at legume

nettle, hop, thistle

report

Castro et al.

1997

Jansen,

Vermeulen et al.

1995

maracuja

Pea, perennial

(Lathyrus odoratus),

sweetpea

report

Case

Case

Giavina-Bianchi,

(Passiflora alata),

1985

Passion flower leaves

“

1

1

1

1

1

1

1/1

1/1

1/1

1/1

1/1

1/1

nd

0/1

nd

1/1

1/1

nd

1/1*

nd

nd

nd

1/1

nd

1/1**

1/1

1/1

1/1***

1/1

1/1

IgE+ with pollen

**SPT+ with whole plant;

off work for 4 weeks;

*Serial PEFR at work and

Greenhouse worker.

to Rhamnus purshiana bark

exposed and co-sensitized

Pharmacy worker co-

Employee in a spice factory

mace and coriander

mace and coriander. See

report

1

1

1

1

1

1

co-sensitized to echinacea,

Herbalist, co-exposed and

van and Dieges

14.3

nd

tetragonum)

1/1

nd

nd

1/1

1/2

1/1

1/1

1/7

1/1

Case

nd

nd

x*

nd

nd

nd

nd

nd

1

Toorenenbergen

1/1

nd

nd

nd

1/2

0/1

1/1

1/1

1/1

Paprika (C.

nd

nd

nd

0/1

nd

nd

nd

nd

1/1

Butcher co-exposed to

1/1

1/1

49.4

28.6

1/1

report

1/1

1/1

233/472

1/1

4/4

–

1/1

2/7

1/1

Case 1

1

13.3

14.3

1/1

et al. 1996

1

1

63/472

1/1

1/4

1/1

1/1

1/7

1/1

Sastre, Olmo

report

van and Dieges

42 (8.9)

1

1

1

1

1 (14.3)

1

leaf and stem

Case

Toorenenbergen

472

1

4

1

1

7

1

1984

“

report

Pozo et al. 1995

(Allium cepa)

2002a

Case

Navarro, del

Onion seeds, red

1994

Case

Sastre, Olmo

Mace (Myristicia

2001

Case

Cartier, Malo

Liquorice roots

9781405157209_4_049.qxd 4/1/08 17:12 Page 1041

%) 1

studied, n 1

type Case

Case report

Reference

Cohen, Forse

et al. 1993

Kraut, Peng

et al. 1992

CAS no.

Pectin (carbohydrate

of plant cells)

“

33

reports

Feo, Martinez

et al. 1997

(Crocus sativus)

sectional

Cross-

report

Hamilton

et al. 1996

Case

Vaswani,

1975

Case

Schoenwetter

series

et al. 1987

Busse and

Case

Cartier, Malo

Saffron pollen

“

“

“

5

26/64

40.6

nd

28.6

nd

nd

nd

nd

1/1*

n/n

%

x*

10**

8/

2/2

1/1

nd

n/n

80

%

2

3

2

1

i(n)

l(n)

SIC reaction

3

5

d(n)

28/64**

10/191

2/2

1/1

1/1

n/n

SPT

43.8

5.2

%

nd

24/166

2/2

0/1

nd

n/n

Spec. IgE

6

14.5

%

Employees of a

and BHR+

symptomatics, sensitized,

individuals only; **SIC in

parameter listed for 10

sensitized; individual LFT

only in symptomatics or

subjects; *LFT and BHR

sectional study of 193

of OA integrated in cross-

hospitals. 4 preceding cases

Health personnel of 4

Housewives

pectin-specific IgG

Candymaker. Positive

and at home for 5 weeks

jam. *Serial PEFR at work

Worker manufacturing

Remarks

50

1

3

5

1 (2)

1

2

3/50

1/1

3/3

5/5

6

8/50

1/1

2/3

5/5

16

nd

nd

nd

1/5

1/1*

nd

nd

4/5

nd

nd

nd

nd

1/1*

nd

2/3

5/5

1

2

3/50

1/1

3/3*

4/4

6

3/50

1/1

nd

5/5

subject; 2/2 conj Ch+

SIC in sensitized asthmatic

Saffron workers. *BHR and

P. ingestion

of anaphylaxis after

Nurse with an episode

and with psyllium husk

firm. *3/3 IC+ with plantain

Workers of pharmaceutical

measured

FEV1 could not be

1/5 had to be intubated

Nurses. 4/5 SIC+ and

listed

sensitized asthmatics not

or inhalative Ch+; **IC;

*18/35 either conj., nasal

9.4

70*

20/

%

SIC

pharmaceutical factory.

6/64

20

2/2

1/1

1/1

n/n

PFT

1975

64

2/

%

BHR

Michaelson

Göransson and

65/197

nd

12.2

Survey

“

24/197 10*

8 (4.1)

sectional

197

Cross-

et al. 1990a

reports

Malo, Cartier

0/2

Psyllium

2/2

et al. 1989

2/2

1/1

0/1

n/n

tuberosum)

2

1/1

%

LFT

Quirce, Gómez

2

1/1

1/1

n/n

n/n 1/1

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

Potato (Solanum

Case

1

ence,

subjects

Study

[synonyms]

1

(preval-

ally exposed

Agents

report

cases, n

asthma

Allergic Occupation-

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1042

report

Case report

Case report

1994b

Vandenplas,

Depelchin

et al. 1996

Helin and

Mäkinen-

Kiljunen 1996

batatas)

Sarsaparilla root dust

(family Liliacea,

order Smilax)

Senna (Cassia

angustifolia,

C. acutifolia or

1

1/1

1/1

4.5

1/1

1/1

9.0

1/1

nd

nd

1/1

nd

nd

1/1

1/1

0/1

nd

nd

1/1

nd

nd

nd

1/1

nd

1/1

1/1

1/1

1/1

66.7

1

1

2

1

1

1

1

1/1

1/1

1/1

1/1

1/1

1/1

7.7

1/1

1/1

1/1

1/1

1/1

1/1

mace. IgE+ with curry,

SPT+ with curry, coriander,

Employee in a spice factory.

SPT+ with wheat, rye

diagnosis of wheat allergy.

Baker with previous

manufacturing hair dyes

Worker in a company

Herbal tea worker

to Banha. See Banha

exposed and co-sensitized

medicine pharmacy co-

Employee of herbal

report

Van der Borght

Case report Case reports

Case reports

Survey

Parra, Lázaro

et al. 1993

Cartier and

Malo 1990

Shirai, Sato et al.

1994

Viegi, Paggiaro

et al. 1986

vulgaris L. cycla)

Tea, dust (Camellia

sinensis)

“, green

Tobacco leaf dust

(Nicotiana tabacum)

et al. 1998

Case

Vandenplas,

Swiss chard (Beta

Sunflower seeds

curry

(C. tetragonum),

223

3

3

1

3

1

10/223

3/3

3/3

1/1

20/223

3/3

2/3

1/1

x*

nd

0/3

0/1

nd

3/3

3/3

1/1

nd

nd

2/3*

nd

nd

3/3

2/3

1/1

3

**

14/182

3/3*

0/3

1/1

nd

3/3**

0/2

1/1

asthmatics not listed

women only; sensitized

**SPT in men and younger

men lower than reference;

younger women and in

older women, FEF75–85 in

cigarette factory. *LFT in

Workers of a cigar and

tea; 1/3 oral Ch+; **PK

SIC+ with oolong and black

gallate; 3/3 IC+ and 1/1

with epigallocatechin

factories. *IC; IC and SIC

Employees in green tea

3 weeks

at work and off work for

Teapackers. *Serial PEFR

of boiling Swiss chard

Housewife inhaling vapors

flour

amylase and SIC+ with

Baker. SPT+ with alpha-

between spices detected

1

1

1/1

0/1

1/1

nd

paprika; no cross-reaction

1985

and other spices:

1

1/1

1/1

1/1

1/1

officinale), paprika

report

van and Dieges

(C. sativum)

1

1/1

1/1

1/1

coriander, mace, ginger,

Case

Toorenenbergen

Spices: Coriander

1

1

1

1

ginger (Zingiber

report

et al. 1996

(Sesame indicum)

1

1

1

mace (M. fragrans),

Case

Alday, Curiel

Sesame seeds

C. senna)

Case

Park, Kim et al.

Sanyak (Dioscorea

9781405157209_4_049.qxd 4/1/08 17:12 Page 1043

Moneo et al.

1988

Vanhanen,

Tuomi et al.

2001

derivative (AHGD)

Animal feed

Enzymes

Flours

Heederik et al.

1998

Alpha-Amylase

Wheat flour

Bakery

allergens

flour/other

Bakery confinement/

Houba,

Cartier et al.

wheat gluten

Storage mites

Lachance,

Alkaline hydrolysis

1987

Hinojosa,

seed dust

report

et al. 1991

Voacanga africana

Case

Picón, Carmona

Vetch (Vicia sativa)

sectional

Cross-

sectional

Cross-

report

Case

report

Case

report

Baur 1993

“

Case

report

et al. 1980

sectional

Gravesen 1988

393

140

1

1

1

1

1

1

1

1

1

8 (50)

16

Cross-

Lander and

“

1

%)

studied, n

type

Reference

CAS no.

Case

ence,

subjects

Study

[synonyms]

Gleich, Welsh

(preval-

ally exposed

“

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

29/393

6/140

1/1

1/1

1/1

1/1

1/1

7.4

4.3

68.8

83/393

22/140

1/1

1/1

–

1/1

1/1

–

n/n

n/n 11/16

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

21.1

15.7

%

nd

nd

0/1

0/1

nd

1/1

nd

x**

n/n

LFT %

nd

nd

0/1

nd

1/1

nd

nd

nd

n/n

BHR %

nd

nd

nd

nd

nd

nd

nd

16*

8/

n/n

PFT %

nd

nd

1/1

1/1

1/1

nd

1/1

nd

n/n

SIC %

1

1

1

i(n)

1

l(n)

SIC reaction d(n)

nd

26/140

7/140

10/140

1/1

1/1

1/1

1/1

1/1

nd

n/n

SPT

18.6

5.0

7.1

%

36/346

26/346

9/140

6/140

3/140

1/1

1/1

1/1*

1/1

1/1

nd

n/n

Spec. IgE

10.4

7.5

6.4

4.3

2.1

%

given

asthmatics with IgE+ not

rhinitis were IgE+;

asthmatics and/or with

Bakery workers. 15/91

factory

Workers of animal feed

cereals; SIC– with wheat

with individual extracts of

producing company. SPT–

Employee of a biscuits-

pharmaceutical plant

a chemist in a

husband’s clothing,

exposed through her

Housewife indirectly

Farmer. *PK

Nasal Ch+

Worker of a tabacco plant.

Tobacco manufacturer

and FEV1

change; **sign. lower FVC

sign. greater diurnal

work for at least 1 week:

*PEFR before and after

Workers of tobacco plant.

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1044

1994

Mixed flour

12.8

4.9

44/234*

31/226

18.8

13.7

22/

nd

9.2

81/

nd

nd

32.5 nd

nd

nd

nd

nd

nd

nd

nd

49/226

17/226

x**

58/355

17/355

18/355

76/180

42.2

nd

nd

nd

nd

Crosssectional

Storaas,

Steinsvag et al.

2005

Acarus siro

Lepidoglyphus

7/183 2/183 6/183 1/183 13/183

Rye flour

Barley flour

Oats flour

Alpha-amylase

9/183

16/183

Wheat flour

destructor

Bakery

37/183*

1/259

Aspergillus

15.0

3/259

Baker’s yeast

fumigatus

6/259

Mould mix

28/259

45/259

62/259

46/259

59/259

58/259

99/259

9/259

27/180

249

14/259

183

239

Wheat grain

et al. 1989

Mixed flour

confusum

Tribolium

putrescentiae

Tyrophagus

longior

Tyrophagus

domesticus

Glycyphagus

destructor

Glycyphagus

Acarus siro

Bakery

40/226*

30/234*

11/226

nd

nd

Storage mites

279

226

26.4

nd

17.3

5.1

5.4

27/210

nd

IgE + to either flour or 11.4

WRS and sensitization to

20/183 18/183 14/183 9/183 4/183

1.1 3.3 0.5 7.1

12/183

15/183

3.8

4.9

8.7

20.2

0.04

1.2

2.3

3.5

5.4

10.8

17.4

23.9

17.8

22.8

22.4

38.2

2.2

4.9

7.7

9.8

10.9

6.6

Glycyphagus domesticus,

17.7

a least one storage mite

Bakery workers. *SPT with

exposure group

Bakers. *WRS in main

putrescentiae

and/or Tyrophagus

Lepidoglyphus destructor

*SPT+ with Acarus siro,

11.9

wheat or alpha-amylase;

Sign. correlation between

Bakers and pastry makers.

available

21.7

8.2

workers. *9/34 asthmatics

4.0

Alpha-amylase; **no data

Supermarket bakery

12.8

L. destructor

asthmatics had SPT+ with

exposure intensity; 5

WRS were related to

occup. exposure to flour;

subjects without previous

workers. *New WRS in

Bakery and flour mill

7.5

nd

nd

24/210

63/239

28.6

8 (3.3)

15.5

92/322

9/210

37/239

14.3

5 (2.1)

9 (3.8)*

5 (1.5)

46/322

27/226

Survey

sectional

et al. 1994

Musk, Venables

Cross-

De Zotti, Larese

et al. 2005

239

344

Wheat flour

barley)

Flours (wheat, rye,

Alpha-amylase

Bakery and pastry

Wheat flour

Alpha-amylase

Bakery

destructor

Lepidoglyphus

Survey

sectional

Lowson et al.

Brant, Berriman

Cross-

Cullinan,

Alpha-amylase

Bakery

9781405157209_4_049.qxd 4/1/08 17:12 Page 1045

Baur, Sauer

57 58/103

92/103

32/83

n/n 38.6

%

76

13/

n/n

LFT

17.1

%

76

10/

n/n

BHR

13.2

%

nd

nd

n/n

PFT %

63/

n/n

SIC %

i(n) l(n)

SIC reaction d(n)

10/88

14/88

25/88*

n/n

SPT

261

24

+*

+* nd

115

30/30

–

nd

nd

nd 33/

nd

17/40*

nd

(asthma, rhinitis and /or

Bakers. *Individual WRS

nd 2/2

6/7

of cereal amylase IgE

Bakers. RAST: level

Rye flour

Wheat flour

Bakery

1983

series

4/7

6/7

6/7

5/7

Bakers

30/30 3/7

16/30

SIC+ with rye were IgE+

29/30

4/7

nd

Wheat flou 3/7

nd

Barley alpha-amylase

5/7

nd

with wheat had IgE+; 34/47

companies; 24/29 SIC+

least one extract of 3

of wheat or barley IgE

5

nd

38/69

30/30

7

nd

13/40*

Barley beta-amylase

Case

30

69

47/

symptoms. *SPT+ with at

correlated with level

Block, Tse et al.

30

34

51

33/51

30/30

series

Case

series

Barley flour

et al. 1994

Sandiford, Tee

et al. 2004

A. oryzae

Alpha-amylase of

Bakery

Rye flour

Wheat flour

Bakery

Wheat flour

149/261

84/261

Soybean flour

components

149/261

Rye flour

Bakers with WR respiratory

Bacillus subtilis protease

A. niger

and 3/261 IgE+ with 26/261

Hemicellulase of

13/261

60/261

also 3/261 IgE+ with papain

Case

nd

sensitized

symptomatics not

bakery allergen; 41/142

Bakers. *with at least 1

Remarks

A. niger

Sander, Merget

nd

19.1

19.1

33.7

52.8

55.1

%

conjunctivitis) not listed;

series

Case

22/104

10/81

51/103

64/104

69/104*

17/89

17/89

30/89

47/89

49/89*

n/n

Glucoamylase of

et al. 1989

1.1

19.3

11.4

15.9

28.4

%

Spec. IgE

A. oryzae

Alpha-amylase of

Bakery

11/103

49/104

62/104*

Soy bean flour

89

25/104

94

94

Alpha-amylase

18/

26/

38/104

series

Rye flour

Wheat flour

Bakery

1/88

37.3

%

Rhinitis

Soy bean flour Case

31/83

n/n

Asthma

Work-related symptoms

Evidence (pathological results)

17/88

survey

%)

ence,

(preval-

cases, n

asthma

Allergic

Alpha-amylase

Rye flour

104

89

Com-

Baur, Degens

Bakery parative

studied, n

type

Reference

CAS no.

et al. 1998a

subjects

Study

[synonyms]

Wheat flour

ally exposed

Occupation-

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1046

report

1996

(Fagopyrum

reports

Case reports

Quirce,

Fernández-Nieto

Soybean trypsin

inhibitor

Case series

Quirce, Hinojosa

et al. 2000

African maple

(Triplochiton

“

“

Obeche, Wawa

Whitewood, Samba,

scleroxylon),

reports

Moneo et al.

1984

Case

Hinojosa,

1986

reports

report

et al. 2000

(Makore)

Case

Case

Obata, Dittrick

African cherry wood

Losada et al.

reports

Hinojosa,

Case

et al. 1976

Abiruana

Booth, LeFoldt

Wood dust

et al. 2002b

Case

et al. 1994

Soybean lecithin

Lavaud, Perdu

esculentum)

Case

reports

et al. 2000

Park and Nahm

Case

Quirce, Polo

Buckwheat

Wheat flour

of soybean

Trypsin inhibitor

(Glycine max)

Soybean flour

of A. oryzae

Alpha-amylase

Bakery

2

4

5

1

2

2

2

1

4

2

4

5

2

2

1

4

4

2/2

4/4

5/5

1/1

2/2

2/2

2/2

1/1

4/4

2/2

4/4

5/5

–

1/2

–

2/2

1/1

4/4

1/2

nd

nd

0/1

nd

nd

–

nd

1/4

nd

nd

nd

1/1

nd

2/2

nd

nd

4/4

nd

nd

2/2*

nd

nd

nd

nd

nd

nd

1/1

4/4

3/3

1/1

2/2*

2/2

2/2

1/1

4/4

1

4

3

1*

1

2

2

1

3

1**

1

1

2/2

4/4

5/5

0/1

2/2**

2/2

2/2

1/1

4/4

2/4

4/4

2/4

2/2

4/4

5/5

nd

nd

2/2

2/2*

1/1

4/4

nd

3/3

2/4

tracheotoma

construction worker with

Woodworker and

also Ramin

reactivity with Ramin; see

carpenter. REIA: cross-

1 woodcarver, and 1

co-exposed to Ramin,

2 sawmill workers

had PFT+

who did not undergo SIC

Woodworkers. *2 subjects

extract

dust; **SIC with aqueous

Carpenter. *SIC with wood

scratch test+

extract; **1/1 IC+, 1/1

factory. *SIC with aqueous

Workers of a furniture

soybean flour

SIC+ with wheat flour and

Bakers. Also 2/2 SPT+, IgE+,

with wheat

soybean flour; 1/2 IgE+

bakery dust; 2/2 IgE+ with

1/2 SPT+ with wheat,

soybean dust, flour, pulp;

additionally 2/2 SPT+ with

with soybean flour;

Bakery employees. *IgE+

with wheat flour

maker. Also IgE+ and SPT+

Buckwheat flour noodle

Bakers, confectioners

9781405157209_4_049.qxd 4/1/08 17:12 Page 1047

et al. 2001

africana or Antiaris

report

Case

3

1

3

1

3/3

1/1

1/1

1/1

1/3

1/1

1/1

1/1

–

2/3

0/1

0/1

0/1

0/1

%

3/3

1/1

1/1

1/1

nd

1/2

n/n

%

3/3

1/1*

1/1

nd

nd

nd

n/n

PFT %

3/3

1/1

1/1

1/1

1/1

2/2

n/n

SIC %

1

2

i(n)

2

l(n)

SIC reaction

1

1

1

1

d(n)

nd

1/1**

nd

1/1

1/1*

2/2

n/n

SPT %

nd

1/1

0/1

1/1

1/1

2/2

n/n

Spec. IgE %

*Serial PEFR at work and

Furniture factory worker.

Furniture-maker

Woodworker

Woodshop worker. *IC

Carpenters

Remarks

and Markos

melanoxylon)

Prescott et al.

1996

Innocenti,

Romeo et al.

(Phoebe porosa),

Imbuia

Cabreuva (Myocarpus

fastigiatus Fr. All.)

and Abboud

1976

doPico 1978

Giavina-Bianchi,

Castro et al.

redwood (Sequoia

semperivirens)

“

Cascara sagrada bark

(Rhamnus purshiana)

1997

Chan-Yeung

California

1991

Jeebhay,

Brazilian walnut

1997

Wood-Baker

Blackwood (Acacia

report

Case

report

Case

reports

Case

report

Case

report

Case

reports

Case

1

1

2

1

1

1

1

1/1

1/1

2/2

1/1

1/1

1/1

1/1

2/2

–

1/1

0/1

1/1

1/2

0/1

1/1

1/1

nd

nd

0/1

nd

nd

nd

nd

nd

x*

1/1

1/1

2/2

1/1

1/1

1

1

2

1/1

0/1

0/2

nd

nd

1/1

nd

nd

nd

0/1

to Passiflora alata

exposed and co-sensitized

Pharmacy worker co-

Retired carpenter

maker

Carpenter and furniture-

Parquet floor layer

pattern

no specific work-related

substantial variability, but

PEFR for 2 weeks showed

Joinery worker. *Serial

makers

Cabinet-maker, furniture-

**SPT–, IC+

Rivas,

excelsior)

1

1

1/1

1/2

n/n

BHR

off work for 2 weeks;

Fernández-

Ash (Fraxinus

report

Case

1

1

%

LFT

et al. 1997

Cartier 1989

americana)

report

Case

1

2/2

n/n

n/n 2/2

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

Pérez-Carral

Malo and

Ash (Fraxinus

toxicana)

Higuero, Zabala

Antiaris (Antiaris

et al. 1978

report

Case

reports

et al. 1994

Yunginger

2

Case

Reijula, Kujala

“

Bush,

%)

studied, n

type

Reference

CAS no.

(Microberlinia)

ence,

subjects

Study

[synonyms]

African zebrawood

(preval-

ally exposed

2

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1048

reports

Schlueter et al.

report

Clayton 1983

walnut (Juglans

25/42

25

59.5

–

6/11

3/3

1/1

2/6

16.7

0/42

2/11

2/3

nd

0/5

nd

0 3/

0/4

nd

1/1

nd

1/1

25 1

1

1

1

1

nd

nd

2/3

0/1*

1/6

0/1

8.3

nd

nd

nd

0/1

nd

nd

changes in PEFR at work as

Sawmill employees. *Sign.

workers

Billiard cue manufacturing

Woodworker. *IC

Workers at joinery works

and mahogany

Carpenters. See also oak

symptomatics

1990

echinata or

report Case report

Olaguibel 1989

Pickering,

Batten et al.

excelsa)

“

1972

Case

Azofra and

Iroko (Cholophora

Guilandia echinata)

*SPT and SIC in

Herrmann

(Caesalpina

1

1

36

1

1 (2.8)

1/1

1/1

9/36

–

1/1

6/36

0/1

0/1

nd

nd

1/1

1/1

nd

nd

nd

nd

1/1

1/1

1/1*

1/1

1*

1

Hausen and

Fernambouc

Survey

1987

1/1

1**

1

1/1

0/1

1/12*

0/1*

nd

nd

0/12

nd

1/1***

crassiflora)

0/1

nd

report

–

1**

1/

1

Marcer et al.

1/1

1/1

1/1*

(Diospyros

1

0/1

Case

–

Maestrelli,

1/1

Ebony wood

1

report

1

Case

et al. 1986

extract; **wood dust

Carpenter. *Aqueous

Carpenter

Music instruments-makers.

Carpenter. *IC

with plicatic acid

western red cedar; ***IgE

plicatic acid and with

for 6 weeks; **SIC with

PEFR at work and off work

Sawmill worker. *Serial

but no changes in FEV1

when exposed to Th. occ.

sign. changes in PC20

these agents, 1 subject had

underwent SIC to both of

cedar (late), 1 subject

1/3 SIC+ with western red

plicatic acid (immediate),

BHR+ only, 1/10 SIC+ with

Cartier, Chan

Sawmill workers. *SIC in

“

12*

sectional

et al. 1994b 42

Cross-

Malo, Cartier

(Thuja occidentalis)

nd

11*

11/

nd

nd

nd

nd

Eastern white cedar

baksiana))

jack pine (Pinus

18/

11

11/

nd

1/1

nd

nd

work

3 (7.1)

11/11

2/3

1/1

6/6

–

compared with periods off

42

11

1

1

2/2

(Abies balsamea),

series

et al. 1986

(Black spruce (Picea

11

3

1

6

2

mariana), balsam fir

Case

Malo, Cartier

reports

Coniferous trees

retusa)

Cocabolla (Dalbergia

Case

Case

Bush and

Central American

Eaton 1973

series

1972

olanchana)

Case

Greenberg

(Cedra libani )

1969

Case

Sosman,

Cedar of Lebanon

Cedar

9781405157209_4_049.qxd 4/1/08 17:12 Page 1049

Schlueter et al.

report

Schlueter et al.

report

Skovsted,

Schlünssen

Pine (Pinus sylvestris)

Losada et al.

bancanus)

Vedal et al.

1984

Chan-Yeung,

(Thuja plicata) sectional

Cross-

reports

Cantalupi et al.

1981

Case

report

Case

reports

Case

Paggiaro,

Western red cedar

Tanganyika aningré

Brooks

saponaria)

et al. 1980

Raghuprasad,

Soapbark (Quillaja

1986

Hinojosa,

Ramin (Gonystylus

et al. 2000

Case

1991

riedelianum)

report

Burches et al.

Case

Basomba,

reports

et al. 1995a

(Balfourodendron

Case

Malo, Cartier

1969

Case

Sosman,

Pau marfin

“

Oak (Quercus robur)

report

Sosman,

Mahogany

1969

Case

et al. 2006

pseudoacacia L.)

reports

Case

Kespohl, Merget

Locust wood (Robinia

652

3

1

2

1

1

3

1

1

2

1 (0.2)

2

1

2

1

1

2

1

1

Case

Ordman 1949

Kejaat (Pterocarpus report

%)

studied, n

type

angolensis)

ence,

subjects

Study

Reference

CAS no.

(preval-

ally exposed

[synonyms]

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

27/652

3/3

1/1

2/2

1/1

1/1

3/3

1/1

1/1

2/2

4.1

–

2/3

1/1

2/2

1/1

1/1

1/3

1/1

–

1/2

–

n/n

n/n 1/1

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

%

x*

1/3

0/1

nd

0/1

1/1

1/1

0/1

1/1

1/2

nd

n/n

LFT %

485

94/

2/2

1/1

nd

nd

nd

3/3

nd

nd

1/1

nd

n/n

BHR

19.4

%

nd

nd

nd

nd

1/1*

nd

1/1

nd

nd

nd

nd

n/n

PFT %

nd

2/3

1/1

2/2

nd

1/1

3/3

1/1

1/1

1/1

nd

n/n

SIC %

2

1

2

1

1

1

1

i(n)

l(n)

SIC reaction

2

1

d(n)

1/652

3/3*

nd

2/2

1/1

1/1*

0/3

0/1

0/1

1/1

1/1*

n/n

SPT

0.15

%

nd

0/3

1/1

nd

nd

1/1

nd

nd

nd

2/2

nd

n/n

Spec. IgE %

FEV1/FVC

BHR+ and *sign. lower

prevalence of asthma and

Exposed had sign. higher

Cedar mill workers.

Woodworkers. *IC

and gum tragacanth

reactivity with gum acacia

worker. RAST: cross-

Saponin-production

maple

Co-exposure to African

Sawmill workers.

PEFR at work and off work

Furniture maker. *Serial

Carpenter. *IC

Carpenters

also cedar and mahogany

Lumber-mill worker. See

cedar and oak

Patternmaker. See also

wood and 1 carpenter

exposure incident to locust

Mechanic with 1 high-

extract of Kejaat; *IC

desensitization with an

symptoms after

Improvement of asthmatic

Wood-machinist.

Remarks

9781405157209_4_049.qxd 4/1/08 17:12 Page 1050

9.4 nd

81.8

14

12

8

54

6

59

22/26*

nd

85

nd

nd

examined after 1–9 years:

125 subjects with OA re-

reports with

and Desjardins

1992

report

Batten et al.

1972

Case

Pickering,

up

follow-

Case

Chan-Yeung

1/1

+*

–

+*

0/1

1/1

1/1

nd

nd

4/4

1/1*

nd

nd

nd

nd

4/8

1/1

4/4*

4

3

1

nd

5/8

0/1

nd

62.5

1/1

nd

nd

0/4*

nd

manufacturing factory.

Employees in a food-

Carpenter

IgE with plicatic acid

Sawmill workers. *SIC and

with plicatic acid; 16/16

Woodworkers. 0/22 SPT+

report

Vuyst et al.

cornucopiae

1991

Case

Michils, De

Pleurotus

1 week

at work and off work for

of mushroom. *Serial PEFR

producing a single type

Worker of a factory

mushroom extract

clinical tests with dried

1

4

0/1

0/4

3/22

*Individual WRS not listed;

1

8

1/1

2/4

22

18/

de Paris

series

et al. 1981

1/1

4/4

nd

plicatic acid

work in SIC+; ***SIC with

work: sign. decrease at

off work and for 3 weeks at

**serial PEFR for 2 weeks

returning to work in SIC+;

decline in PC20 after

Sawmill workers. *Sign.

Champignon

Case

Symington, Kerr

Boletus edulis,

1

4

nd

1

immediate, 5 late, 7 dual)

up

18

12/

13.6

SIC+ with plicatic acid (4

12/22

4

follow-

18/22

nd

with

3

***

**

nd

et al. 1973

22

23

14/

23

13/

series

23*

11/

Case

–

nd

Barton

18/23

Chan-Yeung,

14

series

23

Case

*IC in asthmatics

et al. 1990

1320

Côté, Kennedy

Furniture factory workers.

responsiveness

higher bronchial

diagnosis, and also to

symptoms prior to

of both exposure and

related to longer duration

persistence of symptoms

sectional

nd

125

125/

Cross-

nd

nd

et al. 1973

nd

55

125

Ishizaki, Shida

Psalliata hortensis,

Edible mushrooms

124/

55/

27/

remained asthmatic;

3.4

50/125

37/75 no longer exposed

45/1320

125/125

up

22 (1.7)

125

all 50 still exposed and

1320

125

follow-

series

Lam et al. 1982 with

Case

Chan-Yeung,

MUSHROOMS, MOULDS (FUNGI)

“

“

“

“

“

“

9781405157209_4_049.qxd 4/1/08 17:12 Page 1051

reports

Johannson et al.

3.8

1/1

1/1

2/2

n/n

SIC %

1

1

i(n) 3

l(n)

SIC reaction

1

d(n)

1/1

3/3

n/n

SPT

9.6

%

1/1

3/3

n/n

Spec. IgE %

Baker not sensitized to

housewife

exposed, cook, and

company, indirectly

Office worker in food

Remarks

report

Bardana et al.

1996

common red

report

Case

report

1977

Case

Bardana et al.

report

et al. 1997

Klaustermeyer,

Case

Allmers, Huber

Tarlo, Wai et al.

bread mold

1

25/261

nd

nd

261 original and

with rye

most reactive); SIC–

various molds (Aspergillus

Baker. *IC+ with rye and

1

1

1

1

1

1

1/1

1/1

1/1

1/1

–

–

0/1

nd

0/1

1/1

nd

nd

1/1

nd

nd

nd

1/1

1/1

1

1/1

1/1*

1/1

1/1

nd

1/1

with wood

Logging worker. SPT–

and wheat

Alternaria, IC– with rye

Baker. *IC+ with

aspergillosis)

bronchopumonary

Diagnosis: ABPA (allergic

sensitized to other fungi.

co-exposed and co-

Refuse collection worker

3/10 asthmatics had SPT+

had respiratory WRS;

5/76 new employees

78/261 original and

nd

1/1*

biotechnology plant.

nd

1/1

up

nd

nd

76 new employees of

nd

nd

follow-

–

–

with

10/261

1/1

Survey

3 (1.1)

1

Wales 1994

261

1

for 2 weeks

PEFR at work and off work

other baking allergens/

Seaton and

1977

Case

Klaustermeyer,

Chrysonilia sitophila,

Alternaria

Aspergillus fumigatus

Aspergillus niger

Aspergillus mix

Molds, other fungi

report

baking additives; *serial

%

additives; SIC− with other

1/1*

nd

n/n

PFT

et al. 1996

%

Mora-Gonzalez

1/1

1/1

n/n

BHR

dehydrated yeast

%

Hernandez,

1/1

0/2

n/n

LFT

Belchi-

%

cerevisiae, powdered

1/1

1/3

n/n

n/n 3/3

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

Saccharomyces

1

3

3

Case

Torricelli,

Boletus edulis

1

%)

studied, n

type

Reference

CAS no.

Case

ence,

subjects

Study

[synonyms]

Yeast

(preval-

ally exposed

1997

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1052

report

Case report

Garibaldi et al.

1993

Vandenplas,

Caroyer

discoideum, slime

mould

Monascus ruber

report Case report

et al. 1991

Wenzel

Schaubschläger,

Becker et al.

Plasmopara viticola,

pseudo mildew of

grapevine

report

Jáuregui et al.

1996

Case

Gamboa,

6

42.9

6

24/300

8*

nd

nd

1/1

1/1

1/1

1/1

51.8

Bakers and millers. *3 year

13 days off

for 7 working days and

Pharmacist. *Serial PEFR

moulds

not sensitized to other

Coal miner co-exposed but

to Alternaria

exposed and co-sensitized

Greenhouse worker co-

Plywood factory worker

*SIC+ with red rice

(fermented with M. ruber).

handling Chinese red rice

Food manufacturer

Laboratory worker

“

“

5/24

43/83

Employees of a

(immediate)

decrease in FEV1

1/5 oral Ch induced a 30%

asthmatics underwent SIC;

respiratory symp, not all

workers. *SIC in SPT+ with

Pharmaceutical-industry

asthmatics not listed

cases only; 3/5 nasal Ch+

symptoms listed for index

*Individual allergic

20 additional workers.

31.3

4 index cases and

10/24

26/83

survey

nd

14*

6/

products for bakeries:

nd

nd

with

nd

nd

semimanufactured

5/24

nd

cases)

58.8

factory producing

4/4*

47/80

(index

3/4*

30.0

reports

3

24/80

Case

24

83

Belin 1991

Survey

Brisman and

1992

Hinojosa et al.

Losada,

SPT with flour; sensitized

exposure category; 21/300

incidence; sign. increased symptoms in the highest

nd

1/1

1/1

1/1

1/1

1/1

1/1

PR of 3.0 for chest

nd

1/1

1

1

1

1/1

spective)

nd

1/1*

1/1

0/1

1/1

1/1*

nd

(pro-

28.7* nd

1/1

nd

nd

nd

nd

1/1

et al. 2004

86/300

nd

nd

1/1

1/1

1/1

nd

study

12*

1/1

0/1

0/1

0/1

0/1

nd

Cohort

36/300

1/1

1/1

1/1

1/1

1/1

1/1

Nieuwenhuijsen

300

1

1/1

1/1

1/1

1/1

1/1

Brisman,

report

1994

1

1

1

1

1

Aspergillus oryzae

Case

Ng, Tan et al.

1

1

1

1

1

Alpha-amylase of

Enzymes

Chlorella (algae)

MICROSCOPIC ORGANISMS (PROTOCTISTAE)

Rhizopus nigricans

1994

Case

Neurospora sp.

Côté, Chan

et al. 2000

Case

Gottlieb,

Dictyostelium

9781405157209_4_049.qxd 4/1/08 17:12 Page 1053

4

4

Case reports

Quirce,

Fernández-Nieto

“

1998

Quirce, Cuevas

origin

Aspergillus enzymes

1

l(n)

1

1

1

d(n)

2/2

1/1

1/1

4/5

5/5

x*

1/1*

4/4

n/n

100

%

2/2

1/1

1/1

4/4

5/5

3/3**

1/1

4/4

n/n

100

%

Laboratory workers. SPT–

amylase, lipase)

oryzae enzymes (protease,

*SIC with a mix of A.

Pharmaceutical worker.

1 dual)

with wheat (3 immediate,

IgE+ with wheat; 4/5 SIC+

wheat and with rye; 5/5

Bakers. 4/5 SPT+ with

amylase families

**IgE with 13 alpha-

*3/3 SPT+ with cereals;

Wood factory workers.

flour; SIC– with flour

Baker. *IC; also IC+ with

glucoamylase

hemicellulase; see

plant. 2/4 SPT+ with

of an enzyme-processing

3 bakers and 1 employee

Remarks

Cellulase reports

Kanerva et al.

1991

Case

Tarvainen,

1978

4

2

2

2

3/4

2/2

2/4

2/2

0/4

0/1

2/3

nd

2/4

nd

nd

2/2

3/4

2/2

75

4/4

nd

100

see xylanase

co-sensitized to xylanase;

2 were co-exposed and

and 1 process worker.

3 laboratory assistants

pineapple

also after ingestion of

asthmatic symptoms

workers. 1 subject had

Pharmaceutical laboratory 1

1

3

4

1

2

i(n)

reports

nd

1/1*

%

Rodriguez

nd

nd

4/4

5/5

nd

1/1

3/3

n/n

and IgE– with papain

nd

nd

nd

nd

%

Spec. IgE

Case

nd

nd

5/5

nd

nd

nd

n/n

SPT

Galleguillos and

1/2

–

0/5

nd

%

SIC reaction

“

2/2

1/1

5/5

2/3

nd

4/4

n/n

SIC

reports

2

1

5/5

3/3

%

PFT

et al. 1987

2

1

4

5

3

nd

2/4

n/n

BHR

Gailhofer, Teubl

Case

report

Case

5

3

–

%

LFT

comosus

Baur 1981

series

Case

reports

Case

1/1

4/4

n/n

n/n 4/4

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

Bromelain of Ananas

Aspergillus oryzae

Protease of

Aspergillus oryzae

Amylase of

enzymes

Aspergillus oryzae

Aspergillus niger

Cellulase of

A. oryzae

et al. 1992

Moneo et al.

inhibitors of cereal

Alpha-amylase of

López-Rico,

report

et al. 1988

Alpha-amylase

Case

Birnbaum, Latil

1

%)

studied, n

type

Reference

CAS no.

1

ence,

subjects

Study

[synonyms]

“

(preval-

ally exposed

et al. 2002a

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1054

report

1999

Trichoderma viridae

series

Tuomi et al.

Trichoderma reesei

Case reports

Quirce,

Fernández-Nieto

et al. 2002a

Baur 1981

Glucoamylase

(amyloglucosidase)

of Aspergillus niger

Glucose oxidase of

Crosssectional

Case report

Case series

Park and Nahm

1997

Wiessmann and

Baur 1985

Lysozyme (lysozyme

chloride)

Pancreatin (porcine),

containing trypsin,

reports

Wießmann

et al. 1984

and bovine),

containing trypsin,

report

et al. 1982

Merget,

Bergmann

papaya

“

et al. 1995

Case

Baur, König

Papain of Carica

Survey

report

et al. 1997

(alpha-amylase of

porcine pancreatin)

Case

Aiken, Ward

Pancreatin

alpha-amylase

Case

Baur,

Pancreatin (porcine

amylase, lipase

Lactase of Aspergillus

et al. 1997

report

Muir, Verrall

Aspergillus niger

reports

et al. 1978

Aspergillus niger

Case

Case

Pauwels, Devos

Flaviastase of

2000

Case

Vanhanen,

Cellulase of

moniliform

and Fusarium

Case

Kim, Nahm et al.

Cellulase of

1

33

1

4

14

1

207

1

4

3

11

1

1

12 (36.4)

1

3

11

1

9 (4.4)

1

4

3

7*

1

1/1

15/33

1/1

4/4

14/14

1/1

20/207

1/1

4/4

3/3

8/11

1/1

45.5

9.7

1/1

15/33

–

–

–

1/1

33/207

–

4/4

–

8/11

–

45.5

15.9

1/1

nd

0/1

nd

14

11/

nd

nd

nd

2/4

nd

0/11

nd

50

nd

nd

1/1

nd

1/1

x

nd

4/4

nd

7/11

1/1

100

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

8/9

1/1

nd

8/8

1/1

nd

nd

3/3

nd

8/11

1/1

100

5

8

1

3

7

1

3

1

1

1/1

16/33

nd

nd

12/13

1/1

65/207

1/1*

4/4

nd

10/11

1/1

48.5

92.3

31.4

100

1/1

15/33

nd

3/4

3/4

1/1

nd

1/1

4/4

3/3

8/11

1/1

45.5

75

100

the last 8 years

Grocer, not exposed over

SIC

asthmatics did not undergo

symptomatics; 4 sensitized

elevated SPT+ and IgE+ in

Papain workers. Sign.

Laboratory worker

bovine pancreatin

between porcine and

workers. Cross-reactivity

1 nurse, 3 pharmaceutical

undergo SIC had BHR+

3/6 asthmatics who did not

pharmaceutical company.

Employees of

peptidase; see peptidase

co-sensitization to

worker. Co-exposure and

Pharmaceutical industry

asthmatics sensitized

packaging plant. 9

Employees in a lactase-

Pharmaceutical worker. *IC

hemicellulase

plant. 2/4 SPT+ with

of an enzyme-processing

3 bakers and 1 employee

Pharmacy workers

7/10 SPT+ with xylanase

xylanase; 5/10 IgE+ and

SPT+; co-exposure to

factory. *7/8 SIC+ were

Employees of an enzyme

industry

Employee of the textile

9781405157209_4_049.qxd 4/1/08 17:12 Page 1055

2

2

Case

Hartmann,

Walter et al.

Pectinase of

Aspergillus niger

1

1/1

45.5

1/1

2/3

66

1/1

nd

58.3

%

1/1

nd

nd

n/n

%

nd

3/3*

nd

n/n

PFT %

0/1

nd

nd

n/n

SIC % i(n)

l(n)

SIC reaction d(n)

1/1

nd

1/1*

n/n

SPT

17.5

%

1/1

3/3**

2/2

n/n

Spec. IgE

81.8

%

processing. *Serial PEFR

Employees in fruit salad

production. *Scratch test

mechanic) of pectinase

Employees (secretary and

Remarks

of Bacillus subtilis

Protease, alcalase

“

Aspergillus niger

Phytase of

Serratia ssp.

Peptidase of

“

Pepsin

sectional

sectional

Kollmuss et al.

2.1

22.6

–

35/53

–

1/1

7/12

nd

nd

nd

0/1

nd

nd

nd

1/1

1/1

nd

nd

nd

nd

1/1

nd

nd

nd

1/1

1/1

1642

288/

nd

nd

1/1

1/1

x

15/53

9/11

1/1

1/1

28.3

*Incidence: 34/53

Enzyme-packing worker.

(personal communication)

7 asthmatics sensitized

more frequently IgE+;

High-exposed sign.

husbandry company.

Employees of an animal

and 3 internal controls

subjects; SPT+ in 2 external

compared to non-exposed

elevated IgE of exposed

additives factory. Sign.

Workers of animal feed

lysozyme chloride

co-sensitizazion to

Co-exposure and

Pharmaceutical worker.

with perennial asthma

pharmaceutical company

Employee of a

over the 7-year period

aggravated asthma

19/34 had work-

onset respir. symp. and

34/1642*

12/53

5/11

1/1

1/1

symptomatics had new-

34 (2.1)

7 (13.2)

5 (45.5)

1

1/1

up

with

et al. 1977

1642

53

11

1

1

(immediate)

trichinea. Nasal Ch+

Worker checking meat for

follow-

Survey

Juniper, How

2002

Cross-

Baur, Melching-

et al. 1999

Cross-

Kamminga

report

1997

Doekes,

Case

report

Park and Nahm

Case

et al. 1984b

report

Beyer 1997

Cartier, Malo

Case

Drexler and

of pectinase and glucanase

at work and off work for

1

3/3

nd

n/n

BHR

2 weeks; **IgE with blend

reports

Case

%

LFT

Trichoderma

1998

Aspergillus niger

1/2

n/n

n/n 2/2

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

and glucanase of

Sen, Wiley et al.

Pectinase of

3

%)

studied, n

type

Reference

CAS no.

3

ence,

subjects

Study

[synonyms]

1983

(preval-

ally exposed

reports

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1056

nd

10.7

nd

nd

nd

90 4

nd

65.8

31/667

Detergent enzyme production workers.

4.7 **

report

Case series

Saari 1978

Franz,

McMurrain

parasitica, Suparen®

Subtilisin of Bacillus

subtilis

Case reports

Tarvainen,

Kanerva et al.

Xylanase of

Aspergillus niger

report

Case report

Case report

Sastre, Quirce

et al. 1999

Cephalosporin

(Cefadroxil)

Amoxicillin

et al. 1998

et al. 1979

Case

Newman Taylor

report

et al. 1998

Harries,

Case

Baur, Sander

Jiménez, Antón

Alpha-methyldopa

Drugs

CHEMICALS

“

sectional

et al. 1975

inactivated

1991

Cross-

Colten, Polakoff

Trypsin (porcine),

et al. 1971

Case

Niinimäki and

Rennet of Endothica

1

1

1

1

2

14

38

1

1

1

2

4 (28.6)

22

1

1/1

1/1

1/1

1/1

2/2

4/14

25/38

1/1

28.6

65.8

1/1

1/1

1/1

1/1

1/2

1/14

11/38

–

7.1

28.9

0/1

0/1

nd

1/1

0/2

1/4

3/28

nd

0/1

1/1

0/1

nd

2/2

nd

nd

nd

nd

nd

nd

nd

1/2

nd

nd

nd

1/1

1/1

1/1

1/1

nd

**

3/3

9/10

nd

100

1

1

1

3

0/1

0/1

0/1

1/1

2/2

4/14*

25/38*

1/1*

100

28.6

0/1*

1/1

nd

1/1

2/2

nd

nd

nd

100

(immediate)

22% decrease in FEV1

cephalexin induced a

antibiotics; oral Ch with

*IgE- with ß-lactam

Pharmaceutical worker.

33% decrease in FEV1 (late)

worker. Oral Ch induced a

Pharmaceutic laboratory

Chemist of a drug factory

and wheat flour

alpha-amylase of A. oryzae

with cellulase of A. niger,

Baker. Also SPT+, IgE+

cellulase; see cellulase

co-sensitization to

Co-exposure and

Laboratory assistants.

asthmatics only

*Scratch-test; **SIC in 3

manufacturing plant.

Workers of plastic

*IC; 22/25 asthmatics SPT+

detergent-producing plant;

Employees of enzyme

had WRS

not sensitized, 5 of them

cheesemakers exposed but

test; additional 7

Cheesemaker. *Scratch

**incidence in 10 years

all symptomatics IgE+;

for sensitized subjects only;

Subtilisin-A; *WRS listed

Co-exposure and IgE+) to Alkalase from

**

co-sensitization (70/667

0.9

up

6/667

follow-

**

1981

2.4

10 year

16/667

Thomsen et al.

16 [0.24]

with

Høegh-

of Bacillus subtilis

667

Survey

Zachariae,

Protease, esperase

9781405157209_4_049.qxd 4/1/08 17:12 Page 1057

et al. 1995

Coutts, Dally

(ceftazidime)

Cephalosporins

1

1

Case

1/1

53.8

1/1

nd

56.4

nd

nd

nd

0/1

0/1

%

1/1

nd

1/1

1/1

0/1

nd

1/1

n/n

%

nd

nd

nd

nd

nd

nd

nd

n/n

PFT

31.3

%

1/1

nd

1/1

1/1

1/1

1/1

1/1

1/1

2/2

1/1

n/n

SIC %

1

1

1

1

1

2

1*

i(n)

1

1*

l(n)

SIC reaction

1

d(n)

nd

12/39

0/1

0/1

1/1

1/1

1/1

1/1

2/2

nd

n/n

SPT

15–

30.8

%

nd

12/32**

0/1

0/1

0/1

nd

nd

n/n

Spec. IgE

37.5

%

*Allergic symptoms

ipecacuanha tablets.

Workers packing

diagnosed asthma

with previously physician-

Pharmaceutical worker

production

Workers in ciprofloxacin

worker and chemist

Cephalosporin production

a week apart

worker. *SIC twice,

Ceftazidime packaging

Remarks

report

Wittczak et al.

32**

10/

nd

33***

5–33/

33/33 32/33 29/33

Codeine

Morphine

et al. 1992

Hydrocodone

M-6-HS-HSA

5/33

nd

up

nd

Oxycodone

22/39

33/33

21/39*

follow-

21 (53.8)

Dihydrocodeine

sectional

Bernstein

39

12/33

Cross-

Biagini,

2002

Case

Walusiak,

with

Opiate compounds

Mitoxantrone

100

x***

spec. IgE

****no evidence of

conc. in exposed subjects;

epicutaneus threshold

of >10%; ***sign. lower

cross-shift decline in FEV1

work; 5/30 subjects had

compared to 3 days off

PEFR during workweek

onset asthma; **serial

workers. *10/39 new-

Ethic narcotics manufact.

Nurse

symptomatic

**all IgE+ were

not individually listed;

and chest tightness),

1

nd

1/1

1/1

1/1

1/1

1/1

1/1

nd

0/1

n/n

BHR

(rhinitis, conjunctivitis

1

1

1/2

%

LFT

acuminata)

1984

ipecacuanha

sectional

42

1

2

2/2

1/1

n/n

n/n 1/1

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

and/or Cephaelis

Marshall et al.

(Cephaelis

Cross-

report

Luczynska,

Case

et al. 1990

reports

Case

reports

Case

2

%)

studied, n

type

2

ence,

subjects

report

(preval-

Study

cases, n

ally exposed

asthma

Allergic Occupation-

Perrin, Malo

et al. 1996

Broding, Chen

Ipecacuanha

Hydralazine

(intermediate of C.)

Fluochinolon acid

Ciprofloxacin

Cephalexin

derivative of 7ACA

Tosyalate dihydrate

(7ACA)

alosporanic acid

et al. 1981

Stenton, Dennis

Cephalosporin

7-aminoceph-

Reference

CAS no.

[synonyms]

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1058

Case reports

Case report

et al. 1994

Fawcett, Pepys

et al. 1976

Salbutamol

intermediate-glycyl

31.3 3/48 6.3

6/43

nd

nd

0/1

14.0

0/48

nd

2/2*

nd

nd

nd

3/

1/1

1/1

1/1

2/3

1

1

2

1

1

1

2

2

2/3

3 3

25

75

1

3/4

3/4

1/1

x**

0/1

0/1

1/1

0/4

0/1

nd

nd

nd

nd

nd

0/1

worker. Approx. 18

Salbutamol production

3– 4 weeks

work and off work for

workers. *Serial PEFR at

Pharmaceutical process

Pharmaceutical worker

(late)

a 59% decrease in FEV1

benzyl penicillin induced

(late); 1/1 oral Ch with

36% decrease in FEV1

ampicillin induced a

factory. 1/2 oral Ch with

antibiotics-producing

Workers of a penicillin

Pharmaceutical worker

“

Spiramycin

diacetate

methylacetophenone

hydroxy-3′-hydroxy-

Case report

Pepys 1975

Survey

Davies and

Cartier 1988

Malo and

1

1

***

1/1

1/1

0/1

nd

nd

1/1

12*

1/1

nd

pharmaceutical industry

Engineer of a

diagnosed case

cases include 1 previously

not interpretable; ***4 OA

asthmatics; **SPT results

BHR change and/or

subjects with 2.5-fold

*SIC in BHR+ and/or

2. and 3. assessment;

production period during

in the table reflect the

Symptoms and test results

a pharmac. company.

All 51 employees of

was not tested

21/48

1/1

nd

0/1

nd

1/1

other had symptoms but

18.8

–

1/2

1/1

2/4

1/1

tert-butylamino)-4′-

9/48

1/1

2/2

1/1

3/4

1/1

workers were exposed, one

4 (7.8)

1

1

1

4/4

1/1

2-(N-benzyl-N-

51

1

2

1

4

1

compound powder:

Salbutamol base

Piperacillin

Agius, Davison

reports

et al. 1974

report

Case

Davies, Hendrick

Case

report

et al. 1989

et al. 1995

Case

Lagier, Cartier

Moscato, Galdi

Benzyl penicillin

penicillamic acid)

6APA (6-amino

Ampicillin

Penicillin derivate

Penicillamine

9781405157209_4_049.qxd 4/1/08 17:12 Page 1059

Case report

1989

report

1977

Lee, Wang et al.

Case

Menon and Das

reports

1

1

2

1

1

1/1

1/1

1/1

–

1/2

n/n

n/n 2/2

Rhinitis

Asthma %

Work-related symptoms

Evidence (pathological results)

%

1/1

0/1

0/2

n/n

LFT %

1/1

nd

nd

n/n

BHR %

nd

nd

nd

n/n

PFT %

1/1

1/1*

2/2*

n/n

SIC %

1

1

i(n)

1

l(n)

SIC reaction

2

d(n)

nd

1/1**

0/2**

n/n

SPT %

nd

nd

nd

n/n

Spec. IgE %

factory

Worker of a pharmaceutical

**IC

after SIC, IC and oral Ch;

*FEV1 decrease > 20%

pharmaceutical company.

Mechanic of a

(immediate); **patch test

1/2 SIC+ with adipic acid

with spiramycin base (dual),

adipate (dual), 1/1 SIC+

*2/2 SIC+ with spiramycin

Pharmaceutical workers.

Remarks

result; –: negative test result; x: test done, no individual results listed; nasal Ch: nasal challenge test; conj. Ch: conjunctival challenge test; oral Ch: oral challenge test; HR: histamine release test; PK: Prausnitz–Küstner test.

specific IgE antibody measurement; *, **, ***: for details see column“Remarks”; nd: not done; WR: work-related; WRS: work-related symptoms; OA: occupational asthma; OR: adds ratio; sign., significant; P: pathology; restr. = restrictive ventilation pattern; +: positive test

PEFR showing significant change in follow-up pre-, (during) and post shift; SIC: specific inhalative challenge test; i: immediate; d: dual; l: late response type; SPT: significant positive skin prick test result; IC: significant positive intracutaneous test result; IgE: positive result of

n: number of subjects; n/n: number of subjects with work-related symptoms or positive test results/all investigated subjects; LFT: lung function test showing obstructive ventilation pattern; BHR: bronchial hyperresponsiveness; BD: significant bronchodilator effect; PFT: FEV1 or

Tylosin tartrate

Tetracycline

and adipic acid)

et al. 1984

Case

Moscato, Naldi

(spiramycin base

%)

Spiramycin adipate

ence,

studied, n

type

subjects

Study

Reference

CAS no.

(preval-

ally exposed

[synonyms]

cases, n

Occupation-

asthma

Allergic

Agents

Table 49.1 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:12 Page 1060

9781405157209_4_049.qxd 4/1/08 17:12 Page 1061

CHAPTER 49

In order to compare results of different studies and to initiate targeted preventive measures, it is necessary to attribute clinical and diagnostic findings to well-defined occupational agents. Therefore, the Appendix Table available at www.uke. uni-hamburg.de/institute/arbeitsmedizin lists CAS numbers of causative occupational agents. A prerequisite for their listing in this table was that the individual substance was classified as a respiratory irritant according to ACGIH or by Phrase R37 (“irritating to the respiratory system”) or Phrase R42 (“may cause sensitization by inhalation”) according to EC Directives 67/548/EEC, 2001/59/EC/N, and 2004/73/EC, and ILO/CIS 2005.

Asthma-inducing occupational agents A Medline/PubMed search and several reviews (Alberts et al. 1996; Baur et al. 1998b; Brooks et al. 1998; Malo et al. 1998; van Kampen et al. 1998, 2000; McDonald et al. 2000; Health and Safety Executive 2001; Balmes et al. 2003; Sastre & Quirce 2003; Gautrin et al. 2006a; Malo & Chan-Yeung 2006; Malo et al. 2006; Stevens et al. 2006; Proudhon 2008; CSST 2008) show that more than 450 agents have been reported to cause asthma in the workplace. Those regarded as allergens were subdivided into four groups according to their origin: animal, microorganism, plant, chemical (Table 49.1). Airway irritants are listed separately in alphabetical order (Table 49.2). Because individual causative components in several workplaces with elevated asthma prevalence could not be clearly identified, mixtures or confinements are indicated instead of an agent. If different pathomechanisms were involved, i.e. allergenic as well as irritating ones, the predominant one (based on current knowledge) was chosen to include the respective agent in Table 49.1 or 49.2.

Workplaces and confinements with increased frequencies of occupational airway diseases due to special or mixed exposures Animal confinement facilities An increase in asthma prevalence has been repeatedly found in animal confinement facilities (Hoppin et al. 2003, 2004; Monsó et al. 2004; Portengen et al. 2005; Baur & Schneider 2000), e.g., a recent survey comprising 1824 Norwegian farmers reported a significantly elevated asthma prevalence in cattle and pig farmers (Eduard et al. 2004). In the 1990s, occupational asthma in Finnish farmers was reported to be predominantly due to sensitization to cow allergens. Working in swine confinements (Schwartz et al. 1995b; Cormier et al. 1996; Vogelzang et al. 1998; Radon et al. 2002; Dosman et al. 2004), poultry confinements (Danuser et al. 1988, 2001), poultry slaughterhouses (Perfetti et al. 1997a; Borghetti et al. 2002), or contact with raw poultry (Schwartz 1994) also caused lung function declines and occupational asthma. Recent publications indicate that endotoxins are the predominant cause of obstructive airway diseases in poultry and swine confinement workers (Vogelzang et al. 1998; Hagmar et al. 1990).

Airborne Allergens and Irritants in the Workplace

Bakeries and confectioneries Flour dust is the main cause of baker’s asthma (Musk et al. 1989; Cullinan et al. 1994; Baur et al. 1998a,b; Heederik & Houba 2001; Nieuwenhuijsen et al. 2006). Another important bakery allergen is fungal α-amylase, which is frequently used as a baking additive (Baur et al. 1994; Houba et al. 1996). Further bakery allergens include eggs (also relevant in other food processing facilities; Edwards et al. 1983; Smith et al. 1990; Blanco Carmona et al. 1992), baker’s yeast Saccharomyces cerevisiae (Houba et al. 1996), storage mites (Storaas et al. 2005), and fungi such as Aspergillus and Alternaria spp. (Klaustermeyer et al. 1977). However, it should be mentioned that in a significant number of asthmatic bakers causative agents have not been identified yet (Baur et al. 1998a; Brisman et al. 2004). It can be assumed that irritant gases such as NOx are also important.

Floricultures, greenhouses A variety of fresh or dry flowers and nonflowering green plants elicit dermatitis, rhinitis, and/or bronchial asthma. They include sweet pea (Lathyrus odoratus) (Jansen et al. 1995), baby’s breath (Gypsophila paniculata) (Antepara et al. 1994), freesia (Freesia hybride), paprika (Capsicum annuum) (van Toorenenbergen and Dieges 1984), amaryllis (Amaryllis hippeastrum) (Jansen et al. 1996), spathe flowers (Spathiphyllum wallisii) (Kanerva et al. 1995a), hyacinth (Hyacinthus orientalis) (Piirila et al. 1998), narcissus (Narcissus pseudonarcissus) (Concalo et al. 1987), German statice (Limonium tataricum) (Quirce et al. 1993), G. paniculata (Twiggs et al. 1982), Carthamus tinctorius and Achillea millefolium (Compés et al. 2002), weeping fig (Ficus benjamina) (Axelsson et al. 1985, 1987), carnation (Dianthus caryophyllus) (Sanchez-Guerrero et al. 1999), Easter lily (Lilium longiflorum) (Vidal & Polo 1998; Piirila et al. 1999), mimosa pollen (Acacia floribunda), Compositae such as chamomile (Matricaria chamomilla), Chrysanthemum spp., and Solidago virgaurea (de Jong et al. 1998), sunflower (Helianthus annuus) (Bousquet et al. 1985), Chrysanthemum leucanthemum, Solidago canadensis, bell pepper, statice (Limonium sinuatum) (Ueda et al. 1992), eggplant (Solanum melongena) (Gil et al. 2002), Tetranychus urticae (Navarro et al. 2000), and various decorative flowers (Piirila et al. 1994). A further causative allergen source is the red spider mite (Tetranychus urticae). The predatory mites Amblyseius cucumeris (Groenewoud et al. 2002c), Phytoseiulus persimilis, and Hypoaspis mites (Johansson et al. 2003) have also been reported to cause bronchial asthma among horticulturists working in greenhouses. Furthermore, high indoor temperatures and humidity in greenhouse facilities may result in intensive mold growing, particularly of Cladosporium herbarum, Penicillium, Aspergillus, and Alternaria spp., which have been shown to be associated with an increased asthma prevalence (Monsó et al. 2004).

Grain dust Grain elevator, dock and animal feed industry workers are frequently exposed to high concentrations of grain dust;

1061

et al. 1988

1 1/1

1/1

1/1

1/1

15/51

9/20

1/1

1/1

4/4

29.4

1/1

1/1

+

1/1

x**

4/51

2/20

x*

1/1

nd

n/n

LFT

+

+

+

+

+) (+

one case:

7.8

%

1/1

nd

1/1

nd

9/24

5/20

1/1

1/1

4/4

n/n

BHR

37.5

%

nd

nd

nd

nd

nd

1/4

x*

nd

nd

n/n

PFT %

nd

nd

1/1

nd

nd

nd

nd

nd

nd

n/n

SIC %

1

i(n) l(n)

d(n)

SIC reaction

Profession not mentioned. Co-

pre-existing asthma

1 hour. *Severe deterioration of

Pool cleaner; exposure to agent for

Canning factory worker

**reversible FVC and FEV1 decline

interstitial pneumonitis (P+);

massive spill.*Development of

Maintenance fitter exposed to

Asthmatic symptoms dose-related

Hospital employees exposed to spill.

laboratory

20 workers in a mineral analysis

listed

*Individual data for LFT/PFT not

Profession not mentioned.

fumes

Welder. Co-exposure to welding

fumes of waste acid drums

waste investigators exposed to

1 fire inspector and 3 hazardous

Remarks

diseases

Burge, 1993 (SHIELD)

statistics

Occupational

Gannon and

Acid fluxes

Case report

Boulet, 1988

“, sulphuric 7664-93-9

4

1

4

1

4/4

1/1

+

nd

0/1

nd

x

1/1

nd

x

nd

1989–1991

4/500 occup. asthma cases in

examination 2 yrs later

Cleaner. Normal LFT and SIC- on

after exposure > 2 years

inhalation; duration of asthma

Respir. symptoms after 5 min.

1

1*

1

1*

4 (7.8)

4 (20.0)

1

1

4

%

exposure to sodium hypochlorite.

Case report

1

1

1

51

20

1

1

4

n/n

1994

Deschamps.

“, “

Case report

Case report

Case report

Survey

Survey

Case report

Case report

Case reports

%)

Soler et al.

Boulet, 1988

et al. 1994

Kivity, Fireman

“, hydrochloric 7647-01-0

“, “

Rajan and

“, “

Davies, 1989

Kern, 1991

“, glacial acetic 64-19-7

perchloric, sulphuric)

Musk, Peach

hydrofluoric, nitric,

Broder, 1989

Tarlo and

et al. 1985

Brooks, Weiss

et al. 1994

Kipen, Blume

“, “ (hydrochloric,

“, various

“, “

“, not specified

Acids

Study type

n

Reference

CAS No.

Asthma

or at least

(prevalence,

cases, n

subjects studied,

cases: +

asthma

Agents [synonyms]

RADS all

Irritant

exposed

Work-related symptoms

Evidence (pathological results)

Occupationally

Table 49.2 Irritants reported to cause occupational asthma.

9781405157209_4_049.qxd 4/1/08 17:13 Page 1062

nd nd

1

6

3

2/469 asthma claims between mid-

based case

1

2

Solderer of electronic industry,

(physician-diagnosed). OR 1.8, sign.

15/251 occupational asthma in 1996

Door factory worker

planes

Exposure while making miniature

industry

Forker in loudspeaker production

glue. *Individual data not listed

“

“, methyl metacrylate 80-62-6

“, diacrylate

et al. 1986

Bainbridge

Pickering,

1985

Davison et al.

Lozewicz,

1999

Cartier et al.

Weytjens,

Case report

Case report

Case report

Case reports

1

1

1

4

1

1

1

2

1/1

1/1

1/1

2/4

1/1

0/1

nd

0/1

nd

nd

0/1

0/1

x*

0/1

1/1

nd

nd

nd

nd

nd

nd

1/1

1/1

1/1

2/4

1/1

1/1

1

1

1

1

1

2

1

2 assembly operators in weather

Factory workers: 1 working with

Worker of instrument-manufacture

control model air planes

representative building remote

cement.

Theatre sister handling bone

Dental assistant

rhinitis

Auto body shop worker. Preceding

not listed

with dentin primer. *Individual data

producing dental fillings, 1 working

Savonius,

“, “

1

0/1

2/2

glue, 1 in manufacture of earplugs, 1

1985

137-05-3

1

1/1

2/2

1993

Davison et al.

[methylcyanoacrylate]

Case reports

1

1/2

Keskinen et al.

Lozewicz,

1

0/2

Accountant and computer

et al. 1985

Case report

2/2

Kopp, McKay

“, mecrilate

“

2

strips and rubber-processing factory

2

et al. 2001

Case reports

lampshades

1

1

1

1

Factory employees working with

Quirce, Baeza

3/3

nd

1/1

1/1

1/1

10/11

“

nd

nd

1/1

nd

1/1

nd

2 factory workers assembling

0/3

nd

nd

nd

0/1

x*

1985

nd

nd

1/1

0/1

nd

nd

7085-85-0

3/3

15

1/1

1/1

1/1

10/11

Davison et al.

3

15

1

1

1

10

Lozewicz, 3

15

1

1

1

11

[ethylcyanoacrylate]

Case reports

referent study

Population

et al. 1999)

Case report

Case report

Case report

Case reports

(Toren, Järvholm

et al. 1994)

(Chan, Cheong

2005)

Lemière et al.

(Yacoub,

1985)

Davison et al.

(Lozewicz,

Keskinen 1993)

(Savonius,

and/or BD+

x*

retrospective Review; *2/2 BHR+

nd

(WCB), survey

+

1984 and mid-1988, identified by

2/2

statistics

2

diseases

et al. 1999

2

Occupational

Chatkin, Tarlo

“, ethyl 2-cyanoacrylate

“, “ [loctite®]

“, cyanoacrylate glue

“, “

“, “

“, alkyl cyanoacrylates

“

Acrylates

9781405157209_4_049.qxd 4/1/08 17:13 Page 1063

5.6

+

x**

% x**

n/n

BHR % nd

n/n

PFT % 1/1*

n/n

SIC % 1

1

i(n) l(n)

1

d(n)

SIC reaction

Mechanic in paper mill. *SIC with

Remarks

“, fumes

“

“

“

Case reports

Bernstein and

nd

nd

nd

nd

nd

Solderer of industrial butter plant.

exposure

pulmonary distress after massive

Pulmonary edema with acute

Profession not mentioned.

for 12–32 months

respiratory symptoms persisted

accidental exposure, 4/4 P+,

Profession not mentioned. Massive

and at > 50 mg/m3·yrs (28/73)

reduced in symptomatic nonsmokers

Ammonia workers. *FEV1 sign.

over 12 years

persisting severe airflow obstruction

nd

nd

nd

nd

Massive accidental exposure; P+;

1/1

nd

3/3

nd

follow-up

+

1/1

1/4

x*

with 12 year

1/1

1/1

4/4

33/73

Case report

1

1

4

33 (45.2)

Chemical plant workers. co-

et al. 1992

1

1

4

73

nd

Cable jointers

Leduc, Gris

et al. 1983

Flury, Dines Case report

sectional

et al. 2001

Bernstein, 1989

Cross-

Ali, Ahmed

+

listed

+

asthma; **for LFT no individual data

(SENSOR)

45.2

*6/106 physician-diagnosed occup.

Risks

Ammonia 7664-41-7

(1,5-ca]pyrimidine-2-sulfonamide).

3/6

2

Occupational

6/6

3/3

5-methyl-[1,2,4]triazolol

x**

nd

System of

6/106

nd

exposure to N-(2,6-difluorophenyl)-

6* (5,6)

2/3

indentified by

106

3/3

case series

3

Survey of

3

et al. 2004

Case reports

after SIC

Hnizdo, Sylvain

Cleaner. Subfebrile temperature 7h

Head-on motor vehicle accident

1,2,4-triazole 16691-43-3

1/1

nd

3-Amino-5-mercapto-

1/1

nd

Pickering, 1972

0/1

1/1

Pepys and

nd

nd

111-41-1

1/1

1/1

Amino-ethyl-ethanolamine

1

1

Keskinen, 1994

1

1

Savonius, Case report

Case report

[2-ethanolamine]

2005

Masci et al.

Hambrook,

individual data not listed

(4%), ethanol (10%); **for LFT/BHR

bronze powder (25%), white spirit

2-Aminoethanol

Airbag content

1/1

n/n

LFT

hydroxy-propanolic cid (30%),

1

+) (+

one case:

ink containing acrylic acid (30%),

1

%

1993

Case report

Savonius,

Acrylic acid

n/n

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Keskinen et al.

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1064

Gelfand, 1963

Ammonium thioglycolate

“,”

“, phthalic anhydride 85-44-9

“, maleic anhydride 108-31-6

“, himic anhydride 2746-19-2

“, dioctyl phthalate 117-81-7

“,”

“, various

nd

1/1

1

1

1

Chemical workers. *8/18 dyspnoeic

accidental exposure

manufacturing (Pancoxin). Massive

Worker of poultry-food additive

type listed

diamine. *No individual reaction

monoethanolamine and ethylene

culture industry. Co-exposure to

14 subjects exposed in the beauty

with soldering flux (2 immediate)

(see also zinc chloride). Also 2/2 SIC+

fluxes. Co-exposure to zinc chloride

man; use of soft corrosive soldering

1 tin maker, 1 car radiator repair

3/20

1/1

14/110

nd

1/1

nd

nd

nd

nd

1/1*

nd

nd

2/8*

Chemical plant workers. 3/20

*PFT+: immediate

Bottle stopper production worker.

listed; 16/109 specific IgE+

*Individual reaction type of SIC not

Employees of epoxy resin plant.

1/1

0/1

1/1

nd

1/1

Assistant technician in chemical

8/55

7/36

nd

2/2

2

survey

Welinder et al.

1988

Comparative

Nielsen, 60

5* (8.3)

5/60

nd

nd

nd

nd

4/60 spec. IgE+ exposure-related

bronchitis (6 heavily exposed);

exposed group; 7/60 chronic

6.6 mg/m3. *All cases in heavily

Average conc. 0.4 mg/m3, peaks

and unsaturated polyester resins.

Workers of plants producing alkyde

skin scratch test +; 4/54 specific IgE+

Average conc. 3–13 mg/m3. 3/37

21/118

and/or unsaturated polyester resins.

21 (17.8)

1986

118

Nielsen et al.

Survey

Workers of plants producing alkyde

1

plant

1

Wernfors,

Case report

et al. 1991

Lee, Wang

7/20 rhinitis

3* (15.0)

1

2 (1.8)

wheezing and specific IgE+;

20

1

110

1987

Survey

Case report

Survey

Bernstein et al.

Rosenman,

et al. 1999

Cipolla, Belisario

et al. 1994

Drexler, Weber

IgE+

nd

nd

10/10*

1/1

not listed

1993–1996. *Individual data

7/430 new-onset asthma in

ventilation pattern; 15/92 specific

4/90

nd

nd

2/2

nd

cases

11/90

0/1

nd

2/2

nd

subjects with sign. obstructive

+

nd

2/2

nd

follow-up of

18/92

1/1

14/14

2/2

x*

sectional with

8* (8.7)

1

10

2

(+)

Cross-

92

1

14

2

7/7

et al. 1995

Case report

Case reports

Case report

survey

7

Baur, Czuppon

Freedman, 1976

137-88-2

Anhydrides

Greene and

Amprolium hydrochloride

5421-46-5

et al. 1989

(triple salt) 12125-02-9

statistics

2001

Weir, Robertson

diseases

Harrison et al. (SENSOR),

Occupational

Reinisch,

Ammonium chloride

“

9781405157209_4_049.qxd 4/1/08 17:13 Page 1065

up

Survey with follow-up

Topping et al.

1987

Venables,

Topping et al.

follow-up

Shaughnessy

et al. 1999

anhydride [trimellitic anhydride]

552-30-7

Survey with

Grammer,

4-tricarboxylic acid 1,2-

1978

Babaszak et al.

Schlueter,

“, benzene-1, 2,

“,”

Case series

with follow-

Venables,

“,”

1985

Case series

et al. 1983

Case series

anhydride 117-08-8

“,”

2.7

+

0/7

nd

0/1

2/5

n/n

LFT %

nd

nd

1/1

nd

n/n

BHR %

2/4*

nd

nd

nd

n/n

PFT %

nd

1/1

nd

2/5

n/n

SIC %

1

1

2

i(n) l(n)

2

2

d(n)

SIC reaction

Employees working with epoxy

Chemical foreman. SPT+; spec. IgE+

1,5 years asymptomatic and BHR−

spill. At follow-up after about

Tanker driver exposed to massive

Workers of plastic and paint industry

Remarks

286

5

330

7

7

14* (4.9)

4

9* (2.7)

7

4

14*/286

5/5

9*/330

7/7

7/7

4.9

x**

3/5

nd

0/7

0/7

nd

1/1

nd

5/5

nd

nd

nd

nd

nd

nd

nd

3/5

nd

nd

4/4

2

exposure-related

specific IgE+; IgG sign. disease- and

individual data not listed;18/286

symptoms within 3 years; **for LFT

developed TMA-related respiratory

< 0.00045–1.7 mg/m3.*14/286

industry. Average conc.

Workers of TMA manufacturing

5 plastic industry workers

smoking

to IgE+; 24/300 IgE+, related to

related chest symptoms, not related

Factory workers.*9/396 work-

avoidance

symptoms over 4 years in spite of

IgE+ and SPT+; persistent asthmatic

systems. 4 yrs follow-up; 7/7 specific

Factory workers with epoxy resin

systems. 7/7 specific IgE+

Factory workers with epoxy resin

with FEV1 of 18% decline)

with FEV1 of 15% decline, b.1 late

mixing adhesives (a.1 immediate

a.) already mixed and b.) while

2/7

1/1

1/1

+) (+

one case:

resins. *PFT done while exposed to

2

1

1

5/5

%

(collegues)

7

1

1

2

5

n/n

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

with survey

Case report

Case report

Case report

Howe, Venables

Meadway, 1980

1976

Bernstein et al.

Maccia,

Pahulycz, 1993

Frans and

et al. 1977

“, tetrachlorophthalic

89-32-7

“, pyromellitic dianhydride

“,”

“,”

Case reports

Fawcett,

“,”

Newman Taylor

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1066

2/9

nd

nd

nd

nd

nd

7/9

1/1

1/1

1

1

5

1

1

5 parquet varnishers, 1 spray painter,

Maintenance fitter

3/14 specific IgE+

diseases; 6/14 irritant syndrome;

late-onset asthma; *2/14 both

asthma/rhinitis (immunol.), *4/14

Chemical plant workers. 2/14

28* (18.5)

28/ 151

0/ 28

13/ 28

0/ 11 nd

1

1

1

Profession not mentioned. Average

4/7 SIC+ with rhinitis

Accidental release of fire

single hot tube bathing for 5–10 min.

Non-occupational exposure during

tested 6 years after accident

metasilicate, chloride 18%. Patient

with low-density phosphate, sodium

Acute accidental exposure to fumes,

Profession not mentioned

Worker of chemical factory

manufacturing

Worker in cleaning products

nurses

Medical, surgical and paediatric

Profession not mentioned

(Halon 1301) 75-63-8

Case report

1

1

1/1

+

1/1

1/1

nd

nd

> 7 years; BD+

Obstructive lung patterns for

to a fire extinguishing system).

caused by leak of a tank (belonging

Accidental exposure to fumes,

de la Hoz, 1999

1

Bromotrifluoromethane

1

3

1

subjects individual data not listed

Workers of plastic industry. *For 2/4

with aggravated asthma

nd

nd

nd

nd

1/1

1/1

3/3

2/2

2/2

extinguisher content. 1/4 subjects

nd

nd

nd

1/1

nd

nd

3/3

nd

nd

2004

4/4

2/2

1/1

nd

0/1

nd

2/3

2/2

nd

(Halon 1211) 353-59-3

4/4

nd

+

+

1/1

+

1/1

0/1

nd

nd

2/2

1/2*

Laurence et al.

2/4

2/2

1/1

1/1

1/1

1/1

3/3

2/2

4/4

difluoromethane

3

2/2

1

1

1

1

3

2

4

Matrat, 4

2

1

1

1

3

2

4

Bromochloro-

Case reports

Case reports

Burns and

Bromine, hydrobromic acid

Linden, 1997

Case report

Case report

Boulet, 1988

et al. 1994

Kipen, Blume

Case report

Case report

Case report

Case reports

Case reports

Bleaching agent (fumes)

Bisulfite, SO2 SO2: 7446-09-5

Omodeo et al.

1997

Moscato,

3-one (fumes) 2634-33-5

1994

Stauder et al.

Bernstein,

2000

1, 2-Benzisothiazoline-

“

Kopferschmitt-

Kubler et al.

Purohit,

(fumes) 8001-54-5

et al. 1985

Malo, Pineau

1989

Grange et al.

Normand,

Benzalkonium chloride

“

“

16 late, 6 dual

conc. 2–5 mg/m3. *Asthma type at

151

onset of symptoms: 6 immediate,

Survey

123-77-3

Slovak, 1981

[azodicarbonamide]

Azobisformamide

also dermatitis. 4/7 SIC+ also SPT+;

nd

1/1

nd

2 painters, 1 sales agent. 3 subjects

7/9

1/1

14/14

1995

7

1

6*

Keskinen et al.

9

1

14

Kanerva, Case reports

Case report

Case series

(dust) 64265-57-2

1977

Newman Taylor,

Fawcett,

et al. 1977

Zeiss, Patterson

Aziridine, polyfunctional

“,”

“,”

9781405157209_4_049.qxd 4/1/08 17:13 Page 1067

Case reports

Tarlo and

et al. 1993

thiocarboximide fungicide)

sectional

Crosssectional study

et al. 1996

Mwaiselage,

Bråtveit et al.

2005

“

Cross-

Yang, Huang

“

sectional

Kalacic, 1973

“

Cross-

sectional

Petersen et al.

chromate)

1988

Cross-

Abrons,

Cement dust (see also

2425-06-1

Royce, Wald

Captafol (chlorinated

Case report

sectional

et al. 1988

Broder, 1989

Cross-

Davison, Fayers

Cadmium (fumes)

Calcium oxide 1305-78-8

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

117

412

847

2738

1

2

101

22 (18.8)

36 (8.7)

95 (11.2)

148* (5.4)

1

2

33* (32.7)

22/117

36/412

95

148

1/1

2/2

33/101

18.8

8.7

11.2

5.4

32.7

%)

n

+) (+

one case:

(prevalence,

studied, %

or at least

cases, n

subjects n/n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

22/117

x*

nd

nd

0/1

18.8

nd

nd

nd

nd

1/1

2/2

nd

42.8

33/77

x*

n/n

BHR %

n/n

LFT %

nd

nd

nd

nd

nd

nd

nd

n/n

PFT %

nd

nd

nd

nd

1/1

nd

nd

n/n

SIC %

i(n) l(n)

1

d(n)

SIC reaction

exposure

sign. related to cumulative dust

work-related shortness of breath

vs. 4.8% of controls), COPD and

controls. 22/117 COPD (18.9%

117 cement workers vs. 105

reduced FVC, FEV1, FEF50, FEF75

vs. 6.2% of controls); *LFT sign.

wheezing also sign. Increased (7.6

increased (8.7 vs. 7.2% of controls);

179 controls. Dyspnea sign.

412 portland cement workers vs.

questionnaire

of bias because study based only on

pronounced in smokers); *high risk

(7.4% vs. 3.7% of controls, more

controls); wheezing sign. increased

increased (11.2% vs. 4.3% of

Cement workers. COPD sign.

lung function changes

(4.1% vs. 3% of controls); no sign.

controls); COPD also increased

increased (5.4% vs. 2.7% of

Cement workers. *Dyspnea sign.

Chemical worker

not listed

fumes;*individual data for LFT/PFT

3 years; 1/2 co-exposed to welding

asthmatic symptoms for 1.5 to

Profession not mentioned. Persistent

in 14/75

chest X-ray indicate emphysema

dose-related; lung function and

30% below predicted), cumulative

or FEV1/VC sign. reduced (average

Cadmium workers. *33/101 FEV1

Remarks

9781405157209_4_049.qxd 4/1/08 17:13 Page 1068

et al. 1979

Feinberg and

7080-50-4

“

“

Chlorine 7782-50-5

Chlorhexidine 55-56-1

“

(+)*

0/1

x

nd

x

31.4

1/1

nd

nd

nd

56.9

1/1**

nd

nd

nd

1/1*

nd

nd

nd

1

1

1

Floorer. *SIC with potassium

(20.9% vs. 4.5%)

resp. symptoms: dyspnea 14/67

3%); 9/67 bronchitis; other chronic

*4/67 asthma symptoms (6% vs.

67 cement workers vs. 134 controls.

Asthma sign. exposure level-related

of controls

because inadequate description

bronchitis; *high risk of bias,

8.5% of controls); 14/53 chronic

Asthmatic symptoms (32.1% vs.

Workers of a cement factory.

2/2

1/1

4/5

6/6

(+)*

0/2

1/1

1/5

nd

1/2*

1/1

nd

nd

1/2**

nd

nd

nd

nd

1/2**

1/1

3/3

nd

nd

1/1

nd

1/1*

1

1

1

2

1 nursing auxiliary. *BHR+ and a

Cleaner

4/4 SPT+

2 cleaners, 2 technicians, 1 nurse.

RADS

Co-exposure to halazone; *1/6

removal; 7/7 SPT+ done with clortol

Brewery workers. Recovery after

Spec. IgE+

Worker of metal plating factory.

potassium dichromate

Leather tanning worker. SPT+ with

dichromate

Roofer. *SIC with potassium

exposure

follow-up

Cushman et al.

1994)

Survey with

(Bhérer, 64

15*** (29.4)

58/64

16/51**

29/51**

nd

nd

obstructive lung pattern and BHR+

pulmonary tests; ***15/51 with

**51/58 symptomatics underwent

at follow-up 18 to 24 months

respir. symptoms and possible RADS;

multiple exposures, *71/289 with

289 construction workers. After

1 midwife with **SIC+ and **PFT+

2

1

4

6

nd

nd

nd

nd

nd

nd

nd

nd

1/1

nd

nd

FEV1 decline of 13% after SIC,

2

1

5

6

7/7

1/1

1/1

1/1

1989

Case reports

Case report

Case series

Case series

7

1

1

1

first symptoms after 34 years of

McAlpine et al.

Waclawski,

et al. 1995

Kujala, Reijula

1981

Vooren et al.

Dijkman,

Watrous, 1945

Bourne, Flindt

Chloramine T (powder dust)

1996 7

control

Morimoto,

Case series

1

Case and

“

Shirakawa and

1

1

Case report

Case report

et al. 1998

Leroyer, Malo

Lockman, 2002

“

6.0

15.8

32.1

(17 days at work, 33 days off work);

1/1

4/67

55/348

17/53

dichromate;**PFT done for 50 days

1

4* (6.0)

55 (15.8)

17 (32.1)

et al. 1998

1

67

348

53

Vandecasteele

de Raeve,

Case report

sectional

2001

Cross-

Gomes et al.

sectional

Al-Neaimi,

Cross-

et al. 1997

survey*

Bekele, 1998

AbuDhaise, Rabi

Comparative

Mengesha and

“

“

“

“

“

“

9781405157209_4_049.qxd 4/1/08 17:13 Page 1069

“

“

“

“

“

Survey

Ferris, Burgess

“

15.3

%

nd

nd

n/n

%

nd

nd

n/n

%

1

i(n) l(n)

d(n)

13/278 workers of metal production

*1 asthma (physician-diagnosed)

to sulphur dioxide. *7 COPD,

Pulpmill workers. Co-exposure

Remarks

cohort study

Lefante et al.

sectional

Crosssectional

Case report

et al. 2003

Gautrin, Leroyer

et al. 1995

Lemière, Malo

et al. 1997

Cross-

Andersson, Olin

2003

Retrospective

Glindmeyer,

1

239

385

19601

1

9* (3.8)

12

226

1/1*

38/239**

53/345

x

15.9

+

1/1

x***

nd

nd

1/1

x***

nd

nd

nd

nd

nd

nd

1/1

nd

nd

nd

after accidental inhalation; P+

steroid medication until 5 months

plant.*Persistent symptoms and

Worker of water-filtration

(> 10 puffs)

or frequently exposed workers

increased BHR in symptomatic

FEV1, FVC, FEV1/FVC and/or

chronic bronchitis; sign. reduced

other exposed groups; 3/239

with > 10 puffs compared to

sign. lower/increased in group

*9/239 current asthma; ***LFT/BHR

had persistent shortness of breath,

(puffs) over a 3 year period. 11/239

area with accicendtal exposures

**38/239 workers from the smelting

factor of asthma

sign., for gassing as a strong risk

(Cl2/ClO2, also to SO2). HR 5.6,

department exposed to gassing

and 210 workers of bleachery

*99 workers of paper department

gassed (highly exposed) people

5/447 new asthma cases in group of

vs. 0.13% of controls. Additionally

asthma). **Annual incidence 0.16%

exposure (RR 1.3, sign. to new

with chlorine/chlorine dioxide-

Workers of U.S. pulp/paper mills

deterioration

*3/13 transient FEV1 or BHR

2/13*

nd

n/n

SIC reaction

plant with accidental exposure.

1/13*

%

SIC

case series

(+)

nd

n/n

PFT

follow-up of

13/13

19.4

13/67

+) (+

BHR

et al. 1998

3

8*

%

n/n

LFT

Longitudinal

13

147

%)

(prevalence,

studied, n

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Leroyer, Dewitte

et al. 1966

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1070

et al. 2003

3.4

1

2

3

5/469 asthma claims between mid

*individual data for LFT/PFT not listed

Case report

Novey, Habib

“

et al. 1983

Case report

Joules, 1932

1

1

1

1*

1/1

1/1

1/1

+

nd

nd

1/1

3/5

nd

nd

1/1

5/5*

4/4*

4/4*

nd

nd

1

1

1

1

2

2

1

2 electro-plating employees,

dichromate

industry. *SIC done with potassium

worker, 1 worker of cement

2 metal plating, 1 construction

refridgerator fluid spill

refridgerator maintenance worker;

6 restaurant employees and 1

spill. Respir. symptoms > 2.5 years

Police men exposed to chlorine gas

production plant

139 men of a chlorine gas-

BHR+; **1/5 RADS

1 worker with accidental exposure

potassium dichromate

construction worker. *SIC with

1 tanning, 1 metal plating, 1

2 workerrs of concrete industry,

IgE+ with both agents

2/4 SPT+ with both agents; 1/4 spec.

with nickel sulphate(1dual,1late).

potassium dichromate; 2/4 also SIC+

nd

1/1*

nd

nd

nd

nd

nd

1/1

sulfate (late)

chromium sulfate. 1/1 SIC+ to nickel

Metal plating worker. SIC with

potassium dichromate

subsiding dermatitis; *1/1 SPT+ with

Chromium plating. Asthma with

dichromate

plant.1/1 SPT+ with potassium

1

5/5

nd

2/2

nd

nd

nd

of a chrome pellet manufacturing

1

5

4/4

3/4

3/7

3/3

2/2

1995

Case report

5

nd

nd

0/7

nd

nd

Nakano et al.

Nagasaka,

Basomba, 1989

Olaguibel and Case series

4/4

4/4

+

+

2/58

exposure to nickel. *SIC done with

4

4

5/7

3/3

34/58

1 cement worker, 1 welder. co-

4

4

3

3

2

et al. 2006

Case reports

Case reports

7

3

58

Nieto, Quirce

Fernández-

1994

Case series

“

“

“

“

“

[see also cement]

Park, Yu et al.

Piirilä, Espo

1996

Voshaar et al.

Schönhofer, Case reports

sectional

Gillespie et al.

1969

Cross-

Chester,

(degradation products)

Chromate (not specified)

58.6

nd

asthmatic symptoms for 6 months;

Profession not mentioned. Persistent

accident-related with BD+ and/or

nd

nd

retrospective review. *All 5 cases

x*

x*

(WCB), survey

nd

1/1

1984 and mid 1988 identified by

(+)**

x*

statistics

5/5

1/1

diseases

5

1

et al. 1999

5

1

Occupational

Case report

Chatkin, Tarlo

Broder, 1989

Tarlo and

Chlorofluorocarbons

“

“

“

“

9781405157209_4_049.qxd 4/1/08 17:13 Page 1071

“

Cleansing agents

“

“

“

” (not specified)

”, not specified

Cleaning agents

Bernstein and

CAS No.

control

diseases statistics

Reilly et al.

2003

statistics

2001

1/1

(+)**

1/1

x*

236/236

0/1

x*

nd

nd

nd

n/n

LFT %

nd

nd

nd

1/1

nd

nd

7/40

20/20

n/n

BHR %

nd

nd

nd

nd

nd

nd

nd

nd

n/n

PFT %

nd

nd

nd

nd

nd

nd

nd

20/20

n/n

SIC % 7

4

i(n) l(n) 9

d(n)

SIC reaction

House wife. Symptoms started

not listed

1993–1996. *Individual data

22/430 new-onset asthma in

including; **42 cases of RADS

1993–2001. 189 new-onset asthma

*236/1915 occup. asthma cases

and BHR

workplace cessation of symptoms

up 6 months after leaving the

irritant-induced asthma. At follow-

with cleaning agents. Low-dose

Worker of bottle-filling process

irritant-induced asthma; *BD+

Profession not mentioned. Low-dose

mentioned

**inadequate, because cases not

vs. 5% in non-cleaning workers;

symptoms in cleaning workers 12%

*prevalence of work-related respir.

cleaning workers (OR 1.46);

sign. more prevalent than in non-

domestic cleaning work. Asthma

593 women (13%) employed in

symptoms

related asthmatic and bronchitic

controls; BHR: 18% vs. 3%; bleach-

of controls. *6% COPD vs. 1% of

*24/40 asthma symptoms vs. 0%

Remarks

kitchen drain

cleansing agents to unstop a

1

22/22

236/236

1/1

1/1

x*

24/40

+) (+

one case:

6– 8 weeks after mixing several

1

22

236*

1

1

5.0*, **

12.0 vs.

26*

20/20

%

1976

Case report

survey

22

236

1

1

4521

40

20

20

n/n

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Fairman et al.

Murphy,

diseases

Harrison et al. (SENSOR),

Occupational

Reinisch,

(SENSOR)

Occupational

Case report

Rosenman,

1998

Tabar, Álvarez,

et al. 1994

Kipen, Blume Case report

sectional

Ramón, Zock

et al. 2003

Cross-

Medina-

2005

Nested case

Zock et al.

Review

Study type

Medina-Ramón,

Merget 2006

Reference

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1072

Case referend

Roto, 1980

Linna, Oksa

“

“

“

“

“

“

“

“

“

8/8

15/110

8/8

0/110

x*

5/9

7/8

nd

x**

9/9

nd

nd

nd

nd

nd

8/8*

1/1

6/15**

9/9

9/9**

x*

2

2

4

8

5

2

1

2

Hard metal workers: powder

metal industry; *SIC+ (mostly late)

Largest work population in hard

Workers in shaping, grinding,

confirmed not clearly indicated

questionnaire (sign.), results if cases

suspected asthma cases by

Co-exposure to nickel; *15/110

110 cobalt production workers.

reaction types not listed

1/6 SIC with cobalt dust, individual

**5/6 SIC with cobalt chloride,

definition either LFT+ and/or BHR+;

Cobalt industry workers. *For a case

pattern), 7/9 BHR+, 1/1 SIC+ (late)

(with obstructive ventilation

3 year follow-up: 1/9 still exposed

9 cobalt industry workers.

listed; **9/18 refused SIC

12/12

12/12

nd

12/12

12/12*

5

4

3

Workers in grinding, sintering,

8/8

8/8*

7/8

nd

8/8**

3

3

2

Hard metal plant workers. **SIC

Szeinuk, 2001

Wilk-Rivard and

et al. 1995

Baik, Yoon

diseases

Case report

Case report

(SHIELD)

statistics

Occupational

Burge, 1993

Case reports

Gannon and

1985

Auwerx et al.

Gheysens,

1

1

4

3

1

1

4

3

1/1

1/1

4/4

3/3

nd

0/1

nd

1/3

nd

1/1

nd

2/3

1/1

nd

x

nd

nd

1/1

x

3/3*

1

1

2

Diamond grinder

After SIC also systemic response

Worker of glassware factory.

1989–1991

4/500 occup. asthma cases in

cobalt powder

Diamond workers. *SIC done with

sulfate

IgE+; co-sensitization to nickel

SIC with nickel; 6/8 SPT+; 5/8 spec.

8

with cobalt chloride; *BHR before

8

1990

Case series

Kusaka et al.

Shirakawa,

chloride; 6/12 spec. IgE+

12

powdering. *SIC with cobalt

12

1988

Case series

Kusaka et al.

Shirakawa,

4/8 IgE+

8

15*

21/21

9/9

nd

nd

sintering. *SIC with cobalt chloride;

8

110

6

9

x*

x

followed by toxic lung edema

asthma. Immediate-onset asthma

*22 year old cleaner with preexisting

1989

Case control

Case control

21

9

5.6

nd

1/1

Kusaka et al.

Shirakawa,

et al. 2003

with follow-up

study

Case series

Zedda, 1994

“

18/319

1/1

grinders (6). *Individual data not

18 (5.6)

1*

workers (8), sintering (1), shapers (3),

319

1

et al. 1986

Survey

Review

Case report

Yokoyama

Kusaka,

Merget, 2006

Bernstein and

et al. 2000

Mapp, Pozzato

Pisati and

“

“

Cobalt 7440-48-4

“

9781405157209_4_049.qxd 4/1/08 17:13 Page 1073

“, yarn

“, “

“, “

“, “

“, “

(see also endotoxin)

Cotton dust, raw CNT 750

solder

Colophony: see rosin core

1/1

8.0

+) (+ 1/1

n/n

% 1/1

n/n

13.5

%

nd

n/n

PFT % 1/1*

n/n

SIC % 1

i(n) l(n) d(n)

SIC reaction

Diamond polishing disc former. SIC

Remarks

cohort

Wang et al.

study

Haglind et al.

26.4

nd

12/74**

x**

x*

nd

nd

nd

x*

nd

*74/74 subjects with sign.

Cotton-spinning mill workers.

level and individual FEV1 changes

relationship between cotton-dust

concn. No sign. dose-response-

and symptoms related to endotoxin

in 2/3 groups (8/15); FEV1 declines

cardroom, sign. mean FEV1 declines

cotton samples in experimental

Cotton mill workers. *SIC with

FVC decline

Longitudinal FEV1 decline, also

FEV1 decline is correlated with

67/447 dyspnea; **cross-shift

workers. *25–33% chest symptoms;

15-yr follow-up of 447 cotton textile

sectional

Christiani et al.

Comparative survey

Mengesha and

Bekele, 1998

1987

Cross-

Kennedy,

91

24 (26.4)

x

nd

nd

nd

chronic bronchitis

controls); 25/91 (27.5% vs. 9.5)

symptoms (26.4% vs. 8.5% in

24/91 subjects with asthmatic

Workers of a cotton-yarn factory.

of chronic bronchitis.

dose response to dust exposure

**FEV1 decline cross-shift, sign.

to endotoxin concentration;

and chronic bronchitis sign. related

Cotton workers. *FEV1 reduction

***individual data not listed

**sign. BHR+ increase cross-shift;

x*

x***

nd

nd

2007

24/91

12/150

2/15

x**

FEV1/FVC% decline cross-shift and

*

12** (8.0)

15/15

*

current endotoxin exposure-related;

443

150

15

(25%–33%)*

Latza et al.

sectional

Baur 2003;

15

447

Oldenburg,

Cross-

Oldenburg and

1985

Experimental

Rylander,

2001

Prospective

Christiani,

cobalt chloride

1

%

BHR

with cobalt chloride; SPT+ with

1

n/n

LFT

2005

Case report

Krakowiak,

“

%)

(prevalence,

studied, n

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Dudek et al.

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1074

55.6

+

x*

1/1*

nd

nd

nd

1/1**

nd

nd

1

1

1

1

Assemblers, pressmen and other

turpentine (immediate)

colophony (immediate) and artists’

reodorant (immediate), heated

of 5 months; **also SIC+ with

Toolsetter. *PFT over a period

Profession not mentioned

not listed

with OR 1.9, sign. *Individual data

in 1996 (textile factory workers)

22/294 occupational asthma cases

Hutchcroft

et al. 1990

Graham, Coe

tetrafluoroborate

14239-22-6

“

“

“

Diesel exhaust

statistics

2003

et al. 2006

Hart, Laden

diseases

Derk et al. (SENSOR)

Occupational

Henneberger,

Newman, 1993

Wade and

1994 Case report

Case report

Deschamps,

Dichlorvos (organophosphate)

Questel et al.

sectional

Rezaian, 1997

[mustard gas] +505-60-2

Cross-

Emad and

Dichlorodiethyl sulfide

et al. 1981

study

Luczynska,

Diazonium

Case report

Comparative

et al. 1992

cycloaliphatic (hardener)

Case report

Aleva, Aalbers

Diamine, aliphatic +

propylamine] 109-55-7

536

7

3

1

197

1

45

1

*75

7

3

1

15* (7.6)

1

2

1

nd**

7/7

3/3*

1/1

197/197

1/1

25/45

1/1

+

+

nd**

x

3/3

1/1

15/197

1/1

1/2*

0/1

nd**

x

3/3

1/1

nd

1/1

nd

1/1

nd**

nd

nd

nd

nd

nd

nd

nd

nd**

nd

nd

nd

nd

1/1*

2/2*

1/1

1

relationship

(1.17–2.39)); sign. dose-response

increase in COPD mortality (OR 1.35

of operating trains had a sign.

engineers with exposure ≥ 16 years

workers. *75/536 conductors and

**COPD mortality cases of railroad

1993 and 1995 (7/123 RADS)

7/424 occup. asthma cases between

administration after high exposure

ventilation pattern; twice hospital

2/3 “reversible restrictive”

hours in second locomotive units

a high exposure over several

Railroad workers. RADS after

Cook. Persistent asthma

chronic bronchitis, *89 COPD cases

exposure 10 years ago; 116 with

Iranian veterans. Single massive

*SIC with diazonium chloride

Production of photocopy paper.

*hospital admission 2/45 for LFT/SIC

polymer precursor. 9/43 spec. IgE+;

Workers manufacturing fluorine

covering materials

Salesman selling industrial floor-

symptomatics

cross-week of slightly exposed and

and FEF50 decreases cross-shift and

epoxy resin system; *sign. FEV1

nd

nd

1/1

x*

mould room workers. Use of an

x

1/1

nd

x*

1976

5/25

1/1

1/1

x*

[3-(dimethylamino)

5 (20.0)

1

1

22

Mitchell et al.

25

1

1

22

Sargent,

Survey

Case report

Case report

Case control

propylamine

et al. 1985

Hendy, Beattie

et al. 1994

Kipen, Blume

et al. 1999

Toren, Balder

3-(Diamino-amino)

“

Cutting oil

“, “

9781405157209_4_049.qxd 4/1/08 17:13 Page 1075

et al. 1994

1977

Conrad, Lo

et al. 1998

108-01-0

Diinitrogen tetraoxide

[dinitrogentetroxide]

+

nd

4/6

0/1

4/12

nd

n/n

%

1/1

6/6

1/1

nd

1/1

n/n

%

nd

nd

1/1

10/11

1/1*

n/n

%

1/1

nd

1/1

nd

1/1

n/n

SIC %

1

1

i(n) l(n)

1

1

d(n)

SIC reaction

Laboratory nurse. also SIC+ with

massive exposure

of breath, 151/231 wheezing after

railroad tanker; 207/234 shortness

RADS after massive release from a

6/234 symptomatics developed

Spray painter

and 7 suspected asthma cases

State office building. 7 asthma cases

Steam leak in heating system of a

Metal worker. *PFT for 12 days

Remarks

8/8

x*

x**

nd

nd

nd

1/1

nd

x***

nd

nd

nd

1/1

workers of fiberglass manufacturing.

Maintenance and production

blue (immediate)

Technician. SIC+ also with methyl

Blau V, methylene blue

“ diseases statistics

Harrison et al.

2001 survey

(SENSOR),

Occupational

Reinisch,

listed

1993 and 1996. *Individual data not

8/430 new-onset asthma between

PFT over 10 days

related PEF decline cross-shift with

exposure-related; ***endotoxin-

asthma; **FEV1 and FVC reduction

formaldehyde. *6/37 new-onset

8

14/37

1/1

Co-exposure to phenolics and

8

6*

1

confinement, house dust)

sectional

et al. 1996

cotton dust, swine

37

1

confinement, poultry

Cross-

Case report

Milton, Wypij

Stanescu, 1982

Rodenstein and

Blau V; SPT+ to methyl blue, Patent

1/1

6/6

1/1

14/14

+) (+

PFT

methyl blue (immediate) and Patent

1

6

1

7

%

BHR

1981

1

234

1

14

1/1

n/n

LFT

Nordman et al.

Case report

Case series

Case report

Endotoxin (see also

“

ECG ink

Keskinen,

Cockcroft et al.

[dimethyl ethanolamine]

10544-72-6

Vallières,

2-Dimethylaminoethanol

100-37-8

Gadon, Melius

[diethyl aminoethanol]

et al. 1998

2-Diethylaminoethanol

Case series

1

1

Case report

Piipari,

Diethanolamine 111-42-2

Tuppurainen

%)

n

Study type

(prevalence,

studied,

Reference

or at least

cases, n

subjects one case:

cases: +

asthma Asthma

RADS all

Irritant

exposed

Work-related symptoms

Evidence (pathological results)

Occupationally

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1076

239

117

x**

3.9

x**

x**

x**,*** nd

respiratory work disability; sign. increased asthma PR for workplace ETS OR 1.8; workplace ETS

survey (ECRHS)

“

“

“ sectional

Populationbased case-

et al. 2001

Flodin, Jönsson

et al. 1995

sectional

Büsching et al.

2002

Cross-

Radon,

referent study

Cross-

Janson, Chinn

1890

7882

1562

sectional;

61 (3.9)

x

x

x*

61/1562

nd

x

x*

x*

nd

x

x*

x*

nd

nd

nd

nd

nd

nd

nd

nd

nd

Cross-

nd

et al. 1999

nd

Blanc, Ellbjär

“

nd

ical

x**

et al. 1993

45

Epidemiolog-

Greer, Abbey

“

control

increased

chronic bronchitis (OR 1.9) sign.

home and in the workplace 1.5;

Increased asthma: OR for ETS at

Sample of population-based ECHRS.

asthma cases, OR 1.5 (sign.)

ETS in the workplaces increased

*individual data not listed

dose-related trend with ETS;

and current asthma OR 1.9; BHR+

associated with respir. symptoms

countries. ETS in the workplace

Subjects of 36 centres in 16

*individual data not listed

associated with BHR+ and WRS;

Population-based sample of ECHRS

**individual data not listed

between 1977 and 1987;

*45 subjects with new onset asthma

increases asthma sign., OR 1.45;

again in 1987. ETS in the workplace

Cohort of n = 3914 in 1977 and

for 2 weeks

**no individual data listed; ***PFT

lifetime workplace ETS OR 1.84;

OR 2.21, in total population for

2.16, > 150 cigarette-years adjusted

workplace ETS sign. increased, OR

(n = 487), resp. in asthmatics lifetime

whole working-age population

8% attributable fraction for the

past year (among 239 ETS-exposed).

ETS (workplace + home) during the

*49.2% of asthma attributable to

Incident case

et al. 2003

evidence

evaluation of

Jaakkola, Piipari

relation

Review and

“

Suggested evidence of a causal

Critical

IARC, 2004

“

smoke 1-09-0

Environmental tobacco

9781405157209_4_049.qxd 4/1/08 17:13 Page 1077

Case report

Lambourn,

“, (various)

Farming

“

Ethylene oxide 75-21-8

“

“

2

1

i(n) l(n) d(n)

SIC reaction

6 shellac handlers and 1 rubber

Mould maker and fitter

lung pattern.

n = 47: *42/47(89.4%) obstructive

at work = 7%; subpopulation

attributable fraction of ETS exposure

workplace ETS 1.36); population-

sign. increased (OR for ≥ 23 yrs

Population-based sample. COPD

Remarks

607/1824

1/1

1/1

1/1

x*

nd

1/1

0/1

0/2

nd

nd

1/1

1/1

2/2

nd

nd

nd

nd

nd

nd

1/1*

nd

1/1

2/2

Chemical workers; co-exposure

1

Farmers. 607 dyspnea, 500

*individual reaction type not listed

Factory worker. 1/3 spec. IgE+;

Worker of railway station

(immediate)

SIC+ also with sulphur dioxide

Photography laboratory worker.

2 chemical workers. 2/2 i.c.+

not listed

1 late, 2 dual; **individual data

not listed

wheezing; *sign. declines of FEV1,

607(33.3)

1

1

1

2/2

nd

%

FVC, FEV1/ VCV with individual data

1824

1

1

1

2

nd

7/7*

1/1

nd

n/n

SIC

1987

Survey

Case report

Case report

Case report

2

1/3

%

Graham et al.

Dosman,

et al. 1991

Dugue, Faraut

et al. 1992

Rosenberg

Deschamps,

Yeung, 1980

Lam and Chan-

Matsui, 1990

Nakazawa and Case reports

x**

nd

nd

nd

n/n

PFT

according to onset of symptoms:

3/3*

%

to other amines. *Asthma type

3

nd

0/1

nd

n/n

BHR

1982

3

nd

%

Bellander et al.

Survey

7/7

0/1

42/47*

n/n

LFT

types not listed

“

Hagmar,

7

1/1

+) (+

one case:

industry worker. *Individual reaction

7

1

x

%

107-15-3

Gelfand, 1963

1

2113

n/n

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

y exposed Asthma

RADS all

Irritant

Occupationall

Work-related symptoms

Evidence (pathological results)

[ethylene diamine]

Ethylenediamine

109-55-7

3-(dimethylamino) propylamine

Epoxy resin system; see also

1992

Case series

sectional

et al. 2005

Hayes et al.

Cross-

Eisner, Balmes

“

EPO 60 142443-98

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1078

(Agricultural Health Study 1994–1997)

Umbach et al.

2003; Hoppin,

Umbach et al.

Crosssectional

Portengen,

Preller et al.

“, “

“

“

“

“

50-00-0

Porter, 1975

1995

Desjardins et al.

Lemière,

et al. 1985

Burge, Harries

Case report

Case reports

Case series

case reports

Keskinen et al.

1985

Survey with

Nordman,

et al. 1985

hydrocarbons) (1/D)

Formaldehyde (gas, dust)

Brooks, Weiss

Floor sealant (aromatic

Case report

sectional

et al. 2004

dairy, poultry)

2005

Cross-

Monso, Riu

2004

Survey

Hoppin,

et al. 2004

Eduard, Douwes Survey

1

3

15

230

1

81

105

20468/20898

1614

1

3

7

12

1

36(44.4)

18(17.1)

*

1/1

3/3

5/15

230/230

1/1

81/81

58/105

20 898

3838*/

x*

+

1/1

0/3

nd

2/5*

0/1

x

11/105

nd

nd

nd

3/3

4/14

29/209

1/1

36/78*

nd

nd

nd

x*

46.1

nd

3/3

nd

nd

nd

nd

nd

nd

nd

nd

3/3

7/15

12/230

nd

nd

nd

nd

nd

38/100

1

4

6

23

4

2

1

2

2

15

Polish farmers. SIC with grain

Neurology resident. P+

factory worker

1 chemist, 1 carpenter, 1 wood chips

co-exposed to isocyanates

2 core shop workers; 9/15 subjects

packaging, 1 laminated trray worker,

6 printers/laminators of flexible

medicine packer, 1 farmworker,

formaldehyde-manufacturer, 1

1 plastic moulder, 1 printer, 1

reports (5 subjects)

*Individual data only listed in case

230 formaldehyde workers.

Grocery clerk. RADS for 14 months

with BHR or lower lung function

sign. reduced; endotoxin-associated

(46% vs. 17% of controls); FEV1

Pig farmers. *sign. increased BHR

COPD sign. dust-related

study reported wheezing. 18 COPD;

European never-smoking farmers’

58/105 participants of sample of

(sign.), for solvents 1.16–1.33 (sign.)

OR for driving diesel tractor: 1.31

poultry and number of livestock;

sign. dose–response-related for

(dairy), and 1.70 (eggs); wheezing

wheezing; OR for wheeze 1.26

(pesticide applicators). 3838

20 468, and 20 898 farmers

non-allergic asthma

ammonia pos. associated with

endotoxines, fungal spores and

(OR 1.8 or 1.6); exposure to

elevated in cattle and pig farmers

Norwegian farmers. *Asthma sign.

data not listed

breeding; 47/100 SPT+; *individual

symptoms, cereal farming, animal

x*

feathers; sign. risk factors of respir.

100/100

2004

38(38.0)

dust, animal epidermis, furs and

100

Adamus et al.

Case control

Krawczyk-

Walusiak,

“, animals (pig, beef/veal,

“, “

“, “

“, “

9781405157209_4_049.qxd 4/1/08 17:13 Page 1079

“

“

“

“

11-30-8

Glutaraldehyde [glutaral]

Furan-based binder

Fumigating agent

Freon (heated)

“

“

“

Hendrick and

“

statistics

2003

1/1

1/1

1/1

4/4

65.0

+

1/1

1/1

0/1

nd

x

1/1

nd

n/n

LFT %

1/1

1/1

1/1

nd

x

1/1

nd

n/n

BHR %

1/1

nd

nd

4/4*

nd

nd

nd

n/n

PFT %

1/1*

nd

1/1

4/4*

nd

1/1*

1/1

n/n

SIC %

1

1

1

i(n) l(n)

1

d(n)

SIC reaction

Mold maker. *3 SICs+ (late):

Duration of symptoms for 6 months

Housewife fumigating her kitchen.

2 years

Asthma aggravation at work for

Refrigerator company worker.

*Individual data not listed

4/306 occup. asthma cases in 2002.

24/424 asthma cases in 1993–1995

0.12–0.13 ppm

6 ppm, several short-term-peaks of

challenge; average workplace-conc.:

Textile factory worker. *Workplace

Nurse

Remarks

sectional

diseases

Burge, 1993 (SHIELD)

statistics

Occupational

Gannon and

et al. 1986

Corrado, Osman Case reports

Cross-

et al. 1996

Survey

Curran, Burge

Bound, 1989

Jachuck and

6

4

20

9

6

1

13*

1*(11.1)

6/6

4/4

13/20

1/9*

nd

0/4

nd

nd

nd

nd

nd

nd

x

nd

nd

nd

x

1/4*

7/8*

nd

1

6/500 asthma cases in 1989–1991

alkaline glutaraldehyde

Nurses of endoscopy units. *SIC with

positive IgE, results at high total IgE

asthma cases IgE+; 3/13 false

type not listed; 2/11 occupational

done, individual data on reaction

Hospital employees. *5/13 SIC not

*1subject with breathlessness

Employees of endoscopy unit.

alcohol

furfuryl alcohol combined with

1

1

1

4

24/24

1/1

+) (+

one case:

acid catalyst, sulfuric acid, or butyl

1

1

1

4

24

1

1/1

%

1980

Case report

Case report

Case report

Case reports

24

1

1

1

n/n

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Cartier et al.

Cockcroft,

et al. 1985

Brooks, Weiss

et al. 1984

Malo, Gagnon

Keskinen, 2005

(SENSOR)

disease

Piipari and

Occupational

Derk et al.

Case report

Case report

Study type

Henneberger,

2001

Kim, Song et al.

Lane, 1975

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1080

Hexachlorophene 70-30-4

Hairdressing chemicals

“

green coffee (dust)

“

“

“

“

“

“

“

Grain dust

“

“

survey

sectional Case control

1995

Toren, Balder

35/502

61.0

14.1

nd

nd

2/2

x*

x*

x*

x*

1/7

nd

nd

2/2

x*

45/410

nd

x*

3/7

11.0

nd

nd

nd

nd

nd

x*

x*

7/7

nd

nd

2/2*

nd

nd

nd

x*

7/8

4

2

5

1

2

Workers of endoscopy and X-ray

Grain elevator industry workers. 78

lung function exposure-related

Cross-shift and chronic decreases in

workplace with rye dust

house (SIC with wheat dust); *SIC in

1 farmer, 1 manager of grain ware

listed

OR 4.2, sign.; *individual data not

7/294 occup. asthma cases in 1996.

and sign. endotoxin-dose-related

Grain workers. *FEV1 sign. reduced

4.3% of the survey)

and FVC (FEV1 drop of > 10% in

shift and -week decrease in FEV1

Increased chest symptoms; cross-

Workers of grain elevator terminals.

asthma cases in 1996

74/1765 physician-diagnosed

departments

31*

x*

nd

nd

nd

51 grain elevator workers out of 175

breathlessness and wheezing

4

9/9

10/372

2.6

2/9

x*

nd

nd

nd

nd

4/9

nd

9 coffee industry workers; SIC

decline; 35/362 SPT+; 24/331 IgE+

*exposure-time-related FEV1

7/372 new onset asthma;

1984

Nagy and Orosz,

diseases

Keskinen, 2005

Case report

Finland

statistics

Occupational

Piipari and

1

6

1

6

1/1

x*

nd

nd

1/1

nd

nd

6/6*

1/1

6/6*

Children’s nurse

2002. *Individual data not listed

6/306 occupational asthma cases in

symptomatics SPT+; 4/9 BD+

9

7 (1.8)

done with aqueous extract; 5/9

Case series

(Zuskin,

372

1985)

sectional

et al. 1982)

Kanceljak et al.

Cross-

(Jones, Hughes

32% of controls)

(settled barley dust) (61% vs.

sign. FEV1 decline; 31/51 SPT+

(n = 31) symptoms associated with

31

with respir. symptoms. *Most severe

51

1964

Survey

Williams et al.

Skoulas,

without cough, 20 (4.0) with cough,

(7.0%) wheezing and breathlessness

35

nd

2/2

x*

58

x*

x*

7/7

with work-related breathlessness; 35

502

2

7

58(14.1)

74

7

1964

Survey

Review

2

7

410

610

74

7

Skoulas et al.

Williams,

1992b

Enarson et al.

Chan-Yeung,

et al. 2003

Baur, Preisser Case reports

based cross-

Thorne et al.

et al. 1999

Population

Schwartz,

1980

Comparative

Schulzer et al.

Case series

Case series

Chan-Yeung,

et al. 1997

Ross, Keynes

et al. 1995

Gannon, Bright

9781405157209_4_049.qxd 4/1/08 17:13 Page 1081

Comparative survey

Gamble,

McMichael

Hexamethylene tetramine

100-97-0

et al. 1990

Tarlo and

[hydrochloric acid] 7647-01-0

“

Sahakian, 2003

Bergman,

[hydrofluoric acid] 7664-39-3

Iridium salt

“, “

“, various (HDI, MDI, TDI)

Isocyanates, Isocyanurate

Case report

Franzblau and

39.8

15.4

1/1

x*

0/1

+

x*

0/3

1/1

nd

x*

n/n

+

+

+

+) (+

28.8

%

x*

1/1

1/1

1/1

3/3

1/1

nd

nd

n/n

%

nd

nd

nd

x*

nd

nd

nd

x

n/n

PFT %

nd

nd

nd

nd

nd

nd

7/7*

nd

n/n

SIC %

101 118

i(n) l(n) d(n)

SIC reaction

Worker of elctrochemical factory.

symptoms persistent for > 3 years

stain remover once. Asthmatic

Cleaning with HF containing rust

Profession not mentioned. BD+

for 2 years

Asthmatic symptoms persistent

Profession not mentioned.

Massive exposure by truck accident

Members of police department.

solution; symptoms for 34 months

exposure to pouring of 35%

Power plant utility worker. Massive

7/7 SPT+

Laquer handlers with asthma.

not sign. reduced

tightness, 10/52 dyspnea; *LFT

8/52 wheezing, 14/52 chest

Workers of tire manufacturing plant.

Remarks

247/621

244/244

179/621

x*

nd

3/3**

nd

14/14**

219**

219/

1976–1992. Mainly exposed to

Asthma cases diagnosed

exposure-related COPD; spec.

sectional

vs. 0% (n = 150) asymptomatics

26.4% (14/53) symptomatics SPT+

asymptomatic isocyanate workers);

IgE+ (14% symptomatic vs. 0.3%

companies. 26/247 symptomatics

cross-

et al. 1984

Isocyanate workers of different

impairment of FEV1 Case series/

Baur, Dewair

nd

re-examined in 1995 with sign.

PFT or SIC (mainly); 91 patients

listed; **case diagnosed by BHR or

spray painting; *individual data not

179 (28.8)

236/244

polyurethane foams production or

621

244

follow-up

with

et al. 2000 long-term

Case reports

Piirilä, Nordman

not listed

1/1

1/1

1/1

1/1

3/3

1/1

7/7

8/52

%

BHR

SPT+ (immediate); individual data

1

1

1

1

3

1

7

8 (15.4)

n/n

LFT

1995

1

1

1

1

3

1

7

52

%)

(prevalence,

studied, n

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Svedberg et al.

Case report

Case report

Boulet, 1988

Hydrogen fluoride, as F

Case report

“

Broder, 1989

Promisloff, Phan

Hydrogen chloride

Case reports

Case report

Brooks, Weiss

Hydrazine 302-01-2

et al. 1985

Case series

Gelfand, 1963

“

et al. 1976

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1082

diseases

Keskinen, 2005

(+)**

x*

6/6*

2

7

Workers of foam industry (spray

(1 immediate, 2 late)

4 dual), 3/8 SIC+ with 3rd agent HDI

also SIC+ with MDI (4 immediate,

Profession not mentioned. 8/16 SIC+

54/835 occupational asthma cases in

2002. *Individual data not listed

6/306 occupational asthma cases in

asthma cases in 1996

310/1765 physician-diagnosed

9/465 asthma claims between mid

sectional

Comparative survey

Case series

Korbee et al.

1993

Liss, Bernstein

et al. 1988

Zammit-Tabona,

5873-54-1

“, “

1983

Sherkin et al.

Cross-

Bernstein,

diisocyanate [MDI]

1980–1993

between

(WCB)

statistics,

diseases

78

26

243

6

7 (26.9)

3 (1.2)

12/78

7/26

43/243*

5/11

x*

nd

9/11*

nd

nd

nd

x

7/43**

6/11

nd

nd

spec. IgE−

Foundry workers. 0/6 with SIC+

data not listed

cross-shift; 1/26 IgE+; *individual

workers. *Sign. FEV1 decrease

Current mould and core-room

diagnosed OA 3/7 (BHR 6/7, S1/7)

symptoms. At follow-up, physician-

3/9 OA, 2/4 non-OA, 2/23 free of

and 2/23 controls PFT+ (for 2 weeks):

**3/9 with OA, 2/4 with non-OA

respiratory symptoms, 4 non-OA;

questionnaire: 9 OA, 26 irritant

*Diagnosis of asthma by

Workers of urethane mold plant.

*Individual data not listed

Review of of Ontario Workers’ represented 50% of all OA claims.

x*

occupational

nd

compensation board. Diisocyanates

x*

OA claims of

x*

review of new

x*

2002

425

Retrospective

Tarlo, Liss et al. 425

and/or BHR+; **2/9 RADS

nd

retrospective review. *9/9 BD+

nd

(WCB), Survey

9/9*

1984 and mid 1988 identified by

nd

statistics

9/9

diseases

et al. 1999

9

Occupational

Chatkin, Tarlo 9

*Individual data not listed

nd

6/6*

4

15

11

2003 (54/210 irritant asthma cases).

nd

nd

x*

7

5

statistics

x*

nd

x*

29/62

16/24

diseases

x*

6/6*

x*

nd

nd

Occupational

54

6

x*

46/62

12/24

2005

54

6

x*

14/62

nd

Latza and Baur,

Finland

statistics

Occupational

Piipari and

(SWORD)

statistics

diseases

et al. 1997

310

62/62

24/24

spec. IgE+ and spec. IgG+ Occupational

Ross, Keynes 310

29

16

painters and others. 8/29 SIC+ with

62

24

1989

Case series

Case series

Grammer et al.

Cartier,

et al. 1979

O’Brien, Harries

“, Diphenylmethane

“, “

“, “

“, “

“, “

“, “

“, “

“, “

9781405157209_4_049.qxd 4/1/08 17:13 Page 1083

+

4

2

4

1

34

Workers of woodchip board

spill

Spray painter. RADS after accidental

sign. lower in 93 SIC+ vs. 69 SIC−

Profession not mentioned. *BHR

*Individual data not listed

Workers of a wood products plant.

Foundry workers. 2/78 spec. IgE+

Remarks

1/1

1/1

1/1

nd

1/1

nd

nd

1/1*

nd

10/20*

1

Profession not mentioned; *SIC with

organic solvents; P+

Mechanic; co-exposure to several

7/8 spec. IgE+; 4/8 BD+

(no individual reaction type given);

*2/8 with sign. obstructive pattern

with abnormal chest X-ray

Spray painter; asthma associated

within 1 month

of car (polyurethane) paint twice

after exposed to high concentration

IgE+; *severe asthma symptoms

Gasoline station manager; spec.

with HDI monomer, 5 with both)

days

increasing exposure on following

1

1/1*

nd

0/1

32

d(n)

(interstitial infiltration); *SIC by

1

1

15/20

nd

27

i(n) l(n)

IPDI] 4098-71-9

Aldons, 1981

trimethylcyclohexyl isocyanate

Case report

1

6/20

1/1

2/8*

1/1

%

[isophorone diisocyanate,

Clarke and

et al. 1981

Belin, Hjortsberg Case report

20/20

0/1

nd

nd

93/162

nd

nd

n/n

(4 only with HDI prepolymers, 1 only

10

1/1

1/8

1/1

nd

nd

%

monomer and prepolymers of HDI

20

1

0/8

1/1

93/162*

15/16

nd

n/n

1993)

Case series

1

+

15/93

x*

%

SIC reaction

Cartier et al.

(Vandenplas,

et al. 1996a

Lemière, Malo Case report

8/8

1/1

162/162

18/18

12/78

n/n

SIC

declines of FEV1 and FVC, only

2

1

93

15

nd

%

PFT

manufacturing; 8/8 SIC+ with sign.

8

1

162

18

12/78

n/n

BHR

1993

Case series

Case report

Case series

Case series

12 (15.4)

+) (+

LFT

Malo et al.

Vandenplas,

et al. 2003

Perfetti, Brame

et al. 1988

Boschetto

Mapp,

et al. 1997

Woellner, Hall

“, 3-Isocyanatomethyl-3,5,5-

“, “

“, HDI biuret plus 4035-89-6

822-06-0

plus isodurane diisocyanate

diisocyanate [HDI];

“, Hexamethylene

“, “ prepolymers

“, “

“, “

“, “

sectional

et al. 1985

78

Cross-

Tse, Johnson

“, “

%

%)

n

Study type n/n

(prevalence,

studied,

Reference

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Work-related symptoms

Evidence (pathological results)

Occupationally

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1084

33.3

+

40.0

66.6

37.5

nd

60

1

1

1

Persistently symptomatic residents

Baur, Chen

“, “

nd

nd nd

Exposed residents at bhopal tragedy.

5/10

10/10

nd

10/10

40/40

4

6

sign. difference in FEV1 and BHR.

10/10

nd

up 3–39 months after cessation no

10

x*,**

follow-up

10

x*

series with

40

Case

“, “

40

inflammatory changes in 8/8 (P+)

Biopsy of bronchial mucosa with

Profession not mentioned. At follow-

and FVC of workers with SIC+ late

first diagnosis sign. decline of FEV1

**at follow-up 4–8 years after

*Individual data not listed;

Profession not mentioned.

listed

1967 and 1992.*Individual data not

et al. 1990

6

TDI production employees between

Paint shop workers

Paggiaro, Bacci

25

4

mould factory

polyurethane caster of plastic

1 chemist, 1 foreman, 1

follow-up

9

2

1

1994

nd

6/10

2

*SIC+ workers also BHR+

production workers. 1/6 rhinitis;

1 elastomer, 5 synthetic resin

not listed

questionnaire; *individual data

6 additional cases identified by

*7 cases reported to NIOSH and

section. Co-exposure to MDI;

in production and administrative

Workers of wheel factory employed

respiratory ailments

Bhopal tragedy: mainly mixed

neutrophils (P+)

related to exposure and to BAL

pattern; FEV1/FVC sign. negatively

subjects with obstructive lung

moderately or severely exposed

investigated symptomatic and

Bhopal tragedy. 17 subjects

series with

nd

nd

3/3

3/5*

nd

nd

nd

Case

nd

nd

nd

nd

3/8

nd

nd

Brugnami et al.

x*

4/10

1/3

3/5*

2/3

nd

nd

Marabini,

x*

14/42

2/3

nd

x*

nd

17/54

“, “

19

6 (11.8)

3/3

5/6

17/46

nd

54/54

Ott, Klees et al. 313

42

3

3

13*

(11)

17

2000

cohort study

Survey

3

6

46

41

54

TDI 2,4: 584-84-9; 2,6:91-08-7

et al. 1987

Case reports

Case reports

Survey

Review

Case series

“, Toluene diisocyanate,

Séguin, Allard

isocyanate 9016-87-9

et al. 1979

Harries, Burge

“, Polymethylene polyphenyl

“, “

et al. 1995

et al. 2001

Fuortes, Kiken

diisocyanate [NDI] 3173-72-6

et al. 1990

Mehta, Mehta

Sankaran, 1996

Vijayan and

“, 1,5-Naphthylene

“, “

24/82*

patterns (64/82); 24/82 BD+;

82/82

Predominating restrictive ventilation

24

et al. 1985

82

Mahashur

Case series

97/97 BD+;

restrictive ventilation patterns;

over 24 months; predominating

also gradual FEF25–75 decline

FEV1/FVC decline after 18 months,

lung function fluctuations; *sign.

Kamat,

nd

“, “

nd

1984. 97/113 dyspnea; 32% with

x*

exposed to MIC at Bhopal tragedy

97/97

up of cases

97

study, follow-

et al. 1992

624-83-9

113

Longitudinal

Kamat, Patel

“, Methyl isocyanate [MIC]

9781405157209_4_049.qxd 4/1/08 17:13 Page 1085

nd

34/63

nd

9/13

n/n

%

1

12

5

2

13

*

i(n) l(n)

9

*

d(n)

35 firemen at accidental exposure.

*BHR+ associated with SIC+

63 asthmatic isocyanate workers.

improvement

> 7 years with considerable

Persistent asthmatic symptoms

Police officers exposed to spill.

**LFT not sign.

*4/9 SIC reaction type late or dual;

continuously/intermittently exposed;

study 1973–1975. 89/103 became

103 TDI workers of Longitudinal

Remarks

5/469 asthma claims between mid-

RADS lasting for 1.5 years

et al. 1997

Case report 1

1

1/1

1/1

1/1

1/1

1/1*

1/1

nd

1/1

Bourke, Convery

nd

Isothiazolinone 55965-84-9

1/1

et al. 1997a

1

2451-62-1

1

Case report

onset late)

3 consecutive days with I. (asthma

Chemical plant operator; *PFT on

Laboratory technician. Spec. IgE+

relationship remains unclear

4-year-period. Dose-response-

Piirilä, Estlander

Workers of wood roof industry.

“, Triglycidil isocyanurate

2/2*

Accelerated loss of FEV1 within

nd

review

2/2

Systematic

1/2

et al. 2005

2/2

Wisnewski, Liu

2

monomers negative

2

*SIC with TDI prepolymers, TDI

Case reports

1992

Vandenplas,

reaction type not listed

and/or BHR+; for SIC indiviual

2/5**

nd

retrospective review; 5 /5 BD+

nd

x*

(WCB)

x*

1/1

1984 and mid-1988 identified by

nd

x*

statistics

+

+

diseases

5/5

1/1

Occupational 5

1

et al. 1999

5

1

Chatkin, Tarlo

Broder, 1989

2/32 after 44 months.

asthmatic symptoms after 6 months,

8 hours to 3 weeks (delayed); 7/33

(immediate), additionally 22 within

chest tightness during the fire

small **FEV1/FVC decline; *8/35

Case report

%

Tarlo and

nd

nd

nd

n/n

symptoms 4 years later with

nd

%

20/35 persistent respiratory

x**

32/34*

2/2

8/11

n/n

follow-up

(+)*

nd

1/2

%

SIC reaction

with 4 year

30/35

63/63

+

x**

n/n

SIC

et al. 1976

30

34

2/2

+) (+

PFT

McKerrow

35

63

2

26/89

%

BHR

Case series

Case series

2

9

103

n/n

LFT

Axford,

et al. 1994

Karol, Tollerud

Case reports

%)

(prevalence,

studied, n

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Cartier et al.

“, “

“, “

“, “

“, “

“, “

et al. 1990

Luo, Nelsen

cohort

et al. 1977

“, “

prospective

Butcher, Jones

“, “

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1086

statistics

Henneberger,

Derk et al.

2003

Lubricants (not specified)

et al. 1985

(Dube,

(coating removing chemical)

Metal oxide fume

30

1

1

12 (40.0)

1

1

30/30

1/1

1/1

(+)*

+

5/30

1/1

1/1

12/30

nd

1/1

40.0 nd

nd

nd

nd

nd

nd

1

30 workers of an automobile

associated with metal fume fever

Metal industry worker. RADS

and plastics. RADS for 39 months

Remover of coatings from metals

data not listed

100.0

nd

nd

nd

nd

1/469 asthma claims between mid

aggravated asthma

(*17/20 RADS) plus 10 work-

evaluated. 20 new onset asthma

c. 3,000 exposed subjects medically

Massive spill of pesticide, 197 out of

Audunsson

Block and

Yeung, 1982

Nickel sulphate → anhydrous

7786-81-4 → hexahydrate

et al. 1983

Belin,

Gelfand,1963

Case report

survey

Comparative

Case series

1

48

14

1

13 (27.1)

10

1/1

13/48

14/14

0/1

nd

nd

1/1

8/44

nd

1/1

nd

nd

1/1

nd

10/10*

dermatitis

Metal polisher. SPT+; contact

to isocyanates

1.4-diaza-bicyclo-(2,2,2) octane and

BHR sign. increased; co-exposure to

dyspnea (27 vs. 17% of controls);

workers; 13/48 wheezing and

Polyurethane foam industry

data not listed

ethylene diamine; *individual

ammonium-thioglycolate and

culture industry, co-exposure to

Subjects, exposed in the beauty

*1/1 BD+ and/or BHR+

x*

23/23

retrospective review exposed;

nd

50.0

(WCB)

+

15/30

1984 and mid 1988 identified by

24.3

statistics

nd

48/197

diseases

1

20 (10.1)

Occupational

1

197

et al. 1999

case series

et al. 1994

Chatkin, Tarlo

Retrospective

Cone, Wugofski

7 hypersensitivity pneumonitis

12 OA, 6 industrial bronchitis,

Survey

Case report

Case report

asthma cases = 210); *individual

engine manufacturing plant:

109-02-4

10101-97-0

16.7

8/835 occupational asthma cases in

1993–1995

46/424 occup. asthma cases in

Pharmacist

1998

N-methylmorpholine

dichlorodiethyl sulfide)

Mustard gas (see

141-43-5

Monoethanolamine

Methylmercaptan 74-93-1

Metam sodium 137-42-8

100.0

1

Kadambi et al.

Zacharisen,

2002)

Puruckherr et al.

Brooks, Weiss

Metal coat remover

Metal working fluids [MWF]

+

x*

nd

1/1

5 as irritant asthma (total irritant

nd

nd

1/1

2004. 3 classified as allergic asthma,

nd

x

1/1

statistics

x*

x

nd

diseases

x*

46/46

1/1

Occupational

8

46

1

2005

8

46

1

Latza and Baur,

(SENSOR)

diseases

1994

139-07-1

“

Occupational

Richardson,

ammonium chloride

Case report

Burge and

Lauryl dimethyl benzyl

9781405157209_4_049.qxd 4/1/08 17:13 Page 1087

%

1/1

1/1

n/n

%

1/1

1/1

n/n

%

1

1

1

i(n) l(n)

1

d(n)

SIC reaction

Manual grinding of metal casting.

**PFT for 2 weeks; spec. IgE+

Metal plating factory worker.

Remarks

3

1

1

1

3

1

1

1

3/3

1/1

1/1

1/1

1/3

1/1

nd

1/1

0/3

1/1

nd

nd

2/2*

1/1

nd

nd

3/3

1/1

1/1

1/1

2

1

1

2 life guards of indoor swimming

(low); SPT+

preceding rhinitis; spec. IgE+

Forensic laboratory worker;

dermatitis

Metal plating worker. SPT+; contact

to chromium; spec. IgE+

Metal plating worker. Co-exposure

et al. 1989

Pankow, Hein

et al. 1999

Baur, Schneider

et al. 1994

Kipen, Blume

Perfume agents (research lab)

et al. 1999

Toren, Balder

et al. 1999

Daenen, Rogiers

Broder, 1989

Tarlo and

Case report

Case report

Case report

Case series

Case control

Case report

Case report

1

1

1

80

32

1

1

1

1

1

37

32

1

1

1/1

1/1

1/1

59/80

x*

1/1

1/1

*

1/1

nd

nd

x*

1/1

x*

1/1

nd

nd

nd

x*

1/1

1/1

nd

nd

1/1*

nd

nd

nd

x*

1/1

1/1*

nd

*37/50

nd

1/1

nd

1

1

1

finished, because of incompliance

Hairdresser. *LFT could not be

IgE−

“Must de Cartier”; SPT− and spec.

Saleswoman. *SIC with perfume

dose RADS” ; *BD+

Profession not mentioned. “Low

not listed

dermatitis; individual reaction type

(2% PPD)+: 12/33; 18/80 contact

80 fur industry workers; patch test

data not listed

1996. OR 2.1, sign.;*individual

32/294 occup. asthma cases in

Electric industry worker. SPT+

symptoms persistent for 3 months

Consecutive worker; asthmatic

4 weeks

periods 10–14 years; *PFT over

Case reports

Case report

Case report

Case report

pools, 1 swimming teacher. Latency

Sorrell, 1959

“, not specified

0/1

0/1

n/n

SIC

et al. 2002

106-50-3

Persulphate

0/1

0/1

%

PFT

McCoach

Thickett,

et al. 1997b

Piirilä, Estländer

et al. 1973

McConnell, Fink

et al. 1983

Novey, Habib

Silberman and

“

1/1

1/1

n/n

BHR

IgE+; allergic contact dermatitis

1

1

+) (+

LFT

Co-exposure to chromium; spec.

1

1

%

%)

n n/n

(prevalence,

studied,

1993

Case report

Case report

Study type

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Kanerva et al.

Estlander,

Paraphenylendiamine

Paper dust A111

Palladium 7440-05-3

Paint (fumes)

Nitrogen trichloride

Ninhydrin 485-47-2

“

“

“

Malo, Cartier

“

et al. 1982

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1088

Muñoz, Cruz

et al. 2003

“, Dipotassium

peroxo-peroxodisulfate

et al. 1997

Blainey, Ollier

persulfate] 7727-54-0

“, “

69.1

1/1

10.0

x*

x*

0/1

1/2

0/23

1/1

nd

1/1

nd

6/23

32/53

7/8

1/1

x*

nd

nd

nd

2/2

1/12

nd

nd

nd

1/1

2/2

1/1

nd

4/9

9/41

7/7

1/1

21/47*

x*

28.0

57.0

2

5

1

4

1

1

4

5

1

14

4

1

3

Hairdressers. SIC with ammonium

irritant respir. symptoms; 9/24 SPT+;

Plant workers; 7/24 allergic, 8/24

Hairdresser

Chemical factory workers

peroxodisulfate (3/3 late); 1/23 SPT+

SIC+ also challenged with potassium

Hairdressers. SIC with bleach, 3/4

Hairdressers; 13/54 SPT+

5 hairdressers

3 cosmetic industry workers,

Hairdresser; SPT+, spec. IgE−

associated with SIC+

6/15

nd

“

“

et al. 1972

Pepys, Pickering Case reports

sectional

Bellander

et al. 1984

Cross-

Hagmar,

2

516

2

170 (32.9)

2/2

170/516

32.9

0/2

nd

nd

nd

nd

nd

2/2

nd

2

Chemical industry workers

duration

symptoms sign. related to exposure

12% chronic bronchitis; asthma

27% work-related wheezing,

work-related attacks of dyspnea,

Chemical workers; 33%

bronchitis increased (24/117)

asthma exposure-related; also

had suspected occupational asthma,

additionally 16 former employees

Factory workers; co-exposure to

*individual data not listed

12 late and 1 dual asthma type;

nd

other amines. *According to WRS,

13/130*

et al. 1982

13 (10.0)

Bellander

130

142-64-3

Survey

duration of symptoms 2 years;

Hagmar,

Broder, 1989

Co-exposure to hydrochloric acid;

1

15/24

30.4

3/55

5/6

nd

nd

individual data not listed 1

15

1/1

2/2

7/23

38/55

8/8

1/1

57.1

Tarlo and Case report

24

1

2

4 (17.4)

9 (16.4)

7

1

12/21**

Piperazine dihydrochloride

Phosgene

with controls

and Mathews,

1

2

23

55

8

1

0/21

1973

Case series

Kammermeyer

Case report

Case reports

hydrochloride 39878-87-0

1989

Cuesta et al.

Gamboa, de la

et al. 1979

Baur, Fruhmann

Survey

Phenylglycine acid chloride

“, “

“, “

Vogelmeier

peroxodisulfate [ammonium

et al. 1986

Schwaiblmair,

“, Diammonium

7727-21-1 Survey

cases

1992

[potassium persulfate]

Follow-up of

Parra, Igea et al.

7775-27-1

Case report

47/47

dermatitis; 6/21 BD+; **BHR+ sign.

21

persulfate; 11/21 rhinitis; 8/21

47

2005

Case series

Pignatti et al.

Moscato,

“, Sodium persulfate

“, “

9781405157209_4_049.qxd 4/1/08 17:13 Page 1089

Polyester

Polyamines, aliphatic

“

“

“

“

“

70.0

23/24

nd

%

nd

nd

nd

n/n

PFT %

24/24*

nd

nd

n/n

SIC %

7

2

i(n) l(n)

1

d(n)

SIC reaction

Refinery workers. *Individual

risk of smokers

22/30 SPT+; dose-related increased

24 months; *10/32 PSS with SPT−;

(bronchospasm, rhinitis etc.) within

32/78 new-onset symptoms

New recruits of refinery workers.

new chest symptoms

14/227 SPT+; *6/227 SPT− with

within 5 yrs, smoking-related;

9/14 with new chest symptoms

lung function impairment and BHR+;

Exposure-related respir. symptoms;

Catalyst production employees.

Remarks

follow-up Crosssectional

Case reports

et al. 1989

Baker, Gann

et al. 1990

Pepys and

diseases statistics

Hnizdo et al.

2001

survey Comparative survey

1995

Zuskin,

Mustajbegovic

et al. 1998

Comparative

Ng, Lee et al.

(SORDSA)

Occupational

Esterhuizen,

Pickering, 1972

Survey with

Venables, Dally

400

12

29

16

107

91

4 (1.0)

4

29

10

13

49 (53.8)

4/400

4/12

x

16/16

28/107

49*/91

x*,**

0/12

x

5/16

6/107

nd

nd

nd

x

nd

3/107*

nd

x*

4/12*

nd

nd

nd

nd

nd

nd

x

10/16

nd

nd

**individual data not listed

(32/92); *sign. FEF75 decline;

sign. increased in male workers

Synthetic textile workers. Dyspnea

*PFT for 1 week

Polyamide resin factory workers.

due to platinum in 1997–1999

29/324 occupational asthma cases

SPT+; 7/11 nasal challenges+

Platinum refinery workers; 10/16

cold air

15/107 SPT+; *BHR+ done with

Current workers in metal industry;

related to smoking

Refinery workers; 22/49* SPT+,

19 months (1–77).

after removal from exposure for

no sign. change in BHR and SIC

2/24

7/10*

11/187

n/n

BHR

reaction type not listed; at follow-up

41.0

%

follow-up

23/24

32/78

x

n/n

LFT

series with

24

7 (9.0)

+) (+

one case:

et al. 1994

24

78

9/227

%

Case

cohort study

6*(2.6)

227

n/n

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Merget, Reineke

Prospective

et al. 1995

cohort study

et al. 2000

Calverley, Rees

Prospective

Merget, Kulzer

Platinum salts 7440-06-4

“

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1090

15.1

8.8

nd

1/1

1/1

nd

1/1

1/1

1/1**

1/1*

1/1*

1

1

1

4

1

Plexiglass factory worker. *BD+,

polyethylene (76°C)

Paper packer; *SIC with heated

heated repair tape

Electric cable repairer; *SIC with

Andrasch,

Bardana et al.

Polyvinyl chloride (fume)

9002-86-2

“

“

“

“

Potroom aluminum smelting survey

Wong et al.

7/57

34/57

x

5/7

nd

x**

nd

nd

nd

x**

4/4

nd

1/1*

nd

nd

nd

nd

nd

nd

nd

7/7

3/3*

1/1

3/11

1/1

2

2

1

1

1

3

re-SIC+ (dual) 2 days later

density 6.1/1000 workers; 122/179

2845 person years); incidence

179 cases in 1970–1990 (during

54/227 chronic bronchitis;

*7/227 wheezing and dyspnea;

Workers of aluminum smelter;

duration of exposure

*FEV1 sign. negatively related to

Employees with > 10 yrs of exposure.

worker effect

in potroom. Evidence for healthy

workers with > 50% working time

higher and FEV1 decreased in

controls; respir. symptoms sign.

of controls); **PFT not sign. vs.

*Wheezing (15.1% vs. 10.5%

Workers of aluminum smelter.

nickel chloride proven

SPT+; in 2 subjecs co-sensitization to

Electroplating industry workers. 2/7

BD+ 2/2

Meatwrappers; *SIC at workplace;

PVC dust; *PFT for 3 weeks

Bottle caps factory worker. SIC with

brochospasm at work

Meatwrappers. *33/96 with

Bag factory worker

1989

Welford et al.

O’Donnell, Case series

BD+; **individual data not listed

Workers of aluminium smelter; *7/7

1 year of exposure 57/57

x*

20/227

x*

nd

nd

nd

1/1

nd

1/1

*sign FEV1 decline after more than

34

x

3.1

19.1

x

4/7

2/3

nd

14/14

1/1

et al. 2001 57

179

7/227*

122/641

126/797*

7/7

3/3

1/1

33/96*

1/1

workers at follow up 5 years later;

up

et al. 1998;

179

7 (3.1)

122 (19.1)

126 (15.1)

7

3

1

3

1

de Looff

with follow

de Looff

227

641

797

7

3

1

96

1

Sorgdrager,

Case series

Sorgdrager,

Cvar et al. 1986

Saric, GodnicSurvey

study

Grønnesby

et al. 1990

Longitudinal

Kongerud,

1983

Comparative

Case series

Case reports

Case report

case series

Survey with

Case report

Chan-Yeung,

et al. 1997

chromium;cement)

Bright, Burge

7778-50-9 ( see also

et al. 1973

Sokol, Aelony

1989

Lee, Yap et al.

Potassium dichromate

“, ( fume and dust)

“, (resin dust)

et al. 1994a

250°C 9003-07-0

1976

Malo, Cartier

Polypropylene, heated to

additionally hemoptysis,

1/1*

1/1

1/1

**SIC with plexiglas dust,

1/1

1/1

1/1

1981

1

1

1

Herreros et al.

1

1

1

[plexiglas powder] 9011-14-7

Case report

Case report

Case report

Kennes, Garcia-

et al. 1992

Gannon, Burge

et al. 1989

Stenton, Kelly

Polymethyl-methacrylate

“, heated to 160°C

“, heated to 140°C

Polyethylene 9002-88-4

9781405157209_4_049.qxd 4/1/08 17:13 Page 1091

Powder paints

“,”

“, slaughtery house

“

“

Poultry confinement

“

“

(representative

sectional

Case reports

et al. 1990

Perfetti, Cartier

sectional

Magarolas

sectional

Düzakin-Nystedt

et al. 2005

Cross-

Blomqvist,

et al. 2002

Cross-

Borghetti,

et al. 1997

Cross-

Hagmar, Schütz

sample)

Case series

et al. 2001

Survey

Review

Danuser, Weber

et al. 1988

Danuser, Wyss

Merget 2006

Bernstein &

et al. 2000

Burge, Scott

1994 Case report

Case report

Desjardins,

“

Bergeron et al.

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

118

15

4

23

37

26

1

1

23 (21.9)

1 (7.1)

4

2 (8.6)

37

10 (38.0)

1

1

23/105*

5/14

4/4

2/23

37/37

14/26

x

1/1

1/1

n/n

30.5

35.7

8.6

53.8

%

+) (+

one case:

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

x**

2/5

0/4

0/23

nd

10/26

x

1/1

0/1

n/n

LFT

40.0

38.0

%

nd

nd

nd

nd

nd

nd

1/1

1/1

n/n

BHR %

nd

nd

4/4

x*

nd

nd

0/1*

1/1*

n/n

PFT %

nd

1/5*

nd

nd

nd

nd

x

1/1

nd

n/n

SIC

20.0

%

i(n) l(n)

1

1

d(n)

SIC reaction

questionnaire)

asthmatic symptoms (according to

**no sign. declines in LFT. 32/119

and to triglycidyl isocyanurate;

to various organic acid anhydrides

exposure-related; IgE−; co-exposure

(according to physician) sign.

*Work-related asthmatic symptoms

Employees of powder paint shops.

SIC+ also allergic to storage mite

Farmers’ Project. *Subject with

participated in the European

Spanish poultry farmers who

to chicken feathers

Slaughterhouse workers. 4/4 SPT+

VC and FEV1 declines cross-shift

houses. 2/23 chest symptoms; *sign.

Shacklers of poultry slaughter

OR 2.87, not sign.

Swiss poultry farm workers. Asthma

of occupation

decline sign. related to duration

Swiss poultry farm workers. FEV1

causative agent(s) are unknown

Association to RADS possible. The

workers continue to have asthma.

exposure. About 40% of former

1 week and 10 years after first

First asthma symptoms between

(OASYS-2 score 2.67)

with positive occupational effect

with aluminum chloride; *PFT

Caster of molten aluminium. SIC

3 weeks

Worker of aluminum plant. PFT for

Remarks

9781405157209_4_049.qxd 4/1/08 17:13 Page 1092

4

Korean workers in dye-industry. 55

Workers of textile plants in dye

asymptomatic)

spec. IgE+ (23/53 also SIC+ were

SPT+ ( 5 with SIC+ had SPT−); 53/309

“

“

“

“

“

8050-09-7

decomposition [colophony]

Rosin core solder, thermal

mixture

x*

nd

nd

x*

34/51**

nd

34

*94/1765 physician-diagnosed

with abietic acid (5/6 immediate)

**only sensitized with SIC+; SIC also

asthmatic symptoms before survey,

with occup. asthma and 17/51 with

Electric industry workers. *34/51

intermediate exposure group)

exposure group, 21% in highest and

asthma dose-related (4% in lowest

Solder manufacturers. Prevalence of

Malo, 2006

diseases

Burge, 1993

Review

Systematic

(SHIELD)

statistics

Occupational

Gannon and

(SWORD)

237

41

41

x

41/41

x

nd

nd

x

x

x

1989–1991

41/500 occup. asthma cases in

data not listed

x*

16/31

nd

asthma cases in 1996; *Individual

x*

nd

x

statistics

x*

51/51*

5/45

disease

94

34

5 (11.1)

Occupational

94

51

45

persistent for > 4 years

spill (dust). Asthma symptoms

Millwright in steel mill exposed to

smokers after 10 years

et al. 1997

Case series

Survey

1/1

FEV1 and FVC declines in male

Ross, Keynes

et al. 1980

Burge, Harries

et al. 1981

Burge, Edge

1/1

Forrester, 1997

“, phosphoric acid binder

1

of controls), dyspnea 2° in females

Case report

dyspnea 1° in males (15.7% vs. 2.5%

study

(10.5% vs. 0.0% of controls); sign.

workers; sign respir. symptoms:

Refractory ceramic fibers industry

longitudinal

nd

Worker of textile manufacturing;

blue K-BL; 3/4 spec. IgE+; 4/4 SPT+

sectional and

nd

1

et al. 1998

nd

1/1*

[RCF]

1

nd

anaphylactic reaction; SPT+ x

0/1

brilliant scarlet 3 R, drimaren brilliant

Cross-

x

0/1

Workers of dye industry. *SIC with

Lemasters 742

1/1

4/4*

nd

7

*SIC with lanasal yellow 4G and

1

nd

0/2

1

1992

1

11.1

+

50.0

5

Sulotto et al.

Romano, Case report

3/4

3/6

13/78

brilliant yellow K-GL, cibachrome

nd

13.3

nd

levafix brilliant yellow E-36, drimaren

4/4

2/15

38/78

et al. 1978

4

3.7

nd

Keskinen

4

6/162

78/309

symptoms and LFT+ or BHR+

Alanko, Case reports

4* (2.5)

13 (4.2)

houses. *4 workers with asthma

162

309

et al. 1993

Survey

Survey

Nordlinder

Nilsson,

1991

Park, Lee et al.

Refractory ceramic fibers

“

“

Reactive dyes

9781405157209_4_049.qxd 4/1/08 17:13 Page 1093

et al. 1990

oxybenzene sulphonate

2004

Malo, Cartier

7681-57-4

“

Soldering flux (fumes)

“

Sherson et al.

Stevens, 1976

Korn, 2005

Merget and

et al. 1995b

Madsen,

[metabisulfite sodium]

1990

Thomas et al.

Ferguson,

1988

Connolly et al.

Sodium metabisulfite

“

“

Hendrick,

Stenton, Dennis

Sodium iso-nonanoyl

Case report

Case report

Case report

Case report

Case report

Case report

1

1

1

1

1

1

3

Case reports

397

2

sectional

Case reports

Cross-

et al. 2005

Survey

(SENSOR),

Ekici, Ekici

Weiss, 1996

[SINOS] 123354-92-7

nd

nd

nd

n/n

%

1

3

i(n) l(n) d(n)

23/430 new-onset asthma in

13 months

burning book store. RADS for

Accidental smoke exposure in

Persistent asthma symptoms

1 fire fighter, 2 accidental exposures.

Remarks

1

1

1

1

1

1

3

2

113 (28.5)

1/1

1/1

1/1

1/1

1/1

1/1

3/3

2/2

113/397

0/1

0/1

0/1

1/1

nd

nd

0/3

2/2

x

nd

1/1

0/1

1/1

nd

1/1

2/3

2/2

nd

nd

1/1

nd

nd

nd

0/1

nd

nd

nd

1/1

1/1

1/1

1/1

1/1

1/1

3/3

nd

nd

1

1

1

1

1

exposure

respir. symptoms within days after

Electronic assembler developing

Radiographer

Agricultural producer

Fisherman

over 18 months

Research worker with high exposure

Laboratory technician

Development technicians

> 2 years

massive spill; asthmatic symptoms

Material handlers exposed to

13.6% of controls

women. COPD prevalence 28.5% vs.

Stove smoke-exposed non-smoking

1993–1996; *individual data

nd

nd

%

not listed

nd

1/1

nd

n/n

SIC reaction

statistics

x*

nd

%

SIC

2001

+

+

1/1

n/n

PFT

diseases

34/34

1/1

1/3

+

%

BHR

Occupational 34

1

3/3

n/n

+) (+

LFT

Harrison et al.

34

1

3

3

Case reports

Case reports

%)

n

Study type %

(prevalence,

studied, n/n

or at least

cases, n

subjects one case:

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Reinisch,

et al. 1985

Sodium azide (powder dust)

Smoke, (biomass, indoor)

“

“

Brooks, Weiss

Moisan, 1991

Smoke (fires, pyrolysis

products)

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1094

5/469 asthma claims between mid

1993–1995. 8/131 RADS cases

8/424 occup. asthma cases in

data not listed

1996. OR 2.1, sign.; *individual

38/294 occup. asthma cases in

(ammonium chloride): immediate

see also zinc chloride; 1/1 SIC+

fluxes; co-exposure to zinc chloride,

man. Use of soft corrosive soldering

1 tin maker and 1 car radiator repair

79.7

(+)*

+

nd

nd

0/2

3/3

nd

nd

2/2

3/3

10/69**

nd

nd

nd

nd

nd

2/2

nd

Workers of apricot farms. *55/69

to SO2 gassing

SO2 exposure 63%, and 75% due

fraction of incident asthma due to

rate sign. increased; attributable

Sulfit mill workers. Asthma incidence

Plastic factory workers

1 RADS for 56 months

Painters. 2 RADS for 4 months,

1

1/1

x*

6/9

1/1

4/7

x*

nd

nd

nd

FEV1/ VC

RADS. 6/7 re-examined after 4 and

in pyrite mine in 1977. *7/9 had

Men accidently exposed to SO2

Case report

Boulet, 1988

Sulphuric acid, fume

Case report

Case reports

Broder, 1989

Tarlo and

1

2

1

2

1/1

2/2

0/1

nd

nd

nd

nd

1/1

nd

2/2*

SIC−, BHR+

Cleaner. 2 years after exposure:

not listed

Nurses. *Individual reaction type

listed.

*individual data for LFT/PFT not

to sulphuric acid and chlorine;

asthma symptoms; co-exposure

Profession not mentioned; persistent

pattern.

with persistent obstructive lung

4 subjects with persisting BHR; 2/6

exposure due to pulmonary edema;

1

9/9

13 years; 1 subject died hours post

follow-up

1983; Piirilä,

7

1996

series with

Nordman et al.

9

Nordman et al.

Case

Härkönen,

also as a group in FVC, FEV1,

55/69*

35/674

2/2

3/3

dyspnea; **PFT+ with sign. declines

10 (14.5)

35 (5.2)

2

3

et al. 2003

69

674

2

3

and /or BHR+; **1/5 RADS

Hasanoglu

Koksal,

Survey

sectional

2006

Cross-

Knutsson et al.

Case reports

Case reports

Anderson,

1987

Biscaldi et al.

Moscato,

et al. 1985

Brooks, Weiss

Rosberg, 1946

8014-95-7

5.2

2

1

work-aggravated asthma; *5/5 BD+

nd

nd

1

retrospective review: 4 cases with

nd

nd

nd

2/2

(WCB)

x*

x

nd

2/2

1984 and mid 1988 identified by

nd

x

x*

2/2

statistics

(+)**

+

x*

2/2

diseases

5/5

8/8

x*

2/2

Occupational 5

8

38

2

et al. 1999

5

8

294

2

Chatkin, Tarlo

2003

Derk et al.

Case series

study

et al. 1999

Henneberger,

Case control

Case report

Toren, Balder

et al. 1989

Weir, Robertson

Sulphathiazole 72-14-0

“

“

“

Sulfur dioxide

Styrene monomer 100-42-5

Spray paint

“, “ glue

“, “

Solvents (not specified)

“

9781405157209_4_049.qxd 4/1/08 17:13 Page 1095

nd

nd

nd

1

Intensive swine facility workers.

related; **individual data not listed

respir. symptoms, sign. exposure-

Project. *24.3% with work-related

Pig farmers in the European Farmers’

endotoxin exposure.

declines in FEV1, FVC, and FEF25-75

Pig farmers. *Sign. Longitudinal

exposure

1/1

1/1

1/1

+

1/1**

nd

1/1 nd

nd

1/1

1/1

0/1

1/1

nd

1/1*

nd

1

Teacher with repeated exposure.

for 2 weeks

with Pamtac 1500 (heated); **PFT

Rubber tire industry worker. SIC

Massive exposure

1/1 BD+

Seaton, Cherrie

et al. 1988

Terpene (3-carene)

13466-78-9

Christiani, 1992

Hu and

et al. 2000

Hill, Silverberg

1999

Case report

Case report

Case report

1

1

1

1

1

1

1/1

1/1

1/1

+

nd

1/1

1/1

nd

1/1

nd

0/1

nd

nd

1/1

nd

nd

rubber glove vapours

10 years. Asthma provoked by

Laboratory technician for about

symptoms > 2 years

Massive spill in nightclub; respir.

> 6 months

General and respir. symptoms over

lichenoid dermatitis, fever); SPT+.

generalized symptoms (spongiotic

Prisoner. Massive exposure with

symptoms over 6 months

1

1

1

“Low level RADS”; duration of

1

1

1

Deparis et al.

Case report

“

3/4

nd

nd

Dunglas,

Bayeux-

Tear gas

Case report

Case report

et al. 1996

Cormier, Coll

Tarlo, 1992

“

+

0/4

nd

x**

between 4.5 and 48 months;

4/4

x**

nd

Onset of first respiratory symptoms

4

x

x*

2004

4

2278

x

Lawson et al.

Dosman, Case reports

sectional

Büsching

et al. 2002

Cross-

Radon,

168

73 mL/yr, sign. related to endotoxin

and FEF25-75 decline also related to

Pig farmers. *Sign. FEV1 decline

Remarks

related to cross-shift declines; *FEV1

d(n)

cohort study

554 (24.3)*

i(n) l(n)

longitudinal

%

et al. 1995

nd

n/n

based

%

Donham

nd

n/n

Population-

%

Schwartz,

nd

n/n

SIC reaction

follow-up

x*

%

SIC

1998

82/171

n/n

PFT

study with

%

BHR

Longitudinal

171

n/n

LFT

der Gulden,

Study type

+) (+

one case:

(prevalence,

studied, %)

or at least

cases, n

subjects n

cases: +

asthma

exposed Asthma

RADS all

Irritant

Occupationally

Work-related symptoms

Evidence (pathological results)

Vogelzang, van

Reference

Tall oil

“

“

“

“

“

Swine confinement

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1096

Bittner,

[perchloroethylene] 110-01-0

et al. 1992

Vandenplas,

Delwiche et al.

2000

Burge and

(fungicide)

Tetramethrin

[1-(5-tretrazoly)-4-guanyl-

tetrazene hydrate] 7696-12-0

“

et al. 1992

Savonius,

[carpet fungicide]

Triethanolamine 102-71-6

Tylosin tartrate

Frigas, Filley

“

+

nd

nd

nd

Carpenters. Exposure to dust from

40 open cast miners with persistent

symptoms over 6 months

Stewardess; duration of respir.

decline – 14%)

1/2 borderline immediate (FEV1

urea formaldehyde sawdust with

“,“

“,“

diseases statistics

2001 (SORDSA)

Occupational

Hnizdo et al.

Case reports

Survey

Esterhuizen,

1982

Musk and Tees,

et al. 1991

8

4

11

8

4

7

x

4/4

x*

x

3/4

0/11

x

2/4

7/11

nd

x

due to vanadium in 1997–1999

8/324 occupational asthma cases

listed

*individual asthma symptoms not

power station. BHR sign. increased;

of ashes and clinker in oil-fired

Workers exposed during removal

12/40

nd

1

UF foam exposure

exposure

Pistelli, Pupp

3/40

1/1

1

1

Chemical worker with accidental

Laboratory worker

Tungsten carbide worker

Metal workers

“,“

40/375

nd

2/2*

1/1

nd

1

1

Venipuncture technician

Hospital pharmacist

firm

Worker of insect pest extermination

Farmer

resp. symptoms

12

1/1

nd

nd

nd

1/1

nd

1

1

1

1

1

natural gas; *2/2 BD+

Workers engaged in odorizing

et al. 1999

375

1

2/2

nd

1/1

nd

nd

2/2

1/1

1/1

1/1

1/1

nd

Irsigler, Visser Case series

1

0/2

0/1

nd

1/1

2/2

nd

nd

nd

1/1

nd

1314-62-1

et al. 1996b

Lemière, Malo Case report

2/2

+

nd

nd

1/2

1/1

1/1

1/1

1/1

nd

nd

0/2

0/1

0/1

0/1

0/1

2/2*

formaldehyde; *SIC with cedar

2

1/1

1/1

1/1

1/1

2/2

1/1

1/1

1/1

1/1

2/2

western red cedar chips with urea

2

1

1

1

1

2

1

1

1

1

2

1982

Case reports

1

1

1

1

2

1

1

1

1

2

Hoeppner et al.

Cockcroft,

Case report

Case report

Case report

Case report

Case report

Case report

Case report

Case report

Case report

Case reports

“, divanadium pentoxide

Vanadium 7440-62-2

“

Urea (fume) 57-13-6

et al. 1985

7783-81-5

et al. 1981

Brooks, Weiss

Uranium hexafluoride

et al. 1989a

Bruckner, 1967

Lee, Wang

Tungsten carbide 7440-33-7

1994

Keskinen et al.

Shelton, Urch

Tributyl tin oxide

1994

Richardson,

Honda, Kohrogi

Tetrachloroisophthalonitrile

submitted

Baur and

Tetrachloroethylene

9781405157209_4_049.qxd 4/1/08 17:13 Page 1097

%)

studied, n

i(n) l(n)

d(n)

Remarks

sectional

Aizawa et al.

Occupational diseases statistics Case series with

Karjalainen,

Martikainen

et al. 2002c

Hannu, Piipari

et al. 2006

“

“

“

“

6/6

2/2

0/6

0/2

5/6

0/2

9/15

nd

14/14

18/34

nd

x*

nd

x*

x

x*

x*

x*

nd

1/1*

nd

nd

nd

nd

nd

nd

3/6

2/2**

34/34

nd

nd

nd

nd

nd

9

16

9

3

2

Welders. SIC with mild steel,

electrode; *PFT for 1 week

steel with a nickel/molybdenum

**SIC with SMO steel or duplex

Metal arc welders on stainless steel.

Workers of metal industry

Construction workers

FVC, FEV1, FEF 25 –75% as a group

and sign. spirom. declines in VC,

asthma symptoms (6.3% vs. 1.3%)

Manual steel arc welders. Sign.

related; *individual data not listed

Welders. BHR+ sign. exposure-

not listed

FEV1/ VC decline; individual data

chronic cough (11/143) and with

exposure sign. associated with

Electric arc welders. Cumulative

data not listed

in 1996. OR 2.0, sign.; *individual

26/294 occupational asthma cases

steel; conc. 3.4–150 mg/m3

3

2

34/34

14/14

4/63

x*

x*

x*

stainless steel and/or galvanised

6

2

34

14

4

x*

x*

26

1997

Case series

Case reports

34

14

63

682

143

26

Chan-Yeung,

Contreras and

et al. 2005

follow-up

Case control

Jafari 2004

“

Hannu, Piipari

sectional

et al. 1996

“

Cross-

1998

Cross-

Case control

Nakadate,

et al. 1999

Toren, Balder

(11.9%); sign. FEV1 decline (−8.4%)

5/194 new-onset asthma cases asthma; increase in incidence of BHR

nd

22/333 chronic bronchitis

Employees of vanadium plant;

cohort study

nd

nd

/incidence 3%) plus 1/6 aggravated

46/194

nd

prospective

22/194

nd

logical

14/193

52/326

Epidemio-

5

59/333

et al. 2003

194

59

El-Zein, Malo

Beach, Dennis

“

“

Welding fumes

333

(17.7% vs. 5.1% of controls);

%

work-related asthma sign. increased

n/n

statistics

%

diseases

n/n

Occupational

%

SIC reaction

Rees, 1997

n/n

SIC

Kielkowski and

%

PFT

“,“

n/n

BHR

Study type

+) (+

LFT

Reference %

(prevalence,

subjects n/n

or at least

cases, n

exposed one case:

cases: +

asthma

Occupationally

Asthma

RADS all

Irritant

Work-related symptoms

Evidence (pathological results)

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1098

1

examinated, out of them 2/3 SIC+:

Welder manufacturing automatic

1 immediate, 1 late)

28/835 occupational asthma cases

x*

x*

22/1765 physician-diagnosed

diseases statistics

Harrison et al.

2001

48 [incidence

x

20/123*

+

x

27/151

0/1

nd

24/112

1/1

nd

nd

nd

nd

nd

1/1

RADS (diagnosed 6 months after

exposed fire-fighters developed

17/83 of highly, 3/40 of moderately

Longitudinal study

Banauch, Hall

et al. 2006 (0.4)]

desaster.

in 45 exposed before, in 93 after

disaster in each group; FEV1 < 60%

late-arrival; sign. loss of FEV1 after

number) early- > intermediate- >

arrival-related: severity (i.e., greater

respir. symptoms sign. time of

Exposed FDNY rescue workers;

WTC

of spirometric parameters post

BHR in 55%; *sign. declines

(31%, 10%); after 6 mo. persistent

11 766

20 (16.3)

1/1

disaster). BHR exposure-related

study/review

2003; Banauch,

123

1

than “mild steel”

Stainless steel sign. stronger irritant

2005

cohort

1

3.0–5.0]

about

[incidence

Dhala et al.

Prospective

Alleyne et al.

Case report

Review

Banauch,

1998

Delwiche et al.

Vandenplas,

Merget, 2006

Bernstein and

not listed

6/324 occupational asthma

not listed

1993–1996. *Individual data

9/430 new-onset asthma in

1997–1999. *Individual data

6/6*

nd

(SORDSA)

6/6*

nd

cases due to welding fumes in

nd

nd

statistics

nd

x*

diseases

6/6*

9/9

Occupational

6

9

Keskinen, 2005

6

9

Piipari and

Survey

(SENSOR),

Occupational

Reinisch,

(SWORD)

data not listed

x*

asthma cases in 1996. *Individual

x*

statistics

x*

diseases

22

et al. 1997

22

Occupational

Ross, Keynes

desaster

“

+

1

Metal arc stainless steel welders. (2 years later, 3 subjects re-

cases). *Individual data not listed

x*

1/1*

1

in 2003 (28/210 irritant asthma

nd

nd

1

statistics

nd

1/1

2/3

diseases

x*

1/1

nd

2005

x*

1/1

2/7

Occupational

28

1

0/7*

Latza and Baur, 28

1

5/7

welding on mild steel

Case report

Vandenplas,

5

gates. *SIC with gas metal arc

follow-up

et al. 1980

7

1995

series with

Kalliomäki

Dargent et al.

Case

Keskinen,

World Trade Center

“, aluminium

“

“

“

“

“

“

“

9781405157209_4_049.qxd 4/1/08 17:13 Page 1099

22/119

55/240

x*

37/240

9914

332/

n/n

%

nd

nd

9914

332/

n/n

%

nd

nd

nd

n/n

%

nd

nd

nd

n/n

SIC %

1

2

i(n) l(n)

d(n)

SIC reaction

Clean-up and recovery workers.

to exposure intensity

abnormal spirometry sign. related

Dyspnea and prevalence of

Emergency services police officers.

high, 8% of moderate exposure)

of low exposure), and BHR (23% of

(8% of high, 3% of moderate, 1%

315 dyspnea; dose-related cough

cough; sign. FEV1 and FVC declines;

Firefighters; 332/9914 persistent

Remarks

2

Case reports

Weir, Robertson

et al. 1989

Zinc chloride (fume)

7646-85-7

2

1

1

2

2/2

1/1

1/1

2/2

2/2

1/1

0/1

2/2

nd

2/2

nd

1/1*

nd

1/1

1/1

2/2

2/2

nd

1/1**

2/2

2

chloride)

chloride (see also ammonium

fluxes, co-exposure to ammonium

1 car/truck repairer. Use of soldering

1 worker of tin-making industry and

metal fume fever

Welder. Presenting additionally

**SIC with zinc sulfate; SPT+

Metal plant worker. *PFT+ (late);

metal fever

galvanized metal. 1 subject with

Solderers exposed to fume of

exposure-related

test result; x: test done, no individual results listed; nasal Ch: nasal challenge test; conj.Ch: conjunctival challenge test; oral Ch: oral challenge test; HR: histamine release test; PK: Prausnitz-Küstner test.

specific IgE antibody measurement; *, **, ***: for details see column “Remarks”; nd: not done; WR: work-related; WRS: work-related symptoms; OA: occupational asthma; sign.: significant; P: pathology; restr. = restrictive ventilation pattern; +: positive test result; −: negative

PEFR showing significant change in follow-up pre-, (during) and post shift; SIC: specific inhalative challenge test; i: immediate, d: dual, l: late response type; SPT: significant positive skin prick test result; IC: significant positive intracutaneous test result; IgE: positive result of

n: number of subjects; n/n: number of subjects with work-related symptoms or positive test results/all investigated subjects; LFT: lung function test showing obstructive ventilation pattern; BHR: bronchial hyperresponsiveness; BD: significant bronchodilator effect; PFT: FEV1 or

1

1

2

Case report

Case report

Case report

Kawane, 1988

Cartier, 1993

Malo and

Cartier, 1987

Malo and

prevalence of symptoms

with *6% reduced FEV1 (sign.);

22 (18.5)

55 (18.5)

+

+) (+

PFT

22/119 new-onset wheezing

119

240

332/9914

%

BHR

2005

Survey

Survey

332 (3.3)

n/n

LFT

Frank et al.

Herbstman,

Moosavy, 2004

Salzman,

workers

“

“

Zinc (fume) 7440-66-6

“

“

follow-up of

et al. 2002 exposed

9914

Survey with

Prezant, Weiden

“

%)

(prevalence,

studied, n

or at least

cases, n

subjects one case:

cases: +

asthma Asthma

RADS all

Irritant

exposed

Work-related symptoms

Evidence (pathological results)

Occupationally

Study type

Reference

CAS No.

Agents [synonyms]

Table 49.2 (Cont’d )

9781405157209_4_049.qxd 4/1/08 17:13 Page 1100

9781405157209_4_049.qxd 4/1/08 17:13 Page 1101

CHAPTER 49

Airborne Allergens and Irritants in the Workplace

6–32% of exposed employees develop a febrile syndrome (grain fever), asthma, or an asthma-like syndrome. Crosssectional, as well as longitudinal, studies demonstrate lung function impairment in a dose–response relationship. Since declines in forced vital capacity and forced expiratory volume in 1 s (FEV1) have been consistently reported, grain dustinduced inflammatory reactions obviously involve lung parenchyma. Recent studies indicate that the predominant causative components are endotoxins from Gram-negative bacteria, which are responsible for the febrile syndrome. Further asthma-inducing agents in grain dust are allergens from durum wheat, the storage mites Lepidoglyphus destructor, Acarus siro, and the weevil Sitophilus oryzae (Lunn & Hughes 1967; doPico et al. 1982; Kleine-Tebbe et al. 1992).

reported to have an increased asthma risk, among others, due to the degradation products of polyvinyl chloride (Markowitz 1989).

Drug manufacturing plants

Meat wrapping

Drug manufacturing and application may be associated with airborne dust containing asthma-inducing raw material, intermediate, or end products. These include amoxicillin, amprolium (the latter also causes asthma in poultry feed mixers), ceftazidime, cephalosporins, cimetidine, hydralazine, ipecacuanha, isonicotinic acid hydrazide, methyldopa, mitoxantrone, opiate compounds, penicillamine, penicillins and ampicillin, phenylglycine acid chloride, piperacillin, psyllium, salbutamol intermediate, spiramycin, tetracycline, and tylosin tartrate.

In the 1980s, up to 57% of exposed workers were found to develop asthmatic symptoms. The etiologic agent was thought to be an aerosol or vapor of hydrogen chloride generated by cutting polyvinyl chloride meat-wrapping films using a hot wire. A potential sensitizer in the fume was phthalic anhydride (Brooks & Vandervort 1977; Pauli et al. 1980). A further irritant in the fine particulate fume was reported to be di-2-ethylhexyl adipate (Andrasch & Bardana 1975). Investigations in recent years have not identified a persistent health risk associated with meat wrapping, which may be due to improved hygienic conditions in respective workplaces.

Hairdressing salons Causative hairdressing chemicals comprise persulfate salts, natural latex, paraphenylene diamine, reactive dyes, henna, and other dyes (Gelfand 1963; Pepys et al. 1976; Starr et al. 1982; Blainey et al. 1986; Parra et al. 1992; Bolhaar et al. 2001; Hollund et al. 2001; Muñoz et al. 2003, 2004; Moscato & Galdi 2006).

World Trade Center collapse Firefighters and rescue workers exposed to high concentrations of smoke and alkaline dust including debris, asbestos fibers, and many chemicals developed cough and reactive airways disease syndrome (RADS) in a dose-dependant manner (Weir et al. 1989; Prezant et al. 2002; Banauch et al. 2003, 2005; Salzman et al. 2004; Herbstman et al. 2005). Investigations of World Trade Center rescue workers show some limitations because of the fact that RADS was not established until 6 months later and not within 24 hours after exposure (there was no reporting of inordinate numbers of workers visiting emergency rooms within the first 24 hours). Only approximately 3% of the surveyed workers underwent methacholine challenge, and causative agents were not identified. However, there is no doubt that this heavy exposure elicited acute and chronic airway disorders, mainly cough and irritant asthma. Hitherto rarely recognized similar disorders, especially in firefighters, have to be assumed after comparable single or repeated exposures at other sites (Brooks et al. 1985; Moisan 1991; Reinisch et al. 2001). Firefighters were

Use of cleaning agents Several recent studies demonstrate that cleaners are among the workers with the highest prevalence of occupational asthma (Murphy et al. 1976; Kipen et al. 1994; Tabar et al. 1998; Mapp et al. 2000; Reinisch et al. 2001; Rosenman et al. 2003; Medina-Ramón et al. 2005). The causative agents are mainly unknown and involve lauryl dimethyl benzyl ammonium chloride, detergent enzymes, and bleach chemicals (Sokol et al. 1973; Medina-Ramon et al. 2003; Zock et al. 2004; Gautrin et al. 2006a).

Potrooms Pot fume emissions in aluminum smelting contain various irritants such as gaseous fluorides, hydrofluoric acid, sulfur dioxide, cold tar volatiles, and other particulates. An increased asthma prevalence among potroom workers with an annual incidence of < 1.5% was reported by Norwegian and Dutch investigators (Saric et al. 1986; Wergelund et al. 1987; Robertson et al. 1988; O’Donnell et al. 1989; Kongerud et al. 1990; Desjardins et al. 1994; Sorgdrager et al. 1998; Burge et al. 2000; Gautrin et al. 2006b), but not in former studies performed in other countries (Kaltreider et al. 1972; Discher & Breitenstein 1976). There is evidence for a dose–response relationship of asthma in potroom workers (Chan-Yeung et al. 1983; Kongerud et al. 1990).

Rhinitis-inducing occupational agents From the clinical point of view it is obvious that many asthmainducing agents also affect upper airways. High water solubility and/or large aerodynamic diameters of airborne particles, gases, or vapors predispose to deposition in the upper airways. However, high concentrations of these agents can also affect the lower airways. Recently, Castano and Theriault (2006) defined occupational rhinitis by analogy to occupational asthma/chronic obstructive pulmonary disease as a type of rhinitis characterized by

1101

9781405157209_4_049.qxd 4/1/08 17:13 Page 1102

PART 5

Allergens

intermittent or sometimes permanent nasal airflow limitation due to causes and conditions attributable to the work environment. Work-aggravated rhinitis is considered a separate entity. Occupational rhinitis involves immunologic as well as irritant subtypes, the latter comprising the acute, chronic, and corrosive subgroups (Castano & Thériault 2006). Interestingly, allergic rhinitis frequently precedes involvement of the lower airways, e.g., bronchial asthma in bakers or acid anhydride workers (Baur et al. 1995b; Malo et al. 1997; Cullinan et al. 1999; Cortona et al. 2001; Gautrin 2001a,b, 2006b; Grammer et al. 2002; Karjalainen et al. 2003; Storaas et al. 2005; Walusiak 2006).

Allergen-induced occupational rhinitis It can be assumed that all allergenic occupational asthmainducing agents also elicit allergic rhinitis. Irritant-induced rhinitis including reactive upper airways dysfunction syndrome With regard to airborne irritants, the situation is more complex, especially due to their concentration-dependent effects and their different water solubility, which influences the extent of deposition on airways. Some respiratory irritants such as SO2 and ammonia are very water soluble and provoke irritation and inflammation mainly of the upper airways. Depending on the degree of exposure, they elicit irritantinduced rhinitis, also called reactive upper airways dysfunction syndrome (RUDS). The same is true of chlorine and other agents with intermediate water solubility. At high concentrations, such gases also affect lower airways and may even cause pulmonary edema. Although several publications have summarized approximately four dozen agents that cause irritant-induced rhinitis (Bascom 1993; Bardana 1995; Slavin 1998; Settipane & Lieberman 2001) (Table 49.3), systematic investigation and listing of respective noxae have not yet been performed.

Discussion The European Union directives 1967, 2001, 2004 and/or ACGIH 2007 comprise more than 400 substances classified as respiratory sensitizers and more than 200 substances classified as respiratory irritants. Respiratory irritants are frequently subject to a TWA (time-weighted average) or STEL (short-term exposure limit) in order to protect exposed workers. In comparison with Tables 49.1 and 49.2 (based on medical literature), the more extensive list in the Appendix Table also considers data on the exposure to animals and in vitro experiments as well as respective extrapolation. Together with flour allergens, plant and animal proteins as well as natural organic compounds are the predominant group of occupational asthma-inducing agents in many countries.

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Another important group comprises isocyanates, which mainly elicit occupational asthma due to nonimmunologic mechanisms. They are dominant in several industrialized areas (Table 49.2). Our summary of literature data (Tables 49.1 and 49.2) as well as those of other publications (Chan-Yeung 1986; Mapp et al. 2005; Becklake et al. 2006; Latza et al. 2007) exhibits the following asthma prevalence among exposed subjects: up to 66% by proteolytic enzymes, up to 54% by platinum, up to 25% by cleaning agents, 6–33% by flour, 2–26% by crabs and shrimps, 11–14% by poultry confinements, 4–20% by isocyanates, 6–13% by laboratory animals, 3–18% by acid anhydrides, 7% by egg proteins, approximately 5% by hairdressing agents, and 3–10% by western red/eastern white cedar dust. These figures are evidence for individual differences in the potency of asthma-inducing agents supported by other published “hit lists” of causes of occupational asthma (Gannon & Burge 1993; Sallie et al. 1994; Sallie & McDonald 1996; Rosenman et al. 1997; Ross et al. 1997; Baur et al. 1998b; Chatkin et al. 1999; Karjalainen et al. 2000; Esterhuizen et al. 2001; Kor et al. 2001; Arif et al. 2002; Ameille et al. 2003; Sastre & Quirce 2003; Henneberger et al. 2003; Baur & Latza 2005; Gautrin et al. 2006a; Hauptverband der gewerblichen Berufsgenossenschaften 2006; Malo & Chan-Yeung 2006; Proudhon 2008). It should be mentioned that susceptibility to occupational asthma due to specific agents involves atopy, bronchial hyperresponsiveness, innate and adaptive immune responses determined by the genetic background, and previous environmental exposures including microbial compounds, viral infections and smoking (the latter in asthma due to acid anhydrides or platinum salts) (Gautrin et al. 2001a). As opposed to the frequent clinical picture, there are limited scientific data on rhinitis induced by occupational allergens and irritants. More research in this field is urgently needed as occupational rhinitis (including RUDS) appears to be an important precursor of at least some agent-related occupational asthmatic disorders. The two lists of occupational agents inducing allergic or nonallergic (irritant) asthma may help to identify the causative noxa in a particular case and workplace. However, it must be assumed that many more such agents have not been reported or mentioned in journals nor included on Internet databases. These lists should therefore be continuously updated. The summary of available data on agents that induce occupational asthma, in combination with occupational disease statistics and known dose–response relationships (Baur 2003; Nieuwenhuijsen et al. 2006), will also enable practitioners, primary care physicians, and caregivers involved in diagnosis and/or education to become aware of the frequent occupational origin of airway disorders and promote improvement of corresponding legal regulations and preventive measures.

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Table 49.3 Agents, materials, industrial branches and occupations causing irritant-induced rhinitis. Agents/confinement

Materials, industrial branches, occupations

Acid anhydrides/phthalates Acrylamide Acrylates Ammonia Acid anhydrides Laboratories, chemical industry Arsenic

Plastics/construction workers Resins/plastics manufacturing Resins/plastics manufacturing Tanneries, dye production, bronzing industries,swine confinement facilities

Capsaicin (chilli) Chlorine, chloride, hydrochloric acid Chromium Cleaning agents, bleach, chloramines, glutaral Clothing fragrances Cobalt Cooking odors Cosmetic odors (hairspray) Cotton dust Cyanide, nitriles Detergents Diesel exhaust Endotoxin Environmental tobacco smoke Enzymes Exhaust fumes Flower fragrances Formaldehyde Garden sprays Hairdressing chemicals Inorganic acid vapors/mist Isocyanates Metal oxide fumes Nickel Organic dusts Organophosphide compounds Oxides of nitrogen Ozone Paint fumes/solvents Perfumes Persulfate salts Platinum salts Pollution Pyrethrum Reactive dyes Room deodorizers Rosin core solder thermal decomposition products (colophony) Smoke (fires, pyrolysis products) Solvents Sulfur dioxide Tobacco dust Toluene, xylene, other solvents Vanadium Vinyl chloride Welding fumes Western red cedar (dust, plicatic acid) Wood dust Zinc chloride fume

Glass workers, pesticide or preservative application and handling of respectively treated wood, textiles, etc. Food industry Pulp/paper industry, swimming pool maintenance, waste water treatment, bleach/battery manufacturing Construction/electroplating industry Supermarkets, custodians, health professionals Clothing stores Diamond polishers Restaurants, food manufacturers Cosmetic factories, stores, beauty salons Cotton mills Steel, plastics, herbicide industry Supermarkets Railroad personnel, miners, truck drivers Swine confinement facilities Restaurants Industrial enzyme production Garages, public transportation industry Florist shops, gardening industry Chemical, textile, furniture industries, embalmers Gardening industries Hairdressing salons Laboratories Auto body spray painting, polyurethane foam production Welders Electrolytic plating, nickel mining and refinement Agricultural workers Pesticide industry Chemical industry Welders Department stores, painting, varnishing Beauty salons, department stores Chemical industry, hairdressing salons Platinum refinement Agriculture, custodians, refrigeration workers Insecticide, gardening industries Dye application Department stores, shops Metal, electronics industries Firefighters Construction painters, carpenters Power plants, oil refineries Tobacco workers Petroleum, chemical, paint and printing industries, laboratories Vanadium plants (mill/kiln); manufacture of hard steel alloys, fluorescent light, application of colors PVC production Welders Cedar sawmills Wood workers, furniture industry Military personnel

This information is based on a Medline/PubMed search and on publications by Kup (1985); Chan-Yeung & Lam (1986); Meggs (1994); Slavin (1998); Settipane & Lieberman (2001); Karjalainen et al. (2003); Shusterman (2003); Slavin (2003); Banauch (2005); Custano & Thériault (2006); Gautrin et al. (2006b); and The Collaborative on Health and the Environment (CHE) Toxicant and Disease Database.

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Venables, K.M., Dally, M.B., Nunn, A.J. et al. (1989) Smoking and occupational allergy in workers in a platinum refinery. BMJ 299, 939– 42. Vidal, C. & Polo, F. (1998) Occupational allergy caused by Dianthus caryophillus, Gypsophila paniculata, and Lilium longiflorum. Allergy 53, 995– 8. Viegi, G., Paggiaro, P.L., Begliomini, E., Vaghetti, E., Paoletti, P. & Giuntini, C. (1986) Respiratory effects of occupational exposure to tobacco dust. Br J Ind Med 43, 802–8. Vijayan, V.K. & Sankaran, K. (1996) Relationship between lung inflammation, changes in lung function and severity of exposure in victims of the Bhopal tragedy. Eur Respir J 9, 1977–82. Vogelzang, P.F.J., van der Gulden, J.W.J., Folgering, H. et al. (1998) Endotoxin exposure as a major determinant of lung function decline in pig farmers. Am J Respir Crit Care Med 157, 15–18. Waclawski, E.R., McAlpine, L.G. & Thomson, N.C. (1989) Occupational asthma in nurses caused by chlorhexidine and alcohol aerosols. BMJ 298, 929–30. Wade, J.F. III & Newman, L.S. (1993) Diesel asthma. Reactive airways disease following overexposure to locomotive exhaust. J Occup Med 35, 149–54. Wagner, G.R. & Wegman, D.H. (1998) Occupational asthma: prevention by definition. Am J Ind Med 33, 427–9. Walusiak, J. (2006) Occupational upper airway disease. Curr Opin Allergy Clin Immunol 6, 1– 6. Walusiak, J., Wittczak, T., Ruta, U. & Palczynski, C. (2002) Occupational asthma due to mitoxantrone. Allergy 57, 461. Walusiak, J., Krawczyk-Adamus, P., Hanke, W., Wittczak, T. & Palczynski, C. (2004) Small nonspecialized farming as a protective factor against immediate-type occupational respiratory allergy? Allergy 59, 1294–300. Weir, D.C., Robertson, A.S., Jones, S. & Burge, P.S. (1989) Occupational asthma due to soft corrosive soldering fluxes containing zinc chloride and ammonium chloride. Thorax 44, 220–3. Weiss, J.S. (1996) Reactive airway dysfunction syndrome due to sodium azide inhalation. Int Arch Occup Environ Health 68, 469– 71. Wenzel Schaubschläger, W., Becker, W.-M., Mazur, G. & Gödde, M. (1994) Occupational sensitization to Plasmopara viticola. J Allergy Clin Immunol 93, 457–63. Wergelund, E., Lund, E. & Waage, J.E. (1987) Respiratory dysfunction after potroom asthma. Am J Ind Med 11, 627–36. Wernfors, M., Nielsen, J., Schutz, A. & Skerfving, S. (1986) Phthalic anhydride-induced occupational asthma. Int Arch Allergy Appl Immunol 79, 77–82. Weytjens, K., Cartier, A., Lemière, C. & Malo, J.-L. (1999) Occupational asthma to diacrylate. Allergy 54, 289–90. Wiessmann, K.-J. & Baur, X. (1985) Occupational lung disease following long-term inhalation of pancreatic extracts. Eur J Respir Dis 66, 13–20. Wilk-Rivard, E. & Szeinuk, J. (2001) Occupational asthma with paroxysmal atrial fibrillation in a diamond polisher. Environ Health Perspect 109, 1303–6. Williams, N., Skoulas, A. & Merriman, J.E. (1964) Exposure to grain dust. J Occup Med 6, 319–29. Wisnewski, A.V., Liu, Q., Liu, J. & Redlich, C.A. (2005) Glutathione protects human airway proteins and epithelial cells from isocyanates. Clin Exp Allergy 35, 352–7.

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Zacharisen, M.C., Kadambi, A.R., Schlueter, D.P. et al. (1998) The spectrum of respiratory disease associated with exposure to metal working fluids. J Occup Environ Med 40, 640–7. Zammit-Tabona, M., Sherkin, M., Kijek, K., Chan, H. & Chan-Yeung, M. (1983) Asthma caused by diphenylmethane diisocyanate in foundry workers. Am Rev Respir Dis 128, 226–30. Zeiss, C.R., Patterson, R., Pruzansky, J.J., Miller, M.M., Rosenberg, M. & Levitz, D. (1977) Trimellitic anhydride-induced airway syndromes: clinical and immunologic studies. J Allergy Clin Immunol 60, 96–103. Zock, J.P., Medina-Ramon, M., Kogevinas, M., Sunyer, J. & Anto, J.M. (2004) Asthma and exposure to irritant agents in domestic cleaning women. Eur Respir J 24, Suppl. 48, 367. duakin, E., Valif, F. & Kanceljak, B. (1981) Immunological and respiratory changes in coffee workers. Thorax 36, 9–13. duakin, E., Kanceljak, B., Skurif, Z. & Butkovic, D. (1985) Bronchial reactivity in green coffee exposure. Br J Ind Med 42, 415–20. duakin, E., Mustajbegovic, J., Schachter, E.N., Kern, J., Budak, A. & Godnic-Cvar, J. (1998) Respiratory findings in synthetic textile workers. Am J Ind Med 33, 263–72.

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Allergens from Stinging Insects: Ants, Bees, and Vespids Te Piao King and Rafael I. Monsalve

Summary Allergy to stinging insects is commonly caused by members of ant, bee, and vespid (wasp) families of the order Hymenoptera. The wasp family includes hornets, paper wasps, and yellow jackets. The major venom allergens from these families are proteins of 10–50 kDa. Nearly all have been cloned, and their structures are known. Each family has unique venom allergens, as well as allergens homologous with those of other families. Insects of different species within each family have the same set of homologous venom proteins. Several venom allergens are glycoproteins with similar carbohydrate side chains. Antigenic cross-reactivity of homologous venom allergens can be due to their common protein and/or carbohydrate epitopes. Recombinant venom allergens are available and can be useful reagents for diagnosis and treatment. Insect venom allergens have different biochemical functions, acting as hyaluronidase, phospholipases, phosphatase, and proteases. Their only known common feature is their partial sequence identity with proteins from other sources in our environment. Bee and wasp venoms are rich in cytotoxic peptides, melittin, and mastoparan, respectively. Allergenicity of insect venoms may be due to the combined action of these enzymes and bioactive peptides.

Although many Hymenoptera are capable of stinging, only species belonging to three families (Table 50.1) sting people with a high degree of frequency (Guralnick & Benton 1995). They are the Apidae (bees), Formicidae (ants), and Vespidae (wasps). In this review the term “wasp” or “vespid” is used to designate all members of Vespidae family, and the term “paper wasp” is used to designate members of Polistes genus. A number of insects of the ant, bee, and wasp families have been examined for their venom allergens. The insects common in North America and Europe have been well studied and this is not the case for insects in other areas of the world (Donovan et al. 1993; Matuszek et al. 1994; Costa & Palma 2000; Kim et al. 2001; Pirpignani et al. 2002; Reunala et al. 2005). The importance of venoms as the allergen source in Hymenoptera allergy has been known for some time (Loveless & Fackeler 1959; Lichtenstein et al. 1979). The major venom allergens are generally proteins of 10–50 kDa containing 100–400 amino acid residues. Some venom peptides can be allergens. The structures and/or functions of many of these venom allergens are known. Several of these allergens have been expressed in bacteria, insect, or yeast cells so that they can be useful reagents for studies.

Comparative biochemical data of stinging insect venom allergens Introduction Many insects can cause allergy in humans. People can be exposed to insect body parts or their secretions by inhalation, to their venoms by stinging, and to their salivary gland secretions by biting. Examples of these routes of sensitization are, respectively, allergies to cockroaches of the order Orthoptera, to ants, bee and vespids of the order Hymenoptera, and to flies and mosquitoes of the order Diptera.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Ants, bees, and vespids within each subfamily (Table 50.1) generally have similar venom compositions. The venom allergens of one representative species of each of these insects are given in Table 50.2. These are major allergens, as specific IgEs are present in more than 50% of patients tested. There is one exception, melittin, which is a peptide of 26 amino acid residues active in about one-third of patients (King et al. 1976). Several of the allergens are glycoproteins as indicated in the table. Their sequences, and in several cases their structures, are known. Recombinant proteins of several of these allergens with full biological activities are available.

Bee venom allergens Honeybee (Apis mellifera) venom has six known allergens. Four are proteins, acid phosphatase, hyaluronidase, phospholipase

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Table 50.1 Some reported insects of order Hymenoptera of medical importance. Family/subfamily Apidae Apinae

Formicidae Myrmicinae Myrmicinae Ponrinae

Vespidae Vespinae

Polistinae

?

Genus and species

Common name

Geographic distribution

Apis mellifera Apis cerana Apis dorsata Bombus pennsylvanicus Bombus terrestris

Honeybee Asian honeybee Giant honeybee Bumble bee Bumble bee

Worldwide Australia, Asia

Solenopsis invicta Solenopsis richteri Myrmecia pilosula Myrmecia pyriformis Pachycondyla sennarensis Pachycondyla chinensis

Red imported fire ant Black imported fire ant Jumper ant Bulldog ant Samson ant

Americas Americas Australia Australia Mideast Asia

Vespa crabro Dolichovespula maculata Dolichovespula arenaria Vespula flavopilosa Vespula germanica Vespula maculifrons Vespula pennsylvanica Vespula vulgaris Vespula squamosa Vespula vidua Polistes annularis Polistes dominulus Polistes exclamans Polistes fuscatas

European hornet White-face hornet (bald-face hornet) Yellow hornet (aerial yellow jacket) Yellow jacket Yellow jacket Yellow jacket Yellow jacket Yellow jacket Yellow jacket Yellow jacket Paper wasp Paper wasp Paper wasp Paper wasp

Americas, Europe Americas Americas Americas Americas, Australia, Europe Americas Americas Americas, Asia, Australia, Europe Americas Americas Americas Asia, Americas, Europe Americas Americas

Agelaia pallipes pallipes Polybia scutellaris

Wasp Wasp

South America South America

A2, and protease; and the other two are peptides, melittin with cytotoxic activity and Api m6 of unknown function. Bumble bee has the same protein components as honeybee. In accord with their taxonomy, phospholipase A2 from A. mellifera has a high degree of sequence identity (> 90%) with those of A. cerana and A. dorsata and a low degree (54%) with Bombus pennsylvanicus (Hoffman & Schmidt 2000). As described below, vespid venoms contain hyaluronidase and protease of the same enzymatic specificity as those of bee venom. Bee and vespid hyaluronidases have partial sequence identity of about 50%, and this is also the case for their proteases.

Vespid venom allergens Yellow jacket (Vespula vulgaris) venom has three known allergens: antigen 5 (of unknown biological function), hyaluronidase, and phospholipase A1. Antigen 5 may be a protease because of its sequence homology with a protease in cone snail (Milne et al. 2003). The frequency of patient response to

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Americas Americas, Europe

these yellow jacket venom allergens is as follows: 90% of the 26 patients tested were positive to antigen 5, and 70–80% positive to hyaluronidase and phospholipase (Binder et al. 2002). A fourth allergen, which is a protease, has been identified in several species of paper wasps. Homologs of antigen 5, hyaluronidase, and phospholipase are present in all species of yellow jackets, hornets and paper wasps. Homologs of antigen 5 are present in an Asian hornet Vespa mandarinia (Hoffman & Schmidt 1999) and in a South American wasp Polybia scutellaris (Pirpignani et al. 2002). Another South American wasp Agelaia pallipes pallipes is reported to have hyaluronidase and phospholipase A2 (Costa & Palma 2000). The known vespid phospholipases have the enzymatic specificity of the A1 type with the exception of that for Agelaia pallipes pallipes which is of the A2 type. There is no sequence identity of vespid and bee phospholipases. Two vespid venom allergens are found in multiple forms, antigen 5 from white-faced hornet Dolichovespula maculata

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Table 50.2 Some insect venom allergens with known sequences and structures.

Allergen name*

Common name

Honeybee, Apis mellifera Api m 1 Api m 2 Api m 3 Api m 4 Api m 5 Api m 6

Phospholipase A2 Hyaluronidase Acid phosphatase Melittin Protease

Yellow jacket, Vespula vulgaris Ves v 1 Phospholipase A1 Ves v 2 Hyaluronidase Ves v 5

Fire ant, Solenopsis invicta Sol i 1 Sol i 2 Sol i 3 Sol i 4

Molecular size†

16 kDa 39 kDa 43 kDa 3 kDa 28 kDa 8 kDa

34 kDa 39 kDa

Antigen 5 Protease§

23 kDa 28 kDa

Phospholipase A1

34 kDa 13 kDa 23 kDa 13 kDa

Antigen 5

Glycoprotein

Structure‡

Reference

+ +

++ ++

Dudler et al. (1994) Soldatova et al. (1998) Hoffman (2006) King et al. (1976) Winningham et al. (2004) Kettner et al. (2001)

++

+

+ ++ ++

+

+

King et al. (1996) Kolarich et al. (2005); Skov et al. (2006) Monsalve et al. (1999) Winningham et al. (2004)

Hoffman et al. (2005) Schmidt et al. (1996) Hoffman (1993) Hoffman (1993)

* Allergen names according to an accepted nomenclature system (King et al. 1995). † Several allergens are glycoproteins, and the molecular size given refers only to the protein portion. ‡ ++ and + signs refer, respectively, to structures determined directly or by modeling of structures of homologous proteins. § Protease from V. vulgaris is not known, but it is known for three species from Polistes.

and hyaluronidase from yellow jacket Vespula vulgaris. The two forms of hornet antigen 5 have 77% sequence identity of their 204–205 residues (Fang et al. 1988), and the two forms of yellow jacket hyaluronidase have 58% identity of their 331–335 residues (Kolarich et al. 2005). This degree of sequence identity for two homologous proteins of the same species is about the same as that of different species.

Ant venom allergens Fire ant (Solenopsis invicta) venom contains four known protein allergens: Sol i1 to Sol i4. Sol i1 and Sol i3 are homologous with vespid phospholipase A1 and antigen 5, respectively. Sol i2 and Sol i4 are related and of unknown function. Homologs of these proteins are present in other species of fire ants (Hoffman 1997). There is sequence similarity of ant and vespid phospholipases but there is no sequence similarity with the bee enzyme. Only one allergen of about 12 kDa is known from jumper ant (Myrmecia pilosula) venom, and it was identified by immunoblot with a mercaptoethanol-reduced venom sample (Donovan et al. 1993). Proteins with disulfide bonds usually become denatured on reduction and they may not be detected by patient sera that are specific for the native proteins. The

presence of hyaluronidase, phosphatase, phospholipase, and other enzymes in jumper and bulldog ant venoms has been reported (Matuszek et al. 1994) and it is possible these proteins are allergens.

Structure of venom allergens The structures of bee venom hyaluronidase (Markovic-Housley et al. 2000), melittin (Terwilliger & Eisenberg 1982) and phospholipase A2 (Scott et al. 1990), and those of yellow jacket antigen 5 (Henriksen et al. 2001) and hyaluronidase (Skov et al. 2006), were solved by crystallography. The structure of vespid phospholipase can be obtained by modeling with the homologous porcine pancreatic lipase as its structure is known (Carriere et al. 1994). Using the modeling approach, the structures of nearly all the proteins in Table 50.2 can be obtained. Over 20 antigen 5 homologs from different vespids and fire ants are known. Their sequences vary from as few as several to over 100 residues of their entire molecule of 201–204 residues. When their structures are modeled with that of Ves v5, most of their sequence variations are located on the surface of the molecule. The degree of cross-reactivity of homologous antigen 5s is correlated with how large the areas of their surface identity are.

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Antigenic cross-reactivity of stinging insect venom allergens Insect-allergic patients often have sensitivity to multiple insects by skin test or in vitro test (Lichtenstein et al. 1979). This multiple sensitivity can be due to exposure to different insects and/or antigenic cross-reactivity of different venoms. Cross-reactivity of homologous allergens can be due to their common amino acid residues on the surface of the molecule, and/or due to the carbohydrate side chains for allergens that are glycoproteins (Hemmer et al. 2001). As noted in Table 50.2, several insect allergens are glycoproteins. In contrast to the multitude of amino acid sequences of different insect allergens, the sequence differences in their carbohydrate side chains are limited. The carbohydrate side chain is N-linked to the asparagine residue of the protein by its innermost N-acetylglucosamine residue. The predominant carbohydrate side chain has the sequence: Manα1→6(Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc with one or two fucoses (α1,6 and α1,3) linked to the innermost N-acetylglucosamine residue. This was shown for bee phospholipase (Kubelka et al. 1993), and bee and yellow jacket hyaluronidases (Kolarich et al. 2005). Similar or identical carbohydrate side chains are present in other plant and animal proteins. The glycan in plant proteins contains, in addition to the fucoses, xylose (β1,2) linked to the mannose in the middle position. Tests with patient or animal sera specific for glycans from different sources suggest that the specificity of the cross-reactive carbohydrate determinant resides mainly in the fucose residues for insect proteins, and in the fucose and xylose residues for plant proteins (Bencurova et al. 2004). Indeed horseradish peroxidase can be used to establish whether patient sera cross-reactive to bees and yellow jackets are due to their cross-reactive carbohydrate determinant (Jappe et al. 2006). Bees and vespids have two homologous venom allergens, hyaluronidase and protease. Weak cross-reactivity of honeybee and white-faced hornet hyaluronidases was detected with hyperimmunized mouse sera (Lu et al. 1995). Hornets, yellow jackets, and paper wasps all have homologous antigen 5, hyaluronidase, and phospholipase. Tests with animal sera show that cross-reactivity is readily detectable for homologous vespid hyaluronidases of > 90% sequence identity, and is variable and low for homologous antigen 5s and phospholipases with < 70% sequence identity (King et al. 1985, 1996). These data with animal sera show that the weak crossreactivity of patient sera to multiple insect venoms can be due to their homologous protein antigenic determinants and/or their glycan antigenic determinants. Peptide and carbohydrate antigenic determinants can differ in their epitope density and in their antibody affinity. For

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peptide determinants, the entire accessible surface of a protein represents a continuum of epitopes (Davies & Cohen 1996). For carbohydrate determinants, the number of epitopes is restricted to a few potential glycosylation sites and these sites are not necessarily glycosylated. As an example, yellow jacket hyaluronidase has five potential sites but only two to three sites are glycosylated (Skov et al. 2006). Mediators are released from IgE-bound mast cells or basophils on allergen challenge. This biological activity requires the allergens to have multiple determinants/epitopes so that they can cross-link the bivalent IgE antibodies. Thus, the lowdensity carbohydrate determinants are likely to be of less biological importance than the high-density peptide determinants are. Also, the biological activity of allergens to cause mediator release depends on the affinity of allergen-specific IgEs (Christensen et al. 2006; Fromberg 2006). For these reasons, there is debate as to the importance of glycan-specific IgE in allergic diseases (van Ree 2002; Altmann, 2007). Specific IgEs can be measured by different formats of solution or solid-phase assays with different sensitivities. For example, one commercially available assay, Pharmacia CAP, utilizes solid-phase allergen to capture specific IgEs and another assay, ADVIA- Centaur, utilizes solid-phase anti-IgE to capture IgEs followed by labeled allergen to detect specific IgEs (Ricci et al. 2003; Petersen et al. 2004). As these two assays differ in their sensitivity for antibodies of different affinities, they may provide data of different clinical relevance.

Recombinant insect venom allergens Recombinant allergens have several different applications. First, they can be useful diagnostic reagents for identifying the offending insects. Use of individual recombinant allergens free of glycan side chain in place of venoms can lead to more accurate diagnosis so that the patients can be treated with the proper venom. As an example, recombinant bee phospholipase A2 and vespid antigen 5 can be useful reagents for establishing bee or vespid sensitivity free of interference from other pollen or food sensitizations. Another application is to prepare allergens with reduced allergenicity but retaining their immunogenicity. Such knowledge will improve our understanding of the IgE-binding sites that may be useful as vaccines. If the structure of the allergen of interest is known, one can selectively mutate the surface residues so as to alter its IgE binding (Holm et al. 2004). An alternative approach is to prepare hybrids that contain a small segment of the guest allergen of interest and a large segment of a host protein. The host protein is homologous to the guest allergen, and they are poorly cross-reactive as antigens. The host protein functions as a scaffold to hold the segment of the guest allergen in its native conformation, as homologous proteins of > 30% sequence identity can have closely similar structures. In this way, the hybrids retain the

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discontinuous epitopes of the guest allergen, but at a reduced density. This approach was demonstrated with hybrids of yellow jacket and wasp antigen 5s (King et al. 2001). These two antigens 5s have 59% sequence identity and are poorly crossreactive in patients or in animals. Hybrids with one-quarter of yellow jacket antigen 5 and three-quarters of wasp antigen 5 showed 100- to 1000-fold reduction in allergenicity when tested by histamine release assay in yellow jacket-sensitive patients. These hybrids retained the immunogenicity of antigen 5s for antibody responses specific for the native protein and for T-cell responses in mice. Therefore, the hybrids may be useful vaccines as they may be used at higher doses than the natural allergen.

Biochemical data of low-molecular-weight components of insect venoms In addition to proteins, bee and vespid venoms contain peptides, biogenic amines such as histamine and dopamine, and other low-molecular-weight components (Habermann 1972; Nakajima 1984). The most abundant bee and vespid peptides, about half of venom weight, are melittin and mastoparan, respectively. In contrast to bee and vespids, the major lowmolecular-weight components of fire ant venom are water insoluble alkaloids (Brand et al. 1972). Melittin and mastoparan are basic peptides with 26 and 14–15 residues, respectively. Both peptides are reported to be immunogenic in mice for antibody responses (Kind et al. 1981; King et al. 1984; Ho et al. 1995). Melittin was reported to be an allergen but not mastoparan (King et al. 1976). Mastoparan can induce release of histamine and other mediators from mast cells (Hirai et al. 1979). It binds to cell membranes and it can act as a strong secretagogue for different cell types.

Allergens from Stinging Insects: Ants, Bees, and Vespids

Allergenicity of insect venoms Nearly all venom allergens are enzymes and they have partial sequence identity of 20–50% with homologous proteins from diverse sources (Table 50.3). As an example, homologs of antigen 5 are present in many other organisms, mammals, plants, nematodes, lizards, snakes, and others. The wide distribution of homologs of venom allergens in our environment may contribute to recognition by the host, but this is not likely the only factor of venom allergenicity. Venom enzymes and peptides, by their actions and/or by products of their actions, can act as adjuvants to promote allergen-specific Th2 cell response. Yellow jacket venom phospholipase and mastoparan were both found to stimulate the release of the inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitrous oxide, and prostaglandin (PG) E2 and other mediators from macrophages and peritoneal exudate cells in mice (Wu et al. 1999; King et al. 2003). Also, mastoparan was found to have weak adjuvant activity to enhance IgG1 and IgE responses to yellow jacket antigen 5 in mice (King et al. 2003). Similarly, others found melittin to be an adjuvant for ovalbumin-specific IgE response in mice (Kind et al. 1981). PGE2 is known to be a modulator of immunity by stimulating dendritic cells and act as adjuvant for IgE and IgG responses (Roper et al. 1995; Kalinski et al. 1997). Venom hyaluronidase can act on high-molecular-weight hyaluronic acid (hyaluronan) to generate low-molecularweight fragments. The fragments can bind to Toll-like receptors to act as adjuvants to promote immune response by activation of dendritic cells and T cells (Termeer et al. 2003; Scheibner et al. 2006). Proteases of different specificities can activate cell-surface receptors to promote inflammation with accompanying cytokine release that can alter the

Table 50.3 Insect allergens and homologous proteins. Insect allergens

Other proteins

Reference

Antigen 5

Cone snail protease Mammalian testis protein Human glioma PR protein Hookworm protein Nematode protein Plant leaf PR protein Mexican lizard toxin Snake venom stecrisp

Milne et al. (2003) Kasahara et al. (1989) Murphy et al. (1995) Hawdon et al. (1996) Ravi et al. (2002) Van Loon & Van Strien (1999) Morrissette et al. (1995) Guo et al. (2004)

Hyaluronidase

Mammalian sperm protein

Gmachl & Kreil (1993)

Phospholipase A 1

Mammalian lipases

Carriere et al. (1994)

Phospholipase A 2

Mammalian phospholipases

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course of immune response (Reed & Kita 2004). For example, IgE response to the mite allergen Der p 1 in mice is suggested to be associated with its protease activity (Gough et al. 1999; Kikuchi et al. 2006). Fungal protease was found to function as an adjuvant for allergic response to ovalbumin in the airway (Kheradmand et al. 2002). Thus, the venom hyaluronidase and protease may both have a role in venom allergenicity.

References Altmann, F. The role of protein glycosylation in allergy. (2007) Int Arch Allergy Immunol 142, 99–115. Bencurova, M., Hemmer, W., Focke-Tejki, M., Wilson, I.B. & Altmann, F. (2004) Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 14, 457–66. Binder, M., Fierlbeck, G., King, T.P., Valent, P. & Buehring, H. (2002) Individual hymenoptera venom compounds induce upregulation of the basophil activation marker ectonucleotide pyrophosphatase/ phosphodiesterase 3 (CD203c) in sensitized patients. Int Arch Allergy Immunol 129, 160– 8. Brand, J., Blum, M., Fales, H. & MacConnell, J. (1972) Fire ant venoms: comparative analyses of alkaloidal components. Toxicon 10, 259–71. Carriere, F., Thirstrup, K., Boel, E., Berger, R. & Thim, L. (1994) Structure-function relationships in naturally occurring mutants of pancreatic lipase. Protein Eng 7, 563– 9. Christensen, L.H., Holm, J., Skovsgaard, J. & Lund, K. (2006) The significance of clonality and affinity of individual allergen-specific IgE antibodies for the activation of human basophils. J Allergy Clin Immunol 117, S310. Costa, H. & Palma, M. (2000) Agelotoxin: a phospholipase A(2) from the venom of the neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae). Toxicon 38, 1367–79. Davies, D.R. & Cohen, G.H. (1996) Interactions of protein antigens with antibodies. Proc Natl Acad Sci USA 93, 7–12. Donovan, G.R., Baldo, B.A. & Sutherland, S. (1993) Molecular cloning and characterization of a major allergen (Myr p I) from the venom of the Australian jumper ant, Myrmecia pilosula. Biochim Biophys Acta 1171, 272– 80. Dudler, T., Schneider, T., Annand, R.R., Gelb, M.H. & Suter, M. (1994) Antigenic surface of the bee venom allergen phospholipase A2. Structural functional analysis of human IgG4 antibodies reveals potential role in protection. J Immunol 152, 5514–22. Fang, K.S. Y., Vitale, M., Fehlner, P. & King, T.P. (1988) cDNA cloning and primary structure of a white-face hornet venom allergen, antigen 5. Proc Natl Acad Sci USA 85, 895–9. Fromberg, J. (2006) IgE as a marker in allergy and the role of IgE affinity. Allergy 61, 1234. Gmachl, M. & Kreil, G. (1993) Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc Natl Acad Sci USA 90, 3569–73. Gough, L., Schulz, O., Sewell, H.F. & Shakib, F. (1999) The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response. J Exp Med 190, 1897–902.

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Guo, M., Teng, M., Niu, L., Liu, Q., Huang, Q. & Hao, Q. (2004) Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitorlike fold. J Biol Chem 280, 12405–12. Guralnick, M.W., Benton, A.W. (1995) Entomological aspects of insect sting allergy. In: Levine M.I., Lockey R.F., eds: Monograph on Insect Allergy, 3rd ed. Milwaukee WI: American Academy of Allergy and Immunology, 1995:7. Habermann, E. (1972) Bee and wasp venoms. Science 177, 314–22. Hawdon, J.M., Jones, B.F., Hoffmann, D.R. & Hotez, P.J. (1996) Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. J Biol Chem 271, 6672–8. Hemmer, W., Focke, M., Kolarich, D. et al. (2001) Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinginginsect allergy. J Allergy Clin Immunol 108, 1045–52. Henriksen, A., King, T.P., Mirza, O. et al. (2001) Major venom allergen of yellow jackets, Ves v 5: Structural characterization of a pathogenesis-related protein superfamily. Protein: Structure Function and Genetics 45, 438–48. Hirai, Y., Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M. & Kitada, C. (1979) A new mast cell degranulating peptide “mastoparan” in the venom of Vespula lewisii. Chem Pharm Bull (Tokyo) 27, 1942– 4. Ho, C.L., Lin, Y.L., Chen, W.C. et al. (1995) Immunogenicity of mastoparan B, a cationic tetradecapeptide isolated from the hornet (Vespa basaus) venom, and its structural requirements. Toxicon 33, 1443–51. Hoffman, D.R. (1993) Allergens in hymenoptera venom XXIV: The amino acid sequences of imported fire ant venom allergens II, III, and IV. J Allergy Clin Immunol 91, 71–8. Hoffman, D.R. (1997) J Allergy Clin Immunol 99, S377. Hoffman, D.R. (2006) Hymenoptera venom allergens Clin Rev Allergy Immunol 30, 109–28. Hoffman, D.R. & Schmidt, J.O. (1999) Amino acid sequences of allergens from Vespa mandarinia, an Asian hornet. J Allergy Clin Immunol 103, S164. Hoffman, D.R. & Schmidt, J.O. (2000) Phopholipases from Asian honeybees. J Allergy Clin Immunol 105, S56. Hoffman, D.R., Sakell, R.H. & Schmidt, M. (2005) Sol i 1, the phospholipase allergen of imported fire ant venom. J Allergy Clin Immunol 115, 611–6. Holm, J., Gajhede, M., Ferreras, M. et al. (2004) Allergy vaccine engineering: epitope modulation of recombinant Bet v 1 reduces IgE binding but retains protein folding pattern for induction of protective blocking-antibody responses. J Immunol 173, 5258–67. Jappe, U., Raulf-Heimsoth, M., Hoffmann, M., Burow, G., HubschMuller, C. & Enk, A. (2006) In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 61, 1220–9. Kalinski, P., Hilkens, C.M., Snijders, A., Snijdewint, F.G. & Kapsenberg, M.L. (1997) IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type 2 cytokine production in maturing human naive T helper cells. J Immunol 159, 28–35. Kasahara, M., Gutnecht, J., Brew, K., Spur, N. & Goodfellow, P.N. (1989) Cloning and mapping of a testis-specific gene with sequence similarity to a sperm-coating glycoprotein gene. Genomics 5, 527– 34.

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Kettner, A., Hughes, G., Frutiger, S. et al. (2001) Api m 6: a new bee venom allergen. J Allergy Clin Immunol 107, 914–20. Kheradmand, F., Kiss, A., Xu, J., Lee, S.H., Kolattukudy, P.E. & Corry, D.B. (2002) A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease. J Immunol 169, 5904–11. Kikuchi, Y., Takai, T., Kuhara, T. et al. (2006) Crucial commitment of proteolytic activity of a purified recombinant major house dust mite allergen Der p1 to sensitization toward IgE and IgG responses. J Immunol 177, 1609–17. Kim, S.S., Park, H.S., Kim, H.Y., Lee, S.K. & Nahm, D.H. (2001) Anaphylaxis caused by the new ant, Pachycondyla chinensis: demonstration of specific IgE and IgE-binding components. J Allergy Clin Immunol 107, 1095–9. Kind, L.S., Ramaika, C. & Allaway, E. (1981) Antigenic, adjuvant and permeability enhancing properties of melittin in mice. Allergy 36, 155–60. King, T.P., Sobotka, A.K., Kochoumian, L. & Lichtenstein, L.M. (1976) Allergens of honey bee venom. Arch Biochem Biophys 172, 661–71. King, T.P., Kochoumian, L. & Joslyn, A. (1984) Melittin-specific monoclonal and polyclonal IgE and IgG1 antibodies from mice. J Immunol 133, 2668–73. King, T.P., Joslyn, A. & Kochoumian, L. (1985) Antigenic cross-reactivity of venom proteins from hornets, wasps, and yellow jackets. J Allergy Clin Immunol 75, 621–8. King, T.P., Hoffman, D.R., Lowenstein, H., Marsh, D.G., Platts-Mills, T.A. E. & Thomas, W.R. (1995) Allergen nomenclature. J Allergy Clin Immunol 96, 5–14. King, T.P., Lu, G., Gonzalez, M., Qian, N.F. & Soldatova, L. (1996) Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with their hornet and was phomologs and possible implications for clinical allergy. J Allergy Clin Immunol 98, 588–600. King, T.P., Jim, S.Y., Monsalve, R.I., Kagey-Sobotka, A., Lichtenstein, L.M. & Spangfort, M.D. (2001) Recombinant allergens with reduced allergenicity but retaining immunogenicity of the natural allergens: hybrids of yellow jacket and paper wasp venom allergen antigen 5s. J Immunol 166, 6057–65. King, T.P., Sui, Y.J. & Wittkowski, K.M. (2003) Inflammatory role of two venom components of yellow jackets (Vespula vulgaris): a mast cell degranulating peptide mastoparan and phospholipase A1. Int Arch Allergy Immunol 131, 25–32. Kolarich, D., Leonard, R., Hemmer, W. & Altmann, F. (2005) The Nglycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris. FEBS Journal 272, 5182–90. Kubelka, V., Altmann, F., Staudacher, E. et al. (1993) Primary structures of the N-linked carbohydrate chains from honeybee venom phospholipase A2. Eur J Biochem 213, 1193–1204. Lichtenstein, L.M., Valentine, M.D. & Sobotka, A.K. (1979) Insect allergy: the state of the art. J Allergy Clin Immunol 64, 5–12. Loveless, M.H. & Fackeler, W.R. (1959) Wasp venom allergy and immunity. Ann Allergy 14, 347–66. Lu, G., Kochoumian, L. & King, T.P. (1995) Sequence identity and antigenic cross-reactivity of white face hornet venom allergen, also a hyaluronidase, with other proteins. J Biol Chem 270, 4457–65. Markovic-Housley, Z., Miglierini, G.L.S., Rizkallah, P.J., Muller, U. & Schirmer, T. (2000) Crystal structure of hyaluronidase, a major allergen of bee venom. Structure Fold Des 8, 1025–35.

Allergens from Stinging Insects: Ants, Bees, and Vespids

Matuszek, M.A., Hodgson, W.C., King, R.G. & Sutherland, S.K. (1994) Some enzymic activities of two Australian ant venoms: a jumper ant Myrmecia pilosula and a bulldog ant Myrmecia pyriformis. Toxicon 32, 1543–9. Milne, T.J., Abbenante, G., Tyndall, J.D.A., Halliday, J. & Lewis, R.J. (2003) Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily. J Biol Chem 278, 31105–10. Monsalve, R.I., Gang, L. & King, T.P. (1999) Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast. Protein Expr Purif 16, 410–16. Morrissette, J., Kratzschmar, J., Haendler, B. et al. (1995) Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors. Biophys J 68, 2280–8. Murphy, E.V., Zhang, Y., Zhu, W. & Biggs, J. (1995) The human glioma pathogenesis-related protein is structurally related to plant pathogenesis-related proteins and its gene is expressed specifically in brain tumors. Gene 159, 131–5. Nakajima, T. (1984) Biochemistry of vespid venom. In: Tu, A.T., ed. Handbook of Natural Toxins, Vol. 2, Dekker, New York. pp. 109– 33. Petersen, A.B., Gudmann, P., Milvang-Gronager, P. et al. (2004) Performance evaluation of a specific IgE assay developed for the ADVIA centaur immunoassay system. Clin Biochem 37, 882–92. Pirpignani, M.L., Rivera, E., Hellmann, U. & Biscoglio de Jimenez Bonino, M. (2002) Structural and immunological aspects of Polybia scutellaris Antigen 5. Arch Biochem Biophys 407, 224–30. Ravi, V., Ramachandran, S., Thompson, R.W., Anderse, J.F. & Neva, F.A. (2002) Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae. Mol Biochem Parasitol 125, 73–81. Reed, C. & Kita, H. (2004) The role of protease activation of inflammation in allergic respiratory diseases. J Allergy Clin Immunol 114, 997–1008. Reunala, T., Brummer-Kovenkontio, H., Saarien, K., Rasanen, L., Lestringant, G. & Hoffman, D. (2005) Characterization of IgE-binding allergens in Samsum ant venom. J Allergy Clin Immunol 115, S108. Ricci, G., Capelli, M., Miniero, R. et al. (2003) A comparison of different allergometric tests, skin prick test, Pharmacia UniCAP and ADVIA Centaur, for diagnosis of allergic diseases in children. Allergy 58, 38– 45. Roper, R.L., Brown, D.M. & Phipps, R.P. (1995) Prostaglandin E2 promotes B lymphocyte Ig isotype switching to IgE. J Immunol 154, 162–70. Scheibner, K.A., Lutz, M.A., Boodoo, S., Fenton, M.J., Powell, J.D. & Horton, M.R. (2006) Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J Immunol 177, 1272–81. Schmidt, M., McConnel, T.J. & Hofmann, D.R. (1996) Production of a recombinant imported fire ant venom allergen, Sol i 2, in native and immunoreactive form. J Allergy Clin Immunol 98, 82–8. Scott, D.L., Otwinowski, Z., Gelb, M.H. & Sigler, P. (1990) Crystal structure of bee-venom phospholipase A2 in a complex with a transitionstate analogue. Science 250, 1563–6. Skov, L.K., Seppala, U., Coen, J.J. et al. (2006) Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom. Acta Crystallogr D 62, 595–604. Soldatova, L.N., Crameri, R., Gmachl, M. et al. (1998) Superior biologic activity of the recombinant bee venom allergen hyaluronidase

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expressed in baculovirus-infected insect cells as compared with Escherichia coli. J Allergy Clin Immunol 101, 691–8. Termeer, C., Sleeman, J.P. & Simon, J.C. (2003) Hyaluronan – magic glue for the regulation of the immune response? Trends Immunol 24, 112–4. Terwilliger, T.C. & Eisenberg, D. (1982) The structure of melittin. II. Interpretation of the structure. J Biol Chem 257, 6016–22. Van Loon, L.C. & Van Strien, E.A. (1999) The families of pathogenesisrelated proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55, 85–97.

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van Ree, R. (2002) Carbohydrate epitopes and their relevance for the diagnosis and treatment of allergic diseases. Int Arch Allergy Immunol 129, 189–97. Winningham, K.M., Fitch, C.D., Schmidt, M. & Hoffman, D.R. (2004) Hymenoptera venom protease allergens. J Allergy Clin Immunol 114, 928–33. Wu, T.M., Chou, T.C., Ding, Y.A. & Li, M.L. (1999) Stimulation of TNF-alpha, IL-1beta and nitrite release from mouse cultured spleen cells and lavaged peritoneal cells by mastoparan M. Immunol Cell Biol 77, 476–82.

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Cockroach Allergens, Environmental Exposure, and Asthma Martin D. Chapman and Anna Pomés

Summary Allergic asthma is the most common disease associated with cockroach-infested housing. In the USA, IgE-mediated sensitization and exposure to cockroach allergens are strong risk factors for asthma in inner-city and urban areas, and in suburban and rural homes that sustain cockroach infestation. Lower socioeconomic status and race (African-American or Hispanic) are independent risk factors for cockroach asthma. The association between cockroach allergens and asthma is also found in other parts of the world. The repertoire of cockroach allergens has been defined using allergens cloned from German (Blattella germanica) and American (Periplaneta americana) cDNA libraries. The combined prevalence of IgE reactivity to the most widely studied allergens (Bla g 1, Bla g 2, Bla g 4, Bla g 5, and Per a 7) is ~ 95%, with ~ 60% of patients being sensitized to Bla g 2, a major allergen. These allergenic proteins belong to several distinct protein families with different biological functions, including gut microvillar protein (Bla g 1), inactive aspartic protease (Bla g 2), male reproductive lipocalin (Bla g 4), glutathione transferase (Bla g 5), tropomyosin (Group 7), and troponin (Bla g 6). The high-resolution crystal structure of recombinant Bla g2 revealed substitutions and distortions in the putative aspartic protease catalytic site, which render the molecule enzymatically inactive, as well as the presence of five disulfide bonds and a zinc-binding site. The latter features infer stability on the allergen and may explain why it persists in the environment. Environmental exposure to cockroach allergens has been assessed by measuring Bla g 1 and Bla g 2 in reservoir dust samples from homes, schools, and daycare centers. Exposure to > 8 units/g Bla g 1 is associated with increased asthma morbidity (unscheduled medical visits, missed school days, and hospitalizations) among inner-city children with asthma. This suggests that asthma symptoms could be reduced by cockroach remediation. Several studies report that cockroach counts and cockroach allergen levels in homes can be significantly reduced by use of insecticides (e.g., fipronil,

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

abamectin, hydramethylnon), by professional cleaning and by patient education. A large randomized controlled trial among inner-city children with asthma showed that implementation of these environmental intervention procedures reduced asthma symptoms by ~ 20% over a 2-year period. Reductions in asthma morbidity correlated with reductions in cockroach and mite allergen levels in the children’s bedrooms. Asthma caused by cockroach allergens is a significant public health problem in the USA (and elsewhere). Current evidence suggests that multifaceted and targeted environmental interventions to reduce cockroach allergen exposure improve asthma symptoms and would be expected to have significant public health benefits.

Introduction The chapter on cockroach (CR) allergens and asthma in the first edition of Allergy and Allergic Diseases included a brief history of CR allergy from 1964 until 1997, when the book was published. It described the original observations by Bernton and Brown that patients with asthma living in New York were often sensitized to CR allergens; the extensive studies of Kang and colleagues in Chicago that demonstrated immediate hypersensitivity and bronchial reactivity to CR extracts; and Platts-Mills’ studies in the 1990s that showed that sensitization and exposure to CR allergens was a significant risk factor for admission to hospital emergency departments with asthma (Bernton & Brown 1964; Kang et al. 1979; Gelber et al. 1993; Chapman et al. 1997). A key argument was that asthma was the major disease associated with CR infestation of housing and, as such, was an important public health problem in the USA, and in other parts of the world. This remains true today. The earlier studies showed that CR-induced asthma was strongly associated with persons of lower socioeconomic status living in substandard housing or apartments (which in the USA disproportionately affects African-Americans and Hispanics). By 1997, a panel of allergens from both German CR (Blattella germanica) and American CR (Periplaneta americana) had been identified, cloned and sequenced. Immunoassays to measure environmental exposure to Bla g 1 and Bla g 2 had

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been developed and were used to measure allergen distribution in homes and to define risk levels associated with allergic sensitization (Chapman et al. 1997). Over the past 10 years, there has been a wealth of progress in basic and clinical research on cockroach allergens and allergic disease. In the USA, the focus has been on investigating CR as one of the risk factors associated with childhood asthma in inner-cities, where mortality and morbidity due to asthma is highest. The US National Institutes of Health has funded a series of epidemiologic studies, including the National Cooperative Inner City Asthma Study (NCICAS) and the National Study of Lead and Allergens in Housing (NSLAH) to assess the prevalence of CR allergen exposure and associated health effects. Data on CR allergen exposure has also been gathered from Eastern Europe, former Soviet republics, Asia, and South America. Basic research on allergen structure and function led to the resolution of the first crystal structure of a cockroach allergen (Bla g 2) and, most recently, to the structure of a Bla g 2/monoclonal antibody complex. New families of cockroach allergens have been defined by molecular cloning studies, including tropomyosins, troponins, and myosin, and new diagnostic assays for measuring allergen-specific IgE have been developed. Elegant in situ hybridization and electron microscopy studies have provided strong evidence of the biological functions of allergens such as Bla g 1 and Bla g 4. Significant progress has also been made with trials of CR allergen abatement and remediation procedures. Several studies have reported success in reducing cockroach allergen levels in homes using different insecticides in gel baits, together with professional cleaning and patient education. These culminated in a large controlled trial of allergen control procedures among inner-city children with asthma, which showed a 20% reduction in symptom scores over a 2-year period in the remediation group (Morgan et al. 2004). The success of CR allergen control procedures, coupled with housing improvements designed to reduce dampness and humidity, may be a pragmatic approach to reduce the effects of CR infestation on public health. This chapter reviews progress in the epidemiology and etiology of asthma due to CR; the structural biology and function of CR allergens; prospects for improved diagnosis and treatment; and the efficacy of CR remediation procedures.

Epidemiology of allergic diseases associated with cockroach Sensitization and exposure to cockroach allergens In most epidemiologic studies, sensitization to CR allergens is determined by skin-prick tests using commercial B. germanica or P. americana extracts. A wheal of 3 mm greater than a negative buffered saline control is used as the cutoff for a positive skin-test response. Alternatively, sensitization can be meas-

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ured by in vitro testing for serum IgE antibodies using the radioallergosorbent test (RAST) or by fluorescent enzyme immunoassay using the ImmunoCAP system. The advantages of skin testing are that it provides immediate results and the lancets that are used nowadays are relatively painless, which is important for studies in children. Skin testing provides a good screening test, but it is difficult to quantify the results based on the measurements of weal and erythema. Measurements of allergen-specific IgE in serum are quantitative and can be used to compare IgE responses in different populations or subsets of patients. Once serum is obtained, it can readily be analyzed for IgE to other allergens, or for other immunologic tests, and values obtained in different studies can be directly compared. This results in greater consistency and standardization of outcomes. It is important to consider potential cross-reactions between allergenic extracts when interpreting the results of skin tests and in vitro tests. Mites and CR produce some cross-reactive allergens (e.g., tropomyosin), which may be confounders in populations that are exposed to both allergens. Assessments of allergen exposure can help to determine whether positive IgE responses are the result of cosensitization or of allergenic cross-reactivity. Exposure to indoor allergens is measured by immunoassay of specific allergens in reservoir dust samples or air samples (Platts-Mills et al. 1997). Measurements of Bla g 1 and Bla g 2 by two-site monoclonal antibody-based ELISA have been widely used to assess CR allergen exposure, and risk levels associated with allergic sensitization have been proposed (Pollart et al. 1991a,b). The highest levels of CR allergens are usually found in the kitchen, but significant levels are also found in bedrooms, living rooms, and soft furnishings in CR-infested homes. Typical exposures range from 0.6 to > 100 units/g dust for Bla g 1 and from 0.05 to > 15 μg/g for Bla g 2 (Gelber et al. 1993; Sarpong et al. 1996; Sporik et al. 1999). A common question concerning CR allergen exposure assessment is whether to measure Bla g 1, Bla g 2, or both. A Bla g 1 homolog (Per a 1) is produced by P. americana and there is some cross-reactivity between the two allergens in ELISA. Bla g 1 has been used as a surrogate marker for both species. However, in the USA, B. germanica is the more prevalent species in homes. The prevalence of sensitization to Bla g 2 (∼ 60%) is much higher than Bla g 1 (30–40%), and Bla g 2 is a more highly characterized allergen (Pomés et al. 2002a; Erwin et al. 2005; Satinover et al. 2005). This suggests that measurements of Bla g 2 may be more relevant to health effects (at least in the USA). Of course, the best assessment would be to measure both allergens, a prospect that is becoming more manageable through the development of multiplex suspension arrays for indoor allergen detection. These arrays allow five to ten allergens to be measured simultaneously on a single dust or air sample and have been developed for dust mite, cat, dog, rat, mouse, and CR allergens (Earle et al. 2007).

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Previous studies highlighted the fact that there is a high prevalence of CR sensitization (20–70%) among children and adults with asthma living in inner-cities and urban areas of the USA, e.g., New York, Baltimore, Chicago, Detroit, Atlanta, and Wilmington (DE) (Bernton & Brown 1969; Kang et al. 1979; Pollart et al. 1989; Call et al. 1992; Gelber et al. 1993; Sarpong et al. 1996; Eggleston et al. 1999a). AfricanAmerican or Hispanic race and lower socioeconomic status are independent risk factors for CR sensitization in patients with asthma (Sarpong et al. 1996; Christiansen et al. 1996a,b). In the early 1990s, case–control studies in Charlottesville, Atlanta, and Wilmington showed that sensitization and exposure to CR allergens was an important risk factor for emergency-room admissions with asthma. Exposure to > 2 units/g Bla g 1 or > 2 units/g (or 0.08 μg/g) Bla g 2 was associated with CR sensitization (Gelber et al. 1993; Sporik et al. 1999). Although the highest concentrations of CR allergens were found in kitchens, high levels were also found in bedroom carpets, bedding, and soft furnishings. A subsequent study of inner-city homes in Baltimore found that current exposure to Bla g 1 in children’s bedrooms was more strongly correlated with sensitization (Eggleston et al. 1998). Early exposure to CR allergens was also strongly associated with recurrent wheezing in 1–5-year-old children recruited in Boston in the Epidemiology of Home Allergens and Asthma study (Litonjua et al. 2001, 2002). The first NCICAS study systematically evaluated the relationship between CR sensitization, exposure, and morbidity due to asthma among over 450 children aged 4–9 who were recruited from eight inner-city areas of the USA: Bronx and East Harlem, New York, Baltimore, St Louis, Cleveland, Detroit, Washington DC, and Chicago. In these children, 66% of whom lived in homes below the Federal poverty line (family income < $15 000/year), the prevalence of CR sensitization was 36.8%. The combination of sensitization and exposure to > 8 units/g Bla g 1 was associated with significantly increased, asthma-related, unscheduled medical visits and hospitalizations (P < 0.001) (Rosenstreich et al. 1997) (Fig. 51.1). These children also had increased episodes of wheezing, more missed school days, and more disruption of caregivers daily lives. This was a landmark study because it so clearly demonstrated a relationship between allergen exposure and asthma morbidity among children who were most affected by this disease. A follow-up Inner City Asthma Study (ICAS) enrolled over 900 children (aged 5–11) with moderate to severe asthma living in seven cities (Seattle, Dallas, Tucson, Boston, New York, St Louis, and Chicago). This study compared the prevalence of indoor allergen exposure (mite, CR, pets) with sensitization, symptoms, and geographic location. Remarkably, about 80% of children from New York and Dallas were CR-sensitive, and CR exposure was specifically associated with asthma morbidity (whereas, in this population, dust mite or pet exposure was not) (Gruchalla et al. 2005).

0.4 Hospitalizations in past year

Epidemiology in the USA

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P = 0.001

0.3 0.2 0.1 0.0

Group 1

3 No. of visits in past year

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Group 2

Group 3

Group 4

Group 3

Group 4

P < 0.001

2

1

0

Group 1

Group 2

Fig. 51.1 Rates of hospitalization and unscheduled medical visits amongst inner-city children with asthma. Data from NCICAS: Group 1, no allergy to CR and low levels of exposure; Group 2, no allergy to CR and high exposure; Group 3, CR-allergic children with low-level exposure; Group 4, CR-allergic children with high-level exposure (> 8 units/g Bla g 1). (From Rosenstreich et al. 1997, with permission.)

Cockroach allergy in the USA is not exclusively an innercity problem. Exposure to CR allergens occurs in substandard or deteriorated housing in inner-cities, and also in suburban and rural areas (Garcia et al. 1994; Rauh et al. 2002). The NSLAH study reported elevated levels of Bla g 1 (> 2 units/g) in 13% of US kitchens, which were associated with pre-1940s homes, high-rise apartments, urban areas, and households with incomes of < $20 000/year. High CR allergen levels were linked to visible CR infestation, relative humidity > 60%, moisture problems, and food debris in rooms (Cohn et al. 2006). Similar associations have been found in studies of CR allergen exposure in northeastern states, and with CR infestation in the Salinas Valley in California (Leaderer et al. 2002; Bradman et al. 2005). Public housing apartments in New York City have a high prevalence of CR infestation (77%). More than one-third of these apartments had a resident with asthma, which was associated with high levels of Bla g 2 (and also mouse urinary allergen) (Chew et al. 2006). A surprisingly high prevalence of CR allergen exposure was also reported in suburban middle class homes in the Baltimore area (with household incomes > $50 000/year and 48% of mothers with college degrees). In that study, 33% of suburban-rural homes had kitchen Bla g 1 levels of > 2 units/g dust, and were associated with CR sensitization, although only 5% of homes had visible evidence of CR (Matsui et al. 2003). The results

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suggested that CR allergy was more common in suburban US homes than had previously been expected. There are several reports of CR allergen exposure in schools. Bla g 1 was detected at significant levels (median 2.7–5.2 units/g) in 69% of samples (102/147) in four schools in urban Baltimore, all of which had recognized problems with CR infestation (Sarpong et al. 1997). A subsequent study in Baltimore found a high prevalence of CR allergen (66%) in 12 elementary schools in Baltimore, but lower levels of Bla g 1 and Bla g 2, and concluded that school exposure was likely to be less important for asthma symptoms than exposure in the home (Amr et al. 2003). A study of 11 public high schools from a “major metropolitan area in the northeastern USA” reported Bla g 2 in 71% of dust samples at levels up to 1.1 μg/g in dust. Bla g 2 was also detected in 22% of the school air samples (Chew et al. 2005). A recent study compared allergen levels in 41 public schools from Birmingham (AL), Detroit (MI), and Houston (TX), although, with some exceptions, the allergen levels found in these schools were relatively low (Abramson et al. 2006). Overall, the results of these studies demonstrate that it is possible to make reliable measurements of CR allergen exposure in schools. However, further studies are needed to establish whether school exposures contribute to asthma symptoms.

Cockroach allergy worldwide Cockroaches have been reported to be an important cause of asthma in several other parts of the world, including Taiwan, Thailand, Central and South America, Europe, and South Africa (reviewed in Chapman et al. 1997; Arruda et al. 2001a). The predominant species in Taiwan and Japan are P. americana and P. fuliginosa. Positive skin tests to both P. americana and B. germanica were reported in 55% of children and young adults with asthma and rhinitis living in São Paulo, Brazil (Santos et al. 1999). Significant levels of CR allergens were found in daycare centers and elementary schools in São Paulo, at similar levels to those found in Tampa, Florida (Fernandez-Caldas et al. 2001; Rullo et al. 2002). A high level of sensitization to CR was also reported in subjects with asthma living in Puerto Rico and Venezuela (Sanchez-Borges et al. 2003; Montealegre et al. 2004). The break-up of the Soviet Union and the liberalization of former Eastern Bloc countries has facilitated investigation of CR allergy in these countries. A significant prevalence of CR sensitization has been reported among asthma patients in Zgierz, Poland (24%) and Tallinn, Estonia (35%) (Stelmach et al. 2002; Raukas-Kivioja et al. 2003; Raukas-Kivioja et al. 2006). Reports also suggest that CR allergy is present in Ankara, Turkey, but not in the Trakya region (Yazicioglu et al. 2004; Yilmaz et al. 2004; Uzel et al. 2005). Almost all of these studies have used skin-prick tests to assess CR sensitization and, in most cases, these populations have a high prevalence of mite sensitization. This raises the question as to the extent to which CR sensitization may reflect cross-reactivity with mite or other allergens (e.g.,

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shellfish). A report of 14% prevalence of CR allergy among asthmatic children enrolled in the GAIN study (Genetics of Asthma International Network) in Norway was surprising because there were no previous reports of CR allergy in Scandinavia, nor of CR allergens in housing (Chapman 2002; Lodrup Carlsen et al. 2002). In these circumstances, it is important to confirm the results of skin tests using in vitro IgE assays, to inspect homes for visible evidence of CR infestation and, preferably, to measure exposure to CR allergens.

The repertoire of cockroach allergens Since the molecular cloning of the first CR allergen, Bla g 2, in 1995, a repertoire of eight CR allergens has been described, mostly from B. germanica (Arruda et al. 1995b) (Table 51.1). Bla g 1, Bla g 2, Bla g 4, Bla g 5, Bla g 6, Bla g 7, and Bla g 8 are homologous to gut microvillar proteins, aspartic proteases, lipocalins, glutathione S-transferases, troponin C, tropomyosins, and myosin light chains, respectively (Arruda et al. 1995a,b, 1997; Helm et al. 1996; Pomés et al. 1998; Wu et al. 1998; Jeong et al. 2003; Hindley J et al. 2006). The main allergens in P. americana are Per a 1, Per a 3, and Per a 7 (Wu et al. 1996, 1997, 1998; Melén et al. 1999; Wang et al. 1999; Yang et al. 2000; Diraphat et al. 2003; Jeong et al. 2003) (Table 51.1). Only the Group 1 and Group 7 allergens show antigenic crossreactivity between both CR species. Per a 3 is a homolog of the insect storage proteins arylphorins and the hemocyanins, which are proteins in the hemolymph that bind oxygen through copper (Wu et al. 1996). Experimental evidence suggests that Bla g 2, Bla g 4, and Bla g 5 could be species-specific based on Northern blot analysis and lack of inhibition of IgE antibody binding to Bla g 5 by P. americana extracts (Arruda et al. 1995a,b, 1997). A Per a 2 allergen with 44% identity to Bla g 2 has recently been reported by Chew (Gustchina et al. 2005). Conversely, a Per a 3 homolog was not detected in B. germanica extracts using monoclonal antibodies against Per a 3, but anti-Per a 3 polyclonal antibodies did recognize proteins in a German CR extract, suggesting the presence of low homology variants in German CR (Wu et al. 1990). Other minor CR allergens have been described. The Group 7 allergens are homologs of tropomyosin, a panallergen present in a wide variety of organisms and involved in muscle contraction (Reese et al. 1999). The prevalence of sensitization to rBla g 7 and rPer a 7 was 16% by ELISA and 50% by plaque immunoassay, respectively, in two different populations (Santos et al. 1999; Jeong et al. 2003). A tropomyosin from the dusky brown CR (Periplaneta fuliginosa) with 100% and 98.2% amino acid identity with P. americana and B. germanica, respectively, has also been cloned (Jeong et al. 2004a). Recently, the Group 6 and Group 8 allergens have been described, which show homology to troponin C and myosin light chain, respectively (Hindley et al. 2006) (Table 51.1). Three Bla g 6 isoallergens were cloned with a 14% prevalence of IgE antibodies. Interest-

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Table 51.1 Nomenclature and function of cockroach allergens. Allergens

Molecular weight (kDa)*

Blattella germanica Bla g 1 ∼ 25–90 Bla g 1.0101 46 21 Bla g 1.0102 25–37 Bla g 1.02 56 Bla g 2† 36 Bla g 4 Bla g 5 Bla g 6 Bla g 6.0101 Bla g 6.0201 Bla g 6.0301 Bla g 7 Bla g 8

21 23 17

33 21

Periplaneta americana Per a 1 ∼ 25–45 Per a 1.0101 26 Per a 1.0102 26 Per a 1.0103 45 Per a 1.0104 31 Per a 1.0105 14 Per a 1.02 51 Per a 2 36 Per a 3 46–79 Per a 3.01 79 Per a 3.0201 75 Per a 3.0202 56 Per a 3.0203 46 Per a 6 17 Per a 7 33 Per a 7.0101 Per a 7.0102

Accession number

Function/homology

Reference

Midgut microvilli protein homolog AF072219 AF072221 L47595 AF072220 U28863 U40767 U92412 DQ279092 DQ279093 DQ279094 AF260897 DQ389157

Unusual aspartic protease Lipocalin Glutathione S-transferase Troponin C

Tropomyosin Myosin light chain

Pomés et al. (1998) Pomés et al. (1998) Helm et al. (1996) Pomés et al. (1998) Arruda et al. (1995b); Wünschmann et al. (2005); Gustchina et al. (2005) Arruda et al. (1995a) Arruda et al. (1997) Hindley et al. (2006) Hindley et al. (2006) Hindley et al. (2006) Jeong et al. (2003) Hindley et al. (2006)

Midgut microvilli protein homolog AF072222 U78970 U69957 U69261 AY259514 U69260 AY792947 L40818 L40820 L40819 L40821 AY792950

Inactive aspartic protease Arylphorin/hemocyanin

Troponin C Tropomyosin

Y14854 AF106961

Melén et al. (1999) Wu et al. (1998) Yang et al. (2000) Wu et al. (1998) Diraphat et al. (2003) Wang et al. (1999) Gustchina et al. (2005) Wu et al. (1996) Wu et al. (1996) Wu et al. (1997) Wu et al. (1997) Hindley J et al. (2006) Asturias et al. (1999) Santos et al. (1999)

* Molecular weight calculated from amino acid sequence. † The accession number for Bla g 2 crystal structure in the Protein Data Bank is 1YG9.

ingly, Bla g 6, Per a 6 and Bla g 8 are calcium-binding proteins, and the IgE binding to Bla g 6 has proven to be calcium dependent.

IgE reactivity and immune response The prevalence of IgE antibodies to German CR allergens was measured in several studies of CR allergic patients with asthma (Table 51.2). Results indicate that Bla g 2 and Bla g 5 are the most important allergens, with a prevalence of 57 and 68% to the natural allergens, respectively. These values reached up to 70% for Bla g 2 among patients with high RAST (> 200 RAST units/mL; N = 58) (Arruda et al. 1995b). Measurement of IgE binding to Bla g 1, Bla g 2, Bla g 4 and Bla g 5

accounts for sensitization to B. germanica in 95% of allergic patients (Arruda et al. 1997). Cockroach sequences have been cloned into high-level expression systems for production of recombinant allergens in E. coli (Bla g 5) and in the yeast Pichia pastoris (Group 1, Bla g 2, Bla g 4, Group 7) (Vailes et al. 1998; Arruda et al. 2001b; Pomés et al. 2002b). The recombinant CR allergens were purified using monoclonal antibody or glutathione affinity chromatography to > 90% purity with yields of 10–20 mg/L of culture. The biological activity of purified natural and recombinant CR allergens was demon-strated by intradermal skin testing. Positive immediate skin reactions (> 8 × 8 mm wheal) were obtained using 10–3.5 μg/mL of purified natural Bla g 1 and Bla g 2 and using

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Table 51.2 Prevalence of IgE antibodies to Blattella germanica allergens. Allergens

Technique

Patients

Natural/recombinant

% Total

Reference

Bla g 1

Monoclonal antibody-based RIA Streptavidin CAP Monoclonal antibody-based RIA Streptavidin CAP Plaque assay RIA Streptavidin CAP RIA RIA/ELISA Streptavidin CAP Multiplex array ELISA ELISA

106 115 106 114 73 73 115 40 40 115 104 35 37

Natural Recombinant Natural Recombinant Recombinant Recombinant Recombinant Natural Recombinant Recombinant Recombinant Natural Recombinant

30 26 57 54 60 31–41 17 68 73 37 14 18 16

Arruda et al. (1995b) Satinover et al. (2005) Arruda et al. (1995b) Satinover et al. (2005) Arruda et al. (1995a) Arruda et al. (1995a) Satinover et al. (2005) Arruda et al. (1997) Arruda et al. (1997) Satinover et al. (2005) Hindley J et al. (2006) Jeong et al. (2004b) Jeong et al. (2003)

Bla g 2 Bla g 4

Bla g 5

Bla g 6 Bla g 7

10–3–10–5 μg/mL rBla g 4, and 10–1–10–4 μg/mL rBla g 5. Per a 3 showed a high prevalence of IgE antibodies, 83%, with isoforms that elicited different skin reactions (26–95%) (Wu et al. 1996, 1997). There was a very good quantitative correlation between IgE antibody to natural Bla g 1 and Bla g 2 and to the recombinant proteins, suggesting that recombinant allergens could be used for diagnostic purposes (Chapman et al. 2000; Pomés et al. 2002b). A recent study measured IgE antibodies to rBla g 1, rBla g 2, rBla g 4, rBla g 5 and rPer a 7 in 118 sera from CRsensitized subjects by streptavidin CAP assay. The highest prevalence of IgE antibodies was to rBla g 2 (54%), which was approximately twofold higher than to the other allergens tested: 26% to rBla g 1, 17% to rBla g 4, 37% to rBla g 5 and 13% to rPer a 7 (Satinover et al. 2005). Among sera with high IgE antibody to CR extract (3.5–100 IU/mL), the prevalence of IgE antibodies to rBla g 2 and rBla g 5 was 71% and 58%, respectively. These results confirm previous findings that 42–70% of cockroach-allergic patients produce Bla g 2-specific IgE (Arruda et al. 1995b). Bla g 2 is the most important known cockroach allergen and appears to cause sensitization at exposure levels that are 10–100-fold lower (< 1 μg/g dust) than to cat and mite allergens (Sporik et al. 1999). Bla g 1 and Bla g 2 are used as markers of cockroach allergen exposure in environmental and avoidance studies. Only 16% of CRsensitized subjects with IgE antibodies to housedust mite exhibited IgE antibody binding to rPer a 7. This result suggests that concomitant exposure to housedust mite and CR allergens other than tropomyosin, instead of antigenic crossreactivity, may explain cosensitization to mite and CR (Satinover et al. 2005). Apart from studies on IgE responses, there have been few studies on other aspects of the human immune response to CR allergens. Technical difficulties in measuring total IgG antibody or specific IgG antibody subclasses to CR allergens

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have limited studies on the relationship between IgG and IgE responses in CR-allergic patients. Thus, while high-dose exposure to cat allergen (Fel d 1) was associated with the induction of IgG4 antibodies and a reduction in IgE antibody levels, it has not been possible to investigate whether these “modified Th2 responses,” or forms of tolerance, occur with CR allergens (Platts-Mills et al. 2001a). Similarly, there have been no controlled trials of CR allergen immunotherapy even though, anecdotally, CR immunotherapy appears to be widely practiced by allergists in the USA. The CR extracts used for immunotherapy are nonstandardized and of relatively low potency (Patterson & Slater 2002). However, the relative potency correlated with Bla g 1 and Bla g 2 content, and the US Food Drug Administration has initiated studies to standardize CR extracts, based in part on major allergen measurements. In a prospective birth cohort, exposure to low levels of Bla g 1 and Bla g 2 was associated with wheezing among infants in the first 3 months of life and with increased proliferative T-cell responses to Bla g 2 (Finn et al. 2000). The children who developed atopic disease had lower levels of interferon (IFN)-γ secretion by peripheral blood mononuclear cells in response to either Bla g 2 or Der f 1 than children with no atopic disease (Contreras et al. 2003). No systematic studies of T-cell responses to the available repertoire of CR allergens have been published. This may in part relate to the difficulties associated with recruiting CR-allergic patients on a continuing basis. Mouse models of CR asthma have been developed by sensitizing BALB/c mice with CR extract by inhalation. These mice show airway hyperreactivity, eosinophil accumulation in the lung, and reduced levels of Th2 cytokines. They have been used to investigate the role of proteases, protease receptors, lipopolysaccharide, and candidate pharmacotherapeutics in the asthmatic and control mice (Lundy et al. 2003; Kondo et al. 2004; Thomas et al. 2004; Warner et al. 2004; Berlin & Lukacs 2005; McKinley et al. 2006). However, whether

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these effects are related to specific CR allergens or other antigens, proteins, DNA, or components of CR extracts, has not been established. The CR antigens are described as clinical grade and endotoxin-free, but the use of such heterogeneous extracts is a flaw in these models, made worse by the use of “housedust extract” enriched with cockroach “allergens” (McKinley et al. 2006). Cockroach extracts are known to contain proteases and may contain macromolecules or mediators with other nonspecific pharmacologic or immunologic effects (Esch 1992). It is important that the mouse models of CR asthma are validated by demonstrating that the responses are specific and by identifying the active molecules that are responsible for the immunological and pathological effects.

Molecular structure The primary structure obtained by molecular cloning provides the basis to investigate the tertiary structure of allergens. Protein folding defines the molecular surface of the allergen and its antigenic determinants, including B-cell epitopes. The tertiary structures of Bla g 4 and Bla g 6 were modeled, based on homologous proteins of known structure (Arruda et al. 1995a; Hindley et al. 2006). Recently, the X-ray crystal structure of Bla g 2 has been solved at high resolution (1.3 Å) (Gustchina et al. 2005). The tertiary structure of these allergens provides new insights on structural elements that may influence allergenicity. Bla g 1 and Per a 1 are proteins with tandem repeats of 100 amino acids (Pomés et al. 1998; Melén et al. 1999; Yang et al. 2000). Two consecutive repeats are encoded by one nucleotide repeat, which is generated by gene duplication (Helm et al. 1996; Pomés et al. 1998). Several cDNA clones revealed the existence of isoallergens and variants encoding for Group 1 allergens of different lengths (Table 51.1). Additionally, N-terminal sequencing of the natural allergen showed that the molecules are cleaved after arginine residues by trypsin-like enzymes. This results in several molecular forms containing different number of repeats, which are recognized by IgE antibody (Pomés et al. 2002b). Bla g 4 is an insect homolog of a group of mammalian allergens that belong to the lipocalin family of proteins (Arruda et al. 1995a; Flower et al. 2000). Lipocalins are ligand-binding proteins that transport molecules such as steroids, retinoids, arachidonic acid, and pheromones. Lipocalin allergens include Bos d 2 and Bos d 5 (cow), Can f 1 and Can f 2 (dog), Equ c 1 and Equ c 2 (horse), Fel d 4 (cat), Mus m 1 (mouse) and Rat n 1 (rat). The typical lipocalin structure is very stable and consists of a C-terminal α-helix and a single eight-stranded antiparallel β-barrel enclosing an internal cavity. Amino acid homology as low as 20% identity is enough for these allergens to share the same folding without being cross-reactive. Bla g 6 and Per a 6 share homology with troponin C, which regulates muscle contraction and contains a calcium-binding EF-hand motif (Hindley J et al. 2006). IgE binding to Bla g 6 was highest when calcium was added to calcium-depleted

Cockroach Allergens, Environmental Exposure, and Asthma allergen, indicating that this molecular conformation was prevalent when IgE was produced. Bla g 7 and Per a 7 are homologs of tropomyosin, which has a structure comprising two parallel α-helical molecules wound around each other (Asturias et al. 1999; Santos et al. 1999; Jeong et al. 2003). The high degree of amino acid sequence identity (∼ 80%) explains the allergenic cross-reactivity among tropomyosins from different invertebrate species, including cockroach, mite (Group 10), shrimp (Met e 1, Pen a 1, Pen i 1), and snail (Hel as 1) (Ayuso et al. 2002). The observed crossreactivity may explain reports of adverse reactions to mite immunotherapy among patients who regularly ate shrimp or snails (van Ree et al. 1996a,b). A monoclonal antibody raised against mite tropomyosin reacts with tropomyosins from CR and from the worm Ascaris lumbricoides by immunofluorescence (Fig. 51.2). This monoclonal antibody can also be used for affinity purification of recombinant Per a 7, confirming that it recognizes a common epitope present on tropomyosins from diverse species (Santos et al. 1999; Arruda et al. 2001b).

Tertiary structure of Bla g 2 The recently solved X-ray crystal structure of Bla g 2 revealed that this potent allergen shares the same folding as homologous aspartic protease enzymes (Gustchina et al. 2005) (Fig. 51.3). These enzymes have a bilobal shape, with a cleft between two domains that binds substrates and inhibitors. Two motifs DT/SG from each of the domains are seated at the bottom of the cleft and contain two aspartates that are directly involved in catalysis through the interaction with a water molecule. The substitution of the aspartic protease motifs by DST and DTS in Bla g 2 creates distortions at the level of the catalytic site, so the aspartates are 4.41 Å apart, instead of the usual ∼ 3 Å in standard aspartic proteases. Additionally, there is an insertion of a phenylalanine in position 75a, which provides a mechanism of autoinhibition by blocking the access of substrates to the cleft as in standard enzymes. Bla g 2 strongly resembles a group of pregnancy-associated glycoproteins (PAGs), which also have important amino acid substitutions in the cleft that makes them enzymatically inactive. In the first aspartic protease motif, for example, alanine rather than glycine is present in boPAG-1 and poPAG-1, and glycine instead of aspartate is found in the second motif of ovPAG-1. Ovine PAG-1 has also an extra phenylalanine at an equivalent position (Guruprasad et al. 1996). These modifications from typical aspartic proteases impair enzymatic activity (Wünschmann et al. 2005). Two additional structural features may contribute to the allergenicity of Bla g 2 by conferring stability to the protein: the presence of five disulfide bonds (instead of the usual two or three) and a zinc-binding domain. In the N-terminal domain, cysteines 36–127, 45–50, and 51a–113 form disulfide bonds, and in the C-terminal domain Cys237–245 and 249–282 are involved (Fig. 51.3). Only the bonds between Cys45–50 and Cys249–282 are conserved within the aspartic

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10 mm (a)

(b) Fig. 51.2 Tropomyosin (Per a 7) in cockroach tissue: a cross-reactive allergen. Frozen sections were immunostained with anti-mite tropomyosin. (a) Cockroach muscle fibers; (b) Ascaris. (From Santos et al. 1999, with permission.) (See CD-ROM for color version.)

g 2 in the environment may be influenced by the presence of the five disulfide bonds and the zinc-binding domain, which increase the stability of the allergen, and the likelihood that it will persist in the environment.

protease family of enzymes. The remaining three disulfide bonds do not have structural analogs in the other aspartic proteases described to date. One atom of zinc bound to Bla g 2 was identified by X-ray absorption spectroscopy (EXAFS) near the absorption edges of Ni and Zn, performed on small crystals of the protein mounted in a capillary. Zinc binds through His155, His161, Asp303, and Asp307, keeping together two secondary structure elements (a β-loop and an α-helix) that contain the histidines and aspartates, respectively (Fig. 51.3). Only Asp303 is conserved in standard aspartic proteases, and the other three amino acids involved in zinc binding are only observed in CR sequences from P. americana and Leucophaea maderae. The persistence of Bla

Biological function In some cases, the biological function of allergens can be predicted from amino acid sequence homology to proteins of known function. However, in most cases homology alone is insufficient and functional assays, and/or tissue localization, are needed to confirm a specific biological function. For example, Bla g 2 lacks proteolytic activity despite the homology to aspartic proteases. The function of Group 1 allergens could

His155

N317

2.05 Å 1.97 Å 1.95 Å Zn

N268

Asp303 Zn

249–282

51A–113

1138

D215 2.76 4.41

F75

237–245

His161

Asp307

36–127 D215 D32

1.90 Å

45–50

D32

Fig. 51.3 Crystal structure of rBla g 2 (1yg9.pdb). The structure (at left) shows locations of disulfide bonds, glycosylation sites, the zinc atom and residues at the region of the catalytic site (D215, D32). At right, coordinating residues and interatomic distances of residues that bind zinc (upper panel); aspartate positions in Bla g 2 (red/blue) and in pepsin (orange/yellow). (From Chapman et al. 2007, with permission.) (See CD-ROM for color version.)

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UG U U

Fig. 51.4 Bla g 4 is produced in male cockroach accessory reproductive organs. Blue staining shows deposition of Bla g 4 in conglobate glands (CG) and untricles (U), but not in epididymal pouch (EP), epididymal duct (ED), median sclerite (MS), or uricose gland (UG). (From Chapman et al. 2007, with permission.) (See CD-ROM for color version.)

MS

EP CG

be related to digestion, as these allergens are secreted into the gut lumen and subsequently excreted into the environment (Pomés et al. 1998). Group 1 allergens share homology with ANG12 and AEG12, proteins that are induced after a blood meal in the midgut of adult female mosquitoes (Anopheles gambiae and Aedes aegypti, respectively) (Morlais et al. 2003; Shao et al. 2005). The allergens and mosquito protein AEG12 are homologous to microvillar membrane-associated proteins from the coleopteran Tenebrio molitor. Shao and collaborators elegantly demonstrated the microvillar localization of AEG12 in the midgut microvilli by immunoelectron microscopy. The presence of a putative glycosyl-phosphatidylinositol (GPI) membrane anchor at the C-terminal end of AEG12 and Bla g 1.0101 suggests that these proteins could be membrane proteins, despite the lack of transmembrane domains. Following a blood meal, there would be shortening and loss of microvilli, a phenomenon commonly observed among insects. Similarly, Bla g 1 is most prevalent in the midgut, and the Bla g 1 gene is exclusively expressed in midgut cells (Gore & Schal 2004). Bla g 1 production is related to food intake and adult females excrete significantly more Bla g 1 in their feces than males or nymphs, most likely because females process more food than males or than other life stages of CR (Gore & Schal 2005). These studies indicate that Group 1 allergens could be microvillar proteins whose expression is induced after food intake, as reported for the homologous insect proteins. Bla g 4 has recently been reported to have a male reproductive function. This allergen is only expressed in the adult male reproductive system, specifically in the utricles and the conglobate gland (Fan et al. 2005) (Fig. 51.4). The presence of Bla g 4 in discarded spermatophores expelled by mated females suggests that Bla g 4 may be a component of the spermatophore rather than the seminal secretions, and is transferred to the female within the spermatophore during copulation.

ED

Although the direct effect of these allergens in the lung remains unknown, Bla g 1, Bla g 2, and Bla g 4 are secreted or excreted by CR, and become airborne from the dried secretions. Allergens involved in muscle contraction, such as Bla g 6 and Bla g 7, require CR death and muscle degradation to be released in the environment. The accumulation and persistence of stable CR allergens in the home appears to result in chronic low-level exposure that is associated with asthma.

Clinical trials of cockroach remediation procedures Standard physical recommendations for controlling CR in houses and apartments include reducing access to water sources; removing food and household debris; improving ventilation and reducing humidity; and sealing/caulking CR access and entry points. Insecticides, such as pyrethrins and boric acid, which used to be used for CR control are increasingly being replaced with a new generation of gel baits containing fipronil (0.01%), hydramethylnon (2.15%), abamectin (0.05%), or imidacloprid (2.15%) (in products such as MaxForce, Avert and Pre-Empt, respectively). These powerful insecticides are safe for human use and have been used in clinical trials to: (i) reduce CR and/or allergen levels in homes; and (ii) investigate whether reductions in allergen exposure are associated with improvements in clinical symptoms. An initial intervention study carried out in 48 homes enrolled in the NCICAS and treated with abamectin proved disappointing. Following a short-lived reduction at 2 months, CR allergen levels returned to baseline after 1 year and were higher than 8 units/g Bla g 1 in ∼ 50% of cases (Gergen et al. 1999). Reasons cited for the poor outcome of this study were that most families lived in multifamily apartments, compliance

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3500 Exterminations Bla g 1 (units/g settled dust)

3000 2500 2000 1500 1000 500 0 0

2

4

6

8

10

Months Fig. 51.5 Effect of insecticide treatment on cockroach allergen levels in inner-city homes. The 13 homes received three treatments with abamectin and professional cleaning. Bla g 1 concentrations in kitchen dust samples are shown. (From Eggleston et al. 1999b, with permission.)

with cleaning instructions was poor, and the baseline allergen levels were very high. A more tightly focused study on 13 innercity homes in Baltimore was more promising. This study used repeated applications of abamectin, together with professional cleaning, and reported reductions in CR counts and an 84% reduction in Bla g 1 levels in the kitchen over an 8-month period (Eggleston et al. 1999b) (Fig. 51.5). Both the NCICAS and the Baltimore studies lacked non-intervention control groups. Two subsequent studies compared the effects of insecticide treatment with nonintervention or other control groups. A randomized study in metropolitan regions of North Carolina compared occupant education, professional cleaning, and hydramethylnon gel bait treatment in 16 intervention and 15 control homes. This study had strict enrollment criteria, including that the homes would be occupied by an adult for at least 6 months and that the homes contained 50–500 trapped CR over a 3-day period (Arbes et al. 2003). The results showed dramatic reductions in trapped CR in kitchens, living rooms, and bedrooms in the intervention group, with no CR detected at 6 months. Although decreases in CR counts were also observed in the control group, CR were not eradicated. In parallel with reductions in CR counts, there was also an 83–94% reduction in Bla g 1 levels in beds, bedroom floors, and sofas in the intervention group, which were significant when compared with allergen levels in control homes. The allergen levels before treatment in this study were about nine times higher than in NCICAS. A follow-up study investigated whether the allergen reductions could be maintained by further application of insecticide alone (2.15% hydramethylnon), without professional cleaning (Arbes et al. 2004). The study included a crossover group of previously untreated homes that were enrolled into the intervention group and treated

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with insecticide bait. The reduced allergen levels in the intervention group were maintained from 6 to 12 months by the application of insecticide bait. Furthermore, there was a 67–95% reduction in Bla g 1 and Bla g 2 levels in the crossedover control homes. This study indicated that significant CR allergen reductions could be made by application of insecticide baits alone and questioned whether cleaning measures were essential. Intervention and control groups were also compared in a study of 49 homes in inner-city Los Angeles. This study compared an insecticide treatment (fipronil bait traps and hydramethylnon gel), together with professional cleaning; a sham insecticide group, which received professional cleaning; and an untreated control group. The groups were followed for 11 weeks. The results showed significant reductions in CR counts and levels of Bla g 2 in the insecticide-treated group, but not in untreated control homes. Intensive cleaning, without insecticide, reduced Bla g 2 levels in kitchens that had high initial CR allergen levels (> 5 μg/g Bla g 2), but had no effect on homes with low initial allergen levels (< 0.5 μg/g) (McConnell et al. 2003). The authors concluded that use of professional cleaning to reduce CR allergen exposure in highly infested homes could be beneficial for asthma patients, especially if the cleaning company worked with the patients to introduce long-term CR reduction strategies. However, the data from published studies suggests that insecticide application is essential to obtain sustained reductions in CR. It is difficult to compare the relative efficiency of professional cleaning companies or patient education procedures. Studies that have used effective CR control have generally been most successful in reducing allergen exposure below disease thresholds (Gore & Schal 2007). The largest and most effective demonstration of environmental intervention among inner-city children was ICAS, a randomized controlled study of 937 inner-city children with asthma, which compared exposure and clinical outcomes in a multiallergen intervention over a 2-year period. The intervention used social learning theory to train caregivers about the nature of the problem and to motivate them to implement comprehensive environmental remediation. Environmental counselors were used to evoke behavioral change. The remediation procedures were targeted to dust mite, CR, pets, mold, and passive smoke exposure over a 12-month period, and included allergen-proof bedcovers, high-efficiency particulate air (HEPA) filters, and Terminix insecticide. Over 400 children in the intervention and in the control group completed the 2-year study. The results showed highly significant reductions in symptoms in the intervention group for every 2-week period of the study; 19.5% fewer symptom days, 13.6% fewer unscheduled medical visits, and 20.7% fewer missed school days (Morgan et al. 2004) (Fig. 51.6). These effects on symptoms were comparable to those seen in placebo-controlled trials of inhaled corticosteroids. Moreover, the reductions in asthma morbidity correlated with reductions

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Maximal no. of days with symptoms (no./2 wk)

7 6 5 4

Control

3 Intervention

2 1

6

o M 8 o 10 M o 12 M o 14 M o 16 M o 18 M o 20 M o 22 M o 24

M

4

o

o

M

2 M

o M

Ba

se l

in

e

0

Intervention year

Year after intervention

Fig. 51.6 A home-based environmental intervention reduces symptoms in inner-city children with asthma. Data from the ICAS group. (From Morgan et al. 2004, with permission.)

in CR and mite allergen levels in the children’s bedrooms. Further economic analysis showed that the intervention was cost-effective, especially as the interventions addressed causative factors for disease activity, rather than simply reducing symptoms, and could be expected to have long-term benefits (Kattan et al. 2005).

Conclusions There is abundant evidence that CR produce potent allergens that are an important cause of asthma. The epidemiology of CR asthma has been studied most extensively in the USA, where lower socioeconomic status, race, and poor access to healthcare are important risk factors for CR sensitization and asthma morbidity. Allergic disease caused by CR is most prevalent in inner-cities and urban areas. Cockroach infestation occurs in houses, apartments, public housing, and trailer homes that are in a state of disrepair. Such homes are also found in suburban and rural areas and, while CR allergy has been reported in suburban middle class homes, the disease is primarily associated with poverty and substandard housing conditions. CR allergy is an important public health problem in the USA because many patients are uninsured and use hospital emergency rooms as their source of medical care. The recent ICAS intervention showed that a strong program of patient education, together with comprehensive environmental control procedures, produced sustained, long-term reductions in asthma morbidity among CR allergic inner-city children. The challenge for public health is to develop programs that will be effective in implementing these approaches among high-risk populations. The new generation of insecticides can effectively reduce CR infestation and changes in environmental allergen exposure can be accurately monitored. However, the implementation of public health programs

Cockroach Allergens, Environmental Exposure, and Asthma will also require political will at the federal, state, and local levels to improve housing conditions for lower socioeconomic populations. The repertoire of CR allergens has been defined using molecular cloning techniques and there has been significant progress in determining the structure, biological function, and localization of these allergens. Cockroach allergens are very similar to those of dust mite in that they are strongly associated with asthma, have similar aerodynamic properties, and persist in the environment for long periods of time. The CR and mite airborne particles are 5–10 μm in diameter and only remain airborne for 20–40 min (de Blay et al. 1997). The molecular structures of CR allergens have features that confer stability to these proteins. Strong arguments have been made that the intrinsic proteolytic activity of mite allergens (Der p 1, Der p 3, Der p 9) contributes towards both IgE production and inflammatory responses in the lung, including production of proinflammatory cytokines and disruption of the architecture of bronchial epithelial cells (Gough et al. 1999; Wan et al. 1999). Enzymatic activity could be a contributing factor that explains why mite allergens cause asthma. However, this view is complicated by CR, where none of the allergens that have been cloned are active proteolytic enzymes. This does not entirely rule out a role for enzymes in CR asthma, as CR extracts contain serine proteases which do not appear to be allergens, but may have similar effects on the lung. In contrast to mite and CR, cat and dog allergens lack enzymatic activity and occur on smaller aerodynamic particles, which stay airborne for several hours and are inhaled at much higher cumulative dosages. This persistent exposure to high doses of inhaled allergen may result in the development of tolerance in some individuals and may explain why living with cats can protect against the development of allergy (Platts-Mills et al. 2001b; Ownby et al. 2002). Exposure to mite and cat allergens is generally considered to be ubiquitous and difficult to control for in epidemiologic studies. On the other hand, CR exposure is much more restricted and can often be used as a control in designing studies to investigate risk factors or to assess immunologic responses. The production of recombinant CR allergens in high-level expression systems should facilitate their use in diagnostics and in research studies to develop new allergy vaccines. However, while the molecular biology of CR allergens is as well developed as for other allergen systems, knowledge of human immune responses, particularly T-cell responses and allergen processing, is sparse and clearly warrants further research. New immunotherapy strategies using either purified recombinant allergens or modified allergens (e.g., using CpG-coupled recombinant allergens) are feasible using the allergens that have been identified to date. The quandary from a public health perspective is whether to apply resources toward immunotherapeutic approaches (for which, in the case of CR, there is little evidence of efficacy) and/or to promote environmental and social interventions. The latter have

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shown promising results and, at minimum, demonstrated proof of principle. Ideally, both approaches have merits and should be pursued. However, in an era of evidence-based medicine, the current balance lies in favor of environmental intervention as the most cost-effective and feasible approach to reducing allergic disease caused by CR. A multifaceted approach is needed including improvements in housing conditions (or new housing), strategic application of insecticides, professional cleaning, and patient education. As with all environmental interventions, it is essential to know that the interventions are implemented and maintained on a long-term basis. The recent success of CR intervention provides strong evidence that this is a pragmatic approach, which should reduce CR allergen exposure and associated allergic diseases.

Acknowledgments We are grateful to Drs David Rosenstreich, Karla Arruda, Peyton Eggleston, Chad Gore, Coby Schal, Wayne Morgan, and Herman Mitchell for permission to reproduce their data in this chapter. It has also been a great pleasure to collaborate with Drs Alla Guschina, Mi Li, and Alex Wlodawer (National Cancer Institute) over the past 5 years on the structural biology of Bla g 2.

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Chapman, M.D. (1998) Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem 273, 30801–7. Pomés, A., Chapman, M.D., Vailes, L.D., Blundell, T.L. & Dhanaraj, V. (2002a) Cockroach allergen Bla g 2: structure, function, and implications for allergic sensitization. Am J Respir Crit Care Med 165, 391–7. Pomés, A., Vailes, L.D., Helm, R.M. & Chapman, M.D. (2002b) IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1. Eur J Biochem 269, 3086–92. Rauh, V.A., Chew, G.R. & Garfinkel, R.S. (2002) Deteriorated housing contributes to high cockroach allergen levels in inner-city households. Environ Health Perspect 110 (suppl. 2), 323–7. Raukas-Kivioja, A., Raukas, E., Loit, H.M. et al. (2003) Allergic sensitization among adults in Tallinn, Estonia. Clin Exp Allergy 33, 1342– 8. Raukas-Kivioja, A., Raukas, E.S., Meren, M., Loit, H.M., Ronmark, E. & Lundback, B. (2006) Allergic sensitization to common airborne allergens among adults in Estonia. Int Arch Allergy Immunol 142, 247–54. Reese, G., Ayuso, R. & Lehrer, S.B. (1999) Tropomyosin: an invertebrate pan-allergen. Int Arch Allergy Immunol 119, 247–58. Rosenstreich, D.L., Eggleston, P., Kattan, M. et al. (1997) The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. N Engl J Med 336, 1356–63. Rullo, V.E., Rizzo, M.C., Arruda, L.K., Sole, D. & Naspitz, C.K. (2002) Daycare centers and schools as sources of exposure to mites, cockroach, and endotoxin in the city of Sao Paulo, Brazil. J Allergy Clin Immunol 110, 582–8. Sanchez-Borges, M., Capriles-Hulett, A., Caballero-Fonseca, F. & Fernandez-Caldas, E. (2003) Mite and cockroach sensitization in allergic patients from Caracas, Venezuela. Ann Allergy Asthma Immunol 90, 664–8. Santos, A.B., Chapman, M.D., Aalberse, R.C. et al. (1999) Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens. J Allergy Clin Immunol 104, 329– 37. Sarpong, S.B., Hamilton, R.G., Eggleston, P.A. & Adkinson, N.F. Jr (1996) Socioeconomic status and race as risk factors for cockroach allergen exposure and sensitization in children with asthma. J Allergy Clin Immunol 97, 1393–401. Sarpong, S.B., Wood, R.A., Karrison, T. & Eggleston, P.A. (1997) Cockroach allergen (Bla g 1) in school dust. J Allergy Clin Immunol 99, 486–92. Satinover, S.M., Reefer, A.J., Pomés, A., Chapman, M.D., PlattsMills, T.A. & Woodfolk, J.A. (2005) Specific IgE and IgG antibodybinding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 115, 803–9. Shao, L., Devenport, M., Fujioka, H., Ghosh, A. & Jacobs-Lorena, M. (2005) Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti. Insect Biochem Mol Biol 35, 947–59. Sporik, R., Squillace, S.P., Ingram, J.M., Rakes, G., Honsinger, R.W. & Platts-Mills, T.A. (1999) Mite, cat, and cockroach exposure, allergen sensitisation, and asthma in children: a case-control study of three schools. Thorax 54, 675–80.

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Stelmach, I., Jerzynska, J., Stelmach, W. et al. (2002) Cockroach allergy and exposure to cockroach allergen in Polish children with asthma. Allergy 57, 701–5. Thomas, M.S., Kunkel, S.L. & Lukacs, N.W. (2004) Regulation of cockroach antigen-induced allergic airway hyperreactivity by the CXCR3 ligand CXCL9. J Immunol 173, 615–23. Uzel, A., Capan, N., Canbakan, S., Yurdakul, A.S. & Dursun, B. (2005) Evaluation of the relationship between cockroach sensitivity and house-dust-mite sensitivity in Turkish asthmatic patients. Respir Med 99, 1032–7. Vailes, L.D., Kinter, M.T., Arruda, L.K. & Chapman, M.D. (1998) High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris. J Allergy Clin Immunol 101, 274– 80. van Ree, R., Antonicelli, L., Akkerdaas, J.H., Garritani, M.S., Aalberse, R.C. & Bonifazi, F. (1996a) Possible induction of food allergy during mite immunotherapy. Allergy 51, 108–13. van Ree, R., Antonicelli, L., Akkerdaas, J.H. et al. (1996b) Asthma after consumption of snails in house-dust-mite-allergic patients: a case of IgE cross-reactivity. Allergy 51, 387–93. Wan, H., Winton, H.L., Soeller, C. et al. (1999) Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J Clin Invest 104, 123– 33. Wang, N.M., Lee, M.F. & Wu, C.H. (1999) Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen. Allergy 54, 119–27. Warner, R.L., Lukacs, N.W., Shapiro, S.D. et al. (2004) Role of metalloelastase in a model of allergic lung responses induced by cockroach allergen. Am J Pathol 165, 1921–30.

Cockroach Allergens, Environmental Exposure, and Asthma Wu, C.H., Chiang, B.T., Fann, M.C. & Lan, J.L. (1990) Production and characterization of monoclonal antibodies against major allergens of American cockroach. Clin Exp Allergy 20, 675–81. Wu, C.H., Lee, M.F., Liao, S.C. & Luo, S.F. (1996) Sequencing analysis of cDNA clones encoding the American cockroach Cr-PI allergens. Homology with insect hemolymph proteins. J Biol Chem 271, 17937–43. Wu, C.H., Lee, M.F., Wang, N.M. & Luo, S.F. (1997) Sequencing and immunochemical characterization of the American cockroach per a 3 (Cr-PI) isoallergenic variants. Mol Immunol 34, 1–8. Wu, C.H., Wang, N.M., Lee, M.F., Kao, C.Y. & Luo, S.F. (1998) Cloning of the American cockroach Cr-PII allergens: evidence for the existence of cross-reactive allergens between species. J Allergy Clin Immunol 101, 832–40. Wünschmann, S., Gustchina, A., Chapman, M.D. & Pomés, A. (2005) Cockroach allergen Bla g 2: an unusual aspartic proteinase. J Allergy Clin Immunol 116, 140–5. Yang, C.Y., Wu, J.D. & Wu, C.H. (2000) Sequence analysis of the first complete cDNA clone encoding an American cockroach Per a 1 allergen. Biochim Biophys Acta, 1517, 153–58. Yazicioglu, M., Oner, N., Celtik, C., Okutan, O. & Pala, O. (2004) Sensitization to common allergens, especially pollens, among children with respiratory allergy in the Trakya region of Turkey. Asian Pac J Allergy Immunol 22, 183–90. Yilmaz, A., Tuncer, A., Sekerel, B.E., Adalioglu, G. & Saraclar, Y. (2004) Cockroach allergy in a group of Turkish children with respiratory allergies. Turk J Pediatr 46, 344–9.

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Food Allergens Ricki M. Helm and A. Wesley Burks

Summary At the core of food allergy research is trying to discover what makes an innocuous food protein an allergen. Tremendous strides have been made in resolving the mechanisms leading to an allergic reaction; however, our understanding of the molecular structure of food proteins responsible for allergic sensitization is still incomplete. In this chapter, dietary and dietary-related allergens are reviewed to alert the reader to the advances made, and yet to be made, with regard to what makes a protein an allergen. Recent studies indicate that structural features of dietary proteins could be a precondition for the induction of immediate-type immune responses. Repeated amino acid motifs are but one factor that could lead to innate and adaptive immune responses. For example, peanut allergens preferentially form dimers, trimers and multimers that display repeated surface epitopes. Other conditions include, but not exclusively, relative resistance to gastrointestinal digestion and methods of food processing that lead to altered conformation of the native protein. With respect to allergen cross-reactivity, cross-reactivity among plant profilins suggests a single profilin could be used for diagnosis; however, the fine specificity of IgE directed to variable epitopes by individuals could influence both profilin sensitization and the allergic response. While variable, clinical cross-reactivity is more common among botanically related fruits, among different nuts, mammalian foods, and seafoods than among cereals, grains, and legumes. The “specificity” of food IgE tests is much better when IgEs are directed to unique noncross-reactive food allergens. Unfortunately, neither the presence of food IgE nor its level is predictive of clinical reactivity. To date, no in vitro or in vivo test exists that exhibits full correlation with clinical food allergy. A multitude of recently reported findings and observations indicate that molecular analysis of allergen sensitization pattern may serve to enhance the clinical utility of IgE antibody-based allergy diagnostics. Pure natural and recombinant allergen molecules, as well as panels of synthetic peptides, have been used for this purpose. The success of investigations using food allergen

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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structural bioinformatics to identify potential amino acid sequences that could lead to cross-reactivity will need to be clearly demonstrated. Significantly, the identification of structural similarity that correlates with IgE binding leading to sensitization and the allergic response will be necessary. Toward this end, a continued effort to standardize food allergy animal models could contribute to unraveling the basics for understanding which food protein molecular structures lead to allergenicity and how the immune system responds, and to induce allergen-specific IgE to food proteins at both the innate and adaptive immune site, either locally or systemically.

Introduction Food allergy is an immune system response to a food protein that is mistakenly classified as a potential pathogen. Mechanisms leading to allergic responses have been well described; however, what makes a protein within a food an allergen is unknown. The contribution and interaction of genetics, environment, and protein molecular structure responsible for allergic sensitization are not as well understood. Any food protein is potentially allergenic if its presentation in the appropriate context of the major histocompatibility complex (MHC) class II pathway induces an immunoglobulin switch to antigen-specific IgE and establishes sustaining memory B cells (Aalberse 2006; Aalberse & Stadler 2006; Esch 2006). In simplistic terms, a food allergen is a protein contained within a food that first elicits an IgE antibody response and on subsequent exposures elicits a clinical response. These can take the characteristics of “complete, class 1 or true food” allergens that sensitize and elicit a clinical response or “incomplete, class 2 or nonsensitizing” food allergens that can only elicit but fail to sensitize (Jensen-Jarolim & Untersmayr 2006). Although food allergens, in general, have been identified as water- or saline-soluble (Burks et al. 2001), there are no known unique biochemical or immunochemical characteristics that distinguish them from nonallergen food components. Food-induced allergic reactions result in a variety of symptoms involving the skin, gastrointestinal tract, and respiratory tract that can be caused by IgE-mediated and non-IgE-mediated

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mechanisms. Characterization of food allergens at the molecular level has improved our understanding of underlying disease mechanisms in food allergy, and potentially could lead to better diagnostic and immunotherapeutic approaches as our knowledge evolves about how normal tolerance is abrogated. In an editorial in the Journal of Allergy and Clinical Immunology, Sampson (2005) gives appropriate recognition to the editors for focusing the journal issue to food allergens. In his editorial, Sampson highlights that food allergens are continuing to be recognized, identified, and characterized at the molecular level, and immunologic mechanisms responsible for both tolerance and food hypersensitivity are being defined and refined. Food allergens, in principal, must reach the gastrointestinal tract in immunologically active form in order to exert their allergenic effects. The four main tasks that the gastrointestinal tract and its associated lymphoid tissue structures must simultaneously accomplish are (i) digestion and uptake of nutrients; (ii) avoidance of tissue-damaging immune responses to food antigens; (iii) avoidance of tissue-damaging (inflammatory) immune responses to commensal bacteria; and (iv) generation of protective immune responses to pathogens. Proteins in more than 70 foods have been described as causing food allergies (the informAll database, available at: http://foodallergens.ifr.ac.uk), and yet persistent food allergy remains relatively infrequent within the population. These findings indicate that many childhood food allergies, such as milk and egg, are outgrown. Many of these proteins are comparatively resistant to heat treatment, acid treatment, proteolysis, and digestion, although important exceptions do exist with more than 700 allergen sequences having been identified. Up to 5– 6% of children and 2–4% of adults suffer from clinical symptoms of food allergy (Sampson 2004; Osterballe et al. 2005; Sicherer & Sampson 2006). Within this food allergy-sensitive population, life-threatening anaphylactic shock (49% of life-threatening cases), systemic reactions (33%), laryngeal angioedema (13%), or acute asthma (5%) have an IgE-mediated food allergy (Moneret-Vautrin et al. 2005). Although an individual could be allergic to any food, such as fruits, vegetables, and meats, there are eight foods that account for 90% of all food-allergic reactions. These are milk, egg, peanut, tree nut (walnut, cashew, etc.), fish, shellfish, soy, and wheat, referred to as the “Big Eight” (Teuber et al. 2006). Internationally, the Codex Alimentarius Commission recommended in 1999 that member countries adopt this list of eight common foods and take steps to ensure that manufacturers within member nations list these foods or ingredients derived from these foods on labels (Codex Alimentarius Commission 1999), thus the term “Big Eight.” These eight foods are the focus of subsequent US legislation, the Food Allergen Labeling and Consumer Protection Act (FALCPA) (Public Law 108-282), that took effect January 1, 2006. The FALCPA mandates that foods containing milk, eggs, fish, crustacean shellfish, peanuts, tree nuts, wheat, and soy

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must declare the food in plain language on the ingredient list or via the word “Contains” followed by the name of the major food allergen (milk, wheat, or eggs, for example) or a parenthetical statement in the list of ingredients, e.g., “albumin (egg)”. With the prevalence of food allergy varying in different countries, some countries have added additional foods to the list of ingredients that must be declared on food labels. For example, the European Union has added celery, mustard, and sesame seeds to lists of allergens that must appear on food labels.

Mechanism of food allergy Evidence has emerged supporting the hypothesis that improvements in standards of hygiene, possibly associated with the extensive use of antibiotics, has contributed to the dysregulation of T helper 2 cell (Th2)-type responsiveness that typifies allergy. The gut-associated lymphoid tissue (GALT) is constantly exposed to a variety of antigens and must therefore decipher a large number of distinct signals at all times. Responding correctly to each set of signals is crucial (Smith & Nagler-Anderson 2005). When the GALT receives signals from the intestinal flora or food antigens, it must induce a state of nonresponsiveness (mucosal tolerance). Prioult and Nagler-Anderson (2005) discuss the basic cellular mechanisms of allergic diseases at mucosal surfaces, focusing on allergic responses to food, before examining newer work that suggests the induction of allergic hyperreactivity is due to a deficient immunoregulatory network, a lack of microbial stimulation, or both. More recently, interest has centered on regulatory T cells, which can suppress both Th1 and Th2 cells through the secretion of immunosuppressive cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. T-cell subset pattern identified by cytokine production revealed that food-allergic children’s IL-4 cytokine response is predominantly from CD4+CD45RO+ cells, whereas IL-4 and interferon (IFN)-γ secretion of nonallergic controls was predominantly from mixed CD4+ and CD8+ CD45RO+ populations (Scott-Taylor et al. 2005). Food-specific IgE values did not correspond to cytokine production; however, IL-4 production and IFN-γ reduction relative to normal children were closely associated with total IgE levels. More work in this area is required with respect to cytokine production to T-cell subsets, food allergen epitope specificity, and correlations to IgE levels and clinical responses. Several lines of experimental evidence indicate that all proteins are absorbed across mucosal epithelia by transcellular transport and/or through interstitial spaces of epithelial cells, but not at equal levels. Immunogenic or allergenic potential is given by posttranslational modifications and possibly by unknown structural/conformational alterations. Matsuda et al. (2006) discuss some of the animal models that

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are applicable for assessing the relative allergenic of processed proteins in comparison with that of native proteins. In this review, protein absorption across mucosal epithelia in humans and murine models is cited as well as parameters responsible for animal sensitization. As evidence of membrane transport, the following allergens were cited: egg lysozyme (Hashida et al. 2002), pineapple bromelain (Castell et al. 1997), and cows’ milk lactoglobulin (Lovegrove et al. 1993); in humans with soybean allergy Gly m Bd 30K (Weangsripanaval et al. 2005), lactoferrin (Takeuchi et al. 2004), and egg lysozyme (Nishikawa et al. 2002; Takano et al. 2004); and ovalbumin (Tsume et al. 1996) in mice and rats, with ranges of 1.7 ng/mL to 10.8 μg/ mL in plasma or serum. Mestecky et al. (2005) demonstrated that ingestion, or nasal application, of the neoantigen keyhole limpet hemocyanin primed human volunteers for subsequent humoral and systemic responses induced by systemic immunization, but suppressed cell-mediated responses as evidenced by Tcell proliferation and delayed-type hypersensitivity. Moreover, humoral and cellular immune responses could not be suppressed by subsequent extended ingestion of large doses of the same antigen suggesting that ongoing systemic responses were refractory to induction of mucosal tolerance in humans. Two studies suggest that CD23 is involved in the rapid transepithelial transport of intact allergens, indicating that CD23 opens a protected transport pathway in intestinal epithelial cells (Bevilacqua et al. 2004; Montagnac et al. 2005). Chambers et al. (2004) showed that digestion of peanuts resulted in a large number of protein bodies that were exclusively transported across the epithelium by specialized antigen-sampling cells and delivered to the lymphoid tissue Peyer’s patches, which were cited to favor immune responses rather than oral tolerance. In a murine model, Van Wijk et al. (2005a,b) indicated cytotoxic T lymphocyte antigen 4 signaling was not a crucial factor in preventing sensitization to food allergens, but did play a significant role in regulating the intensity of the peanut-allergic sensitization response.

Food allergen classifications Breiteneder and Radauer (2004) proposed a classification of plant food allergens into families and superfamilies, based primarily on their structural and functional properties (Table 52.1). Proteins were clustered into families with residue identities of 30% or greater, or if their functions and structures were similar (Murzin et al. 1995; Lo Conte et al. 2002). Superfamilies of the cupin (also reviewed by Mills et al. 2002) consist of 7S and 11S seed storage proteins and prolamins (2S albumins, nonspecific lipid transfer proteins, α-amylase/ trypsin inhibitors, and prolamins storage proteins of cereals). Homologous pathogenesis-related proteins and unrelated families of structural and metabolic plant proteins completed the classification (Breiteneder & Radauer 2004) (Tables 52.2 and 52.3). Asero (2005) also provides two very important and useful algorithms for determining sensitizing or triggering allergens in patients with allergy to plant-derived foods: (i) the biological classification of the main vegetable food allergens by grouping these sources according to the cupin and prolamin superfamilies, defense system proteins, and structural/metabolic proteins; and (ii) an algorithm for Roasaceae or tree nuts.

Structure Aalberse (2006) discusses two routes of IgE sensitization: (i) the atopic route; and (ii) a modified Th2 route. Two aspects of allergenic activity, IgE immunogenicity and cross-reactivity, are discussed. IgE immunogenicity is regarded as being accurately determined by factors unrelated to primary structure of the protein and must take into account the food matrix and how the allergen enters the body; essentially, the protein must be an antigen that induces B cells to produce IgE. Crossreactivity on the other hand can be dictated by structural similarity, and thus predictive modeling for IgE immunogenicity

Table 52.1 Allergens from the cupin and prolamin superfamilies. Protein family

Examples

Cupin superfamily Vicilins Legumins

Ara h 1 (peanut), Jug r 2 (walnut) Ara h 3/4 (peanut), Cor a 9 (hazelnut)

Prolamin superfamily 2S albumins nsLTPs Cereal a-amylase/protease inhibitors Cereal prolamins

Ber e 1 (Brazil nut), Ses i 2 (sesame) Pru p 3 (peach), Cor a 8 (hazelnut) Rice dimeric a-amylase inhibitor Tri a 19 (wheat), Sec c 20 (rye)

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Table 52.2 Allergens from the plant defense system. Protein family

Examples

PRs PR-2, endo-b1,3-glucanases PR-3, class I chitanases PR-4, Win-like proteins PR-9, peroxidases PR-10 intracellular PR-proteins PR-14, nsLTPs

Banana glucanase Per s 1 (avocado), Cas s 5 (chestnut) Pru av 2 (cherry), Mal d 2 (apple) Tri a Bd 36K (wheat) Api g 1 (celery), Mal d 1 (apple) See Table 52.1

Proteases Papain-like cysteine proteases Substilisin-like serine proteases

Act c 1 (kiwi), Gly m Bd 30K (soybean) Cuc m 1 (melon)

Protease inhibitors Kunitz-type protease inhibitors Cereal a-amylase/protease inhibitors

Soybean trypsin inhibitor See Table 52.1

Table 52.3 Other allergenic structural and metabolic proteins. Protein family

Examples

Structural proteins Profilins Oleosins

Api g 4 (celery), Pru a 4 (cherry) Peanut oleosin

Storage proteins Patatin

Sola t 1 (potato)

Enzymes Phenylcoumaran benzylic ether reductases Cyclophilins b-Fructofuranosidases Flavin adenine dinucleotide-dependent oxidases

Pyr c 5 (pear) Carrot cyclophilin Lyc e 1 (tomato) Api g 5 (celery)

will be difficult compared with T-cell and B-cell cross-reactivity that can be more easily determined by structural similarities (Aalberse & Stadler 2006).

Epitopes It is widely agreed that T-cell epitopes are largely linear sequences of amino acids that interact with MHC components to direct an immune response, while B-cell epitopes, although in most instances are defined as linear epitopes, are three-dimensional with the ability to accommodate IgE binding. Analysis of T-cell clones from subjects with immediate and nonimmediate-type hypersensitivity specific to ovomucoid revealed either Th1 or Th0 phenotypes (Kondo et al. 2005), suggesting both antigenic and allergenic motifs are in evidence. What discriminates T-cell epitopes that lead to IgE immunogenicity is still largely unknown and requires further investigation (Bohle 2006).

Within the cupin superfamily, homology-based modeling of Ara h 1 (peanut), Len c 1 (lentil), and Pis s 1 (pea), to soybean β-conglycinin revealed that sequential B-cell epitopes characterized on the C-terminus of Ara h 1 were conserved, which could readily account for the IgE-binding cross-reactivity common to vicilin allergens of legume seed allergy (Barre et al. 2005a,b; Kondo et al. 2005). Schimek et al. (2005) undertook an investigation of food allergy to apples, hazelnuts, and celery with birch pollen allergen that is frequently associated with Bet v 1 cross-reactivity. Evaluation of simulated gastric fluid (SGF) on these Bet v 1-related food allergens revealed the following: pepsin completely destroyed IgE binding activity of all allergens within 1 s and trypsin completely destroyed IgE binding of all allergens within 15 min. One exception was, however, noted; hazelnut allergen remained intact for 2 hours. SGF-treated allergens did not induce basophil activation but were capable

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of inducing peripheral blood mononuclear cell proliferation from both allergic and nonallergic individuals. Mal d 1 and Cor a 1.04 activated Bet v 1-specific T cells, whereas digested Api g 1 did not. The results suggest that T-cell epitopes remain intact in birch pollen-related foods and continue to activate pollen-specific T cells; however, failure to activate basophils in parallel suggests the B-cell epitopes have been altered. A significant contribution to the diversity of IgE and IgG4 epitope mapping has been made by Shreffler and colleagues (Shreffler et al. 2005). By designing redundant peptide arrays of different peptide lengths of Ara h 2 it was revealed that IgE from peanut-allergic individuals bound a larger number of peptides; however, IgE and IgG4 epitopes recognized by these subjects were largely the same. A positive association between IgE and IgG4 led the authors to conclude that these two antibodies had a coordinate regulation. Epitope analysis and cluster analysis revealed interpatient heterogeneity and a more detailed map of IgE and IgG4 epitopes that could be useful in future diagnostic and therapeutic treatment regimens. Mittag et al. (2006) used a phage-displayed random 7-mer peptide library to determine cross-reactive IgE to Bet v 1 and homologs Gly m 4, Ara h 8, and Pru v 1. Competitive immunoscreening and fine epitope mapping revealed patient-specific IgE epitope patterns that could be mapped to the surface of the three-dimensional structure of the allergens using a computer-based algorithm. Both of these studies identify basic information of IgE epitope structure that offer significant advances that can be used to identify the heterogeneity of immunoglobulin-binding patterns and patient-specific IgE epitope specificity. What remains to be determined is the clinical significance of the IgE epitopes identified. Endo-β-1,3-glucanase, the major allergen of banana, was modeled to β-1,4-linked glucan trisaccharide (ReceveurBrechot et al. 2006). Well-conserved IgE binding epitopes were localized to the surface of the enzyme, which could account for the IgE-binding cross-reactivity in the latex–fruit syndrome. Structural features and properties of plant food allergens belonging to the cupin and prolamin superfamilies that may play a role in predisposing the sources as allergens have been reviewed by Mills et al. (2004). These authors present an extensive review of these proteins summarizing the allergenic plant food proteins with respect to function, biochemical characteristics, botanical sources, and known allergens. Amino acid sequences are compared, as well as some threedimensional profiling of representative food allergens that is too broad in scope to cover here, and the reader is referred to manuscripts by the noted plant food specialists cited in the review. Although inconclusive, crystallographic studies of allergen molecules show conformational epitopes that preferentially form dimers, trimrers, or multimers that display repetitive IgE epitopes (Untersmayr & Jensen-Jarolim 2006b). Dall’Antonia et al. (2005) purified and crystallized the thaumatin-like pro-

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tein from ripe cherries (Prunus avium, Pru av 2). A database search revealed thaumatin was the structure closest related to Pru av 2 exhibiting 41.5% sequence identity. Using model building and refinement programs such as these will further our understanding of the molecular basis for allergenicity and cross-reactivity among various family-related allergens. A comparison of three-dimensional models of different nonspecific lipid-transfer protein (nsLTP) firstly justified a high degree of conservation of the putative IgE-binding epitopes among proteins of the Rosaceae family (fruits) and, secondly, a significant amino acid replacement in correspondence to the same regions in nsLTPs of botanical species unrelated to Rosaceae (Pasquato et al. 2006). Rupa and Mine (2003, 2006) engineered recombinant ovomucoid in E. coli and subsequent analyses indicated that the recombinant form had more αhelix and less β-structure than the native form, and maintained identical IgE- and IgG-binding activities by immunoblot analysis. However, there were some differences in ELISA studies that suggest that the recombinant conformational epitopes may have been altered.

Individual food allergens Cows’ milk Caseins, lactoglobulins, and bovine serum albumin are the main allergens in cows’ milk. Sensitivity to casein, β-lactoglobulin and α-lactalbumin seem to be highly linked, while sensitivity to bovine serum albumin appears to be completely independent (Wal 2001). Morisset et al. (2003) reported 59 positive double-blind placebo-controlled food challenge(s) (DBPCFCs) for cows’ milk using liquid milk containing 3 mg milk protein in 0.1 mL of milk. One patient reacted at this very low level, but 30% of the patients also showed great sensitivity, reacting to less than 6.8 mL. The lowest provoking dose reported in the literature by double-blind challenge is 1.5 mg cows’ milk protein in an infant formula, and by open challenge just 0.6 mg (Taylor et al. 2002). Children or adults with IgEmediated acute reactions are at risk of developing severe systemic reactions, particularly those with IgE directed towards linear epitopes on the milk proteins, high titers of IgE to cows’ milk, and a history of previous systemic reactions (Sampson 2004). Wal (2004) provides an update on structure, function, and stability of the main milk proteins. Data relevant to clinical and epidemiologic aspects of milk allergy, and the biochemistry and immunochemistry of milk proteins were selected based on the author’s opinion. Of significance to readers, Wal notes that human milk composition continues to change during lactation and does not contain β-lactoglobulin. Wal further concluded that no single allergen or particular structure could account for milk allergenicity. Moreover, was there sufficient evidence to establish an intake threshold below which allergic reactions are not triggered. Both of these conclusions can

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be attributed to the variability in the heterogeneity of the individual human IgE responses to milk proteins.

Eggs The most frequently reported allergens for hen’s egg are ovomucoid, ovalbumin, ovotransferrin, and lysozyme (Poulsen et al. 2001). Walsh et al. (2005) identified four distinct groups of 40 patients that reacted to similar discrete sets of hen’s egg proteins. These four sets included lysozyme and ovalbumin; ovomucoid; ovomucin; and, lastly, ovotransferrin and the yolk proteins apovitelenins I and VI and phosvitin. The results suggest that these four different allergen sets may account for the differences in reported allergic responses to hen’s egg in egg-hypersensitive individuals. It should be noted that the authors used highly purified egg proteins to eliminate any impurities among the allergens that have been previously reported, i.e., contamination of ovalbumin with ovomucoid, which earlier resulted in ovalbumin being identified as the major hen egg allergen.

Peanuts Scurlock and Burks (2004) performed a PubMed search for the years 1980–2004 and highlighted major advances in peanut allergy that will not be further reviewed here. Palmer et al. (2005) showed that Ara h 2 was a more potent allergen than Ara h 1 by ImmunoCap and a functional IgE-crosslinking assay based on degranulation of RBL SX-38 cells. However, using IgE immunoblot analysis with sera from 12 highly sensitive peanut-allergic individuals, Ara h 1 showed a substantially higher radiostained anti-IgE intensity than Ara h 2. Thus, potency prediction cannot be based on a single immunochemical or functional assessment of IgE binding. Size exclusion chromatography of peanut extract showed that approximately 90% of the total protein content eluted as one peak in the exclusion volume with a molecular mass of over 200 kDa in accordance with the 11S plant seed storage proteins signature (Boldt et al. 2005). In a study of glycan structures, Shreffler et al. (2006) found that peanut antigen, but not deglycosylated peanut antigen, activated monocytederived dendritic cells as measured by MHC/costimulatory molecule upregulation, to drive T-cell proliferation. Both peanut antigen and Ara h 1 were found to induce the Erk-1/2 phosphorylation of monocyte-derived dendritic cells, consistent with Th2 adjuvant activity on dendritic cells. This suggested to the authors a similarity with other nonmammalian glycan structures of helminths acting as Th2 adjuvants. Dirks et al. (2005) demonstrated that in peanut-allergic patients who ate or simply chewed and spat out peanut, peanut allergens could be detected in the serum of 8 of 10 these individuals. Biological activity as assessed by histamine release indicated that peanut allergens were readily absorbed across the buccal mucosa; however, caution is warranted regarding extrapolation of this activity to other food allergens in the absence of further detailed investigation.

Food Allergens

Soybeans Soybeans belong to the legume family and, like peanut, have multiple allergens. Specific IgE binding has historically been associated with components identified by ultracentrifugation, namely the 2S, 7S, and 11S fractions, with at least 15 distinct soybean-specific IgE-binding epitopes in the major allergen (Ogawa et al. 2000). The major component is Gly m Bd 30K, an oil-body associated protein (P34) which is a 7S component that has been identified as a major food allergen (Ogawa et al. 1991; Helm et al. 1998). The subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens that belong to the 11S fraction (Beardslee et al. 2000; Helm et al. 2000a,b).

Wheat Wheat proteins have been incriminated as sources of allergens that present with clinical pathologies, including respiratory, contact and food allergies. Major wheat allergens can be identified by IgE binding in water/salt soluble, gluten fractions and wheat-isolated fractions (Battais et al. 2005). Wheatflour allergens are one source of triggers of food-dependent exercise-induced anaphylaxis that merit further investigation as to mechanism of response; it is of interest to note that, independently, exercise or ingestion of wheatflour do not cause food allergy, but in combination there is an as yet undefined interaction resulting in clinical pathophysiology (Beaudouin et al. 2006).

Fish Gad c 1 or allergen M, the calcium-binding protein defined in the parvalbumin family, remains the major allergen of codfish and likely represents the cross-reactivity within different fish species (Poulsen et al. 2001). Considerable research has been performed since Poulsen’s article. Edible seafoods and seafood allergens have been reviewed by Lehrer et al. (2003), defining the three major divisions of sea organisms, Chordata, Mollusca, and Arthopoda, and their respective common names. Wild and Lehrer (2005) reviewed much of what has been learned about allergens in fish and shellfish, reporting that the major allergens responsible for cross-reactivity among distinct species of fish and amphibians are parvalbumins and the major shellfish allergen is tropomyosin. Seafood allergy has been historically reviewed by Chu et al. (2005), with extensive information on allergens in fish, crustacea, and mollusks and associated cross-reactivity among different seafoods and other invertebrate cross-reactive allergens.

Shellfish Shrimp represent the major class of crustacea, with tropomyosins being recognized as the major allergens that are also present in lobster, crab, mollusks, insects, and arachnids (Lehrer et al. 2003). In this review, the major seafood allergens have been identified and will not be repeated here. Secondary structure of natural and recombinant Pen a 1

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revealed that both had α-helical conformation of tropomyosins (Reese et al. 2006). Deduced amino acid sequence and amino acid sequences with allergenic and nonallergenic tropomyosins revealed 80–99% and 51–58%, respectively. Although vertebrate tropomyosins are regarded to be nonallergenic, the basis for differences in allergenicity is not known. Structural studies and investigations of allergenicity in animal models of allergenic and nonallergenic tropomyosins could shed light on the different allergic responses to this common food allergen. Fish have been recognized as a source of potent allergens, both in food and occupational allergy, with new fish allergen sources still being identified. Parvalbumin, a calcium-binding muscle protein that is highly conserved across fish species and amphibians, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. The allergenic crossreactivity between nine commonly edible fish (cod, salmon, pollack, mackerel, tuna, herring, wolffish, halibut, and flounder) was investigated by SDS/PAGE and IgE immunoblotting, IgG ELISA, IgE ELISA inhibition, and skin-prick test (SPT) (Van Do et al. 2005a). Parvalbumins Gad c 1 (cod), Sal s 1 (salmon), The c 1 (pollack), herring, and wolffish were shown to contain the most potent cross-reacting allergens, whereas halibut, flounder, tuna, and mackerel were the least allergenic. Characterization of the Alaska pollack parvalbumin (The c 1) revealed two isoforms that in their native conformation were almost as potent as Gad c 1 parvalbumin of cod. Recombinant isotypes of The c 1 exhibited low IgG and IgE binding (Van Do et al. 2005b). Although the reduced allergenic recombinant The c 1 were regarded to be potential immunotherapeutic agents, conformational changes of the recombinant isotypes could be responsible, and more research into structure is warranted in animal models. Recent studies suggest new methods for immunotherapy of fish-allergic patients. A phage display technique to generate mimotopes mimicking epitopes on previously defined allergenic peptides of cod parvalbumin was used to identify conformational epitopes of parvalbumin relevant for IgE and IgG binding (Untersmayr et al. 2006). The authors concluded that mimotopes are suitable candidates for an epitopespecific immunotherapeutic treatment regimen. This group also showed that codfish allergens have a grossly reduced ability to trigger an intestinal allergic reaction when they are physiologically degraded by simulated gastric fluid digestion (Untersmayr et al. 2005a; Untersmayr & Jensen-Jarolim 2006a). Thus, physiologic digestion of codfish might lower the threshold levels of a food allergen in sensitized patients. Worldwide, fish allergens continue to be identified based on consumption patterns. Lutjanus argentimaculatus (red snapper) and Lutjanus johnii (golden snapper), locally known as merah and jenahak, respectively, are among the most commonly consumed fish in Malaysia (Rosmilah et al. 2005). Although identified only by molecular weight IgE-binding profiles, a heat-resistant protein of ∼ 12 kDa, which is equi-

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valent in size with fish parvalbumin, was demonstrated only as minor allergen for both fishes. Raw, boiled, and fried muscle extracts of four highly consumed Indian fishes, i.e., pomfret, hilsa, bhetki, and mackerel, investigated by ELISA and immunoblot analysis showed that pomfret and hilsa fish allergens are heat-labile, while allergens of bhetki and mackerel maintained strong reactivity even after thermal treatment (Chatterjee et al. 2006).

Tree nuts Roux et al. (2003) reviewed the most common allergic reactions to tree nuts. Hazelnut, walnut, cashew, and almond were regarded to be the most frequent causes of tree nut allergy and pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn as less frequently associated with allergic disorders. Legumins, vicilin, and 2S albumins (the major seed storage proteins) represent major constituents of tree nuts, while panallergens, such as crossreactive allergens, representing lipid transfer proteins, profilins, and members of the Bet v1-related family of allergens, have been identified as minor constituents in tree nuts. Clark and Ewan (2005) present the most recent data on the development, sensitization, and clinical impact of tree allergens. Noteworthy in this review is the claim that an early onset of multiple nut sensitizations and allergies can take place either in utero or soon after birth (Clark & Ewan 2005).

Other allergens The growing literature that continues to reveal new food allergen sources strongly suggests that any food protein can, under appropriate conditions of sensitization, become a food allergen. Most new food allergens have been identified by IgE-binding studies using findings based on reported incidences of eating a food and having some clinical response. For example, Venter et al. (2006) determined the prevalence of food allergy on the Isle of Wight based upon parentally reported food hypersensitivity versus objectively diagnosed food hypersensitivity among 6-year-old children. The rate of sensitization to a panel of predefined food allergens was reported to be 3.6%, while open food challenges or a suggestive history was 2.5% and prevalence based on DBPCFC only 1.6%. This confirms that perception of a food allergy or hypersensitivity is far more prevalent than clinical diagnosis for food allergy. Major food allergens in this study were milk, peanut, and wheat. Continued prevalence studies and reports of abnormal responses to foods are the major sources of information for characterizing and identifying new food allergens. Examples are presented below; however, the list cannot be considered complete nor clinically relevant in all cases.

Sesame seeds Evidence derived from the PubMed database indicated an increased reporting of sesame allergy during the last five decades, with clinical presentation as immediate hypersens-

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itivity and delayed hypersensitivity (Gangur et al. 2005). Both protein and oil components were identified as having the ability to trigger immediate hypersensitivity via IgE.

Lupine Lupine is another emerging food allergy, largely related to its large-scale introduction into processed foods and frequent cross-reactivity to other members of the legume family. Guarneri et al. (2005) using a computer-assisted amino acid sequence homology search, found a significant sequence homology and molecular similarity between Ara h 8 and the pathogenesis-related protein PR-10 of white lupine. Lupine flour has been incorporated into a number of foods with increasing reports of related allergic reactions. A combination of one- and two-dimensional immunoblots identified two lupin(e) allergens (conglutin γ and 11S acidic subunits) that strongly reacted to serum IgE and showed cross-reactivity to other legume species (Magni et al. 2005). In a study on stability, one individual serum used in IgE immunoblotting analysis revealed a previously undetected 70-kDa band following autoclaving for 30 min (Alvarez-Alvarez et al. 2005). The relevance of this allergen has yet to be demonstrated.

Fruits Allergy to kiwi fruit appears to be increasing as indicated by Lucas et al. (2004), with fresh SPT having a good sensitivity (93%), but a poor specificity (45%). CAP sIgE and commercially available skin-test solutions were less sensitive (54%, 75%), but showed better specificity (90%, 67%). Phytocystatin and a thaumatin-like protein were found in both green and gold kiwis, whereas two allergens with homologies to chitinases were identified in gold kiwi, and actinidin exclusively in green kiwi (Bublin et al. 2004). Geographic differences in prevalence and sensitivity were also noted, with Central European patients showing distinct sensitization profiles compared with Austrian and Dutch patients, which could be related to kiwi preferences. Kiwellin, a cysteine-rich 28 kDa protein isolated from kiwi fruit as a specific allergen recognized by IgE of patients with kiwi allergy (Tamburrini et al. 2005). Lucas et al. (2005) compared the IgE-binding patterns of green and gold kiwis by ELISA and western blotting. Western blotting showed differences in allergen patterns, while inhibition of both immunoblots and ELISA revealed extensive IgE inhibitions in each fruit. Thus, despite having different IgE-binding patterns by immunoblot analysis, the two species shared extensive cross-inhibitory IgE profiles that suggest conserved and species-specific allergen. Nonetheless, individuals with allergic responses to one species should take precaution toward consuming many of the new kiwi species entering the market. Apple peel extracts from 10 varieties of apple showed differences in antigenic and allergenic profiles in 22 Spanish patients with oral allergy syndrome after apple ingestion (Carnes et al. 2006). Mal d 3 could be visualized in all extracts

Food Allergens

with a significant variation in content and a considerable degree of different specific IgE values as assessed by skin test and immunoblot analysis. An equivocal allergen in oranges, Cit s 1, identified as a germin-like glycoprotein, could lead to false-positive diagnosis of orange allergy. Ahrazem et al. (2006) identified specific IgE in 63% of 29 individuals from orange-allergic patients, with only 10% of the patients having a positive SPT. Deglycosylation of the allergen resulted in a loss of IgE binding, suggesting that a complex N-linked glycan moiety played a prominent role in IgE binding. IgE immunoblotting and IgE-ELISA reactivity to a group of homologous 9.5–14.5 kDa proteins, identified as napins 2S albumins, were identified in children that reacted positively on SPT with oilseed rape and turnip rape (Puumalainen et al. 2006).

Pollen–food related syndromes Many individuals with pollen or latex allergy and other nonfood-related proteins often experience allergic symptoms when eating certain fruits and vegetables, identified as the pollen–food allergy syndrome (Egger et al. 2006), the Ficus– fruit syndrome (Hemmer et al. 2004) more commonly known as the latex–fruit syndrome (Yagami 2002; Blanco 2003), or the oral allergy syndrome (OAS) (Purohit-Sheth & Carr 2005). Sensitization is commonly associated with allergic reactions to figs and other tropical fruits: kiwi, papaya, avocado, banana, and pineapple. In these syndromes, cross-reactivity occurs between sensitizers, i.e., those capable of peroral sensitization as well as symptom elicitation, and symptom elicitors, i.e., those characterized as antigens that cannot sensitize perorally but which can provoke allergic sensitization in already sensitized subjects. This cross-reactivity is in part mediated by thiol proteases (Hemmer et al. 2004). Investigations into the latex–fruit syndrome point to defense-related plant proteins, class I chitinases that cross react with the major latex allergen, hevein, as panallergens (Blanco 2003). The celery–mugwort-spice syndrome and the mugwort– mustard allergy syndrome described with weed pollinosis can include other associations including mugwort–peach, ragweed–melon–banana, plantain–melon, pelltory–pistachio, goosefoot–fruit, Russian thistle–saffron and the hop–celery associations that are caused by cross-reactive profilins, lipid transfer proteins, and high-molecular weight glycoallergens (Egger et al. 2006).

Exposure Food allergens can present as native food sources or as a component of processed foods, which can result in either allergic reactions or nonallergic reactions, making it important to understand the food matrix that affects sensitization and elicitation of a reaction (Maleki 2004). IgE-binding epitopes

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requiring conformational epitopes depend largely on tertiary structure that can be modified by processing: heating, milling, or fermentation (Poms & Anklam 2004). Some examples from that review are discussed below.

Eggs At 3 months postpartum, ovalbumin was detected in breast milk samples of 35% of women at a level higher than blood circulating levels (Vance et al. 2005). Kato et al. (2001) showed that the ability of ovomucoid to bind IgE was reduced in model pasta composed of durum wheat and egg white, and attributed the difference to be formation of disulfide bonds between ovomucoid and wheat gliadins. Circulating ovalbumin has been detected throughout pregnancy in 20% of women and correlated with both the presence (P < 0.001) and concentration (r = 0.754, P < 0.001) of infant ovalbumin at birth. However, in a later study by Joo and Kato (2006) a study of heat-coagulated ovalbumin performed studies in mice that suggested this treatment does not retain allergenic properties.

Wheat Scibilia et al. (2006) and Weichel et al. (2006) reported that, in adults, more than 25% of patients with confirmed wheat allergy reacted to less than 1.6 g of wheat. Specific IgE was more sensitive than skin test, with predictive values for both tests being low. Serum from wheat-allergic patients was used to determine the allergen profile in cDNA libraries of wheat and maize (Weichel et al. 2006). Allergens belonging to the gliadins, profilins, and β-expansin were identified and, in addition, novel proteins, thioredoxins, were identified by competition ELISA. Other examples of different processing effects include those of egg, soy products, tree nuts, and wheat that show different allergic responses (Besler et al. 2001). Alterations in allergic responses as a result of processing techniques show considerable variability in patient responses that make conclusions as to reduced or increased allergic responses extremely difficult.

Threshold doses Peanuts Ara h 1, Ara h 2, and Ara h 3 peanut allergens following boiling or frying bound significantly less IgE than roasted peanuts (Beyer et al. 2001). Roasted peanuts bound 90-fold higher levels of IgE than did extracts from raw peanuts (Maleki et al. 2000). Although slopes of RAST-inhibition curves did not change for peanut allergens in high-fat (31.5%) versus low-fat (22.9%), the cumulative dose of peanut protein required to elicit DBPCFC in three of four subjects was 12–31 times higher when using the higher fat matrix (Grimshaw et al. 2003; Scholl et al. 2005). The authors concluded that the peanut allergens in the higher fat matrix were not as readily available to react with mediator cells in the gastrointestinal tract. van Wijk et al. (2005b) presented data using the popliteal lymph node assay and an animal model that suggests there is no intrinsic immunogenicity in purified peanut allergens and that the food matrix enhanced immune responses to individual allergens.

Milk Boiling milk reduced IgE binding of α-lactoglobulin and casein by 50–60% and eliminated β-lactoglobulin and serum albumin reactivity in SPT (Besler et al. 2001). Maleki (Maleki & Hurlburt 2004) reaction increased allergenicity of milk in skin tests. Processing of both hard and soft cheeses has resulted in different allergic reactions (Besler et al. 2001). Moreno et al. (2005) showed that physiologically relevant levels of phosphatidylcholine, a surfactant in milk and secreted by the stomach, interact with and protect digestion of α-lactalbumin. Such interactions have twofold importance: matrix effects and slowed digestion can affect allergen stability and promote continued presentation of allergen to the mucosal immune system.

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From the results of a round-table discussion involving 20 clinicians and other interested parties, a consensus protocol for determining threshold levels of allergenic foods was developed (Taylor et al. 2004). Low levels of IgE can be found in healthy nonallergic individuals (Clark & Ewan 2003; Roberts et al. 2005). Indeed 95% diagnostic certainty for allergy is reached only if relatively high levels of food antigen-specific IgE are observed: 6 kU/L for egg, 32 kU/L for milk, 15 kU/L for peanuts, 20 kU/L for fish (Sampson & Ho 1997). Therefore, other immunologic factors (possibly inhibitory antibodies) are probably interfering with the IgE triggering of immune reactions. In children with predominantly atopic dermatitis, Sicherer et al. (2000) reported that 17% reacted to the first dose of 400 or 500 mg. In a series of 124 oral food challenges performed for egg, 16% were reactive at less than 65 mg (Morisset et al. 2003). The lowest observed threshold dose in food challenges for egg white was reported to be less than 2 mg crude egg white. Another group reported results in 36 positive challenges with whole egg. The lowest observed adverse effect level was 0.011 mg in two children, which was the first dose administered, thus, the actual threshold could have been even lower than this (Osterballe & Bindslev-Jensen 2003). In another series of oral controlled challenges, Celik-Bilgili et al. (2005) identified meaningful predictive decision points for hen’s egg of 6.3, 12.6 and 59.2 kU/L in children at 90%, 95%, and 99% predictive probabilities. Only a predictive value of 88.8 kU/L at a 90% predicted probability could be calculated for cows’ milk, and none for soy or for wheat. Two precautions were identified, patient population (age) and specific allergen predictive points were not assessed. One-half of patients reacted after eating 6 mg hazelnut; after

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100 mg of hazelnut all patients reacted (Wensing et al. 2002). No threshold dose studies have been done with the other tree nuts.

Cross-reactivity Based on the extent of cross-reactivity among plant profilins, Radauer et al. (2006) justified the use of a single profilin for diagnosis; however, they caution that the fine specificity of IgE directed to variable epitopes could influence the clinical manifestation of profilin sensitization. This suggestion should apply to all diagnostic and immunotherapeutic applications, as there is a wide spectrum of epitope fine specificity for food allergens. Even though some patients with peanut allergy develop tree nut allergy, to date, this appears to be predominantly due to cosensitization, not classic cross-reactivity. For instance, using sera from several patients highly allergic clinically to both peanut and walnut whose IgE bound the vicilin from both seeds (Ara h 1 in peanut, and Jug r 2 in walnut), there was no evidence of high-affinity cross-reactive IgE between the vicilin from walnut (Jug r 2) and crude peanut extract containing vicilin (Ara h 1) (Teuber et al. 1999). Also, no IgE epitopes were found in common upon comparison of linear epitopes from cashew vicilin (Ana o 1), with published Ara h 1 linear epitopes, even though sera from patients highly allergic to both peanut and cashew were included in the study (Astwood et al. 2002; Wang 2002). From this example of IgE directed toward vicilin allergens in peanut and tree nut at least, it appears that patients undergo separate sensitization events even when allergy to peanuts and tree nuts coexists. However, de Leon et al. (2003) published a small series of four patients who did show some cross-reactivity in vitro between peanut and tree nuts that correlated with clinical reactivity to peanut, almond, Brazil nut, and hazelnut. In a subsequent publication, de Leon et al. (2003) concluded that peanut-specific IgE antibodies that cross-react with tree nut allergens caused effector-cell mediator release that could contribute to the manifestation of tree nut allergy in peanutallergic individuals. The specific proteins involved in the cross-reactivity were not identified, but there will likely be subsets of patients described and defined in more detail who do exhibit allergy due to classic cross-reactivity of IgE towards peanut with other seeds, tree nuts, or pollens as more investigations are performed. Cross-sensitization to other foods include poppy seed and buckwheat flour (Oppel et al. 2006), fruit cross-reactivity (Marzban et al. 2005), and fruit/nut cross-reactivity (Kalogeromitros et al. 2006). Although carbohydrate epitopes can be highly antigenic and cross-reactive, they generally do not elicit allergic reactions; however, the IgE antibodies produced can disturb in vitro IgE tests (Yagami et al. 2002) or lead to false-positives (Ahrazem et al. 2006).

Food Allergens

Respiratory food allergens Inhalant food allergens can elicit symptoms, although the mode of sensitization is still unknown. Respiratory allergens such as flour could possibly sensitize by two routes, the bronchial or gastrointestinal mucosal immune systems. Respiratory food-type allergens can be cleared from the bronchial mucosal linings by mucus clearance and swallowed: this mode of sensitization has not received the interest warranted. Hoffmann et al. (2006) assessed nine respiratory flour-allergic individuals in a DBPCFC demonstrating that, after ingestion of flour, significant increases in serum tryptase and methyl histamine and a decrease in blood basophils were evident with no change in forced expiratory volume at 1 s. A regulatory pathway involving dendritic cells and T cells was suggested that requires further investigation. Seafood allergens aerosolized during food preparation are a source of potential respiratory and contact allergens. Distillates recovered over boiling shrimp confirmed that the primary cross-reactive allergen of shrimp (phylum Arthropoda) and scallops (phylum Mollusca) is the 35–39 kDa heat-stable allergen, previously demonstrated to be muscle tropomyosin in both phyla (Goetz and Whisman 2000).

Digestibility Although digestibility to enzymatic digestion and heat treatment have been regarded as important criteria for determining potentially allergenicity, no validated analytical protocols exist to predict protein allergenicity based on these criteria. For comments, the readers are referred to Teuber (2002) and Taylor (2003). Summarizing these two articles reveals differences in stability assessment based on limited protein/allergens and the effects of food matrixes that should be taken into consideration as well, and also the comparative abundance and pepsin digestion and enzyme/protein ratio.

Processing Pomes et al. (2006) assessed the allergenicity of raw versus roasted peanut extracts using a specific monoclonal antibodybased ELISA. Ara h 1 levels were 22-fold higher in roasted compared with raw peanuts, further suggesting that differences were not associated with conformational changes of the Ara h 1 monoclonal antibody epitopes. No differences were noted in extractability among four cultivars of runner peanuts; however, the efficiency of Ara h 1 extraction from different maturation stages (number 1 compared to jumboand medium-sized peanuts) showed expression to be associated with peanut maturity.

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In cross-reacting birch pollen-related foods, patients are advised to consume Bet v 1-related food allergens following heating. In a study of patients with birch pollen allergy who experienced OAS and exacerbation of atopic dermatitis on the ingestion of fresh apple, celery, or carrot, the subjects were tested in DBPCFCs with the respective food in cooked form (Bohle et al. 2006). Cooked food allergens lost their ability to bind IgE and to induce mediator release but maintained the same potency to activate Bet v 1-specific T cells as native uncooked foods. Cooked food allergen sources lost their ability to induce OAS but continued to cause an exacerbation of atopic eczema. The results suggest the T-cell crossreactivity between Bet v 1 and related food allergens occurs independently of IgE cross-reactivity, warranting caution with respect to recommendations concerning consumption of heated food allergen sources in the pollen-related food allergy syndrome. Model peptides of Ara h 2 sequences responsible for the formation of highly IgE glycation sites have been synthesized, mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes, and subjected to immunologic evaluation (Gruber et al. 2005). Overlapping major epitopes 6 and 7, which did not contain any lysine or arginine moieties, showed a distinctly higher level of IgE binding when subjected to Maillard reaction, providing the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions, and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Further reviews on the Maillard reaction and food allergenicity can be found in presentations by Maleki and Hurlburt (2004) and a review on the metabolism of the Maillard reaction (Tuohy et al. 2006). Digestion of hen’s egg ovomucoid by heat treatment and simulated gastric fluid suggests that the pepsin/ovomucoid ratio in the SGF is important (Takagi et al. 2005). Digestion of ovomucoid revealed that 21% of the patients’ IgE sera retained IgE-binding activity to a small 4.5-kDa fragment, which led the authors to suggest that these subjects would be unlikely to outgrow their egg allergy. No clinical or biological responses were reported to substantiate this; however, evidence for IgE-binding to the 4.5-kDa fragment could be used to follow hen’s egg allergic profile.

Genetically modified allergens (Table 52.4) Aalberse and Stadler (2006) identified two distinct properties of novel proteins that reflect “allergenic potential”: (i) a propensity to induce IgE antibodies; and (ii) the propensity to react with IgE antibodies induced by other proteins. De novo IgE immunogenicity, T- and B-cell reactivity and crossreactivity are highlighted topics. The authors concluded that (i) consensus is lacking for predicting de novo allergenicity; (ii) cross-reactivity at the B-cell level is more predictable

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than allergenicity based upon six, seven, or eight contiguous peptides; and (iii) T-cell cross-reactivity does not appear to contribute significantly to variations in IgE-dependent allergenicity. Ladics et al. (2003) reviewed the various strategies recommended for use in assessing protein allergenicity by a workshop designed to describe the state of knowledge and progress made in development and evaluation of testing strategies, and to identify issues that should be addressed to validate the sensitivity, specificity, and reproducibility of assays being used to determine the allergenicity potential of genetically modified foods. Although significant progress has been made, to the authors’ knowledge, no enzyme digestion or animal model has been validated. In vitro and in vivo assessment of site-directed mutagenesis recombinant Mal d 1 (rMal d 1mut) compared with native rMal d 1 revealed that a moderate decrease in IgE-binding potency translated to potent inhibition of biological activity (Bolhaar et al. 2005). RAST analysis revealed a twofold difference and RAST inhibition a 7.8-fold decrease in IgE binding; SPT and bronchial hyperresponsiveness decreased by 10– 200-fold, with hypoallergenicity being confirmed by DBPCFC. Although the authors suggest that this paves the way for mutant recombinant major food allergen as a safer immunotherapy for the treatment of food allergic patients, clinical trials are warranted to provide further evidence of the safety of recombinant mutated food allergens as hypoallergenic sources. Ogawa et al. (2000) used combined chemical breeding, physicochemical treatment, and enzymatic digestion to modify Gly m Bd 28K. By single or combined application of these techniques, several hypoallergenic soybean products (Tofu, Miso, and soymilk) were shown safe in 80% of soybeansensitive patients. A hypoallergenic form of the third domain of ovomucoid (GMFA) with ablated allergenicity against egg-allergic patients’ serum was used to desensitize domain III-sensitized mice (Rupa & Mine 2006). Suppressed DIII-specific IgE, enhanced specific IgG2a and IgG levels, and low histamine and increased Th1-type cytokines suggest that GMFA can be used to downregulate allergic responses to a Th1 response. Whether this desensitization will be sufficient to suppress IgE-mediated responses to hen’s egg allergens remains to be validated. Jenkins et al. (2005) suggest that it is implicit in the allergenic risk assessment process (FAO-WHO ALINORM 03/04, 2002, www.codexalimentarius.net/web/reports.jsp) that this in silico approach be combined with other factors including environmental factors, such as pollen exposure, dietary habits, the effect of food processing, and matrix, and genetic factors, which could predispose individuals to becoming allergic. This represents a very complex analysis for determining potential allergenic risk; however, with the sensitization mechanism still unknown, this is sound advice to scientists developing

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Table 52.4 FAI-Jaffe Institute Food Allergen Repository. Food protein

cDNA

Vector

Host bacteria

Egg

Ovomucoid Ovalbumin Ovotransferrin

Gal d 1 Gal d 2 Gal d 3

Yes Yes Yes

pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3)

Milk

Caseins as1 as2 b k b-Lactoglobulin a-Lactoalbumin

Bos d 8 Bos d 8 Bos d 8 Bos d 8 Bos d 5 Bos d 4

Yes Yes Yes Yes Yes Yes

pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3)

Peanut

Vicilin (7S) Conglutin (2S-like) Glycinin (11S)

Ara h 1 Ara h 2 Ara h 3

Yes Yes Yes

pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3)

Brazil nut*

2S albumin 11S globulin 7S globulin

Ber e 1 Ber e 2

Yes Yes No

pET 24 BL21 (DE3) pET 24 BL21 (DE3)

Walnut* (black)

2S albumin Vicilin (7S)

Jug n 1 Jug n 2

No No

Hazelnut*

Bet v 1 homolog 11S globulin 7S globulin Heat shock protein Lipid transfer protein Profilin Oleosin

Cor a 1.04 Cor a 9 Cor a 11

Yes Yes Yes Yes No No No

Cor a 8 Cor a 2

Pistachio*

11S globulin 2S albumin 7S globulin

Sesame*

2S albumin 2S albumin 7S globulin 11S globulin-1 11S globulin-2 11S globulin isof.3 11S globulin isof.4 Oleosin Oleosin Oleosin

Ses i 1 Ses i 2 Ses i 3 Ses i 6

Shrimp*

Tropomyosin Shr3 Arginine kinase

Salmon*

Parvalbumins Tropomyosin Collagen

pET 24 BL21(DE3) pET 24 BL21-CodonPlus (DE3)-RIL pET 24 BL21 (DE3) pET 24 BL21-CodonPlus (DE3)-RIL

Yes Yes No Yes Yes Yes Yes Yes No No No No No

pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21-CodonPlus (DE3)-RIL pET 24 BL21 (DE3) pET 24 BL21-CodonPlus(DE3)-RIL

Pen a 1

Yes Yes Yes

pET 24 BL21 (DE3) pET 24 BL21 (DE3) pET 24 BL21 (DE3)

Sal s 1 b1 Sal s 1 b2

Yes Yes Yes No

pET 24 BL21-CodonPlus (DE3)-RIL pET 24 BL21-CodonPlus (DE3)-RIL pET24 BL21 (DE3)

Ses i 4 Ses i 5

* Possess full cDNA library. All recombinant proteins have N-terminal T7-Tag and C-terminal His-Tag.

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and introducing novel or modified proteins into our food supply.

Allergen sequences and databases The International Life Sciences Institute hosted a workshop that consisted of over 40 international experts from academia, industry, and government to assess bioinformatic prediction of novel protein for allergenicity. The results of that workshop are summarized by Thomas et al. (2005a,b), and indicate that workshop participants recognized that the bioinformatics associated with allergy assessment could be helpful in identifying potentially allergenic characteristics of novel protein, but that efforts and standardization for prediction were still evolving. Two important points summarized at this meeting included (i) a review of the allergen concentration to induce specific IgE with > 100 cross-links per cell and an affinity to bind within 100 s identified as primary criteria; and (ii) with more than 150 linear IgE-binding epitopes, ranging from 4 to as many as 23 amino acids in length, a 6-mer sliding amino acid peptide would not provide relevant information for safety assessment of novel protein. Sources for methods to identify potentially allergenic characteristics on novel proteins are briefly discussed below. A number of specialized databases have been developed that include (i) molecular databases focused on protein sequences and structures; (ii) informational databases focused on clinical, biochemical, and epidemiologic data related to protein allergens; and (iii) a database on allergen nomenclature, and other knowledge bases or informational websites that are peripherally related to research on allergens. Examples of each type of database have been reviewed by Gendel and Jenkins (2006). Novel tools to determine the quantitative sequence and three-dimensional relationships between IgE epitopes of major allergens from peanut and other foods have been implemented in a Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/) (Schein et al. 2005). Using physicochemical properties, immunogenicity and IgE reactivity of peanut allergen Ara h 1 could potentially identify arrays of similar antigenic regions on other allergens. In sequence prediction modeling, human IgE reactivity to melon profilin strongly depended on the highly conserved conformational structure, rather than a high degree of amino acid sequence identity or even linear epitope identity (Sankian et al. 2005). Aspects that cannot be taken into account in allergen prediction are solvent-accessible surface-expressed epitopes, neoallergens exposed during digestion, and expression of bacterial/insect/or mammalian recombinant allergen comparisons that do not reach structural identity with native allergens. Query proteins can be compared with a set of prebuilt allergenic motifs that have been obtained from 664 known

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allergen proteins (Riaz et al. 2005). In silico data were used to evaluate sequence homology and three-dimensional modeling according to FAO/WHO guidelines to reveal suspected cross-reaction between lupine and peanut allergens (Guarneri et al. 2005). In both studies by Shreffler and Mittag above, basic information on epitope structure could be used for prediction of cross-reactivity of potentially allergenicity of novel foods; however, their clinical significance would still be suspect.

Animal models The use of animal models to characterize allergen sensitization and potential allergenicity of novel proteins has been extensively reviewed and will only be highlighted here. Summaries of the current animal models under development in rodents, dog, and swine are given by McClain and Bannon (2006). Although animal models that mimic human food allergy are under investigation, no animal model has yet been standardized with respect to allergen and nonallergen potency. Variables affecting model development include allergen concentrations, allergen matrix or source effects, route of exposure, duration of exposure, animal age, use of adjuvants, dose ranges, and genetic background. Animal models suggest that antacid drugs support IgE induction and sensitization to concomitantly consumed food antigens (Scholl et al. 2005; Untersmayr et al. 2003, 2005b). In an observational study of 152 adult patients who were medicated on histamine H2-receptor blockers, 10% of these patients showed an increase in preexisting IgE antibodies and 15% de novo IgE antibodies toward numerous digestible compounds, i.e., milk, potato, celery, carrots, apple, orange, wheat, and rye flour (Untersmayr et al. 2005b). A transdermal exposure to sesame seed extracts resulted in IL-4 activation, IgE response, and clinical sensitization for systemic anaphylaxis in a BALB/c animal model (Navuluri et al. 2006). Using western blot analysis, four allergens were identified that included Ses i 1 and a basic subunit of 11S globulin. The authors further showed an IgE reponse to transdermal exposure in two high IgE-responder mouse strains with disparate MHC confirming the intrinsic allergenicity of sesame seed. In a BALB/c animal model, the Kunitz-type soybean trypsin inhibitor was shown not to adsorb to alum used as an adjuvant; however, alum exerted an adjuvant activity only when coadministered with antigen suggesting that some biochemical effect other than adsorptive capacity enhanced production of antigen-specific IgE (Yamanishi et al. 2003). Intact Gly m Bd 30K and fragments < 20 kDa were shown to survive digestion and could be identified in blood of mice administered dietary fat in association with the allergen. In this study, the authors proposed that coadministration of a 30% corn oil enhanced fat carrier-mediated intestinal transport (Weangsripanaval et al. 2005).

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Conclusions Further identification and characterization of involved allergens is warranted for a better understanding of food allergy. Current usage of nonstandardized allergen extracts continues to cause additional problems for both diagnosis and therapy of food allergic patients, as does the mode of therapy. In the seed storage family of food allergens, vicilins (~ 45 kDa) and conglycinins (~ 65 kDa) appear to be major allergens that share some epitope specificity that accounts for cross-reactivity; however, IgE-binding does not necessarily correlate with clinical manifestations of disease. A higher incidence of allergy to foods would be expected in geographic areas where more is consumed on a regular basis; however, different foods are now reaching distant markets. Early life exposure, as documented for egg allergens, could be one mechanism by which developing immune responses of infants could be modulated. Factors leading to sensitization still remain to be elucidated for food allergens. The importance of diet, food matrix and interaction with other foods, interaction of the local commensal and pathogenic bacteria can all play significant roles in food-allergen sensitization. Although significant information can be gained regarding food-allergen stability to SGF or simulated gastric fluid (SIF) digestion, there are not enough data from nonallergenic food proteins to provide a definitive statement regarding enzyme stability as a reliable marker for potential food allergen identification.

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Chu, K.H., Tang, C.Y., Wu, A. & Leung, P.S. (2005) Seafood allergy, lessons from clinical symptoms, immunological mechanisms and molecular biology. Adv Biochem Eng Biotechnol 97, 205–35. Clark, A.T. & Ewan, P.W. (2003) Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 33, 1041–5. Clark, A.T. & Ewan, P.W. (2005) The development and progression of allergy to multiple nuts at different ages. Pediatr Allergy Immunol 16, 507–11. Codex Alimentarius Commission (1999) Report of the twenty-third session of the Codex Alimentarius Commission. Rome, Alinorm 99/37. Dall’Antonia, Y., Pavkov, T., Fuchs, H., Breiteneder, H. & Keller, W. (2005) Crystallization and preliminary structure determination of the plant food allergen Pru av 2. Acta Crystallograph Sect F Struct Biol Cryst Commun 61, 186– 8. de Leon, M.P., Drew, A.C., Glaspole, I.N. et al. (2005) Functional analysis of cross-reactive immunoglobulin E antibodies: peanutspecific immunoglobulin E sensitizes basophils to tree nuts. Clin Exp Allergy 8, 1056– 64. de Leon, M.P., Glaspole, I.N., Drew, A.C., Rolland, J.M., O’Hehir, R.E. & Suphioglu, C. (2003) Immunological analysis of allergenic crossreactivity between peanut and tree nuts. Clin Exp Allergy 33, 1273– 80. Dirks, C.G., Pedersen, M.H., Platzer, M.H., Bindslev-Jensen, C., Skov, P.S. & Poulsen, L.K. (2005) Does absorption across the buccal mucosa explain early onset of food-induced allergic systemic reactions? J Allergy Clin Immunol 115, 1321– 3. Egger, M., Mutschlechner, S., Wopfner, N., Gadermaier, G., Briza, P. & Ferreira, F. (2006) Pollen-food syndromes associated with weed pollinosis, an update from the molecular point of view. Allergy 61, 461–76. Esch, R.E. (2006) Allergens. Clin Rev Allergy Immunol 30, 71–2. Gangur, V., Kelly, C. & Navuluri, L. (2005) Sesame allergy, a growing food allergy of global proportions? Ann Allergy Asthma Immunol 95, 4–11, quiz 11–13. Gendel, S.M. & Jenkins, J.A. (2006) Allergen sequence databases. Mol Nutr Food Res 50, 633–7. Goetz, D.W. & Whisman, B.A. (2000) Occupational asthma in a seafood restaurant worker, cross-reactivity of shrimp and scallops. Ann Allergy Asthma Immunol 85, 461– 6. Grimshaw, K.E., King, R.M., Nordlee, J.A., Hefle, S.L., Warner, J.O. & Hourihane, J.O. (2003) Presentation of allergen in different food preparations affects the nature of the allergic reaction: a case series. Clin Exp Allergy 33, 1581– 5. Gruber, P., Becker, W.M. & Hofmann, T. (2005) Influence of the maillard reaction on the allergenicity of rAra h 2, a recombinant major allergen from peanut (Arachis hypogaea), its major epitopes, and peanut agglutinin. J Agric Food Chem 53, 2289–96. Guarneri, F., Guarneri, C. & Benvenga, S. (2005) Identification of potentially cross-reactive peanut-lupine proteins by computerassisted search for amino acid sequence homology. Int Arch Allergy Immunol 138, 273–7. Hashida, S., Ishikawa, E., Nakamichi, N. & Sekino, H. (2002) Concentration of egg white lysozyme in the serum of healthy subjects after oral administration. Clin Exp Pharmacol Physiol 29, 79–83. Helm, R., Cockrell, G., Herman, E., Burks, A., Sampson, H. & Bannon, G. (1998) Cellular and molecular characterization of a major soybean allergen. Int Arch Allergy Immunol 117, 29–37. Helm, R.M., Cockrell, G., Connaughton, C. et al. (2000a) A soybean

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G2 glycinin allergen. 1. Identification and characterization. Int Arch Allergy Immunol 123, 205–12. Helm, R.M., Cockrell, G., Connaughton, C. et al. (2000b) A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling. Int Arch Allergy Immunol 123, 213–9. Hemmer, W., Focke, M., Gotz, M. & Jarisch, R. (2004) Sensitization to Ficus benjamina, relationship to natural rubber latex allergy and identification of foods implicated in the Ficus-fruit syndrome. Clin Exp Allergy 34, 1251–8. Hoffmann, H.J., Skjold, T., Raithel, M., Adolf, K., Hilberg, O. & Dahl, R. (2006) Response of respiratory flour allergics in an ingested flour challenge may involve plasmacytoid dendritic cells, CD25+ and CD152+ T cells. Int Arch Allergy Immunol 140, 252–60. Jenkins, J.A., Griffiths-Jones, S., Shewry, P.R., Breiteneder, H. & Mills, E.N. (2005) Structural relatedness of plant food allergens with specific reference to cross-reactive allergens, an in silico analysis. J Allergy Clin Immunol 115, 163–70. Jensen-Jarolim, E. & Untersmayr, E. (2006) Food safety, in vitro digestion tests are non-predictive for allergenic potential of food in stomach insufficiency. Immunol Lett 102, 118–19. Joo, K. & Kato, Y. (2006) Assessment of allergenic activity of a heatcoagulated ovalbumin after in vivo digestion. Biosci Biotechnol Biochem 70, 591–7. Kalogeromitros, D.C., Makris, M.P., Gregoriou, S.G., Katoulis, A.C., Straurianeas, N.G. (2006) Sensitization to other foods in subjects with reported allergy to grapes. Allergy Asthma Proc 27, 68–71. Kato, Y., Oozawa, E. & Matsuda, T. (2001) Decrease in antigenic and allergenic potentials of ovomucoid by heating in the presence of wheat flour, dependence on wheat variety and intermolecular disulfide bridges. J Agric Food Chem 49, 3661–5. Kondo, M., Suzuki, K., Inoue, R. et al. (2005) Characterization of Tcell clones specific to ovomucoid from patients with egg-white allergy. J Invest Allergol Clin Immunol 15, 107–11. Ladics, G.S., Holsapple, M.P., Astwood, J.D. et al. (2003) Workshop overview, approaches to the assessment of the allergenic potential of food from genetically modified crops. Toxicol Sci 73, 8–16. Lehrer, S.B., Ayuso, R. & Reese, G. (2003) Seafood allergy and allergens, a review. Mar Biotechnol (NY) 5, 339– 48. Lo Conte, L., Brenner, S.E., Hubbard, T.J., Chothia, C. & Murzin, A.G. (2002) SCOP database in 2002: refinements accommodate structural genomics. Nucleic Acids Res 30, 264–7. Lovegrove, J.A., Osman, D.L., Morgan, J.B., Hampton, S.M. (1993) Transfer of cow’s milk beta-lactoglobulin to human serum after a milk load, a pilot study. Gut 34, 203–7. Lucas, J.S., Grimshaw, K.E., Collins, K., Warner, J.O. & Hourihane, J.O. (2004) Kiwi fruit is a significant allergen and is associated with differing patterns of reactivity in children and adults. Clin Exp Allergy 34, 1115–21. Lucas, J.S., Lewis, S.A., Trewin, J.B., Grimshaw, K.E., Warner, J.O. & Hourihane, J.O. (2005) Comparison of the allergenicity of Actinidia deliciosa (kiwi fruit) and Actinidia chinensis (gold kiwi). Pediatr Allergy Immunol 16, 647–54. McClain, S. & Bannon, G.A. (2006) Animal models of food allergy, opportunities and barriers. Curr Allergy Asthma Rep 6, 141–4. Magni, C., Herndl, A., Sironi, E. et al. (2005) One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds. J Agric Food Chem 53, 4567–71. Maleki, S.J. (2004) Food processing, effects on allergenicity. Curr Opin Allergy Clin Immunol 4, 241– 5.

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Maleki, S.J. & Hurlburt, B.K. (2004) Structural and functional alterations in major peanut allergens caused by thermal processing. J AOAC Int 87, 1475– 9. Maleki, S.J., Chung, S.Y., Champagne, E.T. & Raufman, J.P. (2000) The effects of roasting on the allergenic properties of peanut proteins. J Allergy Clin Immunol 106, 763– 8. Marzban, G., Mansfeld, A., Hemmer, W., Stoyanova, E., Katinger, H. & da Camara Machado, M.L. (2005) Fruit cross-reactive allergens, a theme of uprising interest for consumers’ health. Biofactors 23, 235– 41. Matsuda, T., Matsubara, T. & Hino, S. (2006) Immunogenic and allergenic potentials of natural and recombinant innocuous proteins. J Biosci Bioeng 101, 203–11. Mestecky, J., Moldoveanu, Z. & Elson, C.O. (2005) Immune response versus mucosal tolerance to mucosally administered antigens. Vaccine 23, 1800– 3. Mills, E.N., Jenkins, J., Marigheto, N., Belton, P.S., Gunning, A.P. & Morris, V.J. (2002) Allergens of the cupin superfamily. Biochem Soc Trans 30, 925– 9. Mills, E.N., Jenkins, J.A., Alcocer, M.J. & Shewry, P.R. (2004) Structural, biological, and evolutionary relationships of plant food allergens sensitizing via the gastrointestinal tract. Crit Rev Food Sci Nutr 44, 379– 407. Mittag, D., Batori, V., Neudecker, P. et al. (2006) A novel approach for investigation of specific and cross-reactive IgE epitopes on Bet v 1 and homologous food allergens in individual patients. Mol Immunol 43, 268–78. Moneret-Vautrin, D.A., Morisset, M., Flabbee, J., Beaudouin, E. & Kanny, G. (2005) Epidemiology of life-threatening and lethal anaphylaxis, a review. Allergy 60, 443– 51. Montagnac, G., Yu, L.C., Bevilacqua, C. et al. (2005) Differential role for CD23 splice forms in apical to basolateral transcytosis of IgE/ allergen complexes. Traffic 6, 230– 42. Moreno, F.J., Mackie, A.R. & Mills, E.N. (2005) Phospholipid interactions protect the milk allergen alpha-lactalbumin from proteolysis during in vitro digestion. J Agric Food Chem 53, 9810–6. Morisset, M., Moneret-Vautrin, D.A., Kanny, G. et al. (2003) Thresholds of clinical reactivity to milk, egg, peanut and sesame in immunoglobulin E-dependent allergies, evaluation by doubleblind or single-blind placebo-controlled oral challenges. Clin Exp Allergy 33, 1046– 51. Murzin, A.G., Brenner, S.E., Hubbard, T. & Chothia, C. (1995) SCOP, a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol 247, 536–40. Navuluri, L., Parvataneni, S., Hassan, H., Birmingham, N.P., Kelly, C. & Gangur, V. (2006) Allergic and anaphylactic response to sesame seeds in mice, identification of Ses i 3 and basic subunit of 11s globulins as allergens. Int Arch Allergy Immunol 140, 270–6. Nishikawa, M., Hasegawa, S., Yamashita, F., Takakura, Y. & Hashida, M. (2002) Electrical charge on protein regulates its absorption from the rat small intestine. Am J Physiol 282, G711–G719. Ogawa, A., Samoto, M. & Takahashi, K. (2000) Soybean allergens and hypoallergenic soybean products. J Nutr Sci Vitaminol (Tokyo) 46, 271– 9. Ogawa, T., Bando, N., Tsuji, H., Okajima, H., Nishikawa, K. & Sasaoka, K. (1991) Investigation of the IgE-binding proteins in soybeans by immunoblotting with the sera of the soybeansensitive patients with atopic dermatitis. J Nutr Sci Vitaminol (Tokyo) 37, 555–65. Oppel, T., Thomas, P. & Wollenberg, A. (2006) Cross-sensitization

Food Allergens

between poppy seed and buckwheat in a food-allergic patient with poppy seed anaphylaxis. Int Arch Allergy Immunol 140, 170–3. Osterballe, M. & Bindslev-Jensen, C. (2003) Threshold levels in food challenge and specific IgE in patients with egg allergy, is there a relationship? J Allergy Clin Immunol 112, 196–201. Osterballe, M., Hansen, T.K., Mortz, C.G., Host, A. & BindslevJensen, C. (2005) The prevalence of food hypersensitivity in an unselected population of children and adults. Pediatr Allergy Immunol 16, 567–73. Palmer, G.W., Dibbern, D.A. Jr, Burks, A.W. et al. (2005) Comparative potency of Ara h 1 and Ara h 2 in immunochemical and functional assays of allergenicity. Clin Immunol 115, 302–12. Pasquato, N., Berni, R., Folli, C. et al. (2006) Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens. J Mol Biol 356, 684–94. Pomes, A., Butts, C.L. & Chapman, M.D. (2006) Quantification of Ara h 1 in peanuts, why roasting makes a difference. Clin Exp Allergy 36, 824–30. Poms, R.E. & Anklam, E. (2004) Effects of chemical, physical, and technological processes on the nature of food allergens. J AOAC Int 87, 1466–74. Poulsen, L.K., Hansen, T.K., Norgaard, A., Vestergaard, H., Stahl Skov, P. & Bindslev-Jensen, C. (2001) Allergens from fish and egg. Allergy 56 (suppl. 67), 39–42. Prioult, G. & Nagler-Anderson, C. (2005) Mucosal immunity and allergic responses, lack of regulation and/or lack of microbial stimulation? Immunol Rev 206, 204–18. Purohit-Sheth, T.S. & Carr, W.W. (2005) Oral allergy syndrome (pollen-food allergy syndrome). Allergy Asthma Proc 26, 229–30. Puumalainen, T.J., Poikonen, S., Kotovuori, A. et al. (2006) Napins, 2S albumins, are major allergens in oilseed rape and turnip rape. J Allergy Clin Immunol 117, 426–32. Radauer, C., Willerroider, M., Fuchs, H. et al. (2006) Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins, an experimental and structure-based analysis. Clin Exp Allergy 36, 920– 9. Receveur-Brechot, V., Czjzek, M., Barre, A. et al. (2006) Crystal structure at 1.45-A resolution of the major allergen endo-beta1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome. Proteins 63, 235–42. Reese, G., Schicktanz, S., Lauer, I. et al. (2006) Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus. Clin Exp Allergy 36, 517–24. Riaz, T., Hor, H.L., Krishnan, A., Tang, F. & Li, K.B. (2005) WebAllergen, a web server for predicting allergenic proteins. Bioinformatics 21, 2570–1. Roberts, G., Lack, G., Northstone, K. & Golding, J. (2005) Prevalence of latex allergy in the community at age 7 years. Clin Exp Allergy 35, 299–300. Rosmilah, M., Shahnaz, M., Masita, A., Noormalin, A. & Jamaludin, M. (2005) Identification of major allergens of two species of local snappers, Lutjanus argentimaculatus (merah/red snapper) and Lutjanus johnii (jenahak/golden snapper). Trop Biomed 22, 171–7. Roux, K.H., Teuber, S.S. & Sathe, S.K. (2003) Tree nut allergens. Int Arch Allergy Immunol 131, 234–44. Rupa, P. & Mine, Y. (2003) Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli. Biotechnol Lett 25, 427–33.

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Latex Allergy Robyn E. O’Hehir, Michael F. Sutherland, Alexander C. Drew and Jennifer M. Rolland

Summary Natural rubber latex is used extensively in the modern world, but perhaps nowhere more so than in the healthcare environment. Allergy occurs to the plant proteins from the rubber tree, Hevea brasiliensis, which are residual contaminants of finished latex products such as medical gloves. Latex allergy emerged as a serious issue in the 1980s triggered by the advent of the AIDS epidemic and the widespread use of universal precautions. Powdered latex gloves are particularly potent sensitizers, through the combination of cornstarch donning powder with highly charged latex allergens and their propagation as allergenic aerosols within the healthcare environment. This, together with the rise in atopy in recent years, produced an epidemic of latex allergy among healthcare workers with resultant contact urticaria, occupational asthma, and anaphylaxis. Other groups of patients were also identified as having an increased risk of latex allergy, including spina bifida patients and patients who have undergone multiple surgical procedures. Important clinical subsets of latex-allergic patients are those with the latex–fruit syndrome, whereby latex allergy subjects report allergy to certain fruits, commonly banana, avocado, and kiwi fruit. These reactions are due to cross-reactive allergens in latex that share significant homology with proteins in certain tropical fruits and vegetables. Thirteen latex allergens have been identified to date and of these the most important for healthcare workers are Hev b 5 and Hev b 6, and for spina bifida children Hev b 1 and Hev b 3. The diagnosis of latex allergy rests on obtaining a history of appropriate clinical symptoms within minutes of exposure to latex proteins together with the demonstration of specific IgE by serum immunoassay or skin-prick testing. Current immunoassays have limitations and skin-prick testing reagents are not widely available, at times hampering diagnosis. Management of affected individuals is based primarily around allergen avoidance. Latex-allergic subjects must stringently avoid personal exposure to latex and, in addition, must not work in environments where powdered latex gloves are used. With these measures, the majority of affected individuals can continue to work in their profession.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Recent epidemiologic studies suggest that the widespread adoption of powder-free, low-allergen gloves has resulted in a decrease in the levels of new sensitization to latex. Nevertheless, latex allergy is increasing in other populations and specific treatments are lacking. Specific immunotherapy for latex allergy has been trialed with evidence of efficacy, but is limited by systemic side effects. The search for safer immunotherapy for latex allergy is currently an area of active research.

Introduction Over the last two decades, allergy to natural rubber latex (NRL) has emerged as a major allergy in certain high-risk occupational and patient groups. Due to its high elasticity, tensile strength, and excellent barrier protection, an estimated 40 000 medical and consumer products contain NRL. Products commonly used in hospitals and the home are listed in Table 53.1 (Crippa et al. 2006; Kelso et al. 2006). NRL products contain highly potent latex allergens, which are very effective at crosslinking effector cell-bound IgE on basophils and mast cells with subsequent inflammatory mediator release. Clinical manifestations of IgE-mediated latex allergy include contact urticaria, rhinitis, conjunctivitis, asthma, food allergy, angioedema, and anaphylaxis. Contact allergy to chemicals utilized in the manufacture of latex products is also a clinical problem. Current understanding of latex allergy, the 13 latex allergens identified to date including observed cross-reactivities with fruits and vegetables, and B- and T-cell epitope identification are addressed in this chapter. The current status of specific immunotherapy for latex allergy will be discussed and possible future approaches to therapy including T-cell epitope peptides and hypoallergenic mutants considered.

A brief history of latex allergy IgE-mediated allergy to NRL was first reported in Germany in 1927 with a case report of a patient suffering from severe generalized urticaria during the use of a rubber dental prosthesis (Stern 1927). The next published case was reported from

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Table 53.1 Products containing natural rubber latex.* Medical products

Other products

Abdominal drain† Balloon connector for CPAP CPAP balloon Defluxion connector ECG electrode Elastic bandage Examination gloves† Fogarty balloon Foley urinary catheter Kehr tube† Malecot† Mask for anesthetics† Oxygen–nitrogen monoxide feeding tube Penrose drain† Sphygmomanometer band† Sphygmomanometer pump† Stethoscope† Surgical gloves† Tape† Thermodilution balloon probe Tourniquet Urinary condom texas†

Diving mask (black rubber) Rubber eraser Baby’s dummy Bike inner tube Car tire† Diving mask strap (black rubber) Diving nose piece† Electric plug cover Feeding bottle sucker† Gas pipe Hot water bottle† Household rubber gloves† Inflatable floating mattress† Latex mattress† Rubber inner sole† Scooter tire† Swim cap†‡ Toy Toy balloon† Truck inner tube Truck tire Unlined rubber gloves for housework† Window wiper† Work gloves†

* Adapted from Crippa et al. (2006) except where stated. † Contains extractable latex allergens. ‡ Kelso et al. (2006).

the UK over 50 years later when a 34-year-old housewife developed contact urticaria to household latex gloves (Nutter 1979). Between 1979 and 1988, there were numerous reports in the European literature of immediate allergic reactions to latex, resulting mainly in contact urticaria (Forstrom 1980; Galinsky & Kleinhans 1982; Meding & Fregert 1984). Intraoperative anaphylaxis in latex-allergic healthcare workers (HCW) was also reported (Axelsson et al. 1987). However, it was not until 1989 that the first reports appeared in the North American literature, when Slater reported two children with spina bifida (SB) who experienced intraoperative anaphylaxis (Slater 1989). By 1997, the US Food and Drug Administration had received over 1700 reports describing allergic reactions experienced by patients or HCW associated with the use of latex gloves or medical products (Ownby 2002; FDA 2003). The majority of case reports involved latex gloves and barium enema catheters. Latex allergy attracted particular attention for its potentially disastrous consequences when mucosal surfaces are breached by latex proteins in allergic individuals, as occurred in the 15 sentinel barium enema deaths (Ownby et al. 1991). Interestingly, follow-up interviews failed to reveal specific risks in

these patients: there were no HCW in the group, and none of the patients reported significant occupational exposure to latex products. However, important epidemiologic clues in some patients identified on history were atopy and active atopic dermatitis. All the patients with atopic dermatitis reported use of latex gloves to protect their hands during housework. Although most cases of latex allergy involve direct mucosal contact, among HCW aerosol transmission of latex proteins was particularly important (Tarlo et al. 1994).

Reasons for the latex allergy epidemic The increase in latex allergy over the last three decades paralleled a remarkable increase in the prevalence of atopic disorders in the western world. This was particularly attributed to changes in the environment as the rapidity of change was too fast for a significant modification of the genetic pool. The popular but controversial “hygiene hypothesis” proposed that the increased prevalence of allergic diseases was associated with changes such as sterile water in infant formula preparation, the early and increased use of antibiotics within the first year

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of life with resultant changes in the gut bacterial flora, and reduced bacterial infections due to cleaner homes, smaller families, and less use of childcare centers (Strachan 1989; Holt et al. 1999). Specifically, the emergence and recognition of latex allergy was linked to the introduction of universal precautions for the minimization of viral infection in the wake of the human immunodeficiency virus and hepatitis epidemics. Occupational health and safety guidelines mandated the protection of HCW by heightened glove usage. The unwitting and widespread use of nonsterile powdered examination gloves of high protein content, resulting in significant aerosolization of latex proteins, is probably the single most important reason for the marked increase in prevalence of latex allergy that occurred in the 1980s (Swanson & Ramalingam 2002). In addition, other high-risk groups identified were SB patients and patients who have high-level latex exposure as a result of multiple surgical procedures. Finally, increased awareness of latex allergy by clinicians together with improved diagnostic techniques is also likely to have resulted in increased identification of affected individuals.

NRL: chemistry and manufacture NRL is produced by anastomized laticifer cells of the rubber tree Hevea brasiliensis, a native of the Amazon basin but now typically grown in plantations in Africa and Malaysia (see reviews by Subramaniam 1995; Cullinan et al. 2003). Tapping and collection of latex is performed by cutting the bark of the rubber tree at an angle, causing liquid latex to flow from the wound into a collection vessel (c. 200 mL over 1–2 hours). Productivity has been enhanced by selective breeding and hormone treatment of trees, both practices thought to enhance the expression of pathogenicity-related proteins including hevein, chitinase, and β-1,3-glucanase (d’Auzac & Jacob 1989). Immediately after collection and before transport, stabilizers such as ammonia are added to prevent premature coagulation of the latex, primarily due to bacterial action on the liquid latex. The rubber hydrocarbon is a cis-1,4-polyisoprene with a molecular mass of 200–600 kDa. The protein content of latex depends on the environmental and seasonal conditions and may be increased by frequent tapping. This may partly explain the increased allergenicity of gloves during the late 1980s when demand outstripped supply (Subramaniam 1995). Different methods are used for the manufacture of dry rubber products, such as tires or solid rubber moldings, compared with dipped rubber products such as gloves, balloons, or condoms (reviewed in Cullinan et al. 2003) (Table 53.2). The former process occurs at far higher temperatures, utilizes acid dipping of rubber and prolonged high temperature leaching, and results in a product with reduced flexibility, less extractable protein and therefore less allergenicity (Yunginger et al. 1994). The manufacture of dipped rubber products entails

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Table 53.2 Dry and dipped latex products. Dry rubber products Cut thread Diver’s flippers Hot water bottle Tires Dipped rubber products Anesthesia rebreathing bags Balloons Bellows Bladders Bulbs Condoms Dental dams Gloves Medical sheaths Probe covers Seals Sphygmomanometer rubbers Swimming hats Tooth protectors Tubes Urinal bags Urinary rubbers

the addition of accelerators (chemicals to speed the crosslinking of the polyisoprene hydrocarbon units to form the elastic polymer in the finished rubber product) and other preservatives (Fig. 53.1). The main accelerators are thiurams, xanthates, and thiazoles (Subramaniam 1995). Antioxidants (phenols and cresols) and antiozonants may be added to the latex compounds depending on the service conditions required. Compounding ingredients such as coloring agents (e.g., dyes), thickeners such as casein milk protein and whiteners such as titanium oxide may be added in latex glove manufacture. Although serum IgE-reactive casein milk protein can be detected readily in some NRL gloves, the clinical importance of residual milk protein in gloves is uncertain (Ylitalo et al. 1999). Finally, drying and vulcanization (the chemical reaction usually mediated by sulfur to cause cross-linking of the rubber polyisoprene hydrocarbon units) of the latex-dipped product is undertaken using high temperature air, initially at 90°C and then finally at 120°C (Subramaniam 1995). During the manufacture of most rubber products and in particular surgical gloves, leaching with water is an integral part of the final production, mainly in an effort to reduce protein content and consequent allergenicity (Subramaniam 1995). Surface treatment of latex products is performed to decrease “stickiness” during glove donning. Powders may be applied, usually talcum powder and bioabsorbable starch. Adsorption of extractable latex proteins to the powder increases allergenicity through the formation of latex protein-coated particles

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Rubber tapping

Non-ammoniated latex (NAL) Ammonia 0.7% Low ammoniated latex (LAL)

Latex Allergy

Extensive efforts have been undertaken to clone and sequence latex allergens, with recombinant proteins now available for the majority. In addition, epitope mapping studies have identified regions of latex allergens responsible for IgE binding and CD4+ T cell reactivity.

Hev b 1 (rubber elongation factor) Stabilization (thiurams, antioxidants) these chemicals cause contact hypersensitivity

Washing, +/– powdering, +/– irradiation

Heating, dipping (glove molds dipped into liquid latex)

Fig. 53.1 Latex glove manufacturing process. (See CD-ROM for color version.)

that are released and easily inhaled (Beezhold & Beck 1992). With the realization of the potential dangers of using powdered latex gloves, other strategies for reducing “stickiness” have been adopted. One approach is chlorination of the surface of the glove, which reduces friction and allergen protein content, but this process must be carefully controlled in order to avoid damage to the latex product. Finally, the latex product may be polymer-coated to prevent contact between the skin and latex proteins (Swanson & Ramalingam 2002).

Latex allergens The functional unit in tapped latex is the rubber particle, a 5 nm to 3 μm diameter spherical polyisoprene droplet. These particles are internally homogeneous but externally coated with protein, phospholipids, and lipid layers that provide structural stability. Prenyltransferase is a key surface protein of the rubber particles but also found free in the cytosol. On ultracentrifugation, fresh latex separates into three discrete fractions: a white “cream” containing all the rubber and a thin band of Frey–Wyssling particles; C-serum, the translucent latex cytosol; and lutoids, the bottom fraction of organelles (d’Auzac & Jacob 1989). Separation of NRL into these fractions has been useful for chemical and immunologic characterization of latex allergens. Hev b 1 was the first identified latex allergen in the mid 1990s (Yeang et al. 1996), and there are now a total of 13 latex allergens, designated Hev b 1 to Hev b 13, recognized by the International Union of Immunological Societies (www.allergen.org; Table 53.3). In addition, five other allergens have been identified by proteomic methods and await further characterization (Yagami et al. 2004). As reports on the frequency of serum IgE binding to latex allergens by well-phenotyped patient groups are published, consensus patterns of reactivity are emerging (Yeang 2004; Wagner & Breiteneder 2005).

Hev b 1 is essential for the formation of rubber polymers by prenyltransferase and in raw latex is bound predominantly to large rubber particles (Yeang et al. 1996). A hydrophobic protein of 137 amino acid residues and molecular mass 14.6 kDa, Hev b 1 was first identified as a tetramer of molecular mass 58 kDa (Czuppon et al. 1993). The importance of Hev b 1 as a latex allergen was established by serum-specific IgE recognition in the majority of SB patients and in some HCW (Chen et al. 1997; Kurup et al. 2000; Wagner, B. et al. 2001; Bernstein et al. 2003). The difference in rates of sensitization between SB children and HCW is likely to represent the differences in the routes and latex product of exposure. The T-cell epitopes of Hev b 1 have been mapped with identification of two immunodominant regions (Raulf-Heimsoth et al. 1998). IgEreactive recombinant Hev b 1 (rHev b 1) has been produced and is capable of inducing histamine release from basophils of latex-allergic subjects (Rihs et al. 2000b).

Hev b 2 (b-1,3-glucanase) Hev b 2, a β-1,3-glucanase of molecular mass 34–36 kDa, is an important defense-related enzyme produced in response to microbial and fungal infection (Yagami et al. 1998). Studies using purified natural Hev b 2 demonstrate reactivity in greater than 60% of latex-allergic glove users (Bernstein et al. 2003; Kurup et al. 2005) with lower reactivity in SB patients (Kurup et al. 2005). The predominant IgE-binding epitopes of Hev b 2 are associated with the carbohydrate components (Yagami et al. 2002), explaining the low frequency of serum IgE reactivity found using rHev b 2 preparations (Yip et al. 2000). Interestingly, Hev b 2 immunologic cross-reactivity with bell pepper and olive has been reported (Barral et al. 2004; Wagner et al. 2004; Palomares et al. 2005).

Hev b 3 (small rubber particle protein) Hev b 3 is tightly attached to small rubber particles in NRL and is a 24-kDa hydrophobic protein that dissociates readily into lower molecular weight polypeptides on storage (Yeang et al. 1996). Like Hev b 1, Hev b 3 is involved in rubber synthesis. Hev b 1 and Hev b 3 share 47% amino acid sequence identity and share IgE-binding epitopes (Wagner et al. 1999; Banerjee et al. 2000). Hev b 3 demonstrates a high frequency of binding to serum IgE from latex-allergic children with SB (Wagner et al. 1999; Kurup et al. 2000). This reactivity may in part but not entirely be due to cross-reactivity between Hev b 1 and Hev b 3 (Wagner et al. 1999). As with Hev b 1, the presence of Hev b 3 on rubber particles and the invasive route of exposure is thought to be responsible for higher sensitization prevalence

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Table 53.3 Latex allergens.

Latex allergen Hev b 1 Hev b 2 Hev b 3 Hev b 4‡

Hev b 5 Hev b 6.01 Hev b 6.02 Hev b 6.03 Hev b 7.01 Hev b 7.02 Hev b 8 Hev b 9 Hev b 10 Hev b 11 Hev b 12 Hev b 13

Recombinant protein produced

Allergen importance for latex glove users*

Allergen importance for spina bifida*

Y

Minor

Major

Y

Y† Y

Minor Minor

Minor Major

Y (lecithinase homolog

Y†

Minor

Minor

C-serum B-serum B-serum B-serum B-serum

Y Y Y Y Y

Major Major Major Minor Minor

Major/minor Major Major Minor Minor

42

C-serum

Y

Minor

Minor

14

C-serum

Y

Minor

Minor

47.6 26

C-serum B-serum

Y Y

Minor Minor

None Minor

33 9.3 43

C-serum Unknown B-serum

Y Y Y†

Minor Minor Major/minor

No data None Major/minor

Protein name

Function

Size (kDa)

Location

Rubber elongation factor b-1,3-Glucanase Small rubber particle protein Component of the lutoid microhelix complex Acidic protein Prohevein Hevein C-terminal fragment Patatin homolog from C-serum Patatin homolog from C-serum Profilin

Rubber biosynthesis

14.6

Antifungal Rubber biosynthesis

34–36 24

Lecithinase and cyanogenic glucosidase

100–115 (53–55 and 57) 16 20 4.7 16 42

Large rubber particles B-serum Small rubber particles B-serum

Unknown Hevein precursor Antifungal Unknown Esterase, rubber biosynthesis inhibitor Esterase, rubber biosynthesis inhibitor Cytoskeletal actin inhibitor Housekeeping enzyme Housekeeping enzyme

Enolase Mn superoxide dismutase Class I chitinase Antifungal Lipid transfer protein Housekeeping protein Early nodule-specific Lipolytic esterase protein homolog

IgE-reactive N-glycans

Y

* Major allergen with ≥ 50% reactivity in most studies. † Recombinant protein non-IgE reactive. ‡ Hev b 4 is a protein complex comprising lecithin homolog and cyanogenic glucosidase.

in children with SB. Hev b 3 is a minor allergen for HCW and other latex-allergic individuals (Kurup et al. 2000; Yip et al. 2000; Bernstein et al. 2003). Several T-cell epitopes of Hev b 3 have been mapped that show no cross-reactivity with Hev b 1 at the T cell level (Bohle et al. 2000).

Hev b 4 (microhelix complex component) Hev b 4 is a protein complex associated with the microhelix of the lutoid bodies of NRL. The complex has a molecular mass of 100–115 kDa, but under reducing conditions dissociates into a 53–55 kDa lecithinase homolog and a 57 kDa cyanogenic glucosidase (Sunderasan et al. 2005). Hev b 4 is usually considered a minor latex allergen for HCW and SB patients, but some studies report higher frequencies of sensitization (Kurup et al. 2000; Bernstein et al. 2003). The lecithinase homolog is highly glycosylated and the carbohydrate moiety is implicated in serum IgE binding (Kolarich et al. 2006). The lecithinase component has been shown to be more allergenic than the cyanogenic glucosidase (Sunderasan et al. 2005).

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Hev b 5 (acidic C-serum protein) Hev b 5 is a highly acidic 16-kDa protein comprising 151 amino acid residues and of unknown biological function (Akasawa et al. 1996; Slater et al. 1996). It is one of the most important latex allergens for latex glove users (Yip et al. 2000; Bernstein et al. 2003). For SB patients, prevalence of Hev b 5 sensitization is generally lower than for HCW (Slater et al. 1996). Hev b 5 is present in low concentrations in NRL but increases in concentration up to 10-fold during latex glove manufacture (Sutherland et al. 2002a) (Table 53.4). To increase diagnostic sensitivity, a rHev b 5-spiked NRL-based assay is now commonly used (Lundberg et al. 2001). Hev b 5 shares high sequence homology with another acidic protein in kiwi fruit (Ledger & Gardner 1994; Akasawa et al. 1996; Slater et al. 1996) and also with an acidic protein in sugar beet (Fowler et al. 2000), but immunologic cross-reactivity has not been demonstrated. Eleven linear IgE-binding epitopes of Hev b 5 have been demonstrated, and modification by alanine substitution of amino acids in eight of these epitopes gave a

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Table 53.4 Relative Hev b5 content of latex gloves. (Modified from Sutherland et al. 2002a, with permission.)

Latex sample

Total protein concentration (mg/mL extract)

rHev b 5 equivalents by mAb inhibition ELISA (mg/mL extract)

rHev b 5 equivalents by IgE inhibition ELISA (mg/mL extract)

% rHev b 5 equivalents by mAb inhibition ELISA

% rHev b 5 equivalents by IgE inhibition ELISA

G1 G2 G3 G4 G5 NAL LAL

274 320 –* –* –* 100 100

6.5 5 –* –* –* 1.8 1

12.5 8 –* –* –* 0.6 1

3 1.6

7.5 2.2

1.8 1

0.6 1

* Levels of total protein of rHev b 5 equivalents below limits of detection. G1, G1, powdered nonsterile utility gloves; G3, G4, G5, sterile surgical gloves.

4500-fold reduction in IgE binding (Beezhold et al. 2001). A subsequent study showed that Hev b 5 contains a repetitive linear IgE-binding epitope, potentially contributing to the high potency of this allergen (Beezhold et al. 2004). T-cell epitope mapping of Hev b 5 in a population of latex-allergic glove users identified two highly reactive regions (de Silva et al. 2000) (Fig. 53.2).

Hev b 6 (hevein precursor) Hev b 6.01 (prohevein) is a 187 amino acid precursor protein of 20 kDa that is cleaved within the rubber plant forming 4.7-kDa hevein (Hev b 6.02; 43 amino acids) and a 14-kDa C-terminal fragment (Hev b 6.03) (Lee et al. 1991; Soedjanaatmadja et al. 1994). Hev b 6.02 is a chitin-binding protein with antifungal properties and a role in latex coagulation (Gidrol et al. 1994). It is the only latex allergen for which the crystal structure has been determined (Rodriguez-Romero et al. 1991). Hev b 6.02 is the most abundant protein in latex extract, with levels 30 times more than Hev b 6.03 and thousands of times higher than some other latex allergens; Hev b 6.03 is rapidly degraded

% responders

100 75

Stimulation index ≥ 2.5

*

*

in NRL (Yeang et al. 2006). It is therefore not surprising that Hev b 6.02 is recognized at high frequency by serum IgE of latex glove users and is considered a major latex allergen, while Hev b 6.03 is recognized at low frequency (Alenius et al. 1996; Rozynek et al. 1998). Hev b 6.02 has striking homology with class I chitinases and has been identified as the principal latex allergen responsible for latex–fruit cross-reactivity (Blanco et al. 1999). Clinical and immunologic cross-reactivity has been demonstrated between Hev b 6.02 and endochitinases of banana (Mikkola et al. 1998; Karisola et al. 2005), avocado (Sowka et al. 1998), chestnut (Diaz-Perales et al. 1998; Sanchez-Monge et al. 2006), and sweet pepper (Gallo et al. 1998). Hev b 6.01 is a tightly folded molecule with a tertiary structure stabilized by seven disulfide bridges. Using a chimera-based IgE epitope mapping strategy, the major conformational IgE-binding epitopes of Hev b 6.02 have been identified (Karisola et al. 2002). Disruption of the first four disulfide bridges of Hev b 6.01 generated a hypoallergenic protein (Drew et al. 2004). T cell-reactive regions of Hev b 6.01 mapped predominantly within the Hev b 6.02 domain (de Silva et al. 2004), consistent with the high content of Hev b 6.02 in latex gloves (Table 53.5). T cell epitopes of Hev b 6.03 have also been mapped (Raulf-Heimsoth et al. 2004).

50

Hev b 7 (patatin-like protein)

25

p16(132–151)

p15(127–146)

p14(118–137)

p13(109–128)

p12(100–119)

p11(91–110)

p10(82–101)

p9(73–92)

p8(64–83)

p7(55–74)

p6(46–65)

p5(37–56)

p4(28–47)

p3(19–38)

p2(10–29)

p1(1–20)

0

Hev b 5 peptide Fig. 53.2 Dominance of T-cell proliferative response* (3H-Tdr uptake) to Hev b 5 p(1–20) and p(46–65) in short-term T-cell lines from latex-allergic subjects (N = 14). (In part, de Silva et al. 2000.) (See CD-ROM for color version.)

Hev b 7 has sequence homology to patatin from potato (Kostyal et al. 1998; Breiteneder et al. 1999), but whether potato patatin shows immunologic and clinical cross-reactivity with Hev b 7 is unclear (Sowka et al. 1999; Seppala et al. 2000). Hev b 7 was initially speculated as a clinically important latex glove allergen, but early studies were confounded by comigration of Hev b 7 with Hev b 13 on SDS-PAGE (Raulf-Heimsoth et al. 2003; Arif et al. 2004). Subsequent studies with both recombinant and purified natural Hev b 7 demonstrated low sensitization rates (Bernstein et al. 2003; Kurup et al. 2005). Two isoforms of Hev b 7 have been cloned and sequenced, Hev b

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Table 53.5 Relative Hev b 6.02 content of latex gloves.

Glove sample

Material

Powdered

Total protein concentration (mg/mL extract)

1 2 3 4 5 6 7 8

Latex Latex Latex Latex Latex Latex Latex Neoprene

Yes No No No Yes Yes No No

570 –* –* 370 250 1280 350 –*

rHev b 6.02 equivalents by IgE ELISA (mg/mL extract)

% rHev b 6.02 equivalents by IgE inhibition ELISA

rHev b 6.02 equivalents by IgE inhibition (ng/g glove)

200 0.016 0.6 0.010 0.08 0.007 0.016 –*

35

180, 180 13 554 5 48 4 13 –*

0.0027 0.032 0.0006 0.0046

* Levels of total protein or rHev b 6.02 equivalents below limits of detection.

7.01 and Hev b 7.02 (Sowka et al. 1999). In a study of German and Portuguese HCW and SB patients, the Hev b 7.02 isoform showed intermediate importance for sensitization compared with other latex allergens (Rozynek et al. 2006).

Hev b 8 (profilin) Profilins are a group of homologous cytoskeletal plant proteins that are well recognized as allergens and a source of allergenic cross-reactivity. The 14-kDa profilin from latex is designated as Hev b 8 and demonstrates low sensitization rates in patients with latex allergy (Rihs et al. 2000a; Ganglberger et al. 2001; Sanz et al. 2006). One study showed no reactivity of Hev b 8-reactive latex-allergic subjects with grass pollen profilins by skin-prick testing (SPT) and serum IgE, suggesting sensitization directly by exposure to latex profilin, but further investigations are required (Rihs et al. 2000a).

Hev b 9 (enolase) Hev b 9 is an enolase of 47.6 kDa with significant sequence homology and limited IgE cross-reactivity with enolases of the molds Cladosporium herbarum and Alternaria alternata (Wagner et al. 2000). Studies indicate that Hev b 9 is a minor allergen among latex-allergic adults and children (Wagner et al. 2000; Sanz et al. 2006).

2003) and children (Sanz et al. 2006), but reactivity of natural Hev b 11 has not been examined.

Hev b 12 (lipid transfer protein) The clinical importance of the 9.3-kDa latex lipid transfer protein, Hev b 12 (Beezhold et al. 2003), is not yet clear but seems low, with one study showing serum IgE reactivity in only 24% of latex glove users (Beezhold et al. 2003) and another showing no reactivity in SB children (Pamies et al. 2005). In a study of patients with latex and fruit allergy, IgE to rHev b 12 was cross-reactive with fruit lipid transfer proteins but had no clinical relevance (Rihs et al. 2006).

Hev b 13 (early nodule-specific protein) Initial reports suggested that Hev b 13 was highly allergenic in HCW and SB patients (Bernstein et al. 2003; Raulf-Heimsoth et al. 2003) but a subsequent study found that Hev b 13 preparations were contaminated with Hev b 6 and that highly purified Hev b 13 was less reactive (Paluso et al. 2007). Full clarification of the clinical importance of Hev b 13 remains to be determined. Predominant IgE reactivity of Hev b 13 resides with the carbohydrate moiety which cross-reacts with the corresponding moiety of potato tuber patatin. Additionally, Hev b 13 shows homology to early nodule-specific protein of legumes (Arif et al. 2004).

Hev b 10 (manganese superoxide dismutase) Hev b 10, another minor latex allergen, is also a member of the “latex-mold” group of allergens. It is a manganese superoxide dismutase (MnSOD) of 26 kDa sharing limited IgE cross-reactivity with the Aspergillus fumigatus homolog (Rihs et al. 2001; Wagner, S. et al. 2001).

Hev b 11 (chitinase) Hev b 11 is a class I chitinase of 33 kDa with significant sequence homology to, but negligible immunologic crossreactivity with, Hev b 6.02 (Rihs et al. 2003). Low IgE reactivity of rHev b11 was reported in latex-allergic adults (Rihs et al.

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Epidemiology The development of latex allergy occurs predominantly in well-defined groups of individuals who have experienced high-level exposure to NRL. Established risk factors include occupation (e.g., HCW, laboratory scientists, oral healthcare professionals), SB, multiple surgical procedures, atopy, and eczema. Additionally, structural homologies between certain allergens in latex and some tropical fruits result in increased prevalence of latex allergy in some subjects with primary

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6. He v

C

b

5 b

MWr (kDa)

He v

fruit allergy. Few studies have investigated possible genetic risk factors for latex allergy. One study showed a significant association between interleukin (IL)-13 and IL-18 promoter polymorphisms and latex allergy (Brown et al. 2005), while another showed an association between particular HLA haplotypes and Hev b 6.02-specific IgE response (Rihs et al. 2002). There are a number of limitations of latex allergy epidemiology studies that must be realized in any assessment of the literature. Firstly, the majority of studies have been prevalence studies, which do not assess change over time. This must be accounted for when comparing historical prevalence rates derived from older studies where latex protein exposure may have been higher, with more recent studies in the era of low allergen or powder-free gloves. Many studies do not report quantified levels of allergen exposure within the HCW environment. There has also been the additional problem of use of a wide variety of diagnostic methods and assay reagents (from glove eluates to standardized skin test reagents), making comparison between studies difficult. A recent metaanalysis summarized the epidemiologic data regarding sensitization to latex among HCW combining studies performed up to 2003 (Bousquet et al. 2006). Latex allergy was found in 4.3% (range 4.0– 4.6%) of HCW, while latexpositive skin-test responses ranged from 6.9–7.8%. This metaanalysis specifically excluded studies where HCW used low-allergen latex gloves, powder-free latex gloves, or both, and as such represents levels of latex allergy seen in the past where powdered latex gloves were widely used. More recent studies suggest that the prevalence of latex allergy among HCW may be decreasing, with the advent of powder-free latex gloves. In a study of HCW at Veteran’s Affairs Hospitals in USA where 91.4% of subjects reported non-powdered glove use, the prevalence of clinical latex allergy (immediate symptoms together with latex-specific IgE antibodies) was only 0.4% (Zeiss et al. 2003). Studies of the incidence of latex allergy are much rarer than those of prevalence. A large prospective Canadian study found an incidence among HCW in hospitals of new cases of latex allergy of 1% (Sussman et al. 1998), while among new dental technicians, a French study found an incidence of 2.5% per person year (Gautrin et al. 2000). Incidence studies are influenced by the ambient levels of latex proteins causing exposure. There is evidence that with the advent of powderfree gloves, latex allergy incidence may be falling. In Germany, where powdered gloves have been banned by legislation in healthcare facilities, the incidence of new cases of contact urticaria suspected to be caused by latex has fallen by 79% since the year of the ban in 1998 (Allmers et al. 2004). Perhaps surprisingly, only 68% of the cases cited were occupationally sensitized with the remaining individuals suspected of sensitization through other contacts. This study was an encouraging reminder of the usefulness of occupational health and safety intervention strategies. Another longitudinal study evaluating avoidance of unnecessary glove use, the use of

02

CHAPTER 53

64 37 26 20 15

8

Fig. 53.3 Detection of Hev b 5 and Hev b 6.02 in powdered utility latex glove extract by immunoblotting. Glove extract stained with Coomassie brilliant blue (C) or probed with monoclonal antibodies to Hev b 5 and Hev b 6.02. (Results from Sutherland et al. 2002a; Drew et al. 2004.) (See CD-ROM for color version.)

nonpowdered latex gloves by all workers and the use of nonlatex gloves by sensitized subjects demonstrated a convincing decrease in latex symptoms and avoidance of new cases of sensitization (Filon & Radman 2006). Interestingly, research has shown clear differences in the amount of residual NRL proteins in comparisons of powdered gloves made for surgical and examination use. Surgical gloves had larger allergenic particles (> 10 μm), while powdered examination gloves released higher levels of total latex allergen of a respirable size range (2.5–10 μm) (Brown et al. 2004). These findings are consistent with those reported by Sutherland and colleagues, who demonstrated that the major latex allergen Hev b 5 was increased in some NRL gloves during manufacture with higher levels in the domestic rubber glove than in the tapped rubber product or the high-quality surgical glove (Sutherland et al. 2002b) (Table 53.4). Hev b 5 and Hev b 6.02 allergens can readily be detected in aqueous extract from powdered utility gloves (Fig. 53.3). Differential expression of latex allergens was also reported on the internal and external surfaces of latex surgical gloves (Peixinho et al. 2006). Moreover, Hev b 5 allergen could readily be detected on particles from latex gloves (Fig. 53.4) and in a bronchoscopy suite on nasal air sampling filters (Mitakakis et al. 2002). Investigation of the potential for latex gloves to leach out allergenic proteins suggested that Hev b 5 and Hev b 13 were strong markers of the allergenicity of latex gloves (Yeang et al. 2004). With the increased awareness of latex allergy and the shift toward a more latex-free environment in hospitals (Allmers

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(a)

(b)

(c) Fig. 53.4 Halogen assay for Hev b 5 on glove particles. Gloves were placed against an adhesive surface and powder probed with an Hev b 5-specific monoclonal antibody (Sutherland et al. 2002a; Mitakakis et al. 2002). Halos around particles represent positive staining. (a) Livingstone-powdered latex examination glove; (b) Uniglove-powdered latex utility glove; (c) vinyl examination glove. (See CD-ROM for color version.)

et al. 2004; Latza et al. 2005), there has been a paradigm shift of latex allergy away from the healthcare industry toward other populations including postal workers, hairdressers, construction workers, food handlers, greenhouse workers, security personnel, and domestic consumers through their

1172

utilization of NRL gloves for barrier protection (Carrillo et al. 1995; Conde-Salazar et al. 2002; Nettis et al. 2003). Countries undergoing a technological revolution including Taiwan (Chen & Lan 2002; Lee et al. 2005), Turkey (Ozkan & Gokdogan 2003), Portugal (Jorge et al. 2006), Thailand (Chaiear et al. 2006), and Poland (Dudek et al. 2005) also report an accompanying increased prevalence of latex allergy and it can be expected that China and India will experience similar consequences. The increased awareness of latex allergy in the community has also shifted diagnosis and management away from allergy specialist centers to primary-care physicians that could result in an underestimation of latex allergy prevalence in hospital-based epidemiologic studies. Multiple surgical procedures heighten the risk of developing latex allergy, particularly in children with SB (Bode & Wahn 1996; Porri et al. 1997; Ylitalo et al. 1997), although there may be other disease-related factors influencing the high rate of latex sensitization in these subjects (Eiwegger et al. 2006). In adults, greater than 10 surgical procedures is significantly associated with a risk of latex allergy (Rueff et al. 2001). The reported prevalence of latex sensitization in SB patients ranges from 4.3% in Venezuela to 40% in Europe (Mazon et al. 2000) and 60% in the USA (Ellsworth et al. 1993). Australian data show a prevalence of 36.9% of latex sensitization among SB patients (Valentine et al. 1999). These patients, through their different route and type of latex allergen exposure, are sensitized to different allergens (mainly Hev b 1 and 3) compared with HCW (mainly Hev b 5 and 6). Women undergoing obstetric and gynecological surgical procedures also show a heightened risk of latex anaphylaxis (Draisci et al. 2007). Atopy, as might be expected, is a risk factor for latex allergy (Grzybowski et al. 1996; Douglas et al. 1997; Leung et al. 1997). Nevertheless, latex allergy is sometimes observed in nonatopic individuals. In one study, up to 19% of individuals with latex allergy were reported to be nonatopic (Aichane et al. 1997). The presence of hand dermatitis is a well-established risk factor for latex allergy, likely through the greater penetrance of latex allergens through the epidermis (Boxer 1996). A study of Japanese children attending allergy outpatient clinics found that the prevalence of latex allergy among those with eczema/dermatitis rose from 6.1% in 2001 to 15.9% in 2003 (Kimata 2005). Primary fruit allergy is a recognized risk factor for latex allergy, due to cross-reactive epitopes on fruit and latex allergens. A study of 57 primary fruit allergic patients showed a rate of subsequent latex allergy of 10.5%, with the most common primary fruit allergies being to melon, peach, and banana (Garcia Ortiz et al. 1998). Only three studies have reported the prevalence of latex allergy in the general population but a metaanalysis of these studies concluded a rate of clinical latex allergy of 1.37% (range 0.43–2.31%) and of latex skin test positivity of 2.08% (range 1.31–2.85%) (Bousquet et al. 2006).

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Clinical features IgE-mediated latex allergy may follow prolonged exposure to Hevea latex proteins. The time between commencement of exposure to rubber proteins (sensitization) and symptoms was found in one study to average 5 years (Allmers et al. 1996). As for other allergic diseases, the symptom constellation of IgE-mediated latex allergy depends on the route of exposure. Latex allergy commonly manifests as hand urticaria (Valsecchi et al. 2000). Hand itching by itself is a poor indicator of latex allergy (Douglas et al. 1997), as this is also consistent with irritant or allergic contact dermatoses, which occur in up to 37.6% of glove users (Uveges et al. 1995). Irritant is by far the most common form of latex dermatitis, accounting for 98.3% of the dermatoses diagnosed in one study (Uveges et al. 1995). Contact allergy associated with latex products is usually due to chemical accelerators used in the manufacture process, particularly thiurams, carbamates, and benzothiazoles (Heese et al. 1991; Conde-Salazar et al. 1993; Cullinan et al. 2003). Added phenols and cresols can also cause contact hypersensitivity (Rich et al. 1991). In some patients, contact latex allergy coexists with IgE-mediated latex allergy, damaged skin perhaps facilitating penetration of the latex allergens (Wakelin & White 1999). Latex allergy is a common cause of allergic rhinoconjunctivitis (Archambault et al. 2001) and occupational asthma (McDonald et al. 2000; Hnizdo et al. 2001). In a South African study, latex was the most common causative agent, followed by isocyanates and platinum salts (Hnizdo et al. 2001). In the UK, a similar study found latex to be a common contributory agent for occupational asthma (Ross et al. 1998). In newly recruited apprentice dental technicians, the cumulative incidence of new cases of occupational asthma due to latex over a 32-month period was 4.5% (Archambault et al. 2001). A Dutch study using inhalation challenge with latex glove powder demonstrated a prevalence of occupational asthma in HCW of 2.5%, with approximately 5% of study subjects having a positive skin test to latex (Vandenplas et al. 1995). Latex-allergic individuals are at appreciable risk of developing anaphylaxis on exposure to latex proteins (Chiu & Kelly 2005), and in one study anaphylaxis was the mode of presentation for 30% of latex-sensitive children (Kwittken et al. 1995). Latex is one of the main causes of intraoperative anaphylaxis (Kelly et al. 1994) and is the most common skin test reagent (including nuts, bee venoms, and antibiotics) associated with an anaphylactic adverse event (Valyasevi et al. 1999). There are numerous reports of anaphylaxis in latex-allergic individuals on exposure to tiny amounts of latex protein in seemingly innocuous everyday activities such as licking a postage stamp (Pumphrey et al. 2001), and eating food prepared by food handlers using latex gloves (Nixon & Lee 2001). The potency of latex allergens is illustrated by the finding that eluates from solid rubber medication vials are

Latex Allergy

sufficient to induce wheal and flare reactions on skin testing of latex allergic individuals (Primeau et al. 2001). Positive skin tests have been reported to a low ammoniated latex (LAL) concentration of 70 pg/mL in highly sensitive individuals (Yip et al. 2000). Up to 52% of latex allergy sufferers have sensitivity to various fruits and vegetables (Blanco et al. 1994). Also, latexallergic individuals have four times the risk of the general population of having food allergy. Food allergy among latexsensitive subjects manifests as anaphylaxis in up to 36% of cases (Blanco et al. 1994). Therefore, the latex–fruit syndrome is of considerable clinical importance. More than 20 foods, fruits or plants are immunologically cross-reactive with latex, most commonly banana, avocado, and kiwifruit (Figs 53.5 and 53.6). Currently, latex-allergic patients are not advised to avoid all such foods unless they have specific symptoms.

(a)

(b) Fig. 53.5 Foods that show clinical cross-reactivity with latex. (a) Fruits (clockwise from top): papaya, mango, bell pepper, banana, fig, tomato, kiwi fruit, and avocado. (b) Vegetables: celery and potato. (See CD-ROM for color version.)

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Family

Viridiplantae (green plants)

Scientific name, Common name

Lauraceae

Persea americana, Avocado

Musaceae

Musa acuminata, Banana

Poaceae

Phleum pratense, Timothy grass

Moraceae

Ficus benjamina, Fig

Passifloraceae

Passiflora edulis, Passion fruit

Euphorbiaceae

Hevea brasiliensis, Latex

Fagaceae

Castanea sativa, Chestnut

Betulaceae

Betula verrucosa, Birch

Caricaceae

Carica papaya, Papaya

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Mangifera indica, Mango

Actinidiaceae Solanaceae Oleaceae Apiaceae

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Apium graveolens, Celery Ambrosia elatior, Ragweed Artemisia vulgaris, Mugwort Cladosporium herbarum

Pleosporaceae

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Actinidia deliciosa, Kiwifruit Solanum lycopersicum, Tomato—Nicotiana tabacum, Tobacco Solanum tuberosum, Potato—Capsicum annuum, Bell pepper Olea europaea, Olive

Fig. 53.6 Phylogenetic relationship between plants and fungi containing allergens that cross-react with latex. Species containing clinically important crossreacting allergens are indicated in bold.

However, it is incumbent upon clinicians to warn patients of the potential for these reactions. Latex allergens shown to cross-react with plant allergens were discussed above, but other potential cross-reactive allergens include Cu/Zn superoxide dismutase, heat-shock protein, and calmodulin (Guarneri et al. 2006). Primary sensitization via fruit exposure has been reported and is a recognized risk factor for latex allergy (Garcia Ortiz et al. 1998). However, it is not always clear whether latex sensitization precedes or follows the development of food allergy (Wagner & Breiteneder 2002).

Diagnosis and management As with all allergic diseases, diagnosis of latex allergy relies on a detailed clinical history consistent with an IgE-mediated reaction confirmed by demonstration of relevant specific IgE reactivity (Sutherland et al. 2002b; Cullinan et al. 2003; Taylor & Erkek 2004; Chiu & Kelly 2005; Bousquet et al. 2006).

In vitro latex-specific IgE assays The three in vitro latex-specific IgE assays for which there are published data on diagnostic efficiency are the Pharmacia UniCAP FEIA (Pharmacia Ltd, Milton Keynes, UK), Immulite/ AlaSTAT (Diagnostic Products Corporation, Euro/DPC Ltd, Caernarfon, UK), and Hycor HY-TECH EIA (Garden Grove, CA, USA) systems. Two large studies evaluated the Pharmacia

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UniCAP latex-specific IgE assay for the diagnosis of latex allergy. Both studies used a physician-administered questionnaire combined with latex SPT as the gold standard for latex allergy diagnosis. The first study in a large sample of 195 latex-allergic individuals, predominantly HCW, found that the Pharmacia latex-specific IgE test had a sensitivity of 76.3% and a specificity of 96.7% (Hamilton et al. 1999). The second study in a population of 60 latex-allergic individuals found a sensitivity of 79.5% and a specificity of 90.2% (Ownby et al. 2000). The diagnostic material used in the above IgE assays has traditionally been nonammoniated latex (NAL). However, HCW and other subjects are generally exposed to the finished latex products and certain allergens may have undergone relative enrichment or alteration during the manufacturing process (Yeang et al. 2006). As mentioned previously, one reason for the imperfect sensitivity of the serum latex-specific IgE assay is that the major latex allergen Hev b 5 is not abundant in NAL (Chen et al. 2000) but is relatively abundant in latex glove proteins (Sutherland et al. 2002a). Consequently, Pharmacia enriched their NAL reagent with rHev b 5, thus enhancing the sensitivity of the latex UniCAP FEIA (Lundberg et al. 2001; Hemery et al. 2005b; Kurup et al. 2005). Another comparative study found that the Immulite system showed similar specificity and sensitivity to the AutoCAP and AlaSTAT assays, but with technological improvements showed enhanced speed and less operator intervention (Yeang et al. 2006).

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Latex Allergy

of surface CD63 or CD203c by flow cytometry to determine basophil activation following in vitro allergen challenge (Sanz et al. 2001; Eberlein-Konig et al. 2006; Ebo et al. 2006). A study of 46 latex-allergic subjects and 33 controls found that this assay had a sensitivity of 84.8% and a specificity of 87.9% in the detection of latex allergy (Hemery et al. 2005a). In another study, using a combination of rHev b 5, rHev b 6.01 and nHev b 6.02 allergens for basophil activation, the assay identified 22 of 23 latex-allergic children (95.6%) (Sanz et al. 2006). The whole blood basophil activation assay has been used to successfully identify hypoallergenic latex preparations for future immunotherapeutic use (Drew et al. 2004) (Fig. 53.7).

Immunoblotting is a useful technique for examining serum IgE reactivity with individual components of a latex extract and confirms Hev b 5 and Hev b 6.01 as important allergens for HCW (Sastre et al. 2006). However, sensitivity is higher than with the CAP system and low-level reactivity may not correlate with clinical outcome. There is a strong relationship between specific IgE level and clinical symptoms of latex allergy (Kim & Safadi 1999). However, in the absence of clinical symptoms of latex allergy, the significance of a positive latex-specific serum IgE result is unclear and more likely reflects cross-reactivity with pollen, fruit, and nut allergens (Blanco et al. 1998; Cullinan et al. 2003).

Other in vitro assays for latex allergy PBMC proliferation and cytokine assays

In vivo latex-specific IgE assays

The utility of peripheral blood mononuclear cell (PBMC) proliferative responses to unfractionated latex glove extracts in the diagnosis of latex allergy was evaluated and found to have low sensitivity as a diagnostic tool, with positive responses in only 20% of latex-allergic subjects (Turjanmaa et al. 1989). Allergen-specific induction of cytokines and chemokines in PBMC and chemokine receptor expression on circulating T cells was evaluated for Hev b 5 and Hev b 6.01 (Lehto et al. 2007).

Skin-prick testing SPT is generally considered the most sensitive assay for the diagnosis of latex allergy and, in the absence of standardized challenge protocols, when combined with an appropriate clinical history, SPT is accepted as the gold standard for the diagnosis of latex allergy. Several skin testing reagents for latex allergy diagnostics are used worldwide including NAL, LAL, and latex glove extracts. In Europe and Australia, the Stallergenes (Antony Cedex, France) LAL extract is widely used. This is a standardized reagent, with a reported 100% specificity and 93% sensitivity when used at the biological potency of 100 Index of Reactivity (IR) Units or 22 μg/mL protein concentration in

Basophil activation test

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a population of 46 latex allergic subjects and 76 nonallergic controls (Turjanmaa et al. 1997). In the USA, an NAL reagent (Greer Laboratories Inc., Lenoir, NC, USA) is used for skin testing. This reagent has a higher protein concentration (1 mg/ mL), with a reported sensitivity of 99% and specificity of 96% at 1 mg/mL and 95% and 100% at 100 μg/mL (Hamilton & Adkinson 1998). The major drawback to the skin test is the risk of systemic reactions. “Mild” reactions such as pruritus and urticaria were reported at a rate of 16.1% using the Greer NAL reagent, and anaphylaxis has been reported with the Stallergenes LAL reagent (Nettis et al. 2001). The risk of anaphylaxis is even higher when latex glove eluates are used in skin testing (Kelly et al. 1993). A diagnostic algorithm is to take a clinical history and then proceed to identification of latex-specific IgE by SPT or in vitro testing depending on the clinical indicators of anaphylactic risk (hand urticaria on glove contact, latex asthma, latex anaphylaxis). In the event of negative in vitro testing, SPT is usually performed. Where both SPT and in vitro tests are negative but there is a high clinical suspicion of latex allergy, it is appropriate to proceed to challenge testing.

Challenge testing for latex allergy Although considered the “gold standard” by many, challenge testing is poorly standardized at present because of the variability of allergen and protein contents in latex gloves. Challenge is usually carried out according to the method of Turjanmaa (Turjanmaa & Reunala 1988), where a powdered latex glove is placed on a wetted finger initially and then hand of the blinded subject, with a nonlatex glove as a control. Two or more patches of urticaria are considered positive (Turjanmaa & Reunala 1988). Standardized inhalation challenge tests using latex glove powder are reported (Kurtz et al. 2001), but are not widely available and can only be considered research tools at present.

Current management of latex allergy

Two years after the introduction of a combined initiative of education and glove regulation for the use of nonpowdered gloves, a German study found a marked reduction in latex asthma (Allmers et al. 2002). A recent metaanalysis concluded: Substitution of powdered latex gloves with low protein powderfree NRL gloves greatly reduces NRL allergens, NRL sensitization, and NRL-asthma in healthcare workers. Evidence in support of this statement is ranked SIGN Level 2+, referring to wellconducted case–control or cohort studies with a low risk of confounding, bias or chance, and a moderate probability that the relationship is causal. (Lamontagne et al. 2006)

A latex aeroallergen level of 0.6 ng/m3 has been proposed as a threshold for sensitization and symptoms in HCW (Baur et al. 1998). It remains unclear whether low-protein powderfree gloves will be sufficient or whether total avoidance of latex (as now happens with SB children) will be necessary to prevent ongoing sensitization. Secondary prevention latex avoidance measures allow latexallergic individuals to safely undergo operations in latex-free theaters (Birmingham et al. 1996). Latex-specific IgE decreases with ongoing strict latex avoidance in latex-allergic SB patients (Niggemann & Wahn 2000) and HCW (Allmers et al. 1998; Yagami et al. 2006). Most latex-allergic HCW can safely return to work if they are provided with nonlatex gloves and any latex gloves worn by others are of low protein and powderfree (Turjanmaa et al. 2002). Use of powder-free, low-protein NRL gloves not only reduces HCW symptoms on exposure to NRL but also additionally has a cost benefit in terms of glove purchases and workers’ compensation claims (Korniewicz et al. 2005). However, a note of caution is raised by a study of latex-sensitized anesthetists where latex-specific IgE rose in those sensitized for 18 months or less if powdered latex gloves were still used by surgical staff despite personal allergen avoidance by the anesthetists (Hamilton & Brown 2000).

Specific immunotherapy in latex allergy Current status of specific immunotherapy

The mainstay of current management for latex allergy is allergen avoidance and symptomatic medication. Patients should be informed of the condition and advised on potentially cross-reactive foods and latex-containing products with appropriate alternatives. Medic-Alert bracelets and anaphylaxis management kits are recommended. Specific immunotherapy (SIT) for latex allergy, with its high level of systemic side effects, is generally considered experimental at this time.

Allergen avoidance in latex allergy Rigorous latex allergen avoidance in high-risk populations can prevent primary sensitization. Several studies have now shown success of primary preventive programs in reducing the incidence of latex sensitization and allergy (Tarlo et al. 2001; Cremer et al. 2002; Nieto et al. 2002; Saary et al. 2002).

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Despite the effectiveness of allergen avoidance in the management of latex allergy, there remains a core group of latexallergic individuals, often with severe allergy and associated food allergy, for whom allergen avoidance is insufficient and for whom safe SIT would be desirable. SIT for grass pollen allergy is long-lasting (Durham et al. 1999) and has been shown to prevent progression of allergic rhinitis to asthma (Moller et al. 2002). There have now been several reported studies of latex immunotherapy, including both subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) (reviewed by Sastre & Quirce 2006). One of the first and most important of these studies was a randomized, placebo-controlled trial conducted by Leynadier and colleagues using the Stallergenes LAL preparation (Leynadier et al. 2000). This preparation is

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the standardized extract used widely in Europe and on a restricted basis in Australia for skin testing in the diagnosis of latex allergy (Turjanmaa 1995). The preparation was tested in a multicenter study in which 17 HCW with latex allergy were randomized to SCIT or placebo (Leynadier et al. 2000). The immunotherapy regimen involved a 2-day rush protocol where patients were up-dosed to their maximum tolerated dose with the 10 IR or 100 IR vial of LAL, depending on sensitivity. This was followed by a 12-month maintenance phase of injections at the maximum tolerated dose, initially fortnightly and then monthly. The active treatment group reported a statistically significant decrease in symptoms of rhinoconjunctivitis and urticaria but not of asthma. It is noteworthy that the placebo group had an increased incidence of asthma at the commencement of the study compared with the active group (Leynadier et al. 2000). Although efficacy for the treatment was shown, side effects were noted. These included a local reaction rate of over 40% in the active treatment group compared with 15% in the placebo group. In addition, 15% of injections in the active group induced episodes of rhinitis, 2.7% asthma, 0.6% angioedema and 0.3% systemic anaphylaxis (Leynadier et al. 2000). Most importantly, these reactions occurred just as frequently in the maintenance phase of injections as compared with the up-dosing phase of treatment. A second study of SCIT in latex allergy was reported in 2003 (Sastre et al. 2003). This was a randomized controlled trial performed in 24 subjects with occupational latex allergy and proven respiratory sensitization. The extract used was a standardized extract from ALK-Abello. Subjects were able to tolerate a mean of 11.1 μg of latex protein. As with Leynadier’s study, systemic reactions were extremely common, being observed in 8% of injections and 68.7% of patients. Despite the high rate of reactions, the extract did show efficacy with significant reduction in cutaneous sensitivity and specific bronchial reactivity to latex (Sastre et al. 2003). In contrast to these two studies, a subsequent controlled trial of 12 months latex-specific SCIT in 23 patients with latex rhinoconjunctivitis showed no significant difference in symptom and medication scores between treated and control groups (Tabar et al. 2006). It was suggested that this result may have been due to a low level of symptoms at baseline and a low maintenance dose of therapy. Again there was a high frequency of systemic reactions. The development of new sensitizations has been reported after 6 months of latex immunotherapy, but levels were low and did not correlate with clinical outcome (Sastre et al. 2006). SLIT for allergic disease is associated with a lower risk of adverse events (Khinchi et al. 2004) and thus is attractive for use with potent allergens such as latex. SLIT for latex allergy has been reported in two major studies and several case reports. In the first large study, a nonrandomized open label study in 24 latex allergic subjects, half of the patients underwent a rush immunotherapy protocol with a sublingual latex

Latex Allergy

extract (ALK-Abello) up to a maximum of 50 μg/mL (Patriarca et al. 2002). No side effects were reported and, after 3 months of latex SLIT, subjects could tolerate wearing latex gloves for 6 hours and undergo medical procedures without latexrelated allergic symptoms, whereas control subjects showed no change in their original allergy. A second open label, uncontrolled study with the same extract in 26 subjects confirmed efficacy with an improved glove use test at 5 days and 10 weeks (Cistero Bahima et al. 2004). However, 25% of doses elicited side effects, although these were mainly local and required treatment in only 10% of cases. In a smaller, open study of 10 latex-allergic children with a history of multiple surgical procedures, reduced symptom scores were observed following 1 year of SLIT with no adverse reactions during the rush phase and only two patients showing mild local symptoms during maintenance treatment (Nucera et al. 2006). These studies indicate some of the inherent difficulties with SIT for latex allergy. Firstly, paradoxically, those patients who are the most latex-sensitive, and thus have the greatest potential benefit from desensitization, are most at risk from therapy. Patients with mild or moderate latex allergy may be managed successfully with allergen avoidance measures alone (Bubak 2000), but those with severe allergy may develop symptoms on exposure to even minute quantities of latex protein. Latex is a ubiquitous material within our modernday environment and these severely affected individuals may be put at risk of anaphylaxis through everyday activities in the community where mildly affected individuals have no difficulty (Fiocchi et al. 2001). Studies to date indicate that there is a high risk of anaphylaxis, or at the least severe local side effects with SCIT using crude latex extracts. Thus, highly latex-sensitive individuals may be exposed to considerable risk by immunotherapy using crude extracts. A negative correlation has been observed between Hev b 6.01-specific serum IgE level at baseline and maximum tolerated dose of latex extract in SCIT (Sastre et al. 2006). SLIT appears to offer the promise of reduced side effects, but further studies are needed before it can be widely recommended. At present, SIT with crude extracts can only be considered experimental and must be performed by an experienced allergist in a hospital setting where intensive care backup is readily available. In view of these difficulties, development of novel approaches such as using peptides, hypoallergenic mutants, or DNA vaccines is warranted.

Future prospects for specific immunotherapy Experimental models of latex allergy have been developed to investigate the influence of routes of sensitization, mechanisms of downregulating the adverse immune response to allergens, and evaluation of new specific treatments (Hardy et al. 2003; Hufnagl et al. 2003; Lehto et al. 2003; Barrios et al. 2006; Farzaneh et al. 2006). Recombinant DNA technology has also allowed the expression of latex allergens for both their immunologic characterization and structural modification

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Fig. 53.8 Approach to latex immunotherapy design. (See CD-ROM for color version.)

via genetic engineering as a prelude to safer latex immunotherapy. Disruption of B-cell epitopes with conservation of T-cell epitopes of recombinant allergens offers the hope of abrogated IgE reactivity but maintenance of efficacy through T-cell activation (Fig. 53.8). This approach has been used successfully in a double-blind placebo-controlled clinical trial of recombinant fragments of the major birch pollen allergen, Bet v 1, where immunization with recombinant proteins safely reduced symptoms in birch pollen-allergic individuals, providing “proof of concept” (Cromwell et al. 2004). In order to develop a latex vaccine for use in HCW, allergens that would need to be included would be Hev b 5 and 6. Hev b 6 is a particularly attractive candidate for such approaches because of its highly conserved tertiary structure, major allergen status and pivotal role in the latex–fruit syndrome. Six single-point mutations of Hev b 6.02 resulted in a mutant with decreased binding to Hev b 6.02-specific IgE in vitro and no reaction on skin testing in vivo (Karisola et al. 2004). A further study used site-directed mutagenesis of four cysteine residues in the Hev b 6.02 molecule to disrupt the disulfide bonds which anchor the tertiary structure (Drew et al. 2004) (Fig. 53.9). The resultant molecule showed markedly reduced IgE binding but maintenance of T-cell reactivity, necessary qualities for a candidate vaccine. Another approach which has been used to treat other allergic diseases, particularly cat allergy, is T cell epitope-based peptide immunotherapy (Larche & Wraith 2005). Short linear peptides based on the known protein sequence of major allergens offer the promise of T-cell activation without the ability to cross-link effector cell-bound IgE. T-cell epitopes have been described for Hev b 1, Hev b 3, Hev b 5 and Hev b 6. Most likely, a cocktail of peptides based on the dominant epitopes of relevant major allergens would be required for this approach (reviewed by Rolland et al. 2005). Other approaches such as conjugating immunostimulatory DNA to allergen vaccines have shown promise, notably for ragweed allergy

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C C CCCCCC

Fig. 53.9 Hev b 6.01 comprises two domains, Hev b 6.02 (hevein) and Hev b 6.03 (the C-terminal fragment). These are cleaved naturally within the rubber plant. Seven disulfide bonds stabilize the structure of Hev b 6.01. Recombinant Hev b 6.01 mutants were created by site-directed mutagenesis, progressively substituting cysteine with alanine amino acids to prevent formation of disulfide bonds in the Hev b 6.02 domain. (See CD-ROM for color version.)

(Creticos et al. 2006), but as yet have not been trialed in latex allergy.

Conclusions The latex allergy epidemic has been a remarkable model of allergic disease that has provided valuable insights into occupational allergy and asthma, industrial hygiene, food allergy, anaphylaxis, and immunotherapy. Overall, much progress has been made. The allergens have been clearly defined, the mode of exposure delineated and, finally, through the widespread application of low allergen powder-free latex gloves, rates of new sensitization are falling in the western world. Moreover, recombinant latex allergens have been manufactured and mutants produced as potential components for specific immunotherapy vaccines. Nevertheless, latex allergy remains a health hazard for atrisk groups. Its prevalence is increasing in developing countries and in industries outside healthcare. In addition there is a small core group of severely latex-allergic HCW who may be unable to work in even latex powder-free conditions, and for whom safe immunotherapy would be a major advance. Suitable specific treatment for latex allergy requires further research and development. Diagnostic methods are still imperfect, with skin testing not always readily available, hampering

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decision-making and advice to patients. Finally, further investigation of the latex–fruit syndrome may yield improved treatment of affected individuals and provide a successful model to aid therapy for other more common food allergies such as peanut allergy.

Recommendations The major recommendation arising from a study of the latex allergy epidemic is that all healthcare institutions should now mandate the use of low-allergen, powder-free latex gloves where possible. This is an effective measure to prevent primary sensitization and, in addition, allows most sensitized individuals, and all but the most severely latex-allergic patients, to continue to work in their environment so long as they themselves avoid all latex stringently and wear synthetic gloves. Such individuals must be monitored carefully to ensure that no increase in their sensitization or decrement in lung function occurs. Advances in the development of suitable specific immunotherapy for latex allergy require identification of suitable allergen formulations for safe and effective use and a better understanding of mechanisms for downregulating adverse immune responses to latex to permit optimal delivery of SIT.

Acknowledgments The work of the authors is supported by the National Health & Medical Research Council of Australia and the Alfred Hospital, Melbourne. The authors are members of the Cooperative Research Centre for Asthma and Airways, Australia.

References Aichane, A., Bouayad, Z., el Outmani, A., Afif, H., Trombati, N. & Bahlaoui, A. (1997) [Latex allergy in a hospital setting. Results of a study in Casablanca.] Rev Mal Respir 14, 451–5. Akasawa, A., Hsieh L.S., Martin B.M., Liu, T. & Lin, Y. (1996) A novel acidic allergen, Hev b 5, in latex. Purification, cloning and characterization. J Biol Chem 271, 25389– 93. Alenius, H., Kalkkinen, N., Reunala, T., Turjanmaa, K. & Palosuo, T. (1996) The main IgE-binding epitope of a major latex allergen, prohevein, is present in its N-terminal 43-amino acid fragment, hevein. J Immunol 156, 1618–25. Allmers, H., Kirchner, B., Huber, H., Chen, Z., Walther, J.W. & Baur, X. (1996) [The latency period between exposure and the symptoms in allergy to natural latex. Suggestions for prevention.] Dtsch Med Wochenschr 121, 823– 8. Allmers, H., Brehler, R., Chen, Z., Raulf-Heimsoth, M., Fels, H. & Baur, X. (1998) Reduction of latex aeroallergens and latex-specific IgE antibodies in sensitized workers after removal of powdered natural rubber latex gloves in a hospital. J Allergy Clin Immunol 102, 841– 6.

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Allmers, H., Schmengler, J. & Skudlik, C. (2002) Primary prevention of natural rubber latex allergy in the German health care system through education and intervention. J Allergy Clin Immunol 110, 318– 23. Allmers, H., Schmengler, J. & John, S.M. (2004) Decreasing incidence of occupational contact urticaria caused by natural rubber latex allergy in German health care workers. J Allergy Clin Immunol 114, 347–51. Archambault, S., Malo, J.L., Infante-Rivard, C., Ghezzo, H. & Gautrin, D. (2001) Incidence of sensitization, symptoms, and probable occupational rhinoconjunctivitis and asthma in apprentices starting exposure to latex. J Allergy Clin Immunol 107, 921–3. Arif, S.A., Hamilton, R.G., Yusof, F. et al. (2004) Isolation and characterization of the early nodule-specific protein homologue (Hev b 13), an allergenic lipolytic esterase from Hevea brasiliensis latex. J Biol Chem 279, 23933–41. Axelsson, J.G., Johansson, S.G. & Wrangsjo, K. (1987) IgE-mediated anaphylactoid reactions to rubber. Allergy 42, 46–50. Banerjee, B., Kanitpong, K., Fink, J.N. et al. (2000) Unique and shared IgE epitopes of Hev b 1 and Hev b 3 in latex allergy. Mol Immunol 37, 789–98. Barral, P., Batanero, E., Palomares, O., Quiralte, J., Villalba, M. & Rodriguez, R. (2004) A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies crossreactivity. J Immunol 172, 3644–51. Barrios, C.S., Kurup, V.P., Rickaby, D.A., Henderson, J.D. Jr, Fink, J.N. & Kelly, K.J. (2006) Gastrointestinal exposure to latex antigens induce allergic responses in mice. Int Arch Allergy Immunol 141, 158–67. Baur, X., Chen, Z. & Allmers, H. (1998) Can a threshold limit value for natural rubber latex airborne allergens be defined? J Allergy Clin Immunol 101, 24–7. Beezhold, D. & Beck, W.C. (1992) Surgical glove powders bind latex antigens. Arch Surg 127, 1354–7. Beezhold, D.H., Hickey, V.L. & Sussman, G.L. (2001) Mutational analysis of the IgE epitopes in the latex allergen Hev b 5. J Allergy Clin Immunol 107, 1069–76. Beezhold, D.H., Hickey, V.L., Kostyal, D.A. et al. (2003) Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein. Ann Allergy Asthma Immunol 90, 439–45. Beezhold, D.H., Hickey, V.L., Sutherland, M.F. & O’Hehir, R.E. (2004) The latex allergen hev B 5 is an antigen with repetitive murine B-cell epitopes. Int Arch Allergy Immunol 134, 334–40. Bernstein, D.I., Biagini, R.E., Karnani, R. et al. (2003) In vivo sensitization to purified Hevea brasiliensis proteins in health care workers sensitized to natural rubber latex. J Allergy Clin Immunol 111, 610–16. Birmingham, P.K., Dsida, R.M., Grayhack, J.J. et al. (1996) Do latex precautions in children with myelodysplasia reduce intraoperative allergic reactions? J Pediatr Orthop 16, 799–802. Blanco, C., Carrillo, T., Castillo, R., Quiralte, J. & Cuevas, M. (1994) Latex allergy, clinical features and cross-reactivity with fruits. Ann Allergy 73, 309–14. Blanco, C., Carrillo, T., Ortega, N., Alvarez, M., Dominguez, C. & Castillo, R. (1998) Comparison of skin-prick test and specific serum IgE determination for the diagnosis of latex allergy. Clin Exp Allergy 28, 971–6. Blanco, C., Diaz-Perales, A., Collada, C. et al. (1999) Class I chitinases as potential panallergens involved in the latex–fruit syndrome. J Allergy Clin Immunol 103, 507–13.

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Latex Allergy

Saary, M.J., Kanani, A., Alghadeer, H., Holness, D.L. & Tarlo, S.M. (2002) Changes in rates of natural rubber latex sensitivity among dental school students and staff members after changes in latex gloves. J Allergy Clin Immunol 109, 131–5. Sanchez-Monge, R., Blanco, C., Lopez-Torrejon, G. et al. (2006) Differential allergen sensitization patterns in chestnut allergy with or without associated latex–fruit syndrome. J Allergy Clin Immunol 118, 705–10. Sanz, M.L., Sanchez, G. Gamboa, P.M. et al. (2001) Allergen-induced basophil activation, CD63 cell expression detected by flow cytometry in patients allergic to Dermatophagoides pteronyssinus and Lolium perenne. Clin Exp Allergy 31, 1007–13. Sanz, M.L., Garcia-Aviles, M.C.. Tabar, A.I. et al. (2006) Basophil Activation Test and specific IgE measurements using a panel of recombinant natural rubber latex allergens to determine the latex allergen sensitization profile in children. Pediatr Allergy Immunol 17, 148–56. Sastre, J. & Quirce, S. (2006) Immunotherapy, an option in the management of occupational asthma? Curr Opin Allergy Clin Immunol 6, 96–100. Sastre, J., Fernandez-Nieto, M., Rico, P. et al. (2003) Specific immunotherapy with a standardized latex extract in allergic workers, a double-blind, placebo-controlled study. J Allergy Clin Immunol 111, 985– 94. Sastre, J., Raulf-Heimsoth, M., Rihs, H.P. et al. (2006) IgE reactivity to latex allergens among sensitized healthcare workers before and after immunotherapy with latex. Allergy 61, 206–10. Seppala, U., Palosuo, T., Kalkkinen, N., Ylitalo, L., Reunala, T. & Turjanmaa, K. (2000) IgE reactivity to patatin-like latex allergen, Hev b 7, and to patatin of potato tuber, Sol t 1, in adults and children allergic to natural rubber latex. Allergy 55, 266–73. Slater, J.E. (1989) Rubber anaphylaxis. N Engl J Med 320, 1126–30. Slater, J.E., Vedvick, T., Arthur-Smith, A., Trybul, D.E. & Kekwick, R.G. (1996) Identification, cloning, and sequence of a major allergen (Hev b 5) from natural rubber latex (Hevea brasiliensis). J Biol Chem 271, 25394–9. Soedjanaatmadja, U.M., Hofsteenge, J., Jeronimus-Stratingh, C.M., Bruins, A.P. & Beintema, J.J. (1994) Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged C-terminal sequences. Biochim Biophys Acta 1209, 144–8. Sowka, S., Hsieh, L.S., Krebitz, M. et al. (1998) Identification and cloning of Prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris. J Biol Chem 273, 28091–7. Sowka, S., Hafner, C., Radauer, C. et al. (1999) Molecular and immunologic characterization of new isoforms of the Hevea brasiliensis latex allergen hev b 7, evidence of no cross–reactivity between hev b 7 isoforms and potato patatin and proteins from avocado and banana. J Allergy Clin Immunol 104, 1302–10. Stern, G. (1927) Uberempfindlichkeit gegen Kautschuk als ursache von Urticaria und quinckeschem. Odem Klin Wochenschr 6, 1096–7. Strachan, D.P. (1989) Hay fever, hygiene, and household size. BMJ 299, 1259–60. Subramaniam, A. (1995) The chemistry of natural rubber latex. Immunol Allergy Clin North Am 15, 1– 20. Sunderasan, E., Bahari, A., Arif, S.A., Zainal, Z., Hamilton, R.G. & Yeang, H.Y. (2005) Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4. Clin Exp Allergy 35, 1490–5.

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Allergens

Sussman, G.L., Liss, G.M., Deal, K. et al. (1998) Incidence of latex sensitization among latex glove users. J Allergy Clin Immunol 101, 171–8. Sutherland, M.F., Drew, A., Rolland, J.M., Slater, J.E., Suphioglu, C. & O’Hehir, R.E. (2002a) Specific monoclonal antibodies and human immunoglobulin E show that Hev b 5 is an abundant allergen in high protein powdered latex gloves. Clin Exp Allergy 32, 583–9. Sutherland, M.F., Suphioglu, C., Rolland, J.M. & O’Hehir, R.E. (2002b) Latex allergy, towards immunotherapy for health care workers. Clin Exp Allergy 32, 667–73. Swanson, M.C. & Ramalingam, M. (2002) Starch and natural rubber allergen interaction in the production of latex gloves, a hand-held aerosol. J Allergy Clin Immunol 110 (suppl. 2), S15–S20. Tabar, A.I., Anda, M., Bonifazi, F. et al. (2006) Specific immunotherapy with standardized latex extract versus placebo in latex-allergic patients. Int Arch Allergy Immunol 141, 369–76. Tarlo, S.M., Sussman, G., Contala, A. & Swanson, M.C. (1994) Control of airborne latex by use of powder-free latex gloves. J Allergy Clin Immunol 93, 985– 9. Tarlo, S.M., Easty, A. Eubanks, K. et al. (2001) Outcomes of a natural rubber latex control program in an Ontario teaching hospital. J Allergy Clin Immunol 108, 628– 33. Taylor, J.S. & Erkek, E. (2004) Latex allergy, diagnosis and management. Dermatol Ther 17, 289– 301. Turjanmaa, K. (1995) Natural rubber latex allergy, the European experience. Immunol Allergy Clin North Am 15, 71– 88. Turjanmaa, K. & Reunala, T. (1988) Contact urticaria from rubber gloves. Dermatol Clin 6, 47– 51. Turjanmaa, K., Rasanen, L., Lehto, M., Makinen-Kiljunen, S. & Reunala, T. (1989) Basophil histamine release and lymphocyte proliferation tests in latex contact urticaria. In vitro tests in latex contact urticaria. Allergy 44, 181– 6. Turjanmaa, K., Palosuo, T., Alenius, H. et al. (1997) Latex allergy diagnosis, in vivo and in vitro standardization of a natural rubber latex extract. Allergy 52, 41– 50. Turjanmaa, K., Kanto, M., Kautiainen, H., Reunala, T. & Palosuo, T. (2002) Long-term outcome of 160 adult patients with natural rubber latex allergy. J Allergy Clin Immunol 110 (suppl. 2), S70– S74. Uveges, R.E., Grimwood, R.E., Slawsky, L.D. & Marks, J.G. Jr. (1995) Epidemiology of hand dermatitis in dental personnel. Mil Med 160, 335–8. Valentine, J.P., Kurinczuk, J.J., Loh, R.K. & Chauvel, P.J. (1999) Latex allergy in an Australian population of children and adolescents with spinal dysfunction. Med J Aust 170, 15–18. Valsecchi, R., Leghissa, P., Cortinovis, R., Cologni, L. & Pomesano, A. (2000) Contact urticaria from latex in healthcare workers. Dermatology 201, 127– 31. Valyasevi, M.A., Maddox, D.E. & Li, J.T. (1999) Systemic reactions to allergy skin tests. Ann Allergy Asthma Immunol 83, 132–6. Vandenplas, O., Delwiche, J.P., Evrard, G. et al. (1995) Prevalence of occupational asthma due to latex among hospital personnel. Am J Respir Crit Care Med 151, 54– 60. Wagner, B., Krebitz, M., Buck, D. et al. (1999) Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida. J Allergy Clin Immunol 104, 1084– 92. Wagner, B., Buck, D., Hafner, C. et al. (2001) Hev b 7 is a Hevea brasiliensis protein associated with latex allergy in children with spina bifida. J Allergy Clin Immunol 108, 621–7.

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Wagner, S. & Breiteneder, H. (2002) The latex–fruit syndrome. Biochem Soc Trans 30, 935–40. Wagner, S. & Breiteneder, H. (2005) Hevea brasiliensis latex allergens, current panel and clinical relevance. Int Arch Allergy Immunol 136, 90–7. Wagner, S., Breiteneder, H., Simon-Nobbe, B. et al. (2000) Hev b 9, an enolase and a new cross–reactive allergen from hevea latex and molds. Purification, characterization, cloning and expression. Eur J Biochem 267, 7006–14. Wagner, S., Sowka, S., Mayer, C. et al. (2001) Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen. Int Arch Allergy Immunol 125, 120–7. Wagner, S., Radauer, C., Hafner, C. et al. (2004) Characterization of cross–reactive bell pepper allergens involved in the latex–fruit syndrome. Clin Exp Allergy 34, 1739–46. Wakelin, S.H. & White, I.R. (1999) Natural rubber latex allergy. Clin Exp Dermatol 24, 245–8. Yagami, A., Suzuki, K., Kano, H. & Matsunaga, K. (2006) Follow-up study of latex-allergic health care workers in Japan. Allergol Int 55, 321–7. Yagami, T., Sato, M., Nakamura, A. et al. (1998) Plant defense-related enzymes as latex antigens. J Allergy Clin Immunol 101, 379–85. Yagami, T., Osuna, H., Kouno, M., Haishima, Y., Nakamura, A. & Ikezawa, Z. (2002) Significance of carbohydrate epitopes in a latex allergen with beta-1,3-glucanase activity. Int Arch Allergy Immunol 129, 27–37. Yagami, T., Haishima, Y., Tsuchiya, T., Tomitaka-Yagami, A., Kano, H. & Matsunaga, K. (2004) Proteomic analysis of putative latex allergens. Int Arch Allergy Immunol 135, 3–11. Yeang, H.Y. (2004) Natural rubber latex allergens, new developments. Curr Opin Allergy Clin Immunol 4, 99–104. Yeang, H.Y., Cheong, K.F., Sunderasan, E. et al. (1996) The 14.6 kd rubber elongation factor (Hev b 1) and 24 kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy. J Allergy Clin Immunol 98, 628–39. Yeang, H.Y., Arif, S.A., Raulf-Heimsoth, M. et al. (2004) Hev b 5 and Hev b 13 as allergen markers to estimate the allergenic potency of latex gloves. J Allergy Clin Immunol 114, 593–8. Yeang, H.Y., Hamilton, R.G., Bernstein, D.I. et al. (2006) Allergen concentration in natural rubber latex. Clin Exp Allergy 36, 1078– 86. Yip, L., Hickey, V., Wagner, B. et al. (2000) Skin prick test reactivity to recombinant latex allergens. Int Arch Allergy Immunol 121, 292– 9. Ylitalo, L., Turjanmaa, K., Palosuo, T. & Reunala, T. (1997) Natural rubber latex allergy in children who had not undergone surgery and children who had undergone multiple operations. J Allergy Clin Immunol 100, 606–12. Ylitalo, L., Makinen-Kiljunen, S., Turjanmaa, K., Palosuo, T. & Reunala, T. (1999) Cow’s milk casein, a hidden allergen in natural rubber latex gloves. J Allergy Clin Immunol 104, 177–80. Yunginger, J.W., Jones, R.T., Fransway, A.F., Kelso, J.M., Warner, M.A. & Hunt, L.W. (1994) Extractable latex allergens and proteins in disposable medical gloves and other rubber products. J Allergy Clin Immunol 93, 836–42. Zeiss, C.R., Gomaa, A., Murphy, F.M. et al. (2003) Latex hypersensitivity in Department of Veterans Affairs health care workers, glove use, symptoms, and sensitization. Ann Allergy Asthma Immunol 91, 539– 45.

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Animal Models of Asthma

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Primate Models of Allergic Asthma Charles G. Plopper, Suzette M. Smiley-Jewell, Lisa A. Miller, Michelle V. Fanucchi, Michael J. Evans, Alan R. Buckpitt, Mark V. Avdalovic, Laurel J. Gershwin, Jesse P. Joad, Radhika Kajekar, Shawnessy D. Larson, Kent E. Pinkerton, Laura S. Van Winkle, Edward S. Schelegle, Emily M. Pieczarka, Reen Wu and Dallas M. Hyde

Summary A number of different species of nonhuman primates, primarily macaque monkeys such as the rhesus monkey, have been used as experimental models of allergic airways disease because (i) all the epithelial and mesenchymal components of the walls of intrapulmonary and extrapulmonary conducting airways that are altered in human asthmatics are present in the lungs of adult macaque monkeys; (ii) a significant portion of lung development (which includes differentiation of these components) occurs postnatally in macaque monkeys as it does in humans; (iii) the principal immunologic, pathophysiologic, and histopathologic features of human asthma are found in the chronic experimental disease in macaque monkeys; and (iv) the response to inhaled allergen challenge also shares the same features in human asthmatics and macaque monkeys with chronic allergic airways disease. Among these shared features are positive skin test to allergen, allergen-specific circulating IgE, specific airway responsiveness to allergen as indicated by pulmonary function tests during allergen challenge, shedding of airway epithelial cells into the airway exudate, increased levels of eosinophils and IgE-positivecells in the airway exudate, increased levels of mucins in the airway exudate, nonspecific airway hyperresponsiveness to methacholine or histamine challenge which is elevated by allergen challenge, mucous cell hyperplasia in conducting airways, increased basement membrane zone thickness, subepithelial fibrosis, and migratory leukocyte (eosinophils, lymphocytes, and dendritic cells) accumulation in the airway wall and lumen. The majority of these features are enhanced or altered by exposure to oxidant air pollutants, especially when exposure occurs during postnatal development.

Introduction The purpose of this chapter is to assess the utility of nonhuman primates as models for allergic airways disease (or asthma) in humans. We address the current status of our understanding Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

of a series of critical issues regarding the utility of this model and its appropriateness for defining mechanisms as they relate to disease processes in human airways, including the biology of the airway within the conceptual framework of the epithelial –mesenchymal trophic unit (EMTU), species differences in the organization of the airway wall in adults, species differences in postnatal development of the airways, airway remodeling associated with asthma, and airway-specific responses to inflammatory agents. Macaque monkeys used as models include rhesus (Macaca mulatta), Japanese (Macaca fuscata), stump-tail (Macaca arctoides), and cynomolgus (Macaca fascicularis) with either allergens from naturally occurring infectious agents or known human allergens (see Coffman & Hessel 2005 for review). This review summarizes data from one model, the rhesus monkey, exposed to a known human allergen, housedust mite. Asthma is thought to be caused by a combination of ongoing lung development, genes (not discussed here) and environmental factors resulting in the alteration of the normal development and function of the lung (Holt et al. 2004). The longer period of human lung maturation (prenatally and postnatally) provides ample time for normal lung development to be challenged and perturbed. Examples of environmental factors associated with disturbance of normal postnatal lung growth and development include respiratory syncytial virus (Lemanske 2004), house-dust mite allergen (Sporik et al. 1992; Richardson et al. 2005; Thorne et al. 2005), endotoxin (Thorne et al. 2005), and environmental air pollutants (Tager et al. 1983; Frischer et al. 1999; Peters et al. 1999a,b). Numerous epidemiologic studies have demonstrated an association between children living in major industrialized urban areas, such as regions of Los Angeles or Mexico City, and the development of childhood respiratory diseases and decreased lung function (Romieu et al. 1996; Peters et al. 1999a,b; Calderon-Garciduenas et al. 2003; Gauderman et al. 2004). This review summarizes how we have used the rhesus monkey and a known human allergen, house-dust mite, as a model to address key questions regarding the potential for the development of allergic airways disease in infants and how polluted air environments may influence susceptibility.

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Animal Models of Asthma Vascular compartment

Interstitial compartment

Epithelial compartment

Attenuated fibroblast

Basement membrane

Immune system Nerves and NEB

The concept of the epithelial–mesenchymal trophic unit The concept of the EMTU (Fig. 54.1) was developed as a framework for defining the cellular and metabolic mechanisms regulating the response to injury in a complex biological structure such as the tracheobronchial airway tree (Evans et al. 1999; Holgate et al. 2000). The basis of this concept is that all of the cellular and acellular compartments within the airway wall have a close interaction through a series of extracellular signaling cascades, which establish a dynamic steady-state. Perturbation of one compartment creates an imbalance in all compartments or, more accurately, a metabolic response in one compartment will produce alterations in the other compartments. Each segment, or airway generation, within the branching tracheobronchial airway tree is addressed as a unique biological entity whose properties may differ from those of neighboring branches and the intervening branch-points. The portions of the airways between branchpoints are treated as separate biological entities from each other and from the intervening branch-points. The epithelial compartment of the airway wall comprises surface epithelium and submucosal glands. The interstitial compartment includes the basement membrane zone, fibroblasts, including the attenuated fibroblast sheath beneath the basement membrane, smooth muscle, cartilage, and the vasculature. The nervous compartment includes the nerve processes which interdigitate between the smooth muscle, the subepithelial matrix, and the epithelium. This includes both afferent and efferent limbs of the nervous system and the central regulating neurons in the brainstem. The vascular compartment includes

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Fig. 54.1 Diagrammatic representation of the epithelial mesenchymal trophic unit of the airway wall illustrating the compartments, including epithelium, interstitium, vasculature, immune cells, and nerves. The basement membrane zone is bordered by the attenuated fibroblasts and epithelium. All these compartments are altered in airway remodeling associated with chronic allergic and inflammatory processes in the airways of humans and rhesus monkeys.

capillaries, arterioles, and venules, primarily from the bronchial circulation, and lymphatic vessels. The immunologic compartment includes both inflammatory cells and migratory cells involved in regulation of immune responses. In the steady state, these compartments establish a baseline trophic interaction that is disrupted during acute injury and repair and is reset by successive cycles of injury, inflammation, and repair characteristic of chronic airway diseases. This paradigm for asthma manifests itself as a complete alteration of not only the cellular compartments directly involved in the interface with noxious agents or allergens, but also in marked changes in the other compartments, e.g., induction of myofibroblasts, smooth muscle hypertrophy, interstitial fibrosis, and thickening of the basement membrane zone (Paige & Plopper 1999). The concept that the cellular populations which organize a conducting airway as a biological entity unique to a particular branch of the airway tree emphasizes the number of factors by which they differ in their pathobiological response to toxic injury, including susceptibility to acute injury, the pattern of repair and the development of tolerance. There are two aspects of microenvironment that are critical when defining the pathobiology of the response to injury. First, all the cellular populations in the airway wall are vital for maintaining differentiated function of the epithelium (Van Winkle et al. 1996a). The three-dimensional configuration of the airway, in other words an intact tubular structure including all components of the wall, is necessary and the absence of airway wall integrity results in a transformation of the epithelial populations. The entire repair process after acute injury, including proliferation and migration to reestablish cell density and differentiation of epithelial phenotypes, occurs in vitro in intact airways under normal growth conditions independent of other extraneous

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factors in the animal (Van Winkle et al. 1996b). Secondly, the injury and the accompanying inflammatory response varies markedly by the position of the target cells within the tracheobronchial airway tree and is true for a wide variety of stressors, including oxidant gases (Hyde et al. 1992; Plopper et al. 1998; Paige & Plopper 1999), bioactivated hydrocarbons (Plopper & Hyde 1992; Plopper et al. 1992; Paige et al. 1997) and allergens (Miller et al. 2005). Regardless of the agent or the phenotype of the target cell population, the sensitivity varies widely between proximal and distal airways. Swiss–Webster mouse

Primate Models of Allergic Asthma

Species differences in tracheobronchial airways in adults Architecture As illustrated in Fig. 54.2, the tracheobronchial conducting airways form a complex series of branching tubes (trachea, bronchi and bronchioles) that extend to the gas-exchange area. Figure 54.2 compares the branching pattern in young adult animals: a Swiss–Webster mouse, a Sprague–Dawley Rhesus monkey

Sprague–Dawley rat

Trachea Primary bronchus Terminal bronchiole Intrapulmonary bronchus

Respiratory bronchiole Alveolar duct

Acinus Alveolar sacs Acinus G1 G2

Terminal bronchiole Respiratory bronchiole Rhesus monkey

G2 G1

Respiratory bronchiole

Alveolar duct

Sprague–Dawley rat Swiss–Webster mouse

Fig. 54.2 Comparison of the architecture of the airspaces in the mammalian respiratory system, including trachea, primary bronchi, intrapulmonary bronchi, bronchioles, and the acinus. The organization of the tracheobronchial airways as represented by silicon casts compare architecture in a Swiss– Webster mouse, Sprague–Dawley rat and rhesus monkey. In addition to

differences in size and branching pattern, the transition zone between the most distal conducting airway, the terminal bronchiole, and the gas-exchange area varies between species with an extensive transition zone represented by multiple generations of respiratory bronchioles in primates and a short or single generation of respiratory bronchioles in laboratory rodents.

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Table 54.1 Comparison of species differences in tracheobronchial airway organization. Parameter

Human

Percent of lung

Monkey

Rat

Mouse

1.8

5.7

11

Cartilage in wall

Trachea to distal bronchiole

Trachea to distal bronchiole

Trachea to lobar bronchi

Trachea to lobar bronchi

Nonrespiratory bronchioles

Several generations

Several generations

Several generations

Several generations

Respiratory bronchioles

Several generations

Several generations

None or one

None or one

Generations to alveolarized bronchiole (axial path)

17–21

13–17

13–17

13–17

Branching pattern

Dichotomous

Dichotomous/trichotomous

Monopodial

Monopodial

Compiled from Mercer & Crapo (1987); Mariassy (1992); McBride (1992); Plopper & Hyde (1992); Tyler & Julian (1992); Miller et al. (1993).

rat, and a rhesus monkey. The tracheobronchial airways occupy approximately 2% of the lung volume in the rhesus monkey, and at least 7% in mice and rats. In humans and nonhuman primates, cartilage is found in the walls of the tracheobronchial airways, from the trachea distally to the smallest bronchioles (Table 54.1). In the distal bronchioles of humans and rhesus monkeys, cartilage is restricted to a small zone in the bifurcation area. By contrast, cartilage ends at the lobar bronchus in mice and rats. All species have a significant number of generations of intrapulmonary airways that are very thin-walled and with minimal cartilage, i.e., nonrespiratory bronchioles. The organization of the zone of transition between conducting airways and the gas-exchange area separates the lungs of primates and carnivores from other mammalian species (Fig. 54.2). In primates and carnivores there is an extensive transition zone, with the walls of the distal airways having a mixture of bronchiolar epithelial subpopulations mixed with the alveolar gas exchange area. The average number of branches from the trachea to the bronchioles is approximately the same for most mammalian species (Table 54.1). Branching itself is relatively unique in primates (including humans and nonhuman primates), with branches separating from the parent airway at closer to a 45° angle and being more nearly equal in size and in diameter (i.e., dichotomous or pseudodichotomous branching).

Cellular composition As summarized in Fig. 54.1, the walls of tracheobronchial airways are highly complex cellular structures. All the compartments that comprise the wall are present to varying degrees in all species. Figure 54.3 compares the histologic composition of conducting airways in the adult Swiss–Webster mouse and the adult rhesus monkey. The full extent of the walls of more proximal airways is not illustrated. All the images are at the same magnification. In all species, the interstitium of the tracheal wall contains C-shaped cartilages and there is a band

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of smooth muscle that joins the open end of the cartilages (Table 54.2). The trachea and proximal airways have extensive submucosal glands beneath the epithelium in rhesus monkeys and humans (see Tables 54.2 and 54.4). These glands are present to a variable extent in smaller laboratory species. As emphasized in Fig. 54.3, there is a substantial difference in the amount of epithelium that lines the luminal surface in the trachea. The thickness of the epithelium in the trachea of rhesus monkeys is approximately twice that of mice and rats, and one-half to one-third that in humans (Table 54.2). Other major differences between species are the composition of the epithelium, the density of cells lining the surface, and the proportion of the epithelium occupied by different cell phenotypes (Table 54.2). Mucus cells are a substantial percentage of the airway in primates, but generally not to a substantial extent in the trachea of healthy pathogen-free mice and rats. The proportion of ciliated cells in the epithelium is relatively similar in all species, yet the proportion of basal cells found in surface epithelium varies by species. As would be expected with the differences in secretory cell populations that line the trachea of different species, there is considerable variation in the carbohydrate content of the secretory product (Table 54.3). Primates, in general, have a more heavily sulfated secretory product that is not usually found in laboratory mammals (Table 54.3). As is illustrated in Fig. 54.3, the organization of the tracheobronchial airways varies by airway generation within the airway tree. The largest, most proximal intrapulmonary bronchi have considerable organizational variation between species (Table 54.1). While smooth muscle is present in the walls of all mammalian species, there is a substantial difference in the extent of cartilage found in the lobar bronchus in laboratory mammals and in the distribution of submucosal glands. Epithelium is reduced in thickness in more distal airways compared with the trachea (compare Tables 54.2, 54.4 and 54.5). There also are major differences in the organization of the surface epithelial population, with mucous and basal cells

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Primate Models of Allergic Asthma

Trachea

Trachea

Proximal bronchus

Proximal bronchus

Midlevel airway

Midlevel airway

Respiratory bronchiole

Respiratory bronchiole

10 mm

10 mm Monkey

Mouse

Fig. 54.3 Differences in cellular composition of the airway wall of rhesus monkeys and Swiss–Webster mice. The thickness of the epithelium in the trachea of rhesus monkeys is approximately twice that of mice and rats and one-third that of humans. The abundance of epithelial cells, including ciliated cells (arrowhead) and secretory cells (*), both mucous goblet and Clara cells, varies by airway generation. The bronchioles of rhesus monkeys have an extensive smooth muscle portion that is arranged in large bundles and is interspersed with extensive connective tissue not observed in rodents. Bar equals 10 mm.

predominating in primates and Clara cells being the principal nonciliated cell population in other laboratory animals (Tables 54.4 and 54.5). In the most distal conducting airways, the bronchioles, the major differences between species are

related primarily to the epithelial surface lining (Table 54.5). In laboratory mammals, the Clara cell is the primary secretory cell phenotype, and there are no mucous cells. The extent of basal cells in the epithelium varies by the extent of

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Table 54.2 Comparison of species differences in cells of the trachea. Parameter

Human

Monkey

Rat

Mouse

Wall Smooth muscle Cartilage Submucosal glands

Present Present Present

Present Present Present

Present Present Present

Present Present Present (proximal one-third)

50–100 303 ± 20 9

20–30 181 ± 51 17 <1 <1 33 42 8

7–10 148 ± 3 <1

11–14 215 <1 <1 49 39 10 1

Epithelium Thickness (mm) Cells/mm b.m. Mucous goblet cells (%) Serous cells (%) Clara cells (%) Ciliated cells (%) Basal cells (%) Other cells (%)

49 33 –

27 20 4

Compiled from Harkema et al. (1991); Hyde et al. (1992); Mariassy (1992); Mercer et al. (1994); Plopper et al. (1998); St George et al. (1988).

alveolorization. The bronchioles of rhesus monkeys have an extensive smooth muscle portion, which is arranged in large bundles and is interspersed with extensive connective tissue not generally observed in laboratory mammals (Fig. 54.3).

Postnatal development During the first year of postnatal development in the rhesus monkey, the airways themselves are in active phases of growth, the epithelium, basement membrane, and smooth muscle of the airways are differentiating, the mucosal immune system of the airways is being established, the nerve networks within the airway are being established, the capillary beds in the airway and the parenchyma are forming, and alveolarization of the parenchyma is active.

Table 54.3 Comparison of species differences in carbohydrate content of Clara (C), serous (S), and mucous (M) cells in tracheal epithelium. Parameter

Human

Monkey

Rat

Mouse

Abundance

+++

++

+⁄−

+⁄−

Periodic acid–Schiff

+ (M)

+ (M)

+ (S) + (M)

+ (M) ±/− (C)

Alcian blue

+

+ (M)

− (S) + (M)

+ (M) − (C)

High iron diamine

+ and − (M)

+ and − (M) and − (M)

− (S) − (M)

− (M) − (C)

Compiled from St George & Wang (1992); St George et al. (1988); Harkema et al. (1991).

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Airway growth The number of conducting airways is completely developed by birth in humans, but airway size increases with lung growth (McBride 1992; Beech et al. 2000). Comparison of lung morphology in macaques and humans shows that there are similarities in segmental arrangement, structure, and branching pattern of airways, arterial structure and in arterial changes after birth (McBride 1992; Tyler & Julian 1992). Although there are differences in the number of lobes, the number of generations of different types of airways and the overall structure in the monkey is more similar to that in

Table 54.4 Comparison of species differences in proximal intrapulmonary airways. Parameter

Human

Monkey

Rat

Mouse

Wall Smooth muscle Cartilage Submucosal glands

Present Present Present

Present Present Present

Present Absent Absent

Present Absent Absent

40–50 ? 10 3

27 175 15 5

13 116 <1 20 <1 53 14 12

8–16 109 <1 <1 61 36 <1 2

Epithelium Thickness (mm) Cells/mm b.m. Mucous goblet cells (%) Serous cells (%) Clara cells (%) Ciliated cells (%) Basal cells (%) Other (%)

37 32 18

47 32 2

Compiled from Plopper et al. (1989, 1998); St George et al. (1988); Harkema et al. (1991); Hyde et al. (1992); Mariassy (1992).

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Table 54.5 Comparison of species differences in terminal bronchioles. Parameter

Human

Monkey

Rat

Mouse

Wall Cartilage Smooth muscle Submucosal glands

Absent (bifurcation) Present Absent

Absent (bifurcation) Present Absent

Absent Present Absent

Absent Present Absent

? ?

? ?

35

∼ 20

52 <1 13

~ 50 ~ 10 ∼5

5–8 ? 0 0 35–60 65–40 <1 0

7–8 ? 0 0 60–80 40–20 <1 0

Epithelium Thickness (mm) Cells/mm Mucous goblet (%) Serous cells (%) Clara cells (%) Ciliated cells (%) Basal cells (%) Other (%)

Compiled from Harkema et al. (1991); Plopper & Hyde (1992); Tyler & Julian (1992); Miller et al. (1993); Mercer et al. (1994); Plopper & ten Have-Opbroek (1994); Plopper et al. (1998).

human than is the structure of the lung of other laboratory animals (Tyler & Julian 1992). In the transition zone, the area where the conducting airways end and the gas-exchange area begins, humans and rhesus monkeys have numerous generations of respiratory bronchioles. In humans, as terminal bronchioles are formed prenatally and only increase in size postnatally, we would expect that the number of respiratory bronchioles would also be formed by birth (Beech et al. 2000). Terminal bronchioles in rhesus monkeys increase by one-third in diameter and by twice in length between 1 and 6 months of age (Tran et al. 2004a).

Epithelial differentiation Epithelial differentiation (especially the secretory cell types and glandular elements) occurs postnatally for both rhesus monkeys (Plopper et al. 1986a,b; Van Winkle et al. 2004) and humans (Bucher & Reid 1961; Thurlbeck et al. 1961). The secretory cell population differentiates in a proximal-to-distal pattern, with nearly mature cells lining proximal airways and immature cells in more distal portions. Glandular mucous cells and serous cells differentiate at different times during prenatal and postnatal development and through a different sequence of events (Plopper et al. 1986b; Van Winkle et al. 2004). This may occur on a different timetable in animals housed in ultraclean environments (Van Winkle et al. 2004).

Basement membrane zone The basement membrane zone (BMZ) develops postnatally in the airways of nonhuman primates (Evans et al. 2002a; Van Winkle et al. 2004) and, apparently, in humans as well (Brewster et al. 1990; Roche et al. 1989). Changes include reorganization of collagen types I, III, and V; a threefold to fourfold increase in thickness; shifts in the deposition of per-

lecan (Fig. 54.1) and its storage of fibroblast growth factor (FGF)-2 (Evans et al. 2002a).

Smooth muscle Smooth muscle develops from myoblasts present in the mesenchyme of the developing lung (Gaultier & Girard 1980; Sward-Comunelli et al. 1997). The bulk of smooth muscle growth and differentiation in large bronchi occurs after birth, where it increases fourfold (Hislop & Haworth 1989; Tran et al. 2004a). The process observed in the rhesus monkey as the airways grow postnatally involves changes in orientation of smooth muscle bundles and increases in total bundle number within an airway branch (Hislop & Haworth 1989); this maintains the relative density of bundle content. Bundle size is also constant and therefore decreases in relation to airway size as each airway grows. Preterm infants with chronic lung disease have significantly increased amounts of smooth muscle in the larger airways (Sward-Comunelli et al. 1997).

Mucosal immune system Newborn infant airways without pulmonary lesions contain few leukocytes. It has been suggested that in healthy infants, leukocyte “seeding” within the airways must be initiated following birth and exposure to environmental stimuli; this process likely continues during the first 2 years of life (Grigg & Riedler 2000). There are no significant changes in lavage cell profile from children between 3 and 8 and 8 and 14 years, suggesting that accumulation of resident immune/inflammatory cells within airways is essentially complete after the age of 3 years (Heaney et al. 1996). In a small study of 18 healthy children (3 months to 10 years), age significantly correlated with the frequency of lymphocytes within lavage, although there was no correlation with other lavage cell phenotypes

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(Riedler et al. 1995). Several groups have reported striking differences in the CD4/CD8 ratio of lavage lymphocytes from children (3 months to 16 years of age) compared with adults; CD4/CD8 ratios ranged from 0.6– 0.8 for children in contrast with 1.8–2.7 for adults (Hunninghake et al. 1981; Costabel et al. 1985; Clement et al. 1987; Ratjen et al. 1995). The lower CD4/ CD8 values for bronchoalveolar lavage in children appear to be due to a higher number of CD8 cells (Ratjen et al. 1995).

Bronchial vasculature Although the postnatal development of the bronchial arterial system has not been described in primates, we do know that in adult rhesus monkeys the subgross anatomic distribution is similar to humans, but there are differences in the supply to the pleura similar to other laboratory animals that have a thin pleura (McLaughlin et al. 1961, 1966).

Innervation Along with anatomic development processes of the lung, the respiratory nervous system proliferates and innervates the developing respiratory tract (Sparrow et al. 2004). We believe that normal sensory innervation of the airway appears first in the large conducting airway and then progresses down the airway tree to more distal airways. In midlevel airways in the rhesus monkey, innervation of the epithelial compartment is a postnatal event. Soon after birth, components of the wall that express markers for nervous tissue consist primarily of neuroendocrine-like cells. By 6 months of age this distribution is lost and a fine arborization of nerve processes can be detected (Larson et al. 2004) .

Alveolar development Stages of human lung development show alveolarization by formation of secondary interalveolar septa from about 36 weeks of gestation to about 1–2 years of age and microvascular maturation by remodeling of interalveolar septa and restructuring of the capillary bed from birth to 2–3 years of age (Thurlbeck 1982; Burri 1997). The majority of alveoli are produced postnatally in humans to reach the adult number of about 450 million alveoli (Ochs et al. 2004). Alveoli increase in number but not volume throughout all the postnatal developmental/growth stages, infant (1–12 months), juvenile (12–24 months), adolescent (2– 4 years), and young adult (4–8 years) (Hyde et al. 2007).

Experimental model for environmental postnatal airways disease To define how exposure to allergen, especially during postnatal life, alters development of the airway EMTU and the establishment of mucosal immunity and how environmental contaminants may influence the ability of developing lungs to resist the impact of allergens and other contaminants, we

1194

have developed a model of allergic airways disease in the adult rhesus monkey (Schelegle et al. 2001) which reflects all the criteria used by the National Heart, Lung, and Blood Institute’s National Asthma Education and Prevention Program Clinical Practice Guidelines. Based on the NIH NHLBI definition we found that asthma could be produced in rhesus monkeys following exposure to house-dust mite allergen (HDMA) (Schelegle et al. 2001). The monkeys developed a positive skin test for HDMA, with elevated levels of IgE in the serum and IgE-positive cells within the tracheobronchial airway walls. The animals exhibited airway obstruction after the inhalation of aerosolized allergen that was associated with cough, rapid shallow breathing, and decreased arterial oxygen saturation, all of which were reversible by treatment with aerosolized albuterol. Further, serum histamine concentrations were elevated in sensitized monkeys following allergen exposure. In sensitized monkeys, shed epithelial cells were detectable in bronchoalveolar lavage immediately after allergen aerosol challenge. As with human asthmatics, the majority of the shed cells were ciliated cells. Immune cells, especially eosinophils, increased markedly in abundance in airway exudates and bronchoalveolar lavage. There were also elevations in CD25 expression on CD4+ lymphocytes in both lavage and serum. The animals also developed nonspecific airways responsiveness, which was reflected as a fourfold reduction in the dose of histamine aerosol required to produce a 150% increase in airway resistance. There was marked mucous cell hyperplasia accompanied by general epithelial hypertrophy in both intrapulmonary and extrapulmonary conducting airways. In most of the intrapulmonary bronchi, the BMZ was markedly thickened. This thickened BMZ appears to be characteristic of conducting airways only in primate asthma models and plays a key role in modulating signaling within the airway wall. There were marked accumulations of eosinophils in both the epithelial and subepithelial matrix compartments as well. All these histologic features were focal and distributed throughout the conducting airway trees. The experimental protocol we used to evaluate the susceptibility of developing lungs in postnatal rhesus monkeys includes beginning exposure early during the postnatal period (30 days after birth) and ending at approximately 6 months of age (Schelegle et al. 2003b). The standard 5-month exposure protocol involves repeated cycles of exposure to ozone, 5 days in succession followed by 9 days in filtered air, at a concentration resembling that of Mexico City (0.5 ppm, 8 hours/day). The allergen exposure, using HDMA, is for approximately 3 days (2 hours per day) followed by 11 days of filtered air. These 3 days of allergen exposure are the last 3 days of ozone exposure. Animals are housed in a filtered air environment and then exposed to ozone, house-dust mite, or a combination for up to 11 cycles. To evaluate the potential for recovery, we have followed the 5 months of exposure with another 6 months in filtered air up to an age of 12 months.

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*

250

Primate Models of Allergic Asthma

12

*

*

% Eosinophils

Baseline Raw, cmH2O/L/s

10 200

150

100

8 6 4 2

50 0 0

FA

HDMA

O3

HDMA + O3

Fig. 54.4 Comparison of baseline airway resistance (Raw) in infant rhesus monkeys (120 days of age) following six cycles of exposure to filtered air (FA), house-dust mite allergen (HDMA), ozone (O3) or both (HDMA + O3). Airway resistance was obtained by the transfer impedance method (Schelegle et al. 2001, 2003b). Mean ± SE. *, P < 0.05 compared to FA; †, P < 0.05 compared to HDMA. (From Schelegle et al. 2003a, with permission.)

12 10 8 6 4

HDMA O3 Treatment

HDMA + O3

Fig. 54.6 Comparison of the percentage of eosinophils found in bronchoalveolar lavage from infant rhesus monkeys (180 days of age) following 11 cycles of exposure to filtered air (FA), house-dust mite allergen (HDMA), ozone (O3) or both (HDMA + O3). Mean ± SE. *, P < 0.05 compared to FA; †, P < 0.05 compared to HDMA. (From Plopper et al. 2007, with permission.)

Airway growth

2 0

FA

et al. 2003b). Ozone with allergen more than doubles baseline resistance. A similar pattern occurs with nonspecific airways hyperresponsiveness to histamine (Fig. 54.5); the combination of ozone and allergen greatly elevates the responsiveness. We have observed increases in eosinophils in bronchoalveolar lavage in response to ozone but not to HDMA in the infant rhesus monkeys (Schelegle et al. 2003b). However, the combination exposure greatly increases the abundance of eosinophils in bronchoalveolar lavage (Fig. 54.6).

14

EC150 Raw (mg/mL)

†

* FA

HDMA O3 Treatment

HDMA + O3

Fig. 54.5 Comparison of the concentration of histamine required to produce a 150% increase in airway resistance (EC150 Raw) in infant rhesus monkeys (180 days of age) following 10 cycles of exposure to filtered air (FA), house-dust mite allergen (HDMA), ozone (O3) or both (HDMA + O3). Airway resistance was obtained by the transfer impedance method (Schelegle et al. 2001, 2003b). Mean ± SE. *, P < 0.05 compared to FA. (From Plopper et al. 2007, with permission.)

Each group also has animals that are housed in a filtered air environment for the entire period of this study.

Response of rhesus monkeys to exposure Physiologic and immunologic responses We have found in the infant animals that allergen exposure alone increases baseline airway resistance (Fig. 54.4) (Schelegle

To evaluate the extent of remodeling, or alterations in airway development and growth, we use a sampling method which requires exposing conducting airway trees in fixed lungs by microdissection, counting each branch and identifying each sample taken from the lung based on its branching history (Plopper 1990). One of the most striking features we have observed with remodeling of the airways in infant rhesus monkeys is changes in normal growth. The airways increase by one-third in diameter and by twice in length between 1 and 6 months in age (Tran et al. 2004a). The growth pattern of distal airways is exacerbated to a mild extent by exposure to allergen alone, but is very markedly changed by exposure to ozone or a combination of allergen and ozone. The combination exposure inhibits growth in diameter yet promotes lengthwise growth. This combination results in longer, narrower airways with higher intrinsic theoretically calculated resistance (Tran et al. 2004a). Exposure to ozone causes infant monkeys to have a decrease in the number of conducting airway generations between the trachea and the gas-exchange area, as represented by the location of the most proximal respiratory bronchiole (Fig. 54.7) (Fanucchi et al. 2006). We have counted the number of generations to the first respiratory

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Airway path to RB (number of branches)

25 20

Airway epithelium

FA HDMA O3 HDMA + O3

15 * *

* 10

*

5 0

R. cranial

R. middle

Fig. 54.7 Comparison of the number of generations of branching from the trachea to the respiratory bronchiole (RB) in infant rhesus monkeys. The axial path of the intrapulmonary airways in the right cranial (R. cranial) and right middle (R. middle) lobes were exposed by microdissection and the branching counted directly in the fixed lung. Rhesus monkeys (180 days of age) were exposed to 11 cycles of filtered air (FA), house-dust mite allergen (HDMA), ozone (O3) or both (HDMA + O3) Mean ± 1 SD. *, P < 0.05 compared to FA. (From Plopper et al. 2007, with permission.)

bronchiole in four different lobes of fixed lungs and found a reduction of as many as six generations of conducting airways following exposure to ozone with or without allergen (Fig. 54.7). A recovery period of an additional 6 months of filtered air does not have a substantial impact on recouping the number of airway generations which have been lost.

Exposure to ozone and allergen during postnatal development tends to modify the conducting airways epithelium by increasing the number and size of mucous cells, and by incorporating a large number of eosinophils into the luminal epithelium (Schelegle et al. 2003b). After 6 months in filtered air, the now 12-month-old animals have reestablished almost a steady-state mucous cell composition in proximal airways but the organization of the airway epithelium is significantly disrupted. In more distal airways the increase in mucous cell abundance is more dramatic in response to exposure to allergen, ozone alone, or the combination (Schelegle et al. 2003b; Fanucchi et al. 2006). This also involves increases in mucous and nonmucous cell hyperplasia in respiratory bronchioles and a disruptive change in the relationship between alveolar tissue and nonalveolarized aspects of the airway wall (Fanucchi et al. 2006).

Basement membrane zone As illustrated in Fig. 54.8, exposure to allergen accelerates the rate at which this material is deposited, such as collagen I (Evans et al. 2002b, 2003, 2006). The epithelial contact surface is a smooth uniform boundary for attachment of basal and columnar cells but the side facing the attenuated fibroblast layer becomes highly irregular. There is a marked change in the chemical composition of the BMZ and in the types

FA

HDMA

O3

O3 + HDMA

1196

Fig. 54.8 Histologic comparison of the basement membrane zone (BMZ) in the trachea of infant rhesus monkeys (180 days of age) following 11 cycles of exposure to filtered air (FA), house-dust mite allergen (HDMA), ozone (O3) or both (O3 + HDMA). The BMZ is identified by indirect immunofluorescence (red) of collagen I. Compared with filtered-air animals, the BMZ of HDMA-exposed animals was much thicker, that of ozone-exposed animals much thinner, and that of animals exposed to both was highly irregular. Bar equals 10 mm. (From Plopper et al. 2007, with permission.) (See CD-ROM for color version.)

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of signaling molecules that are stored there after allergen exposure. In contrast, exposure to oxidant stress via ozone inhalation greatly hampers the development of the BMZ. The combination exposure (ozone plus allergen) completely disrupts differentiation of the BMZ, with many areas being highly thickened and irregular, such as occurs with exposure to allergen alone, and other areas being extremely thin as observed with exposure to ozone alone. During the period between 6 and 12 months of age, the BMZ doubles in size and increases in complexity. Six months of exposure to filtered air subsequent to a 5-month exposure to ozone, allergen, or both does not provide sufficient time for the normal developmental and growth processes of the BMZ to compensate for the previous disruption (Evans et al. 2004).

Epithelial innervation Five months of exposure to ozone, allergen, or both disrupts the organization with a marked reduction in the density and distribution of nerve fibers and an elevation of neuroendocrine-like cells (Fig. 54.9) (Larson et al. 2004). The normal process of further growth and development after 6 months of age is for the density of the nerve fibers to be reduced as the airway increases in size (Kajekar et al. 2006). Six months of exposure to filtered air subsequent to a 5-month exposure to ozone, allergen, or both results in a more than doubling of the density of the nerve fibers within the epithelium and an increase in the neuroendocrine-like cells as compared with unexposed animals (Kajekar et al. 2006).

Airway smooth muscle Exposure to ozone, allergen or both disrupts this developmental process (Fig. 54.10) (Tran et al. 2004b). In terminal bronchioles of filtered-air control monkeys, the majority of the smooth muscle bundles (∼ 74%) around the airway were oriented at an angle less than 15° from perpendicular to the long axis of the airway and only a very small percentage (∼ 3%) of bundles were found at an angle greater than 30° (Fig. 54.10). In ozone-exposed monkeys, only 43% of the terminal bronchiolar smooth muscle bundles were oriented at an angle less than 15° from perpendicular to the long axis of the airway, but there were 12% of bundles oriented at an angle of greater than 30°. There was no significant difference in terminal bronchiolar smooth muscle bundle thickness or abundance between filtered air and ozone-exposed monkeys, respectively. An opposite pattern of smooth muscle bundle orientation was present in the most proximal respiratory bronchiole. Only half of the smooth muscle bundles in the proximal respiratory bronchioles of filtered-air control monkeys were oriented around the airway at an angle less than 15° from perpendicular to the long axis of the airway, and ∼ 16% of bundles were oriented at an angle of greater than 30°. In the first respiratory bronchiole of ozone-exposed monkeys, ∼ 65% of the smooth muscle bundles were oriented at an angle less than 15° from perpendicular to the long axis of the airway and only 5% of

Primate Models of Allergic Asthma

the bundles oriented at an angle of greater than 30°. As in the terminal bronchioles, however, there were no significant differences in smooth muscle bundle thickness or abundance in the first respiratory bronchioles between filtered-air control monkey and ozone-exposed monkeys, respectively (Fanucchi et al. 2006). Six months of exposure to filtered air subsequent to a 5-month exposure to ozone, allergen, or both does not provide sufficient time for the normal developmental and growth processes to adjust bundle size and orientation to match the configuration in unexposed age-matched controls.

Airway vasculature Recent studies have described an increase in bronchovascular density in patients with asthma and asthma-like disease (Hoshino et al. 2001). Bronchial vascular surface area and density were significantly increased at midlevel airways in monkeys exposed to allergen (Avdalovic et al. 2006). Gene expression of vascular endothelial growth factor (VEGF) was also significantly increased at distal airway levels in allergenexposed monkeys. The changes in vascular surface area and density were not as significant in ozone-exposed monkeys, and the combination of ozone and allergen led to similar changes as those seen in allergen-exposed only monkeys (Avdalovic et al. 2006). These results imply that allergen may stimulate an increase in bronchovascular density and that ozone exposure is not additive.

Airway immune system The organization and distribution of immune and inflammatory cells within the infant tracheobronchial airway wall is very specific for the airway branch in which it is evaluated and exposure history (Miller et al. 2005, 2006). Figure 54.6 shows that eosinophil frequency within airway lavage is significantly elevated in response to combined exposure to HDMA and ozone with no significant differences between animal groups exposed to filtered air (sensitized or nonsensitized), allergen alone, and ozone alone (Schelegle et al. 2003b). Comparatively, the density of eosinophil populations within epithelial and interstitial compartments does not necessarily reflect abundance within the airway lumen relative to exposure history. Within the epithelial and interstitial compartments, the volume of eosinophils from allergen-exposed animals is significantly elevated as compared with filtered-air exposed animals, although exposure to both resulted in less abundance than exposure to allergen alone. To add another level of complexity, the distribution of eosinophil populations within epithelial and interstitial compartments is variable, depending on where within the airway tree they are located. After aerosol challenge, serum levels of histamine are elevated in sensitized monkeys. Sensitized monkeys also exhibit increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4+ lymphocytes (Schelegle et al. 2001). In HDMA-challenged monkeys, the

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FA

O3 + HDMA

FA + 6 mo

O3 + HDMA + 6 mo

Fig. 54.9 Comparison of the distribution of nerve fibers and neuronal cells in the epithelium of midlevel airways of infant rhesus monkeys. The nerve fibers and neuronal cells were identified in whole mounts of airways exposed by microdissection using indirect immunofluorescence and an antibody to PGP 9.5. In 6-month-old animals exposed to filtered air for 5 months (FA), nerve fibers were relatively evenly distributed throughout the surface epithelium. In contrast, animals of the same age exposed to ozone and allergen for 11 cycles (O3 + HDMA) had markedly reduced epithelial innervation and clusters of positive epithelial cells. In 1-year-old animals

1198

exposed to filtered air for 11 cycles, nerve fibers that were much reduced in density and distribution maintained the same interwoven pattern after 6 more months of filtered air (FA + 6 mo). In contrast, in 1-year-old animals after 6 months’ exposure to filtered air subsequent to 11 cycles of exposure to ozone and allergen (O3 + HDMA + 6 mo), the intraepithelial innervation was nearly twice as dense and found throughout the epithelial surface interspersed with positive epithelial cells. (From Plopper et al. 2007, with permission.)

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FA

Primate Models of Allergic Asthma

O3 + HDMA

Fig. 54.10 Comparison of the organization of smooth muscle fibers in the proximal respiratory bronchioles of infant rhesus monkeys (180 days of age) following 11 cycles of exposure to filtered air (FA) or both housedust mite allergen and ozone (O3 + HDMA). The smooth muscle fibers are identified in whole mounts of airways exposed by microdissection by epifluorescence using Alexa 568 phalloidin (red), a probe for filamentous actin. Following exposure, smooth muscle bundle thickness increased and orientation was altered. (From Plopper et al. 2007, with permission.) (See CD-ROM for color version.)

volume of CD1a+ dendritic cells, CD4+ T-helper lymphocytes and CD25+ cells, IgE-positive cells, eosinophils, and proliferating cells are significantly increased in the airways. All these cell types accumulate within airways in unique patterns of distribution, suggesting compartmentalized responses with regard to trafficking. Although cytokine mRNA levels are elevated throughout the conducting airway tree of HDMAchallenged animals, the distal airways (terminal and respiratory bronchioles) exhibit the most pronounced upregulation (Miller et al. 2005).

Acknowledgments Development of the rhesus monkey as a model for allergic airways disease was the product of the interactions of all faculty and staff members of the Respiratory Diseases Unit at the California National Primate Research Center, whose members, in addition to the authors of the present paper, include the following: B.K. Tarkington, V.J. Wong, W.F. Walby, J.M. Bric, T.R. Duvall, K.S. Kott, D.R. Morin, A.J. Weir, S.J. Nishio and N.K. Tyler. The support of Primate Services at the California National Primate Research Center for animal handling, care, and coordination and veterinary care, especially the efforts of Dr L.L. Brignolo, Dr K.L. Christe, S.M. Davis and B.E. Rodello were critical to this study and are gratefully acknowledged. The authors thank A.W. Chang for organization and preparation of the manuscript. The work from the University of California at Davis was supported by National Institutes of Health grants NIEHS P01ES00628, NIEHS P01 ES11617 and NCRR RR00169. The University of California at Davis is a National Institute of Environmental Health Sciences Center for Environmental

Health Sciences (ES05707), which supported core facilities used in this study.

References Avdalovic, M.V., Putney, L.F., Schelegle E.S. et al. (2006) Vascular remodeling is airway generation-specific in a primate model of chronic asthma. Am J Respir Crit Care Med 174, 1069–76. Beech, D.J., Sibbons, P.D., Howard, C.V. & van Velzen, D. (2000) Terminal bronchiolar duct ending number does not increase postnatally in normal infants. Early Hum Dev 59, 193–200. Brewster, C.E., Howarth, P.H., Djukanovic, R., Wilson, J., Holgate, S.T. & Roche, W.R. (1990) Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Molec Biol 3, 507–11. Bucher, U. & Reid, L.M. (1961) Development of the mucus-secreting elements in human lung. Thorax 16, 219–25. Burri, P.H. (1997) Postnatal development and growth. In: Crystal, R.G., West, J.B., Weibel, E.R. & Barnes, P.J., eds. The Lung: Scientific Foundations. Lippincott-Raven Publishers, Philadelphia, pp. 1013– 26. Calderon-Garciduenas, L., Mora-Tiscareno, A., Fordham, L.A. et al. (2003) Respiratory damage in children exposed to urban pollution. Pediatr Pulmonol 36, 148–61. Clement, A., Chadelat, K., Masliah J. et al. (1987) A controlled study of oxygen metabolite release by alveolar macrophages from children with interstitial lung disease. Am Rev Respir Dis 136, 1424–8. Coffman, R.L. & Hessel, E.M. (2005) Nonhuman primate models of asthma. J Exp Med 201, 1875–9. Costabel, U., Bross, K.J., Ruhle, K.H., Luhr, G.W. & Matthys, H. (1985) Ia like antigens on T-cells and their subpopulations in pulmonary sarcoidosis and in hypersensitivity pneumonitis. Am Rev Respir Dis 131, 337–42. Evans, M.J., Van Winkle, L.S., Fanucchi, M.V. & Plopper, C.G. (1999) The attenuated fibroblast sheath of the respiratory tract epithelialmesenchymal trophic unit. Am J Respir Cell Mol Biol 21, 655–7.

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Evans, M.J., Fanucchi, M.V., Van Winkle, L.S. et al. (2002a) Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone. Am J Physiol 283, L1263– L1270. Evans, M.J., Van Winkle, L.S., Fanucchi, M.V. et al. (2002b) Fibroblast growth factor-2 in remodeling of the developing basement membrane zone in the trachea of infant rhesus monkeys sensitized and challenged with allergen. Lab Invest 82, 1747–54. Evans, M.J., Fanucchi, M.V., Baker, G.L. et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. Am J Physiol 285, L931–L939. Evans, M.J., Fanucchi, M.V., Baker, G.L. et al. (2004) The remodelled tracheal basement membrane zone of infant rhesus monkeys after 6 months of recovery. Clin Exp Allergy 34, 1131– 6. Evans, M.J., Fanucchi, M.V. & Plopper, C.G. (2006) The basement membrane zone in asthma. Curr Respir Med Rev 2, 331–7. Fanucchi, M.V., Schelegle, E.S., Baker, G.L. et al. (2004) Mucosal delivery of immunostimulatory DNA attenuates airway remodeling in allergic monkeys. Am J Resp Crit Care Med 170, 1153–7. Fanucchi, M.V., Plopper, C.G., Evans, M.J. et al. (2006) Cyclic exposure to ozone alters distal airway development in infant rhesus monkeys. Am J Physiol 291, L664–L650. Frischer, T., Studnicka, M., Gartner, C. et al. (1999) Lung function growth and ambient ozone: a three-year population study in school children. Am J Respir Crit Care Med 160, 390–6. Gauderman, W.J., Avol, E., Gilliland, F. et al. (2004) The effect of air pollution on lung development from 10 to 18 years of age. N Engl J Med 351, 1057– 67. Gaultier, C. & Girard, F. (1980) Normal and pathological lung growth: structure-function relationships. Bull Eur Physiopathol Respir 16, 791–842. Grigg, J. & Riedler, J. (2000) Developmental airway cell biology. The “normal” young child. Am J Respir Crit Care Med 162, S52–S55. Harkema, J.R., Mariassy, A.T., St George, J.A., Hyde, D.M. & Plopper, C.G. (1991) Epithelial cells of the conducting airways: a species comparison. In: Farmer, S.G. & Hay, D.W.P., eds. The Airway Epithelium: Physiology, Pathophysiology and Pharmacology. Marcel Dekker, New York, pp. 3– 39. Heaney, L.G., Stevenson, E.C., Turner, G. et al. (1996) Investigating paediatric airways by non-bronchoscopic lavage: normal cellular data. Clin Exp Allergy 26, 799– 806. Hislop, A.A. & Haworth, S.G. (1989) Airway size and structure in the normal fetal and infant lung and the effect of premature delivery and artificial ventilation. Am Rev Respir Dis 140, 1717–26. Holgate, S.T., Davies, D.E., Lackie, P.M., Wilson, S.J., Puddicombe, S.M. & Lordan, J.L. (2000) Epithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol 105, 193–204. Holt, P.G., Sly, P.D., Martinez, F.D. et al. (2004) Drug development strategies for asthma: in search of a new paradigm. Nat Immunol 5, 695–8. Hoshino, M., Takahashi, M. & Aoike, N. (2001) Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogen immunoreactivity in asthmatic airways and its relationship to angiogenesis. J Allergy Clin Immunol 107, 295– 301. Hunninghake, G.W. & Crystal, R.G. (1981) Mechanisms of hypergammaglobulinemia in pulmonary sarcoidosis. Site of increased antibody production and role of T lymphocytes. J Clin Invest 67, 86–92.

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Hyde, D.M., Hubbard, W.C., Wong, V., Wu, R., Pinkerton, K. & Plopper, C.G. (1992) Ozone-induced acute tracheobronchial epithelial injury: relationship to granulocyte emigration in the lung. Am J Respir Cell Mol Biol 6, 481– 97. Hyde, D.M., Blozis, S.A., Avdalovic, M.V. et al. (2007) Alveoli increase in number but not size from birth to adulthood in rhesus monkeys. Am J Physiol 293, L570–L579. Kajekar, R., Pieczarka, E.M., Smiley-Jewell, S.M., Schelegle, E.S., Fanucchi, M.V. & Plopper, C.G. (2006) Early postnatal exposure to allergen and ozone leads to hyperinnervation of the pulmonary epithelium. Respir Physiol Neurobiol 155, 55–63. Larson, S.D., Schelegle, E.S., Walby, W.F. et al. (2004) Postnatal remodeling of the neural components of the epithelial-mesenchymal trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen. Toxicol Appl Pharmacol 194, 211–20. Lemanske, R.F. (2004) Viral infections and asthma inception. J Allergy Clin Immunol 114, 1023–6. McBride, J.T. (1992) Architecture of the tracheobronchial tree. In: Parent, R.A., ed. Treatise on Pulmonary Toxicology: Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 49–61. McLaughlin, R.L., Tyler, W.S. & Canada, R.O. (1961) A study of subgross pulmonary anatomy in various mammals. Am J Anat 108, 149– 65. McLaughlin, R.F., Tyler, W.S. & Canada, R.O. (1966) Subgross pulmonary anatomy of the rabbit, rat, and guinea pig, with additional notes on the human lung. Am Rev Respir Dis, 94, 380–7. Mariassy, A. T. (1992) Epithelial cells of trachea and bronchi. In: Parent, R.A. ed., Treatise on Pulmonary Toxicology: Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 63–76. Mercer, R.R. & Crapo, J.D. (1987) Three-dimensional reconstruction of the rat acinus. J Appl Physiol 63, 785–94. Mercer, R.R., Russell, M.L., Roggli, V.L. & Crapo, J.D. (1994) Cell number and distribution in human and rat airways. Am J Respir Cell Mol Biol 10, 613–24. Miller, F.J., Mercer, R.R. & Crapo, J.D. (1993) Lower respiratory tract structure of laboratory animals and humans: dosimetry implications. Aerosol Sci Technol 18, 257–71. Miller, L.A., Hurst, S.D., Coffman, R.L. et al. (2005) Airway generationspecific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys. Clin Exp Allergy 35, 894–906. Miller, L.A., Gerriets, J.E., Stovall, M.Y. et al. (2006) Ozone modulates immune cell activation and trafficking in aeroallergen challenged infant monkeys. AJRCCM, submitted. Ochs, M., Nyengaard, J.R., Jung, A. et al. (2004) The number of alveoli in the human lung. Am J Respir Crit Care Med 169, 120–4. Paige, R. & Plopper, C. (1999) Acute and chronic effects of ozone in animal models. In: S.T. Holgate, J.M. Samet, H.S. Koren & R.L. Maynard (eds): London. Air Pollution and Health. Academic Press, pp. 531–57. Paige, R., Wong, V. & Plopper, C. (1997) Dose-related airwayselective epithelial toxicity of 1-nitronaphthalene in rats. Toxicol Appl Pharmacol 147, 224–33. Peters, J.M., Avol, E., Gauderman, W.J. et al. (1999a) A study of twelve Southern California communities with differing levels and types of air pollution. II. Effects on pulmonary function. Am J Respir Crit Care Med 159, 768–75. Peters, J.M., Avol, E., Navidi, W. et al. (1999b) A study of twelve Southern California communities with differing levels and types

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of air pollution. I. Prevalence of respiratory morbidity. Am J Respir Crit Care Med 159, 760–7. Plopper, C.G. (1990) Structural methods for studying bronchiolar epithelial cells. In: Gil, J., ed. Models of Lung Disease: Microscopy and Structural Methods. Marcel Dekker, New York, pp. 537–59. Plopper, C.G. & Hyde, D.M. (1992) Epithelial cells of bronchioles. In: Parent, R.A., ed. Treatise on Pulmonary Toxicology. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 85–92. Plopper, C.G. & ten Have-Opbroek, A.A.W. (1994) Anatomical and histological classification of the bronchioles. In: Epler, G.R., ed. Diseases of the Bronchioles. Raven Press, New York, pp. 15–25. Plopper, C., Alley, J. & Weir, A. (1986a) Differentiation of tracheal epithelium during fetal lung maturation in the rhesus monkey, Macaca mulatta. Am J Anat 175, 59–72. Plopper, C.G., Weir, A.J., Nishio, S.J., Cranz, D.L. & St George, J.A. (1986b) Tracheal submucosal gland development in the rhesus monkey, Macaca mulatta: ultrastructure and histochemistry. Anat Embryol 174, 167–78. Plopper, C.G., Heidsiek, J.G., Weir, A.J., St George, J.A. & Hyde, D.M. (1989) Tracheobronchial epithelium in the adult rhesus monkey: a quantitative histochemical and ultrastructural study. Am J Anat 184, 31– 40. Plopper, C., Macklin, J., Nishio, S., Hyde, D. & Buckpitt, A. (1992) Relationship of cytochrome P-450 activity to clara cell cytotoxicity III. Morphometric comparison of changes in the epithelial populations of terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral administration of napthalene. Lab Invest 67, 553– 65. Plopper, C.G., Hatch, G.E., Wong, V. et al. (1998) Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys. Am J Respir Cell Mol Biol 19, 387–99. Plopper, C.G., Smiley-Jewell, S.M., Miller, L.A. et al. (2007) Asthma/ allergic airways disease: Does postnatal exposure to environmental toxicants promote airway pathobiology? Tox Path 35, 97–110. Ratjen, F., Bredendiek, M., Zheng, L., Brendel, M. & Costabel, U. (1995) Lymphocyte subsets in bronchoalveolar lavage fluid of children without bronchopulmonary disease. Am J Respir Crit Care Med 152, 174– 8. Richardson, G., Eick, S. & Jones, R. (2005) How is the indoor environment related to asthma? Literature review. J Adv Nurs 52, 328–39. Riedler, J., Grigg, J., Stone, C., Tauro, G. & Robertson, C.F. (1995) Bronchoalveolar lavage cellularity in healthy children. Am J Respir Crit Care Med 152, 163– 8. Romieu, I., Meneses, F., Ruiz, S. et al. (1996) Effects of air pollution on the respiratory health of asthmatic children living in Mexico City. Am J Respir Crit Care Med 154, 300–7. Roche, W.R., Beasley, R., Williams, J.H. & Holgate, S.T. (1989) Subepithelial fibrosis in the bronchi of asthmatics. Lancet i, 520– 4. Schelegle, E.S., Gershwin, L.J., Miller, L.A. et al. (2001) Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae). Am J Pathol 158, 333– 41. Schelegle, E.S., Walby, W.F., Alfaro, M.F. et al. (2003a) Repeated episodes of ozone inhalation attenuates airway injury/repair and release of substance P, but not adaptation. Toxicol Appl Pharmacol 186, 127– 42.

Primate Models of Allergic Asthma

Schelegle, E.S., Miller, L.A., Gershwin, L.J. et al. (2003b) Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys. Toxicol Appl Pharmacol 191, 74–85. Sparrow, M.P., Weischselbaum, M., Tollent, J., McFawn, P.K. & Fischer, J.T. (2004) Development of the airway innervation. In: Harding, R., Pinkerton, K.E. & Plopper, C.G., eds. The Lung: Development, Aging and the Environment. Elsevier Academic Press, London, pp. 33–544. Sporik, R., Chapman, M.D. & Platts-Mills, T.A. (1992) House dust mite exposure as a cause of asthma. Clin Exp Allergy 22, 897–906. St George, J.A. & Wang, S. (1992) Secretory glycoconjugates of trachea and bronchi. In: Parent, R.A., ed. Treatise on Pulmonary Toxicology. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 77–83. St George, J.A., Harkema, J.R., Hyde, D.M. & Plopper, C.G. (1988) Cell populations and structure/function relationships of cells in the airways. In: Gardner, D.E., Crapo, J.D. & Massaro, E.J., eds. Toxicology of the Lung. Raven Press, New York, pp. 71–102. Sward-Comunelli, S.L., Mabry, S.M., Truog, W.E. & Thibeault, D.W. (1997) Airway muscle in preterm infants: changes during development. J Pediatr 130, 570–6. Tager, I.B., Weiss, S.T., Munoz, A., Rosner, B. & Speizer, F.E. (1983) Longitudinal study of the effects of maternal smoking on pulmonary function in children. N Engl J Med 309, 699–703. Thorne, P.S., Kulhankova, K., Yin, M., Cohn, R., Arbes, S.J. Jr & Zeldin, D.C. (2005) Endotoxin exposure is a risk factor for asthma: the national survey of endotoxin in United States housing. Am J Respir Crit Care Med 172, 1371–7. Thurlbeck, W.M. (1982) Postnatal human lung growth. Thorax 37, 564–71. Thurlbeck, W., Benjamin, B. & Reid, L. (1961) Development and distribution of mucous glands in the fetal human trachea. Br J Dis Chest 55, 54–64. Tran, M.U., Weir, A.J., Fanucchi, M.V. et al. (2004a) Smooth muscle development during postnatal growth of distal bronchioles in infant rhesus monkeys. J Appl Physiol 97, 2364–71. Tran, M.U., Weir, A.J., Fanucchi, M.V. et al. (2004b) Smooth muscle hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen. Clin Exp Allergy 34, 1627– 33. Tyler, W.S. & Julian, M.D. (1992) Gross and subgross anatomy of lungs, pleura, connective tissue septa, distal airways, and structural units. In: Parent, R.A., ed., Treatise on Pulmonary Toxicology: Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 37–48. Van Winkle, L.S., Buckpitt, A.R., Plopper, C.G. (1996a) Maintenance of differentiated murine clara cells in microdissected airway cultures. Am J Respir Cell Mol Biol 14, 586– 98. Van Winkle, L.S., Isaac, J.M. & Plopper, C.G. (1996b) Repair of naphthalene-injured microdissected airways in vitro. Am J Respir Cell Mol Biol 15, 1– 8. Van Winkle, L.S., Fanucchi, M.V., Miller, L.A. et al. (2004) Epithelial cell distribution and abundance in rhesus monkey airways during postnatal lung growth and development. J Appl Physiol 97, 2355– 63.

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Airway Remodeling in Small Animal Models Clare M. Lloyd

Summary

Airway remodeling

Animal models have been used extensively to unravel the complexities of the many features of asthma in humans. Allergic asthma is a complex disease incorporating airway inflammation as well as changes in airway function. In addition, airway remodeling is increasingly recognized to be a serious consequence of chronic asthma. Although airway eosinophilia and changes in lung function have been modeled extensively in small rodents using a variety of protocols, the structural changes occurring in the lung have only recently been investigated. Investigators have manipulated a variety of different protocols and systems to try to mimic the main pathophysiologic features of the human disease. In this chapter the main systems that have been used to identify the immunologic and pathophysiologic mechanisms that underlie the asthma phenotype are reviewed. The recent efforts to improve models by trying to replicate the structural remodeling that occurs in the lung as a consequence of chronic allergen-driven inflammation are highlighted. The effects of different allergens as well as protocols for sensitization and challenge are discussed. The use of animal models has enabled us to highlight particular pathways and has given us the opportunity to determine the functional contribution of individual pathways to disease parameters. In addition it has been possible to manipulate disease progression to determine whether pathophysiologic features of disease are dependent on each other.

In addition to the airway inflammation and hyperreactivity that is characteristic of asthma, structural changes are also evident within the airways (Bousquet et al. 2000). This phenomenon is termed “airway remodeling” and is thought to occur as a result of an imbalance in regeneration and repair mechanisms resulting in the abnormal regulation of extracellular matrix components (Elias et al. 1999). Remodeling in the lung has been classified as an increase in airway wall thickness, due to multiple factors (Jeffery et al. 1989; Busse et al. 1999). Subepithelial fibrosis is a distinctive feature of airway remodeling and contributes to thickened airway walls due to the deposition of collagen types I, III, and V along with other extracellular matrix proteins such as tenascin and laminin. An increase in myocyte muscle mass is also characteristic of airway remodeling in asthma and may be due to myofibroblast proliferation which may also lead to further collagen production (Brewster et al. 1990). Excessive mucus secretion from hyperplastic goblet cells is a feature of the asthmatic airway and may lead to occlusion of the airways (Wanner 1990). The combination of these processes is thought to lead to airway narrowing and therefore reduced lung function (Fig. 55.1) (Jeffery et al. 1989; Busse et al. 1999). Animal models of the acute allergic response to inhaled antigens have been widely studied in order to elucidate the mechanisms leading to the development of inflammation and airway hyperresponsiveness (AHR) during asthma. In particular many different protocols have been employed to promote sensitization to protein antigens, such as chicken egg ovalbumin (OVA), in rodents, generally mice (summarized in Fig. 55.2). After peripheral sensitization, via the intraperitoneal, intradermal, or subcutaneous route, and subsequent challenge via the airways, either by inhalation or by intranasal instillation, mice exhibit airway hyperreactivity to stimuli such as methacholine. Changes in lung function are accompanied by recruitment of a range of inflammatory cells, predominantly eosinophils but also T cells, to the airway lumen and lung parenchyma. In addition, the majority of models elicit a classic Th2 disease profile, with production of Th2 cytokines such as interleukin (IL)-4, IL-5 and IL-13 together with

Introduction Mouse models of allergic airways disease have long been used to investigate mechanisms of airway inflammation and airway hyperreactivity following allergen challenge. In more recent years, however, investigators have tried to model the more chronic structural changes that occur during the chronic disease in humans. These recent advances are discussed.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Airway Remodeling in Small Animal Models

GENES? ENVIRONMENT? INFECTION?

Mucus production

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Fig. 55.1 Development of airway remodeling in asthma. (See CD-ROM for color version.)

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Fig. 55.2 Summary of mouse models of airway remodeling. (See CD-ROM for color version.)

increased serum production of antigen-specific IgE and IgG1a. However, the clinical relevance of these models to human asthma has been questioned, as the majority involve relatively short-term exposure to aerosolized antigen via the airways (usually 1–2 weeks), and many do not show the chronic inflammatory and epithelial changes or the mucosal inflammation characteristic of human asthma. Moreover, the inflammation and hyperreactivity that are observed generally resolve after

Remodeling

the cessation of allergen challenge. However, efforts have been made to model the chronic aspects of the human disease in rodents. Investigators have tried to incorporate the features of airway remodeling, i.e., deposition of extracellular matrix, changes in epithelial cell hyperplasia, smooth muscle cell proliferation, production of fibrogenic growth factors, mucus production, and matrix dysregulation, in new models of allergic airway disease in mice.

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Animal Models of Asthma ment of airway inflammation and hyperreactivity. In general, repeated allergen challenge over protracted periods is necessary for development of structural abnormalities of the airways. Some of the protocols used to elicit allergen-induced airway remodeling are summarized in Fig. 55.3.

Active immunization models Immunization models rely on the generation of a peripheral immune response before delivery of an antigen, in order to mimic the allergic response to exogenous/innocuous stimuli. This protocol involves sensitization with the allergen before a challenge phase where the allergen is introduced to the target organ, in this case the lung, intranasally as an aerosol or via the trachea. This basic protocol induces pulmonary inflammation, generally in conjunction with an increase in circulating IgE levels. However, there are countless variations to this protocol that have profound consequences for the range of pathophysiologic features developed. This method has been used with a variety of different antigens and protocols, varying from complex microorganisms to simple proteins and chemicals. While all these methods ultimately produce pulmonary inflammation, the type, degree of inflammation, and time-scale of pathophysiology is widely variable. In particular, the protocols required to generate significant airway remodeling are different from those necessary for develop-

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Selection of allergen Allergens used to initiate allergic airways disease range from simple protein antigens to complex microorganisms. The immune response to these different allergens varies and thus affects the ensuing tissue pathology. For example, complex microorganisms such as parasites have a much higher number and range of antigenic epitopes available during T-cell receptor priming compared with those for a soluble protein antigen. These and other factors are important when comparing the effect of particular antigens in a protocol.

Protein allergens Soluble protein antigens are widely used to elicit allergic pulmonary inflammation, and range from simple proteins such as OVA to complex environmentally relevant antigens

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Fig. 55.3 Summary of some of the chronic allergen challenge protocols used to elicit airway remodeling.

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such as cockroach or house-dust mite proteins. The immune response to these antigens is relatively controlled and reproducible, as a defined amount of antigen can be delivered at a particular site. Thus, it is perhaps easier to establish a more stable model than if using an intact biological organism. The most commonly used protein antigen is chicken egg OVA, the use of which models late-phase events such as eosinophilia and, in some protocols, AHR in vivo. OVA has the advantage of reliably inducing in mice antigen-specific IgE responses that are largely Th2-dependent. The majority of investigators have found that sensitization and subsequent challenge with OVA results in a significant increase in the number of eosinophils and lymphocytes to both the peribronchiolar tissue and the bronchiolar lavage. These increases occur in conjunction with airway hyperreactivity to methacholine as well as significantly raised levels of Th2-type cytokines, IL-4, IL-5, and IL-13 in the lung, and serum IgE levels. In order to model structural changes that occur in the lung of chronic asthmatics, investigators have manipulated these acute allergen challenge protocols by using prolonged challenge regimens. In comparison with the shorter acute models (generally several days of allergen challenge), the more chronic models incorporate weeks or months of allergen challenge in sensitized mice. These prolonged models generally result in less inflammation in the lung than the acute models, although eosinophilia is still prominent in the airways and peribronchiolar areas. Importantly, inflammation is accompanied by features of airway remodeling, including increased matrix deposition an increase in smooth muscle cell layer, and goblet cell hyperplasia (Temelkovski et al. 1998; Henderson et al. 2002; Leigh et al. 2002; Cho et al. 2004; McMillan & Lloyd 2004). The use of this type of model by a range of investigators has been successful in defining the role that individual cells and immune pathways play in mediating the inflammatory responses and, more recently, development of airway remodeling. Although all the protocols use the same allergen, the protocols vary in terms of route of administration, the use of adjuvant and the frequency of challenge. The effects of these different regimens on development of pathophysiologic symptoms are discussed below. Other investigators have tried to establish models using more environmentally relevant antigens, such as those proteins derived from cockroaches or house-dust mites. Sensitivity to cockroach antigen is a common problem in inner cities and for those living in crowded lower-socioeconomic areas (Rosenstreich et al. 1997). Intratracheal delivery of cockroach antigens to sensitized mice results in allergen-specific airway eosinophilia concomitant with significantly altered airway physiology (Campbell et al. 1998). Multiple doses of cockroach allergen over a prolonged period also resulted in the development of structural changes to the airway, including peribronchiolar collagen deposition and mucus production (Berlin et al. 2006). Similarly, sensitization and acute challenge of mice with the house-dust mite Dermatophagoides farinae has been

Airway Remodeling in Small Animal Models

found to elicit pulmonary inflammation (Coyle et al. 1996; Yasue et al. 1998; Yu et al. 1999). Intranasal challenge with D. farinae in previously sensitized mice induces pulmonary edema, inflammatory cell recruitment to the lung, eosinophilia, production of cytokines, and AHR (Yu et al. 1999). This eosinophilia was also found to be CD4+ T cell-dependent.

Fungal allergens Allergic airway inflammation may occur after sensitization from spores from fungi such as Aspergillus or Candida (Kauffman et al. 1995; Pacheco et al. 1998). Patients may develop an allergic eosinophilia, or in the case of Aspergillus fumigatus two different forms: as asthma with increased serum IgE titers, or as hypersensitivity pneumonitis with increased serum IgG and low IgE titers (Kurup & Kumar 1991). This pathology can be replicated in mice by sensitization and intratracheal challenge with A. fumigatus, inducing pulmonary eosinophilia, airway IL-4 and IL-5 production, and increased serum IgE titers in conjunction with heightened AHR (Grunig et al. 1997; Shibuya et al. 1999). Moreover, persistent goblet cell hyperplasia together with airway fibrosis and transforming growth factor (TGF)-β expression were observed after airway challenge with fungal spores in A. fumigatus-sensitized mice (Hogaboam et al. 2000). This latter model has been used to define roles for various cytokines and chemokine receptors in the development of airway remodeling in chronic fungal allergic airways disease (Blease et al. 2000a,b, 2001a,b; Schuh et al. 2002a,b).

Aspects of allergen challenge There is a wealth of data to show that sensitization/challenge models reproduce facets of the human asthmatic condition; however, subtle differences in the basic protocol can have drastic effects on the development of pathophysiology, and the interpretation of results becomes critical. This is especially important when inhibitory reagents or genetically modified animals are used to outline the potential functional importance of selected molecules. The route of sensitization with allergen, the inclusion of an adjuvant at sensitization, the dose of allergen used at sensitization and challenge, and the genetic background of the mice have all been found to impact on the development of acute inflammation (reviewed in Lloyd et al. 2001); however, it is less clear how these aspects affect development of airway remodeling. A summary of protocols used to elicit chronic allergen-induced pathology is depicted in Fig. 55.3. The majority of studies have used OVA as an allergen and have immunized at least once peripherally followed by multiple intranasal or aerosol doses of soluble OVA. The regimens last from 4 weeks to 6 months, but all involve intermittent allergen challenge over a protracted period. All these regimens result in increased matrix deposition in peribronchiolar areas, mucus production, epithelial cell hyperplasia, eosinophilic inflammation (although to a lesser degree than that seen in acute protocols), and changes in lung function.

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Animal Models of Asthma

Dose A major factor in the elicitation of an effective T-cell response is in the dose of antigen used in the priming stages. As shown in Fig. 55.3 the amount of OVA used at priming varies 10fold but elicits similar pathology. The amount of OVA given during the allergen challenge is also variable, but results in remarkably similar pathology. However, one study argues that chronic inhalational OVA challenge results in widespread pulmonary parenchymal inflammation or eventual down-regulation of inflammation and airway hyperreactivity (Kumar & Foster 2002). These authors argue that if sensitized mice are exposed by inhalation to carefully controlled mass concentrations of aerosolized allergen, this will minimize the chance of inducing parenchymal inflammation. They found that challenge of sensitized BALB/C mice with 10–20 mg/m3 of aerosolized OVA in a whole-body inhalation exposure box for 30 min/day for 3 days per week resulted in airway specific inflammation and remodeling (Temelkovski et al. 1998). Further analysis revealed that this regimen results in many of the features of chronic asthma in humans: presence of intraepithelial eosinophils, chronic inflammation of the lamina propria, including evidence of a Th2-biased humoral response, AHR, remodeling indicated by epithelial hypertrophy, subepithelial fibrosis, and goblet cell hyperplasia (Foster et al. 2000; Kumar et al. 2000, 2002). However, others using the traditional methods of exposure do not report excessive parenchymal inflammation (Leigh et al. 2002; McMillan & Lloyd 2004). In contrast to the study by Kumar, where pathology is determined in tracheal sections, we and others have looked directly at the bronchial airways. In fact, a recent study has described involvement of the proximal as well as the distal airways after long-term OVA challenge in sensitized mice (Wegmann et al. 2005). Goblet cell hyperplasia and subepithelial fibrosis occurred throughout the bronchial tree. These observations were made in conjunction with persistent bronchial hyperresponsiveness and progressive airflow limitation. One prediction had been that prolonged challenge with OVA would result in induction of tolerance and downregulation of inflammation. However, the majority of investigators have found that although the degree of inflammation is less than that observed at acute stages of disease, eosinophilic inflammation is still apparent after chronic challenge (Wegmann et al. 2005). The degree of inflammation is less but significantly higher than in the control mice. In addition the nature of the inflammatory infiltrates change, with a less profound eosinophilia and higher mononuclear component of infiltrates (McMillan & Lloyd 2004). One particular criticism of mouse models has been that the degree of eosinophilia is exaggerated compared with that in the human disease. We have also found that if T cells are isolated from lung draining lymph nodes after long-term allergen challenge in vivo, significant proliferative responses to OVA stimulation in vitro are retained (Kearley & Lloyd, unpublished data), thus arguing against the establishment of tolerance. The

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key seems to be intermittent challenge with allergen: repeated cycles of allergen stimulation with short periods of recovery are necessary to elicit prolonged inflammation and induction of remodeling. This idea is reinforced by the recent discovery that protective regulatory T cell responses are only maintained with continued allergen challenge (Strickland et al. 2006).

Genetic background Asthma is a complex clinical syndrome that is understood to have a significant genetic component. One can argue that an advantage of using an animal model is that most strains of mice are inbred and are housed in clean environments, which may be beneficial when considering the role of a particular pathway, cell or molecule in isolation. However, it is important to consider that innate tissue responses, as well as primary immune responses are strain dependent. Differences in the strain of mouse used for particular experiments has resulted in some contradictory results in the past (Drazen et al. 1996). One particular facet of the pulmonary allergic response that seems to be genetically restricted is AHR. Not only does allergen-induced AHR vary between different strains of mouse, but it also seems that native AHR is dependent on the background strain of the mouse, consistent with the hypothesis that AHR is a heritable trait (reviewed by Drazen et al. 1999). Several studies have undertaken to compare AHR after acute allergen challenge and the majority have determined that the A/J strain shows the greatest degree of airway responsiveness, while the C57BL/6J, SJL/ and C3H/ HeJ strains are the least responsive (Levitt et al. 1990; Chiba et al. 1995; Wills-Karp & Ewart 1997). Another study contrasts the responses of two commonly used strains in the literature (Zhang et al. 1997). Several OVA-induced allergen challenge protocols were employed to determine the importance of the route of allergen administration and genetic background in modulating the physiologic, inflammatory, and immunologic features characteristic of allergen-induced asthma. C57BL/6 mice showed significantly decreased responses compared with BALB/c mice for all parameters of allergic pulmonary disease examined, with the exception of airspace eosinophilia. The two strains also demonstrated intrinsic differences in airway mechanics, with C57BL/6 mice showing lower basal dynamic compliance than did BALB/c mice. In agreement with earlier studies naïve C57BL/6 mice required higher doses of methacholine to achieve a 50% decrease from the basal value for either lung conductance or dynamic compliance than did BALB/c mice. Following allergen provocation BALB/c mice demonstrated greater hyperreactivity than C57BL/6 mice for both of these parameters. Given that AHR is likely genetically restricted, it is also highly probable that development of airway remodeling also has a genetic component. Although anecdotal evidence suggests that development of airway remodeling is restricted to a proportion of even severe asthmatics, there have been no linkage analysis studies using remodeling as an outcome.

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However, mapping studies have highlighted chromosome 5q31–q33 as it contains the cytokine gene cluster including IL-4, IL-5, IL-9, and IL-13, as well as other growth factors and receptors implicated in asthma pathology (Postma et al. 1995). It is of interest that transgenic models developed using tissue-specific promoters to drive lung expression of IL-9 and IL-13 in particular result in airway remodeling as well as the other common features of the allergic syndrome (Temann et al. 1998; Zhu et al. 1999). Linkage disequilibrium studies identified multiple polymorphisms in the ADAM33 (A disintegrase and metalloproteinase 33) gene that are associated with asthma (Van Eerdewegh et al. 2002). Haplotypes comprised of polymorphisms within the ADAM33 gene were significantly associated with asthma in the case–control samples as well as in family based association tests. ADAM33 is a member of the ADAM subgroup of the zinc-dependent metalloproteinase superfamily, and is a very plausible candidate gene for tissue remodeling in asthma. The gene has been found to be expressed by fibroblasts and smooth muscle cells, as well as epithelial cells in some patients (Lee, J.Y. et al. 2006). In addition, polymorphic variations in ADAM33 predict impaired early life functions (Simpson et al. 2005) and single nucleotide polymorphisms in ADAM33 have been linked with accelerated decline in lung function in the general population (van Diemen et al. 2005). Although its precise function is not fully understood it has similarities to other ADAM genes that possess proteolytic activity and suggest that ADAM33 may play a role in airway remodeling. The development of a mutant mouse either overexpressing ADAM33 or genetically deficient in ADAM33 is eagerly awaited and might reveal the in vivo function of this molecule in development of asthma pathology, including airway remodeling. One study has investigated the effect of genetics on the development of airway remodeling by assessing the responses of commonly used strains of mice to airway allergen challenge in the absence of systemic sensitization (Shinagawa & Kojima 2003). Using this protocol, persistent AHR, eosinophilic inflammation, collagen deposition, and airway wall thickening were all found to be strain-dependent. Changes were minimal in the commonly used BALB/c strain and absent in C57BL/6 and C3H/HeJ mice. Interestingly, the strain that gave consistent remodeling responses to airway intranasal challenge (rather than inhalational) was the A/J mouse which has been shown to exhibit marked methacholine-induced AHR that is independent of allergen sensitization and challenge (De Sanctis et al. 1995). Quantitative locus analysis of the AHR revealed that this naive AHR is conferred by a major locus on A/J chromosome 2, and is an interacting locus on chromosome 6 (Ackerman et al. 2005). Interestingly, chromosome 2 also contains the mouse ortholog of ADAM33.

usually via the peritoneum but also sometimes subcutaneously (Cho et al. 2004). Thereafter, pulmonary inflammation is induced by local challenge to the lung with soluble allergen. Some investigators have used an aerosol to deliver allergen, while others deposit allergen in the lungs intranasally. Both these methods have been used successfully to deliver allergen over prolonged periods in order to promote airway remodeling. There does not seem to be much difference in the degree of pathology caused, although aerosol delivery might arguably cause more intense inflammation than intranasal delivery. However, in one study designed to investigate the development of remodeling in several commonly used strains of mice determined that intranasal delivery of OVA to nonsensitized A/J mice resulted in airway inflammation and remodeling (Shinagawa & Kojima 2003). This was not observed in BALB/c or two other commonly used mouse strains. Moreover, these results were only obtained if the allergen was given intranasally but not by inhalation implying that, under some circumstances, route of delivery is important. All of these studies emphasize the heterogeneity of the response to allergen and the importance of a range of factors in development of pathophysiologic outcome. One of the most valid criticisms of mouse models of allergic asthma has been the fact that it is necessary to immunize the mice systemically, often in the presence of an adjuvant such as aluminum hydroxide (alum) in order to induce inflammation and also remodeling. This has proved necessary because repeated airway challenge of mice with OVA in the absence of prior sensitization has been shown to lead to tolerance (McMenamin & Holt 1993; McMenamin et al. 1994; van Halteren et al. 1997; Stampfli et al. 1998). However, a model has been developed which has eliminated the need for systemic sensitization using an environmental allergen, housedust mite. Johnston and colleagues determined that prolonged intranasal administration of house-dust mite extract resulted in severe and persistent eosinophilia, AHR, and Th2-type inflammation (Johnson et al. 2004). Moreover, mice developed airway remodeling, as shown by goblet cell hyperplasia, collagen deposition, and increased contractile elements. Importantly, although the airway inflammation had resolved by 9 weeks after cessation of house-dust mite exposure, there was only partial resolution of the AHR and no improvement in the airway remodeling. Further investigation revealed that the airway pathology elicited by exposure to intranasal housedust mite was mediated by granulocyte–macrophage colonystimulating factor (GM-CSF) (Cates et al. 2004). Furthermore, exposure to intranasal house-dust mite facilitates a lung microenvironment that allows induction of inflammation in response to an otherwise innocuous antigen such as OVA (Fattouh et al. 2005).

Route of administration

Persistence of remodeling changes

The allergen sensitization models all rely on a primary sensitization phase whereby the mouse is primed with the antigen,

A major criticism of acute models of airway inflammation in mice has been the fact that the pathophysiologic symptoms

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disappear with the cessation of allergen challenge. However, in some of the more recent chronic challenge models, investigators have established that the features of airway remodeling have persisted even in the absence of further allergen challenge (Leigh et al. 2002; Kumar et al. 2004a; McMillan & Lloyd 2004; Wegmann et al. 2005). In contrast there is disagreement between different models as to the persistence of airway inflammation in the absence of allergen challenge. In some models sustained mononuclear and eosinophilic inflammation is reported several weeks after the cessation of allergen challenge (McMillan & Lloyd 2004; Wegmann et al. 2005), while other protocols result in a return to baseline eosinophilia without continued allergen challenge (Leigh et al. 2002; Kumar et al. 2004b). Similarly, there are differences in the persistence of lung dysfunction. In some models this is maintained after allergen challenge is stopped (Leigh et al. 2002), but in other models it resolves and resistance returns to baseline levels (McMillan & Lloyd 2004). Thus, different aspects of sustained airway remodeling, and in some cases dysfunction, existed beyond the resolution of the acute inflammatory events.

Mechanisms of chronic injury These prolonged allergen challenge protocols have been used to investigate mechanisms of chronic injury. Relationships between different cell types and specific mediators and the development of remodeling parameters have been established. Eosinophils and mast cells have been shown to contribute significantly to the development of remodeling (Henderson et al. 2002; Masuda et al. 2003; Humbles et al. 2004). In particular, Th2 cells and the cytokines they produce have been shown to play an important role (Foster et al. 2002; Komai et al. 2003). Abrogation of IL-13 function, either by antibody blockade or use of genetically deficient mice, ameliorates many of the symptoms associated with chronic allergen challenge (Kumar et al. 2002; Foster et al. 2003; Leigh et al. 2004a,b; Yang et al. 2005). Similarly, targeting of IL-5, either by neutralizing antibodies or in IL-5-deficient mice is also beneficial in preventing airway remodeling (Cho et al. 2004; Kumar et al. 2004b). In addition, these blocking experiments have revealed interesting aspects regarding the relationship between parameters of the chronic allergic response. In many instances, investigators have examined the effect of therapy on the development of inflammation (by cytokine production and recruitment of inflammatory cells), development of AHR, and induction of airway remodeling (generally histologic analysis of collagen deposition in the lung). Collectively, these studies reveal that it is possible to affect one parameter without affecting the others, so it is possible to dissociate AHR from either inflammation or remodeling. One study has compared brief versus chronic exposure of mice to OVA and determined that both airway dysfunction and remodeling persisted after the cessation of allergen challenge, although airway inflammation resolved (Leigh et al. 2002). The authors

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determined that sustained airway dysfunction was not associated with ongoing Th2 inflammatory markers such as eosinophilia or IL-13 but occurred as a consequence of airway remodeling. They speculated that initial development of AHR was dependent on acute inflammatory events but sustained airway dysfunction was dependent on structural changes to the smooth muscle layer and increased deposition of extracellular matrix. In contrast we have shown that AHR to inhaled methacholine resolves in the absence of continued allergen challenge, although airway remodeling persists (McMillan & Lloyd 2004). Others have also shown that continued allergen challenge is needed to maintain airway inflammation, but not airway remodeling (Kumar et al. 2004b). Conversely, progressive inflammation (albeit mononuclear) and airflow limitation have been reported in conjunction with airway remodeling even in the absence of continued allergen challenge (Wegmann et al. 2005). It is clear that different allergen challenge regimens coupled with varying methods of measuring lung dysfunction have resulted in conflicting views as to whether parameters of chronic allergen challenge persist even in the absence of continued allergen exposure. In the future, careful analysis of distinct models will be necessary to obtain a definitive answer.

Transgenic models Transgenic technology has been used to generate models where the features of asthma, including airway remodeling, develop spontaneously (Elias 2004). Lung-specific promoters have been used to drive expression of individual cytokines or growth factors and have defined roles for these molecules in some of the pathophysiologic features of asthma. These studies take advantage of a well-characterized lung-specific regulatory element that drives gene expression in the epithelium of adult animals (Hackett & Gitlin 1992). This method exploits the fact that about 50–70% of epithelial cells in the trachea, bronchi, and bronchioles are Clara cells, which produce a 10-kDa protein referred to as CC10 (Rankin et al. 1996). The Th2-type cytokines in particular have exhibited features of remodeling, suggesting that at least some component of the remodeling response is dependent on Th2 cytokines. Many different transgenic models have been generated using lung-specific expression of several key cytokines and growth factors and the resulting phenotypes are summarized in Table 55.1. Pulmonary expression of IL-5 results in spontaneous eosinophilia and AHR (Lee et al. 1997). In addition to epithelial hypertrophy, lymphocytic infiltration of the lungs was accompanied by perturbations of the bronchial epithelium and thickening of the epithelial submucosal region of some bronchioles. Lung-specific expression of IL-13 in particular results in spontaneous airway remodeling characterized by excessive mucus production and increased matrix deposition in the airways, in addition to a mononuclear and

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Table 55.1 Summary of the pathophysiologic changes elicited by lung-specific overexpression of cytokines and growth factors. Gene

Inflammation

Subepithelial fibrosis

Smooth muscle changes

Mucous metaplasia

Airway obstruction/AHR

IL-4 IL-5 IL-6 IL-9 IL-10 IL-11 IL-13 TGF VEGF T-bet

+ + + + + +/– + + + +

– + + + + + + + + +

– – – +/– ? + +/– + + +

+ + – + + – + ? + +

– + – + ? + + ? + +

eosinophilic inflammatory response, eotaxin production, airways obstruction, and nonspecific AHR (Zhu et al. 1999). This model has since been used to define the pathways by which IL-13 mediates fibrosis in the lung, and a range of mediators including the growth factor TGF-β (Lee et al. 2001), chemokine receptors and their ligands such as CCR2 (Zhu et al. 2002a), CCR1 and CCR5 (Ma et al. 2004, 2006), adenosine (Blackburn et al. 2003) and transcription factors (Lee, P.J. et al. 2006) have been implicated in pathogenesis. Similarly, pulmonary overexpression of IL-9 resulted in eosinophil- and lymphocyte-rich pulmonary inflammation, a striking lung mast cell hyperplasia, epithelial cell hypertrophy, and subepithelial collagen deposition (Temann et al. 1998). Although mice showed normal baseline airway resistance, their response to inhaled methacholine was markedly increased. Subsequent analysis of a mouse expressing IL-9 in the lungs under the control of an inducible promoter revealed that IL-9 induces airway inflammation following the production of the Th2 cytokines IL-4, IL-5, and IL-13 (Temann et al. 2002). Moreover, epithelial-derived IL-13 was found to be absolutely required for development of this airway remodeling (Temann et al. 2007). Transgenic mice expressing IL-10 in the lung also develop mucus metaplasia, B-cell- and T-cell-rich inflammation, and subepithelial fibrosis, concomitant with augmented levels of mRNA encoding Gob-5, mucins, and IL-13 (Lee et al. 2002). Interestingly, IL-10 was shown to induce mucous metaplasia tissue inflammation and fibrosis by multiple pathways, with mucous metaplasia being dependent on, and the inflammation and airway fibrosis being independent of, an IL-13/IL-4Rα/STAT-6 activation pathway. IL-11 expression in lung epithelium manifested as nodular peribronchiolar mononuclear infiltrates, with B cells present in larger numbers than T cells, and substantial airway remodeling with subepithelial fibrosis (Tang et al. 1996). A further refinement of the model using a tissue-specific inducible transgenic system was used to show that IL-11 caused abnormalities dependent (large alveoli) and independent (airway remodeling, peribronchiolar nodules) of lung growth and development (Ray et al. 1997).

Direct targeting of classic profibrotic growth factors such as TGF-β and vascular endothelial growth factor (VEGF) to the murine lung also results in phenotypic similarities to those in asthmatic patients. Lung-targeted VEGF resulted in an asthmalike phenotype characterized by inflammation, parenchymal and vascular remodeling, edema, mucous metaplasia, myocyte hyperplasia, and AHR (Lee et al. 2004). Interestingly, these features were shown to develop via IL-13-dependent and IL-13-independent pathways. In contrast, lung-specific expression of TGF-β has been more difficult to achieve as the transgene induces fetal lethality. However, development of a conditional transgenic expression system using a tetracyclinecontrolled transcriptional suppressor and reverse tetracycline transactivator has been designed to circumvent these problems (Zhu et al. 2002b). Analysis of 6-week-old transgene positive and negative mice following activation of expression by intake of doxycycline revealed a complex phenotype (Lee, C.G. et al. 2006). Overexpression of TGF-β in the epithelium induced an early wave of epithelial apoptosis that declines with continued expression of TGF-β. Prominent inflammation was also noted, as well as an airway and parenchymal fibrotic response characterized by increased collagen deposition. Moreover, there was a significant increase in accumulation of myofibroblasts and myocytes. Human asthma is generally considered to be associated with classic Th2-type pathology, so it is interesting that targeted deletion of the Th1 transcription factor T-bet resulted in spontaneous development of the pathophysiologic features of asthma. In addition to AHR and pulmonary eosinophilia, T-bet deficiency was associated with subepithelial collagen deposition, an increase in bronchial myofibroblasts and increased production of TGF-β (Finotto et al. 2002). IL-13 blockade of T-bet-deficient mice led to reduced AHR, eosinophilia, and decreased vimentin, TGF-β and α-smooth muscle actin (αSMA) levels (Finotto et al. 2005). Interestingly, neutralization of TGF-β ameliorated aspects of the chronic airway remodeling phenotype but did not reduce AHR. These data suggest that IL-13 controls lung remodeling via T-bet using a

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Animal Models of Asthma Fibrogenic growth factor production

Mucus production

TGFVEGF PDGF? EDGF? FGF?

Epithelial cell hyperplasia

MMPs TIMPs

Matrix disregulation

Smooth muscle cell proliferation

IL-4 IL-5 IL-9 IL-13

Chemokines

Inflammatory cell recruitment

mechanism that is mediated by TGF-β-dependent as well as TGF-β-independent pathways. Although these transgenic models allow us to determine the particular contribution of individual mediators, one could argue that they may overemphasize the contribution of that molecule, when it is likely that pathologic changes occur as a consequence of a tightly controlled network of mediators. It has been argued that the IL-13 transgenic mouse in particular represents a pathophysiologic picture akin to emphysema rather that asthma. In addition, they do not involve allergen challenge, which likely elicits a distinct array of immunologic pathways that result in chronic inflammation which impacts on lung function.

What can we learn from mouse models? Over the years, mouse models of asthma have proved a valuable tool for investigating the cells and molecular pathways leading to the cardinal pathophysiologic features of the allergic pulmonary response. However, the information that they generate is only valuable if the models are valid. Efforts are constantly being made to refine these models, and recent advances have been the development of models that recapitulate the structural changes observed in lungs of chronic asthmatics. Mouse models reflect the complexity of the human disease and the range of cells and mediators that are involved. Interactions between infiltrating inflammatory cells, as well as resident lung cells (epithelial, smooth muscle, and connective tissue fibroblasts), are likely to be crucial in the remodeling process. The process of matrix regulation is dynamic, being a balance between synthesis and degradation.

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Fig. 55.4 Cells and molecules thought to be involved in the development of allergeninduced airway remodeling during asthma. EDGF, epithelial-derived growth factor; PDGF, platelet-derived growth factor; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases. (See CD-ROM for color version.)

These improved models should provide important information regarding the relationships between airway inflammation and airway dysfunction and remodeling, as well as the contributions of different cells and mediators to these processes (summarized in Fig. 55.4). There are intrinsic differences in lung structure between mice and human, and these will inevitably impact on interpretation of animal studies. Another important consideration when extrapolating results from mice to human is the fact that mice develop airway remodeling relatively quickly, implying that the process of matrix deregulation occurs rapidly. In contrast, the process in humans occurs much more slowly and airway remodeling can take years to develop. Thus, the processes resulting in an imbalance of matrix production versus regulation might be more subtle but prolonged. The limitations of mouse models represent the interpretation and extrapolation of results to human disease. Used with care and in conjunction with well-planned human studies they will be critical to test novel therapeutics, as lessons from previous drug development in asthma have shown that an integrated translational approach is critical.

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Finotto, S., Neurath, M.F., Glickman, J.N. et al. (2002) Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 295, 336–8. Finotto, S., Hausding, M., Doganci, A. et al. (2005) Asthmatic changes in mice lacking T-bet are mediated by IL-13. Int Immunol 17, 993–1007. Foster, P.S., Ming, Y., Matthei, K.I., Young, I.G., Temelkovski, J. & Kumar, R.K. (2000) Dissociation of inflammatory and epithelial responses in a murine model of chronic asthma. Lab Invest 80, 655– 62. Foster, P.S., Yang, M., Herbert, C. & Kumar, R.K. (2002) CD4(+) Tlymphocytes regulate airway remodeling and hyper-reactivity in a mouse model of chronic asthma. Lab Invest 82, 455–62. Foster, P.S., Webb, D.C., Yang, M., Herbert, C. & Kumar, R.K. (2003) Dissociation of T helper type 2 cytokine-dependent airway lesions from signal transducer and activator of transcription 6 signalling in experimental chronic asthma. Clin Exp Allergy 33, 688–95. Grunig, G., Corry, D.B., Leach, M.W., Seymour, B.W., Kurup, V.P. & Rennick, D.M. (1997) Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis. J Exp Med 185, 1089–99. Hackett, B.P. & Gitlin, J.D. (1992) Cell-specific expression of a Clara cell secretory protein-human growth hormone gene in the bronchiolar epithelium of transgenic mice. Proc Natl Acad Sci USA 89, 9079–83. Henderson, W.R.J., Tang, L.O., Chu, S.J. et al. (2002) A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 165, 108–16. Hogaboam, C.M., Blease, K., Mehrad, B. et al. (2000) Chronic airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis during allergic airway disease induced by Aspergillus fumigatus. Am J Pathol 156, 723–32. Humbles, A.A., Lloyd, C.M., McMillan, S.J. et al. (2004) A critical role for eosinophils in allergic airways remodelling. Science 305, 1776–9. Jeffery, P.K., Wardlaw, A.J., Nelson, F.C., Collins, J.V. & Kay, A.B. (1989) Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 140, 1745–53. Johnson, J.R., Wiley, R.E., Fattouh, R. et al. (2004) Continuous exposure to house dust mite elicits chronic airway inflammation and structural remodelling. Am J Respir Crit Care Med 169, 378–85. Kauffman, H.F., Tomee, J.F., van der Werf, T.S., De Monchy, J.G. & Koeter, G.K. (1995) Review of fungus-induced asthmatic reactions. Am J Respir Crit Care Med 151, 2109–115. Komai, M., Tanaka, H., Masuda, T. et al. (2003) Role of Th2 responses in the development of allergen-induced airway remodelling in a murine model of allergic asthma. Br J Pharmacol 138, 912–20. Kumar, R.K. & Foster, P.S. (2002) modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 27, 267–72. Kumar, R.K., Temelkovski, J., McNeil, H.P. & Hunter, N. (2000) Airway inflammation in a murine model of chronic asthma: evidence for a local humoral immune response. Clin Exp Allergy 30, 1486– 92. Kumar, R.K., Herbert, C., Yang, M., Koskinen, A.M., McKenzie, A.N. & Foster, P.S. (2002) Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma. Clin Exp Allergy 32, 1104–11. Kumar, R.K., Herbert, C. & Kasper, M. (2004a) Reversibility of airway inflammation and remodelling following cessation of antigenic

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challenge in a model of chronic asthma. Clin Exp Allergy 34, 1796– 802. Kumar, R.K., Herbert, C., Webb, D.C., Li, L. & Foster, P.S. (2004b) Effects of anticytokine therapy in a mouse model of chronic asthma. Am J Respir Crit Care Med 170, 1043– 8. Kurup, V.P. & Kumar, A. (1991) Immunodiagnosis of aspergillosis. Clin Microbiol Rev 4, 439– 56. Lee, C.G., Homer, R.J., Zhu, Z. et al. (2001) Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 194, 809–21. Lee, C.G., Homer, R.J., Cohn, L. et al. (2002) Transgenic overexpression of interleukin (IL)-10 in the lung causes mucus metaplasia, tissue inflammation, and airway remodeling via IL-13-dependent and -independent pathways. J Biol Chem 277, 35466–74. Lee, C.G., Link, H., Baluk, P. et al. (2004) Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2mediated sensitization and inflammation in the lung. Nat Med 10, 1095–103. Lee, C.G., Kang, H.R., Homer, R.J., Chupp, G. & Elias, J.A. (2006) Transgenic modeling of transforming growth factor-β1: role of apoptosis in fibrosis and alveolar remodelling. Proc Am Thoracic Soc 3, 418–423. Lee, J.J., McGarry, M.P., Farmer, S.C. et al. (1997) Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 185, 2143– 56. Lee, J.Y., Park, S.W., Chang, H.K. et al. (2006) A disintegrin and metalloproteinase 33 protein in patients with asthma: relevance to airflow limitation. Am J Respir Crit Care Med 173, 729–35. Lee, P.J., Zhang, X., Shan, P. et al. (2006) ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo. J Clin Invest 116, 163–73. Leigh, R., Ellis, R., Wattie, J. et al. (2002) Dysfunction and remodeling of the mouse airway persist after resolution of acute allergeninduced airway inflammation. Am J Respir Cell Mol Biol 27, 526–35. Leigh, R., Ellis, R., Wattie, J., Donaldson, D.D. & Inman, M.D. (2004a) Is interleukin-13 critical in maintaining airway hyperresponsiveness in allergen-challenged mice? Am J Respir Crit Care Med 170, 851– 6. Leigh, R., Ellis, R., Wattie, J.N. et al. (2004b) Type 2 cytokines in the pathogenesis of sustained airway dysfunction and airway remodeling in mice. Am J Respir Crit Care Med 169, 860–7. Levitt, R.C., Mitzner, W. & Kleeberger, S.R. (1990) A genetic approach to the study of lung physiology: understanding biological variability in airway responsiveness. Am J Physiol 258, L157–L164. Lloyd, C.M., Gonzalo, J.A., Coyle, A.J. & Gutierrez-Ramos, J.C. (2001) Mouse models of allergic airway disease. Adv Immunol 77, 263–95. Ma, B., Zhu, Z., Homer, R.J., Gerard, C., Strieter, R. & Elias, J.A. (2004) The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodelling. J Immunol 172, 1872– 81. Ma, B., Liu, W., Homer, R.J., Lee, P.J. et al. (2006) Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodelling. J Immunol 176, 4968–78. McMenamin, C. & Holt, P.G. (1993) The natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (MHC) class I-restricted CD8+ T cell-mediated but MHC class II-restricted CD4+ T cell-dependent immune deviation

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resulting in selective suppression of immunoglobulin E production. J Exp Med 178, 889–99. McMenamin, C., Pimm, C., McKersey, M. & Holt, P.G. (1994) Regulation of IgE responses to inhaled antigen in mice by antigenspecific gamma delta T cells. Science 265, 1869–71. McMillan, S. & Lloyd, C. (2004) Prolonged allergen challenge in mice leads to persistent airway remodelling. Clin Exp Allergy 34, 497–507. Masuda, T., Tanaka, H., Komai, M. et al. (2003) Mast cells play a partial role in allergen-induced subepithelial fibrosis in a murine model of allergic asthma. Clin Exp Allergy 33, 705–13. Pacheco, A., Cuevas, M., Carbelo, B. et al. (1998) Eosinophilic lung disease associated with Candida albicans allergy. Eur Respir J 12, 502– 4. Postma, D.S., Bleecker, E.R., Amelung, P.J. et al. (1995) Genetic susceptibility to asthma: bronchial hyperresponsiveness coinherited with a major gene for atopy. N Engl J Med 333, 894–900. Rankin, J.A., Picarella, D.E., Geba, G.P. et al. (1996) Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity. Proc Natl Acad Sci USA 93, 7821–5. Ray, P., Tang, W., Wang, P. et al. (1997) Regulated overexpression of interleukin 11 in the lung. Use to dissociate development-dependent and -independent phenotypes. J Clin Invest 100, 2501–11. Rosenstreich, D.L., Eggleston, P., Kattan, M. et al. (1997) The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. N Engl J Med 336, 1356–63. Schuh, J.M., Power, C.A., Proudfoot, A.E., Kunkel, S.L., Lukacs, N.W. & Hogaboam, C.M. (2002a) Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4–/– mice. FASEB J 16, 1313–15. Schuh, J.M., Blease, K. & Hogaboam, C.M. (2002b) CXCR2 is necessary for the development and persistence of chronic fungal asthma in mice. J Immunol 168, 1447–56. Shibuya, K., Naoe, S. & Yamaguchi, H. (1999) Animal models of A. fumigatus infections. Contrib Microbiol 2, 130– 8. Shinagawa, K. & Kojima, M. (2003) Mouse model of airway remodeling: strain differences. Am J Respir Crit Care Med 168, 959–67. Simpson, A., Maniatis, N., Jury, F. et al. (2005) Polymorphisms in a disintegrin and metalloprotease 33 (ADAM33) predict impaired early-life lung function. Am J Respir Crit Care Med 172, 55–60. Stampfli, M.R., Wiley, R.E., Neigh, G.S. et al. (1998) GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice. J Clin Invest 102, 1704–14. Strickland, D.H., Stumbles, P.A., Zosky, G.R. et al. (2006) Reversal of airway hyperresponsiveness by induction of airway mucosal CD4+/CD25+ regulatory T Cells. J Exp Med 203, 2649–60. Tang, W., Geba, G.P., Zheng, T. et al. (1996) Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction. J Clin Invest 98, 2845–53. Temann, U.A., Geba, G.P., Rankin, J.A. & Flavell, R.A. (1998) Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J Exp Med 188, 1307–20. Temann, U.A., Ray, P. & Flavell, R.A. (2002) Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology. J Clin Invest 109, 29–9.

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Temann, U.A., Laouar, Y., Eynon, E.E., Homer, R. & Flavell, R.A. (2007) IL9 leads to airway inflammation by inducing IL13 expression in airway epithelial cells. Int Immunol 19, 1–10. Temelkovski, J., Hogan, S.P., Shepherd, D.P., Foster, P.S. & Kumar, R.K. (1998) An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax 53, 849– 56. van Diemen, C.C., Postma, D.S., Vonk, J.M., Bruinenberg, M., Schouten, J.P. & Boezen, H.M. (2005) A disintegrin and metalloprotease 33 polymorphisms and lung function decline in the general population. Am J Respir Crit Care Med 172, 329–33. Van Eerdewegh, P., Little, R.D., Dupuis, J. et al. (2002) Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness. Nature 418, 426– 30. van Halteren, A.G., van der Cammen, M.J., Cooper, D., Savelkoul, H.F., Kraal, G. & Holt, P.G. (1997) Regulation of antigen-specific IgE, IgG1, and mast cell responses to ingested allergen by mucosal tolerance induction. J Immunol 159, 3009–15. Wanner, A. (1990) The role of mucus in chronic obstructive pulmonary disease. Chest 97 (suppl. 2), 11S–15S. Wegmann, M., Fehrenbach, H., Fehrenbach, A. et al. (2005) Involvement of distal airways in a chronic model of experimental asthma. Clin Exp Allergy 35, 1263–74. Wills-Karp, M. & Ewart, S.L. (1997) The genetics of allergen-induced airway hyperresponsiveness in mice. Am J Respir Crit Care Med 156, S89– S96.

Airway Remodeling in Small Animal Models

Yang, G., Li, L., Volk, A. et al. (2005) Therapeutic dosing with antiinterleukin-13 monoclonal antibody inhibits asthma progression in mice. J Pharmacol Exp Ther 313, 8– 15. Yasue, M., Yokota, T., Suko, M., Okudaira, H. & Okumura, Y. (1998) Comparison of sensitization to crude and purified house dust mite allergens in inbred mice. Lab Anim Sci 48, 346–52. Yu, C.K., Shieh, C.M. & Lei, H.Y. (1999) Repeated intratracheal inoculation of house dust mite (Dermatophagoides farinae) induces pulmonary eosinophilic inflammation and IgE antibody production in mice. J Allergy Clin Immunol 104, 228–36. Zhang, Y., Lamm, W.J., Albert, R.K., Chi, E.Y., Henderson, W.R. J. & Lewis, D.B. (1997) Influence of the route of allergen administration and genetic background on the murine allergic pulmonary response. Am J Respir Crit Care Med 155, 661–9. Zhu, Z., Homer, R.J., Wang, Z. et al. (1999) Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 103, 779–88. Zhu, Z., Ma, B., Zheng, T. et al. (2002a) IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13induced inflammation and remodelling. J Immunol 168, 2953–62. Zhu, Z., Zheng, T., Lee, C.G., Homer, R.J. & Elias, J.A. (2002b) Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modelling. Semin Cell Dev Biol 13, 121–8.

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56

Are Animal Models of Asthma Useful? Reinhard Pabst

Summary

Introduction

For a meaningful extrapolation from animals to humans in asthma research, a number of structural differences (especially those in mice) must be taken into account. Some examples include the monopodial versus dichotomous branching pattern of the bronchi, the absence or presence of respiratory bronchioles, as well as cell types such as Clara cells which are present in about 50% of mice but rare in human air-conducting parts. The size of particles largely influences the site of deposition in the lung. For some sizes mice and humans are comparable, but for others there are major differences, e.g., 1% in mouse and 30% in human alveoli for 3-μm diameter particles. Many aspects (e.g., air turbulence) influence the distribution and uptake of allergens and are thus of relevance in interpreting the effects of cytokines and chemokines. These are produced by different immune and nonimmune cells in the lung compartments, partly depending on the species studied. The bronchial arteries in humans and most other species supply the bronchial wall with oxygen and transport leukocytes to the bronchial lamina propria in which asthma is infiltrated, e.g., by eosinophils and lymphocyte subsets. However, the mouse lacks bronchial arteries and thus several steps of leukocyte infiltration in the bronchial wall will be completely different. Many experiments are performed in young adult animals. However, age is a major factor influencing the development of lung immune functions. There is growing evidence that the development of asthma depends on prenatal and postnatal exposure to antigens and the induction or suppression of different T-cell responses. Furthermore, major strain differences have become obvious, and even different suppliers of the same strains have given rise to different effects. Animal models are extremely helpful for defining the various steps in the pathophysiology of asthma and for providing ideas for future treatment protocols. However, the structural and functional differences of the lung between experimental animals and humans should always be considered.

Asthma is a term that covers not only allergic asthma but also exertional asthma, diisocyanate asthma (Redlich et al. 2002), and other types which have been well characterized (see pertinent chapters in this book). Therefore, it has to be clearly stated for which type of asthma an animal model is described. Asthma is characterized as a chronic allergic reaction with inflammatory components in both the central air-conducting parts and the more peripheral areas of the lung. The bronchial mucosa is infiltrated by a wide range of heterogeneous inflammatory cells such as eosinophils, neutrophils, lymphocyte subsets, mast cells, macrophages, but also dendritic cells. The role of each of these different leukocyte subsets in the expansion of deleterious Th2 cells in response to normally harmless antigens was summarized a few years ago, but most questions raised then are still unanswered (Wills-Karp 1999). This chronic inflammation leads to remodeling of the airway wall, airway hyperreactivity, and increased mucus production, resulting in partial airway plugging and epithelial desquamation. In humans, an early-phase and a late-phase response can be distinguished. Increased IgE levels are taken as a further hallmark of asthma. All these aspects have their specific time sequence. It is not surprising that for a disease for which the causes and genetic and environmental background are only partly known and in which so many different cells, cytokines and other proteins are involved, there can hardly be a single animal model which is similar in all aspects to the multifaceted human disease, asthma bronchiale. For further details the reader may consult other chapters in this book as well as other extensive monographs (Busse & Holgate 2000; Lambrecht et al. 2003; Mestecky et al. 2005). In the last two decades enormous progress has been made in understanding the molecular biology of immune reactions, e.g., cytokine and chemokine networks and intracellular signal transduction. There are advantages in understanding the sequence of molecular reactions in the different cells of the immune system. However, it is also clear that a transmitter produced by one cell type will only affect cells in the immediate neighborhood, i.e., the microenvironment will define the functional outcome of the molecular reaction of

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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one cell type. Therefore, systemic biology and understanding the compartmentalized micromilieu of the lung are of growing relevance for clarifying the physiology and pathophysiology of diseases such as asthma bronchiale, and finally to developing new strategies for preventing and treating this disease of growing incidence. In addition, not only different compartments of the respiratory tract but also the draining lymph nodes have to be included in concepts of asthma pathophyisology (Pabst 2000; Gajewska et al. 2001; Ritz et al. 2004). The mouse as model species undoubtedly has enormous advantages, with all the transgenic strains and wealth of markers and reagents. However, one has to be aware of the many differences between mice and humans with regard to structure and function of cells and tissues. Mouse models of allergic airway diseases have been critically reviewed (Lloyd et al. 2001; Kumar & Foster 2002) and other articles have summarized the arguments for (Gelfand 2002; Shapiro 2006) and against (Persson 2002; Wenzel & Holgate 2006) the mouse as model species. Other chapters in this book deal with the advantages of primate models (Chapter 54) and rodent models (Chapter 55). Further species, such as the dog asthma model using ragweed as an antigen, are also valid (Redman et al. 2001). There are several critical reviews on the advantages and disadvantages of asthma models in different species (e.g., Bice et al. 2002) and these should be carefully considered before deciding on the most relevant species. A good illustration of the problem of extrapolating from data obtained in mice to treatment protocols in humans is the role of the cytokines interleukin (IL)-4 and IL-5. Following documentation in several mouse experiments (including cytokinedeficient mice) that a reduction in IL-4 or IL-5 had positive effects on asthma symptoms, clinical trials using anti-IL-5 or anti-IL-4 receptor were disappointing (Kumar & Foster 2002). Likewise, another study provided evidence of the effects of IL-5 on eosinophils in the blood but no effects on eosinophil migration to the bronchoalveolar space or on bronchial responsiveness (van Rensen et al. 2001). Thus, great care must be taken when extrapolating from blood leukocyte numbers to the situation in other organs (Blum & Pabst 2006). The main aims of this chapter are to review different model species in rats and mice and elaborate the relevance of species differences with respect to the macroanatomy and microanatomy of the human respiratory tract. Furthermore, the effects of age, strain and breeding conditions on the outcome of asthma research are discussed. When the morphologic and functional species differences are considered, extrapolation from animal experiments will be more cautiously expressed, as is obvious in many papers. With regard to the different aspects of cell type, number, and structure in the respiratory tract compartment of relevance for asthma, mainly reviews will be quoted so readers interested in details should check these.

Are Animal Models of Asthma Useful?

Species differences in structural aspects of the respiratory tract The macroscopically identifiable structures of the lung such as the pleura, thickness of the interlobular septa, and divisions into different numbers of lung lobes are unlikely to have major effects on allergic airway diseases despite the enormous variations, e.g., in numbers of generations of bronchi and nonrespiratory bronchioli (Tyler & Julian 1992). Lung function and deposition of inhaled particles and antigens are largely affected by species differences, which determine where the air-conducting part ends (nonrespiratory bronchiole) and the gas-exchange segments start (respiratory bronchiole, alveolar ducts ending in alveolar air sacs called alveoli). In humans and monkeys, the last nonrespiratory bronchiole is followed by several generations of respiratory bronchioli. In mice, in contrast, respiratory bronchioles are absent and terminal bronchioli open directly into alveolar ducts (reviewed in Tyler & Julian 1992).

Branching pattern of bronchi The well-known branching pattern of bronchi in humans and in many other larger species is called “dichotomous.” This means that one bronchus branches into two bronchi of similar diameter. However, the bronchus following the direction of the more proximal bronchus often has a larger diameter than the other bronchus. This dichotomous branching pattern exists beyond the segmental bronchi (approximately 12 generations). In mice and rats, a different branching pattern (McBride 1992) called “monopodial” is the basic structure, with one bronchus following a main direction and the branches going off in different directions have a much smaller diameter (Fig. 56.1). However, the pattern or structural arrangement of the gas-exchange unit is surprisingly similar. From mice to humans there is an approximately 50-fold increase in acinar number, about 170-fold increase in acinar volume, and a sevenfold increase in acinar diameter (Mercer & Crapo 1992). It has to be stressed that in small rodents cartilage is only found in the bronchial wall near to the hilum, in contrast to humans, pigs and monkeys, in which only bronchioli lack cartilage in the wall (Mariassy 1992).

Cell types in air-conducting segments The epithelium of the air-conducting segments consists of basal cells, mucous or goblet cells, and ciliated cells. The very rare brush cells are not discussed here. The relative frequency of epithelial cell subsets depends on the airway generation. In sheep, there are about 50% mucous cells as peripheral as the 25th airway generation. In rabbits, however, there are only very few mucous cells until the third airway generation (Mariassy 1992). In the review by Evans et al. (2001), the strategic position of basal cells with regard to interactions with other epithelial cells, but also cells of the immune system, as

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(a)

Humans

Dichotomous branching, symmetric = one bronchus branches into two smaller (not always of the same diameter) • cartilaginous rings or plates until bronchioles start • smooth muscles as peripheral as in bronchioles

(b)

Mice

Monopodial branching, asymmetric small branches in different directions from one main bronchus • no cartilage beyond the hilar area • extremely thin muscle layer • tiny lamina propria

• definite lamina propria with structural cells, immune cells, vessels

well as markers for this cell type in different species was summarized, and the enormous number of cellular and molecular characteristics of basal cells in different species listed and their potential functional role mentioned. In mice, up to about half of the airway epithelium consists of Clara cells, which are hardly found in humans (Mariassy 1992). Clara cells are morphologically different from mucous cells, with the dome-shaped luminal end of these columnar cells often extending into the airway lumen. They contain secretory granules and produce a series of proteins (Singh & Katyal 1992). In humans, about 25% of epithelial cells have been characterized as goblet (mucous) cells, while in healthy mice only 1% has been documented (Mariassy 1992; Plopper & Hyde 1992). Goblet cell metaplasia in models of airway disease is often taken as an important parameter, but the enormous species differences in the normal situation neglected. The regulation of one cell type, e.g., the epithelial cell, by one cytokine such as IL-13 can be documented in transgenic mice models. Two typical features of asthma, airway hyperreactivity and mucus overproduction, have been documented with regard to this interaction, although there were no signs of inflammation or remodeling (Kuperman et al. 2002). In a recent review of the expression of mucin genes in chronic inflammatory airway disease, the authors strongly recommend not overlooking the differences between human and murine airways, which might also include the signaling pathways necessary for mucin gene regulation (Voynow et al. 2006). Mucociliary clearance, also relevant for inhaled particulate antigens, is a major component of the lung defense mechanism. Mucus velocity as a measure of mucociliary clearance function has been described for the trachea in many species (Wolff 1992). There is a clear correlation between

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Fig. 56.1 Schematic drawing of the branching pattern of bronchi. (a) “Dichotomous” branching in humans and many nonlaboratory species. (b) “Monopodial” type of branching in small rodents, e.g., mice and rats.

tracheal mucus velocity and body weight, with a correlation coefficient of 0.94 (Wolff 1992). The overproduction of a more viscous mucus is a hallmark of asthma not only in animal asthma models but also in patients. The molecular mechanisms for mucus clearance, including viscosity, mechanical clearance, and the chemical shield of salt and defensins, are still under debate (Knowles & Boucher 2002). All cells have a particular turnover rate in health and this might be increased in disease, e.g., asthma. Basal cells seem to be the local stem cells and an increase in goblet cells after antigen exposure can be due to increased differentiation from basal to goblet cells or loss of ciliated cells. The kinetics of pulmonary cells shows species differences (Shami & Evans 1992) and many aspects that play an important role in the pathophysiology of asthma are still unknown. In this respect, both the role of epithelial stem cells in the lung and the factors regulating the preference of certain cells in the epithelium derived from stem cells will be of major relevance (Otto 2002). Functional aspects of the respiratory tract epithelium, such as the transport of IgG to the lumen, are also significant factors in immune reactions. In the adult lungs of humans, cynomolgus monkeys and mice, transport via FcγRn has been demonstrated, documenting a species-independent mechanism in this respect (Spiekermann et al. 2002). The innervation of the muscles of the bronchial tree is also relevant in asthma as bronchoconstriction is a major symptom and in animal models it is a major criterion. The innervation pattern does not only change from central to peripheral parts of the lung with respect to receptor type and density of nerve endings. Enormous species variation has also been documented including efferent (adrenergic, cholinergic, and nonadrenergic inhibitory) neurons. Furthermore, afferent

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fibers are critical for a great variety of reflexes (Zorychta & Richardson 1992). Antigen uptake and immune reactions to allergens can be affected by an altered or damaged epithelium, which occurs in viral inflammation, and this partly explains the specific interactions between viral and allergic components with epithelial cells (Holtzman et al. 2002).

Bronchial arteries: major species differences In addition to the pulmonary arteries and veins (vasa publica), which are essential for gas exchange, there are bronchial arteries supplying the bronchial wall with oxygen and these arteries derive from the aorta or intercostal arteries in humans (vasa privata). The data on how far peripherally bronchial arteries run in different species are controversial (Kay 1992). The enormous species differences in the origin, diameter, and structure of the muscular layer of the arterial wall and distribution to different segments of the bronchial system have

(a)

(c)

Are Animal Models of Asthma Useful?

been summarized by Kay (1992). Mice, the most often used species to test animal models for asthma, lack bronchial arteries (Mitzner et al. 2000). Thus, the lamina propria of bronchi, which also contains capillaries and veins and which seems to be the site of exit of leukocytes in an asthma attack in humans, does not show similar vessels in mice. In humans, the role of the expression of adhesion molecules after allergen exposure of these vessels in the lamina propria has been documented on biopsy specimens (Hirata et al. 1998). These lamina propria vessels are obviously the exit sites for leukocytes during allergic reactions. Considerable swelling of the bronchial wall, possibly due to dilatation of bronchial blood vessels, followed by congestion and vessel wall edema has often been described in fatal asthma (Jeffery 2004). Changes in the size and density of bronchial vessels during corticoid treatment have been used as criteria for its effectiveness (Chanez et al. 2004; Wanner et al. 2004). As these bronchial vessels do not exist in mice, in contrast to humans, pigs and also rats (Fig. 56.2), the

human

10x

pig

10x

rat

20x

mouse

20x

Fig. 56.2 Histologic cross-sections of bronchi from (a) human, (b) pig, (c) rat, and (d) mouse. Note the width of the bronchial lamina propria in species such as pig and human and also the very thin muscular layers in the mouse and rat. The most interesting aspect is the lack of bronchial arteries and veins in the lamina propria of mice (d). However, the rat does have a bronchial blood supply (arrows). (See CD-ROM for color version.)

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(b)

(d)

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(a)

Bronchus Bronchial lymph node Lymphatics

Alveolar capillaries

Pulmonary artery

phenomena described above do not occur in mice nor can leukocytes use this route. Thus therapeutic strategies that manipulate the adherence and exit of leukocytes in asthma, when the endothelium of bronchial vessels is the target, cannot be tested in mouse models. This is one of the most important drawbacks to using the mouse as a meaningful model for several aspects of human asthma.

Periarterial space: a compartment of the lung often involved in allergic reaction with unknown relevance

Pulmonary vein Periarterial capillaries

(b)

Br

A

Parallel to branches of the pulmonary arteries, a unique capillary network is found in many species (Pabst & Tschernig 2002). This compartment is not only extended in edema, but infiltrated by leukocytes in early phases of the rejection of lung transplantation, and after exposure to bacteria, viruses and allergens such as ovalbumin in asthma models (for review see Pabst 2004). The periarterial accumulation of eosinophils, neutrophils and other leukocyte subsets such as dendritic cells has often been described in addition to peribronchial leukocyte accumulations (Pabst 2004), although the pathophysiologic relevance is still unknown (Fig. 56.3). In a recent study, there were significantly increased numbers of eosinophils and mast cells in this compartment surrounding small pulmonary arteries in the lungs of patients who had died during an asthma attack, but not in the lungs of patients who had died of other causes such as heart attack (Shiang et al. 2006). Thus, the periarterial space is not only affected by leukocyte infiltration in animal models of asthma but also in humans, documenting a similarity between rodents and humans in this aspect. Further studies are needed to understand the relevance of periarterial leukocyte infiltration and to decide whether it is a bystander effect or a constant finding that treatment protocols should aim to reduce.

Gas-exchange region of the lung (c)

Br

This compartment seems to be less relevant in asthma. Although it has to be considered, the broncoalveolar lavage samples fluid and cells not only from the air-conducting parts of the lung but also from the alveolar space. In this respect, it should be remembered that there are enormous species differences in alveolar surface area, capillary surface area,

Br

A

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Fig. 56.3 (left) Schematic drawing of the compartment around a branch of the pulmonary artery, the “periarterial space.” (a) Note the unique pattern of capillaries in this compartment, which are different from the alveolar capillaries essential for the gas-exchange function of the lung. (b) After intraperitoneal sensitization with ovalbumin (OVA) and intratracheal instillation of OVA there is an obvious influx of inflammatory cells into the peribronchial area but the periarterial space is empty of leukocytes. (c) Rat lung after the instillation of sheep red blood cells. There is influx of leukocytes into the periarterial space and partly also in the peribronchial area. A, branch of the pulmonary artery; Br, bronchial branch. (Modified after Pabst 2004, with permission.) (See CD-ROM for color version.)

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etc., as well as the number of alveolar type I and type II cells, which influence surfactant production (Pinkerton et al. 1992).

Pulmonary function tests in small laboratory animals One important criterion for the diagnosis of asthma is the pathologic lung function test. The normal lung shows great variability between species when parameters of ventilation are compared (Lai 1992). It has to be considered whether the animals were under anesthesia or in restraining cages and in prone or supine position (Costa et al. 1992). For mice, the Penh parameter is very often given, although it is still under debate whether Penh results are meaningful (Sly et al. 2005). It remains to be clarified which airway function test in the conscious mouse is most satisfactory. Unrestrained body plethysmography has been advocated as a more relevant technique (Bates & Irvin 2003; Hoymann 2006; Lofgren et al. 2006; Lomask 2006). In systematic airway function tests in mice, it became obvious that strain, sex and age all influenced the outcome (Flandre et al. 2003). Researchers have to be aware of the indications and limitations of lung function tests, particularly in small rodents, when the results are extrapolated to the situation in humans.

Deposition of particles Many allergens consist of particulate matter (Schulz 2003). Therefore the deposition of particles of different sizes in different species is of major importance in studies of asthma models. It is not only the diameter of a particle that influences impaction, sedimentation, and diffusion. Values for deposition in total may be comparable (e.g., < 0.1 μm diameter); however, larger particles can have up to 30% higher deposition rates in humans than in the mouse. Furthermore, the effects of inhaled particles largely depend on where the particles are deposited, e.g., nose versus lung. As an example, inhaled particles of 3-μm diameter are deposited at a rate of only 1% in the alveoli of mice but at a rate of about 30% in humans (for details see Schulz 2003). Deposition experiments using different-sized particles are full of pitfalls when data obtained in rodents are extrapolated to the situation in humans. When BALB/c mice were compared with B6C3F1 mice, particle deposition in the tracheobronchial tree was up to three times greater in BALB/c mice for particles of 2 μm diameter (Oldham & Phalen 2002). In addition, with regard to novel therapeutic approaches using aerosols, it is of major relevance which parts of the lung are accessed by a specific technique in laboratory animals compared with humans (Groneberg et al. 2006). All these examples document the great problem in extrapolating data from one study to another and from one laboratory species to humans.

Are Animal Models of Asthma Useful?

Effect of age, strain and animal supplier on allergic lung reactions Although it is generally accepted that age plays a central role in many structural and functional parameters of the lung, “young adult” animals are mainly used as a model. However, exposure to antigen and the initiation of an allergic reaction resulting in future asthma often happens at a very early age. Therefore, the postnatal growth of the lung in humans (Burri 2006) and in other species has to be considered. Also, aging of the normal lung is of relevance (Sahebjami 1992). When the effect of age was studied in allergen-exposed BN rats at 4 and 13 weeks of age there were impressive differences in, for example, the number of eosinophils, goblet cell hyperplasia, and fibronectin deposition (Palmans et al. 2002). The maturation of the immune system (Th1- and Th2-type reactions) differs between mice and humans, and the effects of respiratory syncytial virus infection at different ages obviously influence this polarity (Holt & Sly 2002). When the variance of gene expression in the human lung was studied using gene array technology, an interesting difference between young (29 years) and old (69 years) organ donors was documented, and the problems in defining a healthy lung were critically discussed (Gruber et al. 2006). The microenvironment in different compartments of the lung has to be studied by modern technologies, for example by combining laser microdissection with molecular biology (Fink et al. 2006). The recently published book on the development, aging, and influence of the environment is recommended for these very relevant aspects (Harding et al. 2004). The extent of allergen-induced airway inflammation is strain-dependent. For example, when ovalbumin was applied as an aerosol after ovalbumin sensitization, the extent of topical morphologic parameters of airway inflammation was more pronounced in Brown-Norway compared with Sprague Dawley rats (Hylkema et al. 2002). Unexpectedly large differences were documented when BN rats of the same strain but from different breeders were compared with respect to lung morphology, despite the fact that all animals were called “SPF rats” (Germann et al. 1998). When animal models for aerogenic recombinant allergens were reviewed, enormous differences between mouse strains and allergen preparations were revealed (Herz et al. 2004).

Immune cells in asthma In all immune reactions in the lung, many different cells interact with each other, e.g., by cytokine release or expressing chemokine receptors (Murray & Discoll 1992; Lukacs & Tekkanat 2000; Lloyd 2002). The enormous advances in understanding the heterogeneity and critical role of the immune reactions of dendritic cells have recently been summarized (Kuipers & Lambrecht 2004; Van Rijt & Lambrecht

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2005). Regulatory T cells (Treg) belong to a cell family with growing heterogeneity and new markers being regularly described. Recently, CD4+CD25+ Treg were documented as major players in the reversal of airway hypersensitivity in a rat model of asthma after chronic aeroallergen exposure (Strickland et al. 2006). Species differences for these cell types will probably be described in future, as was the case a decade ago for macrophages. They are not only found in the lung interstitium and in the bronchoalveolar space but also as intravascular macrophages in some species (Valberg & Blanchard 1992). In many situations it is extremely difficult to differentiate whether infiltrated macrophages have migrated to the lung or are the result of activation of cells already present (Garn 2006). Changes in the local apoptosis of immune cells can also influence the total number, as was recently shown when the number of apoptotic lymphocytes was documented as significantly lower in hypersensitivity pneumonitis compared with controls, which partly explains the higher lymphocyte numbers in the bronchoalveolar lavage of these patients (Laflamme et al. 2003). It has recently been summarized for domestic animals that the initial insult determines the type of inflammation in the lung, which normally maintains cytokines and chemokines in balance (Thacker 2006). For further details, see other chapters in this book.

Conclusions Data obtained in laboratory animals should be extrapolated cautiously to the situation in humans, particularly when only certain parameters have been studied rather than the complete pattern of asthma symptoms. Morphologic aspects with functional relevance should be considered, such as the “monopodial” branching pattern of bronchi in small rodents compared with the dichotomous type in humans. A most important aspect is the lack of bronchial vessels in the mouse, because these are the vessels critical for leukocyte infiltration in human asthma. Not only species but also strains and breeding conditions are of relevance for the outcome of experiments with laboratory animals. In addition to positive results of a specific response in one strain, data which could not be repeated in another strain should also be published. For questions regarding the mechanisms of allergic asthma, genetically manipulated mice can be of great help in understanding the molecular basis of a response. However, one should only extrapolate experimental animal data to the situation in humans with great caution. All too often data obtained in mice have been overinterpreted as of great relevance for pathomechanisms or treatment strategies in humans.

References Bates, J.H.T. & Irvin, C.G. (2003) Measuring lung function in mice: the phenotyping uncertainty principle. J Appl Physiol 94, 1297–306.

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Bice, D., Barrett, E. & Rudolph, K. (2002) Insights from animal models of asthma. In: Heinrich, U. & Mohr, U., eds. Crucial Issues in Inhalation Research. Mechanistic, Clinical and Epidemiologic. Fraunhofer IRB Verlag, Stuttgart, pp. 127–49. Blum, K.S. & Pabst, R. (2006) Lymphocyte numbers and subsets in the human blood. Do they mirror the situation in all organs? Immunol Lett 108, 4551. Burri, P.H. (2006) Structural aspects of postnatal lung development: alveolar formation and growth. Biol Neonate 89, 313–22. Busse, W.W. & Holgate, S.T. (2000) Asthma and Rhinitis. Blackwell Science, Oxford. Chanez, P., Bourdin, A., Vachier, I., Godard, P., Bousquet, J. & Vignola, A.A. (2004) Effects of inhaled corticosteroids on pathology in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1, 184– 90. Costa, D.L., Tepper, J.S. & Raub, J.A. (1992) Interpretations and limitations of pulmonary function testing in small laboratory animals. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 367–99. Evans, M.J., van Winkle, L.S., Fanucchi, M.V. & Plopper, C.G. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Exp Lung Res 27, 401–15. Fink, L., Kwapiszewska, G., Wilhelm, J. & Bohle, R.M. (2006) Lasermicrodissection for cell type- and compartment-specific analyses on genomic and proteomic level. Exp Toxicol Pathol 57 (suppl. 2), 25–9. Flandre, T.D., Leroy, P.L. & Desmecht, D.J. (2003) Effect of somatic growth, strain, and sex on double-chamber plethysmographic respiratory function values in healthy mice. J Appl Physiol 94, 1129– 36. Gajewska, B.U., Swirski, F.K., Alvarez, D. et al. (2001) Temporalspatial analysis of the immune response in a murine model of ovalbumin-induced airways inflammation. Am J Respir Cell Mol Biol 25, 326–34. Garn, H. (2006) Specific aspects of flow cytometric analysis of cells from the lung. Exp Toxicol Pathol 57 (suppl. 2), 21–4. Gelfand, E.W. (2002) Mice are a good model of human airway disease. Am J Respir Crit Care Med 166, 5– 6. Germann, P.G., Häfner, D., Hanauer, G. & Drommer, W. (1998) Incidence and severity of granulomatous pneumonia in Brown Norway (BN) rats: breeder related variations. J Exp Anim Sci 39, 22– 33. Groneberg, D.A., Paul, H. & Welte, T. (2006) Novel strategies of aerosolic pharmacotherapy. Exp Toxicol Pathol 57 (suppl. 2), 49–53. Gruber, M.P., Coldren, C.D., Woolum, M.D. et al. (2006) Human lung project: evaluating variance of gene expression in the human lung. Am J Respir Cell Mol Biol 35, 65–71. Harding, R., Pinkerton, K.E. & Plopper, C.G. (2004) The Lung: Development, Aging and the Environment. Elsevier Academic Press, Amsterdam. Herz, U., Renz, H. & Wiedermann, U. (2004) Animal models of type I allergy using recombinant allergens. Methods 32, 271–80. Hirata, N., Kohrogi, H., Iwagoe, H. et al. (1998) Allergen exposure induces the expression of endothelial adhesion molecules in passively sensitized human bronchus: time course and the role of cytokines. Am J Respir Cell Mol Biol 18, 12–20. Holt, P.G. & Sly, P.D. (2002) Interactions between RSV infection, asthma, and atopy: unraveling the complexities. J Exp Med 196, 1271– 5.

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Holtzman, M.J., Morton, J.D., Shornick, L.P. et al. (2002) Immunity, inflammation, and remodeling in the airway epithelial barrier: epithelial–viral–allergic paradigm. Physiol Rev 82, 19–46. Hoymann, H.G. (2006) New developments in lung function measurements in rodents. Exp Toxicol Pathol 57 (suppl. 2), 5–11. Hylkema, M.N., Hoekstra, M.O., Luinge, M. & Timens, W. (2002) The strength of the OVA-induced airway inflammation in rats is strain dependent. Clin Exp Immunol 129, 390–6. Jeffery, P.K. (2004) Remodelling and inflammation of bronchi in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1, 176– 83. Kay, J.M. (1992) Blood vessels of the lung. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 163–72. Knowles, M.R. & Boucher, R.C. (2002) Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109, 571–7. Kuipers, H. & Lambrecht, B.N. (2004) The interplay of dendritic cells, Th2 cells and regulatory T cells in asthma. Curr Opin Immunol 16, 702– 8. Kumar, R.K. & Foster, P.S. (2002) Modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 27, 267–72. Kuperman, D.A., Huang, X., Koth, L.L. et al. (2002) Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 8, 885– 9. Laflamme, C., Israël-Assayag, E. & Cormier, Y. (2003) Apoptosis of bronchoalveolar lavage lymphocytes in hypersensitivity pneumonitis. Eur Respir J 21, 225–31. Lai, Y.L. (1992) Comparative ventilation of the normal lung. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 217– 39. Lambrecht, B.N., Hoogsteden, H.C. & Diamant, Z. (2003) The Immunological Basis of Asthma. Marcel Dekker, New York. Lloyd, C. (2002) Chemokines in allergic lung inflammation. Immunology 105, 144– 54. Lloyd, C.M., Gonzalo, J.A., Coyle, A.J. & Gutierrez-Ramos, J.C. (2001) Mouse models of allergic airway disease. Adv Immunol 77, 263– 95. Lofgren, J.L., Mazan, M.R., Ingenito, E.P. et al. (2006) Restrained whole body plethysmography for measure of strain specific and allergen-induced airway responsiveness in conscious mice. J Appl Physiol 101, 1495– 505. Lomask, M. (2006) Further exploration of the Penh parameter. Exp Toxicol Pathol 57 (suppl. 2), 13–20. Lukacs, N.W. & Tekkanat, K.K. (2000) Role of chemokines in asthmatic airway inflammation. Immunol Rev 177, 21–30. McBride, J.T. (1992) Architecture of the tracheobronchial tree. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 49– 61. Mariassy, A.T. (1992) Epithelial cells of trachea and bronchi. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 63–76. Mercer, R.R. & Crapo, J.D. (1992) Architecture of the acinus. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 109–19. Mestecky, J., Bienenstock, J., Lamm, M.E., Mayer, L., McGhee, J.R. & Strober, W. (2005) Mucosal Immunology. Elsevier, Amsterdam. Mitzner, W., Lee, W., Georgakopoulos, D. & Wagner, E. (2000) Angiogenesis in the mouse lung. Am J Pathol 157, 93–101.

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Murray, M.J. & Discoll, K.E. (1992) Immunology of the respiratory system. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 725–46. Oldham, M.J. & Phalen, R.F. (2002) Dosimetry implications of upper tracheobronchial airway anatomy in two mouse varieties. Anat Rec 268, 59–65. Otto, W.R. (2002) Lung epithelial stem cells. J Pathol 197, 527– 35. Pabst, R. (2000) Mucosa-associated lymphoid tissue of the lung: localization, numbers and dynamics of lymphoid cells in the five different compartments. In: Busse, W.W. & Holgate, S.T., eds. Asthma and Rhinitis. Blackwell, Oxford, pp. 543–56. Pabst, R. (2004) The periarterial space in the lungs: its important role in lung edema, transplantation and microbial or allergic inflammation. Pathobiology 71, 287–94. Pabst, R. & Tschernig, T. (2002) Perivascular capillaries in the lung: an important but neglected vascular bed in immune reactions? J Allergy Clin Immunol 110, 209–14. Palmans, E., Vanacker, N.J., Pauwels, R.A. & Kips, J.C. (2002) Effect of age on allergen-induced structural airway changes in Brown Norway rats. Am J Respir Crit Care Med 165, 1280–4. Persson, C.G.A. (2002) Mice are not a good model of human airway disease. Am J Respir Crit Care Med 166, 6–7. Pinkerton, K.E., Gehr, P. & Crapo, J.D. (1992) Architecture and cellular composition of the air–blood barrier. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 121–8. Plopper, C.G. & Hyde, D.M. (1992) Epithelial cells of bronchioles. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 85–92. Redlich, C., Wisnewski, A. & Gordon, T. (2002) Mouse models of diisocyanate asthma. Am J Respir Cell Mol Biol 27, 385–90. Redman, T.K., Rudolph, K., Barr, E.B., Bowen, L.E., Muggenburg, A. & Bice, D.E. (2001) Pulmonary immunity to ragweed in a Beagle dog model of allergic asthma. Exp Lung Res 27, 433–51. Ritz, S.A., Cundall, M.J., Gajewska, B.U. et al. (2004) The lung cytokine microenvironment influences molecular events in the lymph nodes during Th1 and Th2 respiratory mucosal sensitization to antigen in vivo. Clin Exp Immunol 138, 213–20. Sahebjami, H. (1992) Aging of the normal lung. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 351–66. Schulz, H. (2003) Aerosole in Medizin und Veterinärmedizin: Partikeldeposition. Pneumologie 57, 167–8. Shami, S.G. & Evans, M.J. (1992) Kinetics of pulmonary cells. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 145–55. Shapiro, S.D. (2006) Animal models of asthma. Pro: allergic avoidance of animal (model[s]) is not an option. Am J Respir Crit Care Med 174, 1171–3. Shiang, C., Mauad, T., Senhorini, A. et al. (2008) Increased mast cell and eosinophilic inflammation in the perivascular space in fatal asthma. submitted. Singh, G. & Katyal, S.L. (1992) Secretory proteins of Clara cells and type II cells. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 93–108. Sly, P.D., Turner, D.J., Collins, R.A. & Hantos, Z. (2005) Penh is not a validated technique for measuring airway function in mice. Am J Respir Crit Care Med 172, 256.

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Spiekermann, G.M., Finn, P.W., Ward, E.S. et al. (2002) Receptormediated immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in the mammalian lung. J Exp Med 196, 303–10. Strickland, D.H., Stumbles, P.A., Zosky, G.R. et al. (2006) Reversal of airway hyperresponsiveness by induction of airway mucosal CD4+CD25+ regulatory T cells. J Exp Med 203, 2649–60. Thacker, E.L. (2006) Lung inflammatory responses. Vet Res 37, 469–86. Tyler, W.S. & Julian, M.D. (1992) Gross and subgross anatomy of lungs, pleura, connective tissue septa, distal airways, and structural units. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 37– 48. Valberg, P.A. & Blanchard, J.D. (1992) Pulmonary macrophage physiology: origin, motility, endocytosis. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 681–723. van Rensen, E.L. J., Stirling, R.G., Scheerens, J. et al. (2001) Evidence for systemic rather than pulmonary effects of interleukin-5 administration in asthma. Thorax 56, 935– 40.

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Van Rijt, L.S. & Lambrecht, B.N. (2005) Dendritic cells in asthma: a function beyond sensitization. Clin Exp Allergy 35, 1125–34. Voynow, J.A., Gendler, S.J. & Rose, M.C. (2006) Regulation of mucin genes in chronic inflammatory airway diseases. Am J Respir Cell Mol Biol 34, 661–5. Wanner, A., Horvath, G., Brieva, J.L., Kumar, S.D. & Mendes, E.S. (2004) Nongenomic actions of glucocorticosteroids on airway vasculature in asthma. Proc Am Thorac Soc 1, 235– 38. Wenzel, S. & Holgate, S.T. (2006) The mouse trap. It still yields few answers in asthma. Am J Respir Crit Care Med 174, 1173–5. Wills-Karp, M. (1999) Immunologic basis of antigen-induced airway hyperresponsiveness. Annu Rev Immunol 17, 255–81. Wolff, R.K. (1992) Mucociliary function. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 659–80. Zorychta, E. & Richardson, J.B. (1992) Innervation of the lung. In: Parent, R.A., ed. Comparative Biology of the Normal Lung. CRC Press, Boca Raton, FL, pp. 157–62.

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Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Genetics of Asthma and Atopic Dermatitis Saffron A.G. Willis-Owen, Miriam F. Moffatt and William O.C. Cookson

Summary The development of asthma and atopic dermatitis (AD, eczema) depends on both genetic and environmental factors. Over the past few years, several genes and genetic loci associated with increased susceptibility to asthma and AD have been described. Modern technology for genome-wide association studies means that novel genes are being identified with increasing frequency and certainty. A surprising number of these new genes are expressed in the mucosa and epidermis, suggesting that events at epithelial surfaces may be driving disease processes. Understanding the mechanisms of innate epithelial immunity may provide new insights for the development of novel treatments for inflammatory epithelial disease.

Introduction Asthma is an inflammatory disease of the small airways of the lung. Currently there is an epidemic of asthma, which now affects more than 10% of children in many Westernized societies. Infantile eczema (atopic dermatitis, AD) is also increasingly common in the developed world, affecting up to 20% of children in some countries. Asthma is present in 60% of children with severe AD, a significant proportion of whom continue to experience problems into adult life. Both diseases are familial and due to an interaction between strong genetic and environmental factors. AD, asthma and hay fever are often considered to be part of a common syndrome of atopic diseases (Johansson et al. 2001). The term “atopy” is variously used, but it most consistently refers to the presence of IgE-mediated skin-test responses to common allergens. Atopic individuals are also typified by the presence of elevated levels of total and allergenspecific IgE in serum.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Given that the basic causes of asthma and AD are unclear, it is proving increasingly helpful to understand their genetic basis. Genetic studies offer a structured approach for identifying unknown genes and pathways. Using this approach, the genes so far identified in asthma and AD are beginning to close some of the gaps in our understanding of these diseases. This chapter describes what is known about the genetics of asthma and AD, and then suggests potential mechanisms to explain the interaction between genes and the environment in disease pathogenesis.

Immunogenetics The study of genetics is the study of polymorphism. However, not all genes are polymorphic, such as those that are crucial to normal development, and not all disease pathways will be discovered using a genetic approach. Nevertheless, survival in a hostile environment characterized by constantly evolving threats from microorganisms depends on spending considerable energy in evolving and maintaining immunity. Consequently, many genes and proteins of the immune system are polymorphic. Although disease can result from mutations in single genes, most common diseases with familial clustering are due to complicated interactions between an unknown number of genes and environmental factors. Genetic research into these “complex diseases” or “complex traits” has begun to make substantial contributions to the understanding of mechanisms for illnesses such as Alzheimer’s disease, diabetes, asthma, and inflammatory bowel disease (IBD). Functional polymorphisms may be expected in many genes that influence immunity, and many of these might show associations with disease if tested carefully enough. It nonetheless seems likely that complex diseases are most influenced by a few polymorphisms that have important effects (Farrall 2004), and it is these polymorphisms that are of particular interest in unraveling disease pathogenesis.

Finding disease genes Susceptibility genes (or more precisely polymorphisms in the genes and their regulatory elements) may be identified

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through a number of strategies. The most simple of these is a candidate gene study in which polymorphisms in a gene of interest are tested for frequency differences in cases and controls. This method is restrained by the choice of candidate gene and does not lead readily to the discovery of novel genetic effects or pathways. In addition, candidate gene studies have frequently been carried out in small samples of subjects, with inadequate control for multiple comparisons, and little attention to whether polymorphism impacts on function through coding changes or through proven effects on transcription. Consequently, the replication of results from such studies has often been poor. Positional cloning is an alternative approach to gene identification and relies on the study of families in which the disease is inherited. The first step in this process is the detection of genetic linkage, or consistent coinheritance of a chromosomal region together with the disease. Linkage is followed by fine mapping of the region with eventual gene identification. Positional cloning was of greatest use when knowledge of the human genome was fragmentary and limited numbers of polymorphisms had been identified. It was difficult and extremely expensive to carry out. However, the Human Genome Project has now identified the whole human genetic sequence and the HapMap project has systematically identified nearly all common human genetic variants. At the same time, advances in technology have meant that it is possible to genotype up to a million single nucleotide polymorphisms (SNPs) in one experiment. Systematic gene identification is now being carried out for many complex diseases through Genome-Wide Association (GWA) studies. This is the equivalent of a candidate gene study of all human genes all at once. Typically, at least 1000 cases and a similar number of controls are genotyped with 100 000–1 000 000 SNPs and any positive results are assessed for replication in larger population samples. At the time of

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writing, leading biomedical journals are being filled with the results of such studies, which are identifying robust and strong genetic effects for many complex disorders.

Genome screens for genetic linkage to asthma Childhood-onset asthma runs strongly in families and studies of twins with asthma or AD show a heritability of approximately 60% (Larsen et al. 1986; Duffy et al. 1990; Schultz Larsen 1993). Many genome screens have been carried out to search for genetic linkage to these illnesses. Genome screens type individuals in families using evenly spaced genetic polymorphisms (typically microsatellite markers) covering all the chromosomes in the genome. Linked regions (loci) are then mapped in detail to identify the underlying disease gene or genes. At least 11 full genome screens have been reported for asthma and its associated phenotypes (reviewed in Cookson 2003; Wills-Karp & Ewart 2004) (Fig. 57.1). These have identified 10 regions of linkage that were replicated between screens and four regions that were statistically significant but not reproduced by other groups (Cookson 2003). It is widely recognized that variations in study design, such as population size, marker density, and effect magnitude, can cause linkage peak location to fluctuate some 20–30 centimorgan (cM) from the true disease locus (Darvasi et al. 1993; Kruglyak & Lander 1995; Williams et al. 1999). It is therefore possible that linkage peaks that are located within a single contiguous 30-cM interval may represent the pleiotropic effects of a common underlying genetic substrate. Several confirmed examples of this are available in the literature, including most recently the reduction of AD and ichthyosis vulgaris QTL to nonsense mutations in the same gene on chromosome 1 (Palmer et al. 2006). Genome screens have also been carried out for many other immune diseases with a genetic basis and have identified regions of linkage that are shared between diseases (Becker

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Fig. 57.1 Genome screens for asthma, AD, and other immune disorders. The results are shown schematically and include only the most significant linkages. The length of the bars indicates imprecision of localization. Clustering of disease susceptibility genes is found for the MHC on chromosome 6p21 as well as in several other genomic regions. (See CD-ROM for color version.)

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et al. 1998). Asthma, for example, consistently shows linkage to the major histocompatibility complex (MHC) (Cookson 2002), in common with many other diseases, and linkage loci for asthma also overlap with loci for ankylosing spondylitis on chromosomes 1p31–36, 7p13, and 16q23; with Type 1 diabetes on 1p32–34, 11q13, and 16q22–24; and with multiple sclerosis and rheumatoid arthritis on 17q22–24 (Cookson 2002). These findings may mean that the susceptibility to different diseases arising from these loci is influenced by individual genes in various forms (alleles). Alternatively, as in the case of the MHC, disease susceptibility may be modified by physical clusters of genes that have a variety of effects on immune responses. In either event, these shared loci are of particular interest to map, because they promise a broad understanding of as yet unknown immune processes.

Genome screens for other epithelial diseases Genome-wide linkage screens based on AD disease status have identified three loci that meet accepted criteria for suggestive linkage, and one locus that meets criteria for significant linkage (Lander & Kruglyak 1995). These loci are positioned, respectively, on chromosomes 1q21 (Cookson et al. 2001), 17q25 (Cookson et al. 2001), 18q11–12 (Haagerup et al. 2002), and on chromosome 3q21 (Lee et al. 2000). In general, the genetics of allergic rhinitis (AR) have been much less investigated than asthma or AD. Two genetic linkage studies have been carried out (Haagerup et al. 2001; Bu et al. 2006), and one locus on chromosome 3q13 has been mapped in detail (Brasch-Andersen et al. 2006). Using a 30-cM coincidence criterion to examine current AD genome-wide mapping data, linkage peak colocalization can be declared in two locations for AD and asthma, chromosomes 1q21 (Cookson et al. 2001; Ferreira et al. 2005) and 18q11–12 (Bradley et al. 2002; Haagerup et al. 2002; Ferreira et al. 2005), with no colocalization observed for AD and AR. If these criteria are relaxed somewhat to include linkage peaks that meet only nominal criteria for significance but which have been replicated to within 30 cM, further evidence of coincidence can be detected on chromosome 3q21 with all three traits demonstrating evidence of linkage to this locus. This level of genomic coincidence (Fig. 57.2) is surprisingly low given the degree of phenotypic comorbidity that is observed in clinical settings. One potential explanation for this is that relative to asthma, genetic analyses of AD and AR are in their infancy. As such, linkage analyses based on asthma phenotypes have identified a comparatively high number of contributory loci, providing a more comprehensive portrait of the underlying genetic architecture (Fig. 57.2). Several genome scans, however, have now been completed in each disease area and, based on the available data, substantially greater overlap has been observed between AD and other phenotypes which are not characterized by atopy. In 2001, when the first genome screen for AD was published (Cookson et al. 2001), a striking pattern of alignment was

Genetics of Asthma and Atopic Dermatitis

Bronchial asthma Atopic dermatitis Allergic rhinitis

Fig. 57.2 Significant genetic linkages for atopic dermatitis (AD), bronchial asthma (BA), and allergic rhinitis (AR). Object size pertains to the number of genome-wide significant or suggestive loci as mapped using a binary measure of disease status. Significance is based on empirical calculations where available, or the widely accepted criteria of Kruglyak and Lander (1995). Data represented in this figure are derived from genome-wide scans only and exclude intermediate or combination phenotypes. There is a generous area of overlap between loci for asthma and AD and BA; loci for AR appear separate, but there have been far fewer genetic studies of this condition. (See CD-ROM for color version.)

observed between AD susceptibility loci and loci mapped for psoriasis, a second chronic inflammatory skin disease. Three of the four suggestive or significant AD loci that have been identified to date lie within just 5.3 cM of a genome-wide significant psoriasis locus (based on the peak marker coordinates within the Marshfield genetic linkage map) (Tomfohrde et al. 1994; Capon et al. 1999; Enlund et al. 1999). For one locus on chromosome 17q25, linkage to the same peak marker (D17S784) was found for both skin diseases (Tomfohrde et al. 1994; Cookson et al. 2001). The chromosome 18q11–12 region for AD also maps to within 4.05 cM of a psoriasis linkage peak, but this peak falls short of genome-wide criteria for suggestive linkage (Asumalahti et al. 2000). These data suggest that mechanisms of epithelial inflammation and immunity may prove central to the pathogenesis of AD in contrast with the historic emphasis on atopy.

Genome-wide association studies of asthma A GWA study of childhood asthma involving approximately 950 children with asthma and 1200 control subjects has been reported (Moffatt et al. 2007). This study identified a single major locus on chromosome 17q12, and identified a novel gene ORMDL3 as conferring asthma susceptibility. The gene encodes a protein with unknown function. It contains four transmembrane regions and may act as a transporter. The gene is only weakly associated with atopy and is unlikely to operate through allergic mechanisms. In large population samples, the gene robustly confers a risk of approximately 1.7.

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Although ORMDL3 dominated the results of this study, numerous other potential peaks of association appeared in the data. These may be validated by combined analyses of other GWAs that are soon to be completed, and by large-scale genetic epidemiology studies in the European population.

Single gene disorders: Netherton’s disease and ichthyosis vulgaris The positional cloning of novel genes from regions linked to complex diseases is a long and difficult business. In contrast, the identification of Mendelian (single gene) disorders is much more straightforward and can sometimes give insight into complex diseases. Netherton’s disease is a rare recessive disorder characterized by a generalized congenital erythroderma. Importantly, children with this syndrome consistently develop symptoms of atopic disease (hay fever, food allergy, urticaria, and asthma) and high levels of serum IgE (Judge et al. 1994; Bitoun et al. 2002). In 2000, mutations in the gene encoding a serine protease inhibitor known as SPINK5 or LEKTI were shown to be causing disease in these patients (Mägert et al. 1999; Chavanas et al. 2000). Subsequent work has shown that common polymorphisms in SPINK5 (particularly Glu420Lys) modify the risk of developing AD, asthma, and elevated serum IgE levels (Walley et al. 2001; Kato et al. 2003; Nishio et al. 2003; Kabesch et al. 2004), indicating that SPINK5 might be involved in an unexpected pathway to the development of atopic disease. The SPINK5 protein contains 13 active protease inhibitor domains, which are joined together by linking domains. The sequence of each of the SPINK5 protease inhibitory domains is slightly different (Mägert et al. 1999), suggesting a polyvalent action against multiple substrates. SPINK5 is expressed in the outer epidermis, the sebaceous glands and around the shafts of hair follicles (Komatsu et al. 2002), indicating that it might be important for the inhibition of environmental proteases such as those that arise from bacteria or allergens. This is consistent with the observation that one-third of patients with AD suffer frequent serious bacterial infections in the skin, and the lesions of over 90% of AD patients are colonized with Staphylococcus aureus (Leyden et al. 1974; Christophers & Henseler 1987). Moreover, nearly all strains of S. aureus from skin lesions of AD have high levels of proteolytic activity (Miedzobrodzki et al. 2002), which is in contrast to the low levels typical of control strains isolated from healthy carriers (Miedzobrodzki et al. 2002). Many children with AD have high titers of IgE specific for allergens from the ubiquitous house-dust mite (HDM) Dermatophagoides pteronyssinus, so named because it feeds on human skin shed from the outermost cornified layer. The major HDM allergens (Der p 1 and Der p 2), present in the fecal pellets, are proteases that have profound effects on epithelial cells, including disruption of intercellular adhesion, increased paracellular permeability, and initiation of cell death (Winton et al. 1998). If these external sources of proteases are important

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in disease pathogenesis, then inhibition of protease activity is a novel approach to the therapy of AD. Indeed, encouraging results have been obtained from a small study in which α1-proteinase inhibitor was effective in the treatment of AD (Wachter & Lezdey 1992), although more comprehensive studies are now required. Ichthyosis vulgaris is another recessive cutaneous disorder caused by loss-of-function homozygous or compound heterozygous mutations, R501X and 2282del4, in exon 3 of the gene encoding filaggrin (FLG) (Smith et al. 2006). Approximately 8% of patients with AD have clinical features of ichthyosis vulgaris (Tay et al. 1999). FLG is major component of the cornified cell envelope of the stratum corneum and the outermost barrier of the skin. The two FLG mutations are strongly associated with AD in populations of European origin (Palmer et al. 2006). This is a robust genetic finding that has been widely reproduced. The variants are carried by 9% of people of European origin (Palmer et al. 2006), and more than double the risk of developing AD (Palmer et al. 2006; Morar et al. 2007). Although it has been claimed that FLG variants may also predispose to asthma (Palmer et al. 2006), asthma is very common in children with AD and the FLG protein is not expressed in the airways. We have found no evidence of an independent association to asthma in a large collection of families with AD (Morar et al. 2007).

The epidermal differentiation complex Genetic linkage studies of AD and psoriasis have both highlighted the importance of chromosome 1q21 (Capon et al. 1999; Cookson et al. 2001). This chromosomal region contains a collection of genes known as the epidermal differentiation complex (EDC) (Mischke et al. 1996). Many of these genes have shown increased expression in AD (Nomura et al. 2003) as well as in psoriasis (Bowcock et al. 2001; Nomura et al. 2003; Zhou et al. 2003). Although polymorphisms in individual EDC genes have not yet been associated with disease, it has been established that disease susceptibility alleles are contained within the cluster (Capon et al. 1999). Several gene families are present within the complex: these encode the small proline-rich proteins (SPRRs), S100A calcium-binding proteins, and late envelope proteins (LEPs) (Mischke et al. 1996; Marshall et al. 2001). The SPRR and LEP genes encode precursor proteins of the cornified cell envelope and are involved in keratinocyte terminal differentiation (Lohman et al. 1997; Marshall et al. 2001). Expression of the EDC proteins occurs late during the maturation of epidermal cells (Hardas et al. 1996) and, like SPINK5, EDC proteins are mainly localized just beneath the cornified envelope (Christiano 1997). Global gene expression studies have been used to investigate the skin lesions of active psoriasis (Bowcock et al. 2001; Zhou et al. 2003). In these studies, 30 of the genes in and around the EDC were differentially expressed when normal and involved skin were compared, with several S100 and

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SPRR family members upregulated in psoriatic skin (Bowcock et al. 2001; Zhou et al. 2003). The expression pattern of particular EDC genes in AD has not yet been described in detail, but it has been observed that SPRR2C is expressed at a level 10 times higher in psoriasis than in AD and that S100A12 expression is three times higher (Nomura et al. 2003). The known functions of some of the EDC gene products indicate that the skin is not functioning as a passive barrier. Accordingly, the S100 calcium-binding proteins are often secreted and have a wide range of immunologic actions (Donato 2001). It is now of interest to investigate the role of these proteins in inflammatory diseases.

Genes identified for asthma and other epithelial diseases Like psoriasis, Crohn’s disease (IBD) affects epithelial surfaces and has a strong genetic component. The positional cloning of susceptibility genes for asthma and both of these other disorders has been remarkably successful recently. Specifically, four completely novel genes underlying asthma have been identified by positional cloning from chromosomes 2q14 (DPP10) (Allen et al. 2003), 7p14 (GPRA) (Laitinen et al. 2004), 13q14 (PHF11) (Zhang et al. 2003), and 20p (ADAM33) (Van Eerdewegh et al. 2002). The functions and activities of these genes are as yet poorly understood, but they certainly do not fit into classical pathways of asthma pathogenesis. ADAM33 is expressed in bronchial smooth muscle, and is thought to alter the hypertrophic response of bronchial smooth muscle to inflammation (a component of a process known as airway remodeling) (Van Eerdewegh et al. 2002). PHF11 encodes a nuclear receptor that is part of a gene complex containing a histone methyltransferase (SETDB2), a regulator of HDAC (RCBTB1), and a nuclear transport molecule (KPNA3). DPP10 encodes a prolyl dipeptidase, which may remove the terminal two peptides from certain inflammatory chemokines (Allen et al. 2003), and GPRA encodes an orphan G protein-coupled receptor named GPRA (G protein-coupled receptor for asthma susceptibility). It is of interest that DPP10 and GPRA are both concentrated in the terminally differentiating bronchial epithelium. The homologous layer in the epidermis is the site of maximal expression of SPINK5 and the genes of the EDC (Fig. 57.3). Many of the genes identified by candidate gene studies may also exert their effects at the mucosa. Polymorphisms in the interleukin (IL)-13 gene influence mucus production as well as serum IgE levels (Kuperman et al. 2002). FCER1B (MS4A2) variants modify the activity of the high-affinity receptor for IgE on mast cells (Traherne et al. 2003) and the receptor for key mast cell signaling factor PTGD2 is also associated with asthma. Microbial pattern recognition receptors of the innate immune system are expressed on dendritic and other cells, and polymorphism in CD14 (Baldini et al. 1999), TLR-2 (Eder et al. 2004), NOD2 (Kabesch et al. 2003), and TIM-1 (McIntire et al. 2003) have all been shown to influence asthma

Genetics of Asthma and Atopic Dermatitis

susceptibility. Other recognized effects are from TNFA (Moffatt & Cookson 1997), which encodes a potent proinflammatory cytokine released by many cells including airway epithelial cells, and TGFβ (Pulleyn et al. 2001), which is an important local regulator of epithelial inflammation. Several other recent observations have indicated the importance of proteins that are expressed by epithelial cells in conferring susceptibility to (or protection against) disease. Terminally differentiating keratinocytes also maximally expresses the psoriasis susceptibility genes (PSORS2: SLC9A3R1) recently cloned from chromosome 17q25 and SLC12A8 from chromosome 3q21 (Hewett et al. 2002) (Fig. 57.3). PSORS1 is another psoriasis susceptibility locus on chromosome 6p. The region is complex and genetic studies have implicated both HLACw*0602 and the neighboring corneodesmosin (CDSN) gene as mediating the susceptibility to psoriasis (Tiilikainen et al. 1980; Ahnini et al. 1999; Asumalahti et al. 2000; Allen et al. 2001). CDSN is also expressed in terminally differentiating keratinocytes and is a key linking component in the stratum corneum (Allen et al. 2001). Susceptibility to IBD is conferred, at least in part, by the NOD2 gene on chromosome 16p (Hugot et al. 2001; Ogura et al. 2001), DLG5 on chromosome 10q23 (Stoll et al. 2004), and OCTN (SLC22A4) on chromosome 5q31 (Peltekova et al. 2004). NOD2 mutants interfere with the function of Paneth cells, which are most numerous in the terminal ileum and are critically important in enteric antibacterial defenses (Hisamatsu et al. 2003; Lala et al. 2003). DLG5 and OCTN are most highly expressed in terminally differentiating epithelial cells (Fig. 57.3). Taken together, these findings emphasize the importance of epithelial defense mechanisms in each of these diseases. Significantly, previous understanding of asthma, AD, psoriasis and IBD has centered on mechanisms in the adaptive immune system, often with an emphasis on the Th1/Th2 paradigm. The results of these genetic studies indicate that understanding innate mechanisms of epithelial defense is essential to the treatment and prevention of these disorders.

Mechanisms of epithelial immunity In evolutionary terms, epithelial surfaces have had to cope with infections and other insults long before the appearance of the adaptive immune system, and most life on earth still exists without the help of an adaptive immune system. Immunity must first have evolved in the primitive epithelium, and the subsequent evolution of specialized immune effector cells of the innate and adaptive immune system has built upon mechanisms initially developed in epithelial cells. It should therefore not be surprising that gene expression studies indicate that epithelial cells are very active immunologically. Keratinocytes in the skin are an extremely wellstudied epithelial cell and are known to produce a wide range of cytokines (Tomic-Canic et al. 1998). Although this activity

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Terminally differentiated keratinocytes

Suprabasal layer

Stratum corneum

Skin

Expression of SPINK5, GPRA, SCL9A3R1 SCL12A8 and EDC genes

Epidermis

Basal layer

Dermis

Mucus

Airway

Goblet cell: IL-13

Outermost epithelial cell layer: DPP10, GPRA and SPINK5

Basal epithelial cell: TNF-a Dendritic cell: CD14, TLR2, TIM-1 and NOD-2 (also expressed by other cells including epithelial cells)

Mast cell: FceR1-b

Smooth muscle cell: ADAM33

has been assumed to be secondary to signaling from classical immune cells (Freedberg et al. 2001), keratinocytes express functional receptors such as CD14 and Toll-like receptor (TLR)4 which recognize lipopolysaccharides, and are capable of inducing inflammatory responses without preinduction by other cells (Song et al. 2002). Pure cultures of pneumocytes also show marked alteration of expression of cytokines, DNAbinding proteins, and NF-κB-regulated genes following exposure to respiratory pathogens (Belcher et al. 2000; Ichikawa et al. 2000). The mechanisms through which the epithelium may interact with the environment include barrier defenses, the recogni-

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Fig. 57.3 Tissue distribution of genes causing susceptibility to asthma, AD, and IBD. DPP10, GPRA, and SPINK5 are found in terminally differentiated airway epithelium. SCL9A3R1, SCL12A8, SPINK5, GPRA, and the genes of the EDC are found in terminally differentiating keratinocytes, and OCTN, DLG5 and GPRA are found in the corresponding layer of the intestinal epithelium. The function of all these positionally cloned genes is uncertain. Patternrecognition receptors for microbial products such as CD14, TLR2, TIM-1, and NOD-2 are found in a variety of cells, including dendritic and epithelial cells. TNFA is released by epithelial cells, as well as effector cells of the immune system. ADAM33 is found in bronchial smooth muscle and is believed to modify bronchial responsiveness to allergens. (See CD-ROM for color version.)

tion of danger, and call for help from specialized inflammatory cells (Fig. 57.4).

Barrier defenses The simplest form of defense consists of erecting a mechanical barrier to bacterial entry and bacterial proteolysis. The ubiquitous presence of bacteria on the surfaces of the body means that an effective barrier needs to be augmented by protease inhibition and by bacteriostatic and antibiotic molecules. Numerous components that make up this barrier have now been defined, many of which are encoded in the EDC on chromosome 1q21.

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Release of active defenses: antimicrobials, proteinase inhibitors and lipase inhibitors

Barrier defenses: stratum corneum (or mucus in the lung and intestine)

Danger Stratum corneum

Recognition of danger or damage through various PRRs

Call for help: release of cytokines, chemokines and inflammatory mediators Fig. 57.4 Defense mechanisms of epithelial cells. Epithelia resist damage through the stratum corneum of the skin or mucus in the airway or intestine. Immune response to external antigens is induced only in the presence of danger and damage. Danger is recognized through patternrecognition receptors, with the resultant release of active defenses such as antimicrobials or antiproteinases, and signaling molecules to recruit specialized immune cells. Different receptors recognize different microbial or other factors, and are capable of modifying the immune signaling milieu appropriately. In this model, Th1 or Th2 responses are driven by the nature and site of the initial injury. (See CD-ROM for color version.)

The location of SPINK5 expression and its polyvalent activity indicate that it may be an important component of the epithelial barrier. Other protease inhibitors with similar roles are encoded by a cluster of genes on chromosome 20q12 (Clauss et al. 2002), close to a region that has shown linkage to AD and asthma (Cookson et al. 2001). These proteins, which have homology to whey acidic protein (WAP) are produced at epithelial surfaces and include elafin and secretory leukocyte protease inhibitor (SLPI) (Clauss et al. 2002). Elafin is a component of the cornified envelope, and has antimicrobial activity against Gram-negative and Gram-positive bacteria (Simpson et al. 1999). SLPI is found in airway surface fluid, where it may play a prominent role in mucosal defenses against microbial attack (Singh et al. 2000). SLPI has been demonstrated to be a potent antimicrobial agent with antiretroviral, bactericidal, and antifungal activity (Tomee et al. 1998). The functions of other members of the WAP cluster have, however, not yet been studied. The importance of airway surface fluid and airway mucus is often neglected in the current understanding of allergic processes. In addition to SLPI, the airway surface fluid contains other small antimicrobial peptides such as defensins

Genetics of Asthma and Atopic Dermatitis

and cathelicidins (Bals et al. 1998a,b; Huttner & Bevins 1999), which are secreted in response to damage and danger signals, and significant antimicrobial activity is also conferred by lysozyme and lactoferrin (Brogan et al. 1975; Singh et al. 2000). While the airway mucosa produces high levels of immunoglobulins, particularly IgA, as shown by microarray analyses of gene expression in the nasal mucosa (Benson et al. 2002), deficiency or low levels of IgA are associated with an increased prevalence of atopic disease (Kaufman & Hobbs 1970; Ludviksson et al. 1992). Failure of nonspecific components of the epithelial barrier, such as IgA or SPINK5, can therefore give rise to increases in serum IgE and the manifestations of atopy. This suggests a hypothesis in which IgE responses to allergens are either the result of an inability to prevent allergens from breaching epithelial surfaces or the secondary penetration of allergens into epithelial surfaces that have already been damaged by other factors. The concept of a general barrier failure in atopic disease may help to explain why more than 95% of the serum IgE in most individuals is directed against unknown antigens, and why many individuals with asthma and AD do not have atopy. Additionally, it could explain why specific immunotherapy and treatment with IgE-specific antibodies do not abolish asthma or AD in patients with atopy.

Danger recognition Before an organism can respond to infection and damage it first has to recognize that danger and damage are present. Microbial infection is recognized by pattern-recognition receptors (PRRs) that respond to a wide variety of pathogenassociated molecular patterns (PAMPs). In addition to the recognition of microbial antigens, the initiation of an immune response also requires “danger” signals produced by injured tissues (Gallucci et al. 1999; Matzinger 2002). Many PRRs, including pulmonary collectins, surfactantassociated proteins A and D, C-reactive protein (CRP) and soluble CD14 (sCD14), act by binding microbial molecules and facilitating their neutralization or degradation by specialized cells (Palaniyar et al. 2002).Other PRRs produce intracellular signals when they bind to particular microbial ligands. The TLRs are the best studied of these and are now known to induce specific reactions to a wide variety of bacterial and fungal components (Kopp & Medzhitov 2003). Intracellular PAMPs, particularly peptidoglycan, are recognized by the NOD/CARD family of proteins (Inohara & Nunez 2003). The NOD/CARD proteins are themselves part of a wider family of NBS-LRR (nucleotide-binding site and leucine-rich repeat) proteins that bind microbial products through their LRRs and bear structural similarities to PRRs in plants (Chamaillard et al. 2003). This family alone contains over 40 members and it is likely that the list of PRRs will continue to grow. The initial immune response to allergens now needs to be examined in the context of these innate mechanisms for

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responding to foreign antigens. The requirement for tissue injury to trigger an immune response means that allergens cannot be simply considered to be soluble proteins that accidentally induce immune responses because they appear in the wrong place. It is more likely that they excite an immune response because they damage epithelial integrity. The toxic effects of Der p 1 and Der p 2 from HDM are well documented (Winton et al. 1998). In addition the observation that Fel d 1, the major cat allergen, degrades denatured collagens and cleaves fibronectin (Ring et al. 2000) and that the major grass allergen Phl p 5 is an RNAase (Bufe et al. 1995) favor the hypothesis of epithelial damage by allergens. If allergen damage to the airways or skin initiates disease through innate pathways, then treatment strategies to circumvent specific adaptive antigendriven immune reactions are unlikely to be successful. Several PRRs have been examined for a role in asthma, usually in studies that have been looking for mechanisms to explain the hygiene hypothesis. While CD14 polymorphisms have been associated with total serum IgE levels (Baldini et al. 1999), TLR4 does not appear to be associated with asthma (Raby et al. 2002; Noguchi et al. 2004), and although TLR2 polymorphisms may show association in children raised on farms (Eder et al. 2004), they do not seem to be associated with asthma in the general population (Noguchi et al. 2004). None of these studies, however, have tested for IgE responses to particular allergens, so systematic studies of PRR activation in asthma and AD are now desirable.

The call for help The recognition of danger is followed by the induction of active defenses against microorganisms and the recruitment of help from specialized cells of the innate and adaptive immune systems. Many molecules secreted by epithelial cells in response to danger have the ability to regulate immune reactions and recruit cells of the innate and adaptive immune systems. These include S100 proteins from the EDC, chemokines, the human cathelicidin cationic antimicrobial peptide LL-37 (Tjabringa et al. 2003), and defensins, which possess structural and signaling similarities to chemokines (Yang et al. 2002). Chemokines have already been widely investigated as targets for therapy of inflammatory disorders. In general, however, they have redundant actions and targeting single cytokines has to date been ineffective in dampening inflammation. In silico studies have suggested that the novel asthma gene DPP10 may serve as a checkpoint in the activation or deactivation of chemokines that carry an X-X-proline-Xserine motif at their N-terminal (Allen et al. 2003). Chemokines which carry this motif include SDF-1, IP10, eotaxin and RANTES (Allen et al. 2003). The nature of these putative substrates tentatively suggests that DPP10 may be a target for the therapy of asthmatic airway inflammation. It is of interest that many of these early signaling molecules also have antimicrobial activities as well as the ability to

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regulate immune reactions. Molecules with this dual function include several S100 proteins (Miyasaki et al. 1993; Brandtzaeg et al. 1995; Gottsch et al. 1999; Steinbakk et al. 1990; Yoshio et al. 2003) and about two-thirds of chemokines (Yang et al. 2003). This duality of function may indicate the evolution of immune signaling from molecules originally secreted purely in defense.

Early innate modulation of Th2 or Th1 responses The nature of the immune response is first influenced by the specific signals that are involved in the early recruitment of immune components to the site of inflammation. As different PRRs are capable of signaling through different pathways, different pathogens or antigens can induce different immune responses (Pulendran et al. 2001; Palaniyar et al. 2002). The nature of the local immune response may also be strongly influenced by tissue-specific factors, and it has been suggested that epithelial cells in general may have a tendency to initiate Th2 rather than Th1 responses (Matzinger 2002). In addition, there is evidence that dendritic cells from the airways have Th2 cytokine production as their default (Stumbles et al. 1998), and that the induction of Th2 or Th1 responses by dendritic cells depends on the stimulus with which they are activated (Mazzoni & Segal 2004). The perception that specific early signals induced by different infections (or damage by different proteins or other entities) may modify the nature of the subsequent immune response has implications for the Th1/Th2 paradigm of atopic disease. One important issue is the timing of establishment of the Th2-cell bias. On the one hand, Th1 or Th2 responses to allergens are fixed at the time of first exposure in early childhood and the bias may be subsequently manipulated by bacterial or other adjuvants. On the other hand, Th1 or Th2 responses may develop as a consequence of activation of particular PRRs by particular PAMPs that are present in allergens. The therapeutic options are different for the two possibilities. If Th2 responses to allergens are the default mechanism, then the ability to modulate the Th2/Th1 balance therapeutically is diminished. Alternative therapeutic possibilities may stem from examination of the specific events that are induced by allergen contact with epithelial surfaces. These may include boosting of the epithelial barrier with protease inhibitors, blocking of particular PRR ligand-binding sites and their downstream signals, or interference with the early signals of inflammation, such as those potentially identified by positional cloning studies.

Microbial infection and atopic disease As mentioned previously, while the opportunistic bacterium S. aureus can be detected on the skin of only around 5–30% of the general population (Williams et al. 1990; Higaki et al. 1999; Tay et al. 1999), up to 93% of AD patients exhibit S. aureus

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colonization (Aly et al. 1977), with the extent of colonization being shown to correlate with disease severity (Williams et al. 1990; Guzik et al. 2005). In addition to enhanced colonization, up to 57% of AD cases also demonstrate type I sensitization towards staphylococcal superantigenic exotoxins (Leung et al. 1993). These are large-molecular-weight protein secretions that have an enhanced capacity to activate T cells and macrophages. Epicutaneous administration of these toxins appears to induce an eczematous reaction in both healthy and atopic skin, characterized by erythema and induration (Stranger et al. 2007). Likewise, a positive linear relationship has been observed between anti-exotoxin IgE in the serum of AD patients and the severity of their disease (Nomura et al. 1999). Consistent with these data, the administration of corticosteroids and antibiotics, either alone or in combination, has been shown to yield significant clinical benefits, particularly in moderate to severe cases of AD (Marzluff et al. 2002). Type I sensitization to the lipophilic Malassezia yeast species (also known as Pityrosporum) appears to also be enhanced in individuals with AD. Although colonization rates and densities appear not to be affected. Type I sensitization as inferred from skin-prick, patch and RAST, (radioallergosorbent) tests can be detected in around 35– 87% of AD patients (Mayser & Gross 2000; Tengvall Linder et al. 2000; Zargari et al. 2001; Johansson et al. 2003). Similar levels of reactivity are not seen in healthy controls or patients with either upper (hay fever) or lower (asthma) airway disease. Recent research has highlighted a specific reactivity toward Malassezia sympodialis, which may be a specific causal factor for AD (Fischer Casagrande et al. 2006). The evidence in favor of defective barrier function in asthma is much less clear than in AD. However, individuals with asthma do appear to carry an enhanced risk for certain respiratory infections. In particular, asthmatics have been shown to carry an increased risk for invasive pneumococcal disease caused by the bacterium Streptococcus pneumoniae (odds ratio 2.4) (Talbot et al. 2005), colonization of the nares by S. aureus (Graham et al. 2006), and atypical bacterial respiratory infections caused by Chlamydia pneumoniae or Mycoplasma pneumoniae. These last two pathogens alter ciliary function and damage ciliated epithelial cells within the bronchial mucosa (reviewed in Johnston et al. 1995). Viral infections of the upper respiratory tract are also recognized as a major precipitant of asthma exacerbations. Hospital admissions for asthma correlate with seasonal variation in the prevalence of viral respiratory infections (Johnston et al. 1995), and respiratory viruses, in particular rhinoviruses, have been associated with 80– 85% of acute exacerbations in school-age children (Johnston et al. 1995) and 44% of exacerbations in adults (Nicholson et al. 1993). Providing a potential mechanism for this association, recent evidence has shown that human rhinoviruses (HRV) can also be isolated from the lower airways of infants with recurrent respiratory

Genetics of Asthma and Atopic Dermatitis

symptoms, and that these viruses may be detected several weeks post infection (Malmstrom et al. 2006). The presence of HRV in the lower airways correlates with impaired airway conductance in these individuals (Malmstrom et al. 2006), and this effect appears to operate independently of other clinical characteristics such as AD disease status and atopy. These data suggest that HRV may be able to invade and persist within the lower airways where it contributes toward inflammation and airway obstruction in asthmatics.

Microbial exposure and protection against atopic disease Current understanding of the hygiene hypothesis rests on the suggestion that microbial stimulation during early life is essential for the normal development of the immune system and to achieve the “correct” cytokine balance (Rook & Stanford 1998). Nevertheless, the evidence described above suggests that damage to the epithelium is most probably the initiating event in atopic disease, and the Th1 or Th2 form of subsequent inflammation may be secondary to the nature of the damage. Alternative mechanisms whereby bacterial products modify the risk of atopic disease include the enhancement of an effective airway barrier by induction of mucus production through IL-13 stimulation (Kuperman et al. 2002), or induction of sufficient polyclonal IgA or IgE to provide nonspecific protection against allergens. Additionally, a protective role for microbes may follow the acquisition of distinct commensal or symbiotic organisms. Once an individual’s commensal microflora is established in the first year of life it remains relatively stable (Hooper & Gordon 2001; Bjorksten 2004). Substantial differences have been observed in the intestinal microflora between neighboring countries with a different prevalence of atopic disease (Sepp et al. 1997), and between atopic and nonatopic children within each of these countries (Bjorksten et al. 1999). As commensal and symbiotic organisms actively manipulate host immunity and the activity of other bacteria (Hooper & Gordon 2001), it should be considered that commensal–host–pathogen interactions might contribute to the increase in prevalence of asthma and AD.

Parent-of-origin effects The risk of transmission of atopic disease from an affected mother is approximately four times higher than from an affected father (Moffatt & Cookson 1998). Similar parent-oforigin effects have been noted in other immunologic diseases, including Type I diabetes (Warram et al. 1984; Bennett & Todd 1996), rheumatoid arthritis (Koumantaki et al. 1997), psoriasis (Burden et al. 1998), IBD (Akolkar et al. 1997), and selective IgA deficiency (Vorechovsky et al. 1999).

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The mechanisms for these parent-of-origin effects are unknown. They may result from immune interactions between the fetus and the mother, which are recognized to take place through the placenta as well as through breast milk (Holt et al. 1999). Alternatively, the maternal effect may be the result of genomic imprinting. Genomic imprinting is the process where the genes from one parent are differentially expressed to the allele derived from the other parent (Hall 1990; Reik & Walter 2001). Imprinting appears important when there is a conflict of interest between maternal and paternal genes, such as those that control fetal growth. It has also been suggested that there is a postnatal phase in which immunologic imprinting in the neonate is determined by maternal immune status (Lemke & Lange 1999), and that this is mediated through the interaction of maternal IgG antibodies and infantile T-cell receptors (Lemke & Lange 2002). There is experimental evidence to show that maternal antibodies regulate clonal development of immune cells (Ono et al. 1974), that sensitivity to particular antigens can be transferred to the fetus (Stern 1976), and that maternal immunization with ovalbumin in mice protects against IgE responses in offspring (Jarrett & Hall 1979, 1983). The observation that the risk of asthma declines with increase in birth order (Strachan 1989; von Mutius et al. 1994; Svanes et al. 1999) may be further evidence of the importance of maternal–fetal immune communication if the maternal immune system is modified by recurrent pregnancies or the affects of aging. Several known genes show parent-of-origin effects on allergic disease. These genes include the FcεRIβ locus on chromosome 11q13 (Cookson et al. 1992; Cox et al. 1998), the SPINK5 gene from chromosome 5q34 (Cookson et al. 2001), and as yet undiscovered genes at loci on chromosomes 4 and 16 (Daniels et al. 1996). Monoallelic expression has been identified for a number of cytokines (Chess 1998) and allelic exclusion is a feature of T-cell receptor and B cell-receptor loci expressed in individual cells (Mostoslavsky et al. 2001). These phenomena are probably the result of the need for individual cells to commit to a consistent pattern of cytokine production or to react to a single epitope. There is no evidence to date that the expressed allele derives preferentially from one parent or another. It is possible, however, that the bias to one parent or another may be incomplete, or that it is most marked at a critical period in development of the immune system. If, as seems likely, the parent-of-origin effect is part of a general phenomenon affecting several immune-related loci and several diseases, it should be assumed that this process is in some way adaptive. Epigenetic markers of imprinting, such as the variable presence of methylation on CpG residues (Reik & Walter 2001), now need to be combined with knowledge of parental atopic status as well as parental genotype.

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Conclusions Although our knowledge of host susceptibility factors to asthma and AD is still incomplete, involvement of proteins in the outermost layers of the skin and mucosa is a consistent theme emerging from genetic studies of these and other epithelial diseases. In addition to specific questions raised by the discovery of individual genes that contribute to asthma susceptibility, such as DPP10 and GPRA, a focus on the epithelium encourages investigation of the mechanisms by which allergens damage the epithelium, and the danger signals and PRRs that they activate. The profound effects of SPINK5 mutations suggest that high IgE levels and symptoms of AD or asthma may result from failure of the epithelial barrier and that damage from nonallergens such as S. aureus in AD may be important in driving disease in some circumstances. The initiation and maintenance of inflammation at epithelial surfaces is induced by local mechanisms that have profound effects on the outcome of the immune response. Genetic studies indicate the presence of many previously unknown or ignored molecules, such as the S100 proteins from the EDC, which may be novel targets for the control and suppression of epithelial inflammation. A central mystery of asthma revolves around the protective effect of microbial exposures in childhood. This effect has primarily been investigated in the context of Th1- or Th2-biased responses, but it may also be explicable by mechanisms confined to epithelial surfaces. Some genetic factors, such as CD14 and TLR2, have been identified that may interact with this microbial environment. The full characterization of genes interacting with microbes in asthma and AD may be expected to shed light on these critical events.

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Song, P.I. et al. (2002) Human keratinocytes express functional CD14 and Toll-like receptor 4. J Invest Dermatol 119, 424–32. Steinbakk, M., Naess-Andresen, C.F., Lingaas, E. et al. (1990) Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet 336, 763– 5. Stern, C.M. (1976) The materno-foetal transfer of carrier protein sensitivity in the mouse. Immunology 30, 443– 8. Stoll, M., Comeliussen, B., Cosiello, C.M. et al. (2004) Genetic variation in DLG5 is associated with inflammatory bowel disease. Nat Genet 36, 476–80. Strachan, D.P. (1989) Hay fever, hygiene, and household size. BMJ 299, 1259–60. Stranger, B.E., Forrest, M.S., Dunning, M. et al. (2007) Relative impact of nucleotide and copy number variation on gene expression phenotypes. Science 315, 848–53. Stumbles, P.A., Thomas, J.A., Pimm, C.L. et al. (1998) Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity. J Exp Med 188, 2019– 31. Svanes, C., Jarvis, D., Chinn, S. & Burney, P. (1999) Childhood environment and adult atopy: results from the European Community Respiratory Health Survey. J Allergy Clin Immunol 103, 415–20. Talbot, T.R., Hartert, T.V., Mitchel, E. et al. (2005) Asthma as a risk factor for invasive pneumococcal disease. N Engl J Med 352, 2082– 90. Tay, Y.K., Khoo, B.P. & Goh, C.L. (1999) The epidemiology of atopic dermatitis at a tertiary referral skin center in Singapore. Asian Pac J Allergy Immunol 17, 137– 41. Tengvall Linder, M., Johansson, C., Scheynius, A. & Wahlgren, C. (2000) Positive atopy patch test reactions to Pityrosporum orbiculare in atopic dermatitis patients. Clin Exp Allergy 30, 122–31. Tiilikainen, A., Lassus, A., Karvonen, J., Vartiainen, P. & Julin, M. (1980) Psoriasis and HLA-Cw6. Br J Dermatol 102, 179–84. Tjabringa, G.S., Aarbiou, J., Ninaber, D.K. et al. (2003) The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol 171, 6690– 6. Tomee, J.F., Koeter, G.H., Hiemstra, P.S. & Kauffman, H.F. (1998) Secretory leukoprotease inhibitor: a native antimicrobial protein presenting a new therapeutic option? Thorax 53, 114–6. Tomfohrde, J., Silverman, A., Barnes, R. et al. (1994) Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q. Science 264, 1141– 5. Tomic-Canic, M., Komine, M., Freedberg, I.M. & Blumenberg, M. (1998) Epidermal signal transduction and transcription factor activation in activated keratinocytes. J Dermatol Sci 17, 167–81. Traherne, J.A., Hill, M.R., Hysi, P. et al. (2003) LD mapping of maternally and non-maternally derived alleles and atopy in FcepsilonRIbeta. Hum Mol Genet 12, 2577– 85. Van Eerdewegh, P., Little, R.D., Dupuis, J. et al. (2002) Association of

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Epidemiology of Asthma, Atopy, and Atopic Disease Debbie L. Jarvis, Seif O. Shaheen and Peter Burney

Summary

Definition of atopy and IgE sensitization

Asthma, atopy, and atopic diseases are very prevalent and becoming still more prevalent in the adult population. Although the increase among children may have become less universal, the epidemic is likely to continue among adults. The hypotheses currently being explored to explain the high prevalence of asthma and atopic disease will probably not explain the whole of the increase or the major part of the variation in prevalence of these conditions. However, it is important that we understand these diseases better if we are to provide effective strategies for managing the epidemic. This understanding will require the combination of good mechanistic studies with high-quality epidemiologic studies involving wide varieties of populations. Confirmation of the findings will only come from large-scale intervention studies.

Pepys (1975) described atopy as “that form of immunological reactivity of the subject in which reaginic antibody now identifiable as IgE antibody is readily produced in response to ordinary exposure to common allergens of the subjects’ environment.” This definition excludes those who have high total IgE levels but no specific IgE, as measured by skin-prick tests or radioallergosorbent test (RAST) to common environmental allergens. Strictly speaking, it also excludes those who have become sensitized to only one uncommon allergen, perhaps as a result of occupational exposure. In 2001, the European Academy of Allergology and Clinical Immunology (EAACI) proposed that atopy was a “personal or familial tendency to produce IgE antibodies in response to low doses of allergens, usually proteins, and to develop typical symptoms such as asthma, rhinoconjunctivitis, and eczema/dermatitis” (Johansson et al. 2001). This definition of atopy combines IgE sensitization with symptoms and may cause some confusion. To avoid this, we will use the term IgE sensitization throughout this chapter and wherever possible avoid the term “atopy.” The presence of IgE antibodies can be assessed by skin tests performed by a variety of methods and allergen extracts and considered positive using different criteria. Comparisons of the prevalence of positive skin-prick tests from different studies conducted at different time points and in different parts of the world are only valid if skin testing has been well standardized between the studies. Clinicians tend to validate skin testing against its prediction of clinical disease but epidemiologists wish to identify individuals as IgE-sensitized regardless of the presence of disease and with minimal bias due to observer variation. For this reason, criteria for test positivity in clinical and epidemiologic practice often differ (Chinn et al. 1996). Allergen-specific IgE in serum can be measured directly. Although skin testing and blood testing (Fig. 58.1) are both indicators of specific IgE, they do not always give the same results in population-based surveys. A positive skin test relies on both the presence of specific IgE and degranulation of sensitized mast cells, and a response in the tissues to the mediators. The lack of agreement between skin and serologic tests may reflect differences in the presentation of allergen or

Introduction The prevalence of asthma, hay fever, and eczema increased substantially during the 20th century. Asthma, hay fever, and eczema are associated with the presence of serum IgE to common aeroallergens, although serum-specific IgE is neither necessary nor sufficient for clinical disease. The rapid increase in disease prevalence over such a short period of time has been attributed to environmental or lifestyle changes that have increased the prevalence of IgE sensitization and/or the manifestation of disease in IgE-sensitized subjects but, as yet, no clear cause for the observed changes has been identified. This chapter describes the time trends in the prevalence of IgE sensitization, asthma, hay fever, and eczema over the last 50 years and presents the epidemiologic evidence for risk factors. Similarities and differences in the epidemiology of clinical disease and IgE sensitization are identified.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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100

Prevalence (%)

Switzerland Japan E. Germany Denmark Finland Greenland

10 1973

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1985

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1995

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Year Fig. 58.1 Increase in population prevalence of positive RAST tests.

the response to antigen–antibody binding or tissue responses. Measurement errors will occur in both tests. Individuals with IgE sensitization often have elevated total IgE (Barbee et al. 1987a). Total IgE levels within a population are normally distributed after logarithmic transformation, with a relatively small proportion of individuals having levels of total IgE below the limits of detection to the test.

Definitions of asthma, hay fever, and eczema Attempts were made to standardize the definition of asthma as long ago as 1958, when the CIBA Guest Symposium defined asthma as “the condition of subjects with widespread narrowing of the bronchial airways which changes in severity over short periods of time either spontaneously or under treatment” (CIBA Guest Symposium 1959). Other definitions have been proposed (American Thoracic Society 1962; American College of Physicians & American Thoracic Society 1975; World Health Organization 1975; National Institutes of Health 1992), but none improves substantially on this early attempt which is little more than a broad description of a disease entity and is open to wide interpretation. Asthma has been identified by symptoms suggestive of disease, by diagnosed disease, and by physiologic measures of airway responsiveness including the bronchial response to histamine, methacholine or exercise, serial measurement of peak flow, and response to bronchodilators. A video questionnaire showing people experiencing symptoms has been developed for use in epidemiologic studies in children and adolescents (Shaw et al. 1992). Results from questionnaire data and the video are not always the same, but the video questionnaire had a high coefficient of repeatability and was as good a predictor of bronchial reactivity (BR) as the International Union Against Tuberculosis and Lung Disease (IUATLD) Bronchial Symptoms Questionnaire. Two large international studies, the European Community Respiratory

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Health Survey (ECRHS) (Burney et al. 1994) and the International Study of Asthma and Allergy in Childhood (ISAAC) (Asher et al. 1995), have developed standardized questionnaires for the assessment of asthma and asthma-like symptoms. These questionnaires have been widely adopted by other epidemiologic groups. More recently, there have been calls for greater recognition and better definition of the different patterns of disease (“phenotypes”) and this may prompt further work in the development of standardized questionnaires (Wenzel 2006). Standardization of testing for BR has been poor and it has been difficult to compare studies performed at different locations and in different geographic areas. Some expert bodies have provided recommendations for standardization (European Respiratory Society 1993; American Thoracic Society 2000), but variations in protocols between studies are common (Chinn 1998). The relationship of BR to clinical disease may differ depending on the agent used (Cockcroft & Davis 2006) and BR to histamine and methacholine is not specific for asthma, being independently associated with age, atopy, and smoking (Burney et al. 1987). BR to methacholine (and histamine) is associated with airway caliber and responses are seen in patients with chronic obstructive pulmonary disease. Responses to adenosine monophosphate (and exercise) are more closely associated with allergic inflammatory responses in the lower airways (de Meer et al. 2002; Holgate 2002) and are more specific for allergic asthma. Rhinitis is “inflammation of the nose” that occurs in response to several agents including infection and environmental allergens. The Allergic Rhinitis and its Impact in Asthma (ARIA) initiative has defined allergic rhinitis clinically as a “symptomatic disorder of the nose induced by an IgE-mediated inflammation after allergen exposure of the membranes lining the nose” with symptoms including rhinorrhea, nasal obstruction, nasal itching, and sneezing (Bousquet et al. 2001). Many epidemiologic surveys, however, ask directly whether subjects have “hay fever” or “nasal allergies” or whether nasal symptoms are present “when you did not have a cold or the flu,”, sometimes with questions on the seasonality of symptoms. Considerable progress has been made in the development of a definition of eczema or “atopic dermatitis” suitable for use in epidemiology. In the UK, a working party derived a minimum set of criteria for diagnosis (Williams et al. 1994a,b,c). This group also developed a structured protocol using photographs to train observers to determine the prevalence of atopic dermatitis in children. Most large epidemiologic studies, including ISAAC and ECRHS, have used questions based on this work.

IgE sensitization and asthma and allergic disease Not all people reporting asthma, rhinitis, or eczema are IgEsensitized, and it is of interest to know the proportion of

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disease that is likely to be attributable to sensitization. Pearce et al. (1999) collated all studies up to 1999 with information on asthma and wheeze and IgE sensitization, and concluded that less than 60% of reported wheeze was explained by IgE sensitization. A similar approach was adopted to examine rhinitis, and a similar proportion of disease was explained by the presence of IgE (Zacharasiewicz et al. 2003). The fraction of asthma attributable to sensitization varies between countries and is higher for more severe disease (Sunyer et al. 2004). The interpretation of population attributable fraction, however, requires caution as it oversimplifies a complex relationship. Spuriously high attributable risks may arise if adults known to be IgE-sensitized are more likely to receive a diagnosis of asthma from their physicians, and the proportion of disease explained by IgE may depend on the presence of other factors that precipitate a clinical response (e.g., exposure to allergen). On the other hand, by looking at the proportion of wheeze attributable to IgE sensitization, many children and adults who would not normally be regarded as asthmatic will be included, so deflating the figure.

Geographic variation The ECRHS has clearly demonstrated substantial geographic variation in the prevalence of IgE sensitization among adults as measured by serum IgE (Burney et al. 1997) (Fig. 58.2) and by skin-prick tests (Bousquet et al. 2007). In general, higher

Epidemiology of Asthma, Atopy, and Atopic Disease

levels of IgE sensitization are seen in English-speaking countries and in mid-Europe compared with northern and southern Europe. Both the ECRHS and others have described large differences in sensitization between children growing up in western Europe compared with former Eastern bloc countries (von Mutius et al. 1992, 1994; von Hertzen et al. 2006). The prevalence of IgE sensitization to outdoor allergens varies (Burney et al. 1997; Bousquet et al. 2007) between areas and the pattern is broadly consistent with the known patterns of vegetation in Europe. The ECRHS has also shown large geographic variation in reported asthma symptoms (European Community Respiratory Health Survey 1996) and in BR (Chinn et al. 1997). In general, these show the same geographic pattern as for specific IgE, although there are some exceptions. ISAAC covers a much wider geographic area than ECRHS, and also shows a higher asthma prevalence in English-speaking countries, and a lower prevalence in many parts of the developing world (International Study of Asthma and Allergies in Childhood Steering Committee 1998). Both ECRHS and ISAAC have also shown substantial variations in the prevalence of “hay fever and nasal allergies” (European Community Respiratory Health Survey 1996) and allergic rhinoconjunctivitis (Strachan et al. 1997a; International Study of Asthma and Allergies in Childhood Steering Committee 1998). In general, higher levels of hay fever are observed in communities with higher levels of asthma and higher levels of IgE sensitization, but such a sweeping

United States

Fig. 58.2 Geographic variation in prevalence of IgE sensitization in adults taking part in ECRHS I. Areas with high (red) and low (blue) prevalence of sensitization to any one of house-dust mite, cat, Timothy grass, or Cladosporium species in ECRHS II (white circles represent areas that participated in the study but which did not have a particularly high or low prevalence). (From Jarvis & Burney 1998, with permission.) (See CD-ROM for color version.)

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generalization masks some important exceptions. For example, in ISAAC, the Nigerian sample of children had one of the highest prevalences of allergic rhinoconjunctivitis (and atopic eczema), while the prevalence of reported asthma symptoms was relatively low. Both ECRHS (Harrop et al. 2007) and ISAAC (International Study of Asthma and Allergies in Childhood Steering Committee 1998) have reported geographic variations in atopic eczema. Even within countries there may be differences in the prevalence of asthma and allergic disease between populations. There has been considerable interest in the difference in prevalence of sensitization and asthma between rural and urban populations in Africa. More than 25 years ago, a substantially lower prevalence of symptoms (Godfrey 1975) and exercise-induced bronchoconstriction (Van Niekerk et al. 1979) was seen in rural compared with urban children living in Gambia and South Africa, respectively, and this has been supported by other observations in Africa (Keeley et al. 1991; Yemaneberhan et al. 1997; Ng’ang’a et al. 1998; Calvert & Burney 2005) and elsewhere (Anderson 1978).

Time trends There are now several population-based studies that have examined changes in IgE sensitization at two time points using the same methodology on each occasion and most show an increase in prevalence. The two earliest were from Switzerland (Gassner 1992) and Japan (Nakagomi et al. 1994), showing that the prevalence of specific IgE to grass and other allergens had increased in children and teenage girls, respectively. Skin tests were used to document increases in IgE sensitization in adults in London between 1974 and 1988 (Sibbald et al. 1990), but one Australian study, which used slightly different methods to assess atopy on each occasion, failed to show an increase in children (Peat et al. 1994) or in adults (Peat et al. 1992) over a 10-year period. With the advent of cheaper and more rapid serum-specific IgE testing, further research has shown increases in serum-specific IgE in Western populations (Linneberg et al. 2000; Kosunen et al. 2002; Krause et al. 2002; Law et al. 2005). One recent study suggests the increases in the prevalence of IgE sensitization that has occurred over the last 50 years may have now slowed down (Braun-Fahrlander et al. 2004). The ECRHS measured specific IgE on two occasions 8 years apart in a cohort of European adults (Jarvis et al. 2005) (Fig. 58.3). There was little evidence of age-related changes in sensitization, but the study showed strong evidence for a cohort-related increase in the prevalence of sensitization in the populations studied. Although in ECRHS this increase was greater for sensitization to grass than to house-dust mite, most studies suggest that the increase in IgE sensitization has occurred for all allergens (Law et al. 2005). From the middle of the 20th century up to the mid-1990s almost all studies that measured prevalence in the same popu-

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40 35 30 25 20 15 10 5 0

20 < 27.5

27.5 < 35

35 < 42.5

42.5 < 50

50+

Age group (years) Fig. 58.3 Age cohort prevalence of sensitization to either house-dust mite, grass, cat or Cladosporium in adults taking part in ECRHS II. (From Jarvis et al. 2005, with permission.)

lation at different times showed an increasing prevalence of asthma and wheezy illness (Burney 2002). This amounted to an approximate doubling of disease every 14 years. More recently, this trend has begun to change. The largest and most recent study of time trends in childhood asthma is the repeat ISAAC survey. This showed that over the previous decade the prevalence of asthma in 6–7 year olds had both increased and decreased in different parts of the world. Furthermore, the pattern of change in older children did not mirror the pattern of change in the younger children (Asher et al. 2006). In the populations taking part in the ECRHS, the prevalence of asthma and treatment for asthma increased over an 8-year follow-up, although the prevalence of wheeze remained relatively stable (Chinn et al. 2004). The number of studies that have examined the change in objective markers of asthma over time is more limited (Wieringa et al. 2001). A study in South Wales showed that over a 15-year period the increase in prevalence of asthma in schoolchildren from 6% to 12% was accompanied by an increase in airway reactivity in response to exercise, suggesting that the observed increase is not merely an artifact of increased awareness of asthma among doctors and their patients (Burr et al. 1989). However, when the study was repeated in 1998, asthma symptoms had increased while there had been a decrease in exercise-induced bronchoconstriction (Burr et al. 2006). This latter observation might be explained by the more widespread use of inhaled corticosteroids amongst the children. Increases in both the prevalence of wheeze and BR have also been noted in children living in New South Wales, Australia (Peat et al. 1994), but when adults living in a coastal area of Western Australia were surveyed in 1981 and 1990, the prevalence of wheeze increased (17.5–28.8%) without any associated increase in the prevalence of BR (Peat et al. 1992). In Belgian conscripts, the prevalence of asthma at medical examination increased from 2.4 to 7.2% between 1978 and 1991, while the proportion of asthmatic individuals with measurable BR to methacholine remained constant, providing evidence that the increase in asthma had been genuine and not related to increased reporting of symptoms or changes in labeling of disease (Dubois et al. 1998).

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Rate per 10 000

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0–4 y 5–14 y

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1 1955

60

65

70

75

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85

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05

Year Fig. 58.4 Hospital admission rates for asthma in England for 1958–2005. Trends for 65+ years are similar to 45–65 years and are omitted for clarity. (From Anderson et al. 2007, with permission.)

Many of the large studies that have shown increases in asthma prevalence have also shown increases in hay fever and eczema (Burr et al. 1989; Ninan & Russell 1992). The ISAAC study has described these changes in many centers across the world in both 6–7-year-old children and 13–14-year-old children (Asher et al. 2006). Further indirect evidence for increases in asthma, hay fever, and eczema comes from health service utilization data. Hospital admission rates for asthma showed a steady increase during the 1980s (Anderson 1989; Vollmer et al. 1993; Hyndman et al. 1994; Anderson et al. 2007) as did general practice consultations (Fleming & Crombie 1987; Gupta et al. 2007) although rates, at least in England, may have fallen over the last decade (Fleming et al. 2000; Gupta et al. 2007) (Fig. 58.4). While genes undoubtedly play a role in the etiology of asthma and allergic diseases, environment and lifestyle are more likely to explain the wide geographic variation in the prevalence of these conditions, and must explain the rapid increases in prevalence.

Personal risk factors Age Cross-sectional studies show a peak in the prevalence of sensitization in early adulthood. This has often been interpreted as reflecting age-related declines in IgE sensitization, but it is more likely that it reflects generational increases in the levels of sensitization. In order to assess the effect of aging on sensitization, cohort studies that use standardized methods of assessment over time are required. There are now several large birth cohorts in Western countries that are collecting such information but most of these children have not yet reached adulthood. Levels of specific IgE increase steadily throughout childhood. Although reversion of positive skin tests has been reported in some individuals taking part in these surveys, it is not clear whether they

Epidemiology of Asthma, Atopy, and Atopic Disease

represent test failures or genuine permanent change in IgE status. As mentioned above, the ECRHS study suggests little change in sensitization over an 8-year period in adults aged between 20 and 50 years, and this probably holds true for older adults as well (Barbee et al. 1987b). Atopic dermatitis is very common in the first 3 years of life (Perkin et al. 2004) but remission after this age is common, although not all studies agree on the likely frequency of remission and it may not be lifelong (Vickers 1980; Williams & Strachan 1998). Hay fever is generally thought to be uncommon before the age of 5 years (Broder et al. 1974; Kulig et al. 2000). The incidence and period prevalence of wheeze and asthma is higher in children than adults (Broder et al. 1974; Anderson et al. 1992; Yunginger et al. 1992). Early childhood wheezing is heterogeneous and the different patterns have different associated risk factors. In the Tucson study, Martinez et al. (1995) proposed three patterns of wheeze in children up to the age of 6 years: transient early childhood wheeze, wheeze starting after the age of 3 years, and persistent wheeze. Remission of symptoms may not be permanent (Kelly et al. 1987) and is unlikely after the age of 30 years (Bronnimann & Burrows 1986). In the 1958 British birth cohort, only onequarter of those who had a history of asthma or wheezy bronchitis by the age of 7 years reported wheeze in the past year at the age of 33 years. Recurrence of wheeze after prolonged remission was associated with other allergic diseases and cigarette smoking (Strachan et al. 1996a). Follow-up of the 1970 British birth cohort showed that, of those who had reported wheeze at the age of 5 years, only 15% had wheeze that persisted to 16 years (Lewis et al. 1995). It is not known if those who have disease that has remitted are at an increased risk of respiratory symptoms or poor lung function in old age.

Gender Most studies suggest that boys are more likely to be IgEsensitized in childhood (Uekert et al. 2006) than girls, and to a large extent this is seen in adults also (Jarvis et al. 1999). There are several studies showing that boys have more wheeze and asthma than girls, a difference that seems to become less apparent as the children get older, and which may even reverse after puberty (Anderson et al. 1992). Some caution should be exercised in the interpretation of these data, as wheeze in early childhood may be a manifestation of lung size, and boys may have smaller lungs than girls at birth (Becklake & Kauffmann 1999). Although Broder et al. (1974) reported more hay fever in boys than girls, others have found little difference between males and females (Huurre et al. 2004). A higher prevalence of eczema has been reported in females compared with males (Mortz et al. 2001; Harrop et al. 2007) at most ages except very early in childhood (Moore et al. 2004), with one study suggesting the excess of eczema in girls aged 5–7 years was related to a relative excess of skin complaints that were not associated with IgE sensitization (Mohrenschlager et al. 2006).

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As there appears to be a difference in the prevalence of IgE sensitization and some allergic disorders between the sexes, and this difference may not be constant throughout the life course, there has been a growing interest in the role of sex hormones in these diseases. Much of the current research in this area is in the area of asthma but the precise nature of these associations is very uncertain (Forbes 2005). However, early onset of puberty, particularly in obese children, is a risk factor for the persistence of asthma into adolescence (Guerra et al. 2004; Varraso et al. 2005), and use of hormonal treatments has been associated with an increase in asthma in older women in cross-sectional (Lange et al. 2001) and longitudinal studies (Barr et al. 2004b).

Race There are inconsistent reports of racial differences in the prevalence of IgE sensitization and asthma, hay fever, and eczema. Where differences have been observed between races it is difficult to determine whether they reflect differences in genetic predisposition, exposure to environmental risk factors or cultural attitude to disease (Barnes 2006). In the USA, disease prevalence in African-American, European American, and Hispanic groups has been examined (Schwartz et al. 1990; Joseph et al. 2000; Litonjua et al. 2005). The comparisons are confounded by differences in social class and material wealth experienced by these groups, which in turn lead to differences in lifestyle and environmental exposures. In Africa, in a wealthy suburb of Harare, the prevalence of exercise-induced bronchoconstriction was similar in the wealthy black population and the wealthy white population (Keeley et al. 1991). In New Zealand, differences in asthma prevalence have been observed between populations of Maori, Pacific Islander, and European descent, these differences having remained despite substantial increases in disease over time (Pattemore et al. 2004). In Leicester, UK, the prevalence of wheeze, but not IgE sensitization or BR, was higher in white children than Asian children (Carey et al. 1996), with the risk of BR and atopy in the Asian children associated with the extent to which they had adopted a Western diet. Confounding by factors other than diet may have occurred, but this study indicates that observed racial differences in disease prevalence may be highly dependent on lifestyle factors. In the UK, Williams et al. (1995) showed a higher prevalence of current eczema, as determined by clinical examination, in black Caribbean children (16.3%) than in white children (8.7%), though prevalence in a small sample of black African children was even lower at 4.7%. Racial differences in atopic dermatitis have also been reported in the USA (Moore et al. 2004) and in Germany (Gruber et al. 2002).

Socioeconomic status The prevalence of asthma in children is higher in wealthy countries (Stewart et al. 2001), but the relation of social class (a marker of personal wealth, at least in the UK) to asthma

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appears to have changed over time, asthma having once been a disease of the more advantaged and becoming more a disease of the disadvantaged (Rona et al. 1999). The reasons behind these associations are complex. Socioeconomic status may be important in disease etiology, disease severity, or labeling and treatment of disease (Rona 2000). Rhinitis and eczema are also more common in wealthy countries, but at an individual level their association with socioeconomic status is not certain and, as for asthma, may be changing. In the early 1990s, eczema in the UK was considered a disease of the advantaged (Williams et al. 1994d), but in Sweden eczema has been linked to lower socioeconomic status (Almqvist et al. 2005). In a South African study, both atopic dermatitis and rhinitis were more common in children who were socially mobile, defined as being poor but attending a school in a wealthier area, than in poor children who were educated near their homes (Mercer et al. 2004). This may reflect differences in the reporting of disease between social groups.

Family structure In 1989, Strachan reported a strong negative association of birth order with the prevalence of hay fever and proposed that exposure to older siblings led to an increased level of infections in early life, which in turn decreased the likelihood of allergic disease (Strachan 1989). This hypothesis was termed the “hygiene hypothesis” and in the following years many studies have examined the association of IgE sensitization and allergic disease with family size, birth order, exposure to other infants in early life, and exposure to infections. Many of these studies have been included in a recent review (Karmaus & Botezan 2002). The negative association of IgE sensitization (most strongly IgE sensitization to grass) with birth order or exposure to other children in early life has been widely reported in both children and adults, and similar associations have been consistently seen for hay fever. This association has been present in populations born since the early 1900s (Kinra et al. 2006). The associations of family structure with total IgE (Svanes et al. 1999; Karmaus et al. 2001), asthma, and eczema are less clear. When protective effects are reported for asthma, asthma has generally been defined by the presence of symptoms with IgE sensitization. Older siblings or daycare attendance may protect against later wheezing (generally thought to be associated with IgE sensitization), but may increase the risk of early childhood wheeze, much of which is related to acute viral infections (Ball et al. 2000). The hypothesis that the protection from allergy by large sibships is due to infection is strengthened by the observation that children from small sibships who attend childcare facilities early in life are similarly protected (Kramer et al. 1999) (Fig. 58.5). A possible alternative explanation has been proposed based on the observation that women who have had more children have less atopy (Sunyer et al. 2001). Although

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Prevalence %

25 20 6 to 11 months 12 to 23 months 24 months or over

15 10 5 0 Small

Large Family size

Fig. 58.5 Prevalence of IgE sensitization in children from small (N = 669) and large (N = 1761) families by age at which first attended daycare. (From Kramer et al. 1999, with permission.)

Epidemiology of Asthma, Atopy, and Atopic Disease

measles infection were much less likely to be IgE-sensitized many years later than children who had been vaccinated and not had measles. However, the children in the measles group had survived a severe epidemic, and differential mortality may be the most likely explanation for the findings. If, among unvaccinated children, those who were already atopic were more likely to suffer severe infection and die, this would have left measles survivors who were less atopic than vaccinated children (Shaheen et al. 1996). Furthermore, a very large population-based study in Finland showed higher rates of atopy in those who had experienced measles compared with those who had been vaccinated against measles (Paunio et al. 2000).

Enteric infections longitudinal studies to assess the influence of successive pregnancies on atopy have produced conflicting results (Harris et al. 2004; Sunyer et al. 2005), this hypothesis reverses the causal pathway, and suggests that part of the birth order effect may reflect changes in the maternal immune system with successive pregnancies. Even if this is the case, it cannot explain the observation that younger siblings are also protective for hay fever, independently of older siblings (Matricardi et al. 1998; Westergaard et al. 2005). There is some evidence, thus far unexplained, that the older sibling effect may be stronger for older brothers than older sisters (Strachan et al. 1997b; Svanes et al. 1999).

Children and adults living in large families are likely to experience higher levels of orofecally transmitted infections, including hepatitis. Matricardi and colleagues examined Italian military recruits and showed that the prevalence of IgE sensitization was lower in those with evidence of previous infection with hepatitis A (Matricardi et al. 1997) and, in another study based in a similar population, in those with previous Toxoplasma gondii and Helicobacter pylori infections (Matricardi et al. 2000). However, other studies in the UK have not replicated these observations (Jarvis et al. 2004; Law et al. 2005).

Bowel flora

The “hygiene hypothesis,” infections, farming, and parasites Acute viral infections in infancy The hygiene hypothesis initially proposed that children exposed to poor hygiene and increased infections in early life had lower levels of IgE sensitization and allergic disease. However, in a large study in Sheffield, England, in which health visitors’ records were examined and the presence of hay fever and positive skin-prick tests assessed in adolescence, no association was seen between symptoms of neonatal infectious disease or infectious disease in the child’s family, and health outcomes (Strachan et al. 1996b). Similarly reported infections in early life did not explain the family size association in a Tasmanian birth cohort (Ponsonby et al. 1999), nor did documented and serologic evidence of infection in a UK birth cohort (Cullinan et al. 2003).

Other viral infections The extensive work by Matricardi and colleagues suggested that orofecal infections were of interest, but he also showed that the virus herpes simplex 1 (which unlike herpes simplex 2 is acquired in early life) was associated with lower levels of IgE sensitization. Other viruses have also been implicated. In a historic cohort study in Guinea-Bissau, children who had had

It seems unlikely that the protective effect of family size or birth order is fully explained by any one of these pathogenic infections. Attention therefore turned to whether commensal bowel flora might have a role in allergic disease through modulatory effects on the immune system. A small Swedish study found marked differences in bowel flora between allergic and nonallergic infants living in Sweden (an area with a high prevalence of allergic conditions) and Estonia (an area with a low prevalence), with lower levels of lactobacilli in allergic children in both countries (Bjorksten et al. 1999). There has also been interest in the use of probiotics to alter bowel flora. However, randomized controlled trials to date have shown little effect on IgE sensitization, although decreases in the prevalence of features characteristic of atopic dermatitis have been lower in those given the active treatment (Kalliomaki et al. 2001) up to the age of 4 years (Kalliomaki et al. 2003). Use of antibiotics, which are known to be associated with changes in bowel flora, has been linked to higher rates of allergic disease in some studies (Farooqi & Hopkin 1998), but not others (Celedon et al. 2002). One birth cohort study has observed positive associations between antibiotic prescriptions and wheeze and seasonal rhinitis, but not IgE sensitization, with the association with wheeze explained by prescriptions for respiratory infections (Harris et al. 2007). It therefore seems likely that the link between antibiotic use and asthma is likely to be explained largely by reverse

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causation, with atopic children possibly having more severe respiratory illness and being therefore more likely to be prescribed antibiotics.

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Anthroposophic lifestyle

30

Farming and proximity to animals Another source of infections may be animals, and the hygiene hypothesis has been proposed as an explanation for the lower levels of atopy seen in farming communities. Following the industrial revolution, an increasing proportion of the European population lived in towns rather than rural areas. This trend continued through the latter half of the 20th century (Sozanska et al. 2007). Children who are brought up on farms have a lower prevalence of IgE sensitization, wheeze, asthma, and hay fever than those who are brought up in the countryside but not on farms (Braun-Fahrlander et al. 1999; Riedler et al. 2001) (Fig. 58.6). This association may, in some part, last into adult life (Leynaert et al. 2001). In the first studies of children on farms, it was observed that allergic outcomes were less common in children who regularly drank unpasteurized milk in the first year of life. Other studies have reported lower levels of total IgE, specific IgE to aeroallergens (Perkin & Strachan 2006), and eczema (Wickens et al. 2002) in those who consume unpasteurized milk, with Perkin and Strachan (2006) observing this association in children of all ages. Riedler et al. (2001) also found lower rates of allergy in children who spent time in the barns or stables at an early age. A large study

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Farming Nonfarming

35 Prevalence (%)

In Sweden, children following an anthroposophic lifestyle are likely to have a high intake of products containing lactobacilli. Steiner schoolchildren in Sweden who follow an anthroposophic lifestyle have lower rates of IgE sensitization, asthma, hay fever, and eczema than children attending non-Steiner schools in the same area (Alm et al. 1999), and prevalence of IgE-related disease is lowest in those who most closely adhered to the anthroposophic lifestyle. However, this lifestyle includes other features such as minimal use of medications and delayed vaccinations in childhood. Steiner schoolchildren living in several countries were examined in PARSIFAL (Prevention of Allergy Risk factors for Sensitisation in Farming and Anthroposophic Lifestyle). The differences in disease prevalence between Steiner and non-Steiner children were not so marked as they had been in the Swedish study, and PARSIFAL suggested that a lower use of antibiotics and of paracetamol was associated with a decreased risk of IgE sensitization (Floistrup et al. 2006). Frequent paracetamol use has been associated with asthma in adults both cross-sectionally (Shaheen et al. 2000; McKeever et al. 2005) and prospectively (Barr et al. 2004a), and in a population-based birth cohort study frequent use of paracetamol in late pregnancy was associated with an increased risk of asthma and elevated total IgE in the offspring (Shaheen et al. 2005). The explanation for the low levels of allergic disease in Steiner schoolchildren is therefore likely to be more complex than originally hypothesized.

40

25 20 15 10 5 0

Asthma

Wheeze

Hay fever

Atopy

Condition Fig. 58.6 Prevalence of symptoms in children of farmers and nonfarmers in Switzerland. (From Braun-Fahrlander et al. 1999, with permission.)

conducted in several countries in Europe has confirmed these findings and suggested that pig farming, feeding silage on the farm, and the child’s involvement in hay-making over prolonged periods are also associated with lower rates of IgE sensitization and asthma in IgE-sensitized individuals (Ege et al. 2007). Levels of endotoxin and other microbial contaminants are high on farms and higher in farm childrens’ mattresses than in those not brought up on farms (Schram et al. 2005), but there is as yet no evidence that these or any other specific contaminants explain the apparent protection afforded by growing up on a farm.

Vaccination and tuberculin sensitivity Concerns have been raised that the increase in asthma and allergic disease is related to the reduced exposure to infections that are prevented by the current extensive vaccination programs. In most observational studies that have examined this, population coverage for vaccination has been high, and because the relatively small proportion of individuals who have not received vaccinations are likely to differ from those who have been vaccinated, confounding by a range of factors (e.g., family history of allergy or social class) is highly likely. This being the case, observational studies reporting more IgE sensitization in those who have had vaccination with pertussis (Odent et al. 1994) should be interpreted with extreme caution. The few randomized controlled trials that have been conducted show little evidence of an important effect (Nilsson et al. 1998) and the public should be reassured that vaccines are safe. In contrast, there has been some interest in the potential benefits of some forms of vaccination. In Japan, children with a strong tuberculin skin reaction had lower rates of allergy (Shirakawa et al. 1997) leading to hypotheses that early administration of the live mycobacterium in the form of BCG may be beneficial. Although there was no evidence that early administration of BCG was associated with lower rates of

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IgE sensitization in children in Sweden (Alm et al. 1998) or Greenland (Krause et al. 2003), or with lower rates of hay fever in the UK (Bremner et al. 2005), other work suggests early administration may be associated with less IgE sensitization in Guinea Bissau (Aaby et al. 2000), and asthma in those with a family history of rhinitis or eczema in Southeast Asian migrants living in Australia (Marks et al. 2003).

house-dust mite (van der Biggelaar et al. 2000). Schistosome antigen-specific concentrations of interleukin (IL)-10 were significantly higher in infected children and were negatively associated with skin-test reactivity to mite.

Exacerbations due to acute viral infections

The current evidence regarding the inheritance of IgE sensitization, asthma, hay fever, and eczema is discussed in depth elsewhere in this book. Rapid advances in the technologies for genotyping mean that samples can be rapidly analyzed and several genes have been identified as being associated with disease. However, many initial findings have not been replicated (Ober & Hoffjan 2006). If the function of a gene is known, and is considered likely to alter the body’s response to a particular lifestyle or environmental exposure, individuals with the relevant genotype may be at substantially increased or decreased risk of disease compared with others. Finding such gene–environment interactions can strengthen the evidence for inferring causal associations between environmental exposures and disease. For example, there is some evidence that a promoter polymorphism in the gene encoding CD14, a receptor for lipopolysaccharide (the major component of endotoxin), may modify the protective effects on atopy of exposure to endotoxin (Simpson et al. 2006) and a farm environment (Leynaert et al. 2006) in early life. Similarly, glutathione S-transferase polymorphisms may modify the effects of prenatal (Gilliland et al. 2002) and postnatal (Kabesch et al. 2004) tobacco smoke exposure on risk of childhood asthma, and the effects of antioxidant supplementation on lung function in asthmatic children (Romieu et al. 2004). Genetic epidemiology may also provide insights into mechanisms and pathogenesis. For example, recent studies have demonstrated that there is greater overlap between genes associated with atopic dermatitis and psoriasis, than between atopic dermatitis and asthma, suggesting that a defective epidermal barrier may play a more important role than atopy in the etiology of atopic dermatitis (Morar et al. 2006). It seems likely that the genetics of asthma and related traits is complex, involving hundreds of genes, each with small effects (relative risks rarely exceeding 1.5). For instance, a recent study from the British 1958 birth cohort showed that the common single nucleotide polymorphisms in the IL-13 gene accounted for only 0.69% of the variance of total IgE (Maier et al. 2006). This means that in order to reliably identify gene associations, sample sizes of several thousand subjects are required, especially if gene–gene and gene–environment interactions are to be detected. The 1958 cohort study, which is of this magnitude, also showed that β2-adrenoceptor polymorphisms were not important determinants of asthma, contrary to previous findings from some much smaller association studies (Hall et al. 2006). This highlights the importance of

Even though the hygiene hypothesis suggests infections in early life may be protective for disease, there is overwhelming evidence that viral upper respiratory tract infections cause exacerbations of asthma in children (Johnston et al. 1995), particularly at the beginning of school term (Johnston et al. 2006), and in adults (Nicholson et al. 1993). In children, infection is associated with wheeze, particularly in children with small lungs (Martinez et al. 1995) and many first episodes of wheeze are associated with an acute infection (Pearson 1958), possibly due to “unmasking” of asthma in susceptible individuals rather than direct causation. Infection with rhinovirus in the first year of life has been associated with the onset of asthma by the age of 3 years and may induce inflammatory mediators that influence airway remodeling and adversely affect lung development (Gern et al. 2005). The relative importance of exposure to allergen and exposure to infections, and the manner of their interaction, is a matter of debate. Adults with asthma and IgE sensitization to common allergens have more prolonged symptoms when they have a rhinovirus infection (Corne et al. 2002), and when infected with rhinovirus the presence of allergen leads to greater morbidity (Green et al. 2002).

Parasites Observations that asthma and allergic disorders are less common in rural African communities have led to investigations of the role of parasitic infection. A recent systematic review suggests that different parasites have different effects, concluding that Ascaris infection was associated with an increased risk of asthma but the opposite was true for hookworm infestation (Leonardi-Bee et al. 2006). To some extent, these conclusions are consistent with a randomized controlled trial in which children were treated with albendazole (an agent used to clear geohelminths including Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale, and Strongyloides stercoralis). This showed no difference in IgE sensitization or allergic diseases between the treatment and placebo groups after 12 months (Cooper et al. 2006). In this study, however, the prevalence of hookworm infestation (A. duodenale) was relatively low in comparison with other infestations. The systematic review did not consider Schistosoma haematobium as there have been relatively few studies investigating its association with allergy. One study has shown that infection with this parasite may decrease the likelihood of having a positive skin-prick test to house-dust mite, even in the presence of serum-specific IgE to

Genetics

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undertaking adequately powered genetic epidemiology studies, to avoid the likelihood of false-positive results, which may be preferentially published.

Exposure to allergen Sensitization Sensitization to allergen requires exposure to allergen. The ECRHS has shown marked geographic variation in Europe in the levels of housedust mite (Zock et al. 2006) and cat allergen (Heinrich et al. 2006) in mattress dust as measured using a standardized method. Der p 1 levels were low in areas with lower winter temperatures (Scandinavian countries) but this was not seen for Der f 1. Levels were also lower in mattresses that had only recently been purchased, in bedrooms that were not on the ground floor and in well-ventilated rooms. Not surprisingly, cat allergen levels were strongly related to the presence of a cat. Among those who do not own a cat, however, they are related to the prevalence of cat ownership within the local community. There is inconsistent evidence that exposure to allergen has increased over the past 30 years. Sporik and colleagues (Sporik et al. 1990) showed no change in housedust mite levels in English homes between 1979 and 1989, but marked increases have been observed in two towns in Australia (Peat et al. 1994) over a 10-year period. The pollen season in London and its immediate surroundings has decreased in length and in severity during the late twentieth century (Emberlin et al. 1993), and in the UK there is little evidence that the number of pets has increased during the past 30 years (Seaton et al. 1994). However, features of modern-day living, with large proportions of time spent in the indoor environment, may have resulted in increased personal exposure to housedust mite and the allergens shed by pets, even if there has been no measurable increase in allergen levels. Despite marked variation in indoor allergen levels there was no evidence in the ECRHS II that the risk of IgE sensitization to housedust mite or cat was higher (or lower) in homes that had more allergen (Heinrich et al. 2006; Jarvis et al. 2007). One reason for this lack of association in adults may be that adult IgE sensitization reflects associations with childhood exposures. However, even if children are studied, associations are far from clear, with reports of paradoxically higher IgE sensitization rates to cat in those with the lowest exposure. This may result from selective avoidance in those children who are at risk but may reflect modified immune responses (Platts-Mills et al. 2001; Hesselmar et al. 2003). Intervention studies of high-risk infants suggest that indoor allergen avoidance does not lead to reduced IgE sensitization in childhood (Corver et al. 2006). In one study, there was even a paradoxical increase in IgE sensitization following mite avoidance (Woodcock 2004). Some studies have suggested that infants born at the time

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of the birch and grass pollen season in Finland (Bjorksten et al. 1980; Bjorksten & Suoniemi, 1981) are more likely than those born in other parts of the year to become sensitized to birch and grass. This would imply that early exposure to allergen is important but these relationships are not consistently seen (Reed & Covallis 1958; Pearson et al. 1977; Morrison Smith & Springett 1979). Reported variation in the prevalence of hay fever by birth month is inconsistent (Anderson et al. 1981; Aberg 1989).

Disease Studies of exposure to house-dust mite have suggested that exposure to high levels of allergen not only increases the risk of sensitization but also increases the risk of clinical disease (Sporik et al. 1990). However, more recent work has not confirmed these associations (Burr et al. 1993; Cullinan et al. 1999; Lau et al. 2000; Marks et al. 2006), and randomized controlled trials of allergen reduction do not show substantial reductions in the development of asthma in early life (Brussee et al. 2005; Marks et al. 2006) or in severity of asthma (Luczynska et al. 2003; Woodcock et al. 2003; Woodcock 2004). Two Cochrane reviews of randomized controlled trials that attempted reductions in indoor allergen exposure as a means of secondary prevention of asthma concluded that, as yet, there was no evidence for beneficial effects of reduction in house-dust mite allergen (Gotzsche et al. 2004) or cat allergen (Kilburn et al. 2001). In the USA, it has been shown that exposure to high levels of cockroach allergen among those sensitized to cockroach greatly increases the likelihood of hospitalization with asthma, and may in part explain the severity of asthma in inner-city children (Rosenstreich et al. 1997). Exposure to outdoor allergen may be an important determinant of severity of disease in asthmatic individuals. Morbidity (Khot et al. 1983) and mortality (Marks & Burney 1997) from asthma in young adults increase in the pollen season in the UK, a seasonal pattern not observed in older adults. Higher death rates in the summer months are not because people are on holiday and a long way from their normal place of healthcare (Nichols et al. 1999). Britton et al. (1988) noted an increase in BR in the south of England between March and June, and a further increase in reactivity in September, but failed to show a relationship between skin sensitivity to pollen and the increased responsiveness in June. In the USA, seasonal variation in attendance at a medical center with asthma associated with specific IgE to ryegrass occurs (Pollart et al. 1988), although sensitization and exposure to other allergens are also important (Pollart et al. 1989). Patients who experienced a respiratory arrest in the US midwest during the season associated with high levels of airborne Alternaria alternata were more likely to be sensitized to A. alternata than those who did not have a respiratory arrest (91% vs. 31%) (O’Hollaren & Yunginger 1991). Epidemics of asthma have occurred in response to high levels of allergen in the air. In Barcelona, these followed the

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release of soybean particles during unloading of soybean cargo at the docks. Case–control studies showed that cases had an increased risk of exposure to high levels of soybean (Sunyer et al. 1992) and appropriate changes to the unloading procedures resulted in cessation of epidemics (Anto et al. 1993). Exposure to castor bean has also been implicated in asthma epidemics (Figley & Elrod 1928; Mendes & Cintra 1953; Ordman 1955) and “thunderstorm asthma” is a well-documented phenomenon (Newson et al. 1998; Marks et al. 2001). A large thunderstorm in southeast England in June 1994, shortly following high recorded levels of pollen, led to a 10-fold increase in attendances at accident and emergency departments with asthma (Venables et al. 1997), and was later attributed to the disruption of pollen grains. Epidemic asthma cases were observed to have IgE to grass and many had a history of hay fever but no previous asthma (Davidson et al. 1996). Exposure to allergen, in particular house-dust mite allergen, has also been implicated in eczema, but the prevalence of eczema in adults taking part in the ECRHS II showed no association of eczema with mattress dust mite level (Harrop et al. 2007). Many randomized controlled trials that have tested allergen avoidance as a primary prevention for asthma have also examined eczema as a secondary outcome and shown no benefit.

Diet Observational studies, mainly cross-sectional, have suggested that a low intake of fruit and vegetables, fish, butter and dairy fat, antioxidants (vitamins C and E, β-carotene, and selenium), magnesium, and n-3 fatty acids, and a high intake of sodium, margarine and n-6 fatty acids may be associated with increased risk of asthma and/or atopy, although evidence is conflicting (Tricon et al. 2006). The hypothesis that a falling intake of n-3 fatty acids relative to n-6 fatty acids (Black & Sharpe 1997) and of antioxidants (Seaton et al. 1994) may have contributed to the rise in asthma and atopy in recent decades in the West has been of particular interest. However, n-3 fatty acid supplementation does not appear to benefit children or adults with established asthma (Thien et al. 2002), and recent trials in asthmatic adults of vitamin C plus magnesium, vitamin E, and selenium, have been disappointingly negative (Fogarty et al. 2003; Pearson et al. 2004; Shaheen et al. 2007). It may be that food-based interventions will hold more promise than supplementation with individual nutrients for the secondary prevention of asthma. Recently, attention has turned to the possible role of nutrition in early life in the inception of asthma and atopic disease (Devereux 2006). Clues are emerging from birth cohort studies to suggest that prenatal nutrition might be important, although evidence to date is not sufficiently compelling to justify a trial in pregnancy aimed at primary prevention. The role of breast-feeding in atopic disease remains controversial,

Epidemiology of Asthma, Atopy, and Atopic Disease

with some observational studies suggesting that it reduces the risk of atopic dermatitis and asthma (Friedman & Zeiger 2005), and others suggesting that it may increase risk. This effect may depend on whether there is a parental history of allergy (Stabell Benn et al. 2004).

Smoking Personal smoking There are several methodologic problems in the identification of associations of smoking with disease, including the “healthy smoker” effect, the tendency for those with diseases such as asthma to avoid smoking (Becklake & Lallo 1990). Total IgE is higher in smokers than nonsmokers (Gerrard et al. 1980; Jarvis et al. 1999), but the association with specific IgE is less certain and may depend on the allergen. Current smokers have been shown to have more IgE to house-dust mite but markedly less specific IgE to cat and to grass (Jarvis et al. 1999). Lower levels of IgE sensitization to grass have also been seen in smokers in Norway (Omenaas et al. 1994) and, in view of this, it is no surprise to note lower levels of hay fever in those who smoke (Wuthrich et al. 1996). On the other hand, occupational studies have shown increased rates of sensitization to occupational allergens including coffee-bean dust (Zetterstrom et al. 1981), ispaghula (Marks et al. 1991), and reactive dyes (Park et al. 1991) in those who smoke. During the Barcelona asthma epidemics, smoking was associated with an increased risk of sensitization to soybean and an increased risk of epidemic asthma (Sunyer et al. 1992). It is well established that smoking is related to chronic bronchitis and to fixed airways obstruction, but whether smoking causes asthma remains highly controversial, some arguing that there is a noncausal association (Piipari et al. 2004) and others that smoking causes increases in asthma severity rather than causing asthma to develop (Siroux et al. 2000). The prevalence of cough and wheeze and bronchial hyperreactivity is increased in those who smoke (Burney et al. 1987) and adults who start to smoke or continue to smoke have greater increases in BR as they age (Chinn et al. 2005).

Passive smoking In 1997, a systematic review and a series of metaanalyses were conducted to assess the health effects of passive smoking on children’s health (Cook & Strachan 1999). There is a very consistent picture of increased respiratory illnesses and symptoms in the children of those who smoke, the risks being greater in young children than older children probably because as children grow up they spend less time in the home with their mother. However, there is little evidence that parental smoking is associated with IgE sensitization. The associations with symptoms may in part be a consequence of in utero exposure to maternal smoking influencing lung growth and making a child more susceptible to wheeze with

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infection. As most mothers who smoke in pregnancy continue to smoke after the child has been born, this effect is difficult to disentangle from the effects of postnatal exposure to tobacco smoke. In adults, respiratory symptoms suggestive of asthma and low lung function have been observed by those who remember that their mother smoked during their childhood (Svanes et al. 2004). Cross-sectional studies show that adults reporting more exposure to other people’s tobacco smoke, particularly in the workplace, have more symptoms suggestive of asthma and more BR (Janson et al. 2001).

Air pollution Air pollution is a general term that represents a wide range of pollutants. The increase in allergy in the West has occurred at a time when the nature of air pollution has changed. In the middle of the 20th century, air pollution was still largely the product of domestic coal burning; 50 years on it is largely due to vehicle emissions. Ishizaki et al. (1987) were the first to notice that Japanese cedar pollenosis was more common in those living near trees in areas of high traffic pollution compared with those living near trees in low traffic areas. However, relatively few studies have assessed objective measures of atopy and traffic-related air pollution at a high level of resolution, and the results so far are not wholly consistent. Hirsch et al. (1999) found no association in an early German study, and Kramer et al. (2000) found an association with sensitivity to airborne allergen with modeled outdoor NO2 exposure, but not with personal NO2 exposure, and then only if a suburban area was excluded from the analysis. However, Wyler et al. (2000) and Nicolai et al. (2003) found an association between traffic counts and pollen sensitization. Penard-Morand et al. (2005) found an association between ozone exposure and pollen sensitization, but no association with the pollutants more directly related to local traffic density (PM10 and NO2) or with SO2. In addition to an influence on allergic responses, air pollution may also affect the airways, altering the expression of all forms of airway disease including asthma. Several studies show that the excess of symptoms associated with air pollution is often confined to those without atopy (Hirsch et al. 1999) or atopic symptoms (McConnell et al. 2006), but not all studies have confirmed this (Modig et al. 2006). Air pollution may have acute effects on asthma. This has been studied in time-series analyses and panel studies. Timeseries studies of asthma mortality and admissions have shown associations with levels of pollution, though there is a great deal of uncertainty about the specific exposures that are important and there are important discrepancies in the evidence. In London, for instance, variations in ozone levels have been associated with daily GP consultations for asthma (Hajat et al. 1999), visits to accident and emergency rooms with

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respiratory complaints (Atkinson et al. 1999), and admissions for asthma (Anderson et al. 1998), as well as total respiratory mortality. However, the data are inconsistent. Neither of the two European multicenter studies of acute air pollution effects using routine health data, Air Pollution and Health: a European Approach (APHEA) I (Sunyer et al. 1997) and APHEA II (Atkinson et al. 2001), found an association between hospital admissions from asthma and ozone levels. APHEA I found associations only with NO2 and APHEA II found associations with small particles. The panel studies have been even less convincing. The largest of these to date, the PEACE study, was a well-conducted international study that had good power to detect effects but was unable to do so (Roemer et al. 1998). Finally, there have been major episodes of air pollution that have been identified as having no clear effect on asthma, including the London smog of 1952 (Fry 1951) and the last major smog epidemic in Europe (Wichmann et al. 1989). The long-term effect of air pollution on asthma outcomes is even less clear. Although the Southern California Children’s Health Study showed that exposure to some pollutants may influence lung growth (Avol et al. 2001; Gauderman et al. 2004), new diagnoses of asthma were associated with highpollutant areas only in those who played three or more team sports (McConnell et al. 2002). In spite of the inconsistency of the findings, there are two indications that there are real effects of air pollution on atopic symptoms and that the underlying mechanism relates to oxidative stress. The first is the experimental evidence that antioxidants can mitigate the effects of air pollutants (Romieu et al. 1998; Grievink et al. 1999), and the second is that those with genetic polymorphisms that make subjects more prone to oxidative stress are more susceptible to ozone (Bergamaschi et al. 2001; Romieu et al. 2004; Romieu et al. 2006). Given the strong biological plausibility that air pollution may affect both atopic disease and asthma, the question arises why the quantification of these effects is so problematic in epidemiologic studies. This may be due to the effects being very small (but, given the almost universal exposure, nonetheless potentially important), the difficulties of assessing exposure of individuals accurately, and the large potential for confounding or effect modification. One specific confounder in the studies of short-term exposure is airborne allergen. This can have a very large effect, as has been shown in a number of epidemics of asthma associated with release of castor bean or soybean allergen into the air (Figley & Elrod 1928; Ordman 1955; Sunyer et al. 1992). Some studies have attempted to deal with this problem by adjusting results for pollens and molds in the air, but this is at best only a partial solution to the problem.

References Aaby, P., Shaheen, S.O., Heyes, C.B. et al. (2000) Early BCG vaccination and reduction in atopy in Guinea-Bissau. Clin Exp Allergy, 30, 644–50.

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areas of West and East Germany. Am J Respir Criti Care Med 149, 358– 64. Wenzel, S.E. (2006) Asthma: defining of the persistent adult phenotypes. Lancet 368, 804–13. Westergaard, T., Rostgaard, K., Wohlfahrt, J., Andersen, P.K., Aaby, P. & Melbye, M. (2005) Sibship characteristics and risk of allergic rhinitis and asthma. Am J Epidemiol 162, 125–32. Wichmann, H.E., Mueller, W., Allhof, P. et al. (1989) Health effects during a smog episode in West Germany in 1985. Environ Health Perspect 79, 89–99. Wickens, K., Lane, J.M., Fitzharris, P. et al. (2002) Farm residence and exposures and the risk of allergic diseases in New Zealand children. Allergy 57, 1171–9. Wieringa, M.H., Vermeire, P.A., Brunekreef, B. & Weyler, J.J. (2001) Increased occurrence of asthma and allergy: critical appraisal of studies using allergic sensitization, bronchial hyper-responsiveness and lung function measurements. Clin Exp Allergy 31, 1553–63. Williams, H.C. & Strachan, D.P. (1998) The natural history of childhood eczema: observations from the British 1958 birth cohort study. Br J Dermatol 139, 834–9. Williams, H.C., Burney, P., Pembroke, A.C. & Hay, R.J. (1994a) The U.K. Working Party’s Diagnostic Criteria for Atopic Dermatitis. III. Independent hospital validation. Br J Dermatol 131, 406–16. Williams, H.C., Burney, P.G., Hay, R.J. et al. (1994b) The U.K. Working Party’s Diagnostic Criteria for Atopic Dermatitis. I. Derivation of a minimum set of discriminators for atopic dermatitis. Br J Dermatol 131, 383–96. Williams, H.C., Burney, P.G., Strachan, D. & Hay, R.J. (1994c) The U.K. Working Party’s Diagnostic Criteria for Atopic Dermatitis. II. Observer variation of clinical diagnosis and signs of atopic dermatitis. Br J Dermatol 131, 397–405. Williams, H.C., Strachan, D.P. & Hay, R.J. (1994d) Childhood eczema: disease of the advantaged? BMJ 308, 1132–5. Williams, H.C., Pembroke, A.C., Forsdyke, H., Boodoo, G., Hay, R.J. & Burney, P.G. (1995) London-born black Caribbean children are at increased risk of atopic dermatitis. J Am Acad Dermatol 32, 212–17. Woodcock, A. (2004) Early life environmental control: effect on symptoms, sensitization, and lung function at age 3 years. Am J Respir Crit Care Med 170, 433–9. Woodcock, A., Forster, L., Matthews, E. et al. (2003) Control of exposure to mite allergen and allergen-impermeable bed covers for adults with asthma. Medical Research Council General Practice Research Framework. N Engl J Med 349, 225–36. World Health Organization (1975) Epidemiology of chronic nonspecific respiratory disease. Bull WHO 52, 251–9. Wuthrich, B., Schindler, C., Medici, T.C., Zellweger, J.P. & Leuenberger, P. (1996) IgE levels, atopy markers and hay fever in relation to age, sex and smoking status in a normal adult Swiss population. SAPALDIA (Swiss Study on Air Pollution and Lung Diseases in Adults) Team. Int Arch Allergy Immunol 111, 396– 402. Wyler, C., Braun-Fahrlander, C., Kunzli, N. et al. (2000) Exposure to motor vehicle traffic and allergic sensitization. The Swiss Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) Team. Epidemiology 11, 450–6. Yemaneberhan, H., Bekele, Z., Venn, A., Lewis, S., Parry, E. & Britton, J.R. (1997) Prevalence of wheeze and asthma and relation to atopy in urban and rural Ethiopia. Lancet 350, 85–90.

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Yunginger, J.W., Reed, C.E., O’Connell, E.J., Melton, L.J., O’Fallon, W.M. & Silverstein, M.D. (1992) A community-based study of the epidemiology of asthma. Incidence rates, 1964–1983. Am Rev Respir Dis 146, 888– 94. Zacharasiewicz, A., Douwes, J. & Pearce, N. (2003) What proportion of rhinitis symptoms is attributable to atopy? J Clin Epidemiol 56, 385–90.

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Zetterstrom, O., Osterman, K., Machado, L. & Johansson, S.G. (1981) Another smoking hazard: raised serum IgE concentration and increased risk of occupational allergy. BMJ 283, 1215–17. Zock, J.-P., Heinrich, J., Jarvis, D. et al. (2006) Distribution and determinants of house dust mite allergen in Europe: The European Community Respiratory Health Survey II. J Allergy Clin Immunol 118, 682–90.

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The Allergy March Ulrich Wahn

Summary The term “allergy march” refers to the natural history of atopic manifestations, characterized by a typical sequence of IgE antibody responses and clinical symptoms that appear during a certain period, persist over years and decades, and often show a tendency for spontaneous remission with age. Knowledge is still limited about the determinants that are modifiable and which may become candidates for preventive intervention. Exposure to indoor allergen during the first 3 years of life may facilitate allergic sensitization, which later is associated with loss of lung function at school age and persistence of asthmatic wheeze throughout adolescence. Prenatal and postnatal exposure to environmental tobacco smoke may increase the risk of allergic sensitization during infancy. Environmental exposure to microbial products such as endotoxins might be considered a protective factor against the development of atopic diseases. Well-controlled intervention studies will be necessary in order to demonstrate where modification of nutritional, environmental, or lifestyle-related factors may be helpful in protecting against the development of atopy and asthma in childhood.

Introduction Atopic diseases such as hay fever, asthma, and eczema are allergic conditions that tend to cluster in families and are associated with the production of specific IgE antibodies to common food or environmental allergens. The process of sensitization may or may not be associated with the induction of clinical symptoms, which are characterized by inflammation and correspond to hyperresponsiveness of skin or mucous membranes. The term “allergy march” refers to the natural history of atopic manifestations, characterized by a typical sequence of IgE antibody responses and clinical symptoms that appear during certain age periods, persist over years and decades, and often show a tendency for spontaneous remission with age. Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Natural history of atopic manifestations Although wide individual variations may be observed, atopic diseases tend to be related to the first decades of life (Bergmann et al. 1994), and thereby to the maturation of the immune system. In general, no clinical symptoms are detectable at birth. Although the production of IgE starts in the eleventh week of gestation, no specific sensitization to food or inhalant allergens as measured by elevated serum IgE antibodies can be detected in cord blood via standard methods. Early findings describing elevated cord blood IgE concentrations as a predictor for clinical manifestations of atopy could not be confirmed (Edenharter et al. 1998). Total serum IgE concentrations after birth increase with age and show distribution over a wide range. The 95th percentile achieved by white children at the age of 1 year is 80 kU/L, is around 400 kU/L at the age of 6 years (Fig. 59.1), and has been demonstrated to peak around puberty. The earliest specific IgE antibody responses directed to food proteins, particularly hen eggs and cows’ milk, may be observed during the first months of life (Nickel et al. 1997). Even in completely breast-fed infants, high amounts of specific serum IgE antibodies to hen eggs can be detected. It has been proposed that exposure to hen egg proteins occurs via the mother’s milk, but this needs further clarification. Sensitization of humans to environmental allergens from indoor and outdoor sources requires more time and is generally observed between the first and tenth year of life. The annual incidence of early sensitization depends on the amount of exposure. In a longitudinal birth cohort study in Germany, a dose–response relationship could be shown between early exposure to cat and mite allergens and the risk of sensitization during the first years of life (Fig. 59.2). It has recently been demonstrated that strong infantile IgE antibody responses to food proteins have to be considered as markers for atopic reactivity in general and are predictors of subsequent sensitization to aeroallergens (Lau et al. 1989, 2000; Kulig et al. 1999a; Illi et al. 2006). Regarding clinical symptoms, atopic dermatitis is generally the first manifestation, with the highest incidence during the first 3 months of life and the highest period prevalence

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1000 Nonatopic and atopic 500

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during the first 3 years (Fig. 59.3). Long-term follow-up studies have demonstrated that 63% of children with infantile eczema were in complete remission by the age of 3, whereas 38% developed an intermittent pattern of disease up to school age (Fig. 59.4) (Illi et al. 2004). The prognosis is mostly determined by the severity and presence of atopic sensitization. Children with atopic dermatitis and wheeze have a marked loss of lung function, suggesting a distinct phenotype rather than a progressive development from atopic dermatitis to asthma (Illi et al. 2004). Seasonal allergic rhinoconjunctivitis is generally not observed during the first 2 years of life, although a minority of children will develop specific IgE antibodies during this early period. Obviously, two seasons of pollen allergen exposure are required before a classical seasonal allergic rhinoconjunctivitis with typical symptoms in association with specific serum IgE antibodies becomes manifest. Prevalence before the end of the first decade in children is around 15% in Central Europe (Kulig et al. 2000). Asthmatic wheezing may be already present during early infancy. The majority of early wheezers turn out to be only transiently symptomatic, whereas a minority may continue to wheeze throughout school age and adolescence. Our understanding of the natural history of childhood asthma is limited, and several datasets support the existence of various asthma subtypes in childhood (Martinez et al. 1995).

Hereditary factors We have known for many years that atopic diseases run in families. The risk of neonates developing atopic symptoms during the first two decades of life is strongly associated with

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6

Fig. 59.1 Percentiles of total serum IgE in children from 1 to 6 years of age as sequentially determined from the prospective birth cohort study MAS. (From Kulig et al. 1999c, with permission.)

manifestations of the disease in their parents and siblings. It is already obvious at the phenotypic level that there is a closer association between specific symptoms like asthma or atopic dermatitis in the child and the same manifestation in parents or siblings than with other atopic manifestations in the family. These clinical observations suggest the presence of phenotypespecific genes (see Chapter 57) (Martinez 1997; Moffatt & Cookson 1998).

Nongenetic factors During the last two decades, two general hypotheses have been proposed in the literature in connection with the observed increases of atopy and asthma in childhood. Firstly, new risk factors that were not known several decades ago might have become relevant in connection with nutrition (Luder et al. 1998; Rook & Stanford 1999), environmental exposure (Wahn et al. 1997; Mahmic et al. 1998; Strachan & Cook 1998a,b), and lifestyle (von Mutius et al. 1994a; Heinrich et al. 1998; Shaheen 1998; Bergmann et al. 2000). Secondly, protective factors related to more traditional lifestyles common in the past might have been lost, which could lead to susceptibility to atopic diseases (Alm et al. 1999).

Lifestyle and development of atopic disease Several studies focusing on differences between the former socialist countries and Western European societies reported lower prevalence rates for atopy in the former Eastern Bloc (von Mutius et al. 1994a). The differences were particularly striking in areas with few genetic differences, such as East and West Germany, where it was found that the critical period during which lifestyle mainly influences the development

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Age (months) Fig. 59.3 Development of atopic dermatitis, asthma, and allergic rhinoconjunctivitis versus age. Data from 1314 children of the prospective birth cohort study MAS. (From Julie Kulig et al. 1999a, with permission.)

Birch Grass

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Fig. 59.2 Prevalences of sensitization to food (a) and inhalant (b, c) allergens by 6 years of age. Cut-off level 0.7 kU/L (CAP Class 2). Data obtained from the prospective birth cohort study MAS. (From Kulig et al. 1999a, with permission.)

of atopy is probably the first years of life. These observations point in the same direction as studies reporting lower prevalence rates for children born into families that have few siblings (Strachan 1997). Recent observations from Germany suggest that within the population of an industrialized country

with a Western lifestyle, high socioeconomic status must be considered a risk factor for early sensitization and the manifestations of atopic dermatitis and allergic airway disease. Turkish migrants living in Germany exhibited higher prevalences of atopy and asthma after cultural assimilation (Grüber et al. 2002a). Differences in the intestinal microflora as a major source of microbial stimulation of the immune system in early childhood has been proposed as a possible explanation for this observation (Sepp et al. 1997). The intestinal microflora has been shown to enhance Th1-type responses (Murosaki et al. 1998; Shida et al. 1998). The results of a comparative study of Estonian and Swedish children demonstrated differences in intestinal microflora. In Estonia, the typical microflora included more lactobacilli and fewer clostridial organisms, which are associated with a lower presence of atopic disease. Intervention studies are needed to demonstrate the relevance of these findings and examine the effects of adding probiotics to infant formulas. In one study from Finland, which unfortunately was not blinded, infants with milk allergy and atopic dermatitis exhibited milder symptoms and fewer markers of intestinal inflammation if their milk formulas were fortified with lactobacilli. A few reports have described an association between the use of antibiotics during the first 2 years of life and increased risks of asthma. It seems too early to draw final conclusions from these publications. Immunizations appear not to influence the risk of early sensitization or development of atopy (Grüber et al. 2002b). Pediatricians should therefore resist questioning successful immunization programs such as those targeting measles.

Domestic environment Allergen exposure No other environmental factor has been studied as extensively as exposure to environmental allergens as a potential

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% AD

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37%

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34%

5 yrs

31%

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36%

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37%

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Persistent

Intermittent

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Fig. 59.4 Natural course of atopic dermatitis (AD) up to age 7 years in children with early manifestations of the disease. Each symbol represents 1% of the children with early AD, and the natural course of each 1% subsample can be traced vertically. Filled squares represent subjects with AD in the respective time period. (From Illi et al. 2004, with permission.)

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Proportion (%) of children sensitized to mites

16

First quartile (< 0.002–0.032 mg/g) Fourth quartile (< 0.9981–240 mg/g)

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risk for sensitization and manifestation of atopy and asthma. From a number of cross-sectional studies performed in children and in adults, it has become obvious that there is a complex association between allergen exposure, particularly in the domestic environment, and sensitization to that specific allergen. On the one hand, longitudinal studies like the MAS study in Germany have clearly demonstrated that during the first years of life there is a dose–response relationship between indoor allergen exposure to dust mite and cat allergens and the risk of sensitization to cat and mites, respectively (Fig. 59.5) (Sporik et al. 1990; Sherrill et al. 1999; Lau et al. 2000). On the other hand, data from the MAAS study in UK involving use of aggressive measures to limit exposure to airborne particulates during infancy have demonstrated the potential for active lowering of indoor allergen exposure levels to increase the risk for sensitization (Woodcock et al. 2004). These seemingly paradoxical findings may be explicable via the bell-shaped nature of the dose–response curve for allergen exposure versus sensitization risk, i.e., the existence of a “tolerogenic” zone toward the high end of the curve (Holt & Thomas 2005). Under such circumstances, the results of intervention or observation studies at different sites involving these two parameters will be largely determined by the baseline exposure levels at the sites under comparison. As far as the manifestations of atopic dermatitis and asthma are concerned, the situation is much less clear. Early studies performed by Sporik et al. (1990) suggested that exposure of sensitized children to dust mite allergens not only determines the risk of asthma, but also the time of onset of the disease. More recent investigations by the same group, however, suggest that other factors besides allergen exposure are important in determining which children develop asthma. It is interesting to note in this context that lung function at age 3 in the active intervention group in the MAAS study was significantly elevated compared with their control subjects, despite higher levels of sensitization (Woodcock et al. 2004). This suggests that agents present in indoor environments other than conventional allergens (e.g., microbial breakdown

14 12 10 8 6 4 2 0 Age (years)

Fig. 59.5 Prevalence of sensitization to house dust and wheeze at ages 1–7 years stratified by highest and lowest quartiles of house-dust mite exposure in infancy. (From Lau et al. 2000, with permission.)

products) may play important roles in the development of asthma-like syndromes. In a comprehensive metaanalysis, Peat and Li (1999) evaluated several environmental factors said to be responsible for the incidence and severity of atopic diseases, particularly asthma. Comparing the strengths of the various effects, they concluded that on the basis of the literature, indoor allergen exposure is the environmental component with by far the

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strongest impact on the manifestation of asthma. In recent years, however, the paradigm that exposure induces asthma with airway inflammation via sensitization has been challenged. In several countries, the prevalence of asthma in children has been increasing independent of allergen exposure. Datasets obtained from the MAS birth cohort suggest that while domestic allergen exposure is a strong determinant for early sensitization in childhood, it cannot be considered a primary cause of airway hyperresponsiveness or asthmatic symptoms as, during the first 3 years of life, the manifestation of wheeze is not related to elevated serum IgE levels or specific sensitization. Studies following up birth cohorts to adolescence have recently indicated that 90% of children with wheeze but no atopy lose their symptoms at school age and retain normal lung function in puberty (Fig. 59.6). By contrast, sensitization to perennial allergens (house-dust mite, cat, and dog) developing in the first 3 years of life was associated with a loss of lung function at school age. Concomitant exposure to high levels of perennial allergens early in life aggravates this process. Such exposures also enhance the development of airway hyperresponsiveness in sensitized children with wheeze. From these data, it can be concluded that impairment of lung function during school age is determined by continuing allergic airway inflammation beginning in the first 3 years of life (Illi et al. 2006). A number of intervention studies to examine the effects of indoor allergen elimination on the incidence of asthma are currently being performed in cohorts followed prospectively from birth. The results will have a strong impact on public health policies because they will determine whether considering indoor allergen elimination an important element of primary prevention of various atopic manifestations is meaningful. Even if the results show that other factors play major roles in determining whether an atopic child will develop asthma, so that allergen elimination as a measure of primary prevention

80

Atopic (n = 94) Non-atopic (n = 59)

Prevalence (%)

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is inefficient, reduction of allergen exposure will still remain a very important element in secondary prevention.

Pollutants and tobacco smoke as adjuvant factors Several environmental factors have attracted the interest of epidemiologists and experimental researchers. Although they do not serve as allergens, these factors are capable of upregulating existing IgE responses or leading to disease manifestation or aggravation of symptoms. Guinea pig and mouse experiments suggested an increase of allergic sensitization to ovalbumin after experimental exposure to trafficor industry-related pollutants. A strong association between allergic rhinitis caused by cedar pollen allergy and exposure to heavy traffic was reported from Japan. Important sociodemographic confounders turned out to be problems when interpreting study results. Other investigators were unable to describe any relationship between traffic exposure and the prevalence of hay fever or asthma. The role of tobacco smoke, a complex mixture of various particles and organic compounds, was studied extensively. Recently reviewed studies consistently demonstrate that the risk of lower airway diseases such as bronchitis, recurrent wheezing in infants, and pneumonia is increased. Whether passive tobacco smoke exposure is causally related to the development of asthma is still disputed (Stick et al. 1996). Until recently, data about the risk of sensitization has been lacking. The prospective birth cohort MAS in Germany reported that increased risks of sensitization were found only in children whose mothers smoked up to the end of their pregnancies and continued to smoke after birth. In this subgroup of the cohort, a significantly increased sensitization rate of IgE antibodies to food proteins, particularly to hen eggs and cows’ milk, was observed only during infancy (Kulig et al. 1999b). Later sensitization rates were no different from those of children who had never been exposed to tobacco smoke. These observations may be related to the fact that the highest urinary cotinine concentrations in children are detected during the first years of life, when children spend most of their time close to their mothers.

60

Early exposure to infections or microbial products?

50

The hypothesis that has attracted the most interest postulates that a decline in certain childhood infections or lack of exposure to infectious agents during the first years of life, associated with smaller families in the middle class environments of industrialized countries, may be causal for the recent epidemic in atopic disease and asthma (Farooqi & Hopkin 1998; Bach 2002). Although this hypothesis is obviously very complex, several pieces of information appear to support it. Studies from several countries provide indirect evidence for the hypothesis that early exposure to viral infections, although triggering lower airway symptoms during early life, may exert long-lasting protective effects. Children born into families with several, particularly older, siblings have been found to

40 30 20 10 0 1

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Age (years) Fig. 59.6 Prevalence of current wheeze from birth to age 13 years in children with any wheezing episode at school age, stratified for atopy at school age. Data obtained from the prospective birth cohort MAS. (From Illi et al. 2006, with permission.)

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have reduced risk of allergic sensitization and asthma at school age (von Mutius et al. 1994b; Strachan 1997). Studies in children who attended daycare centers during infancy support this concept (Krämer et al. 1999). Infections are known to produce long-lasting nonspecific systemic effects on the nature of the immune response to antigens and allergens. For example, recovery from natural measles infection reduces the incidence of atopy and allergic responses to house-dust mites to half the rate seen in vaccinated children (Matricardi et al. 1997; Shaheen et al. 1996; Illi et al. 2001). Observations from Japan suggesting that strong positive tuberculin responses in children predict a lower incidence of asthma, lower serum IgE levels, and Th1-biased cytokine profiles were supported by animal experiments demonstrating that IgE responses to ovalbumin in mice could be downregulated by a previous infection with BCG. Unfortunately, cohort studies from Europe were unable to describe any protective effect of BCG vaccination (Shirakawa et al. 1997; Grüber & Paul 2002). Certain infections induce a systemic and nonspecific switch to Th1 cells, which may be responsible for inhibiting the development of atopy during childhood (Umetsu et al. 2002). Although these observations on the relationship of immune responses to infectious agents, atopic sensitization, and disease expression are stimulating and challenging, conclusions regarding relevance to the atopic march should be drawn with care. In different parts of the world, completely different infectious agents have been addressed in different study settings. It appears to be fashionable to join Rook and Stanford (1999) who, in a recent review article, pleaded “give us this day our daily germs”: which germ, at what time, under which circumstances, and at what price to be paid? The role of endotoxin exposure as a possible element of atopy prevention in early life has recently been discussed. Endotoxins are a family of molecules called lipopolysaccharides (LPS) and are intrinsic parts of the outer membranes of Gram-negative bacteria. LPS and other bacterial-wall components that are found abundantly in stables, where pigs, cattle, and poultry are kept, engage with antigen-presenting cells via CD14 ligation to induce strong interleukin (IL)-12 responses. IL-12, in turn, is regarded as an obligatory signal for the maturation of naive T cells into Th1-type cells. Endotoxin concentrations were recently found to be highest in stables of farming families, and also in dust samples from kitchen floors and mattresses in rural areas in southern Germany and Switzerland where atopy prevalence is extremely low (von Mutius et al. 2000; Braun-Fährländer et al. 2002). These findings support the hypothesis that environmental exposure to endotoxins and other bacterial-wall components is an important protective determinant related to development of atopic diseases. Indeed, endotoxin levels in samples of dust from children’s mattresses were found to be inversely related to the occurrence of hay fever, atopic asthma, and atopic sensitization.

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Conclusion The increasing prevalence of atopic diseases, particularly atopyassociated asthma, has become a major challenge for allergists and public health authorities in many countries. Knowledge of the natural history of the atopic march, including the determinants that are modifiable and which may become candidates for preventive intervention, is still very limited. Information provided by cross-sectional studies can only generate hypotheses that must be supported by prospective longitudinal cohort studies. Ultimately, the results of wellcontrolled intervention studies will identify which nutritional, environmental, or lifestyle-related factors should be considered for early intervention and that might be useful for reversing the current epidemiologic trends.

References Alm, J.S., Swartz, J., Lilja, G., Scheyius, A. & Pershagen, G. (1999) Atopy in children of families with an anthroposophic lifestyle. Lancet 353, 1485–8. Bach, J.-F. (2002) Effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 347, 911–20. Bergmann, R.L., Bergmann, K.E., Lau-Schadendorf, S. & Wahn, U. (1994) Atopic diseases in infancy: German multicenter atopy study (MAS-90). Pediatr Allergy Immunol 5 (suppl. 1), 19–25. Bergmann, R.L., Edenharter, G., Bergmann, K.G., Lau, S. & Wahn, U. (2000) Socioeconomic status is a risk factor for allergy in parents but not in their children. Clin Exp Allergy 30, 1740–5. Braun-Fährländer, C., Riedler, J., Herz, U. et al. (2002) Environmental exposure to endotoxin and its relation to asthma in schoolage children. N Engl J Med 347, 869–77. Edenharter, G., Bergmann, R.L., Bergmann, K.E. et al. (1998) Cord blood IgE as risk factor and predictor for atopic diseases. Clin Exp Allergy 28, 671–8. Farooqi, I.S. & Hopkin, J.M. (1998) Early childhood infection and atopic disorder. Thorax 53, 927–32. Grüber, C. & Paul, K.P. (2002) Tuberculin reactivity and allergy. Allergy 57, 277–80. Grüber, C., Illi, S., Plieth, A., Sommerfeld, C. & Wahn, U. (2002a) Cultural adaptation is associated with atopy and wheezing among children of Turkish origin living in Germany. Clin Exp Allergy 32, 526–31. Grüber, C., Mailschmidt, G., Bergmann, R., Wahn, U. & Stark, K. (2002b) Is early BCG vaccination associated with less atopic disease? An epidemiological study in German preschool children with different ethnic backgrounds. Pediatr Allergy Immunol 13, 177– 81. Heinrich, J., Popescu, M.A., Wjst, M., Goldstein, I.F. & Wichmann, H.E. (1998) Atopy in children and parental social class. Am J Public Health 88, 1319–24. Holt, P. & Thomas, W.R. (2005) Sensitization to airborne environmental allergens: unresolved issues. Nat Immunol 6, 957–60. Illi, S, von Mutius, E., Bergmann, R., Niggemann, B., Sommerfeld, C. & Wahn, U. (2001) Early childhood infectious diseases and the

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development of asthma up to school age: a birth cohort study. Multicenter Allergy Study Group. BMJ 322, 390–5. Illi, S., von Mutius, E., Lau, S., et al. (2004) The natural course of atopic dermatitis from birth to age 7 years and the association with asthma. J Allergy Clin Immunol 113, 925–31. Illi, S., von Mutius, E., Lau, S., Niggemann, B., Grüber, C. & Wahn, U. (2006) Perennial allergen sensitisation early in life and chronic asthma in children: a birth cohort study. Lancet 368, 763–70. Krämer, U., Heinrich, J., Wjst, M. & Wichmann, H.E. (1999) Age of entry to day nursery and allergy in later childhood. Lancet 353, 450– 4. Kulig, M., Bergmann, R., Klettke, U., Wahn, V., Tacke, U. & Wahn, U. (1999a) Natural course of sensitization to food and inhalant allergens during the first 6 years of life. Multicenter Allergy Study Group. J Allergy Clin Immunol 103, 1173– 9. Kulig, M., Luck, W., Lau, S. et al. (1999b) Effect of pre-and postnatal tobacco smoke exposure on specific sensitization to food and inhalant allergens during the first 3 years of life. Multicenter Asthma Study Group. Allergy 54, 220– 8. Kulig, M., Tacke, U., Forster, J., et al. (1999c) Total serum IgE levels during the first 6 years of life. J Pediatr 134, 453–8. Kulig, M., Klettke, U., Wahn, V., Forster, J., Bauer, C.P. & Wahn, U. (2000) Development of seasonal allergic rhinitis during the first 7 years of life. J Allergy Clin Immunol 106, 832–9. Lau, S., Falkenhorst, G., Weber, A. et al. (1989) High mite-allergen exposure increases the risk of sensitization in atopic children and young adults. J Allergy Clin Immunol 84, 718–25. Lau, S., Illi, S., Sommerfeld, C. et al. (2000) Early exposure to housedust mite and cat allergens and development of childhood asthma: a cohort study. Multicenter Allergy Study Group. Lancet 356, 1392–7. Luder, E., Melnik, T.A. & DiMaio, M. (1998) Association of being overweight with greater asthma symptoms in inner city black and Hispanic children. J Pediatr 132, 699–703. Mahmic, A., Tovey, E.R., Molloy, C.A. & Young, L. (1998) House dust mite allergen exposure in infancy. Clin Exp Allergy 28, 1487–92. Martinez, F.D. (1997) Complexities of the genetics of asthma. Am J Respir Crit Care Med 156, 117–22. Martinez, F.D., Wright, A.L., Taussig, L.M., Holberg, C.J., Halonen, M. & Morgan, W.J. (1995) Asthma and wheezing in the first six years of life. N Engl J Med 332, 133– 8. Matricardi, P.M., Rosmini, F., Ferrigno, L. et al. (1997) Cross-sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis A virus. BMJ 314, 999– 1003. Moffatt, M.F. & Cookson, W.O.C.M. (1998) Gene Identification in asthma and allergy. Int Arch Allergy Immunol 116, 247–52. Murosaki, S., Yamamoto, Y., Ito, K. et al. (1998) Heat-killed Lactobacillus plantarum L-137 suppresses naturally fed antigen-specific IgE production by stimulation of IL-12 production. J Allergy Clin Immunol 102, 57– 64. Nickel, R., Kulig, M., Forster, J. et al. (1997) Sensitization to hen’s egg at the age of 12 months is predictive for allergic sensitization to common indoor and outdoor allergens at the age of 3 years. J Allergy Clin Immunol 99, 613–7.

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Peat, J.K. & Li, J. (1999) Reversing the trend: reducing the prevalence of asthma. J Allergy Clin Immunol 103, 1–10. Rook, G.A.W. & Stanford, J.L. (1999) Give us this day our daily germs. Immunol Today 19, 113–17. Sepp, E., Julge, K., Vasar, M., Naaber, P., Björksten, B. & Mikelsaar, M. (1997) Intestinal microflora of Estonian and Swedish infants. Acta Paediatr 86, 956–61. Shaheen, S.O. (1998) Obesity and asthma: a cause for concern? Clin Exp Allergy 29, 291–3. Shaheen, S.O., Aaby, P., Hall, A.J. et al. (1996) Measles and atopy in Guinea-Bissau. Lancet 347, 1792–6. Sherrill, D., Stein, R., Kurzius-Spencer, M. & Martinez F. (1999) On early sensitization to allergens and development of respiratory symptoms. Clin Exp Allergy 29, 905–11. Shida, K., Makino, K., Morishita, A. et al. (1998) Lactobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte cultures. Int Arch Allergy Immunol 115, 278–87. Shirakawa, T., Enomoto, T., Shimazu, S., & Hopkin, J.M. (1997) The inverse association between tuberculin responses and atopic disorder. Science 275, 77–9. Sporik, R., Holgate, S.T., Platts-Mills, T.A.E. et al. (1990) Exposure to house-dust mite allergen (Der p I) and the development of asthma in childhood: a prospective study. N Engl J Med 323, 502– 7. Stick, S.M., Burton, P.R., Gurrin, L., Sly, P.D. & LeSouef, P.N. (1996) Effects of maternal smoking during pregnancy and a family history of asthma on respiratory function in newborn infants. Lancet 348, 1060– 4. Strachan, D.P. (1997) Allergy and family size: a riddle worth solving. Clin Exp Allergy 27, 235–6. Strachan, D.P. & Cook, D.G. (1998a) Health effects of passive smoking. 5. Parental smoking and allergic sensitization in children. Thorax 53, 117–23. Strachan, D.P. & Cook, D.G. (1998b) Health effects of passive smoking. 6. Parental smoking and childhood asthma: longitudinal and case-control studies. Thorax 53, 204–12. Umetsu, D.T, McIntire, J.J., Akbari, O., Macaubas, C. & DeKruyff, R. (2002) Asthma: an epidemic of dysregulated immunity. Nat Immunol 3, 715–20. von Mutius, E., Martinez, F.D., Fritsch, C. et al. (1994a) Prevalence of asthma and atopy in two areas of West and East Germany. Am J Respir Crit Care Med 149, 358–64. von Mutius, E., Martinez, F.D., Fritsch, C. et al. (1994b) Skin test reactivity and number of siblings. BMJ 308, 692–5. von Mutius, E., Braun-Fahrländer, C., Schierl, R., et al. (2000) Exposure to endotoxin or other bacterial components might protect against the development of atopy. Clin Exp Allergy 30, 1230–4. Wahn, U., Lau, S., Bergmann, R. et al. (1997) Indoor allergen exposure is a risk factor for sensitization during the first three years of life. J Allergy Clin Immunol 99, 763–9. Woodcock, A., Lowe, L.A., Murray, C.S. et al. (2004) Early life environmental control: effect on symptoms, sensitization, and lung function at age 3 years. Am J Respir Crit Care Med 170, 433–9.

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Outdoor Air Pollution and Allergic Airway Disease Gennaro D’Amato

Summary Although the role played by outdoor pollutants in allergic sensitization of the airways has yet to be clarified, a body of evidence suggests that urbanization, with its high levels of vehicle emissions, and a westernized lifestyle are linked to the rising frequency of respiratory allergic diseases observed prevalently in most industrialized countries. There is also considerable evidence that subjects affected by asthma are at increased risk of developing obstructive airway exacerbations with exposure to ozone, nitrogen dioxide, sulfur dioxide and inhalable particulate matter. As a consequence, in recent years there has been increasing interest in studies of air pollution and its effects on human health, in particular on the respiratory tract. However, it is not easy to evaluate the impact of air pollution on the timing of asthma exacerbations and on the prevalence of asthma in general. Several manuscripts have been published about air pollution exposure assessment but it is unclear which measurement best reflects the exposure of a subject. As concentrations of airborne allergens and air pollutants are frequently increased contemporaneously in the atmosphere, an enhanced IgE-mediated response to aeroallergens and enhanced airway inflammation could account for the increasing frequency of allergic respiratory allergy and bronchial asthma in atopic subjects. Moreover, the global rise in asthma prevalence and severity that has occurred over recent decades could also be an effect of climate change, including global warming, induced by human activity. Pollen allergy is frequently used to study the interrelationship between air pollution and respiratory allergic diseases such as rhinitis and bronchial asthma. Studies have demonstrated that urbanization, high levels of vehicle emissions, and westernized lifestyle are correlated with an increasing frequency of polleninduced respiratory allergy and people who live in urban areas tend to be more affected by pollen-induced respiratory allergy than people living in rural areas. Climatic factors (temperature, wind speed, humidity, thunderstorms, etc.) can affect both

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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components (biological and chemical) of this interaction. By attaching to the surface of pollen grains and of plant-derived particles of paucimicronic size, pollutants could modify not only the morphology of these antigen-carrying agents but also their allergenic potential. In addition, by inducing airway inflammation, which increases airway permeability, pollutants overcome the mucosal barrier and could be able to “prime” allergen-induced responses. There are also observations that thunderstorms occurring during the pollen season can induce severe asthma attacks in pollinosis patients. After rupture by thunderstorm, pollen grains may release part of their cytoplasmic content, including inhalable, allergen-carrying paucimicronic particles.

Introduction There is extensive scientific literature relating exposure to air pollution with human morbidity and mortality (American Thoracic Society 1996a,b; D’Amato & Holgate 2002; Viegi & Baldacci 2002; European Respiratory Society 2003; WHO 2003; Bernstein et al. 2004; Wong & Lai 2004; Peden 2005; Rom & Samet 2006) and in the past few years, much etiologic and pathogenic research has been carried out in the attempt to study this link. A change in the genetic predisposition is an unlikely cause of the increase in allergic diseases (although it cannot be ruled out) while changes in environmental factors, including indoor and outdoor air pollution may be involved. If the interplay between genetic and environmental factors in the development of allergic respiratory diseases is studied, it appears there is a link between the increase in prevalence of allergic airway diseases and an increase in air pollution. In most industrialized countries, people who live in urban trafficked areas tend to be more affected by allergic respiratory diseases than those of rural areas (Braun-Fahrlander et al. 1999; Riedler et al. 2000). Road traffic with its particulate and gaseous emissions is the main contributor to air pollution in most urban areas and there is evidence that living near highly trafficked roads is associated with impaired respiratory health (Wjst et al. 1993; Edwards et al. 1994; Cacciolla et al. 2002). An individual’s response to air pollution depends on the source

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and components of the pollution, as well as climatic factors. Indeed, some air pollution-related episodes of asthma exacerbation are due to climatic factors that favor the accumulation of air pollutants at ground level and some cities are continuously affected by black smog caused by motor vehicles. A trial carried out in the Netherlands (Boezen et al. 1999) demonstrated that children affected by bronchial asthma are at higher risk of increased bronchial obstruction during episodes of air pollution. Respiratory symptoms in children with bronchial hyperresponsiveness and high levels of serum IgE increased by as much as 139% for every 100 μg/m3 increase in atmospheric particulate matter. Moreover, the study did not take account of the airborne allergen content, which is a potential confounding factor. Air pollution is associated with asthma exacerbations, characterized by increased bronchial hyperresponsiveness, increased medication use, visits to emergency departments, and hospital admissions (Sunyer et al. 1993; Atkinson et al. 1999; Künzli et al. 2000). Time series data show that traffic-related air pollution in urban areas has adverse effects on mortality not only from respiratory disorders but also from cardiovascular disease (Dockery et al. 1993; Pope et al. 1995, 2004; Pekkanen et al. 2002; Sullivan et al. 2003, 2005; Gauderman et al. 2004; Peters et al. 2004). In a study of six US cities, after adjusting for smoking, the mortality rate ratio increased in the most polluted cities compared with the least polluted areas (Pope et al. 1995). The most abundant air pollutants in urban areas with high levels of vehicle traffic are inhalable particulate matter, nitrogen dioxide (NO2), and ozone (O3). The effects of air pollutants on lung function depend on the type of pollutant and its environmental concentration, the duration of exposure, the total ventilation of exposed subjects, and on the interaction between air pollution and aeroallergens such as those derived from pollens and fungal spores (D’Amato et al. 1998, 2005a; Brunekreef et al. 2000; D’Amato 2000a; Barck et al. 2005; Motta et al. 2006). Airborne pollen grains, plant debris of very small size (D’Amato et al. 1998, 2005a; D’Amato 2000a) and cytoplasmic components of pollen grains ruptured during thunderstorms (Celenza et al. 1996; Venables et al. 1997; D’Amato et al. 2005a) can interact with air pollution in producing allergic respiratory symptoms in predisposed subjects. There is a hypothesis that air pollutants promote airway sensitization by inducing changes in the allergenic content of airborne particles carrying antigens (Celenza et al. 1996; Venables et al. 1997; D’Amato et al. 1998, 2005a; Devalia et al. 1998; D’Amato 2000a; Motta et al. 2006). There is also evidence that the airway mucosal damage and impaired mucociliary clearance induced by air pollution may facilitate the penetration and access of inhaled allergens to the cells of the immune system (Rusznak et al. 1996; Devalia et al. 1998; D’Amato et al. 2002, 2005a). Patients affected by asthma frequently experience rhinitis and thus breathe through the mouth, bypassing the nasal

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function and so facilitating the penetration of pollutants and aeroallergens into the lower airways (Corren 1997; D’Amato 2000b).

Outdoor air pollution of urban areas The most abundant pollutants in the atmosphere of urban areas are NO2, O3, and inhalable particulate matter. Sulfur dioxide (SO2) is a component of air pollution prevalently in industrial areas. Aeroallergens are carried and delivered by fungal spores or by plant-derived particles (pollen, components of paucimicronic diameter and of vegetal nature, in some cases soja bean dust, ricinus, etc.).

Ozone O3 is the main component of photochemical oxidants (Holtzman et al. 1983; Beckett et al. 1985; Schlesinger & Driscoll 1987; Molfino et al. 1991; Balmes 1993; Coleridge et al. 1993; Folinsbee et al. 1994; White et al. 1994; Peden et al. 1995; Jorres et al. 1996; Thurston & Gwynn 1997; Kehrl et al. 1999; Peters et al. 1999; Bayram et al. 2001; Vagaggini et al. 2002) and of so-called “summer smog,” because it accounts for up to 90% of total oxidant levels in cities that enjoy a mild sunny climate such as those of the Mediterranean area and California. O3 is generated at ground level by photochemical reactions involving ultraviolet radiation on atmospheric mixtures of NO2 and hydrocarbons deriving from vehicle emissions (Fig. 60.2). However, O3 can be carried long distances by winds and high ozone atmospheric concentrations can be found also in rural areas, where there are no O3 precursors. About 40–60% of inhaled O3 is absorbed in the nasal airways and the remainder reaches the lower airways, and it can affect both the upper and lower respiratory tract. Inhalation of high concentrations of O3 induces deterioration in lung function, increased airway responsiveness to nonspecific and specific bronchoconstrictor agents, and is related to an increased risk of asthma exacerbation in asthmatic patients (Holtzman et al. 1983; Beckett et al. 1985; Schlesinger & Driscoll 1987; Kreit et al. 1989; Devlin et al. 1991; Molfino et al. 1991; Balmes 1993; Coleridge et al. 1993; Folinsbee et al. 1994; White et al. 1994; Horstman et al. 1995; Peden et al. 1995; Jorres et al. 1996; Balmes et al. 1997; Thurston & Gwynn 1997; Kehrl et al. 1999; Peters et al. 1999; Bayram et al. 2001; Vagaggini et al. 2002). Increased air levels of O3 and NO2 have been linked to increases in respiratory morbidity and in hospital admissions for asthma in children and adults (Atkinson et al. 1999; Peters et al. 1999). Children spend greater proportions of time outdoors in exertional activities during the summer months when O3 levels are higher and thus they inhale more polluted air. For these reasons, children are considered to be a greater risk for untoward effects from O3. If normal persons also experience adverse effects due to O3 exposure, subjects with asthma tend

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to experience greater O3-induced respiratory tract injury and inflammation. Bayram et al. (2001) showed that O3 and NO2 modulate airway inflammation in asthma patients by increasing the release of inflammatory mediators from bronchial epithelial cells. It has also been observed that O3 exposure has a priming effect on allergen-induced responses as well as an intrinsic inflammatory effect in the airways of allergic asthmatics (Molfino et al. 1991; Peden et al. 1995; Jorres et al. 1996; Kehrl et al. 1999). O3 also produces an increase in intracellular reactive oxygen species and in epithelial cell permeability, which could facilitate penetration of inhaled allergens and toxins in the airways. There is also increased release of inflammatory mediators such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF). A study in rabbit showed decreased mucociliary clearance times as O3 levels increased (Schlesinger & Driscoll 1987). Vagaggini et al. (2002) showed that ozone’s more dramatic effect in asthmatic subjects is most likely a result of existing chronic inflammation in the lower airways. A study involving a high concentration of O3 (0.4 ppm) and relatively heavy exercise (Kreit et al. 1989) and another involving prolonged exposure (7.6 hours) to a lower O3 concentration (0.16 ppm) (Horstman et al. 1995) showed enhanced responses in asthmatic subjects in comparison with nonasthmatic control subjects. It has been shown that O3 increases asthma morbidity by enhancing airway inflammation. O3 significantly increases levels of inflammatory mediators such as IL-6, IL-8, granulocyte–macrophage colony-stimulating factor (GM-CSF), and fibronectin in bronchoalveolar lavage (BAL) fluid (Devlin et al. 1991; Balmes et al. 1997). Moreover, Basha et al. (1994) and Scannell et al. (1996) found increased neutrophils, cytokine levels, and evidence of epithelial permeability in BAL fluid 18 hours after short-term O3 exposure in subjects with mild asthma versus nonasthmatic subjects. In addition to increased neutrophils, Peden et al. (1996) also found increased eosinophils in BAL fluid 18 hours after O3 exposure. The higher post-O3 cytokine levels in asthmatic subjects are consistent with the hypothesis that preexisting airway inflammation in these subjects facilitates the inflammatory response to O3. Because O3-induced airway inflammation may last several days, O3-related asthma exacerbations can occur several days after exposure. O3 inhalation also reduces exercise tolerance in nonasthmatic athletes (Adams & Schelegle 1983; Folinsbee et al. 1984; Foxcroft & Adams 1986; Schelegle & Adams 1986). Repeated daily, short-term exposures of healthy subjects to O3 attenuates the acute lung function and inflammatory responses (Devlin et al. 1993; Christian et al. 1996). It has been reported that O3 is associated with an increased risk of asthma development among children in California playing outdoor sports. Thus, air pollution and outdoor exercise could contribute to the development of asthma in children by increasing airway inflammation and airway responsiveness (McConnell et al. 2002).

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Controlled studies suggest that O3 inhalation enhances inflammatory responses of airways to inhaled allergens, increasing the risk of allergic sensitization in predisposed subjects. Indeed, by lowering the threshold concentration of allergen able to induce clinical symptoms, O3 can enhance the airway responsiveness of sensitized subjects. Molfino et al. (1991) reported that a 1-hour exposure to 0.12 ppm O3 was able to induce a twofold reduction in the provocation concentration of inhaled antigen required to cause early bronchoconstriction in specifically sensitized asthmatic subjects. The mean provocation dose of ragweed necessary to reduce forced expiratory volume in 1 s (FEV1) by 20% in these allergic asthmatic subjects was significantly reduced to about half the dose of allergen when the patients were preexposed to 0.12 ppm O3 for 1 hour versus preexposure to air. Jorres et al. (1996), utilizing a higher effective dose (0.25 ppm inhaled through a mouthpiece with intermittent exercise) and a longer duration of exposure (3 hours), found that 23 of 24 mild asthmatic subjects after O3 exposure required a lower provocation dose of allergen to cause a 20% decrease in FEV1 (PD20). Ball et al. (1996) used the same approach as Molfino and coworkers but avoided the limitation of the earlier study, i.e., the nonrandom ordering of exposures (filtered air before O3 in 6 of 7 subjects) that could have produced an allergen “priming” effect on the later O3 exposures. These authors were unable to confirm the results of the Molfino study and found that preexposure to O3 versus preexposure to air did not significantly reduce the dose of allergen required to decrease FEV1 by 15%. The disagreement between the two studies was probably due to methodologic differences in sample selection and to repeated exposure to the allergen used in the Ball study. Peden et al. (1995) reported increased nasal inflammatory responses to local allergen challenge after O3 exposure in subjects with allergic rhinitis. Devalia et al. (1998) studied the effect of previous exposure to O3 and NO2 on subsequent allergen-induced changes in the nasal mucosa of patients with seasonal allergic rhinitis or perennial allergic asthma. They found that exposure to these pollutants significantly enhanced the allergen-induced release of eosinophil cationic protein in nasal lavage. In other words, it is possible that exposure to O3 and NO2 may “prime” eosinophils to subsequent activation by inhaled allergen in atopic patients. However, among subjects exposed to O3 inhalation there are subgroups with different susceptibility (Mortimer et al. 2000).

Nitrogen dioxide Like O3, NO2 is an oxidant pollutant, although it is less chemically reactive and thus probably less potent. NO2 is a precursor of photochemical smog, is found in outdoor air in urban and industrial regions, and in conjunction with sunlight and hydrocarbons results in the production of O3. Automobile exhaust is the most significant source of outdoor NO2, although power

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plants and other sources that burn fossil fuels also release NO2 into the environment. The most significant exposure to NO2 occurs indoors in conjunction with the use of gas cookingstoves and kerosene space-heaters. Most ambient NO2 is generated by the burning of fossil-derived fuels. Outdoor levels of NO2 are not usually associated with notable changes in bronchial function in asthmatic patients. Controlled exposure studies of subjects with asthma have produced inconsistent results regarding the ability of NO2 to enhance nonspecific airway responsiveness, with some evidence of a subgroup with increased sensitivity (Hazucha et al. 1983; Bauer et al. 1986; Linn et al. 1986; Moshenin 1987). Epidemiologic and clinical studies have mainly addressed the acute effects of NO2 and only a few population-based studies have evaluated the effects of long-term exposure to NO2, with inconsistent results (Roger et al. 1990; Hirsch et al. 1999). The results of an epidemiologic study showed that exposure to NO2 increases prevalence of asthma and rhinitis, with acute decrements in lung function in asthmatic subjects (De Marco et al. 2002). All eight asthmatic subjects studied by Tunnicliffe et al. (1994) had a greater allergen-induced early bronchoconstrictor response after exposure to 0.4 ppm NO2 for 1 hour while at rest than after sham exposure. Seven of eight asthmatic subjects in a study by Devalia et al. (1998) had a lower PD20 after exposure to 0.4 ppm NO2 or to 0.2 ppm SO2, or to the combination of both pollutants for 6 hours than after sham exposure, but only the combined exposure induced a significant decrease in PD20 in comparison with the sham control. Neither study looked at the effects of pollutants on the late inflammatory response.

Sulfur dioxide SO2 is released into the atmosphere primarily as a result of industrial combustion of high-sulfur-containing coal and oil. It is primarily generated from the burning of sulfur-containing fossil fuel and it has been demonstrated to induce acute bronchoconstriction in asthmatic subjects at concentrations well below those required to induce this response in healthy subjects (Sheppard et al. 1980; Horstman et al. 1986; Balmes et al. 1987; Linn et al. 1987). In contrast to O3, the bronchoconstrictor effect of inhaled SO2 in individuals with asthma occurs after extremely brief periods of exposure, especially with oral breathing and high ventilatory rates, as in exercise (Sheppard et al. 1980; Horstman et al. 1986; Balmes et al. 1987; Linn et al. 1987; Reidel et al. 1988). Significant responses are observed within 2 min, maximal response is seen within 5–10 min. There can also be spontaneous recovery (30 min after challenge) and a refractory period of up to 4 hours, whereas repeated exposure to low levels of SO2 results in tolerance to subsequent exposure. Pharmacologic studies suggest that the effect is a cholinergically mediated neural mechanism. Moreover, SO2 exposure enhances responses to other environmental agents that exacerbate bronchospasm.

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Particulate matter Particulate matter (PM) is the most serious air pollution problem in many cities and towns and it appears to be the component of air pollution most consistently associated with adverse health effects. In other words PM, which is a mixture of solid and liquid particles of different origin, size and composition, is a major component of urban air pollution. Inhalable PM that can reach the lower airways is measured as PM10 (< 10 μm in aerodynamic diameter) and PM2.5 (< 2.5 μm) (Anderson et al. 1994; Churg & Brauer 1997; Rabinovitch et al. 2006). Human lung parenchyma retains PM2.5, whereas particles over 5 μm but below 10 μm only reach the proximal airways, where they are eliminated by mucociliary clearance if the airway mucosa is intact (Anderson et al. 1994; Churg & Brauer 1997; Rabinovitch et al. 2006). Several studies have observed an association between high atmospheric levels of particulate air pollution and enhanced mortality from respiratory and cardiovascular diseases, exacerbation of allergic asthma, chronic bronchitis, respiratory tract infection, cardiovascular diseases, and hospital admissions (Pope et al. 1995, 2004; Salvi & Holgate 1999; Pekkanen et al. 2002; Peters et al. 2004; Barck et al. 2005). The World Health Organization estimates that inhalation of PM is responsible for 500 000 excess deaths each year worldwide (WHO 2003). It has been hypothesized that fine particulate matter found in urban areas, by penetrating deep into airways, is able to induce alveolar inflammation, which is responsible for variation in blood coagulability and release of mediators favoring acute episodes of respiratory and cardiovascular diseases (Seaton et al. 1995). In the context of PM, iron, which generates hydroxyl radicals, seems to be responsible for the adverse respiratory effects (Ghio & Hatch 1993; Smith & Aust 1997). Other transition metals (chromium, cobalt, copper, manganese, nickel, titanium, vanadium, and zinc) derived from various urban or combustion source samples seem to be also correlated with free radical activation and lung injury (Ghio & Hatch 1993; Costa & Dreher 1997).

Diesel exhaust particulate Diesel exhaust particulate (DEP) accounts for most of the airborne PM (up to 90%) in the atmosphere of the world’s largest cities (Takafuji et al. 1987; Rudell et al. 1990; Diesel Working Group 1995; Nauss et al. 1995; Takenaka et al. 1995; Diaz-Sanchez et al. 1996, 1997; Knox et al. 1997; Sydbom et al. 2001; Stenfors et al. 2004; Riedl & Diaz-Sanchez 2005). DEP is characterized by a carbonaceous core in which 18 000 different high-molecular-weight organic compounds are adsorbed. Although diesel engines emit far less carbon dioxide than petrol engines, they emit over 10 times more NO2, aldehydes, and respirable PM than unleaded petrol engines and over 100 times more than engines fitted with catalytic converters (Diesel Working Group 1995). DEP exerts its effect by way of specific activities of chemical agents, i.e., polyaromatic hydrocarbons (see below).

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Acute exposure to diesel exhaust causes irritation of the nose and eyes, headache, lung function changes, respiratory changes, fatigue, and nausea, while chronic exposure is associated with cough, sputum production, and lung function decrements (Diesel Working Group 1995; Sydbom et al. 2001). Experimental studies have shown that DEP causes respiratory symptoms and is able to modify the immune response in atopic subjects (Takafuji et al. 1987; Takenaka et al. 1995; Diaz-Sanchez et al. 1996, 1997; Riedl & Diaz-Sanchez 2005). DEP seems to exert an adjuvant immunologic effect on IgE synthesis in atopic subjects, thereby influencing sensitization to airborne allergens. In addition, DEP can interact with aeroallergens to enhance antigen-induced responses, so that allergen-specific IgE levels are up to 50-fold greater in allergic subjects stimulated with DEP plus allergens than in subjects treated with allergen alone (Riedl & Diaz-Sanchez 2005). Rudell et al. (1990) showed that healthy volunteers exposed to DEP had a greater number of alveolar macrophages, neutrophils, and T lymphocytes in BAL fluid than did controls. Other studies confirmed the effects favoring airway inflammation and demonstrated an atopy-enhancing effect of diesel exhaust (Diaz-Sanchez et al. 1996, 1997). Controlled chamber exposure studies of healthy volunteers exposed to 300 μg/m3 diesel exhaust or diesel particulates for 1 hour showed increased neutrophil counts in sputum and bronchial biopsy specimens and increases in IL-6, IL-8, and growth-related oncogene (GRO)α levels, with minimal changes in lung function (Riedl & Diaz-Sanchez 2005). Diaz-Sanchez et al. (1996, 1997) studied the effect of DEP on antigen in ragweed-sensitive subjects challenged (nasal provocation test) with DEP, the major ragweed allergen (Amb a1), and a combination of DEP and Amb a1. Provocation with ragweed led to an increase in both total and ragweed-specific IgE in nasal lavage fluid measured 18 hours, 4 days, and 8 days post challenge. The DEP challenge increased the concentration of ragweed-specific IgE 16-fold versus concentrations observed after challenge with ragweed alone. The same authors showed that combined exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweedspecific IgE and skews cytokine production to a Th2-type pattern (Diaz-Sanchez et al. 1997). All these results indicate that DEP plays a role increasing allergic and nonallergic inflammatory response (Rudell et al. 1990; Diaz-Sanchez et al. 1997; Knox et al. 1997; Stenfors et al. 2004; Riedl & DiazSanchez 2005). With regard to DEP-related allergic respiratory disease, DEP can adsorb aeroallergens released by pollen grains and can prolong retention of the allergen so as to provide for an enhanced IgE-mediated response (Knox et al. 1997). DEP exerts its effect by way of components such as the hydrophobic polyaromatic hydrocarbons. DEP is deposited on the airway mucous membranes (epithelial cells and macrophages are the first cells to come into contact with inhaled particulate matter) and the aromatic hydrocarbons allow them to diffuse easily through

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cell membranes and bind to a cytosolic receptor complex. Via subsequent action on the nucleus, aromatic hydrocarbons can modify the growth and differentiation programs of cells (Rudell et al. 1990; Diaz-Sanchez et al. 1997). Human epithelial cells and macrophages phagocytose DEP so leading to the production of the inflammatory cytokines IL-6, IL-8, and GM-CSF (Sydbom et al. 2001; Riedl & Diaz-Sanchez 2005). DEP activates chemotaxis of lymphocytes, neutrophils, and eosinophils, and causes histamine release, plasma leakage, smooth muscle contraction of airways, and increased airway hyperresponsiveness. The data on DEP are of particular interest in view of the increasing percentage of new cars with diesel engines in industrialized countries. In Europe, for example, more than 50% of all new cars are diesel-powered thanks to their lower maintenance costs. Diesel-powered cars are usually promoted as being environmentally friendly because they produce up to 25% less carbon dioxide, which is a major contributor to global warming. However, we hope that new diesel engines with more efficient filters will be able to reduce production and release of DEP.

Aeroallergens derived from plants and their relationship with air pollution Pollen grains Respiratory allergy induced by antigens released by pollen grains is very common (Burney et al. 1997; D’Amato et al. 1998; D’Amato 2002a). For instance, between 8 and 35% of young adults in countries of the European Community have IgE serum antibodies to grass pollen allergens (Burney et al. 1997). The cost of pollen allergy in terms of impaired work fitness, sick leave, physician consultations and drugs, is very high. Subjects living in urban areas tend to be more affected by plant-derived respiratory disorders than those living in rural areas (Braun-Fahrlander et al. 1999; Riedler et al. 2000; D’Amato 2002a). Various studies suggest that there is an interaction between air pollutants and allergens that exacerbates the development of atopy and the respiratory symptoms of allergic disease (Knox et al. 1997; D’Amato et al. 1998; TraidlHoffmann et al. 2003). In a time-series study, Brunekreef et al. (2000) found a strong association between the day-to-day variation in pollen concentrations and deaths due to cardiovascular disease, chronic obstructive pulmonary disease, and pneumonia.

Airborne allergen-carrying particles of paucimicronic size and interaction with air pollution Pollen grains are the primary carriers of pollen allergens, which explains why the symptoms typical of hay fever are located in the eyes, nose, and nasopharynx. However, allergic asthma in pollen-sensitive patients is an enigma because

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intact pollen grains, which measure over 10 μm in diameter, are too large to enter the lower airways (Wilson et al. 1973; Solomon et al. 1983; D’Amato et al. 1998; D’Amato 2001; Taylor et al. 2004). Moreover, in many instances peak asthma symptom scores differ temporally from peak pollen counts, and early-morning symptoms sometimes precede later peaks in the daily pollen cycle. Pollen asthma and the discordance between pollen count and bronchial symptoms was partially explained with the identification of pollen allergens in microaerosol suspensions smaller than pollen grains (Wilson et al. 1973; Solomon et al. 1983; Spieksma et al. 1995; D’Amato 2000a, 2001; Taylor et al. 2004; D’Amato et al. 2005a), which could be present in the atmosphere before the start and after the end of the season, so prolonging the respiratory symptoms of sensitized patients. By virtue of their small size, these paucimicronic particles can reach the peripheral airways with inhaled air, so inducing asthma in sensitized subjects (Table 60.1). Thus, parts of an organism (in this case derived from plants) other than pollen grains or spores contain significant allergen concentrations that are readily disseminated via an airborne route. These allergenic paucimicronic particles can act as carriers for the protein agent with antigenic property that causes allergic symptoms. Allergens have been detected in the leaves and stems of allergenic plants (D’Amato et al. 2005a). They may result from elution of allergens from pollen grains with their later dispersion in microdroplets. Moreover, pollen grain allergens could be transferred, by physical contact or by elution, to other small particles present in the atmosphere, for instance DEP, which can penetrate deep into the airways (Knox et al. 1997). Consequently, besides enhancing airway ragweedspecific IgE and skewing cytokine production to a Th2-type pattern in subjects at risk of developing atopy (Diaz-Sanchez et al. 1997), DEP could cause asthma by trafficking allergens into the airways. In the context of paucimicronic particles, there exist allergenic airborne Ubish bodies whose size is optimal for penetration into lower airways. These little grains, with unknown function, are spheroidal structures which develop in the Table 60.1 Airborne small (paucimicronic) allergen-carrying particles. Starch granules and other components of the cytoplasm of pollen grains (released into the atmosphere under wet conditions, and responsible for thunderstorm-associated bronchial asthma) Nonpollen plant parts from inflorescences, leaves or Ubish bodies (spheroidal structures which develop in the anthers of many higher plants and which contain allergens) Nonplant particulate matter (allergens transferred through physical contact or by leaching from the surface of the pollen grain to other airborne small particles) Pollen fragments (can be found prevalently at the beginning of thunderstorms)

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anthers of many higher plants (Pacini & Franchi 1993; D’Amato 2001; Vinckier & Smets 2001). They generally occur in large numbers, are usually only a few micrometers in diameter, and can contain allergens (Jensen et al. 1989; D’Amato et al. 1994).

Thunderstorm asthma during pollen season There is evidence that thunderstorms can be linked with allergic asthma epidemics in pollinosis patients during pollen seasons and there are data in favor of the possibility that thunderstorms concentrate pollen grains at ground level, releasing allergenic particles of respirable size derived from the cytoplasm into the atmosphere (Packe & Ayres 1985; D’Amato et al. 1991, 2005b; Bellomo et al. 1992; Suphioglu et al. 1992; Knox 1993; Murray et al. 1994; Celenza et al. 1996; Davidson et al. 1996; Wallis et al. 1996; Antò & Sunyer 1997; Newson et al. 1997; Venables et al. 1997; Girgis et al. 2000; Marks et al. 2001). There are descriptions of asthma outbreaks associated with thunderstorms which occurred in several cities, particularly in Europe (Birmingham, London, Napoli) and Australia (Melbourne and Wagga Wagga) (Antó et al. 1989; Aceves et al. 1991; D’Amato et al. 1991, 2005b; Bellomo et al. 1992; Suphioglu et al. 1992; Knox 1993; Murray et al. 1994; Davidson et al. 1996; Wallis et al. 1996; Antò & Sunyer 1997; Newson et al. 1997; Girgis et al. 2000; Marks et al. 2001). It has been hypothesized that changes in the weather such as rain or humidity may induce hydration of pollens and fragmentation of these grains which, in turn, generates atmospheric biological aerosols carrying allergens (Suphioglu et al. 1992; Knox 1993). After rupture by osmotic shock during thunderstorms, grass pollen releases large amounts of paucimicronic allergenic particles, i.e., cytoplasmic starch granules containing grass allergens (Suphioglu et al. 1992; Knox 1993; Celenza et al. 1996; Venables et al. 1997). Because of their very small size, starch granules can penetrate the lower airways and induce the appearance of bronchial allergic symptoms. Grass pollen grains, being more than 30 μm in mean diameter, mainly induce allergic rhinitis in sensitized atopic subjects. After rupture induced by thunderstorms, their cytoplasmicderived paucimicronic particles can reach the bronchial airways to induce asthma (Fig. 60.1). As a consequence, during the first phase of a thunderstorm, pollinosis subjects may inhale a high concentration of allergenic material derived from the cytoplasm, which can induce asthmatic reactions and sometimes severe ones. Fortunately, even though it can induce severe asthma, outbreaks associated with thunderstorms are infrequently responsible for respiratory allergic disease. “Thunderstorm-associated asthma” was first recognized over 20 years ago in Britain by Packe and Ayres (1985), who described an association between a thunderstorm and an asthma outbreak with 26 asthmatic subjects treated in Birmingham Hospital in 36 hours, compared with two to three cases at the same time in the days preceding the thunderstorm. Other asthma outbreaks during thunderstorms have

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Fig. 60.1 Pollen grains ruptured in the course of a thunderstorm. (See CD-ROM for color version.)

been described in Melbourne, Australia (Bellomo et al. 1992). This phenomenon was followed by a rapid increase in hospital or general practitioner visits for asthma. No unusual levels of air pollution were noted at the time of these epidemics but there was a strong association with grass pollen (Celenza et al. 1996; Venables et al. 1997). Another asthma outbreak occurred in London coinciding with a heavy thunderstorm on June 24, 1994, when a large increase was observed in the number of visits for asthma at emergency departments in London and the southwest of England; several patients who were not known to be asthmatic or were affected only by seasonal rhinitis experienced an asthma attack (Celenza et al. 1996; Venables et al. 1997). The epidemic had a sudden onset on June 24; 640 patients with asthma or other airways disease were seen during the 30 hours starting from 1800 on June 24, nearly 10 times the expected number (Davidson et al. 1996; Newson et al. 1997; Venables et al. 1997). Other asthma episodes occurred in Wagga Wagga, Australia on October 30, 1997 (Girgis et al. 2000) and in Naples on June 4, 2004 (D’Amato et al. 2005b). During the episode of thunderstorm-associated asthma registered in Naples on June 4, 2004, six adults and a girl of 11 had attacks of severe bronchial asthma; one case was nearly fatal. All patients needed treatment in emergency departments (D’Amato et al. 2005a,b). All subjects were outdoors when the thunderstorm struck and none regularly took antiallergic and/or antiasthma drugs. We found that all seven patients had allergic respiratory symptoms on exposure to Parietaria pollen, an Urticacea widespread in the Naples area (D’Amato et al. 1991, 2005b), and that the concentration of airborne Parietaria pollen grains was particularly high while air pollution levels were relatively low. Subjects sensitive to Parietaria who were indoors in Naples with the windows closed during the night of June 4 did not experience asthma attacks.

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The characteristics of epidemics of thunderstorm-associated asthma can be summarized as follows. 1 There is a link between asthma epidemics and thunderstorm. 2 Epidemics related to thunderstorm are limited to late spring and summer when there are high atmospheric concentrations of airborne allergenic pollens. 3 There is a close temporal association between the start of the thunderstorm and the onset of epidemics. 4 Air pollution is at low levels before and during thunderstorms. 5 The concentration of other aeroallergens such as molds in the atmosphere is low. 6 Subjects with allergic rhinitis but without previous asthma can experience severe bronchoconstriction for the first time during a thunderstorm. 7 Usually, subjects involved in described episodes were not receiving correct antiinflammatory (antiasthma) treatment. 8 Subjects with pollinosis who are indoors with the windows closed during a thunderstorm are not involved. In light of the above, subjects affected by pollen allergy should be alert to the danger of being outdoors during a thunderstorm during the pollen season. They should also consider that global warming favors an increased frequency of thunderstorms in temperate and subtropical areas.

Other plant-derived antigens responsible for epidemic asthma in urban areas Soybean dust could be responsible for outbreaks of severe asthma. Examples of such asthma epidemics have occurred in cities with large industrial port facilities, such as Barcelona (Spain) and Naples (Italy). In Barcelona, from 1981 to 1987, 26 outbreaks of asthma with 11 deaths were registered that did not have any apparent correlation to air pollution (Antó et al. 1989, 1993; Aceves et al. 1991). The etiologic agent was found to be soybean dust released into the air during unloading of cargo into a harbor silo that was not equipped with a dust-control device. Antò and colleagues demonstrated that about 74% of epidemic cases had specific IgE antibodies against a commercial soybean antigen in comparison with 4.6% of control subjects (Antó et al. 1989, 1993). In addition, using assays of urban aerosols collected with high-volume samplers and the RAST inhibition technique, these authors showed highly significant differences in the air content of soybean antigens between days marked by the asthma epidemic and days free of an excess of asthma exacerbations (Antó et al. 1989, 1993). The strong association between airborne soybean dust and asthma outbreaks was reinforced by the results of studies showing high airborne concentrations on epidemic days and low values on nonepidemic days. Moreover, asthma outbreaks were characterized by a “pointsource” epidemic. Protective measures, i.e., cargo unloading after filters were fitted to the grain elevators, progressively

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reduced airborne allergen levels of soybean dust, visits to the emergency room for asthma, and IgE serum levels in exposed subjects (Antò et al. 1993). Other outbreaks of asthma caused by soybean dust pollution have been documented in the Spanish cities of Tarragona and Cartagena (Hernando et al. 1989; Garcia-Ortega et al. 1997). In December 1993, in Naples, more than 100 patients were admitted to hospital for asthma in a single day coinciding with the unloading of a cargo of soybean (D’Amato et al. 2001). The ship was the same involved in the Barcelona epidemics. Interestingly, neither in the Barcelona nor in the Naples outbreaks were there cases of severe asthma attacks in children.

Climate changes and interaction between air pollution and pollinosis We still have much to learn about the effects of other climatic factors that seem to be important to asthma (Shumway et al. 1988; Schwartz 1994; Emberlin 1998; Garty et al. 1998; Bernard et al. 2001; D’Amato 2002b; Beggs 2004). It is well known that exercise in polluted areas results in greater deposition of air pollutants, including allergen-carrying particles, in the lower airways. Exercise increases oral breathing, total ventilation, and inertial impaction of inhaled particles in the airways. The role of climatic factors (e.g., barometric pressure, temperature, humidity) in triggering and/or exacerbating respiratory allergic symptoms in predisposed subjects is still poorly understood (Riedler et al. 2000; D’Amato et al. 2005a) and asthma attacks have been linked with both low and high atmospheric pressure. A variety of direct and indirect evidence suggests that climate changes, e.g., those induced by greenhouse gases, may affect pollen release and consequently pollen-related asthma (Emberlin 1998; Frenguelli & Bricchi 1998; Beggs 2004). Climate variations are likely to influence vegetation with consequent changes in growth and reproductive cycle, and in the production of allergenic pollen (seasonal period and intensity) with a greater proliferation of weed species. Climate changes vary from region to region: some areas will be subject to increases in ultraviolet radiation and/or rainfall frequency and other areas to reductions. In Italy in the 20 years from 1981 to 2000 the average mean temperature has increased by about 0.6°C; this warming has been accompanied by an average 15% reduction in rainfall, and the rain is concentrated in a shorter period causing more violent rainstorms (Ambiente Italia 2001). How are allergenic plants responding to these changes? The increased temperature in winter and spring has brought about early pollination, and the increased summer temperature has resulted in prolongation of the pollination of herbaceous, allergenic plants. Pollen seasons and therefore seasonal allergic symptoms tend to be longer in warmer years. The extension of autumn could prolong the presence of fungal spores in the atmosphere. Because of the “urban climate effect” (heating caused by high

Fig. 60.2 Ozone (O3) is generated at ground level by photochemical reactions involving ultraviolet radiation on atmospheric mixture of nitrogen dioxide (NO2) and hydrocarbons deriving from vehicle emissions. (See CD-ROM for color version.)

building density and soil sealing), pollination can occur 2–4 days earlier in urban than in rural areas. Vegetation reacts with air pollution over a wide range of environmental conditions and pollutant concentrations. Several factors influence the interaction, including type of air pollutant, plant species, nutrient balance, soil conditions, and climatic factors. At low levels of exposure for a given species and pollutant, no significant effect is observed. However, as the exposure level increases, there may be biochemical alterations of the plants (Emberlin 1998; Frenguelli & Bricchi 1998; Beggs 2004) (Table 60.2). Plants can absorb pollutants through the leaves or through the root system. In the latter case, deposition of air pollutants on soils can alter the nutrient Table 60.2 Rationale for the interrelationship between components of air pollution and allergens in inducing respiratory allergy. Air pollution can: Interact with pollen grains, leading to increased release of allergens characterized by modified antigenicity Interact with allergen-carrying paucimicronic particles derived from plants, which are able to reach the lower airways with inhaled air, inducing asthma in predisposed subjects Cause an inflammatory effect on the airways of susceptible subjects, inducing increased epithelial permeability, easier penetration of pollen allergens in the mucous membranes and easier interaction with cells of the immune system. There is also evidence that predisposed subjects have increased airway reactivity induced by air pollution and increased bronchial responsiveness to inhaled pollen allergens Have an adjuvant immunologic effect on IgE synthesis in atopic subjects (demonstrated with diesel exhaust particulates)

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content of soil in the proximity of the plant, thus leading to indirect or secondary effects of air pollutants on vegetation. Metabolic variations affect the plant’s structural integrity and there are probably changes in the pollen proteins, including those acting as allergens. Air pollution can influence plant allergenic content, and by affecting plant growth it can affect both the amount of pollen produced and the amount of allergenic proteins contained in pollen grains (Frenguelli & Bricchi 1998). The pollen of plants stressed by air pollution express enhanced levels of allergenic proteins (Emberlin 1998). Pollen grains collected from roadsides with heavy traffic and from other areas with high levels of air pollution are covered with large numbers of microparticulates (usually less than 5 μm in diameter) and there is a hypothesis that interaction between air pollution components and pollen allergens alters the antigenicity of pollen allergens (Emberlin 1998; D’Amato 2001; Traidl-Hoffmann et al. 2003).

Conclusions and future directions Among the factors implicated in the recent “epidemic” of bronchial asthma are indoor and outdoor air pollution in most industrialized countries. It is not easy to evaluate the effects of single air pollutants because there are limitations for each type of study (laboratory exposure study; time-series analysis of changes in asthma mortality or morbidity; cross-sectional study of comparison of different geographical areas). For example, laboratory exposure frequently uses a single pollutant and this type of exposition is different from real-life urban exposure. However, urbanization with its high levels of vehicle emissions and westernized lifestyle parallels the increase in respiratory allergy in most industrialized countries, and people who live in urban areas tend to be more affected by the disease than those in rural areas. In atopic subjects, exposure to air pollution increases airway responsiveness to aeroallergens and pollinosis seems to be a good model with which to study the interrelationship between air pollution and respiratory allergic diseases. Pollen grains and plant-derived paucimicronic components carrying antigens can produce allergic respiratory symptoms. By adhering to the surface of these airborne allergenic agents, air pollutants could modify their antigenic properties. Several factors influence this interaction, i.e., type of air pollutant, plant species, nutrient balance, climatic factors, degree of airway sensitization, and hyperresponsiveness of exposed subjects. However, the damage to airway mucous membranes and impaired mucociliary clearance induced by air pollution may facilitate the penetration and access of inhaled allergens to the cells of the immune system, and so promote airway sensitization. Consequently, an increased IgE-mediated response to aeroallergens and enhanced airway inflammation favored by air pollution could account for the increasing

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prevalence of allergic respiratory diseases in urban polluted areas. It is not easy to evaluate the impact of air pollution on the timing of asthma exacerbations and on the prevalence of asthma in general, as concentrations of airborne allergens and air pollutants are frequently increased contemporaneously. However, an enhanced IgE-mediated response to aeroallergens and enhanced airway inflammation could account for the increasing frequency of allergic respiratory allergy and bronchial asthma. Pollen allergy is frequently used to study the interrelationship between air pollution and respiratory allergy. Climatic factors (temperature, wind speed, humidity, thunderstorms, etc.) can affect both components (biological and chemical) of this interaction. By attaching to the surface of pollen grains and of plant-derived particles of paucimicronic size, components of air pollution could modify not only the morphology of these antigen-carrying agents but also their allergenic potential. In addition, by inducing airway inflammation, which increases airway permeability, pollutants overcome the mucosal barrier and could be able to “prime” allergen-induced responses. Much remains to be studied using biological, genetic, epidemiologic and clinical approaches to air pollution (Englert 2004; Schwartz 2004; D’Amato et al. 2005a; McCunney 2005; Delfino 2006). However, public health approaches for decreasing the exposure of citizens to air pollution must be implemented bearing in mind the following goals: reducing use of fossil fuels and controlling vehicle emissions; reducing private traffic in towns and improving public transport; favoring pedestrian traffic; and planting nonallergenic trees in cities. Moreover, although there is no general agreement, increasing the antioxidant defenses of the human airways by eating antioxidant foods should be implemented (Fogarty et al. 2003; Allam & Lucane 2004; D’Amato 2004; Pearson et al. 2004).

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D’Amato, G., Spieksma, F.Th.M., Liccardi, G. et al. (1998) Pollenrelated allergy in Europe. Position paper of the European Academy of Allergology and Clinical Immunology. Allergy 53, 567–78. D’Amato, G., Liccardi, G., D’Amato, M. & Cazzola, M. (2001) The role of outdoor air pollution and climatic changes on the rising trends in respiratory allergy. Respir Med 95, 606–11. D’Amato, G., Liccardi, G., D’Amato, M. & Cazzola, M. (2002) Outdoor air pollution, climatic changes and allergic bronchial asthma. Eur Respir J 20, 763– 76. D’Amato, G., Liccardi, G., D’Amato, M. & Holgate, S.T. (2005a) Environmental risk factors and allergic bronchial asthma. Clin Exp Allergy 35, 113–24. D’Amato, G., Liccardi, G., Frenguelli, G. (2007) Thunderstormassociated asthma in pollutinosis patients. Allergy 62, 11–16. Davidson, A.C., Emberlin, J., Cook, A.D. et al. (1996) A major outbreak of asthma associated with a thunderstorm. BMJ 312, 601–4. De Marco, R., Poli, A., Ferrari, M. et al. (2002) The impact of climate and traffic related NO2 on the prevalence of asthma and allergic rhinitis in Italy. Clin Exp Allergy 32, 1405–12. Delfino, R.J. (2006) Who are the children with asthma most susceptible to air pollution? Am J Respir Crit Care Med 173, 1054–5. Devalia, J.L., Rusznak, C. & Davies, R.J. (1998) Allergen/irritant interaction-its role in sensitization and allergic disease. Allergy 53, 335–45. Devlin, R.B., McDonnell, W.F. & Mann, R. (1991) Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung. Am J Respir Cell Mol Biol 4, 72– 81. Devlin, R.B., Folinsbee, L.J., Biscardi, F. et al. (1993) Attenuation of cellular and biochemical changes in the lungs of humans exposed to ozone for five consecutive days. Am Rev Respir Dis 147, A71. Diaz-Sanchez, D., Taieu, A., Casillas, A., Dotson, A.R. & Saxon, A. (1996) Enhanced nasal cytokine production in human beings after in vivo challenge with diesel exhaust particles. J Allergy Clin Immunol 98, 114–23. Diaz-Sanchez, D., Tsien, A., Fleming, J. & Saxon, A. (1997) Combined Diesel exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-specific IgE and skews cytokine production to a T helper cell 2-type pattern. J Immunol 158, 2406–13. Diesel Working Group (1995) Diesel Exhaust: A Critical Analysis of Emissions, Exposure and Health Effects. Health Effects Institute, Cambridge, MA. Dockery, D.W., Pope, C.A., Xu, X. et al. (1993) An association between air pollution and mortality in six US cities. N Engl J Med 329, 1753–9. Edwards, J., Walters, S. & Griffiths, R.K. (1994) Hospital admissions for asthma in preschool children: relationship to major roads in Birmingham, United Kingdom. Arch Environ Health 49, 223–7. Emberlin, J. (1998) The effects of air pollution on allergenic plants. Eur Respir Rev 53, 164– 7. Englert, N. (2004) Fine particles and human health: a review of epidemiological studies. Toxicol Lett 149, 235– 42. European Respiratory Society (2003) European Lung White Book. The First Comprehensive Survey on Respiratory Health in Europe. Gibson, R., Sibille, G.J., eds. Loddenkemper (www.ersnet.org). Fogarty, A., Lewis, S.A., Scrivener, S.L. et al. (2003) Oral magnesium and vitamin C supplements in asthma: a parallel group randomised placebo-controlled trial. Clin Exp Allergy 33, 1355– 9. Folinsbee, L.J., Bedi, J.F. & Horvath, S.M. (1984) Pulmonary func-

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tion changes after a continuous heavy exercise in 0.21 ppm ozone. J Appl Physiol 57, 984–8. Folinsbee, L.J., Horstman, D.H., Kehrl, H.R. et al. (1994) Respiratory responses to repeated prolonged exposure to 0.12 ppm ozone. Am J Respir Crit Care Med 149, 98–105. Foxcroft, W.J. & Adams, W.C. (1986) Effects of ozone exposure on four consecutive days on work performance and VO2max. J Appl Physiol 61, 960–6. Frenguelli, G. & Bricchi, E. (1998) The use of the pheno-climatic model for forecasting the pollination of some arboreal taxa. Aerobiologia 14, 39–44. Garcia-Ortega, P., Rovira, E. & Mora, E. (1997) Soy-seed asthma epidemics in small cities. Med Clin (Barc) 108, 677. Garty, B.Z., Kosman, E. & Ganor, E. (1998) Emergency room visits of asthmatic children, relation to air pollution, weather and airborne allergens. Ann Allergy Asthma Immunol 81, 563–70. Gauderman, W.J., Avol, E., Gilliland, F. et al. (2004) The effect of air pollution on lung development from 10 to 18 years of age. N Engl J Med 351, 1057–67. Ghio, A.J. & Hatch, G.E. (1993) Lavage phospholipid concentration after silica instillation in the rat is associated with complexed (Fe3+) on the dust surface. Am J Respir Cell Mol Biol 8, 403–7. Girgis, S.T., Marks, G.B., Downs, S.H., Kolbe, A., Car, G.N. & Paton, R. (2000) Thunderstorm-associated asthma in an inland town in southeastern Australia. Who is at risk? Eur Respir J 16, 3–8. Hazucha, M.J., Ginsberg, J.F., McDonnell, W.F. et al. (1983) Effects of 0.1 ppm nitrogen dioxide on airways of normal and asthmatic subjects. J Appl Physiol 54, 730–9. Hernando, L., Navarro, C., Marquez, M., Zapatero, L. & Galvañ, F. (1989) Asthma epidemics and soybean in Cartagena (Spain). Lancet i, 502. Hirsch, T., Weiland, S.K., von Mutius, E. et al. (1999) Inner city air pollution and respiratory health and atopy in children. Eur Respir J 14, 669–77. Holtzman, M.J., Fabbri, L.M., O’Byrne, P.M. et al. (1983) Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis 127, 686–90. Horstman, D., Roger, L.J., Kehrl, H. & Hazucha, M.J. (1986) Airway sensitivity of asthmatics to sulfur dioxide. Toxicol Ind Health 2, 289– 98. Horstman, D., Ball, B.A., Brown, J., Gerrity, T. & Folinsbee, L.J. (1995) Comparison of asthmatic and nonasthmatic subjects performing light exercise while exposed to a low level of ozone. Toxicol Ind Health 11, 369–85. Jensen, J., Poulsen, L.K., Mygind, N. & Weeke, E.R. (1989) Immunochemical estimations of allergenic activities from outdoor aeroallergens, collected by a high-volume air sampler. Allergy 44, 52–9. Jorres, R., Nowak, D. & Magnussen, H. (1996) Effect of ozone exposure on allergen responsiveness in subjects with asthma or rhinitis. Am J Respir Crit Care Med 153, 56–64. Kehrl, H.R., Peden, D.B., Ball, B., Folinsbee, L.J. & Horstrom, D. (1999) Increased specific airway reactivity of persons with mild allergic asthma after 76 hours of exposure to 0.16 ppm ozone. J Allergy Clin Immunol 104, 1198–204. Knox, R.B. (1993) Grass pollen, thunderstorms and asthma. Clin Exp Allergy 23, 354–9. Knox, R.B., Suphioglu, C., Taylor, P. et al. (1997) Major grass pollen allergen Lol p1 binds to diesel exhaust particles: implications of asthma and air pollution. Clin Exp Allergy 27, 246– 51.

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Kreit, J.W., Gross, K.B., Moore, T.B. et al. (1989) Ozone-induced changes in pulmonary function and bronchial responsiveness in asthmatics. J Appl Physiol 66, 217– 22. Künzli, N., Kaiser, R., Medina, S. et al. (2000) Public health impact of outdoor and traffic-related air pollution: a European assessment. Lancet 356, 795– 801. Linn, W.S., Shamoo, D.A., Avol, E.L. & Hackney, J.D. (1986) Doseresponse study of asthmatic volunteers exposed to nitrogen dioxide during intermittent exercise. Arch Environ Health 41, 292–96. Linn, W.S., Avol, E.L., Peng, R.C., Shamoo, D.A. & Hackney, J.D. (1987) Replicated dose-response study of sulfur dioxide in normal, atopic and asthmatic volunteers. Am Rev Respir Dis 136, 1127–34. McConnell, R., Berhane, K., Gilliland, F. et al. (2002) Asthma in exercising children exposed to ozone: a cohort study. Lancet 359, 386–91. McCunney, R.J. (2005) Asthma, genes, and air pollution. J Occup Environ Med 47, 1285– 91. Marks, G.B., Colquhoun, J.R., Girgis, S.T. et al. (2001) Thunderstorm outflows preceding epidemics of asthma during spring and summer. Thorax 56, 468– 71. Molfino, N.A., Wright, S.C., Katz, I. et al. (1991) Effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects. Lancet 338, 199– 203. Mortimer, K.M., Tager, I.B., Dockery, D.W., Neas, L. & Redline, S. (2000) The effect of ozone on inner-city children with asthma: identification of susceptible subgroups. Am J Respir Crit Care Med 162, 1838– 45. Moshenin, V. (1987) Airway responses to nitrogen dioxide in asthmatic subjects. J Toxicol Environ Health 22, 371–80. Motta, A.C., Marliere, M., Peltre, G., Sterenberg, P.A. & Lacroix, G. (2006) Traffic-related air pollutants induce the release of allergencontaining cytoplasmic granules from grass pollen. Int Arch Allergy Immunol 139, 294– 8. Murray, V., Venables, K., Laing-Morton, T., Partridge, M. & Williams, D. (1994) Epidemic of asthma possibly related to thunderstorms. BMJ 309, 131– 2. Nauss, K.M., Busby, W.F. Jr, Cohen, A.J. et al. (1995) Critical issue in assessing the carcinogenicity of diesel exhaust: a synthesis of current knowledge. In: Diesel Exhaust: A Critical Analysis of Emission, Exposure and Health Effects. Health Effects Institute, Cambridge, MA, pp. 13–18. Newson, R., Strachan, D., Archibald, E., Emberlin, J., Hardaker, P. & Collier, C. (1997) Effect of Thunderstorms and airborne grass pollen on the incidence of acute asthma in England, 1990–1994. Thorax 52, 680– 5. Pacini, E. & Franchi, G.G. (1993) Role of the tapetum in pollen and spore dispersal. Plant Syst Evol Suppl. 7, 1–11. Packe, G.E. & Ayres, J.G. (1985) Asthma outbreak during a thunderstorm. Lancet ii, 199– 204. Pearson, P.J., Lewis, S.A., Britton, J. & Fogarty, A. (2004) Vitamin E supplements in asthma: a parallel group randomised placebo controlled trial. Thorax 33, 1355– 9. Peden, D.B. (2005) The epidemiology and genetics of asthma risk associated with air pollution. J Allergy Clin Immunol 115, 213–19. Peden, D.B., Setzer, R.W. & Devlin, R.B. (1995) Ozone exposure has both a priming effect on allergen induced responses as well as an intrinsic inflammatory action in the nasal airways of perennial allergic asthmatics. Am J Respir Crit Care Med 151, 1336–45. Peden, D.B., Boehlecke, B., Horstman, D. & Devlin, D.B. (1996) Influx of bronchial neutrophils and eosinophils in asthmatics after

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prolonged exposure to 0.16 ppm ozone. Am J Respir Crit Care Med 153, A700. Pekkanen, J., Peters, A., Hoek, G. et al. (2002) Particulate air pollution and risk of ST-segment depression during repeated submaximal exercise tests among subjects with coronary hearth disease: The exposure and risk assessment for fine and ultrafine particles in ambient air. Circulation 106, 933–8. Peters, A., von Klot, S., Heier, M. et al. (2004) Exposure to traffic and the onset of myocardial infarction. N Engl J Med 351, 1721– 30. Peters, J.M., Avol, E., Gauderman, W.J. et al. (1999) A study of twelve Southern California communities with differing levels and types of air pollution. II. Effects on pulmonary function. Am J Respir Crit Care Med 159, 768–75. Pope, A., Thun, M., Namboodiri, M. et al. (1995) Particulate air pollution as a predictor of mortality in a prospective study of US adults. Am J Respir Crit Care Med 151, 669–74. Pope, C.A., Burnett, R.T., Thurston, G.D. et al. (2004) Cardiovascular mortality and longterm exposure to particulate air pollution: epidemiologic evidence of general pathophysiological pathways of disease. Circulation 109, 71–7. Rabinovitch, N., Strand, M. & Gelfand, E.W. (2006) Particulate levels are associated with early asthma worsening in children with persistent disease. Am J Respir Crit Care Med 173, 1098–105. Reidel, F., Kramer, M., Scheibenbogen, C. & Rieger, C.H.L. (1988) Effects of SO2 exposure on allergic sensitization in the guinea pig. J Allergy Clin Immunol 82, 527–34. Riedl, M. & Diaz-Sanchez, D. (2005) Biology of diesel exhaust effects on respiratory function. J Allergy Clin Immunol 115, 221–8. Riedler, J., Eder, W., Oberfeld, G. & Schrener, M. (2000) Austrian children living on a farm have less hay fever, asthma and allergic sensitization. Clin Exp Allergy 30, 194–200. Roger, L.J., Horstman, D.H., McDonnell, W.F. et al. (1990) Pulmonary function, airway responsiveness and respiratory symptoms in asthmatics following exercise in NO2. Toxicol Ind Health 6, 155–71. Rom, W.N. & Samet, J.M. (2006) Small particles with big effect. Am J Respir Crit Care Med 173, 365–6. Rudell, B., Sandstrom, T., Stjernberg, N. & Heldman, K.B. (1990) Controlled diesel exhaust exposure in an exposure chamber: pulmonary effects investigated with bronchoalveolar lavage. J Aerosol Sci 21, S411–S414. Rusznak, C., Devalia, J.L. & Davies, R.J. (1996) The airway response of asthmatics to inhaled allergen after exposure to pollutants. Thorax 344, 1668–71. Salvi, S. & Holgate, S. (1999) Mechanisms of particulate matter toxicity. Clin Exp Allergy 29, 1187–94. Scannell, C., Chen, L., Aris, R.M. et al. (1996) Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med 154, 24–9. Schelegle, E.S. & Adams, W.C. (1986) Reduced exercise time in competitive simulations consequent to low level ozone exposure. Med Sci Sports Exerc 18, 408–14. Schlesinger, R.B. & Driscoll, K.E. (1987) Mucociliary clearance from the lungs of rabbits following single and intermittent exposures to ozone. J Toxicol Environ Health 20, 125–34. Schwartz, J. (1994) Air pollution and daily mortality: a review and meta-analysis. Environ Res 64, 36–52. Schwartz, J. (2004) Air pollution and children’s health. Pediatrics 113, 1037–43.

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Seaton, A., MacNee, W., Donaldson, K. & Godden, K. (1995) Particulate air pollution and acute health effects. Lancet 345, 176–8. Sheppard, D., Wong, S.C., Uehara, C.D., Nadel, J.A. & Boushey, H.A. (1980) Lower threshold and greater bronchomotor responsiveness of asthmatic subjects to sulfur dioxide. Am Rev Respir Dis 122, 873–8. Shumway, R.H., Azari, A.S. & Pawitan, Y. (1988) Modelling mortality fluctuations in Los Angeles as functions of pollution and weather effects. Environ Res 45, 224– 41. Smith, K.R. & Aust, A.E. (1997) Mobilization of iron from urban particulates leads to generation of reactive oxygen species in vitro and induction of ferritin synthesis in human lung epithelial cells. Chem Res Toxicol 10, 828– 34. Solomon, W.R., Burge, H.A. & Muilenberg, M.L. (1983) Allergen carriage by atmospheric aerosol. I. Ragweed pollen determinants in smaller micronic fractions. J Allergy Clin Immunol 72, 443–7. Spieksma, F.Th.M., Nikkels, B.H. & Dijkman, J.H. (1995) Seasonal appearance of grass pollen allergen in natural, paucimicronic aerosol of various size fractions. Relationship with airborne grasspollen concentration. Clin Exp Allergy 25, 234– 9. Stenfors, N., Nordenhall, C., Salvi, S.S. et al. (2004) Different airway inflammatory responses in asthmatic and healthy humans exposed to diesel. Eur Respir J 23, 82– 6. Sullivan, J., Ishikawa, N., Sheppard, L., Siscovick, D., Checkoway, H. & Kaufman, J. (2003) Exposure to ambient fine particulate matter and primary cardiac arrest among persons with and without clinically recognized heart disease. Am J Epidemiol 157, 501–9. Sullivan, J., Sheppard, L., Schrender, A., Ishikawa, N., Siscovick, D. & Kaufman, J. (2005) Relation between short term fine-particulate matter exposure and onset of myocardial infarction. Epidemiology 16, 41–8. Sunyer, J., Saez, M., Murillo, C. & Castellsague, J.M.F. (1993) Air pollution and emergency room admission for chronic obstructive pulmonary disease: a 5-year study. Am J Epidemiol 137, 701–5. Suphioglu, C., Singh, M.B., Taylor, P. & Knox, R.B. (1992) Mechanism of grass-pollen-induced sthma. Lancet 339, 569–72. Sydbom, A., Blomberg, A., Parnia, S., Stenfors, N., Sandstrom, T. & Dahlén, S.E. (2001) Health effects of diesel exhaust emissions. Eur Respir J 17, 733– 46. Takafuji, S., Suzuki, S., Koizumi, K. et al. (1987) Diesel exhaust particulates inoculated by the intranasal route have an adjuvant activity for IgE production in mice. J Allergy Clin Immunol 79, 639–45. Takenaka, H., Zhang, K., Diaz-Sanchez, D., Tsien, A. & Saxon, A. (1995) Enhanced human IgE production results from exposure to

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Indoor Air Pollution Paul Harrison, Rebecca Slack and Sanjeev Bagga

Summary Many substances occurring within indoor environments, from both natural and anthropogenic sources, exert negative influences on human health. For air pollutants, the predominant effect is respiratory disease, often manifested as changes in lung function, respiratory irritation, allergies, and asthma. Biological particles, whether animal dander, house-dust mite feces, fungal/bacterial cell components and spores, or pollen from external sources, are the primary vectors for indoor allergens and triggers for allergic responses. Other common indoor pollutants such as oxides of nitrogen (NOx), sulfur dioxide (SO2), formaldehyde and, to a lesser extent, ozone, can cause irritation and exert adjuvant effects that may exacerbate respiratory dysfunction. In addition, these and other substances in the indoor environment, for example volatile organic compounds (VOCs), fine particles of anthropogenic origin, environmental tobacco smoke, radon, fibers and heavy metals, can have important impacts on health not mediated by an immune response. Inflammatory change is the most frequent pathologic condition resulting from exposure to indoor air pollutants, which is supported by pharmacologic changes observed in vivo. Epithelial cell damage, airway remodeling and cytokine release are additional physiologic responses to indoor air allergens. Significant indoor sources include fuel-burning appliances, building and decorating materials, furniture and furnishings, and consumer products, as well as biological sources, notably house-dust mites, fungi, and bacteria (frequently associated with dampness and humidity). Various measures can be adopted to reduce indoor air pollutants and their effects, including reducing humidity, increasing ventilation, and removing or reducing the pollutant sources. Efforts to improve the indoor environment can be attempted through building regulations (e.g., ventilation standards), product control, product labeling and individual guideline values for common indoor air pollutants. Guidance for residents

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

is also valuable. However, the links between indoor air quality and allergic airway disease are not yet fully transparent and thus the most effective mitigating method for such impacts, or even whether such mitigation is effective at all, is often not established.

Introduction The effects of air quality on human health have been the focus of much attention and debate for a considerable period of time (Evelyn 1661; Brimblecombe 1987; DuPuis 2004). The development of UK garden cities and model towns, such as Port Sunlight and Saltaire, demonstrated an acknowledgment of the importance of clean air to the health of the population that was further enhanced by the Clean Air Acts of 1956 and 1968 following the London smog of 1952 (Leslie 2000). In the European Union (EU), community legislation on emission sources was first implemented in 1970, with air quality standards following in 1980. The legislation was continuously updated, with the Air Quality Framework Directive (96/62/EC) in 1996 followed by a number of daughter directives. As a consequence of the EU’s 6th Environment Action Programme in 2002, the European Commission adopted a new Strategy on Environment and Health with the aim of reducing diseases caused by environmental factors. Air quality is one of the main issues identified in the Strategy due to its association with pulmonary, allergic, and cardiovascular disease and, at the extreme end of the scale, cancer. The role and significance of indoor air pollution are increasingly being recognized. Although indoor air quality generally reflects the quality of the air outside, there are a number of significant indoor sources, and the confined nature of the indoor environment presents a range of unique problems (Institute of Environment and Health 1996; Harrison & Holmes 2000). This is extremely relevant considering the amount of time spent in indoor environments (at work and at home): it is estimated that people in northern Europe and North America spend more than 70% of their time indoors including time

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spent at home, at work, or in transport (Sneddon 1994). The quality of the indoor air is especially significant for potentially susceptible groups such as the very young, old and chronically ill, who are likely to spend even more time indoors. This chapter presents an introduction to the issue of indoor air pollution, outlining the key contaminants that occur in many homes and nonindustrial workplaces in the developed world and focusing, where relevant, on allergic airway disease. In the developing world, where the main factor influencing indoor air quality is the burning of solid fuel in domestic environments, the contaminants will differ in quantity and composition. The effects of indoor pollutants on health vary, but all exert an influence on the respiratory system through inhalation, potentially affecting respiratory peak flow volumes and causing symptoms of respiratory distress, including wheeze and asthmatic episodes. Sensitization is more prevalent for indoor than outdoor allergens (Boulet et al. 1997) and it is therefore important to consider the consequences for allergic airway disease and other illnesses resulting from poor indoor air quality.

Allergens and allergic airway disease Allergens are foreign substances (antigens) that are recognized as such by the immune system. They have the ability to cause the onset of an allergic response, which initiates a cascade of inflammatory events, both serologic and cellular. Types of allergens vary widely, from foodstuffs (e.g., peanuts, raisins), insects (wasp/bee sting), and drugs to airborne particles such as pollen, dander, dust, and hair. Some allergens result in sensitization, which involves an immunologic response mediated through IgE. The initial immune response can provoke bronchoconstriction and, in more severe cases, inflammation with airway remodeling. Initial allergen contact results in increased synthesis of IgE. This is rapidly transported to target tissues where it combines with effector cells such as mast cells and basophils. This combined IgE fragment is then bound to a specific allergen to release a collection of mediators, mainly inflammatory mediators such as histamine, which contributes to rhinitis and asthma within individuals (Howarth 1998). However, the presence of IgE antibodies is not a direct cause of an allergic disease such as asthma; rather their presence represents a collective immune response, which may result in allergic type conditions in some individuals (Custovic et al. 2005). Asthma is a prime example of a disease that can be impacted by both outdoor and indoor factors. It affects around 10–15% of the population, and has reached epic proportions in western societies, especially over the last two decades (Kay 2001). Asthma is a debilitating and potentially fatal disease constituting a series of physiologic changes, all associated within the respiratory tract. Observed pathologic changes occur as a result of reversible airflow obstruction and airway hyper-

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responsiveness, along with productive cough and wheeze (Bousquet et al. 2000). These combined pathophysiologic alterations within the airways appear to involve a series of mediators, which act by means of subcellular and cytokinedependent mechanisms. Asthma can be intrinsic or extrinsic; in the case of intrinsic asthma, drugs, alcohol, tobacco smoke, and airborne pollution appear to be the usual causal factors of disease propagation, whereas in the extrinsic case, pollen, dust, hair, viruses, or bacteria constitute the triggering factors.

Indoor air quality Indoor air quality is affected by a range of parameters including local climate, season, and building design, construction, function, and use. In developed countries, energy conservation measures, such as improved draught-proofing, have resulted in buildings with less air exchange and thus increased potential for indoor pollutants to accumulate. In developing countries, particularly in less affluent areas, ventilation can be expected to be greater and therefore internal air quality is likely to be predominantly dependent on the external environment as well as the presence of solid fuel stoves. Irrespective of ventilation rates, indoor air quality can differ from the outdoor situation through reduced photolytic reactions due to restricted ingress of ultraviolet light, lower levels of ozone production, and provision of a greater surface area, through the presence of furniture and internal structures, for deposition, adsorption, and chemical reactions. Importantly, there are a number of specific indoor sources of pollution, including for example fuel-burning appliances, building and decorating materials, furniture and furnishings, and consumer products, that liberate a wide range of different pollutants into the indoor air (Institute of Environment and Health 1996). A number of common substances, derived from both natural and artificial sources, can be identified in the air within a typical building, although the concentrations vary significantly between building types and different countries. In addition to particles and biological material (microorganisms and allergens), over 900 different organic compounds have been identified in indoor air in Europe (SCALE 2004). The European Commission’s Scientific Committee on Health and Environmental Risks (SCHER), in a preliminary risk assessment report on indoor air quality, and in line with other work, declared the following substances to be of particular concern in the indoor environment (INDEX 2005; Scientific Committee on Health and Environmental Risks 2007): formaldehyde, carbon monoxide (CO), nitrogen dioxide (NO2), benzene, naphthalene, environmental tobacco smoke (ETS), radon, lead, and organophosphate pesticides. It is possible that the many different types of pollutants give rise to combined health effects, with certain substances initiating or enhancing responses to others. This is particularly important for allergic responses. Some categories of substances

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commonly encountered in the indoor environment are reviewed below.

Particles and ultrafines (nanoparticles) Airborne particulate matter in indoor environments derives from various sources and encompasses a complex group of organic and inorganic substances and mixtures. ETS (see below) can be considered to be the most significant indoor particle source, but emissions from kerosene heaters and wood-burning stoves, particles evolved during cooking (food and fuel combustion), household dust (particularly when disturbed through cleaning activities), pet dander, fungal spores, and outside sources (including traffic emissions, soil and bonfires), all contribute to the indoor particulate load (Institute of Environment and Health 2000). A number of these particle types are considered in more detail below. Various conventions have been developed to define particles, but most widely used definitions use categories based on size distribution. Typically, the main groups used to differentiate between airborne particulates are as follows: • Nanoparticles: less than 100 nm in at least one dimension. These include ultrafine particles that are considered to be less than 100 nm in diameter. • Fine particles: less than 2 μm but greater than 100 nm diameter. Further divided into particles below 0.2 μm diameter (nucleation mode) and particles between 0.2 and 2 μm diameter (accumulation mode). • Coarse particles: greater than 2 μm diameter. Common particle size categories are PM10 (particulate matter with maximum aerodynamic diameter 10 μm, representing the inhalable “thoracic” fraction), PM3.5 (up to 3.5 μm diameter, the “respirable” fraction) and PM2.5 (maximum diameter 2.5 μm, the “high-risk” respirable fraction) (Institute of Environment and Health 2000). Combustion and secondary particles that have a diameter less than 1 μm (PM1.0) are of great concern. They usually contain a wide range of toxic elements (gaseous pollutants, industrial and fuel components) and have the ability to penetrate deep into the lung and alveolar air sacs. The growing use of nanomaterials in household goods, from fabric treatments to cosmetics, is likely to increase the amount and variation of particle exposure in the indoor environment (Rickerby & Morrison 2007). It is important to appreciate that, at the nano scale, substances can demonstrate very different chemical and physical properties than at larger sizes, making translation of current toxicologic data (gained from larger sized particles) difficult. The high surface area to volume ratio of fine particles permits adsorption of volatile organic compounds (VOCs), some of which possess irritant and/or carcinogenic properties, e.g., polycyclic aromatic hydrocarbons (PAHs) (Leslie 2000). Links to incidences of childhood asthma have also been identified and are associated in particular with PM2.5 particles (Weichenthal et al. 2007). Levels of particles measured in indoor environments vary according to sampling

Indoor Air Pollution

category used, sample size, sampling duration, and location (Lazaridis et al. 2006). Early studies in the USA established an average indoor particle level of around 40 μg/m3 for PM10 and PM3.5 and showed that smoking increased the indoor average by a further 20 μg/m3 (Wallace 1996). Personal exposure levels tend to be higher than both indoor and outdoor exposure levels due to the “personal cloud” effect resulting from disturbance in the individual’s immediate vicinity (Harrison 1998).

Environmental tobacco smoke ETS has been referred to as “passive smoking” and includes all emissions from the cigarette tip (sidestream smoke; SS), mainstream smoke (MS), waste smoke (WS) and gases escaping through the paper. ETS consists of a complex array of chemicals produced from incomplete combustion of plant material: 2000 individual compounds have been identified in tobacco leaves resulting, through the processes of pyrolysis, distillation, condensation, filtration, and oxidation, to an estimated 4000 compounds in ETS (Weetman 1994; International Agency for Research on Cancer 2004). The type and concentration of these chemicals varies considerably depending on the type/variety of tobacco plant, location of growth, curing process, manufacture process, etc. It also varies between SS, MS and WS and through the “aging” process, i.e., the time spent in the air, which leads to decay or further chemical reactions or settling of suspended particles. Gas (oxides of nitrogen, sulfur oxides, carbon monoxide, carbon dioxide), vapor (VOCs), and particulate (tar) phases determine the composition of ETS. Particulates are the main health concern, with smoking increasing particle concentrations up to 10 times that of background levels (the increase being less for ETS-associated gases and vapors), although many of the carcinogens associated with cigarette smoke are present in both the gas and particulate phases (Weetman 1994). “Passive” smoking has been cited as a significant cause of lung cancer, pulmonary disease, cardiovascular disease, impaired fetal development, and sudden infant death syndrome (Harrison 1998); however, care must be taken when interpreting epidemiologic evidence owing to the presence of confounders (Wald et al. 1986; Weetman 1994; DiFranza et al. 2004; International Agency for Research on Cancer 2004). ETS is also associated with eye irritation and irritation of the mucous membranes of the nose and mouth, bronchospasm in asthmatics, and increased ear infections/slowed respiratory tract development in children (Tamburlini et al. 2002).

House-dust mites and pet allergens House-dust mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) are ubiquitous in homes in warm temperate regions, preferring warm damp conditions (Harrison 1999). They are not generally believed to be a significant problem in office buildings (Sherwood Burge 1994). There is clear evidence that allergens, principally Der p 1 derived from house-dust mite feces, are one of the major causes of allergic

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sensitization (Korsgaard 1983; Carrer et al. 2001); people who are sensitized to mites are more likely than those not sensitized to manifest symptoms of asthma and other allergies. However, the presence of dust mite allergens, as with all allergens, will not necessarily initiate asthma in an individual, but may cause symptoms to deteriorate (Tavernier et al. 2006). In one UK study, 80% of children displaying asthmatic symptoms also demonstrated an allergic response to house-dust mite (Price et al. 1990). House-dust mites are also linked to allergic rhinitis, a malady with similar symptoms to hay fever, causing inflammation of the nasal passages. The mechanisms causing the allergic response to the Der p 1 allergen provide an example of the cascade of physiologic responses that may result from exposure. In vitro, Der p 1 has been shown to induce proinflammatory cytokine production (King et al. 1998), which could then result in secondary changes such as disruption of epithelial cell layers (Platts-Mills et al. 1998), the release of the type 2 IgE antibody receptor, CD23, thus blocking the inhibitory effect of IgE (Hewitt et al. 1995), and inactivation of the interleukin (IL)-2 receptor on T cells (Schulz et al. 1998). Methods employed to assess house-dust mites and their potential to cause allergic response have included qualitative extraction of mites from dust (to yield species data and determine the effectiveness of mite reduction strategies), monoclonal immunoassays (for more accurate quantification of mite allergens in dust), indirect measurement using an indicator such as guanine (screening tool to determine allergens and the effects of intervention strategies), and measurement of antigens in air samples. Allergen exposures within indoor environments, particularly the home, can vary from trace levels of 0.01–0.03 μg/g dust to 200 μg/g dust, with the highest concentrations associated with mattresses, pillows, upholstered furniture, carpets, and children’s soft toys (Schwartz et al. 1987). The species of mite and their distribution within a house, as well as the quantity of mite allergens, are influenced by temperature and humidity (Building Research Establishment 1996); thus increased ventilation and the use of air conditioning has been shown to reduce levels of mite allergens (Luczynska 1994). Although these and other (e.g., barrier) methods can be employed to reduce exposure to house-dust mite allergen, complete eradication is difficult and a given reduction in exposure will not necessary remove or reduce symptoms in an affected individual. Domestic animals, including cats, dogs, birds, rodents, and even horses, may cause allergic asthma and rhinoconjunctivitis (Carrer et al. 2001; Kim et al. 2007a). In one study, 57% of asthmatic children were allergic to at least one domestic animal (Kjellman & Pettersson 1983). Reports of respiratory illness associated with animals go as far back as Salter (1864) and Blackley (1873). Allergens are associated with dander, hair, saliva, and urine. Dander is the most important source of cat allergen, with the allergen present in concentrations of

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250–1140 ng/m3 in households with at least one cat (Carrer et al. 2001). The allergen is particularly associated with particles of 1–10 μm in size (Swanson et al. 1989). Such particles remain airborne for long periods, increasing the likelihood of exposure and frequently initiating an allergic response in sensitized individuals immediately on entry into an affected indoor space. Ventilation, humidity, and even building quality have negligible effects on pet allergen levels; a regular household cleaning regimen has been shown to be the predominant method of controlling pet allergen levels. Pet-derived allergens are easily transported between indoor environments, with cat allergens detected in schools and offices where no animals were present (Liccardi et al. 2000; Carrer et al. 2001).

Fungi, bacteria and viruses Damp and mold are commonly found in northern European houses and have been a cause of concern regarding possible effects on health from exposure to, principally, fungal spores and, to a lesser extent, bacteria. Many species of these microorganisms are associated with various forms of organic matter including the surface coating of the walls, wood, fabrics, and foodstuffs. Some species are particularly associated with dampness, which is known to correlate with respiratory symptoms in some circumstances, although the direct causal relationship between damp buildings and illness is not established due to the large number of external factors, which may play a part in promoting disease. The 1996 Building Research Establishment study in British homes identified a number of fungal species, including Penicillium, Cladosporium, Aspergillus, and Mycelia sterile (Building Research Establishment 1996). A large number of fungi are linked to extrinsic allergic alveolitis. Stachybotrys chartarum has been the highlight of many recent cases of complaints with respect to indoor exposure (Kuhn & Ghannoum 2003). The association between Stachybotrys and human disease was first noted during equine epidemics when individuals who came into contact with musty straw developed dermatologic and respiratory symptoms (Drobotko 1945). However, a definitive link between exposure to Stachybotrys in indoor environments and human disease remains unconfirmed. Potential mechanisms in humans of respiratory-related disease from fungal exposure have been hypothesized, but not proved experimentally. One possible trigger may be mycotoxins, biologically active species produced during fungal growth that have been shown to possess carcinogenic, neurotoxic, and teratogenic properties (Samson 1992). However, positive skin-prick tests (SPT) used to determine allergic response to individual allergens or allergenic sources have found no significant correlation between positive SPT, mold abundance, and mold “allergy” despite prevalence of molds within homes (Katz et al. 1999). Bacteria are also present within indoor environments,with Gram-positive bacteria dominating over Gram-negative bacteria (Sherwood Burge 1994). Endotoxins are lipopolysaccharides

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present as components of the cell wall of Gram-negative bacteria, and are associated with asthma responses in exposed individuals (Carrer et al. 2001). Endotoxins have a multifunctional effect in the human body; immunotoxicity appears to be the major issue but other intracellular reactions are involved, such as the release of inflammatory mediators (leukotrienes, prostaglandins), histamine and blood-clotting factors, which can result in bronchoconstriction and airway inflammation. Indoor endotoxin exposure is elevated in homes where pets reside and also where individuals have regular contact with farm animals. One group of bacteria, Legionella species, causes the pneumonic illness Legionnaires’ disease and has been responsible for a number of deaths in buildings around the world (Kurtz 1994). Thriving in warm damp conditions, it is an environmental disease caused by the inhalation of aerosols containing the organisms. Domestic hot water systems, usually in large buildings or institutions such as hotels and hospitals, are the most frequent habitats of the Legionella bacteria causing the disease, with other building water sources or areas of stagnant water collection (heating ducts, water cooling systems, swimming pools/spas, showers and taps, garden sprinklers, etc.) also acting as breeding grounds. Bacterial contamination may be minimized through good maintenance practices, the avoidance of stagnant water in pipes and avoidance of temperature fluctuations. Tuberculosis (caused by Mycobacterium tuberculosis) has also been known to spread from person to person, especially among children, in indoor environments via ventilation pathways or as a consequence of poor ventilation. Viruses, such as the common cold, spread easily in indoor environments and favor high humidity levels and poor ventilation. Virus infections are known to initiate and prolong airways disease, in particular asthma. Like bacteria, viruses can cause the onset of a complex set of intracellular reactions, releasing various enzymes that have a collaborative effect resulting in inflammation and airways obstruction. Respiratory syncytial virus (RSV) infection has been studied in depth and is thought to enhance the development of allergic inflammatory responses in the presence of allergens (Zeldin et al. 2006). It also interacts with the neuroimmune pathway, which has been postulated to precipitate cycles of inflammation and obstruction in the airways (Piedimonte 2001). Ventilation systems may actually provide routes by which viruses can spread, for example the last outbreak of smallpox occurred in an indoor environment and spread from a scientific laboratory via a ventilation system. It is possible that the health effects attributed to the incidence of microorganisms in indoor environments may be associated with a combination of microbes rather than one individual species (Sherwood Burge 1994; Institute of Environment and Health 1996). Microorganisms have also been suggested as a factor responsible for sick building syndrome (SBS), alongside other potential causes such as VOCs (see below) and ETS.

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Fibers Although now banned for use in buildings in many countries, the natural mineral fibers collectively known as asbestos have been widely used in indoor environments for a number of functions (heat and electrical insulation, flooring/roofing tiles, older textured paints, etc.) due to their inert nature. Several forms of asbestos are known: white asbestos or chrysotile, and the amphibole materials of which blue or crocidolite and brown or amosite asbestos are the most common. Asbestos materials, particularly the amphibole minerals, are recognized carcinogens on inhalation and other noncarcinogenic conditions (Doll 1955; Wagner et al. 1960; Peto et al. 1982). However, while occupational asbestos exposure can cause pulmonary disease (asbestosis) as well as mesothelioma, it is unlikely that undisturbed and undamaged asbestos (in walls, ceilings, etc.) will result in significant exposures in indoor environments (Institute of Environment and Health 1997). Calcium sulfate is frequently fibrous and, due to common use in plaster and plaster board, provides the commonest source of fibers in indoor air. Synthetic mineral fibers, collectively called mineral wools (glass wool, rock wool, etc.), are often similar in size to natural fibers (length or diameter) and used for loft and cavity wall insulation. However, there is a tendency for synthetic mineral fibers to split transversely (rather than longitudinally as is the case for asbestos fibers) into shorter segments that are more likely to be removed from the lung by macrophages, although to regard synthetic mineral fibers as being “without concern” is inadvisable (Brown & Hoskins 1992; Institute of Environment and Health 1997). Synthetic zeolites, used in phosphate-free laundry powders, also fall into this category and, as they possess similar properties to the natural zeolite erionite which has recognized carcinogenic properties (by inhalation), may be a cause for concern.

Formaldehyde Formaldehyde is a ubiquitous chemical with a multitude of sources, from vehicle exhausts and tobacco smoke to foodstuffs (particularly fruit and vegetables). While classed as a VOC, it is often considered separately because of its high concentration in internal environments. Principal indoor sources include urea formaldehyde foam insulation (UFFI), urea formaldehyde resin in particle boards/plywoods used in construction and furniture, and as a component/preservative in furnishings and cleaning agents. Increased levels are associated with new homes, homes with integral garages, and new furnishings and decorations (Building Research Establishment 1996). More energy-efficient homes have higher levels due to the increased use of insulating materials and lower air exchange rates. Indoor levels have been found to be 10 times those found outdoors. Overall levels of formaldehyde (0.01– 0.1 mg/m3 and 0.1–0.8 mg/m3 in two separate studies) tend to be similar around the world, although mobile/condominium homes can be higher (3.7 mg/m3) (Bernstein et al. 1984;

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European Collaborative Action: Indoor Air Quality 1990). Formaldehyde can cause irritation to the eyes and nose, with chronic exposure resulting in sensitization and respiratory allergenicity in some individuals. High temperature, high humidity, and low ventilation rates have resulted in acutely irritant indoor concentrations of formaldehyde vapor (Harrison 1998). The accepted “safe” level is 0.1 mg/m3 for a maximum 30-min average (World Health Organization 2000).

Other volatile organic compounds VOCs are a varied collection of substances representing most organic families (aliphatic and aromatic hydrocarbons, halogenated compounds, and aldehydes) incorporated into adhesives, solvents, and flame retardants in building materials, paints and other DIY products, furnishings, furniture, cleaning agents, cosmetics, and tobacco smoke, and also produced by the occupants themselves. Common VOCs include benzene, toluene, ethylbenzene, and xylenes (the so-called BTEX compounds). Concentrations in the indoor environment are influenced by age of source material, level of ventilation, temperature, and humidity. Data concerning levels of VOCs and total VOCs (TVOCs) vary depending on the definition used and the methodology of study. A number of studies have evaluated exposure to VOCs in the home, including the 1996 BRE study, the United States Environment Protection Agency’s (US-EPA) Total Exposure Assessment Methodology (TEAM) studies, and a study by the Committee of the Health Council in the Netherlands (Wallace 1986; Building Research Establishment 1996; Health Council of the Netherlands 2000). The former identified over 200 VOCs with mean TVOC concentrations in different rooms varying only slightly from 0.2 to 0.4 mg/m3, with indoor levels several times higher than those measured outdoors and levels highest during home decoration activities. The US-EPA studies gave similar results, with TVOC samples exceeding 1 mg/m3 in about 60% of samples and 5 mg/m3 in 10% of samples, with the higher concentrations accounted for by personal activities such as smoking and painting/decorating and house cleaning. The Dutch study estimated the maximum tolerable pollution of indoor air by VOCs to be 0.2–3.0 mg/m3, with a cumulative limit value of 0.2 mg/m3. Another study reported more than 350 VOCs in indoor air at levels of 0.2 μg/m3 but all were far below their threshold limit value (Sterling 1985). Certain VOCs have recognizable odors that are perceived to compromise air quality even at concentrations below the threshold for irritation/discomfort, and may be linked to SBS, although the putative causative agents for this condition are various and debatable (Harrison 1998). VOCs have varied health effects, from central nervous system (CNS) depression to liver dysfunction, though the scale of effects associated with indoor exposures is unclear (Fiedler et al. 2005; Laumbach et al. 2005). While VOCs have been considered to possess adjuvant allergy-enhancing effects, no consistent association has been found between indoor VOC exposure other than

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formaldehyde and respiratory disease/asthma (Nielsen et al. 2007). Microbial volatile organic compounds (MVOCs) are metabolic byproducts of bacteria and fungi, responsible for the musty odor often found in damp buildings. MVOCs consist of a range of VOCs (including ketones, aldehydes, alcohol, aromatic and chlorinated hydrocarbons, sulfur-based compounds, amines, and terpenes), with 150 volatile compounds produced by 12 fungal species alone (Filer et al. 2001; Foruk et al. 2001). As the musty odor is often present before any visible evidence of bacterial/fungal growth, it is a clear indicator of potential indoor contamination or even damp problems, linking to the potential health effects that can arise from the inhalation of fungal spores. MVOCs can also have a direct irritant effect, for example β1→3 glucan used as a biomarker for total fungal biomass has been shown to exhibit inflammatory properties.

Plasticizers (including phthalates) Plasticizers are worthy of separate consideration and have been found in internal environments to be correlated with respiratory symptoms in school children (Kim et al. 2007b). These authors measured the plasticizers TMPD-MIB (2,2,4trimethyl-1,3-pentanediol monoisobutyrate, Texanol) and TMPD-DIB (2,2,4-trimethyl-1,3-pentanediol diisobutyrate, TXIB) at concentrations of 0.89 and 1.64 g/m3 at schools in Sweden, finding an association with new buildings. Another group of plasticizers, the phthalates, have been identified in housedust and in indoor air (Rudel et al. 2003). The phthalates di-(2-ethylhexyl) phthalate (DEHP) and butyl benzyl phthalate (BBP) are particularly associated with house dust. One study has found an association between DEHP and asthma, with BBP correlating with rhinitis in children at high exposure levels (Bornehag et al. 2004). However, phthalates are unlikely to be present above the lowest levels of exposure associated with respiratory dysfunction (Nielsen et al. 2007).

Nitrogen dioxide Nitrogen dioxide (NO2) is a product of fossil fuel combustion and occurs where gas cookers (approximately 50% of UK homes) and other unflued combustion appliances are present, as well as being a component of tobacco smoke (Institute of Environment and Health 1996; Harrison 1997). While not an allergen in itself, NO2 is an irritant gas that can increase susceptibility and responsiveness to allergens (through adjuvant effects) or aggravate existing lung problems, and may be a factor in chronic pulmonary disease. Laboratory animals have demonstrated morphologic changes to lung tissue after prolonged exposure to NO2, leading to reduced lung function and impaired ability to remove particles from alveoli (World Health Organization 1977). Overall, the published evidence on health effects of indoor NO2 points most to a hazard of respiratory illness in children, perhaps resulting from increased susceptibility to infection (Institute of Environment and Health

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1996). Studies in UK homes with and without gas cookers have shown 1- or 2-week averages ranging from 25 to 70 μg/m3 and from 13 to 40 μg/m3, respectively (Melia et al. 1990; Raw & Coward 1992; Ross 1994; Building Research Establishment 1996). Continuous monitoring in kitchens with gas cookers have shown 1-hour average levels of up to 1115 μg/m3; this compares with the World Health Organization (WHO) 1-hour guideline value of 400 μg/m3 (latterly reduced to 200 μg/m3) (World Health Organization 2000). These limited data suggest that in many homes using gas for cooking, levels in the kitchen (and possibly in other rooms) approach or exceed the WHO guideline value. Indoor NO2 levels can be two to three times the outdoor concentration in winter but are generally similar in summer.

Indoor Air Pollution

uninhabited indoor spaces for a number of years and are increasingly being made available to the consumer for use within their own home. The WHO’s Air Quality Guidelines Global Update 2005 report suggested a guideline limit of 100 μg/m3 for a daily maximum 8-hour mean (World Health Organization 2005). Ozone is highly reactive and oxidizes terpenes commonly found in indoor environments such as limonene and pinene (Wainman et al. 2000; Sarwar et al. 2004; Wolkoff et al. 2006). The reaction will reduce/remove indoor terpenes and ozone levels but may lead to an increase in particulates and “indoor smog” with possible irritant effects. The effects of reactions between other VOCs and ozone are poorly studied.

Other contaminants of concern Carbon monoxide Carbon monoxide (CO) is one of the most common and potentially most dangerous indoor pollutants, being associated with deaths and hospital admissions from exposure to accidentally elevated levels (Institute of Environment and Health 1998). CO poisoning, through the displacement of oxygen in oxyhemoglobin to form the more stable compound carboxyhemoglobin (COHb), results primarily as a consequence of the blockage of and/or leakage from flues of gas heating appliances. Other heating appliances, gas cookers, tobacco smoke, the presence of an attached garage, and proximity to heavily trafficked roads also affect indoor CO levels (Institute of Environment and Health 1998). A UK study found CO concentrations (maximum 1-hour averages) in kitchens with gas appliances ranging from 1.9 to 24.5 mg/m3 (Harrison 1998). A maximum 1-hour concentration of 57 mg/m3 was recorded in one instance due to a malfunctioning boiler. The equivalent WHO guideline is 30 mg/m3 for a maximum 1-hour average and 10 mg/m3 for a daily maximum 8-hour mean (World Health Organization 2000). As well as the more obvious acute effects of high-level exposure to CO, there are concerns about the health consequences of long-term low level exposure (Institute of Environment and Health 1998).

Ozone Ozone (O3) is a secondary product produced by the dissociation of NO2 and VOCs in sunlight. It is a lung irritant and exposure to high levels results in a reduction in lung capacity (Leslie 2000). Ozone is particularly hazardous to asthmatics, affecting the lower airways probably as a result of chronic inflammation (Vagaggini et al. 2002). It can be present in indoor environments as a consequence of high outdoor levels but can also be produced within the home through the operation of electrical equipment. However, significant indoor levels will only occur in the presence of high-voltage appliances such as photocopiers and laser printers, items that are unlikely to be found in the home but will be a factor in workplace environments. Air purifiers, and even air fresheners, that emit low levels of ozone have been used to sanitize and deodorize

Sulfur dioxide (SO2), generated from the combustion of fossil fuels such as coal and petroleum products, is a principal concern in the outdoor environment. Levels within homes will vary depending upon proximity to main roads and traffic, and the presence/absence of a coal fire. Human exposure to high levels of sulfur dioxide results in rapid bronchoconstriction in both healthy and asthmatic subjects, irritation with reflex coughing, wheezing and tightness of the chest. Airways resistance is enhanced through lung inflammation and in individuals who suffer from other respiratory problems such as asthma. Heavy metals such as arsenic are also associated with the burning of coal (sourced from particular geographic locations), and the past use of lead-containing substances in building and decorating materials (notably leaded paint), as well as lead additives in petrol, have resulted in contamination of indoor environments. Pesticides resulting from indoor insecticide treatments of soft furnishings, fungicidal paint and from outdoor sources also occur within home and work environments (Grey et al. 2006). Organophosphate pesticides are considered to be of particular concern (Scientific Committee on Health and Environmental Risks 2007). Most pesticides have a low volatility and hence are unlikely to be available for inhalation, although fumigation spraying will introduce droplets/aerosols into the air. A number of pesticides bind to house dust, prolonging their presence within the indoor environment. In Europe, many pesticides have been withdrawn from the market or are now unavailable for use by the amateur user (Slack et al. 2004). Radon is a recognized lung carcinogen of concern in some geographic areas and may be a common problem in parts of Europe (Darby et al. 2005). Radon gas diffuses through the bedrock and into the building. Increased ventilation creates a chimney effect and results in the drawing up of more radon, but a range of technical procedures can be used to counteract the effects. Exposure to plant allergens from external sources, notably pollen, can lead to asthma, allergic rhinitis (hay fever) and

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pulmonary hypersensitivity. Household plants, including the weeping fig (Ficus benjamina), can also be considered to be sources of allergen (Axelsson 1995). Cotton lint is a major component of house dust and allergic response to this may be indistinguishable from house-dust mite allergy. Similarly, insect secretions and feces can initiate an allergic response, with cockroaches a significant source of allergens in poor sanitary conditions (Chapman 1993). New substances and mixtures of substances are frequently a feature of consumer product development but many are untested for behavior in the unique environment presented by homes and workplaces. In addition, new sources of “old” pollutants may arise, particularly with regard to the increase in VOC-containing products. Isothiazolinines have been used in air-conditioning systems and have been linked to sensitization in workers in affected offices (Clark 1987). Water vapor, while not normally considered a pollutant, can increase the pollutant load of an internal space through the provision of a damp habitat for bacteria, fungi and housedust mites, as discussed above.

Mitigating pollution and associated health problems As external air quality has improved, so there have been improvements in indoor air quality, particularly in industrialized urban areas. However, an increase in consumer goods, a wider array of furnishing materials, a more extensive variety of cleaning products, improved thermal insulation, and lower rates of ventilation have resulted in a considerable number of substances within the indoor environment. Concern regarding indoor pollution and the consequences for respiratory diseases, asthma, and allergies has increased steadily over the last 15 years and there is ongoing evaluation of the need for effective management strategies. Review papers and scoping reports stress the need for more extensive toxicologic research and more focused epidemiologic studies into the effects of indoor pollutants on human health (and the environment), particularly the interaction of the different pollutants and their behavior in variable moisture and ventilation regimes (Harrison 1999). Although more work is required, the risk pathways, if not necessarily the risks, are generally already well recognized. However, for a number of social and political as well as practical reasons, regulating the home environment through legislation is unlikely to be easily achieved, although building regulations and product controls do have important roles to play (Harrison & Holmes 2000). Also, each potential contaminant is generated in different quantities in different indoor environments, making it difficult to assess the individual or combined risks posed to human health. Organizations such as the WHO have established guideline values, (as referenced in the above sections) that aim to inform about “safe” exposure levels to air pollutants

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generally, and some individual countries have established specific indoor air guidelines for some pollutants (Harrison 2002). California, for example, has successfully introduced nonmandatory guidelines to reduce VOCs in new buildings as well as making informative guidance documents available to the public on how to reduce exposures to formaldehyde and combustion pollutants within the home (Alevantis 2000). A recommendation to develop indoor air quality guidelines for the UK was made by the House of Commons Select Committee in 1991 (House of Commons Environment Committee 1991; Harrison 2002), culminating eventually in guidance published by the Committee on the Medical Effects of Air Pollution (Committee on the Medical Effects of Air Pollution 2004). However, this falls far short of introducing emission standards applicable to household equipment, materials and consumer products. Perhaps the most important step that can be taken is through education. Many indoor air quality problems are associated with ETS, poor levels of ventilation, and humidity issues. If residents are provided with appropriate information relating to the potential health effects arising from these issues, they are more likely to make informed decisions concerning their indoor environments. More information on packaging of consumer goods, such as high VOC paints and air fresheners, may alter usage patterns leading to an improvement in indoor air. A “common sense approach” would allow the individual and society in general to consider provision of adequate ventilation/ air exchange capacity, restricting levels of humidity, temperature control, use of extractor fans when cooking, not smoking indoors, use of water-based rather than solvent-based paints, low-formaldehyde particle board/carpets, alternatives to organochlorine/organophosphate pesticides, outdoor activities (domestic bonfires, vehicle activity close to homes, etc.), allergen avoidance, and so on, to reduce their individual exposures to indoor pollutants. It is, however, rare for high levels of any indoor pollutant to be present in an average household/ office workplace, and an overzealous use of hazard symbols on packaging may increase wariness and distrust of everyday consumer products. The so-called “hygiene hypothesis” is relevant here, with recent observations indicating that early exposure of very young children to endotoxin associated with Gram-negative bacteria and other potential allergens may mitigate development of allergy and asthma, with the possibility that large exposures early enough prevent sensitization to low-dose exposures later in life (Strachan 1989; Liu 2004). In addition to the well-researched aspects discussed above, there are new and emerging issues regarding pollution of the indoor environment, including electromagnetic fields (EMF) associated for example with Wi-Fi networks, mobile phone masts, and electricity pylons. While very unlikely to be a significant causative factor for allergic airway disease, the health consequences of prolonged exposure to EMF radiation are currently much debated, with more work needed to resolve the frequently conflicting reports published to date

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(International Commission on Non-Ionizing Radiation Protection 1998).

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Piedimonte, G. (2001) Neural mechanisms of respiratory syncytial virus-induced inflammation and prevention of respiratory syncytial virus sequelae. Am J Respir Crit Care Med 163, 18S–21S. Platts-Mills, T.A., Wheatley, L.M. & Aalberse, R.C. (1998) Indoor versus outdoor allergens in allergic respiratory disease. Curr Opin Immunol 10, 634–9. Price, J.A., Pollock, I., Little, S.A., Longbottom, J.L. & Warner, J.O. (1990) Measurement of airborne mite antigen in homes of asthmatic-children. Lancet 336, 895–7. Raw, G.J. & Coward, S.K.D. (1992) Proceedings of Unhealthy Housing: The Public Health Response. University of Warwick, Coventry. Rickerby, D.G. & Morrison, M. (2007) Nanotechnology and the environment: a European perspective. Sci Technol Adv Materials 8, 19– 24. Ross, D. (1994) Continuous and Passive Monitoring of Nitrogen Dioxide in UK Homes. BRE Note N109/94. Building Research Establishment, Watford. Rudel, R.A., Camann, D.E., Spengler, J.D., Korn, L.R. & Brody, J.G. (2003) Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrine-disrupting compounds in indoor air and dust. Environ Sci Technol 37, 4543–53. Salter, H.H. (1864) On Asthma: Its Pathology and Treatment. Blanchard & Lea, Philadelphia. Samson, R. (1992) Mycotoxins: a mycologist’s perspective. J Med Vet Mycol 30, 9–18. Sarwar, G., Olson, D.A., Corsi, R.L. & Weschler, C.J. (2004) Indoor fine particles: the role of terpene emissions from consumer products. J Air Waste Management Assoc 54, 367–77. SCALE (2004) SCALE Baseline Report on Research Needs in the Framework of the European Environment and Health Strategy. Technical Working Group on Research Needs in Environment and Health, Brussels. http://www.brussels-conference.org/Download/Baseline_report_ TWG_Research_Needs_fin.pdf Scientific Committee on Health and Environmental Risks (2007) Preliminary Report on Risk Assessment on Indoor Air Quality. SCHER, Health & Consumer Protection Directorate-General. European Commission, Brussels. Schulz, O., Sewell, H.F. & Shakib, F. (1998) Proteolytic cleavage of CD25, the alpha subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity. J Exp Med 187, 271–5. Schwartz, B., Lind, P. & Lowenstein, H. (1987) Level of indoor allergens in dust from homes of allergic and nonallergic individuals. Int Arch Allergy Appl Immunol 82, 447–9. Sherwood Burge, P. (1994) Bacteria, fungi and other micro-organisms. In: Leslie, G.B. & Lunau, F.W., eds. Indoor Air Pollution: Problems and Priorities. Cambridge University Press, Cambridge, pp. 29–61. Slack, R.J., Gronow, J.R. & Voulvoulis, N. (2004) Hazardous components of household waste. Crit Rev Environ Sci Technol 34, 419– 45. Sneddon, J.M. (1994) The oxides of nitrogen. In: Leslie, G.B. & Lunau, F.W., eds. Indoor Air Pollution: Problems and Priorities. Cambridge University Press, Cambridge, pp. 63–76. Sterling, D.A. (1985) Volatile organic compounds in indoor air: an overview of sources, concentrations and health effects. In: Gammage, R.B. & Kaye, S.V., eds. Indoor Air and Human Health. M.I. Lewis, Chelsea, pp. 387–402. Strachan, D.P. (1989) Hay-fever, hygiene, and household size. BMJ 299, 1259–60.

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Swanson, M.C., Campbell, A.R., Klauck, M.J. & Reed, C.E. (1989) Correlations between levels of mite and cat allergens in settled and airborne dust. J Allergy Clin Immunol 83, 776–83. Tamburlini, G., von Ehrestein, O.S. & Bertollini, R. (2002) Children’s Health and Environment: A Review of Evidence. WHO and EEA Environmental Issue Report 29. European Environment Agency, Copenhagen. Tavernier, G., Fletcher, G., Gee, I. et al. (2006) IPEADAM study: indoor endotoxin exposure, family status, and some housing characteristics in English children. J Allergy Clin Immunol 117, 656–62. Vagaggini, B., Taccola, M., Cianchetti, S. et al. (2002) Ozone exposure increases eosinophilic airway response induced by previous allergen challenge. Am J Respir Crit Care Med 166, 1073–7. Wagner, J.C., Sleggs, C.A. & Marchand, P. (1960) Diffuse pleural mesotheliomas and asbestos exposure in North Western Cape Province. Br J Ind Med 17, 260–71. Wainman, T., Zhang, J.F., Weschler, C.J. & Lioy, P.J. (2000) Ozone and limonene in indoor air: a source of submicron particle exposure. Environ Health Perspect 108, 1139– 45. Wald, N.J., Nanchahal, K., Thompson, S.G. & Cuckle, H.S. (1986) Does breathing other peoples’ tobacco-smoke cause lung-cancer? BMJ 293, 1217–22. Wallace, L. (1996) Indoor particles: a review. J Air Waste Management Assoc 46, 98–126. Wallace, L.A. (1986) Personal exposures, indoor and outdoor air

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concentrations, and exhaled breath concentrations of selected volatile organic-compounds measured for 600 residents of New-Jersey, North-Dakota, North-Carolina and California. Toxicol Environ Chem 12, 215–36. Weetman, D.F. (1994) Environmental tobacco smoke. In: Leslie, G.B. & Lunau, F.W., eds. Indoor Air Pollution: Problems and Priorities. Cambridge University Press, Cambridge, pp. 193–236. Weichenthal, S., Dufresne, A. & Infante-Rivard, C. (2007) Indoor ultrafine particles and childhood asthma: exploring a potential public health concern. Indoor Air 17, 81–91. World Health Organization (1977) Environmental Health. Volume 4: Oxides of Nitrogen. WHO, Geneva. World Health Organization (2000) Air Quality Guidelines for Europe, WHO Regional Publications, European Series, No. 91. WHO Regional Office for Europe, Copenhagen. World Health Organization (2005) WHO Air Quality Guidelines Global Update 2005. Report on a Working Group meeting, Bonn, Germany, 18– 20 October 2005. WHO, Geneva. Wolkoff, P., Wilkins, C.K., Clausen, P.A. & Nielsen, G.D. (2006) Organic compounds in office environments: sensory irritation, odor, measurements and the role of reactive chemistry. Indoor Air 16, 7–19. Zeldin, D.C., Eggleston, P., Chapman, M., Piedimonte, G., Renz, H. & Peden, D. (2006) How exposures to biologics influence the induction and incidence of asthma. Environ Health Perspect 114, 620–6.

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Molecular Immunopathology of Allergic Disease Susan Foley and Qutayba Hamid

Summary Rapid progress is being made in understanding the basic immunopathology of allergic diseases. However, the precise mediators and cellular interactions that result in allergic inflammation are far from clear. For many years, conventional methods such as histology and electron microscopy have been the principal tools used to study the pathology of allergic disease. In situ hybridization and immunocytochemistry remain powerful tools currently used in cytokine research that give further insight into the pathogenesis of disease. With advances in fluorescent and confocal microscopy techniques we have gained further insight into the pathogenesis of allergic disease. Polymerase chain reaction continues to advance and its use as a quantitative measurement of mediators complements more conventional morphology techniques. Although there was a lot of hope with the emergence of microarray technology, it remains too early to say if it will be of benefit in the future in improving our understanding of the molecular pathology of disease. Currently, allergic disease is considered to result from an inappropriate balance between allergen activation of regulatory T cells and effector Th2 cells in susceptible individuals, a process in which dendritic cells are key players. This lack of regulation in the immune response leads to an ongoing inflammatory process, in which different immune processes follow one another and accumulate over time. The presence of the allergen not only induces the activation of an effector response that is responsible for the clinical manifestations, but also promotes an immunomodulatory process, which may determine the evolution of the disease. In addition, the genetic and environmental susceptibility of each patient plays a role in the activation of the inflammatory response, leading to a highly variable inflammatory process and ultimately a wide range of clinical manifestations. The identification of effector molecules that drive the inflammatory process in allergy has led to the development of biological therapies that specifically block allergic pathways or enhance their natural regulatory mechanisms (Holgate 2004;

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Poole et al. 2005). However, therapeutic efficacy of these molecules is still under study. The recent identification of novel cytokines such as interleukin (IL)-17, IL-25, or thymic stromal lymphopoietin, highly implicated in the immune process underlying allergy, also offers new targets to control allergic inflammation. Current research strategies seek to exploit recent knowledge in relation to Treg and dendritic cells in order to induce specific tolerance against allergens that would allow longer-lasting control of allergic diseases.

Introduction Allergic disease has currently reached epidemic proportions, with a high percentage of individuals in the developed world exhibiting an allergic response after exposure to some common environmental factors. Key factors in the development of disease are the genetic predisposition of the individual and exposure to allergen. At the molecular and cellular levels, multiple mediators, cytokines, cell types, and pathways are involved in the allergic inflammatory cascade that ensues after allergen exposure. Allergic diseases are characterized by the IgE-dependent release of mast cell-derived mediators and cellular infiltration particularly of activated eosinophils and T lymphocytes. Although the clinical manifestations of the allergic response vary depending on the tissue and antigen involved, the allergic reaction consists of both early and late-phase responses. The early-phase response primarily involves mast cell degranulation and is accompanied by the release of a wide variety of mediators. Depending on the amount of allergen, these immediate hypersensitivity reactions are followed by late-phase reactions, characterized by the migration of inflammatory cells from the circulation, which then slowly resolves. Currently both upper and lower airway diseases are considered to be parts of the same syndrome, but in different locations (Simons 1999; Togias 2003). This has led to the concept of the united airways disease (Watson et al. 1993; Passalacqua et al. 2000). Several hypotheses have been put forward as possible reasons to account for the increasing incidence of allergic disease. Presently, it has been proposed that allergy is the result

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of an improper balance between tolerance and immunity to harmless antigens (Umetsu et al. 2002; Perez-Machado et al. 2003; Akdis et al. 2004). The recent identification of T regulatory (Treg) cells as key regulators in peripheral tolerance to allergens (Ling et al. 2004; Akdis et al. 2005; Hawrylowicz & O’Garra 2005), and the role that dendritic cells have on their generation (Grunig et al. 2005), open up new perspectives for the understanding of allergy pathogenesis and to the development of more efficient therapies. A number of molecular techniques have been invaluable in aiding our understanding of the pathology of allergic disease. The most widely used techniques to identify cytokine expression are in situ hybridization (ISH) and immunocytochemistry (ICC). Other techniques include real-time reversetranscriptase polymerase chain reaction (RT-PCR), microarray, and more recently genetically engineered tissue. Most of the studies to date have been performed on biopsies or body fluid (lavages or blood samples). The use of genetically engineered tissue may also become a reality in the near future. We present some photographs of these techniques throughout the text.

Molecular techniques used in allergic disease Immunolocalization of cytokines ICC has been used extensively to detect the presence of immunoreactive protein within cells or tissues and thus provides evidence that translation from mRNA to protein has occurred by means of antigen–antibody reaction. It aids characterization of the phenotype of cells that are infiltrating the mucosa and as such has enhanced our understanding of disease processes. Given the rapid release of cytokines, their short halflife and the presence of high-affinity receptors, the technique of immunolocalizing cytokines with specific antibodies has been particularly informative in the pathogenesis of allergic diseases. ICC may also localize the secretion of cytokines from specialized storage granules, which can occur in the absence of apparent mRNA production. For example, mast cells and eosinophils have the capacity to store cytokines intracellularly and both acute release and storage of these mediators can be detected by ICC (Bradding et al. 1992; Lamkhioued et al. 1995). Although many methods are available, the principal three are direct, indirect, and unlabeled antibody enzymatic methods. The presence of cytokine immunoreactivity in particular cells does not necessarily indicate that the cell actually synthesizes the protein; cells such as macrophages and epithelial cells have the ability to endocytose protein from surrounding media. The use of ICC to detect cytokine immunoreactivity requires an intrinsic understanding of the basic principles combined with an appreciation of cytokine biology. Given the appropriate reagents and conditions, ICC provides a powerful tool in aiding our awareness of immunoregulation.

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In situ detection of cytokines ISH has been used extensively for the intracellular localization of cytokine mRNA in tissue sections and cytospin preparations from normal and diseased individuals (Kay et al. 1991; Hamid et al. 1991; Robinson et al. 1992). ISH can be defined in general as the cellular localization of specific nucleic acid sequences using a labeled complementary strand. The technique is of particular importance in cytokine research as there is in vitro evidence suggesting that cytokines are synthesized de novo and secreted very rapidly; they are not stored within the cell. The localization of cytokine mRNA at the tissue level indicates the expression and activation of the gene and the potential ability of the cell to produce cytokines. The general principle of ISH is based on the fact that labeled single-stranded RNA or DNA containing complementary sequences is hybridized intracellularly to mRNA under appropriate conditions, thereby forming a stable hybrid. This is detected according to the type of labeling of the probe. Different probes are available to detect mRNA including double- and single-stranded DNA (Herrington & McGee 1990), oligonucleotides and single-stranded RNA probes (Cox et al. 1984). Single-stranded RNA probes have been used extensively in recent years for the detection of cytokine mRNA by both isotopic and nonisotopic methods. The advantages of RNA probes lies in their high specificity and sensitivity compared with other types of probes (Cox et al. 1984). The demonstration of mRNA within a cell provides valuable information about gene expression and indicates possible synthesis of the corresponding protein. Hybridization signals can be detected according to the label that has been incorporated into the probe. For radiolabeled probes, standard autoradiographic techniques with an emulsion can be used. For nonradiolabeled probes, the RNA hybrid is usually detected by immunocytochemical methods in which an antibody (e.g., anti-digoxigenin) is used and developed by chromogens (Ying et al. 1993, 1994).

Polymerase chain reaction PCR refers to the enzymatic amplification of a cDNA template using specific primers. As nucleic amplification methods remain fundamentally important tools in basic science research and molecular diagnostics (Schweitzer & Kingsmore 2001), efforts are continuously made to improve current methodologies as well as to develop novel technologies. Currently, RT-PCR-based assays are the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations (Orlando et al. 1998). The fluorescence-based real-time RT-PCR is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine, and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. Target gene mRNA levels are measured and are normalized to

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reference housekeeping genes to control for cDNA loaded into the reaction. This results in the reporting of each gene expression measurement as a numerical value that allows direct comparison between experiments carried out within the same or different laboratories. However, when using this technique for analysis of biopsy samples, it is often necessary to pool the cDNA extracted from several biopsies from the same individual in order to increase the yield of cDNA.

Microarray technology The measurement of gene expression using microarrays is relevant to many areas of biology and medicine. This technology can be used to identify disease genes by comparing gene expression in diseased and normal cells. A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip forming an array for the purpose of expression profiling, monitoring expression levels for thousands of genes simultaneously. The affixed DNA segments are known as probes or reporters, thousands of which can be placed in known locations on a single DNA microarray. The technology evolved from Southern blotting. Single nucleotide polymorphism (SNP) microarrays are a particular type of DNA microarray used to identify genetic variation in individuals and across populations. Short oligonucleotide arrays can be used to identify the SNPs thought to be responsible for genetic variation and the source of susceptibility to genetically caused diseases. Generally termed “genotyping applications,” DNA microarrays may be used in this fashion for rapidly discovering or measuring genetic predisposition to disease or identifying DNA-based drug candidates. Although microarray has been used on DNA extracted from tissues, its use in defining morphology is limited and it is used mainly to search for differences in gene expression associated with a particular disease.

local levels of IgE only after degranulation of the eosinophil (Smith et al. 2000). Although IgE production has long been ascribed to B cells in secondary lymphoid tissue, bone marrow, and blood, recent studies have demonstrated that this phenomenon may also occur locally in allergic tissue. A resident population of B cells is present in the nasal mucosa and the factors required for induction of isotype switching to IgE are expressed within this tissue in individuals with allergic rhinitis (Davidsson et al. 1994). Increased numbers of ε-heavy chain germline (Iε) and ε-heavy chain constant region (Cε) ribonucleic acid-positive (RNA+) cells, in the absence of a change in B-cell number, have been observed in allergic nasal mucosa after allergen challenge and natural allergen exposure. To confirm that these increases were not merely the result of B-cell infiltration, Cameron et al. (2000a) cultured nasal mucosal explants with allergen and demonstrated an increased number of Iε and Cε RNA+ cells in resident B cells (Figs 62.1 and 62.2). They also

(a)

(b)

IgE and receptors The IgE molecule plays a central role in the pathogenesis of immediate hypersensitivity reactions because of its capacity to bind specifically to high-affinity IgE receptors on mast cells or basophils by the α chain of Fcε receptor type 1 (FcεRIα) (Turner & Kinet 1999). In addition to its ability to activate mast cells and basophils resulting in the release of an array of inflammatory mediators, IgE can bind to the low-affinity IgE receptor (CD23 or FcεRII) on B cells to augment cellular and humoral immune responses in allergic disease (Delespesse et al. 1992; Waldschmidt & Tygrett 1992). FcεRI has also been found on antigen-presenting cells, where it can facilitate the IgE-dependent trapping and presentation of allergen to T cells (Maurer et al. 1996). Eosinophils also possess FcεRIα, but in these cells the receptor is intracellular and helps regulate

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(c) Fig. 62.1 B lymphocytes identified on the basis of CD20 immunoreactivity within allergic nasal mucosal explant cultured for 24 hours in the presence of specific allergen (500 PNU/mL). B cells were observed just beneath the basement membrane (a), infiltrating the epithelial layer (b), and clustering within the submucosa in groups of three or four cells (c). (See CD-ROM for color version.)

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Fig. 62.2 Expression of eRNA transcripts within explanted nasal mucosal tissue. Significantly higher numbers of Ie (a) (N = 13) and Ce (b) (N = 13) RNA-positive cells were observed in allergen-stimulated (Ag; 500 PNU/mL) compared with unstimulated [medium alone (MA)] allergic tissue (open circles), but not within tissue obtained from nonallergic patients (closed

circles; N = 9, P > 0.05). The number of Ce RNA-positive cells minus Ie RNA-positive cells (ΔCe) within allergic tissue cultured in allergen-treated medium was significantly higher than the number of Ce RNA-positive cells in unstimulated tissue (c) (n = 10, *P < 0.05). Circles alongside the data represent median values.

confirmed that ε-germline transcription (and therefore IgE synthesis) occurs locally in the nasal mucosa (Cameron et al. 2000a) and may be regulated by the local T-cell and mast cell production of IL-4 and IL-13.

influencing mainly terminal differentiation of CD34/CD33+ progenitor cells (Ema et al. 1990). They are the only human leukocytes that express membrane-bound receptors specific to IL-5, demonstrating the importance of this cytokine in the development of eosinophilia (Migita et al. 1991; Egan et al. 1996). Eosinophils are the predominant cell in the chronic inflammatory process characteristic of the late-phase allergic response (Fig. 62.3). They release an array of proinflammatory mediators, including cysteinyl leukotrienes, cationic proteins, eosinophil peroxidase (EPO), and major basic protein (MBP), and might serve as a major source of IL-3, IL-5, GMCSF, and IL-13. They are a potent source of leukotriene (LT)C4. This latter mediator is elevated in nasal lavage fluid of patients with seasonal allergic rhinitis after allergen challenge and has been suggested to be of greater importance than histamine for nasal blockage. Increased levels of eosinophil-derived proteins such as MBP and eosinophil cationic protein (ECP) are also present within nasal mucosa and secretions in allergic rhinitis, which have been shown to cause degranulation of other inflammatory cells as well as epithelial cell damage. The presence of eosinophils within mucosal tissue of allergic airways disease is believed to be due to de novo infiltration of mature cells from the bone marrow. However, recent studies by Cameron et al. (2000b), using an explant system of human allergic nasal mucosa, provide strong evidence that a subpopulation of eosinophils may undergo local differentiation within the mucosa itself. Following ex vivo stimulation with specific allergen or recombinant human (rh) IL-5, more MBP-immunoreactive and IL-5 mRNA+ cells, along with fewer CD34/IL-5Rα cells, were found in nasal mucosal tissues

Inflammatory cells in allergic disease Inflammation in allergic diseases appears to involve a cascade of cells that become activated, including eosinophils, basophils, mast cells, T cells, epithelial cells, neutrophils, and even smooth muscle cells. Although each inflammatory cell has its proponents and the interaction of these cells adds further complexity to the pathogenesis of allergic airway disease, substantial attention has been directed toward eosinophils and T cells. ICC has been the principal technique used for identification of the phenotype and state of activation of these inflammatory cells.

Eosinophils Increased numbers of inflammatory cells, particularly eosinophils, in the tissues is a feature common to allergic diseases. Eosinophils have been associated with the characteristic pathologic features of allergic mucosa, namely epithelial injury and desquamation, subepithelial fibrosis, and hyperresponsiveness (Bousquet et al. 1990). Eosinophils arise predominantly in the bone marrow from CD34+ pluripotent progenitor cells in the presence of IL-3, IL-5, and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Denburg et al. 1985; Clutterbuck et al. 1989; Gibson et al. 1990; Shalit et al. 1995). IL-5 is critical in their development

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(a)

(a)

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(b) Fig. 62.3 Immunostaining for MBP showing increased numbers of eosinophils in allergic rhinitis (a) and the asthmatic lung (b) after antigen challenge. (See CD-ROM for color version.)

(Fig. 62.4) (Cameron et al. 2000b). The process was found to be highly IL-5-dependent, implying that it might be regulated in vivo by endogenous production of sIL-5Rα. As the α subunit of the IL-5 receptor (IL-5Rα) is expressed almost exclusively by eosinophils, the colocalization of CD34 immunoreactivity with IL-5Rα is considered to be a marker for precursor eosinophils (CD34/IL-5Rα+) (Sehmi et al. 1997). Such precursors have been found in nasal polyps, and in the presence of locally produced IL-5, likely differentiate into mature eosinophils (Kim et al. 1999). Similarly, eosinophil precursors and IL-5 mRNA have been identified by Robinson et al. (1999) in the lungs of atopic asthmatic patients, and correlate with the number of major basic protein (MBP+) cells, indicating that a similar process of local differentiation of eosinophils may occur within the asthmatic lung. Eosinophils are major effectors in allergic tissue reactions. They have the capacity to synthesize and store cytokines, particularly of the Th2 type, which may lead to increased cell

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(c) Fig. 62.4 Photos of nasal mucosal explant tissue 24 hours following culture. The presence of CD34+ progenitor cells within tissue cultured in medium alone (a). Under dark-field illumination, cells coexpressing both CD34 immunoreactivity and IL-5Ra mRNA are also seen within unstimulated tissue; these cells appear both orange and illuminated (b, arrowhead). The considerable number of MBP-positive cells within tissue cultured with ragweed allergen (c). Colocalization of CD34 and IL-5Ra mRNA has been suggested as a marker of eosinophil precursors. (See CD-ROM for color version.)

survival within tissues (Kay et al. 1997). IL-4 and IL-5 production by eosinophils may amplify local allergic inflammatory responses. Eosinophils are also a potent source of LTC4 (Holgate et al. 1996), which is reported to be elevated in the nasal lavage fluid of subjects with seasonal allergic rhinitis after allergen challenge (Volovitz et al. 1988). Increased levels of eosinophil-derived proteins such as MBP and ECP are also present within the sinus mucosa in allergic rhinitis. These proteins have been shown to cause degranulation of other inflammatory cells and to promote epithelial cell damage (Hisamatsu et al. 1990; Moy et al. 1990; Oddera et al. 1996). Hamilos et al. (1993) found tissue eosinophilia to be a prominent

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feature of both allergic and nonallergic chronic hyperplastic sinusitis/nasal polyposis, correlating in both groups with the density of GM-CSF and IL-3 mRNA+ cells. In most asthma phenotypes, there are increases in eosinophils in the tissues, blood, and bone marrow and, in general, raised numbers correlate with disease severity. It is thought therefore that the eosinophil is the central effector cell responsible for ongoing airway inflammation. The cell has the potential to cause damage to the airway mucosa and associated nerves through the release of granule-associated basic proteins, which damage nerves and epithelial cells, lipid mediators (which cause bronchoconstriction and mucus hypersecretion), and reactive oxygen species (which generally injure mucosal cells).

T lymphocytes T lymphocytes play an important role in allergic airway inflammation by coordinating and mediating the adaptive immune response that the body elicits in response to foreign antigen. Allergic subjects have increased numbers of T lymphocytes that have been associated with both onset and severity of disease. Increased lymphocyte numbers are a frequent finding within the bronchial mucosa of patients with all forms of asthma, from newly diagnosed to severe disease (Dunnill 1960; Jeffery et al. 1989). T lymphocytes are broadly subdivided into two distinct subsets according to their cell-surface markers and distinct effector functions. T cells expressing the CD4 antigen are involved in humoral immunity and are termed T helper cells (Th cells), whereas those expressing the CD8 antigen are referred to as T cytotoxic/suppressor cells (Tc cells). CD8+ cytotoxic T cells orchestrate the cell-mediated response and interact with endogenously processed antigen presented in conjunction with major histocompatibility complex (MHC) class I. In contrast, CD4+ lymphocytes are capable of recognizing foreign antigens processed in association with MHC class II on the surface of professional antigen-presenting cells. As such, CD4+ cells have attracted considerable attention in the pathogenesis of asthma and allergic rhinitis because of their ability to drive antigen-specific inflammatory responses and regulate immunoglobulin production. CD4+ T helper cells are further divided into T-helper 1 (Th1) and T-helper 2 (Th2) based on the cytokines they produce (Mosmann et al. 1986). Naive Th cells (CD4+ T cells never exposed to antigen) when activated by antigen-presenting cells produce cytokines from both subsets and are known as Th0 cells (Kelso 1995). If given the proper stimulus, naive Th cells can differentiate into the biased Th1 or Th2 subsets. Th1 cells produce IL-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. They are inducers of cell-mediated immunity or delayed-type hypersensitivity reactions. The major Th2 cytokines are IL-4, IL-5, and IL-13. They are helpers for Bcell antibody secretion, particularly IgE (Romagnani 1991). During the last 15 years, it has largely been shown that aller-

Molecular Immunopathology of Allergic Disease

gic inflammation is caused by activated Th2 lymphocytes, leading to IgE production and eosinophil activation. Another subset of T-helper cells, which secrete high levels of transforming growth factor (TGF)-β but low levels of Th1 and Th2 cytokines, if any, are known as Th3 cells (Weiner 2001). Th3 cells can suppress both Th1 and Th2 cytokine production and are thought to have a role in mucosal tolerance to antigen but their role in atopy is unclear. There is a marked increase in the number of CD4+ T lymphocytes seen in nasal biopsy tissue obtained from individuals with allergic rhinitis 24 hours after allergen challenge outside the pollen season (Varney et al. 1992). Furthermore, ISH with antisense complementary riboprobes has enabled detection of increased amounts of the Th2-type cytokines IL-3, IL-4, IL-5, and IL-13, as well as GM-CSF mRNA+ cells within allergic nasal mucosa after local allergen provocation (Sobol et al. 2001). These lymphocytes, both in bronchial and nasal mucosa, appear to be “activated” as determined by their expression of the IL-2 receptor (CD25), suggesting that these cells are a cardinal feature of allergic airways inflammation (Bradley et al. 1991). Acute atopic dermatitis (AD) skin lesions are also associated with increased numbers of CD4+ cells (Fig. 62.5) that infiltrate the epithelium and an increased number of Th2 cells expressing mRNA for IL-4 and IL-13 compared with IFN-γ mRNA- and IL-12 mRNA-expressing cells. The Th2 cells are memory cells that also express cutaneous lymphocyteassociated antigen, a homing receptor to direct the cells to the skin. Th2-associated cytokines are thought to promote IgE production and thus allergen sensitization. Chronic lichenified AD skin lesions, in contrast to the acute lesions, demonstrate less IL-4 and IL-13 mRNA-expressing cells but have increased numbers of IL-5, GM-CSF, IL-12 and IFN-γ mRNA-expressing cells. These cytokines support the growth of eosinophils and macrophages and promote the Th1-type inflammation more characteristic of chronic disease.

Fig. 62.5 Immunohistochemistry showing large numbers of infiltrating CD4+ cells stained with fast red by using the alkaline phosphatase method in skin lesions of patients with acute atopic dermatitis. (See CD-ROM for color version.)

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CD8+ T cells constitute a minority of CD3+ lymphocytes found in the bronchial mucosa and thus it is likely that they provide only a modulatory influence over CD4+-driven inflammation. Although both CD4 and CD8 T lymphocytes in bronchoalveolar lavage (BAL) fluid from asthmatic subjects expressed CD25, only the numbers of activated CD4+ cells correlated with numbers of BAL eosinophils and disease severity (Walker et al. 1991). CD4+ T lymphocytes, on the other hand, are the predominant cells in allergic mucosal inflammation and help perpetuate the allergic response by providing a source of cytokines and amplifying and regulating local immune responses. CD8-cytotoxic T cells, although present in the nasal mucosa, are mainly associated with cell-mediated immune responses to viral infections. Some studies have shown that CD8 T cells can produce IL-4 and stimulate B cells to produce IgE and IL-5 and are able to induce tissue eosinophilia (Coyle et al. 1995; Meissner et al. 1997). CD8 T cells may not play as great a role in controlling allergic inflammation as Th2 cells but they may help promote and maintain it.

Treg cells and allergic inflammation The recent identification of the CD4+CD25+ Treg cells as key regulators in peripheral tolerance to allergens (Akdis et al. 2005), and the role that dendritic cells have on their generation (Grunig et al. 2005), open up new perspectives for the understanding of allergy pathogenesis and to the development of more efficient therapies. Treg cells produce high levels of IL-10 and are involved in the maintenance of self-tolerance and in the suppression of the immune responses. The expression of CD25 antigen, the IL-2 receptor α-subunit, is induced in T cells on antigen stimulation, but Treg cells express high levels constitutively (Baecher-Allan et al. 2001). First discovered in the mid-1990s, when depletion of CD25+ cells in mice led to multiple organ autoimmunity (Sakaguchi et al. 1995), these cells account for 5–10% of peripheral CD4+ T cells. Production of IL-10 by these cells can inhibit Th1 and Th2 cytokine production and proliferation of other CD4+ T cells (Cottrez et al. 2000; Annacker et al. 2001). A recently described transcription factor, Forkhead Box P3 (Fox p3), is associated with Treg cells (Brunkow et al. 2001). Mutations in Fox p3 in mice lead to lethal autoimmune syndromes as a consequence of deficiency of Treg cells (Fontenot et al. 2003; Khattri et al. 2003). In humans, mutations in Fox p3 lead to the immunodysregulation, polyendocrinopathy, and enteropathy (IPEX) syndrome (Wildin et al. 2001). Treg cells are present in both atopic and nonatopic individuals, but those from atopic patients have a significantly lowered activity in inhibiting proliferation and Th2 cytokine production of other cells, especially during allergen exposure (Grindebacke et al. 2004; Ling et al. 2004).

Mast cells Mast cells are key participants in allergic inflammation and contain a potent array of inflammatory mediators. They develop

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from bone marrow progenitor cells and are found in increased numbers in the airways of subjects with allergic rhinitis and asthma (Bradding et al. 1995; Castells et al. 1995). They have unique characteristics and possess high-affinity IgE receptors. The cross-linking of bound IgE to FcεRI by allergen triggers mast cell activation and their resultant explosive degranulation, resulting in the release of a complex cascade of both preformed and newly generated mediators that have synergistic effects on resident cells in tissues (Turner & Kinet 1999; Williams & Galli 2000; Gelfand 2004). Mast cell degranulation is the critical initiating event of acute allergic symptoms. During allergen assault in allergic rhinitis, mediators such as histamine, tryptase, chymase, prostaglandin (PG)D2, PGF2α, and bradykinin are released rapidly and these mediators cause edema and mucus secretion, thus inducing vasodilation, vascular permeability, and cellular adhesion. Cytokines, including TNF-α, IL-4, IL-5, IL-6, TGF-β, and IL-13, may also be released. Mast cells also synthesize a large number of proinflammatory cytokines (including IL-4, IL-5, and IL-13), that regulate both IgE synthesis and the development of eosinophilic inflammation and several profibrotic cytokines including TGF-β and basic fibroblast growth factor (FGF)-2 (Bradding & Holgate 1999; Bradding 2000). There is evidence that mast cells localize to the airway smooth muscle (ASM) in asthma, as well as airway mucous glands and the bronchial epithelium. This facilitates specific interactions between these cells and ASM in terms of both localized mediator release and direct cell-to-cell contact. The mechanism of mast cell recruitment by asthmatic ASM involves the CXCL10/CXCR3 axis and several mast cell mediators have profound effects on ASM function. It is probable that these mast cells contribute to the development of hypertrophy and hyperplasia, smooth muscle dysfunction expressed as bronchial hyperresponsiveness, and variable airflow obstruction (Bradding et al. 2006). Mast cells are also a potential source of products that stimulate migration and proliferation of fibroblasts (Ruoss et al. 1991; Nagata et al. 1992).

Basophils Although basophils are granulocytes that express FcεRI, Th2 cytokines, and histamine, they are believed to be a separate lineage from mast cells (Denburg et al. 1985). They develop in the bone marrow and represent less than 1% of peripheral blood leukocytes. Due to their low levels in blood, it has been difficult to ascribe a physiologic function to the basophil but they may promote phase inflammation by Th2 cytokine production and may also promote IgE class switching as they also express CD40L (Gauchat et al. 1993). Similar to mast cells, basophils bind allergen by IgE/FcεRI and release histamine on activation (Iliopoulos et al. 1992). As the level of histamine, but not of tryptase and PGD2, is increased during late-phase response, it is attributed to basophil activation rather than to secondary mast cell degranulation (Bascom et al. 1988). Basophils produce many other lipid mediators

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and cytokines similar to mast cells, such as LTC4, IL-4, and IL13 (Schroeder et al. 2001). Conversely, PDG2 and IL-5 are not produced by basophils (Prussin & Metcalfe 2003). Several studies have demonstrated infiltration of basophils in human allergen-induced late responses occurring in the nose and bronchi (Prat et al. 1993; Guo et al. 1994). These late asthmatic responses are accompanied by increases in tissue basophils, a proportion of which express IL-4 mRNA and protein in the bronchial mucosa at 24 hours after challenge (Nouri-Aria et al. 2001). Basophil numbers are also increased in nasal lavage fluid obtained 24 hours after allergen challenge (Pawankar et al. 1997). To date, none of the products released by basophils are unique to this cell type. In searching for a unique product of basophils, several basophil-specific monoclonal antibodies have been identified: Bsp-1, which recognizes a cell-surface marker, and 2D7 and BB1, which appear to react with a granule constituent (Bodger et al. 1987; Kepley et al. 1995; McEuen et al. 1999). Using 2D7 monoclonal antibody, Kepley et al. (2001) recently showed that basophil infiltration was significantly increased in lungs from individuals who died of asthma, suggesting that this cell type is involved in the pathogenesis of fatal asthma.

Epithelial cells The epithelium has long been considered to act mainly as a barrier participating in mucociliary clearance and removal of noxious agents. Loss of integrity of the epithelial layer, a feature of both allergic rhinitis and asthma, may promote exposure of the mucosa by allergen and promote further inflammation. Apart from its barrier functions, however, the epithelium has been shown more recently to participate in inflammation by producing a wide array of mediators, including cytokines, chemokines, eicosanoids, peptidases and matrix proteins. Cytokines produced by epithelial cells include IL-1β, IL-6, IL-11, IL-13, IL-16, GM-CSF, and TGF-β. The chemokine eotaxin-1 (CCL-1) (Ponath et al. 1996; Lilly et al. 1997), produced by the epithelium, recruits cells from the blood for inflammatory reactions in the tissues. Inducible nitric oxide synthase (iNOS) is synthesized in epithelial cells (Guo et al. 1995). NOS has vasodilating and bronchodilating effects and plays an important role in neurotransmission, immune defense, cytotoxicity, ciliary beat frequency, and mucus secretion (Naclerio & Baroody 1998). Epithelial cells also perform an immune function through their capacity to express human leukocyte-associated antigen (HLA)-DR and present antigen (Bousquet et al. 2000). In asthma, epithelial cells are likely to be important in repair processes. They release extracellular matrix proteins, including fibronectin which appears important in cell regeneration (Shoji et al. 1990; Campbell et al. 1993; Harkonen et al. 1995).

Neutrophils Neutrophils are important effector cells in host defense and

Molecular Immunopathology of Allergic Disease

in the inflammatory response to antigen. Although the precise role of neutrophils in allergic airways disease remains unclear, there is increasing evidence of neutrophil participation in asthma and the allergic process. Neutrophils have been recovered in the sputum of patients with exacerbations of asthma (Fahy et al. 1993), but they are found in relatively low numbers in BAL and bronchial biopsies of asthmatic subjects (Jeffery et al. 1989; Bradley et al. 1991; Lacoste et al. 1993). Increased numbers of neutrophils in the airways have also been confirmed in nocturnal asthma (Martin et al. 1991), patients with longstanding asthma (Foresi et al. 1990), corticosteroid-dependent asthma (Tanizaki et al. 1993), and cases of status asthmaticus (Lamblin et al. 1998). As neutrophils have been shown to be relatively steroid-resistant, this increased neutrophilic inflammation may explain the poor response to steroids in these patients (Schleimer et al. 1989; Wenzel et al. 1999). Neutrophils have the capacity to synthesize and release various proinflammatory mediators, proteases, and cytotoxic compounds, as well as cytokines which may be involved in the events central to the pathogenesis of asthma such as bronchoconstriction, tissue damage, and chronic airways inflammation (Boshetto et al. 1989; Lloyd & Oppenheim 1992). Upon activation, they release myeloperoxidase (MPO) together with other granule enzymes. Monteseirín et al. (2001) showed that there is elevated secretion of MPO by neutrophils from asthmatic patients suggesting persistent neutrophil activation in asthma. This may indicate that these cells may be primed by allergens. They also demonstrated a significant inverse relationship between MPO levels released by neutrophils from allergic patients and lung function, as assessed by forced expiratory volume in 1 s (FEV1). In that study, immunotherapy was associated with a significant reduction in MPO release in vitro by neutrophils from allergic asthmatic patients to levels equal to those from nonallergic subjects. To date, little is known about neutrophils and their role in upper allergic airways diseases but their presence has been detected in nasal lavage from patients with allergic rhinitis obtained 6–24 hours after allergen challenge which correlated with the levels of LTB4 (Miadonna et al. 1999). The expression of the high-affinity receptor for IgE (FcεRI) on neutrophils from asthmatic patients provides further evidence for a role for neutrophils in allergic disease (Gounni et al. 2001) (Fig. 62.6). These data suggest that neutrophils may play a role in allergic inflammation through IgE-dependent mechanisms.

Cytokines in allergic disease Interleukin-4 Originally described in 1986 as a factor capable of inducing B cell, T cell, and mast cell proliferation (Yokota et al. 1986), IL-4 is located in the so-called Th2 cytokine gene cluster on

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(d) Fig. 62.6 Detection of FceRIa chain mRNA in human PMNs by in situ hybridization. (a) Positive signal was detected using antisense FceRIa chain riboprobe in PMNs from an asthmatic patient. The FceRIa chain-positive cells (dark-field, arrowheads in b) show neutrophil morphology with phase-contrast microscopy (d). (c) No specific signal was detected with sense probe. Magnification: (a) ×200; (b–d) ×400. The data are representative of nine experiments. (See CD-ROM for color version.)

chromosome 5 (5q31.1) alongside IL-3, IL-5, IL-9, IL-13, and GM-CSF (Le Beau et al. 1989; Morgan et al. 1992). It is mainly produced by T cells, particularly Th2 cells but also by mast cells (Bradding et al. 1992), basophils (Schroeder et al. 2001), and eosinophils (Nonaka et al. 1995). Its actions are mediated through the IL-4 receptor composed of two subunits, IL-4 receptor α-subunit (IL-4Rα) and the IL-2 receptor common γ subunit (γc) (Idzerda et al. 1990; Kondo et al. 1993). Within allergic airways, IL-4 is presumed to be critical for the development of Th2 cells. It also inhibits the expression of the β2 subunit of the IL-12 receptor (IL-12Rβ2) in T cells (Temann et al. 1997). IL-4 has been shown to induce naive T cells to commence the production of Th2 cytokines, the pro-

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duction of NO, and the secretion of other proallergic cytokines (Schleimer et al. 1992; Sornasse et al. 1996; Guo et al. 1997; Temann et al. 1997). IL-4 is critical in the switching of B cells to IgE production, although this effect can be enhanced by other cytokines, including IL-5, IL-10, and TNF-α (Pene et al. 1988). After switching occurs, IL-4 potentiates IgE production. Furthermore, IL-4 enhances the IgE-mediated response by upregulating IgE receptors on inflammatory cells within the airway such as mast cells (Vercelli et al. 1988; Toru et al. 1996). IL-4 has also been shown to promote goblet cell metaplasia, mucus hypersecretion, and vascular cell adhesion molecule (VCAM)-1 expression in endothelial cells resulting in the recruitment of eosinophils. Also, inhalation of IL-4 causes

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the development of sputum eosinophilia and increased airway hyperresponsiveness (AHR) (Shi et al. 1998). The major cellular sources of IL-4 mRNA within the airways of allergic subjects are CD4+ T cells and to a lesser extent CD8+ T cells, eosinophils and mast cells (Robinson et al. 1992; Ying et al. 1995).

Interleukin-5 IL-5 is expressed by activated eosinophils, Th2 cells, and activated mast cells. This cytokine is critical in the development of eosinophils in the bone marrow influencing mainly terminal differentiation of CD34/CD33+ progenitor cells (Clutterbuck et al. 1989; Ema et al. 1990). IL-5 releases both mature and immature eosinophils from the bone marrow, regulates the expression of the transmembrane isoform of its own receptor, and is essential for the terminal differentiation of committed eosinophil precursors. In addition to hematopoietic properties, IL-5, though a weak eosinophil chemoattractant, primes these cells for recruitment by other chemotactic agents such as eotaxin. IL-5 also stimulates the production of IgA from plasma cells, which are extremely efficient at degranulating eosinophils when bound to antigen. IL-5 mRNA expression has been identified by Robinson et al. (1999) in the lungs of asthmatic patients and has been shown to be closely related to lung function in asthmatics (Lamkhioued et al. 1997). Studies with explanted airways have shown that allergen challenge results in an increased number of IL-5 mRNA+ cells suggesting that at least part of the cytokine production is from resident inflammatory cells (Eidelman et al. 1996). IL-5, a critical cytokine in eosinophil survival, also plays a role in eosinophil differentiation, maturation, recruitment, and activation at sites of inflammation. It is therefore the most important Th2 cytokine associated with eosinophils. Mice that are genetically engineered to express high levels of IL-5 have lifelong eosinophilia in their lungs (Mould et al. 2000). In addition, intravenous injections of antibodies that block IL-5 lead to a decrease in asthmatic sputum eosinophilia (Leckie et al. 2000). IL-5 promotes eosinophil survival by blocking apoptosis (Rothenberg et al. 1989). It is also capable of activating eosinophil secretion, cytotoxicity, and chemotaxis (Yamaguchi et al. 1988). IL-5 activates eosinophils to upregulate integrin receptor expression, which promotes adhesion to VCAM-expressing endothelial cells and eosinophil accumulation in the tissues (Walsh et al. 1990, 1991). IL-5 is produced by Th cells, cytotoxic T lymphocytes, and mast cells, but a major source of IL-5 is the eosinophil itself (Ohnishi et al. 1993).

Interleukin-9 IL-9, a Th2 cytokine, is expressed and released by a variety of cells such as lymphocytes, eosinophils, mast cells, and neutrophils. It has pleiotropic activities on Th2 lymphocytes, mast cells, B cells, eosinophils, and airway epithelial cells. It promotes the proliferation and differentiation of mast cells and

Molecular Immunopathology of Allergic Disease

hematopoeitic progenitors and it enhances the production of IgE by B cells (Demoulin & Renauld 1998; Dong et al. 1999; Levitt et al. 1999). The production of IL-9 by human CD4+ T cells is dependent on T-cell activation and may occur in vivo through stimulation by IL-2 or a combination of cytokines. Human peripheral eosinophils are capable of IL-9 production (Levitt et al. 1999). Eosinophils from asthmatic subjects express high levels of biologically active IL-9 that is enhanced by TNF-α and IL-1β (Gounni et al. 2000). Like other Th2 cytokines, IL-9 has been linked to bronchial asthma (Nicolaides et al. 1997). The locus of the IL-9 gene is associated with AHR and elevated levels of serum IgE. Bronchial biopsies of asthmatic patients show higher numbers of IL-9 mRNA+ cells (Fig. 62.7) (Shimbara et al. 2000; Ying et al. 2002). The cells were identified as CD3+ lymphocytes (68%), eosinophils (16%), and neutrophils (8%) (Shimbara et al. 2000). Total numbers of IL-9 mRNA+ cells correlated with FEV1 and PC20 in those asthmatic patients (Shimbara et al. 2000). In atopic asthmatics, segmental allergen challenge led to increased production of IL-9 (Erpenbeck et al. 2003). BAL lymphocytes were identified as the major source of IL-9 in that study. In humans, birch allergy in children has been associated with persistent IL-4 and IL-5 production as well as IL-9 mRNA expression in peripheral blood mononuclear cells (Bottcher et al. 2002). Selective overexpression of the IL-9 gene within the lungs of transgenic mice results in massive airway inflammation with eosinophils and lymphocytes, as well as in the release of CC chemokines from airway epithelial cells (Temann et al. 1998; Dong et al. 1999). This reaction may be caused by the increase in other Th2 cytokines (IL-4, IL-5, IL-13) in response to IL-9, as other features seen in this model were mast cell hyperplasia, epithelial cell hypertrophy with mucus accumulation, subepithelial collagen deposition, and AHR. Studies from systematically expressing transgenic mice showed that the overexpression of IL-9 enhanced an eosinophilic inflammation during antigen challenge (McLane et al. 1998). Intratracheal instillation of IL-9 in mice led to lung eosinophilia and increased serum IgE levels (Levitt et al. 1999). Blockade of IL-4 and IL-5 after IL-9 induction reduced airway eosinphilia, whereas neutralization of IL-13, which has been shown to be a potent inducer of eotaxin expression and concomitant eosinophilia, completely blocked lung inflammation (Li et al. 1999; Temann et al. 2002). The receptor for IL-9 (IL-9R) is expressed by ASM from asthmatic subjects but not normal controls (Gounni et al. 2004), and IL-9 may play a direct role in the production of eosinophilia through induction of eotaxin 1/CCL11 in human ASM although the reports are conflicting. IL-9 has also been implicated in other allergic diseases. Patients with nasal polyposis and asymptomatic bronchial hyperresponsiveness are reported to have an airway eosinophilia comparable to the asthmatic patients as assessed by BAL and bronchial biopsies (Lamblin et al. 1999). Those with nasal polyposis without bronchial hyperresponsiveness have

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(d) Fig. 62.7 Representative examples of in situ hybridization of IL-9 mRNA using 35S-labeled cRNA probes and immunocytochemistry for IL-9 in bronchial biopsy specimens. (a) In situ hybridization of IL-9 mRNA from asthmatic subject (original magnification ×200) (positive cells indicated by arrowheads) and (b) high power of (a) (original magnification ×400). IL-9 immunoreactivity in bronchial biopsy specimen from atopic asthmatic subject (c) and from nonatopic healthy control subject (d). (See CD-ROM for color version.)

no airway eosinophilia. As IL-9 and bronchial eosinophilia are related, IL-9 may play a role in patients with nasal polyposis who develop AHR.

Interleukin-13 IL-13 was first described in 1993 as a cytokine produced after activation of T cells (Minty et al. 1993). There is mounting

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evidence that it is a key mediator of several allergic disorders in humans including asthma, atopic rhinitis, allergic dermatitis, and chronic sinusitis. IL-13 has 70% sequence homology with IL-4 and has many similar actions because it shares the IL-4Rα subunit for its high-affinity receptor formation. It induces IgE production and the expression of VCAM-1 on endothelial cells and activates eosinophils by inducing the

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expression of CD69. IL-13 has been shown to downregulate the transcription of IFN-γ and IL-12 and thus may modulate the cytokine environment at the time of antigen presentation. Due to redundancy in IL4Rα binding, both IL-4 and IL-13 exhibit some degree of functional overlap. As in the case of IL-4, overexpression of IL-13 within the lungs results in the production of IgE, eotaxin, inflammation, mucus hyperecretion, eosinophilia, and upregulation of VCAM-1 (Zurawski & de Vries 1994). However, in animal models of antigen challenge, IL-13 can cause hyperreactivity to contractile agonists (Wills-Karp et al. 1998). IL-13 is able to perpetuate the effector arm of the Th2 immune response. Although it does not appear to have direct effects on T-cell differentiation, it may impact on or perpetuate the Th2 response via several pathways. Firstly, it induces the expression of several chemokines that are thought to selectively recruit Th2 cells, namely thymus- and activationregulated chemokine (TARC) and macrophage-derived chemokine (MDC) (Zhu et al. 2002). Secondly, it can recruit additional dendritic cells to the site of allergen exposure via the induction of matrix metalloproteinase (MMP) 9 and TARC (Vermaelen et al. 2003). Furthermore, it appears to promote its own production via regulation of several mediators, such as adenosine and histamine, which in turn stimulate cells such as eosinophils, mast cells, basophils, and smooth muscle cells to produce more IL-13. Through stimulation of these pathways, IL-13 may be an important contributor to the chronicity of allergic disease. Thus, blockade of IL-13 may have the added benefit of breaking this vicious Th2-promoting positive feedback loop. IL-13 is thought to be closely associated with the pathophysiology of allergic airways disease; the increased expression of IL-13 mRNA has been demonstrated in both asthma and allergic rhinitis (Ghaffar et al. 1997; Naseer et al. 1997). Numerous studies have shown that both message and protein levels of IL-13 are elevated in bronchial biopsy specimens and BAL cells from subjects with allergic asthma, compared with those of control subjects (Huang et al. 1995; Humbert et al. 1997; Ying et al. 1997). IL-13 is also a critical factor for mucin synthesis, a characteristic feature of asthma. Studies in murine models of allergeninduced airway inflammation have shown that blocking IL-13 results in the complete abrogation of mucin synthesis in airway epithelial cells (Grunig et al. 1998; Wills-Karp et al. 1998). In another murine study, administration of IL-13 was sufficient to induce AHR and administration of soluble IL13Rα2 completely reversed IL-13-mediated AHR in albino Jackson (A/J) mice (Wills-Karp et al. 1998). There is mounting evidence that genetic variants of the IL-13 gene and/or its signaling components contribute at least in part to genetic susceptibility to allergic airway disease. Polymorphisms in the IL-13 gene have been reported to be associated with various features of the asthmatic phenotype (van der Pouw Kraan et al. 1999; Graves et al. 2000; Heinzmann et al.

Molecular Immunopathology of Allergic Disease

2000; Celedon et al. 2002). In addition, there is strong evidence that polymorphisms in the IL-4Rα chain are associated with susceptibility to allergic disorders (Hoffjan & Ober 2002). However, further studies are required to fully elucidate the functional consequences of polymorphisms in the IL-13 gene. Evidence continues to support the importance of IL-13 in asthma pathogenesis and has pointed toward exciting new mechanisms of disease. Specifically, through the induction of a complex array of genes in a variety of cell types resident in the airway, such as epithelial cells, smooth muscle cells, fibroblasts, and monocyte/macrophages, IL-13 can induce all the pathologic features of asthma independently of traditional effector cells such as mast cells and eosinophils. These cells may provide an explanation for the chronicity of allergic disease.

Interleukin-16 IL-16, previously known as “lymphocyte chemoattractant factor,” is an immunomodulatory cytokine that acts as a chemoattractant for such CD4-expressing cells as T cells, eosinophils, dendritic cells, and monocytes, and contributes to their recruitment and activation at sites of inflammation associated with allergic disease (Cruikshank et al. 1998). It exerts its biological activities through direct stimulation of the surface molecule CD4, which is present on a number of inflammatory cells including eosinophils and Th cells (Cruikshank et al. 2000), although other receptors independent of CD4 may also be involved (Mathy et al. 2000). Increased levels of IL-16 have been demonstrated in the bronchial mucosa of the lung in mild asthma (Laberge et al. 1997), where the levels of the cytokine within the bronchial epithelium correlated with CD4+ cell infiltration, including Th2 cells (Laberge et al. 1998). Increased levels of IL-16 have been shown in both asthmatics and subjects with allergic rhinitis after specific allergen challenge. IL-16 was primarily localized to the epithelium and CD3+ T cells and, to a lesser extent, eosinophils and mast cells. There is evidence that IL-16 plays an important role during different types of cutaneous inflammatory responses, including allergic contact dermatitis and AD. Increased serum levels of IL-16 have been reported in adult-type AD; serum levels were shown to be a predictor of the eosinophil count (Masuda et al. 2003). In that study, circulating IL-16 levels decreased significantly in patients with AD after topical treatment with corticosteroids or tacrolimus. These findings provide evidence that IL-16 plays a role in the exacerbation of chronic adult AD. IL-16 expression is complex and controlled at both the transcriptional and posttranslational levels (Bannert et al. 1999). Recently, a SNP was described in the region of the promoter of the IL-16 gene. It had initially been hypothesized that this functional polymorphism (T–295C) may be associated with altered levels of gene expression (Nakayama et al. 2000) and may partially account for the increased levels of IL-16 seen in the asthmatic airways. However, recent studies are conflicting. The IL-16 T–295C promoter polymorphism

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might also influence susceptibility to contact allergy (Reich et al. 2003).

Interleukin-17 There has been increasing interest in the IL-17 cytokine because of its essential roles in inflammation and numerous diseases (Kolls & Linden 2004). Six members of the IL-17 family have been identified to date: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E/IL-25, and IL-17F (Yao et al. 1995; Li et al. 2000; Shi et al. 2000; Lee et al. 2001; Starnes et al. 2001, 2002). IL-17A is important in reinforcing a sustained mobilization of neutrophils in host defense in the lungs and in the gastrointestinal system. In the lungs, IL-17A seems to exert its neutrophil-orchestrating activity mainly indirectly, through the induced release of several chemokines and growth factors in local resident cells (Kolls & Linden 2004; Linden et al. 2005). It has been termed by some as an upstream remodeling factor, acting either through accumulated neutrophils or through the release of other cytokines, cytokines that per se are believed to generate classical hallmarks of airway remodeling in asthma and/or chronic obstructive pulmonary disease (COPD) (Linden 2006). IL-17A stimulates the production and release of a number of cytokines implicated in the remodeling process. Human airway epithelial cells cultured in vitro respond to IL-17A by releasing cytokine IL-6, a cytokine known to increase elastase release from human neutrophils in vitro (Bank et al. 1995; Fossiez et al. 1996; Kawaguchi et al. 2001; Laan et al. 2001). IL-17A also stimulates the production and release of colonystimulating factors, including GM-CSF, in human airway epithelial cells in vitro. There is now evidence that IL-17A stimulates the release of the proangiogenic factor vascular endothelial growth factor (VEGF) in lung fibroblasts, generated by in vitro experiments using primary cells from mice (Numasaki et al. 2004). In a study on bronchial fibroblasts from patients with asthma, IL-17A stimulated the production and release of IL-11, and this IL-11-release was glucocorticoid-sensitive (Kawaguchi et al. 2001). According to the one study on this phenomenon, the IL-11 response tends to be higher in bronchial fibroblasts from patients with asthma than in nonasthmatic controls subjects. TNF-α and IL-1β are also released from activated fibroblasts and macrophages by IL-17A. Despite the ongoing research, the causative role of IL-17A in human lung disease still remains unknown and there are yet no clinical studies evaluating IL-17A versus remodeling parameters. Detectable levels of IL-17A mRNA have been found in human peripheral blood T cells (Yao et al. 1997). Molet et al. (2001) demonstrated upregulation of IL-17 expression in sputum and BAL fluid of asthmatic patients, and that eosinophils and CD4+ T cells are the source of its production. There is also data suggesting that patients with mild asthma have a modest increase in the concentration of free, soluble IL-17A protein in the airways (Kawaguchi et al. 2001). Finally,

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there is a recently published clinical study demonstrating increased protein expression of IL-17A in the nasal mucosa of patients with nasal polyps, compatible with IL-17A being linked to atopic airway disease (Molet et al. 2003).

Interleukin-25 IL-25 is a novel Th2 cytokine of the IL-17 family that plays a key role in allergic inflammation. It appears to have biological activities that are different from the other family members. Although it was originally thought to be exclusively expressed in polarized Th2 cells (Fort et al. 2001), IL-25 mRNA has since been detected in multiple other tissues such as colon, stomach, small intestine, kidney, uterus, and lung (Lee et al. 2001; Hurst et al. 2002). Systemic administration of IL-25 results in eosinophilia through the production of IL-5 (Fort et al. 2001; Pan et al. 2001; Hurst et al. 2002), while other IL-17 family cytokines induce predominantly neutrophilia (Schwarzenberger et al. 1998; Linden et al. 2000; Shi et al. 2000). Cheung et al. (2006) found that the viability of IL-25-treated eosinophils was greatly enhanced when compared with untreated ones, indicating that IL-25 could delay eosinophil apoptosis and therefore promote eosinophilia in allergic inflammation. IL-25 also induces increased gene expression of IL-4 and IL-13 in tissues leading to elevated IgE levels and typical Th2-associated pathologic changes in the tissues (Fort et al. 2001; Pan et al. 2001; Hurst et al. 2002). Animal studies have demonstrated IL-25-induced Th2 cytokine production occurs even in mice lacking T cells (Fort et al. 2001; Pan et al. 2001). This suggests that IL-25 is capable of promoting an allergic inflammatory response, even in the absence of Th2 cells. Mast cells have been shown to be potent IL-25 producers and may play a role in prolonging the Th2-type immune response. While the physiologic effects of IL-25 have been well illustrated in animal models (Fort et al. 2001; Hurst et al. 2002; Kim et al. 2002), the cell populations that express the IL-25 receptor are still poorly recognized in humans. The receptor for IL-25 was first identified as IL-17B receptor (IL-17BR) (Shi et al. 2000), also called IL-17 receptor homolog 1 (IL-17Rh1) and Evi27 (Tian et al. 2000; Lee et al. 2001). Wong et al. (2005) showed that human eosinophils constitutively express protein of IL-17BR. They also found that the median plasma concentration of IL-25 in allergic asthmatic patients was significantly higher than that of normal control subjects. Evidence is therefore accumulating for a role of IL-25 in allergic diseases, particularly in the enhancement and/or prolongation of Th2 cell-mediated allergic diseases such as asthma and allergic rhinitis, and has implicated IL-25 as a possible future therapeutic target in these diseases.

Interleukin-33 IL-33, a very recently described member of the IL-1 family, is the latest cytokine to be implicated in the immunopathology

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of allergic (or Th2) diseases (Dinarello 2005). The human gene, located on chromosome 9p24.1, is the specific ligand for the IL-1 receptor ST2 (Schmitz et al. 2005), a receptor that had been termed an “orphan receptor” since its discovery more than 16 years ago. IL-33 mediates its biological effects via this ST2 receptor by activation of the NF-κB and MAP kinase pathways and drives production of Th2-associated cytokines from in vitro polarized Th2 cells. Murine studies have shown that the dominant effect of IL-33 in vivo is the induction of IL-4, IL-5, and IL-13 with the subsequent development of severe pathologic changes in mucosal organs (Schmitz et al. 2005). The changes reported were of eosinophilic infiltration of the bowel, arterial walls and lung, and elevated serum immunoglobulin levels as well as copious mucus secretion in the upper airways and bowel lumen. IL-33 plays an important role in Th2-associated immunology. However, the mechanism by which its receptor, ST2, contributes to Th2 responses has remained unclear. All IL-1 family members are tightly regulated, either at the ligand or receptor level or both (Dinarello 1997; Dunne & O’Neill 2003). The well-known soluble form of ST2 may play an important role in regulating the biological activity of IL-33. The identification of this novel cytokine and its receptor paves the way for more intense research in our attempts at understanding the complex mechanisms of the immune system.

Chemokines in allergic inflammation Chemokines undoubtedly play an important role in the establishment and persistence of allergic disease. Along with their receptors, they are involved at many levels in the allergic process. As well as their role in the recruitment of leukocytes, recent research suggests that chemokines can also influence the immune response by activating and differentiating different cell populations involved in allergic diseases, such as Th1 and Th2 cells. Their spectrum of activities is broad and encompasses effects on morphogenesis, proliferation, and recruitment/activation of inflammatory cells. Chemokines are a family of structurally related peptides and are subdivided into four families (C, CXC, CC, CX3) on the basis of their cystine residues. These families also differ in their biological activity according to the various types of cells on which they act. Members of the CC family include the majority of the chemokines that stimulate movement of cells associated with allergic responses, including monocytes, lymphocytes, basophils, and eosinophils. Thus, these cells are the most responsive to RANTES (regulated upon activation, normal T cell expressed and secreted); eotaxin types 1, 2, 3; monocyte chemotactic protein (MCP) types 2, 3 and 4; and macrophage inflammatory protein (MIP) types 1α, 1β, 3α and 3β. Following antigen challenge, the nasal mucosa of subjects

Molecular Immunopathology of Allergic Disease

with seasonal allergic rhinitis exhibits increased numbers of RANTES, eotaxin, and MCPs. Colocalization studies have demonstrated that, within the submucosa, mRNA coding for RANTES, MCP-3, MCP-4, and eotaxin is expressed mainly by macrophages, T cells, and eosinophils (Sobol et al. 2001). Furthermore, these chemokines are induced by the cytokines IL-4, IL-13, and TNF-α. IL-8 is also expressed within the nasal mucosa by leukocytes, as well as the epithelium, after allergen exposure. This is a CXC chemokine and its expression by the epithelial layer has been suggested to mediate the recruitment of mast cells and eosinophils toward this layer, as observed in seasonal and perennial rhinitis. Aside from being induced by cytokines, the epithelial expression of chemokines may also be a direct result of allergen–epithelial cell interaction, either from the enzymatic activity of the allergen or IgE cross-linkage. Of the CC chemokines, eotaxin has attracted the most attention by far because of the potency and specificity of its actions on eosinophils. Originally described in the BAL fluid of allergen-challenged guinea pigs, eotaxin is described as a novel eosinophil chemoattractant (Jose et al. 1994). Both eotaxin mRNA and protein have been detected at increased levels in nasal, as well as bronchial, epithelium and in submucosal inflammatory cells in patients with allergic rhinitis and asthma, respectively (Lamkhioued et al. 1997; Minshall et al. 1997a). Many chemokines are upregulated in atopic dermatitis to help recruit cells to sites of injury or irritation and promote the development of lesions. CCL5, MCP-4, and CCL11 are all increased in the skin lesions of patients with AD, and possibly help in the recruitment of T cells, macrophages, and eosinophils into the skin (Fig. 62.8) (Taha et al. 2000). Microarray analysis of lesions from patients with AD has revealed an increase in other chemokines such as CCL-18 (pulmonary and activation-regulated chemokine; PARC) and CCL27 (cutaneous and T cell-attracting chemokine; CTACK), both of which are T-lymphocyte chemoattractants (Nomura et al. 2003). It is necessary for better characterization of key chemokines involved in AD in order to better elucidate the disease process and timing of events in this condition. Structural cells, such as epithelial cells, endothelial cells, smooth muscle, and fibroblasts are also sources of chemokines within the airways (Ghaffar et al. 1999; Teran et al. 1999). CCL5 (RANTES), CCL11 (eotaxin 1), and CCL13 (MCP-4), important chemoattractants for eosinophils, were strongly upregulated in epithelial cells of asthmatics compared with healthy controls (Devalia et al. 1999). In vitro studies have shown that TNF-α and IL-1, two cytokines that are present in the epithelial environment in association with infection, can induce chemokine production by epithelial cells. TNF-α and IFN-γ have been shown to induce the production of eotaxin 1 from endothelial cells isolated from nasal mucosa of subjects with allergic rhinitis. This cytokine-induced production of eotaxin

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(b) Fig. 62.8 (a) In situ hybridization with digoxigenin-labeled antisense probe of eotaxin mRNA-positive cells stained in red in the epidermal layer of the skin obtained from subjects with chronic atopic dermatitis (AD). Note the presence of some positive cells within the subepithelial layer (arrow). (b) Negative control from subjects with chronic AD with use of digoxigenin-labeled sense probe. (See CD-ROM for color version.)

was higher in nasal mucosal endothelial cells of atopic subjects than atopic controls (Terada et al. 1996). MCP-4, another eosinophil chemoattractant, has been described in the airways of asthmatic individuals. Moreover, using the nasal allergen challenge model, MCP-4’s expression was shown to increase after allergen provocation (Fig. 62.9) (Christodoulopoulos et al. 1999). As well as producing chemokines in response to inflammation, structural cells may be themselves activated by certain chemokines. Joubert et al. (2005) reported that the expression of CCR3 by ASM cells is increased in asthmatics, and that a CCR3 ligand such as eotaxin induces migration of ASM cells in vitro. These results may suggest that eotaxin could be involved in the increased smooth muscle mass observed in asthmatics through the activation of CCR3. Airway epithelial cells in asthmatics also express CCR3 and react to CCR3 ligands (Stellato et al. 2001). Teran et al. (1999) have previously shown that fibroblasts stimulated the migration of eosinophils by the release of CCL5 and CCL11 following stimulation with TNF-α in vitro. Furthermore, CCL2, a chemokine that recruits macrophages and basophils, is upregulated by IL-4 and IL-13, two Th2 cytokines closely involved in allergic disease (Laberge & El Bassam 2004), signaling its important contribution to the asthmatic phenotype.

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Adhesion molecules Leukocyte and vascular endothelial adhesion molecules (VCAMs) play an important role in the migration of inflammatory cells into tissues during allergic reactions. This response involves a sequence of events that includes margination of leukocytes along the walls of the microvasculature, adhesion to the endothelium, transmigration through the vessel walls, and migration along a chemotactic gradient within the extravascular compartment. These events are mediated by cell adhesion molecules, including integrins, selectins, and members of the immunoglobulin gene superfamily. Initial interactions between leukocytes and endothelial cells during allergic responses are regulated through the L, P, and E types of selectins. Studies have shown that eosinophil accumulation is reduced in the airways of P selectin-deficient mice after antigen challenge (De Sanctis et al. 1997; Broide et al. 1998). In contrast to P selectin, neutrophils bind with greater avidity to E selectin than eosinophils (Sriramarao et al. 1996). Firmer adhesion is regulated by VCAMs and intracellular adhesion molecules (ICAMs) including ICAM-1 and VCAM-1, which can be upregulated by IL-1 and TNF-α. ICAM-1, a ligand for the β2 integrin molecules LFA-1 and Mac-1, which are present on the surface of leukocytes, mediates the attachment

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(b) Fig. 62.9 Representative examples of immunostaining for MCP-4 in nasal biopsy specimens from placebo-treated (a) and steroid-treated (b) patients with allergic rhinitis after allergen challenge. Note a high expression of MCP-4 after allergen challenge in the nasal biopsy section from a subject given placebo (a). In comparison, the expression of MCP-4 in the nasal mucosa after challenge was inhibited in subjects pretreated with steroids

(b). The insert in (a) shows the colocalization of MCP-4 immunoreactivity (brown) to CD3+ T cells (red) in the nasal tissue after challenge. Nasal biopsy specimen after allergen challenge stained with a nonspecific mouse immunoglobulin (negative control), showing no cellular staining (c). (See CD-ROM for color version.)

of all classes of leukocytes to endothelial cells. ICAM-1 is known to be constitutively expressed on vascular endothelial cells and can be induced by inflammatory mediators (Staunton et al. 1989; Bevilacqua & Nelson 1993). As with ICAM-1, VCAM-1 is a member of the immunoglobulin gene superfamily and supports the adhesion of leukocytes to endothelial cells through interactions with the integrin molecule very late antigen (VLA)-4 (α4β1) which is present on the surface of lymphocytes, monocytes, eosinophils, and basophils, but not neutrophils (Schleimer et al. 1992). VCAM-1 expression can be selectively induced on the endothelium by treatment with IL-4 or IL-13. Inflammatory cell infiltration of the nasal mucosa is regulated by the endothelial expression of selectins and adhesion

molecules as well as a chemokine gradient within the tissue. The endothelium of allergic nasal mucosa expresses E and P-selectin, as well as increased levels of the adhesion molecules ICAM-1 and VCAM-1 (Montefort et al. 1992). Circulating inflammatory cells bind to these selectins and come in contact with chemokines on the surface of the endothelium, leading to leukocyte activation and expression of surface molecules such as lymphocyte function antigen (LFA)-1 and VLA-4 (Lee et al. 1994). In vitro, IL-4, IL-13, and TNF-α upregulate endothelial VCAM-1, which is the counterligand for VLA-4 expressed by eosinophils, basophils, and lymphocytes. Interaction between ligand–receptor pathways, such as VCAM-1/ VLA-4, facilitates firm adhesion of the inflammatory cell to the vascular endothelium.

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Within the nasal mucosa of patients with allergic rhinitis, there is a tendency for inflammatory cell accumulation within the epithelial layer, particularly mast cells and eosinophils. This has been attributed to the ability of the epithelial cells to generate chemokines, particularly the CC chemokines (Minshall et al. 1997a; Christodoulopoulos et al. 1999), with chemotactic activity for eosinophils and T lymphocytes (GarciaZepeda et al. 1996a,b; Kuna et al. 1998). Together these molecules, selectins, adhesion molecules, and chemokines, coordinate the rolling, firm adhesion and extravasation of inflammatory cells into the inflammatory site.

Transcription factors Within the past decade, there have been major insights into the mechanisms by which genes are regulated and this research is now being applied to the task of deciphering the control of cytokine production in allergic diseases. Central to the control of gene transcription are transcription factors, proteins that act to facilitate or inhibit RNA production. These transcription factors operate by binding to specific recognition sites, usually located in the upstream promoter region of the gene. Activation of the upstream elements controlling the phosphorylation state of the transcription factors, results in translocation of the factors to the nucleus. Specific DNA binding by transcription factors results in the activation or repression of gene expression. Studies examining the expression of transcription factors in allergic disease are beginning to emerge. Both Th1 and Th2 cytokine responses in allergic disease are associated with transcription factors that control gene expression and lineage commitment by promoting transcription and chromatin remodeling (Agarwal & Rao 1998). Chromatin remodeling is a process where DNA is made more or less accessible for transcription by modification of DNA and proteins, and is coordinated by transcription factors specific to lineage. The transcription factors activated protein (AP)-1 and NFκB are particularly important in allergic diseases. They have been shown to be responsible for the gene transcription of a wide range of cytokines implicated in allergic disease including IL-1β, TNF-α, IL-2, IL-6, GM-CSF, MCP-1, and RANTES (Du et al. 1995; Baldwin 1996). In addition, one of the mechanisms by which steroids are thought to act is by sequestra-

Fig. 62.10 (opposite) Representative examples of in situ hybridization of GATA-3 mRNA in bronchial biopsy specimens and BAL preparations from atopic asthmatic subjects and normal control subjects by using complementary RNA probes. (a) In situ hybridization (dark-field illumination) of GATA-3 mRNA in a bronchial biopsy specimen from an asthmatic subject. Note the presence of positive signals within infiltrating cells but not in the epithelial or endothelial cells. (b) GATA-3 mRNA expression under dark-field illumination in a bronchial biopsy specimen from a normal control subject. (c) In situ hybridization for GATA-3 in BAL cells from a subject with asthma

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tion of AP-1 (Yang-Yen et al. 1990; Demoly et al. 1992; Hart et al. 1998). c-Maf, a basic region/leucine zipper transcription factor has also been implicated in Th2-specific gene transcription. It is expressed in Th2 but not Th1 clones and is induced during normal murine precursor cell differentiation along a Th2, but not a Th1, lineage. To date, c-Maf has been reported to activate the IL-4 promoter (Ho et al. 1996). Expression of c-Maf was first demonstrated in human asthmatic airways 5 years ago, suggesting a role in allergic inflammation (Christodoulopoulos et al. 2001). A more recent study by Erpenbeck et al. (2006) showed that segmental allergen challenge in asthmatics leads to increased GATA-3, c-Maf and T-bet expression in BAL cells, but not in bronchial biopsies.

GATA-3 GATA-3 has a major role to play in the development of Th2 reactions. Naive, freshly activated T cells, and Th2 cells (but not Th1 cells, macrophages, or B cells), express high levels of GATA-3 (Marine & Winoto 1991; Zheng & Flavell 1997). The role of GATA-3 in the development of Th2 reactions cannot be shown by simple deletion of the gene as knockout mice die 11–12 days after conception (Pandolfi et al. 1995). Inhibition of GATA-3 with antisense oligonucleotides showed a crucial function for this transcription factor in activation of Th2 cytokines (Zheng & Flavell 1997). GATA-3 is critical for the production of IL-5 and IL-13, but not for IL-4 (Zhang et al. 1997, 1998; Zhu et al. 2004). It controls the expression of Th2 cytokines IL-4, IL-5, and IL-13, and other factors (Yamashita et al. 2004). GATA-3 is increased in allergic asthma in humans and in allergen sensitization models in mice (Nakamura et al. 1999; Zhang et al. 1999; Christodoulopoulos et al. 2001). GATA-3 mRNA is elevated in bronchial biopsies of atopic asthmatic subjects (Fig. 62.10) and in the nasal mucosa of subjects with allergic rhinitis compared with controls. This suggests a significant role for GATA3 in allergic disorders by biasing T cells in favor of Th2type cytokine production (Nakamura et al. 1999, 2000). The current consensus is that GATA-3 is a master regulator of Th2 cell generation.

STAT-6 Signal transducer and activator of transcription (STAT)-6 is another transcription factor that has attracted considerable

and (d) a normal control subject. (e) Combined in situ hybridization– immunocytochemistry localizing GATA-3 mRNA to CD3-immunoreactive cells in BAL fluid from an asthmatic subject. A GATA-3 mRNA+/CD3+ cell is indicated (arrowhead). (f) Coexpression of GATA-3 mRNA and IL-5 mRNA in BAL cells from an asthmatic subject by double in situ hybridization. GATA-3 probes were labeled with 35S, and IL-5 probes were labeled with biotin. Signals were developed by autoradiography and diaminobenzidine, respectively. (See CD-ROM for color version.)

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Etiology and Pathology In vitro, STAT-6 activation is induced by the interaction of IL-4 with its cell-surface receptor in target cells (Keegan et al. 1994; Takeda et al. 1997). Th2 commitment is associated with the transcription factors STAT-6 and GATA-3 (Kaplan et al. 1996; Zheng & Flavell 1997; Kurata et al. 1999). STAT-6 is activated by IL-4 and IL-13 and it controls transcription of GATA-3 (Kurata et al. 1999).

T-bet One of the main transcription factors associated with Th1 cells is T-bet (T-box expressed in T cells), which transactivates and controls IFN-γ production in Th1 cells (Szabo et al. 2000, 2002). It has the unique ability to redirect fully polarized Th2 cells into Th1 cells as demonstrated by simultaneous induction of IFN-γ and repression of IL-4 and IL-5 (Szabo et al. 2000; Grogan et al. 2001). Expression of T-bet is decreased in airways of patients with asthma (Finotto et al. 2002) compared with T cells from airways of nonasthmatic subjects, concordant with decreased Th1 cytokine production in asthma. This suggests that loss of T-bet might be associated with asthma (Finotto et al. 2002). Mice with targeted deletion of the T-bet gene and those with severe combined immunodeficiency receiving CD4+ cells from T-bet knockout mice spontaneously demonstrated multiple physiologic and inflammatory features characteristic of asthma. Thus, T-bet deficiency in the absence of allergen exposure induces a murine phenotype of asthma. GATA-3 and STAT-6 are increased in the upper and lower airways in asthma and allergies (Nakamura et al. 1999, 2000; Christodoulopoulos et al. 2001). Th1- and Th2-associated transcription factors can further bias cytokine responses by inhibiting gene expression. T-bet represses Th2 lineage commitment by interfering with GATA-3 activity (Hwang et al. 2005). STAT-6 and GATA-3 also inhibit IFN-γ production in Th1 cells and functions (Ouyang et al. 1998; Ferber et al. 1999).

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Remodeling in allergic disease (c) Fig. 62.11 Representative example of STAT-6 immunoreactivity in bronchial biopsy specimens from a patient with atopic asthma (a), a patient with nonatopic asthma (b), and a healthy control subject (c). (See CD-ROM for color version.)

attention in the pathogenesis of allergic diseases. Expression of STAT-6 is increased in the nasal mucosa of patients with allergic rhinitis and in bronchial biopsies of asthmatic subjects (Ghaffar et al. 2000; Christodoulopoulos et al. 2001) (Fig. 62.11). The importance of the STAT-6 pathway was recently demonstrated when STAT-6 knockout mice were protected from developing AHR and from producing excess mucus in response to allergen challenge. These mice also failed to mount allergen-specific IgE responses (Kuperman et al. 1998).

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Remodeling refers to a dynamic process of matrix deposition and degradation in response to insult, leading to the reconstruction of damaged tissue. It is a process critical to the wound-repair process. However, in the asthmatic airways, dysregulation in the balance of deposition versus degradation results in an increasingly thick sub-basement membrane layer of collagen and extracellular matrix protein, the end result of which is an altered structure referred to as airway remodeling (Bousquet et al. 2000). Synthesis of these matrix products is attributed at least partially to increased activity of fibroblasts and myofibroblasts (Gizychi et al. 1997). Cytokines such as TGF-β, GM-CSF, and IL-11 have been suggested to stimulate these cell types (Elovic et al. 1994; Ohno et al. 1995; Gizychi et al. 1997).

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Myofibroblasts are fibroblasts expressing α-actin and are thought to be the cells of origin for nascent smooth muscle cells. These potentially contractile cells are increased in number in biopsy samples from atopic asthmatic subjects and have been associated with thickening of the reticular layer (Brewster et al. 1990). These structural changes are thought to result in the irreversible component of airway obstruction and the persistent AHR seen in asthmatic patients. Although these inflammatory mediators may be contributing to the remodeling of the airways, whether myofibroblasts are responsible for alterations in airway function remains to be addressed. Further details on airway remodeling in asthma may be found in another chapter (see Chapter 79). Studies have shown that skin lesions in chronic atopic dermatitis are associated with remodeling, similar to that seen in asthma. Levels of profibrotic cytokines, including TGF-β1, IL-11, and IL-17 are all significantly elevated in acute and chronic skin lesions from subjects with AD compared with uninvolved skin from subjects with AD and healthy skin from control subjects (Toda et al. 2003). In addition, IL-11 is also correlated with collagen 1 deposition and eosinophil numbers, a cell type capable of producing significant amounts of profibrotic cytokines.

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References

(b) Fig. 62.12 Remodeling in the airways. Van Gieson staining of a bronchial biopsy (a) showing subepithelial fibrosis and increased collagen deposition in a severe asthmatic subject. Immunohistochemistry showing vascular remodeling in allergic rhinitis (b). (See CD-ROM for color version.)

Allergic diseases such as rhinitis and asthma are chronic inflammatory processes in which modification of the histologic and functional structure of tissue leads to remodeling (Fig. 62.12). Minimal information exists regarding the remodeling process within the nasal mucosa. Increased subepithelial deposition of type I and II collagen has been demonstrated within the bronchial submucosa of nonasthmatic individuals with allergic rhinitis compared with healthy control subjects (Chakir et al. 1996). Many studies, however, have focused on fibrosis and remodeling in asthma. Subepithelial fibrosis is the most invariable finding within the asthmatic airways, attributed to the deposition of collagen types III and V, fibronectin, tenascin, and proteoglycans (Roche et al. 1989; Minshall et al. 1997b; Huang et al. 1999). This thickening of the reticular lamina is thought to be an early and fundamental change within the airways, the result of fibroblast and myofibroblast activation, and can be observed in newly diagnosed patients with asthma (Jeffery et al. 1989).

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Molecular Immunopathology of Allergic Disease

Zhang, D.H., Cohn, L., Ray, P., Bottomly, K. & Ray, A. (1997) Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J Biol Chem 272, 21597–603. Zhang, D.H., Yang, L. & Ray, A. (1998) Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3. J Immunol 161, 3817–21. Zhang, D.H., Yang, L., Cohn, L. et al. (1999) Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11, 473–82. Zheng, W. & Flavell, R.A. (1997) The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–96. Zhu, J., Min, B., Hu-Li, J. et al. (2004) Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat Immunol 5, 1157–65. Zhu, Z., Ma, B., Zheng, T. et al. (2002) IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL13-induced inflammation and remodeling. J Immunol 168, 2953– 62. Zurawski, G. & de Vries, J.E. (1994) Interleukin 13, an interleukin 4like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today 15, 19–26.

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Diagnosis of Allergic Disease

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Principles and Practice of Diagnosis and Treatment of Allergic Disease Anthony J. Frew and A. Barry Kay

Summary This chapter complements the individual contributions in the clinical volume of the textbook by giving an overview of principles and practice of allergic diseases. We have emphasized the importance of a thoughtful and thorough clinical history. Despite advances in technology, allergy tests still support, rather than make, an allergy diagnosis. Allergy is a mechanism rather than a disease and in some clinical conditions, such as asthma, may be only one of many triggers. The conditions usually managed by allergists/allergologists include summer hay fever, perennial rhinitis, asthma, allergy to stinging insects, drug allergy, and allergic skin diseases (urticaria, atopic dermatitis, contact dermatitis), as well as adverse reactions to foods. The importance of distinguishing food allergy from food intolerance and the effect of dietary manipulation is emphasized. We also discuss the special considerations of allergy in children. An evaluation of the role of allergy in nonspecific polysymptomatic illness is also given. The basis of treatment of allergic diseases is allergen avoidance, antiallergic medication, and specific immunotherapy. The major pharmacologic agents used in the treatment of allergic diseases, for either relief or prevention, are given, as is a summary of the use of specific immunotherapy. Some general requirements for “good allergy practice” in terms of staff and facilities are suggested and we also give a view on the role of alternative/complementary medicine.

Introduction This chapter on diagnosis and treatment complements the many other contributions throughout these two volumes that discuss the various individual allergic diseases in detail. Allergy is now a major problem throughout the developed world and is increasing in importance in developing countries. While different countries organize and deliver healthcare in a variety of different ways, the basic principles of diagnosis and therapy

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

of allergic disorders are common worldwide. In all areas of medicine, the key principles are to identify diseases correctly, to inform and advise patients of the options available to them, and to provide an appropriate and effective therapy to relieve the symptoms and prevent complications. In the case of children, and also in adults who are not competent to make informed decisions, these processes will obviously involve their families and carers as well. In relation to allergic disorders, the principles are only different in that we seek to identify conditions that may have an allergic basis, determine whether there are any specific allergic triggers, and provide appropriate advice on avoidance or therapy. When consulted by patients with diseases that may have an allergic basis, physicians and other healthcare workers therefore need to be aware of the possible role of allergic triggers in causing the patient’s symptoms, and need to know how to investigate, advise, and treat appropriately (Royal College of Physicians 2003). Because of the very large number of patients who are affected by allergic disorders, all healthcare workers need some understanding of allergy. General practitioners, pharmacists, emergency care staff, general pediatricians, and general hospital physicians all see unselected patients and need to be allergy-aware. Those working in more specialized areas of medicine where allergic conditions are common, need a detailed knowledge of the role of allergy in the type of conditions that they deal with (examples include ENT, respiratory medicine, dermatology, and ophthalmology). These organbased specialists also need to appreciate that allergy can affect other organs outside their particular area of expertise. This is true whether they feel competent to address the other manifestations or would need to refer to another specialist for assessment. More complex allergic disorders may require assessment by a specialist who has particular training expertise in allergy. For historical reasons, different countries have devised various different training programs for this group of specialists, but the common ground is that every healthcare system needs to make provision for the care of patients with complex allergic disorders (Bonini et al. 2006). In the UK, allergic diseases affect at least 15% of the population and are the cause of much ill-health. The commonest atopic allergic disorders are those that produce sneezing, wheezing, itching, and digestive disorders. Allergy should therefore

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Asthma

Table 63.1 Categories of allergy and its mimics. Category 1: atopic allergic disease Allergic rhinitis (including hay fever) Allergic (atopic) asthma Immediate (IgE-mediated) reactions to foods Category 2: non-IgE-mediated allergic disease Contact dermatitis Extrinsic allergic alveolitis (e.g., farmer’s lung, bird fancier’s lung) Category 3: conditions attributable to external agents but which do not involve allergic sensitization (e.g., food intolerance) Food-induced migraine Reactions to sulfites and nitrites Category 4: conditions incorrectly attributed to allergy Chronic fatigue syndrome Symptoms associated with certain psychologic disturbances

be considered in any patient presenting with symptoms suggestive of summer hay fever (seasonal, or intermittent, rhinoconjunctivitis), perennial rhinoconjunctivitis, asthma (including suspected occupational asthma), reactions to stinging insects (especially wasps and bees), reactions to drugs, allergy-related skin disorders (e.g., urticaria, angioedema and atopic eczema), suspected food allergy and food intolerance, and acute generalized anaphylaxis. In addition, allergologists may be asked to assess patients presenting with conditions that are often incorrectly attributed to allergy, such as chronic fatigue syndrome and symptoms associated with certain psychologic disturbances. The range of conditions associated with allergy and their mimics are shown in Table 63.1. It is well recognized that in some of these conditions, particularly asthma, chronic nasal symptoms (rhinitis), eczema (dermatitis), and urticaria (itchy skin blotches or hives), allergy plays a role in some patients but not all. Moreover, in asthma, allergy is just one of the many factors that can trigger an attack (Fig. 63.1). The importance of allergy may also change with age, as in eczema, where allergic factors seem to play a larger role in children than in adults.

Assessment of the allergic patient History and physical examination The cornerstone of allergy diagnosis is the clinical history. Key features include the relationship of symptoms to the time of year (e.g., depending on the country, hay fever may occur in spring and early summer), their occupation (e.g., occupational asthma and dermatitis), and any contact with animals (particularly cats, dogs, and horses). In relation to house-dust mite (the commonest allergy in many parts of the world) it is worth asking about the home and any exacer-

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Rhinitis Reactions to drugs

Allergic mechanisms

Reactions to foods

(Food intolerance)

Urticaria and angioedema Eczema/contact dermatitis

Anaphylaxis

“Multiple chemical sensitivity” Fig. 63.1 Diagrammatic representation of the role of allergy in various diseases. Allergy plays an important role in some asthmatics for some of the time. In the majority of cases of eczema and urticaria, allergy probably only plays a small role. Rhinitis (inflammation of the nose) can have both allergic and nonallergic causes. Seasonal allergic rhinitis (hay fever) is entirely due to allergy. In chronic allergic rhinitis, the allergens are usually the house-dust mite and animal danders. However, many cases of chronic rhinitis have no allergic cause (this is sometimes called “vasomotor rhinitis”). In patients with food intolerance and drug reactions, allergy is often unproven. There is no evidence that multiple chemical sensitivity has an allergic basis.

bation during cleaning activities, making beds, etc. Listening to the patient is critical: the physician should try to establish when and where symptoms first began and what the patient believes are the important causative agents or triggers. However, it is important to keep an open mind about possible links between exposure and symptoms as patients may have made false connections between particular exposures and their symptoms. Some patients will have modified their diets either spontaneously or on the basis of laboratory tests. The history should therefore include details of any prior investigations and any response to prior intervention. When taking a history from patients who have nonspecific symptoms or who are polysymptomatic, it may be worthwhile to encourage them to avoid using the term “allergy” when describing their complaint(s). This is sometimes helpful in correcting patients who make an erroneous self-diagnosis of allergy. Non-IgE-mediated allergic diseases such as contact dermatitis and celiac disease are often more difficult to diagnose and treat than the common atopic disorders. In the UK, these disorders would usually be managed by the relevant organ specialist, while in other countries allergologists may have a role. In all cases the critical issue is that the person undertaking diagnosis and treatment should have appropriate training to enable them to practice effectively and safely, while at the same time meeting all local licensing regulations. The extent and nature of physical examination will vary according to the particular symptoms reported by the patient. Generally it is useful to record height and weight, particularly

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in children and in anyone with suspected asthma. Specific aspects of examination are discussed below in the sections on specific conditions.

Tests for diagnosis of allergic disease Although the diagnosis of allergy is often straightforward, some form of further investigation such as skin or laboratory tests for specific IgE may be needed to confirm the nature of the condition and the range of likely triggers (see Chapter 65). Laboratory tests are of little value on their own and must always be interpreted in the light of the clinical history. Advice printed on laboratory result forms can easily lead to an incorrect diagnosis if the patient’s clinical history is not considered at the same time. Positive skin or blood tests confirm the presence of specific IgE directed against the target allergen, but this does not necessarily mean the patient has an allergic disease. Indeed, population surveys show that about half of those sensitized to grass pollen have no reported symptoms during the hay fever season. Similarly, while the probability of having clinical symptoms increases with the size of the skin reaction to peanut extract, there is no simple cutoff point above which all patients react and below which none react (Fig. 63.1). For most common allergens, a simple skin-prick test is usually sufficient to confirm sensitization. Skin-prick tests are easy and quick to perform, with results available in clinic within 10–15 min. A soluble extract of the suspected allergen is placed on the skin and then introduced into the epidermis with a 1-mm lancet point. Positive responses are seen as a wheal and flare response. These tests are safe and consistent, but positive and negative controls must always be included. The negative control validates the positive results, by showing that the wheals are not simply due to scratching the skin, while the positive control validates the negative results, by showing that if histamine was released, then it would be visible. In some parts of the world, particularly in North America, intradermal tests are used. This technique involves injecting allergen solutions directly into the skin and observing whether the intradermal lump expands by more than 5 mm. This technique uses lower concentrations of allergen than the skin-prick method and is useful for allergens where only a dilute concentration can be obtained, including some venoms and drugs. Several laboratory tests have been devised to measure allergen-specific IgE in blood. Although still often called RAST (radioallergosorbent test), current tests do not involve radioactivity but use ELISA (enzyme-linked immunosorbent assay) or chemiluminescence technology. These techniques are relatively expensive and are normally only necessary in special circumstances (see below). In diseases not associated with specific IgE, other types of tests are required to identify the allergen. For example, in contact dermatitis patch testing is used. The test substances are applied to normal skin, covered and left in place for 48 hours. A positive reaction produces an area of eczema. This

test may help to identify “contact sensitizers,” i.e., substances encountered either at home or in the workplace. In farmer’s lung (and other forms of extrinsic allergic alveolitis) it is IgG, rather than IgE antibodies that are implicated and can be detected by blood test. On rare occasions, special provocation tests are needed to confirm the diagnosis of allergy or identify the allergen. For example, inhalation tests can confirm suspected causes of occupational asthma, and double-blind placebo-controlled tests with suspected foods in disguised forms may help identify or disprove food intolerance. In summary, the diagnosis of atopic allergic diseases is usually straightforward and does not involve expensive tests or hazardous procedures. The situation with food intolerance, drug allergies and certain skin diseases is more complex, as in most of these conditions precise diagnostic tests are lacking. In all cases, the physician or healthcare worker performing tests and advising patients needs to understand the limitations of the tests and the critical importance of interpreting their results in the context of the clinical history.

Specific clinical conditions Hay fever Hay fever (seasonal allergic rhinoconjunctivitis) is caused by allergy to pollen, or more specifically the soluble substances that leach out of pollen grains when they touch the mucous membrane of the nose and eyes (see Chapters 45 and 67). Hay fever is common: 15–20% of the British population are now affected and the incidence of hay fever seems to be increasing, especially in the younger age groups. The rise in hay fever is not due to increased exposure: pollen counts have been steadily falling for many years for reasons that are not entirely clear, and the amount of time spent outdoors has also reduced. Most hay fever sufferers have their “really bad days” at the height of the summer when vast clouds of grass pollens become airborne. In the UK, pollination of grasses is usually well established by the first week in June and is at its peak by mid-June and early July. Some hay fever sufferers are also sensitive to tree and weed pollens. Depending on latitude, tree pollination takes place from February to May. Clinically relevant trees include hazel and alder, which are the first to pollinate, with silver birch peaking in April, followed by oak and plane in May. Weed pollination is usually at its peak in late July and early August. Parietaria (wall pellitory) is an important weed allergen in southern Europe, and its range extends into southern England. Ragweed is the most important weed in the USA, where it causes “fall rhinitis.” Significant ragweed pollen counts are found in Central Europe, especially in Hungary, but the plant does not currently grow in northern or western Europe. Finally, a few patients have seasonal allergy to mold spores, which generally peak in late summer or early autumn (August/September). Grass

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pollen ripens and is shed in the southwest of England about 5 weeks earlier than in the north of Scotland. A warm spring will tend to bring the pollen season forward by 1–2 weeks. The common British grasses include rye, timothy, cocksfoot, meadow fescue, bent, and Yorkshire fog. Large areas of the UK are permanent grassland for both agricultural and amenity purposes. Pollen grains are disseminated by airstreams for many miles. Most pollens remain airborne for long periods and are sometimes carried up to heights of 3000 m. Pollen counts are usually very high on warm, dry, sunny days, and fall during cold and wet periods. Light rainfall removes pollens from the air, but thunderstorms can bring down large amounts of intact and fragmented pollen, which can precipitate acute allergic symptoms. Grass pollen is released in the morning and, as the air heats up, it is carried high into the atmosphere during the middle of the day, descending again as the air cools in the late afternoon or early evening. The highest pollen counts at ground level usually occur midmorning and again in late afternoon. Cities stay hotter for longer, so the pollen count often stays high well into the evening, whereas it falls earlier in rural areas. Pollen counts of 50 grains/m3 usually cause symptoms in susceptible people, but a few very sensitive people will respond to concentrations as low as 1 grain/m3. Fungal-spore and weed-pollen counts are often elevated in late summer. These may be significant allergens in individuals whose symptoms persist after the end of the grass-pollen season. The diagnosis of seasonal hay fever is usually easy: a typical seasonal history of sneezing, runny nose, blocked nose, itching, and streaming eyes. Some wheeziness often coexists, especially when the pollen counts are very high (seasonal allergic asthma). The range of pollens involved can be confirmed by skin-prick testing. This is not essential in all hay fever patients, but is mandatory if treatment by desensitization (immunotherapy) is being considered. Oilseed rape (canola) has been suggested as a possible cause of hay fever (Parratt et al. 1990). However, in common with most brightly colored flowering plants, its pollen is not very aerodynamic, and there is no convincing evidence that this is a common allergic problem.

Perennial rhinitis Chronic symptoms of a blocked runny nose and sneezing are common and can be very troublesome. Infective, structural, and nonallergic causes need to be considered, as well as allergies, and a small minority of patients have underlying immunodeficiency (see Chapter 67). In the UK, allergy to the house-dust mite (Dermatophagoides spp.) and animal-derived allergens are the most important causes of chronic perennial allergic rhinitis. Allergic sensitization to house-dust mite is also commonly found in people who are asymptomatic. Patients with minor symptoms often cope with their condition by simple measures, but when these fail, further management requires consideration of both allergic and structural

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causes. Some clinics offer a combined approach while, in other areas, patients may need two separate assessment by allergy and rhinology specialists. House-dust mites flourish best in sites that offer the most warmth, moisture, and food (skin scales), such as soft furnishings, carpets, mattresses, bedding, sofas, and soft toys (see Chapter 47). The ideal conditions for mite population growth seem to be about 75–80% relative humidity at 25°C. Mites do not flourish in the cold, in very dry climates or at high altitudes. The climatic conditions of most of continental Europe and the UK, eastern North America, Australia, and New Zealand are ideal for them, although they are found throughout the world. The quantity of mite allergens in houses and other locations can be measured, and this may help in deciding on antimite measures. Allergens derived from animals (especially cats, dogs, and horses, but also pet rodents such as mice, rats, guinea pigs, hamsters, rabbits, and gerbils) are important causes of perennial rhinitis, and chronic allergic asthma (see Chapter 48). A high percentage of atopic patients are sensitized to animal-derived allergens. The pet population seems to be increasing and therefore allergy to domestic animals, particularly cats, may become more prevalent. Atopic individuals readily develop allergy to any furry mammal after exposure. Some animal allergens are more potent than others: the worst are the epidermal scales (termed scurf or dander), hair, feathers, saliva, and urine. Cat allergens seem to be a common cause of asthma in sensitized subjects. Kittens and puppies may be more allergenic than adult cats and dogs. The entire home where pets are kept will be contaminated; and symptoms can occur without direct contact. Moreover, reactions to cats and dogs may continue for many months or years after an animal has been removed. Some short-haired dogs shed more dander than long-haired breeds and the amount of allergen present in dander may vary between breeds. Horse dander is a particularly potent sensitizer and is often a problem in people who ride for leisure, as well as for stable and racecourse employees and agricultural workers. Mice, rats, guinea pigs, hamsters, and gerbils can all cause allergic sensitization, both in people who keep them as pets but also in those exposed in a laboratory environment, i.e., animal house attendants and research workers. These workers may also develop allergies to insects such as cockroaches and locusts. Birds can also give rise to allergic disease, although the most dangerous form of hypersensitivity to birds is extrinsic allergic alveolitis, caused by IgG antibody and cellular responses to their feces. A few patients are allergic to feathers, but most patients who are worse after sleeping with feather pillows or duvets are reacting to the house-dust mites they harbor rather then the feathers themselves. Chronic rhinitis can be due to allergic and/or nonallergic mechanisms, including nasal polyposis with or without aspirin intolerance. From the history, examination of the nose and

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other investigations as appropriate, allergy can be diagnosed or excluded and advice given on nonallergic causes. Where allergens such as the house-dust mite and animals are identified, advice should be given on allergen avoidance. If standard drug management by antihistamines, local corticosteroids, and leukotriene antagonists do not relieve obstruction, an ENT opinion is always worth considering.

Asthma and allergy Asthma is among the commonest chronic diseases of the western world and accounts for much ill-health and time off work (see Chapters 76 and 80). It is a condition characterized by episodes of wheezy breathlessness, although it may present as isolated cough, particularly in children. The cause of asthma is obscure, but the basic pathology is inflammation of the airways (bronchi) and the consequence is an irritable or “twitchy” airway, in which airflow obstruction results from exposure to a variety of nonspecific irritants (bronchial hyperresponsiveness). There is a wide clinical spectrum, ranging from mild occasional wheezing through to severe intractable symptoms that may be resistant to long-term systemic corticosteroid therapy. The pathophysiology of asthma is driven by airways inflammation. A wide variety of triggers can trigger symptoms, including allergens, viral infections, exercise, exposure to fumes and other irritants, and certain drugs (especially aspirin and related compounds) (Table 63.2). Food allergens and additives are rarely responsible but can occasionally be implicated. When considering the cause of attacks, it is important to try to distinguish between poor asthma control and specific exposures, but this is sometimes difficult, and the exact role of allergy in asthma is often uncertain. In a few cases, the role of allergy is obvious: for example, in patients who wheeze when the pollen count is high, but not at other times of year. Allergic sensitization is an important risk factor for developing asthma, but it is less clear whether ongoing exposure

Table 63.2 Asthma triggers. Viral infection Allergens Small molecular weight occupational agents Airway cooling/heat loss Cold air Exercise Hyperventilation Irritants and fumes Drugs, e.g., aspirin (and other nonsteroidal antiinflammatory drugs), beta-blockers Food and food additives Emotion, stress Hormonal influences, e.g., menstrual irregularities, pregnancy, menopause Esophageal reflux

to house-dust mite or domestic animals is important in maintaining chronic asthma. In practice, allergy is usually only one of many trigger factors for asthmatic reactions. A significant proportion of asthmatics (about 25%) do not show positive skin-prick tests to common airborne allergens. These patients are called “intrinsic” or “nonatopic” asthmatics. Their disease often starts in later life and can be more severe than those who have childhood-onset allergic asthma. The importance of a specific allergen may be apparent from the clinical history. For example, house-dust mite allergy should be considered if symptoms occur after vacuuming and bed making, or when the patient stays in old, dusty, or damp premises. Asthmatic symptoms related to animal dander are usually obvious; in severe cases they may be provoked by contact with the clothes of someone who has handled or ridden a horse. Sometimes the clinical history does not point towards allergy. For instance, many mold spores are allergenic but may be difficult to incriminate because they are so common. Mold allergy may be suspected if symptoms are worse in the late summer and autumn. Molds are often common in damp and poor housing conditions and may be easier to identify by visiting the home than by extensive laboratory screening tests. Nevertheless, the precise relationship between damp, poor housing, and respiratory symptoms is still unclear and more research is needed. In longstanding chronic asthma, it is often impossible to tell what proportion of symptoms are due to specific allergens. The precise contribution of allergy, as opposed to other triggers, is of practical importance where the allergic substance or substances can be easily identified and avoided. Assessment of asthmatic patients should also include other important potential triggers or precipitating factors such as stress and hormonal influences, esophageal reflux, and nasal disease. Thus, the treatment of patients with chronic asthma involves a thorough evaluation of the patient and their environment as well as appropriate use of antiasthma medication. Except in very special circumstances, specific immunotherapy (desensitization or hyposensitization) should not be prescribed in patients whose primary problem is asthma.

Occupational asthma Occupational asthma due to sensitization to agents occurring in the workplace is an important cause of adult-onset asthma and is described in detail in Chapter 82. Substances that cause occupational asthma include low-molecular-weight chemicals, wood dusts, and a wide range of vegetable and organic dusts. An accurate diagnosis of occupational asthma requires a careful history as well as familiarity with the chemical and other substances involved and how they are used in the workplace. The diagnosis may be confirmed by serial peak flow measurements, but sometimes requires specific inhalation challenges that need to be done in specialized units.

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Some patients with work-related asthma do not have true allergies but have irritant responses to chemicals encountered in the workplace. Irritant-induced asthma can be caused by a wide variety of chemicals and irritants: diagnosis usually requires specialist assessment and advice.

Allergy to stinging insects Anaphylactic reactions to stinging insects are relatively rare but can be fatal (see Chapters 50 and 96). Most wasp and bee stings are a temporary irritation, but a few unlucky people are sensitized to the venom and will experience anaphylaxis after a sting. Bee-sting allergy is common in beekeepers, their relatives, and neighbors. Bumblebees rarely sting, but with their increased use as pollinators in intensive agriculture, there have been reports of occupational anaphylaxis. Any patient who has suffered a generalized reaction after a bee or wasp sting should be assessed by someone who is familiar with venom allergy and given appropriate advice. Usually this will include a first aid package (an epinephrine auto-injector and oral antihistamines), but some patients will need to be desensitized. Fortunately, there is a gradual resolution of the allergy over time, so that by 10 years after the last sting the risk of anaphylaxis will have reverted to a background level (Golden et al. 2004). Patients with large local reactions do not benefit from specific immunotherapy.

Drug allergy Adverse reactions to drugs are common and pose a serious medical problem (see Chapter 94). In most instances, drugs act as haptens, which bind to large molecules (such as plasma proteins) and are then recognized by the body as a foreign complex allergen. Certain drugs are particularly prone to cause allergic reactions, most of which are urticaria or measles-like rashes. The β-lactam antibiotics (penicillins and cephalosporins) account for about 75% of all drug rashes. Skin tests with the various metabolic products of penicillin are useful in assessing patient with a doubtful history of antibiotic-associated anaphylaxis. Allergic reactions to anesthetic agents are potentially fatal, and should always be investigated. Muscle relaxants are the commonest cause of adverse reactions but induction agents, opiates, antibiotics, latex, and blood products can all be responsible. True allergy to local anesthetics is rare, but many patients report adverse reactions following dental work or other local anesthetic procedures. Skin testing is usually negative and can help reassure clinicians and patients. Occasionally, preservatives of latex may be the cause of the problem. Some patients develop acute severe reactions to aspirin, with rhinorrhea, nasal obstruction, asthma, and urticaria. Often, there will be co existent asthma and nasal polyps. When all three are present, this syndrome is called “Samter’s triad.” Hypersensitivity reactions to aspirin and related compounds result from an unusual susceptibility to the pharmacologic action of the drugs rather than a true allergy. A few very

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sensitive individuals also react to the natural aspirin-like substances (salicylates) in food. In addition, substances added to pills or medicines (including tartrazine or benzoates) can sometimes cause adverse reactions in susceptible individuals. It is often difficult to obtain evidence of specific antibody or sensitized T lymphocytes in patients with drug allergy. Generally speaking, the clinical history is the most important method of evaluating adverse reactions to drugs, and if in doubt, avoidance of the suspect drug may be the best course of action.

Allergy and the skin Although swelling, itching and redness are found in many different skin disorders, it is often very difficult to establish a clear association between allergy and skin problems. While most cases of contact dermatitis are clearly allergic, the relationship between allergy and angioedema or atopic eczema is much less certain.

Urticaria Urticaria and angioedema are common disorders that affect about one in four people at some stage in their lives (see Chapter 90). Acute urticaria is most often due to viral infection, but sometimes is caused by food allergy, especially to peanuts and treenuts or shellfish. When physical triggers such as pressure, cold, or heat induce symptoms, the condition is much more likely to be endogenous than allergic in origin. Chronic urticaria (by definition lasting intermittently or continuously for more than 3 months) is generally endogenous, but disability, if severe, has been shown to be comparable with coronary artery disease. Angioedema in the absence of urticaria can be endogenous as well; however, C1 inhibitor deficiency can be hereditary or acquired, the latter associated with lymphoma or connective diseases. The most common cause of severe angioedema seen in emergency rooms in the USA is due to angiotensin-converting enzyme inhibitors. These disorders are mediated by bradykinin rather than histamine.

Atopic dermatitis Atopic dermatitis (or atopic eczema) is a chronic recurrent inflammation of the skin characterized by intense itching, which tends to affect specific parts of the body, particularly the flexures (creases), at different ages (see Chapter 88). The condition is often associated with other forms of atopic allergy, but it is currently thought that the dermatitis develops first and predisposes to the development of allergic antibodies rather than the other way around. In childhood, symptoms are sometimes triggered by egg or cows’ milk, but in adulthood food allergy is rarely, if ever, responsible. Patients with atopic eczema often have other atopic diseases such as allergic rhinitis, asthma, or both. Patients often have raised titers of IgE antibodies against a variety of airborne or food allergens, but there is little evidence that allergen avoidance alters the course of the disease, especially in older children and adults.

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Atopic eczema may be triggered or made worse by a number of different external influences, including emotional stress, irritation of the skin by wool or nylon, infections, vaccinations, and eating certain foods. Dietary elimination would seem sensible in young children where eczema is troublesome and where cows’ milk or egg allergy are suspected (Atherton et al. 1978). If improvement follows, it may then be helpful to see what happens when the food is reintroduced. Management consists of breaking the “itch– scratch cycle,” reducing T-cell inflammation in the skin with steroids and calcineurin inhibitors, and the use of emollients and nonirritant cleansers.

Contact dermatitis Contact dermatitis may be due to allergic sensitization, irritation or a combination of both (see Chapter 89). Irritant contact dermatitis is the more common form, especially in the occupational setting, but many people have contact allergy to nickel, lanolin, cosmetic ingredients, etc. Contact dermatitis presents with blisters, thickening of the skin (lichenification), or scaly eruptions at sites of exposure. The pattern and shape of the skin changes may suggest the likely cause. For example, contact dermatitis due to plants such as primula has a typical linear appearance where the plant has brushed against the skin. Common “sensitizing” agents causing allergic contact dermatitis include nickel, preservatives such as ethylene diamine, perfumes, chromium, resins, formaldehyde, dyes, and many other chemical compounds. The standard method for testing for allergic contact sensitivity is patch testing. Some forms of contact allergy only occur with concurrent exposure

to sunlight (photoallergic dermatitis), for example gentamicin ointment, phenothiazine tranquilizers (e.g., chlorpromazine), and some sunscreen preparations.

Adverse reactions to foods Food allergy is one of the most controversial subjects in the practice of allergy (see Chapter 93). Confusion has arisen because of lack of a universal agreement on definitions and diagnostic criteria. As many adverse reactions to foods do not involve specific sensitization processes, the term “food intolerance” or “food sensitivity” is preferred when describing adverse reactions to foods. In this definition, food allergy constitutes a subset of food intolerance (Table 63.3). It is particularly important not to diagnose food allergy without clear and strong evidence, as unnecessary dietary restrictions can seriously disrupt not only the patient’s life but also the whole family. In extreme cases this can lead to severe malnutrition (May 1980; Young et al. 1994). As in all areas of allergy practice, a well-taken history is the cornerstone of diagnosis. Food allergy is a cause of symptoms, not a diagnosis in its own right. In the more severe forms of food allergy, there will be a clear relationship between exposure to the suspect food and the onset of symptoms. In almost all cases, there will be some symptoms in and around the mouth. This means that isolated asthma or rhinitis, occurring without oral or perioral symptoms, is not caused by food allergy. Similarly, if the symptoms begin several hours after exposure, it is much less likely to be food allergy than if the symptoms start within an hour of exposure. Most acute food allergies can be easily diagnosed from the history, and

Table 63.3 Food allergy, intolerance and aversion. Group 1 Food allergy involving IgE (“food anaphylaxis”): examples include immediate, sometimes violent, reactions to peanuts, tree nuts, eggs, milk, fish and shellfish Oral allergy syndrome Group 2 Food allergy, not involving IgE, but where there is strong evidence that there is specific (immunologically mediated) hypersensitivity. Examples are celiac disease and cows’ milk protein enteropathy Group 3 Food intolerance reactions affecting certain susceptible individuals where allergic sensitization is not involved. Examples include some types of irritable bowel syndrome, food-induced migraine, reactions to sulfites or nitrites, lack of digestive enzymes (hypolactasia, low aldehyde dehydrogenase) or other types of reaction (e.g., to tartrazine, red wines, cheeses) Group 4 Established and well-recognized intestinal diseases where the role of foods in causation is sometimes suspected but remains unproven. Examples include ulcerative colitis and Crohn disease Group 5 Food aversion, and subjects with nonspecific symptoms to foods not confirmed by double-blind testing (this includes, for example, individuals with chronic fatigue who believe, erroneously, that their symptoms are due to foods)

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confirmed on skin-prick tests. Sometimes acute food poisoning can mimic anaphylaxis. This occurs particularly with scombrotoxic poisoning, which is due to bacterial spoilage of scombroid fish (e.g., tuna, mackerel) and produces histamine and related pharmacologic agents. Symptoms include diarrhea, flushing, headache, sweating, and sometimes nausea, abdominal pain, and burning in the mouth. Blood tests for specific IgE may be useful to confirm negative skin tests before reassuring patients. In a few cases it may be necessary to proceed to food challenges before allowing the patient to eat the suspect food. Other forms of food intolerance (i.e., those that do not involve allergy) are more difficult to diagnose. In a true food sensitivity, the symptoms should disappear when foods are eliminated from the diet and should reappear when the food is eaten again even when given in disguised form. If critical decisions are to be made about diet, the relationship between food and symptoms should be assessed by a double-blind, placebo-controlled food challenge. These are often quite difficult to perform, particularly if food has to be disguised for several weeks. Food challenges should not be performed to confirm allergy in patients who have a clear-cut clinical history of immediate or acute allergic (anaphylactic) reactions to certain foods. The management of patients with food allergy varies with the severity of the reaction and the risk of exposure to the culprit food. This is discussed in more detail below. Although patients with allergic rhinitis, asthma, and atopic eczema frequently have positive skin-test reactions to foods, eating these foods does not always cause symptoms. When such foods provoke symptoms, they can cause asthma or rhinitis alone, or as part of a more widespread response. Sneezing bouts, blocked nose, or asthma, can also occur after taking wine or other alcoholic drinks because of the irritant effect of sulfite preservatives, or other components such as fermentation products. These are not allergic reactions, but result from the pharmacologic actions of the compound in question. Similarly, many patients with atopic eczema find that certain foods provoke a transient red and blotchy rash, especially if the food contains vasoactive amines. However, eliminating these from the diet rarely improves atopic eczema in adults. A relatively recently described allergic condition is the oral allergy syndrome, which consists of swelling or itching of lips, mouth, tongue, or throat immediately after contact with certain foods. It occurs in tree, grass, weed, and latex allergy sufferers, and is due to cross-reacting proteins in pollens and foods. Reactions to the same foods when cooked are less likely but can sometimes occur. In its mildest form, as many as 75% of birch-allergic patients may be affected. Typically these individuals have a sensation in the lips or tongue after eating raw apples. Nonallergic forms of food intolerance are frequently reported, and probably form the largest category of suspected food reactions. The range of adverse reactions to food is wide

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and only selected examples are given here. Both coffee and coffee withdrawal can provoke migraine in susceptible people. Many Oriental people are intolerant of alcohol because compared with white people they have lower levels of aldehyde dehydrogenase, the enzyme that breaks down acetaldehyde, a toxic product derived from alcohol. Other foods preserved by sulfites, particularly white wine, dried fruit, and fruit salads sold in restaurants and supermarkets, sometimes provoke asthma by the release of sulfur dioxide. Nitrites used as flavor enhancers and preservatives can cause symptoms due to direct dilatation of blood vessels (typically migraine or flushing). Certain cheeses cause headaches: this may be due to the tyramine content, which increases as the cheese matures. The tyramine content of red and white wines is similar but red wine causes headaches in susceptible people because of a group of chemicals called congeners, of which the most important are phenolic flavonoids. Monosodium glutamate (MSG) has been reported to cause chest pain and sweating but similar symptoms can be provoked by other foods, including spicy tomato dishes, suggesting this may be an irritant effect. The so-called “Chinese restaurant syndrome” is also associated with nausea, dizziness, fainting, and tingling of the lips and face in susceptible individuals. However, in formal double-blind studies, these symptoms occurred with both MSG and placebo and so the cause of this syndrome remains unclear (Kenney 1986). Although reactions to food additives do occur, they are not as common as many people believe (Young et al. 1987). Certain additives such as benzoates, tartrazine (E102) and metabisulfites may cause symptoms in a few susceptible individuals but, by and large, the evidence for this is unconvincing.

The role of allergy in other conditions An allergic etiology has been suggested for a whole range of clinical disorders that fall outside the generally accepted cluster of atopic disease. In some of these conditions there is evidence that food and other external triggers may precipitate symptoms, but there is little evidence of specific immunologic sensitization. In other conditions, the link between exposure and symptoms is tenuous or nonexistent. It is quite common for patients and their healthcare advisers to use the term allergy to describe these conditions, and this has led to disagreements and difficulties. A detailed discussion of all the issues involved is beyond the scope of this chapter, but the most commonly encountered issues are outlined below.

Hyperactivity (hyperkinesis) A particularly controversial area is the association between foods, food additives, and hyperactivity (or hyperkinesis) in children. Thirty years ago it was claimed that a diet free of preservatives, salicylates, and artificial flavors might benefit

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70% of hyperactive children (Feingold 1975). The assumption that food additives play a major role has been challenged (David 1987), but others have reported improvements in carefully controlled studies of dietary restriction (Egger et al. 1985; Pollock & Warner 1990). It is important to recognize that hyperactive behavior has many causes, which risk being neglected if undue attention is focused on the diet. Parents who suspect food additive intolerance in their child may nevertheless insist on maintaining children on restrictive diets, even when dietary challenges are negative (David 1987; Pollock & Warner 1990). The importance of gathering a good history and interpreting diagnostic tests in context cannot be overemphasized. With a few exceptions (e.g., eggs, nuts and fish), skin and blood tests for food allergens are unreliable or misleading as (i) patients may have IgE antibodies but no symptoms; and (ii) foods can cause nonspecific (“irritant”) positive skin reactions.

Specific intestinal diseases Food intolerance may contribute to several common chronic intestinal diseases. Their mechanisms are obscure but they do not involve immediate (IgE-mediated) allergic responses. In celiac disease (gluten-sensitive enteropathy) there is impaired food absorption (malabsorption) with diarrhea, weight loss, and malnutrition. Glutens are proteins found in wheat, barley, oats, and rye. Patients with celiac disease react against gluten, and develop damage to the intestinal lining (atrophy of the jejunal mucosa). Antibodies can be detected against endomysial proteins, but the main cause of the condition is T cell-mediated hypersensitivity to gluten. Adhering to a strict gluten-free diet leads to restoration of normal jejunal mucosal architecture and resolution of symptoms. Milk proteins can also cause a malabsorption syndrome similar to celiac disease (Walker-Smith 1986). Cows’ milk protein enteropathy mainly occurs in babies, who fail to thrive because they have diarrhea due to malabsorption and they may even have intestinal bleeding and colitis. Symptoms disappear when cows’ milk is removed from the diet. Reintroduction of cows’ milk causes recurrence of the gastrointestinal symptoms and failure to thrive. After a viral gastrointestinal infection, cows’ milk may also be poorly tolerated for a while because of a temporary inability to digest lactose, which is the sugar in milk. Thus, in babies and small children with chronic gastrointestinal symptoms and failure to gain weight, trials (under medical supervision) of milk exclusion (or gluten elimination) are justified, and the diagnosis can usually be confirmed by food challenge. Recovery often occurs within a few months. There is no evidence that soya-based formulas are less likely than cows’ milk formulas to cause allergies. Some soya preparations, especially those containing sucrose, may actually cause diarrhea. However, infants who cannot tolerate cows’ milk may benefit from a change to soya provided

that the preparation used is appropriately supplemented with L-methionine, calcium, and vitamins. Many adults have mild reactions that may be caused or exacerbated by milk. Because strict milk-free diets are very difficult to achieve and maintain, they should not be introduced lightly. Double-blind challenge tests may be needed to test the diagnosis. It does not follow that every individual with cows’ milk intolerance has an allergy and some can tolerate milk products in small amounts. Many adults, especially if they are not white-skinned, cannot digest lactose because of a constitutional deficiency of the enzyme lactase. In these subjects, undigested sugar is fermented in the lower bowel, causing diarrhea and wind. Much of the wind is hydrogen gas, and this is the basis for the simple “breath hydrogen” diagnostic test. Lactose intolerance is rare in children of European descent, but affects up to 90% of adult Africans and Orientals. It can occur as a transient result of gastroenteritis and even as a secondary effect of cows’ milk protein intolerance. This can cause confusion in diagnosis unless a lactose challenge is performed separately from a cows’ milk protein challenge. Goat and sheep milk contain similar proteins and sugars to cows’ milk, but their fat content is distinct. Patients who are truly allergic to cows’ milk will not be able to tolerate other ruminant milks, but those who have difficulties digesting the fat content may find goat or sheep milk a useful alternative. Irritable bowel syndrome (IBS) is a descriptive term applied to patients with irregular bowel habit, typically alternating constipation and diarrhea, abdominal bloating and, in many cases, colicky abdominal pain (Agrawal & Whorwell 2007). In some patients, constipation predominates (“spastic colon”) and gastrointestinal transit times are greatly increased. Most of these cases do not involve food intolerance but a relationship to specific foods can be demonstrated in a minority, usually those with predominant diarrhea, with some bloating and pain (Jones & Hunter 1986; Price 1987; Nanda et al. 1989). In one study, 72 of 189 patients who improved on a restricted diet were then able to identify foods such as dairy products and cereal grains that provoked a recurrence of symptoms when they were reintroduced and they remained well while avoiding these foods (Nanda et al. 1989). Similar symptoms can be caused by lactase deficiency, allergic gastroenteritis, and conditions in which an excess of dietary fiber or other unabsorbed food residues are fermented in the lower bowel. Patients who seek help in the alternative therapy sector for this condition usually get advised to reduce their wheat and dairy intake, whatever else they are told. The advice is generically sound, even if the tests done are doubtful. This can lead to inappropriate conclusions about other so-called triggers identified by the alternative therapy tests (see below). Food allergy is not thought to play any part in ulcerative colitis or Crohn disease, but dietary measures can be of

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benefit in Crohn disease. Here it is the consistency of the food (e.g., liquid, solid, high residue, low residue) rather than the chemical nature of the food that seems to be important.

can lead to anaphylaxis. In others, there is IgE antibody to a specific food, but no clinical reactivity to it except if the food is eaten prior to exercise and anaphylaxis ensues.

Diet and allergy in children

Evaluating the role of allergy in nonspecific/polysymptomatic illness

Current evidence suggests that many children who become allergic nowadays would not have been allergic if they had been born 30 or 50 years ago (see Chapter 97). Considerable effort has been invested in determining what it is about the modern environment that increases the risk of children becoming allergic, and to identify measures that may prevent or reduce that risk. It seems that the window of opportunity for intervention is very early, probably during the first year of life or even in utero. When advising interventions for children who are otherwise healthy, it is even more important that the measures suggested are safe than when treating children with established disease. Measures that seem to be helpful include the following. • Breast-feeding, at least for the first 4– 6 months. • Early weaning should be avoided, especially with foods such as egg and peanut butter. • Nursing mothers should eat a balanced diet. There is no firm evidence that consuming peanuts, eggs or milk will increase the risk of the child developing food allergy. • Parents should not smoke during pregnancy or the child’s first year (as allergies are more common in children of people who smoke).

Anaphylaxis Anaphylaxis is defined as an acute, severe, generalized allergic reaction (see Chapter 92). This is a serious and potentially life-threatening condition. Common triggers include foods (especially peanut, treenuts, and shellfish), wasp and bee stings, drugs, blood products, and radiocontrast media. The cause of the anaphylactic episode will usually be clear from the history, although other causes of sudden loss of consciousness (e.g., cardiac arrhythmias or vasovagal attacks) sometimes need to be excluded. There is also a group of individuals who have anaphylaxis for which no specific cause can be identified. The reason for such unexplained and serious attacks may not become apparent until a later date and these patients need careful assessment, advice and follow-up. Where the history is clear and an obvious provoking factor is identified, avoidance is usually effective. However, some foods can turn up in unexpected places, so first-aid advice is also needed. Usually this will involved self-administered epinephrine and antihistamines. For patients in whom a trigger factor cannot be clearly defined, advice should also be given on emergency treatment, together with the opportunity to return for reassessment if the attacks change in character or frequency. Subpopulations of patients with exercise-induced anaphylaxis have symptoms related to food ingestion. In some, eating, regardless of food type, within 4–5 hours before exercise

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Allergists can provide an important service in evaluating the possible role of allergy as a cause of nonspecific or polysymptomatic illness. In addition, the general practitioner (or healthcare purchaser) may face a request for referral to a specialist by a patient who has a large number of poorly defined symptoms or by a patient who is convinced that symptoms are related to an underlying “allergic” process, often given misleading titles such as multiple chemical sensitivities, environmental illness, or total allergy syndrome. In some instances, a relationship can be established (e.g., between caffeine and headaches) but often there is no evidence of any link between food and the symptoms. Some patients may “somatize,” i.e., they experience psychologic distress as physical symptoms for which they seek medical advice (Stewart 1990). Some may have recognizable disorders such as depression, but have been resistant to seeking or accepting psychiatric help. These patients require early diagnosis, sympathetic and tactful handling, and the institution of appropriate treatment and/or psychiatric referral to avoid inappropriate and costly medical consultations and tests (Howard & Wessely 1993).

Treatment of allergic diseases including asthma Allergen avoidance The identification of relevant allergens, in the home and workplace, and the implementation of avoidance measures may improve prognosis and appreciably cut the cost of drugs used to suppress symptoms. Thus, allergen avoidance should be the first line of treatment in allergic disease and is sometimes relatively easy (e.g., avoidance of pets), provided patients wish to take the necessary action. On the other hand, the benefits of major changes such as moving house are generally too uncertain to be recommended. Pollen grains are clearly difficult to avoid but some simple measures can reduce exposure. Hay fever sufferers should avoid outdoor exercise in the morning and late afternoon on sunny days in the pollen season, and should drive with their car windows closed. Sunglasses and spectacles give some protection from pollen settling in the eyes. People who are allergic to a domestic animal find it takes several months to free the household of fur, especially cat dander. Boarding a pet with friends may help; if the patient improves significantly the family often decides not to take the animal back. Avoidance of allergens is more difficult with work-related allergies, but good work practices can minimize exposure.

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Veterinary and bakery workers may need to change their jobs, and individuals exposed to allergens in factories may benefit from relocation to another working area. Every effort must be made to remove the individual from the allergyprovoking environment. The emission of noxious industrial fumes and dust should be corrected by appropriate ventilation and good industrial hygiene. Avoiding exposure to dust mites is helpful and can reduce the symptoms of allergic rhinitis and asthma. House-dust mite avoidance measures are particularly appropriate in bedrooms but, to be effective, they must be carried out vigorously and repeatedly. Mattresses pillows and duvets may be encased in tightly woven covers and bedding, including pillows and duvets, should be washed at temperatures above 60°C on a regular basis. In bedrooms, exposure can be reduced by removing carpets, heavy curtains, upholstered furniture, books, and woolly toys. Regular vacuuming with a filtered cleaner is usually helpful, but few people will agree to remove all their fitted carpets and sofas. The benefit of acaricidal agents in reducing symptoms has not been clearly established and more controlled trials are needed.

Drugs for allergy treatment In the UK, all licensed drugs used in the treatment of allergy have been tested for quality, efficacy, and safety. Years of research and development are required before a new drug is given a product license and marketed. Granting of a product license does not guarantee safety, and as in all other branches of medicine, the benefit–risk ratio of a particular preparation must always be carefully considered for each patient.

Drugs for hay fever Antihistamines The introduction of nonsedating antihistamines (selective H1 antagonists) in the early 1980s was a substantial advance in treating hay fever and allied disorders. As these compounds do not cross the blood–brain barrier, they rarely impair alertness and do not potentiate the effects of alcohol or tranquilizers. Examples include fexofenadine, cetirizine, loratadine, and desloratadine. Older antihistamines such as chlorphenamine and hydroxyzine often cause drowsiness and inattention and should no longer be used (see Chapter 25). Severe chronic urticaria/angioedema is an exception where sedating antihistamines, side effects notwithstanding, are recommended if these can be employed rather than low-dose corticosteroids or cyclosporin A when a double dose of a nonsedating antihistamine is ineffective (see Chapter 90). A variety of antihistamines are also available for ocular use.

Corticosteroids Topical corticosteroids are particularly effective in controlling nasal symptoms. They reduce allergic inflammation and swelling of the lining of the nose (nasal mucosal edema), but are less effective against congestion than other nasal symp-

toms. Topical corticosteroid sprays and drops are remarkably free from side effects, even after many years of seasonal or continuous use (see Chapter 69).

Sodium cromoglycate Eye symptoms of hay fever are not always controlled by antihistamines. Sodium cromoglycate drops (2%) are often very effective but the preservative in them sometimes causes unacceptable stinging. One or two drops are applied to each eye four times daily. Very occasionally, corticosteroid eye drops (which should be preservative-free to minimize irritation) are prescribed for pollen-induced allergic conjunctivitis. In these circumstances, an ophthalmologist’s opinion should be sought before prescribing them, as there are dangers in giving topical corticosteroids in the presence of ophthalmic infections or glaucoma. Decongestants Topically applied nasal decongestants quickly relieve nasal obstruction and can be used when a completely blocked nose prevents penetration of corticosteroid nasal sprays. Their effect is temporary and they do not relieve nasal inflammation. Sympathomimetic nasal decongestants such as xylometazoline should be restricted to a maximum of 2 weeks, because longer use often leads to rebound congestion when they are stopped. They should be prescribed with caution in patients with heart or thyroid disease. Systemic corticosteroids Systemic corticosteroids should only be considered in patients with severe continuous symptoms who have not been helped by combinations of antihistamines and local steroids. Single depot injections of corticosteroids have often been used for summer hay fever in special circumstances, for example students sitting their summer examinations. Although these drugs seem effective, there have been few (if any) controlled studies of efficacy. There is little rationale for their use, as short courses of oral prednisolone allow better adjustment of the dose and duration of therapy while minimizing side effects. Prednisolone may be given at a dose of 20 mg/day for no more than 7 days to control very severe hay fever at the height of the pollen season.

Drugs for asthma Many drugs are available for the treatment of asthma (see Chapter 80). In general, they are used either for prevention of recurrent attacks or for the relief of acute wheeze. Prescriptions for individual patients may vary considerably and several different preparations may be needed to achieve optimal control of symptoms. For these reasons, clinicians (and ideally patients as well) should have a basic knowledge of the mode of action of the various antiasthma drugs so that they can be used to best advantage.

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β Agonists The most effective drugs for the relief of symptoms are the βadrenergic agonists, for example salbutamol and terbutaline. These are mainly used by inhalation (as a pressurized aerosol, powder, or nebulizer) but can be taken orally, subcutaneously, or intravenously in selected cases. They act principally by relaxing the muscle (bronchial smooth muscle) of both large and small airways. The β agonists are particularly useful for the relief of wheeze and are also quite effective in protecting against exercise-induced asthma. Patients requiring an inhaled β agonist more then twice daily should be prescribed inhaled corticosteroid for regular use. The bronchodilator is then used as required, as “rescue” medication. Xanthines Xanthines, such as theophylline and aminophylline, are phosphodiesterase inhibitors that can relax airways smooth muscle and may help to stabilize nocturnal asthma. Their precise mode of action in asthma is uncertain, and they have little marginal effect when added to β agonists. They are toxic to the gut and CNS, and their chief disadvantage is the relatively narrow margin between the effective and toxic doses. In recent years, they have largely been relegated to use as third- or fourth-line drugs. Xanthines are also helpful in the emergency treatment of very severe acute asthma attacks. If the patient is already taking xanthines by mouth, intravenous aminophylline should never be given without first checking blood levels. Anticholinergic drugs Anticholinergic drugs, such as ipratropium bromide, have a longer duration of action than β agonists but rarely produce greater bronchodilatation. They are of value in acute attacks, in some cases of troublesome nocturnal asthma, and in a few patients with severe chronic asthma. They are more useful in patients with COPD than those with pure asthma. Inhaled corticosteroids Corticosteroids are very effective treatments for most types of asthma, but they have significant side effects when taken by mouth for long periods. The introduction of inhaled corticosteroids in the early 1970s was a major advance, enabling many patients who were previously dependent on oral corticosteroids to control their asthma without systemic side effects. Since then, intermittent or long-term treatment with inhaled corticosteroids has become the mainstay of the therapy in all but the mildest cases of asthma. Sodium cromoglycate and nedocromil sodium Sodium cromoglycate was originally marketed as a mast cell stabilizer. It is now believed to have antiinflammatory and other properties, probably through the inhibition of chloride channels. Nedocromil sodium is a similar compound, but has a wider range of effects on inflammatory cells. In some patients, they are highly effective prophylactic agents, although it

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is difficult to predict which patients will benefit. Children and young adults are more likely to derive benefit from cromones, but they are worth trying in older asthmatics, especially those with a clear history of allergen-induced wheeze. Both drugs have an excellent safety record with few side effects, apart from some occasional local irritation.

Omalizumab A monoclonal antibody directed against IgE (omalizumab, Xolair) is available for the treatment of asthma and there have also been successful trials in allergic rhinitis and food anaphylaxis. In the UK, the use of omalizumab is restricted to patients with severe chronic asthma who have had severe exacerbations/hospital admissions. A full account of treatment with anti-IgE is found in Chapter 81.

Immunotherapy (desensitization or hyposensitization) Specific immunotherapy (SIT) involves the administration of increasing amounts of allergen, in order to deviate the allergic response and thereby alleviate the symptoms of allergic disease (see Chapter 73). SIT was first developed in the late 19th century and has evolved gradually over the last 100 years. SIT is a useful treatment for selected patients with IgE-mediated allergies, especially those with a limited range of sensitivities. Because it is possible to demonstrate a reduction in sensitivity of the target organ (nose, eye or bronchi) after symptom provocation, SIT is sometimes called desensitization (or hyposensitization). The first controlled studies of SIT were performed in the 1950s, and subsequent studies have shown that SIT is effective in allergic rhinitis (Frankland & Augustin 1954; Varney et al. 1991), mild allergic asthma (Bousquet et al. 1990) and allergy to stinging insects (Lockey 1990; Valentine et al. 1990). However, patients with moderate to severe asthma are at greatly increased risk of adverse side effects, including the precipitation of fatal asthmatic attacks. Current UK indications for asthma immunotherapy are (i) patients with seasonal allergic rhinitis due to grass pollen or tree pollen (hay fever), who have failed to respond adequately to antiallergic drugs; (ii) patients with anaphylaxis due to wasp or bee venom hypersensitivity; and (iii) patients with rhinitis due to cat or house-dust mite where the symptoms are definitely due to the allergen, and avoidance measures are impractical or ineffective. In practical terms, it has been difficult to persuade UK healthcare purchasers to commission SIT, as there is only a limited number of clinics with experience in the technique. This contrasts with most other parts of Europe and North America, where there are many more allergists and the role of SIT is more widely accepted. Only a few SIT vaccines have full product licences in the UK, but further clinical trials are currently in progress which should lead to a range of extracts becoming available soon. SIT should be administered only by doctors with experience

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and training in immunotherapy, and at present this means either in hospitals or in specialized clinics. Epinephrine should always be immediately available and there should be easy access to resuscitative facilities. Attendant staff should be trained in resuscitative techniques. Patients should be kept under close supervision for the first 30 min after each injection. This period should be extended if the patient has any generalized symptoms, however mild. A severe or prolonged adverse reaction may necessitate hospital admission. Immunotherapy is not recommended in nonallergic rhinitis, nonallergic asthma, atopic dermatitis, chronic urticaria, food hypersensitivity, or drug and chemical hypersensitivities. In practice, allergen injection immunotherapy is usually well tolerated. With standard depot preparations, mild adverse reactions occur after about 1 in 25 injections and systemic adverse reactions after about 1 in 500 injections. In the UK, treatment is usually continued for 3 years. There is no advantage in going on beyond this, although it is difficult to find hard evidence due to the difficulty in performing long-term follow-up studies. In recent years, there has been considerable interest in giving SIT by the sublingual route. This technique uses high doses of allergens, and should not be confused with homeopathic approaches to allergy (where infinitesimal dilutions are used). Sublingual immunotherapy (SLIT) is particularly popular in Italy and France, where it now comprises the vast majority of SIT (see Chapter 74). New standardized tablets have been developed that allow patients to start SLIT without having to build up gradually. Levels of benefit on symptoms and medication usage are similar to those achieved by injection SIT, although there is still only limited evidence for the durability of benefits achieved by SLIT. The precise mechanism(s) of action of SIT is still uncertain. Overall, SIT damps down allergic inflammation and acute hypersensitivity of the mucous membranes of the eyes and the nose. After several years of immunotherapy, serum IgE antibody concentrations eventually begin to decline, but this occurs long after the effects of SIT are achieved. Allergenspecific IgG antibodies are induced, but again they tend to follow the clinical improvement rather than precede it. Successful SIT is associated with induction of regulatory T cells, which produce interleukin (IL)-10 and promote the production of IgG4 rather than IgE. The allergen-specific IgG response to SIT may thus reflect the induction of T regulatory cells and is not generally thought to be the mechanism of benefit. Further work is ongoing to identify the precise mode of action, so that this can be achieved as quickly and safely as possible, perhaps using modified allergens or different adjuvants.

Pediatric allergy Allergic diseases are particularly important in children, and all pediatricians need to be aware of the possible role of

allergy in their area of specialization (see Chapter 77). In an ideal world, all children should be diagnosed and managed by doctors who have specialist training in pediatrics. However, in individual cases, it may be appropriate for a child with complex allergies to be seen by someone whose primary interest is in adults with allergic disease. Correspondingly, it is important that adult allergists should have some knowledge of the way that allergies present in children, and that they should work closely with their pediatric and primary care colleagues. The progression of allergies in children differs from adults as children may “grow out” of some food allergies. Drug treatments are different for children both in dosage and side effects. The inherent dangers of unnecessary dietary restriction for treatment of some types of allergic diseases are far greater in children because of disturbances in nutrition and growth, and expert pediatric dietetic advice is usually required. Many members of the general public believe that allergies can be involved in a diverse range of children’s diseases such as hyperactivity, behavioral disorder, recurrent abdominal pain, and chronic headaches. Generally speaking, there is little hard evidence that this is true. It is essential that such children are seen by pediatricians within the framework of a comprehensive children’s department, rather than seeking advice from adult allergists or complementary therapists.

Unconventional (“alternative”) allergy Allergy, particularly “alternative allergy,” is a popular subject with the media. Unfortunately many members of the public and even many doctors are often uncertain as to which forms of diagnosis and treatment have been validated by objective studies. There are many reasons for the high level of interest in alternative medicine. These include limitations in the provision of conventional allergy services, distrust of authority, distrust of science and especially the scientific approach to medicine, and a current tide of enthusiasm for “natural” remedies. Several different strands of alternative medicine are at work under the banner of alternative allergy. Firstly there is unconventional therapy for disorders that everyone agrees are allergic in origin, e.g., homeopathic and other unconventional treatments for allergic rhinitis (hay fever). Second is the use of unconventional diagnostic methods to identify triggers of disorders that can be allergic in origin, e.g., electrical testing to identify food allergies as a cause of asthma. And third is the use of various techniques to identify external causes of conditions that are not normally thought to have an allergic basis whatsoever, e.g., allergy testing for headaches, fatigue, or alleged reactions to Candida or dental amalgam. Patients often complain that conventional allergists are unwilling to undertake these unconventional forms of diagnosis and treatment through the NHS, but until these diagnostic and therapeutic methods have been evaluated by reputable, randomized, double-blind, placebo-controlled trials, they cannot

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be accepted into routine clinical practice. This is particularly important as symptoms in chronic allergic disease are known to fluctuate over time and can often improve when treated with placebo. The more frequently used methods of alternative allergists have been assessed in detail and the conclusions published elsewhere (Royal College of Physicians and Royal College of Pathologists 1994). Although a few trials of alternative therapies have shown marginal benefit in rhinitis and asthma, larger scale trials have not confirmed their efficacy. For now, patients who wish to use these practices do so at their own cost and at their own risk.

Conclusion This chapter has reviewed the principles and practice of diagnosis and treatment of allergic diseases, with particular reference to the UK perspective, although many of these points apply equally elsewhere. It is important to emphasize that allergy is a mechanism rather than a disease. Some cases of “allergic disorders” are not in fact due to allergy: patients and nonexpert clinicians can compound this by, for example, talking about acute urticaria as an “allergic reaction” or by talking about atopic eczema on the basis of physical appearances rather than allergic etiology. In other conditions, allergy is a risk factor for developing the condition, but once the condition is established, both allergic and nonallergic triggers can set off attacks (e.g., asthma). In evaluating, a good clinical history is essential, and diagnostic tests should be used to support or refute an allergic basis for the history. Using tests as a primary screening tool will lead to misdiagnosis and mismanagement. Allergy is so common that every healthcare professional needs to understand something about it. Patients expect their advisers to know how to assess and investigate their conditions, including knowing how to refer on for help when the condition falls outside their competence. Healthcare purchasers need to distinguish between good conventional practice and “alternative allergy,” as well as ensuring that appropriate resources are made available for the care of patients with allergic disease.

References Agrawal, A. & Whorwell, P.J. (2007) Irritable bowel syndrome: diagnosis and management. BMJ 332, 280–3. Atherton, D.J., Sewell, M., Soothill, J.F., Wells, R.S. & Chilvers, C.E.D. (1978) A double-blind controlled cross-over trial of an antigen-avoidance diet in atopic eczema. Lancet i, 401–3. Bonini, S., Ansotegui, L.J., Durham, S., et al. (2006) Allergy and clinical immunology services in Europe. Allergy 61, 1191–6. Bousquet, J., Hejjaoui, A. & Michel, F.-B. (1990) Specific immunotherapy in asthma. J Allergy Clin Immunol 86, 292–305. David, T.J. (1987) Reactions to dietary tartrazine. Arch Dis Child 62, 119–22.

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Egger, J., Carter, C.M., Graham, P.J., Gumley, D. & Soothill, J.F. (1985) Controlled trial of oligoantigenic treatment of the hyperkinetic syndrome. Lancet i, 540– 4. Feingold, B. (1975) Hyperkinesis and learning disabilities linked to artificial food flavors and colors. Am J Nursing 75, 797–803. Frankland, A.W. & Augustin, R. (1954) Prophylaxis of summer hay fever and asthma. A controlled trial comparing crude grass pollen extracts with the isolated main protein component. Lancet i, 1055–7. Golden, D.B., Kagey-Sobotka, A., Norman, P.S., Hamilton, R.G. & Lichtenstein, L.M. (2004) Outcomes of allergy to insect stings in children, with and without venom immunotherapy. N Engl J Med 351, 668–74. Howard, L.M. & Wessely, S. (1993) The psychology of multiple allergy. BMJ 307, 747–8. Jones, V.A. & Hunter, J.O. (1986) Food intolerance. In: Pounder, R.E., ed. Recent Advances in Gastroenterology. Churchill Livingstone, Edinburgh, pp. 281–300. Kenney, R.A. (1986) The Chinese restaurant syndrome: an anecdote revisited. Food Chem Toxicol 24, 351–4. Lockey, R.F. (1990) Immunotherapy for allergy to insect stings. N Engl J Med 323, 1627–8. May, C.D. (1980) Food allergy: material and ethereal. N Engl J Med 302, 1142–3. Nanda, R., James, R., Smith, H., Dudley, C.R.K. & Jewell, D.P. (1989) Food intolerance and the irritable bowel syndrome. Gut 30, 1099– 104. Parratt, D., Thomson, G., Saunders, C., McSharry, C. & Cob, S. (1990) Oilseed rape as a potent antigen. Lancet 335, 121–2. Pollock, I. & Warner, J.O. (1990) Effect of artificial food colours on childhood behaviour. Arch Dis Child 65, 74–7. Price, J.F. (1987) Paediatric allergy. In: Lessof, M.H., Lee, T.H. & Kemeny, D.M., eds. Allergy: An International Textbook. John Wiley, Chichester, pp. 423–53. Royal College of Physicians (2003) Allergy: the unmet need. A blueprint for better patient care. A report of the Royal College of Physicians Working Party on the provision of allergy services in the UK. Royal College of Physicians, London. Royal College of Physicians and Royal College of Pathologists (1994) Good allergy practice: standards of care for providers and purchasers of allergy services within the National Health Service. Royal College of Physicians, London. Reprinted in Clin Exp Allergy (1995) 25, 586–95. Stewart, D.E. (1990) The changing faces of somatization. Psychosomatics 31, 153–8. Valentine, M.D., Schuberth, K.C., Kagey-Sobotka, A. et al. (1990) The value of immunotherapy with venom in children with allergy to insect stings. N Engl J Med 323, 1601–3. Varney, V., Gaga, M., Frew, A.J., Aber, V.R., Kay, A.B. & Durham, S.R. (1991) Usefulness of immunotherapy in patients with summer hay fever uncontrolled by antiallergic drugs. BMJ 302, 265– 9. Walker-Smith, J.A. (1986) Milk intolerance in children. Clin Allergy 16, 183–90. Young, E., Patel, S., Stoneham, M., Rona, R. & Wilkinson, J.D. (1987) The prevalence of food additives in a survey population. J R Coll Physicians Lond 21, 241. Young, E., Stoneham, M.D., Petruckevitch, A., Barton, J. & Rona, R. (1994) A population study of food intolerance. Lancet 343, 1127– 30.

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Skin Testing in Diagnosis and Management of Respiratory Allergic Diseases Pascal Demoly, Anaïs Pipet and Jean Bousquet

Skin

Summary Since the recognition that IgE-mediated allergic diseases are caused by exposure to allergens, the diagnosis of respiratory allergies is based on skin testing and/or specific serum IgE measurements. Skin tests have evolved over the years and optimized. They follow a proper methodology, utilize negative and positive controls, have positivity criteria, and should always be interpreted in the context of the clinical history. Indeed, falsenegative and false-positive skin tests are well known and should be taken into account.

Skin tests Mast cell Blood Y

Histamine release Basophil

Mast cell

Introduction The diagnosis of allergic rhinoconjunctivitis and asthma is based on the coordination between a typical history of allergic symptoms and diagnostic tests. Since the recognition that IgEmediated respiratory allergic diseases are caused by exposure to allergens, it has been a common practice to establish a diagnosis by reexposure of the individual to the suspected allergens. Thus, in vivo and in vitro tests used in the diagnosis of allergic diseases are directed toward the detection of free or cell-bound IgE (Fig. 64.1). Skin tests have represented the primary diagnostic tool in allergy since their introduction in 1865 by Blackley (1880). The intracutaneous test proposed by Mantoux in 1908 was rapidly applied to immediate allergy. Some years later, Lewis and Grant (1924) described the prick test. These methods were subsequently refined, standardized, and further validated. The diagnosis of respiratory allergy has also been improved by allergen standardization providing satisfactory extracts for most inhalant allergens as well as by recombinant allergens. Skin tests have many uses other than the diagnosis of IgEmediated allergy, such as epidemiologic and pharmacologic

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Measurement of serum-specific IgE

Basophil activation

Provocation tests: nasal, bronchial, oral, conjunctival challenges

Mucosa Fig. 64.1 Diagnosis of IgE-mediated respiratory allergy. (See CD-ROM for color version.)

studies. Indeed, changes in skin sensitivity are used for the standardization of allergen extracts and for pharmacologic studies. Skin tests and methods derived from them can also be used to better understand the pathophysiology of the allergic reaction, as well as helping to evaluate the mechanisms of antiallergic treatments.

Techniques of skin tests Skin testing methods Several methods of skin testing are available. Before initiating any skin test, some precautions should be taken (Table 64.1). Note that scratch tests should no longer be used because of poor reproducibility and possible systemic reactions.

Prick and puncture tests Prick and puncture tests are usually recommended for the diagnosis of immediate-type allergy. The prick test was first

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Table 64.1 Skin testing precautions.

Table 64.2 Common errors in prick testing.

Skin tests should never be performed unless a physician is available immediately to treat systemic reactions Have emergency equipment readily available, including epinephrine Be careful with patients with current allergic symptoms Determine the value of allergenic extracts used and assess their stability Be certain that the test concentrations are appropriate Include a positive and a negative control solution Perform tests in normal skin Evaluate the patient for dermographism Determine and record medications taken by the patient and time of last dose Record the reactions at the proper time

Tests are placed too close together (< 2 cm), and overlapping reactions cannot be separated visually Induction of bleeding, leading possibly to false-positive results Insufficient penetration of skin by puncture instrument, leading to falsenegative results. This occurs more frequently with plastic devices Spreading of allergen solutions during the test or when the solution is wiped away

described by Lewis and Grant in 1924, but became widespread in the 1970s after its modification by Pepys (1975). The modified prick test is performed by placing a small drop of each test extract and control solution on the volar surface of the forearm (or occasionally on the back). The drops are placed 2 cm or more apart to avoid false-positive reactions (Nelson et al. 1996). A disposable hypodermic needle (25 or 26 gauge) is passed through the drop and inserted into the epidermal surface at a low angle with the bevel facing up. The needle tip is then gently lifted upward to elevate a small portion of the epidermis without inducing bleeding. The needle is then withdrawn and the solution gently wiped away with a paper tissue approximately 1 min later. A separate needle must be used for each test to avoid mixing solutions. Using the same needle or lancet wiped with dry cotton wool (Piette et al. 2002) or cotton moistened with 75% ethanol (Kupczyk et al. 2001) between tests when undertaking several prick tests, with the aim of saving time as well as money, provokes an unacceptable number of false-positive results. In addition, the Occupation Safety and Health Administration of the US Department of Labor alerted its personnel to the risk of a blood-borne pathogen exposure incident, because the technician could unintentionally prick himself or herself with the device in wiping it between tests (Occupation Safety and Health Administration 1995). Furthermore, smearing test solutions on adjacent test sites must be avoided. Some common errors in prick testing are listed in Table 64.2. Other prick-puncture test methods, in which the test instrument is inserted perpendicular to the skin, have been proposed to decrease the variability of the prick when performed by different investigators (Demoly et al. 1991). The most popular instruments are the Morrow Brown standardized needle (Brown et al. 1981), the Pricker (Osterballe & Weeke 1979), the Stallerpointe (Bousquet et al. 1985a), and the Phazet (Chanal et al. 1988). These puncture tests can also be performed with other devices (Santilli et al. 1980; Ownby & Anderson 1982; Corder et al. 1996). Another variant in prick testing is to carry a drop of extract from the bottle with the

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lancet and then prick the skin, the application of the extract and the puncture occurring in one step (Malling 1985; Demoly et al. 1991). Skin-prick testing can be carried out with multiheaded devices, reducing technician time. In a head-to-head prospective study comparing the performance of eight commonly used skin test devices (four single-headed and four multiheaded) on both the arms and back of each subject, statistically significant differences among the devices were found in all areas (Carr et al. 2005). However, all of them, except the multiheaded Greer Track, had sensitivities of 86–97% and specificities of 98% or greater. Single devices had larger reactions on the arm, whereas multidevices had larger ones on the back. Multiheaded devices showed significant intradevice variability and were more painful than single devices. With regard to food extracts, it has been proposed that the so-called prick-prick test be used (Dreborg 1988), in which first the fresh food and then the skin is pricked with the same device. The prick-puncture test appears to be safe, and exceptional systemic reactions have been observed with noncommercial extracts (Novembre et al. 1995; Devenney & Falth-Magnusson 2000). As an example, the overall rate of generalized reactions with fresh food was 0.52% in one pediatric study (Devenney & Falth-Magnusson 2000) and 0.26% in another pediatric and adult study (Codreanu et al. 2006). No fatalities have been reported (Codreanu et al. 2006; Lin et al. 1993; Reid et al. 1993; Devenney & Falth-Magnusson 2000).

Intradermal tests The intradermal tests described by Mantoux (1908) are still used in clinical practice. The allergen extract is injected intracutaneously from a 0.5 or 1.0 mL tuberculin syringe through a 26- or 27-gauge needle. Before injection, all bubbles should be carefully eliminated to avoid “splash” reactions that could be interpreted mistakenly. An effort should be made to avoid penetrating the subepidermal capillary bed of the skin. The syringe placed at an angle of 45° to the skin, the bevel of the needle downward, facing the skin, and penetrating entirely, but not going deeper than, the superficial layers of the skin. A volume of approximately 0.03–0.05 mL is gently injected to produce a small superficial bleb approximately 4 mm in diameter. Intradermal tests may elicit pain, which can be reduced by the use of a topical anesthetic cream such as the

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eutectic mixture of local anesthetics (EMLA) (Lander et al. 2006), which reduces the flare but not the wheal (Sicherer & Eggleston 1997). Intradermal tests may provoke a low rate of very large local (both immediate and late) and systemic reactions, ranging from 0.02% (Lin et al. 1993) to 1.4% (Lockey et al. 1989) of the tested patients. Some fatalities have been reported (Lockey et al. 1987; Reid et al. 1993). Thus, although intradermal tests may be done by a nurse or a technician, a physician should always be nearby. A waiting period of 20 min in the office of the physician before the patient is released has been recommended, and this period of time may be extended for high-risk patients (American Academy of Allergy and Immunology 1990). Particular care should be taken in patients treated with beta-blocking agents, which may increase the risk of fatal systemic reactions. Performing prick-puncture tests prior to intradermal tests, as well as utilizing serial 10-fold dilutions of the usual test concentration, especially in patients with histories of anaphylaxis, are useful ways to avoid adverse local and systemic reactions in routine skin testing (Co-Minh et al. 2006). In case of generalized anaphylactic reactions, a rubber tourniquet should be placed above the test site on the arm and 1 in 1000 aqueous epinephrine administered intramuscularly in the lateral thigh (vastus lateralis) (Lieberman 2006). Because of the risk of infectious bacterial or viral diseases, allergy testing of multiple patients should not be performed with a common intradermal skin test syringe (Lutz et al. 1984; Shulan et al. 1985).

Comparison of prick/puncture tests and intradermal tests As a general rule, the starting dose of intracutaneous extract solutions in patients with a preceding negative prick test should range between 100- and 1000-fold dilutions of the concentrated extract used for prick-puncture tests (Bernstein & Storms 1995). With potent standardized allergen extracts [100 000 AU (allergy units) per mL], the range for starting intradermal skin tests, in patients with a negative prickpuncture test, is between 10 and 100 AU. For less potent allergens at 10 000 AU concentration, the range for starting intradermal skin tests, in patients with a negative prickpuncture test, is between 100 and 1000 AU. The concentration of allergen extract required to elicit a positive reaction with intradermal testing is 1000 to 30 000 times lower than that necessary for a positive prick-puncture test. With standardized and/or potent extracts, the prick-puncture test appears to have several advantages over the intradermal test. These include economy of time, patient comfort, safety, and a steeper dose–response curve that is more useful in titrated skin testing. Prick testing also uses extracts in 50% glycerin that have greater stability. Intradermal testing cannot use this diluent, because it results in an irritant or false-positive response. It is generally accepted that prick-puncture tests are less sensitive and less reproducible but more specific than intradermal tests. However, it is questionable whether

the increased sensitivity of intradermal tests, at the strength customarily performed, is clinically necessary or simply increases the chance of a false-positive response (Wood et al. 1999). It is indeed considered that prick-puncture tests correlate better with symptoms (Dreborg et al. 1989), although in patients with a low sensitivity, intradermal skin tests may be the only positive test. In effect, skin prick-puncture tests are recommended as the primary test for the diagnosis of IgE-mediated allergic diseases and for research purposes by the European Academy of Allergology and Clinical Immunology (1993) and the US Joint Council of Allergy Asthma and Immunology (Bernstein & Storms 1995).

Negative and positive control solutions Because of interpatient variability in cutaneous reactivity, it is necessary to include negative and positive controls in every skin-test study. The negative control solutions are the diluents used to preserve the allergen extracts. All negative controls should be totally negative. The rare dermographic patient will show wheal-and-erythema reactions to the negative control. The negative control will also detect traumatic reactivity induced by the skin test device (with a wheal that may approach a diameter of 3 mm with some devices) and/or the technique of the tester (Bernstein & Storms 1995). Any reaction at the negative control test sites will make interpretation of the allergen sites much more difficult (Bernstein & Storms 1995). Positive control solutions are used to detect (i) suppression by medication or disease; (ii) the exceptional patients who are poorly reactive to histamine; and (iii) differences in technician performance. In the USA, the usual positive control for prick-puncture testing is histamine phosphate, used at a concentration of 5.43 mmol/L (or 2.7 mg/mL, equivalent to 1 mg/mL of histamine base). Wheal diameters with this preparation range from 2 to 7 mm. However, a 10-fold greater concentration may be more appropriate, with a mean wheal size ranging between 5 and 8 mm. For the intradermal test, the concentration routinely used is 0.0543 mmol/L. The mean wheal size elicited ranges from 10 to 12 mm. Mast cell secretagogues such as codeine phosphate (9% solution) may also be used as positive controls.

Grading of skin tests Measurement Skin tests should be read at the peak of their reaction. Whatever the method, the immediate skin test induces a response that reaches a peak in 8–10 min for histamine, 10–15 min for mast cell secretagogues, and 15–20 min for allergens. Late-phase reactions are not often recorded, because their exact significance is not known (Agarwal & Zetterstrom 1982; Bernstein & Storms 1995). Skin tests should be read in a standard manner (European Academy of Allergology and Clinical Immunology 1993; Bernstein & Storms 1995). When the reactions are mature,

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the size of each reaction is measured with a millimeter rule. The largest and smallest diameter of the wheal and/or erythema are measured. As the reactions are often oval or irregular in shape, the diameters measured are necessarily at right angles to each other. Both diameters are recorded, summed, and divided by two. To obtain a permanent record, the size of the reaction may be outlined with a pen, blotted onto cellophane tape, and stored on paper. The area of the cutaneous response may be estimated by planimetry (Sussman et al. 1982), by weighing the tape with a precision scale, or by scanning with a computer and measuring the surface areas with commercially available softwares (Poulsen et al. 1993). Ultrasonic measurements permit assessment of other parameters (Bousquet et al. 1990a), such as the wheal thickness and wheal volume, which might allow different tests to be better differentiated when performing the end-point titration. The quantification may also be done by ultrasonic measurements (Serup 1984), which quantify wheal thickness and wheal volume or with laser Doppler flowmetry (Serup & Staberg 1985), which determines the blood flow in the wheal and erythema.

Criteria of positivity The wheal and the erythema have been considered elements for assessing the positivity of skin tests. Using the prickpuncture test, Pepys (1975) suggests that when control sites are completely negative, small wheals of 1–2 mm with flare and itching are likely to represent a positive immunologic response and the presence of specific IgE antibodies. Though significant in immunologic terms, small positive reactions do not necessarily indicate the presence of a clinically relevant allergy. Using prick-puncture tests, reactions generally regarded as indicative of clinical allergy are usually over 3 mm in wheal diameter (corresponding to a wheal area of 7 mm2) and over 10 mm in flare diameter (Adinoff et al. 1990). Another criterion is the ratio of the size of the reaction induced by the allergen to the size of that elicited by the positive control solution.

Grading systems Several grading systems have been proposed. For prickpuncture tests, the sizes of the wheal and erythema often are not seriously considered, although a grading system based on the ratio between the reactions induced by the allergen and the histamine reference (5.43 mmol/L) has been used in Scandinavia for many years (Table 64.3) (Dreborg et al. 1989). Due to the log/log relationship between changes in skin prick-puncture test response and allergen concentrations, only large differences in skin sensitivity will be detected using a fixed concentration of allergen. More information is usually obtained if a threshold dilution titration skin test is performed employing serial dilutions (Bousquet et al. 1985b). End-point titrations estimated as the concentration of allergen giving a wheal size comparable with that of a positive control solution

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Table 64.3 Grading of skin-prick test by comparison to histaminepositive control (Dreborg et al. 1989).

Grade

Per cent of the area of the wheal induced by histamine reference (5.43 mmol/L)

– + ++ +++ ++++

Same size as negative 25 50 100 200

have been performed, but they should be replaced by methods based on parallel line bioassay or on medial slope (Dreborg et al. 1989). For intradermal tests, one of the most widely used grading systems derives from Norman et al. (1973) (Table 64.4). Although a single concentration of a specific extract may be selected for testing and the reaction graded for positivity, more information is obtained if a threshold dilution titration skin test is performed employing a threefold or tenfold dilution series. The grading of the reaction may be studied using the wheal and/or the erythema. The size of a wheal for a single allergen dose is not accurate, as identical reactions may be observed for tests performed with allergenic extracts whose potencies differ 100-fold. Several methods of evaluation have therefore been proposed. The lowest dilution required for 1+ or 2+ reactions is considered the end point. Other investigators have considered the end-point titer to be the dilution of extract that gives a wheal identical to the histamine-positive control. Norman et al. (1973) introduced the midpoint method by establishing a dose–response curve and determining the dose of allergen extract producing a wheal 7 mm in diameter. They found that the midpoint skin test was correlated with serum IgE levels, leukocyte histamine release, provocative challenges and symptoms (Bruce et al. 1974). Van-Metre et al. (1990) examined the effect of immunotherapy with ragweed extracts on skin sensitivity and found no effect using the end-point titration, whereas use of the midpoint one showed a significant decrease in skin sensitivity. Finally, Turkeltaub et al. (1982) used the erythema diameter, not the wheal diameter, because the slope of the regression line of the former is steeper. For routine diagnosis

Table 64.4 Grading of intradermal test (Norman et al. 1973). Grade

Erythema

Wheal

0 ± 1+ 2+ 3+ 4+

< 5 mm 5–10 mm 11–20 mm 21–30 mm 31–40 mm > 40 mm

< 5 mm 5–10 mm 5–10 mm 5–10 mm 10–15 mm or with pseudopods > 15 mm or with many pseudopods

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of allergy, a single allergen concentration is usually sufficient, but for research purposes more sophisticated techniques are required.

recombinant allergen panels covering the most important allergenic structures present in a given complex allergenic extract.

Number of skin tests and frequency of skin testing

Area of the body

Number of skin tests

The site of skin testing may affect the results. The back as a whole is more reactive than the forearm (Nelson et al. 1996). The mid and upper back are more reactive than the lower back. The antecubital fossa is the most reactive portion of the arm, whereas the wrist is the least reactive. The ulnar side of the arm is more reactive than the radial. It is recommended that tests not be placed in areas 5 cm from the wrist or 3 cm from the antecubital fossae (Bernstein & Storms 1995).

The number of skin tests to use varies according to the following. 1 Age of the patient: fewer prick-puncture tests need to be performed in infants, especially with food allergens, housedust mites, indoor molds, indoor insects, and animal danders as opposed to pollens (Bernstein & Storms 1995). 2 Geographic location and mobility of the patient. 3 History of the allergic disease (persistent versus intermittent symptoms, clear causative factors).

Frequency of skin testing Skin tests may be repeated for a variety of reasons (Bernstein & Storms 1995), including the following. 1 Age of the patient: allergic children acquire new sensitivities, beginning with foods and indoor allergens and then pollens and outdoor molds. 2 The patient’s exposure to new allergens (acquisition of a new pet, geographic relocation), particularly if their symptomatology has changed. 3 Immunotherapy treatment; however, except for venom immunotherapy, routine periodic skin-test titration is not recommended.

Factors affecting skin tests

Age, gender and race Skin reactions vary with age. Infants react predominantly with a large erythematous flare and a small wheal. Using prick-puncture tests it has been observed that a significant wheal was detectable after 3 months of age in most infants tested with either histamine, codeine phosphate, or allergen extracts (Menardo et al. 1985). Skin-test wheals increase in size from infancy to adulthood and then often decline after the age of 50 (Skassa-Brociek et al. 1987). There is no clearcut gender difference in skin-test reactivity and the variation of whealing reactivity during the menstrual cycle (weakest on the first day and around the 20th day, strongest at midcycle) has no clinical significance (Weinmann et al. 1987). The whealing reactivity to histamine is significantly greater in healthy nonatopic black subjects with darkly pigmented skin than it is in whites with light skin pigmentation (VanNiekerk & Prinsloo 1985). The flare is difficult to measure in patients with pigmented skin.

Allergenic extracts Skin reactions are dependent on a number of variables, among which the quality of the allergen extract is of major importance. Some false-negative reactions are due to the lack of allergens in some nonstandardized extracts. When possible, it is advisable to use allergen extracts standardized by biological methods and labeled in biological units (Bernstein & Storms 1995). The use of mixes of unrelated allergens is not recommended, because they may result in false-negative responses due to some overdiluted allergenic epitopes in some mixes (Bernstein & Storms 1995) or to enzyme degradation. Considering the difficulties in preparing consistently standardized extracts from natural raw material, new technologies have been tried. DNA technology has allowed production of pure biochemically characterized recombinant allergens of various pollens, molds, mites, latex, and various food allergens which have been used for skin testing in more than 1500 allergic and control individuals (Schmid-Grendelmeier & Crameri 2001). They have proved to be highly specific and safe; however, the diagnostic sensitivity of single recombinant allergens is generally lower than those obtained with allergen extracts, but can be considerably increased by using

Seasonal variations Seasonal variations related to specific IgE antibody synthesis have been demonstrated in pollen (Haahtela & Jokela 1980; Oppenheimer & Nelson 1993) and house-dust mite allergy (Nahm et al. 1997). Skin sensitivity, especially for tree pollen allergy (Sin et al. 2001), increases after the pollen season and then declines until the next season. This effect has some importance in patients with a low sensitivity and/or if allergen extracts are of weak potency. Ultraviolet B radiation significantly reduces wheal intensities (Vocks et al. 1999).

Pathologic conditions As a rule, it seems reasonable not to perform skin tests in areas where there is any skin lesion that might interfere with skin reactivity. Patients with chronic renal failure and/or using chronic hemodialysis usually have decreased skin reactivity and the texture of their skin makes testing difficult (Bousquet et al. 1988). Some, but not all, patients with cancer have decreased skin reactivity, which is more pronounced on the flare than on the wheal (Bousquet et al. 1991). Patients with spinal cord injuries (Rebhun & Botvin 1980) or peripheral

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nerve abnormalities such as a diabetic neuropathy show decreased flare reactivity.

Drugs Some drugs can affect the sensitivity of skin tests, modifying either the wheal or the flare response and making the interpretation of skin tests difficult (Table 64.5). H1 antihistamines (Cook et al. 1973; Harvey & Schocket 1980; Phillips et al. 1983; Long et al. 1985; Almind et al. 1988; Juniper et al. 1988; Simons et al. 1988, 1990; Watson et al. 1989; Bousquet et al. 1990a, 1996, 1998; Heykants et al. 1995; Nelson et al. 1995; Purohit et al. 2001; Pearlman et al. 2003) inhibit the wheal-and-flare reaction to histamine, allergen and mast cell secretagogues. The duration of the inhibitory effect appears to be linked to the pharmacokinetics of the drug and its metabolites. First generation H1 antihistamines reduce skin reactivity for up to 24 hours (Cook et al. 1973; Phillips et al. 1983; Almind et al. 1988; Simons et al. 1990). Azelastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, and mizolastine can block skin reaction for 3–10 days. Astemizole has an effect for up to 60 days (Long et al. 1985; Juniper et al. 1988). Although some antihistamines inhibit skin tests more than others (Simons et al. 1990; Purohit et al. 2001), skin-test reactivity to allergen or histamine is not predictive of the clinical efficacy of these drugs during seasonal allergic rhinitis (Bousquet et al. 1998; Persi et al. 1999). Topical H1 antihistamines such as levocabastine (Heykants et al. 1995) and azelastine (Pearlman et al. 2003) may reduce responsiveness to skin tests (Heykants et al. 1995). H2 antihistamines used alone have a limited inhibitory activity on skin tests (Harvey & Schocket 1980; Meyrick-Thomas et al. 1985). Ketotifen suppresses skin-test responses for a period of over 5 days (Phillips et al. 1983; Esau et al. 1984). Tricyclic antidepressants exert a significant and sustained decrease of skin reactions to histamine (Sullivan 1982). This effect may last for a few weeks. Tranquilizers and antiemetic agents of the phenothiazine class have H1 antihistamine activity and can reduce skin test reactivity (Wolfe & Fontana 1964). Topical doxepin hydrochloride has been shown to block skin reactivity after 1–3 days of therapy and up to 11 days after cessation of the latter (Karaz et al. 1995). Short-term (less than 1 week) administration of corticosteroids at the therapeutic doses used in asthmatic patients does not modify cutaneous reactivity to histamine, compound 48/80, or allergens (Slott & Zweiman 1974). Long-term corticosteroid therapy does not alter histamine-induced vascular reactivity in skin, but affects skin mast cell responses (Olson et al. 1990) and modifies skin texture, thereby making the interpretation of immediate skin tests difficult in some cases. However, it has been shown that allergen-induced skin tests can be accurately carried out in asthmatic patients receiving long-term oral corticosteroid treatment (Des-Roches et al. 1996). Inhaled corticosteroids have not been tested, but

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because therapeutic doses produce fewer systemic effects than oral corticosteroids, it may be predicted that these drugs will not modify skin tests. Application of topical dermal corticosteroids for a period of 1 week reduces both the immediate- and the late-phase skin reaction induced by allergens (Andersson & Pipkorn 1987; Pipkorn et al. 1989). Theophylline slightly reduces skin-test reactivity (Chipps et al. 1980; Fine et al. 1980), but it does not need to be stopped prior to skin testing. Short-acting inhaled β2 agonists at doses used for the treatment of asthma do not usually reduce allergen-induced skin test reactivity (Imbeau et al. 1978; Spector 1978). For long-acting inhaled β2 agonists such as formoterol (Gronneberg & Zetterstrom 1992) and salmeterol definitive results are lacking. Inhaled cromones do not alter the skin whealing response to skin tests with allergens or degranulating agents (Ting et al. 1983). Other drugs such as dopamine (Casale et al. 1984) and clonidine (Miadonna et al. 1989) have been shown to decrease skin test reactivity. Nifedipine (Fernandez-Rivas et al. 1990) and montelukast (Simons et al. 2001) have no effect on skin reactivity. Angiotensin-converting enzyme (ACE) inhibitors moderately increase skin reactivity to allergen, histamine, codeine, and bradykinin (Anderson & deShazo 1990). Topical pimecrolimus does not seem to modify skin reactivity (Weissenbacher et al. 2006). A decreased wheal-and-flare reaction has been noticed in patients undergoing specific immunotherapy (Bousquet & Michel 1995). However, these effects were seen mostly when skin tests were carried out using several dilutions.

Interpretation of skin tests False-positive and false-negative skin tests False-positive skin tests may be provoked by impurities, contaminants, and nonspecific mast cell secretagogs in the extract, as well as by dermographism, respiratory syncytial virus infection, and hyperresponsiveness due to a nearby strong reaction (Voorhorst 1980; Terho et al. 1987; Skoner et al. 2006). False-negative skin tests can be caused by (i) extracts of poor initial potency or subsequent loss of potency (Dreborg et al. 1989); (ii) drugs that modify the allergic reaction; (iii) diseases attenuating the skin response; (iv) decreased reactivity of the skin in infants and elderly patients; (v) improper technique (no or weak puncture); (vi) ultraviolet exposure; (vii) too short/too long time interval from reaction; (viii) organ allergy; and (ix) non-IgE-mediated mechanism. The use of positive control solutions may overcome some of the false-negative results.

Positive skin tests in a population without clinical allergy The occurrence of positive skin tests does not necessarily imply that the patient is allergic, because many studies have shown

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Table 64.5 Inhibitory effect of various treatments on IgE-mediated skin tests. Drug H1 antihistamines Astemizole (Almind et al. 1988; Nelson et al. 1995) Azelastine (Almind et al. 1988) Cetirizine (Almind et al. 1988; Watson et al. 1989) Chlorphenamine (Phillips et al. 1983; Long et al. 1985) Clemastine (Phillips et al. 1983) Cyproheptadine (Almind et al. 1988; Long et al. 1985) Desloratadine Diphenhydramine (Long et al. 1985) Doxepin (Karaz et al. 1995) Ebastine (Nelson et al. 1995) Hydroxyzine (Long et al. 1985) Levocabastine (Heykants et al. 1995) Levocetirizine Loratadine (Almind et al. 1988) Mequitazine Mizolastine (Bousquet et al. 1996) Promethazine (Long et al. 1985) Terfenadine (Almind et al. 1988) Tripelennamine (Long et al. 1985) H2 antihistamines Cimetidine (Harvey & Schocket 1980) Ranitidine (Meyrick-Thomas et al. 1985) Ketotifen (Phillips et al. 1983; Esau et al. 1984) Imipramines (Sullivan 1982) Phenothiazines (Wolfe & Fontana 1964) Corticosteroids Systemic, short term (Slott & Zweiman 1974) Systemic, long term (Olson et al. 1990; Des-Roches et al. 1996) Inhaled Topical skin (Andersson & Pipkorn 1987; Pipkorn et al. 1989) Theophylline (Fine et al. 1980; Chipps et al. 1980) Cromolyn (Ting et al. 1983) b2 Agonists Inhaled (Imbeau et al. 1978; Spector 1978; Chipps et al. 1980; Gronneberg & Zetterstrom 1992) Oral, injection (Imbeau et al. 1978; Chipps et al. 1980) Formoterol (Gronneberg & Zetterstrom 1992) Salmeterol Dopamine (Casale et al. 1984) Clonidine (Miadonna et al. 1989) Montelukast (Simons et al. 2001) Specific immunotherapy (Bousquet & Michel 1995)

Degree

Duration

Clinical significance*

++++ ++++ ++++ ++ +++ 0 to + ++++ 0 to + ++ ++++ +++ Possible ++++ ++++ ++++ ++++ ++ ++++ 0 to +

30–60 days 3–10 days 3–10 days 1–3 days 1–10 days 1–8 days 3–10 days 1–3 days 3–11 days 3–10 days 1–10 days

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

0 to + + ++++ ++++ ++ 0 Possible 0 0 to ++ 0 to + 0

3–10 days 3–10 days 3–10 days 3–10 days 1–3 days 3–10 days 1–3 days

> 5 days > 10 days

No No Yes Yes Yes

Yes Yes No

0 to +

No

0 to ++ Unknown Unknown + ++ 0 0 to ++

No

No

* Clinical significance for skin testing.

that skin tests can be positive to common aeroallergens in 8– 30% of asymptomatic subjects. In some cases, the presence of irritants or nonspecific mast cell secretagogues may explain positive responses (Roane et al. 1968). In most other cases, positive skin tests probably detect the presence of specific IgE antibodies to environmental allergens, although their presence

may not always coincide with clinically significant allergic disease. However, the presence of positive skin tests in asymptomatic subjects may predict the onset of allergic symptoms (Hagy & Settipane 1971; Horak 1985). The optimal cutoff values for prick test responses to five common aeroallergens, in order to distinguish patients with symptomatic sensitivity

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from those without, were a wheal area of, respectively, 32.4 mm2 for seasonal intermittent allergens and 31.2 mm2 for Dermatophagoides pteronyssinus (Pastorello et al. 1995).

Correlation with other diagnostic tests used in allergy diagnosis Comparisons between skin tests and serum specific IgE assays depend on the quality and standardization of allergens used in both tests (Dreborg et al. 1989). Using standardized extracts, the concordance between specific IgE assays and skin prickpuncture tests ranges between 85 and 95% depending on the allergens (Bousquet et al. 1990b; Ewan & Coote 1990; Crobach et al. 1998; Wood et al. 1999). However, skin tests appear to be more sensitive but less specific than IgE assays (van-der-Zee et al. 1988). When there is a suggestive history and strongly positive skin tests, the correlation between skin tests and bronchial or nasal challenges is highly significant (European Academy of Allergology and Clinical Immunology 1993). Poor correlations are often observed with unstandardized allergenic extracts or when there is a discrepancy between histories and skin tests.

Diagnostic value of skin tests The position papers on skin tests by the European Academy of Allergology and Clinical Immunology (1993) and the US Joint Council of Allergy Asthma and Immunology (Bernstein & Storms 1995) agree that, when properly performed, prickpuncture tests are generally considered to be the most convenient and least expensive screening method for detecting respiratory allergic patients. Positive skin tests with a history suggestive of clinical sensitivity strongly incriminate the allergen as a cause of the disease. As an example, Crobach et al. (1998) have demonstrated (with reference to advice by experts) that the predictive value of the clinical history alone for the diagnosis of allergic rhinitis was 82– 85% for intermittent seasonal allergens (and at least 77% for persistent allergens) and this increased to 97–99% when skin-prick tests (or specific IgE assays) were performed. Conversely, a negative skin test with a negative history indicates a nonallergic disorder. Interpretation of skin tests that do not correlate with the clinical history is more difficult and, in this case, specific IgE assays and provocative challenges are of interest. As an example, Wood et al. (1999) have demonstrated that all patients with negative skin-prick tests to cat allergens had negative radioallergosorbent tests and that, with reference to a cat exposure model, the negative predictive values of both tests were almost identical (72–75%) for the diagnosis of cat allergy. For occupational sensitizers, skin tests are often unreliable, except in cases of high-molecular-weight compounds such as natural rubber latex (Sicard et al. 1996; Blanco et al. 1999), enzymes (Sarlo et al. 1990), and flour. In one study (Blanco et al. 1999), the diagnostic sensitivity of different latex extracts ranged from 90 to 98% and the specificity was 100%. In

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bronchopulmonary aspergillosis, skin tests (mainly intradermal) with Aspergillus fumigatus antigen represent one of the diagnostic tools and produce both immediate and late-phase reactions. However, some patients with Aspergillus-induced asthma or aspergilloma may also have such biphasic skin response, which in such cases is not pathognomonic of bronchopulmonary aspergillosis. Recombinant allergens can be used (Moser et al. 1994).

Conclusions Skin tests provide useful confirmatory evidence for a diagnosis of specific allergy that has been made on clinical grounds. Their characteristics (simplicity, rapidity of performance, low cost, and high sensitivity) explain the key position in respiratory allergy diagnosis. However, when improperly performed, skin tests can lead to falsely positive or negative results. The main limitation of skin tests is that positive reactions do not necessarily mean that patients will experience symptoms, as symptom-free subjects may have allergen-specific IgE. Therefore, adequate interpretation requires trained investigators who will consider all factors that might modify the results and provide sufficient information for the patient.

References Adinoff, A.D., Rosloniec, D.M., McCall, L.L. & Nelson, H.S. (1990) Immediate skin test reactivity to Food and Drug Administrationapproved standardized extracts. J Allergy Clin Immunol 86, 766–74. Agarwal, K. & Zetterstrom, O. (1982) Diagnostic significance of late cutaneous allergic responses and their correlation with radioallergosorbent test. Clin Allergy 12, 489–97. Almind, M., Dirksen, A., Nielsen, N.H. & Svendsen, U.G. (1988) Duration of the inhibitory activity on histamine-induced skin weals of sedative and non-sedative antihistamines. Allergy 43, 593–6. American Academy of Allergy and Immunology (1990) The waiting period after allergen skin testing and immunotherapy. J Allergy Clin Immunol 85, 526–7. Anderson, M.W. & deShazo, R.D. (1990) Studies of the mechanism of angiotensin-converting enzyme (ACE) inhibitor-associated angioedema: the effect of an ACE inhibitor on cutaneous responses to bradykinin, codeine, and histamine. J Allergy Clin Immunol 85, 856– 8. Andersson, M. & Pipkorn, U. (1987) Inhibition of the dermal immediate allergic reaction through prolonged treatment with topical glucocorticosteroids. J Allergy Clin Immunol 79, 345–9. Bernstein, I.L. & Storms, W.W. (1995) Practice parameters for allergy diagnostic testing. Joint Task Force on Practice Parameters for the Diagnosis and Treatment of Asthma. The American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology. Ann Allergy Asthma Immunol 75, 543–625. Blackley, C. (1880) Hay Fever: Its Causes, Treatment and Effective Prevention; Experimental Researches, 2nd edn. Bailliere, Tindal & Cox, London.

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Blanco, C., Carrillo, T., Ortega, N., Alvarez, M., Dominguez, C. & Castillo, R. (1999) Comparison of skin-prick test and specific IgE determination for the diagnosis of latex allergy. Clin Exp Allergy 29, 133– 4. Bousquet, J. & Michel, F. (1995) Specific immunotherapy in allergic rhinitis and asthma. In: Busse, W. & Holgate, S., eds. Asthma and Rhinitis. Blackwell Scientific Publications, Oxford, pp. 1309–24. Bousquet, J., Djoukadar, F., Hewitt, B. et al. (1985a) Comparison of the stability of a mite and a pollen extract stored in normal conditions of use. Clin Allergy 15, 29–35. Bousquet, J., Calvayrac, P., Guerin, B. et al. (1985b) Immunotherapy with a standardized Dermatophagoides pteronyssinus extract. I. In vivo and in vitro parameters after a short course of treatment. J Allergy Clin Immunol 76, 734– 44. Bousquet, J., Maurice, F., Rivory, J.P. et al. (1988) Allergy in longterm hemodialysis. II. Allergic and atopic patterns of a population of patients undergoing long-term hemodialysis. J Allergy Clin Immunol 81, 605–10. Bousquet, J., Chanal, I., Skassa-Brociek, W. et al. (1990a) Lack of subsensitivity to loratadine during long-term dosing during 12 weeks. J Allergy Clin Immunol 86, 248–53. Bousquet, J., Chanez, P., Chanal, I. & Michel, F.B. (1990b) Comparison between RAST and Pharmacia CAP system: a new automated specific IgE assay. J Allergy Clin Immunol 85, 1039–43. Bousquet, J., Pujol, J.L., Barneon, G. et al. (1991) Skin test reactivity in patients suffering from lung and breast cancer. J Allergy Clin Immunol 87, 1066–72. Bousquet, J., Chanal, I., Murrieta, M. & Stalla-Bourdillon, A. (1996) Lack of subsensitivity to mizolastine over 8-week treatment. Allergy 51, 251– 6. Bousquet, J., Czarlewski, W., Cougnard, J., Danzig, M. & Michel, F.B. (1998) Changes in skin-test reactivity do not correlate with clinical efficacy of H1-blockers in seasonal allergic rhinitis. Allergy 53, 579– 85. Brown, H.M., Su, S. & Thantrey, N. (1981) Prick testing for allergens standardized by using a precision needle. Clin Allergy 11, 95–8. Bruce, C., Rosenthal, R., Lichtenstein, L. & Norman, P. (1974) Diagnostic tests in ragweed-allergic asthma. A comparison of direct skin tests, leukocyte histamine release, and quantitative bronchial challenge. J Allergy Clin Immunol 53, 320– 8. Carr, W.W., Martin, B., Howard, R.S., Cox, L. & Borish, L. (2005) Immunotherapy Committee of the American Academy of Allergy, Asthma and Immunology. Comparison of test devices for skin prick testing. J Allergy Clin Immunol 116, 341–6. Casale, T.B., Shelhamer, J.H., Parrillo, J.E. & Kaliner, M.A. (1984) Dopamine inhibition of histamine-mediated cutaneous responses. J Allergy Clin Immunol 73, 837– 41. Chanal, I., Horst, M., Segalen, C. et al. (1988) Comparison between modified skin prick test with standardized allergen extracts and Phazet. J Allergy Clin Immunol 82, 878– 81. Chipps, B.E., Sobotka, A.K., Sanders, J.P. et al. (1980) Effect of theophylline and terbutaline on immediate skin tests. J Allergy Clin Immunol 65, 61– 4. Codreanu, F., Moneret-Vautrin, D.A., Morisset, M. et al. (2006) The risk of systemic reactions to skin prick-tests using food allergens: CICBAA data and literature review. Allerg Immunol (Paris) 38, 52–4. Co-Minh, H.B., Bousquet, P.J., Fontaine, C., Kvedariene, V. & Demoly, P. (2006) Systemic reactions during skin tests with βlactams: a risk factor analysis. J Allergy Clin Immunol 117, 466–8.

Cook, T., McQueen, D., Wittig, H. et al. (1973) Degree and duration of skin test suppression and side effects with antihistamines. A double blind controlled study with five antihistamines. J Allergy Clin Immunol 51, 71–6. Corder, W.T., Hogan, M.B. & Wilson, N.W. (1996) Comparison of two disposable plastic skin test devices with the bifurcated needle for epicutaneous allergy testing. Ann Allergy Asthma Immunol 77, 222–6. Crobach, M.J.J.S., Hermans, J.O., Kaptein, A.A., Ridderikhoff, J., Petri, H. & Mulder, J.D. (1998) The diagnosis of allergic rhinitis: how to combine the medical history with the results of radioallergosorbent tests and skin prick tests. Scan J Prim Health Care 16, 30–6. Demoly, P., Bousquet, J., Manderscheid, J.C. et al. (1991) Precision of skin prick and puncture tests with nine methods. J Allergy Clin Immunol 88, 758–62. Des-Roches, A., Paradis, L., Bougeard, Y. et al. (1996) Long-term oral corticotherapy does not alter the results of immediate type allergy skin prick tests. J Allergy Clin Immunol 98, 522–7. Devenney, I. & Falth-Magnusson, K. (2000) Skin prick tests may give generalized allergic reactions in infants. Ann Allergy Asthma Immunol 85, 429–30. Dreborg, S. (1988) Food allergy in pollen-sensitive patients. Ann Allergy 61, 41–6. Dreborg, S., Backman, A., Basomba, A. et al. (1989) Skin tests used in type I allergy testing. Position paper of the European Academy of Allergology and Clinical Immunology. Allergy 44 (suppl. 10), 1–69. Esau, S., del-Carpio, J. & Martin, J.G. (1984) A comparison of the effects of ketotifen and clemastine on cutaneous and airway reactivity to histamine and allergen in atopic asthmatic subjects. J Allergy Clin Immunol 74, 270–4. European Academy of Allergology and Clinical Immunology (1993) Position paper. Allergen standardization and skin tests. Allergy 48, 48– 82. Ewan, P.W. & Coote, D. (1990) Evaluation of a capsulated hydrophilic carrier polymer (the ImmunoCAP) for measurement of specific IgE antibodies. Allergy 45, 22–9. Fernandez-Rivas, M., Puyana, J., Quirce, S. et al. (1990) Effect of nifedipine on skin prick tests. Allergol Immunopathol (Madr) 18, 79– 82. Fine, S.R., Fogarty, M., Goel, Z. & Grieco, M.H. (1980) Correlation of serum theophylline levels with inhibition of allergen and histamine-induced skin tests. Int Arch Allergy Appl Immunol 61, 241–4. Gronneberg, R. & Zetterstrom, O. (1992) Inhibitory effects of formoterol and terbutaline on the development of late phase skin reactions. Clin Exp Allergy 22, 257–63. Haahtela, T. & Jokela, H. (1980) Influence of the pollen season on immediate skin test reactivity to common allergens. Allergy 35, 15–21. Hagy, G. & Settipane, G. (1971) Prognosis of positive allergy skin tests in an asymptomatic population. A three year follow-up of college students. J Allergy 48, 200–5. Harvey, R.P. & Schocket, A.L. (1980) The effect of H1 and H2 blockade on cutaneous histamine response in man. J Allergy Clin Immunol 65, 136–9. Heykants, J., Van-Peer, A., Van-de-Velde, V. et al. (1995) The pharmacokinetic properties of topical levocabastine. A review. Clin Pharmacokinet 29, 221–30.

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Horak, F. (1985) Manifestation of allergic rhinitis in latent-sensitized patients. A prospective study. Arch Otorhinolaryngol 242, 239–45. Imbeau, S.A., Harruff, R., Hirscher, M. & Reed, C.E. (1978) Terbutaline’s effects on the allergy skin test. J Allergy Clin Immunol 62, 193–6. Juniper, E.F., White, J. & Dolovich, J. (1988) Efficacy of continuous treatment with astemizole (Hismanal) and terfenadine (Seldane) in ragweed pollen-induced rhinoconjunctivitis. J Allergy Clin Immunol 82, 670–5. Karaz, S.S., Moeckli, J.K., Davis, W. & Craig, T.J. (1995) Effect of topical doxepin cream on skin testing. J Allergy Clin Immunol 96, 997–8. Kupczyk, M., Kuprys, I., Gorski, P. & Kuna, P. (2001) Not one lancet for multiple SPT. Allergy 56, 256–7. Lander, J., Weltman, B. & So, S. (2006) EMLA and Amethocaine for reduction of children’s pain associated with needle insertion. Cochrane Database Syst Rev 3, CD004236. Lewis, T. & Grant, R. (1924) Vascular reactions of the skin to injury. Part II. The liberation of a histamine-like substance in injured skin, the underlying cause of factitious urticaria and of wheals produced by burning; and observations upon the nervous control of certain skin reactions. Heart 209, 1924. Lieberman, P. (2006) Anaphylaxis. Med Clin North Am 90, 77–95. Lin, M.S., Tanner, E., Lynn, J. & Friday, G. Jr. (1993) Nonfatal systemic allergic reactions induced by skin testing and immunotherapy. Ann Allergy 71, 557– 62. Lockey, R.F., Benedict, L.M., Turkeltaub, P.C. & Bukantz, S.C. (1987) Fatalities from immunotherapy (IT) and skin testing (ST). J Allergy Clin Immunol 79, 660–77. Lockey, R.F., Turkeltaub, P.C., Olive, C.A. et al. (1989) The Hymenoptera venom study. II: Skin test results and safety of venom skin testing. J Allergy Clin Immunol 84, 967–74. Long, W.F., Taylor, R.J., Wagner, C.J. et al. (1985) Skin test suppression by antihistamines and the development of subsensitivity. J Allergy Clin Immunol 76, 113–17. Lutz, C.T., Bell, C. Jr, Wedner, H.J. & Krogstad, D.J. (1984) Allergy testing of multiple patients should no longer be performed with a common syringe. N Engl J Med 310, 1335–37. Malling, H.J. (1985) Reproducibility of skin sensitivity using a quantitative skin prick test. Allergy 40, 400– 4. Mantoux, C. (1908) Intradermoréaction de la tuberculose. CR Acad Sci 147, 355. Menardo, J.L., Bousquet, J., Rodiere, M. et al. (1985) Skin test reactivity in infancy. J Allergy Clin Immunol 75, 646–51. Meyrick-Thomas, R.H., Browne, P.D. & Kirby, J.D. (1985) The effect of ranitidine, alone and in combination with clemastine, on allergen-induced cutaneous wheal-and-flare reactions in human skin. J Allergy Clin Immunol 76, 864–9. Miadonna, A., Tedeschi, A., Leggieri, E. et al. (1989) Clonidine inhibits IgE-mediated and IgE-independent in vitro histamine release from human basophil leukocytes. Int J Immunopharmacol 11, 473–7. Moser, M., Crameri, R., Brust, E., Suter, M. & Menz, G. (1994) Diagnostic value of recombinant Aspergillus fumigatus allergen I/a for skin testing and serology. J Allergy Clin Immunol 93, 1–11. Nahm, D.H., Park, H.S., Kang, S.S. & Hong, C.S. (1997) Seasonal variation of skin reactivity and specific IgE antibody to house dust mite. Ann Allergy Asthma Immunol 78, 589– 93. Nelson, H.S., Bucher, B., Buchmeier, A. et al. (1995) Suppression of the skin reaction to histamine by ebastine. Ann Allergy Asthma Immunol 74, 442–7.

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Nelson, H.S., Knoetzer, J. & Bucher, B. (1996) Effect of distance between sites and region of the body on results of skin prick tests. J Allergy Clin Immunol 97, 596–601. Norman, P., Lichtenstein, L. & Ishizaka, K. (1973) Diagnostic tests in ragweed hay fever. A comparison of direct skin tests, IgE antibody measurements, and basophil histamine release. J Allergy Clin Immunol 52, 210–24. Novembre, E., Bernardini, R., Bertini, G., Massai, G. & Vierucci, A. (1995) Skin-prick-test-induced anaphylaxis. Allergy 50, 511–3. Occupation Safety and Health Administration (1995) Hazard Information Bulletin. Occupation Safety and Health Administration, Washington, DC, September 21. Olson, R., Karpink, M.H., Shelanski, S. et al. (1990) Skin reactivity to codeine and histamine during prolonged corticosteroid therapy. J Allergy Clin Immunol 86, 153–9. Oppenheimer, J.J. & Nelson, H.S. (1993) Seasonal variation in immediate skin test reactions. Ann Allergy 71, 227–9. Osterballe, O. & Weeke, B. (1979) A new lancet for skin prick testing. Allergy 34, 209–12. Ownby, D.R. & Anderson, J.A. (1982) An improved prick skin-test procedure for young children. J Allergy Clin Immunol 69, 533–5. Pastorello, E.A., Incorvaia, C., Ortolani, C. et al. (1995) Studies on the relationship between the level of specific IgE antibodies and the clinical expression of allergy: I. Definition of levels distinguishing patients with symptomatic from patients with asymptomatic allergy to common aeroallergens. J Allergy Clin Immunol 96, 580–7. Pearlman, D.S., Grossman, J. & Meltzer, E.O. (2003) Histamine skin test reactivity following single and multiple doses of azelastine nasal spray in patients with seasonal allergic rhinitis. Ann Allergy Asthma Immunol 91, 258–62. Pepys, J. (1975) Skin testing. Br J Hosp Med 14, 412–25. Persi, L., Demoly, P., Harris, A.G., Tisserand, B., Michel, F.B. & Bousquet, J. (1999) Comparison between nasal provocation tests and skin tests in patients treated with loratadine and cetirizine. J Allergy Clin Immunol 103, 591–4. Phillips, M.J., Meyrick-Thomas, R.H., Moodley, I. & Davies, R.J. (1983) A comparison of the in vivo effects of ketotifen, clemastine, chlorpheniramine and sodium cromoglycate on histamine and allergen induced weals in human skin. Br J Clin Pharmacol 15, 277–86. Piette, V., Bourret, E., Bousquet, J. & Demoly, P. (2002) Prick tests to aeroallergens: is it possible to simply wipe the device between tests? Allergy 57, 940–2. Pipkorn, U., Hammarlund, A. & Enerback, L. (1989) Prolonged treatment with topical glucocorticoids results in an inhibition of the allergen-induced wheal-and-flare response and a reduction in skin mast cell numbers and histamine content. Clin Exp Allergy 19, 19–25. Poulsen, L.K., Liisberg, C., Bindslev-Jensen, C. & Malling, H.J. (1993) Precise area determination of skin-prick tests: validation of a scanning device and software for a personal computer. Clin Exp Allergy 23, 61–8. Purohit, A., Duvernelle, C., Melac, M., Pauli, G. & Frossard, N. (2001) Twenty-four hours of activity of cetirizine and fexofenadine in the skin. Ann Allergy Asthma Immunol 86, 387–92. Rebhun, J. & Botvin, J. (1980) Histamine flare, a neurovascular response. Ann Allergy 45, 59–62. Reid, M.J., Lockey, R.F., Turkeltaub, P.C. & Platts-Mills, T.A. (1993) Survey of fatalities from skin testing and immunotherapy 1985– 1989. J Allergy Clin Immunol 92, 6–15.

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Skin Testing in Diagnosis and Management of Respiratory Allergic Diseases

Roane, J., Crawford, L., Triplett, F. & Brasher, G. (1968) Intradermal tests in nonatopic children. Ann Allergy 26, 443. Santilli, J. Jr, Potsus, R.L., Goodfriend, L. & Marsh, D.G. (1980) Skin reactivity to purified pollen allergens in highly ragweed-sensitive individuals. J Allergy Clin Immunol 65, 406–12. Sarlo, K., Clark, E.D., Ryan, C.A. & Bernstein, D.I. (1990) ELISA for human IgE antibody to subtilisin A (Alcalase): correlation with RAST and skin test results with occupationally exposed individuals. J Allergy Clin Immunol 86, 393– 9. Schmid-Grendelmeier, P. & Crameri, R. (2001) Recombinant allergens for skin testing. Int Arch Allergy Immunol 125, 96–111. Serup, J. (1984) Diameter, thickness, area, and volume of skin-prick histamine weals. Measurement of skin thickness by 15 MHz Amode ultrasound. Allergy 39, 359– 64. Serup, J. & Staberg, B. (1985) Quantification of weal reactions with laser Doppler flowmetry. Comparative blood flow measurements of the oedematous centre and the perilesional flare of skin-prick histamine weals. Allergy 40, 233–7. Shulan, D.J., Weiler, J.M., Koontz, F. & Richerson, H.B. (1985) Contamination of intradermal skin test syringes. J Allergy Clin Immunol 76, 226–7. Sicard, H., Turjanmaa, K., Palosuo, T. et al. (1996) Latex allergy diagnosis: standardization of a natural rubber latex extract. J Allergy Clin Immunol 97, 323. Sicherer, S.H. & Eggleston, P.A. (1997) EMLA cream for pain reduction in diagnostic allergy skin testing: effects on wheal and flare responses. Ann Allergy Asthma Immunol 78, 64–8. Simons, F.E., Watson, W.T. & Simons, K.J. (1988) Lack of subsensitivity to terfenadine during long-term terfenadine treatment. J Allergy Clin Immunol 82, 1068–75. Simons, F.E., McMillan, J.L. & Simons, K.J. (1990) A double-blind, single-dose, crossover comparison of cetirizine, terfenadine, loratadine, astemizole, and chlorpheniramine versus placebo: suppressive effects on histamine-induced wheals and flares during 24 hours in normal subjects. J Allergy Clin Immunol 86, 540–7. Simons, F.E.R., Johnston, L., Gu, X. & Simons, K.J. (2001) Suppression of the early and late cutaneous allergic responses using fexofenadine and montelukast. Ann Allergy Asthma Immunol 86, 44–50. Sin, B.A., Inceoglu, O., Mungan, D., Celik, G., Kaplan, A. & Misirligil, Z. (2001) Is it important to perform pollen skin tests in the season? Ann Allergy Asthma Immunol 86, 382–6. Skassa-Brociek, W., Manderscheid, J.C., Michel, F.B. & Bousquet, J. (1987) Skin test reactivity to histamine from infancy to old age. J Allergy Clin Immunol 80, 711–16. Skoner, D.P., Gentile, D.A., Angelini, B. & Doyle, W.J. (2006) Allergy skin test responses during experimental infection with respiratory syncytial virus. Ann Allergy Asthma Immunol 96, 834–9. Slott, R. & Zweiman, B. (1974) A controlled study of the effect of corticosteroids on immediate skin test reactivity. J Allergy Clin Immunol 54, 229–34. Spector, S.L. (1978) Effect of beta-adrenergic agents on skin test responses and bronchial challenge responses. Chest 73, 976–7.

Sullivan, T.J. (1982) Pharmacologic modulation of the whealing response to histamine in human skin: identification of doxepin as a potent in vivo inhibitor. J Allergy Clin Immunol 69, 260–7. Sussman, G.L., Harvey, R.P., Schock, A.L. (1982) Evaluation of skin test response using two techniques of measurement. Ann Allergy 48, 75–7. Terho, E.O., Husman, K., Vohlonen, I. & Heinonen, O.P. (1987) Atopy, smoking, and chronic bronchitis. J Epidemiol Community Health 41, 300–5. Ting, S., Zweiman, B. & Lavker, R.M. (1983) Cromolyn does not modulate human allergic skin reactions in vivo. J Allergy Clin Immunol 71, 12–17. Turkeltaub, P.C., Rastogi, S.C., Baer, H. et al. (1982) A standardized quantitative skin-test assay of allergen potency and stability: studies on the allergen dose-response curve and effect of wheal, erythema, and patient selection on assay results. J Allergy Clin Immunol 70, 343–52. van-der-Zee, J.S., de-Groot, H., van-Swieten, P. et al. (1988) Discrepancies between the skin test and IgE antibody assays: study of histamine release, complement activation in vitro, and occurrence of allergen-specific IgG. J Allergy Clin Immunol 82, 270–81. Van-Metre Te, J., Adkinson, N. Jr, Kagey-Sobotka, A. et al. (1990) Immunotherapy decreases skin sensitivity to ragweed extract: demonstration by midpoint skin test titration. J Allergy Clin Immunol 86, 587–8. Van-Niekerk, C.H. & Prinsloo, A.E. (1985) Effect of skin pigmentation on the response to intradermal histamine. Int Arch Allergy Appl Immunol 76, 73–5. Vocks, E., Stander, K., Rakoski, J. & Ring, J. (1999) Suppression of immediate-type hypersensitivity elicitation in the skin prick test by ultraviolet B irradiation. Photodermatol Photoimmunol Photomed 15, 236–40. Voorhorst, R. (1980) Perfection of skin testing technique. A review. Allergy 35, 247–50. Watson, W.T., Simons, K.J., Chen, X.Y. & Simons, F.E. (1989) Cetirizine: a pharmacokinetic and pharmacodynamic evaluation in children with seasonal allergic rhinitis. J Allergy Clin Immunol 84, 457– 64. Weinmann, G.G., Zacur, H. & Fish, J.E. (1987) Absence of changes in airway responsiveness during the menstrual cycle. J Allergy Clin Immunol 79, 634–8. Weissenbacher, S., Traidl-Hoffmann, C., Eyerich, K. et al. (2006) Modulation of atopy patch test and skin prick test by pretreatment with 1% pimecrolimus cream. Int Arch Allergy Immunol. 140, 239– 44. Wolfe, H.I. & Fontana, V.J. (1964) The effect of tranquilizers on the immediate skin wheal reaction. J Allergy 35, 271. Wood, R.A., Phipatanakul, W., Hamilton, R.G. & Eggleston, P.A. (1999) A comparison of skin prick tests, intradermal skin tests, and RASTs in the diagnosis of cat allergy. J Allergy Clin Immunol 103, 773–9.

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Allergy Testing in the Laboratory Steven O. Stapel and Jörg Kleine-Tebbe

Summary Diagnostic allergy testing in the laboratory is based on various methods: detection of total IgE, allergen-specific IgE (RAST-type assays), allergen-specific IgG (in particular cases) in patient serum, and allergen-induced IgE-mediated basophil stimulation. Currently applied in vitro assays for allergen-specific IgE in serum generally give similar (but not always identical) results, if values are obtained from interpolation to a World Health Organization (WHO) standard for total IgE. The recently launched multiallergen (“chip”-like tests) may give different results, due to varying assay designs (e.g., incubation schedules for serum IgE and detecting anti-IgE) and the IgE-binding properties of the solid phase used in the assay. Inhibition of IgE binding by heterologous allergens has proven to be a valuable tool for the elucidation of IgE cross-reactivity, for instance between insect venoms and among foods, but also between food components and inhalant allergens. The clinical relevance of frequently observed IgE cross-reactivities varies and should be considered on an individual basis. Testing for IgG4 against foods has no diagnostic value and is therefore not recommended. Increased production of IgG4 to inhalant allergens, induced by immunotherapy, and to insect venom allergens may indicate immunologic tolerance, induced by regulatory T cells. Basophil-based ex vivo techniques, demonstrating the presence of allergen-specific cell-bound IgE indirectly, either by the release of mediators or by upregulation of activation markers (CD63, CD203c), are analytically extremely sensitive, even in case of low (total) serum IgE. Due to their complexity in performance and interpretation, basophil tests present complementary diagnostic tools for particular allergic sensitizations in selected cases. Positive test results, generated either by direct IgE detection with RAST techniques or indirectly with cellular basophil-based methods, indicate allergic sensitizations, which are only clinically relevant in the context of corresponding symptoms.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Introduction In a time when the various mechanisms involved in allergic disease are gradually becoming clear, we are also witnessing an outbreak of allergies in the western industrialized world. This is most probably the result of a changing lifestyle that no longer challenges the immune system to abandon the Th2 pathway, the default setting of the system after birth, which favors allergic status. It remained unclear for a long time which factors are involved in the etiology of these illnesses, and it took until the early 1920s before Prausnitz and Küstner showed, by their serum transfer experiments, that a serum component (“reagin”) was responsible. The fact that this reagin is, under normal circumstances, present in only minute amounts in plasma hampered its identification for decades, but eventually Ishizaka and Ishizaka (1967) and Johansson and Bennich (1967) were able to perform this task. They identified this component as an immunoglobulin, and designated it IgE. Their achievement was a major breakthrough for the study of IgE-mediated disease, and has also prompted the design of sensitive in vitro allergy tests for the detection and quantification of total IgE (present in plasma at a concentration around 100 000-fold less than IgG), and allergen-specific IgE in patient serum. Diagnosis in allergy can be performed in relation to the different aspects of the allergic reaction shown in Fig. 65.1. This includes testing for the presence of allergens, for allergeninducing T-cell activity, for the presence of allergen-specific IgE in serum, for IgE-mediated mast cell and basophil degranulation and for the presence of their mediators (histamine, leukotrienes) in serum or urine, and by provocation with allergen. This chapter only deals with procedures that are performed in laboratory settings, i.e., in vitro assays (measurement of total and allergen-specific IgE/allergen-specific IgG in serum or plasma) and ex vivo assays (detection of activation markers and release of mediators from basophils). Test indications and limitations, technical aspects, and interpretation of results are also discussed.

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Allergen detection

Allergen

Indoor allergens Der p 1, Der f 1, Fel d 1 T cell T cell function APC

CD

40 C IL4/ D1 5 IL13 4

Lymphocyte stimulation test Cytokine assays

IgE detection

IgE

Allergenspecific IgE total IgE

B cell

Allergens

Mastcell Basophil Leukocyte

Cellular in vitro tests Histamine release Leukotriene production CD63/CD203c expression

Mediator detection

Mediators, e.g., histamine

Histamine, tryptase, ECP

Receptor binding

Challenge tests Skin, nose, bronchi

Blood vessels Nerve fibers

Muscles Glands

Fig. 65.1 Mechanisms of the allergic reaction. APC, antigen-presenting cell.

In vitro IgE testing Total IgE When the appropriate reagents became available, measurement of total IgE in patient serum became a standard procedure, initially by inhibition radioimmunoassay, later mainly by reliable sandwich tests, where serum IgE is captured by anti-IgE immobilized on solid phase and subsequently detected with monoclonal or polyclonal anti-IgE, labeled with either biotin or with some radioactive, fluorescent, or enzymatic marker molecule (Fig. 65.2a). Results for total IgE are commonly expressed as kilounits per liter (kU/L), 1 unit representing 2.4 ng of IgE antibody.

Allergic inflammation

Interpretation of total IgE results requires some consideration (as outlined in Table 65.1). Since the publication of age-dependent cutoff IgE values (Kjellman et al. 1976), total IgE values < 100 kU/L in adults have been interpreted as “negative,” classifying subjects with these lower values as “nonallergic.” However, it is very important to realize that a total IgE serum concentration of, for instance, 20 kU/L is certainly sufficient to produce a positive result in a number of allergen-specific IgE tests, which may well be related to serious clinical problems. The age-dependent cutoff values have merely statistical importance, although testing for total IgE does provide information about the immunologic status of the patient: higher values for total IgE are positively correlated with the risk for IgE-mediated allergic disease. Thus if

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Labeled anti-IgE

Labeled allergen

Labeled anti-IgE

Serum IgE Serum IgE Serum IgE Allergen (mixture)

Anti-IgE

Anti-IgE

(a)

Solid phase Total IgE

(b)

Solid phase Classic RAST

(c)

Solid phase Reversed RAST

Fig. 65.2 In vitro IgE testing. (a) Total IgE sandwich test. (b) Classic RAST: IgE captured by immobilized allergen. (c) Reversed RAST: IgE captured by immobilized anti-IgE. (See CD-ROM for color version.)

a patient has a high total IgE value and testing for allergenspecific IgE has failed to give positive results, then the possibility should be considered that the correct allergen has not yet been discovered. It is therefore important to stress that testing for total IgE has only relative value as a diagnostic tool, and is mainly relevant in epidemiologic investigations. Obviously, there will be no allergen-specific IgE demonstrable if there is no total IgE. Almost everyone, atopic or not, shows demonstrable total IgE values (it is very uncommon to find total IgE < 10 kU/L in serum from adults), and nonatopic subjects are, in principle, capable of producing antigen-specific IgE, as can be observed when testing for tetanus toxoid-specific IgE after immunization (Aalberse 1991); however, not everybody shows IgE against common allergens. This brings up the question: why is specific IgE relatively rare, while demonstrable total IgE is common? Demonstrable total IgE may have to be ascribed, at least partly, to external factors capable of invoking an increase in polyclonal IgE, as can be observed, in a transient manner, after bone marrow transplantation. For instance, it is generally recognized that smoking or, more dramatically, parasitic

infection can enhance IgE production. High IgE values are found in the hyper-IgE (Job) syndrome, a disease of unknown etiology. However, there may be other (hypothetical) clues for the presence of IgE in serum from apparently nonallergic subjects: among the Toll-like receptors (TLRs), which are instrumental in responses against microbial infections, there is one representative, TLR2, which is capable of inducing Th2biased immunoresponsiveness, associated with the induction of IgE synthesis (Redecke et al. 2004). It may therefore be noteworthy that Staphylococcus aureus residing on the skin contains ligands that are able to switch on TLR2 signaling, leading to a rise in polyclonal IgE production. Also, a staphylococcal toxin, TSST-1, is capable of causing increased IgE by acting as a superantigen, stimulating the immune system in an antigen-independent manner (Hofer et al. 1999). Also other, presently unidentified, environmental components may influence immunomodulation in an IgE-associated direction, as has been observed for Schistosoma glycolipids (van der Kleij et al. 2002), or for pholcodine, a neuromuscular blocking agent present in some types of cough medicines, which dramatically stimulates IgE production (Florvaag et al. 2006).

Table 65.1 Clinical utility of total IgE testing in relation to allergic disease. Total serum IgE

Reasons and consequences

Elevated serum concentrations indicate atopic disposition

Levels in subjects with atopic eczema/dermatitis syndrome are usually more elevated than in allergic rhinoconjunctivitis or allergic asthma

Total IgE may be low in subjects with allergic disorders (“false-negative”)

Predictive value of total IgE for the diagnosis of IgE-mediated allergy is poor

Levels of total IgE in subjects without atopic disorders are usually low or in the normal range

Low total IgE indicates absence of atopic risk

High total IgE may occur in subjects without allergic disorders (“false-positive”)

Total IgE testing is not recommended for definitive exclusion of an IgE-mediated allergy

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Allergen-specific IgE “Classic RAST” and related tests For diagnosis of IgE-mediated hypersensitivity, two types of testing are commonly used: skin-prick (in vivo) tests or RAST-type (in vitro) assays. Skin testing is very sensitive, measures the results of histamine release from mast cells in the skin, and provides a convincing “on-the-spot” result for the patient. In some cases, however, it cannot be employed, for instance when (i) the patient is treated with antihistamines; (ii) there is reason for concern about possible severe reactions; (iii) an infant patient would not tolerate largescale skin testing; or (iv) the condition of the skin, i.e., due to dermographic urticaria or other skin diseases, makes interpretation of the test cumbersome. RAST, although generally somewhat less sensitive than skin testing, is an appropriate alternative with some specific advantages: testing is not influenced by antihistamines; many allergens can be assayed with a limited amount of serum, which can be stored for subsequent testing; and testing is, apart from the need to draw blood, not invasive. It should be stressed, however, that RAST-type assays only provide information about the presence of allergen-specific IgE (sensitization), but not about the clinical status of the patient. The following deals exclusively with in vitro IgE testing. The classic laboratory test for detection of allergen-specific IgE proceeds basically as follows: allergen, immobilized to solid phase, catches allergen-specific antibodies (if present) in patient serum to be tested. Nonbound serum components are removed by washing, and IgE from serum, bound to immobilized allergen, is detected by addition of labeled anti-IgE antibody which specifically recognizes human IgE (Fig. 65.2b). This test, first described by Wide et al. (1967), was originally performed using dextran polymer as solid phase and 125Iradiolabeled anti-IgE. It has acquired worldwide fame as the radioallergosorbent test, and is generally referred to by its acronym RAST. Allergen-specific IgE is either expressed in RAST classes, based on the amount of solid phase-bound anti-IgE, or in kilounits per liter (kU/L), when test results are read from a total IgE calibration curve, as is at present common practice. In vitro allergy testing in the current diagnostic laboratory setting is still based on this original principle, although variations have been introduced with regard to the solid-phase material used for antibody catching (allergosorbent): the once widely used CNBr-activated paper disks are now often replaced by other allergen carriers, such as cellulose polymers (ImmunoCAP, Phadia) or polystyrene ELISA plates. Anti-IgE antibodies, either polyclonal or monoclonal, are nowadays labeled with different types of detector molecules, and in most laboratories the use of radioactive markers has been abandoned in favor of enzymatic, fluorescent, or chemoluminescent markers, which significantly enhances handling convenience. Tests performed with anti-IgE not labeled by radioactive tracers should, for strictly semantic reasons, not be designated

Allergy Testing in the Laboratory

as “RAST,” but this appellation has gained such wide acceptance for specific IgE testing that it is also often used for test formats in which enzymatic (EAST, Pharmacia) or fluorescent (FAST, Pharmacia) markers are used. In this chapter the label “RAST” or “RAST-type test” will be used to designate all nonmultiplex (see below) specific IgE tests, including the reversed RAST test mentioned below.

Solid phase (allergosorbent) The first step in the classic RAST is to capture allergen-specific antibodies from the serum using allergen bound to solid phase. This solid phase makes it possible to perform washing steps between the incubation steps in order to remove nonbound components. In order to guarantee appreciable allergenspecific IgE binding, it is essential that all relevant IgEbinding components are represented on the solid phase. In this context, it is important to note that allergen-specific non-IgE immunoglobulin in the serum, e.g., allergen-specific IgG, which may have been induced as a result of allergenspecific immunotherapy, will compete with specific IgE for binding to immobilized allergen. Allergen extracts that contain only one or two different IgE-binding components, such as extracts from birch pollen, where mainly two proteins are important IgE binders (Bet v 1 and profilin), and cat dander extract, also with two major allergenic components (Fel d 1 and feline serum albumin), will generally pose few problems when preparing solid phases, provided that the allergenic compounds are represented in sufficient concentration in the extract. More complex allergen extracts, with many minor allergenic proteins, may need more attention. Examples of such complex allergens are house-dust mite and mold extracts. There are several ways to immobilize allergen on a solid phase. Adsorption This is the rationale when polystyrene (ELISA plates) or nitrocellulose (dot blots) are used for allergen immobilization. It should be realized that the mechanisms involved in protein binding to these types of solid phase are not completely understood, although protein charge and hydrophilicity will be important factors. This means that when noncovalent binding is used, some proteins may be more efficiently bound to the solid phase than others, which in the case of complex extracts may be problematic. This type of allergen immobilization may also lead to (partial) denaturation of the protein, resulting in loss of binding of specific IgE, as well as in falsepositive binding of IgE and other antibodies. When it can be assumed that the compound of interest binds properly to the solid phase, for instance by comparing test results with those obtained using a high-binding solid phase (e.g., ImmunoCAP or Sepharose), then ELISA technology seems an appropriate and convenient option, for instance as performed for component-resolved testing using recombinant allergens from carrot (Ballmer-Weber et al. 2005).

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Chemical binding Chemical binding includes polysaccharide solid phases, such as the paper disks introduced in the early days of RAST testing, agarose (Sepharose-4B, Amersham Biosciences), and the cellulose polymers nowadays applied in the automated ImmunoCAP technology (Phadia, formerly Pharmacia). An advantage of this type of allergen binding is the fact that allergens in the test are presented in a more hydrophilic environment, and that binding is more robust, although one should always remain aware of “bleeding” of allergens from the solid phase, and of the fact that coupling to solid phase may theoretically render important epitopes unavailable for IgE binding. A major advantage of the Sepharose system is that solid-phase capacity problems can easily be overcome by increasing the amount of agarose beads per test. This is not possible in the ImmunoCAP system, but the cellulose sponges are well known for their high total protein-binding capacity (about 20 μg per ImmunoCAP; personal communication from Phadia, The Netherlands). Ligand binding Ligand binding uses solid phase-bound monoclonal antibody, or some other ligand, to capture a particular allergen from a complex extract. The ligand-bound allergen can subsequently be used in a RAST procedure. This set-up is termed “indirect RAST” and has proven to work satisfactorily, for instance for detection of IgE against tropomyosin, a cross-reactive allergenic muscle protein in dust mites, crustaceans and insects (Witteman et al. 1994), and for profilin, a pollen allergen, which can easily be purified by immobilized poly-L-proline (Valenta et al. 1991). It is important to note that ligand– allergen binding, like covalent binding, may shield an IgEbinding site, in which case RAST results will be lower. Since the early 1980s, allergenic extracts have been extensively studied on a molecular basis, and a vast array of IgE-inducing proteins from all kinds of sources has been identified, purified, and in many cases cloned. This makes it possible, in principle, to obtain unlimited amounts of wellcharacterized allergenic proteins for solid phase preparation and in vitro allergy testing.

RAST variants A variation of the RAST-type procedure is the reversed RAST technique (Fig. 65.2c). A typical example of this technology is the Advia-Centaur test, launched by Bayer Diagnostics. In this type of assay, serum is incubated with solid phase-bound anti-IgE. After IgE binding, biotinylated allergen is added and the presence of allergen-specific IgE demonstrated by adding labeled streptavidin. For IgE testing by reversed RAST, where it will be desirable to capture up to 5000 kU/L IgE/mL (0.1 μg of IgE per 10 μL serum), about 0.2– 0.5 μg anti-IgE per test is required. This implies that for an efficient IgE-catching solid phase, purified anti-IgE should be used. Monoclonal anti-IgE, in principle

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available in unlimited amounts, is then an attractive option. However, it should be noted that not all anti-IgE monoclonals will bind all IgE allotypes (van Lochem et al. 1984). An advantage of this test is that washing before allergen is added will avoid interference by serum components, such as allergen-specific IgG. After IgE catching, immobilized IgE should capture labeled allergen. Saturation of IgE in the femtomole range requires a substantial concentration of allergen, typically 10 times the Kd (dissociation constant of the binding reaction); for a Kd of 10–9 mol/L, about 10–8 mol/L (about 100 ng/mL for a 10-kDa protein) is needed. As it can be assumed that lower allergen concentrations, especially for mixtures of allergens, are present, it is to be expected that this assay will be more selective for high-affinity IgE antibody. As it cannot be excluded that this high-affinity IgE may be of increased clinical importance, the interesting question arises whether reversed RAST testing provides more clinically relevant information than classic RAST assays. A somewhat different RAST variant is the Immulite technology, marketed by Diagnostic Products Corporation (DPC), where patient serum and biotinylated polymerized allergen are simultaneously added to a streptavidin-coated polystyrene bead. IgE binds to allergen and will therefore, through the allergen, become immobilized to the bead. This allergenspecific IgE is detected by alkaline phosphatase-labeled antiIgE in combination with a chemiluminescent substrate. A special feature of this test is the fact that IgE and allergen meet each other in solution, which may better reflect physiologic circumstances. Both systems mentioned above are marketed as automated tests, which makes them serious competitors of the automated ImmunoCAP test from market leader Phadia.

Multiallergen testing The recent increase in allergic disease, and especially the increase in the number of samples to be tested, has stimulated the development of a type of allergy testing that aims at performing as many tests as possible, using as little patient serum as possible, in the shortest time possible. These tests are generally designed to make use of the many purified and recombinant allergenic proteins that are currently available. Multiallergen testing has been performed for decades in the format of “multi-RASTs,” where a mixture of several allergens is immobilized on the solid phase. However, this classic multiallergen RAST does not give separate test results for individual allergens, so that after a positive result for an allergen mixture all constituting components should be tested separately. This set-up was the basis of the widely applied Phadiatop (Pharmacia) test from the late 1980s, which was used to discriminate between IgE-negative and IgE-positive patients, initially only for inhalant allergens but later also for foods. In the early days of this assay, a positive result for the Phadiatop test put the user in an awkward position, as the

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manufacturer initially did not wish to reveal which allergens were provided in the mixture. At present there is, as in the techniques currently used in genomics, much interest in the development of rapid, smallscale, multiplex testing, giving direct separate simultaneous results for specific IgE against many allergens in one single run. An archetypical predecessor of such a test (although not the only one) is the QAS (Quidel Allergy Screen), marketed at the end of the 1980s, which was designed for simultaneous testing for IgE against nine different allergens, and is basically a derivative of the dot-blot assay, where allergen is immobilized on nitrocellulose. Although the QAS is not specially relevant, and certainly has not become a commercial success, some aspects of the test are discussed more extensively as they are still representative for the practicalities encountered when using modern multiplex assays. Although this type of test still seems attractive because of its convenience, it should be noted that its results are mainly qualitative, showing only limited quantitative values. In this respect it is also similar to the IgE immunoblot. The test, which resembles the nitrocellulose-based dotblot test, was meant to be used in the doctor’s office and was therefore formatted as a “dipstick,” using paper-adsorbed allergen for binding specific IgE from patient serum. Subsequent IgE detection was by alkaline phosphatase-conjugated anti-IgE in combination with an enzyme substrate that was converted into an insoluble colored product that precipitated on the paper. The amount of color observed was related to the amount of allergen-specific IgE bound to the paper-adsorbed allergen. A rather remarkable feature of the test was the choice of house-dust extracts among the selected immobilized allergens. However, house dust should be considered an illdefined entity, only to be used in situations where other tests have failed (see autologous dust RAST). In comparison with skin testing, sensitivity of the QAS for some allergens was reported to be poor (Iwamoto et al. 1990), although it should be taken into consideration that the investigations of comparative testing by these authors were hampered by the fact that not all allergens on the dipstick were available for skin testing. Another paper on this test also reported a rather low sensitivity (45.1%) but a reasonable specificity (95.4%) compared to skin-prick testing. In this case the authors were able to use the same extracts for skin testing as the ones applied in the QAS test (Twiggs et al. 1989). However, a clearly attractive feature of this test was its (rather modest) “multiplex” character, so that several allergens could be tested simultaneously, giving separate, directly interpretable, results. After the QAS test (and comparable variations on this theme), new multiplex tests have been presented. There are essentially two formats for multiplex tests. The first uses single-surface multiple allergens, designated “chips” (following the terminology adopted from present DNA technology). Some examples of this format include the test described by

Allergy Testing in the Laboratory

Suck et al. (2002), where nitrocellulose membranes are used to host spots of adsorbed allergen; the assay by Kim et al. (2002), who immobilized crude allergen extracts to silica solid phase; the multiallergen chip test described by Hiller et al. (2002), in which a wide range of purified natural and recombinant allergens are microarrayed on a glass slide; and a similar microarray test presented by Fall et al. (2003). The nitrocellulose system presented by Suck and colleagues looks promising, especially if, as suggested by the authors, the use of chemiluminescent markers for detection is introduced. In the present format, however, sensitivity was at best 1.5 kU/L. So far, no new updates of this test have been presented. The silica-based test appears to suffer from a similar lack of sensitivity. The glass chip technology seems interesting, since 94 different allergens are tested with 40 μL of serum. The authors reported a positive correlation between the results of their assay and the clinical status of the 20 patients tested, although the results did not strictly match their skin-test findings. No comparisons with RAST were shown, and no indication of the sensitivity of the test is given; the test by Fall and colleagues claims high sensitivities for IgE against recombinant Bet v 1 (birch pollen) and PLA2 (bee venom). The other multiplex format involves multiple surfaces, single allergens. A very attractive example of such a test design is the Luminex assay, which employs different batches of colored latex beads (“microspheres”). To each batch of microspheres, an analyte can be covalently immobilized. Batches with different analytes are mixed and incubated with a small amount of serum. Antibodies to different analytes are bound by the different beads in the incubate, which are individually counted by a counting apparatus. With this procedure it is possible to compose mixtures of up to 94 different types of differently colored microspheres, and consequently antibodies against 94 different analytes, possibly allergens, may be measured using only 50 μL of serum. So far, however, no reports on successful allergy testing using this technique have been published, possibly because of the same problem we encountered in our own laboratory when exploring this technology: low sensitivity due to inefficient binding of allergens to the microspheres.

Antibody binding in RAST-type assays Crucial for IgE antibody testing and subsequent expression of results in weight per volume (such as kU/L) is undoubtedly the question of whether the participating components in the system will reach binding equilibrium. IgE binding is reversible (Fig. 65.3), and possible effects of incomplete binding equilibrium are demonstrated by experiments presented in Figs 65.4 and 65.5. Figure 65.3 demonstrates that serum IgE can, after binding to solid phase, subsequently “leak away” from immobilized allergen. Grass pollen-positive sera (RAST class 2, 3, 4 and 5) are incubated with solid phase (grass pollen extract coupled to Sepharose). After 24 hours the solid phase is washed, and

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left to stand for another 24 hours. After centrifugation, supernatant is retested on grass pollen Sepharose, resulting in a positive supernatant RAST for the RAST class 5 sample. This means that initially bound IgE has leaked from the allergosorbent; IgE binding has a dynamic character. Figure 65.4 demonstrates what happens when variable incubation times are used in the RAST procedure for binding of serum IgE from four grass pollen-positive patients, and for radiolabeled anti-IgE. “0.5/0.5” indicates that both incubations lasted for half an hour; “3/3” indicates that IgE and anti-IgE incubation each lasted for 3 hours, etc.; and “24/48” means that serum incubation was 24 hours and anti-IgE incubation 48 hours. Figure 65.4a demonstrates the impact of different incubation regimens on the chimeric IgE calibration system (see below for description of chimeric IgE as a calibration tool), Fig. 65.4b shows patient IgE binding to grass pollen Sepharose, and Fig. 65.4c the results when these binding percentages are converted into kU/L using the matching calibration curve. It is obvious that longer incubations lead to higher binding results, and it seems that calculation of results in kU/L gives more variable results when allergenspecific IgE in serum is higher. It is also important to note that when RAST results are expressed in RAST classes, based on the amount of solid phase-bound (radio)label, very different results may be found, depending on the incubation regimen employed. Figure 65.5 shows test results for three grass pollenpositive patients when serum incubation times were varied, while the incubation time for detecting anti-IgE is kept the same in all tests (24 hours). As can be concluded from Fig. 65.5a (binding of the calibration chimeric antibody to immobilized Der p 2) and from Fig. 65.5b, which reflects binding of specific IgE to grass pollen solid phase, this binding becomes more efficient the longer the incubation time. Figure 65.5c, in which specific IgE results are expressed as kU/L, again underlines the importance of longer incubations and more efficient IgE binding, as higher test results are observed.

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Fig. 65.4 Effect of variable incubation times for IgE and anti-IgE on RAST results. (a) Chimeric IgE calibration curves. (b) RAST results as percent of bound radiolabeled anti-IgE. (c) RAST results as kU/L.

These observations stress the importance of appropriate test parameters when quantitative results are required, and it remains questionable if the novel chip-like formats, with their small (and virtually unknown) amounts of allergen on various types of solid phase, minimal amounts of serum employed, and short incubation regimens, will be able to

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have been attractive enough to be adopted by a major manufacturer for upscaling and commercialization.

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Allergy Testing in the Laboratory

Fig. 65.5 Effect of variable incubation times for IgE on RAST results. (a) Chimeric IgE calibration curves. (b) RAST results as percent of bound radiolabeled anti-IgE. (c) RAST results as kU/L.

provide adequate results in kU/L. For qualitative testing, however, they may function satisfactorily. Over the last few years many multiplex-type IgE tests have been presented to the allergologic community, but up to now none seems to

Sensitivity of RAST-type assays Skin testing is generally more sensitive, but poses high demands on the quality of the extracts used for testing. This is clearly demonstrated by the observations of van der Veen et al. (1996), who tested dog dander skin-test material, obtained from five different manufacturers, for the amount of major dog allergen Can f 1, and for the presence of contaminating house-dust mite allergen, by determination of mite allergen Der p 2. The investigators found that this mite compound was present in all dog dander extracts tested, and that the ratio of Can f 1 to Der p 2 showed wide variation in these dog dander preparations. In all extracts, sufficient mite allergen appeared to be present for the induction of positive skin tests in mite-allergic patients. This means that false-positive skin test results will be found when dog-negative, but mitepositive, subjects are tested with these dog dander extracts. The same may be true for other mammalian dander preparations. In the less sensitive RAST-type assay, however, this type of false positivity is less likely to pose a problem. RAST values lower than 0.35 kU/L are considered negative. This value stems from the Pharmacia birch pollen calibration system, which has served for many years as the standard for quantitative allergen-specific IgE testing. From our own experience we know that RAST tests are able to detect about 50 pg of specific IgE per test, which corresponds to approximately 0.15 kU/L, when 10 μL of serum are used. This is very convenient when only small amounts of serum (or plasma) are available and several allergens have to be tested. The practical applicability of this idea has been confirmed by Eysink et al. (2004), who investigated IgE conversion in young children who visited the general practitioner because of prolonged coughing complaints. Capillary blood samples were obtained by fingerprick and adsorbed to filter paper and, after elution from the paper, tested for total IgE and for IgE against a number of inhalant allergens, using approximately 10 μL of plasma per test. The manufacturers of the Immulite test (DPC) have now lowered their test cutoff to 0.1 kU/L, and stress the necessity of sensitive IgE tests in general, stating that detection failure of low specific IgE values in children may lead to suboptimal medical treatment, based on the observations by Sasai et al. (1996). However, it should not be expected that competing tests such as ImmunoCAP will not be able to measure specific IgE with similar sensitivity. It must be emphasized that high sensitivity testing may occasionally lead to unwanted results: irrelevant positive test values may be encountered, partly due to technical reasons, for instance when assaying serum samples with high total IgE (> 2000 kU/L). Another possible setback may be the clinically irrelevant or false-positive results that may be found due to IgE against cross-reactive

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or contaminating components: it is to be expected that high sensitivity testing will lead to clinically false-positive cat dander RASTs due to IgE against lipocalin, evoked by dog dander. The same considerations hold true for the abovementioned contamination of dog dander extracts with housedust mite. In the case of increased sensitivity testing, the quality of the allergenic extract used in the test will be of vital importance. For these reasons it is unclear whether demonstrating minute amounts of allergen-specific IgE (< 0.2 kU/L) in serum substantially contributes to the quality of allergy diagnosis. More data are needed to shed some light on this matter.

Chimeric IgE as a RAST calibration tool All major test manufacturers nowadays apply total IgE calibration curves for the expression of RAST results in units per volume. From a technical point of view, this is not completely accurate, as the type of binding of the labeled anti-IgE is not fully comparable in assays for total IgE (where the Fc portion of patient IgE is sandwiched between anti-IgE on solid phase and labeled anti-IgE) and for specific IgE (where the Fc portion of allergen-captured patient IgE is free for binding of detecting anti-IgE). For this reason, it seems better to employ a true RAST-type calibration system in which the test procedure is similar to the RAST and in which the amount of specific IgE applied is exactly known. In our laboratory, we have such a system at our disposal: a chimeric IgE antibody, composed of the heavy chain variable domains and light chains of a murine monoclonal antibody directed against house-dust mite allergen Der p 2 and a human IgE heavy chain (Schuurman et al. 1997), is used in a RAST-type set-up in combination with immobilized recombinant Der p 2. As the amount of chimeric IgE can be properly determined (by testing for total IgE, based on a WHO standard), the unique situation is established where the amount of specific IgE in the sample equals the amount of allergenspecific IgE. Testing for specific IgE in patient samples using this calibration system therefore results in more accurate values. In addition, chimeric allergen-specific IgE antibody has proven its unique value as a test sample for quality testing among different laboratories. Autologous dust RAST Application of a “personalized” house-dust RAST proved its utility when an allergic patient was suspected of being allergic to some unknown indoor allergenic component. RAST assays for common indoor allergens, such as house-dust mite, cat and dog dander, were negative. In such cases, it can be helpful to make an extract of the patient’s own house dust and couple this to solid phase. RAST testing with serum from the patient and such a solid phase gave positive results (RAST class 4), indicating the presence of some IgE-inducing component in the patient’s dust. Moreover, it appeared that this positive RAST could be inhibited (see RAST inhibition and

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IgE cross-reactivity) with house-dust extracts from other people’s homes, and eventually it was found that the presence of this allergenic compound could be shown in one of five Dutch dust samples. This means that there must exist some relevant dust allergen that has, until now, remained unidentified (Aalberse 2000).

RAST inhibition and IgE cross-reactivity One particular variant of the RAST test has proven to be very useful for the investigation of quality and allergenic similarity (IgE cross-reactivity) of allergenic extracts. When serum, prior to incubation with immobilized allergen on solid phase, is incubated with the same allergen in solution, specific antibodies will bind to fluid-phase allergen and will thus be prevented from binding to the solid phase; in this set-up RAST results will end up negative after detection with labeled antiIgE. This is called homologous RAST inhibition: inhibiting allergen is the same as solid phase-bound allergen, inhibition should be complete. However, when serum is preincubated with an extract which is partially similar to the immobilized extract, i.e., the two extracts share some but not all IgEbinding epitopes, partial inhibition will be observed. RAST inhibition can therefore provide relevant information on the degree of IgE-binding similarity and cross-reactivity of different allergens. Application of RAST inhibition techniques has provided a wealth of information. It has shown, for instance, that IgE cross-reactivity among common grasses is very high, rendering it superfluous to test patients with extracts from many different grass pollen species belonging to the same subfamily (in western Europe mostly Pooideae). However, it may be indicated for testing grasses that are less closely related to the Pooideae, such as Bermuda grass (Cynodon dactylon, subfamily Chloridae), which might induce species-specific noncrossreactive IgE in the areas where this species is endemic. RAST inhibition has also revealed surprising examples of unexpected clinically relevant cross-reactivity. It was found that IgE, supposedly induced by latex, was responsible for the anaphylactic reaction observed in two patients after consumption of buckwheat-containing pancakes. Serum from these patients was preincubated with latex extract (inhibitor) in solution, prior to adding it to immobilized Sepharosebound buckwheat extract and, in a parallel experiment, with buckwheat extract before incubation with immobilized latex. Buckwheat extract could hardly inhibit the RAST for latex, while very small amounts of latex extract were capable of complete inhibition of the buckwheat RAST. It is therefore most probable that latex had acted as the “inducing” allergen, evoking IgE that cross-reacted with buckwheat components, in these cases leading to severe clinical problems (de MaatBleeker & Stapel 1998). Another remarkable example in which RAST inhibition assays proved to be very useful was in the course of investigations on the etiology of allergic disease. Calkhoven (1989)

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Cat serum Fel d 1 Pig serum 50

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tested longitudinal series of serum from young children for IgE against cat allergens Fel d 1 (the major allergenic protein in cat dander) and cat serum. In early samples, 5 of 213 randomly selected children showed a positive RAST for cat serum but not for Fel d 1, as exemplified by the results obtained with serum MK from a patient born in November 1981 (Fig. 65.6). This anti-cat serum IgE response declined over time, while at the same time the response to Fel d 1 increased. It seems remarkable that IgE against one cat allergen decreases, while IgE against the other increases over time. It was noted that the longitudinal response against cat serum showed a striking resemblance to the patterns commonly observed when sample series from young children are tested for IgE against foods, in which case a similar decrease in IgE response is frequently found. This led the investigators to the hypothesis that the positive IgE test for cat serum might well be the result of IgE cross-reaction with some food component. A possible candidate for induction of cross-reactive IgE could be meat, which also contains serum proteins. When testing serum from these children in RAST inhibition, it was found that the RAST for cat serum could be inhibited completely by preincubation of patient serum with pig serum. Longitudinal testing for IgE to pig serum showed the same pattern as was observed for cat serum. This may indicate that consumption of pork had induced IgE that was cross-reactive with cat serum. The gradual rise in Fel d 1-specific IgE could then (hypothetically) be explained as follows: when these children encounter skin flakes from cats, these flakes will, after inhalation, land on the nasal mucosa. Some of the cat serum protein present on these skin flakes will solubilize in the moist mucosal environment and encounter IgE, which was previously induced by consumption of pork. Since this antimeat IgE is cross-reactive with cat serum proteins, it will bind to these proteins, leading to histamine release and a small local allergic reaction, resulting in tissue-disturbing processes. As a result of these reactions, the mucosa will locally

Allergy Testing in the Laboratory

become more permeable, and Fel d 1, the main cat allergen, which will certainly also be present on the above-mentioned skin flakes, gains access to the immune system and will thus be able to induce an IgE response. Although this model contains some highly hypothetical assumptions, it is an attractive and instructive example of the potential relevance of IgE cross-reaction, and it also shows the applicability and effectiveness of RAST inhibition techniques. It should be noted that the principles of this inhibition technique can also be applied in IgE immunoblotting, where, after electrophoresis and nitrocellulose transfer of componentresolved allergen extract, preincubation of serum with a second allergen extract provides qualitative information about the presence of common IgE-binding components in both extracts. Also, for foods, many IgE cross-reactions have been identified by RAST inhibition assays: one of the first observed cross-reactions was ascribed to the presence of glycan groups in pollens and vegetable foods. These sugars, which were designated as cross-reactive carbohydrate determinants (CCDs) by Aalberse et al. (1983a), appeared to be not only present in vegetable allergens but also in extracts from invertebrate animals and insect venoms. Instrumental in IgE binding are two monosaccharides, xylose and fucose (van Ree et al. 2000). There has been a longstanding debate about the clinical relevance of IgE against these sugars, but it has now become clear that IgE, solely directed to CCD, does not induce clinical problems (Mari et al. submitted). Another extensively cross-reactive IgE-binding protein is profilin, found in pollen from grasses, trees and weeds, and also in extracts from vegetable foods. The clinical relevance of anti-profilin IgE is still under discussion. However, a number of other vegetable proteins are clinally relevant, although their clinical impact is different. A major component in birch pollen is allergen Bet v 1, which has structurally similar counterparts in stony fruits. A well-known clinical result of IgE cross-reactivity is the “oral allergy syndrome,” where IgE, initially generated against Bet v 1, induces itching or burning sensations in the oral cavity, of the lips or the palate after consumption of, for instance, apples and cherries. The culprit proteins in these fruits are Bet v 1 homologs, such as Mal d 1 in apple. However, these components are relatively thermolabile; their clinical impact is usually mild. Also, in other vegetable foods, such as soybean and hazelnut, the presence of Bet v 1 homologs has been demonstrated, leading occasionally to severe oral allergy symptoms or even to systemic reactions depending on the amount of ingested protein. Some vegetable IgE-cross-reactive proteins can induce serious, sometimes even anaphylactic symptoms. It concerns small pepsin-resistant components, such as lipid transport protein (LTP), thaumatin-like protein (TLP), 2-S albumins, and oleosins. It is not unlikely that their clinical impact is associated with their low susceptibility to stomach enzymes. This type of component is responsible for the occasionally

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Table 65.2 Clinical utility of allergen-specific IgE testing. Allergen-specific IgE

Reasons and consequences

Indicates IgE-mediated sensitization

Elevated serum IgE leads subsequently to distribution of IgE in tissues and high-affinity binding to FceRI of mast cells, basophils, and other cells

Is clinically relevant in case of corresponding symptoms

Full interpretation only in the context of clinical findings

Can be false-negative

Due to extremely low total IgE (< 10 kU/L, i.e., nonatopics with insect venom or occupational allergy) Due to competition with allergen-specific IgG (only in “classical” allergen solidphase assays, not in “reverse type” assays using anti-IgE capture as first test step) Due to low abundance of the sensitizing molecule in the extract mixture (i.e., Gly m 4 in soy extracts)

Can be false-positive (preferred term “clinically irrelevant”)

Due to binding to irrelevant extract components (i.e., binding to CCD, binding to monomeric epitopes) Due to cross-reactivity Due to extremely high total IgE Due to enhanced assay sensitivity

severe, sometimes even lethal reactions after ingestion of peanut (where Ara h 2, another small pepsin-resistant protein, plays an important role) and hazelnut.

Interpretation of RAST results General guidelines for the interpretation of results obtained by specific IgE testing are given in Table 65.2. The most important issue to be aware of when looking at RAST results is that in vitro testing only gives information about sensitization (the presence of IgE directed to a certain allergen) but not possible clinical consequences. It is therefore not acceptable to burden a patient with some diet solely based on a positive RAST for a certain food. When looking at positive RAST results, one should always keep the possibility of IgE cross-reactivity in mind. Clinically irrelevant results can be obtained due to IgE to CCD. This is reflected in a large number of positive tests when vegetable and/or invertebrate and/or insect venom allergens are assayed. It has been shown that one-third of grass pollen-positive patients had a clinically irrelevant peanut-positive RAST as a result of IgE against CCD (van der Veen et al. 1997). Direct information on the presence of IgE against these sugar groups can be obtained by testing for CCD, either by a RAST for bromelain or horseradish peroxidase (both CCD-rich proteins), or for proteinase K-treated grass pollen. Many positive RASTs for allergens of vegetable origin also point to IgE against profilin, a ubiquitous vegetable protein. The clinical relevance of this component has not yet been unequivocally determined. “Pattern recognition” also helps: it is very uncommon to find a positive RAST for apple and a negative RAST for birch pollen, at least in northern European countries, where positive tests for apple are almost exclusively the result of crossreactivity with Bet v 1 from birch pollen. When RAST is used to determine the proper insect venom for immunotherapy,

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it should be noted that the RAST for vespid venom and bee venom can both be positive due to cross-reactivity, either by CCD, or by phospholipase A2. In the latter situation, RAST inhibition should be used to identify the culprit insect. Other explanations for a clinically irrelevant positive RAST could be (i) IgE binding to a single allergen epitope, while essentially two epitopes are needed for the induction of clinical consequences, i.e., triggering mediator release from mast cells or basophils; or (ii) (low) positive RAST tests by nonspecific IgE binding to solid phase in samples with very high total IgE. A matter of debate is the practical use of quantitative food RAST values in the prediction of clinical problems. Sampson investigated both retrospectively (Sampson & Ho 1997) and prospectively (Sampson 2001) whether RAST could provide values which would predict (with at least 95% probability) positive food challenge tests, and found RAST cutoff values of 6, 32, 15, and 20 kU/L (ImmunoCAP) for hen’s egg, cows’ milk, peanut and fish, respectively. Adopting these values would eliminate the need for time-consuming, expensive, double-blind, placebo-controlled food challenges. Shek et al. (2004) reported that the rate of decrease in food-specific IgE over time was predictive for the probability of developing tolerance to milk and egg. In contrast, other investigators (Celik-Bilgili et al. 2005) were able to establish such cutoff values only for egg (95% probability level) and milk (90% probability level), but not for wheat and soy. Eigenmann (2005) states that this has to be ascribed to differences in the patient populations studied and the prevalence of the disease in the population. Therefore, predictive values for a certain group cannot easily be applied to other populations; corrections are needed for each specific situation. False-negative RASTs are less common, and can often be attributed to low-quality allergosorbents. RAST testing

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inhaled antigen (allergen) in the alveoli, and are commonly designated “precipitins.” For decades, the classic Ouchterlony double diffusion assay, a straightforward precipitation test, has been used as a standard in vitro diagnostic tool for allergic bronchopulmonary aspergillosis, bird fancier’s lung, farmer’s lung, and related alveolar diseases. Allergens in type III allergy are in most cases inhaled organic dusts, such as mold spores and bird feces. In the early 1980s, however, the use of ELISA testing for diagnosis of Aspergillus-induced type III hypersensitivity was proposed (Kauffman et al. 1983). In this comparative investigation, precipitating antibodies did not always correspond with the IgG ELISA titers. However, the observations by Kauffman and colleagues are not fully compatible with the results obtained by Gugnani et al. (1990) who found a positive correlation for the two test procedures. At present Phadia is promoting and marketing a RAST-type test for diagnosis of type III allergic disease, using immobilized antigen on solid phase and labeled anti-IgG for antibody detection. This test can be performed on ImmunoCAP automates. Because it seems logical to assume that the precipitating nature of the antigen-specific antibodies is typically involved in the etiology of the disease, we also decided to compare RAST/ELISA-type testing with immunoprecipitation. Pigeon serum-specific total IgG and IgM were determined by ELISA, and antigen-specific IgG4 levels were assayed by IgG4 RAST (van der Giessen et al. 1976; Calkhoven et al. 1991), using 24 serum samples sent to our diagnostic department for assay of precipitating antibodies against pigeon serum. The results, for ELISA and RAST testing expressed in arbitrary units/mL and for double diffusion testing as the number of precipitation lines obtained, are shown in Fig. 65.7. It is obvious that these different test formats measure different phenomena: precipitin-negative serum samples occasionally show high titers of antigen-specific IgG or IgM antibody. These results are not in accordance with earlier findings by Lopata et al.

for apple has, for a long time, been a notorious example of a cumbersome test due to the use of deteriorated apple extracts. An important aspect of the interpretation of specific IgE results is also the fact that the observed results should be considered in the context of possible exposure to the allergen for which sensitization has been found: when a patient shows IgE to birch pollen while living in the south of Spain where birch trees are seldom encountered, it will be obvious that such a finding is of less importance than the same finding for an inhabitant of some Scandinavian country. Another equally important parameter to take into account is the reactivity of the end organ: patients with increased nonspecific bronchial hyperreactivity might suffer more from IgE-mediated airway responses than those with a rather normal nonspecific bronchial hyperreactivity. In conclusion, in vitro IgE testing using RAST-type assays has shown its applicability, utility, and validity for many years now, and will undoubtedly continue to do so, provided that well-defined allergens are used that conform to state of the art standards.

IgG testing IgG also has a role in allergic disease, and different actions, beneficial as well as detrimental, are ascribed to this antibody class. Testing for allergen-specific IgG is therefore occasionally indicated.

IgG in type III allergy IgG plays an important role in type III hypersensitivity, characterized by alveolar lung damage as a result of immune complex-induced complement activation. In this type of disease, antibodies, mainly belonging to the IgG (but also IgM) class, are responsible for the formation of complexes with

sIgG4 Total sIgG sIgM

100 000

Arbitrary units/mL

10 000

1000

100

10 Fig. 65.7 Pigeon serum precipitin-positive patients in IgG4 and IgM RAST/ELISA. sIgG4, antigen-specific IgG4; sIgM, antigen-specific IgM.

1 0

0

0

0

0

0

0

0

0

0

0

0

1

2

2

3

3

3

3

3

3

4

4

4

Number of precipitation lines

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(2004), who compared ELISA and precipitin testing for antibird IgG and who claim that ELISA results correlate well with precipitin testing. The reasons for these discrepancies are as yet unknown. Unexpectedly, high levels of antigen-specific IgG4 do not seem to prevent the formation of immune complexes (van der Zee et al. 1986).

Is IgG4 instrumental in disease? Although it has been convincingly established that IgE is the guilty immunoglobulin in the etiology of immediate (type I) allergic disease, some investigators suspect IgG(4) of being able to act as an inducer of histamine release. Arguments for this are based on studies that show the presence of allergenspecific IgG in serum, mainly directed against foods. This has led to the situation where in many countries commercial laboratories are offering serologic screening of IgG(4) against an array of foods, welcomed by patients who declare they are food hypersensitive or intolerant and who are disappointed with the results of regular IgE-based diagnostics. It should be emphasized, however, that the allegations against IgG are mainly based on associative observations about the presence of specific IgG and occurrence of disease; convincing arguments for a role of IgG in type I allergic disease have not been shown so far. Of the four subclasses of IgG, IgG1 is normally the most abundant subclass in human serum and IgG4 the least abundant. An IgG1 response is mainly involved in protection against invading pathogens, and capable of activating the complement system, which results in destruction of IgG1opsonized microorganisms. In the 1970s it was discovered, by serum transfer experiments, that IgG in monkeys was capable of inducing histamine release in the skin. This IgG was designated “short-term sensitizing IgG” (Parish 1970), but at that time the subclass of this specific type of antibodies remained unclear. Soon after these findings, however, the IgG4 subclass was identified, and it was this novel subclass that became suspected of histamine-releasing activity. In the 1990s, it was demonstrated that IgG4 production was, like IgE production, under control of Th2 cytokines, like interleukin (IL)-4 and IL-13, which made the accusations against IgG4 even more serious. Measurement of IgG against foods was considered useful for antigen avoidance in irritable bowel syndrome (Atkinson et al. 2004), but the evidence for this conclusion was strongly challenged on the basis of poor study design (Hunter 2005). Also, testing for IgG against wheat gliadin is occasionally used in the diagnosis of celiac disease, but the information obtained is of poor specificity and sensitivity, and this test should only be applied in the case of IgA deficiency. Sletten et al. (2006) recently suggested a disease-inducing role for IgG4 in non-IgE-mediated cows’ milk allergy; this conclusion was based on observations on increased IgG4 titers to βlactoglobulin in cows’ milk IgE-negative patients with hypersensitivity to milk. The suggested detrimental role for IgG4

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was in this paper was merely founded on associative results; mechanistic arguments were not given. IgG4 is a remarkable antibody. It does not activate complement, and forms, in contrast to IgG1, only small complexes in combination with antigen (Van der Zee 1986). This has to be ascribed to the most notable feature of this subclass: its ability to exchange half molecules, resulting in bispecific IgG4 molecules (Aalberse & Schuurman 2002) that are not equipped for the formation of large immune complexes; IgG4 in serum should therefore be considered as “functionally monovalent.” Convincing evidence on the histamine-releasing potential of the IgG4 subclass has not been published. As yet there is only one well-documented, although highly artificial, example of histamine release in which allergen-specific IgG4 is involved: Schuurman et al. (1998) investigated a possible role for IgG1 and IgG4 in histamine release from basophils, using chimeric mouse/human monoclonal IgG1, IgG4 and IgE directed against house-dust mite allergen Der p 2. Their conclusion was that, under these conditions, a bivalent chimeric IgG4 antibody against Der p 2 was able to induce histamine release from basophils by indirect cross-linking of Der p 2specific, basophil-immobilized, chimeric IgE. It should be noted, however, that IgG1 performed equally well under these conditions; that the IgG4 chimeric antibody used in this test was functionally bivalent and that, without Der p 2specific IgE on the basophil, no histamine release could be demonstrated. These results therefore do not argue for a “natural” histamine-releasing role for IgG4 in plasma. However, it is gradually being accepted that testing for allergen-specific IgG4 can be of interest in the context of IgE-mediated disease, particularly as a serologic parameter for monitoring immunologic effects of allergen-specific immunotherapy. It is well known that application of relatively high amounts of allergen, for instance by subcutaneous injection, leads to the formation of allergen-specific IgG. Initially, specific IgG1 is found, but in the course of ongoing therapy the IgG response becomes gradually dominated by allergenspecific IgG4 (Aalberse et al. 1983b). This phenomenon is also observed under natural circumstances: experienced beekeepers, after repeated bee stings, show an IgG response to bee venom component phospholipase A2 (PLA2) mainly consisting of PLA2-specific IgG4. However, this allergen-specific IgG4 response should be considered as beneficial, as IgG4 does not activate the complement cascade, with its possibly detrimental results. Moreover, IgG4 has been shown to possess another remarkable feature: it is capable of the inhibition of IgE-mediated facilitated antigen presentation (van Neerven et al. 2006). In the past few years it has become clear that IgG4 formation is a consequence of the activity of allergen-specific regulatory T (Treg) cells, which are involved in dampening possible overreactions of the immune system (Satoguina et al. 2005). These T cells are characterized by the production of IL-10,

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which can evoke a pronounced IgG4 response against the antigen challenging the immune system. As at present the rise in allergen-specific Treg cells and IL-10 production is generally considered to be associated with the induction of tolerance (Jutel et al. 2006), it is tempting to hypothesize that an allergen-specific IgG4 response should also be considered a marker of tolerance. Testing for the allergen-specific IgG4 response in the course of immunotherapeutic treatment may thus provide a relevant parameter for evaluation of the immunologic status of the patient.

Testing for allergen-specific IgG for monitoring immunotherapy To investigate if allergen-specific immunotherapy has triggered the immune system in an allergen-specific manner, measuring of specific IgG is a sensible option. This can be performed in several ways.

Allergy Testing in the Laboratory

However, it should be realized that a high-capacity anti-IgG4 solid phase is needed in order to bind sufficient total IgG4 to enable the detection of the (initially) small fraction of allergen-specific IgG4. This is even more true for the antigenbinding test for detection of total allergen-specific IgG, where it is desirable to bind about 50 μg of IgG from serum, prior to detection of the allergen-specific IgG fraction. Sepharoseimmobilized protein A or protein G have proven to be adequate solid phases for this purpose. It is important to note that the relevance of the test results is influenced by the choice of labeled allergenic protein. In our laboratory grass pollen allergen Lol p 1, widely known as a major IgE-binding compound, was initially used for monitoring immunologic responses to grass pollen immunotherapy in patient sera, until it became clear that Lol p 5 was a superior candidate. Levels of IgG4 against Lol p 5, but not Lol p 1, correlated positively with clinical improvement (Nouri-Aria et al. 2004).

RAST/ELISA-type testing The most obvious approach is application of a RAST- or ELISA-type assay, using immobilized allergen extract, and detection of allergen-bound antibodies by application of labeled anti-IgG4. However, it should be realized that in this set-up IgG against nonallergenic (clinically irrelevant) components in the immobilized extract is also detected, as patients are commonly injected with complete allergen extract and will therefore generate an IgG response against non-IgEbinding proteins as well. When this type of test is applied, using complete allergen extract on solid phase, this clinically irrelevant IgG response will also be measured.

Reversed IgG RAST (“antigen-binding test”) In this type of assay, IgG from test serum is captured by immobilized anti-IgG; detection of allergen-specific IgG is carried out by incubation with labeled allergen. It is advisable to use labeled purified single allergenic protein in this test, as this warrants measurement of IgG against a clinically relevant, IgE-inducing, component. This test has been propagated by Aalberse et al. (1983b) and shows some advantages in addition to high specificity (assaying for IgG against a relevant compound) and sensitivity (detection of 25 ng/mL of specific IgG using 1 μL serum per test is possible), since a 250-fold rise in allergen-specific IgG titer can easily be measured. This test, which in principle is similar to the IgE-detecting reversed RAST (Fig. 65.2c), uses very small amounts of purified allergen (approximately 1 ng of protein per test) and therefore measures high-affinity antibodies preferentially. Testing for IgG4 against a single allergenic protein is carried out as follows: patient serum is incubated with immobilized anti-IgG4 monoclonal antibody, followed by removal of nonbound serum components by washing, incubation with labeled purified allergenic protein, and a second washing step to remove nonbound (radio)label. By using this test format, interference by antibodies of other (sub)classes is avoided.

Basophil stimulation tests for allergy diagnosis Basic mechanisms of immediate-type hypersensitivity reactions have been extensively studied using human basophils. These cells, like mast cells, are equipped with receptors (FcεRI), binding IgE with high affinity. FcεRI receptors are, on allergen stimulation, cross-linked via allergen-specific IgE, which leads to receptor aggregation, intracellular signaling, cellular activation, and the release of a variety of mediators (histamine, LTC4, IL-4, IL-13) (Schroeder et al. 2001; Prussin & Metcalfe 2006). Subsequently, diagnostic tools have been introduced that quantify histamine release, leukotriene production, or the upregulation of surface markers (CD63 and CD203c) from these cells after allergen stimulation. An increasing number of diagnostic studies have promoted the utility of these cellular basophil-based tests in demonstrating allergic sensitization to allergens and compounds that might impose diagnostic difficulties in other diagnostic tests, like skin tests or IgE assays.

Heterogeneity of basophil responses due to complex basic variables During the last decades, the basics of IgE-dependent stimulation have largely been uncovered by detailed histamine release studies (Osler et al. 1968; Kagey-Sobotka et al. 1982; Lichtenstein et al. 1983). Broad variability in basophil responses exists between different basophil donors and different antigens within one donor. This manifests itself in terms of reactivity (maximum response of activation or secretion), based on the intracellular signaling of the basophils studied, and in terms of sensitivity (Fig. 65.8) (MacGlashan et al. 2002; Kleine-Tebbe et al. 2006). The latter is governed by the affinity of specific IgE for its ligand (the allergen), the number

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Spec. IgE

Read-out

Total serum IgE

#1 Total IgEreceptor cell surface density

(a) #6 Biochemical and structural features of the allergen

Read-out

Basophil reactivity

Basophil sensitivity (b) C

Read-out

B

(c)

A

Log allergen (i.e., ng/mL)

Fig. 65.8 Concept of basophil sensitivity and reactivity. (a) IgE/FceRImediated basophil functions demonstrate highly variable dose–response curves between different donors or within the same donor using different stimuli (i.e., allergens). (b) Basophil reactivity (maximum of the basophil response) and basophil sensitivity, i.e., the (allergen) concentration inducing a distinct response, are used to analyze IgE/FceRI-mediated basophil responses. (c) Both variables, basophil reactivity (in this example C > B > A) and basophil sensitivity (C < B < A), are independent of each other.

of IgE receptors per basophil, the ratio of allergen-specific IgE to total IgE, and the number of cell-surface allergen-specific IgE molecules for half-maximal responses, designated as “intrinsic sensitivity.” These variables (Fig. 65.9) give rise to shifts in the dose–response curves of a magnitude of several logs that, in a diagnostic setting where only a single allergen concentration is employed, may produce false-negative data. Thus, in order to utilize the current basophil activation tests for diagnostic purposes, each allergen should be preevaluated separately, in order to determine a suitable stimulation range or an appropriate concentration, if, despite interindividual variation, only a single concentration will be applied.

Principles of flow cytometric analysis of basophil activation Multicolor flow cytometry enables the analysis and quantification of basophils on a single-cell basis. Upregulation of

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#5 Cellular basophil reactivity (maximum of the response)

#2 Fraction of membranebound specific IgE #7 Nature of the aggregates (dimers, trimers, oligomers) #3 Antibody avidity #4 Intrinsic basophil sensitivity

Fig. 65.9 Variables determining basophil responses. Arrows indicate direct influence of one variable on another. Straight lines indicate independence of neighboring variables from each other. (Modified from Kleine-Tebbe et al. 2006, with permission.)

particular activation markers will provide indirect information on the stimulus applied. Subsequently, allergens and specific stimuli can be identified which are able to cross-link allergenspecific IgE bound to its high-affinity receptor (FcεRI), leading to activation and anaphylactic degranulation of the cell. CD63 and CD203c (Knol et al. 1993; Bühring et al. 2001) represent two activation markers most commonly used in diagnostic studies using basophils. Both markers have particular features with functional consequences on activation (Table 65.3). Basophil- and mast cell-specific CD203c is constitutively expressed and upregulated on stimulation with FcεRI-dependent stimuli, but also by priming stimuli like IL3. In contrast, CD63, being not as basophil-specific as CD203c and anchored in the granule membrane of resting basophils, appears on the outer membrane only on activation and will more closely parallel degranulation. If CD63 is applied, basophils have to be stained by another basophil-selective marker for successful identification. AntiIgE antibodies are commonly used; competing signals because of binding to IgE on monocytes or dendritic cells can be circumvented by removing these cells from the flow cytometric analysis on the basis of their scatter characteristics. When basophils are activated via FcεRI, reduced binding of anti-IgE

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Table 65.3 CD63 and CD203c have particular features with functional consequences on activation. CD63

CD203c

Synonyms

Gp53, lysosome-associated membrane protein (LAMP)-3

Neural cell-surface differentiation antigen E-NPP3, PD-Ib, B10, gp130RB13–6

(Super)family

Transmembrane-4 superfamily (tetraspanins)

Pyrophospatase/phosphodiesterases (E-NPPs)

Unstimulated basophils Localization

Barely detectable Intracellular granule membrane

Constitutively expressed Surface membrane

FceRI-mediated activation Expression pattern Localization

Upregulation starts within 10 min Expressed with high density Surface membrane

Upregulation starts within 5 min and peaks within 10–15 min Expression less profound compared with CD63 Surface membrane

IL-3 priming

No upregulation without FceRI or IgE stimulation

Upregulation without further FceRI stimulation

Specificity of expression

Also expressed by macrophages and platelets

Exclusively expressed on basophils

Similar surface markers

CD107a

CD13, CD164

to these activated basophils may be a point of concern. Another approach is labeling of CD203c, as a selective marker for peripheral blood basophils, in addition to CD63.

Experimental considerations and technical aspects Use of whole blood or isolated basophils Flow cytometric analysis of basophils can be performed with whole blood cells, after dextrane sedimentation, or with purified basophils. Whole-blood experiments represent the physiologic cellular environment more closely, but interference with serum components like IgG antibodies, anti-IgE or antireceptor antibodies can possibly occur. In contrast, separation and purification of basophils requires experience and can lead to cell loss and accidental activation.

Enhanced signals by prestimulation with IL-3 Preactivation with the priming cytokine IL-3 at concentrations of 2 ng/mL has been introduced to enhance basophil signals after weak stimulation. However, since IL-3 affects downstream rather than early signaling events (Vilarino et al. 2005), it will lead only to a moderate shift of the resulting dose–response curves, reflecting the generally enhanced responses to various stimulus concentrations.

Positive and negative control The diagnostic window of basophil stimulation tests is defined by a positive and a negative control. The latter is usually the diluent of the stimuli to be tested. For IgE-dependent stimulation, a positive control, such as anti-IgE or anti-FcεRIα antibodies, has been introduced. Currently, the best anti-IgE antibody is not a mouse monoclonal antibody, but rather a polyclonal anti-IgE antibody preparation. IgE-independent stimuli, such as formyl-methionyl-leucylphenylalanine (fMLP) combined with C5a, cannot be considered an appropriate positive control in diagnostic settings

looking for IgE-dependent reactions (Kleine-Tebbe et al. 2006). They can be used as additional positive controls to verify cell functionality and basophil test performance. This approach is particularly useful in the case of a negative response to anti-IgE or anti-FcεRIα, indicating nonresponsiveness to IgEmediated stimulation occurring in 10–20% of donors due to a defect in early signaling, particularly Syk phosphorylation (Kepley et al. 2000; Vilarino et al. 2005).

Allergen selection and challenge concentration Features of the allergens, most commonly natural extracts, will provide the basis for successful IgE-mediated activation of basophils, usually expressed as the percentage of activated basophils. Similar to other allergen-based diagnostic methods, like IgE assays or skin tests, the quality of the allergen preparations is of paramount importance for obtaining valid results after basophil stimulation. Variable compositions and potencies and the presence of additional stimulants or inhibitory components (endotoxins, lectins, preservatives) can profoundly influence the diagnostic result. Bell-shaped allergen dose–response curves from basophil mediator release experiments can differ in the ascending part from the rather sigmoid curves generated with activation markers like CD63 and CD203c. Therefore, suitable allergen concentrations should not be simply deduced from histamine or leukotriene studies but checked in dose-finding experiments for each individual allergen, in order to define a range for submaximal and maximal responses. The shape of the allergendriven dose–response curves will depend on the number of IgE-binding epitopes applied in the test. Therefore, complex extracts will usually generate broad dose–response curves spanning several logs, while single allergens and molecules with limited IgE-binding sites will generally show a considerably smaller range of basophil-activating concentrations (Abuaf et al. 1999).

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Table 65.4 Basophil-based assays, read-out and manufacturers. Brand name

Assay principle

Manufacturer

Flow-CAST,* FAST Basotest No brand name available CAST ELISA*

CD63, flow cytometry CD63, flow cytometry CD203c, flow cytometry ELISA for sulfidoleukotrienes (LTC4, LTD4, LTE4) Fluorometric assay for histamine RIA for histamine, ELISA for histamine

Bühlmann Laboratories AG, Switzerland Orpegen Pharma, Heidelberg, Germany Miltenyi Biotec, Germany Bühlmann Laboratories AG, Switzerland

HR-test Histamine RIA, histamine ELISA, histamine EIA

RefLab, Copenhagen, Denmark Several manufacturers

* A combined assay is marketed as Combi CAST. HR, histamine release.

Read-out for the analysis of basophil activation CD63-based methods Basophil-based stimulation tests are commonly performed by identifying cells with anti-IgE and determining their activation by applying anti-CD63 to washed cells (Bühlmann Laboratories, Basel, Switzerland; Orpegen Pharma, Heidelberg, Germany) or whole blood (Pharmingen, Biosciences, Erembodegen-Aalst, Belgium) (Table 65.4). Technical aspects of the anti-IgE/anti-CD63 protocol have been extensively described (Ebo et al. 2005a). Potential activation of basophils by the anti-IgE reagent, potential binding of the anti-IgE to other IgE-bearing cells like monocytes and dendritic cells, and additional expression of CD63 on thrombocytes have been some of the concerns regarding this method. These issues can be resolved (Ebo et al. 2004), but have to be taken into account during planning of the individual experiment (i.e., application of a stimulus or a drug which alters thrombocyte adherence or aggregation). Another approach is labeling of CD203c, a selective marker for peripheral blood basophils, in addition to CD63. An additional option for identifying basophils is the use of anti-CD123 and anti-HLA-DR, targeting the population of CD123+/HLA-DR cells (Sturm et al. 2004; Ebo et al. 2005a,b).

CD203c-based methods Because of the exclusive expression of CD203c on basophils, CD203c-based methods circumvent the need of anti-IgE or anti-CD123/anti-HLA-DR for basophil identification, and enable the establishment of a single-color basophil activation assay (Kahlert et al. 2003; Bühring et al. 2004). Pitfalls can be related to various issues: difficulties in detecting nonstimulated basophils in the case of low constitutive expression of the marker; less pronounced upregulation in comparison with CD63; and preactivated basophils with spontaneous CD203c expression may fail to show further activation with other stimuli. Additional markers with similar behavior to CD203c include CD13 and CD164. Another basophil antigen resembles

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CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), which is combined with CD3-negative staining and is offered in a CRTH2/CD3/CD203c protocol (Beckman Coulter, Mijdrecht, The Netherlands).

Mediator-based methods Some laboratories use the detection of histamine by fluometric techniques (Siraganian & Hook 1980; Skov et al. 1985), often expressed as percent released histamine of the total histamine content of a parallel sample, or immunoassay (Immunotech, Marseille, France; Hycor Biomedical Inc., Garden Grove, CA 92841) after basophil activation, providing histamine concentrations in ng/mL. They can be transformed to percent histamine release (HR) by the following formula (Siraganian & Hook 1980). HR (%) = (HRsample/HRdiluent)/(total histamine content/HRdiluent) × 100 Most of the fundamental findings with regard to antigen– FcεRI interaction and signal–response coupling stems from histamine release studies using human leukocytes (Oliver et al. 2000; MacGlashan 2005). Production of sulfidoleukotrienes (given in pg/mL), which can be monitored with a sensitive ELISA (Bühlmann Laboratories, Basel, Switzerland) (see Table 65.2), has been utilized as a read-out for basophil activation using various stimuli (de Weck & Sanz 2004). Individual and stimulus-dependent variability, the complexity of the cellular test performance, and difficulties in interpretation have hampered wider use of this method in the diagnostic allergy laboratory.

Clinical results with basophil tests Various studies have been performed with basophils from donors allergic to inhalants, latex, foods, hymenopteran venoms, drugs, and related compounds. The results, mostly obtained in recent years with the activation markers CD63

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Table 65.5 Diagnostic performance of flow cytometric analysis of basophils after IgE-dependent activation ex vivo. (Modified from Ebo et al. 2006a, with permission.) Stimulus

Reference test

Sensitivity (%)

Specificity (%)

N*

Reference

House-dust mite (HDM) Dactylis glomerata Cypress pollen HDM and Lolium perenne Latex Latex Latex Wasp venom Wasp venom Wasp venom Bee venom b-Lactam b-Lactam Metamizol Aspirin and NSAID NBA NBA NBA Carrot–celery–hazelnut Apple Apple (Mal d 1) Celery (Api g 1) Carrot (Dau c 1)

H + IgE and/or ST H + IgE and/or ST H + ST + PT H + IgE + ST H + IgE + ST H + ST H + IgE and/or ST H + IgE and ST H H H H + ST H + ST ± IgE + PT H + PT H + PT H H + ST H H (OAS) H (OAS) H (OAS)

56–78 73–100 91 93 93 93 80 84 92 85 91 50 49 42 15–5 64 54 79 85–90 90 75 65 75

91–100 100 100 98 92 100 97 100 80 83 90 93 91 100 74–100 93 100 100 80–90 100 64 86 82

20 20 75 128 102 73 79 94 70 87 87 88 110 55 90 50 60 24 40 59 54 54 54

Cozon et al. (1999)† Cozon et al. (1999)† Paris-Köhler et al. (2000) Sanz et al. (2001) Ebo et al. (2002) Sanz et al. (2003) Hemery et al. (2005) Ebo et al. (2006c) Erdmann et al. (2004) Sturm et al. (2004) Sturm et al. (2004) Sanz et al. (2002) Torres et al. (2004) Gamboa et al. (2003) Gamboa et al. (2004) Abuaf et al. (1999) Monneret et al. (2002) Sudheer et al. (2005) Erdmann et al. (2003) Ebo et al. (2005a) Erdmann et al. (2005) Erdmann et al. (2005) Erdmann et al. (2005)

* Total number of patients and controls. † In the study by Cozon et al. (1999), sensitivity and specificity vary according to different IL-3 preactivation protocols. H, history; NBA, neuromuscular blocking agent; NSAID, nonsteroidal antiinflammatory drug; PT, provocation test; ST, skin test.

and CD203c, have been thoroughly reviewed (Sanz et al. 2002; Ebo et al. 2004, 2006a) and are listed in Table 65.5. Some of the studies are based on small groups, single-stimulus concentrations without documented dose-finding experiments, or inappropriate positive controls like fMLP (Paris-Köhler et al. 2000; Sanz et al. 2001; Ebo et al. 2002; Erdmann et al. 2003, 2004; Torres et al. 2004). Under these conditions, it may be difficult to calculate diagnostic performance characteristics as sensitivity, specificity, and predictive values. In addition, allergy diagnoses obtained by such tests were rarely confirmed by provocation tests, leaving a considerable degree of uncertainty concerning the clinically relevant symptoms of the study population. Interesting results with successful basophil activation tests have been obtained in difficult cases with rare allergens (Ebo et al. 2006b) and stimuli (Ebo et al. 2005b).

Indications, performance and interpretation Indications Basophil-based tests may provide additional and important information in particular diagnostic situations (Kleine-Tebbe et al. 2006).

• Suggestive history of IgE-mediated allergy, negative skin test, and negative IgE. Due to their extremely high analytical sensitivity, basophil-based tests can indirectly indicate allergenspecific IgE on the cell, especially if allergen-specific serum IgE levels are low (under threshold). This is rarely the case in atopic individuals, but can be encountered in nonatopic individuals with IgE-mediated sensitizations to nonatopic allergens (hymenopteran venom, IgE-inducing drug allergens, some occupational allergens). • History of IgE-mediated allergy with important therapeutic implications, discordance between skin test and IgE measurement. A basophil-based test can serve as another indirect indicator (besides skin tests) for IgE-mediated reactions and can subsequently confirm important therapeutic decisions (i.e., for occupational allergens or drug allergens with questionable skin test or IgE results). • History of IgE-mediated reaction, if no appropriate allergen for IgE detection is available. In some instances, an allergen preparation is not available for allergen-specific IgE measurements, but an aqueous solution for skin tests and subsequent basophil tests may exist. Positive test results could indirectly suggest IgE-mediated sensitization. Additional appropriate

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negative control results with nonaffected individuals have to be demonstrated. This is particularly important in the case of noncharacterized, possibly contaminated, allergen preparations with irritative potential, as basophil tests represent bioassays based on live cells.

Performance Diagnostic tests with basophils consist of three steps: preanalytical handling; stimulation of the basophils, including appropriate controls; and read-out of the activation signal. Basophil-containing samples should be handled with care. Reactivity and viability will continuously decline within hours; therefore transportation and subsequent experimentation should be carefully organized, the latter ideally performed within a few hours. Proper stimulation requires experience in handling basophil leukocytes. Inadequate steps like vigorous vortexing, delayed work-up and incorrect temperatures can easily ruin the experiment. Similar to other diagnostic tests in allergy, the source and quality of the applied stimuli (i.e., allergens) will be crucial for the validity of the result.

Interpretation Different outcomes are to be expected after basophil-based tests (Kleine-Tebbe et al. 2006). • A positive test indicates IgE-mediated sensitization, provided that appropriate allergens have been applied, that negative control values with the same allergens and nonaffected individuals are available, and that positive parallel controls (anti-IgE or anti-FcεRI antibodies) have demonstrated a positive response. • A negative test can exclude IgE-mediated sensitization, provided that appropriate allergens have been applied, which have demonstrated a positive response in other affected individuals; and that positive parallel controls with anti-IgE or anti-FcεRI antibodies have demonstrated a positive response. • Inconclusive results have to be considered, i.e., if positive parallel controls (anti-IgE or anti-FcεRIα antibodies) did not induce a positive response, regardless of the response to the allergen in question, while IgE-independent controls (fMLP plus C5a) demonstrated a positive response (indicating sufficient cell viability and robust general test performance). • No results (due to preanalytical or laboratory failure) can be considered, i.e., if all positive controls (anti-IgE or anti-FcεRIα antibodies and fMLP plus C5a) have failed to induce a positive response, indicating insufficient cell viability or failure in performing the test.

Perspectives Basophil-based tests represent laboratory ex vivo methods with extraordinarily high analytical sensitivity. Subsequently, trace amounts of antigens can be compared for potency and IgE-binding capacity (Kahlert et al. 2003). Basophil-based tests are valuable research tools for better understanding signal–response coupling, i.e., IgE-mediated signaling, activa-

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tion, and degranulation. Novel insights into both stimulatory and inhibitory signals after FcεRI-mediated activation might establish new treatment options for allergic reactions and diseases (Oliver et al. 2000). Compared with the detection of IgE in serum, basophil tests have the advantage of demonstrating functional responses: IgE-mediated activation will only occur after successful crosslinking of two IgE molecules by an allergenic molecule, and not by incomplete antigens with only one IgE-binding epitope. However, the handling of basophils and the extraordinary variability of their responses represent inherent difficulties, limiting the potential of basophil-based tests to complement diagnostic tools in selected cases.

References Aalberse, R.C. (1991) The IgE response and atopy. Eur Respir J 4 (suppl. 13), 78–84. Aalberse, R.C. (2000) Autologous dust RAST. A neglected tool to detect idiopathic sources of allergens in the home. Clin Rev Allergy Immunol 18, 301–9. Aalberse, R.C. & Schuurman, J. (2002) IgG4 breaking the rules. Immunology 105, 9–19. Aalberse, R.C., Koshte, V. & Clemens, J.G. (1983a) Immunoglobulin E that cross-react with vegetable foods, pollen and Hymenoptera venom. J Allergy Clin Immunol 68, 356–64. Aalberse, R.C., van der Gaag, R. & van Leeuwen, J. (1983b) Serologic aspects of IgG4 antibodies. 1. Prolonged immunization results in an IgG4-restricted response. J Immunol 130, 722–6. Abuaf, N., Rajoely, B., Ghazouani, E. et al. (1999) Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy. J Allergy Clin Immunol 104, 411–18. Atkinson, W., Sheldon, A., Shaath, N. & Whorwell, P.J. (2004) Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial. Gut 53, 1459–64. Ballmer-Weber, B.K., Wangorsch, A., Bohle, B. et al. (2005) Component-resolved in vitro diagnosis in carrot allergy: does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure? Clin Exp Allergy 375, 970–8. Bühring, H.J., Seiffert, M., Giesert, C. et al. (2001) The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3. Blood 97, 3303–5. Bühring, H.J., Streble, A. & Valent, P. (2004) The basophil-specific ectoenzyme E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis. Int Arch Allergy Immunol 133, 317–29. Calkhoven, P.G. (1989) The role of food antigens in the development of inhalant allergy. Thesis. University of Amsterdam. Calkhoven, P.G., Aalbers, M., Koshte, V.L. et al. (1991) Relationship between IgG1 and IgG4 antibodies to foods and the development of IgE antibodies to inhalant allergens. I. Establishment of a scoring system for the overall food responsiveness and its application to 213 unselected children. Clin Exp Allergy 21, 91–8. Celik-Bilgili, S., Mehl, A., Verstege, A. et al. (2005) The predictive value of specific immunoglobulin E levels in serum for the outcome of oral food challenges. Clin Exp Allergy 35, 268–73.

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Cozon, G., Ferrandiz, J., Peyramond, D. & Brunet, J. (1999) Detection of activated basophils using flow cytometry for diagnosis in atopic patients. Allergol Immunopathol (Madr) 27, 182–7. de Maat-Bleeker, F. & Stapel, S.O. (1998) Crossreactivity between latex and buckwheat. Allergy 53, 538–9. de Weck, A.L. & Sanz, M.L. (2004) Cellular allergen stimulation test (CAST) 2003: a review. J Invest Allergol Clin Immunol 14, 253–73. Ebo, D.G., Lechkar, B., Schuerwegh, A.J., Bridts, C.H., De Clerck, L.S. & Stevens, W.J. (2002) Validation of a two-color flow cytometric assay detecting in vitro basophil activation for the diagnosis of IgEmediated natural rubber latex allergy. Allergy 57, 706–12. Ebo, D.G., Hagendorens, M.M., Bridts, C.H., Schuerwegh, A.J., De Clerck, L.S. & Stevens, W.J. (2004) In vitro allergy diagnosis: should we follow the flow? Clin Exp Allergy 34, 332–9. Ebo, D.G., Hagendorens, M.M., Bridts, C.H., Schuerwegh, A.J., De Clerck, L.S. & Stevens, W.J. (2005a) Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy. Cytometry B Clin Cytom 64B, 28–33. Ebo, D.G., Wets, R.D., Spiessens, T.K., Bridts, C.H. & Stevens, W.J. (2005b) Flow-assisted diagnosis of anaphylaxis to patent blue. Allergy 60, 703– 4. Ebo, D.G., Sainte-Laudy, J., Bridts, C.H. et al. (2006a) Flow-assisted allergy diagnosis: current applications and future perspectives. Allergy 61, 1028–39. Ebo, D.G., Bridts, C.H., Mertens, M.H. & Stevens, W.J. (2006b) Coriander anaphylaxis in a spice grinder with undetected occupational allergy. Acta Clin Belg 61, 152– 6. Ebo, D.G., Hagendorens, M.M., Schuerwegh, A.J. et al. (2007) Flowassisted quantification of in vitro activated basophils in the diagnosis of wasp venom allergy and follow-up of wasp venom immunotherapy. Cytometry B Clin Cytom 72, 196–203. Eigenmann, P.A. (2005) Are specific immunoglobulin E titres reliable for prediction of food allergy? Clin Exp Allergy 35, 247–9. Erdmann, S.M., Heussen, N., Moll-Slodowy, S., Merk, H.F. & Sachs, B. (2003) CD63 expression on basophils as a tool for the diagnosis of pollen-associated food allergy: sensitivity and specificity. Clin Exp Allergy 33, 607–14. Erdmann, S.M., Sachs, B., Kwiecien, R., Moll-Slodowy, S., Sauer, I. & Merk, H.F. (2004) The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy. Allergy 59, 1102–9. Erdmann, S.M., Sachs, B., Schmidt, A. et al. (2005) In vitro analysis of birchpollen-associated food allergy by use of recombinant allergens in the basophil activation test. Int Arch Allergy Immunol 136, 230– 8. Eysink, P.E.D., Bottema, B.J.A.M., ter Riet, G., Aalberse, R.C., Stapel, S.O. & Bindels, P.J.E. (2004) Coughing in pre-school children in general practice: when are RASTs for inhalation allergy indicated? Pediatr Allergy Immunol 15, 394–400. Fall, I.B., Eberlein-Konig, D., Behrendt, H., Niessner, R., Ring, J. & Weller, M.G. (2003) Microarrays for the screening of allergenspecific IgE in human serum. Anal Chem 75, 556–62. Florvaag, E., Johansson, S.G.O., Öman, H., Harboe, T. & Nopp, A. (2006) Pholcodine stimulates a dramatic increase of IgE in IgEsensitized individuals. A pilot study. Allergy 61, 49–55. Gamboa, P.M., Sanz, M.L., Caballero, M.R. et al. (2003) Use of CD63 expression as a marker of in vitro basophil activation and leukotriene determination in metamizol-allergic patients. Allergy 58, 312–17.

Allergy Testing in the Laboratory

Gamboa, P., Sanz, M.L., Caballero, M.R. et al. (2004) The flowcytometric determination of basophil activation induced by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is useful for in vitro diagnosis of the NSAID hypersensitivity syndrome. Clin Exp Allergy 34, 1448–57. Gugnani, H.C., Reijula, K.E., Kurup, V.P. & Fink, J.N. (1990) Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay. Mycopathologia 109, 33–40. Hemery, M.L., Arnoux, B., Dhivert-Donnadieu, H. et al. (2005) Confirmation of the diagnosis of natural rubber latex allergy by the Basotest method. Int Arch Allergy Immunol 136, 53–7. Hiller, R., Laffer, S., Harwanegg, C. et al. (2002) Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment. FASEB J 16, 414–16. Hofer, M.F., Harbeck, R.J., Schliefert, P.M. & Leung, D.Y.M. (1999) Staphylococcal toxins augment specific IgE responses by atopic patients exposed to allergen. J Invest Dermatol 112, 171–6. Hunter, J.O. (2005) Food elimination in IBS: the case for IgG testing remains doubtful. Gut 54, 1203. Ishizaka, K. & Ishizaka, T. (1967) Identification of gamma-E antibodies as a carrier of reaginic antibody. J Immunol 99, 1187–98. Iwamoto, I., Yamazaki, H., Kimura, A., Ochiai, K., Tomioka, H. & Yoshida, S. (1990) Comparison of a multi-allergen dipstick IgE assay to skin-prick test and RAST. Clin Exp Allergy 20, 175–8. Johansson, S.G.O. & Bennich, H. (1967) Immunological studies of an atypical (myeloma) immunoglobulin. Immunology 13, 381– 94. Jutel, M., Akdis, M., Blaser, K. & Akdis, C.A. (2006) Mechanisms of allergen specific immunotherapy T cell tolerance and more. Allergy 61, 796–807. Kagey-Sobotka, A., MacGlashan, D.W. & Lichtenstein, L.M. (1982) Role of receptor aggregation in triggering IgE-mediated reactions. Fed Proc 41, 12–16. Kahlert, H., Cromwell, O. & Fiebig, H. (2003) Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c. Clin Exp Allergy 33, 1266–72. Kauffman, H.F., Beaumont, F., Meurs, H., van der Heide, S. & de Vries, K. (1983) Comparison of antibody measurements against as pergillus fumigatus by means of double-diffusion and enzymelinked immunosorbent assay (ELISA). J Allergy Clin Immunol 72, 255– 61. Kepley, C.L., Youssef, L., Andrews, R.P., Wilson, B.S. & Oliver, J.M. (2000) Multiple defects in FcεRI signaling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion. J Immunol 165, 5913–20. Kim, T.E., Park, S.W., Cho, N.Y. et al. (2002) Quantitative measurements of serum allergen-specific IgE on protein chip. Exp Mol Med 34, 152–8. Kjellman, N.-I.M., Johansson, S.G.O. & Roth, A. (1976) Serum IgE levels in healthy children quantified by a sandwich technique (PRIST). Clin Allergy 6, 51–9. Kleine-Tebbe, J., Erdmann, S., Knol, E.F., MacGlashan, D.W. Jr, Poulsen, L.K. & Gibbs, B.F. (2006) Diagnostic tests based on human basophils: potentials, pitfalls and perspectives. Int Arch Allergy Immunol 141, 79–90. Knol, E.F., Verhoeven, A.J. & Roos, D. (1993) Stimulus secretion coupling in human basophilic granulocytes. Clin Exp Allergy 23, 471–80.

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Diagnosis of Allergic Disease

Lichtenstein, L.M., Schleimer, R.P., Peters, S.P. et al. (1983) Studies with purified human basophils and mast cells. Monogr Allergy 18, 259–64. Lopata, A.L., Schinkel, M., Potter, P.C. et al. (2004) Qualitative and quantitative evaluation of bird-specific IgG antibodies. Int Arch Allergy Immunol 134, 173– 8. MacGlashan, D.W. Jr. (2005) IgE and FcεRI Regulation. Ann NY Acad Sci 1050, 73–88. MacGlashan, D.W. Jr, Schroeder, J.T., Lichtenstein, L.M., Saini, S.A. & Bochner, B.S. (2002) Mediator release from basophils and mast cells and its relationship to FceRI expression and IgE-suppressing therapies. In: Fick, R.B. Jr & Jardieu, P.M., eds. IgE and Anti-IgE Therapy in Asthma and Allergic Disease. Marcel Dekker, New York, pp. 39–68. Mari, A., Ooievaar-de Heer, P., Scala, E., Bethel, D. & van Ree, R. Production of pharmaceutical proteins in plants: glycosylation does not create a risk of allergic reactions. Submitted for publication. Monneret, G., Benoit, Y., Debard, A.L., Gutowski, M.C., Topenot, I. & Bienvenu, J. (2002) Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs. Clin Immunol 102, 192–9. Nouri-Aria, K.T., Wachholz, P.A., Francis, J.N. et al. (2004) Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity. J Immunol 172, 3252–9. Oliver, J.M., Kepley, C.L., Ortega, E. & Wilson, B.S. (2000) Immunologically mediated signaling in basophils and mast cells: finding therapeutic targets for allergic diseases in the human FcvarepsilonR1 signaling pathway. Immunopharmacology 48, 269–81. Osler, A.G., Lichtenstein, L.M. & Levy, D.A. (1968) In vitro studies of human reaginic allergy. Adv Immunol 8, 183–231. Parish, W.E. (1970) Short-term anaphylactic IgG antibodies in human serum. Lancet ii, 591–2. Paris-Köhler, A., Demoly, P., Persi, L., Lebel, B., Bousquet, J. & Arnoux, B. (2000) In vitro diagnosis of cypress pollen allergy by using cytofluorimetric analysis of basophils (Basotest). J Allergy Clin Immunol 105, 339– 45. Prussin, C. & Metcalfe, D.D. (2006) 5. IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol 117, S450– S456. Redecke, V., Häcker, H., Datta, S.K. et al. (2004) Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J Immunol 172, 2739–43. Sampson, H.A. (2001) Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 107, 891–6. Sampson, H.A. & Ho, D.G. (1997) Relationship between foodspecific IgE concentrations and the risk of positive food challenges in children and adolescents. J Allergy Clin Immunology 100, 441– 51. Sanz, M.L., Sanchez, G., Gamboa, P.M. et al. (2001) Allergen-induced basophil activation: CD63 cell expression detected by flow cytometry in patients allergic to Dermatophagoides pteronyssinus and Lolium perenne. Clin Exp Allergy 31, 1007–13. Sanz, M.L., Gamboa, P.M., Antepara, I. et al. (2002) Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate-type reactions to betalactam antibiotics. Clin Exp Allergy 32, 277– 86. Sanz, M.L., Gamboa, P.M., Garcia-Aviles, C. et al. (2003) Flowcytometric cellular allergen stimulation test in latex allergy. Int Arch Allergy Immunol 130, 33– 9.

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Measurement of Markers of Inflammation in Induced Sputum and Exhaled Air Ian D. Pavord and Dominick E. Shaw

Summary

Measurement of airway inflammation

Induced sputum is safe in both children (Venge et al. 1977) and adults (Gibson et al. 2002; Green et al. 2002a), repeatable, inexpensive, and provides a clinically relevant marker of airway inflammation in the assessment and treatment of asthma. It can also be used to measure a variety of cells, effector mediators, cellular markers, and cytokines and its use has led to new insights into the pathophysiology and management of asthma and other airways disease. FENO is a reasonably reliable marker of eosinophilic airway inflammation that can be measured simply and noninvasively. The clinical value of measuring FENO has not been explored extensively but it could be a valuable tool for diagnosis and monitoring of corticosteroid-responsive airways disease in primary care.

Airway inflammation can be assessed by the measurement of cells and biomarkers in induced sputum or blood and by assessment of the content of exhaled gases or condensate. More invasive techniques include bronchial biopsy, bronchial wash, and bronchoalveolar lavage. These techniques can provide information on airway structure and the site of the inflammatory response as well as its nature. Table 66.1 summarizes some of the characteristics of the various approaches used to assess airway inflammation. The bulk of the work done to date on the validation and clinical application of measures of airway inflammation has been with induced sputum and exhaled nitric oxide (FENO); this chapter focuses particularly on these techniques. Sputum induction has been shown to be safe and applicable across a variety of clinical situations. The technique of sputum processing is inexpensive, although training and experience are required to obtain reliable results. The most clinically important results are the differential inflammatory cell counts; these have been shown to be repeatable and responsive in a variety of clinical situations (Pizzichini et al. 1996a; Pavord et al. 1997). Induced sputum has the advantage of providing measurements of the type of airway inflammation (eosinophilic versus neutrophilic) as well as its severity. The main limitation is that results are not available immediately, limiting the application of the technique in asthma monitoring. FENO has the advantage of being simple to measure and provides an immediate result, but it cannot provide accurate information on the nature of the inflammatory response. The different strengths and weaknesses of the techniques suggest that they may find different roles, with FENO being used mainly in primary care to facilitate diagnosis and to titrate corticosteroid therapy and induced sputum used in secondary and tertiary care, where more detailed information on the type of lower airway inflammation is necessary.

Introduction The goals of asthma management are the accurate diagnosis and effective control of symptoms, prevention of exacerbations, and the achievement of best possible pulmonary function with minimal side effects (Anon. 2003). It has not been standard practice to assess and control the airway inflammation that is thought to be the basis for many of the clinical manifestations of the disease. However, the recent development and validation of noninvasive methods to assess airway inflammation suitable for use in clinical practice has made it possible to do this. In this chapter we review methodologic aspects of the assessment of airway inflammation and discuss how the measurement of airway inflammation has informed our understanding of the link between airway dysfunction and airway inflammation. Finally, we review evidence that measurement of airway inflammation provides additional, clinically important information that is necessary for the optimum management of the patient with airway disease.

Assessment of airway inflammation using induced sputum Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Methodology Sputum induction using nebulized hypertonic saline is used to collect respiratory secretions from the airways of patients

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Table 66.1 Comparison of methods measuring airway inflammation. Ease of performing technique

Ease of analyzing result

Induced sputum

Simple sputum sample

Blood eosinophil count

Time to result

Cost

Influence on outcome proved

Potential use

Simple, but requires experience

3–4 hours

Moderate

Yes

Secondary care

Simple blood test

Simple

30 min

Inexpensive

May help differentiate atopic and intrinsic asthma (Venge et al. 1977)

Secondary care

Eosinophil cationic protein

Urine test/sputum test/blood test

Simple

3–4 hours

Moderate

Not proven conclusively

Research

Exhaled nitric oxide

Very easy breathing test

Easy

Immediate

Expensive

Not proven

Research

Carbon monoxide

Electrochemical sensor/ (laser spectrophotometer/ near-infrared analyzer)

Easy (can be influenced by hydrogen)

Immediate

Inexpensive

Studies awaited

Research

Breath condensate (hydrocarbons)

Cooling/freezing of exhaled air

Simple

Moderate

Inexpensive

Studies awaited

Research

BAL, bronchial wash and biopsy

Biopsy-invasive

Complex

2 days

Moderate

Yes

Secondary care

who do not expectorate spontaneously. It is generally agreed that the central airways are sampled with induced sputum. This view is supported by studies showing a greater proportion of granulocytes in both sputum and bronchial samples compared with bronchoalveolar lavage (Pizzichini et al. 1998a; Alexis et al. 2000; Moodley et al. 2000), and by the demonstration that sputum induction results in greater clearance of radiolabeled aerosol from the central airways than the peripheral airway (Alexis et al. 2001). There is evidence that increasing the duration of sputum induction leads to sampling of more distal airways although, as yet, the clinical utility of this technique has not been explored (Gershman et al. 1999). The precise mechanism leading to production of secretions is not known, but it may involve both direct and indirect mechanisms. The increased osmolarity of the airway lining fluid during induction is thought to precipitate production of mucous by the submucosal glands; it may also lead to increased mucus production by increasing the vascular permeability of the bronchial mucosa and by provoking release of proinflammatory mediators. A variety of protocols for sputum induction have been published and shown to be safe provided patients are pretreated with bronchodilators and monitored carefully (Hunter et al. 1999; Paggiaro et al. 2002). Risk factors for bronchoconstriction include a low baseline forced expiratory volume in 1 s (FEV1) percent predicted (de la Fuente et al. 1998), overuse of short-acting β2 agonists (ten Brinke et al. 2001), and poor asthma control (ten Brinke et al. 2001). Theoretically, higher nebulizer output, higher concentration of inhaled saline, longer duration of saline inhalation, and reduced frequency

and timing of safety assessment by FEV1 or peak expiratory flow (PEF) might also influence safety. Higher output nebulizers have also been associated with the development of sputum neutrophilia 24 hours after sputum induction (Pavord 1998). Whether this is seen with the low-output nebulizers is unknown. A common protocol is to use a relatively lowoutput ultrasonic nebulizer (output 0.7–0.9 mL/min) as there is wide experience with this method and it has been shown to be successful in various settings (Brightling et al. 2001; Green et al. 2002a,b). Furthermore there are theoretical reasons to suggest that that the risk of bronchoconstriction and the effect of sputum induction on neutrophil counts might be less (Pavord 1998). The method is summarized in Fig. 66.1. Once expectorated, sputum should be processed within 2 hours. There is evidence that sputum can be stored for up to 9 hours in a refrigerator at 4°C or that sputum can be snap frozen for longer without affecting cell counts (Efthimiadis et al. 2002), although experience with these techniques is limited. The whole expectorate or selected sputum plugs can be processed. The latter approach has the advantage of producing better quality cytospins and more repeatable differential cell counts (Ward et al. 1999). Sputum plugs are selected and centrifuged with dithiothreitol (Fig. 66.2). The total cell count, cell viability, and squamous cell contamination are assessed using a hemocytometer. Differential cell counts are determined by counting 400 leukocytes on an appropriately stained cytospin. Other biomarkers can also be measured in the sputum supernatant. Some of the molecular markers of airway inflammation that have been successfully measured in sputum are shown in Table 66.2.

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Measure FEV1 – 3 times

Process at 4°C within 2 hours of expectoration Select sputum (if necessary using inverted microscope)

Salbutamol 200 mg – by MDI with spacer Weigh and incubate with 4 × volume 0.1% dithiothreitol (DTT) Remeasure FEV1 after 20 minutes – 3 times Gently aspirate with Pasteur pipette, vortex for 15 seconds Administer 3% saline using an ultrasonic nebulizer for 5 minutes Rock on bench rocker for 15 minutes on ice Blow nose, rinse mouth and swallow water

Mix with equal volume (to DTT) of Dulbecco’s phosphate-buffered saline (D-PBS)

Expectorate sputum Vortex for 15 seconds ≥ 10%, < 20% fall in FEV1 Filter through 48 mm nylon gauze (pre-wet with D-PBS)

Remeasure FEV1 (3 times if less than best post bronchodilator value) < 10% fall in FEV1

≥ 20% fall in FEV1 or troublesome symptoms

Centrifuge 790 g 10 minutes

Store supernatant at –70°C

Resuspend cell pellet in D-PBS

Measure fluid phase

Discontinue

Repeat procedure with 4 and 5% saline

Perform total cell count and viability by trypan blue exclusion method in Neubauer hemocytometer

Fig. 66.1 Protocol for sputum induction using nebulized hypertonic saline. Adjust cell suspension to 0.5–0.75 × 106 cells/mL with D-PBS

Measurement characteristics of induced sputum Induced sputum is well validated (Pavord et al. 1997) and normal ranges have been published for a large adult population (Belda et al. 2000; Spanevello et al. 2000; Thomas et al. 2004) (Table 66.3). Age has been shown to influence differential sputum neutrophil counts, with the higher values occurring in the older age groups (Thomas et al. 2004). Up to 80% of corticosteroid-naive patients (Pizzichini et al. 1996a;

Table 66.2 Cell types and molecular markers that have been successfully measured in induced sputum. Cells Eosinophils Neutrophils Macrophages Lymphocytes Epithelial cells Effector mediators LTC4/LTD4/LTE4 PGD2 Histamine Cellular markers Eosinophilic cationic protein Neutrophil elastase Cytokines/chemokines IL-8

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Prepare cytospins by placing 50 mL or 75 mL cell suspension in cups of cytocentrifuge and centrifuge at 450 rpm for 6 minutes

Fig. 66.2 Protocol for processing of sputum.

Pavord et al. 1997) and 50% of corticosteroid-treated patients (Louis et al. 2000; Green et al. 2002b) with current asthmatic symptoms have a sputum eosinophil count above the normal range. There is good evidence that the sputum differential eosinophil, macrophage and neutrophil counts and the sputum supernatant concentration of eosinophilic cationic protein (ECP), cystinyl leukotrienes, prostanoids, and interleukin (IL)-8 can be measured repeatably in asthma (in’T Veen et al. 1996; Pizzichini et al. 1996a; Pavord et al. 1999a) (Table 66.3) and in chronic obstructive pulmonary disease (COPD) (Brightling et al. 2001). The differential lymphocyte and epithelial cell count and the total cell count are less repeatable (Pizzichini et al. 1996a). The sputum eosinophil count is responsive in that it increases when asthma worsens (e.g., after allergen challenge and following relevant occupational exposures) (Pizzichini et al. 1996b; Lemiere et al. 1999) and decreases when asthma improves with inhaled corticosteroid treatment (Pavord et al. 1999b). Sputum and bronchoscopy studies with corticosteroids (Bentley et al. 1996; Pavord et al. 1999b) and anti-IL-5 (Leckie et al. 2000; Flood-Page et al. 2003) suggest that the sputum eosinophil count is more

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Table 66.3 Normal ranges and within-subject repeatability of induced sputum cell counts in adults. Normal ranges

Repeatability

Belda et al. (2000)

Spanevello et al. (2000)

Thomas et al. (2004)

Pizzichini et al. (1996a)

Efthimiadis et al. (2002)

Number (male) Characteristics

96 (54) Healthy

96 (46) Healthy

66 (24) Healthy

21 (10) Asthma

Mean age TCC (×106/g) Eosinophils (%) Neutrophils (%) Macrophages (%) Lymphocytes (%) Epithelial cells (%)

Not recorded 4.1 (4.8) 0.4 (0.9) 37.5 (20.1) 58.8 (21.0) 1.0 (1.1) 1.6 (3.9)

Not recorded 2.7 (2.5) 0.6 (0.8) 27.3 (13.0) 69.2 (13.0) 1.0 (1.2) 1.5 (1.8)

46 (25) 2.1 (2.4) 0.3 (0.6) 47.0 (27.0) 49.0 (25.2) 1.0 (1.4) 2.5 (3.2)

39 (20) Asthma (19) Healthy (10) Smokers (10) 39 0.35 0.94 (0.75*)† 0.81 (14.0) 0.71 0.25

24 (4) 0.85 (6.2) 0.57 (15.5) 0.64 (7.7) 0.76 (3.9) 0.64 (7.7)

Ward et al. (1999) 88 (44) Asthma (53) Healthy (19) Rhinitis (16) 38 0.44 0.84 (0.17*) 0.75 0.76 0.39 0.56

Figures represent mean (SD). Repeatability expressed as intraclass correlation coefficient (within-subject SD or *log within-subject SD). Repeatability was assessed over 2 days (Efthimiadis et al. 2002), 6 days (Pizzichini et al. 1996a), and 1 week (Ward et al. 1999). † Within-subject log standard deviation of sputum eosinophil count overly influenced by small absolute differences in normals; in subjects with asthma it was 0.25.

responsive than tissue eosinophil counts. The sputum differential neutrophil count is reduced following treatment with the fixed-dose combination inhaler Seretide in patients with COPD (Barnes et al. 2006). There are theoretical reasons to suggest that the total neutrophil count may be a more responsive measure than the differential count as the relationship between them becomes relatively flat over a differential neutrophil count of 80% (Neale et al. 2002). Based on what is known about the responsiveness of the sputum eosinophil count, a twofold change is regarded as clinically significant (Pavord et al. 2002). A reduction of the sputum neutrophil differential cell count of 13% or more has been regarded as significant, as longitudinal observational studies suggest that a difference of this magnitude is associated with a clinically important reduction in the rate of decline in lung function in patients with COPD (Stanescu et al. 1996).

Assessment of airway inflammation using exhaled nitric oxide

euvers since the values are affected by repeated spirometric maneuvers and by bronchodilator therapy (Kharitonov et al. 1997). Measurements should be made at an exhaled rate of 50 mL/s maintained within 10% for > 6 s, and with an oral pressure of 5–20 cm H2O to ensure velum closure. Results are expressed as the NO concentration in ppb (equivalent to nanoliters/liter) based on the mean of three values within 10%. It has been suggested that the mean of two measurements produces equally valid results and the need for three measurements may be rethought (Kharitonov et al. 2003). NO output can be calculated as the product of the exhaled NO concentration (nL/L) and exhalation flow (L/min). Other measures (notably alveolar NO concentration) can be derived by analysis of NO output at multiple exhalation flows (Tsoukias & George 1998). Alveolar NO may reflect distal lung inflammation; it is increased in patients with refractory asthma and is reduced by oral, but not inhaled, corticosteroids (Berry et al. 2005a). The clinical value of measuring alveolar NO is unclear.

Methodology Measurement of FENO requires expensive equipment and is only available in specialist laboratories. However, cheap portable FENO analyzers are becoming available and it is likely that this technique will become available more widely. Methodologic aspects of the measurement of FENO have been reviewed by a European Respiratory Society (ERS) task force in 1997 (Kharitonov et al. 1997), by the American Thoracic Society (ATS) task force in 1999 (American Thoracic Society 1999), and by a joint ATS/ERS task force in 2005 (ATS/ERS 2005). FENO is best measured before other spirometric man-

Measurement characteristics Robust estimates of FENO measured using the above methods are available in children (Buchvald et al. 2005) and, to a lesser extent, adults (Kharitonov et al. 2003). FENO tends to increase in healthy children to the age of 17; thereafter the values are similar to adults. There is no age effect in adults. There is conflicting data on the effects of gender (Kharitonov et al. 2003; Buchvald et al. 2005). One study suggested fluctuations in FENO in healthy premenopausal women related to the menstrual cycle, with peak values midcycle (Kharitonov

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et al. 1994a). A reasonable estimate of the normal ranges of FENO in adults is < 30 ppb (Kharitonov et al. 2003; Buchvald et al. 2005); this is reduced to < 24 ppb when outliers are removed from the analysis and < 22.4 ppb when outliers and atopics are removed. The between-subject standard deviation of FENO is around 25 ppb in asthma and 8 ppb in normal controls (Kharitonov et al. 2003). The within-subject standard deviation has been estimated at 1.6–2 ppb in a range of subjects (Kharitonov et al. 2003; Buchvald et al. 2005). FENO is reduced twofold to fourfold by corticosteroids in patients with asthma (Massaro et al. 1995; Kharitonov et al. 1994b; Jatakanon et al. 1999) and is increased (by about 60%) by during the late response to allergen in subjects with atopic asthma (Kharitonov et al. 1995). There is a dose-related effect of low-dose inhaled corticosteroids on FENO, but no additional benefit above a dose of budesonide of 400 μg/day (Jatakanon et al. 1999; Jones et al. 2002; Kharitonov et al. 2002). One corticosteroid reduction study has suggested that a 60% increase in FENO has a positive predictive value for identifying subsequent loss of asthma control of 83% (Jones et al. 2001). Based on what is known about the effect of inhaled corticosteroids on FENO and the predictive value of a change in FENO (Jones et al. 2001), a reasonable minimally important difference is a 60% change either way. FENO is a reasonably robust measure of the presence of eosinophilic airway inflammation across a wide range of patients differing in atopic status and medication (Berry et al. 2005b). The association is lost in current smokers (Berry et al. 2005b). There is accumulating evidence of an important degree of dissociation between eosinophilic airway inflammation and symptoms/disordered airway function in patients with asthma (Crimi et al. 1998; Rosi et al. 1999; Green et al. 2002b) so FENO is likely to provide information about the disease that is not available otherwise. There is evidence that a raised FENO is related to a beneficial short-term response to corticosteroid therapy irrespective of the clinical context in which it is present (Smith et al. 2005a). One corticosteroid reduction study has shown that a raised FENO is predictive of loss of asthma control (Jones et al. 2001), but two others have shown less convincing evidence for such an effect (Leuppi et al. 2001; Deykin et al. 2005).

Other methods to measure airway inflammation Exhaled condensate The collection of exhaled breath condensate (EBC) and assay of inflammatory markers is the most recent development in noninvasive asthma monitoring technologies. Water vapor and respiratory droplets and particles are cooled and collected and used for conventional assay or inflammatory markers such as leukotrienes (Montuschi & Barnes 2002), 8isoprostane (Mondino et al. 2004), pH (Jobsis et al. 1998), and hydrogen peroxide (Dohlman et al. 1993; Antczak et al. 1997; Horvath et al. 2004). The current methods for the collection of EBC vary primarily in the type of condensers that are

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employed. The physical surface properties of each condenser system may influence the condensate collected. Therefore, it is possible that there may be variation between systems in terms of the particles collected. Methodologic aspects of EBC collection and analysis have been reviewed by an ERS working party (Horvath et al. 2005). The influence of salivary contamination on EBC values may be considerable as many of the mediators assayed are found in high concentration in saliva (Montuschi et al. 1999). Assessment of airway inflammation using EBC is noninvasive and feasible in almost all patients. It has the potential to provide information about different aspects of the airway inflammatory response and this information may be clinically important. However, results are not available immediately, limiting the clinical utility of the technique. Future technologic advances may overcome this problem. There is evidence of significant variability in the concentration of some EBC markers and the reported relationship between the concentration of EBC markers and other inflammatory or clinical measures is variable between studies, raising the possibility of unresolved methodologic issues and a need for further validation work.

Serum ECP Serum ECP concentrations are higher than EDTA plasma concentration, probably because blood eosinophils continue to produce ECP ex vivo in the absence of additives. Serum ECP concentrations are thought to better at discriminating health from disease and are preferred. A standardized collection, processing, and testing method has been described (Venge et al. 1999). As with other markers of eosinophilic airway inflammation, serum ECP increases with spontaneous allergen exposure or after a laboratory allergen challenge and decreases following allergen avoidance and inhaled corticosteroid therapy. However, serum ECP is a less responsive measure than the sputum eosinophil count and FENO (Kips & Pauwels 1998; Currie et al. 2003). Compared with eosinophil counts, ECP measurements in either induced sputum or serum failed to reflect treatmentrelated changes in chronic asthma (Aldridge et al. 2002), in keeping with the view that serum ECP is not a sensitive or reliable means of evaluating eosinophilic airway inflammation. Moreover, serum ECP was found to be insensitive marker in titrating and monitoring therapy with inhaled corticosteroids over a wide dose range in childhood asthma (Visser et al. 2002) and it does not help predict a response to corticosteroid therapy (Meijer et al. 2002). Finally, a randomized trial that compared a serum ECP-based algorithm with a conventional algorithm for managing asthma found no improvement in symptom scores, despite increased doses of inhaled corticosteroids (Lowhagen et al. 2002). These findings suggest that serum ECP is an imperfect and insensitive measure of eosinophilic airway inflammation, which provides no particular advantages over the other techniques.

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Relationship between eosinophilic airway inflammation and airway dysfunction Asthma has been traditionally viewed as a condition where eosinophilic airway inflammation causes airway hyperresponsiveness, which in turn leads to variable airflow obstruction and symptoms. This hypothesis is deeply embedded, to the point where it is incorporated into recent definitions of asthma (Global Initiative for Asthma 2002). However, several observations made from cross-sectional and longitudinal studies of airway inflammation using sputum suggest that this hypothesis requires modification.

Poor correlation between eosinophilic airway inflammation and airway dysfunction Our view of the importance of eosinophilic airway inflammation in the pathogenesis of asthma has been heavily influenced by bronchoscopy studies performed over the last 20 years (Djukanovic et al. 1990). These, by necessity, were largely limited to young volunteers with mild disease. The development of a noninvasive technique to assess airway inflammation has made it possible to relate the presence of airway inflammation to objective measures of disordered airway function in larger and more heterogeneous populations than was possible with bronchoscopy studies. In general, these studies have contradicted findings in the earlier bronchoscopy studies in that they have not found a correlation between the sputum eosinophil count and various markers of airway dysfunction (Crimi et al. 1998; Rosi et al. 1999; Green et al. 2002b). One surprising observation has been that a subset of symptomatic asthmatics does not have sputum evidence of eosinophilic airway inflammation (Pavord et al. 1999b; Green et al. 2002b). Many have a sputum neutrophilia. This sputum profile is evident in corticosteroid-naive as well as corticosteroid-treated subjects suggesting it is not always an artifact related to treatment. Importantly, patients with noneosinophilic asthma respond less well to inhaled budesonide than a group with more typical sputum features (Pavord et al. 1999b; Green et al. 2002b). Similar sputum findings have been reported in patients with more severe asthma (Gibson et al. 2001), and Wenzel et al. (1999) have identified a subgroup of patients with refractory asthma who have bronchoscopic evidence of neutrophilic airway inflammation, normal eosinophil counts, and normal basement membrane thickness. These findings suggest the presence of a distinct asthma phenotype characterized by a predominantly neutrophilic airway inflammatory response and relative corticosteroid resistance across the range of asthma severity. However, they are based on single observations, and in a variable disease there is a clear need to establish whether this asthma phenotype and the associated impaired response to corticosteroid treatment persists in the longer term.

Thus, cross-sectional studies suggest that, to a large extent, disordered airway function and eosinophilic airway inflammation appear to be independently regulated, suggesting that our earlier paradigm of a simple causal relationship between them needs to be modified. The real patient examples shown in Fig. 66.3 illustrate this point well.

Findings with anti-IL-5 Whether changes in eosinophilic airway inflammation are causally linked to changes in airway function has been called into question by recent findings with humanized monoclonal antibodies to IL-5. One study has shown that the antibody caused a profound and long-lasting reduction in blood and induced sputum eosinophil numbers, but had no effect on airway responsiveness, lung function, or symptoms before or after allergen challenge (Leckie et al. 2000). In another study, there was no evidence of improvement in traditional markers of asthma control in a cohort of patients with more severe asthma who were symptomatic and had disordered airway function despite treatment with high-dose inhaled corticosteroids (Kips et al. 2003). One problem in interpreting these studies is that the anti-IL-5 antibody only partially reduces the tissue eosinophilia (Flood-Page et al. 2003) although the effects seen were significant, and probably equivalent to the effects of inhaled and oral corticosteroids on tissue eosinophilia. The findings with anti-IL-5 monoclonal antibodies therefore suggest that changes in airway function and eosinophilic airway inflammation are independent. They do not exclude the possibility that the abnormalities of airway function seen in asthma are causally linked to other aspects of the inflammatory response that, although closely linked to eosinophilic airway inflammation, can be disassociated from it (Brightling et al. 2002).

Presence of eosinophilic airway inflammation in the absence of asthma A third unexpected observation made following the widespread use of sputum to assess inflammation in the clinical assessment of airways disease is the presence of eosinophilic airway inflammation in airway conditions other than asthma (Fig. 66.4). Up to 40% of patients with nonasthmatic chronic cough (Carney et al. 1997; Brightling & Pavord 2000a) and a similar proportion of subjects with COPD (Brightling et al. 2000b) have a raised sputum eosinophil count. The clinical characteristics of patients with or without eosinophilic airway inflammation are similar. Eosinophilic bronchitis is a condition characterized by a corticosteroid-responsive cough and a sputum eosinophilia occurring in the absence of variable airflow obstruction or airway hyperresponsiveness (Brightling & Pavord 2000b). The dissociation between eosinophilic airway inflammation and airway hyperresponsiveness is therefore clearly seen in patients with eosinophilic bronchitis. Closer study of this condition may be particularly informative as any difference in pathology between the two conditions is likely

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WEEK 2

DATE:

12/10

13/10

14/10

15/10

16/10

17/10

18/10

Daytime asthma:

0

0

0

0

0

0

0

Night-time wakening:

0

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0

PEAK FLOW

AM

440

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440

440

430

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430

Best of 3

PM

500

490

470

470

470

470

460

Number of puffs of Ventolin or Bricanyl per 24 hours

WEEK 1

DATE:

22/11

23/11

24/11

25/11

26/11

27/11

28/11

Daytime asthma:

2

2

2

2

3

1

1

Night-time wakening:

3

3

2

3

3

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PEAK FLOW

AM

280

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400

Best of 3

PM

300

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11

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13

12

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8

6

Number of puffs of Ventolin or Bricanyl per 24 hours

Fig. 66.3 Diary card recordings and induced sputum cytospin preparation from a 42-year-old man with severe corticosteroid-dependent but currently stable asthma who had been previously ventilated on three occasions (top) and a 19-year-old woman with severe symptoms but no serious asthma-related outcomes (bottom). Despite apparently good clinical

80

%

control at the time of study, the patient shown in the top panels has markedly raised numbers of eosinophils in the sputum (note the eosinophilic staining of the cytoplasm and characteristic bilobed nucleus). In contrast, the patient shown in the bottom panels has no eosinophilic airway inflammation. (See CD-ROM for color version.)

philia and thickening of the basement membrane and lamina reticularis, are also features of eosinophilic bronchitis and are therefore unlikely to be critical factors causing airway hyperresponsiveness or variable airflow obstruction (Brightling et al. 2003). The only difference observed in a detailed comparison of the two conditions was increased mast cells within the airway smooth muscle in asthma (Brightling et al. 2002). These findings suggest that localization of mast cells within the airway wall, rather than the presence of eosinophils in the airway mucosa, is the crucial determinant of the functional associations of airway inflammation (Fig. 66.5).

100

60 40 20

Normal

Cough

COPD

Asthma

Fig. 66.4 Approximate prevalence of eosinophilic airway inflammation (defined as sputum eosinophil count > 1.9%) in different patient populations. Each dot represents an estimate of prevalence from one study. (Adapted from Gibson et al. 2002, with permission.) (See CD-ROM for color version.)

to give important clues about the features that are relevant to the different functional associations. In a recent study it was found that several of the traditional characteristics of the immunopathology of asthma, including submucosal eosino-

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2 puffs 2 puffs 2 puffs 2 puffs 2 puffs 2 puffs 2 puffs

How might eosinophilic airway inflammation contribute to the pathophysiology of asthma? Both eosinophilic bronchitis and asthma are associated with cough and it is possible that eosinophilic airway inflammation is directly responsible for this aspect of the asthmatic process. The previous demonstration of a significant correlation between the improvement in cough reflex sensitivity and fall in induced sputum eosinophil count following treatment of subjects with eosinophilic bronchitis with inhaled

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EOSINOPHILIC BRONCHITIS

ASTHMA

T-cell CD4+ MAST CELL

Fig. 66.5 Mast cell and eosinophil microlocalisation in asthma and eosinophilic bronchitis. In asthma mast cells localise to the airway smooth muscle whereas in eosinophilic bronchitis they localise to the epithelium. (See CD-ROM for color version.)

corticosteroids (Brightling et al. 2000a) would be consistent with a causal association. It is also possible that the increased rate of decline in FEV1 and the development of fixed airflow obstruction are related to persistent eosinophilic airway inflammation (Berry et al. 2005c). Finally, exacerbations are an important feature of asthma, which might be closely linked to eosinophilic airway inflammation and associated airway mucosal edema, mucus hypersecretion, and impaction of the airway lumen with cellular debris as well as airway smooth muscle contraction. Recent studies have shown that the exacerbation frequency does not relate closely to symptoms and measures of disordered airway function, suggesting that the mechanisms responsible for these features of asthma are different (Reddel et al. 1999; Kips 2001). This view is supported by the findings of the FACET study which showed that higher-dose inhaled corticosteroids had a marked beneficial effect on exacerbation frequency but relatively less effect on symptoms and PEF, whereas with the addition of longacting β2 agonists the opposite was true (Pauwels et al. 1997). The beneficial effect of corticosteroids on exacerbation frequency would be consistent with the view that eosinophilic airway inflammation is particularly important in the genesis of exacerbations. In keeping with this, four recent studies have shown that the sputum eosinophil count is an independent variable predicting the occurrence of an asthma exacerbation after inhaled corticosteroid withdrawal (Pizzichini et al. 1999a; Leuppi et al. 2001; Jatakanon et al. 2000; Deykin et al. 2005). Moreover, significant increases in the sputum eosinophil count occur well before the onset of exacerbations (Pizzichini et al. 1999a). However, it remains a possibility that airway eosinophilia is a surrogate marker of another airway abnormality, and that these other corticosteroid-responsive abnormalities are more important in the pathogenesis of asthma exacerbations. A key remaining question is whether anti-IL-5 therapy has any effect on exacerbation frequency.

Th2 cytokines IL-4, 5, 13

EOSINOPHIL

Potential clinical role for monitoring airway inflammation Role in diagnosis None of the currently available diagnostic tests are sufficiently sensitive to rule out asthma (Hunter et al. 2002; Smith et al. 2004) with the result that treatment trials are often instigated without good evidence of variable airflow obstruction, airway hyperresponsiveness, or airway inflammation. One study has shown that out of 263 subjects referred to a tertiary referral center with suspected asthma, 160 received an alternative diagnosis (Joyce et al. 1996). Many of these had received prolonged treatment with potentially toxic therapy before the correct diagnosis was reached. Even in tertiary referral centers the diagnosis of refractory asthma can be difficult to make with certainty (Robinson et al. 2003). The presence of a sputum eosinophilia or raised FENO in asthma is sufficiently common to suggest that these findings may have a role in the diagnosis of asthma. Two studies have directly addressed this question. Hunter and colleagues showed that the validity of a sputum eosinophil count outside the normal range in identifying asthma (defined as consistent symptoms with objective evidence of abnormal variable airflow obstruction) was around 80%, significantly better than PEF amplitude percent mean and the acute bronchodilator response, and approached the sensitivity and specificity of measurement of airway responsiveness (Hunter et al. 2002). Smith et al. (2004) have reported similar findings; in this study a high exhaled nitric oxide concentration achieved a similarly high diagnostic accuracy. Arguably it matters more to patients what can be done to help them than the diagnostic label attached to them. Thus, testing strategies that identify patients who are going to respond well to corticosteroid and provide guidance on the

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dosing of corticosteroids might be particularly helpful. There is increasing evidence that the presence of a sputum eosinophilia may predict corticosteroid responsiveness. This was first clearly demonstrated by Morrow-Brown in the 1950s who showed that patients with airway disease and a sputum eosinophilia responded to corticosteroid treatment whereas those without a sputum eosinophilia did not (MorrowBrown 1958). It has since been shown that patients with noneosinophilic asthma respond less well to inhaled budesonide than a group with more typical sputum features (Pavord et al. 1999b; Green et al. 2002b). This is also the case with longer-term corticosteroid treatment in patients with more severe asthma (Green et al. 2002a). A sputum eosinophilia is a predictor of a steroid response irrespective of the clinical context: patients with chronic cough respond well to inhaled corticosteroids if there is a sputum eosinophilia (Pizzichini et al. 1999b; Brightling et al. 2000a) and patients with COPD with sputum eosinophilia respond better to systemic and inhaled corticosteroids than those without (Pizzichini et al. 1998b; Brightling et al. 2000b, 2005).

Role in monitoring asthma The clear implication of the studies discussed above is that eosinophilic airway inflammation is more closely related to the genesis of asthma exacerbations than day-to-day symptoms and airway caliber. Thus, the clinician who relies on an assessment of symptoms and simple tests of lung function might have a limited ability to predict the extent of eosinophilic airway inflammation and, by implication, the exacerbation risk. There might also be a danger of unnecessary use of high-dose inhaled and oral corticosteroids in a patient who has noneosinophilic corticosteroid-resistant disease. Recent studies have directly tested the hypothesis that a management approach that measures and attempts to normalize eosinophilic airway inflammation, as well as minimize symptoms and maximize lung function, might be particularly effective in preventing exacerbations. The first study was a randomized controlled trial of 74 subjects attending outpatients with moderate to severe asthma (Green et al. 2002a). Asthmatics were randomized to treatment by British Thoracic Society guidelines or using a management strategy where treatment was adjusted according to the sputum eosinophil counts. In the sputum management group, decisions about antiinflammatory treatment were made in accordance with an algorithm based on maintenance of a sputum eosinophil count at or below 3% with a minimum dose of antiinflammatory treatment. The 3% cutoff was chosen because this was previously shown to identify individuals with corticosteroid-responsive asthma. If the sputum eosinophil count was less than 1%, antiinflammatory treatment was reduced irrespective of asthma control. If the eosinophil count was 1–3%, no changes to antiinflammatory treatment were made; if the eosinophil count was greater than 3%, antiinflammatory treatment was increased. Decisions about

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changes in bronchodilator treatment were based on individual patients’ symptoms, PEF readings, and use of rescue β2 agonists compared with baseline using the same criteria as in the BTS management group. In the patient examples shown in Fig. 66.3, the patient depicted in the top panels would have been advised to increase antiinflammatory treatment and reduce bronchodilator treatment if randomized to the sputum management group; in contrast, the patient shown in the bottom panels would have been advised to reduce antiinflammatory treatment and increase bronchodilator treatment. Management decisions were made by an independent individual who was unaware of the clinical characteristics of the patient, and who recorded separate treatment plans to be followed depending on whether the patient’s asthma was poorly or well controlled. The strategy based on sputum eosinophil counts achieved significantly better control of eosinophilic-related airway inflammation over the 12 months of the trial. There was also an improvement in methacholine PC20. Both management strategies achieved equivalent control of symptoms, quality of life, and disordered airway function. However, in the sputum management group there was a marked reduction in severe asthma exacerbations and significantly fewer hospital admissions with asthma exacerbations (Fig. 66.6). The intervention group used no more inhaled or oral corticosteroid than the control group, and overall, the sputum eosinophil guided management strategy was cost-effective. Jayaram et al. (2006) reported very similar findings. They evaluated the effects of a sputum eosinophil-guided management strategy in a larger population with a wider spread of severity studied over 2 years and found a halving of severe asthma exacerbations in the intervention population. In this study, the benefits of inflammation-guided management were more striking in patients with more severe asthma and in patients taking long-acting β2 agonists, perhaps because

120 Severe exacerbations (cumulative number)

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*P = 0.01

100

BTS guidelines 6 asthma admissions

80 60 40

Sputum guidelines

20

1 asthma admission

0 0 1 2 3 4 5 6 7 8 9 10 11 12 Time (months)

Fig. 66.6 Comparison of effects of two treatment strategies on rates of severe exacerbations of asthma. One strategy (BTS guidelines) utilized standard guidelines of the British Thoracic Society and the other (sputum guidelines) adjusted the antiinflammatory treatment with corticosteroids based on the eosinophil counts (see text for criteria). (From Green et al. 2002a, with permission.)

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Table 66.4 Characteristics of an ideal monitoring tool for assessing airway inflammation.

Easy to measure Acceptable to patients Immediate result Well validated Does not require technical training Can be done cheaply Discriminates different types of inflammation

Induced sputum

FENO

No Yes No Yes No Yes Yes

Yes Yes Yes Yes Yes Yes No

disociation between eosinophilic airway inflammation and the clinical expression of asthma is more marked in these patients. Experience with asthma management guided by the sputum eosinophil count is limited but even so there is sufficient evidence to suggest that it provides additional important information that is necessary for the optimum management of patients with more severe asthma. Induced sputum has important limitations as a tool for monitoring asthma, including the absence of an immediate result and the requirement for technician time and expertise. In contrast, assessment of FENO has many off the attributes required for a monitoring tool (Table 66.4). Three studies (Pijnenburg et al. 2005; Smith et al. 2005b; Shaw et al. 2007) have explored the use of FENO as a tool to facilitate asthma management. None showed that FENO-guided management was associated with a reduction in the frequency of asthma exacerbations. In one, marginally better control of exacerbations was achieved with a 40% lower dose of inhaled corticosteroid (Smith et al. 2005b); in another study involving children, FENO-guided management resulted in improved airway responsiveness (Pijnenburg et al. 2005). More work is required, particularly in more severe asthma, before FENO can be recommended as a monitoring tool in asthma.

Future directions Recently, it has become possible to investigate the contribution of the distal lung in the inflammatory process in asthma and other airway diseases by measurement of FENO at different expiratory flows (Tsoukias & George 1998). Lehtimaki and colleagues have used this method to demonstrate that patients with nocturnal symptoms have elevated alveolar NO concentrations (Lehtimaki et al. 2002) and that alveolar NO concentration does not fall in response to treatment with inhaled corticosteroids (Lehtimaki et al. 2001). Patients with refractory asthma have a raised alveolar NO concentration, which is reduced by oral but not inhaled corticosteroids (Berry et al. 2005a), suggesting that it reflects corticosteroid-

responsive inflammation in a site that cannot be accessed by inhaled corticosteroids. Induced sputum may also have a role in investigating proximal and distal airway inflammation as there is evidence that distal airways are sampled with increasing output and duration of nebulized hypertonic saline (Gershman et al. 1999). The clinical utility of these methods has not been explored. They could provide useful information on the need for a systemic rather than an inhaled approach to antiinflammatory treatment with agents such as prednisolone, anti-IgE, and perhaps anti-IL-5. The presence of a sputum eosinophilia in COPD is related to corticosteroid-responsive disease in COPD (Pizzichini et al. 1998b; Brightling et al. 2000b, 2005) and chronic cough (Pizzichini et al. 1999b; Brightling et al. 2000a) and it is possible that the sputum eosinophil count could be used to monitor corticosteroid treatment in these conditions. There is initial evidence that such a management strategy is associated with a 60% reduction in hospital admissions with exacerbations of COPD (Siva et al. 2005). Very little is known about FENO in COPD and its relationship with eosinophilic airway inflammation. Potentially this might be a useful technique for identifying corticosteroid-responsive disease and to direct therapy. One of the major limitations of sputum induction is that the induction and processing are labor-intensive. A further problem is that it is not possible to get an immediate result, which limits the clinical utility of the technique. There is an important need for simpler measures that might be applicable in primary care settings. FENO might be a satisfactory alternative in many situations but a simplified sputum assay capable of discriminating eosinophilic and neutrophilic airway inflammation might also be of value.

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Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Definition and Classification of Allergic Rhinitis and Upper Airways Diseases Wytske Fokkens and Jean Bousquet

Summary Allergic rhinitis is the most common form of noninfectious rhinitis and is associated with an IgE-mediated immune response against allergens. It is often associated with ocular symptoms. Several nonallergic conditions can cause similar symptoms: infections, hormonal imbalance, physical agents, anatomic, anomalies and the use of certain drugs. The diagnosis of allergic rhinitis is often easy, but in some cases it may cause problems and many patients are still underdiagnosed, often because they do not perceive the symptoms of rhinitis as a disease impairing social life, school, and work. The diagnosis of allergic rhinitis is based on the coordination between a typical history of allergic symptoms and diagnostic tests. Typical symptoms of allergic rhinitis include rhinorrhea, sneezing, nasal obstruction, and nasal pruritus. Ocular symptoms are common, particularly in patients allergic to outdoor allergens. Diagnostic tests are based on the demonstration of allergen-specific IgE in the skin (skin tests) or the blood (specific IgE). The measurement of total IgE is not useful in the diagnosis of allergic rhinitis. Many asymptomatic subjects can have positive skin tests and/or detectable serum specific IgE. Many patients have positive tests which are irrelevant. In some countries, the suspicion of allergic rhinitis may be raised in the pharmacy. Patients with persistent and/or moderate/severe symptoms of rhinitis should be referred to a physician. Most patients with rhinitis are seen in primary care, and in developed countries allergy tests are available to screen for allergy. Patients with persistent and/or moderate/severe symptoms of rhinitis need a detailed allergy diagnosis.

ing of the nose. In rhinitis, these symptoms occur for two or more consecutive days for more than one hour on most days (International Rhinitis Management Working Group 1994). Allergic rhinitis is the most common form of noninfectious rhinitis. It is often associated with ocular symptoms. Several nonallergic conditions can cause similar symptoms: infections, hormonal imbalance, physical agents, anatomic anomalies, and the use of some drugs (Fokkens 2002). Rhinitis is therefore classified as shown in Table 67.1 (Bousquet et al. 2001). The differential diagnosis of rhinitis is presented in Table 67.2 (Bousquet et al. 2001). Since the nasal mucosa is continuous with that of the paranasal sinuses, congestion of the ostia may result in sinusitis that does not exist without rhinitis, suggesting that the term “rhinosinusitis” should replace “sinusitis” (Fokkens et al. 2005). Finally, there are structural causes of rhinitis, including nasal polyps.

Table 67.1 Classification of rhinitis. (From Bousquet et al. 2001, with permission.) Infectious Viral Bacterial Other infectious agents Allergic Intermittent Persistent Occupational Intermittent Persistent

Introduction Rhinitis is defined as an inflammation of the lining of the nose characterized by nasal symptoms including anterior or posterior rhinorrhea, sneezing, nasal blockage, and/or itch-

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Nonallergic rhinitis Drug-induced: aspirin, other medications Hormonal Other causes NARES Irritants Food Emotional Atrophic Idiopathic

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Rhinitis

Table 67.2 Differential diagnosis of allergic rhinitis. (From Bousquet et al. 2001, with permission.) History nasal discharge blockage sneeze/itch

Polyps Mechanical factors Deviated septum Hypertrophic turbinates Adenoidal hypertrophy Anatomic variants in the ostiomeatal complex Foreign bodies Choanal atresia Tumors Benign Malignant Granulomas Wegener’s granulomatosis Sarcoid Infectious Malignant: midline destructive granuloma Ciliary defects Cerebrospinal rhinorrhea

“sneezers and runners”

“blockers”

Fig. 67.1 Diagnosis of allergic rhinitis in the clinic. (From International Consensus Report 1994.) (See CD-ROM for color version.)

Definition for epidemiologic studies

Allergic rhinitis Definition Clinical definition Allergic rhinitis is clinically defined as a symptomatic disorder of the nose induced by IgE-mediated inflammation after allergen exposure of the membranes lining the nose (Bousquet et al. 2001). Symptoms of rhinitis include rhinorrhea, nasal obstruction, nasal itching and sneezing which are reversible spontaneously or under treatment (Fig. 67.1 and Table 67.3). It is subdivided into intermittent or persistent disease. The severity of allergic rhinitis can be classified as mild or moderate/severe (Bousquet et al. 2001).

The clinical definition of rhinitis is difficult in epidemiologic settings of large populations, where it is not possible either to visit every person or to obtain laboratory evidence of the immune response. Initial epidemiologic studies have assessed allergic rhinitis on the basis of simple working definitions. Various standardized questionnaires have been used to this effect (Sibbald & Strachan 1995; Charpin et al. 1996). • The first questionnaires that aimed at assessing seasonal allergic rhinitis dealt with “nasal catarrh” (Medical Research Council 1960) and “runny nose during spring” (Brille et al. 1962). • Successive questions introduced the diagnostic term “seasonal allergic rhinitis” and patients were asked “Have you ever had seasonal allergic rhinitis?” or “Has a doctor said that you suffer from seasonal allergic rhinitis?” • In the European Community Respiratory Health Survey (ECRHS) full-length questionnaire, the question asked about rhinitis was “Do you have any nasal allergies including seasonal allergic rhinitis?” (Burney et al. 1994). In order to identify the responsible allergen, the ECRHS study has included potential triggers of the symptoms. • A score including most features (clinical symptoms, season of the year, triggers, parental history, individual medical history,

Table 67.3 Clinical classification of allergic rhinitis. (From Bousquet et al. 1994, 2001, with permission.) “Sneezers and runners”

“Blockers”

Sneezing

Especially paroxysmal in bouts

Little or none

Rhinorrhea

Always present: watery, anterior and sometimes posterior

Variable, can be thick mucus, and generally more posterior

Nasal itching

Yes, often

No

Nasal blockage

Variable

Often severe

Diurnal rhythm

Worse on awakening, improves during the day and usually worsens again in the evening

Constant day and night, may be worse at night and is often severe

Conjunctivitis

Often present

None

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perceived allergy) of allergic rhinitis has recently been proposed (Annesi-Maesano et al. 1998). Taking the doctor’s diagnosis (based on questionnaire, examination and skin tests to common aeroallergens) as a gold standard, such a score had good positive and negative predictive values (84% and 74%, respectively) in identifying patients suffering from allergic rhinitis. Perennial rhinitis has been defined as frequent nonseasonal nasal or ocular symptoms (“rhinoconjunctivitis”). • In the study by Pariente et al. (1997), the duration of the disease was also taken into consideration in order to differentiate perennial rhinitis from “common cold” (viral upper respiratory infections). Objective tests for the diagnosis of IgE-mediated allergy (skin-prick test, serum specific IgE) can also be used (Tollerud et al. 1991; Vervloet et al. 1991; Droste et al. 1996). The diagnostic efficiency of IgE, skin-prick tests, and Phadiatop was estimated in 8329 randomized adults from the Swiss Study on Air Pollution and Lung Diseases in Adults (SAPALDIA). The skin-prick test had the best positive predictive value (48.7%) in comparison to Phadiatop (43.5%) or total serum IgE (31.6%) for the epidemiologic diagnosis of allergic rhinitis (Tschopp et al. 1998). Future working definitions are intended to encompass, besides clinical symptoms and immune response tests, nasal function and eventually specific nasal challenge (Annesi-Maesano 1999).

Intermittent and persistent allergic rhinitis Previously, allergic rhinitis was subdivided, based on the time of exposure, into seasonal, perennial and occupational (1994; Dykewicz & Fineman 1998; van Cauwenberge et al. 2000; Dykewicz 2003). Perennial allergic rhinitis is most frequently caused by indoor allergens such as dust mites, molds, insects (cockroaches), and animal danders. Seasonal allergic rhinitis is related to a wide variety of outdoor allergens such as pollens or molds. However, this classification is not entirely satisfactory for the following reasons. • There are some places where pollens and molds are perennial allergens, e.g., grass pollen allergy in southern California and Florida (Bucholtz et al. 1991) or Parietaria pollen allergy in the Mediterranean area (D’Amato & Lobefalo 1989). • Symptoms of perennial allergy may not always be present all year round. This is particularly the case for a large number of patients allergic to house-dust mites and who only suffer from mild or moderate/severe intermittent rhinitis (PlattsMills et al. 1987; Ciprandi et al. 1995; Bauchau & Durham 2004, 2005). This is also the case in the Mediterranean area where levels of house-dust mite allergens are low in the summer (Passalacqua et al. 1998). • The majority of patients are sensitized to many different allergens and therefore exposed throughout the year (Sibbald & Rink 1991; Bauchau & Durham 2004; Arbes et al. 2005; Bousquet et al. 2005a; Ciprandi et al. 2005a). In many patients, perennial symptoms are often present and patients present seasonal exacerbations when exposed to pollens or molds. It

appears therefore that this classification does not represent real life. • Many patients allergic to pollen are also allergic to molds and it is difficult to define the pollen season (Bruce et al. 1977). • Some patients only sensitized to a single pollen species present perennial symptoms (Kirmaz et al. 2005). • Due to the priming effect on the nasal mucosa induced by low levels of pollen allergens (Connell 1969; Juliusson & Bende 1988; Wachs et al. 1989; Naito et al. 1993; Koh et al. 1994) and minimal persistent inflammation of the nose in patients with symptom-free rhinitis (Knani et al. 1992; Ciprandi et al. 1995; Ricca et al. 2000), symptoms do not necessarily occur strictly in conjunction with the allergen season. • Nonspecific irritants such as air pollution may aggravate symptoms in symptomatic patients and induce symptoms in asymptomatic patients with nasal inflammation (Riediker et al. 2001). Thus, a major change in the subdivision of allergic rhinitis was proposed in the ARIA (allergic rhinitis and its impact on asthma) document with introduction of the terms “intermittent” and “persistent” (Bousquet et al. 2001). It was shown that the classic types of seasonal and perennial rhinitis cannot be used interchangeably with the new classification of intermittent/persistent, as they do not represent the same stratum of disease. Thus, “intermittent” and “persistent” are not synonymous with “seasonal” and “perennial” (Bauchau & Durham 2004; Demoly et al. 2003; Bousquet et al. 2005a; Bachert et al. 2006; Van Hoecke et al. 2006). In the original ARIA document, the number of consecutive days used to classify patients with persistent rhinitis was more than 4 days a week (Bousquet et al. 2001). However, it appears that patients with persistent rhinitis usually suffer almost every day (Bousquet et al. 2006a). However, some patients may suffer from mild persistent rhinitis during most months of the year and have more severe symptoms when exposed to high concentrations of allergens. This is particularly the case for patients allergic to indoor allergens and pollens or cats. Since most patients are polysensitized, it appears that the ARIA classification is closer to patient’s needs than the previous one (Walls et al. 2005). Moreover, persistent rhinitis may not have an allergic origin (Bachert 2004). Other conditions associated with allergic rhinitis include asthma, sinusitis, otitis media, nasal polyposis, and lower respiratory tract infections (Hellings & Fokkens 2006).

Assessment of severity of allergic rhinitis Classical signs and symptoms Allergic rhinitis is characterized by subjective symptoms, namely rhinorrhea, nasal obstruction (Ciprandi et al. 2005b), nasal itching and sneezing, that are difficult to quantify because they depend largely on the perception of the patient. Post-nasal drip mainly occurs either with profuse anterior rhinorrhea in allergic rhinitis (Doyle et al. 1995) or without significant

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anterior rhinorrhea in chronic rhinosinusitis (CRS) (Knutson & Slavin 1995; Wreesmann et al. 2001).

Symptoms associated with social life, work and school It is now recognized that allergic rhinitis comprises more than the classic symptoms of sneezing, rhinorrhea, and nasal obstruction. It is also associated with impairments in how patients function in day-to-day life. Impairment of quality of life is seen in adults (Bousquet et al. 1994; Dykewicz & Fineman 1998; Leynaert et al. 2000a) and in children (Juniper et al. 1994; Berdeaux et al. 1998; Nascimento Silva et al. 2001; Roberts et al. 2003). Patients may also be troubled by sleep disorders and emotional problems, as well as by impairment in activities and social functioning (Juniper et al. 1999). Poorly controlled symptoms of allergic rhinitis may contribute to sleep loss or disturbance (Lavie et al. 1981; Olsen et al. 1981; Zwillich et al. 1981; Houston et al. 1982; McNicholas et al. 1982; Young et al. 1997; Craig et al. 1998, 2003, 2004; Gosepath et al. 1999). Moreover, sedation in patients with allergic rhinitis may be increased by using sedative treatments (Passalacqua et al. 1996; Casale et al. 2003). Although sleep apnea syndrome has been associated with nasal disturbances (Rubinstein 1995; Kushida et al. 1997; Houser et al. 2002), it is unclear whether allergic rhinitis is associated with sleep apnea (McNicholas et al. 1982; Kushida et al. 1997; Kramer et al. 2001). It has been shown that patients with severe symptoms of intermittent or persistent allergic rhinitis have an impaired sleep pattern compared with normal subjects and patients with mild rhinitis (Leger et al. 2006). It is also commonly accepted that allergic rhinitis impairs work (Dykewicz & Fineman 1998; Blanc et al. 2001; Demoly et al. 2002; Bousquet et al. 2006a) and school performance (Vuurman et al. 1993; Simons 1996; Blaiss 2004). In several studies, the severity of allergic rhinitis assessed using qualityof-life measures, work productivity questionnaires or sleep questionnaires was found to be independent of duration (Bousquet et al. 2005a, 2006a; Leger et al. 2006).

Objective measures of severity Objective measures of the severity of allergic rhinitis include: • visual analog scales (VAS) (Spector et al. 2003); • measurements of nasal obstruction, such as peak inspiratory flow, acoustic rhinometry, and rhinomanometry (Clement & Gordts 2005; Starling-Schwanz et al. 2005; Ragab et al. 2006); • measurements of inflammation, such as nitric oxide (NO), cells and mediators in nasal lavages, cytology and nasal biopsy (Ragab et al. 2006; Struben et al. 2006); • reactivity measurements, such as provocation with histamine, methacholine, allergen hypertonic saline, capsaicin or cold dry air (Litvyakova & Baraniuk 2001); • measurements of smell (Moll et al. 1998).

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Table 67.4 Classification of allergic rhinitis according to ARIA. (From Bousquet et al. 2001, with permission.) Intermittent Symptoms are present: less than 4 days a week, or for less than 4 weeks Persistent Symptoms are present: almost all days, and for more than 4 weeks Mild None of the following are present: sleep disturbance impairment of daily activities, leisure and/or sport impairment of school or work troublesome symptoms Moderate–severe One or more of the following are present: sleep disturbance impairment of daily activities, leisure and/or sport impairment of school or work troublesome symptoms

Measurements using VAS, nasal obstruction and smell are used in clinical practice. The other measurements are primarily used in research.

ARIA classification of allergic rhinitis In the ARIA classification, allergic rhinitis can be classified as mild and moderate/severe, depending on the severity of symptoms and their impact on social life, school and work (Table 67.4). The severity of allergic rhinitis is independent of treatment. In asthma, the control level is also independent of asthma medications (Bateman et al. 2004; Li et al. 2005; Roche et al. 2005; Bateman 2006). Although such an independent relationship was suspected in a study of allergic rhinitis (Bousquet et al. 2005a), this very important finding was confirmed in a recent study in which it was found that the severity of rhinitis is independent of treatment (Bousquet et al. 2000).

Comorbidities and complications of allergic rhinitis Asthma The nasal airways and their closely associated paranasal sinuses are an integral part of the respiratory tract (Bousquet et al. 2001; Togias 2003). The nasal and bronchial mucosa are similar and one of the most important concepts regarding nose–lung interactions is the functional complementarity (Togias 2003). Epidemiologic studies have consistently shown that asthma and rhinitis often coexist in the same patients. The prevalence of asthma in subjects without rhinitis is usually less than

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2% (Linneberg et al. 2002; Leynaert et al. 2004; Bousquet et al. 2005a). The prevalence of asthma in patients with rhinitis varies from 10 to 40% depending on studies. The majority of patients with asthma present with rhinitis symptoms (Linneberg et al. 2000a,b; Kivity et al. 2001; Terreehorst et al. 2002; Peroni et al. 2003; Banac et al. 2004; Yu et al. 2005; Georgy et al. 2006; Janahi et al. 2006; Kuyucu et al. 2006). It appears that patients with seasonal and perennial allergies are more prone to have asthma as a comorbidity than those with seasonal or perennial allergic rhinitis (Leynaert et al. 2004). Similarly, persistent rhinitis is more often associated with asthma than is intermittent rhinitis (Bousquet et al. 2005a). However, in many instances, symptoms may predominate in one organ and be hidden or unrecognized in other organs even though they exist. Rhinitis is a factor independent of allergy in the risk for asthma (Leynaert et al. 2000b; Bousquet et al. 2001). Adults with asthma and documented concomitant allergic rhinitis experience more asthma-related hospitalizations and general practitioner visits, and incur higher asthma drug costs than do adults with asthma alone (Bousquet et al. 2005b; Price et al. 2005; Thomas et al. 2005). Although differences exist between rhinitis and asthma, the upper and lower airways may be considered a unique entity influenced by a common and probably evolving inflammatory process (Chanez et al. 1999; Gaga et al. 2000), which may be sustained and amplified by intertwined mechanisms. However, there are differences in remodeling in the nose and the lower airways (Bousquet et al. 2004a). According to the ARIA guidelines (Bousquet et al. 2001), patients with persistent allergic rhinitis should be evaluated for asthma by history, chest examination and, if possible and when necessary, the assessment of airflow obstruction before and after bronchodilator. Patients with asthma should be appropriately evaluated (history and physical examination) for rhinitis. A combined strategy should ideally be used to treat upper and lower airway diseases in terms of efficacy and safety.

Conjunctivitis Ocular symptoms occur in a large proportion of patients with rhinitis. However, the prevalence of the association between rhinitis and conjunctivitis cannot be easily defined, since conjunctival symptoms are often considered of minor importance (Bonini & Bonini 1987), and possibly not spontaneously reported by patients with rhinitis and/or asthma in medical interviews or in questionnaire-based epidemiologic studies such as the International Study of Asthma and Allergies in Childhood (ISAAC) and ECRHS (Burney et al. 1994; Asher et al. 1995). Accordingly, the association between rhinitis and conjunctivitis is largely underestimated in epidemiologic studies. A second even more relevant reason, which makes several clinical studies on the prevalence of conjunctivitis in rhinitis patients of limited value, is the heterogeneity of ocular symp-

toms usually referred to as “conjunctivitis.” In fact, these symptoms can be caused by allergic and nonallergic agents. Moreover, allergic eye diseases represent a heterogeneous entity including different forms of conjunctivitis with different signs, symptoms, pathophysiology, degree of severity, and response to treatment (Allansmith & Ross 1988; Allansmith 1990; Bonini & Bonini 1998). Allergic conjunctivitis is usually classified as acute, seasonal or seasonal allergic, perennial, vernal, and atopic. An immunologic mechanism has also been postulated for conjunctival symptoms in contact lens wearers. • Acute allergic conjunctivitis is an acute hypersensitivity reaction with hyperemia and chemosis accompanied by intense tearing, itching and burning of the eye, caused by accidental exposure to several substances such as gas and liquid “irritants” or animal danders. • Seasonal allergic conjunctivitis is the typical conjunctival reaction in seasonal allergic rhinitis rhinoconjunctivitis or following exposure to seasonal pollen allergens in sensitized subjects. • Perennial allergic conjunctivitis is a less intense but continuous conjunctival reaction related to exposure to perennial allergens such as house-dust mite. • Vernal conjunctivitis is a severe bilateral eye condition of children with frequent involvement of the cornea (vernal keratoconjunctivitis) characterized by conjunctival hypertrophy and mucus excess. • Atopic conjunctivitis is a keratoconjunctivitis associated with eczematous lesions of the lids and skin. • Contact lens conjunctivitis is a giant-papillary conjunctivitis observed in hard and soft contact-lens wearers. The prevalence of rhinitis in patients with atopic and contact lens conjunctivitis is similar in allergic and nonallergic patients (Bonini & Bonini 1987). From surveys on a large number of patients with “allergic conjunctivitis” (Bonini & Bonini 1987), the prevalence of the association between rhinitis and “conjunctivitis” appears to be different depending on the type of allergic conjunctivitis. Allergic conjunctivitis is more common with outdor allergens than with indoor allergens. In some studies of pollen allergy, conjunctivitis is sometimes present in over 75% of patients suffering from rhinitis.

Rhinosinusitis The role of allergy in sinus disease is still unclear (Baraniuk & Maibach 2005; Fokkens et al. 2005; Hellings & Fokkens 2006). It has been speculated that nasal inflammation induced by IgE-mediated mechanisms favors the development of acute and/or chronic sinus disease. Similar inflammation is observed in the nose and sinuses of patients with allergic rhinitis (Hamilos et al. 1993, 1995, 1998; Demoly et al. 1994, 1997, 1998). Moreover, sinus involvement has been observed by computed tomography (CT) in allergic patients during the ragweed

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pollen season (Naclerio et al. 1997). Nasal challenge with allergen induces a sinus reaction demonstrated by CT (Piette et al. 2004). Total IgE serum levels correlate with the sinus mucosal thickness on CT (Baroody et al. 1997). However, at present, it remains incompletely understood whether, and if so via which mechanisms, the presence of allergic inflammation in the nose predisposes the individual to the development of sinus disease. However, epidemiologic studies are inconclusive and so far there are no published prospective reports on the incidence of infectious rhinosinusitis in populations with and without clearly defined allergy. Several epidemiologic studies report a high prevalence of sensitization to inhalant allergens in patients with both acute rhinosinusitis (Savolainen 1989) and CRS (Benninger 1992; Karlsson & Holmberg 1994). Prevalence ranges up to 84 % of patients undergoing revision sinus surgery (Emanuel & Shah 2000). Compared with the general population, where CRS is estimated to be found in up to 6 % of subjects (Gordts et al. 1996; Greisner & Settipane 1996; Collins 1997; Chen et al. 2003), patients sensitized to inhalant allergens seem to present more often with sinus complaints. However, on the basis of these epidemiologic observations, one may not conclude that allergic rhinitis predisposes to the development of CRS as these studies include a large referral bias. A predominance of allergy to perennial versus seasonal allergens was found in patients with chronic sinusitis at the time of indication for surgery (Emanuel & Shah 2000). Moreover, epidemiologic studies failed to demonstrate a higher incidence of sinus disease during the pollen season in pollen-sensitized patients (Karlsson & Holmberg 1994). The role of molds in CRS is unclear. Fungal elements were suspected to be one of the causative agents of CRS, possibly by an allergic mechanism (Cody et al. 1994; Ponikau et al. 1999; Khan et al. 2000; Taylor et al. 2002), but controversy has accumulated (Braun et al. 2003; Luong & Marple 2004) and the benefits of topical amphotericin therapy were not confirmed (Ebbens et al. 2006). Only a limited number of studies have examined the effect of antiallergic therapy in atopic patients with sinus disease. Loratadine, as an adjunctive therapy of atopic patients with acute sinusitis, was found to modestly improve sneezing and nasal obstruction (Braun et al. 1997). Well-conducted clinical trials showing beneficial effects of antihistamine therapy in patients with CRS are lacking. Notwithstanding the lack of precise insight into mechanisms, symptoms of IgE-mediated allergic inflammation should be requested during history-taking in patients with CRS, and skin-prick tests or specific IgE should be performed in the case of clinical suspicion. Despite limited evidence regarding the effectiveness of antiallergic therapy in patients with chronic sinus disease, it would seem logical to add antiallergic therapy to the treatment scheme of patients with chronic sinus disease and concomitant allergy.

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Nasal polyps Nasal polyps are considered a chronic inflammatory disease of the sinonasal mucosa, being part of the spectrum of chronic sinus pathology (Fokkens et al. 2005). The role of allergy in the generation of nasal polyps is even more unclear than in CRS (Hellings & Fokkens 2006). Both allergic rhinitis and nasal polyps are characterized by an inflammatory response that shows many similarities. However, until now, no clear epidemiologic data support a role of allergy in nasal polyposis. Further studies are needed to elucidate the relevance of local IgE production in nasal polyposis and in perpetuation of the inflammatory cascade.

Adenoid hypertrophy The adenoid, the peripheral lymphoid organ located in the nasopharynx, is part of Waldeyer’s ring and contributes to the development of immunity against inhaled microorganisms in early life (Hellings et al. 2000). Many triggers, including microbial stimuli such as molds (Huang & Giannoni 2001) or external irritants like cigarette smoke (Gryczynska et al. 1999), have been related to the enlargement of adenoid tissue and hence to the development of symptoms. Symptoms related to adenoid hypertrophy range from nasal obstruction, rhinolalia clausa, open-mouth breathing and snoring, to the so-called “adenoid facies.” In children, both allergic rhinitis and adenoid hypertrophy may give rise to similar symptoms, and therefore need to be differentiated at the time of the consultation. Little is known about the correlation between allergic rhinitis and adenoid hypertrophy in children. The presence of sensitization to inhalant allergens has been reported to alter the immunology of adenoid tissue. CD1a+ Langerhans cells and eosinophils are increased in the adenoids of allergic children (Vinke et al. 1999a; Nguyen et al. 2004). Similarly, eosinophils and interleukin (IL)-4 and IL-5 mRNA-positive cells are increased in the adenoids of atopic children (Nguyen et al. 2004). Furthermore, atopy is associated with increased numbers of IgE-positive cells in adenoids irrespective of the presence of adenoid hypertrophy (Papatziamos et al. 1999). However, no correlation is observed between the atopic state and the degree of adenoid hypertrophy (Cassano et al. 2003). Although the role of allergy in adenoid hypertrophy is unclear, allergy should be investigated in children with symptomatic adenoid hypertrophy. Properly-conducted clinical trials on antihistamines in allergic children with allergic rhinitis and adenoid hypertrophy are lacking. In contrast, nasal steroids are capable of reducing adenoid-related symptoms (Demain & Goetz 1995; Criscuoli et al. 2003; Cengel & Akyol 2006) with no differences in response between atopic and nonatopic children (Cengel & Akyol 2006). In these studies, the effects of nasal steroids on symptoms of allergic inflammation in the nose and adenoid cannot be dissociated from their antiinflammatory effects on the adenoid itself. Recently, a short treatment with oral steroids followed by prolonged oral antihistamine and nasal

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steroid spray therapy was found to reduce adenoid volume and associated symptoms (Georgalas et al. 2005).

Tubal dysfunction The Eustachian tube exerts a major function in middle-ear homeostasis via its role in the ventilation and protection of the middle ear and mucociliary clearance. In line with the concept of global airway allergy, the Eustachian tube is lined with respiratory epithelium and may be involved in the allergic response following allergen inhalation. The mucosal lining in the tubarian tube, i.e,. the nasopharyngeal orifice of the Eustachian tube, contains an allergic inflammatory infiltrate in allergic rhinitis patients (Nguyen et al. 2004). It is therefore not surprising that allergic inflammation with concomitant mucosal swelling may impair the function of the Eustachian tube. Allergic rhinitis patients have a higher risk of Eustachian tube dysfunction assessed by tympanometry than nonallergic subjects, particularly during childhood (Lazo-Saenz et al. 2005). Nasal challenge with house-dust mite induces nasal obstruction and tubal dysfunction in allergic individuals (Skoner et al. 1986). At present, it remains to be elucidated whether nasal allergen inhalation leads to the deposition of allergens in the tubarian tube with induction of a local allergic response, or whether it gives rise to a systemic immune response involving the airway mucosa at the site of the tubarian tube. Both mechanisms may be involved in the generation of allergic inflammation and swelling of the tubarian tube, ultimately leading to otitis media with effusion in predisposed patients.

Otitis media with effusion During the last few decades, the etiologic relationship between rhinitis and otitis media, especially the role of allergy in otitis media with effusion (OME), has been the subject of much controversy (Hellings & Fokkens 2006). OME is an inflammatory disease of the middle-ear mucosa and remains a significant problem in the pediatric population. It is estimated that more than 80% of all children have at least one episode of otitis media by the age of 3 years and that 40% will have three or more episodes (Teele et al. 1989). The nose and middle ear are situated in a system of contiguous organs. Both cavities are covered by respiratory mucosa and there is an anatomic continuation between these two cavities through the Eustachian tube. However, it is not fully understood whether inflammation, infection or obstruction in the nose influences or promotes otitis media. There are several controversies with regard to the etiology and pathogenesis of OME, one of which is the relationship between allergy and OME. In view of the concept of global airway allergy, it can be expected that an allergic inflammatory response can also take place in the middle ear. Indeed, all cells and mediators that contribute to allergic inflammation are present in the middle-ear fluid of OME patients (Hurst &

Venge 2000; Wright et al. 2000). The middle-ear fluid of atopic patients with OME contains more eosinophils and IL-4 and IL-5 mRNA-positive cells than in nonatopic patients with OME (Nguyen et al. 2004), suggesting a role for allergic inflammation in OME. IgE sensitization and respiratory allergy symptoms are independent risk factors for the development of OME (Chantzi et al. 2006). It is possible that children with atopic dermatitis present a higher prevalence of OME than nonatopic children (Van Cauwenberge & Ingels 1993). In this large study, asthma and rhinitis were not predisposing factors for the development of OME. However, the number of OME episodes may be greater in atopic children than in nonatopic children (Irander et al. 1993). It remains difficult to interpret epidemiologic data as we cannot estimate to what extent the enhanced prevalence of allergy in OME patients reported by some authors (Rylander & Megevand 2000; Alles et al. 2001) represents a true finding or rather a referral bias. Many important questions still need to be answered. • Does the presence of rhinitis predispose an individual to the development of otitis? • Does nasal dysfunction cause otitis to worsen? • Can OME be cured by treating the underlying nasal or sinus infection? • Can the middle-ear mucosa be targeted directly by allergens? It is proposed that children with recurrent OME should be tested for allergy (Miceli Sopo et al. 2004; Tewfik & Mazer 2006).

Cough and bronchial symptoms without asthma Chronic cough can be caused by a number of factors, including allergic rhinitis, infections, rhinosinusitis, asthma, and environmental stimuli (Millqvist & Bende 2006). In rhinosinusitis, it is associated with post-nasal drip.

Other causes of rhinitis Infectious rhinitis Usually the term “rhinosinusitis” is preferred for infectious rhinitis. Rhinosinusitis is an inflammatory process involving the mucosa of the nose and one or more sinuses. The mucosa of the nose and sinuses forms a continuum and thus more often than not the mucous membranes of the sinus are involved in diseases primarily caused by inflammation of the nasal mucosa. For this reason infectious rhinitis is discussed under rhinosinusitis.

Occupational rhinitis Occupational rhinitis arises in response to an airborne agent present in the workplace and may be due to an allergic reaction or nonallergic hyperresponsiveness (Gautrin et al. 2006). Many occupational agents are irritant. Causes include laboratory animals (rats, mice, guinea pigs, etc.) (Heederik

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et al. 1999), wood dust, particularly hard woods (mahogany, western red cedar, etc.) (Fernandez-Rivas et al. 1997), mites (Groenewoud et al. 2002), latex (Bousquet et al. 2006b), enzymes (Sarlo & Kirchner 2002), grain (bakers and agricultural workers) (Baur 1999; Gautrin et al. 2002), and chemicals such as acid anhydrides, platinum salts (Malo 2005), glues and solvents (Schiffman & Nagle 1992). Occupational rhinitis is frequently underdiagnosed due to underreporting and/or a lack of physician awareness (Hytonen et al. 1997; Gautrin et al. 2006). Diagnosis is suspected when symptoms occur in relation to work. Differentiating between immunologic sensitization and irritation may be difficult. Given the high prevalence of rhinitis in the general population from all causes, objective tests confirming the occupational origin are essential (Hytonen & Sala 1996). Measures of inflammatory parameters via nasal lavage and objective assessment of nasal congestion both offer practical means of monitoring responses. Growing experience with acoustic rhinometry and peak nasal inspiratory flow suggests that these methods may have a role in monitoring and diagnosis (Pirila et al. 1997). Recent findings indicate that work-related rhinitis is to some extent preventable. Surveillance of sensitized workers may allow early detection of occupational asthma.

Drug-induced rhinitis Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) commonly induce rhinitis and asthma (Table 67.5). In a population-based random sample, aspirin intolerance was more frequent among subjects with allergic rhinitis than among those without (2.6% vs. 0.3%) (Hedman et al. 1999). In about 10% of adult patients with asthma, aspirin and other NSAIDs that inhibit cyclooxygenase (COX)-1 and COX-2 precipitate asthmatic attacks and naso-ocular reactions (Szczeklik et al. 2004). This distinct clinical syndrome, called aspirininduced asthma, is characterized by a typical sequence of symptoms, intense eosinophilic inflammation of nasal and bronchial tissues, combined with overproduction of cysteinyl leukotrienes (Szczeklik & Stevenson 2003) and other prostanoids (Kowalski et al. 2005; Ying et al. 2006). After ingestion of aspirin or other NSAID, an acute asthma attack occurs within 3 hours, usually accompanied by profuse rhinorrhea, conjunctival injection, periorbital edema and sometimes a scarlet flushing of the head and neck. Aggressive nasal polyposis and asthma run a protracted course, despite the avoidance of aspirin and cross-reacting drugs (Kowalski 2000). Blood eosinophil counts are raised and eosinophils are present in nasal mucosa and bronchial airways. Specific antiCOX-2 drugs are usually well tolerated in aspirin-sensitive patients (Szczeklik & Stevenson 2003) but many are no longer marketed. A range of other medications is known to cause nasal symptoms, including reserpine (Girgis et al. 1974), guanethidine (Bauer et al. 1973), phentolamine (Gerth-van-Wijk & Dieges 1991), methyldopa (Bauer et al. 1973), angiotensin-converting

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Table 67.5 NSAIDs that cross-react with aspirin in respiratory reactions. (From Bousquet et al. 2001, with permission.) Generic name

Brand name

Indomethacin Piroxicam Ibuprofen Naproxen Fenoprofen Ketoprofen Diclofenac Diflunisal Tolmetin Mefenamic acid Flurbiprofen Sulindac Ketoralac Etodolac Nabumetone Oxaprozin Metamizol Noramidopyrine Aminophenazone Propylphenazone Oxyphenbutazone Klofezon

Indocid, Metindol Feldene Motrin, Rufen, Advil Naprosyn, Anaprox, Aleve Nalfon Orudis, Oruval Voltaren, Cataflam Dolbid Tolectin Ponstel, Mefacit Ansaid Cilnoril Toradol Lodine Relafen Daypro Analgin Novalgin Isalgin Pabialgin, Saridon Tanderil Perclusone

Paracetamol is well tolerated by the majority of patients, especially in doses not exceeding 1000 mg/day. Nimesulide and meloxicam in higher doses might precipitate nasal and bronchial symptoms.

enzyme inhibitors (Proud et al. 1990), α-adrenoceptor antagonists, intraocular ophthalmic preparations such as beta-blockers (Kaufman 1986), chlorpromazine, and oral contraceptives. The term “rhinitis medicamentosa” (Scadding 1995; Graf 1997) applies to the rebound nasal obstruction which develops in patients who, for some reason, use intranasal vasoconstrictors chronically. Rhinitis medicamentosa can be a contributing factor to a basic nonallergic noninfectious rhinitis, which is often the reason why the patient uses the vasoconstrictor. The pathophysiology of the condition is unclear; however, vasodilatation and intravascular edema have both been implicated. Management of rhinitis medicamentosa requires withdrawal of topical decongestants to allow the damaged nasal mucosa to recover, followed by treatment of the underlying nasal disease (Graf 2005). Cocaine sniffing is often associated with frequent sniffing, rhinorrhea, diminished olfaction, and septal perforation (Schwartz et al. 1989; Dax 1990). For most multiuse aqueous nasal, ophthalmic, and otic products, benzalkonium chloride is the preservative of choice. Intranasal products containing this preservative appear to be safe and well tolerated for both long- and short-term clinical use (Marple et al. 2004).

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Hormonal rhinitis Changes in the nose are known to occur during the menstrual cycle (Ellegard & Karlsson 1994), puberty, pregnancy (Mabry 1986; Ellegard & Karlsson 1999), and in specific endocrine disorders such as hypothyroidism (Incaudo & Schatz 1991) and acromegaly (Fatti et al. 2001). Hormonal imbalance may also be responsible for the atrophic nasal change in postmenopausal women. A persistent hormonal rhinitis or rhinosinusitis may develop in the last trimester of pregnancy in otherwise healthy women. Its severity parallels the blood estrogen level (Ellegard 2004). The symptoms will disappear at delivery. In a woman with perennial rhinitis, the symptoms may improve or deteriorate during pregnancy (Schatz 1998).

cluding headache and nose irritation (rhinorrhea, nasal congestion, postnasal drip, and sneezing) (Jinot & Bayard 1996). Tobacco smoke can alter mucociliary clearance (Bascom et al. 1995) and can cause an eosinophilic and “allergic”-like inflammation in the nasal mucosa of nonatopic children (Vinke et al. 1999b). Tobacco smoking or passive smoking can, in some rhinitis patients, induce symptoms of rhinitis (Willes et al. 1992). In many individuals it is thought that smoking results in the same clinical picture of rhinitis, rhinorrhea and nasal obstruction. However, in normal subjects, smoking was not found to impair nasal quality of life (Bousquet et al. 2004b). NARES might be caused by passive smoking which induced an “allergy-like” inflammatory response (Villar & Holgate 1995).

Other causes NARES and eosinophilic rhinitis

Food-induced rhinitis

Persistent nonallergic rhinitis with eosinophilia is a heterogeneous syndrome consisting of at least two subgroups: NARES and aspirin intolerance (Fokkens 2002). Nonallergic rhinitis with eosinophilia syndrome (NARES) was defined in the early 1980s (Mullarkey et al. 1980; Jacobs et al. 1981). Although it probably does not represent a disease entity on its own, it may be regarded as a subgroup of idiopathic rhinitis characterized by the presence of nasal eosinophilia and persistent symptoms of sneezing, itching, rhinorrhea, and occasionally a loss of sense of smell in the absence of demonstrable allergy. It occurs in children and adults. Asthma appears to be uncommon but around 50% of the patients show bronchial nonspecific hyperreactivity (Leone et al. 1997). It has been suggested that NARES represents an early stage of aspirin sensitivity (Moneret-Vautrin et al. 1990). NARES responds usually but not always favorably to intranasal glucocorticosteroids (Blom et al. 1997a).

Food allergy is a very rare cause of isolated rhinitis (Bousquet et al. 1997). However, nasal symptoms are common among the many symptoms of food-induced anaphylaxis (Bousquet et al. 1997). On the other hand, foods and alcoholic beverages in particular may induce symptoms by unknown nonallergic mechanisms. Gustatory rhinitis (hot spicy food such as hot red pepper) (Raphael et al. 1989) can induce rhinorrhea, probably since it contains capsaicin. This is able to stimulate sensory nerve fibers, inducing them to release tachykinins and other neuropeptides (Lacroix et al. 1991). Dyes and preservatives as occupational allergens can induce rhinitis (Quirce et al. 1994), but in food they appear to play a role in very few cases (Bousquet et al. 1997).

Nasal symptoms related to physical and chemical factors Physical and chemical factors can induce nasal symptoms that may mimic rhinitis in subjects with sensitive mucous membranes, and even in normal subjects if the concentration of chemical triggers is high enough (Shusterman et al. 1998; Leroyer et al. 1999). Skier’s nose (cold dry air) (Silvers 1991) has been described as a distinct entity. However, the distinction between a normal physiologic response and disease is not clear and all rhinitis patients may exhibit an exaggerated response to nonspecific physical or chemical stimuli. Little information is available on the acute or chronic effects of air pollutants on the nasal mucosa (Calderon-Garciduenas et al. 1992). In multiple chemical sensitivities, nasal symptoms such as impaired odor perception may be present (Ojima et al. 2002).

Rhinitis in smokers In smokers, eye irritation and odor perception are more common than in nonsmokers (Bascom et al. 1996). Moreover, some smokers report a sensitivity to tobacco smoking in-

Rhinitis of the elderly Rhinitis of the elderly, or senilic rhinitis as it is called in the Netherlands, is a characteristic clinical picture of an elderly patient suffering from a persistent clear rhinorrhea without nasal obstruction or other nasal symptoms. Patients often complain of the classical drop on the tip of the nose. The first treatment option is intranasal ipratropium bromide (up to six times daily) generally with a good clinical result, suggesting overactivity of the parasympathetic nervous system.

Emotions Stress and sexual arousal are known to have an effect on the nose probably due to autonomic stimulation.

Atrophic rhinitis Primary atrophic rhinitis is characterized by progressive atrophy of the nasal mucosa and underlying bone (Goodman and De Souza 1973), rendering the nasal cavity widely patent but full of copious foul-smelling crusts. It has been attributed to infection with Klebsiella ozaenae (Henriksen & Gundersen 1959), although its role as a primary pathogen is not determined. The condition produces nasal obstruction, hyposmia, and a constant bad smell (ozena) and must be distinguished from secondary atrophic rhinitis associated with chronic

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granulomatous conditions, excessive nasal surgery, radiation and trauma.

task force attempted to accommodate these needs by allocating definitions adapted to different situations (Aukema et al. 2005; Fokkens et al. 2005).

Unknown etiology (idiopathic rhinitis) Sometimes termed “vasomotor rhinitis,” patients with this condition manifest upper respiratory hyperresponsiveness to nonspecific environmental triggers such as change in temperature and humidity, exposure to tobacco smoke, and strong odors. The limited data available suggest that these patients might present as follows (van Rijswijk et al. 2005): (i) nasal inflammation (in a small number of patients); (ii) an important role for C-fibers, although direct observations explaining this mechanism are lacking; (iii) parasympathetic hyperreactivity and/or sympathetic hyporeactivity; and/or (iv) glandular hyperreactivity. Some persons consider even slight nasal symptoms to be abnormal and seek consequent medical advice. Inquiry into the number of hours spent with daily symptoms may help to determine a distinction between a normal physiologic response and disease. Also, the use of a daily record card to score symptom duration and intensity can be combined, if appropriate, with peak nasal inspiratory flow measurements to give the physician more insight into the severity of the disease. Marked discrepancies can be found between the description of the problem at the first visit and data from these daily measurements (Blom et al. 1995, 1997b).

Rhinosinusitis Sinusitis and rhinitis usually coexist and are concurrent in most individuals; thus, the correct terminology for sinusitis is now “rhinosinusitis.” The diagnosis of rhinosinusitis is made by a wide variety of practitioners, including allergologists, otolaryngologists, pulmonologists, primary care physicians and many others. Therefore, an accurate, efficient and accessible definition of rhinosinusitis is required. Attempts have been made to define rhinosinuisitis in terms of pathophysiology, microbiology, radiology, and severity of symptoms and their duration (Shapiro & Rachelefsky 1992; Williams & Simel 1993; Lund 1997). Until recently, rhinosinusitis was usually classified, based on duration, into acute, subacute and chronic (Lund 1997). This definition does not incorporate severity of the disease. Also, due to the long timeline of 12 weeks in CRS, it can be difficult to discriminate between recurrent acute rhinosinusitis and CRS with or without exacerbations. Because of the large variety of techniques for the diagnosis and treatment of rhinosinusitis/nasal polyps by ENT specialists and nonspecialists, subgroups should be differentiated. Epidemiologists need a workable definition that does not impose too many restrictions when studying large populations, whereas researchers need a set of clearly defined items to describe their patient population accurately. The EPOS

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Clinical definition Rhinosinusitis (including nasal polyps) is defined as follows. 1 Inflammation of the nose and paranasal sinuses characterized by two or more symptoms, one of which should be either nasal blockage/obstruction/congestion or discharge (anterior/posterior nasal drip): (a) blockage/congestion; (b) discharge (anterior/posterior nasal drip); (c) facial pain/pressure; (d) reduction or loss of smell; and either 2 Endoscopic signs: (a) polyps; and/or (b) mucopurulent discharge from middle meatus; and/or (c) edema/mucosal obstruction primarily in middle meatus; and/or 3 CT changes: mucosal changes within ostiomeatal complex and/or sinuses. CT of the paranasal sinuses has emerged as the standard test for the assessment of CRS, as evidenced by the emergence of several CT-based staging systems. Despite its central role in the diagnosis and treatment planning for CRS, the sinus CT represents a “snapshot in time.” In CRS, the correlation between CT and symptoms is low to nonexistent (Basu et al. 2005; Wabnitz et al. 2005). The most frequently used scoring system for CT in CRS is the Lund–Mackay score (Lund & Mackay 1993). Overall, the Lund–Mackay score in the general population is not zero. A Lund score ranging from 0 to 5 may be considered within an incidentally “normal” range, and should be factored into clinical decision-making (Ashraf & Bhattacharyya 2001).

Severity of the disease The disease can be divided into mild, moderate or severe based on total severity VAS (0–10 cm), where mild is 0–3, moderate 3–7, and severe 7–10. To evaluate total severity the patient is asked to indicate where their symptoms lie on a 10-cm line, with “not troublesome” at one end of the line and “extremely troublesome” at the other. Severity of rhinosinusitis can also be assessed using quality-of-life questionnaires (Metson & Gliklich 2000; Birch et al. 2001; Senior et al. 2001; van Agthoven et al. 2001; Kay & Rosenfeld 2003; Wang et al. 2003; Atlas et al. 2005; Chen et al. 2005; Wabnitz et al. 2005). However, these different methods of evaluation of rhinosinusitis severity are not always correlated (Ragab et al. 2004; Wabnitz et al. 2005).

Duration of the disease • Acute: < 12 weeks; complete resolution of symptoms. • Chronic: > 12 weeks; incomplete resolution of symptoms.

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Definition for epidemiologic studies Acute rhinosinusitis Acute rhinosinusitis is defined as sudden onset of two or more of the symptoms, one of which should be either nasal blockage/obstruction or nasal discharge: • blockage/congestion; • discharge (anterior/posterior nasal drip); • facial pain/pressure; • reduction/loss of smell. Symptoms should be present for less than 12 weeks, with validation by telephone or interview. Questions on allergic symptoms, i.e., sneezing, watery rhinorrhea, nasal itching and itchy watery eyes, should be included. Common cold/acute viral rhinosinusitis Common cold/acute viral rhinosinusitis is defined by a duration of symptoms of less than 10 days. Children have six to ten common colds per year, adults usually two to three. The most important viruses involved are rhinovirus (> 50%), coronavirus (15– 20%), respiratory syncytial virus (RSV), adenoviruses, and parainfluenza virus. Infection with influenza virus (’flu) usually causes a more severe acute rhinosinusitis on top of the symptoms described above and characterized by high fever and feeling of illness. Rhinosinusitis caused by influenza more often results in complications and high-risk groups need to be vaccinated. Acute nonviral (bacterial) rhinosinusitis Acute nonviral (bacterial) rhinosinusitis is defined as increase in symptoms after 5 days or persistent symptoms after 10 days with duration less than 12 weeks. The most common cause is bacterial infection, of which Streptococcus pneumoniae (20–35%) and Haemophilus influenzae (5– 25%) are the most common (Gwaltney et al. 1992; Van Cauwenberge et al. 1997). However, a range of other organisms, including Moraxella catarrhalis, Staphlyococcus aureus, and anaerobic bacteria, are also found. Chronic rhinosinusitis/nasal polyps 1 Inflammation of the nose and paranasal sinuses characterized by two or more symptoms, one of which should be either nasal blockage/obstruction/congestion or discharge (anterior/posterior nasal drip): (a) blockage/congestion; (b) discharge (anterior/posterior nasal drip); (c) facial pain/pressure; (d) reduction or loss of smell. 2 Nasal congestion/obstruction/blockage with facial pain/ pressure, or discolored discharge (anterior/posterior nasal drip), or eeduction/loss of smell. Symptoms should be present for more than 12 weeks, with validation by telephone or interview. Questions on allergic symptoms, i.e., sneezing, watery rhinorrhea, nasal itching and itchy watery eyes, should be included.

Table 67.6 Presenting symptoms of CRS. (Adapted from Meltzer et al. 2004, with permission.)

Presenting symptom

Percentage of patients with symptom

Nasal obstruction Nasal discharge Facial congestion Facial pain/pressure/fullness Loss of smell Fatigue Headache Ear pain/pressure Cough Halitosis Dental pain Fever

94 82 85 83 68 84 83 68 65 53 50 33

For epidemiologic studies, the definition is based on symptomatology without ENT examination or imaging. However, it has to be realized that considerable overestimation of the disease can be observed when a definition of rhinosinusitis based only on symptomatology without ENT examination or imaging is used (Blomgren et al. 2002, 2005; Varonen et al. 2003). The presenting symptoms are indicated in Table 67.6.

Definition for research For research purposes, CRS is the major finding and nasal polyposis is considered to be a subgroup of this entity. For the purposes of a study, the differentiation between CRS and nasal polyposis must be based on outpatient endoscopy. The research definition is based on the presence of polyps and prior surgery.

Definition when no previous sinus surgery has been performed • Polyposis: bilateral, endoscopically visualized in the middle meatus. • CRS: bilateral, no visible polyps in the middle meatus, if necessary following decongestant. This definition accepts that there is a spectrum of disease in CRS which includes polypoid change in the sinuses and/or middle meatus but excludes those with polypoid disease presenting in the nasal cavity to avoid overlap. Definition when sinus surgery has been performed Once surgery has altered the anatomy of the lateral wall, the presence of polyps is defined as pedunculated lesions as opposed to cobblestoned mucosa over 6 months after surgery on endoscopic examination. Any mucosal disease without overt polyps should be regarded as CRS.

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Chronic rhinosinusitis Nasal polyps

0.5–1.5% of patients with positive skin-prick tests for common allergens (Caplin et al. 1971; Settipane & Chafee 1977). Nasal polyposis has also been associated with cystic fibrosis.

Acknowledgments Fig. 67.2 The spectrum of chronic rhinosinusitis and nasal polyps. (See CD-ROM for color version.)

Nasal polyposis Nasal polyposis and CRS are often grouped as one disease entity, because it seems impossible to clearly differentiate them (Ichimura et al. 1996; Dykewicz 2003; Steinke et al. 2003). Nasal polyposis is considered a subgroup of CRS (Fig. 67.2). The question remains as to why “ballooning” of mucosa develops in polyposis patients but not in all rhinosinusitis patients. Nasal polyps have a strong tendency to recur after surgery even when aeration is improved (Vento et al. 2000). This may reflect a distinct property of the mucosa of polyp patients which has yet to be identified. Some studies have tried to divide CRS and nasal polyps based on inflammatory markers (Hamilos et al. 1995, 1996; Bachert & Van Cauwenberge 1997; Rudack et al. 1998) Although these studies point to a more pronounced eosinophilia and IL-5 expression in nasal polyps than found in patients with CRS, these studies also point to a continuum in which differences might be found at the ends of the spectrum, but at the moment no clear-cut division can be made. Nasal polyps appear as grape-like structures in the upper nasal cavity, originating from the ostiomeatal complex. They consist of loose connective tissue, edema, inflammatory cells, and some glands and capillaries and are covered with varying types of epithelium, mostly respiratory pseudostratified epithelium with ciliated cells and goblet cells. Eosinophils are the most common inflammatory cells in nasal polyps, but neutrophils, mast cells, plasma cells, lymphocytes, and monocytes are also present, as well as fibroblasts. IL-5 is the predominant cytokine in nasal polyposis, reflecting activation and prolonged survival of eosinophils (Bachert et al. 1997). The reason why polyps develop in some patients and not in others remains unknown. There is a definite relationship in patients with Fernand Widal triad: asthma, NSAID sensitivity, and nasal polyps. However, not all patients with NSAID sensitivity have nasal polyps, and vice versa. In the general population, the prevalence of nasal polyps is 4% (Hedman et al. 1999). In patients with asthma, a prevalence of 7–15% has been noted, whereas in NSAID sensitivity nasal polyps are found in 36– 60% of patients (Settipane & Chafee 1977; Larsen 1996). It had long been assumed that allergy predisposed to nasal polyps because the symptoms of watery rhinorrhea and mucosal swelling are present in both diseases, and eosinophils are abundant. However, epidemiologic data provide no evidence for this relationship: polyps are found in

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Both authors are members of the GALEN (Global Allergy and Asthma European Network), supported by EU Framework program for research, contract number FOOD-CT-2004–506378.

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Allergic Rhinoconjunctivitis and Immunotherapy

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Pathophysiology of Allergic Rhinitis Peter H. Howarth

Summary Allergic rhinitis describes an abnormal state of mucosal inflammation in the nose that arises in sensitized individuals due to the tissue interaction between specific IgE and environmental allergens. This inflammation involves many cell types, both infiltrating and structural, and characteristically leads to the generation of symptoms such as itch, sneeze, rhinorrhea, and congestion. These symptoms can be related to the local release of mediators and their interaction with end-organ receptors on neural and vascular structures within the nose. Histamine is a prominent mediator and many of its effects can be related to stimulation of H1-receptors at these sites, although it is clear that there is also likely to be involvement of other histamine receptor subtypes and that a range of other mediators such as prostanoids, leukotrienes, kinins and proteases are also involved. Some mediators may be derived from infiltrating cells, such as eosinophils and basophils, in addition to those linked with mast cell degranulation. The tissue recruitment of these circulating leukocytes involves systemic signaling to promote bone marrow progenitor generation and maturation, endothelial cell activation with upregulation of specific leukocyte endothelial adhesion molecules that permit their recruitment from the circulation, and the directed migration of these cells toward the epithelium and nasal lumen. This directed migration can be linked to chemokine synthesis by epithelial cells and the epithelial accumulation of mast cells, dendritic cells, T cells, basophils, and eosinophils is evident in active disease. Dendritic cells can sample allergen from the lumen and present antigen to T cells, directing T-cell development through costimulatory signals that favor, in allergic disease, the development of the Th2 phenotype. The local generation of Th2 cytokines explains the nature of the allergic inflammatory profile as well as the IgE-isotype switch by B cells. The process of nasal inflammation alters nasal reactivity, so that nonallergenic stimuli such as temperature change, environmental pollutants and strong odors induce nasal symptoms in those with rhinitis at thresholds lower than

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that in the normal nose. In addition nasal inflammation does not exist in isolation, with common mucosal events also being evident within the conjunctiva, lower airways, and sinuses. The impact of the disease is thus more extensive than might be suspected from the nasal symptoms alone and significantly impacts on well-being and quality of life.

Introduction Rhinitis is clinically recognized by the anterior nasal symptoms of itch, nasal discharge, sneezing and nasal stuffiness. It may be a manifestation of a range of underlying disorders of which allergic and infective causes are most common. Allergic rhinitis describes an inflammatory condition of the nasal mucosa, which is induced by an IgE-mediated response (Howarth 1998). In addition, there may be an associated loss of sense of smell and inability to taste. This is most common in chronic rhinitis. The symptoms are periodic with seasonal disease, occurring in temporal relationship to the presence of seasonal allergens in individuals who are appropriately sensitized. The predominant allergens causing seasonal rhinitis are aeroallergens, commonly outdoor allergens, such as tree, grass or weed pollens. The time of pollination and hence the “season” for different allergens will vary from country to country depending on the climate. In temperate climates the typical pattern would be for tree pollination to occur in early and late spring, grass pollination in late spring and throughout the early summer months, and for weed pollens, such as ragweed, to be present in the air in late summer and autumn. Perennial disease, which may be present all year round, relates to sensitization to perennial allergens. These are typically indoor aeroallergens, such as those related to house-dust mites, cockroaches, pets (cats, dogs) or, in certain climates, molds. Individuals with perennial rhinitis may of course also be sensitized to outdoor allergens and experience worsening of their disease during the appropriate seasonal exposure. Recent guidelines for the management of rhinitis have classified the disease as intermittent or persistent, rather than seasonal and perennial (Bousquet et al. 2001). There are several reasons for this. Firstly polysensitization is common,

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with indviduals sensitized to pollen allergens, such as those from trees, grass and weeds, having long-lasting disease that potentially extends from early spring to late autumn in the temperate climates of the world. Their disease is thus more persistent than that in an individual with typical seasonal allergic rhinitis due to a single sensitization. In the more tropical climates pollen sensitization may lead to disease all year round, as there may be several periods of pollination and as such typical seasonal allergens may give rise to perennial disease. Conversely, sensitization to the typical perennial allergens does not always give rise to perennial disease. Depending on the level of exposure and the degree of sensitization, the rhinitis may be intermittent or focused at one particular time of year. In those sensitized to animal allergens who do not own pets, the exposure and symptoms may only be intermittent and transient so the subdivision into intermittent and persistent disease is now preferred. Intermittent rhinitis is defined on the basis of symptoms that are present for less than 4 days per week and for less than 4 weeks in duration. If symptoms are present for more than 4 days per week or have lasted more than 4 weeks, regardless of the number of days per week, then the disease is classified as persistent. However, much of the information on the pathophysiology of the disease has been gained from those with allergic rhinitis who have been categorized as having either seasonal or perennial disease. It is probable that much of the information previously gained from naturally occurring disease reflects persistent disease, whereas detailed studies on allergen challenge, while not necessarily physiologic, provide information relating to acute intermittent allergic disease. Studies of the distribution of intermittent and persistent rhinitis among those with rhinitis have suggested that within the community 71% is intermittent and 29% persistent (Bauchau & Durham 2005), whereas in those seeing a specialist on account of troublesome rhinitis, 73% is persistent (Bousquet et al. 2005). Within the latter group, 81% of those with persistent rhinitis had moderate/severe disease and 19% mild disease. These studies also highlighted that intermittent and persistent disease does not equate to seasonal and perennial disease respectively, with the community study identifying that over 50% of those with persistent disease had grass pollen allergen sensitization, a finding endorsed by the study of specialist referrals. However, in the later study it was evident that monosensitization to grass pollen was more evident within the intermittent group and monosensitization to house-dust mite allergens was more commonly associated with persistent disease. In addition to allergic triggers, nasal symptoms may be induced in allergic rhinitis by nonallergic factors due to the development of nasal hyperreactivity. Nasal hyperreactivity describes an increased sensitivity of the nasal mucosa to irritants and nonspecific stimuli, such as changes in temperature and strong odors (Gerth-van-Wijk 1991; Nathan et al. 2005). Nasal challenge with a variety of stimuli, including methacholine, histamine, kinins, capsaicin and cold air, has been used

Pathophysiology of Allergic Rhinitis

to explore nasal reactivity (Gerth-van-Wijk 1991; Nathan et al. 2005; Sarin et al. 2006). These studies identify increased reactivity in allergic rhinitics as compared to nonrhinitic controls (Borum 1979; Gerth-van-Wijk & Dieges 1987; Gerthvan-Wijk 1991; Nathan et al. 2005; Sarin et al. 2006). The outcome with these challenges differs depending on the stimulus with histamine predominantly inducing sneezing, methacholine rhinorrhea, and cold air nasal obstruction. While all these challenges demonstrate differences between allergic rhinitis and healthy controls, suggestive of the importance of allergic inflammation in the generation of these reactivity changes, they have not been so clearly discriminating in nonallergic rhinitis (Gerth-van-Wijk & Dieges 1991; Lacroix et al. 1991; Braat et al. 1998), a condition in which fumes and odors also induce symptoms (Chih-Feng & Baraniuk 2002; Sarin et al. 2006). Such differences likely reflect differences in the underlying disease process and the involvement of separate pathways for disease pathogenesis, as clearly with histamine nasal challenge the tests indicate greater reactivity in allergic as compared to nonallergic rhinitic subjects, whereas with cold air challenge such differences are not so marked (Gerth-van-Wijk & Dieges 1987, 1991). Although there is considerable overlap in measures, making clinical measurement of nasal reactivity in rhinitis less valuable diagnostically than bronchial responsiveness recordings in lower airways disease such as asthma, end-point titration of the reflex-mediated response to histamine (sneezing and secretion) but not the nasal obstructive response has been shown to differentiate disease from nondisease (Gerth-vanWijk & Dieges 1987) and to correlate with clinical symptom scores (Gerth-van-Wijk 1991). The presence of nasal hyperreactivity means that individuals with rhinitis experience enhancement of their nasal symptoms when exposed to ambient factors within the air, such as tobacco smoke, strong odors, perfumes, car exhaust pollutants, and photochemical pollutants for example. The exaggerated response to these common environmental exposures may limit the lifestyle of subjects with rhinitis and thus have an additional impact on their quality of life. In addition, when considering the pathophysiology of allergic rhinitis, it must also be appreciated that disease is not only limited to the nasal mucosa and that it is usual for there to be more extensive disease, with mucosal abnormalities typically evident within the conjunctiva, lower airways and sinuses that may manifest by clinical symptoms referable to these sites. Thus conjunctivitis, asthma and sinusitis are often comorbidities that are evident in those with rhinitis. In addition in allergic rhinitis there is evidence of systemic signaling that influences bone marrow function and individuals with rhinitis often feel systemically unwell, an important factor that contributes to their impaired quality of life. Thus while considerations of disease pathophysiology largely focus on the generation of nasal symptoms, an appreciation of the systemic nature of the disease and how that links to the nasal

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disease is also of relevance to disease understanding and therapeutic intervention.

Disease pathophysiology An appreciation of the basis for disease expression in rhinitis depends on an understanding of the normal nasal anatomy and physiology, how an atopic tendency coupled with relevant allergen exposure alters the cellular components within the nose, and how such cellular changes associated with cell activation interacts with the normal nasal structure to induce clinical disease expression.

Anatomy and physiology of the normal nose The nasal cavity is divided into two separate air passages by the nasal septum. These air passages start at the nostrils (anterior nares) and remain separate until they unite in the posterior nares prior to joining the nasopharynx. The nasal air passages are structured laterally by a compliant nasal vestibule anteriorly and a bony cavum posteriorly. The lateral wall of the bony component is lined by the turbinates (inferior, middle and superior). The nasal cavity narrows from around 90 mm2 at its orifice (anterior vestibule) to a slit of 30 mm2 approximately 3 cm into the nostrils, at the anterior end of the inferior turbinate. This point of narrowing, termed the nasal valve, is the major site of airways resistance within the nose (Haight & Cole 1983) and contributes two-thirds of the total resistance to airflow within the airways (Ferris et al. 1964). Beyond this point the nasal cavity expands to approximately 130 mm2 and the airstream bends through 90°. During quiet breathing the nasal airflow is predominantly directed around the inferior turbinate along the floor of the nasal cavity. Relatively little airflow is directed upward toward the middle and superior turbinates, although this is achieved at higher inspiratory flow, as with sniffing, when greater turbulence of flow directs air toward these turbinates and the olfactory region in the upper part of the nasal cavity. A thin layer of mucus, consisting of a low-viscosity sol phase and a viscous gel phase, covers the nasal epithelium and is constantly transported to the nasopharynx by ciliary movements. Nasal secretions have multiple sources, such as the submucosal glands, goblet cells, tears and exudation from blood vessels, and consist of albumin, immunoglobulins, proteolytic and bacteriolytic enzymes, mediators and cells, forming a generalized protection against infection. Mucociliary transport is dependent on the correct consistency of the mucus and on the effective movement of the cilia, which beat about 1000 times per minute, moving the superficial gel layer and the debris trapped therein with a speed of about 3–25 mm/min. Impacted particles adhere to the nasal epithelial mucous layer and are cleared from the nasal passages by coordinated ciliary movement. The direction of mucociliary clearance is predominantly toward the nasopharynx, where

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cleared mucus is swallowed, with only lesser clearance toward the anterior vestibule. However, the anterior clearance does protect the majority of the nasal mucosal epithelium from anteriorly impacted particles and gaseous pollutants. The epithelium of the anterior vestibule is stratified squamous epithelium while that of the main respiratory area within the nostrils is pseudostratified ciliated columnar epithelium (Mygind et al. 1982). There is a specialized epithelium, with ciliated receptor cells, within the olfactory area. The epithelial cell types of the nasal respiratory epithelium, which overlie a continuous basement membrane, are basal cells, goblet cells, and columnar cells (ciliated and nonciliated). The ratio of columnar cells to goblet cells is about 5 : 1. The goblet cells are not under autonomic innervation and it is probable that local factors regulate the secretion of mucin by these cells. It has been calculated that the density of goblet cells within the normal nose is 10 000/mm2 (Tos 1983). Seromucous glands are present deep within the lamina propria and are of the compound alveolar type, consisting of both mucous and serous glands in a ratio of approximately 1 : 8 (Widdicombe & Wells 1982). These glands are under parasympathetic cholinergic regulation and neural stimulation induces a watery nasal secretion (Eccles & Wilson 1993). The secretion from these glands flows onto the epithelial surface via large ducts. There are approximately 100 000 seromucous glands within the nose. The density of these glands is greatest in infants and decreases during progression to adulthood: 34 glands/mm2 in the infant to 8.3 glands/mm2 in the adult nose (Tos 1983). This difference explains the greater tendency for physiologic rhinorrhea in small infants and children. Immediately beneath the epithelium is an almost cell-free zone composed of a network of fine fibrils, which have been shown to be composed of fibronectin and collagens type III and V. These merge with the underlying connective tissue that contains blood vessels and is populated by resident cells such as fibroblasts and mast cells and infiltrating cells such as lymphocytes and neutrophils (Busuttil et al. 1977; Janke 1978; Wilson & Howarth 1995).

Nasal vasculature The nasal vascular anatomy is complex, with both resistance and capacitance vessels. The resistance vessels are predominantly small arteries, arterioles, and arteriovenous anastomoses (Fig. 68.1). These are fed by a number of vessels, including the anterior and posterior ethmoid arteries, both branches of the ophthalmic artery, the sphenopalatine artery, a branch of the external carotid, and the facial artery. Blood flow to the nasal mucosa is regulated by constriction and dilatation of these vessels (Dawes & Pritchard 1953; Slome 1995–6). This is under sympathetic control, with stimulation inducing vascoconstriction and a reduction in nasal mucosal blood flow (Malma 1977). Arterial blood reaches the venous drainage system either via superficial capillaries or via arteriovenous anastomoses. It has been estimated that up to 60% of the

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Nasal lumen Epithelium

Superficial capillary network Fig. 68.1 Schematic representation of the nasal vasculature showing the arterial supply and how it connects to the venous drainage, with the potential to bypass the superficial capillary network via the arteriovenous anastomoses. The state of engorgement of the venous sinusoids determines turbinate size and hence nasal patency within each nostril. The direct effects of inflammatory mediators on receptors colocalized to the resistance and capacitance vessels determine the magnitude of nasal obstruction. (See CD-ROM for color version.)

Arteriovenous anastomosis Resistance vessels

Venous sinusoids

Capacitance vessels

Glandular capillaries Arteriovenous anastomosis

Venous drainage

Arterial supply

nasal mucosal blood flow is shunted through these anastomoses, thereby bypassing the superficial vessels (Angaard 1974). This system allows alterations in nasal blood flow without changes in nasal resistance, so that temperature control can be facilitated. The superficial capillaries beneath the basement membrane and around the glandular tissue are unusual in being fenestrated, with the fenestrations facing the respiratory epithelium (Cauna & Hindere 1969; Cauna 1970; Greves & Herrman 1987). This fenestration allows unimpeded extravasation of plasma proteins. The fenestrations are holes in the vascular basement membrane not covered by endothelial cells and indicate that plasma extavasation is an important intrinsic property of the vessels at this site (Watanabe et al. 1980). Extravasated plasma contains albumin and immunoglobulins as well as factors involved in the kinin, complement, coagulation, and fibrinolytic systems. It is probable that this exudation is essential both for epithelial nutrition and for local immune defense. This exudation is in contrast to vascular exudation at most other tissue sites, which is via gaps in endothelial cells at postcapillary venules and depends on the local regulation of endothelial cell behavior (McDonald 1990). The dominant determinant of exudation within the nasal capillaries is arteriolar tone. Arteriolar dilatation promotes exudation, by increasing capillary blood flow and increasing capillary vascular pressure. The balance between arteriovenous anastomotic flow and capillary flow will determine the magnitude of exudation. The arterial blood drains into a venous system composed of a labyrinth of valveless venous sinusoids within the lamina propria that are particularly prominent within the turbinates (Cauna 1970). These drain into muscular veins that empty into the veins of the palate and nasopharynx as well as into

the ethmoidal veins, which connect with the sagittal sinus. The large collapsable venous sinusoids are the capacitance component of the system. Vascular congestion of the venous sinusoid results in engorgement of the turbinates, an increase in nasal airways resistance, and a reduction in nasal airflow. Venous engorgement is under sympathetic regulation and is determined by the tone of the arteriolar, arteriovenous anastomotic and muscular venous draining vessels. An increase in sympathetic tone decreases nasal congestion by reducing flow and facilitating sinus emptying. The vascular tone is also regulated locally by neuropeptides (see below) and in response to locally released mediators. Nasal blockage is thus a vascular phenomenon that is a reflection of the local neurohumoral environment.

Neural innervation The innervation of the nasal mucosa is well defined (Eccles 1982; Lundblad 1984; Lundberg et al. 1987). The olfactory and trigeminal nerves predominantly supply the sensory innervation of the nose. The olfactory nerves enter the nose through the cribriform plate and form a distinct olfactory area that acts as a chemoreceptor to sample inspired air and provide an appreciation of odors and facilitate taste. The ophthalmic and maxillary branches of the trigeminal nerve contain the sensory nerves from the nasal mucosa and nasal vertibule and service the sensations of touch, pain, temperature and itch as well as the appreciation of nasal airflow. These sensory nerves consist of both myelinated and nonmyelinated fibers. The unmyelinated fibers are slow conducting and, in the vast majority, belong to the nociceptor C-fiber type. Some of the thinnest among the myelinated fibers belong to the Aδ category, which may also have nociceptive functions. Sensory neural responses are conducted centrally

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Allergic Rhinoconjunctivitis and Immunotherapy Nasal insufflation with substance P and also NKA but not CGRP has been shown to cause nasal blockage and increase vascular permeability in subjects with rhinitis but to have little effect in the normal nose (Devillier et al. 1988; Braunstein et al. 1991; Guarnaccia et al. 1994). Activation of the sensory nerves, in addition to a vasodilator action through axonal reflexes, leads to vasodilatation by modification of intrinsic sympathetic tone, through downregulation of ganglionic neurotransmission at the trigeminal and sphenopalatine ganglia. These neuropeptides have also been implicated in a range of cellular effects, involving cells such as lymphocytes, eosinophils, mast cells, and macrophages (Fajac et al. 1995; James & Nijkamp 1999; O’Connor et al. 2004). UCN may act as a locally expressed proinflammatory factor and induce mast cell degranulation and cytokine secretion and trigger vascular permeability, mediated by CRF receptors in peripheral tissues (Kim et al. 2007). Both parasympathetic and sympathetic nerve fibers supply the efferent neural pathways to the nasal mucosa (Fig. 68.2). The parasympathetic system largely regulates nasal glandular secretion, while sympathetic fibers regulate nasal flow and the state of engorgement of the venous erectile tissue. The parasympathetic fibers relay in the sphenopalatine ganglion and the postganglionic nerves reach the nasal glands via branches of the posterior nasal nerve. The primary

on afferent pathways and give rise to the recognition of the sensory neural response as, for example, pain or itch. In addition, an important aspect of the nonmyelinated C fibers is that their dendrites can be antidromically stimulated by action potentials that originate at different terminals of the same neuron. This results in the release of inflammatory neuropeptides from peripheral neurosecretory varicosities. Sensory C fibers from the trigeminal ganglion contain the tachykinins substance P, neurokinin A (NKA), and calcitonin gene-related peptide (CGRP). In addition, gastrin-releasing peptide has also been identified in the human nasal mucosa (Baraniuk et al. 1990a) and more recently urocortin (UCN), a member of the corticotropin-releasing factor (CRF) neuropeptide family (Kim et al. 2007). Immunohistochemical analysis reveals that these neuropeptide-containing nerves are present in nerve endings around the sphenopalatine ganglion cells, around blood vessels, and beneath or within the epithelium. Receptors for these neuropeptides are also found in a similar distribution, colocalized to neural ganglia, glands, and arterial and venous blood vessels (Baraniuk et al. 1990b, 1991; Kim et al. 2007). It is probable that in the normal nose these neuropeptides exert a fine-tuning role in the regulation of glandular secretion and vascular tone, as in animal models release of neuropeptides by sensory nerve endings produces vasodilatation and increased microvascular permeability.

Sensory nerve stimuli Histamine Bradykinin PGF2a

Afferent pathway

Glandular secretion

SP

Glands SP CGRP NKA UCN

Efferent pathway

NKA SP CGRP NKA UCN

CGRP

Glands Ganglia H3 Sympathetic

Ach Parasympathetic VIP, PHI Ach, VIP, NO

Noradrenaline NPY Vasodilatation

Vasodilatation Vasoconstriction

Fig. 68.2 Schematic representation of the neural connections within the nose. Classically, sensory neural stimulation by inflammatory mediators in allergic rhinitis activates afferent pathways that connect centrally to efferent parasympathetic pathways, such that unilateral nasal stimulation induces bilateral glandular secretion. Parasympathetic responses promote glandular secretion through release of acetylcholine (Ach) and to a considerably lesser extent vasoactive intestinal polypeptide (VIP). The release of VIP, Ach and the neural generation of nitric oxide (NO) may influence vascular tone to promote vasodilatation and engorgement of the venous sinusoids. The state of vascular tone is also influenced by sympathetic tone,

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with the release of norepinephrine (noradrenaline) and neuropeptide Y (NPY) inducing vasoconstriction and thereby maintaining nasal patency. Sympathetic neurotransmission is potentially regulated at the ganglia with histamine, acting on H3-receptors, and the neuropeptides substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) all reducing neurotransmission and thereby indirectly promoting nasal congestion due to reduction in the vasoconstrictor tone. Neuropeptides may be released antidromically following sensory neural stimulation and can also act locally to modify glandular secretion and alter vascular tone. (See CD-ROM for color version.)

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parasympathetic postganglionic neurotransmitter is acetylcholine, which acts on muscarinic receptors. These are now recognized to number five different subtypes that are distributed on glands, arteries, veins, and epithelial cells. The M3 subtype is the most abundant and the predominant effect of physiologically released acetylcholine is to stimulate glandular secretion through action on M3 receptors. The cholinergic nerves also contain and release vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) (Baraniuk et al. 1990c). VIP has vasodilatory properties and has also been shown to stimulate serous but not mucous cell secretion in human nasal mucosal explants (Baraniuk et al. 1990c). However, the role of VIP in parasympathetic reflexes within the normal nose may be small as atropine pretreatment is very effective in inhibiting reflex-mediated nasal secretion in vivo (Konno & Togawa 1979). The sympathetic nerves reach the nasal mucosa via the superior cervical ganglion and are distributed to the nasal blood vessels via the nerves of the pterygoid canal and by branches of the trigeminal nerve. The primary neurotransmitter is norepinephrine (Pernow 1988). This has vasoconstrictor actions through actions on α-adrenergic receptors. Sympathetic neurons contain postjunctional α1- and α2-adrenoceptors, both mediating vasoconstriction, and prejunctional α2-adrenoceptors (Lacroix 1989). Through constriction of the smooth muscle of resistance vessels, sympathetic stimulation will reduce nasal blood flow and prevent blood pooling in the venous sinusoids. Although the primary sympathetic response in vasoconstrictor, it has been suggested from animal models that β1- and β2-adrenoceptors are also present on the vasculature of the nasal mucosa, and that their stimulation results in vasodilatation of resistance vessels and thereby an increase in blood flow (Malm 1977; Wang & Lung 2003). In addition it has been indicated that β-adrenoceptors have an influence on glandular activity (Hall & Jackson 1968). Sympathetic nerves also contain the vasoconstrictor peptide neuropeptide Y (NPY). NPY nerve fibers are present in the walls of arterioles, arteriovenous anastomoses, and other vessels within the nasal mucosa and NPY receptors have been colocalized to these vascular sites, suggestive of the relevance of NPY to the regulation of vascular tone (Baraniuk et al. 1992; Cervin et al. 1999). Consistent with this, NPY has been shown in human nasal mucosal tissue biopsy specimens to cause dose-dependent vasoconstriction (Fischer et al. 1993). A rich adventitial plexus of nerve fibers containing sympathetic, parasympathetic, and nonadrenergic noncholinergic neurons thus surrounds the arteries and veins of the nasal mucosa. The local regulation of vascular tone determines the nasal cycle, which involves reciprocal oscillations between the nostrils of congestion and decongestion related to the state of engorgement of the venous sinusoids (Eccles 1996). Nasal airflow is not symmetrical between both nostrils and it is appreciated that airflow is usually more dominant in one nostril than the other at any one time. This dominance is not

Pathophysiology of Allergic Rhinitis

fixed within the normal nose but oscillates from side to side over several hours. The basis for these cyclical changes relates to alterations in the neural regulation of vascular tone. Studies evaluating the distribution of axonal and transmitter immunoreactivity have identified that these are predominantly located in the arterial part of the human nasal vascular system (Riederer et al. 2002). In the venous capacitance vessels there is only a sparse innervation, although there is a rich nerve supply in the subendothelial muscular bolsters of the cushion veins. Besides the adventitial plexus, which supplies the circular muscle of the tunica media, there are additional adrenergic axons that supply the longitudinal subendothelial musculature. These subendothelial cushions regulate venous sinusoidal drainage. Alterations in local sympathetic regulation of the arterial supply and these muscular “cushion” veins are considered to underlie the nasal cycle (Eccles 1996). A dual (endothelial and neuronal) control exists in arterioles, whereas the control in the subendothelial muscular swellings of the cushion veins appears to be mainly neuronal. Nasal obstruction due to nasal engorgement arises because of simultaneous relaxation of all smooth muscle cells, which leads to dilatation of arteries as well as venous sinuses. The drainage of the vascular bed is reduced by the venous muscular bolsters protruding into the lumen of the venous sinuses. Conversely, sympathetic stimulation will induce contraction of all smooth muscle cells leading to contraction of the arteries and, consecutively, to a reduction of blood supply. Simultaneously the muscular bolsters are contracted out of the lumen of venous sinusoids, allowing blood drainage to be increased. Alterations in sympathetic tone thus underlie the nasal cycle. Sensory neural stimulation and reflex neural responses are thus important in relationship to nasal itch, sneezing, rhinorrhea, and nasal blockage (Fig. 68.2) and alterations in threshold responses for stimulation or distribution and balance of neuropeptides may well be relevant to the induction and maintenance of nasal hyperresponsiveness. Many mediators stimulate either the sensory nerves or can act directly on the nasal vasculature, including those linked to the development of allergic rhinitis, such as histamine, leukotrienes, prostaglandins, and kinins (Howarth et al. 2000).

IgE, antigen presentation and rhinitis The basis for the development of allergic rhinitis is the overexpression of IgE and the interaction between IgE and allergen. A significant relationship has been demonstrated in community epidemiologic studies between the level of specific IgE in the serum and the likelihood of expressing clinical rhinitis (Burrows et al. 1989; Droste et al. 1996; Braun-Fahrlander et al. 1997; Arshad et al. 2000, 2002). For example, the SCARPOL community study in schoolchildren identified that sensitization to any allergen was strongly associated with reported seasonal allergic rhinitis [odds ratio (OR) 5.7], nose problems accompanied by itchy-watery eyes (OR 4.4), symptoms occurring only during pollen season (March through September)

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P < 0.001 12

12

10

10 No. IL-4 mRNA+ve cells/mm2

(OR 4.9), and a combination of these latter two (OR 5.8). Other studies have reported that a positive skin-prick test to grass pollen increase the likelihood of hay fever, with ODs of 3.62 and 5.85 being reported in two separate studies involving 3985 and 1218 children respectively (Arshad et al. 2000, 2002). The nature of the rhinitis depends on the sensitization, with specific pollen-related IgE against trees, grasses or weeds giving rise to seasonal-related symptoms and that against the common indoor allergens, such as those related to house-dust mites, pets or cockroaches, often leading to perennial disease. IgE is generated by B lymphocytes under the regulation of cytokines generated by T lymphocytes (de-Vries et al. 1992). This is determined by key cytokines, such as interleukin (IL)-4 (Pene et al. 1988) and IL-13 (Punnonen et al. 1993), in addition to copromoting cytokines such as IL-6 and specific receptor–ligand interactions during cell-to-cell contact between the two populations of lymphocytes. These signaling interactions involve cognate interactions between B-cell major histocompatibility complex (MHC) class II and the Tcell receptor/CD3 complex (Clark & Ledbetter 1994), as well as noncognate interactions involving B cell-expressed CD40 and its complementary ligand (CD40L) expressed on activated T cells (Vercelli et al. 1989; Spriggs et al. 1992). This CD40/ CD40L interaction is pivotal in the induction of B-cell switching leading to IgE synthesis. Upon activation, T cells transiently express CD40L and this induces resting B cells to express CD80, leading to a subsequent interaction between CD80 and its ligand CD28, which enhances cytokine production, and to CD40L expression by T cells. This series of mutual activations of T and B cells, via interactions between CD28/CD80 and CD40/CD40L respectively, is thought to be short-lived, as a result of the transient expression of CD40L on activated T cells, and as a consequence the resulting induction of IgE synthesis is only transient. Rodent studies have also indicated the relevance of cytotoxic T-lymphocyte antigen (CTLA)-4. Not only does engagement of CTLA-4 reduce Th2 cell development but also identify that CTLA-4 is expressed on B cells and that its activation on B cells can also reduce IgE synthesis by these cells. Both mast cells and basophils can also generate IL-4 and IL-13 (Bradding et al. 1993; MacGlashan et al. 1994; Mueller et al. 1994; Bradding et al. 1995a; Pawankar et al. 1997; Wilson et al. 2000) and nasal mast cells from subjects with perennial allergic rhinitis have been shown to express CD40L and have been found, in the presence of IL-4, to be able to induce IgE synthesis by B cells (Pawankar et al. 1997; Pawankar & Ra 1998). This is of particular interest in relationship to allergic rhinitis, as there is now evidence for B-cell IgE synthesis locally within the nasal mucosa. In situ hybridization studies have identified increased mRNA expression for the IgE heavy chain (Cε) and IgE heavy chain promoter (Iε) that can be localized to B cells within nasal biopsies taken 24 hours after nasal allergen challenge (Durham et al. 1997). In the same

No. Ce RNA+ve cells/mm2

PART 9

P < 0.001

8

6

4

8

6

4

2

2

0

0 Placebo

(a)

BS

Placebo PS

(b)

BS

PS

mm2)

Fig. 68.3 Changes in the number of cells (per expressing mRNA for (a) IgE heavy chain (Ce) and (b) interleukin (IL)-4 in nasal biopsies from subjects with grass pollen-sensitive seasonal allergic rhinitis when biopsied in season before nasal allergen challenge and 24 hours after nasal allergen challenge while receiving placebo therapy. The level of significance indicates the difference between before stimulation (BS) and post stimulation (PS). (Adapted from Cameron et al. 1998, with permission.)

study the challenge was associated with increased mRNA expression for IL-4, which was localized predominantly to tissue T lymphocytes and to mast cells (Fig. 68.3). Both of these cell populations could thus be linked to this local IgE generation. The induced increase in local mRNA for IL-4 and heavy chain for IgE following allergen challenge has been shown to be inhibited by pretreatment with intranasal glucocorticosteroids (Durham et al. 1997). Such treatment has also been shown to prevent IgE class switching locally within the nasal mucosa in subjects with naturally occurring seasonal allergic rhinitis (Cameron et al. 1998). The local generation of IgE provides a ready mechanism for IgE availability to bind to tissue cells and provides an explanation for the occasional individual with a classical history of seasonal rhinitis but in whom skin-prick tests are negative. Most atopic subjects have a systemic manifestation of their sensitization and B-cell IgE synthesis is primarily considered to take place in draining lymph nodes, with the antigen having been presented to T cells in a modified format by antigen-presenting cells within the nasal mucosa. At this site, it is the Langerhans or dendritic cell (DC) that appears the most capable antigen-presenting cell (Lambrecht 2001) and it is the signals delivered by these cells to T lymphocytes during antigen presentation that directs the development of Th1 tolerance or Th2 hypersensitivity (McWilliam

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et al. 1995). These DCs have been shown to express the highaffinity IgE receptors, at levels approximately one-hundredth that of mast cells and basophils (Has et al. 1997). This facilitates allergen uptake and processing prior to presentation to T cells. Immunohistochemical staining of nasal mucosal biopsies has confirmed the presence of these cells and has demonstrated that they increase in number in naturally occurring seasonal allergic rhinitis (Fokkens et al. 1989), in house-dust mite sensitive perennial rhinitis (Hartmann et al. 2006), as well as following repeated laboratory allergen challenge out of season (KleinJan et al. 2006). Under these circumstances there is a preferential relocation into the epithelial compartment. The epithelium has been shown to express the DC chemoattractant stromal cell-derived factor (SDF)-1 that acts through the chemokine receptor CXCR4 (Godthelp et al. 1996). This chemokine receptor has enhanced expression in allergic rhinitis and has been shown to be upregulated by bradykinin and IL-1β (Eddleston et al. 2002). The binding of SDF-1 (CXCL12) to the CXCR4 receptor initiates intracellular activation pathways relevant to epithelial cell activation. The localization of DCs to this superficial airway location is important for environmental sampling and access. It has been demonstrated within the nose in allergic rhinitis that HLA-DR- and CD11c-positive DCs can express claudin-1 and penetrate beyond the occludin-positive tight junctions in the epithelium (Takano et al. 2005). This enables these antigen-presenting cells to sample the airway lumen without jeopardizing epithelial barrier function. Interestingly such luminal sampling by DCs was not evident within the normal nose. The cell-surface expression of CD11c is evident on DCs of the myeloid lineage. In contrast to these findings

Pathophysiology of Allergic Rhinitis

with myeloid DCs, an alternative population of DCs, the plasmacytoid dendritic cells, has been reported to be reduced in number within the epithelium in allergic rhinitis, in comparison to those numbers identified at this site within the healthy nose (KleinJan et al. 2006). The subsequent presentation of antigen to T cells and CD4 lymphocyte activation requires the interaction of a specific T-cell receptor with peptide–MHC class II complexes on an antigen-presenting cell and requires the ligation of costimulatory receptors of the CD28 family on T cells by B7 family members of costimulatory molecules (CD80 and CD86) on antigen-presenting cells (Eisenbarth et al. 2003; Itano & Jenkins 2003). A higher percentage of CD11c+ cells in the nasal mucosa lamina propria have been shown to express the critical T-cell costimulatory molecule CD86 in allergic rhinitis than within the normal nose, and have been found in disease to exist in close approximation with mucosal and epithelial T lymphocytes (KleinJan et al. 2006).

Cellular basis of allergic rhinitis Nasal biopsy studies reveal activation of a range of cell populations, involving both infiltrating cells and resident tissue cells (Howarth et al. 2000). The initial response to the allergen involves activation of immune cells. In this respect, DCs, which present antigen to the T cell, the T cell itself, and the mast cell are all important orchestrators of the subsequent cellular response, through their ability to be activated by allergen and to release cytokines that modify the activity of other cell populations (Fig. 68.4). Within the tissue, the endothelial cells become activated. The endothelial activation is associated with upregulation of leukocyte–endothelial

Symptoms Allergens

Mediators

End organ receptors

Mast cells Mediators

Dendritic cells

T-lymphocytes

Cytokines (Th2 profile)

Endothelial cell activation

B-lymphocytes Epithelial cell activation

Eosinophil and basophil tissue recruitment and activation

IgE production Fig. 68.4 Schematic representation of the cellular events in allergic rhinitis. (See CD-ROM for color version.)

Stimulation of bone marrow progenitors

Circulating progenitors

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Mediators and cytokines

Inflammatory cell recruitment

Inflammatory cell recruitment

Stimulate progenitor cells and enhance maturation

adhesion molecules, which are involved in the recruitment of circulating cells, such as eosinophils and basophils, from the circulation. Once within the tissue, these cells, along with mast cells and T cells, accumulate due to inhibition of their natural apoptosis, and are subject to directed accumulation superficially within the nasal mucosa, in response to epithelial derived signals. The epithelium can generate a range of products, including a range of chemokines, which are involved in the attraction and activation of cells to this site. This represents an important process in allergic mucosal inflammation. In addition to these local events within the nose there is also a systemic component to the allergic response, with stimulation of the synthesis and maturation of bone marrow precursors for eosinophils, basophils, and mast cells. These cells, in an altered state of activation within the circulation, are primed for tissue recruitment to sites of ongoing allergic inflammation. Consistent with this concept is the experimental finding that nasal allergen challenge can increase inflammatory cell recruitment within the lower airways and that lower airway allergen challenge enhances upper airway cell recruitment in allergic rhinitis (Braunstahl et al. 2001a,b). There is thus an important systemic component to allergic disease and this may be one factor contributing to the greater impact of disease on quality of life than can purely be explained by the presence of nasal symptoms (Fig. 68.5). Knowledge of the sequence of events and the cellular processes in allergic rhinitis has come both from laboratory allergen challenge studies and from studies in naturally occurring disease. It appears that the initial response to allergen represents mast cell activation due to cross-linkage of IgE, which is bound to the cell surface of mast cells, a process that leads to mast cell degranulation.

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Fig. 68.5 Schematic representation of the systemic signaling evident in upper and lower airway inflammation that influences the generation of progenitor cell production and maturation within the bone marrow and increases the availability of circulating progenitors and mature leukocytes that are then available to be recruited at sites of allergic tissue inflammation. Through such systemic signaling nasal inflammation may worsen lower airway inflammation and vice versa. (See CD-ROM for color version.)

Mast cells Mast cells are derived from CD34+ hematopoietic progenitor cells (Kirshenbaum et al. 1991), which migrate to and mature in the peripheral tissues (Li & Krilis 1999). Interactions between the tyrosine kinase receptor, c-kit, expressed on the surface of mast cells and mast cell precursors, and the c-kit ligand, stem cell factor (SCF), are essential for normal mast cell development and survival (Gali et al. 1994). SCF is expressed on the plasma membrane of a variety of structural cells such as fibroblasts and vascular endothelial cells, with the extracellular domain of SCF being released from these cells by proteolytic cleavage (Costa et al. 1996). The cytoplasm of mast cells contains granules packaged with preformed mediators, such as the amine histamine, the proteases tryptase and chymase, cytokines, such as IL-4, IL-5, IL-8, IL-13, granulocyte–macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α, and the proteoglycan heparin (Krishnaswamy et al. 2001) (Fig. 68.6). Human mast cells have been categorized into two phenotypically distinct subpopulations, based on the type of neutral proteases they express, namely MCT that contain only tryptase, and MCTC that contain chymase, cathepsin G and carboxypeptidase in addition to tryptase (Irani et al. 1986). The cytokine profile of these subsets differ in that IL-4, while present in both subsets, is predominantly colocalized to the MCTC subset, whereas IL-5 and IL-6 are almost completely restricted to the MCT subset (Bradding et al. 1995b). When activated by an IgE-dependent or independent mechanism, mast cells release these granule products into the extracellular environment and also generate arachidonic acid products, such as prostaglandins and leukotrienes, from the phospholipid cell membrane (Fig. 68.7). The major

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Mediators: Histamine Tryptase Prostaglandin D2 Leukotriene C4, D4 Heparin

Cytokines: IL-4 IL-5 IL-6 IL-8 IL-13 GM-CSF TNF-a

Fig. 68.6 Transmission electron micrograph of a mast cell in a nasal biopsy from an individual with active allergic rhinitis illustrating the ongoing mast cell degranulation, with vacuolation of the granules. Some intact granules still exist and the boxed text highlights the mediators that are either stored preformed within the mast cell granule or which are generated de novo on cell activation from arachidonic acid within the phospholipid membrane. The cytokines associated with the mast cell are also highlighted and these are also stored preformed within the mast cell granules ready for rapid release. See text for definition of abbreviations. (See CD-ROM for color version.)

Nuclear phospholipid membrane Arachidonic acid Cyclooxygenase 5-Lipoxygenase

Phospholipase A2 5(S)-HETE 5(S)-HPETE

FLAP

5-Lipoxygenase LTA4 hydrolase LTB4

LTA4

LTC4 synthase

g-glutamyl transpeptidase

PGG2 PGE2 PGF2a

LTC4

PGI2

LTD4

6-keto-PGF1a

Olipeptidases

PGH2

TXA2

PGD2

TXB2

9a, 11b-PGF2a

LTE4 Fig. 68.7 Schematic representation of the arachidonic acid cascade with details of the enzymes involved and the products associated with the 5-lipoxygenase and cyclooxygenase pathways. LT, leukotriene; PG, prostaglandin; TX, thromboxane; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; FLAP, 5-lipoxygenase-activating protein. (See CD-ROM for color version.)

prostanoid generated by mast cells is PGD2. Sequential nasal lavage following nasal allergen challenge in allergic rhinitis identifies the local release of histamine, tryptase, PGD2, LTB4, and LTC4 in association with development of nasal puritus, sneezing, rhinorrhea, and nasal blockage (Naclerio et al. 1983; Creticos et al. 1984; Miadonna et al. 1987; Castells & Schwartz 1988; Lebel et al. 1988). The increase in these medi-

Pathophysiology of Allergic Rhinitis

ators is rapid, peaking within 10–15 min of allergen exposure. Although histamine, LTB4 and LTC4 are not specific markers for mast cell activation, the parallel identification of elevations in tryptase and PGD2 is indicative of a mast cell source and is consistent with mast cell degranulation. In addition to these mediators, elevations in lavage levels of the kinins kallidin (lysyl-bradykinin) and bradykinin have been reported (Proud et al. 1983; Baumgarten et al. 1985). Kallidin and bradykinin are potent vasoactive peptides, formed as cleavage products by the action of kallikrein on low and high molecular weight kininogens respectively. The mast cell protease tryptase possesses weak kallikrein-like activity and could thus contribute to the kinin generation identified under such circumstances, although kinins are generated extracellularly with inflammation, in association with endothelial activation and plasma protein extravasation, so there is no specific requirement to link specific mast cell proteases with local kinin generation to explain their appearance in association with mast cell degranulation (see Chapter 11). Evidence exists for mast cell activation and changes in mast cell populations within the nasal mucosa during naturally occurring allergic rhinitis. Mast cells are constitutive cells of the normal nasal mucosa, but are not normally found superficially within the airway epithelium. Immunohistochemical staining of nasal biopsies with monoclonal antibodies against mast cell tryptase identifies an increase in mast cells within the airway epithelium in both seasonal and perennial allergic rhinitis, in comparison with biopsy findings in nonatopic nonrhinitic subjects (Bentley et al. 1992; Bradding et al. 1993, 1995a; Salib et al. 2004). This has also been reported with electron-microscopic evaluation of nasal biopsies in seasonal allergic rhinitis (Enerback et al. 1986). In addition CD34-positive, tryptase-negative and IgE-negative cells expressing the c-kit receptor have been reported superficially within the nasal mucosa and are likely to represent mast cell progenitor cells awaiting maturation in response to local environmental stimuli (Kawabori et al. 1997). The local airway factors influencing these human cells are poorly defined. The local synthesis of SCF, which is produced mainly by structural cells such as epithelial cells, fibroblasts and endothelial cells, with which mast cells interact closely, will obviously be important. In this respect cell–cell contact would seem to be crucial, even though SCF can be secreted. In mice that secrete soluble SCF but which are homozygous for a mutation in the gene encoding the membrane-bound form of this cytokine, the Steel–Dickie allele (Sld), there is a deficiency of tissue mast cells, which suggests that the SCF expressed on the cell membrane is most important for mast cell development. Consistent with the relevance of SCF to mast cell growth and differentiation, a relationship has been reported between the expression of SCF mRNA within nasal biopsies and mast cell numbers and histamine content within the tissue (Otsuka et al. 1998). There is also evidence that SCF is necessary for optimal IgE/antigen-induced mast cell

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degranulation and cytokine production, although direct evidence for this in relationship to nasal mast cells is deficient. There is no good evidence for the direct involvement of neurally derived factors, such as substance P, in nasal mast cell degranulation and the mast cell activation changes in allergic rhinitis are explainable by IgE-dependent degranulation. The activation of mast cells in naturally occurring disease is evidenced by the identification of elevated levels of the mast cell mediator tryptase and PGD2 in nasal lavage fluid in both seasonal allergic disease (Sugimoto et al. 1994; Lorenzo et al. 1997; Wilson et al. 1998) and perennial allergic disease (Trotter & Orr 1973; De Graaf-In et al. 1996), along with the identification of ultrastructural changes of degranulation on electron microscopic examination of nasal biopsies (Friedman & Kaliner 1985; Knani et al. 1992). In seasonal disease, analysis of prostaglandin levels in nasal lavage fluid by the combination of microcolumn high-performance liquid chromatography and a He/Cd laser-induced fluorescence system found elevated levels of PGD2 (1.33 ± 0.17 nmol/mL) and PGE2 (0.8 ± 0.11 nmol/mL) in allergic rhinitis as compared to their respective levels in healthy controls of 0.23 ± 0.16 nmol/mL and 0.29 ± 0.19 nmol/mL (Sugimoto et al. 1994). There was no difference between the groups in measures of nasal lavage levels of PGF2α or 6-keto PGF1α. In naturally occurring disease, increased leukotriene levels in nasal lavage fluid are also evident (Baumgarten et al. 2002). Mast cell activation may contribute to such changes but these leukotrienes, which are generated from arachidonic acid, may be derived from a variety of cell sources including eosinophils and basophils, as well as mast cells (Creticos et al. 1984). While allergen-induced mast cell degranulation is IgE-dependent, it is appreciated that a range of other stimuli can also lead to mast cell degranulation. In this respect, the report by Bradding et al. (1995c) that β-endorphin, a derivative of proopiomelanocortin, which is released during stress, is able to potentiate the histamine release induced by nasal allergen exposure is of interest. It provides a mechanism as to how stress could influence the severity of allergic disease and potentially under other circumstances contribute to nonallergic disease expression. In addition to the effects of acute mast cell degranulation on immediate symptom generation, mast cell degranulation will contribute to the eosinophilic and basophilic mucosal inflammation evident in rhinitis. Mast cells within the nose have been demonstrated to contain preformed cytokines, particularly IL-4, IL-5, IL-6, IL-13 and TNF-α (Bradding et al. 1993, 1995a,b; Iademarco et al. 1995). Both IL-5 and TNF-α have actions relevant to eosinophil activation and recruitment, while IL-4, IL-5 and IL-13 will potentiate the effects of TNF-α on the expression of vascular cell adhesion molecule (VCAM)-1 on the vascular endothelium (Montefort et al. 1993; Saito et al. 1994; Bochner et al. 1995). VCAM-1 is a leukocyte endothelial adhesion molecule relevant to the tissue recruitment of eosinophils, basophils and lymphocytes through its interaction with the ligand very late antigen

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3H4

AA1

Fig. 68.8 Immunohistochemical staining of adjacent 1-mm nasal biopsy sections with a monoclonal antibody against IL-4 (3H4) and a monoclonal antibody against tryptase (AA1) to define mast cells. The arrows highlight mast cells that express the 3H4 antibody on their cell surface consistent with IL-4 secretion from their granules and pericellular presentation. (See CD-ROM for color version.)

(VLA)-4. The immunoreactivity for IL-4 within nasal mucosal biopsies exists in two patterns when stained with different antibodies that recognize separate epitopes. The antibody 49D stains cytoplasmic IL-4 and this pattern is evident in both rhinitic and nonrhinitic biopsies, whereas the antibody 3H4 gives a peripheral ring staining to the cells and is considered to recognize a secretory form of IL-4 (Fig. 68.8). The IL4 remaining cell associated due to the limited diffusion of exocytosed mast cell proteoglycan heparin. The 3H4 pattern of immunoreactivity is increased in seasonal and perennial rhinitis in comparison to the findings in the normal nose (Bradding et al. 1993, 1995a), indicative of ongoing cytokine secretion by mast cells in active disease. The release of cytokines from mast cells and from T lymphocytes will influence the recruitment of these leukocytes through their activation of endothelial and epithelial cells in addition to any systemic activity on the bone marrow precursors.

T lymphocytes The T lymphocyte represents a significant nonstructural infiltrating cell within the nasal mucosa and epithelial accumulation in CD4+ T cells has been described in rhinitis in both seasonal disease (Calderon et al. 1994; Karlsson et al. 1994; Pawankar et al. 1995a) and perennial disease (Pawankar et al. 1995a,b). In perennial rhinitis, there is also an increase in CD4+ T memory cells, CD4+ T cells and B cells in the nasal mucosa (Pawankar et al. 1995b). This is associated with an increased number of IL-4, IL-5 and IL-13 mRNA-positive cells, suggestive of a Th2 pattern of activation (Varga et al. 1999; Wright et al. 1999). The overexpression of Th2 cytokines is associated with downregulated gene expression and production of the Th1-related cytokine IL-12 in allergic rhinitis

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(Varney et al. 1992; Riccio et al. 2002). The dysregulation of the Th1/Th2 balance in allergic disease is now considered to reflect changes in the regulatory T-cell population that is characterized by CD25 cell-surface receptor expression and which expresses the transcription factor Foxp3. Consistent with this, a recent report has suggested that there are less Foxp3-positive lymphocytes and decreased Foxp3 mRNA in nasal tissue in allergic rhinitis as compared to the findings in the healthy nose (Xu et al. 2007). With nasal allergen challenge, there is an enhanced Th2 cytokine profile within the tissue at 24 hours, with increased mRNA levels in the nasal mucosa of IL-3, IL-4, IL-5 and GM-CSF (Durham et al. 1992). The T cell is the major source of these cytokines and the relevance of this cell to tissue inflammation is suggested by the correlation between the increase in CD4+ T cells during the late-phase allergic reaction and the number of infiltrating eosinophils in the mucosa (Durham et al. 1992). Ex vivo studies have identified that it is T cells expressing the γδ T-cell receptor, which specifically increase within the epithelial compartment in perennial allergic rhinitis, that are the subpopulation of T cells that generate the Th2 cytokines in allergic as opposed to infective rhinitis (Pawankar et al. 1995a, 1996). This accumulation of Th2-specific T cells may be explained by the epithelial generation of CCL-17, previously known as thymus and activationregulated chemokine (TARC). This chemokine is known to facilitate the recruitment, activation and development of Th2 polarized cells and has been shown to have enhanced epithelial cell expression in allergic rhinitis (Terada et al. 2001a). Studies on epithelial cells in culture have identified that both IL-4 and IL-13, when applied with TNF-α, exert a synergistic induction of CCL-17 expression. Stimulation had a greater effect on CCL-17 in epithelial cells cultured from subjects with allergic rhinitis than those from healthy control subjects (Terada et al. 2001a). These studies suggest an important interaction between epithelial cells and both mast-cell and T-cell derived cytokines and the localized epithelial recruitment of Th2-specific T cells. There is also a report of an increase in CXCR1+CD4+ T cells in allergic rhinitis suggestive of the relevance of this chemokine receptor to their tissue recruitment and retention (Francis et al. 2004).

Tissue and luminal cell recruitment: involvement of structural cells Cytokines generated by mast cells and T cells can initiate the process of leukocyte tissue cell recruitment. This involves endothelial cell activation and the adherence, activation and transendothelial migration of leukocytes, in particular eosinophils but also basophils and T lymphocytes, and then their subsequent migration under a chemotactic stimulus toward the epithelium. The activation of the epithelium as part of the allergic inflammatory response and the generation of chemokines from these cells can be implicated in this directed migration.

Pathophysiology of Allergic Rhinitis

Endothelial activation is an early event in the recruitment of circulating cells (Montefort et al. 1993). The initial aspect of this process is the histamine-related upregulation of P-selectin (Asako et al. 1994; Kubes & Kanwar 1994) on the endothelial surface which, along with the cytokine-upregulated E-selectin, induces rolling margination (Fig. 68.9). This brings leukocytes into contact with chemokines, such as CCL-5 [RANTES (regulated upon activation, normal T-cell expressed and secreted)], CCL-3 [macrophage inflammatory protein (MIP)-1α] and CXCL-8 (IL-8), presented on the endothelial glycocalyx and activates these cells (Terada et al. 1996a). Activation results in upregulated expression or functional conformational changes in cell-surface expressed ligands, such as the β2 integrins. These integrins recognize specific ligands that allow firm adherence to the endothelial surface. In this respect, the expression of the β2 integrin, VLA-4, by eosinophils, basophils and T lymphocytes, but not neutrophils, confers some specificity in the recruitment, as Th2 cytokines such as IL-4, IL-5, IL-13 and TNF-α upregulate the leukocyte endothelial cell adhesion molecule VCAM-1 (Montefort et al. 1993; Bochner et al. 1995; Iademarco et al. 1995), which provides firm adherence through its interaction with VLA-4. Endothelial VCAM-1 is overexpressed in seasonal and perennial allergic rhinitis (Montefort et al. 1992; Karlsson & Hellqvist 1996) and its presence can be related to the number of infiltrating eosinophils and T cells (Montefort et al. 1992; Jahnsen et al. 1995; Terada et al. 1996b). Nasal allergen challenge has also been shown to increase endothelial VCAM-1 expression at 24 hours post challenge (Lee et al. 1994). The epithelium forms an interface between the internal and external environment and these lining cells are activated in rhinitis. There is increased expression of the epithelially expressed intercellular adhesion molecule (ICAM)-1 in naturally occurring seasonal and perennial allergic rhinitis in both nasal smear and nasal biopsy samples (Devalia et al. 1992; Ciprandi et al. 1994, 1995). Cultured human airways epithelial cells have been shown to synthesize GM-CSF, IL-6, IL-8 and RANTES (Cromwell et al. 1992; Becker et al. 1993; Kenney et al. 1994) and the presence of enhanced IL-6, TNF-α, IL-8, GM-CSF and RANTES immunoreactivity has been demonstrated within the airway epithelium in nasal biopsies taken from individuals with allergic rhinitis (Nonaka et al. 1996; Wang et al. 1996; Saito et al. 1997, 2000). This activation is also indirectly reflected by the identification of elevated levels of nitric oxide (NO) in nasal luminal air in seasonal and perennial allergic rhinitis (Martin et al. 1996; Baraldi et al. 1998; Gratziou et al. 2001). NO is generated by nitric oxide synthase (NOS) from the semi-essential amino acid L-arginine and there is evidence of increased epithelial expression of the inducible form of NOS in allergic airways disease (Kawamoto et al. 1999). At present the role of NO within the nasal airways is incompletely defined (al-Ali & Howarth 1998) and its specificity for nasal events in doubt as although it is appreciated that measurement will partly reflect nasal mucosal

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Endothelial cells Histamine IL-6, IL-8 eotaxin

FLOW

A-4 VL

Selectins (P + E)

Chemokine exposure

Rolling margination Histamine LTC4

LFA-1

ICAM-1 + VCAM-1

Ligand expression Histamine

Firm adherence IL-4, IL-5, IL-13 TNF-a

Diapedesis

Fig. 68.9 Schematic representation of the processes involved in the tissue recruitment of circulating leukocytes as part of the allergic reaction. Mast-cell release of histamine and leukotrienes will initially upregulate cell-surface expression of P-selectin from endothelial storage sites. This plus the induced expression of E-selectin promotes rolling margination of leukocytes that will then be exposed to chemokines expressed on the endothelial surface whose expression has coincidentally been upregulated by the ongoing mast cell degranulation. These chemokine exposures upregulate cell-surface ligands that enable the cells to firmly adhere to other leukocyte endothelial cell adhesion molecules. The cell-surface ligand lymphocyte function associated antigen (LFA)-1 recognizes and

binds to intracellular adhesion molecule (ICAM)-1 while very late antigen (VLA)-4 binds to vascular cell adhesion molecule (VCAM)-1. The Th2 cytokines IL-4, IL-5 and IL-13 along with tumor necrosis factor (TNF)-a favor the expression of VCAM-1 and as eosinophils, basophils and lymphocytes express VLA-4 but neutrophils do not the local release of Th2 cytokines leads to selective cell recruitment. Continuing mediator and cytokine release in association with ongoing allergic inflammation will maintain this activation. Under these circumstances the events initiated by mast cell degranulation will continue to be supported by that process but will also be supplemented by activation and mediator and cytokine release from other cell populations. (See CD-ROM for color version.)

inflammation, it is also appreciated that NO derived from other sources, such as the paranasal sinuses, has a significant impact on nasal measures (Lundberg et al. 1995). This is suggested by the report identifying that nasal NO measures increase once osteomeatal obstruction improves and that humming, which improves sinus ventilation, increases nasal measures of NO (Colantonio et al. 2002; Weitzberg & Lundberg 2002). Both these findings suggest that the reservoir of NO within the nasal sinuses is a major contributor to nasal NO measures. However, the nasal mucosa itself can also contribute and this contribution is likely to be increased in disease, as the intranasal administration of eotaxin (CCL-11) has been shown to increase both nasal eosinophil influx and nasal NO (Hanazawa et al. 2000). Under nonchallenge situations the natural production of chemokines by the nasal airway epithelium can account for the inflammatory cell recruitment to this site and within the nasal airway lumen (Fig. 68.10). Nasal epithelial cells express RANTES and eotaxin, two chemokines relevant to airway eosinophil recruitment. Epithelial cells, cultured from allergic rhinitic subjects, have an enhanced ability in vitro to release RANTES on stimulation, in comparison to epithelial cells from healthy controls (Calderon et al. 1997) and nasal allergen challenge has been shown to stimulate RANTES mRNA

expression (KleinJan et al. 1999). Nasal challenge with RANTES induces the tissue accumulation of basophils, eosinophils, and T cells (Kuna et al. 1998) consistent with its known properties in vitro. Eotaxin is released into the lumen following nasal allergen challenge (Minshall et al. 1997; Pullerits et al. 2000; Greiff et al. 2001; Terada et al. 2001b), while eotaxin mRNA and protein expression is upregulated in the nasal tissue of patients with allergic rhinosinusitis (Minshall et al. 1997; Pullerits et al. 2000) and this is increased further following intranasal allergen challenge (Minshall et al. 1997). Nasal challenge with eotaxin has been found to induce nasal eosinophilic inflammation (Hanazawa et al. 2000; Górski et al. 2002). Two studies have reported an increase in eotaxin in nasal lavage fluid after allergen challenge (Greiff et al. 2001; Terada et al. 2001b). In one study eotaxin was found to increase in both the early- and late-phase responses to challenge (Terada et al. 2001b). These changes paralleled lavage increases in both the eosinophil activation marker, eosinophil peroxidase (EPX), and in eosinophils. There was a significant correlation between nasal lavage eotaxin levels and the number of eosinophils (r = 0.73) and the level of EPX (r = 0.67). In the other study, in which daily nasal allergen challenge was undertaken for 6 days, a significant increases in nasal lavage levels of eotaxin, the eosinophil granule protein eosinophil

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Pathophysiology of Allergic Rhinitis

Epithelium Lumen

TGF-b Fig. 68.10 Schematic representation of factors generated by epithelial cells and their relevance to the local accumulation of inflammatory cells at this site, enabling their interface with the environment. These include transforming growth factor (TGF)-b, stromalcell derived factor (SDF)-1, RANTES (regulated upon activation, normal T-cell expressed, and secreted), macrophage inflammatory protein (MIP)-1a, monocyte chemotactic protein (MCP)-1, interleukin (IL)-8 alone or coupled with secretory immunoglobulin A (sIgA) a process that alters it from a neutrophil to an eosinophil chemoattractant, thymus and activation-regulated chemokine (TARC), and eotaxins 1, 2 and 3. (See CD-ROM for color version.)

Chemoattractant for: Mast cells

Eotaxins CCL11, CCL24, CCL26

SDF-1 CXCL12 IgA

Chemoattractant for: Dendritic cells RANTES CCL5 Chemoattractant for: T-cells MIP-1a CCL3 Basophils MCP-1 CCL2 Mast cells Chemoattractants Eosinophils and activators for: Basophils Macrophages

cationic protein (ECP), and eosinophils was also present (Greiff et al. 2001). Baseline analysis in this study identified a significant correlation between nasal lavage eotaxin concentrations and those of ECP and α2-macroglobulin. These relationships were lost, however, after the period of allergen exposure and at the end of the study there was no relationship between nasal lavage eotaxin levels and nasal mucosal tissue eosinophil numbers, suggestive of the involvement of other processes in eosinophil recruitment with repeated allergen exposure. This suggests that eotaxin is relevant to eosinophil recruitment in association with acute immediate nasal responses but that other factors are more relevant to tissue eosinophil recruitment with sustained allergen exposure. The involvement of eotaxin in the acute response is indicated by the influence of pretreatment with an antieotaxin monoclonal antibody, CAT-213, prior to nasal allergen challenge. Intranasal pretreatment with this antibody has been found to reduce the number of eosinophils within the tissue 6 hours post challenge in comparison to placebo pretreatment. No such studies exist for persistent disease but it is understood that, in natural disease, nasal lavage fluid concentrations of eotaxin-1 are significantly higher in both perennial and seasonal (in-season) allergic rhinitis, as compared with nonrhinitic controls, and significantly relate to both the percentage of lavage eosinophils and α2-macroglobulin concentrations (Salib et al. 2005), suggestive of the link between the release of eotaxin in naturally arising disease and airway eosinophil recruitment and the associated exudative processes. The CCR3 receptor represents the major CC chemokine receptor on eosinophils and one that is acted on by eotaxin. A study investigating the effects of a CCR3blocking monoclonal antibody on the eosinophil chemotactic response to RANTES (CCL5), eotaxin (CCL-11), MCP-2 (CCL-8), MCP-3 (CCL-7), and MCP-4 (CCL-13) indicated that greater than 95% of the eosinophil chemotactic activity

Chemoattractant for: Eosinophils Mast cells Basophils T-cells

Secretory component TARC CCL17 IL-8 CXCL8

Chemoattractant for: Neutrophils

Chemoattractant for: Th2 T cells IL-8 sIgA Chemoattractant and activator for: Eosinophils

of all these CC chemokines was mediated through the CCR3 receptor (Heath et al. 1997). There does not appear to be a CCR5 receptor on resting eosinophils and the CCR1 or CCR4 level of expression is about 1–5% that of CCR3 (Daugherty et al. 1996; Heath et al. 1997). A further chemokine, MCP-1 (CCL-2), also from the same subclass, may be generated from macrophages and is present within the nasal mucosa in rhinitis (Fujikura & Otsuka 1998) and elevated levels in nasal lavage fluid are evident during naturally occurring seasonal allergic rhinitis (Fujikura & Otsuka 1998). This chemokine can activate monocytes and basophils and has been implicated in eosinophil, T-cell and monocyte cell influx and accumulation. The persistence of cells within the epithelium, in particular eosinophils and mast cells, is not only a reflection of tissue recruitment and localization but also a reflection of the local generation of inhibitors of apoptosis and of growth factors. Epithelial cells generate GM-CSF (Cromwell et al. 1992), which prolongs eosinophil survival, and SCF (Kuna et al. 1996; Kim et al. 1997), which is an important mast cell mitogen. Cultured nasal epithelial cells have been shown to generate SCF in vitro and elevated levels of SCF have been found to be present in nasal lavage fluid in seasonal allergic rhinitis (Otsuka et al. 1998). The SCF levels correlated with nasal lavage mast cell chemotactic activity. In support of this, a further study reported that SCF mRNA expression correlated with both mast cell numbers and histamine content within the nasal mucosa (Otsuka et al. 1998). Other cytokines within the microenvironment, including T lymphocyte-derived cytokines, may also influence mast cell development and studies on rodent cells have highlighted the relevance of IL-3 and IL-9. Such studies have also indicated the relevance of transforming growth factor beta (TGF)-β (Nilsson et al. 1998). This growth factor is particularly potent in inducing the development of mucosal mast cells from mouse bone marrow-derived mast cells and significantly enhances the expression of mouse

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mast cell proteases (Funaba et al. 2005, 2006). TGF-β can also increase mast cell integrin expression, in particular α7, which promotes mast cell adhesion to laminin-1, an interaction relevant to mast cell–epithelial interactions (Rosbottom et al. 2002). TGF-β is also recognized to be a potent mast cell chemoattractant. This has been demonstrated with both a human mast cell line (HMC-1) and with primary human mast cells cultured from gut tissue (Olsson et al. 2000). These cells express the TGF-β serine/threonine type I and type II receptors and exhibit a characteristic bell-shaped chemotactic response curve to the different TGF-β isoforms, with high dose inhibition of chemotaxis. Of the three different human isotypes, TGF-β3, with an EC50 of 0.01 pmol/L, is marginally more chemotactic than either TGF-β1 or TGF-β2. All three TGF-β isoforms are also expressed within the airway epithelium in allergic rhinitis, with TGF-β2 being the most highly expressed isoform (Salib et al. 2004). There is significantly greater expression at this site in both perennial and seasonal allergic rhinitis than in the normal nose (Salib et al. 2004). Within the rhinitic subjects, the extent of epithelial immunoreactivity for TGF-β positively correlated with the number of intraepithelial mast cells and mast cells were demonstrated to express TGF-β receptors. Although correlations do not prove cause and effect, it would thus seem reasonable to suspect that in allergic rhinitis the local generation of TGF-β by the epithelium contributes to the selective accumulation of mast cells in this location. The role of chemokines in the terminal differentiation and particularly the directed accumulation of tissue mast cells is incompletely understood. Their importance is suggested by the recognition that mast cells express chemokine receptors (Juremalm & Nilsson 2005). Immature mast cell progenitors express the chemokine receptors CXCR2, CCR3, CXCR4 and CCR5, although with maturation only the CCR3 receptor remains. However, this is the receptor for eotaxin-1 (CCL-11), eotaxin-2 (CCL-24), and eotaxin-3 (CCL-26) and recognizes other chemokines such as RANTES (CCL-5), MIP-1α (CCL-3), and monocyte chemotactic proteins-2 (CCL-8), -3 (CCL-7) and -4 (CCL-13). As fibroblasts and the airway epithelium expresses both eotaxin and RANTES, two chemokines of the CC class that are known to be mast cell chemoattractants, it would appear probable that chemokine–CCR3 interactions will contribute to mast cell recruitment in allergic airways disease, even though these chemokines are several log orders less potent than TGF-β as mast cell chemoattractants (Olsson et al. 2000). Studies in vitro have, however, indicated that the interaction between eotaxin and CCR3 on immature mast cells favors the development of the MCTC phenotype, rather than the MCT phenotype whose number increases in allergic inflammation. This might suggest that eotaxin–CCR3 interactions, in conjunction with SCF–c-kit ligand interactions, are more relevant to the normal recruitment and development of tissue mast cells rather than a disease-directed pro-

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cess. However, the situation is more complex in that MCTC subtypes provide a source of IL-4 and IL-13, two cytokines of relevance to allergic airway inflammation, and that eotaxin stimulation of mast cells activated immunologically, by crosslinkage of cell-surface expressed high-affinity Fc receptors for IgE (FcεRI), leads to enhanced IL-13 generation (Price et al. 2003). This interaction is linked and coordinated, as under quiescent conditions CCR3 is found predominantly intracellularly in mast cells, colocalized to secretory granules, and is mobilized to the cell surface with FcεRI cross-linking. This change in cell-surface expression permits interaction with eotaxin to promote amplification of the immune response. Murine studies in knockout animals suggest that this more subtle interaction is more pertinent to the behavior of airway mast cells in allergic airways disease than a CCR3-directed epithelial accumulation. Wild-type mice sensitized intraperitoneally with ovalbumin and subsequently challenged with aerosolized antigen develop eosinophilic airway inflammation, intraepithelial mast cell accumulation, and airway hyperreactivity. In CCR-3 deficient mice the same process is not associated with tissue eosinophilia, linking CCR-3 to tissue eosinophil recruitment (Humbles et al. 2002). Surprisingly, however, these CCR-3 deficient mice exhibit an increase in intraepithelial mast cell accumulation and an enhancement in the induced airway hyperreactivity under such circumstances (Humbles et al. 2002). Thus paradoxically CCR-3 ligand interactions would appear, at least in rodents, to protect against intraepithelial mast cell residency and hence other interactions, such as that described with TGF-β, appear more relevant to the clinical disease process. The epithelial accumulation of mast cells in naturally occurring disease, such as in seasonal allergic rhinitis, is considered to underlie the phenomenon of “priming” under such circumstances, a situation whereby there is greater symptom expression for an equivalent pollen exposure later in the season than at its onset. The recruitment of mast cells superficially toward the airway lumen provides more immunologically primed cells to interact with ambient inhaled allergen. TNF-α is an important cytokine regulating epithelial cell activation and is present in mast cell granules and has been shown to be released into the nasal cavity during the immediate allergic response (Bradding et al. 1995c). TNF-α will also activate endothelial cells and structural mesenchymal cells. Nasal challenge with TNF-α has been shown to increase recovery within nasal lavage of α2-macroglobulin, IL-8 and ECP (Widegren et al. 2007). In addition, it is now apparent that allergens, which are proteolytic enzymes, can directly activate epithelial cells independent of IgE (Asokananthan et al. 2002) and lead to cytokine release (Vignola et al. 1995; Tomee et al. 1998). This process is enhanced by local generation of the proteases, tryptase and chymase, as well as histamine released in association with mast cell activation and by corelease of IL-4 and IL-13 from these cells, as these mediators and Th2 cytokines have been shown to upregulate

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epithelial cell activation (Andersson et al. 1989; Kato et al. 1998). These findings further add to the importance of epithelial cell–mast cell interactions in rhinitis.

Infiltrating leukocytes Nasal biopsies taken from the normal nose or from monoallergic pollen-sensitive subjects outside the pollen season reveal no evidence of eosinophilic airway inflammation and nasal smears reveal very few if any basophils or eosinophils in these circumstances. The appearance of these cells within the nasal mucosal tissue and within nasal luminal samples is associated with clinical disease expression. Activation of these cells leads to mediator release and it is probable that they contribute, along with mast cell-derived mediators, to the severity of symptom expression.

Eosinophils Eosinophils are not a normal cellular constituent of the nose. Immunohistochemical staining of nasal mucosal biopsies shows that eosinophils are evident in nasal biopsies within the submucosa and epithelium in active rhinitis (Bentley et al. 1992; Bradding et al. 1993, 1995a) and their recovery is increased in nasal luminal samples (Pipkorn et al. 1988; Grieff et al. 1998). Transmission electron microscopy examination of nasal biopsies from patients with rhinitis reveals evidence of ultrastructural granular changes of eosinophil activation and there is also evidence of eosinophil lysis (Egesten et al. 1994). The normal eosinophil has granules that on electron microscopy have an electron-dense core surrounded by a less dense matrix (Fig. 68.11). The electron-dense core is due to the presence of crystals of major basic protein (MBP), which comprise approximately 55% of the granular protein content. In addition to MBP, the eosinophil granules contain, within the matrix, ECP, eosinophil-derived neurotoxin (EDN) and EPX, as well as numerous enzymes such as ribonucleases, histaminase and arylsulfatase (Svensson et al. 1990). Eosinophils in nasal biopsies in rhinitis have been demonstrated to also contain IL-5 (Wilson et al. 2000). Eosinophil activation is associated with both the release of granule components and the de novo generation of arachidonic acid products. The major lipoxygenase arachidonic-acid cleavage product is LTC4 and eosinophils, primed by chemotactic factors and cytokines such as IL-3, IL-5 and GM-CSF, have an enhanced state of activation and exhibit exagerated LTC4 generation and release. An elevation in immunoreactive LTC4 is evident in nasal lavage fluid in seasonal allergic rhinitis (Knani et al. 1992). However, this is not a specific activation marker for eosinophils as in addition to an eosinophil source, LTC4 may also be derived following either mast cell or basophil activation. Markers that are more specific for eosinophil activation are the granule proteins ECP and EPX. Both ECP and EPX show increased concentrations in nasal lavage in active rhinitis compared with those in healthy controls (Svensson et al. 1990; Knani et al. 1992; De Graaf-In et al. 1996; Lorenzo et al.

Fig. 68.11 Transmission electron micrograph of an eosinophil in a nasal biopsy from a patient with active allergic rhinitis illustrating the presence of granules that are intact with an electron-dense core as well as many that show reversal of this electron density consistent with activation.

1997; Wilson et al. 1998). There are clear increases in EPX levels in nasal lavage when evaluated in the same subjects with grass pollen sensitization out of season and in-season. The increase in nasal lavage ECP has been reported to correlate with symptom scores (rs = 0.53) in naturally occurring seasonal allergic rhinitis (Knani et al. 1992). Elevated nasal lavage ECP levels are also present in perennial allergic rhinitis (De Graaf-In et al. 1996; Wilson et al. 1998) as are those for EPX (Knani et al. 1992). Studies of nasal allergen challenge reveal an early increase in eosinophils 30–60 min post challenge, in recovered nasal lavage fluid, and a second increase 6–10 hours post challenge (Terada et al. 2001b) which is sustained up to 24 hours post challenge but not beyond (Miadonna et al. 1998). This change in eosinophils is paralleled by changes in nasal lavage EPX levels (Terada et al. 2001b). The early increase in eosinophils and EPX may be a reflection of the enhanced plasma protein exudation washing resident airway cells and their free granules out into the airway lumen, as an increase in ECP in nasal lavage has also been reported after nasal histamine challenge (Meyer et al. 1999). The latter increase reflects the stimulated process of tissue cell recruitment as part of the allergic inflammatory process. Eosinophils also generate chemokines, such as eotaxin, and the generation of this CC chemokine along with IL-5 may serve an autoregulatory function. Nasal challenge with IL-5 induces nasal eosinophil influx (Terada et al. 1992). As

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discussed in the section on structural cells, the epithelium is an important source of eosinophilic chemokines and the generation of both eotaxin and RANTES by these cells have been implicated in tissue eosinophil recruitment. Eosinophils have also been shown to be the major source of preformed nerve growth factor (NGF) in allergic rhinitis and this cellular source may account for the elevated levels of NGF in naturally occurring disease and contribute to the increase in nasal lavage fluid levels identified after nasal allergen challenge.

Basophils Circulating basophil and eosinophil progenitors are evident in peripheral blood in association with allergic rhinitis (Linden et al. 1999). These cells are derived from CD34+ progenitor cells within the bone marrow. The lack of a good monoclonal antibody specific for basophils has until recently hampered assessment of tissue accumulation of basophils, and their identification has largely depended on ultrastructural evaluation of cells in nasal smear or lavage by transmission electron microscopy. There have been reports of basophils increasing in nasal secretions in active rhinitis (Okuda & Otsuka 1977). The development of basophil-specific antibodies, such as BB1 and 2D7, has allowed more detailed evaluation of the localization of basophils within the nasal mucosa. These cells are sparse in the normal nose and have to been found to increase in the submucosa in naturally occurring seasonal allergic rhinitis (Wilson et al. 2001). With intranasal allergen challenge an increase in basophils has been reported in both the epithelium and lamina propria as early as 1 hour after challenge, with the cellular accumulation within the lamina propria persisting for up to a week after challenge (KleinJan et al. 2000; Braunstahl et al. 2001a). Basophils posses fewer and larger granules than mast cells and differ from the mast cell in that they contain less histamine. On immunologic activation the basophil only releases 20–30% of the histamine released from a comparable number of mast cells. Basophils posses different proportions of proteoglycans, predominantly chondroitin sulfate A and E rather than heparin, contain only 1–3% of the tryptase content of mast cells, do not generate PGD2 and, unlike the mast cell, store a small quantity of MBP and EDN (Howarth 1989). However, the basophil does generate a comparable amount of LTC4 as the mast cell following immunologic activation (60 ng/106 cells) (Howarth 1989). Nasal allergen challenge is associated with an early increase in nasal lavage histamine levels followed, after a return toward baseline levels, by a further late increase in lavage histamine recovery (Naclerio et al. 1985). The early increase is accompanied by an increase in PGD2, consistent with mast cell degranulation (Naclerio et al. 1983; Lebel et al. 1988). This is not the case with the late increase and this difference can be explained by basophil accumulation and activation accounting for the late-phase histamine response. Consistent with this, a late increase in basophil numbers has been identified in nasal lavage fluid

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following intranasal allergen challenge (Bascom et al. 1988). The percentage of basophils in nasal lavage fluid is small, rising from 0.2% of cells at baseline to 0.7% during the late reaction, although this increase is a reflection of an approximate 12-fold increase in total basophil numbers. Significant correlations have been reported between nasal resistance measurements and the numbers of basophils 8 hours after nasal allergen challenge (Pastorello et al. 1994) and between the number of basophils and the cumulative levels of histamine in nasal lavage 3–11 hours post allergen challenge (Bascom et al. 1988). A number of cytokines have been reported to prime basophils in vitro for increased mediator release in response to subsequent stimulation. These stimuli include IL-1, IL-3, IL-5 and GM-CSF (Massey et al. 1989; Bischoff et al. 1990a; Pastorello et al. 1994). In general, these cytokines are unable to induce mediator release directly (Subramanian & Bray 1987; Bischoff et al. 1990b) although this has not been a consistent finding. A number of other cytokines have more recently been identified that may be of greater relevance to basophil activation in rhinitis as they are more potent and not solely restricted in their action to only a proportion of donors. These are “histamine-releasing factors” (HRFs), a terminology used to describe products released from activated mononuclear cells that are capable of inducing basophil histamine release (Baeza et al. 1989). HRFs have been identified in nasal lavage (Thueson et al. 1979). The CC chemokines are now appreciated to account for most of the activity and the most potent is MCP-1, known previously as monocyte chemotactic and activating factor (Kuna et al. 1992). Several other members of the chemokine family are also known to account for a proportion of this activity, including connective tissue activating peptide (CTAP)-III and its derivative neutrophil activating peptide (NAP)-2, RANTES, and MIP-1α (Baeza et al. 1990; Kuna et al. 1992; Reddigari et al. 1992; Sim et al. 1992). The relevance of these observations to clinical rhinitis is as yet undetermined but RANTES has been shown to be increased within the airways epithelium in rhinitis and to be released following nasal challenge. In addition, basophils themselves are now also understood to be capable of synthesising the cytokines IL-4 and IL-8 (Elmedal et al. 1993; Kuna et al. 1993; MacGlashan et al. 1994; Mueller et al. 1994). The relevance of this cell population to cytokine synthesis within the tissue is uncertain as preformed IL-4 predominantly colocalizes to mast cells in nasal biopsies (Wilson et al. 2000). However, there is a report that the level of basophil activation, as evaluated from peripheral blood samples, correlates with disease severity in seasonal rhinitis (Winther et al. 1999).

Mediators, receptors and symptom expression The accumulated cells are in an activated state and it is the mediators released by the cells that lead to symptom generation through stimulating specific receptors on sensory nerves

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and blood vessels within the nasal mucosa. Stimulation of sensory nerves leads to the sensation of itch, sneezing and via reflex stimulation of efferent vagal pathways to glandular secretion and hence anterior rhinorrhea. In addition, the local release of neuropeptides such as substance P and CGRP from nonadrenergic noncholinergic nerves within the nasal mucosa can modify the state of nasal vascular engorgement, either through a direct action on the vessels, promoting vasodilatation, or indirectly by modifying sympathetic ganglionic neurotransmission (see Fig. 68.2). The nasal vasculature is under sympathetic tone; the sympathetic neurotransmitter norepinephrine promotes vasoconstriction and maintains nasal airway patency. The ganglionic reduction in sympathetic tone by neuropeptides will thus lead indirectly to nasal congestion. In addition to these neural effects, mediators generated from mast cells, basophils and eosinophils, such as histamine, tryptase, kinins, prostaglandins and leukotrienes, will have direct effects on the nasal vasculature, through their interaction with specific receptors and induce nasal blockage and plasma protein extravasation. Plasma protein exudation is an importance hallmark of the airway inflammation in allergic rhinitis (Persson et al. 1998; Howarth et al. 2005). Nasal challenge studies have been undertaken with these mediators to explore their potential effects. Nasal insufflation with histamine produces a full range of nasal symptoms, including nasal blockage. Unilateral nasal challenge induces bilateral rhinorrhea, indicative of the contribution of neural reflexes in this response. This efferent response is cholinergic, in that it can be blocked in the unchallenged nostril by the topical administration of the antimuscarinic agent ipratropium bromide. The nasal itch, sneeze and rhinorrhea are completely abolished by H1-receptor antagonists, indicative of the involvement of H1-receptor stimulation in initiation of the response (Mygind et al. 1993). However, the nasal blockage is only partially modified by H1-receptor antagonism. H2-receptor antagonists can also modify the obstructive response but there is no additional effect to that of H1antihistamines when they are coadministered, indicating that their protective effects are sequential in the same process (Wood-Baker et al. 1996). Thus histamine-induced nasal blockage is incompletely modified by H1 or H2 receptor blockade. It was thus hypothesized that additional histamine receptors, potentially the H3-receptors (Ishikawa & Sperelakis 1987), may also contribute to nasal obstruction by modulating sympathetic tone. Animal studies suggest that there may be validity to this hypothesis, as H3-receptor antagonists have been found to regulate sympathetic neurotransmitter release and vascular tone in porcine nasal mucosal samples (Varty & Hey 2002). Histamine also acts through H4-receptors and studies on neural plexi in the gut suggest that such actions could have implications for the neural effects of histamine within the nose (Breunig et al. 2007). This is strongly supported by animal models in which histamine and a selective

Pathophysiology of Allergic Rhinitis

Nerves

Vasculature • Vasodilation • Plasma protein extravasation • P-selectin expression • E-selectin expression • IL-6, IL-8 generation

Sensory neural stimulation Smooth muscle Contraction Histamine

• ICAM-1 expression • IL-6, IL-8, GM-CSF, TNF-a generation • H1-receptor expression

Dendritic cells • IL-1b, IL-6, IL-18 MCP-1b, RANTES production • Dendritic cell maturation

Epithelial cells

PBMC • IL-1, IL-6, IL-18 IFN-g production

Monocytes/macrophages IL-6, IL-10 production

Fig. 68.12 The wide range of biological activities of histamine and the relevant cells on which it exerts an influence. ICAM-1, intracellular adhesion molecule 1; IL, interleukin; GM-CSF, granulocyte–macrophage colonystimulating factor; H, histamine; IFN-g, interferon g; MCP-1b, monocyte chemotactic protein-1b; PBMC, peripheral blood mononuclear cells; RANTES, regulated upon activation, normal T-cell expressed, and secreted; TNF-a, tumor necrosis factor a. (See CD-ROM for color version.)

histamine H4-receptor agonist caused scratching responses in mice, which were almost completely attenuated in histamine H4-receptor knockout mice or by pretreatment with the selective histamine H4-receptor antagonist JNJ 7777120. Pruritus induced by allergic mechanisms was also potently inhibited with histamine H4-receptor antagonist treatment or in histamine H4-receptor knockout mice (Dunford et al. 2007). These findings help explain the incomplete effect of H1-receptor antagonism in alleviating nasal pruritic responses in allergic rhinitis. Histamine is able to exert a range of cellular effects relevant to allergic disease additional to its actions on the neural and vascular structures (Fig. 68.12), indicative of the importance of this mediator to rhinitis. Nasal blockage can be induced by nasal insufflation with kinins, PGD2, and LTC4 and LTD4 (Howarth 1997). Thus these mediators will also contribute in allergic disease. Nasal challenge with kinins induces a painful nasal sensation, different from histamine-induced itch, as well as glandular secretion and plasma protein extravasation (Rajakulasingam et al. 1991, 1992). These effects are mediated through the kinin B2 receptor, as the B2 agonist kallidin, but not the B1 agonist [des-Arg9]-bradykinin, mimics the increase in nasal airways resistance and plasma protein exudation induced by bradykinin (Rajakulasingam et al. 1991, 1993). However, studies on bradykinin receptor mRNA expression in nasal mucosa in allergic rhinitis have demonstrated disease-related differences in B1-receptor expression, with an increase in allergic rhinitis associated with functional differences (Christiansen et al. 2002). Kinin stimulation of B1 receptors in allergic rhinitics but not healthy controls activates extracellular signal-related kinase, which in epithelial cells in culture upregulates the activity of the transcription factor AP-1. This disease-related difference is not seen with B2-receptor stimulation. Thus

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although the acute nasal effects of kinins are mediated by B2 receptors, stimulation of B1 receptors, which have also been shown to be upregulated by allergen, may contribute to the chronicity or persistence of disease through epithelial cell activation. The effects of PGD2, LTC4 and LTD4 are more limited, with their predominant effect being nasal blockage. The leukotrienes are also potent mediators of vascular permeability and induce plasma protein exudation (Howarth 2000). The effects of PGD2 are mediated through the vascular D prostanoid (DP) receptor, as opposed to the common thromboxane receptor (Harrison et al. 1990; Johnston et al. 1993). The presence of DP and CRTH2 (chemoattractant receptorhomologous molecule expressed on Th2 cells) receptors have been explored within the nasal mucosa, along with evaluation of the expression of hematopoietic-type PGD2 synthase (h-PGDS) and lipocalin-type (l)-PGDS, enzymes involved in the synthesis of PGD2 (Okano et al. 2006). The h-PGDS but not l-PGDS was clearly expressed in nasal mucosa. The expression of h-PGDS in allergic patients was significantly higher than in control patients without mucosal hypertrophy. A variety of infiltrating cells including mast cells, eosinophils, macrophages, and lymphocytes as well as constitutive cells such as epithelial cells and fibroblasts were found to express h-PGDS. Both DP and CRTH2 receptors were present within the nasal mucosa. The level of expression of CRTH2 but not DP was highly and significantly correlated with the number of eosinophils infiltrating into nasal musosa, consistent with the known involvement of this receptor in tissue eosinophilia. With respect to leukotrienes, the CysLT1 receptor is involved in their vascular effects within the nose. Studies using in situ hybridization to localize gene expression for the CysLT1 receptor in human nasal mucosa have revealed that receptors can be localized to endothelial cells (Shirasaki et al. 2002). There is also receptor localization to eosinophils, mast cells, macrophages, and neutrophils but little related to epithelial cells or glands. There is also gene expression for the CysLT2 receptor within nasal tissue but to date this has not been colocalized. Intervention with a CysLT1-receptor antagonist in seasonal allergic rhinitis is reported to improve a wider profile of symptoms other than just nasal blockage (Nayak et al. 2002; Philip et al. 2002). Thus although the nasal challenge studies identify an effect confined to the nasal vasculature, it would appear likely that leukotrienes either have interactions with other mediators in vivo, that expand their profile of influence, or that their known effect on the marrow precursor generation reduces nasal inflammatory cell recruitment and thus indirectly reduces the expression of all nasal symptoms. It seems probable that leukotrienes modify the neural responses to histamine and in other systems it is appreciated that bradykinin responses can also be modified by leukotrienes. The cointeractions between mediators and the relevance of interactive inflammatory processes in the induction of nasal hyperresponsiveness remains to be better

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explored. The current understanding of nasal reactivity and the relevance of neural interactions not solely confined to the nose has recently been reviewed (Sarin et al. 2006).

Systemic cell signaling The involvement of the bone marrow in allergic responses is crucial as without stimulation of the generation and maturation of progenitor cells there would not be the relevant circulating cells to be available for recruitment to tissue sites of allergic inflammation. As eosinophils represent a hallmark cell involved in allergic inflammation, most attention has focused on circulating eosinophils and markers of their presence. In seasonal allergic rhinitis there is evidence of bone-marrow stimulation as reflected by circulatory changes in eosinophils, with evidence for an increase in eosinophil and basophil progenitors in the circulation (Denburg et al. 1985; Otsuka et al. 1986), an increase in the number of circulating eosinophils (Bhandari & Baldwa 1976; Urmil et al. 1984), and changes in eosinophil function and behavior consistent with activation (Shult et al. 1986; Frick et al. 1988; Kohi et al. 1990). The change in eosinophil number with seasonal allergen exposure is variable and although several studies have revealed higher mean peripheral blood eosinophil counts, in comparisons between seasonal allergic rhinitic populations and healthy control populations, the range of values is large with considerable population overlap. Thus for an individual the measurement of a peripheral blood eosinophil count is of little diagnostic value and does not clearly relate to the clinical severity of the disease (Shult et al. 1986). Measurement has also been made of ECP levels in serum as an ex vivo measurement of eosinophilic activation. There are reports of seasonal increases in serum ECP consistent with priming/activation of peripheral blood eosinophils (D’Amato et al. 1996; Klimek et al. 1996). It has also been reported that there is an increase in hypodense eosinophils within the circulation in seasonal allergic rhinitis (Shaw et al. 1985). Normal peripheral blood eosinophils are dense cells and can be separated by density gradient centrifugation. The change in density of eosinophils to a hypodense phenotype has been taken to be an indicator of cell activation, as hypodense eosinophils have been shown to release more proinflammatory products such as LTC4 (Khalife et al. 1986) and platelet-activating factor (Lee et al. 1984), to release more tissue toxic products such as EPX (Wingqvist et al. 1982), to have increased oxidative metabolism (Kohi et al. 1990), and to possess more IgG and complement receptors (Prin et al. 1983), as well as exhibiting more cytotoxic activity for antibody-coated targets (Prin et al. 1983). On the basis of this, Fukuda and Gleich (1989) concluded in their editorial on the heterogeneity of eosinophils that the hypodense state is itself a product of eosinophil activation. There has been some debate as to whether this change is an ex vivo occurrence, as a product of the separation process, or whether it is an in vivo

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event (Kaufmann et al. 1991). Nevertheless it does reveal an alteration in eosinophil behavior in the disease, in eosinophils recovered from the peripheral circulation. In one study in seasonal allergic disease there was an increase in the percentage of hypodense eosinophils in the peripheral blood to 30%, as opposed to 10% for controls, with the percentage of hypodense eosinophils returning to normal when their disease was quiescent outside the pollen season (Frick et al. 1988). Not all studies have confirmed a seasonal change in peripheral blood eosinophil activation and this may relate to the level of seasonal allergen exposure. One report assessing circulatory changes in relationship to seasonal pollen exposure identified an increase in peripheral blood eosinophils, an increase in intracellular ECP as well as an increase in the cell-surface activation markers CD9 and CD11b during a high pollen season in patients with allergic rhinitis (Fernvik et al. 1996). These changes were not evident in a low pollen season. This influence of allergen exposure level may thus account for some of the variability within the literature in relationship to peripheral blood eosinophil counts. There are close parallels between these findings and nasal findings. Not all studies have revealed an increase in tissue or lavage eosinophils in seasonal allergic rhinitis (Lim et al. 1995; Nielsen et al. 1996). While this may relate to the site of biopsy or the use of tinctorial stains, which may be less robust than specific immunohistochemical analysis, it may also relate to the level of pollen exposure. Recent studies in animals have investigated the mechanisms of allergen challenge-induced bone marrow precursor stimulation (Kung et al. 1994; Wooley et al. 1994; Denburg et al. 1995; Ignez et al. 1997). One study specifically assessing nasal allergen challenge identified that within 2 hours of challenge there was an increase in IL-3 sensitive progenitors that yielded eosinophil-bearing colonies (Ignez et al. 1997). The bone marrow of these animals demonstrated increased IL-2 and to a greater extent IL-5 eosinophil differentiation. This increased responsiveness was only evident 24 hours following challenge. While IL-5 was partly responsible, as assessed by the in vivo effect of the anti-IL-5 monoclonal antibody TRFK-5, the majority of the bone marrow stimulatory effect in this rodent model was due to an unidentified circulatory factor. How these studies relate to human eosinophil differentiation is unclear as an IL-5 monoclonal antibody has not been assessed under similar circumstances. Such studies are likely to be required as investigation of these mechanisms in humans using a nasal allergen challenge model has proved difficult in that the nasal stimulus, despite producing nasal eosinophil recruitment, was too small to produce circulatory changes (Wilson et al. 2005). Clinical studies consistently identify that montelukast, a CysLT1-receptor antagonist, reduces circulating eosinophil numbers providing indirect evidence for the involvement of leukotrienes in the systemic bone marrow stimulatory process (Nayak et al. 2002; Philip et al. 2002).

Pathophysiology of Allergic Rhinitis

Therapeutic implications Allergic rhinitis is thus an inflammatory nasal disorder in which a range of cells and mediators contribute, in a coordinated complex network that underlies clinical disease expression. Drug therapy is currently directed toward receptor antagonism of mediators, functional antagonism of end-organ responses, and inhibition of cell accumulation and/or activation. Histamine is a major contributor and, as such, receptor antagonists of H1-receptors have played a prominent role in management guidelines. The appreciation that other histamine receptors may also be involved in the nasal allergic response partly explains the incomplete effect with H1-antihistamines. In addition, histamine is not the only player. Inhibition of other mediators is likely to confer additional benefit and combination therapy with CysLT1-receptor antagonists and an H1-receptor antagonist has shown a greater effect on quality of life than either agent administered as sole therapy (Meltzer et al. 2000), although it has been difficult to clearly show statistical improvement of the combination over the individual drugs given alone (Meltzer et al. 2000; Nayak et al. 2002). Antagonists of kinins and PGD2 are developmental projects. More novel therapies such as an anti-IgE monoclonal antibody has been shown to significantly reduce symptom expression (Casale et al. 2001) and would also be anticipated to have benefit in exerting antiinflammatory effects when used prophylactically. At present, however, the predominant antiinflammatory therapy recommended for the treatment of allergic rhinitis in guidelines is topical corticosteroids (Bousquet et al. 2001). However, the effects of such treatment are predominantly local, although they do modify conjunctival symptoms when nasally administered and those that are systemically available have also been reported to modify lower airway disease. Such treatment, however, does not encompass the systemic nature of allergic disease and the only currently available treatment that truly modifies disease at all sites in allergic rhinitis is allergen-specific immunotherapy. This is most effective for pollen-related allergy and represents the only form of treatment that can fundamentally alter the immune response to allergen and thus the natural history of the disease. Immunotherapy induces its beneficial effects through promoting regulatory T-cell activity.

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Terada, N., Hamano, N., Kim, W.J. et al. (2001b) The kinetics of allergen-induced eotaxin level in nasal lavage fluid. Am J Respir Crit Care Med 164, 575–9. Thueson, D.O., Speck, L.S., Lett-Brown, M.A. & Grant, J.A. (1979) Histamine-releasing activity (HRA). 1. Production of a histamine releasing lumphokine by mitogen or antigen stimulated human mononuclear cells. J Immunol 123, 623–32. Tomee, J.F., van-Weissenbruch, R., de Monchy, J.G. & Kauffman, H.F. (1998) Interactions between inhalant allergen extracts and airway epithelial cells: effect on cytokine production and cell detachment. J Allergy Clin Immunol 102, 75–85. Tos, M. (1983) Distribution of mucous producing elements within the respiratory tract. Differences between upper and lower airways. Eur J Respir Dis Suppl 128, 269–79. Trotter, C.M. & Orr, T.S.C. (1973) A fine structure of some cellular components in allergic reaction. 1. Degranulation of human mast cells in allergic asthma and perennial rhinitis. Clin Exp Allergy 3, 411–25. Urmil, G., Bazaz-Malik, G. & Mohindra, S.K. (1984) Significance and comparison of blood, nasal secretion and mucosal eosinophils in nasal allergy. Ind J Pathol Microbiol 27, 27–32. Varga, E.M., Jacobson, M.R., Till, S.J. et al. (1999) Cellular infiltration and cytokine mRNA expression in perennial allergic rhinitis. Allergy 54, 338–45. Varney, V.A., Jacobson, M.R., Sudderick, R.M. et al. (1992) Immunohistology of the nasal mucosa following allergen-induced rhinitis. Identification of activated T lymphocytes, eosinophils, and neutrophils. Am Rev Respir Dis 146, 170–6. Varty, L.M. & Hey, J.A. (2002) Histamine H3 receptor activation inhibits neurogenic sympathetic vasoconstriction in porcine nasal mucosa. Eur J Pharmacol 452, 339–45. Vercelli, D., Jabara, H.H., Arai, K. & Geha, R.S. (1989) Induction of human IgE synthesis requires IL-4 and T/B cell interactions involving the T cell receptor/CD3 complex and MHC class II antigens. J Exp Med 169, 1295–307. Vignola, A.M., Crampette, L., Mondain, M. et al. (1995) Inhibitory activity of loratadine and descarboethoxyloratadine on expression of ICAM-1 and HLDA-DR by nasal epithelial cells. Allergy 50, 200–3. Wang, J.H., Devalia, J.L., Xia, C., Sapsford, R.J. & Davies, R.J. (1996) Expression of RANTES by human bronchial epithelial cells in vitro and in vivo and the effects of corticosteriods. Am J Respir Cell Mol Biol 14, 27–35. Wang, M. & Lung, M. (2003) Adrenergic mechanisms in canine nasal venous systems. Br J Pharmacol 138, 145–55. Watanabe, K., Saito, Y., Watanabe, I. & Mizahira, V. (1980) Characterisics of capiallary permeability in the nasal mucosa. Ann Otol 89, 377–82. Weitzberg, E. & Lundberg, J.O. (2002) Humming greatly increases nasal nitric oxide. Am J Respir Crit Care Med 166, 144–5. Widdicombe, J.G. & Wells, U.M. (1982) Airway secretions. In: Proctor, D.F. & Andersen, I., eds. The Nose, Upper Airway Physiology and the Atmospheric Environment. Elsevier Biomedical Press, Amsterdam, pp. 215–44. Widegren, H., Korsgren, M., Andersson, M. & Greiff, L. (2007) Effects of TNFalpha on the human nasal mucosa in vivo. Respir Med 101(9), 1982–7. Wilson, A.M., Duong, M., Crawford, L. & Denburg, J. (2005) An evaluation of peripheral blood eosinophil/basophil progenitors

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following nasal allergen challenge in patients with allergic rhinitis. Clin Exp Allergy 35, 39– 44. Wilson, D.R., Irani, A.M., Walker, S.M., Jacobson, M.R., Macka Schwartz, L.B. & Durham, S.R. (2001) Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium. Clin Exp Allergy 31, 1705–13. Wilson, S.J. & Howarth, P.H. (1995) Collagen phenotypes and subepithelial collagen band thickness in the allergic nasal mucosa. Am J Respir Crit Care Med 151, A563. Wilson, S.J., Lau, L. & Howarth, P.H. (1998) Inflammatory mediators in naturally occurring rhinitis. Clin Exp Allergy 28, 220–7. Wilson, S.J., Shute, J.K., Holgate, S.T., Howarth, P.H. & Bradding, P. (2000) Localisation of interleukin (IL)-4 but not IL-5 to human mast cell secretory granules by immunoelectron microscopy. Clin Exp Allergy 30, 493–500. Wingqvist, I., Olofsson, T., Olsson, I., Persson, A. & Hallberg, T. (1982) Altered density, metabolism, and surface receptors of eosinophils in eosinophilia. Immunology 47, 531–9.

Pathophysiology of Allergic Rhinitis

Winther, L., Moseholm, L., Reimert, C.M., Stahl, S.P., Kaergaard, S. & Poulson, L. (1999) Basophil histamine release, IgE, eosinophil counts, ECP, and EPX are related to the severity of symptoms in seasonal allergic rhinitis. Allergy 54, 436–45. Wood-Baker, R., Lau, L.C.K. & Howarth, P.H. (1996) Histamine and the nasal vasculature: the influence of H1 and H2 histamine receptor antagonism. Clin Otolaryngol 21, 348–52. Wooley, M.A., Denburg, J.A., Ellis, R., Dahlback, M. & O’Byrne, P.M. (1994) Allergen-induced changes in bone marrow progenitors and airway responsiveness in dogs and the effect of inhaled budesonide on these parameters. Am J Respir Cell Mol Biol 11, 600–6. Wright, E., Christodoulopouslos, P., Frenkiel, S. & Hamid, Q. (1999) Expression of interleukin (IL)-12 (p40) and IL-12 (β2) receptors in allergic rhinitis and chronic sinusitis. Clin Exp Allergy 29, 1320– 6. Xu, G., Mou, Z., Jiang, H. et al. (2007) A possible role of CD4+CD25+ T cells as well as transcription factor Foxp3 in the dysregulation of allergic rhinitis. Laryngoscope 117, 876–80.

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Management and Treatment of Allergic Rhinitis Jean Bousquet and Michael A. Kaliner

Summary

Introduction

The management of allergic rhinitis is well established and many guidelines have been issued. Drugs for the treatment of allergic rhinitis can be administered intranasally or (for some) orally. Many drugs are safe and effective for the treatment of allergic rhinitis, including oral and intranasal H1-antihistamines, intranasal corticosteroids (the most effective drug), antileukotrienes, and cromones. Intranasal anticholinergics are only effective in rhinorrhea. Oral first-generation H1antihistamines should be replaced by second-generation ones. Long courses of oral corticosteroids and depot preparations of corticosteroids are not recommended due to side effects. The treatment of allergic rhinitis should consider the severity and duration of the disease, patient preferences, as well as efficacy, availability and costs of drugs. A stepwise approach that considers the severity and duration of rhinitis is proposed. A tailored approach is needed for each individual patient. Not all patients with moderate/severe allergic rhinitis are controlled despite optimal pharmacotherapy. Allergen avoidance for tertiary prevention of allergic rhinitis is difficult to evaluate. It has no effect in nonselected patients whereas it may be effective in some highly selected ones. Allergen-specific immunotherapy is classically administered via the subcutaneous route but local routes are now available. Specific immunotherapy needs a precise diagnosis of IgE-mediated allergy. Subcutaneous immunotherapy is effective in adults and children for pollen and mite allergy, but it is burdened by the risks of side effects. Exceptionally, these reactions may be life-threatening. Sublingual immunotherapy is recommended for the treatment of pollen and mite allergy. Allergen-specific immunotherapy can alter the natural course of allergic diseases. Most data are available for the subcutaneous route. Many patients who use complementary and alternative medicine appear to be satisfied but there are no convincing data supporting its effect.

Allergic rhinitis is a symptomatic disorder of the nose induced by IgE-mediated inflammation of the nasal membranes in response to allergen exposure (Bousquet et al. 2001). Predominant symptoms include rhinorrhea, nasal obstruction, nasal itching and sneezing, which can improve spontaneously or with treatment (International Rhinitis Management Working Group 1994; van Cauwenberge et al. 2000; Bousquet et al. 2001). The high prevalence of allergic rhinitis and its effect on quality of life have led to its classification as a major chronic respiratory disease (Strachan et al. 1997; Bousquet et al. 2001). It is reported to affect between 10 and 40% of the global population and its prevalence is increasing in both children and adults. Allergic rhinitis can significantly reduce quality of life (Leynaert et al. 2000; Tripathi & Patterson 2001), impairing sleep and adversely affecting leisure, social life, school performance (Simons 1996), and work productivity (Blanc et al. 2001; Bousquet et al. 2006a). The direct and indirect financial costs of allergic rhinitis are substantial. Indirect costs include sick leave, school and work absenteeism as well as loss of productivity (Sullivan & Weiss 2001; Weiss & Sullivan 2001; Bousquet et al. 2005a). Asthma and rhinitis are common comorbidities, suggesting the concept of “one airway, one disease” (Simons 1999; Bousquet et al. 2001, 2003a; Bachert et al. 2004a). In addition, allergic rhinitis is associated with conjunctivitis and sinusitis (Meltzer et al. 2004; Kaliner 2005). Recent advances in our understanding of the mechanisms underlying inflammation of the upper and lower airways have led to improved therapeutic strategies for the management of allergic rhinitis. Practice guidelines incorporating these advances have been developed (van Cauwenberge et al. 2000; Bousquet et al. 2001). In addition, a new classification of allergic rhinitis aids the establishment of an appropriate initial treatment strategy based on the duration and intensity of the patient’s symptoms and lifestyle limitations (Bousquet et al. 2001, 2002). Many patients suffering from allergic rhinitis do not recognize the process as such, do not consult a physician (Dykewicz

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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et al. 1998a; Bousquet et al. 2002) and only use over-thecounter drugs. Others commonly seek self-treatment for the relief of symptoms and use unproven therapies. It is therefore very important to recognize the signs and symptoms suggestive of severe rhinitis that may require urgent medical management (Anon. 2004a).

Comorbidity and complications Allergic inflammation does not necessarily limit itself to the nasal airway. Multiple comorbidities have been associated with rhinitis.

Asthma The nasal and bronchial mucosa share many similarities (Bousquet et al. 2003a, 2004a; Bachert et al. 2004a). Epidemiologic studies have consistently shown that asthma and rhinitis often coexist in the same patients (Sibbald & Rink 1991; Wright et al. 1994; Leynaert et al. 1999; Yawn et al. 1999). Most patients with allergic and nonallergic asthma have rhinitis. Allergic rhinitis correlates with and constitutes a risk factor for asthma (Guerra et al. 2002). Many patients with allergic rhinitis have increased nonspecific bronchial hyperreactivity (Townley et al. 1975; Leynaert et al. 1997). There is a temporal relationship between the onset of allergic rhinitis and asthma, with rhinitis frequently preceding the development of asthma. In normal subjects, the structure of the airway mucosa in the nose and the bronchi is similar. There are also differences. In the nose, there is a large blood supply, and changes in the vasculature can lead to severe nasal obstruction (Holmberg et al. 1988). On the other hand, smooth muscle is present from the trachea to the bronchioles and accounts for the bronchoconstriction in asthma. It is also probably linked with the regulation of bronchial remodeling in asthma. Pathophysiologic studies suggest that a strong relationship exists between rhinitis and asthma. The recent progress achieved in the cellular and molecular biology of airway diseases has documented that the inflammation of nasal and bronchial mucosa plays a critical role in the pathogenesis of asthma and rhinitis and present many similarities. On the other hand, airway remodeling exists microscopically in most if not all asthmatics (Bousquet et al. 2000) but may not be so obvious in rhinitis patients (Bousquet et al. 2004a). Several mechanisms have been proposed to link uncontrolled allergic rhinitis and the occurrence or worsening of asthma (Corren 1997). Although nasal challenge with allergen does not induce airflow limitation of the lower airways, it may cause nonspecific bronchial responsiveness (Aubier et al. 1991; Corren et al. 1992). These data have led to the concept that the upper and lower airways may be considered as a unique entity influenced by a common, evolving inflammatory process, which may be sustained and amplified by interconnected mechanisms. Patients with claimed nasal symptoms usually have less

Management and Treatment of Allergic Rhinitis

controlled asthma than those without symptoms (Bousquet et al. 2005b). Therefore, when considering a diagnosis of rhinitis or asthma, an evaluation of both the lower and upper airways should be made.

Other comorbidities These include sinusitis, nasal polyposis, and conjunctivitis (Fokkens et al. 2005). The association between allergic rhinitis and otitis media is less well understood (Hellings & Fokkens 2006).

Diagnosis and assessment of severity The tests and procedures listed below represent the spectrum of investigations, which may be used in the diagnosis of allergic rhinitis. However, only a certain number of these are routinely available or applicable to each individual patient (Table 69.1).

Table 69.1 Diagnostic tests for allergic rhinitis. (Adapted from Bousquet et al. 2002, with permission.) Routine tests History General ENT examination Allergy tests Skin tests Serum specific IgE Endoscopy Rigid Flexible Nasal secretions Cytology Nasal challenge Allergen Lysine aspirin Radiology CT Optional tests Nasal biopsy Nasal swab Bacteriology Radiology CT, MRI Mucociliary function Nasal mucociliary clearance Ciliary beat frequency Electron microscopy Nasal airway assessment Nasal inspiratory peak flow Rhinomanometry (anterior and posterior) Acoustic rhinometry Olfaction Nitric oxide measurement

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Symptoms suggestive of allergic rhinitis

Symptoms usually NOT associated with allergic rhinitis

Unilateral symptoms + + + + Nasal obstruction without other symptoms Mucopurulent rhinorrhea Posterior rhinorrhea (post-nasal drip) – With thick mucus – And/or no anterior rhinorrhea – Pain – Recurrent epistaxis – Anosmia

2 or more of the following symptoms for > 1 hour on most days – Watery rhinorrhea – Sneezing, especially paroxysmal – Nasal obstruction – Nasal pruritus ± Conjunctivitis

– – – –

Classify and assess severity

Fig. 69.1 Symptoms of allergic rhinitis (ARIA 2004). (See CD-ROM for color version.)

History and general ENT examination A clinical history is essential for an accurate diagnosis of rhinitis and for assessment of its severity and response to treatment (see Table 67.2, p. 1384). Patients with allergic rhinitis often suffer from sneezing and a runny nose. Those classified as “blockers” do not have allergic rhinitis. Many patients do not consult a physician for nasal symptoms. However, some symptoms require urgent investigation (Fig. 69.1). In patients with mild intermittent allergic rhinitis, a nasal examination is optimal. However, all patients with persistent allergic rhinitis should undergo nasal examination. Anterior rhinoscopy, using a speculum and mirror, provides limited information and nasal endoscopy is more useful. Many patients with pollen-induced rhinitis present with eye symptoms,

Symptoms suggestive of allergic conjunctivitis

Allergy diagnosis The diagnosis of allergic rhinitis is based on a typical history of allergic symptoms and diagnostic tests. In vivo and in vitro tests used to diagnose allergic diseases are directed toward the detection of free or cell-bound IgE. The diagnosis of allergy has been improved by allergen standardization, which has provided satisfactory diagnostic vaccines for most inhalant allergens. Immediate hypersensitivity skin tests are widely used to demonstrate an IgE-mediated allergic reaction and represent a major diagnostic tool in the field of allergy. If properly performed, they yield useful confirmatory evidence for the

Symptoms NOT suggestive of allergic conjunctivitis

1 or more of the following symptoms for > 1 hour on most days – Symptoms associated with rhinitis – Bilateral eye symptoms – Eye itching – Watery eyes – Red eyes – NO photophobia

Do the symptoms concern the patient or the pharmacist?

and in this case it is important to distinguish between allergic and nonallergic symptoms (Fig. 69.2).

1 (or more) of the following symptoms – Symptoms NOT associated with rhinitis – Unilateral conjunctivitis – NO eye itching – BUT eye burning – Dry eyes – Photophobia

Yes

Refer the patient to a doctor

no Oral H1-blocker*/$ or ocular H1-blocker*/£ or ocular cromone*/£ *: Depending on drug availability AND not in preferred order $: Non-sedating H1-blockers are preferred £: Formulations without preservatives are better tolerated

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If after 7–15 days no improvement

Fig. 69.2 Symptoms of allergic conjunctivitis. (See CD-ROM for color version.)

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diagnosis of a specific allergy. As there are many complexities in their performance and interpretation, it is recommended that implementation should be carried out by trained health professionals (Demoly et al. 1998; Nelson 2004). The measurement of total serum IgE has poor predictive value for allergy screening in rhinitis and should not be used as a diagnostic tool (Dykewicz & Fineman 1998). In contrast, the measurement of allergen-specific IgE in serum is of importance and has a value similar to that of skin tests (Bousquet et al. 1990; Pastorello et al. 1992). Some in vitro specific IgE methods use either a mixture of several allergens in a single assay (Eriksson 1990) or test several different allergens during a single assay. These tests can therefore be used by specialized doctors and nonallergists as screening tests for the diagnosis of allergic diseases. Nasal challenge tests with allergens are used in research and, to a lesser extent, in clinical practice. However, they are important in the diagnosis of occupational rhinitis. A subcommittee of the International Committee on Objective Assessment of the Nasal Airways has proposed guidelines relating to the indications, techniques and evaluation of nasal provocation tests (Malm et al. 1999).

Management and Treatment of Allergic Rhinitis

rhinitis symptoms are independent of treatment (Bousquet et al. 2005c).

Rhinitis treatments The management of allergic rhinitis encompasses patient education, pharmacotherapy and allergen-specific immunotherapy. Surgery may be used as an adjunctive intervention in a few highly selected patients (Bousquet et al. 1998, 2001, 2004b). Environmental control is more controversial (Schmidt & Gotzsche 2005). Evidence-based medicine is an increasingly important concept that has become a new paradigm in medicine (Sackett et al. 1996). Its increasing influence, due partly to the work of the Cochrane Collaboration, has led the way in setting new standards for preparing clinical recommendations (Bousquet et al. 2004b). We recommend a strategy combining the treatment of both upper and lower airway disease in terms of efficacy and safety (Bousquet et al. 2001).

Environmental control Allergen avoidance

Diagnosis of asthma The diagnosis of asthma may be difficult due to the transient nature of the disease and the reversibility of the airflow obstruction spontaneously or following treatment. Guidelines for the recognition and diagnosis of asthma have been published by the Global Initiative for Asthma (GINA 2006) and have been recommended in the WHO/NHLBI Workshop Report (1995). As already identified by GINA, measurement of lung function and confirmation of the reversibility of airflow obstruction are essential steps in the diagnosis of asthma. An update of the 1995 document is being prepared and will represent state of the art of the management of asthma (Bateman et al. 2008).

Assessment of the severity of rhinitis For asthma, there are objective measures of severity such as pulmonary function tests and well-defined criteria for symptom severity WHO/NHLBI Workshop Report (1995). For atopic dermatitis, there are clinical scores of severity such as SCORAD (Consensus Report of the European Task Force on Atopic Dermatitis 1993). However, for rhinitis, there is no accepted objective measure for nasal obstruction. Nasal inspiratory peak flow has been extensively studied but the results are not consistent among the different studies (Holmstrom et al. 1990; Clarke & Jones 1994; Prescott & Prescott 1995). Moreover, the correlation between the objective measurement of nasal resistance and subjective reports of nasal airflow sensation is usually poor. Simple measures such as the visual analog scale (VAS) appear to be sufficient to assess the severity of rhinitis symptoms (van Cauwenberge et al. 2000; Bousquet et al. 2003b; Spector et al. 2003). As in asthma, the severity of

A range of allergens have been associated with allergic rhinitis, of which house-dust mite is the most important and most investigated (Wahn et al. 1997; Platts-Mills 2001). Most allergen avoidance studies have dealt with asthma symptoms and very few have studied rhinitis symptoms. Unfortunately, the majority of interventions have failed to achieve a sufficient reduction in allergen load to enhance any clinical improvement. Allergen avoidance is not recommended as part of the management of asthma (GINA 2006). A systematic review of dust mite avoidance measures for perennial allergic rhinitis was published in 2003 (Sheikh & Hurwitz 2003). A similar review was published for asthma in 2004 (Gotzsche et al. 2004). Only four rhinitis trials satisfied the inclusion criteria, all of which were small and judged to be of poor quality. There was no beneficial effect from physical or chemical interventions. Subsequent to this metaanalysis, a large study investigated the effectiveness of mite allergenimpermeable encasings in mite-sensitized patients with perennial rhinitis and a positive nasal challenge test to mite extract (Terreehorst et al. 2003, 2005). The active covers reduced the level of mattress Der p1 to approximately 30% of the baseline level, whereas the placebo covers had no effect. However, there was no difference between groups in any of the outcome measures. Two small studies have addressed the effects of pet allergen control measures in rhinitis. In a randomized controlled trial of the efficacy of high-efficiency particulate air (HEPA) filters, nasal symptoms did not differ between active intervention and the placebo group (Wood et al. 1998). In another study, a set of allergen control measures (washing all walls and floors, removing carpeting from bedrooms, applying tannic acid,

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Table 69.2 Effectiveness of avoidance measures in rhinitis and asthma for certain indoor allergens. (Adapted from Custovic & Wijk 2005, with permission.)

Measure

Evidence of effect on allergen levels

House-dust mites Encase bedding in impermeable covers

Some

Wash bedding on hot cycle (55–60°C) Replace carpets with hard flooring Acaricides and/or tannic acid Minimize objects that accumulate dust Vacuum cleaners with integral HEPA filter and double-thickness bags Remove, hot wash or freeze soft toys Pets Remove cat/dog from the home Keep pet from main living areas/bedrooms HEPA filters Wash pet Replace carpets with hard flooring Vacuum cleaners with integral HEPA filter and double-thickness bags Set of allergen control measures

Evidence of clinical benefit

Some Some Weak None Weak

None (adults): evidence A Some (children): evidence B None: evidence A None: evidence A None: evidence A None: evidence B None: evidence B

None

None: evidence B

Weak Weak Some Weak None None

None: evidence B None: evidence B None: evidence B None: evidence B None: evidence B None: evidence B

Some

Some: evidence B

washing bedding, replacing duvets and pillows, using impermeable covers, washing the cat every 2 weeks, etc.) resulted in a fall in the Fel d1 level to 6.8% of the baseline and in a significant improvement in nasal symptoms and nasal peak flow (Bjornsdottir et al. 2003). Although the general consensus is that allergen avoidance should lead to an improvement in symptoms, there is very little evidence to support the use of single physical or chemical methods (Table 69.2). Recommendations proposing their use are at variance with the current evidence (Custovic & Wijk 2005; Schmidt & Gotzsche 2005). The use of mattress encasings or HEPA filters as a single intervention for housedust mite and pet allergy in adults with asthma or rhinitis cannot be advocated. Considering the management of allergy, current evidence suggests that interventions in children (either single or multifaceted) may be associated with at best a minor beneficial effect on asthma control. However, no conclusive evidence exists regarding rhinitis or eczema. Conversely, there is little evidence to support the recommendation of allergen avoidance methods in adults with asthma and rhinitis. There is a need for an adequately designed trial assessing the effects of a multifaceted intervention in this age group (Marinho et al. 2006).

Other measures Many occupational agents are involved in the development of rhinitis and asthma. However, no study has convincingly demonstrated the efficacy of environmental control measures

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due to methodologic problems. This is particularly the case for latex allergy (evidence level B) (Bousquet et al. 2006b). Indoor and outdoor air pollutants are commonly associated with nonallergic rhinitis and may exacerbate symptoms in patients with allergic rhinitis. On the other hand, tobacco smoke does not appear to aggravate the symptoms of allergic rhinitis. Again, no existing study demonstrates that environmental control measures are beneficial due to methodologic problems (evidence level B).

Drug treatment Pharmacologic treatment should take the following factors into account: efficacy, safety and cost-effectiveness of medications; the patient’s preference; the objective of the treatment (Marinker 1998; Kaliner 2001a,b; Bousquet et al. 2006c); likely adherence to recommendations (Bousquet et al. 2003b); severity of the disease; and the presence of comorbidities. Medications used for rhinitis are most commonly administered intranasally or orally (Table 69.3). The efficacy of medications may differ between patients. Medications have no long-lasting effect when stopped. Therefore, in persistent disease, maintenance treatment is required. Tachyphylaxis does not usually occur with prolonged treatment. The efficacy and safety of each of these medications has been documented in other parts of this book and only a summary is given here. Certain studies have compared the relative efficacy of these medications and have found that intranasal corticosteroids are most effective (Weiner et al. 1998). A review of medications

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Table 69.3 Glossary of medications used in allergic rhinitis. (Adapted from Bousquet et al. 2002, with permission.) Name

Generic name

Mechanism of action

Side effects

Comments

Oral H1-antihistamines

New generation Acrivastine Azelastine Cetirizine Desloratadine Ebastine Fexofenadine Levocetirizine Loratadine Mequitazine Mizolastine Rupatadine

Blockage of H1 receptor Some antiallergic activity New-generation drugs can be used once daily No development of tachyphylaxis

New generation No sedation for most drugs No anticholinergic effect No cardiotoxicity Acrivastine has sedative effects Mequitazine has anticholinergic effect Oral azelastine may induce sedation and a bitter taste

New-generartion oral H1antihistamines should be preferred for their favorable efficacy/safety ratio and pharmacokinetics Rapidly effective (< 1 hour) on nasal and ocular symptoms Poorly effective on nasal congestion Cardiotoxic drugs are no longer marketed in most countries

Old generation Sedation is common And/or anticholinergic effect

Old generation Chlorpheniramine Clemastine Hydroxyzine Ketotifen Oxatomine Cardiotoxic* Astemizole Terfenadine Local H1-antihistamines (intranasal, intraocular)

Azelastine Levocabastine

Blockage of H1 receptor Some antiallergic activity for azelastine

Minor local side effects Azelastine: bitter taste

Rapidly effective (< 30 min) on nasal or ocular symptoms

Intranasal corticosteroids

Beclomethasone Budesonide Flunisolide Fluticasone Mometasone Triamcinolone

Potently reduce nasal inflammation Reduce nasal hyperreactivity

Minor local side effects Wide margin for systemic side effects Growth concerns with some molecules only In young children consider the combination of intranasal and inhaled drugs

Most effective pharmacologic treatment of allergic rhinitis Effective on nasal congestion Effective on smell Effect observed after 12 hours but maximal effect after a few days

Oral/i.m. corticosteroids

Dexamethasone Hydrocortisone Methylprednisolone Prednisolone Prednisone Triamcinolone

Potently reduce nasal inflammation Reduce nasal hyperreactivity

Systemic side effects common, particularly for i.m. drugs Depot injections may cause local tissue atrophy

When possible, intranasal corticosteroids should replace oral or i.m. drugs However, a short course of oral corticosteroids may be needed if severe symptoms

Local cromones (intranasal, intraocular)

Cromoglycate Nedocromil Naaga

Mechanism of action poorly known

Minor local side effects

Intraocular cromones are very effective Intranasal cromones are less effective and their effect is short-lasting Overall excellent safety

Oral decongestants

Ephedrine Phenylephrine Phenylpropanolamine Pseudoephedrine

Sympathomimetic drugs Relieve symptoms of nasal congestion

Hypertension Palpitations Restlessness Agitation Tremor Insomnia Headache Dry mucous membranes Urinary retention Exacerbation of glaucoma or thyrotoxicosis

Use oral decongestants with caution in patients with heart disease Oral H1-antihistamine/decongestant combination products may be more effective than either product alone but side effects are combined

Continued p. 1436

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Table 69.3 (Cont’d ) Name

Generic name

Mechanism of action

Side effects

Comments

Intranasal decongestants

Oxymetazoline Others

Sympathomimetic drugs Relieve symptoms of nasal congestion

Same side effects as oral decongestants but less intense Rhinitis medicamentosa is a rebound phenomenon occurring with prolonged use (> 10 days)

Act more rapidly and more effectively than oral decongestants Limit duration of treatment to < 10 days to avoid rhinitis medicamentosa

Intranasal anticholinergics

Ipratropium

Anticholinergics block rhinorrhea almost exclusively

Minor local side effects Almost no systemic anticholinergic activity

Effective in allergic and nonallergic patients with rhinorrhea

Cysteinyl leukotriene antagonists

Montelukast Pranlukast Zafirlukast

Block CysLT receptor

Excellent tolerance

Effective on rhinitis and asthma Effective on all symptoms of rhinitis and on ocular symptoms

* Removed from most markets due to side effects.

for the treatment of allergic rhinitis has recently been published and details on drugs are provided (Bousquet et al. 2006c).

Routes of administration Medications used for rhinitis are administered in the majority of cases intranasally or orally. Intranasal medications offer several advantages since high concentrations can be delivered directly into the nose, avoiding or minimizing systemic effects. However, problems are encountered with intranasal medications. Many patients with allergic rhinitis also have conjunctivitis and/or asthma, and medications need to be administered to various target organs. The intranasal distribution of medication is not optimal for some patients, but in many the local effects are beneficial and the speed and convenience of local administration is an advantage. In exceptional circumstances, corticosteroids may be administered intramuscularly due to their unfavorable efficacy/safety ratio.

Advantages of intranasal administration • High concentrations can be delivered directly into the target organ avoiding or minimizing systemic effects. • Some of the drugs (e.g., cromones) used for the treatment of rhinitis should be administered only via the intranasal route as they are not adequately absorbed when given orally. • Some drugs have systemic effects when administered orally (e.g., glucocorticosteroids and atropine derivatives). • The onset of action of an intranasal drug is usually faster than that of an oral drug (e.g., vasoconstrictors and some H1antihistamines). • Nasal administration places the medication directly on the affected organ and may be more effective than oral administration.

1436

Problems of intranasal administration • Many patients with allergic rhinitis present also with conjunctivitis and/or asthma. Thus, there is a need for multiple administrations in target organs. By using as-needed treatment, however, overall dosing may be reduced by local administration only to affected organs. • An irritant or cilia toxic effect from added preservatives may be observed with some intranasal drugs. • Other local side effects are medication-dependent. Prolonged use of an intranasal vasoconstrictor results in the risk of developing rhinitis medicamentosa (Scadding 1995). The use of intranasal ipratropium bromide can cause an unpleasant feeling of nasal dryness and also produce blood-tinged mucus. • Intranasal medication cannot be given when the nose or nostril is completely blocked. • Patient compliance may be greater with oral than with topical drugs, especially if multiple target organs are to be treated. Education about the advantages of topical treatment would probably improve compliance.

Oral H1-antihistamines H1-blockers or H1-antihistamines are medications that block histamine at the H1-receptor level (neutral antagonists or inverse agonists) (Leurs et al. 2002). Some also possess additional antiallergic properties (Bousquet et al. 2003c). Over the past 20 years, pharmacologic research has produced compounds with minimal sedative effect and impairment: the so-called second-generation H1-antihistamines, as opposed to the first-generation H1-antihistamines (Bousquet et al. 2003c). The term “third-generation” should be reserved for an H1antihistamine with novel properties (Holgate et al. 2003).

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Oral H1-antihistamines were introduced over half a century ago (Bovet et al. 1944). They are effective against symptoms mediated by histamine (rhinorrhea, sneezing, nasal itching and eye symptoms) but are less effective on nasal congestion (Simons 2004). They improve the patient’s quality of life (Bousquet et al. 1996; Bachert et al. 2004b; Guadano et al. 2004). One double-blind, placebo-controlled, long-term trial found that levocetirzine was cost-effective in the treatment of persistent rhinitis (Bachert et al. 2004b; Bousquet et al. 2005a). First-generation oral H1-antihistamines possess significant side effects due to their sedative and anticholinergic properties. Newer antihistamines induce no (Skassa-Brociek et al. 1988; Howarth et al. 1999; Hindmarch & Shamsi 2001; Simons et al. 2003) or little (Berman 1990; Bachert et al. 2004b) sedation or impairment. They are not anticholinergic. Long-term treatment (years) with oral H1-antihistamines is safe. Some, but not all, oral H1-antihistamines undergo hepatic metabolism via the cytochrome P450 system and are prone to drug interactions (Simons et al. 1991). Although cardiotoxicity is not a class effect (Passalacqua et al. 2002), major concerns have existed about the arrhythmogenic action of terfenadine, astemizole and high doses of diphenhydramine which have rarely been associated with fatalities. H1-antihistamines have also been approved for young children (Simons 2002). Cetirizine, when compared with placebo, delayed or in some cases prevented the development of asthma in a subgroup of infants with atopic dermatitis sensitized to grass pollen and, to a lesser extent, house-dust mite (Warner 2001). Further studies are required to substantiate this finding and should focus specifically on sensitized groups. Several properties should be met by oral H1-antihistamines (Table 69.4) (Bousquet et al. 2003d). Although first-generation oral H1-antihistamines are effective, they cannot be recommended when second-generation drugs are available because of their sedative and anticholinergic effects (Plaut & Valentine 2005). Only safe second-generation antihistamines should be prescribed due to their favorable efficacy/safety ratio. As a result of the over-the-counter introduction of loratadine in the USA, health plans have attempted to determine the best policy for incorporating this change within their existing drug benefit structure for second-generation H1-antihistamines (Sullivan & Nichol 2004). These important changes need to be taken into consideration for optimal cost-effectiveness. The doubling of copayments was associated with reductions in the use of eight therapeutic classes. The largest decreases occurred for nonsteroidal antiinflammatory drugs (NSAIDs) (45%) and antihistamines (44%) (Goldman et al. 2004).

Topical H1-antihistamines Intranasal H1-antihistamines are effective at the site of their administration in reducing itching, sneezing, runny nose, and nasal congestion (Schata et al. 1991; McNeely & Wiseman 1998; LaForce et al. 2004). Given ocularly, they are effective

Management and Treatment of Allergic Rhinitis

Table 69.4 Requirements for oral H1-antihistamines. Pharmacologic properties Potent and selective H1-receptor blockade Additive antiallergic activities (see below) No clinically relevant pharmacokinetic interference by foods, medications or intestinal transport proteins No known interaction with cytochrome P4503A (CYP3A) No known interaction with disease to avoid toxic reactions Efficacy Effective in the treatment of intermittent and persistent rhinitis as defined in the ARIA document Effective for all nasal symptoms including nasal obstruction Improvement of eye symptoms If a claim for asthma is made: improvement of asthma symptoms (short-term studies) reduction of asthma exacerbations (long-term studies) improvement of pulmonary function tests, even though FEV1 and peak flow rates are usually not altered in pollen-induced bronchial symptoms If a claim for a preventive effect is proposed, appropriate trials should be conducted Studies should be carried out in young children and elderly patients to assess efficacy Side effects No sedation, no cognitive or psychomotor impairment No anticholinergic effects No weight gain No cardiac side effects Possible use in pregnancy and breast-feeding Studies should be carried out in young children and elderly patients in order to assess safety Prospective postmarketing safety analyses should be conducted Pharmacodynamics Rapid onset of action Long duration of action (at least persistence of clinical effects at the end of the 24-hour dosing period) so that the drug can be administered once a day No likelihood of development of tolerance (tachyphylaxis)

in allergic eye symptoms (Odelram et al. 1989; Katelaris et al. 2002). They can be effective within 20 min of administration. Topical H1-antihistamines require twice-a-day dosing. In general, topical H1-antihistamines are well tolerated but, in some patients, azelastine induces an unpleasant taste and odor. Moreover, while intranasal glucocorticosteroids are significantly more effective than oral antihistamines for the treatment of allergic rhinitis and, in particular, nasal congestion, recent data suggest that nasal azelastine is equally effective as compared with nasal fluticasone over a 14-day trial and that the combination of nasal corticosteroid plus azelastine acts synergistically in relieving symptoms (Yanez & Rodrigo 2002). It may be that the combination of topical nasal corticosteroid

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and topical antihistamine such as azelastine will be the preferred treatment for rhinitis, as azelastine is quite effective in treating both nonallergic rhinitis and allergic rhinitis. Nasal azelastine works within a few minutes of administration while nasal corticosteroids take hours to days to be effective. Thus, the combination effectively relieves nasal symptoms, including congestion, and has few systemic effects, especially when used at one spray each twice a day. Direct comparisons between nasal and oral H1-antihistamines demonstrate that nasal azelastine is effective in patients who do not respond adequately to either oral fexofenadine (LaForce et al. 2004) or loratadine (Berger & White 2003). Direct head-to-head comparisons between intranasal azelastine and oral cetirizine indicate that azelastine has a much faster onset of effects and that patients respond with both reduced nasal symptoms and quality of life (Corren et al. 2005). Thus, intranasal azelastine has some qualities that make it an attractive treatment option in both allergic and nonallergic vasomotor rhinitis.

Intranasal glucocorticosteroids Intranasal glucocorticosteroids are the most efficacious medication available for the treatment of allergic and nonallergic rhinitis (Weiner et al. 1998). The rationale for using intranasal glucocorticosteroids in the treatment of allergic rhinitis is that high drug concentrations can be achieved at receptor sites in the nasal mucosa with a minimal risk of systemic adverse effects. These medications are effective in improving all symptoms of allergic rhinitis as well as some ocular symptoms (Bernstein et al. 2004). If nasal congestion is present or symptoms are frequent, an intranasal glucocorticosteroid is the most appropriate first-line treatment, especially if combind with an intranasal antihistamine. Due to their mechanism of action, efficacy appears 7–8 hours after dosing, but maximum efficacy may require up to 2 weeks. However, the onset of action of intranasal corticosteroids may be shorter than previously thought (Jen et al. 2000; Dykewicz et al. 2003). Fluticasone propionate aqueous nasal spray improves some of the nasal symptoms of seasonal allergic rhinitis when used as needed (Jen et al. 2000; Dykewicz et al. 2003). Patients who require fast onset of relief or who wish to use mediations only as needed will respond more reliably to oral or nasal antihistamines than to nasal steroids. Intranasal glucocorticosteroids are well tolerated, and adverse effects are few in number, mild in severity, and have the same incidence as placebo. The current intranasal preparations are well tolerated and can be used on a long-term basis without atrophy of the mucosa (Holm et al. 1998; Laliberte et al. 2000). Evidence shows that the long-term use of intranasal glucocorticosteroids is free of the concerns associated with the long-term use of oral glucocorticosteroids. Growth has been a concern in children treated with inhaled corticosteroids. The rate of growth was slightly reduced in children regularly treated with intranasal beclomethasone over 1 year (Skoner

1438

Table 69.5 Requirements for intranasal corticosteroids. Pharmacologic properties Potent action on transcription factors First-pass hepatic metabolism Efficacy Effective in the treatment of intermittent and persistent rhinitis as defined in the ARIA document Effective for all nasal symptoms Improvement of eye symptoms If a claim for asthma is proposed: improvement of asthma symptoms (short-term studies) reduction of asthma exacerbations (long-term studies) improvement in pulmonary function tests, even though FEV1 and peak flow rates are usually not altered in pollen-induced bronchial symptoms If a claim for nasal polyposis or sinusitis is proposed, the adequate appropriate trials should be conducted If a claim for a preventive effect is proposed, appropriate trials should be conducted Side effects Minimal local side effects No effects on hypothalamic–pituitary–adrenal axis, especially in children and in association with the inhaled (intrabronchial) form No long-term effect on growth in children Possible use in pregnancy Pharmacodynamics Assessment of the onset of action Long duration of action, at least 24 hours, so can be administered once a day If a claim for p.r.n. use is proposed, additional appropriate trials should be conducted

et al. 2000). However, no growth retardation has been observed in 1-year follow-up studies of children treated with fluticasone propionate (Allen et al. 2002) or mometasone furoate (Schenkel et al. 2000a; Fink et al. 2002; Daley-Yates & Richards 2004). Moreover, a pharmacokinetic/pharmacodynamic model of the relationship between systemic corticosteroid exposure and growth velocity has been proposed and may be useful for the development of future local corticosteroids (Fink et al. 2002; Daley-Yates & Richards 2004). Nonetheless, even the possibility of systemic effects in growing children dictates that nasal steroids be used at the lowest possible dose and that other medications be used to reduce exposure to nasal steroids in growing children. Several properties should be met by intranasal glucocorticosteroids (Table 69.5) (Bousquet et al. 2003d).

Antileukotrienes Several pivotal studies have been carried out on seasonal allergic rhinitis comparing montelukast and placebo. In some studies, the combination of montelukast and loratadine was

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also used (Nayak et al. 2000; Philip et al. 2002; van Adelsberg et al. 2003a,b; Chervinsky et al. 2004). It was consistently found that montelukast was more effective than placebo for all nasal and ocular symptoms and that there was no significant difference between montelukast and loratadine, even for nasal obstruction. Moreover, in contradistinction with the first study (Meltzer et al. 2000), the combination of montelukast and loratadine did not provide any additive beneficial effect over the two drugs alone. Montelukast is equally effective in patients exposed to low and high pollen counts (Chervinsky et al. 2004). In a study carried out on patients with seasonal allergic rhinitis and asthma, montelukast was found to improve nasal and bronchial symptoms (Philip et al. 2004). The use of β agonists was also reduced with montelukast. The combination of montelukast and cetirizine, when started 6 weeks before the pollen season, was effective in preventing symptoms of allergic rhinitis and reduced allergic inflammation in nasal mucosa during natural allergen exposure (Kurowski et al. 2004). In one study of perennial rhinitis, montelukast was found to be superior to placebo (Patel et al. 2005). Overall, it appears that leukotriene receptor antagonists are less effective than intranasal corticosteroids in allergic rhinitis and have an efficacy similar to that of oral H1-antihistamines (Pullerits et al. 2002; Di Lorenzo et al. 2004; Wilson et al. 2004). Montelukast does not modify skin-prick test results (Simons et al. 2001; Hill & Krouse 2003) and does not need to be discontinued before skin testing.

Cromones Cromoglycate and nedocromil are available as intranasal or ocular preparations. They are modestly effective in nasal symptoms and more effective in ocular symptoms. They are particularly safe (Bousquet et al. 2001).

Management and Treatment of Allergic Rhinitis

In many countries, the combination of oral H1-antihistamines and decongestants represents a large market share (Storms et al. 1989; Bertrand et al. 1996; Sussman et al. 1999). The objectives of these combinations are to improve nasal obstruction, which shows little change using oral H1-antihistamines. Since pseudoephedrine is used, the combination has all the side effects of the vasoconstrictor, and food intake may alter pharmacokinetics (Nomeir et al. 1996). There are many overthe-counter drugs combining sedative oral antihistamines with decongestants.

Anticholinergic agents Double-blind placebo-controlled studies have shown that ipratropium bromide is effective in controlling watery nasal discharge, but that it does not affect sneezing or nasal obstruction in perennial allergic and nonallergic (vasomotor) rhinitis (Borum et al. 1979; Mygind & Borum 1989; Wagenmann & Naclerio 1992). Topical side effects, due to the anticholinergic action, are uncommon and obviously dose-dependent in their severity (Bousquet et al. 2001).

Systemic glucocorticosteroids In rare cases, patients with severe symptoms who do not respond to other drugs or those intolerant of intranasal drugs may need to be treated with systemic glucocorticosteroids (Plaut & Valentine 2005). There is a lack of comparative studies on the preferred dose, route of administration, and the dose–response relationship. Glucocorticosteroids can be given orally (e.g., prednisolone, starting dose 20–40 mg/day) or as a depot injection (e.g., methylprednisolone 40–80 mg/injection) (Borum et al. 1987). The long-term use (a few weeks) of oral drugs and intramuscular glucorticosteroids bears the wellrecognized risks of systemic glucorticosteroids. The latter drugs should be avoided.

Decongestants In the treatment of nasal obstruction in both allergic and nonallergic rhinitis, intranasal decongestants are effective in the short term (Johnson & Hricik 1993; Johannssen et al. 1997). However, they do not improve nasal itching, sneezing, or rhinorrhea. Prolonged use (> 10 days) of intranasal vasoconstrictors may lead to tachyphylaxis, rebound swelling of the nasal mucosa, and “drug-induced rhinitis” (rhinitis medicamentosa) (Graf et al. 1995; Graf & Hallen 1996). Oral vasoconstrictors such as ephedrine, phenylephrine, phenylpropanolamine and, especially, pseudoephedrine are the most commonly used oral decongestants (Broms & Malm 1982; Kanfer et al. 1993; Simons et al. 1996). Systemic side effects are not rare with oral drugs and include irritability, dizziness, headache, tremor and insomnia, as well as tachycardia and hypertension (Bousquet et al. 2001). Patients with glaucoma or hyperthyroidism and elderly men with urinary retention due to prostate enlargement are also at risk when using oral sympathomimetic decongestants. Pseudoephedrine was recently banned for Olympic athletes.

Other medications The NSAID ketorolac is modestly effective when used in ophthalmic preparations (Yaylali et al. 2003).

Allergen-specific immunotherapy: therapeutic vaccines for allergic diseases Several guidelines for specific immunotherapy with inhalant allergens have been published. These have endorsed the conclusions of previous guidelines from the World Health Organization (WHO) (Bousquet et al. 1998) and the European Academy of Allergology and Clinical Immunology (EAACI) (Malling et al. 1998) and serves as an update using newly published, randomized, double-blind and placebo-controlled trials.

Subcutaneous immunotherapy The clinical efficacy of subcutaneous immunotherapy is well established for both rhinitis and asthma, and a metaanalysis of its efficacy on asthma is available (Abramson et al. 1999)

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Table 69.6 Indications for subcutaneous immunotherapy. Patients with symptoms induced predominantly by allergen exposure Patients with clinical symptoms due to a single or few allergens Patients with a prolonged season or with symptoms induced by succeeding pollen seasons Patients with rhinitis and symptoms from the lower airways during peak allergen exposure Patients in whom antihistamines and moderate-dose topical glucocorticoids insufficiently control symptoms Patients who do not want to be on constant or long-term pharmacotherapy Patients in whom pharmacotherapy induces undesirable side effects

with a recent update (Abramson et al. 2003). Since the publication of the ARIA workshop report, several studies have confirmed these findings. Clinical efficacy (reduction of symptoms and/or need for medications) has been confirmed with grass (Winther et al. 2000a,b; Leynadier et al. 2001; Walker et al. 2001; Corrigan et al. 2005; Frew et al. 2006), birch (Winther et al. 2000a,b; Rak et al. 2001; Arvidsson et al. 2002; Bodtger et al. 2002), Parietaria (Polosa et al. 2003), mites (Grembiale et al. 2000; Pichler et al. 2001; Pifferi et al. 2002; Varney et al. 2003; Maestrelli et al. 2004), cat (Nanda et al. 2004), and ragweed (Mirone et al. 2004). It should be noted that two of these studies (Mirone et al. 2004; Nanda et al. 2004) clearly demonstrated that the clinical effect is dose-dependent. One study showed that recombinant grass pollen vaccines were effective on rhinitis symptoms (Jutel et al. 2005). Subcutaneous immunotherapy raises contrasting efficacy and safety issues. In many of the recently published studies, systemic side effects were still noticed using standardized extracts or recombinant allergens. Thus, the use of an optimal dose of vaccines labeled either in biological units or in mass of major allergens has been proposed. For most allergen vaccines, the optimal dose of major allergens is 5–20 μg (Bousquet et al. 1998). The indications for this form of immunotherapy are similar to those published by Bousquet et al. (1998) (Table 69.6). Subcutaneous immunotherapy alters the natural course of allergic diseases (Des-Roches et al. 1997; Durham et al. 1999; Pajno et al. 2001) and may prevent the development of asthma in patients with rhinitis (Moller et al. 2002; Niggemann et al. 2006).

this form of therapy has gained little acceptance in the USA. It was suggested to be ineffective, of concern (Frew 2003), or possibly effective but with many unanswered questions (Cox et al. 2006). Wilson et al. (2005) have published a Cochrane Collaboration metaanalysis of SLIT in rhinitis. Overall there was a significant reduction in both symptoms (standardized mean difference (SMD) −0.42, 95% confidence interval −0.69 to −0.15; P = 0.002) and medication requirements (SMD −0.43, 95% confidence interval −0.63 to −0.23; P = 0.00003) following immunotherapy. The Cochrane metaanalysis of Wilson et al. (2005) was followed by several studies which accorded with the results of the review (Pajno et al. 2003a; Bowen et al. 2004; Bufe et al. 2004; Marogna et al. 2004, 2005; Rolinck-Werninghaus et al. 2004a; Smith et al. 2004; Sopo et al. 2004; Tonnel et al. 2004; Dahl et al. 2006a; Durham et al. 2006; Passalacqua et al. 2006a). Moreover, pivotal trials have been carried out very recently and the results on over 600 patients showed convincingly that in grass pollen allergy, SLIT is safe and effective (Dahl et al. 2006b). A recent metaanalysis in children showed that sublingual delivery of allergen vaccination constituted a safe and effective alternative to the injectable route in reducing allergy respiratory symptoms and drug intake (Olaguibel & Alvarez Puebla 2005). In a metaanalysis on SLIT in asthma, 25 studies involving 1706 patients were evaluated (Calamita et al. 2006). Immunotherapy was seen to significantly reduce asthma severity when parameter compositions were all analyzed by categorical outcomes. The safety of SLIT has been demonstrated in adults and children in several papers (Andre et al. 2000; Pajno et al. 2003b), phase I trials (Kleine-Tebbe et al. 2006), and by postmarketing surveillance data (Di Rienzo et al. 1999; Canonica & Passalacqua 2003). SLIT may also impact the natural course of the disease (Di Rienzo et al. 2003; Novembre et al. 2004), but more data are needed to confirm this. The indications for SLIT are given in Table 69.7.

Anti-IgE The recombinant humanized monoclonal anti-IgE antibody (omalizumab) forms complexes with free IgE, blocking its interaction with mast cells and basophils and lowering free IgE levels in the circulation (Holgate et al. 2005). In a large pivotal trial, omalizumab decreased serum-free IgE levels and provided clinical benefit in a dose-dependent fashion in patients with seasonal allergic rhinitis (Casale et al. 2001;

Sublingual immunotherapy

Table 69.7 Indications for high-dose sublingual immunotherapy.

Sublingual immunotherapy (SLIT) is currently marketed in several European countries (Passalacqua et al. 2004) and is also available in other countries (e.g., Argentina, Brazil, Gulf States, South Africa). Most extracts are standardized either biologically or immunologically and for most preparations the microgram content of major allergen(s) is also available. However, SLIT has been controversial for many years and

Carefully selected patients with rhinitis, conjunctivitis and/or asthma caused by pollen and mite allergy Patients insufficiently controlled by conventional pharmacotherapy Patients who have presented with systemic reactions during injection specific immunotherapy Patients showing poor compliance with or refusing injections

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Kaliner 2004). In adults and adolescents, omalizumlab was found to decrease all nasal symptoms and to improve rhinoconjunctivitis quality of life questionnaire (RQLQ) in patients with rhinitis induced by birch and ragweed pollens as well as in those with sensitization to outdoor allergens (Adelroth et al. 2000; Chervinsky et al. 2003). Moreover, the treatment was safe and well tolerated (Berger et al. 2003; Nayak et al. 2003). In patients with asthma and rhinitis, omalizumab improved nasal and bronchial symptoms and reduced unscheduled visits due to asthma (Vignola et al. 2004). The clinical benefit of treatment with omalizumab is associated with an antiinflammatory effect on cellular markers in blood and nasal tissue (Plewako et al. 2002; Bez et al. 2004) as well as with a reduction in mast cell FcεRI expression and function (Beck et al. 2004). Omalizumab inhibits allergen challenge-induced nasal response (Hanf et al. 2004). It also rapidly decreases nasal allergic response and FcεRI on basophils (Lin et al. 2004). The relative efficiency of this treatment compared with H1antihistamines and intranasal glucocorticosteroids needs to be established. Omalizumab pretreatment decreases acute reactions after rush immunotherapy for ragweed-induced seasonal allergic rhinitis (Casale et al. 2006). The coseasonal administration of omalizumab after preseasonal specific immunotherapy decreases ocular and nasal symptom scores and rescue medication in grass pollen-allergic children (Kuehr et al. 2002; Rolinck-Werninghaus et al. 2004b). This combination might prove useful for the treatment of allergic rhinitis, particularly for polysensitized patients.

Management and Treatment of Allergic Rhinitis

substances (Niggemann & Gruber 2003; Bielory 2004; Saper et al. 2004), such as the ephedrine-containing remedies that have been banned in the USA (Anon. 2004b). A mandatory prerequisite for evaluating herbal remedies/mixtures is that the method of preparation, doses, components, and active ingredients should be clearly defined, according to WHO guidelines (1993, 1996). The therapeutic efficacy of CAM is not supported by currently available evidence (Passalacqua et al. 2006b). More data from randomized, double-blind, placebo-controlled trials are required. In addition, CAMs may not be devoid of side effects and some of them may interact with other medications (Niggemann & Gruber 2003; Bielory 2004).

Other treatments Saline douche is a simple and inexpensive treatment which was shown to bear some efficacy (Spector et al. 1982; Taccariello et al. 1999; Ragab et al. 2004; Passali et al. 2005). Physicochemical approaches have been proposed. Rhinophototherapy is effective (Koreck et al. 2005), but more data using simpler equipment are needed. Nasal filters (O’Meara et al. 2005) or pollen blocker creams (Schwetz et al. 2004) during natural exposure to ragweed and grass pollen can reduce nasal symptoms.

Practical guidelines for the treatment of allergic rhinitis and comorbidities Evidence-based treatment Development of guidelines for rhinitis

Complementary and alternative medicine Complementary/alternative medicines (CAM) are extensively used in the treatment of allergic rhinitis and asthma, but evidence-based recommendations are difficult to propose due to methodologic problems in many trials (i.e., not randomized, not controlled, not blinded, and with no quantitative measurement) (Linde et al. 2001a–d; Passalacqua et al. 2006b). CAM is widely practiced and many patients who use this treatment appear to be satisfied. From a scientific viewpoint, there is no definitive or convincing proof of efficacy for most CAMs in rhinitis or asthma. Considering the randomized controlled trials, there is no clear evidence of the efficacy of acupuncture in rhinitis and asthma. Some positive results have been described in rhinitis using homeopathy in good-quality trials, but an equal number of negative studies counterbalance the positive ones. It is therefore impossible to provide evidencebased recommendations for the use of homeopathy in the treatment of allergic rhinitis, and further randomized controlled trials are needed. Some herbal remedies have proved effective in the treatment of rhinitis (Schapowal 2002, 2005), but the studies are too few to make any firm recommendations. There are also safety and drug interaction concerns associated with these remedies. In fact, herbal remedies are not usually sufficiently standardized and can also contain harmful

The 1994 International Consensus for Rhinitis guidelines (International Rhinitis Management Working Group 1994) followed a stepwise approach in the treatment of allergic and nonallergic rhinitis, because this seemed to be the most practical approach for the general practitioner and for the specialist. In 1999, the EAACI proposed new guidelines (van Cauwenberge et al. 2000) and, unlike the 1994 guidelines, not only the mild and moderate cases were considered but also the severe ones. In the ARIA guidelines, the suggestions were made by a panel of experts and were based on an extensive review of the literature available up to December 1999 (Bousquet et al. 2001). Papers for the review were extracted from Medline using PubMed and Embase. A consensus was reached on all the material presented in this position paper. The panel recognized that the suggestions it put forward were valid for the majority of patients within a particular classification but that individual patient responses to a particular treatment may differ from the suggested therapy. It was assumed that a correct diagnosis was achieved before treatment. The statements of evidence for the development of these guidelines has followed WHO rules and are based on Shekelle et al. (1999). The statements of evidence for the different treatment options of allergic rhinitis have been examined by the report panel.

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Availability of the treatment

The ARIA update is ongoing and some papers have been published (Bousquet et al. 2006c; Passalacqua et al. 2006b). It is also evidence-based and the proposals are listed in Table 69.8. Since most trials were carried out before the new classification of allergic rhinitis has been made, Table 69.8 reports seasonal and perennial rhinitis.

The guidelines are made on the presumption that the suggested treatments are available and affordable to the patient. WHO has published a list of essential drugs. It is important that all the drugs which are of importance in the treatment of rhinitis should be available worldwide.

Table 69.8 Level of evidence of different interventions in allergic rhinitis.* Intervention Allergen avoidance House-dust mites Cats, dogs Cockroaches Outdoor allergens Latex H1-antihistamines Oral Intranasal Intraocular Glucocorticosteroids Intranasal Oral Intramuscular Cromones Intranasal Intraocular Naaga intranasal† Decongestant Intranasal Oral Oral decongestant + H1-antihistamine Anticholinergic (intranasal) Antileukotriene Homeopathy Acupuncture Chiropractic medicine Phytotherapy Other alternative medicine Specific immunotherapy Subcutaneous Asthma Rhinitis + conjunctivitis Intranasal‡ Rhinitis + conjunctivitis Sublingual‡ Rhinitis + conjunctivitis Asthma

Seasonal: adults

Seasonal: children

Perennial: adults

Perennial: children

D D D D

D D D D

D D D

D D D

D A A A

A A A

A A

A A

A A* A*

A

A

A

A* A* A*

A* A*

A* A*

D A A

D

D

D

A

A A

A

A A

A

A A

A A

A A

A

A

A

A A

A B

A A

A B

* The strength of recommendation was made according to Shekelle et al. (1999). Adapted from Bousquet et al. (2001); Custovic & Wijk (2005); Bousquet et al. (2006c); Passalacqua et al. (2006b). † Based on a single study. More data are needed. ‡ Recommendation only applied to high-dose vaccine. A, level of evidence 1b with double-blind, placebo-controlled studies; A*, level of evidence 1b with double-blind studies.

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Table 69.9 Level of evidence of different interventions in patients with concomitant rhinitis and asthma. Oral H1-antihistamines are effective in rhinitis Oral H1-antihistamines are poorly effective in asthma Intranasal corticosteroids are inconsistently effective in asthma Intranasal corticosteroids reduce asthma exacerbations Role of intrabronchial corticosteroids in rhinitis is unclear Oral leukotriene receptor antagonists are effective in rhinitis and asthma Allergen-specific immunotherapy is effective in rhinitis and asthma Anti-IgE monoclonal antibody is effective in rhinitis and asthma

The guidelines do not take into account the costs of the treatment. They are made on the presumption that all treatments are readily available and financially affordable to the patient (on health insurance). However, most patients may need to buy drugs, and cost-effectiveness is therefore of importance.

Recommendations for the management of allergic rhinitis Depending on the classification of allergic rhinitis (seasonal and perennial or intermittent and persistent), several algorithmguided therapeutic schemes can be proposed (International Rhinitis Management Working Group 1994; Dykewicz et al. 1998a; van Cauwenberge et al. 2000; Bousquet et al. 2001). However, most guidelines are in general agreement (Plaut & Valentine 2005) (Table 69.9) and usually follow progressive management (Kaliner 2003). It has been shown that in seasonal allergic rhinitis guideline-led treatment is more effective than free treatment choice by general practitioners (Bousquet et al. 2003b). ARIA has proposed the recommendations shown in Fig. 69.3 (Bousquet et al. 2001). Allergen avoidance was proposed as a primary measure but new data no longer favor this recommendation. Pharmacologic treatment based on guidelines (International Rhinitis Management Working Group 1994) is not effective in all patients (Bousquet et al. 2003b). Around 50% of patients with severe symptoms are uncontrolled and around 25% still present severe symptoms, particularly conjunctivitis and nasal obstruction.

Treatment of rhinitis and asthma Although asthma and allergic rhinitis commonly occur together, treatments for one of the conditions could potentially alleviate the coexisting condition (Table 69.9). Medications for asthma and rhinitis can be administered via local (intranasal, intraocular or inhaled (intrabronchial)), oral and parenteral routes. There are advantages as well as some drawbacks when administering the drug directly into the target organ (Bousquet et al. 2001). Moreover, some drugs like cromoglycate or nedocromil are not absorbed when given orally and are only effective when administered locally. In patients suffering from asthma and rhinitis, local administra-

Evidence A Evidence A Evidence A Evidence B Evidence B Evidence A Evidence A Evidence A

tion of drugs requires the use of both nasal and bronchial routes and this may decrease compliance to treatment, which is low in asthma and rhinitis. Glucocorticosteroids are the most effective drugs when used topically in the nose and the bronchi for the treatment of rhinitis and asthma. The intranasal treatment of rhinitis using glucocorticosteroids was found to improve asthma at best moderately in some but not all studies (Taramarcaz & Gibson 2003; Dahl et al. 2005). Symptoms and pulmonary function tests were inconsistently improved. However, a number of aspects, such as the extent to which the pathophysiology of the two diseases overlaps and whether treating one will affect the other, still remain to be clarified. Less is known about the effects on nasal disease of inhaled (intrabronchial) treatment with glucocorticosteroids. One study examined the effects on nasal allergic disease of inhaled budesonide (avoiding nasal deposition of the drug) in patients with seasonal allergic rhinitis but without asthma (Greiff et al. 1998). During the birch pollen season, budesonide reduced the seasonal eosinophilia both in the circulation and in the nose and resulted in an attenuation of seasonal nasal symptoms. However, this study was not confirmed (Dahl et al. 2005). Three studies have shown that treating allergic rhinitis reduces healthcare utilization for comorbid asthma (Adams et al. 2002; Crystal-Peters et al. 2002; Corren et al. 2004). A first retrospective cohort study was carried out on 4944 patients with both allergic rhinitis and asthma, aged 12–60 years, who were continuously enrolled and had no evidence of chronic obstructive pulmonary disease (Crystal-Peters et al. 2002). The risk of an asthma-related event (hospitalizations and emergency department visits) for the treated group was about half that for the untreated group. Another retrospective cohort study was carried out on 13 844 asthmatics (over 5 years of age) of a managed care organization (Adams et al. 2002). Patients who received intranasal corticosteroids had a reduced risk of emergency department visits by comparison to those who did not receive this treatment. The safety of intranasal glucocorticosteroids has been established (Wilson et al. 1998). Large doses of inhaled (intrabronchial) glucocorticosteroids can induce side effects (Lipworth 1999). One of the problems of dual administration includes

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Diagnosis of allergic rhinitis (history ± skin prick tests or serum specific IgE) Allergen avoidance

Intermittent symptoms

Mild

Persistent symptoms

Moderate severe

Mild

Moderate severe Intranasal CS

Not in preferred order Oral H1 blocker Intranasal H1-blocker And/or decongestant

Review the patient after 2–4 wks

Not in preferred order Oral H1 blocker Intranasal H1-blocker And/or decongestant Intranasal CS (chromone)

In persistent rhinitis review the patient after 2–4 wks

Improved

Failure

Step-down and continue treatment for ? 1 month

Review diagnosis review compliance query infections or other causes

If failure: step-up If improved: continue for 1 month

Increase intranasal CS dose

Rhinorrhea add ipratropium Itch/sneeze add H1 blocker

Blockage add decongestant or oral CS (short term)

Failure

Surgical referral If conjunctivitis Add Oral H1-blocker Or intraocular H1-blocker Or intraocular chromone (or saline) Consider specific immunotherapy Fig. 69.3 ARIA recommendations for the stepwise treatment of adolescents and adults. (See CD-ROM for color version.)

the possible additive side effects. In one study, it was found that the addition of intranasal formulations to inhaled formulations did not produce any further significant suppression of mean values but that there were more individual abnormal cortisol values associated with dual therapy (Wilson & Lipworth 1999). However, more data are needed. Drugs administered orally may have an effect on both nasal and bronchial symptoms. Oral H1-antihistamines repres-

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ent first-line treatment of allergic rhinitis. However, although some studies have found some effect on asthma symptoms (Baena-Cagnani 2001), these drugs are not recommended for the treatment of asthma (van Ganse et al. 1997). The association of oral H1-antihistamines and decongestants was found to be more effective on asthma symptoms (Corren et al. 1997). Antileukotrienes are effective for both asthma and rhinitis (Philip et al. 2004). The anti-IgE antibody omalizumab is

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effective in both asthma and rhinitis in the same patients (Vignola et al. 2004). The indications for specific immunotherapy in allergic asthma and rhinitis have been separated in some guidelines (Bousquet et al. 1998). This artificial separation has led to unresolved issues (Barnes 1996; Norman 1996) possibly because the allergen-induced IgE-mediated reaction has not been considered a multiorgan disease. It is therefore important to consider specific immunotherapy based on the allergen sensitization rather than on the disease itself since most patients with allergic asthma also present with rhinitis or rhinoconjunctivitis. It has been shown in the same population that asthma, rhinitis and conjunctivitis are controlled by specific immunotherapy (Bousquet et al. 1987, 1988, 1989). It has been proposed that the prevention or early treatment of allergic rhinitis may help to prevent the occurrence of asthma or the severity of bronchial symptoms, but more data are needed.

Management of rhinitis in developing countries In developing countries, the management of rhinitis is based on medication affordability and availability (Ait-Khaled et al. 2000) and on cultural differences (Enarson & Ait-Khaled 1999). The rationale for treatment choice in developing countries is based on level of efficacy, low drug cost affordable for the majority of patients, and inclusion in the WHO essential list of drugs (only chlorpheniramine and beclomethasone are listed). It is hoped that new drugs will become available on this list. Immunotherapy is not usually recommended in developing countries because many allergens in developing countries are not well identified, specialists must prescribe desensitization, and immunotherapy must be administered by doctors because of possible side effects. A stepwise medical treatment was proposed in the ARIA workshop report (Bousquet et al. 2001) as follows. • Mild intermittent rhinitis: oral or nasal H1-antihistamines. • Moderate/severe intermittent rhinitis: intranasal corticosteroids (equivalent beclomethasone 300–400 μg daily) should be prescribed. If needed, after 1 week of treatment, oral or nasal H1-antihistamines and/or oral corticosteroids should be added. • Mild persistent rhinitis: treatment with oral or nasal H1antihistamines or a low dose of intranasal corticosteroid (equivalent beclomethasone 100–200 μg) should be sufficient. • Moderate/severe persistent rhinitis: a high dose of intranasal corticosteroids (equivalent beclomethasone 300–400 μg) should be prescribed. If symptoms are severe, add oral or nasal H1-antihistamines and/or oral steroids at the beginning of the treatment. Asthma management for developing countries was proposed in the IUATLD Asthma Guide. The affordability of inhaled steroids is usually low in developing countries. If it is affordable for the patient to treat the two manifestations of the disease, it is recommended to add the treatment of allergic rhinitis to the asthma management plan.

Management and Treatment of Allergic Rhinitis

Symptoms of allergic rhinitis

Mild intermittent

Mild persistent Moderate–severe intermittent

Moderate–severe persistent

Oral H1-blocker*/$ Or nasal H1-blocker* Or decongestant* Or nasal cromone* Or nasal saline

Oral H1-blocker*/$ Or nasal H1-blocker* And/or decongestant* Or nasal steroid*/£ Or nasal cromone*

Refer to doctor

If after 7–15 days no improvement *: Depending on drug availability AND not in preferred order $: Nonsedating H1-blockers should be preferred £: If nasal obstruction predominates, intranasal corticosteroids are the first line treatment Fig. 69.4 Management of allergic rhinitis in the pharmacy. (See CD-ROM for color version.)

Over-the-counter management of allergic rhinitis Worldwide, pharmacists receive sophisticated clinical training. Given the well-known and well-publicized recognition of iatrogenic disease, pharmacists’ skills represent an enormous potential resource in maximizing the benefits and minimizing the adverse events associated with pharmacotherapy (Pincus et al. 2002). Pharmaceutical care includes the prevention, treatment or cure of a disease (Dessing 2000). Interest and expectation that pharmacists provide broader “pharmaceutical care” services has therefore increased (Strom & Hennessy 2002). Pharmaceutical care for the patient is likely to be optimal when there is collaboration between pharmacists, patients, and other healthcare professionals, specifically physicians. As trusted healthcare professionals in the community, pharmacists are well placed to identify the symptoms of allergic rhinitis and to recommend appropriate treatment by: • understanding the effect of treatment on rhinitis and comorbidities; • determining whether management in the pharmacy is appropriate (Fig. 69.4); • initiating an appropriate treatment and monitoring plan; • proposing appropriate preventive measures.

Specific considerations Pediatric aspects Allergic rhinitis is part of the “allergic march” during childhood (Kjellman 1994; Wahn et al. 1997) but intermittent allergic rhinitis is unusual before 2 years of age. Allergic rhinitis is most prevalent during school age years. The principles of

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treatment for children are the same as those for adults, but special care has to be taken to avoid the side effects typical in this age group (Passali & Mösges 1999; van Cauwenberge et al. 2000). Doses of medication have to be adjusted and special considerations followed. Few medications have been tested in children under the age of 2 years. Rhinitis in children under 4 years is a difficult problem to treat safely and effectively and few studies have been performed (Fokkens & Scadding 2004). Oral and intramuscular glucocorticosteroids should be avoided in the treatment of rhinitis in young children. Intranasal glucocorticosteroids are an effective treatment for allergic rhinoconjunctivitis, although the possible effects of some but not all intranasal glucocorticosteroids on growth is of concern (Skoner et al. 2000). It has been shown that the recommended doses of intranasal mometasone (Schenkel et al. 2000b) and fluticasone do not affect growth in children with allergic rhinoconjunctivitis. Disodium cromoglycate is commonly used to treat allergic rhinoconjunctivitis in children because of the safety of the drug.

Pregnancy Rhinitis is often a problem during pregnancy since nasal obstruction may be aggravated by pregnancy itself (Ellegard & Karlsson 1999). Caution must be taken when administering any medication during pregnancy, as most drugs cross the placenta (Demoly et al. 2003). For most drugs, limited studies have been performed on only small groups without long-term analysis (Ciprandi et al. 1997; Schatz 1999). Moreover, there are differences in regulations between countries. A study of 53 pregnant women showed no effect of fluticasone propionate on fetal growth or pregnancy outcome (Ellegard et al. 2001). Although safe in pregnant women, it was not very effective for this condition.

Elderly people With aging, various physiologic changes occur in the connective tissue and vasculature of the nose which may predispose or contribute to chronic rhinitis (Edelstein 1996). Some drugs may induce specific side effects in elderly patients (Kaliner 2002). In the elderly, intranasal corticosteroids, at the recommended dose, have not been associated with an increased risk of fractures (Suissa et al. 2004).

Education Education of the patient and/or the patient’s caregiver about the management of rhinitis is essential. Such education is likely to maximize compliance and optimize treatment outcomes (Dykewicz et al. 1998b). However, the benefit of education has never been tested in terms of treatment efficacy, compliance and effectiveness in allergic rhinitis.

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Acknowledgments Jean Bousquet is a member of the GALEN (Global Allergy and Asthma European Network), supported by EU Framework programme for research, contract number FOOD-CT2004–506378.

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Nasal Polyps and Rhinosinusitis Wouter Huvenne, Paul Van Cauwenberge and Claus Bachert

Summary Sinusitis, defined as inflammation of the paranasal sinuses, has a large socioeconomic impact as it is one of the three most common healthcare complaints and causes loss of school or working days in both children and adults. Because sinusitis usually coexists with rhinitis, and isolated sinusitis is rare, the more correct term to use now is “rhinosinusitis,” covering a group of disorders characterized by inflammation of the mucosa of the nose and the paranasal sinuses. The diagnosis is based on symptoms and clinical findings in acute rhinosinusitis, and includes nasal endoscopy and computed tomography (CT) in chronic rhinosinusitis. Because of its high prevalence, interest in the pathophysiology of different forms of sinusitis as the basis for better treatment modalities is still growing. Chronic rhinosinusitis (CRS) is an umbrella term covering different disease entities. Clinical heterogeneity of rhinosinusitis is very likely, pointing toward different hypotheses for respective subgroups of CRS. Whereas CRS without nasal polyps is perceived as a neutrophilic disease, nasal polyps (also referred to as CRS with nasal polyps) are dominated by eosinophilic inflammatory mechanisms and may be accompanied by aspirin sensitivity and asthma. A better differentiation between sinusitis subgroups might be facilitated by investigation of cytokine and growth factor patterns. This may not only help to enlarge our knowledge of the pathophysiology of sinusitis but also lead to new therapeutic options. Recently, the role of bacterial infection in CRS has been challenged, with claims of a modifying role of colonizing Staphylococcus aureus in CRS with nasal polyps. Nasal polyps are more frequently colonized with S. aureus, combined with a local immune response that consists of IgE formation and eosinophilic inflammation, compared with control patients. Interestingly, this phenomenon was only rarely found in CRS without polyps, again separating the two disease entities. Above all, the presence of a local immune response to superantigens, characterized by IgE formation to S. aureus enterotoxins, is strongly associated with asthma in patients with nasal polyps.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Concerning remodeling, further research is needed to unravel whether the specific remodeling patterns in CRS are a consequence of inflammation or are independent processes in chronic sinus disease. Future therapeutic approaches might focus on this remodeling process. We conclude that CRS is clinically heterogeneous and requires different diagnostic and therapeutic tools. Staphylococcus aureus enterotoxins are potent modulators of CRS disease features.

Definition and classification Inflammation of the paranasal sinuses, or sinusitis, is one of the three most common healthcare complaints (Moss & Parsons 1986) and accounts for high socioeconomic costs and lost school or working days in both children and adults. The high prevalence has recently led to increased interest in the pathophysiology of different forms of sinusitis as the basis for better treatment modalities. Because sinusitis usually coexists with rhinitis, and isolated sinusitis is rare, the more correct term to use now is “rhinosinusitis.” Rhinosinusitis is a group of disorders characterized by inflammation of the mucosa of the nose and the paranasal sinuses. The diagnosis is based on symptoms and clinical findings in acute rhinosinusitis, and includes nasal endoscopy and CT in chronic rhinosinusitis (CRS). Acute rhinosinusitis in most cases is of viral origin, but may also be caused by bacterial infections; in these cases, the disease is normally more severe and may lead to complications. Whether recurrent acute rhinosinusitis is a prerequisite for the development of chronic sinus, and would result in persistent obstruction of the ostiomeatal complex, is speculative (Stammberger 1986; Baraniuk 1994; Gwaltney 1994). CRS is an umbrella term, and clinical heterogeneity of rhinosinusitis is very likely, implying different hypotheses for respective subgroups of CRS. Whereas CRS without nasal polyps is perceived as a neutrophilic disease, nasal polyps (CRS with nasal polyps) are dominated by eosinophilic inflammatory mechanisms and may be accompanied by aspirin sensitivity and asthma (Meltzer et al. 2004). Recently, the role of bacterial infection in CRS has been challenged, with claims of a modifying role

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of colonizing Staphylococcus aureus in CRS with nasal polyps. Furthermore, underlying diseases such as cystic fibrosis, immune deficiency, or primary cilia dyskinesia may give rise to sinusitis complaints. Thus, CRS is clinically heterogeneous, and requires different diagnostic and therapeutic tools. Finally, it needs to be understood whether the specific remodeling patterns in CRS are a consequence of inflammation or are independent processes in chronic sinus disease. Table 70.1 summarizes the clinical definition and the definition for research. In the past, research activities mostly focused on functional parameters, microbiology, and morphology of the sinuses. This knowledge led to the development of concepts of sinus physiology, which stressed the importance of the ostiomeatal complex for ventilation and drainage of the sinuses, and was followed by the introduction of sophisticated surgical techniques that took functional and anatomic aspects into

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account (Stammberger 1986). However, failure of treatment or recurrence of disease after restitution of ventilation and drainage still occur, and are obviously linked to underlying inflammatory processes. Recently, an increasing body of knowledge on the role of cytokines, chemokines and adhesion receptors has emerged from studies of different models of nasal inflammation such as viral or allergic rhinitis. It is only within the last few years that studies of similar biological factors were performed in sinusitis and nasal polyposis. The investigation of cytokine and growth factor patterns may help not only to enlarge our knowledge of the pathophysiology of sinusitis and possibly lead to new therapeutic options, but also to differentiate between sinusitis subgroups (Bachert et al. 1998; Van Zele et al. 2006). Furthermore, it will link diseases of the upper airways to those of the lower respiratory tract (Bachert et al. 2006).

Table 70.1 Rhinosinusitis/nasal polyps: clinical definition and definition for research. Clinical definition Inflammation of the nose and the paranasal sinuses characterized by two or more of the following symptoms: Blockage/congestion Discharge (anterior/posterior nasal drip) Facial pain/pressure Reduction or loss of smell and either Endoscopic signs: Polyps Mucopurulent discharge from middle meatus Edema/mucosal obstruction primarily in middle meatus and/or CT changes: Mucosal changes within ostiomeatal complex and/or sinuses Severity of disease The disease can be divided into mild and moderate/severe categries based on total visual analog scale (VAS) score (0–10 cm): Mild, VAS 0–4 Moderate/severe, VAS 5–10 Duration of disease Acute/intermittent: < 12 weeks of symptoms with complete resolution of symptoms Chronic/persistent: > 12 weeks of symptoms with incomplete resolution of symptoms Definition for research Definitions when no earlier sinus surgery has been performed Polyposis: bilateral; endoscopically visualized in middle meatus Chronic rhinosinusitis (CRS): bilateral; no visible polyps in middle meatus, if necessary following decongestant This definition accepts that there is a spectrum of disease in CRS that includes polypoid change in the sinuses and/or middle meatus but excludes those with polypoid disease presenting in the nasal cavity to avoid overlap Definitions when sinus surgery has been performed Once surgery has altered the anatomy of the lateral wall, the presence of polyps is defined as pedunculated lesions as opposed to cobblestoned mucosa > 6 months after surgery on endoscopic examination. Any mucosal disease without overt polyps should be regarded as CRS

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Physiologic /anatomic considerations Basic anatomy Nose and nasal cavity The supporting structure of the nose consists of bone, cartilage and connective tissue. The shape, position and properties of the bone and cartilage of the nose influence considerably the form and aesthetic harmony of the face and the function of the nasal cavity. The interior of the nose is divided by the nasal septum into two cavities, which are usually unequal in size. Each side may be divided into the nasal vestibule and the nasal cavity itself. The nasal vestibule is covered by epidermis containing hairs (vibrissae) and sebaceous glands. The outline of the lateral wall of the nasal cavity is more complex than that of the medial wall. It contains several structures that are important in the function of the nose and nasal cavity. Figure 70.1 shows that the nasal lateral wall comprises three nasal turbinates, the ostia of the nasal sinuses, and the opening of the nasolacrimal duct. The superior, middle and inferior meatus lie inferior to the three turbinates, and the sinuses as well as the nasolacrimal duct open into them. Thus, they are of diagnostic and therapeutic significance. • The inferior meatus, lying between the floor of the nose and the insertion of the lower turbinate, does not contain a sinus ostium, but does have the opening of the nasolacrimal duct lying about 3 cm posterior to the external nasal opening.

FS ST A MT AV

IT NV

ET

B

Crosssection A

Crosssection B

C

Crosssection C

Fig. 70.1 Nasal cavity anatomy – Lateral nasal wall. NV, nasal vestibule; IT, inferior turbinate; MT, middle turbinate; ST, superior turbinate; FS, frontal sinus; ET, eustachian tube; AV, adenoid vegetation.

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• The middle meatus, between the inferior and middle turbinate, is of clinical importance because the nasofrontal duct, the anterior ethmoid cells and the maxillary antrum open into it. • The superior meatus, between the middle and superior turbinate, contains the opening for the posterior ethmoid cells. The sphenoid ostium lies on the anterior wall of the sphenoid sinus at the level of the superior meatus.

Paranasal sinuses The paranasal sinuses are prolongations of the nasal cavity into the neighboring bone of the skull. They are only partially present at birth (maxillary and ethmoidal sinuses), but develop in childhood from the ethmoidal sinuses into otherwise compact bone of the forehead and the sphenoidal region. Their presence and size is therefore dependent on healthy conditions during this period of life, and is very variable. Each sinus is lined with ciliated pseudostratified columnar epithelium with goblet cells that transport mucus into the nasal cavities via rather small ostia. These ostia also guarantee ventilation of the sinuses, which furthermore is dependent on unobstructed airflow in the nasal cavity. The complex and variable anatomic structures of the sinuses and their terminology have repeatedly been discussed and renamed (Stammberger & Kennedy 1995). The ostiomeatal complex, consisting of the maxillary infundibulum (opening), the frontal recess (a cell leading to the frontal sinus), the ethmoidal bulla, and the middle meatus have been identified as key regions for the ventilation and drainage of the maxillary, anterior ethmoidal cells and frontal sinuses. The maxillary sinus is the largest sinus, with an average size of 15 mL. The paired sinuses are often developed asymmetrically, and the resulting different thickness of the bony wall may give rise to incorrect radiologic diagnoses. The sinus usually consists of one chamber only, but it may have niches and indeed may even contain separate loculi. These may cause difficulties in diagnosis and treatment. The ostium of the maxillary sinus lies in the superior part of the medial wall of the sinus and opens into the nose in the middle meatus. This position of the opening does not favor spontaneous emptying of the cavity since it does not lie at the deepest point of the cavity in the upright position, but indeed lies almost at the top of the cavity. The frontal sinus varies in form and extent more than the maxillary sinus. The average frontal sinus has a capacity of 4–7 mL. The difference in size between the right and left cavities is often considerable in the same person. The frontal sinuses may be completely absent on one or both sides in 3–5% of subjects, but they may also be very extensive and demonstrate loculi. The latter favor the development of inflammatory complications. The bony nasofrontal recess runs a curved course to open in the nose under the head of the middle turbinate in the infundibulum of the hiatus semilunaris. The ethmoid labyrinth consists of six to ten air-containing

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cells of a total volume of 2–3 mL. Clinically, anterior and posterior ethmoidal cell groups may be distinguished. There are communications between all the ethmoidal cells of one side and often also common ostia for the anterior ethmoid complex in the middle meatus and for the posterior ethmoid complex in the superior meatus. The sphenoid is the most posterior of the sinuses. It lies in the skull base, bilaterally at the junction of the anterior and middle fossae in the body of the sphenoid bone. There are marked individual variations in shape and size, the capacity of the sinus being 0.5–3 mL. The sphenoid sinuses may be entirely absent in 3–5% of all subjects. The ostium of the sphenoid sinus lies on the anterior wall of the body of the sphenoid bone in the sphenoethmoidal recess behind and somewhat above the superoir turbinate. A series of CT images of a patient without sinusitis is shown in Fig. 70.2. The blood supply of the nasal cavity and the nasal sinuses is provided by both the internal and external carotid artery and their accompanying veins. Nerve supply consists of both sensory and autonomic (secretory and vasomotor) fibers, with a special sensory function for the olfactory nerve. The sensory nerve supply is provided by the first and second branches of the trigeminal nerve. The complex autonomic innervation for secretion and vasomotor supply consists of parasympathetic (secretion and vasodilatation) and sympathetic (vasoconstriction and inhibition of secretion) fibers.

Basic physiology The nose is both a sense organ and a respiratory organ. In addition, the nose performs an important function for the entire body by providing both physical and immunologic protection from the environment. Finally, it is also important in the formation of speech sounds.

Olfaction The human sense of smell is poorly developed compared to most mammals and insects. Despite that, it is still very sensitive in the human and is almost indispensable for the individual. For example, taste is only partially a function of the taste buds since these can only recognize the qualities of sweet, sour, salt, and bitter. All other sensory impressions caused by food such as aroma and bouquet are mediated by olfaction. This gustatory olfaction is due to the fact that the olfactory substances of food or drink pass through the olfactory cleft during expiration while eating or drinking. The sense of smell can stimulate appetite but can also depress it. It also provides warning of rotten or poisonous foods and also of toxic substances, e.g., gas. Olfactory disorders can be classified into quantitative disorders (anosmia, hyposmia, hyperosmia) and qualitative disorders (dysosmia, phantosmia). Presbyosmia is the term sometimes used to designate the age-related impairment of smell perception. Another classification divides olfactory disorders into three general classes (Rombaux et al. 2005):

Nasal Polyps and Rhinosinusitis

Table 70.2 Causes of olfactory disturbance. Drugs and medications Endocrine/metabolic Adrenocortical insufficiency Cushing syndrome Hypothyroidism Pseudohypoparathyroidism Industrial products, dusts, metals, volatile compounds Acetone Benzene Chromium Paint solvents Spices Trichloroethylene Rhinologic disease Inflammatory disease Allergic rhinitis Chronic rhinosinusitis with nasal polyposis Atrophic rhinitis Postviral olfactory loss Posttraumatic olfactory loss Post surgery Intracranial neoplasms Gliomas Olfactory meningioma Intranasal neoplasms Esthesioneuroblastoma Adenocarcinoma Neurologic disease Alzheimer disease Parkinson disease Psychiatric Nutritional, metabolic Cirrhosis of liver Renal insufficiency Gout

• conductive or transport impairment as a result of obstruction of the nasal passage; • sensorineural impairment as a result of damage to the neuroepithelium; • central olfactory neural impairment as a result of central neural damage. Table 70.2 shows the possible causes of olfactory disturbance.

Respiration and protection In the human the only physiologic respiratory pathway is via the nose. Mouth breathing is unphysiologic and is only brought into play in an emergency to supplement nasal respiration. During normal respiration, the inspired air is warmed, moistened and purified during its passage through the nose. The protective function of the nose consists of highly differentiated, efficient and polyvalent resistance mechanisms against environmental influences on the body. A basic

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FS

AE PE

UP

MT

MS

IT

AE PE

SS

Fig. 70.2 A series of CT images of a patient without sinusitis. AE, anterior ethmoid; FS, frontal sinus; IT, inferior turbinate; MS, maxillary sinus; MT, middle turbinate; PE, posterior ethmoid; SS sphenoid sinus; UP, uncinate process.

element of this defensive system is the mucociliary apparatus, i.e., the functional combination of the secretory film and the cilia of the respiratory epithelium by which the colloidal secretory film is transported continuously from the nasal introitus toward the choana. The efficiency of this system

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depends on several factors, such as pH, temperature, condition of the colloids, humidity, width of the nose, and toxic gases. Disturbances of the composition or physical characteristics of the mucosal blanket or ciliary activity can have marked influences on the physiology of the nasal cavity.

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Physiology of the paranasal sinuses The biological purpose of the paranasal sinuses is largely speculative. It is obvious that the pneumatized cavities of the bone of the skull reduce weight, while at the same time increasing the superficial extent of the bones of the skull. The existence of the ostia causes particular pathophysiologic problems affecting ventilation and drainage. Ostial obstruction interrupts the self-cleaning mechanism of the affected sinus and therefore secretions stagnate and change in composition. The retained secretions form an ideal medium for saprophytic bacteria, which are often present in normal sinuses. Acute sinusitis may originate from or be perpetuated by local or systemic factors predisposing to sinus ostial obstruction and infection (Hamilos 2000). These factors include anatomic or inflammatory factors leading to sinus ostial narrowing, disturbances in mucociliary transport, and immune deficiency. Sinus ostial narrowing may be caused by acute viral upper respiratory infection or chronic allergic inflammation. A similar set of factors contributes to sinusitis chronicity, but a key role is dedicated to the host immune–microbial interaction.

Nasal polyposis Epidemiology Nasal polyps represent edematous semi-translucent masses in the nasal and paranasal cavities, mostly originating from the mucosal linings of the sinuses and prolapsing into the nasal cavities. Nasal polyposis represents a great challenge to the otorhinolaryngologist and allergist: etiology and pathophysiology are only partly understood, we lack a valid classification of polyp subgroups to allow prediction of outcome after medical or surgical therapy, and recurrences are frequent regardless of treatment, making repeated surgical interventions necessary. Mucosal wound healing after these surgical interventions may be impaired as a result of ill-defined factors in the inflamed mucosa, leading to unsatisfactory healing and causing complications due to scar formation. Figure 70.3 shows massive nasal polyps in the left nostril. Nasal polyps are linked to comorbidities such as asthma and aspirin sensitivity or may represent part of a systemic disease such as cystic fibrosis. A hallmark of bilateral nasal polyposis in white adults is the abundant number of eosinophils within the tissue, which can be found in about 70–90% of cases. However, polyps also may present as “neutrophilic” in cystic fibrosis, or as adult bilateral polyps in the Asian population.

Incidence and prevalence The prevalence of nasal polyps in the general population is commonly considered to be low (Hosemann et al. 1994). Valid epidemiologic studies are lacking, and the fact that an endoscopic examination would be necessary for diagnosis

Fig. 70.3 Massive nasal polyps in the left nostril. (See CD-ROM for color version.)

further complicates such approaches. A postal questionnaire survey of a population-based random sample of 4300 adult women and men aged 18–65 years was recently performed in southern Finland (Hedman et al. 1999). The prevalence of nasal polyposis was 4.3%, and nasal polyposis and aspirin sensitivity were associated with an increased risk of asthma. The prevalence of doctor-diagnosed aspirin sensitivity was 5.7%. The incidence is higher in men than in women and significantly increases after the age of 40 years (Moloney 1977). Nasal polyps are associated with asthma, aspirin sensitivity, and cystic fibrosis (Settipane 1999). Asthma is frequently found in nasal polyp patients; in particular, nonallergic asthma is significantly more frequently linked to polyps compared with allergic asthma. Aspirin sensitivity is found in 15% of polyp patients, and 40–80% of all cases of aspirinsensitive patients suffer from polyps. In a large series of patients with cystic fibrosis, nasal polyposis occurred in 37–48% (di Sant’agnese & Davis 1979; Hadfield et al. 2000a). When polyps occur in children and adolescents, cystic fibrosis should always be considered. Other conditions associated with nasal polyps are Churg–Strauss syndrome and Kartagener syndrome. Table 70.3 shows the prevalence of nasal polyposis in population subgroups (Settipane 1996).

Genetics An interesting observation is that nasal polyps are frequently found to run in families, suggestive of a hereditary or shared environmental factor. Rugina et al. (2002) found that 52% of 224 patients with nasal polyps had a positive family history of nasal polyposis. The presence of nasal polyps was considered when nasal polyposis had been diagnosed by an ENT practitioner or the patients had undergone sinus surgery for nasal polyposis. A lower percentage (14%) of familial occurrence of nasal polyposis was reported earlier by Greisner and

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Table 70.3 Prevalence of nasal polyposis in population subgroups. Aspirin hypersensitivity Adult asthma IgE-mediated Non-IgE-mediated Chronic sinusitis IgE-mediated Non-IgE-mediated Childhood asthma/sinusitis Cystic fibrosis Children Adults Allergic fungal sinusitis

36–72% 7% 5% 13% 2% 1% 5% 0.1% 10% 50% 66–100%

Settipane (1996) in smaller group (50) of adult patients with nasal polyposis. Thus, these results strongly suggest the existence of a hereditary factor in the pathogenesis of nasal polyposis. In this regard, recent genetic studies have found a significant correlation between certain human leukocyte antigen (HLA) alleles and nasal polyps. Luxenberger et al. (2000) reported an association between HLA-A74 and nasal polyps, whereas Molnar-Gabor et al. (2000) reported that subjects carrying HLA-DR7-DQA1*0201 and HLA-DR7DQB1*0202 haplotypes had a two to three times increased odds ratio of developing nasal polyps.

Mechanisms The eosinophilic mediators in nasal polyp tissue have been the subject of many studies, demonstrating that different cell types generate these mediators. Denburg et al. (1987) and Xaubet et al. (1994) postulated that differentiated progenitor cells stimulated by soluble hemopoietic factors derived from mucosal cell populations may at least partly cause the eosinophilic accumulation in nasal polyps. Increased synthesis of granulocyte–macrophage colony-stimulating factor (GM-CSF) by epithelial cells, fibroblasts, monocytes, and eosinophils was later suggested (Ohno et al. 1991; Hamilos et al. 1993; Mullol et al. 1995). Different pathomechanisms have been suggested for allergic versus nonallergic polyps by Hamilos et al. (1995). Using in situ hybridization, they found tissue densities of GM-CSF, interleukin (IL)-3, IL-4, and IL-5 transcripts to be higher in patients with “allergic” polyps than in controls, whereas patients with nonallergic polyps had higher tissue densities of GM-CSF, IL-3, and interferon (IFN)-γ transcripts. However, our studies involving protein measurements in tissue homogenates could not support these findings (Bachert et al. 1997, 1998). In contrast, IL-3 and GM-CSF protein was found in only a small number of polyp and control turbinate samples, whereas IL-5 was found to be significantly increased in nasal

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polyps compared with healthy controls. Moreover, the concentration of IL-5 was independent of the atopic status of the patient and the highest concentrations of IL-5 were actually found in subjects with nonallergic asthma and aspirin sensitivity. Furthermore, eosinophils were positively stained for IL-5, suggesting a possible autocrine role for this cytokine in the activation of eosinophils, and a strong correlation between concentrations of IL-5 protein and eosinophilic cationic protein (ECP) was later demonstrated (Bachert et al. 2001). The key role of IL-5 was supported by the finding that treatment of eosinophil-infiltrated polyp tissue with neutralizing antiIL-5 monoclonal antibody, but not anti-IL-3 or anti-GM-CSF monoclonal antibodies in vitro, resulted in eosinophil apoptosis and decreased tissue eosinophilia (Simon et al. 1997). Summarizing, these data suggest that the atopic status of the patients is detached from the increased production of IL5, which likely influences the predominance and activation of eosinophils in nasal polyps. Several other studies meanwhile have supported the lack of difference in the amounts of cytokines detected in polyps from allergic or nonallergic patients (Min et al. 1997; Lee et al. 1998). Furthermore, Wagenmann et al. (2000) demonstrated that both Th1- and Th2-type cytokines were upregulated on a cellular level in eosinophilic nasal polyposis, irrespective of allergen skin test results. Eosinophil chemoattraction was showed by Bartels et al. (1997), who demonstrated that expression of eotaxin and RANTES mRNA, but not monocyte chemotactic protein (MCP)-3 mRNA, was elevated in nonatopic and atopic nasal polyps, when compared with normal nasal mucosa. Jahnsen et al. (1999) demonstrated similar results, with increased mRNA expression for eotaxin, eotaxin-2, and MCP-4. The expression of eotaxin-2, another CCR3-specific chemokine, was found to be the most prominent of the three chemokines investigated. Eotaxin, rather than RANTES, in cooperation with IL-5 plays a central role in chemoattraction and activation of eosinophils in nasal polyp tissue according to our data (Bachert et al. 1997, 2000, 2001). These findings are supported by an extensive study of about 950 nonallergic or allergic polyp patients, which has also suggested that nasal polyp eosinophilic infiltration and activation may correlate mainly with increased eotaxin gene expression, rather than with RANTES expression (Shin et al. 2000). Studies of cell adhesion molecules are relatively few. Early studies by Symon et al. (1994) demonstrated that intercellular adhesion molecule (ICAM)-1, E-selectin and P-selectin were well expressed by nasal polyp endothelium, whereas vascular cell adhesion molecule (VCAM)-1 expression was weak or absent. However, an elegant study by Jahnsen et al. (1995), employing three-colour immunofluorescence staining, has demonstrated that both the number of eosinophils and the proportion of vessels positive for VCAM-1 are significantly increased in nasal polyps compared with the turbinate mucosa of the same patients. Moreover, treatment with top-

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Table 70.4 Influence of glucocorticosteroids (GCS) on nasal polyp (NP) tissue.

IL-5 (pg/mL) Eotaxin (pg/mL) ECP (mg/L) TGF-b1(pg/mL) Albumin (g/L)

Controls

vs.

Untreated NP

vs.

Oral GCS NP

vs. controls

Not detectable 78.0 666.5 9952 8.576

P = 0.008 P = 0.003 P < 0.001 P < 0.001

159.4 965.9 5134.8 2585 19.646

NS P = 0.036 NS P = 0.02

1 of 5 detectable 403.2 1035.4 5203 12.477

NS NS NS NS

ECP, eosinophil cationic protein; TGF, transforming growth factor; NS, not significant.

ical glucocorticosteroids decreases the density of eosinophils and the expression of VCAM-1 in polyps (Tingsgaard et al. 1999). The interaction between very late antigen (VLA)-4 on eosinophils and VCAM-1 on endothelial cells may not only be of particular importance for transendothelial migration of eosinophils, but may also modify their activation and effector functions (Palframan et al. 1998). Eosinophils, the hallmark of nasal polyp disease, predominantly express transforming growth factor (TGF)-β1 and TGF-β2. Several studies report on their putative effects on fibroblast activity and the pathogenesis of nasal polyps (Elovic et al. 1994; Coste et al. 1998). Again looking at protein levels in tissue homogenates of polyp patients, who were either untreated or treated with oral corticosteroid, and control subjects, untreated polyp samples and controls showed significantly higher concentrations of IL-5, eotaxin, ECP and albumin and significantly lower concentrations of TGF-β1. In contrast, corticosteroid treatment significantly reduced IL-5, ECP and albumin concentrations, whereas TGF-β1 was increased (Bachert et al. 2000). Table 70.4 shows the influence of glucocorticosteroids on nasal polyps. These observations suggest that IL-5 and TGF-β1 represent cytokines with counteracting activities, with a low TGF-β protein concentration in IL-5-driven nasal polyps. Furthermore, polyp formation due to the deposition of albumin and other plasma proteins and regulated by subepithelial eosinophilic inflammation is supported by these observations, possibly pointing toward a pathogenic principle of polyp formation. TGF-β1 is a potent fibrogenic cytokine that stimulates extracellular matrix formation, acts as a chemoattractant for fibroblasts, but which inhibits the synthesis of IL-5 and abrogates the survival-prolonging effect of hematopoietins (IL-5 and GM-CSF) on eosinophils (Alam et al. 1994). Staining of nasal polyp tissue shows that TGF-β1 is mainly bound to the extracellular matrix, where it is found in its latent inactive form.

Etiology and pathogenesis Nasal polyps and asthma Lower respiratory tract disorders such as asthma and nonspecific bronchial hyperreactivity are frequently associated

with nasal polyposis (Lamblin et al. 1997), as up to 70% of patients with nasal polyposis suffer from asthma (Settipane et al. 1982; Drake-Lee et al. 1984; Larsen 1996). This link between upper and lower airway diseases is reflected in several findings: first of all there is evidence of bronchial hyperreactivity in patients with nasal polyposis, but no history of asthma, especially in nonatopic patients (Kordash et al. 1978; Miles-Lawrence & Kaplan 1982). In line with these findings, a long-term follow-up study confirmed that the incidence of subsequent clinically significant bronchial asthma was much higher than in the general population (Kordash et al. 1978). This leads to a classification into two groups of polyps: those with current or future lower airway disease features and those without. This finding is supported by the fact that patients with nasal polyposis and asymptomatic bronchial hyperreactivity have an eosinophilic bronchial inflammation similar to that observed in asthmatic patients with nasal polyposis, whereas patients with nasal polyposis without bronchial hyperreactivity do not have eosinophilic lower airways inflammation. The link between upper and lower airways is again reflected in the fact that medical or surgical treatment of nasal polyposis may impact on the control of asthma in those subjects. A study involving 205 patients with asthma and aspirin sensitivity indicated that surgery improves asthma for relatively long periods of time (English 1986). However, there are no data concerning the evolution of asymptomatic bronchial hyperreactivity in patients with nasal polyposis, and individual patients may develop asthma symptoms after surgery. This development may represent the natural course of the disease rather than a true shift from upper to lower airways disease.

Nasal polyps and allergy Although elevated total IgE was found in polyp fluid, there was no difference between polyps from allergic and nonallergic subjects (Drake-Lee & McLaughlan 1982). However, it was noted that total IgE was higher in polyp fluid than in the corresponding serum in both allergic and nonallergic polyp subjects. Local IgE production could also be demonstrated in nasal polyps associated with negative skin tests and serum

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RAST (Small et al. 1985). It was recently demonstrated that skin-prick tests do not predict total IgE levels in polyp homogenates (Bachert et al. 2001). In contrast, high total IgE concentrations locally are most likely due to local production of Staphylococcus aureus enterotoxins (SAEs), acting as superantigens and inducing polyclonal IgE formation. The major features of nasal polyps point toward an allergic etiology: clinical symptoms similar to allergic rhinitis, the association with late-onset asthma and elevated local IgE in polyp fluid as well as a pronounced tissue eosinophilia. However, polyps were found in only 0.5% of 3000 consecutive atopic patients examined by Caplin et al. (1971). Even today, an increased risk for allergic subjects to develop nasal polyps could not be demonstrated. In contrast, in a retrospective study by Settipane and Chafee (1977), polyps were present in 2.8% of atopic patients, but 5.2% of nonatopic subjects. In another study, there was a positive association between the blood eosinophil count and the presence of asthma with the number of polypectomies, but not with positive skin tests for different allergens (Wong et al. 1992). Seasonal allergen exposure in patients with nasal polyps also did not enhance symptoms or markers of eosinophilic inflammation such as eosinophil percentage or ECP concentration in nasal secretions (Keith et al. 1994).

Aspirin sensitivity (or aspirin-exacerbated respiratory disease) The symptom triad of aspirin sensitivity, steroid-dependent asthma, and nasal polyposis (rhinosinusitis) described by Widal in 1922 was made known by Samter and Beers in 1968. Aspirin-sensitive rhinosinusitis (ASRS) is characterized by increased eosinophils in the nasal and bronchial mucosa, and elevated cysteinyl leukotriene concentrations in the tissue and urine, which further increases after aspirin exposure (Kowalski et al. 1996; Szczeklik & Stevenson 2003). Initially, the symptoms mostly develop after a prolonged common cold episode in the third or fourth decade of life with nasal congestion, rhinorrhea, post-nasal drip, and hyposmia, based on persistent mucosal inflammation. Within a few years, nasal polyposis and bronchial asthma develop, until aspirin sensitivity is suspected due to a typical respiratory reaction and eventually is diagnosed by oral provocation test (Szczeklik et al. 2000). Asthma and rhinitis attacks are caused by ingestion of aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) that share the ability to inhibit cyclooxygenase (COX-1 and COX-2). About 15% of patients with aspirininducible asthma and rhinitis are unaware of aspirin sensitivity, indicating that aspirin challenge is necessary to fully diagnose the disease. About 50% of patients need systemic steroid treatment on top of inhaled corticosteroids, emphasizing the severity of the disease in the upper and lower respiratory tract. Interestingly, the course of disease is independent from aspirin intake, indicating that the disease is driven by so far unknown agents and, with few exceptions,

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aspirin sensitivity remains lifelong. No validated laboratory test is available, and the diagnosis is based on oral, bronchial or nasal provocation tests (Schapowal et al. 1995). Although ASRS is often associated with allergy and highly elevated local IgE levels (Bachert et al. 2001), an IgEmediated mechanism has not been demonstrated, and atopy does not seem to influence the risk of developing aspirin sensitivity (Szczeklik et al. 2000). However, alterations in arachidonic acid metabolism, resulting in an alteration of the cellular response to aspirin, have been suggested (Szczeklik & Stevenson 2003). Blockade of COX-2 reduces asthma symptoms and cysteinyl leukotriene release, in contrast to COX-1 inhibition which precipitates asthma attacks (Szczeklik et al. 2001). Additionally, it has been found that COX-2 expression is downregulated in nasal mucosa of aspirin-intolerant patients. COX-2 mRNA expression is regulated by cytokines, which activate the nuclear factor (NF)-κB transcription factor. Further studies are needed to explain whether an alteration in NF-κB and/or other regulatory mechanism is responsible for the abnormal expression of COX-2 mRNA in patients with aspirin-exacerbated respiratory disease (Picado et al. 1999). LTC4 synthase has been demonstrated to be overexpressed in eosinophils and mast cells (Cowburn et al. 1998), resulting in overproduction of cysteinyl leukotrienes, which may be released into the airways after aspirin challenge, causing typical symptoms (nasal congestion, rhinorrhea, bronchoconstriction), as well as constantly inducing eosinophilia. In a subgroup of nasal polyp patients with the clinical history of asthma and aspirin sensitivity, a marked tissue eosinophilia, increased IL-5 and eotaxin expression, as well as LTC4/LTE4 overproduction have been linked to an immune reaction to SAEs, also inducing a local multiclonal IgE response (Bachert et al. 2001). However, the mechanisms of the direct effect of S. aureus on arachidonic metabolism still need to be defined. It seems that aspirin sensitivity and immune reactions to SAEs are independently related to eosinophilic inflammation (Perez-Novo et al. 2004). Conservative treatment possibilities consist of (i) avoidance of aspirin and other NSAIDs, which does prevent exacerbations but does not prevent progression of disease; (ii) oral and/or topical glucocorticosteroids; (iii) eventually leukotriene receptor antagonists or synthesis inhibitors; and (iv) in selected cases, aspirin desensitization. To prevent exacerbations, the ingestion of aspirin and COX-inhibiting NSAIDs has to be avoided, while acetaminophen, nimesulide (dose-dependently) and selective COX-2 inhibitors (celecoxib, rofecoxib) may be tolerated (Szczeklik & Stevenson 2003). Although systemic steroids have been proven effective, but may cause side effects in long-term usage, antileukotriene drugs deserve further trials to find their place in the treatment regimen. Aspirin desensitization consists of administering incremental oral doses, to reach a maintenance dose of > 650 mg daily, inducing a refractory period of a few days. Continuous treatment over years may lead to a significant

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reduction in numbers of sinus infections per year, hospitalizations for treatment of asthma per year, improvement in olfaction, and reduction in use of systemic corticosteroids (Stevenson et al. 1996). Furthermore, the number of sinus operations per year was significantly reduced. However, due to gastrointestinal side effects of aspirin and a relapse of risk in case of noncompliance, this therapy is not widely accepted. Furthermore, aspirin desensitization does not seem to change the long-term course of the disease. Sinus surgery is indicated in patients resistant to conservative therapy; however, single cases have shown that early surgical intervention may alter the course of disease. A retrospective study of 80 patients with an average follow-up of 3 years demonstrated a significant improvement in sinusitis symptoms, relief of asthma complaints, and need for medication in more than 80% of subjects (McFadden et al. 1997). However, there was a significant incidence of revision surgery, suggesting a conservative surgical approach and close longterm follow-up with intense medical management.

Cystic fibrosis As mentioned above, nasal polyps represent a chronic form of upper airway inflammation, occurring in a considerable part of the general population and linked to comorbidities such as asthma and aspirin sensitivity, at least in adults. However, in children suffering from nasal polyp formation systemic diseases such as cystic fibrosis have to be considered. The vast majority (about 80%) of nasal polyps in adults are characterized by a predominant infiltration of eosinophils. In contrast, nasal polyps in patients with cystic fibrosis (CF) are marked by an apparent neutrophilic inflammation. In addition, nasal polyps in CF and in those without CF differ not only in terms of inflammatory cell and cytokine patterns, but also in terms of expression of innate markers (Claeys et al. 2005). Nasal polyps in CF are associated with the upregulation of both human β defensin 2 (HBD2) and Toll-like receptor (TLR)2, while expression of the macrophage mannose receptor (MMR) dominates the innate defense in non-CF nasal polyps. These apparent differences point toward a variable inflammatory background in nasal polyps in CF and non-CF. CF (McFadden et al. 1997) is the most common fatal inherited disease among white people, affecting approximately 1 in 2000 live births. The basic metabolic derangement is related to a mutation in the gene regulating chloride transport in epithelial cells (Yamaya et al. 1991). The position of the apical membrane-bound protein, called cystic fibrosis transmembrane conductance regulator (CFTR), is affected leading to a decrease in apical chloride transport, thickening of mucous secretions, and impaired mucociliary clearance. Although bacterial infection is widely accepted to be a major factor in the pathogenesis of acute exacerbations and chronic progression of lung disease in CF, it remains unclear if the CF-specific sinonasal pathogens, of which S. aureus, Pseudomonas

Nasal Polyps and Rhinosinusitis

aeruginosa, Haemophilus influenzae and anaerobes are the most common, play a particular role in the pathogenesis of nasal polyps in CF. Bacterial infection is present in 95% of all cases, the predominant organisms being Pseudomonas aeruginosa, S. aureus, Haemophilus influenzae and anaerobes. Unfortunately, the response to antimicrobial therapy often is suboptimal when compared with non-CF patients. Tobramycin, given once daily as nasal irrigation, was shown to reduce the growth of Pseudomonas organisms (Davidson et al. 1995). In a recent prospective, randomized, double-blind trial, reductions in polyp size in CF have been demonstrated when nasal corticosteroids were used (Hadfield et al. 2000b). Chronic lung disease and pancreatic insufficiency dominate the clinical picture and determine mortality. Involvement of the nose and sinuses commonly occurs in patients with CF, although sinus problems very rarely contribute to mortality. Sinusitis is almost always detected in radiologic investigations of CF patients, even if many of these patients have no sinonasal complaints (Isaacson & Yanagisawa 1998). The incidence of nasal polyps in CF varies from 6 to 48% in different studies (Cepero et al. 1987), being found in children of 5 years of age and older. A recent study of 211 CF adults reported that 37% of subjects suffered from nasal polyps (Hadfield et al. 2000a). Half of the children between 4 and 16 years of age presenting with nasal polyps have CF (Schramm & Effron 1980). The sweat chloride test (increased electrolytes) remains the simplest and the most reliable laboratory procedure for the screening and diagnosis of CF. Because of the ubiquitous and persistent nature of the disease and the often transient effect of surgery (Batsakis & El-Naggar 1996), sinus surgery should only be performed in cases of sufficient symptoms or before lung transplantation. The development of functional endoscopic sinus surgery has decreased the morbidity of sinus surgery and reduced the recurrence of nasal polyposis in CF (Jones et al. 1993; Coste et al. 1997; Gysin et al. 2000). A careful postoperative followup is mandatory, but often difficult in young patients. Nasal irrigations with saline solution may help to clean the cavities after surgery.

Fungal disease Fungi have been increasingly recognized as important pathogens in sinusitis. Fungal infection, mainly by molds, can impose a severe acute and chronic sinusitis in the immunocompromised host. In contrast, fungi are regarded as frequent innocent bystanders when cultured from the respiratory tract of immunocompetent hosts (Uffredi et al. 2003). Fungal sinusitis can be divided into four primary categories (Morpeth et al. 1996): acute/fulminant (invasive), chronic/indolent (invasive), fungus ball, and allergic fungal sinusitis. Of these, allergic fungal sinusitis is the most common form. The fungus ball, also referred to as mycetoma, is mostly a unilateral symptomatic or asymptomatic chronic maxillary

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sinusitis with little mucosal tissue reaction in an immunocompetent host. CT and magnetic resonance imaging (MRI) findings may be suggestive (Zinreich et al. 1988), showing heterogeneous opacification, calcification with increased attenuation in CT, and hypointense signal characteristics on T2-weighted MRI due to the presence of calcium in the fungal concretion. Within the sinus, creamy or clay-like secretions are typically found. A possible dentogenic pathway has to be excluded, and sinus surgery represents the treatment of choice. Fungi associated with the development of allergic fungal rhinosinusitis (AFS) are ubiquitous and predominantly of the dematiaceous family (Aspergillus, Rhizopus, Alternaria, Curvularia, Bipolaris specifera and others) (Marple 2001). However, exposure alone to these fungi appears to be insufficient to initiate the disease. AFS mostly develops in atopic young hosts, with the immunologic reaction to the presence of fungi giving rise to the development of nasal polyps, and positive fungal-specific immediate hypersensitivity skin tests to the fungus may be found with an elevated serum total IgE and fungal-specific IgG (Schubert & Goetz 1998a; Manning & Holman 1998). The sinus mucosa shows a characteristic eosinophilic inflammation, with allergic mucin filling the sinuses (thick gluey brownish eosinophilic mucus with fungal hyphae and Charcot–Leyden crystals) (Katzenstein et al. 1983). Extracellular major basic protein (MBP) as well as neutrophil elastase is found in high quantities in the mucin, indicating that eosinophil and neutrophil activation occurs in the disease (Khan et al. 2000). AFS is unilateral in more than 50% of the cases, but may involve several sinuses bilaterally, and bone erosion and extra-sinus extension have been reported. About half of the patients may also suffer from asthma (Bent & Kuhn 1994). Interestingly, there is geographic variation, with a higher incidence in southern American practices compared to northern locations (Ferguson et al. 2000). Both surgical intervention and the use of systemic and long-term topical steroids are recommended in the treatment of allergic fungal sinusitis (Kuhn & Javer 2000; Schubert 2000). Monitoring of total serum IgE can be helpful in the clinical follow-up of these patients, as an increase in total serum IgE was found to have significant predictive value for recurrent surgical intervention (Schubert & Goetz 1998b). Invasive forms have to be differentiated based on presentation, either as indolent, chronic, slowly destructive disease, which is endemic in Sudan and mostly caused by Aspergillus flavus, or as the afulminant, acute, necrotizing form in immunocompromised hosts (e.g., AIDS < 50 CD4 cells/mL, or neutrophils < 1000/mL), mostly caused by Aspergillus fumigatus and often lethal within days because of hematogenous dissemination. Although fungi have long been specified as the main etiologic agent of different forms of CRS in immunocompetent patients (Ponikau et al. 1999), fungal elements are nowa-

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days regarded as innocent bystanders. The concept of fungal involvement in CRS should be that ubiquitous airborne fungi become entrapped in sinonasal mucus, are attacked by eosinophils, and cause, via the release of toxic granules from eosinophils, secondary mucosal inflammation in susceptible individuals. If true, fungal eradication using intranasal antifungals should improve the course of the disease. However, in a double-blind, placebo-controlled, multicenter trial no additional benefit of amphotericin nasal lavages over intranasal steroids and irrigation in patients with CRS with or without nasal polyps with a previous history of endoscopic sinus surgery was shown (Ebbens et al. 2006). These results confirm the concept of extramucosal fungi being innocent bystanders in the upper respiratory tract and playing no demonstrable role in the pathophysiology of CRS in immunocompetent patients. In another study patients with CRS and extensive nasal polyps were recruited to allow recognition of medicationrelated improvement. The authors concluded that neither 8 weeks of topical treatment with amphotericin nor the fungal state before and after treatment had any significant effect on nasal cell activation markers in CRS with nasal polyps (Weschta et al. 2006). Using terbinafine as antifungal therapy in patients with CRS, no improvement of the symptoms or radiographic appearance of CRS was seen, even when nasal irrigation samples were positive for fungus on culture (Kennedy et al. 2005).

Role of Staphylococcus aureus enterotoxins Early studies have shown that tissue IgE concentrations and the number of IgE-positive cells may be raised in nasal polyps, suggesting the possibility of local IgE production (Donovan et al. 1970). This was confirmed by a study demonstrating that concentrations of IL-5, eotaxin, LTC4/LTD4/ LTE4, sCD23, ECP, and total IgE were significantly higher in polyp tissue compared with nonpolyp tissue. Furthermore, IL-5, eotaxin, LTC4/LTD4/LTE4, sCD23, ECP and tissue eosinophils were significantly correlated with tissue total IgE. A detailed analysis of IgE indicated for the first time that specific IgE to SAEs was present in the tissues and that this was associated with severe local eosinophilic inflammation (Bachert et al. 2001). Furthermore, polyclonal specific IgE formation to inhalant allergens, high levels of tissue total IgE, and a high prevalence of asthma and aspirin sensitivity were demonstrated in polyps with specific IgE to SAEs. These studies indicate that SAEs possibly act as superantigens, through unconventional interaction with the T-cell receptor, as has been shown for atopic dermatitis (Leung 1999). These superantigens are potent activators of T cells, induce the synthesis of IgE in B cells, and have direct effects on proinflammatory cells, such as eosinophils. Moreover, SAE IgE antibodies have also been described in the sera of patients with asthma, and are linked to severity of disease and steroid insensitivity. Therefore, SAEs may be important in the pathogenesis of

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Epithelial damage (barrier dysfunction) Chronic microbial trigger

Massive polyclonal lymphocyte activation B Hyper IgE ↑

T Cytokines ↑

Polyclonal IgE

Albumin Superantigens

Eosinophils ↑ (↓ apoptosis)

IL-5

Chemokines Eotaxin

ECP

Fig. 70.4 Impact of Staphylococcus aureus superantigens on the immune reaction in nasal polyps. (See CD-ROM for color version.)

nasal polyps due to their potential role as disease modifiers. The impact of S. aureus superantigens on the immune reaction in nasal polyps is shown in Fig. 70.4. Recently, we found increased colonization of nasal polyps with S. aureus, combined with a local immune response that consists of IgE formation and eosinophilic inflammation, compared with control patients (Van Zele et al. 2004). Interestingly, this phenomenon was only rarely found in CRS without polyps, separating the two disease entities. In subjects with nasal polyps and comorbid asthma or aspirin sensitivity, rates of colonization and IgE response in nasal tissue homogenates were further increased, paralleled by increases in ECP and total IgE. Above all, the presence of a local immune response to superantigens, characterized by IgE formation to SAEs, is strongly associated with asthma in patients with nasal polyps. When comparing these findings with nasal polyps in subjects from southern China, again an upregulation of IgE production is found, being related to atopy and the impact of SAEs (Zhang et al. 2006). In contrast, a less severe eosinophilic inflammatory pattern is noted, compared with polyps from white subjects. TGF-β1, an important antiinflammatory and profibrotic factor, was significantly downregulated in polyps from Chinese subjects compared with control tissues, and within the polyp group TGF-β1 was especially low in the subgroup with IgE antibodies to SAEs. These data confirm that immune responses to SAEs can be found in polyp disease in China and may elicit immune modulatory activities. The involvement of bacterial superantigens like SAEs in the regulation of inflammatory pathways such as the arachidonic acid cascade has been suggested in the past. Incubation of permeabilized human polymorphonuclear granulocytes with staphylococcal toxic shock syndrome toxin (TSST)-1 and streptococcal erythrogenic toxin A resulted in the upregulation of signal transduction pathways (release of LTB4) in these cells, affecting immune-modulatory functions (Hensler et al. 1993). From previous research from our group we have suggested that the presence of an S. aureus-induced immune

Nasal Polyps and Rhinosinusitis

response may increase eosinophilic inflammation in nasal polyp patients, and that the finding of IgE antibodies to SAEs was significantly increased in aspirin-sensitive subjects (PerezNovo et al. 2004; Van Zele et al. 2004). We also showed that upregulation of the arachidonic aced cascade runs parallel with the severity of inflammation and disease (Perez-Novo et al. 2005). Starting from these findings we recently demonstrated that tissue leukotriene and lipoxin synthesis is significantly upregulated in nasal polyp patients with specific IgE to SAEs (Perez-Novo et al. 2006). In addition, this increase of eicosanoids appears to be correlated to the inflammatory reaction derived by the immune response to superantigens. These findings implicate a possible impact of S. aureus infection or colonization in the regulation of inflammatory mechanisms like the eicosanoid pathway. In conclusion, the above findings suggest that eradication of S. aureus colonization in the nose may provide an effective means for the management of polyps at least in a subgroup of patients. A first antibiotic DBPCR trial in nasal polyps has confirmed this notion (Bachert et al. unpublished data).

Pathology Histomorphologic characterization of polyp tissue reveals frequent epithelial damage, a thickened basement membrane, and edematous to sometimes fibrotic stromal tissue, with a reduced number of vessels and glands but virtually no neural structure (Taylor 1963; Kakoi & Hiraide 1987; Mygind & Lildholdt 1997). The stroma of mature polyps is mainly characterized by its edematous nature and consists of supporting fibroblasts and infiltrating inflammatory cells, localized around “empty” pseudocyst formations. Among the inflammatory cells, EG2+ (activated) eosinophils are a prominent and characteristic feature in about 80% of polyps (Stoop et al. 1993), whereas lymphocytes and neutrophils are the predominant cells in CF and primary ciliary dyskinesia. Eosinophils are localized around the vessels and glands, and directly beneath the mucosal epithelium (Kakoi & Hiraide 1987). In small polyps, not larger than 5 mm, growing on normallooking mucosa of the middle turbinate in patients with bilateral polyposis, early processes of polyp growth have been studied (Bachert et al. 2000). Numerous subepithelial EG2+ eosinophils were present in the luminal compartment of the early stage polyp, forming a cap over the central pseudocyst area. In contrast, mast cells were scarce in the polyp tissue, but were normally distributed in the pedicle and the adjacent mucosa, which had a normal appearance. This contrasts with mature polyps, where mast cells and eosinophils are diffusely distributed in the polyp tissue. Fibronectin deposition was noticed around the eosinophils in the luminal compartment of the early stage polyp, and formed a networklike structure in the polyp center and within the pseudocysts. The presence of myofibroblasts was limited to the central pseudocyst area. Interestingly, albumin and probably other plasma proteins were deposited within the pseudocysts,

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adjacent to the eosinophil infiltration. These observations suggest a central deposition of plasma proteins, regulated by the subepithelial eosinophilic inflammation, as a pathogenetic principle of polyp formation and growth. The extravasated plasma – for reasons of distance, binding force, or by extracellular matrix abnormality – may not find its way to the airway surface (Persson 1991).

Clinical features and investigations The diagnosis of nasal polyps is based on the finding of pale-grey, semi-translucent, round or bag-shaped mucosal protrusions from the sinuses into the nasal cavity, filled with gelatinous or watery masses. Most nasal polyps arise from the clefts of the middle nasal meatus and ethmoidal cells, prolapsing into the nose, with some polyps originating in the maxillary, sphenoid or frontal sinuses (Stammberger 1999). Polyps originating from the middle and superior turbinates may be seen in more severe disease, and those from the inferior turbinate are extremely rare. Depending on the extent of polyp masses within the nasal cavities, patients develop various symptoms and complaints. The typical history is a “cold” that has persisted over months or years, with nasal obstruction and discharge as the most prominent symptoms. With time, hyposmia or anosmia develop, and additional complaints such as the feeling of a “full head” are present (Drake-Lee et al. 1984). Anosmia is a typical symptom of nasal polyposis, differentiating it from chronic sinusitis without polyposis, and may serve as a valid marker for estimating the duration and extent of disease. Interestingly, whereas chronic sinusitis is often associated with headache and facial pain, nasal polyposis itself rarely causes pain despite the fact that most of the sinuses, including the frontal sinuses, are opacified. Viral infections frequently cause prolonged episodes of severely obstructed nasal passages and colored secretions, probably due to the fact that ventilation and drainage of the sinuses are decreased by the polyp masses, with subsequent bacterial infection. According to our own investigations, infections may also cause temporary growth of the polyps and, if persistent, accelerate the course of disease. Inhalant allergens do not seem to induce additional complaints. Patients also often report nasal congestion and discharge due to alcoholic beverages. As nasal polyps may represent part of a systemic disease, adequate questions and further investigations may be necessary. Asthma and other lung diseases, aspirin sensitivity, Churg–Strauss syndrome, inhalant allergies and CF have to be considered. With the introduction of rigid endoscopes into daily practice, nasal polyps are discovered at earlier stages today compared with 10 years ago. While anterior rhinoscopy may detect large polyps, it is not considered sufficient to exclude polyps. In particular, endoscopic investigation of the nose after topical decongestion is necessary for the differential diagnosis. To investigate the extent of disease within the

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sinuses, a CT scan with coronary sections is performed with special reference to mucosal structures and the delicate anatomy of the sinuses (Zinreich 1993). A CT scan is mandatory before sinus surgery may be considered, and has to be available during surgery to inform the surgeon about anatomic variations. An additional MRI may be helpful for the diagnosis of fungal disease, tumor, or if intracranial extension of disease is suspected. Functional tests such as a smell test and acoustic rhinometry, a cytologic examination of nasal secretions for eosinophils and a blood eosinophil count, skin-prick testing for inhalant allergens, endoscopically guided microbiology from the middle meatus, or a biopsy may provide additional information. Measurement of nitric oxide requires little patient collaboration and is quick and easy to perform. On the other hand, the availability of measuring equipment at present limits its use. Nitric oxide is found in the upper and lower respiratory tract and is a sensitive indicator of the presence of inflammation and ciliary dysfunction. The majority of nitric oxide is made in the sinuses and therefore may be low even in the presence of normal activity if the sinus ostia are blocked, e.g., nasal polyposis (Colantonio et al. 2002). However, it may be a useful tool as an outcome measurement after therapy (Ragab et al. 2004).

Differential diagnosis As the symptomatology of nasal polyps is rather nonspecific, nasal endoscopy needs to be performed to confirm the diagnosis and exclude other sinister diseases. Nasal obstruction may also be caused by foreign bodies, allergy, turbinate hypertrophy, CRS, adenoid hypertrophy, or Wegener granuloma. Although nasal polyps have a characteristic appearance when investigated by nasal endoscopy, inverting papillomas and occasionally benign or malignant tumors or even meningoencephaloceles may be mistaken for nasal polyps. Any unilateral obstruction, nose bleeding, or crusting should be intensively investigated. Biopsy should be carried out if necessary.

Treatment The management of nasal polyps may involve medical approaches, mainly based on the use of topical or systemic corticosteroids, and surgical procedures, ranging from the extraction of polyps within the nasal lumen to radical ethmoidectomy, which tries to eradicate all polyp tissue. However, as nasal polyposis is a chronic disease with a high rate of recurrence in about one-hird of patients, surgical overtreatment and its sequelae should be avoided. Instead, a combined treatment strategy is recommended for long-term control of the disease. Figure 70.5 shows the therapeutic scheme in nasal polyposis. The symptomatic efficacy of intranasal corticosteroids in patients with nasal polyps is well documented. Symptoms such as nasal blockage, rhinorrhea and occasionally hyposmia are

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Diagnosis: nasal polyposis Not obstructive

Obstructive, anosmia, asthma

Topical GCS b.i.d., after symptom control OD for 3 months

Oral GCS (decreasing scheme), topical GCS b.i.d., evtl. antibiotics

Symptom control? Yes Long-term therapy with topical GCS

No Surgery No

Combination treatment necessary

Yes Fig. 70.5 Therapeutic scheme in nasal polyposis. GCS, glucocorticosteroids. (See CD-ROM for color version.)

reduced during the period of treatment, but recurrence of symptoms occurs within weeks to months (Mygind et al. 1975; Lund et al. 1998; Tos et al. 1998; Blomqvist et al. 2001). The effects on nasal obstruction and polyp masses could also be documented by objective methods such as peak nasal inspiratory flow, rhinomanometry, rhinometry, and smell tests (Ruhno et al. 1990; Lildholdt et al. 1995; Holmberg et al. 1997; Lildholdt et al. 1997; Keith et al. 2000). Topical corticosteroids may also reduce the incidence of polyp recurrences after surgery (Virolainen & Puhakka 1980; Karlsson & Rundcrantz 1982; Hartwig et al. 1988). However, topical corticosteroids may be insufficient in severe bilateral polyps (Krouse et al. 1983), and polyp growth may be observed despite treatment (Fig. 70.6).

Before treatment: right side

Nasal Polyps and Rhinosinusitis

Systemic corticosteroids, for example beginning with 32 mg methylprednisolone and reducing the dose stepwise during a 14–20 day oral course, are extremely effective in reducing polyp size and symptoms (van Camp & Clement 1994). The suppression of gene transcription of many cytokines is a prominent action of glucocorticosteroids (Barnes & Adcock 1993; Schwiebert et al. 1996; Rudack et al. 1999; Bolard et al. 2001), including IL-5 and eotaxin. Because of these effects, recruitment and localization of inflammatory cells into polyp tissue is inhibited, as well as their activation and protein synthesis. This has a prominent effect on numbers of nasal eosinophils, eosinophil products and survival, and may also affect plasma protein retention (Bachert et al. 2000). However, polyps will recur rapidly in the severe group of patients, and there is little evidence so far that the natural course of the disease is influenced by long-term low-dose treatment regimens. Current studies focus on the effect of anti-IL-5 treatment in severe polyposis to possibly circumvent the side effects induced by long-term steroid treatment (see below). Based on theoretical considerations, it has been proposed that antileukotriene therapy would be successful in patients with aspirin sensitivity or recurrences after surgery (Ulualp et al. 1999; Parnes & Chuma 2000). However, large-scale placebo-controlled studies have not yet been reported. Antibiotics are indicated in the case of superimposed bacterial infection. Recently, macrolide antibiotics have been suggested to not only decrease the virulence of colonizing bacteria, but also to have antiinflammatory activities, leading to a significant reduction in polyp size paralleled by a decrease in local IL-8 (Yamada et al. 2000; Anders 2001). Again, large-scale placebo-controlled studies have to be performed

Left side

Symptoms: obstructed nasal passage, secretion, impaired sense of smell

After treatment: right side

Left side

Symptoms: mild secretion, nasal passage free Fig. 70.6 Effect of 3 months of topical steroid treatment on nasal polyps. (See CD-ROM for color version.)

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to formally test this hypothesis. However, the recent finding of a possible role of SAEs in the pathomechanism of nasal polyps may suggest the long-term use of antibiotics for primary treatment (Bachert et al. 2001). Sinus surgery, today referred to as functional endoscopic sinus surgery (FESS), is a standard treatment with good functional results in patients resistant to medical treatment and avoids radical surgical procedures (Lanza & Kennedy 1992; Mygind & Lildholdt 1996; Stammberger 1999). The aim is to remove polyp tissues in the nose and sinuses with preservation of anatomic structures and healthy mucosa. Extensive postoperative care and follow-up is required to preserve the postoperative results and to prevent regrowth of polyps. In fact, an individualized management for nasal polyposis may combine long-term topical steroids, short-term systemic steroids, and surgery. In a 20-year follow-up study of 41 patients with nasal polyps, 85% of patients still suffered from the disease, with anosmia present in 61% (Vento et al. 2000). Eight subjects, including seven with aspirin sensitivity, had undergone 11 or more surgical operations during the 20-year period. This study as well as others showing the high recurrence rate in nasal polyposis clearly indicate the chronicity of the disease at least in this subgroup of patients and suggest a reserved surgical approach. Eradication of disease by surgery is an exception.

Course and prognosis Inflammatory mechanisms do not only play a role in the disease process, but also participate in the healing process after sinus surgery. We know from clinical experience that impaired healing may occur after FESS and seems to be linked to postoperative infection, but may also be related to changes in the mucosal immunology caused by the original disease process. For example, wound healing in CF patients or subjects with massive polyps and underlying pathology such as asthma and aspirin sensitivity often leads to a much thicker and less functional sinus mucosa compared with wound healing in uncomplicated chronic sinusitis. Wound healing is a highly coordinated process involving clot formation, inflammatory reaction, immune response and, finally, tissue remodeling and maturation. Thus, in nasal polyps (Bachert et al. 2000), the deficit in production of TGF-β1, a potent chemoattractant for fibroblasts and stimulus for extracellular matrix formation, could have a severe impact on healing.

Future trends Consistent with current knowledge of the pathophysiology of nasal polyposis, new therapeutic approaches could focus on eosinophilic inflammation, eosinophil recruitment, the T cell as the orchestrating cell and IgE antibodies, as well as on tissue destruction and remodeling processes. Recently, the introduction of monoclonal humanized antibodies has opened new perspectives. However, these molecules are still

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intensively studied in upper airway disease, but could offer solutions if they demonstrate more effectiveness and better tolerability than corticosteroids.

Possible future treatment modalities IL-5 antagonists IL-5 is an eosinophil differentiation factor that increases the sensitivity of eosinophils toward other stimuli and delays their cell death. High-affinity IL-5 receptors are exclusively expressed by eosinophils and basophils, but no other human cells. Therefore, neutralization of IL-5 appeared to be an obvious approach for treating eosinophilic disorders, since no major side effects, due to effects on other cells, are expected (Simon 2002). Results from animal experiments suggested that IL-5 monoclonal antibody may have antiasthmatic activities (Danzig & Cuss 1997), which makes IL-5 an interesting molecular target for pharmacologic intervention in eosinophilic diseases. Its role as a central nonredundant player in eosinophil function in vivo has been very well documented over the past years. Recent observations point toward an additional function on airway smooth muscle cells, suggesting that IL-5 may directly contribute to bronchial hyperresponsiveness. Yet, initial studies using humanized anti-IL-5 monoclonal antibodies in asthma have been disappointing. The role of eosinophils in nasal polyposis is strongly suggested by observational studies and by in vitro data, although never firmly proven. Indeed, the increased concentrations of IL-5 and severe tissue eosinophilia were found in nasal polyp tissue and treatment of eosinophil-infiltrated polyp tissue with neutralizing anti-IL-5 monoclonal antibody, but not anti-IL-3 or anti-GM-CSF monoclonal antibodies, resulted in eosinophil apoptosis and decreased tissue eosinophilia in vitro (Simon et al. 1997). As nasal polyps are characterized by eosinophilic inflammation and high IL-5 levels, antagonizing the effect of IL-5 is a potential new treatment strategy in patients with nasal polyps (Gevaert et al. 2006). We therefore performed a double-blind, placebo-controlled, randomized, two-center safety and pharmacokinetic study, where 24 subjects with bilateral nasal polyps were randomized to receive a single intravenous infusion of reslizumab, a humanized anti-human IL5 monoclonal antibody, at 3 mg/kg or 1 mg/kg or placebo. This resulted in a reduction of blood eosinophil numbers and ECP concentrations up to 8 weeks after treatment in serum and nasal secretions. Individual nasal polyp scores improved in only half of the verum-treated patients for up to 4 weeks. When carefully analyzing responders and nonresponders in a post-hoc analysis, only those nasal polyp patients with increased levels of IL-5 (> 40 pg/mL) in nasal secretions before treatment seemed to benefit from anti-IL-5 treatment. Furthermore, nasal IL-5 levels decreased only in the responders, whereas they increased in the nonresponders. These data show that at least in 50% of the nasal polyps, IL-5 and eosinophils play a key role (IL-5 dependent) in sustaining

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polyp size, whereas in the rest eosinophilia might be dependent on other factors (IL-5 independent). Future approaches might exist in combination therapy with anti-IL-5 and a CCR3 antagonist because this would have the advantage of inhibiting both bone marrow maturation (primarily IL-5 dependent) and tissue accumulation (mainly a CCR3-dependent effect).

Anti-CCR3, anti-eotaxin CC-chemokine receptor 3 (CCR3)-stimulating chemokines are likely to have an important in vivo role in the regulation of eosinophil, basophil and potentially Th2 and mast cell recruitment. Several CC chemokines including eotaxin and RANTES are potent eosinophil chemotatic and activating peptides acting through CCR3. As eosinophils have also been implicated in the pathogenesis of nasal polyposis, and RANTES and eotaxin have been identified as eosinphil chemoattractants in polyp tissue, antagonism of CCR3 could have a therapeutic role in this disease. The development of small nonpeptide molecule CCR3 antagonists currently offers advantages over antichemokine (i.e., eotaxin) antibodies in terms of a broader approach, affecting several chemokines and effector cells, and favorable pharmacokinetic, targeting the receptors on peripheral blood cell surfaces and being independent from tissue penetration. Compared with placebo, pretreatment with antieotaxin reduces nasal obstruction, eosinophil influx and mast cells when anti-eotaxin antibodies are administrated intranasally in grass pollen-sensitive subjects (Taylor-Clark et al. 2003). Studies in nasal polyps have not been reported yet.

Nasal Polyps and Rhinosinusitis

IgE antagonism Therapies antagonizing IgE could be relevant, as IgE appears to be important in the regulation of chronic inflammation. We have showed evidence for marked local IgE production in nasal polyposis and its relation to severity of disease (Bachert et al. 2001). Treatment of allergic asthma and rhinitis with omalizumab, a humanized anti-IgE monoclonal antibody, causes a marked reduction in circulating free IgE levels. Treatment has been shown to reduce symptoms and exacerbations, and decrease the need for other medication in patients with these allergic diseases. Until now, no studies in nasal polyposis have been published to investigate whether high concentrations of IgE antibodies within the polyp tissue can be targeted with success. At the moment, anti-IgE therapy in patients suffering from nasal polyposis is being evaluated.

Immunosuppression Severe and corticosteroid-resistant eosinophilic airway inflammation including severe asthma and nasal polyposis remain difficult to manage. Cyclosporin A has been used as immunosuppressive therapy for many years in organ transplantation, because of its effectiveness in inhibiting T-cell proliferation and attenuation of inflammatory processes. New cyclosporin A analogs and other molecules that inhibit T-cell proliferation and IL-5 production in peripheral blood mononuclear cells from asthmatic individuals at physiologic concentrations (Powell et al. 2001) can be used as new therapeutic strategies. Clinical studies with immunosuppressive agents are lacking in nasal polyposis.

IL-4 and IL-13 antagonists The closely related Th2 cytokines IL-4 and IL-13 share biological functions that are considered important in the development of airway inflammation, including induction of the IgE isotype switch, increased expression of VCAM-1, promotion of eosinophil transmigration across the endothelium, stimulation of mucus production, and Th2 cell differentiation, leading to release of IL-4, IL-5, IL-9, IL-13 and eotaxin (Bachert et al. 2005). Furthermore, elevated levels of IL-4 at a site of injury could result in the development of fibrosis by enhancing fibroblast subset proliferation and collagen synthesis. The overlap of their functions results from the IL-4Rα chain forming an important functional signaling component of both the IL-4 and IL-13 receptors. IL-4 and IL13 mRNA-positive cells have been described in polyps, and the number of cells expressing IL-4 mRNA was shown to be increased compared with healthy mucosa independent of the atopic status. It can be expected that strategies to antagonize IL-4/IL-13 would also reduce inflammation in nasal polyps, although no specific studies have been performed. Early studies with inhaled recombinant human soluble IL-4R in adult asthmatics have shown promising results, and further progress may be expected from combined IL-4/IL-13 antagonists, based on recent data from murine asthma models.

Matrix metalloproteinase inhibitors Recent studies on tissue remodeling processes have focused on the role of matrix metalloproteinases, particularly MMP9, in nasal polyposis (Lechapt-Zalcman et al. 2001; Watelet et al. 2004). Besides the natural inhibitors of metalloproteinases, TIMP-1 and TIMP-2, synthetic broad-spectrum inhibitors of MMPs and antibodies to MMP-9 and MMP-2 have been developed. In vitro evidence has shown a reduction of MMP-9 activity; however none of these agents was studied further.

Acute and chronic rhinosinusitis Epidemiology Prevalence Sinusitis is an inflammatory disease with a prevalence that has been estimated at 10–30% in Europe and 15% in the American population (Moss & Parsons 1986; Gwaltney 1996; Kaliner et al. 1997). When reviewing the literature it becomes clear that the estimation of the prevalence of CRS remains speculative, because of the heterogeneity of this group of disorders and the diagnostic uncertainties.

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Acute rhinosinusitis is the most common health complaint, affecting more than 1 billion persons annually in the USA, and the prevalence of CRS exceeds that of other chronic conditions. In a survey on the prevalence of chronic conditions, it was estimated that CRS, defined as having “sinus trouble” for more than 3 months in the year before the interview, affects 15.5% of the total population in the USA (Collins 1997), ranking this condition second in prevalence among all chronic conditions. Later, the high prevalence of CRS was confirmed by another survey suggesting that 16% of the adult US population has CRS (Blackwell et al. 2002). In a Canadian study, the prevalence increased with age, with a mean of 2.7% in the group aged 20–29 years and 6.6% in the group aged 50–59 years. After the age of 60 years, prevalence levels of CRS leveled off to 4.7% (Chen et al. 2003). In Belgium, Gordts et al. (1996) reported that 6% of subjects in the general population suffered from chronic nasal discharge.

Genetics Although chronic sinus disease has been observed in family members, no genetic abnormality has been linked to CRS. However, the role of genetic factors in CRS has been implicated in patients with CF (McFadden et al. 1997) and primary ciliary dyskinesia (Kartagener syndrome). CF is one of the most frequent autosomal recessive disorders of the white population, caused by mutations of the CFTR gene on chromosome 7. The most common mutation, ΔF508, is found in 70–80% of all CFTR genes in northern Europe (Kerem et al. 1989; Cuppens et al. 1993). Upper airway manifestations of CF patients include CRS and nasal polyps, which are found in 25–40% of CF patients above the age of 5 years (De Gaudemar et al. 1996). Interestingly, Jorissen et al. (1999) reported that ΔF508 homozygosity represents a risk factor for paranasal sinus disease in CF.

Mechanisms The incidence of acute viral rhinosinusitis (common cold) is very high. It has been estimated that adults suffer two to five colds per year, and schoolchildren may suffer seven to ten colds per year. The exact incidence is difficult to measure because most patients with common cold do not consult a doctor. More reliable data are available on acute rhinosinusitis. Acute nonviral rhinosinusitis is defined as an increase of symptoms after 5 days or persistent symptoms after 10 days after a sudden onset of two or more of the symptoms of blockage/congestion, discharge, anterior/posterior nasal drip, facial pain/pressure, and/or reduction/loss of smell. It is estimated that only 0.5–2% of viral upper respiratory tract infections are complicated by bacterial infection; however, the exact incidence is unknown given the difficulty distinguishing viral from bacterial infection without invasive sinus-puncture studies. Bacterial culture results in suspected cases of acute community-acquired sinusitis are positive in only 60% of

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cases (Gwaltney et al. 1995). Signs and symptoms of bacterial infection may be mild and often resolve spontaneously. Normal sinuses are free of bacterial growth (Kern 1984). Although nasal smears, even when taken from the middle nasal meatus, are mostly contaminated and therefore not meaningful, cultures by sinus puncture from both adults and children with acute sinusitis predominantly grow Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis (Wald et al. 1981; Gwaltney 1994). In chronic maxillary sinusitis, however, anaerobic bacteria alone or mixed infections with facultative anaerobes and aerobes are predominant: S. aureus, coagulase-negative Staphylococcus, Pseudomonas aeruginosa and anaerobes. Unfortunately, studies show incredible variation, with anywhere from 0 to 100% of samples showing growth of anaerobes (Wald et al. 1981; Goldenhersh et al. 1990; Ramadan 1995; Klossek et al. 1998; Rontal et al. 1999; Brook et al. 2000). It is unclear, however, whether bacterial infections truly contribute to chronic sinusitis pathophysiology or whether they are just secondary to the local milieu in an obstructed sinus encouraging bacterial growth (Wald 1995). Thus, together with the CT-based finding of anterior ethmoidal cells and the ostiomeatal complex being obstructed (Stammberger 1986), it has been suggested that the development of chronic sinusitis is a two-step process initially, involving infection and mucosal remodeling with obstruction.

Etiology and pathogenesis A fundamental role in the pathogenesis of rhinosinusitis is played by the ostiomeatal complex, a functional unit that comprises maxillary sinus ostia, anterior ethmoid cells and their ostia, ethmoid infundibulum, hiatus semilunaris, and middle meatus. The key element according to current understanding is the maintenance of ostial patency. Specifically, ostial patency significantly affects mucus composition and secretion; moreover, an open ostium allows mucociliary clearance to easily remove particulate matter and bacteria eventually come in contact with the sinus mucosa. Problems occur if the orifice is too small for the amount of mucus, if mucus production is increased, for instance during an upper respiratory tract infection, or if ciliary function is impaired. Stasis of secretions follows and bacterial export ceases, causing or exacerbating inflammation of the mucosa while aeration of the mucosa is decreased, causing even more ciliary dysfunction. This vicious circle can be difficult to break, and if the condition persists it can result in CRS. The role of ostium occlusion in CRS seems to be less pronounced than that in acute rhinosinusitis. Figure 70.7 shows the vicious circle of CRS.

Pathology When studying the cytokine profile in patients with naturally acquired viral rhinitis, Bachert and colleagues found a significant upregulation of IL-1β, IL-6 and IL-8 levels in nasal

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Ostium Occlusion Inhibition of ventilation and drainage

Increased mucosal thickness

Stagnation of secretion

Inflammation of the lamina propria

Change in the composition and pH of secretion

Change of the host milieu bacteria become pathogenic

Change of the mucosal gas metabolism

Ciliary and epithelial damage

Fig. 70.7 The vicious circle of chronic rhinosinusitis.

lavage fluid from viral rhinitis patients compared with that from control subjects (Roseler et al. 1995). IL-4 was not measurable in any of the samples, suggesting that cytokines such as IL-1β, IL-6 and IL-8, but not IL-4, are involved in the pathophysiology of the common cold. Looking at the cellular level in a mouse model of viral rhinosinusitis, a significant increase in T-suppressor and T-regulatory cells is seen, after resolution of the acute infection (Klemens et al. 2006). Intranasal inoculation with Sendai virus resulted in an acute infection that resolved spontaneously within 10 days. Intranasal inoculation with reovirus in mice resulted in reovirus-like immunoreactivity in the septa and paranasal sinus mucosa in mice as early as day 2, with peak intensity seen on day 4, and scant staining seen on day 7 (Ramadan et al. 2002). Complete absence of viral staining was seen by day 10. By day 10, a large mucosal influx of B cells was observed, with a moderate influx of macrophages and smaller influx of T cells. By day 14, there was a peak in the number of B cells with a corresponding but less pronounced peak in T cells, while macrophages began to decline at this point. By day 21, the panel of immune markers returned to near normal levels. The results of this study suggest that the immune system continues to produce a response as long as 2 weeks after clearance of viral antigens. One proposed mechanism for this phenomenon is that local factors such as cytokines are released continually after infection, even in the absence of persistent viruses or bacteria. As sinus mucosal tissue from subjects with acute bacterial rhinosinusitis (ARS) is difficult to sample, there is a relative lack of studies on cytokines and mediators in ARS. One of the first studies reported 10 subjects undergoing surgery for complications, with mucosal tissue sampled from the maxillary sinus, which demonstrated significantly elevated protein concentrations of IL-8 compared with seven controls (Rudack et al. 1998a). Similar results, though not reaching

Nasal Polyps and Rhinosinusitis

significance, were obtained for IL-1β and IL-6, whereas other cytokines such as GM-CSF, IL-5 and IL-4 were not upregulated. Recently, IL-8 and also TNF-α and total protein content were increased in nasal lavage from subjects with ARS compared with controls and allergic rhinitis subjects (RepkaRamirez et al. 2002). Proinflammatory cytokines such as IL-1β, IL-6 and TNF play a prominent role in ongoing inflammatory reactions by activating endothelial cells, T lymphocytes and others, inducing the expression of cell adhesion molecules and the release of other cytokines such as IL-8. IL-8 belongs to the CXC chemokine group and is a potent neutrophil chemotactic protein, which is constantly synthesized in the nasal mucosa (Bachert et al. 1995). The cytokine pattern found in ARS resembles that found in lavage fluid in naturally acquired viral rhinitis (Roseler et al. 1995). In the sinus fluid of patients with chronic sinusitis undergoing surgery, inflammatory cells are predominantly neutrophils, as is observed in acute sinusitis, but a low percentage of eosinophils, mast cells and basophils may also be found (Stierna & Carlsoo 1990; Georgitis et al. 1995). The mucosal lining in chronic sinusitis is characterized by basement membrane thickening, goblet cell hyperplasia, subepithelial edema, and mononuclear cell infiltration. In a recent study evaluating the percentage of eosinophils (out of 1000 inflammatory cells counted per vision field), 31 patients with untreated chronic sinusitis without nasal polyps all had less than 10% eosinophils (overall mean 2%), whereas in 123 untreated nasal polyp specimens 108 samples showed more than 10% eosinophils (overall mean 50%) (Jankowski et al. 2002). These observations suggest that tissue eosinophilia is not a hallmark of chronic sinusitis without polyp formation, and that there are major differences in the pathophysiology of both sinus diseases. A highly potent chemoattractant for neutrophils, IL-8 has been demonstrated in chronic sinusitis tissue (Takeuchi et al. 1995) and IL-8 protein concentrations in nasal discharge from chronic sinusitis patients were significantly higher than in allergic rhinitis patients in a study also involving immunohistochemistry and in situ hybridization (Suzuki et al. 1996). In a study measuring cytokine protein concentrations including IL-3, IL-4, IL-5, IL-8 and GM-CSF in tissue homogenates, IL-8 was found to be significantly increased in acute sinusitis, whereas IL-3 was increased in chronic sinusitis mucosa compared with inferior turbinate samples (Rudack et al. 1998b). IL-3 might be involved in local defense and repair of chronically inflamed sinus mucosa by supporting various cell populations and indirectly contributing to fibrosis and thickening of the mucosa (Persson et al. 1997). With respect to the findings in nasal polyposis described above, we recently investigated sinus mucosa homogenates of subjects with chronic sinusitis and nasal polyposis versus normal controls. In nasal polyp samples we found an increased concentration of IL-5 and total IgE, but these parameters did not differ from controls in the chronic sinusitis group (Van

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Zele et al. 2006). However, there was a clear increase in IFN-γ concentrations in CRS without polyps, as well as an increase in neutrophil chemoattractants (IL-8). Compared with controls, higher levels of TGF-β1 were found in CRS homogenates, consistent with the remodeling features of fibrosis in CRS and edema in nasal polyps.

Comorbidity The association between sinusitis and asthma has long been appreciated. Up to 70% of patients with asthma also present with sinusitis as assessed by sinus radiography (AnnesiMaesano 1999). There is circumstantial evidence that CRS may be linked to chronic lung disease, especially severe asthma. This includes studies showing that abnormal sinus radiographs are frequently found in children (Rossi et al. 1994) and adults (Fuller et al. 1994; Peters et al. 1999) with asthma exacerbations, and that drug management of sinusitis (Friedman et al. 1984) or sinus surgery (Mings 1988; Parsons & Phillips 1993; Nishioka et al. 1994; Park et al. 1998; Senior et al. 1999) results in a significant improvement in asthma symptoms and exacerbations, and reduction of corticosteroid use. However, the mechanisms by which sinusitis influences asthma are not well understood. Many pathogenic hypotheses have been proposed to explain the link between sinus disease and asthma. At first, activation of the pharyngobronchial reflex might be involved (Rolla et al. 1997). A second hypothesis explaining the link between asthma and sinus disease is the silent dripping of material containing mediators from the nose and aspiration into the bronchial tree. Although radionuclides placed into the maxillary sinuses of patients with sinusitis and asthma could not be retrieved in the lungs after 24 hours, pulmonary aspiration of radionuclide-labeled nasal secretions during sleep has been described. However, there was no difference in aspirated amounts between the asthma/chronic sinusitis group and the control group (Bardin et al. 1990; Ozcan et al. 2003). Following the concept of rhinosinusitis, allergic and nonallergic rhinitis may also give rise to sinusitis. Naclerio et al. (1997) presented several hypotheses to explain the link be-

tween allergy, tissue edema and vascular congestion causing obstruction of sinus drainage and finally nasal and sinus inflammation. There is, however, no evidence of a change in ostial patency or of an increased incidence of purulent sinusitis due to seasonal rhinitis. It was therefore suggested that perennial allergic (and nonallergic) rather than seasonal rhinitis may predispose to sinusitis (Berrettini et al. 1999). Furthermore, allergy represents a poor prognostic factor for the outcome of sinus surgery in some studies (Kennedy et al. 2000). In patients with chronic obstructive pulmonary disease (COPD), van Manen et al. (2001) showed that comorbid sinusitis was present significantly more than in controls. Adjusted odds ratios were also significant for sinusitis, indicating a higher risk for this disease in COPD patients. CRS represents a common disorder of multifactorial origin. Indeed several factors are associated with CRS: ciliary impairment, allergy, asthma, immunocompromised state, genetic factors, pregnancy and endocrine state, local host factors, microorganisms as bacteria and fungi, osteitis, environmental factors and iatrogenic factors (Fokkens et al. 2005). All these factors are, to a certain degree, etiologically linked to CRS.

Clinical features Signs and symptoms of ARS include nasal congestion, nasal discharge, purulent discharge, postnasal drainage, facial pain, headache, pressure or a feeling of fullness in the head, hyposmia, cough, fever, and referred dental and ear pain. In acute bacterial infections, facial pain, fever, pain on palpation and headache are often predominant, with varying locations depending on the sinuses affected, and partial or complete opacification of one or more sinuses is found by radiographic imaging. A scheme of the diagnosis of ARS is shown in Fig. 70.8. Major and minor symptoms associated with the diagnosis of chronic sinusitis have been defined (Lanza & Kennedy 1997). In chronic sinusitis, symptoms are generally the same as in acute disease; the most common symptoms are headache, facial pain, nasal obstruction, and discharge. However, in

Symptoms

Viral rhinitis/common cold Acute rhinosinusitis/increase in symptoms after 5 days Acute rhinosinusitis/symptoms exceed 10 days

0

5

10 Days

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15

Fig. 70.8 Common cold or acute rhinosinusitis. Acute rhinosinusitis may be diagnosed when a common cold gets more symptomatic after 5 days or persists longer than 10 days. Less than 4% of patients have a bacterial form of ARS; most ARS episodes are viral. (See CD-ROM for color version.)

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some patients, symptoms may be diffuse, may just consist of a single symptom such as headache (e.g., isolated sphenoiditis) or postnasal drip, or the patient may not be aware of sinus involvement, especially in subjects with concomitant asthma. Therefore, a comprehensive diagnostic evaluation should be considered in every patient with nasal complaints or lower airway disease, and sinus CT imaging is mandatory for the diagnosis and eventually any surgical approach.

Diagnosis/differential diagnosis Evaluation of the duration and course of disease, the character, severity and location of symptoms, comorbidities, underlying pathologies as well as earlier management are essential to provide a working diagnosis. Anterior rhinoscopy may show hyperemia and swelling of the inferior turbinates, structural deformities of the septum, and even purulent secretions from the sinuses. Rigid endoscopy, mostly performed with a rigid endoscope after nasal decongestion, is a rapid and easy method to evaluate the middle nasal meatus and the ostiomeatal complex, posterior nasal structures, and nasopharynx (Stammberger 1986). In children, fiberoptic rhinoscopy is a convenient alternative to rigid endoscopy and also permits examination of the pharynx and larynx. Standard radiographs may be used for the diagnosis of acute frontal or maxillary sinusitis, but often do not provide additional information over history alone. Ultrasound is of limited value, but may be used in pregnant women to avoid exposure to radiation. CT has its indications in orbital or cerebral complications of sinusitis, in the preoperative evaluation of chronic sinusitis, and in the diagnosis of all sinister pathologies such as tumor, meningoceles or mucoceles (Zinreich et al. 1988; Zinreich 1993). CT helps to unravel anatomic abnormalities, the extent of the disease, and changes in the ostiomeatal complex and provides a “map” for surgery. MRI is particularly sensitive in the evaluation of fungal sinusitis, tumor, and extension of disease into the brain. As nasal cultures do not give an adequate picture of the organisms responsible for sinusitis, the indication to sample secretions from the sinus itself by puncture is limited to patients with infections resistant to treatment or in immunocompromised hosts (HIV, chemotherapy, diabetes, intensive care units). In daily practice, nasal obstruction and headache are frequently encountered. Differential diagnosis of facial pain and headache includes migraine, tension, cluster and rebound headaches, cranial neuralgia, and atypical facial pain. Furthermore, eye diseases and problems with accommodation may cause periorbital pain sensations. When indicated, a neurologic and/or ophthalmologic examination should be performed to exclude other causes. Unilateral nasal obstruction, possibly with pain or bloody discharge, is always suggestive of a sinister pathology developing in the nasal, paranasal or nasopharyngeal cavities.

Nasal Polyps and Rhinosinusitis

Treatment/management Patients with ARS (worsening of symptoms after 5 days, or persistence over 10 days) do not need antibiotic treatment, but may be treated symptomatically if disease is mild (decongestant, pain relief) or with antiinflammatory drugs (topical steroid) if symptoms are moderate to severe. Medical treatment of ARS aims to reopen the ostiomeatal complex, restore ventilation and drainage, and decrease the inflammatory process and to relieve the patient from symptoms such as pain and nasal obstruction. With Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis representing the most frequent bacteria associated with acute sinusitis, first-choice antibiotic treatment includes amoxicillin, cephalosporins and eventually amoxicillin plus clavulanic acid. For these antibiotics, a significant and similar therapeutic efficacy versus placebo has been demonstrated (Wald et al. 1986; Camacho et al. 1992; Lindbaek et al. 1996; van Buchem et al. 1997; Clement & de Gandt 1998; de Ferranti et al. 1998; Sparandero 2000), although the frequency of side effects is higher with amoxicillin plus clavulanic acid compared to nonpenicillins (Sterkers 1997). Antibiotics only should be indicated, if a bacterial form of ARS is suspected, as listed in Table 70.5. Although topical and oral decongestants have been shown to decongest the nasal cavities, there is no evidence of such an effect on the ostiomeatal complex or the sinus mucosa. There is, however, evidence that topical corticosteroids may have such effects as adjunctive treatment to antibiotics (Meltzer et al. 1993; Barlan et al. 1997; Meltzer et al. 2000) and reduce nasal symptoms including obstruction as well as headache significantly better than antibiotics alone. In allergic subjects suffering from sinusitis, added relief may also be provided by antihistamine treatment (Braun et al. 1997). Other drugs including secretolytics and homeopathics have not been shown to be effective. Most of the randomized controlled trials studying nasal and antral irrigation with isotonic or hypertonic saline in the treatment of acute/intermittent and chronic/persistent rhinosinusitis offer evidence that nasal washouts or irrigations with isotonic or hypertonic saline are beneficial in terms of alleviation of symptoms, endoscopic findings, and healthrelated quality-of-life improvement in patients with persistent CRS. Hypertonic saline is preferred to isotonic treatment for rhinosinusitis by some authors in the USA, mostly based on a paper indicating that it significantly improves nasal

Table 70.5 Indications for antibiotics in acute rhinosinusitis. Severe illness, fever Severe unilateral (especially frontal) headache/facial pain Beginning or manifest complications of acute rhinosinusitis Compromised immune status

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mucociliary clearance measured by saccharine test in healthy volunteers (Talbot et al. 1997). In chronic sinusitis, the role of bacteria has been challenged (Wald 1995; Van Cauwenberge et al. 1997) and the benefit of antibiotic treatment is questionable (Otten & Grote 1988; Legent et al. 1994); however, antibiotics are indicated for treating acute exacerbations. One study showed that long-term topical corticosteroid treatment has beneficial effects on nasal and pain symptoms, and also helped to avoid surgery during the treatment period (Qvarnberg et al. 1992). Antifungals are only indicated in invasive forms of sinus mycosis or in immunocompromised hosts. Surgery is indicated for acute sinusitis in case of complications, sinusitis resistant to adequate medical treatment, and in chronic sinusitis after unsuccessful drug treatment. Today, FESS is the standard procedure for chronic sinus disease, and has been proven to reduce symptoms as well as to increase quality of life, with decreased morbidity compared to former, more invasive procedures (Messerklinger 1970; Kennedy 1992; Lund & Scadding 1994; Stammberger 1994; Terris & Davidson 1994; Metson & Gliklich 1998). The aim is to restore sinus ventilation and drainage by opening the key areas and preserve sinus mucosa, which has the potential to regenerate. Possible complications of FESS include severe bleeding, orbital trauma and cerebrospinal fluid leaks with secondary complications such as meningitis or cerebral damage; however, in experienced hands, complications are not more frequent with endonasal approaches compared with former open procedures.

adequate antibiotics; however, orbital complications may develop in children and adolescents, with bacterial infections penetrating from the ethmoid or the frontal sinus through the thin bone of the orbital frame within the first days of infection (Fig. 70.9). The first sign is typically a reddish swelling of the medial upper eyelid (cellulitis), which may develop into a subperiostal abscess, an intraorbital or eyelid abscess, or an orbital phlegmon. Such an abscess has to be urgently drained by surgical intervention, and whenever there is even partial loss of vision, the patient needs immediate surgical care and intravenous antibiotic treatment. From an orbital phlegmon, thrombosis of the cavernous sinus may develop, with possible intracranial infection and complete loss of vision (apex orbitae syndrome). In adults, an empyema of the frontal sinus may lead to meningitis or an epidural or subdural brain abscess with typical signs of meningitis and increased brain pressure. In these rare cases, an immediate combined rhinologic and neurosurgical approach is indicated. Osteomyelitis of the frontal bone also needs surgical intervention and long-term antibiotic treatment. Recurrent episodes of meningitis may arise from bony defects in the frontal skull base or the sphenoid sinus, and requires closure of the defect to prevent future risks. Fungal disease, if invasive, can invade the bony structures, and penetrate into the orbit, cheek and brain. Chronic closure of the frontal, ethmoidal or sphenoidal sinuses may furthermore lead to the development of a pyo-mucocele, which should be drained into the nose.

Future trends Course and prognosis Complications of bacterial sinusitis are rare nowadays, because most patients with acute bacterial sinusitis are treated with

As CRS was historically considered to be predominantly an infectious process, it has been treated with antibiotics and surgical drainage. However, a significant proportion of CRS

Fig. 70.9 Orbital complication of acute ethmoiditis in a 9-year-old girl: subperiostal abscess. (See CD-ROM for color version.)

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patients continues to suffer from disease, despite adequate surgical and/or antibiotic therapy. An antiinflammatory approach in those patients might be indicated, as often an inflammatory infiltrate is found in the sinus mucosa of such patients. Macrolide therapy and immunomodulatory therapy are examples of such approaches.

Macrolide therapy Recent studies suggest that long-term, low-dose macrolide therapy might have a place in the treatment of CRS (Hashiba & Baba 1996; Cervin et al. 2002). The precise mechanisms by which they suppress inflammation remain unclear, although the antiinflammatory effect appears to be separate from the antimicrobial effect. Macrolides inhibit production of proinflammatory cytokines, decrease airway mucus secretion, and inhibit inflammatory cell chemotaxis (Kawasaki et al. 1998; Wallwork et al. 2002; Tamaoki 2004). As suggested by recent studies the proinflammatory transcription factor NF-κB may be the target, as clarithromycin reduced the DNA-binding activity of NF-κB in human nasal epithelial cells and fibroblasts (Miyanohara et al. 2000). In vitro evidence shows decreased expression of TGF-β and again NF-κB (Wallwork et al. 2004) and, compared with prednisolone, clarithromycin reduces IL-5, IL-8 and GM-CSFin cultured nasal mucosa from CRS patients (Wallwork et al. 2002). Much of the evidence concerns the antiinflammatory effect of macrolides on neutrophilic inflammation, and the effect of macrolides on eosinophilic inflammation is much less clear. The benefit of long-term, low-dose macrolide treatment seems to be that it is, in selected cases, effective when steroids fail. Placebo-controlled studies should be performed to establish the efficacy of macrolides if this treatment is to be accepted as evidence-based medicine.

Immunomodulatory therapy Treatment with filgrastim, recombinant human G-CSF, was tested in a randomized controlled trial in a group of patients with persistent rhinosinusitis refractory to conventional treatment, which did not confirm significantly improved outcomes after such expensive treatment (van Agthoven et al. 2001). A pilot study with IFN-γ suggested that this treatment may be beneficial in treating persistent rhinosinusitis, but the number of patients was not adequate to provide evidence justifying such treatment (Jyonouchi et al. 2003). Certain groups of antibiotics may be regarded as immunomodulators, like quinolones and macrolides.

Future research topics Although much work has been done in CRS and nasal polyposis, many questions remain unanswered. The following suggestions should highlight some areas of interest or further research (Fokkens et al. 2005).

Nasal Polyps and Rhinosinusitis

• Prospective population-based study on the impact of allergy on CRS/nasal polyposis. • Long-term follow-up cohort studies in CRS or nasal polyposis to study the natural history of the condition. • Studies of the benefit of long-term macrolide/antibiotic therapy in patients with CRS/nasal polyposis. • Studies on nasal steroids as a single-modality treatment in CRS. • Comparison of surgical and medical treatment modalities in CRS/nasal polyposis.

References Alam, R., Forsythe, P., Stafford, S. & Fukuda, Y. (1994) Transforming growth factor beta abrogates the effects of hematopoietins on eosinophils and induces their apoptosis. J Exp Med 179, 1041–5. Anders, C. (2001) The anti-inflammatory effect of erythromycin and its derivatives, with special reference to nasal polyposis and chronic sinusitis. Acta Otolaryngol 121, 83–92. Annesi-Maesano, I. (1999) Epidemiological evidence of the occurrence of rhinitis and sinusitis in asthmatics. Allergy 54 (suppl. 57), 7–13. Bachert, C., Wagenmann, M. & Hauser, U. (1995) Proinflammatory cytokines: measurement in nasal secretion and induction of adhesion receptor expression. Int Arch Allergy Immunol 107, 106–8. Bachert, C., Wagenmann, M., Hauser, U. & Rudack, C. (1997) IL-5 synthesis is upregulated in human nasal polyp tissue. J Allergy Clin Immunol 99, 837–42. Bachert, C., Wagenmann, M., Rudack, C. et al. (1998) The role of cytokines in infectious sinusitis and nasal polyposis. Allergy 53, 2–13. Bachert, C., Gevaert, P., Holtappels, G., Cuvelier, C. & van Cauwenberge, P. (2000) Nasal polyposis: from cytokines to growth. Am J Rhinol 14, 279–90. Bachert, C., Gevaert, P., Holtappels, G., Johansson, S.G.O. & van Cauwenberge, P. (2001) Total and specific IgE in nasal polyps is related to local eosinophilic inflammation. J Allergy Clin Immunol 107, 607–14. Bachert, C., Watelet, J.-B., Gevaert, P. & Paul Van, C. (2005) Pharmacological management of nasal polyposis. Drugs 65, 1537–52. Bachert, C., Patou, J. & Van Cauwenberge, P. (2006) The role of sinus disease in asthma. Curr Opin Allergy Clin Immunol 6, 29–36. Baraniuk, J. (1994) Physiology of sinusitis. In: Druce, H.M., ed. Sinusitis: Pathophysiology and Treatment. Marcel Dekker, New York, pp. 19–39. Bardin, P.G., Van Heerden, B.B. & Joubert, J.R. (1990) Absence of pulmonary aspiration of sinus contents in patients with asthma and sinusitis. J Allergy Clin Immunol 86, 82–8. Barlan, I.B., Erkan, E., Bakir, M., Berrak, S. & Basaran, M.M. (1997) Intranasal budesonide spray as an adjunct to oral antibiotic therapy for acute sinusitis in children. Ann Allergy Asthma Immunol 78, 598– 601. Barnes, P.J. & Adcock, I. (1993) Anti-inflammatory actions of steroids: molecular mechanisms. Trends Pharmacol Sci 14, 436–41. Bartels, J., Maune, S., Meyer, J.E. et al. (1997) Increased eotaxinmRNA expression in non-atopic and atopic nasal polyps: comparison to RANTES and MCP-3 expression. Rhinology 35, 171–4.

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Powell, N., Till, S., Bungre, J. & Corrigan, C. (2001) The immunomodulatory drugs cyclosporin A, mycophenolate mofetil, and sirolimus (rapamycin) inhibit allergen-induced proliferation and IL-5 production by PBMCs from atopic asthmatic patients. J Allergy Clin Immunol 108, 915–17. Qvarnberg, Y., Kantola, O., Salo, J., Toivanen, M., Valtonen, H. & Vuori, E. (1992) Influence of topical steroid treatment on maxillary sinusitis. Rhinology 30, 103–12. Ragab, S.M., Lund, V.J. & Scadding, G. (2004) Evaluation of the medical and surgical treatment of chronic rhinosinusitis: a prospective, randomised, controlled trial. Laryngoscope 114, 923–30. Ramadan, H.H. (1995) What is the bacteriology of chronic sinusitis in adults? Am J Otolaryngol 16, 303–6. Ramadan, H.H., Meek, R.B., Dawson, G.S., Spirou, G.A., Cuff, C.F. & Berrebi, A.S. (2002) Histologic and immunologic observations of viral-induced rhinosinusitis in the mouse. Am J Rhinol 16, 61–7. Repka-Ramirez, S., Naranch, K., Park, Y.J., Clauw, D. & Baraniuk, J.N. (2002) Cytokines in nasal lavage fluids from acute sinusitis, allergic rhinitis, and chronic fatigue syndrome subjects. Allergy Asthma Proc 23, 185–90. Rolla, G., Colagrande, P., Scappaticci, E. et al. (1997) Damage of the pharyngeal mucosa and hyperresponsiveness of airway in sinusitis. J Allergy Clin Immunol 100, 52–7. Rombaux, P., Collet, S., Eloy, P., Ledeghen, S. & Bertrand, B. (2005) Smell disorders in ENT clinic. B-ENT Suppl. 1, 97–107; quiz 8–9. Rontal, M., Bernstein, J.M., Rontal, E. & Anon, J. (1999) Bacteriologic findings from the nose, ethmoid, and bloodstream during endoscopic surgery for chronic rhinosinusitis: implications for antibiotic therapy. Am J Rhinol 13, 91–6. Roseler, S., Holtappels, G., Wagenmann, M. & Bachert, C. (1995) Elevated levels of interleukins IL-1 beta, IL-6 and IL-8 in naturally acquired viral rhinitis. Eur Arch Otorhinolaryngol 252 (suppl. 1), S61–S63. Rossi, O.V., Pirila, T., Laitinen, J. & Huhti, E. (1994) Sinus aspirates and radiographic abnormalities in severe attacks of asthma. Int Arch Allergy Immunol 103, 209–13. Rudack, C., Hauser, U., Wagenmann, M., Bachert, C. & Ganzer, U. (1998a) [Cytokine pattern in various forms of sinusitis.] Laryngorhinootologie 77, 34–7. Rudack, C., Stoll, W. & Bachert, C. (1998b) Cytokines in nasal polyposis, acute and chronic sinusitis. Am J Rhinol 12, 383–8. Rudack, C., Bachert, C. & Stoll, W. (1999) Effect of prednisolone on cytokine synthesis in nasal polyps. J Interferon Cytokine Res 19, 1031–5. Rugina, M., Serrano, E., Klossek, J.M. et al. (2002) Epidemiological and clinical aspects of nasal polyposis in France; the ORLI group experience. Rhinology 40, 75–9. Ruhno, J., Andersson, B., Denburg, J. et al. (1990) A double-blind comparison of intranasal budesonide with placebo for nasal polyposis. J Allergy Clin Immunol 86, 946–53. Samter, M. & Beers, R.F. Jr (1968) Intolerance to aspirin. Ann Intern Med 68. Schapowal, A.G., Simon, H.U. & Schmitz-Schumann, M. (1995) Phenomenology, pathogenesis, diagnosis and treatment of aspirinsensitive rhinosinusitis. Acta Otorhinolaryngol Belg 49, 235–50. Schramm, V.L., Jr & Effron, M.Z. (1980) Nasal polyps in children. Laryngoscope 90, 1488–95. Schubert, M.S. (2000) Medical treatment of allergic fungal sinusitis. Ann Allergy Asthma Immunol 85, 90–7; quiz 7–101.

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Ocular Allergy Avinash Gurbaxani, Virginia L. Calder and Susan Lightman

Summary

Introduction

The spectrum of ocular allergy ranges from the nonsightthreatening conditions such as seasonal allergic conjunctivitis (SAC) (accounts for as much as 90% of all ocular allergies), perennial allergic conjunctivitis (PAC) and giant papillary conjunctivitis (GPC) to the more severe vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). Theses conditions are increasing in prevalence and can affect up to one-fifth of the population. The vast majority of ocular allergies affect the conjunctiva, the mucous membrane of the eye. Various factors have been implicated in causing ocular allergy, including infections, genetics, air pollution in urban areas, pets, and childhood. Common ocular clinical features include redness, itching and tearing. The most striking difference within this group of ocular diseases is that SAC and PAC remain self-limited without ocular surface damage, while AKC and VKC can compromise the cornea, causing ulcers and scarring, and can ultimately lead to vision loss. Most ocular allergies are IgE-mediated type I reactions (or have at least a type I component) characterized by involvement of specific humoral factors: IgE and histamine release from mast cells. In addition to the mast cell response, output of cytokines by conjunctival epithelium T cells leads to multiple cytokine appearance in tears in SAC, and also VKC and AKC. Treatment options range from simple measure like allergen avoidance and cold compresses, antihistamines, mast cell stabilizers to topical steroids and cyclosporin A in the more severe forms of disease. An understanding of the various clinical presentations and pathophysiology is vital in recognizing the similarities and differences among these diseases. In this chapter we provide an overview of these diseases. A quick revision of the relevant anatomy and immunology is followed by detailed descriptions of the clinical features, pathophysiology and treatment of the diseases. We hope to make the reader familiar with the disease mechanisms as well as provide a practical insight into their management.

Ocular allergy is a common disorder that affects 15–20% of the population in developed nations (Butrus & Portela 2005) and its incidence is increasing. Ocular involvement in allergy was first described by Charles Blackley in 1873 as part of his original description of hay fever (Bonini 2006). A large proportion of patients visit their general practitioner with allergic eye disease and it is the most frequent ocular disease seen in general practice after infective conjunctivitis (Dart et al. 1986; Sheldrick et al. 1993). Recent studies have also reported allergic eye disease to occur more frequently among certain immigrant populations in the UK (Singh et al. 2003). Ocular allergy describes a group of conjunctival disorders (Table 71.1) including seasonal allergic conjunctivitis (SAC) where the inciting allergens are often outdoor allergens such as pollens, perennial allergic conjunctivitis (PAC) where the allergens are indoor allergens such as house-dust mite (HDM), pets and cockroaches, and the more severe atopic keratoconjunctivitis (AKC) and vernal keroconjunctivitis (VKC) where sight-threatening sequelae can occur. More recently, studies have identified that this classification is too simplistic as many patients are sensitized to both seasonal and perennial antigens and have suggested a more realistic classification of intermittent and persistent disease (Ciprandi et al. 2005). The clinical severity of allergic eye disease varies according to the pathogenesis and involves different cell types in the different clinical entities. Recent progress in understanding allergic mechanisms and inflammation has brought new insights into the pathophysiology of allergic eye disease and suggests a reclassification in accordance with the multifactorial pathogenesis (Bonini 2006).

Anatomy of the outer eye Eyelids

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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The lids extend from the orbital rim to the palpebral aperture (Fig. 71.1). The anterior lamella consists of skin overlying the orbicularis oculi muscle, which is responsible for lid closure and the posterior lamella of the tarsal plate which consists of

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Table 71.1 Classification of ocular allergic diseases.

Disease

Timing

Age group

Prevalance

Keratopathy

Sight threatening

Course

SAC

Seasonal

Majority children and young adults

Very common

No

No

Mild, nonprogressive, often resolves

PAC

Perennial

Adult

Common

Common

No

Not serious, nonprogressive

AKC

Perennial

Adult

Rare

Yes

Yes

Serious and progressive, vision often reduced

VKC

Seasonal or perennial if severe

Children

Uncommon

Yes

Yes

Serious, but usually resolves in 2–10 years with good outcome if well managed. Can change into AKC

GPC

Related to exposure to precipitant

Adults

Uncommon

Occasionally, minimal

No

Reversible with removal of cause

AAC

Single or episodic

Any

Fairly common

No

No

Spontaneous resolution

AAC, acute allergic conjunctivitis. Lid crease (insertion of levator palpebrae superioris muscle)

Orbital portion of upper eyelid

Orbital septum

Bone Pre-aponeurotic fat pad

Skin

Palpebral aperture

Tarsal portion of upper eyelid

Levator palpebrae superioris muscle

Orbicularis oculi

Medial canthus Müller’s muscle

Fig. 71.1 Surface anatomy of the eyelids.

dense connective tissue containing meibomian glands tightly adherent to conjunctiva (Fig. 71.2). The orbital septum is a fibrous sheet running from the orbital rim to the tarsal plate which separates the lid from the orbit. The levator palpebrae superioris, the main upper lid retractor, originates at the orbital apex and its aponeurosis passes through the orbital septum and inserts into the lid skin and the tarsal plate. Müller’s muscle, another upper lid retractor, arises from the inferior surface of levator palpebrae and inserts in the upper border of the tarsal plate. In the lower lid a sheet of fibrous tissue analogous to the levator aponeurosis arises from the sheath of the inferior ocular muscles, and the inferior tarsal muscle is analogous to Müller’s muscle in the upper eyelid. The mucocutaneous junction (known as the gray line) runs horizontally along the lid margin. Anterior to the gray line are the lash follicles,

Tarsal plate containing meibomian glands

Conjunctiva

Fig. 71.2 Cross-sectional anatomy of the upper eyelid.

glands of Zeis (modified sebaceous glands associated with the lash follicles), and the ducts of glands of Moll (modified sweat glands). Posterior to it are the meibomian gland orifices (Fig. 71.3a,b). Approximately 25 of these modified sebaceous glands lie vertically in the tarsal plate and secrete the lipid component of the precorneal tear film. The eyelids are highly vascular and have a rich network of arteries. The lateral and medial palpebral arteries, which

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Posterior surface Meibomian gland orifice

Cilia in follicle

Gray line (mucocutaneous junction)

Gland of zeis

Meibomian gland orifice Conjunctiva

Conjunctiva

Skin Skin Eyelash follicle (a)

Orbicularis oculi

Anterior surface (b)

originate from the internal carotid artery and ophthalmic artery, supply the marginal and peripheral vascular arcades. There are also many anastomoses with branches of the external carotid artery. Venous return is conveyed by the ophthalmic, angular and superficial temporal veins. Lymphatic drainage is to the submandibular and preauricular lymph nodes. Lid sensation is supplied by branches of the ophthalmic division of the trigeminal nerve in the upper lids. The lower lids are supplied by branches of the maxillary division of the trigeminal nerve. The orbicularis oculi muscle is supplied by the facial nerve and the levator muscle by the oculomotor nerve. Müller’s muscle and the inferior tarsal muscle are supplied by sympathetic nerves arising from the superior cervical ganglion.

Tarsal plate containing meibomian glands

Fig. 71.3 Anatomy of the lid margin. (a) Cross-sectional anatomy of the lower margin. (b) Surface anatomy of the lid margin.

13 mm

5 mm

9 mm

Fig. 71.4 Surface markings of the conjunctiva.

Conjunctiva The conjunctiva is a thin, transparent, vascular mucous membrane lining the inner lid surfaces and the anterior sclera. It is involved in host defense against infection and trauma and helps maintain a suitable environment for the cornea. The conjunctiva is divided into palpebral, forniceal, and bulbar portions (Fig. 71.4). The palpebral conjunctiva covers the posterior aspects of the upper and lower lids and is firmly adhered to the tarsal plate. The bulbar portion extends from the limbus, and covers the anterior sclera, being loosely attached to the underlying tissues. In the fornices, the conjunctiva is loose and is reflected back toward the lids. The conjunctiva is continuous with the corneal epithelium at the limbus and with the skin at the gray line. The normal conjunctiva consists of a nonkeratinizing squamous epithelium between two and ten cell layers thick, overlying a basement membrane and a substantia propria of loose, highly vascular connective tissue containing lymphoid tissue. The accessory glands of Wolfring

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and Krause in the submucosa supplement the aqueous tear layer, the majority of which is produced by the lacrimal gland. Goblet cells in the epithelium secrete mucus which is hydrophilic and adheres to microvilli on the surface of the epithelial cells. This stabilizes the tear film by converting the corneal epithelium from a hydrophobic to a hydrophilic surface (Zhang et al. 2003). The tarsal and forniceal conjunctival tissues are supplied by two palpebral vascular arcades and the bulbar conjunctiva by the anterior ciliary artery via a superficial and deep plexus. The veins accompany the arteries and drain into the palpebral veins or the superficial and inferior ophthalmic veins. Lymphatic drainage parallels that of the lids. Sensation of the superior conjunctiva is mediated by the branches of the ophthalmic division of the trigeminal nerve and that of the inferior conjunctiva by the maxillary division of the trigeminal nerve. Sympathetic fibers arise from the superior

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Epithelium Bowman’s layer

Basement membrane of epithelium Stroma

Descemetic membrane Endothelium Fig. 71.5 Diagrammatic representation of the normal corneal histology.

cervical ganglion and vasomotor parasympathetic fibers from the pterygopalatine ganglion.

Cornea The cornea is the transparent anterior part of the outer coat of the eye which allows light to enter the eye. It is also the main structure responsible for the refraction of light entering the eye and the curvature of the cornea is greater than the rest of the eyeball. In an emmetropic eye the cornea is circular posteriorly with a diameter of 11.6 mm but anteriorly the cornea is more elliptical with a vertical diameter of 10.5 mm. The cornea is aspherical, with a smaller radius of curvature centrally than peripherally and consists of five layers: epithelium, Bowman’s membrane, stroma, Descemet’s membrane, and endothelium (Fig. 71.5). The corneal epithelium is stratified and consists of one or two layers of superficial cell layers: one layer of superficial, flattened, nucleated, nonkeratinized squamous cells that are shed into the tears and which are replaced from below; two layers of wing cells; and a single layer of columnar cells lying on the basement membrane. Bowman’s membrane is a dense acellular anterior portion of the corneal stroma and measures 8–12 μm. The stroma comprises 90% of the total corneal thickness and is made up of parallel lamellae of collagen fibrils surrounded glysosaminoglycans, extracellular matrix and scattered keratocytes. Descemet’s membrane is the basement membrane of the endothelium. The endothelium is a single layer that maintains the relative dehydration of the cornea and hence its transparency via an active pump mechanism. These endothelial cells cannot replicate and cell loss is compensated for by enlargement of neighboring cells. The cornea is avascular and metabolic requirements are met by diffusion from the atmosphere, tear film, and from the aqueous. Anastomoses of the ophthalmic nerves with the conjunctival nerves form a pericorneal plexus from which a superficial and deep group of nerves run into the cornea. The nerves become demylinated 1–2 mm into the cornea and some fibers of the superficial group enter the epithelium and run between the cells.

Ocular Allergy

Conjunctival immune tissue A component of the mucosal-associated lymphoid tissue (MALT), conjunctival lymphoid tissue (T and B lymphocytes) is present in the submucosal layer of the conjunctiva. MALT is important in the defense of mucosal body surfaces against exogenous pathogens (Smolin 1987). The predominant inflammatory cells in the conjunctiva are the lymphocytes. T-cell numbers are higher than B cells and CD8+ T cells outnumber CD4+ T cells (Hingorani et al. 1997). Leukocytes are frequent in the normal noninflamed conjunctiva. The mean number of leukocytes in the epithelium is 20 × 109/L (range 0–50 × 109/ L) and in the submucosa 154 × 109/L (range 10–355 × 109/L) (Allansmith et al. 1978). In the normal epithelium only lymphocytes, neutrophils and macrophages are found, whereas in the normal submucosa lymphocytes, neurophils, macrophages, plasma cells, and mast cells are present (Hingorani et al. 1997). Mast cells lie mainly in relation to blood vessels and meibomian glands and 95% are of the tryptase- and chymasecontaining type (MCTC) or connective-tissue type (Irani et al. 1990; Morgan et al. 1991a). Eosinophils and basophils are not components of normal conjunctiva (Allansmith et al. 1979). Human leukocyte antigen (HLA)-DR-positive dendritic cells are found in the epithelium and stroma and are probably Langerhans cells (Bhan et al. 1982; Irani et al. 1990). These, together with HLA-DR-positive cells of the monocyte/macrophage lineage (Hamrah et al. 2004), are thought to present antigen to T cells in the MALT and local lymph nodes (Foster et al. 1991). Immunoglobulins are present in normal human tears, IgA being predominant (IgA > IgG > IgE > IgM), with more IgA plasma cells present than other plasma cells (Little et al. 1969; McClellan et al. 1973; Sompolinsky et al. 1982). At least some of the IgA and IgE is produced locally and this proportion increases in allergic disease (Aalders-Deenstra et al. 1985; Somos et al. 2001). As in other mucosal secretions, tear IgA is dimeric, with a secretory component added by conjunctival epithelial cells (Sompolinsky et al. 1982). Several proinflammatory chemokines and cytokines are detected at low levels in normal tears (Leonardi et al. 2006a; Sack et al. 2006) which are selectively upregulated during both acute and chronic forms of conjunctivitis (Leonardi et al. 2006b). Both conjunctival fibroblasts and epithelial cells have been shown to produce similar cytokine profiles to those detected in tear fluids, suggesting those cells as potential sources of cytokines detected in tears. The presence of low levels of cytokines in normal tears is thought to be involved in maintaining “immune surveillance” at the ocular surface (Sathe et al. 1997).

Overview of pathophysiology and disease mechanisms (Fig. 71.6) Ocular allergy was traditionally classified as a type I hypersensitivity reaction. While this still probably holds true for some ocular allergy (acute conjunctivitis of the seasonal and

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Ag

APC MHC class II

Epitope

T cell

-5 IL-4 IL -13 IL IL-6

B cell IgE

IL-3 I GM L-5 -CS F

IL-3 IL-4 EO MC

Feedback/ amplification

Histamine, tryptase LT, PG Chymase Cytokines

Granule proteins LT, PG O2 metabolites Cytokines

perennial type; Bonini et al. 1989), it is no longer tenable for the more severe forms of disease such VKC and AKC (Bonini 2006). The understood mechanism where allergen cross-links mast cell membrane-bound IgE leading to mast cell degranulation together with eosinophilic infiltration corresponds with the type of cellular and immunoglobulin response seen in SAC and PAC. Mast cell degranulation and eosinophilic infiltration are also seen in histologic specimens of other types of ocular allergy (Allansmith et al. 1979; Abelson et al. 1983a; Dart et al. 1986; Foster et al. 1991; Tuft et al. 1991). Experimental models of acute ocular allergy have been utilized to investigate the early conjunctival responses such as mast cell degranulation, while more protracted models of ocular allergy are being developed to examine the role of tissue resident cells and the chemical mediators involved (Calonge et al. 2003; Groneberg et al. 2003). The conjunctival allergen challenge model, in which the ocular surface is challenged with an allergen to artificially induce an allergic response, is performed in previously sensitized individuals (Friedlander 2004). The symptoms resemble those found in SAC and therefore the model is useful for investigating the early- and late-phase allergic responses

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Fig. 71.6 Overview of the pathogenetic mechanisms in allergic eye disease.

at the ocular surface. An increase in histamine and tear tryptase can be detected in tears during the early phase (20 min), suggesting the infiltrating cell population to be predominantly mast cells, whereas in the late phase (6 hours) histamine and eosinophil cationic protein levels are increased but not tryptase, suggesting that basophils and eosinophils are the predominating cell types (Bacon et al. 2000). This allergen challenge model is often used for testing the efficacy of eye drops. Another model of allergic conjunctivitis has also been established in mice (Magone et al. 2000) by an initial footpad sensitization with short ragweed pollen followed, 7–10 days later, by conjunctival allergen challenge, in genetically susceptible hosts. An infiltration of mast cells, neutrophils and eosinophils occurs, as well as increased conjunctival chemosis and lid edema. Interestingly in both interferon (IFN)-γ- and interleukin (IL)-12-deficient mice, there was significantly less conjunctival inflammation, supporting a role for Th1 cytokines in this model (Magone et al. 2000, Stern et al. 2005). Stimulation of mast cell degranulation using compound 48/80 in the rabbit is followed by a dramatic conjunctival eosinophil infiltrate, the density of which correlates with the

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degree of mast cell stimulation (Abelson et al. 1983b). Injection of antigen into the ocular adnexa in order to induce “ocular anaphylaxis” in animals produces a massive mast cell degranulation, which returns to baseline in 24 hours (Allansmith et al. 1983). One of the major mast cell mediators, histamine, causes vasodilation, increased postcapillary permeability, chemokinesis of eosinophils and neutrophils, and includes the release of substance P by afferent nerve fibers via antidromic reflex (Schwartz 1988). These models are therefore useful for understanding the cells and mediators involved during the conjunctival inflammatory response. Considerable understanding of the effects of histamine at the ocular surface has come from the histamine conjunctival provocation test, which mimics ocular allergic signs, including itching, redness, watering, chemosis, and mucus production (Abelson & Udell 1981), and involves both H1 and H2 receptors. In humans the H1 receptor appears to be important in vasodilation, itch and burning sensation, all of which can be induced and blocked specifically by H1 agonists and antagonists, respectively. A selective H2 agonist has been shown to lead to human conjunctival vasodilation without inducing mucus production, chemosis or itch (Abelson & Udell 1981). Raised total or specific IgE is frequently seen in tears or serum, with a high rate of positive skin-prick tests (Ballow & Mendelson 1980; Baryishak et al. 1982; Donshik & Ballow 1983, Samra et al. 1984; Kari et al. 1985; Dart et al. 1986). The pathophysiologic features of SAC and PAC are mainly type I hypersensitivity reactions, involving mast cells, eosinophils and neutrophils. In sensitized individuals, the allergen cross-links IgE antibodies on the surface of mast cells and basophils, resulting in degranulation of these cells with release of mediators including histamine, prostaglandins, leukotrienes, kinins, chemokines, and cytokines. However, in chronic ocular allergy such as giant papillary conjunctivitis (GPC), VKC and AKC, other immune mechanisms are also involved as the cellular infiltration is more mixed, involving T cells as well as eosinophils, mast cells and neutrophils. Both VKC and AKC can severely compromise the cornea, with ulcers and scarring that may ultimately lead to visual loss by immunopathogenic mechanisms involving a combination of IgE and T cell-mediated responses (Calder et al. 1999; Leonardi et al. 1999; Maggi et al. 1991; Montan et al. 2002). Proinflammatory cytokines such as IL-4, IL-5 and IL-13 are produced by Th2 lymphocytes. Th2 cytokines and chemokines increase the number of activated eosinophils which, in turn, release proinflammatory and epitheliotoxic mediators found in tears and tissues of patients with AKC and VKC (Trocme et al. 1993; Abu El-Asrar et al. 2000, 2003; Leonardi et al. 2000; Futjitsu et al. 2005; Leonardi et al. 2006a,b). Although a predominately Th2 cytokine expression was found in VKC (Matsuura et al. 2004), IFN-γ-secreting (Th1) CD4+ T cells were also identified by flow cytometry (Leonardi et al. 1999) in tears from patients with active VKC and from conjunctival biopsy specimens in AKC (Calder et al. 1999).

Ocular Allergy

Cytokines derived from conjunctival T cells have been reported to have stimulatory effects on fibroblasts (Leonardi et al. 2003; Fujitsu et al. 2005) including increased IL-6, IL-8 and adhesion molecule expression, particularly intercellular adhesion molecule (ICAM)-1 (Maggi et al. 1991; Leonardi et al. 2006a). These adhesion molecules, often expressed by stromal cells, interact with inflammatory cells, favoring their persistence and activation in connective tissue and leading to a perpetuation of conjunctival inflammation. Experimental results have shown that both Th1 and Th2 cytokines are produced and expressed in tear-derived cells during the active phase of severe ocular allergy as a consequence of immune-mediated disease. Thus it is now thought that in GPC, VKC and AKC, types I, II and IV hypersensitivity responses are involved, affecting T cells, eosinophils, mast cells, and neutrophils. During type IV hypersensitivity responses, T cells are activated, leading to cytokine production. Antigen binding to the T-cell receptor on the T cell stimulates cells to secrete cytokines, macrophages become activated, and the synthesis and secretion of cytokines by T cells takes up to 72 hours (delayed-type hypersensitivity). Atopic individuals have a defect in immunomodulatory T cells responsible for regulating IgE production to allergens. However, type IV hypersensitivity is only one of the mechanisms involved in the pathogenesis of AKC. The immunopathologic characteristics of AKC specimens are similar to those in cicatricial pemphigoid and others, emphasizing that fibroblast activation, proliferation and production of cicatrization may result from a variety of chronic conjunctival inflammatory responses involving T cells, macrophages and mast cells. The histopathologic findings of the conjunctiva of patients with AKC suggest that AKC is characterized by mast cell, eosinophil and T-cell infiltration, along with chronic mononuclear cell infiltration prominent in the substantia propria. In VKC, type I hypersensitivity responses play a role as well as other mechanisms, particularly type IV hypersensitivity. The histopathology of the conjunctival papillae shows large collections of mononuclear cells, fibroblasts and newly secreted collagen. It has been demonstrated that the conjunctiva of patients with VKC contains mainly CD4+ T cells which secrete IL-4 on stimulation. The etiology of GPC is yet to be understood, but is thought to include type IV hypersensitivity reactions against the contact lens material itself, protein deposits coating the lens or lens solutions and, due to trauma to the tarsal conjunctiva, results in release of neutrophil chemotactic factors and other inflammatory mediators. Most of these patients are not atopic.

Seasonal allergic conjunctivitis The most common type of ocular allergy, SAC, is recurrent and often seen during the pollen season. It is currently increasing in prevalence and along with PAC is often underdiagnosed

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Table 71.2 Incidence of atopy in ocular allergic disease. Disease

Prevalence (%)

SAC PAC VKC AKC GPC

84 79 75 100 No increased rate

and undertreated. (Bielory 2006) It is common in both adults and children who become sensitized to outdoor antigens such as tree, grass and ragweed pollen. These allergens initiate an immune response by cross-linking mast cell-bound specific IgE to cause mast cell degranulation. However, many patients may also be sensitive to indoor antigens such as from pet hair and HDM and they may have symptoms which are not purely seasonal and have a mixed picture. In addition some plants have long pollination seasons and this may be variable in different countries. The pollen count varies according to the location of the patients, i.e., whether city or rural, and warm dry weather also tends to worsen symptoms. A personal and/or family history of atopy is common (Table 71.2) and often there is associated seasonal rhinitis. In patients with seasonal allergic rhinitis, 65% had ocular symptoms (Ciprandi et al. 2005). Eye symptoms usually occur in both eyes and include itching, burning, redness, watering, and eyelid swelling. Patients can sometimes present with lid edema which may be dramatic (Friedlander 1993, 2004). Slight conjunctival hyperemia and edema cause milky or pinkish appearance, with chemosis occurring infrequently. Examination of the tarsal conjunctiva reveals a diffuse micropapillary reaction (papillae < 0.3 mm in diameter). Ocular examination outside the pollen season is completely normal unless sensitization to other antigens has occurred.

Pathology Conjunctival scrapings have been shown to contain eosinophils or eosinophil granules (Abelson et al. 1983a; Dart et al. 1986). Histologic examination reveals the migration of mast cells into the epithelium, and an increase in stromal connective tissue mast cells has also been noted (Irani et al. 1990; Morgan et al. 1991a; Bacon et al. 2000).

Immunoglobulins There have been several reports on the level of total and specific immunoglobulins in SAC patients. Dart et al. (1986) found raised levels of total IgE in the serum of 78% and in the tears of 98% of patients. Serum IgE reactivity profiles can also vary between different European populations (Moverare et al. 2002) and systemic changes in IgE have been found even when symptoms are local (Szabo et al. 2000). Aalders-Deenstra

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et al. (1985) found the increase in total tear IgE to be a function of being atopic rather than of the eye disease itself. There is also an increased frequency of pollen-specific IgE in the tears in SAC but this varies. In addition many patients have low levels of locally produced pollen-specific IgG in their tears (Ballow et al. 1983). It is thought most likely that the pollen antigens which are airborne dissolve in tears, diffuse into the conjunctiva, and initiate an immune response, thereby crosslinking mast cell-bound specific IgE and causing mast cell degranulation.

Mediators Release of preformed histamine and newly generated mediators derived from mast cells are involved in the generation of the seasonal allergic response (leukotrienes, prostaglandins, cytokines, etc.). Allergen challenge in the presence of active SAC produces a rapid increase in tear histamine in two-thirds of patients, and a clinically detectable response occurs in 100% (Kari et al. 1985). In the conjunctiva, histamine has been shown to induce various physiologic and immunologic changes through both H1 and H2 receptor stimulation. Histamine binding to conjunctival H1 receptors (through the phospholipase C-dependent inositol phosphate pathway) leads to the symptom of pruritus while histamine stimulation of conjunctival H2 receptors has been indirectly shown to cause vasodilation (Bielory & Ghafoor 2005). There is also a significantly greater proportion of individuals with raised levels of tear tryptase in active SAC than in controls (Butrus et al. 1990). The actions of platelet-activating factor (PAF) include vasodilation, alteration of vascular permeability, platelet aggregation, monocyte, neutrophil and eosinophil chemotaxis, and eosinophil activation, degranulation and secretion (Wardlaw et al. 1986; Kroegel & Matthys 1993). PAF may play a role in chronic stages of allergic conjunctivitis by inducing eosinophil mediator release (Kato et al. 2003). Eosinophil chemotactic protein, a marker of eosinophilic activation, was found to be increased in tears and serum in SAC, although levels did not correlate with clinical signs and symptoms (Acar et al. 2003). Cytokines and chemokines are either stored preformed within mast cells, released immediately upon degranulation, or are synthesized on mast cell activation. These cytokines and chemokines are thought to play an important role in recruiting other cell types to the conjunctiva, including eosinophils and neutrophils, during SAC and PAC. Cytokine levels in tears can be a useful indicator of immune mechanisms occurring during allergic conjunctivitis (reviewed by Cook et al. 2004). Tear levels of eotaxin-1, a chemokine for eosinophils, were significantly upregulated in SAC and correlated with clinical scores (Eperon et al. 2004), and IL-1β, IL-2, IL-5, IL-6, IL-12, IL-13, and macrophage chemotactic factor (MCP)-1 levels were also found to be elevated in SAC in comparison with controls (Leonardi et al. 2006a). Conjunctival biopsies following allergen conjunctival provocation test in patients with SAC outside the pollen season

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showed a significant increase in the numbers of mast cells, eosinophils, neutrophils, macrophages, and basophils at 6 hours post challenge (Bacon et al. 2000). Although the clinical signs are biphasic, histology suggests a continuous process (Bonini et al. 1989). While this test is reproducible, there is no correlation with seasonal symptom severity (Radcliffe et al. 2006).

Treatment Simple measures such as reducing antigenic exposure by avoiding grassy fields, trees and flowers, keeping windows shut and staying indoors on high pollen count days decrease symptoms. Nonspecific measures, including cold compresses or conjunctival irrigation with normal saline or artificial tears, aid comfort and may dilute allergen or reduce time of allergen– conjunctiva contact. In SAC and PAC management begins with avoidance of known allergens. Iced artificial tears or cold compresses may suffice to relieve mild symptoms. Oral and topical antihistamines with multiple histamine receptor binding activities provide an improved treatment paradigm for the various signs and symptoms of ocular allergy (Table 71.3). The receptor affinities of ketotifen, pyrilamine, and epinastine appear to have the strongest H1 and H2 affinities (Bielory & Ghafoor 2005) among the histamine H1, H2 and H3 receptors. Mast cell stabilizers act by preventing calcium influx across mast cell membranes thus inhibiting degranulation, and also have some inhibitory effects on neutrophils and eosinophils. They should be used prophylactically for maximal effectiveness. More recently, the development of topical antiallergic drugs, possessing both mast cell stabilizing and antihistaminic properties, has led to improved treatment options. Excep-

Table 71.3 Management of SAC. Nonspecific Cold compresses Saline irrigation Topical lubricants, e.g., hypromellose four times daily (q.d.s.) Allergen-related Immunotherapy Systemic specific Oral antihistamines, e.g., terfenadine, cetirizine, fexofenadine, loratadine Topical specific Antihistamine/vasoconstrictor combinations, e.g., antazoline/ naphazoline q.d.s. New antihistamine: levocabastine 0.05% b.d. to q.d.s. Mast cell stabilizers Sodium cromoglycate 2% or 4% q.d.s. ± occasional cromoglycate nocte Nedocromil 2% q.d.s. Lodoxamide 0.1% q.d.s. New combination drugs: Relestat, Patanol NSAID: ketorolac tromethamine 0.5% q.d.s.

Ocular Allergy

tionally in severe cases short-term topical corticosteroids may be used with ophthalmic supervision but their long-term use is discouraged as they can be associated with cataracts, glaucoma, and superinfections of the ocular surface. Vasoconstrictors (e.g., naphazoline) are effective in reducing symptoms and signs, particularly of hyperemia (Abelson et al. 1990). There has been concern that chronic vasoconstrictor use may lead to tolerance, rebound vasodilation, and long-term vessel dilation. However, vasoconstrictors are no longer recommended as they are nonspecific in their action and do not target the inflammatory cascade (Leonardi 2005). Topical antihistamine, used in combination with a vasoconstrictor, is commonly used for relief of symptoms. These components have been demonstrated to suppress the conjunctival reaction to histamine or allergen (Abelson et al. 1990). The widespread use of sodium cromoglycate (SCG) in SAC is based on strong evidence of its efficacy. The mechanism of action in allergic disease was previously ascribed to mast cell membrane stabilization via alteration in calcium flux, despite the fact that it is only a moderately effective inhibitor of mast cell degranulation (Cox 1971; Spataro & Bosman 1976; Kay et al. 1987; Leonardi et al. 2003). Data suggest that SCG has other important actions, such as inhibition of chemotaxis, activation, degranulation and cytotoxicity of human neutrophils, eosinophils and monocytes (Kay et al. 1987; Beauvais et al. 1989; Bruijnzeel, et al. 1990). Open trials have shown SCG to be effective in symptom reduction in SAC (Leino & Tuovinen 1980) and many randomized double-blind trials have confirmed a significantly greater relief of symptoms, as assessed by patients and clinicians, by SCG compared with placebo for both the 2% (Lindsay-Miller 1979; Nizami 1981; Kray et al. 1986) and 4% (Greenbaum et al. 1977; Olkowska & Cavanagh 1978; Welsh et al. 1979; Friday et al. 1983) eye drops delivered four times daily. Levocabastine is a topical H1 receptor-specific antagonist that has rapid onset of action and is extremely potent (Parys et al. 1992). Levocabastine was more effective than artificial tears in controlling acute symptoms of allergic conjunctivitis, demonstrating that the selective H1 receptor antagonist action of this drug is rapidly effective in a clinical setting in a recent study (Fujishima et al. 2006). In grass and birch pollen SAC, levocabastine has been shown to be significantly more effective than placebo or conventional topical antihistamine/ vasoconstrictor preparations in the relief of ocular symptoms and was associated with a reduction in the use of “rescue medications” (Pipkorn et al. 1985; Bende & Pipkorn 1987; Azevedo et al. 1991; Davies & Mullins 1993). Also, the onset of action of levocabastine is more rapid and it is very well tolerated (Azevedo et al. 1991; Janssens & Blockhuys 1993). Oral antihistamines, such as terfenadine and clemastine, are frequently used. Newer second-generation antihistamines (cetirizine, fexofenadine, loratadine, desloratadine) are preferred over older first-generation antihistamines in order to

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avoid the sedative and anticholinergic effects associated with first-generation agents (Bielory et al. 2005). Nedocromil sodium 2% is a high-potency topical mast cell stabilizer with other antiallergic actions, including inhibition of granulocyte and monocyte chemotaxis, activation and mediator release, and possibly inhibition of mediator action (Gonzalez & Brogden 1987; Moqbel et al. 1988; Bruijnzeel et al. 1989; Dahlen et al. 1989). There have been a number of controlled, masked trials comparing nedocromil sodium with placebo in SAC, and in all nedocromil sodium has shown significant superiority in the relief of symptoms and in patient satisfaction (Hirsch et al. 1988; Jarmoszuk et al. 1988; Stockwell & Easty 1988; Leino et al. 1990; Blumenthal et al. 1992; Migloir et al. 1993; Bielory et al. 2005). Lodoxamide (0.1% drops four times daily) is a mast cell inhibitor that is also effective in treating SAC. One randomized controlled trial of 4 weeks’ duration compared the use of lodoxamide tromethamine 0.1% with placebo for the treatment of allergic conjunctivitis in adults. Those using lodoxamide (N = 14) reported significantly fewer symptoms of lacrimation, burning and itching, photophobia and eyelid swelling compared with those using placebo (Cerqueti et al. 1994; Bielory et al. 2005). Fahy et al. (1992) found lodoxamide to have an earlier onset of action and a greater clinical improvement in SAC compared with SCG 2% and with very few side effects (mild stinging only). Most studies show improvement in symptoms post provocation, and in allergic conjunctivitis, especially for symptoms of itching (the hallmark symptom of allergic conjunctivitis), in those treated with antihistamines compared with those given placebo. There is no evidence from the randomized controlled trials identified to support the use of one type of topical antihistamine over another. In a three-armed trial of two antihistamines and one corticocorticosteroid conducted after induction of allergy by a conjunctival provocation test, global score was reduced significantly more for the eyes treated with mequitazine and levocabastine than dexamethasone at all evaluation times. The randomized, double-masked, intraindividual trial assessed efficacy of 0.05% mequitazine versus 0.05% levocabastine versus 0.1% dexamethasone on clinical signs/symptoms (hyperemia, itching, tearing, chemosis, and palpebral edema) with a global score obtained by measuring hyperemia and itching intensity (Richard et al. 2005) A randomized, double-masked, contralateral study following conjunctival allergen challenge was performed to compare efficacy of 0.05% levocabastine versus olopatadine 0.1% (Abelson & Greiner 2004). Ocular discomfort was graded on clinical symptoms/signs (ocular itching and redness scores) and results found that olopatadine-treated eyes had both significantly lower itching (at 3 and 10 min post challenge) and redness scores (at all time points post challenge). A two-armed, prospective, double-masked, randomized, contralateral eye study of levocabastine 0.05% versus emedas-

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tine 0.05% or emedastine versus placebo was conducted after conjunctival allergen challenge (Netland et al. 2000). Emedastine 0.05% was found to be significantly more efficacious at reducing ocular itching at 10 min and 2 hours, although they were statistically equivalent in reducing conjunctival redness. Abelson et al. (2004) conducted a randomized, placebocontrolled, double-masked, environmental study during the spring allergy season, appraising the efficacy of olopatadine 0.2%. Simultaneous analysis of the pollen levels (frequency based) and pollen period (severity based) were performed to account for the influence of environmental variables on clinical differences in ocular itching and redness between treatment groups. This study showed that olopatadine 0.2% was associated with significant reduction in both calculated mean scores for ocular itching and redness by pollen level and by pollen period. Dual-action agents like ketotifen fumarate 0.025% ophthalmic solution were well tolerated and effective in reducing the signs and symptoms of SAC, and in preventing their recurrence (Kidd et al. 2003). Ketotifen consistently showed the best efficacy in comparison with both placebo and levocabastine. This indicates that ketotifen eye drops are a valuable treatment option for this condition. There has been interest in the use of nonsteroidal antiinflammatory drugs (NSAIDs) in the treatment of allergic eye disease. Two trials with ketorolac tromethamine 0.5% in SAC have shown a significantly better reduction in symptoms and signs measured by patients and clinicians than with placebo (Ballas et al. 1993; Tinkelman et al. 1993). Corticosteroids, which are highly effective suppressors of allergic inflammation, are not indicated in the treatment of SAC, as the serious nature of the side effects of treatment far outweigh the problems of the disease. In a randomized controlled trial comparing epinastine with levocabastine, epinastine-treated patients reported significantly less ocular itching than those receiving vehicle (P = 0.045). Ocular itching and hyperemia scores were similar between the epinastine and levocabastine groups. No clinically or statistically significant between-group differences were seen in slit-lamp biomicroscopy findings, changes in visual acuity from baseline, or the incidence of treatment-related adverse effects (Whitcup et al. 2004). Immunotherapy, either parenterally or orally, has been shown to be effective in reducing symptoms and conjunctival provocation test reactivity in patients with SAC and rhinitis (Bjorkstein et al. 1986; Moller et al. 1986), without detectable alterations in immune responses (Rolinck-Werninghaus et al. 2005). In a recent randomized, placebo-controlled, doseescalation trial, patients (N = 43) out of season were given grass allergen tablets sublingually for 28 days and the tablets were well tolerated and safe (Calderon & Essendrop 2006). However, immunotherapy is usually reserved for the most serious cases due to the potential risks and long duration of therapy required.

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Acute allergic conjunctivitis Acute allergic conjunctivitis (AAC) is an acute urticarial reaction in the lids and conjunctiva. It is moderately common but is usually mild and often patients will not be seen by an ophthalmologist; if associated with systemic effects, then patients may present to a physician.

Clinical features The patient complains of sudden-onset itching with mild redness, watering and swelling of the lids and conjunctiva. The ocular signs may be unilateral or bilateral. The lids are edematous, slightly hyperemic, and perhaps vesiculated and the conjunctiva is chemosed and mildly hyperemic (Figs 71.7 and 71.8). If there is a systemic reaction, there may be dyspnea and wheeze, skin urticaria, swelling of the lips and, in the worst cases, hypotension, laryngeal edema and cardiac arrhythmias. AAC will spontaneously resolve within 24 hours provided there is no more exposure to the offending allergen.

Ocular Allergy

In cases where there have been serious systemic effects, no diagnostic exposure to the allergen should be performed. Radioallergosorbent test (RAST) may help, but since it measures free and not mast cell-bound IgE, false negatives occur regularly (Lawlor 1993). In the milder cases, the diagnosis may be made by controlled allergen exposure, using the rub test or patch testing, with a short interval of exposure (20 min), looking for a wheal-and-flare or vesicular reaction at 45–60 min. No work exists on ocular and lid histology, but in the skin there is marked stromal edema and a variable perivascular infiltrate of lymphocytes, neutrophils and eosinophils. Elsewhere in the stroma, degranulating mast cells can be seen, surrounded by degranulating eosinophils, with major basic protein (MBP) deposition (Ormerod 1993).

Pathogenetic mechanisms The reaction is due to type I hypersensitivity and mast cell degranulation, with effects mediated by histamine, tryptase, phosphorylcholine, and leukotrienes; kinins and complement are activated and substance P and other proinflammatory neuropeptides may be involved (Ormerod 1993). Both systemic allergens (e.g., food, drugs, insect bites) and local allergens (e.g., topical medication, food transferred via hands) can precipitate the reaction. Occasionally it can occur after minor trauma, such as rubbing the eyes, or exercise (Lawlor 1993).

Treatment A systemic reaction will need urgent treatment with parenteral epinephrine, antihistamine, volume-expanding intravenous agents, and resuscitative measures. In a purely ocular reaction, drugs may not be necessary but, if severe, oral or topical antihistamines may be required.

Perennial allergic conjunctivitis Fig. 71.7 Unilateral eyelid edema causing mechanical ptosis in acute allergic conjunctivitis. (See CD-ROM for color version.)

Fig. 71.8 Localized area bulbar conjunctival chemosis in acute allergic conjunctivitis. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

In PAC the main allergens involved are indoor allergens such as from HDM, pet hairs and cockroaches. However, many patients are sensitized to other allergens such as grass pollens and may have a seasonal element as well. As with SAC, a family and/or personal history of atopy is common and many patients also have allergic rhinitis (see Table 71.2). In one recent study 46% of patients with perennial allergic rhinitis and 47% of those with mixed allergic rhinitis had symptoms of PAC (Ciprandi et al. 2005). PAC is seen fairly frequently in general practice and usually starts in the early teens (Dart et al. 1986; Butrus & Portela 2005). It is more often misdiagnosed than SAC as the clinical features may be less obvious and may also be confused with vasomotor conjunctivitis (Bielory 2006). Patients complain of perennial ocular itching, burning and discomfort, watering and redness. There may be a slight mucoid discharge and mild to moderate eyelid swelling. Approximately 79% have some seasonal exacerbation and

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Fig. 71.9 Marked chemosis in hay fever conjunctivitis. (See CD-ROM for color version.)

be responsible (Donshik 1988). Many patients give a history of exacerbation on exposure to dust; skin-prick tests to HDM are often positive (71%); and there are locally produced specific anti-HDM IgE antibodies present in 78% of tear samples (Dart et al. 1986; Bloch-Michel 1988). Proinflammatory cytokines are likely present in various combinations during ocular allergic disease. A study has demonstrated the importance of the individual and combined effects of proinflammatory cytokines, namely tumor necrosis factor (TNF)-α, IL-1β, and IFN-γ, present in allergic conjunctivitis on conjunctival epithelial cell activation (Stahl et al. 2003). TNF-α and IL-1β had similar and synergistic effects on increasing expression of ICAM-1, whereas IL-1β was a more potent upregulator of the release of IL-8 than was TNF-α. Allergenmediated mast cell activation is a key feature of ocular allergic diseases as well as eosinophil-derived mediators in tears and conjunctival biopsy specimens. Mediators released from conjunctival mast cells promote eosinophil adhesion to conjunctival epithelial cells as well as eosinophil degranulation (Cook et al. 2004).

Pathology There are increased numbers of mast cells and IgE-positive plasma cells (Bloch-Michel 1988). Eosinophils may be seen on conjunctival scrape cytology but this is negative in up to 50–75% (Abelson et al. 1983a; Dart et al. 1986). Some T cells have also been observed, which might reflect the chronicity of the response, but there is very little known about the role of these cells in PAC. Fig. 71.10 Papillary reaction in upper tarsal conjunctiva in perennial allergic conjunctivitis. (Courtesy of Mr S. Tuft.) (See CD-ROM for color version.)

it may be possible from the history to correlate an increase in symptomatology with exposure to a particular allergen. Approximately one-third have perennial rhinitis (Dart et al. 1986). In general, PAC is less severe than SAC, and the clinical signs may be minimal. The lids may show some edema, the bulbar conjunctiva may be injected and hyperemic (Fig. 71.9), or micropapillae may be seen in the tarsal conjunctiva (Fig. 71.10). There is no associated corneal disease.

Pathogenetic considerations Over two-thirds of patients have other atopic diseases and, as in SAC, a type I hypersensitivity reaction is believed to initiate the inflammatory events. Total levels of serum IgE are raised in 79% of patients and total tear IgE in all patients (Dart et al. 1986), related to atopy rather than atopic eye disease per se (Aalders-Deenstra et al. 1985). HDM (Dermatophagoides pteronyssinus) is the most common allergen thought to be involved. The mechanisms and mediators involved are considered to be broadly the same as in the sensitizing allergen, although molds, animal dander and other antigens may

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Treatment The management of PAC is similar to that of SAC and is based on the same body of experimental and clinical evidence. Few studies have addressed management questions concerning PAC alone. Allergen reduction is laborious but may be helpful: in HDM sensitivity, removal of the bedroom carpet, vacuuming of the mattress, covering the pillows and mattress in impermeable fabric; in mold sensitivity, dehumidifying devices (Christiansen 1988). Topical lubricants or saline and cold compresses will relieve itching. Vasoconstrictor/antihistamine combinations or levocabastine can be employed, but oral antihistamines are of little use. One study found levocabastine to be faster in effect and slightly better than SCG (Tiszler Cieslik et al. 1994). Anti-mast cell agents, particularly SCG but also nedocromil sodium and lodoxamide, are the most useful therapeutic agents, with minimal side effects. In comparisons of nedocromil sodium with placebo, the active preparation gives significantly better control of symptoms and a higher level of clinician and patient satisfaction (El-Guindi & El-Zawahry 1994; Van Bijsterveld et al. 1994). A larger and longer study found nedocromil sodium four times daily better than nedocromil sodium twice daily and both were better than placebo in reducing symptoms (Adenis 1994). Topical azelastine progressively improved itching and conjunctival redness in PAC

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patients compared with placebo and was at least as effective as levocabastine (Canonica et al. 2003). Corticosteroids are not indicated, and other drugs, such as human IgE pentopeptide (HEPP) and NSAIDs, are of uncertain benefit.

Vernal keratoconjunctivitis VKC is a rare but serious ocular allergy that accounts for approximately 0.1– 0.5% of ocular disease and usually affects boys particularly between the ages of 3 and 25 years (Allansmith & Ross 1988a; Buckley 1988a). In 80% the onset occurs before 14 years and 85% are male (Allansmith & Ross 1988a; Buckley 1988a). In some cases it is associated with other “allergic” disorders such as atopic dermatitis, asthma and allergic rhinitis (Tanaka et al. 2004).

Clinical features VKC is a chronic bilateral inflammation of the conjunctiva characterized by hyperemia, chemosis, photophobia, and filamentous and sticky mucous discharge (Bonini et al. 2006). Conjunctival signs are maximal in the upper tarsal area may be quite asymmetrical. There is hyperemia, edema and infiltration of the conjunctiva, with a papillary response including giant (cobblestone) papillae (diameter > 1 mm) (Fig. 71.11). Reticular subepithelial scarring may be seen (Fig. 71.12) and tenacious mucus can make conjunctival examination difficult. At the limbus, gelatinous nodules, thickening, broadening and opacification may occur and Trantas dots (small white focal collections of eosinophils) are seen at the tips of limbal papillae (Figs 71.13 and 71.14). Itching, photophobia, burning, and tearing are the major ocular symptoms. Patients also complain of frequent conjunctival redness after exposure to nonspecific stimuli. This finding supports previous reports suggesting the presence of conjunctival hyperreactivity when sun, dust, wind, and other general climactic factors or nonspecific stimuli come in contact with the conjunctival mucosa (Bonini et al. 1992). In the cornea, superficial keratopathy especially superiorly is common and there may be shield ulcers (occurring in approximately 3–11%) and neovascularization can also occur (Bonini et al. 2000). Several stages have been identified (Table 71.4) (Buckley 1981). Blepharitis is frequently associated and eczema or maceration of the lids can be observed. Cataract and corticosteroidinduced glaucoma can also occur (Tabbara 1999). With correct management, the majority will resolve spontaneously after 2–10 years or at about 16–25 years of age without major corneal scarring. Occasionally, this disease metamorphoses into AKC in adulthood.

Histology The submucosal infiltrate consists of eosinophils, mast cells, basophils, plasma cells, lymphocytes and some neutrophils

Fig. 71.11 Upper tarsal giant papillae in vernal keratoconjunctivitis. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

Fig. 71.12 Scarring and giant papillae in upper tarsal conjunctiva in vernal keratoconjunctivitis. (See CD-ROM for color version.)

and fibroblasts. (Morgan 1971; Collin & Allansmith 1977; Allansmith & Baird 1978; El-Asrar et al. 1989); an increased number of activated macrophages are also present (Metz et al. 1997). Large papillae are obvious, immature lymphoid follicles occur, and there is marked collagen deposition (Morgan 1971; Collin & Allansmith 1977; El-Asrar et al. 1989). Endothelial cell swelling and death are visible in submucosal

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Fig. 71.13 Active limbal inflammation in vernal keratoconjunctivitis. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

Fig. 71.14 Limbal nodules in vernal keratoconjunctivitis. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

Table 71.4 Classification of vernal keratopathy. Stage 1 Punctate epithelial keratitis: discrete or coalescent, usually seen as a gray speckling of the epithelium with fluorescein staining Stage 2 Epithelial macroerosion (a discrete epithelial defect) Stage 3 Plaque ulcer: shallow; round or oval “shield-like” with poor wetting by the tears and overlying mucus/epithelial debris (Fig. 71.15) Stage 4 Subepithelial scarring: ring scar, peripheral tongue-shaped scar, punctate scars Stage 5 Pseudogerontoxon: a limited arcus-like lesion, in the shape of a crescent, which waxes and wanes and is related to, but separate from, the superior limbus

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Fig. 71.15 Vernal plaque ulcer stained with rose bengal. (See CD-ROM for color version.)

blood vessels, with extravasation of red blood cells and fibrin (Collin & Allansmith 1977). The epithelium shows cellular proliferation with outgrowths down into the submucosa and occasional keratinization. Epithelial cells at the tips of papillae undergo mucinous degeneration and there is an increase in the number of goblet cells in the crypts between papillae (Morgan 1971). Inflammatory cells, including mast cells, eosinophils, basophils and plasma cells, migrate into the epithelium and there are many dendritic cells (Collin & Allansmith 1977; Allansmith & Baird 1978; Allansmith et al. 1979; El-Asrar et al. 1989). Since it is possible to see a large number of leukocytes in the normal conjunctiva, Allansmith et al. (1979) suggested certain findings (found in VKC and, to a lesser extent, in GPC) that can always be regarded as pathologic: eosinophils, basophils, mast cells and plasma cells in the epithelium and eosinophils and basophils in the submucosa. Mast cells in the epithelium and submucosa of VKC conjunctiva stain for surface IgE (El-Asrar et al. 1989) and although there are greater numbers of both MCTC and tryptase-containing (MCT) mast cells, the percentage of MCT cells is markedly raised (Irani et al. 1990; Baddeley et al. 1995). Ultrastructural studies have shown extensive mast cell degranulation throughout the conjunctiva (Henriquez et al. 1981). Lymphocytes are mainly of the CD4 T-cell type, although there are more CD8 cells than normal, and the CD4 cells appear to be activated, with increased surface HLA-DR (Bhan et al. 1982; Magrini et al. 1993). In addition, the expression of adhesion molecules, including ICAM-1, E-selectin, and vascular cell adhesion molecule (VCAM)-l, in conjunctival vascular endothelium is dramatically upregulated, correlating with the number of infiltrating cells (Saiga et al. 1994; Tabbara et al. 1996; Bacon et al. 1998). Histologic examination of excised vernal plaque material (Rahi et al. 1985) has shown a laminated structure tightly

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adherent to Bowman’s membrane. This is composed of a mixture of mucopolysaccharides and proteins rich in tyrosine and sulfur-containing amino acids, immunoglobulins, complement, and young and old fibrin. Cell debris, necrotic epithelium and a few inflammatory cells are also present.

Pathogenetic considerations Approximately 75– 80% of patients have a personal history and 65% a family history of atopic disease (Frankland & Easty 1971; Allansmith & Ross 1988a; Buckley 1988a) (see Table 71.2). Interestingly, patients from countries such as the Middle East and Israel lack the association with atopy and the seasonality of symptoms (Neumann et al. 1959; Hyams et al. 1975; El-Hennawi 1980). Biopsy specimens from patients with VKC in Kenya contained increased B-cell clustering, mast cell and eosinophil infiltration of the epithelium than those from patients with VKC in more temperate regions (Tuft et al. 1998). Studies in different ethnic groups from the same locality suggest this may have a genetic rather than an environmental basis (Dahan & Appel 1983; Tuft et al. 1989). Mean serum total IgE is significantly raised and a high serum IgE occurs more frequently in VKC than in normal controls (Allansmith et al. 1976; Easty et al. 1980; Samra et al. 1984), although this has not been a consistent result in all series (Sompolinsky et al. 1982; Ballow et al. 1983). In a case series of 195 VKC patients, conjunctival eosinophil infiltration was a constant histolopathologic finding and the larger the giant papillae, the more persistent the disease (Bonini et al. 2000). Specific IgE was present in the serum in 56% of 406 VKC cases in Italy, and 43% had positive skin-prick tests (Leonardi et al. 2006a), but this could not be related to the severity of the disease or the presence of atopy (Allansmith & Frick 1963). In the tears, total IgE is often but not always raised (Brauringer & Centifano 1971; Allansmith et al. 1976; Baryishak et al. 1982; Sompolinsky et al. 1982; Ballow et al. 1983). There are also raised levels of pollen-specific IgE in the tears in VKC and these are locally produced (Ballow & Mendelson 1980; Easty et al. 1980). Other tear immunoglobulins, including total and specific IgG and total IgM, are also at higher than normal levels and again with local production (McClellen et al. 1973; Ballow et al. 1983). Conjunctival biopsy has shown more IgE plasma cells with higher levels of extravascular IgE in the submucosa, but there are also increased numbers of IgA and IgD plasma cells (Allansmith et al. 1976).

Inflammatory mediators Investigations into tear inflammatory mediators have shown increased levels of histamine (Abelson et al. 1977, 1980b), tryptase (Butrus et al. 1990), and prostaglandin (PG)F (Dhir et al. 1979) compared with those with nonallergic ocular inflammatory conditions or normal controls. Raised levels of tear C3 and factor B components of complement (Ballow et al. 1985; Mondino & Phinney 1988) and a slight reduction

Ocular Allergy

in lactoferrin (Ballow et al. 1987a) have been documented in active VKC. Both eotaxin-1 and TNF-α were detected at significantly increased levels in cell-free VKC tear specimens (N = 18), which were undetectable in control and other allergic tears, suggesting a VKC-specific inflammatory response (Leonardi et al. 2006a). Interestingly, the chemokine profile secreted by conjunctival fibroblasts closely mirrored that detected in VKC tears, supporting these cells as the main source of tear-derived chemokines. Levels of matrix metalloproteases (MMP) have also been found to be significantly raised in VKC tears (N = 14) in comparison with healthy controls, in particular MMP-1 and MMP-9, in contrast to tear trypsin inhibitory capacity, which could explain the prolonged conjunctival inflammation in VKC (Ghavami et al. 2007). The concept of the role played by eosinophils in allergic inflammation has undergone a radical change in the last decade and eosinophils are now regarded as central to the process rather than acting purely as an effector cell (Venge & Hakansson 1991; Hansel et al. 1993). There is a heavy eosinophil infiltrate in VKC and attempts have been made to correlate the presence of eosinophils and eosinophil products to the severity of the disease. In particular, the well-recognized epithelial toxicity of eosinophil granule proteins in other tissues has led to the suggestion that differences in the release or deposition of such proteins may explain why corneal pathology is seen in only some of the ocular allergies (Venge et al. 1988; Trocme et al. 1989). In a masked assessment of conjunctival biopsies, a significantly higher deposition of MBP was seen in patients with VKC than in controls (Trocme et al. 1989). Extracellular MBP is found in plaque material or inflammatory debris overlying deepithelialized areas of cornea (Trocme et al. 1993). Both MBP and Charcot–Leyden crystal protein are significantly raised in the tears of patients with VKC compared with other ocular inflammatory conditions, atopics without eye disease or normal controls, and these levels directly correlate with severity of the disease (Udell et al. 1981). Eosinophilic cationic protein (ECP) is found in higher concentrations in the tears and conjunctiva in VKC compared with normal controls, SAC and PAC, and tear ECP levels parallel the disease state, decreasing with treatment (Saiga et al. 1991; Montan & van Hage-Hamsten 1994; Borghesan et al. 1995).

Overview of disease mechanisms Type I, type IV and basophil hypersensitivity have all been postulated to describe the inflammatory processes in VKC (Abelson & Allansmith 1987). The evidence for a type I mechanism includes the association with atopy; raised IgE levels in the tears; the increase in IgE plasma cells in the conjunctiva; the high levels of histamine and tryptase in the tears; the eosinophilic and mast cell infiltration; and the good response to SCG. However, evidence of a more complex situation includes the increased total and allergen-specific

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tear IgG; the increase in plasma cells secreting most classes of immunoglobulin; the mixed-cell infiltrate; collagen deposition in the conjunctiva; the poor response to antihistamines; and the lack of asociation with atopy in certain hot countries. VKC has been reported in association with the hyper-IgE syndrome, suggesting IgE-mediated disease but these patients have also been shown to have a T-cell bias toward a Th2 phenotype with markedly reduced IFN-γ production by their T cells (Netea et al. 2005). Similar to the bronchial hyperactivity seen in asthma (Kay 1991), an increased conjunctival reactivity to nonspecific stimuli has been reported in VKC. These patients develop more conjunctival hyperemia and have a reduced threshold of response to topical histamine compared with normal patients (Bonini et al. 1992). Whether this is of relevance or simply a function of ongoing conjunctival inflammation is unclear. T cell-mediated inflammation is suggested by the increase in CD4+ T cells (Bhan et al. 1982; Magrini et al. 1993). Early evidence suggests that T-cell cytokine production may be important. In transgenic mice overexpressing IL-4 (and with consequently high serum IgE), ocular surface inflammation is seen, with accumulation of monocytes, eosinophils and mast cells (i.e., similar to VKC lesions) (Tepper et al. 1990). Clones derived from T cells that accumulate in the conjunctiva in VKC produce high levels of IL-4 and very little or no IFN-γ, and are able to promote IgE synthesis from allogenic B cells; this is a Th2-like pattern, reminiscent of the type of T cells found in asthma (Maggi et al. 1991; Robinson et al. 1992). Immunohistochemical methods have localized IL-4 to mast cells (Baddeley et al. 1995) and in situ hybridization reveals an increase in IL-3, IL-4 and IL-5 mRNA in VKC conjunctiva (Metz et al. 1997).

Treatment Medical therapy (Table 71.5) Although the majority of affected children resolve spontaneously at the onset of puberty, up to half will have had persistent symptoms over a 5-year period and up to 5% can suffer visual compromise from corneal scarring. Management should be symptomatic and initially cold compresses and topical lubricants may aid comfort. A cool moist atmosphere (e.g., by air-conditioning) is helpful and avoiding specific inducing allergens may alleviate mild cases. SCG and the other antiallergic compounds such as lodoxamide tromethamine, nedocromil sodium, spaglumic acid, and topical antihistamines are effective in reducing signs and symptoms of the disease (Bonini et al. 1997). Use of unpreserved solutions may reduce the risk of local hypersensitivity to preservatives. Mast cell stabilizers are found to be most useful in seasonal management of VKC as they truncate the signal that would otherwise result in an increased physiologic response to allergens and proteins respectively. NSAIDs also produce a beneficial effect on the course of VKC (D’Angelo et al. 2003). Topical corticosteroids remain the

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Table 71.5 Treatment of vernal keratoconjunctivitis. General Cool and moist atmosphere Cold compresses Conjunctival saline irrigation Topical lubricants Mucolytics, e.g., acetylcysteine 5–20% q.d.s. Specific Antihistamine: usually unhelpful Mast cell stabilizers Sodium cromoglycate 2%/4% q.d.s., ointment nocte Nedocromil 2% q.d.s. Lodoxamide 0.1% b.d. or q.d.s. Steroids If keratopathy/severe disease activity: dexamethasone 0.1% every 1–2 h or prednisolone 1% every 1–2 h If mild keratopathy/high disease activity: prednisolone 0.1–0.3% o.d. or q.d.s. Oral aspirin/topical NSAID Cyclosporin A 0.005–2% Surgical Superficial keratectomy Lamellar corneal graft Penetrating corneal graft Excimer laser

Table 71.6 Ocular side effects of topical steroid use. (Data from Havener 1980.) Infective Bacterial and fungal keratitis Herpes simplex keratitis Intraocular pressure Ocular hypertension Glaucoma Lens Cataract (posterior subcapsular) Healing Abnormal corneal wound healing Inhibits epithelial regrowth

most effective form of treatment, but unfortunately have potentially sight-threatening side effects, with a quoted 2% incidence of glaucoma (Table 71.6) (Havener 1980; Bonini et al. 2000). Ideally, corticosteroid treatment is limited to short, rapidly tapering courses during periods of high disease activity or episodes of keratitis. However, this ideal may not be realistic, as maintenance of a normal life and regular schooling have to be considered and the minimum dose of topical corticosteroid for as short a duration as possible

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should be used. When there is a breach in the corneal epithelium (macroerosion or plaque), topical antibiotics should be used as antiinfective prophylaxis, especially since corticosteroid use predisposes to infection. Topical cyclosporin A 0.5–2% is effective in controlling the inflammatory process in VKC, blocking Th2 lymphocyte proliferation and IL-2 production. Furthermore, it inhibits histamine release from mast cells and basophils and, via reduction of IL-5 production, may reduce the recruitment and effects of eosinophils on the conjunctiva (Avunduk et al. 2001; Pucci et al. 2002). Cyclosporin A is effective in reducing conjunctival fibroblast proliferation rate and IL-1β production, which further contributes to reducing this lymphoproliferative disorder (Leonardi et al. 2001). More recently, short-term low-dose topical mitomycin C has been shown in one study to be an effective and safe drug for controlling acute exacerbations in patients of severe VKC refractory to conventional treatment (Jain & Sukhija 2006). In a few patients with VKC, systemic immunosuppressive treatment may be required. Oral antihistamines can reduce the generalized hyperreactivity but have little or no effect on VKC, while aspirin treatment (Chaudhary 1990) as well as oral administration of montelukast (5–10 mg once daily), an antileukotriene drug usually used in mild asthma, have been demonstrated to be effective in reducing signs and symptoms of VKC (Lambiase et al. 2003).

Surgical therapy In the past, surgical attempts to relieve conjunctival inflammation have included excision of papillae (Shimken 1938; Tse et al. 1983), cryotherapy (Amoils 1975; Singh 1982), repeated radiotherapy (Jackson 1918; Neumann et al. 1959), mucous membrane grafting (Cross 1959; Butrus & Abelson 1984), partial tarsectomy (Sugar 1962), and excision of tarsal conjunctiva with forniceal conjunctival advancement to cover the bare area (Shimken 1938), and all have been reported to be successful. However, these procedures are not widely endorsed and may cause conjunctival scarring and distortion (Rice et al. 1971; Buckley 1988a). Surgery is predominantly limited to those with corneal plaque or severe corneal scarring. In one series, 71% of those with plaque required surgery, and reepithelialization was much more rapid than with medical therapy alone (Buckley 1981). The conjunctiva should be medically quietened preoperatively and the plaque removed by superficial keratectomy under general anesthesia. This aids healing, probably by removing the nonwetting surface and by reducing the load of toxic chemicals, such as eosinophil granule proteins, which inhibit epithelial ingrowth. If the corneal structure is compromised further, a lamellar graft may be indicated. If perforation occurs or there is deep scarring reducing vision, a penetrating corneal graft may be necessary. The excimer laser is currently being evaluated for the management of superficial corneal scar tissue (Cameron et al. 1995).

Ocular Allergy

Atopic keratoconjunctivitis AKC is a potentially serious chronic blepharokeratocojunctivitis associated with atopic dermatitis (AD) and is the least common and the most poorly understood of the ocular allergic diseases. It was first described by Hogan in 1952. It is a disease of adults, with onset in the late teens and the maximum age range lying between 20 and 60 years. A male sex predominance is generally quoted (Jay 1981; Buckley 1988b; Tuft et al. 1992), although another study did not confirm this (Foster & Calonge 1990). It represents about 1% of all ocular allergy.

Clinical features Symptoms may have a seasonal component (Foster & Calonge 1990) but normally occur all year and consist of intense itching, burning, heavy mucoid discharge, tearing, and blurred vision. There is usually eczema on the face and elsewhere and often a history of other atopic disease in the patient or the family (Foster & Calonge 1990). The condition can be categorized into two types, which usually remain distinct over time. In atopic blepharoconjunctivitis (ABC) the cornea is unaffected and in AKC there is keratopathy; however, AKC is often used in the clinical context to describe both entities. The lids show eczematous changes, with thickening, induration, erythema, dryness, scaling, and scratch marks (Fig. 71.16). Lid-margin disease is usually present (Tuft et al. 1991); anterior lid margin disease manifests as erythema and scaling, sometimes with small areas of ulceration, at the base of the lashes; posterior lidmargin disease is seen as rounding and irregularity of the posterior margin, with hyperemia, dilated blood vessels and abnormal meibomian gland secretions and orifices (Fig. 71.17). There may be marked lash abnormalities and the lid anatomy may be distorted by marginal notches, eversion of the punctum, ectropion, and entropion. The conjunctiva is hyperemic, edematous and shows a papillary reaction, which may include giant papillae (Fig. 71.18). A thick mucous exudate is seen. The limbus may be involved, with infiltration, papillae and, occasionally, Trantas dots (Jay 1981). Cicatrization of the conjunctiva also occurs, appearing clinically as subepithelial fibrotic scars on the tarsus, symblepharon and shortened fornices (Fig. 71.19). Corneal involvement may be extensive and progressive. Punctate epithelial keratopathy, keratoconjunctivitis sicca, and filamentary keratopathy are the mildest manifestations and may progress to epithelial macroerosion, which can be persistent. Gradual neovascularization with or without lipid infiltrate, progressive scarring and occasionally plaque may cause severe reduction in vision secondary to corneal opacification (Fig. 71.20). Corneal complications include bacterial keratitis (especially staphylococcal), herpes simplex keratitis (which may be bilateral, severe and difficult to treat) (Easty

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Fig. 71.18 Conjunctival thickening and giant papillae in atopic keratoconjunctivitis. (See CD-ROM for color version.)

Fig. 71.19 Subepithelial conjunctival scarring in atopic keratoconjunctivitis. (See CD-ROM for color version.) Fig. 71.16 Atopic dermatitis affecting the face and eyelids. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

Fig. 71.17 Posterior lid margin disease showing irregular posterior lid margin, hyperemia, abnormal meibomian gland secretions, and some abnormally directed eyelashes. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

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Fig. 71.20 Corneal scarring in atopic keratoconjunctivitis. (See CD-ROM for color version.)

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et al. 1980), and keratoconus. One study showed reduction in visual acuity due to keratopathy in 40% of AKC patients (Tuft et al. 1991); in another, 75% of patients had developed a severe keratopathy of some kind (Foster & Calonge 1990). AKC usually does not resolve spontaneously. It is potentially blinding and, due to the high frequency of corticosteroid requirement with its consequent risks and the possible need for surgical intervention, the patients will require long-term ophthalmologic management.

Histology There is a dense cellular infiltrate throughout the conjunctiva. This consists of mast cells, eosinophils, T cells (mainly of the CD4+ subtype, with some expressing IL-2 receptor), macrophages, dendritic cells in the epithelium and submucosa and, in addition, there are neutrophils, B cells and plasma cells in the submucosa, with occasional granuloma formation (Foster et al. 1991; Morgan et al. 1991b; Bacon et al. 1993; Metz et al. 1996). Goblet cell proliferation and pseudotubular formation are seen in the epithelium, collagen deposition occurs in the submucosa, and there is a significant increase in HLA class II and adhesion molecules ICAM-1 and VCAM-1 (Foster et al. 1991; Metz et al. 1996). There appears to be a denser infiltrate of mast cells, eosinophils and neutrophils in AKC compared with ABC (Bacon et al. 1993). Conjunctival scrapings may reveal eosinophils or free eosinophil granules (Abelson et al. 1983a; Friedlander et al. 1984), and slightly raised levels of ECP have been measured in tears (Montan & van Hage-Hamsten 1994).

Pathogenetic mechanisms The incidence of any ocular involvement in AD is approximately 40% (Garrity & Liesegang 1984) and this includes keratoconus, lens opacities, and other manifestations apart from AKC (Garrity & Liesegang 1984; Rich & Hanifin 1985) (Table 71.7). It is difficult to give an accurate figure for the percentage of those with AD who have AKC, or to know why this occurs; while the lids are common sites for eczematous Table 71.7 Ocular complications of atopic dermatitis. Lids Lid dermatitis Herpes simplex infection Anterior segment Atopic blepharoconjunctivitis Atopic keratoconjunctivitis Herpes simplex keratitis Keratoconus Cataracts Anterior/posterior subcapsular fleck opacities Posterior segment Retinal detachment

Ocular Allergy

disease (Garrity & Liesegang 1984), AKC itself is uncommon. Staphylococcal colonization of the lid margins is a frequent finding in AD and it has been suggested that bacteria or their toxins could play an etiologic role in AKC via direct effects or through hypersensitivity reactions (Foster & Calonge 1990). However, no significant difference could be found in the presence of staphylococci or their products, or in the presence of antistaphylococcal immunity, between AD patients with or without ocular disease (Tuft et al. 1992). By definition, AKC has a 100% association with atopic disease and there is a family history of atopy in more than 50% of patients (Jay 1981; Tuft et al. 1991). AKC is often referred to as the adult version of VKC (Buckley 1988b) and, similarly, mast cells, eosinophils and T cells have been implicated in its pathogenesis (Foster & Calonge 1990). The concentrations of both serum and tear total IgE are significantly raised in AKC, but this is also true in those with AD and no eye disease (Tuft et al. 1991). Surprisingly, conjunctival biopsy has shown few IgE plasma cells but greater numbers of plasma cells producing other classes of immunoglobulin (Foster et al. 1991). However, plasmapheresis has been used successfully to treat a small number of AKC patients with high serum levels of IgE (Aswad et al. 1988). Pathologic investigations (see below) show a heavy T-cell infiltrate and increased expression of IL-3, IL-4, IL-5 and IL-2 in these cells, and a significant increase in CD3+ T-cell expression of IFN-γ, suggesting a role for cell-mediated immunity in this form of allergic eye disease (Metz et al. 1997). In both AKC and VKC, the T cell is considered the primary effector cell although different patterns of T-cell cytokine profiles are seen for each disease, a Th2-like profile in VKC and a shift toward a Th1-like profile in AKC. Conjunctival T-cell lines from AKC produced selective increases in IFN-γ, IL-10 and IL-13 while those from VKC produced increased IL-5. There was moderate to strong expression of IFN-γ in five of six T-cell lines from AKC (Calder et al. 1999).

Treatment Treatment involves a multidisciplinary approach (Table 71.8). Facial and lid eczema need to be controlled with emollients, topical corticosteroids and, in some cases, systemic therapy, and this is best performed in conjunction with a dermatologist. Lid-margin disease should be aggressively treated with lid hygiene, topical antibiotics, topical low-dose corticosteroid or combination corticosteroid/antibiotic ointments, and 3– 6 month courses of systemic low-dose antibiotics (usually tetracyclines). If a particular allergen seems to exacerbate the condition (e.g., dust or animal dander), then measures should be taken to reduce antigen exposure. Topical tear substitutes may help symptoms and are also indicated if the tear film is poor. Mucolytics such as acetylcysteine drops may be helpful in cases with a heavy mucoid discharge. Systemic antihistamines can be used to relieve itching and may be particularly helpful at night (Foster & Calonge 1990).

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Table 71.8 Drugs used in the treatment of ocular allergy. Drug

Trade name

Preparation

Dose

Class

Actions

Hypromellose

Drops

0.3% q.d.s.

Tear substitutes

Placebo effect; dilute and wash away antigen, soothing

Polyvinyl alcohol

Drops

1.4% q.d.s.

Acetylcysteine

Ilube

Drops

5–20% q.d.s.

Mucolytic

Dissolves and thins mucus

Antazoline/xylometazoline

Otrivine-Antistin

Drops

0.05–0.5% q.d.s.

Antihistamine/ vasoconstrictor combination

Competitive antagonism of histamine receptor plus sympathomimetic constriction of blood vessels

Antazoline/naphazoline

Vasocon A

Drops

0.05–0.5% q.d.s.

Antihistamine/ vasoconstrictor combination

Drops

0.05% b.d. or q.d.s.

Antihistamine

High-potency H1 receptor blockade

Sodium cromoglycate

Opticrom

Drops

2 or 4% q.d.s.

Mast cell stabilizer

Inhibits mast cell degranulation and activity of granulocytes

Lodoxamide

Alomide

Ointment Drops

4% t.d.s. or nocte 0.1% q.d.s.

Mast cell stabilizer

Inhibits mast cell degranulation

Nedocromil sodium

Tilavist Rapitil

Drops

2% q.d.s.

Mast cell stabilizer

Inhibits mast cell degranulation and activity of granulocytes

Azelastine

Optivar

Drops

0.05%

Mast cell stabilizer and antihistamine

H1 inhibitor

Epinastine

Elestat

Drops

0.05%

Mast cell stabilizer and antihistamine

H1 and H2 inhibitor

Ketotifen

Zaditor

Drops

0.025%

Mast cell stabilizer and antihistamine

H1 inhibitor

Olopatadine

Patanol

Drops

0.1%

Mast cell stabilizer and antihistamine

H1 inhibitor

Emadastine

Emadine

Drops

0.05%

Antihistamine

H1 inhibitor

Levocabastine

Livostin

Drops

0.025%

Antihistamine

H1 inhibitor

Levocabastine

SCG given continuously has been shown to significantly reduce symptoms and signs (although the onset of action is slow) and to reduce the corticosteroid requirement compared with placebo (Ostler et al. 1977). Jay (1981) performed an open trial, which showed a good response to SCG, followed by a placebo-controlled double-blind trial in which SCG produced a positive effect in 66% of patients. Additionally, nedocromil, lodoxamide and topical NSAIDs have been used, as well as an array of antihistamine and mast cell stabilizers, in AKC. Most of these patients will require topical corticosteroids at some point; ideally this should be in short courses, but some will require longer-term treatment (Ostler et al. 1977; Tuft et al. 1991). These patients are at a higher risk of some of the side effects of corticosteroids, namely herpetic or bacterial keratitis, glaucoma and cataracts due to long-term administration of corticosteroids. All other avenues of therapy should be explored to minimize the corticosteroid dose. Topical cyclosporin A (2% in maize oil) was found to be

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effective as a corticosteroid-sparing therapy (Hingorani et al. 1999). The newer 0.05% preparation also may alleviate signs and symptoms of severe AKC refractory to topical corticosteroid treatment (Akpek et al. 2004). Other treatments that have been used include mitomycin C and tacrolimus (Stumpf et al. 2006). The high incidence of corneal and sight-reducing complications means that procedures such as therapeutic soft contact lens fitting, cyanoacrylate tissue glue use, and lamellar and penetrating corneal grafts may be required. Again, postoperative topical corticosteroids must be used with caution.

Giant papillary conjunctivitis GPC is a foreign-body-associated papillary conjunctivitis. It occurs due to chronic inflammation after persistent conjunctival minor trauma. It is uncommon and can be seen at any age and in either sex. It is seen in 1–5% of those wearing rigid

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gas permeable lenses and 10–15% of those wearing hydrogel contact lens (Greiner 2001). A review of contact lens chemistry and the occurrence of GPC show that it can occur with any type of contact lens (Donshik 2003), but the severity of GPC and symptoms are severely reduced with the use of daily disposable soft lenses and better cleaning solutions.

Clinical features The early stages of GPC are often asymptomatic. Symptoms usually precede signs and the onset is very variable, ranging from 3 weeks to 5 years after beginning contact lens wear and occurring earlier in soft than in hard contact lens wear (Allansmith et al. 1977). The patients complain of itching, especially on lens removal, discomfort or sometimes pain, watering, blurring of vision, reduced lens tolerance, lens decentration (usually upwards), and mucus discharge and accumulation on the lens. The symptoms usually decrease with a new lens or cessation of lens wear. Clinical findings are most marked in the upper tarsal area. There is a papillary reaction, with hyperemia, edema and infiltration (Fig. 71.21); giant papillae (sometimes with fluorescein staining of the tips) may be seen but are not necessary for the diagnosis (Fig. 71.22). Strands of mucus are seen on the conjunctiva and adherent

Fig. 71.22 Giant papillae in giant papillary conjunctivitis. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

to the lens. If severe, limbal involvement may be seen. The cornea is usually not affected, although occasionally there may be mild superior punctate fluorescein staining. GPC is a completely reversible condition provided the provoking factor can be removed. With appropriate modifications of the contact lens or prosthesis and their care and wearing schedules and, in some cases, topical therapy, the vast majority will be able to resume satisfactory use of their device.

Histology The conjunctiva is thrown up into large papillae with irregular epithelial thickening and extensions of epithelium into the submucosa. There are fewer goblet cells at the papillary tips and hyperplastic goblet elements in the interpapillary crypts (Allansmith et al. 1977). The conjunctiva is infiltrated with mast cells, eosinophils, basophils, neutrophils, lymphocytes and plasma cells. There is collagen deposition in advanced disease (Allansmith et al. 1977). The histopathologic features of GPC are qualitatively similar to those in VKC but occur to a lesser degree. Conjunctival scrapings frequently show eosinophils or free eosinophil granules (Friedlander et al. 1984) and there is a significant increase in MBP deposition in the conjunctiva (Trocme et al. 1989), although MBP deposition in the contact lens is an unusual finding (Trocme et al. 1990). The epithelial mast cells are MCTC type but there is no increase in stromal mast cell numbers (Irani et al. 1990). Submucosal and epithelial mast cells are degranulated (Allansmith & Baird 1981; Henriquez et al. 1981).

Pathogenetic considerations

Fig. 71.21 Diffuse upper tarsal macropapillary reaction in giant conjunctivitis. (Courtesy of Mr R.J. Buckley.) (See CD-ROM for color version.)

The condition may be due to a combination of chronic lowgrade trauma and a hypersensitivity reaction, to the foreign material itself or, more probably, to surface biodeposits. Other reported causes include ocular prostheses (Srinavas et al. 1979), exposed monofilament sutures (Nirankari et al. 1983), extruded scleral buckles, and cyanoacrylate glue (Buckley

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1993). The probability of developing GPC during contact lens wear is increased by long daily wear time, long-term consistent wear, larger lens diameter, soft lens wear, heavy lens deposition, and idiosyncratic reactivity to certain lens materials (Allansmith & Ross 1988b). Atopy is found no more frequently than in control populations (Allansmith et al. 1977). However, the total tear IgE is raised in GPC (particularly in the worse eye) compared with contact lens wearers without GPC; tear IgG and IgM are also higher, but IgA is not (Donshik & Ballow 1983). These higher levels are due to local production. However, there is no deposition of IgE on contact lenses in GPC (Richard et al. 1992), nor is there any significant difference in the degree of lens deposition of lactoferrin, lysozyme or other immunoglobulin classes, except for IgM, as compared with nonsymptomatic lens wearers (Richard et al. 1992). There are increased levels of histamine and tryptase in the tears of patients with GPC (Allansmith & Baird 1981; Butrus et al. 1990). Tear C3 and factor B are higher in active GPC (Ballow et al. 1985; Mondino & Phinney 1988) and there are lower concentrations of lactoferrin (Ballow et al. 1987a). Deposits occur on all contact lenses during wear, and lysozyme, lactoferrin, albumin, PMFA (protein fraction migrating faster than albumin), IgA, IgG and protein G are all seen on the lenses of normal wearers (Lin et al. 1991). These are not completely removed by routine cleaning (Fowler & Gaertner 1990). The condition can be alleviated by regular enzymatic protein removal or new lenses, which implies that lens deposition is involved in the pathogenesis. Monkeys have been shown to mount an increased GPC-like ocular surface response to contact lenses from patients with GPC when compared to lenses from asymptomatic wearers (Ballow et al. 1987b, 1989). While eosinophils have been associated with GPC, a recent study found that eotaxin and eotaxin-mediated eosinophil recruitment did not seem to play a major role in the immunopathology of chronic GPC associated with an ocular prosthesis (Sarac et al. 2003). There was no significant difference in tear eotaxin levels between patients with GPC and healthy subjects, and in fact in patients with chronic GPC the tear eotaxin levels were significantly lower, suggesting no major role for eosinophils.

Treatment If the offending foreign material is removed, a dramatic improvement is usually seen within days (Allansmith et al. 1977) and the condition will resolve. This is the treatment in those with protruding sutures or explants but, in the case of contact lenses or prostheses, most patients will wish to continue wearing the device. Usually a short period without lens or prosthesis wear is recommended until the acute findings have subsided. The contact lens hygiene regimen may need to be modified; careful and thorough hygiene is essential and cleaning/soaking solutions containing preservatives should

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be avoided where possible. Regular and frequent protein removal will also help. Alterations to the lens/prosthesis itself can be beneficial, such as providing a new (nondeposited) lens, improving the fit, improving the surface quality (e.g., by polishing), changing the edge profile, reducing the diameter, or changing the material of the lens (e.g., change soft to rigid lenses). The wearing time may need to be reduced temporarily or permanently. SCG is widely prescribed and is thought to have a beneficial effect (Molinari 1982); one small open trial showed a helpful effect in reducing the symptoms and signs with 2% or 4% SCG (Meisler et al. 1982). In a double-blind trial, nedocromil sodium reduced itch and mucus production in the first 3 weeks only; results in increasing lens tolerance were equivocal (Bailey & Buckley 1993). Topical suprofen 1% four times daily has been evaluated against placebo in 75 patients and found to significantly suppress symptoms and clinical findings, but it is not in common use for this indication (Wood et al. 1988). Corticosteroid drops can only be justified in those with a blind eye who are using a prosthesis long term, in whom risks to the sight can be discounted.

Investigations and differential diagnosis in ocular allergy Clinical features The differential diagnosis of ocular allergic conditions includes a wide range of ocular surface and inflammatory disorders: infective conjunctivitis (viral, bacterial and Chlamydia D), anterior and posterior lid-margin disease, keratoconjunctivitis sicca, mucous membrane pemphigoid, foreign body (especially subtarsal), superior limbic keratoconjunctivitis, episcleritis, and scleritis. It is possible to mistake conjunctival granulomas or neoplasms for giant papillae in diseases such as sarcoidosis, papilloma or carcinoma of the conjunctiva or its glands. Finally, in any case of chronic ocular surface disorder with no obvious explanation, factitious disease must be considered (Abelson & Allansmith 1987; Friedlander 1993; Jackson 1993). A number of clues may be elicited from the history and examination. The age of onset for the majority of ocular allergies is under 30 years and characteristic symptoms are itch, eye-rubbing, and ropy nonpurulent discharge. Tearing, discomfort and photophobia occur but are nonspecific. A history of the timing of symptoms may provide useful information. Seasonal symptoms classically relate to SAC, with grass pollenosis worst in the spring and summer and ragweed pollenosis (in the USA) worst in the autumn; VKC is worse in the spring and summer. These symptoms can fluctuate daily with the pollen count, humidity and temperature. AKC and PAC may also have seasonal components. Complaints that are perennial suggest a sensitivity to HDM, animal dander or mold spores, or may be a function of the chronic nature of

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the disorder (e.g., AKC). Enquiries should also be made into diurnal variations; for instance, night and early morning are the worst times in HDM sensitivity because of exposure to the allergen in bed linen and the mattress (Christiansen 1988). The relationship of location to symptomatology should be investigated. Those with SAC will be worse outdoors and better inside or with air-conditioning. If the problems are more marked within the home environment, this could relate to high levels of HDM, pets or a hobby; if within the workplace, there could be sensitivity to allergens such as industrial chemicals, and a full occupational history should be taken. On enquiry into other provocative factors, most will be nonspecific irritants, such as smoke or wind, but occasionally a particular precipitating factor may be discovered, for instance a food. The use of cosmetics, soaps and shampoos, topical medications, and hand and face products should be queried. If the patient is a contact lens wearer, the duration of total and daily wear, the age of the present lenses, and the hygiene system should be closely examined. The ocular surface and lids have a limited spectrum of responses to a wide range of injuries and insults, and many findings are common to a range of disease processes. However, some signs point toward an allergic pathology. In the lids, marked edema, allergic shiner, induration, and scaly or dermatitic changes are typical. Chemosis of the bulbar conjunctiva, papillary reaction (especially giant papillae or with limbal Trantas dots), pale pink thickening of the tarsal conjunctiva, and stringy exudation are indicative of allergy. Corneal plaque, keratoconus and atopic lens opacities should be looked for. Systemic examination may reveal other atopic diseases such as asthma and eczema.

Laboratory investigations Conjunctival cytology or, occasionally, biopsy may be useful. Conjunctival scraping is performed with a disposable plastic spatula to produce a smear on a glass slide which is stained with Giemsa and examined for inflammatory cells, particularly eosinophils; as an alternative, conjunctival impression cytology may be performed. Conjunctival scrape cytology is a simple and rapid procedure, but by no means infallible (Butrus & Abelson 1988). The likelihood of a true positive result (i.e., seeing eosinophils) is only 45% in SAC, 25% in PAC, 25% in AKC, and 63% in VKC, even when there are eosinophils present in the epithelium and submucosa on the corresponding biopsy (Abelson et al. 1983a,b). If free eosinophil granules are included in the search, the overall likelihood of making the diagnosis rises significantly, but a negative result does not exclude allergy (Kari et al. 1985). Conjunctival biopsy may be used in diagnosis but is invasive and seldom indicated. Tears may be collected, by using a capillary tube or by soaking a cellulose sponge in the inferior fornix, and analyzed for IgE levels or cytology. Tear mediator levels can be measured but are not at present useful clinically. Total IgE

Ocular Allergy

levels are probably the most sensitive test; measurements of greater than the normal range of 6–16 IU/mL are seen in 98% of SAC, 100% of PAC, 63–100% of VKC, and 87% of AKC and are very suggestive of ocular allergy (Brauringer & Centifano 1971; Baryishak et al. 1982; Sompolinsky et al. 1982; Samra et al. 1984; Dart et al. 1986; Tuft et al. 1991). However, such results are not uncommon in atopy without eye disease, and there is a significant rate of false positives and false negatives. The presence of specific IgE in the tears can only be used as a guide, but if a clinical diagnosis of SAC or PAC has been made, then it may be possible to deduce the offending allergen from the specific IgE in the tears. High total-serum IgE may indicate atopy, but this is neither a sensitive nor a specific test for ocular allergy. In general, serum-specific IgE levels are not measured (by RAST) unless there is some reason why skin-prick testing is not feasible (Christiansen 1988). Skin-prick testing, to determine the presence and approximate levels of systemic specific IgE, may be performed with aeroallergens introduced intradermally to produce a whealand-flare response (within 20 min). The size of the response is used to estimate the level of antibody. However, skin reactions may be misleading, since allergens that produce a positive result in the skin may not correlate with those that do so in the eye. Occasionally, conjunctival allergen challenge is performed to elicit an early-phase response (within 20 min) (Friedlander & Sweet 1988). In the main, this is an experimental tool and only occasionally, in a persistent and severe case of PAC, is it indicated. It should be remembered that any type of allergen challenge can have serious and even fatal consequences and therefore the investigators must have the appropriate resuscitation skills and facilities. Finally, skinpatch testing is used for detecting sensitization of known allergens (Rich & Hanifin 1985; Friedlander 1993).

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Aswad, M.L., Tauber, J. & Baum, J. (1988) Plasmapheresis treatment in patients with severe atopic keratoconjunctivitis. Ophthalmology 95, 444–7. Avunduk, A.M., Avunduk, M.C., Erdol, H., Kapicioglu, Z. & Akyol, N. (2001) Cyclosporine effects on clinical findings and impression cytology specimens in severe vernal kerato-conjunctivitis. Ophthalmology 215, 290–3. Azevedo, M., Castel-Branco, M.G., Oliveira, J.E. et al. (1991) Double-blind comparison of levocabastine eye drops with sodium cromoglycate and placebo in the treatment of seasonal allergic conjunctivitis. Clin Exp Allergy 21, 689– 94. Bacon, A.S., Tuft, S.J., Metz, D.M. et al. (1993) The origin of keratopathy in chronic allergic eye disease: a histopathological study. Eye 7 (suppl.), 21–5. Bacon, A.S., McGill, J.I., Anderson, D.F., Baddeley, S., Lightman, S.L. & Holgate, S.T. (1998) Adhesion molecules and relationship to leukocyte levels in allergic eye disease. Invest Ophthalmol Vis Sci 39, 322– 30. Bacon, A.S., Ahluwalia, P., Irani, A.M., et al. (2000) Tear and conjunctival changes during the allergen-induced early- and latephase responses. J Allergy Clin Immunol 106, 948–54. Baddeley, S.M., Bacon, A.S., McGill, J.I., Lightman, S.L., Holgate, S.T. & Roche, W.R. (1995) Mast cell distribution and neutral protease expression in acute and chronic allergic conjunctivitis. Clin Exp Allergy 25, 41–50. Bailey, C.S. & Buckley R.J. (1993) Nedocromil sodium in contact lens-associated papillary conjunctivitis. Eye 7 (suppl.), 29–33. Ballas, Z., Blumenthal, M. & Tinkelman, D. (1993) Clinical evaluation of ketorolac tromethamine 0.5% ophthalmic solution for treatment of seasonal allergic conjunctivitis. Surv Ophthalmol 38 (suppl.), 141–8. Ballow, M. & Mendelson, L. (1980) Specific IgE antibodies in tear secretions of patients with vernal conjunctivitis. J Allergy Clin Immunol 66, 112–18. Ballow, M., Donshik, P.C., Mendelson, L. et al. (1983) IgG specific antibodies to rye grass and ragween pollen antigens in the tear secretions of patients with vernal conjunctivitis. Am J Ophthalmol 95, 161–8. Ballow, M., Donshik, P.C. & Mendelson, L. (1985) Complement proteins and C3 anaphylotoxin in the tears of patients with conjunctivitis. J Allergy Clin Immunol 76, 473–6. Ballow, M., Donshik, P.C., Rapacz, P. & Samartino, L. (1987a) Tear lactoferrin levels in patients with external inflammatory ocular disease. Invest Ophthalmol Vis Sci 28, 543–5. Ballow, M., Maenza, R., Yamase, H. & Donshik, P.C. (1987b) An animal model for contact lens-induced giant papillary conjunctivitis. Invest Ophthalmol Vis Sci 28, 39. Ballow, M., Donshik, P.C., Rapacz, P. et al. (1989) Immune response in monkeys to lenses from patients with contact lens-induced giant papillary conjunctivitis. CLAO J 15, 64–70. Baryishak, YR., Zavaro, A., Monselise, M. et al. (1982) Vernal keratoconjunctivitis in an Israeli group of patients and its treatment with sodium cromoglycate. Br J Ophthalmol 66, 118–22. Beauvais, E., Hieblot, C. & Benveniste, J. (1989) Effect of sodium cromoglycate and nedocromil sodium on anti-IgE-induced and anti-IgG4–induced basophil degranulation. Drugs 37 (suppl. 1), 4–8. Bende, M. & Pipkorn, U. (1987) Topical levocabastine, a selective HI antagonist in seasonal allergic rhinoconjunctivitis. Allergy 42, 512–15.

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Rolinck-Werninghaus, C., Kopp, M., Liebke, C., Lange, J., Wahn, U. & Niggemann, B. (2005) Lack of detectable alterations in immune responses during sublingual immunotherapy in children with seasonal allergic rhinoconjunctivitis to grass pollen. Int Arch Allergy Immunol 136, 134–41. Sack, R.A., Conradi, L., Krumholz, D., Beaton, A., Sathe, S. & Morris, C. (2006) Membrane array characterization of 80 chemokines, cytokines, and growth factors in open- and closed-eye tears: angiogenin and other defense system constituents. Invest Ophthalmol Vis Sci 46, 1228–38. Saiga, J., Ueno, N. & Shimizu, Y. (1991) Deposition of eosinophil cationic protein in conjunctival tissues and tears in vernal keratoconjunctivitis. Invest. Ophthalmol. Vis. Sci., 32 (Suppl.), 682. Saiga, T., Win, T., Shumizu, Y. et al. (1994) Immunohistochemical study of the adhesion molecules in the conjunctiva of vernal keratoconjunctivitis. In: XVth International Congress on Allergology and Clinical Immunology 1994. Hogrefe & Huber, Seattle, p. 14. (Abstracts). Samra, I., Lavaro, A., Barishak, Y. & Sompolinsky, D. (1984) Vernal kerato-conjunctivitis: the significance of IgE levels in tears and serum. Int Arch Allergy Appl Immunol 74, 158–64. Sarac, O., Erdenet, U., Irkec, M., Us, D. & Gungen, Y. (2003) Tear eotaxin levels in giant papillary conjunctivitis associated with ocular prosthesis. Ocul Immunol Inflamm 11, 223–30. Sathe, S., Sakata, M., Beaton, A.R. et al. (1997) Polymorphonuclear leukocyte cells and elastase in tears. Curr Eye Res 16, 810–19. Schwartz, L.B. (1988) Preformed mediators of human mast cells and basophils. In: Holgate, S.T., ed. Mast Cells Mediators and Disease. Kluwer Academic Publishers, Lancaster, pp. 129–48. Sheldrick, J.H., Wilson, A.D., Vernon, S.A. & Sheldrick, C.M. (1993) Management of ophthalmic disease in general practice. Br J Gen Pract 43, 459–62. Shimken, N. (1938) Anteposito conjunctivae fornicis: operation in severe cases of spring catarrh. Br J Ophthalmol 22, 287–95. Singh, G. (1982) Cryosurgery in palpebral vernal catarrh. Ann Ophthalmol 14, 252–4. Singh, S., Awasthi, N., Egwuagu, C.E. & Wagner, B.J. (2003) Immunoproteasome expression in a nonimmune tissue, the ocular lens. Arch Biochem Biophys 405, 147–53. Smolin, G. (1987) Immunology. In: Smolin, G. & Thoft, R.A., eds. The Cornea, 2nd edn. Little, Brown, Boston, pp. 99–302. Somos, S., Schneider, I. & Farkas, B. (2001) Immunoglobulins in tears and sera in patients with atopic dermatitis. Allergy Asthma Proc 22, 81–6. Sompolinsky, D., Samra, Z., Zavaro, A. & Banshak, R.A. (1982) Contribution to the immunopathology of vernal keratoconjunctivitis. Doc Ophthalmol 53, 61–92. Spataro, A.E. & Bosman, H.B. (1976) Mechanism of action of disodium cromoglycate-mast cell calcium ion reflux after a histaminereleasing stimulus. Biochem Pharmacol 25, 505–10. Srinavas, B.D., Jakobiec, F.A., Iwamoto, T. & DeVoe, A.G. (1979) Giant papillary conjunctivitis with ocular prostheses. Arch Ophthalmol 97, 892–5. Stahl, J.L., Cook, E.B., Graziano, F.M. & Barney, N.P. (2003) Differential and cooperative effects of TNF alpha, IL-1 beta, and IFN gamma on human conjunctival epithelial cell receptor expression and chemokine release. Invest Ophthalmol Vis Sci 44, 2010–15. Stern, M.E., Siemasko, K., Gao, J. et al. (2005) Role of interferon-γ in a mouse model of allergic conjunctivitis. Invest Ophthalmol Vis Sci 46, 3239–46.

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Stockwell, A. & Easty, D. (1988) Double-blind comparative trial of 2% nedocromil sodium eye drops with placebo eye drops in the treatment of seasonal allergic conjunctivitis. Invest Ophthalmol Vis Sci 29 (suppl.), 229. Stumpf, T., Luqmani, N., Sumich, P., Cook, S. & Tole, D. (2006) Systemic tacrolimus in the treatment of severe atopic keratoconjunctivitis. Cornea 25, 1147– 9. Sugar, H.S. (1962) Tarsectomy for proliferative palpebral vernal conjunctivitis. Am J Ophthalmol 53, 429– 35. Szabo, Z., Szilasi, M., Brugos, L. et al. (2000) Differences in the changes of allergen-specific IgE serum levels and the chemiluminescence of peripheral blood phagocytes in patients with allergic rhinoconjunctivitis during ragweed season. Immunol Lett 74, 201–5. Tabbara, K.F. (1999) Ocular complications of vernal keratoconjunctivitis. Can J Ophthalmol 34, 88– 92. Tabbara, K.F., El-Asrar, A.M., Geboes, S. et al. (1996) Adhesion molecules in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 37, 4695. Tanaka, M., Dogru, M., Takano, Y. et al. (2004) The relation of conjunctival and corneal findings in severe ocular allergies. Cornea 23, 464– 7. Tepper, R.I., Levinson, D.A., Stanger, B.Z. et al. (1990) IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell 62, 457– 67. Tinkelman, D., Rupp, G., Kaufman, H. et al. (1993) Double-masked paired-comparison clinical study of ketorolac tromethamine 0.5% ophthalmic solution compared with placebo eyedrops in the treatment of seasonal allergic conjunctivitis. Surv Ophthalmol 38 (suppl.), 133– 40. Tiszler Cieslik, E., Nowak, W. & Gluza, J. (1994) A comparison of levocabastine and sodium cromoglycate in allergic conjunctivitis due to house dust mite. In: XVth International Congress on Allergology and Clinical Immunology 1994. Hogrefe & Huber, Seattle, p. 16. (Abstracts). Trocme, S.D., Kephart, G.M., Allansmith, M.R. et al. (1989) Conjunctival deposition of eosinophil granule major basic protein in vernal keratoconjunctivitis and contact lens associated giant papillary conjunctivitis. Am J Ophthalmol 108, 57–63. Trocme, S.D., Kephart, G.M. & Bourne, W.M. (1990) Eosinophil granule major basic protein in contact lenses of patients with giant papillary conjunctivitis. CLAO J 16, 219–22. Trocme, S.D., Kephart, G.M., Bourne, W.M., Buckley, R.J. & Gleich, G.J. (1993) Eosinophil granule major basic protein deposition in

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corneal ulcers associated with vernal keratoconjunctivitis. Am J Ophthalmol 115, 640–3. Tse, D.T., Mendelbaum, S., Epstein, E. et al. (1983) Mucous membrane grafting for severe palpebral vernal conjunctivitis. Arch Ophthalmol 101, 1879. Tuft, S.J., Dart, J.K.G. & Kemeny, M. (1989) Limbal vernal keratoconjunctivitis: clinical characteristics and IgE expression compared with palpebral vernal. Eye 3, 420– 7. Tuft, S.J., Kemeny, M.D., Dart, J.K.G. & Buckley, R.J. (1991) Clinical features of atopic keratoconjunctivitis. Ophthalmology 98, 150–8. Tuft, S.J., Ramakrishnan, M., Seal, D.Y. et al. (1992) Role of Staphylococcus aureus in chronic allergic conjunctivitis. Ophthalmology 99, 180– 9. Tuft, S.J., Cree, I.A., Woods, M. & Yorston, D. (1998) Limbal vernal keratoconjunctivitis in the tropics. Ophthalmology 105, 1489–93. Udell, I.J., Gleich, G.J., Allansmith, M.E. et al. (1981) Eosinophil granule major basic protein and Charcot-Leyden crystal protein in human tears. Am J Ophthalmol 92, 824–8. Van Bijsterveld, O.P., Terpstra, G.K., Moons, I. et al. (1994) Nedocromil sodium in PAC not fully controlled by sodium cromoglycate. In: XVth International Congress on Allergology and Clinical Immunology 1994. Hogrefe & Huber, Seattle, p. 14. (Abstracts). Venge, P. & Hakansson, I. (1991) Current understanding of the role of the eosinophil granulocyte in asthma. Clin Exp Allergy 21 (suppl. 3), 31–7. Venge, P., Dahl, R., Fredens, K. & Peterson, C.G.B. (1988) Epithelial injury by human eosinophils. Am Rev Respir Dis 138, 554–7. Wardlaw, A.J., Moqbel, R., Cromwell, O. & Kay, A.B. (1986) Platelet activating factor: a potent chemotactic and chemokinetic factor for human eosinophils. J Clin Invest 78, 1701–6. Welsh, P., Yunginger, J.W., Tani, D.G. et al. (1979) Topical ocular administration of cromolyn sodium for treatment in seasonal ragweed conjunctivitis. J Allergy Clin Immunol 64, 209–15. Whitcup, S.M., Bradford, R., Lue, J., Schiffman, R.M. & Abelson, M.B. (2004) Efficacy and tolerability of ophthalmic epinastine: a randomized, double-masked, parallel-group, active- and vehiclecontrolled environmental trial in patients with seasonal allergic conjunctivitis. Clin Ther 26, 29–34. Wood, T.S., Steward, R.H. & Bowman, R.W. (1988) Suprofen treatment of contact lens associated GPC. Ophthalmology 96, 822–6. Zhang, Y.L., Craster, R.V. & Matar, O.K. (2003) Surfactant driven flows overlying a hydrophobic epithelium: film rupture in the presence of slip. J Colloid Interface Sci 264, 160–75.

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Mechanisms in Allergen Injection Immunotherapy Stephen J. Till and Stephen R. Durham

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Immune deviation in favor of Th1 cells

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Specific allergen injection immunotherapy is an effective treatment for IgE-mediated diseases, particularly pollen-induced allergic rhinitis and systemic hypersensitivity to wasp and bee venoms. As the only antigen-specific desensitizing treatment in current clinical use there is considerable interest in the immune regulatory mechanisms invoked by injecting escalating doses of allergens. Treatment results in rapid inhibition of allergeninduced late responses, with a slower and proportionately smaller decline in early responses. Biopsies taken from skin and nasal mucosa have revealed reductions in inflammatory cell numbers, including mast cells, basophils and eosinophils. Around 6–8 weeks after starting weekly immunotherapy updosing injections, increases in allergen-specific IgG, particularly of IgG4 isotype, are observed. These antibodies block IgE effector mechanisms including basophil histamine release and IgE-facilitated antigen presentation to T cells. Induction of allergen-specific IgA is also observed and these antibodies can induce monocytic cells to produce interleukin (IL)-10, an immunoregulatory cytokine. These humoral responses likely reflect modulation of allergen-specific T-cell responses. Immunotherapy modifies peripheral and mucosal Th2 responses to allergen in favor of Th1 cytokine and IL-10 production (Fig. 72.1). The latter may be a key early event and IL-10-producing T cells are detectable within a few weeks of the first injection. IL-10 inhibits mast cell, eosinophil and T-cell responses, as well as acting on B cells to favor IgG4 production. These IL-10-producing T cells may be so-called TR1-type inducible regulatory T cells. The mechanism leading to development of these cells has yet to be elucidated, though similar populations can be experimentally induced by tolerogenic dendritic cells. Novel approaches in current clinical use include combining allergens with immunomodulatory adjuvants to potentiate responses. These include bacterially derived modified lipid compounds or CpG-rich immunostimulatory oligodeoxynucleotides which act through Toll-like receptors 4 and 9, respectively. Alternative strategies include the use of

allergen-derived peptides or modified recombinant allergen vaccines. These aim to maintain the beneficial effects of vaccines while minimizing the immediate IgE-dependent complications that currently require immunotherapy to be conducted cautiously and under specialist supervision.

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Fig. 72.1 Summary of the effects of immunotherapy on T-cell responses: immunotherapy redresses the balance between Th2/Th1 responses in favor of Th1 responses. An increase in IL-10-producing T cells, possibly regulatory T cells (TR), is also seen. The relationship between these events remains controversial. These pathways suppress allergic responses by blocking infiltration and function of effector cells and inhibit IgE-dependent pathways of effector cell activation. (See CD-ROM for color version.)

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Early response

Late response

Immunotherapy is highly effective in appropriately selected patients with allergic disease (see Chapter 73). Whereas conventional vaccination strategies are employed to initiate and then boost immunologic memory, allergen injection immunotherapy aims to subdue established pathologic immune responses mediated by IgE and allergen-specific memory T cells through regular exposure to the offending allergen. Since subcutaneous injection of sufficient native allergen to invoke immunoregulatory mechanisms can trigger unwanted IgEmediated reactions, the amounts of allergen contained with injections are increased incrementally from low levels until a safe but sufficient maintenance dose can be achieved. Of the immunomodulatory strategies commonly employed in clinical practice, allergen immunotherapy (in both injection and sublingual forms) represents a uniquely antigen-specific treatment for an immunologic disease. Injection immunotherapy is effective for venom anaphylaxis and in rhinoconjunctivitis and asthma due to inhalant allergens (Bousquet et al. 1998; Lockey 2001), particularly seasonal disease induced by pollen (Varney et al. 1991; Frew et al. 2006). For example, grass pollen injection immunotherapy improves seasonal asthma symptoms, inhibits seasonal increases in bronchial hyperresponsiveness, and improves quality of life in hay fever sufferers (Walker et al. 2001). The clinical benefits are dependent on the final maintenance dose of allergen administered (Frew et al. 2006) and are maintained for at least 3 years following discontinuation (Durham et al. 1999; Golden et al. 2004). In children, immunotherapy has been shown to prevent onset of new sensitizations (Pajno et al. 2001) and reduce progression of rhinitis to physiciandiagnosed asthma up to at least 2 years after discontinuation (Moller et al. 2002; Niggemann et al. 2006). While current treatment regimens are effective, refinement of immunotherapy in terms of both efficacy and safety profile remains an important goal. In addition, the possibility of developing immunoregulatory vaccines for nonallergic immune diseases ensures considerable ongoing interest into underlying mechanisms.

The allergic response The natural course of an experimental allergen exposure in the nose, eyes or bronchi is the immediate development of mast cell-dependent sneeze, pruritis, watery discharge and bronchospasm, maximal at 15–30 min and resolving within 1–3 hours (Fig. 72.2). This “early response” is triggered by IgE-dependent activation of mast cells through high-affinity IgE receptor (FcεRI) resulting in release of numerous mediators, including histamine, tryptase, TAME-esterase, bradykinin, leukotrienes (including LTC4, LTD4, and LTE4), prostaglandins

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Fig. 72.2 In susceptible individuals allergen exposure results in the production of allergen-specific IgE. Subsequent exposure to allergen leads to mast cell activation, the early response and immediate symptoms. The infiltration of effector cells is delayed and results in a second peak of symptoms during the late response. (See CD-ROM for color version.)

including PGF2α and PGD2 (specific for mast cells), and plateletactivating factor. These mediators collectively induce vasodilatation, increased vascular permeability, mucosal edema, increased mucus production from submucosal glands and goblet cells within the respiratory/gastric epithelium, and smooth muscle contraction (particularly in the lower respiratory tract). A proportion of subjects develop a late response, manifest in both nose and lung as airflow obstruction. Intradermal challenge testing provokes analogous cutaneous early and late responses characterized by wheal and flare with delayed onset but prolonged edema and tenderness. Classically, late responses are maximal at 6–12 hours and resolve within 24 hours (Fig. 72.2). The late response is accompanied by recruitment, activation and persistence of eosinophils, basophils, activated T cells and possibly dendritic cells at the sites of allergen exposure. Inflammatory mediators secreted by these cells are presumed to contribute to pathogenesis. Of particular interest are Th2 cytokines such as interleukin (IL)4, IL-5, IL-9 and IL-13 produced by T cells. These cytokines have specific proallergic properties through direct effects on other inflammatory cells, adhesion molecule expression, and B-cell IgE synthesis. However, it is likely that IgE–mast cell mechanisms also contribute as anti-IgE therapy (omalizumab) is associated with inhibition of cutaneous allergen-induced

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Fig. 72.3 Time-course analysis of clinical and immunologic measurements during the first year of grass pollen immunotherapy. The dose of grass pollen allergen given at each immunotherapy visit is represented in the top left panel. Early and late skin responses were assessed at 15 min and 24 hours following intradermal challenge with grass pollen allergen. Allergenspecific IgG4 was measured in serum by enzyme-linked immunosorbent assay. Immunotherapy serum was tested for inhibition of FceRI-mediated allergen-induced basophil histamine release in whole blood, with histamine

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PhI p-specific serum IgG4 (arbitrary units/mL)

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of the early skin response was predictive of prolonged suppression following discontinuation of immunotherapy requires confirmation in a prospective study (Des Roches et al. 1996). The time of onset of early and late response inhibition has been recently examined in relation to a conventional grass pollen immunotherapy regimen (Francis et al. 2008) (Fig. 72.3). The striking finding was that late responses to intradermal challenge testing were reduced 2 weeks into treatment, by which time patients had received less than 1% of the total cumulative allergen dose administered weekly during the 2-month updosing phase. Since low-dose immunotherapy is known to be ineffective, this raises the possibility that efficacy is not dependent on late-response suppression. In contrast, inhibition of early responses was more modest but occurred over a time-frame more in keeping with clinical protection from symptoms.

Inhibition of histamine release (%)

late responses in addition to early responses (Ong et al. 2005). The immunopathologic changes in the mucosa on chronic allergen exposure (e.g., during the pollen season) resemble those seen during the late response. A characteristic feature of immunotherapy is its ability to inhibit late responses in the skin (Pienkowski et al. 1985), nose (Iliopoulos et al. 1991), and lung (Warner et al. 1978), but it is not clear whether suppression of the late response is predictive of clinical improvement following immunotherapy. The effects of immunotherapy on the early response after antigen exposure have been modest and somewhat variable: some studies have reported inhibition of the early response in the skin, whereas others have shown only temporary inhibition (Walker et al. 1995) and no inhibition in the lung (Warner et al. 1978). The interesting discovery, within a group of house-dust mite-sensitive children, that suppression

Inhibition of allergen/IgE binding to B cells (%)

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being measured by enzyme-linked immunosorbent assay. Serologic inhibitory activity against FceRII/CD23-dependent IgE-facilitated allergen binding was measured by incubating B cells with grass pollen-specific IgE, grass pollen allergen and sera from immunotherapy patients. Surfacebound IgE was quantified by flow cytometry. Data are expressed as mean ± standard error. The grey line represents pollen counts in London, UK. *, P < 0.05 versus preimmunotherapy value. (Adapted from Francis et al. 2008, with permission.) (See CD-ROM for color version.)

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P = 0.001 P = 0.0000

c-Kit+ mast cells/mm2

200

Fig. 72.4 Effects of natural seasonal exposure to grass pollen and allergen immunotherapy on numbers of c-kit-positive mast cells in the nasal mucosa. Data shown are for out-of-season (pre-treatment) and peak-season (after immunotherapy or placebo) nasal biopsy specimens. Horizontal bars represent median values. Right panel shows an example of a nasal biopsy section immunostained for c-kit. (Adapted from Nouri-Aria et al. 2005, with permission.) (See CD-ROM for color version.)

P = 0.03 (285) (238) (215)

150

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0 PrePost PrePost treatment treatment treatment treatment peak season peak season Placebo

Effector cell recruitment Nasal scrapings obtained before and after house-dust mite immunotherapy in children showed reductions in metachromatic cells, which were presumed to be mast cells (Otsuka et al. 1991). Seasonal allergic rhinitis is also associated with migration of tryptase-positive mast cells into nasal mucosa as identified by immunocytochemistry, though grass pollen immunotherapy was not associated with reductions (Wilson et al. 2001a). In a further study, mast cell numbers were reexamined using the c-kit/stem cell factor receptor transmembrane tyrosine kinase as a marker. This revealed a marked recruitment of c-kit-positive cells during the pollen season that was suppressed by immunotherapy (Nouri-Aria et al. 2005) (Fig. 72.4). The same study also reported that nasal mucosal expression of IL-9, a growth factor for mast cells, was also lower in treated subjects. Basophils have also been examined in the nasal mucosa of grass pollen immunotherapy patients during natural exposure using the 2D7 monoclonal antibody (Wilson et al. 2001a). Whereas treatment did not appear to affect basophil numbers in the lamina propria, infiltration into the epithelium could be demonstrated in approximately 35% of placebo-treated rhinitics but only 5% of immunotherapy patients. This suggests that immunotherapy may act to reduce the seasonal recruitment of both mast cells and basophils into the nasal mucosa. Immunotherapy has also been associated with reduced eosinophil recruitment into tissue after allergen challenge. For example, after grass pollen immunotherapy, reductions in the cutaneous late response to allergen provocation were accompanied by a trend for lower eosinophil numbers in skin biopsies (Varney et al. 1993). Ragweed immunotherapy was also associated with lower eosinophil numbers in nasal

Immunotherapy

lavage fluid 24 hours after nasal ragweed challenge (Furin et al. 1991). Using the nasal biopsy model, mucosal eosinophils were also examined after grass pollen immunotherapy. Treatment was reported to inhibit eosinophil recruitment during both the allergen-induced late response (Durham et al. 1996) and natural seasonal exposure (Wilson et al. 2001b). In contrast, neutrophil numbers did not appear to be affected by treatment. The effect of immunotherapy on lower airway eosinophilia was also examined in subjects with birch polleninduced seasonal asthma (Rak et al. 1988). Eosinophils and eosinophil cationic protein (ECP) were measured in bronchoalveolar lavage collected by fiberoptic bronchoscopy during the birch pollen season. In contrast to untreated control subjects, the immunotherapy patients developed less lung symptoms on exposure with lower measurable bronchial hyperactivity, eosinophil counts, and ECP concentrations.

Immunoglobulin responses Conventional pollen immunotherapy appears to have little effect on allergen-specific serum IgE concentrations (Gehlhar et al. 1999), although increases in IgE during the pollen season are blunted (Lichtenstein et al. 1973). Whole extracts used for immunotherapy usually contain multiple allergenic proteins. Following treatment with birch pollen extract, serum immunoblotting analysis revealed that a proportion of individuals mounted IgE responses to previously unrecognized proteins (Moverare et al. 2002). However, the concentrations of IgE produced were relatively low and the clinical implications of these new sensitizations are unknown. As early as 1935 Cooke was able to demonstrate that sera from immunotherapy patients inhibited allergic responses in vivo. Some 40 years later, Platts-Mills and colleagues reported

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that IgG and IgA antibodies isolated from nasal washings following immunotherapy could inhibit histamine release from basophils in vitro (Platts-Mills et al. 1976). Increases in serum allergen-specific IgG1, IgG4 and IgGA isotypes have all been reported following immunotherapy (Gehlhar et al. 1999; Jutel et al. 2003). The effect of immunotherapy on immunoglobulin avidities as well as quantities has received some attention. Svenson et al. (2003) examined this in a small group of birch-allergic patients before and 5 years into a conventional immunotherapy regimen. Allergen binding to the whole IgG fraction of sera was measured using radiolabeled Bet v 1 and increased between 6.2 and 32-fold. In contrast, the binding avidity to IgG (Kd in range 0.35–0.70 ng/ mL) showed minimal changes. Another group examined the binding avidities of Bet v 1-specific IgG1, IgG4 and IgE using surface plasmon resonance (Biacore) in 10 immunotherapytreated subjects and 10 untreated controls (Jakobsen et al. 2005). No significant differences were detected for each isotype after purification from sera. Of the isotypes that increase during immunotherapy, IgG4 consistently shows the greatest proportionate increase, albeit from extremely low pretreatment levels. IgG4 antibodies have little or no measurable inflammatory activity in humans and have therefore been proposed as “blocking antibodies.” According to this model, allergen-specific IgG4 antibodies compete for allergen binding with cell surface-bound IgE (i.e., bound to FcεRI high-affinity receptors), thereby inhibiting cross-linking and cell activation. For example, IgG4 induced by immunotherapy blocks allergen-induced IgE-dependent histamine release by basophils in vitro (Garcia et al. 1993). More recently, the effects of immunotherapy on IgA antibodies have been examined (Pilette et al. 2007). Highly significant increases in serum allergen-specific IgA2 and polymeric IgA were observed, with a lesser but significant rise in IgA1. Cross-linking of polymeric IgA2 was shown to be a potent inducer of IL-10 expression by monocytes. IL-10 favors B-cell production of IgG4 as well as being a potentially a key immunoregulatory cytokine (see below). In addition to preventing cross-linking of FcεRI-bound IgE, blocking antibodies also inhibit formation of IgE–allergen complexes in the liquid phase. It appears that formation of these multivalent complexes is a requirement for IgE binding to low-affinity FcεRII (CD23) IgE receptors. Thus allergen and IgE are mutually dependent for binding to FcεRII. FcεRII is expressed by antigen-presenting cells (APCs) including B cells, dendritic cells and monocytes. Complex formation appears to be important in allowing these cells to bind extremely low concentrations of allergen. The complexes are then internalized to endosomal compartments where the allergen is processed and presented to T cells on human leukocyte antigen (HLA) class II molecules. This process, known as “facilitated antigen binding,” thus enables APCs to activate allergen-specific T cells at low allergen concentrations. Van Neerven et al. (1999) demonstrated that serum

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from patients allergic to birch pollen facilitated antigen binding to B cells and ensuing activation of birch-specific T cells. This process was inhibited by sera from birch pollen immunotherapy patients and the inhibitory activity resided within the IgG fraction. Subsequent studies have confirmed these findings in grass pollen immunotherapy (Wachholz et al. 2003; Van Neerven et al. 2004) and further shown that IgG4 coelutes with this inhibitory activity (Nouri-Aria et al. 2004). In contrast, IgA fractions did not demonstrate significant blocking activity in cellular assays (Pilette et al. 2007). When the development of grass pollen allergen-specific IgG4 and corresponding blocking activity were studied over a detailed time-course, it was apparent that this functional activity took 6–8 weeks to achieve significant levels (Francis et al. 2008) (Fig. 72.3). In summary, blocking antibodies including allergen-specific IgG4 appear to inhibit FcεRI- and FcεRIIdependent IgE-mediated events such as histamine release and T-cell activation, respectively. Coaggregation of inhibitory FcγRIIb IgG receptors with high-affinity FcεRI IgE receptors theoretically offers another mechanism by which allergen-specific IgG could mediate an antiinflammatory effect (Daeron et al. 1995). Phosphorylation of FcγRIIb receptors by FcεRI-associated kinases causes the former to bind intracellular phosphatases that mediate reciprocal inhibition of FcεRI signaling (Malbec et al. 1998). Thus, FcεRI is able to mediate negative feedback inhibition on itself in a mechanism involving juxtaposition of FcγRIIb and FcεRI receptors. Potentiation of FcγRIIb–FcεRI receptor interaction by a recombinant chimeric Fcε–Fcγ construct resulted in inhibition of allergen–IgE dependent histamine release by human basophils (Zhu et al. 2002). On the other hand, blockade of FcγRIIb signaling by a monoclonal antibody did not reverse the inhibitory effect of IgG from birch pollen immunotherapy patients on Bet v 1-induced basophil activation (Ejrnaes et al. 2006). The conventional objection to the blocking antibody model is the weak correlation between allergen-specific IgG concentrations and the clinical response to treatment (Djurup & Malling 1987; Ewan et al. 1993; Bodtger et al. 2005). For example, immunotherapy in “rush” protocols is effective long before any changes in antibody synthesis are detected. Data from our group has shown that 2 years following discontinuation of grass pollen immunotherapy, grass pollen allergen-specific IgG4 levels decline by approximately 80% (unpublished observations). This was discordant with blocking activity in vitro which persisted for up to 2 years, during which time patients remained in remission. The possibility that a population of long-lived high-avidity memory IgG-positive B cells selectively persists following immunotherapy withdrawal, perhaps due to low-dose natural antigen exposure, merits further evaluation. Additionally, the surface plasmon resonance studies described above (Jakobsen et al. 2005) showed statistically significant negative correlations in immunotherapy-treated subjects between both IgG1 and

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IgG4 binding affinities for Bet v 1 and visual analog symptom scores. These observations suggest that measuring activity of allergen-specific IgG, as a blocking antibody or otherwise, may be more clinically relevant than quantifying absolute antibody levels in sera.

Effects on T-cell responses T-cell differentiation is a critical decision step in regulating downstream effector mechanisms. A considerable research effort has therefore been directed towards characterizing Tcell responses to immunotherapy vaccines. The major themes that have emerged include deviation of Th2 responses in favor of a Th0/Th1 phenotype (assumed to be less pathogenic) and suppression of allergen-specific T cells by newly induced regulatory T cells or through inhibition of antigen presentation by IgG.

Immune deviation Although there is a consensus that immune deviation plays an important role in successful immunotherapy, there is no consistent pattern of cytokine production changes that is common to all studies. Investigations on peripheral blood mononuclear cells (PBMC) from patients receiving immunotherapy have identified reductions in proliferative responses to allergen, with others additionally reporting shifts away from Th2 toward Th1 cytokine production (Jutel et al. 1995; Bellinghausen et al. 1997; Ebner et al. 1997; Benjaponpitak et al. 1999). In contrast, other investigators were unable to reproduce these findings, reporting no changes in proliferative responses or cytokine production following allergen immunotherapy (Till et al. 1997; Klimek et al. 1999; Wachholz et al. 2002; Francis et al. 2003). There are many potential reasons for the discrepancies between these studies, including wide variations in laboratory methodologies between different investigators and lack of standardization of allergen extracts. A possible explanation is that inhibition of peripheral T-cell proliferation and Th2 cytokine production is not the fundamental event in immunotherapy. Cultures of PBMC may provide only a crude reflection of immune interactions and responses in lymphoid and/or mucosal tissues. The effects of immunotherapy on the cytokine profile of T cells recruited into tissue (skin and nose) has also been studied in some detail (Klimek et al. 1999; Wachholz et al. 2002). Increases in numbers of cells expressing mRNA encoding interferon (IFN)-γ were found in the nasal mucosa during the allergen-induced late response of grass pollen immunotherapy patients (Durham et al. 1996). The inverse correlation between the numbers of these cells and clinical symptom scores recorded during the pollen season supports the hypothesis that a mucosal IFN-γ response may be biologically relevant. Among the signals able to induce IFN-γ expression by allergen-specific mucosal T cells is IL-12 (Varga

Mechanisms in Allergen Injection Immunotherapy

et al. 2000) and there is evidence that this mechanism may operate in grass pollen immunotherapy. Skin biopsies collected taken 24 hours after cutaneous allergen challenge were examined for IL-12 mRNA by in situ hybridization (Hamid et al. 1997). In immunotherapy patients there was concomitant late-response suppression and IL-12 mRNA expression. The latter correlated directly with IFN-γ and the principal source of IL-12 mRNA was CD68+ macrophages. Similar studies have demonstrated that immunotherapy significantly inhibits seasonal increases in IL-5 and IL-9 mRNAexpressing cells in the nasal mucosa (Wilson et al. 2001b; Nouri-Aria et al. 2005). Collectively, these studies highlight the importance of studying cytokine responses in mucosal tissue rather than the peripheral blood, particularly for inhalant allergies such as allergic rhinitis.

IL-10 and regulatory T cells Several studies have suggested that immunotherapy modifies T-cell response to allergens through the induction of regulatory mechanisms. Naturally occurring regulatory cells, first identified as CD4+CD25+ cells by Sakaguchi et al. (1995), arise in the thymus and express the transcription factor Foxp3 (Hori et al. 2003). Numerous studies confirmed the inhibitory properties of human CD4+CD25+ cells in vitro and in suppressing inflammatory responses in vivo (Sakaguchi 2005). Regulatory T-cell function is thought to be mediated, at least in vitro, by cell to cell contact. Functional roles for membrane CTLA-4 (Takahashi et al. 2000), surface-bound transforming growth factor (TGF)-β (Nakamura et al. 2001), and glucocorticoid-induced tumor necrosis factor receptor (GITR) (McHugh et al. 2002) have been described. Suppression of allergen-specific responses by these regulatory cells may represent the normal “healthy” response to allergen. In subjects with grass pollen-induced allergic rhinitis, purified CD4+CD25+ T cells failed to suppress activation of CD4+CD25– T cells by allergen, unlike in nonallergic controls (Ling et al. 2004). In addition to naturally occurring regulatory T cells, a separate population of “inducible” cells with similar suppressive properties has been described. Inducible regulatory T cells have been generated in vitro through stimulation (Levings et al. 2001; Barrat et al. 2002; Sundstedt et al. 2003) and are classically described as not expressing Foxp3 (Vieira et al. 2004), suggesting that these cells represent a subset distinct from the naturally occurring regulatory T cells. However, this may be overly simplistic since human CD4+CD25– T cells activated by allogeneic stimuli, recall antigens or polyclonal stimuli transiently express Foxp3 and show suppressive activity in vitro (Pillai et al. 2007). Inducible regulatory T cells can produce cytokines such as IL-10 and TGF-β, both of which have important immunomodulatory properties. IL-10-producing regulatory T cells are frequently referred to as TR1 cells. Over the last decade, expression of IL-10 by T cells after immunotherapy has emerged as a remarkably consistent

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Allergic Rhinoconjunctivitis and Immunotherapy markers. Somewhat surprisingly only 15% of mRNA signals colocalized to CD3+ T cells, with a further 35% being accounted for by CD68+ tissue macrophages (Nouri-Aria et al. 2004). However, although in situ hybridization facilitates the analysis of cytokine responses within target organ tissues, the numbers of cells expressing IL-10 mRNA may not correlate with overall secretion of bioactive IL-10 protein. Thus it is possible that the overall contribution of T cells to IL-10 production is underestimated by such techniques. In peripheral blood studies of bee venom immunotherapy, monocytic cells (which can differentiate into macrophages under certain conditions) as well as B and T cells were identified as sources of IL-10 (Akdis et al. 1998). The endogenous IL10 produced in these cultures suppressed CD4+ T-cell responses. Subsequently, T-cell phenotypic subsets were analyzed for IL-10 production by flow cytometry. This methodology is based on immunostaining of permeabilized T cells for intracellular cytokine protein in combination with extracellular surface markers using specific monoclonal antibodies. Since T cells usually rapidly secrete cytokine protein with little or no intracellular storage, this method requires disablement of the Golgi apparatus with inhibitors such as monensin or brefeldin A. Using this approach, IL-10 production could be shown to be associated with CD4+CD25+ T cells following immunotherapy with bee venom (Akdis et al. 1998), grass pollen (Francis et al. 2003), or house-dust mite (Jutel et al. 2003). In the case of house-dust mite immunotherapy, these CD4+CD25+ T cells produced both IL-10 and TGF-β and suppressed immune responses to allergen in vitro. Reconciling this phenotype with our current perspective of regulatory T cell subsets (i.e., “naturally occurring” CD4+CD25+ versus “inducible” TR1 regulatory cells) represents something of a challenge. However, CD25 is not exclusively a marker of naturally occurring regulatory T cells, being expressed by virtually all recently activated T cells. Indeed, in several of the studies described above T cells were stimulated in vitro with allergen to elicit measurable IL-10 production and therefore CD25 expression could be viewed as an artifactual outcome of the assay. However, one study did separate CD4+CD25+

finding (Bellinghausen et al. 1997; Akdis et al. 1998; Francis et al. 2003). This cytokine is expressed by a variety of immune cells including both Th1 and Th2 cells, B cells, monocytes, dendritic cells, mast cells, and eosinophils. In mouse models, IL-10 has been associated with inflammatory suppression in diverse models include airway immunopathology (Tournoy et al. 2000). In the presence of IL-4, IL-10 acts on B cells to induce production of IgG4 (Jeannin et al. 1998) and also directly inhibits mast cell activation by IgE (Royer et al. 2001). In human T cells, IL-10 also suppresses production of Th2 cytokines such as IL-5 (Francis et al. 2003) and induces a state of hyporesponsiveness or “anergy” (Groux et al. 1996). The proposed mechanism involves IL-10 receptor-dependent blockade of CD28 phosphorylation and therefore inhibition of essential costimulatory signals received by T cells from APCs (Akdis et al. 2000). IL-10-producing cells have been described following immunotherapy with bee venom (Bellinghausen et al. 1997), house-dust mite (Jutel et al. 2003), and grass pollen (Francis et al. 2003) (Fig. 72.5). Beekeepers who have become tolerized against anaphylaxis through repeated stings also produce IL-10 in response to T-cell stimulation with bee venom allergen (Akdis et al. 1998). IL-10 production is also evident within tissue: in nasal biopsies of grass pollen immunotherapy patients, increased numbers of IL-10 mRNA-expressing cells were described (Nouri-Aria et al. 2004). This expression was dependent on current antigen exposure as IL-10 mRNApositive nasal mucosal cells were identified neither in immunotherapy patients outside the pollen season nor untreated patients during the pollen season. Additionally, this did not represent a restoration of the “normal” mucosal immune response as IL-10 mRNA expression was minimal in nonallergic control subjects. IL-10 mRNA has also been described during the late response in skin biopsies after venom injection in wasp immunotherapy patients (Nasser et al. 2001). Several studies have attempted to characterize the phenotype of IL-10-producing cells after immunotherapy. In the nasal mucosa this was achieved by colocalizing IL-10 mRNA signals with immunocytochemical cell-surface phenotypic

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Fig. 72.5 IL-10 production by allergenstimulated peripheral blood mononuclear cells (PBMC) is higher in hay fever subjects treated with immunotherapy. PBMC were obtained from the same individuals during and after the 2001 UK grass pollen season and stimulated with Timothy grass extract for 6 days. IL-10 concentrations in culture supernatants are shown, as measured by enzyme-linked immunosorbent assay. (Adapted from Francis et al. 2003, with permission.) (See CD-ROM for color version.)

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and CD4+CD25– T cells from immunotherapy subjects before in vitro stimulation and IL-10 production compartmentalized to the former subset (Jutel et al. 2003 ). However, since these individuals had been treated with repeated allergen injections over several months it is possible that these IL-10-producing T cells were activated in vivo by allergen. Antigen-specific activation may even be a necessary step for the induction of these cells (Pillai et al. 2007).

Initiation of IL-10 responses Of considerable importance is the mechanism by which immunotherapy vaccines interact with the immune system to induce IL-10-producing T cells (i.e., putative TR1 cells). Although mouse models have provided some insights, few have specifically reproduced the tolerogenic effects of immunotherapy in mature Th2 responses with established mucosal inflammation (Tournoy et al. 2006). Additional important differences include the common use of adjuvants such as alum to achieve initial allergen sensitization, usually via the intraperitoneal route. Allergic inflammation in the respiratory tract is subsequently induced by repeated mucosal challenge with allergen. Tolerizing animals by oral or intranasal exposure to ovalbumin prior to intraperitoneal sensitization induced regulatory T cells with characteristics of TR1 cells (Akbari et al. 2001; Zhang et al. 2001). In the intranasal tolerance model, a series of adoptive transfer experiments revealed that induction of TR1 cells was dependent on pulmonary lymph node dendritic cells expressing IL-10 and the costimulatory molecule ICOS ligand. Indeed, adoptive transfer of these dendritic cells or TR1 cells themselves conferred tolerance on the recipient animals (Akbari et al. 2001, 2002). Such mechanisms may be relevant to human TR1 responses. Human peripheral blood plasmacytoid dendritic cells stimulated with a Toll-like receptor (TLR)9 agonist express ICOS ligand and induce differentiation of TR1 cells from naive T cells in vitro (Ito et al. 2007). Cross-linking of the high-affinity IgE receptor (FcεRI) on plasmacytoid dendritic cells by allergen also induces IL-10 expression (Novak et al. 2004). Although these mechanisms have not been shown to operate in immunotherapy, they do indicate that dendritic cells are strong candidates for directing T-cell IL-10 responses following allergen vaccination. Further research is needed to address this possibility and identify any molecular signals that might result in induction of “tolerogenic” dendritic cells.

Novel strategies for immunotherapy Immunotherapy performed with modern extracts prepared to pharmaceutical standards is a relatively safe form of treatment. Nevertheless, the subcutaneous administration of native allergen in IgE-sensitized individuals has the potential to result in both local and systemic reactions. One consequence is that immunotherapy regimens are necessarily cau-

Mechanisms in Allergen Injection Immunotherapy

tious, involving numerous injections of gradually increasing allergen doses performed under specialist supervision. Novel immunotherapy strategies that reduce the scope for side effects are also likely to translate into high-dose therapies with fewer injections for patients and lower demands on health services. The majority of novel therapies under investigation are based on two hypotheses: firstly, that the effect of immunotherapy is primarily mediated through mechanisms involving T-cell stimulation at high antigen doses, presumably leading to regulatory T-cell induction. A corollary of this hypothesis is that IgE-dependent mechanisms are primarily responsible for immunotherapy side effects but are not necessary for vaccine efficacy. The second hypothesis is that by adding novel adjuvants, the immunologic effects of vaccination with native allergens can be potentiated. Within the first category, strategies tested include genetically modified allergen proteins with reduced IgE binding but native T-cell epitopes. For example, recombinant fragments of major birch pollen allergen Bet v 1 have been generated with minimal allergenicity in cutaneous and nasal challenge models (Nopp et al. 2000; Hage-Hamsten et al. 2002). These proteins were adsorbed to aluminum hydroxide (as with conventional whole extract vaccines) and successfully elicited Bet v 1-specific IgG1 and IgG4 responses that blocked basophil histamine release triggered by exposure to wild-type Bet v 1 (Niederberger et al. 2004). Based on the same rationale, allergen-derived peptides that stimulate T cells but which cannot cross-link IgE and cause mast cell activation have also been tested in small clinical studies with promising results (this subject is comprehensively covered in Chapter 75). Finally, a recent randomized, double-blind, placebo-controlled trial assessed the value of pretreatment with a humanized anti-IgE monoclonal antibody (omalizumab) on side effects in a rapid updosing (“rush”) protocol of ragweed immunotherapy for allergic rhinitis (Casale et al. 2006). Such accelerated immunotherapy regimens balance increased convenience and compliance with a higher risk of systemic reactions. However, the addition of omalizumab resulted in a fivefold reduction in the risk of anaphylaxis during the 1-day rapid updosing period. Within the second category are a number of adjuvants principally developed to potentiate Th2 to Th1 immune deviation, but which might also have effects on regulatory T-cell responses. The type-B immunostimulatory phosphorothioate oligodeoxynucleotide 5′-TGACTGTAACGTTCGAGATGA (ODN-1018) has been tested both in vitro and in vivo. In ragweed-stimulated PBMC responses, ODN-1018 promoted Th1 cytokine and IL-12 responses at the expense of Th2 cytokine production, a property which was further enganced by direct conjugation with the major ragweed allergen Amb a 1 (Marshall et al. 2001). Another type-B phosphorothioate oligodeoxynucleotide (ODN-2006; 5′TCGTCGTTTTGTCGTTTTGTCGTT) with equivalent activity in the PBMC model was separately shown to activate human

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Aug. 1 Aug. 16 Sept. 1 Sept. 16 Oct. 1 Oct. 16 Nov. 1 2001 Ragweed season Fig. 72.6 Effects of immunotherapy with ODN-1018/Amb a1 major ragweed allergen conjugate on nasal symptoms during the 2001 ragweed season. The vaccine was administered in six preseasonal injections. Mean daily nasal symptom diary scores are shown for subjects receiving active (AIC) or placebo therapy. Nasal symptoms were scored over a range of 0–20, with higher scores indicating more symptoms. The broken vertical lines indicate the beginning and end of the full season. The long horizontal line shows the mean preseasonal nasal symptom diary score. P-values shown are for treatment effect. (From Creticos et al. 2006, with permission.) (See CD-ROM for color version.)

plasmacytoid dendritic cells to induce regulatory T cells (Moseman et al. 2004; Ito et al. 2007), presumably acting through the TLR9 pathway. A recent randomized, doubleblind, placebo-controlled phase 2 trial examined the effects of six weekly injections of the ODN-1018/Amb a 1 conjugate on ragweed-induced allergic rhinitis (Creticos et al. 2006). Treatment was associated with improvement in peak season visual analog, nasal and quality of life symptom scores (Fig. 72.6). However, whether conjugation with ODN-1018 confers additional clinical efficacy over Amb a 1 alone remains to be proven. An alternative immunomodulatory adjuvant that has been clinically tested is a derivative of monophosphoryl lipid A (MLA) from lipopolysaccharide of Salmonella minnesota. A structurally modified product, MPL, was associated with reduced pyrogenicity while the TLR4 agonist activity needed for adjuvant activity was retained (Persing et al. 2002). MPL promotes Th1 responses in human PBMC responses to allergen through induction of IL-12 production (Puggioni et al. 2005). In a randomized, double-blind, placebocontrolled, multicenter study of 141 subjects, four preseasonal injections of a vaccine containing MPL and tyrosine-adsorbed glutaraldehyde-modified extracts of pollens from 12 different grasses (Pollinex Quattro) reduced hay fever symptoms and medication requirements, and increased allergen-specific IgG (Drachenberg et al. 2001).

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It is clear that greater understanding of the mechanisms of immunotherapy has provided impetus for the development of novel approaches that result in effective immune deviation and/or induction of regulatory responses, while at the same time reducing the potential for IgE-mediated side effects. This new knowledge is also likely to enable the development of effective biomarkers in order to predict those patients who are likely to respond to immunotherapy, when to start, when to stop and to predict relapse following discontinuation, and the need for a further course of immunotherapy. Measurement of the biological activity of so-called “blocking” antibodies holds promise in this regard (Shamji et al. 2006). The sublingual route has emerged as an effective and safe alternative (Wilson et al. 2005) and is covered elsewhere. Meanwhile the subcutaneous route using standardized natural allergens remains the gold standard against which to test putative biomarkers and novel immonomodulatory approaches.

References Akbari, O., DeKruyff, R.H. & Umetsu, D.T. (2001) Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2, 725–31. Akbari, O., Freeman, G.J., Meyer, E.H. et al. (2002) Antigen-specific regulatory T cells develop via the ICOS–ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med 8, 1024– 32. Akdis, C.A., Blesken, T., Akdis, M., Wuthrich, B. & Blaser, K. (1998) Role of interleukin 10 in specific immunotherapy. J Clin Invest 102, 98–106. Akdis, C.A., Joss, A., Akdis, M., Faith, A. & Blaser, K. (2000) A molecular basis for T cell suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding. FASEB J 14, 1666–8. Barrat, F.J., Cua, D.J., Boonstra, A. et al. (2002) In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)and Th2-inducing cytokines. J Exp Med 195, 603–16. Bellinghausen, I., Metz, G., Enk, A.H., Christmann, S., Knop, J. & Saloga, J. (1997) Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift, and changes surface marker expression in venom-allergic subjects. Eur J Immunol 27, 1131–9. Benjaponpitak, S., Oro, A., Maguire, P., Marinkovich, V., DeKruyff, R.H. & Umetsu, D.T. (1999) The kinetics of change in cytokine production by CD4 T cells during conventional allergen immunotherapy. J Allergy Clin Immunol 103, 468 –75. Bodtger, U., Ejrnaes, A.M., Hummelshoj, L., Jacobi, H.H., Poulsen, L.K. & Svenson, M. (2005) Is immunotherapy-induced birch-pollenspecific IgG4 a marker for decreased allergen-specific sensitivity? Int Arch Allergy Immunol 136, 340–6. Bousquet, J., Lockey, R. & Malling, H.J. (1998) Allergen immunotherapy: therapeutic vaccines for allergic diseases. A WHO position paper. J Allergy Clin Immunol 102, 558–62. Casale, T.B., Busse, W.W., Kline, J.N. et al. (2006) Omalizumab pretreatment decreases acute reactions after rush immunotherapy for

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ragweed-induced seasonal allergic rhinitis. J Allergy Clin Immunol 117, 134– 40. Creticos, P.S., Schroeder, J.T., Hamilton, R.G. et al. (2006) Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis. N Engl J Med 355, 1445–55. Daeron, M., Malbec, O., Latour, S., Arock, M. & Fridman, W.H. (1995) Regulation of high-affinity IgE receptor-mediated mast cell activation by murine low-affinity IgG receptors. J Clin Invest 95, 577– 85. Des Roches, A., Paradis, L., Knani, J. et al. (1996) Immunotherapy with a standardized Dermatophagoides pteronyssinus extract. V. Duration of the efficacy of immunotherapy after its cessation. Allergy 51, 430–3. Djurup, R. & Malling, H.J. (1987) High IgG4 antibody level is associated with failure of immunotherapy with inhalant allergens. Clin Allergy 17, 459– 68. Drachenberg, K.J., Wheeler, A.W., Stuebner, P. & Horak, F. (2001) A well-tolerated grass pollen-specific allergy vaccine containing a novel adjuvant, monophosphoryl lipid A, reduces allergic symptoms after only four preseasonal injections. Allergy 56, 498–505. Durham, S.R., Ying, S., Varney, V.A. et al. (1996) Grass pollen immunotherapy inhibits allergen-induced infiltration of CD4+ T lymphocytes and eosinophils in the nasal mucosa and increases the number of cells expressing messenger RNA for interferongamma. J Allergy Clin Immunol 97, 1356– 65. Durham, S.R., Walker, S.M., Varga, E.M. et al. (1999) Long-term clinical efficacy of grass-pollen immunotherapy. N Engl J Med 341, 468–75. Ebner, C., Siemann, U., Bohle, B. et al. (1997) Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phl p 1, a major grass pollen allergen. Clin Exp Allergy 27, 1007–15. Ejrnaes, A.M., Svenson, M., Lund, G., Larsen, J.N. & Jacobi, H. (2006) Inhibition of rBet v 1-induced basophil histamine release with specific immunotherapy-induced serum immunoglobulin G: no evidence that FcgammaRIIB signalling is important. Clin Exp Allergy 36, 273– 82. Ewan, P.W., Deighton, J., Wilson, A.B. & Lachmann, P.J. (1993) Venom-specific IgG antibodies in bee and wasp allergy: lack of correlation with protection from stings. Clin Exp Allergy 23, 647–60. Francis, J.N., Till, S.J. & Durham, S.R. (2003) Induction of IL10+CD4+CD25+ T cells by grass pollen immunotherapy. J Allergy Clin Immunol 111, 1255– 61. Francis, J.N., James, L.K., Paraskevopoulos, G. et al. (2008) Grass pollen immunotherapy: IL-10 induction and suppression of late responses precedes IgG4 inhibitory antibody activity. In press. Frew, A.J., Powell, R.J., Corrigan, C.J. & Durham, S.R. (2006) Efficacy and safety of specific immunotherapy with SQ allergen extract in treatment-resistant seasonal allergic rhinoconjunctivitis. J Allergy Clin Immunol 117, 319–25. Furin, M.J., Norman, P.S., Creticos, P.S. et al. (1991) Immunotherapy decreases antigen-induced eosinophil cell migration into the nasal cavity. J Allergy Clin Immunol 88, 27–32. Garcia, B.E., Sanz, M.L., Gato, J.J., Fernandez, J. & Oehling, A. (1993) IgG4 blocking effect on the release of antigen-specific histamine. J Invest Allergol Clin Immunol 3, 26–33. Gehlhar, K., Schlaak, M., Becker, W. & Bufe, A. (1999) Monitoring allergen immunotherapy of pollen-allergic patients: the ratio of

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allergen-specific IgG4 to IgG1 correlates with clinical outcome. Clin Exp Allergy 29, 497–506. Golden, D.B., Kagey-Sobotka, A., Norman, P.S., Hamilton, R.G. & Lichtenstein, L.M. (2004) Outcomes of allergy to insect stings in children, with and without venom immunotherapy. N Engl J Med 351, 668–74. Groux, H., Bigler, M., de Vries, J.E. & Roncarolo, M.G. (1996) Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J Exp Med 184, 19–29. Hage-Hamsten, M., Johansson, E., Roquet, A. et al. (2002) Nasal challenges with recombinant derivatives of the major birch pollen allergen Bet v 1 induce fewer symptoms and lower mediator release than rBet v 1 wild-type in patients with allergic rhinitis. Clin Exp Allergy 32, 1448–53. Hamid, Q.A., Schotman, E., Jacobson, M.R., Walker, S.M. & Durham, S.R. (1997) Increases in IL-12 messenger RNA+ cells accompany inhibition of allergen-induced late skin responses after successful grass pollen immunotherapy. J Allergy Clin Immunol 99, 254–60. Hori, S., Nomura, T. & Sakaguchi, S. (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–61. Iliopoulos, O., Proud, D., Adkinson, N.F. Jr et al. (1991) Effects of immunotherapy on the early, late, and rechallenge nasal reaction to provocation with allergen: changes in inflammatory mediators and cells. J Allergy Clin Immunol 87, 855– 66. Ito, T., Yang, M., Wang, Y.H. et al. (2007) Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand. J Exp Med 204, 105–15. Jakobsen, C.G., Bodtger, U., Poulsen, L.K. & Roggen, E.L. (2005) Vaccination for birch pollen allergy: comparison of the affinities of specific immunoglobulins E, G1 and G4 measured by surface plasmon resonance. Clin Exp Allergy 35, 193–8. Jeannin, P., Lecoanet, S., Delneste, Y., Gauchat, J.F. & Bonnefoy, J.Y. (1998) IgE versus IgG4 production can be differentially regulated by IL-10. J Immunol 160, 3555–61. Jutel, M., Pichler, W.J., Skrbic, D., Urwyler, A., Dahinden, C. & Muller, U.R. (1995) Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures. J Immunol 154, 4187–94. Jutel, M., Akdis, M., Budak, F. et al. (2003) IL-10 and TGF-beta cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 33, 1205–14. Klimek, L., Dormann, D., Jarman, E.R., Cromwell, O., Riechelmann, H. & Reske-Kunz, A.B. (1999) Short-term preseasonal birch pollen allergoid immunotherapy influences symptoms, specific nasal provocation and cytokine levels in nasal secretions, but not peripheral T-cell responses, in patients with allergic rhinitis. Clin Exp Allergy 29, 1326–35. Levings, M.K., Sangregorio, R. & Roncarolo, M.G. (2001) Human CD25(+)CD4(+) T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med 193, 1295–302. Lichtenstein, L.M., Ishizaka, K., Norman, P.S., Sobotka, A.K. & Hill, B.M. (1973) IgE antibody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy. J Clin Invest 52, 472–82. Ling, E.M., Smith, T., Nguyen, X.D. et al. (2004) Relation of CD4+CD25+ regulatory T-cell suppression of allergen-driven T-cell

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activation to atopic status and expression of allergic disease. Lancet 363, 608–15. Lockey, R.F. (2001) ARIA: Global guidelines and new forms of allergen immunotherapy. J Allergy Clin Immunol 108, 497–9. McHugh, R.S., Whitters, M.J., Piccirillo, C.A. et al. (2002) CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16, 311–23. Malbec, O., Fong, D.C., Turner, M. et al. (1998) Fc epsilon receptor I-associated lyn-dependent phosphorylation of Fc gamma receptor IIB during negative regulation of mast cell activation. J Immunol 160, 1647–58. Marshall, J.D., Abtahi, S., Eiden, J.J. et al. (2001) Immunostimulatory sequence DNA linked to the Amb a 1 allergen promotes T(H)1 cytokine expression while downregulating T(H)2 cytokine expression in PBMCs from human patients with ragweed allergy. J Allergy Clin Immunol 108, 191–7. Moller, C., Dreborg, S., Ferdousi, H.A. et al. (2002) Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study). J Allergy Clin Immunol 109, 251–6. Moseman, E.A., Liang, X., Dawson, A.J. et al. (2004) Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J Immunol 173, 4433–42. Moverare, R., Elfman, L., Vesterinen, E., Metso, T. & Haahtela, T. (2002) Development of new IgE specificities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP System. Allergy 57, 423–30. Nakamura, K., Kitani, A. & Strober, W. (2001) Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J Exp Med 194, 629– 44. Nasser, S.M., Ying, S., Meng, Q., Kay, A.B. & Ewan, P.W. (2001) Interleukin-10 levels increase in cutaneous biopsies of patients undergoing wasp venom immunotherapy. Eur J Immunol 31, 3704–13. Niederberger, V., Horak, F., Vrtala, S. et al. (2004) Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc Natl Acad Sci USA 101 (suppl. 2), 14677–82. Niggemann, B., Jacobsen, L., Dreborg, S. et al. (2006) Five-year follow-up on the PAT study: specific immunotherapy and longterm prevention of asthma in children. Allergy 61, 855–9. Nopp, A., Hallden, G., Lundahl, J. et al. (2000) Comparison of inflammatory responses to genetically engineered hypoallergenic derivatives of the major birch pollen allergen bet v 1 and to recombinant bet v 1 wild type in skin chamber fluids collected from birch pollen-allergic patients. J Allergy Clin Immunol 106, 101–9. Nouri-Aria, K.T., Wachholz, P.A., Francis, J.N. et al. (2004) Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity. J Immunol 172, 3252–9. Nouri-Aria, K.T., Pilette, C., Jacobson, M.R., Watanabe, H. & Durham, S.R. (2005) IL-9 and c-Kit+ mast cells in allergic rhinitis during seasonal allergen exposure: effect of immunotherapy. J Allergy Clin Immunol 116, 73–9. Novak, N., Allam, J.P., Hagemann, T. et al. (2004) Characterization of FcepsilonRI-bearing CD123 blood dendritic cell antigen-2 plasma-

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cytoid dendritic cells in atopic dermatitis. J Allergy Clin Immunol 114, 364–70. Ong, Y.E., Menzies-Gow, A., Barkans, J. et al. (2005) Anti-IgE (omalizumab) inhibits late-phase reactions and inflammatory cells after repeat skin allergen challenge. J Allergy Clin Immunol 116, 558–64. Otsuka, H., Mezawa, A., Ohnishi, M., Okubo, K., Seki, H. & Okuda, M. (1991) Changes in nasal metachromatic cells during allergen immunotherapy. Clin Exp Allergy 21, 115–19. Pajno, G.B., Barberio, G., De Luca, F., Morabito, L. & Parmiani, S. (2001) Prevention of new sensitizations in asthmatic children monosensitized to house dust mite by specific immunotherapy. A six-year follow-up study. Clin Exp Allergy 31, 1392–7. Persing, D.H., Coler, R.N., Lacy, M.J. et al. (2002) Taking toll: lipid A mimetics as adjuvants and immunomodulators. Trends Microbiol 10 (10 suppl.), S32–S37. Pienkowski, M.M., Norman, P.S. & Lichtenstein, L.M. (1985) Suppression of late-phase skin reactions by immunotherapy with ragweed extract. J Allergy Clin Immunol 76, 729–34. Pilette, C., Nouri-Aria, K.T., Jacobson, M.R. et al. (2007) Grass pollen immunotherapy induces an allergen-specific IgA2 antibody response associated with mucosal TGF-beta expression. J Immunol 178, 4658–66. Pillai, V., Ortega, S.B., Wang, C.K. & Karandikar, N.J. (2007) Transient regulatory T-cells: a state attained by all activated human T-cells. Clin Immunol 123, 18–29. Platts-Mills, T.A., von Maur, R.K., Ishizaka, K., Norman, P.S. & Lichtenstein, L.M. (1976) IgA and IgG anti-ragweed antibodies in nasal secretions. Quantitative measurements of antibodies and correlation with inhibition of histamine release. J Clin Invest 57, 1041–50. Puggioni, F., Durham, S.R. & Francis, J.N. (2005) Monophosphoryl lipid A (MPL) promotes allergen-induced immune deviation in favour of Th1 responses. Allergy 60, 678–84. Rak, S., Lowhagen, O. & Venge, P. (1988) The effect of immunotherapy on bronchial hyperresponsiveness and eosinophil cationic protein in pollen-allergic patients. J Allergy Clin Immunol 82, 470– 80. Royer, B., Varadaradjalou, S., Saas, P., Guillosson, J.J., Kantelip, J.P. & Arock, M. (2001) Inhibition of IgE-induced activation of human mast cells by IL-10. Clin Exp Allergy 31, 694–704. Sakaguchi, S. (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6, 345–52. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. & Toda, M. (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155, 1151–64. Shamji, M.H., Wilcock, L.K., Wachholz, P.A. et al. (2006) The IgE-facilitated allergen binding (FAB) assay: validation of a novel flow-cytometric based method for the detection of inhibitory antibody responses. J Immunol Methods 317, 71–9. Sundstedt, A., O’Neill, E.J., Nicolson, K.S., Wraith, D.C. (2003) Role for IL-10 in suppression mediated by peptide-induced regulatory T cells in vivo. J Immunol 170(3), 1240–8. Svenson, M., Jacobi, H.H., Bodtger, U., Poulsen, L.K., Rieneck, K. & Bendtzen, K. (2003) Vaccination for birch pollen allergy. Induction of affinity-matured or blocking IgG antibodies does not

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Vieira, P.L., Christensen, J.R., Minaee, S. et al. (2004) IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J Immunol 172, 5986–93. Wachholz, P.A., Nouri-Aria, K.T., Wilson, D.R. et al. (2002) Grass pollen immunotherapy for hayfever is associated with increases in local nasal but not peripheral Th1:Th2 cytokine ratios. Immunology 105, 56–62. Wachholz, P.A., Soni, N.K., Till, S.J. & Durham, S.R. (2003) Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 112, 915–22. Walker, S.M., Varney, V.A., Gaga, M., Jacobson, M.R. & Durham, S.R. (1995) Grass pollen immunotherapy: efficacy and safety during a 4-year follow-up study. Allergy 50, 405–13. Walker, S.M., Pajno, G.B., Lima, M.T., Wilson, D.R. & Durham, S.R. (2001) Grass pollen immunotherapy for seasonal rhinitis and asthma: a randomized, controlled trial. J Allergy Clin Immunol 107, 87–93. Warner, J.O., Price, J.F., Soothill, J.F. & Hey, E.N. (1978) Controlled trial of hyposensitisation to Dermatophagoides pteronyssinus in children with asthma. Lancet ii, 912–15. Wilson, D.R., Irani, A.M., Walker, S.M. et al. (2001a) Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium. Clin Exp Allergy 31, 1705–13. Wilson, D.R., Nouri-Aria, K.T., Walker, S.M. et al. (2001b) Grass pollen immunotherapy: symptomatic improvement correlates with reductions in eosinophils and IL-5 mRNA expression in the nasal mucosa during the pollen season. J Allergy Clin Immunol 107, 971–6. Wilson, D.R., Lima, M.T. & Durham, S.R. (2005) Sublingual immunotherapy for allergic rhinitis: systematic review and metaanalysis. Allergy 60, 4–12. Zhang, X., Izikson, L., Liu, L. & Weiner, H.L. (2001) Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration. J Immunol 167, 4245–53. Zhu, D., Kepley, C.L., Zhang, M., Zhang, K. & Saxon, A. (2002) A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation. Nat Med 8, 518–21.

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Allergen Injection Immunotherapy: Indications and Practice Hans-Jørgen Malling

Summary Allergen-specific immunotherapy is the administration of gradually increasing doses of the allergen that induces clinical disease. Subcutaneous immunotherapy has, in carefully selected patients, the capacity to reduce disease severity of a magnitude comparable to optimal pharmacotherapy and in addition has long-term and preventive effects (disease-modifying specific intervention). In contrast to pharmacotherapy, immunotherapy has documented clinical efficacy after termination and may decrease the risk of progression of disease severity and the development of further sensitization to other allergens. The most important aspect in obtaining high clinical efficacy and optimal safety is to select patients with disease predominantly induced by the allergen sensitivity and without chronic irreversible structural alterations in the airway mucosa. For patients suffering from inhalation allergy, the indication is based on severity of the disease, duration of clinical symptoms, response to drug treatment, and the patient’s attitude to the disease and treatment. For patients allergic to Hymenoptera venom, immunotherapy is the only life-saving intervention. The major problem related to subcutaneous immunotherapy is the risk of inducing anaphylactic side effects. The very principle of administering allergens to an IgE-sensitized individual always involves a risk of eliciting anaphylaxis. The art of making subcutaneous immunotherapy a safe treatment is highly dependent on practice and simple guidelines. The guidelines relate to how treatment is organized and especially the immediate availability and proper use of rescue facilities. However, the optimal situation is to organize treatment in such a way that side effects are avoided or reduced to a minimum. This can be achieved by selecting patients carefully, focusing on optimally drug-treated patients without allergic inflammation, and selecting allergen extracts with documented clinical efficacy and safety profiles based on clinical trials. The practical performance of the treatment has to be carried out following evidence-based standards in relation to induction regimens and top dose. The careful preinjection evaluation of patients aims at

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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identifying risk factors for inducing anaphylactic reactions and avoiding injections in patients at risk. The staff administering immunotherapy injections should be familiar with and regularly trained in recognizing patient conditions that increase this risk. Dose adjustments should balance the importance of administering a dose sufficiently high to ensure clinical efficacy and at the same time avoid side effects. The rigorous monitoring before injection and during the observation period and prompt recognition and treatment of anaphylactic reactions make this treatment acceptable as a safe routine treatment of allergy.

Successful immunotherapy Allergen-specific immunotherapy is the practice of administering gradually increasing quantities of an allergen to an allergic subject in order to ameliorate the symptoms associated with subsequent exposure to the causative allergen. Allergenspecific immunotherapy is a well-documented treatment in allergic diseases (Malling & Weeke 1993; Bousquet et al. 1998, 2001). Subcutaneous immunotherapy induces clinical and immunologic tolerance, has long-term efficacy, and may prevent the progression of allergic disease. Additionally, the treatment also improves the quality of life of allergic patients. Successful immunotherapy relates primarily to clinical efficacy. Other important issues relate predominantly to practical aspects like the organization of treatment, patient information, serviceable settings in the clinic, physician experience, steps taken to avoid or minimize side effects, rescue facilities, and prompt and adequate treatment of possible side effects (Malling & Weeke 1993; AAAI Board of Directors 1994). Scientific data relating to practical aspects like dosage regimens and dose modifications are rather scarce, and primarily based on personal experiences. The present guidelines are based predominantly on the European Academy of Allergology and Clinical Immunology Immunotherapy Position Papers (Malling & Weeke 1993; Alvarez-Cuesta et al. 2006) and the joint guidelines from the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology (Li et al. 2003) summarizing the experiences of European and American specialists.

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Table 73.1 Treatment strategy for patients referred with allergic symptoms. Control of symptoms using an optimal pharmacologic treatment Allergy diagnostic procedures (to evaluate the possibilities of instituting disease-modifying specific treatment) Disease-modifying treatment Allergen avoidance (reduction) Allergen-specific immunotherapy Stepping down pharmacologic treatment to the lowest possible dose, keeping the patient adequately controlled Follow-up with education and adjustment of pharmacologic treatment until a stable situation is obtained

Treatment strategy Available treatments for allergic rhinoconjunctivitis and asthma include four elements: (i) allergen avoidance, (ii) pharmacotherapy, (iii) allergen-specific immunotherapy, and (iv) patient education. There are few studies that directly compare the relative advantages of these interventions. However, it seems logical that their optimal combination for each individual patient should improve the clinical outcome. Allergen avoidance should be considered as a first-line intervention and even when not completely effective may reduce disease severity and the need for additional treatment (Gotzsche et al. 2004). Drug treatment is for most patients the next step to reduce disease severity. In patients who need regular pharmacotherapy, it is often recommended that immunotherapy be started early while the disease still remains without chronic irreversible structural alterations and when it is possible to prevent deterioration of disease (Malling & Weeke 1993; Bousquet et al. 1998, 2001). Although drugs are often effective and for most without significant side effects, drugs represent a symptomatic treatment, while immunotherapy represents the only treatment that might alter the natural course of the disease (Durham et al. 1999; Niggemann et al. 2006). Using an appropriate allergen product and a correct indication, immunotherapy can significantly reduce the severity of the allergic disease, reduce the need for antiallergic drugs (Malling 1998), and improve the quality of life for allergic patients (Alvarez-Cuesta et al. 2005). A schematic strategy for the treatment of patients referred with allergic symptoms is shown in Table 73.1.

Indications and contraindications Indications Immunotherapy is indicated for confirmed IgE-mediated disease using standardized products with documented clinical efficacy and safety: grass, birch, ragweed, olive, Parietaria, cypress, cat, house-dust mites (Dermatophagoides pteronnysinus, D. farinae), and Hymenoptera venoms (vespids and honeybee)

Allergen Injection Immunotherapy: Indications and Practice (Bousquet et al. 1998; Bonifazi et al. 2005). Immunotherapy for inhalant allergies is indicated as a supplement to allergen avoidance and pharmacotherapy. For allergy to Hymenoptera, it is the only lifesaving long-term intervention. Normally subcutaneous immunotherapy is restricted to patients above 5 years of age. Below this age inhalation allergens play a less important role. Furthermore, when subcutaneous allergenspecific immunotherapy is prescribed below 5 years of age, it is critical that the physician responsible for the injections has experience in identifying and treating emerging signs of anaphylaxis in this age group. In addition nurses and other staff administrating allergen injections to children must be specially trained to treat this age group. Except for insect venom allergy, subcutaneous immunotherapy is rarely used after the age of 60 years. Due to lack of efficacy and the risk of severe side effects immunotherapy is not indicated for the treatment of food allergy. In atopic eczema the demonstration of IgE sensitization is mostly not linked to the disease.

Indications in rhinoconjunctivitis and asthma Immunotherapy is indicated in the following groups of patients: • those with symptoms induced predominantly by allergen exposure; • those with clinical symptoms due to a single or few allergens; • those with a prolonged season or with symptoms induced by succeeding pollen seasons; • those with rhinitis and symptoms from the lower airways during peak allergen exposure; • those in whom antihistamines and moderate-dose topical glucocorticoids insufficiently control symptoms; • those who do not want to be on constant or long-term pharmacotherapy; • those in whom pharmacotherapy induces undesirable side effects. It is advocated that the indication follow the guidelines of the European Academy of Allergology and Clinical Immunology (EAACI) Immunotherapy Position Paper (Malling & Weeke 1993). The indication for allergen-specific immunotherapy should be explicitly defined in relation to (i) disease and disease severity; (ii) allergens and the importance of allergen sensitization; (iii) the need for and effect of symptomatic treatment; (iv) risks of the disease and the treatment; (v) psychologic factors; and (vi) the patient’s attitude to the disease and treatment. The indication for immunotherapy in allergic rhinoconjunctivitis and/or asthma is related to both the severity of the disease and the duration of symptoms (Malling & Weeke 1993; Bousquet et al. 1998). Mild symptoms that respond adequately to as-needed oral or topical antihistamines (rhinitis) or to a modest consumption of β2 agonists (asthma) is no argument for beginning immunotherapy. However, a need for

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repeated courses of topical or inhaled corticosteroids (applying to both a short or a long season), or symptoms lasting several months (even if these symptoms are rather mild and respond adequately to pharmacologic treatment), may be argument in favor of initiating immunotherapy (Malling & Weeke 1993). The intensity of drug treatment, in relation to both the doses needed to reduce symptoms and the frequency of daily administrations, and the number of organs needing treatment constitute an important part of the rationale for adding immunotherapy to the general treatment strategy (in an attempt to reduce the requirement for drugs optimally to an as-required basis). A general misconception is to reserve immunotherapy to patients who do not respond adequately to drug treatment or who present with side effects during drug treatment (Malling & Weeke 1993). Optimal results of immunotherapy are obtained in patients with mild disease, i.e., requiring a rather modest pharmacologic treatment (Bousquet et al. 1998). It seems that young patients (children) respond better to immunotherapy than adults (Hejjaoui et al. 1990; Malling & Weeke 1993). This may be related to the actual age of the patient, but is more likely related to the duration of the disease, implying that attempts to interfere with the natural course of the disease should be introduced at a time where the patient has the capacity to respond positively, i.e., before the disease deteriorates to a chronic nonreversible condition (Bousquet et al. 1992). Risk–benefit assessments in relation to both the disease and possible treatments constitutes an important aspect in evaluating the indication for immunotherapy (Malling & Weeke 1993; Bousquet et al. 1998). Generally, allergic rhinitis is a boring but in no way life-threatening disease. The disabling nature of rhinitis in relation to diminishing performance capacity in school, work, and social situations is of extremely high importance for the suffering patient (quality of life) (Juniper et al. 1996). Another aspect is that a considerable number of rhinitic patients develop asthma during the course of the disease (Sly 1996; Strachan et al. 1996). As asthma is a more severe disease, in relation to acute attacks, hospital admissions, days off work, and development of chronic pulmonary insufficiency, inadequate treatment is associated with a considerably increased risk (Peat et al. 1987; Bousquet et al. 1992). The hazards of immunotherapy are strictly related to the risk of inducing anaphylactic reactions. The rate of severe systemic reactions in rhinitics treated with injections of high-potency extracts is approximately 5% (Tabar et al. 1993; Rugose et al. 1997), primarily in the induction phase (Malling & Weeke 1993). In asthmatics, the risk of systemic reactions is slightly higher, primarily due to bronchial obstruction. Monitoring the lung function of asthmatic patients before the injections is therefore mandatory, as is ensuring optimal antiasthmatic pharmacologic treatment (Malling & Weeke 1993; Bousquet et al. 1998). Systemic reactions represent a general limitation for the use of immunotherapy, and there-

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fore the indication and possibly also the practical treatment has to be performed by a specialist who is aware of the risks of immunotherapy and who is capable of dealing with systemic reactions (Malling & Weeke 1993; Bousquet et al. 1998). Psychological factors include compliance with drug intake and the fixation of the patient’s character in a diseasedependent state due to the constant intake of drugs. Studies of drug compliance in asthmatic patients have shown that only approximately half the prescribed drugs are actually being taken (Alessandro et al. 1994; Yeung et al. 1994). Drug compliance may be higher in rhinitis, due to the shorter duration of symptoms in seasonal hay fever, but patients often tend to tolerate some symptoms (in a considerable number of patients, rather severe symptoms) without substituting the as-required use of systemic or topical antihistamines by the more effective topical corticosteroids (Bronsky et al. 1996). An important issue in treating allergic disease is to be aware of the patient’s individual perception of disease severity and the psychological motivation and scientific understanding of the rationale for treating allergic inflammation in contrast to only diminishing the inconvenience of symptoms. Despite a massive amount of information on the safety of topical corticosteroids, “steroid phobia” still represents a problem that excludes patients from effective drug treatment. Because a growing number of patients fear the risks of taking synthetic drugs, many consult their physicians with requests for a treatment that has the potential to interrupt the natural course of their disease (immunotherapy) instead of continuing to take drugs that only reduce their symptoms. In the discussion of the treatment of allergic rhinitis and asthma, several factors have to be taken into consideration. • A considerable number of patients with rhinitis (about 10%) have symptoms from the lower airways, often without these symptoms being considered (and treated) as asthma. • Patients presenting with symptoms only from the upper airways run a significant risk of developing clinically manifest asthma with time. • The less intensive the symptoms, the less symptomatic treatment is needed, but on the other hand intervention in the natural course of the disease is more successful. Generally speaking, no definite rules for instituting immunotherapy in rhinitis and asthma can be stipulated. The indication is based on a careful balancing of advantages and disadvantages, taking into consideration the patient’s attitude to symptoms and possible treatments (Malling & Weeke 1993). In this way immunotherapy is not an ultimate treatment principle, but represents a supplement to drug treatment used in the early phase of the disease (Malling & Weeke 1993; Bousquet et al. 1998). In asthma, the important issue is to exclude patients with primarily nonallergic-induced nonspecific hyperreactivity. Asthmatic patients who are candidates for immunotherapy should have normal lung function, a limited range of allergen sensitization, and symptoms predominantly induced by the allergen (Malling & Weeke 1993;

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Table 73.2 Indications for immunotherapy based on patient profiling. Age of patient and duration of disease (in years) Organs involved Severity and duration of symptoms Allergens responsible for clinical symptoms Importance of allergen sensitization vs. nonspecific triggers Allergen exposure and effect of allergen avoidance Clinical response to pharmacotherapy Number and types of drugs needed Risk incurred by pharmacotherapy vs. immunotherapy Impact of disease and treatment on quality of life Patients’ attitude to and expectations of treatment Each individual item is evaluated for conditions that argue for or against immunotherapy, and only patients whose overall disease profile favors immunotherapy should be offered the treatment.

Allergen Injection Immunotherapy: Indications and Practice

Contraindications The contraindications for subcutaneous allergen-specific immunotherapy with inhalant and Hymenoptera venom allergens include (Bousquet et al. 1998) the following.

Absolute contraindications • Serious immunologic diseases, major cardiovascular disease (except in cases of serious insect venom allergies), cancer, chronic infections. • Severe asthma with persistently reduced lung function (forced expiratory volume in 1 s, FEV1) below 70% of predicted despite optimal pharmacologic treatment. • Treatment with beta-blockers (including topical) (see Hymenoptera immunotherapy). • Lack of compliance and severe psychological disorders.

Relative contraindications Bousquet et al. 1998). The likelihood of immunotherapy rendering asthmatic patients completely symptom-free is modest, but many patients prefer this treatment as an attempt to interfere with the natural progression of the disease and to reduce the need for prophylactic drugs. As successful immunotherapy in allergic rhinitis and asthma is based on a multitude of factors that favor or disfavor the use of immunotherapy, it may be advantageous to base the indication for immunotherapy on patient profiling (Table 73.2), balancing pros and cons of each individual item in order to make a comprehensive disease profile of the patient. Patients offered immunotherapy should be those whose profile points to a greater likelihood of high clinical effect and low risk of side effects.

Indication in Hymenoptera venom allergy Venom immunotherapy is indicated in both children and adults with a history of severe systemic allergic reactions, including respiratory and/or cardiovascular symptoms and documented sensitization to the respective insect as determined by either skin tests and/or specific serum IgE tests (Müller & Mosbech 1993; Bonifazi et al. 2005). With regard to systemic reactions that are not life-threatening (urticaria, erythema, pruritus), other factors may influence the decision to initiate venom immunotherapy. These include availability of immediate access to medical care, occupations and/or hobbies where the risk of exposure is high, concomitant cardiovascular disease, the presence of other pathologies such as mastocytosis, and psychological factors arising from anxiety, which can seriously impair patient quality of life. Recent guidelines do not recommend immunotherapy in children below the age of 15 years with exclusively cutaneous reactions (urticaria). Immunotherapy is not recommended for large local reactions or unusual reactions, such as non-IgEmediated hypersensitivity reactions like vasculitis, nephrosis or thrombocytopenia (Müller & Mosbech 1993).

• Pregnancy (no documentation of teratogenic risk), but a risk of anaphylactic reactions during induction phase and consequent damage to the fetus. It is recommended not to start induction during or in case of planned pregnancy. Uncomplicated maintenance treatment may be continued after careful information and acceptance by the patient. The treatment should be terminated by the slightest patient insecurity or the development of complications of immunotherapy. • Severe atopic eczema.

Hymenoptera venom immunotherapy In contrast to inhalation allergen immunotherapy, immunotherapy for Hymenoptera venom allergy is often indicated in elderly patients with coexisting cardiovascular disease, who are at special risk of developing severe or even fatal reactions. Such patients are commonly on beta-blocker treatment. In this situation the risk of stopping the drug must be carefully balanced against the risk of abandoning venom immunotherapy (Bonifazi et al. 2005). In coronary heart disease or severe ventricular arrhythmia the risk of stopping the beta-blocker can be unacceptable. If highly exposed to the relevant insect, venom immunotherapy may be carried out in patients with ongoing beta-blockade but under careful supervision, including monitoring of blood pressure and ECG and with expertise and remedies at hand if severe side effects with resistance to treatment due to beta-blockade should occur (Müller & Haeberli 2005).

Preventive and disease-modifying capacity Clinical studies have documented that subcutaneous immunotherapy has disease-modifying capacities. The capacity to suppress the development of new sensitization has been investigated in three nonrandomized studies in monosensitized patients (Des Roches et al. 1997; Pajno et al. 2001; PurelloD’Ambrosio et al. 2001). In an open retrospective study

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Purello-D’Ambrosio et al. (2001) followed up 7182 patients monosensitized to different allergens and treated with subcutaneous immunotherapy for 4 years and off immunotherapy for 3 years. The control group consisted of 1214 matched patients followed for 7 years. The development of sensitization to new allergens showed a clinically relevant and statistically significant difference at the 4-year follow-up, with values of 68% in the control group versus 24% in the immunotherapy group; at the 7-year follow-up, the figures were 78% and 27%, respectively. Pajno et al. (2001) followed 75 subcutaneous immunotherapy-treated children only sensitized to housedust mites and 63 comparable controls treated pharmacologically for 6 years. In the immunotherapy-treated group, 74% continued to be monosensitized versus 33% in the control group. Although these studies are of interest, prospective randomized controlled studies are needed. Subcutaneous immunotherapy might prevent the progression of rhinitis into asthma. A multicenter pediatric study investigated the capacity of immunotherapy in children with allergic rhinitis to downregulate the development of asthma (Niggemann et al. 2006). Children allergic to birch and grass pollen and no symptoms of lower airway hyperreactivity were randomized to receive either immunotherapy or an optimal pharmacologic treatment. After 3 years of treatment and 2 years off immunotherapy, the number of children experiencing clinical asthma was statistically reduced in the immunotherapy group. The development of asthma was 20% in immunotherapy-treated children versus 43% in the drug-treated group, indicating that the high risk of developing symptoms from the lower airways in allergic rhinitic children may be diminished by immunotherapy. Bronchial hyperresponsiveness to methacholine decreased significantly in immunotherapy-treated children, but only 1 of 36 children with asthma at inclusion was free of asthma after 5 years (in the control group asthma disappeared in six children). The results clearly indicate that immunotherapy has a greater capacity for preventing than for curing asthma. Further studies are needed to clearly define the preventive capacity of subcutaneous immunotherapy in the treatment of allergic rhinoconjunctivitis and asthma.

Organization of immunotherapy Appropriate organization of the practical aspects of immunotherapy (e.g., office facilities, opening hours) should not be neglected, as these may be of major importance in ensuring optimal patient compliance. Physicians and nurses giving injections and organizing immunotherapy should be regularly updated about recent scientific knowledge in immunotherapy and trained in practical skills, e.g., injection technique, supervision, and emergency treatment (Alvarez-Cuesta et al. 2006). Patient management plans should be based on a quality-assurance program, e.g., a systematic description of

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elements and processes related to the diagnosis and treatment of patients offered allergen-specific immunotherapy. The practical organization has to take into consideration the situation in individual countries. The evaluation of patients with regard to disease, severity of symptoms, diagnostic tests, and indication for immunotherapy should be undertaken by a specialist in allergic diseases (Malling & Weeke 1993). Inappropriate evaluation of the patient and indication for immunotherapy may well be the major reason for insufficient response to immunotherapy: “To minimize risk and improve efficacy, subcutaneous immunotherapy needs to be prescribed by specialists and administered by or under the close supervision of physicians trained to manage acute systemic reactions if anaphylaxis occurs” (Malling & Weeke 1993; Alvarez-Cuesta et al. 2006). Physicians, nurses and healthcare personnel should be offered a systematic theoretical and practical education before being involved in the treatment and must be trained and regularly updated in subcutaneous immunotherapy including the observation and rescue treatment of systemic anaphylactic reactions (“minimal patient risk”). As a minimum requirement, a competent physician must always be present when subcutaneous immunotherapy is carried out and be responsible for the treatment. In addition, one extra person should be available for proper management of serious adverse events. All staff participating actively in subcutaneous immunotherapy should have clearly defined responsibilities and have received education to deal adequately with the responsibility, and furthermore to accept the responsibility. As the risk of inducing side effects is highest during the induction phase of immunotherapy, it is recommended that injections are administered under the auspices of a specialist until the maintenance dose is reached (Malling & Weeke 1993). Maintenance treatment may be continued by the specialist or taken over by the general practitioner in close collaboration with the specialist.

Rescue facilities The facilities for injection immunotherapy have been debated, based on the British Committee on the Safety of Medicines (1986) requirement for full rescue facilities. A basic list of drugs and resuscitation equipment for the treatment and monitoring of systemic anaphylaxis based on European and American guidelines includes the following (Li et al. 2003; Alvarez-Cuesta et al. 2006): • epinephrine (1 mg/mL) for injection; • antihistamine, corticosteroids, and a vasopressor for injection or oral treatment; • syringes, needles, tourniquet, and equipment for infusion; • saline for infusion; • oxygen and suction equipment; • silicone mask and equipment for manual ventilation; • equipment for measurement of blood pressure; • forms for recording the course and treatment of anaphylaxis.

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In settings remote from intensive care facilities, equipment for direct laryngoscopy, DC cardioversion, tracheotomy, and intracardiac injection may be optional, but the rare situation in which these procedures might be essential does not justify that they be immediately available for subcutaneous immunotherapy. It is recommended that the responsibility for checking the presence and function of equipment be delegated to a specified person, and that verification be documented in a logbook.

Patients Information The patient should receive verbal and written information about immunotherapy, including a description of efficacy, possible complications and inconvenience (Alvarez-Cuesta et al. 2006), practicalities about the duration of treatment, observation, cost benefits, and who is administering the injections. The information to the patient should be given in such a way that the patient feels quite at ease with the treatment and is capable of deciding whether or not to accept the treatment. The information to the patient should focus on immunotherapy as a treatment that aims to increase the patient’s clinical tolerance to an allergen by regular subcutaneous injection of successively increasing doses of the allergen and on the disease-modifying capacities in terms of long-term efficacy and preventive prospects (Malling & Weeke 1993; Alvarez-Cuesta et al. 2006). In addition, it is important that some fundamental aspects of immunotherapy are included in the information. 1 Immunotherapy aims at administering the optimum maintenance dose (high clinical efficacy and low risk of side effects). 2 Immunotherapy is a supplement to drug treatment and to allergen avoidance (Bousquet et al. 1998). 3 Immunotherapy is potentially dangerous because allergen material is injected in a sensitized person (Lockey et al. 1987; Reid et al. 1993) and therefore a 30-min observation period after each injection together with observation of delayed reactions is essential (Malling & Weeke 1993; Bousquet et al. 1998).

Observation The American guidelines (Li et al. 2003) imply a 20–30 min observation period, but the observation period can be extended to more than 30 min in high-risk patients, e.g., asthmatics and patients with previous systemic reactions. However, the 30-min observation period seems appropriate as a gold standard as several surveys have shown that most side effects, and especially the severe life-threatening anaphylactic reactions, occur within 30 min (Reid et al. 1993; Stewart & Lockey 1992; Frew 1994; Löfkvist et al. 1994; Malling 2000).

Allergen Injection Immunotherapy: Indications and Practice

Diagnostic procedures It is important that diagnostic procedures to ensure that patients suffer from an allergic disorder are performed before initiating subcutaneous immunotherapy. IgE sensitization may be assessed by skin testing and/or in vitro determination of serum concentrations of allergen-specific IgE antibodies. Allergen challenge tests may occasionally be indicated in cases of uncertainty. Immunotherapy should be considered when positive tests correlate with suspected triggers and the patient’s known exposures. Immunotherapy should not be given to patients with negative diagnostic tests or those with positive tests that do not correlate with suspected triggers, clinical symptoms, or exposure (the presence of specific IgE antibodies does not necessarily indicate clinical sensitivity). Clinically relevant exposure to allergens like house-dust mites and animal dander may need substantiation by quantifying the actual level in matrasses and furniture to which the patient is exposed. In asthmatic patients it is critical to ensure that the majority of clinical symptoms are induced by allergen exposure and that nonspecific hyperreactivity only partially contributes to the total sum of symptoms (Bousquet et al. 1998).

Allergen products The quality of allergen products used for immunotherapy is crucial in order to obtain high clinical efficacy and minimize side effects (Bousquet & Michel 1992; Tabar et al. 1993). Extracts used for routine immunotherapy should be standardized (Dreborg & Frew 1993; American Academy of Allergy, Asthma and Immunology 1997) and subject to quality control (Allergen Standardization Subcommittee 1983; Anon. 1985; Nordic Council on Medicines 1989; Anon. 1991). In daily clinical practice, only allergen extracts for which clinical efficacy and safety have been documented by controlled studies should be used (Malling & Weeke 1993). Commercial allergen extracts may be delivered as aqueous, depot, or modified extracts. Aqueous extracts may be used for administration of multiple injections like “rush” or cluster immunotherapy. The disadvantages of aqueous extracts are the rapid degradation of allergen, and the high frequency of side effects (Stewart & Lockey 1992; Tamir et al. 1992; Reid et al. 1993). The apparently increased risk of using aqueous extracts is related to the direct availability of allergen epitopes to IgE-bearing subcutaneous mast cells, but also mainly to the preferential use of aqueous extracts in accelerated dose-increase regimens (Hejjaoui et al. 1992). On the other hand, a relative advantage of aqueous extracts may be that severe systemic side effects are elicited within 30 min after injection, i.e., while the patient is still under medical supervision (Malling & Weeke 1993). Depot extracts involve binding of allergens to a carrier in order to diminish the degradation and removal of allergens at the injection site. This may imply

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a reduction in the frequency of side effects (Pécoud et al. 1990) and potentially better efficacy (Norman 1990; Stewart & Lockey 1992). The disadvantages are that one (few) injection(s) may only be administered in one sequence and that side effects may be observed after 30 min. Life-threatening anaphylactic reactions do occur within 30 min and late occurring side effects are rarely life-threatening (Varney et al. 1991) and only rarely require specific intervention (BSACI 1993). Modified extracts involve either a physical or chemical alteration of allergen structure in order to reduce the allergenicity with retained immunogenicity (Norman 1990). Reduction in allergenicity often results in a need for higher doses in order to obtain clinical efficacy, thereby running the same risk of inducing side effects as with native allergen extracts (Mosbech et al. 1990; Stewart & Lockey 1992; Malling & Weeke 1993). Furthermore, modified extracts present tremendous problems for standardization, as this cannot be based on IgE-binding assays. Allergen mixtures of unrelated allergens are not recommended in immunotherapy (Malling & Weeke 1993; AlvarezCuesta et al. 2006). This is because of possible interactions between nonrelated allergens and a risk of enzymatic degradation, problems in standardizing mixtures, and a lowering of the optimal dose of each individual allergen, leading to lowdose immunotherapy for some of the allergens in a mixture (the allergen to which the patient is most sensitive will define the top dose for all allergens in a mixture) (Adkinson et al. 1997; Valenta & Kraft 2002). If allergen mixtures of nonrelated allergens are to be used, data on stability should be provided. Mixtures of cross-reacting allergens like grasses, mites, etc. could be used, but seldom have advantages over single allergens due to the high sequence homology for major epitopes. Clinical studies only show minimal difference in clinical response between single allergens and mixtures of cross-reacting allergens (Frostad et al. 1983; Möller et al. 1987; Wihl et al. 1988). In some patients, based on knowledge of allergen extract composition and the outcome of diagnostic tests, mixtures may be preferable due to a shortage of relevant epitopes in single allergen extracts. On the other hand, immunotherapy may be less effective in patients sensitive to multiple pollen species compared with patients only reacting to one pollen (Bousquet et al. 1991).

Dose-increase regimens Much mystery seems related to dose-increase regimens, but the fact is that the induction regimen is of little or no importance in relation to the long-term clinical efficacy of immunotherapy. The induction phase of immunotherapy simply represents a titration to the dose essential for an immunologic response (Malling 1994). The reason for using different induction regimens is to attain an appropriate balance between time needed to reach the top dose and the

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risk of eliciting side effects (Malling & Weeke 1993; AlvarezCuesta et al. 2006). Induction schedules are not fixed proposals, but should be adjusted according to the responses of the patient. Side effects in the dose-increase phase are related to the disease treated, severity of the disease, allergen extract (species, composition, type), regimens used during induction treatment, top dose, and dose increments (Malling 1998, 2000). Conventional dose-increase schedules involve one (rarely two) injection weekly until the maintenance dose is reached. The advantage is that the slow induction of immunologic tolerance results in a rather low frequency of side effects compared with more aggressive regimens, but on the other hand is highly time-consuming (induction regimens of 15–20 weeks are not unusual) with a risk of low compliance (Cohn & Pizzi 1993). The ultimate alternative to conventional dose-increase regimens is “rush” immunotherapy. The principle involves continuous injections at intervals of 30–60 min until the top dose is reached. This regimen may be highly efficient as the maintenance dose is normally reached in 3–5 days (Hejjaoui et al. 1992), although some schedules reach maintenance dose within 2.5 hours. The disadvantages are that it is often impossible to reach an appropriate maintenance dose so that the rush phase only represents a short part of the induction phase; the remaining part has to be completed by a conventional dose-increase regimen. Furthermore, the risk of inducing severe side effects is high, implying that rush immunotherapy should only be performed on hospitalized patients (Malling & Weeke 1993). The compromise between these two extremes is cluster immunotherapy, which involves administration of two to four injections at 30-min intervals in weekly sequences. The advantage is a reduction in time to reach the maintenance dose at the expense of a slightly increased risk of inducing side effects compared with conventional immunotherapy (Malling & Weeke 1993).

Top dose Clinical efficacy is related to the allergen dose, although few dose–efficacy studies have been performed (Johnstone 1957; Golden et al. 1981; Hirsch et al. 1981; Bousquet et al. 1990; Turkeltaub et al. 1990; Haugaard et al. 1993; Frew et al. 2006). After summarizing available data, low-dose immunotherapy (like Rinkel therapy) is ineffective (Creticos 1992), whereas extreme high-dose immunotherapy results in an unacceptably high frequency of side effects (Malling et al. 1986). Moderate-dose treatment may show a slower onset of action with respect to clinical efficacy, i.e., maximal efficacy will first be achieved after 2–3 years of treatment (a faster onset is obtained in high-dose immunotherapy) but with a more acceptable frequency of side effects (Haugaard et al. 1993). Some years ago, extreme high-dose studies were performed based on the concept of administering the maximal tolerated

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dose, i.e., a dose resulting in systemic reactions in a high number of patients (Norman & Lichtenstein 1986). Some extreme high-dose studies show systemic side effects in 100% of patients (Malling et al. 1986; Sundin et al. 1986). However, it should be noticed that the aim of the study of Malling et al. (1986) was to investigate the absolute upper limit of allergen dosing based on continuing dosage increase until the occurrence of systemic reactions. During recent years a new approach has evolved that involves administering more moderate doses during the induction phase in order to minimize the risk of inducing anaphylactic reactions (Malling 1994; Alvarez-Cuesta et al. 2006).

Risk factors in immunotherapy The risk of inducing systemic reactions depends on a number of factors. Knowledge of these might avoid initiating immunotherapy in high-risk patients. Asthmatics seem to be a highrisk group in relation to experiencing systemic reactions compared with hay fever patients. In a prospective study of immunotherapy involving 419 patients (Tabar et al. 1993) that followed the guidelines of EAACI Immunotherapy Subcommittee (Malling & Weeke 1993), 84% of patients who developed systemic side effects were asthmatics (asthma patients constituted 57% of the study population). In immunotherapy fatalities, asthma likewise has been identified as a risk factor (Reid et al. 1993), and the risk is further increased when the asthmatic patient is steroid-dependent, has required recent hospitalization or visits to the accident and emergency department, has unstable asthma, or has complicating cardiovascular disease(s) (Malling & Weeke 1993). The severity of asthma is a key factor. Bousquet et al. (1989) showed lung function persistently below 70% of predicted in patients on optimal drug therapy to be highly associated with side effects and furthermore to indicate poor clinical efficacy. The age of the patient is also of importance in relation to the frequency of systemic reactions. Children below the age of 5 years experience more side effects (Hejjaoui et al. 1990). Some studies have identified a slightly increased risk in females but, in practice, gender is of minimal importance as a risk factor (Reid et al. 1993). High sensitivity as identified by skin test or RAST score is a risk factor for anaphylaxis (Lockey et al. 1987; Mosbech et al. 1990), whereas allergy to Hymenoptera venom per se does not seem to increase the risk (Reid et al. 1993). Treatment during allergen season has been identified as a risk factor for anaphylaxis (Reid et al. 1993) and, based on this, dose escalation should not be performed during the season (Malling & Weeke 1993; Alvarez-Cuesta et al. 2006). Treatment with beta-blockers is considered a risk factor by some authors; however, the risk is not related to an increased risk of inducing systemic reactions (Hepner et al. 1990) but rather is related to an increase in the severity of a systemic reaction or to a reduction in the

Allergen Injection Immunotherapy: Indications and Practice effect of adrenergic drugs used to treat a systemic reaction (Toogood 1988; Javeed et al. 1996). Patients with other diseases or conditions which complicate the treatment of anaphylaxis, like symptomatic coronary heart disease and severe hypertension, also have an increased risk of possible systemic reactions that may end in fatality (Malling & Weeke 1993). During ongoing immunotherapy recent heavy intakes of alcohol might, due to interaction with diamine oxidase, lower the threshold of allergen tolerance and thereby a systemic reaction may be induced in patients with longlasting well-tolerated immunotherapy (Wantke et al. 1993). Although used by many physicians to modify doses (Varney et al. 1991; Löfkvist et al. 1994), controlled studies of the predictive value of local reactions do not indicate that the size of local reactions could be used to predict systemic side effects (Malling et al. 1986; Mosbech et al. 1990).

Safety of immunotherapy When investigating side effects in immunotherapy, it is important to distinguish the almost inevitable large local reactions, i.e., diffuse swellings that may persist for a day or more. These local reactions are annoying, but in no way are reactions due to symptoms from organs distant from the site of injection. Systemic side effects may vary from a few sneezes to fulminant anaphylactic shock and even death. Severity is related to how rapidly symptoms develop after injection. Itching in the palms, soles, and on hairy body parts, and rapid onset of erythema and urticaria, rhinitis or asthma occurring within minutes after injection will often progress to anaphylaxis and require treatment without delay. Systemic reactions are categorized into immediate systemic reactions (occurring within 30 min) and late systemic reactions (> 30 min after injection). A system for grading systemic reactions based on the rate of onset and severity of reactions has recently been proposed by the EAACI (Alvarez-Cuesta et al. 2006) (Table 73.3). Classification of the reaction, in addition to type and magnitude of reaction, should also include the time of onset following injection and the rate of subsequent development. In grading the severity of symptoms, any reaction where the time factor indicates prompt treatment with epinephrine (and consequent management of the symptoms) should be classified as a grade IV reaction (anaphylaxis), even if the patient does not progress to obvious shock. The response to treatment is also of importance, e.g., late-occurring angioedema or generalized urticaria only requiring minimal treatment is classed as a garde II reaction.

Frequency of systemic reactions According to surveys, severe systemic reactions are rare in routine immunotherapy. In a study involving a large number of patients (20 000 patients and 150 000 injections), Vervloet

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Table 73.3 Grading of systemic side effects. (Based on Alvarez-Cuesta et al. 2006, with permission.) 0

No symptoms or nonspecific symptoms

I

Mild systemic reactions: localized urticaria, rhinitis or mild asthma (PF < 20% decrease from baseline)

II

Moderate systemic reactions: slow onset (> 15 min) of generalized urticaria and/or moderate asthma (PF < 40% decrease from baseline)

III

Severe (nonlife-threatening) systemic reactions: rapid onset (< 15 min) of generalized urticaria, angioedema, or severe asthma (PF > 40% decrease from baseline)

IV

Anaphylactic shock: immediate evoked reaction of itching, flushing, erythema, generalized urticaria, stridor (angioedema), immediate asthma, hypotension

PF, peak flow.

et al. (1980) observed systemic side effects following 0.1% of injections (155 patients) including two cases of anaphylactic shock. In his work on anaphylaxis, Frankland (1980) using Allpyral found one fatality with 750 000 injections. A similar study found no mortalities following more than 4 million Allpyral injections (Ancill & Wainscott 1980). Despite the fact that most deaths are due to a failure to comply with a number of safety regulations and inadequate early treatment of the systemic reaction, they serve as a warning. Particularly in asthma patients, immunotherapy with high-potency extracts is not without risk, and therapists must be prepared to immediately tackle the early signs of anaphylactic shock (Perkin & Anas 1985; Anderson et al. 1986). About 60% of all systemic reactions with routine immunotherapy have no discernible cause, while 27% are due to change of ampoule and 14% to an error (ampoule or dosage) and negligence of increased intervals between injections (Vervloet et al. 1980). The frequency of systemic side effects in studies of experimental high-dose immunotherapy and accelerated dose-increase regimens is high, but this risk does not apply to standard immunotherapy. The noncritical systemic reactions (mild asthma and rhinitis) are noted more frequently, and in some studies with aggressive dosing are reported following 20% of injections (Malling et al. 1986). A study of rush immunotherapy in children, showed 100% systemic reactions following doses of 10 000–100 000 SQ-U/mL (Sundin et al. 1986). The life-threatening systemic reactions (severe asthma, angioedema and anaphylactic shock) seldom occur in newer studies, the rate ranging from zero or very few per cent (D’Souza et al. 1973; Greenberg et al. 1986; Nelson et al. 1986) to up to 34% (Bousquet et al. 1989). The frequency of systemic side effects is related to the dosage regimen, with “rush” the most dangerous, and to high-dosage regimens in

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highly sensitive subjects (Stewart & Lockey 1992). In routine immunotherapy following the guidelines of the EAACI Immunotherapy Position Paper (Malling & Weeke 1993), systemic reactions were observed in 5% of patients (in 9500 injections, no anaphylactic shock or life-threatening reactions were registered) (BSACI 1993). Even in pregnant women immunotherapy for allergic diseases seems safe (Shaikh 1993). Immune complexes have been associated with immunotherapy, but rarely with long-term adverse effects (Taylor 1991). The prevalence of more severe systemic reactions has been analyzed recently in the UK (Committee on the Safety of Medicines 1986) and in the USA (Lockey et al. 1987; Reid et al. 1993). The UK survey indicates 26 fatalities during a 31-year period, with a tendency to increase during the last 10 years. The first US retrospective study (Lockey et al. 1987) reviewed 24 fatalities from immunotherapy in the period 1959–1984. Not included in these figures are 16 additional fatalities from skin testing and immunotherapy, with information not sufficiently detailed to permit further analysis. However, the risk of a fatal reaction to immunotherapy is low, as 7–10 million allergen injections are administered annually in USA (Lockey et al. 1987). The second US survey (Reid et al. 1993) reports 17 fatalities for the years 1985–1989. The most important factors associated with death were asthma (76%), high sensitivity (71%), and dose-increase phase (56%). Other factors were changing to a new vial, dosing error or inappropriate dose adjustment, allergen season, symptoms before injection, not waiting after injection, and home injection. Although fatal reactions to immunotherapy are rare, it is completely unacceptable to expose patients with modest afflictions to the risk of anaphylaxis and even death. Strict guidelines about emergency resuscitation equipment and trained personnel as well as the observation period after injection and dosage adjustment is essential in order to reduce the risk to a minimum (Malling & Weeke 1993; Li et al. 2003; Alvarez-Cuesta et al. 2006).

Dosage and dose modifications The maintenance dose achieved should ideally be the highest dose not eliciting side effects but still ensuring clinical efficacy, i.e., the optimum dose (Hejjaoui et al. 1992; Stewart & Lockey 1992; Malling & Weeke 1993). Based on a number of studies with proven efficacy, doses are in the range of 5–20 g of major allergen (Norman 1990; Bousquet et al. 1998). The lower doses are most appropriate as a guide to an effective but safe dosage, as some of the high side-effect figures are from studies designed to administer the maximal tolerated dose (Norman 1990). The maintenance dose may be below the one recommended by the manufacturer, but it may also be higher. Dosage schedules supplied by the manufacturers should therefore be used as a rough guide only (Malling & Weeke 1993). In sensitive patients this must be modified, which implies smaller

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maintenance doses, repetition of doses, and/or reduced increments. Highly sensitive patients may need smaller initial and top doses. No optimal dosage schedule exists as tolerance to injection therapy depends on nonstandardized patients with a variety of factors like disease, subclinical exposure to allergens, and nonspecific hyperreactivity (Alvarez-Cuesta et al. 2006). The scheduled dose should be modified to produce a reduced dose, the same dose as the previous injection (i.e., no increment), or omitting the dose in case of adverse reactions. Some guidelines may be recommended with respect to dose modification (Malling & Weeke 1993; Alvarez-Cuesta et al. 2006), but it is crucially important not to follow general guidelines but rather to adjust allergen dose in relation to the individual, and evaluation of the patient, including sensitivity and allergy state, is obligatory before each injection. Generally, the safety of the patient is the most important aspect, and it is advisable to reduce the dose in case of uncertainty (Malling & Weeke 1993). The magnitude of the dose reduction is dependent on the severity of the reaction. American guidelines (Li et al. 2003) stipulate the magnitude of allergen dose reduction with respect to immediate and late

Table 73.4 Dose guidelines for subcutaneous immunotherapy: induction phase. (Based on Alvarez-Cuesta et al. 2006, with permission.) Omit injection in case of: Infection in airways or other disease during the last 3 days Deterioration of allergic symptoms or increased needs for drugs during the last 3 days Peak flow < 80% of individual best value Terminate treatment session Local immediate reaction > 5 cm Systemic reaction Injection interval 2 weeks Dose escalation according to schedule 2–4 weeks Repeat preceding dosing 4–6 weeks Dose reduction 1 step 6–8 weeks Dose reduction 2 steps 8 weeks Treatment reinstituted Local immediate reaction at preceding injection (30 min) < 5 cm Dose escalation according to schedule 5–8 cm Repeat preceding dosing > 8 cm Dose reduction 1 step Local delayed reaction at preceding injection (first day) Repeat preceding dose if the reaction has been inconvenient for the patient Mild systemic reaction at preceding injection (mild urticaria, rhinitis, asthma) Dose reduction 1–2 steps Severe systemic reaction Confer on continuous treatment

Allergen Injection Immunotherapy: Indications and Practice local reactions, systemic reactions, and exceeding a time interval between injections. Tables 73.4 and 73.5 show the EAACI guidelines (Alvarez-Cuesta et al. 2006).

Dose modifications • Postpone injections in patients with airway infections or other significant diseases within the last 3 days. • Postpone injections in patients with deterioration of allergy symptoms or increased need for antiallergic drugs due to recent allergen exposure within the last 3 days. • Postpone injections in patients with decreased lung function < 80% of personal best value. In asthmatic patients, measuring lung function before each injection is mandatory (peak flow is sufficient). • Reduce the scheduled dose if the interval between injection sessions has been exceeded. The magnitude of reduction

Table 73.5 Dose guidelines for subcutaneous immunotherapy: maintenance treatment. (Based on Alvarez-Cuesta et al. 2006, with permission.) Definition of maintenance dose Optimal dose defined from clinical studies Individual optimal dose (based on patient response) Intervals between injections at shift to maintenance treatment 2 weeks (max. 3 weeks) 4 weeks (max. 5 weeks) 8 weeks (max. 10 weeks) Maintenance treatment Omit injection in case of: Infection in airways or other disease during the last 3 days Deteriorations of allergic symptoms or increased need for drugs during the last 3 days Peak flow < 80% of normal value Injection intervals in maintenance treatment 10 weeks Unchanged dosing 10–12 weeks Dose reduction 20% 12–16 weeks Dose reduction 40% 16 weeks Treatment reinstituted Local immediate reaction at preceding injection (30 min) < 8 cm Unchanged dosing > 8 cm Dose reduction 20% Local delayed reaction at preceding injection (first day) Dose reduction 20% if the reaction has been inconvenient for the patient Mild systemic reaction Dose reduction 20–40% Severe systemic reaction Confer on continuous treatment Dose increases after reduction of maintenance dose 20% Full dose after 4 weeks and then after 8 weeks > 20% Weekly injections to maintenance, then 2–4–8 weeks

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depends on the degree of excess and should be defined in the clinical guidelines. • Reduce the scheduled allergen dose in case of a systemic reaction at the preceding visit. The magnitude of reduction depends on the severity of the reaction and should be defined in the clinical guidelines. In case of anaphylactic and other life-threatening reactions the continuation of subcutaneous immunotherapy should be carefully evaluated (except in cases of Hymenoptera venom allergy, in which it actually reinforces the indication for immunotherapy). • Traditionally the late local reaction at the injection site has been used to adjust allergen dosing at the next allergen administration. Several studies have indicated that the late local reaction at the preceding injection is not related to the risk of developing a systemic reaction at the next injection (Malling 2000).

Dosing during allergen season It is recommended not to start induction treatment during allergen seasons. During allergen seasons injections should not be given if the patient has clinical symptoms (AlvarezCuesta et al. 2006). As a general safety precaution, a routine reduction in allergen dose during allergen seasons is commonly used, but if the patient is symptom-free the dose does not need to be reduced. In symptomatic patients, the injection should be postponed, symptomatic treatment instituted (intensified), and a reduced allergen dose given when the patient is asymptomatic.

Monitoring The primary target for monitoring of immunotherapy is related to safety monitoring (Malling & Weeke 1993). Safety monitoring is undertaken with the aim of reducing the risk of immunotherapy-induced systemic reactions, and includes evaluation of the patient before, after, and between injections (Malling & Weeke 1993; Malling 1994; Li et al. 2003; Alvarez-Cuesta et al. 2006). Safety monitoring should include verbal and written information and instructions involving patients in the monitoring of immunotherapy, with the aim of making patients more responsible for their own safety. Monitoring should be supervised by the staff. Special record forms for registering the actual state of the patient, amount of allergen injected, lung function, and possible side effects are recommended (Fig. 73.1a), as well as a uniform registration of motivations for dose reductions and grading of side effects (Fig. 73.1b). Special attention should be focused on reactions to previous injections, deterioration of allergic disease, and intercurrent infections. In patients with bronchial asthma, including those having allergic rhinitis with bronchial asthma, it is essential that all regular antiasthmatic drugs are continued (Malling & Weeke 1993). This is of special importance on the day of injection. In asthmatics,

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peak flow should be measured before and 30 min after the injection. Injections should be postponed in patients with peak flows below 80% of normal. Likewise, caution is needed, and the injection postponed, when there are signs of an ongoing or recent viral infection, recent allergen exposure, or when the patient has forgotten to use routine drugs. It is mandatory that the patient waits in the physician’s office or waiting room for 30 min after the injection. The patient is instructed to immediately report any symptoms appearing during the observation period. The injection site has to be inspected before the patient leaves the office. In order to obtain valid information on the frequency, severity, treatment, and outcome of side effects, a record form adapted for computer registration is recommended. An example is shown in Fig. 73.2. The patient should be informed to consult a physician in case of a (serious) late reaction. The patient also has to be instructed to avoid intense physical exercise, prolonged hot bathing, and contact with relevant allergens (especially pets) on the day of injection. The patient should be carrying antiasthma medication, i.e., inhaled β2 agonists. It is recommended to separate allergen injections from other vaccinations for infectious diseases by at least 1 week.

Prevention and treatment of side effects Prevention of side effects In order to minimize the risk of eliciting side effects, the patient should be assessed carefully and the appropriate dose decided on before the injection is given. Particular attention should be paid when there is a history of increased exposure to relevant allergens, recent viral or bacterial infections, intakes of drugs increasing the risk of immunotherapy (especially in asthmatic patients), recent history of exercise-induced asthma, and increased need for antiallergic drugs. Concerning the use of antiallergic drugs, it is advisable to continue the administration of these drugs until clinical efficacy has been obtained. Meticulous care should be taken to avoid mistakes involving patients, allergen extracts, allergen concentration, and volume injected (Malling & Weeke 1993; Alvarez-Cuesta et al. 2006). In some centers prophylactic antihistamines are used prior to injections to reduce the number and severity of side effects. A study of pretreatment with terfenadine in honeybee venomallergic patients showed a reduction in cutaneous symptoms but no reduction in the more severe respiratory and cardiovascular symptoms (Berchtold et al. 1992). In a placebocontrolled double-blind study of inhalant immunotherapy (Nielsen et al. 1996), pretreatment with loratadine showed a reduction in the number of patients experiencing systemic side effects of more than 50% and also a significant reduction in the severity of systemic reactions. A further advantage of antihistamine pretreatment was a diminished frequency of dose reductions and thereby a reduction in the number of injections to reach the maintenance dose. The advantages

Fig. 73.1 (a) Front page of an example of an immunotherapy record form used by the author.

(a)

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Fig. 73.1 (Cont’d ) (b) Back page of the immunotherapy record form grading systemic reactions and motivations for dose reductions. AH, antihistamine; BP, blood pressure; PEFR, peak expiratory flow rate; PF, peak flow.

(b)

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(a) Fig. 73.2 (a) Front page of a record form for standardized registration of systemic reactions used by the author.

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(b) Fig. 73.2 (Cont’d ) (b) Back page of a systemic reaction record form containing information on severity, onset, and duration of systemic reactions, treatment, complications, and outcome.

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of antihistamine pretreatment in routine immunotherapy needs further evaluation before it can be recommended for general use.

Treatment of mild to moderately severe adverse reactions Mild adverse reactions include local swelling, mild urticaria or rhinitis, and these may require either no treatment or treatment with oral or parenteral antihistamines. Large local swellings may be treated successfully with ice bags or topical glucocorticosteroids. In case of immediate, mild or moderately severe bronchial obstruction, especially in patients treated with depot extracts, it is advisable not only to use a β2 agonist but also to add oral corticosteroids to prevent late reactions. In case of severe bronchial obstruction (FEV1 < 50% of predicted) some centers prefer to use epinephrine given by inhalation. Patients with urticaria should be observed carefully as this may be an early warning of an impeding more serious reaction (Malling & Weeke 1993).

Treatment of serious reactions Anaphylactic reactions may progress rapidly, terminating in death within a few minutes (Lockey et al. 1987; Reid et al. 1993). The early signs and symptoms of anaphylaxis include pruritus of the palms and scalp, intense erythema of the skin, conjunctival injection, and cough. Angioedema, laryngeal edema, severe asthma, hypotension, and shock may follow rapidly. Subcutaneous or intramuscular epinephrine should be injected immediately, and the patient should be placed in the supine position. An intravenous line should be set up, and intravenous fluids should be administered rapidly. Steroids and antihistamines should be given and antiasthmatic drugs and oxygen may also be required. In some patients local injection of epinephrine around the site of the allergen injection combined with a tourniquet may be helpful. In severe cases of anaphylactic reactions further doses of epinephrine may be required. In the event of poor peripheral circulation and no response to subcutaneous or intramuscular epinephrine, intravenous injections of diluted epinephrine may be needed. Patients should be observed in hospital for at least 24 hours due to the risk of delayed anaphylactic reactions.

Algorithm and annotations Figure 73.3 summarizes essential elements related to successful immunotherapy. The algorithm has been adapted from Li et al. (2003) and tries to extract the best from the joint American “practice parameters” and the European “standards” (Alvarez-Cuesta et al. 2006). The annotations represent statements for the appropriate use of immunotherapy and also a summary of this chapter. 1 Immunotherapy is clinically effective in selected patients with predominantly IgE-mediated allergic rhinitis, asthma

Allergen Injection Immunotherapy: Indications and Practice and Hymenoptera venom allergy. Furthermore, subcutaneous immunotherapy has disease-modifying capacity in terms of long-term efficacy and prevention of new sensitization and progression of disease. With regard to rhinitis, patients with moderate or severe disease are candidates, especially if asthma complicates the upper airway symptoms. With regard to asthma, the most important issue is allergen sensitization and allergen exposure (allergen-induced symptoms should be dominating, and nonspecific hyperreactivity should be responsible for only a limited part of the disease). With regard to Hymenoptera venom-allergic patients, those with a systemic reaction and positive IgE tests are candidates. 2 Demonstration of IgE sensitization by means of skin tests or in vitro quantitation of specific IgE antibodies should be performed. Immunotherapy may be indicated when positive diagnostic tests correlate with clinical symptoms and the patient is exposed to the relevant allergen. 3 Patients with negative skin test or serum specific IgE are not candidates for immunotherapy. Immunotherapy should not be based on sensitization not resulting in clinical symptoms (the presence of specific IgE antibodies does not necessarily indicate clinical sensitivity). Likewise, sensitized patients not exposed to the allergen should not be offered immunotherapy. 4 To identify the optimal candidate for immunotherapy a careful assessment of the patient is essential. The assessment should include an evaluation of the risks and potential benefits of various treatment options like pharmacotherapy and allergen reduction versus immunotherapy. It is important to take into consideration the severity of the disease, including duration of clinical symptoms, number of organs involved, and type and number of medication needed to control clinical symptoms. The impact on quality of life is highly individual and not necessarily related to objective disease severity parameters. Preferences for treatment should also be part of the assessment. We have to accept that a considerable number of patients do not want to use pharmacotherapy for emotional reasons and the risk of side effects. It is essential that clinical symptoms can be abolished pharmacologically. It is a fundamental mistake to restrict immunotherapy only to patients not responding to drugs. Immunotherapy is indicated in patients with treatable disease, i.e., completely reversible symptoms. Finally, patients should be assessed for contraindications. 5 Taking the results of patient profiling (Table 73.2) into consideration, the decision for or against immunotherapy should be taken by mutual agreement between the patient and the physician. Each element should be carefully considered with respect to benefits, risks, and costs of possible interventions. Good candidates are patients with moderate to severe rhinitis in whom antihistamines and moderatedose topical corticosteroids control symptoms insufficiently. With regard to asthma, candidates are patients with mild to moderate symptoms despite allergen avoidance requiring

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Patient presents with clinical manifestations (allergic rhinitis, asthma, or Hymenoptera venom allergy) for which immunotherapy might be indicated

1

Demonstrated IgE-sensitization? Test results correlate with clinical symptoms and allergen exposure?

NO

Not candidate for immunotherapy

3

2

Assess • Risks and benefits of available management options – Immunotherapy – Pharmacotherapy – Allergen exposure reduction • Disease severity and impact on quality of life • Patients’ treatment preferences • Response to previous treatment • No contraindications

Is immunotherapy recommended for the patient?

4

NO

Immunotherapy not given

6

5

• Inform the patient about the benefits and risks of immunotherapy • Educate the patient about immunotherapy principles and safety monitoring • Obtain informed consent

Identify • Specific allergen product • Risk factors requiring special starting dose, induction regimen, and maintenance dose • Risk of continuous low-grade allergen exposure

Administer immunotherapy • Safety equipment and procedures in place • Preinjection safety monitoring • Peak flow monitoring of rhinitis and asthma patients before and after injections • Observation 30 minutes after immunotherapy

Systemic reactions to immunotherapy injections?

7

8

9

YES

10 NO Assess at follow-up • Clinical response to immunotherapy (reduction in symptoms and need for medication) • Administration of appropriate top dose, reactions, compliance with treatment • Continuation/discontinuation of immunotherapy

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12

Manage reaction • Assess factors related to systemic reaction • Consider top dose or schedule adjustment • Consider discontinuing immunotherapy 11

Fig. 73.3 Algorithm for safe and effective subcutaneous allergen-specific immunotherapy. For further details see annotations in the text.

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medication that may result in side effects. In Hymenoptera venom-allergic patients with severe anaphylactic reactions, immunotherapy is the only life-saving treatment and should be strongly recommended. 6 Patients who do not comply with the indications or have contraindications should not be offered immunotherapy “just to see if it will work.” 7 Before immunotherapy is started the patient should be carefully informed about the benefits and risks of immunotherapy. Careful education of the patient about the principles of the treatment including safety monitoring is essential before starting injections. Active involvement of the patient in safety monitoring may improve safety and increase patient compliance with treatment. Depending on local legal regulations, it is recommended to obtain informed consent. 8 The prescribing physician identifies the allergen product and, based on evaluation of specific risk factors, defines the starting dose, induction regimen, and maintenance dose. It is recommended that the induction phase be carried out by an experienced allergist as the risk of systemic reactions is highest during this phase. Physicians, nurses and healthcare personnel must be trained and regularly updated in subcutaneous allergen-specific immunotherapy, including the observation and rescue treatment of systemic anaphylactic reactions. As a minimum requirement, a competent physician must always be present when subcutaneous immunotherapy is carried out and be responsible for the treatment. In addition, one extra person should be available for proper management of serious adverse events. In patients treated with perennial allergens, it is important to be aware of low-grade allergen exposure and ensure monitoring of ongoing inflammation. Particularly in asthmatic patients this may enhance the sensitivity of the patient, and consequently increase the risk of inducing systemic reactions. 9 Immunotherapy should be administered in a setting that permits the prompt recognition and management of side effects. Essential rescue equipment (see p. 1526) must be available and be functioning, and the staff trained in identifying the early signs of an anaphylactic reaction and be able to administer emergency treatment without any delay. Preinjection monitoring should ensure that the patient is well, with no signs of infection, no actual allergic symptoms or increased intakes of antiallergic drugs. Preinjection monitoring of patients also includes a check of any drug intake that may either increase the risk of systemic side effects or render the treatment of anaphylactic reactions more difficult (betablockers being the most important). Monitoring lung function before and after injections is mandatory in asthmatics but also recommended in rhinitis patients. Peak flow below 80% of normal is a risk factor for a systemic reaction and should result in postponement of the injection. Patients should be under observation for 30 min after each injection (may be extended in cases of systemic reactions and after the treatment of systemic reactions). The patient should be informed

Allergen Injection Immunotherapy: Indications and Practice not to leave the office during the observation period and to immediately inform the staff if early symptoms of a systemic reaction occur. 10 Subcutaneous injection of allergen products may frequently induce local reactions. In general these swellings are to be expected, are well tolerated, and require no specific therapy. Systemic reactions are any symptoms from organs distant from the site of injection. Systemic side effects may vary from a few sneezes to full-blown anaphylactic shock and even death. Severity is related to how rapidly the symptoms develop after the injection. Itching in the palms, soles, and on hairy body parts, and rapid onset of erythema and urticaria, rhinitis or asthma occurring within minutes after injection will often progress to anaphylaxis and require treatment without delay. 11 Systemic reactions are treated according to severity and speed of onset. Reactions starting shortly after injection have an increased risk of progressing to severe anaphylaxis and require immediate treatment with epinephrine. Epinephrine should be administered intramuscularly and in a sufficient dosage. To prevent late symptoms corticosteroids are helpful, but neither antihistamines nor corticosteroids should replace epinephrine because of their slow onset of action and lack of immediate vascular effects. An audit of the systemic reaction is recommended in order to identify risk factors related to the event (mistakes, insufficient monitoring, etc.). Based on the indication, the severity of the reaction and possible explanations, reevaluation of the top dose or adjustment of the schedule if during induction treatment should be considered. Severe systemic reactions with inhalant allergens normally result in discontinuation of treatment. In Hymenoptera venom-allergic patients, systemic reactions during immunotherapy reinforce the indication and may necessitate a higher top dose. 12 In order to ensure optimal treatment, patients should be regularly followed up at least once yearly. Based on clinical effect (reduction in symptoms and/or need for medication) in relation to eventual side effects or other problems related to injections, the prescribing allergist has to decide on continuation/discontinuation of treatment. For patients not treated in the prescribing physician’s office, the assessment also includes control of the administration of an appropriate allergen dose, evaluation of reactions to treatment, and patient compliance. The recommended duration (in responding patients) is 3–5 years. In order to ensure long-term efficacy, too short a duration of treatment is insufficient. No convincing tests are useful for determining when to stop treatment.

References AAAI Board of Directors (1994) Guidelines to minimize the risk from systemic reactions caused by immunotherapy with allergenic extracts. J Allergy Clin Immunol 93, 811–12.

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Adkinson, N.F. Jr, Eggleston, P.A., Eney, D. et al. (1997) A controlled trial of immunotherapy for asthma in allergic children. N Engl J Med 336, 324–31. Alessandro, F., Vincenzo, Z.G., Marco, S., Marcello, G. & Enrica, R. (1994) Compliance with pharmacologic prophylaxis and therapy in bronchial asthma. Ann Allergy 73, 135– 40. Allergen Standardization Subcommittee (1983) Report on behalf of International Union of Immunologic Societies (IUIS). Arbeiten Paul-Ehrlich-Institut 78, 41– 8. Alvarez-Cuesta, E., Aragoneses-Gilsanz, E., Martín-Garcia, E. et al. (2005) Immotherapy with depigmented glutaraldehyde-polymerized extracts: change in quality of life. Clin Exp Allergy 35, 572–8. Alvarez-Cuesta, E., Bousquet, J., Canonica, G.W., Durham, S.R., Malling, H.-J. & Valovirta, E. (2006) Standards for practical allergen-specific immunotherapy. Allergy 61 (suppl. 82), 1–20. American Academy of Allergy, Asthma and Immunology (1997) Position Statement. The use of standardized allergen extracts. J Allergy Clin Immunol 99, 583– 6. Ancill, R. & Wainscott, G. (1980) Anaphylactic reaction to desensitisation. BMJ 281, 1429. Anderson, J.A., Chai, H., Claman, H.N. et al. (1986) Personnel and equipment to treat systemic reactions caused by immunotherapy with allergenic extracts. J Allergy Clin Immunol 77, 271–3. Anon. (1985) Biological products: allergenic extracts: implementation of efficacy review. Federal Register, Food and Drug Administration; 21 CRF Parts 600; 610 and 680 (Docket no. 81N-0096). Anon. (1991) Rules governing medical products in the European Community. Vol. III. Note for guidance on allergen products. CPMP/BWP/243/96. Berchtold, E., Maibach, R. & Müller, U. (1992) Reduction of side effects from rush immunotherapy with honeybee venom by pretreatment with Terfenadine. Clin Exp Allergy 22, 59–65. Bonifazi, F., Jutel, M., Bilo, B.M., Birnbaum, J. & Muller, U. (2005) EAACI Interest Group on Insect Venom. Hypersensitivity Group. Prevention and treatment of hymenoptera venom allergy: guidelines for clinical practice. Allergy 60, 1459–70. Bousquet, J. & Michel, F.-B. (1992) Advances in specific immunotherapy. Clin Exp Allergy 22, 889–96. Bousquet, J., Hejjaoui, A., Dhivert, H., Clauzel, A.M. & Michel, F.B. (1989) Specific immunotherapy with a standardized Dermatophagoides pteronyssinus extract. III. Systemic reactions during the rush protocol in patients suffering from asthma. J Allergy Clin Immunol 83, 797–802. Bousquet, J., Hejjaoui, A., Soussana, M. & Michel, F.-B. (1990) Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids. IV. Comparison of the safety and efficacy of two dosages of a high-molecular-weight allergoid. J Allergy Clin Immunol 85, 490–7. Bousquet, J., Becker, W.M., Hejjaoui, A. et al. (1991) Differences in clinical and immunologic reactivity of patients allergic to grass pollens and to multiple-pollen species. II. Efficacy of a double-blind, placebo-controlled, specific immunotherapy with standardized extracts. J Allergy Clin Immunol 88, 43–53. Bousquet, J., Chanez, P., Lacoste, J.Y. et al. (1992) Asthma: a disease remodeling the airways. Allergy 47, 3–11. Bousquet, J., Lockey, R.F. & Malling, H.-J. (eds) (1998) WHO Position Paper. Allergen immunotherapy: therapeutic vaccines for allergic diseases. Allergy 53 (suppl. 44), 1– 42.

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Bousquet, J., van Cauwenberge, P. & Khaltaev, N. (2001) Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol 108, 147–336. Bronsky, W.A., Dockhorn, R.J., Meltzer, E.O. et al. (1996) Fluticasone propionate aqueous nasal spray compared with terfenadine tablets in the treatment of seasonal allergic rhinitis. J Allergy Clin Immunol 97, 915–21. BSACI (1993) Position paper on immunotherapy. Clin Exp Allergy 23, 1– 44. Cohn, J.R. & Pizzi, A. (1993) Determinants of patient compliance with allergen immunotherapy. J Allergy Clin Immunol 91, 734–7. Committee on the Safety of Medicines (1986) SMS update: desentizing vaccines. BMJ 293, 948. Creticos, P.S. (1992) Immunotherapy with allergens. JAMA 268, 2834–9. Des Roches, A., Paradis, L., Ménardo, J.-L., Bouges, S., Daurès, J.-P. & Bousquet, J. (1997) Immunotherapy with a standardized Dermatophagoides pteronyssinus product. VI. Specific immunotherapy prevents the onset of new sensitizations in children. J Allergy Clin Immunol 99, 450–3. Dreborg, S. & Frew, A. (1993) EAACI Allergen Standardization and Skin Test Position Paper. Allergy 48, 49–83. D’Souza, M.F., Pepys, J., Wells, I.D. et al. (1973) Hyposensitization with Dermatophagoides pteronyssinus in house dust allergy: a controlled study of clinical and immunological effects. Clin Allergy 3, 177–93. Durham, S.R., Walker, S.M., Varga, E.M. et al. (1999) Long-term clinical efficacy of grass-pollen immunotherapy. N Engl J Med 341, 468–75. Frankland, A.W. (1980) Anaphylactic reaction to desensitisation. BMJ 281, 1429. Frew, A.J. (1994) Conventional and alternative allergen immunotherapy: do they work? Are they safe? Clin Exp Allergy 24, 416–22. Frew, A.J., Powell, R.J., Corrigan, C.J. & Durham, S.R. (2006) Efficacy and safety of specific immunotherapy with SQ allergen extract in treatment-resistant seasonal allergic rhinoconjunctivitis. J Allergy Clin Immunol 117, 319–25. Frostad, A.B., Grimmer, Ø., Sandvik, L., Moxnes, A. & Aas, K. (1983) Clinical effects of hyposensitization using a purified allergen preparation from timothy pollen as compared to crude aqueous extracts from timothy pollen and a four-grass pollen mixture respectively. Clin Allergy 13, 337–57. Golden, D.B.K., Kagey-Sobotka, A., Valentine, M.D. & Lichtenstein, L.M. (1981) Dose dependence of Hymenoptera venom immunotherapy. J Allergy Clin Immunol 67, 370–4. Gotzsche, P.C., Johansen, H.K., Schmidt, L.M. & Burr, M.L. (2004) House dust mite control measures for asthma. Cochrane Database Syst Rev 4, CD001187. Greenberg, M.A., Kaufman, C.R., Gonzalez, G.E., Rosenblatt, C.D., Smith, L.J. & Summers, R.J. (1986) Late and immediate systemicallergic reactions to inhalant allergen immunotherapy. J Allergy Clin Immunol 77, 865–70. Haugaard, L., Dahl, R. & Jacobsen, L. (1993) A controlled doseresponse study of immunotherapy with standardized, partially purified extract of house dust mite: clinical efficacy and side effects. J Allergy Clin Immunol 91, 709–22. Hejjaoui, A., Dhivert, H., Michel, F.B. & Bousquet, J. (1990) Immunotherapy with a standardized Dermatophagoides pteronnysinus

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extract. IV. Systemic reactions according to the immunotherapy schedule. J Allergy Clin Immunol 85, 473–9. Hejjaoui, A., Ferrando, R., Dhivert, H., Michel, F.B. & Bousquet, J. (1992) Systemic reactions occurring during immunotherapy with standardized pollen extracts. J Allergy Clin Immunol 89, 925–33. Hepner, M.J., Ownby, D.R., Anderson, J.A., Rowe, M.S., Sears-Ewald, D. & Brown, E.B. (1990) Risk of systemic reactions in patients taking beta-blocker drugs receiving allergen immunotherapy injections. J Allergy Clin Immunol 86, 407–11. Hirsch, S.R., Kalbfleisch, J.H., Golbert, T.M. et al. (1981) Rinkel injection therapy: a multicenter controlled study. J Allergy Clin Immunol 68, 133–55. Javeed, N., Javeed, H., Javeed, S. et al. (1996) Refractory anaphylactoid shock potentiated by β-blockers. Cathet Cardiovasc Diagn 39, 383– 4. Johnstone, D.E. (1957) Study of the role of antigen dosage in the treatment of pollenosis and pollen asthma. Am J Dis Child 94, 1–5. Juniper, E.F., Guyatt, G.H., Griffith, L.E. & Ferrie, P.J. (1996) Interpretation of rhinoconjunctivitis quality of life questionnaire data. J Allergy Clin Immunol 98, 843–5. Li, J.T., Lockey, R.F., Bernstein, I.L., Portnoy, J.M. & Nicklas, A. (2003) Allergen immunotherapy: a practice parameter. Ann Allergy Asthma Immunol 90, 1– 40. Lockey, R.F., Benedict, L.M., Turkeltaub, P.C. & Bukantz, S.C. (1987) Fatalities from immunotherapy (IT) and skin testing (ST). J Allergy Clin Immunol 79, 660–77. Löfkvist, T., Agrell, B., Dreborg, S. & Svensson, G. (1994) Effects of immunotherapy with a purified standardized allergen preparation of Dermatophagoides farinae in adults with perennial allergic rhinoconjunctivitis. Allergy 49, 100–7. Malling, H.-J. (1994) Immunotherapy in Europe. Clin Exp Allergy 24, 515–21. Malling, H.-J. (1998) Immunotherapy as an effective tool in allergy treatment. Allergy 53, 461–72. Malling, H.-J. (2000) Minimising the risks of allergen-specific injection immunotherapy. Drug Saf 23, 323–32. Malling, H.-J. & Weeke, B. (1993) EAACI Immunotherapy position papers. Allergy 48 (suppl. 14), 9–35. Malling, H.-J., Dreborg, S. & Weeke, B. (1986) Diagnosis and immunotherapy of mould allergy. V. Clinical efficacy and side effects of immunotherapy with Cladosporium herbarum. Allergy 41, 507– 19. Möller, C., Dreborg, S. & Einarsson, R. (1987) Cross reactivity between trees during immunotherapy. II. Specific IgE and IgG responses. Clin Allergy 17, 551– 62. Mosbech, H., Dirksen, A., Dreborg, S. et al. (1990) Hyposensitization in asthmatics with mPEG-modified and unmodified house dust mite extract. IV. Occurrence and prediction of side effects. Allergy 45, 142–50. Müller, U. & Haeberli, G. (2005) Use of beta-blockers during immunotherapy for Hymenoptera venom allergy. J Allergy Clin Immunol 115, 606–10. Müller, U. & Mosbech, H. (1993) EAACI Position Paper: immunotherapy with Hymenoptera venoms. Allergy 48, 36–46. Nelson, B.L., Dupont, L.A. & Reid, M.J. (1986) Prospective survey of local and systemic reactions to immunotherapy with pollen extracts. Ann Allergy 56, 331– 4. Nielsen, L., Johnsen, E.R., Mosbech, H. et al. (1996) Antihistamine

Allergen Injection Immunotherapy: Indications and Practice premedication in specific cluster immunotherapy: a double-blind, placebo-controlled study. J Allergy Clin Immunol 97, 1207–13. Niggemann, B., Jacobsen, L., Dreborg, S. et al. (2006) Five year follow-up on the PAT-study: specific immunotherapy and longterm prevention of asthma in children. Allergy 61, 855–9. Nordic Council on Medicines (1989) Registration of allergenic preparations. Nordic Guidelines, 2nd edn. NLN Publications no. 23, pp. 1–34. Norman, P.S. (1990) Immunotherapy. In: Melillo, G., Norman, P.S. & Marone, G., eds. Respiratory Allergy. B.C. Decker, Toronto, pp. 3–15. Norman, P.S. & Lichtenstein, L.M. (1986) The great debate: immunotherapy and asthma. Clin Allergy 16, 269–71. Pajno, G.B., Barberio, G., de Luca, F.R., Morabito, L. & Parmiani, S. (2001) Prevention of new sensitizations in asthmatic children monosensitized to house dust mite by specific immunotherapy. A six-year follow-up study. Clin Exp Allergy 31, 1392–7. Peat, J.K., Woolcook, A.J. & Cullen, K. (1987) Rate of decline of lung function in subjects with asthma. Eur J Respir Dis 70, 171–9. Pécoud, A., Nicod, L., Badan, M., Agrell, B., Dreborg, S. & Kolly, M. (1990) Effects of one-year hyposensitization in allergic rhinitis. Comparison of two house dust mite extracts. Allergy 45, 386–92. Perkin, R.M. & Anas, N.G. (1985) Mechanisms and management of anaphylactic shock not responding to traditional therapy. Ann Allergy 54, 202–8. Purello-D’Ambrosio, F., Gangemi, S., Merendino, R.A. et al. (2001) Prevention of new sensitizations in monosensitized subjects submitted to specific immunotherapy or nor. A retrospective study. Clin Exp Allergy 31, 1295–302. Reid, M.J., Lockey, R.F., Turkeltaub, P.C. & Platts-Mills, T.A.E. (1993) Survey of fatalities from skin testing and immunotherapy 1985–1989. J Allergy Clin Immunol 92, 6–15. Rugose, F.V., Passalacqua, G., Gambardella, R. et al. (1997) Nonfatal systemic reactions to subcutaneous immunotherapy: a 10-year experience. Invest Allergol Clin Immunol 7, 151–4. Shaikh, W.A. (1993) A retrospective study of the safety of immunotherapy in pregnancy. Clin Exp Allergy 23, 857–60. Sly, R.M. (1996) Managed care: the key to quality of management of asthma. Ann Allergy 76, 161–3. Stewart, G.E. II & Lockey, R.F. (1992) Systemic reactions from allergen immunotherapy. J Allergy Clin Immunol 90, 567–78. Strachan, D.P., Butland, B.K. & Anderson, H.R. (1996) Incidence and prognosis of asthma and wheezing illness from early childhood to age 33 in a national British cohort. BMJ 312, 1195–9. Sundin, B., Lilja, G., Graff-Lonnevig, V. et al. (1986) Immunotherapy with partially purified and standardized animal dander extracts. I. Clinical results from a double-blind study on patients with animal dander asthma. J Allergy Clin Immunol 77, 478–87. Tabar, A.I., Garcia, B.E., Rodriguez, A. et al. (1993) A prospective safety-monitoring study of immunotherapy with biologically standardized extracts. Allergy 48, 450–3. Tamir, R., Levy, I., Duer, S. & Pick, A.I. (1992) Immediate adverse reactions to immunotherapy in allergy. Allergy 47, 260–3. Taylor, R.J. (1991) Hypersensitivity vasculitis occuring in a patient receiving immunotherapy. J Allergy Clin Immunol 88, 889–90. Turkeltaub, P.C., Campbell, G. & Mosimann, J.E. (1990) Comparative safety and efficacy of short ragweed extracts differing in potency and composition in the treatment of fall hay fever. Use of allergenically bioequivalent doses by parallel line bioassay to evaluate comparative safety and efficacy. Allergy 45, 528–46.

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Valenta, R. & Kraft, D. (2002) From allergen structure to new forms of allergen-specific immunotherapy. Curr Opin Immunol 14, 718– 27. Varney, V.A., Gaga, M., Frew, A.J., Aber, V.R., Kay, A.B. & Durham, S.R. (1991) Usefulness of immunotherapy in patients with severe summer hay fever uncontrolled by antiallergic drugs. BMJ 302, 265–9. Vervloet, D., Khairallah, E., Arnaud, A. & Charpin, J. (1980) A prospective national study of the safety of immunotherapy. Clin Allergy 10, 59– 64. Wantke, F., Demmer, C.M., Götz, M. & Jarisch, R. (1993) Inhibition

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of diamine oxidase is a risk in specific immunotherapy. Allergy 48, 552. Wihl, J.Å., Ipsen, H., Nüchel-Petersen, B., Munch, E.P., Janniche, H. & Løwenstein, H. (1988) Immunotherapy with partially purified and standardized tree pollen extracts. II. Results of skin prick tests and nasal provocation tests from a three-year double-blind study of patients treated with pollen extracts either of birch or combination of alder, birch and hazel. Allergy 43, 363–9. Yeung, M., O’Connor, S.A., Parry, D.T. & Cochrane, G.M. (1994) Compliance with prescribed drug therapy in asthma. Respir Med 88, 31–5.

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Sublingual Immunotherapy G. Walter Canonica and Giovanni Passalacqua

Summary Sublingual immunotherapy (SLIT) is currently accepted as a viable option to injection immunotherapy and is widely used in many European countries, although several issues regarding efficacy and safety still need to be resolved. A considerable amount of new data concerning clinical, immunologic and practical aspects of SLIT has been published and there have been confirmatory metaanalyses of the efficacy of SLIT in rhinitis and asthma, as well as data in children. Moreover, several postmarketing surveys have confirmed acceptable safety, including in children below the age of 5 years. Because of the good safety profile it may be possible to give SLIT without the updosing phase and, in fact, the most recent clinical trials have been performed with a steady-dosage regimen. Nevertheless, treatment with SLIT still requires a precise diagnosis, detailed patient information, and strict follow-up. Additional encouraging information is now available on patient compliance measured in both adults and children. One pharmacoeconomic study showed that in pollinosis, SLIT was more advantageous than drug treatment alone. Furthermore, as with the injection route, SLIT can prevent the onset of new sensitizations and the onset of asthma. These findings need to be confirmed by larger studies, as does the long-lasting effect after discontinuation of treatment. The mechanisms of action are only beginning to be investigated in detail and the biodistribution of sublingual allergens also requires further study. More and more new data on SLIT are rapidly appearing in the international literature that confirm the value of this treatment and show that SLIT is a viable and useful form of immunotherapy. Nevertheless, patient selection and optimal dose regimens still need to be defined unequivocally.

Introduction Allergen-specific immunotherapy or allergy vaccination is the practice of administering to allergic subjects increasing

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

amounts of allergen(s) (the allergenic extract or vaccine) in order to achieve hyposensitization and to reduce symptoms occurring during natural exposure to the allergen(s). Since its first description (Noon 1911), immunotherapy (IT) has been given via subcutaneous injection (SCIT). Nevertheless, other routes of administration were proposed and investigated during the 20th century. In some cases, the approach was to specifically desensitize the target organs (nose or bronchi) by giving the allergen either as a nasal spray (local nasal immunotherapy) or by inhaled aerosol (bronchial immunotherapy). Alternatively systemic desensitization could be induced by administering the allergen orally. These routes have been variously named “alternative,” “nonparenteral,” “noninjection,” or “local.” Presently, it is agreed that the most proper terms are “local” and “noninjection,” which are equivalent, whereas the word “alternative” has been abandoned since it might cause confusion with nonconventional medicines. Sublingual administration was proposed at the beginning of the 1980s, and the first double-blind placebo-controlled study was published in 1986 (Scadding & Brostoff 1986). An impressive number of clinical trials with sublingual immunotherapy (SLIT) have been published in less than 20 years and metaanalyses on efficacy are now available and additional data on the safety, compliance and mechanisms also published. So far, SLIT is routinely used in many European countries and clinical trials have started in the USA.

Historical perspective (Fig. 74.1) The idea of administering allergenic extracts via noninjection routes is not so recent as commonly believed; in fact, the first clinical attempts were made at the beginning of the 20th century (Black 1927, 1928). During the same century, other routes of administration were proposed, i.e., local bronchial in the 1950s (Herxeimer 1951; Herxeimer & Prior 1952), local nasal in the 1970s (Taylor & Shivalkar 1972; Metha & Smith 1975), and the oral route at the beginning of the 1980s (Rebien et al. 1980; Taudorf & Weeke 1983). Except for local nasal immunotherapy, which has been extensively studied since the 1970s, all other approaches were substantially anecdotal, since the subcutaneous route was well established. In 1986,

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1970s ORAL IT

1986 Scadding 1st DBPC trial

1998 WHO SLIT is accepted

2001 ARIA document

1998 first tablet SLIT

2004 1st META ANALYSIS

2005–2006 large randomized controlled trials studies on the mechanism of action

the UK Committee on the Safety of Medicines (1986) reported several deaths certainly caused by SCIT, and raised serious concerns about the safety and the risk–benefit ratio of immunotherapy, especially as inexpensive and effective drugs were then available. Indeed, it was subsequently demonstrated that, in most cases, life-threatening events were due to avoidable human errors (incorrect unstandardized extracts, poor patient selection) (Reid et al. 1993; Aaronson & Gandhi 2004; Bernstein et al. 2004). Nonetheless the interest in noninjection routes of immunotherapy rapidly increased. The sublingual route immediately appealed as a promising therapeutic option, especially because of the good safety profile (Scadding & Brostoff 1986). The original rationale for administering immunotherapy sublingually was to achieve prompt and rapid absorption of the vaccine, in order to avoid possible gastrointestinal degradation. Although it was recently demonstrated that no relevant direct absorption through the sublingual mucosa occurs, SLIT proved effective in several clinical trials and rapidly became the most widely used noninjection route for immunotherapy in Europe. In 1998 a panel of experts of the World Health Organization, based on an extensive review of the literature, concluded that SLIT was a viable alternative to SCIT (Bousquet et al. 1998) and this statement was then confirmed in a position paper of the European Academy of Allergology and Clinical Immunology (Malling 1998) and in the ARIA document (Bousquet & Van Cauwenberge 2001) that extended the indications for SLIT to children. Finally, a recent comprehensive review by the AAAAI, despite skepticism on some points, acknowledged the clinical value of SLIT (Cox et al. 2006).

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1993 SLIT is mentioned in an EAACI pos pap

1997 Tari, 1st pediatric trial

2004 preventive effect compliance

2005 SLIT in children below the age of 5

Fig. 74.1 A brief history of the development of SLIT.

Practical aspects of SLIT In European countries, SLIT is currently marketed by several manufacturers. Obviously, the administration schedules and the amount of allergen(s) vary depending on the producer. The vaccines commercialized in Europe are standardized, either biologically or immunologically and, similarly to SCIT, the extracts are labeled in different units such as allergen units (AU), index of reactivity (IR), biological units (BU), and standard units (STU). Recently, for many extracts the content in micrograms of the major allergens has become available. SLIT is given as soluble tablets or drops to be kept under the tongue for 1–2 min and then swallowed (sublingualswallow mode). Indeed, in some studies a different method was adopted: the allergen was kept under the tongue and then spat out (sublingual-spit). Presently, based on clinical results and pharmacokinetic considerations, only the sublingualswallow route is used, and thus the acronym SLIT usually indicates the sublingual-swallow modality. SLIT can be administered either preseasonally (treatment stops at the beginning of the season), pre-coseasonally (treatment stops at the end of the season), or continuously. Pre-coseasonal schedules are commonly used for pollen allergy, whereas for perennial allergens continuous treatments are preferred. The amount of allergen given during a course of SLIT is usually higher than an equivalent SCIT schedule and for this reason the treatment has also been termed high-dose SLIT. SLIT involves a build-up phase (with gradually increasing doses) and a maintenance phase, where the maximum dose

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is administered either daily or on alternate days. The buildup phase usually lasts about 4– 6 weeks, and the vaccine is prepared in separate vials (or in separate blisters of tablets) at increasing concentrations. Based on the good safety profile of SLIT, accelerated updose or no-updose schedules with oncea-day maintenance have been proposed and used in clinical trials (see below). The use of very simplified schedules of administration is clearly advantageous for patients. There is no controlled study in the literature dealing with the optimal duration of a SLIT course. Therefore, based on what we know from subcutaneous immunotherapy, a duration of 3–5 years is usually recommended. Identically, as happens with injection immunotherapy, SLIT should be discontinued if no efficacy is seen after 2 years or after two pollen seasons. Concerning the evaluation of efficacy in everyday practice, none of the surrogate markers proposed in clinical trials (e.g., nasal cells and mediators, organ-specific responsiveness to challenge, systemic immunologic changes) can replace clinical evaluation. Thus the judgment of clinical efficacy of SLIT, as for SCIT, is made on a clinical basis (symptoms and use of rescue medications). In the case of asthmatic patients, the measurement of respiratory function or bronchial nonspecific hyperresponsiveness may be useful for evaluating the effects of SLIT. Since SLIT is self-administered at home, regular follow-up of the patient is always recommended, for instance with a visit every 3– 4 months. As a general rule, patients should be instructed in detail about the use of SLIT, the possible side effects and how to manage them. Written instructions are usually appreciated by patients. Currently, the indications for SLIT do not differ from those of SCIT (Bousquet et al. 1998; Bousquet & Van Cauwenberge 2001) with the addition of those patients who refuse injections or who experience intolerable adverse reactions with SCIT (Table 74.1).

Table 74.1 Factors to be considered in prescribing SLIT. (Modified from Bousquet et al. 1998, with permission.) Indications Allergic rhinitis and/or asthma Patients who refuse injections Patients who experienced previous adverse reactions with SCIT Severe uncontrolled asthma is a prudential contraindication In isolated mild intermittent rhinitis, SLIT may not be cost-effective Carefully evaluate IgE-mediated nature of the disease is clearly demonstrated Role of allergen(s) in eliciting symptoms is well documented Response to allergen avoidance and pharmacologic treatment Costs Ability and willingness of the patient to comply Availability of standardized extracts with documented efficacy

Sublingual Immunotherapy

Clinical efficacy Rhinitis and asthma Table 74.2 summarizes a number of randomized, doubleblind, placebo-controlled trials with SLIT (Tari et al. 1990; Sabbah et al. 1994; Feliziani et al. 1995; Troise et al. 1995; Hirsch et al. 1997; Clavel et al. 1998; Horak et al. 1998; Hordijk et al. 1998; Passalacqua et al. 1998, 1999, 2006a; Vourdas et al. 1998; Bousquet et al. 1999; Purello D’Ambrosio et al. 1999; La Rosa et al. 1999; Pradalier et al. 1999; Caffarelli et al. 2000; Guez et al. 2000; Pajno et al. 2000, 2003a; Ariano et al. 2001; Bahcecilier et al. 2001; Voltolini et al. 2001; Lima et al. 2002; Andrè et al. 2003; Ippoliti et al. 2003; Mortemousque et al. 2003; Wuthrich et al. 2003; Bowen et al. 2004; Bufe et al. 2004; Rolinck-Werninghaus et al. 2004; Smith et al. 2004; Tonnel et al. 2004; Dahl et al. 2006a,b; Durham et al. 2006; Lue et al. 2006; Niu et al. 2006; Valovirta et al. 2006). The majority have confirmed clinical efficacy of SLIT in allergic rhinitis caused by grasses, trees, ragweed, Parietaria and mites, whereas others studies with mites (Hirsch et al. 1997; Guez et al. 2000) and grasses (Lima et al. 2002) failed to demonstrate a significant difference between active and placebo groups. In the study by Smith et al. (2004), a significant effect was reported only in a subset of patients with more severe disease. When compared to inhaled fluticasone propionate, SLIT produced no additional benefit in allergic asthma but improved nonbronchial symptoms (Pajno et al. 2003a). In two recent large trials, the magnitude of the effect over placebo on symptoms and drug use was reported to be respectively 16% and 28% (Durham et al. 2006) and 30% and 38% (Dahl et al. 2006b). In the largest study available so far, including more than 800 patients, a clear dose dependence of the clinical effect was also demonstrated (Durham et al. 2006). A metaanalysis of 22 trials and 979 patients up to and including September 2002 concluded that SLIT was significantly “greater than” placebo in allergic rhinitis (Wilson et al. 2005). Another metaanalysis of the treatment of allergic rhinitis with SLIT in pediatric patients (aged 4–18 years), involving 10 trials, showed that SLIT was effective as assessed by reductions in symptom scores and rescue medications (Penagos et al. 2006). The large majority of the studies were conducted in rhinitis and, in fact, the first metaanalysis (Wilson et al. 2005) stated that there were too few studies on the use of SLIT in allergic asthma to perform an evaluation. Indeed, several trials have reported a beneficial effect of SLIT on lung symptoms (Bousquet et al. 1999; Caffarelli et al. 2000; Pajno et al. 2000; Bahcecilier et al. 2001; Lue et al. 2006; Niu et al. 2006) and a significant effect on bronchial hyperresponsivenss was also reported in doubleblind (Pajno et al. 2004) and open (Lombardi et al. 2001a; Marogna et al. 2005) trials. A metaanalysis in asthma was recently repeated, including 25 trials (either open or blinded) involving more than 1000 adults and children (Calamita et al. 2006). This demonstrated a significant effect of SLIT for most

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Table 74.2 Summary of double-blind placebo-controlled studies with SLIT.

Reference

Age range (years)

Patients (A/P)*

Tari et al. (1990) Sabbah et al. (1994) Feliziani et al. (1995) Troise et al. (1995) Hirsch et al. (1997) Passalacqua et al. (1998) Vourdas et al. (1998) Clavel et al. (1998) Horak et al. (1998) Hordijk et al. (1998) Bousquet et al. (1999) Passalacqua et al. (1999) Pradalier et al. (1999) La Rosa et al. (1999) Purello et al. (1999) Pajno et al. (2000) Guez et al. (2000) Caffarelli et al. (2000) Ariano et al. (2001) Bahcecilier et al. (2001) Voltolini et al. (2001) Lima et al. (2002) Mortemousque et al. (2003) Andre et al. (2003) Ippoliti et al. (2003) Pajno et al. (2003a) Wuthrich et al. (2003) Tonnel et al. (2004) Bufe et al. (2004) Smith et al. (2004)

5–12 13–51 14–48 17–60 6–16 15–46 7–17 8–55 16–48 18–45 15–37 15–42 6–25 6–14 14–50 8–15 6–51 4–14 19–50 7–15 15–52 16–48 6–60 6–55 5–12 8–14 6–13 7–45 6–13 18–60

Rolinck-Werninghaus et al. (2004) Bowen et al. (2004) Dahl et al. (2006a) Niu et al. (2006) Passalacqua et al. (2006a) Durham et al. (2006)

3–14 6–52 18–64 6–12 14–56 18–66

30/28 19/29 18/16 15/16 15/15 10/9 33/31 62/28 18/16 30/27 15/15 15/15 59/61 20/21 14/16 12/12 24/18 24/20 10/10 8/7 24/13 24/22 26/19 48/51 47/39 15/15 11/11 15/17 68/74 45 1 year, 46 2 years, 46 placebo 39/38 36/40 61/32 49/48 29/29 416/439

Valovirta et al. (2006)

Dahl et al. (2006b) Lue et al. (2006)

6–14

23–35 6–12

* A, active; P, placebo. † A, asthma; R, rhinitis; C, conjunctivitis.

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88

274/272 10/10

Allergen

Duration

Cumulative dose

Disease†

Mites Grasses Grasses Parietaria Mites Mites (monoid) Olive Grasses Birch Grasses Mites Parietaria Grasses Parietaria Parietaria Mites Mites Grasses Cypress Mites Trees Grasses Mites Ragweed Mites Parietaria Grasses Mites Grasses Grasses

18 months 17 weeks 4 months 10 months 1 year 2 years 2 years 6 months 4 months 6 months 2 years 8 months 4 months 6 months 8 months 2 years 2 years 3 months 8 months 6 months 24 months 18 months 24 months 7 months 6 months 13 months 2 years 2 years 3 years 1 year 2 years

365 STU 4500 IR 25 BU 105 BU 570 mg Der p 1 10 000 AU 4 mg Ole e 1 28 mg Phl p 5 250 STU 300 000 BU 104 000 IR (4.2 mg) Der p 1 265 BU (16 mg) Par j 1 11 000 IR (0.935 mg) Phl p 5 75 000 IR (52.5 mg) Par j 1 200 BU (12 mg) Par j 1 360 mg Der p 1 90 000 IR (2.2 mg) Der p 1 37 000 AU (monoid) 250 000 RU 7000 IR (0.56 mg) Der p 1 250 000 RU 0.9 mg Phl p 5 per month 90 000 IR (2.2 mg) Der p 1 Variable 57 mg Der p 1 23 mg Par j 1 NA 1.28 mg Der p 1 2 650 000 AU (9.6 mg) Phl p 5 6.2 mg Lol p 1, 3.6 mg Dac g 1 per year

R/A R R R R/A R R/A R/A R R/A A R/A R/A R/A R/A A R R/A R/A R/A R R C R R/A R/A/C R/A R R/A R

Grasses Ragweed Grass Mites Mites Grasses

3 years 4 months 6 months 6 months 2 years 18 weeks

Hazelnut, birch, elm (two doses) Grass Mite

18 months

7 months 6 months

NS Cumulative 2.7 mg Phl p 5 Cumulative 1.7 mg Der p and 3 mg Der f Allergoid 2500 SQ (0.06 mg) Phl p 5 per 4 months 25 000 SQ (0.6 mg) Phl p 5 per 4 months 75 000 SQ (1.8 mg) Phl p 5 per 4 months Weekly dose of Bet v 1, Cor a 1, Aln g 1 Group 1, 3.6 mg; Group 2, 30 mg 15 mg Phl p 5 daily Cumulative 1.7 mg Der p

R R R/C A R

R/C

R/C A

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of the considered outcomes including symptoms plus medications, pulmonary function, and overall improvement. Finally, we performed another metaanalysis on SLIT in asthmatic children (aged 4–18 years). This included 10 randomized controlled studies with 445 patients and concluded that SLIT was significantly better than placebo in reducing symptom scores and rescue medication usage (Penagos et al. 2007).

Comparison with SCIT When comparing two different routes of administration, the gold standard methodology is the double-blind, doubledummy trial. Indeed there are two double-dummy studies published as full papers (Quirino et al. 1996; Khinchi et al. 2004), both failing to detect a significant clinical difference between the two routes. Nevertheless, the study by Quirino et al. (1996) raised some concerns as it was not placebo-controlled. On the other hand, the study by Khinchi et al. (2004), comparing SCIT and SLIT in birch pollinosis, was double blind, placebo controlled, and also involved a detailed power analysis. In this study symptoms and drug intake were reduced by about one-third in the SLIT group and by half in the SCIT group, with no significant difference evident between treatments. On the other hand there were six grade 3 and 4 reactions in the SCIT group and none in the SLIT group. Some other comparative studies, with variable results, can be found in literature with Alternaria (Bernardis et al. 1996), grasses (Ongari et al. 1995), and mites (Piazza & Bizzarro 1993; Mungan et al. 1999) but they were conducted in an open fashion and therefore the results cannot be assumed as conclusive. One of these studies (Mungan et al. 1999) showed that clinical improvement is more prompt with the subcutaneous route, especially for asthma symptoms, although SLIT controlled rhinitis symptoms well. Despite being placebo-controlled, there was a striking imbalance in asthma symptoms between the groups at baseline.

Efficacy in other conditions It is likely that the good safety profile of SLIT will allow expansion of its indications to conditions other than respiratory allergy. There is one randomized controlled trial investigating the effects of SLIT in isolated allergic conjunctivitis due to mites (Mortemousque et al. 2003). Significant clinical efficacy was demonstrated in the second year of therapy and an increase in the conjuntival provocation threshold was also seen in the first year. One recent study demonstrated that SLIT is clinically effective in the treatment of IgE-mediated food allergy to hazelnut as testified by the increase in the threshold oral provocation dose (Enrique 2005). Encouraging reports are also available on the use of SLIT in latex allergy (Cistero Bahima et al. 2004; Bernardini et al. 2006), but this aspect needs further studies. Another possible field of investigation is desensitization for nickel allergy, but in this case there are only basic studies in animal models (Roelofs-Haarhuis et al. 2004).

Sublingual Immunotherapy

Clinical safety Randomized controlled trials The main rationale of SLIT is minimizing the risks of adverse events and therefore particular attention has been paid to safety. The most frequently and commonly reported side effect is the onset of oral/sublingual itching shortly after taking the dose. This phenomenon was always described as mild and self-resolving. In a single study (La Rosa et al. 1999) a significant rate of gastrointestinal complaints was also reported, but in this study the amount of allergen was as high as 375 times the amount usually administered in a standard SCIT course. Headache, rhinorrhea, constipation or urticaria were reported only sporadically and their incidence did not differ from the placebo groups. Noticeably, no fatal adverse event has been reported in the literature. The most recent review of the existing literature (Cox et al. 2006) reported a total occurrence of 14 severe adverse events (mainly asthma) in 20 years of clinical trials. So far there are two reports of anaphylaxis probably due to SLIT (Antico et al. 2006; Dunsky et al. 2006). In one case the diagnosis of anaphylaxis was not made by a health professional and the SLIT used was a mixture of six different allergens standardized as weight/volume. In the other case the reaction with a standardized latex extract occurred at the maximum recommended dose. Andre et al. (2000) reviewed the safety aspects of the trials performed with the vaccines of a single manufacturer; 690 subjects were enrolled (347 active and 343 placebo), with 218 children (103 active and 115 placebo). The large majority of events were mild. All events had a similar incidence in active and placebo groups, with the exception of the oral and gastrointestinal side effects. The occurrence of side effects and dropouts was similar in adults and children. A controlled dose-finding study of safety (Kleine-Tebbe et al. 2006) involved 48 grass-allergic patients outside pollen season. They received SLIT for 28-day periods at progressively increasing doses, up to 200 μg Phl p 5 allergen, i.e., about 40 times the amount given with one injection. The overall incidence of side effects was 74%, all of mild or moderate intensity. The most frequently reported events were irritation of the throat and oral itching. The possible effects of the sublingual administration of allergens was investigated by measuring the mucosal level of tryptase and eosinophil cationic protein and no change in these mediators could be detected at all, even in one patient reporting oral itching after SLIT intake (Marcucci et al. 2001).

Postmarketing surveys The information on safety provided by the controlled trials are valuable, but in these trials patients are highly selected and the administration is supervised. This situation is therefore profoundly different from that occurring in clinical

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reality. In this regard, postmarketing surveys would provide more realistic information on the safety in everyday clinical practice. Several postmarketing surveys conducted in both children (Di Rienzo et al. 1999; Pajno et al. 2003b; Agostinis et al. 2005; Di Rienzo et al. 2005; Fiocchi et al. 2005) and adults (Lombardi et al. 2001b) are now available. Based on these large surveys the overall rate of side effects ranged between 3 and 18% of patients and was invariantly less than 1 reaction per 1000 doses. In recent years great attention has been paid to safety in children. It is well known that the age of 5 years is considered as a relative contraindication for SCIT, mainly because in young children any reaction may be more severe and more difficult to treat than in adults. Recently, several postmarketing surveys have been conducted in the pediatric age group, some of them also involving children aged between 3 and 5 years (Pajno et al. 2003b; Agostinis et al. 2005). Also in the case of young children the safety profile was confirmed to be optimal. In fact the rate of all adverse events was never reported greater than 10%, but it is important to notice that at least 80% of the adverse events were local, mild, and selfresolving. The most troublesome side effects, reported in only a minority of patients, were gastrointestinal complaints such as stomach-ache or nausea.

Side effects and dose of the allergen When looking at the controlled trials published in the last 20 years, the relation between allergen dose and side effects is not immediately clear (due to the small samples of patients). A review of the available trials suggested that local side effects are even more frequent with low doses of allergen, whereas the gastrointestinal complaints are clearly dose related (Gidaro et al. 2005). In this review it was also suggested that the overall occurrence of all adverse events is not related to the dose administered. On the other hand, the more recent randomized controlled trials (Dahl 2006a; Durham et al. 2006), which involved hundreds of patients treated with the same standardized extract, reported that side effects (local and systemic) are dose dependent. This agrees with what is well known to occur with SCIT.

New modalities of administration The favorable safety profile of SLIT is generally accepted and for these reasons it has been proposed that a slow updosing phase is not necessary. This would result in a treatment that is more patient-friendly and convenient to manage. Indeed, there have been some reports with “ultra-rush” (< 2 hours) updosing showing that the rapid increase in doses does not lead to an increase in side effects (Rossi & Monasterolo 2005; Tripodi et al. 2005). Subsequently, preliminary experience with no updosing (i.e., starting SLIT with the maintenance dosage)

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confirmed the feasibility of this administration (Grosclaude et al. 2002; Guerra et al. 2006). The most recent large randomized trials (Dahl et al. 2006a,b; Durham et al. 2006) were all performed with the no-updosing regimen and their results in terms of safety, in addition to efficacy, were as favorable as those of the studies performed via the traditional route. There is also a randomized trial that compared the safety of the traditional updosing regimen with no updosing (Rodriguez et al. 2006) in 135 patients. This study found no difference in the rate and type of adverse events between the two groups of patients. As mentioned before, there are different ways to give SLIT, i.e., drops from vials, monodose vials, predosed dispensers, and tablets. From a historical point of view, soluble tablets were introduced in Italy during the 1980s and immediately proved to be convenient and easy to manage (Passalacqua et al. 1998). During the last 5 years the idea of tablets has been reconsidered and have been used in the more recent large trials (Wilson et al. 2005; Durham et al. 2006). Tablets have the advantages of simple use, of avoiding possible dosing errors, and their time of dissolution in the oral cavity can be precise. On the other hand, soluble tablets cannot be divided and therefore adjusting or reducing the dose may represent a problem. Nonetheless, taking into account the advantages and the possible disadvantages it is likely that soluble tablets will be the most suitable way of administration in the future.

Additional effects of SLIT SCIT has been shown to modify the natural history of the disease. This is testified by the fact that SCIT maintains longlasting efficacy after discontinuation, prevents the onset of new skin sensitizations, and reduces the risk of asthma onset in rhinitis patients (Durham et al. 1999; Pajno et al. 2001; Moller et al. 2002). Of course, robust evidence for such effects are still lacking for SLIT, since it has been used routinely only in the last 10 years. Nevertheless, some data are beginning to appear in the literature. Di Rienzo et al. (2003) performed a prospectic nonrandomized controlled study in 60 children (mean age 8.5 years) suffering from allergic asthma/rhinitis due to mite. They found in the SLIT group a significant difference versus baseline for the presence of asthma and the use of asthma medications 5 years after discontinuation of the treatment. Marogna et al. (2004) reported that the occurrence of new skin sensitization at 3 years was 5.9% in the SLIT group and 40% in the placebo group. In an open randomized trial of patients with grass pollen allergy, Novembre et al. (2004) found that after 3 years of SLIT, 8 of 45 actively treated subjects and 18 of 44 controls developed asthma, with a relative risk for untreated patients of developing asthma of 3.8. These figures are quite close to those reported in an analogous study performed with SCIT (Moller et al. 2002).

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The patient’s viewpoint: adherence, costs, quality of life SLIT is self-administered and managed at home by patients themselves, and thus concerns about compliance with SLIT have been raised. In this sense SCIT is believed to be superior, since it is given by a physician with virtually full compliance. Indeed, looking at the few studies that have assessed compliance, we can observe that the rate of discontinuation with SCIT ranges between 10 and 34% (Lower et al. 1993) and that up to 50% of patients are noncompliant because of side effects (Cohn & Pizzi 1993). In the recent years, some studies have attempted to quantify adherence to therapy in the case of SLIT by means of unscheduled telephone interviews. This could be done as the treatments were prepared as tablets or single-dose vials so that it was easy to count the remaining dose and to calculate adherence. In one study, involving 126 adult patients receiving SLIT in tablets, the compliance was reported greater than 90% over a 1-year period (Lombardi et al. 2004). In another observational study of 442 patients the compliance measured at 3 and 6 months was reported higher than 75% in 86% of the patients (Passalacqua et al. 2006b). A similar study was conducted in a population of 71 children (Passalacqua et al. 2006c) and the results did not substantially differ from those in adults. In fact, compliance data were available for all children at 3 months and for 56 children at 6 months. At 3 months, 85% of subjects had a compliance rate of greater than 75% (69% of them adhered > 90%). At 6 months, 84% had a compliance rate of greater than 75% (66% of them adhered > 90%). Concerning costs, it is true that the cumulative dose of allergen given via the sublingual route is higher than in SCIT and therefore the cost of the vaccine is higher as well. Nonetheless, a gross estimate shows that the cost of the extract is effectively balanced by the reduced need for medical and nursing time, so that the global cost of SLIT is even less than that of SCIT (Canonica & Passalacqua 2003). More recently, a formal cost–benefit analysis was conducted using a validated pharmacoeconomic model (Berto et al. 2006). In this study it was shown that SLIT for pollinosis in patients with rhinitis and/or asthma leads to significant savings (in terms of direct and indirect costs). Quality of life is now accepted as an essential part of the evaluation of the efficacy of a treatment. Nonetheless, there are very few studies where quality of life during immunotherapy was assessed (Oude Elberink et al. 2002; Radcliffe & Lewith 2003; Alvarez-Cuesta et al. 2005). Concerning SLIT, an early study (Bousquet et al. 1999) performed in asthmatic patients reported significant improvement as assessed with the generic SF-20 questionnaire. Another study performed in mild rhinitis (Passalacqua et al. 2006a) failed to detect a change in the generic SF-36 questionnaire items because all

Sublingual Immunotherapy

patients had a normal response. Nonetheless in this study it was shown that SLIT significantly modified the overall perception of the disease. Finally, in the study by Durham et al. (2006), SLIT significantly improved in a dose-dependent manner the quality of life measured with the disease-specific RQLQ. It is recommended that quality of life be included as a mandatory outcome in future studies on SLIT and immunotherapy in general.

Mechanisms of action Much is known about the putative mechanisms of action of SCIT (Till et al. 2004), including the modulation of Th1/Th2 balance possibly via the action of Treg cells (Romagnani 2006). On the other hand, since SLIT has been studied mainly from a clinical point of view, investigations on mechanisms of action have only appeared in the last few years. SLIT is associated with some qualitatively similar events to SCIT, including a modest increase in IgG, a transient increase in IgE, suppression of eosinophil recruitment, and activation in target organs, but these effects are not constant and reproducible (Marcucci et al. 2005; Rolinck-Werninghaus et al. 2005; Dehlink et al. 2006). Moreover, some studies failed to demonstrate changes in systemic T-cell and cytokine responses (Rolinck-Werninghaus et al. 2005; Dehlink et al. 2006) or local changes in T cells or effector cells within the sublingual mucosa (Lima et al. 2002). On the other hand, some studies have reported an increase in interleukin (IL)-10 production (Ciprandi et al. 2005; Cosmi et al. 2006) and one showed suppression of allergen-specific T-cell esponses after 12 months of grass immunotherapy (Fanta et al. 1999). One in vitro study demonstrated that SLIT reduced the expression of IL-5 and enhanced the expression of IL-10 in peripheral blood mononuclear cells stimulated with the allergen (Savolainen et al. 2006). Finally, one study in children reported a significant decrease in serum IL-13, an important Th2 cytokine (Ippoliti et al. 2003). Although increasing evidence is accumulating, it is generally acknowledged that further studies are needed to determine whether the same or alternative mechanisms are important for the effects of SLIT (Moingeon et al. 2006). It is reasonable to expect that the biodistribution of allergens following sublingual administration play a crucial role in the mechanism of action. The pharmacokinetics of SLIT was assessed in humans using radiolabeled purified allergens and nuclear medicine techniques (Bagnasco et al. 1997). The first studies were carried out with the Par j 1 allergen. After sublingual administration of the 123I-radiolabeled allergen, it was observed that no direct absorption of the allergen through the mucosae occurs. In fact, plasma radioactivity increased only after the allergen was swallowed. Interestingly, the allergen was retained for hours at mucosal level and gastrointestinal

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absorption was described, but no trace of the native allergen could be detected in the bloodstream. In contrast, if a modified allergen (allergoid) was used, it could be detected partially unmodified in the plasma (Bagnasco et al. 2001). No significant difference in terms of the biodistribution between the sublingual-swallow and the sublingual-spit methods was seen (Passalacqua et al. 2001). These experiments were replicated with a different allergen, namely Der p 2 (Bagnasco et al. 2005), and superimposable results were obtained. Thus, the biodistribution of SLIT seems to be independent of the protein used. Taken together, these data suggest that contact of the allergen with the oral mucosa is critical and that the allergen is not absorbed in the mouth.

Unmet needs Despite the amount of data available concerning the clinical aspects of SLIT, there are still several critical points that need to be clarified or defined. The main points are as follows. • The optimal dose of allergen is probably the most important aspect to be defined. Clinical trials have been conducted with largely variable doses of allergen extracts, usually labeled in arbitrary units. On the one hand, some trials do not report the content of allergens in micrograms. On the other, both positive and negative results have been reported with doses ranging from 3 to 300 times higher than the doses used in a corresponding SCIT course. Thus, so far it is not possible to provide guidelines on the optimal dose to be administered. Indeed, one recent study with grass allergen SLIT (Durham et al. 2006) in tablets compared the effects of three different doses labeled as 2500, 25 000 and 75 000 SQ-T, corresponding respectively to 0.5, 5, or 15 μg Phl p 5. The results clearly showed a dose dependence of the effect and that the higher dose (15 μg Phl p 5) was the most effective with a good safety profile. This is the first rigorous dose-ranging trial performed with SLIT. Although complex, expensive and time-consuming, this represents the ideal model to be applied in future studies to define the optimal dose of each allergen. • The optimal duration to achieve maximum benefit and possibly to achieve long-term benefit plus a preventive effect has yet to be determined. • Long-lasting effect has so far been demonstrated in a single open nonrandomized trial and therefore confirmatory data with more rigorous designs are needed. • As with SCIT, there is a single randomized, open, controlled trial demonstrating a preventive effect on the onset of asthma. Further studies are needed to confirm the effect and its magnitude, and to identify which children would benefit. • The mechanisms of action are overall poorly known. Some data would suggest that SLIT may act via T regulatory cells, but other studies also show that mechanisms of action are partially different from those of SCIT. Detailed knowledge of

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the molecular events leading to the clinical effect is mandatory to improve the treatment. • Finally, it is clear that SLIT (identically to SCIT) has variable efficacy within subjects. Criteria for the selection of patients likely to obtain most benefit from this treatment need to be established.

Conclusions The treatment of respiratory allergy is based on allergen avoidance, pharmacologic treatment, and immunotherapy. Immunotherapy is an allergen-oriented immunomodulator that affects the immune response to allergens and whose action develops over long periods (months). SLIT represents a significant advance because of the safety and good acceptance profile. Nonetheless, the self-administration itself requires careful instruction and detailed follow-up of the patients. Its prescription must be made only by a specialist, after a detailed diagnosis has been established and the expected benefit–cost ratio has been carefully evaluated. The clinical efficacy of SLIT in both asthma and rhinitis is now supported by a large number of controlled trials and metaanalyses. After a period of justified skepticism, due to the few studies available, in very recent years there has been a gradual change in the general opinion and SLIT has became increasingly accepted (Bousquet 2005; Bieber 2006; Cox et al. 2006). As per guidelines SLIT is indicated in patients with rhinitis or asthma or both, with low adherence or previous severe adverse reactions to SCIT (Fig. 74.2). Although there is no evidence of an increased risk, for prudential reasons and with analogy to SCIT, SLIT it is not recommended in patients with severe asthma. Also, the cost–efficacy and clinical value of SLIT in mild intermittent rhinitis is a matter of discussion.

Not cost? Mild intermittent

RHINITIS Moderatesevere intermittent

Mild persistent

Moderate– severe persistent

SLIT

Intermittent

Mild

Moderate

Severe

ASTHMA HIGH RISK?

Fig. 74.2 Indications for SLIT according to disease severity.

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Certainly, SLIT should not be considered a last-choice treatment but a complement to drug treatment. It is important to remember that SLIT must not be regarded as a substitute for SCIT, but rather as an additional choice or therapeutic tool. Similarly to SCIT, SLIT is not an alternative to drugs for controlling symptoms, but must be used in combination with them.

Acknowledgment This work has been partially supported by ARMIA (Associazione Ricerca Malattie Immunologiche e Allergiche).

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Committee on the Safety of Medicines (1986) CSM update. Desensitizing vaccines. BMJ 293, 948. Cosmi, L., Santarlasci, V., Angeli, R. et al. (2006) Sublingual immunotherapy with Dermatophagoides monomeric allergoid downregulates allergen-specific immunoglobulin E and increases both interferon-gamma-and interleukin-10 production. Clin Exp Allergy 36, 261–72. Cox, L.S., Linnemann, D.L., Nolte, H., Weldon, D., Finegold, I. & Nelson, H.S. (2006) Sublingual immunotherapy: a comprehensive review. J Allergy Clin Immunol 117, 1021–35. Dahl, R., Stender, A. & Rak, S. (2006a) Specific immunotherapy with SQ standardized grass allergen tablets in asthmatics with rhinoconjunctivitis. Allergy 61, 185– 90. Dahl, R., Kapp, A., Colombo, G. et al. (2006b) Efficacy and safety of sublingual immunotherapy with grass allergen tablets for seasonal allergic rhinoconjunctivitis. J Allergy Clin Immunol 118, 434–40. Dehlink, E., Eiwegger, T., Gerstmayr, M. et al. (2006) Absence of systemic immunologic changes during dose build-up phase and early maintenance period in effective specific sublingual immunotherapy in children. Clin Exp Allergy 36, 32– 9. Di Rienzo, V., Pagani, A., Parmiani, S., Passalacqua, G. & Canonica, G.W. (1999) Post-marketing surveillance on the safety of sublingual swallow immunotherapy in children. Allergy 54, 1110–13. Di Rienzo, V., Marcucci, F., Puccinelli, P. et al. (2003) Long-lasting effect of sublingual immunotherapy in children with asthma due to house dust mite: a ten year prospective study. Clin Exp Allergy 33, 206–10. Di Rienzo, V., Musarra, A., Sambugaro, R. et al. (2005) Post marketing survey on the safety of sublingual immunotherapy in children below the age of 5 years. Clin Exp Allergy 35, 560– 4. Dunsky, E., Goldstein, M.F., Dvorin, M.J. & Belecanech, G. (2006) Anaphylaxis due to sublingual immunotherapy. Allergy 61, 1235. Durham, S.R., Walker, S.M., Varga, E.M. et al. (1999) Long-term clinical efficacy of grass-pollen immunotherapy. N Engl J Med 341, 468–75. Durham, S.R., Yang, W.H., Pedersen, M.R., Johansen, N. & Rak, S. (2006) Sublingual immunotherapy with once-daily grass-allergen tablets: a randomised controlled trial in seasonal allergic rhinoconjunctivitis. J Allergy Clin Immunol 117, 802– 9. Fanta, C., Bohle, B., Hirt, W. et al. (1999) Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy. Int Arch Allergy Immunol 120, 218– 24. Enrique, E., Pineda, F., Malek, T. et al. (2005) Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract. J Allergy Clin Immunol 116(5), 1073–9. Feliziani, V., Lattuada, G., Parmiani, S. & Dall’Aglio, P.P. (1995) Safety and efficacy of sublingual rush immunotherapy with grass allergen extracts. A double blind study. Allergol Immunopathol 23, 173–8. Fiocchi, A., Pajno, G., La Grutta, S. et al. (2005) Safety of SLIT in children aged 3 to 7 years. Ann Allergy Asthma Immunol 95, 254–8. Gidaro, G.B., Frati, F., Sensi, L., Marcucci, F., Incorvaia, C. & Ciprandi, G. (2005) The safety of sublingual-swallow immunotherapy: an analysis of published studies. Clin Exp Allergy 35, 565–71. Grosclaude, M., Bouillot, P., Alt, R. et al. (2002) Safety of various dosage regimens during induction of sublingual immunotherapy. Int Arch Allergy Immunol 129, 248– 53. Guerra, L., Compalati, E., Rogkakou, A., Pecora, S., Passalacqua, G.

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& Canonica, G.W. (2006) Randomized open comparison of the safety of SLIT in a no updosing and traditional updosing schedule in patients with parietaria allergy. Allergol Immunopathol (Madr) 34, 82–3. Guez, S., Vatrinet, C., Fadel, R. & Andre, C. (2000) House dust mite sublingual swallow immunotherapy in perennial rhinitis: a double blind placebo controlled study. Allergy 55, 369–75. Herxeimer, H. (1951) Bronchial hypersensitization and hyposensitization in man. Int Arch Allergy Appl Immunol 40, 40–57. Herxeimer, H. & Prior, E.N. (1952) Further observations in induced asthma and bronchial hyposensitization. Int Arch Allergy Appl Immunol 3, 159– 61. Hirsch, T., Sahn, M. & Leupold, W. (1997) Double blind placebo controlled study of sublingual immunotherapy with house dust mite extracts in children. Pediatr Allergy Immunol 8, 21–7. Horak, F., Stubner, U.E., Berger, U., Marks, B., Toth, J. & Jager, S. (1998) Immunotherapy with sublingual birch pollen extract: a short term double blind study. J Invest Allergol Clin Immunol 8, 165–71. Hordijk, G.J., Antwelink, J.B. & Luwema, R.A. (1998) Sublingual immunotherapy with a standardized grass pollen extract: a double blind placebo controlled study. Allergol Immunopathol (Madr) 26, 234–240. Ippoliti, F., De Sanctis, W., Volterrani, A. et al. (2003) Immunomodulation during sublingual therapy in allergic children. Pediatr Allergy Immunol 14, 216–21. Khinchi, M.S., Poulsen, L.K., Carat, F., Andrè, C., Hansen, A.B. & Malling, H.-J. (2004) Clinical efficacy of sublingual and subcutaneous birch pollen allergen specific immunotherapy. A randomized placebo controlled, double blind, double dummy study. Allergy 59, 33– 44. Kleine-Tebbe, J., Ribel, M. & Herold, D.A. (2006) Safety of a SQstandardised grass allergen tablet for sublingual immunotherapy: a randomized, placebo-controlled trial. Allergy 61, 181–4. La Rosa, M., Ranno, C., Andre, C. et al. (1999) Double blind placebo controlled evaluation of sublingual swallow immunotherapy with standardized Parietaria judaica extract in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 104, 425–32. Lima, M.T., Wilson, D., Pitkin, L. et al. (2002) Grass pollen sublingual immunotherapy for seasonal rhinoconjunctivitis: a randomized controlled trial. Clin Exp Allergy 32, 507–14. Lombardi, C., Gargioni, S., Venturi, S., Zoccali, P., Canonica, G.W. & Passalacqua, G. (2001a) Controlled study of preseasonal immunotherapy with grass pollen extract in tablets: effect on bronchial hyperreactivity. J Invest Allergol Clin Immunol 11, 41–5. Lombardi, C., Gargioni, S., Melchiorri, A. et al. (2001b) Safety of sublingual immunotherapy with monomeric allergoid in adults: multicenter post-marketing surveillance study. Allergy 56, 989–92. Lombardi, C., Gani, F., Landi, M. et al. (2004) Quantitative assessment of the adherence to sublingual immunotherapy. J Allergy Clin Immunol 113, 1219–20. Lower, T., Hensy, J., Mandik, L., Janosky, J. & Friday, G.A. Jr (1993) Compliance with allergen immunotherapy. Ann Allergy 70, 480–2. Lue, K.H., Lin, Y.H., Sun, H.L., Lu, K.H., Hsieh, J.C. & Chou, M.C. (2006) Clinical and immunologic effects of sublingual immunotherapy in asthmatic children sensitized to mites: a double-blind, randomized, placebo-controlled study. Pediatr Allergy Immunol 17, 408–15. Malling, H.J. (ed.) (1998) EAACI Position Paper on local immunotherapy. Allergy 53, 933.

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Marcucci, F., Sensi, L., Frati, F. et al. (2001) Sublingual tryptase and ECP in children treated with grass pollen sublingual immunotherapy (SLIT): safety and immunologic implications. Allergy 56, 1091–5. Marcucci, F., Sensi, L., Di Cara, G. et al. (2005) Three-year follow-up of clinical and inflammation parameters in children monosensitized to mites undergoing sub-lingual immunotherapy. Pediatr Allergy Immunol 16, 519– 26. Marogna, M., Spadolini, I., Massolo, A., Canonica, G.W. & Passalacqua, G. (2004) Randomized controlled open study of SLIT for respirtory alllergy in real life: clinical efficacy and more. Allergy 59, 1205–10. Marogna, M., Spadolini, I., Massolo, A., Canonica, G.W. & Passalacqua, G. (2005) Clinical, functional, and immunologic effects of sublingual immunotherapy in birch pollinosis: a 3-year randomized controlled study. J Allergy Clin Immunol 115, 1184–8. Metha, S.B. & Smith, J.M. (1975) Nasal hyposensitization and hayfever. Clin Allergy 5, 279– 84. Moingeon, P., Batard, T., Fadel, R., Frati, F., Sieber, J. & Van Overtvelt, L. (2006) Immune mechanisms of allergen-specific sublingual immunotherapy. Allergy 61, 151–65. Moller, C., Dreborg, S., Ferdousi, H.A. et al. (2002) Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study). J Allergy Clin Immunol 109, 251– 6. Mortemousque, B., Bertel, F., De Casamayor, J., Verin, P. & Colin, J. (2003) House-dust mite sublingual-swallow immunotherapy in perennial conjunctivitis: a double-blind, placebo-controlled study. Clin Exp Allergy 33, 464– 9. Mungan, D., Misirligil, Z. & Gurbuz, L. (1999) Comparison of the efficacy of subcutaneous and sublingual immunotherapy in mite sensitive patients with rhinitis and asthma: a placebo controlled study. Ann Allergy Asthma Immunol 82, 485–90. Niu, C.K., Chen, W.Y., Huang, J.L., Lue, K.H. & Wang, J.Y. (2006) Efficacy of sublingual immunotherapy with high-dose mite extracts in asthma: a multi-center, double-blind, randomized, and placebocontrolled study in Taiwan. Respir Med 100(8), 1374–83. Noon, L. (1911) Prophylactic inoculation against hay fever. Lancet i, 1572– 3. Novembre, E., Galli, E., Landi, F. et al. (2004) Coseasonal sublingual immunotherapy reduces the development of asthma in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 114, 851–7. Ongari, S., Domeneghetti, P. & Parmiani, S. (1995) Comparison among drugs, injective IT and sublingual IT in grass allergic patients. Allergy 50, 358. Oude Elberink, J.N., De Monchy, J.G., Van Der Heide, S., Guyatt, G.H. & Dubois, A.E. (2002) Venom immunotherapy improves health-related quality of life in patients allergic to yellow jacket venom. J Allergy Clin Immunol 110, 174– 82. Pajno, G.B., Morabito, L., Barberio, G. & Parmiani, S. (2000) Clinical and immunological effects of longterm sublingual immunotherapy in asthmatic children sensitized to mite: a double blind study. Allergy 55, 842– 9. Pajno, G.B., Barberio, G., De Luca, F., Morabito, L. & Parmiani, S. (2001) Prevention of new sensitizations in asthmatic children monosensitized to house dust mite by specific immunotherapy. A six-year follow-up study. Clin Exp Allergy 31, 1392–7. Pajno, G., Vita, D., Caminiti, D., Parmiani, S. & Barberio, G. (2003a) Impact of sublingual immunotherapy on seasonal asthma and skin reactivity in children allergic to Parietaria pollen treated with inhaled fluticasone propionate. Clin Exp Allergy 33, 1641–7.

Sublingual Immunotherapy

Pajno, G.B., Peroni, D.G., Vita, D., Pietrobelli, A., Parmiani, S. & Boner, A.L. (2003b) Safety of sublingual immunotherapy in children with asthma. Paediatr Drugs 5, 777–81. Pajno, G.B., Passalacqua, G., Vita, D., Parmiani, S. & Barberio, G. (2004) Sublingual immunotherapy abrogates seasonal bronchial hyperresponsiveness in children with parietaria-induced respiratory allergy: a randomized controlled trial. Allergy 59, 883–7. Passalacqua, G., Albano, M., Fregonese, L. et al. (1998) Randomised controlled trial of local allergoid immunotherapy on allergic inflammation in mite induced rhinoconjunctivitis. Lancet 351, 629–32. Passalacqua, G., Albano, M., Riccio, A.M. et al. (1999) Clinical and immunological effects of a rush sublingual immunotherapy to parietaria species: a double blind placebo controlled trial. J Allergy Clin Immunol 104, 964–8. Passalacqua, G., Villa, G., Altrinetti, V. et al. (2001) Sublingual swallow or spit? Allergy 56, 578. Passalacqua, G., Pasquali, M., Ariano, R. et al. (2006a) Randomized double-blind controlled study with sublingual carbamylated allergoid immunotherapy in mild rhinitis due to mites. Allergy 61, 849– 54. Passalacqua, G., Musarra, A., Pecora, S. et al. (2006b) Quantitative assessment of the compliance with a once daily sublingual immunotherapy in real life. J Allergy Clin Immunol 117, 946–8. Passalacqua, G., Musarra, A., Pecora, S. et al. (2006c) Quantitative assessment of the compliance with a once-daily sublingual immunotherapy regimen in children. Pediatr Allergy Immunol 18(1), 58–62. Penagos, M., Compalati, E., Tarantini, F. et al. (2006) Efficacy of sublingual immunotherapy in the treatment of allergic rhinitis in children. Meta analysis of randomized controlled trials. Ann Allergy Asthma Immunol 97, 141–8. Penagos, M., Passalacqua, G., Compalati, E. et al. (2007) Meta-analysis of the efficacy of sublingual immunotherapy in the treatment of allergic asthma in pediatric patients, 3 to 18 years of age. Chest Oct 20; [Epub ahead of print]. Piazza, I. & Bizzarro, N. (1993) Humoral response to subcutaneous, oral and nasal immunotherapy for allergic rhinitis due to Dermatophagoides pteronyssinus. Ann Allergy 71, 461–9. Pradalier, A., Basset, D., Claudel, A. et al. (1999) Sublingual swallow immunotherapy (SLIT) with a standardized five grass pollen extract (drops and sublingual tablets) versus placebo in seasonal rhinitis. Allergy 54, 819–28. Purello, D., Ambrosio, F., Gangemi, S., Isola, S. et al. (1999) Sublingual immunotherapy: a double blind placebo controlled trial with Parietaria judaica extract standardized in mass units in patients with rhinoconjunctivitis, asthma or both. Allergy 54, 968–73. Quirino, T., Iemoli, E., Siciliani, E. & Parmiani, S. (1996) Sublingual vs injective immunotherapy in grass pollen allergic patients: a double blind double dummy study. Clin Exp Allergy 26, 1253–61. Radcliffe, M.J. & Lewith, J.T. (2003) Enzyme potentiated desensitisation in treatment of seasonal allergic rhinitis: double blind randomised controlled study. BMJ 327, 251–4. Rebien, W., Wahn, U., Puttonen, E. & Maasch, H.G. (1980) Comparative study of immunological and clinical efficacy of oral and subcutaneous hyposensitization. Allergologie 3, 101–9. Reid, M.J., Lockey, R.F., Turkeltaub, P.C. & Platt-Mills, T.A.E. (1993) Survey of fatalities from skin testing and immunotherapy. J Allergy Clin Immunol 92, 6–15. Rodriguez, F., Boquete, M., Ibanez, M.D., de la Torre-Martinez, F. & Tabar, A.I. (2006) Once daily sublingual immunotherapy without

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updosing: a new treatment schedule. Int Arch Allergy Immunol 140, 321–6. Roelofs-Haarhuis, K., Wu, X. & Gleichmann, E. (2004) Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J Immunol 173, 1043– 50. Rolinck-Werninghaus, C., Wolf, H., Liebke, C. et al. (2004) A prospective, randomized, double-blind, placebo-controlled multi-centre study on the efficacy and safety of sublingual immunotherapy (SLIT) in children with seasonal allergic rhinoconjunctivitis to grass pollen. Allergy 59, 1285– 93. Rolinck-Werninghaus, C., Kopp, M., Liebke, C., Lange, J., Wahn, U. & Niggemann, B. (2005) Lack of detectable alterations in immune responses during sublingual immunotherapy in children with seasonal allergic rhinoconjunctivitis to grass pollen. Int Arch Allergy Immunol 136, 134– 41. Romagnani, S. (2006) Regulatory T cells: which role in the pathogenesis and treatment of allergic disorders? Allergy 61, 3–14. Rossi, R. & Monasterolo, R.E. (2005) A pilot study of feasibility of ultra-rush (20–25 minutes) sublingual-swallow immunotherapy in 679 patients (699 sessions) with allergic rhinitis and/or asthma. Int J Immunopathol Pharmacol 18, 277– 85. Sabbah, A., Hassoun, S., Le Sellin, J., Andre, C. & Sicard, H. (1994) A double blind placebo controlled trial by the sublingual route of immunotherapy with a standardized grass pollen extract. Allergy 49, 309–13. Savolainen, J., Jacobsen, L. & Valovirta, E. (2006) Sublingual immunotherapy in children modulates allergen-induced in vitro expression of cytokine mRNA in PBMC. Allergy 6, 1184– 90. Scadding, K. & Brostoff, J. (1986) Low dose sublingual therapy in patients with allergic rhinitis due to dust mite. Clin Allergy 16, 483– 91. Smith, H., White, P., Annila, I., Poole, J., Andre, C. & Frew, A. (2004) Randomized controlled trial of high-dose sublingual immunotherapy to treat seasonal allergic rhinitis. J Allergy Clin Immunol 114, 831–7. Tari, M.G., Mancino, M. & Monti, G. (1990) Efficacy of sublingual immunotherapy in patients with rhinitis and asthma due to house

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Novel Approaches to Allergen Immunotherapy Mark Larché

Summary The development of novel forms of allergen immunotherapy has been facilitated by recent improvements in technology, such as recombinant DNA techniques. The use of defined recombinant allergens for subcutaneous and sublingual immunotherapy may supersede traditional extract-based formulations. Improved safety may be achieved through the use of hypoallergenic proteins, which may be naturally occurring or engineered in the laboratory. Synthetic T-cell epitopes may also offer a safer alternative to conventional therapy. Coadministration or conjugation of allergen molecules, or derivatives, with stimulators of innate immunity such as the Toll-like receptors may result in enhanced efficacy and prolonged duration of effect. Further clinical and mechanistic studies are required to determine the optimal approach to safe and efficacious allergen immunotherapy.

an increase in the Th1/Th2 balance of the response; reduction in the number of mast cells, eosinophils, and release of mediators; and an increase in allergen-specific IgG antibodies, particularly IgG4, which is believed to exert its effect by sequestering allergen and blocking IgE-facilitated allergen presentation to T cells (reviewed in Larché et al. 2006). SIT is currently routinely performed with allergen extracts that are only partially characterized/standardized and contain variable mixtures of allergens together with contaminating nonallergen proteins including bacterial endotoxin (Trivedi et al. 2003). As a result preparations differ considerably from one manufacturer to the next (compounded by different extraction techniques) with only a few major proteins being specifically quantified. In recent decades several approaches for improving the safety and efficacy of SIT have been pursued. In addition to improving standardization and characterization of allergen extracts, a major focus of effort has been to reduce the allergenicity of preparations while retaining their ability to stimulate modulation of the response away from Th2/IgE toward protective responses.

Introduction Allergen-specific immunotherapy (SIT) is an effective form of treatment for allergic diseases caused by inhalant allergens and insect venom (Durham et al. 1999; Golden et al. 2000). SIT is disease modifying rather than merely palliative and has been shown to prevent the progression of rhinitis to asthma (Moller et al. 2002) and the onset of sensitization to new allergens (Des Roches et al. 1997). However, treatment involves administering allergens to allergically sensitized individuals and, as a result, allergic side effects or adverse events ranging from local reactions at the site of injection to systemic and potentially life-threatening reactions such as anaphylactic shock are common. Several immunologic pathways appear to be involved in establishing functional immunologic tolerance leading to clinical efficacy, including the induction of allergen-specific regulatory T cells; immune deviation, with

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Recombinant allergens for SIT Genes encoding a significant percentage of common allergens have been cloned and expressed, resulting in the isolation of pure allergens produced as recombinant proteins (Fig. 75.1). Recombinant allergens have been used to establish diagnostic tests to determine molecular reactivity profiles (Hiller et al. 2002) and also for diagnostic in vivo provocation testing (Schmid-Grendelmeier & Crameri 2001). More recently, recombinant aeroallergens have been produced in order to generate molecularly defined vaccines for birch and grass pollen sensitivity. Recombinant grass pollen allergens have been successfully generated for the treatment of allergic rhinitis. In a randomized, double-blind, placebo-controlled trial, approximately equimolar amounts of the five timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6) were adsorbed to aluminum hydroxide and administered by subcutaneous depot injection (Jutel et al. 2005). The recombinant allergen vaccine preparation contained substantially less endotoxin

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Allergen source

RNA

Fel d 1

cDNA

Phl p 5

Protein purification

Gene cloning Expression vector

Transformation of bacteria (E. coli)

than conventional allergen extracts despite being produced in bacteria. The study spanned two pollen seasons and commenced with a preseasonal updosing regimen culminating in maintenance doses of 40 μg total protein which was halved during the pollen season. After the first 18 months of treatment there was a statistically significant reduction in combined symptom and medication scores. This was associated with a significant improvement in all fields of a validated rhinitis quality-of-life questionnaire and a trend toward improved allergen tolerance threshold in conjunctival provocation testing. Substantial increases in serum allergen-specific IgG antibodies were observed, with approximately 60-fold increases in IgG1 which peaked shortly after the initiation of therapy and up to 4000-fold increases in IgG4 which continued to increase over the duration of the study. No significant changes in allergen-specific IgE concentrations were observed (Jutel et al. 2005). Hypoallergenic variants of grass pollen allergens are currently under development for future therapy (Schramm et al. 1999). Recombinant Bet v 1, the major allergen of birch pollen, is currently being developed for clinical trials. Clinical grade recombinant (r)Bet v 1a was produced in Escherichia coli and compared to purified natural (n)Bet v 1a. The recombinant protein was recognized by both patient sera and anti-Bet v 1 antibodies generated in mice, indicating that the tertiary structure of the molecule was retained. Both rBet v 1a and nBet v 1a efficiently released histamine from sensitized peripheral blood basophils obtained from birch pollen-allergic donors. T-cell reactivity to both natural and recombinant proteins appeared to be equivalent, albeit in stimulation of Tcell lines derived from a small group of allergic subjects (Batard et al. 2005).

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Fig. 75.1 Cloning of allergen genes and production of recombinant allergen proteins. RNA is isolated from cells of the allergen source. RNA is reverse-transcribed to complementary DNA (cDNA) which is then ligated into an expression vector. Ligation of one cDNA into each vector molecule and subsequent transformation of bacterial cells allows cloning of individual cDNA molecules. Proteins expressed by the bacteria are subsequently purified for diagnosis and therapy. (See CD-ROM for color version.)

Hypoallergenic recombinant allergens While native recombinant allergens improve the standardization and quality control of allergen preparations for SIT, they still retain the native IgE-binding protein epitopes and therefore the risk of allergic adverse events. A potential solution for this problem may be the use of hypoallergenic and/or engineered recombinant allergens that occur naturally in the environment or that have been mutated, recombined or restructured in order to decrease allergenicity, increase immunogenicity (immunomodulation), or a combination of the two (Valenta 2002). Using in vitro site-directed mutagenesis, multiple amino acid residues of Bet v 1a were substituted with those present in the same positions of low IgE-binding isoforms. Thus, a Bet v 1 mutant carrying six point mutations was produced that displayed extremely low IgE-binding activity for all patients tested (Ferreira et al. 1998). In vivo (skin-prick) tests indicated that the potency of the six-point mutant to induce typical urticarial skin reactions in allergic individuals was dramatically reduced (100- to 1000-fold) compared with Bet v 1a. The hypoallergenic Bet v 1 molecule showed reduced IgE binding and basophil histamine release and was able to induce IgG antibodies in mice that blocked interaction between human IgE and allergen (Ferreira et al. 1996). Investigation of the IgE-binding activity of isoallergens led to the identification of naturally occurring Bet v 1 hypoallergens (Arquint et al. 1999). Isoforms Bet v 1d, Bet v 1g and Bet v 1l were found to be highly antigenic in T-cell proliferation assays and poorly allergenic in vitro and in vivo. The crystal structure of the hypoallergenic isoform Bet v 1l was recently determined and shown

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not to differ significantly from the high IgE-binding isoform Bet v 1a (Markovic-Housley et al. 2003). Thus, the low IgEbinding activity of certain isoforms is not due to problems in recombinant production leading to unfolded proteins. Such well-characterized molecules would be excellent candidates for specific immunotherapy. However, naturally occurring hypoallergens have not been identified for other allergen families. Instead, genetic engineering has been widely used to generate low IgE-binding variants. The most advanced studies to date have focused on two formulations of the major birch pollen allergen Bet v 1. Two fragments constituting the entire sequence of the allergen were expressed as proteins in E. coli (Vrtala et al. 1997) and evaluated in clinical trials together with a recombinant trimer (Vrtala et al. 2001). Both preparations consisted of native allergen sequence but the rearrangement of structure resulted in favorable characteristics for therapy. The recombinant trimer showed increased immunogenicity and induced a Th1-biased immune response in vitro and in vivo. The trimer displayed approximately 100-fold lower histamine-releasing potential and de novo leukotriene synthesis in basophils from birch pollen-allergic individuals (Hage-Hamsten et al. 1999). On injection into mice and rabbits, the trimer induced IgG antibodies that inhibited human IgE binding to Bet v 1 monomer (Vrtala et al. 2000). A number of potential explanations were proposed to explain why the IgE-binding Bet v 1 trimer showed reduced anaphylactic potential including a lower affinity for IgE binding, steric changes in IgE epitopes preventing crosslinking of FcεRI-bound IgE antibodies, and microaggregation causing concealment of IgE epitopes required for IgE cross-linking. The trimer was evaluated in a multicenter, double-blind, placebo-controlled trial together with the two recombinant fragments encompassing the entire sequence of the Bet v 1 molecule (Niederberger et al. 2004). A total of 124 patients were treated at three centers but detailed immunologic analysis was only performed on a 66 patient subset treated at one site. Both the trimer and the fragments induced a Th1biased immune response in vivo. Serum levels of allergenspecific IgG increased significantly after one preseasonal treatment period, with levels of IgG1, IgG2 and IgG4 being elevated in both serum and nasal secretions following treatment. A more modest increase in allergen-specific IgA and IgM were also observed (Niederberger et al. 2004). Objective nasal allergen challenge in a subgroup of individuals demonstrated that treatment was associated with reduced sensitivity to allergen challenge and that this improvement in sensitivity correlated with IgG4 levels in nasal lavage (Reisinger et al. 2005). Serum from treated patients was able to inhibit allergenspecific histamine release from sensitized basophils by approximately 10-fold. The cumulative dose of allergen trimers or fragments correlated with the induction of allergen-specific IgG1 and this in turn correlated with improvements in symptom scores. An attenuated seasonal boost of allergen-specific IgE was observed during the birch pollen season, suggesting

Novel Approaches to Allergen Immunotherapy

that treatment also modified the IgE memory B-cell response to allergen (Niederberger et al. 2004). In a subgroup analysis of patients treated at a second study site, treatment with recombinant Bet v 1 trimers was associated with significant increases in allergen-specific IgG (IgG1, IgG2 and IgG4) and a significant decrease in the frequency of allergen-specific interleukin (IL)-5 and IL-13-secreting cells (also a trend for reduction in IL-4-secreting cells and a trend for an increase in IL-12-secreting cells). Treatment was associated with a significant reduction in immediate skin reactivity to allergen in the treatment group but not in the placebo group (Gafvelin et al. 2005). Further subgroup analysis demonstrated induction of IgG1 and, to a lesser extent, IgG4 antibodies induced by treatment with the engineered Bet v 1 molecules that were able to recognize Bet v 1 cross-reactive food allergens Api g 1 (celery), Dau c 1 (carrot), and Mal d 1 (apple). Functional activity of the crossreactive antibodies was demonstrated by inhibition of sensitized basophil histamine release, which was as high as 94% inhibition with Api g 1 and 28% with Mal d 1 (Niederberger et al. 2007). Many other examples of hypoallergenic engineered allergen molecules have been reported. To date, these have not been evaluated in clinical studies, but the clinical efficacy described above with both native recombinant allergens and hypoallergens suggests that further clinical studies in a broader range of allergens will be forthcoming. An exhaustive review of all these approaches is beyond the scope of this chapter. However, examples of some of these molecules are given below. Point mutation of calcium binding EF-hand motifs of the Brassica pollen allergen Bra r 1 resulted in reduced IgE reactivity when screened with sera from two highly sensitized subjects (Okada et al. 1998). Immunization of mice with a variety of mutant proteins resulted in the production of IgG that was still capable of recognizing the native protein, suggesting that such mutants may be efficacious in human immunotherapy. Mutations to the EF-hand calcium-binding domain of the timothy grass pollen allergen Phl p 7 resulted in a mutant with markedly reduced IgE reactivity (77% reduction with sera from 40 allergic individuals), moderately reduced basophil histamine-release profile, and almost equivalent skin-prick test reactivity to the native molecule, highlighting the difficulty of extrapolating in vitro findings into the clinical arena (Westritschnig et al. 2004). Two unmutated peptide fragments of the molecule demonstrated much more impressive reductions in reactivity in all three tests. Similarly reduced IgE binding was demonstrated in a calcium-binding deficient mutant of Bet v 4 (Engel et al. 1997; Ferreira et al. 1999). Swoboda et al. (2002) generated nine mutants of the ryegrass group 5 allergen Lol p 5 containing single or multiple substitutions. A number of mutants displayed reduced IgEbinding activity, although mutations in the C-terminus of the molecule had little effect. Screening of three of the mutants

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for reduced histamine-releasing activity in blood basophils demonstrated heterogeneous effects with only one showing reduced histamine release in all eight allergic subjects screened. In this particular case the reduction in histamine release was only 10-fold, which may not be clinically relevant for the purposes of immunotherapy. These results highlight the fact that in many cases it may prove difficult to identify mutations that render a protein fully hypoallergenic in all subjects. Mutation of cysteine residues in the Hev b 6.02 domain of the major latex allergen Hev b 6.01 were generated to create a hypoallergenic molecule suitable for immunotherapy. Serum IgE from 43 latex-allergic subjects demonstrated markedly reduced reactivity to each of four mutants containing variable numbers of cysteine mutations. Mutant Hev b 6.01 proteins and a mutant of the Hev b 6.02 peptide were able to inhibit IgE binding to the native protein although results were variable. Basophil activation assays (upregulation of CD63) showed similar reductions in allergenicity of the mutants, although variability between basophil donors was also detected (Drew et al. 2004). A total of 24 point mutations and a 10-amino acid deletion were introduced into the bee venom allergen Api m 1 resulting in substantially reduced IgE and IgG binding. Murine antibodies induced by the mutant were only poorly crossreactive with the native molecule and the mutant was shown to have an unstable tertiary structure. T-cell reactivity of the mutant molecule with murine T-cell hybridomas was retained (Buhot et al. 2004).

Allergen A

Allergen hybrids Molecular engineering has been used to generate singlemolecule vaccines consisting of the most important allergens of a given source (Linhart et al. 2002) (Fig. 75.2). A number of hybrid venom allergens suitable for immunotherapy were created by inserting Ves v 5 sequences into a structurally homologous, but poorly cross-reactive, host molecule, Pol a 5 (King et al. 2001). The homologous allergens from yellow jacket venom Ves v 5 and from paper wasp Pol a 5 (59% sequence identity) show very limited cross-reactivity to antibodies from sensitized patients. Hybrids retained discontinuous B-cell epitopes and were able to compete for both murine and human Ves v 5 in inhibition studies. Furthermore, hybrids containing 10–49 residues of Ves v 5 showed 100- to 3000fold reduction in allergenicity in histamine release assay with basophils from yellow jacket-sensitized patients. Linhart et al. (2005) generated a hybrid grass allergen molecule by polymerase chain reaction (PCR)-based recombination of cDNAs encoding the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5 and Phl p 6. B-cell epitopes of the parent molecules were retained since the hybrid was able to block the binding of allergen-specific IgE, derived from patients’ sera, to the individual molecules and to an allergen extract. The hybrid induced stronger proliferative responses in peripheral blood mononuclear cells (PBMC) than did equimolar mixtures of the individual molecules and PBMC responses were associated with reduced IL-5 production and

Allergen B

Allergen gene A

Allergen gene B

Hybrid allergen construct

Hybrid protein for immunotherapy

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Fig. 75.2 Allergen hybrids. Genes or gene fragments encoding allergen molecules are cloned and ligated to each other to create novel gene sequences encoding chimeric proteins containing native T-cell epitope sequences in polypeptides with altered three-dimensional structure. Disrupted structure results in reduced allergenicity through reduced IgE binding. (See CD-ROM for color version.)

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enhanced IL-10 and interferon (IFN)-γ responses. Similarly, immunization of mice with the hybrid or mixtures of the individual proteins resulted in responses of greater magnitude. Antisera to the hybrid molecule was cross-reactive with major allergens from nine major grass species and completely inhibited β-hexosaminidase release from IgE-primed rat basophil leukemia cells incubated with allergen. A hybrid molecule containing two major bee venom allergens, Api m 1 and Api m 2, displayed reduced reactivity with human and murine antibodies specific for the conformational determinants of the native molecules (Kussebi et al. 2005). The molecule was similarly hyporeactive in basophil histamine release and leukotriene synthesis assays. T-cell reactivity, assessed by proliferation and cytokine production of PBMC and Api m 1-specific T-cell clones, appeared to be retained. Skin-prick test reactivity to the hybrid molecule was substantially reduced, with 12 of 13 individuals testing negative to the hybrid while 12 of 13 individuals tested positive to an equimolar preparation of Api m 1. Additionally, the hybrid prevented sensitization to native allergen in a prophylactic model of immunotherapy. A similar hybrid composed of three major bee venom proteins, Api m 1, Api m 2 and Api m 3, has also been constructed by molecular engineering (Karamloo et al. 2005). Overlapping noncontiguous sequences of Api m 1 and Api m 2 were generated together with the entire sequence of Api m 3 (melittin) in a single molecule. The hybrid displayed substantially reduced IgE reactivity in dot blot, Western blot, and ELISA assays. Both histamine release and generation of leukotrienes from human peripheral blood basophils was reduced. T-cell reactivity appeared to be retained in PBMC cultures and T-cell clones. In common with the two-allergen hybrid, the threeallergen hybrid showed reduced skin reactivity in allergic individuals in skin-prick test assays. Prophylactic treatment

Novel Approaches to Allergen Immunotherapy

of mice prevented sensitization to native allergens. Treatment of mice with established allergen-specific IgE responses resulted in enhanced IgE responses together with substantial IgG2a responses, such that the ratio of IgG2a to IgE was markedly enhanced (Karamloo et al. 2005).

T-cell peptide epitopes Synthetic peptides containing T-cell epitopes of allergens have been employed to target epitope-specific CD4+ T cells (Fig. 75.3). Peptide therapy in both in vitro and in vivo experiments has been shown to induce functional tolerance to the allergen protein(s). The use of peptides for the treatment of allergic and autoimmune diseases has recently been reviewed in detail (Larché & Wraith 2005). Early in vitro experiments demonstrated that peptides could induce antigen-specific hyporesponsiveness. For example, Lamb and colleagues cultured T-cell clones with high doses (50 μg) of peptide and induced transient (lasting about 1 week) nonresponsiveness. Subsequently, prophylactic and therapeutic application of peptides in vivo has been evaluated in experimental models of allergy, autoimmunity, and transplantation. In recent human studies peptides from two allergens in particular (Fel d 1 and Api m 1) have been studied.

Cat allergy The majority (> 90%) of individuals with a clinical history of cat allergy are sensitized to Fel d 1, the major allergen of cats. The protein is widespread in the environment and can be found in homes and in public places, including schools; the protein is readily transported on clothing (Custovic et al. 1998). Epitope mapping studies have identified a variety of peptides that have been evaluated in clinical studies of Antigen uptake + presentation

No mast cell activation

Fig. 75.3 Peptide immunotherapy. Short synthetic peptides, designed to be incapable of cross-linking IgE molecules (by virtue of length), do not activate mast cells and basophils. In this way, the allergenicity of peptide vaccines is lower than conventional allergen extract-based approaches. Peptides are taken up by antigenpresenting cells under neutral noninflammatory conditions leading to T-cell recognition of peptide–MHC complexes without activation of the innate immune system and a subsequent regulatory T-cell response. (See CD-ROM for color version.)

IgE

Presentation to T cells T-cell activation ? IgG/G4 synthesis Treg IL-10

IL-10

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peptide immunotherapy (Counsell et al. 1996). Early studies evaluated the safety and clinical efficacy of two 27-amino acid sequences, IPC-1 and IPC-2, in clinical trials. The efficacy of IPC-1/IPC-2 was evaluated by Norman et al. (1996) in a double-blind placebo-controlled trial. Four weekly subcutaneous injections of placebo or peptides at doses of 7.5, 75, or 750 μg were administered. Modest improvements in symptom and medication scores were observed in the highest-dose group, 6 weeks after treatment. Individual results were heterogeneous, with some individuals experiencing marked improvements in symptoms and others not. Treatment was associated with a significant number of early and late adverse events, including chest tightness, nasal congestion and flushing. These occurred a few minutes to several hours after administration of the peptides. In an associated study, a decrease in IL-4 production (but not proliferation) by IPC-1/IPC-2-specific T-cell lines from subjects in the highdose group was demonstrated (Marcotte et al. 1998). In an inhaled allergen challenge study, allergen PD20 was performed before and after a variable cumulative dose of peptides (Pene et al. 1998). After therapy, PD20 was not significantly different between the treated and placebo groups. However, in the middle- and high-dose groups, there was a significant increase in allergen tolerance between baseline and posttreatment days (statistically significant with the groups but not between treatment and placebo). IL-4 release was significantly reduced in the high-dose group, but there was no change in IFN-γ production. In a randomized, double-blind, parallel-group study (Simons et al. 1996), 40 cat-allergic subjects received subcutaneous injections of 250 μg of IPC-1/IPC-2 weekly for 4 weeks. The magnitude of early- and late-phase skin reactivity to whole cat extract up to 24 weeks after the last injection showed no change. No modulation of cat antigen-specific cytokine production was observed. Frequent adverse events were reported, including symptoms of asthma, rhinitis and pruritus. Treatment with IPC-1/IPC-2 was also associated with significant clinical improvement in pulmonary function (forced expiratory volume in 1 s, FEV1) in individuals with reduced baseline FEV1 in a multicenter, randomized, double-blind, placebo-controlled study of 133 cat-allergic patients (Maguire et al. 1999). Subjects received subcutaneous injections of either 75 or 750 μg of peptides (total of eight injections). A significant improvement in the subjective ability to tolerate cats was also observed. Subjects recruited to this study had moderate to severe disease with high IgE levels. Approximately 30% had failed previous whole allergen immunotherapy and more than 40% of the group had asthma. Adverse events were common and consisted of occasional IgE-mediated acute reactions and more frequent late-onset symptoms of asthma which declined with successive doses. More recently, a number of clinical studies have been performed with shorter peptides from Fel d 1 administered intradermally to cat-allergic asthmatic subjects with mild to

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moderate disease severity. Significant reductions in the magnitude of the cutaneous late-phase reaction to intradermal allergen challenge were reported together with reductions in T-cell proliferation, Th2 cytokines, and IFN-γ and a concomitant increase in IL-10. In a double-blind placebo-controlled study, 24 cat-allergic asthmatic subjects were subjected to cutaneous allergen challenge, inhaled methacholine PC20, and inhaled allergen PD20. Subjects received incremental divided doses totaling 90 μg of each of 12 peptides administered at 3–4 day intervals (Oldfield et al. 2002). Significant reductions in both earlyand late-phase cutaneous reactions to intradermal challenge with allergen were observed. Subjects treated with peptides felt significantly better able to tolerate exposure to cats after therapy. No significant improvements were observed in PD20 or PC20, although the study was not sufficiently powered to detect such differences. In a related study (Alexander et al. 2005a), a lower dose (41.1 μg) of peptides delivered at 2-week intervals led to a significant improvement in PC20. A significant reduction in the magnitude of the cutaneous late-phase reaction to allergen challenge was also observed. Studies evaluating upper and lower airway outcomes following a higher-dose peptide therapy protocol have also been performed (Alexander et al. 2005b). Treatment significantly reduced cutaneous late-phase reactions to intradermal allergen challenge as in previous studies. The effect of peptide therapy on late asthmatic responses was evaluated by comparison of bolus inhaled allergen challenge before and after treatment. No effect was observed on the early asthmatic reaction but a significantly reduced late asthmatic reaction was observed within the group who developed a dual asthmatic response to inhaled allergen extract (FEV1; area under curve 2–8 hours post challenge). Nasal allergen challenge revealed a significant reduction in outcome scores (sneezing, weight of nasal secretions, and nasal blockage) in individuals who displayed only a single early response to inhaled allergen extract at baseline challenge. No change in the suppressive activity of CD4+CD25+ cells was observed following peptide immunotherapy, suggesting that CD4+CD25+ regulatory T cells may not play a significant role in the mechanism of action (Smith et al. 2004). However, antigen-specific inducible regulatory T cells were induced. CD4+ cells isolated after peptide immunotherapy could suppress the proliferative response of baseline CD4− cells (Verhoef et al. 2005). These data provide evidence that peptide immunotherapy induces a population of CD4+ T cells with regulatory activity.

Bee venom allergy Peptides from the major bee venom allergen Api m 1 have been evaluated in venom-allergic individuals. Five subjects received divided incremental doses (cumulative dose 397.1 μg) of a mixture of three peptides at weekly intervals (Muller et al. 1998). After treatment, subcutaneous challenge with 10 μg

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of Api m 1 was tolerated by all subjects; three of five tolerated a wild bee sting challenge without reaction, while the remaining two subjects developed mild systemic allergic reactions. No changes were observed in levels of allergen-specific serum IgE or IgG4 during therapy but allergen after treatment resulted in a sharp increase in serum concentrations of IgE and IgG4. Four similar peptides (total dose of 431.1 μg) were administered to 12 subjects in a controlled, open-label, single-blind study (Tarzi et al. 2006). Treatment was well tolerated and no adverse events were observed. Allergen-specific T-cell proliferative responses and IL-13 and IFN-γ responses to allergen were reduced following treatment whereas IL-10 was increased. Late-phase cutaneous reactions to allergen were significantly reduced. In a third study, venom-allergic subjects were treated according to a “rush” protocol with three polypeptides (long synthetic peptides; LSP) encompassing the Api m 1 molecule (Fellrath et al. 2003). No significant change in skin sensitivity to allergen challenge was observed, but a transient increase in T-cell proliferation to the peptides was seen. IFN-γ and IL10 levels but not Th2 cytokines increased. Allergen-specific IgG4 levels increased throughout the study period. Some local and mild systemic skin reactions were observed.

CpG oligonucleotides in SIT Targeting of allergens and Toll-like receptor (TLR) ligands has been proposed as a strategy for modulating immune responses to allergens resulting in a shift in the Th1/Th2 balance (Horner et al. 2004) (Fig. 75.4). The genetic material of prokaryotes is rich in sequences containing unmethylated cytosinephosphoguanosine (CpG) dinucleotide motifs that are relatively less common in mammals. Synthetic oligonucleotides (ODNs) containing CpG motifs are able to activate both innate and acquired immune responses through TLR9 (Krieg 2006). Distinct immunostimulatory profiles can be observed with different ODNs depending on the sequence, length, number, and location of CpG motifs in an ODN. CpG ODNs are currently being developed for a variety of indications including oncology, allergy and infectious diseases. CpG ODN sequences tend to induce Th1-like cytokine response. CpG ODN as an adjuvant may be injected together with natural allergens (coadministered or conjugated) or may be incorporated into a vector together with an allergen cDNA. CpG ODN, administered in conjunction with antigen, was effective in reducing established Th2 responses in murine models (Broide et al. 1998; Kline et al. 1998). Efficacy was associated with the induction of the Th1 cytokines IFN-γ and IL-12. CpG-containing ODNs (referred to as immunostimulatory sequences; ISS) were covalently linked to Amb a 1, the major ragweed allergen, to generate allergen immunostimulatory conjugates (AIC) (Tighe et al. 2000). In addition to interaction with TLR9, ODN sequences reduced the allergenicity of

Novel Approaches to Allergen Immunotherapy IFN-g

IL-12

Tn

Th1

APC IFN-g TLR9

TLR9 CpG-conjugated allergen

Fig. 75.4 Immune deviation through Toll-like receptor (TLR) activation. Allergen is conjugated with synthetic unmethylated DNA motifs that interact with intracellular TLR9 receptors following uptake by antigenpresenting cells (APC). TLR9 activation leads to activation of IL-12 transcription, which directs differentiation of allergen-specific naive T cells (Tn) toward Th1 cells that antagonize Th2 responses through production of interferon (IFN)-g. (See CD-ROM for color version.)

Amb a 1 in skin-prick tests, probably through masking of IgE epitopes. Allergen–ISS conjugates (AIC) were more effective (than allergen alone) in inducing protective immune responses (Th1 cytokines and IgG antibody response) in preclinical studies in a mouse model and in human PBMC (Spiegelberg et al. 1998). Ragweed-allergic individuals were treated preseasonally with escalating doses of AIC at weekly intervals. Symptom and medication scores were followed for two consecutive pollen seasons. No significant clinical improvement was observed after the first year but a significant reduction in chest symptoms during the peak pollen season was reported in the second season. A trend toward improved nasal symptoms and overall symptom and medication scores was also observed, but these failed to achieve statistical significance. In contrast to clinical findings, nasal biopsies showed significant reductions in eosinophils and IL-4 mRNA-positive cells and an increase in IFN-γ mRNA-positive cells following allergen challenge at the end of the first pollen season compared with baseline challenge (Tulic et al. 2004). In a randomized, double-blind, placebo-controlled pilot study, 25 ragweed-allergic adults were treated with either AIC or placebo (Creticos et al. 2006). Treatment was administered before the ragweed season of 2001 and subjects were monitored during and after the 2001 season and the 2002 season, although no treatment was given in 2002. Treatment consisted of a course of six weekly subcutaneous injections with weekly dose elevation as follows: 0.06, 0.3, 1.2, 3.0, 6.0 and 12 μg/mL of AIC. The primary outcome of the study was vascular permeability in the nose following allergen challenge.

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This outcome was not changed by treatment. However, the total nasal challenge score was significantly improved along with post nasal drip. There was a trend for a reduction in nasal itch and the sneeze count after allergen challenge was significantly reduced in the treated group. Rhinitis qualityof-life questionnaire analysis revealed a significant overall mid-season improvement in symptoms during the first ragweed season. Treatment was also associated with improved peak-season nasal symptoms and visual analog scores. The treatment effect was maintained through the second ragweed season despite the lack of treatment. Again subjects reported significantly improved peak-season nasal symptom and visual analog scores. Treatment with AIC blunted seasonal increases in allergenspecific IgE in both seasons. AIC induced a transient increase in Amb a 1 and ragweed-specific IgG which, at its peak, represented an approximate doubling of baseline levels. Specific IgG levels appeared to be waning 2 months after the end of the first ragweed season and had returned to normal by the end of the second. No subclass analysis was performed. Functional analysis of the ability of posttreatment serum IgG to block IgE–allergen complex binding to B cells (facilitated antigen presentation, FAP) (Wachholz et al. 2003) showed no activity, suggesting that the transient induction of allergenspecific IgG did not contribute to the mechanism of efficacy. Therapeutically effective conventional allergen immunotherapy has been shown to induce much greater levels of specific IgG, which is active in FAP analysis. Sensitivity to allergen skin tests was analyzed by measuring changes in immediate (skin-prick test) and late-phase (intradermal measured at 24 hours) responses. In both cases, AIC treatment was associated with a reduction in the magnitude of the reactions but the differences failed to achieve statistical significance between groups. There was a significant decrease in IL-4-positive basophils after treatment. No significant changes in levels of secreted or intracellular IL-4 or IFN-γ were found in activated peripheral blood T cells (CD4+CD69+). IL-10 levels appeared to be increased in PBMC by seasonal allergen exposure but not modulated by treatment. Levels of mRNA expression of a panel of 184 cytokine and chemokine-associated genes were not modified by treatment. In none of these analyses of peripheral blood cells were allergen-specific T cells selected or activated with allergen prior to analysis and thus any treatment-induced changes in T-cell responses would be difficult to detect above background. The results of more recent clinical trials have yet to be formally reported in the literature. Oral congress presentations suggest that clinical efficacy in these studies was lower than expected.

Conclusions Specific allergen immunotherapy (SIT) is an efficacious, disease-modifying form of treatment for allergic disease that

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has been used for nearly a century. Strategies to improve the safety of SIT focus on both reducing the allergenicity of the treatment preparation whilst maintaining or enhancing the immunogenicity, and refining the composition of materials for treatment to include only relevant molecules. Recombinant DNA technology has enabled the cloning and expression of recombinant allergens and the engineering of hypoallergenic variants. Both native and engineered recombinants have recently been evaluated for the first time in clinical trials and exhibited promising therapeutic activity. Deconstruction of the conformation of allergen molecules, and thus their ability to activate mast cells and basophils has been achieved by the identification and synthesis of peptides representing T cell epitopes. Peptide immunotherapy has been evaluated in a number of clinical studies and trials and shown to improve surrogate outcomes related to allergic disease. Conjugation of allergen to synthetic bacterial DNA motifs has been proposed as a strategy both for reducing IgE reactivity, and achieving activation of the innate immune response leading to the induction of antagonistic Th1 responses to allergen. Each of these approaches remains at an early stage of clinical development and further studies are required to determine both safety and efficacy profiles.

References Alexander, C., Ying, S., Kay, B. & Larché, M. (2005a) Fel d 1-derived T cell peptide therapy induces recruitment of CD4+ CD25+; CD4+ interferon-gamma+ T helper type 1 cells to sites of allergen-induced late-phase skin reactions in cat-allergic subjects. Clin Exp Allergy 35, 52–8. Alexander, C., Tarzi, M., Larché, M. & Kay, A.B. (2005b) The effect of Fel d 1-derived T-cell peptides on upper and lower airway outcome measurements in cat-allergic subjects. Allergy 60, 1269–74. Arquint, O., Helbling, A., Crameri, R., Ferreira, F., Breitenbach, M. & Pichler, W.J. (1999) Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen. J Allergy Clin Immunol 104, 1239–43. Batard, T., Didierlaurent, A., Chabre, H. et al. (2005) Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy. Int Arch Allergy Immunol 136, 239– 49. Broide, D., Schwarze, J., Tighe, H. et al. (1998) Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice. J Immunol 161, 7054–62. Buhot, C., Chenal, A., Sanson, A. et al. (2004) Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity. Protein Sci 13, 2970– 8. Counsell, C.M., Bond, J.F., Ohman, J.L. Jr, Greenstein, J.L. & Garman, R.D. (1996) Definition of the human T-cell epitopes of Fel d 1, the major allergen of the domestic cat. J Allergy Clin Immunol 98, 884–94. Creticos, P.S., Schroeder, J.T., Hamilton, R.G. et al. (2006) Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis. N Engl J Med 355, 1445–55.

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Custovic, A., Simpson, A., Pahdi, H., Green, R.M., Chapman, M.D. & Woodcock, A. (1998) Distribution, aerodynamic characteristics, and removal of the major cat allergen Fel d 1 in British homes. Thorax 53, 33– 8. Des Roches, A., Paradis, L., Ménardo, J.-L., Bouges, S., Daurès, J.-P. & Bousquet, J. (1997) Immunotherapy with a standardized Dermatophagoides pteronyssinus extract. VI. Specific immunotherapy prevents the onset of new sensitizations in children. J Allergy Clin Immunol 99, 450– 3. Drew, A.C., Eusebius, N.P., Kenins, L. et al. (2004) Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity. J Immunol 173, 5872–9. Durham, S.R., Walker, S.M., Varga, E.M. et al. (1999) Long-term clinical efficacy of grass-pollen immunotherapy. N Engl J Med 341, 468– 75. Engel, E., Richter, K., Obermeyer, G. et al. (1997) Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains. J Biol Chem 272, 28630– 7. Fellrath, J.M., Kettner, A., Dufour, N. et al. (2003) Allergen-specific T-cell tolerance induction with allergen-derived long synthetic peptides: results of a phase I trial. J Allergy Clin Immunol 111, 854–61. Ferreira, F., Rohlfs, A., Hoffmann-Sommergruber, K. et al. (1996) Modulation of IgE-binding properties of tree pollen allergens by site-directed mutagenesis. Adv Exp Med Biol 409, 127–35. Ferreira, F., Ebner, C., Kramer, B. et al. (1998) Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J 12, 231–42. Ferreira, F., Engel, E., Briza, P., Richter, K., Ebner, C. & Breitenbach, M. (1999) Characterization of recombinant Bet v 4, a birch pollen allergen with two EF-hand calcium-binding domains. Int Arch Allergy Immunol 118, 304– 5. Gafvelin, G., Thunberg, S., Kronqvist, M. et al. (2005) Cytokine and antibody responses in birch-pollen-allergic patients treated with genetically modified derivatives of the major birch pollen allergen Bet v 1. Int Arch Allergy Immunol 138, 59–66. Golden, D.B., Kagey-Sobotka, A. & Lichtenstein, L.M. (2000) Survey of patients after discontinuing venom immunotherapy. J Allergy Clin Immunol 105, 385– 90. Hage-Hamsten, M., Kronqvist, M., Zetterstrom, O. et al. (1999) Skin test evaluation of genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1: results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season. J Allergy Clin Immunol 104, 969– 77. Hiller, R., Laffer, S., Harwanegg, C. et al. (2002) Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment. FASEB J 16, 414–16. Horner, A.A., Redecke, V., & Raz, E. (2004) Toll-like receptor ligands: hygiene, atopy and therapeutic implications. Curr Opin Allergy Clin Immunol 4, 555– 61. Jutel, M., Jaeger, L., Suck, R., Meyer, H., Fiebig, H. & Cromwell, O. (2005) Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 116, 608–13. Karamloo, F., Schmid-Grendelmeier, P., Kussebi, F. et al. (2005) Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes. Eur J Immunol 35, 3268–76. King, T.P., Jim, S.Y., Monsalve, R.I., Kagey-Sobotka, A., Lichtenstein,

Novel Approaches to Allergen Immunotherapy

L.M. & Spangfort, M.D. (2001) Recombinant allergens with reduced allergenicity but retaining immunogenicity of the natural allergens: hybrids of yellow jacket and paper wasp venom allergen antigen 5s. J Immunol 166, 6057–65. Kline, J.N., Waldschmidt, T.J., Businga, T.R. et al. (1998) Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 160, 2555–9. Krieg, A.M. (2006) Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov 5, 471–84. Kussebi, F., Karamloo, F., Rhyner, C. et al. (2005) A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy. J Allergy Clin Immunol 115, 323–9. Lamb, J.R., Skidmore, B.J., Green, N. et al. (1983) Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J Exp Med 157, 1434–47. Larché, M. & Wraith, D.C. (2005) Peptide-based therapeutic vaccines for allergic and autoimmune diseases. Nat Med 11, (4 suppl.), S69– S76. Larché, M., Akdis, C.A. & Valenta, R. (2006) Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol 6, 761–71. Linhart, B., Jahn-Schmid, B., Verdino, P. et al. (2002) Combination vaccines for the treatment of grass pollen allergy consisting of genetically engineered hybrid molecules with increased immunogenicity. FASEB J 16, 1301–3. Linhart, B., Hartl, A., Jahn-Schmid, B. et al. (2005) A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination. J Allergy Clin Immunol 115, 1010–16. Maguire, P., Nicodemus, C., Robinson, D., Aaronson, D. & Umetsu, D.T. (1999) The safety and efficacy of ALLERVAX CAT in cat allergic patients. Clin Immunol 93, 222–31. Marcotte, G.V., Braun, C.M., Norman, P.S. et al. (1998) Effects of peptide therapy on ex vivo T-cell responses. J Allergy Clin Immunol 101, 506–13. Markovic-Housley, Z., Degano, M., Lamba, D. et al. (2003) Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier. J Mol Biol 325, 123–33. Moller, C., Dreborg, S., Ferdousi, H.A. et al. (2002) Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study). J Allergy Clin Immunol 109, 251–6. Muller, U., Akdis, C.A., Fricker, M. et al. (1998) Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol 101, 747–54. Niederberger, V., Horak, F., Vrtala, S. et al. (2004) Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc Natl Acad Sci USA 101 (suppl. 2), 14677–82. Niederberger, V., Reisinger, J., Valent, P. et al. (2007) Vaccination with genetically modified birch pollen allergens: immune and clinical effects on oral allergy syndrome. J Allergy Clin Immunol 119, 1013–16. Norman, P.S., Ohman, J.L. Jr, Long, A.A. et al. (1996) Treatment of cat allergy with T-cell reactive peptides. Am J Respir Crit Care Med 154, 1623– 8. Okada, T., Swoboda, I., Bhalla, P.L., Toriyama, K. & Singh, M.B. (1998) Engineering of hypoallergenic mutants of the Brassica pollen allergen, Bra r 1, for immunotherapy. FEBS Lett 434, 255–60.

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Oldfield, W.L., Larché, M. & Kay, A.B. (2002) Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: a randomised controlled trial. Lancet 360, 47– 53. Pene, J., Desroches, A., Paradis, L. et al. (1998) Immunotherapy with Fel d 1 peptides decreases IL-4 release by peripheral blood T cells of patients allergic to cats. J Allergy Clin Immunol 102, 571–8. Reisinger, J., Horak, F., Pauli, G. et al. (2005) Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity. J Allergy Clin Immunol 116, 347– 54. Schmid-Grendelmeier, P. & Crameri, R. (2001) Recombinant allergens for skin testing. Int Arch Allergy Immunol 125, 96–111. Schramm, G., Kahlert, H., Suck, R. et al. (1999) Allergen engineering: variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity. J Immunol 162, 2406–14. Simons, F.E., Imada, M., Li, Y., Watson, W.T. & HayGlass, K.T. (1996) Fel d 1 peptides: effect on skin tests and cytokine synthesis in cat-allergic human subjects. Int Immunol 8, 1937–45. Smith, T.R., Alexander, C., Kay, A.B., Larché, M. & Robinson, D.S. (2004) Cat allergen peptide immunotherapy reduces CD4(+) T cell responses to cat allergen but does not alter suppression by CD4(+) CD25(+) T cells: a double-blind placebo-controlled study. Allergy 59, 1097–101. Spiegelberg, H.L., Tighe, H., Roman, M., Broide, D. & Raz, E. (1998) Inhibition of IgE formation and allergic inflammation by allergen gene immunization and by CpG motif immunostimulatory oligodeoxynucleotides. Allergy 53 (45 suppl.), 93– 7. Swoboda, I., De Weerd, N., Bhalla, P.L. et al. (2002) Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy. Eur J Immunol 32, 270– 80. Tarzi, M., Klunker, S., Texier, C. et al. (2006) Induction of interleukin-

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10 and suppressor of cytokine signalling-3 gene expression following peptide immunotherapy. Clin Exp Allergy 36, 465–74. Tighe, H., Takabayashi, K., Schwartz, D. et al. (2000) Conjugation of immunostimulatory DNA to the short ragweed allergen Amb a 1 enhances its immunogenicity and reduces its allergenicity. J Allergy Clin Immunol 106, 124–34. Trivedi, B., Valerio, C. & Slater, J.E. (2003) Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 111, 777–83. Tulic, M.K., Fiset, P.O., Christodoulopoulos, P. et al. (2004) Amb a 1: immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal inflammatory response. J Allergy Clin Immunol 113, 235–41. Valenta, R. (2002) The future of antigen-specific immunotherapy of allergy. Nat Rev Immunol 2, 446– 53. Verhoef, A., Alexander, C., Kay, A.B. & Larché, M. (2005) T cell epitope immunotherapy induces a CD4+ T cell population with regulatory activity. PLoS Med 2, e78. Vrtala, S., Hirtenlehner, K., Vangelista, L. et al. (1997) Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy. J Clin Invest 99, 1673–81. Vrtala, S., Akdis, C.A., Budak, F. et al. (2000) T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies. J Immunol 165, 6653–9. Vrtala, S., Hirtenlehner, K., Susani, M. et al. (2001) Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1. FASEB J 15, 2045–7. Wachholz, P.A., Soni, N.K., Till, S.J. & Durham, S.R. (2003) Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 112, 915–22. Westritschnig, K., Focke, M., Verdino, P. et al. (2004) Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7. J Immunol 172, 5684–92.

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Asthma and its Treatment

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Definition, Clinical Features, Investigations, and Differential Diagnosis of Asthma Piero Maestrelli, Gaetano Caramori, Francesca Franco and Leonardo M. Fabbri

Summary Asthma is a chronic inflammatory disorder of the airways that affects subjects of all ages and which usually starts in early life. Usually asthma is reasonably easy to control with adequate treatment but, if uncontrolled, can place severe limits on daily life and even be fatal. The aim of this chapter is to present the reader with an update on the definition, classification, risk factors, clinical features, and diagnosis of the disease. Particular reference will be made to phenotypes such as cough variant asthma, brittle asthma, near-fatal asthma, and severe refractory asthma. Finally, a discussion of the overlap between asthma and chronic obstructive pulmonary disease is presented. The most recent guidelines from the Global Initiative for Asthma (GINA), integrated with recent literature on particular aspects of the disease, represent the backbone of the chapter. Particular importance is now devoted to the concept of asthma control. The clinical manifestations of asthma, i.e., symptoms, sleep disturbances, limitation of daily activity, impairment of lung function, and use of rescue medications, can be controlled with appropriate treatment. GINA guidelines now recommend classification of asthma by level of control to reflect the understanding that asthma severity involves not only the severity of the underlying disease but also its responsiveness to treatment, and that severity is not an unvarying feature of an individual patient’s asthma but may change over months or years. The mechanisms whereby host and environmental risk factors influence the development of asthma are complex and interactive. In addition, developmental aspects, such as the maturation of the immune response and the timing of infectious exposures during the first years of life, are emerging as important factors modifying the risk of asthma in the genetically susceptible person. A correct diagnosis of asthma is essential if appropriate drug therapy is to be given. Asthma symptoms may be intermittent and their significance may be overlooked by patients and physicians or, because they are nonspecific, they may result

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

in misdiagnosis, particularly in children, leading to inappropriate treatment. The classical clinical features for diagnosis are presented, including symptoms, cough variant asthma, and physical examination findings. Lung function testing, including spirometry or peak expiratory flow monitoring and variability, continues to be strongly recommended as an aid to diagnosis, assessment of severity, and evaluation of control. In contrast, measurement of airway hyperresponsiveness and reversibility testing are recommended only for differential diagnosis in difficult cases.

Definition Asthma was originally and clinically defined in terms of symptoms and lung function, i.e., reversible airflow limitation and airway hyperresponsiveness (American Thoracic Society 1962). Advances in the understanding of the mechanisms involved in asthma have made this definition unsatisfactory. The “Global strategy for asthma management” prepared by a panel convened by the National Institutes of Health and the World Health Organization (GINA 2006) has proposed a definition of asthma based on pathology and its functional consequences in addition to its clinical characteristics, recognizing that asthma is associated with chronic inflammation of the airways. According to this definition, the interaction of symptoms, airway limitation, inflammation, and bronchial hyperresponsiveness is believed to determine the various phenotypic patterns of asthma, its severity, and the response to treatment. Thus, based on the functional consequences of airway inflammation, the most recent operational definition of asthma proposed (GINA 2006) is as follows: Asthma is a chronic inflammatory disorder of the airways, in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable, airflow limitation within the lung that is often reversible, either spontaneously or with treatment.

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This definition is not substantially different from the working definition proposed in the third revision of the guidelines for diagnosis and management of asthma by the Expert Panel Report of the National Heart, Lung and Blood Institute (2007) except for a more precise characterization of inflammation and the concept that reversibility of airflow limitation may be incomplete in some patients with asthma, which are underlined in the latter report. Although airway inflammation may be variable in degree and distinct phenotypes of asthma exist (e.g., intermittent, persistent, exercise-associated, aspirin-sensitive, or severe asthma), the immunohistopathologic features exhibit a consistent pattern, characterized by infiltration of eosinophils, lymphocytes (Th2-like cells), and neutrophils (especially in sudden-onset fatal asthma exacerbations), mast-cell activation, and epithelial cell injury. Recently, natural killer T cells have raised a lot of interest. This family of cells with immunoregulatory properties were found to be important in the biology of allergic airway inflammation in animal models, so there was interest about their potential role in the pathogenesis of asthma in humans. At present the question remains unresolved. Akbari et al. (2003) reported that about 60% of the CD4+ T cells in bronchoalveolar lavage (BAL) fluid obtained from 14 patients with moderate-to-severe persistent asthma met the criteria for natural killer T cells that also expressed the CD4 molecule, whereas recent work by Vijayanand et al. (2007) showed that the numbers of natural killer T cells were not increased in samples of BAL fluid obtained from patients with asthma, as compared with samples obtained from healthy volunteers or patients with chronic obstructive pulmonary disease (COPD). Further work on the precise role of this potent group of immunoregulatory cells in airway inflammation is needed to fully understand how immune cells are regulated in the lungs and how defects in immunoregulation might lead to airway inflammation and manifest in clinical disorders such as asthma (Ho 2007). The paradigm of asthma has been modified over the last 10 years with the recognition that airway remodeling may occur in some patients. These persistent changes in airway structure include subepithelial fibrosis, mucus hypersecretion, smooth muscle hypertrophy, and angiogenesis. Airway remodeling may explain the incomplete response to current available asthma treatments in some situations (Bateman et al. 2004; Holgate & Polosa 2006; O’Byrne & Parameswaran 2006). If one accepts the most recent definitions, any lung disease that manifests itself with asthma symptoms and which is associated with airway inflammation, reversible airflow limitation, and airway hyperresponsiveness may be labeled as asthma. However, airway inflammation is not easy to measure and thus to use in clinical practice. Also, airway hyperresponsiveness, which can be measured with standardized methods (Sterk et al. 1993; Crapo et al. 2000) (i) does not accurately reflect the severity of the disease nor the response to treatment (GINA

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2006; Expert Panel Report of the National Heart, Lung and Blood Institute 2007); (ii) may not be present at all times in all asthmatics; and (iii) may be present in asymptomatic subjects as well as in subjects with other obstructive diseases of the airways (Boulet 2003). Special mention should be made of asymptomatic airway hyperresponsiveness (AAHR), a common phenomenon which should be differentiated from the situation where symptoms attributable to mild asthma are unrecognized. AAHR is more frequently observed in subjects with atopy, in members of families with asthma, in those individuals exposed to tobacco smoke, and in women. It is associated with airway inflammation and remodeling, although these features are not as severe as observed in asthma. AAHR is associated with an increased risk for later development of asthma, suggesting that it is a marker of a pathologic process that could lead to asthma (Boulet et al. 2006). In conclusion airway hyperresponsiveness is no longer recommended for the assessment of severity and monitoring of treatment in asthma, and it is not even recommended for the diagnosis of asthma, with the exception of cough variant asthma and subjects with symptoms of asthma and normal lung function (GINA 2006). Thus from a clinical standpoint, the most reliable, simple objective method to confirm the diagnosis of asthma in a subject with symptoms of asthma (see below) remains the demonstration of reversible airflow limitation. While the spontaneous reversibility of airflow limitation may be assessed by monitoring peak expiratory flow (PEF), the reversibility of the airflow limitation induced by pharmacologic treatment may be assessed by measuring forced expiratory volume in 1 s (FEV1) before and after a single dose of a bronchodilator, or before and after a course of full antiasthma treatment including glucocorticosteroids (GINA 2006; Expert Panel Report of the National Heart, Lung and Blood Institute 2007). Genetic and environmental components are important to the development and expression of asthma. Atopy, the genetic predisposition to mount an IgE-mediated response to allergens, and exposure to common allergens are the most important predisposing factors for development of asthma (Apter 2007). Of the environmental factors, viral respiratory infections represent one of the most important causes of asthma exacerbation and may also contribute to the development of asthma (Johnston 2005; Tan 2005). However, since the relative contribution of host and environmental factors and the precise interaction between them that leads to the initiation and persistence of the disease are not fully established, the working definition of asthma remains descriptive.

Classification Asthma may be classified according to etiology, asthma severity or asthma control.

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Etiology Many attempts have been made to classify asthma according to etiology, particularly with regard to environmental sensitizing agents. However, such a classification is limited by the existence of patients in whom no environmental cause can be identified. Despite this, an effort to identify an environmental cause for asthma (e.g., occupational asthma) should be part of the initial assessment to enable the use of avoidance strategies in asthma management. Describing patients as having allergic asthma is usually of little benefit, since single specific causative agents are seldom identified (GINA 2002, 2006).

Asthma severity Asthma may greatly differ in term of overall severity and of severity of its exacerbations. No single test is able to precisely classify the severity of the disorder. Characterization of the severity of asthma and of its exacerbations is based on the combination of symptom scores, repeated measurement of lung function, and the amount of medication required to control asthma. The description of levels of disease severity based on a combination of the level of symptoms as well as objective measurements of lung function has differed little for many years between the guidelines produced in various countries. In fact, previous GINA documents subdivided asthma by severity into four categories: intermittent, mild persistent, moderate persistent, and severe persistent (GINA 2006). This classification of asthma by severity is useful when decisions are being made about management at the initial assessment of a patient (GINA 2006). However, asthma severity involves both the severity of the underlying disease and its responsiveness to treatment (GINA 2006). In addition, severity is not an unvarying feature of an individual patient’s asthma, but may change over months or years (GINA 2006). Because of these considerations, the classification of asthma severity provided in Table 76.1, which is based on expert opinion rather than evidence, is no longer recommended for clinical practice but only for research (GINA 2006). Its main limitation is its poor value in predicting what treatment will be required and what a patient’s response to the treatment might be. For this purpose, a periodic assessment of asthma control is more relevant and useful (GINA 2006).

Asthma control Asthma control may be defined in a variety of ways. In general, the term “control” may indicate disease prevention, or even cure. However, in asthma, where neither of these is realistic options at present, it refers to control of the manifestations of disease. Ideally this should apply not only to clinical manifestations, but also to laboratory markers of inflammation and pathophysiologic features of the disease as well. There is evidence that reducing inflammation with controller therapy

Table 76.1 Classification of asthma severity by clinical features before treatment. (From GINA 2006, with permission.) Intermittent Symptoms less than once a week Brief exacerbations Nocturnal symptoms not more than twice a month FEV1 or PEF ≥ 80% predicted PEF or FEV1 variability < 20% Mild persistent Symptoms more than once a week but less than once a day Exacerbations may affect activity and sleep Nocturnal symptoms more than twice a month FEV1 or PEF ≥ 80% predicted PEF or FEV1 variability < 20–30% Moderate persistent Symptoms daily Exacerbations may affect activity and sleep Nocturnal symptoms more than once a week Daily use of inhaled short-acting b2 agonist FEV1 or PEF 60–80% predicted PEF or FEV1 variability > 30% Severe persistent Symptoms daily Frequent exacerbations Frequent nocturnal asthma symptoms Limitation of physical activities FEV1 or PEF ≤ 60% predicted PEF or FEV1 variability > 30%

achieves clinical control, but because of the cost and/or general unavailability of tests such as measurement of sputum eosinophils and exhaled nitric oxide or even endobronchial biopsy, it is recommended that treatment be aimed at controlling the clinical features of disease, including lung function abnormalities. Table 76.2 provides the characteristics of controlled, partly controlled, and uncontrolled asthma. This is a working scheme based on current opinion and has not been validated. Complete control of asthma is commonly achieved with treatment, the aim of which should be to achieve and maintain control for prolonged periods with due regard to the safety of treatment, potential for adverse effects, and the cost of treatment required to achieve this goal. The classification of asthma by level of control reflects an understanding that asthma severity involves not only the severity of the underlying disease but also its responsiveness to treatment, and that severity is not an unvarying feature of an individual patient’s asthma but may change over months or years (GINA 2006). Furthermore, the most recent guidelines stress that the achievement and maintenance of clinical control is the goal of asthma treatment, thus reflecting the importance of classifying asthma by level of control (GINA 2006).

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Table 76.2 Levels of asthma control. (From GINA 2006, with permission.)

Characteristic

Controlled (all of the following)

Partly controlled (any measure present in any week)

Daytime symptoms

None (twice or less/week)

More than twice/week

Limitation of activities Nocturnal symptoms/awakening Need for reliever Lung function (PEF/FEV1)* Exacerbations

None None Twice or less/week Normal None

Any Any More than twice/week < 80% predicted or personal best One or more/year†

Uncontrolled Three or more features of partly controlled asthma in any week

One in any week‡

* Lung function is not a reliable test for children 5 years and younger. † Any exacerbation should promote review of maintenance treatment to ensure that it is adequate. ‡ By definition, an exacerbation in any week makes that an uncontrolled asthma week.

Risk factors Factors that influence the risk of asthma can be divided into those that cause the development of asthma and those that trigger asthma symptoms; some do both. The former include host factors (which are primarily genetic) and the latter are usually environmental factors. However, the mechanisms whereby they influence the development and expression of asthma are complex and interactive. In addition, developmental aspects, such as maturation of the immune response and the timing of infectious exposures during the first years of life, are emerging as important factors modifying the risk of asthma in the genetically susceptible person. The lack of a clear definition for asthma presents a significant problem in studying the role of different risk factors in the development of this complex disease, because the characteristics that define asthma (e.g., airway hyperresponsiveness, atopy, allergic sensitization) are themselves products of complex gene–environment interactions and are therefore both features of asthma and risk factors for the development of the disease (Yang et al. 2007).

Host factors Genetic Asthma has a heritable component, but it is not simple. Current data show that multiple genes may be involved in the pathogenesis of asthma, and different genes may be involved in different ethnic groups (Le Souef 2006; Holloway & Koppelman 2007) The search for a specific gene (or genes) involved in susceptibility to atopy or asthma continues, as results to date have been inconsistent. In addition to genes that predispose to asthma there are genes that are associated with the response to asthma treatments (Hall 2006; Tantisira & Weiss 2006; Weiss et al. 2006). Genetic markers will likely become important not only as

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risk factors in the pathogenesis of asthma but also as determinants of responsiveness to treatment.

Obesity Obesity has also been shown to be a risk factor for asthma. Certain mediators such as leptins may affect airway function and increase the likelihood of asthma development (Weiss 2005).

Sex Male sex is a risk factor for asthma in children. Prior to the age of 14, the prevalence of asthma is nearly twice as great in boys as in girls. As children get older the difference between the sexes narrows and by adulthood the prevalence of asthma is greater in women than in men (Melgert et al. 2007). The reasons for this sex-related difference are not clear. However, lung size is smaller in males than in females at birth but larger in adulthood (GINA 2006).

Environmental factors There is some overlap between environmental factors that influence the risk of developing asthma and factors that cause asthma symptoms, for example occupational sensitizers belong in both categories. However, there are some important causes of asthma symptoms (e.g., air pollution and some allergens) which have not been clearly linked to the development of asthma.

Allergens Although indoor and outdoor allergens are well known to cause asthma exacerbations, their specific role in the development of asthma is still not fully resolved. Birth-cohort studies have shown that sensitization to housedust mite allergens, cat dander, dog dander, and Aspergillus mold (Hogaboam et al. 2005) are independent risk factors for asthmalike symptoms in children up to 3 years of age. However, the

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relationship between allergen exposure and sensitization in children is not straightforward. It depends on the allergen, the dose, the time of exposure, the child’s age, and probably genetics as well (Hogaboam et al. 2005; Sharma et al. 2007). For some allergens, such as those derived from house-dust mites and cockroaches, the prevalence of sensitization appears to be directly correlated with exposure. However, although some data suggest that exposure to house-dust mite allergens may be a causal factor in the development of asthma, other studies have questioned this interpretation. Cockroach infestation has been shown to be an important cause of allergic sensitization, particularly in inner-city homes. In the case of dogs and cats, some epidemiologic studies have found that early exposure to these animals may protect a child against allergic sensitization or the development of asthma, but others suggest that such exposure may increase the risk of allergic sensitization. This issue remains unresolved. The prevalence of asthma is reduced in children raised in a rural setting, which may be linked to the presence of endotoxin in these environments (Ege et al. 2007).

Infections During infancy, a number of viruses have been associated with inception of the asthmatic phenotype. Respiratory syncytial virus (RSV) and parainfluenza virus produce a pattern of symptoms including bronchiolitis that parallel many features of childhood asthma. A number of long-term prospective studies of children admitted to the hospital with documented RSV have shown that approximately 40% will continue to wheeze or have asthma into later childhood. On the other hand, evidence also indicates that certain respiratory infections early in life, including measles and sometimes even RSV, may protect against the development of asthma. The data do not allow specific conclusions to be drawn (Singh et al. 2007). The “hygiene hypothesis” of asthma suggests that exposure to infections (Singh et al. 2007) early in life influences the development of a child’s immune system along a “nonallergic” pathway, leading to a reduced risk of asthma and other allergic diseases. Although the hygiene hypothesis continues to be investigated, this mechanism may explain observed associations between family size, birth order, daycare attendance, and the risk of asthma (de Meer et al. 2005). The interaction between atopy and viral infections appears to be a complex relationship, in which the atopic state can influence the lower airway response to viral infections, viral infections can then influence the development of allergic sensitization, and interactions can occur when individuals are exposed simultaneously to both allergens and viruses.

Occupational sensitizers Over 300 substances have been associated with occupational asthma, which is defined as asthma caused by exposure to an agent encountered in the work environment. These

substances include highly reactive small molecules such as isocyanates, irritants that may cause an alteration in airway responsiveness, known immunogens such as platinum salts, and complex plant and animal biological products that stimulate the production of IgE. Occupational asthma arises predominantly in adults and occupational sensitizers are estimated to cause about 1 in 10 cases of asthma among adults of working age. Asthma is the most common occupational respiratory disorder in industrialized countries. Occupations associated with a high risk for occupational asthma include farming and agricultural work, painting (including spray painting), cleaning work, and plastic manufacturing. Most occupational asthma is immunologically mediated and has a latency period of months to years after the onset of exposure. IgE-mediated allergic reactions and cell-mediated allergic reactions are involved. Levels above which sensitization frequently occurs have been proposed for many occupational sensitizers. However, the factors that cause some people but not others to develop occupational asthma in response to the same exposures are not well identified. Very high exposures to inhaled irritants may cause “irritant induced asthma” (formerly called the reactive airways dysfunctional syndrome) even in nonatopic persons. Atopy and tobacco smoking may increase the risk of occupational sensitization to certain substances, but screening individuals for atopy is of limited value in preventing occupational asthma. The most important method of preventing occupational asthma is elimination or reduction of exposure to occupational sensitizers (Mapp et al. 2005; Beach et al. 2007; Boulet et al. 2007).

Tobacco smoke Tobacco smoking is associated with accelerated decline of lung function in people with asthma, increases asthma severity, may render patients less responsive to treatment with inhaled and systemic glucocorticosteroids, and reduces the likelihood of asthma being controlled. Exposure to tobacco smoke both prenatally and after birth is associated with measurable harmful effects, including a greater risk of developing asthma-like symptoms in early childhood. However, evidence of increased risk of allergic diseases is uncertain. Distinguishing the independent contributions of prenatal and postnatal maternal smoking is problematic (Pattenden et al. 2006). However, studies of lung function immediately after birth have shown that maternal smoking during pregnancy has an influence on lung development. Furthermore, infants of smoking mothers are four times more likely to develop wheezing illnesses in the first year of life. In contrast, there is little evidence that maternal smoking during pregnancy has an effect on allergic sensitization. Exposure to environmental tobacco smoke (passive smoking) increases the risk of lower respiratory tract illnesses in infancy and childhood (Carlsen & Lodrup Carlsen 2005).

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Outdoor/indoor air pollution The role of outdoor air pollution in causing asthma remains controversial. Children raised in a polluted environment have diminished lung function, but the relationship of this loss of function to the development of asthma is not known. Outbreaks of asthma exacerbations have been shown to occur in relation to increased levels of air pollution, and this may be related to a general increase in the level of pollutants or to specific allergens to which individuals are sensitized. However, the role of pollutants in the development of asthma is less well defined. Similar associations have been observed in relation to indoor pollutants, e.g., smoke and fumes from gas and biomass fuels used for heating and cooking, molds, and cockroach infestations (Sarnat & Holguin 2007; Sharma et al. 2007).

chial hyperreactivity should not be prescribed ophthalmic β-adrenergic blockers (Lama 2002). Despite their widespread use for the treatment of asthmatic exacerbations, systemic glucocorticoids (particularly hydrocortisone, prednisone, and methylprednisolone) may very rarely cause worsening of asthma control and even anaphylaxis possibly via an IgE-mediated mechanism. This event is more common in asthmatics with a history of aspirininduced asthma (Sheth et al. 2006). Conversely, there is little evidence that any of these drugs can cause asthma.

Contributing factors Contributing factors may increase the probability of asthma exacerbation upon exposure to a causal factor or even the susceptibility to developing asthma.

Diet

Respiratory infections

The role of diet, particularly breast-feeding, in relation to the development of asthma has been extensively studied and, in general, the data reveal that infants fed formulas of intact cow’s milk or soy protein have a higher incidence of wheezing illnesses in early childhood compared with those fed breast milk. Some data also suggest that certain characteristics of Western diets, such as increased use of processed foods and decreased antioxidant (in the form of fruits and vegetables), increased n-6 polyunsaturated fatty acid (found in margarine and vegetable oil), and decreased n-3 polyunsaturated fatty acid (found in oily fish) intakes have contributed to the recent increases in asthma and atopic disease (Devereux & Seaton 2005; Friedman & Zeiger 2005).

It is well established that viral respiratory infections can trigger exacerbations in asthmatic patients, particularly in children but also in adults. Clinical studies performed using polymerase chain reaction suggest that the proportion of virusinduced asthma exacerbations is likely be around 80–85% in school-age children and 60–75% in adults of the total number of exacerbations, rhinovirus being the virus type most frequently identified, followed much more rarely by influenza, enterovirus, and RSV (Caramori et al. 2006). Respiratory viral infections not only contribute substantially to asthma morbidity in mild-moderate asthma, but they also appear to be associated with more severe exacerbations. In fact, upper respiratory viral infections are strongly associated in time with hospital admissions for asthma in both adults and children (Caramori et al. 2006). Winter peaks in asthma mortality have been observed in adults over 45 years of age, suggesting that respiratory virus infections, which are more frequent in winter, may also precipitate asthma deaths in these age groups (Caramori et al. 2006). Respiratory viruses can act synergistically with other factors that cause asthma exacerbations such as allergic sensitization and air pollution (Contoli et al. 2005). RSV, rhinovirus, coronavirus, and influenza virus have been implicated, with rhinovirus being involved in the majority of the exacerbations of asthma in children and adults (Caramori et al. 2006). As opposed to the important role of viral infection in exacerbations of asthma, there is no evidence that viral respiratory infections can actually cause the development of asthma (Lambrecht & van Rijt 2006). However, there is some evidence that viral respiratory infections are temporarily associated with the development of asthma, particularly in childhood (Oh 2006). Increased asthma hospitalization rates of children and adults, particularly in the early fall, have been observed to follow school vacations. A recent study by Johnston et al. (2006) sought to determine the sequence of timing of September asthma hospitalization epidemics in children and adults and whether school-age children were the primary source of

Drugs In addition to aspirin and nonsteroidal antiinflammatory agents, many other drugs (e.g., β-adrenergic blockers, angiotensin-converting enzyme inhibitors, opiates, Nacetylcysteine, intravenous adenosine, amiodarone, inhaled amphotericin, metoclopramide, pentamidine, dipyridamole; for the most recent list please consult http://www.pneumotox. com/index.php?lg=en&nf=) can exacerbate asthma in some susceptible patients and rarely even cause asthma death. Aspirin-induced asthma in adults is more prevalent than previously suggested (Stevenson & Szczeklik 2006). When there is clinical necessity to use aspirin or a nonsteroidal antiinflammatory drug and there is uncertainty about safety, oral provocation testing should be performed (Jenkins et al. 2004). Cardioselective beta-blockers do not usually produce clinically significant adverse respiratory effects in patients with mild to moderate asthma. Given their demonstrated benefit in conditions such as heart failure, cardiac arrhythmias, and hypertension, cardioselective beta-blockers should not be withheld from patients with mild to moderate asthma if they is not causing an objective worsening of asthma control (Salpeter et al. 2002). In contrast, patients with bron-

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transmission of agents that caused them. September epidemics of asthma hospitalizations in Canada were found to have a precise relationship to school return after the summer vacation and the authors speculated that school-age children may transmit the agents responsible for the epidemic to adults. The role of bacterial infections in asthma remains unclear, but there are some data for a potential role of Mycoplasma pneumoniae (Martin 2006) and Chlamydia pneumoniae (Johnston 2006) as triggers of exacerbations in asthmatic patients and in the development of chronic airflow limitation (Johnston & Martin 2005).

underlined that successful short-term treatment with a proton pump inhibitor in patients suspected of having gastroesophageal reflux disease does not confidently establish the diagnosis when the disease is defined by currently accepted reference standards (Numans et al. 2004). For these reasons the diagnosis of GER and its relationship to asthma worsening must be established in each individual patient by simultaneous 24-hour monitoring of esophageal pH and pressure and PEF.

Clinical features Rhinitis, nasal polyposis and sinusitis Upper airway diseases such as allergic rhinitis, nasal polyposis, and chronic sinusitis are sometimes associated with asthma and are considered risk factors for exacerbations of asthma (GINA 2006). Treatment of allergic rhinitis is often associated with a concomitant improvement of asthma (Slavin 2006; Thomas 2006). The IgE-mediated airway inflammation of allergic rhinitis is now considered as one airway disease, with manifestation of symptoms in upper, lower or global airway, the so-called atopic march, as highlighted in the “Allergic Rhinitis and its Impact on Asthma” (ARIA) document (Bachert et al. 2002; Spergel 2005). For this reason patients with allergic rhinitis have an increased risk of developing asthma (Koh & Kim 2003). Nasal polyposis is often associated with allergic rhinitis and asthma, and particularly with aspirin-induced asthma. Such subjects are usually nonatopic and older than 40 years (Corren & Kachru 2007). The role played by chronic sinus disease in asthma is only partially understood, largely because of deficits in the clinical classification and in knowledge of the pathophysiologic pathways (Bachert et al. 2006). Chronic sinusitis is sometimes associated with the presence of asthma. Effective treatment of chronic sinusitis has been reported to be associated with improvement of asthma (Smart 2006).

Gastroesophageal reflux Gastroesophageal reflux (GER) occurs in a large majority of patients with asthma, particularly those with severe disease. However, the reverse does not seem to be true: asthma is not more prevalent among individuals with GER compared with the normal population (Mathew et al. 2004). Furthermore because some antiasthma drugs such as theophylline and β2 agonists may cause GER, it remains unclear whether GER is the cause or the consequence of asthma and/or of its treatment (Ruigomez et al. 2005; Hancox et al. 2006; Nordenstedt et al. 2006). A strong criticism against a linkage between asthma and GER is that most studies have shown that medical management of GER with antireflux therapy did not consistently improve pulmonary functions, asthma symptoms, nocturnal asthma, or result in any decrease in the use of asthma medication (Mathew et al. 2004). Also it should be

The common disease asthma is probably not a single disease, but rather a complex of multiple separate syndromes that overlap. Although clinicians have recognized these different clinical phenotypes for many years, they are still poorly characterized in their pathophysiology (Wenzel 2006).

Symptoms The characteristic symptoms of asthma are episodic breathlessness and wheezing, often associated with cough and chest tightness, worse particularly at night and in the early morning (Sutherland 2005). The most recent guidelines stress that the clinical manifestations of asthma can be controlled with appropriate treatment (GINA 2006). Other symptoms of asthma include wheezing and cough induced by exercise (Bonini et al. 2006) or by thermic stimuli, such as exposure to cold dry air or fog (Cockcroft & Davis 2006), cleaning products (Rosenman 2006), active or passive tobacco smoke (Thomson et al. 2004), air pollutants (Wong & Lai 2004), odors (Opiekun et al. 2003) or by emotional responses, such as fear, crying and laughing (Rosenkranz et al. 2005). These are signs of bronchial hyperresponsiveness (Cockcroft & Davis 2006) and should also be investigated. Some asthmatic patients have no symptoms until the lower airways are severely narrowed. Thus, breathlessness and wheezing cannot be the only subjective diagnostic criteria, since individual tolerance to a given degree of dyspnea is variable. Decreased levels of perception of airflow limitation may be a risk factor associated with lifethreatening asthma (Scano & Stendardi 2006). The onset, duration, and frequency of asthmatic symptoms should be carefully investigated.

More clinical phenotypes of asthma Cough variant asthma A group of patients with asthma have chronic, usually nonproductive, cough as their predominant, if not only, symptom (Abouzgheib et al. 2007). Frequently cough occurs at night, whereas examinations during the day are normal. Documentation of variability in lung function, of sputum eosinophilia, and a bronchoprovocation challenge to detect airway hyperresponsiveness are particularly important. The beneficial effect of nocturnal administration of long-acting bronchodilators

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may also help to confirm the diagnosis. This clinical presentation is called cough variant asthma and it is a common cause of persistent cough in the general population (Dicpinigaitis 2006; Abouzgheib et al. 2007). However, it should be noted that patients with cough variant asthma may, over time, develop wheezing and the other asthma symptoms (Matsumoto et al. 2006). Cough variant asthma should be not confused with chronic nonasthmatic eosinophilic bronchitis. These rare patients complain of chronic productive cough and have sputum eosinophilia but normal lung function, normal PEF variability, and normal airway responsiveness (Brightling 2006a).

Brittle asthma The term “brittle asthma” was first used to describe patients with asthma who maintained a wide variation in PEF despite treatment with high doses of inhaled glucocorticoids (TurnerWarwick 1977). Then brittle asthma was defined as diurnal PEF variability (amplitude as percent maximum) of more than 40% for more than 50% of the time (e.g., 16 days a month) despite maximal medical treatment, namely high doses of inhaled glucocorticoids with repeated doses of inhaled bronchodilator, often by nebulizer, and maintenance or courses of oral glucocorticoids (O’Driscoll et al. 1988; Ayres et al. 1998). Premenstrual asthma may be one such example as some women develop a marked drop in PEF in the few days before menstruation and sometimes these premenstrual exacerbations are so severe that they necessitate ventilation (Beynon et al. 1988; Ayres et al. 1998). To allow for this, the following classification of brittle asthma has been suggested. 1 Type 1 brittle asthma: characterized by maintained wide PEF variability (> 40% diurnal variation for > 50% of the time over a period of at least 150 days) despite considerable medical therapy including a dose of inhaled glucocorticoids of at least 1500 μg of beclomethasone (or equivalent). 2 Type 2 brittle asthma: characterized by sudden acute exacerbations occurring in less than 3 hours without an obvious trigger on a background of apparently normal airway function or well-controlled asthma (Ayres et al. 1998). However, this definition does not take into account those patients who are subject to sudden severe life-threatening exacerbations often on a background of apparently good asthma control. Near-fatal asthma (NFA) represents the most severe clinical phenotype of asthma, outside of fatal asthma. There are no universally agreed diagnostic criteria for NFA, which is usually defined by the appearance of various events such as cardiorespiratory arrest, hypercapnia, acidemia, the need for orotracheal intubation and mechanical ventilation, or admission to an intensive care unit. However, an episode of hypercapnia occurs 10 to 20 times more frequently than the need for orotracheal intubation and mechanical ventilation and does not necessarily imply a poor prognosis. Similar to fatal asthma, most of these patients have slow-onset NFA and, on average, no more than one-third of the patients have

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rapid-onset NFA. When compared with mild to moderate asthmatics, a recent study carried out on the ENFUMOSA cohort found that, in stable conditions, patients with an NFA attack in the previous 5 years could not be distinguished from patients with mild-to-moderate asthma, while they were different from severe asthmatics in terms of both lung function and airway inflammation. The risk factor that characterizes this group of patients is reduced usage of prophylactic corticosteroids (Romagnoli et al. 2007).

Overlap between asthma and COPD Both asthma and COPD are major chronic obstructive airway diseases that involve underlying airway inflammation. COPD is characterized by airflow limitation that is not fully reversible, is usually progressive, and is associated with an abnormal inflammatory response of the lungs to noxious particles or gases. Individuals with asthma who are exposed to noxious agents (particularly cigarette smoking) may develop fixed airflow limitation and a mixture of “asthma-like” inflammation and “COPD-like” inflammation. Thus, even though asthma can usually be distinguished from COPD, in some individuals who develop chronic respiratory symptoms and fixed airflow limitation, it may be difficult to differentiate the two diseases (GINA 2006). From the functional point of view, nonsmoking asthmatic patients may have a significantly greater decline in FEV1 compared with nonasthmatic subjects (Lange et al. 1998) and may develop chronic irreversible (fixed) airflow limitation (Fabbri et al. 1998, 2003). This has been related to the physiologic consequences of chronic airway inflammation causing airway remodeling, a collective term to include large airways epithelial basement membrane thickening, airway wall edema, and airway smooth muscle hyperplasia and hypertrophy (Postma & Timens 2006; Hackett & Knight 2007). However, these lesions are all potentially reversible and their long-term clinical consequences, particularly in nonsmokers, are unknown. There is little radiologic evidence for lung destruction (pulmonary emphysema), which is potentially irreversible, in nonsmoking asthmatics, including in patients with severe persistent asthma (Gelb et al. 2002; Jensen et al. 2002; Fabbri et al. 2003) and a large longitudinal study suggests that FEV1 does not decline more rapidly in asthmatics or in patients with asthma and COPD compared with nonasthmatics (Sherrill et al. 2003). Furthermore, asthma is an uncommon cause of severe chronic respiratory failure necessitating long-term domiciliary oxygen therapy in nonsmokers (Caramori et al. 2005). Globally these data support the current consensus that asthma and COPD are different diseases with differing stages of severity (Global Initiative for Chronic Obstructive Lung Disease 2001; GINA 2006). However, some patients with chronic persistent asthma, both smokers and nonsmokers, may exhibit an irreversible component of airflow limitation and fail to achieve normal lung function despite adequate therapy with systemic and

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inhaled glucocorticoids (Fabbri et al. 2003). For these reasons a correct differential diagnosis between asthma and COPD requires methodical collection of a previous history of asthma and a test of reversibility to both bronchodilators and inhaled glucocorticoids (GINA 2002, 2006; Fabbri et al. 2003). Active cigarette smoking is a well-recognized environmental factor that could be involved in explaining the variability in individual therapeutic responses to drugs used to treat asthma, in addition to adherence to therapy and genetic factors (Thomson 2007). A recent multicenter, placebocontrolled, double-blind, double-dummy, crossover trial aimed to determine if the response to an inhaled corticosteroid or a leukotriene receptor antagonist was attenuated in individuals with asthma who smoked. The study showed that in subjects with mild asthma who smoked, the response to inhaled corticosteroids was attenuated, suggesting that adjustments to standard therapy may be required to attain asthma control. A greater improvement was seen in some outcomes in smokers treated with montelukast, suggesting that leukotrienes may be important in this setting (Lazarus et al. 2007). This study confirms the presence of corticosteroid insensitivity in patients with asthma who smoke and suggests that leukotriene modifiers may be beneficial in these patients. Randomized trials are needed to further investigate how to best manage this group of patients (Lazarus et al. 2007).

Severe refractory asthma The term “severe refractory asthma” (SRA) in adults applies to patients who remain difficult to control despite extensive reevaluation of diagnosis and management and following an observational period of at least 6 months by an asthma specialist. Factors that influence asthma control, such as environmental exposures, comorbidities, adherence, and inhalation technique, should be recognized and adequately addressed prior to confirming the diagnosis of SRA. Validated health status and asthma control questionnaires, lung function, assessment of airway inflammation, and history of exacerbations should be recorded regularly during follow-up (Chanez et al. 2007). The majority of patients with asthma have mild to moderate disease that can be well controlled with standard treatment, including the regular use of inhaled corticosteroids combined with short- or long-acting inhaled β2 agonists. There is, however, a subset of asthmatic patients in whom even high doses of these drugs fail to control the disease. Indeed it is well recognized that some asthma patients have more severe disease than others and early descriptions identified a subset of asthmatic patients whose disease was apparently unresponsive to corticosteroids. In the literature these patients are labeled as having “difficult-to-treat asthma,” “therapy-resistant asthma,” “steroid-dependent asthma,” “brittle asthma,” and so on. In 1999, a European Respiratory Society (ERS) Task Force adopted the term “difficult asthma,” which was defined as “asthma, poorly controlled in terms of chronic symptoms, with episodic exacerbations, persistent and variable airway

obstruction and continued requirement for short-acting beta2-agonists and a reasonable dose of inhaled corticosteroids.” In 2000, the American Thoracic Society (ATS) defined “refractory asthma” using one or two major and two or more minor criteria, which included medication requirements, asthma symptoms, frequency of asthma exacerbations, and degree of airflow limitation. The ERS and ATS definition differ from the recently updated GINA guideline for asthma management and prevention in that they include medication criteria. According to the ATS definition, patients who need oral corticosteroids or high-dose inhaled corticosteroids to remain under control, as well as patients with ongoing asthma symptoms despite appropriate maintenance therapy, should be regarded as having severe asthma. All these definitions include an assessment of disease control, exacerbating factors/ comorbidities, and response to treatment, which may influence control and treatment requirements. Inhaled corticosteroids and bronchodilators are the mainstay of treatment for SRA. Despite intensive multidrug treatment, many patients with SRA remain uncontrolled and there is urgent need for new and more effective medications (Chanez et al. 2007).

Measuring the level of asthma control outside the exacerbation Examples of validated instruments for assessing the level of asthma control are the Asthma Control Test (ACT) (www. asthmacontrol.com) (Nathan et al. 2004), the Asthma Control Questionnaire (ACQ) (www.qoltech.co.uk/Asthma1.htm) (Juniper et al. 1999), the Asthma Therapy Assessment Questionnaire (ATAQ) (www.ataqinstrument.com) (Vollmer et al. 1999), and the Asthma Control Scoring System (Boulet et al. 2002). The most recently released GINA guidelines have proposed a working scheme to measure the level of asthma control in a given week, as summarized in Table 76.2, incorporating both symptoms, lung function, and rescue medications (GINA 2006). This scheme is based on expert opinion and has not been validated (GINA 2006).

Asthma exacerbations Exacerbations of asthma (asthma attacks or acute asthma) are episodes of progressive increase in shortness of breath, cough, wheezing, or chest tightness, or some combination of these symptoms. Respiratory distress is common. Exacerbations are characterized by decreases in expiratory airflow that can be quantified by measurement of lung function (PEF or FEV1). These measurements are more reliable indicators of the severity of airflow limitation than is the degree of symptoms. However, the degree of symptoms may be a more sensitive measure of the onset of an exacerbation because the increase in symptoms usually precedes the deterioration in peak flow rate. Still, a minority of patients perceive symptoms poorly, and may have a significant decline in lung function without a significant change in symptoms. This situation especially affects patients with a history of NFA and also appears

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to be more likely in males. Strategies for treating exacerbations, though generalizable, are best adapted and implemented at a local level. Severe exacerbations are potentially lifethreatening, and their treatment requires close supervision. Patients with severe exacerbations should be encouraged to see their physician promptly or, depending on the organization of local health services, to proceed to the nearest clinic or hospital that provides emergency access for patients with acute asthma. Close objective monitoring (PEF) of the response to therapy is essential. The primary therapies for exacerbations include (in the order in which they are introduced, depending on severity) repetitive administration of rapid-acting inhaled bronchodilators, early introduction of systemic glucocorticosteroids, and oxygen supplementation. The aims of treatment are to relieve airflow obstruction and hypoxemia as quickly as possible, and to plan the prevention of future relapses. Patients at high risk of asthma-related death require closer attention and should be encouraged to seek urgent care early in the course of their exacerbations. These patients include those: • with a history of NFA requiring intubation and mechanical ventilation; • who have had hospitalization or an emergency care visit for asthma in the past year; • currently using or have recently stopped using oral glucocorticosteroids; • not currently using inhaled glucocorticosteroids; • overdependent on rapid-acting inhaled β2 agonists, especially those who use more than one canister of salbutamol (or equivalent) monthly; • with a history of psychiatric disease or psychosocial problems, including the use of sedatives; • with a history of noncompliance with an asthma medication plan. Response to treatment may take time and patients should be closely monitored using clinical as well as objective measurements. The increased treatment should continue until measurements of lung function (PEF or FEV1) return to their previous best (ideally) or plateau, at which time a decision to admit or discharge can be made based on these values. Patients who can be safely discharged will have responded within the first 2 hours, at which time decisions regarding patient disposition can be made. The severity of the exacerbation determines the treatment administered. Indices of severity, particularly PEF (in patients older than 5 years), pulse rate, respiratory rate, and pulse oximetry, should be monitored during treatment. Fig. 76.1 and Fig. 76.1 provide a guide to the severity of an exacerbation of asthma at the time the examination is made (GINA 2006). Assessment of the severity of asthma exacerbations in the hospital requires information about potential triggering events, all current medication, and prior hospitalizations and emergency department visits for asthma. The physical examination will assess severity of the exacerbation (Fig. 76.1)

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and the presence of complications (e.g., pneumonia, atelectasis, pneumothorax, or pneumomediastinum). PEF and/or FEV1 should be measured at least hourly, with initial measurement made before treatment, if possible. Arterial blood gas measurement provides important information for assessing the severity of asthma exacerbations. Gas exchange abnormalities, as reflected by arterial hypoxemia, are common in asthma patients during a severe exacerbation. The degree of ventilation–perfusion mismatching correlates poorly with clinical findings and indices of airflow limitation (FEV1 or FEF25–75), and thus alterations of arterial blood gases may be present even when symptoms are mild and/or spirometric indices are normal or borderline. During a mild asthma exacerbation, the usual pattern consists of a normal PaO2, associated with hypocapnia and slight respiratory alkalosis due to compensatory hyperventilation. In additon to the invasive measurement of arterial blood gases (Gunen et al. 2006), the noninvasive measurement of pulse oximetry (SO2) may also be useful, both in the community and in the hospital, in assessing the severity and in predicting the outcome of asthma exacerbations, both in children and adults (Cunningham & McMurray 2006). Normocapnia or hypercapnia associated with normoxemia or hypoxemia are poor prognostic evidence of a severe asthmatic exacerbation. Respiratory failure with hypoxemia, hypercapnia and respiratory acidosis occurs in a minority of patients (McFadden 2003). Assessment of the severity of asthma exacerbations is also used for instructing patients to manage their own asthma, and particularly for modifying therapy according to degree of control of asthma. Interestingly, a simple self-management plan based on a PEF records or symptoms score given to the patient in a credit card improves overall symptoms and lung function and reduces exacerbations (D’Souza et al. 1994). Optimal self-management allowing for optimization of asthma control by adjustment of medications may be conducted by either self-adjustment with the aid of a written action plan or by regular medical review. Individualized written action plans based on PEF are equivalent to action plans based on symptoms. Reducing the intensity of self-management education or level of clinical review may reduce its effectiveness (Gibson et al. 2003; Powell & Gibson 2003; Gibson & Powell 2004).

Diagnosis To establish the diagnosis of asthma, the clinician should determine that symptoms compatible with asthma are present, airflow limitation is at least partially reversible, and alternative diagnoses are excluded. The minimal requirements to establish the diagnosis of asthma are represented by symptoms (see Clinical features) and by measures of airflow limitation. The most recent guidelines stress that lung function testing by spirometry or PEF continues to be recommended

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Table 76.3 Severity of asthma exacerbations. The presence of several parameters, but not necessarily all, indicates the general classification of the exacerbation. (From GINA 2006, with permission.) Respiratory arrest imminent

Mild

Moderate

Severe

Breathless

Walking Can lie down

Talking Infant: softer shorter cry; difficulty feeding Prefers sitting

At rest Infant stops feeding Hunched forward

Talks in

Sentences

Phrases

Words

Alertness

May be agitated

Usually agitated

Usually agitated

Respiratory rate

Increased Increased Normal rates of breathing in awake children < 2 months: < 60/min 2–12 months: < 50/min 1–5 years: < 40/min 6–8 years: < 30/min

Often > 30/min

Accessory muscles and suprasternal retractions

Usually not

Usually

Usually

Paradoxical thoracoabdominal movement

Wheeze

Moderate, often only end expiratory

Loud

Usually loud

Absence of wheeze

Pulse/min

< 100 100–120 Guide to limits of normal pulse rate in children Infant (2–12 months): < 160/min Preschool (1–2 years): < 120/min School age (2–8 years): < 110/min

> 120

Bradycardia

Pulsus paradoxus

Absent < 10 mmHg

May be present 10–25 mmHg

Often present > 25 mmHg (adult) 20–40 mmHg (child)

Absence suggests respiratory muscle fatigue

PEF after initial bronchodilator (% predicted or % personal best)

Over 80%

Approximately 60–80%

< 60% predicted or personal best (< 100 L/min adults) or response lasts < 2 hours

Pao2 (on air)*

Normal; test not usually necessary

> 60 mmHg

< 60 mmHg Possible cyanosis

and/or Paco2*

< 45 mmHg

< 45 mmHg

> 45 mmHg; possible respiratory failure (see text)

Sao2 (on air)*

> 95% 91–95% < 90% Hypercapnia (hypoventilation) develops more readily in young children than in adults and adolescents

Drowsy or confused

* Kilopascals are also used internationally; conversion would be appropriate in this regard.

as an aid to diagnosis and monitoring. Measuring the variability of airflow limitation is given increased prominence, as it is key to both asthma diagnosis and the assessment of asthma control (GINA 2006). Recommended methods for establishing the diagnosis are a detailed medical history, physical examinatin, spirometry

and additional studies when it is necessary to exclude alternate diagnoses.

Medical history Several attempts have been made to find the most valid symptoms-based questions to diagnose asthma, particularly

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Initial Assessment • History, physical examination (auscultation, use of accessory muscles, heart rate, respiratory rate, PEF or FEV1, oxygen saturation, arterial blood gas if patient in extremis)

• • • •

Initial Treatment Oxygen to achieve O2 saturation ≥ 90% (95% in children) Inhaled rapid-acting b2-agonist continuously for one hour Systemic glucocorticosteroids if no immediate response, or if patient recently took oral glucocorticosteroid, or if episode is severe Sedation is contraindicated in the treatment of an exacerbation

Reassess after 1 Hour Physical examination, PEF, O2 saturation and other tests as needed

Criteria for Severe Episode: • History of risk factors for near fatal asthma • PEF < 60% predicted/personal best • Physical exam: severe symptoms at rest, chest retraction • No improvement after initial treatment

Criteria for Moderate Episode: • PEF 60–80% predicted/personal best • Physical exam: moderate symptoms, accessory muscle use Treatment: • Oxygen • Inhaled b2-agonist and inhaled anticholinergic every 60 min • Oral glucocorticosteroids • Continue treatment for 1–3 hours, provided there is improvement

Treatment: • Oxygen • Inhaled b2-agonist and inhaled anticholinergic • Systemic glucocorticosteroids • Intravenous magnesium

Reassess after 1–2 Hours

Good Response within 1–2 Hours: • Response sustained 60 min after last treatment • Physical exam normal: no distress • PEF > 70% • O2 saturation > 90% (95% children)

Incomplete Response within 1–2 Hours: • Risk factors for near fatal asthma • Physical exam: mild to moderate signs • PEF < 60% • O2 saturation not improving

Poor Response within 1–2 Hours: • Risk factors for near fatal asthma • Physical exam: symptoms severe, drowsiness, confusion • PEF < 30% • PCO2 > 45 mmHg • PO2 < 60 mmHg

Admit to Acute Care Setting • Oxygen • Inhaled b2-agonist ± anticholinergic • Systemic glucocorticosteroid • Intravenous magnesium • Monitor PEF, O2 saturation, pulse

Admit to Intensive Care • Oxygen • Inhaled b2-agonist + anticholinergic • Intravenous glucocorticosteroids • Consider intravenous b2-agonist • Consider intravenous theophylline • Possible intubation and mechanical ventilation

Reassess at intervals Improved: Criteria for Discharge Home • PEF > 60% predicted/personal best • Sustained on oral/inhaled medication Home Treatment: • Continue inhaled b2-agonist • Consider, in most cases, oral glucocorticosteroids • Consider adding a combination inhaler • Patient education: Take medicine correctly Review action plan Close medical follow-up

Poor Response (see above): • Admit to intensive care Incomplete response in 6–12 hours (see above) • Consider admission to intensive care if no improvement within 6–12 hours

Improved (see opposite)

Fig. 76.1 Management of asthma exacerbations in an acute-care setting. (From GINA 2006, with permission.)

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for epidemiologic studies. A postal questionnaire developed by the International Union Against Tuberculosis and Lung Diseases and suitable for epidemiologic studies of asthma is shown below (Burney et al. 1989). Five simple questions are included in the questionnaire, which could be used also in the single patient. 1 Have you had wheezing or whistling in your chest at any time? 2 Have you had an attack of shortness of breath that came on following strenuous activity at any time? 3 Have you woken up with an attack of wheezing at any time? 4 Have you woken up with an attack of cough at any time? 5 Have you had an attack of shortness of breath that came on during the day when you were at rest at any time? Self-reported asthma from a questionnaire has a sensitivity of 36% and a specificity of 94% if one uses airway hyperresponsiveness as the gold standard of asthma. The sensitivity increases to 68% and the specificity to 94% if one uses the clinical diagnosis as the gold standard of asthma (Toren et al. 1993). The relationship between the answers to a questionnaire and airway hyperresponsiveness has been reported (Shaw et al. 1992; Venables et al. 1993). The questions which more strongly predict the presence of airway hyperresponsiveness regard the appearance of symptoms in response to exercise or dusty environment and/or development of symptoms at night or early in the morning. This questionnaire has a good sensitivity (65– 91%) and specificity (85–96%). Even if these questionnaires have been designed for epidemiologic research, the use of some questions on current respiratory symptoms can be useful to assess the diagnosis of asthma in the single individual.

Physical examination Although physical examination remains quite an important diagnostic tool in the diagnosis and assessment of severity of asthma exacerbations, it is of little help in the diagnosis of intermittent and mild asthma. Furthermore, some patients with asthma may have normal auscultation but significant airflow limitation when measured by spirometry. Tachypnea, hyperinflation of the chest, prolonged expiration, use of accessory respiratory muscles, hyperresonance of percussion note, inspiratory and/or expiratory rhonchi, and wheezing reflect the presence of airflow limitation. However it should be stressed that these signs are neither sensitive enough to exclude a diagnosis of asthma nor specific to confirm a diagnosis of asthma by their presence alone (Maitre et al. 1995; Meslier et al. 1995; Yernault & Bohadana 1995). Interestingly, properly standardized assessment of lung sounds may detect morphologic changes in the airways occurring in asthma (Schreur et al. 1994). Physical findings that increase the probability of asthma can be detected in the upper respiratory tract and skin. Since asthma is often associated with rhinitis, increased nasal

secretion, blocked nose, mucosal swelling, and nasal polyps may be present. Atopic eczema and other manifestations of an allergic skin condition should be investigated.

Measures of airflow limitation The common symptoms described above are not in themselves diagnostic. Patients’ perception of airflow limitation is highly variable and lung function tests sometimes reveal limitation much more severe than that which would have been estimated from the medical history. On the other hand, physical examination is not a reliable means of assessing the degree of airflow limitation or predicting whether the limitation is reversible (Expert Panel Report of the National Heart, Lung and Blood Institute (2007). The objective assessment of airflow limitation greatly enhance diagnostic confidence. However, pulmonary function measures often do not correlate directly with symptoms or other measures of disease control (Expert Panel Report of the National Heart, Lung and Blood Institute (2007). It is therefore important to use multiple parameters for a comprehensive assessment of asthma. It is recommended that spirometry is used for diagnostic purposes over measurements by a peak flow meter because there is wide variability in the published predicted PEF reference values and normative brand-specific values currently are not available for most brands.

Spirometry Among the wide range of different methods used to assess the degree of airflow limitation, the most common indices are the volume of air forcibly exhaled in 1 s from full inspiration (FEV1), forced vital capacity (FVC), forced expiratory volume in 6 s (FEV6), and the ratios FEV1/FVC or FEV1/FEV6. Lung volumes and flow can be measured with different types of spirometers (American Thoracic Society 1991; Quanjer et al. 1993; Miller et al. 2005; Wanger et al. 2005). FEV1 is the single best measure for assessing severity of airflow limitation. The FEV1/FVC ratio provides an early and sensitive indication of airflow limitation. The FEV1/FVC ratio is increasingly used as a measure for diagnosis because it distinguishes between restrictive and obstructive disease (American Thoracic Society 1991; Quanjer et al. 1993; Miller et al. 2005; Wanger et al. 2005). Spirometry is generally valuable in children over age 4. Healthy young children complete exhalation of their entire vital capacity in a few seconds, but there is evidence that indices derived from exhalations with forced expiratory times of less than 1 s may have clinical usefulness. At present, there are insufficient data to recommend the use of FEV0.5 or FEV0.75. Complete exhalation can take much longer in older patients especially in those who have airflow limitation. In these patients, sustaining a maximal expiratory effort for the time necessary to meet the ATS/ERS criteria for acceptability may be uncomfortable or associated with light-headedness (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). This accounts for the

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interest in measurement of FEV6 as a substitute for FVC. In adults, FEV6 has been shown to be equivalent to FVC in identifying obstructive and restrictive patterns, using the ATS algorithm, and to be more reproducible and less physically demanding than FVC (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). Airflow limitation is indicated by a reduction in the values of both FEV1 and the FEV1/FVC (or FEV1/FEV6) relative to reference or predicted values. In the USA, ethnically appropriate National Health and Nutrition Exhamination Survey (NHANES) III reference equations are recommended for those aged 8–80 years. For children aged under 8 years, the equations of Wang et al. (1993) should be used. Currently there is no specific set of equations recommended for use in Europe (ATS/ERS Task Force) (Pellegrino et al. 2005). Spirometry is recommended in the initial assessment of most patients with suspected asthma, and periodically in selected patients to confirm home PEF measurements made with a peak flow meter, but subsequent measurements of PEF are sufficient to follow most adult patients in assessing the severity of symptoms and making therapeutic decisions. Indeed FEV1 and PEF are strongly correlated and have almost identical variability in asthma (Troyanov et al. 1994). However, particularly in children, PEF may underestimate the severity of asthma exacerbations (Sly et al. 1994), and most peak flow meters are inaccurate (Quanjer et al. 1997). Consequently, patients should be advised of these potential biases, and spirometry should be performed in all subjects in whom PEF values do not fit with the clinical severity of asthma.

Reversibility of airflow limitation induced by a single dose of a rapid-acting bronchodilator When pulmonary function measures are obtained, measuring pulmonary function before and after bronchodilator treatment to determine reversibility is recommended. The degree of airway reversibility correlates with airway inflammation, as measured by sputum eosinophilia and fractional exhaled nitric oxide (FENO) (Expert Panel Report of the National Heart, Lung and Blood Institute 2007), with the risk of developing fixed airflow limitation and with the loss of lung function. In addition, the postbronchodilator measures can be used to follow lung growth patterns over time (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). The response to a single dose of rapid-acting bronchodilator should be measured. Usually a β2 agonist is used (e.g., four puffs of salbutamol, equivalent to 400 μg of salbutamol); alternatively, an anticholinergic (e.g., ipratropium) is used. Lung function measurements are usually repeated 15–30 min after inhalation of the bronchodilator. A 12% increase in FEV1 and > 200 mL in absolute value is usually assumed to be evidence of reversible airflow limitation (Global Initiative for Chronic Obstructive Lung Disease 2001). In some patients, and particularly young patients and athletes with a history of symptoms, a bronchodilator reversibility test is recommended

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even when baseline lung function is normal, because their normal lung function might be significantly higher than the predicted value. In these cases, a marked improvement in lung function might reveal airflow limitation even if the FEV1/FVC ratio is within normal limits. Some patients who have symptoms and signs of asthma may not demonstrate reversibility until after a 2–3 week course of oral corticosteroid treatment is administered (see below). In addition, spirometry measured after a short course of corticosteroids and acute administration of a bronchodilator may not indicate the patient’s best achievable lung function. Therefore, follow-up spirometric assessment is indicated when asthma control improves. Office-based physicians who care for asthma patients should have access to spirometry, which is useful in both diagnosis and monitoring of the disease. Assessment in a specialized pulmonary function laboratory should be performed when office spirometry shows severe abnormalities or if there are questions regarding test accuracy or interpretation.

Additional lung function tests In subjects with atypical history and symptoms, or in subjects with symptoms of asthma and normal spirometry or airflow limitation not reversible by a single dose of a rapid-acting bronchodilator, additional confirmatory tests are recommended, including the measurement of airway responsiveness to bronchoconstrictor stimuli and the reversibility of airflow limitation after a course of glucocorticoids. PEF is less reliable than spirometry in diagnosing asthma and has not been found useful for classifying severity of impairment due to asthma, but it may serve as a useful tool for monitoring asthma over time (Expert Panel Report of the National Heart, Lung and Blood Institute 2007).

Measurement of airway responsiveness Airway hyperresponsiveness is an exaggerated response to a large variety of physical, chemical and pharmacologic bronchoconstrictor stimuli (Sterk et al. 1993; Cockcroft & Davis 2006). In other words airways narrow too easily and too much to a large variety of stimuli (Woolcock 1994). Because in both asthmatic and nonasthmatic subjects airflow limitation may itself be the cause of an exaggerated response to bronchoconstrictor stimuli, the measurement of airway hyperresponsiveness for asthma diagnosis is usually restricted to subjects with normal lung function. The degree of airway responsiveness can be measured in the laboratory using various stimuli and methods. Because the airways respond to a variety of stimuli which, at variance with allergens and sensitizing agents, may cause airflow limitation even in normal subjects, airway hyperresponsiveness is sometimes referred to as nonspecific. However, even nonspecific stimuli act through very specific mechanisms and some of these stimuli (e.g., exercise, ultrasonically nebulized distilled water) cause airflow limitation almost exclusively in

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asthmatics, thus the term “nonspecific” should be avoided. Airway responsiveness should be referred to the stimulus used to measure it. Bronchoconstrictor stimuli may be classified as causing airflow limitation by directly stimulating airway smooth muscle (e.g., methacholine and histamine; Cockcroft & Davis 2006) or indirectly by releasing pharmacologically active mediators (e.g., exercise, nebulized hypotonic or hypertonic solutions) or stimulating sensory nerves (e.g., metabisulfite, bradykinin), or a combination of both mechanisms (Joos et al. 2003). Unlike inhalation challenges with indirect stimuli, those with histamine and methacholine have been properly standardized and validated in patients with asthma and are safe, and thus should be preferred in the clinical evaluation of patients (Sterk et al. 1993; Crapo et al. 2000). Airway responsiveness is usually expressed as the amount of bronchoconstrictor stimulus able to change expiratory flows, i.e., the concentration or dose of the bronchoconstrictor agent that provokes (PC or PD) a significant decrease (usually 20% starting from the lower value between baseline or phosphate buffer saline inhalation) of FEV1 (PC20FEV1 or PD20FEV1). A PC20FEV1 methacholine or histamine greater than 16 mg/ mL or a PD20FEV1 methacholine or histamine greater than 16 mmol or 1.4 mg is usually considered to be normal (Sterk et al. 1993; Crapo et al. 2000). Although airway hyperresponsiveness is present in the large majority of asthmatics, a negative inhalation challenge with methacholine or histamine does not exclude the diagnosis of asthma, particularly in subjects with occupational asthma (Mapp et al. 1986). Patients with a history of asthma and normal airway responsiveness to methacholine or histamine should be kept under regular control. In a followup study, 33 (9%) of 334 subjects with suspected asthma but normal airway responsiveness developed asthma within 10 years. In contrast, up to 10% of normal asymptomatic subjects have an increased response to bronchoconstrictor stimuli and no evidence of asthma. In a 2-year follow-up study, up to 45% of asymptomatic subjects with clear-cut hyperresponsiveness to methacholine (PD20 < 3.2 mmol) developed asthma within 2 years, suggesting that airway hyperresponsiveness, in addition to being a feature of asthma, might be a risk factor for the development of asthma (Cockcroft & Davis 2006). Finally airway responsiveness may not reflect the severity of asthma and so far it has been disappointing in predicting exacerbation (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). Thus, it is not recommended as a relevant outcome measurement for the monitoring of asthma (Josephs et al. 1989; Muller et al. 1994; Perin et al. 1994).

Reversibility of airflow limitation induced by a course of glucocorticoids In patients with airflow limitation not relieved by a single dose of a rapid-acting bronchodilator in the past, a short (1–2 weeks)

trial with high doses of systemic glucocorticoids has been often used in the differential diagnosis of asthma and COPD (Callahan et al. 1991; Vestbo et al. 1999; Fabbri et al. 2003). Some patients with moderate or severe persistent asthma, and up to 10% of patients with stable COPD, show a significant improvement in airflow limitation that confirms the presence of an asthmatic component of airflow limitation (Callahan et al. 1991). It is possible that these patients have concomitant asthma, since both diseases are very common. In fact a clinical trial has shown that COPD patients with high BAL eosinophil counts and a thickened basement membrane improved their FEV1 by > 12% after 2 weeks of prednisolone (Chanez et al. 1997). Furthermore, in a trial of high-dose inhaled beclomethasone dipropionate (3 mg per day for 4 weeks) only those subjects with an asthma component showed any improvement in FEV1 (Nishimura et al. 1999). Although eosinophils may be detected in asthmatic and COPD airways, the proportion of patients with airway eosinophilia is greatly different in the two diseases. In both asthma and COPD the presence of airway eosinophilia is predictive of a better response to corticosteroid treatment. Most of the benefit of corticosteroids is found in severe exacerbations of both diseases, suggesting a similar role of these cells in airway diseases that are, in general, phenotypically dissimilar (Maestrelli 2007). However, evidence from the ISOLDE study suggests that prednisolone testing is an unreliable predictor of the benefit from inhaled fluticasone propionate in individual patients and that patients with COPD cannot be separated into discrete groups of corticosteroid responders and nonresponders (Burge et al. 2003). In conclusion, assessment of response to therapy is important, but there is inconsistency about its definition and measurement. When response is defined solely by FEV1, it can be influenced by disease activity independent of the intervention (Expert Panel Report of the National Heart, Lung and Blood Institute 2007).

PEF monitoring PEF is the highest flow obtained during a forced expiration starting immediately after a deep inspiration at total lung capacity. PEF can be measured using inexpensive and portable peak flow meters which can be mechanical or electronic. Daily monitoring of PEF over a period of time provides a simple, quantitative, and reproducible assessment of the existence and severity of airflow limitation. PEF measurements have some limitations. PEF is effort dependent, and proper training is required to obtain the best reproducible measurements from the individual. Also, PEF mainly reflects the caliber of large airways, and it may underestimate the degree of airflow limitation present in peripheral airways. Predicted values of PEF are related to gender, race, age and height, and normal values as well as normal limits of diurnal variability are available in the literature, but only for few brands (Quackenboss et al. 1991; Quanjer et al. 1997). Because of the wide interindividual variability, some subjects have PEF

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values very different from the average values of subjects with the same demographic characteristics, and some subjects with long-lasting asthma or other COPD may develop irreversible airflow limitation. For these two reasons, the patient’s personal best PEF measured during a period of monitoring while the patient is under effective antiasthma treatment is the most appropriate reference value for the patient’s action plan. PEF should be measured at least twice a day, in the morning on awakening and 12 hours later. Further measurements should be recommended when symptoms develop, or to investigate specific allergen or workplace exposure. The monitoring of PEF has been used in clinical trials for adjusting therapy and assessing its effect, and for detecting early signs of deterioration. In some patients, objective measurement with a PEF meter may help to distinguish whether the symptoms are caused by airflow limitation or by other factors. In clinical practice, the results of PEF monitoring are not consistent enough for this tool to be recommended uniformly for all asthma patients (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). Thus, the relative usefulness of PEF monitoring needs to be individualized. Long-term PEF monitoring should be considered in patients who have moderate to severe persistent asthma or who have history of severe exacerbations, while its utility is questionable in mild asthma or in individuals who have histories of rapid onset of severe airflow limitation. In addition, PEF monitoring may be useful in individuals who have poor ability to perceive signs and symptoms of severe asthma (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). PEF utility decreases in preschool children and in the elderly, while minority and poor children exhibit greatest benefit (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). Finally, home PEF monitoring has to be considered during exacerbations for patients who have a history of severe exacerbation, moderate to persistent asthma, or difficulty perceiving signs of worsening disease.

Measurement of lung volume and evaluation of flow–volume loop Additional pulmonary function studies may be indicated, if there are questions about possible coexisting COPD, a restrictive defect, vocal cord disfunction (VCD), or central airway limitation. In addition, assessment of carbon monoxide diffusing capacity may be helpful in differentiating asthma from emphysema in older patients and smokers, who are at risk for both diseases.

Evaluation of risk factors Once the diagnosis of asthma is established, the potential risk factors should be investigated, in particular atopy and allergic sensitization.

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History The presence of asthma or other allergic (atopic) disorders in family medical history should be investigated (Koppelman 2006). History also provides important information about lifestyle and occupation (which both influence exposure to allergens, sensitizing agents, and irritants), the duration of symptoms and the factors possibly involved in the onset of asthma, the frequency of asthmatic exacerbations, the exacerbation of asthmatic symptoms in relation to exposure to allergens or irritants, physical exercise and respiratory infections, and the evolution of asthma over the years. One should then establish the relationship between exposure to one or more allergens and the occurrence of asthma symptoms and/or ocular and/or nasal symptoms. In addition, one should determine if there is a relationship with certain months of the year (seasonal pollen asthma), with the presence of pets in the home or during housecleaning, or with the workplace. Natural rubber latex sensitization should be enquired about, particularly in healthcare workers (Bousquet et al. 2006). The identification of other triggers of asthma exacerbations (e.g., exercise, occupational agents, drugs, cold air, emotions) is of paramount importance for the management of asthma, because it provides the necessary information for prevention and/or pharmacologic prophylaxis (e.g., pretreatment with β2 agonists or antileukotrienes before exercise) (GINA 2006).

Allergy: in vivo and in vitro tests The choice of the most appropriate test to perform in a patient with suspected allergic asthma depends on the kind of sensitization and the nature of the allergen to be tested. No allergy test by itself can prove that one or more allergens are the cause of asthma. Only the combination of history, skin-prick tests and, in few cases, in vitro measurement of allergen-specific serum IgE antibodies provides the necessary information to establish the importance of a given allergen in the pathogenesis of asthma. In a very few patients in whom these tests are not conclusive, allergen bronchial inhalation challenges may be required to evaluate specific airway responsiveness to a given allergen (Hansel et al. 2002).

Skin tests Skin tests using all relevant allergens found in the subject’s geographical area represent the first-choice test in diagnosis of allergy, because they are simple, easy and rapid to perform, and are highly sensitive and specific. The method most frequently used is skin-prick testing. Skin-prick testing relies on the introduction of a very small amount (a drop) of allergen extract into the epidermis using a standardized method to ensure reproducibility and comparability of results. The results of skin-prick testing are read at 10 min for the positive control (histamine dihydrochloride or codeine) and 15 min for the allergen, and the diameter of the resulting wheal is recorded in two dimensions. By convention, a positive test is

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one in which the mean of the two wheal diameters is at least 3 mm greater than the negative control (saline), although if the reaction is as small as this, the relevance of the response is in question. Positive and negative controls are critical to enable interpretation of test results (Dreborg & Frew 1993; Douglass & O’Hehir 2006). Skin-prick tests are reproducible and very seldom provoke systemic reactions, but fatalities have been described (Bernstein et al. 2004; Liccardi et al. 2006). Currently, skin-prick testing is performed using crude allergen extracts, which allow the identification of the allergen-containing source but not the disease-eliciting molecules. Crude extracts of allergens have many shortcomings that might produce unexpected results. With the introduction of recombinant allergens produced by molecular biology techniques, a large panel of allergenic molecules has become available and may improve the specificity of skin-prick testing in the future (Mothes et al. 2006).

In vitro tests The in vitro measurements of allergen-specific IgE antibodies by immunoenzymatic assay is much more expensive than skin-prick testing. It should be used only when skin-prick testing cannot be performed. In fact, when performed correctly, skin-prick testing with aeroallergens (e.g., house-dust mite allergen, pollens, domestic pet allergens) shows good correlation with blood allergen-specific IgE testing in a semiquantitative manner (Wood et al. 1999). The measurement of total serum IgE provides additional information in selected severe asthmatics to be treated with anti-IgE treatment (Strunk & Bloomberg 2006). The measurement of other serum immunoglobulins (e.g., IgG4) does not provide additional clinically relevant information.

Inhalation challenges with allergen, drugs, and chemical sensitizers Inhalation challenge with allergens is sometimes required to establish the relevance of single allergens for asthma and for research purpose (Hansel et al. 2002). Because these tests are risky and demand skilful interpretation, they should be performed only in specialized centers (Sterk et al. 1993). Atopic individuals with airway hyperresponsiveness to methacholine or histamine often also respond to allergens to which they are positive in skin-prick testing but which may not be relevant for their asthma, and thus the real value of allergen provocation in the management of the single patient remains unclear. In general, because inhalation challenge tests with allergens are risky and do not provide real useful information in the individual patient, they should not be used in clinical practice. In contrast, inhalation challenge with drugs (e.g., aspirin) or low-molecular-weight chemical sensitizers (e.g., isocyanates), which cause asthma through non-IgE-mediated mechanisms (and thus cannot be used for skin-prick testing or in vitro tests), may provide the only method which objectively confirms

occupational asthma (see Chapter 82). The cost–benefit ratio of performing inhalation tests with allergens or other sensitizing agents should be carefully examined in each patient, taking into account the high cost and the potential risk involved (Fish 1993; Sterk et al. 1993).

Ancillary tests Some minimally invasive markers such as induced sputum, exhaled gases, and exhaled breath condensate are increasingly used in clinical research, but will require further evaluation before they can be recommended as a clinical tool for routine asthma management. The use of serum and/or BAL fluid levels of circulating lymphocyte activation markers, adhesion molecules such as intercellular adhesion molecule-1 or vascular cell adhesion molecule-1, inflammatory mediators such as eosinophil cationic protein and myeloperoxidase, or urinary LTE4 in the monitoring of asthma control and severity is of unproven clinical utility (Wennergren 2000; Venge 2004). Although these markers of asthmatic inflammation may provide quite useful information when used in controlled clinical studies or for research purposes, they are at present of no relevant value in clinical practice because they rarely provide information specific to asthma, and it is still unclear what they may add to history and measurement of lung function. Apart from a few exceptions where differential diagnosis needs to be established (see below), bronchoscopy and related techniques (bronchial biopsies, BAL) have no clinical indications in asthma.

Induced sputum Induced sputum may provide a simple noninvasive method of assessing airway inflammation in asthma. Many reports indicate that collecting sputum expectorated by subjects after they inhale hypertonic saline solution (induced sputum) represents a safe and minimally invasive method for obtaining airway secretions from asthmatic subjects, who are unable to expectorate sputum spontaneously (Hargreave et al. 1993). Various cells and mediators can be analyzed in induced sputum and may provide useful information in asthma. The most robust parameter in asthma is the number or proportion of eosinophils. Sputum eosinophilia is good at distinguishing between patients who have and those who do not have asthma and in predicting responsiveness to starting or withdrawing inhaled corticosteroid treatment (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). The presence of sputum eosinophilia is associated with the presence of significant reversibility of airflow limitation (Pizzichini et al. 1998; Papi et al. 2000; Chavannes et al. 2006). The numbers of eosinophils in sputum decrease after treatment with glucocorticoids, reflecting an improvement in asthma (Brightling 2006b). Measuring sputum eosinophilia may be particularly useful in the noninvasive monitoring of asthmatic inflammation in children (Zacharasiewicz et al. 2006). A controlled

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prospective study has demonstrated that adjusting corticosteroid treatment to reduce sputum eosinophilia, as opposed to controlling symptoms, bronchodilator use and pulmonary function, significantly reduced both the rate of exacerbation and the cumulative dose of corticosteroids (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). The principal drawbacks of induced sputum are the difficulties in standardizing the methods and the demands on the time of highly trained technical staff for obtaining, preparing, and analyzing the samples (Expert Panel Report of the National Heart, Lung and Blood Institute 2007).

Fractional exhaled nitric oxide There is good evidence that exhaled nitric oxide (NO) is produced in increased amounts in the lower airways of stable asthmatics and is further increased during exacerbations (ATS/ ERS 2005). Increases in FENO are thought to reflect the intensity of eosinophilic inflammation of the bronchial mucosa. Like sputum eosinophil counts, measurement of FENO distinguishes patients who have or do not have asthma, is repeatable, is associated with other markers of asthma severity and, in some but not all studies, predicts responsiveness to starting or withdrawing corticosteroid treatment (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). The presence of an elevated level of exhaled NO in patients with chronic airflow limitation is a marker of an asthmatic component (Papi et al. 2000). Exhaled NO has also been suggested as an additional guide for adjusting the dose of inhaled glucocorticoids in patients with mild-to-moderate asthma, although this indication is still controversial (Corbetta & Fabbri 2005; Smith et al. 2005). A device for measuring FENO has been approved by the US Food and Drug Administration.

Differential diagnosis On a patient’s admission to hospital, an asthma exacerbation is usually easily identified. However, in a few cases a differential diagnosis with other pathologic conditions which manifest themselves with wheezing and dyspnea needs to be established. For this reason we should always remember the old adage of Chevalier Jackson (1965) that “all that wheezes is not asthma.” Upper airway limitation due to laryngeal edema, VCD (Newman & Dubester 1994), laryngeal or tracheal stenosis (Ravenna et al. 2002) (Fig. 76.2) or malacia, primary or secondary tracheal neoplasm (Fig. 76.3), and foreign bodies should always be considered. In addition to a suggestive history, patients present with stridor, and physical examination reveals harsh respiratory sounds in the area of trachea. Laryngoscopy or bronchoscopy may sometimes be useful. Dyspnea, cough, and chest tightness are frequent manifestations of VCD (Newman & Dubester 1994; Mikita & Mikita 2006). A high degree of clinical suspicion is required to recognize VCD and diagnosis is made most confidently by laryngoscopy. Bronchial stenosis, neoplasm, or aspiration of foreign body (Swanson & Edell 2001) may all mimic asthma. The patient usually complains of paroxysms of cough, and continuous wheezing is heard in a localized area. Patients with COPD may have partial reversibility of airflow limitation (see above) and may develop acute airflow limitation during exacerbations. A history of heavy smoking

Exhaled breath condensate The condensate is collected by passing exhaled air through a cold tube for 10–20 min. Several studies have shown differences in concentrations of various compounds in exhaled breath condensate (EBC) of healthy persons and those who have asthma, like hydrogen ion (pH), ammonia, isoprostanes, leukotriene metabolites, and products of nitrosylation (Expert Panel Report of the National Heart, Lung and Blood Institute 2007). There is evidence that abnormalities in EBC composition may reflect biochemical changes of airway lining fluid. Among the different biomarkers, pH can be measured with less expensive devices and represents the most studied parameter. Acidification of EBC has been demonstrated in acute asthma and COPD. More investigation are needed to establish the range of normal values, repeatability, association with other markers of asthma severity, and responsiveness to treatment. In addition, the rate of dilution of respiratory droplets in water and potential contamination with ammonia and other substances generated in the mouth need to be considered in the interpretation of the data (Kharitonov & Barnes 2006).

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Fig. 76.2 Spiral CT of the thorax with three-dimensional reconstruction showing the presence of a short trachea giving early origin to the main bronchi. (See CD-ROM for color version.)

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5 cm

Fig. 76.3 CT of the chest before surgery: the arrow indicates a solid mass in the upper portion of the trachea. Final pathologic diagnosis on the surgically resected sample was adenoid cystic carcinoma of the trachea.

is suggestive of COPD (Global Initiative for Chronic Obstructive Lung Disease 2001). When asthma and COPD occur in the same patient, they should be recognized and treated appropriately (Global Initiative for Chronic Obstructive Lung Disease 2001; GINA 2002, 2006). Heart failure with consequent cardiogenic venous pulmonary hypertension may both mimic asthma in nonasthmatic subjects with cardiac disease, and trigger asthma exacerbations in asthmatic subjects with cardiac disease (Regnard 2003). History, physical examination (signs of left and/or right ventricular failure, including basilar rales, gallop rhythms and jugular congestion, peripheral edema), and radiologic signs of cardiomegaly, or additional tests such as echocardiography and serum B-type natriuretic peptide (BNP) assay (Mueller et al. 2006) may be required for the differential diagnosis. Acute coronary syndrome should be considered sometimes in the differential diagnosis of subjects presenting with symptoms of asthma, particularly if the onset of symptoms occurs after the age of 40 in heavy smokers (Thibault 1994). Pulmonary microembolism or recurrent episodes of pulmonary embolism may mimic an asthmatic exacerbation because of the sudden development of dyspnea (Thibault 1995). The development of dyspnea is usually unexpected, is not associated with acute airflow limitation, and occurs in patients at risk of pulmonary embolism (e.g., deep venous thrombosis of the legs, surgery). Contrast-enhanced chest computed tomography (CT) may be necessary to exclude pulmonary embolism in patients at risk. Pneumothorax may also cause acute breathlessness, but it is accompanied by acute chest pain and the absence of airflow limitation, and ultimately chest radiography will provide the diagnosis. Eosinophilic lung disease (Allen & Davis 1994) (see Chapter 86), some pulmonary vasculitides such as Churg–Strauss syndrome (Anon. 1992; Richeldi et al. 2002) (Fig. 76.4), allergic bronchopulmonary fungal disease (Iber 1993; Tillie-Leblond & Tonnel 2005) (see Chapter 84), and some helminthic infections such as Ascaris suum (Phills et al. 1972), Strongyloides

Fig. 76.4 Eosinophilic vasculitis in the lung of a patient with Churg–Strauss syndrome. Videothoracoscopy lung biopsy. Hematoxylin and eosin, magnification × 200. (See CD-ROM for color version.)

stercoralis (Pansegrouw 1994), trichinellosis (Bourèèe 1991) and visceral larva migrans (Feldman & Parker 1992) (see Chapter 86) may all be associated with asthma, and therefore should be properly investigated. Algal blooms may cause outbreaks of asthma-like symptoms (Gallitelli et al. 2005). It is still unknown if they can trigger an asthmatic exacerbation only in preexisting disease or also in nonasthmatic subjects. Carcinoid tumors may mimic asthma symptoms. Patients with carcinoid may develop the carcinoid syndrome, i.e., flushing of the face and other portions of the body, hyperperistalsis of the gut with diarrhea, hypotension and tachycardia sometimes associated with wheezing. Facial rash and diarrhea, in addition to the increase in urinary 5-hydroxyindoleacetic acid are usually guidelines for a correct diagnosis (Creutzfeldt & Stockmann 1987). Cystic fibrosis should be considered if the asthma is atypical in its course and is associated with frequent chest infections (Woolcock 1994). Aortic arch anomalies can present as exercise-induced asthma (Bevelaqua et al. 1989).

Acknowledgments The authors are supported by Ministry of University, PRIN grant, Rome, and the University of Padova.

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Ravenna, F., Caramori, G., Panella, G.L. et al. (2002) An unusual case of congenital short trachea with very long bronchi mimicking bronchial asthma. Thorax 57, 372–3. Regnard, J. (2003) [Cardiac asthma.] Rev Mal Respir 20, S50–S54. Richeldi, L., Rossi, G., Ruggieri, M.P., Corbetta, L. & Fabbri, L.M. (2002) Churg–Strauss syndrome in a case of asthma. Allergy 57, 647–8. Romagnoli, M., Caramori, G., Braccioni, F. et al. (2007) Near-fatal asthma phenotype in the ENFUMOSA Cohort. Clin Exp Allergy 37, 552–7. Rosenkranz, M.A., Busse, W.W., Johnstone, T. et al. (2005) Neural circuitry underlying the interaction between emotion and asthma symptom exacerbation. Proc Natl Acad Sci USA 102, 13319–24. Rosenman, K.D. (2006) Cleaning products-related asthma. Clin Pulm Med 13, 221–228. Ruigomez, A., Rodriguez, L.A., Wallander, M.A., Johansson, S., Thomas, M. & Price, D. (2005) Gastroesophageal reflux disease and asthma: a longitudinal study in UK general practice. Chest 128, 85–93. Salpeter, S., Ormiston, T. & Salpeter, E. (2002) Cardioselective betablockers for reversible airway disease. Cochrane Database Syst Rev CD002992. Sarnat, J.A. & Holguin, F. (2007) Asthma and air quality. Curr Opin Pulm Med 13, 63– 6. Scano, G. & Stendardi, L. (2006) Dyspnea and asthma. Curr Opin Pulm Med 12, 18– 22. Schreur, H.J., Vanderschoot, J., Zwinderman, A.H., Dijkman, J.H. & Sterk, P.J. (1994) Abnormal lung sounds in patients with asthma during episodes with normal lung function. Chest 106, 91–9. Sharma, H.P., Hansel, N.N., Matsui, E., Diette, G.B., Eggleston, P. & Breysse, P. (2007) Indoor environmental influences on children’s asthma. Pediatr Clin North Am 54, 103–20, ix. Shaw, R.A., Crane, J., Pearce, N. et al. (1992) Comparison of a video questionnaire with the IUATLD written questionnaire for measuring asthma prevalence. Clin Exp Allergy 22, 561–8. Sherrill, D., Guerra, S., Bobadilla, A. & Barbee, R. (2003) The role of concomitant respiratory diseases on the rate of decline in FEV1 among adult asthmatics. Eur Respir J 21, 95–100. Sheth, A., Reddymasu, S. & Jackson, R. (2006) Worsening of asthma with systemic corticosteroids. A case report and review of literature. J Gen Intern Med 21, C11–C13. Singh, A.M., Moore, P.E., Gern, J.E., Lemanske, R.F. Jr & Hartert, T.V. (2007) Bronchiolitis to asthma: a review and call for studies of gene-virus interactions in asthma causation. Am J Respir Crit Care Med 175, 108–19. Slavin, R.G. (2006) Allergic rhinitis: managing the adult spectrum. Allergy Asthma Proc 27, 9–11. Sly, P.D., Cahill, P., Willet, K. & Burton, P. (1994) Accuracy of mini peak flow meters in indicating changes in lung function in children with asthma. BMJ 308, 572– 4. Smart, B.A. (2006) Is rhinosinusitis a cause of asthma? Clin Rev Allergy Immunol 30, 153– 64. Smith, A.D., Cowan, J.O., Brassett, K.P., Herbison, G.P. & Taylor, D.R. (2005) Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 352, 2163–73. Spergel, J.M. (2005) Atopic march: link to upper airways. Curr Opin Allergy Clin Immunol 5, 17– 21. Sterk, P.J., Fabbri, L.M., Quanjer, P.H. et al. (1993) Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Report Working Party Standardization of Lung Function Tests, European Community

for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl 16, 53– 83. Stevenson, D.D. & Szczeklik, A. (2006) Clinical and pathologic perspectives on aspirin sensitivity and asthma. J Allergy Clin Immunol 118, 773–86; quiz 787–8. Strunk, R.C. & Bloomberg, G.R. (2006) Omalizumab for asthma. N Engl J Med 354, 2689–95. Sutherland, E.R. (2005) Nocturnal asthma. J Allergy Clin Immunol 116, 1179–86; quiz 1187. Swanson, K.L. & Edell, E.S. (2001) Tracheobronchial foreign bodies. Chest Surg Clin North Am 11, 861–72. Tan, W.C. (2005) Viruses in asthma exacerbations. Curr Opin Pulm Med 11, 21–6. Tantisira, K.G. & Weiss, S.T. (2006) The pharmacogenetics of asthma therapy. Curr Drug Targets 7, 1697–708. Thibault, G.E. (1994) Clinical problem-solving. The appropriate degree of diagnostic certainty. N Engl J Med 331, 1216–20. Thibault, G.E. (1995) Clinical problem-solving. Diagnostic strategy: the shotgun versus the arrow. N Engl J Med 332, 321–5. Thomas, M. (2006) Allergic rhinitis: evidence for impact on asthma. BMC Pulm Med 6 (suppl. 1), S4. Thomson, N.C. (2007) Smokers with asthma. Am J Respir Crit Care Med 175, 749–753. Thomson, N.C., Chaudhuri, R. & Livingston, E. (2004) Asthma and cigarette smoking. Eur Respir J 24, 822–33. Tillie-Leblond, I. & Tonnel, A.B. (2005) Allergic bronchopulmonary aspergillosis. Allergy 60, 1004–13. Toren, K., Brisman, J. & Jarvholm, B. (1993) Asthma and asthmalike symptoms in adults assessed by questionnaires. A literature review. Chest 104, 600–8. Troyanov, S., Ghezzo, H., Cartier, A. & Malo, J.L. (1994) Comparison of circadian variations using FEV1 and peak expiratory flow rates among normal and asthmatic subjects. Thorax 49, 775–80. Turner-Warwick, M. (1977) On observing patterns of airflow obstruction in chronic asthma. Br J Dis Chest 71(2), 73–86. Venables, K.M., Farrer, N., Sharp, L., Graneek, B.J. & Newman Taylor, A.J. (1993) Respiratory symptoms questionnaire for asthma epidemiology: validity and reproducibility. Thorax 48, 214–19. Venge, P. (2004) Monitoring the allergic inflammation. Allergy 59, 26– 32. Vestbo, J., Sorensen, T., Lange, P., Brix, A., Torre, P. & Viskum, K. (1999) Long-term effect of inhaled budesonide in mild and moderate chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 353, 1819–23. Vijayanand, P., Seumois, G., Pickard, C. et al. (2007) Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease. N Engl J Med 356, 1410–22. Vollmer, W.M., Markson, L.E., O’Connor, E. et al. (1999) Association of asthma control with health care utilization and quality of life. Am J Respir Crit Care Med 160, 1647–52. Wang, X., Dockery, D.W., Wypij, D., Fay, M.E. & Ferris, B.G.J. (1993) Pulmonary function between 6 and 18 years of age. Pediatr Pulmonol 15, 75– 88. Wanger, J., Clausen, J.L., Coates, A. et al. (2005) Standardisation of the measurement of lung volumes. Eur Respir J 26, 511–22. Weiss, S.T. (2005) Obesity: insight into the origins of asthma. Nat Immunol 6, 537– 9. Weiss, S.T., Litonjua, A.A., Lange, C. et al. (2006) Overview of the pharmacogenetics of asthma treatment. Pharmacogenomics J 6, 311–26.

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Wennergren, G. (2000) Inflammatory mediators in blood and urine. Paediatr Respir Rev 1, 259– 65. Wenzel, S.E. (2006) Asthma: defining of the persistent adult phenotypes. Lancet 368, 804–13. Wong, G.W. & Lai, C.K. (2004) Outdoor air pollution and asthma. Curr Opin Pulm Med 10, 62– 6. Wood, R.A., Phipatanakul, W., Hamilton, R.G. & Eggleston, P.A. (1999) A comparison of skin prick tests, intradermal skin tests, and RASTs in the diagnosis of cat allergy. J Allergy Clin Immunol 103, 773–779. Woolcock, A.J. (1994) Asthma. In: Murray, J. & Nadel, J., eds. Textbook of Respiratory Medicine, 2nd edn. W.B. Saunders, Philadelphia, pp. 1030–68. Yang, I.A., Savarimuthu, S., Kim, S.T., Holloway, J.W., Bell, S.C. & Fong, K.M. (2007) Gene-environmental interaction in asthma. Curr Opin Allergy Clin Immunol 7, 75– 82. Yernault, J.C. & Bohadana, A.B. (1995) Chest percussion. Eur Respir J 8, 1756–60. Zacharasiewicz, A., Erin, E.M. & Bush, A. (2006) Noninvasive monitoring of airway inflammation and steroid reduction in children with asthma. Curr Opin Allergy Clin Immunol 6, 155– 60.

Appendix Examples of the questions and scales used in the symptom diary (A) and weekly symptom questionnaire (A, B, C). Subjects rated responses using the severity scale (A) and symptom transition scale (C). Questions were repeated for wheeze, breathlessness, chest tightness and overall asthma symptoms (Gibson et al. 1992).

Cough A How much discomfort or distress did you have this week

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as a result of cough? Please indicate how much discomfort or distress you had as a result of cough by choosing one of the options from the card in front of you. 1 Virtually unbearable, the most severe discomfort or distress ever. 2 A very great deal of discomfort or distress. 3 A great deal of discomfort or distress. 4 A good deal of discomfort or distress. 5 Moderate amount of discomfort or distress. 6 Mild discomfort or distress. 7 Very mild discomfort or distress. 8 Barely perceptible, hardly aware of discomfort or distress. 9 Absent, none at all. B Overall, has there been any change in your cough since you saw us one week ago? Please indicate if there has been any change in your cough by choosing one of the options from the card in front of you. Has your cough been: 1 worse 2 about the same 3 better. If 1 or 3, go to Question C. C How much worse (or better) would you say your cough has been since the last time you saw us one week ago? Please choose one of the options from the card in front of you. 1 Almost the same, hardly worse (better) at all. 2 A little worse (better). 3 Somewhat worse (better). 4 Moderately worse (better). 5 A good deal worse (better). 6 A great deal worse (better). 7 A very great deal worse (better).

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Asthma in Infancy and Childhood John O. Warner

Summary

Historical introduction

Asthma is the commonest chronic disease of childhood, affecting upward of 20% of the whole childhood population in developed countries. Its prevalence and therefore health economic burden has increased progressively over the last 30–40 years. Understanding the early life origins and underlying immunopathology will aid management. Many infant wheezers do not go on to develop asthma and therefore their management should be symptomatic alone. However, once it can be established that there is persistent disease with evidence of underlying inflammation, particularly in association with allergy, then prophylaxis as well as symptomatic relief is required. The majority of acute exacerbations are due to a combination of virus infection against a background of allergic sensitization and exposure to allergen. The mechanisms which underlie susceptibility to virus-induced exacerbation are now being clarified. Management involves a combination of nonpharmacologic approaches including avoidance of triggers, of which allergens are a major factor, and employment of pharmacotherapy in a stepwise fashion based on frequency of problems and severity. The majority of children can be effectively managed with a combination of low-dose inhaled corticosteroids and rescue treatment with inhaled β agonists. For more persistent and severe disease add-on therapy with either leukotriene receptor antagonists or long-acting β agonists constitutes the next step in the algorithm. A few subjects have relatively steroid-insensitive asthma often associated with neutrophil rather than eosinophil mediated airway inflammation. Here the evidence base for management is scanty or nonexistent but involves the employment of a range of immunosuppressives. The exact place of allergen immunotherapy remains to be established despite a good evidence base for efficacy. Finally education and attention to psychosocial issues is also an important component of the management.

Asthma was recognized as a distinct entity in ancient texts such as the Egyptian Ebers papyrus and ancient Chinese texts such as Mah Huang. The condition was described by Hippocrates who recognized that it could occur in children: “Children are liable to convulsions and asthma which are regarded as divine visitations and the disease itself as sacred.” However, many subsequent texts tended to ignore asthma in childhood as exemplified by Aurelianus Caelius who observed that “asthma occurs oftener in men than in women, in middle age than in children or old men and in the delicate rather than the strong.” Thus virtually all subsequent descriptions were of the condition in adults and quite frequently physicians described their own disease such as Sir John Floyer in his Treatise of the Asthma published in 1698 where he observes “I have suffered under the tyranny of the asthma, at least 30 years.” It must therefore have had its onset in childhood (Brewis 1990). In 1769 John Millar described a child who had died of asthma, with “an abundance of gelatinous secretions obstructing the bronchi.” He also reported on another child who had died with asthma but whose lungs were said to be perfectly normal (Millar 1769). While descriptions of the histopathology of asthma have primarily come from adult bronchial biopsy studies, the very first publication to emphasize that airway inflammation may occur in mild asthma came from a pediatric study of ultrastructure of open lung biopsies from two asthmatic children during clinical remission. The appearances were compared with lung tissue from two children who had died of asthma. All four asthma cases had submucosal cellular infiltrates, especially with eosinophils and with denudation of epithelium (Cutz et al. 1978). This paper appeared in 1978, some time before the seminal publication on the pathology of asthma by Laitinen et al. (1985). John Millar also made very perceptive observations about the potential causes of childhood asthma: “It is chiefly incident in children, especially such as have been lately weaned, and that it has been most prevalent in spring and autumn, moist seasons, changeable weather and when the mercury stood low in the barometer.” He suggested that “disease must

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Epidemiology There has been a dramatic increase in the prevalence and severity of asthma in childhood. While one or two recent studies have suggested that the asthma epidemic is beginning to subside, there are still recent publications, such as one from South Wales using identical ascertainment among 12 year olds, which has shown a change in point prevalence of asthma between 1973, 1988 and 2003 from 5.5% to 12% to 27.3% (Burr et al. 2006). This has been mirrored by similar increases in eczema and rhinitis (Fig. 77.1). This increase has occurred in diverse environments and across all continents, although the absolute prevalence rates vary considerably as demonstrated by the International Study

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30 25 20 15

1973 1988 2003

10 5 0 Eczema

Rhinitis

Asthma

Wheeze

Fig. 77.1 The prevalence of atopic diseases in 12-year-old children surveyed using identical ascertainment in schools in South Wales showing continuing increases between 1973, 1988 and 2003. Eczema, rhinitis and asthma have increased in prevalence at an equivalent rate. However, there is a dramatic narrowing of the gap between prevalence of wheezing and diagnosed asthma in 2003 compared with 1973 and 1988. (From Burr et al. 2006, with permission.) (See CD-ROM for color version.)

of Asthma and Allergies in Childhood (ISAAC), being higher in English-speaking communities with reducing rates progressing south through Europe and into Africa and east through Asia (ISAAC Steering Committee 1998). Studies of migrating populations have suggested that early-life environment is critical to the expression of disease. Thus South Asian immigrants to the UK retain a low prevalence rate for asthma if their arrival in the UK was after 5 years of age (Kuehni et al. 2007). Being born in the UK or arriving before 4 years of age conferred the highest risk of developing asthma (Fig. 77.2). There was also a relationship to the family adopting western lifestyle changes as represented by the use of the English language at home.

18 Asthma prevalence (% of population)

depend greatly on the state of the atmosphere” (Millar 1769). Through the 19th century it became apparent that exposure to pollen was a cause of exacerbation and this was brilliantly demonstrated by Charles Harrison Blackley who performed skin tests and nasal, conjunctival and bronchial provocation tests on himself to demonstrate the effects (Blackley 1873). Finally, in the 20th century the range of allergens involved in aggravating the disease were identified, with house-dust mite being seen as the major allergen in both children and adults in temperate climates throughout the world (PlattsMills et al. 1997). However, it has been in the last 30 years that more attention has focused on the early-life origins of asthma as it has become apparent that there has been an increase in the prevalence of disease particularly focused on children (von Mutius 1999). Given the magnitude of the modern asthma epidemic, which appears to have exceeded any others in previous generations, one might question why this is associated with what we all believe to be a dramatic improvement in the understanding of the pathology and management of the disease. It is a remarkable paradox that a controllable chronic disease has had increasing morbidity, and in some circumstances mortality, during the last four decades. While we focus on deficiencies in management strategies as the cause for this, we should not neglect the very strong probability that the asthma epidemic has been caused by a change in lifestyle and environment which is likely to be particularly relevant to the fetus and young infant. The best prospect for prevention may well lie in environmental modification and immune modulation in early life rather than by the employment of pharmacotherapy. While it is attractive to consider that early diagnosis and effective early intervention is likely to modify long-term outcomes, hitherto it has not been possible to demonstrate any effects of for instance employing inhaled corticosteroids (ICS) at a very early stage in disease evolution (Guilbert et al. 2006). With the possible exception of allergen immunotherapy, no treatment has been shown to modify the natural course of the disease and certainly no cure has been identified.

% of 12 year olds

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16 14 12 10 8 6 4 2 0 Born in UK

Migrated to UK aged 0–4 years 5–14 years > 15 years

Fig. 77.2 Asthma prevalence rates in South Asian women based on the timing of migration to UK. Prevalence is significantly higher in those who were either born in the UK or arrived in the UK in the first 4 years of life. This suggests that early-life environment has a greater effect on disease manifestation than genotype. (From Kuehni et al. 2007, with permission.) (See CD-ROM for color version.)

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Economic burden It was estimated that in 2000 the cost to the UK health service of asthma was £754 million, of which 79% was for prescribed medications, 13% for general practitioner consultations, and 8% for hospital admissions, most of which were emergencies (Gupta et al. 2004). It is difficult to disentangle the relative costs in relation to children compared with adults. However, it is clear that children contribute proportionately more both to primary and secondary care costs, with admission rates being significantly higher in children per head of population. Economic evaluations fail to take account of the education and support required to optimize medication use or the medical equipment and consumables such as nebulizers, spacers and peak flow meters, and the indirect costs to patients and their families are not accounted. The latter particularly relates to school absence and of course work absence for the parents who need to stay at home to deal with their wheezing child as well as the costs of frequent visits for primary care or hospital consultations in relation to travel, parking and child care. The long-term costs of school absence and its impact on education, qualifications, and career attainment also require consideration as well as the adverse effects on quality of life. One study estimated that the mean annual cost for a family with a young child with asthma to be £562 (Stevens et al. 2006). This comprised a mixture of lost salary for work absence, health service costs, and increased family expenditure for extra heating, child minding and transport. Finally none of the health economic studies have taken account of comorbidities. Many children with asthma have associated allergic rhinitis and have had or continue to have atopic eczema. These impose very considerable additional burdens on the child and family as well as impacting on health economics with additional visits to dermatologists, ENT surgeons as well as other direct health service costs.

Asthma deaths Deaths from asthma are rare, particularly in children under 15 years of age. Thus in 2004 in England and Wales there was only one reported death due to asthma in a child under 4 and 37 in those between 5 and 14 years of age. Factors associated with risk of death from asthma in childhood are very similar to those in adults and relate to disease severity, reduced concordance with recommended therapy, failure to access medical services, and a range of adverse psychosocial factors (Fletcher et al. 1990). However, it has been suggested that on occasions children with ostensibly mild disease can still succumb during an acute episode (Robertson et al. 1992). Whether this is a consequence of a failure to appreciate more severe disease or is genuinely a catastrophic episode in

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a previously mild asthmatic is impossible to ascertain. The other possibility is that these were misidentified anaphylactic deaths. It has been well established that food allergy is overrepresented in children suffering very severe acute exacerbations requiring intensive care (Roberts et al. 2003). As a proportion of children and indeed adults who have been identified as dying from acute asthma have minimal or no chronic airway inflammation (as suggested by Millar in 1769), it is possible that they have actually died as a consequence of an acute anaphylactic reaction in which wheezing is a common feature. One study has shown that key factors in children who have died of asthma are delayed treatment in the final attack associated with psychosocial factors, poor perception of airflow limitation, and heightened bronchial hyperresponsiveness (Fletcher et al. 1990). Another study suggested that one potential marker of fatality proneness is the measurement of soluble interleukin (IL)-2 receptor in the serum (Warner et al. 1998). However, at present there are no reliable identifying factors which characterize the fatalityprone asthmatic.

Natural history of asthma William Osler made the apocryphal and presumed incorrect statement that “the asthmatic pants on into old age.” This is assumed to indicate that asthma deaths do not occur. However, it could be taken to indicate that this is a chronic condition which is far more likely to persist into old age than to remit. Sadly there is still a prevailing view that childhood asthma is a self-limiting disorder which tends to improve spontaneously during adolescence. This is a misleading and inaccurate generalization. It would be truer to say that asthmatics outgrow their pediatrician rather than outgrowing their asthma. Many longitudinal studies from childhood to adulthood have shown that a significant percentage persist and even among those who remit in adolescence a high percentage have a recurrence of problems later in adult life. The Melbourne prospective study of asthma has the longest follow-up of all. This study demonstrated that intermittent wheezing in mid-childhood was predominantly self-limiting. whereas children who had persistent asthma rarely remitted. The prognosis was less favorable in those with an early age of onset associated with frequent, severe or prolonged attacks in the first year after onset and/or the presence of infantile eczema (Williams & McNicol 1969). A Dutch study has shown that reduced lung function and heightened bronchial hyperresponsiveness in 7-year-old asthmatics was associated with a greater degree of lung function deficit and bronchial hyperresponsiveness in the same individuals at 21 years of age (Roorda et al. 1994). The conclusions that can be drawn from longitudinal asthma cohort studies is that the more severe the problem at its onset, the greater the probability it will persist and remain severe.

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The male to female distribution for mild intermittent asthma in childhood is 1 : 1. However, in childhood progressively more severe and persistent disease is associated with a male predominance, reaching 4 : 1 at the severe end of the spectrum. In adulthood, the gender ratio is much closer to 1 : 1, indeed with a female predominance for severe asthma. The implication from these observations is that during adolescence more males are likely to improve or remit while more females are likely to have persistent problems or develop asthma for the first time. There is some indirect evidence to suggest that this is indeed the case (Kaur et al. 1998). Smoking in adolescence decreases the probability of an improvement in asthma symptoms among patients who have wheezed since early childhood and the uptake of smoking in early adulthood increases the risks of a remitted asthmatic subsequently relapsing (Martin et al. 1982).

Infant wheezing and asthma Wheezing in infancy is very common. However, the relationship with subsequent asthma and also chronic obstructive pulmonary disease in late adult life remains incompletely understood. However, large birth cohort studies have identified a number of distinct phenotypes each with rather different prognoses. Transient early wheezing occurring before 3 years of age comprises 60% of all infant wheezers, and is associated with remission of symptoms by 5–7 years of age. The symptoms occur almost exclusively in association with viral infections and some studies have identified that the infants most at risk have reduced lung function even before the first symptoms occur (Martinez & Godfrey 2003). These in turn are most strongly associated with maternal smoking during pregnancy, which has a clear effect on fetal lung development (Dezateux et al. 1999). Males are more strongly represented in this group, which may be a consequence of differences in respiratory physiology. Breast-feeding may decrease the risks of transient infant wheezing, although this effect may be confounded by other factors. Early admission to daycare with greater exposure to viruses increases the prevalence. Low birth weight, whether due to intrauterine growth retardation or prematurity, also significantly increases with risks of developing the transient early wheezing phenotype. Some of the risk factors for transient virus-associated wheeze of infancy have also been related to chronic obstructive pulmonary disease in later adult life. There is therefore the potential that these transient wheezers could reappear with respiratory problems as adults. However, this has yet to be confirmed. The second phenotype is nonatopic persistent wheezing, which constitutes 20% of infant wheezers. Such infants wheeze through early to mid childhood, although they still have an excellent prognosis with many remitting in later

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childhood. The most common association is with an acute respiratory illness in the first year of life, frequently respiratory syncytial virus (RSV) bronchiolitis. Such infants retain a minor lung function abnormality even at 11 years of age irrespective of whether they have continuing wheezing at that age (Stein et al. 1999). Therefore there is potential for these children also to have problems in late adult life. The factors that underlie the development of RSV bronchiolitis and postbronchiolitic wheezing include low birth weight, reduced lung function prior to the RSV infection, and preexisting underlying immunologic, cardiovascular or respiratory problems. There is a suggestion that the infant who develops bronchiolitis, as distinct from just an upper respiratory tract infection, on exposure to RSV has an impaired Th1 response to the virus and therefore does not generate interferon (IFN)-γ but rather responds with a Th2 pattern involving the release of IL-4 (Legg et al. 2003). This pattern of response is also clearly associated with allergy. Some studies, notably from Scandinavian countries, have suggested there is a strong link between RSV bronchiolitis and subsequent allergy (Sigurs et al. 2005). However, UK studies have suggested that this is not the case. They have shown that persistent wheezing through childhood is common after bronchiolitis, but atopy occurs no more frequently than in the unaffected population (Henderson et al. 2005). There is little or no evidence that ICS have any impact on transient virus-associated wheeze of infancy or on nonatopic persistent wheezing. However, many of these categories of wheezing do respond to bronchodilators and a recent trial of montelukast, a leukotriene receptor antagonist, has demonstrated a beneficial impact on virus-induced wheeze in the preschool age group (Bisgaard et al. 2005). The third category, which constitutes 20% of infant wheezers, is associated with allergy and virtually all have persistent wheezing identified as atopic asthma. Such infants have problems that persist through childhood and into adolescence, with a high percentage continuing into adulthood. The presence of allergy in the form of eczema and/or positive skin-prick tests confers a poorer prognosis with regard to persistence of wheezing through to adulthood. Thus the presence of allergy is one factor that can be used clinically to aid decisions on therapy. The nonallergic infant wheezer should be treated with circumspection and perhaps the use of only a bronchodilator as necessary. However, in the presence of allergy, early introduction of ICS is more appropriate. One study from Oslo in Norway has suggested that a combination of lung function measurement and assay of serum eosinophil cationic protein as a marker of allergic inflammation might discriminate the future atopic wheezers. The authors found a strong positive correlation between the level of serum eosinophil cationic protein and the degree of responsiveness to nebulized salbutamol (Lodrup-Carlsen et al. 1995).

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Immunopathology of asthma in childhood Airway remodeling is considered to be a characteristic feature of asthma. It is postulated that it results from damage to the airway epithelium as a consequence of inflammation with subsequent hypertrophy of airway smooth muscle and deposition of collagen in the lamina reticularis below the true epithelial basement membrane as well as increased collagen in the lamina propria. Bronchial biopsy studies from children with asthma have shown identical changes to those seen in adult asthmatics, with shedding of the suprabasal epithelial layer and cellular infiltration dominated by eosinophils with increases also in mast cells and lymphocytes (Cokugras et al. 2001). Thickening of the lamina reticularis has been observed in children aged 5–14 years of age with moderately severe asthma but without any correlation with the duration of disease or allergic status. Similar changes of remodeling have been seen in the biopsies of young children shortly after they first presented with wheezing (Pohunek et al. 2005), although it has not been seen to the same extent in predominantly nonatopic wheezing infants (Saglani et al. 2005). It is therefore unclear whether remodeling occurs only some time after the disease is established, or as a premorbid event. However, the two pathologic features in bronchial biopsies in children when they first present with wheezing that predict persistence of problems (in other words asthma) are the presence of eosinophilic inflammation and increased thickness of the lamina reticularis (Pohunek et al. 2005). There is also no correlation between the duration of asthma and the thickness of the lamina reticularis (Pohunek et al. 2005; Kim et al. 2007). This suggests that the remodeling process is not a consequence of eosinophilic inflammation but is occurring in

parallel with the inflammation and may conceivably occur before first symptoms are manifest (Fig. 77.3). In support of the concept that the immunopathology of asthma antedates the onset of symptoms, it has now been shown that diminished lung function and heightened bronchial hyperresponsiveness at 4 weeks of age is associated with asthma at 6 and 11 years of age from birth cohort studies in Perth (Turner et al. 2004). However the Tucson cohort studies have tended to suggest that diminished lung function in early infancy is associated with virus-associated wheeze and nonatopic wheeze but not with atopic asthma (Martinez et al. 1995). Indeed, in this study the true asthmatics had normal lung function in infancy but showed progressive deterioration in lung function by 6 years of age. The conflicting information will only be resolved by larger longitudinal studies of form and function but these are fraught with ethical issues and information will accrue only slowly. The acquisition of biopsy specimens are only justified when there is a clear-cut clinical indication for the procedure in the individual patient. Airway remodeling can occur independently of eosinophilic inflammation, as for instance described in elite cross-country skiers who have no allergy but recurrent wheezing and primarily neutrophilic rather than eosinophilic inflammation (Karjalainen et al. 2000). Many asthmatics particularly those with more severe disease both in infancy and adulthood have a predominant neutrophil infiltration. Furthermore, the number of neutrophils correlates with levels of the major neutrophil chemokine IL-8, which is released by host lung cells including macrophages and airway epithelium when stressed (Marguet et al. 1999, 2000 and 2001). There are in addition raised levels of matrix metalloproteinase (MMP)-9, which is predominantly generated by neutrophils in a high-molecularweight form that renders it resistant to the effects of its

Infant who subsequently developed asthma

Fig. 77.3 Bronchial biopsies from two children who presented with recent onset of respiratory symptoms. At subsequent follow-up, (a) developed clear asthma and (b) did not. The child who subsequently had asthma already had both inflammation and remodeling at presentation. The latter includes increased collagen deposition below the true basement membrane (1), increased collagen in the lamina propria (2), and smooth muscle hypertrophy (3). (See CD-ROM for color version.)

(a)

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Infant with no subsequent asthma

(b)

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inhibitor, TIMP-1 (Turner et al. 2004). The author’s group has found raised levels of TIMP-1 to predict persistence of wheezing in infant wheezers followed for at least 1 year. Furthmore, raised TIMP-1 correlates with the degree of airwall thickening in asthma (Matsumoto et al. 2005). It is possible that the raised levels of TIMP-1 are generated in a frustrated attempt to counteract the effects of neutrophil MMP-9. While neutrophil MMP-9 will degrade extracellular matrix, TIMP-1 will inhibit matrix turnover thereby leading to accumulation of collagens, as seen in the remodeled airway of asthma. The other pivotal factor found in excess in the airways of asthmatics and even in infant wheezers is the cytokine transforming growth factor (TGF)-β. This is found in the airway matrix and is released in a free form as the matrix is degraded by MMP-9. It has a potent effect in stimulating fibroblast activity and the generation of myofibroblasts, which may contribute to airway smooth muscle hypertrophy (Blobe et al. 2000). These observations suggest that neutrophils may have a more important role in the genesis of asthma than has hitherto been considered the case. It is for instance interesting to note that neutrophils appear in the airway at the onset of the late allergen-induced response some 4 hours after exposure while eosinophils are only evident when the late reaction is well established some 12–24 hours after challenge (Smith & Deshazo 1993). It also implies that many airway insults such as infection and pollution as well as allergic reactions could initiate the remodeling process. The pivotal question is to establish whether the pathologic changes do antedate the onset of symptoms. This will have an appreciable effect on therapeutic strategies. Hitherto the main emphasis has been on earlier and earlier employment of ICS after the onset of symptoms. However, as is implicit from the different asthma phenotypes, this cannot be based on symptoms alone as many young wheezers do not go on to have established asthma. Many attempts have been made to identify surrogate markers for the pathologic changes in the airway and hitherto none have been found to be totally satisfactory in predicting outcomes. Measuring markers of eosinophilic inflammation have proved disappointing. Measurements of bronchial hyperresponsiveness and even bronchodilator responsiveness has not necessarily discriminated the good-prognosis nonatopic wheezers from the persistent asthmatics. More recent studies of exhaled nitric oxide and measurement of mediators in exhaled breath condensates have looked promising but have yet to be established as clinical tools in this situation (Pijnenburg et al. 2006).

Viral infections and asthma It has long been known that viral respiratory infections are commonly associated with exacerbations of wheezing in asthmatic children (Friedlander & Busse 2005) (Fig. 77.4). This is particularly true in infants but it is apparent that virus infec-

1596

100 82 75 %

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50

44 25

25

0 6 weeks

6 months

Fig. 77.4 In this study 82% of children admitted to hospital with acute asthma had rhinovirus RNA in nasal lavage; 6 weeks and 6 months later rhinovirus was detectable in 44 and 25% respectively. This either represents a failure to eliminate the virus or susceptibility to repeated infection. Both are possible given improved understanding of the mechanisms of rhinovirus infectivity and clearance. (From Kling et al. 2005, with permission.) (See CD-ROM for color version.)

tion is the major trigger for exacerbation at all ages. While in infancy RSV is the most common cause of wheezing, its relationship to subsequent asthma remains uncertain as indicated above. Once asthma is manifest rhinovirus is the leading cause of exacerbations. Up to 80% of acute exacerbations of asthma in childhood are associated with identification of rhinovirus in the airway. Asthmatics would appear to be not only more susceptible to rhinovirus infection but also have difficulties in clearing the virus such that it can be detected in airway secretions sometimes for many months after an acute exacerbation (Kling et al. 2005). Rhinovirus gains access to cells via intercellular adhesion molecule (ICAM)-1, which is notably upregulated in asthma. Indeed, a soluble component of this molecule, sICAM-1, is raised in both serum and bronchoalveolar lavage from young children with asthma (Marguet et al. 2000). Furthermore, serum levels of this marker are higher in infant wheezers who have persistent problems and go on to develop asthma compared with those who do not. Once the rhinovirus has gained access to the cell it upregulates the production of inflammatory mediators including its own receptor, ICAM-1. This explains the susceptibility of the asthmatic to rhinovirus. Recently it has been shown that there is impaired production of IFN-β in cultured bronchial epithelial cells harvested from adult asthmatics and then infected with rhinovirus. This results in a failure of the cell to enter apoptosis, which would normally limit viral replication. The consequence of failed apoptosis is cell disruption with release of the virus into the airway where it can extend its infectivity (Wark et al. 2005). Whether this defect is present in younger asthmatics remains to be established but it is very likely to be the case. This impaired innate immune response explains why the virus persists and causes a greater intensity and persistence of airway inflammation.

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Odds ratio for asthma admission

20

Asthma in Infancy and Childhood

The allergy/virus vicious cycle and asthma

18 16 Increased ICAM-1

14

Increased rhinovirus

12

Upset MMP9:TIMP1 ratio

TGFrelease

Neutrophil influx

Remodeling

Deficient IFN-

10 8 6

Allergy and allergen

4

Epithelial damage

2 0

Sensitized Exposed Virus

+ − −

− − +

+ + −

+ − +

+ + +

Fig. 77.5 This study in children illustrates the cumulative effect of allergen sensitization and exposure combined with virus infection on the risk of admission to hospital with an acute asthma attack. (From Murray et al. 2006, with permission.) (See CD-ROM for color version.)

There is a strong interaction between allergy, asthma and virus infection. Thus the risk of acute exacerbation requiring hospital admission has an odds ratio of 19.4 if an allergic asthmatic is exposed to a relevant inhalant allergen and then has a viral infection (Murray et al. 2006) (Fig. 77.5). A number of mechanisms have been postulated to explain this, including the production of virus-specific IgE. It is intriguing that in adults respiratory rhinovirus infection not only enhances airway hyperresponsiveness but also promotes the development of a late asthmatic response and increased eosinophilic airway inflammation in response to allergen (Lemanske et al. 1989). Thus a vicious cycle can be created whereby the allergic asthmatic when exposed to allergen has an increase in expression of ICAM-1 which increases frequency and susceptibility to rhinovirus infection, which in turn promotes airway inflammation in response to allergen (Fig. 77.6).

Eosinophil influx

IL-8 release

Fig. 77.6 Hypothetical synthesis of the mechanisms of airway inflammation and remodeling that links allergy and viral infections with eosinophil- and neutrophil-mediated processes. Thus allergy and allergen exposure increases intercellular adhesion molecule (ICAM)-1, which facilitates rhinovirus access to airway epithelium and together with allergeninduced eosinphil influx causes epithelial damage. Deficient interferon (IFN)-b in epithelial cells leads to virus persistence and spread with release of interleukin (IL)-8, a major neutrophil chemokine. Activated neutrophils by releasing matrix metalloproteinase (MMP)-9 releases transforming growth factor (TGF)-b from the intercellular matrix, which in turn stimulates fibroblasts with consequent remodeling of the airway. Both neutrophils and eosinophils are more likely to persist in the altered airway matrix. (See CD-ROM for color version.)

Demonstration of bronchial hyperresponsiveness is achieved by a variety of provocation strategies including exercise, inhalation of cold dry air, and inhalation of a variety of pharmacologically active factors such as histamine, methacholine and adenosine. The correlation in the degree of hyperresponsiveness using different challenges is significant but there is considerable variation. Furthermore, there is little association between the demonstration of exercise-induced bronchial hyperresponsiveness in the laboratory and the presence of exercise-induced asthma clinically.

Allergy Bronchial hyperresponsiveness The old definitions of asthma incorporated bronchial hyperresponsiveness as the sine qua non of the condition. Certainly hyperresponsiveness is present in most children with asthma and tends to subside in adolescence when asthma remits. There is a weak correlation between the degree of hyperresponsiveness and severity of asthma. However, not only do some asthmatics not have demonstrable hyperresponsiveness but also a number of nonasthmatic conditions such as cystic fibrosis can be associated with hyperresponsiveness. Furthermore, there are inconsistencies in the way that bronchial responses are modified by drugs compared with the response to such drugs clinically (Boner & Warner 1988).

Allergy is one of the major risk factors for the development of asthma. Infants with eczema have a 40% chance of developing asthma, particularly if there is a family history of allergic disease (Bergmann et al. 1998). However, if such infants also show evidence of allergic sensitization to egg, house-dust mite, grass pollen or cat, their risk of developing asthma within the next 3 years is increased further to somewhere between 60 and 80% (Warner 2001) (Fig. 77.7). When infants first develop wheezing, the main consistent factor predicting ongoing disease is the presence of allergy. Furthermore early-onset allergy in children who have already developed asthma is associated with a far higher probability of its persistence through adolescence and into adulthood (Peat et al. 1990)

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Raised Normal 40.7%

IgE Egg

1.4

55.0% 48.3%

IgE Milk

52.8%

IgE Grass pollen

46.3%

IgE Housedust mite

44.4%

0.9–1.4

1.7

1.4–2.1

79.4%

43.4%

1.6

1.3–1.9

1.5

1.2–1.9

66.2%

Fig. 77.7 Data from a follow-up study of infants with atopic eczema demonstrating that sensitization (specific IgE > 0.35 IU/mL) to egg, grass pollen, house-dust mite, and cat but not milk significantly increased the risk of subsequently developing asthma over 3 years of follow-up. (From Warner 2001, with permission.) (See CD-ROM for color version.)

50 40

%

30 20 10 0

Current wheeze Early-onset atopy < 8 years

Bronchial hyper-responsive

Late-onset atopy 10–14 years

Non-atopic by 14 years

Fig. 77.8 A citation classic showing that early-onset allergic sensitization (8 years) is associated with a greater risk of persistent wheeze and bronchial hyperresponsiveness at 14 years. Later-onset allergy has an intermediate effect compared with never being allergic. (From Peat et al. 1990, with permission.) (See CD-ROM for color version.)

(Fig. 77.8). Finally, the degree of allergy, as reflected by total IgE levels to a certain extent, correlates with the severity of asthma clinically (Carroll et al. 2006) (Fig. 77.9). Direct organ challenge with an allergen has been used in studies of bronchial asthma for many years. The initial response to allergen is similar to that of a nonspecific provoking agent such as histamine or methacholine. However the response is somewhat slower to develop and lasts a little longer, although it is self-limiting and at maximum lasts for an hour. It is totally prevented by β agonists but unaffected by steroids. This therefore is only representative of mild episodic disease. However, many individuals after an allergen challenge have a late response commencing 3– 4 hours after exposure that

1598

600

P < 0.001

500 400 300 200 100

70.6%

IgE Cat Dander

P < 0.001

700

1.1–1.7

1.1

Yes No

P < 0.001

800

Relative risk 95% CI for RR Total IgE IU/mL

PART 10

0 FEV1 < 80%

Hospital admission in last yr.

ICS prescribed

Fig. 77.9 Significantly higher total IgE levels in children with more severe asthma as represented by lower forced expiratory volume in 1 s (FEV1), hospital admission in the last year, and requirement for inhaled corticosteroid (ICS). (From Carroll et al. 2006, with permission.) (See CD-ROM for color version.)

lasts for a minimum of 12 hours and sometimes up to 48 hours. The late reaction is more severe than the immediate response and its lung function characteristics are those of severe disease. Furthermore, the presence of dual immediate and late reactions is associated with more severe clinical disease. The late reaction is less responsive to bronchodilators but can be completed abolished by pretreatment with steroids (Warner 1976) (Fig. 77.10). It has been shown in adults and children that an allergen challenge will enhance nonspecific bronchial hyperresponsiveness and this can last for some days or even a week after a single allergen exposure. The presence of a late bronchial reaction is likely to reflect a greater degree of allergen sensitivity and can be generated in the vast majority of patients if a large enough dose of allergen is administered. It has been shown in adults that the onset of the late reaction is associated with an influx of neutrophils and by 12 hours an intense eosinophilic infiltrate (Smith & Deshazo 1993). Thus a late reaction is very much representative of more severe and persistent disease. Allergen avoidance studies in established asthma have shown that it is possible to achieve improvements in control with associated reductions in bronchial hyperresponsiveness and the need for concomitant asthma therapy. This has been best demonstrated in children resident at high altitude for prolonged periods where exposure to house-dust mite allergen is minimal (Peroni et al. 1994). Such children have a progressive reduction in treatment requirements and nonspecific bronchial hyperresponsiveness. Furthermore, after 9 months in a house-dust mite-free environment, the house-dust mite bronchial challenge produces a much less severe bronchial response with coincident falls in specific IgE levels, reflecting a decrease in allergen sensitivity. However, this effect is transient in that a period of sustained reexposure to allergen results in a relapse in all features of the disease (Fig. 77.11).

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Lung function as a % of predicted

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100

90

80

(a) Lung function as a % of predicted

Effect of -agonist

0 10 20 30 Minutes

1 4 Hours

8

12

0 10 20 30 Minutes

4 1 Hours

8

12

100

90

80

(b)

Fig. 77.10 Allergen bronchial challenges inducing both an immediate and late reaction showing the effects of b agonists in abolishing the immediate reaction but having much less effect on the late response (a) while inhaled corticosteroids (ICS) only affect the late reaction (b). (See CD-ROM for color version.)

80 400

60 40

200

20

some physical measures, such as the use of bed barrier systems, removal of carpets and dehumidification, may be of benefit. It is felt that combinations of strategies will be required to achieve meaningful reductions in mite levels sufficient to improve disease to the extent that is observed when resident at high altitude (van Schayck et al. 2007). Finally, as a proof of principle that allergy is important in relation to asthma, there are two modalities of treatment directed specifically at allergy which have been shown in many trials to achieve clinically meaningful improvements. Thus allergen-specific immunotherapy administered by subcutaneous injection has been shown in Cochrane-based systematic reviews to generate a progressive reduction in sensitivity; this achieves improvements in symptoms, decreases in drug requirements, and improvements in bronchial hyperresponsiveness using extracts of house-dust mite, grass pollen, cat and dog (Abrahmson et al. 2001). The second modality of treatment that has achieved significant improvements is the use of antiIgE, which reduces circulating IgE levels and expression of IgE receptor on mast cells and basophils. Successive trials have shown that this therapy can have very particular value at the very severe end of the spectrum of disease (Holgate et al. 2005). Notwithstanding all the above comments, it is clear from epidemiologic studies that at most only 50% of asthma may be attributable to allergy (Pearce et al. 1999; Sunyer et al. 2004). This highlights the fact that there are additional abnormalities in airway structure and function that are important cofactors in the overall disease process (Fig. 77.12).

Clinical features

600 Methacholine PD20 (mg)

Total IgE (IU/mL)

100

Asthma in Infancy and Childhood

Childhood asthma is characterized by recurrent episodes of wheezing. This is commonly associated with episodes of paroxysmal coughing particularly at night. However, it is 45 40

January Total IgE

June

October

35

PD20

Fig. 77.11 Effect of residence at high altitude for 9 months (between October and June), with no exposure to house-dust mites, on mean methacholine provocation dose produces a 20% fall in lung function (PD20) and total IgE in 23 asthmatic children with house-dust mite allergy. There are considerable improvements but relapses over the 3-month period between June and October when the children return to their homes. (From Warner & Boner 1988, with permission.) (See CD-ROM for color version.)

Unfortunately, dust mite avoidance is not easily achieved in a normal domestic environment. Indeed, Cochrane metaanalysis of single-intervention house-dust mite avoidance strategies have suggested that there is little or no benefit to be achieved (Gotzsche et al. 2001). However, it is admitted that

30 %

October

25

Attributable fraction

20 15 10 5 0 Estonia France

Italy

UK

USA Australia

Fig. 77.12 The attributable fraction of asthma due to allergy in several countries. The highest are below 50% in UK, USA and Australia where asthma and allergy have the highest prevalence rates. In Estonia allergy contributes virtually nothing, where infection and possibly pollution have a greater impact. (From Sunyer et al. 2004, with permission.) (See CD-ROM for color version.)

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important to emphasize that paroxysmal cough alone in the absence of wheeze is most commonly not due to asthma (Faniran et al. 1998). Additional symptoms include a sensation of chest tightness, and breathlessness on exertion, particularly outdoors in cold weather. Sometimes there is a history of reaction on exposure to allergens but this is often not an obvious feature. There may be seasonal variation in the problems and most commonly more frequent symptoms at night disturbing sleep. Acute exacerbations are almost invariably associated with intercurrent viral infections. However, the more persistent asthmatic will have wheezing episodes associated with a range of triggers, including exercise, emotional disturbance, laughing, exposure to nonspecific irritant dust and fumes, and inhalation of cold or dry air. In taking a history from a parent it is important to be aware that the word “wheeze” is used very loosely. Thus studies have shown that up to 30% of parents will use other words to describe the wheeze and conversely 30% will label other sounds as a wheeze. Using a video representation of wheezing in childhood, the label can be changed in up to 39% of cases (Cane & McKenzie 2001; Elphick et al. 2001). More recently a study has shown that children with parentalreported wheezing have normal lung function equivalent to that of nonwheezing controls (Lowe et al. 2004). However, when the wheeze has been recorded by a doctor there is a significant difference in lung function. It is also important to be aware that there are many other causes of wheezing in early life, including recurrent milk aspiration, cystic fibrosis, primary ciliary dyskinesia syndrome, foreign body aspiration, and bronchomalacia, bronchogenic cysts and other mediastinal lesions which compress the airway. Features suggesting that a wheeze is not due to asthma are a neonatal onset, associated failure to thrive, production of purulent sputum, a monophonic wheeze, or where the wheezing occurs in association with feeds. When there is any doubt about the diagnosis further investigation is required. Positive features for asthma include association of an allergic disease in the child or family, episodes occurring in the absence of infection such as during exercise, and evidence of significant and rapid improvement when asthma therapy is administered (Table 77.1). Examination of sputum can be particularly useful for identifying an increase in eosinophils. Aspiration may be detected by examining macrophages for fat content by performing a lipid laden macrophage index. The measurement of exhaled nitric oxide (NO) has proved to be useful in aiding diagnosis. There is a strong correlation between raised levels and increased eosinophilic inflammation in the airways (Payne et al. 2001). Levels of exhaled NO may also be used to predict responsiveness to steroids and may be used as a monitor of progress (Pijnenburg et al. 2005). Lung function tests will indicate the presence of airflow limitation, which should be measured frequently to demonstrate variability, and this will be very consistent with the diagnosis. Detection of an immediate

1600

Table 77.1 Features of recurrent wheezing associated with asthma or alternative diagnoses. Wheeze possibly not due to asthma Neonatal onset Associated failure to thrive Purulent sputum Monophonic wheeze Associated with feeds Following an acute choking episode Maternal smoking Wheeze suggestive of asthma Associated eczema and/or positive allergy tests Family history of allergy Exercise induced Episodes without infection Improves with asthma therapy

response to bronchodilator also aids diagnosis. Simple lung function tests can be performed on most children beyond the age of 5 years and there are now techniques for making measurements even in uncooperative younger children using forced oscillation or the so-called interrupter technique for measuring resistance (Stocks et al. 1996). While bronchial hyperresponsiveness has been considered a cardinal feature of the disease, detecting its presence has not been particularly useful in aiding diagnosis. It is sometimes present in nonasthmatics and very variable in patients with established disease. On examination it is worthwhile looking for the stigmata of allergic disease, such as allergic shiners with discolored swollen eyelids, a transverse nasal crease because of constant nose rubbing, and signs of mouth breathing. Serous otitis media with a conductive hearing deficit is common. Atopic eczema occurs in up to 50% of children and may lead to areas of depigmentation, hyperpigmentation, or chronic lichenification even after the eczema has resolved. The highly compliant thoracic cage of the child commonly results in the asthmatic developing significant chest deformity with pectus carinatum associated with Harrison’s sulci and sometimes a degree of spinal kyphosis. Children with more severe disease may be underweight and have some degree of growth delay. Puberty can be dramatically delayed particularly in boys, though for the overwhelming majority the adult height achieved is normal (Balfour-Lynn 1986).

Investigations As part of the differential diagnosis a chest radiograph is necessary for the majority of patients other than those with infrequent episodic disease. Skin-prick testing to demonstrate allergy is a safe and satisfactory way of highlighting the

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atopic constitution. Employing house-dust mite, cat fur and grass pollen extracts together with a positive and negative control will identify 85% of atopic cases in mid-childhood. As indicated above, lung function testing and measurement of exhaled NO can be of value. Currently there is no reliable circulating marker of disease beyond detecting a raised IgE and specific IgE antibodies. The key issue in relation to the clinical assessment of a child with asthma is to establish whether there is episodic or persistent disease. The former will be associated with normal lung function and no symptoms between acute exacerbations and this is obviously often a nonatopic phenomenon with a good prognosis. However, if there is any degree of lung function deficit between episodes or evidence of minor wheezing or paroxysmal coughing particularly at night or with some restriction in exercise tolerance, then this must be classified as persistent disease and mandates the use of preventive therapy.

Management of asthma The management must be tailored to the individual’s requirements based on severity, triggers and age. To some extent the ideal recipe is achieved by a process of trial and error using a rational sequence of therapeutic approaches. Most guidelines have been based on this principle, involving a progressive step-up in treatment until disease control is attained with subsequent reductions in therapy being effected with great circumspection. With experience it is often possible to assess severity and identify the therapeutic strategy immediately without resorting to a series of therapeutic trials. The prerequisite therefore for effective application of these management principles is accurate diagnosis and assessment of severity in the first place. There is now evidence not only that asthma is undertreated as a consequence of inappropriate diagnosis but also sometimes overtreated when the disease is infrequent and only episodic. The goals of treatment must be to return the child to a normal existence with participation in all normal childhood activities, which is possible in many but not all cases. Decisions about the therapeutic strategy must be based on an understanding of the natural history of the disease and its various manifestations as well as an understanding of the pharmacokinetics of drugs to be administered, which will vary with age. A pediatrician must also understand the complexities of the child in his or her environment and should be equipped to give appropriate support to families and all other agencies involved day by day with the child.

Nonpharmacologic management Before launching into pharmacotherapy it is important to be aware that there are a host of nonpharmacologic approaches that have equal importance in the overall strategy. Many

Asthma in Infancy and Childhood

transcend the traditional severity ratings and are applicable to all stages of the disease. This of course involves the avoidance of triggers, of which allergens are prominent, and attention to psychosocial issues. While Cochrane reviews have suggested that house-dust mite avoidance is ineffective, there is a feeling that some physical methods may reduce asthma symptoms. Thus there is a justification for recommending complete barrier bedcovering systems, removal of carpets and soft toys from the bedroom and soft toys from the bed, high-temperature washing of bed linen, and the employment of procedures to reduce indoor humidity. While cat and dog allergy are common it is unfortunately very difficult to avoid these allergens completely. It has been shown that children exposed in school via the clothing of other children who have a pet can increase bronchial hyperresponsiveness and increase abnormalities in lung function and symptoms. Furthermore, cat and dog allergen can be detected in dust from public places such as cinemas and bus seats. (Warner 2000). Removing a pet from the home leads to only very slow reductions in animal allergen levels. Nevertheless it would seem prudent to recommend removal of the pet if a child is clearly allergic to it. A randomized controlled trial of a high-efficiency vacuum cleaner used in homes that did not contain a cat or dog but with a cat- or dog-allergic child showed significant improvements in bronchial hyperresponsiveness (Popplewell et al. 2000). Paradoxically, however, continued high exposure to animal allergens might actually induce a degree of tolerance (Platts-Mills et al. 2001). Sadly there are no good controlled trials in relation to any allergens other than house-dust mite and cockroach in US deprived communities (Gergen et al. 1999). Avoidance of other environmental triggers is clearly important. Exposure to environmental tobacco smoke increases respiratory symptoms and has been identified as causal in increasing exacerbations of asthma in preschool children (Committee on the Assessment of Asthma and Indoor Air 2000). Studies have shown that if parents stop smoking this can result in a decrease in the severity of their child’s asthma (Murray & Morrison 1993). Furthermore, starting smoking as a teenager increases risks of persistence and therefore parents must be encouraged to stop smoking in order to set an appropriate example to their child. There are no studies investigating in detail whether reductions in other domestic pollutants such as nitrogen oxides and volatile organic compounds can have an effect in improving control of asthma, although there is limited evidence that exposure to these factors may increase asthma severity (Asher & Dagli 2004). Allergen immunotherapy as indicated above does have clear-cut benefits in asthma (Abrahmson et al. 2001). However, there remain concerns about potential for severe side effects including fatal anaphylaxis. There are no controlled trials which have made direct comparisons between what

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is perceived as much safer conventional pharmacotherapy compared with allergen immunotherapy. However, in the presence of undoubted benefits from this approach in allergic asthmatics, there remains a need to do further studies to identify vaccines which are safe while retaining efficacy. There is little doubt that this will be achieved with various forms of modification which may well raise the profile of this form of therapy. Treatment of gastroesophageal reflux, which is relatively common in asthmatics, does not achieve any benefit in terms of improving control of asthma symptoms or lung function where the two conditions coexist, although there is some reduction in cough particularly at night. Thus gastroesophageal reflux should be treated in its own right but this approach cannot be expected to impact on the asthma (Cockland et al. 2001). A host of other treatments have at various times been recommended for management, including acupuncture, homeopathy, hypnosis and herbal treatments as well as physical and breathing exercises. None have achieved a sufficient evidence base from which to make any recommendations (Warner 2003).

Pharmacotherapy All guidelines now divide asthma into persistent and episodic and then grade the severity of each. The International Paediatric Consensus Statement employed severity categories in the following order: infrequent episodic disease, frequent episodic disease, and then persistent asthma divided into mild, moderate and severe. An algorithm can then be constructed to indicate a stepwise approach of increasing therapy depending on the frequency and severity of disease (Warner & Naspitz 1998) (Fig. 77.13). In infrequent episodic asthma, where there is normal spirometry between episodes, treatment may be entirely symptomatic employing intermittent short-acting β-agonist inhalations alone. There is no evidence that employing this approach in episodic asthma will lead to deterioration in lung function. With more frequent episodic disease where coughing and wheezing occur more often than once every 4 weeks, it is appropriate to consider the introduction of prophylaxis even if spirometry is normal between episodes. The choice rests between the use of low-dose ICS or a leukotriene receptor antagonist (LTRA). A recent trial has suggested that an LTRA, montelukast, unlike ICS will reduce the frequency of virusinduced wheezing in infancy and early childhood (Bisgaard et al. 2005). Concordance with a single dose of therapy orally is significantly greater than for frequent inhalations, such as the four times daily dose frequency required for cromones. However, low-dose ICS remains the most commonly employed strategy in this situation. Indeed, the START (Inhaled Steroid Treatment As Regular Therapy in Early Asthma) trial has demonstrated the value of once-daily budesonide in individuals (both adults and children) with asthma of recent onset

1602

Infrequent intermittent

Inhaled short-acting beta-agonist as required

Frequent intermittent

Low-dose ICS or LTRA

Mild persistent

ICS mandatory with titration of dose

Moderate persistent

Add LTRA, slow-release theophylline or long-acting beta-agonist

Steriod insensitive

Consider methotrexate, cyclosporin and Anti-IgE (Omalizumab) etc.

Fig. 77.13 Therapeutic algorithm for the management of childhood asthma, incorporating a stepwise sequence taking account of the pattern and severity of disease. (See CD-ROM for color version.)

compared with placebo. The active treatment improved overall asthma control and reduced severe exacerbations and the need for systemic steroids (Pauwels et al. 2003). Among the patients with normal lung function at recruitment (> 80% FEV1), there were equally beneficial effects. There was, however, a small but significant effect on growth in the children. Thus at all times a balance must be achieved between the consequences of the disease and its treatment. As soon as a patient is identified as having any degree of lung function abnormality between episodes, then the classification is of persistent asthma and ICS are mandatory. There is no evidence that starting with very high doses of ICS and stepping down confers any additional benefit over a progressive step-up approach. However, the aim is to achieve good control with the lowest possible dose of ICS. Provided this is equal to or less than 400 μg/day of beclomethasone dipropionate (BDP) or equivalent then there are no major concerns about growth or any other significant side effects (Sharek et al. 2005). Once it is not possible to control of the problem with BDP 400 μg/day or equivalent, which would be indicated by the need for inhaled short-acting β-agonist rescue treatment more frequently than three times a week, then either the dose of ICS is increased or an additional therapeutic intervention is required. This latter may be either an LTRA, long-acting inhaled β agonist (LABA), or an oral slow-release theophylline. The choice will depend on availability, suitability, concordance issues, patient preferences, cost, and individual clinician’s experience. The evidence base for the use of add-on therapy with LABA or LTRA is strong, but this is not the case for theophylline. However, its very low cost may influence decisions

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where resource is limited. SMART (Salmeterol Multicentre Asthma Research Trial) found a higher incidence of death and near-death episodes in patients taking the LABA salmeterol, but this was primarily in the Afro-Carribean population. It was also notable that among those with a clear asthma death, ICS had not been prescribed (Nelson et al. 2006). This has prompted the US Food and Drug Administration to issue a black box warning about the use of LABAs. As the disease becomes progressively more severe with poor response to ICS, patients are classified as having steroidinsensitive asthma. Alternative strategies have included a range of treatments such as immunosuppression with methotrexate or cyclosporin A, continuous subcutaneous infusions of β agonists, omalizumab (anti-IgE), and intravenous immunoglobulin. However, as the disease becomes more difficult to control it is imperative to review the clinical diagnosis in order to establish whether there is reasonable concordance with therapy and to identify whether there are any trigger factors that might be contributing to the more severe disease. In adolescents, dysfunctional laryngeal breathing may be superimposed on asthma and make it appear very much more severe (Niggemann 2002). Detailed investigation is required to elaborate this issue, which can sometimes be effectively handled by the use of either psychotherapy or intervention from a speech and language therapist. Bronchoscopy, bronchoalveolar lavage, and bronchial biopsy are often indicated in these difficult cases. This can establish whether there is ongoing inflammation and whether this is predominantly with eosinophils or neutrophils (Wenzel & Szefler 2006). Persistent eosinophilic inflammation despite steroids may be treated with either alternative immune suppression or, in the presence of a raised IgE, may now be treated with the anti-IgE antibody omalizumab. If the inflammation is predominantly with neutrophils, it is worthwhile attempting treatment with a prolonged course of a macrolide antibiotic such as clarithromycin or azithromycin (Hatipoglu & Rubinstein 2004).

Inhaled medications It has become established dogma that there are advantages in employing the inhaled route for medication administration because this delivers the drug directly to the source of the problem in the airways at high concentrations. However, the total body dose is very much lower. There is often a much more rapid onset of action, such as with the use of shortacting β agonists, and systemic side effects are minimized. However, it is evident from concordance studies that the oral route is preferred by patients (Kelloway et al. 1994). There is an enormous array of inhalation devices that can cause significant confusion, and attention needs to be devoted to education and training in the use of devices. In general it is important to stick to one variety of device in any one patient to avoid confusion. Thus the preventer and reliever medica-

Asthma in Infancy and Childhood

tion should be administered by the same variety of device. Where LABAs are administered with ICS it is now accepted that it is preferable to administer the medications in combination from a single device (Nelson et al. 2003). The National Institute for Clinical Excellence in the UK has issued guidelines for inhaler devices to be used in the management of chronic asthma in childhood. Based on a systematic review of evidence, the recommendation is that in addition to the therapeutic need for a particular drug at a particular dose there are other factors which need to be taken into account when choosing the inhaler device for a child. These include (i) the ability of the child to develop and maintain an effective technique with a specific device, (ii) the suitability of the device for individual children and their carers, and (iii) the child’s preference for, and willingness to use, the device (National Institute for Clinical Excellence 2002). Having selected a device, demonstration and training in its efficient use is essential. Mistakes in inhaler technique are common and there is no doubt that training improves this (Pedersen & Mortensen 1990). This should be checked on a regular basis. Thus not only should symptoms and lung function be monitored but also inhaler technique. In general, the press and breathe pressurized metered-dose inhaler (pMDI) with a suitable spacer device is the first choice for administration of ICS. However, for β agonists a wide range of devices may be considered to take into account frequency of use, portability, and personal preference. It is worthwhile noting that for the administration of β agonists the use of a pMDI and spacer is at least as good as a nebulizer during acute attacks in children aged over 5 years (Dewar et al. 1999). There is now a range of spacer devices. Evidence would suggest that the large-volume spacer (750 mL) is best for children over 3 years of age and the majority will be able to use the mouthpiece. Under 2 years of age there are no adequate trials. However, the low-volume spacer (250 mL) has at least been shown to have some beneficial effects for the administration of β agonists (Wildahaber et al. 1997). There is some suggestion that the pMDI and spacer for administration of ICS reduces oropharyngeal deposition and therefore oral candidiasis. In general, dry powder inhalers (DPI) cannot be reliably used in young children who are unable to generate a sufficient peak respiratory flow rate to disaggregate the particles and achieve airway deposition. However, DPIs can achieve excellent airway deposition in older children and being smaller and therefore more portable may well be preferred. Breathactivated pMDIs are also sometimes preferred by older children. However, while actuation is automatic, thus removing some coordination difficulties, the sound of the device and the sensation of the actuation sometimes hampers continuing inspiration of the aerosol. The use of nonchlorofluorocarbon (CFC) propellants for pMDIs is creating additional confusion. The dose equivalence of β agonists from a CFC or non-CFC inhaler is a 1 : 1 ratio. However, this is not necessarily the case for ICS. At least one

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company now markets a hydrofluoroalkane propellant for the delivery of BDP which is associated with a smaller particle size and therefore greater airway deposition. This leads to higher systemic activity as well as potency (Pedersen et al. 2002). It is therefore imperative if changing to a new propellant for ICS that the doses are retitrated to establish the lowest compatible with good control. It is also important to be mindful of the differing potencies of the range of ICS. Fluticasone is twice as potent as BDP and budesonide and also twice as likely to produce systemic effects. BDP and budesonide are equipotent in terms of antiinflammatory effect. However, budesonide administered as a DPI by Turbohaler is more effective and systemically active than either medication by pMDI (Harrison et al. 2001).

Education and management of psychosocial issues A recent study has highlighted a number of factors that are barriers to good asthma control in children (Walders et al. 2006) (Fig. 77.14). Preeminent in the list is concordance problems. Thus enormous efforts are required to facilitate good concordance by appropriate education in the optimal use of the treatments. However, it is important to be aware that there are other barriers, including concerns around schooling, other emotional factors, economic factors, and lack of understanding about the disease and its treatment. Intervention to deal with these components can achieve significant improvements. The study by Walders et al. (2006) identified the barriers to management then implemented an intervention strategy to address the issues. There was a significant reduction in emer-

80

% of asthmatic children

70 60

Asthma risk profile

50 40 30 20 10 0 Concordance with Rx

School concerns

Emotional factors

Limited knowledge

Economic factors

Fig. 77.14 Factors that inhibit effective control of asthma, with concordance problems predominating. Focusing on these issues can lead to significant improvements in outcomes as shown in Fig. 77.15. (From Walders et al. 2006, with permission.) (See CD-ROM for color version.)

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45

P = 0.05

40 35 30 %

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Intervention Comparison group

25 20 15 10 5 0 ↓ Symptoms

ER visits in 6 mths

ER visits in 12 mths

Fig. 77.15 Changes in effectiveness of asthma control as a consequence of addressing the barriers itemized in Fig. 77.14. (From Walders et al. 2006, with permission.) (See CD-ROM for color version.)

gency room visits over the subsequent 12 months compared with a comparator group where there was no intervention (Fig. 77.15). Facilitating effective management in schools by appropriate training of school personnel and of the children themselves in the school setting can also improve asthma management significantly (McCann et al. 2006).

References Abrahmson, M., Puy, R. & Viner, J. (2001) Allergen immunotherapy for asthma (Cochrane review). Cochrane Library 4. Asher, I. & Dagli, E. (2004) Environmental influences on asthma and allergy. Chem Immunol Allergy 84, 36–101. Balfour-Lynn, L. (1986) Growth and childhood asthma. Arch Dis Child 61, 1049–55. Bergmann, R.L., Edenharter, G., Bergmann, K.E. et al. (1998) Atopic dermatitis in early infancy predicts allergic airway disease at five years. Clin Exp Allergy 28, 965–70. Bisgaard, H., Zielen, S., Garcia, M.L. et al. (2005) Monteleukast reduces asthma exacerbations in 2–5 year old children with intermittent asthma. Am J Respir Crit Care Med 171, 315–22. Blackley, C.H. (1873) Experimental Researches on the Causes and Nature of Catarrhus Aestivus. Bailliere Tindall Cox, London . Blobe, G.C., Schiemann, W.P. & Lodish, H.E. (2000) Role of transforming growth factor beta in human disease. N Engl J Med 342, 1350–8. Boner, A.L. & Warner, J.O. (1988) Allergy and childhood asthma. In: A.B. King, ed. Clinical Immunology and Allergy: The Allergic Basis of Asthma. Bailliere Tindall, London, pp. 217–29. Brewis, R.A.L. (1990) Classic Papers in Asthma Volume 1. The Evolution of Understanding. Science Press, London. Burr, M.L., Wat, D., Evans, C. et al. (2006) Asthma prevalence in 1973, 1998 and 2003. Thorax 61, 296–9. Cane, R.S. & McKenzie, S.A. (2001) Parents interpretations of childrens respiratory symptoms on video. Arch Dis Child 84, 31–4. Carroll, W.D., Lenney, W., Child, F. et al. (2006) Asthma severity and atopy: how clear is the relationship? Arch Dis Child 91, 401–9.

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of airway inflammation and remodelling in ski athletes with and without bronchial hyperresponsiveness to methacholine. Am J Respir Crit Care Med 161, 2086–91. Kaur, B., Anderson, H.R., Austin, J. et al. (1998) Prevalence of asthma symptoms, diagnosis and treatment in 12–14 year old children across Great Britain (international study of asthma and allergies in childhood, UK). BMJ 316, 118–24. Kelloway, J.S., Wyatt, R.A. & Adlis, S.A. (1994) Comparison of patients compliance with prescribed oral and inhaled asthma medications. Arch Intern Med 154, 1349–52. Kim, E.S., Kim, S.H., Kim, K.W. et al. (2007) Basement membrane thickening and clinical features of children with asthma. Allergy 62, 635–40. Kling, S., Donninger, H., Williams, Z. et al. (2005) Persistence of rhinovirus RNA after asthma exacerbation in children. Clin Exp Allergy 35, 672–8. Kuehni, C.E., Strippoli, M.-P.F., Low, N. & Silverman, M. (2007) Asthma in young south asian women living in the United Kingdom: the importance of early life. Clin Exp Allergy 37, 47–53. Laitinen, L.A., Heino, M., Laitinen, A., Carver, T. & Hachtera, T. (1985) Damage to the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131, 599–606. Legg, J.P., Hussain, I.R., Warner, J.A. et al. (2003) Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med 168, 633–9. Lemanske, R.F., Dick, E.C., Swenson, C.A. et al. (1989) Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions. J Clin Invest 83, 1–10. Lodrup-Carlsen, K.C., Ragnhild, H., Ahlstedt, S. & Carlsen, K.-H. (1995) Eosinophil cationic protein and tidal flow volume loops in children 0–2 years of age. Eur Respir J 8, 1148–54. Lowe, L., Murray, C.S., Martin, L. et al. (2004) Reported versus confirmed wheeze and lung function in early life. Arch Dis Child 89, 540– 3. McCann, D.C., McWhirter, J., Coleman, H. et al. (2006) A controlled trial of a school based intervention to improve asthma management. Eur Respir J 27, 921–8. Marguet, C., Jouen-Boedes, F., Dean, T.P. & Warner, J.O. (1999) Bronchoalveolar cell profiles in children with asthma, infantile wheeze, chronic cough or cystic fibrosis. Am J Respir Crit Care Med 159, 1533–40. Marguet, C., Dean, T.P. & Warner, J.O. (2000) Soluble intercellular adhesion molecule-1 (sICAM-1) and interferon-gamma in broncho alveolar lavage fluid from children with airway diseases. Am J Respir Crit Care Med 162, 1016–22. Marguet, C., Dean, T.P., Basayau, J.P. & Warner, J.O. (2001) Eosinophil cationic protein and interleukin-8 levels in bronchial lavage fluid from children with asthma and infantile wheeze. Pediatr Allergy Immunol 12, 27–33. Martin, A.J., Landau, L.I. & Phelan, P.D. (1982) Predicting the course of asthma in childhood. Aust Pediatr J 18, 84–7. Martinez, F.D. & Godfrey, S. (2003) Epidemiology of wheezing in infants and preschool children. In: Wheezing Disorders in the Preschool Child. Martin Dunitz, New York, pp. 1–19. Martinez, F.D., Wright, A.L., Taussig, L.M. et al. (1995) Asthma and wheezing in the first six years of life. N Engl J Med 332, 133–8. Matsumoto, H., Niimi, A., Takemura, M. et al. (2005) Relationship of airway wall thickening to an imbalance between matrix metaloproteinase 9 and its inhibitor in asthma. Thorax 60, 277–81.

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Millar, J. (1769) Observations on the Asthma and on the Whooping Cough. T. Cadell, London, chapter VII, pp. 58–73. Murray, A.B. & Morrison, B.J. (1993) The decrease in the severity of asthma in children of parents who smoke since the parents have been exposing themselves to less cigarette smoke. J Allergy Clin Immunol 91, 102–10. Murray, C.S., Poletti, G., Kebadze, T. et al. (2006) Study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children. Thorax 61, 376– 82. National Institute for Clinical Excellence (2002) Inhaler devices for routine treatment of chronic asthma in older children (aged 5– 15 years). Technology appraisal guidance number 38. National Institute for Clinical Excellence, London. Nelson, H.S., Chapman, K.R., Pyke, S.D. et al. (2003) Enhanced synergy between fluticasone propionate and salmeterol inhaled from a single inhaler versus separate inhalers. J Allergy Clin Immunol 112, 29–36. Nelson, H.S., Weiss, S.T., Bleeker, E.R. et al. (2006) The salmeterol multi-centre asthma research trial: a comparison of usual pharmacotherapy for asthma or usual pharmacotherapy plus salmeterol. Chest 129, 15–26. Niggemann, B. (2002) Functional symptoms confused with allergic disorders in children and adolescents. Pediatr Pulmonol 13, 312– 18. Pauwels, R.A., Pedersen, S., Busse, W.W. et al. (2003) Early intervention with budesonide in mild persistent asthma: a randomised double-blind trial. Lancet 361, 1071– 6. Payne, D.N.R, Adcock, I.M., Wilson, N.M. et al. (2001) Relationship between exhaled nitric oxide and mucosal eosinophilic inflammation in children with difficult asthma, after treatment with oral prednisalone. Am J Respir Crit Care Med 164, 1376–81. Pearce, N., Pekkanen, J. & Beasley, R. (1999) How much asthma is really attributable to atopy? Thorax 54, 268– 72. Peat, J.K., Salome, C.M. & Woolcock, A.J. (1990) Longitudinal changes in atopy during a 4 year period. J Allergy Clin Immunol 85, 65–74. Pedersen, S. & Mortensen, S. (1990) Use of different inhalation devices in children. Lung 168 (suppl.), 653– 7. Pedersen, S., Warner, J.O., Wahn, U. et al. (2002) Growth, systemtic safety and efficacy during one year of asthma treatment with different beclamethasone dipropionate formulations: an open label randomised comparison of extra fine and conventional aerosols in children with asthma. Pediatrics 109, 1–10. Peroni, D.G., Bonner, A.L., Valone, G. et al. (1994) Effective allergen avoidance at high altitude reduces allergen induced bronchial hyperresponsiveness. Am J Respir Crit Care Med 149, 1442–6. Pijnenburg, M.W., Bakker, E.M., Hop, W.C. & De Jongste, J.C. (2005) Titrating steroids with exhaled nitric oxide in children with asthma. A randomised controlled trial. Am J Respir Crit Care Med 172, 831–6. Pijnenburg, M.W., Flaw, S.E., Hop, W.C. & De Jongste, J.C. (2006) Daily ambulatory exhaled nitric oxide measurements in asthma. Pediatr Allergy Immunol 17, 189– 93. Platts-Mills, T.A.E, Vervloet, D., Thomas, W.R. et al. (1997) Indoor allergens and asthma: report of the Third International Workshop. J Allergy Clin Immunol 100, S2–S24. Platts-Mills, T.A.E., Vaughan, J., Squillace, S. et al. (2001) Sensitisation, asthma and a modified Th2 response in children exposed to cat allergens. A population based cross sectional study. Lancet 357, 752–6.

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Pohaunek, P., Warner, J.O., Turzikova, J. et al. (2005) Markers of eosinophilic inflammation and tissue remodelling in children before clinical diagnosed bronchial asthma. Pediatr Allergy Immunol 16, 43–51. Popplewell, E.J., Innes, V.A., Lloyd-Hughes, S. et al. (2000) The effect of high efficiency and standard vaccuum cleaners on mite, cat and dog allergen levels and clinical progress. Pediatr Allergy Immunol 11, 142– 8. Roberts, G., Patel, N., Levi-Schaffer, F., Habibi, P. & Lack, G. (2003) Food allergy as a risk factor for life-threatening asthma in childhood: a case controlled study. J Allergy Clin Immunol 112, 168–74. Robertson, C.F., Rubinfeld, A.R. & Bowes, G. (1992) Pediatric asthma deaths in Victoria: the mild are at risk. Pediatr Pulmonol 13, 95– 100. Roorda, R.J., Gerritsen, J., van Aalderen, W.M. et al. (1994) Follow up of asthma from childhood to adulthood: influence of potential childhood risk factors on the outcome of pulmonary function and bronchial hyperresponsiveness in adulthood. J Allergy Clin Immunol 93, 575–84. Saglani, S., Malmström, K., Pelkonen, A.S. et al. (2005) Airway remodelling and inflammation in symptomatic infants with reversible airflow obstruction. Am J Respir Crit Care Med 171, 722–7. Sharek, P.J., Bergmann, D.A. & Ducharme, F. (2005) Beclomethasone for asthma in children: effects on linear growth. Cochrane Library 3. Sigurs, N., Gustafsson, P.M., Bjarnason, R. et al. (2005) Severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13. Am J Respir Crit Care Med 171, 137–41. Smith, D.L. & Deshazo, R.D. (1993) Bronchoalveolar lavage in asthma: an update and perspective. Am Rev Respir Dis 148, 523–32. Stein, R.T., Sherrill, D., Morgan, W.J. et al. (1999) Respiratory syncitial virus in early life and the risk of wheeze and allergy by 13. Lancet 353, 541–5. Stevens, C.A., Turner, D., Kuehni, C.E., Couriel, J.M. & Silverman, M. (2006) The economic impact of preschool asthma and wheeze. Eur Respir J 21, 1000–6. Stocks, J., Sly, P.D., Tepper, R.S. & Morgan, W.J. (1996) Infant Respiratory Function Testing. Wiley Liss, New York. Sunyer, J., Jarvis, D., Pekkanen, J. et al. (2004) Geographic variations in the effect of atopy on asthma in the European community respiratory health study. J Allergy Clin Immunol 114, 1033–9. Turner, S.W., Palmer, L.J., Rye, P.J. et al. (2004) The relationship between infant airway function, childhood airway responsiveness and asthma. Am J Respir Crit Care Med 169, 921–7. van Schayck, O.C.P, Maas, T., Kapper, J. et al. (2007) Is there any role for allergen avoidance in the primary prevention of childhood asthma? J Allergy Clin Immunol 119, 1323–8. von Mutius, E. (1999) Environmental factors and rising time trends in prevalence and severity. In: Holgate, S., Boushey, H. & Fabbri, L., eds. Difficult Asthma. Martin Dunitz, London. Walders, N., Kercsmar, C., Schluchter, M. et al. (2006) An interdisciplinary intervention for undertreated pediatric asthma. Chest 129, 292–9. Wark, P.A.B., Jonhston, S.L., Bucchieri, F. et al. (2005) Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. J Exp Med 201, 937–47. Warner, J.A. (2000) Controlling indoor allergens. Pediatr Allergy Immunol 11, 209–19. Warner, J.O. (1976) The significance of late reactions following bronchial challenge with house dust mite. Arch Dis Child 51, 905–11.

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Warner, J.O. (2001) A double blind randomised placebo controlled trial of cetirizine in preventing the onset of asthma in children with atopic dermatitis: 18 months treatment and 18 months post treatment follow up. ETAC study group. J Allergy Clin Immunol 108, 929– 37. Warner, J.O. (2003) Guidelines for treatment of asthma. In: Leung, D.Y.M., Sampson, H.A., Geha, R.S. & Szefler, S.J., eds. Pediatric Allergy Principles and Practice. Mosby, St. Louis, Missouri, pp. 350–6. Warner, J.O. & Naspitz, C.K. (1998) Third international paediatric consensus statement on management of childhood asthma. Pediatr Pulmonol 25, 1–17.

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Warner, J.O., Nikolaizic, W.H., Besley, C.R. & Warner, J.A. (1998) A childhood asthma death in a clinical trial; potential indicators of risk. Eur Respir J 11, 229–33. Wenzel, S. & Szefler, S.J. (2006) Managing severe asthma. J Allergy Clin Immunol 112, 508–11. Wildahaber, J.H., Devadason, S.G., Heydon, M.J. et al. (1997) Aerosol delivery to wheezy infants: a comparison between a nebuliser and two small volume spacers. Pediatr Pulmonol 23, 212–16. Williams, H. & McNicol, K.N. (1969) Prevalence, natural history, and relationship of wheezing bronchitis and asthma in children. BMJ 4, 321–5.

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Pathogenesis of Asthma Stephen T. Holgate

Summary While asthma is considered an inflammatory disorder of the conducting airways, it is becoming increasingly apparent that the disease is heterogeneous with respect to immunopathology, clinical phenotypes, response to therapies, and natural history. Once considered purely an allergic disorder dominated by Th2type lymphocytes, IgE, mast cells, eosinophils, macrophages, and cytokines, the disease also involves local epithelial, mesenchymal, vascular and neurologic events that are involved in directing the Th2 phenotype to the lung and through aberrant injury-repair mechanisms to remodeling of the airway wall. Structural cells provide the necessary “soil” upon which the “seeds” of the inflammatory response are able to take root and maintain a chronic phenotype and upon which are superimposed acute and subacute episodes usually driven by environmental factors such as exposure to allergens, microorganisms, pollutants or caused by inadequate antiinflammatory treatment. Greater consideration of additional immunologic and inflammatory pathways are revealing new ways of intervening in the prevention and treatment of the disease. Thus increased focus on environmental factors beyond allergic exposure (such as virus infection, air pollution, and diet) are identifying targets in structural as well as immune and inflammatory cells at which to direct new interventions.

tributions of smooth muscle contraction, edema and remodeling of the formed elements of the airways. Heterogeneity of asthma also relates to the different response to therapies. Asthma is considered a good example of gene–environment interactions, although no single gene or environmental factor accounts for the disease. This chapter focuses on the pathophysiologic events underlying the inflammatory and remodeling response.

Airway inflammation is fundamental to asthma pathogenesis Airway inflammation in asthma is a multicellular process involving mainly eosinophils, neutrophils, CD4+ T lymphocytes and mast cells, with eosinophilic infiltration being the most striking feature (Kay 2005) (Fig. 78.1). The inflammatory process is largely restricted to the conducting airways but as the disease becomes more severe and chronic the inflammatory infiltrate spreads both proximately and distally to include the small airways and in some cases adjacent alveoli

Introduction Asthma is a disorder of the conducting airways, which contract too much and too easily spontaneously and in response to a wide range of exogenous and endogenous stimuli. This airway hyperresponsiveness is accompanied by enhanced sensory irritability of the airways and increased mucus secretion. The different clinical expressions of asthma involve varying environmental factors that interact with the airways to cause acute and chronic inflammation, and the varying con-

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Fig. 78.1 Cross-section of a small asthmatic airway showing abundant inflammation both inside and outside the smooth muscle and remodeling as evidenced by the deposition of new matrix beneath the epithelium and in all layers of the airway. (Holgate & Polosa 2006) (See CD-ROM for color version.)

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(Kraft et al. 1999). The inflammatory response in the small airways appears to be predominantly outside the airway smooth muscle (Fig. 78.1), whereas in the large airways inflammation of the submucosa dominates (Haley et al. 1998). This Th2 type of inflammation is common to chronic allergic inflammatory responses at multiple tissue sites and indeed is seen at these sites in patients with asthma who frequently express comorbidities such as chronic rhinitis, sinusitis, atopic dermatitis, and food allergy (Kay 2001). There continues to be debate about the specific contribution of individual cell types in these other diseases, but at least in asthma with an allergic component a common theme is now emerging. Other chapters have provided considerable detail of the potential individual roles of these cells and their mediators in the allergic tissue response, so only brief mention is made of them here with respect to their relevance to human asthma.

The immune response A fundamental feature of asthma associated with allergic sensitization is the ability of the airway to recognize common environmental allergens and to generate a Th2 cytokine response to them. Recognizing that in excess of 40% of the Western population is atopic (i.e., have elevated IgE to common environmental allergens), only about 7% express their atopy in the form of asthma (Beasley et al. 2001). Therefore, a crucial question to ask is what mechanisms account for the specific expression of atopy in the conducting airways and why some patients despite being highly atopic have no evidence of asthma? One explanation is the way that the immune response to allergens is regulated at the surface of the airways. A fundamental feature of allergen sensitization is the uptake and processing of inhaled allergens by dendritic cells situated in the airway epithelium and submucosa and which extend their processes to the airway surface (von Garnier et al. 2005; Hammad & Lambrecht 2006). Uptake of allergen is enhanced by IgE bound to high-affinity receptors on dendritic cells that facilitate allergen internalization (Kitamura et al. 2007). Once inside the dendritic cell, processing of allergens by cathepsin S and the subsequent selection of peptides loaded onto and presented by HLA molecules (MHC class II) is fundamental to the ability of these cells to serve as antigen-presenting cells to T lymphocytes (Riese & Chapman 2000). Once the dendritic cell has engaged allergen, it receives signals to migrate to local lymphoid collections where antigen presentation takes place. Its specific chemokine receptors, including CCR7 and its ligands CCL19 and CCL21 (and to a lesser extent CXCR4 and its ligand CXCL12), are involved in this chemotactic migration to enable contact with naive T cells (Humrich et al. 2006; Pease & Williams 2006) (see Chapter 8). Presentation of a selected antigen peptide to the T-cell receptor initiates sensitization and the subsequent immune response to the specific allergen

Pathogenesis of Asthma

(Smit & Lukacs 2006). The nature of this immune response depends on whether engagement of selective costimulatory molecules occurs in parallel. For efficient antigen-dependent T-cell activation engagement of either CD80 (B7.1) or CD86 (B7.2) on the dendritic cells with CD28 on T cells leads to sensitization, whereas lack of, or inefficient, engagement of these costimulatory molecules may lead to anergy (Larche et al. 1998; van Rijt et al. 2004). An alternative method of preventing sensitization and rendering T cells anergic is engagement of a second costimulatory molecule, cytotoxic T-lymphocyte antigen (CTLA)4, which has a higher affinity than either CD80 or CD86 for CD28 and can therefore prevent CD80/CD86 costimulation (Jaffar et al. 1999a,b). This is the basis of the successful clinical application of the CTLA4–immunoglobulin fusion protein abatacept, used as an immunomodulatory agent in such diseases as rheumatoid arthritis and in an animal model of allergen-induced airway inflammation (Weinblatt et al. 2006). In more severe asthma the relative importance of CD28 signaling in supporting the inflammatory response is reduced (Lordan et al. 2001). Under these circumstances other costimulatory pathways are thought to be engaged in T-cell activation, including ICOS and its multiple possible ligands B7-RP1, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3 and B7-H4 (B7-S1) (Greenwald et al. 2005) as well as OX40 (CD134) and its ligand OX40L (CD252) (Salek-Ardakani et al. 2003; Burgess et al. 2005). The capacity of dendritic cells to generate interleukin (IL)12 determines the balance between Th1 and Th2 responses, IL-12 polarizing T-cell differentation in favor of a Th1 response (Kuipers et al. 2004). However, while IL-12 is able to counteract Th2 sensitization, it is also able to contribute to maximal expression of allergic airway disease post sensitization (Meyts et al. 2006). Once sensitized, T cells not only migrate back to the airways to the site of antigen presentation under the influence of the chemokines CCL11, CCL24, CCL26, CCL7, CCL13, CCL17 and CCL22 (which interact with their reciprocal receptors including CCR3, CCR4, CCR5, CCR6, CCR7 and CCR8) (Garcia et al. 2005; Kallinich et al. 2005), but these cells also become potent producers of a range of cytokines, the majority of which are expressed on the long arm of chromosome 5, namely IL-3, IL-4, IL-5, IL-6, IL-9, IL-13 and granulocyte– macrophage colony-stimulating factor (GM-CSF) (Kay 2006; Ryu et al. 2006). IL-1β produced by macrophages, monocytes, dendritic cells, and smooth muscle and epithelial cells in large amounts (Schmitz et al. 2003; Dragon et al. 2006) and IL-2 produced by T cells further enhance antigen-induced T-cell proliferation and maturation (Anderson 2002). There is now persuasive evidence that at least in mild to moderate asthma Th2-type cells dominate the T-cell repertoire in the airways (Anderson 2002). Through cytokine production, they have the capacity to recruit secondary effector cells such as macrophages, basophils and eosinophils into the inflammatory zone where these cells become primed and

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IgE FceRI B cell Plasma cell Allergen

Mast cell Basophil

IL-4 IL-13

Histamine Leukotrienes Prostaglandins Cytokines Basic proteins & enzymes

Allergic asthma

Fig. 78.2 Schematic representation of the inflammatory cascade in allergic asthma. (Bradding et al. 2006) (See CD-ROM for color version.)

IL-5 Antigen-presenting cell

Th2 cell

Eosinophil

subsequently activated for mediator secretion (Fig. 78.2). There has also been recent interest in the potential role of IL-4- and IL-13-secreting, CCR9+ natural killer (NK) T cells in orchestrating the inflammatory response in chronic asthma (Sen et al. 2005; Akbari et al. 2006; Umetsu & Dekruyff 2006) (see Chapter 3) (Pham-Thi et al. 2006; Thomas et al. 2006), although initial findings of their primacy has been challenged (Ho 2007; Vijayanand et al. 2007). Overall, it is the Th2-type T cell bearing the CCR4 chemokine receptor that is the cell which dominates the allergic immune response and may be the cell most probably responsible for contributing to the ongoing chronic inflammatory response. Indeed, asthma severity is reported to be associated with an increase in CCR4+ T cells (Ishida et al. 2006). Thus, inhibitors of CCR4 (e.g., the antibodydependent cell cytotoxic monoclonal antibody KM2760) could be highly effective in the treatment of asthma by inactivating or removing CCR4+ Th2 cells (Ishida et al. 2006). While Th2-type T cells may be important in the pathogenesis of mild to moderate asthma, as the disease becomes more severe and chronic Th1-type T cells are recruited that have the capacity to secrete tumor necrosis factor (TNF)-α and interferon (IFN)-γ (Truyen et al. 2006). This more complicated T-cell profile may help to explain the aggressive and tissuedamaging aspects of the immune response in more severe disease. Although these Th1-type cells, as well as CD8+ T cells, have been incriminated in both more severe asthma (Hamzaoui et al. 2005) and during asthma exacerbations (especially following virus infection) (O’Sullivan 2005), the precise mechanisms whereby they achieve this is still largely unknown. Although the T lymphocyte has been given primacy with regard to the orchestration of the inflammatory response in asthma, there have been no studies using selective T-cell-depleting strategies with either monoclonal antibodies or drugs in mild to moderate asthma (Kon & Kay 1999; Bharadwaj & Agrawal 2004). However, in a small proof of concept study, a single infusion of anti-CD3 improved lung function in patients with severe disease (Kon et al. 1998). The immunosuppressant approach to asthma treatment has not been pursued largely due to

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difficulties in developing calcineurin inhibitors such as cyclosporin A and tacrolimus in an inhaled form and problems with side effects.

Mast cells The mast cell has long been associated with asthma. The early asthmatic reaction following inhaled allergen provocation is mast-cell dependent and drugs such as sodium cromoglycate and nedocromil sodium are believed to mediate their effects by inhibiting mast cell mediator secretion (Holgate 1996). For many years it was thought that the mast cells present in the airway epithelium and submucosa were fundamental to the contribution that these cells make to asthma (Shahana et al. 2005), but recent studies have indicated that mast cells deeper in the airway wall are also important. While undoubtedly mucosal-type mast cells (tryptase positive, chymase negative) under the control of T lymphocytes (specifically IL-3, IL-4 and IL-9) are highly responsive to inhaled allergens (and possibly other stimuli such as hypertonicity) in causing bronchoconstriction, there has been recent interest in mast cells present deeper in the airway wall and in the more peripheral airways as being more fundamental to some of the chronic inflammatory responses in asthma (Bradding et al. 2006). Of particular interest has been the discovery that in chronic asthma (but apparently not in eosinophilic bronchitis), mast cells are markedly increased in association with airway smooth muscle in both the large and the small airways (Brightling et al. 2002). It is at this site that mast cells interact with airway smooth muscle through the action of autacoid mediators such as leukotriene (LT)D4, prostaglandin (PG)D2 and histamine, but also contributing to fibrogenesis and an increase in smooth muscle as part of the “remodeling” response (Kaur et al. 2006; Plante et al. 2006). Although many factors are involved in the regulation of mast cell function in the submucosa and deeper in the airway wall, mast cells in the airway smooth muscle differ from mucosal mast cells in the being of the connective

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tissue-type (tryptase positive, chymase positive, and carboxypeptidase positive) and in being more dependent on stem cell factor (SCF) for their survival. SCF (c-kit ligand) is produced by the epithelium, smooth muscle and fibroblasts and is upregulated in asthma (Al Muhsen et al. 2004; Plante et al. 2006). In addition, CXCL8 and CXCL10 produced by airway smooth muscle itself are not only important in the recruitment of mast cells into this compartment by interacting with their receptors CXCR3 and CXCR2 respectively, but also in their priming for enhanced mediator secretion (Woodman et al. 2006; Scott & Bradding 2005). In the reverse direction, mast cells secrete CCL19 that, through its receptor CCR7, stimulates airway smooth muscle migration and probably contributes to the smooth muscle hyperplasia (Kaur et al. 2006). Thus, airway smooth muscle is partly dependent on mast cells for its survival and enhanced contractility, whereas mast cells are dependent on smooth muscle factors for their survival and activation. On activation, irrespective of their subtype, mast cells release preformed granule-associated mediators such as histamine, tryptase and other proteases, heparin and some cytokines, as well as newly formed eicosanoids that include PGD2, thromboxane (TX)A2, and the cysteinyl leukotreines (LTC4 and LTD4) (Bradding et al. 2006). These mediators are potent smooth muscle contractile agents and also increase microvascular permeability. Both PGD2 and LTD4 interact with cell-surface receptors on eosinophils, macrophages, basophils and mast cells where they serve as chemoattractant as well as priming agents (Ogawa & Calhoun 2006). Thus, cysteinyl leukotriene antagonists such as montelukast and pranlukast are not only able to block the acute effects of leukotrienes on the formed elements of the airway, but also exert some antiinflammatory action. The mast cell is also well endowed with neutral proteases (Reed & Kita 2004). One that has attracted particular interest is tryptase, a tetrameric neutral protease that has a preferential action on G protein-coupled protease-activated receptor type 2 (PAR2), which is present on many cell types including epithelial cells, fibroblasts, smooth muscle cells, endothelial cells, and on a number of inflammatory cells (Cairns 2005; Hallgren & Pejler 2006). The exact role of the PAR2 receptor in asthma has yet to be clearly defined, but there is increasing evidence that its activation is involved in mesenchymal cell proliferation and airway wall remodeling (Berger et al. 2001). Mast cells and eosinophils are also an important source of the zinc-dependent matrix metalloproteinases (MMP)-3 and MMP-9, which through their interaction with matrix proteins and proteogylcans have also been incriminated in airway wall remodeling (Dahlen et al. 1999; Wenzel et al. 2003). Activation of mast cells, particularly through the highaffinity IgE receptor (FCεRI), leads to the release of certain cytokines that are packaged within mast cell granules, including TNF-α, IL-4 and IL-5 (Bradding et al. 1995; Wilson et al. 2000), but also induces transcription of these and other cytokines

Pathogenesis of Asthma

and chemokines that are then secreted over a period of up to 72 hours following cell perturbation (Okayama et al. 1995, 2003). These cytokines and chemokines undoubtedly contribute to the ongoing inflammatory response in asthma and may be partly responsible for the allergen-induced late-phase inflammatory response characteristic of allergen provocation. Blockade of IgE using the monoclonal antibody omalizumab leads to marked attenuation of both the early and late phase allergen-induced bronchoconstrictor and skin inflammatory responses, linking both to mast cell activation (Fahy et al. 1997; Ong et al. 2005). However, the precise mechanisms through which late-phase bronchoconstriction and the associated inflammatory cell influx with neutrophils and eosinophils lead to bronchoconstriction is not fully established, although its partial attenuation with antileukotriene drugs suggests at least some role for cysteinyl leukotrienes (Leigh et al. 2002).

Eosinophils A very prominent cell in the inflammation of allergic asthma is the eosinophil leukocyte, which is present not only in the airway wall but in uncontrolled asthma is also found in large numbers in the sputum and bronchoalveolar lavage fluid (Kay 2005; Lemiere et al. 2006). These cells are in large part initially recruited from the bone marrow as CD34 precursors, following the release of PGD2, cysteinyl leukotrienes, cytokines and chemokines from the asthmatic airway. The developing eosinophils then pass from the circulation via the microvascular compartment into the airway wall. There has been much research on the mechanisms involved in this recruitment, including the role of specific chemokines and adhesion molecules (reviewed in detail elsewhere in this book). Suffice to say that IL-3 and GM-CSF and eotaxins 1–3 are crucial to the early derivation of eosinophils from CD34+ bone marrow precursor cells, with IL-5 being responsible for their maturation and recruitment into the airways (Robinson et al. 1999; Sehmi et al. 2003) (Fig. 78.3). Eosinophils are a rich source of granule basic proteins, such as major basic protein, eosinophil peroxidase, and eosinophil cationic protein, and also have the capacity to generate eicosanoids such as prostacyclin (PGI2) and cysteinyl leukotrienes and release potentially tissue-damaging superoxide and a range of cytokines and chemokines (Kariyawasam & Robinson 2006). The dramatic reduction in sputum and tissue eosinophils that occurs on treatment of asthma with inhaled or oral corticosteroids associated with clinical improvement has led to the idea that eosinophils are fundamental to airway dysfunction in asthma and are the principal target for this drug class (Djukanovic et al. 1992, 1997). Undoubtedly, eosinophils play an important role in the allergic inflammatory response, but recently their primacy in the inflammatory milieux of asthma has been challenged

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CD34+ precursor Th2 cell

IL-3, GM-CSF

IL-5

Commitment

IL-5

IL-5

IL-5

Immaturity

Survival Activation IL-3, GM-CSF

Maturity

IL-3, GM-CSF

(Flood-Page et al. 2003a). Following allergen challenge in moderately severe asthma, the administration of a humanized blocking IgG monoclonal antibody directed to IL-5 resulted in a dramatic reduction (> 80%) in circulating and sputum eosinophils. However, this was not accompanied by any inhibitory effect on the allergen-induced late-phase reaction in the airways or skin or acquired airway hyperreactivity (Leckie et al. 2000; Phipps et al. 2004). In a large clinical study three injections of anti-IL-5 given 2 weeks apart also had a dramatic effect on circulating and sputum eosinophils but paradoxically did not affect any of the clinical outcome measures of asthma including baseline lung function (Fig. 78.4). These studies questioned either the primacy given to the eosinophil as the dominant inflammatory cell of asthma or the effectiveness of anti-IL-5 to deplete eosinophils. Thus, in a series of subsequent bronchial biopsy studies, anti-IL-5 antibody therapy only reduced airway eosinophils by about 50% and it has been suggested that the remaining 50% of eosinophils could account for ongoing asthma symptomology (FloodPage et al. 2003a) (Fig. 78.4). Anti-IL-5 monoclonal antibody also induced bone marrow eosinophil maturation arrest and decreased CD34+ eosinophil progenitors in the bronchial mucosa of atopic asthmatics (Flood-Page et al. 2003b). Further studies have demonstrated that eosinophil depletion using this approach was able to modify certain matrix proteins in the subepithelial basement membrane such as tenascin C, lumican and procollagen 3 (Flood-Page et al. 2003b) (Fig. 78.4). Based on these findings, it has been suggested that the eosinophil may be playing a more important role in airway remodeling than has hitherto been recognized (Kay et al. 2004). Persistence of some eosinophils in asthmatic airway tissue in

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Fig. 78.3 Th2 cytokines involved in the maturation and recruitment of eosinophils from CD34+ precursors in the bone marrow and airway wall. Note the prominent contribution of IL-5. See text for definition of abbreviations. (See CD-ROM for color version.)

the presence of IL-5 blockade might be explained by some loss of the IL-5 receptor from such cells when eosinophils are recruited into the airways (Liu et al. 2002). A positive contribution of eosinophils to tissue remodeling is supported by their capacity to generate transforming growth factor (TGF)β1 and support fibroblast proliferation, collagen synthesis and myofibroblast maturation (Williams & Jose 2000). Attraction of eosinophils to the site of inflammation is largely mediated through eotaxins 1, 2 and 3, macrophage chemotactic factor (MCP)-3, MCP-4, and RANTES (Williams & Jose 2000). These chemokines interact with the CCR3 and, to a lesser degree, CCR5 receptors on eosinophils and, as with other chemokines, not only provoke their directed migration but also their priming. Therefore, it is somewhat disappointing that despite showing activity in mouse models (Das et al. 2006), CCR3 receptor antagonists have so far proven disappointing when tested in the clinic. There is also considerable debate about how eosinophils are cleared from the airways in asthma. At one time it was thought that programmed cell death, mediated by withdrawal of specific cytokines such as IL-5 and GM-CSF, resulted in accelerated programmed cell death (Meagher et al. 1996) and uptake by macrophages and epithelial cells (Sexton et al. 2004). Similarly, it was believed that an important component of corticosteroid action was through acceleration of this apoptotic response (Meagher et al. 1996). However, detailed examination of asthmatic tissue has demonstrated little evidence of eosinophil apoptosis; rather eosinophils display an unusual form of cytolysis that leaves their eosinophil granules intact within the airway wall even though the cell membrane and cytoplasm have disappeared (Uller et al. 2004). Corticosteroids

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750 mg

0.3

0.2

0.2

0.1

0.0 Baseline2

0.1

4 5

8

9

12

16

20

0.0 Endpoint

Week

(a)

109/L

15

10 5 0

Pre Post 52% suppression

10 5 0

Pre

Post

0.4 0.3 0.2 0.1 0.0

Blood

Pre Post 88% suppression Placebo

0.4 0.3 0.2 0.1 0.0

Pre

(b)

Post

Bronchial eos/mm2

0.3

Bone marrow

Bronchial eos/mm2

0.4

% of eos in BM

0.4

15

% of eos in BM

Blood eosinophils (109/L)

Mepolizumab anti-IL-5 0.5

Blood eos

250 mg

109/L

Placebo

Blood eos

Treatment 0.5

Pathogenesis of Asthma

75 50 25 0

50 25 0

Post

Mild asthmatic pre-anti-IL-5

Post

P = 0.08 2400 Density x thickness

2400 Density x thickness

Normal control subject

Pre

Tenascin C P = 0.004

1800 1200 600 0

(c)

Pre Post 55% suppression

75

P = 0.01 Pre

Bronchial mucosa

Mild asthmatic post-anti-IL-5

(d)

1800 1200 600 0

Pre Post Anti-IL-5

Pre

Post Placebo

Fig. 78.4 Effect of intravenous injections of anti-IL-5 monoclonal antibody (mepolizumab) on (a) circulating eosinophils in over 100 moderately severe asthmatic patients; (b) comparative effect on eosinophils at different sites; (c) immunoreactivity to tenascin C; (d) tenascin C quantified by image analysis. (Reproduced with permission from (b & c) Flood-Page et al. 2003b (a & d) Flood-Page et al. 2007). (See CD-ROM for color version.)

have also been shown to increase clearance of airway eosinophils through egression into the airway lumen as well as by inhibiting chemokine production (Uller et al. 2006). In an animal model, activation of apoptosis through the Fas receptor has been shown to magnify rather than resolve asthmatictype inflammation (Uller et al. 2005). Thus, while much has been learnt about eosinophils in asthma, there still remain many unanswered questions. The role of neutrophils is discussed later in this chapter.

to play an increasingly important role and may well account for the ongoing chronic inflammation associated with their preferential infiltration into the airway wall in patients with longstanding corticosteroid-resistant disease (Sher et al. 1994; Loke et al. 2006). It is clear that more attention focused on these cells is required, especially how they fit into the overall Th2 paradigm and whether they represent a separate component of the immune and inflammatory response which has not yet been adequately explored.

Monocytes and macrophages

Basophils

Monocytes are able to differentiate into macrophages and dendritic cells in the presence of GM-CSF (Gajewska et al. 2003), the latter requiring IL-4 (Webb et al. 2007). In chronic asthma both monocytes and macrophages are prominent cells in the airway mucosa and undoubtedly play an important role in disease pathogenesis. While these cells are an important source of cysteinyl leukotrienes, reactive oxygen and a variety of lysosomal enzymes, their precise role in mediating tissue damage and contributing to the overall airway pathology of asthma is largely unknown. In corticosteroidrefractory asthma, monocytes and macrophages are thought

Although basophils have largely been thought of as circulating IgE-triggered inflammatory cells, in certain types of immune response they do accumulate in tissues. The discovery of unique basophil-specific markers such as basogranulin (Mochizuki et al. 2003; Agis et al. 2006) has enabled their identification in the airways of subjects with asthma (Macfarlane et al. 2000; Kepley et al. 2001). However, at this time, it is not clear what their precise role is in either acute or chronic disease, although it is known that they share many of their recruitment mechanisms with eosinophils and are likely to be accompaniments of eosinophil infiltration (Gangur et al. 2003).

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Inflammatory cell products environmental agents

Nonallergic (late-onset or intrinsic) asthma Although the majority of asthma is associated with atopy and has its onset in early childhood across all ages and especially after the age of 40 years, there are forms of asthma that appear to be independent of atopy (Humbert 2000). These nonallergic forms of the disease have been subject to careful comparative investigation but so far no clear pathogenic pathways have been identified (Humbert et al. 1999; Corrigan 2004). There is some evidence to suggest that local IgE mechanisms may be involved with the detection of IgE isotype switching in airway biopsies, but the clinical significance of this has yet to be determined (Ying et al. 2001; Jayaratnam et al. 2005). The immunopathology of late-onset nonallergic asthma appears to be very similar to that of allergic asthma, although there have been some differences reported in the relative proportion of the various inflammatory cells present (Ying et al. 1997; Ying et al. 1999; Strek 2006). It is important to recognize that some forms of late-onset asthma have an occupational cause due to non-IgE-dependent sensitization to chemicals in the workplace (Malo 2005; Slavin 2005). In addition to immunologic sensitization to chemicals, both intrinsic asthma (Nahm et al. 2002) and asthma caused by diisocyanate exposure are associated with antibodies directed to epithelial components (Ye et al. 2006a). Other autoantibodies in asthma include those directed to heat-shock protein (HSP)-70 (Yang et al. 2005), CD28 (Neuber et al. 2006), and α-enolase (Nahm et al. 2006). Whether such autoantibodies are truly pathogenetic or related more to tissue damage and inflammation remains to be established (Ye et al. 2006b). Whatever the mechanisms, this type of asthma is likely to be highly heterogeneous, exhibiting overlap with chronic obstructive pulmonary disease. It is clearly an area where much more careful phenotyping is required as well as the application of novel cell and molecular methodology to determine underlying mechanisms.

The airway epithelium in asthma The airway epithelium, while playing an important role as a physical barrier, is now known to be fundamental to asthma pathogenesis. Bronchial biopsies in anything but the mildest forms of asthma show areas of epithelial metaplasia and damage, thickening of the subepithelial basal lamina, increased number of myofibroblasts, and other evidence of airway remodeling such as hypertrophy and hyperplasia of airway smooth muscle, mucous gland hyperplasia, angiogenesis and an altered deposition and composition of extracellular matrix proteins and proteoglycans (Knight & Holgate 2003; Holgate et al. 2004). While these pathologic features have been commonly reported in asthma deaths and in bronchial biopsies from patients with asthma of varying severity, more recently similar findings have been found in the airways of children in

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Structural damage

Stress

Epithelial damage

TGF-b FGFs PDGF

Eosinophil recruitment & activation

MYOFIBROBLAST ACTIVATION Fig. 78.5 Schematic diagram of epithelial injury and activation of subepithelial mesenchymal cells through the release of growth factors creating a chronic wound scenario. See text for definition of abbreviations. (See CD-ROM for color version.)

relation to the onset of asthma (Fedorov et al. 2005; Saglani et al. 2005; Barbato et al. 2006). Evidence of epithelial damage with upregulation of epidermal growth factor receptors (EGFRs) and features of impaired proliferation, e.g., reduced expression of proliferative markers such as Ki67 and proliferating cell nuclear antigen (PCNA) and upregulation of the cell cyclin inhibitor, nuclear p21wat, suggest that, as in adult asthma (Puddicombe et al. 2003), the epithelium is chronically injured and unable to repair properly (Bucchieri et al. 2002; Kicic et al. 2006) (Fig. 78.5). One important feature of the asthmatic epithelium is its capacity to defend itself against oxidant injury (Bucchieri et al. 2002; Comhair et al. 2005), a feature that may partly explain why asthmatic subjects are so sensitive to oxidant pollutants such as ozone, environmental tobacco smoke and ambient air particulates (Truong-Tran et al. 2003). Under normal circumstances the epithelium forms a highly regulated and almost impermeable barrier through the formation of tight junctions (Godfrey 1997). These protein complexes at the apex of the columnar cells (Mullin et al. 2005) comprise a series of proteins that includes claudins and transmembrane adhesion proteins that connect adjacent cells. Structural integrity is also maintained through cell-cell and cell–extracellular matrix interactions that includes involvement of E-cadherin, desmosomes and hemidesmosomes (Roche et al. 1993). Although at one time thought to be an artifact of sample handling (Ordonez et al. 2000), it is now clear in asthma that the epithelium is more fragile with easy loss of the columnar cells due to disruption of both tight junctions and desmosomal attachments (Montefort et al. 1992; Barbato et al. 2006; Shahana et al. 2006) (Fig. 78.6). This abnormality is also apparent in the epithelium of nasal polyps from asthmatics compared with those from nonasthmatic subjects (Shahana et al. 2005). Using differentiated epithelial cells in culture brushed from normal and asthmatic airways, it has been shown that the permeability (leakiness) of the asthmatic epithelium is greatly increased, leading to greater

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Normal

(a)

Pathogenesis of Asthma

Mild asthma

Severe asthma

(b)

Solutes

ZO-1

Occludin

ZO-2 ZO-3

Claudins

ZO-1

Actin

Cingulin

JAM E-cadherin Catenin Cell 1

Cell 2

(c)

(d) Fig. 78.6 Role of the epithelium in chronic asthma. (a) Extensive epithelial damage and mucus plugging of an airway from a patient who has died from asthma. (b) Progressive epithelial damage and disposition of matrix in asthma relative to disease severity. (c) Infiltration of the asthmatic epithelium with eosinophils associated with damage. (d) Molecular components of tight junctions that control intracellular permeability. (See CD-ROM for color version.)

access of inhaled allergens, pollutants and other irritants to basal cells and the underlying airway tissue. This increase in epithelial permeability has also been observed in vivo using inhaled radiolabeled impermanent probes, such as technetiumlabeled DTPA (Ilowite et al. 1989). A reduction in the ability of the airway epithelium to exclude inhaled environmental agents may partly explain why certain atopic individuals go on to develop asthma whereas those with good barrier function do not. This loss of barrier function may reflect a broader abnormality in affecting other organs such as the skin, conjunctiva and gut that are foci for other atopic disorders (Hijazi et al. 2004; Liu et al. 2005; Hughes et al. 2006; Proksch et al. 2006). A recent development has been the discovery of a number of genes encoded on chromosome 1q, including filaggrin and

the S100 proteins, that are involved in maintaining epithelial integrity in both the skin and the airways (Marshall et al. 2001) (Fig. 78.7). The association of genetic polymorphism of the pro-filaggrin gene on chromosome 1q13 with atopic dermatitis and asthma (Palmer et al. 2006; Ying et al. 2006) is of considerable interest and refocuses the possible origins of asthma more toward the epithelium and formed elements of the airways, rather than the immune response alone (Hudson 2006). Another example of an environmental injury targeting the airway epithelium in asthma is the effect of respiratory virus infections. It has long been known that common cold viruses such as rhinovirus are associated with exacerbations of asthma in both children and adults (Johnston et al. 1995; Corne et al.

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0kb Exon 1

S100A10

THH

Exon 2

Family 1 I

500kb II

II

I

SPRR3

1q21

2282del4/ wt

2282del4/ wt

II

2282del4/ 2282del4/ wt 2282del4 AD AD

2282del4/ 2282del4/ R501X wt/wt wt/wt R501X R501X wt PFT AD AD AD PFT PFT PFT Family 6 “isolated case”

Family 5 I

I AD

Family 3 ?

I

Family 4 INV

(b)

2282del4/ 2282del4/ wt wt

3702delG/ wt/wt R501X AD

1000kb

3‘UTR

Family 2 I

R501X/ 3702delG/ wt wt AD

FLG

p

Exon 3

2282del4/ wt

?

? R501X/ wt

R501X/ wt

SPRR1B SPRR2A 1500kb

LOR S100A9 S100A8

q

II

II

II 2282del4/ R501X AD PFT

AD

2282del4/ R501X AD

R501X/ R501X AD (c)

S100A7 S100A6 S100A5 S100A4 2000kb

S100A3 S100A2 S100A1

(a) Healthy skin

IV:R501X/R501X

(d)

Fig. 78.7 Molecules involved in regulating epithelial integrity. (a) Gene cluster on chromosome 1q encoding a series of genes involved in maintaining epithelial integrity, including filaggrin (FLG), involucrin (INV), loricin (LOR), and the S100 proteins. (b) Genomic architecture of the filaggrin gene. The arrows denote single nucleotide polymorphisms in exon 3 associated with loss of function. (c) Six family pedigrees with

ichthyosis vulgaris (homozygotes shown as symbols and heterozygotes as symbols); note the strong association with atopic dermatitis (AD). (d) Immunohistochemical localization of filaggrin to superficial layer of the skin and its absence in a patient homozygous for the R501X genotype of ichthyosis vulgaris. (Reproduced from Palmer et al. 2006, with permission.) (See CD-ROM for color version.)

2002). Bronchial biopsy studies have shown that the airway epithelium is the preferential site for these viruses to enter the airway tissue (Papadopoulos et al. 2000). Infection of asthmatic epithelial cells compared with normal epithelial cells in vitro has revealed that the former lack the ability to generate IFN-β and IFN-λ, cytokines essential for eliminating viruses partly through induction of apoptosis (Wark et al. 2005; Contoli et al. 2006). In asthma the viruses continue to replicate until they kill the epithelial cells cytotoxically, leading to massive virus shedding and infection of adjacent cells, as well as the release of mediators from the damaged cells. By adding IFN-β back into the cell cultures, resistance to rhinovirus infection is restored (Wark et al. 2005), suggesting that this

may be a new approach to the prevention and treatment of acute asthma exacerbations (Holgate 2005). A second type of injury that targets the epithelium is air pollution, asthma worsening at times of air pollution episodes. A number of studies have shown that the antioxidant defenses mounted by the airway epithelium in asthma are markedly reduced and are associated with reductions in superoxide dismutase (Comhair et al. 2005) and glutathione peroxidase (Qujeq et al. 2003; Misso & Thompson 2005). In not being able to defend itself adequately against oxidant damage, the airway epithelium is damaged more easily (Rahman et al. 2006). The association of asthma with ozone and particle pollution episodes can be explained on this basis (Morrison et al. 2006).

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Pathogenesis of Asthma

phenotype (Polosa et al. 2002; Hamilton et al. 2005; CasalinoMatsuda et al. 2006) and an altered inflammatory response involving neutrophils (Hamilton et al. 2003), which are all characteristic of more chronic and severe asthma. Other epithelialderived factors include chemokines attracting neutrophils and other inflammatory cells, as well as a range of growth factors including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-1, FGF-2 and TGF-β that are active on fibroblasts and smooth muscle (Fig. 78.5). The secretion of latent TGF-β and its activation by proteolytic enzymes are fundamental to the subsequent airway remodeling that accompanies all but the most mild asthma (Boxall et al. 2006; Howell & McAnulty 2006). TGF-β itself is able to further impair epithelial repair responses while at the same time promoting the differentiation of fibroblasts into myofibroblasts (Leung et al. 2006). Bronchial biopsies from asthma have revealed that myofibroblasts are present in increased numbers in the subepithelial and submucosal region of asthmatic patients and increase in proportion to disease chronicity and severity (Choe et al. 2006; Wicks et al. 2006). The precise role of these

A third example is the inflammatory and immune cascade initiated by activation of Toll-like receptors (TLRs). The recent discovery that a novel cytokine, thymic stromal lymphopoietin (TSLP), is generated by airway and skin epithelial cells following activation of TLR3, TLR6, TLR7, TLR8 and TLR9 and the capacity of this cytokine to generate Th2-polarized and mast cell activation responses places this cytokine in a unique position to orchestrate the airway inflammatory response of asthma (Ying et al. 2005; Liu 2006). When damaged, the airway epithelium needs to repair but, as referred to above, the repair process is compromised in asthma. The airway epithelium enters into a chronic “wound scenario” with consequent production of a variety of cytokines and growth factors in an attempt to repair the “wound” (Fig. 78.8). One group of growth factors central to epithelial repair and its altered phenotype in asthma is epidermal growth factor (EGF) and related molecules (HB-EGF, amphiregulin) (Puddicombe et al. 2000) that, by interacting through their tyrosine kinase receptors, promote repair. EGFR stimulation of the damaged epithelium also generates a mucus-secretory

P = 0.043 P = 0.0001 P = 0.0001

Collagen band thickness ( m)

7 6 5 4 3 2 1 0 Control

Moderate

(a)

Severe Asthma P = 0.002

Moderate

80 Severe

60 40 20 0 Normal

(b)

Moderate Asthma

Severe

(c)

P = 0.02

P = 0.0001 30

100 p21% intact epithelium

% epithelial area

100

Ki-67 (MIB-1) % intact epithelium

P = 0.0001 P = 0.025

120

P = 0.002

P = 0.001

P = 0.01

25 20 15

ns

10 5 0

80 60 40 20 0

Normal

Moderate Asthma

Severe

Control

Moderate

Severe

Asthma

Fig. 78.8 Evidence for epithelial injury and impaired repair in childhood asthma. Bronchial biopsies in asthma show increased thickening of the lamina reticularis (a), increased expression of the epidermal growth factor receptor (b), reduced biomarkers of cell proliferation and increased nuclear translocation of the cell cycle inhibitor p21 (c). (Reproduced with permission of Fedorov et al. Thorax 2005). (See CD-ROM for color version.)

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Asthma and its Treatment AIRWAY PATHOLOGY IN ASTHMA Th2 cytokines

Environmental agents Inflammatory cell products Dendritic cell

Th2 inflammation

Initiation Th2 cell IL-3, IL-5 GM-CSF

IL-9, IL-4

(myo) fibroblasts

Amplification

IL-3 Mast cell

Propagation

Eosinophil

Airway microenvironment

Mucus

Smooth muscle Basophil

Blood vessels THE EPITHELIAL–MESENCHYMAL TROPHIC UNIT IN ASTHMA

Chemoattractants, proinflammatory mediators Fig. 78.9 Schematic representation of the interactions between airway inflammation and remodeling in clinical asthma involving the epithelial–mesenchymal trophic unit. (Reproduced with permission of Holgate et al. 2004). (See CD-ROM for color version.)

cells in airway wall remodeling has yet to be proven, but it is highly likely that they do play a key role, both with respect to making good the damage caused to the epithelium through the deposition of matrix in the lamina reticulosa of the basement membrane and also as a result of interactions with recruited inflammatory cells such as eosinophils and mast cells, thereby helping sustain the chronic inflammatory response (Fig. 78.9). The conducting airways contain an epithelium that is stratified, the upper layer containing a mixture of ciliated, goblet and Clara cells. In chronic asthma the number of goblet cells that secrete viscous mucus increases, with a parallel reduction in ciliated cells. Since mucus production is fundamental to the pathogenesis of chronic asthma, this metaplastic change in the airway epithelium is of great importance, particularly since this has been shown to occur in the more peripheral airways which are normally devoid of goblet cells (Ordonez et al. 2001; Perez-Vilar 2006). The factors responsible for goblet cell metaplasia include the Th2 cytokines IL-4, IL-9 and IL-13 as well as TNF-α, which are all capable of directing differentiation to a mucus-secreting phenotype and interact by inducing EGFR-mediated goblet cell metaplasia through receptor transduction and induction of EGFR ligands such as HB-EGF and amphiregulin (Cohn 2006; Temann et al. 2007).

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In asthma it is mucin 5AC that dominates the mucin glycoproteins that are secreted and it is this mucin which is largely responsible for the unusual viscoelastic properties observed in asthmatic sputum that lead to difficulties in its expectoration (Morcillo & Cortijo 2006; Voynow et al. 2006). Recently IL-13 has been shown to regulate a chloride channel (GOB5; CLCA3), which is intimately involved in the regulation and secretion of mucin from goblet cells (Thai et al. 2005; Long et al. 2006). However, the identification of multiple forms of GOB5 and its precise role in asthma has yet to be fully understood, though it does represent a therapeutic target. One interesting idea that may partly account for the onset of asthma in genetically susceptible children is the fact that the airway epithelium is fundamentally abnormal both in its response to environmental injury and its repair. A recent study (COAST) in children born of asthmatic and atopic parents has shown that those who develop more persistent wheezing at the age of 4 years are those who had frequent symptomatic virus infections in early infancy, particularly rhinovirus (Lemanske et al. 2005). Impaired innate immunity, possible due to a genetic defect in interferon production in response to virus infection, may initiate the chronic injury– repair cycle associated with the onset of chronic disease.

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80

Atopic (n = 94) Non-atopic (n = 59)

70 Prevalence %

60 50 Atopic

40 30 20

Non-atopic

10 0 1

2

3

4

5

6 7 8 Age years

9

10 11 12 13

Fig. 78.10 Influence of atopy on the persistence of asthma after 5 years of age. (Reproduced from Illi et al. 2006). (See CD-ROM for color version.)

Longitudinal cohort studies have also demonstrated that while atopy failed to predict persistent wheezing up to the age of 5 years, beyond this age those who persistently wheeze are the atopic children whereas those who lose their wheezing (“grow out of” asthma) are the nonatopics (Illi et al. 2006) (Fig. 78.10). Together, these observations point to environmental factors operating independently at different stages during the evolution of asthma and again places the airway epithelium at the boundary where environmental interactions take place to generate the asthma phenotype.

Airway wall remodeling Although difficult to define functionally, from a structural standpoint there is ample evidence that, beyond airway inflammation, changes to the formed elements of the airway contribute significantly to the pathophysiology of asthma (James & Carroll 2000) (Figs 78.1 and 78.9). The most obvious change is in the airway smooth muscle, which not only increases in amount due to hypertrophy and hyperplasia, but also spreads both up and down the airways (Fig. 78.1). In asthma the increase in muscle and its altered function most likely underlies an important component of hyperresponsiveness that characterizes this disease. However, intensive studies have tried to identify cell and molecular abnormalities in the airway smooth muscle that may account for its abnormal behavior in this disease, but as yet there are no clear mechanisms that explain this (James 2005). What has emerged is that in chronic asthma the airways become thickened, not only due to an increase in airway smooth muscle, but also as a consequence of the laying down of new matrix proteins including collagen fibers, increased proliferation of microvessels along with vascular leakage and deposition of proteoglycans, with their ability to sequestrate water. In both children and adults, high-resolution computed tomography (HRCT) has revealed an association between airway wall thickness and disease chronicity and severity that can best be explained on the basis of remodeling (Bumbacea et al. 2004; Jain et al.

Pathogenesis of Asthma

2005). Paradoxically, however, airway wall thickness is inversely correlated with airway hyperresponsiveness, suggesting that this thickening with deposition of matrix proteins may be a protective response against frequent smooth muscle contraction (Shaw et al. 2004). Thus in patients who are highly hyperreactive with brittle asthma, the airway remodeling response is minimal, whereas in those with chronic disease who exhibit some degree of fixed airflow obstruction, remodeling is more prominent (Paganin et al. 1996). Remodeling as a series of interacting processes is complex. For example, while immunostaining for proteoglycans such as biglycan, lumican, versican and decorin is increased in both moderate and severe asthma, with no differences in the amount present in the subepithelial layer, deposition of biglycan and lumican is significantly greater in the smooth muscle in moderate compared with severe asthma, suggesting a compensatory role (Pini et al. 2007). One molecule that has emerged as involved in airway remodeling is ADAM33. This susceptibility gene for asthma was first identified by positional cloning (Van Eerdewegh et al. 2002) and has been replicated in a number of studies (Blakey et al. 2005). In being preferentially expressed in airway mesenchymal cells (fibroblasts and smooth muscle), it has been shown to be involved in the pathogenesis of airway hyperresponsiveness and decline in lung function over time (Jongepier et al. 2004; van Diemen et al. 2005). ADAM33 has metalloproteinase, fusagenic, adhesion and intracellular signaling activities and exist in at least six alternatively spliced isoforms (Van Eerdewegh et al. 2002). Although the full-length molecule (120 kDa) is expressed as a transmembrane protein, a soluble form (∼ 55 kDa) has also recently been identified whose levels increase in proportion to disease severity (Lee et al. 2006; Foley et al. 2007). Of the many potential biological actions that ADAM33 possesses, its proteolytic activity, which is also present in the soluble form, is likely to be important in generating growth factors that influence mesenchymal cell number and or maturation (Holgate et al. 2006). Persistent airway inflammation may be an important factor that contributes to airway wall remodeling, including the secretion of mediators and growth factors such as TGF-β1 from eosinophils. Kariyawasam et al. (2007) have recently shown that, in those with both early and late asthmatic responses to allergen challenge, markers of airway wall remodeling such as tenascin, procollagens 1 and 3, HSP-47 and α-smooth muscle actin (marker of myofibroblasts) were all elevated beyond the 7-day time point when the inflammatory response had resolved. However, as discussed above, epithelial injury, impaired repair, and the secretion of growth factors from this structure may also contribute to the ongoing repair response characteristic of remodeling. Since these changes have been described in the absence of eosinophil infiltration, it may be that epithelial injury and remodeling is a necessary precursor to the onset of asthma, the altered microenvironment in the airway then providing the opportunity for inhaled

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environmental insults to gain access and generate the characteristic chronic inflammatory response involving atopy. The various growth factors involved in airway wall remodeling are similar to those involved in branching morphogenesis of the lung in the developing fetus (Bousquet et al. 2004; Holgate et al. 2006) and this has led to use of the term “epithelial mesenchymal trophic unit” to describe the interaction between the epithelium and the underlying mesenchymal cells in generating this altered structural response in chronic disease (Holgate et al. 2001; Knight et al. 2004) (Fig. 78.9). Regarding asthma not only as an inflammatory disease but also one in which there is abnormal signaling between the epithelium and mesenchymal cells has created an opportunity to search for novel therapeutics that act on this aspect of the disease. If epithelial injury and impaired repair are important as pathophysiologic mechanisms, then new treatments targeted to these pathways will represent a real advance. When applied to a damaged asthmatic epithelium in vitro, both EGF and keratinocyte growth factor are able to restore full barrier function as well reestablishing the protection of the epithelium against environmental injuries (Berlanga et al. 2002; Basuroy et al. 2006). Clinical trials are also currently in progress to investigate the effect of surfactant as a potential “barrier treatment” for chronic asthma, with the idea that by excluding environmental agents from penetrating the airway wall, continued aggravation of the underlying airway inflammation may be reduced (Babu et al. 2003). The relative importance of airway inflammation and epithelial mesenchymal signaling in asthma pathogenesis has recently been highlighted by three studies that have demonstrated that inhaled corticosteroids, when administered to children born of asthmatic and atopic parents for 1–4 years, has no effect on the natural history of asthma (Bisgaard et al. 2006; Guilbert et al. 2006; Murray et al. 2006). Two previous trials of inhaled corticosteroids administered over prolonged periods to older children also failed to influence the course of the disease (Childhood Asthma Management Program Research Group 2000; Pauwels et al. 2003). Thus, if airway inflammation (and by implication Th2 driven-immunity) was the cause of the asthmatic process and the airway dysfunction that is associated with it, then its prevention at the time of disease onset should influence the natural cause, whereas in these trials it did not.

Vascular remodeling One aspect of airway remodeling that is frequently neglected is the role of the vasculature. In both adults and children recent biopsy studies have demonstrated a large increase in the number of microvessels present in the airways of patients with chronic asthma, with some evidence that the endothelium of these vessels is undergoing proliferation (Vrugt et al.

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2000; Wilson & Robertson 2002; Barbato et al. 2006). Vascular remodeling has also been shown to occur in a chronic allergen exposure rat model (Tigani et al. 2006). Since microvascular leakage as well as recruitment of inflammatory cells is fundamental to asthma pathogenesis, a greater understanding of the cellular and molecular mechanisms involved in new vessel formation in the airways is important. The discovery of vascular endothelial growth factor (VEGF) in the damaged airway epithelium in asthma as an important mediator contributing to microvascular leak, proliferation, and vascular remodeling is being pursued with much interest (Chetta et al. 2005; Abdel-Rahman et al. 2006; Bhandari et al. 2006). An additional mediator that has recently been discovered in association with the T-cell-dependent component of the allergen-induced late asthmatic response is calcitonin generelated peptide, a potent vasodilator (Kay et al. 2007). It is likely that, as with fibroblasts and smooth muscle, this aspect of the remodeling response involves interaction of a wide range of different mediators including factors that act simultaneously on all three cell types (e.g., TGF-β).

Neural remodeling An increase in neural networks in the asthmatic airway is also key to disease pathogenesis. Nerve growth factors or neurotrophins are clearly of importance in promoting the altered neural regulation that undoubtedly occurs in asthma (Nassenstein et al. 2006) but the precise mechanisms have yet to be defined. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) as well as other members of this family are produced by epithelial cells and smooth muscle as well as a range of inflammatory cells including mast cells and eosinophils (Olgart et al. 2002). Circulating levels of NGF correlate with asthma severity (Bonini et al. 1996). The neurotrophins activate two receptor subtypes: TrkA (tropomyosin receptor kinase A) and p75 NTR (neurotrophin receptor) in the death receptor family (Frossard et al. 2004). The neurotrophins not only promote neural growth and maturation but are also important mast cell growth factors, enhance eosinophil survival, and augment human airway smooth muscle responsiveness (Kassel et al. 2001; Nassenstein et al. 2003; Frossard et al. 2005; Hahn et al. 2006) and therefore have the capacity to enhance asthmatic airway inflammation (Quarcoo et al. 2004). Bronchoconstriction provoked by irritants such as sulfur dioxide and ozone are also likely to operative via this abnormal neural network, with the release of acetylcholine and neuropeptides that have multiple effects on inflammatory cells, smooth muscle, blood vessels, and mucus-secreting cells. Although neuropeptide antagonists have so far been disappointing in asthma, the pleiotropic effects of the neurotrophins and their receptors make them more attractive therapeutic targets (de Vries et al. 2006; Watson et al. 2006).

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Heterogeneity of asthma Asthma is a heterogeneous disorder (Wenzel 2006). Although different types of asthma have long been recognized, such as that triggered by exposure to allergens, occupational chemicals, nonsteroidal antiinflammatory drugs (NSAIDs) and nonallergic “intrinsic” asthma, there has never been a cellular or molecular basis for these different manifestations of intermittent airflow obstruction other than exposure to a particular environmental stimulus. With the advent of fiberoptic bronchoscopy and the ability to biopsy different parts of the bronchial tree, noninvasive lung imaging such as HRCT and the development of therapeutics that target single cells or pathways, it is now clear that asthma is not a single disease but a wide range of different disorders that share some common phenotypes such as reversible airflow obstruction and associated symptoms. For the last 50 years we have only had corticosteroids, β2-adrenoceptor agonists and other bronchodilators (such as xanthines), and chromone-like drugs to treat asthma and this has led to the reinforcement of asthma as a “single disease.” Using noninvasive markers of airway inflammation suggests the presence of at least four distinct “phenotypes”: eosinophilic, neutrophilic, mixed inflammatory, and paucigranulocytic asthma (Wenzel 2006).

Eosinophilic asthma Bronchial biopsy studies of patients with mild asthma have shown that eosinophilic airway inflammation is highly characteristic regardless of whether patients were atopic, nonatopic, aspirin-sensitive, or had occupational asthma (Brightling et al. 2003; Bochner & Busse 2005). A broad correlation between clinical asthma severity and the degree of airway eosinophilia has been recorded, especially when eosinophils appear to be activated. With the advent of sputum induction it appears that there is a complex relationship between eosinophilic inflammation and other markers of asthma, including lung function and airway hyperresponsiveness. In contrast eosinophilic airway inflammation appears to be much more closely related to the risk of severe asthma exacerbation (Green et al. 2002). This particular phenotype would be worth investigating for a potential therapeutic effect of anti-IL-5 therapy which, as referred to earlier, has failed to modify baseline asthma even though it has modifying effects on indices of remodeling.

Pathogenesis of Asthma

dominantly neutrophilic airway inflammation and absence of eosinophils (Nguyen et al. 2005). Intense neutrophilic inflammation has also been reported in patients ventilated for acute severe asthma (Tonnel et al. 2001) and in those who died suddenly of asthma (Carroll et al. 1996). In general, asthma associated with neutrophils tends to be a more aggressive disease possibly with more tissue destruction and airway remodeling (Holgate & Polosa 2006). Intervention with the anti-TNF p75 fusion protein etanercept over a period of 10– 12 weeks has shown clinical benefit in such patients, suggesting that as the disease becomes more chronic and severe the inflammatory phenotype changes from Th2 more toward a Th1 type (Kumar et al. 2006) (Fig. 78.11). Neutrophilic and mixed eosinophilic and neutrophilic asthma has also been associated with Mycoplasma pneumoniae and/or Chlamydia pneumoniae infection, with a beneficial response to macrolide antimicrobials (Johnston et al. 2006; Kraft & Hamid 2006). Whether the chronic bacterial infection is primary or secondary to the underlying asthmatic response in such patients is not known, but since asthmatic airway epithelial cells are known to be deficient in their ability to mount a primary interferon response following infection with common respiratory viruses such as rhinoviruses (Wark et al. 2005; Contoli et al. 2006), it is possible that defective innate immunity may be fundamental to the origins and progression of chronic disease. Indeed, longitudinal cohort studies have shown that children with early onset and persistent wheezing in the first 3–5 years of life and classified as severe childhood asthma tend to maintain this phenotype throughout life (Stein & Martinez 2004) and it is these individuals who appear to have increased susceptibility to virus infection early in life (Lemanske et al. 2005) and possibly are those who are also susceptible to chronic bacterial infection (Chaudhuri et al. 2005, 2006; Singh et al. 2007). Tobacco smoking is also associated with a greater neutrophil component and, importantly, corticosteroid refractoriness in both the airways (Chaudhuri et al. 2003; Tomlinson et al. 2005) and systemically (Livingston et al. 2007). One possible explanation for this is the effect of smoking and oxidative stress in reducing histone deacetylase activity in the nuclear chromatin, thereby diminishing the opportunity for corticosteroids to access antiinflammatory genes (Adcock et al. 2005).

Paucigranulocytic asthma Noneosinophilic asthma The use of induced sputum as well as lavage and fiberoptic bronchoscopy has revealed that some patients with asthma have a sputum neutrophilia in the absence of eosinophils (Wenzel et al. 1999; Tsoumakidou et al. 2006). Other studies have noted neutrophilic inflammation in some patients with severe asthma and during virus-induced exacerbations (Wark et al. 2002). In addition, patients with severe asthma treated with oral (but not inhaled) corticosteroids also exhibit a pre-

Although sudden asthma death has been recorded in the absence of airway inflammation, this is highly unusual. Asthma in the absence of either neutrophils or eosinophils (paucigranulocytic asthma) has been described in which MMP-9 levels in sputum disease were normal (as opposed to elevated levels in patients with eosinophilic asthma; Simpson et al. 2005), suggesting that an abnormal epithelium or underlying mesenchyme and/or smooth muscle may itself lead to an asthma phenotype without the presence of obvious inflammation.

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P < 0.0003

10 000 Relative mRNA expression

Epithelium 1000 100 10 1

Mast cells

0.1

Eosinophils

0.01

Mild asthma (n = 14)

(a)

Severe asthma (n = 14)

(b)

P = 0.03

(c)

1 0.1 0.01

Week 1

Week 12

10 1 0.1

35

35

30

30

Symptom scores

10

Symptom scores

100 Methacholine PC20

Methacholine PC20

100

25 20 15 10 5

0.01

Week 1

Week 12

(d)

0

P < 0.001

25 20 15 10 5

Week 1

Week 12

0

Week 1

Week 12

Fig. 78.11 Role of tumor necrosis factor (TNF)-a in severe asthma. (a) Relative expression of TNF-a mRNA in bronchial biopsies from patients with mild and severe corticosteroid-refractory asthma. (b) Immunohistochemical staining for TNF-a in severe asthmatic biopsy. (c) Effect of etanercept twice weekly for 12 weeks on methacholine airway responsiveness. (d) Effect of etanercept on asthma symptoms in severe asthma. (Reproduced with permission of Howarth et al. 2005). (See CD-ROM for color version.)

Refractory asthma as a distinct inflammatory phenotype Much recent attention has focused on patients with chronic asthma taking high doses of inhaled and oral corticosteroids, long-acting β2-adrenoceptor agonists, and other antiasthma therapies and yet who remain symptomatic. This refractory asthmatic phenotype is associated with upregulation of the TNF-α pathway with increased expression of membranebound TNF-α, TNF-receptor 1, and TNF-α converting enzyme by peripheral blood monocytes (Berry et al. 2005). This is consistent with the finding of increased mucosal TNF-α expression in severe asthma compared with patients with mild disease (Howarth et al. 2005; Silvestri et al. 2006) and the identification of a phenotype that is responsive to anti-TNF therapy (Russo & Polosa 2005) (Fig. 78.8). A range of abnormalities has been described that are said to be associated with corticosteroid-refractory asthma including defects in nuclear histone acetylation (Barnes 2006), overexpression of the β-isoform of the corticosteroid receptor serving a dominant negative function in the presence of corticosteroids (Lewis-Tuffin & Cidlowski 2006), a defect in vitamin D signaling (Xystrakis et al. 2006), and increased airway wall remodeling leading to a degree of fixed airflow obstruction (Bai & Knight 2005).

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A new approach to categorizing asthma subphenotypes Recognizing that asthma is a complex disease involving varying components of airway inflammation, hyperresponsiveness, variable airflow obstruction, and a range of symptoms it has been possible to apply statistical methods in an attempt to dissect this complexity into subphenotypes within a population of patients with severe asthma (Wardlaw et al. 2005). Cluster analysis is one such statistical tool that seeks to organize information about variables so that heterogeneous groups of subjects can be classified into relatively homogeneous “clusters.” Haldar et al. (2005) have used this technique of cluster analysis to identify subphenotypes within a population of patients with severe disease. In an analysis of over 270 patients with severe asthma they identified four distinct phenotypes. 1 Patients with relatively well-controlled symptoms and minimal airway inflammation. 2 A group with early-onset atopic asthma with severe symptoms, persistent airway inflammation, and markedly variable airflow obstruction. 3 A group of mainly females who have late-onset asthma with

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marked symptoms but minimal eosinophilic inflammation, many of whom are obese. 4 A group with male predominance who have late-onset asthma characterized by persistent eosinophilic inflammation in the absence of symptoms. It may be that these different subphenotypes have therapeutic implications but clearly much larger studies are needed to answer this question.

Asthma: a disorder involving variable airway inflammation and remodeling As we learn more about asthma subphenotypes and the immunopathology of asthma it is becoming increasingly apparent that the disorder is a spectrum of pathophysiologic processes that to some degree map onto the different therapeutic responses (Fig. 78.9). At the mildest end of the spectrum, i.e., allergic asthma associated with other allergic comorbidities such as rhinitis and atopic dermatitis, the disease is dominated by Th2-mediated inflammatory responses that are usually (but not always) responsive to corticosteroids. Also in this category is intermittent asthma associated with seasonal changes in aeroallergens, which in addition to being responsive to inhaled corticosteroids may also benefit from allergen-specific immunotherapy. In patients with anything more than mild disease evidence of altered epithelial–mesenchymal communication is found that might explain the more chronic nature of asthma as it evolves from childhood into adult disease and also its variable nature in response to a variety of environmental factors in addition to allergens such as air pollutants, infectious agents and certain drugs. Alterations in the epithelium or underlying mesenchyme leading to the generation of growth factors and cytokines that sustain inflammation can be thought of as generating a “fertile soil” in which the “seeds” of inflammation are able to take hold and persist (Davies et al. 2003; Holgate et al. 2004). The third type of asthma is one in which the epithelial–mesenchymal component becomes dominant, leading to progressive airflow obstruction, a component of which is irreversible with β2-adrenoceptor agonists and corticosteroids (James et al. 2005; Bergeron & Boulet 2006). It is in these patients that tissue damage and remodeling becomes prominent along with increased mucus secretion and the “plugging” of peripheral airways. Superimposed on these three broad pathophysiologic phenotypes are a range of environmental factors that individuals with asthma will respond to. Thus, asthma associated with NSAIDs is characterized by increased cysteinyl leukotriene production and in general a more beneficial response to antileukotriene therapy (Kumlin et al. 1992; Dahlen et al. 2002). Patients with severe allergic asthma in which IgE plays a predominant role in driving the disease are responsive to the IgE-blocking monoclonal antibody omalizumab, with attendant reductions in IgE-associated airway inflammation (Holgate et al. 2005). The more aggress-

Pathogenesis of Asthma

ive disease associated with increasing neutrophilic inflammation may respond to macrolide antimicrobials, but it is in this group that anti-TNF therapy may be beneficial. Additional factors that can influence the clinical expression of asthma include female hormone status (Beck 2001; Stanford et al. 2006), obesity (Appleton et al. 2006; McLachlan et al. 2006), infection with Aspergillus (bronchopulmonary allergic aspergillosis) (Gibson 2006), and in rare cases, pulmonary vasculitis (Churg–Strauss syndrome) (Keogh & Specks 2006).

Identification of novel mechanisms through genetics With increased knowledge asthma appears more and more complex in its pathophysiology, although genetic studies are beginning to reveal molecules and pathways that may underlie the origins of this disease (Holloway & Holgate 2004; Meurer et al. 2006). It is of particular interest that the majority of the novel genes identified through positional cloning that increase susceptibility to asthma are preferentially expressed in either the epithelium or the mesenchyme and smooth muscle rather than the immune or inflammatory pathways (Cookson 2004; Vendelin et al. 2005; Holgate et al. 2006) (Fig. 78.12). It is also of note that many of the molecules that appear to be involved in disordered epithelial–mesenchymal signaling in asthma are also utilized in fetal branching morphogenesis of the lung, suggesting that asthma at least in some of its manifestations has morphogenetic origins (Vendelin et al. 2005; Holgate et al. 2006). Babies whose airways are unable to adequately resist the early-life environmental insults such as environmental tobacco smoke, air pollutants, infections and allergens may be the children who go on to develop asthma, not because he or she has any fundamental abnormality in their adaptive immune system, but because their airways are inadequately equipped to defend themselves against environmental insults (Eder et al. 2006). Thus, in planning new therapeutic strategies it is interesting to speculate whether a novel approach to asthma might be to increase the intrinsic resistance of the airways to the environment rather than concentrating all our efforts in suppressing inflammation or manipulating the immune response.

Concluding comments The recognition that asthma is a highly heterogeneous disorder in terms of associated environmental factors, clinical expression, response to different therapies, and natural history has opened up the disease beyond allergic sensitization. This has important implications, moving us back to considering asthma as a disease whose origins lie in the airways. Human genetics linked to environmental epidemiology through epigenetics is revealing entirely novel mechanisms and

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Inflammation Environmental factors, viruses, air pollution, diet, allergens

Barrier/host defense

DDP10 TNF-a IL-4 IL-13 IL-4Ra STAT6 cysLTs

SPINK5 GST-P1,M1 TLRs PCDH1

Mucus ESE 3 MUC 8 IL-13 IL-4 IL-4Ra IL-9 STAT6

(myo) fibroblasts

Mast cell

IL-4 IL-13 IL-4Ra STAT6 Remodeling ADAM33 GPRA TGF-b

Smooth muscle BHR

Blood vessels

ADAM33

approaches to treatment. The application of cell and molecular biology to different phenotypes of asthma at different points in their natural history will eventually reveal why atopy in some individuals evolves into asthma while in others it does not. An important aspect of this relates to factors that downregulate immune and inflammatory reactions such as regulatory T cells. These are considered in detail in Chapter 4. Although at one time asthma was considered a relatively simple disease, treated with a limited range of drugs, it is now realized that all of these suppress rather than cure the disease. What is now needed is a concerted effort to understand why certain people develop asthma and others do not. It is only with this increased understanding that a real chance of cure and prevention will occur.

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Fig. 78.12 Asthma susceptibility genes expressed in the epithelium and mesenchyme. (See CD-ROM for color version.)

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Pathology of Asthma Peter K. Jeffery, A. Barry Kay and Qutayba Hamid

Summary

Introduction

Asthma is a heterogeneous disease varying in clinical phenotype and severity. There is damage to airway surface epithelium, thickening of the epithelial reticular basement membrane in all forms of asthma, and airway wall fibrosis when severe. The percentage of bronchial wall occupied by bronchial smooth muscle increases in mild and especially fatal asthma. Bronchial goblet cell hyperplasia and submucosal gland enlargement, dilatation and increase of bronchial mucosal blood vessels, congestion and wall edema may occur. These structural changes, or “remodeling,” contribute to airway wall thickening, reduced luminal patency, and airway hyperresponsiveness. Asthma is a chronic inflammatory condition of the bronchi that may, when severe, affect small airways, adjacent arteries, and surrounding alveoli. The activated T lymphocyte and its associated interleukins are considered to control and perpetuate the chronic inflammation of asthma. While the association between tissue eosinophilia and asthma is a strong one, the degree of tissue eosinophilia varies and neutrophils and their chemoattractants increase in exacerbations and as asthma increases in severity. Mast cell infiltration of airway smooth muscle is seen in asthmatics and this “mast cell myositis” may be a key feature of asthma per se. Dendritic cells form an interface between innate and adaptive immunity and their number, maturity and function have been shown to be altered by smoking, a habit in asthmatics associated with refractoriness to corticosteroid therapy. There are a number of explanations for the association of upper and lower airway inflammation and these are the basis for the current “united airways theory.” Biopsy studies of airway tissue demonstrate that the characteristic pathologic features of asthma in adults and school-aged children may develop in preschool children with confirmed wheeze between the ages of 1 and 3 years, at a time when intervention may modify the natural history of asthma.

The characteristic features of most asthma variants, including allergic asthma, are airway inflammation, airway hyperresponsiveness (AHR) (increased sensitivity or “irritability” of the bronchi), excessive airway mucus production due to goblet cell hyperplasia, and mucous gland hypertrophy. These changes, associated with increased thickness of the airway wall, are often referred to as remodeling and considered as consequent to excessive repair processes following repeated airway injury. Contributing to the greater thickness are increases in airway smooth muscle mass and mucus-secreting glands, deposition of matrix proteins, new blood vessel formation, and expansion of the adventitia, all of which reduce lumen diameter and increase resistance to airflow (Bai et al. 2000) (Fig. 79.1). This chapter describes the salient structural and inflammatory changes of mild, moderate and severe asthma in the adult and explores when these changes appear in the child. Occlusion of the airway lumen by tenacious secretions, tissue eosinophilia, loss of airway surface epithelium, thickening of the reticular basement membrane (also referred to as the lamina reticularis), and enlargement of bronchial smooth muscle and submucosal gland mass have been consistently reported in cases of fatal asthma in studies conducted postmortem. Loss of surface epithelium, tissue eosinophilia, and thickening of the reticular basement membrane have also been reported in bronchial biopsy samples taken by flexible fiberoptic bronchoscopy from subjects with mild atopic asthma in its relatively stable phase (Laitinen et al. 1985; Beasley et al. 1989; Jeffery et al. 1989; Azzawi et al. 1990, 1992; Jeffery 1992). Tissue eosinophilia and eosinophil activation are also key features of the late-phase reaction (LPR) following allergen challenge (Diaz et al. 1984; deMonchy et al. 1985; Bentley et al. 1993). Infiltration of the mucosa by inflammatory cells, including eosinophils, is a feature of the airway mucosa in all forms of asthma, whether mild or severe, allergic (also called extrinsic or atopic) or nonallergic (intrinsic or nonatopic), or due to occupation (e.g., exposure to isocyanates) (Azzawi et al. 1990; Bradley et al. 1991; Bentley et al. 1992a,b; Di Stefano et al. 1993; Bel 2004). The role of structural cells including

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Normal

Asthma

Fig. 79.1 Diagrammatic representation of the differences between the airways of asthmatic and normal subjects: reduction of airflow results from thickening of the airway wall with narrowing of the lumen and increased secretions.

fibroblasts, epithelial cells and smooth muscle cells in the initiation and persistence of airway remodeling is also of importance and is considered in Chapters 16, 18 and 41 as well as briefly below.

Appearance of the lung at postmortem Postmortem examination of cases of fatal asthma has shown that the lungs are red, edematous, and hyperinflated (Fig. 79.2) and remain so on opening the pleural cavities, due to the widespread presence of markedly tenacious plugs in the conducting airways (Fig. 79.3). It is not uncommon to observe focal depressions in the lung bounded by interlobular septa in the pleural surface, representing areas of subsegmental atelectasis caused by bronchiolar obstruction. Cut sections of the lung characteristically show the bronchi and the bronchioles to be plugged with viscid tenacious mucus, which in some cases extends into the trachea. Focal cystic bronchiectasis, often in the upper and middle lobes, has been reported in some patients (although allergic bronchopulmonary aspergillosis

should be excluded). Mucous plugs are most frequent in bronchi and membranous bronchioles (Fraser et al. 1999). They are composed of both mucoid and proteinaceous material and typically fill the airway lumen. Scattered within them is a variable number of cells, most of which are eosinophils or epithelial cells. The latter can occur singly or in clusters of up to 100 cells; such clusters are termed Creola bodies (Naylor 1962). Also present in the both airway plugs and sputum are Charcot–Leyden crystals (see Chapter 1) and Curschmann spirals. The former are variably sized crystals ranging from 20 to 40 μm in length and from 2 to 4 μm in width and are believed to be composed predominantly of phospholipase derived from eosinophil granules. The crystals appear bright yellow-green on fluorescent microscopy. Curschmann spirals are convoluted strands of mucus having a relatively compact central core surrounded by numerous delicate fibrils. They are typically microscopic in size but may be up to 2 mm in length. Although they are generally believed to be formed by inspissations of secretions within small airways, similar structures have been seen in pleural and peritoneal fluid specimens and the precise mechanism by which they are formed

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Fig. 79.2 Gross pathology of lung of an asthmatic patient who died of status asthmaticus. The lungs appear hemorrhagic, edematous, and there is hyperinflation due to obstruction of the conducting airways. (See CD-ROM for color version.)

Fig. 79.4 Hematoxylin and eosin stained tissue section of part of the bronchial wall in a patient who died of status asthmaticus showing (left to right) the classic pathologic features of asthma: plug with inflammatory cells; variable loss of surface epithelium with only basal cells remaining attached to a thickened hyaline reticular basement membrane; in the subepithelium, there is an eosinophilic inflammatory infiltrate and enlargement of the smooth muscle mass. (See CD-ROM for color version.)

propria), submucosa (including airway smooth muscle and mucus-secreting glands), and adventitia (the interface between airway and surrounding lung parenchyma) (Huber & Koessler 1922; Hogg 1993) (Fig. 79.4). Information on the pathologic changes in asthma has been obtained from studying airway sections from asthma deaths, patients who had asthma but died from nonasthma-related causes, from surgically resected lung, and from endoscopic biopsies of mild, moderate and severe asthmatic subjects. Tissue from transbronchial biopsies of severe asthmatics has also been studied for inflammation and remodeling. Fig. 79.3 In fatal asthma, the airways in transverse section are seen to be plugged by tenacious secretions composed of mucus and an eosinophilic inflammatory exudate in which there may be sloughed epithelial cells.

is not clear. Although characteristic of asthma, neither Charcot– Leyden crystals nor Curschmann spirals are pathognomonic of asthma. On intrabronchial inflation with fixative, even a 1.5-m head of pressure hardly moves these sticky airway plugs (Dunnill 1960; Dunnill et al. 1969) (see Fig. 79.3). The arrangement of the cellular elements of the plug is often concentric and multiple, suggesting several episodes leading to their formation rather than a single terminal event. The nonmucinous proteinaceous (eosinophilic) contribution is the result of increased vascular permeability and includes a fibrinous component. Interaction of constituents of serum and mucin is likely to lead to increased viscosity of the plug.

Histopathology of asthma Histopathologic changes in the bronchial and bronchiolar walls in asthma involve the mucosa (i.e., epithelium and lamina

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Epithelial changes Histologically, damage and shedding of airway surface epithelium are prominent in asthma, both in fatal asthma and in biopsy specimens of patients with mild disease (see Fig. 79.4) (Laitinen et al. 1985; Beasley et al. 1989; Jeffery et al. 1989; Amin et al. 2000). However, the epithelial loss is highly variable and others have not found this alteration in mild asthma (Lozewicz et al. 1990; Soderberg et al. 1990; Ordonez et al. 2000a). In normal healthy individuals loss of superficial epithelium is accompanied by mitotic activity in the remaining epithelial cells and this is associated with rapid restitution of the barrier, followed by regeneration of normal ciliated and goblet cell phenotypes (Erjefalt et al. 1995; Holgate et al. 1999). In experimental studies the entire process normally takes about 2 weeks (Ayers & Jeffery 1988). However, there are reports that such mitotic responses are deficient in asthma. Thus, rather than increased destruction, defective repair of epithelium in asthma may lead to the observed disruption and loss (Holgate et al. 1999; Holgate 2000). The greater the loss of surface epithelium in biopsy specimens, the greater appears to be the degree of AHR (Beasley et al.1989; Jeffery et al. 1989).

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However, it is recognized that there is inevitably artifactual loss of surface epithelium during the taking and preparation of such small endobronchial biopsies. It is an unreliable end point and interpretation of this alteration is highly controversial. In the study by Jeffery et al. (1989), the greatest loss of surface epithelium was found in asthmatics symptomatic at the time of the bronchoscopy. The interpretation made was that the observed loss reflected fragility of the epithelium in vivo, which was present even in mild asthma. The interpretation is supported by the frequent appearance of clusters of sloughed epithelial cells in sputa (Naylor 1962) and the increased presence of bronchial epithelial cells in bronchoalveolar lavage (BAL) of asthmatics with mild disease (Beasley et al. 1989). Such fragility of the surface may involve disruption of the tight junction by pollutants, microorganisms, and allergen-derived proteases (Elia et al. 1988; Godfrey et al. 1992a; Wan et al. 1999). Squamous metaplasia may often be present in severe asthma, even in the absence of a history of smoking. Tight junctions act as a selective barrier to the passage of ions, molecules and water between cells; this may enhance stimulation of intraepithelial nerves, leading to axonal reflexes, stimulation of secretion by mucous glands, vasodilatation and edema through the release of sensory neuropeptides (i.e., neurogenic inflammation) (Barnes 1986; Jeffery 1994a). Experimentally, there is also evidence that the sensitivity of bronchial smooth muscle to substances placed in the airway lumen correlates strongly with the integrity of the surface epithelium (Sparrow & Mitchell 1991). Loss or damage of surface epithelium would thus lead to a reduction in the concentration of factors normally relaxant to bronchial smooth muscle, with resultant increased sensitivity and “reactivity” (Hogg & Eggleston 1984; Van Houtte 1989). Apart from their role as stem cells, the basal cells of normal pseudostratified surface epithelium have been suggested to act as a bridge, enhancing the adhesion of “superficial” cells to epithelial basement membrane (Evans & Plopper 1988). When superficial cells are lost in asthma, the preferential plane of cleavage appears to be between superficial and basal cells (Montefort et al. 1992), leaving basal cells still attached to their basement membrane (Figs 79.4 and 79.5). Epithelial cells also act as effector cells, synthesizing and releasing a wide variety of agents including cytokines and chemokines. Disruption of the epithelium and attempts at repair may increase production of proinflammatory cytokines by cells that remain. Epithelial cells are also reported to express receptors for a number of cytokines and chemokines as well as Toll-like receptors. As reviewed in Chapters 16 and 21, it is clear that epithelial cells are a major player in host defense and in regulating inflammatory responses in asthma and other immune-mediated lung diseases.

Reticular basement membrane and subepithelial matrix deposition Thickening of the reticular basement membrane (i.e., lamina reticularis), observed by light microscopy, has long been recog-

Pathology of Asthma

Fig. 79.5 Scanning electron micrograph of a patient with 25 years’ history of asthma but who died postoperatively of another cause. The fracture plane shows disrupted surface epithelium, beneath which there is a thickened reticular basement membrane (arrow) with strands of interstitial collagen below. (Magnification ×1010.)

nized as a consistent change in all forms of asthma (Crepea & Harman 1955; Dunnill 1960; Nowak 1969; Callerame et al. 1971; Sobonya 1984; Jeffery et al. 1989; Roche et al. 1989) (Figs 79.4 and 79.5). While there may also be focal and variable thickening in chronic obstructive pulmonary disease (COPD) and other inflammatory chronic diseases of the lung such as bronchiectasis and tuberculosis (Crepea & Harman 1955) and in association with transplant, the lesion, when homogeneous and hyaline in appearance, is highly characteristic and present in both fatal and mild asthma and in patients with a long history of asthma. In contrast, there appears to be little or no alteration to the “true” epithelial basement membrane (i.e., basal lamina), which consists mainly of type IV collagen, glycosaminoglycans, and laminin. The thickened reticular basement membrane (RBM) is immunopositive for collagen types III and V, together with fibronectin but not laminin, and thus the thickening has sometimes been referred to as “subepithelial fibrosis” (Roche et al. 1989). However, a relatively recent study that examined the bronchial mucosa of children and adults with asthma by electron microscopy has demonstrated that the fibrils comprising the RBM in asthma are ultrastructurally dissimilar to those comprising normal interstitial collagen and those increased in fibrosis, i.e., fibrils of the RBM from asthmatics are significantly thinner and lack the characteristic periodic (65 nm) banded appearance of interstitial collagen. Moreover, compared to the norm, the ratio of fibril to matrix remains unchanged in asthma, favoring the hyopothesis that there is “more of the same” rather than a relative increase of the fibrillary component as would be expected of a fibrotic process (Saglani et al. 2006) Also, the thickening does not appear to be progressive (as in fibrosis) as it is maximal early on in childhood, when asthma frequently first develops, and already similarly thickened to that seen in adults with asthma. Its

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Fig. 79.6 Van Giesen staining of a tissue section from a subject with severe asthma demonstrating extensive subepithelial fibrosis extending to the smooth muscle. (Magnification ×400.) (See CD-ROM for color version.)

thickening also does not increase significantly with age, duration, or severity of disease (Payne et al. 2003). Certainly the subepithelial (deeper zone) of asthmatic airway wall contains more collagen I, III and V as well as alteration of the orientation and fiber thickness of collagen I (Minshall et al. 1997). There are also increases in asthma of tenascin, lumican, biglycan, versican, laminin β2 and α1, as well as elastase (Altraja et al. 1996; Laitinen et al. 1997; Vignola et al. 1998; Westergren-Thorsson et al. 2002). There is controversy as to whether the subepithelial fibrosis described deeper in the wall of severe asthmatics (Fig. 79.6) is related to severity of the disease (Minshall et al. 1997; Chu et al. 1998). Studies showing a correlation between the extent of subepithelial fibrosis and pulmonary function have also demonstrated a positive correlation with the severity of asthma (Wan et al. 1999). These studies have also suggested that excessive deposition of extracellular matrix proteins can lead to a substantial degree of fixed airway obstruction. However, other studies have suggested a protective role for subepthelial “fibrosis” through its increasing the stiffness of the airways, which would be expected to oppose bronchospasm and airway luminal occlusion (Wiggs et al. 1997). It is generally believed that RBM thickening and subepithelial “fibrosis” is the result of uncontrolled chronic inflammation and the inability of steroids to alter the function of the fibroblasts and myofibroblasts that lie close to the RBM and thought to contribute to its production (Brewster et al. 1990; Gizycki et al. 1997) (Fig. 79.7). Whereas the presence of RBM thickening in a pediatric population was reported to occur in the absence of chronic eosinophilic inflammation (Fedorov et al. 2005), a positive association between the presence of tissue eosinophilia and thickening of the RBM has emerged in experimental studies (Flood-Page et al. 2003; Humbles et al. 2004) in both adults and children with asthma (Saglani et al. 2008) and in adults with eosinophilic bronchitis, in which AHR is not a feature (Brightling et al. 2002).

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Fig. 79.7 An ultrathin tissue section of an endobronchial biopsy from a subject with mild atopic asthma, visualized by transmission electron microscopy. There is a lymphocyte (at the bottom of the picture) with an eosinophil above, releasing its electron-dense granules and cytotoxic contents: these are considered to damage the epithelium whose basal cells (b) are shown attached to the underlying reticular basement membrane. Factors such as transforming growth factor-b may stimulate adjacent fibroblasts to produce additional reticulin. (Magnification ×2535.)

A number of growth factors, particularly members of the transforming growth factor (TGF)-β superfamily, have been implicated in enhancing fibrosis in asthma. The TGF-β superfamily of ligands includes TGF-β1–3, activins, and bone morphogenetic proteins (BMP). These ligands signal via a constitutively active serine/threonine kinase-specific type II receptor that complexes with a type I receptor which then propagates the signal downstream by phosphorylating receptorregulated Smads (R-Smads). R-Smads then translocates to the nucleus in association with the common Smad4 to initiate gene transcription. Type I receptors TGF-β1–3 and activin-A are predominantly ALK-1/ALK-5 and ALK-4 respectively, whereas type II receptors are TβRII and ActRIIA/RIIB respectively. BMP signaling is predominantly through the type II receptor BMPRII, and three type I receptors, ALK-2, ALK-3 and ALK-6. Activin-A has potent fibrogenic properties whereas

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BMPs, particularly BMP-7 (also known as osteogenic protein 1 or OP-1), are antifibrotic factors and can antagonize the effects of TGF-β1 and so inhibit fibrotic processes. Kariyawasam et al. (2008) found that asthmatic airways had marked differences from normals in the expression of all BMP ligand signaling pathway components, suggesting that dysregulated BMP signaling may contribute to disease manifestations. Kariyawasam et al. (2007) also found that TGF-β3 and activinA, and respective type I and II receptors, were increased compared with the normal airway, suggesting abnormal signaling in asthma. TGF-β superfamily members and other growth factors involved in matrix deposition are derived largely from mesenchymal epithelial cells and inflammatory cells such as eosinophils (Duvernelle et al. 2003). As shown by depletion studies with anti-interleukin (IL)-5, eosinophils may contribute to subepithelial fibrosis, at least at certain stages of disease development (Flood-Page et al. 2003). Fibrosis is not only due to excessive deposition of collagen fibers, but might be also due to defects in the degradation of matrix. Mechanical strain of fibroblasts within the airways may be an additional factor in the excessive deposition of extracellular matrix (Ludwig et al. 2004).

marks and the correlate of mucus hypersecretion in chronic bronchitis. These alterations may also occur in fatal asthma (Dunnill et al. 1969), when they contribute to excess of mucus and airway blockage (Wanner 1988). There is dilatation of gland ducts, referred to as bronchial gland ectasia (Cluroe et al. 1989). In chronic bronchitis, there is loss of serous acini of the submucosal glands, which normally contain antibacterial substances, such as lysozyme, lactoferrin and a low-molecularweight antiprotease; the reduction in these factors favors bacterial colonization and proteolytic damage to airways. Such loss of serous acini is not reported in asthma (Glynn & Michaels 1960). While increased bronchial epithelial mucous cell number (i.e., goblet cell hyperplasia) may be a feature of both asthma (Ordonez et al. 2001; Aikawa et al. 1992) and bronchitis, the appearance and increase in number of newly differentiated goblet cells in bronchioli of less than 2 mm diameter (referred to as mucous metaplasia) is debated in asthma but is highly characteristic of small-airway disease in COPD (Jeffery & Saetta 2003). One suggestion is that mucus present at this distal site in asthma may have been aspirated from larger airways. Moreover, goblet cell hyperplasia in the larger airways is not common when the epithelial cells undergo squamous metaplasia, especially in severe asthma.

Alterations to mucus-secreting cells

Increase in bronchial smooth-muscle mass

Bronchial goblet cell hyperplasia (Fig. 79.8) and submucosal gland enlargement have been reported as the histologic hall-

The percentage of bronchial wall occupied by bronchial smooth muscle shows a striking increase in fatal asthma (Dunnill et al. 1969) (Figs 79.9 and 79.10). The increase in muscle mass is reported to be particularly marked in large intrapulmonary bronchi of lungs obtained following a fatal attack, as compared with that in asthmatic subjects dying of other causes (Carroll et al. 1993). Muscle mass enlargement is an important contributor to the thickening of the airway wall and hence to increased airflow resistance (Moreno et al. 1986; James

Fig. 79.8 Endobronchial biopsy of a subject with severe asthma showing marked goblet cell hyperplasia and a moderate infiltrate by inflammatory cells. (See CD-ROM for color version.)

Fig. 79.9 Hematoxylin and eosin stained section from a subject with severe asthma showing thickening of the airway wall due mainly to an increase in airway smooth muscle mass. Note the short distance between the smooth muscle bundle and the epithelium. (See CD-ROM for color version.)

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M

M M

V

V

Fig. 79.10 Scanning electron micrograph of a longitudinal section of part of the bronchial wall in a case of fatal asthma. The mucosa is thickened by marked dilatation of bronchial vessels (V) and an increase in the mass of bronchial smooth muscle (M). The surface epithelium has been completely lost. (Magnification ×160.)

et al. 1989; Wiggs et al. 1990, 1992). Using a morphometric technique, Dunnill et al. (1969) showed that approximately 12% of the wall in segmental bronchi obtained from cases of fatal asthma was comprised of muscle, whereas the figure was about 5% in normals. Hogg (1993) has confirmed this trend in airways larger than 2 mm diameter and demonstrated a threefold to fourfold increase over normal in the area of the wall occupied by bronchial smooth muscle. The increase in muscle mass could also be observed in airways of less than 2 mm in diameter.

Hyperplasia versus hypertrophy Whether the increase in muscle mass in asthma is caused by muscle fiber proliferation (i.e., hyperplasia) or hypertrophy is unclear, albeit both may contribute (Heard & Hossain 1983; Ebina et al. 1990, 1993). Two patterns of distribution of increased muscle mass have been described in asthma: (i) an increase occuring throughout the airways (both large and small), and (ii) increase restricted to the largest airways (Ebina et al. 1993). In the first pattern, muscle fiber hypertrophy may be present throughout the bronchial tree, whereas hyperplasia alone may distinguish the second pattern. Evidence favoring myocyte hypertrophy has come from examination of endobronchial biopsies in cases of severe asthma, where greater than normal areas of smooth muscle are found to be located in the mucosa (Benayoun et al. 2003). Such changes have also been described in milder asthma, where the fraction of bronchial biopsy occupied by smooth muscle is increased by 50%; in this study myocytes were not increased in size but rather had increased in number by about twofold, without evidence of a hypercontractile phenotype (Woodruff et al. 2004). These data favoring an increased proliferative response of bronchial smooth muscle in asthma are supported

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Fig. 79.11 Transmission electron micrograph of a bronchial biopsy taken at the time of an allergen-induced late-phase response, showing the presence of one of many myofibroblasts each about twice the size of a fibroblast and with irregular outline. There are bundles of longitudinally oriented cytoplasmic filaments running along the edge of the cell as well as many profiles of dilated rough endoplasmic reticulum, indicating that this cell has both contractile and secretory features.

by mechanistic studies of asthmatic airway smooth muscle in vitro (Johnson et al. 2001; Borger et al. 2006).

Muscle plasticity The extent of airway smooth muscle plasticity, its potential for secretion, and the role of its connective tissue matrix in regulating these changes is an intensive area of current research (Hirst 1996; Halayko et al. 1999; Hirst et al. 2000; Chan et al. 2006). Accordingly, a newly proposed mechanism for the increase in muscle mass involves dedifferentiation of existing smooth muscle bundles. In support of this, cells that have ultrastructural features of both a contractile and a secretory phenotype have been found in substantial numbers during the LPR to allergen challenge (Gizycki et al. 1997; Kelly et al. 2006) (Fig. 79.11). With repeated exposure to allergen, these cells may contribute to an increased mass of bronchial smooth muscle by a process of differentiation of existing smooth muscle and its migration to a subepithelial site where new muscle is formed. The capacity for smooth muscle cells to migrate has been demonstrated (Poliakov et al. 1999; Parameswaran et al. 2002) and the mechanisms involved in this response are likely to be similar to those occurring in atherosclerosis, in which there is vascular smooth muscle dedifferentiation and migration to form a neointima (Jeffery 1994b; Hirst 1996; Halayko et al. 1999). Not only can the “secretory” form of muscle cell release proinflammatory mediators (John et al. 1997), but it may also contribute directly to increases in extracellular matrix including the RBM (Hirst 1996; Halayko et al. 1999). Increase in smooth muscle mass has been consistently shown to correlate with disease severity and it is possibly a major cause of fixed airway obstruction. Pepe at al. (2005) reported that airway smooth muscle area was greater in subjects with severe compared to moderate asthma and that distance between the epithelial and airway smooth muscle

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layers was significantly less in the severe group than in the moderate group. As discussed in Chapters 21 and 41, smooth muscle, like other mesenchymal cells, have the capacity to generate a range of proinflammatory cytokines and chemokines that could help support chronic inflammation. Smooth muscle cells also produce several extracellular matrix proteins and express receptors for most of the cytokines and chemokines. For example, Kaur et al. (2006) showed that the CCL19/CCR7 axis may play an important role in the development of airway smooth muscle. Airway smooth muscle, myofibroblasts, and fibroblasts all express CCR7, and CCL19 but not CCL21 was highly expressed in bronchial biopsies by mast cells and vessels in asthma of all severities as well as airway smooth muscle in severe disease. Airway smooth muscle cells also express receptors for IgE and can respond to IgE stimulation (Gounni et al. 2005).

Pathology of Asthma

smooth muscle shortens, even normally (Moreno et al. 1986; Wiggs et al. 1990, 1992). The extent to which muscle shortening can occur is determined, in part, by the force opposing it, this being largely due to lung elastic recoil. Airway wall edema has been suggested to uncouple muscle from its surrounding tissues, thus removing the normal restraints on its contraction and resulting in a greater force and maximal contraction (Ding et al. 1987; Ward et al. 2001). In a similar way, destruction of alveolar attachments to bronchioli in smoker’s COPD (Saetta et al. 1985; Jeffery 1990) may lead to reduction in forced expiratory volume in 1 s (FEV1) and the development of apparent AHR. Loss of lung elastic recoil in asthma would be predicted to have a similar effect, but studies of the elastic fiber content of asthmatic lung and bronchi have shown conflicting results (Godfrey et al. 1992b; Bousquet et al. 1996; Mauad et al. 1999).

Bronchial vasculature, congestion and edema The increase in thickness of the bronchial wall in asthma is unlikely to be accounted for by the increase in bronchial smooth-muscle and mucous gland mass alone. Dilatation of bronchial mucosal blood vessels, congestion, and wall edema are also features of fatal asthma (Fig. 79.10). Vascular remodeling and increased expression of associated growth factors such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF) are well-recognized features of asthma (Li & Wilson 1997; Vrugt et al. 2000; Hoshino et al. 2001a,b; Asai et al. 2003; Chetta et al. 2003, 2005; Feltis et al. 2006). Such vascular remodeling is likely to be functionally important because enhanced vascularity in the inner wall of the medium airways correlates with airflow obstruction in asthma (Hashimoto et al. 2005). Moreover, Siddiqui et al. (2007) showed that vascular remodeling was associated with airflow obstruction but not AHR. Airway vascularity is more fully addressed in Chapter 17. Subepithelial edema has been suggested as responsible for lifting of the overlying surface epithelium (Dunnill 1960). The onset of vasodilatation, congestion, and mucosal edema in response to a variety of mediators of inflammation (Widdicombe 1993) can be rapid and, equally, can be relatively rapidly reversed. James et al. (1989) have shown that airway wall thickening (due to one or more of the above changes) need only be relatively minor to have dramatic consequences on airflow limitation. The increased wall thickness in airways greater than 2 mm diameter was recognized in fatal asthma by Huber and Koessler as early as 1922. Benson (1975) suggested that, for a given degree of smooth-muscle shortening, the effect on luminal narrowing and hence resistance to airflow (to the power 4) would be considerably greater if the airway wall were thickened. The relationship between airway geometry and AHR has been confirmed by computer modeling. The model predicts that when the airway wall is thickened there will be only moderate effects on baseline airflow resistance; in contrast, there will be profound effects when bronchial

Inflammation It is now accepted that asthma is a chronic inflammatory condition, and evidence of inflammation can be observed in mild, moderate and severe disease, albeit the relative magnitude, type of inflammatory cells, and site of the inflammatory infiltrate may differ. In fatal asthma, there is a marked infiltrate throughout the airway wall and also in the occluding plug, lymphocytes are abundant, eosinophils are characteristic, and neutrophils are often sparse (Dunnill et al. 1969; Saetta et al. 1991; Azzawi et al. 1992). The inflammation may spread to surrounding alveolar septa and affect small airways and adjacent arteries (Saetta et al. 1991). The association between tissue eosinophilia and asthma is a strong one, but the degree of tissue eosinophilia varies with each case and the duration of the terminal episode (Gleich et al. 1980; Azzawi et al. 1992; Sur et al. 1993). There is now a considerable amount of knowledge on the “living pathology” of asthma as biopsies can be readily obtained by flexible fiberoptic bronchoscopy or at open lung biopsy. Eosinophils (Fig. 79.12), activated T cells and neutrophils, as well as a few basophils, are usually readily detectable in such tissue. The role of the eosinophil in asthma is considered in detail in Chapter 12. Studies with anti-IL-5, which partially depletes eosinophils from the airway wall, indicate that this cell may play a role in TGF-β-dependent remodeling synthesis and secretion (Flood-Page et al. 2003). The role of the activated T lymphocyte in controlling and perpetuating chronic inflammation in asthma has received much attention (Larché et al. 2003). In some studies T-cell activation could be related to measures of asthma severity, such as the degree of airway narrowing or AHR, as well as the bronchial eosinophil response (Robinson et al. 1993a). Similarly, after the description of the Th2/Th1 dichotomy, mRNA-positive cells for the signature Th2 cytokines IL-4 and IL-5 were detected in airway samples from atopic asthmatics

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Fig. 79.12 Transbronchial biopsy of a small airway in a subject with severe asthma showing much folding of the mucosa and consequent compression of the airway lumen. There is an eosinophilic inflammatory infiltrate within the epithelium and in the subepithelium. (Hematoxylin and eosin; magnification ×400.) (See CD-ROM for color version.)

(Robinson et al. 1992). This attractively linked IgE synthesis through IL-4, and eosinophilic airway inflammation through IL-5, together with IL-3 and granulocyte–macrophage colonystimulating factor (GM-CSF). Many other T cell-related cytokines with potential relevance to asthma have been described. IL-9, IL-11, IL-13, IL-16, IL-17, and IL-19 (Del Prete et al. 1993) have been linked to asthma, IL-25 acts in Th2 differentiation, while IL-12, IL-18, IL-23, and IL-27 are involved in Th1 development and interferon (IFN)-γ production, which may be deficient in asthma. Bronchial biopsies from nonatopic asthmatics and individuals with occupational asthma have revealed remarkable histopathologic similarities to individuals with atopic asthma (Azzawi et al. 1990; Bentley et al. 1992a,b). Thus, eosinophil infiltration and cells bearing markers of T-cell activation such as CD25 were present in increased numbers in bronchial biopsies from all three forms of disease. Similarly, the cytokine profile in the airway of nonatopic asthmatics also showed prominence of IL-4, IL-5 and IL-13, with no increase in IFN-γ compared with nonasthmatic control volunteers (Humbert et al. 1996). Some studies also showed Th1 cytokines in BAL from asthmatics, particularly during exacerbations (Krug et al. 1996), although most studies confirm Th2 predominance in stable disease. Mast cells, basophils and eosinophils are themselves potential sources of Th2-type cytokines (Robinson et al. 1993b). Indeed, immunohistochemical staining for cytokines in bronchial biopsies suggests that these cytokines are localized mainly to non-T cells (Ying et al. 1997). However, these findings likely reflect storage of cytokines in mast cells, eosinophils and basophils, as mRNA for IL-4 and IL-5 localized predominantly to T cells with minor contributions from mast cells, basophils and eosinophils. IL-13 is upregulated in asthmatic airways and several animal

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models have shown that IL-13 plays a key role in the development of AHR and mucus production (Grunig et al. 1998; Wills-Karp et al. 1998). Mattes et al. (2002) provided evidence that eosinophils could modulate IL-13 production from CD4+ cells, possibly via IL-18. IL-16 is a CD4+ lymphocyte chemoattractant, while IL-17 is a T cell-derived cytokine that can induce fibroblast production of proinflammatory cytokines including GM-CSF (Molet et al. 2001). IL-25 is a more recently characterized cytokine with homology to IL-17 and drives Th2 IL-4, IL-5 and IL-13 production in murine T cells (Fort et al. 2001). All these cytokines have been reported to be overrepresented in the airways of asthmatic subjects compared with nonasthmatic control volunteers (Humbert et al. 1997; Laberge et al. 1997; Ying et al. 2002), and IL-11 was detected in severe asthma, where it may act in remodeling (Minshall et al. 2000). Mast cells are usually found adjacent to blood vessels in the lamina propria in normal human airways, but in asthma they are observed in the airway epithelium (Bradding et al. 1994), airway mucous glands (Carroll et al. 2002), and airway smooth muscle (Brightling et al. 2002; Bradding 2007; Ammit et al. 1997). Bronchial mucosal biopsies of asthmatics compared with those from patients with eosinophilic bronchitis (which is characterized by cough without reversible airway narrowing or AHR) showed no difference in mucosal eosinophil or T-cell infiltration. However, mast cell infiltration of smooth muscle was seen in the asthmatics, but not in the patients with eosinophilic bronchitis, and it was therefore suggested that AHR and airway narrowing in asthma were more related to smooth muscle and mast cell interaction than the eosinophil–T cell axis (Brightling et al. 2002; Bradding, Walsh & Holgate 2006). The significant correlation between airway smooth muscle mast cell number and bronchial hyperresponsiveness within the asthmatic group also supported the view that the mast cell myositis was of functional relevance. Most asthmatic airway mast cells contain both tryptase and chymase, and express IL-4 and IL-13 but not IL-5. No T cells or eosinophils were found in the smooth muscle of asthma, eosinophilic bronchitis or normals. Neutrophilic inflammation is found in severe persistent asthma (Jatakanon et al. 1999), asthma exacerbations (Fahy et al. 1995; Ordonez et al. 2000b; Qiu et al. 2007), sudden-onset fatal asthma (Sur et al. 1993), occupational asthma (Anees et al. 2002), nocturnal asthma (Martin et al. 1991), and even in childhood asthma (McDougall & Helms 2006). Neutrophils could have a role in the pathophysiology of airway disease through their release of reactive oxygen species, cytokines, lipid mediators, and enzymes including elastase, cathepsin G and myeloperoxidases and nonenzymatic defensins (Borregaard & Cowland 1997). Neutrophil-derived serine proteinases and defensins markedly affect the integrity of the epithelial layer, decrease ciliary beat frequency, and induce the synthesis of epithelium-derived mediators that may influence the amplification and resolution of inflammation (Hiemstra et al.

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1998). Neutrophil proteases, especially neutrophil elastase, can also activate eosinophils (Liu et al. 1999) and are important mucin secretagogues for goblet cells and submucosal gland cells (Nadel et al. 1999) and, by inference, likely to contribute to mucus hypersecretion in asthma. Neutrophil products may also be mediators of increased vascular permeability (Persson 1986) and may directly contribute to AHR (Anticevich et al. 1996). Dendritic cells and their subsets are key antigen-presenting cells, responding rapidly to antigenic challenge with kinetics similar to neutrophils (Moller et al. 1996). They form an interface between innate and adaptive immunity and orchestrate both primary and secondary immune responses (Masten et al. 2006). They are present throughout the respiratory tree and within the epithelium, numbering about 500/mm2 (Holt et al. 1990). New markers to identify subsets (e.g., BDCA1, BDCA2) of these cells and of cell maturity (e.g., CD83) have recently become available and their identification and quantification in asthma, their rapid recruitment in response to allergen challenge (Moller et al. 1996; Jahnsen et al. 2001), and their identification in COPD (Demedts et al. 2005) has begun. Recent data indicate that the numbers of mature bronchial mucosal dendritic cells, B lymphocytes and key regulatory cytokines are reduced in asthmatic smokers as compared with asthmatic never-smokers (Tsoumakidou et al. 2007). There are also data indicating that the effects of smoke include reduced dendritic cell function (Vassallo et al. 2005). Macrophages may also increase in number in asthma, particularly in intrinsic asthma (Bentley et al. 1992b).

Airway wall nerves Airway wall innervation and its relationship to asthma is considered in Chapter 38. There are data showing that in fatal asthma there is an absence of (relaxant) vasoactive intestinal polypeptide (VIP)-containing nerve fibers and an increase in the numbers of substance-P-containing fibers (stimulatory to bronchial smooth muscle), contrasting markedly with the innervation of control lungs taken at resection from chronic smokers (Ollerenshaw et al. 1991; Ollerenshaw & Woolcock 1993). However, this reduction has not been confirmed in examination of bronchial biopsies in mild asthma (Howarth et al. 1995). While Sharma and colleagues have described a reduction in airway VIP and β-adrenoceptors in cystic fibrosis, the densities of both VIP receptors and β-adrenoceptors are reported to be similar in asthma to those of grossly normal tissue of lungs resected for carcinoma (Sharma & Jeffery 1990a,b).

Pathology of distal lung in asthma Original physiologic studies in the Meakins-Christie Laboratories suggested that the distal airways and the lung par-

Pathology of Asthma

enchyma are likely to play a major role in asthma (Macklem & Mead 1967; Brown et al. 1969). However, it is only fairly recently that the technology has been available to study these peripheral structures directly (Romero et al. 1992; Tepper et al. 1992; Sato et al. 1993; Suki et al. 1995). Thus molecular pathology data obtained from surgically resected lung tissue (Hamid et al. 1997; Minshall et al. 1998; Taha et al. 1999), postmortem lung specimens (Carroll et al. 1997; Faul et al. 1997; Christodoulopoulos et al. 2000), and transbronchial biopsies (Kraft et al. 1996, 1999a) have revealed severe inflammatory and structural changes in both the distal lung (Carroll et al. 1996; Synek et al. 1996; Kraft et al. 1999a) and in the lung parenchyma of asthmatic patients (Kraft et al. 1996). The early studies on the pathophysiology of the peripheral or distal airways in asthma originated from autopsy studies (Carroll et al. 1996, 1997; Christodoulopoulos et al. 2000). These studies showed that the entire length of the airway was involved, not only the central airways as originally proposed. Carroll and colleagues carefully examined the distribution of inflammatory cells throughout the bronchial tree of both fatal and nonfatal cases of asthma and demonstrated increased numbers of lymphocytes and eosinophils, uniformly distributed, throughout the large and distal airways of mild and severe asthmatics when compared to control cases (Christodoulopoulos et al. 2000). Similar infiltration of T cells, macrophages and eosinophils into the proximal and distal lung tissues has also been reported in rare cases of sudden asthma death (dying within 1 hour after the onset of their symptoms) (Sur et al. 1993) and in soybean dust-induced asthma (Synek et al. 1996). Using surgically resected lung specimens from asthmatic and nonasthmatic patients, it has also been demonstrated that the inflammatory response in asthma is not restricted to the proximal airways but is also seen in the distal lung (Hamid et al. 1997). Thus there was evidence of increased number of CD3+ T cells and major basic protein (MBP)-positive eosinophils in not only the large airways (> 2 mm internal diameter) but also in the distal airways (< 2 mm internal diameter) from asthmatic patients when compared with healthy controls. In fact, a greater number of activated eosinophils was seen in the airways < 2 mm internal diameter than in the airways > 2 mm internal diameter, suggesting that a similar but more severe inflammatory process is present in the distal airways (Kraft et al. 1999a). In the same cohort of patients, increased IL-5 and IL-4 mRNA-positive cells were observed in the distal airways of asthmatic subjects compared with nonasthmatic controls (Minshall et al. 1998); moreover, the expression of IL-5 mRNA was increased in the distal airways compared with the large airways. The numbers of cells expressing IL-2 and IFN-γ in these subjects was no different to controls. Furthermore, simultaneous in situ hybridization and immunocytochemistry indicated that 85% of the IL-5 mRNA-positive cells in the distal airways were CD3+ T cells, i.e., similar to the proportion found in the large airways (81%) (Minshall et al.

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1998). In addition, there was increased eotaxin and monocyte chemotactic protein (MCP)-4 mRNA expression in the distal airway epithelium of asthmatics compared with nonasthmatics and the number of chemokine-positive cells in the distal airways of these patients correlated with the number of MBP-positive eosinophils at this same site (Taha et al. 1999). Haley et al. (1998) measured the distribution of CD45+ cells within the subepithelial regions of the airway wall. They showed that the majority of their CD45+ leukocytes were associated with the distal asthmatic airways and that eosinophils were mainly in the “outer” airway wall region (“outer” defined as the area between the smooth muscle and the alveolar attachments). However, in the large asthmatic airways the greatest density of eosinophils was in the “inner” airway wall region (“inner” defined as the area between the epithelial RBM and the smooth muscle). These differences highlight the regional variations in inflammatory cell distribution within the asthmatic airway wall and these differences appear to be disease specific since such variations were not observed in patients with cystic fibrosis (Haley et al. 1998). The different regional organization of inflammatory cells throughout the tracheobronchial tree may be attributed to the different mechanisms of inflammatory cell recruitment and/or differences in chemokine and cytokine production between these tissue regions. Such differences may influence the physiologic response resulting from local production of proinflammatory mediators. In support of this claim, increased expression of eotaxin, a potent eosinophil chemoattractant, has been documented in the airway epithelial layer of the peripheral airways of asthmatics (Taha et al. 1999). The smooth muscle itself has been proposed as a site of chemokine production in the distal airways (Haley et al. 1998). In patients who died of asthma, inflammation extends well beyond the airway smooth muscle and is still significant around the pulmonary arterioles (Saetta et al. 1991). Kraft et al. (1996) have shown significant alveolar inflammation in patients with nocturnal asthma (NA) compared with patients with nonnocturnal asthma (NNA). In these studies, both proximal airway endobronchial and distal alveolar tissue transbronchial biopsies were performed in the same patient at 4.00 p.m. and 4.00 a.m. Patients with NA had increased number of eosinophils per lung volume in their lung parenchyma at 4.00 a.m. compared with patients without NA, and the NA patients had a greater number of eosinophils and macrophages in their alveolar tissue at 4.00 a.m. than at 4.00 p.m. Moreover, in NA patients, only alveolar but not central airway eosinophilia correlated with overnight reduction in lung function (Kraft et al. 1996) and was associated with increased number of CD4+ cells in alveolar tissue of NA patients at 4.00 a.m. compared with NNA patients (Kraft et al. 1999a). Although the number of CD4+ cells in the endobronchial lamina propria was higher than in the alveolar tissue, once again only the alveolar CD4+ lymphocytes correlated with

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predicted lung function at 4.00 a.m. and with the number of activated alveolar EG2+ eosinophils (Kraft et al. 1999a). In this same group of patients, NA was associated with reduced glucocorticoid receptor (GR)-binding affinity, reduced proliferation of peripheral blood mononuclear cells, and decreased responsiveness to steroids when compared with NNA patients (Kraft et al. 1999b). Those studies have demonstrated that the increased numbers of CD4+ cells in alveolar tissue in NA patients, as well as reduced GR-binding affinity and reduced steroid responsiveness, may be responsible for orchestrating eosinophil influx and exacerbations of symptoms in patients with NA. One of the proposed mechanisms that may be responsible for this phenomenon is upregulation in the expression of GR-β, previously reported in steroid-insensitive subjects with severe asthma (Christodoulopoulos et al. 2000). Christodoulopoulos and colleagues have characterized GR-β expression within the peripheral airways in cases of severe fatal asthma. They have demonstrated that the main cells expressing GR-β were CD3+ T lymphocytes and, to a lesser extent, eosinophils, neutrophils and macrophages (Christodoulopoulos et al. 2000). Those results suggest that increased numbers of GR-β cells in the distal airways of patients with fatal asthma may be associated with steroid resistance, contributing to asthma mortality. Distal airway inflammation has been reported in severe symptomatic, steroid-dependent asthmatics. Using endobronchial and transbronchial biopsies, Wenzel and colleagues reported persistent proximal and distal airway inflammation in these patients (Wenzel et al. 1997; Kraft et al. 1999b). Although the number of eosinophils was similar in severe asthmatics and normal controls, severe asthmatics had high numbers and percentages of neutrophils in their BAL and endobronchial and transbronchial biopsy specimens when compared with mild-to-moderate asthmatics, despite aggressive treatment with steroids. In cases of fatal asthma, inflammatory cells make up a higher proportion of the exudates that occlude the smaller airways than the exudates from the larger airways (Kraft 1999). From this data it is concluded that the inflammatory cell density in the distal airways of severe asthmatics may relate to the peripheral airway obstruction characteristic of this disease. The distal airway inflammation may cause uncoupling of the parenchyma and airways due to the mechanical interdependence between these two compartments leading to changes in overall lung mechanics in asthmatics.

Pathology of upper airways in asthma (united airways theory) The majority (80%) of asthmatics have allergic rhinitis and up to 50% of rhinitics have asthma (Demoly & Bousquet 2006). In patients with rhinitis there is evidence of subclinical inflammation in the lower airways including an increase in the number of eosinophils and T cells and high expression of

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IL-5 even in the absence of asthma symptoms (Chakir et al. 2000; Gaga et al. 2000). Similarly, a number of reports have demonstrated evidence of inflammation in the upper airways in asthmatic subjects under baseline conditions and following segmental allergen challenge (Braunstahl et al. 2000). The association between asthma and sleep apnea has been attributed to inflammation outside the lung, in the uvula and posterior pharynx and larynx. Sobol et al. (2002) have shown in allergic individuals that more than 50% of children with otitis media have no evidence of infection. These investigators demonstrated that the middle ear and the lower part of the Eustachian tube have inflammatory infiltrates similar to that found in asthma and rhinitis. There are a number of explanations for the association of upper and lower airway inflammation including neuronal response, bone marrow stimulation, and mechanical strain. These observations are the basis for the “united airways theory” that is currently an area of active investigation and which has been reviewed (Jeffery & Haahtela 2006).

Childhood and infant “asthma” Wheezy symptoms often develop early in infants: these may be transient or, in some, they may persist to become asthma in later childhood. Those who develop difficult asthma experience continuing symptoms despite maximal conventional treatment. Their lives are severely disrupted, with frequent hospital visits, school absence, and limitation of normal activities. Persistent airflow limitation is present in probably less than 5% of all children with asthma, but their contribution to morbidity and use of health service resources is disproportionately large, as is the case in adult asthma. Cutz et al. (1978) were among the first to report the lung pathology of children with asthma. They investigated lung tissue of an 11-year-old asthmatic child who had received an open lung biopsy because of persistently abnormal chest radiography with pulmonary nodularity and suspected fibrosis. Although the child had been in remission for 3 months, the airways still showed plugging by mucus and exudates. This case, and two further pediatric cases of status asthmaticus of about the same age examined at autopsy, also demonstrated the presence of airway plugging and, in addition, there was tissue eosinophilia, loss of surface epithelium, thickening of the RBM, enlargement of bronchial smooth muscle mass, bronchial goblet cell hyperplasia, and submucosal gland hypertrophy, i.e., all the features described above in adults with asthma. Among the key questions in asthma are the following. 1 When does the inflammation and remodeling of asthma begin in those who will go on to develop asthma? 2 Which of these comes first, inflammation or remodeling, and what is their timing and relationship with the clinical signs, symptoms and lung functional changes of asthma?

Pathology of Asthma

3 Are the pathologic changes already present at birth or is there a “window of opportunity” after birth during which appropriate intervention may slow their progression? Improvements in bronchoscopy and reaffirmation of its safety when used in experienced hands have recently allowed the sampling of the airways of wheezy children with and without asthma. There are data coming from examination of sputa (Cai et al. 1998), BAL (Stevenson et al. 1997; Krawiec et al. 2001), and exhaled NO (Mattes et al. 1999; Payne et al. 2001) demonstrating variable sputum eosinophilia and loss of epithelial cells, and increases in BAL total cells and inflammatory cells of all phenotypes, particularly eosinophils and mast cells in atopic asthmatic children, but not in children with virus- associated wheeze or atopy alone. In acute exacerbations of asthma, studies of sputa demonstrate three overall patterns of inflammation: (i) increased eosinophils, (ii) eosinophilic and neutrophilic infiltration, and (iii) a noneosinophilic pattern (Gibson et al. 1999). There appear to be associations between exhaled NO and sputum eosinophils and biopsy tissue eosinophils in patients with symptoms that persist despite treatment with prednisolone. The challenges of biopsy in both adults and children have been discussed (Bush & Pohunek 2000; Jeffery et al. 2003; Midulla et al. 2003). Because bronchoscopy in children can only be justified for clinical indications, of necessity most (but not all) of these studies have been in children at the severe end of the clinical spectrum, i.e., with persistent symptoms and unresponsive to conventional therapy. A case series of six children with oral corticosteroid-dependent asthma reported thickening of the RBM and minimal cellular inflammation in endobronchial biopsy specimens (Jenkins et al. 2003). de Blic et al. (2004) examined endobronchial biopsy specimens in groups of asthmatic children with or without persistent airflow limitation despite high-dose inhaled corticosteroids and long acting β2 agonist: greater numbers of intraepithelial eosinophils and neutrophils were seen in the subgroup with persistent symptoms than in those with few symptoms. Most studies report that tissue eosinophilia is highly variable and one study reports increased numbers of CD4+ T cells and their inverse relationship with percent FEV1 (Payne et al. 2004). RBM thickening appears to be a consistent finding in all studies of difficult asthma in children. The RBM thickening appears to be maximal in asthmatic children of about 10 years median age, with no relationship to age, duration, or severity of disease (Cokugras et al. 2001; Payne et al. 2003). The thickening is even present on examination of children with relatively mild asthma who were undergoing bronchoscopy for clinical indications other than asthma (Barbato et al. 2003). RBM thickening begins during the early evolution of their disease at a time prior to the diagnosis of asthma (Pohunek et al. 2005; Saglani et al. 2008). However, in recurrent wheezy infants of median age 12 months with reversible airflow obstruction (determined by plethysmography) it was expected that the early changes of asthma would be detected. Interestingly,

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despite their severe symptoms, their biopsies showed an absence of RBM thickening and no tissue eosinophilia. Thus RBM thickening, a key feature of asthma in older children and adults, is not yet developed in a group of symptomatic infants under 2 years of age with reversible airflow obstruction, even in the presence of atopy (Saglani et al. 2005). In contrast, RBM thickening and tissue eosinophilia is detected in preschool children of median age 29 months, when a positive relationship between eosinophilia and RBM thickening becomes apparent (Saglani et al. 2008). Thus, biopsy studies of airway tissue demonstrate that the characteristic pathologic features of asthma in adults and school-aged children develop in preschool children with confirmed wheeze between the ages of 1 and 3 years, at a time when intervention may modify the natural history of asthma.

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Barnes, P.J. (1986) State of art: neural control of human airways in health and disease. Am Rev Respir Dis 134, 1289–314. Beasley, R., Roche, W., Roberts, J.A. & Holgate, S.T. (1989) Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 139, 806–17. Bel, E.H. (2004) Clinical phenotypes of asthma. Curr Opin Pulm Med 10, 44–50. Benayoun, L., Druilhe, A., Dombret, M.C., Aubier, M. & Pretolani, M. (2003) Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 167, 1360–8. Benson, M.K. (1975) Bronchial hyperreactivity. Br J Dis Chest 69, 227–39. Bentley, A.M., Maestrelli, P., Saetta, M. et al. (1992a) Activated T lymphocytes and eosinophils in the bronchial mucosa in isocyanate-induced asthma. J Allergy Clin Immunol 89, 821–9. Bentley, A.M., Menz, G., Storz, C. et al. (1992b) Identification of Tlymphocytes, macrophages and activated eosinophils in the bronchial mucosa in intrinsic asthma: relationship to symptoms and bronchial responsiveness. Am Rev Respir Dis 146, 500–6. Bentley, A.M., Meng, Q., Robinson, D.S., Hamid, Q., Kay, A.B. & Durham, S.R. (1993) Increases in activated T lymphocytes, eosinophils and cytokine messenger RNA expression for IL-5 and GMCSF in bronchial biopsies after allergen inhalation challenge in atopic asthmatics. Am J Respir Cell Mol Biol 8, 35– 42. Borger, P., Tamm, M., Black, J.L. & Roth, M. (2006) Asthma: is it due to an abnormal airway smooth muscle cell? Am J Respir Crit Care Med 174, 367–72. Borregaard, N. & Cowland, J.B. (1997) Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503–21. Bousquet, J., Lacoste, J.-Y., Chanez, P., Vic, P., Godard, P. & Michel, F.-B. (1996) Bronchial elastic fibers in normal subjects and asthmatic patients. Am J Respir Crit Care Med 153, 1648–54. Bradding, P. (2007) Mast cell regulation of airway smooth muscle function in asthma. Eur Respir J 29, 827–30. Bradding, P., Roberts, J.A., Britten, K.M. et al. (1994) Interleukin-4, -5, and -6 and tmour necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am J Respir Cell Mol Biol 10, 471–80. Bradding, P., Walls, A.F. & Holgate, S.T. (2006) The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol 117, 1277–84. Bradley, B.L., Azzawi, M., Jacobson, M. et al. (1991) Eosinophils, Tlymphocytes, mast cells, neutrophils and macrophages in bronchial biopsies from atopic asthmatics: comparison with atopic non-asthma and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol 88, 661–74. Braunstahl, G.J., Kleinjan, A., Overbeek, S.E., Prins, J.B., Hoogsteden, H.C. & Fokkens, W.J. (2000) Segmental bronchial provocation induces nasal inflammation in allergic rhinitis patients. Am J Respir Crit Care Med 161, 2051–7. Brewster, C.E.P., Howarth, P.H., Djukanovic, R., Wilson, J., Holgate, S.T. & Roche, W.R. (1990) Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 3, 507–11. Brightling, C.E., Bradding, P., Symon, F.A., Holgate, S.T., Wardlaw, A.J. & Pavord, I.D. (2002) Mast cell infiltration of airway smooth muscle in asthma. N Engl J Med 346, 1699–705. Brown, R., Woolcock, A.J., Vincent, N.J. & Macklem, P.T. (1969) Physiological effects of experimental airway obstruction with beads. J Appl Physiol 27, 328–35.

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Pathology of Asthma

Wanner, A. (1988) Airway mucus and the mucociliary system. In: Middleton, E., Reed, C.E., Ellis, E.F., Adkinson, N.F. & Uunginer, J.W., eds. Allergy: Principles and Practice. C.V. Mosby, St Louis, pp. 541–8. Ward, C., Johns, D.P., Bish, R. et al. (2001) Reduced airway distensibility, fixed airflow limitation, and airway wall remodeling in asthma. Am J Respir Crit Care Med 164, 1718–21. Wenzel, S.E., Szefler, S.J., Leung, D.Y., Sloan, S.I., Rex, M.D. & Martin, R.J. (1997) Bronchoscopic evaluation of severe asthma. Persistent inflammation associated with high dose glucocorticoids. Am J Respir Crit Care Med 156, 737–43. Westergren-Thorsson, G., Chakir, J., Lafreniere-Allard, M.J., Boulet, L.P. & Tremblay, G.M. (2002) Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects. Int J Biochem Cell Biol 34, 1256–67. Widdicombe, J. (1993) New perspectives on basic mechanisms in lung disease. 4. Why are the airways so vascular? Thorax 48, 290–5. Wiggs, B.R., Moreno, R., Hogg, J.C., Hilliam, C. & Pare, P.D. (1990) A model of the mechanics of airway narrowing. J Appl Physiol 69, 849– 60. Wiggs, B.R., Bosken, C., Pare, P.D., James, A. & Hogg, J.C. (1992) A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 145, 1215–18. Wiggs, B.R., Hrousis, C.A., Drazen, J.M., Kamm, R.D. (1997) On the mechanism of mucosal folding in normal and asthmatic airways. J Appl Physiol 83, 1814–21. Wills-Karp, M., Luyimbazi, J., Xu, X. et al. (1998) Interleukin-13: central mediator of allergic asthma. Science 282, 2258–61. Woodruff, P.G., Dolganov, G.M., Ferrando, R.E. et al. (2004) Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 169, 1001–6. Ying, S., Humbert, M., Barkans, J. et al. (1997) Expression of IL-4 and IL-5 mRNA and protein product by CD4+ and CD8+ T cells, eosinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics. J Immunol 158, 3539–44. Ying, S., Meng, Q., Kay, A.B. & Robinson, D.S. (2002) Elevated expression of interleukin-9 mRNA in the bronchial mucosa of atopic asthmatics and allergen-induced cutaneous late-phase reaction: relationships to eosinophils, mast cells and T lymphocytes. Clin Exp Allergy 32, 866–71.

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Management of Chronic Asthma Peter J. Barnes

Summary There are now highly effective treatments available for the management of chronic asthma. Short-acting β2 agonists are used as required for relief of symptoms but inhaled corticosteroids (ICS) should be given as a regular treatment for all patients who have symptoms three or more times a week. Other controlled treatments, including antileukotrienes and theophylline, are less effective. For patients who are not controlled on a low dose of ICS, addition of a long-acting β2 agonist is recommended. This treatment is most conveniently given as a combination inhaler. Theophylline is another add-on therapy that is useful in patients with more severe disease, whereas antileukotrienes appear to benefit some patients when used as an add-on therapy. A few patients are not controlled on maximal inhaled therapy and may require daily oral corticosteroids, but the lowest dose needed to control asthma should be used. Some patients with severe asthma and fixed airway obstruction may benefit from an inhaled anticholinergic. Steroid-sparing therapies are not usually effective. Anti-IgE therapy (omalizumab) is indicated only in patients who are not controlled with maximal inhaled therapy or a low dose of oral corticosteroids and who have frequent exacerbations. Allergen avoidance provides no convincing benefit for most patients and allergen immunotherapy is not recommended. Alternative therapies are ineffective but may be used if the patient wishes as long as conventional therapy is continued. New therapies are needed for patients with refractory asthma.

Introduction The treatment of asthma is relatively straightforward and the majority of patients are now managed by general practitioners with effective and safe therapies. There are several aims of therapy (Table 80.1). Most emphasis has been placed

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Table 80.1 Aims of asthma therapy. Minimal (ideally no) chronic symptoms, including nocturnal Minimal (infrequent) exacerbations No emergency visits Minimal (ideally no) use of as-required b2 agonist No limitations on activities, including exercise PEF circadian variation < 20% (Near) normal PEF Minimal (or no) adverse effects from medicine PEF, peak expiratory flow.

in drug therapy, but several nonpharmacologic approaches have also been used. The main drugs for the management of chronic asthma can be divided into bronchodilators, which give rapid relief of symptoms mainly through relaxation of airway smooth muscle, and controllers, which inhibit the underlying inflammatory process and prevent the development of symptoms. Asthma is usually managed according to guidelines which adopt a stepwise approach to therapy (British Thoracic Society 2003; Global Initiative for Asthma 2006).

Bronchodilators Bronchodilators act primarily on airway smooth muscle to reverse the bronchoconstriction of asthma. This gives rapid relief of symptoms but has little or no effect on the underlying chronic inflammatory process. Thus, bronchodilators are not sufficient to control asthma in patients with persistent symptoms. There are three classes of bronchodilator in current use: β2-adrenergic agonists, anticholinergics, and theophylline; of these, β2 agonists are by far the most effective.

b2 Agonists

β2 Agonists activate β2-adrenergic receptors, which are widely expressed in the airways. The β2-adrenergic receptors are coupled through a stimulatory G protein to adenylyl cyclase, resulting in increased intracellular cyclic AMP, which relaxes smooth muscle cells and inhibits certain inflammatory cells.

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Mode of action

Safety

The primary action of β2 agonists is to relax airway smooth muscle cells of all airways, where they act as functional antagonists, reversing and preventing contraction of airway smooth muscle cells by all known bronchoconstrictors. This generalized action is likely to account for their great efficacy as bronchodilators in asthma. There are also additional nonbronchodilator effects that may be clinically useful, including inhibition of mast cell mediator release, reduction in plasma exudation, and inhibition of sensory nerve activation. Inflammatory cells express small numbers of β2 receptors, but these are rapidly downregulated with β2-agonist activation so that, in contrast to corticosteroids, there are no effects on inflammatory cells in the airways and there is no reduction in airway hyperresponsiveness (AHR).

The safety of β2 agonists has been an important issue. There is an association between asthma mortality and the amount of SABA used, but careful analysis demonstrates that the increased use of rescue SABA reflects poor asthma control, which is a risk factor for asthma death (Ernst et al. 1993). Recently concerns have been raised about the safety of LABAs in the management of asthma and this is clearly particularly relevant to the use of a LABA in treating exacerbations (Salpeter et al. 2006). A controlled trial of salmeterol versus placebo in over 26 000 patients with asthma showed a small but significant excess of asthma mortality and life-threatening events in the LABA-treated patients, raising concerns that this treatment may be causally related to the increased deaths (Nelson et al. 2006). Subgroup analysis showed that the great majority of these deaths occurred in inner city AfricanAmericans and it is very likely that this can be explained by failure to use concomitant ICS, as recommended in clinical practice. It is also possible that this could be explained by genetic differences in β2-adrenergic receptors in this group. A metaanalysis that included this study with other smaller studies, including studies with formoterol treatment, concluded that LABAs may increase severe exacerbations and mortality but did not analyze whether this effect was not seen if patients were treated with concomitant ICS (Salpeter et al. 2006). The likely cause of increased mortality associated with increased use of β2 agonists is overreliance on a bronchodilator that does not treat the underlying inflammatory disease, which requires ICS therapy, and the fact that there is confounding by severity, so patients with more severe asthma and exacerbations take higher doses of inhaled β2 agonists. The problem of safety should be resolved by using concomitant corticosteroids in the form of a fixed combination inhaler. There are extensive data on the safety of combination inhalers, often in high doses, over periods of up to 12 months in asthmatic patients of varying severity. These studies have shown a further reduction in severe exacerbations compared with corticosteroids alone and there are no safety concerns in terms of adverse effects (Pauwels et al. 1997; O’Byrne et al. 2001; Bateman et al. 2004).

Clinical use β2 Agonists are usually given by inhalation to reduce side effects. Short-acting β2 agonists (SABAs), such as salbutamol and terbutaline, have a duration of action of 3–6 hours. They have a rapid onset of bronchodilatation and are therefore used as needed for symptom relief. Increased use of SABAs indicates that asthma is not controlled. They are also useful in preventing exercise-induced asthma if taken prior to exercise. SABAs are used in high doses by nebulizer or via a metered-dose inhaler with a spacer. Long-acting β2 agonists (LABAs) include salmeterol and formoterol, both of which have a duration of action over 12 hours and are given twice daily. LABAs have replaced the regular use of SABAs, but LABAs should not be given in the absence of inhaled corticosteroids (ICS) as they do not control the underlying inflammation. However, they do improve asthma control and reduce exacerbations when added to ICS, which allows asthma to be controlled at lower doses of corticosteroids (Greenstone et al. 2005). This observation has led to the widespread use of fixed combination inhalers that contain a corticosteroid and a LABA, which have proved to be highly effective in the control of asthma (Barnes 2002).

Side effects Adverse effects are not usually a problem with β2 agonists when given by inhalation. The commonest side effects are muscle tremor and palpitations, which are seen more commonly in elderly patients. There is a small fall in plasma potassium due to increased uptake by skeletal muscle cells, but this effect does not usually cause a clinical problem.

Tolerance Tolerance is a potential problem with any agonist given chronically, but while there is downregulation of β2-adrenergic receptors, this does not reduce the bronchodilator response as there is a large receptor reserve in airway smooth muscle cells (Grove & Lipworth 1995). In contrast, mast cells become rapidly tolerant, but this may be prevented by concomitant administration of ICS (O’Connor et al. 1992).

Anticholinergics Muscarinic receptor antagonists, such as ipratropium bromide, prevent cholinergic nerve-induced bronchoconstriction and mucus secretion (Gross 2006). They are much less effective than β2 agonists in asthma therapy as they inhibit only the cholinergic reflex component of bronchoconstriction, whereas β2 agonists prevent all bronchoconstrictor mechanisms as they are functional antagonists. Anticholinergics are therefore only used as an additional bronchodilator in patients with chronic asthma that is not controlled on other inhaled medications. The long-acting anticholinergic tiotropium bromide given once is preferred to short-acting drugs such as ipratropium bromide. Side effects are not usually a problem as there is little or

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no systemic absorption. The most common side effect is dry mouth; in elderly patients, urinary retention and glaucoma may also be observed.

Theophylline Theophylline was widely prescribed as an oral bronchodilator several years ago, especially as it was inexpensive. It has now fallen out of favor as side effects are common and inhaled β2 agonists are much more effective as bronchodilators. The bronchodilator effect is due to inhibition of phosphodiesterases in airway smooth muscle cells, which increases cyclic AMP, but doses required for bronchodilatation commonly cause side effects that are mediated mainly by phosphodiesterase inhibition. There is increasing evidence that theophylline at lower doses has antiinflammatory effects, and these are likely to be mediated through different molecular mechanisms (Barnes 2003). There is evidence that theophylline activates the key nuclear enzyme histone deacetylase 2, which is a critical mechanism for switching off activated inflammatory genes, and thus enhances the antiinflammatory effect of corticosteroids, which suppress inflammation through the same mechanism.

Side effects Oral theophylline is well absorbed and is largely inactivated in the liver. Side effects are related to plasma concentrations; measurement of plasma theophylline may be useful in determining the correct dose. The commonest side effects are nausea, vomiting, and headaches and are due to phosphodiesterase inhibition Diuresis and palpitations may also occur; at high concentrations, cardiac arrhythmias, epileptic seizures, and death may occur due to adenosine receptor antagonism. Theophylline side effects are related to plasma concentration and are rarely observed at plasma concentrations below 10 mg/ L. Theophylline is metabolized by cytochrome P450 in the liver, and thus plasma concentrations may be elevated by drugs that block cytochrome P450, such as erythromycin and allopurinol (Table 80.2). Other drugs may also reduce clearance by other mechanisms leading to increased plasma concentrations. At low doses, giving plasma concentrations of 5–10 mg/L, the drug is well tolerated.

Controller therapies Inhaled corticosteroids

Clinical use Oral theophylline is usually given as a slow-release preparation once or twice daily as this gives more stable plasma concentrations than normal theophylline tablets. It may be used as an additional bronchodilator in patients with severe asthma when plasma concentrations of 10–20 mg/L are required, although these concentrations are often associated with side effects. Low doses of theophylline, giving plasma concentrations of 5–10 mg/L, have additive effects to ICS and are particularly useful in patients with severe asthma (Evans et al. 1997). Indeed, withdrawal of theophylline from these patients may result in marked deterioration in asthma control (Kidney et al. 1995). Theophylline is less effective than LABAs as an add-on therapy to ICS (Wilson et al. 2000; Kankaanranta et al. 2004).

ICS are by far the most effective controllers for asthma, and their early use has revolutionized asthma therapy. ICS are now first-line therapy for all patients with persistent asthma (see Chapter 31).

Clinical use ICS are by far the most effective controllers in the management of asthma and are beneficial in treating asthma of any severity and age (Barnes et al. 1998). ICS are usually given twice daily, but some may be effective once daily in mildly symptomatic patients. ICS rapidly improve the symptoms of asthma, and lung function improves over several days. They are effective in preventing asthma symptoms, such as exercise-induced asthma and nocturnal exacerbations, but also prevent severe exacerbations. ICS reduce AHR, but maximal improvement

Table 80.2 Levels of asthma control. (Adapted from Global Initiative for Asthma 2006, with permission.)

Characteristic

Controlled (all of the following)

Partly controlled (any measurement in any week)

Daytime symptoms

None (twice or less/week)

More than twice/week

Limitation of activities Nocturnal symptoms/awakening Need for reliever Lung function (PEF/FEV1) Exacerbations

None None Twice or less/week Normal None

Any Any More than twice/week < 80% predicted or personal best One or more/year

PEF, peak expiratory flow; FEV1, forced expiratory volume in 1 s.

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Uncontrolled Three or more features of partly controlled asthma in any week

One in any week

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may take several months of therapy (Juniper et al. 1990). However, ICS appear to control airway inflammation in asthma within a few hours (Gibson et al. 2001). Early treatment with ICS appears to prevent irreversible changes in airway function that occur with chronic asthma (O’Byrne et al. 2006). Withdrawal of ICS results in slow deterioration of asthma control, indicating that they suppress inflammation and symptoms but do not cure the underlying condition. ICS are now given as first-line therapy for patients with persistent asthma, but if they do not control symptoms at low doses, it is usual to add a LABA as the next step.

They are less effective than ICS in controlling asthma and have less effect on airway inflammation, but are useful as an add-on therapy in some patients not controlled with low doses of ICS, although they are less effective than LABAs based on controlling symptoms and reducing exacerbations (Ducharme et al. 2006). They are given orally once (montelukast) or twice (zafirlukast) daily and are well tolerated. Some patients show a better response than others to antileukotrienes, but this has not been convincingly linked to any consistent genomic differences in the leukotriene pathway (Wechsler & Israel 2002).

Side effects

Cromones

Local side effects include hoarseness (dysphonia) and oral candidiasis, which may be reduced with the use of a largevolume spacer device. There has been concern about systemic side effects from lung absorption, but many studies have demonstrated that ICS have minimal systemic effects. At the highest recommended doses, there may be some suppression of plasma and urinary cortisol concentrations, but there is no convincing evidence that long-term treatment leads to impaired growth in children or to osteoporosis in adults. Indeed effective control of asthma with ICS reduces the number of courses of oral corticosteroids needed and thus reduces systemic exposure to ICS.

Sodium cromoglycate and nedocromil sodium (collectively known as cromones) are asthma controller drugs that appear to inhibit mast cell and sensory nerve activation, and are therefore effective in blocking trigger-induced asthma such as exercise-induced asthma, and allergen- and sulfur dioxideinduced symptoms (Norris 1996). Cromones have relatively little benefit in the long-term control of asthma in adults or children due to their short duration of action (at least four times daily by inhalation) (Wouden et al. 2003). They are very safe and were popular in the treatment of childhood asthma, although now low doses of ICS are preferred as they are more effective and have a proven safety profile (Guevara et al. 2006).

Systemic corticosteroids A short course of oral corticosteroids (usually prednisone or prednisolone 30– 45 mg o.d. for 5–10 days) is used to treat acute exacerbations of asthma; no tapering of the dose is needed. Approximately 1% of asthma patients may require maintenance treatment with oral corticosteroids; the lowest dose necessary to maintain control needs to be determined. Systemic side effects, including truncal obesity, bruising, osteoporosis, diabetes, hypertension, gastric ulceration, proximal myopathy, depression, and cataracts, may be a major problem, and steroid-sparing therapies may be considered if side effects are a significant problem. If patients require maintenance treatment with oral corticosteroids, it is important to monitor bone density so that preventive treatment with bisphosphonates or estrogen in postmenopausal women may be initiated if bone density is low. Intramuscular triamcinolone acetonide is a depot preparation occasionally used in noncompliant patients (ten Brinke et al. 2004), but proximal myopathy is a major problem with this therapy.

Antileukotrienes Cysteinyl leukotrienes are potent bronchoconstrictors, cause microvascular leakage, and increase eosinophilic inflammation through the activation of CysLT1 receptors (Leff 2001). These inflammatory mediators are produced predominantly by mast cells and, to a lesser extent, eosinophils in asthma. Antileukotrienes, such as montelukast and zafirlukast, block CysLT1 receptors and provide modest clinical benefit in asthma.

Steroid-sparing therapies Various immunomodulatory treatments have been used to reduce the requirement for oral corticosteroids in patients with severe asthma who have serious side effects with this therapy. Methotrexate, cyclosporin A, azathioprine, gold, and intravenous gammaglobulin have all been used as steroidsparing therapies, but none of these treatments has any longterm benefit and each is associated with a relatively high risk of side effects (Davies et al. 2000; Evans et al. 2001a,b; Dean et al. 2004).

Anti-IgE Omalizumab is a blocking antibody that neutralizes circulating IgE without binding to cell-bound IgE, and thus inhibits IgE-mediated reactions. This also inhibits the production of IgE from B lymphocytes, resulting in a marked downregulation of high-affinity IgE receptors on mast cells (Fahy 2006). This treatment has been shown to significantly reduce the number of exacerbations (by up to 50%) in patients with severe asthma and may improve asthma control and reduce the need for corticosteroids, while having little effect on AHR and lung function (Walker et al. 2006). However, the treatment is very expensive and is only suitable for highly selected patients who are not controlled on maximal doses of inhaled therapy and have circulating IgE within a specified range. Patients should be given a 3–4 month trial of therapy to show objective benefit. The treatment is only likely to be

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cost-effective in patients who have frequent hospitalizations for acute severe asthma. Omalizumab is usually given as a subcutaneous injection every 2– 4 weeks and appears to have no significant side effects.

Immunotherapy Specific immunotherapy using injected extracts of pollens or house-dust mite has not been very effective in controlling asthma and may cause anaphylaxis. A metaanalysis of immunotherapy in asthma compared with placebo shows modest clinical efficacy (Abramson et al. 2003). One problem is that most asthma patients are sensitized to several environmental allergens, but immunotherapy against multiple allergens has not been successful (Adkinson et al. 1997). Side effects may be reduced by sublingual dosing but sublingual immunotherapy is rather poorly effective in asthma for the same reason (Calamita et al. 2006). Specific immunotherapy is not recommended in most asthma treatment guidelines because of lack of evidence of clinical efficacy and lack of studies comparing it with traditional therapy, particularly ICS.

Management of chronic asthma There are several aims of chronic therapy in asthma (Table 80.1). It is important to establish the diagnosis objectively using spirometry or peak expiratory flow (PEF) measurements at home. A recent study showed that PEF monitoring was no more useful than symptom monitoring, at least in elderly patients (Buist et al. 2006). However, patients who have poor perception of asthma worsening may be helped by objective measurements. Triggers that worsen asthma control, such as allergens or occupational agents, should be avoided, whereas triggers such as exercise and fog, which result in transient symptoms, provide an indication that more controller therapy is needed. There may be a compromise between the degree of control and the amount of medication needed to achieve this. Total control of symptoms is difficult to achieve without high doses of inhaled therapy that may have side effects (Bateman et al. 2004).

Allergen avoidance Alternative therapies Nonpharmacologic treatments, including hypnosis, acupuncture, chiropraxy, breathing control, yoga, and speleotherapy, may be popular with some patients. However, placebocontrolled studies have shown that each of these treatments lacks efficacy and cannot be recommended (Slader et al. 2006). However, they are not detrimental and may be used as long as conventional pharmacologic therapy is continued.

Although allergens, particularly house-dust mites, are important in activating chronic airway inflammation in asthma, allergen avoidance has not proved to be of significant clinical value in the control of asthma (O’Connor 2005). This may be because of the inefficiency of methods that are practical at home, including mite-impermeable mattress covers. Food allergens are very rarely of importance in asthma so special diets are not beneficial.

Future therapies

Stepwise pharmacologic therapy

It has proved very difficult to discover novel pharmaceutical therapies, particularly as current therapy with corticosteroids and β2 agonists is so effective in the majority of patients. However, there is a need for the development of new therapies for patients with refractory asthma who have side effects with systemic corticosteroids. Several new therapies for asthma are now in development (Barnes 2004) (see Chapter 83). Antagonists of specific mediators have little or no benefit in asthma, apart from antileukotrienes, which have a rather weak effect, presumably reflecting the fact that multiple mediators are involved. Antagonists of chemokine receptors, particularly CCR3, are in development and may be more effective. Novel antiinflammatory treatments that are in clinical development include inhibitors of phosphodiesterase-4, NF-κB, p38 MAP kinase, and phosphoinositide 3-kinase. However, these drugs, which act on signal transduction pathways common to many cells, are likely to have troublesome side effects, necessitating their delivery by inhalation. Safer and more effective immunotherapy using T-cell peptide fragments of allergens or DNA vaccination are also being investigated. Bacterial products, such as CpG oligonucleotides that stimulate Th1 immunity or regulatory T cells, are also currently under evaluation.

Inhaled drug therapy is the mainstay of therapy for chronic asthma (Global Initiative for Asthma 2006). For patients with mild intermittent asthma, a SABA such as salbutamol or terbutaline is all that is required (Fig. 80.1) (O’Byrne & Parameswaran 2006). There is some evidence that formoterol, but

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OCS anti-IgE LABA

LABA

ICS high dose

ICS high dose

LABA ICS low dose

ICS low dose

Short-acting b2-agonist as required for symptom relief Mild intermittent Step 1

Mild persistent Step 2

Moderate persistent Step 3

Severe persistent Step 4

Very severe persistent Step 5

Fig. 80.1 Stepwise approach to asthma therapy according to the severity of asthma and ability to control symptoms. ICS, inhaled corticosteroid; LABA, long-acting b2 agonist; OCS, oral corticosteroid. (See CD-ROM for color version.)

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Controlled

Treatment action Reduce

Level of control

Uncontrolled Exacerbation

Maintain and find lowest controlling step Consider stepping up to gain control

Partly controlled Increase

not salmeterol, may be used as a reliever since it has a rapid onset of bronchodilatation and does not have cumulative prolonged side effects (Tattersfield et al. 2001; Pauwels et al. 2003). Formoterol appears to be more effective as a reliever than SABAs but is considerably more expensive so is not routinely used. However, use of a reliever medication more than three times a week indicates the need for regular controller therapy. The treatment of choice for all patients is ICS given twice daily. It is usual to start with an intermediate dose (e.g., 200 μg b.d. beclomethasone dipropionate or equivalent) and to decrease the dose if symptoms are controlled after 3 months. If symptoms are not controlled, a LABA should be added, which is most conveniently given by switching to a combination inhaler. The dose of controller should be adjusted accordingly, as judged by the need for a rescue inhaler. Low doses of theophylline or an antileukotriene may also be considered as an add-on therapy, but these are less effective than LABAs. In patients with severe asthma, low-dose oral theophylline is also helpful, and when there is irreversible airway narrowing the long-acting anticholinergic tiotropium bromide may be tried. However, there are no controlled trials demonstrating its efficacy in asthma. If patients are still not controlled despite good compliance and use of inhaler devices, maintenance treatment with an oral corticosteroid (prednisolone is preferred) may be needed and the lowest dose that maintains control should be used. As discussed above, corticosteroidsparing therapies are not effective and have a high risk of side effects. Occasionally, omalizumab may be tried (3–4 month trial) in steroid-dependent asthmatics who are not well controlled and who have frequent exacerbations.

Management of Chronic Asthma

Step up until controlled Treat as exacerbation

Fig. 80.2 Treatment of asthma based on level of control. (Adapted from Global Initiative for Asthma 2006, with permission.) (See CD-ROM for color version.)

at the time of need appears to suppress the increasing airway inflammation during the evolution of an exacerbation (Barnes 2007).

Asthma control Recently more emphasis has been placed on asthma control rather than arbitrarily assigning a disease severity (Global Initiative for Asthma 2006) (Table 80.2). This is because it is difficult to estimate disease severity, especially when patients are already taking regular therapy. Changing treatment based on the level of control is more logical and more useful in clinical practice. Asthma therapy is then stepped up until control is achieved and stepped down to find the minimal therapy that maintains control (Fig. 80.2). Exacerbations need to be treated via a different strategy.

Inhaler devices Combination inhalers Fixed combination inhalers containing a corticosteroid and a LABA are very effective in controlling chronic asthma. They are more convenient for patients than using separate inhalers and this has been shown to increase compliance with therapy (Stoloff et al. 2004). In most counties combination inhalers are less expensive than using the two inhalers together. The conventional approach is to use the combination inhaler (fluticasone/salmeterol or budesonide/formoterol) in a fixed dose twice daily and to increase or decrease the dose according to how well asthma is controlled (Bateman et al. 2004). The two currently available combination inhalers give a similar degree of control of asthma. However, budesonide/formoterol can also be used as a reliever instead of the conventional SABA. This approach using a single inhaler for maintenance and relief of asthma improves overall control and markedly reduces the number of severe exacerbations (O’Byrne et al. 2005). This is partly explained by the fact that formoterol is more effective as a reliever, but is mostly accounted for by the fact that patients take an increased dose of ICS when their symptoms increase and this plays a major role in the efficacy of this approach (Rabe et al. 2006). Increasing the dose of ICS

If asthma is not controlled despite the maximal recommended dose of inhaled therapy, it is important to check compliance and inhaler technique (Dolovich et al. 2005) . Some patients find it difficult to use pressurized metered-dose inhalers because of poor coordination between activation and inhalation and may find it easier to switch to a dry powder inhaler or to use a large-volume spacer chamber (Crompton et al. 2006). Nebulizers are not recommended in the routine management of chronic asthma and they lead to overuse of bronchodilator therapy and there is little advantage to giving corticosteroids by nebulizer. The cost of therapy is significantly greater with nebulized therapy.

Step-down Once asthma is controlled, it is important to slowly decrease therapy in order to find the minimal dose required to control symptoms (Hawkins et al. 2003). After adequate control has been achieved, the dose of ICS may be slowly reduced by about 25% every month to maintain stability. It is important not to make changes in dose too quickly as it may take some time after reducing the dose before asthma deteriorates (Juniper et al. 1991; Jatakanon et al. 2000). It is sometimes possible to

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withdraw treatment completely, but ICS may need to be restarted if symptoms recur, for example following an upper respiratory tract viral infection.

Education Patients with asthma need to understand how to use their mediations, and the difference between reliever and controller therapies. Education may improve compliance, particularly with ICS (FitzGerald & Gibson 2006). All patients should be taught how to use their inhalers correctly. In particular, they need to understand how to recognize worsening of asthma and how to step up therapy. Written action plans based on either symptoms or PEF recordings have been shown to reduce hospital admissions and morbidity in adults and children, and are recommended particularly in patients with unstable disease who have frequent exacerbations (Bhogal et al. 2006).

Refractory asthma Although most patients with asthma are easily controlled with appropriate medication, a small proportion of patients (approximately 5% of asthmatics) are difficult to control despite maximal inhaled therapy (Heaney & Robinson 2005; Wenzel 2005). Some of these patients will require maintenance treatment with oral corticosteroids. In managing these patients, it is important to investigate and correct any mechanisms that may be aggravating asthma. There are two major patterns of difficult asthma: some patients have persistent symptoms and poor lung function despite appropriate therapy, whereas others may have normal or near-normal lung function but intermittent severe (sometimes life-threatening) exacerbations. The commonest reason for poor control of asthma is noncompliance with medication, particularly ICS. Compliance with ICS may be low because patients do not feel any immediate clinical benefit or may be concerned about side effects. Compliance with ICS is difficult to monitor as there are no useful plasma measurements that can be made. Compliance may be improved by giving ICS in combination with a LABA that gives symptom relief. Compliance with oral corticosteroids may be measured by suppression of plasma cortisol and the expected concentration of prednisone/prednisolone in plasma. There are several factors that may make asthma more difficult to control, including exposure to high ambient levels of allergens or unidentified occupational agents. Severe rhinosinusitis may make asthma more difficult to control, so upper airway disease should be vigorously treated. Gastroesophageal reflux is common among asthmatics due to bronchodilator therapy, but there is little evidence that it is a significant factor in worsening asthma, and treatment of the reflux is not usually effective at improving asthma symptoms. Some

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patients may have chronic infection with Mycoplasma pneumoniae or Chlamydia pneumoniae and benefit from treatment with a macrolide antibiotic (Kraft et al. 2002). Drugs such as β-adrenergic blockers, aspirin, and other cyclooxygenase inhibitors may worsen asthma. Some women develop severe premenstrual worsening of asthma that is unresponsive to corticosteroids and requires treatment with progesterone or gonadotrophin-releasing factors. Few systemic diseases make asthma more difficult to control, but hyperthyroidism and hypothyroidism may increase asthma symptoms and should be investigated if suspected. Some patients who apparently have difficult-to-control asthma have vocal cord dysfunction, resulting in wheezing or stridor. This symptom is thought to be an attention-seeking hysterical conversion syndrome and may lead to escalating doses of asthma therapy with some patients taking high doses of oral corticosteroids. It may be recognized by the characteristic discrepancy between tests of forced expiration, such as forced expiratory volume in 1 s (FEV1) and PEF, and relatively normal airway resistance. Direct inspection by laryngoscopy may confirm adduction of the vocal cords at the time of symptoms. This condition is usually difficult to manage, but it is important that patients be weaned off oral and inhaled corticosteroids. Speech therapy is sometimes beneficial. Some patients with chronic obstructive pulmonary disease (COPD) may be diagnosed as asthmatic and may show the characteristic poor response to corticosteroids and bronchodilators, but this situation is complicated by the fact that some patients with COPD also have concomitant asthma.

Corticosteroid-resistant asthma A few patients with asthma show a poor response to corticosteroid therapy and may have various molecular abnormalities that impair the antiinflammatory action of corticosteroids. Complete resistance to corticosteroids is extremely uncommon and affects less than 1 in 1000 patients. It is defined by a failure to respond to a high dose of oral prednisone/prednisolone (40 mg o.d. over 2 weeks), ideally with a 2-week run-in with matched placebo. More common is reduced responsiveness to corticosteroids where control of asthma requires oral corticosteroids (corticosteroid-dependent asthma). In all patients with poor responsiveness to corticosteroids, there is a reduction in the response of circulating monocytes and lymphocytes to the antiinflammatory effects of corticosteroids in vitro, and reduced skin blanching in response to topical corticosteroids. There are several mechanisms that have been described, including an excess of the transcription factor AP-1, an increase in the alternatively spliced form of the glucocorticoid receptor GR-β, an abnormal pattern of histone acetylation in response to corticosteroids, a defect in interleukin (IL)-10 production, and a reduction in histone deacetylase activity (as in COPD) (Adcock & Lane 2003). These observations suggest that there are likely to be heterogeneous mechanisms for corticosteroid

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resistance, but whether these mechanisms are genetically determined has yet to be discovered.

Brittle asthma Some patients show chaotic variations in lung function despite taking appropriate therapy. Some show a persistent pattern of variability and may require oral corticosteroids or, at times, continuous infusion of β2 agonists (type 1 brittle asthma), whereas others have generally normal or near-normal lung function but precipitous unpredictable falls in lung function that may result in death (type 2 brittle asthma) (Ayres et al. 1998). These latter patients are difficult to manage as they do not respond well to corticosteroids, and the worsening of asthma does not reverse well with inhaled bronchodilators. The most effective therapy is subcutaneous epinephrine, which suggests that the worsening is likely to be a localized airway anaphylactic reaction with edema. In some of these patients, there may be allergy to specific foods. These patients should be taught to self-administer epinephrine and should carry a medical warning accordingly.

Management Refractory asthma is difficult to control, by definition. It is important to check compliance and the correct use of inhalers, and to identify and eliminate any underlying triggers. Low doses of theophylline may be helpful in some patients and theophylline withdrawal has been found to worsen many patients. Most of these patients will require maintenance treatment with oral corticosteroids, and the minimal dose that achieves satisfactory control should be determined by careful dose titration. Steroid-sparing therapies are rarely effective. In some patients with allergic asthma, omalizumab is effective, particularly when there are frequent exacerbations (Walker et al. 2006). There is some evidence that antitumor necrosis factor (TNF) therapy may be effective, but this is controversial and is very expensive (Berry et al. 2006). A few patients may benefit from infusions of β2 agonists (Ayres et al. 1998). New therapies are needed for these patients, who currently consume a disproportionate amount of healthcare spending.

Special considerations Although asthma is usually straightforward to manage, there are some situations that may require additional investigation and different therapy.

Aspirin-sensitive asthma A small proportion (approximately 1%) of asthmatics become worse with aspirin and other cyclooxygenase inhibitors, although this is much more commonly seen in severe patients and in those with frequent hospital admission (Szczeklik & Stevenson 2003). Aspirin-sensitive asthma is a well-defined

Management of Chronic Asthma

subtype of asthma that is usually preceded by perennial rhinitis and nasal polyps in nonatopic patients with late onset of the disease. Aspirin, even in small doses, characteristically provokes rhinorrhea, conjunctival irritation, facial flushing, and wheezing. There is a genetic predisposition to increased production of cysteinyl leukotrienes with functional polymorphism of cysteinyl leukotriene synthase. Asthma is triggered by cyclooxygenase inhibitors, but is persistent even in their absence. All nonselective cyclooxygenase inhibitors should be avoided, but selective cyclooxygenase type 2 inhibitors are apparently safe to use when an antiinflammatory analgesic is needed (Martin-Garcia et al. 2002). Aspirin-sensitive asthma responds to usual therapy with ICS. Although antileukotrienes should be effective in these patients, they are no more effective than in allergic asthma (Stevenson et al. 2000). Occasionally, aspirin desensitization is necessary, but this should only be undertaken in specialized centers.

Asthma in the elderly Asthma may start at any age, including in elderly patients. The principles of management are the same as in other asthmatics, but side effects of therapy may be a problem, including muscle tremor with β2 agonists and more systemic side effects with ICS (Barua & O’Mahony 2005). Comorbidities are more frequent in this age group, and interactions with drugs such as beta-blockers, cyclooxygenase inhibitors, and agents that may affect theophylline metabolism need to be considered. COPD is more likely in elderly patients and may coexist with asthma. A trial of oral corticosteroids may be very useful in documenting the steroid responsiveness of asthma.

Pregnancy Approximately one-third of asthmatic patients who are pregnant improve during the course of a pregnancy, one-third deteriorate, and one-third are unchanged. It is important to maintain good control of asthma as poor control may have adverse effects on fetal development. Compliance may be a problem as there is often concern about the effects of antiasthma medications on fetal development (Namazy & Schatz 2005). The drugs that have been used for many years in asthma therapy have now been shown to be safe and without teratogenic potential. These drugs include SABAs, ICS, and theophylline; there is less safety information about newer classes of drugs such as LABAs, antileukotrienes, and anti-IgE. If an oral corticosteroid is needed, it is better to use prednisone rather than prednisolone as it cannot be converted to the active prednisolone by the fetal liver, thus protecting the fetus from systemic effects of the corticosteroid. There is no contraindication to breast-feeding when patients are using these drugs.

Cigarette smoking Approximately 20% of asthmatics smoke, which may adversely affect asthma in several ways. Smoking asthmatics have

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more severe disease, more frequent hospital admissions, a faster decline in lung function, and a higher risk of death from asthma than nonsmoking asthmatics (Thomson et al. 2004). There is evidence that smoking interferes with the antiinflammatory actions of corticosteroids, necessitating higher doses for asthma control. Smoking cessation improves lung function and reverses the steroid resistance, and thus vigorous smoking cessation strategies should be used (Chaudhuri et al. 2006). Some patients report a temporary worsening of asthma when they first stop smoking, which could be due to the loss of the bronchodilating effect of nitric oxide in cigarette smoke.

Surgery If asthma is well controlled, there is no contraindication to general anesthesia and intubation. Patients who are treated with oral corticosteroids will have adrenal suppression and should be treated with an increased dose of oral corticosteroid immediately prior to surgery. Patients with FEV1 less than 80% of their normal levels should also be given a boost of oral corticosteroids prior to surgery (Tirumalasetty & Grammer 2006). High maintenance doses of corticosteroids may be a contraindication to surgery because of increased risks of infection and delayed wound healing. There should be no problems with general anesthesia providing asthma is well controlled.

Bronchopulmonary aspergillosis Bronchopulmonary aspergillosis is uncommon and results from an allergic pulmonary reaction to inhaled spores of Aspergillus fumigatus and occasionally other Aspergillus species. A skin-prick test to A. fumigatus is always positive, whereas serum Aspergillus precipitins are low or undetectable. Characteristically, there are fleeting eosinophilic infiltrates in the lungs, particularly in the upper lobes. Airways become blocked with mucoid plugs rich in eosinophils and patients may cough up brown plugs and may have hemoptysis. Bronchopulmonary aspergillosis may result in bronchiectasis, particularly affecting central airways, if not suppressed by corticosteroids. Asthma is controlled in the usual way by ICS, but it is necessary to give a course of oral corticosteroids if there is any sign of worsening or pulmonary shadowing is found (Gibson 2006). Treatment with the oral antifungal itraconazole is beneficial in preventing exacerbations and improving lung function and may be indicated in some patients (Wark et al. 2004).

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Ayres, J.G., Miles, J.F. & Barnes, P.J. (1998) Brittle asthma. Thorax 53, 315–21. Barnes, P.J. (2002) Scientific rationale for combination inhalers with a long-acting β2-agonists and corticosteroids, Eur Respir J 19, 182– 91. Barnes, P.J. (2003) Theophylline: new perspectives on an old drug. Am J Respir Crit Care Med 167, 813–18. Barnes, P.J. (2004) New drugs for asthma. Nat Rev Drug Discov 3, 831–44. Barnes, P.J. (2007) Scientific rationale for using a single inhaler for asthma control. Eur Respir Dis 29, 587–95. Barnes, P.J., Pedersen, S. & Busse, W.W. (1998) Efficacy and safety of inhaled corticosteroids: an update. Am J Respir Crit Care Med 157, S1–S53. Barua, P. & O’Mahony, M.S. (2005) Overcoming gaps in the management of asthma in older patients: new insights. Drugs Aging 22, 1029–59. Bateman, E.D., Boushey, H.A., Bousquet, J. et al. (2004) Can guidelinedefined asthma control be achieved? The Gaining Optimal Asthma Control Study. Am J Respir Crit Care Med 170, 836–44. Berry, M.A., Hargadon, B., Shelley, M. et al. (2006) Evidence of a role of tumor necrosis factor alpha in refractory asthma. N Engl J Med 354, 697–708. Bhogal, S., Zemek, R. & Ducharme, F.M. (2006) Written action plans for asthma in children. Cochrane Database Syst Rev 3, CD005306. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58 (suppl. 1), 1–90. Buist, A.S., Vollmer, W.M., Wilson, S.R., Frazier, E.A. & Hayward, A.D. (2006) A randomized clinical trial of peak flow versus symptom monitoring in older adults with asthma. Am J Respir Crit Care Med 174, 1077–87. Calamita, Z., Saconato, H., Pela, A.B. & Atallah, A.N. (2006) Efficacy of sublingual immunotherapy in asthma: systematic review of randomized-clinical trials using the Cochrane Collaboration method. Allergy 61, 1162–72. Chaudhuri, R., Livingston, E., McMahon, A.D. et al. (2006) Effects of smoking cessation on lung function and airway inflammation in smokers with asthma. Am J Respir Crit Care Med 174, 127–33. Crompton, G.K., Barnes, P.J., Broeders, M. et al. (2006) The need to improve inhalation technique in Europe: A report from the Aerosol Drug Management Improvement Team. Respir Med 100, 1479–94. Davies, H., Olson, L. & Gibson, P. (2000) Methotrexate as a steroid sparing agent for asthma in adults. Cochrane Database Syst Rev 2, CD000391. Dean, T., Dewey, A., Bara, A., Lasserson, T.J. & Walters, E.H. (2004) Azathioprine as an oral corticosteroid sparing agent for asthma. Cochrane Database Syst Rev CD003270. Dolovich, M.B., Ahrens, R.C., Hess, D.R. et al. (2005) Device selection and outcomes of aerosol therapy: evidence-based guidelines. American College of Chest Physicians/American College of Asthma, Allergy, and Immunology. Chest 127, 335–71. Ducharme, F.M., Lasserson, T.J. & Cates, C.J. (2006) Longacting beta2-agonists versus anti-leukotrienes as add-on therapy to inhaled corticosteroids for chronic asthma. Cochrane Database Syst Rev CD003137. Ernst, P., Habbick, B., Suissa, S. et al. (1993) Is the association between inhaled beta-agonist use and life-threatening asthma because of confounding by severity? Am Rev Respir Dis 148, 75–9.

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Evans, D.J., Taylor, D.A., Zetterstrom, O., Chung, K.F., O’Connor, B.J. & Barnes, P.J. (1997) A comparison of low-dose inhaled budesonide plus theophylline and high-dose inhaled budesonide for moderate asthma. N Engl J Med 337, 1412–18. Evans, D.J., Cullinan, P. & Geddes, D.M. (2001a) Cyclosporin as an oral corticosteroid sparing agent in stable asthma. Cochrane Database Syst Rev 2, CD002993. Evans, D.J., Cullinan, P. & Geddes, D.M. (2001b) Gold as an oral corticosteroid sparing agent in stable asthma. Cochrane Database Syst Rev CD002985. Fahy, J.V. (2006) Anti-IgE: lessons learned from effects on airway inflammation and asthma exacerbation. J Allergy Clin Immunol 117, 1230– 2. FitzGerald, J.M. & Gibson, P.G. (2006) Asthma exacerbations. 4: Prevention. Thorax 61, 992– 9. Gibson, P.G. (2006) Allergic bronchopulmonary aspergillosis. Semin Respir Crit Care Med 27, 185– 91. Gibson, P.G., Saltos, N. & Fakes, K. (2001) Acute anti-inflammatory effects of inhaled budesonide in asthma: a randomized controlled trial. Am J Respir Crit Care Med 163, 32– 6. Global Initiative for Asthma (2006) Global strategy for asthma management and prevention. NHLBI/WHO Workshop Report. Available at www ginasthma com. Greenstone, I.R., Ni Chroinin, M.N., Masse, V. et al. (2005) Combination of inhaled long-acting beta2-agonists and inhaled steroids versus higher dose of inhaled steroids in children and adults with persistent asthma. Cochrane Database Syst Rev CD005533. Gross, N.J. (2006) Anticholinergic agents in asthma and COPD. Eur J Pharmacol 533, 36– 9. Grove, A. & Lipworth, B.J. (1995) Tolerance with beta 2-adrenoceptor agonists: time for reappraisal. Br J Clin Pharmacol 39, 109–18. Guevara, J.P., Ducharme, F.M., Keren, R., Nihtianova, S. & Zorc, J. (2006) Inhaled corticosteroids versus sodium cromoglycate in children and adults with asthma. Cochrane Database Syst Rev CD003558. Hawkins, G., McMahon, A.D., Twaddle, S., Wood, S.F., Ford, I. & Thomson, N.C. (2003) Stepping down inhaled corticosteroids in asthma: randomised controlled trial. BMJ 326, 1115. Heaney, L.G. & Robinson, D.S. (2005) Severe asthma treatment: need for characterising patients. Lancet 365, 974–6. Jatakanon, A., Lim, S. & Barnes, P.J. (2000) Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med 161, 64–72. Juniper, E.F., Kline, P.A., Vanzieleghem, M.A., Ramsdale, E.H., O’Byrne, P.M. & Hargreave, F.E. (1990) Effect of long-term treatment with an inhaled corticosteroid (budesonide) on airway hyperresponsiveness and clinical asthma in nonsteroid-dependent asthmatics. Am Rev Respir Dis 142, 832– 6. Juniper, E.F., Kline, P.A., Vanzielegmem, M.A. & Hargreave, F.E. (1991) Reduction of budesonide after a year of increased use: a randomized controlled trial to evaluate whether improvements in airway responsiveness and clinical asthma are maintained. J Allergy Clin Immunol 87, 483– 9. Kankaanranta, H., Lahdensuo, A., Moilanen, E. & Barnes, P.J. (2004) Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review. Respir Res 5, 17. Kidney, J., Dominguez, M., Taylor, P.M., Rose, M., Chung, K.F. & Barnes, P.J. (1995) Immunomodulation by theophylline in asthma: demonstration by withdrawal of therapy. Am J Respir Cri Care Med 151, 1907–14.

Management of Chronic Asthma

Kraft, M., Cassell, G.H., Pak, J. & Martin, R.J. (2002) Mycoplasma pneumoniae and Chlamydia pneumoniae in asthma: effect of clarithromycin. Chest 121, 1782–8. Leff, A.R. (2001) Regulation of leukotrienes in the management of asthma: biology and clinical therapy. Annu Rev Med 52, 1–14. Martin-Garcia, C., Hinojosa, M., Berges, P. et al. (2002) Safety of a cyclooxygenase-2 inhibitor in patients with aspirin-sensitive asthma. Chest 121, 1812–17. Namazy, J.A. & Schatz, M. (2005) Treatment of asthma during pregnancy and perinatal outcomes. Curr Opin Allergy Clin Immunol 5, 229– 33. Nelson, H.S., Weiss, S.T., Bleecker, E.R., Yancey, S.W. & Dorinsky, P.M. (2006) The Salmeterol Multicenter Asthma Research Trial: a comparison of usual pharmacotherapy for asthma or usual pharmacotherapy plus salmeterol. Chest 129, 15–26. Norris, A.A. (1996) Pharmacology of sodium cromoglycate. Clin Exp Allergy 26 (suppl. 4), 5–7. O’Byrne, P.M. & Parameswaran, K. (2006) Pharmacological management of mild or moderate persistent asthma. Lancet 368, 794–803. O’Byrne, P.M., Barnes, P.J., Rodriguez-Roisin, R. et al. (2001) Low dose inhaled budesonide and formoterol in mild persistent asthma: the OPTIMA randomized trial. Am J Respir Crit Care Med 164, 1392– 7. O’Byrne, P.M., Bisgaard, H., Godard, P.P. et al. (2005) Budesonide/ formoterol combination therapy as both maintenance and reliever medication in asthma. Am J Respir Crit Care Med 171, 129–36. O’Byrne, P.M., Pedersen, S., Busse, W.W. et al. (2006) Effects of early intervention with inhaled budesonide on lung function in newly diagnosed asthma. Chest 129, 1478–85. O’Connor, B.J., Aikman, S.L. & Barnes, P.J. (1992) Tolerance to the non-bronchodilator effects of inhaled b2-agonists. N Engl J Med 327, 1204–8. O’Connor, G.T. (2005) Allergen avoidance in asthma: what do we do now? J Allergy Clin Immunol 116, 26–30. Pauwels, R.A., Lofdahl, C.-G., Postma, D.S. et al. (1997) Effect of inhaled formoterol and budesonide on exacerbations of asthma. N Engl J Med 337, 1412–18. Pauwels, R.A., Sears, M.R., Campbell, M. et al. (2003) Formoterol as relief medication in asthma: a worldwide safety and effectiveness trial. Eur Respir J 22, 787–94. Rabe, K.F., Atienza, T., Magyar, P., Larsson, P., Jorup, C. & Lalloo, U.G. (2006) Effect of budesonide in combination with formoterol for reliever therapy in asthma exacerbations: a randomised controlled, double-blind study. Lancet 368, 744–53. Salpeter, S.R., Buckley, N.S., Ormiston, T.M. & Salpeter, E.E. (2006) Meta-analysis: effect of long-acting beta-agonists on severe asthma exacerbations and asthma-related deaths. Ann Intern Med 144, 904–12. Slader, C.A., Reddel, H.K., Jenkins, C.R., Armour, C.L. & BosnicAnticevich, S.Z. (2006) Complementary and alternative medicine use in asthma: who is using what? Respirology 11, 373–87. Stevenson, D.D., Simon, R.A., Mathison, D.A. & Christiansen, S.C. (2000) Montelukast is only partially effective in inhibiting aspirin responses in aspirin-sensitive asthmatics. Ann Allergy Asthma Immunol 85, 477–82. Stoloff, S.W., Stempel, D.A., Meyer, J., Stanford, R.H. & Carranza, R. Jr (2004) Improved refill persistence with fluticasone propionate and salmeterol in a single inhaler compared with other controller therapies. J Allergy Clin Immunol 113, 245–51.

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Szczeklik, A. & Stevenson, D.D. (2003) Aspirin-induced asthma: advances in pathogenesis, diagnosis, and management. J Allergy Clin Immunol 111, 913– 21. Tattersfield, A.E., Lofdahl, C.G., Postma, D.S. et al. (2001) Comparison of formoterol and terbutaline for as-needed treatment of asthma: a randomised trial. Lancet 357, 257– 61. ten Brinke, A., Zwinderman, A.H., Sterk, P.J., Rabe, K.F. & Bel, E.H. (2004) “Refractory” eosinophilic airway inflammation in severe asthma: effect of parenteral corticosteroids. Am J Respir Crit Care Med 170, 601– 5. Thomson, N.C., Chaudhuri, R. & Livingston, E. (2004) Asthma and cigarette smoking. Eur Respir J 24, 822–33. Tirumalasetty, J. & Grammer, L.C. (2006) Asthma, surgery, and general anesthesia: a review. J Asthma 43, 251– 4. Walker, S., Monteil, M., Phelan, K., Lasserson, T.J. & Walters, E.H.

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(2006) Anti-IgE for chronic asthma in adults and children. Cochrane Database Syst Rev CD003559. Wark, P.A., Gibson, P.G. & Wilson, A.J. (2004) Azoles for allergic bronchopulmonary aspergillosis associated with asthma. Cochrane Database Syst Rev CD001108. Wechsler, M.E. & Israel, E. (2002) Pharmacogenetics of treatment with leukotriene modifiers. Curr Opin Allergy Clin Immunol 2, 395–401. Wenzel, S. (2005) Severe asthma in adults. Am J Respir Crit Care Med 172, 149–60. Wilson, A.J., Gibson, P.G. & Coughlan, J. (2000) Long acting betaagonists versus theophylline for maintenance treatment of asthma. Cochrane Database Syst Rev CD001281. Wouden, J.C., Tasche, M.J., Bernsen, R.M., Uijen, J.H., Jongste, J.C. & Ducharme, F.M. (2003) Inhaled sodium cromoglycate for asthma in children. Cochrane Database Syst Rev CD002173.

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Anti-IgE in Persistent Severe Allergic Asthma Marc Humbert, Stephen T. Holgate, Howard Fox and Jean Bousquet

Summary Asthma causes substantial mortality and morbidity and has a considerable economic impact. The burden of asthma is greatest in patients with severe asthma, which is often inadequately controlled despite guideline-based therapy. IgE plays a central role in the allergic inflammatory cascade of asthma of all severities. Allergen binds to and cross-links IgE bound to high-affinity IgE receptors (FcεRI) on the surface of basophils and mast cells, triggering the release of preformed proinflammatory mediators and newly synthesized cytokines and chemokines that underlie the allergic asthmatic inflammatory cascade. Omalizumab, an anti-IgE antibody, binds all forms of circulating IgE and prevents subsequent IgE-mediated events. By reducing free IgE, omalizumab also downregulates FcεRI on basophils, mast cells and dendritic cells. In patients with allergic asthma, omalizumab inhibits both the early- and late-phase asthmatic response and significantly reduces the number of circulating, sputum and bronchial submucosal eosinophils. The efficacy of omalizumab has been demonstrated in seven clinical studies in patients with predominantly severe persistent allergic asthma, including the INNOVATE study, which enrolled patients with inadequately controlled severe persistent allergic asthma despite high-dose inhaled corticosteroids plus longacting β2 agonist (LABA) (with additional controller medication if required). In the INNOVATE study, add-on omalizumab significantly reduced clinically significant exacerbation rate (26%), severe exacerbation rate (50%), and total emergency visit rate (43%), and significantly improved asthma symptoms, lung function, and quality of life compared with add-on placebo. These findings were supported by pooled analyses, which also demonstrated efficacy irrespective of oral corticosteroid or leukotriene modifier use. Analysis of data from completed Phase I, II and III studies involving more than 7500 patients with asthma, rhinitis or related conditions has demonstrated a clinical safety and tolerability profile of omalizumab that was similar to placebo, with no evidence of an increased risk of hypersensitivity reactions, malignant neoplasia or parasitic infection.

In the European Union (EU), omalizumab is indicated as addon therapy to improve asthma control in patients (≥ 12 years) with severe persistent allergic asthma despite daily high-dose inhaled corticosteroids plus an LABA. As analyses have found that it is difficult to predict which patients in the label population will receive greatest benefit based on pretreatment characteristics, all eligible patients should receive an initial 16-week course of omalizumab and the decision to continue therapy should be based on whether a marked improvement in overall asthma control has been achieved, as specified in the EU label. In this patient population, omalizumab has been shown to be cost-effective.

Introduction Asthma affects an estimated 300 million people worldwide and this number is predicted to rise to 400 million by 2025 (Masoli et al. 2004). Asthma is a cause of substantial mortality (World Health Organization 2002) and morbidity (Masoli et al. 2004), and significantly impairs physical, emotional and social aspects of daily living (Juniper 1998; Hooi 2003) and increases psychological distress (Adams et al. 2004). In addition, asthma has a considerable economic impact (Beasley 2002; Redd 2002; ERS/ELF 2003). The Asthma Insights and Realities (AIR) surveys estimated that approximately 20% of patients with asthma have severe asthma (Rabe et al. 2000, 2004). However, estimates of the prevalence of severe asthma differ depending on the definition. It seems likely that approximately 10–20% of patients with asthma have severe disease. The burden of asthma is greatest in patients with severe asthma, which is often inadequately controlled despite guideline-based therapy (Bateman et al. 2004; Partridge et al. 2006). The purpose of this chapter is to review the clinical profile of omalizumab, an anti-IgE antibody, in the treatment of severe persistent allergic asthma.

Burden of severe asthma Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Asthma mortality and hospitalization Patients with severe asthma have a higher risk of asthmarelated hospitalization or death than patients with less severe

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asthma, with an estimated 80– 85% of asthma deaths occurring in patients with severe and poorly controlled disease (Papiris et al. 2002). The relationship between increasing asthma severity and asthma mortality is well established (Rea et al. 1986; Crane et al. 1992; Tough et al. 1998; Guite et al. 1999; Hartert et al. 2002). Factors indicative of severe asthma (e.g., hospital admission) significantly increase the risk of asthma mortality (Rea et al. 1986; Crane et al. 1992). In a case controlled study in the UK, asthma mortality was increased sixfold in patients with a history of clinically severe asthma (Guite et al. 1999) and previous hospital admission was found to increase the risk of asthma mortality 10-fold in a Canadian study (Tough et al. 1998). In a retrospective survey of adults with asthma (65 years and over), patients with the most severe disease had the highest incidence of recurrent hospital admission and death within 1 year of their initial visit (Hartert et al. 2002). Healthcare utilization also increases with asthma severity. In a 3-year, multicenter, observational study of almost 5000 patients with severe or difficult-to-treat asthma in the USA (TENOR study), patients with severe asthma had considerably higher levels of healthcare utilization than patients with mild or moderate asthma (Dolan et al. 2004). For example, emergency department visits were needed by 6% of patients with mild asthma, 11% of patients with moderate asthma, and 21% of patients with severe asthma. Similarly, 10% of patients with severe asthma were hospitalized, compared with 3% of those with moderate asthma and 1% of those with mild asthma. The TENOR study also demonstrated that a prior hospitalization or exacerbation was a significant risk factor for a future hospitalization or exacerbation (Miller et al. 2007). Similar findings were reported in a Brazilian study, which found that emergency department visits for asthma were more frequent in patients who had a history of hospital admissions (Dalcin et al. 2004).

Impact of severe asthma on quality of life and daily living The impact of asthma on patients’ quality of life is most marked in patients with severe asthma (Andersson et al. 2003; Schmier et al. 2003; Juniper et al. 2004). A large cross-sectional observational study has been conducted to assess the effect of asthma on the quality of life of patients with severe persistent asthma (Turk et al. 2005a,b). In total, 965 patients from France, Germany, Italy, Spain and the UK were recruited by primary care physicians and respiratory specialists and their severity classified according to the GINA guidelines. The impact of asthma on quality of life was assessed using the Mini-AQLQ (Juniper et al. 1999). Quality of life significantly deteriorated as asthma severity increased from moderate to severe asthma and from severe controlled to severe inadequately controlled asthma, both in terms of Mini-AQLQ overall score and individual domain scores. Even though there was a large decrease in overall Mini-AQLQ score (0.86-point), as severity increased from moderate to severe, a further decrease (0.46-point) in

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overall score was seen as severity increased from severe controlled to severe inadequately controlled disease. The impact of uncontrolled asthma on the lives of patients was illustrated in the European Fighting for Breath survey of 1300 people with current treated asthma at the severe end of the spectrum (Dockrell et al. 2007). Two-thirds of patients reported that their sleep was disturbed at least once a week over the year prior to the survey. For one in four patients, the severity of their night-time symptoms was more problematic, with their sleep being disturbed more often than one night a week. Two-thirds of patients experienced wheezing attacks at least once a week. Speech-limiting attacks occurred at least once a week for one in four patients. Asthma had a major impact on activities and most patients reported having limitations to their lifestyles as a consequence of the symptoms of severe asthma. Almost 70% of patients reported that physical activity was restricted, 50% were restricted from having pets, 30% from taking holidays, and 30% felt that job opportunities or promotion were limited by their asthma.

Economic impact of severe asthma Studies conducted in France (Godard et al. 2002), Spain (SerraBatlles et al. 1998), Germany (Graf von der Schulenburg et al. 1996), and Italy (Antonicelli et al. 2004) have consistently shown that asthma-related costs increase with asthma severity. In the Italian study, total costs of asthma were greatest in patients with GINA-defined severe persistent asthma, accounting for > 50% of total asthma costs (Antonicelli et al. 2004). It is also notable that direct costs accounted for 48% of total costs, whereas indirect costs (e.g., work days lost) accounted for the remaining 52% of total costs. The relationship between costs and GINA-defined asthma severity was also assessed using data from Italy, France and Spain (Van Ganse et al. 2006). This study also found that patients with severe persistent asthma accounted for the majority of total asthma-related costs (approximately 60%). The costs of severe asthma are greatest in patients with inadequately controlled asthma (Hoskins et al. 2001; McCowan et al. 2002; Van Ganse et al. 2002; Schwenkglenks et al. 2003; Turk et al. 2005b). In a cross-sectional observational study, patients with inadequately controlled severe persistent asthma had significantly more asthma exacerbations requiring a primary care visit or emergency hospital treatment and spent significantly more time in hospital than patients with severe controlled asthma (Turk et al. 2005b).

Treatment options for severe persistent asthma Historically, asthma was classified by severity based on the level of symptoms, airflow limitation and lung function variability into four categories: intermittent, mild persistent, moderate persistent, or severe persistent. Classification by severity is

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Table 81.1 Classification of asthma severity by clinical features before treatment (GINA 2006). Intermittent Symptoms less than once a week Brief exacerbations Nocturnal symptoms not more than twice a month FEV1 or PEF ≥ 80% predicted PEF or FEV1 variability < 20% Mild persistent Symptoms more than once a week but less than once a day Exacerbations may affect activity and sleep Nocturnal symptoms more than twice a month FEV1 or PEF ≥ 80% predicted PEF or FEV1 variability < 20–30% Moderate persistent Symptoms daily Exacerbations may affect activity and sleep Nocturnal symptoms more than once a week Daily use of inhaled short-acting b2 agonist FEV1 or PEF 60–80% predicted PEF or FEV1 variability > 30% Severe persistent Symptoms daily Frequent exacerbations Frequent nocturnal asthma symptoms Limitation of physical activities FEV1 or PEF ≤ 60% predicted PEF or FEV1 variability > 30%

useful when decisions are being made about assessment and management of a patient, but it is important to recognize that asthma severity involves both the severity of the underlying disease and its responsiveness to treatment (Martinez et al. 1995). Asthma can present with severe symptoms and be classified as severe persistent, but respond fully to treatment

Anti-IgE in Persistent Severe Allergic Asthma

and then be classified as moderate persistent. In addition, severity is not an unvarying feature of an individual patient’s asthma, but may change over months or years. Classification by severity is no longer recommended as the basis for ongoing treatment decisions, but retains its value as a means of characterizing a group of patients with asthma who are not on inhaled corticosteroid (ICS) treatment e.g., in selecting patients for inclusion in an asthma study (GINA 2006) (Table 81.1). A periodic assessment of asthma control is more relevant and useful in predicting what treatment will be required and what a patient’s response to that treatment might be. Asthma control refers to control of the manifestations of disease, ideally not only clinical manifestations but also laboratory markers of inflammation and pathophysiologic features. Because of the cost and/or general unavailability of laboratory tests, it is recommended that treatment be aimed at controlling the clinical features of disease, including lung function abnormalities. Table 81.2 provides the characteristics of controlled, partly controlled, and uncontrolled asthma. This is a working scheme based on current opinion and has not been validated (GINA 2006). Complete control of asthma is commonly achieved with treatment the aim of which should be to maintain control for prolonged periods (Gotzsche et al. 2004) with due regard to the safety of treatment, potential for adverse effects, and costs. The patient’s current level of asthma control and current treatment determine the selection of pharmacologic treatment, with treatment being stepped up until control is achieved. If control has been maintained for at least 3 months, treatment can be stepped down with the aim of establishing the lowest step and dose of treatment that maintains control. If asthma is partly controlled, an increase in treatment should be considered (Fig. 81.1). For patients with occasional daytime symptoms of short duration, as-needed reliever medication such as a rapid-acting β2 agonist is recommended (GINA step 1). Suggested alternatives are inhaled anticholinergic, shortacting oral β2 agonist or short-acting theophylline, although

Table 81.2 Levels of asthma control (GINA 2006).

Characteristic

Controlled (all of the following)

Partly controlled (any measure present in any week)

Daytime symptoms

None (twice or less/week)

More than twice/week

Limitation of activities Nocturnal symptoms/awakening Need for reliever Lung function (PEF/FEV1)* Exacerbations

None None Twice or less/week Normal None

Any Any More than twice/week < 80% predicted or personal best One or more/year†

Uncontrolled Three or more features of partly controlled asthma in any week

One in any week‡

* Lung function is not a reliable test for children 5 years and younger. † Any exacerbation should promote review of maintenance treatment to ensure that it is adequate. ‡ By definition, an exacerbation in any week makes that an uncontrolled asthma week.

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Reduce

Management approach based on control For children older than 5 years, adolescents and adults Level of control Controlled

Treatment action Maintain and find lowest controlling step Consider stepping up to gain control

Increase

Partly controlled Uncontrolled Exacerbation

Reduce Step 1

Step up until controlled Treat as exacerbation

Treatment steps Step 2

Step 3

Increase Step 4

Step 5

Asthma education Environmental control As needed rapidacting b2-agonist

As needed rapid-acting b2-agonist Select one Low-dose inhaled ICS*

Controller options

Leukotriene modifier 0

Select one

Add one or more

Add one or both

Oral Low-dose ICS plus Medium- or high-dose ICS plus long-acting glucocorticosteroid long-acting b2-agonist (lowest dose) b2-agonist Medium- or high-dose ICS

Leukotriene modifier

Low-dose ICS plus leukotriene modifier

Sustained release theophylline

Anti-IgE treatment

Low-dose ICS plus sustained release theophylline * ICS = inhaled glucocorticosteroids 0 = Receptor antagonist or synthesis inhibitors Alternative reliever treatments include inhaled anticholinergics, short-acting oral b2-agonists, some long-acting b2-agonists, and short-acting theophylline. Regular dosing with short- and long-acting b2-agonists is not advised unless accompanted by regular use of an inhaled glucocorticosteroid.

they have a slower onset of action and a higher risk of side effects. Steps 2–5 combine as-needed reliever with regular control treatment. GINA step 2 recommends low-dose ICS as controller treatment or, as an alternative for those who experience intolerable side effects from ICS, leukotriene modifiers. Other options (sustained-release theophylline and cromones) are not recommended as initial or first-line controllers. GINA step 3 control treatment combines low-dose ICS with one additional controller, either inhaled long-acting β2 agonist (LABA), leukotriene modifier, or sustained-release theophylline. Alternatively, increasing to medium- or highdose ICS is given as an option. GINA step 4 treatment recommends medium- or high-dose ICS plus LABA, with add-on leukotriene modifier or sustained-release theophylline. GINA step 5 treatment recommends the addition of oral corticosteroids or anti-IgE therapy, or both, to existing step 4 therapy. Addition of oral corticosteroids to other controller medications may be effective (Mash et al. 2001) but is associated with severe side effects (Ayres et al. 2004a) and should only be considered if the patient’s asthma remains severely uncon-

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Fig. 81.1 GINA 2006 guidelines for asthma treatment (GINA 2006).

trolled on step 4 medications. Addition of anti-IgE treatment to other controller medications has been shown to improve control of allergic asthma when control has not been achieved on combinations of other controllers including high-dose ICS or oral corticosteroids (Milgrom et al. 1999; Busse et al. 2001; Bousquet et al. 2004a; Djukanovif et al. 2004; Holgate et al. 2004; Humbert et al. 2005). While the majority of asthma patients can obtain the targeted level of control, some patients will not do so even with the best available therapy (Bateman et al. 2004). Patients who do not reach an acceptable level of control at step 4 can be considered to have difficult-to-treat asthma (Wenzel 2005). When reasons for lack of treatment response (incorrect diagnosis, noncompliance, smoking, comorbidities, and psychological and psychiatric issues) have been considered and addressed, a compromise level of control may need to be accepted and discussed with the patient to avoid futile overtreatment (with its attendant cost and potential for adverse effects). The objective is then to minimize exacerbations and need for emergency medical interventions while achieving as high a

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level of clinical control with as little disruption of activities and as few daily symptoms as possible (GINA 2006). Although treatment with ICS is a key component of asthma management, there is a recognized plateau effect with ICS, where increasing doses are not matched by further improvements in therapeutic outcomes (Barnes et al. 1998; Holt et al. 2001). Adding a LABA to ICS improves treatment outcomes, but asthma control is often not achieved with ICS/LABA therapy in patients with severe disease. For example, the Gaining Optimal Asthma Control (GOAL) study investigated whether treatment with fluticasone propionate or fluticasone/ salmeterol combination therapy could achieve guideline-based asthma control in patients with uncontrolled asthma (Bateman et al. 2004). In patients with the most severe asthma, asthma remained inadequately controlled in 38% of patients despite optimized treatment with fluticasone/salmeterol. When a course of oral steroids was added to fluticasone/salmeterol at the end of this 1-year study, asthma remained inadequately controlled in 31% of patients. Similar findings were reported in the International Asthma Patient Insight Research (INSPIRE) survey of 3145 patients (≥ 16 years) with asthma (Partridge et al. 2006). In the INSPIRE study, despite all patients receiving regular maintenance therapy with ICS (30% of patients) or ICS plus LABA (70% of patients), 74% of all patients used short-acting β2 agonists daily and only 28% of all patients were classified by the Asthma Control Questionnaire (ACQ) as having well-controlled asthma. In addition, of those patients whose asthma was not well controlled according to the ACQ, 87% classed their asthma control as relatively good and 55% of patients classified as having uncontrolled asthma rated their asthma control as relatively good. For patients who fail to achieve control despite ICS plus LABA, the GINA guidelines recommend add-on therapy with leukotriene modifiers, sustained-release theophylline, oral β2 agonists, oral corticosteroids, or anti-IgE. Zafirlukast, a leukotriene modifier, has been shown to improve asthma control in patients receiving high-dose ICS (Virchow et al. 2000). Leukotriene modifiers used as add-on therapy may reduce the dose of ICS required by patients with moderate to severe asthma (Ownby et al. 2002), and may improve asthma control in patients whose asthma is not controlled with low or high doses of ICS (Martinez et al. 1995; Strachan & Cook 1998; Dezateux et al. 1999; Isolauri et al. 2001). Leukotriene modifiers may be particularly beneficial in patients with severe asthma who are aspirin-sensitive (Gibbs et al. 2002; ENFUMOSA 2003; Dolan et al. 2004; Kupczyk et al. 2004). Theophylline is also a possible add-on therapy for use in patients with uncontrolled asthma, but has limited evidence of efficacy and significant side effects such as gastrointestinal and cardiovascular effects (GINA 2006). The use of short-acting oral β2 agonists is associated with a higher prevalence of adverse effects such as tremor and tachycardia than inhaled formulations (GINA 2006). Regular treatment with systemic corticosteroids is associated with significant side effects, including

Anti-IgE in Persistent Severe Allergic Asthma

weight gain, glaucoma, cataracts, cardiovascular disease, osteoporosis, and growth retardation in the young (Wei et al. 2004; Angeli et al. 2006; GINA 2006). There is also increasing evidence that short-term rescue use of systemic cortiocosteroids (steroid bursts) can increase mood disturbances (Brown et al. 2002), the risk of atrial fibrillation (Huerta et al. 2005), and the risk of osteoporosis (McKee et al. 2001). Anti-IgE (omalizumab), the most recent addition to the guidelines, is a treatment option limited to patients with elevated serum levels of IgE. Its current EU indication is for patients with severe allergic asthma (Humbert et al. 2005) who are uncontrolled on ICS plus LABA, although the dose of concurrent treatment has varied in different studies. Several studies have indicated that anti-IgE appears to be safe as add-on therapy (Holgate et al. 2004; Corren et al. 2005; Humbert et al. 2005). Omalizumab is discussed in greater detail in subsequent sections, with a focus on patients with severe persistent allergic asthma. Experimental agents such as methotrexate (Davies et al. 2000), azathioprine (Dean et al. 2004), chloroquine (Dean et al. 2003), cyclosporin A (Evans et al. 2001a), trolandeomycin (Evans et al. 2001b), and gold compounds (Evans et al. 2001c) have failed to show an acceptable risk–benefit ratio. Early studies of anti-tumor necrosis factor (TNF)-α treatment (etanercept) have reported promising effects on asthma symptoms, lung function, and bronchial hyperresponsiveness in patients with severe asthma (Howarth et al. 2005; Berry et al. 2006; Erin et al. 2006). Large clinical trials are in progress to confirm these preliminary efficacy findings. The safety profile of anti-TNF-α treatment will also require careful scrutiny.

Omalizumab in the treatment of severe persistent allergic asthma Discovery of IgE Prausnitz and Küstner first performed passive transfer of a positive allergy skin test (the PK test) in 1921, after a report of passive transfer of allergy to horse dander via blood transfusion. Over the next 40 years however, little progress was made in isolating and characterizing the responsible “reagin.” In 1964, Ishizaka and colleagues reported that this reagin was an antibody belonging to the gA immunoglobulin class (Ishizaka et al. 1964). Further work led them to claim in 1967 to have an antiserum that could precipitate the reagin, resulting in a decrease in reaginic activity, and could also bind to a mixture of reaginic serum and radiolabeled allergen (Ishizaka & Ishizaka 1967). Independently of this work, in 1965 Bennich and colleagues detected an atypical myeloma protein with similar physicochemical characteristics to the reagin. Studies using radioimmunoassay in patients with allergic asthma found that they had a high concentration of immunoglobulin ND (IgND). It was also shown that the PK reaction in humans could be

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inhibited with isolated IgND or the Fc fragments of the ND protein (Stanworth et al. 1967). In 1967, after exchange of reagents between the two laboratories, it was found that Ishizaka’s antiserum reacted with isolated ND protein and that purified ND protein could block the reaction of the antiserum. These results, as well as immunologic reagent, including purified ND protein, were submitted to the World Health Organization Immunoglobulin Reference Centre (Bennich et al. 1968). In 1968 the WHO International Immunoglobulin Reference Centre officially declared the discovery of IgE based on the work of Bennich, Ishizaka and Johansson. The importance of IgE in allergy is such that the definition of allergic disease is based on the presence of allergen-specific IgE. Research in immunology increased enormously, leading to the discovery and characterization of leukotrienes, cytokines

Rationale for targeting IgE in patients with allergic asthma IgE is an antibody, and like other antibodies comprises two identical light chains and two identical heavy chains, which are covalently linked by disulphide bonds (Fig. 81.2a,b). The IgE molecule has an antigen-binding fragment (Fab) and a crystallizable fragment (Fc). Antigen binds to the Fab region of IgE via infinitely variable amino acid sequences known as the complementarity-determining region (CDR), which binds specific epitopes of the antigen. The Cε3 domain in the Fc region of IgE binds to high-affinity (FcεRI) cell-surface receptors, mainly expressed on mast cells and basophils (Blank et al. 1989;

Fab unit

Constant region (C) domains determine secondary biological function (e.g., cell surface binding)

Variable domain (V) binds antigen Variable regions

Antigenbinding site

Antigenbinding site

Carbohydrate chain

Constant regions

CL

VL

s s

VH Receptor binding site

Fab unit

(a)

Peptide loops containing hypervariable regions or complementarity determining regions (CDR)

Fc unit

CL s s Disulfide bond CL Ce2 Ce3 Ce4

s

Heavy chain

Heavy chain IgE domains

s

VH VL

CL

Antigen-binding fragments (Fab)

Antigenbinding site

Light chain

Light chain

Binds to FceRI and FceRII cell surface receptors

Fc fragment (c = crystallizable)

(b) Allergen-binding site VH

VL

IgE

CL

Ce1

Ce2 Ce3 FceRI

a2

Ce4 a1 Out Cell membrane In

(c)

a

b

g

g

Fig. 81.2 (a) Molecular structure and (b) schematic diagram of IgE. (c) Binding of IgE to the FceRI receptor. (From Holgate 1998, with permission from Oxford University Press.) (See CD-ROM for color version.)

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Gounni et al. 1994), and a low-affinity cell-surface receptor (FcεRII) mainly expressed on B cells (Kehry & Yamashita 1989; Conrad 1990). The best-characterized IgE-mediated response is the interaction between IgE and the FcεRI receptor on mast cells (Holgate 1998) (Fig. 81.2c). FcεRI receptors have also been detected on eosinophils (Rajakulasingam et al. 1998), neutrophils (Gounni et al. 2001), platelets (Joseph et al. 1997), epithelial cells (Campbell et al. 1998), and airway smooth muscle cells (Gounni 2006), although the clinical significance remains to be established. FcεRI consists of four polypeptide chains, γβα2. The α-chain binds to a chain of five amino acids (330– 335) of the Cε3 domain of the Fc segment of IgE (Fig. 81.2). FcεRI is expressed in trimeric form (γα2) on antigen-presenting cells, such as monocytes and dendritic cells. Allergic asthma is characterized by the presence of IgE antibodies against common allergens such as house-dust mite, animal dander, and molds (Ehnert et al. 1992; Custovic et al. 1996; Woodcock & Custovic 1998). It has been known for many decades that the risk of developing asthma increases with increasing levels of serum IgE (Burrows et al. 1989). However, asthma severity is poorly associated with total IgE levels (Bousquet et al. 2007). The association between specific IgE and asthma severity is currently under investigation. It is estimated that two-thirds of patients with asthma have allergic asthma (Novak & Bieber 2003). While it has long been thought that patients with severe asthma are less likely to be allergic, approximately 60% of patients with severe asthma tested skin-prick test positive for one or more common aeroallergens in the European Network For Understanding Mechanisms of Severe Asthma study (ENFUMOSA 2003). Although there are no visible asthma effects, large amounts of the corresponding IgE antibody are produced on first exposure to an allergen (sensitization). This allergen-induced response begins with the uptake and processing of allergen by antigen-presenting cells. Development of naive T cells is then directed towards the T helper (Th)2-type, which produce interleukin (IL)-4, IL-5 and IL-13 (Mosmann et al. 1986; Novak & Bieber 2003; Prussin & Metcalfe 2003). The cytokines secreted by T cells stimulate production of allergenspecific IgE antibodies by B cells. Differentiation of B cells into IgE-producing plasma cells requires two signals: (i) IL-4 and IL-13 (Lebman & Coffman 1988; Jabara et al. 1990a; Jelinek 2000); and (ii) an interaction of the costimulatory antigen CD40 on the surface of B cells with CD40 ligand on T-cell surfaces (Vercelli et al. 1989; Jabara et al. 1990b). Subsequent allergen exposure then causes the release of IgE, which binds to FcεRI on mast cells triggering degranulation and release of mediators such as histamine, prostaglandin (PG)D2, leukotriene (LT)C4, and tumor necrosis factor (TNF)-α (Novak & Bieber 2003; Prussin & Metcalfe 2003). Subsequent release of inflammatory mediators such as IL-4, IL-5 and IL-13 promotes adhesion and infiltration of circulating inflammatory cells (primarily eosinophils, basophils, and Th2 cells) into the

Anti-IgE in Persistent Severe Allergic Asthma

tissues and results in the characteristic symptoms of asthma (Novak & Bieber 2003; Prussin & Metcalfe 2003). The role of the low-avidity IgE receptor (FcεRII) is less clearly established; it appears to be implicated in the synthesis of IgE and in IgE-mediated immune and inflammatory functions (Gosset et al. 1999; Oettgen & Geha 1999; Tsicopoulos & Joseph 2000).

Development of anti-IgE (omalizumab) An overview of the early development of anti-IgE follows. For a more comprehensive review, please refer to Heusser and Jardieu (1997). Various therapeutic strategies targeting the production and action of IgE have been considered, including IL-4 antagonists and IL-4 antibodies to inhibit its production (Paul 1997), STAT-6 protein inhibition to stop B-cell switching (Shimoda et al. 1996), anti-FcεRII antibodies, allergen modification to shift the immune response to IgG production, IgEderived peptides and oglionucleotides to prevent IgE binding (Hamburger 1979; Burt & Stanworth 1987; Helm et al. 1989; Wiegand et al. 1996), and a soluble α-subunit of FcεRI to block IgE binding to mast cells (Haak-Frendscho et al. 1993). However, the most promising approach has been the development of antibodies directed against the region of IgE involved in the interaction with its receptors, thereby blocking the binding of IgE to the FcεRI receptor without cross-linking IgE and triggering mast cell degranulation. Early studies demonstrated the inhibition of IgE binding to the FcεRI receptor using anti-IgE antibodies in mice (Baniyash & Eshhar 1984), but with cross-linking. Further anti-IgE antibodies were developed that did not induce degranulation in mice or in human basophils (Baniyash et al. 1988; Hakimi et al. 1990; Heusser et al. 1991; Presta et al. 1994) indicating potential therapeutic application. Reduction of serum IgE levels by polyclonal antibodies was reported (Bozelka et al. 1982, 1985), with experiments showing that B cells and secondary IgE responses could be inhibited, but the memory IgE response was not (Haba & Nisonoff 1994). Chimeric and humanized antibodies were developed to overcome the problem of antigenicity in humans (Morrison et al. 1984; Jones et al. 1986). CGP 51901 (Davis et al. 1993), CGP 56901 (Kolbinger et al. 1993), and rhuMAb-E25 (omalizumab) (Presta et al. 1993) had comparable activity to their parent murine antibodies, but less than 5% of their CDRs were mouse derived. In vitro studies showed inhibition of IgE binding to basophils and mast cells without histamine release (Davis et al. 1993; Shields et al. 1995), and studies in human tissue (Fox et al. 1996) and cynomolgus monkeys (Meng et al. 1996) further supported the safety of rhuMAb-E25 and CGP 56901/CGP 51901 in vivo. RhuMAb-E25 reduced skin reactivity to ragweed in five of six ragweed-sensitized cynomolgus monkeys after a single administration and in all six monkeys after a second administration, despite continued allergen injections, demonstrating that reduced serum IgE levels would in turn reduce cutaneous allergic reactions.

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Following in vitro and animal studies, it was hypothesized that the administration of anti-IgE to humans would be safe and effective in reducing levels of circulating IgE. Phase I studies showed that these drugs reduced serum-free IgE in a dose-dependent manner, and were well tolerated. A doubleblind multidose study in 33 pollen-sensitive subjects with raised serum IgE who received either CGP 51901 or placebo showed that a single infusion of CGP 51901 caused an immediate fall in serum free IgE at all doses (Corne et al. 1997). Administration of 100 mg CGP 51901 suppressed serum free IgE by more than 96%, with a mean recovery time to 50% of baseline levels after 39 days. Total IgE levels (comprising free and complexed IgE) increased as both stored and newly synthesized IgE bound to CGP 51901. IgE–CGP 51901 complexes were slowly cleared with a half-life of 10–14 days. Only one subject showed a weak antibody response to CGP 51901. Clinical studies have been carried out to determine the effect of anti-IgE on rhinitis symptoms in adults with seasonal allergic rhinitis to ragweed (Zweiman 1993; Casale et al. 1997), the hypothesis being that reduction in serum IgE would reduce these symptoms. Results showed a significant reduction in overall nasal symptom scores, reduced rescue medication usage, and enhanced patient feelings of well-being compared with placebo, demonstrating the efficacy of anti-IgE in allergic rhinitis. Examining the effect of anti-IgE on allergic airways response, rhuMAb-E25 has been found to inhibit the early and latephase response to inhaled allergen (Fahy et al. 1997). The mean maximal fall in forced expiratory volume in 1 s (FEV1) was significantly reduced from baseline in the rhuMAb-E25treated group (N = 9) compared with placebo (N = 9) during the early response (30 ± 10% at baseline to 18.8 ± 8% vs. 33 ± 8% at baseline to 34 ± 4%; P = 0.01) and late response (24 ± 20% at baseline to 9 ± 10% vs. 20 ± 17% at baseline to 18 ± 17%; P = 0.047) to allergen challenge. In a similar study in 19 patients with allergic asthma (Boulet et al. 1997), rhuMAb-E25 was found to inhibit the early asthmatic response to inhaled allergen. Measured on days 27, 55 and 77, the median allergen PC15 (the concentration of allergen causing a 15% reduction in FEV1) significantly increased by 2.3, 2.2 and 2.7 doubling doses from baseline in the anti-IgE group compared with −0.3, +0.1 and −0.8 in the placebo group (P ≤ 0.002). Methacholine PC20 improved slightly after rhuMAb-E25, but only became statistically significant (P < 0.05) after 11 weeks compared with the placebo group where no change was seen. The central role of IgE in the allergic inflammatory cascade also provided an attractive target for the development of new treatment options for severe persistent allergic asthma. Interrupting the interaction between IgE and FcεRI would be expected to block subsequent events in the cascade, preventing asthma exacerbations. Targeting IgE is now a reality following the development of omalizumab, a humanized anti-IgE monoclonal antibody, which is the first treatment to target IgE. Omalizumab comprises a human IgG framework onto

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which is grafted the CDR from a murine anti-IgE antibody (Presta et al. 1993). This “humanization” process ensures that residues of murine origin constitute less than 5% of the omalizumab molecule, thereby minimizing the potential for an immune response. Omalizumab binds all forms of circulating IgE, whatever its allergen specificity, and prevents subsequent IgE-mediated responses. It competes for the same region of the IgE molecule that interacts with IgE receptors on effector cells, i.e., the Cε3 domain, and cannot bind to IgE that is already bound to the high-affinity IgE receptor (FcεRI) (Presta et al. 1994). This is an important feature as it means that omalizumab cannot cross-link FcεRI receptors and activate effector cells.

Mechanism of action Omalizumab binds free IgE and inhibits mast cell degranulation. By reducing free IgE, omalizumab also downregulates FcεRI on basophils and mast cells (MacGlashan et al. 1997; Holgate et al. 2005a). These dual effects of omalizumab are important, since without FcεRI downregulation almost complete removal of free IgE would be necessary to elicit functional consequences on mast cells and basophils. In patients with allergic asthma, omalizumab significantly reduces both the early-phase and late-phase asthmatic response to allergen challenge (Fahy et al. 1997). The effects of omalizumab on early- and late-phase asthmatic responses to allergen were assessed by measuring mean maximal decreases in FEV1 within 1 hour (early response) or after 2–7 hours (late response) in a study of 19 patients with mild allergic asthma after 9 weeks of omalizumab treatment. The early asthmatic response was reduced by 85% (P = 0.01) and the late asthmatic response by more than 65% (P = 0.047) compared with placebo. Omalizumab also produced a progressive reduction in both the early phase (15 min) and late phase (6 hours) allergen-induced skin response model, with a greater effect on the late-phase response (Ong et al. 2005). Patients received omalizumab or placebo for 12 weeks, with allergen challenges performed at 2-weekly intervals. The median reduction in early-phase response was 24% with omalizumab compared with 3% for placebo (P = 0.005 vs. placebo). The median reduction in late-phase response was 63% compared with 6% for placebo (P < 0.001 vs. placebo). Definitive evidence of the antiinflammatory action of omalizumab was reported in a study by Djukanovif et al. (2004). In this study, 45 patients with corticosteroid-naive, mild-tomoderate persistent asthma underwent bronchial biopsy and induced sputum sampling. After 16 weeks of treatment with omalizumab or placebo, there were significant changes from baseline in several cell types in the bronchial submucosa of omalizumab recipients. Importantly, treatment with omalizumab led to marked depletion in eosinophils in both sputum and biopsy specimens. In the omalizumab group, the percentage of eosinophils in induced sputum decreased from 4.8% to 0.6% (P = 0.05 vs. placebo) (Fig. 81.3a) and there was

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P = 0.05

Eosinophils (% of inflammatory cells)

40

40 P = 0.16

30

30 P < 0.001

20 10

20 10

5.8

4.8

2.3

0.6

0

0 Baseline

Post-treatment

Baseline

Omalizumab (n = 19)

(a)

Post-treatment Placebo (n = 22)

P = 0.03 80

80

P = 0.81

60

60 P < 0.001

40

40

20

20 8.0

0

(b)

1.5 Baseline Post-treatment Omalizumab (n = 14)

6.3

6.4 0

Baseline

Post-treatment Placebo (n = 14)

Fig. 81.3 Eosinophils in (a) induced sputum and (b) bronchial submucosa during 16 weeks of treatment with omalizumab or placebo. (From Djukanovic´ et al. 2004, with permission from the American Thoracic Society.)

a significant reduction in tissue eosinophils in the bronchial submucosa from 8.0 to 1.5 cells/mm2 (P = 0.03 vs. placebo) (Fig. 81.3b). In addition to reducing eosinophil infiltration, omalizumab significantly reduced staining for IL-4 compared with baseline and placebo (P < 0.001 for both). There were also significant reductions in numbers of T cells (CD4+ and CD8+) and B cells in the submucosa in patients receiving omalizumab (P ≤ 0.05 vs. placebo). Surprisingly, there was no improvement in airway hyperresponsiveness to methacholine challenge, indicating that IgE and eosinophils may not mediate methacholine responsiveness in mild to moderate asthma. One study analyzed the effect of omalizumab on bronchoconstriction induced by methacholine (acting directly on smooth muscle) or adenosine 5-monophosphate (AMP; which acts on receptors on “primed” mast cells causing mast cell degranulation and the release of histamine and leukotrienes with subsequent smooth muscle contraction) in patients with mild to moderate asthma. After 12 weeks the mean AMP PC20 (provocative concentration required to reduce FEV1 by 20%) values were significantly increased in the omalizumab group (1.91 doubling concentrations; P < 0.001) compared withthe placebo group (1.01 doubling concentrations; P = 0.16).

Anti-IgE in Persistent Severe Allergic Asthma

Changes in methacholine PC20 values were not significantly different between the omalizumab and placebo groups (Prieto et al. 2006). A recent study examined the effect of omalizumab on passive sensitization-induced hyperresponsiveness, alterations in IgE-positive inflammatory cells, and mast cell degranulation within the bronchial wall (Berger et al. 2007). Dissected human bronchi were incubated in normal or asthmatic serum, containing various concentrations of omalizumab, and their contractile response to histamine or house-dust mite (Dermatophagoides pteronyssinus) extract was measured. Specific bronchial hyperresponsiveness to D. pteronyssinus and nonspecific bronchial hyperresponsiveness to histamine were both significantly inhibited by omalizumab. Omalizumab also reduced passive sensitization-induced increase in IgE-positive cells in a concentration-dependent manner, and inhibited mast cell degranulation. Mast cell degranulation in passively sensitized bronchi positively correlated with in vitro hyperresponsiveness to histamine and to D. pteronyssinus. Researchers concluded that omalizumab blocks specific and nonspecific bronchial hyperresponsiveness. In another study, omalizumab decreased the number of circulating eosinophils and Th2 type cytokines (IL-13, IL-5 and IL-8) in patients with moderate-to-severe allergic asthma (Noga et al. 2003). Omalizumab has also been shown to induce eosinophil apoptosis and reduces the number of lymphocytes producing proinflammatory cytokines in patients with coexisting moderate-to-severe allergic asthma and rhinitis (Noga et al. 2006). In addition to its effects on antiinflammatory cells and mediators, omalizumab has also been shown to downregulate FcεRI expression on precursor dendritic cells in a study of 24 patients with ragweed-sensitive seasonal allergic rhinitis (Prussin et al. 2003). This suggests that omalizumab may alter allergen presentation to T cells, resulting in a reduction in Th2 cell activation and Th2 cytokine generation. Through this mechanism it is possible that omalizumab may have immunomodulatory effects on dendritic cells at a stage of development preceding their trafficking into tissues and maturation and may have a unique capacity to block both sensitization (acute) and effector (chronic) phases of the allergic inflammatory process. The proposed mechanism of action of omalizumab is depicted in Fig. 81.4.

Clinical pharmacology Omalizumab is absorbed slowly after subcutaneous administration, reaching peak serum concentrations after an average of 7–8 days (Novartis Pharmaceutical Corporation 2004). When bound to IgE, omalizumab forms small biologically inert, noncomplement-fixing complexes (predominantly trimers) with a molecular mass of approximately 500 kDa (Liu et al. 1995; Fox et al. 1996). Omalizumab–IgE complexes are cleared via interactions with Fcγ receptors of the hepatic sinusoidal endothelial cells of the reticuloendothelial system (Mariani

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Anti-IgE decreases free-IgE and reduces FceRI expression Dendritic cell (DC) Clinical effects B cell

Mast cell/basophil ?

Clinical effects

T cell Anti-IgE reduces DC FceRI expression and could decrease presentation of allergen to T cells

Eosinophil Anti-IgE reduces eosinophil influx Tissue infiltration

& Strober 1990; Ghetie et al. 1996). Clearance is slow (mean 2.4 ± 1.1 mL/kg per day), with a terminal half-life of 26 days. No clinically important changes in the pharmacokinetics of omalizumab were observed as a result of differences in age, sex, or race (Novartis Pharmaceutical Corporation 2004). Treatment with omalizumab results in dose-dependent reductions in serum free IgE concentrations within 1 hour after dosing. Mean maximal decreases in serum free IgE in phase III studies were greater than 96% of baseline (Novartis Pharmaceutical Corporation 2004). Studies in patients with IgE-mediated asthma or rhinitis showed that clinical benefits with omalizumab are observed when serum free IgE levels are reduced to ≤ 50 ng/mL (20.8 IU/mL). The ability of omalizumab to reduce free IgE to such levels is dependent on dose, the patient’s weight, and baseline total IgE level (Hochhaus et al. 2003). Dosing tables were developed to facilitate calculation of the omalizumab dose required to achieve the target reduction in free IgE based on serum total IgE levels measured before treatment initiation and the patient’s body weight (Table 81.3). Once baseline serum IgE levels have been measured, there is no need to retest IgE levels during omalizumab treatment. This is because total IgE levels are elevated due to the persistence of IgE bound to omalizumab. Dose adjustments may be required if there is a significant change in the patient’s body weight. In the event of such a dose adjustment, the pretreatment serum IgE level and new body weight should be used to recalculate the dose. The dosing tables for omalizumab are based on a maximum dose of 750 mg every 4 weeks, which precludes treatment of patients with IgE levels above 700 IU/ mL. Studies are being conducted to investigate the treatment of patients with higher IgE levels (up to 1300 IU/mL). The potential for reducing the dose of omalizumab during therapy was recently explored. Simulations to assess whether, after 28 weeks’ treatment, consistent suppression of free IgE to the target level (< 50 ng/mL) could be achieved with lower doses than specified in the omalizumab dosing table found

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Fig. 81.4 Proposed mechanisms of action of omalizumab (Reprinted from Holgate et al. 2005a, with permission from Elsevier). Omalizumab decreases free IgE and reduces FceRI receptor expression on mast cells and basophils. This results in decreased mast cell activation and sensitivity, leading to a reduction in eosinophil influx and activation. Anti-IgE treatment with omalizumab may result in decreased mast cell survival. Omalizumab also reduces dendritic cell FceRI receptor expression. (See CD-ROM for color version.)

that lower omalizumab doses would result in an inability to maintain free IgE at the target level. The effect of discontinuing omalizumab treatment on clinical outcomes was also evaluated in a 16-week follow-up period of a 28-week randomized placebo-controlled trial (Slavin et al. 2007). After cessation of omalizumab therapy, asthma symptoms, morning peak expiratory flow (PEF), and rescue medication use gradually returned to pretreatment levels and there was good correlation between the percentage of average steady-state omalizumab concentration and worsening asthma control. Based on these data, reductions in omalizumab dose after 6 months of treatment cannot be recommended. The ongoing Epidemiological Study of Xolair Evaluating Clinical Effectiveness and Long-Term Safety in Patients with Moderate to Severe Asthma (EXCELS) study will provide additional information on the potential for dose reduction following long-term treatment (up to 5 years) with omalizumab (Miller et al. 2006).

Efficacy of omalizumab in patients with severe persistent allergic asthma The efficacy of omalizumab has been extensively evaluated in clinical studies in patients with predominantly severe persistent allergic asthma (Table 81.4). The efficacy of omalizumab was assessed in patients with inadequately controlled severe persistent allergic asthma despite high-dose ICS plus LABA (with additional controller medication if required) in a 28week randomized placebo-controlled trial (INNOVATE study) (Humbert et al. 2005). Efficacy was also assessed in a 1-year, randomized, controlled, open-label study which enrolled patients with poorly controlled moderate-to-severe allergic asthma (94% severe persistent asthma) and compared omalizumab plus current asthma therapy with current asthma therapy alone (ETOPA study) (Ayres et al. 2004b). Five additional randomized trials in patients with moderate-to-severe or severe asthma have also been conducted (Busse et al. 2001;

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Table 81.3 Omalizumab dosing tables (EU). (a) Omalizumab doses (mg/dose) administered by subcutaneous injection every 4 weeks. (b) Omalizumab doses (mg/dose) administered by subcutaneous injection every 2 weeks. (a) Body weight (kg) Baseline IgE (IU/mL) ≥ 30–100

> 20–25

> 25–30

> 30–40

> 40–50

> 50–60

> 60–70

> 70–80

> 80–90

> 90–125

> 125–150

300

300

75

75

75

150

150

150

150

150

> 100–200

150

150

150

300

300

300

300

300

> 200–300

150

150

225

300

300

> 300–400

225

225

300

> 400–500

225

300

> 500–600

300

300

> 600–700

300

ADMINISTRATION EVERY 2 WEEKS SEE (b)

(b) Body weight (kg) Baseline IgE (IU/mL)

> 20–25

> 25–30

> 30–40

> 40–50

> 50–60

> 60–70

> 70–80

> 80–90

> 90–125

> 125–150

225

300

300

375

≥ 30–100 > 100–200

ADMINISTRATION EVERY 4 WEEKS SEE (a)

> 200–300 > 300–400

225

225

225

225

225

225

300

300

375

375

> 400–500

225

225

300

300

> 500–600

225

300

300

375

225

300

375

> 600–700

225

DO NOT ADMINISTER

Table 81.4 Details of omalizumab controlled studies.

Study

No. of patients with severe persistent asthma* (%)

Treatment arms†

Duration (weeks)

1 2 3 4 5 6 7

419 (100) 294 (94.2) 364 (89.9) 523 (99.6) 537 (98.4) 315 (92.4) 1556 (88.4)

Omalizumab + CAT vs. placebo + CAT Omalizumab + CAT vs. CAT alone Omalizumab + CAT vs. placebo + CAT Omalizumab + CAT vs. placebo + CAT Omalizumab + CAT vs. placebo + CAT Omalizumab + CAT vs. placebo + CAT Omalizumab + CAT vs. CAT alone

28 52 28 52‡ 52‡ 32 24

INNOVATE study ETOPA study SOLAR study Busse study Solèr study Holgate study ALTO study

* GINA 2002 classification. † All patients were receiving ICS; long-acting b2 agonists (LABAs) were not used in studies 4 and 5. ‡ 24-week core study and 24-week extension. CAT, current asthma therapy; ICS, inhaled corticosteroids.

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Solèr et al. 2001; Buhl et al. 2002; Lanier et al. 2003; Holgate et al. 2004; Vignola et al. 2004; Genentech, data on file). Four studies were randomized placebo-controlled trials (Busse et al. 2001; Solèr et al. 2001; Buhl et al. 2002; Lanier et al. 2003; Holgate et al. 2004; Vignola et al. 2004) and one study was a randomized open label safety study, which also evaluated efficacy outcomes (Genentech data on file).

INNOVATE study The INNOVATE study was a randomized, placebo-controlled, double-blind study conducted in 419 patients with inadequately controlled severe persistent allergic asthma. All patients enrolled in the INNOVATE study were receiving high-dose ICS (> 1000 μg/day beclomethasone dipropionate equivalent) plus an LABA and approximately 60% were taking additional controller medication including oral corticosteroids (22% of patients). Despite high levels of medication use, 67% were considered to be at high risk of asthma-related death (previous intubation or emergency room visit/hospitalization in past year). In addition, asthma had a meaningful impact on patients’ quality of life and an average of 31 school/work days had been missed in the past year due to asthma. Patients included in the INNOVATE study represent the asthma population with the most severe disease and the greatest unmet medical need. The primary efficacy variable in the INNOVATE study was the rate of clinically significant exacerbations, which was defined as a worsening of asthma symptoms requiring treatment with systemic corticosteroids. Other efficacy variables included the following. • Severe exacerbation rate: FEV1 or PEF < 60% of personal best and requiring treatment with systemic corticosteroids. • Total emergency visit rate: hospital admissions, emergency room visits, unscheduled doctor’s visits. • Asthma-related quality of life: change from baseline in Asthma Quality of Life (AQLQ) overall score (Juniper et al.

Severe exacerbation rate

Total emergency visit rate D–43.9%

D–50.0% 0.6

0.6

P = 0.002 0.48

0.5

1992) and the proportion of patients with ≥ 0.5-point, ≥ 1.0point and ≥ 1.5-point improvements in AQLQ overall score (Juniper et al. 1994). • Lung function. • Asthma symptom scores. • Patient’s overall assessment. • Physician’s overall assessment. The physician’s overall assessment of asthma control is a composite measure that encompasses multiple aspects of response. It is based on clinical assessments including patient interviews, review of medical notes, spirometry and diaries of symptoms, rescue medication use and PEF. The physician’s overall assessment was graded in a five-level evaluation of asthma control: complete control; marked improvement in control; discernible but limited control; no appreciable change; worsening in control. In addition, patients made an overall assessment using the same five-level evaluation. Add-on omalizumab therapy resulted in a significant 26.2% reduction in the rate of clinically significant exacerbations (adjusted post hoc for an observed imbalance in exacerbation history) (0.68 vs. 0.91, P = 0.042). Severe exacerbation rate was significantly reduced by 50% (0.24 vs. 0.48, P = 0.002) and the total emergency visit rate was significantly reduced by 44% (0.24 vs. 0.43, P = 0.038) compared with placebo (Fig. 81.5). Omalizumab treatment significantly improved patients’ quality of life. Change from baseline in AQLQ overall score was 0.91 in omalizumab compared with 0.46 in the placebo group (P < 0.001). Significant (P < 0.01) improvements were seen in all individual domains (Fig. 81.6). A significantly greater proportion of omalizumab-treated patients achieved a clinically meaningful (≥ 0.5-point) improvement in AQLQ overall score compared with placebo (60.8% vs. 47.8%, P = 0.008). Similarly, a significantly greater proportion of omalizumabtreated patients achieved moderate 1.0-point improvements (45.1% vs. 24.9%, P < 0.001) and large 1.5-point improvements (27.5% vs. 17.1%, P = 0.011).

P = 0.038

0.5 0.43 0.4

0.4

0.3

0.3

0.24

0.24 0.2

0.2

0.1

0.1 0

0 Omalizumab (n = 209)

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Placebo (n = 210)

Omalizumab (n = 209)

Placebo (n = 210)

Fig. 81.5 Adding omalizumab significantly reduces severe exacerbation and total emergency visit rates in patients with inadequately controlled severe persistent allergic asthma despite high-dose ICS and an LABA. (From Humbert et al. 2005, with permission from Blackwell Publishing.) (See CD-ROM for color version.)

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Change from baseline in AQLQ score, LSM

Omalizumab

P < 0.001 1.0

Anti-IgE in Persistent Severe Allergic Asthma

0.91

P = 0.002 0.95

Placebo

P < 0.001

P < 0.001

P < 0.001

0.90

0.89

0.91

0.8 0.57

0.6 0.46

0.40

0.4 Fig. 81.6 Adding omalizumab significantly improves quality of life in patients with inadequately controlled severe persistent allergic asthma despite high-dose ICS and a LABA. (Humbert et al. 2005.) (See CD-ROM for color version.)

0.44

0.46

0.2 0 Activities

Emotions

Symptoms

Environment

Overall

AQLQ = Asthma Quality of Life Questionnaire LSM = Least squares mean

Overall change from baseline in mean morning PEF was significantly greater for omalizumab-treated patients than for placebo (P = 0.042). FEV1 (percent predicted) was significantly improved with omalizumab compared with placebo at study completion (P = 0.043). Mean change from baseline in total asthma symptoms score was significantly greater in omalizumab-treated patients compared with placebo during the overall treatment period (P = 0.039). Omalizumab was evaluated more favorably than placebo to a similar degree by both patients and physicians, with statistically significant (P < 0.001) overall difference for both evaluations. For the physician’s evaluation, 60.5% of omalizumab-treated patients achieved complete asthma control or marked improvement in control compared with 42.8% for placebo. For the patient’s evaluation, 64.3% of omalizumab-treated patients achieved complete asthma control or marked improvement in control compared with 43.3% for placebo. The role of the physician’s overall assessment in identifying patients who respond to omalizumab is discussed below in the section on selection of patients for omalizumab treatment.

ETOPA study In the 1-year open-label ETOPA study (Ayres et al. 2004b), adding omalizumab to current asthma therapy significantly reduced exacerbation rates (worsening of asthma requiring systemic corticosteroids) compared with current asthma therapy alone (1.12 vs. 2.86, P < 0.001). Similar data were reported in an additional analysis of a subgroup of patients with severe persistent allergic asthma (1.24 vs. 3.0, P < 0.001) (Bousquet et al. 2004b). An analysis has also been performed that assessed the efficacy of omalizumab in a subgroup of 164 patients who were receiving high-dose ICS plus LABA (Niven et al. 2007). Of the 312 patients (omalizumab 206; placebo 106) included in the original study (Ayres et al. 2004b), 164 patients (omalizumab 115; placebo 49) were receiving high-dose ICS (> 1000 μg/day beclomethasone dipropionate equivalent)

plus an LABA. Mean annual rate of asthma exacerbations was significantly lower in the omalizumab-treated group compared with the control group (1.26 vs. 3.06); rate ratio (95% CI) for omalizumab to control was 0.410 (0.288, 0.583) (P < 0.001), which equates to a 59% reduction with omalizumab.

Pooled analysis A pooled analysis of omalizumab efficacy has also been performed including data from INNOVATE and six additional randomized trials. In total, 4308 patients were included in the analysis (2511 patients received omalizumab) and 93% of patients met GINA 2002 criteria for severe persistent asthma (Bousquet et al. 2005a). In all studies, omalizumab was added to current asthma therapy and compared with current asthma therapy plus placebo or current asthma therapy alone. Add-on omalizumab consistently reduced asthma exacerbation rates across the seven studies (Table 81.5). Overall, asthma exacerbation rate was reduced by 38% in the pooled population (P < 0.001). Total emergency visit rates were significantly reduced by 47% in the omalizumab group compared with the control group (0.332 vs. 0.623, P < 0.001); hospital admissions were reduced by 51% (P < 0.05), emergency room visits by 60% (P < 0.05), and unscheduled doctor’s visits by 43% (P < 0.001) (Bousquet et al. 2005a). Pooled analysis of data from the double-blind placebo-controlled studies produced similar data (Novartis, data on file). One analysis of the five double-blind placebo-controlled studies showed exacerbation rates to be significantly reduced by 38% (0.832 vs. 1.348, P < 0.0001), while analysis of data from four of these studies (due to differences in efficacy variables) showed total emergency visits to be significantly reduced by 46% (0.213 vs. 0.396, P < 0.0001). Quality of life was measured in 1221 patients receiving omalizumab and 1032 receiving placebo/control in a pooled analysis of data from six clinical trials (Chipps et al. 2006). Patients receiving omalizumab experienced consistent improvements

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Table 81.5 Consistent reduction in exacerbation rates across all seven studies. (From Bousquet et al. 2005a, with permission from Blackwell Publishing.) Annualized exacerbation rate Study

Omalizumab

Control

Treatment difference

Percent reduction

P value

1 INNOVATE study* 1 INNOVATE study† 2 ETOPA study 3 SOLAR study 4 Busse study 5 Solèr study 6 Holgate study 7 ALTO study Pooled‡

1.36 1.55 0.98 0.49 0.59 0.51 1.18 1.02 0.910

1.85 1.93 2.47 0.79 0.99 1.21 1.60 1.20 1.474

0.49 0.37 1.49 0.29 0.40 0.70 0.42 0.18 0.56

26.2 19.2 60.4 37.5 40.3 57.6 26.5 15.3 38.3

0.042 0.156 < 0.001 0.027 < 0.001 < 0.001 0.165 0.077 < 0.001

* Adjusted for a baseline imbalance in exacerbation history. † Unadjusted. ‡ Includes unadjusted exacerbation data from the INNOVATE study.

in quality of life with significantly greater improvements in AQLQ overall scores (1.01 vs. 0.61, P < 0.001) and individual domains (P < 0.001) achieved in omalizumab-treated patients compared with placebo/control. The proportion of omalizumab-treated patients in the pooled population recording a ≥ 0.5 point improvement in AQLQ total score (66.3% vs. 52.4%, P < 0.001), a ≥ 1.0-point improvement (49.0% vs. 31.2%, P < 0.001) and a ≥ 1.5-point improvement (32.8% vs. 18.2%, P < 0.001) was significantly greater than for control. In an additional pooled analysis of data from four studies, quality of life was assessed in 759 patients (omalizumab 420, control 339) with severe persistent allergic asthma despite receiving high-dose ICS plus an LABA (Beeh et al. 2006). Omalizumab recipients had a mean increase in AQLQ overall score of 0.99 points compared with 0.56 in the control group (P < 0.001). In addition, significantly more patients achieved clinically meaningful improvements in AQLQ score in the omalizumab group (62.6%) than in the control group (47.5%, P < 0.001). Using data from all seven studies, omalizumab significantly improved FEV1 compared with control (Holgate et al. 2005b). At study end, percent predicted FEV1 in the omalizumab group was 71.18% vs. 68.93% in the control group while change from baseline in least squares mean (LSM) percent predicted FEV1 in the two groups was 0.91 and −1.34, respectively (LSM difference 2.24; 95% CI 1.35, 3.14; P < 0.001 for both comparisons). FEV1 changes were classified as clinically meaningful improvement (≥ 200 mL increase) or clinically meaningful worsening (≥ 200 mL decrease) and net benefit (percentage with improvement – percentage with worsening) within omalizumab and control groups calculated. Among omalizumab recipients, 29.1% had ≥ 200 mL increase in FEV1, and 17.5% had ≥ 200 mL decrease. In the control group, 26.4% had ≥ 200 mL increase in FEV1, and 26.1% had ≥ 200 mL decrease.

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Omalizumab-treated patients thus had a net benefit of 11.6% versus a net benefit of 0.3% for control (P < 0.001). While the improvements in FEV1 are relatively modest, this is not surprising considering that this is a patient population with long-established asthma and patients were receiving multiple therapies including an LABA in many cases.

Efficacy in patients receiving oral corticosteroids or leukotriene modifiers Oral corticosteroids Four clinical trials, comprising two placebo-controlled (Holgate et al. 2004; Humbert et al. 2005) and two open label (Ayres et al. 2004b; Genentech data on file), included patients who were receiving regular oral corticosteroids (Wenzel et al. 2007). Overall, 446 patients were receiving oral corticosteroids and 2386 were not receiving oral corticosteroids. Omalizumab significantly reduced exacerbation rates by 34.9% (2.18 vs. 3.34, P = 0.001) in patients who required oral corticosteroids and by 29.4% in patients who were not receiving oral corticosteroids (1.14 vs. 1.62, P < 0.001) and significantly reduced emergency visit rates by 57.6% (0.84 vs. 1.99, P = 0.036) in patients taking oral corticosteroids and by 35.8% (0.43 vs. 0.67, P < 0.001) in patients not taking oral corticosteroids. Significant improvements in quality of life and FEV1 were also seen with omalizumab irrespective of oral corticosteroid use. Although INNOVATE (Humbert et al. 2005) was not powered for oral corticosteroid subgroup analysis, the effect of add-on omalizumab therapy on the clinically significant exacerbation rates was evaluated according to baseline oral corticosteroid use (Bleecker et al. 2005). Of 419 patients included in this analysis, 91 required oral corticosteroid at baseline. Addon omalizumab therapy reduced the annualized exacerbation rate by 35% compared with placebo in patients requiring oral corticosteroids (0.79 vs. 1.22, P = 0.111) and by 24% compared

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with placebo in patients not receiving maintenance oral corticosteroid (0.70 vs. 0.90, P = 0.134). In the ETOPA study, subgroup analysis found that exacerbation rates were significantly reduced in the omalizumab plus current asthma therapy group compared with the current asthma therapy alone group irrespective of oral corticosteroid use (Ayres et al. 2004b). These data indicate that add-on omalizumab therapy significantly reduces asthma exacerbations and emergency visits and improves quality of life and FEV1 in patients with severe persistent allergic asthma, irrespective of oral corticosteroid use.

Leukotriene modifiers The effect of leukotriene receptor antagonist (LTRA) use on the efficacy of omalizumab was assessed in a pooled analysis of data from the INNOVATE (Humbert et al. 2005) and ETOPA (Ayres et al. 2004b) studies (Massanari et al. 2005). Of the 731 patients included in this pooled analysis, 236 (32.3%) were receiving LTRAs at baseline (omalizumab, 134; control, 102). The relative risk of asthma exacerbations versus control was similar irrespective of LTRA use: LTRA subgroup 0.62 (95% CI 0.42– 0.91); non-LTRA subgroup 0.5 (95% CI 0.35–0.72). Similar results were reported in a separate pooled analysis of data from INNOVATE (Humbert et al. 2005), ETOPA (Ayres et al. 2004b), and ALTO (Genentech data on file), which assessed the need for systemic steroid bursts (Zeldin et al. 2006). In total, 46% of patients (1199/2630) took LTRAs. In non-LTRA users (N = 1199), omalizumab reduced the need for steroid bursts by 51% [relative risk (omalizumab vs. control) 0.49; 95% CI 0.34–0.71; P < 0.001]. In LTRA users (N = 1431), omalizumab reduced the need for steroid bursts by 53% (relative risk 0.47; 95% CI 0.32– 0.68; P < 0.001). A subgroup analysis of data from the ETOPA study also found that exacerbation rates were significantly reduced in the omalizumab plus current asthma therapy group compared with current asthma therapy alone group irrespective of concomitant use of leukotriene modifiers (Ayres et al. 2004b). The results of these subgroup analyses indicate that add-on omalizumab therapy is efficacious irrespective of concomitant use of leukotriene modifiers.

Anti-IgE in Persistent Severe Allergic Asthma

The ability of omalizumab to reduce oral corticosteroid requirements has also been assessed in 35 patients with moderate-to-severe allergic asthma (Milgrom et al. 1999). During a steroid-reduction phase, the dose of oral corticosteroid was tapered by up to 20% at 1-week intervals, with further reductions only if there was no significant deterioration. Patients receiving omalizumab had a reduced requirement for oral corticosteroids. In the group receiving higher doses of omalizumab (5.8 μg/kg per ng IgE/mL), 33% of patients were able to discontinue oral corticosteroids, compared with 17% in the placebo group. In addition, 78% of these omalizumabtreated patients were able reduce their oral corticosteroid dose by at least 50%, compared with 33% of placebo recipients (P = 0.04). The capacity of omalizumab to reduce maintenance oral corticosteroid use is currently being evaluated. In a pooled analysis of the seven clinical trials, the relative risk of systemic corticosteroid bursts was 43% lower in omalizumabtreated patients than in the control group (relative risk 0.57; 95% CI 0.48–0.66; P < 0.001) (Maykut et al. 2006).

Efficacy in patients with coexiting rhinitis Asthma and rhinitis frequently coexist and there is strong evidence that the two conditions share a common allergic inflammatory cause centered on IgE (Bousquet et al. 2003). Studies have shown that patients with asthma and documented concomitant allergic rhinitis experience significantly more asthma-related hospitalizations, visit a physician more frequently, and incur higher asthma-related drug costs compared with patients with asthma alone (Bousquet et al. 2005b; Price et al. 2005). Omalizumab has also been shown to provide clinical benefits for patients with coexisting asthma and rhinitis. In a study of patients with coexisting moderateto-severe allergic asthma and rhinitis, omalizumab significantly improved rhinitis outcomes compared with placebo (Vignola et al. 2004). Although omalizumab is not indicated for the treatment of allergic rhinitis, it may provide additional benefits in patients with severe persistent allergic asthma and concomitant rhinitis.

Steroid-sparing effects

Safety and tolerability

In three studies, ICS doses were adjusted to provide the lowest dose consistent with asthma control (Busse et al. 2001; Solèr et al. 2001; Holgate et al. 2004). The “core” treatment period of the three studies consisted of two phases: a 16-week “steroid-stable” phase during which patients received omalizumab or placebo treatment in addition to a constant dose of ICS, and a 12-week (Busse et al. 2001; Solèr et al. 2001) or 16-week (Holgate et al. 2004) “steroid-reduction” phase. Patients who received omalizumab had a median 75% (Busse et al. 2001), 83% (Solèr et al. 2001), and 60% (Holgate et al. 2004) reduction in ICS dose, compared with 50% (Busse et al. 2001; Solèr et al. 2001; Holgate et al. 2004) in the placebo group (P < 0.01).

The clinical safety and tolerability of omalizumab has been assessed using data from completed phase I, II and III studies involving more than 7500 patients with asthma, rhinitis or related conditions (Corren et al. 2005). The long-term safety of omalizumab is being evaluated in a 5-year prospective openlabel observational cohort study in patients with moderateto-severe asthma and a positive skin-prick test, currently underway in the USA (EXCELS) (Miller et al. 2006). EXCELS was initiated in 2004 and completed enrolment of 5000 patients receiving omalizumab and 2500 control patients in 2006. Analyses of adverse events (AEs) were performed on three large datasets derived from the omalizumab development

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program: (i) six placebo-controlled phase IIB/III studies in adults and adolescents (≥ 12 years) with predominantly severe persistent allergic asthma enrolled in placebo-controlled studies (2342 patients); (ii) two large, controlled, open-label, standard asthma therapy studies in adults and adolescents (≥ 12 years) with predominantly severe persistent allergic asthma (2004 patients); and (iii) all controlled studies in all indications, including children, adults and adolescents with allergic asthma as well as other indications, such as seasonal allergic rhinitis, perennial allergic rhinitis and atopic dermatitis (6130 patients).

In the all-controlled studies population (omalizumab, 3678; control, 2452), AEs were reported in 74.8% of patients receiving omalizumab and 75.2% of control group patients. Serious AEs were reported by 4.2% of patients receiving omalizumab and by 3.8% of patients receiving controls. The most frequent AEs (≥ 3% in any group) in placebo-controlled and standard therapy controlled allergic asthma trials are shown in Table 81.6. The most frequently reported AEs were upper respiratory tract infection, nasopharyngitis, sinusitis, and headache in the omalizumab and control groups. Any differences between groups were small and not indicative of

Table 81.6 Most common adverse events (≥ 3% in any group) in placebo-controlled and standard therapy-controlled allergic asthma studies. Values are number of events, with percentages in parentheses. (Novartis data on file.) Placebo-controlled studies

Standard therapy controlled studies

MedDRA Organ Class Preferred term

Omalizumab (N = 1192)

Placebo (N = 1150)

Omalizumab (N = 1338)

Control (N = 666)

Infections and infestations Nasopharyngitis Upper respiratory tract infection Sinusitis Influenza Bronchitis Gastroenteritis Pharyngitis Viral infection Lower respiratory tract infection

277 (23.2) 210 (17.6) 150 (12.6) 110 (9.2) 77 (6.5) 49 (4.1) 54 (4.5) 45 (3.8) 58 (4.9)

274 (23.8) 214 (18.6) 168 (14.6) 114 (9.9) 86 (7.5) 36 (3.1) 53 (4.6) 46 (4.0) 55 (4.8)

151 (11.3) 209 (15.6) 160 (12.0) 43 (3.2) 96 (7.2) 28 (2.1) 33 (2.5) 33 (2.5) 34 (2.5)

58 (8.7) 83 (12.5) 74 (11.1) 19 (2.8) 45 (6.8) 8 (1.2) 10 (1.5) 12 (1.8) 24 (3.6)

98 (8.2) 81 (6.8) 57 (4.8) 43 (3.6) 25 (2.1)

98 (8.5) 103 (9.0) 53 (4.6) 35 (3.0) 37 (3.2)

61 (4.6) 56 (4.2) 16 (1.2) 25 (1.9) 20 (1.5)

13 (1.9) 13 (1.9) 17 (2.5) 12 (1.8) 9 (1.3)

Nervous system disorders Headache

230 (19.3)

229 (19.9)

121 (9.0)

25 (3.7)

Gastrointestinal disorders Diarrhea Nausea Dyspepsia

59 (4.9) 59 (4.9) 44 (3.7)

53 (4.6) 47 (4.1) 65 (5.6)

41 (3.1) 46 (3.4) 16 (1.2)

Musculoskeletal and connective tissue disorders Back pain Arthralgia Myalgia Pain in extremity

96 (8.0) 72 (6.0) 52 (4.4) 40 (3.4)

92 (8.0) 56 (4.9) 48 (4.2) 27 (2.3)

44 (3.3) 41 (3.1) 22 (1.6) 18 (1.3)

10 (1.5) 4 (0.6) 3 (0.4) 3 (0.4)

General disorders and administration site conditions Pyrexia

43 (3.6)

39 (3.4)

13 (1.0)

4 (0.6)

Psychiatric disorders Insomnia

29 (2.4)

37 (3.2)

11 (0.8)

4 (0.6)

Injury, poisoning, and procedural complications Joint sprain

40 (3.4)

25 (2.2)

17 (1.3)

8 (1.2)

Respiratory, thoracic and mediastinal disorders Pharyngolaryngeal pain Cough Rhinitis Nasal congestion Rhinitis allergic

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5 (0.7) 8 (1.2) 2 (0.30)

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specific toxicity. In studies where local symptoms and signs of injection site reactions were evaluated prospectively after each dose, the overall frequency of injection site reactions was similar in the omalizumab group (45%) and the placebo group (43%).

Adverse events of special interest The risk of specific hypothetical safety concerns that might be associated with a monoclonal antibody targeting an immune system mediator (e.g., hypersensitivity reactions, malignant neoplasia, parasitic infections) as well as on issues identified during preclinical investigation (e.g., thrombocytopenia) have also been assessed.

Hypersensitivity As omalizumab is a protein, it might be expected to be associated with hypersensitivity reactions and related immunologic effects. However, as residues of murine origin constitute less than 5% of the omalizumab molecule and omalizumab cannot cross-link FcεRI receptors and activate effector cells, omalizumab has low anaphylactogenic potential. This has been confirmed by an analysis of AEs that were suggestive of hypersensitivity reactions (such as anaphylaxis, urticaria or serum sickness-like syndrome), which showed that there was no evidence of an increased risk of immune complexmediated or other hypersensitivity reactions with omalizumab (Corren et al. 2005). The incidence of other AEs suggestive of hypersensitivity reactions in the all-controlled studies population was 4.2% in the omalizumab group and 4.7% in the control group. In the all-controlled studies population, the frequency of skin rash (any type) including pruritus was 6.9% in the omalizumab group and 5.3% in the control group. Urticaria occurred infrequently (omalizumab 1.3%, control 1.3%). The incidence of anaphylaxis was uncommon (omalizumab 0.14%, control 0.07%) and there were no AEs reported as serum sickness or serum sicknesslike syndrome in any treatment groups and none of the omalizumab-treated patients developed measurable antiomalizumab antibodies. Nevertheless, as with all recombinant DNA-derived humanized monoclonal antibodies, the possibility of developing antiomalizumab antibodies cannot be excluded and recently a boxed warning has been added to the drug label in the US based on the incidence of anaphylactic reactions from postmarketing surveillance (0.2%: Limb et al. 2007).

Malignant neoplasia During the omalizumab clinical program, a number of cases of cancer were reported, with a slight numerical imbalance between those arising in the omalizumab-treated group (25 cases; 0.5%) and those arising in the control group (5 cases; 0.18%). Analyses of malignant neoplasia were therefore performed on data from 35 phase I, II and III multiple dose studies of omalizumab in all indications (N = 7869) (Fernández

Anti-IgE in Persistent Severe Allergic Asthma

et al. 2005). The overall incidence and type of malignant neoplasms in the omalizumab and control groups were recorded and malignancy rates were calculated. A comparison of cancer rates with the National Institutes of Health (NIH) Surveillance, Epidemiology, and End Results (SEER) database was undertaken and the expected number of cancers was calculated by applying the SEER gender- and age-specific cancer rates to the time at risk using the indirect age standardization method. The actual number of neoplasms (excluding nonmelanoma skin cancer) in the omalizumab group (15) was consistent with the expected value (15.21) derived from the SEER database, resulting in a standardized incidence ratio (SIR) of observed to expected number of events of 0.99 (95% CI 0.55, 1.63). In contrast, the incidence of neoplasms (excluding nonmelanoma skin cancer) in the control group (2) was lower than the expected value of 6.54, resulting in an SIR of 0.31 (95% CI 0.04, 1.11). These data should be interpreted with caution because of the very small number of malignant neoplasms reported, particularly in the control group. The relative risk estimates (omalizumab/control) of malignant neoplasia in phase I, II and III trials ranged from 0.77 to 5.14 and are consistent with estimates in the asthmatic populations reported in the literature of 0.78–2.1 (Reynolds & Kaplan 1987; McWhorter 1988; Mills et al. 1992; Kallen et al. 1993; Vesterinen et al. 1993; Eriksson et al. 1995). Overall, clinical data do not suggest a causal link between omalizumab therapy and cancer. The incidence of malignancies is being assessed in the EXCELS study (Miller et al. 2006).

Parasitic infection Parasitic helminth infection induces production of IgE, which is regarded as an important immune effector mechanism against such infection (Jarrett & Miller 1982). There is therefore a theoretical possibility that treatment with omalizumab could increase susceptibility to helminth infection. The risk of parasitic infection was therefore investigated in a 52-week, randomized, double-blind, placebo-controlled study in 137 adults and adolescents with allergic asthma or allergic rhinitis at high risk of intestinal helminth infection (Cruz et al. 2007). Of the omalizumab subjects, 50% (34/68) experienced at least one intestinal geohelminth infection compared with 41% (28/69) of placebo subjects [odds ratio 1.47, 95% CI 0.74–2.95; one-sided P = 0.14; odds ratio (adjusted for study visit, baseline infection status, gender and age) 2.2, 95% CI 0.94–5.15; one-sided P = 0.035], providing some evidence for a potentially increased incidence of geohelminth infection in subjects receiving omalizumab. There were no clinical or laboratory AEs indicative of morbidity due to helminth infection during omalizumab treatment. The clinical severity of parasite infections was similar between treatment groups and all appropriate anthelmintic treatments administered for infections during the study resulted in cure in both intervention groups.

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In the all-controlled studies population, parasitic infections were rare in both treatment groups (omalizumab 0.19%, control 0.16%) and all events were mild or moderate. In the entire omalizumab clinical trials program, there was only one (0.03%) reported helminthic infection (pinworm) from 1934 patient-years of omalizumab treatment in 3678 treated patients. However, caution may be warranted in patients at high risk of helminth infection, particularly when travelling to areas where helminthic infections are endemic.

Thrombocytopenia Reduced platelet counts were observed in juvenile cynomolgus monkeys receiving omalizumab at doses 3.7- to 20-fold greater than the highest dose recommended in humans. This observation was made after initiation of the phase III clinical trials and led to implementation of increased surveillance of platelet counts in all ongoing and subsequent clinical studies. In all completed studies, there was no evidence of a clinically relevant impact on platelet count during omalizumab treatment. Additional platelet testing is not a requirement of the US or EU label for omalizumab.

Selection of patients for omalizumab treatment Identifying patients who will achieve greatest benefit from omalizumab based on pretreatment clinical characteristics Analyses of data from the INNOVATE study were conducted to determine whether pretreatment baseline characteristics could be used to identify patients likely (or unlikely) to benefit from treatment with omalizumab (Bousquet et al. 2007). Baseline total IgE was the only variable in that study with broad predictive value, with lower baseline IgE associated with a smaller treatment benefit. In light of these findings, subgroup analyses were conducted in the pooled population of patients enrolled in seven clinical trials (including INNOVATE) according to baseline total IgE (Bousquet et al. 2007). Omalizumab reduced asthma exacerbation rates across all IgE quartiles (0– 75 IU/mL, 76–147 IU/ mL, 148–273 IU/mL, and ≥ 274 IU/mL), reaching statistical significance in each of the three upper IgE quartiles. Similarly, total emergency visit rates and AQLQ improvements and FEV1 net benefit favored omalizumab-treated patients in the three upper IgE quartiles. However, for other outcomes there were benefits across all four quartiles, including severe exacerbation rates (statistically significant differences in quartiles 1, 3 and 4) and physician’s overall assessment (statistically significant benefits in all IgE quartiles). These analyses indicate that it is difficult to predict which patients in the label population will have the greatest benefit based on pretreatment characteristics. The potential predictive value of specific IgE is currently being evaluated.

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Identifying patients who respond to omalizumab following a course of treatment The best method of identifying responders to omalizumab following treatment was explored by assessing the ability of various clinical response criteria to identify responders and discriminate patient exacerbation and other outcomes in randomized placebo-controlled omalizumab trials (Bousquet et al. 2007). Response criteria assessed included: • physician’s overall assessment (complete control of asthma or marked improvement); • ≥ 0.5-point improvement in total AQLQ score (Juniper et al. 1992, 1994); • ≥ 200-mL improvement in FEV1 (American Thoracic Socisety 1999); • ≥ 1.0-point reduction in daytime symptom score (4-point scale, from 0, no symptoms, to 4, major discomfort) (Solèr et al. 2001); • ≥ 1.0-point reduction in nocturnal symptom score (4-point scale, from 0, no symptoms, to 4, major discomfort) (Solèr et al. 2001); • reduction ≥ 1/week and by at least 50% in night awakenings. Analysis of INNOVATE data found that the physician’s overall assessment was able to identify 61% of patients as responders to omalizumab. In responders, exacerbation rates (worsening of asthma requiring treatment with systemic corticosteroids), severe exacerbation rates (FEV1 or PEF < 60% of personal best and requiring treatment with systemic corticosteroids), and other outcome measures were markedly reduced compared with nonresponders (Table 81.7). In omalizumab-treated responders, rates of clinically significant exacerbations were reduced by 60% (0.34 vs. 0.85, P < 0.001 vs. placebo) in the 28-week treatment period, which compares to a 26% reduction in the overall omalizumab-treated population (Humbert et al. 2005). Severe exacerbation rates were reduced by 76% (0.13 vs. 0.54, P < 0.001 vs. placebo) and emergency visit rates by 76% (0.098 vs. 0.412, P < 0.001) (Fig. 81.7). These data compare with 50% and 44% reductions, respectively, in the overall omalizumab-treated population (Humbert et al. 2005). Similar findings were observed using pooled data from INNOVATE and four additional randomized placebo-controlled trials. The physician’s overall assessment was also able to identify a high proportion of patients who were classified as responders by the other response criteria. AQLQ also identified 61% of patients as responders and discriminated clinically significant exacerbation response, but was not discriminative for severe exacerbation response. In the ETOPA study, 70% of patients were classified as responders (≥ 0.5-point improvement in Mini-AQLQ overall score) (Niven et al. 2007). In responders, exacerbation rates, compared with control, were reduced by a similar degree (64%) to that seen in the INNOVATE study.

Economic implications The economic burden of asthma is most notable in patients with inadequately controlled, severe asthma (Turk et al. 2005b;

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Table 81.7 Annualized exacerbation rates, unscheduled healthcare utilization and other asthma control measures by physician’s overall assessment of responders and nonresponders to omalizumab in the INNOVATE study. (From Bousquet et al. 2007, with permission from Elsevier.) Responder Clinically significant exacerbations Rate, mean (SD) Severe exacerbations Rate, mean (SD) Hospitalizations* Patients hospitalized in treatment phase (%) Rate, mean (SD) Emergency room visits* Rate, mean (SD) Unscheduled physician visits* Rate, mean (SD) Any unscheduled healthcare utilization Rate, mean (SD) Asthma symptom score, mean (SD)† Night awakenings due to asthma, per week mean (SD)† Daily rescue medication use, puffs mean (SD)† FEV1 (mL) mean (SD)† AQLQ improvement ≥ 0.5-point, % of patients

Nonresponder

0.6 (1.31)

2.6 (6.39)

0.2 (0.6)

1.4 (6.1)

2.5 0.03 (0.22)

9.1 0.10 (0.35)

0.02 (0.17)

0.17 (0.80)

0.11 (0.44)

0.49 (1.31)

0.20 (0.61) −1.24 (1.82) −1.23 (2.22) −2.32 (3.93) 252 (521) 78.8

1.50 (6.14) −0.47 (1.72) −0.28 (2.74) −0.17 (3.79) 87 (445) 34.7

* Rates in the previous year were similar for responders and nonresponders. † Values are changes from baseline.

Severe exacerbation rate 0.6

D–76.3%

Total emergency visit rate 0.6

P < 0.0001

Fig. 81.7 Omalizumab significantly reduces severe exacerbation and total emergency visit rates in responders. (Bousquet et al. 2007 and Novartis data on file.) (See CD-ROM for color version.)

D–76.2% P < 0.0001

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0

0 Omalizumab (n = 118)

Van Ganse et al. 2002). Due to limited healthcare resources it is useful for a new treatment to offer improved economic value as well as increased clinical efficacy. Data from the ETOPA study have been analyzed to calculate the incremental costeffectiveness ratio (ICER) of omalizumab added to standard therapy, to determine the cost-effectiveness of omalizumab in severe persistent allergic asthma (Brown et al. 2007). The ICER was calculated to be a31 209. This compares favorably with a similar analysis carried out on data from the INNOVATE study in which the ICER was calculated to be a56 091

All placebo (n = 210)

Omalizumab (n = 118)

All placebo (n = 210)

(Dewilde et al. 2006). The result from the ETOPA data may be more representative of what would be expected in clinical practice due to the extended length of the trial and its naturalistic setting. Brown et al. (2007) concluded that add-on omalizumab therapy significantly reduces the economic cost of inadequately controlled severe persistent allergic asthma.

Indication Omalizumab was approved in the USA in 2003 for the treatment of patients with moderate-to-severe persistent allergic

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asthma despite treatment with ICS. In the EU (approved 2005), omalizumab is indicated as add-on therapy to improve asthma control in adult and adolescent patients (12 years of age and above) with severe persistent allergic asthma who have a positive skin test or in vitro reactivity to a perennial aeroallergen and who, despite receiving daily high-dose ICS plus an LABA, have the following characteristics: reduced lung function (FEV1 < 80%), frequent daytime symptoms or night-time awakenings, and multiple documented severe asthma exacerbations. Omalizumab treatment should only be considered for patients with convincing lgE-mediated asthma. Prescribing physicians should ensure that patients with IgE below 76 IU/mL have unequivocal in vitro reactivity (RAST) to a perennial allergen before starting therapy. Not all patients respond to omalizumab, and as specified in the EU label, only patients who are judged to have achieved a marked improvement in asthma control after an initial trial of omalizumab should continue therapy beyond 16 weeks. As there is currently limited experience with self-administration, omalizumab should be administered by a healthcare provider. The dose of omalizumab should be calculated from dosing tables (see Fig. 81.5).

Future direction Pediatric asthma The efficacy and tolerability of adding omalizumab to ICS has also been assessed in 334 children (6–12 years) with moderate-to-severe allergic asthma in a 28-week phase III clinical trial with a similar design to trials conducted in adult and adolescent patients (Milgrom et al. 2001). After a 1-week enrollment and screening period, there were three phases: (i) run-in phase (4– 6 weeks), (ii) stable-steroid phase (16 weeks), and (iii) steroid dose-reduction phase (12 weeks). Over the 28-week treatment period, the incidence (percentage of patients with exacerbation) and the frequency (number of episodes per patient) of asthma exacerbations requiring treatment with doubling of beclomethasone dipropionate dose or systemic corticosteroids were lower in the omalizumab group. Treatment differences were statistically significant during the steroid dose-reduction phase (18.2% vs. 38.5%, P < 0.001; 0.42 vs. 2.72, P < 0.001, respectively). Omalizumab-treated children required fewer unscheduled physician visits than placebo-treated children (12.9% vs. 30.3%, P = 0.001) and a significantly greater proportion of omalizumab-treated children were able to reduce their ICS dose (P = 0.002 vs. placebo). Median reduction in ICS dose was significantly greater in the omalizumab-treated group than in the placebo group (100% vs. 67%, P = 0.001). A significantly greater proportion of patients were able to discontinue ICS treatment in the omalizumab group compared with placebo (55% vs. 39%; P = 0.004). At the end of the steroid-reduction phase, patients in the omalizumab-treated group reported significant improve-

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ments (P < 0.05) in Pediatric Asthma Quality of life Questionnaire (PAQLQ) overall scores and significantly more patients in the omalizumab group achieved clinically relevant (≥ 0.5) changes in PAQLQ scores compared with placebo (46.9% vs. 33.7%, P < 0.05) (Lemanske et al. 2002). In a 24-week extension to the core trial, the reduction in ICS with omalizumab was maintained and AEs were comparable to those seen in the placebo group (Berger et al. 2003). A further clinical trial of omalizumab in children (6–11 years) is currently underway in the USA. Although data are encouraging, omalizumab is not currently approved for children below 12 years of age.

Other IgE-mediated diseases Several studies have shown that omalizumab is effective in patients with allergic rhinitis (Ädelroth et al. 2000; Casale et al. 2001; Chervinsky et al. 2003; Vignola et al. 2004). Omalizumabtreated patients with seasonal allergic rhinitis (Ädelroth et al. 2000; Casale et al. 2001) or perennial allergic rhinitis (Chervinsky et al. 2003) had significant reductions in daily nasal symptom severity scores, significantly improved quality of life, and decreased rescue antihistamine use compared with the placebo group. In addition, omalizumab significantly improved rhinitis outcomes seen in omalizumab-treated patients with concomitant asthma and rhinitis (Vignola et al. 2004). Studies examining the possible effectiveness of omalizumab therapy in treating other IgE-mediated conditions are ongoing. Areas under investigation include latex allergies (Leynadier et al. 2004), severe eye allergies (Williams 2004; Williams & Sheppard 2005), and atopic dermatitis (Vigo et al. 2006). The efficacy of an investigational anti-IgE monoclonal antibody (TNX-901) in some subjects with peanut allergy has been demonstrated (Leung et al. 2003), although as an experimental drug, confirmation of these results in additional studies is required.

Conclusions The burden of asthma is greatest in patients with severe persistent asthma, particularly those patients with inadequately controlled asthma. Patients with severe asthma are at high risk of life-threatening exacerbation, hospitalization, and mortality (Rea et al. 1986; Crane et al. 1992; Tough et al. 1998; Guite et al. 1999; Hartert et al. 2002) and are most affected in terms of quality of life (Andersson et al. 2003; Schmier et al. 2003; Juniper et al. 2004; Turk et al. 2005a,b). Similarly, the economic burden of asthma increases with asthma severity (Graf von der Schulenburg et al. 1996; Serra-Batlles et al. 1998; Godard et al. 2002; Antonicelli et al. 2004; Van Ganse et al. 2006) and is greatest in patients with inadequately controlled severe persistent asthma (Hoskins et al. 2001; McCowan et al. 2002; Van Ganse et al. 2002; Schwenkglenks et al. 2003; Turk et al. 2005b).

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Despite the development of treatment guidelines, and treatment in accordance with these guidelines, many patients with severe asthma remain inadequately controlled despite intensive therapy (Bateman et al. 2004; Partridge et al. 2006). There is a significant unmet need for an effective treatment for patients with severe persistent allergic asthma that remains inadequately controlled despite high-dose ICS and an LABA. Omalizumab, an anti-IgE antibody, binds all forms of circulating IgE, whatever its allergen specificity, and prevents subsequent IgE-mediated responses. By reducing IgE, omalizumab treatment also leads to the downregulation of FcεRI receptors (MacGlashan et al. 1997; Holgate et al. 2005a). By downregulating FcεRI receptors on dendritic cells (Prussin et al. 2003), omalizumab may alter allergen presentation to T cells. In patients with allergic asthma, omalizumab inhibits both the early- and late-phase asthmatic response to allergen (Fahy et al. 1997), significantly reduces sputum eosinophils, submucosal eosinophils and submucosal IL-4, T cells and B cells (Djukanovif et al. 2004), significantly reduces circulating eosinophils and proinflammatory cytokines (Noga et al. 2003), induces eosinophil apoptosis, and reduces the number of lymphocytes producing proinflammatory cytokines (Noga et al. 2006). Omalizumab has demonstrated efficacy in patients with severe persistent allergic asthma. In the INNOVATE study (Humbert et al. 2005), which exclusively enrolled patients with severe persistent allergic asthma who remained inadequately controlled despite high-dose ICS and an LABA plus additional controller medication if required, omalizumab significantly reduced exacerbations and total emergency visit rates and significantly improved quality of life, lung function and asthma symptoms. These data are supported by results from the subgroup analysis of patients taking high-dose ICS and LABA in the ETOPA study (Niven et al. 2007) and by the results of pooled analyses (Bousquet et al. 2005a; Chipps et al. 2006). In addition, omalizumab is effective in patients with severe persistent allergic asthma irrespective of concomitant use of oral corticosteroids (Ayres et al. 2004b; Bleecker et al. 2005; Wenzel et al. 2007) or leukotriene modifiers (Massanari et al. 2005; Zeldin et al. 2006) and is efficacious in patients with coexisting allergic asthma and rhinitis (Vignola et al. 2004). Omalizumab has demonstrated a good safety and tolerability profile in clinical trials involving more than 7500 adults and adolescents with asthma, rhinitis and related conditions. The frequency and nature of adverse events were similar in the omalizumab and control treatment groups and there is no evidence of an increased risk of hypersensitivity reactions, malignant neoplasia, parasitic infections, or thrombocytopenia with omalizumab. The benefits of omalizumab are considerably greater in patients who are judged by physicians to have responded to therapy. In omalizumab-treated responders in the INNOVATE study, rates of clinically significant exacerbations were reduced by 60%, severe exacerbations by 76%, and emer-

Anti-IgE in Persistent Severe Allergic Asthma

gency visits by 76% (Bousquet et al. 2007) compared with 26%, 50% and 44% reductions, respectively, in the overall omalizumab-treated population (Humbert et al. 2005). A similar reduction in exacerbation rates compared with control (64%) was also reported in patients who responded to omalizumab in the ETOPA study (Niven et al. 2007). In the EU, omalizumab is indicated for patients with severe persistent allergic (pretreatment total IgE 30–700 IU/mL) asthma that remains inadequately controlled despite highdose ICS plus LABA. Analyses of patients treated in clinical trials have shown that it is difficult to predict which patients, within the label population, will derive greatest benefit from omalizumab based on pretreatment patient characteristics. Treatment should be initiated in eligible patients and the response evaluated by the physician after 16 weeks of therapy. Treatment should be continued in patients who are judged by the physician to have achieved a marked improvement or complete asthma control. Targeting these patients will minimize unwarranted drug exposure and maximize the efficacy and cost-effectiveness of omalizumab. In summary, targeting IgE with omalizumab represents a major advance in the treatment of severe persistent allergic asthma and has a potential role in the treatment of pediatric asthma and other IgE-mediated diseases.

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Occupational Asthma Paul Cullinan and Anthony J. Newman Taylor

Summary Around 350 workplace agents have been reported to cause occupational asthma and many more are implicated in the provocation of preexisting disease. Epidemiologic studies suggest that some 10% of all new or relapsed asthma in adulthood has a workplace etiology, and thus is amenable to primary prevention. Many industries continue to pose a high risk of occupational asthma for their workforces and in most economically developed nations the disease is the most common form of occupational respiratory disease. The primary determinant of risk is the level of atmospheric exposure in the workplace to the causative allergen(s) but innate susceptibility, perhaps related at least partly to genetic factors, appears to be important. Other factors such as cigarette smoking may also, variously, be significant. At an individual clinical level, the identification of an occupational origin provides an unusual opportunity to effect a cure for asthma; at the same time the misattribution of an occupational cause can lead to a great deal of unnecessary hardship. Thus an accurate diagnosis is essential. In most cases this can be achieved through a careful history, the appropriate use of immunologic testing, and the use of serial peak flow measurements. Less often, specific inhalation testing is required. Once a (confident) diagnosis of occupational asthma has been made, case management is usually centered around the avoidance of further exposure, although the effectiveness of this approach is variable.

Definitions Asthma may be related to exposures in the workplace in several ways, and some dozen relationships between asthma and occupation have been described (Harber 1992). A more parsimonious approach limits these to two broad categories (Table 82.1). First, asthma may be induced, or initiated de novo, by exposure to a respiratory sensitizing or irritant agent encountered at work. This may take place in a previously well individual or in one with preexisting asthma from another

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

cause. Alternatively, preexisting asthma (or bronchial hyperreactivity) may be provoked by occupational exposure(s) to “inciting” agents, which include irritant fumes or dusts, cold air and physical exercise. These two forms differ in their etiologies, mechanisms and severities but each carry similar and serious social and economic consequences for affected individuals (Cannon et al. 1995). It has been argued (Wagner & Wegman 1998) that preventive efforts should embrace both kinds of asthma but a more general application of this approach has been criticized on the grounds of unworkability (Malo & Chan-Yeung 1999). Thus the description “occupational asthma” is generally confined to disease that is induced or initiated by an exposure at work (Bernstein, I.L. et al. 1993; Newman Taylor 1994). In almost all recognized cases it arises as a consequence of an immune response to an airborne sensitizing agent encountered in the workplace, and has the clinical and immunologic characteristics of an acquired hypersensitivity response. These include a latent (sensitizing) period between first exposure and the onset of symptoms (usually weeks or months) and, once sensitization has occurred, the provocation of symptoms by very low exposures. Although immunologic mechanisms have been identified for many of the responsible agents, in most cases involving the production of specific IgE antibodies, the nature and even existence of an immunologic response to others, particularly low-molecular-mass chemicals, is still lacking. A small proportion of cases of occupational asthma arise following high, generally single exposures to respiratory irritants; the mechanism of this response is not primarily immunologic. Originally termed reactive airways dysfunction syndrome (RADS) (Brooks et al. 1985), it is often referred to as “irritantinduced” asthma. The question of whether more frequent, lower intensity exposures can similarly induce asthma remains open. Occasionally sensitization to occupationally encountered agents may occur vicariously, or indirectly, as in the response to agents carried on the clothing of directly exposed workers or, occasionally, to external emissions from factory sites (Carroll et al. 1976). It has even been suggested that wheeze in childhood might be related to maternal occupation (Magnusson et al. 2006).

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Table 82.1 Types of work-related asthma. Preexisting asthma

Agent type

Mechanism

Latent period

Examples of causative agents

Label

No (Yes) No Yes

“Inducer” (sensitizing) “Inducer” (irritant) “Inciter”

Type I hypersensitivity “Irritation” “Irritation”

Yes No No

Flour, enzyme, colophony fume Chlorine fume (high dose), SO2 Chlorine fume (low dose), cold air

Occupational asthma Occupational asthma Work-exacerbated asthma

Occupational respiratory sensitization is one of the few wellestablished etiologies for adult asthma, with a clear pattern of induction, clinical response and, frequently, improvement when exposure is avoided. For this reason it is potentially preventable, with clear public-health and legislative implications. It also appears to be an expensive disease. A report commissioned by the UK Health and Safety Executive estimates that the total lifetime costs of those cases of occupational asthma reported to a national surveillance scheme each year are £72–100 million; since recognized cases are believed to represent only one-third of the true incidence, the real costs could be as high as £133.5 million (HSE 2006). Most of these costs are borne by the state rather than the employer. Finally the academic study of identifiable groups of workers exposed to (often) high intensities of single and wellcharacterized respiratory sensitizers, which may be relatively easily measured, provides a potentially useful model for understanding the responses of human airways to other, nonoccupational agents.

Causes of occupational asthma Around 350 agents have been reported to cause occupational

asthma. The list grows annually but reasonably comprehensive information is available in textbooks (Malo & Chan-Yeung 2006) and on websites (http://www.asmanet.com; http:// www.asthme.csst.qc.ca; http://www.eaaci.net). Responsible agents are divided, somewhat arbitrarily, into those of high and low molecular mass; some commonly encountered agents, categorized by occupational group are displayed in Tables 82.2 and 82.3. This list is not intended to be exhaustive but provides a perspective on the wide variety of biological and chemical agents which may cause occupational asthma.

Epidemiology of occupational asthma Frequency From its beginnings in the second half of the 20th century, research into occupational asthma has moved rapidly from case reports identifying and characterizing sensitizing agents to the study of disease frequency and determinants among populations of exposed workers. More recently still, interest has developed in the frequency of work-related asthma in broad population-based samples. Broadly speaking, estimates of the frequency of occupational asthma are derived in three ways.

Table 82.2 Selected high-molecular-mass causes of occupational asthma by occupational group. Occupational group

Agent(s)

Bakers and millers

Flour (wheat, barley, rye, oat, soya), fungal a-amylase, egg proteins, milk proteins, storage mites

Laboratory technicians, research scientists, animal handlers

Small animal proteins (urine, dander, serum): rats, mice, guinea-pigs, ferrets, etc. Insect proteins: cockroach, locust, housefly, fruit fly, gypsy moth, mealworm, etc. Other animal proteins, latex

Farmers, farmworkers and agriculturists

Storage mites, mealworms, spider mite, poultry mite, cow dander, cow b-lactoglobulin, pig urine, mink urine, insect larvae, poultry feathers, honeybee dust, silkworm larvae, fruit, vegetable and flower pollens, fungi, grain dust

Food processors (nonbaking/milling)

Linseed, green coffee bean, castor bean, tea dust, tobacco leaf, rosehip, shellfish proteins, fish proteins, milk proteins, egg proteins, cocoa proteins, proteolytic enzymes

Nurses, dental workers, other healthcare workers

Latex

Detergent powder manufacturers

Detergent enzymes: protease, amylase, lipase, cellulose

Florists, botanists

Pollens, weeping fig, baby’s breath, spider mite, vine weevil

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Table 82.3 Selected low-molecular-mass causes of occupational asthma by occupational group. Occupational group

Agent(s)

Spray painters

Hexamethylene diisocyanate, toluene diisocyanate, dimethylethanolamine, other amines

Chemical processors

Azodicarbonamide, phthalic anhydride, trimellitic anhydride, maleic anhydride, hexavalent chromium

Plastics workers

Diphenylmethane diisocyanate, toluene diisocyanate, monomer acrylates, various amines

Food processors (nonbaking/milling)

Chloramine-T, metabisulfite

Nurses, dental workers, other healthcare workers

Glutaraldehyde, formaldehyde, monomer acrylates, antibiotics, psyllium, hexachlorophane, pancreatic extracts, N-acetylcysteine

Pharmaceutical workers, pharmacists

Psyllium, ispaghula, methyldopa, penicillins, cephalosporins, tetracycline, sulfathiazole, spiramycin, isoniazid, piperazine, cimetidine, dichloramine, ipecacuanha, bromelin, morphine and other opiates

Welders, solderers, electronic workers

Colophony fume, stainless steel welding fume, aminoethylethanolamine, cyanoacrylates, toluene diisocyanate, persulfate salts

Woodworkers

Hardwood dusts: western red cedar, iroko, African maple, mahogany, manosonia, obeche, etc.

Metal refiners

Complex platinum salts, hexavalant chromium, nickel, vanadium, fufuryl alcohol

Hairdressers

Persulfate salts, henna

Textile/fabric workers

Reactive dyes, gum acacia

Routine surveillance In several countries, notably Finland, the UK, France and parts of the USA and Australia, there are established surveillance schemes for occupational lung diseases including asthma. They are likely to become more widespread. In some instances these schemes are embedded in programs that cover all occupational diseases. In each case they measure disease recognized and reported by more or less specialized physicians, generally in occupational or respiratory medical practice. Where denominators are available, occupation-specific incidence rates may be estimated although these are often crude. Examples are provided in Table 82.4. Surveillance of this nature sacrifices sensitivity (completeness of disease capture) for practicality, rapidity and uniformity. There is also evidence of clear differences between schemes in different countries. While these presumably reflect, in part, differing industrial exposures, they are likely also to result from variations in diagnostic practice and national legal/ compensatory requirements. In any case, surveillance of this kind generally underestimates the true incidence of occupational asthma, perhaps by as much as twofold or threefold, and especially so when the workforce denominators are nonspecific (Draper et al. 2003). Nonetheless surveillance schemes provide valuable information on the approximate size and distribution of the problem at a national level. Alternative surveillance methods rely on the enumeration of compensation claims or mandatory notifications, by

industry, for occupational asthma. The completeness of these methods is dependent on a variety of external factors and is poorly understood. In general they provide importantly different estimates from the clinical schemes above.

Population-based studies Using a stochastic (probabilistic) approach to causation, a number of epidemiologic studies have attempted to estimate the proportion of adult asthma that can be “attributed” to occupational exposures. The reasoning is importantly different from the individual approach used in clinical practice and in the surveillance schemes described above. Epidemiologic approaches measure the frequency of asthma (usually self-reported) in a representative sample of the community under study and then relate any excess risks of asthma in separate occupational groups to the risk in a referent group, generally office workers. The sum of increased occupationspecific risks is used to generate an estimate of the total proportion of communal disease attributable to occupational exposures. The totality of such studies is usefully summarized by Blanc and Toren and by the American Thoracic Society (Blanc & Toren 1999; Balmes et al. 2003). Their systematic reviews include metaanalyses suggesting that between 10 and 15% of all incident or recurrent cases of adult asthma are caused by workplace exposures. Methods like these are useful in a public health context and perhaps also in the identification

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Table 82.4 Estimated annual incidence rates of occupational asthma in the UK (SWORD, Meredith 1993), France (ONAP, Ameille et al. 2003) and Finland (FIOH, Karjalainen et al. 2000) in selected occupational groups, as reported to national surveillance schemes. Occupational groups are defined differently in different countries. SWORD 1989–90

Occupational group Coach and spray painters Chemical processors Plastics workers Bakers Metal treatment workers Laboratory workers Welders and electronic assemblers Food processors (not bakers) Hairdressers Painters (not spray) Woodworkers Farmers Healthcare workers Veterinary surgeons Cleaners Teachers All groups

No. of cases

Annual incidence per million workers

65 54 47 50 43 50 78 35 16 28 51 23 22

658 364 337 334 267 188 175 108 81 66 54 28 16

15

1985

ONAP 1996–99

No. of cases

FIOH 1989–95* Annual incidence per million workers

111

326

410

No. of cases

Annual incidence per million workers

683

45 12 18 101

223 146 68 444

138

308

52 68 7 1

116 76 99 33

89

218

213

41

10

74

55

591 10 5 1 6

120 5 171 4 5

20

2178

24

2602

17

* Men only.

of increased risks of asthma among occupational groups not traditionally recognized in clinical practice (Kogevinas et al. 1999; Jaakkola et al. 2003). Population-based methods cannot easily distinguish occupational from work-exacerbated asthma. Furthermore, it is not always clear whether the apparently high rates of asthma in some occupations arise from a risk inherent to a job or whether people with asthma migrate to that job. Remarkably little is known about the processes that determine job selection by people with asthma (Radon et al. 2006).

Workplace studies Estimates of the prevalence, and more occasionally incidence, of occupational asthma within a workplace or industrial group are made using standard cross-sectional or cohort epidemiologic techniques or by analysis of records from routine health surveillance. The latter technique may underestimate the true frequency of disease if the methods of surveillance (generally questionnaires and spirometry) are insensitive. Estimates of asthma prevalence, and of exposure effects, from cross-sectional surveys may be distorted by survivor effects, including the movement of affected persons out of situations of high exposure. The sum effect of such movements probably leads to an underestimation of both disease

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frequency and the magnitude of any exposure–response relationship: those who accumulate exposure are those who survive to do so. This “healthy worker” effect has been demonstrated in other occupational respiratory diseases though less often in the study of occupational asthma (Cullinan et al. 1994a). On first principles, since occupational asthma is an acute response to an identifiable environmental agent and the latent period is generally short, workforce selection would be expected to be significant. Despite these reservations, and the further question of how far findings from one workplace can be generalized to others, estimates of disease frequency in a variety of occupational workforces provide much of our knowledge of the epidemiology of the disease.

Determinants of occupational asthma Among the potential determinants of occupational asthma, three factors have received particular attention. First, and probably most important, is the level of exposure in the workplace to the sensitizing agent. The other factors, atopic status and genotype, relate to individual susceptibility. A closer understanding of the roles and interactions of these in occupational asthma should lead to improvements in primary disease prevention and possibly also to an improved understanding of the etiology of asthma outside the workplace.

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Exposure Exposure may conveniently be partitioned into duration and intensity. It is not clear what combination(s) of these elements are important in the development of respiratory sensitization. Given the immunologic model believed to underlie most cases of hypersensitivity-induced occupational asthma, the intensity of exposure necessary for initial sensitization is probably higher than that required to provoke symptoms in a sensitized subject. Nor is it clear whether these two parameters of exposure have an equivalent force, i.e., whether for example a high-intensity exposure for a brief duration has an equivalent effect to a longer lower-level exposure. Studies of exposure–response relationships based on crosssectional occupational populations should be interpreted with caution: they are prone to an unquantifiable bias from survivor effects and, since disease incidence is difficult to measure, it may be difficult retrospectively to identify and quantify the causative exposure. Furthermore, very few studies of whatever design have attempted to measure levels of sensitizing agents directly in exposed workforces and fewer still have characterized the nature (in terms of particle size, lung deposition, etc.) of these exposures. Sandiford et al. (1994) used a cascade impactor to determine the size of airborne allergen particles in a British bakery and flour mill and estimated that in dusty areas up to 20% were of a diameter which would allow deposition in bronchial airways and alveoli. Larger particles may deposit in the nose, also a site of sensitization. With similar methods, Platts-Mills et al. (1986) demonstrated that 28% of rat urinary aeroallergen in animal laboratories was carried on particles of under 5 μm diameter. Direct relationships between symptoms consistent with occupational asthma and allergen exposure at work have been identified for several different agents. These include a number of high-molecular-mass agents including flour allergens (Musk et al. 1989; Cullinan et al. 1994b, 2001; Houba et al. 1998; Brisman et al. 2000; Heederik & Houba 2001) in bakery employees, enzymes (Weill et al. 1971; Juniper et al. 1977; Cathcart et al. 1997; Vanhanen et al. 1997; Cullinan et al. 2000) in detergent manufacturers, laboratory animal allergens (PlattsMills et al. 1987; Kruize et al. 1997; Cullinan et al. 1999) in pharmaceutical company and university research staff, shellfish proteins (McSharry et al. 1994; Ortega et al. 2001) in seafood processors, and green coffee and castor bean (Osterman et al. 1982). Similar studies have been limited to evidence of allergic sensitization alone (Houba et al. 1996; Vanhanen et al. 1997; Nieuwenhuijsen et al. 1999; Heederik et al. 1999a). There are fewer studies of workers exposed to low-molecularmass agents but positive exposure–response relationships have been described in those working with diisocyanates (Tarlo et al. 1997; Meredith et al. 2000; Petsonk et al. 2000), acid anhydrides (Liss et al. 1993; Grammer et al. 1994; Barker et al. 1995), colophony fume (Burge et al. 1981), pharmaceuticals (Coutts et al. 1984; Hagmar et al. 1984), complex platinum salts

Occupational Asthma

(Calverley et al. 1995), and western red cedar (Brooks et al. 1981). Only one published study has examined the relationship between exposure and response in irritant-induced asthma (Kern 1991); the frequency of RADS in a group of hospital workers with high exposure to a spill of acetic acid was greater (21%) than that in those with medium (3%) or low (0%) exposures, providing strong evidence for a causal relationship. Concurrent cigarette smoking has been shown to increase the risk of sensitization or of respiratory symptoms consistent with asthma in some workforces. These include platinum refinery workers (Venables et al. 1989a), laboratory animal workers (Venables et al. 1988a), snow-crab process workers (Cartier et al. 1984), and those occupationally exposed to tetrachlorophthalic anhydride (Venables et al. 1985) and ispaghula (Zetterstrom et al. 1981). It is not always clear that these effects are independently attributable to smoking.

Atopy Atopy, defined as an innate tendency to produce elevated levels of specific IgE antibody after exposure to a sensitizing agent, is identified by positive responses to skin-prick testing with ubiquitous inhalant allergens or by the measurement of serum specific IgE antibodies to the same allergens. In Europe these generally include a house-dust mite allergen, a pollen mixture, and cat or dog fur. Workers who are atopic have been shown to be at increased risk of developing occupational asthma from a number of workplace allergens; a systematic examination of this issue is provided by Nicholson et al. (2005). The most consistent evidence is for allergens that routinely induce a specific IgE response as is the case for most of those of high molecular mass. Thus atopy increases the risk of asthma in workers exposed to bakery allergens, laboratory and other animals, detergent enzymes, and some reactive dyes. Other studies have reported associations between atopy and specific occupational sensitization in workers exposed to various enzymes, green coffee and castor bean, bakery allergens, crab, prawn, and acid anhydrides. Evidence for a similar association in those working with low-molecular-mass allergens is either absent (western red cedar, glutaraldehyde, complex platinum salts) or inconsistent (diisocyanates). As above, the findings from crosssectional surveys need to be interpreted with care since selection and survival pressures, perhaps operating more strongly among atopic employees, may be responsible for reported associations, negative or positive. Care also needs to be taken when assessing the independent roles of atopy and cigarette smoking in the development of occupational sensitization, since there may be a degree of inverse confounding between them. Furthermore, neither smoking nor atopy alone is a sufficiently discriminatory risk marker for occupational asthma especially when used in a program of preemployment screening. Atopic status, for example, has been used to select employees for a variety of

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workforces, including that in the precious metal refining industry. Yet the majority of atopic subjects will develop neither sensitization nor symptoms and their exclusion, notwithstanding issues of equity, precludes access to employees of some 30–40% of the general population. Analysis of data from a study of occupational asthma in laboratory workers suggests that seven atopic employees would need to be excluded from employment to prevent one case (Cullinan et al. 1994a).

Genotype Not all those who work with an occupational allergen at suprathreshold exposures develop respiratory hypersensitivity, although in some instances the proportion of affected employees can be very high (Gibbons et al. 1965; Cullinan et al. 2000). Individual, innate susceptibility is probably, at least in part, a reflection of genotype. Two classes of gene have been studied in the context of occupational asthma: those that determine (and restrict) antigenspecific responsiveness, i.e., genes of the human leukocyte antigen (HLA) class II complex, and those that determine respiratory antioxidant responses. In each instance, the presence or absence of candidate genes has been compared between employees with (“cases”) or without (“controls”) occupational asthma. Some authors have attempted to control for relevant allergen exposure, generally through matching; this is far easier in workplace populations than it is in the general population. Studies such as these require large numbers of cases and most have probably been underpowered; on the other hand, occupational asthma (or sensitization) provides a specificity of phenotype that is difficult to achieve in other types of asthma. Associations between HLA genes and specific sensitization have been reported in populations working with trimellitic, but not phthalic, anhydride acid (Young et al. 1995), laboratory animal proteins (Sjostedt et al. 1996; Jeal et al. 2003), western red cedar (Horne et al. 2000), and complex platinum salts (Newman Taylor et al. 1999). The last of these suggested an interaction between genotype and allergen exposure whereby the effects of the former were more marked among those who had acquired specific sensitivity at relatively low intensities of exposure. A larger number of studies have been of employees exposed to diisocyanates but the findings have not been entirely consistent. Positive associations between HLA-DQB1*0503 (and negative associations with DQB1*0501) and asthma induced by toluene diisocyanate (TDI) have been reported in three studies (Bignon et al. 1994; Balboni et al. 1996; Mapp et al. 2000), but not a fourth (Rihs et al. 1997). Genes involved in respiratory tract antioxidation, i.e., those of the glutathione S-transferase and N-acetyltransferase groups, have been described in association with occupational asthma from a variety of diisocyanates (Piirila et al. 2001; Mapp et al. 2002; Wikman et al. 2002). In some studies gene–gene interactions have been apparent (Piirila et al. 2001; Wikman et al. 2002).

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Outcome of occupational asthma Once removed from further exposure to the causative allergen, most patients with occupational asthma improve, and many recover completely. However, this is not always the case and in some, asthmatic symptoms and airway hyperreactivity do not resolve. The frequency and determinants of recovery have been systematically reviewed (Rachiotis et al. 2007). A metaanalysis of 39 publications with a median length of follow-up of 31 months suggested that only one-third of patients recovered completely from their asthma; however, estimates ranged from 0 to 100%. Twenty eight publications detailing recovery in nonspecific bronchial hyperresponsiveness were identified; the overall prevalence of persistent hyperreactivity was 73%. The same review examined a number of potential determinants of recovery (Fig. 82.1). Complete symptomatic recovery was significantly lower with increasing age at diagnosis and in studies where patients were identified in specialist clinics (P = 0.053). Those with the longest durations of employment, or of symptomatic exposure, were least likely to recover, although the differences were not statistically significant. In 10 of 11 reports that examined the effect of the duration of symptomatic exposure, subjects who were asymptomatic at follow-up had shorter durations of symptoms than those who remained symptomatic; in half of these studies the association was statistically significant. No studies examined the independent effects of age and exposure duration. There were no clear differences in recovery between patients whose disease had been caused by agents of different molecular mass or between those who were or were not cigarette smokers. Estimates of recovery were higher in studies with complete follow-up (40%) rather than incomplete follow-up (27%). Studies of the outcome of occupational asthma need to be interpreted with care. Most are of patients seen and followed in specialist centers whose experience may not be representative of the wider picture; a potential source of bias in populations such as these is the probable tendency for patients with more severe occupational asthma to be referred to specialist clinics. Many study reports are of relatively poor quality with little information on the selection or follow up of participating patients. The issue of publication bias also needs to be considered. It is not known why or how disease persists after exposure avoidance. In a longitudinal study of 31 snow-crab workers with occupational asthma (Malo et al. 1988), any recovery in lung function and bronchial hyperreactivity appears to take place in the first 2 years after exposure ceases. Persistence of longer duration is associated with histologic evidence of airway inflammation, characterized by continuing eosinophil and lymphocyte infiltration, and may be related to fixed airway remodeling (Paggiaro et al. 1990; Saetta et al. 1992, 1995), which itself may reflect age and/or the duration of allergen exposure. Retention of allergen or hapten within the lung, or extrapulmonary tissue, has been suggested as a mechanism for

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6–31 Follow-up (months)

P = 0.506 (trend)

32–56 57–240

31–38 Age at diagnosis (years)

P = 0.019 (trend)

39–43 44–53

Duration of employment (months)

Duration of symptomatic exposure (months)

6–76 P = 0.764 (trend)

77–138 139–217

≤ 26

P = 0.120

> 26

Clinic

P = 0.053

Setting Workplace

LMW Agent

P = 0.694

HMW Various

Fig. 82.1 Systematic review (39 studies) of the determinants of full recovery from occupational asthma after avoidance of exposure. The x-axis indicates the proportion of subjects with complete remission of their asthma. (From Rachiotis et al. 2007, with permission.)

Europe Country

P = 0.274

USA P = 0.168

Canada

continued IgE production after the cessation of external exposure to acid anhydrides (Venables et al. 1987); in an animal model, IgE (and IgG) production has been demonstrated even long after allergen exposure has stopped (Holt et al. 1984). Unfortunately very little other work on the determinants of persistent disease is available and nor is it clear to what extent, if any, treatment affects this prognosis. Just one study has examined, in a controlled and randomized fashion, the effects of medical treatment. Small but statistically significant improvements in some symptoms, peak flow and quality of life were reported for treatment with inhaled corticosteroids after exposure avoidance (Malo et al. 1996). Other, not directly functional, outcomes are common in occupational asthma; these may be equally or even more important to the sufferer. Surveys of patients, from a limited number of countries, suggest that about one-third of them are unemployed after diagnosis, although it is not clear how far this figure departs from that among other adults with asthma (Axon et al. 1995; Cannon et al. 1995; Larbanois et al. 2002). The rate of unemployment appears to fall with increasing time after

0%

20%

40%

60%

80%

100%

diagnosis (Ross & McDonald 1998). The loss of employment following diagnosis is commonly associated with a loss of income, and in comparison with other adult asthmatics those with work-related disease find employment more difficult (Venables et al. 1989b; Cannon et al. 1995; Larbanois et al. 2002).

Mechanisms of occupational asthma Asthma induced by exposure to a sensitizing agent encountered at work This, the most common sort of occupational asthma, characteristically displays the features of an acquired hypersensitivity reaction. 1 Sensitization and asthma develop only in a proportion (usually a minority) of exposed subjects. Longitudinal studies of laboratory workers, for example, have demonstrated that positive skin tests to rat urine develop at a rate of approximately 2.5 new cases per 1000 person-months, the remaining employees escaping sensitization.

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2 Both symptoms and serologic evidence of sensitization develop only after an asymptomatic (latent) period of exposure; patients with occupational asthma will frequently describe such a period lasting several weeks or months, and occasionally years. The duration of this latent interval may depend on the agent, the characteristics of exposure, and individual biological variability. In many studies the risk of occupational asthma is highest in the first year or two of exposure and considerably lower thereafter (Slovak 1981; Agrup et al. 1986; Venables et al. 1989a; Calverley et al. 1995; Cullinan et al. 1999; Gautrin et al. 2001). 3 Once asthma has developed, it is provoked by low concentrations of the sensitizing agent, concentrations that do not provoke symptoms in nonsensitized workers and which the affected individual would previously have tolerated. Workers sensitized to TDI, for example, may develop asthma after exposure to concentrations of TDI as low as 0.001 ppm (Henschler et al. 1962). These are characteristics of an immunologic response and indeed a wide variety of occupational sensitizing agents are believed to act through an IgE-associated immune mechanism; these include most if not all of the high-molecular-mass as well as some low-molecular-mass agents. The former group, the great majority proteins of biological origin, are complete allergens and it is unsurprising that they are capable of stimulating a Th2 lymphocyte IgE-associated immune response. Specific IgE antibodies may be detected either in the serum of persons sensitized to these agents or by skin testing with water-soluble extracts of the responsible allergen. On the other hand, low-molecular-mass chemicals are “incomplete” allergens and where they induce an immune response it is believed that this is as haptens in combination with one or more human proteins. Specific IgE antibodies against a hapten–protein conjugate may be detected, for example with the acid anhydrides and in some cases the diisocyanates and reactive dyes and, using skin-prick testing, complex platinum salts. Studies that have failed repeatedly to demonstrate a correlation between disease and specific IgE production suggest either that the relevant allergen has yet to be identified or that an alternative mechanism is responsible for the clinical picture. For example, it has not been possible to demonstrate a consistent relationship between specific IgE levels and symptoms in one of the most important groups of occupational sensitizing agents, the diisocyanates. Specific IgE to a hapten– protein conjugate is detectable in only a minority of cases (Tee et al. 1998) and careful study of patients with a confirmed asthmatic response to controlled exposures failed to detect either specific IgE antibodies or upregulation of IgE receptors (Jones et al. 2006). In part this may reflect the highly reactive nature of these chemicals and the possibility that a variety of hapten–protein conjugates may be formed after inhalation through the respiratory tract. A similar lack of association currently exists for two other important causes of occupa-

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tional asthma, plicatic acid (Tse et al. 1982) and colophony (Malo & Bernstein 1992). Even when “specific” IgE antibodies are detected it is not clear that they have any clinical or pathologic specificity (Di Stefano et al. 2001). Consequently, alternative immunologic mechanisms have been investigated. It has been suggested for example that CD8+ cells are important in diisocyanate-induced asthma; most T-cell clones obtained at bronchial biopsy of patients with this disease show a CD8 phenotype and produce interferon (IFN)-γ and interleukin (IL)-5, with few clones producing IL-4 (Maestrelli et al. 1994). These T cells may represent a CD3+/CD4–/CD8+ population with expression of a γ/δ T-cell receptor specific for the diisocyanate (Wisnewski et al. 2003). Occupational asthma induced by low-molecular-mass agents is also associated with an increased number of cells producing proinflammatory cytokines (Bernstein et al. 2002). The relative difficulty of delineating an immunologic mechanism for several important causes of occupational asthma has stimulated the search for nonimmunologic pathways. Pharmacologic mechanisms, for example, have been implicated for plicatic acid, which has been shown to be capable of directly activating complement (Chan-Yeung et al. 1982), and for TDI which may have direct actions on bronchial smooth muscle, either as a bronchoconstrictor or as a β2-adrenergic blocking agent (Davies et al. 1977; McKay et al. 1981). Interest has also focused on the possibility that some occupational agents may act through a variety of neurogenic mechanisms. TDI again has been shown, in animal models, to stimulate neuropeptide release and to inhibit neutral endopeptidase (Mapp et al. 1990; Sheppard et al. 1990) and so to modulate neurogenic inflammation. Although such nonimmunologic mechanisms may have important secondary roles in the clinical picture of occupational asthma, it is difficult to understand how they account for the clinical pattern of hypersensitivityinduced asthma.

Irritant-induced asthma Asthma developing shortly after exposure to high concentrations of inhaled respiratory irritants is distinguished from hypersensitivity-induced asthma by the absence of an appreciable latent period, and is probably the outcome of nonimmunologic mechanisms. The small number of bronchial biopsies carried out in subjects with irritant-induced asthma (Brooks et al. 1985; Bernstein et al. 1989) have documented bronchial wall damage and inflammation with lymphocytes and plasma cells but not eosinophils. Submucosal inflammation and sub-basement membrane collagen deposition have also been described. It is important to note that histologic data prior to the initiating incident are unavailable in all cases reported to date. These findings suggest that the clinical picture may be a manifestation of epithelial cell injury and activation of nonadrenergic noncholinergic networks with consequent transmitter release and inflammatory response.

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Identifying occupational asthma The early identification of an occupational cause of asthma has several important consequences. First, in patients with new symptoms consequent on an occupational respiratory sensitization, it may explain the onset of new asthma in adult life and make a search for alternative causes unnecessary. Second, it allows a rational approach to nonpharmacologic treatment and even prevention. Studies of the effects of continuing exposure on the prognosis of occupational asthma are somewhat inconsistent in their findings but the available evidence accords well with clinical impressions that prolonged exposures (or a higher age at diagnosis) increase the risk of chronic disease. Accuracy in diagnosis is critically important. A diagnosis of occupational asthma usually leads to a change in occupation, and not infrequently to the loss of current employment or a major switch in careers. Misdiagnosis, i.e., the false attribution to an occupational cause, can lead to a lack of improvement in symptoms and to unnecessary socioeconomic hardship once exposure has ceased. Guidelines to the diagnosis and management of patients with occupational asthma, derived from systematic review of the published literature, are available both in print (Nicholson et al. 2005) and electronically (http://www.bohrf.org.uk/ downloads/asthevre.pdf). Other guidelines, developed by consensus, are also available (Pauli et al. 1986; Chan-Yeung 1988; Burge 1989; Canadian Thoracic Society 1989; Committee on Occupational Lung Disease 1989). An outline of available methods is shown in Fig. 82.2.

Occupational Asthma

Adult asthma: • Onset in working life • Relapse in working life • Deterioration in working life

Symptoms (severity) work-related? • Worse at/after work • Better away from work Yes Consider work-related asthma

Detailed clinical history • Timing of symptoms • Nature of symptoms • Previous asthma

Detailed occupational history • Agents • Tasks • Dates • Previous jobs • Occupational health service?

Immunology available? • Skin prick test • Serum specific IgE • Other

Asthma? Work-related? • Serial measurement of peak flow • NSBHR (serial)

Specific provocation testing

History

Fig. 82.2 Summary of diagnostic approaches to occupational asthma.

Patients seen in specialist clinics with hypersensitivity-induced occupational asthma frequently describe a characteristic history with the onset of asthmatic symptoms a short period (several weeks or months) after first exposure to a recognized respiratory sensitizing agent. Clearly a low threshold of suspicion and familiarity with common sensitizing agents, and the occupations in which they are encountered, is important for the clinician but patients may of course develop sensitivity to a rare or previously unrecognized agent. The relationship between the symptoms and current exposure may be clear, with cough and/or chest tightness occurring typically at or after periods of exposure at work and improving during weekends or holidays. The complications caused by shift patterns should be remembered as should symptoms which develop only after leaving work, during the evening or the following night, or both. Typically, with repeated exposure, patients with occupational asthma take longer to recover so that improvement may not be noticeable, for example over a 2-day weekend but only after a holiday of several days or more. Symptoms characteristic of nonspecific bronchial hyper-

reactivity may also occur, provoked by a variety of irritants encountered at and away from work, including cold air, tobacco smoke, exhaust fumes, and the vapors of paint or perfumes. Associated work-related upper respiratory symptoms of nasal obstruction, sneezing and discharge, and itching or running of eyes, as well as itching of the skin, may also develop. The extent and nature of any relationship between occupational asthma and other (nose, eye and skin) symptoms is disputed. Occupational rhinitis is up to three times more frequent than occupational asthma and the two conditions frequently occur together; 76–92% of patients with occupational asthma also have rhinitis (Siracusa et al. 2000). One population-based study reported that occupational rhinitis was strongly associated with asthma (Karjalainen et al. 2003), especially in farmers and wood workers and that the highest risk of asthma was in the first year after rhinitis was reported. Little evidence for such a temporal relationship was found in a longitudinal study of a similar workforce (Cullinan et al. 1994a). The intensity of

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nasal symptoms may be greater in the case of high-molecularmass agents (Malo et al. 1997). A history of preexisting or coexisting respiratory disease, though not precluding a diagnosis of occupationally induced asthma, should alert the clinician to the possibility of aggravation of preexisting asthma or other chronic obstructive airways disease by the considerable number of respiratory irritants encountered at work. The distinction is clinically important since there is no evidence that continuing exposure, at low levels, to such irritants in these patients is detrimental to the prognosis of their disease; frequently they may be managed by reducing, rather than eliminating, the relevant exposure(s). The history of a patient with irritant-induced asthma (RADS) is very different, partly through constraints imposed by the current disease definition. Symptoms develop within minutes or hours after a recognizable, high (and often dramatic) exposure to a respiratory irritant, frequently in an individual without previous respiratory disease. Unlike those with occupational sensitization, patients with irritant-induced asthma can usually identify precisely the time of onset of their disease, which may be many years after the onset of employment and of exposure, at lower levels, to the responsible agent.

Investigations The diagnosis of occupational asthma rests first on the identification of variable airflow obstruction, second on exposure to a sensitizing agent at work, and third on a causal relationship between the two. Relevant investigations are of two sorts: those aimed at establishing a relationship between airway function and specific occupational exposure, and those aimed at identifying evidence of a specific immunologic response to the putative agent. The presence of specific IgE antibodies may only reflect exposure rather than determine attribution but the absence of such in a category of occupational asthma where an IgE-associated mechanism is well established is strong evidence against the diagnosis.

Functional tests of work-related airflow limitation Patterns of airflow limitation attributable to workplace exposures are most easily investigated by serial, self-recorded measurements of peak flow; at present these are the most appropriate indices for self-measurement, although portable techniques for measuring others, for example forced expiratory volume in 1 s (FEV1), are increasingly available. Patients under investigation are supplied with a suitable peak-flow meter and invited to make repeated measurements over a period of time including periods at and away from work. In the UK, 2-hourly measurements during waking hours over 4 weeks are widely used and are both acceptable and reliable with adequate records being provided by most subjects. Less frequent measurements have been shown to have lower diagnostic sensitivity and may mask important variations (Blainey et al. 1986; Malo et al. 1993). Importantly, patients should be instructed to record repeatable measurements and

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are usually advised to record the best of three readings on each occasion, two at least to be within 20 L/min of each other. The potential confounding effects of antiasthmatic treatment should be appreciated, particularly when this is increased because of increased severity of asthma on days at work; confusion can be avoided if patients are asked to maintain the same treatment throughout the measurement period. In some cases, and especially those where the occupational asthma is of long standing, it can be helpful to include, in the period of measurement, a prolonged absence from work (such as a holiday of several days or more) in which case a series of longer than 4 weeks’ duration may be required. Examples of peak flow measurements analyzed in this way are shown in Fig. 82.3. The essence of this technique is examination of any correlation between peak flow and periods at work. The results of such measurements are best expressed as the maximum, minimum and mean readings from each day, plotted graphically and serially. Currently there are no agreed criteria to determine what constitutes a diagnostically positive series and the interpretation of such plots is usually made visually; agreement between experienced clinicians is high but there is significant variability among less-experienced observers. Statistical analyses of peak flow series, aimed at producing a more objective diagnostic test, have proved disappointing and little more valid or reliable than visual inspection (Perrin et al. 1992; Cote et al. 1993; Leroyer et al. 1998). There is a single report of a computed method of analysis, including 67 records with 32 cases of occupational asthma; a sensitivity of 75% and specificity of 94% were reported (Gannon et al. 1996). The assessment of serial peak-flow measurements has a very high diagnostic sensitivity and (particularly) specificity when compared with specific inhalation testing or expert opinion (Cote et al 1990; Liss & Tarlo 1991; Perrin et al. 1992; Malo et al. 1993; Leroyer et al. 1998; Bright et al. 2001). Difficulties arise in subjects with asthma in whom the variations between work and rest days are small; these may be overcome with a repeated series of measurements that includes a longer absence from exposure. Confusion and even misdiagnosis may also arise if shift patterns are not accounted for, particularly if these result in readings being taken at different times on work and rest days (Venables et al. 1989c). Further difficulty arises in using serial peak flow measurements to distinguish immunologically mediated occupational asthma from asthma exacerbated by workplace exposure to respiratory irritants. It is doubtful whether such a distinction can be made reliably, although plots which demonstrate increasing variability in peak flow only after several days of exposure (for example toward the end of a week) may be more indicative of immunologic-type asthma. Less time-consuming techniques are also widely used but have been shown to have lower specificity and sensitivity (i.e., more false positives and false negatives). These include the measurement of FEV1 before and after shifts, or the longitudinal measurement of FEV1 and forced vital capacity (FVC),

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Overall mean: 441 Ls/minute

Occupational Asthma

Predicted mean: 439 Ls/minute

Completeness: 100%

% variability: 2

4

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Fig. 82.3 Serial peak flow records of patients with (a) no asthma, (b) occupational asthma and (c) asthma unrelated to work. In each case the shaded columns represent days at work. On each day the average, maximum, and minimum peak flow measurements only are plotted. (See CD-ROM for color version.)

at intervals of several months or years, frequently used in surveillance programs. A high proportion of workers subsequently shown to have occupational asthma may have, at single readings, a normal FEV1 (Burge 1982a; Chan-Yeung et al. 1982), or at least one that is not different from similar, asymptomatic workers. Likewise, cross-shift measurements of FEV1 have failed, in colophony (Burge 1982b) and pharmaceutical (Bardy et al. 1987) workers, to distinguish between those with and those without occupational asthma. The comparison of nonspecific bronchial hyperreactivity either side of a work shift is sometimes used as a diagnostic tool but has limited sensitivity and specificity, with values around

65% and 55% respectively (Cote et al. 1990; Tarlo & Broder 1991; Perrin et al. 1992). It is important to note that bronchial responsiveness may be normal in patients with confirmed occupational asthma, especially if measurements are made outside periods of exposure to the causative allergen.

Specific inhalational testing The observed response to carefully controlled inhalation of a suspected sensitizing agent has generally been accepted as the gold standard of diagnostic tests for occupational asthma, although occasional difficulties with the procedure can mean it is not wholly sensitive. Tests such as these have been carried

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Overall mean: 498 Ls/minute

Predicted mean: 583 Ls/minute

Completeness: 99%

% variability: 23

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Fig. 82.3 (Cont’d )

out in the investigation of occupational asthma since 1970 (Pickering 1972), being based on tests originally used for other occupational lung diseases (Pepys & Jenkins 1965; Hargreave et al. 1966). Indications for specific inhalation testing vary between centers and many are conducted as a necessary step in the process of establishing appropriate compensation. In the UK, and in many other European countries, it is not necessary for this purpose and the indications for its use are more strictly clinical. In practice, inhalational testing is usually used when other, simpler diagnostic techniques have failed to establish the diagnosis with sufficient accuracy; frequently this is the case when exposure to the suspected agent has ceased (usu-

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ally at the behest of the patient’s employers) but an accurate diagnosis is still required to assist in decisions about continuing employment. In these cases serial measurement of lung function during periods of exposure is impossible. Alternatively, the patient may be exposed to more than one sensitizing agent at work and the identification of which (if any) is responsible for disease is considered important, or the suspected agent may be previously unrecognized as a respiratory sensitizer. Occasionally the symptoms provoked by exposure at work have been too severe or unpleasant to justify a return to the workplace and the serial measurement of peak flow, in which case judicious inhalational testing can be an appropriate alternative. Specific inhalation testing is potentially

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Overall mean: 308 Ls/minute

Occupational Asthma

Predicted mean: 452 Ls/minute

Completeness: 96%

% variability: 22

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Fig. 82.3 (Cont’d )

hazardous and should be undertaken only by experienced staff in specialist centers. For this reason alone the indications for testing should be carefully considered in each case, and should be used primarily to inform the patient about prognosis and future employment; testing solely in support of a legal claim is difficult to justify. Over the past 35 years variations in the technique of inhalation testing have been developed. The originally described methodology aimed to recreate as closely as possible exposures encountered by the patient at the workplace, and this remains a straightforward and appropriate technique that is still widely practiced (Fig. 82.4). Employers are asked to provide a sample of the suspected agent and, under close supervision in special-

ized laboratories, patients are exposed to it in an appropriate manner. Volatile agents, for example glutaraldehyde, may be painted onto a flat surface and dusts (flour, enzymes, pharmaceuticals) mixed with an inert excipient (usually lactose) and tipped between trays. Patients with suspected sensitivity to hexamethylene diisocyanate (HDI) (a hardening agent in spray paints) may be asked to spray an HDI-containing paint mixture, and those with possible colophony asthma to undertake some soldering with a resin-cored solder. More recently, some centers have developed more sophisticated methods of delivery, involving mechanized dust and fume generation, though the diagnostic advantages offered by these over the simpler techniques are unclear.

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Asthma and its Treatment stable baseline measurement. FEV1, recorded at 5-min intervals for up to 1 hour and hourly thereafter, is commonly used, although peak expiratory flow is a suitable, if less reliable, alternative. In addition, changes in nonspecific bronchial hyperreactivity may be measured using standard histamine or methacholine challenge testing. The details of the test protocol will vary between diagnostic centers; at the Royal Brompton Hospital in London, inert and active challenges at increasing concentrations are carried out on consecutive days with responses measured until the patient (an inpatient) goes to bed. In order to increase the specificity of the test, reproducibility is assessed by repeating active and control tests on one or more occasions (Fig. 82.5). Interpretation of the results requires an experienced clinician, although diagnostic rules (such as a 15% or greater fall in baseline FEV1) may be applied. Several patterns of response are taken to be indicative of sensitization. An “immediate” or “early” reaction is distinguished by a fall in baseline lung function whose onset and recovery occur within 1 or 2 hours of exposure; these are not necessarily associated with an increase in nonspecific bronchial hyperreactivity and may indicate either respiratory sensitization or reflex irritation. On the other hand, “late” reactions develop only several hours after exposure and may persist for 24 hours or longer. They are frequently accompanied by an increase in bronchial hyperreactivity from baseline, either at 3 hours (Durham et al. 1987) or 24 hours after challenge; this too may persist, sometimes for days after the exposure. Early and late (“dual”) reactions may occur in the same individual and “recurrent nocturnal” reactions may develop after a single test exposure (Newman Taylor et al. 1979). Occasionally, there are difficulties with a confident interpretation of a test result. False-positive results may be apparent in patients with unstable baseline asthma and it may be

Fig. 82.4 Woman undergoing specific provocation testing using a heatsealer provided by her bakery employers. (See CD-ROM for color version.)

Naturally, care must be taken over the doses used in these challenges; in general these should be lower than those experienced in the workplace and several orders lower than recommended occupational safety limits. Centers that carry out large numbers of these tests will have established protocols which determine the delivered concentrations; these may be easier to regulate with automated delivery systems (Cloutier et al. 1989). Tests should also be carried out using inert or control materials (Stenton et al. 1994) with the patient uninformed of the nature of each; ideally a double-blind method should be used, although this is often difficult to organize. Responses to the challenge are assessed by an index of lung function measured at regular intervals after challenge and compared with a

Control Amylase 1% 15

Amylase 0.1% 15 Amylase 2.5% 10

30 Pre-challenge 24 hrs post challenge

% Change in baseline

15 Control

> 16 > 16

Amylase 0.1% 15

> 16 13.5

0 –15 –30

Amylase 2.5% 10

–60 BL 0

4.8 0

–45

5 10 15 30 60 ~ (Mins)

2

3

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5

Time after challenge

6

7

8

9 10 11 12 (Hours)

Fig. 82.5 Specific inhalation challenge to bakers’ fungal amylase: positive result. (See CD-ROM for color version.)

1700

13.5 11.7 > 16

Amylase 1% 15

5 10 15 Histamine PC20 mg/mL

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difficult to distinguish an irritant from a hypersensitivity response. Negative results, in sensitized individuals, may occur when patients have been away from workplace exposures for prolonged periods (Carroll et al. 1976; Cartier et al. 1984), particularly if the inhalation testing has been carried out an insufficient number of times. Negative results may also be obtained if an inappropriate form of the test material is used, or indeed the wrong material altogether.

Immunologic tests The identification of specific IgE antibodies in a patient suspected of having occupational asthma provides useful evidence in support of the diagnosis, although as discussed above it is neither necessary nor sufficient. The presence of antibodies may only confirm that exposure has taken place and that immunologic “sensitization” has occurred. In practice very high antibody levels accompanying a characteristic history are often taken to be sufficient evidence for a diagnosis of occupational asthma. The situations where standard immunologic evidence is either unavailable or unhelpful in the diagnosis have been discussed above (see Mechanisms of occupational asthma) and are listed in Table 82.3. Measurement of specific IgE antibodies may be made using skin-prick tests with water-based extracts of soluble antigens or by standard techniques (commonly the ImmunoCap, Phadia, Sweden (CAP) test in serum samples. In some cases (e.g., laboratory rat- and latex-induced asthmas) the major allergens in the responsible proteins have been identified, although purified antigens are rarely used in practice. For a few low-molecular-mass chemicals, specific IgE bound to an albumen conjugate may be assayed; this is the case for occupational asthma attributed to acid anhydrides (Maccia et al. 1976; Zeiss et al. 1977; Howe et al. 1983) and to reactive dyes (Luczynska & Topping 1986).

Examples of occupational asthma Diisocyanates Diisocyanates are bifunctional molecules that polymerize polyhydroxyl and polyglycol compounds, a reaction exploited commercially to produce polyurethanes. Diisocyanates also react exothermically with water to produce carbon dioxide and are so used in the manufacture of polyurethane foam. TDI and HDI, both with high vapor pressures, evaporate under the exothermic conditions of these urethane reactions and may be inhaled as a vapor. Low vapor pressure compounds, including methylene diphenyldiisocyanate (MDI) and naphthalene diisocyanate (NDI), evaporate and may be inhaled after heating. The industrial use of diisocyanates is widespread and exposure may occur in a variety of settings, including the manufacture of flexible and rigid polyurethane foams (e.g., car head rests), the application of two-part polyurethane paints (aircraft and car spray painting), and in packaging where

Fig. 82.6 Spray painting of industrial components. The coatings often contain a diisocyanate or epoxy resin, both recognized as causes of occupational asthma. (See CD-ROM for color version.)

diisocyanates may be used in inks and laminating adhesives. The number of workers currently exposed to diisocyanates is unknown but may be increasing; it is likely that many are employed in situations where control and surveillance techniques are rudimentary. Each of the commonly encountered diisocyanates has been demonstrated to cause occupational asthma. In Europe, HDI used in spray paints probably accounts for the single largest category of diisocyanate-induced occupational asthma (Meredith et al. 1991) (Fig. 82.6). In the past TDI and MDI were more widely used and probably of greater importance than is currently the case. Inhaled diisocyanates may also cause other respiratory reactions. 1 Inhalation of high “toxic” concentrations of TDI may result in respiratory mucosal irritation (Henschler et al. 1962) and, after a single inhalation, has been reported to cause an “irritantinduced” asthma (Luo et al. 1990). 2 Extrinsic allergic alveolitis has been reported in workers exposed to MDI (Zeiss et al. 1980; Malo & Zeiss 1982) and HDI (Malo et al. 1983). 3 Airflow limitation, manifest as an accelerated decline in FEV1, has been demonstrated in nonsmokers working with TDI (Diem et al. 1982).

Laboratory animals Laboratory animal allergy is an important cause of occupational asthma in countries with a large scientific or pharmaceutical workforce; Draper et al. (2003) estimated that around 15 000 workers in the UK are regularly exposed to laboratory animals, especially rats and mice. The allergens responsible for laboratory animal allergy have been extensively studied and include proteins found in the urine (Newman Taylor et al. 1977), pelt (Walls & Longbottom 1983), hair (Longbottom & Price 1987), and serum (Wahn et al. 1980) of a variety of species. Characterization of the allergens present in rat excreta, by analysis of serum from sensitized subjects, has identified

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17 kDa (α2-microglobulin), 21 kDa (prealbumin), and 23 kDa proteins as major allergens. These are all present in urine from rats of both sexes and of all ages, but primarily in postpubertal males (Gordon et al. 1993), and to a less predictable extent in their epithelium (hair) and in the dust from their cages. The prevalence of work-related respiratory symptoms has been estimated in several cross-sectional studies of exposed workforces (Cockcroft et al. 1981; Slovak & Hill 1981; Beeson et al. 1983; Agrup et al. 1986; Venables et al. 1988b). A small number of longitudinal studies have also been reported (Botham et al. 1987; Renstrom et al. 1994; Cullinan et al. 1999). The incidence of sensitization in the first year of employment is about 15%, and of asthma around 2% (Davies et al. 1983); rates are higher among those employees who are atopic (Botham et al. 1995; Cullinan et al. 1999). The relationships between work-related chest or skin symptoms, and of skin sensitization to an extract of rat urine, and the intensity of exposure to rat urinary aeroallergen exposure may be modified by atopy and cigarette smoking (Cullinan et al. 1994a).

Baker’s asthma Occupational asthma among bakers has a venerable history, with an early description in 1713 (Ramazzini 1713). In several European countries it is the most common cause of highmolecular-mass occupational asthma reported in national surveillance schemes (Meredith & Nordman 1996; Baur et al. 1998; Kopferschmitt-Kubler et al. 2002). A variety of agents encountered by those involved in bread production have been demonstrated to be capable of causing respiratory sensitization and asthma; these include water-soluble cereal flour proteins, α-amylase, and various flour-associated mites and molds (Fig. 82.7).

Wheat, rye, barley, oat and other flours derived from Triticaceae species are all implicated and immunochemical studies suggest an important degree of cross-reactivity among these (Hoffman 1975) and among other grass or cereal proteins (Blands et al. 1976). Studies of the IgE-binding properties of various wheat-derived proteins have suggested that watersoluble albumins and globulins are the most allergenic fractions of wheat flour (Baldo & Wrigley 1978) and specification of these has led to the identification of a variety of low and medium molecular mass allergenic proteins (Gomez et al. 1990; Pfeil et al. 1990; Franken et al. 1991; Sanchez-Monge et al. 1992; Armentia et al. 1993; Sandiford et al. 1995). Sensitization to α-amylase, derived from the industrial culture of Aspergillus oryzae and added to dough mixtures to improve their rising, has also been shown, by immunologic measurement and specific challenge testing, to be an important cause of occupational asthma in bakers (Baur et al. 1986; Brant et al. 2005a). The frequency of sensitization among baking populations varies widely and may reflect different levels or types of exposure to the relevant allergens in different industrial settings. The prevalence of work-related respiratory symptoms and of specific sensitization is higher in workers who are exposed to high concentrations of total dust, much of it measured or assumed to be flour dust (Musk et al. 1989; Cullinan et al. 1994b, 2001; Houba et al. 1998; Brisman et al. 2000; Heederik & Houba 2001). Similar relationships have been shown with exposure to α-amylase (Nieuwenhuijsen et al. 1999; Brisman et al. 2004). Respiratory symptoms are commonly reported at survey by bakery workers; in a proportion they are not accompanied by evidence of specific sensitization to flour or amylase. This suggests that such symptoms may be mediated by nonimmunologic mechanisms.

Latex

Fig. 82.7 Working in an industrial bakery: a common cause of occupational asthma. (See CD-ROM for color version.)

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Natural rubber latex (cis-1,4-polyisoprene) is processed from the milky sap (latex) of the rubber tree Hevea brasiliensis; a variety of additives and thermochemical processes renders the latex elastic and heat-stable. The uses of natural rubber latex are widespread and include the production of adhesives and foam or carpet backings in addition to the very number large number of rubber goods. “Dipped” products such as medical gloves, condoms and balloons can contain high concentrations of natural rubber latex. Immediate-type hypersensitivity to latex was first described by Stern (1927) but an apparent epidemic of the disease was reported in the 1980s and 1990s. Rates were particularly high among healthcare workers, attributable it is believed to the huge increase in the use of latex gloves for control o f cross-infection (Bousquet et al. 2006). A large number of allergens have been identified in natural rubber latex (Jaeger et al. 1992; Slater & Chhabra 1992; Alenius et al. 1994); exposure may be through inhalation of airborne particles or through direct mucosal contact, as in those with indwelling surgical

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rubber devices such as urinary catheters, and possibly through the skin. Atopy is a major risk factor for latex sensitization in an exposed individual; a high rate of glove-changing has also been reported to be associated with increased risk. Persons with sensitivity to natural rubber latex are at high risk of immediate-type allergic reactions during invasive surgery; these may include life-threatening anaphylaxis (Gerber et al. 1989; Leynadier et al. 1989). Sensitivity to a wide variety of cross-reacting plant proteins has also been described and exposure to several foods including avocados and bananas, and to other plants such as the weeping fig (Ficus benjamina), may provoke symptoms in latex-sensitive subjects. Healthcare workers may be exposed to a variety of other potential respiratory sensitizers and a great number of irritants (Kern 1991; see Table 82.1) and the prevalence of occupational asthma or sensitization in the very large population of exposed healthcare workers is high (Ross et al. 1998).

Colophony Colophony is pine wood resin whose major industrial use is in a solder flux in the assembly of electronic goods. At soldering temperatures, a fume of resin acids and aldehydes (primarily abietic and pumaric acids) is generated. Asthma initiated by inhalation of this fume was first described in 1976 (Fawcett et al. 1976) but to date no convincing evidence for a specific immunologic mechanism for this reaction has been identified. Other occupational uses of colophony have been reported to cause asthma; these include its use in coolant oil (Hendy et al. 1985), bitumen mixtures (Burge et al. 1986), and in poultry processing (So et al. 1981). A cross-sectional survey of 446 employees in an electronics factory demonstrated that workers in shop-floor areas were more likely (22%) to report work-related respiratory symptoms than were those working in other parts of the factory (16%) and that sensitization had occurred in employees with only indirect exposure to colophony fume (Burge et al. 1979a). Further examination of the workforce from the same factory suggested that a high proportion of those leaving employment in the 3.5 years prior to the survey also had work-related respiratory symptoms (Perks et al. 1979). In a study of a colophony-flux manufacturing plant where atmospheric concentrations of colophony fume were available, Burge et al. (1981) found an association between the current intensity of exposure and the reported prevalence of workrelated respiratory symptoms; 21% of employees in the highintensity group reported symptoms, compared with 4% of those in low-intensity occupations.

Complex platinum salts The complex halide salt of platinum, ammonium hexachloroplatinate, is strongly allergenic and has been recognized to cause sensitization in humans since 1945 (Hunter et al. 1945) and possibly since 1911 (Karasek & Karasek 1911). Exposure to the powdered salt is primarily in the precious-metal refin-

Occupational Asthma

ing industry, although chemical and pharmaceutical workers may also be exposed. Platinum oxide, used extensively and increasingly in the manufacture of automobile catalytic converters, is not considered to be a sensitizing agent. Probably only a few thousand workers worldwide are exposed to sensitizing platinum salts. The pathogenesis of platinum-salt asthma is believed to be an IgE-associated hypersensitivity and specific IgE antibodies to ammonium hexachloroplatinate–protein conjugates can be demonstrated by skin-prick testing. The diagnosis of occupational asthma may be readily confirmed by inhalation challenge testing (Pepys et al. 1972). A retrospective longitudinal study of a cohort of 91 workers in a UK refinery revealed a high period prevalence (24%) of work-related respiratory disease and specific sensitization (Venables et al. 1989a); similarly high rates have been reported in cross-sectional surveys of refinery workers in other countries (Biagini et al. 1985; Baker et al. 1990). The risk of sensitization is greatest in the first year of employment and is increased among workers who smoke (Venables et al. 1989a). Platinum refinery workers are also potentially exposed to high levels of other respiratory irritants, including chlorine and sulfur dioxide fumes, which may similarly modify the immunologic response to platinum salts.

Enzymes Biological enzymes have been widely used in the detergent industry and in food processing for many years. Recent advances in recombinant DNA technology have allowed the largescale production of enzymes; α-amylase, for example, used in the baking industry is harvested from the commercial culture of adapted Aspergillus oryzae. Although sensitivity to detergent enzymes was reported in a small number of consumers (Belin et al. 1970; Zetterstrom & Wide 1974) before the advent of enzyme granulation, the highest risks of sensitization are experienced by those employed in the manufacture of enzymes and detergent products. Specific IgE antibodies to enzymes may be demonstrated by skin-prick testing and by in vitro serum assay. Occupational respiratory sensitization among detergent manufacturers was first described in 1969 (Flindt 1969) and subsequently shown to be a common problem in a series of cross-sectional surveys of exposed populations undertaken in the UK (Greenberg et al. 1970; Newhouse et al. 1970; Cullinan et al. 2000), USA (Weill et al. 1971), and Australia (Mitchell & Gandevia 1971). Occupational exposures to other enzymes has also been found to induce asthma. Baur and Fruhmann (1979a) reported asthma and rhinitis, accompanied by specific IgE antibodies, in 7 of 11 kitchen workers using papain as a meat tenderizer. Bromelin (an enzyme extract of pineapple) induced respiratory disease in pharmaceutical workers (Baur & Fruhmann 1979b) and blood-laboratory workers (Gailhofer et al. 1988). It is probable that any airborne and respirable enzyme, or

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indeed protein of any kind, is capable of inducing occupational asthma.

Management of occupational asthma The importance of precision in diagnosis has been emphasized earlier; the social and economic consequences of occupational asthma can be profound and incorrect attribution of asthma to a workplace exposure can lead to unnecessary changes in, and even loss of, employment and livelihood. In the established case the aim is the control of symptoms and prevention of the development of chronic asthma. In general this requires complete avoidance of exposure to the causative allergen; there is, as above, reasonably consistent evidence that the outcome of occupational asthma is worse in those with the longest periods of exposure before diagnosis. Standard medical treatments for asthma are generally ineffective in the face of continuing allergen exposure. Avoidance may be possible through a change of work-practice or a move within the same company or industry to a nonexposed area; unhappily it frequently requires a more major change in job or even occupation. Once a diagnosis is established, decisions about future employment are often made by the employers concerned. In some cases, particularly those who are self-employed or who have invested significantly in a current occupation (e.g., research scientists), there is considerable resistance to change (Cannon et al. 1995). While further allergen exposure cannot be advised, support for those determined to continue can be provided by monitoring of their asthma and the prevention, where possible, of exposure at levels high enough to incite symptoms or changes in lung function. Repeated serial measurements of peak flow during periods both at work and away are helpful in these situations, as is good pharmacologic control of symptoms while suitable alternative employment is found. Enhanced respiratory protective equipment is often considered where a change of job is not feasible. It is only effective if worn appropriately and if the correct procedures are followed for removal, storage and maintenance. A few studies have examined its effectiveness in small numbers of workers or under artificial conditions. These suggest that air-fed helmet respirators can improve or abolish symptoms in some but not all employees who continue to work where there is a potential for allergen exposure (Slovak et al. 1985; Pisati et al. 1993; Laoprasert et al. 1998; Muller-Wening & Neuhauss 1998; Taivainen et al. 1998; Obase et al. 2000). Symptomatic treatment is as for nonoccupational asthma; little or no treatment may be required once allergen exposure has ceased. In a hospital-based case series, lung function and nonspecific bronchial hyperreactivity continued to improve for up to 2 years after the last exposure; there was little improvement after this period (Malo et al. 1988). Although

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information such as this is useful in providing a general prognosis to patients, it should be cautiously extrapolated to all types of occupational asthma and to all levels of exposure prior to diagnosis. There is only limited evidence that treatment with antiinflammatory agents (e.g., inhaled steroids) improves the prognosis in occupational asthma, whether allergen exposure continues or otherwise.

Prevention of occupational asthma Primary prevention of occupational asthma aims to reduce disease incidence by the elimination of exposure to sensitizing agents at work, or to reduce it to levels at which sensitization does not occur; exposure to other determinants of occupational asthma may also be eliminated or reduced. There is a sufficiently large body of knowledge relating the risks of occupational asthma directly to workplace exposure intensity to suppose that controls in exposure are an effective way to reduce disease incidence (Nicholson et al. 2005). However, the detailed nature of any determined exposure–response relationship has not been examined and it is not known for example whether the relationship is a linear one or whether a threshold of zero incidence at low exposures exists. Nonetheless, a practical approach to the prevention of occupational asthma would be to maintain exposures (both average and occasional “peak” exposures) at as low an intensity as possible, and below that which incites symptoms in sensitized subjects; this is the basis for much current legislation. Useful summaries of this and other preventative approaches are available elsewhere (Venables 1994; Heederik et al. 1999b; Cullinan et al. 2003). A variety of hygienic techniques exists for the primary prevention of allergenic exposures at work (Corn 1983), including elimination or substitution of the responsible agent and, if these are not feasible, various methods for reducing exposures such as isolating or ventilating hazardous processes. Where these are not possible then personal protective devices may be a suitable alternative, although their use is frequently impractical. There are several demonstrations of the effectiveness of primary preventive strategies in occupational asthma. These include disease caused by acid anhydrides (Liss et al. 1993; Drexler et al. 1999), latex (Levy et al. 1999; Tarlo et al. 2001; Allmers et al. 2002), laboratory animals (Botham et al. 1987; Fisher et al. 1998), diisocyanates (Venables et al. 1985; Tarlo et al. 2002), and detergent enzymes (Juniper et al. 1977; Cathcart et al. 1997). In most cases these involved improvements in exposure control alongside other procedures such as targeted preemployment screening, enhanced surveillance, and the increased use of personal protective equipment. With complex preventive programs it is generally difficult to separate the effects of the component strategies. Moreover, most studies have measured only newly identified cases with no

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reference to the denominator at-risk population. Thus it is not always clear that true disease incidence has fallen following the introduction of a program of prevention. Attention to other, nonexposure, determinants of occupational asthma has focused on restrictions on employment of perceived “high-risk” persons. In some industries, notably the detergent manufacturing and precious metal refining industries, applicants at increased risk of allergy may be denied employment; in practice this is determined by assessment of allergic status using skin-prick testing to common inhalant or occupational allergens, and personal history of previous allergic disease. As a screening technique the discrimination of such an approach is poor. Increasing interest in the genetic determinants of occupational asthma may result in the extension of such techniques. At present, given the uncertainty about the relative importance of various risk factors and concerns over inequitable discrimination, the use of screening procedures such as these would seem premature and may lead to an undesirable reduced emphasis on the management of sensitizing exposures. Other potential risk factors, particularly those relating to concomitant exposures to respiratory irritants, have been less well explored. Workplace programs to discourage cigarette smoking, shown to have an important modifying role in several types of occupational sensitization, may have wide-reaching preventive implications. Secondary and tertiary prevention aims at early detection of a premorbid state of disease in order to minimize progression and so optimize outcome. In occupational asthma this generally entails medical surveillance of at-risk workforces and the early recognition of sensitization or of work-related symptoms, and the assumption that early detection and effective intervention will improve prognosis. Observational data suggest that continued symptomatic exposure to the causative allergen results in chronic asthma but, as discussed above, the evidence for this supposition is limited. Nonetheless, there exists a wide body of legislation in many countries requiring the periodic examination of workers exposed to potentially sensitizing agents. The most widely used methods for surveillance are probably the questionnaire and the regular assessment of lung function. The screening characteristics of these tools (in particular their predictive values, sensitivity and specificity) have been incompletely evaluated, but it is probable that self-completed questionnaires result in underreporting of respiratory symptoms (Brant et al. 2005b). Routine measurement of basic lung function is probably insensitive also since most employees with occupational asthma will have normal spirometry, especially if it is assessed when they are not directly exposed to the causative allergen. In any case spirometry detects very few cases that would not otherwise have been recognized (Bernstein, D.I. et al. 1993) and produces a high rate of false-positive cases (Kraw & Tarlo 1999). Immunologic surveillance is routinely used in some industries, either throughout the workforce as in some platinum refineries and detergent powder factories or on a targeted

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basis in employees who report respiratory symptoms (Brant et al. 2005b). It is of course not necessarily specific or predictive for occupational asthma but in carefully selected instances can be a useful adjunct to other methods; it is likely to be increasingly used in the future.

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Franken, J., Stephan, U., Neuber, K., Bujanowski-Weber, J., Ulmer, W.T. & Konig, W. (1991) Characterisation of allergenic components of rye and wheat flour (Secale, Triticum vulgaris) by Western blot with sera of bakers: their effects on CD23 expression. Int Arch Allergy Appl Immunol 96, 76–83. Gailhofer, G., Wilders-Truschnig, M., Smolle, J. & Ludvan, M. (1988) Asthma caused by bromelain: an occupational allergy. Clin Allergy 18, 445–50. Gannon, P.F.G, Newton, D.T., Belcher, J. et al. (1996) Development of OASYS-2: a system for the analysis of serial measurement of peak expiratory flow in workers with suspected occupational asthma. Thorax 51, 484–9. Gautrin, D., Infante-Rivard, C., Ghezzo, H. et al. (2001) Incidence and host determinants of probable occupational asthma in apprentices exposed to laboratory animals. Am J Respir Crit Care Med 163, 899–904. Gerber, A.C., Jorg, W., Zbinden, S., Seger, R.A. & Dangel, P.H. (1989) Severe intraoperative anaphylaxis to surgical gloves: latex allergy, an unfamiliar condition. Anesthesiology 71, 800–2. Gibbons, H.L., Dille, J.R. & Cowley, R.G. (1965) Inhalant allergy to the screwworm fly. Arch Environ Health 10, 424–30. Gomez, L., Martin, E., Hernandez, D. et al. (1990) Members of the α-amylase inhibitors family from wheat endosperm are major allergens associated with baker’s asthma. FEBS Lett 261, 85–8. Gordon, S., Tee, R.D. & Newman Taylor, A.J. (1993) Analysis of rat urine proteins and allergens by sodium dodecyl-polyacrylamide gel electrophoresis and immunoblotting. J Allergy Clin Immunol 92, 298–305. Grammer, L.C., Shaughnessy, M.A., Lowenthal, M. & Yarnold, P.R. (1994) Risk factors for immunologically mediated respiratory disease from hexahydrophthalic anhydride. J Occup Med 36, 642– 6. Greenberg, M., Milne, J.F. & Watt, J. (1970) Survey of workers exposed to dust containing derivatives of Bacillus subtilis. BMJ 2, 629–33. Hagmar, L., Bellander, T., Ranstam, J. & Skerfving, S. (1984) Piperazine-induced airway symptoms: exposure–response relationships and selection in an occupational setting. Am J Ind Med 6, 347–57. Harber, P. (1992) Assessing occupational disability from asthma. J Occup Med 34, 120–8. Hargreave, F.E., Pepys, J., Longbottom, J.L. & Wraight, D.G. (1966) Bird breeder’s (fancier’s) lung. Lancet i, 445–9. Heederik, D. & Houba, R. (2001) An exploratory quantitative risk assessment for high molecular weight sensitizers: wheat flour. Ann Occup Hyg 45, 175–85. Heederik, D., Venables, K.M., Malmberg, P. et al. (1999a) Exposure– response relationships for work-related sensitization in workers exposed to rat urinary allergens: results from a pooled study. J Allergy Clin Immunol 103, 678–84. Heederik, D., Doekes, G. & Nieuwenhuijsen, M.J. (1999b) Exposure assessment of high molecular weight sensitisers: contribution to occupational epidemiology and disease prevention. Occup Environ Med 56, 735–41. Hendy, M.S., Beattie, B.E. & Burge, P.S. (1985) Occupational asthma due to an emulsified oil mist. Br J Ind Med 42, 51–4. Henschler, D., Assman, W. & Meyer, K.O. (1962) The toxicology of toluene diisocyanate. Arch Toxicol 19, 364–87. Hoffman, D.R. (1975) The specificities of human IgE antibodies combining with cereal grains. Immunochemistry 12, 535–8.

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to inahles acid anhydrides. Am J Respir Crit Care Med 151, 219– 21. Zeiss, C.R., Patterson, R., Pruzansky, J.J., Miller, M.M., Resonberg, M. & Lebitz, D. (1977) Trimellitic anhydride induced airways syndrome: clinical and immunological studies. J Allergy Clin Immunol 6, 96–103. Zeiss, C.R., Kanellaks, T.M., Bellone, T.D., Levitz, D., Pruzansky, J.J. & Patterson, R. (1980) Immunoglobulin E-mediated asthma and hypersensitivity pneumonitis with precipitating antibodies due to diphenyl methane diisocyanate (HDI) exposure. J Allergy Clin Immunol 65, 347–52. Zetterstrom, O. & Wide, L. (1974) IgE antibodies and skin test reactions to a detergent enzyme in Swedish consumers. Clin Allergy 4, 273–80. Zetterstrom, O., Osterman, K., McHardo, L. & Johansson, S.G. (1981) Another smoking hazard: raised serum IgE concentration and risk of occupational allergy. BMJ 283, 1215–17.

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New Drugs for the Treatment of Allergy and Asthma Trevor T. Hansel, Ed Erin, Onn Min Kon and Peter J. Barnes

Summary There are considerable ongoing efforts from academic scientists and the pharmaceutical industry to develop new therapies for the treatment of allergy and asthma (Barnes 2004; Barnes & Hansel 2004; Bochner & Busse 2005; Buhl & Farmer 2005; Ballow 2006; Barnes 2006a) (see Fig. 83.1). Since the early 1970s asthma therapy has been dominated by use of inhaled synthetic β2-adrenoceptor agonists and corticosteroids, and the Global Initiative on Asthma (GINA) guidelines reflect the importance of using a combination long-acting β2 agonist (LABAs) and inhaled corticosteroid (ICS) in a single inhaler (National Institutes of Health 2005). Novartis and TheravanceGSK are involved in the development of once-daily “ultra” LABAs (Cazzola et al. 2005; Beeh et al. 2007). Novel recently licensed ICS include mometasone (Bousquet et al. 2000) and ciclesonide (Pearlman et al. 2005), while GSK 685 698 (Allermist) is a once-daily corticosteroid for allergic and nonallergic rhinitis submitted for marketing approval in July 2006. A humanized anti-IgE monoclonal antibody (Xolair, Novartis-Genentech) is the first biotechnology product to be licensed for the treatment of severe persistent allergic asthma that is not responding to high-dose inhaled ICS and LABAs (Holgate et al. 2005). There are a variety of novel IgEdirected approaches (Poole et al. 2005a), including anti-CD23 (low-affinity IgE receptor, FcεRII) (Rosenwasser et al. 2003; Poole et al. 2005b). In the field of allergen immunotherapy, sublingual grass pollen immunotherapy has recently been licensed (Grazax, ALK-Abello) and peptide immunotherapy is also being assessed (Dahl et al. 2006; Durham et al. 2006). Thymic stromal lymphopoietin (TSLP) has attracted considerable attention as a target for allergy since it is produced by keratinocytes and may represent an important switch for dendritic cell control of allergic inflammation (Liu 2006). Specific biological therapy is under development to counteract individual cytokines, chemokines and adhesion molecules involved in allergy and asthma. Soluble interleukin (IL)-4

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receptor (Nuvance, Immunex) was promising in preliminary studies involving abrupt withdrawal of inhaled steroids in patients with asthma (Borish et al. 1999; Borish et al. 2001) but larger-scale clinical studies in both mild and moderate asthma were discontinued in 2001 due to lack of efficacy. IL-13 has related but distinct properties and remains an attractive target for novel asthma therapy (Blanchard et al. 2005). IL-5 acts on the final stages of eosinophil maturation and release from the bone marrow, and humanized antibodies against IL-5 diminish eosinopoiesis and blood eosinophilia, but have no effect on responses to inhaled allergen (Gleich 2000). However, a bronchial biopsy study suggests that anti-IL-5 is not removing eosinophils from the airway wall, and that additional or alternative therapy will be required to ablate the eosinophil from the airways (Flood-Page et al. 2003a). It has been reported that anti-IL-5 was not effective in a clinical trial in severe asthma (Kips et al. 2003), but a study is ongoing on the effect of anti-IL-5 on asthma exacerbations in severe eosinophilic asthma. There is increasing evidence of a role for tumor necrosis factor (TNF)-α in severe asthma. A soluble TNF receptor construct (Nuvance, Immunex) has been found effective in a small study in severe asthma (Berry et al. 2006), while a monoclonal antibody (Remicade, Centocor) decreased exacerbations in less severe asthma (Erin et al. 2006). The chemokine CC receptor (CCR)3 is expressed on a range of cells including eosinophils and activated Th2 cells, and is an important target in asthma for a range of synthetic low-molecular-weight chemicals (Erin et al. 2002). VLA-4 antagonists have been considered for development in asthma, but there are concerns about Tysabri (natalizumab) being associated with progressive multifocal leukoencephalopathy (Berger & Koralnik 2005). Among specific receptor antagonists there are novel histamine H3 and H4 receptor antagonists (Leurs et al. 2005; Dunford et al. 2007) and prostaglandin (PG)D2 receptor antagonists (Yoshimura-Uchiyama et al. 2004). There has been extensive activity in developing inhibitors of cell signaling and transcription. Cilomilast (Ariflo, GSK) and roflumilast (Daxas, Nycomed) are oral tablets that inhibit phosphodiesterase type 4 (PDE4) (Lipworth 2005). Roflumilast has been shown to be effective in decreasing the early and late asthmatic reactions after inhaled allergen challenge (van Schalkwyk et al. 2005), and also in the treatment of asthma (Bateman et al. 2006; Bousquet et al. 2006). A range of kinase

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inhibitors have been developed primarily for the treatment of malignancies and some of these have potential for treatment of allergy and asthma (Adcock et al. 2006; Barnes 2006b). Targets include receptor tyrosine kinase inhibitors (c-kit, EGFR and VEGFR), Syk kinase inhibitors, phosphoinositide 3-kinase (PI3K) inhibitors, p38 MAP kinase inhibitors, and IKK2 (NF-κB) inhibitors. Among approaches directed against transcription factors and nuclear receptors, we consider glucocorticoid receptors and peroxisome proliferator-activated receptor (PPAR) agonists and therapy directed against JNK and AP-1. Of especial promise for the future is the advent of nucleic acid therapy (antisensense oligodeoxynucleotides) and interference RNA that targets specific mRNA (Popescu 2005; Sel et al. 2006; Ulanova et al. 2006) and can be delivered topically. Based on better understanding of the genetics and molecular pathophysiology that underlies allergy and asthma, novel rational biotechnology therapies are currently being tested in clinical trials. Proof-of-concept studies with highly specific protein therapeutics in novel clinical models are currently ongoing, and these studies should answer fundamental questions about the mechanisms of airway diseases, and enable the rational development of a range of novel synthetic chemical antiinflammatory therapies.

Overview

New Drugs for the Treatment of Allergy and Asthma

Symbicort SMART In the Formoterol and Corticosteroids Enabling Therapy (FACET) study it was demonstrated that addition of formoterol to budesonide decreased the rate of asthma exacerbations (Pauwels et al. 1997). Furthermore, scrutiny of 425 severe exacerbations in the FACET study demonstrated that most exacerbations were preceded for a few days by an increase in symptoms (Tattersfield et al. 1999). Hence there is a warning period, and this is the logic that symptomatic patients can use to increase therapy with Symbicort (combined budesonide and formoterol in a single inhaler), as opposed to taking a short-acting β2 agonist such as salbutamol or terbutaline. This simplified approach is possible because formoterol is as rapid in onset as salbutamol (Seberova & Andersson 2000) and has been extensively used as a reliever (Pauwels et al. 2003; Rubinfeld et al. 2006). Use of Symbicort as Maintenance and Reliever Therapy (SMART regimen) for asthma has been studied in five large clinical trials: STEP in 1890 asthmatics aged 11–80 years (Scicchitano et al. 2004); STAY in 2760 asthmatics aged 4– 80 years (O’Byrne et al. 2005); STEAM in 341 asthmatic children aged 4–11 years with asthma (Bisgaard et al. 2006); SMILE in 3394 asthmatics over 12 years (Rabe et al. 2006); and COMPASS in 3335 adult and adolescent asthmatics (Kuna et al. 2007). Based on these studies Symbicort SMART was licensed in the European Union (EU) in October 2006.

Current asthma therapies Asthma has become one of the most common chronic diseases in industrialized countries and its frequency is predicted to increase throughout the world over the next decade, particularly in developing countries. Twenty years ago, asthma was viewed as a disease of bronchoconstriction and treated predominantly with bronchodilators. However, at present it is considered an inflammatory disease of the airways, and the mainstay of modern management is treatment with inhaled corticosteroids (ICS). It is also recognized that, particularly in more severe asthma, there are structural changes in the airway that might reduce its reversibility and response to therapy. The inflammation of the airway in asthma is characterized by activation of mast cells, infiltration of eosinophils, and an increased number of activated T helper 2 (Th2) cells, which orchestrate allergic inflammation. ICS have revolutionized the management of asthma, leading to better control of asthma, reduced hospital admissions, and reduced mortality. Corticosteroids and long-acting β2 agonists (LABAs) in fixed-combination inhalers are currently the most effective therapy for asthma and are increasingly used in patients with persistent symptoms. However, there is still concern about the use of ICS because patients fear long-term side effects such as osteoporosis and stunting of growth in children. Furthermore, these effective medications have to be taken by inhalation, although oral medications are generally preferred by patients.

The need for new therapies Although ICS are effective, compliance with this medication is surprisingly poor (Barnes 2006c). Even when taken regularly, ICS do not seem to modify the course of the disease significantly and are not curative because asthma symptoms and inflammation rapidly recur when the treatment is discontinued. Also, although this therapy is effective for the majority of patients, there is a small percentage of patients (∼ 5%) who are not responsive and have severe asthma (Heaney & Robinson 2005; Wenzel 2005; Holgate & Polosa 2006; Moore & Peters 2006; Wenzel 2006). These patients account for a disproportionate amount of healthcare spending because they are frequently admitted to hospital, take many medications, and miss time from work. This has led to the search for novel or improved therapies for asthma, driven by the prospect of large sales for antiasthma medications globally. It is now recognized that distinct therapeutic approaches might have effects on different aspects of the inflammatory process, resulting in a change in different outcome measurements (Fig. 83.1). For example, some treatments might have a major impact on exacerbation frequency, whereas others might predominantly improve lung function. This means that several outcome measures might need to be assessed in the clinical development of new treatments for asthma. Moreover, better understanding of mechanisms might lead to improved design in clinical trials. Here, some of the new

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Allergen Immunotherapy • Specific AG • T cell peptides • BCG, CpG

New corticosteroids New bronchodilators PDE4 inhibitors Transcription factor & kinase inhibitors • NF-kB • NF-AT • GATA3 • P38 MAP kinase • JNK • Syk

Epithelial denudation subepithelial fibrosis

Anti-allergy drugs • anti-IgE • anti-CD23

Mast cell

Macrophage/dendritic cell

TNF-a inhibitors

Cell adhesion blockers • ICAM-1 • VLA4 Mediator antagonists • antihistamines • leukotriene • prostaglandins • endothelin • adenosine • tryptase • NO

Interleukin inhibitors • IL-5, 4, 13 • IL-9 • IL-1

CD4+ Th2 cell

Eosinophil

AHR

Cytokines • IL-10 • IFN-g • IL-12/18 Chemokine receptor inhibitors • CCR2, 3, 4 & 8

Vasodilation & angiogenesis

Fig. 83.1 Novel therapeutic targets: asthma. (From Barnes 2004.) (See CD-ROM for color version.)

therapeutic targets and treatments for asthma that are currently under clinical development are discussed. A major problem that new drug development is facing is the fact that existing therapies for asthma, particularly combination inhalers, are highly effective, inexpensive and reasonably safe. There is a strong scientific rationale for this approach to asthma therapy (Barnes 2002). This poses a hurdle that has to be overcome in providing treatments that improve on existing therapy. Another problem is that animal models of asthma are poorly predictive of efficacy of treatment in asthmatic patients (Kumar & Foster 2002; Wenzel & Holgate 2006); indeed, most drugs that have proved effective in preclinical models have failed in clinical trials, an especially important example being monoclonal antibody directed against IL-5 (Kips et al. 2003; Leckie 2003). The types of new drugs that are needed for asthma include: (i) new classes that are effective in severe poorly controlled asthma, both for prevention of exacerbations and treatment of exacerbations (Holgate et al. 2006); (ii) an oral treatment that is as effective as ICS without any side effects (Barnes 2006c); or (iii) drugs that modify or even cure allergy and asthma. Up to now the approaches that have been taken include improving existing treatments, such as β2 agonists or corticosteroids, or finding drugs against novel targets that

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have been identified by better understanding of the disease, such as blockers of cysteinyl leukotrienes or interleukin (IL)-5. For 30 years asthma therapy has been dominated by use of inhaled synthetic β2-adrenoceptor agonists and corticosteroids, but the last decade has seen the introduction of highly effective combinations of LABAs and ICS in a single inhaler. Nevertheless, significant numbers of patients do not comply with this combination therapy, often due to reluctance to have treatment with corticosteroids, or remain symptomatic despite this treatment. Hence, there are considerable ongoing efforts by the scientists and the pharmaceutical industry to develop new therapies for allergy and asthma, and there is a large range of drugs in clinical development (Barnes 2004; Barnes & Hansel 2004; Buhl & Farmer 2005; Effros & Nagaraj 2007).

Safety in clinical studies Safety is of paramount importance in clinical trials, and Good Clinical Practice (GCP) is a legal requirement for all clinical studies (European Parliament 2001). The Salmeterol Multicenter Asthma Research Trial (SMART) has recently shown an increase in respiratory-related and asthma-related deaths (Nelson et al. 2006), and a metaanalysis of deaths also made the controversial claim that LABAs should not be given

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without ICS for asthma (Ernst et al. 2006; Hasford & Virchow 2006; Salpeter et al. 2006). In hindsight, giving a safety study the acronym “SMART” was perhaps unwise, especially when there is potential confusion with Symbicort SMART therapy (see above). Two deaths in the USA have had a profound influence on the conduct of clinical trials: the death in 1999 of 18-year-old Jesse Gelsinger in a gene transfer trial at the University of Pennsylvania, and the death in 2001 of 24-year-old Ellen Roche in a Johns Hopkins study involving inhalation of hexamethonium (Ramsay 2001; Steinbrook 2002). These incidents highlight the importance of adequate institutional review board or ethics committee assessment, and the provision of accurate information to potential volunteers. On March 13, 2006 a fully humanized monoclonal antibody directed against CD28, produced by the German biotechnology company TeGenero (TGN 1412), was first administered to human subjects. CD28-directed therapy had been studied in rats where it caused selective activation of T regulator cells at low doses, and had been suggested as a potential therapy for multiple sclerosis and other autoimmune disorders (Beyersdorf et al. 2005). However, at higher doses TGN 1412 is a CD28 superagonist, and because it was given at too high a dose in humans, it induced adverse effects related to this activity. Following infusion of six subjects with the monoclonal antibody and two with placebo, six previously healthy young males developed a systemic inflammatory response with induction of proinflammatory cytokines at the Parexel Clinical Pharmacology Research Unit in Harrow, northwest London (Suntharalingam et al. 2006). Within 90 min after the single intravenous infusion individuals suffered headache, myalgia, nausea, diarrhea, erythema, vasodilation, and hypotension. Within 12–16 hours, the subjects became critically ill, with lung infiltrates, renal failure, and disseminated intravascular coagulation. Two subjects required intensive organ support for 8 and 16 days. All subjects survived, but one individual has permanent ischemic damage to his toes and fingertips, while all six subjects may be vulnerable to develop malignancies and autoimmune disease. In retrospect, TGN 1412 probably caused widespread activation of T cells leading to a cytokine storm, this leading to a capillary leak syndrome and organ system failure.

Translational medicine Translational medicine involves the transfer of scientific insights obtained by study of human specimens and animal models of disease into rational drug discovery and novel effective treatments for patients. A large variety of challenge models and clinical trial designs are possible for studying new therapies for asthma and allergy, and regulatory guidance is available on development of new drugs for asthma. Genetic and phenotypic studies are important in defining targets for new therapy, and development of validated noninvasive biomarkers in breath and condensate is proving challenging.

New Drugs for the Treatment of Allergy and Asthma

Inhaled allergen challenge The inhaled allergen challenge represents the classical method of assessing antiinflammatory therapies for asthma in humans. A large number of studies with new chemical entities as well as established asthma medications have been assessed in this model (Hansel et al. 2002). In particular a single dose of an ICS given 30 min before inhaled allergen challenge causes a profound inhibition of the late asthmatic reaction (Parameswaran et al. 2000).

Nasal allergen challenge The nose is much more accessible than the airways for assessing the effects of antiinflammatory therapy. Hence, it is possible to obtain repeated samples of nasal exudates and mucosa cells before and after nasal allergen challenge (NAC) in a relatively noninvasive way by techniques such as nasal lavage, filter paper, and nasal brushing and scraping. A comprehensive review of the extensive clinical research experience with these nasal methodologies has recently been published by Howarth et al. (2005a). Following NAC it is possible to measure symptoms, employ acoustic rhinomanometry, and measure levels of cells and mediators to evaluate new drugs for allergic rhinitis and asthma (Naclerio et al. 2002; Barnes 2004; Wang et al. 2004). It has long been recognized that there is a strong functional and immunologic relationship between the nose and bronchi (American Thoracic Society Workshop 1999; Chanez et al. 1999), especially in terms of infiltrating leukocytes and inflammatory mediators when comparing allergic rhinitis and allergic asthma (Gelfand 2004). The upper and lower airways have related respiratory epithelium and similar responses to allergen challenge. Indeed, allergic rhinitis and asthma commonly coexist (Corren 1997), since allergy is a systemic disorder that can affect various organs within the unified immune system (Togias 2003; Braunstahl 2005). This is in line with the WHO Initiative on Allergic Rhinitis and its Impact on Asthma (ARIA), which stresses the concept of a single airway disease (Bousquet et al. 2001). However, the nasal model involves a different vasculature to that in the airways, while the bronchi have added airway smooth muscle. At a pathologic level, the extent of nasal remodeling in allergic rhinitis seems to be much less than that in the bronchi of asthmatic patients (Davies et al. 2003; Bousquet et al. 2004). Topical allergen challenge increases the levels of mucosal mRNA of IL-5 and IL-13 (Ghaffar et al. 1997; Masuyama et al. 1998) but nasal cytokines and chemokines may be produced at low concentration in nasal secretions, and may be undetectable when employing conventional enzyme-linked immunosorbent assays. A single dose of topical corticosteroid has been shown to reduce levels of granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-5 detected by absorption with filter paper following nasal challenge with grass pollen in allergic rhinitis (Masuyama et al. 1998; Linden et al. 2000).

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In order to sample nasal exudates for allergic inflammatory mediators, a commonly employed method is that of nasal lavage (Naclerio et al. 1983; Greiff et al. 1990; Grunberg et al. 1997). An alternative method is to sample nasal secretions by the use of filter paper strips placed on the turbinates to absorb nasal secretions (Alam et al. 1992). The nasal filter paper method has the advantage of directly sampling nasal secretions which are less diluted and can therefore detect protein signals below the detection limits of nasal lavage. The matrix or filter paper method has been used to measure chemokines and cytokines after NAC (Sim et al. 1994, 1995; Weido et al. 1996; Linden et al. 2000). In 1985 a pollen challenge environment was described by Davies that gave grass pollen at specified levels (grains/m3) to individuals (Davies 1985). The Vienna Challenge Chamber (VCC) developed by Friedrich Horak and colleagues is a controlled experimental allergen exposure unit that can challenge up to 20 individuals under controlled and reproducible conditions, and that has proved useful in the assessment of new therapies for allergic rhinitis (Horak & Jager 1987; Horak et al. 1993, 2002, 2003). The Environmental Exposure Unit (EEU) was developed by James Day and associates in Kingston, Ontario and adapted for allergy research, and has a capacity for up to 160 subjects (Day & Briscoe 1999; Day et al. 2000, 2004). Data from environmental chamber studies is acceptable within the latest draft guidance for clinical testing from the FDA (US Department of Health and Human Services 2000). Attenuation of airway responses to inhaled allergen has been the classical model to detect effects of novel potential antiinflammatory asthma therapy (Inman et al. 1995; Hansel et al. 2002). Indeed inhaled allergen challenge was used in early studies to demonstrate the potential of systemic anti-IgE therapy (Fahy et al. 1997, 1999). Inhaled steroids are especially potent, even at single doses, in inhibiting the late asthmatic reaction following inhaled allergen challenge (Parameswaran et al. 2000). The use of bolus dose allergen challenge for repeated tests in the same patient is a safe and validated method for administering inhaled allergen in clinical trials with valid responses compared with incremental dose allergen challenge (Arshad 2000; Taylor et al. 2000). NAC is an elegant model that is increasingly used to assess both topical and systemic new therapies because of features such as ease of recruitment, reproducibility, possibility for multiple crossover designs, safety of challenge, and potential for repeat sampling of a range of mediators. Use of nasal challenge models coupled to sensitive biomarkers and clinical end points can readily be used to establish clinical efficacy in small-scale studies. Novel therapies have the potential to selectively inhibit various cell types and particular mediators involved in diverse inflammatory diseases. We believe that NAC has advantages over inhaled allergen challenge: easy recruitment, safety, and repeat noninvasive sampling. In the future this will ensure that nasal challenge will play a growing

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role in clinical pharmacology assessment of antiinflammatory therapy. Direct analysis of nasal epithelial cells has great potential in terms of translational research.

Whole blood assays Whole blood stimulation assays can be employed in vitro early in drug development to screen drug activities, In phase II clinical pharmacology studies in humans, assessment of the pharmacodynamic (functional) activity in human blood ex vivo is often complementary to assessment of pharmacokinetics (drug concentration). Pharmacodynamic assessment assists in rational selection of dose magnitude and interval. Blood samples may be collected at varying intervals following dosing and stimulated ex vivo to monitor magnitude and duration of pharmacologic activity. In this manner it is possible to optimize doses as well as treatment intervals during early clinical development. The approach of using human peripheral blood to assess pharmacodynamic potential has general utility, not only for testing chemokine receptor antagonists but also other antiinflammatory agents (Davis et al. 2000). A human whole blood eosinophil shape change assay was previously developed by our group by identifying eosinophils on the basis of their characteristic autofluorescence and then measuring their change in forward scatter as a reflection of shape change or chemotactic potential (Bryan et al. 2002), and employed in studies of CCR3 antagonism in humans (Erin et al. 2002) as well as in studies on monkeys (Zhang et al. 2002). We have developed different whole blood assays, assessing neutrophil CD11b upregulation and shape change to document the activity of a CXCR2 antagonist in vitro by addition of SB-656933 to human blood and then stimulation with CXCL1 (Nicholson et al. 2007). CD11b (Mac-1, αMβ2) is found both preformed in cytoplasmic granules and on the cell surface of leukocytes, and on cellular activation the cytoplasmic stores are mobilized and cause rapid upregulation of CD11b levels on the cell surface. CD11b/CD18 acts as a leukocyte cell adhesion molecule and also as a complement receptor (CR3).

Biomarkers in exhaled breath and sputum A major therapeutic advance would be to have biomarkerdriven definition of asthma phenotypes and monitoring of asthma therapy (Deykin 2006). Exhaled breath nitric oxide (FENO) and assessment of sputum eosinophils have proved useful in clinical management (Green et al. 2002; Menzies et al. 2006), but there remains a need for circulating biomarkers of severe asthma (Holgate 2006). FENO in adults with asthma has been modified to measure alveolar NO and in this way distal inflammation has been demonstrated in refractory asthma (Berry et al. 2005; van Veen et al. 2006). Exhaled NO has been the subject of an American Thoracic Society (ATS)/ European Respiratory Society (ERS) Task Force (ATS/ERS 2005) and has widespread potential application in asthma

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(Smith et al. 2005; Taylor et al. 2006). There is a special need for more discriminatory markers of small airways function in both asthma and chronic obstructive pulmonary disease (COPD) (Battaglia et al. 2005), and also for better imaging techniques (Ueda et al. 2006). A range of measures of airway hyperresponsiveness (AHR) has increased understanding of airways disease (Van Schoor et al. 2005; Cockcroft & Davis 2006). The technical issues involved in exhaled breath condensate (EBC) analysis have been the subject of a recent review by an ATS/ERS Task Force (Horvath et al. 2005). Within this document there is a list of the many factors which affect the collection of EBC. The review also considers the source of EBC. Much of EBC consists of water vapor that may arise from the respiratory tract and ambient air, and mixing droplets of proteinaceous fluid with large volumes of water may cause proteins to precipitate. Volatiles are largely present as gaseous vapors, while proteins are present in droplets, that may be derived from the large airways and oropharynx. Two studies on EBC hydrogen peroxide have shown the need for appropriate controls when studying patients with COPD (Dekhuijzen et al. 1996; van Beurden et al. 2002). The later paper looked at changes during the day, and demonstrated variability in levels of hydrogen peroxide, which doubled from 9 a.m. to 3 p.m. In addition, in contrast to the earlier report, the later paper showed that patients with stable COPD do not have elevated EBC hydrogen peroxide compared with appropriate controls. A major issue with EBC is that of contamination with saliva and oral flora (Effros et al. 2002, 2004, 2005) and effects of breathing pattern and inspired air on condensate volume have recently been noted (McCafferty et al. 2004). Dietary factors, oral bacterial flora, and saliva contribute to EBC nitrite (Marteus et al. 2005). Standardized methodology for sputum induction and processing has been generated by an ERS Task Force (Djukanovic et al. 2002). A treatment strategy aimed at normalizing sputum eosinophils in patients with moderate to severe asthma demonstrated that this approach reduces asthma exacerbations without the need for additional antiinflammatory therapy (Green et al. 2002). A particular problem in measuring levels of chemokines and cytokines in sputum supernatants is that dithiothreitol used to liquefy sputum causes breakage of disulfide bonds. In many cases this causes denaturation of proteins and loss of detectable levels.

Genetics There is current evidence that most polymorphisms related to asthma determine risk for disease in the context of other genes and the environment (Martinez 2007). Positional cloning has identified genes such as ADAM33 (Shapiro & Owen 2002; Holgate et al. 2004a), PHF11, DPP10, HLA-G, and G protein-related receptor for asthma (GPRA) (Kormann et al. 2005; Postma & Koppelman 2005). Airway epithelium may be important for barrier defense with DPP10, GPRA and

New Drugs for the Treatment of Allergy and Asthma

SPINK5 (Cookson 2004; Lilly & Palmer 2005). Nucleotidebinding oligomerization domain protein (NOD)-1 polymorphisms have also been associated with asthma (Hysi et al. 2005) and atopic eczema (Weidinger et al. 2005). In addition, pharmacogenetics has the potential to optimize therapy for individual patients with asthma.

Microarray Microarray technology has exciting potential for research into mechanisms of allergy and lung disease (Sheppard & Roger 2002; Benson et al. 2004). Gene profiling has been performed on nasal cells in allergic rhinitis (Benson et al. 2005), in skin lesions from patients with atopic dermatitis and psoriasis (Nomura et al. 2003), and in asthma (Yuyama et al. 2002; Zimmermann et al. 2003). Gene expression profiling techniques have identified arachidonate 15-lipoxygenase (ALOX 15) and fractalkine receptor (CX3CR1) in asthma (Hansel & Diette 2007).

New drugs for asthma and allergy New bronchodilators Bronchodilators prevent and relieve bronchoconstriction in asthma and COPD. The major advance in bronchodilator therapy in the past 20 years has been the introduction of the LABAs salmeterol and formoterol, the action of which lasts for up to 12 hours. These drugs have complementary actions to corticosteroids. Fixed-combination inhalers together with a corticosteroid are now the most effective available therapy for asthma. “Ultra-long acting” β2 agonists, which bronchodilate for at least 24 hours, include indacaterol, carmoterol and GSK159797 (Cazzola et al. 2005). Indacaterol is a chirally pure inhaled β agonist (Davies & Castaner 2005) that has been shown in studies on primates to be fast in onset, cause prolonged bronchodilation, and have an improved cardiovascular safety profile (Battram et al. 2006). A 28-day study has demonstrated the safety and tolerability of indacaterol in patients with COPD (Beier et al. 2006). Indacaterol has also been characterized on isolated human bronchi (Naline et al. 2007), and recently shown to cause 24-hour bronchodilation in asthma patients (Beeh et al. 2007). A once-daily anticholinergic bronchodilator, tiotropium bromide, is now available but is less effective in asthma than β2 agonists and is used predominantly in COPD (Koumis & Samuel 2005). However, tiotropium could prove useful in severe and emergency asthma, and have protective effects on mucociliary clearance (Hasani et al. 2004), AHR (Terzano et al. 2004), fibrosis (Matthiesen et al. 2006), and airway smooth muscle remodeling (Gosens et al. 2005; Kanazawa 2006). Novel classes of bronchodilators have proved difficult to develop and new drugs, such as analogs of vasoactive intestinal

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peptide (VIP) (Groneberg et al. 2006) and potassium-channel openers, have had side effects due to the fact that they are more potent vasodilators than bronchodilators. Nevertheless, an inhaled VIP analog is an effective bronchodilator in asthma (Linden et al. 2003) and has potential for the treatment of primary pulmonary hypertension (Petkov et al. 2003). However, the recent demonstration of novel β2-adrenoceptor signaling pathways, indeed activation of multiple effector pathways, both cyclic AMP-dependent and -independent, may permit development of novel β2 agonists selected for efficacy and safety (Giembycz & Newton 2006). Transient receptor potential (TRP) channels are a gene family of cation channels with broad therapeutic potential (Li et al. 2005a,b).

Although corticosteroids are effective in most patients with asthma, they have to be given in high doses in the minority of patients with a severe form of the disease, and there are still concerns about systemic side effects of inhaled steroids. This has prompted the search for alternative antiinflammatory therapies, particularly treatments that are effective orally because this might also treat associated allergic diseases such as rhinitis and atopic dermatitis. In severe asthma, many patients seem to have reduced responsiveness to corticosteroids so that nonsteroidal antiinflammatory drugs might be indicated. There are several new classes of treatments now at various stages of development as asthma therapies; many of them act on signaltransduction mechanisms, such as kinases (Barnes 2006b).

New corticosteroids

Leukotriene and prostanoid antagonists

ICS are the most effective antiinflammatory therapy for asthma and are effective in most patients (Barnes & Adcock 2003). However, all currently available ICS are absorbed by the lungs and therefore might have systemic effects. This has led to a concerted effort to find safer corticosteroids that have reduced oral bioavailability, are less absorbed by the lungs or are inactivated in the circulation. Ciclesonide, a newly introduced steroid, is a prodrug that becomes activated (desciclesonide) by the action of esterases in the lung. This corticosteroid seems to have less systemic effects than currently available corticosteroids; this might be due to long-term retention in the lung, no oral bioavailability, and a high degree of binding to circulating proteins (Reynolds & Scott 2004; Pearlman et al. 2005). In addition, mometasone (Bousquet et al. 2000) is a novel licensed ICS, while GSK 685 698 (Allermist) is a once-daily corticosteroid for allergic rhinitis submitted for marketing approval for rhinitis in Europe in 2006. Another approach is to develop dissociated steroids that have separate side-effect mechanisms and antiinflammatory mechanisms. This is theoretically possible because side effects are largely mediated by genomic effects and binding of glucocorticoid receptors to DNA, whereas antiinflammatory effects are largely mediated by inhibition of transcription factors through a nongenomic effect (Barnes 2006d). Some novel corticosteroids have a greater effect on the nongenomic than genomic effect (dissociated steroids) and thus might have a better therapeutic ratio and might even be suitable for oral administration (Schacke et al. 2004). Nonsteroidal glucocorticoid-receptor activators such as AL-438 have recently been discovered and are under clinical development (Rosen & Miner 2005). Corticosteroids switch off inflammatory genes by recruiting the nuclear enzyme histone deacetylase-2 (HDAC2) to the activated inflammatory gene initiation site so that activators of this enzyme can also have antiinflammatory effects or enhance the antiinflammatory effects of corticosteroids (Barnes 2006d). There may be additional mechanisms for the antiinflammatory effects of corticosteroids that might also be targeted in the future.

Since a large spectrum of mediators are involved in the complex inflammatory process present in asthma, blocking the synthesis or the receptor of a single mediator will only be effective if this factor operates in an early or pivotal part of the inflammatory reaction. Investigation of the metabolism of arachadonic acid permitted the discovery of the leukotrienes (Samuelsson 2000; Holgate et al. 2003; Ogawa & Calhoun 2006) (Fig. 83.2). The only mediator antagonists currently used in asthma and allergy therapy are the antileukotrienes, which block cysteinyl leukotriene receptors (Capra et al. 2006; Peters-Golden et al. 2006; Polosa 2007). However, a range of drugs that act against leukotriene receptors and lipoxygenases are emerging for asthma and cancer therapy (Poff & Balazy 2004). The involvement of prostaglandins and cyclooxygenase (COX)-2 in inflammatory lung diseases is complex because mediators may variously have proinflammatory or inflammatory actions (Moore & Peebles 2006; Park & Christman 2006).

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Histamine, adenosine and other mediator antagonists Inhibitors of other mediators and their receptors have all proved to be largely ineffective in asthma (Barnes et al. 1998). This includes histamine, prostaglandins, platelet-activating factor (PAF), bradykinin and tachykinins (Abraham et al. 2006; Schelfhout et al. 2006), and adenosine (Jacobson & Gao 2006). There has been the recent introduction of secondgeneration H1-receptor antagonists with a lower incidence of side effects (Devillier 2006) (Fig. 83.3), H3 agonists and antagonists have been proposed for asthma and allergic rhinitis (Leurs et al. 2005), and H4 antagonists have been effective in experimental pruritis (Dunford et al. 2007) while H4 receptors mediate allergic airway inflammation in mice (Dunford et al. 2006). Nerve growth factor (NGF) has recently been demonstrated to play a role in asthma (van den et al. 2004), small-molecule antagonists of the vanilloid receptor TRPV1 have been identified (Rami et al. 2006; Szallasi et al. 2006), and cannabinoid receptor agonists inhibit sensory nerve activation in guinea pig airways (Yoshihara et al. 2004).

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Stimulus Phospholipids PGI2 prostacyclin

Phospholipase A2 Arachidonic acid

Cyclooxygenase COX-1: constitutive COX-2: inducible

5-Lipoxgenases

5-HPETE LTA4

LTC4 LTD4 LTE4

LTB4

Peptidyl LTs

Fig. 83.2 Leukotrienes and prostaglandins. (See CD-ROM for color version.)

Adenosine receptor biology in human allergic disease is a controversial area (Fozard & Hannon 1999, 2000; Fozard 2003; Jacobson & Gao 2006). There are pronounced differences in adenosine receptors between animals and human, differences in human allergic and nonallergic states, and

PGD2

PGE2

PGF2a

PGI2

TXA2

DP CRTH2

EP1-4

FP

IP

TP

receptors can have opposite actions on different inflammatory cells. A1 receptors are widespread in distribution, and in asthmatic individuals there are increased levels of A1 receptors on smooth muscle and in allergic subjects A1 may be present on mast cells (Obiefuna et al. 2005). A2a receptors are present

ACUTE IMMEDIATE SYMPTOMS

Granule release: Histamine, heparin, b-tryptase

Allergen Pollen grain timothy grass pollen Phleum pretense

PGH2

Specific PG synthases

Nerve reflex

Mediator synthesis: prostaglandin D2, leukotrienes

Vascular receptors

Mast cell

Sneezing Itchiness Rhinorrhoea Congestion

IgE antibody production

Nasal respiratory epithelium

CHRONIC LATE SYMPTOMS

B cell

Nasal obstruction Mucosal hyperreactivity –

Dendritic cell Langerhans cell Antigen-presenting cell (APC)

– Th0 Uncommitted

Eosinophil – SLIT Grazax (ALK-Abello)

Th2 T helper 2 cell IL-4, -5, -13 T Regulatory (TReg) IL-10, TGF-b

Fig. 83.3 Pathogenesis of mast cell and eosinophil disease. (See CD-ROM for color version.)

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on neutrophils and mast cells (Matera & Polosa 2007), but an A2a/A3 receptor antagonist was ineffective in nasal allergen challenge (Rimmer et al. 2007). A2b along with A1 receptors are present on smooth muscle cells and mast cells, and are responsible for mast cell degranulation (Matera & Polosa 2007). A3 receptors are present on eosinophils and mast cells, and A3 agonists inhibit eosinophil activation but may activate the mast cell (Walker et al. 1997). CGH 2466, a combined adenosine receptor antagonist with kinase and phosphodiesterase (PDE)4 activity, has potent antiinflammatory activities (Trifilieff et al. 2005).

IgE-directed therapy Most asthmatic patients are atopic, hence treatments that target the underlying allergic inflammation are a logical approach, particularly because these treatments might also treat associated allergic conditions. Indeed, even in patients with nonatopic asthma the same inflammatory mechanisms seem to be operative. The humanized anti-IgE monoclonal antibody (Xolair, Novartis-Genentech) is the first biotechnology product to be licensed for the treatment of severe persistent refractory allergic asthma in patients already on high-dose ICS and LABAs (Holgate et al. 2005; Poole et al. 2005a). Anti-IgE was demonstrated to be effective when given intravenously in inhibiting the early and late asthmatic reactions after inhaled allergen challenge (Fahy et al. 1997), but this therapy was ineffective when given by inhalation (Fahy et al. 1999). In a landmark study, anti-IgE was shown to be effective in decreasing symptoms in severe allergic asthma, and especially in terms of steroid sparing during a withdrawal phase (Milgrom et al. 1999). Subcutaneous injections of recombinant human anti-IgE (rhuMAb-E25) have now completed a range of studies in severe asthma in patients on combined ICS and LABA (Ayres et al. 2004; Holgate et al. 2004b; Bousquet et al. 2005; Humbert et al. 2005). Omalizumab is injected twice or four times per week and is a useful add-on therapy in some patients who are affected by very severe asthma with frequent exacerbations and who are not responsive even to high doses of ICS and LABAs (Strunk & Bloomberg 2006). It is an expensive treatment, so patients must be selected carefully for a trial of therapy. Omalizumab is effective in allergic asthma (Holgate et al. 2005) and improves quality of life (Buhl et al. 2002; Finn et al. 2003) and decreases exacerbations (Busse et al. 2001; Soler et al. 2001). Omalizumab decreases acute reactions after “rush” immunotherapy with ragweed pollen (Casale et al. 2006), and aids other forms of immunotherapy (Kuehr et al. 2002), and permits increased exposure to peanuts in sensitized patients (Leung et al. 2003). More recently, a monoclonal antibody directed against the low-affinity IgE receptor (FcεRII, CD23) (lumiliximab, Biogen Idec) has been reported to be well tolerated and inhibit allergen-induced responses in antigen-producing cells and T cells (Poole et al. 2005a,b). Allergens bind to a low-affinity IgE receptor (FcεRII, also known as CD23) and the high-

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affinity receptor FcεRI on several immune cells, including T and B cells (Rosenwasser & Meng 2005). An anti-CD23 antibody (lumiliximab) was well tolerated and reduced IgE concentrations in patients with mild asthma, but its clinical efficacy has not been reported (Rosenwasser et al. 2003). In addition, oral drugs that might inhibit IgE signaling are also of potential value.

Immunotherapy and microbial products A considerable variety of approaches have been employed to treat allergic disease by allergen immunotherapy, although indications for these forms of therapy remain controversial (Bousquet et al. 1998; Casale 2004; Platts-Mills 2004; Devereux 2006). Efforts have been made to develop novel allergens (Larché et al. 2006), and Grazax (ALK-Abello) is a form of grass pollen sublingual immunotherapy that has recently been licensed, and encouraging results have been obtained in clinical studies in patients with hay fever (Dahl et al. 2006; Durham et al. 2006). The hygiene hypothesis proposes that exposure to microbes in early life may promote innate responses that protect against or suppress atopy (Racila & Kline 2005; Bloomfield et al. 2006; Vercelli 2006). However, this may be relevant for atopic respiratory as opposed to skin disease (Ring et al. 2004). Indeed some viruses may initiate asthma or cause exacerbations (Schaller et al. 2006; Morris et al. 2007), while other microbes (hepatitis A, Helicobacter pylori, Toxoplasma) may protect against asthma and allergies (von Mutius 2004). Another hypothesis is that nutrition and neonatal immune responses may have an influence on the development of allergy (Amoudruz et al. 2005; Devereux 2006; Tricon et al. 2006). A number of attempts have been made to develop adjuvants, some of which comprise nonpathogenic microbial products that act as immune response modifiers (Racila & Kline 2005). These include a ragweed pollen antigen conjugated to a Toll-like receptor (TLR)9 immunostimulatory sequence that contains a CpG motif (Jain et al. 2003; Silverman & Drazen 2003; Creticos et al. 2006; Kitagaki et al. 2006), bacterial products (Beasley et al. 2002a; Matricardi & Yazdanbakhsh 2003), NOD-1, and chitinases (Elias et al. 2005). Various approaches using CpG oligodeoxynucleotides, which target TLR9, are currently being explored as potential therapies for asthma (Krieg 2006). However, in a human study a synthetic oligonucleotide-containing immunostimulatory CpG motif did not alter the late asthmatic reaction to inhaled allergen, despite inducing interferon (Gauvreau et al. 2006). NOD-1 polymorphisms have also been associated with asthma (Hysi et al. 2005) and atopic eczema (Weidinger et al. 2005). This is a link with Gram-negative bacteria, since NOD-1 is a cytosolic receptor for a muropeptide found in their peptidoglycans. The homopentameric B-subunit of Escherichia coli heatlabile enterotoxin (EtxB) is immunogenic and an adjuvant. EtxB abrogates oral tolerance, promoting mainly Th2-type immune responses (Plant et al. 2003). Indeed, nasal delivery

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of antigen with EtxB augments antigen-specific T-cell clonal expansion (Apostolaki & Williams 2004), and causes CD8+ T-cell apoptosis (Salmond et al. 2002, 2003). In addition, manipulation of nutritional status may be a future gentle means of decreasing the development of allergy, and nutrition and allergic disease has been the subject of a recent authoritative review (Tricon et al. 2006).

Dendritic cells, antigen-specific T cells, and T regulator cells ICS and LABA do not have long-term effects on airway inflammation or remodeling but are only temporarily palliative, with asthma returning on cessation of therapy, and therefore are not disease-modifying or curative. The nearest thing we have to curative therapy is immunotherapy for allergic disease, through restoring Th1 predominance or Treg function (Wohlleben & Erb 2001; Akdis et al. 2005). However, the long-term consequences of these approaches need to be carefully evaluated, particularly as they would probably need to be used in children at the onset of disease. The role of other TLRs in asthma is uncertain, with beneficial and detrimental effects reported in different animal models (Feleszko et al. 2006). Dendritic cells are implicated in the pathogenesis of asthma (Lambrecht et al. 2004), and thymic stromal lymphopoietin (TSLP) is a cytokine that triggers dendritic cell-mediated Th2 inflammatory responses, thus switching on allergic inflammation (Liu 2006). TSLP is a novel IL-7-like cytokine that

Toll-like receptor (TLR) ligands • Bacterial LPS • Poly I:C • CpG • R848 CD40L

New Drugs for the Treatment of Allergy and Asthma

is highly expressed in airway epithelial cells of asthmatic patients. It activates dendritic cells to orchestrate an allergic pattern of inflammation through activation of Th2 cells (Liu 2006). Furthermore, proinflammatory and Th2 cytokines then induce TSLP on human skin keratinocytes (Bogiatzi et al. 2007). Hence, there is intense interest in blocking TSLP as a new therapeutic approach to allergic diseases. The cytokine macrophage migration inhibitory factor (MIF) has been shown to have proinflammatory effects in a range of diseases (Ishiguro et al. 2006; Morand et al. 2006), and induces macrophage recruitment via CCL2 (MCP-1) (Gregory et al. 2006). MIF is a potent immunomodulator that can amplify the response to lipopolysaccharide (Kudrin et al. 2006), and amplify IL-1 and tumor necrosis factor (TNF) receptor experession (Toh et al. 2006). Deficiency of MIF impairs murine allergic inflammation (Wang et al. 2006) and MIF is amplified in tears from patients with severe atopic dermatitis (Kitaichi et al. 2006). MIF may influence glucocorticoid sensitivity, and may be a useful target for steroid sparing (Aeberli et al. 2006). A variety of approaches have centered on selective trageting of the Th2 cell (Fig. 83.4) (Larché et al. 2003; Georas et al. 2005; Heijink & Van Oosterhout 2005; Romagnani 2006). Recently the second PGD2 CRTH2 (chemoattractant receptorhomologous molecule expressed on Th2 cells) has been shown to play important roles in allergy through stimulation of Th2 cells (Tanaka et al. 2004; Yoshimura-Uchiyama et al. 2004; Pettipher et al. 2007), eosinophils and basophils (Nagata & Hirai 2003). Indeed, CRTH2 has a major role in mediating

TSLP Epithelial cell & stromal cells

Dendritic cell

IL-12, -27 . . . IFN-g IL-18 . . . IFN-g IL-23 . . . ThIL-17

MDC, TARC OX40L CCL25

Naïve CD4+ T cell

Foxp3 IL-4 IL-17E = IL-25 OX40L iNK T cells CCR9+

Th1 Th2 STAT1 STAT4 STAT12 T-bet

IL12R

Treg

STAT6 GATA-3 Socs3

CD4 CD25

– IL-10, TGF-b1

CrTh2 (PGD2) CCR3 (eotaxin, RANTES, MCP-3/4) CCR4 (MDC/TARC) CCR8 (I-309) IL-4R (IL-4) CD4 (IL-16)

IFN-g IL-4, -5, -9, -13, -17E (25), 31 TNF-a Fig. 83.4 T-cell maturation. (See CD-ROM for color version.)

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chemotaxis of Th2 cells in response to mast cell supernatants (Gyles et al. 2006). Cyclosporin A is a T cell-directed therapy that has been shown to be effective in inhaled allergen challenge (Sihra et al. 1997; Khan et al. 2000), as well as in patients with chronic severe asthma (Alexander et al. 1992; Lock et al. 1996), and a monoclonal antibody directed against CD4 (keliximab, SKB) also showed efficacy in severe asthma (Kon et al. 1998). Blocking IL-15 prevents the induction of allergen-specific T cells in mice (Ruckert et al. 2005), and IL-15 is associated with pediatric asthma (Bierbaum et al. 2006). T-cell costimulatory molecules also offer targets for monoclonal antibodies to treat allergic disease (Kroczek & Hamelmann 2005). Regulatory T cells (Treg) have a key role in orchestrating immunity. There is evidence for a defect in Treg function in patients with asthma that might be linked to increased numbers of Th2 cells. There is convincing evidence that specific immunotherapy increases IL-10 production from Treg cells (Tr-1 cells) (Akdis et al. 2005). Vaccines that enhance Tr-1cell function and increase IL-10 release are now under development. T-cell peptides are also under development as a safer form of immunotherapy.

Cytokine inhibitors There has been particular interest in cytokines as targets for new asthma therapies because of their key role in chronic inflammation (Barnes 2003; Holgate 2004; Yamagata & Ichinose 2006). Many cytokines are now implicated in asthmatic inflammation and airway wall remodeling, and some cytokine inhibitors have already been tested in asthma (Barnes 2003). IL-5 is of crucial importance for eosinophilic inflammation and a blocking antibody to IL-5 depletes eosinophils from the circulation and sputum of asthmatic patients but, disappointingly, has no effect on the response to inhaled allergen, AHR symptoms, lung function, or exacerbation frequency in asthmatic patients (Leckie et al. 2000; Kips et al. 2003). In addition, it has been reported that anti-IL-5 was not effective in clinical trials in severe asthma (Kips et al. 2003). However, it has recently been found that three injections of anti-IL-5 causes a 90% decrease in blood and bronchoalveolar lavage eosinophils, but only a 50% decrease in bronchial mucosal biopsy eosinophils (Flood-Page et al. 2003a), so that additional or alternative therapy is required to ablate the eosinophils from the airways. Although apparently ineffective in treating severe stable asthma (Kips et al. 2003), eosinophil-directed therapy may be effective in controlling airway remodeling (Flood-Page et al. 2003b) and preventing exacerbations of asthma, for which clinical trials are ongoing. IL-4 and IL-13 have a range of activities on eosinophils, IgE production, and AHR. Soluble IL-4 receptor antagonist (Nuvance, Immunex) binds IL-4, suppressing IgE production and eosinophil migration into the airways. Nuvance was promising in preliminary studies involving abrupt with-

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drawal of inhaled steroids in patients with asthma (Borish et al. 1999, 2001) but larger-scale clinical studies in both mild and moderate asthma were discontinued in 2001 due to lack of efficacy. Nevertheless, IL-13 has related but distinct properties, and remains an attractive target for novel asthma therapy (Taube et al. 2002; Blanchard et al. 2005). CAT-354 (Cambridge Antibody Technology) is an anti-IL-13 monoclonal antibody that inhibits respiratory inflammation induced in mice by human IL-13 (Blanchard et al. 2005). Also favoring therapy directed against IL-13 is that there are larger amounts of IL-13 compared with IL-4 in asthmatic airways (Wills-Karp 2004). Aerovant (Aerovance Inc., Berkeley, CA) is a recombinant human IL-4 variant that is an inhibitor of both IL-4 and IL-13 receptors. An inhaled allergen challenge after Aerovant (28 days of twice-daily inhaled therapy in 30 patients) caused an impressive 72% reduction in the late asthmatic reaction (Aerovance Press Release, Jan 3, 2007). TNF-α-directed therapy has proved very effective for some patients with severe rheumatoid arthritis, ankylosing spondylitis, and inflammatory bowel disease (Vilcek & Feldmann 2004). In addition, there is increasing evidence of a role for TNF-α in severe asthma, systemic cachectic COPD, and sarcoidosis (Baughman et al. 2006). A soluble TNF receptor construct (Nuvance, Immunex) and a monoclonal antibody (infliximab, Remicade, Centocor) are licensed for use in severe rheumatoid arthritis and have been studied in severe asthma (Howarth et al. 2005b; Berry et al. 2006; Erin et al. 2006). Anti-TNF-α therapy (infliximab) is less effective in less severe asthmatics compared with severe asthmatics (Erin et al. 2006), perhaps reflecting the fact that TNF-α levels are increased only in severe asthma. Should efficacy be demonstrated in proof-ofconcept studies, synthetic low-molecular-weight chemicals could be used to target TNF-α (He et al. 2005). A recent study with Remicade failed to show efficacy in patients with stable moderate-to-severe COPD, but there was an alarming excess of patients developing malignancy in the treated versus placebo group (Rennard et al. 2007). IL-9 can be induced by allergen in humans (Devos et al. 2006) and is produced in the lungs of infants with severe respiratory syncytial virus (RSV) bronchiolitis (McNamara et al. 2004), while IL-10 expression can be induced by peptide immunotherapy (Tarzi et al. 2006). IL-15 has been a target for autoimmune disease (Baslund et al. 2005) and cancer (Waldmann 2006), and an association has been found with pediatric asthma (Bierbaum et al. 2006). Blocking IL-15 prevents induction of allergic inflammation in mice (Ruckert et al. 2005). IL-17 is a T-cell product released in response to CD23 (Kawaguchi et al. 2004; Bowman et al. 2006; Iwakura & Ishigame 2006) that has a putative role in airway remodeling (Guyatt et al. 1987; Letuve et al. 2006; Linden 2006). IL-17 is produced by pathogeneic T helper cells (Iwakura & Ishigame 2006) and is a survival factor for airway macrophages in

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allergic airway inflammation (Sergejeva et al. 2005). IL-17A is a major orchestrator of sustained neutrophil mobilization (Linden et al. 2005), while IL-17E upregulates lung fibroblasts and eosinophilia (Letuve et al. 2006). A promoter variant for IL-18 is a genetic risk factor for atopic asthma (Imboden et al. 2006), and IL-18 has three gene polymorphisms relating to allergic rhinitis (Sebelova et al. 2007). IL-18 is a Th1-inducing proinflammatory cytokine that causes interferon (IFN)-γ synthesis and is part of the IL-1 family (Dinarello 1999). IL-18 enhances antigen-induced eosinophil recruitment into mouse airways (Kumano et al. 1999). Elevated levels of IL-18 have been found in plasma and skeletal muscle in COPD (Petersen et al. 2007), while levels of IL-18 are reduced by smoking in sputum from asthmatics and normal subjects (McKay et al. 2004), and decreased levels of IL-18 occur in plasma in childhood asthma exacerbations (Cebeci et al. 2006). IL-21 is a newly described cytokine produced by activated T helper cells, and can modulate both innate and adaptive immunity (Fina et al. 2007). Monoclonal antibody directed against CD23 has demonstrable clinical activity in Crohn disease (Burakoff et al. 2006) and autoimmune encephalomyelitis (Chen et al. 2006) and has been proposed for the treatment of a variety of autoimmune diseases (Bowman et al. 2006). IL-23 induced the differentiation of naive CD4+ T cells into pathogeneic T helper cells that produce IL-17 (Iwakura & Ishigame 2006).

Antiinflammatory cytokines Some cytokines are inhibitory to the inflammatory process and might therefore be considered as therapy. For example, IL-10 has a broad spectrum of antiinflammatory effects and its secretion is defective in asthma, especially in patients with more severe symptoms (Barnes 2003). IL-10 is effective in some animal models of asthma, but its efficacy has not yet been demonstrated in humans. IL-10 needs to be injected on a daily basis and it is likely to have unacceptable side effects; thus, it is probably not useful. IL-12 is a cytokine that regulates the balance between Th1 and Th2 cells by suppressing Th2 cells, thus reducing eosinophilic inflammation and IgE levels. Although repeated IL-12 injections decrease circulating eosinophils in asthmatic patients, they do not reduce the response to inhaled allergen or AHR, as with IL-5 inhibitors (Bryan et al. 2000). In addition, this cytokine has unacceptable side effects, including malaise and occasional dangerous cardiac arrhythmias.

Chemokine receptor antagonists Chemokines are small peptides that attract inflammatory cells, including mast cells, eosinophils and Th2 cells, into the airways and are therefore appropriate targets for new therapies for asthma and eczema, particularly because they signal via G protein-coupled receptors (GPCRs) for which small-

New Drugs for the Treatment of Allergy and Asthma

molecule inhibitors can be developed (Gonzalo et al. 1998; Lukacs et al. 2005; Charo & Ransohoff 2006; Homey et al. 2006; Pease & Williams 2006; Smit & Lukacs 2006; Steinke & Borish 2006). Chemokine receptors comprise a large family of GPCRs that are tractable targets in terms of developing low-molecular-weight oral antagonists (Owen 2001; Jin et al. 2004; Pease 2006; Wells et al. 2006). The major focus of interest has been the chemokine (CC motif) receptor 3 (CCR3), which is predominantly expressed on eosinophils and mediates the chemotactic response to the CC-chemokine eotaxin, which is secreted in asthma (Bryan et al. 2002; Erin et al. 2002; Zhang et al. 2002). CCR3 is also expressed on mast cells and some Th2 cells. Several smallmolecule inhibitors of CCR3 are under clinical development but their effects in asthma have not yet been reported. In addition, monoclonal antibodies and solubilized receptors can target chemokines such as eotaxin (Main et al. 2006). Other chemokines and chemokine receptors that could also be targeted for asthma therapy are CCR2 on monocytes and T cells, MCP-1 synthesis by lung fibroblasts (Hogaboam et al. 1998), CXCR2 on neutrophils (Jin et al. 2004), CCR4 on Th2 cells (Nouri-Aria et al. 2002), the CCL19/CCR7 axis putatively involved in airway smooth muscle hyperplasia (Kaur et al. 2006), and CCR9 on variant natural killer T cells (Sen et al. 2005).

Adhesion molecule blockade Another approach to inhibit inflammation is to block the adhesion molecules involved in the recruitment of inflammatory cells from the circulation into the airways (Bochner 2004; Vanderslice et al. 2004). Selectin antagonists have therapeutic potential in asthma (Romano 2005), and bimosiamose (Encysive Pharmaceuticals) is a pan-selectin inhibitor presently being developed by Revotar Biopharmaceuticals (Meyer et al. 2005; Romano 2005; Beeh et al. 2006; Kranich et al. 2007). Bimosiamose in a single intravenous dose did not inhibit the early or late asthmatic reaction (Avila et al. 2004), but attenuated the late asthmatic reaction when inhaled (Beeh et al. 2006). Intravenous (Meyer et al. 2005) and inhaled (Meyer et al. 2007) tolerability of bimosiamose has been described separately. Although many different adhesion molecules have been identified, there has been particular interest in very late antigen-4 (VLA-4, α4β1), which is involved in the recruitment of eosinophils and T cells (Singh et al. 2004). Small-molecule inhibitors have been effective in animal models and are currently being tested in asthma patients, although there have been major safety concerns about progressive multifocal leucoencephalopathy (Berger & Koralnik 2005; Drazen 2005; Kleinschmidt-DeMasters & Tyler 2005; Langer-Gould et al. 2005; Steinman 2005). The VLA-4 antagonist IVL 745 did not affect the early and late asthmatic responses in humans (Norris et al. 2005), but WAY 103 inhibited eosinophil vascular

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cell adhesion molecule (VCAM)-1-dependent functions (Sedgwick et al. 2005).

Phosphodiesterase inhibitors The most advanced of the new inhibitors of intracellular events are PDE4 inhibitors (Giembycz 2002). PDE4 inhibitors have a wide spectrum of antiinflammatory effects, inhibiting T cells, eosinophils, mast cells, airway smooth muscle, epithelial cells, and airway nerves. They have been shown to be highly effective in various animal models of asthma (Lipworth 2005), and can be assessed for efficacy on human whole blood (Ouagued et al. 2005). PDE4 inhibitors inhibit MUC5AC expression, proliferation of pulmonary artery smooth muscle (Growcott et al. 2006), cell migration (Sanz et al. 2005), and neutrophil function (Sato et al. 2002; Jones et al. 2005), and these effects could be beneficial in COPD and severe asthma (Mata et al. 2005). The identification of the crystal structure of PDE4D and inhibitor complex will assist rational drug design (Xu et al. 2000; Lee et al. 2002; Card et al. 2004). Inhibitors of PDE4 have been the subject of extensive clinical development for COPD and asthma. Cilomilast (Ariflo, GSK) and roflumilast (Daxas, Altana-Nycomed) are oral tablets that inhibit PDE4 (Lipworth 2005). Cilomilast looked initially promising in COPD (Compton et al. 2001), but has been disappointing in phase III studies (Kips et al. 2003), and has received little study in asthma. An oral PDE4 inhibitor, roflumilast, has an inhibitory effect on allergen-induced responses in asthma (van Schalkwyk et al. 2005). Furthermore, in a therapeutic trial, roflumilast reduced symptoms and lung function in a comparable way to low doses of inhaled steroids (Bousquet et al. 2006). Roflumilast has proven to be effective in the treatment of mild-to-moderate asthma (Bateman et al. 2006; Bousquet et al. 2006), exercise-induced asthma (Timmer et al. 2002), inhaled allergen challenge (van et al. 2005) and allergic rhinitis (Schmidt et al. 2001), but was disappointing in COPD (Rabe et al. 2005). Both cilomilast and roflumilast have relatively minor clinical effects, and have significant adverse events including nausea, vomiting, headache, and gastrointestinal disturbance. These side effects are also mediated by PDE4 and therefore it has been difficult to avoid them in new PDE4 inhibitors. The antiinflammatory effects of PDE4 are mediated by the 4B isoenzyme, but there may be nonredundant function of PDE4D and 4B in neutrophil recruitment (Ariga et al. 2004). However, nausea and vomiting seem to be mediated by the 4D isoenzyme (Robichaud et al. 2002), suggesting that PDE4B selective inhibitors might be better tolerated (Houslay et al. 2005). Nevertheless, PDE4D plays a critical role in smooth muscle contraction in mice (Mehats et al. 2003). Another approach is to deliver PDE4 inhibitors by inhalation (Kuss et al. 2003), and a combined PDE4 and mitogen-activated protein (MAP) kinase inhibitor has been identified (Trifilieff et al. 2005). PDE7A is a new therapeutic target for chronic inflammatory lung disease (Giembycz & Smith 2006).

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Airway remodeling Study of pulmonary fibrosis has provided insight into mechanisms of airway remodeling in asthma (Chapman 2004). For more severe asthma it would be useful to prevent and treat airways remodeling (Beasley et al. 2002b; Beckett & Howarth 2003; Holgate & Polosa 2006). Transforming growth factor (TGF)-β has an important pathogenic role causing increased smooth muscle mass and deposition of extracellular matrix proteins (Howell & McAnulty 2006). In addition, vascular endothelial growth factor (VEGF) causes increased vascularity (Feltis et al. 2006; Lee et al. 2006; Papaioannou et al. 2006), fibrosis and proteases (Nagase et al. 2006; Turk 2006). Imatinib (see next section) has the potential to be effective in treating airway remodeling in asthma, and epidermal growth factor (EGF) receptor inhibitors for lung cancers may prove useful in asthma (Burgel & Nadel 2004; Casalino-Matsuda et al. 2006; Heymach et al. 2006; Ingram & Bonner 2006). There are inhibitors of EGF receptor as well as platelet-derived growth factor (PDGF) receptor tyrosine kinases (Ingram & Bonner 2006), and IL-13 and IL-1β promote lung fibroblast growth through upregulation of PDGFAA and PDGF-Rα (Ingram et al. 2004).

Transcription factor and kinase inhibition Following receptor-mediated activation, there is commonly a cascade of kinase activation before activation of transcription factors (Adcock et al. 2006; Barnes 2006b; O’Neill 2006) (Fig. 83.5). Transcription factors have a crucial role in regulating the expression of inflammatory genes in asthma. In particular, there has been interest in the role of nuclear factor (NF)-κB, which is activated in asthmatic airways and activates many of the inflammatory genes that are switched on in asthma (Barnes 2006e; D’Acquisto & Ianaro 2006). Small-molecule inhibitors of the key enzyme inhibitor of NFκB kinase (IKK2) block inflammation induced by NF-κB activation and are now under preclinical testing (Karin et al. 2004; Birrell et al. 2005, 2006a). P38 MAP kinase activates inflammatory genes similar to those activated by NF-κB, and several small-molecule inhibitors are now under clinical development for the treatment of other inflammatory diseases (Kumar et al. 2003; Birrell et al. 2006b). An antisense molecule that was able to block p38 MAP kinase in a murine model demonstrated marked efficacy in suppressing pulmonary inflammation (Duan et al. 2005). One general concern about all these novel kinase inhibitors is that they might have side effects because they target mechanisms that are present in many cell types. It might therefore be necessary to develop inhaled formulations for use in asthma in the future, as for corticosteroids. Inhibitors of the enzyme spleen tyrosine kinase (Syk), which is involved in the activation of mast cells, is currently under development (Wong et al. 2004; Berton et al. 2005). An antisense inhibitor of Syk is effective in an animal model of asthma (Stenton et al. 2002), and the small-molecule inhib-

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TCR

BCR

Syk

FceRI

Syk

New Drugs for the Treatment of Allergy and Asthma Cytokine

Syk

IP3

PI3K DAG

EGF-R

STAT GATA-3

PLC

Chemokine

STAT

Src

Ras

Rac

Raf

MEKK1-4

PDK1

Itk/Btk Akt

PKC Ca2+/CaM

CARMA1

PLCg

PKCz IKK

MEK1/2

MKK3/6

MKK4/7

ERK1/2

p38

JNK

IKK/IkB NFAT NFkB

P70S6K

AP-1, Elk-1, ATF2, c-Jun

Gene Transcription: Allergic Airway Inflammation Fig. 83.5 Receptors, kinases and transcription factors. (From Wong Curr Opinion Pharmacol 2006, with permission from Elsevier.) (See CD-ROM for color version.)

itor R112 given nasally reduces nasal symptoms in hay fever patients (Meltzer et al. 2005). As with other kinase inhibitors, there might be side effects with systemic administration so that inhalation might be the preferred route of delivery. Stem-cell factor (SCF) is a key regulator of mast-cell survival in the airways and acts via the receptor c-kit on mast cells (Brightling et al. 2003; Akin & Metcalfe 2004; Lennartsson & Ronnstrand 2006; Okayama & Kawakami 2006; Reber et al. 2006). Blockade of SCF or c-kit is effective in animal models of asthma, suggesting that this pathway might be a good target for new asthma therapies (Jensen et al. 2007). Imatinib (Novartis, Gleevec) is a potent inhibitor of a number of kinases including c-kit (Capdeville et al. 2002), which is itself a transmembrane receptor kinase. This type of therapy has been used effectively in systemic mastocytosis (QuintasCardama et al. 2006), the hypereosinophilic syndrome (Cools et al. 2003), and dermatologic disorders (Scheinfeld 2006). Encouragingly, imatinib attenuates peribronchial remodeling in chronic cockroach allergen-induced asthma in mice (Berlin et al. 2006), and SCF has been found in nasal lavage fluid (Nilsson et al. 1998). Peroxisome proliferator-activated receptor (PPAR)-γ is a transcription factor that is a regulator of lung inflammation and repair (Standiford et al. 2005; Belvisi et al. 2006). PPAR-γ agonists have potential as antiinflammatory agents for both asthma and COPD (Becker et al. 2006; Spears et al. 2006). In addition, class IB phosphatidylinositol 3-kinase p110γ

(PI3K-γ) has gained increasing attention as a promising antiinflammatory target (Ruckle et al. 2006).

Nucleic acid therapy: antisense oligodeoxynucleotides and interference RNA Antisense oligodeoxynucleotides (AS-ODNs) and interference RNA (RNAi) represent highly specific therapies that can be directed against synthesis of inflammatory proteins (Popescu 2005; Bumcrot et al. 2006; Sel et al. 2006; Ulanova et al. 2006). These therapies can be rapidly developed for a variety of targets in inflammatory disease, and have the advantage of low cost of goods. Topical administration gives a targeted approach and the respiratory tract may be particularly amenable to uptake of nucleic acids. There is active development of nucleic acid therapy for the treatment of influenza (Bitko et al. 2005) and RSV infection (Musiyenko et al. 2007). When using nucleic acid therapy in clinical studies it will be a challenge to establish the right molecular target or range of targets. In addition, there must be evidence of selective inhibition (specificity) of the targeted mRNA without “offtarget silencing,” thus causing a minimum of nontarget effects (Li & Cha 2007). There must be reasonable uptake of a correctly formulated nucleic acid formulation (liposomes and lipid complexes), with optimal stability and minimal degradation (Griesenbach et al. 2006; Li & Huang 2006; Aagaard & Rossi 2007). When carrying out clinical trials with nucleic acid-based

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therapy, it is important to have an effective delivery device, which may involve a choice of nebulizer. In clinical studies the dose magnitude and interval should be established, and this can be performed in a phase II program with topical nasal delivery and nasal allergen challenge in patients with allergic rhinitis out of the allergy season. Phase IIB studies can be done in wild-type symptomatic disease, and may precede large-scale investment in phase III clinical trials. There must not be side effects due to nucleic acids acting on TLRs and causing cytokine release and an influenza-like syndrome (Agrawal & Kanimalla 2004; Senn et al. 2005; Gauvreau et al. 2006; Sioud 2006), and adenosine should not be released to cause bronchoconstriction (Holgate 2002). Strategies for posttranscriptional modification of gene expression using nucleic acids are a rapidly advancing area of modern therapeutics, and have the potential to be a novel class of therapy for asthma and allergy (Ball et al. 2004; Popescu 2005; Sel et al. 2006; Ulanova et al. 2006). This type of therapy is highly specific, has been shown to work in vitro and in animals, has the potential for rapid generation of different candidate molecules, and can be given topically for respiratory diseases. Nucleic acid-based therapy can be broadly divided into three types of approach. • AS-ODNs: generally consist of 15–25 nucleotides of singlestranded DNA, and include respirable RASONS (Popescu 2005). • Immunostimulatory sequences (ISS): oligodeoxynucleotides containing CpG motifs (Racila & Kline 2005; Gauvreau et al. 2006). • RNAi: small interfering RNA (siRNA), consisting of short segments of double-stranded RNA (Bumcrot et al. 2006). This engages Dicer enzyme and then RNA-induced silencing complex (RISC). The importance of nucleic acid-based therapy is reflected by the way that major pharmaceutical companies have undertaken interactions with biotechnology companies involved in AS-ODN and RNAi technology. Merck have recently acquired Sirna Therapeutics (October 2006), who have an alliance with GSK for the development of siRNA for respiratory diseases. Alnylam is involved in development of inhaled and nasal RNAi to prevent RSVinfection, and has collaborations with Merck and a project on pandemic ’flu with Novartis (February 2006) (www.alnylam.com). In April 2007 there was large media interest because it was announced that using RNAi can be used to sensitize cancer cells to paclitaxel concentrations 1000-fold lower than normally required (Whitehurst et al. 2007).

Antisense oligodeoxynucleotides Aerosolized adenosine A1 receptor AS-ODN or RASON (EPI2010 from Epigenesis) attenuates adenosine- and allergen-

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induced AHR in rabbits and primates (Wright et al. 1983; Nyce & Metzger 1997; Ali et al. 2001; Tanaka & Nyce 2001; Nyce 2002; Sandrasagra et al. 2002; Ball et al. 2003). ASM 8 is an AS-ODN mix that has been developed by Topigen to target the common β-chain of the receptor for GM-CSF, IL-3 and IL-5 (Allam & Renzi 2001) as well as the eotaxin receptor CCR3 (Allakhverdi et al. 2006; Fortin et al. 2006). Hence ASM 8 is a multitargeted approach being developed by inhalation for asthma (Allakhverdi et al. 2006) (www.topigen.com). Intratracheal administration of Topigen ASM 8 in rats caused inhibition of eosinophil influx and AHR (Allakhverdi et al. 2002). It is important to emphasize the large number of studies carried out in animal allergic/asthmatic models with a variety of AS-ODNs: GATA-3 (Finotto et al. 2001a), Syk (Stenton et al. 2000), c-kit (Finotto et al. 2001b), p38 MAP kinase (Duan et al. 2005), NF-κB (Choi et al. 2004), FcεRI (Kim et al. 1998), CD86 (Crosby et al. 2007), VLA-4 (Lofthouse et al. 2005), IL-5Rα (Lach-Trifilieff et al. 2001), IL-13 (Yang et al. 2006), IL-5 (Karras et al. 2000), IL-4Rα (Karras et al. 2007), and IL-4 (Fiset et al. 2003).

CpG Unmethylated CpG oligodeoxynucleotides are immunostimulatory sequences that have been extensively studied act in animal models where they suppress allergic and asthmatic responses (Brown et al. 1993; Broide et al. 1998; Spiegelberg et al. 1998; Kline et al. 1999; Shirota et al. 2000; Jain et al. 2003; Kitagaki et al. 2006). However, a recent inhaled allergen study in humans demonstrated that an ISS containing CpG motifs did not have effects on the early or late phase asthmatic reaction, despite inducing interferon and interferoninducible genes (Gauvreau et al. 2006).

Interference RNA Inhibition of arginase I activity by RNAi in mice decreases STAT6 activation of Th2 cells and IL-13-mediated AHR (Yang et al. 2006). Nasally administered siRNA for RSV (Bitko et al. 2005; Barik & Bitko 2006; Musiyenko et al. 2007) was found safe and well tolerated in two phase I trials in over 100 healthy adult volunteers (Bumcrot et al. 2006). There has been a recent report of inefficient cationic lipid-mediated RNAi transfer to airway epithelial cells in vivo (Griesenbach et al. 2006).

Gene therapy and stem cells Gene therapy has attracted a great deal of interest for the treatment of genetic disorders and, despite initial disappointing results, gradual progress is being achieved in the development of gene therapy for cystic fibrosis (Davies & Alton 2005). Stem cells of the alveolar epithelium have potential for the treatment of emphysema and cystic fibrosis (Conese & Rejman 2006), but remain a very experimental approach (Griffiths et al. 2005). In the future, stem cell therapy may

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hold promise for patients with severe asthma and extensive remodeling.

Future perspectives Based on better understanding of the genetics and molecular pathophysiology that underlie asthma and allergy, novel rational biotechnology therapies are currently being tested in clinical trials. Proof-of-concept studies with highly specific protein therapeutics are currently ongoing, and these studies should answer fundamental questions about the mechanisms of airway diseases, and enable the rational development of a range of novel synthetic chemical antiinflammatory therapies. It has proved remarkably difficult to discover novel therapies for asthma, despite intense effort and investment. We already have effective therapies that are also safe, placing an additional demand on drug discovery. Despite the availability of highly effective therapies, asthma is often poorly controlled because of poor adherence. Strategies to increase adherence or better ways to monitor compliance with regular therapy should be sought in the future. Asthma is a highly complex disease and therefore it is unlikely that targeting a single receptor or mediator will be effective. Corticosteroids are effective because they suppress multiple inflammatory mechanisms at the same time. It is possible that targeting a key upstream event in the complex inflammatory process might be effective, such as anti-TNF therapy in rheumatoid arthritis. However, these targets are usually only identifiable by trial and error. More selective targeting of drugs to patients with particular subtypes of asthma might be possible in the future with the development of discriminatory biomarkers and genetic profiling. Animal models have proved to be misleading and there is now pressure to do earlier proof-ofconcept studies in humans. A major unmet need in asthma is to treat patients with severe asthma who are relatively corticosteroid-resistant more effectively. These patients share several characteristics with patients who have COPD who are also steroid-resistant. This means that drugs under evaluation for COPD might also be effective in treating severe asthma (Barnes & Hansel 2004). The mechanism of corticosteroid resistance in COPD seems to be defective function of HDAC2 (Barnes 2006f) and similar abnormalities might also be found in severe asthma (Cosio et al. 2004). HDAC2 activity might be restored by low doses of theophylline (Parr & Stockley 2004), and identification of the pathways involved might lead to new approaches to restore steroid sensitivity in severe asthma (Barnes 2005). The other unmet need in asthma is to develop an effective oral therapy for patients with mild and moderate disease. However, this has proved to be a major challenge because it is likely that any therapy that will be effective has side effects and is therefore disadvantageous compared with current inhaled therapy.

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New Drugs for the Treatment of Allergy and Asthma

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Eosinophil-associated Disease and Hypersensitivity Pneumonitis

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Allergic Bronchopulmonary Aspergillosis André-Bernard Tonnel, Stéphanie Pouwels-Frys and Isabelle Tillie-Leblond

Summary Clinical, biological, pathologic, and radiologic features related to Aspergillus fumigatus differ depending on the type of disease: saprophytic infestation, invasive diseases, or allergic diseases such as hypersensitivity pneumonitis, Aspergillus-sensitive asthma and allergic bronchopulmonary aspergillosis (ABPA). ABPA occurs in nonimmunocompromised patients. It is considered that 1–2% of asthmatics and 7–10% of patients with cystic fibrosis develop ABPA. Major criteria for ABPA diagnosis are asthma, detection of A. fumigatus-specific IgE and IgG, elevated total serum IgE levels, and central bronchiectasis. Pulmonary infiltrates or blood eosinophilia may be only present at the moment of an exacerbation or during the acute phase of the disease. In cystic fibrosis patients, ABPA is a common complication, occurring in approximately 10% of cases; it is associated with a poorer condition. Diagnosis of ABPA in cystic fibrosis patients is difficult as several of the criteria used for ABPA diagnosis are common manifestations of cystic fibrosis and patients may have immune responses to Aspergillus in the absence of ABPA. The recommendation is that cystic fibrosis patients should be screened for ABPA from 6 years of age, once a year or in response to clinical suggestions of ABPA. Treatment appears to require two types of therapy: corticosteroids to treat the inflammatory response and antifungal agents, mainly itraconazole, to suppress or limit the proliferation of A. fumigatus but also to participate in bronchial inflammation control.

Introduction Aspergillus organisms are extremely resilient and ubiquitous in the domestic and rural environment. Although the pathophysiology of the various pulmonary manifestations related to Aspergillus infection remains complex and poorly understood, the severity of these conditions seems to depend mainly on the quantity of Aspergillus inhaled and on the status of the

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

host defense. Clinical, biological, pathologic, and radiologic features differ depending on the type of disease, i.e., saprophytic infestation, invasive diseases, or allergic diseases such as hypersensitivity pneumonitis, Aspergillus-sensitive asthma and allergic bronchopulmonary aspergillosis (ABPA). ABPA occurs in nonimmunocompromised patients in the absence of invasive aspergillosis, and is defined as a hypersensitivity disorder induced by an Aspergillus species. The activities of polymorphonuclear leukocytes and alveolar macrophages, two cell types involved in host defense against Aspergillus, are not impaired in patients with ABPA. Most patients with ABPA have either asthma or cystic fibrosis: it is considered that 1–2% of asthmatics and 7–10% of patients with cystic fibrosis develop ABPA. Inhalation of spores from the environment is followed by growth of hyphae in the mucus of the bronchial tree, which stimulates an immune response involving Th2 CD4+ T cells and IgE and IgG antibody production. This disease, first reported in 1890, was later described by Hinson et al. (1952) in 12 asthmatics with recurrent pulmonary infiltrates, eosinophilia (blood and sputum), and Aspergillus hyphae in their sputum. Precipitating antibodies to Aspergillus were identified by Pepys in 1959 (Pepys 1969).

Aspergillus Aspergillus fumigatus is involved in the majority of ABPA cases. However, clinical and radiologic features, similar to those observed in A. fumigatus ABPA cases, are occasionally seen in association with other fungi (such as Stemphylium lanuginosum, Helminthosporium spp., Candida spp., Curvularia spp., Schizophyllum commune, Dreschslera hawaiiensis, Fusarium vasinfectum) and other species of Aspergillus (A. niger, A. flavus, A. nidulans, A. oryzae and A. glaucis) (Crompton 1990; Lake et al. 1990; Greenberger 1994). This review focuses on A. fumigatus, the Aspergillus species that most frequently infects humans. In the mycelium phase, Aspergillus exists in the form of 7–10 μm long, septate, uniform hyphae with dichotomus branching at an angle of 45°. The hyphae can be identified using PAS and Grocott’s stains. Reproduction is characterized by the formation of

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conidiophores with terminal vesicles producing multiple chains of spores. The spores measure 2– 4 μm in diameter, are thermotolerant (grow at temperatures ranging from 15 to 53°C), and are able to develop on Sabouraud dextrose agar slants (Latge 1999). It takes about 12–14 hours for A. fumigatus to germinate at 37°C on simple media and only 4–5 hours on rich media. Moreover, hydrocortisone accelerates the linear growth rate by 30– 40%. Conidiophores and spores may be seen together, mainly in structures in contact with the ambient atmosphere. Aspergillus is ubiquitous, existing in water, decaying organic materials, soil spaces, wood chips, mown vegetation, and basement/indoor air, walls and ceilings, particularly when these environments contain moisture. Aspergillus-related diseases are most common in individuals working in the farming industry, where they are recognized as an occupational disease. Identification of Aspergillus in cultures derived from the sputum of individuals with ABPA does not necessarily mean that the fungus is implicated in the disease. A relationship between the level of exposure to Aspergillus and the occurrence of ABPA has not been clearly identified, although Radin et al. (1983) suggested that high levels of exposure may be associated with APBA exacerbations. Inhalation of conidia is followed by airway colonization. In some individuals, proliferation of the fungus in the airway lumen results in chronic bronchial inflammation and an IgE-mediated hypersensitivity response, blood eosinophilia, and production of local and bloodprecipitating antibodies (Patterson & Roberts 1974; Patterson et al. 1977; Tonnel et al. 1987; Greenberger 1988; Leser et al. 1992). Aspergillus spores also bind to activated epithelial cells and basement membrane components. Epithelium activation occurs in individuals with asthma and in those with cystic fibrosis, and may facilitate Aspergillus penetration of the bronchial mucosa. A positive correlation has been identified between impaired pulmonary function and the presence of serum antibodies to A. fumigatus in cystic fibrosis patients.

Genetic abnormalities

CFTR gene mutation Immune predisposition to allergy

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IgE

Eosinophils

Activation of B cells

Increased proliferation of A. fumigatus

Increase of the Th2 CD4+ immune response to A. fumigatus

IL-5

• Alterations of bronchial epithelium • Increased mucus production

Factors underlying the development of ABPA remain unclear. The bronchopulmonary response to inhalation of Aspergillus spores involves three associated factors: fungal proliferation, hypersensitivity, and abnormalities of the bronchial epithelium (Fig. 84.1). After A. fumigatus spores are inhaled into the bronchial airways, they are usually trapped by the luminal mucus and then destroyed by mechanisms of local defense. However, proliferation of A. fumigatus with mycelia formation can occur in individuals with abnormalities of the immune response (local defense with development of an aspergilloma inside a residual cavity devoid of alveolar macrophages and neutrophils, or in chronic obstructive pulmonary disease, or in systemic defense in cases of invasive aspergillosis). The second response mechanism is hypersensitivity likely to induce either an IgG response with complement activation (rare cases of hypersensitivity pneumonitis) or, in atopic patients, an IgE response consecutive to sensitization to A. fumigatus (allergic asthma). ABPA is different and is characterized by markedly elevated Aspergillus-specific IgE, IgA and IgE antibodies, eosinophilic pulmonary infiltrates, bronchiectasis, and fibrosis. Several associated concomitant factors favor ABPA development. Among these factors, the respective roles of genetics and preactivation of epithelial cells (as well as the extent to which this activation facilitates bronchial penetration of the fungus), but also of the immune response (bronchial/bronchiolar inflammation, remodeling and destruction of the bronchi), are not yet fully understood. Indeed, mechanisms involved in ABPA development are complex (Fig. 84.2). Pepys suggested that ABPA was the result of type I and type III immunologic responses, classified according to Gell and Coombs. However, this classification probably provides a too restricted view of ABPA pathogenesis.

Specific Th2 CD4+ immune response

IL-4, IL-13

• HLA-DR restriction • IL-4 hypersensitivity • SNPs IL-4Ra chain

Pathophysiology

IgE/IgA/IgG Fig. 84.1 Pathogenesis of ABPA: genetic abnormalities and development of a specific Th2 CD4+ immune response. (See CD-ROM for color version.)

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Allergic Bronchopulmonary Aspergillosis

Hypersensitivity to A. fumigatus

Proliferation of A. fumigatus IgE + IgG

IgG/complement (precipitins)

IgE

Abnormalities of the immune defense

Systemic

Local

Invasive aspergillosis

Aspergilloma

Fig. 84.2 Aspergillus fumigatus and lung disease: infection and hypersensitivity disorders. (See CD-ROM for color version.)

Genetic factors +

CD4 Th2 lymphocytes from ABPA patients are restricted to six major histocompatibility complex (MHC) class II HLA-DR subtypes. Genetic studies suggest that HLA-DR molecules (DR2, DR5, and possibly DR4 or DR7) are associated with susceptibility to ABPA, whereas HLA-DQ2 molecules are associated with resistance (Chauhan et al. 1997, 2000). The combination of these data with mutations of the cystic fibrosis transmembrane conductor regulator (CFTR) gene might determine the outcome of ABPA in patients with cystic fibrosis and asthma (Knutsen et al. 2002). Marchand et al. (2001) found that the frequency of CFTR gene mutations was higher in patients with ABPA than in those with allergic asthma, even though both groups showed normal sweat chloride concentrations. This indicates that CFTR gene mutations are likely to participate in the development of ABPA. In addition, Saxena et al. (2003) identified an association between polymorphism in the collagen region of pulmonary surfactant protein-A2 and a predisposition to ABPA and severity of the disease.

Characteristics of the A. fumigatus microorganism Aspergillus fumigatus antigens have the capacity to interact with epithelial cells of the bronchial mucosa by releasing proteolytic enzymes that cause epithelial detachment and facilitate transport of antigens and allergens across the epithelial barrier. In addition, Tomee et al. (1997) showed that human bronchial and alveolar epithelial cell lines produce large amounts of proinflammatory cytokines and chemokines [interleukin (IL)-6, IL-8 and MCP-1] when incubated with A. fumigatus proteases, inducing additional epithelial activation. This activation process is partly dependent on proteaseactivated receptor (PAR)2 (Kauffman et al. 2000). Moreover, the spectrum of response to recombinant A. fumigatus allergens suggests that some of them appear to be more strongly implicated in both Aspergillus sensitization and ABPA (Kurup et al. 2000).

ABPA

Hypersensitivity pneumonitis

A. fumigatus asthma A. fumigatus sensitization

Cystic fibrosis Abnormalities of the bronchial mucosa

Induction of a Th2 CD4+ T-cell response ABPA is characterized by a strong Th2 CD4+ response, with two main consequences: the release of large amounts of IL-5, which explains the influx of eosinophils, mainly during exacerbation episodes, but also activation of B cells (Slavin et al. 1978; Kauffman et al. 1989; Knutsen & Slavin 1989; Walker et al. 1989; Knutsen et al. 1994; Chauhan et al. 1997). Lymphocyte activation has been shown in humans and in animal models (Slavin et al. 1978; Kauffman et al. 1989; Knutsen & Slavin 1989; Walker et al. 1989; Knutsen et al. 1994; Chauhan et al. 1997). In mice challenged with A. fumigatus, there is accumulation of pulmonary Th2 cells, with a higher number of granulocyte–macrophage colonystimulating factor (GM-CSF)-, IL-4- and IL-5-positive cells in the ABPA murine model than in controls (Chu et al. 1996), while IL-10 seems to act as a natural suppressor of the proinflammatory reaction (Grunig et al. 1997; Schuyler 1998). In patients with ABPA, peripheral blood mononuclear cells showed increased sensitivity to IL-4 (Khan et al. 2000). IL-4 is involved in IgE production, but also eosinophil activation, via upregulation of VLA-4 and CCR3 expression (Kurup et al. 1997a,b; Khan et al. 2000). Elevated blood sIL-2 receptor concentrations and higher levels of CD23 expression on B cells were found in ABPA patients (Brown et al. 1995). Compared with A. fumigatus asthmatics without ABPA, the increase in CD23 expression is also partly mediated by IL-4 (Khan et al. 2000). Recently, Hartl et al. (2006) evaluated proTh2 chemokines in ABPA in patients with cystic fibrosis. Th2 cells preferentially expressed the chemokine receptor CCR4 and were attracted by the corresponding chemokines TARC (thymus and activation-regulated chemokine) and MDC (macrophage-derived chemokine). The levels of TARC were elevated in ABPA compared with atopic controls or cystic fibrosis without ABPA: these values increased significantly during acute exacerbations of ABPA, in parallel with total IgE levels. It is interesting to relate these data to the experimental study by Schuh et al. (2002): the CCR4 knockout mouse

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model exhibited reduced bronchial hyperresponsiveness and more rapid clearance of A. fumigatus, indicating a major role for CCR4 and TARC in the immune response to A. fumigatus. Another indication of increased IL-4 sensitivity in ABPA was provided by Knutsen et al. (2006). They studied the presence of IL-4R α-chain single-nucleotide polymorphisms (SNPs) in ABPA and showed that IL4R α-chain SNPs were observed in 95% of ABPA patients, with a predominance of the extracellular IL-4Rα SNP Ile75Val, present in 88% of those with ABPA. The latter study suggested that this polymorphism might be a genetic marker of ABPA risk. Thus, IL-4 sensitivity is clearly involved in the amplified IgE response in ABPA.

Amplification of the B-cell response The T-cell response in ABPA patients is associated with B-cell activation and the presence of large amounts of IgE, IgA and IgG in the blood and bronchial lumen (Greenberger et al. 1988). Divergent results have been obtained in studies of blood and bronchoalveolar lavage (BAL) fluids concerning the production of immunoglobulins directed against A. fumigatus. Such inconsistencies may be explained by differences in the detection methods used: some authors evaluated precipitating antibodies, whereas others used enzyme-linked immunosorbent assay or radioimmunoassay methods. Moreover, the quality of antigen extracts differed considerably between studies. The use of recombinant antigens should improve detection rates and make the results of these studies more reproducible and reliable. The IgE response is largely, but not exclusively, directed toward A. fumigatus epitopes (Patterson et al. 1977). Indeed, mold A. fumigatus contains abundant carbohydrates, including glycan, chitin and galactomannan. In mice sensitized with A. fumigatus extracts treated by sodium periodate, which destroys carbohydrates, a significant decrease in both total and specific IgE was obtained, as well as a reduction in eosinophil recruitment. Indeed, carbohydrates present in ABPA play a key role as internal adjuvants in the total IgE response (Yamashita et al. 2002).

Pathology Although pathologic specimens are obviously not necessary for diagnosis, when bronchial samples were studied the bronchial tree was dilated and filled with mucus plugs containing macrophages, eosinophils, Charcot–Leyden crystals, and sometimes hyphae or hyphal fragments (Jelihovsky 1983; Slavin et al. 1988). Bronchial walls were infiltrated with inflammatory cells (eosinophils, lymphocytes and plasma cells); thickening of the basement membrane along with epithelial abrasion were also found (Fraser et al. 1999). The pathology of the peribronchial areas and parenchyma is sometimes different from that described above: bronchocentric granulomatosis with bronchial remodeling and dilation has been described as being a complication of ABPA (Hanson et al. 1977; Jelihovsky 1983). However, bronchocentric granulomatosis is clearly a particular entity associated with a different pseudotumoral radiologic pattern (Fraser et al. 1999), and possibly with other conditions such as tuberculosis, inflammatory disease of the bowel, and rheumatoid arthritis (Katzenstein et al. 1975; Koss et al. 1981; Hellems et al. 1983; Berendsen et al. 1985). Infiltration of the parenchyma with mononuclear cells, eosinophils and lymphocytes leads to inflammation that mimics or is associated with patterns observed in individuals with other forms of interstitial disease such as granulomatous bronchiolitis, exudative bronchiolitis, or obliterans bronchiolitis (Fraser et al. 1999). Microabscesses with Aspergillus hyphae and granulocytes have also been described in the parenchyma of ABPA patients, demonstrating that the frontier between invasive and allergic manifestations is sometimes poorly delimited.

Diagnosis ABPA occurs mainly in asthmatics and patients with cystic fibrosis.

Tissue damage

Asthma

Tissue damage (bronchiectasis formation) occurs in ABPA patients as a consequence of the local influx of neutrophils and eosinophils. Sputum eosinophil and neutrophil counts are higher in ABPA patients with bronchiectasis than in those without bronchial destruction (Wark et al. 2000). The extent of bronchiectasis, detected by high-resolution computed tomography (CT), correlates with the number of eosinophils and neutrophils in the sputum, but not with total IgE levels in the serum (Wark et al. 2000). Recently, Gibson et al. (2003) demonstrated that IL-8 gene expression and IL-8 protein levels in the sputum were higher in ABPA patients than in controls, and the extent of these two parameters correlated with the degree of bronchial neutrophilia and airway obstruction. Thus, IL-8 may be a key mediator of tissue damage in ABPA.

In asthmatic patients, the diagnosis of ABPA is based on the presence of a combination of clinical, biological, and radiologic criteria. The prevalence of ABPA is difficult to establish. When screening was performed in patients with persistent asthma, the prevalence was between 1 and 2% (Schwartz & Greenberger 1991). The role of Aspergillus or other molds in moderate to severe persistent asthma remains unclear. About 20–25% of patients with persistent asthma have sensitization to one or more molds (Schwartz & Greenberger 1991; Boulet et al. 1997; Mari et al. 2003; Maurya et al. 2005). Patients with severe asthma admitted to hospital for acute attacks (at least twice a year) more frequently have sensitization to Aspergillus, Penicillium, Cladosporium, Alternaria, or Candida compared with patients with mild to moderate asthma

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Table 84.1 Criteria for diagnosis of ABPA (without cystic fibrosis). Asthma Immediate cutaneous reaction to A. fumigatus Total serum IgE concentration (> 1000 ng/mL) Elevated A. fumigatus-specific serum IgE levels Precipitating antibodies to A. fumigatus in serum Peripheral blood eosinophilia (not essential for diagnosis) Chest radiographic infiltrates (not essential for diagnosis) Central bronchiectasis

(respectively 76% and 16–19%; P = 0.0001) (O’Driscoll et al. 2005). Among 1132 asthmatics, sensitization to Alternaria or Cladosporium increased the risk of severe asthma (Zureik et al. 2002). The major criteria for diagnosis of ABPA are listed in Table 84.1. Eight criteria were initially identified, but only some of them are crucial. The nonessential criteria, for example pulmonary infiltrates or blood eosinophilia, may only be present at the moment of exacerbation or during the initial phase of the disease. Bronchiectasis involving the more central segmental bronchi is a strong diagnostic criterion but is not always present in patients during follow-up or at the time of diagnosis. Greenberger et al. (1993) identified two groups for differentiating ABPA patients with and without bronchiectasis: ABPA with central bronchiectasis, and seropositive ABPA without bronchiectasis. When a patient with asthma does not have bronchiectasis, the following criteria are sufficient for a diagnosis of ABPA: high total serum IgE levels associated with an immediate cutaneous reaction to Aspergillus, elevated Aspergillus-specific IgE (or IgG) levels, and the presence of precipitating Aspergillus antibodies in the serum. In other cases, and particularly in the absence of systemic corticosteroids, elevated blood eosinophil counts, a marked increase in precipitating Aspergillus antibodies, or pulmonary infiltrate can enable ABPA diagnosis (Backman et al. 1995). Several other criteria can also be taken into consideration: mucoid impactions have been described in 14–54% of ABPA patients (McCarthy & Pepys 1971a; Jelihovsky 1983) and in some cases A. fumigatus has been found in the sputum, particularly in the plugs (Pepys 1969; Jelihovsky 1983).

Cystic fibrosis In cystic fibrosis patients, ABPA is a common complication of this disease, occurring in approximately 10% of cases. Diagnosis of ABPA in cystic fibrosis patients is difficult for several reasons. Some of the criteria used for ABPA diagnosis are common manifestations of cystic fibrosis. Cystic fibrosis patients often present with exacerbations with bronchial obstruction, pulmonary infiltrate, and bronchiectasis (Knutsen & Slavin 1992; Stevens et al. 2003). In addition, cystic fibrosis patients may have immune responses to Aspergillus (IgE, IgA, IgG antibody production, elevated total serum IgE levels) in

Allergic Bronchopulmonary Aspergillosis

the absence of ABPA. The boundary separating these biological responses from those involved with ABPA is extremely difficult to define (Silverman et al. 1978; Zeaske et al. 1988; Nikolaizik et al. 1995). In 2001, the Cystic Fibrosis Foundation proposed a new set of criteria for ABPA diagnosis in cystic fibrosis patients: • clinical deterioration (coughing, wheezing, increased sputum production, exercise intolerance, decrease in pulmonary function); • immediate hypersensitivity to A. fumigatus (positive skin test or IgE response); • total serum IgE concentration > 1000 kIU/L; • precipitating antibodies to A. fumigatus; • abnormal chest radiography (infiltrate, mucus plugs or unexplained changes compared with previous chest X-ray). These criteria are particularly valuable for diagnosis in cases where the condition of the patient has only slightly improved, or not improved at all, after treatment for bacterial bronchial infection. It is recommended that cystic fibrosis patients be screened for ABPA from 6 years of age onward, once a year or in response to clinical suggestions of ABPA. The Epidemiologic Register of Cystic Fibrosis reported that ABPA prevalence was 7.8% (ranging from 2.1% in Sweden to 13.6% in Belgium). The prevalence of ABPA was low in patients under 6 years of age. ABPA was more common in patients with a poorer clinical condition, i.e., lower forced expiratory volume in 1 s (FEV1), higher rate of microbial colonization, poor nutritional status. Most of these patients had a ΔF508/ΔF508 genotype (Mastella et al. 2000). Due to this strong association between cystic fibrosis and ABPA, it may be useful to perform sudoral tests on patients showing signs of ABPA. In some patients, cystic fibrosis was diagnosed at the same time as ABPA (Coltey et al. 2001).

Other conditions Although rare, cases of ABPA have been reported in patients without asthma (Glancy et al. 1981; Berkin et al. 1982; Ricketti et al. 1983). ABPA has been described in patients with other chronic obstructive pulmonary diseases and in association with allergic fungal sinusitis, bronchocentric granulomatosis, hyper-IgE syndrome (Buckley), and chronic granulomatous disease. In this situation of neutrophil disorders, differentiating between ABPA and an invasive disease related to Aspergillus is sometimes difficult. When ABPA is diagnosed, invasive aspergillosis can be fatal in patients with hyper-IgE syndrome or chronic granulomatous disease, in so far as systemic corticosteroids may accelerate tissue damage and invasive fungal infections. Two additional associations with ABPA have been reported. • Allergic Aspergillus sinusitis (Shah et al. 2001): a symptomatic search for sinus localization is recommended. In a series of 95 patients with ABPA, Shah et al. (2001), in 22 cases, showed radiologic evidence of sinusitis and confirmed, by antral wash or a surgical approach, the presence of Aspergillus

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in seven cases. The same group (Shah & Panjabi 2006) recently reported the coexistence of ABPA, allergic Aspergillus sinusitis, and aspergilloma in the same patient, an association that we also observed in two patients some years ago (personal unpublished data). • Another manifestation, different from ABPA, is represented by Aspergillus bronchitis in cystic fibrosis. Patients demonstrated the presence of A. fumigatus in sputum, responsible for respiratory exacerbations, but they did not fulfill the criteria of ABPA. Nevertheless, treatment with antifungal agents led to improvement (Shoseyov et al. 2006).

Clinical characteristics and stages ABPA onset can occur in childhood (Slavin et al. 1970), but is more frequent in young adults. Most patients have other allergic disorders, such as rhinitis, conjunctivitis, atopic dermatitis, and sensitization to common pneumallergens and trophallergens. ABPA appears at the time of, or frequently after, asthma onset and is usually associated with transformation of mild asthma into corticosteroid-dependent asthma, with unusual symptoms such as malaise, fever (body temperatures reaching 38.5°C), presence of sputum plugs and purulent sputum, coughing or increased coughing, chest pains, and hemoptysis (Tonnel et al. 1987). Pulmonary consolidation without bacterial infection has been observed. Physical examination does not provide useful information. In patients with consolidation or fibrosis, crackles may be heard on breathing. In cystic fibrosis patients, exacerbation may be associated with weight loss and a marked increase in productive coughing. ABPA progresses in five stages (Patterson et al. 1982) listed in Table 84.2. Treatment differs depending on the ABPA stage. Patients with acute exacerbations respond to corticosteroids, and early treatment of pulmonary infiltrates with these drugs can pre-

vent bronchial or bronchiolar destruction. Long-term treatment with corticosteroids is not recommended because this does not prevent the emergence of new infiltrates or progression to fibrosis. Measuring total serum IgE levels is helpful for monitoring the treatment regimen (Table 84.2). Total serum IgE levels are high during the acute and exacerbation phases of ABPA. By the end-stage, prognosis and treatment resemble those for management of cystic fibrosis: patients have extensive bronchial destruction and the bronchial tree may be colonized by Staphylococcus aureus and/or Pseudomonas aeruginosa. Response to corticosteroids is limited at this stage. However, progression from stage I to stage V is not unavoidable and progression from stage IV to V is particularly uncommon. Kumar (2003) studied the characteristics of ABPA patients and found that they could be divided into three groups: ABPA with positive serology (ABPA-S), ABPA with central bronchiectasis (ABPA-CB), and ABPA with central bronchiectasis and other radiologic features (ABPA-CB-ORF). Pulmonary function abnormalities were mild in the ABPA-S group, moderate in the ABPA-CB group, and severe in the ABPACB-ORF group. Absolute eosinophil counts rose in each group, but were highest (1233/mm3) for the ABPA-CB-ORF group. The levels of A. fumigatus-specific IgE followed the same pattern, with a maximum of 47.91 kIU/L for the ABPACB-ORF group. Symptom scores were also higher for the ABPA-CB-ORF group than for the other groups. Thus, the ABPA-S group probably comprised patients at an early stage or with a less aggressive form of ABPA. Studies by Greenberger et al. (1993) and Greenberger (2002) led them to suggest that early recognition and treatment of ABPA may prevent progression to end-stage ABPA. A recent study from India (Agarwal et al. 2006) evaluated 564 patients with asthma screened for Aspergillus with skin tests; 223 (39.5%) had positive A. fumigatus skin tests. ABPA was diagnosed in 126 patients (27%). This population comprised 27% ABPA-S,

Table 84.2 Stages of ABPA (according to Patterson et al. 1982). Stage

Clinical characteristics

Biology

Radiology

I: Acute

Fever, cough, chest pain, hemoptysis, sputum

Elevated total serum IgE levels (+++) (+/– blood eosinophilia)

Pulmonary infiltrate(s) (upper/middle lobes)

II: Remission

Asymptomatic/stable asthma

Normal or elevated total serum IgE levels

No infiltrates, in the absence of systemic corticosteroid therapy for > 6 months

III: Exacerbation

Symptoms mimicking the acute stage or asymptomatic

Elevated total serum IgE levels (+++) (+/– blood eosinophilia)

Pulmonary infiltrate(s) (upper/middle lobes)

IV: Corticosteroid-dependent asthma

Persistent severe asthma

Normal or elevated total serum IgE levels (+)

With or without pulmonary infiltrate(s)

V: Fibrosis (end-stage)

Cyanosis, severe dyspnea

Normal or elevated total serum IgE levels (+)

Cavitary lesions, extensive bronchiectasis, fibrosis

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33% ABPA-CB, and 40% ABPA-CB with radiologic findings. An interesting point is that there was no difference between the stage of ABPA and the severity of asthma, the duration of illness, or serologic findings. In this population, tuberculosis diagnosed in 46.8% in the past represents a particular point (Agarwal et al. 2006).

Biology Nearly all ABPA patients show an immediate cutaneous reaction to skin prick-tests with an Aspergillus mixture. A dual reaction is rare, involving about 16–33% of patients (McCarthy & Pepys 1971b; Rosenberg et al. 1977). Patients may also have sputum and/or blood eosinophilia, particularly at the time of diagnosis or when exacerbations occur during periods when they are not receiving corticosteroids. In these situations, blood eosinophil levels may be unusually high (1500–3000 × 106/L (McCarthy & Pepys 1971a). Aspergillus fumigatus can be detected in the sputum of 50% of ABPA patients (Schwartz & Greenberger 1991). The most reliable diagnostic tests are measurements of total serum IgE and serum IgE and IgG specific antibodies as well determination of the presence of A. fumigatus antibody precipitins (results expressed as the number of precipitation lines). Some Aspergillus antigens (catalase, trypsin, chymotrypsin) are essential for these reactions. These enzyme activities can be detected after gel diffusion and, as these antigens appear to be specific to A. fumigatus, may be useful for diagnosis (Dessaint et al. 1976). Variations in levels of specific antibodies are a function of treatment, age and stage of ABPA (Ricketti et al. 1984; Apter et al. 1989; Greenberger et al. 1989). Total serum IgE levels are high in ABPA patients and decrease when they are in remission as a result of corticosteroid treatment. This decrease usually occurs within 2 months after initiation of corticosteroid treatment. Total serum IgE levels sometimes return to within the normal range during the end-stage (Ricketti et al. 1984). Approximately 40 epitopes able to bind the IgE molecule have been identified from A. fumigatus, alongside more than 20 recombinant allergens (named Asp f1 to Asp f22) (Greenberger 2002). Studies suggest that some of the recombinant allergens may be useful for discriminating between individuals with ABPA and those with A. fumigatus-sensitized asthma (Kurup et al. 2000). These authors assessed the ability of recombinant Aspergillus allergens (Asp f1, Asp f2, Asp f3, Asp f4, and Asp f6) from the sera of ABPA patients and A. fumigatussensitive asthmatics to bind to IgE: the number of recombinant allergens able to bind to the IgE antibody was higher in sera from patients with ABPA than in those of the asthmatics. Asp f2, Asp f4, and Asp f6 interacted with IgE in all ABPA patients tested. Such binding tests could therefore be used in ABPA diagnosis. In contrast, IgE antibody binding to Asp f1 and Asp f3 was not specific. Nevertheless, the usefulness

Allergic Bronchopulmonary Aspergillosis

of recombinant allergens in the diagnosis of ABPA remains difficult to assess. Several studies have compared the respective responses in terms of specific IgE antibodies toward recombinant A. fumigatus antigens (mainly Asp f2, Asp f 3, Asp f4, Asp f6 and Asp f16) in asthmatics sensitized to A. fumigatus, and in ABPA patients with or without cystic fibrosis. Results presently at our disposal show some discrepancies. Crameri et al. (1998) reported higher IgE levels to rAsp f4 and rAsp f6 in ABPA patients when compared with asthmatics sensitized to Aspergillus. The same group (Crameri et al. 2006) showed that specific IgE to rAsp f1 and rAsp f 3 represented a marker for sensitization, while specific IgE antibodies to rAsp f4 and rAsp f6 gave an indication for ABPA that was clinically confirmed. Another study (Kurup et al. 2006) analyzed the position of different recombinant antigens in their binding to IgA, IgG and IgE antibodies in patients with cystic fibrosis alone or associated with ABPA. Indeed, present data suggest that no antigen (rAsp f1, rAsp f 2, rAsp f 3, rAsp f4, rAsp f6), antibody isotype or method is capable of differentiating cystic fibrosis with or without ABPA, although some allergens show a higher prevalence of strong reactions. Schwienbacher et al. (2005) showed that rAsp f6 is specifically expressed in hyphae, which might explain a preferential IgE response to rAsp f6 in ABPA patients. Two additional points must be discussed in patients with ABPA: amplification of the total IgE response appears to be associated with the presence of abundant amounts of carbohydrates present in the mold Aspergillus, but carbohydrates only act as an adjuvant and are not the target, properly speaking, of the induced IgE response (Yamashita et al. 2002). Similarly, the kinetics of the antibody response to recombinant antigens, in ABPA patients, showed 16–18 times higher serum levels of specific IgE to rAsp f4 and rAsp f6, but follow-up of the specific IgE response was of limited value for guiding therapy. In fact, they are not yet adapted to clinical routine (Casaulta et al. 2005). Concerning the use of recombinant antigens in cutaneous testing, Hemmann et al. (1999) showed that skin-prick tests with rAsp f4 and rAsp f6 provoked immediate skin reactions in patients with ABPA, but not in controls, and therefore enabled discrimination between ABPA and sensitization to A. fumigatus. Banerjee et al. (2001) showed that 70% of patients with ABPA had high levels of serum IgE antibodies to Asp f16, a 43-kDa protein, whereas patients with positive A. fumigatus skin test results did not. However, at the time of the study, recombinant allergens were only available for research purposes and the data obtained need to be confirmed.

Radiology and pulmonary function tests Radiographic analyses have been carried out on chest X-rays and high-resolution CT scans. Some abnormalities tend to be transient, such as pulmonary infiltrates, the presence of fluid in the bronchi, and lobar or segmental collapse linked to

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Fig. 84.3 Mucoid impaction of the left upper bronchi during an exacerbation of ABPA.

mucus plugs (Tillie-Leblond et al. 2002). Permanent patterns included bronchiectasis, most frequently in the upper lobes in the segmental and subsegmental bronchi, and cavities. Bronchiectasis occurs at more central sites in ABPA patients than in those with other bronchial diseases. However, this central location is suggestive only of ABPA, as bronchiectasis has been reported in the peripheral airways in some cases (Reiff et al. 1995). Analysis using plain film revealed that most patients (19/20; 95%) had upper lobe abnormalities, but 9/20 had both upper and lower lobe involvement (Mintzer et al. 1978). Descriptions of “glove–finger” opacities are common and correspond to bifurcating opacities caused by the bronchial distribution resulting from mucoid impaction (Fig. 84.3). The collapse of a lobe segment, or entire lobes, has been described and is often associated with clinical exacerbation. Recurrence of mucoid impaction in these segments is not rare and may predispose the patient to bronchial damage. Pleural effusion or calcifications of mucoid impactions are rare but have been reported (Murphy & Lane 1981). Pulmonary fibrosis, pneumothorax and cavities occur during end-stage ABPA (Fig. 84.5) (Mintzer et al. 1978; Neeld et al. 1990; Reiff et al. 1995). High-resolution CT is more sensitive than chest radiography for the detection of transient pulmonary infiltrate or bronchiectasis (Figs 84.5– 84.6). Bronchiectasis patterns are described as cylindrical in most cases, but have also been referred to as cystic or varicose (Neeld et al. 1990). The extent of bronchectasis is also usually defined. Several studies have compared abnormalities in ABPA patients with those in Aspergillus-sensitive asthmatics (Neeld et al. 1990; Angus et al. 1994; Ward et al. 1999; Eaton et al. 2000; Mitchell et al. 2000). One of these studies showed that high-resolution CT is more

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Fig. 84.4 Infiltrates and bronchocele during an exacerbation of ABPA.

Fig. 84.5 End-stage of ABPA with bronchiectasis and parenchymal destruction.

sensitive than chest radiography for diagnosing bronchiectasis (Angus et al. 1994). In this study, bronchiectasis was identified in 14 of 17 ABPA patients (82%), pleural thickening in 14 (82%), and atelectasis in 9 (64%) (Angus et al. 1994).

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Fig. 84.6 Bilateral proximal bronchiectasis in a 42-year-old man with ABPA.

Respiratory function tests (expiratory flow rates, lung volumes and diffusion capacities) are useful for diagnosis and during follow-up, but alone are not sufficient for monitoring treatment. Obstruction and restriction are both aggravated during acute exacerbations. Reductions in lung volume and diffusion capacity have been observed during exacerbations and in patients with end-stage ABPA (Nichols et al. 1979). The severity of the obstruction in corticosteroid-dependent asthma (stage IV) varies depending on the patient (Greenberger et al. 1980; Basich et al. 1981; Patterson et al. 1987). Deterioration of lung function also differs between ABPA patients; in some individuals lung function remains stable, whereas in others functional parameters progressively deteriorate (Lee et al. 1987). Malo et al. (1977) compared the results of lung function tests of 20 asthmatic patients with ABPA with those of 20 asthmatics, paired in terms of sex, age and duration of asthma. All patients with ABPA and 75% of patients with asthma alone showed significantly reduced FEV1. In contrast, FEV1 reversibility was more frequent in patients with asthma alone (50%) than in those with ABPA (31%), and the extent of this reversibility was also statistically higher in patients with asthma compared to those with ABPA.

Treatment The long-term prognosis of ABPA is usually favorable, with most patients maintaining good respiratory status. Nevertheless, patients with “refractory asthma” or bronchial destruction may develop permanent airflow obstructions and/or severe restrictions. Detecting exacerbations is essential for limiting airway destruction, but the long-term use of systemic corticosteroids is not recommended, as there is no proof that

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this treatment prevents progressive bronchial destruction. In addition, exacerbations have been described in ABPA patients receiving high doses of oral corticosteroids (Middleton et al. 1977), indicating that bronchial inflammation sensitive to corticosteroids is not the only factor involved in ABPA (Capewell et al. 1989; Wark et al. 2003a). Bronchial colonization by fungal microorganisms represents an additional factor justifying the use of antifungal therapies. The goals of the treatment are: • to limit exacerbations (requiring systematic testing for pulmonary infiltrates, which may or may not be associated with clinical symptoms); • to eradicate colonization and/or proliferation of A. fumigatus within the airway lumen and inside bronchiectasis and mucus plugs; • to manage corticosteroid-dependent asthma and fibrosis. Thus, treatment appears to require two types of therapy: corticosteroids to treat the inflammatory response and antifungal agents to suppress or limit the proliferation of A. fumigatus and reduce bronchial inflammation (Wark et al. 2003a).

Oral corticosteroids Systemic corticosteroids are currently the most effective treatment for the acute phase of ABPA. The recommended dose is 0.5 mg/kg daily for the first 2 weeks, followed by a progressive decrease in dose over the next 6–8 weeks. The treatment is monitored by assessing symptoms (fever, chest pain, hemoptysis, acute wheezing, sputum production). However, monitoring must also include chest radiography or high-resolution CT, as infiltrates do not lead to clinical manifestations in one-third of cases (Safirstein et al. 1973). Repeated measurement of total IgE serum levels is also recommended every 6–8 weeks during the first year after diagnosis in order to determine a baseline value for each patient. Increases in total IgE serum levels of more than 100% above this base-line value indicate that the patient is at high risk of an exacerbation. A recent Indian study (Agarwal et al. 2006) suggests that higher doses and longer duration of oral corticosteroids may be associated with better outcome. The lung function tests recommended for asthma patients must also be performed, as reductions in lung volume, diffusing capacity, or exercise tolerance may be associated with exacerbation. Long-term systemic corticosteroid therapy is not recommended, and thus assessment of these parameters is necessary for monitoring the treatment. If the patient has no new exacerbation within 6 months, he or she is judged to be in remission (stage II). Stage IV patients have severe asthma, which is corticosteroid-dependent. In these cases, the minimal dose required to stabilize the patient must be determined. Treatment preventing corticosteroid-induced osteroporosis must also be proposed if necessary. The extent of bronchial destruction in stage V patients makes the prognosis poor. In addition, these patients suffer from recurrent infections

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(the majority of which involve Pseudomonas) and respiratory insufficiency with limited exercise tolerance. Treatment with corticosteroids is generally proposed, but is poorly efficient. Lee et al. (1987) assessed 17 patients with stage V ABPA (fibrotic stage) for a mean observation period of 5 years. Roentgenographic infiltrates reoccurred in only one patient after initial diagnosis. All patients required long-term prednisone therapy for controlling asthma. The prognosis was poor for patients with FEV1 of less than 0.8 L after the initial corticosteroid treatment.

Antifungal drugs Several antifungal agents (amphotericin, ketoconazole, clotrimazole, nystatin, natamycin) have been proposed as treatments for ABPA. However, no significant beneficial effects were observed with these medications and in several cases they were even responsible for severe adverse effects (Fournier 1987). In contrast, the new orally administered antifungal agent itraconazole appears to be an effective adjunctive therapy for ABPA. Some years ago, we conducted a retrospective clinical study comparing the outcome of 1-year itraconazole treatment with that of 2-year therapy with normal treatment involving corticosteroids alone; 14 patients were included in this study and follow-up lasted for a period of 3 years. The following characteristics were compared: symptom scores, number of exacerbations, pulmonary function tests, total and A. fumigatus-specific serum IgE levels, amount of corticosteroid required during the first 2 years by patients treated with these drugs alone and the amount required during the 1-year study period by patients treated with the itraconazole/corticosteroid combination. The number of exacerbations was lower for the itraconazole-treated group compared with the group treated with corticosteroids alone. Daily corticosteroid requirements decreased from 22 to 6.5 mg, although the dose required differed substantially between patients (Salez et al. 1999). Later, the results of a 16-week randomized double-blind trial of twice-daily treatment with either 200 mg itraconazole or a placebo showed that itraconazole prevented disease progression in corticosteroid-dependent ABPA patients without any toxic effects (Stevens et al. 2000). A positive response was defined as a reduction of at least 50% in corticosteroid dose, a decrease of at least 25% in serum IgE concentration, and one of the following: improvement of at least 25% in exercise tolerance or pulmonary function tests, or partial clearance or absence of pulmonary infiltrates. In a second phase of the same trial, consisting of an open-label study, all patients received 200 mg itraconazole per day for 16 additional weeks. In the double-blind phase of the trial, 46% of patients in the itraconazole group responded to treatment compared with 19% in the placebo group (P = 0.04). About one-third (36%) of the patients who did not respond during the double-blind phase responded to treatment in the

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open-label phase of the trial, and none of the patients who responded in the double-blind phase of the trial had a relapse (Stevens et al. 2000). The mechanisms underlying this treatment remain unclear. However, several data from a separate study suggested that itraconazole had an antiinflammatory effect in the ABPA patients tested (Wark et al. 2003a): in this double-blind placebo-controlled trial involving ABPA patients with stable symptoms (N = 29) and receiving either 400 mg itraconazole (N = 15) or a placebo (N = 14) for 16 weeks, results demonstrated that itraconazole treatment reduced eosinophilic airway inflammation, systemic immune activation, and the number of exacerbations (Wark et al. 2003a). These results confirm that itraconazole can be used as a useful treatment for ABPA. Metaanalysis of the data available (mainly three prospective, randomized, controlled studies) led to the conclusion that itraconazole modifies the immunologic activation associated with ABPA and improves clinical outcome, at least over a period of 16 weeks (Cochrane Airways Group Asthma Trial register) (Wark et al. 2003b). Some adverse events have been reported: adrenal suppression caused by inhalation of corticosteroids associated with itraconazole is a potential concern. Some additional side effects must be considered, mainly alterations in liver function. On the other hand, the hypothesis that long-term prescription of antifungal therapy may lead to resistance is subject to debate. Indeed, treatment with this antifungal agent reduces bronchial inflammation and may prevent bronchial destruction and exacerbation in stable ABPA patients. It also improves the clinical status of corticosteroid-dependent ABPA patients. Trials validating the use of itraconazole in ABPA patients used a dose of 200 mg/day, administered for a duration of 16 weeks (Wark et al. 2003b). Voriconazole has no indication for ABPA treatment, although it has been a useful adjunctive therapy for ABPA in cystic fibrosis patients (Hilliard et al. 2005). In 13 children with cystic fibrosis, voriconazole led to significant improvement in clinical and functional parameters. Adverse effects were reported, such as a photosensitivity reaction, nausea, a rise in hepatic enzymes, and hair loss (Hilliard et al. 2005). However, data concerning the efficacy and safety of this drug are currently not available and thus it cannot be indicated in ABPA. The impact of long-term exposure to Aspergillus present in the environment is uncertain, but direct exposure to high concentrations of this fungus should be avoided. Fiberoptic bronchoscopy may be necessary to remove the mucoid impaction responsible for atelectasis in rare cases in which it is refractory to corticosteroid treatment.

Conclusion ABPA is a common manifestation in chronic allergic asthma and cystic fibrosis patients. Despite the high frequency of the

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disease among these patients, diagnoses are not generally made until long after the onset of the asthmatic disease. When clinical, radiologic and biological criteria for ABPA appear in combination and the diagnosis is made, then treatment would need to include both corticosteroids and the antifungal agent itraconazole. However, treatment regimens for this antifungal therapy have yet to be definitively established.

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with increased levels of total IgE antibodies and eosinophilia in patients with allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 111, 1001–7. Schuh, J.M., Power, C.A., Proudfoot, A.E., Kunkel, S.L., Lukacs, N.W. & Hogaboam, C.M. (2002) Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4–/– mice. FASEB J 16, 1313–15. Schuyler, M. (1998) The Th1/Th2 paradigm in allergic bronchopulmonary aspergillosis. J Lab Clin Med 131, 194–6. Schwartz, H.J. & Greenberger, P.A. (1991) The prevalence of allergic bronchopulmonary aspergillosis in patients with asthma, determined by serologic and radiologic criteria in patients at risk. J Lab Clin Med 117, 138–42. Schwienbacher, M., Israel, L., Heesemann, J. & Ebel, F. (2005) Asp f6, an Aspergillus allergen specifically recognized by IgE from patients with allergic bronchopulmonary aspergillosis, is differentially expressed during germination. Allergy 60, 1430–5. Shah, A. & Panjabi, C. (2006) Contemporaneous occurrence of allergic bronchopulmonary aspergillosis, allergic Aspergillus sinusitis, and aspergilloma. Ann Allergy Asthma Immunol 96, 874–8. Shah, A., Panchal, N. & Agarwal, A.K. (2001) Concomitant allergic bronchopulmonary aspergillosis and allergic Aspergillus sinusitis: a review of an uncommon association. Clin Exp Allergy 31, 1896–905. Shoseyov, D., Brownlee, K.G., Conway, S.P. & Kerem, E. (2006) Aspergillus bronchitis in cystic fibrosis. Chest 130, 222–6. Silverman, M., Hobbs, F.D., Gordon, I.R. & Carswell, F. (1978) Cystic fibrosis, atopy, and airways lability. Arch Dis Child 53, 873–7. Slavin, R.G., Laird, T.S. & Cherry, J.D. (1970) Allergic bronchopulmonary aspergillosis in a child. J Pediatr 76, 416–21. Slavin, R.G., Fischer, V.W., Levine, E.A., Tsai, C.C. & Winzenburger, P. (1978) A primate model of allergic bronchopulmonary aspergillosis. Int Arch Allergy Appl Immunol 56, 325–33. Slavin, R.G., Bedrossian, C.W., Hutcheson, P.S. et al. (1988) A pathologic study of allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 81, 718–25. Stevens, D.A., Schwartz, H.J., Lee, J.Y. et al. (2000) A randomized trial of itraconazole in allergic bronchopulmonary aspergillosis. N Engl J Med 342, 756–62. Stevens, D.A., Moss, R.B., Kurup, V.P. et al. (2003) Allergic bronchopulmonary aspergillosis in cystic fibrosis: state of the art. Cystic Fibrosis Foundation Consensus Conference. Clin Infect Dis 37 (suppl. 3), S225–S264. Tillie-Leblond, I., Scherpereel, A. & Iliescu, C. (2002) Aspergillose broncho-pulmonaire allergique. Rev Fr Allergol Immunol Clin 42, 231–40. Tomee, J.F., Wierenga, A.T., Hiemstra, P.S. & Kauffman, H.K. (1997) Proteases from Aspergillus fumigatus induce release of proinflammatory cytokines and cell detachment in airway epithelial cell lines. J Infect Dis 176, 300–3. Tonnel, A.B., Gosset, P. & Wallaert, B. (1987) Allergic bronchopulmonary aspergillosis. In: Michel, F.B., Bousquet, J. & Godard, P., eds. Highlights in Asthmology. Springer Verlag, XXXX, pp. 58–65. Walker, C.A., Fitzharris, P., Longbottom, J.L. & Taylor, A.J. (1989) Lymphocyte sensitization to Aspergillus fumigatus in allergic bronchopulmonary aspergillosis. Clin Exp Immunol 76, 34–40. Ward, S., Heyneman, L., Lee, M.J., Leung, A.N., Hansell, D.M. & Muller, N.L. (1999) Accuracy of CT in the diagnosis of allergic bronchopulmonary aspergillosis in asthmatic patients. AJR Am J Roentgenol 173, 937–42.

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Yamashita, Y., Okano, M., Yoshino, T. et al. (2002) Carbohydrates expressed on Aspergillus fumigatus induce in vivo allergic Th2-type response. Clin Exp Allergy 32, 776–82. Zeaske, R., Bruns, W.T., Fink, J.N. et al. (1988) Immune responses to Aspergillus in cystic fibrosis. J Allergy Clin Immunol 82, 73–7. Zureik, M., Neukirch, C., Leynaert, B., Liard, R., Bousquet, J. & Neukirch, F. (2002) European Community Respiratory Health Survey. Sensitisation to airborne moulds and severity of asthma: cross sectional study from European Community respiratory health survey. BMJ 325, 411–14.

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Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis Michael C. Zacharisen and Jordan N. Fink

Summary Extrinsic allergic alveolitis (EAA) or hypersensitivity pneumonitis is a non-IgE T-cell mediated inflammatory pulmonary disease with systemic symptoms that occurs after repeated inhalation of antigens. These antigens typically include bacterial or fungal microorganisms at the workplace or bird antigens encountered predominantly in recreational hobbies. An increasing number of cases of EAA are being attributed to indoor residential mold exposures. Diverse and unrelated medications may induce an EAA-like syndrome. Some antigens that cause asthma can also induce EAA. The prevalence of EAA varies and is related to the particular antigen and the host immune response. Studies have shown that a minority of individuals exposed develop disease. Cytokine gene polymorphisms in the tumor necrosis factor (TNF)-α promoter region appear to be host susceptibility factors. EAA has been diagnosed in infants and children, but is significantly more common in adults. Despite the antigen implicated, the clinical response is the same. Clinical forms of EAA include acute, subacute, and chronic. The acute form can easily be misdiagnosed as acute viral pneumonia, while the chronic form presents similar to usual interstitial fibrosis. Establishing the diagnosis is challenging, requiring a high index of suspicion, a thorough history, careful examination, complete pulmonary function tests, and radiographic studies. Highresolution chest computed tomography provides excellent detail. Laboratory evaluation for elevated serum precipitins to environmental antigens may establish significant exposure but not necessarily disease. Skin testing has poor predictive value as most antigens are not well characterized. Lung function testing reveals a restrictive ventilatory defect either in a biphasic or delayed-onset pattern in the acute form. In chronic disease, an obstructive component may be identified. This is accompanied by a decline in diffusing capacity and elevation in white blood cell count and fever. If performed, bronchoalveolar lavage and lung biopsy can eliminate other possible disorders and histology reveals a lymphocytic infiltrate with a predominance of CD8+ lymphocytes, “foamy” alveolar macrophages, and noncaseating granulomas consistent with nonspecific interstitial pneumonia.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Microbial cultures are negative. Early identification of patients with EAA with subsequent avoidance of the causative antigen is the key to a successful outcome. An index case at a workplace is a sentinel event that should prompt evaluation for other affected workers and assessment of working conditions, identification of causative antigens, and prompt remediation of exposures. Pharmacologic treatment for acute EAA is limited to oxygen and oral corticosteroids. Oral steroids may not affect the long-term outcome. The prognosis is generally favorable if intervention takes place before pulmonary fibrosis occurs.

Definition/classification Extrinsic allergic alveolitis (EAA), also known as hypersensitivity pneumonitis, is a non-IgE T-helper cell type 1 (Th1)mediated inflammatory pulmonary disease with systemic symptoms that is caused by the repeated inhalation and sensitization to any of a number of antigens primarily inhaled as organic dusts. These antigens incite an inflammatory cascade resulting in acute, subacute, and chronic lymphocytic alveolitis and bronchiolitis that may progress to fibrosis. Despite the abundance of knowledge concerning the immunopathologic mechanisms of EAA, much remains to be elucidated. The symptoms of EAA overlap with other pulmonary diseases and a careful history and thorough environmental and immunologic evaluation are needed to arrive at the diagnosis. Early recognition and intervention is the key to successful outcomes.

Epidemiology Ramazzini, the father of occupational medicine, identified pulmonary disease in grain workers in 1713. In 1932, Campbell published a clinical description of farmer’s lung (Campbell, 1932). Early reports postulated that infectious agents or toxins were responsible for inducing the disease. In 1963, Pepys documented thermophilic actinomycetes within moldy hay as a cause of farmer’s lung (Pepys et al. 1990). EAA has subsequently been diagnosed in men, women and children. The vast majority of cases are related to occupational exposures

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such as farming (hay, grain, mushroom, tobacco), bird raising (chickens, turkeys), woodworking, stucco plastering, machining, and chemical and polyurethane producing industries. Epidemiologic studies of pigeon breeders demonstrate that only 8% of exposed individuals develop clinical disease while up to 50% develop specific antibodies to the antigen but remain asymptomatic (Elgefors et al. 1971; Rodriguez de Castro et al. 1993). In contrast, up to 29% of workers at a metal working facility developed EAA (Bracker et al. 2003). The prevalence of farmer’s lung varies between countries and within the same country depending on climatic conditions and agricultural practices. Ranges had been 2.3–8.6%, but with improved farming methods and increased awareness of this disease, the incidence is decreasing (Boyd 1971; Grant et al. 1972; Madsen et al. 1976). Heavily contaminated air conditioning systems may affect 15–60% of building inhabitants (Banaszak et al. 1970). National surveillance of occupational lung disease in the UK from 1992 to 2001 identified 414 cases of acute EAA. Cases were related to agriculture, forestry or fishing, with 83% due to inhalation of organic dust, of which 66% was bacterial or fungal and 33% animal related. Various chemicals particularly isocyanates accounted for the remaining 17% (McDonald et al. 2005). Of 36 cases of chronic EAA in Japan, 10 had summer-type, seven bird related, five home-related, five isocyanate exposure, four farmer’s lung, and five due to other causes (Yoshizawa et al. 1999). More than 74% of all cases of EAA in Japan are summer-type EAA (Ando et al. 1991). The low prevalence of EAA may be an underestimation as mild or subclinical disease may go undetected, undiagnosed, or misdiagnosed. EAA in children is rarely reported and may be an underrecognized cause of interstitial lung disease. Less than 100 cases have been reported since 1960 and 80% of these are related to pet birds or raising pigeons (Fan 2002; Ettlin et al. 2006). Farmer’s lung and indoor mold exposures account for the majority of the rest of the cases. Most cases are males and 25% of the time other family members had EAA. Although deaths have been reported, most children had a rapid recovery after removal from the exposure and corticosteroid therapy.

Genetics Although many individuals are exposed to sensitizing organic antigens, typically only a small fraction become sensitized and even fewer develop disease. This observation suggests that host susceptibility factors play a role. Human leukocyte antigen (HLA) haplotypes have not been consistently associated with an increased risk of disease. With recent advances in technology, rapid and accurate identifications of gene polymorphisms associated with pulmonary illness have been determined. Tumor necrosis factor (TNF)-α has been shown to be an important mediator in EAA (Dai et al. 2005). Cytokine gene polymorphisms are associated with varying levels of

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cytokine release. A hay dust challenge resulted in an increase in TNF serum bioactivity in patients with farmer’s lung but not in asymptomatic exposed antibody-positive controls. Farmer’s lung was associated with the TNFA2 polymorphism in the promoter region of the TNF-α gene, with high TNF production (Schaaf et al. 2001). Polymorphisms in the 5′ promoter region of the TNF-α gene on chromosome 6 in pigeon breeders that were associated with susceptibility to EAA included increased frequency of HLA-DRB1*1305 and HLADRQB1*0501 and decreased frequency of HLA-DQB1*0402 alleles (Camarena et al. 2001). There also was an increase in DRB1-BQB1 haplotypes in patients with EAA and increased allele frequency of the TNF-2–308 promoter region. When subjects with summer-type and bird fancier’s lung EAA were compared with controls using restriction fragment length polymorphism analysis, no significant differences in allele frequency or genotype distribution were detected for cytokine polymorphisms for TNF-α, interleukin (IL)-10, transforming growth factor (TGF)-β1, and IL-6 (Kondoh et al. 2006). Gene expression patterns using oligonucleotide DNA microarrays able to assess an estimated 46 000 gene cluster was implemented to compare patients with EAA to idiopathic pulmonary fibrosis (IPF). The gene expression signature for EAA was enriched for genes functionally associated with inflammation, T-cell activation, and immune responses. IPF signatures were characterized by genes expressing tissue remodeling, and epithelial and myofibroblast activation (Selman et al. 2006). Additional application of genetic fingerprinting may eventually be able to predict susceptibility to EAA and differentiate it from IPF.

Etiology Multiple antigens small enough to reach the distal airway have been identified as inducing EAA (Table 85.1). A wide variety of inhaled antigens, including microorganisms (bacteria, fungi, amoeba), animal proteins, drugs and chemical compounds, can trigger similar immunologic reactions. Each year new antigens are recognized as causing EAA. Because of this wide variety of offending agents, EAA can occur after exposures at work, home or recreational hobbies.

Microbial antigens Bacteria and fungi represent the most common causes of EAA.

Bacteria Many bacteria have been implicated in EAA including multiple species of thermophilic actinomycetes. These are the classic and well-known causes of farmer’s lung (Campbell 1932; Pepys et al. 1990). Other causes include organisms such as Thermoactinomyces sacchari in moist sugarcane leading to the EAA termed “bagassosis” (Salvaggio et al. 1969; Lacey 1971).

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Table 85.1 Selected causative antigens in extrinsic allergic alveolitis. Antigen Animal proteins Avian: feather bloom or droppings

Cat hair, animal pelts Rodent proteins Shell proteins Bacteria Bacillus cereus, Klebsiella oxytoca Bacillus subtilis Cephalosporium acremonium Cytophaga Thermophilic actinomycetes Faeni rectivirgula Thermoactinomyces vulgaris Thermoactinomyces saccharii Pseudomonas fluorescens, Acinetobacter calcoaceticus, Mycobacterium immunogenum Sphingobacterium spiritivorum Mycobacterium avium complex Chemicals Diphenylmethane diisocyanate (MDI)

Source

Disease

Pigeon, duck, goose, owl, chicken, turkey, love bird, dove, parrot, parakeet, canary, pheasant

Bird fancier’s disease, pigeon breeder’s disease, budgerigar’s disease, duck fever, plucker’s lung Furrier’s lung Laboratory worker’s lung Oyster shell lung

Fur dust Laboratory rat or gerbil urine Oyster/mollusks

Ultrasonic cool mist humidifier Enzyme dust, house dust Wood floors Air conditioning system Hay, grain, compost, silage

Humidifier lung Detergent worker’s lung Floor finisher’s lung

Sugarcane (bagasse) Humidifier/air conditioner Used metal-working fluids

Bagassosis Ventilation pneumonitis Machine operator’s lung

Contaminated steam iron Hot tub, showers

Hot tub lung

Farmer’s lung

Polyurethane elastomers (rollers, packing, rubber vibration insulators, synthetic leather), spandex fibers, rubber shoe soles Polyurethane foam, varnish, lacquer, foundry casting Laboratory reagent Dyes, insecticides, plasticizers Insecticide Dental prosthesis production

Chemical worker’s lung

Wood worker’s lung Malt worker’s lung Stipatosis Compost lung Tobacco worker’s disease

Aureobasidium pullulans Botrytis cinerea Candida spp. Cephalosporium spp.

Wood dust Brewer’s malt dust Esparto grass in stucco Compost Tobacco Oxygen humidifier, soy sauce Contaminated HVAC system Grapes Reed Sewer-water in basement

Cladosporium spp. Cryptostroma corticale Epicoccum nigrum

Contaminated sauna Maple bark Basement shower

Fusarium napiforme Graphium spp., Alternaria, A. pullulans Lycoperdon puffballs Paecilomyces variotii, P. nivea Penicillium frequentans

Moldy house Redwood dust Puff ball spores Oil fan heater Cork dust

Toluene diisocyanate (TDI) Pauli’s reagent Phthalic anhydride Pyrethrum Methylmethacrylate Fungi Alternaria spp. Aspergillus fumigatus, A. clavatus

Paint refinisher’s disease, bathtub refinisher’s lung Pauli’s hypersensitivity pneumonitis Plastic worker’s lung Pyrethrum lung

Air-conditioner lung Wine grower’s lung Saxophonist’s lung Cephalosporium hypersensitivity pneumonitis Sauna taker’s lung Maple bark stripper disease Basement shower hypersensitivity pneumonitis Sequoiosis Lycoperdonosis Suberosis Continued p. 1760

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Table 85.1 (Cont’d ) Antigen

Source

Disease

Penicillium caseii and P. roqueforti Penicillium brevicompactum, Fusarium, Absidia corymbifera, Wallemia sebi Penicillium expansum, P. cyclopium, P. chrysogenum Penicillium camembertii, P. nalgiovense, P. chrysogenum Penicillium and Monocillium spp. Paecilomyces spp. Pleurotus osteatus, Hypsizigus marmoreus basidiospores Pezizia domiciliana Rhizopus and Mucor spp. Rhodotorula rubra Sacchoromonospora viridis Streptomyces albus

Cheese mold Hay or cowshed fodder

Cheese worker’s lung Farmer’s lung

Trichosporum cutaneum, T. ovoides, Cryptococcus albidus

Japanese house dust

Insect proteins Sitophilus granarius Silkworm larvae Amoebae Naegleria gruberi Acanthamoeba castellani Plants Soybean hull Coffee and tea dust Pine and cabreuva wood

Wood dust, moldy house Salami seasoning

Salami worker’s lung

Peat moss Moldy oak and maple trees Commercial indoor mushroom cultivation

Peat moss processor’s lung Woodman’s disease Mushroom worker’s lung

Flooded California house Wood trimmings Cellar/bathroom walls Dried grasses and leaves Contaminated fertilizer

El Niño lung Wood trimmer’s disease

Infested wheat flour Cocoon fluff

Thatched-roof disease Streptomyces albus hypersensitivity pneumonitis Summer-type hypersensitivity pneumonitis

Wheat weevil disease Sericulturist’s lung disease

Contaminated ventilation system Ventilation pneumonitis

Veterinary feed Coffee bean or tea leaf dust Sawdust

Tea worker’s lung Wood worker’s lung

Medications and drugs Amiodarone, beta-blockers, chlorambucil clozapine, gold, cyclosporin A, fluoxetine, HMG-CoA reductase inhibitor, hydroxycarbamide (hydroxyurea), intranasal heroin, intravesicular BCG, leflunomide, loxoprofen, mesalamine, methotrexate, minocycline, nitrofurantoin, procarbazine, sirolimus, sulfasalazine, tocainamide, trofosfamide

Contaminated ventilation systems grow Thermoactinomyces vulgaris while cool mist humidifiers have grown Bacillus cereus and Klebsiella oxytoca in environments of patients with EAA (Kane et al. 1993). Workers exposed to the Gram-negative Cytophaga-contaminated air conditioning in a nylon plant developed EAA or humidifier fever (Nordness et al. 2003). Workers exposed to Bacillus subtilis in the enzyme industry have developed either asthma or EAA (Tripathi & Grammer 2001). Atypical mycobacteria including Mycobacterium immunogenum are responsible for machinist’s lung (Wallace et al. 2002). Other bacteria that thrive in water-based machining fluids/coolants and which are resistant to current biocides and

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which appear to cause disease in machinists include Gramnegative rods such as Pseudomonas (Bernstein et al. 1995; Fishwick et al. 2005) and Acinetobacter (Zacharisen et al. 1998). As the composition of the metal working fluids has changed over the decades, so has the propensity for the fluids to form an airborne mist. Sphingobacterium spiritivorum has been implicated in disease from inhaling water from a steam iron. This Gram-negative rod has occasionally been isolated from human blood or urine specimens but rarely causes respiratory disease (Kampfer et al. 2005). Mycobacterium avium complex has been linked to respiratory symptoms after using indoor hot tubs and showers (Embil et al. 1997; Marras et al. 2005).

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Fungi Fungi have become the most commonly reported cause of EAA and fungal antigens can be found both indoors and outdoors in diverse locations worldwide. Fungal spores less than 3 μm in diameter are small enough to become airborne and inhaled into the peripheral airways and alveoli. Changes in farming practices have reduced thermophilic bacterial contamination yet farmers continue to be at risk for EAA caused by fungi. Hay analyses in France identify Absidia corymbifera and Eurotium amstelodami (Roussel et al. 2004) as causative organisms. Candida albicans was confirmed in farmer’s lung using inhalation challenge while the environmental isolate was Candida guiliermondii (Ando et al. 1994). Three Canadian farmers died from chronic pulmonary disease due to Penicillium brevicompactum and Penicillium olivicolor (Nakagawa-Yoshida et al. 1997). A 5-year-old child was diagnosed with acute EAA due to Aspergillus exposure in a hay barn and an organic compost container (Aebischer et al. 2002). Occupations associated with fungal-related EAA, other than farmers, include food workers in the malt, cheese, sausage, and soy sauce industries and commercial mushroom growing. Malt-workers in Scotland had a 5.2% incidence of EAA but with enclosed malting systems and conveyor belts, they do not need to shovel or plow on open malting floors, thus decreasing worker exposure and the incidence of EAA (Blyth et al. 1977). Cheese workers in the USA and France have developed EAA due to Pencillium roqueforti while making blue cheese (Campbell et al. 1983) and Gruyère cheese, respectively (Dalphin et al. 1990). Salami factory workers exposed to airborne spores while brushing off excess mold developed EAA from Pencillium camemberti (Marchisio et al. 1999; Rouzaud et al. 2001). A Japanese soy sauce brewer developed acute EAA to Aspergillus oryzae; this organism produces a protease used in soybean fermentation (Tsuchiya et al. 1993) and is also used in producing glucose dipsticks. Mushroom worker’s lung was originally reported to be due to thermophilic actinomycetes from compost used to cultivate mushrooms (Bringhurst et al. 1959; Sakula 1967). Grown as commercial crops, mushrooms such as the basidiospores of Shiitake, Pholiota nameko and Lyophyllum aggregatum are themselves responsible for causing EAA (Nakazawa & Tochigi 1989; Matsui et al. 1992). Similarly Pleurotus ostreatus, Hypsizigus marmoreus, and Maitake mushrooms have been reported in outbreaks of EAA where indoor cultivation is practiced (Mori et al. 1998; Tanaka et al. 2000, 2004). Subacute EAA was described in women harvesting L. aggregatum mushrooms (Tsushima et al. 2001). Nonfood-related industries associated with mold-induced EAA include cork makers, peat moss growers, plasterers, and woodworkers. Cork obtained from the cork tree Quercus suber is stored in dark humid conditions until moldy, then sliced, punched and polished. Inhalation of cork dust contaminated with Penicillium frequentans was considered the first case of

Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

suberosis in 1955. Other molds colonizing cork such as Aspergillus, Mucor, and Rhizopus species as well as suberin (noncontaminated cork antigen) are additional causes of suberosis (Morell et al. 2003). Peat moss harvested from sphagnum moss is used as a soil additive, home-heating fuel, and in the fabrication of organic filters. While EAA has only been reported in one worker, three seropositive asymptomatic workers had lymphocytic alveolitis (Cormier et al. 1998). In Spain, esparto grass (Stipa tenacissima) is used for manufacturing ropes and baskets and in producing plaster for stucco. Stucco makers developed acute EAA to Aspergillus fumigatus and thermophilic actinomycetes. Esparto grass itself has been implicated and the disease termed stipatosis (Gamboa et al. 1990; Moreno-Ancillo et al. 2003). Tree trimmers, lumber/wood pulp and sawmill workers exposed to mold-contaminated wood dust have developed EAA. The fungi implicated are Penicillium spp., Paecilomyces spp., Aspergillus niger, Rhizopus (Belin 1987; Dykewicz et al. 1988), Cryptostroma corticale (Emanuel et al. 1966), and Alternaria (Schlueter et al. 1972). In contrast, pine and cabreuva wood dust without mold contamination can induce EAA (Malmstrom et al. 1999; Baur et al. 2000). Contamination of homes with fungus from water incursion or contaminated vaporizers, saunas, cool mist humidifiers, or air-conditioning systems is another important cause of EAA. Areas with high humidity and poor ventilation, such as basement showers and wooden floors, are the most widely described. Leucogyrophana pinastri, the discomycete mushroom Pezizia domiciliana, and the red yeast Rhodotorula rubra were implicated in EAA in homes (Stone et al. 1989; Park et al. 1994; Hogan et al. 1996; Wright et al. 1999). In Japan, inhalation of house dust contaminated with Trichosporon spp. is responsible for summer-type EAA, the most prevalent type of EAA in Japan accounting for 75% of cases (Kawai et al. 1984). Cryptococcus albidus and Humicola fuscoatra have also been identified as etiologic agents in homes where Trichosporon was not isolated (Miyagawa et al. 2000; Kita et al. 2003). Ultrasonic and cool mist humidifiers disperse droplets ranging from 0.5 to 3 μm in size and have been identified as sources responsible for EAA. Although the specific antigen is frequently not identified (Shiue et al. 1990; Volpe et al. 1991; Suda et al. 1995), specific fungi that have been implicated include Klebsiella (Kane et al. 1993), Rhodotorula (Alvarez-Fernandez et al. 1998), and Debaryomyces hansenii (Yamamoto et al. 2002). The growth of thermophilic actinomycetes can be supported in furnace humidifiers and air heating systems and implicated in humidifier lung (Banaszak et al. 1970; Kohler et al. 1976; Burke et al. 1977). Moldy books in a museum were responsible for acute EAA (Kolmodin-Hedman et al. 1986) while chronic EAA occurred in a teacher who occupied a school contaminated with multiple molds caused by roof and plumbing leaks. She had precipitating antibodies to Alternaria tenuis, A. fumigatus, Botrytis cinerea, Cladosporium herbarum, Penicillium notatum, Pullullaria pullulans, and Rhizopus rhizopodiformis (Thorn et al. 1996).

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Ultrasonic misting fountains used for domestic decoration, aromatherapy, and air humidification have been associated with acute EAA in 9 of 11 adult patients. The other two patients had “humidifier fever” likely related to endotoxin. Culture of the water in the fountains yielded fungi and bacteria (Koschel et al. 2005). Metal-working fluids have also been contaminated with Fusarium.

Animal emanations Avian antigens are the most common animal proteins associated with EAA. The antigens include feather bloom or dustcontaining droppings with fecal enzymes and proteins such as avian albumin, γ-globulin, intestinal mucin, and IgA (Edwards et al. 1970, 1969; Berrens & Maesen 1972). Live bird exposure is not required as exposure to feather decorations and feather duvets has been reported (Haitjema et al. 1992). Primarily domesticated birds have been implicated but wild birds have been causative as well (Reed et al. 1965; Hargreave et al. 1966; Stiehm et al. 1967; Korn et al. 1968; Riley & Saldana 1973; Cunningham et al. 1976; Choy et al. 1995; Saltoun et al. 2000). A bird fancier may keep a single bird, while pigeon breeders may keep several hundred in a coop. During cleaning of the coop, large amounts of antigen may be inhaled in contrast to an indoor bird where low amounts of antigen are continuously inhaled. Occupational EAA to domestic fowl has been reported for workers exposed to chickens, turkeys, and pheasants (Boyer et al. 1974; Partridge et al. 2004). Bird breeders disease is also reported in children (Ettlin et al. 2006). Other animal proteins include wheat wheevil, rat and gerbil, dust from mollusk shells, cat dander, and fur pelts.

and trofosfamide (Franzen & Pettersson 1983; Lewis 1984; Ridley et al. 1988; Akoun et al. 1991; Leino et al. 1991; Guillon et al. 1992; Lombard et al. 1993; Crestani et al. 1994; Kopp et al. 2004). Leflunomide, a disease-modifying antirheumatic agent, alone or in combination with methotrexate was linked to pneumonitis (Savage et al. 2006). Sirolimus, a potent immunosuppressant for solid organ transplants, was associated with a CD4+ pulmonary infiltrate (Howard et al. 2006). Other routes of drug administration that have caused EAA include intranasal heroin and intravesical bacillus Calmette-Guèrin (BCG) for treatment of a bladder tumor (Reinert & Sybrecht 1994).

Immunopathogenesis The immunopathogenesis of EAA is complex and despite extensive research much remains undefined. Inhalation of low-molecular-weight chemical or small organic antigens into the distal respiratory tract incites inflammation through a series of inflammatory cells and mediators with both protective and promoting factors that affects the lung parenchyma and small airways (Fig. 85.1) (Girard et al. 2004). Despite the variety of antigen triggers, the resulting patterns of clinical phenotype are similar and the sequence of immunopathologic events is common to all antigens. It remains unclear why the majority of exposed persons do not develop disease and why many individuals develop antibodies to EAA antigens yet do not develop the disease, although gene polymorphisms appear to increase the risk of developing disease.

Low-molecular-weight chemical compounds

Inflammatory cells

Low-molecular-weight chemicals such as isocyanates used in the production of polyurethane foams, elastomers, adhesives, and paints have been implicated in EAA (Fink & Schlueter 1978; Patterson et al. 1982; Malo & Zeiss 1985; Vandenplas et al. 1993). At least 1% of isocyanate workers appear to be sensitized and symptomatic (Baur 1995) Another group of reactive chemicals known as acid anhydrides used in the production of dyes, insecticides and plastics can result in an EAA-like syndrome with anemia (Patterson et al. 1982). Inhalation of phthalic anhydride used in epoxy resin can result in EAA. Rare causes of EAA include the insecticide pyrethrum, Pauli’s reagent (sodium diazobenzene), and methyl methacrylate (Scherpereel et al. 2004).

The inhaled antigens become soluble and complex with IgG antibodies resulting in complement cascade activation and the release of complement C5. C5 and the antigen activate alveolar macrophages. Alveolar macrophages are the primary cell involved in EAA, likely due to their strategic location in the encounter of allergens inhaled into the alveoli. The alveolar macrophages increase in number and become activated, releasing large amounts of TNF-α, a pleiotropic cytokine with broad immune regulation functions. Cytokines and chemokines then attract neutrophils into the airway. Neutrophils release proinflammatory mediators that contribute to the inflammation. This is followed by an influx of primarily CD8+ lymphocytes that exhibit prolonged survival likely related to antiapoptotic cytokines (Laflamme et al. 2003). While lymphocytes are clearly implicated in acute disease, their impact on chronic fibrosis is questionable as a murine model using adoptive transfer of Th1 clones resulted in reversible EAA but not fibrosis (Irifune et al. 2003). Eosinophils attracted to the distal airway by neuropeptides such as neurokinin A may contribute to the inflammatory cascade (Tiberio et al. 2003). Peripheral eosinophilia is less common. Histamine and mast cells have been identified in the bronchoalveolar

Medications Reports of various unrelated medications administered orally have been associated with pulmonary disease resembling EAA. These drugs include amiodarone, gold, celiprolol (betablocker), clozapine, cyclosporin A, dextropropoxyphene (analgesic), fluoxetine, sulfasalazine, nitrofurantoin, chlorambucil, minocycline, procarbazine, hydroxycarbamide (hydroxyurea), flecainide, mesalamine, methotrexate, mexiletine, tocainide,

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ALVEOLAR SPACE Antibody

Antigen (Ag) Ag + Ab Low glutathione, catalase, MnSOD

Abnormal surfactant

NORMAL

Alveolar macrophage (AM)

B7 C5a IL-8

CD8+

Activated AM

CD4+ Th1

CD28 PMN

Eos AM

O3– OH–

Cytokines Increased: IL-2, 8, 12, 16 MIF, IFN-g, NGF Decreased: IL-10

B cell

Cytokines IL-1, 8, 12, TNF-a Chemokines MIP1a, MCP-1 CCL18 AA products – O3, OH–, NO

Plasma cell

Activated endothelium VASCULAR SPACE

Antibody (Ab)

Fig. 85.1 Pathogenesis of extrinsic allergic alveolitis (EAA). This simplified diagram illustrates the proposed mechanism of EAA. After a period of sensitization, inhaled antigens combine with specific antibody leading to the activation of complement and the release of C5a, which attracts polymorphonuclear cells (PMN) into the airway. These cells release toxic oxygen species. Antigens are also engulfed by alveolar macrophages (AM), thus activating the cell to release multiple proinflammatory cytokines and chemokines resulting in an influx of various cells such as eosinophils, mast cells, and lymphocytes (CD8+ > CD4+). Activated alveolar macrophages interact with CD4+ T lymphocytes, resulting eventually in the production

of specific antibody. Activated alveolar macrophages also interact with CD8+ T lymphocytes, again releasing multiple chemical mediators that predominantly promote airway inflammation, activate endothelium, and may lead to collagen synthesis with secretion of glycoproteins resulting in airway fibrosis. Low levels of antioxidant enzymes, abnormal surfactant, viral infections, and smoking can modulate and promote the development of EAA in genetically susceptible individuals. MnSOD, manganese superoxide dismutase. See text for definition of other abbreviations. (See CD-ROM for color version.)

lavage (BAL) fluid of patients with EAA (Soler et al. 1987). Even asymptomatic patients with previous EAA have been shown to have abnormal lung scans using inhaled 99mTclabeled diethylene triamine pentaacetic acid (DTPA) and elevated BAL eosinophils, suggesting continued alveolar disease (Schmekel et al. 1990; Hakansson & Schmekel 1992).

clarify the role of persistence of lymphocytic inflammation (Navarro et al. 2002). Interferon (IFN)-γ produced by Th1 lymphocytes is further stimulated and increased by the addition of IL-12 but reduced with IL-10 (Costabel & Guzman 2001). Cysteinyl leukotrienes and cyclooxygenase-dependent prostaglandin (PG)D2 are released from alveolar macrophages and mast cells during acute EAA and urinary eicosanoids can be used as a biomarker. Following steroid therapy, urinary eicosanoids significantly decrease. In the same patients, serum surfactant protein D, a biomarker of interstitial lung disease, was elevated and decreased following steroid therapy (Higashi et al. 2005). Nerve growth factor (NGF), a neurotrophic cytokine, synthesized by mitogen-activated peripheral blood lymphocytes is higher in asymptomatic pigeon fanciers than in normal control subjects (McSharry et al. 2006). NGF correlated with levels of serum IgG antibody against avian antigens. Serum NGF was normal. Mitogen-stimulated NGF may be an important biomarker and a potential pathway for therapeutic intervention. Chemotactic factors such as IL-8, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α play a role in the inflammatory pattern in EAA (Denis 1995). IL-8 attracts T cells and neutrophils into the alveoli, while MCP-1 engages monocyte/macrophage

Mediators Cytokines, chemokines and adhesion molecules appear to play a part in the pathogenesis of EAA. The alveolitis of EAA has been linked to many endogenous mediators such as IL-1α, IL1β, and TNF-α released from activated alveolar macrophages. These immunoregulatory cytokines upregulate intracellular adhesion molecule (ICAM)-1 through its ligand lymphocyte function-associated antigen (LFA)-1, thereby leading to enhanced antigen-presenting capacity of the alveolar macrophages. The interaction between T cells and alveolar macrophages is enhanced by upregulation of the B7 costimulatory molecules CD80 and CD86 on alveolar macrophages and CD28 on T cells (Israel-Assayag et al. 1999). L-selectin is an adhesion molecule expressed on leukocytes that participates in accumulation of T cells at sites of inflammation. BAL fluid from patients with EAA has upregulated L-selectin. This may

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infiltration. MIP-1α, with its proinflammatory properties, attracts CD8+ T cells and promotes differentiation of CD4+ Th0 cells to Th1 cells. The lymphocyte chemoattractant CCL18 produced by macrophages is found in high levels in subacute and chronic EAA and is likely responsible for T-cell recruitment (Pardo et al. 2001). IL-12 from activated macrophages further skews the development from Th0 to Th1 cells. Antiinflammatory cytokines also participate in the pathogenesis of EAA. IL-10 appears to downregulate the severity of disease activity by modulating the effect of IFN-γ, TNF-α and IL-1. IL-10 was decreased in patients with diisocyanateinduced EAA compared with asymptomatic individuals (Sumi et al. 2003). Other factors that may modulate the severity and persistence of disease include elevations of free radicals and reactive oxygen species, surfactant abnormalities, and depressed levels of antioxidant enzymes. Increased production of nitric oxide by alveolar macrophages occurs during the acute but not the chronic phase of EAA (Lakari et al. 2002). Nitric oxide has direct toxic effects on cells that contribute to lung inflammation. Glutathione is an antioxidant found in high levels in the lungs. Reduced glutathione levels in lung lavage fluid was found in a patient with farmer’s lung after a hay challenge compared with exposed yet asymptomatic farmers (Behr et al. 2000). Furthermore, the radical-scavenging antioxidant enzymes catalase and manganese superoxide dismutase together may protect the lung from progression of interstitial lung disease (Lakari et al. 2000). Pulmonary surfactant has a general suppressive effect on lung lymphocytes and alveolar macrophages. However, with lung injury, surfactant is biochemically and physically modified such that it has less effect on mitogen-induced proliferation, thereby allowing the alveolitis to persist (Israel-Assayag & Cormier 1997; Gunther et al. 1999).

Extracellular matrix Lung lavage fluid from patients with acute EAA has demonstrated elevated levels of glycoproteins such as vitronectin and fibronectin and increased collagen metabolite, procollagenIII peptide. These interstitial lung matrix proteins may be early markers of fibrosis. Thrombin-activable fibrinolysis inhibitor is produced and secreted by lung cells, resulting in reduced plasmin activity leading to excessive accumulation of extracellular matrix in the lung (Fujimoto et al. 2003). The adhesive glycoproteins may participate in tissue remodeling in the extracellular matrix (Teschler et al. 1993).

Antibodies High levels of complement-fixing precipitating antibodies of the IgG and IgM type are the serologic hallmark of EAA. They are believed to be markers of exposure rather than directly involved in disease. A patient with common variable immunodeficiency who was unable to make specific antibodies developed EAA (Schkade & Routes 1996). The role of

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immune complexes is still unclear as skin reactivity induced by antigens may result in a delayed arthus-type reaction with antigen, antibody and complement found on biopsy. However, immune complexes have not been found in serum or lung lavage fluid. Vasculitis and involvement of other organ systems is absent.

Modulating factors Viral infections Viral infections may act as a promoting factor in the development of EAA. Acute symptoms are suggestive of viral infection and many patients report a preceding ’flu-like illness with purulent sputum. Influenza A viral proteins have been observed in high concentrations in BAL macrophages of patients with EAA compared with healthy volunteers (Dakhama et al. 1999). Viral infection can augment the inflammatory response in a mouse model of EAA (Gudmundsson et al. 1999).

Cigarette smoking The incidence of EAA is lower in smokers than in nonsmokers. The effect of inhaling cigarette smoke may influence the immunologic processes in the lungs by affecting phagocytosis by alveolar macrophages. Alveolar macrophages of smokers have lower expression of B7 costimulatory molecules and decreased ability to produce IL-1 and TNF-α (Yamaguchi et al. 1993). While smoking appears to “protect” against the acute form of EAA, individuals with a history of smoking tend to have more severe chronic disease (Ohtsuka et al. 1995). Patients with EAA from metal-working fluids who were cigarette smokers are less likely to develop lung rales, have elevated erythrocyte sedimentation rates, and restrictive spirometry compared with nonsmokers with EAA (Dangman et al. 2004). In a murine model, nicotine had an antiinflammatory effect by decreasing lung tissue inflammation, lymphocyte number, and expression of CD80, TNF-α and IFN-γ production (Blanchet et al. 2004). This downregulatory effect may prevent acute EAA, but subject cigarette smokers to the insidious form that progresses to irreversible lung damage with a poor prognosis.

Clinical features The clinical features of EAA depend on (i) the nature of the organic dust such as particle size, solubility, and antigenicity; (ii) the frequency and intensity of inhalation exposure; and (iii) the immune response of the exposed individual. There is overlap between the various forms and they could represent a spectrum of disease in varying stages of inflammation and repair.

Acute Within 4–12 hours after a heavy exposure to the antigen, high fever (40°C), chills, body aches, nonproductive cough,

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exertional breathlessness, myalgia, and malaise occur. These ’flu-like symptoms may dominate the respiratory symptoms and last up to 24 hours and resolve spontaneously with antigen avoidance. After each subsequent antigen exposure, symptoms will occur but severity may be variable. The acute form of recurrent episodes can develop into a chronic pattern of respiratory and systemic signs and symptoms. Even after exposure ceases, chronic changes and symptoms may persist. Between acute episodes the examination may be normal, but following an antigen exposure the patient appears acutely ill with fever, tachypnea and dyspnea, but no conjunctivitis, rhinitis and pharyngitis. Fine bibasilar end-inspiratory rales or less commonly diffuse wheezing are auscultated and may last for weeks after the fever resolves. Lymphadenopathy, musculoskeletal abnormalities, abdominal symptoms, and rash are absent.

Subacute The subacute form of EAA is subtle and indolent and occurs following repeated low-level antigen exposures over weeks to months. Systemic symptoms are less prominent or even absent, and the pulmonary symptoms predominate. Patients have progressive dyspnea and/or cough with fatigue, malaise, myalgia and anorexia but high fever is lacking. Eventually, severe respiratory distress and cyanosis may occur. Examination typically reveals bilateral dry rales.

Chronic This form presents after months or years of antigen exposure as either insidious onset of slowly progressive dyspnea related to low-level but prolonged antigen exposure or as progressive symptoms following recurrent acute episodes. Typically the patient lacks high fever, chills and sweats but reports dyspnea, fatigue, weakness, anorexia, and weight loss. The patient may appear chronically ill, exhibit fine dry rales or wheezing, and rarely has nail clubbing. In children this form is marked by weight loss hypothesized to be related to malabsorption and steatorrhea associated with swallowing the antigen.

Investigations The diagnosis of EAA is challenging and established through a combination of clinical features and environmental exposures supported by laboratory, radiographic, and lung function data (Table 85.2). EAA should be considered in patients with recurrent acute, chronic or progressive lower respiratory symptoms, recurrent pneumonia, interstitial lung disease, recurrent fever, or weight loss (Fink et al. 2005). When the diagnosis is unclear, invasive procedures such as BAL and/or histopathology is useful in ruling out other disorders. The single most important process is obtaining a detailed and thorough history of exposures at home and work and during

Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

Table 85.2 Investigations into extrinsic allergic alveolitis. History Occupation: farming, forestry, chemicals, factory Home: mold incursion/dampness, pets (birds) Hobby: birds, composting, woodworking Medications Examination Fever, weight loss, rales, clubbing Radiology Chest X-ray High-resolution computed tomography Pulmonary function testing Spirometry Lung volumes Diffusion of carbon monoxide Arterial blood gas after exercise Laboratory Serum precipitins, quantitative immunoglobulins Complete blood count Erythrocyte sedimentation rate, C-reactive protein, lactate dehydrogenase Antinuclear antibody Bronchoscopy/bronchoalveolar lavage Cell phenotyping: CD8+ > CD4+ Specific IgG and IgA antibody Gram stain Culture Lung biopsy Open, transthoracic, transbronchial Challenge Hospital Natural

any hobbies (Jacobs et al. 2005). The diagnosis can be made clinically if it includes at least two of the following criteria: 1 symptoms compatible with EAA; 2 evidence of exposure to an antigen either by history or by detection of antibody in the serum or lung lavage fluid; 3 chest X-ray or high-resolution computed tomography (HRCT) with compatible findings such as ground glass infiltrates; 4 lymphocytosis in lung lavage fluid (if bronchoscopy is performed); 5 compatible histopathologic changes (if lung biopsy is performed); 6 reproduction of symptoms, laboratory and lung function abnormalities after exposure to the suspect environment. Other criteria that nearly all patients exhibit, but which also overlap with other interstitial lung diseases, include dyspnea on exertion, bibasilar dry inspiratory rales, recurrent fever,

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decreased lung diffusion capacity, and arterial hypoxemia at rest or with exercise. In contrast to previous guidelines, precipitating antibodies and lymphocytic alveolitis with reduced CD4+/CD8+ ratio are not required for diagnosis although they are frequently present (Richerson et al. 1989; Schuyler & Cormier 1997).

Laboratory No single test is specific for EAA, but symptomatic patients typically have findings consistent with pulmonary inflammation and systemic symptoms. In the acute form, a significant leukocytosis with left shift may be seen with eosinophilia up to 20%. Many patients have elevated erythrocyte sedimentation rate, C-reactive protein, and serum lactate dehydrogenase but their usefulness in monitoring disease activity is unclear. Rheumatoid factor may be positive, but antinuclear antibody is usually negative. Quantitative serum immunoglobulins (IgG, IgM, IgA) may be elevated except for IgE, unless there is concomitant atopy. Serum complement levels are normal or slightly increased. In the acute form, arterial blood gases reveal hypoxemia with respiratory alkalosis, while hypoxemia with exertion occurs in the chronic form. The characteristic immunologic finding in EAA is serum precipitating antibodies directed against the specific offending antigen as identified by Ouchterlony gel technique or other immunoassays (Fig. 85.2). The presence of serum precipitins confirms exposure to an antigen but can be detected in up to 50% of similarly exposed asymptomatic individuals. It is unclear if the antibody is involved in pathogenesis of the disease or is simply a marker of exposure. While precipitin assays are relatively insensitive and detect larger concentrations of specific IgG, newer ELISA immunoassays can detect smaller amounts of antibody. A negative precipitating antibody panel in a clinically confirmed case of EAA may be the result of nonstandardized antigens, incorrect antigens, or low serum concentrations of IgG antibody in the patient. Serum precipitins may disappear over time if antigen exposure has ceased. Lymphocyte transformation studies using the specific antigen are positive in symptomatic patients but can also be positive in up to 15% of asymptomatic exposed individuals.

Fig. 85.2 Serum precipitins: serum precipitating antibodies as demonstrated by Ouchterlony double immunodiffusion gel system. The intensity of the stained bands between the central wells containing antigen and top wells containing patient serum indicates the presence of precipitating antigen–antibody complexes.

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In a large percentage of patients with bird breeder’s disease, elevated levels of macrophage migration inhibition factor (MIF) can be found compared with asymptomatic bird breeders. Potential new biomarkers involved in the immunologic pathogenesis are serum surfactant protein D (SP-D) and urinary eicosanoid (8-isoPGF2α) (Higashi et al. 2005). Skin-prick testing would be unnecessary as the immunopathogenesis is non-IgE-mediated. Although nitric oxide is believed to play a role in EAA pathogenesis, no data are available on breath analysis of exhaled nitric oxide. Intradermal testing has been evaluated, but is not routinely performed due to both falsepositive and false-negative skin reactions.

Radiologic features The radiographic features do not necessarily correlate with duration of disease and all but honeycombing can be completely or partially reversible.

Chest radiography In the acute form, a plain chest radiograph typically reveals bilateral diffuse ground-glass patchy opacification and interstitial infiltrates or a fine nodular or reticulonodular pattern (Fig. 85.3) (Glazer et al. 2002). Findings notably absent include pleural effusion, lymphadenopathy, and single nodular lesions. Between acute exacerbations, the chest radiograph can appear normal. During the subacute phase the reticulonodular pattern becomes more prominent, with general coarsening of the

Fig. 85.3 Chest radiograph of acute EAA. (Courtesy of Dr Lawrence Goodman, Milwaukee, Wisconsin.)

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Fig. 85.4 A 56-year-old with chronic farmer’s lung EAA. Chest radiograph shows large lung volumes with bilateral coarse fibrosis with an apex to base gradient. (Courtesy of Dr David Edmondson, Wausau, Wisconsin.)

bronchovascular markings. In the chronic form, fibrosis with volume loss, honeycombing, upper lobe contractions and reticular opacities can be seen (Fig. 85.4).

High-resolution chest computed tomography The greater sensitivity of HRCT compared with plain radiographs makes this the method of choice. A variety of patterns can be seen, including ground-glass opacification, centrilobular nodules, fibrosis, mosaic attenuation/air trapping, and emphysema (Fig. 85.5). In contrast to plain chest radiography, up to 50% of patients have small mediastinal lymphadenopathy with nodes less than 20 mm. The ground-glass opacification is a hazy pattern seen during times of antigen exposure (acute, subacute or chronic) that can be patchy or diffuse and predominate in the middle lung zones. This can resolve with removal from antigen exposure. Ground-glass opacification is believed to be related to alveolar inflammation or fine fibrosis and is observed with a restrictive ventilatory pattern on pulmonary function tests and impaired gas exchange. Centrilobular nodules are small poorly defined nodules less than 5 mm in diameter and found throughout the lung but which tend to cluster in the mid to lower lung zones. They are frequently seen with ground-glass attenuation and resolve with removal from antigen exposure similar to ground-glass attenuation. In the chronic form, and occasionally in the subacute form, varying degrees of fibrosis can be identified as irregular linear opacities, traction bronchiectasis, honeycombing, and lobar volume loss. There can be a mid-zone

Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

Fig. 85.5 High-resolution chest computed tomography scan of acute EAA. (Courtesy of Dr Lawrence Goodman, Milwaukee, Wisconsin.)

predominance, but the upper and lower lobes can also be involved. Emphysematous changes can be more common than fibrosis in the chronic forms of EAA in bird fancier’s lung and farmer’s lung (Fig. 85.6). The mosaic pattern is a patchwork of ground-glass attenuation and air-trapping combinations and is commonly found in EAA. The air trapping seen on

Fig. 85.6 High-resolution chest computed tomography scan of chronic EAA. (Courtesy of Dr Lawrence Goodman, Milwaukee, Wisconsin.)

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0 % Change from baseline

DLCO

50

FVC: heavy lines FEV1: light lines

Biphasic response, dashed Iines; Late-onset response, solid lines

Temp

2

4

Antigen exposure

6

8

10

24

Time in hours

Fig. 85.7 Pulmonary function changes in acute EAA illustrating the spirometric biphasic response and late-onset response to inhaled antigen. Both forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) decrease consistent with a restrictive ventilatory defect. The diffusion capacity to carbon dioxide (DLCO) decreases. Also during this acute reaction, the total white blood cell count increases and fever develops. Within 24 hours of exposure the parameters gradually return to normal if further antigen exposure is avoided.

HRCT correlates with airway obstruction on pulmonary function testing (Glazer et al. 2002).

Pulmonary function Complete pulmonary function tests including spirometry should be obtained. A variety of patterns can be observed (Fig. 85.7). A restrictive ventilatory defect with a decrease in both forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) is classically observed 4–6 hours after exposure to the antigen. Peak flow rates are generally normal. A decrease in diffusion capacity as measured by DLCO is seen. Arterial blood gases demonstrate hypoxemia occasionally at rest but frequently with exercise. Another pattern is a biphasic response, with an immediate decrease in FVC, FEV1, and peak flow, followed by a late reaction as just described. This has been reported more frequently in pigeon breeder’s disease as opposed to farmer’s lung. In chronically ill patients a mixed obstructive and restrictive pattern can be observed that is variably responsive to bronchodilators.

CD8+ ratio in most (Costabel & Guzman 2001). In contrast, sarcoidosis is classically associated with lymphocytosis and a high ratio of CD4+/CD8+ cells. In EAA, activated alveolar macrophages, mast cells, and natural killer cells are observed. The Th1 cytokine IFN-γ is produced from the lymphocytes (Yamasaki et al. 1999). Soluble TNF-α receptors 1 and 2 are increased and this correlates with BAL lymphocyte percentage. This finding can also be seen in active pulmonary tuberculosis. Large numbers of neutrophils are seen early in acute EAA episodes, leading to oxidative stress with decreased concentrations of oxidized and reduced glutathione. Specific IgG and IgA antibody is found but this can also be seen in asymptomatic but exposed individuals. Elevated IgM in BAL is found in symptomatic patients. Cultures for microbial organisms are negative.

Histopathology In some cases lung biopsy is necessary to determine the diagnosis or aid in eliminating other diseases in the differential diagnosis. Open lung biopsy is generally preferred as it yields better biopsy specimens than transbronchial biopsies, although the transthoracic approach is a suitable alternative. The classic triad of EAA histopathology is cellular bronchiolitis, lymphoplasmocytic interstitial infiltrate, and poorly formed nonnecrotizing granulomas (Fig. 85.8). Unfortunately, this triad is frequently not present and the findings depend on the stage of the disease and the intensity of antigen exposure. Typical findings are patchy interstitial infiltrates with peribronchiolar accentuation of plasma cells and lymphocytes, nonnecrotizing granulomas with epithelial histiocytes, and foci of bronchiolitis obliterans (Katzenstein 1997). The mechanisms that may underlie the pathologic findings are reviewed below.

Acute form The alveolar and interstitial inflammation has a marked lymphocyte predominance. There are activated macrophages

Bronchoalveolar lavage Consultation with a pulmonary specialist for bronchoscopy with BAL is a standard diagnostic procedure for the majority of patients presenting with interstitial lung disease. Various studies can be helpful in excluding causes of pneumonitis and assessing the immunologic milieu. BAL in nonexposed asymptomatic individuals reveals low numbers of cells with mostly alveolar macrophages and CD4+ lymphocytes. In EAA patients and asymptomatic exposed individuals, BAL reveals a striking lymphocytosis with an absolute increase in both CD4+ and CD8+ cells. In symptomatic individuals there are predominantly CD8+ T cells resulting in a decreased CD4+/

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Fig. 85.8 Low-power view of an open lung biopsy specimen from a patient with chronic EAA revealing noncaseating granulomas and lymphocytic infiltrate. (See CD-ROM for color version.)

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(described as having “foamy” cytoplasm), plasma cells and neutrophils present. Granulomas are only seen in 60–70% of acute cases (Yoshizawa et al. 1999). Lung epithelial cells and activated alveolar macrophages release large amounts of proinflammatory cytokines and chemokines that recruit a variety of cells into the interstitium. MIP-1α contributes to the CD8+ lymphocyte predominance, while neutrophils are attracted by CXCL8/IL-8 and TNF-α. IL-1 and TNF-α (which have similar biological effects) mediate leukocyte adhesion through increased expression of ligands on endothelial cells. The lymphocyte predominance is the result of recruitment and/or local proliferation of CD8+ T cells. IFN-γ is likely responsible for the granuloma formation.

Subacute form During this stage, there is usually a relatively mild usually peribronchiolar interstitial infiltrate frequently accompanied by poorly formed interstitial noncaseating granulomas or isolated giant cells. The interstitial process becomes more fibroblastic with obliteration of the alveolar space. During this phase, the likely mechanism is continued effect of IFN-γ contributing to granulomas and increased percentages and absolute numbers of CD8+ T cells with regulatory activity (suppressor and cytotoxic activity) due to chemokines such as IL-1 and IL-12 secreted from activated macrophages (Agostini et al. 2004). Simultaneously, activated macrophages are facilitating repair by secreting growth factors that stimulate fibroblast proliferation and collagen synthesis. The isolated multinucleated giant cells may represent fusion of activated macrophages.

Chronic form At this stage there are intraalveolar foamy macrophages with less prominent lymphocytic alveolitis. Mast cells and plasma cells dominate the interstitial inflammation. There are noncaseating granulomas in less than 50% of cases, intraalveolar buds, and interstitial fibrosis with collagen-thickened bronchiolar walls. A usual interstitial pneumonitis (UIP)-like pattern, nonspecific interstitial pneumonitis (NSIP)-like pattern, and an irregular peribronchiolar pattern have been described (Churg et al. 2006). These patterns did not all occur in the same biopsy specimen. This obstructive bronchiolitis may lead to destruction of alveolar walls, resulting in fibrosis, honeycombing and/or cystic changes. Findings of connective tissue destruction or vasculitis should prompt an investigation for other etiologies. Immunofluorescent studies of lung biopsy specimens have detected antigen in alveolar septae and in the interstitium. Immunoglobulin and complement are rarely found in lung biopsies. The mechanism at this stage is consistent with a chronic delayed-type hypersensitivity reaction, with connective injury being replaced by fibrosis possibly related to continued production of cytokines and growth factors. While the presence of plasma cells, mature antibody-secreting B cells, is expected and the antibody de-

Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

tected in BAL fluid or serum, the role of mast cells involved in type I immediate hypersensitivity reactions involving IgE is uncertain. Ongoing antigen exposure may lead to persistent traffic of activated neutrophils that show intense immunoreactive collagenase-2 and gelatinase B staining. This could play a role in the evolution of fibrosis as in other interstitial lung diseases (Agostini et al. 2004).

Antigen challenge Purposeful challenges can be conducted using two methods. First, the patient can return to the environment suspected of harboring the antigen, thus undergoing a “natural” challenge. Laboratory, clinical, and lung function measurements are assessed before and after exposure. Further investigation of the site with cultures can be performed if the challenge is positive. Secondly, an intentional hospital challenge to a suspect antigen using a nebulizer is typically reserved for unique cases where symptoms and clinical abnormalities are vague or undefined, in clinical research, or when a new antigen is under investigation. Testing is limited by a lack of standardized antigens. Material used for testing may be contaminated with nonspecific irritant. Challenges should be performed by qualified personnel in research centers with experience. Usually a transient inflammatory response is observed. Methacholine or other challenges assessing for bronchial hyperresponsiveness are ineffectual in distinguishing asthma from EAA as some patients with EAA will be positive.

Other If improvement occurs spontaneously after the patient is removed from the suspect environment, this is suggestive of EAA. Similarly, a rapid response to parenteral steroids should alert one to EAA.

Differential diagnosis A variety of interstitial lung diseases in addition to EAA should be considered when evaluating patients with recurrent progressive pulmonary symptoms (Table 85.3). The disorders to be considered should take into account patient age and type of presentation such as acute, subacute or chronic.

Infections Acute EAA is frequently misdiagnosed as an acute lower respiratory tract infection. Outpatient treatment with antibiotics has no effect as long as the patient continues to be exposed to the antigen. If the patient is hospitalized and thus removed from the antigen source, symptoms will resolve spontaneously. Acute infections that may mimic symptoms include legionellosis, Mycoplasma pneumonitis, bacterial pneumonia, psittacosis, Q fever, and malaria. Chronic EAA should be differentiated from disseminated tuberculosis and Mycobacterium avium complex.

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Table 85.3 Differential diagnosis of extrinsic allergic alveolitis. Acute form Infections Pneumonia: viral, bacterial, Mycoplasma Pontiac fever (Legionella) Psittacosis (Chlamydia psittaci) Q fever (Coxiella burnetii ) Pneumocystis carinii Malaria Organic dust toxic syndrome Grain fever, animal house fever, toxic alveolitis, humidifier fever, pulmonary mycotoxicosis Textile dust-related disease Byssinosis: cotton dust and endotoxin Mill fever (tannins in cotton mill dust, kapok) Weaver’s cough (tamarind seed powder) Toxic fume bronchiolitis Nitrogen and sulfur dioxides, chlorine, ozone, phosgene and ammonia Silo unloader’s disease (nitrogen dioxide) Collagen vascular diseases Dermatomyositis, scleroderma, Sjögren syndrome, polymyositis, rheumatoid arthritis Medication reactions Gefitinib, rituximab, sirolimus Chronic form Chronic granulomatous infections Disseminated tuberculosis Mycobacterium avium complex Inorganic respiratory dust syndromes Berylliosis, silicosis, asbestosis Coal worker’s pneumoconiosis Idiopathic interstitial pneumonitis UIP, DIP, LIP, NSIP, BOOP, RB-ILD and cryptogenic organizing pneumonia Textile dust-related disease Byssinosis (cotton, hemp, flax, jute, sisal) Nylon flock Other conditions Radiation pneumonitis Sick building syndrome Pulmonary sarcoidosis Chronic ammonia inhalation “Lifeguard lung” Alveolar proteinosis Pulmonary embolism Histiocytosis X Pediatrics: lipid storage disease, UIP of infancy, familial DIP, surfactant deficiency, BPD, persistent tachypnea of infancy, CIP of infancy BOOP, bronchiolitis obliterans organizing pneumonia; BPD, bronchopulmonary dysplasia; CIP, cellular interstitial pneumonitis; DIP, desquamative interstitial pneumonitis; LIP, lymphocytic interstitial pneumonitis-associated with Sjögren syndrome; NSIP, nonspecific interstitial pneumonitis; RB-ILD, respiratory bronchiolitis-associated interstitial lung disease; UIP, usual interstitial pneumonia (formerly idiopathic pulmonary fibrosis).

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Building-related illnesses (“sick building syndrome”) Because EAA is frequently linked to exposures at the workplace, building-related illness should be considered. Immunologic, infectious, toxic or irritant mechanisms may account for symptoms in buildings with inadequate ventilation. Levels of bioaerosols and volatile organic compounds such as aldehydes may increase in airtight buildings engineered to conserve energy or those workplaces with inadequate engineering design, faulty installation, poor maintenance, antiquated equipment, and crowding. Other environmental factors that appear to be associated with sick building syndrome include increased vibration and sound and lighting that is high in intensity and glare. This tends to affect many workers with vague nonspecific and subjective complaints related to ocular, respiratory and neurobehavioral symptoms such as headache, memory loss, depression, and dizziness. The affected individuals tend to have more job-related tension and lower job satisfaction (Barrios et al. 2004).

Humidifier fever When Gram-negative organisms contaminate water used in humidification and cooling, endotoxin can be released when the water is sprayed into the air, resulting in fever, malaise, and mild cough within 4– 8 hours (Pickering 1982; Rylander et al. 1989). Chest radiography is normal and symptoms clear within a few hours. Pulmonary function tests may show mild airway obstruction but normal diffusing capacity. Direct instillation of endotoxin into the lungs of normal humans results in an early phase influx of neutrophils, cytokines and chemokines in BAL. At 24 – 48 hours, increased numbers of neutrophils, macrophages, monocytes, and lymphocytes are present (O’Grady et al. 2001). Outbreaks of respiratory disease with granulomatous pneumonitis occurred after exposure to indoor swimming pools with extensive spray features contaminated with pathogenic bioaerosols with elevated endotoxin levels. This condition, designated “lifeguard lung,” was associated with a CD4+ lymphocytosis in lung lavage fluid, high attack rate, and short duration of exposure (Rose et al. 1998).

Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

Disorders with lymphocytic infiltrates Other lung disorders associated with a lymphocytic BAL include pulmonary sarcoidosis, chronic beryllium disease, tuberculosis, connective tissue disorders, drug-induced pneumonitis, malignant infiltrates, silicosis, Crohn disease, primary biliary cirrhosis, HIV infection, and viral pneumonia. Pulmonary sarcoidosis is characterized by hilar lymphadenopathy, elevated angiotensin-converting enzyme level, CD4+ alveolitis, and a positive gallium scan with involvement of the parotid gland, lymph nodes, and extrathoracic tissues.

Diffuse parenchymal lung diseases Recent advances in the diagnosis of interstitial pneumonitis have clarified important differences in the clinical features, therapeutic responses, and long-term outcome of the heterogeneous group of disorders comprising UIP (formerly known as idiopathic pulmonary fibrosis), NSIP, desquamative interstitial pneumonitis, and others (Martinez & Keane 2006). UIP is differentiated from EAA by clubbing on examination, predominance of neutrophils in BAL, biopsy consistent with UIP with less ground-glass attenuation, and a poor outcome. The finding of NSIP has been associated with EAA and various connective tissue diseases such as polymyositis, primary Sjögren syndrome and rheumatoid arthritis. Neonates and young children with severe interstitial disease should be evaluated for surfactant protein C deficiency.

Textile dust-related interstitial lung disease Nylon flock is pulverized synthetic or natural fibers applied to fabrics to create a velvet-like surface. In plants where nylon flock is processed, workers have developed pulmonary inflammation with clinical features similar to EAA but lacking immunologic abnormalities (Eschenbacher et al. 1999). Byssinosis refers to the disease resulting from the inhalation of dust in textile and cotton mills. Affected workers have fever and respiratory symptoms at the beginning of the work week, with cough and decreased FEV1 and FVC. Evaluation of cotton dust has yielded endotoxin, proteases and fungi. Long-term exposure results in substantial declines in lung function and persistence of symptoms (Wang et al. 2005).

Organic dust toxic syndromes For agricultural workers, organic dust toxic syndrome is a noninfectious febrile illness occurring after workers are exposed to high levels of bacteria and fungi. This syndrome has been colorfully named as grain fever, silo unloader’s disease, and pulmonary mycotoxicosis. It typically affects a majority of individuals exposed. Lung lavage fluid reveals increased total cells including neutrophils, spore and fungal elements. After the first week, a CD8+ lymphocytic alveolitis is present (Reymenants et al. 1990). Similarly, exposure to ammonia, endotoxin, and sulfur dioxide in animal confinement buildings can trigger symptoms without evidence of sensitization and with complete resolution after a brief period of symptoms. This has been coined “animal house fever.”

Treatment/management Avoidance Similar to other allergic lung diseases, the best treatment for EAA is avoidance of the specific antigen trigger. This alone may be sufficient intervention. In occupational EAA, job retraining or changing professions may be required, although this results in substantial financial and emotional stress. In industrial exposures, enclosing a specific work process, changing working materials, and instituting personal respiratory protection may be helpful. In an effort to prevent farmer’s lung, treating hay with buffered propionic acid significantly

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decreased the concentration of fungi and thermophilic actinomycetes without harmful effects on machinery or cattle (Reboux et al. 2002). Hay conditioners and dryer systems may be effective, but implementation can be cost prohibitive (Dalphin et al. 1991). In residential mold-induced EAA, remediation measures by professionals may include dehumidification, removal of carpet, wallboard, wood and insulation, and decontamination with bleach or other fungicides. In Japan preventive measures have decreased summer-type EAA (Yoshida et al. 1989).

cyclosporin A has failed to achieve effective concentrations in the lower respiratory tract in sarcoidosis (Garcia et al. 1998). Studies in humans are lacking. Allergen immunotherapy with the specific antigens has not been reported and is not recommended due to the potential adverse effects from toxins or the development of vasculitis. Although eicosanoids and leukotrienes participate in the pathogenesis of EAA, there are no reports evaluating the effects of leukotriene-receptor antagonist therapy.

Pharmacotherapy

Course and prognosis

Treatment alternatives are limited and inadequately studied. Systemic corticosteroids are recommended for acute, severe, or progressive forms and generally relieve the symptoms in dramatic fashion. They may not necessarily change the longterm prognosis. The dose of oral steroids ranges from 40 to 80 mg daily for 1–2 weeks for acute disease, while chronic EAA may require similar doses but a gradual reduction over several months based on clinical and laboratory improvement. Supplemental oxygen is indicated in ill patients with hypoxemia. Other treatments have been described in case reports. After oral steroids, inhaled beclomethasone 400 μg daily in hydrofluoroalkane-134a (HFA) propellant successfully treated mild EAA. The extra-fine mist associated with HFA propellant is hypothesized to improve drug delivery to the distal airways and alveoli (Tanaka et al. 2004). Farmer’s lung was successfully treated with five courses of intravenous methylprednisolone (15 mg/kg on three consecutive days) 1 month apart followed by inhaled budesonide (Chen et al. 2003). Although the hypoxemia resolved quickly, the patient had persistent BAL lymphocytosis. Inhaled cromolyn sodium prevented symptoms and albuterol reversed an acute fall in FEV1 in laboratory challenge setting but there is no evidence that either can prevent disease during natural exposure. There is insufficient evidence on using inhaled corticosteroids alone, although they may be helpful if an obstructive component is established. Similarly, short-acting bronchodilators may be helpful if reversible airway obstruction is identified. Several treatments have been studied in vitro. In cases of hot-tub lung associated with Mycobacterium avium complex, antimycobacterial drugs have been administered (Marras et al. 2005). Pentoxifylline, a nonselective phosphodiesterase inhibitor used for treating vascular insufficiency, decreased cytokine production (TNF-α and IL-10) from alveolar macrophages in patients with EAA (Tong et al. 2004). Long-term use of macrolide antibiotics has antiinflammatory effects in subjects with chronic lower respiratory tract inflammation. In a mouse model of EAA, erythromycin suppressed the early influx of neutrophils, inhibiting the upregulated expression of ICAM-1 (Miyajima et al. 1999). Cyclosporin A has been shown to inhibit basal secretion of TNF-α from human alveolar macrophages in vitro, but systemic administration of

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Although the prevalence of EAA appears low, the impact it has on individuals is a major concern. After a period of exposure ranging from months to years, the individual will become sensitized and develop symptoms on subsequent exposures. Left untreated and with continued exposure, disease progression can result in irreversible pulmonary damage. The duration of exposure and nature of the antigen both play a role: their long-term effect on pulmonary function is still unclear. Early treatment of acute or subacute disease results in complete recovery for nearly all patients, while those with chronic EAA may develop permanent sequelae such as progressive interstitial fibrosis, emphysema or asthma-like symptoms. The clinical course is variable and even a single severe acute episode may result in sequelae, while patients with subacute or chronic disease can progress despite avoidance or remain stable notwithstanding continued exposure (Zacharisen et al. 2002). These data stem primarily from pigeon breeder’s disease and farmer’s lung patients and direct comparisons may not be appropriate. Oral corticosteroid therapy can improve symptoms and reduce hypoxemia, but has not been shown to affect the long-term prognosis (Kokkarinen et al. 1992). Patients with EAA and pulmonary fibrosis are more likely to be older, exhibit a greater restrictive lung defect, and have a diminished median survival of 7.1 years after diagnosis compared to those without fibrosis (Vourlekis et al. 2004). Fatal cases of EAA have been reported and long-term mortality rates range from 1 to 10% (Zacharisen & Schoenwetter 2005). Although smokers are less likely to develop acute EAA, the outcome in smokers may be poor due to the progression of inflammation resulting in chronic EAA.

Prevention The single most important factor in managing EAA is preventing further exposure to the antigen. This can be accomplished by either removing the antigen from the environment or removing the patient from the area or environment containing the antigen. Where avoidance of the antigen is not fully possible, reducing inhalation exposure by wearing a well-fitted mask may help.

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If the antigen is identified, mechanisms to remove the antigen from the patient’s environment are undertaken. For example, if fungus is identified in an individual’s home, measures to decrease fungal proliferation and remediate the problem may include the following. 1 Increase ventilation by opening windows and using exhaust fans. 2 Maintain humidity < 50% by operating air conditioners and/or dehumidifiers. 3 Avoid indoor items such as aquariums, fountains, clothes hampers, hot tubs, plants and humidifiers/vaporizers that increase air moisture. 4 Regularly treat stagnant water sources such as water traps, garbage disposals, and commode tanks with bleach. 5 Remove garbage daily. 6 Install and frequently clean high-efficiency filters. 7 Regularly clean air-conditioning systems and refrigerator condensation pans and water-prone areas beneath and behind appliances. 8 Repair water damage to the structure. If bird fancier’s lung is identified, it is imperative to remove the patient from the premises until a thorough cleaning of the area can be accomplished. The bird(s) should be removed from the house and patient should abstain from cage-cleaning. New methods of farming such as mechanical feeding systems, efficient drying of hay and cereals, and improved ventilation of farm buildings has markedly decreased the incidence of farmer’s lung (Zejda et al. 1993). Chemically treating hay or sugarcane waste may reduce the proliferation of thermophilic bacteria. For machinists exposed to contaminated metal-working fluid, personal respiratory protection, improved ventilation with better general air handling (eliminating mist cooling and increasing dilutional ventilation), and enclosing selected automated machining operations may help. The affected coolant systems should be promptly drained and steam cleaned (Bracker et al. 2003). After the system is recharged with new coolant, an improved fluid management program using phenolic biocides may control mycobacteria but also have chemical toxicity. Regular monitoring of the chemical, biological and physical properties of metal-working fluid may prove helpful, but current quantitative exposure assessment tools are of limited utility. With good metal-working fluid practices that not only minimize skin and inhalation exposure but keep the fluid as clean as possible before systems are heavily contaminated, worker health should improve (Stear 2005). If the antigen is not specifically confirmed but a relationship to an environment is established, the patient should be removed from that environment. When a person has been diagnosed with EAA, this sentinel event should prompt urgent and further investigation of worker practices and additional monitoring of other individuals with similar exposures.

Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

Needs and opportunities for research in EAA There remain major unanswered questions relevant to our understanding of EAA (Fink et al. 2005). The relative importance and contribution of innate immunity, humoral immunity, and adaptive T-cell responses in the pathogenesis of EAA is unknown. The role of host susceptibility factors on the occurrence, development and severity of EAA is in the early stages of elucidation through the identification of genetic polymorphisms. Similarly, the role of environmental promoting factors such as viral infections, smoke, and endotoxin in the development of EAA is still unclear. These major questions will form the basis for future research into EAA. Further characterization of the nature of the antigen through detailed molecular analysis with production of purified recombinant antigen will enable the development of specific and sensitive assays. Additional research is needed to define the actions of cytokines and chemokines on granuloma formation and lung tissue injury at a molecular level. Continued development of animal models and human experimental challenge studies should be designed and implemented. Improved assays for T-cell proliferative responses using cytokine production are needed to overcome the high background responses presently encountered. These high background responses are likely due to the presence of multiple immunostimulatory factors in the complex antigen mixtures. The role of regulatory T cells in preventing the development of EAA in exposed but asymptomatic individuals is an area with excellent opportunities. Continued study of markers of susceptibility will aid in the identification of individuals at high risk of developing disease or developing severe disease. The availability of genomics and proteomics, including tissuelevel microarray analysis, may help distinguish EAA from other interstitial lung diseases, thus assisting in rapid diagnosis and direction of treatment options. Additional methods of pharmacologic management need to be explored particularly for patients with chronic unremitting disease not improved with corticosteroids. To obtain adequate numbers of patients with EAA, these studies will require the collaboration of a network of researchers from a variety of fields such as basic science, clinical research, and epidemiology. This collaboration could result in improved recognition, diagnosis and management of EAA and a storehouse for serum, tissue and radiographic images to promote clinical and laboratory-based research.

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Pulmonary Eosinophilia Jean-François Cordier and Vincent Cottin

Summary Eosinophilic lung diseases encompass a wide and heterogeneous spectrum of disorders characterized by prominent infiltration of the lungs by polymorphonuclear eosinophils. Peripheral blood eosinophilia greater than 1 × 109/L (and preferably > 1.5 × 109/L) is present in most cases of eosinophilic pneumonia, and rapidly orientates the diagnosis when present. However, blood eosinophilia may be lacking, especially in the early phase of idiopathic acute eosinophilic pneumonia, or in patients already receiving oral corticosteroids. High eosinophilia at bronchoalveolar lavage (> 25%, and preferably > 40%) is currently the method of choice to establish the diagnosis of eosinophilic pneumonia in a compatible setting. There is very little indication, if any, for pulmonary biopsy in the diagnostic process of the eosinophilic lung diseases. Eosinophilic lung diseases may manifest clinically by a pulmonary disease of varying severity, ranging from chronic or transient infiltrates with mild symptoms (Löffler syndrome) to the acute severe eosinophilic pneumonia resembling acute respiratory distress syndrome and necessitating mechanical ventilation. The most common presentation is that of idiopathic chronic eosinophilic pneumonia, with chronic onset of dyspnea, cough, weight loss, fatigue, mild fever, and patchy alveolar infiltrates on chest imaging, predominating in the upper lobes and in the peripheral areas of the lungs, and possibly migratory. The etiologic diagnosis of eosinophilic lung diseases is crucial, and includes a systematic search for potential causes, especially drug intake, illicit drugs, toxics, and infection with parasites or fungi. Laboratory investigations for parasitic causes must take into account the specific epidemiology of parasites. Biological investigations for allergic bronchopulmonary aspergillosis should be prompted by, but not restricted to, the presence of proximal bronchiectasis in patients with asthma or cystic fibrosis. When no cause is found, the eosinophilic lung disease is considered idiopathic. Systemic eosinophilic diseases such as Churg–Strauss syndrome or the hypereosinophilic syndrome are suspected in the presence of extrathoracic manifestations; the clinical phenotypes and biological correlates of both these disorders are currently being revised.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Treatment of a determined cause may provide improvement (e.g., drug withdrawal in drug-induced disease). Corticosteroids remain the cornerstone of symptomatic treatment for eosinophilic disorders, and these are necessary in idiopathic chronic or acute eosinophilic pneumonia, flares of allergic bronchopulmonary aspergillosis, and Churg–Strauss syndrome (with the addition of immunosuppressants in patients with poor prognosis factors). Despite dramatic response to corticosteroids in idiopathic chronic eosinophilic pneumonia and in most cases of Churg–Strauss syndrome, relapses often occur when tapering the doses or after stopping treatment. Imatinib, a tyrosine kinase inhibitor, has recently proven very effective in the treatment of steroid-resistant forms of the myeloproliferative variant of hypereosinophilic syndrome.

Introduction The eosinophilic lung diseases are defined by prominent infiltration of the lung by eosinophils, especially eosinophilic pneumonia, often associated with concomitant marked blood eosinophilia (> 1 × 109/L, and especially > 1.5 × 109/L). Diseases where eosinophils are considered to play a significant role but not fitting the above criteria are not discussed in this chapter (e.g., the eosinophilic phenotype of asthma).

The eosinophilic pneumonias Eosinophilic pneumonia is a pneumonia where eosinophils are the most prominent inflammatory cells on histopathologic examination. Although other inflammatory cell types (especially lymphocytes and neutrophils) are often associated, the eosinophils clearly predominate. Eosinophilic pneumonias manifest as chronic, subacute, or acute syndromes (Table 86.1). In the vast majority of idiopathic cases, the response to corticosteroid treatment is dramatic, and healing leaves no significant sequelae. The eosinophilic pneumonias may be separated into two main etiologic categories: those where a definite cause is found, and those of undetermined origin (i.e., idiopathic), with the eosinophilic pneumonia being either solitary or part of a systemic syndrome such as Churg–Strauss syndrome

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Table 86.1 Classification of the eosinophilic pneumonias. Eosinophilic lung disease of undetermined cause Idiopathic eosinophilic pneumonias Idiopathic chronic eosinophilic pneumonia Idiopathic acute eosinophilic pneumonia Churg–Strauss syndrome Hypereosinophilic syndrome Myeloproliferative variant Lymphocytic variant Unclasssified Eosinophilic lung disease of determined cause Eosinophilic pneumonias of parasitic origin Eosinophilic pneumonias of other infectious causes Allergic bronchopulmonary aspergillosis and related syndromes Allergic bronchopulmonary aspergillosis Other allergic bronchopulmonary mycoses Bronchocentric granulomatosis Drug-, toxic agent-, and radiation-induced eosinophilic pneumonias Drugs Toxic agents and illicit drugs Eosinophilic pneumonia induced by radiation therapy to the breast Miscellaneous lung diseases with possible associated eosinophilia “Simple” eosinophilic asthma Eosinophilic bronchitis Langerhans cell granulomatosis Organizing pneumonia Other lung diseases with occasional eosinophilia Idiopathic interstitial pneumonias and interstitial pneumonia in the connective tissue diseases Sarcoidosis Lung transplantation Paraneoplastic eosinophilic pneumonia

(CSS). A definite cause must be systematically investigated in any patient with eosinophilic pneumonia because it usually has practical consequences (e.g., stopping a drug responsible for iatrogenic eosinophilic pneumonia or treating a parasitic infection). An initial improvement may occur on corticosteroid treatment whatever the cause of the eosinophilic pneumonia (including infections), and thus response to treatment should not be considered a diagnostic test for the idiopathic origin of eosinophilic pneumonia. The pathologic features of eosinophilic pneumonia have mainly been studied in patients with idiopathic chronic eosinophilic pneumonia (ICEP) (Carrington et al. 1969; Liebow & Carrington 1969; Jederlinic et al. 1988) which used to be routinely diagnosed by open lung biopsy. These features may be considered as a common denominator of all eosinophilic pneumonias. Some features specific of a cause have been reported, such as a distinctive distribution of lesions (e.g., bronchocentric) or the presence on pathologic examination of causative agents such as parasites or fungal hyphae. The global architecture of the lung remains intact, without necrosis or fibrosis. The alveolar spaces are filled with eosino-

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phils. The distribution of the lesions of eosinophilic pneumonia is generally diffuse. However, it may be more focal or patchy in some cases, and the lesions may have an angiocentric or bronchiolocentric distribution in some etiologic groups of eosinophilic pneumonia. Some multinucleated giant cells are scattered in the infiltrate; eosinophilic granules or Charcot–Leyden crystals reflecting local eosinophilic degranulation may be present (Carrington et al. 1969). Some eosinophilic microabscesses may be observed. Morphologic and immunohistochemical studies have demonstrated that eosinophil degranulation takes place in eosinophilic pneumonias (Olopade et al. 1995). The associated interstitial inflammatory cellular infiltrate comprises eosinophils, lymphocytes, plasma cells, and histiocytes. A mild vasculitis involving both small arteries and venules is common, with perivascular cuffing and a few cells infiltrating the arterial media. In “simple” eosinophilic pneumonia, this vasculitis is nonnecrotizing when present. The hilar lymph nodes associated with ICEP contain many eosinophils, and lymphoid hyperplasia is present (Carrington et al. 1969). Some organization of the alveolar inflammatory exudate is a rather common finding, well identified in the early reported cases of ICEP (Carrington et al. 1969). Bronchiolitis obliterans of the proliferative type may be associated. These pathologic findings suggest some possible overlap between ICEP and cryptogenic organizing pneumonia. However, intraluminal organization of the distal airspaces is only sparse and never prominent in ICEP. Mucus plugs obstructing the small airways may be present (Carrington et al. 1969). In idiopathic acute eosinophilic pneumonia (IAEP) the pathologic pattern includes intraalveolar and interstitial eosinophilic infiltrates, diffuse alveolar edema, intraalveolar fibrinous exudates, organizing pneumonia, and nonnecrotizing vasculitis (Tazelaar et al. 1997). Pathologic examination of the lung is the gold standard for defining eosinophilic pneumonia, but lung biopsy is seldom necessary in clinical practice. Transbronchial lung biopsy may show characteristic features of eosinophilic pneumonia, but the small size of the specimen usually does not allow morphologic evidence of an etiologic process. In particularly complex cases, video-assisted thoracoscopic lung biopsy (which is a safe procedure) may be warranted.

Diagnosis of eosinophilic pneumonia The diagnosis of eosinophilic pneumonia requires characteristic clinical and radiologic features (dyspnea with alveolar opacities at chest imaging) and the demonstration of eosinophilia (preferably in the lung, or in the peripheral blood). The eosinophilic pneumonias may manifest by different clinical– radiologic syndromes, namely Löffler syndrome, chronic eosinophilic pneumonia, or acute eosinophilic pneumonia. Although usually safe, surgical lung biopsy in order to obtain a large piece of lung tissue is rarely necessary. Thus the

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diagnosis of eosinophilic pneumonia is still usually accepted when both radiographic pulmonary opacities and peripheral blood eosinophilia are present. For example, Löffler syndrome as it occurs in ascariasis is defined by rather mild and nonspecific symptoms with cough and wheezes, transient pulmonary infiltrates, and blood eosinophilia. However, the finding of peripheral blood eosinophilia obviously does not prove that the observed pulmonary opacities correspond to eosinophilic pneumonia, and the lower limit of peripheral eosinophilia for a confident diagnosis of eosinophilic pneumonia is not established. Furthermore, in IAEP, peripheral blood eosinophilia is often absent at presentation. Diagnosing eosinophilic pneumonia solely on the finding of blood eosinophilia and pulmonary opacities therefore requires markedly elevated eosinophilia (> 1 × 109/L and preferably 1.5 × 109/L) together with typical clinical–radiologic features. Bronchoalveolar lavage (BAL) has become a widely accepted noninvasive surrogate of lung biopsy for the diagnosis of eosinophilic pneumonia, although no study has established a correlation between increased eosinophils at differential cell count and the finding of eosinophilic pneumonia at pathologic examination of the lung. In normal controls, BAL eosinophilia is lower than 1% of cells at differential count. BAL eosinophilia of 3% or greater was found in 13.3% of a series of 1084 BAL procedures (Velay et al. 1987). Values between 3 and 40% (and especially between 3 and 9%) were found in various conditions, mainly including idiopathic pulmonary fibrosis, interstitial lung disease associated with connective tissue disorders, hypersensitivity pneumonitis, sarcoidosis, radiation pneumonitis, asthma, pneumoconioses, and infection. BAL eosinophilia greater than 40% was found mainly in patients with chronic eosinophilic pneumonia. A conservative cutoff of ≥ 40% eosinophils at BAL differential cell count has been adopted for the diagnosis of eosinophilic pneumonia (Marchand et al. 1998, 2003). The proposed cutoff for the diagnosis of IAEP was 25% (Pope-Harman et al. 1996). For clinical practice, our current recommendation is that a diagnosis of eosinophilic pneumonia be supported by alveolar eosinophilia when the eosinophils (i) are the predominant cell population (macrophages excepted) and (ii) represent more than 25 and preferably 40% of differential cell count. In clinical practice, eosinophilic pneumonia is suspected mostly in a patient presenting with respiratory symptoms, pulmonary infiltrates on chest radiograph, and peripheral blood eosinophilia. BAL is useful in most cases to establish the diagnosis of eosinophilic pneumonia, although in typical cases BAL may not be always mandatory (such as in a patient with migratory pulmonary opacities and high peripheral eosinophilia suggesting ICEP in the appropriate context). However demonstration of alveolar eosinophilia at BAL excludes alternative diagnoses such as bacterial or parasitic pneumonia, or pulmonary infiltrates related to malignancies (e.g., Hodgkin disease) which may cause both pulmonary opacities and peripheral blood eosinophilia. Alternatively,

Pulmonary Eosinophilia

eosinophilic pneumonia may present with acute-onset dyspnea and pulmonary infiltrates, with negative blood and BAL cultures, sometimes in the absence of blood eosinophilia; cytologic examination of BAL is thus mandatory in such cases to establish the diagnosis of acute eosinophilic pneumonia. Lung biopsy is seldom necessary. Once the diagnosis of eosinophilic pneumonia has been established, a thorough evaluation must be conducted to exclude potential causes such as parasite infection, or drug or toxic exposure, to investigate for nonrespiratory manifestations, and to diagnose one of the possible clinical entities (Table 86.2). Occasionally, difficult

Table 86.2 Diagnostic approach to the eosinophilic pneumonias. Suspect eosinophilic pneumonia Respiratory symptoms Pulmonary infiltrates on chest radiograph Peripheral blood eosinophilia > 1 × 109/L and especially 1.5 × 109/L (may be absent) Establish eosinophilic pneumonia Bronchoalveolar lavage eosinophilia > 25% (and especially > 40%) Search for potential causes Parasites: history of travel in areas endemic for parasites; cutaneous and/or gastrointestinal symptoms; serologies for cosmopolitan parasites (e.g., Toxocara canis); repeated search for parasites in feces Drugs, toxics, and radiation therapy: medical history, systemic record of drug exposure ABPA: history of asthma; proximal bronchiectasis; cultures of sputum and endobronchial aspirates or BAL; IgG and IgE specific for Aspergillus; immediate and late skin reaction to Aspergillus Assess pulmonary function Obstructive ventilatory defect and/or asthma: ICEP, ABPA, CSS Restrictive ventilatory defect: ICEP Search for nonrespiratory manifestations CSS: mononeuropathy multiplex; myocarditis, pericarditis (echocardiography), coronary artery disease (ECG); palpable purpura, subcutaneous nodules, livedo reticularis; central nervous system manifestations; polyarthralgia, arthritis; gastrointestinal tract or renal involvement HES: cutaneous papules or urticarial plaques, hepatomegaly, splenomegaly, mucosal ulcerations; endomyocardial fibrosis (echocardiography) Drug-related eosinophilic disease: skin (or renal) involvement Parasites: cutaneous and/or gastrointestinal symptoms Biology Parasites: appropriate serologies HES: lymphocytic or myeloproliferative variant (see Table 86.4) Classify into clinical entities See Table 86.1 ABPA, allergic bronchopulmonary aspergillosis; CSS, Churg–Strauss syndrome; HES, hypereosinophilic syndrome; ICEP, idiopathic chronic eosinophilic pneumonia.

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cases may not fit the proposed classification of eosinophilic pneumonia.

Eosinophilic lung disease of undetermined cause Idiopathic eosinophilic pneumonias The idiopathic eosinophilic pneumonias may be either chronic or acute. Whereas ICEP develops progressively within a few weeks with cough, increasing dyspnea, malaise, and weight loss, IAEP presents as an acute pneumonia with frequent respiratory failure necessitating mechanical ventilation.

Idiopathic chronic eosinophilic pneumonia ICEP was proposed as a distinct entity by Carrington et al. (1969) and occurs predominantly in women (female/male ratio of 2 : 1) (Jederlinic et al. 1988; Marchand et al. 1998). ICEP may occur in young people (Jederlinic et al. 1988; Wubbel et al. 2003). The incidence of ICEP peaks in the fifth decade (Marchand et al. 1998). A majority of patients with ICEP are nonsmokers (Jederlinic et al. 1988; Naughton et al. 1993; Marchand et al. 1998), suggesting that smoking might be protective. A prior history of atopy is found in about half of the patients, with allergic rhinitis in about 10–20% (Jederlinic et al. 1988; Marchand et al. 1998), drug allergy in about 10% (Jederlinic et al. 1988; Marchand et al. 1998), nasal polyposis in about 10% (Jederlinic et al. 1988; Marchand et al. 1998), urticaria in 10% (Marchand et al. 1998), and eczema in 5% (Marchand et al. 1998). Although ICEP is usually not mentioned as a complication of asthma, prior asthma is present in up to two-thirds of the patients (Liebow & Carrington 1969; Jederlinic et al. 1988; Naughton et al. 1993; Hayakawa et al. 1994; Marchand et al. 1998; Marchand et al. 2003). It may also occur concomitantly with the diagnosis of ICEP patients, or develop after ICEP (Marchand et al. 2003). The presentation of ICEP is similar in asthmatics and nonasthmatics. Higher total IgE levels are seen in asthmatics (Marchand et al. 2003). ICEP may develop while asthmatic patients are on a desensitization course, but there is no evidence of a causative link. Asthma often gets more severe after the occurrence of ICEP and requires longterm oral corticosteroid treatment (Marchand et al. 2003). The onset of ICEP is progressive over several weeks, with a mean interval between onset of symptoms and diagnosis of 4 months (Marchand et al. 1998). The most frequent respiratory symptoms are nonspecific with cough, dyspnea, and chest pain (Jederlinic et al. 1988; Marchand et al. 1998). Dyspnea is usually not severe, and mechanical ventilation is rarely required (Libby et al. 1980). Hemoptysis occurs in about 10% of cases (Jederlinic et al. 1988; Marchand et al. 1998). Wheezes at physical examination are found in one-third of patients (Jederlinic et al. 1988), and crackles in the same proportion (Marchand et al. 1998). Pleural effusion is uncommon and

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should point toward another etiology, especially CSS-associated cardiac disease. Manifestations of chronic rhinitis or sinusitis are present in about 20% of patients with ICEP (Marchand et al. 1998). Systemic symptoms are often prominent, with fever and weight loss (10 kg or more in about 10%), as well as asthenia, malaise, fatigue, anorexia, and night sweats. The imaging features of ICEP may be characteristic enough to evoke the diagnosis. Peripheral infiltrates are almost always present at chest X-ray (Carrington et al. 1969; Gaensler & Carrington 1977; Jederlinic et al. 1988; Mayo et al. 1989; Ebara et al. 1994; Marchand et al. 1998; Johkoh et al. 2000) and are migratory in about one-quarter of the cases (Marchand et al. 1998). High-resolution computed tomography (HRCT) accurately identifies the characteristic imaging features of ICEP. The opacities are almost always bilateral. They predominate in the upper lobes (Jederlinic et al. 1988; Mayo et al. 1989; Marchand et al. 1998), are peripheral, and generally associated with ground-glass and consolidation opacities (Ebara et al. 1994; Marchand et al. 1998; Arakawa et al. 2001) (Figs 86.1 and 86.2). The main differential diagnosis is with organizing

(a)

(b) Fig. 86.1 CT of the chest in a patient with idiopathic chronic eosinophilic pneumonia showing alveolar infiltrates and ground-glass opacities. (a) Upper lobes; (b) lower lobes.

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Fig. 86.2 Bilateral patchy subpleural opacities (consolidation) in a patient with idiopathic chronic eosinophilic pneumonia.

pneumonia. Streaky or band-like opacities parallel to the chest wall may also be present (Ebara et al. 1994). With corticosteroid treatment, opacities rapidly decrease in both size and extent, with possible evolution from consolidation to groundglass opacities or inhomogeneous opacities, and later to streaky or band-like opacities (Ebara et al. 1994). Small-size pleural effusion is present in up to 10% of cases at HRCT, and mediastinal lymph node enlargement in 17% (Marchand et al. 1998). Peripheral blood eosinophilia is almost always present in ICEP. However, since peripheral blood eosinophilia is often a diagnostic criterion of ICEP, the proportion of patients with ICEP and normal peripheral blood count is actually unknown. In our series (with eosinophilic pneumonia defined by blood eosinophilia > 1.0 × 109/L and/or eosinophil percentage > 40% at BAL differential count), the mean blood eosinophilia was 5.5 × 109/L (Marchand et al. 1998). BAL eosinophilia is a characteristic feature in ICEP (Pesci et al. 1988; Marchand et al. 1998), with a mean of about 60% at differential cell count (Marchand et al. 1998). It may be associated with an increase in neutrophils, lymphocytes and mast cells (Marchand et al. 1998). Sputum eosinophilia has been reported in ICEP (Crofton et al. 1952; Hayakawa et al. 1994). BAL eosinophils of patients with ICEP are activated and release eosinophil proteins, which are taken up by macrophages (Janin et al. 1993). Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) (Jorens et al. 1996) are increased in the BAL fluid of patients with ICEP. In ICEP, the expression of HLA-DR on most alveolar eosinophils in contrast to few blood eosinophils suggests compartmentalization of eosinophilic activation (Beninati et al. 1993). BAL lymphocytes in ICEP are characterized by an accumulation of CD4+ T cells (Mukae et al. 1995) expressing activation

Pulmonary Eosinophilia

surface antigens of memory T cells (CD45RO+, CD45RA–, CD62L–). Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels are increased (Jederlinic et al. 1988; Naughton et al. 1993; Marchand et al. 1998). Total blood IgE level is increased in about half of cases, and greater than 1000 kU/L in 15% (Marchand et al. 1998). Markedly increased urinary EDN levels, indicating active eosinophil degranulation, is found in patients with ICEP (Cottin et al. 1998). Some manifestations outside the respiratory tract have been occasionally reported in ICEP, overlapping with systemic eosinophilic disorders especially CSS. These include one of the following: arthralgias, repolarization (ST–T) abnormalities on the ECG, pericarditis, altered liver biological tests, eosinophilic infiltration at liver biopsy, mononeuritis multiplex, diarrhea, skin nodules, and eosinophilic enteritis (Carrington et al. 1969; Weynants et al. 1985; Marchand et al. 1998). In addition, ICEP may be a presenting feature of overt CSS (Hueto-Perez-De-Heredia et al. 1994; Steinfeld et al. 1994). Patients with ICEP often receive corticosteroid treatment, which may avoid the development of systemic vasculitis. In a series of ICEP with a rather high prevalence of extrapulmonary signs (30%), none of the patients treated with corticosteroids developed characteristic CSS (Weynants et al. 1985). Lung function tests in ICEP show an obstructive ventilatory defect in half the cases (Jederlinic et al. 1988; Marchand et al. 1998) and a restrictive ventilatory defect in the other half (Marchand et al. 1998). PaO2 of 75 mmHg or less was present in about two-thirds of patients in a series (Marchand et al. 1998). A CO transfer factor of less than 80% predicted was present in 52%, with a transfer coefficient less than 80% predicted in only 27%. The lung function tests normalize with treatment in most patients (Jederlinic et al. 1988). However, a fixed airflow obstruction may develop in some patients, especially those with a markedly increased BAL eosinophilia at initial evaluation (Durieu et al. 1997; Marchand et al. 1998). Spontaneous resolution of ICEP may rarely occur (Jederlinic et al. 1988; Marchand et al. 1998), and death resulting from ICEP seems extremely rare. The response of ICEP to corticosteroids is more dramatic than in any other pneumonia (this is even a characteristic feature of this disorder). Symptoms improve within 48 hours in about 80% of cases (Marchand et al. 1998), with a fall of blood eosinophil level within 24– 48 hours (blood differential cell count must therefore be done on suspicion of ICEP before corticosteroid treatment is started). The optimal dose of corticosteroids is not established. Our current recommendation is to start with prednisone 0.5 mg/kg daily. Pulmonary opacities disappear rapidly with treatment (within 1 week in about 70% of patients treated with a mean initial dose of 1 mg/kg daily) (Marchand et al. 1998). The long-term follow-up chest X-ray is normal in almost all patients (Marchand et al. 1998). Despite the remarkable efficacy of corticosteroids, most patients require prolonged treatment (i.e., more than 6 months)

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because of relapse while decreasing or after stopping the corticosteroid treatment. Recurrence occurs in 50–60% of cases after corticosteroids are discontinued, or while being tapered especially below 10 mg prednisone per day (Jederlinic et al. 1988; Marchand et al. 1998). Relapses may occur in the same or in different areas of the lung (Marchand et al. 1998). They respond quite well to corticosteroid treatment. Only 31% of patients had been weaned off treatment at the last followup visit in our series, with a mean 6.2 years of follow-up (Marchand et al. 1998). Relapses of ICEP are less frequent in patients with asthma, possibly because they often receive inhaled corticosteroids after stopping oral corticosteroids (Marchand et al. 1998, 2003). Inhaled corticosteroids may thus help in reducing the maintenance oral corticosteroid dose.

Idiopathic acute eosinophilic pneumonia IAEP (Allen et al. 1989; Hayakawa et al. 1994; Cheon et al. 1996; Pope-Harman et al. 1996; King et al. 1997; Tazelaar et al. 1997; Philit et al. 2002) presents as an acute lung disease with hypoxemia and marked eosinophilic alveolitis at BAL, contrasting with the usual lack of blood eosinophilia. The diagnostic criteria proposed by Pope-Harman et al. (1996) included onset of any symptoms within 7 days before presentation, and response to corticosteroid therapy. However, patients seen between 7 and 31 days after the first symptoms have a similar presentation, and a favorable outcome may occur without corticosteroid therapy (Philit et al. 2002). The diagnostic criteria we currently propose for IAEP (Cottin & Cordier 2005) are listed in Table 86.3. The mean age at presentation is about 30 years (PopeHarman et al. 1996; Philit et al. 2002), with male predominance. Despite a history of atopy in some patients (Hayakawa et al. 1994), IAEP is not associated with asthma. Some activities leading to inhalation of peculiar dusts or gases within the days before onset of disease have been reported, such as cave exploration, plant repotting, wood pile moving, smoke-house cleaning, motocross racing in dusty conditions (Pope-Harman et al. 1996), indoor renovation work, gasoline tank cleaning,

Table 86.3 Diagnostic criteria for idiopathic acute eosinophilic pneumonia. (Adapted from Tazelaar et al. 1997; Pope-Harman et al. 1996; Philit et al. 2002.) Acute onset of febrile respiratory manifestations (≤ 1 month duration before consultation) Bilateral diffuse infiltrates on chest radiography Hypoxemia, with PaO2 on room air < 60 mmHg, and/or PaO2/FIO2 ≤ 300 mmHg, and/or SaO2 on room air < 90% Lung eosinophilia, with > 25% eosinophils on BAL differential cell count (or eosinophilic pneumonia at lung biopsy) Absence of infection or of other known causes of eosinophilic lung disease (especially exposure to drug known to induce pulmonary eosinophilia)

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explosion of a tear gas bomb (Philit et al. 2002), dust-containing asbestos fibers, fly ash, and large particle-size silicates (Rom et al. 2002). IAEP has developed soon after the initiation of smoking (especially of large quantities) in some patients (Nakajima et al. 1998, 2000; Shintani et al. 2000a,b; Kawayama et al. 2003) and challenge with cigarette smoking was positive in some of them (Nakajima et al. 1998, 2000; Shintani et al. 2000a,b), with tolerance developing in some patients who resume smoking (Nakajima et al. 2000; Shintani et al. 2000a,b). Inhalation of tobacco smoke or of any dust or gas may thus favor the development of IAEP in some individuals. The onset of IAEP is acute, although it may develop subacutely over a few weeks. Symptoms at presentation consist of cough, dyspnea, fever, and chest pain. Abdominal pain and myalgias have been reported (Pope-Harman et al. 1996). Tachypnea and tachycardia are usually present, with crackles on auscultation. Chest X-ray shows diffuse infiltrative opacities, with mixed alveolar or interstitial opacities (Cheon et al. 1996; Pope-Harman et al. 1996; King et al. 1997; Philit et al. 2002), often associated with bilateral pleural effusion and Kerley B lines (Pope-Harman et al. 1996). On HRCT, airspace consolidation and ground-glass opacities are the most common parenchymal patterns; poorly defined nodules, interlobular septal thickening, and pleural effusion usually bilateral (Cheon et al. 1996; Pope-Harman et al. 1996; Johkoh et al. 2000; Philit et al. 2002) are present in a majority of patients (Fig. 86.3). When done in less severe cases, lung function tests have shown a mild restrictive ventilatory defect with normal FEV1/FVC ratio and reduced transfer factor (Ogawa et al. 1993). Lung function tests performed after recovery are normal in most patients (Buchheit et al. 1992; Chiappini et al. 1995; Pope-Harman et al. 1996). Lung biopsy is not necessary for the diagnosis of IAEP. When performed, it has shown a pattern of acute and organizing diffuse alveolar damage, together with interstitial alveolar and bronchiolar infiltration by eosinophils, intraalveolar eosinophils, and interstitial edema (Pope-Harman et al. 1996; Tazelaar et al. 1997; Kawayama et al. 2002). At presentation the peripheral blood eosinophil cell count in IAEP is only rarely higher than 0.3 × 109/L; instead, increased leukocytosis with a predominance of neutrophils is common. However, the eosinophil count may rise during the course of disease (Hayakawa et al. 1994; Pope-Harman et al. 1996; Philit et al. 2002) despite corticosteroid treatment, a feature considered so characteristic of IAEP that it should raise the suspicion of acute eosinophilic pneumonia in undiagnosed pneumonia. BAL is the key to the diagnosis of IAEP, showing an average differential eosinophil count of about 35–55% (Pope-Harman et al. 1996; Philit et al. 2002). BAL eosinophilia may persist for several weeks (Taniguchi et al. 1999). Eosinophilia greater than 25% at BAL in a characteristic context may obviate lung biopsy in nonimmunocompromised patients. Eosinophilia is also present at pleural fluid differential cell count (Ogawa et al. 1993;

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Pulmonary Eosinophilia

ally started when a diagnosis of IAEP is made. Corticosteroids ensure improvement within 48 hours and possible weaning from the ventilator. There are no significant clinical or imaging sequelae and, in contrast with ICEP, no relapse occurs after stopping corticosteroid treatment. Before a diagnosis of IAEP is confirmed, a careful search for a potential cause of eosinophilic pneumonia with acute onset is mandatory. Two main etiologic categories must especially be considered: drug reactions and infections (infectious agents are systematically searched in BAL by culture and appropriate staining).

Churg–Strauss syndrome (a)

(b) Fig. 86.3 CT of the chest in a patient with idiopathic acute eosinophilic pneumonia showing alveolar consolidation of the right lower lobe associated with bilateral pleural effusion. (a) Lung windows; (b) mediastinal windows.

Pope-Harman et al. 1996; Shintani et al. 2000b), and in the sputum of patients with IAEP (Hayakawa et al. 1994). IgE level is increased in some patients (Ogawa et al. 1993). Hypoxemia may be severe (Allen et al. 1989; Philit et al. 2002), and a majority of patients match either the definition of acute lung injury (acute onset, bilateral infiltrates on chest X-ray, pulmonary artery wedge pressure ≤ 18 mmHg or no evidence of left atrial hypertension, and PaO2/FIO2 ≤ 300 mmHg) or the definition of acute respiratory distress syndrome (acute lung injury and PaO2/FIO2 ≤ 200 mmHg). A majority of patients require mechanical ventilation, either noninvasively or with intratracheal intubation (Pope-Harman et al. 1996; Philit et al. 2002). Shock is exceptional in IAEP (Buddharaju et al. 1999; Kawayama et al. 2002) and extrapulmonary organ failure does not occur (a marked difference with acute respiratory distress syndrome). Although spontaneous recovery without corticosteroid treatment has been reported, corticosteroid treatment is usu-

CSS belongs to the group of small-vessel vasculitides as defined in the Chapel Hill Consensus Conference on the Nomenclature of Systemic Vasculitis (Jennette et al. 1994). It is defined as an eosinophil-rich and granulomatous inflammation involving the respiratory tract, and a necrotizing vasculitis affecting small to medium-sized vessels, and associated with asthma and eosinophilia. Whereas the original description of the syndrome by Churg and Strauss in 1951 reported widespread necrotizing granulomatous vasculitis and granulomatous extravascular lesions (mainly in autopsied cases), the pathologic lesions currently found in CSS only rarely comprise all the typical features on a single biopsy of any organ (Garrell 1960; Churg 2001), especially because diagnosis is now made at an earlier stage. Vasculitis, involving especially medium-sized vessels, may be necrotizing or not. Extravascular lesions consist of eosinophilic infiltration with granulomas comprising palisading histiocytes and giant cells. Eosinophilic infiltration of the tissues with perivascular eosinophilia but in the absence of overt vasculitis is commonly found at the early stage of CSS. CSS is rare among other vasculitides. It occurs in middleaged adults (Lanham et al. 1984; Mouthon et al. 2002; Keogh & Specks 2003), although it has been occasionally reported in children and adolescents. There is no sex predominance. The evolution of CSS typically follows three stages: asthma and rhinitis; tissue eosinophilia; and eventually eosinophilic disease with vasculitis. Asthma is almost always present. It becomes progressively more severe and corticosteroid-dependent than isolated asthma. It usually precedes the onset of vasculitis by less than 9 years (Chumbley et al. 1977; Lanham et al. 1984; Guillevin et al. 1999; Keogh & Specks 2003), but the interval may be longer (Chumbley et al. 1977) or they may develop simultaneously (Keogh & Specks 2003). Asthma may attenuate when the vasculitis develops and further increase when vasculitis recedes and corticosteroid treatment is decreased or stopped (Chumbley et al. 1977; Lanham et al. 1984). Pulmonary infiltrates represent the most typical abnormalities on chest X-ray and have been reported in 37% (Guillevin et al. 1999) to 72% (Lanham et al. 1984) of cases. The pulmonary infiltrates are found especially at presentation, but the

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chest X-ray may be normal throughout the course of the disease. The pulmonary opacities are usually ill-defined opacities of varying density, sometimes migratory (Chumbley et al. 1977; Degesys et al. 1980; Lanham et al. 1984; Choi et al. 2000), which rapidly resolve with corticosteroid treatment. Pleural effusion (usually mild) and phrenic nerve palsy may be present. On thin-section CT, the pulmonary opacities mainly consist of areas of ground-glass attenuation (Fig. 86.4) or airspace consolidation, with peripheral predominance; other less common imaging features include centrilobular nodules, bronchial wall thickening or dilatation, interlobular septal thickening, hilar or mediastinal lymphadenopathy, pleural effusion or pericardial effusion (Fig. 86.5) (Worthy et al. 1998; Choi et al. 2000; Johkoh et al. 2000). Rhinitis, generally of the allergic type, is present in CSS in a majority of patients (Liebow et al. 1972; Olsen et al. 1995). It is often accompanied by the presence of polyps. Crusty rhinitis may be occasionally present; however, rhinitis in CSS is much less severe than in Wegener granulomatosis, and septal nasal

Fig. 86.4 CT of the chest in a patient with Churg–Strauss syndrome showing subpleural patchy areas of ground-glass opacities.

Fig. 86.5 CT of the chest in a patient with Churg–Strauss syndrome and cardiomyopathy showing bilateral pleural and pericardial effusion.

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perforation and saddle nose deformation are exceptional in CSS. Paranasal sinusitis has been reported in about 60% of patients (Guillevin et al. 1999). Blood eosinophilia is generally 5–20 × 109/L, but may reach higher values (Chumbley et al. 1977; Lanham et al. 1984; Guillevin et al. 1999). Blood eosinophilia often disappears dramatically within a few days after corticosteroid treatment has been initiated. Eosinophilia sometimes greater than 60% is found on BAL differential cell count (Wallaert et al. 1993) and in the pleural fluid when present (Erzurum et al. 1989). CSS is one of the pulmonary anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides, together with Wegener granulomatosis and microscopic polyangiitis. However, ANCA is not constantly present in CSS, being reported in 48% (Guillevin et al. 1999) to 73% (Keogh & Specks 2003). ANCAs are mainly perinuclear with myeloperoxidase specificity (much less often cytoplasmic ANCA with proteinase 3 specificity) (Guillevin et al. 1999; Keogh & Specks 2003). IgE levels are usually markedly increased. The ESR and CRP are increased and anemia is common. High levels of urinary EDN might represent an activity index of disease (Cottin et al. 1995). Asthenia, weight loss, fever, arthralgias, and myalgias often precede the development of the numerous extrapulmonary manifestations of CSS. Mononeuritis multiplex (or asymmetrical polyneuropathy) is the most common neurologic manifestation, present in about three-quarters of patients (Guillevin et al. 1999). Cranial nerve palsies and central nervous system involvement are rare. Heart involvement in CSS results from eosinophilic myocarditis and/or less commonly coronary arteritis. It may be severe and lead to death (Chumbley et al. 1977; Lanham et al. 1984; Reid et al. 1998; Guillevin et al. 1999; Keogh & Specks 2003). It was the primary cause of death in recent series (Sable-Fourtassou et al. 2005). Cardiac involvement is often asymptomatic for long periods, and thus diagnosed only when left ventricular failure and dilated cardiomyopathy have developed. Heart failure may lead to heart transplantation; eosinophilic vasculitis may recur in the transplanted heart. Mild myocardial impairment as well as coronary arteritis may markedly improve with corticosteroid treatment, thus necessitating a strict cardiac evaluation in any patient with suspected CSS in order to precisely characterize the severity of disease. Pericarditis with limited effusion is common in CSS, but tamponade is rare. Gastrointestinal tract involvement, present in one-third of cases (Guillevin et al. 1999), usually manifests as isolated abdominal pain, and may be difficult to diagnose. Other digestive manifestations include diarrhea, ulcerative colitis, gastroduodenal ulceration, perforation (esophageal, gastric, intestinal), digestive hemorrhage, and cholecystitis. Cutaneous manifestations are present in about half of patients (Guillevin et al. 1999). They consist especially of palpable purpura of the extremities, subcutaneous nodules (especially of the scalp and extremities), erythematous rashes, and urticaria. Biopsy

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of the cutaneous lesions is the most common and simple procedure to obtain pathologic evidence of vasculitis. Renal involvement resulting from glomerular vasculitis present in 26% of cases is usually mild, and is rarely responsible for chronic renal failure (in contrast to Wegener granulomatosis) (Guillevin et al. 1999). Some triggering or adjuvant factors (such as vaccines or desensitization) have been suspected to play a role in CSS (Guillevin et al. 1987; Mouthon et al. 2001). The possible role of allergy as a risk factor is suspected, because a family history of atopy is common, and allergic rhinitis is present in many patients with CSS. Other possible triggering factors include Aspergillus, candidiasis, Ascaris, bird exposure, or cocaine. Druginduced eosinophilic vasculitis with pulmonary involvement has been reported with macrolides (Hubner et al. 1997) and diphenylhydantoin (Yermakov et al. 1983). The possible responsibility of leukotriene-receptor antagonists (montelukast, zafirkukast, pranlukast) in the development of CSS is debated (Lilly et al. 2002; Keogh & Specks 2003). The association may be coincidental: some cases of smoldering CSS may flare because of reducing oral or inhaled corticosteroids and/or adding leukotriene receptor antagonists; alternatively, these drugs might really exert a role in the pathogenesis of the vasculitis. Although no causative role of leukotriene-receptor antagonists has been established, these should be avoided in any asthma patient with eosinophilia. There is currently no consensus on diagnostic criteria for CSS. Lanham et al. (1984) have proposed three diagnostic criteria, including (i) asthma, (ii) eosinophilia exceeding 1.5 × 109/L, and (iii) systemic vasculitis of two or more extrapulmonary organs. These diagnostic criteria were proposed before ANCAs were available. When present (with specificity for myeloperoxydase or proteinase 3 on ELISA), ANCAs may probably be considered a major diagnostic criterion (Cottin & Cordier 1999). The skin, nerve, and muscle are the most common sites where a pathologic diagnosis of vasculitis may be obtained (Guillevin et al. 1999). Lung biopsy is not indicated. Transbronchial biopsies usually do not show characteristic features of vasculitis or granulomas. A pathologic diagnosis is not mandatory in patients with characteristic clinical features and marked eosinophilia. The diagnosis of CSS is particularly difficult in the so-called formes frustes of CSS, without overt vasculitis involving several organs. Two recent studies have analyzed the clinical manifestations of CSS according to the status of ANCA antibodies (SableFourtassou et al. 2005; Sinico et al. 2005). In both these studies, ANCA antibodies were present in 38% of the patients. Interestingly, the presence of ANCA was significantly associated with an increased frequency of extracapillary glomerular lesions, peripheral neuropathy, and biopsy-proven vasculitis. Conversely, cardiac involvement and fever were more frequently encountered in patients with CSS but no ANCA. These observations suggest two distinct phenotypes of CSS: (i) CSS, vasculitic phenotype, with ANCA; and (ii) CSS, eosinophilic

Pulmonary Eosinophilia

tissular disease phenotype, without ANCA (Kallenberg 2005; Sinico et al. 2005). Corticosteroids represent the core treatment of CSS, and suffice to control disease in a large number of cases (Lanham et al. 1984; Abu-Shakra et al. 1994; Guillevin et al. 1996). An initial bolus of methylprednisolone is useful in the most severe cases. Corticosteroid treatment is prolonged for several months with progressive tapering of doses. Relapses are common. Asthma often reappears when corticosteroid doses are reduced or stopped. Distinguishing relapse of CSS or mere persistence of simple asthma needs careful evaluation in order to avoid overtreatment or undertreatment with oral corticosteroids. Treatment with corticosteroids alone is reserved for patients without manifestations associated with mortality or severe morbidity (Langford 2001). A retrospective study of patients with either polyarteritis nodosa or CSS (Guillevin et al. 1996) identified parameters with significant pronostic value and responsible for higher mortality and which warranted associated immunosuppressive treatment when present: proteinuria greater than 1 g/day; renal insufficiency with serum creatinine greater than 15.8 mg/L; and gastrointestinal tract involvement. Cardiomyopathy and central nervous system (CNS) involvement were associated with a relative risk of mortality of 2.2 and 1.8 respectively (not statistically significant). The addition of immunosuppressive agents to corticosteroids improves disease control, despite associated infections (Chow et al. 1989; Guillevin et al. 1992). Mortality was associated with disease severity, and treatment with cytotoxic agents did not prevent relapses (Gayraud et al. 2001). Therefore, corticosteroid treatment alone is considered sufficient for CSS patients without poor prognostic factors at onset, with most patients achieving complete remission without relapse (Ribi et al. 2008). In patients with mild or severe relapses, under 20 mg of prednisone per day or greater, or in patients with poor prognostic factors at onset, immunosuppressive treatment (such as azathioprine or cyclophosphamide) is warranted in addition to corticosteroids in patients with poor prognosis factors at onset, and also in patients experiencing relapses while receiving 20 mg of prednisone per day or greater. Subcutaneous interferon (IFN)-α has been successfully used mainly in CSS patients with severe disease (Tatsis et al. 1998). High-dose intravenous immunoglobulins and cyclosporin A have been occasionally used successfully. More recently, rituximab has been used in cases of CSS refractory to conventional therapy (Kaushik et al. 2006; Koukoulaki et al. 2006). Of note, the anti-IgE agent omalizumab should be used with caution in asthmatic patients suspected of having CSS, as CSS has developed a few months after starting omalizumab for asthma in a patient (Winchester et al. 2006). The prognosis of CSS has considerably improved over time, with 79% of patients alive at 5 years (Guillevin et al. 1996). In fact, a diagnosis of CSS unexpectedly did not confer increased mortality in one series (Keogh & Specks 2003).

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Hypereosinophilic syndrome The initial definition of “idiopathic” hypereosinophilic syndrome (HES) was as follows (Chusid et al. 1975): (i) a persistent eosinophilia greater than 1.5 × 109/L for longer than 6 months, or death before 6 months associated with the signs and symptoms of hypereosinophilic disease; (ii) lack of evidence for parasitic, allergic, or other known causes of eosinophilia; and (iii) presumptive signs and symptoms of organ involvement, including hepatosplenomegaly, organic heart murmur, congestive heart failure, diffuse or focal CNS abnormalities, pulmonary fibrosis, fever, weight loss, or anemia. Idiopathic HES further proved heterogeneous, although patients with typical chronic disease shared common complications, especially cardiac involvement. Recent studies have demonstrated that HES may result from clonal proliferation of lymphocytes producing eosinophilopoietic chemokines (lymphocytic variant), or from the clonal proliferation of the eosinophil precursors themselves (myeloproliferative variant) (Bain 1996; Brito-Babapulle 1997; Oliver et al. 1998; Chang et al. 1999; Schoffski et al. 2000; Bigoni et al. 2000) (Table 86.4), so that the term “idiopathic” might be abandoned in the classification of HES (Roufosse et al. 2003). There still remains a proportion of cases that cannot be classified in either category. In the lymphocytic variant of HES, polymorphonuclear eosinophils accumulate in tissues as a consequence of the production of chemokines, especially interleukin (IL)-5, by clonal Th2 lymphocytes often bearing an aberrant immunologic phenotype (such as CD3–CD4+). Serum levels of IgE are elevated (Simon et al. 1999; Roufosse et al. 2000, 2003). Most

patients present with skin manifestations (papules or urticarial plaques infiltrated by lymphocytes and eosinophils). Serum levels of IL-5 and TARC (thymus and activation-regulated chemokine) are increased (Kakinuma et al. 2001). A cutaneous T-cell lymphoma or the Sézary syndrome may develop, and the lymphocytic variant of HES may thus be considered as a premalignant T-cell disorder (Cogan et al. 1994; Simon et al. 1999; Roufosse et al. 2000). The myeloproliferative variant of HES is the consequence of a constitutively activated tyrosine kinase fusion protein created by fusion of Fip1L1 and PDGFR-α resulting from a chromosomal interstitial deletion in the long arm of chromosome 4 (4q12) (Cools et al. 2003; Griffin et al. 2003). Serum tryptase levels are increased. The constitutively active tyrosine kinase activity transforms hematopoietic cells and is inhibited by imatinib (2-phenylaminopyrimidine-based tyrosine kinase inhibitor) originally used to treat chronic myelogenous leukemia. Imatinib further proved efficient in the treatment of other chronic myeloproliferative diseases and also gastrointestinal stromal tumors. Imatinib proved efficient for several months in treating HES in patients with disease refractory to corticosteroids, hydroxycarbamide (hydroxyurea), and/or IFN-α. Pulmonary involvement in patients with HES has not been studied precisely in the two variants. Lung or pleural involvement has been mentioned (Hirshberg et al. 1999; Simon et al. 1999; Roufosse et al. 2000) in patients with clonal lymphoid proliferations. Reconsideration of the following classical manifestations according to the lymphocytic or myeloproliferative variant will be necessary.

Table 86.4 Distinctive features of the lymphocytic and myeloproliferative variants of hypereosinophilic syndrome. (Adapted from Cottin & Cordier 2005.) Lymphocytic variant

Myeloproliferative variant

Pathogeny

T-cell clone producing IL-5

Fip1L1–PDGFR-a fusion protein (interstitial deletion on 4q12)

Distinctive clinical features

Cutaneous papules or urticarial plaques

Male predominance Endomyocardial fibrosis Hepatomegaly, splenomegaly Mucosal ulcerations variant

Distinctive biological features

Elevated serum IgE Polyclonal hypergammaglobulinemia Clonal peripheral lymphocytes bearing an aberrant CD3–CD4+ surface phenotype Elevated serum IL-5 and TARC

Anemia, thrombocytopenia Increased serum vitamin B12 Increased leukocyte alkaline phosphatase Circulating leukocyte precursors Increased serum tryptase levels Fip1L1–PDGFR-a fusion protein (interstitial deletion on 4q12)

Main treatment considerations

Corticosteroids Interferon-a Anti-IL-5 (mepolizumab)?

Imatinib mesylate Hydroxycarbamide Interferon-a Anti-IL-5 (mepolizumab)?

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HES is much more common in men than in women (9 : 1), mostly aged between 20 and 50 years (Weller & Bubley 1994). The onset is generally insidious, with eosinophilia discovered incidentally in about 10% of patients (Fauci et al. 1982). The mean eosinophil count at presentation was about 20 × 109/L in one series (Spry et al. 1983) but values up to 100 × 109/L have been reported (Chusid et al. 1975). The main presenting symptoms are weakness and fatigue, cough, and dyspnea (Fauci et al. 1982). Cardiovascular involvement, present in about 60% of patients (Weller & Bubley 1994), is characterized by endomyocardial fibrosis (Roberts & Ferrans 1975; Fauci et al. 1982), a feature dictinct from cardiac involvement in CSS. Endomyocardial fibrosis is preceded by an initial acute necrosis followed by a thrombotic process (Weller & Bubley 1994). Cardiac manifestations include dyspnea, congestive heart failure, mitral regurgitation, and cardiomegaly (Fauci et al. 1982; Lefebvre et al. 1989). Echocardiography demonstrates the classic features of HES, consisting of mural thrombus, ventricular apical obliteration, and involvement of the posterior mitral leaflet (Ommen et al. 2001). Nervous manifestations (thromboemboli, CNS dysfunction, peripheral neuropathy) and cutaneous manifestations (erythematous pruritic papules and nodules, urticaria, angioedema) may be encountered (Kazmierowski et al. 1978; Weller and Bubley 1994). Pulmonary involvement is present in about 40% of patients (Chusid et al. 1975; Fauci et al. 1982) and includes cough, pleural effusion, and interstitial infiltrates. Severe coughing attacks were present in 40% of the cases in one series (Spry et al. 1983). Reported CT findings consisted of small nodules with or without a halo of ground-glass attenuation and focal areas of ground-glass attenuation mainly in the lung periphery (Kang et al. 1997). CT findings are poorly specific among eosinophilic diseases (Johkoh et al. 2000). It is likely that a proportion of the pulmonary imaging abnormalities may actually reflect cardiac disease in HES. “Pulmonary fibrosis,” as mentioned in the historic description of the disease, has been very poorly described and is not reported in recent cases. Less than half of patients with HES respond well to corticosteroids as a first-line therapy (Parrillo et al. 1979; Lefebvre et al. 1989). Other treatments have included hydroxycarbamide, vincristine, etoposide, cyclosporin A (Parrillo et al. 1979; Spry et al. 1983; Weller & Bubley 1994), and IFN-α either as monotherapy (Butterfield & Gleich 1994a,b; Ceretelli et al. 1998; Yoon et al. 2000) or in association with hydroxycarbamide, particularly in the myeloproliferative variant (Coutant et al. 1993; Demiroglu & Dundar 1997). Imatinib has now become the treatment of choice in patients with the myeloproliferative variant of HES and unresponsive to corticosteroids. Anti-IL-5 may be promising, although a paradoxical rebound of symptoms and circulating eosinophilia has been observed in the weeks following the first treatment (Klion et al. 2004). In contrast with a 3-year survival of only 12% in the first published series (Chusid et al. 1975), the prognosis has subse-

Pulmonary Eosinophilia

quently improved, with about 80%, 70%, and 42% survival at 5, 10, and 15 years respectively (Fauci et al. 1982; Lefebvre et al. 1989).

Eosinophilic lung disease of determined cause Eosinophilic pneumonias of parasitic or infectious origin Parasite infestation (mostly by helminths) represents the most common cause of eosinophilic pneumonia in the world, especially in tropical areas with endemic filariasis (Malavige 2003).

Tropical eosinophilia Tropical eosinophilia was initially described (Weingarten 1943) as a syndrome with severe spasmodic bronchitis (with dry, hacking cough especially during night-time) and peripheral blood eosinophilia, often associated with dyspnea, wheezing, weight loss, and anorexia. The clinical manifestations predominate in the second and third decades of life, with male predominance. The presence of wheezing may lead to misdiagnosis of tropical eosinophilia as asthma. Patients usually do not have symptomatic lymphatic filariasis. It is caused by the filarial nematodes Wuchereria bancrofti and Brugia malayi, which cause endemic lymphatic filariasis mostly in the tropical and subtropical areas of coastal regions of Asia, southern and western Pacific, and Africa. Humans become infected by larvae through mosquito bites. While the adult worms reside in the lymphatic vessels, leading to lymphatic obstruction with subsequent elephantiasis, most respiratory manifestations are thought to be related to an immune response of the host to larvae or microfilariae released by the adult female worms in the bloodstream and which become trapped in the lung vasculature (Ottesen et al. 1979; Chitkara & Sarinas 1997; Ong & Doyle 1998). Several stages of pulmonary disease have been described. The early phase (first 2 weeks) is characterized by high blood eosinophilia, dense infiltration of the lung with histiocytes, but no prominent eosinophilic infiltration. Eosinophilic pneumonia occurs later (1–3 months), with the formation of eosinophilic abscesses and granulomatous lesions with giant and epithelioid cells, and prominent eosinophilic infiltration at the periphery of the granuloma. When left untreated for several years, the disease may evolve to pulmonary fibrosis. Imaging features include irregular basilar opacities (Rom et al. 1990), and nonspecific disseminated bilateral opacities on chest CT, that may be associated with bronchiectasis, air trapping, and mediastinal lymphadenopathy (Sandhu et al. 1996). Lung function tests show a restrictive ventilatory defect, with a reversible obstructive ventilatory defect and hypoxemia in about one-quarter of the patients (Udwadia 1967; Poh 1974). Peripheral blood eosinophilia over 2 × 109/L is constant and may reach 60 × 109/L (Rohatgi & Smirniotopoulos 1991);

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similarly high alveolar eosinophilia is found in BAL, with a mean of 54% of eosinophils, marked eosinophil degranulation (Pinkston et al. 1987), and increased levels of EDN, an eosinophil-derived protein likely implicated in the pathogenesis of pulmonary tropical eosinophilia (O’Bryan et al. 2003). Eosinophils, and sometimes Charcot–Leyden crystals, are present in the sputum. IgE levels are increased (Ray & Saha 1978). Microfilariae are usually not found in the blood or the lung, and parasite infestation is demonstrated by the presence of antifilarial IgG antibodies in the serum. BAL and blood eosinophils decrease within 2 weeks of treatment with diethylcarbamazine (Pinkston et al. 1987; Vijayan et al. 1991a,b). The diagnosis of tropical eosinophilia is confirmed by the combination of persistent blood eosinophilia greater than 3 × 109/L, IgE levels exceeding 10 000 ng/mL, and markedly increased antifilarial IgG. Diagnosis is further supported by clinical improvement in the weeks following treatment (Neva & Ottesen 1978; Rohatgi & Smirniotopoulos 1991; Kazura 1997; Cooray & Ismail 1999) in a patient who has been living for several months in an endemic area. Diethylcarbamazine is the treatment of choice for tropical pulmonary eosinophilia because it kills both microfilariae and adult-stage worms. Corticosteroids may be beneficial in addition to diethylcarbamazine (Kazura 1997).

Ascaris pneumonia Infection by the nematode Ascaris lumbricoides is the most common helminthic infection in humans (Salata 1997a). It is especially common in children in the tropical and subtropical areas, who become infected through food or water contaminated by human feces and containing eggs. Once swallowed, the infective eggs develop into larvae in the small intestine, then penetrate through the intestinal wall, and migrate via the venous circulation to the lungs where they break out into the alveoli. Larvae migrate from alveoli to the bronchi and trachea, and mature into adult worms in the small intestine after being swallowed. Mature females in human intestine then release large numbers of eggs expulsed with stools which may survive for several months or years. Pulmonary manifestations mainly occur during migration of the larvae through the lungs. Symptoms are usually mild, and spontaneously resolve in a few days. Most patients manifest with Löffler syndrome, with cough, wheezing, possible fever, transient pulmonary infiltrates, and blood eosinophilia. Nonspecific pruritic eruption may be present at the time of respiratory symptoms. The presence of peripheral blood eosinophilia rapidly suggests the diagnosis. It may be as high as 22 × 109/L (Gelpi & Mustafa 1968), and may remain elevated for several weeks. Although larvae may occasionally be found in the sputum or gastric aspirates, the diagnosis is readily obtained by the finding of the worm or ova in the stool. Because of the life cycle of the parasite, positivity of this test may be delayed by 3 months after the pulmonary manifestations (Salata 1997a).

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Treatment for the transient pulmonary manifestations may not be necessary; intestinal ascariasis is treated with mebendazole for 3 days, or pyrantel palmoate and albendazole (Khuroo 1996; Salata 1997a; Sarinas & Chitkara 1997).

Eosinophilic pneumonia due to toxocariasis (larva migrans syndrome) Toxocariasis (or visceral larva migrans) (Beaver et al. 1952) is a zoonotic infection occurring predominantly in children, who become infected when playing in contaminated areas, especially when they practice geophagia. Eggs of the parasite Toxocara canis may contaminate the soil in all temperate and tropical areas of the world, especially playgrounds in urban areas, after eggs are released by female worms in feces of infected dogs. Ingested eggs hatch in the intestine. Larvae then migrate through the portal circulation, and invade the liver, lung, and other organs. However, in humans the development of the parasite is blocked at the larval stage. The majority of infected children are asymptomatic, and the disease often remains undiagnosed. When present, symptoms may include fever, seizures, and fatigue, and respiratory manifestations including cough, wheezes, and dyspnea. Wheezes or crackles are present at pulmonary auscultation. Pulmonary infiltrates at chest X-ray may contribute to the diagnosis when present, but they may be lacking even in patients with pulmonary symptoms (Beaver et al. 1952; Schantz & Glickman 1978). Although predominating in children, toxocariasis may be encountered in adults (Roig et al. 1992; Bartelink et al. 1993). Although usually mild, pulmonary manifestations may occasionally be severe (about 17% of cases) and necessitate mechanical ventilation. Such cases may benefit from corticosteroids (Beshear & Hendley 1973). The presence of peripheral blood eosinophilia orientates the diagnosis, but may develop only in the days following pulmonary manifestations in some patients. When performed, BAL differential cell count demonstrates increased eosinophils. The diagnosis of toxocariasis is made by serology. Visceral larva migrans usually requires only symptomatic treatment. The use of antihelminthics is controversial (Iwai et al. 1994).

Strongyloides stercoralis infection Strongyloidiasis or human infection with the intestinal nematode Strongyloides stercoralis is widely distributed in the tropics and subtropics (Evans 1998). It may be acquired by direct skin contact with the contaminated soil of beaches or mud that contains infective larvae released in the stools of infected individuals. Larvae pass through the skin to the bloodstream, then pass to the lungs where they break into alveoli, ascend the trachea, are swallowed, and reside in the small intestine. Larvae then mature into adult worms in the intestine, where adult females deposit eggs which hatch into larvae. Clinical manifestations include mild or moderate pulmonary and intestinal symptoms in recently infected immuno-

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competent individuals, and possibly severe autoinfection in immunocompromised patients. Symptoms in immunocompetent patients are nonspecific. Most patients manifest with Löffler syndrome, which occurs when larvae migrate through the lungs after acute infection. Pneumonia, bronchospasm, or bronchitis, and abdominal pain or diarrhea may be present. Concomitant respiratory and intestinal symptoms may suggest strongyloidiasis in patients living, or having traveled, in endemic areas. Peripheral blood eosinophilia is usually present at this stage. The diagnosis is obtained by the demonstration of larvae in the feces, sputum, or BAL fluid, and further confirmed by serologic methods (ELISA) (Salata 1997b). Because of the risk of severe disseminated strongyloidiasis when immunosupppression from any cause occurs, anthelminthic treatment with thiabendazole is recommended in all infected patients diagnosed with strongyloidiasis, symptomatic or not (Salata 1997b). Severe disseminated strongyloidiasis is a severe autoinfection resulting from persistence of the parasite in the human host for years, and triggered by immunosuppression, whatever its cause. Patients with coexisting chronic lung disease and receiving corticosteroid or immunosuppressive treatment are at risk of this complication, and may represent 20% of hospitalized patients with strongyloidiasis (Davidson 1992). This massive larval infection (hyperinfection syndrome) may affect all organs including the lungs. Patients may manifest with fever, abdominal pain, ileus or small bowel obstruction, jaundice, meningitis, cough, wheezing, dyspnea, or acute respiratory failure due to alveolar hemorrhage (Mora et al. 2006). Bilateral patchy infiltrates are found on the chest radiograph. Peripheral blood eosinophilia is often absent in disseminated disease (Igra-Siegman et al. 1981). The diagnosis of severe disseminated strongyloidiasis may be obtained by the recovery of rhabditiform larvae by BAL, bronchial washing, or sputum (Harris et al. 1980; Williams et al. 1988; Jamil & Hilton 1992).

Eosinophilic pneumonias in other parasitic and infectious diseases A typical Löffler syndrome may be caused by the dog hookworm Ancylostoma braziliense, the causative agent of cutaneous helminthiasis. Transient migratory pulmonary infiltrates may develop after the seventh day of cutaneous eruption in half the cases, associated with peripheral eosinophilia. Diagnosis is not straightforward, since extrapulmonary manifestations may be absent (Ambrus & Klein 1988). Löffler syndrome may also be caused by the human hookworms Ancylostoma duodenale and Necator americanus. Human schistosomiasis is caused by either Schistosoma haematobium or S. mansoni. The acute stage of the disease may comprise eosinophilia and transient multiple small pulmonary nodules on chest imaging (Barrett-Connor 1982; King 1997; Schwartz et al. 2000). In chronic schistosomiasis, granulomas composed of lymphocytes, eosinophils, and giant cells

Pulmonary Eosinophilia

may develop within the lungs as a reaction to embolization of ova in small arteries in the lungs, with ensuing occlusion of pulmonary arteries and possible progressive pulmonary hypertension. Eosinophilic pneumonia (also called lung shift, verminous pneumonia, or reactionary Löffler-like pneumonitis) may develop following anthelminthic treatment (Davidson et al. 1986), possibly resulting from parasitic antigen release. Other parasites that may rarely cause pulmonary manifestations with eosinophilia include Paragonimus westermani, Trichomonas tenax, Capillaria aerophila, and Clonorchis sinensis. Human infection by the filarial parasite of dog Dirofilaria immitis may occasionally manifest as solitary pulmonary nodules or masses with or without eosinophilia, and rarely eosinophilic pulmonary infiltrates (Gershwin et al. 1974; Feldman et al. 1992). Pulmonary infection with eosinophilia has been reported with the fungi Coccidioides immitis, Bipolaris australiensis and Bipolaris spicifera. BAL eosinophilia may occur in patients with Pneumocystis-jiroveci pneumonia (Fleury-Feith et al. 1989). Eosinophilic pneumonia may occasionally be caused by bacterial and viral pulmonary infections (e.g., tuberculosis, brucellosis, respiratory syncytial virus infection).

Allergic bronchopulmonary mycosis Pulmonary manifestations due to the fungus Aspergillus are varied and include aspergilloma, chronic necrotizing aspergilloma, invasive pulmonary aspergillosis, allergic asthma, and allergic bronchopulmonary aspergillosis (ABPA). ABPA is secondary to a complex allergic and immune reaction to the presence of Aspergillus spp. colonizing the airways. It may be associated with allergic Aspergillus sinusitis (Shah et al. 1990, 1993), a condition with similar fungus-driven inflammatory pathophysiology and resulting in so-called sinobronchial allergic aspergillosis (Shah et al. 1993; Torres et al. 1996; Schubert & Goetz 1998; Ponikau et al. 1999; Leonard et al. 2001; Venarske & Deshazo 2002). ABPA occurs mainly in patients with asthma (prevalence 1–2%) where the asthma may have been present for several years, and in 2–7.8% of patients with cystic fibrosis (Geller et al. 1999; Mastella et al. 2000). Interestingly, the prevalence of heterozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is increased in patients with ABPA or sinobronchial allergic mycosis, in the absence of overt cystic fibrosis (Marchand et al. 2001; Venarske & Deshazo 2002). CFTR gene mutations may thus somehow facilitate colonization of the airways by Aspergillus and be involved in the development of ABPA. In addition, ABPA may be occasionally facilitated by some environmental or occupational exposures. For example, a high prevalence of ABPA has been reported in workers in the bagasse-containing sites in sugarcane mills (Mehta & Sandhu 1983). Prevalence of infection with Mycobacterium abscessus may be increased in patients with ABPA and cystic fibrosis (Mussaffi et al. 2005).

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The pathophysiology of ABPA is complex and results from both an immune and inflammatory reaction in response to antigens from Aspergillus growing in mucus plugs in the airways of asthmatics (see Chapter 84 for further pathophysiologic description). Both type I hypersensitivity (mediated by IgE antibodies) and type III hypersensitivity (involving IgG and IgA antibody responses) are involved in the immune process. The inflammatory and immunologic response of the host predominates in the bronchial wall, where growth of the fungus takes place, and results in bronchial wall inflammation and bronchiectasis. Tissue damage extends from the bronchial wall to the adjacent pulmonary parenchyma (Kauffman et al. 1995; Patterson & Grammer 1995). ABPA is characterized by asthma, eosinophilia, and bronchopulmonary manifestations with bronchiectasis. Five stages of ABPA have been described, although they may overlap: acute, remission, recurrent exacerbations, corticosteroiddependent asthma, and fibrotic end stage (Patterson & Roberts 1995). The diagnosis if most often made in patients presenting with recurrent exacerbations of asthma, with sputum, fever, transient infiltrates due to eosinophilic pneumonia and proximal bronchiectasis on chest imaging, and peripheral blood eosinophilia (which rapidly resolves with corticosteroid treatment). Chest imaging may also reveal mucus plugging, with a typical V-shaped opacity with the vertex pointing toward the hilum, and segmental, lobar, or even whole lung atelectasis (Mintzer et al. 1978; Sulavik 1988). Proximal bronchiectases, well visualized on CT and predominating in the upper lobes (Bosken et al. 1988; Angus et al. 1994; Panchal et al. 1997), are a hallmark of ABPA, and should raise suspicion of the disease when present in an asthmatic patient. However, typical proximal bronchiectasis may be absent in a proportion of patients with ABPA, such cases being designated ABPA-seropositive (Greenberger 2002a). Nevertheless, the combination of proximal bronchiectasis, centrilobular nodules, and mucoid impaction on CT (Ward et al. 1999) is highly characteristic and allowed a correct diagnosis to be made in 84% of cases of ABPA among other eosinophilic lung diseases (Johkoh et al. 2000). Eosinophils and Charcot–Leyden crystals may be found in sputum and expectorated mucus plugs. Blood eosinophilia is generally greater than 1 × 109/L especially during the acute phase and exacerbations of the disease. IgG- and especially IgE-mediated hypersensitivity to Aspergillus antigens may be documented. Approximately 40 antigenic components may be targeted by IgE antibodies, of which 22 recombinant Aspergillus allergens (Asp f1 to Asp f 22) have been made available (Greenberger 2002a). A syndrome of allergic bronchopulmonary mycosis similar to ABPA may be associated with several other fungi or yeasts, including Pseudallescheria boydii, Cladosporium herbarum, Candida albicans, Stemphylium sp., Torulopsis sp., Curvularia lunata, Bipolaris sp., Rhizopus sp., Trichosporon terrestre, Fusarium vasinfectum,

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Table 86.5 Diagnostic criteria for allergic bronchopulmonary aspergillosis (ABPA). (Adapted from Greenberger 2002a,b.) Minimal criteria Asthma Immunologic hypersensitivity to Aspergillus fumigatus Immediate reaction to skin-prick test Elevated specific IgE Elevated serum IgE Central bronchiectasis* Other criteria Pulmonary infiltrates on chest radiograph Serum precipitating antibodies to A. fumigatus Peripheral blood eosinophils > 1 × 109/L Other common findings Expectoration of mucus plugs Presence of Aspergillus in sputum Late skin reactivity to Aspergillus antigen * When absent, ABPA is called ABPA-seropositive.

and Helminthosporium sp. (Cordier 2003). Demonstration of IgE-mediated hypersensitivity to fungi other than Aspergillus is difficult as commercially available tools are lacking. The suggested diagnostic criteria of ABPA (Table 86.5) have not been prospectively validated. They include a combination of asthma, pulmonary infiltrates, proximal bronchiectasis, elevated serum IgE, and immunologic hypersensitivity to A. fumigatus (including immediate reaction to skin-prick test for Aspergillus antigen, precipitating antibodies against A. fumigatus, and elevated specific IgE against A. fumigatus) (Rosenberg et al. 1977; Patterson et al. 1995). In addition, patients may manifest with recurrent expectoration of mucus plugs, with possible culture of Aspergillus spp. When documented, late skin reactivity to Aspergillus antigen also contributes to the diagnosis (Rosenberg et al. 1977). On rare occasions, eosinophilic pneumonia has been documented pathologically in resection specimens from patients with ABPA and chronic pulmonary consolidation (Mccarthy et al. 1970; Bosken et al. 1988), but diagnostic lung biopsy is not indicated. The diagnosis of ABPA in cystic fibrosis may be particularly difficult, and specific diagnostic criteria have been suggested (see Chapter 84). The treatment of ABPA mainly relies on both corticosteroids and itraconazole. When possible, the use of oral corticosteroids is limited to the treatment of recurrent exacerbations of the disease; management of episodes of pulmonary consolidation may prevent the progression of ABPA to the fibrotic end stage (Patterson et al. 1987). Long-term corticosteroids may be necessary in patients with frequent symptomatic attacks and/or evidence of progressive lung damage (Wardlaw & Geddes 1992; Greenberger 1995). Inhaled corticosteroids may reduce the need for long-term oral corticosteroids.

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Oral itraconazole allows reduction of the doses of oral corticosteroids and is therefore a useful adjunct to corticosteroids (Salez et al. 1999; Stevens et al. 2000; Wark & Gibson 2001) (see Chapter 84). In a double-blind, randomized, placebo-controlled study, immunologic and physiologic criteria were improved in patients treated with itraconazole, although no significant effect was found on pulmonary infiltrates (Stevens et al. 2000). Treatment with itraconazole also improves several surrogate immunologic markers of the disease (sputum eosinophilia, sputum ECP levels, serum IgE levels, serum IgG levels to A. fumigatus) and reduces the rate of acute exacerbations in patients with ABPA (Wark et al. 2003). Care should be given to the potential interactions of itraconazole with many medications and possible induction of adrenal insufficiency (Granger & Houn 1991; Parmar et al. 2002). The newer agent voriconazole has been used in ABPA only in isolated case reports (Mulliez et al. 2006). Improvement in symptoms and normalization of lung function has been reported in a child with ABPA and cystic fibrosis and severe corticosteroid-related adverse events treated with an anti-IgE recombinant antibody, allowing stopping of steroids (Van Der Ent et al. 2007).

Bronchocentric granulomatosis Bronchocentric granulomatosis (Liebow 1973) is an inflammatory bronchiolocentric process, which begins within the bronchiolar walls and further extends into the surrounding lung parenchyma. Tissue damage parallels the anatomic distribution of the lesions (Katzenstein & Askin 1997), which predominate in the bronchiolar and peribronchiolar area, with progressive destruction of both the mucosa and bronchiolar walls by the granulomatous inflammatory process. The necrotic areas resulting from the destroyed bronchioles are often surrounded by palisading histiocytes. The cellular infiltrate is mainly composed of polymorphonuclear eosinophils, especially in asthmatic patients. Infiltration of the peribronchial tissue by inflammatory mononuclear cells is usually limited but may occasionally extend more distally in the parenchyma. Scattered fungal hyphae may be demonstrated by Grocott silver stain in some patients; an infectious cause may be found in some of the cases, especially in nonasthmatic patients. Vascular inflammation and mucoid impaction may be found in some cases (Katzenstein et al. 1975; Katzenstein & Askin 1997). Clinical and radiologic manifestations of bronchocentric granulomatosis are nonspecific, and the diagnosis is seldom evoked before pathologic analysis of a lung specimen. In about half the cases, bronchocentric granulomatosis occurs in asthmatic patients, with fever, cough, and peripheral blood eosinophilia (generally greater than 1 × 109/L) (Katzenstein et al. 1975; Katzenstein & Askin 1997). Most of these patients fulfill the diagnostic criteria for ABPA. Imaging features of bronchocentric granulomatosis are varied. When present in a patient with asthma and peripheral blood eosinophilia, lung

Pulmonary Eosinophilia

masses may allow differentiation from ABPA and suggest the diagnosis of bronchocentric granulomatosis. Imaging features may also include lung consolidation, alveolar infiltrates, or reticulonodular opacities. Radiologic abnormalities predominate in the upper lobes and are unilateral in a majority of patients (Robinson et al. 1982; Ward et al. 2000). Corticosteroids represent the mainstay of treatment, with good clinical efficacy and excellent prognosis, although recurrences are common (Katzenstein & Askin 1997).

Eosinophilic pneumonias induced by drugs, toxic agents, and radiation Drug-induced eosinophilic pneumonia Eosinophilic pulmonary infiltrates may be reportedly caused by more than 80 drugs (Table 86.6), although causality has been definitely established for only a minority of them. The list of potential causative agents is still open (Espelata et al. 2007; Hayes et al. 2007). The most consistent reports of druginduced eosinophilic pneumonia implicate either nonsteroidal antiinflammatory drugs or antibiotics; cholesterol-lowering agents (statins) are also common causative agents. As a result, all drugs taken in the weeks or months preceding the symptoms must be systematically recorded and a potential iatrogenic cause must be systematically considered in patients with eosinophilic pneumonia, before the diagnosis of ICEP or IAEP is made. Drug-induced eosinophilic lung disease may present as (i) transient cough and wheezes with interstitial infiltrates and peripheral blood eosinophilia (Löffler syndrome), which may be missed if chest imaging is not performed; (ii) chronic eosinophilic pneumonia, with progressive dyspnea, cough and mild fever, that may occur after a drug has been taken for several months, or years for the treatment of a chronic disease; or (iii) acute eosinophilic pneumonia sometimes requiring mechanical ventilation. In addition, asymptomatic eosinophilic pneumonia may be incidentally discovered by systematic chest X-ray. Systemic eosinophilic vasculitis involving the lung (and thus closely resembling CSS) has also been reported (Rich 1942; Yermakov et al. 1983). Eosinophilic pneumonias due to drug exposure are usually indistinguishable from their idiopathic counterparts; however, pleural effusion and cutaneous rash may be more frequently encountered. Absolute proof that a drug is responsible for the eosinophilic pneumonia has been demonstrated in some cases by its reintroduction with ensuing relapse of pneumonia. This unethical approach may be dangerous and is not recommended. Since most patients with suspected drug-induced eosinophilic pneumonia are given corticosteroids concomitantly with drug withdrawal, the responsibility of the drug may not be ascertained in the majority of cases. In some cases with moderate disease, the incriminated drug may be withdrawn as the sole therapeutic procedure; subsequent regression of the eosinophilic pneumonia (which may take several weeks) is a convincing clue for the iatrogenic origin of the lung disease.

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Table 86.6 Drugs that may cause eosinophilic pneumonia.* (Adapted from Cordier 2003.) Drugs that typically cause pulmonary eosinophilia Acetylsalicylic acid Captopril Diclofenac Ethambutol Fenbufen Granulocyte–macrophage colony-stimulating factor Ibuprofen L-Tryptophan Minocycline Naproxen Para (4)-aminosalicylic acid Penicillins Phenylbutazone Piroxicam Pyrimethamine Sulindac Sulfonamides Tolfenamic acid Trimethoprim–sulfamethoxazole

monia include the eosinophilia–myalgia syndrome, which was related to the intake of contaminated preparations of Ltryptophan. Respiratory manifestations (dyspnea and cough) were associated with interstitial lung disease or infiltrates in only 13% on chest CT (Hertzman et al. 1995). The outcome was favorable, especially in patients receiving corticosteroids (Tazelaar et al. 1990; Strumpf et al. 1991; Hertzman et al. 1995). Similarly, the toxic-oil syndrome, which was caused by the ingestion of denatured cooking oil (Alonso-Ruiz et al. 1993). Although both conditions are not encountered any more, physicians should remain aware of the possibility of toxic or illicit drug-induced eosinophilic pneumonia in either isolated cases or apparently epidemic cases.

Eosinophilic pneumonia after radiation therapy

Drugs that occasionally cause pulmonary eosinophilia Bleomycin Carbamazepine Chlorpromazine Cocaine Desipramine Dapsone Febarbamate Gold salts Heroin Imipramine Isoniazid Loxoprofen Mephenesin Methotrexate Methylphenidate Nitrofurantoin Nomifensine Pentamidine Perindopril Phenytoin Propranolol Sulfasalazine (Parry et al. 2002) Trimipramine

Chronic eosinophilic pneumonia has been reported in women treated by radiation therapy for breast cancer (Cottin et al. 2004). All patients had a history of asthma, allergy, or both. The eosinophilic pneumonia followed the completion of radiation therapy by a median time of 3.5 months (range 1–10 months). Clinical and imaging features were indistinguishable from those of ICEP, with dyspnea and cough being the main presenting symptoms. Pulmonary opacities on chest radiograph were unilateral (irradiated lung) or bilateral, and migratory in some cases. All patients had blood eosinophilia of 1.0 × 109/L or greater and/or eosinophilia greater than 40% at BAL differential cell count. The search for other known causes of eosinophilic pneumonia was negative. As in other cases of eosinophilic pneumonia, oral corticosteroids was followed by rapid clinical and radiologic improvement without sequelae, although relapse occurred in some patients after treatment withdrawal. Radiation therapy may also be considered as a potential cause or trigger in patients with relapsing eosinophilic pneumonia (Cottin & Cordier 2006; Miranowski & Ditto 2006). Although the pathophysiology of this uncommon response to radiation therapy is not well understood, this syndrome compares with the now wellrecognized occurrence of bronchiolitis obliterans organizing pneumonia syndrome primed by radiation therapy to the breast (Crestani et al. 1998). It is hypothesized that eosinophilic pneumonia may develop preferentially in patients with preexisting asthma or atopy, with a shift of lymphocyte response toward a Th2 response phenotype facilitated by yet unidentified genetic and/or environmental factors.

* See also www.pneumotox.com

Eosinophilic asthma and eosinophilic bronchitis Eosinophilic asthma

Eosinophilic pneumonia due to toxic agents Physicians should systematically enquire about the possible intake of illicit drugs such as cocaine and heroin (often denied by the patient), which represent well-documented causes of eosinophilic pneumonia. Other causes of eosinophilic pneu-

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Eosinophil cell counts may be moderately elevated (usually 0.5–1 × 109/L) in bronchial asthma, in the absence of eosinophilic pneumonia, and especially with lack of criteria for the diagnosis of ICEP, ABPA, or CSS. Known causes of eosinophilia and eosinophilic pneumonia must be systematically investigated in such patients. When performed, eosinophil differential cell count at BAL may also show mildly increased

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levels of eosinophils (usually fewer than 5%) in asthmatics (Van Vyve et al. 1992). Although prospective studies of patients with isolated eosinophilic asthma are lacking, it is considered that such patients should be carefully monitored, given their potential propensity to further develop CSS or formes frustes of CSS. Oral corticosteroids may be required for asthma in a proportion of the patients not controlled despite high-dose inhaled corticosteroids and bronchodilators. The polymorphonuclear eosinophil plays a major role in the pathogenesis of asthma (Bousquet et al. 1994), as described elsewhere in this book. Eosinophilic infiltration in the bronchial wall of asthmatics has been correlated with the severity of asthma (Bousquet et al. 1990). Interestingly, infiltration of the airways by eosinophils is very common in eosinophilic pneumonias, and bronchial asthma would theoretically be expected in the majority of these patients (in whom asthma is actually rather inconstant).

Eosinophilic bronchitis Eosinophilic bronchitis (without asthma) is defined by an increased percentage of eosinophils (up to 40%) in sputum. The observed values of sputum eosinophils are often much higher in eosinophilic bronchitis than in asthma (Niimi et al. 1998). Spirometry is normal, and bronchial hyperreactivity is lacking. Eosinophilic bronchitis is now recognized as a cause of chronic cough responsive to corticosteroid treatment (Gibson et al. 1989, 2002; Niimi et al. 1998). Cough may also respond to prolonged inhaled corticosteroids. Eosinophilic bronchitis may evolve over time to bronchial asthma (Park et al. 2004) or to irreversible airflow obstruction without asthma (Brightling et al. 1999), suggesting that eosinophilic bronchitis may in some cases represent an early stage of obstructive lung disease. Eosinophilic bronchitis with chronic cough induced by latex gloves has been reported in a nurse (Quirce et al. 2001). A single case of eosinophilic bronchitis revealing the myeloproliferative variant of chronic HES with the fusion protein Fip1L1–PDGFR-α has been reported (Chung et al. 2006).

Other lung diseases with associated eosinophilia Peripheral blood and/or alveolar mild eosinophilia may be present in several pulmonary disorders where eosinophilic pneumonia is not a major finding.

Organizing pneumonia Organizing pneumonia is defined pathologically by the presence of buds composed of inflammatory cells, fibroblasts, and connective tissue within the lumen of distal airspaces. Clinical and imaging features of organizing pneumonia (especially cryptogenic organizing pneumonia) may resemble those of

Pulmonary Eosinophilia

ICEP, with chronic or subacute onset of dyspnea and cough, and patchy alveolar infiltrates that may be migratory on chest radiograph (Cordier 2004). Eosinophilia usually less than 20% at BAL differential cell count may be present (the BAL differential cell count usually shows a mixed pattern, with increase in lymphocytes, neutrophils and eosinophils). The diagnosis of organizing pneumonia may be obtained by transbronchial or video-assisted thoracoscopic lung biopsy. On pathologic examination, some overlap may exist between organizing pneumonia and ICEP, with possible eosinophils in organizing pneumonia, and foci of organizing pneumonia in ICEP respectively (in the rare event when a lung biopsy is performed in eosinophilic pneumonia). In addition, organizing pneumonia may represent the evolution of untreated ICEP in some cases (Cordier & Cottin 2003).

Idiopathic pulmonary fibrosis and interstitial pneumonia in connective tissue disease Mildly increased levels of eosinophils (together with increased levels of neutrophils) may be found at BAL differential cell count in idiopathic pulmonary fibrosis, and is associated with a poor clinical response to corticosteroids, in studies that antedate the current classification of idiopathic interstitial pneumonias (Haslam et al. 1980; Rudd et al. 1981; TurnerWarwick & Haslam 1987). In addition, focal eosinophilic pneumonia has been reported in cases of usual interstitial pneumonia/idiopathic pulmonary fibrosis (Yousem 2000). Mildly increased levels of eosinophils at BAL may also be observed in interstitial pneumonia in connective tissue disease especially scleroderma.

Langerhans cell granulomatosis and sarcoidosis The pathologic pulmonary lesions of Langerhans cell granulomatosis (also designated eosinophilic granuloma or pulmonary histiocytosis X) consist of stellate-shaped nodules of proliferating Langerhans cells, associated with variable numbers of eosinophils, plasma cells, and lymphocytes. Eosinophils, usually present in the initial active stage of disease, contribute to the “eosinophilic granuloma” and are located at the periphery of the lesions. Eosinophils are rare or absent in lung biopsies taken during the chronic stage of disease (Basset et al. 1978; Friedman et al. 1981; Powers et al. 1984; Travis et al. 1993; Lieberman et al. 1996). Mild peripheral blood eosinophilia and tissue eosinophilia may be present in sarcoidosis (Renston et al. 2000). Sarcoidosis with blood eosinophilia developed in a patient with a history of asthma and eosinophilic pneumonia (Anon. 1989). Pulmonary eosinophilia has been described after transplantation for sarcoidosis (Gerhardt et al. 2003). ICEP may overlap with sarcoidosis and Behçet disease (Shijubo et al. 1995).

Other conditions with occasional eosinophilia Pulmonary eosinophilia in lung transplant recipients may result from infection due to agents such as Aspergillus, Pseudomonas,

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or coxsackie virus (Yousem 1992), or may be associated with acute rejection (Yousem 1992; Bewig et al. 1999; Mogayzel et al. 2001; Gerhardt et al. 2003). Eosinophilic pneumonia was reported in a patient with gastric cancer producing granulocyte–macrophage colonystimulating factor and IL-5 (Horie et al. 1996), and in a patient with papillary adenocarcinoma of the lung (Valdivia-Arenas & Khayat 2007).

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Hypereosinophilic Syndromes Hans-Uwe Simon

Summary The hypereosinophilic syndromes (HES) are a heterogeneous group of disorders characterized by the presence of marked blood and tissue eosinophilia resulting in a variety of clinical manifestations. Corticosteroids are the first-line therapy for most HES patients, although difficulties may arise due to side effects and/or primary or secondary unresponsiveness. Recently, there has been significant progress in the understanding of the pathogenesis of at least some HES subgroups, leading to novel strategies for the treatment. For instance, anti-interleukin (IL)-5 monoclonal antibody treatment has been shown to have a significant corticosteroid-sparing effect. Moreover, an HES subgroup was identified, in which platelet-derived growth factor receptor α (PDGFRA) plays a key role in the pathogenesis, that can be successfully treated with a tyrosine kinase inhibitor. Additional work is required to understand the pathogenesis of other HES subgroups, to define markers for diagnosis and treatment responses, as well as to develop new therapeutic approaches for these disorders.

(a)

Definition/classification Three criteria define hypereosinophilic syndrome (HES): (i) sustained blood eosinophilia > 1.5 × 109/L present for longer than 6 months (Fig. 87.1); (ii) other apparent etiologies for eosinophilia must be absent; and (iii) patients must have signs of organ involvement. These criteria were established by Chusid et al. (1975). However, the general scientific community finds it increasingly difficult to follow these criteria (Table 87.1). For instance, it is unlikely that an HES patient with symptoms would remain untreated for 6 months, with no therapeutic effort made to improve the patient’s condition. Therefore, there are current limitations in establishing the diagnosis of HES and it is therefore important to suggest a

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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(b) Fig. 87.1 Blood eosinophilia as a characteristic feature of patients suffering from hypereosinophilic syndrome (HES). (a) Blood smear from a patient with HES. Multiple eosinophils are seen following staining with Giemsa–May–Grünwald. (b) Purified blood eosinophils are used for in vitro experiments (see, for example, Simon et al. 2003a; Vassina et al. 2006). (See CD-ROM for color version.)

more practicable definition. Within the International Eosinophil Society (http://www.eosinophil-society.org/eosinophil/), plans are currently underway to develop such a new definition. Another problem is the exclusion of some eosinophilic diseases that are associated with blood eosinophil numbers in excess of 1.5 × 109/L and no evidence for an infectious or

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Table 87.1 Diagnostic criteria of hypereosinophilic syndromes. Blood eosinophilia > 1.5 × 109/L for at least 6 months No antigenic or other known trigger of eosinophilia (allergic disorders, parasitic helminth infections, HIV, drug hypersensitivity, cancer) Pathologic eosinophil infiltration of tissues/organs associated with dysfunction

allergic disease. It is unclear why we consider HES a heterogeneous group of disorders with unknown pathogenesis, but do not consider certain eosinophilic diseases that fulfill all the criteria of HES. Examples are eosinophilic pneumonia and Churg–Strauss syndrome since neither has an apparent etiology different from that of HES. Moreover, there are several eosinophilic skin diseases with some specific clinical features, and providing no infectious, allergic, or other causative trigger can be identified and blood eosinophil numbers exceed 1.5 × 109/L, these diseases fulfil the criteria of HES.

(a)

Epidemiology HES comprises a group of uncommon disorders. It is a predominantly male condition where the male-to-female ratio averages 3 : 1. The onset of the disease commonly occurs between the ages of 20 and 50 years, but may also occur in childhood or adolescence. It is estimated that approximately 5000 patients suffer from HES in the USA (Wilkins et al., 2005). However, exact data on its prevalence or incidence are currently not available. This might be due to the many different subgroups treated by different medical specialists, but also because of the problems with the HES definition. However, some data do exist for specific HES subgroups. For instance, HES associated with an immunophenotypically abnormal T-cell clone was found in approximately 25% of HES patients suffering from dermatologic symptoms (Simon et al. 1999). Moreover, the FIP1L1–PDGFRA gene fusion was seen in 14% of HES patients with a myeloproliferative disease (Pardanani et al. 2004).

Physiology Eosinophils originate from hematopoietic stem cells. Under the influence of interleukin (IL)-5 and, to a lesser extent, IL-3 and granulocyte–macrophage colony-stimulating factor (GMCSF), hematopoietic progenitor cells differentiate into mature eosinophils in the bone marrow (Rothenberg & Hogan 2005). In HES patients, the generation of eosinophils in the bone marrow is accelerated (Fig. 87.2). Mature eosinophils are predominantly tissue-dwelling cells and only a small proportion circulates in the peripheral blood as leukocytes. Eosinophils display a striking affinity for tissues having an epithelial inter-

(b) Fig. 87.2 Bone marrow eosinophilia due to increased eosinophil production in patients suffering from hypereosinophilic syndrome (HES). Bone marrow sections were stained with anti-eosinophil cationic protein antibody. (a) Normal bone marrow; (b) HES bone marrow. (See CD-ROM for color version.)

face with the environment. Nevertheless, under physiologic conditions, with the exception of hematopoietic and lymphatic tissues, eosinophils are almost exclusively limited to the digestive tract mucosa (Straumann & Simon 2004). Little is known about the physiologic lifespan of mature eosinophils in tissues. Ex vivo studies using nasal polyp explants suggest that eosinophils survive for up to 2 weeks under inflammatory conditions (Simon et al. 1997).

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Genetics Eosinophilia seems to be under genetic control and linkage peaks have been mapped to the 5q31–33 locus (Martinez et al. 1998; Rioux et al. 1998). However, no data are currently available regarding HES, except for familiar eosinophilia (see below). An acquired genetic alteration has been described, leading to overexpression of the PDGFRA gene in a subgroup of HES patients (PDGFRA-associated HES) (Cools et al. 2003).

Etiology According to the current definition, an apparent etiology for eosinophilia in HES must be absent (Chusid et al. 1975). However, in some HES subgroups, new molecular and immunologic markers that are largely driving the syndrome have been described (Roufosse et al. 2003). This is another reason why the current HES definition is limited in value, and therefore the Hypereosinophilic Syndromes Working Group of the International Eosinophil Society has suggested that the term “idiopathic” should no longer be used in association with the term “HES” (Klion et al. 2006).

Pathogenesis Increased expression of the eosinophil hematopoietins IL-3 and IL-5, but not GM-CSF, is associated with HES (Owen et al. 1989; Simon et al. 1999; Vassina et al. 2006). In most cases, IL-5 appears to be the driving force of hypereosinophilia, since the therapeutic use of an anti-IL-5 monoclonal antibody rapidly decreases eosinophil numbers in blood (Plötz et al. 2003; Klion et al. 2004a). Many different pathologic events may lead to increased IL-5 expression. One recognized HES subgroup is associated with immunophenotypically abnormal clonal T cells overexpressing IL-5 (Cogan et al. 1994; Simon et al. 1996, 1999). This subgroup is often termed “lymphocytic variant HES” (Roufosse et al. 2003). However, HES may also involve T cells that have a normal immunophenotype or do not represent a T-cell clone. Therefore, the term “lymphocytic variant HES” should not necessarily be restricted to the HES subgroup associated with an IL-5-producing T-cell clone. Another recognized HES subgroup is familiar eosinophilia (Klion et al. 2006). Genetic studies suggested that this disorder is also cytokine driven, since it has been mapped to the cytokine gene cluster located at the 5q31–33 locus (Rioux et al. 1998). Activation of IL-5 or IL-3 genes may also occur as a consequence of a chromosomal translocation (Meeker et al. 1990; Malbrain et al. 1996; Luciano et al. 1999). The central role of IL-5 in the pathogenesis of many forms of HES is also underlined by observations made in experi-

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mental animal models. Overproduction of IL-5 in transgenic mice results in profound eosinophilia (Dent et al. 1990), and deletion of the IL-5 gene causes a marked reduction in eosinophil numbers (Kopf et al. 1996). Moreover, neutralization of IL-5 using specific antibodies also reduced eosinophil numbers in multiple experimental systems (Simon 2002). However, eosinophilia may also develop, at least partially, in an IL-5-independent manner. For instance, PDGFRAassociated HES is the consequence of a gene fusion in pluripotent hematopoietic stem cells. Therefore, some authors consider this disorder a chronic eosinophilic leukemia or a chronic myeloid leukemia associated with eosinophilia (Simon & Simon 2007). The gene fusion can be identified in multiple hematopoietic lineages, including neutrophils, monocytes, lymphocytes, and mast cells in such patients. Since PDGFRA is a tyrosine kinase, the treatment approach in this subgroup of HES is different. Indeed, the tyrosine kinase inhibitor imatinib has been shown to be effective in these patients (Gleich et al. 2002; Cools et al. 2003; Klion et al. 2004b). There are other chronic myeloid leukemias associated with eosinophilia/ myeloproliferative variants of HES that result from known mutations in pluripotent hematopoietic stem cells or multipotent myeloid stem cells (Simon & Simon 2007). Moreover, there are other patients diagnosed as HES who are responsive to imatinib but have no evidence for a known genetic rearrangement. This raises the possibility that such patients have a rearrangement, or at least overexpression, of a gene encoding a tyrosine kinase.

Clinical features HES can cause multiple symptoms depending on the location and the strength of the eosinophilic inflammatory response. In principle, every organ can be affected (Fig. 87.3). It has been reported that the eosinophilic infiltration may affect the cardiovascular system (58%), peripheral and central nervous system (54%), skin (56%), respiratory system (49%), spleen (43%), liver (30%), as well as the gastrointestinal tract (23%) (Weller & Bubley 1994). Other tissues, for instance bone or fat, can also be infiltrated by eosinophils, although less frequently (Fig. 87.4). The possible clinical symptoms depending on the affected organ/system are listed in Table 87.2. HES typically has a gradual onset. At the beginning, patients often complain of general symptoms such as anorexia, fatigue, weight loss, fever, abdominal pain, and night sweats. Throughout the course of the disease, the clinical manifestations and prognosis depend on the involved organs. Patients with cardiac involvement have a rather poor prognosis with the risk of fatal outcome, whereas those with skin disease generally endure a milder course (Roufosse et al. 2003). On the other hand, there are HES patients without symptoms as well as symptomatic HES patients with normal eosinophil counts on therapy, suggesting that, besides eosinophil infiltration,

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Bone marrow

Blood IL-5 GM-CSF IL-3

Table 87.2 Organ involvement in patients suffering from hypereosinophilic syndromes and associated symptoms.

Tissue IL-5 GM-CSF IL-3 •Skin •Heart •Brain

IL-5 GM-CSF IL-3

•Lungs •Liver •Intestine

Differentiation

Adhesion/migration or apoptosis

Hypereosinophilic Syndromes

Activation and increased survival

Fig. 87.3 Production, tissue infiltration, activation, and lifespan of eosinophils in tissues are influenced by eosinophil hematopoietins, in particular by IL-5. In principle, eosinophils can infiltrate into any organ in patients suffering from hypereosinophilic syndromes. (See CD-ROM for color version.)

Hematologic Eosinophilia in blood and bone marrow, leukocytosis Thrombocytosis or thrombocytopenia Anemia Increased serum vitamin B12 Splenomegaly Increased circulating myeloid precursors Dysplastic eosinophils Increased serum tryptase Spindle-shaped mast cells in bone marrow Myelofibrosis Cardiovascular Dyspnea Angina pectoris Heart insufficiency Endomyocardial fibrosis Damage of atrioventricular valves Cardiomegaly Intracavitary thrombi Thromboembolic phenomena

an eosinophil activation process is required for eosinophilmediated damage (Simon et al. 2003a). At the moment, no biomarker suitable for clinical monitoring of eosinophil activity is available (Klion et al. 2006).

Cutaneous Pruritic erythroderma Papules Urticarial plaques Angioedema Mucosal ulcerations

Differential diagnosis

Ocular Visual symptoms, often blurring

Hypereosinophilia is a common finding in clinical practice. Parasitic infections, atopic diseases, and allergic drug reactions account for most cases of hypereosinophilia. Moreover, hematopoietic or solid tumors can represent the underlying cause. Churg–Strauss syndrome, eosinophilic pneumonia, and eosinophilic gastroenteritis are also often associated with hypereosinophilia, but are not considered HES. These and other underlying diseases must be ruled out before HES can be considered. Some of the HES subgroups can be distinguished from other conditions associated with eosinophilia through a combination of routine and specialized testing (Roufosse et al. 2003; Klion et al. 2006). Besides clinical investigation including blood count and tissue(s) analysis, efforts should be made to understand the molecular mechanisms responsible for the clinical condition of patients suffering from HES. Since IL-5 plays a central role in multiple subgroups of HES, detectable levels of this cytokine in blood are suggestive of an IL-5driven mechanism. The analysis of other eosinophil hematopoietins (IL-3, GM-CSF) might also be helpful, since not all cytokine-driven HES may be caused by IL-5. However, in particular, sample collection for cytokine measurements is not standardized, making it difficult to compare results

Gastrointestinal Dysphagia Diarrhea Hepatitis Pulmonary Cough Pulmonary infiltrates Lung fibrosis Bronchospasm (seldom) Neurologic Embolic strokes or transient ischemic episodes Encephalopathy Peripheral neuropathy Rheumatologic Arthralgias Raynaud phenomenon Myalgia

between different laboratories at present. It should be noted that an undetectable level of IL-5 in blood does not exclude IL-5-driven HES. The source of increased eosinophil hematopoietin production differs between subgroups and patients with HES.

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Skin

Fat tissue

(b)

(a) Stomach

Bone

(c)

(d) Fig. 87.4 Examples of eosinophil tissue infiltration in patients suffering from hypereosinophilic syndromes. The tissue sections were stained with Giemsa–May–Grünwald (a, b, and d; almost all infiltrating cells represent eosinophils) or anti-eosinophil cationic protein antibody (c). (Tissue sections in a, b, and d kindly provided by Dr S.G. Plötz, Munich.) (See CD-ROM for color version.)

The most common cells producing IL-5 are T cells. In one subgroup of HES, patients demonstrate circulating IL-5producing clonal T cells with abnormal immunophenotypes. This represents the subgroup called lymphocytic variant HES. The T cells exhibit abnormal expression of lineage-associated surface markers, such as CD2, CD3, CD4, CD5, CD6, CD7, and CD8 (Cogan et al. 1994; Simon et al. 1996, 1999). In some cases, they do not express the death receptor Fas, although the T cells show evidence for activation (positive for HLA-DR and CD25, as well as production of IL-5). It is possible, but not proven, that lack of Fas expression contributes to the expansion of the T-cell clone (Simon et al. 1996). To establish the diagnosis, T cells are analyzed by flow cytometry and T-cell clonality is demonstrated by Southern blot analysis and/or reverse transcriptase-polymerase chain reaction (RTPCR). These patients also have high levels of TARC (thymus and activation-regulated chemokine) in blood (de Lavareille et al. 2002), a phenomenon that can be used in the diagnostic work-up. Patients suffering from lymphocytic variant HES

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often exhibit skin symptoms (Cogan et al. 1994; Simon et al. 1996, 1999, 2001; de Lavareille et al. 2002), although gastrointestinal symptoms may also occur (Bauer et al. 1996; Simon et al. 1996, 1999). On the other hand, the molecular abnormality driving eosinophil expansion may be localized within the eosinophil itself, causing a clonal eosinophil disorder. This subgroup of HES is termed “myeloproliferative variant HES” (Roufosse et al. 2003; Klion et al. 2006). These patients have clinical and biological features reminiscent of chronic myelogenous leukemia and other myeloproliferative syndromes. Laboratory abnormalities include increased serum vitamin B12 levels, altered leukocyte alkaline phosphatase score, chromosomal abnormalities, anemia and/or thrombocytopenia, hepatomegaly, splenomegaly, and circulating leukocyte precursors. One subgroup belonging in this group of patients is PDGFRAassociated HES, which is additionally characterized by increased serum tryptase, myelofibrosis, and dysplastic mast cells or megakaryocytes in bone marrow (Cools et al. 2003;

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Table 87.3 Diagnostic evaluation of patients suffering from hypereosinophilic syndromes. Symptom-oriented diagnosis Imaging techniques (abdominal ultrasound, echocardiogram, cardiac MRI) Organ functions Biopsies Exclusion of known triggers of eosinophilia Infections (parasites, fungi, HIV, bacteria) Allergy Drug hypersensitivity Cancer Peripheral blood Differential blood count Thrombocyte numbers Serum tryptase Serum TARC Serum vitamin B12 Serum IgE, IgG, IgA, IgM Leukocyte alkaline phosphatase score Lymphocyte phenotyping T-cell receptor gene rearrangement analysis Conventional cytogenetic analysis Cytokine profile of T cells FIP1L1–PDGFRA gene fusion (RT-PCR or FISH) Bone marrow Bone marrow smear and biopsy (tryptase and reticulin stain) Conventional cytogenetic analysis FISH, fluorescence in situ hybridization; RT-PCR, reverse transcriptasepolymerase chain reaction.

Klion et al. 2003). Since the FIP1L1–PDGFRA gene fusion also affects mast cells, these patients need to be distinguished from patients suffering from systemic mastocytosis. The FIP1L1– PDGFRA gene fusion can be detected by RT-PCR or fluorescence in situ hybridization. Distinction of an underlying myeloid or lymphocytic disorder is important for follow-up and treatment. A summary of the clinical and laboratory diagnostic procedures discussed above is presented in Table 87.3.

Treatment/management Corticosteroids are the first-line treatment for most patients, although the most appropriate initial dose and the duration of therapy has not been evaluated. Usually, patients are treated with a high dose (> 40 mg prednisone equivalent) initially and then the dose is gradually reduced. Using this approach, most, but not all, HES patients respond. Patients receiving long-term treatment have increased risks of developing opportunistic infections (e.g., Pneumocystis-induced pneumonia)

Hypereosinophilic Syndromes

and bone loss. Corticosteroid-mediated adverse effects often become limiting and the use of alternative therapies must be considered. Cytotoxic therapies may be used in corticosteroid-refractory HES. Hydroxycarbamide (hydroxyurea) has been successfully applied at doses of 1–3 g/day (Parrillo et al. 1978). In contrast to the rapid reduction of eosinophil numbers after application of corticosteroids, a decrease in the eosinophil count is generally not seen until 2 weeks after starting hydroxycarbamide therapy. Higher doses are associated with unwanted hematologic and gastrointestinal effects. Low doses (0.5 g/day) may act synergistically with interferon (IFN)-α (Butterfield 2005). Low-dose IFN-α (1–3 million units daily) has been reported to control HES for prolonged periods of time. Similarly to hydroxycarbamide, eosinophil numbers do not decline rapidly, but a reduction can be expected within 2–4 weeks (Simon et al. 2003b; Butterfield 2005). IFN-α, even at low doses, has multiple unwanted systemic effects. The already mentioned combination with hydroxycarbamide might allow reduction of the doses of both drugs. Anti-IL-5 monoclonal antibody therapy reduces eosinophil numbers in HES patients within 1 day of administration (Plötz et al. 2003; Klion et al. 2004a). This response is sustained and lasts for at least 4 weeks after a single infusion. In a large, double-blind, placebo-controlled study, neutralization of IL-5 using an anti-IL-5 antibody (mepolizumab; GlaxoSmithKline) has been shown to be successful in reducing the need for systemic corticosteroids (Rothenberg et al. 2006). This antibody treatment was extremely well tolerated. However, it should be noted that stopping treatment may lead to a rebound of eosinophilia and symptoms (Kim et al. 2004). Imatinib, a tyrosine kinase inhibitor, is an effective drug in PDGFRA-associated HES. The imatinib response rate in these patients is nearly 100%, with only two reported cases of acquired drug resistance (as a consequence of a T674I mutation in PDGFRA) (Gleich et al. 2002; Cools et al. 2003; Klion et al. 2004b). A reduction in eosinophil numbers is seen within 1 week of starting therapy. Clinical improvement is seen within 1 month. Low-dose imatinib (100 mg/day) is usually sufficient to control eosinophilia and symptoms. The use of imatinib in HES patients in whom PDGFRA does not play a role is controversial. In general, there is no or a slower response and patients require higher imatinib doses. Multiple other immunomodulatory and/or immunosuppressive therapies have been reported. These include cyclosporin A, methotrexate, anti-CD52 monoclonal antibody, intravenous immunoglobulin preparations, and several anticancer drugs (Klion et al. 2006). However, the available clinical material is not sufficient and does not allow a general conclusion at present. Although eosinophil numbers are usually used to control the response to treatment, it is, as mentioned above, difficult to predict eosinophil-mediated tissue damage. No other marker,

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including eosinophil-derived basic proteins, has been validated. However, in subgroups of HES patients, the treatment response can be monitored precisely. For instance, the presence or absence of the abnormal fusion protein in PDGFRAassociated HES (Klion et al. 2004b) or of immunophenotypically abnormal T-cell clones in lymphocytic variant HES (Simon et al. 2001) allows monitoring of therapeutic effects. In other cases, however, eosinophil numbers and clinical presentation remain the only parameters that are used at present.

Prognosis The severity of eosinophil-mediated organ damage determines the prognosis of HES patients. In particular, heart involvement and development of malignancy limit the lifespan of an affected patient. Currently, 70% of HES patients live 10 years and longer, thanks to an increased awareness of the necessity of lowering blood eosinophil numbers to prevent organ damage. Moreover, the surgical management of cardiac complications has improved. Cardiac complications are preferentially observed in patients with elevated tryptase levels (Klion et al. 2003). Most of these patients belong to the subgroup of PDGFRA-associated HES. In myeloproliferative variant HES, progression into leukemia may occur (Griffin et al. 2003; Vandenberghe et al. 2004). Lymphocytic variant HES is considered to have a better prognosis, since the disease appears to be stable over years (Simon et al. 2001). However, the clonal T cells may transform into a T-cell lymphoma (Simon et al. 1999, 2001). Therefore, monitoring of the numbers of abnormal T cells over time is recommended.

Acknowledgments The work in the laboratory of the author is supported by grants from the Swiss National Science Foundation (310000107526), the Stanley Thomas Johnson Foundation (Bern), and OPO-Foundation (Zurich).

References Bauer, S., Schaub, N., Dommann-Scherrer, C.C., Zimmermann, D.R., Simon, H.-U. & Wegmann, W. (1996) Long-term outcome of idiopathic hypereosinophilic syndrome: transition to eosinophilic gastroenteritis and clonal expansion of T-cells. Eur J Gastroenterol Hepatol 8, 181–5. Butterfield, J.H. (2005) Interferon treatment for hypereosinophilic syndromes and systemic mastocytosis. Acta Haematol 114, 26–40. Chusid, M.J., Dale, C.D., West, B.C. & Wolff, S.M. (1975) The hypereosinophilic syndrome: analysis of fourteen cases with review of the literature. Medicine (Baltimore) 54, 1–27. Cogan, E., Schandene, L., Crusiaux, A., Cochaux, P., Velu, T. &

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Goldman, M. (1994) Brief report: clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome. N Engl J Med 330, 535–8. Cools, J., DeAngelo, D.J., Gotlib, J. et al. (2003) A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med 348, 1201–14. de Lavareille, A., Roufosse, F., Schmid-Grendelmeier, P. et al. (2002) High serum thymus and activation-regulated chemokine levels in the lymphocytic variant of the hypereosinophilic syndrome. J Allergy Clin Immunol 110, 476–9. Dent, L.A., Strath, M., Mellor, A.L. & Sanderson, C.J. (1990) Eosinophilia in transgenic mice expressing interleukin 5. J Exp Med 172, 1425–31. Gleich, G.J., Leiferman, K.M., Pardanani, A., Tefferi, A. & Butterfield, J.H. (2002) Treatment of hypereosinophilic syndrome with imatinib mesilate. Lancet 359, 1577–8. Griffin, J.H., Leung, J., Bruner, R.J., Caligiuri, M.A. & Briesewitz, R. (2003) Discovery of a fusion kinase in EOL-1 cells and idiopathic hypereosinophilic syndrome. Proc Natl Acad Sci USA 100, 7830–5. Kim, Y.J., Prussin, C., Martin, B. et al. (2004) Rebound eosinophilia after treatment of hypereosinophilic syndrome and eosinophilic gastroenteritis with monoclonal anti-IL-5 antibody SCH55700. J Allergy Clin Immunol 114, 1449–55. Klion, A.D., Noel, P., Akin, C. et al. (2003) Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness. Blood 101, 4660– 6. Klion, A.D., Law, M.A., Noel, P., Haverty, T.P. & Nutman, T.B. (2004a) Safety and efficacy of the monoclonal anti-interleukin 5 antibody, SCH55700, in the treatment of patients with the hypereosinophilic syndrome. Blood 103, 2939–41. Klion, A.D., Robyn, J., Akin, C. et al. (2004b) Molecular remission and reversal of myelofibrosis in response to imatinib mesylate treatment in patients with the myeloproliferative variant of hypereosinophilic syndrome. Blood 103, 473–8. Klion, A.D., Bochner, B.S., Gleich, G.J. et al. (2006) Approaches to the treatment of hypereosinophilic syndromes: A workshop summary report. J Allergy Clin Immunol 117, 1292–302. Kopf, M., Brombacher, F., Hodgkin, P.D. et al. (1996) IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses. Immunity 4, 15–24. Luciano, L., Catalano, L., Sarrantonio, C., Guerriero, A., Califano, C. & Rotoli, B. (1999) AlphaIFN-induced hematologic and cytogenetic remission in chronic eosinophilic leukemia with t(1;5). Haematologica 84, 651–3. Malbrain, M.L., van den Bergh, H. & Zachee, P. (1996) Further evidence for the clonal nature of the hypereosinophilic syndrome: complete haematological and cytogenetic remission induced by interferon-alpha in a case with a unique chromosomal abnormality. Br J Haematol 92, 176–83. Martinez, F.D., Solomon, S., Holberg, C.J., Graves, P.E., Baldini, M. & Erickson, R.P. (1998) Linkage of circulating eosinophils to markers on chromosome 5q. Am J Respir Crit Care Med 158, 1739–44. Meeker, T.C., Hardy, D., Willman, C., Hogan, T. & Abrams, J. (1990) Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia. Blood 76, 285–9.

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Owen, W.F., Rothenberg, M.E., Petersen, J. et al. (1989) Interleukin 5 and phenotypically altered eosinophils in the blood of patients with the idiopathic hypereosinophilic syndrome. J Exp Med 170, 342– 8. Pardanani, A., Brockman, S.R., Paternoster, S.F. et al. (2004) FIP1L1– PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia. Blood 104, 3038– 45. Parrillo, J.E., Fauci, A.S. & Wolff, S.M. (1978) Therapy of the hypereosinophilic syndrome. Ann Intern Med 89, 167–72. Plötz, S.G., Simon, H.-U., Darsow, U. et al. (2003) Use of an antiinterleukin-5 antibody in the hypereosinophilic syndrome with eosinophilic dermatitis. N Engl J Med 349, 2334–9. Rioux, J.D., Stone, V.A., Daly, M.J. et al. (1998) Familial eosinophilia maps to the cytokine gene cluster on human chromosomal region 5q31– q33. Am J Hum Genet 63, 1086–94. Rothenberg, M.E. & Hogan, S.P. (2005) The eosinophil. Annu Rev Immunol 24, 147–74. Rothenberg, M.E., Klion, A.D., Roufosse, F.E. et al. (2008) Treatment of hypereosinophilic syndrome with mepolizumab. N Engl J Med, in press. Roufosse, F., Cogan, E. & Goldman, M. (2003) The hypereosinophilic syndrome revisited. Annu Rev Med 54, 169–84. Simon, D. & Simon, H.-U. (2007) Eosinophilic disorders. J Allergy Clin Immunol 119, 1291–1300. Simon, H.-U. (2002) The neutralization of interleukin-5 as a therapeutic concept in allergic inflammation. Sarcoidosis Vasc Diffuse Lung Dis 19, 25– 8. Simon, H.-U., Yousefi, S., Dommann-Scherrer, C.C. et al. (1996) Expansion of cytokine-producing CD4–CD8– T cells associated with abnormal Fas expression and hypereosinophilia. J Exp Med 183, 1071– 82.

Hypereosinophilic Syndromes

Simon, H.-U., Yousefi, S., Schranz, C., Schapowal, A., Bachert, C. & Blaser, K. (1997) Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia. J Immunol 158, 3902–8. Simon, H.-U., Plötz, S.G., Dummer, R. & Blaser, K. (1999) Abnormal clones of T cells producing interleukin-5 in idiopathic eosinophilia. N Engl J Med 341, 1112–20. Simon, H.-U., Plötz, S.G., Simon, D., Dummer, R. & Blaser, K. (2001) Clinical and immunological features of patients with interleukin5-producing T cell clones and eosinophilia. Int Arch Allergy Immunol 124, 242–5. Simon, H.-U., Plötz, S., Simon, D. et al. (2003a) Interleukin-2 primes eosinophil degranulation in hypereosinophilia and Wells’ syndrome. Eur J Immunol 33, 834–9. Simon, H.-U., Seelbach, H., Ehmann, R. & Schmitz, M. (2003b) Clinical and immunological effects of low-dose IFN-α treatment in patients with corticosteroid-resistant asthma. Allergy 58, 1250– 5. Straumann, A. & Simon, H.-U. (2004) The physiological and pathophysiological roles of eosinophils in the gastrointestinal tract. Allergy 59, 15–25. Vandenberghe, P., Wlodarska, I., Michaux, L. et al. (2004) Clinical and molecular features of FIP1L1–PDGFRA(+) chronic eosinophilic leukemias. Leukemia 18, 734–42. Vassina, E.M., Yousefi, S., Simon, D., Zwicky, C., Conus, S. & Simon, H.-U. (2006) cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis. Eur J Immunol 36, 1975–84. Weller, P.F. & Bubley, G.J. (1994) The idiopathic hypereosinophilic syndrome. Blood 83, 2759–79. Wilkins, H.J., Crane, M.M., Copeland, K. & Williams, W.V. (2005) Hypereosinophilic syndrome: an update. Am J Hematol 80, 148– 57.

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Atopic Dermatitis Julia D. Proelss and Thomas Bieber

Summary

Introduction

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in daily clinical routine. As a rule, AD starts in infancy and is considered as the starting point of the so-called atopic march, i.e., followed by allergic rhinitis and allergic asthma. The clinical spectrum of AD varies with age, including all features of acute or chronic dermatitis with intense pruritus being an essential hallmark. While much progress in understanding its pathogenesis has been achieved in recent years, it has become clear that AD is a genetically complex disease involving gene–gene and gene–environment interactions. Lessons from genetic studies have shown a linkage to genes related to epidermal barrier function as well as to candidate genes of the immune system. Among the possible triggering factors, exposure to aeroallergens or food allergens as well as hygienic factors, bacterial or viral infections, and stress has been described as initiating or exacerbating AD. Although the “hygiene hypothesis” is controversial, the importance of lifestyle and environment in the mechanisms of atopic disease is now well accepted. AD develops on a background of generalized Th2-deviated immune response but the skin condition itself demonstrates a biphasic course. The initial phase of disease is characterized by a Th2 response while chronic lesions harbor Th0/Th1 cells. Moreover, T cells with regulatory activity have been shown to be altered in AD. Most importantly the innate immune system in the skin seems to be profoundly disturbed, providing the basis for increased microbial complications. The principal goal in the management of AD resides in control of the skin inflammation, the elimination of triggering factors, and restoration of skin barrier function in order to potentially prevent the emergence of sensitization.

Atopic dermatitis (AD) is the most common chronic and relapsing inflammatory skin condition. Due to its increasing prevalence, it represents a major public-health problem, mostly in industrialized countries. The pathophysiology of AD is complex. Parental atopy, particularly AD, is significantly associated with the manifestation and severity of early AD in children. Other pathogenetic factors include environmental, immunologic, genetic, and pharmacologic features. As a rule, AD starts during the first year of life and presents with patches, plaques, papules, and vesicles. During the course of the disease, excoriations, bacterial infections, and lichenification will usually occur. Further atopic disorders, such as allergic rhinoconjunctivitis or allergic bronchial asthma, may develop during the active course or remission phase of AD, supporting the concept of the atopic march.

Definition The disease has puzzled generations of physicians and researchers and resulted in a multitude of different names: atopic dermatitis and atopic eczema are currently the most widely used; Besnier’s prurigo, disseminated neurodermitis, or neurodermitis constitutionalis sive atopica, and constitutional eczema are less frequently chosen. In some countries like the UK, the disease is simply named “eczema.” The variation of terms arose from different understandings of the pathogenesis of AD. In order to be consistent, the term “atopic dermatitis” is used in this chapter as it is currently the most widely accepted name, particularly since the recent consensus nomenclature provided by the World Allergy Organization (Johansson et al. 2004).

Atopy Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

In 1923, Coca and Cooke coined the term “atopy” to designate a group of patients suffering from one or several distinct

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disorders, namely skin rush (eczema), asthma bronchiale, and reactions due to pollen (hay fever, rhinitis, and conjunctivitis). The term atopy was adapted from the Greek word atopia, meaning “out of place.” In 1967, IgE antibodies were first discovered and associated with AD as a sign of dysregulation of the immune system. Due to advances in the pathophysiology of AD and the need for a unified definition in the context of globalized medicine and biomedical research, a consensual nomenclature became mandatory. In 2004 the World Allergy Organization proposed a revised terminology for atopy and atopic diseases, defining atopy only in association with IgE sensitization, i.e., atopic diseases due to IgE-mediated pathophysiology. According to this, the term “atopy” should only be applied in combination with documented specific IgE antibodies in serum or with a positive skin-prick test (Johansson et al. 2004). This current terminology substitutes the former term of “extrinsic AD” and includes only patients who show IgE sensitization against inhalant and/or food allergens in skin tests or serum. A small group comprising approximately 20–30% of affected patients show clinical signs of AD without any IgE sensitization (Novak & Bieber 2003). The term “nonatopic eczema” is now applied to this group, replacing the previous term “intrinsic AD.” Patients with nonatopic eczema lack IgE mediation and exhibit negative skin tests. Both forms of eczema have associated eosinophilia.

Prevalence With a lifetime prevalence of 15–30% in children and 2– 10% in adults, the incidence of AD has increased twofold to threefold during the past three decades in industrialized countries (Asher et al. 2006). AD usually presents during early infancy and childhood but may also persist or start in adulthood. The 12-month prevalence in 11-year-old children has been shown to vary from 1 to 20%, with the highest prevalence typically found in northern Europe (International Study of Asthma and Allergies in Childhood) (Asher et al. 2006). In 45% of children the onset of AD occurs during the first 6 months of life, 60% of children are affected during the first year of life, and 85% are affected before the age of 5 years. The prevalence of AD in rural areas is significantly lower, emphasizing the importance of lifestyle and environment in the mechanisms of atopic disease. The so-called “hygiene hypothesis” (Strachan 1989) emphasizes that the propensity to atopic-associated diseases is due to reduced microbial exposure in early life. However, AD seems to be more complex than this, relying on the interplay of several diverse factors. Due to modern advances in genetics and immunology, much progress has been made in elucidating the pathophysiology of AD, yet the hygiene hypothesis is still one important and hotly debated feature (Williams & Flohr 2006).

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Clinical features and course Symptoms of AD vary with age and may differ during the course of disease (reviewed in Williams 2005). The clinical spectrum of AD is wide, ranging from mild forms such as pityriasis alba (dry depigmented patches) to major forms with erythrodermic rash (Table 88.1). The eczema is polymorphic, with three forms: acute (oozing, crusted, eroded vesicles or papules on erythematous plaques), subacute (thick pale excoriated plaques), and chronic (lichenified, slightly pigmented, excoriated plaques). Abnormally dry skin and a lowered threshold for itching are important features of AD. All stages of disease are associated with or caused by pruritus. Pruritus results in scratching, prurigo papules, and lichenification, which themselves cause pruritus. Because of constant rubbing, the fingernails become polished (glossy nails). Although pruritus can occur throughout the day, it generally worsens during the evening; these paroxysmal attacks of itching cause insomnia, exhaustion, and impaired ability to work. Exacerbation of pruritus and scratching can be caused by diverse trigger factors, such as heat and perspiration, wool, emotional stress, foods, alcohol, upper respiratory infections, and house-dust mites (Table 88.2). Subjective relief is obtained by excoriation and bleeding of the skin. Since the pathophysiology of pruritus in AD is still not fully understood, satisfactory therapy is still lacking; some relief is achieved by the use of antihistamines but this is more due to sedation of the patient than to an antihistamine effect.

Table 88.1 Clinical features of atopic dermatitis. Major features Pruritus Flexural eczema Chronic or relapsing dermatitis Associated features Xerosis Personal or family history of atopy Cutaneous infections Nonspecific hand and foot dermatitis Elevated serum IgE concentrations Positive immediate-type allergy skin tests Early age onset Other features Ichthyosis, palmar hyperlinearity, keratosis pilaris Nipple eczema Pityriasis alba White dermatographism Dennie-Morgan infraorbital folds, periorbital darkening Facial erythema or pallor Anterior subcapsular cataracts, keratoconus

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Atopic Dermatitis

Table 88.2 Triggers of itch in atopic dermatitis. Xerosis Irritants Disinfectants Soaps Contact with food: fresh fruits and juices, meats, vegetables Contact allergens and aeroallergens House-dust mites Pets Pollens Molds Human dander Microbial agents Viral infections Staphylococcus aureus (pathogen or “superantigen”) Others Foods Temperature/climate Psyche Hormones

Fig. 88.1 Atopic eczema in a neonate with milk crust. (See CD-ROM for color version.)

Table 88.3 Differential diagnosis of atopic dermatitis.

Infants First signs of inflammation typically occur during the third month of life. Infants present with facial and patchy or generalized body eczema. Lesions generally first appear on the cheeks and are characterized by dry and erythematous skin with papulovesicular lesions. Scratching of the skin results in inflammatory and crusty erosions. Perioral and paranasal areas are spared in the beginning, but as lip licking progresses, oozing, crusting, and scaling on the lips and perioral skin occurs. The term “milk crust” or “milk scurf” refers to the occurrence of yellowish crusts on the scalp that resemble scaled milk (Fig. 88.1). During this stage, differentiation from seborrheic dermatitis may be sometimes difficult (Table 88.3). Due to the persisting pruritus, the infant is uncomfortable and becomes restless and agitated during sleep. Once the infant becomes more mobile and begins crawling, exposed areas, such as the inner and outer parts of the arms and legs, may also be affected. The diaper area is usually spared. Itching is very intense and promotes a great tendency to bacterial secondary infection (impetiginization). The lesions may run a chronic or relapsing course and may be influenced by events such as teething, respiratory infections, and adverse emotional stimuli. In about 50% of patients, lesions heal by the end of the second year of life; in some cases they gradually lose their original exudative character and turn into chronic lesions, characterized by lichenification.

Chronic dermatoses Seborrheic dermatitis Ichthyosis Contact dermatitis (allergic or irritant); nummular eczema Psoriasis Congenital disorders Netherton syndrome Familial keratosis pilaris Scabies HIV-associated dermatitis Dermatophytosis Malignant disease Cutaneous T-cell lymphoma (mycosis fungoides, Sézary syndrome) Letterer–Siwe disease Immunologic disorders Dermatitis herpetiformis Pemphigus foliaceus Graft-versus-host disease Dermatomyositis Immunodeficiencies DiGeorge syndrome Wiskott–Aldrich syndrome Hyper-IgE syndrome Severe combined immunodeficiency Metabolic disorders Phenylketonuria Multiple carboxylase deficiency

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Fig. 88.2 Typical flexural eczema in childhood. (See CD-ROM for color version.)

Fig. 88.3 Atopic eczema in an adult patient. (See CD-ROM for color version.)

Children In childhood, from 18 to 24 months onwards, favored sites for eczema include flexural areas (antecubital fossae, neck, wrists, and ankles) and the nape of the neck, dorsum of the feet, and hands (Fig. 88.2). The eczema can either develop from the preceding neonatal phase or arise de novo. Rashes usually begin with papules that become hard and lichenified with inflammatory infiltration when they are scratched. The skin around the lips may be inflamed and constant licking of the area may lead to small painful cracks in the perioral skin. Constant scratching and manipulation of affected skin cause destruction of melanocytes, resulting in areas of hypopigmentation when the inflammation subsides (postinflammatory hypopigmentation). During childhood, eczema may disappear completely for long periods, leaving sensitive dry skin.

Adolescents and adults (Figs 88.3 and 88.4) For unknown reasons, AD may relapse during puberty. As in the childhood phase, localized inflammation with lichenification of the flexural areas is the most common pattern seen in adolescents and adults. Skin lesions usually present symmetrically. Favored sites include the neck, upper chest, large joint flexures, and backs of the hands. Facial skin is usually affected on the forehead, eyelids, and perioral region. Scalp involvement is possible and may lead to a diffuse loss of hair. The remaining hair is dry and dull; the lateral parts of the eyebrows are thinned, known as Hertoghe’s sign. During adulthood, dry skin continues to be a persistent problem, especially in winter months.

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Fig. 88.4 Atopic eczema in an adult patient. (See CD-ROM for color version.)

More localized lesions such as hand dermatitis, nummular eczema or inflammation around the eyes are subsumed as minimal variants. These variants are often associated with AD, though they are not specific and can be likewise found in patients without any signs of AD. The noneczematous skin findings are considered nonessential for the diagnosis of AD, although they often give a first diagnostic lead.

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Minimal variants of AD Palmar and plantar dermatitis Hand dermatitis is the most common expression of the adult atopic diathesis. Approximately 20–30% of all cases show typical signs as erythema, vesicles, scaling, and fissuring, as well as lichenification of the hands. Erythematous and scaled lesions with fissures occur on the palms while erythematous plaques with blisters may also occur on the back of the hands; the fingers are usually inflamed. Nails are often affected, resulting in coarse pitting and ridging. Additionally, almost half of patients show eczema of the feet with similar symptoms; aggravation of the symptoms can often be seen during winter months. If there are no clinical or anamnestic signs of AD, hand and foot eczema can also be related to a dyshidrosis and is then termed “dyshidrotic eczema.” It is sometimes very difficult to distinguish dyshidrotic eczema from atopic palmoplantar dermatitis since clinical signs are nearly identical. However, dyshidrotic eczema is often additionally associated with a chronic contact dermatitis, mycosis, or nicotine abuse. In addition to antiinflammatory agents, treatment should include that of the primary disease.

Numular eczema Nummular eczema is characterized by erythematous, scaly, coin-shaped lesions of 2–10 cm in diameter. The clinical picture of AD may evolve into different forms of eczema. Lesions may be uniformly red at first, than clear in the center to form a ring. Nummular eczema is characterized by erythema, scaling, and lichenification. The outbreak may begin with one or more lesions usually located on the lower and upper extremities as well as on the trunk.

Eyelid dermatitis With an incidence of about 10–20%, eyelid dermatitis is a frequent symptom of AD patients. The occurrence is supported by the vulnerability of the thin eyelid skin, constant exposure to irritants or allergens, and chronic scratching of this area. Eyelid dermatitis presents as symmetrical, pruritic, scaly erythematous, lichenified plaques of the medial eyelid. The entire orbital skin becomes involved due to constant irritation and may result in loss of eyelashes and/or eyebrows.

Atopic Dermatitis

cheilitis sicca with severe itching. Inflammation is caused by frequent licking to rehydrate the lips (vicious circle).

Nipple eczema Approximately 10–15% of young patients with AD are affected with sensitive areolar skin which is irritated by the slightest manipulation (e.g., friction of clothing). Symmetrical, scaly, papulovesicular patches around the nipple or vermillion are typical signs of this condition.

Pityriasis alba Pityriasis alba is characterized by hypopigmented, poorly demarcated, macular plaques with fine scaling. Sites of predilection include the face, neck and upper trunk. Lesions generally become more apparent after ultraviolet (UV) exposure. Pityriasis alba is caused by lack of differentiation of melanocytes to keratinocytes, explaining the hypopigmentation of the lesions.

Atopic stigmata Atopic stigmata include specific clinical features that are highly characteristic for AD (Przybilla et al. 1991). These characteristic signs can be found at any age and any stage of disease. Atopic stigmata are characteristic of AD, but lack specificity since they can likewise be present in other diseases as well. Atopic stigmata include the following symptoms.

Xerosis Xerosis (dry skin) is an important feature of the atopic state. Xerosis may appear at any age and is recognized as fine scaling on clinically noninflamed skin. Dry skin is sensitive, easily irritated by external stimuli and, more importantly, itchy. Symptoms are worse during winter months, when scaling skin becomes cracked and fissured. Perennial fissuring of the infraauricular, retroauricular, and infranasal areas is perennially characteristic of AD skin. It is often the result of extremely dry skin with bacterial superinfection. Xerosis is predominantly located on the extensor surfaces of the legs and arms, but may involve the entire cutaneous surface in susceptible individuals.

Keratosis pilaris

Lichenified atopic eczema of the anogenital area is characterized by very severe pruritus, lichenification, chronicity, and a tendency to recurrence. Persistent scratching causes inflammation which may last for years since treatment is often difficult. Because of self-medication, allergic or irritant contact dermatitis should always be considered.

Keratosis pilaris is characterized by small, rough, follicular papules that appear at any age. It is regarded as a defect of keratinization in the xerotic hair follicles. Keratosis pilaris is frequently noted during childhood and peaks during adolescence, improving thereafter. Frequently involved are the posterolateral aspects of the upper arms and anterior thighs, but any area, with the exception of the palms and soles, may be involved.

Cheilitis

Palmar and plantar hyperlinearity

A typical stigma in adolescence is a persistent scaliness of the lips, which often occurs during cold weather as exfoliative

Palmar and plantar hyperlinearity is recognized as an accentuation of the major skin creases of the palms. This

Lichenification of the anogenital area

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hyperlinearity becomes more prominent as age and disease severity increases. Although markings become more visible on dry skin, it is not believed to be a result of xerosis.

Dennie-Morgan lines Dennie-Morgan lines are symmetrical folds that extend from the medial aspect of the lower lid. In general, being a sign of Down syndrome, it can be seen in 60– 80% of atopic individuals. They are first noticed at birth and persist for life, but can equally appear in persons who present no signs of atopy.

Anterior neck folds Anterior neck folds are noted as horizontal folds running across the middle of the anterior neck of patients with AD. Like Dennie-Morgan lines, they are asymptomatic without any clinical significance.

Hertoghe’s sign Hertoghe’s sign describes the absence or thinning of the lateral eyebrows and has no clinical significance.

White dermographism Erythematous dermographism is a normal reaction in about 5% of the population. After a physical stimulus is applied to the skin (i.e., scratching), the path of irritation is observed as a red line. This response occurs within a few minutes after the insult; wheals are absent. White dermographism can be observed in patients with AD and allergic contact dermatitis. The reaction is thought to be due to a paradoxical skin reaction to histamine, cholinergic agents, and nicotinates. It has been suggested that white dermographism represents an inflammatory epiphenomenon rather than a disorder of atopy itself.

Periorbital darkening Periorbital darkening is a typical but not very specific sign of AD. The skin around the eyes appears gray to violet, while the rest of the facial skin is rather pale. These discolorations are termed “allergic shiners” and are due to increased pressure on nasal and paranasal venous plexi, caused by constant rhinitis, conjunctivitis, and eye rubbing. No clinical significance is associated.

Diagnostic criteria The diagnosis of AD is based on clinical findings and is historically based on the criteria of Hanifin and Rajka (1980) (Table 88.4). Evidence of itchy dry skin and a history of asthma or hay fever usually gives the first lead. The diagnosis becomes probable when at least three major and at least three minor criteria are evident. Since Hanifin and Rajka’s minor criteria lack specificity and may also be found in patients without any atopic history, a revision of the diagnostic criteria was published by Williams et al. (1994). Of the given

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Table 88.4 Criteria for diagnosis of atopic dermatitis. (Adapted from Hanifin & Rajka 1980.) Major features (must have three or more) Pruritus Flexural lichenification in adults Facial and extensor involvement in infants and children Chronic dermatitis Personal or family history of asthma, allergic rhinitis, or atopic dermatitis Minor features (must have three or more) Cataracts Conjunctivitis Cheilitis Eczema Facial pallor/facial erythema Hand dermatitis Ichthyosis Food intolerance Elevated IgE levels Immediate skin test reactivity Staphylococcus aureus, herpes simplex infections Dennie-Morgan line Sweat-triggered itching Keratoconus Keratosis pilaris Nipple dermatitis Orbital darkening Palmar hyperlineartiy Pityriasis alba White dermographism Xerosis

clinical criteria, four have to be fulfilled for a diagnosis of AD (Table 88.5). The SCORAD (Score in atopic dermatitis) index has been introduced to evaluate the severity of AD objectively by assessing the area and severity of involved skin (Anon. 1993) and is often used in daily practice but most importantly for clinical trials. There is no one unique sign or laboratory parameter for diagnosis of AD, rather than multiple diagnostic criteria should be fulfilled. Allergologic, skin, and in vitro tests such as RAST

Table 88.5 Criteria for the diagnosis of atopic dermatitis. (Adapted from Williams et al. 1994.) The diagnosis of AD requires evidence of itchy skin plus three or more of the following: History of involvement of the skin creases (e.g., fronts of elbows, backs of knees) History of asthma or hay fever History of generally dry skin in the past year Onset in a child under 2 years of age (criterion not used if the child is under 4 years of age) Visible flexural dermatitis

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(radioallergosorbent test) are reserved for patients with relevant clinical symptoms in order to detect specific IgE antibodies against an increasing number of allergens. Besides clinical evaluation, allergologic testing should be performed when clinical symptoms are present. RAST is performed to determine specific IgE antibodies against allergens. Results of RAST are presented qualitatively (classes 1–6) and quantitatively (kU/L). IgE sensitization has to be positive (> 150 kU/L) to meet the general criteria of AD. Provocation tests are additionally performed to determine the clinical significance of positive laboratory tests, since skin tests and in vitro testing should complement one another yet do not always have to be concordant. The atopy patch test (APT), a provocation test, is used to provoke an eczematous reaction through the application of aeroallergens and food allergens epicutaneously to a patient’s back (Ring et al. 1991). Its major usefulness resides in its specificity rather than its sensitivity (Darsow et al. 1995, 1999).

Histology Histology has no fundamental impact on the diagnosis of AD; clinical features should always be predominant in the diagnosis of AD. All signs of chronic or acute AD or eczema are combined in the histopathology of AD. Spongiosis (intercellular edema of the epidermis) and infiltration of lymphocytes into the epidermis and upper dermis can be seen. Chronic lesions are characterized by acanthosis (abnormal but benign thickening of the epidermis) and hyperkeratosis and parakeratosis (hypertrophy of the stratum corneum/retention of nuclei in the cells of the stratum corneum). Even clinically unaffected skin shows such histologic alterations as perivascular infiltrates of lymphocytes, strongly suggesting that minimal inflammation is still present.

Trigger factors AD is a multifactorial disease that can be aggravated by several factors (Morren et al. 1994) (see Table 88.2). Exogenous and endogenous aggravating factors have to be identified and should be eliminated for successful management. The most important trigger factors are discussed below.

Stress Stress is one of the major trigger and pathophysiologic factor in AD and is addressed in detail in the section on pathogenesis. The exact mechanisms of stress-induced eczema are yet not well understood (see below); neuroimmunologic factors such as neuropeptides and adenyl cyclase/cyclic AMP are thought to be essential for the development of AD. However, since the severity of skin lesions affects the personality, there is always the question as to which comes first. Patient informa-

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tion about the impact of stress on the genesis of AD is essential in the management of AD.

Allergens Inhalant allergens as well as food allergens are further important trigger factors in the genesis of AD. Food allergies are frequently seen, especially during the childhood phase. Eggs, milk, wheat, soy, and peanuts are the most common allergens and may induce eczematoid skin rashes of the child. Up to 40% of exacerbated AD is thought to be due to food allergies. Food allergen-specific T cells have been cloned from skin lesions of patients with AD, providing direct evidence that foods are responsible for skin immune responses. After being orally sensitized with foods, mice with AD developed eczematous skin lesions on repeat of food challenges. It has been shown that food allergies are transient during the course of disease; milk and egg allergies seem more important during the infant stage, while allergies to peanuts may continue throughout life. It is not fully understood why circulating T cells respond to food antigens and home to the skin to generate eczematous processes. It is hypothesized that food-derived antigens activate immature T cells to become skin-homing, or that lymphocytes stimulated by food allergens reach the skin via the circulation. Interestingly, even direct contact of food with the skin can provoke an aggravation of eczema (e.g., in the preparation of meals or APT). Food allergens become less frequent after the third year of life and inhalant allergies emerge. Pruritus and skin lesions may develop after intranasal or bronchial inhalation of aeroallergens such as house-dust mite, weeds, animal dander, or molds. House dust is the most important aeroallergen in AD. Even the penetration of mites through the skin may provoke exacerbations of AD. Epicutaneous application of aeroallergens on uninvolved skin as in the APT elicits eczematoid reactions in 30–50% of AD patients (Darsow et al. 1999). It has been shown that actions to reduce house-dust mites are associated with amelioration of eczema and should be included as an important feature of therapeutic management.

Microbial agents Staphylococcus aureus is the predominant skin microorganism in AD. Binding of S. aureus to skin causes the secretion of bacterial toxins which act as so-called superantigens. Most AD patients present specific IgE antibodies against staphylococcal superantigens, which correlate with severity of skin disease (Leung et al. 1993). Treatment with antistaphylococcal antibiotics has been shown to be effective and results in reduction of skin inflammation. The association of fungi with AD is controversial. Pityrosporum orbiculare, a member of the normal cutaneous flora, is frequently associated with subforms of AD, such as head and neck dermatitis. Pityrosporum orbiculare, also denoted Pityrosporum ovale or Malassezia furfur, preferentially colonizes the sebum-rich head, neck and upper trunk areas. Pityrosporum

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orbiculare-specific IgA, IgG and IgM antibodies can be found in both healthy individuals and patients with skin diseases, while IgE against P. orbiculare is only detected in atopic individuals, particularly those with AD (Tengvall Linder et al. 1998). An antifungal treatment is indicated in affected patients, although no causality to the pathogenesis of AD has been documented.

Complications Bacterial and viral infections The most important complications of AD are due to secondary infections. In the light of recent progress in innate immunity, it has become clear that deficiency in antimicrobial peptides is responsible for the increased bacterial and viral complications in AD (see below). Staphylococci, and to a lesser extent streptococci, present an integral part of the clinical picture, frequently provoking impetiginization of lesions (Fig. 88.5). Superinfection results in yellow impetigo-like crusting with a typical smell. Patients with AD are at increased risk for fulminant herpes simplex virus infections (eczema herpeticum) (Fig. 88.6). The course of disease may be severe with high fever and widespread eruptions. Clinically, numerous vesicles in the same stage of development is a characteristic sign. Patients feel ill and diagnosis should be accomplished rapidly, e.g., by polymerase chain reaction (PCR). It is unclear whether viral warts or molluscum contagiosum are likewise more prevalent in AD (Fig. 88.7).

Fig. 88.6 Eczema herpeticum. (See CD-ROM for color version.)

Fig. 88.7 Molluscum contagiosum of a child with AD. (See CD-ROM for color version.)

Impact on quality of life

Fig. 88.5 Secondary infection (impetiginization) of a child with AD. (See CD-ROM for color version.)

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AD may have a profound impact on patients’ lives and the lives of their families. Social interactions, psychologic adjustments, work success, sexual relationships, and quality of life often are dependent on the course of disease. Fatigue and loss of concentration due to insomnia can provoke behavioral

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difficulties in childhood. Constant pruritus has a great affect on the personality and may influence the progress of children. Depression and anxiety seem to be the most important factors in adolescence and adult patients due to frustrating therapies and the lack of a “cure.” It has long been known that the emotions of an individual are capable of triggering AD (see Table 88.2). Stress has been increasingly recognized as an important trigger of AD. Stressful events have often been observed before exacerbations of AD.

Growth delay Growth delay is significantly correlated with the degree of eczema. It is usually seen in severe cases. Long-term therapy with topical or systemic agents has been discussed and may contribute to a growth delay in any child (Bode 1980).

Differential diagnosis The diagnosis of AD usually causes no difficulties and is made according to clinical features. However, a number of alternative diagnoses may have to be considered (Table 88.3). Differentiation has to be made between several forms of eczema/dermatitis. The World Allergy Organization recently introduced a revised nomenclature for intrinsic/extrinsic dermatitis, now denoted nonatopic and atopic eczema/dermatitis respectively. According to this, the term “atopy” or “atopic” denotes exclusively the genetic predisposition to become IgE sensitized to allergens and has to be associated with increased IgE levels (> 150 kU/L). IgE sensitization occurs to all allergens commonly found in the environment but not to less common allergens such as Hymenoptera sting. The term “nonatopic eczema/dermatitis” is reserved for patients without detectable IgE-mediated reactions against typical allergens, e.g., aeroallergens or food allergens. This form is also not associated with asthma or rhinitis and presents with low total IgE (< 150 kU/L). Clinical distinction between atopic eczema/dermatitis and nonatopic eczema/dermatitis is not possible at present. In this context, 80% of adult patients suffer from the atopic form, while 10–20% are affected by a nonatopic form. From a diagnostic point of view, especially during the neonatal period, seborrheic dermatitis but also scabies and pyoderma always have to be excluded. In adults the differentiation of lichen simplex chronicus, nummular (microbial) eczema, chronic lichenified contact eczema, or nonallergic chronic eczema may cause problems. AD of the hands and feet must be differentiated from palmoplantar psoriasis, dyshidrotic eczema, and tinea. Several rare syndromes have to be taken into account such as phenylketonuria, Wiskott– Aldrich syndrome, hyper-IgE syndrome, Netherton syndrome, DiGeorge syndrome, and ataxia telangiectasia (see Table 88.3).

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Pathogenesis Much insight has been achieved regarding the complex pathogenesis of AD in recent years. Genetic predisposition, immunologic abnormalities, and environmental factors are the main factors involved in the development of this chronic condition. Progress in understanding the pathophysiologic basis of AD is especially important for comprehending other chronic diseases, such as psoriasis, and is essential for the development of new therapeutic strategies.

Genetics The genetic basis of AD is complex. Parental atopy, particularly AD, is significantly associated with the development of early AD in children (Schultz Larsen & Holm 1985). Other parental atopic diseases such as allergic asthma or allergic rhinitis seem to be minor factors in the development, suggesting the existence of genes specific to AD. So far, four genome-wide screens have been performed to identify these chromosomal regions (Cookson 2001; Cookson et al. 2001). In this approach, families with at least two affected individuals are included and their DNA was screened in a search for disease-related chromosomal regions/loci. The regions of highest linkage could be found on chromosomes 1q21 and 17q25 (Lee et al. 2000; Cookson et al. 2001; Bradley et al. 2002). Interestingly these regions correspond partially to gene loci found in patients with psoriasis, suggesting a genetic coherency of both chronic skin diseases (Cookson et al. 2001). This is especially surprising, since both diseases were thought to be pathogenetically and clinically quite different. However, recent publications have disproved the assumption that psoriasis-associated genes account for the linkage between AD and psoriasis on the chromosome 17q25 region (Morar et al. 2006). Similar findings have been observed on chromosomes 3q21 and 20p that suggested an association between chronic inflammatory diseases and variants of these loci. Disposition to atopy and asthma seem to be linked on chromosome 20p and 16q, assuming different genetic factors of both diseases. In addition to genome-wide screens, candidate gene studies have been performed to determine a possible contribution of genetic variants in known candidate gene regions. These genetic variants or polymorphisms are caused by the exchange of single base pairs (single nucleotide polymorphisms, SNP). Several candidate genes have been identified in recent years, notably on chromosome 5q31–33, all containing genes for the Th2 cytokines interleukin (IL)-3, IL-4, IL-5, IL-13, and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Forrest et al. 1999). Further studies identified variants of the IL-13-encoding region, functional mutations of the promoter region of the chemokine RANTES (regulated on activation,

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normal T-cell expressed and secreted) (17q11), and gain-of function polymorphisms in the α subunit of the IL-4 receptor (16q12) (Hershey et al. 1997). Polymorphisms of the IL-4 subunit may have an influence on the IL-4 receptor-related synthesis of IgE. This could be linked to the incidence of nonatopic (formerly intrinsic) eczema, which occurs without IgE sensitization. The long-known hypothesis of a dysbalance between Th1 and Th2 immune responses in AD may be elucidated by the detection of polymorphisms of the IL-18 gene, which result in Th2 predominance. Peripheral blood mononuclear cells showing these polymorphisms react, after being stimulated by superantigens, with upregulation of IL-18 (Novak et al. 2005). This upregulation results in a downregulation of IL-12 and consequently in a Th2 predominance. Particularly severe courses of disease with colonization of S. aureus might be associated with an SNP of the Toll-like receptor (TLR)2 gene (Ahmad-Nejad et al. 2004).

Hygiene hypothesis The “hygiene hypothesis” was first proposed by Strachan (1989). He postulated that infections during early childhood are important for the prevention of allergic diseases. In fact, environmental factors such as increased air pollution, indoor exposure to house-dust mite antigens, and dietary changes have been implicated in the pathomechanisms of AD. Many clinical studies have been performed to study this hypothesis and determine the importance of hygiene in the pathogenesis of AD. However, this concept is currently much debated, some authors questioning an association between hygiene and the incidence of AD (Zutavern et al. 2005; Williams & Flohr 2006). Earlier studies analyzing the risk of environmental factors, antibiotic therapies, or economic standards, resulted in contradictory data. Overall, exposure to infections during early childhood seems to be important for the development of immunity. Recent studies pointed out that atopic symptoms seem to occur less frequently following infections with hepatitis A virus, Helicobacter pylori or Toxoplasma gondii (Matricardi & Ronchetti 2001). It has been postulated that due to infection with these organisms the dysbalance between Th1 and Th2 immune response is diminished since bacterial and viral inflammation underlie a Th1-associated pattern. This might support the assumption that limited exposure to infectious pathogens during infancy may prevent the establishment of regulatory T cells (Treg). Treg are responsible for downregulation of Th1 and Th2 cells and are therefore closely related with the pathogenesis of AD. The characteristics of Treg are discussed subsequently. Antiinflammatory cytokines during a helminth infection are considered to suppress atopic symptoms. Although all these studies present contradictory data, there is indeed some evidence that endotoxin exposure in a farming environment, daycare attendance, or pet-keeping has a

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protective effect for AD. However, this protection might be connected with chronic infection rather than to an infection per se.

Psychological factors Psychological stress is known to exacerbate AD. The exact mechanisms are poorly understood, although neuroimmunologic factors such as neuropeptides have been thought to play an important role. Neuropeptides mediate different actions such as vasodilatation, edema, itch and pain or sweat gland secretion and have a minor ability to regulate T-cell activation (Gordon et al. 1997). Neuropeptides can be detected in blood and within epidermal nerve fibers in close association with epidermal Langerhans cells, suggesting an interaction between the immune and nervous systems (Hosoi et al. 1993). Recent studies have demonstrated increased levels of nerve growth factor and substance P in plasma of AD patients that correlated positively with disease activity (Toyoda et al. 2002). Further neuroimmunologic factors resulted in an increase in eosinophil granulocytes in patients under stress. Enhanced levels of brain-derived neurotrophic factor (BDNF) have been detected recently in sera and plasma of patients with AD. BDNF is responsible for a reduction in eosinophil apoptosis while enhancing chemotaxis of eosinophils in vitro, which might explain the generally found eosinophilia of patients with AD (Raap & Kapp 2005).

Skin barrier dysfunction AD is characterized by dry and harsh skin affecting lesional and nonlesional skin areas. An altered skin barrier function (Proksch et al. 2006), resulting in transepidermal water loss, is typical of this condition and may be the reason for the cumulative infiltration of allergens, bacteria, and viruses. The loss of skin ceramides, which serve as the major waterretaining molecules in the extracellular space of the so-called cornified envelope, has been thought to cause this modification of the skin barrier (Sator et al. 2003). Furthermore, it has been shown that variations of stratum corneum pH may impair lipid metabolism in the skin (Rippke et al. 2004). Overexpression of enzymes such as chymase is also likely to contribute to the breakdown of the AD epidermal barrier. Further considerations include a Δ6-desaturase deficiency and decreased conversion of ω6-linoleic acids to prostaglandin in affected patients. Studies underline the importance of a primary epithelial barrier defect in the pathogenesis of AD. It has been shown that mutations of filaggrin (R510X and 2282del4), a key protein in terminal differentiation of the epidermis, seem to be an important risk factor for AD and AD in combination with asthma (Palmer et al. 2006; Weidinger et al. 2006; Nomura et al. 2007). The skin barrier may be impaired due to an alteration in keratin aggregation and constitutes an important trigger factor for the penetration of allergens, even aeroallergens.

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Immunologic mechanisms Innate immunity It is widely accepted that the immune system consists of two branches: innate and adaptive immunity. Adaptive immunity relies on antigen-presenting cells to capture and present antigens to T and B cells. Innate immunity is characterized by an immediate response to pathogens through genetically encoded receptors and antimicrobial proteins. The innate immune system of the epidermis is the first line of defense against cutaneous infections with microbes. Once the epidermis is invaded by microorganisms, antimicrobial peptides are activated and form part of this defense system (McGirt & Beck 2006). Three antimicrobial peptides are known: the β-defensins HBD-2 and HBD-3, and the cathelicidin hCAP18/LL-37. All show different spectra of activity. HBD-2 is effective against Gram-negative organisms such as Escherichia coli, Pseudomonas aeruginosa, and yeasts. HBD-3 and cathelicidin are more potent, broad-spectrum antibiotics that kill both Gram-positive and Gram-negative organisms as well as Candida albicans. Immunostaining and measurement of specific mRNA for HBD-2, HBD-3, and cathelicidin in lesions from patients with AD detected a significant decrease in expression of antimicrobial peptides, explaining the susceptibility of AD patients to bacterial infections (Ong et al. 2002; Nomura et al. 2003; Howell et al. 2006). The innate skin defense system of patients with AD may be further reduced by deficiency of dermcidin-derived antimicrobial peptides in sweat, which correlates with infectious complications (Rieg et al. 2005). The peptide dermcidin is specifically expressed in sweat glands in the dermis of skin and has a broad spectrum of activity against a variety of pathogenic microorganisms. After sweating, there is a reduction in bacteria in healthy subjects, while in patients with AD this response is lacking. The reduced production of antimicrobial peptides is a major factor contributing to the susceptibility to bacterial infections in atopic patients.

Acquired immunity T cells The importance of T cells becomes evident when observing that T cell-related immunodeficiency disorders are frequently associated with elevated serum IgE levels and eczematous skin lesions, which clear after successful bone marrow transplantation (Baud et al. 2001). In animal models of AD, eczematous rashes do not occur in the absence of T cells (Spergel et al. 1999), and treatment with specific T cell-mediated medication such as calcineurin inhibitors significantly reduces eczema. Furthermore, it has been shown that Th1 and Th2 cytokines play an important role in the skin inflammatory response. Mice with overexpression of IL-4 develop AD-like skin lesions, suggesting that local skin expression of Th2 cytokines plays a critical role in the genesis of AD. Treg cells have been the center of attention in different research areas (Ziegler 2006). These diverse and complex

Atopic Dermatitis

cells have been studied for their role in transplantation, tumor immunology, and allergy since they have the ability to suppress T cells (Th1 and Th2). Specific surface markers are still lacking but activation of the nuclear transcription factor Foxp3 is characteristic of these cells (Ochs et al. 2005). It has been shown that mutations of Foxp3 result in hyperIgE, food allergy, and eczema (part of the IPEX syndrome). In addition, several studies have documented an association between atopy and the loss of Treg function (Ou et al. 2004; Verhagen et al. 2006). A revised version of the hygiene hypothesis proposes loss of Treg cells due to limited exposure to infectious pathogens during infancy. This may explain the rapid increase in the prevalence of allergies in developed countries.

Cytokines and chemokines As with all skin diseases, lesions are the expression of a distinct and specific inflammatory micromilieu. This is the result of a complex constellation of cytokines and chemokines produced by resident cells (such as keratinocytes, endothelial cells or fibroblasts) and by nonresident cells (T cells, eosinophils, antigen-presenting cells). A predominant systemic Th2 dysbalance with increased IgE levels and eosinophilia is widely accepted as involved in the pathogenesis of AD. The production of Th2-mediated cytokines, notably IL-4, IL-5, and IL-13, can be detected in lesional and nonlesional skin during the acute phase of disease. IL-4 and IL-13 are implicated in the initial phase of tissue inflammation by mediating isotype switching to IgE synthesis and upregulation of adhesion molecule expression on endothelial cells (Hamid et al. 1994). IL-5 seems to increase the survival of eosinophils, and a systemic eosinophilia with increased eosinophil cationic protein is characteristic of high disease activity in AD (Simon et al. 2004). Although Th2-mediated cytokines seem to be predominant in the acute phase of AD, they are less important during its chronic phase. In chronic AD skin lesions, increased levels of interferon (IFN)-γ and IL-12 as well as IL-5 and GM-CSF can be detected, being characteristic of Th0/Th1 dominance (Grewe et al. 1998). The maintenance of chronic AD later involves the production of the Th1-like cytokines IL-12 and IL-18, as well as several remodeling-associated cytokines such as IL-11, IL-17 and transforming growth factor (TGF)β1 (Toda et al. 2003). Interestingly, IL-31 has been shown to be expressed in high levels in AD but also in allergic contact dermatitis and may be responsible for itching in these diseases (Neis et al. 2006; Sonkoly et al. 2006). Thymic stromal lymphopoietin (TSLP) represents another important cytokine in the pathogenesis of AD. TSLP is an IL-7-like cytokine cloned from a murine thymic stromal cell line (Soumelis et al. 2002). TSLP is expressed primarily by epithelial cells, including keratinocytes, in acute and chronic lesions of AD and is associated with the activation and migration of dendritic cells within the dermis (Liu 2006). TSLP is

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thought to prime naive CD4+ T cells to differentiate into Th2 cells, which ultimately contribute to the induction of allergic inflammation. TSLP-activated cutaneous dendritic cells prime T helper cells to produce the proallergic cytokines IL-4, IL-5, IL-13, and tumor necrosis factor (TNF)-α. However, expression of the antiinflammatory cytokine IL-10 and the Th1 cytokine IFN-γ are inhibited. These features suggest that TSLP represents a critical mediator in uncontrolled allergic inflammation. A number of chemokines have lately gained additional interest in the pathology of AD. Large amounts of chemokines like MIP-4/CCL18, TARC/CCL17, MDC/CCL22, and CCL1 seem to be involved in the development of acute and chronic skin manifestations (Homey et al. 2006). These chemokines play an important role in the amplification of allergic reactions through bacteria or allergens. CC chemokines (MCP4, RANTES, eotaxin) contribute to the infiltration of macrophages, eosinophils, and T cells into acute and chronic AD skin lesions. An increasing number of chemokines have been shown to be upregulated or downregulated in AD patients; their exact role in the pathogenesis is still not fully resolved.

Dendritic cells Dendritic cells (DC) are highly specialized antigen-presenting cells that are essential for allergen uptake and presentation to T cells but may be distinguished by their respective functional capacities (Shortman & Naik 2007). Myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) have been delineated. In lesional skin of AD, only mDC have been reported while, in contrast to other inflammatory skin conditions, pDC are almost absent (Wollenberg et al. 2002). The prototype of mDC, the Langerhans cell, and inflammatory dendritic epidermal cells (IDEC) are assumed to play a central role in this disease (Novak & Bieber 2005). Both cell types express the high-affinity receptor for IgE (FcεRI) (Bieber et al. 1992; Wollenberg et al. 1996) in lesional skin of AD but not in other conditions (Wollenberg et al. 1995). IDEC are absent in healthy skin. Langerhans cells play a predominant role in the initiation of the allergic immune response and prime naive T cells to differentiate into Th2 cells (Novak et al. 2004a). In contrast, the stimulation of FcεRI on IDEC leads to (i) the release of large amounts of proinflammatory signals and (ii) the secretion of IL-12 and IL-18, which contribute to the switch from Th2 to Th1 and potentially contribute to amplification of the allergic immune response. The APT can be used as an experimental model for AD. Skin biopsy specimens from APT lesions have demonstrated the kinetic interplay of DC subtypes and skin migration (Kerschenlohr et al. 2003); 72 hours after allergen challenge, high numbers of IDEC invade the epidermis. pDC play a major role in the defense against viral infections by producing type-1 interferons, i.e., IFN-α and IFN-β. pDC in the peripheral blood of AD patients express high

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amounts of FcεRI (Novak et al. 2004b). Activation of this receptor on pDC leads to altered surface expression of major histocompatibility complex (MHC) molecules, enhanced apoptosis of pDC, but most importantly to reduced secretion of type-1 interferons. This observation, combined with the fact that pDC are almost absent in the skin of patients with AD (Wollenberg et al. 2002) and the absence of antimicrobial peptides, might contribute to the susceptibility of patients to viral skin infections such as herpes simplex-induced eczema herpeticum.

Microbial agents Staphylococcus aureus is the predominant skin microorganism in AD lesions and is found in over 90% of patients affected with AD (Aly et al. 1977; Bibel et al. 1977). Up to 50–60% of S. aureus found in atopic patients is toxin producing. Infections often provoke exacerbation or aggravation of lesional skin while nonaffected areas are significantly less affected. Today, S. aureus enterotoxins are gaining increasing importance in the pathogenesis of AD (Cardona et al. 2006). Staphylococcal enterotoxins A, B, C, and D are frequently detected in patients and are supposed to provoke IgE sensitization (Leung et al. 1993) and to influence the APT. Additionally, it has been shown that S. aureus enterotoxins act as so-called superantigens (Strickland et al. 1999). Superantigens interact directly with the MHC–T-cell complex on antigen-presenting cells and provoke proliferation of T cells without clonal specificity. This results in inflammation and may lead to the typical eczematoid skin reactions of AD patients. Specific IgE antibodies directed against staphylococcal superantigens can be detected in most AD patients, which correlate with skin disease severity. Additionally, it has been shown that binding of S. aureus to the skin is significantly enhanced by AD skin inflammation. An altered composition of fibrin and fibrinogen of AD skin is thought to be responsible for this phenomenon. Scratching probably even enhances S. aureus binding by disturbing the skin barrier. Isolated S. aureus has been shown to possess increased activity of ceramidase and this may be responsible for aggravating the skin barrier. Further skin fungal colonization includes Pityrosporum orbiculare and, less frequently, Candida subclasses.

Atopic dermatitis: an autoimmune disease? It has been proposed that autoimmunity may play a role in AD since adult patients with severe AD display IgE responses to autoallergens (Mittermann et al. 2004). These allergens represent an increasing group of proteins to which the immune system produces IgE autoantibodies because of their homology with environmental allergens and molecular mimicry. Several autoallergens have been identified, including the

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transcription factor LEDGF/DSF70 (Sugiura et al. 2007), the atopy-related autoantigens Hom s 1 to Hom s 5 produced by keratinocytes (Valenta et al. 1998), and the manganese superoxide dismutase (MnSOD) to which patients with AD produce specific IgE. This sensitization is induced by skin colonization with Malassezia sympodialis, which causes, due to its high homology, sensitization against the human MnSOD (Schmid-Grendelmeier et al. 2005). This phenomenon is predominantly observed in patients with head and neck dermatitis. Recent findings have pointed out that IgE-related autoimmunity develops during the first years of life (Mothes et al. 2005). Whether this early IgE production against autoallergens is beneficial or deleterious for the subsequent course of the disease remains unclear.

Treatment Management of AD presents a clinical challenge (Akdis et al. 2006). The main goals of treatment for AD are elimination of inflammation and infection, preservation and restoration of the stratum corneum barrier, antipruritic management, and control of exacerbating factors. Successful management requires a multipronged approach; education of the patient is as important as further therapeutic strategies.

Basic treatment Based on disease severity multiple therapeutic agents can be used as basic treatment and may even be combined. A key feature of AD is severe dryness of the skin due to dysfunction of the skin barrier with increased transepidermal water loss. The regular use of emollients and skin hydration is very important for preventing intense pruritus and inflammation. Emollients should be applied even if no actual inflammatory skin lesion is obvious. The choice of emollients depends on individual skin status. Water-in-oil or oil-in-water emulsions may be substituted to support skin barrier function. Polidocanol can be additionally applied to reduce pruritic symptoms. Urea allows intensive hydration of the skin, while salicylic acid can be added to an emollient for the treatment of chronic hyperkeratotic lesions. Irritants such as soaps, clothing made from occluding or irritating synthetic or wool material, and hot water should be avoided or reduced to a minimum (Morren et al. 1994). The patient should be educated adequately to avoid provocation factors. Specific provocation factors, such as airborne and food allergens, have to be considered and identified by serum tests for allergen-specific IgE or skin-prick tests. Extensive diets, which can be nutritionally deficient, are useless. If patients clearly identify aggravating foods, avoidance should be proposed. Dietary assessment should be performed to confirm adequate nutrition. Sensitized patients should avoid house-dust mites; appropriate measures include house-dust

Atopic Dermatitis

mite-proof encasings on pillows, mattresses and boxsprings. Bedding should be washed every week; the removal of carpets from bedrooms is important. Patients sensitized to animal dander should be discouraged from keeping household pets. Other defined trigger factors should be avoided. In selected patients hospitalization may be of great benefit, especially in centers using a multidisciplinary approach.

Topical glucocorticosteroids Topical steroids are safe and effective medications when used properly. Anxiety among the general public and family doctors is often well out of proportion to the true risk. Aside from the antiinflammatory effect, topical steroids contribute to a reduction of skin colonization with S. aureus (Stalder et al. 1994). The strength and mode of application depends on disease activity and severity, locations to be treated, and the age of the patient. Only mild to moderately potent preparations should be used on genital, facial, or intertriginous skin areas. Less potent topical steroids can also be employed in children less than 1 year old. Application should be limited to short-term therapy of about 2 weeks no more than twice a day. New steroid preparations and adequate application protocols have reduced the risk of adverse effects over the last few years. Different therapeutic schemes have been established: initial treatment should be with moderate- to highpotency steroids followed by dose reduction or change to a lower-potency preparation. Combined therapy with emollients should be routine during the course of treatment.

Topical calcineurin inhibitors Pimecrolimus and tacrolimus are topical immunosuppressant medications that inhibit a calcium-activated phosphatase called calcineurin. They have been proved to be efficient and safe in the management of AD (Alomar et al. 2004; Iskedjian et al. 2004; Breuer et al. 2005). Their antiinflammatory potency is similar to a corticosteroid of moderate potency. Pimecrolimus cream (1%) and tacrolimus ointment (0.03%) are available for treatment of children 2 years of age and older. Tacrolimus ointment (0.1%) is only approved in adults. Side effects such as dermal atrophy are not seen; treatment of the face is therefore possible. Treatment for up to 1 year with pimecrolimus and up to 4 years with tacrolimus should not be exceeded. Side effects include a transient burning sensation of the skin. While using calcineurin inhibitors, excessive exposure to natural or artificial sunlight (tanning beds or UVA/B treatment) should be avoided. Long-term safety studies analyzing the evidence for a causal link between cancer and calcineurin inhibitors as well as an increased incidence of viral infections are ongoing.

Wet-wrap therapy In cases of exacerbated AD the employment of a wet layer of cotton dressing, which is applied over emollients, has been

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shown to be beneficial for AD patients. Emollients can be used in combination with antiseptics or topical steroids in cases of superinfection. The dressings should be used overnight or changed every 12 hours. This regimen is designed as a shortterm treatment and requires close supervision.

Erythromycin is less useful because of frequent high resistance rates. In cases of allergy, clindamycin or oral fusidic acid are possible alternatives. Randomized trials found no benefits for prescribing floxacillin continually for 4 weeks compared with placebo; the risk of methicillin-resistant strains was even more common in those who were prescribed antibiotics.

Topical antimicrobial therapy Inflammation is often seen in patients with AD due to deficient skin barrier function. The skin is heavily colonized with S. aureus, even on nonaffected skin areas. Exudation and pustule formation imply superinfection of the skin, which has to be treated. Topical antiseptics such as triclosan or chlorhexidine have low sensitizing potential and show low resistance rates. They can be used in emollients or syndets or as part of an additional “wet-wrap” dressing. The combination of topical steroid and topical antimicrobial therapy shows great benefit for patients compared with topical steroids alone. Due to high resistance rates of S. aureus to erythromycin, fusidic acid is preferentially used in the treatment of bacterial infections. Topical fusidic acid has been proven to be very effective against S. aureus because of its low minimal inhibitory concentration and good tissue penetration. However, long-term therapy with fusidic acid should be abandoned due to increasing resistance rates. Therapy should be restricted to 2 weeks. Intranasal eradication of methicillinresistant S. aureus, frequently found in AD patients, can be achieved by the topical use of mupirocin. In general, signs of secondary infection should only be treated if clinical signs are present. Prophylactic treatment only increases resistance rates and has no benefit on the course of disease. The use of silver-coated textiles and silk fabric with a durable antimicrobial finish is supposed to reduce S. aureus colonization and eczema (Juenger et al. 2006). This option is still under investigation, but seems to be promising, especially for children.

Systemic corticosteroids Oral corticosteroids have a limited but definite role in the treatment of severe exacerbations of AD. A brief course may be used to control severe disease, but ongoing use of systemic corticosteroids leads to significant adverse effects. After discontinuation of the medication, severe relapses have been noted. Data from randomized clinical trials are lacking.

Cyclosporin A Cyclosporin A (CyA) inhibits calcineurin-dependent pathways, resulting in reduced levels of proinflammatory cytokines. Ongoing treatment with systemic immunosuppressive agents such as CyA should be reserved for very severe cases of disease not responding to other measures (Griffiths et al. 2006; Hijnen et al. 2007). Multiple studies have shown a positive effect in children and adults. Despite effectiveness, side effects, especially renal toxicity with hypertension and renal impairment, are of particular concern. Close monitoring of creatinine, blood pressure, and CyA serum levels is important. Exacerbation can be seen after discontinuation, yet posttreatment disease severity often does not return to baseline levels. Treatment should follow a body weight-dependent dosing regimen, with 3–5 mg/kg daily as high-dose and 2.5 mg/ kg daily as low-dose treatment. The lowest dose for minimal side effects should be applied. In children it should be remembered that vaccinations may not be effective under immunosuppression.

Mycophenolate mofetil

Systemic treatment Antihistamines Itch is often the most severe symptom for patients and is difficult to treat. Currently there is no specific antipruritic treatment except local applications of antiinflammatory preparations and emollients. Histamine H1-receptor antagonists (alimemazine and promethazine) are mainly used for their sedative effect and should be given 1 hour before bedtime. Most studies conclude that nonsedating antihistamines seem to have little or no value in the treatment of atopic eczema (Wahlgren et al. 1990).

Systemic antibiotic agents In the case of widespread bacterial secondary infection (primarily S. aureus) systemic antibiotic treatment is indicated. A short course of treatment (3–7 days) with cephalosporins (cefalexin) or semisynthetic penicillins (flucloxacillin and amoxicillin-clavulanate) has been shown to be effective.

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Mycophenolate mofetil is a purine biosynthesis inhibitor used as an immunosuppressant for the treatment of moderate to severe AD (Murray & Cohen 2007). The drug is generally well tolerated when given as short-term oral treatment (2 g/ day). After 5 weeks the dosage can be reduced to 1 g/day and continued for another 3 weeks. Occasional herpes retinitis has been recorded and should be treated.

Azathioprine Azathioprine is another immunosuppressant drug sometimes used for the treatment of severe AD (Meggitt & Reynolds 2001; Meggitt et al. 2006). It affects purine nucleotide synthesis and metabolism and has antiinflammatory and antiproliferative effects. Controlled trials are lacking; side effects are high, including myelosuppression, hepatotoxicity, gastrointestinal disturbances, increased susceptibility to infections, and possible development of skin cancer. As azathioprine is metabolized by thiopurine methyltransferase, deficiency of this enzyme

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should be excluded before starting oral immunosuppression with azathioprine.

Biologics The anti-IgE antibody omalizumab was the first biologic used in AD but the results are controversial (Krathen & Hsu 2005; Lane et al. 2006). This is mostly due to the very high levels of serum IgE observed in patients with severe forms. Efalizumab (anti-CD11a) has been introduced as a promising alternative to current immunosuppressive therapies, although further double-blind placebo-controlled studies are needed to test its efficacy and safety (Hassan et al. 2007; Takiguchi et al. 2007). Infliximab (ant-TNF-α) has been also reported to be successful in a few case reports (Chan et al. 2004; Jacobi et al. 2005; Cassano et al. 2006). However, one has to bear in mind that the side effects of biologics may be serious and need further evaluation.

Phototherapy UVB (280–320 nm), narrow-band UVB (311–313 nm), UVA (320–400 nm), medium- and high-dose UVA1 (340–400 nm), PUVA, and Balneo-PUVA have undergone trials for the treatment of AD (Dawe 2003; Silva et al. 2006). UVA1 irradiation seems to be superior to conventional UVA/UVB phototherapy in patients with severe AD. It has been established as a standard second-line treatment only for adults with or without additional topical or systemic treatment. In children UV therapy should be restricted since data about long-term side effects (e.g., skin cancer) are still not available. It has been suggested that there is a correlation between UV irradiation and photoaging, skin carcinogenesis, or melanoma induction, so therapy should be limited to about 4–6 weeks.

Atopic Dermatitis

ivitis. Only very few studies on AD have been reported (Mastrandrea et al. 2000; Pajno et al. 2003). Side effects of SLIT, especially with high doses of allergens, seem to be very few, and the sublingual application constitutes a less invasive form of treatment that could be easily administered to children. Nevertheless, there is no further information about any correlations between SLIT and improvements in AD.

Diet restriction Food hypersensitivity affects about 10–40% of children with AD. In children 90% of reactions are caused by only five allergens: eggs, milk, peanuts, soy, and wheat. A dietary restriction is only of value in patients with these allergies when they have been properly diagnosed (Hon et al. 2006). Diets should be closely supervised by a pediatric dietitian to ensure they are nutritionally adequate. Hydrolyzed cow’s milk formula consists of predigested peptides of whey and cysteine. Nutritional values are equivalent to normal milk, but it has a reduced capacity to induce IgE-mediated reactions.

Education Education of especially young patients and their parents has proved of great importance (Staab et al. 2006). Knowledge about the disease and its management will lead to higher compliance as well as psychological stability. Acceptance of the disease and its chronic course by the patient and the patient’s environment will significantly improve quality of life. Adequate educational programs are additionally of great value when offered by dermatologists as well as pediatricians, dietitians, psychologists, and nursing staff to patients and their families.

Immunotherapy

Future perspectives

Inhalant allergens with detectable specific IgE play a predominant role in the development of AD. Allergen-specific immunotherapy has been proposed for almost a century as a means of reducing immune responses in atopic patients. Unfortunately with regard to AD only limited, and often contradictory, information is available. Double-blind controlled trials have reported no or only limited benefit from allergenspecific desensitization, leaving the role of this treatment uncertain or negative (Bussmann et al. 2006). A recently published study that reexamined the efficacy of subcutaneous immunotherapy in atopic patients sensitized to house-dust mites demonstrated effectiveness in reducing eczema and allergic sensitization to mites (Werfel et al. 2006). The improvement in eczema was accompanied by a reduction in topical corticosteroids needed to treat eczema. Although promising data were presented, further studies to verify these results are needed. Since the mid-1980s, sublingual immunotherapy (SLIT) as an alternative to subcutaneous immunotherapy has been widely discussed for the treatment of allergic rhinoconjunct-

Skin reactions of patients with AD often present as the first signs of a lifetime disease of allergy and asthma. Chronic diseases such as AD are always a distressing condition for patients and their families. Unfortunately therapeutic options are limited, and a “cure” has yet to be found. Advances have been made in the field of research and look promising for future strategies that are more specifically targeted at pathogenic mechanisms. Monoclonal antibodies targeting IgE (omalizumab) already represent a new option in the treatment of asthma. Furthermore, antibodies against interleukins, namely IL-5 or IL-4, could be promising for future management of AD. Cytokine modulation (e.g., TNF inhibitors), blockade of inflammatory cell recruitment, and inhibition of T-cell activation likewise need further investigation. The principal goal in management of AD resides in the control of skin inflammation, elimination of triggering factors, and restoration of skin barrier function in order to potentially prevent the emergence of sensitization.

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Toyoda, M., Nakamura, M., Makino, T., Hino, T., Kagoura, M. & Morohashi, M. (2002) Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis. Br J Dermatol 147, 71–9. Valenta, R., Natter, S., Seiberler, S. et al. (1998) Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients. J Invest Dermatol 111, 1178–83. Verhagen, J., Akdis, M., Traidl-Hoffmann, C. et al. (2006) Absence of T-regulatory cell expression and function in atopic dermatitis skin. J Allergy Clin Immunol 117, 176–83. Wahlgren, C.F., Hagermark, O. & Bergstrom, R. (1990) The antipruritic effect of a sedative and a non-sedative antihistamine in atopic dermatitis. Br J Dermatol 122, 545–51. Weidinger, S., Illig, T., Baurecht, H. et al. (2006) Loss-of-function variations within the filaggrin gene predispose for atopic dermatitis with allergic sensitizations. J Allergy Clin Immunol 118, 214–19. Werfel, T., Breuer, K., Rueff, F. et al. (2006) Usefulness of specific immunotherapy in patients with atopic dermatitis and allergic sensitization to house dust mites: a multi-centre, randomized, dose-response study. Allergy 61, 202–5. Williams, H.C. (2005) Clinical practice. Atopic dermatitis. N Engl J Med 352, 2314–24. Williams, H. & Flohr, C. (2006) How epidemiology has challenged 3 prevailing concepts about atopic dermatitis. J Allergy Clin Immunol 118, 209–13. Williams, H.C., Burney, P.G., Hay, R.J. et al. (1994) The U.K. Working Party’s Diagnostic Criteria for Atopic Dermatitis. I. Derivation of a minimum set of discriminators for atopic dermatitis. Br J Dermatol 131, 383–96. Wollenberg, A., Wen, S.P. & Bieber, T. (1995) Langerhans cell phenotyping: a new tool for differential-diagnosis of inflammatory diseases. Lancet 346, 1626–7. Wollenberg, A., Kraft, S., Hanau, D. & Bieber, T. (1996) Immunomorphological and ultrastructural characterization of Langerhans cells and a novel, inflammatory dendritic epidermal cell (IDEC) population in lesional skin of atopic eczema. J Invest Dermatol 106, 446–53. Wollenberg, A., Wagner, M., Gunther, S. et al. (2002) Plasmacytoid dendritic cells: a new cutaneous dendritic cell subset with distinct role in inflammatory skin diseases. J Invest Dermatol 119, 1096–102. Ziegler, S.F. (2006) FOXP3: of mice and men. Annu Rev Immunol 24, 209–26. Zutavern, A., Hirsch, T., Leupold, W., Weiland, S., Keil, U. & Von Mutius, E. (2005) Atopic dermatitis, extrinsic atopic dermatitis and the hygiene hypothesis: results from a cross-sectional study. Clin Exp Allergy 35, 1301–8.

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Contact Dermatitis David I. Orton and Carolyn M. Willis

Summary Contact dermatitis is a dermatitis invoked as a result of an exogenous agent. It is characteristically divided into two types, allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) which differ in the underlying causative mechanism. However, in clinical practice they may coexist. ACD is considered to be a prototype of delayed hypersensitivity requiring two phases, induction and elicitation. The amount of allergen per skin surface area is the key factor that determines the risk of induction. Exposure-related factors that influence the elicitation phase include allergen concentration (dose), duration and frequency of exposure, the matrix, the presence of irritants, and the region of application. However, ICD differs fundamentally from ACD in that there is no prior requirement for sensitization and no evidence of immunologic memory by T cells. In general, ICD is more frequently encountered in clinical practice than ACD. The acute type of ICD usually has a single cause and is often indistinguishable from ACD. In contrast, the chronic form (“wear and tear dermatitis”) is usually a multifactorial disease, resulting from repetitive injury to the skin. The diagnosis is sometimes only made by exclusion, after patch testing. A diagnosis of contact dermatitis therefore requires careful evaluation of a patient’s clinical history, physical examination, and various types of skin testing (patch testing, photopatch testing, repeat open application tests, skin-prick tests). The diagnosis of ACD can only be confirmed by patch testing and is always required to exclude contact allergy as a complicating factor in stubborn cases of eczematous diseases, as well as cases where ACD is suspected from the pattern or distribution of eczema. It is particularly important in chronic cases of dermatitis that are unresponsive to traditional treatments. During the last few decades much effort has been put into standardization of allergens, vehicles, concentrations, tapes, and scoring of test reactions. The method today is considered both accurate and reliable. However, there are a number of variables that may all influence the result and frequent errors are therefore possible.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

The most difficult part includes distinguishing allergic from irritant patch test reactions and ascribing the relevance of an allergic reaction. Here, both the investigator’s skill and experience are crucial factors in providing accurate information to patients. In this way, the best management plan for each individual case can be formulated. This may include allergen/irritant avoidance and substitution measures, and treatment of inflammatory flares.

Definition Contact dermatitis is a dermatitis invoked as a result of an exogenous agent. It is characteristically divided into two types, allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) which differ in the underlying causative mechanism. However, in clinical practice they may coexist. This chapter discusses the immunologic and clinical features of both types, but concentrates on the investigation and management of ACD. Although the terms “dermatitis” and “eczema” are often used synonymously, where a difference is implied “eczema” refers to a more precise pattern of clinical and histologic response and “dermatitis” may be used to encompass a greater and broader range of reaction patterns. In some countries, dermatitis more often implies external causation.

Introduction and historical perspective The knowledge that certain agents in our environment can be harmful to the skin and that some individuals appear to react whereas others do not predates by many decades the concepts of allergy and idiosyncrasy. Proof of sensitization (Bloch & Steiner-Woerlich 1926) was of more recent origin, as was the finding that simple chemicals must combine with protein in order to sensitize (Landsteiner & Jacobs 1936; Lachapelle 2006). Although the Langerhans cell (LC) and LC–monocyte apposition play a central role in both the induction and elicitation of allergic contact sensitivity (Silberberg-Sinakin et al.

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1976), one has to be careful not to imply specific immune mechanisms to all the events reported in ACD, since most such changes can also be seen with irritant (nonimmune) contact dermatitis (Willis et al. 1986). It is also important to recognize that not all eczematous skin reactions are allergic in origin. Some cases of eczema or dermatitis are of unknown origin, although constitutional and physiologic factors often play a part. In many other cases of eczema, exogenous irritants are the principal factors. In reality, allergy may be responsible for only a minority of cases. Nevertheless, for these cases it can often have a significant socioeconomic impact. Despite many advances in our understanding of allergy and immunology, the patch test, when performed and interpreted correctly, still remains the best method of detecting and confirming ACD. The technique was first established by Jadassohn (1896). For many years, however, there was often no clear separation between irritant and allergic responses and the test was mostly used to confirm the reproducibility of dermatitis with certain substances. However, Jadassohn and others did recognize the phenomenon of “specific intolerance” or “idiosyncrasy.” However, it was not until the works of Bloch (1929) and Bonnevie (1939) that the concept of testing patients with a standard series of common allergens became established and not until Marcussen (1962) and the formation of the International Contact Dermatitis Research Group (ICDRG) in 1967 that the importance of epidemiology and standardization in patch testing was recognized. From then on, the European Environmental and Contact Dermatitis Research Group (EECDRG) was established in 1985, heralding the formation of the European Society of Contact Dermatitis (ESCD) in 1998. The EECDRG continues to publish multicenter studies evaluating new allergens and optimizing the patch-test concentration of new and established allergens as well as guiding legislation to protect consumers. More recently a working party within the ESCD was formed to provide epidemiologic patch-test data across European centers, the European Surveillance System of Contact Allergies (ESSCA). (The website addresses of these organizations can be found in the Further reading section.)

Epidemiology Contact dermatitis is a variable disease in which the symptoms alter both in presentation and severity over time. Therefore the term “prevalence” should be used judiciously, restricted to defined populations and a defined point in time. Publications based on the number of patients visiting dermatology clinics and/or patch-testing units have to be carefully interpreted, since no information on population-based incidence or prevalence rates can be derived from these data. However, within a population of clinic patients, systematic collection and registration of data can be the basis of a meaningful analysis, especially if it is on an international basis (ESSCA). The

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guidelines for publication and analysis of such data have been published (Uter et al. 2004). Prevalence studies strongly suggest that age and sex are not risk factors for contact dermatitis in themselves, but that these characteristics are associated with certain exposures in occupational and household activities. Furthermore, epidemiologic reviews conclude that age-dependent immunologic reactivity is less important than differences in exposure between age groups, and that the differences in sensitization patterns between the sexes seems to be caused by different exposure patterns (Menné et al. 1987; Coenraads et al. 2006). Occupational disease registries available in many countries provide national incidence data based on the notification of occupational skin diseases. It is estimated that eczema or contact dermatitis accounts for about 90–95% of all occupational skin diseases. The average incidence rate of registered occupational contact dermatitis in some countries lies between 0.5 to 1.9 cases per 1000 full-time workers per year (Dickel et al. 2002). Since 2001, the EPIDERM project in the UK, in combination with OPRA (Occupational Physicians Reporting Activity), requires dermatologists in a number of centers to report confirmed or suspected cases of occupational skin disease, including the occupation of the patient concerned (Cherry et al. 2000). Overall results suggest that the annual incidence of occupational contact dermatitis is 12.9 cases per 100 000 workers (Meyer et al. 2000). The highest incidence rates were seen in hairdressers (Shum et al. 2003). Agents accounting for the highest number of ACD cases were rubber, nickel, epoxies and other resins, aromatic amines, chromate, fragrances, cosmetics, and preservatives. However, soap, wet work, petroleum products, solvents, and cutting oils were the most frequently cited agents in cases of irritant dermatitis (Meyer et al. 2000). It is well documented that certain individuals, especially atopics (Keil & Shmunes 1983; Rystedt 1985), are particularly at risk of ICD. The level of sensitivity in a population constantly changes; allergens come and go and the prevalence of sensitivity to an individual substance will vary, depending on the past and present environmental levels of allergens to which the population has been exposed. When reporting the results of patch tests in any population, important demographic characteristics may have a profound impact on the observed spectrum of ACD. The MOAHLFA index (males, occupational relevance, atopic dermatitis, hand dermatitis, leg dermatitis, face dermatitis, age at patch testing > 40 years) (Coenraads et al. 2006) is an extension of the original MOHL index (Wilkinson et al. 1980) and the later MOAHL index (Andersen & Veien 1985). For example, a high proportion of occupational contact dermatitis cases (“O”) will evidently raise the frequency of positive reactions to “occupational” allergens depending on the spectrum of local industries. In this way, it is possible to compare patch-test results among different groups more meaningfully.

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Moreover, authors have concluded that when evaluating multicenter patch-test studies, the patch-test application time, the amount of the allergens applied on the chambers, the reading time, and the reading scale should also be taken into account (Andersen 1998).

Cellular mechanisms Allergic contact dermatitis ACD is considered to be a prototype of delayed hypersensitivity. Although humoral antibody-mediated responses may play a part, the condition depends primarily on the activation of allergen-specific T cells. Since, in general, the allergens themselves are nontoxic at the doses encountered, ACD may be regarded as an aberrant reaction, cell-mediated immunity having evolved principally to help eradicate microorganisms and toxins (Rustemeyer et al. 2006). ACD consists of two distinct phases: induction (sensitization) which lasts between 3 days and several weeks, and effector phase (elicitation) which takes 1–2 days to develop. During induction, allergen (hapten) penetrates the skin and binds to major histocompatibility complex (MHC) proteins, present in abundance on epidermal LCs. Allergen-carrying LCs become activated, releasing interleukin (IL)-1β which in turn stimulates the release of tumor necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor (GMCSF). Together, these cytokines facilitate the migration of LCs away from the skin, via the afferent lymphatic vessels, to the regional lymph nodes. Here, they settle as interdigitating cells (IDCs) in the T cell-rich paracortical areas, encountering and binding with naive T cells which specifically recognize allergen–MHC complexes. Supported by IDC-produced IL-1, activation of the allergen-specific T cells then occurs, leading to the release of a number of growth factors, including IL-2. Receptors for IL-2 are simultaneously upregulated, resulting in an autocrine cascade that leads to vigorous blast formation and proliferation within a few days. Released via the efferent lymphatics into the circulation, the majority of the expanded allergen-specific T-cell progeny, consisting of both effector and memory cells, then migrate into peripheral tissues, a process facilitated by the upregulation of several homing receptor molecules. In the absence of further allergen exposure, the numbers of these specific T cells gradually decline, but nevertheless remain higher than those of naive individuals. On renewed skin contact with the same or a closely related allergen, the effector phase begins. LCs and keratinocytes become haptenized once more, and are rapidly met, within the epidermis, by extravasated hapten-specific effector T cells displaying lowered activation thresholds. Proinflammatory cytokines and chemokines are released locally, leading to the recruitment of further specific and nonspecific T cells and other mononuclear cells to the site, which amplify local mediator release. Additionally, in response to mediators such as inter-

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feron (IFN)-γ, upregulation of cellular adhesion molecules on the surface of epidermal and dermal cells occurs, which further promotes the local inflammatory reaction. Reaching a peak at 24–72 hours, macroscopically detectable signs of cutaneous inflammation, namely erythema, vesiculation and induration, develop at the contact site. Thereafter, downregulatory mechanisms, most notably those mediated through keratinocyte-, macrophage- and T cell-derived IL-10, begin to predominate, leading to a gradual decline in the level of inflammation.

Irritant contact dermatitis Although ICD was once regarded as a nonimmunologically mediated condition, it is now accepted that the immune system does play a significant role. However, ICD differs fundamentally from ACD in that there is no prior requirement for sensitization and no evidence of immunologic memory by T cells (Lisby & Baadsgaard 2006). Despite this, the histopathologic changes, at least in the acute forms of both, are very similar, reflecting commonalities in the inflammatory signals and responding cell types. Our knowledge of the pathophysiologic changes that take place during the development of chronic ICD, the more commonly encountered problem in clinical practice, remains relatively scant. Topical exposure to irritants leads to a variety of specific and nonspecific cellular changes that depend on the chemical structure and concentration of the irritant, as well as the duration and frequency of contact (Willis et al. 1989). During penetration through the stratum corneum, there may be removal of lipids and naturally occurring hygroscopic materials, denaturation of keratin, and the extraction of proteins and amino acids. All of this leads to impairment of the skin barrier, which facilitates further penetration by the irritant and renders the skin more susceptible to the effects of other chemical and physical agents. Importantly, the changes also precipitate measures designed to restore the barrier, including the release of the proinflammatory mediators IL-1α, IL1β, TNF-α, and GM-CSF (Wood et al. 1992). Many irritants will then go on to directly damage epidermal keratinocytes below the stratum corneum, by disrupting cell membranes and lysosomes, altering DNA and phospholipid metabolism, and modifying triglyceride synthesis. These changes lead to further cytokine and chemokine production, together with the release of arachidonic acid-derived inflammatory mediators, including prostaglandins and leukotrienes. In common with allergic reactions, cellular adhesion molecules such as intercellular adhesion molecule (ICAM)-1 on keratinocytes and E-selectin on endothelial cells are also upregulated, facilitating the extravasation of a variety of white blood cells into the dermis and epidermis of the affected skin site (Willis et al. 1991; Basketter et al. 1999a). The role of epidermal LCs, so pivotal in ACD, is somewhat uncertain in irritant reactions. Changes in morphology and density do occur, but it is perhaps more likely that nonspecific

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mechanisms, such as cytokine release, simply contribute to the overall cellular response (Willis et al. 1990). Within 48–72 hours of acute exposure to irritants, mediators with predominantly antiinflammatory properties, such as IL-10, begin to exert their effect, gradually dampening down the inflammation over the next 2–3 weeks. However, affected sites remain vulnerable for much longer than this, possibly because of incomplete local barrier repair and/or the continued low-level presence of certain immune cells and cellular products.

Chemical considerations Allergic contact dermatitis Most contact allergens are highly chemically reactive molecules, with a molecular mass of less than 1000 Da. Being intrinsically too small to act as antigens in their own right (and hence often referred to as haptens), they must have the ability to penetrate the stratum corneum of the skin and bind with epidermal proteins to produce a “nonself” antigen in order to generate an immune response. As a general rule, the greater the ease with which a chemical reacts with proteins, the greater its skin sensitization potential (Lepoittevin 2006). Since many haptens are electrophiles (electron-poor), protein conjugation is typically by way of covalent bonding with nucleophilic (electron-rich) groups on amino acids, the most frequently cited of which are lysine and cysteine. Chemical reactivity patterns toward these nucleophilic amino acids depend on the electron density at the reactive site. Molecules of different structure but similar molecular shape can display similar chemical reactivity, thereby activating the same T-cell receptors. This is the basis of the so-called cross-reaction phenomenon, seen for example between nickel and palladium. However, many apparent cross-reactions are due to concomitant sensitization. Although bonding between haptens and proteins is most commonly covalent in nature, weaker interactions, including hydrophobic bonding, may play a role in the reactivity of very lipophilic antigens such as poison ivy or poison oak. Some haptens are directly reactive, while others, often referred to as pro-haptens or pre-haptens, may require some form of structural transformation to become reactive. This may occur within the skin itself as a result of enzymatic metabolic or detoxification processes; those based on monoamine oxidases and peroxidases are believed to be particularly relevant. Alternatively, sensitivity to heat, light and/or oxygen may chemically modify the hapten during storage and handling. The potential for a chemical to sensitize is dependent not only on its reactivity but also on aspects of its hydrophobicity, such as log P (where P is the octanol/water partition coefficient). Predicting the sensitization potential of new compounds is an important aspect of their development and risk assessment, and, to this end, a variety of in vitro, animal and

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Table 89.1 Classification of haptens (allergens) based on functional groupings. Hapten (allergen)

Example

Acids Aldehydes Amines Diazo compounds Esters Ethers Epoxides Halogenated compounds Quinones Metals Unsaturated compounds

Maleic acid Formaldehyde Paraphenylenediamine Bismarck brown Benzocaine Benzyl ether Epoxy resin Dinitrochlorobenzene Hydroquinone, primin Ni2+, Co2+, Cr2+, Hg2+, etc. Turpentine

human tests, and computer-based models, have been devised (Basketter et al. 1999b). Table 89.1 gives examples of the different classes of haptens based on functional groupings.

Irritant contact dermatitis In order to act as an irritant, a chemical must be able to penetrate the stratum corneum, a property governed by size and/or shape parameters, such as molecular volume and inertial axes, the presence of charged groups and, as a measurement of hydrophobicity, the partition coefficient. Other important factors include the pKa, a measure of acidity/basicity, and an aspect of reactivity known as the dipole moment (Basketter et al. 1999a). Of the wide variety of chemicals that cause irritation, surfactants are among the most commonly encountered in daily life, with the cationic variants displaying the highest irritancy potential. Other important classes of irritants include the organic solvents, acids and alkalis, reducing agents such as thioglycollates, oxidizing agents such as benzoyl peroxide, and various topical medicaments including dithranol. Predictive tests for the irritancy potential of a chemical are carried out using combinations of in vitro models, such as the human keratinocyte neutral red assay (Borenfreund & Puerner 1985), animal models, including the Draize rabbit test, and human models, of which conventional patch testing, either single or repeated, is the most widely used.

Histopathology Allergic contact dermatitis Positive allergic patch test reactions (effector phase) are characterized by spongiosis, resulting from the accumulation of fluid around individual keratinocytes and, as a consequence, the stretching of intercellular desmosome complexes. This intercellular edema may be extensive at times, involving all layers of the epidermis, with rupturing of the intercellular

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Fig. 89.1 Toluidine blue-stained 1-mm plastic section of a 48-hour patch-test reaction to nickel, showing spongiosis and exocytosis of primarily mononuclear cells in the epidermis (original magnification ×400). (See CD-ROM for color version.)

Fig. 89.2 Toluidine blue-stained 1-mm plastic section of a 48-hour patch-test reaction to the irritant benzalkonium chloride (a cationic detergent) showing an area of necrosis within the upper epidermis (original magnification ×400). (See CD-ROM for color version.)

connections to produce vesicles. White blood cells, responding to proinflammatory mediators, migrate into the epidermis from the dermis, in a process known as exocytosis (Fig. 89.1). Most of these cells are lymphocytes, but macrophages are also present, with occasional polymorphonuclear neutrophils and eosinophils being seen. Within the dermis, a dense, largely mononuclear, perivascular infiltrate accumulates, accompanied by dilated papillary blood vessels and sometimes by dilated lymphatic vessels. Dermal edema is usually evident, with deposits of acid mucopolysaccharides also being present (Lachapelle & Marot 2006). Immunopathologic characterization of the inflammatory infiltrate has revealed that the majority of the cells are CD3+/ CD4+ T lymphocyes, approximately 30% of which coexpress CD25 and CTLA-4, a phenotype consistent with either activated effector or regulatory T cells. Allergen-specific CD8+ cells are also represented. In addition, sizeable numbers of CD1a+ dendritic cells and CD1c+ dendritic cells, together with CD123+, CD45RA+, BDCA-2+, CLA+ and CD62L+ plasmacytoid dendritic cells contribute to the infiltrate (Willis et al. 1986; Bangert et al. 2003).

irritant-dependent patterns of keratinocyte damage were demonstrated; the anionic detergent sodium lauryl sulfate, for example, produced marked parakeratosis indicative of increased epidermal cell turnover, while the cationic detergent benzalkonium chloride induced predominantly spongiosis and exocytosis with focal necrotic damage (Willis et al. 1989) (Fig. 89.2). Other epidermal changes that have been described include karyopyknosis, cytolysis, dyskeratosis, acanthosis, intraepidermal or subepidermal vesicle and bulla formations, acantholysis, and accumulation of polymorphonuclear cells within the epidermis (Lachapelle & Marot 2006). Changes within the dermis of patch-test reactions generally share greater similarity between irritants, with a perivascular infiltrate of leukocytes becoming evident within 24 hours. In common with ACD, mononuclear cells, in particular T lymphocytes of the CD4+ subset, predominate, although the overall numbers are generally somewhat less. Dermal edema is less apparent than in ACD. Despite considerable effort, it has not, as yet, proved possible to separate irritant from allergic reactions by immunocytochemistry in respect of either LC behavior (Kanerva et al. 1984; Willis et al. 1990) or composition of the infiltrate (Avnstorp et al. 1987; Willis et al. 1993). However, it is conceivable that with further refinements of technique, some differences may be seen early on in the reaction, perhaps with regard to the response of memory T cells (Brasch et al. 1990), the expression of activation markers (Scheynius & Fischer 1986; Lange Vejlsgaard et al. 1989; Willis et al. 1991), or the characterization of very early cytokine release.

Irritant contact dermatitis The differing types of chemicals that can induce acute skin irritation exhibit varying mechanisms of action, and this is reflected in the range of histopathologic changes observed at affected sites, particularly within the epidermis. Irritant concentration, duration of contact, severity of reaction, time of biopsy, and possibly individual constitutional factors also influence the histologic appearance. Some irritants, such as croton oil, mimic ACD exactly both clinically and histologically. In other cases, the epidermal changes are very pleomorphic, with characteristic changes being seen with some irritants (Willis et al. 1989). In a systematic study of 48-hour ICD patch-test reactions, distinct

Clinical features of contact dermatitis While the most commonly encountered adverse reaction to contactants results in a dermatitis or eczematous tissue

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response, other clinical manifestations have also been described (Armstrong et al. 1997; Evans et al. 2003). These include erosions, ulcerations, urticaria, erythema multiforme, purpura, lichenoid eruptions, exanthems, erythroderma, allergic contact granuloma, lymphocytoma, sarcoidal reactions, toxic epidermal necrolysis, and pigmented contact dermatitis.

Irritant contact dermatitis In general, ICD is more frequently encountered in clinical practice than ACD. With irritants, the same chemical may cause a different response in different persons or a different reaction in the same person according to the concentration at which the chemical is applied (Gollhausen & Kligman 1985; Anderson 1990). The response may also vary according to site and other factors listed in Table 89.2. Irritation and ICD occur more easily at extremes of temperature and humidity and when there is occlusion. Twin studies suggest that genetic factors may also influence susceptibility to irritants (Holst & Möller 1975) and that atopics (Rystedt 1985) and those with fair skins may be more easily irritated (Kligman & Epstein 1975). Patients who have suffered from severe atopic dermatitis in childhood are likely to experience ICD later in life, particularly on the hands (Rystedt 1985). Ultraviolet (UV) light may also combine with some chemicals to produce a phototoxic irritant response. The most common causative irritants and occupations in which they are encountered are listed in Tables 89.3 and 89.4. The clinical spectrum of ICD listed in Tables 89.5 and 89.6 is much wider than that of ACD. Severe damage may lead to

Table 89.2 Factors influencing the response to an irritant. Chemical Type of exposure Site Susceptibility Concentration Duration of contact

Table 89.3 Common occupational irritants. Wet work Detergents and solvents Alkalis and acids Cutting oils Oxidizing and reducing agents Biological agents Dust/soil/desiccation Physical Low ambient humidity Friction Microtrauma, etc.

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Table 89.4 High-risk occupations for irritant contact dermatitis. Hairdressing Healthcare Cleaning Food processing and catering Printing Mechanical engineering Construction Agriculture, horticulture and forestry

Table 89.5 Spectrum of irritant response. Subjective Subclinical Transient Dermatitis Caustic burn

Table 89.6 Irritant reactions. Subjective Immediate stinging Delayed stinging Pruritus Pain (burning and smarting) Dysesthesia Tightness Dryness Cosmetic intolerance Objective Contact urticaria/erythema Acute irritant reaction Chronic irritant reaction Acute irritant contact dermatitis Cumulative irritant dermatitis Caustic burn

chemical burns and ulceration (Fig. 89.3) while less severe injury may result in acute toxic inflammatory reactions, including transient erythema or whealing, or mild dermatitis (Fig. 89.4) or more severe dermatitis with edema and vesiculation (Fig. 89.5). Most uncomplicated cases of ICD will settle within 2 weeks if further irritant exposure is avoided. However, the barrier function, and normal skin reactivity, may take many further weeks to completely return to normal. Although the range of responses to irritant chemicals is broader and more variable than that produced by ACD, the actual pattern of response is often rather monomorphic as opposed to the more polymorphic picture seen in ACD, where there is synchronous presence of macules, papules and vesicles.

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Acute ICD

Fig. 89.3 Caustic burns from cement. (See CD-ROM for color version.)

The acute type usually has a single cause and is often indistinguishable from ACD. The diagnosis is sometimes only made by exclusion, after patch testing. In practice, the commonest causes include cosmetics and reactions to medicaments. In the general population, adverse reactions to cosmetics are reported to be quite common (12.2%), but only a minority of reactions (approximately 10%) were shown to be allergic in origin (De Groot 1988). In a more recent UK epidemiologic study on sensitive skin, 23% of women and 13.8% of men reported an adverse reaction to a personal care product in the preceding year, with higher overall rates of 51.4% and 38.2% over a lifetime (Willis et al. 2001). Nonimmunologic contact urticaria (Maibach & Johnson 1975) and the purely subjective forms of immediate and delayed stinging that may be encountered when applying various substances to the skin (especially the face) are also forms of an irritant response (Frosch & Kligman 1977). Acute ICD results from a single (or short series) of exposures to an irritant (Fig. 89.6). Differentiation between an acute ICD and a chemical burn is not always possible. Most of the latter episodes occur as a result of accidents at work and the dermatitis normally heals quickly, unless there is skin necrosis. There is normally a sensation of burning or stinging, followed by inflammation and varying degrees of damage to the skin.

Chronic ICD

Impairment (log scale)

Fig. 89.4 Irritant reaction on the forearm of a hairdresser. (See CD-ROM for color version.)

In contrast, the chronic form (“cumulative insult dermatitis”/ “wear and tear dermatitis”) is usually a multifactorial disease. It results from chronic or repetitive injury to the skin (Fig. 89.7). Although irritants are the principal cause, physical factors such as friction, microtrauma, cold, heat, the desiccating effects

Clinically apparent disease Subliminal damaging effect

Time Exposure to an irritant at sufficient dose and concentration as to cause acute irritant contact dermatitis Exposure to a clinically subirritant dose of the same chemical or physical agent Clinically apparent disease

Fig. 89.5 Severe irritant hand eczema. (See CD-ROM for color version.)

Fig. 89.6 Acute irritant contact dermatitis. The damaging effects on the skin of acute (noncumulative) exposure to irritants.

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Impairment (log scale)

Allergic contact dermatitis Clinically apparent disease

Subliminal damaging effect

a

c

c b

c c

c c

Time

Insult a

Major but not clinically apparent insult

b

Major and apparently “causal” factor

c

Minor perpetuating or conditioning factors

Sensitization may occur following a single exposure to a strong allergen, but usually several or many exposures are required before sensitization ensues and dermatitis results. The severity of dermatitis is determined by the intensity of exposure and level of sensitivity. The clinical appearance also partly depends on the site of dermatitis and the nature of the causative agent. ACD normally presents as erythema and swelling, with subsequent development of papules and papulovesicles, which can progress to vesiculation and exudation. Pruritus may be intense. With time, the skin may become dry and scaly and in chronic cases there may be lichenification and fissuring. It can mimic almost any type of eczema (Figs 89.9–89.12).

Clinically apparent dermatitis Fig. 89.7 Cumulative irritant contact dermatitis.

of low humidity, and frequent exposure to water (“wet work”) are also important. Atopics and those with recently healed eczema or with eczema elsewhere are also more susceptible. With cumulative-insult dermatitis, even minor irritants may act as important perpetuating factors. The prime localization of this type of eczema is on the hands, and clinically it may present as dryness, erythema or chapping (Fig. 89.8) or as an eczematous reaction indistinguishable from allergic or constitutional eczema.

Fig. 89.9 Vesicular eczema due to sensitivity to “Bends” isothiazolinone (an industrial biocide). (See CD-ROM for color version.)

Fig. 89.8 Chronic irritant reaction. (See CD-ROM for color version.)

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Fig. 89.10 Constitutional-looking palmar vesicular/hyperkeratotic hand eczema due to sensitivity to isothiazolinone in a shampoo. (See CD-ROM for color version.)

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Fig. 89.11 A “seborrheic dermatitis”-like pattern of facial eczema in an individual allergic to cosmetics. (See CD-ROM for color version.)

Contact Dermatitis

of exposure, the matrix, the presence of irritants (Heydorn et al. 2003; Pedersen et al. 2004), and the region of application. A cumulative effect of exposures has also been demonstrated, so that repeating exposures can cause elicitation in more individuals (Andersen et al. 2001; Hextall et al. 2002). Anatomic regions also appear to differ in their sensitivity. The upper arm has been shown to be more sensitive than the forehead and ventral aspect of the lower arm in usage tests, the axilla more sensitive than the outer aspect of the upper arm (Johansen et al. 1998), and recently it has been shown that the neck and face are more sensitive than the outer aspect of the upper arm (Zachariae et al. 2004). ACD appears to be more common among women than men. This probably relates more to exposure patterns than to gender (Modjtahedi et al. 2004). Children have been thought to develop ACD less often than adults. However, reviews suggest that the pattern of sensitization is similar to that of adults and is common in children (Weston & Weston 1984; Romaguera & Vilaplana 1998). Elderly persons frequently develop ACD from substances in topical medicaments, fragrance chemicals, and balsam of Peru (Uter et al. 2002). There is some question as to the exact relationship between atopy and contact sensitization. It has been suggested that atopics become sensitized less often than nonatopics (Jones et al. 1973), but most authors conclude that it is just as likely, and that it is equally important to patch test atopic subjects under the right clinical circumstances (Whitmore 1994).

Specific types of ACD Fig. 89.12 Lichenified eczema from allergic contact dermatitis to nickel from spectacle frames. (See CD-ROM for color version.)

The diagnosis of ACD is therefore based on a careful history, combined with the knowledge of common allergens in the patient’s environment and confirmed by patch testing. If patch testing is not performed, many cases of ACD will be missed: the history and pattern of dermatitis alone are insufficient to make a diagnosis. A diagnosis of constitutional or cumulative irritant dermatitis should therefore only be made by exclusion. While ACD may be only part of a multifactorial etiology for the dermatitis, exposure to the sensitizer(s) may be the only factor that can be altered and permanently change the severity of the eczema. Several studies have shown that detailed patch testing is beneficial for patients and improves their quality of life (Rajagopalan & Anderson 1997; Thomson et al. 2002; Woo et al. 2003). ACD depends on two phases, induction and elicitation. The amount of allergen per skin surface area is the key factor that determines the risk of induction (Rees et al. 1990). Exposure-related factors that influence the elicitation phase include allergen concentration (dose), duration and frequency

The site and pattern of dermatitis often suggests a range of possible allergens and, combined with the history, may prove a convenient starting point for the investigation of contact dermatitis. For instance, with plants, the rash may be linear and sometimes bullous (phytophoto ACD) and may fade leaving postinflammatory pigmentation (Fig. 89.13).

Hands Approximately two-thirds of all cases of contact dermatitis involve the hands, which is the most important site for both ICD and ACD. It is particularly important in the occupational setting where the correct diagnosis is essential for further management. Patch testing should be undertaken in all chronic or recurrent cases of hand eczema, since allergens are otherwise easily missed. Both chromate allergy and ICD can cause discoid or nummular patches of eczema on the backs of hands (Fig. 89.14) and irritants generally affect the backs of the hands and webs of the fingers (Fig. 89.15) or under a ring due to the accumulation of water and detergents. However, ACD to rubber accelerator chemicals may also present with a similar appearance (Fig. 89.16). ACD can also present as a pompholyx-like eczema, and ingested nickel and a few other allergens are also reported to “cause” vesicular palmar eczema.

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Fig. 89.15 Irritant pattern hand dermatitis. (See CD-ROM for color version.)

Fig. 89.13 Acute phytophotodermatitis following contact with phototoxic plants. (See CD-ROM for color version.)

Fig. 89.16 Hand eczema resulting from allergic contact dermatitis to rubber chemicals in household gloves. (See CD-ROM for color version.)

Fig. 89.14 Discoid hand eczema. (See CD-ROM for color version.)

Face The face is often involved in ACD from biocides or other cosmetic ingredients (Fig. 89.17). The eyes and ears are also commonly involved in cases of allergy to medicaments. Fragrance sensitivity may involve the neck and face, as part of a volatile-type exposure. The eyelids are also more susceptible to the effect of irritants.

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Fig. 89.17 Acute facial contact dermatitis from allergy to ingredients in a cosmetic. (See CD-ROM for color version.)

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Allergy to hair dye, Primin, biocides, fragrance chemicals and nail varnish may also affect the eyelids, face and neck (Figs 89.18– 89.20). Nickel sensitivity often first becomes apparent on the ear lobes (Fig. 89.21), and may also involve the eyelids.

Fig. 89.18 Allergic contact dermatitis affecting the eyelids. (See CD-ROM for color version.) Fig. 89.21 Nickel allergy from ear studs. (See CD-ROM for color version.)

Fig. 89.19 Acute allergic contact dermatitis affecting the eyelids and mimicking angioedema resulting from allergic contact dermatitis to paraphenylenediamine. (See CD-ROM for color version.)

Fig. 89.22 Typical distribution of eczema in a patient with Compositae dermatitis. (See CD-ROM for color version.)

The face may also be involved in photosensitivity reactions. The skin under the chin, under the eyebrow, and under the lobe of the ear may be spared in such dermatoses, but tends to be involved when the dermatitis is due to volatile allergens or dust. In both volatile and photosensitive eczema, there is often a clear demarcation line at the neck (Fig. 89.22). Apart from fragrance and plant allergens (sesquiterpene lactones from Compositae species and primin from Primula spp.), other volatile agents that may give a volatile pattern of dermatitis include dust from exotic woods, epoxy hardeners and many other agents.

Fig. 89.20 Allergic contact dermatitis around the neck in a patient allergic to fragrance chemicals in a shampoo. (See CD-ROM for color version.)

Other patterns of contact dermatitis The feet are often involved with allergy to shoe components (Fig. 89.23) that may have been caused by chromate (used in

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Fig. 89.23 Allergic contact dermatitis to footwear. (See CD-ROM for color version.)

tanning leather) or allergy to glues, rubber chemicals or dyes. Textile dermatitis (sometimes secondary to textile dyes or formaldehyde finishes) will tend to spare the skin covered by undergarments, but tends to involve sweaty sites of friction such as the body flexures and also the apex of the axillae (Fig. 89.24). Dermatitis of the axillary vault is often either irritant or due to fragrance allergy in a deodorant. The lips and perioral area may be involved in an allergy to a lip cosmetic, and “chapping” or “lip lick” dermatitis commonly develops around the mouth in atopics during the winter months. The nail plate may become dystrophic and the nail folds may become eczematous with an allergy to acrylates from artificial nails (Fig. 89.25). It is obviously not possible in this chapter to cover all the rarer and more unusual patterns of eczema and for more detailed information on contact dermatitis, readers are referred elsewhere (Kanerva et al. 2000; Rietschel & Fowler 2001; Frosch 2006).

Fig. 89.25 Periungual allergic contact dermatitis from acrylates in artificial nails. (See CD-ROM for color version.)

Contact urticaria and protein contact dermatitis Many foods and other agents (latex, animal products, drugs, semen, etc.) may cause an urticarial reaction on contact with the skin. In many cases, this is an IgE-mediated response. In some cases there is direct histamine release (e.g., cinnamaldehyde, nicotinic acid, dimethylsulfoxide). In both types of contact urticaria, the reaction develops in minutes and usually subsides within a few hours. The term “protein contact dermatitis” was introduced by Hjorth and Roed-Petersen (1976). It is caused by proteins, mainly foodstuffs, animal danders and other animal products. Handling such foods often causes immediate irritation and visible erythema sometimes accompanied by urticaria. It may then progress to cause dermatitis (commonly including the fingertips) in a pattern that is indistinguishable from other types of contact dermatitis. Many individuals have a positive prick test or scratch-patch test to the protein, along with an eczematous response if the causative protein is applied to previously affected skin. This diagnosis must not be overlooked in chefs and sandwich-makers, veterinarians, and those working in abattoirs.

Mucosal contact dermatitis

Fig. 89.24 Characteristic pattern of dermatitis in a patient sensitive to clothing dye (disperse blue 124). (See CD-ROM for color version.)

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Allergic reactions on mucosal surfaces are uncommon, exposure via mucosal surfaces more often inducing tolerance (Van Hoogstraten et al. 1989). Mucosal reactions when they do occur are often secondary to skin sensitization. Contact dermatitis in the mouth normally presents as erythema and swelling and both vesiculation and itching are uncommon. Sometimes the only sign of allergy may be eczematous

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Table 89.7 Noneczematous contact reactions. Type of reaction

Example

Erythema multiforme-like Pigmented purpuric eruption Lichenoid eruption Granulomatous reactions Scleroderma-like Pigmented contact dermatitis Leukodermic contact dermatitis

Exotic woods and plants, some medicaments Black rubber, some dyes Photographic color developers Zirconium, aluminum-containing vaccines, tattoos Solvents Dyes and fragrances Phenols, catechols, hydroquinone

changes on the adjacent skin. Mercury amalgam may cause a lichenoid reaction. Contact reactions have also been reported to nickel, gold, and methylmethacrylate. Some flavorings and medicaments have also been reported as causing allergic reactions.

There is also immune and nonimmune contact urticaria. Other noneczematous contact reactions are listed in Table 89.7.

Photocontact dermatitis

A diagnosis of contact dermatitis requires careful evaluation of a patient’s clinical history, physical examination, and various types of skin testing (patch testing, photopatch testing, repeat open application tests, skin-prick tests). A thorough knowledge of the clinical features of the skin’s reactions to various contactants is important in making the correct diagnosis of contact dermatitis, as well as being able to differentiate other skin conditions, e.g., the Köbner reactions on the hands of patients with psoriasis can mimic both ICD and ACD. Therefore patch testing may best be performed by a clinician with a sound basis of dermatology who has undergone specific training in the investigation and management of contact dermatitis (Aberer et al. 1993).

Certain substances are transformed into irritants or sensitizers by the action of UV light. Phototoxicity is the result of direct cellular damage caused by a nonimmunologic inflammatory mechanism, which is initiated by a phototoxic agent and subsequent irradiation. In contrast, photoallergic reactions represent delayed or cell-mediated or type IV hypersensitivity responses, which require the specific sensitization of an individual to a photoactivated allergen. The mechanisms are poorly understood but involve covalent allergen–protein photobinding (haptenization) leading to the formation of a complete photoantigen (Mang et al. 2006). Phototoxic reactions can be elicited in most individuals even after the first exposure, if the concentration of chemical and amount of radiation is sufficient. These can occur following systemically administered drugs, such as nonsteroidal antiinflammatory drugs (NSAID) and sulfonamides, or contact with psoralen-containing plants, such as giant hogweed and other Umbelliferae. Clinically, they resemble exaggerated sunburn reactions. Phytophotodermatitis is one rather distinctive form of phototoxic reaction (Fig. 89.13). In contrast, photoallergy requires prior sensitization and only these individuals will react, often to relatively small amounts of allergen and UV light. As a consequence, photoallergic reactions occur less frequently, and have been described with sunscreen chemicals, topical antimicrobials, NSAIDs, psychiatric medications, and some fragrance materials. Patients allergic to Compositae are often affected by severe chronic dermatitis with associated light sensitivity, termed chronic actinic dermatitis (Ducombs et al. 1990).

Noneczematous contact reactions Not all contact reactions are eczematous in nature. Some may be purely subjective, e.g., immediate stinging from alcohol or delayed stinging from substances such as lactic acid.

Investigation of ACD

Clinical history A clinician must have a high level of suspicion that the dermatitis has been caused or exacerbated by an external agent. The patient is often convinced of this but usually thinks it to be allergic in nature, whereas most contact dermatitis is irritant in nature. Furthermore, most ICD occurs as a result of cumulative exposure but the patient only recognizes the final insult in a whole series of irritant exposures. Patients are often preoccupied with occupational sources of exposure while domestic or leisure activities may also be wholly or partly responsible for the dermatitis. The exposure to irritants may be more damaging to those with a constitutionally compromised skin, such as found in atopic subjects. The history should therefore include all contributory factors: • personal history of eczema and other atopic manifestations; • family history of atopy; • details of other skin conditions, past and present; • known allergies; • current occupation and previous occupations; • hobbies and leisure activities; • ages of children (in those with childcare duties);

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• topical and systemic medications used (prescribed and over the counter); • use of cosmetics and skin-care products on themselves or others.

Clinical diagnostic tests Patch testing This bioassay is always required to exclude contact allergy as a complicating factor in stubborn cases of eczematous disease such as atopic dermatitis, stasis dermatitis, seborrheic dermatitis, and vesicular hand dermatitis, as well as cases where ACD is suspected from the pattern or distribution of eczema (Table 89.8). It is particularly important in chronic cases of dermatitis that are unresponsive to traditional treatments, where patients might have developed allergy to medicaments (e.g., steroids, antibiotics) or vehicles (e.g., emulsifying agents/biocides). Apart from its use to confirm suspected ACD, the patch-test procedure can also be used for recommending safe alternative medicaments, cosmetics, gloves, etc. It is a relatively cheap and safe procedure, but it takes skill and experience to interpret the results and to attribute relevance to any positive findings. Overreading is the most common mistake (Aberer et al. 1993). The history and examination of a patient will usually give clues to the possible sensitizers and should guide the choice of patch-test materials. Unfortunately, it is not sufficient to patch test with only suspected sensitizers, for unsuspected ones frequently turn out to be relevant (e.g., in cases of connubial dermatitis). During the last few decades much effort has been put into standardization of allergens, vehicles, concentrations, patch-test materials, tapes, and the scoring of test reactions. The method today is considered both accurate and reliable. A series of papers (Bousema et al. 1991; Belsito et al. 1992; Brasch et al. 1994) has demonstrated good reproducibility of patch-test results. The skin condition, vehicle, concentration and volume of the test allergen, as well as the size of the test chamber, test site, application time, and number of readings may all influence the result, and frequent errors are therefore possible.

The technique involves the cutaneous application of a small amount of the suspected allergen in a suitable concentration and vehicle. Allergens are obtainable from the following manufacturers (although there may be local distributors in some countries): • Hermal Kurt Herman, Scholtzstr. 3, D-21465 Reinbeck, Germany (available at http://www.hermal.de/her/pages/unternehmen/english/trolab.php) • Chemotechnique Diagnostics, Modemgatan 9, S-235 39 Vellinge, Sweden (available at http://www.chemotechnique.se/) • TRUE TEST Panels 1, 2, 3 and 4. Mekos laboratories (available at http://www.truetest.com/). For the most part, allergens are dispersed in white soft paraffin and are supplied in labeled syringes with the name and concentration of the substance on the label, together with an expiry date. They should be stored at 4°C in the dark. Over 300 test allergens are available from suppliers, and others can be made up from the patient’s own materials. If the patient’s own products are manufactured for direct application to the skin, so-called “leave on products,” they can be tested “as are” without the need to dilute them. However, certain other products that are designated “rinse off” products such as soaps and detergents should be diluted in water to a concentration of 1–10%. Other substances can be made up for patch testing, but care should be taken to find the appropriate concentration and vehicle (De Groot 1986). Fabric should be steamed before testing and paper should have a drop of ethanol applied. This helps to release all the potential allergens. Some identified plant materials can also be applied directly. These should be 1-cm squares of leaf or 1-cm lengths of stem, root, etc. The sample should be bruised lightly with an orange-stick. Many plants are irritant, e.g., Brassicaceae family, and there is little point in patch testing with known irritants (Benezra et al. 1986). However, allergens are pure chemicals and if the original offending agent was an impurity, metabolite, or degradation product, the cause will be missed. A supplementary test with the patient’s own working materials should be done in these cases where the test with the screening series was negative

Table 89.8 Patterns of dermatitis where patch testing might be indicated (with possible suspect allergens included in parentheses). Facial dermatitis including eyelid dermatitis (cosmetic allergens: fragrance, chemicals/biocides, medicaments) Chronic otitis externa (ear medicament allergens) Cheilitis (cosmetic allergens, flavorings) Flexures (cosmetic allergens, textile dyes) Hands (cosmetic allergens, rubber chemicals, plant allergens) Anogenital (medicament and cosmetic allergens) Feet (footwear allergens including chromate, rubber chemicals) Photosensitive dermatitis (sunscreen chemicals, cosmetic allergens) Airborne dermatitis (volatile cosmetic allergens including fragrance chemicals, volatile occupational allergens including epoxy resin, plant allergens)

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but the suspicion of ACD remains. A patient’s own cosmetics may also be tested. It is sometimes necessary to obtain constituent ingredients directly from the cosmetic manufacturer in order to identify the causative allergen, if it is not explained by testing to the selected test allergens. In this way, new allergens may be identified for further evaluation. Test concentrations are generally given as percentages. A better way of expressing concentration would be as both percentage and molality (i.e., number of moles per 1000 g of solvent or vehicle) (Benezra et al. 1978). Test concentrations have been carefully selected to elicit an allergic response in those previously sensitized and to cause no reaction in those who are not sensitive. However, this is unfortunately not always the case, as some allergens have elicitation concentrations close to their irritant threshold, e.g., chromate (Burrows et al. 1989). For ease of clinical practice, groups of test allergens are arranged into test series or “batteries.” An experienced clinician might be able to correctly guess the relevant contact allergen(s) in some patients based on the history and the clinical appearance of the eczema. However, this failure to guess correctly explains why a “standard series” of test allergens should be applied in the evaluation of all patients suspected of having a contact dermatitis. A sensitizer is suggested for inclusion in the standard series when routine patch testing of patients with suspected contact dermatitis results in a contact allergy rate exceeding 0.5–1.0% (Bruynzeel et al. 1995; Bruze et al. 1999; Isaksson et al. 2000). The current European standard series, as recommended by EECDRG, is listed in Table 89.9. It detects approximately 75– 80% of all contact allergies (Menné et al. 1992). The European standard series is dynamic and subject to continual modification depending on population exposures and prevalence of contact allergy. It can be extended to include allergens of local importance to specific dermatology departments. The advantages and disadvantages of using a standard series of patch tests have been discussed by Lachapelle and Maibach (2003). The commonest system used to occlude and apply the allergens is the Finn chamber system (supplied by Epitest Ltd, Oy, Finland; http://www.epitest.fi) on Scanpor tape (supplied by Norgesplaster, Norway; http://www.norgesplaster.com). These are also available from local distributors. The chambers are supplied in strips of five or ten and consist of small aluminum disks mounted on nonocclusive tape that has been chosen for its excellent adhesive properties and hypoallergenic acrylic-based adhesive. Other systems consist of square polyethylene chambers on hypoallergenic tape (IQ chambers, Chemotechnique; van der Bend chambers, Brielle, The Netherlands). Prepackaged tests (TRUE TESTS) are also available for a limited number of test allergens, and contain homogeneously dispersed allergens in a hydrophilic gel base (cellulose derivatives) mounted on an acrylic-based adhesive tape. They are a portable and convenient method for those wishing to test only a limited number of allergens.

Contact Dermatitis

Table 89.9 Currently recommended European standard series of test allergens. Potassium dichromate Neomycin sulfate Thiuram mix Paraphenylenediamine free base Cobalt chloride Benzocaine Formaldehyde Colophony Clioquinol Balsam of Peru Wool alcohols Mercapto mix Epoxy resin Parabens mix N-Isopropyl-N-phenyl-4-phenylenediamine Paratert phenol formaldehyde resin Fragrance mix 1 Quaternium 15 Nickel sulfate Cl-Me-isothiazolinone (aq.) Mercaptobenzothiazole Sesquiterpene lactone mix Primin Budesonide Tixocortol-21-pivalate Methyldibromoglutaronitrile

0.5% (pet.) 20.0% (pet.) 1.0% (pet.) 1.0% (pet.) 1.0% (pet.) 5.0% (pet.) 1.0% (aq.) 20.0% (pet.) 5.0% (pet.) 25.0% (pet.) 30.0% (pet.) 2.0% (pet.) 1.0% (pet.) 16.0% (pet.) 0.1% (pet.) 1.0% (pet.) 8.0% (pet.) 1.0% (pet.) 5.0% (pet.) 0.01% (aq.) 2.0% (pet.) 0.1% (pet.) 0.01% (pet.) 0.01% (pet.) 0.1% (pet.) 0.5% (pet.)

A small amount or “snake” of each allergen (approximately 5 mm) is deposited from the syringe into the chamber such that it fills the well of the disk but does not extrude when the patch is applied to the back. For aqueous-based allergens, small filter-papers are placed in the well and these will hold around 15 μL of liquid. Allergens are applied on the patient’s back (Fig. 89.26), and left in place for 48 hours. For a small number of allergens, for example when retesting, the outer aspect of the upper arm is also acceptable. False-negative test results can be obtained when testing on the lower back or on the volar forearms. Even the sequence of allergens should be carefully selected so that those frequently causing strong, cross or concomitant reactions are not adjacent to each other. The order given in the catalogs produced by Chemotechnique Diagnostics (2006) and Trolab Hermal (2006) can usually be followed. These catalogs list all commercially available allergens in the European and international standard series, tables of allergen mixes, as well as information on the occurrence of allergens and cross-reactivity. The basic concept of using mixes of allergens instead of single allergens is to save time and space. Once the patches are in place, the disk positions are marked with an indelible marking system that will last for the duration of the test, e.g., a violet marker pen or dihydroxyacetone

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Fig. 89.26 Application of patch tests. (See CD-ROM for color version.)

solution 15% in acetone. Patients should be instructed not to bath or shower for the duration of the test and to avoid exercise that is likely to dislodge the disks. The test should be deferred if the patient has recently acquired a suntan over the back, and the patches should not be exposed to the sun or other sources of UV light (Sjovall 1988). Both topical and oral corticosteroids (O’Quinn & Isbell 1969; Sukanto et al. 1981) and other immunosuppressive drugs should be stopped (where this is feasible) before patch testing. This information is better given to patients before they book their appointments. An example of a patient information sheet that can be given to patients may be found on the British Contact Dermatitis Society (BCDS) website (http:// www.bcds.org.uk/). Patch testing should ideally not be carried out in patients with active eczema since it may reduce the threshold of reactivity and cause nonspecific irritant reactions (Bruynzeel et al. 1981).

Reading patch tests It is strongly recommended that two readings are carried out, the first after removal of the patches (on day 2) and the second 2–5 days later (Rietschel et al. 1988). In one study (MacFarlane et al. 1989) paired readings on days 4 and 7 were found to be more reliable than those on days 2 and 4. From studies with repeated readings it is obvious that the

same patch-test preparation can produce lost as well as found reactions (Geier et al. 1999; Dickel et al. 2000). Neomycin, corticosteroids, and gold are often-quoted examples of allergens with late appearance (“slow” allergens) while others (fragrance mix, balsam of Peru) are classified as “early” allergens. Multiple readings are thus highly recommended and the importance of readings beyond day 2 must be stressed (Uter et al. 1996). If practical or geographical circumstances permit only one reading, the present accepted compromise is reading on day 3 (72 hours), i.e., 24 hours after removal of the patches. However, studies by Shehade et al. (1991) and Todd et al. (1996) suggested that a single reading on day 4 would have been most useful. Patients must also be instructed to report any late reactions to the patch test clinic. The internationally accepted recording system for reading patch tests was originally developed by the ICDRG (Wilkinson et al. 1970) and is shown in Table 89.10. Difficulties in discriminating weak allergic from irritant reactions frequently occur. Such gray zones need to be handled by supplementary tests such as dose–response tests, serial dilutions, and repeat open application tests. In the final decision they must also be related to the clinical history. Other drawbacks with this system are that it confuses morphology with interpretation. The ideal methodology is to record what is seen at days 2 and 4 (Fig. 89.27) and then to decide if this represents an allergic or irritant response, combining morphology with knowledge of the substance, skin type, concentration, controls, etc. The next step involves deciding whether the allergen is of current relevance to the patient’s presenting problem or whether it represents an “immunologic scar” from a previous problem. For example, the patient may have a nonhealing leg ulcer and be allergic to the topical antibiotic neomycin, which is of current relevance to the use of topical medications. The same patient may also have a nickel-positive reaction, which is of past relevance to a problem experienced many years ago with costume jewellery. Sometimes it is impossible to attribute relevance, e.g., in an isolated epoxy resin-positive reaction with no history of previous exposure, and this is then recorded as unknown relevance. It is not possible to apply absolute rules for determining the relevance of an allergic reaction. However, based on the presence of a putative allergen in someone’s occupational or leisure environment, the distribution of the skin lesions,

Table 89.10 Recording of patch-test reactions according to the International Contact Dermatitis Research Group. +? + ++ +++ IR NT

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Doubtful reaction: faint erythema only Weak positive reaction: erythema, infiltration, possibly papules Strong positive reaction: erythema, infiltration, papules, vesicles Extreme positive reaction: intense erythema, infiltration and coalescing vesicles Irritant reaction of different types Not tested

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Table 89.11 Causes of false-positive reactions. Too high a concentration Impure substance (contaminants) Irritant vehicle Excess allergen applied Uneven dispersion Current or recent dermatitis at patch-test site Pressure effect of hard materials Adhesive-tape reactions “Angry-back” reaction causing intensification of weak irritants Artifact

Table 89.12 Causes of false-negative reactions.

Fig. 89.27 Positive patch-test reactions (allergic). (See CD-ROM for color version.)

and the effect of elicitation to exposure and healing from avoidance, positive patch-test reactions can be classified as “possible,” “probable,” or “certain relevance.” This is always the most difficult and intricate part of the test procedure and the dermatologist’s skill and experience are crucial factors. Apart from patch tests, use tests, chemical analyses, and work-site visits may also be of help in discriminating allergic from irritant reactions and the relevance of an allergic response.

Difficulties in interpretation and complications of patch testing Apart from the difficulties in distinguishing allergic from irritant reactions and ascribing the relevance of an allergic reaction, there are still some further pitfalls that the investigator should be aware of. Some allergic reactions are “crossreactions” to other allergens, e.g., paraphenylenediamine, used as a hair dye reagent, may cross-react with other substances that have an amino group in the para position such as benzocaine, a local anesthetic. This occurs when the allergenic part of the molecule is similar in different substances and forms a similar hapten that is indistinguishable by the immune system. Atopics are also particularly prone to nonspecific (erythematous) patch-test reactions (Klas et al. 1996) and the irritant reactions themselves have several different morphologic types. Sometimes there may be false-positive (Table 89.11) and false-negative (Table 89.12) reactions which are dose dependent. There are also occasional complications from patch testing (Table 89.13).

Too low a concentration Insufficient amount applied Wrong vehicle Allergen not in active form, e.g., insufficiently oxidized for some fragrance chemicals Insufficient occlusion Poor adhesion of patches Patches applied at wrong site Substance degraded Pretreatment of patch-test site with UV light, topical corticosteroids or immunomodulators Systemic treatment with corticosteroids or immunomodulators Failure to perform delayed readings

Table 89.13 Possible complications of patch testing. Active sensitization Irritant reactions from patient’s own products inadequately diluted Flare of dermatitis at site of patch testing Flare of dermatitis at previous contact sites Pigmentation or depigmentation

Enhanced skin reactivity can be seen in patients with both active dermatitis as well as strong positive allergic patch-test reactions. Mitchell (1975) coined the term “angry back syndrome” which describes multiple false-positive patch-test reactions that are not reproducible when repeated separately. The nonreproducibility is blamed on the presence of other strong positive tests and/or inflamed skin elsewhere. Since hyperreactivity is not restricted to the back, “excited skin syndrome” may be a more appropriate term. The term “compound allergy” is used to describe patients who are patch test positive to formulated products, usually cosmetic creams or topical medicaments, but test negative to all the ingredients when tested individually (Kelett et al. 1986). This phenomenon can sometimes be explained by irritancy of the original formulation, but in some cases it has

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been demonstrated that reactivity was due to the combination of constituent ingredients forming reaction products (Smeenk et al. 1987). Another reason might be that the ingredients were patch tested at usage concentrations, which are too low for many allergens (Bashir et al. 2000). In cases where there is strong suspicion of ACD yet negative patch tests to individual analytical-grade constituents, it may be preferable to obtain the actual manufacturing grade ingredients direct from the manufacturer. There are several case reports in the literature where this has been necessary (Orton & Shaw 2001). One of the most serious adverse reactions from patch testing is sensitization induced by the procedure itself (active sensitization). However, this is considered to be a very rare occurrence. Traditionally, patch-test sensitization is detected by a flare-up reaction at the test site 10 days after application (Cronin 1980). On repeat testing, the reaction is usually already positive by day 2– 4. It is important to avoid patch testing with unknown substances or formulations, since scarring, necrosis, pigmentary change, systemic effects, and active sensitization must be avoided. When patients bring suspected products or materials from their (work) environment, it is recommended that adequate material safety data sheets or lists of ingredients are requested from the manufacturer prior to testing. In this way, reference books, research papers, and chemical analysis can be used to identify the optimal patch-test concentration and vehicle (De Groot 1986) for individual chemicals. However, it is still very important to test a patient’s own materials (including cosmetics) to provide the optimal circumstances for detecting allergens, provided that certain procedures are undertaken (Uter et al. 2005).

Photopatch tests Certain substances are transformed into sensitizers by the action of UV light. To perform photopatch tests, suspected allergens are applied in duplicate on symmetrical body sites (usually on the left- and right-hand sides of the lower back). At the 2-day reading, the sites are inspected and read as usual. One set is then photoirradiated with UVA. The optimal dose is 50% of the minimal erythema dose (MED) of UVA. This is important if patients are light sensitive, as they have reduced MED. However, for other patients, a standard dose of 5 J/cm2 UVA is recommended (Hasan & Jansen 1996; British Photodermatology Group 1997; Bruynzeel et al. 2004). The light source is usually a bank of fluorescent tubes, as commonly found in a hand and foot photochemotherapy (PUVA) unit. The light source intensity must be monitored regularly as the tubes deteriorate with time and exposure time will have to increase to compensate for this in order to maintain the same dose. Care must be taken to avoid irradiation by natural light or by the light source on the control set of allergens. A positive photoallergic patch test is recorded when an allergic reaction occurs only on the irradiated side and not on

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the control. The commonest photoallergens in recent reports have been the chemical sunscreens, which are UV lightabsorbing agents (Darvay et al. 2001; Bryden et al. 2006). By their nature they absorb light in a specific waveband. Rarely it may be necessary to perform a photopatch test with more specific wavebands (e.g., UVB) using a monochromator or solar simulator. A recent review has concluded that irradiation after 48 hours’ occlusion will be more sensitive at detecting photoallergens than after 24 hours’ occlusion (Batchelor & Wilkinson 2006).

Repeat open application tests These are very useful when patch tests with suspected allergens and products continue to give negative results, or when the relevance of a patch-test reaction is in question (Johansen et al. 2006). Repeat open application tests should not be used with “rinse off” products because these could result in falsepositive reactions.

Management of contact dermatitis Contact dermatitis can only be properly managed when it is first suspected and recognized and after the causative agent(s) has been identified. This usually requires patch testing. Negative investigations, if done correctly, can still be informative and subsequently help with the management of the patient. It is important to recognize that dermatitis often occurs as a result of a combination of constitutional, irritant, and allergic factors. If irritant or allergic factors are identified, then a patient must exclude himself or herself as far as possible from the source of exposure, with a change in working practice or relevant personal protective equipment. It is also just as important to address any mechanical factors such as heat, sweating or friction. The principles of management are outlined in Table 89.14. However, the medical literature suggests that the prognosis for contact dermatitis is worse for atopics, for those with well-established irritant dermatitis, and for those allergic to chromate and nickel.

Advice and counseling Patients require a clear explanation of any allergies, where the allergen is likely to be encountered, and the relevance of any positive test. Patient information sheets concerning the

Table 89.14 Principles of management. Prevention: human/environmental factors Avoidance: irritants and sensitizers Protection Replacement

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more common allergens and sources of exposure can often be helpful. These can be obtained from certain organizations including the BCDS (http://www.bcds.org.uk/) and Derm Net (http://www.dermnetnz.org/). Patients may also need advice about alternative products that they can safely use, and about complicating factors, for example atopics and those with recent eczema will need to understand their continuing susceptibility to irritants. Some patients will require help in creating a “low-risk” environment or with allergen replacement. Others may require advice about gloves or protective clothing. Sometimes a change of employment may have to be considered, but employers may be able to relocate individuals within the workplace. Occupational ACD deserves particular mention since studies indicate that a change in occupation does not always result in a significant improvement in prognosis (Cahill et al. 2004). In those with chronic ICD, efforts should be made to reduce the irritant load, both at home and at work. A period off work to let the skin heal may also be needed. Primary prevention and career advice for atopics is also very important, but should be considered as a last resort. There is some good evidence that primary prevention strategies may make a significant impact on reducing contact dermatitis (Dickel et al. 2002; Loffler et al. 2006).

Legislative changes By analyzing patch-test data it has become apparent that legislation can be an effective tool in the prevention of contact dermatitis. Equally, the corollary of analyzing patch-test data and recognizing trends has also resulted in changes of legislation. The aim of ESSCA is the continuous analysis (surveillance) of pooled data for several purposes. This includes the recognition of allergens for consumer protection, as well as time-trend analyses, and subgroup analyses providing a valuable starting point for lowering the incidence of contact allergies. For industry, such analyses can serve the purpose of postmarketing surveillance of product safety. The nickel directive, adopted by the European Parliament and Council in 1994 (European Parliament and Council Directive 94/2/EEC), has resulted in reduced sensitization rates to nickel among younger age groups in Denmark (Johansen et al. 2000). In Scandinavia, legislation that regulated the content of hexavalent chromium and the addition of ferrous sulfate to cement has effectively reduced the prevalence of chromate allergy in construction workers. Similar legislation has recently been adopted by the European Union (EU) (Bock et al. 2003). While the 6th amendment of the EU cosmetics directive, passed in 1996, legislated for the labeling of constituent ingredients according to the International Nomenclature of Cosmetic Ingredient (or INCI names), under the 7th amendment of the European Cosmetics Directive (European Council Directive 76/768/EEC) the public can obtain information from cosmetic companies via the European Directory for Public

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Access. This information includes the full INCI listing of international ingredients, as well as information concerning undesirable effects from the use of these cosmetics. Each company participating in the directory is responsible for this information (http://www.european-cosmetics.info). However for continued consumer safety, it is vital that toxicologists develop risk tools to predict allergen skin sensitization risks and that manufacturers take responsibility to limit known sensitizers, find suitable alternatives, and correctly label their products as completely as possible. It is also imperative for dermatologists to accurately record data of relevant allergic reactions and for this information to be coordinated to allow effective surveillance.

Clinical treatment of contact dermatitis Apart from minimizing exposure to irritants and removing exposure to correctly identified allergens, mild forms of dermatitis may be sufficiently controlled by the regular use of emollients/moisturizers. Severe relapsing forms require topical corticosteroids. These remain the principal treatment for those with contact dermatitis. In severe cases, a short course of systemic steroids may also be required, with topical or systemic antibiotics when there is evidence of secondary infection. Wet soaks or compresses may also be helpful for more acute cases. Severe cases may require topical phototherapy or treatment with other systemic immunosupressants. The reader is referred to the relevant sections of the recommended textbooks for a more comprehensive account of specific treatment modalities (Kanerva et al. 2000; Rietschel & Fowler 2001; Frosch 2006).

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Bryden, A.M., Moseley, H., Ibbotson, S.H. et al. (2006) Photopatch testing of 1155 patients: results of the U.K. multicentre photopatch study group. Br J Dermatol 155, 737–47. Burrows, D., Andersen, K.E., Camarasa, J.G. et al. (1989) Trial of 0.5% versus 0.375% potassium dichromate. European Environmental and Contact Dermatitis Research Group (EECDRG). Contact Dermatitis 21, 351. Cahill, J., Keegel, T. & Nixon, R. (2004) The prognosis of occupational contact dermatitis in 2004. Contact Dermatitis 51, 219–26. Cherry, N., Meyer, J.D., Adisesh, A. et al. (2000) Surveillance of occupational skin disease: EPIDERM and OPRA. Br J Dermatol 142, 1128–34. Coenraads, P.-J., Diepgen, T., Uter, W., Schnuch, A. & Gefeller, O. (2006) Epidemiology. In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin, 135– 63. Cronin, E. (1980) Contact Dermatitis. Churchill Livingstone, Edinburgh. Darvay, A., White, I.R., Rycroft, R.J., Jones, A.B., Hawk, J.I. & McFadden, J.P. (2001) Photoallergic contact dermatitis is uncommon. Br J Dermatol 145, 597–601. De Groot, A.C. (1986) Patch Testing: Test Concentrations and Vehicles for 2800 Allergens. Elsevier, Amsterdam. De Groot, A.C. (1988) Adverse reactions to cosmetics. Thesis, State University of Groningen. Dickel, H., Taylor, J.S., Evey, P. & Merk, H.F. (2000) Delayed readings of a standard screening patch test tray: frequency of “lost”, “found”, and “persistent” reactions. Am J Contact Dermatitis 11, 213–17. Dickel, H., Bruckner, T., Bernhard-Klimt, C., Koch, T., Scheidt, R. & Diepgen, T.L. (2002) Surveillance scheme for occupational skin disease in the Saarland, FRG. First report from BKH-S. Contact Dermatitis 46, 197–206. Ducombs, G., Benezra, C., Talaga, P. et al. (1990) Patch testing with the “sesquiterpene lactone mix”: a marker for contact allergy to Compositae and other sesquiterpene-lactone-containing plants. A multicentre study of the EECDRG. Contact Dermatitis 22, 249–52. European Council Directive 76/768/EEC. Official Journal of the European Communities 27-09-1976. No. L262 (cosmetics). European Parliament and Council Directive 94/2/EEC. Official Journal of the European Communities 22-07-1994. No. L188/1-2 (nickel). Evans, A.V., Banerjee, P., McFadden, J.P. & Calonje, E. (2003) Lymphomatoid contact dermatitis to para-tertyl-butyl phenol resin. Clin Exp Dermatol 28, 272–3. Frosch, P.J. (2006) In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin see under further reading. Frosch, P.J. & Kligman, A.M. (1977) A method of assessing the stinging capacity of topically applied substances. J Soc Cosmet Chem 28, 197–209. Geier, J., Gefeller, O., Wiechmann, K. & Fuchs, T. (1999) Patch test reactions at D4, D5 and D6. Contact Dermatitis 40, 119–26. Gollhausen, R. & Kligman, A.M. (1985) Human assay for identifying substances which induce non-allergic contact urticaria: the NICU test. Contact Dermatitis 13, 98–106. Hasan, T. & Jansen, C.T. (1996) Photopatch test reactivity: effect of photoallergen concentration and UVA dosaging. Contact Dermatitis 34, 383–6. Hextall, J.M., Alagaratnam, N.J., Glendinning, A.K. et al. (2002) Dose-time relationships for elicitation of contact allergy to paraphenylenediamine. Contact Dermatitis 47, 96–9. Heydorn, S., Andersen, K.E., Johansen, J.D. & Menne, T. (2003) A stronger patch test elicitation reaction to the allergen hydroxy-

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citronellal plus the irritant sodium lauryl sulphate. Contact Dermatitis 49, 133–9. Hjorth, N. & Roed-Petersen, J. (1976) Occupational protein contact dermatitis in food handlers. Contact Dermatitis 2, 28– 42. Holst, R. & Möller, H. (1975) One hundred twin pairs patch tested with primary irritants. Br J Dermatol 93, 145–9. Isaksson, M., Brandao, F.M., Bruze, M. & Goossens, A. (2000) Recommendation to include budesonide and tixocortol pivalate in the European standard series. Contact Dermatitis 43, 41–2. Jadassohn, J. (1896) Zur Kenntins der Afzueiexanatheme. Arch Dermatol Forschr 34, 103. Johansen, J.D., Rastogi, S.C., Bruze, M. et al. (1998) Deodorants: a clinical provocation study in fragrance-sensitive individuals. Contact Dermatitis 39, 161–5. Johansen, J., Menne, T., Christophersen, J., Kaaber, K. & Vien, N. (2000) Changes in the pattern of sensitisation to common contact allergens in Denmark between 1985–86 and 1997–98, with a special view to the effect of preventive strategies. Br J Dermatol 142, 490–5. Johansen, J.D., Frosch, P.J. & Menne, T. (2006) Allergic contact dermatitis in humans. Experimental and quantitative aspects. In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin, pp. 189–98. Jones, H.E., Lewis, C.W. & McMarlin, S.L. (1973) Allergic contact sensitivity in atopic dermatitis. Arch Dermatol 107, 217–22. Kanerva, L., Ranki, A. & Lauharanta, J. (1984) Lymphocytes and Langerhans cells in patch tests: an immunocytochemical and electron microscopic study. Contact Dermatitis 11, 150–5. Kanerva, L., Elsner, P., Wahlberg, J.E. & Maibach, H.I. (2000) Handbook of Occupational Dermatology. Springer, Berlin. Keil, J.E. & Shmunes, E. (1983) The epidemiology of work-related skin disease in South Carolina. Arch Dermatol 118, 650–4. Kelett, J.K., King, C.M. & Beck, M.H. (1986) Compound allergy to medicaments. Contact Dermatitis 14, 45– 8. Klas, P.A., Corey, G., Storrs, F.J., Chan, S.C. & Hanifin, J.M. (1996) Allergic and irritant patch test reactions and atopic disease. Contact Dermatitis 34, 121– 4. Kligman, A.M. & Epstein, W. (1975) Updating the maximisation test for identifying contact allergens. Contact Dermatitis 1, 231–9. Lachapelle, J.-M. (2006) Historical aspects. In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin, pp. 1–7. Lachapelle, J.-M. & Maibach, H.I. (2003) Patch Testing, Prick Testing. A Practical Guide. Springer, Berlin. Lachapelle, J.-M. & Marot, M. (2006) Histopathological and immunohistopathological features of irritant and allergic contact dermatitis. In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin, pp. 107–115. Landsteiner, K. & Jacobs, J. (1936) Studies on the sensitization of animals with simple chemical compounds. J Exp Med 64, 629–39. Lange Vejlsgaard, G., Ralfkiaer, E., Avnstorp, C. et al. (1989) Kinetics and characterization of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes in various inflammatory skin lesions and malignant cutaneous lymphomas. J Am Acad Dermatol 20, 782. Lepoittevin, J.-P. (2006) Molecular aspects of allergic contact dermatitis. In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin, pp. 45– 60. Lisby, S. & Baadsgaard, O. (2006) Mechanisms of irritant contact dermatitis. In: Frosch, P.J., Menné, T. & Lepoittevin, J.-P., eds. Contact Dermatitis. Springer, Berlin, pp. 69–79.

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Shum, K.W., Meyer, J.D., Chen, Y., Cherry, N. & Gawkrodger, D.J. (2003) Occupational contact dermatitis to nickel: experience of the British dermatologists (EPIDERM) and occupational physicians (OPRA) surveillance schemes. Occup Environ Med 60, 954–7. Silberberg-Sinakin, I., Thorbecke, J., Baer, R.L. et al. (1976) Antigenbearing Langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell Immunol 25, 137–51. Sjovall, P. (1988) Ultraviolet radiation and allergic contact dermatitis: an experimental and clinical study. Thesis, University of Lund, Sweden. Smeenk, G., Kerckhoffs, H.P.M. & Schreurs, P.H.M. (1987) Contact allergy to a reaction product in Hirudoid® cream: an example of compound allergy. Br J Dermatol 116, 223–31. Sukanto, H., Nater, J.P. & Bleumink, E. (1981) Influence of topically applied corticosteroids on patch test reactions. Contact Dermatitis 7, 180–5. Thomson, K.F., Wilkinson, S.M., Sommer, S. & Pollock, B. (2002) Eczema: quality of life by body site and the effect of patch testing. Br J Dermatol 146, 627–30. Todd, D.J., Handley, J., Metwali, M., Allen, G.E. & Burrows, D. (1996) Day 4 is better than day 3 for a single patch test reading. Contact Dermatitis 34, 402– 4. Uter, W.J.C., Geier, J. & Schnuch, A. (1996) Good clinical practice in patch testing: readings beyond day 2 are necessary: a confirmatory analysis. Am J Contact Dermatitis 7, 231–7. Uter, W., Geier, J., Pfahlberg, A. & Effendy, I. (2002) The spectrum of contact allergy in elderly patients with and without lower leg dermatitis. Dermatology 204, 266–72. Uter, W., Schnuch, A. & Gefeller, O. (2004) Guidelines for the descriptive presentation and statistical analysis of contact allergy data. Contact Dermatitis 51, 47–56. Uter, W., Balzer, C., Geier, J., Frosch, P.J. & Schnuch, A. (2005) Patch testing with patient’s own cosmetics and toiletries: results of the IVDK, 1998–2002. Contact Dermatitis 53, 226–33. Van Hoogstraten, M.W., Andersen, K.E., von Bloomberg, B.M.E. et al. (1989) Preliminary results of a multicentre study on the incidence of nickel allergy in relationship to previous oral and cutaneous contacts. In: Frosch, P.J., Dooms-Goossens, A., La Chapelle, J.-M. et al., eds. Current Topics in Contact Dermatitis. Springer, Berlin, pp. 178–83. Weston, W.L. & Weston, J.A. (1984) Allergic contact dermatitis in children. Am J Dis Child 138, 932– 6. Whitmore, S.E. (1994) Should atopic individuals be patch tested? Dermatol Clin 12, 491–9. Wilkinson, D.S., Fregert, S., Magnusson, B. et al. (1970) Terminology of contact dermatitis. Acta Dermatol Venereol 50, 287–92. Wilkinson, J.D., Hambly, E.M. & Wilkinson, D.S. (1980) Comparison of patch test results in two adjacent areas of England. II. Medicaments. Acta Derm Venereol 60, 245–9.

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Willis, C.M., Young, E., Brandon, D.R. & Wilkinson, J.D. (1986) Immunopathological and ultrastructural findings in human allergic and irritant contact dermatitis. Br J Dermatol 115, 305–16. Willis, C.M., Stephens, C.J.M. & Wilkinson, J.D. (1989) Epidermal damage induced by irritants in man: a light and electron microscopic study. J Invest Dermatol 93, 695–9. Willis, C.M., Stephens, C.J.M. & Wilkinson, J.D. (1990) Differential effects of structurally-unrelated chemical irritants on the density and morphology of epidermal CD1a cells. J Invest Dermatol 95, 711–16. Willis, C.M., Stephens, C.J.M. & Wilkinson, J.D. (1991) Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis. J Invest Dermatol 96, 505–11. Willis, C.M., Stephens, C.J.M. & Wilkinson, J.D. (1993) Differential patterns of epidermal leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants. J Invest Dermatol 101, 364. Willis, C.M., Shaw, S., De Lacharriere, O. et al. (2001) Sensitive skin: an epidemiological study. Br J Dermatol 145, 258–63. Woo, P.N., Hay, I.C. & Ormerod, A.D. (2003) An audit of the value of patch testing and its effect on quality of life. Contact Dermatitis 48, 244–7. Wood, L.C., Jackson, S.M., Elias, P.M., Grunfeld, C. & Feingold, K.R. (1992) Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice. J Clin Invest 90, 482–7. Zachariae, C., Hall, B., Cottin, M., Andersen, K.E. & Menne, T. (2004) Formaldehyde allergy: clinically relevant threshold reactions [Abstract]. Contact Dermatitis 50, 136.

Further reading and websites Frosch, P.J., Menné, T. & Lepoittevin, J.-P. (eds) (2006) Contact Dermatitis. Springer, Berlin. Kanerva, L., Elsner, P., Wahlberg, J.E. & Maibach, H.I. (eds) (2000) Handbook of Occupational Dermatology. Springer, Berlin. Rietschel, R.L. & Fowler, J.F. (eds) (2001) Fisher’s Contact Dermatitis, 5th edn. Lippincott, Williams & Wilkins, Philadelphia. European Environmental and Contact Dermatitis Research Group (EECDRG) http://orgs.dermis.net/content/e05eecdrg/index_ ger.html [Date accessed 15 Feb 2008] European Society of Contact Dermatitis http://www.escd.org [Date accessed 15 Feb 2008] European Surveillance System on Contact Allergies http://www. essca-dc.org/ British Contact Dermatitis Society http://www.bcds.org.uk/ Derm Net http://www.dermnetnz.org/

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Urticaria and Angioedema Allen P. Kaplan

Summary Acute urticaria with or without angioedema is commonly seen as a result of IgE-mediated hypersensitivity to drugs or foods and can be diagnosed based on history and skin test. The reaction can vary from days to a few weeks and may present as intermittent episodes. Physical urticarias are diagnosed based on the nature of the stimulus causing a reaction, the location and shape of the lesions, the presence of hives that last (individually) less than 2 hours, and responsiveness to antihistaminics but not to corticosteroid. The exception is delayed pressure urticaria which is the only one with long-lasting lesions that can be refractory to antihistamines but which responds to steroid. Urticaria with or without angioedema is considered chronic if present daily for over 6 weeks. An exogenous allergenic cause is then unlikely. Between 40 and 45% represent chronic autoimmune urticaria and 55–60% chronic idiopathic urticaria. The autoimmune group is due to IgG anti-IgE receptor antibodies and occasionally IgG anti-IgE antibodies which degranulate cutaneous mast cells, fix complement, release C5a, and lead to a cellular infiltrate resembling a late-phase reaction, but with increased neutrophils and monocytes and both Th1 and Th2 lymphocytes. There is also a higher incidence of antithyroid antibodies. Treatment may require high-dose antihistaminics (both H1 and H2 antagonists) and leukotriene receptor antagonists. If severe, even a double dose of nonsedating antihistaminics may not suffice for antihistamine therapy and significant improvement can be obtained with 25–50 mg hydroxyzine q.i.d. If truly refractory, cyclosporin A or low-dose daily or alternate-day corticosteroid may be employed. Methotrexate or intravenous gammaglobulin can be effective with a lesser success rate. Recurrent angioedema in the absence of urticaria can be due to angiotensin-converting enzyme inhibitors, or hereditary or acquired C1 inhibitor deficiency; the swelling in each of these is mediated by bradykinin. Idiopathic angioedema responds to antihistamines, short courses of corticosteroid (2–3 days for an acute episode) but prophylaxis for those refractory to antihistaminics can include leukotriene synthesis inhibitors or tranexamic acid.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Urticarial vasculitis due to immune complex-dependent activation of complement may occur as an isolated entity or as a manifestation of a connective tissue disorder, and may respond to agents such as hydroxychloroquine (particularly the hypocomplementemic urticarial vasculitis syndrome), dapsone, or colchine, none of which are reliably effective for nonvasculatic urticaria.

Introduction Urticaria is characterized by the appearance of pruritic, erythematous, and elevated cutaneous lesions that blanch with pressure, indicating the presence of dilated blood vessels and intracutaneous edema. In its simplest form, it is envisioned to represent the same sort of wheal-and-flare reaction observed when histamine is injected into the skin. The flare reaction augments the area of redness by release of substance P from afferent nerve endings that mediate “itch” with spread to recruit additional nerve fibers by antidromic (reverse) conduction. Biopsy of acute urticarial lesions reveals dilation of small venules and capillaries located in the superficial dermis with widening of the dermal papillae, flattening of the rete pegs, and swelling of collagen fibers. Angioedema is caused by the same or similar pathologic alterations that occur in the deep dermis and subcutaneous tissue. Thus an area involved with angioedema has swelling as the prominent manifestation, and the appearance of the overlying skin may be normal. Because angioedema occurs in deeper skin layers where there are fewer mast cells and sensory nerve endings, the lesions have little or no associated pruritus, and the swelling may be described as painful or burning. Urticaria may occur on virtually any part of the body, whereas angioedema (in the absence of hives) often involves the face, tongue, extremities, or genitalia. In contrast to other forms of edema, angioedematous swellings do not characteristically occur in dependent areas, are asymmetrically distributed, and are transient, typically resolving within 2–5 days. Of course, urticaria and angioedema frequently occur together. This chapter first examines the biochemical mechanisms that lead to the development of urticaria and angioedema and then summarizes

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the presentation, pathogenesis, and treatment of various clinical entities, as well as the diagnostic studies indicated.

Pathogenesis Mast cell and histamine release

Epidemiology Urticaria and angioedema are common. Age, race, sex, occupation, geographic location, and season of the year may be implicated in urticaria and angioedema only insofar as they may contribute to exposure to an eliciting agent. Of a group of college students, 15–20% reported having experienced urticaria, while 1–3% of the patients referred to hospital dermatology clinics in the UK noted urticaria and angioedema. In the National Ambulatory Medical Care Survey data from 1990 to 1997 in the USA, women accounted for 69% of patient visits. There was a bimodal age distribution in patients aged birth to 9 years and 30–40 years (Henderson et al. 2000). Urticaria/angioedema is considered to be acute if it lasts less than 6 weeks. Most acute episodes are due to adverse reactions to medications or foods or in children, to viral illnesses. Episodes of urticaria/angioedema persisting beyond 6 weeks are considered chronic and are assumed to be present daily or at least most days of the week. These are divided into two major subgroups: chronic autoimmune urticaria (45%) and chronic idiopathic urticaria (55%) with a combined incidence in the general population of 0.5% (Gaig et al. 2004). Individual lesions last over 4 hours and typically remain 12–24 hours. I have not included physically induced urticaria/angioedema in this definition because their inclusion inflates the data regarding the incidence where a “cause” for chronic hives is found and their pathogenic mechanisms have little or no resemblance to that operative in either chronic idiopathic or chronic autoimmune urticaria, with the possible exception of delayed pressure urticaria. Many types of physical urticaria/angioedema may persist for years, but are intermittently present and are not really chronic. However, dermatographism and delayed pressure urticaria can be present daily or close to it. Individual lesions of all the physical urticarias last fewer than 2 hours except for delayed pressure urticaria. Whereas 85% of children experience urticaria in the absence of angioedema, 40% of adult patients with urticaria also experience angioedema. About 50% of patients with chronic urticaria (with or without angioedema) are free of lesions within 1 year, 65% within 3 years, and 85% within 5 years; fewer than 5% have lesions that last for more than 10 years. A protracted course with an increased incidence of recurrence is seen in those patients who have an autoimmune cause of the urticaria compared with those still considered idiopathic. There are no data regarding the remission rate in patients with idiopathic angioedema, i.e., recurrent angioedema, without identifiable etiology, in the absence of urticaria. The hereditary group is considered to be lifelong once the diagnosis becomes clinically manifest.

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The mast cell is the major effector cell in most forms of urticaria and angioedema, although other cell types undoubtedly contribute. Cutaneous mast cells adhere to fibronectin and laminin through the very late activation (VLA) β1 integrins VLA-3, VLA-4, and VLA-5 and to vitronectin through αvβ3 integrin. Cutaneous mast cells, but not those from other sites, release histamine in response to compound 48/80, C5a, morphine, and codeine. The neuropeptides substance P (SP), vasoactive intestinal peptide (VIP), and somatostatin (but not neurotensin, neurokinins A and B, bradykinin, or clacitonin gene-related peptide) activate mast cells for histamine secretion. Dermal microdialysis studies of the application of SP on skin indicate that it induces histamine release only at 10–6 mol/L or more which suggests that after physiologic nociceptor activation, SP may not contribute significantly to histamine release (Weidner et al. 2000). Not all potential biological products are produced when cutaneous mast cells are stimulated. For example, SP releases histamine from cutaneous mast cells above 10–6 mol/L but does not generate prostaglandin (PG)D2. Vascular permeability in skin is produced predominantly by H1 histamine receptors (85%), although H2 histamine receptors account for the remaining 15%. The participation of the mast cell in vivo in urticaria and angioedema has been studied by analysis of morphologic alterations of mast cells and by identification and quantitation of mast cell products in tissues or biological fluids. Aspirates of experimental suction blisters generated over lesional and control skin have provided biological fluid for analysis (Kaplan et al. 1978). This skin chamber model has been useful particularly because the presentation of antigen into the chamber is controlled, and the mast cell products appearing in the fluid can be assessed quantitatively and serially. Scanning laser Doppler imaging has been used to study dermal blood perfusion, and dermal microdialysis has been used to detect the presence of biochemical mediators and the consequences of their actions (Petersen et al. 1997). The current hypothesis regarding cellular infiltration that follows mast cell degranulation suggests that the release of mast cell products (histamine, leukotrienes, cytokines, chemokines) leads to alterations in vasopermeability, upregulation of adhesion molecules on endothelial cells, and rolling and attachment of blood leukocytes, followed by chemotaxis and transendothelial cell migration. Various forms of physical urticaria/angioedema have provided experimental models for the study of urticaria/angioedema by allowing observation of the elicited clinical response, examination of lesional and normal skin biopsy specimens, assay of chemical mediators released into the blood or tissues, and characterization of peripheral leukocyte responses (Gorevic & Kaplan 1980; Soter 1987). The intracutaneous injection of

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specific antigen in sensitized individuals has provided an experimental model for analysis of the role of IgE and its interaction with the mast cell. In many subjects, the challenged cutaneous sites demonstrate a biphasic response, with a transient, pruritic, erythematous wheal-and-flare reaction followed by a tender, deep, erythematous, poorly demarcated area of swelling that persists for up to 24 hours. This is the late-phase response with recruitment of variable numbers of neutrophils, prominent eosinophils, monocytes, small numbers of basophils, and CD4+ T lymphocytes of the Th2 subclass (Zweiman et al. 2000). In a study of the histamine-induced wheal-and-erythema reaction with dermal microdialysis and scanning laser Doppler imaging, the wheal began to develop at 1–2 min and reached a maximum at 10 min. The flare reached a steady state between 7 and 10 min. Although the rate of development of the flare was independent of the histamine concentration, the steady-state flare area at 10 min was concentrationdependent. The fact that histamine was detected in the wheal but not in the flare implies that the flare is a neurogenic “axon reflex” (Petersen et al. 1997) as described above.

Autoimmunity and chronic urticaria: historical observations The first suggestion that patients with chronic urticaria and angioedema might have an autoimmune diathesis was the observation that there is an increased incidence of anti-thyroid antibodies in such patients relative to the incidence in the population at large (Leznoff et al. 1983). These include anti-microsomal (peroxidase) and anti-thyroglobulin antibodies, as seen in patients with Hashimoto thyroiditis (Leznoff & Sussman 1989). Patients may have clinical hypothyroidism, but a small number might be hyperthyroid if inflammation is at an early stage when thyroid hormone is released into the circulation. This atypical presentation should be distinguished from the occasional patient with Graves disease. Nevertheless, most patients are euthyroid. The incidence of anti-thyroid antibodies in chronic urticaria, as reported in the literature, varies between 15 and 24% (Kaplan & Finn 1999; Kikuchi et al. 2003), but the most recent data are closer to the latter figure (Kikuchi et al. 2003) and demonstrate segregation of anti-thyroid antibodies with chronic autoimmune urticaria rather than chronic idiopathic urticaria. However, the association is not absolute. The incidence was 27% in the chronic autoimmune urticaria subgroup and 11% in the chronic idiopathic urticaria subgroup, while the incidence in the population at large is 7– 8%. Gruber et al. (1988) considered the possibility that patients might have circulating anti-IgE antibodies that are functional and did indeed find these in about 5–10% of patients. Grattan et al. (1986, 1998) sought antibodies reactive with skin mast cells by performing an autologous skin test and found a 30% incidence of positive reactions in patients with chronic urticaria. There were only rare positive reactions in healthy control subjects or patients

Urticaria and Angioedema

IgE IgE IgG anti receptor antibody

Secretion MAST CELL IgE

“Late phase” reaction

Infiltrative hive Fig. 90.1 Activation of mast cells by anti-IgE receptor antibody. Although not shown, IgG anti-IgE will cross-link IgE and produce the same result. (See CD-ROM for color version.)

with other forms of urticaria. Subsequently, this level of positivity was shown by Hide et al. (1993) to be due to an IgG antibody reactive with the α subunit of the IgE receptor; in addition a 5–10% incidence of functional anti-IgE antibodies was confirmed (Grattan et al. 1992). Augmented histamine release observed when IgE was stripped from basophils substantiated the presence of anti-receptor antibody, whereas a prominent decrease in histamine release on IgE stripping indicated the presence of anti-IgE. The antibody was of the IgG class, with only rare instances of IgM antibody; this is depicted in Fig. 90.1. The anti-receptor antibody interacts with unoccupied IgE receptors in most instances, although some exceptions do exist. This was shown by means of inhibition of basophil histamine release stimulated by sera from patients with chronic urticaria if the basophils were preincubated with an IgE myeloma protein. Thus IgE antibody or IgG anti-α might bind to the same epitope on the α chain, or bound IgE sterically interferes with the ability of the autoantibody to interact with the α chain.

Functional assays of the anti-IgE receptor Assays for anti-IgE receptor antibody have included in vivo methods and in vitro assays. The autologous skin test (Grattan et al. 1986; Greaves 2000), as noted above, provided one of the first clues that a subset of patients with chronic urticaria have an autoimmune disorder. Basophils are frequently used as an in vitro surrogate for mast cells, and secretion of histamine as a result of incubation with patient sera or purified IgG was readily demonstrated (Fiebiger et al. 1995; Zweiman et al. 1996; Tong et al. 1997) but was not observed if the source of basophils was from a “nonreleaser,” i.e., basophils with an abnormality in the signal transduction molecules Lyn or Syc, which are unresponsive to signals through the IgE receptor but which are normally responsive to other secretagogues such as the cytokine macrophage chemotactic factor (MCP)-1 (Kikuchi & Kaplan 2001). The incidence of a positive assay result was generally higher than that observed with the autologous skin test; reported values varied between

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35 and 50% (Fiebiger et al. 1995; Tong et al. 1997; Ferrer et al. 1998). Absorption of sera, with cloned α subunit, decreased the percentage of histamine release as the amount of added α subunit was increased (Kikuchi & Kaplan 2001), confirming reactivity with this receptor subunit. The assay could be made more sensitive by preincubating basophils with interleukin (IL)-3 (Ferrer et al. 2003), although the percentage of positive reactions was not significantly affected. Release of leukotriene (LT)C4 and IL-3 along with histamine was also demonstrable (Wedi et al. 2000; Ferrer et al. 2002). Although more cumbersome, activation of cutaneous mast cells could also be performed in vitro by using either thin skin slices (Niimi et al. 1996) or partially purified mast cells derived from foreskin samples (Ferrer et al. 1999).

Binding assays for anti-receptor antibody Attempts to quantitate IgG antibody to the α subunit have, in general, been unsuccessful. Initial attempts to demonstrate the presence of antibody by means of immunoblotting (Ferrer et al. 1998; Fiebiger et al. 1998) have not proved useful because positive reactions can be seen in other autoimmune disorders (Fiebiger et al. 1998) or occasionally with sera (Ferrer et al. 1998) from subjects with no history of urticaria. In addition, studies of the relationship of a positive immunoblot result with histamine release did not yield a significant correlation (Kikuchi & Kaplan 2001); sera with a positive blot result but negative histamine release were frequently seen. However, sera with positive histamine release and a negative blot result did have demonstrable IgG anti-α because absorption of such sera with the α subunit inhibited histamine release. Thus lack of sensitivity of the immunoblot explained part of the discrepancy, but the cause of the false-positive blot results was not apparent. Subclass analysis of the pathogenic IgG has been helpful in further delineating the relationship (or lack thereof) of binding assays versus functional methods. An early report suggested that much of the anti-receptor antibody exists within the IgG1 and IgG3 subclasses, but these data were based on immunoblot analysis (Fiebiger et al. 1998). Thus we employed affinity chromatography to purify IgG subclasses from the sera of patients with chronic urticaria to determine which have functional anti-receptor antibody. When we isolated IgG2 antibody from nine patients with chronic urticaria, none of them released histamine from human basophils, irrespective of whether a positive IgG2 anti-α antibody immunoblot result was seen. Much of the histamine release does appear to be due to IgG1 and IgG3, and rarely IgG4 (Soundararajan et al. 2005). Recently, a series of publications have demonstrated that normal serum has natural antibody to the α subunit of the IgE receptor with germline variable region sequences that is primarily IgM (Horn et al. 1999, 2001; Pachlopnik et al. 2004). Such antibodies can be functional but appear to require

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stripping of IgE from donor basophils. Evidence to suggest cross-reactivity of anti-α antibody with tetanus toxoid was reported, which implied immunization as a cause of such antibodies; however, we have been unable to absorb sera with tetanus toxoid and diminish histamine release (unpublished observations), and if such antibodies are part of our innate repertoire, no external stimulus is needed to explain their presence. But some unknown stimulus or abnormal control mechanism might lead to pathogenic IgG antibody, such as we have seen.

Role of complement The notion that complement might contribute to the histamine release observed with sera from patients with chronic urticaria was suggested by studies in which complement depletion or inactivation appeared to diminish histamine release (Zweiman et al. 1996; Fiebiger et al. 1998). A series of reports by Ferrer et al. (1999) and Kikuchi and Kaplan (2001, 2002) then documented not only a role for complement but also more specifically activation of the classical pathway and generation of C5a. First, it was demonstrated that the addition of purified patient IgG to normal serum (as a source of complement) but not sera deficient in C2 or C5 augmented cutaneous mast cell histamine release (Ferrer et al. 1999). This was confirmed with basophils (Kikuchi & Kaplan 2001, 2002). C5-deficient serum could also be reconstituted with purified C5 to recover the augmentation of histamine release provided by serum. Then inhibition of the complement contribution to histamine release was demonstrated by using an antibody to the C5a receptor (Kikuchi & Kaplan 2002). It is not clear why the presence of a functional anti-IgE receptor would cause symptoms limited to the skin, but among the differences between pulmonary and cutaneous mast cells is the absence of C5a receptors on lung mast cells. On the other hand, it is also not clear why the IgG2 anti-IgE receptor antibody, which is often demonstrable by means of immunoblotting, does not activate basophils or cutaneous mast cells because IgG2 anti-FcεRIα should cross-link α subunits, as might any IgG subclass antibody directed to the α subunit. The difference might have to do with antibody affinity or differing α subunit epitopes with which these antibodies react. The geometry of binding is also a factor because the Fc region of two adjacent IgG molecules must each be able to bind to one of the globular heads of C1q to initiate complement activation (Fig. 90.2). It is theoretically possible to have binding of IgG to the IgE receptor in the absence of cross-linking (e.g., the receptors are too far apart) and still observe histamine release if the two Fc regions are sufficiently close together to activate C1. If receptors are sparse, the antibody can bind without activating either the cells or complement, whereas receptors saturated with IgE might preclude binding. These considerations may account for the marked heterogeneity in patient manifestations and severity.

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PHYSIOLOGY AND MOLECULAR MECHANISM OF AUTOIMMUNE URTICARIA C3

C4 + C2

C1

C3b

C4b2a3b

C4b2a

C5

Activated C1

C5b

C5a

Antigen–antibody (IgG) complex

Y Y

Y

Y

Anti-FceRI IgG

Fig. 90.2 Schematic diagram of the activation of cutaneous mast cells by IgG anti-receptor antibody, followed by activation of complement, release of C5a, and augmentation of mast cell release. (See CD-ROM for color version.)

C5a receptor

C5a Cell activation Mediator release Histamine Leukotrienes Cytokines Chemokines

IgE receptor

Cellular infilitrate Mast cell degranulation certainly initiates the inflammatory process in autoimmune chronic urticaria and is assumed to also do so in idiopathic chronic urticaria. Evidence for increased number of mast cells in chronic urticaria has been presented (Elias et al. 1986; Nettis et al. 2001) but there are also publications indicating no significant differences from normal (Smith et al. 1995); these studies did not discriminate the autoimmune from the idiopathic groups. However, no alternative mechanisms for mast cell degranulation in the idiopathic groups have been suggested to date. Yet the histology of the two groups differs only in minor ways. Common to all biopsy specimens is a perivascular infiltrate that surrounds small venules within the superficial and deep venular plexus, with a prominence of CD4+ T lymphocytes and monocytes and virtually no B cells (Natbony et al. 1983; Elias et al. 1986). Granulocytes are quite variable but are plentiful if the lesion undergoes biopsy early in its development. Neutrophils and eosinophils are both present (Haas et al. 1998a; Sabroe et al. 1999a), although the degree of eosinophil accumulation varies greatly (Natbony et al. 1983). Even when eosinophils are not evident, major basic protein can be identified within lesions (in at least two-thirds of patients), which most likely represents evidence of prior eosinophil degranulation (Peters et al. 1983). The presence of basophils has also been recently demonstrated by using an antibody (BB1) that is specific for this cell type (Sabroe et al. 1999a). Thus the infiltrate re-

sembles that of an allergic late-phase reaction, as suggested previously (Grattan et al. 1990), although the percentage of each cell type differs, with neutrophils and monocytes being relatively more prominent in urticaria. The recruitment of these cells requires release of cytokines and chemotactic factors active on each cell type and activation and recruitment of adhesion molecules on the migrating cell, as well as the endothelial cell. This combination of integrins and selectins mediates various aspects of cell migration, rolling along the endothelial cell surface, firm adhesion, and then transmigration. Endothelial cell activation is suggested by the presence of intercellular adhesion molecule (ICAM)-1 and E-selectin in biopsy specimens of urticarial lesions (Haas et al. 1998b). Sources of chemokines include the mast cell and the activated endothelial cell; the latter cells are stimulated not only by cytokines or monokines, such as IL-4, IL-1, and tumor necrosis factor (TNF)-α, but also by the vasoactive factors (e.g., histamine and leukotrienes) released from activated mast cells (Lee et al. 2002). Complement activation and the release of C5a results not only in augmented mast cell (and basophil) histamine release, but C5a is also chemotactic for neutrophils, eosinophils, and monocytes. The presence of C5a is one of the factors that would distinguish this lesion from a typical allergen-induced cutaneous late-phase reaction. The particular chemokines released in chronic urticaria have not been studied. The presence of increased plasma IL-4 levels (Ferrer et al. 2002) in patients with chronic urticaria provides indirect

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Fig. 90.3 Skin biopsy of typical patient with chronic urticaria demonstrating a nonnecrotizing perivascular infiltration with mononuclear cells (CD4+ lymphocytes and monocytes) and small numbers of neutrophils, eosinophils, and basophils. (See CD-ROM for color version.)

evidence of lymphocyte activation, basophil activation, or both, and isolated CD4+ lymphocytes of patients were shown to secrete greater amounts of both IL-4 and interferon (IFN)γ compared with that seen in healthy control subjects on stimulation with phorbol myristate acetate. A direct comparison between cutaneous late-phase reactions and the histology of chronic urticaria revealed that infiltrating cells had characteristics of both Th1 and Th2 cells (Fig. 90.3), with production of IFN-γ by the former cells and IL-4 and IL-5 by the latter (Ying et al. 1999). Alternatively, this might represent activated Th0 cells (i.e., activated CD4+ lymphocytes that have not differentiated into Th1 or Th2 cells). When the histology of autoimmune and idiopathic chronic urticaria was compared (Sabroe et al. 1999a), the autoimmune subgroup had greater prominence of granulocytes within the infiltrate, whereas other infiltrating cells were quite similar, with a small increment in cytokine levels in the autoimmune group and greater tryptase positivity (less degranulation?) in the autoantibody-negative group. The patients with autoimmune chronic urticaria generally had more severe symptoms than those with idiopathic chronic urticaria (Sabroe et al. 2002).

Basophil releasibility The basophils of patients with chronic urticaria have been shown to be hyporesponsive to IgG anti-IgE (produced in rabbits by immunization with human IgE myeloma protein), an observation made by Kern and Lichtenstein (1976) long before there were any clues to the pathogenesis of this disorder. These findings were confirmed (Sabroe et al. 1998) and appeared to be associated with basopenia (Grattan et al. 1997) and were thought to segregate with the autoimmune subgroup. One possible interpretation is that there is in vivo

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desensitization of basophils in the presence of circulating anti-IgE or anti IgE receptor antibodies. However, we have found a paradoxical result when the isolated basophils of patients with chronic urticaria were activated and compared with the basophils of healthy control subjects. Although the basophils of patients with urticaria were clearly less responsive to anti-IgE, they demonstrated augmented histamine release when incubated with serum and it did not matter whether the sera were taken from normals, other patients with chronic urticaria, or was their own (autologous) (Luquin et al. 2005). This augmented responsiveness of basophils was evident when basophils were isolated from patients with chronic idiopathic urticaria, as well as from those with chronic autoimmune urticaria, and might represent a cellular abnormality of basophils present in patients with all types of chronic urticaria. The nature of this augmented reactivity and the nature of the serum factor that appears to stimulate these cells is unknown. A more recent publication found hyporesponsiveness to anti-IgE in only half their patients with chronic urticaria. However it did not segregate to the autoimmune subgroup and was associated with decreased levels of the Src-homology 2-containing inositol phosphatases (SHIP) (Vonakis et al. 2007). Another publication suggests an underlying abnormality in signal transduction in patients with chronic urticaria, including increased expression of the p21ras protooncogene and low expression of the regulatory protein “son of sevenless” (HSOS1) (Confino-Cohen et al. 2002).

Additional considerations A recent publication describes a mechanism for histamine release in which antibody to the low-affinity IgE receptor on eosinophils induces the release of cationic proteins capable of causing histamine release from basophils and/or mast cells. However, the antibody has not been purified so as to clearly distinguish it from antibody to the high-affinity IgE receptor (anti-α chain) and the results have not yet been confirmed (Puccetti et al. 2005). Another study reexamined the autologous skin test employing citrated plasma rather than serum and reported an incidence of 90%, suggesting that some other permeability factor or antibody is being missed (or destroyed during coagulation) using the serum test (Asero et al. 2006). The factor(s) has not been isolated or identified in any way and, here too, confirmation of the finding is needed. These authors clearly demonstrated activation of the extrinsic (tissue factor) coagulation pathway with release of thrombin (as assessed by the presence of a prothrombin cleavage product) and propose a mechanism for histamine release that is thrombin dependent via thrombin (PAR) receptors. Alternatively, perhaps activation products of mast cells, which clearly activate endothelial cells, may also induce expression of tissue factor to cause thrombin formation. Thus thrombin formation might be a consequence of mast cell (basophil) activation rather than the cause of it.

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Bradykinin: role in angioedema Kinins are low-molecular-weight peptides that participate in inflammatory processes by virtue of their ability to activate endothelial cells and, as a consequence, lead to vasodilatation, increased vascular permeability, production of nitric oxide, and mobilization of arachidonic acid. Kinins also stimulate sensory nerve endings to cause a burning dysesthesia. Thus the classical parameters of inflammation (i.e., redness, heat, swelling, and pain) can all result from kinin formation. Bradykinin is the best characterized of this group of vasoactive substances. There are two general pathways by which bradykinin is generated and the plasma pathway is considered in considerable detail in Chapter 20. The simpler of the two has only two components: an enzyme, tissue kallikrein (Margolius 1998), and a plasma substrate, low-molecular-weight kininogen (LK) (Jacobson & Kritz 1967; Muller-Esterl et al. 1985). Tissue kallikrein is secreted by many cells throughout the body; however, certain tissues produce particularly large quantities. These include glandular tissues (salivary and sweat glands and pancreatic exocrine gland) and the lung, kidney, intestine, and brain. The enzyme is processed intracellularly from a precursor, prokallikrein, to produce tissue kallikrein; however, the enzyme responsible for this conversion has not been identified. Tissue kallikrein is secreted and digests LK to yield a 10-amino-acid peptide, lysyl-bradykinin (kallidin), with the sequence Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-PheArg. A plasma aminopeptidase cleaves the N-terminal Lys from it, and the 9-amino-acid peptide bradykinin results. The second pathway for bradykinin formation is far more complex and is part of the initiating mechanism by which the intrinsic coagulation pathway is activated (Kaplan et al. 2002) and is summarized here. Factor XII is the initiating protein that binds to certain negatively charged macromolecular surfaces and autoactivates (autodigests) to form factor XIIa (Silverberg et al. 1980; Tankersley & Finlayson 1984). There are two plasma substrates of factor XIIa, namely prekallikrein (Mandle & Kaplan 1977) and factor XI (Bouma & Griffin 1977; Kurachi & Davie 1977), and each of these circulates as a complex with high-molecular-weight kininogen (HK) (Mandle et al. 1976; Thompson et al. 1977). These complexes also attach to initiating surfaces, and the major attachment sites are on two of the domains of HK, thereby placing both prekallikrein and factor XI in optimal conformation for cleavage to kallikrein (plasma kallikrein) and factor XIa, respectively. It is important to note that plasma kallikrein and tissue kallikrein are separate gene products and have little amino acid sequence homology, although they have related functions (i.e., cleavage of kininogens). Tissue kallikrein prefers LK but is capable of cleaving HK, whereas plasma kallikrein cleaves HK exclusively. The two kininogens have an identical amino acid sequence starting at the N-terminus and continuing to 12 amino acids beyond the bradykinin moiety (MullerEsterl et al. 1985) but differ in C-terminal domains because

Urticaria and Angioedema

of alternative splicing at the transcriptional level (Kitamura et al. 1985; Takagaki et al. 1985). The enzymes that destroy bradykinin consist of kininases I and II. Kininase I is also known as plasma carboxypeptidase N (Erdos & Sloane 1962), which removes the C-terminal Arg from bradykinin or kallidin to yield des-Arg9-bradykinin or des-Arg10-kallidin, respectively (Sheikh & Kaplan 1986a). It is the same enzyme that cleaves the C-terminal Arg from the complement anaphylatoxins C3a and C5a. Kininase II is identical to angiotensin-converting enzyme (ACE) (Yang & Erdos 1967). Kininase II is a dipeptidase that cleaves the C-terminal Phe-Arg from bradykinin to yield a heptapeptide, which is cleaved once again to remove Ser-Pro to leave the pentapeptide Arg-Pro-Pro-Gly-Phe (Sheikh & Kaplan 1986b). If the C-terminal Arg of bradykinin is first removed with kininase I, then ACE functions as a tripeptidase to remove Ser-Pro-Phe to leave the above pentapeptide (Sheikh & Kaplan 1989). Bradykinin and kallidin stimulate constitutively produced B2 receptors (Vavrek & Stewart 1985), whereas desArg9-bradykinin or des-Arg10-Lys-bradykinin both stimulate B1 receptors (Regoli & Barabe 1980), which are induced as a result of inflammation. Stimuli for B1 receptor transcription include IL-1 and TNF-α (Davis & Perkins 1994; Marceau 1995). For further details and diagrams depicting the pathways for generation and degradation of bradykinin, see Chapter 20.

Approach to the patient (Fig. 90.4) When urticaria has been present for days or weeks at a time (but less than 6 weeks), or occurs recurrently for similar intervals, the main considerations are allergic reactions (IgEmediated) to food or drugs and careful history regarding possibilities is essential. Skin testing can corroborate IgEmediated hypersensitivity to foods or can provide suspects where the history is unrevealing. Sometimes it is done in patients with chronic urticaria to demonstrate that suspected foods are in fact not responsible for the urticaria. Doubleblind placebo-controlled food challenge can demonstrate clinical relevance where the role of a food is uncertain. NonIgE-mediated causes of urticaria include adverse reactions to nonsteroidal antiinflammatory drugs (NSAIDs) and opiates. Any of these can be associated with concomitant angioedema or, less commonly, present as angioedema in the absence of urticaria. Children may have acute urticaria in association with viral illnesses; it is unclear whether infection with bacteria such as streptococci can do so as well but neither occurs in adults with the exception of urticaria in association with infectious mononucleosis (Epstein–Barr virus) or as a prodome to hepatitis B infection. In each of these circumstances, individual lesions last anywhere from 4 to 24 hours and fade without associated purpura. If hives last less than 2 hours, the cause is usually a “physical urticaria,” the most common being dermatographism, cholinergic urticaria, and cold

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History: Recurrent transient hives or swelling

Clinical appearance: wheals, angioedema

Wheals ± angioedema

Angioedema only

Duration of individual hive

Drugs, ACE inhibitor, other family history

30 min. to 2 h

History physical stimulus

C4 level

24–48 h with either bruising, severe arthralgia, fever, ↓ C4

4 h to 36 h

Course < 6 weeks

Course > 6 weeks

Consider drugs, foods, food skin testing, infection (particularly in children), other identifiable stimulus

Thyroid function tests, anti-microsomal antibody, antithyroglobulin antibody, autologous skin test, in vitro – anti-IgE receptor

C1 inhibitor by protein and function Normal

Abnormal

Idiopathic angioedema

Hereditary angioedema a. C1 INH protein and function abnormal – Type I

Physical challenge

Physical urticaria

Acute urticaria/ angioedema

Positive

Chronic autoimmune urticaria

Negative

Chronic idiopathic urticaria

Skin biopsy

Normal C1Q

Positive

b. C1 INH protein normal or elevated, function abnormal – Type II Acquired C1 INH deficiency, depressed C1Q level

Urticarial vasculitis Search for lymphoma, connective tissue disease, Type I

Anti-C1 INH, Type II

Overlap situation Fig. 90.4 Approach to the patient with urticaria and angioedema.

urticaria. The main exception is delayed pressure urticaria where lesions typically last 12–36 hours and first appear 3– 6 hours after the initiating stimuli. Once urticaria is present for over 6 weeks (particularly if present for many months or years) chronic urticaria is present, which is now divided into chronic idiopathic urticaria where a cause has not yet been found and chronic autoimmune urticaria. Angioedema accompanies chronic urticaria in 40% of such cases and is more problematic in the autoimmune subgroup. Swelling in association with chronic urticaria can affect hands, feet, eyes, cheeks, lips, tongue, and pharynx, but not the larynx. When angioedema is present in the absence of an identifiable antigen or exogenous stimulus, the main entities to consider are C1 inhibitor deficiency (hereditary or acquired), ingestion of an ACE inhibitor, or idiopathic angioedema. About 0.5% of patients have an urticarial vasculitis with palpable purpura or other stigmata of a possible vasculitis such as fever, elevated crythocyte sedimentation rate, petechae or purpura, elevated white cell count, or lesions of unusual duration (36–72 hours).

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Clinical entities Immunologic: IgE- and IgE receptor-dependent urticaria/angioedema Atopic diathesis Episodes of acute urticaria/angioedema that occur in individuals with a personal or family history of asthma, rhinitis, or eczema are presumed to be IgE-dependent. An evaluation for allergy to foods or drugs is required. In clinical practice, however, urticaria/angioedema rarely actually accompanies an exacerbation of asthma, rhinitis, or eczema. The prevalence of chronic urticaria/angioedema is not increased in atopic individuals.

Specific antigen sensitivity Common examples of specific antigens that provoke urticaria/ angioedema include foods (e.g., shellfish, nuts, and chocolate), drugs and therapeutic agents (notably penicillin), aeroaller-

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gens, and Hymenoptera venom (Fig. 90.5). Urticaria in patients with helminthic infestations has been attributed to IgE-dependent processes; however, proof of this relationship is often lacking. Most cases will have an elevated IgE level and eosinophilia. Specific allergens and nonspecific stimuli may activate local reactions termed “recall urticaria” at sites previously injected with allergen immunotherapy.

not associated with atopy. The peak prevalence occurs in the second and third decades. In one study, the duration of dermographism was greater than 5 years in 22% of individuals and greater than 10 years in 10%. Most are transient, lasting 6 months to 2 years. Elevations in blood histamine levels have been documented in some patients after experimental scratching, and increased levels of histamine (Garafalo & Kaplan 1981), tryptase, SP, and VIP, but not calcitonin gene-related peptide, have been detected in experimental suction-blister aspirates. The dermatographic response has been passively transferred to the skin of normal subjects with serum or IgE (Soter et al. 1977). Delayed dermatographism develops 3–6 hours after stimulation, either with or without an immediate reaction, and lasts 24–48 hours. The eruption is composed of linear red nodules. This condition may be associated with delayed pressure urticaria and these may in fact represent the same entity. Cold-dependent dermatographism is a condition where marked augmentation of the dermatographic response occurs when the skin is chilled (Kaplan 1984). In rare instances the dermatographic response is delayed, i.e., appearing after 15– 30 min; too soon for “delayed” dermatographism but considerably longer then is usual, and can be readily missed.

Physical urticaria/angioedema

Pressure urticaria

Fig. 90.5 Patient with chronic autoimmune urticaria. (See CD-ROM for color version.)

(Gorevic & Kaplan 1980; Soter 1987)

Dermatographism Dermatographism is the most common form of physical urticaria and is the one most likely to be confused with chronic urticaria. It appears as a linear wheal with a flare at a site where the skin is briskly stroked with a firm object (Fig. 90.6). A transient wheal appears rapidly and usually fades within 30 min; however, the patient’s normal skin is typically pruritic so that an itch–scratch sequence may appear. In two studies, the prevalence of dermatographism in the general population was found to be 1.5 and 4.2%; its prevalence in patients with chronic urticaria is 22%. It is

Fig. 90.6 Topical dermatographic response to scratching the skin. (See CD-ROM for color version.)

Delayed pressure urticaria appears as erythematous, deep, local swellings, often painful, that arise from 3 to 6 hours after sustained pressure has been applied to the skin (Estes & Yung 1981; Dover et al. 1988). Spontaneous episodes are elicited after sitting on a hard chair, under shoulder straps and belts, on the feet after running, and on the hands after manual labor. The peak prevalence occurs in the third decade. Delayed pressure urticaria may occasionally be associated with fever, chills, arthralgias, and myalgias, as well as with an elevated erythrocyte sedimentation rate and leukocytosis. In one study, it accompanied chronic urticaria in 37% of patients. This is far more commonly seen than patients with pressure urticaria and no spontaneously occurring hives. Thus delayed pressure urticaria in the absence of seemingly spontaneously occurring hives is a rare disorder. Although an IgE-mediated mechanism has not been demonstrated, histamine and IL-6 have been detected in lesional experimental suction-blister aspirates and in fluid from skin chambers, respectively (Kaplan et al. 1978; Lawlor et al. 1993; Barlow et al. 1994). LTB4, 12- and 15-hydroxyeicosatetraenoic acids, and IL-1 were absent. Immediate pressure urticaria is a rare idiopathic disorder. It has been described in patients with the hypereosinophilic syndrome, and can be diagnosed by thumb pressure on the back for 30 s with no movement of the thumb. A hive the shape of the thumbprint is seen within a few minutes. A positive dermatographic response may also be seen but dermatographic patients are negative when tested this way if there is no motion of the thumb other than pressing down.

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Vibratory angioedema Vibratory angioedema may occur as an acquired idiopathic disorder, in association with cholinergic urticaria, or after several years of occupational exposure to vibration (Weiner et al. 1983). It has been described in families, with an autosomal dominant pattern of inheritance (Ting et al. 1983). The heritable form is often accompanied by facial flushing. An increase in the level of plasma histamine was detected during an experimental attack in patients with the hereditary form and in patients with acquired disease (Ting et al. 1983; Weiner et al. 1983). A typical symptom is hives across the back when toweling off after a shower (in the absence of dermatographism).

Cold urticaria There are both acquired and inherited forms of cold urticaria/ angioedema, although the familial form is rare. Idiopathic or primary acquired cold urticaria may be associated with headache, hypotension, syncope, wheezing, shortness of breath, palpitations, nausea, vomiting, and diarrhea. Attacks occur within minutes after exposures that include changes in ambient temperature and direct contact with cold objects. The elicitation of a wheal after the application of ice has been called a diagnostic cold contact test (Fig. 90.7). If the entire body is cooled (as during swimming), hypotension and syncope are potentially lethal events in this situation (drowning). In rare instances, acquired cold urticaria has been associated with circulating cryoglobulins, cryofibrinogens, cold agglutinins, and cold hemolysins, especially in children with infectious mononucleosis (Costanzi & Coltman 1967; Costanzi et al. 1969; Gorevic 1995). Passive transfer of cold urticaria by intracutaneous injection of serum or IgE to the skin of normal recipients has been documented (Houser et al. 1970; Wanderer et al. 1971). Histamine, chemotactic factors for eosinophils and neutrophils, PGD2, cysteinyl leukotrienes, platelet-activating factor, and TNF-α have been released into the circulation after

experimental challenge (Kaplan et al. 1975; Soter et al. 1976a; Wasserman et al. 1977, 1982; Grandel et al. 1985; Ormerod et al. 1988a; Tillie-Leblond et al. 1994). Histamine, SP, and VIP, but not calcitonin gene-related peptide have been detected in experimental suction-blister aspirates. Histamine has been released in vitro from chilled skin biopsy specimens that have been rewarmed (Kaplan et al. 1981). Neutrophils harvested from the blood of an experimentally cold-challenged arm manifested an impaired chemotactic response. Whereas complement has no role in primary acquired cold urticaria, cold challenge of patients with cold urticaria who have circulating immune complexes, (such as cryoglobulins) can provoke a cutaneous necrotizing venulitis with complement activation (Eady & Greaves 1978; Soter et al. 1978; Eady et al. 1981; Wanderer et al. 1983). Rare forms of acquired cold urticaria that have been described mainly in case reports include systemic cold urticaria (Kaplan 1984), localized cold urticaria (Kurtz & Kaplan 1990), cold-induced cholinergic urticaria, cold-dependent dermatographism (Kaplan 1984), and localized cold reflex urticaria (Czarnetzki et al. 1981; Ting & Mansfield 1985). Two forms of dominantly inherited cold urticaria have been described. Familial cold urticaria, which has been termed familial cold autoinflammatory syndrome, is considered a type of periodic fever (Hoffman et al. 2001a). It is an autosomal dominant disorder with genetic linkage to chromosome 1q44. The responsible gene has been identified as CIASI, which codes for a protein, cryopyrin, involved in regulation of inflammation and apoptosis (Hoffman et al. 2001b). The eruption occurs as erythematous macules and infrequent wheals and is associated with burning or pruritus. Fever, headaches, conjunctivitis, arthralgias, and a neutrophilic leukocytosis are features of attacks. The delay between cold exposure and onset of symptoms is 2.5 hours, and the average duration of an episode is 12 hours. Renal disease with amyloidosis occurs infrequently. Skin biopsy specimens show mast cell degranulation and an infiltrate of neutrophils. The cold contact test and passive transfer with serum have been negative. Serum levels of IL-6 and granulocyte colony-stimulating factor (GCSF) were elevated in one patient. Delayed cold urticaria occurs as erythematous, edematous, deep swellings that appear 9– 18 hours after cold challenge. Lesional biopsy specimens show edema with minimal numbers of mononuclear cells; mast cells are not degranulated; and neither complement proteins nor immunoglobulins are detected. Cold immersion does not release histamine, and the condition cannot be passively transferred.

Cholinergic urticaria

Fig. 90.7 Positive ice cube testing a patient with cold urticaria. (See CD-ROM for color version.)

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Cholinergic urticaria develops after an increase in core body temperature, such as during a warm bath, prolonged exercise, or episodes of fever (Grant et al. 1935). The highest prevalence is observed in individuals aged 23–28 years. The eruption appears as distinctive, pruritic, small, 1–2 mm wheals that are

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variant in which typical “cholinergic”-looking lesions appear with exercise, but only if the person is chilled, e.g., exercise outside on a winter’s day. The ice cube test and methacholine skin test are both negative (Kaplan & Garofalo 1981).

Local heat urticaria

Fig. 90.8 Lesions of cholinergic urticaria observed in a patient after 15 min of exercise in a warm room. (See CD-ROM for color version.)

Local heat urticaria is a rare form of urticaria in which wheals develop within minutes after exposure to locally applied heat. An increased incidence of atopy has been reported. Passive transfer has been negative. Histamine, neutrophil chemotactic activity, and PGD2 have been detected in the circulation after experimental challenge (Grant et al. 1981). A familial delayed form of local heat urticaria in which the urticaria occurred 1–2 hours after challenge and lasted up to 10 hours has been described.

Solar urticaria surrounded by large areas of erythema (Fig. 90.8). Occasionally, the lesions may become confluent, or angioedema may develop. Systemic features include dizziness, headache, syncope, flushing, wheezing, shortness of breath, nausea, vomiting, and diarrhea. An increased prevalence of atopy has been reported. The intracutaneous injection of cholinergic agents, such as methacholine chloride, produces a wheal with satellite lesions in approximately one-third of patients (Herxheimer 1956; Commens & Greaves 1978). Alterations in pulmonary function have been documented during experimental exercise challenge (Soter et al. 1980) or after the inhalation of methacholine, but most are asymptomatic. Recent data indicate that a major subpopulation of patients with cholinergic urticaria have a positive skin test and in vitro histamine release due to autologous sweat (Fukunaga et al. 2005). It is not clear whether this is IgE-mediated (or not) and any antigen present in sweat is unidentified. This is the same subpopulation with a positive methacholine skin test with satellite lesions and a nonfollicular distribution of the wheals. The remaining patients were negative to an autologous sweat skin test or in vitro histamine release. The methacholine skin test is negative for satellite lesions and the hives tend to be follicular in distribution. Familial cases have been reported only in men in four families (Onn et al. 1996). This observation suggests an autosomal dominant pattern of inheritance. One of these individuals had coexisting dermatographism and aquagenic urticaria. After exercise challenge, histamine and factors chemotactic for eosinophils and neutrophils have been released into the circulation (Kaplan et al. 1975; Soter et al. 1980). Tryptase has been detected in lesional suction-blister aspirates. The urticarial response has been passively transferred on one occasion; however, most other attempts to do so have been unsuccessful. Cold urticaria and cholinergic urticaria are not uncommonly seen together (Sigler et al. 1979; Ormerod et al. 1988b) and cold-induced cholinergic urticaria represents an unusual

Solar urticaria occurs as pruritus, erythema, wheals, and occasionally angioedema that develop within minutes after exposure to sun or artificial light sources. Headache, syncope, dizziness, wheezing, and nausea are systemic features. Most commonly, solar urticaria appears during the third decade (Uetsu et al. 2000). In one study, 48% of patients had a history of atopy. Although solar urticaria may be associated with systemic lupus erythematosus and polymorphous light eruption, it is usually idiopathic. The development of skin lesions under experimental conditions in response to specific wavelengths has allowed classification into six subtypes; however, individuals may respond to more than one portion of the light spectrum. In type I, elicited by wavelengths of 285–320 nm, and in type IV, elicited by wavelengths of 400–500 nm, the responses have been passively transferred with serum suggesting a role for IgE antibody. In type I, the wavelengths are blocked by window glass (Harber et al. 1963; Sams et al. 1969). Type VI, which is identical to erythropoietic protoporphyria, is due to ferrochelatase (heme synthetase) deficiency (Bonkowsky et al. 1975). There is evidence that an antigen on skin may become evident once irradiated with the appropriate wavelength of light followed by complement activation and release of C5a (Gigli et al. 1980; Lim et al. 1981, 1984). Histamine and chemotactic factors for eosinophils and neutrophils have been identified in blood after exposure of the individuals to ultraviolet (UV)A, UVB, and visible light (Soter et al. 1979; Hawk et al. 1980). In some individuals, uncharacterized serum factors with molecular weights ranging from 25 to 1000 kDa, which elicit cutaneous wheal-anderythema reactions after intracutaneous injection, have been implicated in the development of lesions.

Exercise-induced anaphylaxis Exercise-induced anaphylaxis is a clinical symptom complex consisting of pruritus, urticaria, angioedema (respiratory distress), and syncope that is distinct from cholinergic urticaria (Sheffer & Austen 1980; Lewis et al. 1981; Sheffer et al. 1983;

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Casale et al. 1986). In most patients, the wheals are not punctate and resemble the hives seen in acute or chronic urticaria. It is not readily reproduced by exercise challenge as is cholinergic urticaria. There is a high prevalence of atopic diathesis. Some cases are food dependent, i.e., exercise will lead to an anaphylactic-like episode only if food was ingested within 5 hours of the exercise. The food dependency is subdivided into two groups: in the first group the nature of the food eaten is not relevant, while in the second it is a specific food to which there is IgE-mediated hypersensitivity that must be eaten for hives to appear (Maulitz et al. 1979; Kidd et al. 1983; Novey et al. 1983; Kivity et al. 1988). Yet in this instance, eating the food without exercise does not result in urticaria. The food-dependent group is easier to treat because avoidance of food (or a specific food) for 5–6 hours prior to exercise prevents episodes. Those not related to food require therapy for acute episodes and prophylaxis with high-dose antihistaminics or avoidance of exercise. Analysis of a questionnaire study of individuals with exercise-induced anaphylaxis for more than a decade (Shadick et al. 1999) disclosed that the frequency of attacks had decreased in 47% and had stabilized in 46%. 41% had been free of attacks for 1 year. Rare familial forms have been described. In exercise-induced anaphylaxis, pulmonary function tests are normal, biopsy specimens show mast cell degranulation, and histamine and tryptase are released into the circulation.

Adrenergic urticaria Adrenergic urticaria occurs as wheals surrounded by a white halo that develop during emotional stress. The lesions can be elicited by the intracutaneous injection of norepinephrine.

Aquagenic urticaria and aquagenic pruritus Contact of the skin with water of any temperature may result in pruritus alone or, more rarely, urticaria. The eruption consists of small wheals that are reminiscent of cholinergic urticaria. Aquagenic urticaria has been reported in more than one member in five families (Luong & Nguyen 1998). Aquagenic pruritus without urticaria is usually idiopathic but also occurs in elderly persons with dry skin and in patients with polycythemia vera, Hodgkin disease, myelodysplastic syndrome, and hypereosinophilic syndrome. Patients with aquagenic pruritus should be evaluated for the emergence of a hematologic disorder. After experimental challenge, blood histamine levels were elevated in subjects with aquagenic pruritus and with aquagenic urticaria. Mast cell degranulation was present in lesional tissues. Passive transfer was negative.

Other forms of urticaria/angioedema Contact urticaria Urticaria may occur after direct contact with a variety of substances. It may be IgE-mediated or nonimmunologic. The transient eruption appears within minutes, and when it is IgE-mediated it may be associated with systemic manifesta-

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tions. Passive transfer has been documented in some instances. Proteins from latex products are a prominent cause of IgEmediated contact urticaria (Poley & Slater 2000). Latex proteins may also become airborne allergens, as demonstrated by allergen-loaded airborne glove powder used in inhalation challenge tests. These patients may manifest cross-reactivity to fruits, such as bananas, avocado, and kiwi (Kurup & Fink 2001). Associated manifestations include rhinitis, conjunctivitis, dyspnea, and shock. The risk group is dominated by biomedical workers and individuals with frequent contact with latex, such as children with spina bifida. Agents such as stinging nettles, arthropod hairs, and chemicals may release histamine directly from mast cells.

Papular urticaria This occurs as episodic, symmetrically distributed, pruritic, 3–10 mm urticarial papules that result from a hypersensitivity reaction to insect bites, such as mosquitoes, fleas, and bedbugs. This condition appears mainly in children. The lesions tend to appear in groups on exposed areas such as the extensor aspects of the extremities (Millikan 2004). They are prominently pruritis and are typically present for 1–2 weeks.

Reactions to the administration of blood products Urticaria/angioedema may develop after the administration of blood products. It is usually the result of immune complex formation and complement activation that leads to direct vascular and smooth muscle alterations and, indirectly, via anaphylatoxins to mast cell mediator release. This mechanism has been delineated clearly in the case of urticarial or anaphylactic reactions to blood, plasma, or immunoglobulin in patients with antibodies to IgA, which may arise after transfusion or by placental transfer. These IgG antibodies form complexes with donor IgA and may activate the complement system. However, urticarial or anaphylactic reactions to the administration of immunoglobulin do not always depend on antibody to IgA. Aggregates of IgG may activate mast cells by binding to FcγRIII or by activating complement. The intradermal administration of aggregated IgG to humans causes swelling and erythema within 10 min that becomes tender and persists up to 24–48 hours. Biopsy specimens of these reactions show neutrophilic infiltrates at 6 and 24 hours, with a mononuclear infiltrate appearing at 24 hours. The implication that aggregated IgG is responsible for most human reactions is strengthened by the fact that the administration of IgG from which aggregates have been removed is not associated with urticaria or anaphylaxis. An uncommon mechanism for the development of urticaria after the administration of blood products is the transfusion of IgE of donor origin directed toward an antigen to which the recipient is subsequently exposed. Another mechanism may be the transfusion of a soluble antigen present in the donor preparation into a previously sensitized recipient.

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Infections Episodes of acute urticaria can be associated with upper respiratory tract viral infections, most commonly in children (Mortureux et al. 1998). The acute urticaria resolves within 3 weeks. Hepatitis B virus infection has been associated with episodes of urticaria lasting up to a week that are associated with fever and arthralgias as part of the prodrome. The mechanism is analogous to that seen in serum sickness-like reactions with viral-antibody immune complexes. A similar brief urticarial episode may be seen with infection with Epstein–Barr virus manifest as infectious mononucleosis.

Urticaria/angioedema after direct mast cell degranulation Various therapeutic and diagnostic agents have been associated with urticaria/angioedema. Up to 8% of patients receiving radiographic contrast media experience such reactions, which occur most commonly after intravenous administration. Decreased serum alternative pathway complement protein levels and increased serum histamine levels have been detected in patients receiving radiocontrast media. The suggested sequence is complement activation and depletion of select components, anaphylatoxin formation, and histamine release. However, some of these agents may cause histamine release directly. The osmolality of the agents is a critical parameter and newer agents that are osmotically neutral rarely cause reactions. Opiate analgesics, polymyxin B, curare, and D-tubocurarine release histamine from mast cells and basophils. A greater risk for urticaria/angioedema and anaphylactic-like reactions in women is suggested by one study in which 70% of those experiencing such a reaction were women.

Urticaria/angioedema relating to abnormalities of arachidonic acid metabolism Intolerance to aspirin manifest as urticaria/angioedema occurs in otherwise normal individuals or in patients with allergic rhinitis and/or bronchial asthma. Urticaria/angioedema in response to aspirin and NSAIDs occurred in approximately 10–20% of individuals referred to a hospital dermatology clinic in the UK. Patients intolerant of aspirin may also react to indomethacin and to other NSAIDs. Reactions to aspirin are shared with other NSAIDs because they reflect inhibition of prostaglandin endoperoxide synthase 1 (PGHS-1, cyclooxygenase 1) (Szczeklik et al. 1975) as well as inhibition of the inducible PGHS-2 (cyclooxygenase 2). Sodium salicylate and choline salicylate are generally well tolerated because of their weak activity against PGHS-1. PGHS-2 inhibitors are generally well tolerated in those with NSAIDinduced urticaria (Sanchez Borges et al. 2001; Mastalerz et al. 2004). Reactions to NSAIDs increase the levels of cysteinyl leukotrienes (Israel et al. 1993), which may relate to the appearance of urticaria, although their role in NSAID-induced asthma is better characterized. Skin-prick tests are of no diagnostic value, passive transfer reactions are negative, and

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neither IgG nor IgE antibodies have been associated with clinical disease. The clinical manifestations elicited by aspirin challenge of aspirin-intolerant patients are blocked when such patients are protected with a cysteinyl leukotriene receptor blocker or biosynthetic inhibitor; this finding confirms a pathobiological role for the cysteinyl leukotrienes.

Chronic idiopathic urticaria and idiopathic angioedema Because this clinical entity is common, has a capricious course, and is recognized easily, it is frequently associated with concomitant events. Such attributions must be interpreted with caution. Although infections, food allergies, adverse reactions to food additives, metabolic and hormonal abnormalities, malignant conditions, and emotional factors have been claimed as causes, proof of their etiologic relationship is often lacking. The latest consideration is chronic urticaria as a consequence of infection with Helicobacter pylori. Articles both supporting (Di Campli et al. 1998; Liutu et al. 1998; Wedi et al. 1998) and denying (Becker et al. 1998; Valsecchi & Pigatto 1998; Schnyder et al. 1999; Hidvegi et al. 2001) a relationship are numerous and a definite answer is not available. However, the H. pylori infection rate in the population at large is far greater than the incidence of chronic urticaria and in my opinion the association is spurious. The controversy has been put in perspective by Greaves (2001). Idiopathic angioedema is diagnosed when angioedema is recurrent, in the absence of urticaria, and when no exogenous agent or underlying abnormality is identifiable. It is often stated that when one considers chronic urticaria and angioedema, 40% have chronic urticaria in the absence of angioedema, 40% have urticaria and angioedema, and 20% have only angioedema. This assumes that the underlying etiology of these is similar, but idiopathic angioedema may actually represent a separate entity. It is more common in men and the incidence of anti-thyroid antibodies and/or antibody to the IgE receptor is far less than is seen in the group with chronic urticaria. Angioedema can affect the lips, tongue, pharynx, cheeks, eyes, hands, feet, penis, and scrotum. An extensive review of angioedema has been recently published (Kaplan & Greaves 2005). Cyclic episodic angioedema has been associated with fever, weight gain, absence of internal organ damage, a benign course, and peripheral blood eosinophilia (Gleich et al. 1984). Biopsy specimens of tissues show eosinophils, eosinophil granule proteins, and CD4 lymphocytes exhibiting HLA-DR. Blood levels of IL-1, soluble IL-2 receptor and IL-5 are elevated.

Miscellaneous Muckle–Wells syndrome consists of urticaria, amyloidosis, and nerve deafness and is due to the same gene defect as seen in familial cold urticaria (Hoffman et al. 2001a; Lipsker et al. 2001). Schnitzler syndrome is a chronic urticaria associated with IgM monoclonal protein, osteosclerosis, and has been shown to have antibody to IL-1α.

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Hereditary angioedema and acquired C1 inhibitor deficiency Hereditary angioedema is an autosomal dominant disorder caused by the absence of C1 inhibitor (INH) (Donaldson & Evans 1963), in which patients may have attacks of swelling involving almost any portion of the body. A traumatic episode may initiate an attack. However, such a triggering event may not be evident, and the swelling appears to occur spontaneously. It is not associated with urticaria, and patients with both urticaria and angioedema without a family history invariably have normal C1 INH. In addition to the family history, the presence of visceral involvement suggests the hereditary disorder. The most severe complication is laryngeal edema, which has been a major cause of mortality in this disease. Patients can also have abdominal attacks lasting 1– 3 days, consisting of vomiting, severe abdominal pain, and guarding in the absence of fever, leukocytosis or abdominal rigidity. This can occasionally be difficult to distinguish from an acute abdominal condition; however, the attacks are selflimited and are caused by edema of the bowel wall. The ultrastructural lesion seen in tissues of patients with hereditary angioedema consists of gaps in the postcapillary venule endothelial cells, edema, and virtually no cellular infiltrate, consistent with the release of a vasoactive factor such as a kinin. Patients with hereditary angioedema have measurable ¯ levels of the activated first component of complement (C1), although this protein generally circulates as an unactivated enzyme. The serum level of C4 is diminished even when the patient is free of symptoms and is usually undetectable during an attack. A C4 determination is therefore the simplest test for diagnosing hereditary angioedema. C2, the other substrate of C1, is usually within normal limits when the patient is asymptomatic, but its concentration also is diminished during an attack. When a diminished C4 level is obtained, a direct assay of protein C1 INH should be performed. A diminished or absent level of C1 INH confirms the diagnosis. However, approximately 15–20% of patients have a normal or elevated level of C1 INH protein. In these cases the protein is not functional and often has an abnormal electrophoretic mobility. A functional C1 INH assay is then necessary to determine whether the diagnosis is really hereditary angioedema. The pathogenesis of the swelling involves primarily the plasma kinin-forming pathway, although the complement cascade is clearly activated as well. Direct demonstration of such a kinin-like peptide on interaction of C1¯ with C4 and C2 or C2 alone is lacking. Published evidence regarding such a peptide suggested that cleavage of C2b by plasmin generates a kinin, but attempts to confirm this experiment have failed (Curd et al. 1983; Fields et al. 1983). The only identifiable kinin seen in these latter studies was bradykinin and bradykinin is now considered to be the cause of the swelling (Kikuchi et al. 2003). Twenty-four hour urinary histamine excretion is also increased during attacks of angioedema, suggesting that C3a, C4a, or C5a is being generated. Although the plasma levels of

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C3 and C5 are normal in this disorder, C3 turnover is enhanced. However, the lesions are not pruritic, and antihistamines have no effect on the clinical course of the disease. C1 INH also inhibits activated Hageman factor, kallikrein, and plasmin. Therefore, it is also an important modulator of bradykinin generation. Patients appear to be hyperresponsive to injections of kallikrein, and there is evidence that the C1 activation observed in hereditary angioedema may be Hageman factor-dependent (Donaldson 1968). Thus some Hageman factor-dependent enzyme may be initiating the classic complement cascade. Plasmin is capable of activating C1s and may represent one such enzyme. However, Ghebrehiwet et al. (1983) have demonstrated that Hageman factor fragment can directly initiate the classic complement cascade by activating C1. This may represent a critical link between the intrinsic coagulation kinin cascade and complement activation. The presence of kallikrein-like activity in the induced blisters of patients with hereditary angioedema supports this notion (Curd et al. 1980), as does the progressive generation of kinin on incubation of hereditary angioedema plasma in plastic (noncontact-activated tubes) (Fields et al. 1983) and the low prekallikrein and HK levels seen during attacks (Schapira et al. 1983). More recent data support these indirect observations regarding bradykinin as the critical pathogenic peptide and encompass both hereditary angioedema and acquired C1 INH deficiency as well. One unique family has been described in which there is a point mutation in C1 INH (Ala443→Val) leading to inability to inhibit the complement cascade but normal inhibition of factor XIIa and kallikrein (Zahedi et al. 1995). There is prominent activation of the complement pathway but no activation of the kinin-forming cascade and no family member with this type II mutation has had angioedema. Plasma bradykinin levels are increased during attacks of swelling in both hereditary and acquired C1 INH deficiency (Cugno et al. 1996; Nussberger et al. 1998) and local bradykinin generation has been documented at the site of the swelling (Nussberger et al. 1999). Treatment for attacks of hereditary angioedema may include intermittent administration of subcutaneous epinephrine; however, this is one of the few forms of angioedema that may not respond to it. Attacks usually abate in 3–4 days even if no medication is given. A tracheostomy is indicated if laryngeal edema occurs, and mild analgesics may be used to relieve the discomfort of severe swelling and abdominal pain. Intravenous fluids may be necessary if the patient is unable to eat or drink. Acute administration of purified C1 INH concentrate (where available) or fresh frozen plasma (to supply C1 INH) can abort acute episodes. Successful prevention has been reported with the administration of androgens (Davis et al. 1974) and large doses of antifibrinolytic agents such as aminocaproic acid (Frank et al. 1972) and tranexamic acid (Sheffer et al. 1972). The precise mechanism of action of these last agents is unknown, but there are multiple possible effects. Plasmin, can activate C1 as well as Hageman factor (Kaplan

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& Austen 1971), and it also degrades C1 INH. Therefore inhibition of plasmin by the last two agents is beneficial. Nevertheless, the drugs of choice are androgen derivatives such as danazol and stanozolol, which not only prevent attacks of swelling in patients with hereditary angioedema but also induce synthesis of normal C1 INH and cause the C4 level to return toward normal (Gelfand et al. 1976). Where available, however, prophylactic administration of C1 INH purified concentrate can prevent episodes and is the preferred approach. Yet these drugs may also have other, as yet unknown, effects because many patients respond to low doses that are insufficient to raise levels of C4 and C1 INH (Sheffer et al. 1987). Alternatively, tissue levels may be more important than plasma levels. Patients with hereditary angioedema are heterozygotes and therefore possess one normal gene. However, levels may be less than 25% of normal rather than the expected 50% because of depletion by binding to activated enzymes or inhibition of the one normal gene. Patients with a functionally abnormal C1 INH also respond to androgen therapy (Gadek et al. 1979). Such patients possess single amino acid substitutions at the active site of the inhibitor. With treatment, normal C1 INH is synthesized along with the abnormal protein, suggesting that such patients possess one gene that manufactures an abnormal protein (structural gene defect) and a second gene that codes for a normal product but whch is inhibited or the product catabolized excessively (Quastel et al. 1983). A proposed model for the pathogenesis of hereditary angioedema is given in Fig. 20.2. An acquired form of this disease was initially described in patients with lymphoma who have circulating low-molecularweight IgM and depressed C1 INH levels. This entity has an unusual complement utilization profile because C1q levels are low and C4, C2, and C3 are depleted. The low C1q level distinguishes this condition from the hereditary disorder (Schreiber et al. 1976). The depressed C1 INH level may be caused by depletion secondary to C1 activation by immune complexes or C1 interaction with the tumor cell (Geha et al. 1985). In the latter instance, C1 fixation and C1 INH depletion are caused by an antiidiotypic antibody bound to immunoglobuin on the surface of a B-cell lymphoma. Other patients with connective tissue disorders (primarily systemic lupus erythematosus) (Donaldson et al. 1977), or cryoglobulinemia, or carcinoma have acquired C1 INH deficiency and respond to androgen therapy, which is helpful in these disorders by enhancing C1 INH synthesis. Subsequently, an additional form of acquired C1 INH deficiency was described which results from synthesis of an autoantibody (IgG) directed to C1 INH (Alsenz et al. 1987). Like the aforementioned acquired forms of C1 INH deficiency, there are low levels of C4, C1q, and C1 INH and no family history. Under normal circumstances C1 INH is a substrate for the enzymes it inactivates; that is, the enzyme cleaves C1 INH, which exposes the active site in the inhibitor. The cleaved C1 INH then binds stoichiometrically to the enzyme

Urticaria and Angioedema

and inactivates it. When antibody to C1 INH is present, C1 INH is cleaved but the antibody inhibits its ability to inactivate the enzyme. Cleaved functionless C1 INH then circulates (Zuraw & Curd 1986), and unopposed activation of the complement and kinin-forming cascades takes place (Malbran et al. 1988). Therapeutic modalities other than androgen therapy include the use of plasmapheresis and immunosuppressive agents. Most recently a new form of Hereditary Angioedema with normal C1 INH has been described (Binkley & Davis 2000) occurring primarily in females. It is estrogen dependent and has dominant inheritance. Some families have a mutation in Factor XII in which the activity of the enzyme (Factor XIIa) is greater than normal (Dewald G and Bork K, 2006).

Angiotensin-converting enzyme inhibitors Angioedema has been associated with the administration of ACE inhibitors (Sabroe & Black 1997). The frequency of angioedema occurring after ACE inhibitor therapy is 0.1–0.7%. Angioedema develops during the first week of therapy in up to 72% of affected individuals and usually involves the head and neck, including the mouth, tongue, pharynx, and larynx (Fig. 90.9). Urticaria occurs only rarely. Cough and angioedema of the gastrointestinal tract are associated features. It has been suggested that therapy with ACE inhibitors is contraindicated in patients with a prior history of idiopathic angioedema, hereditary angioedema, and acquired C1 INH deficiency (Kaplan & Greaves 2005). It appears that this swelling is also a consequence of elevated levels of bradykinin (Nussberger et al. 1998); however, the accumulation of bradykinin is due to a defect in degradation rather than excessive production. ACE, being identical to kininase II, is the major enzyme responsible for bradykinin degradation (see Fig. 20.1), and although it is present in plasma, the vascular endothelium of the lung appears to be its major site of action

Fig. 90.9 Patient with airway obstruction requiring intubation in the intensive care unit caused by an ACE inhibitor given for treatment of hypertension. (See CD-ROM for color version.)

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(Alabaster & Bakhle 1992). The action of ACE always leads to the formation of degradation products with no activity, whereas kininase I alone yields the des-Arg products, which are capable of stimulating B1 receptors. The excessive accumulation of bradykinin implies that production is ongoing or that some event leads to activation of the plasma cascade or release of tissue kallikrein is present with faulty inactivation of bradykinin that leads to swelling. Continuous turnover of the cascade is implied by data demonstrating activation along the surface of cells and cellular expression or secretion of a prekallikrein activator other than factor XIIa (Joseph et al. 2002; Shariat-Madar et al. 2002).

Urticaria and vasculitis Patients with an immune complex disease such as serum sickness or systemic lupus erythematosus (Provost et al. 1980) can have urticaria as a manifestation of the underlying disease. Biopsy of such lesions shows evidence of a necrotizing vasculitis involving the small venules (hypersensitivity angiitis), and immunofluorescent studies demonstrate deposition of immunoglobulin and complement (Fig. 90.10). In patients with serum sickness, IgE-dependent histamine release, as well as histamine release secondary to complement activation, may contribute to the urticaria. Patients having urticaria secondary to cutaneous vasculitis (Soter et al. 1976b) could not be readily classified as having an underlying collagen vascular disease. An elevated erythrocyte sedimentation rate, arthralgias, and myalgias, as well as fever and leukocytosis, were the main associated abnormalities. Like patients with palpable purpura associated with a variety of diseases, patients could be divided into a group with serum hypocomplementemia and a group in whom serum complement was normal. The group with hypocomplementemia had lesions characterized by infiltration with neutrophils, fibrin deposition, and prominent nuclear debris. The normocomplementemic group had lesions in which lymphocytes predominated, and there was promin-

Fig. 90.10 Skin biopsy of patient with urticarial venulitis (cutaneous vasculitis) demonstrating leukocytoclastic angiitis with a neutrophilic infiltrate, vessel wall destruction, and fragmented cells. (See CD-ROM for color version.)

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ent perivenular fibrin deposition. In each, there was evidence of mast cell degranulation and true vessel wall necrosis. Other isolated cases of chronic urticaria have also been reported to involve cutaneous vasculitis and hypocomplementemia (McDuffie et al. 1973; Marder et al. 1976). This syndrome is reminiscent of connective tissue diseases, but it does not fall into one of the standard diagnostic entities and is termed “hypocomplementemic vasculitic urticarial syndrome.” It is now appreciated that other organ involvement (e.g., glomerulonephritis, pseudotumor cerebri, angioedema, arthralgias, and myalgias) can occur. Low-molecular-weight precipitins that interact with C1q and thereby lower C1q levels appear to be characteristic (Agnello et al. 1971). Recent studies indicate that the binding protein is IgG and may therefore be an autoantibody directed to C1q (Wisnieski & Naff 1989). Thus it is an immune complex-mediated disorder in which activation of the classic complement pathway is seen with tissue deposition of immune complexes and inflammation. The urticaria, in particular, may therefore be a result of anaphylatoxin generation and cutaneous mast cell degranulation. This disorder is unique because the hives are often responsive to hydroxychloroquine (Plaquanil) at 200– 400 mg/day.

Laboratory findings The evaluation of patients with urticaria/angioedema (Table 90.1) begins with a comprehensive history, with particular emphasis on the recognized causes, and a physical examination. Some varieties of urticaria may be identified by their characteristic appearance, such as the small wheals with a large erythematous flare in cholinergic urticaria , the linear wheals in dermatographism, and the localization of lesions to exposed areas in light- or cold-induced urticaria. If suggested by the history, the physical examination in all patients with urticaria should include tests for physical urticaria, such as a brisk stroke to elicit dermatographism, the use of a weight to elicit delayed pressure urticaria, and application of a cold or warm stimulus for cold-induced urticaria and localized heat urticaria, respectively. Exercise, such as running in place, may elicit cholinergic urticaria and, in some instances, exerciseinduced anaphylaxis. Phototests to elicit solar urticaria are usually performed in referral centers, as are challenges for exercise-induced anaphylaxis. In most patients with chronic urticaria/angioedema, no underlying disorders or causes can be discerned. Diagnostic studies should be based on findings elicited by the history and physical examination. Evaluation of chronic urticaria/ angioedema should include thyroid function tests, assays for anti-microsomal and anti-thyroglobulin antibodies, and the autologous skin test can be done, even in an office setting (Sabroe et al. 1999b). Routine screening laboratory tests are of little value. The histamine release assay for anti-IgE receptor or anti-IgE antibodies remains a research tool. Serum

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Table 90.1 Testing procedures for urticaria and angioedema. Food and drug reactions

Elimination of offending agent, challenge with suspected foods, lamb and rice diet, special diets eliminating natural salicylates and food additives

Inhalant allergens

Skin tests, in vitro histamine release from human basophils, radioallergosorbent test

Collagen vascular diseases

Skin biopsy; CH50, C4, C3, factor B, immunofluorescence of tissue

Malignancy with angioedema

CH50, C1q, C4, C1 INH determinations

Cold urticaria

Ice cube test

Solar urticaria

Exposure to defined wavelengths of light, red cell protoporphyrin, fecal protoporphyrin and coproporphyrin

Dermatographism

Stroking with narrow object (e.g., tongue blade, fingernail)

Pressure urticaria

Application of pressure for defined time and intensity

Vibratory angioedema

Vibration with laboratory vortex for 4 min

Aquagenic urticaria

Challenge with tap water at various temperatures

Urticaria pigmentosa

Skin biopsy, test for dermatographism

Hereditary angioedema

C4, C2, C1 INH by protein and function

Familial cold urticaria

Challenge by cold exposure, measurement of temperature, white blood cell count, sedimentation rate, and skin biopsy

C3b inactivator deficiency

C3, factor B, C3b inactivator determinations

Idiopathic/autoimmune

Skin biopsy, immunofluorescence (negative), autologous skin test, anti-thyroglobulin and anti-microsomal antibodies

hypocomplementemia is not present in chronic idiopathic urticaria or chronic autoimmune urticaria and mean levels of serum IgE are no different from those of the general population where incidence of atopy is 20%. Cryoproteins should be sought in patients with acquired cold urticaria. An antinuclear antibody test should be obtained in patients with solar urticaria. Assessment of serum complement proteins may be helpful in identifying patients with urticarial venulitis or serum sickness (C4, C3, C1q binding assay for circulatory immune complexes), as well as those with hereditary and acquired forms of C1 INH deficiency (C4, C1 INH by protein and function, C1q level). Skin biopsy of chronic urticarial lesions should be undertaken to identify urticarial venulitis or to assess rashes where the urticarial nature is not clear. There is little role for routine skin-prick testing or the radioallergosorbent test (RAST) in the diagnosis of specific IgE-mediated antigen sensitivity in chronic urticaria/angioedema. Inhalant materials are uncommon causes of urticaria/ angioedema, and food skin tests may be difficult to interpret. The tests for drugs are limited to penicillin but cannot be performed in patients with dermatographism. The RAST should be reserved for those in whom skin testing is contraindicated, unavailable, or unrevealing despite a highly suspect history.

The release of histamine from peripheral basophilic leukocytes has supported the diagnosis of anaphylactic sensitivity to a variety of antigens, including pollens and insect venom.

Treatment The autologous skin test with serum can be made more reliable and a better mirror of the findings seen with basophil histamine release (A.P. Kaplan, unpublished observations) if 50 μL are used (rather than 20 μL), and readings are made at 5, 10, 20, and 30 min. When compared with the saline control there should be an increasing diameter of the wheal with time, there should be associated erythema, and 3 mm (rather than 1.5 mm) is taken as a significant difference at 30 min. Treatment of acute urticaria employs antihistamines and if symptoms are mild nonsedating agents such as fexofenodine, cetirizine, or desloratidine can be employed. However, if these are insufficient to control symptoms, hydroxyzine or diphenhydramine at 25–50 mg q.i.d. can be employed (Kaplan 2002). A course of corticosteroid can be added, particularly if there is also prominent angioedema, e.g., 60–80 mg/day for 3 days and then tapered by 5–10 mg/day. Epinephrine can relieve severe symptoms (generalized urticaria, severe pruritus,

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accelerating angioedema) and is indicated if laryngeal edema is present. Edema of the posterior tongue and/or pharyngeal edema can be confused with it, and are far less likely to cause asphyxiation. Nevertheless, when extreme they can be obstructing; the patient can choke on secretions and/or aspirate and epinephrine can be helpful here as well. The potential for laryngeal edema or severe tongue/pharyngeal edema of this sort is greatest with anaphylaxis, the use of ACE inhibitors, or the various types (hereditary/acquired) of C1 INH deficiency. Physical urticarias can be treated with antihistaminics (except delayed pressure urticaria which is typically refractory); nonsedating agents may be tried first, then the older first-generation agents if this is not effective. Most types are unresponsive to corticosteroids; again delayed pressure urticaria is the exception. Although many antihistamines should be theoretically effective for cold urticaria, cryproheptadine (Wanderer et al. 1977) appears to be particularly effective. Escalating doses up to 8 mg q.i.d. may be needed, employing 4-mg tablets. Familial cold urticaria (cold autoinflammatory syndrome) responds to IL-1 receptor antagonist (Anakina), indicating that the pathogenesis of this genetic disorder is very much dependent on IL-1. The drugs of choice for cholinergic urticaria are cetirizine 10–20 mg daily and then hydroxyzine 25–50 mg q.i.d. if cetirizine is ineffective

(Kaplan et al. 1975). No specific agents are recommended for local heat urticaria, dermatographism, solar urticaria or aquagenic urticaria. Sunscreens can effectively block UV light and prevent reactivity in patients with type I solar urticaria. Delayed pressure urticaria can require low-dose corticosteroid therapy as described below for chronic urticaria and cyclosporin A may also be effective and a good alternative. Leukotriene receptor antagonists or a leukotriene synthesis inhibitor can be tried, and in some cases may be steroid sparing. However, the data suggesting efficacy are anecdotal rather than properly controlled studies in large numbers of patients. An approach to therapy for chronic urticaria/angioedema is given in Fig. 90.11 and a literature review has been published (Kaplan 2002). Nonsedating antihistamines such as fexofenadine (180 mg daily), loratidine (10 mg daily), or cetirizine (10 mg daily) are first-line agents and can decrease the number of urticarial lesions and the frequence of eruptions and diminish pruritus. Mild cases of chronic urticaria can be successfully treated with these agents solely. For more severe symptoms, the dose can be doubled or even tripled with increasing effect, although issues of cost and/or insurance cover may be problematic. An example of such a combination would be fexofenadine 180 mg or loratidine 10 mg in the morning plus cetirizine 10 mg at midday and bedtime.

1. Non-sedating antihistaminics a. One agent b. Combinations at 2–3 times the usual dose Response

Inadequate response 1. Other antihistamines to maximal dose e.g. hydroxazine or diphenhydramine 25–50 mg q.i.d. 2. Addition of H2 antagonist ± leukotriene antagonist

Response Maintain at dose where urticaria is “mild” – need not be eliminated

Insufficient response Add

Presents with history of taking considerable corticosteroid or has hypertension, diabetes, osteoporosis, severe stria, morbid obesity Add

1. Low dose daily (10 mg prednisone or equivalent) steroid or alternate day steroid (20–25 mg q.o.d.) with slow taper employing 5 mg and 1 mg prednisone tablets 2. Cyclosporine as an alternative to #1

Cyclosporin – concomitant steroid is maximum of 15 mg/day – taper gradually with goal of eliminating it. Cyclosporin is ineffective or side effects prohibitive a. Low-dose steroids as noted above b. Weekly methotrexate c. Intravenous gamma globulin d. Plasmapheresis for autoimmune subgroup

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Good response with steroid

Insufficient response to steroids

a. Taper dose every 2–3 weeks as tolerable. Goal is mild urticaria; rare angioedema

Cyclosporin – once response is obtained, eliminate corticosteroid

* Agents expected to be effective rarely, if ever: hydroxychloraquine, cholchine, dapsone, sulfasalazine, mycofenolate mofitil. Hydroxychloroquine is, however, the drug of choice for the hypocomplementamic urticarial vasculitis syndrome. Urticarial vasculitis may respond to dapsone or cholchicine.

Fig. 90.11 Treatment of chronic idiopathic or autoimmune urticaria/angioedema.

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Nevertheless, severe chronic urticaria may not be responsive to such combinations, but further diminution of lesions and symptom relief can be obtained with hydroxyzine or diphenhydramine at 200 mg/day (25-mg or 50-mg tablets) divided into three or four doses. The dose is based on the severity of the urticaria and the patient’s tolerance to the somnolent effect of the drug. Most patients adapt to these effects within a week. When prescribing the drug, one should stress that it must be taken as prescribed and not just as needed. Daily administration minimizes or prevents outbreaks; the use of antihistamines after the onset of lesions occurs is too late. When considering such an approach it is important to note that it is the ratio of histamine to antihistamine at the cutaneous endothelial cell H1 receptor that determines the response and that if the histamine level exceeds that of the antihistamine, therapy will be ineffective. This occurs far more often in the skin than the nose or conjunctiva. Sedation is certainly of concern and studies of single-dose or brief courses of firstgeneration antihistaminics reveal impairment in performance even if the person claims not to be sedated. However, no study has ever been done on patients with chronic urticaria with hives, swelling, severe pruritis and sleep impairment due to symptoms. The benefits of antihistamines used in this fashion far outweigh the negatives if the next choice is addition of chronic steroid or agents such as cyclosporin A. Doxepin is favored by some because its ability to block H1 receptors is particularly high when tested in vitro and expressed on a molar basis and because it also possesses some H2 receptor antagonism (Goldsobel et al. 1986). However, it is particularly sedating and might best be used as a nighttime dose of 25–50 mg to supplement nonsedating antihistaminics. Nevertheless, patients with severe chronic urticaria may not respond satisfactorily to any of the regimens suggested above. Patients with IgG anti-IgE or IgG anti-IgE receptor antibodies appear to be more severe than those without (Sabroe et al. 1999c) and may therefore be more difficult to treat. If urticaria involves the face (particularly with facial angioedema) or when hives are florid and pruritus is severe, additional therapy may be required. Some patients obtain relief when an antihistamine that blocks histamine H2 receptors is added to one of the regimens. Cimetidine (Tagamet) 300–400 mg twice a day or ranitidine (Zantac) 150 mg twice a day as used in the treatment of ulcer disease may therefore be added (Kaplan 2002). The rationale is that the cutaneous vasculature possesses not only a large number of H1 receptors but also a small number of H2 receptors, estimated to be about 15% based on inhibition of intracutaneous histamine (Harvey & Schocket 1980). Once H1 receptors have been blocked, the addition of an antihistamine that binds to H2 receptors augments the antihistamine effect. Use of an H2receptor antagonist alone has no effect on urticaria; only with significant H1-receptor blockade is any effect observable. My policy is to discontinue H2 blockers within 3 weeks unless their use has clearly improved the urticaria. The use

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of leukotriene antagonists in this setting is uncertain, with equal numbers of articles for and against. The toxicity of these agents is very low and may be worth trying. If antihistamines alone are insufficient to control chronic urticaria, the addition of a corticosteroid is indicated (Kaplan 2002); cyclosporin A is a good alternative. It is necessary to determine the lowest possible effective dosage. I usually start with prednisone, giving 40 mg in one morning dose for the first 3 days and then decreasing the dosage by 5 mg/day. When the dosage is down to 20 mg/day, it is decreased by 5 mg on alternate days. Thus the daily dosage becomes 20, 15, 20, 10, 20, 5 mg and so on until a dosage of 20 mg every second day is achieved. This alternate-day regimen is effective in the majority of patients. The steroid should then be slowly withdrawn, decreasing the dosage 2.5–5.0 mg every 2–3 weeks. The decision to keep decreasing the dosage depends on the achievement of considerable improvement with the 20-mg alternate-day dosage and no obvious worsening of symptoms as the dosage is decreased. Optimally, close to 3 months would be needed to discontinue the steroid. Antihistamines are continued with the corticosteroid and should probably not be tapered until the patient no longer requires the steroid. Sometimes the duration of steroid therapy must be extended because the drug cannot be tapered below a certain dosage. This is maintained for a while (usually 1–2 months), and then the physician should try again to taper the dosage. A common difficulty is achievement of good control of hives during the steroid “on” day and prominent exacerbation during the “off” day. I approach this situation by giving the prednisone in two doses (i.e., 15 mg in the morning and 5 mg in the evening every second day). Once the urticaria is under reasonable control, the evening dose is gradually decreased and the increment added to the morning dose until the evening dose has been discontinued. At this point, steroids are tapered as previously indicated. An alternative approach is to use daily steroid, although we do not exceed 15 mg/day and taper by 1–2.5 mg every 7–14 days; 1-mg prednisone tablets and half a 5-mg tablet can be used for this purpose. Rarely there is no response to prednisone regardless of the dosage used. In this case an equivalent dosage of methylprednisolone (Medrol) can be tried. Occasionally, a patient may be unable to metabolize prednisone to prednisolone; in such a patient a low dose of methylprednisolone is often effective, whereas a high dosage of prednisone is not. Finally, a small number of patients have severe urticaria that does not respond to corticosteroids within the range suggested. Some of these patients have cutaneous vasculitis with urticarial lesions, but this is not true of most patients. At present, the histologic pattern seen from skin biopsy specimens does not correlate with the severity of urticaria, nor can it be used as a guide to therapy. In view of data indicating that chronic urticaria may result from a circulating autoantibody (Hide et al. 1994), plasmapheresis, intravenous gammaglobulin, or other specific antiinflammatory regimens such as

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methotrexate or cyclosporin A (Fradin et al. 1991; Toubi et al. 1997) can be considered next. The most promising of these is cyclosporin A, which is often a steroid-sparing agent at an adult dose of 200–300 mg/day (Grattan et al. 2000). Two double-blind placebo-controlled studies have shown efficacy of close to 75% in those with chronic autoimmune urticaria, and in my experience 50% of patients with chronic idiopathic urticaria will also respond. Blood pressure, blood urea nitrogen, creatinine, and urinalysis should be followed every 6 weeks since renal toxicity and hypertension are the major possible side effects. The response to 10–15 mg methotrexate taken weekly is significantly less, perhaps 20–30%. Use of hydroxychloroquine, colchicine, or dapsone should be limited to urticarial vasculitis; they rarely have efficacy for chronic urticaria and angioedema. However, hydroxychloroquine is the drug of choice for the hypocomplementemic urticarial vasculitis syndrome. Although controversial, I do not support the use of thyroid hormone as therapy for the urticaria when anti-thyroid antibodies are present. I have not observed any significant effect and most studies have insufficient numbers of patients and are not properly controlled. Therapy of idiopathic angioedema is usually attempted with high dosages of antihistamines such as diphenhydramine and corticosteroids, as outlined for chronic urticaria if the frequency and severity of attacks warrant it. Infrequent episodes can be treated with 2–3 days of high-dose steroid (plus diphenhydramine) without any taper. The nonhistaminergic group may respond to tranexamic acid as reported in studies in Europe where it is available (Cicardi et al. 1999); ε-aminocaproic acid may be tried as an alternative.

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Leznoff, A., Josse, R., Denburg, J. et al. (1983) Association of chronic urticaria and angioedema with thyroid autoim. Arch Dermatol 119, 636– 40. Lim, H., Perez, H., Poh-Fitzpatrick, M. et al. (1981) Generation of chemotactic activity in serum from patients with erythropoietic protoporphyria and porphyria cutanea tarda. N Engl J Med 304, 212–16. Lim, H., Poh-Fitzpatrick, M. & Gigli, I. (1984) Activation of the complement system in patients with porphyrias after irradiation in vivo. J Clin Invest 74, 1961–5. Lipsker, D., Veran, Y., Grunenberger, F. et al. (2001) The Schnitzler syndrome. Four new cases and review of the literature. Medicine 80, 37– 44. Liutu, M., Kalimo, K., Uksila, J. et al. (1998) Etiologic aspects of chronic urticaria. Int J Dermatol 37, 515–19. Luong, K. & Nguyen, L. (1998) Aquagenic urticaria: report of a case and review of the literature. Ann Allergy Asthma Immunol 80, 483–5. Luquin, E., Kaplan, A. & Ferrer, M. (2005) Increased responsiveness of basophils of patients with chronic urticaria to sera but hyporesponsiveness to other stimuli. Clin Exp Allergy 35, 456–60. McDuffie, F., Sams, W.J., Maldonado, J. et al. (1973) Hypocomplementemia with cutaneous vasculitis and arthritis. Possible immune complex syndrome. Mayo Clin Proc 48, 340–8. Malbran, A., Hammer, C., Frank, M. et al. (1988) Acquired angioedema: observations on the mechanism of action of autoantibodies directed against C1 esterase inhibitor. J Allergy Clin Immunol 81, 1199–204. Mandle, R.J. & Kaplan, A. (1977) Hageman factor substrates. Human plasma prekallikrein: mechanism of activation by Hageman factor and participation in Hageman factor-dependent fibrinolysis. J Biol Chem 252, 6097–104. Mandle, R., Colman, R. & Kaplan, A. (1976) Identification of prekallikrein and high-molecular-weight kininogen as a complex in human pl. Proc Natl Acad Sci USA 73, 4179–83. Marceau, F. (1995) Kinin B1 receptors: a review. Immunopharmacology 30, 1–26. Marder, R.J., Rent, R., Choi, E. et al. (1976) C1q deficiency associated with urticarial-like lesions and cutaneous vasculitis. Am J Med 61, 560–5. Margolius, H. (1998) Tissue kallikreins structure, regulation, and participation in mammalian physiology and disease. Clin Rev Allergy Immunol 16, 337– 49. Mastalerz, L., Setkowicz, M., Sanak, M. et al. (2004) Hypersensitivity to aspirin: common eicosanoid alterations in urticaria and asthma. J Allergy Clin Immunol 113, 771–5. Maulitz, R., Pratt, D. & Schocket, A. (1979) Exercise-induced anaphylactic reaction to shellfish. J Allergy Clin Immunol 63, 433–4. Millikan, L. (2004) Papular urticaria. In: Greaves, M. & Kaplan, A., eds. Urticaria and Angioedema. Marcel Dekker, Basel, pp. 251–8. Mortureux, P., Leaute-Labreze, C., Legrain-Lifermann, V. et al. (1998) Acute urticaria in infancy and early childhood: a prospective study. Arch Dermatol 134, 319–23. Muller-Esterl, W., Rauth, G., Lottspeich, F. et al. (1985) Limited proteolysis of human low-molecular-mass kininogen by tissue kallikrein. Isolation and characterization of the heavy and the light chains. Eur J Biochem 149, 15–22. Natbony, S., Phillips, M., Elias, J. et al. (1983) Histologic studies of chronic idiopathic urticaria. J Allergy Clin Immunol 71, 177–83.

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Nettis, E., Dambra, P., Loria, M. et al. (2001) Mast-cell phenotype in urticaria. Allergy 56, 915. Niimi, N., Francis, D., Kermani, F. et al. (1996) Dermal mast cell activation by autoantibodies against the high affinity IgE receptor in chronic urticaria. J Invest Dermatol 106, 1001–6. Novey, H., Fairshter, R., Salness, K. et al. (1983) Postprandial exerciseinduced anaphylaxis. J Allergy Clin Immunol 71, 498–504. Nussberger, J., Cugno, M., Amstutz, C. et al. (1998) Plasma bradykinin in angioedema. Lancet 351, 1693–7. Nussberger, J., Cugno, M., Cicardi, M. et al. (1999) Local bradykinin generation in hereditary angioedema. J Allergy Clin Immunol 104, 1321–2. Onn, A., Levo, Y. & Kivity, S. (1996) Familial cholinergic urticaria. J Allergy Clin Immunol 98, 847–9. Ormerod, A., Kobza Black, A., Dawes, J. et al. (1988a) Prostaglandin D2 and histamine release in cold urticaria unaccompanied by evidence of platelet activation. J Allergy Clin Immunol 82, 586–9. Ormerod, A., Kobza-Black, A., Milford-Ward, A. et al. (1988b) Combined cold urticaria and cholinergic urticaria: clinical characterization and laboratory findings. Br J Dermatol 118, 621–7. Pachlopnik, J., Horn, M., Fux, M. et al. (2004) Natural antiFcepsilonRIalpha autoantibodies may interfere with diagnostic tests for autoimmune urticaria. J Autoimmun 22, 43–51. Peters, M., Schroeter, A., Kephart, G. et al. (1983) Localization of eosinophil granule major basic protein in chronic urticaria. J Invest Dermatol 81, 39–43. Petersen, L.J., Church, M.K. & Skov, P.S. (1997) Histamine is released in the wheal but not the flare following challenge of human skin in vivo: a microdialysis study. Clin Exp Allergy 27, 284–95. Poley, G.J. & Slater, J. (2000) Latex allergy. J Allergy Clin Immunol 105, 1054–62. Provost, T., Zone, J., Synkowski, D. et al. (1980) Unusual cutaneous manifestations of systemic lupus erythematosus: I. Urticaria-like lesions. Correlation with clinical and serological abnormalities. J Invest Dermatol 75, 495–9. Puccetti, A., Bason, C., Simeoni, S. et al. (2005) In chronic idiopathic urticaria autoantibodies against Fc epsilonRII/CD23 induce histamine release via eosinophil activation. Clin Exp Allergy 35, 1599–607. Quastel, M., Harrison, R., Cicardi, M. et al. (1983) Behavior in vivo of normal and dysfunctional C1 inhibitor in normal subjects and patients with hereditary angioneurotic edema. J Clin Invest 71, 1041–6. Regoli, D. & Barabe, J. (1980) Pharmacology of bradykinin and related kinins. Pharmacol Rev 32, 1–46. Sabroe, R. & Black, A. (1997) Angiotensin-converting enzyme (ACE) inhibitors and angio-oedema. Br J Dermatol 136, 153–8. Sabroe, R., Francis, D., Barr, R. et al. (1998) Anti-Fc(episilon)RI auto antibodies and basophil histamine releasability in chronic idiopathic urticaria. J Allergy Clin Immunol 102, 651–8. Sabroe, R., Poon, E., Orchard, G. et al. (1999a) Cutaneous inflammatory cell infiltrate in chronic idiopathic urticaria: comparison of patients with and without anti-FcepsilonRI or anti-IgE autoantibodies. J Allergy Clin Immunol 103, 484–93. Sabroe, R., Grattan, C., Francis, D. et al. (1999b) The autologous serum skin test: a screening test for autoantibodies in chronic idiopathic urticaria. Br J Dermatol 140, 446–52. Sabroe, R., Seed, P., Francis, D. et al. (1999c) Chronic idiopathic urticaria: comparison of the clinical features of patients with and without anti-FcepsilonRI or anti-IgE autoantibodies. J Am Acad Dermatol 40, 443–50.

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Wisnieski, J. & Naff, G. (1989) Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome. Arth Rheum 32, 1119–27. Yang, H. & Erdos, E. (1967) Second kininase in human blood plasma. Nature 215, 1402–3. Ying, S., Robinson, D., Meng, Q. et al. (1999) C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES). J Immunol 163, 3976–84. Zahedi, R., Bissler, J., Davis, A. et al. (1995) Unique C1 inhibitor dysfunction in a kindred without angioedema. II. Identification of an Ala443→Val substitution and functional analysis of the recombinant mutant protein. J Clin Invest 95, 1299–305. Zuraw, B. & Curd, J. (1986) Demonstration of modified inactive first component of complement (C1) inhibitor in the plasmas of C1 inhibitor-deficient patients. J Clin Invest 78, 567–75. Zweiman, B., Valenzano, M., Atkins, P. et al. (1996) Characteristics of histamine-releasing activity in the sera of patients with chronic idiopathic urticaria. J Allergy Clin Immunol 98, 89–98. Zweiman, B., Haralabatos, I., Pham, N. et al. (2000) Sequential patterns of inflammatory events during developing and expressed skin late-phase reactions. J Allergy Clin Immunol 105, 776–81.

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Mastocytosis Nataliya M. Kushnir-Sukhov, Dean D. Metcalfe and Jamie A. Robyn

Summary

Pathology

Mastocytosis is characterized by the abnormal growth and accumulation of mast cells in one or more organ systems and may vary significantly in clinical presentation and severity. The disease is unusual, is known to affect both males and females in all age groups, and in most cases is not inherited. While knowledge of mast cell growth and differentiation has advanced tremendously since this cell was discovered by Ehrlich in 1878, the explanation for the basis of mastocytosis in all its variants is only partially understood. In most instances, however, systemic mastocytosis is now considered a clonal disorder of the hematopoietic system, and in the majority of cases follows a benign course. The diagnosis of systemic mastocytosis is challenging, especially in the absence of cutaneous involvement. Manifestations of the disease are provoked in part by the resultant increase in mast cell-derived mediators, which leads to a variety of local and systemic effects. Most patients with mastocytosis have indolent disease and are managed symptomatically using treatments directed toward mast cell mediator-related symptoms. Such patients have a normal lifespan with minimal limitations. Cases of aggressive mastocytosis, on the other hand, may lead to disability or even death. Cases of mast cell disease diagnosed in childhood often resolve by adulthood, while adult-onset mastocytosis usually persists. Mastocytosis is variable in respect to the organ systems involved, clinical presentation, symptoms, and association with other hematologic diseases. This has suggested the need for an improved classification scheme to allow assessment of prognosis and therapy. The heterogeneity of the disease patterns in mastocytosis strongly suggests that more than one biological lesion may occur in the developmental sequence that leads to placement of mast cells in tissues. The diagnosis is now aided by new surrogate markers. Therapy of mastocytosis is mainly symptomatic and palliative. At the molecular level, recent studies have reinforced the role of activating mutations in Kit in the etiology of mastocytosis. These findings provide a conceptual basis for the development of new therapeutic strategies.

Mast cell hyperplasia and mediator release contribute to the clinical signs and symptoms of mastocytosis (Fig. 91.1). Mediators include preformed substances stored in mast cell granules, as well as newly formed mediators produced following mast cell activation. Cytokine, protease, bioamine and prostaglandin overproduction result in both acute and chronic findings, including flushing, pruritus, nausea, vomiting, diarrhea, abdominal pain, headache, heart palpitations, dyspnea, and syncope. Acute symptoms are mainly attributed to histamine and arachidonic acid metabolites

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Headaches, Cognitive dysfunction, Depression

Urticaria pigmentosa

Flushing, Pruritus

Osteoporosis, Fractures

Hypotension, Vascular instability, Increased vascular permeability GERD, Gastric hypersecretion, Peptic ulcers Hepatomegaly, Splenomegaly, Ascites Abdominal discomfort, Diarrhea Muscle spasms, Fatigue

Mast cell hyperplasia, Fibrosis, Eosinophilia

Fig. 91.1 Signs and symptoms of mastocytosis. GERD, gastroesophageal disease. (See CD-ROM for color version.)

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(Roberts et al. 1982). Hepatic and splenic involvement, including fibrosis, has been reported to occur in patients with all variants of systemic mastocytosis. Tissue eosinophilia may be present.

Mast cell biology Mast cells arise from CD34+ pluripotent cells in the bone marrow. Committed mast cell precursors exit the bone marrow into the blood, migrate to the peripheral tissues and mature into mast cells (Kirshenbaum et al. 1991). Under normal conditions, mast cells are found throughout vascularized tissues and are especially abundant in proximity to the epithelial surfaces of the skin, respiratory tract, gastrointestinal tract, and genitourinary tract. Terminal differentiation of mast cells occurs in vascular tissues under the influence of stem cell factor (SCF), in addition to cytokines including inter-

Mastocytosis

leukin (IL)-6, IL-9, and nerve growth factor (NGF). The one obligatory growth factor for human mast cells is SCF and the development of mast cells from CD34+ progenitors is dependent on SCF produced principally by stromal cells. SCF is the ligand for Kit (Fig. 91.2). Thrombopoietin alone or in combination with SCF has been shown to support growth of CD117low/CD110+ mast cells (Kirshenbaum et al. 2005), while granulocyte–macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ both inhibit development of mast cells. Mast cells synthesize and release mediators necessary to maintain tissue homeostasis and integrity (Artuc et al. 1999). When malignant transformation occurs, clonal expansion of mast cells leads to an increase in the mast cell burden within tissues and, in severe cases, may lead to the displacement of other cell types. Currently, it is not clear whether tissue mast cell hyperplasia is due to an influx of progenitors that subsequently mature or is due to local proliferation of progenitors

SCF dimer

Extracellular domain

Transmembrane domain

V560

Juxtamembrane domain

G560

P

P

P

P

Grb2 SOS

SHIP

RAS 1st catalytic domain GTP Tyrosine kinase domain

D816

ATP

P

GDP

Autophosphorylation

ATP

2nd catalytic domain

V816

P

P

P PI3K

P

Tail

MAPKs

P

JAKs (a)

(b)

STATs Growth, differentiation, migration

Fig. 91.2 Structure of human Kit. (a) Structural portions of Kit affected by mutations. (b) Binding of stem cell factor (Kit substrate) leads to Kit dimerization and activation of signaling pathways. (See CD-ROM for color version.)

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and mature mast cells (Bischoff et al. 1999; Kambe et al. 2001). Mast cells are commonly known as effector cells in allergic reactions. Cross-linking of IgE bound to high-affinity IgE receptors (FcεRI) with antigen causes release and generation of bioactive mediators, leading to allergic reactions including systemic anaphylaxis in sensitized patients. Note, however, that IgE-mediated allergy is not increased in patients with mastocytosis (Muller et al. 1990). Other factors are also known to trigger mast cell degranulation, such as temperature, mechanical stimuli, and the anaphylatoxins C3a and C5a.

Epidemiology The prevalence of mastocytosis is not known. Two-thirds of all cases present in childhood with a second peak of onset in the late third to early fourth decade. The most cited published report estimates that 1 in 1000 to 1 in 8000 of new dermatologic patients have some form of mastocytosis (Golkar & Bernhard 1997). Although there are more than 50 cases of familial cutaneous mastocytosis reported, most patients report no family history of mastocytosis. One pedigree analysis in a rare instance of familial mastocytosis reported a probable autosomal dominant inheritance with incomplete penetrance (Chang et al. 2001). The male to female ratio is approximately

1 : 1. In one recent report of 180 pediatric patients, a 1.5 : 1 ratio was reported (Ben-Amitai et al. 2005).

Classification The classification of variants of mastocytosis has been updated periodically at consensus conferences as knowledge of the disease evolves. The most recent classification system for mastocytosis is shown in Table 91.1 (Valent et al. 2001). Mast cell hyperplasia confined to the skin is termed cutaneous mastocytosis (CM). There are three subvariants: maculopapular CM (also called urticaria pigmentosa, UP), diffuse cutaneous mastocytosis, and solitary mastocytoma of skin. Systemic mastocytosis (SM) is characterized by variable multiorgan involvement, including the bone marrow, spleen, liver, lymph nodes, and gastrointestinal tract. The most frequent form of SM is indolent systemic mastocytosis (ISM) which tends to follow a benign course. The term “smoldering mastocytosis” has now been introduced to define patients with extensive systemic disease but no evidence of aggressive mastocytosis or associated nonmast cell clonal disease (Akin et al. 2001; Valent et al. 2002). SM with associated hematologic nonmast-cell lineage disease, aggressive systemic mastocytosis, and mast cell leukemia have a more guarded prognosis.

Table 91.1 WHO classification of mastocytosis. (From Valent et al. 2001, with permission.) Variant (term)

Abbreviation

Subvariants

Cutaneous mastocytosis

CM

Urticaria pigmentosa (UP): same as maculopapular CM (MPCM) Diffuse CM (DCM) Mastocytoma of skin

Indolent systemic mastocytosis

ISM

Smoldering SM Isolated bone marrow mastocytosis

Systemic mastocytosis with an associated clonal hematologic nonmast-cell lineage disease

SM-AHNMD

SM-AML SM-MDS SM-MPD SM-HES SM-CMML SM-NHL

Aggressive systemic mastocytosis

ASM

Lymphadenopathic SM with eosinophilia

Mast cell leukemia

MCL

Aleukemic MCL

Mast cell sarcoma Extracutaneous mastocytoma AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; HES, hypereosinophilic syndrome; MDS, myelodysplastic syndrome; MPD, myeloproliferative disease; NHL, non-Hodgkin lymphoma.

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Mastocytosis

Skin involvement

Clinical symptoms Clinical symptoms follow patterns of organ system involvement, including the skin, gastrointestinal tract, lymph nodes, liver, spleen, and bone marrow (Fig. 91.1). The respiratory, reproductive, and endocrine systems are not usually involved. Patients with cutaneous and systemic disease may experience flushing and/or life-threatening episodic hypotension. Hypotension may occur spontaneously without an obvious associated trigger or on a patient-specific basis may be related to ingestion of alcohol, insect stings, infection, certain medications, and contrast materials. An increased incidence of bacterial, fungal, and viral infections has not been reported.

Among patients with ISM approximately 80% show CM. Thus, some patients with mastocytosis first present with an unexplained skin “rash.” CM is often first reported in the axilla, later spreading to the trunk and extremities. CM usually spares the palms, soles, and face. Episodic flushing and pruritis are common. UP is the most frequent form of skin lesion (Figs 91.3–91.5). It is composed of characteristic fixed hyperpigmented macules or papules that urticate upon physical irritation. Darier sign, a reaction of local whealing, may be provoked by scratching or rubbing of the rash, and is associated with a lesion (Fig. 91.3b). CM is often associated with pruritus that may be exacerbated by changes in temperature, local mechanical irritation, ingestion of hot beverages or

(a)

(b)

(c)

(d) Fig. 91.3 (a) 9-year-old girl with urticaria pigmentosa (UP) demonstrating skin erythema on accidental rubbing (arrows). (b) Urtication and flare can be provoked by mechanical irritation of the UP lesion (arrow) and is characteristic of the Darier sign. (c) Blistering lesion in 8-month-old girl diagnosed with UP shortly after birth (arrow). (d) Mastocytoma lesion on the plantar surface of foot of 3-year-old boy (arrow). (See CD-ROM for color version.)

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(a)

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(b)

Fig. 91.4 (a) 4.5-year-old boy with cutaneous mastocytosis. Note irregular-shaped brown lesions of various size. (b) 9-month-old girl with crusting of the aseptic blister. (See CD-ROM for color version.)

(a)

(b)

(c)

(d) Fig. 91.5 (a–d) 56-year-old man with systemic disease. Skin lesions consist of small multiple brown lesions primarily distributed on the trunk and extremities, almost confluent in the areas of mechanical irritation. (See CD-ROM for color version.)

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spicy foods, ethanol, and certain drugs. CM may be present at birth, but more often presents within the first 6 months of life. The pigmented lesions associated with CM in childhood are generally more variable in size compared to those found in adults (Fig. 91.4). Blisters and bullae may form in children (Figs 91.3c and 91.4b) (Hartmann & Metcalfe 2000). Telangiectasia macularis eruptiva perstans (TMEP) is a rare form of CM and is described as occurring mainly in adults. TMEP lesions appear as generalized red telangiectatic macules with a background color from tan to brown. Individual lesions are approximately 2– 6 mm in diameter without sharply demarcated borders. Sites usually do not become edematous if rubbed. TMEP should not be confused with UP with associated telangiectasia.

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the medullary cords (13%), and the sinuses (6%). Mast cells were frequently found in a perivascular location, and associated eosinophilia was common.

Musculoskeletal symptoms Musculoskeletal pain is frequently reported. Bone pathology in patients with mastocytosis ranges from osteoporosis to osteosclerosis (Johansson et al. 1996). Pelvic bones, skull, ribs, hands, spine and femur are frequently affected. Bone scans may demonstrate multifocal abnormalities and in some cases show a diffuse increase in uptake (Chen et al. 1994). Vertebral compression fractures may occasionally be the first presentation of mastocytosis. It has been reported that patients with less severe disease and lower serum tryptase levels are more likely to show osteopenia (Kushnir-Sukhov et al. 2006).

Gastrointestinal symptoms Abdominal pain is the most common gastrointestinal symptom, followed by diarrhea, nausea, and vomiting (Cherner et al. 1988). The pathogenesis of abdominal symptoms appears multifactorial. Various reports have attributed abdominal complaints to peptic ulcer disease, edema, urticarial lesions of the gastrointestinal tract, or to a hypermotility or motility disorder. Approximately 70% of patients with dyspeptic types of abdominal pain have evidence of gastric acid hypersecretion. Some evidence of malabsorption is found in up to one-third of patients with SM due to diffuse small intestinal mucosal dysfunction. Malabsorption is usually not severe and is manifested primarily as mild steatorrhea with impaired absorption of D-xylose or vitamin B12. Hepatomegaly is common in patients with longstanding SM, and may be accompanied by normal or mildly elevated levels of liver enzymes. However, aggressive mastocytosis may lead to fibrosis, cirrhosis, ascites, and portal hypertension (Metcalfe 1991). Similarly, splenomegaly is reported in systemic disease and is more pronounced in aggressive forms of mastocytosis.

Hematologic and lymphoid involvement Cytopenias including anemia, thrombocytopenia, and neutropenia are common in aggressive forms of SM. Neutrophilia, monocytosis, and eosinophilia may also be seen (Horny et al. 1990). In a prospective study of 46 patients with systemic disease, univariate analysis identified hematologic variables such as thrombocytopenia, elevated lactate dehydrogenase level, anemia, qualitative peripheral blood smear abnormalities, and elevated alkaline phosphatase level as factors increasing the risk of death (Lawrence et al. 1991). Central and peripheral lymphadenopathy may occur in aggressive forms, or during longstanding ISM. Mast cell infiltration can be focal or diffuse with generally preserved architecture of the lymph nodes. In one study, 23 lymph node specimens from 19 patients with SM were reviewed (Travis & Li 1988). Mast cell infiltrates affected the paracortex (88%), the parafollicular region (50%), the follicles (25%),

Cardiovascular and respiratory problems Hypotensive episodes with or without syncope may be the sole clinical presentation of patients with systemic disease. The severity and frequency of such events differ significantly from patient to patient, occurring as often as daily or as infrequently as once a year. The episodes may be preceded by “auras” or lightheadedness. Severe cases are considered a medical emergency and fatalities have been reported. Patients with mastocytosis do not have an increase in asthma or other chronic lung diseases compared with the normal population (Muller et al. 1990). Several agents and situations have been reported that may provoke systemic mediator-related symptoms including hypotension and shock (resembling anaphylaxis) in some patients with mastocytosis. There is no evidence that all patients must avoid these agents and situations. • Drugs/drug classes: aspirin and other nonsteroidal antiinflammatory drug (NSAIDs), opiates, morphine, morphine derivatives, contrast media. • Inhalants or foods in patients allergic to these substances. • Surgery, endoscopy. • Infection (bacteria, viruses). • Insect stings. • Alcohol. • Physical stimuli: friction, heat, exercise. If use of any of the above listed medications/procedures is required or an adverse event encountered, it is best to consult a specialist.

Generalized complaints Fatigue, weight loss, fever, and sweats have been reported by patients with longstanding forms of mastocytosis, but may also be the presenting symptoms for aggressive disease or SM with an associated hematologic disorder. Fatigue, usually mild, is the most frequent symptom. Fever and sweats are less frequent. Headache is a frequent complaint. Chronic symptoms variably reported include decreased attention span, forgetfulness, irritability and depression. Poor

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motivation, confusion, anxiety, lethargy, and sleepiness have also been noted (Rogers et al. 1986). EEG abnormalities are nonspecific in such patients.

Diagnosis and diagnostic criteria for systemic mastocytosis Skin biopsy Suspected lesions with characteristic Darier sign suggestive of CM (see section on skin involvement above; Fig. 91.3b) and/or macroscopically resembling a form of CM should be biopsied. A positive skin biopsy usually demonstrates multifocal or diffuse aggregates of mast cells in the papillary dermis extending into the reticular dermis (Lewis et al. 1995). Perivascular areas are frequently involved. Typically, mast cells are increased tenfold or more in lesions of UP compared with normal skin. Note that a mild increase in mast cell number

does not confirm a diagnosis of UP (Garriga et al. 1988). The recommended method of evaluation of mast cells in tissues is immunohistochemical staining with tryptase (Horny et al. 1997). This method is more sensitive and reliable than metachromatic stains such as Giemsa or toluidine blue. It is important to determine whether a patient presenting with cutaneous lesions also has systemic disease. CM in the absence of systemic disease is common in patients with early pediatric-onset disease. In contrast, patients who experience onset of lesions after age 2 years are less likely to present with skin-restricted disease. The work-up for suspected SM should include measurement of total serum tryptase and a bone marrow aspirate and biopsy as described below.

Bone marrow aspirate and biopsy Examination of the bone marrow biopsy and aspirate in patients with suspected mastocytosis provides valuable information for diagnosis and for evaluating extent of disease (Fig. 91.6).

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(d) Fig. 91.6 Bone marrow biopsy in systemic mastocytosis. (a) Multifocal dense infiltrate of mast cells in the bone marrow (between arrows) (hematoxylin and eosin, ×20). (b) Closer view of mast cells (between arrows) (hematoxylin and eosin, ×40). (c) Positive tryptase stain of mast cell infiltrate (×20). (d) Abnormal spindle-shaped mast cell positively stained for tryptase (arrow). (See CD-ROM for color version.)

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Examination of the aspirate also allows evaluation for dysplasia or a nonmast-cell lineage hematologic disorder. Ideally, the marrow examination should include screening of the bone marrow aspirate for c-kit codon 816 mutations using restriction fragment length polymorphism (RFLP) analysis or a technique of similar sensitivity. If this screen is negative in a bone marrow sample from a patient who meets criteria for SM, sequence analysis to test for other c-kit mutations may be required. A sequence analysis may yield falsenegative results because of the level of sensitivity. However, a false-negative result using RFLP is unusual unless mast cell infiltrates in the bone marrow are very small or in situations where mast cells are outnumbered by leukemic (D816Vnegative) cells (Fritsche-Polanz et al. 2001). In such cases, the mutation may only be detected with enrichment of mast cells (sorted or microdissected). Peripheral blood in patients with coexisting eosinophilia should be examined for the presence of the FIP1L1–PDGFRA fusion gene (Cools et al. 2004). It may be appropriate to measure the numbers of colony-forming progenitor cells in bone marrow samples of patients with suspected associated hematologic disorders (Valent et al. 2005). While cytogenetic studies show a normal karyotype in the majority of patients with SM, a number of translocations have been described in patients who have a myeloid disorder in addition to SM (Valent et al. 2005). In the pediatric population, considering the less frequent occurrence of systemic disease, a bone marrow biopsy is generally reserved for patients with late onset of skin lesions (i.e., after 2 years of age) and those with hepatomegaly, splenomegaly, unexplained lymphadenopathy, abnormalities in complete blood counts, or baseline serum total tryptase levels above 20 ng/mL (Valent et al. 2001).

Mastocytosis

Diagnostic criteria World Health Organization (WHO) criteria for the classification of mastocytosis variants are shown in Table 91.1, and criteria to establish the diagnosis of SM in Table 91.2. In order to satisfy the diagnosis of SM, a patient must meet the major criterion along with at least one minor criterion or, in the absence of the major criterion, at least three minor criteria.

Major criterion The major criterion for diagnosis of SM is a finding of multifocal dense infiltrates of mast cells in bone marrow or other extracutaneous tissues, the most specific pathologic feature of mastocytosis. Mast cells should be observed in aggregates of 15 or more, and confirmed by tryptase immunohistochemistry or metachromatic stains such as Giemsa or toluidine blue. In bone marrow, mast cell aggregates can be observed in paratrabecular and perivascular locations and may be associated with a benign lymphoid aggregate consisting of B and T cells (Sperr et al. 2001) which have been shown to bear the Kit-activating mutation D816V (Taylor et al. 2004). Eosinophils are often present in variable numbers in these lesions. The decalcification process necessary for sectioning of the paraffin-embedded bone marrow tissue often interferes with metachromatic staining of the mast cells. Immunohistochemical staining for mast cell tryptase is thus more reliable and has largely replaced metachromatic staining for the diagnosis of mast cell disease. If the disease is at an early stage, or if the bone marrow biopsy sample is inadequate, the major criterion may be absent. In this case, three of the four minor criteria must be fulfilled for diagnosis.

Table 91.2 Criteria for the diagnosis of systemic mastocytosis (SM). Major Multifocal dense infiltrates of mast cells in the bone marrow or in other extracutaneous organ(s) (> 15 mast cells per aggregate) detected by tryptase immunohistochemistry Minor Mast cells show abnormal morphology: atypical mast cells (> 25%) in bone marrow smears, or are spindle-shaped cells (> 25%) in compact infiltrates in extracutaneous organ(s) Kit mutation* at codon 816 in extracutaneous organ(s) (bone marrow is the recommended tissue for screening) Kit-positive mast cells in the bone marrow or in another extracutaneous organ express CD2 and/or CD25 (multicolor flow cytometry is the recommended technique for testing; for CD25, immunohistochemistry is also acceptable provided that the cells are clearly identified as Kit+ mast cells) Serum total tryptase > 20 ng/mL (does not count in patients who have AHNMD-type disease in addition to SM) * Other activating mutations at codon 816 also meet this minor criterion (Valent et al. 2003). AHNMD, associated clonal hematologic nonmast-cell lineage disease.

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Minor criteria Atypical mast cell morphology Mast cells in bone marrow from patients with SM may exhibit a number of phenotypic abnormalities: spindle shape, cytoplasmic projections, hypogranulation, multilobular nucleus, or an eccentric nucleus. Mast cells are often found in paratrabecular locations in bone marrow specimens from mastocytosis patients (Fig. 91.6a). In order to fulfill this minor criterion, at least 25% of all mast cells in the biopsy sections or aspirate smears must have aberrant morphology (Fig. 91.6b–d). An aggressive variant of the disease (such as mast cell leukemia) must be considered if the percentage of mast cells exceeds 20% of all nucleated cells in the bone marrow aspirate. Mast cell leukemia is a rare subentity of SM where SM criteria are fulfilled and there is a diffuse leukemic infiltration of hematopoietic tissues by immature neoplastic mast cells.

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Serum tryptase measurement Tryptase is a well-established and important disease-related marker, which should be determined in patients with suspected mastocytosis. In healthy individuals, total serum tryptase levels are generally reported to range between 1 and 15 ng/mL (Horny & Valent 2001; Akin & Metcalfe 2002). In patients with CM without systemic involvement, total serum tryptase levels are normal to slightly elevated. The same is true for most cases with “isolated” bone marrow mastocytosis without multiorgan involvement, where tryptase is believed to correlate with the burden of neoplastic mast cells (Sperr et al. 2002a). Higher tryptase values increase the likelihood of multiorgan involvement. Thus, in patients with SM, total serum tryptase levels usually exceed 20 ng/mL. Notably,

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Immunophenotype of mast cells in mastocytosis Multicolor flow cytometric analysis of the bone marrow aspirate is used to demonstrate the aberrant immunophenotype of mast cells in mastocytosis (Fig. 91.7). First, all mast cells express a characteristic cell-surface antigen phenotype. Mast cells are thus distinguished from other cells by bright staining with CD117 (Kit), the presence of surface IgE, and by high side-scatter properties. The immunophenotype of normal bone

marrow mast cells as determined by multiparametric flow cytometry shows expression of CD9, CD44, CD45, CD33, CD63, CD68, and CD117. However, normal mast cells do not express CD2, CD14, CD15, CD16, CD25, or T and B cell-related antigens (Escribano et al. 2004). Abnormal expression of at least one of two aberrant antigens on mast cells (CD25 or CD2) is employed as a minor criterion for diagnosis of SM. Expression of CD2 and CD25 in bone marrow mast cells can be investigated by flow cytometry (Fig. 91.7) or by immunohistochemistry. CD25 is the more sensitive parameter in either technique (Sotlar et al. 2004).

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Fig. 91.7 Flow cytometry. Bone marrow aspirate demonstrates a population of aberrant mast cells (within circles) by positive coexpression of CD117/FceRI and CD2, CD25, CD35, CD45. Genotyping was positive for the D816V mutation in c-kit. (See CD-ROM for color version.)

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however, elevated total tryptase levels have not only been detected in SM but also in association with systemic anaphylaxis and with certain myeloid neoplasms, especially acute and chronic myeloid leukemias and myelodysplastic syndromes without mastocytosis (Sperr et al. 2002b). Thus, mast cell tryptase alone cannot be regarded as a disease-specific diagnostic marker of SM. Further, because total tryptase levels elevate transiently in serum or plasma in association with anaphylaxis, the serum tryptase level in patients with suspected mastocytosis must be obtained when the patient is in a baseline state of health and not soon after or during an anaphylactic episode. Based on these limitations, a persistently elevated serum total tryptase level in excess of 20 ng/mL is employed as a minor criterion of SM. In the presence of an associated hematologic nonmast-cell lineage disease, the serum tryptase does not count as a criterion of SM.

Codon 816 c-kit mutation Acquired somatic point mutations in codon 816 (most commonly Asp816Val) of the protooncogene c-kit have been detected in the lesional tissues (skin or bone marrow) of most adult patients with SM examined to date, and in some children with severe disease. In patients with extensive disease, the mutation may usually be detectable in peripheral blood cells, most frequently in monocytes and B cells. As stated above, there are several techniques that allow sensitive screening of bone marrow mononuclear cells for the c-kit D816V mutation, including RFLP analysis, pyrosequencing and PNA (peptide nucleic acids). The presence of either the D816V c-kit mutation or another known activating mutation in the kit gene is considered a minor criterion for the diagnosis of SM.

Laboratory investigation As mentioned above, Kit (Fig. 91.2) is the main mast cell growth factor receptor; hence it is a focus of interest in mastocytosis research. c-kit is the homolog of its viral counterpart, v-kit, which was found in the genome of the Hardy– Zuckerman 4 feline sarcoma virus. The c-kit gene is located on chromosome 4q11–12 in humans and on chromosome 5 (W locus) in mice (Yarden et al. 1987; Gokkel et al. 1992). In addition to mast cells, which express Kit at all stages of maturation, it is also expressed by germ cells, melanocytes, hematopoietic progenitor cells, gastrointestinal cells of Cajal, and neurons (Rolle et al. 2002). Several mast cell lines have been generated and have proven useful for exploring mast cell biology. The HMC-1 cell line established in 1988 has been used for many years, despite two limitations: (i) the HMC-1 cell line is growth factor independent, and (ii) HMC-1 cells exhibit inconsistent degranulation to IgE-dependent signals. Two novel stem cell-

Mastocytosis

dependent cell lines, LAD 1 and LAD 2, were established from a patient with mast cell sarcoma/leukemia in 2003 and have proven useful in studies of mast cell function as they consistently express receptor for IgE. Two strains of mast cell-deficient mice (W/Wv and Sl/Sld) have been traditionally used in the evaluation of Kit–SCF interactions. W/Wv mice are known to have macrocytic anemia, lack hair pigmentation, to be sterile, and to have reduced numbers of thymic and gastrointestinal pacemaker cells. Transplantation of normal bone marrow cells into W/Wv mice cures the anemia and reconstitutes mast cells in tissues (Galli & Kitamura 1987; Nakayama et al. 1988). Sl/Sld mice share almost identical phenotypic features with W/Wv mice, including mast cell deficiency. However, one remarkable difference between these two mouse strains is that the mast cell deficiency in the Sl/Sld mice is not correctable by bone marrow transplantation. These studies led to the discovery that Sl/Sld mice have normal hematopoietic/mast cell precursor cells but lack a factor driving their maturation, while the defect in W/Wv mice is in the hematopoietic/mast cell precursor cells themselves. The mutant genes responsible for these phenotypes have been identified as c-kit in W/Wv mice (Chabot et al. 1988; Geissler et al. 1988) and Kit ligand (SCF) in Sl/Sld mice (Williams et al. 1990). The Kit–SCF interaction is also known to be essential for regulation of the proliferation, survival, and migration of melanocytes (Wehrle-Haller 2003). In human studies, SCF has been shown to be essential for in vitro generation of mast cells from CD34+ progenitors (Kirshenbaum et al. 1992). Cultured human mast cells undergo rapid apoptosis if SCF is omitted from the culture medium (Metcalfe et al. 1995; Mekori et al. 2001). The Kit protein contains extracellular, transmembrane, and intracellular portions (Fig. 91.2a). The intracellular section contains a kinase enzymatic domain. The region between the tyrosine kinase domain and the transmembrane portion is called the juxtamembrane domain and regulates the enzymatic activity of the tyrosine kinase domain (Gilfillan & Tkaczyk 2006). The extracellular portion of the molecule has five immunoglobulin-like domains. The first two of these domains are involved in binding of Kit to its ligand, SCF. Binding of ligand (homodimer) causes dimerization of two Kit receptors through the immunoglobulin-like domain 4, which in turn activates the intrinsic tyrosine kinase enzymatic activity of the intracellular portion and results in autophosphorylation of the receptor (Fig. 91.2b). Phosphorylated receptor then becomes a docking site for downstream signal transduction and regulatory and adaptor proteins. Figure 91.2b shows a simplified diagram of the intracellular pathways involved in mast cell growth and differentiation. Tyrosine kinase domain mutations of c-kit directly affecting the ATP-binding ability of the molecule are detected in SM, core factor binding leukemias, sinonasal lymphomas,

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and seminomas (Akin & Metcalfe 2004). The D816V c-kit mutation is the most common mutation found in neoplastic mast cells in SM. c-Kit mutations are detected in acute myeloid leukemia (AML) only in the context of additional well-defined chromosomal abnormalities (Nanri et al. 2005). The hypothesis that additional genetic events might be involved in the pathogenesis of mastocytosis is supported by the fact that c-kit D816V mutations occur in a wide clinical spectrum of mast cell disease, ranging from a self-limited form to an aggressive form with decreased life expectancy (Worobec et al. 1998). Mutations in the extracellular portion of Kit have been described in myeloproliferative disorders, AML, and gastrointestinal stromal tumors (GISTs). Exon 8 mutations corresponding to immunoglobulin-like domain 5, resulting in deletion of codon 419, have been described in approximately 20% of AMLs associated with the chromosomal abnormalities inv(16) ort(8;21) (Gari et al. 1999). The transmembrane domain of Kit anchors the molecule to the cell membrane. It consists of a 23-amino-acid segment encoded by codons 521–543. Mutations in this domain have been described in AML and SM. A codon 530 point mutation has been detected in a patient with core factor binding leukemia. c-Kit exon 8 mutations were shown to be a significant factor adversely affecting relapse rate (Care et al. 2003). A germline activating mutation at codon 522 in the transmembrane domain of Kit has been reported to be associated with a rare variant of mastocytosis. The mast cells in this patient had a well-differentiated phenotype and lacked the codon 816 mutation typically associated with SM (Akin et al. 2004). The juxtamembrane domain of Kit is thought to exert a negative regulatory role on tyrosine kinase activation of the molecule, and thus mutations in this domain might result in loss of this negative regulation. Such mutations have been described in GISTs, sinonasal lymphomas, and less commonly in SM (Beghini et al. 2001; Longley et al. 2001). Exon 11 is the most common site of c-kit mutations in GISTs, although exon 9 c-kit mutations are found in approximately 5–10% of these tumors.

Differential diagnosis Recognition of mastocytosis can be difficult, especially in patients who do not have the characteristic skin lesions and Darier sign. In addition, many clinicians may be unfamiliar with the disease and thus may fail to consider the diagnosis. Even when mast cell disease is suspected, confirmatory work-up should be meticulous to rule out other mimicking diseases. Due to a variety of secreted mediators, symptoms may be nonspecific, and attributed to chronic fatigue or stress. Although mastocytosis is uncommon and heterogeneous, it is highlighted by certain combinations of signs and symptoms that

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Table 91.3 Differential diagnosis of mastocytosis. Skin disorders mimicking cutaneous mastocytosis Scalded skin syndrome Erythema multiforme Benign nevus, freckles, nonspecific pigment changes Contact dermatitis Atopic dermatitis Bullous disorders of childhood Chronic urticaria Benign dermatographism Benign cutaneous flushing Systemic disorders mimicking cell mediator effects Carcinoid syndrome Pheochromocytoma Vasoactive intestinal peptide-secreting tumors Gastrointestinal disorders with persistent or intermittent diarrhea such as Crohn disease, ulcerative colitis; gastroesophageal reflux, gastric acid hypersecretion Disorders/reactions associated with mast cell activation Allergic disorders Idiopathic anaphylaxis Certain drug reactions Local mast cell hyperplasia Cutaneous tumors (melanomas, basal cell carcinoma) Inflammatory lesions Myelomastocytic overlap syndromes Myelomastocytic leukemia (myeloid neoplasm with increase in mast cells but criteria to diagnose SM not fulfilled) Tryptase-positive acute myeloid leukemia (AML) Kit-positive AML with blast cells expressing CD2 (FAB AML-M4eo, some M3) AML with aberrant expression of c-kit point mutations at codon 816 Chronic myeloid leukemia with accumulation of tryptase-positive immature cells Idiopathic myelofibrosis with focal accumulation of mast cells Acute or chronic basophilic leukemia

should trigger a mastocytosis work-up. The major conditions and diseases that should be considered in the differential diagnosis are listed in Table 91.3. Patients with a myeloproliferative form of hypereosinophilic syndrome that is associated with elevated serum tryptase, mast cell dysplasia, and poor prognosis have been described. This disease appears to be different from mastocytosis, despite the fact that the laboratory findings in many of these patients fulfill the currently accepted diagnostic criteria for mastocytosis. This clinical subtype is negative for the D816V Kit mutation and appears to correlate with the presence of a recently described fusion tyrosine kinase, FIP1L1–PDGFRA, that is a therapeutic target of imatinib in myeloproliferative hypereosinophilic syndrome.

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Treatment Approaches to the treatment of mastocytosis can be divided into two basic strategies: those intended to control symptoms due to mediators released from mast cells and those aimed at reducing mast cell burden and /or the treatment of associated hematologic disorders. An outline of suggested therapeutic approaches is summarized in Table 91.4. Therapy should be individualized to each patient’s clinical presentation and prognosis. The mainstay of treatment for

Mastocytosis

most indolent categories of mastocytosis is H1 and H2 antihistamine blockade for prophylaxis of hypotensive episodes, control of cutaneous manifestations such as pruritus and flushing, and gastric hypersecretion. Addition of an H2 antihistamine may be beneficial in cases where insufficient symptom control is afforded by the use of an H1 antihistamine alone. Neither cytoreductive therapy nor splenectomy has any role in the treatment of cutaneous or indolent forms of mastocytosis. For cutaneous manifestations of mastocytosis, which may be quite diverse, other therapies may be beneficial in addition

Table 91.4 Suggested treatment of symptoms and variants of mastocytosis. Hypotension/anaphylactoid reactions Epinephrine (intramuscular, e.g., EpiPen) self-administration More severe, frequent episodes: consider scheduled use of H1, H2 antihistamines ± glucocorticoids Recurrent episodes of abdominal pain, diarrhea H1, H2 antihistamines, consider sodium cromolyn, glucocorticoids, leukotriene antagonists Cutaneous disease (symptomatic) Antihistamines: H1 ± H2 Topical corticosteroids Recalcitrant disease: PUVA Laser therapy: consider for TMEP Gastrointestinal disease Peptic ulcer disease/gastroesophageal reflux: H2 antihistamines, omeprazole Abdominal cramping: H2 antihistamines, cromolyn sodium, leukotriene antagonists Diarrhea: cromolyn sodium, omeprazole, anticholinergics, and/or leukotriene antagonists Malabsorption: glucocorticoids Ascites: glucocorticoids; consider a portacaval shunt Bone disease Osteopenia/osteosclerosis: calcium supplementation ± vitamin D; bisphosphonates. Estrogen therapy for postmenopausal women. Testosterone replacement in men with low testosterone levels. Those with severe osteoporosis at risk for pathologic bone fractures: interferon-a2b Radiotherapy: severe, localized bone pain Mastocytosis with an associated clonal nonmast-cell lineage disease Interferon-a2b, splenectomy when indicated. Treatment of the associated hematologic disorder. Imatinib mesylate (Gleevec) for patients without D816V mutation Smoldering systemic mastocytosis Interferon-a2b Aggressive mastocytosis Interferon-a2b, cladribine. Chemotherapy if nonresponsive or intractable side effects of interferon-a2b or cladribine. Splenectomy when indicated. Imatinib mesylate (Gleevec) for patients without D816V mutation. Consider clinical trials. Rapidly progressive aggressive mastocytosis or mast cell leukemia Treat life-theatening emergency (severe hypotensive episodes, coagulopathy, and gastrointestinal bleeding) as appropriate. Interferon-a2b plus chemotherapy Mast cell sarcoma Chemotherapy, local radiotherapy. Palliative surgery TMEP, telangiectasia macularis eruptiva perstans.

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to antihistamines. UP has been shown responsive to topical corticosteroids but recurs once therapy is stopped. Oral methoxypsoralen therapy with psoralen ultraviolet A (PUVA) therapy may provide transient relief of pruritus and fading of skin lesions in some patients. Treatment of TMEP is somewhat more complicated in that lesions are chronic and tend to be unresponsive to currently available therapies. Neither corticosteroids nor PUVA have been shown to afford significant long-term improvement in TMEP (Kolde et al. 1984). A limited body of literature indicates that laser therapy may have some utility in the treatment of this recalcitrant disorder (Monahan & Petropolis 2003). Disodium cromoglycate has no effect on the cutaneous symptoms of CM. Management of gastrointestinal symptoms should address the type and severity of these symptoms. H2 antihistamines are used to treat gastric hypersecretion and peptic ulcer disease associated with oversecretion of gastric acid stimulated by histamine (Johnson et al. 1980). Omeprazole may be effective in decreasing diarrhea, in addition to controlling gastric acid hypersecretion (Buhl & Clearfield 1990). In severe cases involving refractory malabsorption or ascites, corticosteroids may be indicated. Anticholinergics and orally administered cromolyn sodium may also be useful for control of diarrhea, especially in children (Horan et al. 1990). Epinephrine is employed to treat acute episodes of hypotension (Shaffer et al. 2006). Treatment of refractory hypotension and shock requires immediate evaluation by medical professionals and fluid resuscitation measures, along with additional pharmacologic intervention. Osteoporosis in those with mastocytosis may be unrecognized and hence undertreated, especially in patients with milder forms such as ISM (Kushnir-Sukhov et al. 2006). It is important to utilize dual energy X-ray absorptiometry (DEXA) scanning in the evaluation of patients with mastocytosis. Musculoskeletal pain may be related in some cases to osteoporosis, and is often chronic and difficult to manage. Recommended approaches to the treatment of osteoporosis include calcium supplementation, consideration of estrogen replacement in postmenopausal women, and use of bisphosphonates. Narcotic analgesics may potentiate mast cell degranulation, and thus should be used with care, particularly at high doses. Radiotherapy may have a palliative role in decreasing bone pain in localized areas (Johnstone et al. 1994). Interferonα2b may have some efficacy in decreasing musculoskeletal pain and improving bone mineralization in patients with extensive bony involvement (Lehmann et al. 1996). The decision to initiate treatment with interferon-α therapy should take into consideration potentially debilitating side effects such as fever, malaise, nausea and hypothyroidism, along with the small but well-described risk for anaphylaxis. For patients with aggressive disease, interferon-α and 2-chloro-2-deoxyadenosine (cladribine) are potential firstand second-line therapeutic options, respectively (Worobec 2000). The successful treatment of a patient with SM, who

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was positive for the D816V mutation, has been reported, with a decrease in mast cells and normalization of mast cell mediators. In another study, five aggressive patients with SM were treated with either interferon-α2b alone (N = 4) or in combination with prednisolone (N = 1) (Hauswirth et al. 2004). During therapy, two of the five patients showed a major response, defined as complete resolution of symptoms, one had a partial response, and one had no response. In one patient, progression to mast cell leukemia was seen after 3 months. Cladribine, a nucleoside analog, does not appear to require cells in active cell cycle to exert its cytotoxic activity, and thus appears beneficial in slowly progressing neoplastic processes (Gleixner et al. 2006). In a pilot study, the efficacy of cladribine was studied in 10 patients with SM with severe symptoms (Kluin-Nelemans et al. 2003). Nine patients received six courses, while one patient stopped because of toxicodermia. All patients experienced some symptom relief and a decrease in serum tryptase and urinary histamine metabolite excretion, although none achieved a complete remission. Bone marrow transplantation (BMT) has been investigated as a treatment option for patients with advanced categories of mastocytosis associated with poor survival. BMT has been performed to treat a hematologic disorder associated with mastocytosis in only a handful of reported instances (RonnovJessen et al. 1991; Przepiorka et al. 1998). Although these studies reported favorable responses of the associated hematologic disorders, the overall effect on mast cell hyperplasia was poor. A trial of nonmyeloablative transplantation for three patients with aggressive forms of SM did not yield promising results (Nakamura et al. 2006). Imatinib mesylate (Gleevec; Novartis, Basel, Switzerland), approved by the US Food and Drug Administration for treatment of chronic myelogenous leukemia and Kit-positive GISTs, has a fairly specific inhibition profile that includes bcrabl, Kit, and platelet-derived growth factor-receptor tyrosine kinases. In vitro studies investigating the ability of imatinib mesylate to inhibit various mutant forms of kit revealed that although the drug effectively inhibited wild-type Kit and Kit-bearing juxtamembrane activating mutations (similar to those found in GISTs), it failed to inhibit Kit bearing codon 816 mutations associated with most common forms of SM (Akin et al. 2003). This has been attributed to a conformational change in Kit bearing the codon 816 mutation that interferes with the association of the drug with the ATP-binding domains of the receptor. Consistent with these observations, imatinib mesylate showed a strong in vitro cytotoxic effect on mast cells bearing wild-type Kit, whereas mast cells bearing a codon 816 mutation isolated from bone marrow aspirates of patients with mastocytosis were relatively resistant to the drug. These studies suggest that imatinib mesylate is unlikely to be an effective therapy for patients who carry codon 816 mutations. However, imatinib mesylate has been shown to be of value in unusual clinical presentations of mastocytosis

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associated with novel mutations of c-kit (Akin et al. 2004). A careful mutational analysis of a sample enriched for lesional mast cells appears essential before contemplating therapy with imatinib mesylate. In vitro studies with other tyrosine kinase inhibitors such as dasatinib (Shah et al. 2006), PKC412 (Gleixner et al. 2006) and AMN107 (Gleixner et al. 2006) demonstrate promising results for patients with the D816V mutation, and clinical trials are in progress. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinoid ansamycin antibiotic that binds to heat-shock protein (HSP)-90, causing destabilization of various HSP-90dependent kinases involved in proliferation and survival of malignant cells and resulting in preferential cytotoxicity against neoplastic human mast cells treated ex vivo (Fumo et al. 2004). A dose-dependent reduction in mast cell percentage, determined by flow cytometry, was observed in mast cells cultured from two patients with ISM and HMC-1 human neoplastic mast cell line.

Mastocytosis

mastocytosis seem to express the activating mutations seen in adults (Hartmann & Metcalfe 2000). The prognosis for infants with CM appears to depend partly on whether they exhibit bullae early in the neonatal period or if bullae are delayed relative to the appearance of their skin lesions. Children who manifest diffuse CM prior to bullous eruptions appear to have a better chance of gradual improvement in their disease (Kettelhut & Metcalfe 1994). Several reports of the occurrence of acute lymphocytic leukemia in rapidly progressive and late-onset pediatric mastocytosis suggest the possibility of a predilection for the development of a hematologic malignancy in these patients (Lewis et al. 1995).

Acknowledgments This work was supported by the National Institute of Allergy and Infectious Diseases Division of Intramural Research, National Institutes of Health.

Pediatric mastocytosis Pediatric-onset mastocytosis often follows a benign course. The disease in children is less likely to have a systemic component. The most common manifestation is a solitary mastocytoma (Fig. 91.3d), with UP being the next most frequent manifestation (Ben-Amitai et al. 2005). The most common initial presenting symptom of pediatric mastocytosis is pruritus. In a report of 67 children with UP, 83.7% developed lesions in the first year of life, and the average duration of disease was 9.4 years (Azana et al. 1994). Approximately onethird had improvement at an average of 6.1 years follow-up. In another report of 180 patients, UP was recorded in 65% of patients (Ben-Amitai et al. 2005). It was present at birth in 20% and appeared during the first year in the remaining 80%. The majority of lesions were distributed over the trunk and limbs. Associated symptoms were variable. Prognosis in general was good. The skin lesions thus appear to resolve in approximately half of the patients by adolescence (Kettelhut & Metcalfe 1991; Ben-Amitai et al. 2005). Children whose mastocytosis persists into adulthood may experience progression to systemic involvement. There are reports that suggest that children at potential risk for experiencing shock or sudden death include those with extensive bullous cutaneous involvement, those with symptoms of vasodilatation, flushing and hypotension, and those with early onset of disease (Murphy et al. 1999). Syncope and anaphylaxis as the first presentation of mastocytosis in a pediatric patient with UP has also been reported (Shaffer et al. 2006). Pediatric and adult mastocytosis may often be based on different pathogenic mechanisms. Adult patients usually express activating mutations of c-kit. Most children lack these mutations (Buttner et al. 1998). Only children with progressive

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Givol, D. (1992) Structural organization of the murine c-kit protooncogene. Oncogene 7, 1423–9. Golkar, L. & Bernhard, J.D. (1997) Mastocytosis. Lancet 349, 1379– 85. Hartmann, K. & Metcalfe, D.D. (2000) Pediatric mastocytosis. Hematol Oncol Clin North Am 14, 625–40. Hauswirth, A.W., Simonitsch-Klupp, I., Uffmann, M. et al. (2004) Response to therapy with interferon alpha-2b and prednisolone in aggressive systemic mastocytosis: report of five cases and review of the literature. Leuk Res 28, 249–57. Horan, R.F., Sheffer, A.L. & Austen, K.F. (1990) Cromolyn sodium in the management of systemic mastocytosis. J Allergy Clin Immunol 85, 852–5. Horny, H.P. & Valent, P. (2001) Diagnosis of mastocytosis: general histopathological aspects, morphological criteria, and immunohistochemical findings. Leuk Res 25, 543–51. Horny, H.P., Ruck, M., Wehrmann, M. & Kaiserling, E. (1990) Blood findings in generalized mastocytosis: evidence of frequent simultaneous occurrence of myeloproliferative disorders. Br J Haematol 76, 186–93. Horny, H.P., Ruck, P., Krober, S. & Kaiserling, E. (1997) Systemic mast cell disease (mastocytosis). General aspects and histopathological diagnosis. Histol Histopathol 12, 1081–9. Johansson, C., Roupe, G., Lindstedt, G. & Mellstrom, D. (1996) Bone density, bone markers and bone radiological features in mastocytosis. Age Ageing 25, 1–7. Johnson, G.J., Silvis, S.E., Roitman, B., Blumenthal, M. & Gilbert, H.S. (1980) Long-term treatment of systemic mastocytosis with histamine H2 receptor antagonists. Am J Gastroenterol 74, 485–9. Johnstone, P.A., Mican, J.M., Metcalfe, D.D. & DeLaney, T.F. (1994) Radiotherapy of refractory bone pain due to systemic mast cell disease. Am J Clin Oncol 17, 328–30. Kambe, N., Kambe, M., Kochan, J.P. & Schwartz, L.B. (2001) Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes. Blood 97, 2045–52. Kettelhut, B.V. & Metcalfe, D.D. (1991) Pediatric mastocytosis. J Invest Dermatol 96, 15S–18S. Kettelhut, B.V. & Metcalfe, D.D. (1994) Pediatric mastocytosis. Ann Allergy 73, 197–202; quiz 202–7. Kirshenbaum, A.S., Kessler, S.W., Goff, J.P. & Metcalfe, D.D. (1991) Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells. J Immunol 146, 1410–15. Kirshenbaum, A.S., Goff, J.P., Kessler, S.W., Mican, J.M., Zsebo, K.M. & Metcalfe, D.D. (1992) Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells. J Immunol 148, 772–7. Kirshenbaum, A.S., Akin, C., Goff, J.P. & Metcalfe, D.D. (2005) Thrombopoietin alone or in the presence of stem cell factor supports the growth of Kit (CD117) Low/MPL (CD110) human mast cells from hematopoietic progenitors. Exp Hematol 33, 413–21. Kluin-Nelemans, H.C., Oldhoff, J.M., Van Doormaal, J.J. et al. (2003) Cladribine therapy for systemic mastocytosis. Blood 102, 4270– 6. Kolde, G., Frosch, P.J. & Czarnetzki, B.M. (1984) Response of cutaneous mast cells to PUVA in patients with urticaria pigmentosa: histomorphometric, ultrastructural, and biochemical investigations. J Invest Dermatol 83, 175–8. Kushnir-Sukhov, N.M., Brittain, E., Reynolds, J.C., Akin, C. & Metcalfe, D.D. (2006) Elevated tryptase levels are associated with

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greater bone density in a cohort of patients with mastocytosis. Int Arch Allergy Immunol 139, 265–70. Lawrence, J.B., Friedman, B.S., Travis, W.D., Chinchilli, V.M., Metcalfe, D.D. & Gralnick, H.R. (1991) Hematologic manifestations of systemic mast cell disease: a prospective study of laboratory and morphologic features and their relation to prognosis. Am J Med 91, 612–24. Lehmann, T., Beyeler, C., Lammle, B. et al. (1996) Severe osteoporosis due to systemic mast cell disease: successful treatment with interferon alpha-2B. Br J Rheumatol 35, 898–900. Lewis, H.M., Winter, E., Darbyshire, P., Yoong, A., Marsden, J.R. & Moss, C. (1995) Urticaria pigmentosa and acute lymphoblastic leukaemia. J R Soc Med 88, 530P–531P. Longley, B.J., Reguera, M.J. & Ma, Y. (2001) Classes of c-KIT activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk Res 25, 571–6. Mekori, Y.A., Gilfillan, A.M., Akin, C., Hartmann, K. & Metcalfe, D.D. (2001) Human mast cell apoptosis is regulated through Bcl-2 and Bcl-XL. J Clin Immunol 21, 171– 4. Metcalfe, D.D. (1991) The liver, spleen, and lymph nodes in mastocytosis. J Invest Dermatol 96 (3 suppl.), 45S–46S. Metcalfe, D.D., Mekori, J.A. & Rottem, M. (1995) Mast cell ontogeny and apoptosis. Exp Dermatol 4, 227–30. Monahan, T.P. & Petropolis, A.A. (2003) Treatment of telangiectasia macularis eruptiva perstans with total skin electron beam radiation. Cutis 71, 357–9. Muller, U., Helbling, A., Hunziker, T. et al. (1990) Mastocytosis and atopy: a study of 33 patients with urticaria pigmentosa. Allergy 45, 597–603. Murphy, M., Walsh, D., Drumm, B. & Watson, R. (1999) Bullous mastocytosis: a fatal outcome. Pediatr Dermatol 16, 452–5. Nakamura, R., Chakrabarti, S., Akin, C. et al. (2006) A pilot study of nonmyeloablative allogeneic hematopoietic stem cell transplant for advanced systemic mastocytosis. Bone Marrow Transplant 37, 353–8. Nakayama, H., Kuroda, H., Fujita, J. & Kitamura, Y. (1988) Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin. Development 102, 107–16. Nanri, T., Matsuno, N., Kawakita, T. et al. (2005) Mutations in the receptor tyrosine kinase pathway are associated with clinical outcome in patients with acute myeloblastic leukemia harboring t(8;21)(q22;q22) Leukemia 19, 1361– 6. Przepiorka, D., Giralt, S., Khouri, I., Champlin, R. & Bueso-Ramos, C. (1998) Allogeneic marrow transplantation for myeloproliferative disorders other than chronic myelogenous leukemia: review of forty cases. Am J Hematol 57, 24– 8. Roberts, L.J. II, Turk, J.W. & Oates, J.A. (1982) Shock syndrome associated with mastocytosis: pharmacologic reversal of the acute episode and therapeutic prevention of recurrent attacks. Adv Shock Res 8, 145–52. Rogers, M.P., Bloomingdale, K., Murawski, B.J., Soter, N.A., Reich, P. & Austen, K.F. (1986) Mixed organic brain syndrome as a manifestation of systemic mastocytosis. Psychosom Med 48, 437–47. Rolle, U., Piotrowska, A.P., Nemeth, L. & Puri, P. (2002) Altered distribution of interstitial cells of Cajal in Hirschsprung disease. Arch Pathol Lab Med 126, 928–33. Ronnov-Jessen, D., Lovgreen Nielsen, P. & Horn, T. (1991) Persistence of systemic mastocytosis after allogeneic bone marrow

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transplantation in spite of complete remission of the associated myelodysplastic syndrome. Bone Marrow Transplant 8, 413–15. Shaffer, H.C., Parsons, D.J., Peden, D.B. & Morrell, D. (2006) Recurrent syncope and anaphylaxis as presentation of systemic mastocytosis in a pediatric patient: case report and literature review. J Am Acad Dermatol 54 (5 suppl.), S210–S213. Shah, N.P., Lee, F.Y., Luo, R., Jiang, Y., Donker, M. & Akin, C. (2006) Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis. Blood 108, 286–91. Sotlar, K., Horny, H.P., Simonitsch, I. et al. (2004) CD25 indicates the neoplastic phenotype of mast cells: a novel immunohistochemical marker for the diagnosis of systemic mastocytosis (SM) in routinely processed bone marrow biopsy specimens. Am J Surg Pathol 28, 1319–25. Sperr, W.R., Escribano, L., Jordan, J.H. et al. (2001) Morphologic properties of neoplastic mast cells: delineation of stages of maturation and implication for cytological grading of mastocytosis. Leuk Res 25, 529–36. Sperr, W.R., Jordan, J.H., Fiegl, M. et al. (2002a) Serum tryptase levels in patients with mastocytosis: correlation with mast cell burden and implication for defining the category of disease. Int Arch Allergy Immunol 128, 136–41. Sperr, W.R., Stehberger, B., Wimazal, F. et al. (2002b) Serum tryptase measurements in patients with myelodysplastic syndromes. Leuk Lymphoma 43, 1097–105. Taylor, M.L., Sehgal, D., Raffeld, M. et al. (2004) Demonstration that mast cells, T cells, and B cells bearing the activating kit mutation D816V occur in clusters within the marrow of patients with mastocytosis. J Mol Diagn 6, 335– 42. Travis, W.D. & Li, C.Y. (1988) Pathology of the lymph node and spleen in systemic mast cell disease. Mod Pathol 1, 4–14. Valent, P., Horny, H.P., Escribano, L. et al. (2001) Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res 25, 603–25. Valent, P., Akin, C., Sperr, W.R., Horny, H.P. & Metcalfe, D.D. (2002) Smouldering mastocytosis: a novel subtype of systemic mastocytosis with slow progression. Int Arch Allergy Immunol 127, 137–9. Valent, P., Akin, C., Sperr, W.R., Horny, H.P. & Metcalfe, D.D. (2003) Mast cell proliferative disorders: current view on variants recognized by the World Health Organization. Hematol Oncol Clin North Am 17, 1227–41. Valent, P., Akin, C., Sperr, W.R. et al. (2005) Mastocytosis: pathology, genetics, and current options for therapy. Leuk Lymphoma 46, 35– 48. Wehrle-Haller, B. (2003) The role of Kit-ligand in melanocyte development and epidermal homeostasis. Pigment Cell Res 16, 287–96. Williams, D.E., Eisenman, J., Baird, A. et al. (1990) Identification of a ligand for the c-kit proto-oncogene. Cell 63, 167–74. Worobec, A.S. (2000) Treatment of systemic mast cell disorders. Hematol Oncol Clin North Am 14, 659– 87, vii. Worobec, A.S., Semere, T., Nagata, H. & Metcalfe, D.D. (1998) Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis. Cancer 83, 2120–9. Yarden, Y., Kuang, W.J., Yang-Feng, T. et al. (1987) Human protooncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J 6, 3341–51.

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Anaphylaxis and Allergy to Food and Drugs

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Anaphylaxis M. Rosario Caballero, Stephen J. Lane and Tak H. Lee

Summary Anaphylaxis represents the most serious allergic reaction that is rapid in onset and may cause death. Fatal allergic reactions have been described for at least 4000 years, but it was not until the early 20th century, when the term “anaphylaxis” was introduced by Portier and Richet. Since then, several advances have been made in the understanding the etiology, mechanism, diagnosis and treatment, despite difficulties resulting from a wide range of possible clinical presentations and the lack of a universally accepted clinical definition of anaphylaxis. The history of anaphylaxis critically reflects the changing pattern of the host environment over time. In the 1940s, the advent of penicillin heralded another source of widely administered foreign material, which is one of the commonest causes of systemic anaphylaxis yet. The most common causes of anaphylaxis are foods, insect stings, medications, allergen immunotherapy injections, and idiopathic anaphylaxis. Interestingly, although the pattern of foreign agents capable of inducing anaphylaxis continues to change, the incidence of anaphylaxis by Hymenoptera venom remains constant throughout time. In the 1990s, an unexpected development was the epidemic of allergic reactions to natural rubber latex. Recently, a major interest is anaphylactic reactions during the perioperative period and during medical procedures. In general surgery, muscle relaxants are known to be the cause of up to 60% of the anaphylactic reactions which occur during anesthesia. A meticulous medical history focusing on prior adverse reactions and, if necessary, skin testing to diagnose drug and latex allergy are crucial to prevent further anaphylaxis reaction anesthesia. In the last few years, new advances in laboratory tests have become available to support the clinical diagnosis of anaphylaxis. Serum tryptase, serum histamine, and urinary histamine are metabolites that might be increased during an anaphylactic reaction. Also, a ratio of total tryptase (α plus β) to β-tryptase is helpful for distinguishing between anaphylaxis related or unrelated to systemic mastocytosis.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

The treatment of anaphylaxis has changed little since the introduction of epinephrine in the early 20th century and the development of antihistamines in the mid-20th century, although new recommendations have been made about epinephrine administration.

Historical perspective (Table 92.1) Systemic anaphylaxis represents the most serious allergic reaction that is rapid in onset and may cause death. Although fatal allergic reactions have been described for at least 4000 years, since the hieroglyphic recording of the death of King Menes of Egypt from an insect sting, the biological mechanisms underlying anaphylaxis only began to be unraveled in the early 20th century (Fernberg et al. 1956). The history of anaphylaxis critically reflects the changing pattern of the host environment over time. The term was introduced in 1902 by Portier and Richet to describe the paradoxical catastrophic reaction seen in dogs that were injected with an extract of sea anemone in an attempt to induce tolerance or resistance to the administered antigen. The extract was well tolerated on the first injection but induced a rapidly fatal reaction on subsequent reinjection several weeks later. Instead of the anticipated protective or prophylactic effect of these immunizations, an increased sensitivity was identified and referred to as “anaphylaxis” (from the Greek ana, meaning backward, and phylaxis, meaning protection). Although others had documented the occurrence of similar reactions in the 19th century, Richet was awarded the Nobel Prize in Medicine and Physiology in 1913 for his pioneering work in this field (Cohen & Zelaya-Quesada 2002). It became apparent in 1921 that the anaphylactic response was acquired and not necessarily due to a toxin (Arthus 1921). A wide variety of injected foreign proteins and low-molecular-weight substances (e.g., drugs) were found to induce similar reactions after a delay of several weeks from the first exposure. With the increasing use of hyperimmune horse serum as a source of antitoxins against tetanus and diphtheria toxins and as antisera to organisms such as pneumococcus, meningococcus and the tubercle bacillus, a substantial increase in the reported fatalities

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Table 92.1 The changing pattern of anaphylaxis. 4000 BC First recorded case of insect anaphylaxis 1902 Anaphylaxis defined by Portier and Richet after inducing fatal allergic shock in a dog due to sensitization to sea anemone 1921 Arthus discovers that anaphylaxis can be induced by nontoxic proteins and small molecules 1936 Increasing use of hyperimmune horse serum as antitoxin and antisera now accounts for 50% of reported fatalities from anaphylaxis 1949 First recorded fatality from anaphylaxis to penicillin 1972 Penicillin accounts for 75% of all recorded fatalities due to anaphylaxis 1980s Increases in reported fatalities due to radiocontrast media, now second only to penicillin as a cause of fatal reactions 1990s Latex anaphylaxis recognized as an occupational hazard for healthcare workers and for their patients 1990s Peanut allergy recognized as the major cause of food-induced anaphylaxis 2000s Muscle relaxants recognized as the major cause of anaphylaxis during general anesthesia

from anaphylaxis was seen to occur (Lamson 1924, 1929). A literature review of 1936 attributed 50% of the fatalities to serum therapy and the remainder to other, mainly proteinaceous, foreign materials (Vaughan & Pipes 1936). Serious reactions to horse serum were seen to occur in those patients previously sensitized to horse serum antitoxin therapy, with prior ingestion of horse meat or milk, and in horse dander-sensitive asthmatic subjects. Their sensitivity was such that skin testing with horse dander antigen could induce life-threatening reactions. Horse serum is now rarely used therapeutically except in the case of antivenom therapy for snake bite. The advent of penicillin in the 1940s heralded another source of widely administered foreign material, which was subsequently to develop into one of the commonest cause of systemic anaphylaxis seen today. The first case was described in 1949 by Waldblott and by the 1960s the mortality was estimated at between 100 and 500 per year in the USA (Parker 1963, 1972). By the 1970s penicillin was estimated to account for approximately 75% of all cases of severe anaphylaxis (Delage & Irey 1972; Parker 1972). Of penicillin courses in the USA, 1–2% are complicated by systemic allergic reactions and 10% of these reactions are serious or lifethreatening. Although serious reactions occur about twice as frequently after intravenous administration, oral administration can also cause anaphylaxis and death (Anderson 1986). Other administered agents that commonly cause anaphylaxis today include radiocontrast media, plasma substitutes, narcotics, depolarizing agents, and local anesthetic reagents. However, only a very small percentage of patients claiming to have allergic reactions to local anesthetics are confirmed when they are tested. An important cause of anaphylaxis, the incidence of which has probably remained constant throughout time, is the venom of Hymenoptera, which accounts for 40– 80 deaths per year

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after field stings in the USA (Patterson & Valentine 1982; Kaliner 1987; Jerrard 1996). Immunotherapy and skin testing to venom can also cause anaphylaxis and a study by Lockey et al. (1987) identified 45 fatalities due to immunotherapy or skin-prick testing since 1945. Of the 330 patients on whom sufficient data were available, 24 deaths occurred during immunotherapy and six during skin testing. Five of the six deaths during skin testing occurred in patients in whom intradermal tests were not accompanied by skin tests. The pattern of foreign agents capable of inducing anaphylaxis continues to change. In the 1990s, an unexpected development was the epidemic of allergic reactions to natural rubber latex. Since then, several epidemiologic and mechanistic studies have been designed which have contributed to identifying at-risk groups and to reducing anaphylactic events in healthcare facilities. It is therefore apparent that although anaphylactic reactions have been described for many centuries, the pattern of etiologic agents has changed, especially over the last century. Recently, a major interest is anaphylactic reactions during the perioperative period and during medical procedures. The estimated incidence of anaphylaxis during anesthesia has been reported to range from 1 in 4000 to 1 in 25 000 and is estimated to be as high as 6% (Yocum et al. 1999). Clark et al. (1975) reported a mortality rate of 4.3% during anesthesia. Further studies have shown similar incidence rates (Sage et al. 1981; Moscicki et al. 1990; Pepys et al. 1994). Muscle relaxants are the main cause for intraoperative anaphylaxis, with an incidence up to 60%. Latex is the next most frequent cause for intraoperative reactions, with an incidence reported to be higher than 17%, although it might be decreasing. Extensive studies are currently underway by experts from different disciplines to identify and evaluate perioperative reactions in order to reduce the risk of anaphylaxis during anesthesia.

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Epidemiology of anaphylaxis Race, sex, occupation, geographical location, and season of the year do not affect the risk of anaphylactic reactions, except that such factors may influence the availability of, and exposure of individuals to, the offending agents. Data regarding the incidence and prevalence of anaphylaxis remain limited. Nevertheless, in the last few years, some epidemiologic studies have been carried out. In 1998, an international study on anaphylactic and anaphylactoid reactions showed approximately 154 annual fatal episodes per million hospitalized subjects (International Collaborative Study of Severe Anaphylaxis 1998). Other studies revealed that the estimated risk of anaphylaxis per person in the USA is 1–3% (Yocum et al. 1999). Other than previous exposure, there are no known epidemiologic characteristics that reliably identify those who may be at risk from anaphylactic sensitivity. Among the known causes of anaphylactic reactions to drugs, antibiotics, especially β-lactam antibiotics, remain the most common causes of serious anaphylactic events (Ansell et al. 1980; Weiss & Adkinson 1988; Atkinson & Kaliner 1992; Kemp & Lockey 2002). The prevalence of reactions to drugs has been estimated at 15–40 per 10 000 patients (Boston Collaborative Drug Surveillance Program 1973; Giansiracus & Upchurch 1985), whereas fatal reactions to penicillin, the most common agent, occur at the rate of one fatality for every 7.5 million injections (Idsoe et al. 1968). Radiologic contrast media provoked anaphylactic reactions in 1 in 600 patients (Lasser et al. 1987) and anesthetic drugs in 1 in 20 000 (Fisher & Baldo 1984). Serious reactions to Hymenoptera stings occur in 0.4– 0.8% of the population (Settipane et al. 1972). Mortality has been estimated by Parish (1965) at 1 in 6.5 million stings. Factors associated with reaction severity in different studies are age, cardiovascular and respiratory comorbidities (Tunonde-Lara et al. 1992; Brown 2004). Asthmatic subjects appear

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to be at greater risk from a fatal outcome than nonasthmatic subjects, although most studies suggest that an atopic person is at no greater risk than a nonatopic for the development of IgE-mediated anaphylaxis in response to drugs or to insect stings. Moreover, Brown et al. (2003) found that age is a major determinant of reaction severity in Hymenoptera venom allergy and iatrogenic anaphylaxis.

Definition and etiology (Tables 92.2 and 92.3; Fig. 92.1) Interestingly, there is still no universally accepted clinical definition of anaphylaxis. Some clinicians define it as “a syndrome of one or more systemic signs and symptoms, without precise features alone or in combination” (Project Team of the Resuscitation Council UK 1999). Others grade symptoms or severity of reactions from grade I to grade IV (Stewart 1996). In order to achieve a universal definition of anaphylaxis, the National Institute of Allergy and Infectious Disease (NIAID) and the Food Allergy and Anaphylaxis Network (FAAN) has started to work toward an international agreement. They have described anaphylaxis as a severe, potentially fatal, systemic allergic reaction that occurs suddenly after contact with an allergy-causing substance. In 2006, participants at the symposium agreed a general definition: “Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death.” Proposed criteria for recognizing anaphylaxis are summarized in Table 92.2. The most common causes of anaphylaxis are foods, insect stings, medications, allergen immunotherapy injections, and idiopathic anaphylaxis (Stewart & Lockey 1992; Kemp et al. 1995; Lieberman 1998; Yocum et al. 1999; Brown et al. 2001). Anaphylaxis to peanuts or tree nuts is of special concern because of its life-threatening potential, especially in subjects who suffer from asthma and the tendency for lifelong sensitivity to these foods.

Table 92.2 Clinical criteria for diagnosing anaphylaxis. (From National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network Symposium 2006, with permission.) Anaphylaxis is highly likely when any one of the following three criteria is fulfilled 1 Acute onset of illness (minutes to several hours) with involvement of the skin, mucosal tissue, or both, and at least one of the following: (a) Respiratory compromise (b) Reduced blood pressure (BP) or associated symptoms of end-organ dysfunction 2 Two or more of the following that occur rapidly after exposure to a likely allergen for that patient (minutes to several hours): (a) Involvement of the skin/mucosal tissue (b) Reduced BP or associated symptoms (c) Persistent gastrointestinal symptoms 3 Reduced BP after exposure to known allergen for that patient (minutes to several hours): (a) Infants and children: low systolic BP (age specific) or greater than 30% decrease in systolic BP (b) Adults: systolic BP < 90 mmHg or greater than 30% decrease from that person’s baseline

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Table 92.3 Mechanisms and agents responsible for systemic anaphylactic reactions. Mechanism

Agents

Examples

IgE-mediated reaction against native proteins

Venoms Foods Enzymes Hererologous sera Human proteins Others Immunotherapy

Hymenoptera, snake Peanuts, milk, fish Chymopapain, streptokinase Antilymphocyte globulin, tetanus and botulinum antitoxin Insulin, ACTH, seminal fluid Protamine, latex rubber, alcuronium Venom, inhalant allergens

IgE-mediated reactions against protein–hapten conjugates

Antibiotics Disinfectants

Penicillins, cephalosporins, sulfonamides Ethylene oxide

Non-IgE, immune complexmediated associated with IgA deficiency

Blood products

Blood, gammaglobulin, cryoprecipitate

Non-IgE complement-mediated

Radiocontrast media Cuprammonium cellulose Metrizamide

Activation of coagulation

Collagen, endotoxin

Direct mast cell activation

Osmotic stimuli Other

Mannitol, dextrose, radiocontrast media Opiates, muscle relaxants, dextran

Modulation of arachidonic acid metabolism

NSAID Dyes Preservatives

Aspirin Tartrazine Benzoates

Multiple mechanisms

Radiocontrast media

Unknown mechanisms

Sulfite sensitivity Exercise Exercise and food Catamenial Idiopathic anaphylaxis

Bisulfite

There are now three well-established means by which exposure to a foreign substance can lead to anaphylaxis, and the list of the more common causative agents is extensive and growing continually with the introduction of new therapeutic and diagnostic materials. The necessary components of the anaphylactic response are: 1 a sensitizing antigen, usually administered parenterally, either in its native state or as a hapten after covalent attachment to a carrier protein; 2 an IgE-class antibody response resulting in systemic sensitization of mast cells and basophils; 3 reintroduction of the antigen, usually systemically; 4 mast cell degranulation secondary to antigen-induced cross-linking of the high-affinity IgE receptor on the surface of the mast cell and basophils, with subsequent mediator release, generation or both;

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5 production of several pathologic responses by the mast cell-derived mediators and manifest as clinical anaphylaxis. Why only certain persons have such a response remains unclear. Mast cells can also be activated by means other than via the high-affinity IgE receptor in situations where prior exposure to the offending agent is not necessary, resulting in the same clinical manifestations of anaphylaxis and release of identical chemical mediators as occurs in response to IgEmediated mast cell activation. Some authors reserve the term “anaphylaxis” only for IgE-dependent events and the term “anaphylactoid” for IgE-independent reactions that are otherwise clinically indistinguishable. In these reactions, immune complexes or other agents activate the complement cascade, resulting in the formation of anaphylatoxins such as the complement protein fragments C3a and C5a, which can bind to the surface of the mast cell and trigger the release of medi-

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Anaphylaxis

IgE-mediated

Sensitization IgE response Re-exposure

Non-IgE-mediated

Complement activation

Non-complementmediated

Immune complex Direct activation of complement Kinin-mediated Coagulation pathwaymediated

Dextran Mannitol Opiates

Mast cell activation

Direct mast cell activation

Mediator release, e.g., histamine, tryptase

Clinical anaphylaxis Fig. 92.1 Classification of anaphylaxis based on underlying pathogenic mechanisms.

ators, bypassing the high-affinity IgE receptor. In addition, certain agents, such as hyperosmolar solutions (e.g., mannitol and radiocontrast media), can stimulate the release of mediators directly by an as yet unknown mechanism independent of IgE or complement. Additional causes without any clear mechanism have been identified that are associated with clinical anaphylaxis and increases in mast cell-derived mediators.

IgE-mediated anaphylaxis Antibiotics and other drugs By far the most frequently implicated drug agent in anaphylaxis is penicillin, with reported reactions of severity ranging from 1 to 10% of treated patients (Parker 1972), independent of atopic status. Of the patients who have a systemic reaction 10% are serious or life-threatening. Some 400–800 deaths per year in the USA can be attributed to penicillin anaphylaxis (Kaliner 1987). These drugs are of low molecular weight and are not antigenic in their own right, requiring carrier proteins, such as serum globulins, before they are capable of inducing an immune response. Such molecules are called haptens. Penicillin and related antibiotics are the most important haptens. The higher the affinity of the hapten to its carrier protein, the more likely it is to induce an allergic reaction. Although Baldo and Fisher (1983) have suggested that some

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small molecules are able to stimulate IgE responses without the necessity of binding the carrier proteins, the antigens that characteristically trigger anaphylactic reactions are large molecules of greater than 10 kDa. They occur most frequently in the 40–49 year age group and are common and more severe with parenterally administered drugs. Penicillin hypersensitivity declines with time and the greater the interval since a previous reaction, the less likely the sensitivity is to exist. In addition, atopy is not a predisposing factor. Almost all patients treated with penicillin mount an immunologic response (de Weck & Blum 1965; Levine 1966), although only a small proportion develop adverse reactions. Specific IgE is directed either against the major metabolic product, the benzylpenicilloyl poly-L-lysine group (called major determinant or PPL), or against minor determinants that include crystalline penicillin, sodium penicilloate, and sodium benzylpenicilloylamine. The presence of IgE-blocking antibody specific for the major determinant prevents it from triggering mast cells. The minor determinants, unlike the major determinant, do not induce specific IgG-blocking antibodies and the presence of specific IgE antibodies directed against these minor determinants appears to be responsible for penicillin anaphylaxis (Levine & Redmond 1969). As all penicillin derivatives possess the same 6-amino-penicillanic acid nucleus, there is a high degree of cross-reactivity between them. Any patient with a history of a severe reaction to one penicillin should be considered at risk of similar reactions to any other penicillin (Steward 1962; Green & Rosenblum 1971). In 2003, the interest group on drug hypersensivity of the European Network for Drug Allergy (ENDA) and the European Academy of Allergology and Clinical Immunology (EAACI) published guidelines for diagnosis of immediate reaction to β-lactam antibiotics (Torres et al. 2003). After taking a careful clinical history, a blood sample is drawn to perform in vitro tests, basically a noncompetitive fluorescent enzyme immunoassay (CAP-FEIA; Pharmacia Diagnostics, Uppsala, Sweden). Then skin-prick tests (SPT) are performed to different dilutions with the following reagents: minor determinants mixture, benzylpenicilloyl poly-L-lysine (PPL), amoxicillin, ampicillin, and cephalolosporin. Subsequently, if the SPT is negative, intradermal tests are performed with the same reagents and dilutions. If any of the skin and in vitro tests is positive, the patient is considered allergic. If SPT, intradermal tests, and CAP-FEIA results are negative, a drug provocation test with the apparent culprit drug should be considered. If the patient tolerates the drug, the patient could be considered nonallergic. In some cases, when the history of an allergy reaction is clear and there is a long interval between the last reaction and the drug challenge, the same study should be performed 2–4 weeks afterwards. Patients may lose sensitivity and become negative over time, but the percentage of cases that will become resensitized after one or further contact with a β-lactam is unknown. As regards the sensitivity of penicillin skin testing, some studies have shown

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that PPL is the most important determinant, with sensitivity higher than 70% in patients with IgE-mediated clinical reactions to penicillin. However, Torres et al. (2001) found, in a study carried out with 290 patients, that the sensitivity of skin testing for any single hapten was low, with values between 22 and 43%. The combination of all four haptens gave a sensitivity of 70%. The specificity of skin testing has demonstrated to be very high (97–99%) (Torres et al. 2001). Cephalosporins share the same β-lactam ring structure and similar metabolic pathways as penicillins and consequently cross-react immunologically (Batchelor et al. 1966). Early estimates of clinical cross-reactivity were as high as 30%, but subsequent reviews (Petz 1978; Saxon et al. 1987) lowered this figure to approximately 8% after performing SPT and intradermal testing with the specific cephalosporin. If skin testing is positive to penicillin, and cephalosporin is needed, then desensitization should be performed using the particular cephalosporin. Another alternative is to perform a graded challenge with the cephalosporin, but the risk of anaphylaxis, although low, must be recognized. If the history is not consistent with penicillin allergy, a graded challenge may be considered without previous skin testing. In a recent review (Gruchalla & Pirmohamed 2006), among 12 cases of fatal anaphylaxis caused by antibiotics, six cases occurred after the first dose of a cephalosporin, and three of the six patients were known to have penicillin allergy. There is not cross-reaction between monobactams and other β-lactams, except for ceftazidime, with which aztreonam shares an identical R-group side chain (Saxon et al. 1984). Therefore, patients allergic to β-lactams (except for ceftazidime) might tolerate aztreonam and otherwise. On the other hand, skin test studies show cross-reactivity between carbapenems and penicillins, although no definitive clinical challenge studies in patients have been performed (Saxon et al. 1988).

Hymenoptera venom Hymenoptera stings are also a common cause of IgE-mediated anaphylaxis. Between 0.5 and 4% of the population will experience a systemic reaction after being stung and at least 40 subjects die each year in the USA as a result of such stings. In recent studies, the prevalence of anaphylaxis caused by Hymenoptera venom among all emergency department visits was estimated to be 0.09–1% but these numbers are thought to be gross underestimates (Klein & Yocum 1995; Clark et al. 2005). Golden et al. (1989) studied 269 subjects and found the incidence of systemic reactions to insect sting to be 3.3%; 26.5% had IgE antibodies to venom demonstrated by radioallergosorbent test (RAST) or skin test. Asymptomatic sensitization was seen in 15% of subjects with no history of an allergic sting reaction. Systemic allergic reactions and asymptomatic sensitization to venom therefore appear to be quite common, with most subjects never seeking medical advice. Positive venom tests were more frequent in men, in subjects

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aged 20–29 years, and in those with positive skin tests to inhalant allergens. In addition, a positive venom test was more frequent in subjects who had been stung within the previous 3 years than in those stung more than 3 years before. The Hymenoptera venom study of 3236 venom-sensitive patients defined male sex, older age (average age of reaction 30 years), bee-keeping, and atopy as being risk factors for systemic reactions (Lockey et al. 1988). Over the 3-year study period there was an average of 2.7 stings per patient and 51% of the reactions occurred within 10 min of the sting. The order Hymenoptera contains two families: Apidae (bees) and Vespidae (wasps, hornets and yellow jackets). The genus Apis contains only the honeybee (Apis mellifera), the genus Vespula contains hornets and yellow jackets, and Polistes contains the wasps. In the UK, Hymenoptera venom allergy is usually caused by stings from the honeybee or the wasp. The vast majority of UK patients with bee-venom allergy are bee-keepers and their relatives and neighbors as they have close contact with bees and are therefore at high risk of being stung (Ewan 1984, 1985). Wasp-venom allergy usually occurs in patients who have been stung infrequently, often at long intervals over many years. A small number of patients appear to be at risk of recurrent wasp stings, for example, fruit farm or bakery workers. The major allergens in both bees and wasps are different types of the enzyme phospholipase A. In addition to this protein, bee venom contains kinin-like molecules including tachykinins and a number of biologically active mediators, including dopamine, norepinephrine, a neurotoxin apamin, mellitin (which causes hemolysis), and peptide 401 (which causes mast cell degranulation), plus a variety of enzymes including hyaluronidase and acid phosphatase. Wasp venoms contain similar but not identical toxins and different phospholipase A. Hypersensitivity reactions may be either local or generalized. Local reactions present as large painful pruritic reactions close to the site of injection, which may persist for days. Generalized reactions are remote from the site of the sting and vary from mild rashes to life-threatening anaphylaxis. Onset can vary from a few minutes to 30 min and common clinical features include erythema, urticaria, angioedema, laryngeal edema, bronchospasm, and hypotension. Patients may also develop acute rhinoconjunctivitis, diarrhea, fits, or visual disturbances. Asthmatic patients do not have an increased risk of developing systemic reactions to Hymenoptera stings; however, they do demonstrate significantly more severe reactions, manifested by acute dyspnea (Settipane et al. 1980). Vasovagal symptoms occur after stings and can sometimes be difficult to differentiate from true anaphylaxis. The diagnosis of insect-sting allergy is made primarily on the history. The nature of each reaction should be carefully documented, noting severity, rapidity of onset, the number of stings causing the reaction, the treatment required, the response to therapy, and the likelihood of future exposure. The frequency of stings, the interval between stings, and the

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number of stings not causing a reaction must be recorded in order to obtain an accurate picture of the natural history and severity of reactions. The demonstration of specific IgE can be assessed, using pure venom extract for skin testing or specific serum IgE tests. There is a high spontaneous cure rate, as shown in studies of the natural history of venom allergy and in the placebo groups of controlled trials of venom immunotherapy. Settipane and Chafee (1979) examined 119 patients who had a generalized reaction to bee sting and who subsequently had a history of being restung before immunotherapy was commenced. Of these patients 44.5% had an improved grade of reaction, 42.9% had the same grade of reaction, and 12.6% had a worse grade of reaction. Those patients who were restung within 2 weeks (anergic period) or over 5 years after a generalized reaction to a sting had a significantly improved response. Ewan (1984) demonstrated a subsequent generalized reaction in 74% of wasp-allergic patients and in 80% of bee-allergic patients who had previously suffered a generalized reaction. A more dramatic spontaneous improvement was seen by Hunt et al. (1978) in that 40% of patients on the placebo limb of an immunotherapy trial had no reaction to a challenge sting. Unfortunately, despite these studies, it still remains very difficult to predict the severity of reaction to the next sting. Selecting patients who need venom immunotherapy is mainly based on the patient’s natural history of insect sting allergy. It is indicated in patients with a history of severe systemic reactions including respiratory and cardiovascular symptoms and positive test results to the respective insect with either skin tests and/or specific serum IgE tests. In the case of reactions that are not life-threatening, other factors such as occupations and/or hobbies where the risk of exposure is high, the culprit insect itself, concomitant cardiovascular diseases, other pathologies (mastocytosis), or psychological factors arising from anxiety that can seriously impair patient quality of life may influence the decision to initiate venom immunotherapy (Bilò et al. 2005). The natural course of children with a history of mild generalized reaction (urticaria, erythema, pruritus) is particularly good, with up to 95% of these children not reacting to a subsequent sting (Valentine et al. 1990).

Latex Since its resurgence in 1979, several epidemiologic and mechanistic studies have been performed which have contributed to knowledge about at-risk groups and prevent the number of anaphylactic events in healthcare facilities. The estimated incidence of latex sensitivity in the general population ranges from 0.8 to 6.5%, but has been reported as high as 72% in patients who required several surgical operations, especially patients with spina bifida or urinary congenital malformation (Gerald et al. 2000; Lieberman 2002). Healthcare workers, mainly those who work in operating rooms, are also

Anaphylaxis

a group that is at risk. Interestingly, the incidence of intraoperative anaphylaxis caused by latex is increasing and is probably the second most important cause of intraoperative anaphylaxis after muscle relaxants. Obstetric and gynecologic procedures are the most frequent type of surgical procedures associated with reactions to latex, with a frequency of 50% of all reactions. Abdominal operations represent approximately 20% of reactions to latex and orthopedic operations 10%. Other medical procedures, such as the insertion of catheters, dental procedures and childbirth, are also associated with reactions to latex. Atopy condition associated with latex allergy. When genetic predisposition is combined with exposure, the risk is greatest. Moreover, the latex–fruit syndrome is a well-defined disorder that affects 20–60% of patients with latex allergy, depending on the study considered (Blanco 2003). It is now well established that many patients with latex allergy are also sensitized to certain plant foods. Banana, avocado, chestnut, and kiwi are the main fruits implicated, but tomato, papaya, potato, and other plant foods seem to be involved also. Latex sensitivity is covered in more detail in Chapter 53.

Foods In the last decade, several studies have been carried out on food allergy, with a significant improvement in the diagnosis and management of food allergy. Food allergy is one of the leading causes of anaphylaxis and, like other atopic disorders, it appears to be increasing. Munoz-Furlong et al. (2004) found in a recent study in the USA that 3.5–4% of the population suffered from IgE-mediated food allergies. Food allergies are one of the most common causes of anaphylaxis and, depending on the study, the single most common cause. It is estimated that about 100 fatal cases of food-induced anaphylaxis occur in the USA each year (Sampson 1999). Interestingly, most food-induced anaphylactic reactions are not associated with major increases in serum tryptase. Factors associated with severe food reactions include asthma, history of previous severe reactions, denial of symptoms, and delay to initiation of therapy. Severe respiratory compromise is associated with more severe anaphylactic reactions. Approximately 6% of young children and 3.7% of adults in the USA have a food allergy (Sicherer & Sampson 2006). The most common causal foods in children are cow’s milk (2.5%), egg (1.3%), peanut (0.8%), and wheat, soy, tree nuts, fish and shellfish (all < 0.4%). About 80% of early childhood allergies (milk, egg, soy, and wheat) resolve by age 5 years. Peanut, tree nut, and seafood allergies are usually permanent, but 20% of young children with peanut allergy may outgrow it by school age. In adults, the most common causal foods are shellfish (2%), peanut (0.6%), tree nuts (0.5%), and fish (0.4%). Approximately 5% of adults develop fruit and/or vegetable allergy, but it is usually not severe. As seeds are being increasingly used in food manufacture, reactions to

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them are more frequently reported. Anaphylaxis to carmine dye, reported as an occasional reaction to the red coloring in foods, is often overlooked . Peanut is one of the leading causes of food-allergic reactions in the USA, and in the UK allergy clinics also report an increasing number of peanut-allergic patients. Peanut (Arachis hypogaea) is a member of the prolific legume family and is closely related to peas, beans, soybeans, and kidney beans, although cross-reactivity is rare. Peanuts of the genus Arachis have been cultivated since 2000–3000 BC, but during the last decade demand for peanuts has steadily increased, because of their value as a source of easily digestible proteins and their versatility. Peanut kernels contain 45% oil, 25% protein, 5% moisture, 3% fiber, and 2.5% ash. The peanut skin contains 49% carbohydrate and 19% fiber. Peanut proteins are classified as water-soluble albumins and saline-soluble globulins. The globulins are subdivided into arachin and conarchin fractions, which comprise the major storage proteins. The components of the albumin fraction are agglutinins, lectin-reactive glycoproteins, protease inhibitors, α-amylase inhibitors, and phospholipases. The major fractionated allergens, as confirmed by skin testing, basophil histamine release, RAST inhibition and immunoblotting, are peanut 1, concanavalin-A-reactive protein, Ara h 1 and Ara h 2. Biologically active peanut 1 glycoprotein was isolated by Sachs et al. (1981) and found to account for some of the allergenicity of peanuts by RAST inhibition assays. It comprises two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with apparent molecular masses of 20 and 30 kDa. The concanavalin-A-reactive glycoprotein is biologically active and has a monomeric molecular mass of 65 kDa. Ara h 1 and Ara h 2 are two biologically active allergens of molecular masses 63.5 and 17 kDa, respectively. Unfortunately, death due to peanut allergy, even when recognized, is not separately registered by the Office of Population Censuses and Surveys, and accurate statistics of the dangers that may face peanut-allergy sufferers in the UK are not available. From 1992 to 1994, there were at least six cases of known peanut-allergy sufferers who died after eating peanuts (Chief Medical Officer 1994). Assem et al. (1990) described four cases of severe anaphylactic reactions to peanuts, two of whom died, and Sampson et al. (1992) demonstrated that peanut was involved in three of six cases of fatal food allergies and in one in seven cases of near-fatal food allergies. In a survey of Colorado emergency departments, Bock (1992) found that peanuts accounted for one-third of anaphylactic reactions to foods. For the vast majority of the population, peanuts pose no problems, but their wide availability and the fact that peanuts are used in many foods may help to account for the apparent increase in peanut allergy and associated illnesses. Lack et al. (2003) found that sensitization to peanut protein may occur in children through the

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application of peanut oil to inflamed skin, and the association with soy protein could arise from cross-sensitization through common epitopes. Unlike other food sensitivities, patients with severe peanut hypersensitivity rarely outgrow their allergy, which often causes a more severe reaction than other food allergies (Sampson 1990). Peanuts are legumes but cross-reactivity to other legumes such as soybeans seem to be rare; however, multiple nut sensitivity is more common in this group and should be tested for. The wide range of reactions to peanuts may depend on whether the reaction is caused by contaminated hands or by eating or inhaling peanuts or products that contain peanuts. As regards peanut oil, although most patients with peanut allergy avoid ingesting peanut oil, highly processed oils can be tolerated by peanutallergic patients since they do not contain peanut protein. Nevertheless, cold-pressed or extruded peanut oils might contain peanut protein and induce allergic reactions (Sampson 2002). In sensitive subjects, a minute quantity of allergen can lead to a life-threatening response. The commonest symptoms in the very young are acute abdominal pain and vomiting. This may be followed by oral edema and swelling of the face, generalized rash, hypotension, difficulty in breathing, and anaphylaxis. Similar symptoms may occur at any age, but hypotension and anaphylaxis can occur without warning. All patients suspected of suffering from a peanut allergy should be referred to a specialist clinic and should on no account be advised to test their reaction by eating peanuts. The diagnosis is based on clinical history, demonstration of an IgE-mediated reaction and, if crucial, food challenge. It is important to note that challenge or provocative trial with peanut is contraindicated if the patient’s history indicates severe anaphylactic reactions. History alone is unreliable except for that of major immediate anaphylactic reactions. Elimination diets and food diaries are not as helpful as one might wish. SPT is more reliable in children and is positive in 50–70% of patients presenting a confirmed allergic reaction. SPT is very sensitive and has an excellent negative predictive value (i.e., if the SPT is negative, then the patient rarely has an IgE-mediated food allergy to that antigen), but has poor specificity and positive predictive value. Treatment is supportive (see below) and as yet there is no place for desensitization in peanut-allergic patients, although novel therapies for IgE-mediated food allergy are being studied. In a multicenter study, injections of anti-IgE antibodies (TNX-901) for treatment of patients with peanut allergy showed an increase in the average amount of peanut tolerated, although 25% of the group showed no improvement (Leung et al. 2003). Other studies of traditional Chinese herbs showed efficacy in a murine model of peanut-induced anaphylaxis (Li et al. 2001). Standard immunotherapy for pollen-induced rhinitis might improve pollen-food allergy syndrome (Oppenheimer et al. 1992). Bolhaar et al. (2004) also studied the effect of birch-pollen immunotherapy for

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decreasing allergy to foods containing Bet v 1-homologous allergen. Confirmation studies in these fields are needed. Several approaches for delaying or preventing food-induced allergy through dietary manipulation have been studied, although they remain inconclusive. Food allergens are passed to the newborn through breast milk, but we lack knowledge about the tolerizing or sensitizing effects on the newborn’s immune system. A conservative approach was suggested by the American Academy of Pediatrics (AAP Committee on Nutrition 2000). They recommended that children with a high risk of allergy should delay the introduction of solids beyond the sixth month of life in order to prevent food sensitization, dairy products after the age of 1 year, eggs until 2 years, and peanuts, nuts and seafood after the age of 3 years. However, this dietary recommendation has been recently amended due to the lack of evidence about the preservative effect of dietary restrictions after the age of 4–6 months (Høst et al. 2008). Avoidance of peanuts and peanut products is the best way to prevent known peanut allergy, but is often difficult to achieve. Food manufacturers and shopkeepers are becoming more aware of the problem and are now more ready to give helpful advice on avoidance. Eating out remains a problem and, in unfamiliar surroundings, the best policy is to speak with the person preparing the food. No special advice is necessary during pregnancy but if a mother who is not affected herself already has a child with peanut sensitivity, it seems reasonable to avoid peanuts during pregnancy and lactation and to select infant foods that do not contain peanuts. One explanation to account for a proportion of sudden unexplained postperinatal deaths (sudden infant death syndrome or SIDS) is the so-called “anaphylaxis hypothesis.” The basic premise of this hypothesis is that infants become sensitized to protein components of cow’s milk while feeding and that during sleep the fatal reaction occurs following gastroesophageal reflux and aspiration of a small amount of recently ingested cow’s milk into the lungs (Parish et al. 1960a, 1964; Coombs & Holgate 1990). Until recently, evidence for this hypothesis was largely circumstantial, although there are compelling features which command attention. Direct support for the operation of an anaphylactic reaction in cot death was hampered by the inability to obtain adequate evidence of mast cell activation, either by assay of specific chemical markers or by assessment of cell ultrastructure (Coombs & Holgate 1990). However, some evidence for the anaphylaxis hypothesis comes from studies in an animal model, in which guinea pigs fed cow’s milk in their drinkingwater can, if challenged with very small quantities of cow’s milk (e.g., 0.1 mL), be shown to undergo a fatal anaphylactic reaction that bears certain similarities to sudden unexplained death in infants (Parish et al. 1960, 1963; Devey et al. 1976). Holgate et al. (1994) and Platt et al. (1994) were able to show that serum concentrations of the specific mast cell marker

Anaphylaxis

tryptase were elevated in SIDS, suggesting that mast cell degranulation, possibly as a result of anaphylaxis, may have occurred around the time of death in some instances. An immunohistologic study by Howat et al. (1994), who showed an increase in peribronchial mast cells in SIDS, was consistent with this view. However, new data on infant death suggests morphophysiologic abnormalities as predisposing factors for SIDS (Ozawa & Takashima 2002).

Proteins used in therapy Immunotherapy for allergic diseases is among the most common triggers of mild anaphylaxis, in that this treatment is based on progressively increasing the dose of antigens given to sensitive individuals, and before commencing treatment it is necessary to assess the risk–benefit ratio and to consider both the incidence of side effects and the spontaneous cure rate. Allergen immunotherapy vaccines and intradermal and skin testing are, in general, safe procedures if carried out by experienced personnel in the presence of facilities for emergency resuscitation. In a study of fatalities from immunotherapy and skin testing since 1945, Lockey et al. (1987) identified 30 cases in which there was sufficient information for analysis. Risk factors identified were administration during seasonal exacerbations or when symptomatic from underlying asthma, errors of administration, and the concomitant use of beta-blocking agents. Of the 24 fatalities associated with immunotherapy, four had experienced previous reactions, 11 manifested a high degree of sensitivity, and four had been injected with newly prepared extracts. Of the six reactions associated with skin testing, five were due to intradermal testing without prior puncture testing. Allergic systemic reactions are associated with positive venom skin tests but are unusual and rarely severe. In the Hymenoptera venom study, of 3236 patients who underwent skin testing, 1.4% suffered systemic reactions, of which 0.25% were considered severe (Lockey et al. 1989). Other authors have reported a higher incidence of side effects. In a prospective study in the UK, systemic reactions affected 23% of all wasp-venom injections and 10% of bee-venom injections, although many of these reactions were of a mild nature (Ewan & Stewart 1993). Side effects are more likely to occur during the induction phase of treatment and, in general, are commoner with bee venom.

Rare IgE-mediated agents Anaphylaxis can potentially occur to any foreign protein. From time to time, novel or unusual anaphylaxis triggers are reported. A thorough clinical history and investigations is helpful to document possibly novel anaphylaxis triggers. Due to their more frequent use, there is an increased incidence of reactions to anticancer chemotherapy drugs, such as cisplatin and carboplatin, and/or the solvent in which these drugs are prepared. Reactions to Cremophor-L has increased in the last few years (Moreno-Ancillo et al. 2003). Skin testing to study

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allergy to these agents and desensization protocols, if necessary, are currently offered by specialists (Markham et al. 2003). Dialysis membranes may also cause anaphylaxis by an IgEdependent mechanism. Grammer and colleagues have demonstrated increased levels of IgE directed against ethylene oxide-altered human serum albumin in the sera of 24 patients who experienced anaphylaxis during hemodialysis compared with 41 patients who did not experience such reactions (Grammer et al. 1985; Grammer & Patterson 1987). Ethylene oxide is used to sterilize membranes. Dialysis membranes also directly activate the plama bradykinin-forming cascade. (Shulman et al. 1993). Mittman et al. (1990) have reported postcoital anaphylaxis in a 24-year-old woman that was prevented by the use of condoms. SPT confirmed sensitization to five Sephadex G-100-separated fractions of her husband’s seminal plasma. Leukocyte histamine release studies revealed 100% release to one fraction and 37% release to a second fraction. These fractions were therefore pooled and used for rapid immunotherapy over a 2-day period and maintenance immunotherapy was continued three times weekly for 4 months until the patient could tolerate 100 mg of both fractions. At the end of this time, coitus was safely resumed and immunotherapy was discontinued.

Non-IgE-mediated (anaphylactoid) reactions The mechanisms of non-IgE-mediated anaphylactoid reactions are not well understood. There are a number of possible pathways whereby substances may bypass the antibody– antigen reaction and directly or indirectly stimulate mast cells. Some anaphylactoid reactions may be mediated by other inflammatory mechanisms that are independent of mast cells. Examples of possible mechanisms include (i) immune complexmediated; (ii) activation of coagulation or fibrinolysis systems, with the resultant generation of kinins, such as bradykinin, and the anaphylatoxins C5a and C3a; (iii) activation of the complement cascade to generate anaphylatoxins; and (iv) modulation of arachidonic acid metabolism in favor of the generation of proinflammatory mediators.

Immune complexes Anaphylaxis may occur during the infusion of blood, gammaglobulin or serum products such as cryoprecipitate (Ellis & Henney 1969; Burman et al. 1973). Such reactions are thought to be associated with immune complex formation and subsequent complement activation via the alternative and classical pathway. The clinical and physiologic signs of anaphylaxis are closely mimicked by acute complement activation, with the release of vasoactive amines, oxygen radicals, mast cell degranulation, increased arachidonic acid and leukotriene metabolism (Bolt & Herman 1983), and altered vascular permeability. Immune complexes, especially those containing IgA, are known to activate the alternative complement pathway, and IgG aggregates are able to mimic

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clinical anaphylaxis (Christian 1960). Patients who lack IgA (1 in 700 of the population) form IgG antibodies against IgA in about 50% of individuals. Such antibodies may also occur in patients lacking only a subclass of IgA and in patients who have been previously transfused (Vyas et al. 1975). If such patients are transfused with blood containing IgA, or therapeutic preparations of IgG, which may also be contaminated with IgA, immune complexes of recipient IgG and donor IgA form and are demonstrable in these individuals in association with anaphylactic reactions (Wells et al. 1977).

Complement-mediated reactions The alternative pathway of complement may be activated by IgA-containing immune complexes (see above). Complement may also be activated by cuprammonium cellulose, a material used for hemodialysis membranes, with the generation of C3a and C5a and mast cell degranulation (Hakim et al. 1984). Radiocontrast media are also known to activate the complement cascade, both in vivo and in vitro, with the consequent generation of anaphylatoxins. This is a short-lived phenomenon, with demonstrable loss of complement components occurring immediately after administration and with complement levels returning to normal levels within 30 min (Freyia et al. 1982). Unlike the activation produced by immune complexes, that induced by radiocontrast media does not progress in an orderly sequential manner and is independent of calcium and magnesium. Several components may be cleared simultaneously and there is evidence for the secondary generation of plasmin-like activity (Lieberman et al. 1987). Strongly ionic contrast media appear to act by altering the conformation of C3 and C4 through disruption of their thiol–ester bond, resulting in conformations similar to C3b and C4b, with a similar ability to activate further components. Nonionic agents may act in different ways. Metrizamide interferes with normal inhibition of the complement pathway and also acts directly on C2 (von Zabern et al. 1984). Complement activation is also seen in other clinical syndromes that share some features with anaphylaxis. Hereditary angioedema is inherited as an autosomal dominant trait. A deficiency in the activity of C1 esterase inhibitor, either due to absence or to a functionally inactive inhibitor, results in elevated bradykinin levels, the mediator of the swelling, which cause unpredictable acute episodes of angioedema and may result in asphyxia and death due to laryngeal edema in up to 30% of patients (Frank et al. 1976; Fields et al. 1983).

Activation of the coagulation pathways Activation of factor XII (Hageman factor) by complex insoluble materials such as proteoglycans containing heparan sulfate and chondroitin sulfate have been described (Kaplan et al. 2002). Such activation results in the generation of kinins, primarily bradykinin, which is a potent vasodilator, as well as activation of the complement cascade, generating C5a and C3a. This mechanism of mediator release has been demonstrated to

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occur with radiocontrast media (Lieberman et al. 1987) and has been postulated as a cause of some anaphylactoid mechanisms.

Anaphylaxis

if restaurants or processed foods are implicated. Challenges with oral sulfiting agents indicate that, in contrast to asthma, anaphylaxis is a rare consequence.

Direct activation of mast cells A variety of agents have been shown to cause histamine release from mast cells and basophils by pharmacologic mechanisms. Both hypoosmotic and hyperosmotic stimuli induce histamine release (Lasser et al. 1987). Hyperosmolar solutions, such as mannitol, 50% dextrose, and radiocontrast media (Rice et al. 1983), have also been shown to cause histamine release in vitro. The risk of anaphylaxis may be reduced by decreasing the infusion rate of hypertonic solutions. Other agents recorded as causing histamine release directly include opiates (Schoenfeld 1960), muscle relaxants (Fisher 1975), and dextran (Hedin et al. 1976).

Modulation of arachidonic acid metabolism The ability of aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) to induce bronchospasm, urticaria or anaphylactic reactions has been recognized for some time and has been estimated to occur in approximately 1% of individuals (Chafee & Settipane 1974; Szczeklik 1987). The ability of NSAIDs to induce anaphylaxis appears to be related to their potency as inhibitors of the enzyme cyclooxygenase rather than to similarities of structure. This may affect the balance of proinflammatory mediators in two ways: (i) by preferential metabolism of arachidonic acid by the lipoxygenase pathways to form leukotrienes, which are potent proinflammatory mediators, causing bronchoconstriction and increased vascular permeability; and (ii) by decreasing production of prostaglandin (PG)E2, which acts as a bronchodilator.

Sulfite sensitivity Sulfiting agents (sodium and potassium sulfites, bisulfites, metabisulfites and gaseous sulfur dioxide) are added to foods as preservatives in order to prevent discoloration. Foods to which these substances are added in the highest concentrations include leafy green salad (particularly at salad-bar restaurants); light-coloured fruits and vegetables (particularly dried fruits, such as apples or golden raisins, and instant potatoes); wine, beer, dehydrated soups, fish and shellfish (particularly shrimp); and rapidly perishable foods, such as avocados. Sulfites are also used as preservatives in a variety of medications. Ingestion of sulfites may produce asthma and anaphylaxis in susceptible persons (Nicklas 1989). The mechanisms may involve conversion of the sulfites in the acid environment of the stomach to SO2 and H2SO3, which are then inhaled. Asthmatic individuals can develop bronchospasm to concentrations of SO2 below 1 ppm. The magnitude of the problem is unclear, but provocation challenges indicate that less than 10 mg of potassium metabisulfite can cause asthma and collapse in a proportion of the asthmatic population (< 5%). Sulfite sensitivity should be suspected in individuals who relate asthmatic symptoms to eating, particularly

Multiple mechanisms Some agents causing anaphylaxis may work via more than one mechanism. One example is the anaphylaxis during general anesthesia, the intraoperative period, and the postoperative period. Due to the use of several drugs during general anesthesia, it is difficult to differentiate between immune/ nonimmune, mast cell, and pharmacologic effects. For example, radiocontrast media may cause mast cell activation by a combination of direct osmotic effects, complement activation and activation of the bradykinin-forming cascade, and possibly immunologic mechanisms. Any of these mechanisms may contribute to the final common pathway of mast cell degranulation and mediator release. Yocum et al. (1999) has reported the incidence of anaphylaxis during anesthesia to be between 1 in 4000 and 1 in 25 000. The mortality from anaphylaxis related to anesthesia is estimated to be up to 6%. In general surgery, the most common cause of anaphylaxis during anesthesia is muscle relaxants, with an incidence as high as 60%. This is followed by latex, with an estimated incidence of 17%. Interestingly, latex-induced reactions are usually delayed compared with muscle relaxant reactions, which typically occur during the earlier stage of the general anesthesia procedure. Other causes of anaphylaxis or anaphylactoid reactions related to anesthesia are antibiotics (β-lactam), induction agents or hypnotics, opioids, colloid blood products, protamine, isosulfan blue dye for lymph node dissection, gelatin solution, chlorhexidine, ethylene oxide, radiocontrast media, streptokinase, methylmethacrylate, and chymopapain (Mertes et al. 2003; Thong & Yeow-Chan 2004). Life-threatening muscle reactions are usually caused by specific IgE, although most reactions occur because of direct mast cell activation and are less severe. A meticulous medical history focusing on prior adverse reactions and, if necessary, skin testing to study allergy to general anesthetic drugs and latex are crucial.

Anaphylaxis of unknown mechanism Exercise Strenuous exercise may lead to anaphylaxis in susceptible individuals (Sheffer & Austen 1980; Sheffer et al. 1983). This reaction can be differentiated from exercise-induced asthma and cholinergic urticaria by the frequency with which the responses follow exercise and by the symptom complex initiated. In the asthmatic individual, exercise (especially in the cold) regularly causes asthma, whereas individuals with exercise-related anaphylaxis (EIA) experience the reaction only intermittently. Cholinergic urticaria usually appears following any stimulus which causes the patient to sweat. Lesions are small punctate pruritic rashes that may spread all over the body. Cholinergic urticaria appears rapidly and lasts

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30–60 min, and is easily reproducible by exercise challenge test. In contrast, patients suffering from EIA syndrome may develop pruritus and giant hives, followed by hypotension and cardiovascular collapse if they do not receive emergency treatment. The response resembles anaphylaxis in every respect, including elevated urine and plasma histamine levels and serum tryptase levels. It is usually hard to reproduce by exercise challenge test. Attacks are more likely in hot humid weather and may be precipitated by exercise ranging from tennis warm-ups and jogging to sprinting in track events. Food is associated with EIA in about 37% of all cases (Shadick et al. 1999). Approximately 50% of individuals are atopic and some familiar occurrences have been reported (Sheffer et al. 1983). Food-dependent EIA often necessitates the prior ingestion within 2–4 hours of certain substances for its development, but when the food intake and the exercise are not associated, there are no symptoms. Factors associated with food-dependent EIA are positive skin testing to the food which triggers the reaction, asthma, and/or other atopic diseases. It is twice as common in females as males. Wheat, milk, shellfish, fruit, celery, fish and other foods have been implicated as triggers, although the mechanism of this disorder is still unknown (Sampson 1999).

Idiopathic anaphylaxis A group of subjects who recurrently experience anaphylaxis with no apparent recognized cause has been identified (Wiggins et al. 1988). Idiopathic anaphylaxis symptoms are indistinguishable from other anaphylactic reactions. The diagnosis of idiopathic anaphylaxis is a diagnosis of exclusion. Therefore, a detailed clinical history and appropriate laboratory tests are crucial. Other causes of anaphylaxis (e.g., food allergy) and/or underlying diseases, such us mastocytosis, should be ruled out. Serum tryptase level may be helpful for excluding systemic mastocytosis, as the baseline level of total tryptase can be increased (Schwartz & Irani 2000). Other rare cause of repeated episodes of anaphylaxis without a readily identifiable etiology include catamenial anaphylaxis, a syndrome of hypersensitivity to endogenous progesterone secretion (Meggs et al. 1984; Slater et al. 1987). Some, but not all, patients with catamenial anaphylaxis exhibit a cyclical pattern of attacks that intensifies during the luteal phase of the menstrual cycle. These patients have positive skin tests to medroxyprogesterone, experience systemic reactions to infusions of luteinizing hormone-releasing hormone (LHRH), and respond favorably to ovarian suppression with LHRH agonists or oophorectomy (Slater et al. 1987).

Pathogenesis The essential common factor in anaphylaxis, whether IgE- or non-IgE-mediated, is activation of mast cells and circulating

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basophils, with the release of biochemical mediators and chemotactic substances. Biochemical mediators include preformed granule-associated substances, such as histamine, tryptase, chymase, and heparin; histamine-releasing factor and other cytokines; and newly generated lipid-derived mediators such as PGD2, leukotriene (LT)B4, platelet-activating factor (PAF), and the cysteinyl leukotrienes LTC4, LTD4, and LTE4. The mast cell is capable of releasing a wide variety of mediators with effects on bronchial and vascular smooth muscle and on vascular permeability. In addition, many of these mediators are chemotactic and proinflammatory for other inflammatory cells, such as eosinophils, which are prominent in allergic reactions. Once recruited, these cells are, in turn, able to amplify the inflammatory response by secondary generation of mediators. Newly generated mediators are not present in the stored form but are rapidly generated following cell activation. Their in vitro actions as proinflammatory and chemotactic mediators are coupled to their action on smooth muscle, mucus secretion, and vascular permeability. Histamine was the first preformed mast cell mediator to be recognized and has been identified in increased amounts in the blood of patients with anaphylaxis (Sheffer et al. 1983). Histamine activates H1 and H2 receptors which might cause pruritus, rhinorrhea, tachycardia, bronchospasm, headache, flushing, and hypotension. Subcutaneous injection of histamine increases vascular permeability in postcapillary venules. An infusion of histamine in normal volunteers can lead to a number of pathophysiologic changes that can mimic anaphylaxis. Flushing, tachycardia, and headache are seen as serum levels of histamine approach 4 ng/mL (Kaliner et al. 1981, 1982). Higher levels are needed to induce hypotension and lower systemic vascular resistance, although the development of bronchospasm does not appear to correlate with plasma histamine levels. Serum histamine levels correlate with the severity and persistence of cardiopulmonary manifestations and gastrointestinal symptoms, but not with urticarial symptoms (Lin et al. 2000a). Histamine binding to H1 receptors during anaphylaxis also stimulates endothelial cells to convert the amino acid L-arginine into nitric oxide (NO), a potent autacoid vasodilator (Mitsuhata et al. 1995). Enhanced NO production decreases venous return, which contributes to the vasodilation that occurs during anaphylaxis. Tryptase plasma levels correlate with the clinical severity of anaphylaxis (Schwartz et al. 1994). β-Tryptase levels in blood are considered a measure of mast cell activation; in contrast, α-tryptase appears to be secreted constitutively. Unlike the half-life of histamine in the blood, the half-life of tryptase is in the order of several hours, making it more useful for the retrospective detection of anaphylactic events. A human study of insect sting anaphylaxis due to bee venom demonstrated efficacy of venom immunotherapy rather than the whole body extract and showed not only massive release of histamine and other products of mast cell

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and basophil degranulation but also total digestion of plasma high-molecular-weight kininogen so as to release about one-third of the total plasma bradykinin available as well as consumption coagulopathy (Smith et al. 1980). This may be occurring in all severe anaphylactic reactions. Recent studies with murine models have revealed two distinct mechanisms inducing anaphylaxis (Finkelman et al. 2005). The first classical pathway results from activation of IgE bound to mast cell FcεRI causing the rapid release of vasoactive mediators. These mediators increase vascular permeability and deplete intravascular volume. A second pathway is IgE independent and cause systemic anaphylaxis in mice by forming complexes with IgG that cross-link macrophage FcγRIII, stimulating PAF release, and occurs even in the absence of mast cells, FcεRI, and IgE. These results cannot be extrapolated simply to humans and further research is required.

Clinical and biochemical features (Table 92.4 and Fig. 92.2) Anaphylaxis is the most serious emergency in allergic disease. Although individuals may vary greatly in onset, clinical manifestations, and time-course, the hallmark of the anaphylactic reaction is the onset of symptoms within seconds or minutes of exposure to the precipitating agent, depending on the sensitivity of the person and on the route, quantity and rate of administration of the antigen. The initial symptoms and signs are often sensations of warmth, pruritus and tingling, especially of the hands, feet, groins and axillae, pharyngeal edema, tongue swelling, accompanied by a generalized flush. Subjects commonly feel an overwhelming sense of impendTable 92.4 Clinical features of acute systemic anaphylaxis: acute temporal relationship between exposure and clinical symptoms and signs. Initial symptoms Warmth, pruritus, tingling of extremities and groin Feeling of doom, extreme anxiety Abdominal cramps and diarrhea Faintness Cutaneous manifestations Generalized erythema Generalized urticaria Angioedema Respiratory manifestations Upper airway obstruction (stridor) Lower airway obstruction (asthma) Cyanosis and eventual asphyxia Cardiovascular manifestations Circulatory shock due to lowered peripheral resistance and depressed myocardial function

Anaphylaxis

Dizziness, seizures, loss of consciousness, death as a result of hypotension

Patients frequently report severe prodromal anxiety described as a sense of impending doom

Fatal arrhythmias and infarcts have been documented Nausea, vomiting, abdominal cramps and diarrhea

Urticarial eruptions are common during the acute phase

Swelling of lips and tongue can impede breathing or swallowing

Laryngeal edema may cause upper airway obstruction Acute bronchoconstriction of lower airways can cause severe hypoxemia and contribute to hypotension

Tingling of hands and feet is a common warning symptom of impending anaphylaxis

Fig. 92.2 Clinical features of anaphylaxis.

ing doom and may complain of abdominal cramps and a feeling of faintness. Cutaneous effects are not life-threatening and may progress from a flush through to generalized urticaria or angioedema over several hours. Such effects are transient and will resolve within 24 hours. Gastrointestinal disturbance may progress from the initial cramping pains and nausea to profuse vomiting and diarrhea. The most serious manifestations of acute anaphylaxis are those affecting the respiratory and cardiovascular systems. Together with the initial flush and angioedema, these form the most common manifestations of anaphylaxis, with one series of 276 patients studied by Fisher and Baldo (1988) finding cardiovascular collapse in 92%, erythema in 48%, bronchospasm in 29%, and angioedema in 24%. In another series, Delage and Irey (1972) found that 70% of fatalities were due to respiratory complications and 24% due to respiratory collapse. Respiratory tract involvement may take the form of upper airway obstruction due to edema of the larynx, epiglottis, or surrounding tissues. Lower airway obstruction is due to bronchospasm and edema. Initial symptoms and signs of upper airway obstruction include hoarseness, dysphagia, a sense of fullness or constriction of the throat, and the development of respiratory stridor. Lower respiratory tract manifestations

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include wheezing, coughing, chest tightness, and increasing shortness of breath. Both may progress to asphyxia. Cardiovascular collapse is often rapid in onset, with a feeling of faintness and retrosternal pain, followed by syncope, due either to a combination of vasodilation and reduced cardiac output or to asphyxia. Myocardial infarction is a recognized phenomenon of anaphylactic shock (Levine 1976). It is important to note that up to 20% of patients treated for an initial episode of anaphylaxis may have a further life-threatening event up to 8 hours after the apparent remission of symptoms, so-called biphasic reactions (Stark & Sullivan 1986). This phenomenon may reflect the release of secondary mediators from recruited inflammatory cells, a situation similar to the late-phase asthmatic response seen after bronchial antigen challenge in sensitized subjects. It has also become clear that a number of factors may increase the severity of anaphylaxis or interfere with resuscitative therapy. Ongoing treatment with beta-blocking drugs or the presence of bronchial asthma worsens the response of the airways in anaphylaxis and may make resuscitation attempts more difficult. In addition, the use of epinephrine in patients taking beta-blockers may lead to unopposed αadrenergic effects and result in severe hypertension (Toogood 1987). Other factors that may increase the risk of severe anaphylactic reactions include rapid intravenous infusion of an allergen and preexisting cardiac disease. In the last few years, the clinical diagnosis of anaphylaxis can be supported by different laboratory tests. For instance serum tryptase, serum histamine, and urinary histamine metabolites may be increased during an anaphylactic reaction. Serum tryptase seems to be more helpful than histamine levels since it can be raised 1– 6 hours after the onset of the reaction, compared with 15 min for histamine (Tannenbaum et al. 1975; Smith et al. 1980; Lin et al. 2000a). There are two main types of mast cell tryptase, α-tryptase and β-tryptase. α-Protryptase is secreted constitutively from mast cells as an inactive proenzyme and is the major form of tryptase found in the blood of normal subjects. β-Tryptase is released from mast cells in parallel with histamine, although on some occasions only one of these mediators is increased. In both anaphylaxis and systemic mastocytosis, serum β-tryptase levels may be increased. To distinguish between these two events, a ratio of total tryptase (α plus β) to β-tryptase of 10 or less is indicative of an anaphylactic episode and not related to systemic mastocytosis, whereas a ratio of 20 or greater is consistent with systemic mastocytosis (Lieberman et al. 2005). High tryptase concentrations can be found in serum obtained from patients up to 3 days after death from suspected anaphylaxis. It has been reported that postmortem levels of β-tryptase are elevated in victims of SIDS. However, postmortem level of serum tryptase by itself cannot be used to diagnose anaphylactic death with no other clinical evidence as it might be increased in death related to trauma or other illness (Edston & van Hage-Hamsten 2003). The value of

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Table 92.5 Differential diagnosis. Vasovagal attacks Hereditary angioedema Systemic mastocytosis Cholinergic urticaria Cold urticaria Panic attacks Autonomic epilepsy Pheocromocytoma Carcinoid syndrome Thyroid tumours Scombroid fish poisoning

other biomarkers, such as mast cell carboxypeptidase, is also being investigated (Zhou et al. 2006).

Differential diagnosis (Table 92.5) The combination of symptoms and signs rarely leaves the diagnosis in doubt in the acute situation, given the constellation of an acute exposure to a provocative agent followed, usually within minutes, by the evolution of multisystem manifestations, including flushing, urticaria, pruritus and edema. However, in some cases, the initial manifestation may be loss of consciousness and, in these cases, the possibility of an arrhythmia, pulmonary embolus, acute respiratory obstruction due to inhalation, acute anxiety, seizures and/or hypoglycemia must be considered. Vasovagal attacks are probably the condition most commonly confused with anaphylactic and anaphylactoid reactions, but they are not accompanied by urticaria or any evidence of respiratory obstruction, such as stridor or wheeze. In addition, there is pallor rather than evidence of vasodilatation, the heart rate is typically bradycardic, and symptoms are rapidly relieved by lying flat. However, distinction between the two is important, e.g., in the setting of medical procedures, such as the administration of radiocontrast agents, as the inappropriate administration of atropine can worsen the progression of anaphylaxis. Most reactions to local anesthetics fall into this category of vasovagal attacks. If laryngeal edema is the presenting problem, hereditary angioedema must be considered. This disorder is usually inherited (although a smaller proportion of the population may acquire the defect) and is accompanied by painless (and pruritus-free) angioedema, gastrointestinal cramps and distension, recurrent attacks and, usually, a family history of similar attacks, sudden death or both. Hereditary angioedema is not associated with flushing, asthma or urticaria, is of slower onset and, in the absence of severe airway obstruction, is not a cause of hypotension. Systemic mastocytosis is a generalized disease of mast cells that may represent an isolated overgrowth of mast cells or be

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associated with another hematologic abnormality, frequently leukemia. Urticaria pigmentosa is a frequently encountered dermal expression of the generalized overgrowth of mast cells, usually seen with the benign form of the disease. In either case, the mast cells may degranulate, generally producing local effects or, more rarely, causing systemic effects exactly like anaphylaxis. Indeed, some patients originally diagnosed as having idiopathic anaphylaxis have later been found to have systemic mastocytosis on bone-marrow biopsy. Currently, discrimination between mature β-tryptase and total serum tryptase is helpful to differentiate anaphylaxis in the setting of systemic mastocytosis, in which the baseline levels of α-protryptase-by measuring the difference between total tryptase and β-tryptase-tend to increase. Degranulation of mast cells can occur spontaneously and after exposure to NSAIDs, alcohol, narcotics, and other nonimmunologic mast cell-degranulating agents. Suspicion of the diagnosis should be raised with the recognition of the classic reddish-brown macular to low papular skin lesions, which urticate on trauma (Darier sign), the history of flushing attacks, evidence of bone involvement (pain, abnormal bone scans, abnormal radiographs), gastrointestinal pain and peptic ulcers, histaminuria, histaminemia, and increased urinary PGD2 metabolites. Bonemarrow biopsy is usually diagnostic. Patients with cold urticaria who develop diffuse reactions after cold-water immersion or similar incidents can present a picture identical to that of systemic anaphylaxis. The only distinguishing feature would be the history of a generalized reaction occurring after cold exposure. Other diseases which involve flushing episodes may mimic an anaphylaxis event and should be considered in the differential diagnosis, such us pheocromocytoma, the carcinoid syndrome, and thyroid tumours. In these conditions, the appropriate laboratory tests to study these diseases are valuable to establish the diagnosis (Lieberman et al. 2005). An important condition to include in the differential diagnosis is scombroid fish poisoning, a toxic reaction to decomposing scombroid fish. The clinical picture may be very similar to anaphylaxis as it is an acute syndrome resulting from consumption of fish containing high levels of histamine, although

Anaphylaxis

serum tryptase levels are normal and other people might be affected at the same time. Local urticaria may be reproduced by applying an ice cube to the forearm for a short time. Some patients with cholinergic urticaria can develop diffuse reactions in response to extreme changes in temperature, which can present a picture identical to that of systemic anaphylaxis, and passive heat challenges are valuable in differentiating this from other forms of anaphylaxis (see above) (Casale et al. 1986).

Management (Fig. 92.3) General principles (Table 92.6) The treatment of anaphylaxis has changed little since the introduction of epinephrine in the early 20th century and the development of antihistamines in the mid-20th century. As is true for all allergic diseases, avoidance of exposure and preventive measures for persons identified as being at high risk continue to be the mainstay of therapy. A careful history often indicates the substance involved, after which sensitivity can be determined by SPT or RAST, although these tests are not completely reliable. In cases of insect-venom sensitivity, patients should be advised to avoid areas in which there is an increased likelihood of insect encounters, always to wear shoes when outside, and to avoid hair sprays, perfumes, after-shave lotions and flowered or brightly colored clothing. Patient education plays an important role in antigen avoidance and, in addition, high-risk patients should be provided with easily injectable epinephrine via a suitable portable system in the outpatient setting and instructed in its effective use. Epinephrine is the treatment of choice for anaphylaxis: it stimulates α-adrenoceptors, reversing peripheral vasodilation and reducing edema and urticaria; as a β-agonist, it dilates the airways, is positively inotropic, and suppresses basophil histamine and leukotriene release. Because the symptoms develop so rapidly and are lifethreatening, controlled trials are difficult to carry out. Treatment should be started early, i.e., within 20–30 min of the challenge and before the more severe and life-threatening features have developed. Soreide et al. (1988) showed in a

Table 92.6 Prevention: general measures. Identify at-risk patients and causative agents by history, skin test, RAST Avoid exposure Patient education Supply with portable and easily injectable epinephrine, e.g., EpiPen autoinjector Adult dose 0.3–1 mg or 0.3–1 mL of epinephrine injection 1 in 1000 (1 mg/mL) Pediatric dose 0.01 mg/kg or 0.01 mL/kg of epinephrine 1 in 1000 (1 mg/mL) Advise Medic-Alert enrolment Optimize management of concurrent diseases, particularly asthma Substitute beta-blockers with another suitable agent Oral treatment with drugs is always preferable to intravenous route Always give intravenous injections slowly

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Assess severity: Airway Breathing Circulation Level of consciousness Stable

Unstable

Observation (individualized) Portable epinephrine Clinical follow-up

Low BP

LAO

UAO

i.m. epinephrine (every 5–15 minutes × 2)

Oxygen i.m. epinephrine (every 5–15 minutes × 2)

Oxygen i.m. epinephrine

R

NR

Observe

NR Plasma expansion (crystalloids and/or colloids)

R

Pulseless: CPR Inotropic support Dobutamine i.v. epinephrine i.v. norepinephrine

Observe

Intubation/ tracheostomy tube +i.v. antihistamines +i.v. epinephrine +i.v. hydrocorstisone

NR i.v. aminophylline NR i.v. epinephrine NR Intubation/ventilation

retrospective study of 27 patients with anaphylaxis occurring outside hospital that all patients treated within 30 min of the onset of the reaction recovered. In contrast, two patients for whom treatment was delayed by more than 45 min died. The UK consensus panel on emergency guidelines and the international consensus guidelines for emergency cardiovascular care both recommend intramuscular epinephrine injections for anaphylaxis (Project Team of the Resuscitation Council UK 1999; Cummins et al. 2000). In a study in children not experiencing anaphylaxis, Simons et al. (1998) demonstrated more rapid absorption and higher plasma epinephrine levels when epinephrine was administered intramuscularly in the anterior lateral thigh compared with values after subcutaneous administration. Similarly, in a study performed in adults not experiencing anaphylaxis, Simons et al. (2001) demonstrated higher and quicker peaking of plasma epinephrine

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NR

Observe

Inhaler/i.v. salbutamol i.v. hydrocortisone

R NR

R

R

Fig. 92.3 Flow diagram outlining acute management of anaphylaxis. BP, blood pressure; LAO, low airway obstruction; UAO, upper airway obstruction; R, responsive to intervention; NR, nonresponsive to intervention; CPR, cardiopulmonary resuscitation.

concentrations after intramuscular injection of epinephrine into the thigh than after epinephrine was injected intramuscularly or subcutaneously into the deltoid muscle. Therefore, the participants of the National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network Symposium (2006) recommended the administration of intramuscular epinephrine in the anterior lateral thigh. However, as noted below, intravenous epinephrine might be preferred in some cases if an intravenous line is in place (e.g., during surgery). For adults, the recommended dose range is 0.3–1 mg, i.e., 0.3–1 mL of epinephrine injection 1 in 1000 (1 mg/mL) (Fisher 1992). For children, the dose is based on body weight and is 0.01 mg/kg, i.e., 0.01 mL/kg of epinephrine 1 in 1000 (1 mg/mL). For most patients, only a single injection is needed, but up to 10% of patients need a second injection and occasionally several doses may be necessary. Second and subse-

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quent doses are given at 5–15 min intervals and are repeated until the patient’s symptoms improve and blood pressure is maintained. Two prefilled syringe preparations are currently available for the self-administration of epinephrine: Min-IJet epinephrine 1 in 1000 is available as a 1-mL disposable syringe with a 25-gauge short (0.25 inch) needle for subcutaneous use and 21-gauge short needle for intramuscular use. The patient has to measure the dose needed, since a full syringe delivers 1 mg. The second type of prefilled syringe is the EpiPen autoinjector, which has a spring-activated concealed needle and is designed to deliver a single 0.3-mg dose of epinephrine subcutaneously (0.3 mL of a 1 in 1000 injection) when the pen is pushed firmly against the outer thigh. This system may reduce fear of self-injection felt by some patients. A child’s version contains a dose of 0.15 mg epinephrine (0.3 mL of a 1 in 2000 injection) (EpiPen Jr). Epinephrine should be stored at room temperature and protected from the light and patients should be aware of the expiry date and be sure that they get a replacement beforehand. In 1997, a self-administered epinephrine aerosol inhaler, Medihaler-Epi, licensed in the UK as an adjunct to the treatment of anaphylaxis, was withdrawn from the market. If venous access is difficult, epinephrine can be given by nebulizer or via an endotracheal tube, but this is still an experimental approach. When treatment has been delayed and shock or severe dyspnea is present, epinephrine can be given intravenously (see below). Furthermore, patients should enroll with Medic-Alert and wear an identity bracelet clearly stating their condition. Concurrent diseases, such as asthma or underlying cardiac disease, should be optimally managed and beta-blockers should be substituted by other agents when clinically indicated. Anaphylactic reactions occurring in patients receiving propranolol have been described as being unusually severe and as having a “sluggish” response to epinephrine (Toogood 1987). In these patients receiving beta-blockers with poor response to epinephrine, intravenous glucagon infusion 1–5 mg (in children 20–30 μg/kg, maximum 1 mg) and isotonic volume expansion should be considered. Greenberger et al. (1986) have shown that the use of beta-blockers or calcium antagonists per se did not increase the incidence of anaphylaxis in a large group of patients who received radiocontrast media while undergoing coronary arteriography. As a general rule, oral treatment should be used in preference to intravenous therapy if possible and intravenous medications should be administered slowly.

Specific preventive measures (Table 92.7) In some situations, the administration of agents known to cause anaphylaxis to persons at high risk becomes medically necessary. Several protocols have been developed to prevent or reduce the severity of reactions in situations in which anaphylaxis appears likely. These protocols involve pharmacologic pretreatment, short-term desensitization, and longerterm desensitization employing immunotherapy. Depending

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Table 92.7 Prevention: specific measures. Pharmacologic prophylaxis: radiocontrast media Short-term desensitization: penicillins Long-term desensitization: Hymenoptera

on the agent involved, each of these protocols has been successfully employed.

Pharmacologic pretreatment Pharmacologic pretreatment has been used in patients sensitive to radiographic contrast media (RCM) (Miller et al. 1975). Pretreatment regimens for prevention of repeat anaphylactoid reactions have consisted basically of oral glucocorticosteroids, H1 and H2 antihistamines, and occasionally ephedrine. A protocol commonly recommended in the past has been 50 mg of prednisone given orally 13, 7, and 1 hour before administration of RCM; 50 mg of diphenhydramine given orally or intramuscularly 1 hour before the administration of RCM; and 25 mg of ephedrine given orally 1 hour before RCM administration. An emergency pretreatment regimen that is used successfully includes hydrocortisone 200 mg i.v. immediately and every 4 hours until RCM is administered, and diphenhydramine 50 mg i.m. 1 hour before RCM (Erffmeyer et al. 1985; Greenberger et al. 1986).

Short-term desensitization Short term desensitization can be tried in cases of penicillin anaphylaxis in those patients in whom it is clinically indicated and there is no alternative drug or in patients with numerous antibiotic reactions (Sullivan 1985). Stark et al. (1987) examined the safety, efficacy and mechanisms of penicillin desensitization in patients with debilitating and life-threatening infections that required therapy with a β-lactam drug. Increasing oral doses of phenoxymethylpenicillin were administered at 15-min intervals to a cumulative dose of 1.3 million units, at which point parenteral therapy with the β-lactam drug of choice was instituted. Most of the patients were successfully desensitized and went on to receive full dose therapy. Side effects occurred in 30% and ranged from pruritus to serum sickness, but therapy had to be discontinued in only one patient with cystic fibrosis, because of worsening wheeze. Skin tests to one or all penicillin determinants became negative in 11 of 15 patients retested after acute desensitization. Chronic desensitization was maintained in seven patients for 3 weeks to 2 years.

Long-term desensitization (immunotherapy) In the UK, long-term desensitization is primarily carried out in patients who are at risk of fatal reactions to bee or wasp venom and in patients with severe treatment-resistant seasonal pollen-induced hay fever. In 1986 the Committee on the Safety of Medicines (CSM) conducted a review of the safety

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of desensitizing vaccines and recommended that desensitization should only be conducted in hospitals or clinics where facilities for cardiopulmonary resuscitation were immediately available and that patients should be monitored for 2 hours after each injection. In the light of recent studies demonstrating that anaphylaxis develops within 30 min and bronchospasm within 1 hour of injection, the CSM now requires patients to be monitored for only 1 hour after injection, providing no signs of hypersensitivity remain. It is recommended that desensitizing vaccines should be avoided in patients who are pregnant, under 5 years old, or taking beta-blockers. Immunotherapy as a means of preventing serious reactions to insect stings was first described in the Proceedings of the Witwatersand Medical Society, published in the South African Medical Record (Braun 1925). The indication for treatment in this case was unavoidable and recurrent stings were associated with severe systemic reactions. The agent used for diagnosis and for treatment consisted of an extract of the macerated portion of the posterior one-third of wholebody extracts. Unfortunately, therapy had to be stopped because of repeated unpleasant reactions to the injections themselves. In the early 1970s, it became apparent that although high venom concentrations were directly toxic for cells in vitro, the blood basophils of venom-allergic patients released histamine in a fashion characteristic of IgE-mediated reactions when exposed to lower, nontoxic concentrations of venom. Very soon thereafter, purified venoms became available for skin testing and for immunotherapy and were found to be much more effective in inducing tolerance to further stings than whole-body extracts. There is now a consensus that venom immunotherapy should be offered to any patient with a history of prior systemic reaction of a potentially life-threatening nature (which, in practice, is equated with any reaction that objectively or symptomatically involves hypotension, airway compromise, or bronchospasm). It is well established that venom immunotherapy using pure venom extracts is highly effective (Hunt et al. 1978; Muller et al. 1979). Numerous placebo-controlled studies between 1978 and 1990 using postintervention sting challenge have demonstrated a reduction in generalized reactions to between 3 and 23% in patients with previous reactions (British Society of Allergy and Clinical Immunology 1993). It has been established in adults and in children that stopping venom therapy after 5 uninterrupted years of treatment is associated with persistent immunity to sting challenges, regardless of the quantities of serum IgE or IgG antibodies present and independent of the state of venom skin-test reactivity (Golden et al. 1986). In addition 200 stings over a 3– 6-year period in 49 children undergoing long-term venom immunotherapy resulted in only four mild systemic reactions (98% efficacy), which argues favorably for the safety of prolonged venom desensitization (Graft et al. 1987). In view of the spontaneous remission rate, some physicians advocate sting challenge before a patient is considered for a

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course of immunotherapy (Muller et al. 1990). Immunotherapy is usually confined to those patients with severe systemic reactions (respiratory and/or cardiovascular reactions) or to those with moderate systemic reactions (urticaria, angioedema, mild asthma, nausea, light-headedness, etc.), depending on the frequency of reactions, likelihood of future exposure, and access to medical services providing venom-specific IgE can be demonstrated. At present, there is no evidence to suggest that venom immunotherapy is indicated in the treatment of large local reactions or unusual systemic reactions such as vasculitis or fever. In children, venom immunotherapy is also confined to those with severe systemic reactions (respiratory and/or cardiovascular reactions) and positive test results to the respective insect with either skin tests and/or specific serum IgE tests. However, venom immunotherapy should not be offered to children with a history of mild generalized reactions (urticaria, erythema, pruritus) since up to 95% of these children do not react to a subsequent sting (Valentine et al. 1990). Elderly patients tend to have more severe reactions and a greater fatality rate from Hymenoptera stings; however, they also have a higher rate of adverse reactions to immunotherapy and respond less well to epinephrine. Except for bee-keepers, the risk of further stings in the elderly is probably lower than that of the general population and therefore immunotherapy is usually not indicated. Immunotherapy should not be started in pregnancy. The position paper of the British Society for Allergy and Clinical Immunology (1993) concluded that it was generally inadvisable to continue maintenance injections during pregnancy, since there is a risk, albeit small, of inducing an anaphylactic reaction with each maintenance injection. If immunotherapy is stopped, it must be recommenced at the lowest dose of venom. In the USA, pregnancy is not regarded as a contraindication to maintenance treatment. Although there are no data about it, one approach is to give half or one-third of the maintenance dose and build back to full dose after delivery. For a patient in the build-up phase, immunothery should be stopped and started after delivery, or if substantially into the build-up phase, maintain half the last dose during pregnancy and build up after delivery.

Management of the acute attack Management of the acute attack can be conveniently divided into management of the airway and cutaneous reactions and management of the cardiovascular reactions. Full cardiopulmonary resuscitation (CPR) should be instigated if the patient is pulseless.

Airway and cutaneous reactions Initial therapy for airway reactions involves supportive and specific therapy for reversing hypoxemia. Severe hypoxemia may occur due to a combination of upper and lower airway obstruction. Oxygen should be given via a face mask if possible; however, intubation and mechanical ventilation may

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be necessary in a severely affected patient. Intubation may not be possible in cases of severe angioedema or laryngeal edema, and a tracheostomy tube may need to be temporarily inserted in order to facilitate oxygen delivery. The percentage of inhaled oxygen should be increased correspondingly in order to maintain PaO2 above 8 kPa. Epinephrine is the drug of first choice for severe reactions and is indicated for severe bronchospasm, laryngeal edema, severe urticaria, and angioedema. A dose of 0.3–1 mg i.m. or s.c. is given, and 0.5 mg is often sufficient. In adults, the drug can be given by slow intravenous injection of 3–5 mL of adrenaline 1 in 10 000 given over 5 min or by continuous infusion using 1 mL of adrenaline 1 in 1000 diluted in 500 mL of 5% dextrose and infused at a rate of 0.25–2.5 mL/min (Bochner & Lichtenstein 1991). For children, the dose is 0.01 mg/kg by slow intravenous injection or by infusion of 0.1–1 μg/kg per min. Heart rate and rhythm should be carefully monitored during intravenous administration, in view of the increased incidence of arrhythmias, myocardial ischemia and infarction. In a study of 227 patients who developed anaphylaxis in hospital after anesthesia, four patients given intravenous epinephrine developed supraventricular tachycardia, which progressed to ventricular fibrillation (Fisher 1986). The goal of therapy is to maintain airway patency and reduce fluid extravasation and pruritus. Drugs of second choice include intravenous salbutamol, aminophylline, corticosteroids, and antihistamines. Salbutamol can be nebulized using an oxygen mask and a disposable jet nebulizer unit. The nebulizing dose is 2.5–5 mg every 2–4 hours. A minimum initial reservoir volume of 4 mL (with additional diluent) and an oxygen flow rate of 6–8 L/min are advocated to optimize nebulizer output. Salbutamol can be given intravenously to patients with more severe symptoms. It is given as a bolus dose of 100–300 μg, or a loading infusion up to 500 μg over 1 hour, followed by an infusion of 5–20 μg/min. The rate of infusion is limited by side effects, particularly tachycardia and tremor. Lactic acidosis, hyperglycemia, and hypokalemia may be associated with both intravenous epinephrine and salbutamol. Lactate levels respond within hours to reduction of the rate of infusion. Aminophylline 5–6 mg/ kg i.v. can also be given slowly over 20–30 min if bronchospasm is unresponsive to epinephrine and/or salbutamol. This can be followed by an infusion of 0.5–1 mg/kg per hour thereafter, remembering to monitor levels 1–2 hours after the loading dose and 24-hourly thereafter, due to the narrow toxic/therapeutic ratio of this drug, i.e. 5–20 mg/L (30– 110 μmol/L). Toxic effects become more frequent as the serum level approaches 220 mg/L and include headache, nausea, vomiting, and restlessness. Life-threatening arrthymias and convulsions may occur at concentrations above 40 mg/L (200 μmol/L). The dose must be reduced in patients with cirrhosis, congestive cardiac failure, chronic obstructive pulmonary disease or acute fevers, or in patients receiving cimetidine, erythromycin or antiviral vaccines. The dose may

Anaphylaxis

need to be increased in young patients and in regular alcohol consumers. In order to block or reduce prolonged late-phase reactions, corticosteroids are usually given at a dose of 250 mg hydrocortisone or 50 mg methylprednisolone intravenously every 6 hours for two to four doses. However, Stark and Sullivan (1986) found that 10 of 12 patients with persistent or biphasic anaphylaxis had been given oral or parenteral corticosteroids during treatment of the initial reaction, and yet still had severe prolonged or recurrent symptoms within 12 hours. In addition, antihistamines, e.g., diphendydramine 25–50 mg or chlorpheniramine 10 mg, can be given 6–8 hourly orally, intravenously, or intramuscularly in order to reduce pruritus and antagonize the H1 effects of histamine. Although still controversial, several studies have reported the combination of H1 and H2 antagonist to be more effective than H1 antagonist alone (Schoning et al. 1982; Runge et al. 1992; Lin et al. 2000b). Ranitidine might be better tolerated than cimetidine, causing fewer drug interactions.

Cardiovascular reactions Initial therapy for cardiovascular reactions consists of plasma expansion, in the form of intravenous fluids, in order to correct hypotension. Plasma expanders are given rapidly to correct the hypovolemia and consequent hypotension and reduced cardiac output resulting from acute vasodilation and leakage of fluid from the intravascular space. Crystalloid solutions might be preferred in the first instance, but in anaphylaxis with persistent hypotension both colloids and crystalloids might be required (Schierhout & Roberts 1998). Very large volumes of fluid may be needed (i.e., 1 L every 20–30 min, as needed), and central venous pressure (CVP) monitoring and measurement of hematocrit are helpful. Infusions should be continued if pulmonary capillary wedge pressure (PCWP) is less than 6 mmHg or CVP is less than 5 cmH2O and the patient remains hypotensive. If CVP or PCWP is elevated to greater than 15 cmH2O or 18 mmHg, respectively, inotropic agents should be used in order to prevent congestive cardiac failure and pulmonary edema. Dopamine produces selective splanchnic vasodilation at 2– 3 μg/kg per min and α- and β-adrenergic effects with increasing doses. A dose of 5–10 μg/kg per min is useful for renal dilation and for managing hypotension. Dobutamine at a dose of up to 10 μg/kg per min results in better blood-pressure control than dopamine in moderate to low doses and therefore it is often used with low-dose dopamine in order to combine splanchnic dilation with peripheral vasoconstriction. If blood pressure is unresponsive, intravenous epinephrine as outlined above should be commenced. Because of the risk for potentially lethal arrhythmias, epinephrine should be administered intravenously only during cardiac arrest or to profoundly hypotensive subjects who have failed to respond to intravenous volume replacement and several injected doses of epinephrine. If blood pressure

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remains severely refractory to epinephrine, norepinephrine, alone or in combination with the above, can be used. The dose is 4 mg diluted in 1 L of 5% dextrose and administered at an hourly rate of 2–12 μg (0.5–3 mL)/min. If the patient has been receiving beta-blockers and is resistant to the above adrenergic agents, a glucagon infusion can be started, since it can produce inotropic and chronotropic effects on the heart despite beta-blockade (Bochner & Lichtenstein 1991). The dose is 1 mg diluted in 1 L of 5% dextrose and given intravenously at a rate of 5–15 μg (5–15 mL)/min; the main side effects include nausea and hyperglycemia.

Prognosis Recovery from anaphylaxis is usually rapid and complete and occurs within a few hours, although in a minority it may persist or recur within 12–24 hours. The outcome of an individual attack depends on the age of the victim, the activity of concurrent diseases, particularly asthma, whether or not the patient is taking beta-blockers and, most importantly, how soon after the inciting incident epinephrine is administered. Long-term sequelae are rare, unless cardiac or neurologic damage is incurred in the initial episode. The severity of further anaphylactic attacks appears to be related to the cause of the anaphylaxis.

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Food Allergy and Eosinophilic Gastroenteropathies Scott H. Sicherer and Hugh A. Sampson

Summary

Definition/classification

Food allergies are defined as an adverse immune response to food proteins. Food allergies may manifest as acute reactions, for example anaphylaxis, or they may contribute to chronic disease such as atopic dermatitis or eosinophilic gastroenteropathies. Significant food allergies affect as many as 6% of young children and 3–4% of adults and appear to be increasing in prevalence. Both IgE-mediated and non-IgE-mediated (cellular) mechanisms result in a spectrum of food-allergic disease affecting target organs such as the skin and the respiratory and gastrointestinal tracts. Food allergy results from an abrogation of normal oral tolerance, and the phenotypic manifestations are influenced by host factors such as target organ reactivity and the degree and type of immune response, and by features of food allergens, such as stability during heating or digestion. Almost any food may provoke a reaction, but relatively few foods are responsible for the majority of significant food allergic reactions: milk, egg, peanuts, tree nuts, fish, and shellfish. Disease manifestations of IgE-mediated reactions may include specific symptoms such as urticaria, angioedema, wheezing, vomiting, abdominal pain, and hypotension/shock. Disorders that are IgE antibody mediated include pollen-food related syndrome (oral pruritus from pollen-homologous proteins in raw fruits/ vegetables), anaphylaxis, and food-associated exercise-induced anaphylaxis. Disorders that are cell-mediated and rarely associated with detection of food-specific IgE include infantile proctocolitis, enterocolitis and enteropathy. Several chronic disorders often responsive to elimination diets are variably associated with foodspecific IgE: eosinophilic esophagitis and gastroenteritis, and atopic dermatitis. Diagnosis requires a careful history followed by laboratory studies, elimination diets, and often physiciansupervised oral food challenges to confirm a diagnosis. Many food allergens have been characterized at a molecular level, which has increased understanding of the immunopathogenesis of food allergy and may soon lead to novel diagnostic and therapeutic approaches. However, current management requires educating the patient to avoid ingesting the responsible allergen and to initiate therapy, for example with injected epinephrine, in the event of unintended ingestion leading to anaphylaxis.

“Food allergy” is a term used to indicate an adverse reaction to food protein that is mediated by immune responses. As such, food allergy is one of several types of adverse reactions to foods. A task force of the European Academy of Allergology and Clinical Immunology published a revised nomenclature for allergy (Johansson et al. 2001) in which the term “food hypersensitivities” includes any exaggerated abnormal reaction resulting from the ingestion of a food, and therefore includes nonimmunologic/nonallergic (also commonly referred to as food intolerance) and immunologic (food allergy) etiologies. Food intolerances are adverse responses due to physiologic characteristics of the host, typically attributable to a metabolic or digestive disorder. Lactase deficiency, resulting in lactose intolerance, is a common example. Toxic reactions are distinct from hypersensitivities because they may occur in anyone who ingests a sufficient quantity of tainted food (e.g., bacterial food poisoning). Scombroid fish poisoning is an example of a toxic reaction that may mimic a food allergy because histamine-like chemicals in spoiled dark-meat fish induce symptoms such as flushing and abdominal pain. Pharmacologically active substances, such as caffeine in various beverages or tyramine in aged cheese, may also cause symptoms categorized as toxic reactions. Food aversions may mimic various adverse food reactions and are associated with psychological factors, but are not reproducible in blinded feeding studies. Neurologic responses such as gustatory rhinitis, i.e., vasomotor rhinitis induced by spices or hot foods, may also mimic a food allergic reaction. Though somewhat artificial, it is conceptually helpful to broadly classify food allergic disorders by immunopathology, among those that are or are not associated with detectable food-specific IgE antibodies (Sicherer 2002a; Sicherer & Sampson 2006). This classification, based on presumed etiology, also parallels the clinical pattern of food-allergic disorders and is therefore diagnostically useful. Disorders with an acute onset of symptoms following ingestion, for example anaphylaxis, are typically mediated by IgE antibodies that are detectable by skin-prick tests or serum immunoassay. Another group of food-allergic disorders, affecting primarily

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Table 93.1 A classification of adverse reactions to foods with clinical examples. Intolerance (nonallergic hypersensitivity), e.g., lactose intolerance due to lactase deficiency Pharmacologic, e.g., caffeine causing palpitations and muscle twitch Toxins, e.g., bacterial food poisoning Food allergy (allergic hypersensitivity, an adverse immune response) e.g. IgE-mediated: urticaria Non-IgE-associated: food protein-induced proctocolitis of infancy Mixed IgE/non-IgE: atopic dermatitis, eosinophilic esophagitis Masqueraders of food allergy e.g. Neurologic response such as auriculotemporal syndrome (facial flush with salivation) or gustatory rhinitis (rhinitis from hot spicy foods) Toxic response with allergic symptoms such as scombroid fish poisoning Emotional or psychiatric response such as anorexia nervosa, anxiety, food refusal

the gastrointestinal tract, are subacute or chronic and are characteristically not associated with detection of food-specific IgE antibodies. These non-IgE antibody associated disorders are presumed to be primarily mediated by T cells, resulting in chronic and subacute inflammation. Chronic inflammatory disorders such as atopic dermatitis and eosinophilic gastroenteropathies (characterized by, and named according to, segmental infiltration of gut segments) are often attributed to immune responses to foods. This third group of food-allergic disorders are variably associated with detectable IgE antibody (IgE associated/cell-mediated disorders), though the inflammation may also be a result of causes other than foods. Table 93.1 summarizes the classification of adverse reactions to foods with clinical examples.

Epidemiology Atopic disease appears to be increasing worldwide and food allergy is apparently included in this rise. In the European Community Respiratory Health Survey administered to 17 280 adults among 15 countries (Woods et al. 2001), 12% of respondents reported a food allergy/intolerance, ranging from 4.6% in Spain to 19.1% in Australia. A population-based study of 33 110 persons in France defined food allergy as selfreported typical allergic symptoms and found a rate of 3.5% (Kanny et al. 2001). The foods responsible in childhood were primarily egg and milk with atopic dermatitis a primary manifestation. In adulthood, seafood, fruits and vegetables were the common offenders with angioedema/urticaria as the most frequent symptoms. Perceived food allergies are often not verified when standardized procedures, such as doubleblind placebo-controlled oral food challenges (DBPCFCs), are used to confirm adverse reactions. A population-based study in the UK involving 20 000 persons included oral food challenges to eight selected foods in a small subset of subjects with reported reactions; the prevalence of food allergy was 1.8% (Young et al. 1994). The prevalence rate is likely to be an underestimate because half of the implicated foods were

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not formally evaluated and the study was undertaken prior to the apparent recent rise in atopic disease. With regard to teenagers, a cohort study on the Isle of Wight, UK evaluated 11-year-old (N = 757) and 15-year-old (N = 775) children (Pereira et al. 2005). Food avoidance rates were 16% and 19% for the 11 and 15 year olds, respectively. By means of objective assessment, 2.3% of both 11- and 15year-old children had food allergy. Studies of younger children typically show the highest rate of food allergies. Using DBPCFCs, 6% of 520 consecutive newborns evaluated in a health clinic in the USA to age 3 years reacted on challenge to suspected foods. Bock 1987). Additional information about epidemiology is evident from studies of allergies to specific foods. Population-based studies of cow’s milk allergy confirmed by challenge in infants/young children document a prevalence of 1.9–3.2% (Jakobsson & Lindberg 1979; Host & Halken 1990; Schrander et al. 1993; Eggesbo et al. 2001a) Egg is another common allergen in childhood with an estimated cumulative prevalence of 2.6% by age 2.5 years (Eggesbo et al. 2001b). Population-based questionnaire studies in the USA and UK estimate peanut allergy in 0.5–0.6% (Emmett et al. 1999; Sicherer et al. 1999) and tree nut allergy (e.g., hazel, walnut) in 0.5% (Sicherer et al. 1999). Studies in the USA and UK have indicated at least a doubling in the rate of peanut allergy in young children within the past decade (Grundy et al. 2002; Sicherer et al. 2003). These and other studies (e.g., Kagan et al. 2003) are indicating a rate of peanut allergy of 1% or more in schoolaged children. It is not known whether this pattern of rising prevalence is exclusive to peanut. Overall, the common or “major” allergens of infancy and early childhood are egg, milk, peanut, wheat, and soy, while allergens responsible for severe reactions in older children and adults are primarily caused by peanut, tree nuts, and seafood. However, virtually all foods have been implicated to cause an allergy for at least some individuals (Hefle et al. 1996). Reactions to fruits and vegetables are particularly common (∼ 5%), but usually not severe, and reactions to seeds (e.g., sesame, poppy), including severe reactions, are being reported with increasing frequency

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(Derby et al. 2005). Allergy to additives and preservatives, though often suspected, are uncommon (< 1%) (Simon 2003).

Genetics Food allergy is a complex trait whose various phenotypic expressions are undoubtedly determined by multiple genes with major and minor influences impacted by gene–gene and gene–environment interactions. It may be surmised that the genes linked to allergic inflammation (T helper type 2 or Th2 responses) are partly responsible for food allergy and include those involved in the expression of cytokines, chemokines, IgE antibody, and T-cell receptors (Barnes 2000; Cookson & Moffatt 2000). Evidence for this includes the observation that food allergy is a strong predictor of other atopic disease, and occurs more frequently among persons with a family history of atopy. In a study of a birth cohort of 1218 children on the Isle of Wight, UK (Tariq et al. 1998), family history of atopy was the best predictor of atopic disease and those with infantile egg allergy were more likely at age 4 years to have sensitization to aeroallergens (59.3% vs. 18.2%; P < 0.0001) and asthma/rhinitis [odds ratio (OR) 5.0, 95% CI 1.1–22.3; P < 0.05]. Further, a study from the German multicenter allergy birth cohort study showed that children persistently sensitized to food had a 5.5-fold higher risk of developing asthma than infants who were only transiently food sensitized (Kulig et al. 1998). While genes relevant to atopy are evidently relevant for expression of food allergy (Barnes 2000; Cookson & Moffatt 2000), it is also likely that food allergy is influenced by targetorgan and food-specific genes. For example, a strong genetic influence on peanut allergy was demonstrated because the concordance rate is 64% among identical twins compared with 7% among fraternal twins who on average share only 50% of their genes (Sicherer et al. 2000a). For responses to specific food proteins, HLA class II are candidate genes that have shown weak (Howell et al. 1998) and poorly reproducible (Shreffler et al. 2006) associations with expression of peanut allergy. With regard to broad manifestations of foodallergic disease, candidate genes include those responsible for expression of target-organ specific manifestations such as interleukin (IL)-5 and eotaxin as they relate to eosinophilic esophagitis (Blanchard et al. 2006), or genes associated with gut antigen processing, or genes that may be associated with metabolic processes (e.g., polymorphisms in N-acetyltransferase 2) (Gawronska-Szklarz et al. 2001; Sicherer 2002b).

Mechanisms/etiology The specific manifestations of food allergy follow from a variety of immune mechanisms, and target organ responses (Table 93.2). IgE-mediated reactions result from overexpres-

Food Allergy and Eosinophilic Gastroenteropathies

Table 93.2 Food-allergic disorders and symptoms categorized according to apparent pathophysiologic (immune) basis. IgE antibody-associated symptoms/disorders Urticaria/angioedema Immediate gastrointestinal reaction (gastrointestinal anaphylaxis) Oral allergy syndrome (pollen-related) Rhinitis Asthma Anaphylaxis Food-associated exercise-induced anaphylaxis Mixed IgE antibody-associated/cell-mediated chronic disorders Atopic dermatitis Eosinophilic gastroenteropathies Non-IgE-associated disorders Dietary protein enterocolitis Dietary protein proctitis Dietary protein enteropathy Celiac disease/dermatitis herpetiformis Contact dermatitis Pulmonary hemosiderosis (Heiner syndrome)

sion of food-specific IgE antibodies, which arm tissue mast cells and basophils in the bloodstream. Reexposure to the allergen results in mediator release and acute symptoms. The manifestation of the clinical response depends on many factors, including target organ reactivity, immune factors (degree and specificity of IgE response), and features of the causal food protein. For example, persons with asthma and food allergy are at higher risk of experiencing severe or fatal anaphylaxis (Sampson et al. 1992), presumably because of heightened target organ responsiveness. Food proteins that are labile and prone to degradation by heat or digestion such as pollen-related proteins in fruits/vegetables are less likely to induce severe reactions than stable proteins, such as seed storage proteins in peanuts and tree nuts that may more readily gain access to the bloodstream in an “immunologically intact” form. Reduced digestive capabilities, which may occur for example when using acid blockers, may enhance reactions to the resulting intact proteins (Untersmayr et al. 2005a,b). The specificity of the IgE antibody response, for example to particular epitopes of food proteins, may further influence the expression of IgE-mediated allergy. For example, persons with peanut allergy whose IgE antibodies bind numerous epitopes experience on average more severe reactions than persons whose IgE antibodies bind fewer ones (Shreffler et al. 2004, 2005). Another indicator of severity may be the specific proteins that are recognized (Astier et al. 2006). Whether IgE recognizes epitopes that are sequential and resistant to digestion, or those that depend on threedimensional conformational structures that are sensitive to heat and digestion, may further distinguish persons with more persistent and severe allergy in the former situation,

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from persons with mild or transient allergy in the latter situation (Cooke & Sampson 1997). Several food-allergic disorders are characteristically not associated with detectable food-specific IgE antibodies. These non-IgE-mediated disorders primarily comprise gastrointestinal disorders of infancy and are presumed to result from cell-mediated responses to food proteins. For example, food protein-induced enterocolitis syndrome (FPIES) of infancy is associated with detection of antigen-specific T-cell elaboration of tumor necrosis factor (TNF)-α (Benlounes et al. 1996, 1999). Chung et al. (2002) examined the presence of TNF-α in duodenal biopsy specimens using immunostains in infants with FPIES. Semiquantitative analyses revealed higher staining for TNF-α in affected infants with villous atrophy compared to those without atrophy and in normal controls. They also noted that receptors for the regulatory T-cell cytokine transforming growth factor (TGF)-β1 were decreased in the biopsies. While more work is needed to elucidate the immunologic basis of this disorder, a deficit in TGF-β1 response and excessive TNF-α response may be important factors. Celiac disease is an extensive enteropathy leading to malabsorption, and is associated with an immune response to gliadin found in wheat, rye and barley; it is associated with HLA-DQ2, which is present in over 90% of patients (Farrell & Kelly 2002). Though celiac disease may be categorized as a food allergy, it is not considered further in this chapter. The etiologic role of food allergy in atopic dermatitis and eosinophilic gastroenteropathies, eosinophilic esophagitis in particular, is complex because persons with these disorders are not uniformly responsive to food elimination/challenge, and when they are, the triggering food is variably associated with detection of IgE antibodies to the causal protein. Studies using DBPCFCs show that approximately one in three young children with moderate to severe atopic dermatitis has food allergy (Eigenmann et al. 1998; Sicherer & Sampson 1999a). Food-responsive T cells that bear the skin-homing receptor cutaneous lymphocyte antigen (CLA) may be important for the expression of the allergy in the skin (Abernathy-Carver et al. 1995; Reekers et al. 1999). With regard to eosinophilic esophagitis, it appears that more than 90% will have resolution of eosinophilia when all food allergens are eliminated from the diet (Kelly et al. 1995; Liacouras et al. 2005) but some may be responsive primarily to airborne allergens such as pollen (Fogg et al. 2003) and some may have an intrinsic immune disorder not responsive to allergen elimination.

Pathogenesis There are several ways in which an adverse immune response to food proteins may occur. There may be a direct breach in oral tolerance to foods while they are being ingested (traditional or class 1 food allergy), or an allergic response may result from sensitization to allergens presented dur-

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ing inhalation (class 2 food allergy), bypassing oral tolerance altogether (Breiteneder & Ebner 2000; Chehade & Mayer 2005). In accordance with the immunopathogenesis, class 1 food allergy typically occurs to food proteins that are generally heat and acid stable, being ingested by infants or children during a presumed window of immunologic immaturity and weakened gut barrier. Typical examples include milk, egg and peanut allergy. In contrast, class 2 food allergy results from sensitization through the respiratory route to proteins that are typically labile; this route of exposure avoids digestion. The classic example of respiratory exposure resulting in food allergy involves pollen proteins that are homologous to ones in foods (e.g., pollen-food related or oral allergy syndrome). Molecular and biochemical aspects of various food allergens are described in Chapter 52. Respiratory sensitization may be just one example of how a food protein may bypass tolerance induction through the gastrointestinal route of exposure. Murine studies (Hsieh et al. 2003) and circumstantial evidence from human epidemiologic studies (Lack et al. 2003) indicate that allergens such as egg and peanut typically associated with class 1 food allergy may also abrogate oral tolerance by initial sensitizing exposure through the skin. Avoidance of class 1 allergy requires an intact and immunologically active gastrointestinal barrier and the induction of oral tolerance to food proteins. The gastrointestinal mucosal surface presents a physical barrier composed of a single layer of epithelial cells joined by tight junctions and covered with a thick mucus layer that traps particles, bacteria and viruses, trefoil factors which help strengthen and promote restoration of the barrier, and luminal and brush border enzymes, bile salts and extremes of pH which, in addition to destroying pathogens, renders antigens less immunogenic. Innate immune responses (NK cells, polymorphonuclear leukocytes, macrophages, epithelial cells and Toll-like receptors) and adaptive immune responses (intraepithelial and lamina propria lymphocytes, Peyer’s patches, sIgA, and cytokines) provide an active barrier to foreign antigens. Immaturity of various components of the gut barrier and immune system may reduce the efficiency of the infant mucosal barrier, increasing the potential for allergy (Weaver et al. 1987). For example, enzymatic activity is suboptimal in the newborn period and the sIgA system is not fully mature until 4 years of age. It is presumed that this immature state of the mucosal barrier plays a role in the increased prevalence of food allergy seen in the first few years of life, and the gradual resolution of certain food allergies as the barrier matures in early childhood. Studies in the 1920s by Brunner and Walzer (1928) using passive sensitization showed that even in the face of an intact and mature gut barrier in adults, about 2% of ingested food antigens are absorbed and transported in the bloodstream in a form that can be recognized by IgE antibodies. It is apparent that gastrointestinal exposure to diverse immunologically intact food proteins does not normally result in adverse

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immune responses. In contrast, a natural state of oral tolerance is induced. Husby (2000) demonstrated that oral feeding leads to immunologic tolerance induction in humans. The underlying immunologic mechanisms involved in oral tolerance have not been fully elucidated, but studies suggest that various antigen-presenting cells, especially intestinal epithelial cells and various dendritic cells, and regulatory T cells play a central role (Mowat 2003; Chehade & Mayer 2005). Several regulatory T cells have been identified in conjunction with intestinal immunity: Th3 cells, a population of CD4+ cells that secrete TGF-β; Tr1 cells, CD4+ cells that secrete IL-10; CD4+CD25+ regulatory T cells; CD8+ suppressor T cells; and γδ T cells. Intestinal epithelial cells can process luminal antigen and present it to T cells on major histocompatibility complex (MHC) class II complex, but lack a “second signal,” thus suggesting their potential to play a major role in tolerance induction to food antigens as nonprofessional antigen-presenting cells. Dendritic cells residing within the lamina propria and noninflammatory environment of Peyer’s patches express IL-10 and IL-4, which favor tolerance (Frossard et al. 2004; Chehade & Mayer 2005). In addition to host factors, properties of antigens, dose and frequency of exposure influences tolerance induction. Murine models indicate that “high-dose” tolerance involves deletion of effector T cells, while “low-dose” tolerance is mediated by activation of regulatory T cells with suppressor functions (Chehade & Mayer 2005). It has been suggested that T cells primed in the local mucosal environment lead to tolerance induction, whereas T cells primed in the mesenteric lymph nodes, either from antigen reaching the node in the lymph or carried there by circulating dendritic cells, differentiate and travel to the mucosa where they induce local immune responses (Mowat 2003). Commensal gut flora may play a key role in normal processes leading to oral tolerance, as suggested by the observation that mice raised in a germ-free environment from birth fail to develop normal tolerance (Sudo et al. 1997). Mice treated with antibiotics and then exposed to a sensitizing regimen of exposure to peanut were more prone to developing peanut allergy than those not treated with antibiotics (Bashir et al. 2004). The potential benefit of particular gut flora was also demonstrated in human studies where lactating mothers and their offspring fed Lactobacillus GG (probiotics) experienced less atopic dermatitis (Kalliomaki et al. 2003), possibly due to enhanced Th1 cytokine response (interferon-γ) (Pohjavuori et al. 2004). The pathogenesis of eosinophilic esophagitis is being increasingly elucidated. Murine models reveal that eosinophilic esophagitis can be elicited by intranasal and bronchial sensitization and challenge to Aspergillus fumigatus (Mishra et al. 2001). The response was diminished in mice with a deficiency in the eosinophil chemoattractant eotaxin-1 and further eliminated in transgenic mice lacking the eosinophilopoietic cytokine IL-5. Intratracheal installation of the Th2 cytokine IL-13 (Mishra & Rothenberg 2003) resulted in both brochial

Food Allergy and Eosinophilic Gastroenteropathies

and esophageal eosinophilia that was dependent on IL-5, eotaxin-1, and STAT-6 (signal transducers and activators of transcription). In human studies utilizing gene microarray analysis of esophageal tissue, the gene encoding eotaxin-3 was the most highly induced gene compared with healthy controls, esophageal eotaxin-3 mRNA and protein levels correlated with tissue eosinophlia, and a single-nucleotide polymorphism in the eotaxin-3 gene was associated with disease susceptibility (Blanchard et al. 2006). These studies and the observation of food responsiveness of eosinophilic esophagitis in most patients indicates the possibility of various pathogenic elements including an allergen-specific or nonspecific relationship to respiratory allergy/asthma, and a crucial role for specific inflammatory pathways.

Food allergens, cross-reactivity, and food additives The major food allergens identified as class 1 allergens are water-soluble glycoproteins 10–70 kDa in size and relatively stable to heat, acid and proteases. Examples of common class 1 food allergens are caseins and β-lactoglobulin in cow’s milk, vicillins and conglutin in peanut, egg ovomucoid and ovalbumin, fish parvalbumin, and nonspecific lipid transfer proteins found, for example, in apple (Mal d 3) or corn (Zea m 14). Class 2 allergens are relatively labile, and examples include proteins that are homologous with pathogen-related protein 10 in birch pollen (Bet v 1) such as Mal d 1 in apple and Dau c 1 in carrot. An individual may mount immune responses to various proteins in a specific food, and toward various segments (epitopes) of a particular protein and this may correlate with expression of disease. For example, major class 1 allergens in peanut include Ara h 1, Ara h 2 and Ara h 3 which are associated with severe peanut allergy, while Ara h 8 is a Bet v 1 homolog class 2 peanut allergen that is less likely to be associated with severe clinical reactions (Mittag et al. 2004). Many botanically related proteins or homologous animal proteins may show cross-reactivity on allergy testing, but clinical evidence of cross-reactivity is less common (Sicherer 2001). Bernhisel-Broadbent and Sampson (1989) specifically addressed the issue of legume cross-reactivity by performing open or DBPCFCs in 69 highly atopic children with at least one positive skin test to a legume. Oral challenges to the five legumes (peanut, soybean, pea, lima bean, green bean) resulted in 43 reactions in 41 patients (59%). Only 2 of 41 (5%) with any one positive challenge reacted to more than one legume. Reactions to multiple types of fish in an individual with fish allergy are common (> 50%), but not the rule (BernhiselBroadbent et al. 1992; Hansen et al. 1997). Several reports demonstrate that isolated allergy to a single species of fish, e.g., tropical sole (Asero et al. 1999a) or swordfish (Kelso et al. 1996), occurs and usually does so in the relative absence of IgE antibody to common fish allergens (parvalbumin). Processing fish for canning may reduce its allergenicity (BernhiselBroadbent et al. 1992). Reactions to multiple types of shellfish

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and to a lesser extent to mollusks and bivalves in an individual with crustacean shellfish allergy is common, but not the rule (Sicherer 2001). The dominant allergen is tropomyosin (Leung et al. 1996), a pan-allergen with significant sequence homology identified in crustaceans such as shrimp (Daul et al. 1994), crab (Leung et al. 1998a) and lobster (Leung et al. 1998b), mollusks such as oyster, scallop and squid (Leung et al. 1996), and parasites such as Anisakis (Asturias et al. 2000). There are few clinical studies addressing clinical cross-reactivity but it is estimated that over 50% of persons will react to multiple types (Waring et al. 1985). Cereal grains (e.g., wheat, rye, barley, oat) share homologous proteins with grass pollens and each other (Donovan & Baldo 1990; Jones et al. 1995). This may account for the high rate of cosensitization to these foods (Jones et al. 1995), but among those with reactions to one grain, 80% are tolerant of all other grains. Allergy to sesame, poppy, and mustard are being reported with increasing frequency (Sporik & Hill 1996; Beyer et al. 2002; Morisset et al. 2003; Agne et al. 2004). Homologous seed storage proteins are being identified as the causal allergenic protein (Asero et al. 1999b; Rance et al. 2000; Beyer et al. 2002). IgE antibody-mediated reactions to fruits/vegetables are the most common type of food allergy reported by adults, and are often associated with sensitization to homologous pollen proteins (Kanny et al. 2001). Crespo et al. (2002) evaluated 65 adults diagnosed with clinical allergy to one or more fruits for allergy to other related foods. Of those tested, 34 (52%) were found to be clinically allergic to more than one fruit. Food challenges with potential cross-reactive foods uncovered 18 further reactions in 14 (22%) out of 65. Only 8% (18/223) of positive results for allergy tests to potential cross-reactive foods investigated were clinically relevant. Therefore, elimination of related fruits without testing or based on test results could have resulted in unnecessary restriction of 205 foods in the 65 people studied. However, 18 food reactions in one-fifth (14/65) of the patients could have been missed if oral challenges/evaluations were not pursued. In vitro studies show extensive cross-reactivity among sheep, cow and goat milk (Spuergin et al. 1997) and among cow, ewe, goat and buffalo milk with no significant binding to camel’s milk (Restani et al. 1999). Oral challenge studies of goat’s milk show this to be unsafe for patients with cow’s milk allergy: 92% of 26 patients reacted (Bellioni-Businco et al. 1999). However, only 4% of 25 children with cow’s milk allergy reacted to mare’s milk (Businco et al. 2000). Unfortunately, most of the readily available animal milks are problematic for those with cow’s milk allergy. A variety of food additives are used for flavor, preservation, color, or texture enhancement. Nonprotein substances added to foods are unlikely to cause IgE-mediated allergic reactions. These chemical additives (e.g., tartrazine, sunset yellow, erythrosine, ponceau 4R and many others) are not likely to cause IgE-associated allergic reactions, but some may have drug effects that cause adverse reactions, includ-

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ing symptoms that are allergy-like or may invoke immune responses. Sulfites are added to foods as a preservative, antibrowning agent, or for a bleaching effect. Sulfites can, in sensitive persons, induce asthma, and very rarely cause more significant allergic-like responses. Sulfites are used to preserve some drugs, including epinephrine; however, the low amounts used in epinephrine preparations have never been reported to cause a reaction. The term “spice” describes a variety of flavoring agents such as basil, cardamom, cinnamon, garlic, pepper, rosemary, saffron, tumeric and many others. These “spices” represent a huge array of foods that have proteins that share features with other items from nature such as various foods and pollens. Annatto is obtained from the seed of the South American tree Bixa orellana and provides an orange or yellow color to foods. It has rarely been associated with anaphylactic reactions (Nish et al. 1991). Carmine (cochineal) imparts a red color to food and is derived from dried bodies of a female insect that lives as a parasite on cactus. This protein has also rarely caused immediate-type allergic reactions (Chung et al. 2001). Gelatin is derived most often from beef or pork, and sometimes from fish and may induce allergic reactions (Sakaguchi et al. 1996; Wang & Sicherer 2005). Gelatin may also be used to stabilize vaccines. Usually, an individual with allergy to beef or pork gelatin can tolerate fish gelatin (Sakaguchi et al. 1999, 2000). However, there may be fish proteins in fish gelatin, a potential issue for those with fish allergy (Sakaguchi et al. 2000).

Clinical features and pathology Food-allergic disorders may affect one or several organ systems. The specific symptoms may be chronic, acute or subacute in nature, depending on the immunopathophysiology of the specific disorder and frequency of allergen ingestion.

Gastrointestinal food-allergic disorders (Table 93.3) Pollen–food allergy syndrome (oral allergy syndrome) This mild form of food allergy is characterized by oral pruritus and sometimes mild lip angioedema triggered by raw fruits and vegetables. Symptoms may rarely (∼ 1%) be more severe (Ortolani et al. 1988). Initial respiratory sensitization to pollen proteins results in IgE antibodies that bind to homologous proteins in the foods. For example, the birch pollen protein Bet v 1 shares homology with apple Mal d 1. Birch pollenallergic patients may develop symptoms following the ingestion of raw potatoes, carrots, celery, apples, pears, cherries, hazelnuts and kiwi, ragweed-allergic patients may react to fresh melons and bananas, and grass pollen-allergic patients may develop symptoms when ingesting raw tomatoes. Causal proteins are presumably heat-labile since cooking the food typically abolishes reactions. Commercial extracts used for

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Table 93.3 Gastrointestinal food-allergic disorders. Disorder

Epidemiologic features

Clinical/pathologic features

Oral allergy syndrome (pollen food-related syndrome)

About 50% with pollen allergy experience syndrome Anaphylaxis is uncommon Birch related: apple, peach, cherry Ragweed related: melons

Oral pruritus, oral edema Raw forms, not cooked Seasonal variation

Gastrointestinal anaphylaxis

Uncommon as a sole manifestation

Acute onset of nausea, vomiting, pain associated with sensitization to food trigger

Allergic eosinophilic gastroenteritis

Associated with multiple food allergy Uncommon

Symptoms: vomiting, diarrhea, growth failure, edema, obstruction, protein loss Symptoms vary by degree and locationof eosinophilic inflammation

Eosinophilic esophagitis

Associated with multiple food allergy, possibly respiratory allergens Associated atopic disease in majority

Reflux symptoms with prominence of dysphagia, impaction > 20 eosinophils per high power field

Proctocolitis

Breast-fed infants, no evidence of IgE antibodies, cow’s milk most common allergen

Mucus-laden bloody stools without other symptoms Eosinophilic inflammation of rectum

Food protein-induced enterocolitis syndrome

Triggers: cow’s milk, soy, occasionally solids (oat, rice, poultry)

Chronic exposure: vomit, diarrhea, dehydration, growth failure, acidemia Reexposure: 2-hour delay until repetitive vomit, later diarrhea, possible acidemia, methemoglobinemia, shock

Enteropathy

Rare presentation with protein loss associated with milk allergy

Vomiting and malabsorption. Villous blunting

skin testing may lack the triggering proteins due to denaturation, and so use of fresh fruit juices for allergy skin testing has been suggested (Ortolani et al. 1989). The disorder must be distinguished from mild oral reactions to stable proteins and oral reactions that may be a first symptom of a more progressive allergic response. The same foods causing this oral syndrome may induce systemic reactions in persons reactive to stable proteins in them (e.g., lipid transfer proteins) (Scheurer et al. 2004).

Gastrointestinal anaphylaxis This is a term used to indicate acute onset of IgE antibodyassociated allergic reactions that are primarily isolated to gastrointestinal responses (Sampson & Anderson 2000). Symptoms may include acute nausea, colicky abdominal pain, and vomiting. Persons may have reactions in other organ systems at other times of exposure and so this is primarily a descriptive category.

Eosinophilic gastroenteropathies The eosinophilic gastrointestinal disorders may be due to IgE- and/or non-IgE-mediated food allergy and are characterized by infiltration of the esophagus, stomach and/or intest-

inal walls with eosinophils, basal zone hyperplasia, papillary elongation, absence of vasculitis, and peripheral eosinophilia in up to 50% of patients (Sampson et al. 2001). The disorders are named according to the primary site of inflammation (Sampson & Anderson 2000).

Allergic eosinophilic gastroenteritis This disorder may occur at any age, including young infants, where it may present as pyloric stenosis with gastric outlet obstruction and postprandial projectile emesis. Weight loss or failure to thrive is a hallmark of this disorder. Depending on the extent and location of the inflammatory involvement, patients may present with abdominal pain, vomiting, diarrhea, blood loss in the stools, iron-deficiency anemia, and protein-losing enteropathy (Moon & Kleinman 1995). The spectrum of eosinophilic gastroenteritis was described in a series by Talley et al. (1990). Food intolerance or allergy was reported by half of the 23 patients with mucosal disease, but not by any patients with muscle layer or subserosal disease. However, formal studies to determine the role of food allergy were not undertaken. Eosinophilic gastroenteritis with protein-losing enteropathy has been described in a small group of young children presenting with primarily vomiting,

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diarrhea, failure to thrive, and edema; these children all responded to elemental diets devoid of allergen and to treatment with systemic steroids (Chehade et al. 2006).

Eosinophilic esophagitis Eosinophilic esophagitis (EE) is characterized by significant eosinophilic inflammation (typically > 20 eosinophils/high power field of the most involved field) that is not responsive to therapy with acid blockade (Rothenberg 2004). Reflux disease may result in an eosinophilic inflammatory process, but the number of eosinophils is not typically as high. Patients with EE often have normal pH probes. Endoscopy and biopsy is required for diagnosis and the surface of the esophagus may disclose furrows, rings or white plaques (eosinophilic pustules). Early symptoms may include vomiting and reflux symptoms, but dysphagia and food impaction, which often present in the pre-teenage, teenage and adult years, are symptoms that are more specific for EE (Rothenberg et al. 2001; Noel et al. 2004). There is a strong impression that the disease is increasing in frequency (Straumann & Simon 2005). Patients are typically atopic and asthmatic, indicating a possible systemic allergic response or a reaction to inhaled or other allergens. The role of food as a trigger for EE was first determined by Kelly et al. (1995), who showed complete resolution of inflammation when children were placed on a nonallergenic amino acid-based formula (Fig. 93.1). Subsequent studies showed that this treatment is almost uniformly effective (Liacouras et al. 2005), although prescription of a broader diet eliminating only the causal food proteins for an individual is the goal. Causal foods are variably associated with detection of IgE antibodies and so trial elimination diets and refeeding followed with serial biopsies is required to confirm causal foods. Treatment with systemic steroids is effective, but side effects are problematic and so the use of topical steroids, e.g. swallowing inhaled steroids, is a treatment option (Teitelbaum et al. 2002; Aceves et al. 2005). The effectiveness of cromolyn or antileukotrienes is currently unclear, and one study showed promise for therapy with anti-IL-5 (Garrett et al. 2004). The long-term prognosis of EE has not been clearly delineated, but a study of adults indicates that the disorder is chronic, persistent, and debilitating and may result in stricture formation requiring dilation therapy (Teitelbaum 2004). However, neoplastic transformation has not been reported.

Infantile food protein-induced proctocolitis This cell-mediated disorder generally presents in the first few months of life with mucus-laden bloody stools due to an immune response associated primarily with cow’s milk proteins passed in maternal breast milk or with infant formula (Lake 2000). Infants typically appear healthy and grow well. Lesions are confined to the distal large bowel and consist of mucosal edema with infiltration of eosinophils in the epithelium and lamina propria. Empiric treatment with dietary

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(a)

(b) Fig. 93.1 Histologic appearance of eosinophilic esophagitis (a) before and (b) following dietary therapy. (a) Biopsy of distal esophageal mucosa (before treatment). There are abundant intraepithelial eosinophils, which tend to localize in the superficial layers of the epithelium (superficial layering) and form occasional eosinophilic microabscesses. The maximum high-power (×400) field contains 52 eosinophils. The epithelium shows reactive basal zone hyperplasia (hematoxylin and eosin stain, original magnification ×200). (b) Biopsy of distal esophageal mucosa (after dietary treatment). The appearance of the epithelium has normalized, with dramatic shrinkage of the basal zone. Only rare eosinophils are now evident within the epithelium (the maximum high-power field contains 4 eosinophils) (hematoxylin and eosin stain, original magnification ×200). (Courtesy of Margret S. Magid MD, Mount Sinai School of Medicine, New York.) (See CD-ROM for color version.)

elimination of milk, and sometimes other common allergens, from the maternal diet while breast-feeding is usually effective; persistent bleeding may warrant biopsy to determine alternative causes (Xanthakos et al. 2005).

Food protein-induced enterocolitis syndrome This is a cell-mediated hypersensitivity disorder most commonly seen in infants, who present with failure to thrive, vomiting, and diarrhea prior to 3 months of age (Powell 1978;

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Sicherer 2005). Symptoms are most commonly provoked by cow’s milk or soy protein-based formulas, but may be due to other foods in older infants, e.g., oat, rice and poultry (Nowak-Wegrzyn et al. 2003). Chronic symptoms resolve with dietary elimination. Reexposure typically results in a delayed (1–3 hour) onset of repetitive vomiting, followed later by diarrhea that may be bloody. There is also a characteristic elevation of the peripheral blood polymorphonuclear leukocyte count. About 15% of these infants develop hypotension, and acidemia and methemoglobinemia may occur (Murray & Christie 1993). Infantile FPIES is a diagnosis that is generally made clinically; therefore, there are no series in which biopsies are performed solely in patients with this diagnosis. However, several case series include patients who fulfill criteria for a diagnosis of FPIES and these describe varied and nonspecific histologic features that overlap other inflammatory disorders and are not diagnostic (Gryboski 1967; Fontaine & Navarro 1975; Halpin et al. 1977; CoelloRanurez & Larrosa-Haro 1984; Jenkins et al. 1984; Goldman & Provjanksy 1986). Colonic biopsies in symptomatic patients reveal crypt abscesses and a diffuse inflammatory cell infiltrate with prominent plasma cells; small bowel biopsies reveal edema, acute inflammation and mild villus injury. In some cases, focal erosive gastritis and esophagitis is found with prominent eosinophilia and villous atrophy.

Food Allergy and Eosinophilic Gastroenteropathies

Other gastrointestinal disorders associated with food allergy Infantile colic is due to food hypersensitivity in a minority of infants presenting with this disorder. Infantile colic is a syndrome of paroxysmal fussiness characterized by inconsolable crying that generally develops in the first 2–4 weeks of life and persists through the third to fourth month of life and which may respond to dietary alterations, such as elimination of cow’s milk (Hill & Hosking 2000). Recalcitrant constipation and reflux has also been attributed to milk allergy (Iacono et al. 1996, 1998) although a pathophysiologic basis is not clear.

Cutaneous food-allergic reactions (Table 93.4) Acute urticaria and angioedema Immediate urticarial reactions from ingestion of food is among the most common symptoms of IgE-mediated food-allergic reactions. Acute contact urticaria due to food that may be tolerated when ingested is common among young children. Food allergy, as previously defined, is infrequently the cause of chronic urticaria and angioedema (e.g., symptoms lasting greater than 6 weeks) (Greaves & Kaplan 2004); however, there are reports that nonallergic hypersensitivity to food additives or food components plays a role for a subset of patients (Zuberbier et al. 2002; Di Lorenzo et al. 2005).

Atopic dermatitis Dietary protein-induced enteropathy (excluding celiac disease) This generally presents in the first several months of life with diarrhea (mild to moderate steatorrhea in about 80%) and poor weight gain (Savilahti 2000). Biopsy reveals a patchy villous atrophy, a prominent mononuclear round cell infiltrate, and few eosinophils. Milk is the most common trigger and the disorder usually resolves in early childhood.

Also termed atopic eczema dermatitis syndrome, this is a form of eczema that generally begins in early infancy and is characterized by typical distribution, extreme pruritus, and chronically relapsing course (Sicherer & Sampson 1999a). Approximately 35% of children with moderate to severe atopic dermatitis have food allergy (Eigenmann et al. 1998). Ingestion of specific foods in patients with food allergy has been shown to provoke a markedly pruritic, erythematous,

Table 93.4 Cutaneous food allergic reactions/disorders. Disorder

Epidemiologic features

Clinical/pathologic features

Urticaria, angioedema

Most common acute symptom of food-allergic reaction Chronic urticaria not typically associated with food allergy

Transient wheal/flare

Atopic dermatitis

About 35% of children with moderate–severe atopic dermatitis have food allergy About 90% associated with milk, egg, wheat, soy

Usually associated with IgE Elimination and rechallenge may be needed to confirm diagnosis

Contact dermatitis

Typically associated with occupational exposure

Similar to other causes of allergic contact dermatitis

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morbilliform rash. Following prolonged avoidance of the causal food, there may be increased risk for an acute reaction if tolerance has not developed (David 1984; Flinterman et al. 2006a). The rate of food allergy in adults with atopic dermatitis may be underappreciated because in one study about 45% of adult patients with atopic dermatitis and birch pollen allergy were found to develop worsening of their eczema within 48 hours of ingesting Bet v 1-containing foods (e.g., raw apples, carrots, celery), even in the absence of noticeable immediate oral symptoms (Reekers et al. 1999). Food-specific IgE antibody is often detected but oral food challenges may be needed to determine the relevance of a positive test to clinical reactions.

Food-induced contact dermatitis This cell-mediated disorder is often seen among food handlers, especially among those who handle raw fish, shellfish, meat, and eggs (Judd 1994).

Dermatitis herpetiformis This is a chronic blistering skin disorder associated with a gluten-sensitive enteropathy and characterized by a chronic, intensely pruritic papulovesicular rash symmetrically distributed over the extensor surfaces and buttocks (Nicolas et al. 2003). IgA deposition and activation of the alternative complement pathway is prominent.

Respiratory food hypersensitivity reactions (Table 93.5)

Asthma and rhinoconjunctivitis Respiratory reactions from foods due to IgE-mediated reactions usually occur in conjunction with other symptoms during a systemic reaction (Sampson & Scanlon 1989). However, chronic asthma or chronic rhinoconjunctivitis alone is rarely the result of a food-allergic reaction. Airway hyperreactivity and worsening of asthma may also be induced in the absence of marked bronchospasm following the ingestion of small

amounts of food allergens in sensitized subjects (James et al. 1996). Inhalation of airborne food particles, usually during active cooking or in industrial settings, may also trigger respiratory symptoms (Roberts et al. 2002). This is in distinction to food odors that are not associated with a high airborne content of protein (e.g., peanut butter) (Simonte et al. 2003). Food-induced asthmatic symptoms should be suspected in patients with refractory asthma and a history of atopic dermatitis, gastroesophageal reflux, food allergy or feeding problems as an infant, or a history of positive skin tests or reactions to a food.

Heiner syndrome This rare form of food-induced pulmonary hemosiderosis is caused by cow’s milk protein and is associated with milkspecific precipitating (IgG) antibodies (Heiner & Sears 1960).

Systemic reactions (anaphylaxis) (Table 93.6) Generalized anaphylaxis due to food allergies accounts for at least one-third of anaphylaxis cases seen in hospital emergency departments (Yocum et al. 1999). In addition to variable expression of cutaneous, respiratory, and gastrointestinal symptoms, patients may develop cardiovascular symptoms including hypotension, vascular collapse, and cardiac dysrhythmias (Pumphrey 2004). In contrast to other forms of anaphylaxis, serum tryptase is not typically elevated in foodinduced anaphylaxis (Sampson et al. 1992). In a survey of 32 fatalities caused by food-induced anaphylaxis (Bock et al. 2001) common themes included (i) reactions to peanut or tree nuts, (ii) victims were adolescents or young adults usually with a known food allergy (not necessarily severe), (iii) asthma, and (iv) the availability of epinephrine for use at the time of their reaction in only 10% of cases. Food-associated exercise-induced anaphylaxis is a form of anaphylaxis that occurs only when the patient exercises within several hours of ingesting food, usually a specific food to which sensitization is demonstrable by skin testing or serum IgE antibody

Table 93.5 Respiratory food hypersensitivity reactions/disorders. Disorder

Epidemiologic features

Clinical/pathologic features

Asthma

Bronchospasm often a component of systemic reactions Chronic asthma uncommon sole presentation of food allergy Occupational exposure/inhalation may induce symptoms (e.g., baker’s asthma)

Wheezing associated with exposure, reproducible with food elimination/challenge Airway hyperreactivity may be increased without active wheezing

Allergic rhinitis

Often a component of a systemic reaction Rarely a sole manifestation of food allergy

Responsive to elimination/ challenge

Heiner syndrome

Rare disorder presents in infants Associated with milk

Pulmonary hemosiderosis Elevated milk-specific IgG antibodies

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Table 93.6 Systemic reactions (anaphylaxis). Disorder

Epidemiologic features

Clinical/pathologic features

Anaphylaxis

Food is most common trigger for community anaphylaxis Common triggers: peanut, tree nuts, seafood Fatalities more common if: delayed therapy, comorbid asthma, teenagers

Serum tryptase may not be elevated

Food-associated exercise-induced anaphylaxis

May occur with any meal or more commonly associated with a specific food (e.g., wheat, celery, seafood)

Exercise is tolerated unless preceded by causal food/foods

Investigations The clinical investigation of an adverse reaction to food depends on a careful history and physical examination to determine the possible trigger and the type of adverse response, e.g., toxic, intolerance, pharmacologic, or allergic. If allergy is suspected, then determination of a likely pathophysiology, for example IgE-mediated reactions or non-IgE-associated ones, is needed to direct further inquiry (Sicherer & Teuber 2004). Important factors to consider include the types of symptoms, the chronicity, reproducibility, and alternative reasons for symptoms. If symptoms indicate a nonimmune response is likely, additional evaluation would follow the specific suspicion. For example, lactose intolerance may be confirmed through breath hydrogen testing. For chronic disorders such as atopic dermatitis and eosinophilic gastroenteritis, the identification of suspect foods is difficult because food is ingested throughout the day and symptoms are often chronic with a waxing and waning course. Symptom diaries are helpful but rarely diagnostic. In addition, individuals with these disorders often test positive to multiple foods that may not be causing illness. Care in selecting and interpreting the tests is paramount. For determination of food-specific IgE antibodies, skin-prick tests (performed using a probe to introduce food protein into the superficial skin layer) or serum tests are generally sensitive (∼ 75–95%) and specific (∼ 30– 60%) (Sicherer & Teuber 2004). Intradermal skin tests should not be used for evaluation of food allergy because they are overly sensitive and may induce systemic allergic reactions. Though commercial extracts are available for performing skin-prick tests for many foods, fresh extracts, particularly when testing fruits and vegetables

whose proteins are prone to degradation, may be more sensitive. Skin-prick tests are typically considered positive if there is a mean wheal diameter of 3 mm or greater, after subtraction of the saline control. Another means of detecting food-specific IgE antibody is commonly referred to as the radioallergosorbent test (RAST), although the term is antiquated because modern assays typically do not use radiolabels. A positive skin-prick test or serum IgE test merely indicates that food-specific IgE is present; it does not itself confirm an allergy. Increasingly larger wheal diameters or increasing concentrations of IgE antibodies are associated with increasingly higher chances that the test reflects clinical reactions (Sampson 2001; Roberts & Lack 2005). Figure 93.2 represents the typical probability curves generated by studies of test results against outcomes of physician-supervised oral food challenges. These increasing concentrations of foodspecific IgE antibodies do not necessarily reflect increasing severity of a reaction. Studies indicate that the curves may 100 Probability of a clinical reaction

determination. In the absence of exercise, the patient can ingest the food without any apparent reaction. While many foods have been implicated (celery, fish), ω5 gliadin found in wheat has been shown to be a major cause of food-dependent exercise-induced anaphylaxis (Palosuo et al. 2001).

80 60 40 20 0 Increasing skin test wheal or serum food-specific IgE concentration

Fig. 93.2 Probability of a clinical reaction compared with degree of specific food sensitization. Shown here is an illustration of the general concept that increasingly larger skin test wheal or increasing concentration of foodspecific IgE antibody is associated with increased risk of a clinical reaction. The exact curves vary by food, age, and test reagents among other variables. It should be noted that negative tests may occur when clinical reactions, even severe ones, are noted during oral food challenges (see text). (See CD-ROM for color version.)

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vary according to the food and study population. It should be noted that negative tests may occur in persons who react to a food on challenge, including persons with anaphylaxis. The reason for this may relate to deficiencies in test reagents (Hauswirth & Burks 2005; Leduc et al. 2006) or other factors. This is why the history is a crucial diagnostic modality for determining prior probability of an allergy to a specific food. For example, a positive test may confirm an allergy when there is a convincing history, but a negative test may not exclude an allergy if the history is highly suggestive. For these reasons, obtaining “panels” of food allergy tests without consideration of the history is not a good practice because numerous irrelevant positive results may occur. In a limited number of studies of a few foods in infants and/or children, diagnostic values associated with very high (≥ 95%) predictive values for reactions have been determined, though not universally confirmed. A study using skin-prick tests in young children revealed that when wheals were particularly large (≥ 8 mm for milk and peanut and ≥ 7 mm for egg) clinical reactions were virtually certain (Sporik et al. 2000). Studies determining the concentration of specific IgE antibody measured using a particular method (CAP-RAST FEIA or UniCap reported in arbitrary units, kUA/L) showed that food-specific IgE concentrations of ≥ 7 kUA/L to egg, ≥ 15 kUA/L to milk, and ≥ 14 kUA/L to peanut were 95% predictive for a reaction among 5-year-old children (Sampson 2001). These results are not always applicable to different study populations (Celik-Bilgili et al. 2005). For children under age 2 years, the values where most reacted were lower (e.g., ≥ 2 kUA/L for egg or milk) (Boyano-Martinez et al. 2002). It must be emphasized that diagnostic concentrations are undetermined for many foods, allergic disorders, and age groups. Food-specific IgE typically declines as a food allergy resolves (Sicherer & Sampson 1999b; Shek et al. 2004). Additional diagnostic tests are being investigated. The atopy patch test (APT), which is performed by placing the food allergen on the skin under occlusion for 48 hours and assessing for a delayed rash at 24–72 hours after removal, shows some promise for non-IgE-mediated disorders (Joint Task Force of the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology 2006). In one study of children with atopic dermatitis (Roehr et al. 2001), the APT had the highest positive predictive value for allergy to egg and milk compared with skin-prick test or RAST for both immediate and delayed reactions. However, the results vary widely among workers since much lower positive predictive accuracy (40–63%) and specificities (0.71– 0.87) have been reported (Majamaa et al. 1999). Spergel et al. (2005) showed the utility of this test for detection of food allergies in children with EE and food protein-induced enterocolitis syndrome, but confirmation by other investigators is needed. There are a host of tests that have been touted for the diagnosis of food allergy, but which have never been found useful in blinded studies. These include

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measurement of IgG4 antibody, provocation-neutralization (drops placed under the tongue or injected to diagnose and treat various symptoms), and applied kinesiology (muscle strength testing) (Joint Task Force of the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology 2006). For evaluation of chronic disease, such as atopic dermatitis and EE, the amelioration of symptoms during dietary elimination of suspected foods provides presumptive evidence of causality. Elimination diets can be undertaken by removing foods suspected to be causing symptoms, removing all but a defined group of foods that are rarely allergenic (oligoantigenic diet) or by giving an elemental diet of only a hypoallergenic extensively hydrolyzed formula or a nonallergenic amino acid-based formula. The elemental diet provides the most definitive trial. The type of elimination diet selected will depend on the clinical scenario, a priori reasoning concerning offending foods, and the results of tests for IgE antibody/APT. For EE, the length of trial is usually 6 weeks or more. Oral food challenges are required when the history and simple tests have not confirmed an allergy, or when tolerance is suspected (Joint Task Force of the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology 2006). An oral food challenge is performed by feeding gradually increasing amounts of the suspected food under physician observation over hours or days. They are performed either openly or blinded by camouflaging the food in a carrier food or opaque capsules. The DBPCFC method is least prone to bias and is considered the “gold standard” for diagnosing food allergy (Bock et al. 1988). This can be used to evaluate any type of suspected adverse response to foods. The procedure is most often utilized in disorders with chronic symptoms when several foods are under consideration, tests for specific IgE are positive, and elimination results in resolution of symptoms. The challenge setting also provides a safe means to introduce foods highly suspected of causing severe reactions despite negative skin tests or IgE tests. There are clinical circumstances when DBPCFC is not relevant or is contraindicated (Sicherer 1999). Recent severe anaphylaxis to a suspect allergen, with a positive test for specific IgE antibody to the suspected food is one example of a relative contraindication because the diagnosis is already clear. If the food being eliminated is not a major part of the diet (i.e., a rarely ingested fruit), then challenge may be optional. If several members of a food family are being eliminated, but the food family is not a major part of the diet (e.g., tree nuts), these same approaches may apply, although individualizing is an option. Risk assessments with regard to reaction risk and severity of symptoms in case a reaction occurs should be made prior to performing an oral food challenge. These assessments are typically based on prior reaction histories, test results, and comorbidity such as asthma. Higher-risk challenges may

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warrant a hospital setting and intravenous access prior to commencing a feeding. Except in the uncommon circumstances where risk of a severe reaction is essentially absent, oral food challenges are undertaken under medical supervision in a monitored setting. It must be appreciated that oral challenges can elicit severe anaphylactic reactions, so the physician must be comfortable with this potential and be prepared with emergency medications and equipment to promptly treat such a reaction (American Academy of Allergy and Immunology 1986; Joint Task Force of the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology 2006). A study by Perry et al. (2004a) reviewed risks of oral food challenges in children typically assessed to have a 50% risk or less of a reaction prior to challenge. Of the 584 challenges completed, 253 (43%) were positive to milk (90), egg (56), peanut (71), soy (21), and wheat (15). Of patients who failed, there were 197 (78%) cutaneous, 108 (43%) gastrointestinal, 66 (26%) oral, 67 (26%) lower respiratory, and 62 (25%) upper respiratory reactions. Despite presumptions about certain foods causing more severe reactions (e.g., peanut) than others (e.g., egg, milk), there was no difference between foods in the severity of failed challenges or the type of treatment required to reverse symptoms. For preparing blinded challenges, allergens may be hidden in masking agents such as fruit juices, infant formulas, mashed potatoes, and other vehicles. Certain flavoring agents such as mint, tuna or garlic can mask odors. Opaque capsules may be convenient, but may mask early symptoms otherwise detectable when food allergens are mixed with foods for normal oral exposure. Equipment for food preparation includes paper plates, cups, disposable utensils, mixing bowls, scale, mortor/pestle, blender, and a microwave. Certain preparation methods (canning, dehydration) may alter the allergens (Bernhisel-Broadbent et al. 1992; Chatterjee et al. 2006), so an open challenge with a meal-sized portion of the food prepared in its natural state for consumption following a negative DBPCFC is essential. It is preferable not to use fatty foods as vehicles since they can delay gastric absorption (Grimshaw et al. 2003). Prior to food challenge, risks and benefits should be reviewed and informed consent obtained/documented. Patients avoid the suspected food(s) for at least 2 weeks, antihistamines and medications that may alter reaction symptoms are discontinued according to their elimination half-life, and chronic asthma medications are reduced as much as possible prior to undertaking the challenge. Beta-agonists are eliminated for a relevant time period before challenges are undertaken. Patients are kept nil by mouth for at least 2 hours prior to challenge. Children may require specific preparation to facilitate the procedure such as videos, games, familiar utensils/ dishware, and other distractions. No single dosing regimen is universally established. The general approach is to select a regimen that is unlikely to

Food Allergy and Eosinophilic Gastroenteropathies

result in a severe reaction by cautiously and gradually increasing the amount provided/weighed against practical considerations of time constraints and mimicking natural exposure (e.g., aiming to consume a meal-sized portion). One approach used for DBPCFC is to administer a total of 8–10 g of the dry food or 100 mL of wet food (double amount for meat/fish) in gradually increasing doses at 10–15 min intervals or longer (Joint Task Force of the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology 2006). The whole challenge may be distributed, for example, in portions such as 1%, 4%, 10%, 20%, 20%, 20%, and 25% of the total. For example, 1% of the milk challenge is 1 mL. However, a variety of other challenge regimens have been used (lower starting doses including 10-fold or lower doses for potentially highly sensitive persons, variations in the degree of dosing increases, different time intervals, etc.). For IgE-mediated reactions, two challenges may be performed each day with several hours between challenges (one is placebo and one is active, so one food is tested each day). The practice of interspersing placebo and active food proteins during a single challenge (i.e., random ordering of sequential doses that may or may not contain the causal protein) should be discouraged because it may be difficult to differentiate whether a symptom followed a placebo or was delayed from true allergen. It is important to follow negative DBPCFCs with open feeding of a meal-sized portion of the food prepared in the manner relevant to the patient’s history (e.g., cooked or raw) to confirm that the food is tolerated. If such an open feeding induces a reaction, consideration may be given to a repeat DBPCFC using larger doses or different methods of food preparation. The selection of a starting dose should be one unlikely to trigger a reaction. To be cautious in persons who may be extremely sensitive, one could argue for starting doses that begin under the thresholds reported to induce reactions, but these are extremely small amounts (Taylor et al. 2004). The published thresholds vary by logarithmic differences among studies and data are not available for most foods. However, reactions are usually not reported under 0.25 mg of protein for peanut (about 1/1000th of 1 mL of peanut butter), 0.13 mg for egg (similar to the volume of peanut) and 0.6 mg for milk (about 0.02 mL) (Taylor et al. 2002). Measuring small amounts may be difficult in an office setting, so mixing and diluting the food in a carrier vehicle may be required. Labial food challenge, i.e., placing the food extract on the lower lip for 2 min and observing for local or systemic reactions in the ensuing 30 min (Rance & Dutau 1997), has been suggested as a starting point before an oral food challenge. However, the utility of this approach has not been extensively studied. For non-IgE-mediated reactions, the DBPCFC is usually the only means of diagnosis. DBPCFC for enterocolitis syndrome can induce severe reactions. For enterocolitis syndrome, the recommended challenge dose is 0.15 mg/kg of

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protein and obtaining intravenous access prior to the challenge is recommended for treatment in the event of a severe reaction. Prior to commencing an oral food challenge, patients should be examined carefully prior to challenge to confirm that they are not already having chronic symptoms and to determine their “baseline.” For persons with severe atopic dermatitis, hospitalization may be necessary to treat acute disease and establish a stable baseline prior to challenges. At baseline and during oral food challenges, symptoms are recorded and frequent assessments are made for symptoms affecting the skin, gastrointestinal tract, and/or respiratory tract (Bock et al. 1988). The false-positive and false-negative rate for DBPCFC based primarily on studies in children with atopic dermatitis is 0.7% and 3.2%, respectively (Sampson 1999; Caffarelli & Petroccione 2001). False-negative tests can be diminished by ensuring that open feeding, under supervision, of a meal-sized portion of the tested food prepared in its usual manner is tolerated following any negative DBPCFC (May 1976; Metcalfe & Sampson 1990; Niggemann et al. 1994). When one is evaluating subjective symptoms, there is a greater likelihood that false-positive or negative determinations would occur. Increasing the number of challenges (additional placebo and true foods) helps to diminish the possibility of a random association, but this can be a very labor-intensive approach (Briggs et al. 2001). A general scheme for the diagnosis of food allergy is shown in Fig. 93.3.

Differential diagnosis Symptoms of food allergy may mimic other allergic disorders as well as disorders that are not immunologic in nature. The history is paramount in considering alternative diagnoses. For example, acute allergic reactions may have been triggered by alternative allergens (e.g., insect stings, medications). Atopic dermatitis may flare from irritants and allergens. Chronic gastrointestinal symptoms may result from reflux, infection, anatomic disorders, metabolic abnormalities, and other causes. Chemical effects and irritant effects of foods may mimic allergic reactions. For example, gustatory rhinitis may occur from hot or spicy foods due to neurologic responses to temperature or capsaicin. Tart foods may trigger an erythematous band on the skin of the cheek along the distribution of the auriculotemproal nerve in persons with gustatory flushing syndrome (Sicherer & Sampson 1996). Food poisoning, namely scombroid poisoning caused by spoiled dark-meat fish such as tuna and mahi-mahi, can mimic an allergic reaction. For persons with eosinophilic gastrointestinal disease, alternative and concomitant diagnoses such as parasite infections, gastroesophageal reflux disease, systemic eosinophilic disorders, and vasculitis should be considered. Behavioral and mental disorders may result in food aversion (anorexia nervosa). Pharmacologic effects of foods and food additives have been attributed to attention disorders (Bateman et al. 2004), but more studies are needed.

History/physical examination – determine likely type of adverse reaction – identify suspect foods – if likely allergy, categorize likely pathophysiology

Consistent with non-allergic hypersensitivity/ pharmacologic/ neurologic/toxic

Pursue confirmatory tests, as indicated, e.g., breath hydrogen

Consistent with non-IgEmediated allergy

Confirmatory diagnostic tests if indicated (e.g., endoscopy)

Elimination diets and oral food challenges to confirm/identify triggers, if indicated

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Chronic disease likely associated with food allergy (e.g., significant atopic dermatitis, eosinophilic esophagitis)

Limited tests (IgE/Patch) based upon history and epidemiology. Elimination diets and oral challenges to confirm, as indicated

Consistent with IgE-mediated disorder (e.g., acute reaction)

Tests for food-specific IgE to suspect foods

Elimination of suspect food. Oral food challenge if history and test results are not otherwise adequately conclusive (see text)

Fig. 93.3 General diagnostic scheme for food allergy. See text for additional details.

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Treatment/management The primary therapy for a diagnosed food allergy is to avoid the causal food. This instruction appears simple, but has a tremendous impact on quality of life (Cohen et al. 2004) and is prone to errors. Studies indicating the amount of food capable of triggering an allergic reaction are incomplete, but typically reveal that trace amounts, for example less than a milligram, of milk, egg, or peanut can induce symptoms (Taylor et al. 2002, 2004). Thresholds of clinical reactivity vary by individual; for example one person may react to trace amounts, another may not experience a reaction until ingesting over 10 g (Sicherer et al. 2000b; Flinterman et al. 2006b). Clinical experience shows that some individuals with egg or milk allergy tolerate these foods in low amounts as a minor ingredient baked into products such as cake or cookies, but the impact on the immune system of this exposure is currently unknown. Certain types of mild allergy may not require strict avoidance (e.g., pollen food-related syndrome/oral allergy syndrome) (Ma et al. 2003). Education of patients about food avoidance is required for successful treatment. Laws regarding what a manufacturer is required to present on an ingredient statement of a prepared food varies by country (Taylor & Hefle 2006). Concerns for a food-allergic consumer include disclosure of all ingredients, disclosure of potential cross-contact of allergen with the final food product, and manufacturing procedures that provide consistent ingredients (e.g., no inadvertent inclusion of an undisclosed allergen). Education is also required with regard to meal preparation at home. When an allergen is used in a home, caution is needed to prevent ingestion by the allergic individual. Principles of avoidance of cross-contact must be reviewed with patients/families. For example, utensils, cookware, glassware, storage containers, and other food preparation materials in contact with an allergen should not be shared unless thoroughly cleaned before being used to prepare an allergen-free meal. Though only investigated for peanut butter, cleaning with typical means such as liquid or bar soap and running water or commercial wipes (but not simply antibacterial gels) for hands, or wet washes of tabletops, until visibly clean should be sufficient (Perry et al. 2004b). Care for allergen avoidance extends to restaurant meals where crosscontact with allergens is particularly problematic. Allergic individuals must also be educated about the use of food proteins in items not ingested, for example vaccines, craft items (modeling dough), and cosmetics. Exposure to allergens from skin contact is not likely to induce severe reactions (Simonte et al. 2003). Inhalational reactions typically result in rhinitis or asthma symptoms and result when foods become aerosolized, as during cooking or when powdered forms are used (Roberts & Lack 2003). In contrast, inhalational reactions to oily forms, such as peanut butter, are unlikely (Simonte et al. 2003). Casual contact can be more problematic

Food Allergy and Eosinophilic Gastroenteropathies

when saliva is exchanged, as by kissing or sharing utensils (Maloney et al. 2006). For persons with potentially life-threatening anaphylaxis, self-injectable epinephrine should be prescribed (Sampson et al. 2006). Judgment is needed to determine those at risk, but candidates include food-allergic patients with previous severe reactions, allergy to foods commonly causing severe reactions, and food-allergic patients with underlying asthma (Sicherer & Simons 2005). Adjunctive therapies that cannot be depended on in anaphylaxis include antihistamines and inhaled bronchodilators. The technique of using a self-injector should be reviewed as mistakes are common. The indications for use of epinephrine should be reviewed; it should be noted that fatal food-induced anaphylaxis has occurred without skin symptoms (Sampson et al. 1992). Patients must be instructed that following the administration of the medications, prompt transportation to an emergency facility (i.e., ambulance) should be sought where the patient should be observed for a prolonged period (> 4 hours), since recurrence of severe symptoms is possible, i.e., a biphasic or late-phase response. The administration of activated charcoal has been proposed since an in vitro study showed the ability of activated charcoal to bind peanut proteins (Vadas & Perelman 2003). However, the clinical utility of this approach has not been studied so general use cannot currently be recommended. Patients should obtain medical emergency bracelets identifying their allergy, and be reminded to update expired and expended epinephrine injectors. For children, an important component of the school and camp management of food allergy is to have a clear emergency plan in place with written instructions that are reviewed, medications readily available, and school personnel trained in recognizing and treating reactions. Potential future therapies for food allergy are under investigation (Nowak-Wegrzyn & Sampson 2004). In a double-blind placebo-controlled study of monthly injections of anti-IgE antibodies (TNX-901; 450 mg/month), peanut-allergic patients tolerated significantly greater amounts of peanut protein to elicit symptoms compared with before treatment (about 1 /2 to 9 peanut kernels; P = 0.001) (Leung et al. 2003). However, the response was not uniform, as nearly 25% of the group treated at the highest dose failed to tolerate any more peanut than they had at baseline. Therefore, additional studies are needed. Theoretically, anti-IgE antibody therapy should be protective against multiple food allergens, although it would have to be administered indefinitely. Another therapy that has shown promise in a murine model of anaphylaxis is traditional Chinese herbs (Li et al. 2001). This preparation completely protected the mice during subsequent peanut challenges and reduced peanut-specific IgE and Th2 responsiveness. Additional treatments that are specific to a particular food are under investigation. Early studies suggested that standard immunotherapy for persistent food allergy, specifically peanut allergy, was effective but the side effects were

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unacceptable (Nelson et al. 1997). Approaches to more safely alter allergic responses include the use of engineered proteins that lack IgE-binding sites, or utilize small overlapping peptides, to avoid activation of IgE while T-cell tolerance can be induced. Th1-promoting adjuvants such as immunostimulatory DNA sequences, heat-killed Escherichia coli or Listeria (Li et al. 2003; Frick et al. 2005) may help promote the efficacy of such vaccination. Another way to avoid side effects of injection immunotherapy is to administer treatment with native proteins orally or sublingually. Enrique et al. (2005) showed preliminary success in adults with hazelnut allergy who were treated with sublingual immunotherapy; mean hazelnut quantity provoking objective symptoms increased from 2.29 to 11.56 g (P = 0.02) in the treatment group while the placebo group experienced no significant improvement (3.49 to 4.14 g). Another potential immunotherapeutic approach would be the use of a chimeric protein, e.g. peanut Ara h 2/Fcγ, that could form complexes with allergen-specific IgE bound to mast cells and basophils to inhibit mast cell and basophil function (Zhu et al. 2005).

Course and prognosis With regard to IgE-associated food allergies, most (~ 85%) children lose their sensitivity to most allergenic foods (egg, milk, wheat, soy) within the first 3–5 years of life (Wood 2003). Even children with multiple severe allergies usually achieve tolerance (Hill et al. 1999). In contrast, adults with food allergy may have long-lived sensitivity. Rate of allergy resolution appears to slow significantly after the age of 6 years (Saarinen et al. 2005). Sensitivity to peanut, tree nuts, and seafood is rarely lost. The notion that peanut and tree nut allergy is permanent derives, in part, from the observation that it is an allergy that affects adults (Emmett et al. 1999; Sicherer et al. 1999) and longitudinal studies of school-age children have not documented resolution (Bock & Atkins 1989). However, it is now apparent that about 20% of peanut-allergic children under age 2 years may achieve tolerance by school age (Skolnick et al. 2001). In addition, about 8% lose their tree nut allergy by school age (Fleischer et al. 2005). Children who outgrow these allergies tend to have small or negative skin-prick tests, a long (over 3 years) interval since the last reaction, and few additional food allergies. Recurrence of peanut allergy has been noted when peanut was tolerated during a food challenge but the food was not incorporated into the diet (Busse et al. 2002; Fleischer et al. 2004), apparently indicating that patients should be instructed to continue to eat the foods to which they have achieved tolerance. Infants with non-IgE-mediated reactions to foods usually develop tolerance by 2–3 years of age (Saarinen et al. 2005). The course of childhood EE is not well studied, but the disease appears to persist (Straumann et al. 2003).

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Table 93.7 Prevention strategies. Well established Exclusive breast-feeding to 4–6 months If not breast-fed or supplemented, infants at increased risk (family history) should receive a hypoallergenic infant formula (shown to reduce atopic disease) rather than soy or cow’s milk formula Controversial/uncertain Maternal dietary alteration during pregnancy or lactation Avoidance of solid foods beyond 4–6 months Avoidance of specific allergenic foods beyond time of exclusive breast-feeding

Prevention Prevention or delay of food allergy and atopy through dietary means has primarily focused on elimination of major allergens from the diet. These approaches have been the subject of several reviews and metaanalyses resulting in consensus statements (American Academy of Pediatrics 2000; Gdalevich et al. 2001a,b; Muraro et al. 2004). Studies in this area are limited by the inability to randomize families to breast-feeding, specific formula and foods. Studies suggest a beneficial role for exclusive breast-feeding of infants at “high risk” for atopic disease for the first 3–6 months of life, and for avoiding supplementation with cow’s milk or soy formulas in favor of hypoallergenic formulas if breast-feeding is not possible. It is not clear if maternal exclusion diets during breast-feeding are additionally protective (Friedman & Zeiger 2005). For solid food introduction, waiting until age 6 months has been recommended in general, but experts recommend different strategies with regard to timing of introduction of specific allergens such as milk, egg, peanut, nuts, and seafood. A few studies have suggested that waiting too long to introduce certain allergens may be associated with an increased risk of allergy and atopic disease (Zutavern et al. 2004; Poole et al. 2006) but it is difficult to control for reverse causation in these studies. A summary of recommendations for allergy prevention is shown in Table 93.7. Prospective interventional studies will be needed to determine risks of introducing allergenic foods at different ages.

Conclusions Food allergies affect approximately 6% of young children and 3–4% of adults. For the clinician, recognizing clinical symptoms, syndromes, pathophysiology (IgE-mediated and non-IgE-mediated mechanisms), and epidemiology is important to aid in obtaining a correct diagnosis. Our understanding

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of the immune mechanisms of food allergy is increasing. For now, limited tests and oral food challenges are required for diagnosis and treatment is primarily reactionary: to avoid causal foods and treat reactions. Future modalities may allow improved diagnosis and more definitive treatments.

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Food Allergy and Eosinophilic Gastroenteropathies

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Drug Hypersensitivity Werner J. Pichler

Summary Drug hypersensitivity reactions represent a major clinical problem. Although they are rather frequent, occurring in 2–4% of hospitalized patients, they are still quite rare for each single drug. Their clinical characteristics are very heterogeneous as drugs can actually elicit all types of immune reactions. Thus, they can also imitate many different diseases. The antigenicity of drugs relies on the fact that small molecules can bind covalently to carrier proteins, which become modified and then behave like foreign antigenic proteins. Such modification of soluble or cell-bound proteins can induce humoral and T cell-mediated immune reactions, particularly if the drug or the metabolite is stimulating the innate immune system as well or is given into an already activated immune system. In addition, drugs can directly interact with immune receptors like the highly polymorphic αβ T-cell receptors and thereby stimulate some cells of the specific immune system via their surface receptors for antigen. This new concept is named pharmacologic interaction with immune receptors (p-i concept). Humoral immune responses involve formation of drugspecific IgE and can lead to symptoms like urticaria and anaphylaxis. Rather frequently and unexpectedly, such reactions can occur at first exposure, without known previous exposure to the drug. Quite rare are IgG-mediated reactions, which cause cell destruction like thrombocytopenia or anemia or immune complex deposition with vasculitis or serum sickness. The most common forms of drug hypersensitivity reactions involve drug-specific T cells: these T cells elicit an inflammatory response by secreting various cytokines and chemokines, which recruit and activate different inflammatory cells, namely macrophages, eosinophils, or neutrophils. Clinically, different forms of exanthema or interstitial nephritis can be observed. Quite importantly, drug-specific cytotoxic T cells are involved in all these reactions to a variable degree. The strong participation of drug-specific CD8+ T cells is associated with more severe diseases, e.g., severe bullous skin diseases. Every clinician using drugs has to be aware of these reactions, since severe drug hypersensitivity reactions can be lethal. This can occur via IgE-mediated degranulation of mast cell

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

and basophils (immune-mediated anaphylaxis) and via T cellmediated reactions, leading to severe bullous skin diseases or severe systemic inflammation involving internal organs (hepatitis, colitis, etc.). These severe reactions can in most instances be traced back to certain drug groups, in particular β-lactam antibiotics, pyrazolones, neuromuscular-blocking agents, and radiocontrast media for anaphylaxis, and antiepileptics and antibiotics for severe delayed-type reactions. The diagnosis of drug hypersensitivity is still a controversial and difficult area. In general, a positive skin or in vitro test may be indicative of sensitization, which may be clinically relevant. Due to the limited sensitivity of most tests, a negative test can often not completely rule out a drug hypersensitivity. In milder cases provocation tests can be applied. Treatment relies on stopping the drug, symptomatic treatment of the acute events, and in avoiding the drug and its class completely in the future. For optimal advice to the patient, careful documentation of the acute event is obligatory, as the history is still the most important factor for diagnosis and further management of the hypersensitivity. Under certain circumstances desensitization can be attempted.

Introduction Drug-induced adverse reactions are common and normally classified as type A or type B reactions. Type A reactions represent predictable side effects due to the pharmacologic action of the drug. An example would be sleepiness with some of the first- and second-generation antihistamines. Type B reactions are not predictable and comprise idiosyncratic reactions due to some individual predisposition (e.g., an enzyme defect) or hypersensitivity reactions (Naisbitt et al. 2000). Drug hypersensitivity reactions account for about one-sixth of all adverse drug reactions. They comprise allergic and so-called pseudoallergic reactions (nonallergic hypersensitivities), the latter of which may directly stimulate effector cells of the immune response, and thus imitate an allergic reaction, but without detectable reactions of the adaptive immune system. Drug hypersensitivity can result in a great variety of diseases, some of which are severe and even fatal (Lazarou et al. 1998; Roujeau & Stern 1994). The most common allergic reactions occur in the skin and are observed in about 2–3% of

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hospitalized patients (Bigby et al. 1986; Hunziker et al. 1997). Any drug is assumed to be able to elicit hypersensitivity reactions. Antibiotics, nonsteroidal antiinflammatory drugs (NSAIDs), and antiepileptics are the drugs most frequently causing them. The risk of sensitization and the severity of clinical symptoms depend on various factors. • State of immune activation of the individual: an underlying disease may have previously stimulated the immune system and thus lowered the threshold of reaction to a drug (which might be one factor explaining the frequent involvement of antibiotics in drug hypersensitivity, as infection may have previously stimulated the immune system). • Dose: important in all drug hypersensitivity reactions, both for sensitization and elicitation. Sometimes even minute amounts can elicit symptoms. • Duration of treatment: some drug hypersensitivity reactions appear only if the treatment is given for days to weeks. • Female sex: can increase the frequency, e.g., the welldocumented case of gemifloxacin-induced exanthema (Schmid et al. 2006). • New data have shown that the immunogenetic predisposition (in particular HLA-B alleles) is a very good predictor, although only for a certain drug, type of reaction, and in specific populations (e.g., Han Chinese), while a pharmacogenetic predisposition has been detected quite rarely. • Epicutaneous application of a drug clearly increases the probability of sensitization compared with oral or parenteral treatment. • Atopy: defined as the genetic predisposition to mount an IgE response to inhaled or ingested innocuous proteins. It is not normally associated with a higher risk of drug hypersensitivity, but an atopic predisposition may prolong the detectability of drug-specific IgE in the serum (Manfredi et al. 2004).

Immune recognition of drugs The hapten and prohapten concept An immune response may only arise if the antigen–drug complex has the ability to stimulate the innate immune system and to form antigenic determinants for specific immune receptors (B- and T-cell receptors for antigen, BCR and TCR, respectively). The ability to activate the innate immune system and thus to initiate an immune response is called immunogenicity, and supplements antigenicity, which is the provision of antigenic determinants for specific immune receptors. A full antigen requires both antigenicity and immunogenicity. It is a dogma in immunology that small molecules (< 1000 Da) are not antigenic per se. Thus, the recognition of small molecules like drugs by B and T cells is usually explained by the hapten concept (Naisbitt et al. 2000; Pichler 2003). This states that a small molecule becomes antigenic if it binds to a larger molecule (antigenicity). Haptens are chemically react-

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ive small molecules that are able to undergo stable covalent binding to a larger protein or peptide (Fig. 94.1). Modification can affect soluble autologous proteins (e.g., albumin), cellbound proteins (e.g., an integrin), or the peptide embedded in the major histocompatibility complex (MHC) molecule itself. An exception to this rule is nickel, which is a major cause of contact dermatitis. Nickel stimulates the T-cell immune system in a very peculiar way: it forms a coordination complex, where nickel complexes with two regions of the MHC/peptide molecule and two sites on the TCR. The molecular structure of some nickel-specific T-cell clones and their binding to the TCR and MHC/peptide molecules involved has been clarified (Weltzien 2007). Chemical haptens often have a tendency to bind covalently to a certain amino acid within a protein. Consequently, many different new antigenic determinants are formed by hapten modification of different proteins, resulting in a broad array of immune responses to a hapten. The type, location and abundance of the molecules modified has a big influence on the type of immune response arising (Fig. 94.1a). This can lead to a great heterogeneity of immune responses and clinical pictures, based on formation of antibodies (IgG, IgE) directed to the soluble or cell-bound proteins. T cells are almost always involved, reacting with processed haptenmodified peptide fragments. Occasionally, an exclusive T-cell response is generated without antibody formation. One reason might be modification of an MHC-associated peptide itself (Fig. 94.1a). Alteration of the MHC molecule directly is also possible, but data from a mouse model using TNP suggest that this causes an immune response less frequently (Martin & Weltzien 1994). The great variability of possible immune responses explains why drug hypersensitivity reactions nowadays represent the great imitator of diseases, having taken over this role from syphilis, the great disease imitator a century ago. The immunogenic potential of drugs/chemicals causing contact dermatitis is often associated with some toxic effect, as many contact sensitizers can also induce some type of toxic/irritative reaction, which initiates some inflammatory reaction. The toxic effect of a substance may be related to adduct binding to proteins or DNA/RNA, induction of signaling, inhibition of cell function, etc. This causes cell damage and thus “danger” signals. The major antigen-presenting cells (dendritic cells) may be activated by these danger signals, or the chemical/drug may directly affect their function by binding covalently to internal or cell-surface structures (Vital et al. 2004). At present many groups are trying to decipher the capacity of drugs to stimulate the innate immune system, by measuring upregulation of CD86 or CD40 on dendritic cells for example. While this is important for hapten-induced contact dermatitis, it is questionable whether a toxic effect of a drug is needed to elicit a generalized immune response as it occurs in drug hypersensitivity (see p-i concept below). A typical hapten is benzylpenicillin. It tends to bind cova-

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O C H2

Hapten

Penicillin G H

H N

1)

N O a)

2) 3)

Processing

Hapten (penicillin G) Binding to 1) soluble proteins or CH3 2) membrane–bound proteins ONa or 3) the MHC–peptide complexes O (I & II) directly binding a) via b-lactam-ring forming penicilloyl (PPL-PLL) or b) via thiaolidin structure.

S b) CH3

3)

Drug Hypersensitivity

Clinic: “everything” 1 & 2) (binding to cell-bound and soluble proteins) → IgE or IgG to hapten–protein: anaphylaxis, hemolytic anemia, thrombocytopenia 3) MHC class I and II modification: T-cell reaction with exanthem, hepatitis, interstitial lung disease, contact dermatitis, AGEP, TEN. . . .

(a) Prohapten NH2 or NO

SMX (-NO)

N O (b)

O S O NH

R NH SO

R NH SO

NHOH

R NH SO

N S H

NO

H3C

p-i concept

SMX NH2

SMX

N O (c)

1) MHC class I

O S O NH

T cell MHC class II

2)

Metabolism-dependent hapten formation (e.g., sulfamethoxazole, SMX) Uptake of the non–hapten drug SMX in cells able to metabolize it, generation of a hapten (SMX-NO), which can bind to intracellular proteins: presentation of processed modified peptides and binding to extracellular soluble proteins ( → both, T- and B-cell responses might develop). The metabolism may also induce co-stimulatory molecules on antigenpresenting cells.

Clinic: “everything” potentially immunogenic for B and T cells; Immunogenicity and clinical manifestation might be restricted to the liver (hepatitis!) or kidney (interstitial nephritis!), where metabolism occurs

p-i concept The drug happens to fit into some TCR (1) with sufficient affinity to cause a signal. This drug–TCR interaction is supplemented by MHC interaction (2). The T cells react and proliferate. No metabolism of drugs required. The reacting T cells are probably preactivated and have an additional peptide specificity.

Clinic: only T cells An exclusive T-cell response might develop with exanthems, hepatitis, etc. Whether B cells (by drug binding to Ig) can similarily be stimulated, remains unclear.

H3C

Fig. 94.1 Hapten and prohapten concept and noncovalent drug presentation to T cells. (a) Haptens are chemically reactive compounds like benzylpenicillin: they bind to lysines in proteins (as penicilloyl, shown) or to SH groups in the thiazolidine ring (not shown). This covalent binding can modify soluble or cell-bound molecules. They can even bind directly to the immunogenic major histocompatibiliy complex (MHC)/peptide complex on antigen-presenting cells (APC), either to the embedded peptide or to the MHC molecule itself. Thus, the chemical reactivity of haptens can lead to the formation of many distinct antigenic epitopes, which can elicit both humoral and cellular immune responses. Some examples of a B-cell or T-cell-mediated immune response are listed on the right-hand side. (b) Other drugs are prohaptens if they require metabolic activation to become haptens (i.e., chemically reactive). The metabolism leads to formation of a chemically reactive compound, e.g., from sulfamethoxazole (SMX) to the

chemically reactive form SMX-NO. The metabolite may modify cell-bound or soluble proteins, similar to a real hapten. If this metabolite is available in the liver or kidney only, a localized immune response may develop. If it is available throughout the body, a generalized reaction might ensue. (c) The p-i concept (pharmacologic interaction with immune receptors): drugs are often designed to fit into certain proteins/enzymes to block their function. Some drugs may happen to bind some of the available and highly polymorphic T-cell receptors (TCR) for antigen. Under certain conditions (see text), this drug–TCR interaction may lead to stimulation of the T cell. For full T-cell stimulation by such a chemically inert drug (no hapten-like features), an additional interaction of the TCR with the MHC molecule is required. This type of drug stimulation would result in exclusive T-cell stimulation. A similar model is feasible for immunoglobulins, but has never been shown. (See CD-ROM for color version.)

lently to lysine groups within soluble or cell-bound proteins, thereby modifying them (Fig. 94.1a). The binding can occur via the β-lactam ring, by binding as penicilloyl to lysine. Alternatively, the binding can occur via the SH group of the thiazolidine ring. It is feasible (but not documented) that penicillins affect costimulatory molecules on dendritic cells, thus also having immunogenic features. As a consequence, B- and T-cell reactions to penicillins may develop. It might also be possible that the hapten binds directly to the im-

munogenic peptide presented by the MHC molecule itself, although the likelihood for this seems to be low. In this case no processing is required (Fig. 94.1a). Many drugs are not chemically reactive but are still able to elicit immune-mediated side effects. The prohapten hypothesis tries to reconcile this phenomenon with the hapten hypothesis by stating that a chemically inert drug may become reactive upon metabolism (Griem et al. 1998; Naisbitt et al. 2000; Pichler 2003) (Fig. 94.1b). Sulfamethoxazole (SMX) is

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a prototype of such a prohapten. It is not chemically reactive itself but gains reactivity and thus antigenicity by intracellular metabolism. Cytochrome P450-dependent metabolism (CYP2C9) in the liver leads to sulfamethoxazole-hydroxylamine (SMX-NHOH) which can be found in the urine and is easily converted to the highly reactive sulfamethoxazolenitroso (SMX-NO) by oxidation. The latter is chemically highly reactive and binding of SMX-NO to intracellular proteins creates neoantigenic determinants (Fig. 94.1b). Moreover, toxic effects of SMX appear above a threshold level. Thus, SMX seems to have indirect antigenic and immunogenic features. Since many different proteins may be modified, the resulting clinical picture might be as variable as with real haptens and SMX is indeed known to cause many different types of diseases affecting many organs (e.g., exanthems, anaphylaxis, Stevens–Johnson syndrome, hepatitis). These side effects are mediated by antibodies and/or T cells. On the other hand, the conversion of a prohapten to the reactive hapten may occur exclusively in the liver or kidney and may thus cause an isolated hepatitis or interstitial nephritis (Spanou et al. 2006).

The p-i concept Recently, a third possibility, namely the pharmacologic interaction of drugs with immune receptors (p-i concept; Fig. 94.1c) has been proposed (Zanni et al. 1998; Pichler 2002). According to this concept, chemically inert drugs, unable to covalently bind to peptides or proteins, can nevertheless directly activate particular T cells if they are able to match with sufficient affinity some of the more than 1012 different TCRs. This interaction is akin to the interaction with other “pharmacologic” receptors. It can result in nonrecognition, blocking, or partial or full activation. The latter results in selective stimulation of T cells, which expand, infiltrate the skin and other organs, and organize orchestrated inflammation. The final result is similar to the activation of T cells elicited by hapten or peptide/MHC stimulation. The p-i mechanism of T-cell stimulation does not require biotransformation of the inert drug to a chemically reactive compound and no generation of a drug (hapten)-specific immune response. Consequently, according to the p-i concept, no involvement/ stimulation of the innate immune system is required (Pichler 2005), since drug binding to the TCR can activate only previously primed effector and memory T cells, as these cells have a substantially lower threshold for activation than naive T cells. Effector and memory T cells, in contrast to naive T cells, are less dependent on costimulation by CD28 for activation, and thus the signal via TCR may suffice. This threshold of T-cell activation might be further lowered by massive immune stimulation of T cells, as occurs during a generalized herpes or HIV infection but also during exacerbations of autoimmune diseases, as the generalized inflammation accompanies high cytokine levels and increased expression of MHC and costimulatory molecules. Consequently, T cells are preactivated and more ready to react to a minor signal like binding of a drug to

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their TCRs. This would explain the high occurrence of drug hypersensitivity in these diseases. The p-i concept is a radical change in our way of explaining drug-induced hypersensitivity reactions, as it actually states that drug allergies are pharmacologic reactions. The target structure for the drug is not only the selected ligand (e.g., some enzyme pocket or surface receptor) but the drug also matches some of the many TCRs. The evolving immune response is therefore actually due to cross-reactivity of the TCRs, which react with the drug and an unknown peptide structure. The p-i concept is well accepted for sensitized T cells, as many in vitro studies have shown that drug-specific T-cell clones are stimulated directly via the p-i mechanism (reviewed in Pichler 2002). In fact, drug hypersensitivity due to the p-i concept is very likely more frequent and relevant for generalized drug hypersensitivity reactions like maculopapular, bullous, pustular exanthemas and associated diseases than the hapten/prohapten mechanism (which is probably more relevant for contact dermatitis). On the other hand, the role of the p-i mechanism in inducing drug hypersensitivity is still disputed (Sieben et al. 2002). However, the p-i concept does not imply the induction of a specific immune response but postulates that it is the consequence of a cross-reactivity and is only able to activate already primed immunologically competent cells (Pichler 2005) (Table 94.1). Thus, the old dogma that small chemicals are not full antigens is still valid, but drugs have other ways to stimulate the immune system. Recent data suggest that the drug binds first to the TCR, but for full activation additional MHC interaction with the TCR is required. The p-i concept can thus explain the following. 1 Symptoms at first encounter with the drug (especially if there were no sensitization phase) would be explained by many T cells being stimulated by the drug. If there were a time interval before symptoms arose, T cells would have to expand to reach the clinically relevant level. 2 The higher risk of drug hypersensitivity in generalized viral infections. 3 Some peculiar in vitro and in vivo features of the drugelicited immune response, which is more reminiscent of superantigen stimulation leading to massive overstimulation than a coordinated immune response (Pichler 2002). 4 Some drugs frequently involved in drug hypersensitivity and shown to be stimulatory via the p-i mechanism (sulfamethoxazole, lamotrigine, carbamazepine) may also form some hapten-like compounds after metabolism. Thus, the hapten and p-i mechanism might occur simultaneously (Zanni et al. 1998).

Classification of drug hypersensitivity reactions Drug hypersensitivity reactions can cause many different diseases. To account for this heterogeneity and to better explain

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Table 94.1 The p-i concept postulates direct interaction of drugs with the T-cell receptor (Gerber & Pichler 2004). Fixed antigen-presenting cells, unable to process antigens, can still present the drug and stimulate specific T cells Many drug-specific T cells are stimulated only if the drug is constantly present. Washing removes the drug, while covalently bound haptens are not washed away Drug-reactive T-cell clones react to the drug within seconds or minutes, long before metabolism and processing can take place Some T-cell reactivity to drugs can already be observed in the absence of antigen-presenting cells The p-i concept has been found to be relevant for such different drugs as sulfamethoxazole, lidocaine, mepivacaine, celecoxib, lamotrigine, carbamazepine and p-phenylendiamine causing MPE, DiHS/DRESS, AGEP and SJS/TEN AGEP, acute generalized exanthematous pustulosis; DiHS/DRESS, drug-induced hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms; MPE, maculopapular exanthem; SJS/TEN, Stevens–Johnson syndrome/toxic epidermal necrolysis.

the various clinical pictures, Gell and Coombs classified drug hypersensitivity, as well as other immune reactions, into four categories, I–IV (Fig. 94.2) (see below). This classification has recently been revised to better account for the heterogeneity of T-cell functions so well described in modern immunology (Pichler 2003). It relies on IgE, complement-fixing antibodies, and T-cell reactions, which orchestrate different forms of inflammations. These reactions are tightly connected, for example the maturation of B cells to IgE- or IgG-producing plasma cells depends on the help of T cells. Thus, for example, type I and type IVb as well as type II or III with type IVa reactions often occur together, and the clinical picture is probably dominated by the prevalent immune reaction.

Antibody-mediated drug hypersensitivity reactions As outlined above (Fig. 94.1), hapten-like features of a drug allow the modification of soluble and cell-bound proteins. The natural reaction of the immune system to such antigens is the development of a humoral immune response. Consequently, if a humoral immune response does develop, the eliciting drug should have hapten-like features, forming hapten–carrier complexes, or be itself a protein bearing “foreign” determinants (e.g., chimeric antibodies). It should induce a B- and T-cell response, the T cells orchestrating the type of reaction by secreting certain cytokines and thus contributing to immunoglobulin isotype switch. Indeed, as shown in Table 94.2, drugs able to elicit IgE-mediated allergies are often haptens or they contain foreign antigenic structures. However, there is a growing number of drugs and related case series where IgE (or IgG) responses are implied, without previous exposure and sensitization phase. Examples are anaphylaxis to neuromuscular blocking agents or radiocontrast media, but also others. Immediate skin-prick tests and occasionally in vitro drug-specific IgE are positive (particularly in severe reactions). Other patients with the same symptoms to these drugs have no detectable IgE and are usually class-

ified as having “pseudoallergy” (see below). Researchers have tried to explain these puzzling reactions by invoking crosssensitization to a related compound, but some explanations seem to be far fetched and can only explain certain cases. The origin of preformed drug-reactive IgE often remains enigmatic.

Type I (IgE-mediated) allergies The IgE system is geared to react to small amounts of antigen. It achieves this extraordinary sensitivity by the ubiquitous presence of mast cells armed with high-affinity Fc-IgE receptors (FcεRI), to which allergen/drug-specific IgE is bound. Very small amounts of a drug are apparently sufficient to interact and stimulate these receptor-bound IgE molecules, as occasionally even the small amounts of drugs used for skin tests can elicit systemic reactions. On cross-linking FcεRI various mediators, e.g., histamine, tryptase, leukotrienes, prostaglandins, tumor necrosis factor (TNF)-α, are released that cause the symptoms. IgE-mediated reactions to drugs are usually thought to depend on the prior development of an immune response to a hapten/carrier complex: B cells need to mature into IgE-secreting plasma cells, and T cells help in this process by interacting with B cells (CD40–CD40L interaction) and by releasing interlukin (IL)-4/IL-13, which are switch factors for IgE synthesis. This sensitization phase is asymptomatic and may have occured during an earlier drug treatment. At renewed contact with the drug, a hapten–carrier complex is formed again, which then cross-links preformed drug-specific IgE on mast cells and causes degranulation. The drug itself is normally too small to cross-link two adjacent IgE molecules, which is the reason why when skin testing for IgE against penicillin the hapten (penicilloyl) is coupled to a 8–10 lysinecontaining peptide (penicilloyl-polylysine). These reactions were erroneously considered to be dose independent, as sometimes very small amounts can cause severe reactions. However, further diminishing the dose, as is done in desensitization

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Type I

Type II

Type III

Type IV a

Type IV b

Type IV c

Type IV d

Immune reactant

IgE

IgG

IgG

IIFN-g, TN-Fa (Th1 cells)

IL-5, IL-4/IL-13 (Th2 cells)

Perforin/ granzyme B (CTL)

CXCL-8. GM-CSF (T cells)

Antigen

Soluble antigen

Cell- or matrixassociated antigen

Soluble antigen

Antigen presented by cells or direct T-cell stimulation

Antigen presented by cells or direct T-cell stimulation

Cell-associated antigen or direct T-cell stimulation

Soluble antigen presented by cells or direct T-cell stimulation

Effector

Mast-cell activation

FceR+ cells (phagocytes, NK cells)

FceR+ cells complement

Macrophage activation

Eosinophils

T cells

Neutrophils

Immune complex Blood vessel Platelets

Th2 IFN-g

Th1 IL-4, IL-5

Ag

Eotaxin CTL

CXCL8 GM-CSF

PMN

Eosinophil

Example of hypersensitivity reaction

Allergic rhinitis, asthma, systemic anaphylaxis

Hemolytic anemia, thrombocytopenia (e.g., penicillin)

Serum sickness, arthus reaction

Chemokines, cytokines, cytotoxins

Cytokines, inflammatory mediators

Tuberculin reaction, contact dermatitis (with IVc)

Chronic asthma, chronic allergic rhinitis Maculopapular exanthema with eosinophilia

Cytokines, inflammatory mediators Contact dermatitis Maculopapular and bullous exanthema hepatitis

AGEP Behçet disease

Fig. 94.2 Revised Gell and Coombs classification of drug reactions. Drugs can elicit all types of immune reactions. In fact, all reactions are T-cell regulated, but effector function relies mainly on antibody-mediated effector functions (types I–III) or T cell/cytokine-dependent functions (types IVa–IVd). Type I reactions are IgE-mediated reactions. Cross-linking of IgE molecules on high-affinitiy IgE receptors (FceRI) on mast cells and basophilic leukocytes leads to degranulation and release of mediators, which cause a variety of symptoms (vasodilatation, increased permeability, bronchoconstriction, itch, etc.). Type II reactions are IgG mediated, and cause cell destruction due to complement activation or interaction with Fc-IgG receptor-bearing killer cells. Type III reactions are also IgG mediated. Complement deposition and activation in small vessels and recruitment of neutrophilic granulocytes via Fc–IgG receptor interaction leads to local vascular inflammation. Type IVa

reactions correspond to Th1 reactions with high IFN-g/TNF-a secretion and involves monocyte/macrophage activation. Often, one can also see CD8 cell recruitment (type IVc reaction). Type IVb reactions correspond to eosinophilic inflammation and to a Th2 response with high IL-4/IL-5/IL-13 secretion; they are often associated with an IgE-mediated type I reaction. In type IVc reactions, the cytotoxic reactions (by both CD4 and CD8 cells) rely on cytotoxic T cells themselves as effector cells. They seem to occur in all drug-related delayed hypersensitivity reactions. Type IVd reactions correspond to a T-cell-dependent, sterile neutrophilic inflammatory reaction. It is clearly distinct from the rapid influx of polymorphonuclear leukocytes in bacterial infections. It seems to be related to high CXCL-8/ GM-CSF production by T cells (and tissue cells). See text for definition of abbreviations. (See CD-ROM for color version.)

procedures, illustrates that also these reactions are clearly dose dependent. In sensitized individuals the reaction can start within seconds after contact with the parenterally applied drug, and minutes after oral uptake (Fig. 94.3a). Anaphylactic shock may occur within 1–15 min, and asphyxia due to laryngeal edema often between 15 and 60 min. Initial symptoms may include a palmar, plantar, genital or axillary itch, which should be seen as an alarm sign, as it often heralds a possibly severe anaphylactic reaction following rapidly within minutes. The skin becomes red (diffuse generalized erythema),

often first on the trunk, later over the whole body. In the next 30–60 min urticaria may appear, together with swelling of the periorbital, perioral and sometimes genital area. Asphyxia may account for 60% of anapylaxis-related deaths: Laryngeal swelling may be suspected if the voice becomes hoarse and it is more difficult to inspire than to expire. The patient has difficulty speaking and swallowing when the tongue is swollen. The patient may also complain about chest tightness and dyspnea, having signs of acute bronchospasm with wheezing and prolonged expiration. Some patients develop gastrointestinal symptoms (nausea, cramps, vomiting,

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Table 94.2 Drugs involved in IgE-mediated and “pseudoallergic” reactions.* Drugs involved in IgE-mediated reactions b-lactam antibiotics: penicillins, cephalosporins Pyrazolones Foreign proteins (chimeric antibodies, immunglobulin preparations) Muscle relaxants Quinolones Platinum-containing cytostatic drugs (carboplatin, oxaliplatin, etc.) Drugs causing “pseudoallergic” reactions (Radio)contrast media Pyrazolones NSAIDs: acetylsalicylic acid, diclofenac, mefenamic acid, ibuprofen Muscle relaxants Quinolones Plasma expanders Vancomycin

(a)

* Not complete, only main groups mentioned.

diarrhea, and fecal incontinence). The blood pressure may collapse, due to either massive vasodilatation or a shift of volume into the extravascular space; more severely, a cardiac arrhythmia may develop. The full syndrome is termed “anaphylactic shock,” which is lethal in about 1% of all cases. Risk factors for a severe course are fulminant appearance (as seen preferentially by parenteral application), preexisting (undertreated) asthma, and older age, as myocardial infarction or cerebral hypoxia and damage can lead to death days after the acute event. Anaphylaxis is a severe event, and survivors often have some cognitive or intellectual impairment. Table 94.2 summarizes the main drugs causing immediate reactions (within 1 hour). Most IgE-mediated reactions to drugs are less severe, and often only urticaria, angioedema, or a local wheal around the injection site may develop (Fig. 94.3a). However, any IgEmediated drug allergy can be potentially life-threatening, as the mild symptoms might be due to a relatively low dose, and each new treatment might also boost the IgE response.

Pseudoallergy (nonimmune-mediated hypersensitivity) including anaphylactoid reactions An unsolved but common problem in allergology and specifically hypersensitivity to drugs are so-called “pseudoallergic” reactions (nonimmune-mediated hypersensitivities), which are in fact as frequent as true immune-mediated reactions. The pathomechanism of these reactions is not well understood and may rely on different mechanisms. The majority of these reactions imitate the clinical features of immediate reactions (erythema, urticaria, angioedema appearing within 1 hour after drug intake) and most are not dangerous. However, some reactions cause anaphylaxis and can be lethal.

(b) Fig. 94.3 Immunoglobulin-mediated drug hypersensitivity reactions. (a) IgE-mediated urticaria after pyrazolone. (b) IgG-mediated vasculitis induced by sulfamethoxazole treatment. Skin manifestations mainly affect the legs. For details see text. (See CD-ROM for color version.)

The IgE-like reactions are also called anaphylactoid reactions, if no drug-specific IgE (or T cells) can be detected using skin or in vitro tests (however, the available tests are not very sensitive). The symptoms can appear at first encounter with the drug. It is questionable whether one can differentiate them clinically from “real” IgE-mediated reactions. It has been suggested that some appear less rapidly (often > 15–30 min after oral administration) than true IgE-mediated allergies, they may require higher doses, and the typical initial symptoms for IgE-mediated anaphylaxis, namely palmar and/or plantar itch, are perhaps less common. High tryptase levels after some reactions underline the role of mast cell degranulation, at least in some of these reactions. “Pseudoallergic” reactions can be elicited by many drugs, but some drugs seem to elicit them more often (Table 94.2). In vitro, these drugs do not release mediators from basophils or mast cells from normal individuals. On the other hand, an elevated basophil activation test has been described with some NSAIDs using blood of affected individuals (asthma, polyposis nasi, urticaria), while persons not affected did not

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show such a response (Sanz et al. 2005). Confirmation of this finding is needed, in particular as the mechanism of action of NSAIDs on basophil activation is unclear. Pseudoallergy is normally associated with immediate reactions involving mast cell and/or basophil degranulation. However, there are also delayed reactions, like macular exanthema after certain drugs, where all drug-specific tests and even provocation tests remain negative. It is usually explained by a concomitant viral infection or a combined effect of drug and virus infection. Some, often female, individuals seem to show a higher susceptibility to react to a variety of xenobiotics with distinct chemical and pharmacologic features with macular exanthema, urticaria and, rarely, more severe symptoms. Some people even complain about intolerance to generally harmless substances like colorants or stabilizers in food. Double-blind provocation tests are often negative, so the real relevance of these complaints remains unclear. While some people may indeed attribute different complaints to drug use, some have repeated and well-documented skin symptoms. A subgroup may have chronic urticaria, whereby exacerbations might be triggered by drug intake (often NSAIDs); another group has constantly elevated tryptase levels without other signs of mast cell expansion (borderline mastocytosis). Some reactions can be suppressed by pretreatment with antihistamines. A rather common problem in clinical practice is the occurrence of adverse reactions to local anesthetics of the amide type (e.g., lidocaine). It occurs often, but not exclusively, during dental treatment, with some individuals experiencing repeated episodes. The symptoms comprise local swelling, urticaria, dizziness, heart palpitations, nausea, and sometimes short-lasting collapse. Most reactions are interpreted as vasovagal reactions, as all allergy tests and even provocation tests are negative. Some are attributed to comedication, in particular epinephrine. An IgE-mediated allergy can hardly ever be demonstrated, with the exception of some reactions to local anesthetics of the ester type (procaine), which are rarely used nowadays. In a minority of cases with delayed (> 2– 4 hours after injection) generalized urticaria and local swellings after treatment with amidetype local anesthetics, a T-cell reaction to local anesthetics can be demonstrated (Orasch et al. 1999). The majority of these patients became sensitized by the use of ointments for hemorrhoids, which contain local anesthetics. Another subgroup of patients with repeated reactions may have an idiosyncratic reaction to local anesthetics, which may cause vasodilatation by an unknown mechanism. This vasodilatation can sometimes even be observed with skin tests to local anesthetics.

IgG-mediated reactions (cytotoxic mechanism, type II) Type II and type III reactions rely on the formation of complement-fixing IgG antibodies (IgG1, IgG3). Occasionally, IgM is involved. These reactions are similar, as both depend on the formation of immune complexes and interaction with

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complement and Fc-IgG receptor (FcγRIIa and FcγRIIIa)bearing cells (on macrophages, natural killer cells, granulocytes, platelets), but the target structures and physiologic consequences are different. In type II reactions, the antibody can be directed to cell structures on the cell membrane (rarely) or immune-complex activation occurs on the cell surface. Both events can lead to cell destruction or sequestration. Affected target cells include erythrocytes, leukocytes, platelets, neutrophilic granulocytes, and probably hematopoietic precursor cells in the bone marrow. The mechanism of type II reaction is not as clear as originally thought, since a clear hapten-specific immune reaction can often not be documented . The reaction can be observed in vitro only in the presence of the drug, which can be washed away (Aster 1999; Arndt & Garratty 2005). It is important to differentiate between the following. 1 Development of an IgG immune reaction to the hapten– carrier complex, mostly after longer duration of high-dose treatment. It is rather rare and best documented for highdose penicillin and cephalosporin treatment. The immune reaction is due to complement-fixing antibodies (IgG1, IgG3), occasionally due to IgM antibodies, which can cause intravascular hemolysis. Some antibody reactivity may be directed to the carrier molecule itself (i.e., autoantibodies). This autoimmune form (e.g., induced by methyldopa) is less abrupt but longer lasting, as it persists for weeks instead of days after cessation of the drug. 2 Nonspecific adherence with autoantibody induction can occur when a drug or metabolite is somehow adsorbed to the erythrocyte or thrombocyte membrane, creating a new antigenic complex in combination with the cell membrane. For example, quinine-induced immune thrombocytopenia is caused by a remarkable class of IgG and/or IgM immunoglobulins that react with selected epitopes on platelet membrane glycoproteins, usually GPIIb/IIIa (fibrinogen receptor) or GPIb/IX (von Willebrand factor receptor) only when the drug is present in its soluble form (Aster 1999). Well-documented cases are due to quinine, quinidine, or sulfanilamide antibiotics. The antibodies are clearly not hapten-specific, and it remains enigmatic how a soluble drug can promote binding of an otherwise innocuous antibody to a membrane glycoprotein to cause platelet destruction. The antibody-coated cells will be sequestrated to the reticuloendothelial system in liver and spleen by Fc or complement receptor binding. More rarely, intravascular destruction may occur by complement-mediated lysis. Hemolytic anemia has been attributed to penicillin and its derivatives, cephalosporins, levodopa, methyldopa, quinidine, and some antiinflammatory drugs (Table 94.3). Today cephalosporins (mainly cefotetan and ceftriaxone) are the main cause. The clinical symptoms of hemolytic anemia are insidious and may be restricted to symptoms of anemia (fatigue, paleness, shortness of breath, tachycardia) and jaundice with dark urine. Laboratory investigation may reveal reduced erythrocyte and hemoglobin levels, increased reticulocytes, a positive direct

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Table 94.3 Drugs causing blood cell dyscrasias. Hemolytic anemia Cephalosporins* Penicillin Levodopa Methyldopa Quinine NSAIDs Thrombocytopenia Heparin* Abciximab* b-Lactam antibiotics Sulfanilamide antibiotics Disease-modifying drugs (DMARD in rheumatic disorders?) Neutropenia Clozapine* Ibuprofen Indomethacin Sulfopyridine Sulfasalazine Sulfamethoxazole Carbimazole Propylthiouracil Procainamide Gold salts Ticlopidine * Relatively frequent.

and (if the drug is present during the test) indirect Coombs test. Indirect bilirubin levels are elevated, and haptoglobulin decreased. Urinary hemoglobin and hemosiderin are increased (Arndt & Garratty 2005). Thrombocytopenia is a relatively common side effect of drug treatment. Acute, sometimes severe and life-threatening thrombocytopenia is a recognized complication of treatment with quinine, quinidine, sulfanilamide antibiotics, and many other medications (Table 94.3). It is a not infrequent complication of treatment with biologicals, which often contain human Fc fractions themselves. Drug-induced immune thrombocytopenia usually develops after 5–8 days of exposure to the sensitizing medication or after a single exposure in a patient previously exposed to the same drug. It is mostly due to sequestration to Fc-IgG receptor-bearing cells in the spleen and liver. Patients with this condition often present with widespread petechial hemorrhages in the skin and buccal mucosa, sometimes accompanied by urinary tract or gastrointestinal bleeding. Intracranial hemorrhage is rare, but examples have been reported. After discontinuation of the provocative medication, platelet counts usually return to normal within 3–5 days. A special, intermediate form between type II and type III reactions is heparin-induced thrombocytopenia. Platelets contain low-affinity (FcγRIIa) receptors that are capable of

Drug Hypersensitivity

binding immune complexes, leading to platelet activation (Greinacher & Warkentin 2006). Heparin is a high-molecularweight, sulfated, linear polysaccharide that inhibits blood coagulation by activating regulatory proteins such as antithrombin III. About 50% of patients anticoagulated with heparin for at least 7 days produce antibodies that recognize complexes consisting of heparin and platelet factor (PF)4, a CXC chemokine normally stored in platelet α granules. When a patient with such an antibody is given heparin, heparin– PF4 complexes are formed that react with antibodies to form immune complexes, which bind to platelet FcγRIIa receptors, leading to platelet activation, additional PF4 release and, eventually, platelet destruction. Thrombocytopenia occurs in about 5% of patients given heparin and is rarely severe enough to cause bleeding. However, about 10% of affected patients experience paradoxical thrombosis, which can be life-threatening. Abciximab is a chimeric (human/mouse) Fab fragment that binds with high affinity to an epitope on the IIIa component of GPIIb/IIIa close to a site that is essential for fibrinogen binding. Abciximab-induced thrombocytopenia can be observed in 1–2% of patients treated the first time, but in about 10% after second exposure. It is caused by antibodies specific for the murine component of the chimeric abciximab molecule (Artoni et al. 2004). Idiosyncratic drug-induced neutropenia is an unpredictable side effect of many medications. Epidemiologic studies suggest that up to three-quarters of all cases of severe idiosyncratic neutropenia are presumably caused by drug hypersensitivity (Kaufman et al. 1996). There are no tests available capable of identifying the responsible drug in individual cases. Many drugs have been associated with neutropenia (Table 94.3), but in a relatively low frequency (∼ 10 cases per 1 million users). Clozapine is exceptional in causing neutropenia in 2–3% of patients and complete failure of neutrophil production (agranulocytosis) in 0.5–1% within the first 6 months of treatment. Some drugs may interfere with protein synthesis or mitosis in susceptible individuals, while in others antibodies to neutrophils were detected in the presence of the drug, strongly suggesting an immunologic mechanism as for thrombocytopenia or anemia. Typically, patients will have taken the provocative drug for a week or more before presenting with signs of infection such as pharyngitis, stomatitis, pneumonia or sepsis. Blood studies demonstrate neutropenia but platelet levels and hematocrit are usually normal. Bone marrow findings range from total absence of identifiable neutrophil precursors to maturation arrest in the neutrophil series without abnormalities in megakaryocytes or erythroid precursors (Aster 2007).

IgG-mediated reactions (immune complex deposition, type III) Formation of immune complexes is a common event in the frame of a normal immune response and does normally not

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cause symptoms. Immune complexes may also be formed during drug treatment as well, either if the drug forms a hapten–carrier complex and thus gives rise to an immune reaction, or if the drug is a (partly) foreign protein that elicits an immune reaction itself (e.g., chimeric antibodies). Such immune complexes will normally be rapidly cleared, either by FcγRI or complement receptor 1 (CR1) binding on reticuloendothelial cells. No symptoms arise, but the efficiency of treatment decreases. Why under certain circumstances an immune complex disease develops is not clear. Responsible mechanisms might include (i) very high immune complex levels, (ii) relative deficiency of some complement components (C1, C4, C2), (iii) imbalance between local complement activation with C3a/C5a formation but reduced capacity to transport and eliminate immune complexes via erythrocytes, which bear complement receptors, or (iv) aberrant FcγR function. Recently, a low copy number of FcγRIII was associated with another immune complex disease, namely glomerulonephritis (Aitmann et al. 2006). The clinical symptoms of a type III reaction may be a hypersensitivity, small-vessel vasculitis and/or serum sickness. Serum sickness was first described with the use of heterologous or foreign serum for passive immunization. Antibodies are generated within 4–10 days, which react with the antigen, forming soluble circulating immune complexes. Complement (C1q)-containing immune complexes are deposited in the postcapillary venules and attract leukocytes by interacting with their FcγRIII receptors (Stokol et al. 2004), which thereby release proteolytic enzymes that can mediate tissue damage. Currently, nonprotein drugs are the most common cause of serum sickness. Hypersensitivity vasculitis (Fig. 94.3b) has an incidence of 10–30 cases per million per year. Most reports concern cefaclor, followed by trimethoprim–sulfamethoxazole, cephalexin, amoxicillin, NSAIDs, and diuretics. The main symptoms of immune complex diseases are arthralgia, myalgia, fever, and vasculitis. This may be localized mainly to the skin as “palpable purpura,” purplish red spots usually found on the legs. In children, it is often referred to as Henoch–Schönlein purpura, not infrequently with arthritis. Lesions may coalesce to form plaques and these may ulcerate in some instances. The internal organs most commonly affected are the gastrointestinal tract, kidneys, and joints. The prognosis is good when no internal involvement is present. The disorder may be acute or chronic. Histology can reveal IgA-containing immune complexes, and the histology of kidney lesions is in fact identical to that of IgA nephropathy, a major cause of chronic renal failure.

T-cell-mediated delayed drug hypersensitivity reactions Mechanism The Gell and Coombs classification was established before a detailed analysis of T-cell subsets and functions was available. Meanwhile, immunologic research has revealed that the three

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antibody-dependent types of reactions require involvement of helper T cells. Moreover, T cells can orchestrate different forms of inflammations. Therefore T-cell-mediated type IV reactions were further subclassified into type IVa–IVd reactions (see Fig. 94.2) (Pichler 2003). This subclassification considers the distinct cytokine production by T cells and thus incorporates the well-accepted Th1/Th2 categorization of T cells; it includes the cytotoxic activity of both CD4 and CD8 T cells (IVc); and it emphasizes the participation of different effector cells like monocytes (IVa), eosinophils (IVb), or neutrophils (IVd), which are the cells causing the inflammation and tissue damage.

Type IVa reactions Type IVa reactions correspond to Th1-type immune reactions. Th1 cells activate macrophages by secreting large amounts of interferon (IFN)-γ, drive the production of complementfixing antibody isotypes involved in type II and III reactions (IgG1, IgG3), and are costimulatory for proinflammatory responses (TNF, IL-12) and CD8+ T-cell responses. The T cells promote these reactions by secreting IFN-γ and possibly other cytokines (TNF-α, IL-18). An in vivo correlate would be, on the one hand, monocyte activation, e.g., in skin tests to tuberculin or even granuloma formation as seen in sarcoidosis; on the other hand, these Th1 cells are known to activate CD8 cells, which might explain the common combination of IVa and IVc reactions (e.g., in contact dermatitis). Type IVb reactions Type IVb reactions correspond to the Th2-type immune response. Th2 cells secrete the cytokines IL-4, IL13 and IL-5, which promote B-cell production of IgE and IgG4, macrophage deactivation, and mast cell and eosinophil responses. The high production of the Th2 cytokine IL-5 leads to an eosinophilic inflammation, which is the characteristic inflammatory cell type in many drug hypersensitivity reactions (Pichler 2003). In addition, there is a link to type I reactions, as Th2 cells boost IgE production by IL-4/IL-13 secretion. Thus, type IVb reactions may actually be involved in the late-phase allergic (IgE-initiated) inflammation of the bronchi or nasal mucosa (asthma and rhinitis). Another in vivo correlate might be infestation with nematodes, eosinophil-rich maculopapular exanthems, or other T cell-dependent diseases with hypereosinophilia. Type IVc reactions T cells themselves can also act as effector cells. They emigrate to the tissue and can kill tissue cells like hepatocytes or keratinocytes in a perforin/granzymeB and FasL-dependent manner (Fig. 94.4) (Schnyder et al. 1998; Nassif et al. 2002). Such reactions are occurring in most drug-induced delayed hypersensitivity reactions, mostly together with other type IV reactions (monocyte, eosinophil or polymorphonuclear recruitment and activation). Cytotoxic T cells thus play an

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Keratinocyte

ICD541 MHC II

Fig. 94.4 Mechanism of cytotoxicity by cytotoxic CD4+ (mainly in maculopapular exanthem, MPE) or CD8+ (mainly in bullous) skin reactions: drug-reactive T cells kill keratinocytes, which express MHC class II molecules and thus strengthen the drug–TCR signal. The reactive T cell has cytotoxic activity. (a) Perforin-positive cells in epidermis during MPE. (b) Drug-specific CD4 and T cells. (See CD-ROM for color version.)

important role in maculopapular or bullous skin diseases as well as in neutrophilic inflammation (acute generalized exanthematous pustulosis, AGEP), and in contact dermatitis. Type IVc reactions appear to be dominant in bullous skin reactions like Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), where activated CD8+ T cells kill keratinocytes (Schnyder et al. 1998; Nassif et al. 2002; Pichler 2003), but may also be the dominant cell type in hepatitis or nephritis (Fig. 94.4).

Type IVd reactions Rather neglected was the possibility that T cells could coordinate (sterile) neutrophilic inflammation as well. Typical examples would be sterile neutrophilic inflammation of the skin, in particular AGEP. In this disease CXCL8 and granulocyte–macrophage colony-stimulating factor (GM-CSF)producing T cells recruit neutrophilic leukocytes via CXCL8 release and prevent their apoptosis via GM-CSF release (Britschgi et al. 2001). Besides AGEP, such T-cell reactions are also found in Behçet disease and pustular psoriasis (Keller et al. 2005). The relationship of these T cells to the recently described IL-17-producing T cells needs to be clarified. Tolerance mechanism Most patients can take drugs without developing immunemediated side effects. One could argue that they lack precursor cells able to interact with the drug. However, the great heterogeneity of the immune response to drugs (Pichler 2003), a high precursor frequency in sensitized patients (Beeler et al. 2006), and the finding that 2– 4% of the normal population but 30–50% of HIV-infected patients react to sulfamethoxazole suggest that, rather than a lack of precursor cells, other

Perforin granzyme B TCR LFA-1

(a)

(b)

factors like underlying immune status (activation of effector and/or memory T cells) and “regulatory” mechanisms may be important. Thereby “regulation” may occur on different levels and may be different for drugs stimulating the immune system via hapten and the p-i concept (Table 94.4).

Preferential skin involvement in drug hypersensitivity How is the immune system activated by drugs in exanthems: via p-i or hapten mechanisms? The skin harbors an enormous number of T cells (Clark et al. 2006). These represent sentinel cells, ready to react if antigenic/immunogenic agents penetrate this barrier. It is still debated how these sentinel T cells are best characterized by surface markers. One hypothesis postulates that they are CCR8+ T cells, responsive to CCL1 and characterized by an enhanced readiness to react to skin-penetrating aggressors (Schaerli et al. 2004). This T-cell activation in the skin is rapid and efficient, and probably facilitated by the high number of cutaneous dendritic cells. On activation they secrete TNF-α, IFN-γ, and also CXCL8 (Keller et al. 2005). These cytokines and chemokines stimulate keratinocytes and endothelial cells to produce more cytokines and chemokines, particularly CCL20, the ligand for CCR6. Due to these cutaneous events, circulating CCR6+ effector T cells, previously primed in the lymph nodes, are stimulated to follow the CCL20 gradient and emigrate into the skin, where they are reactivated and coordinate the inflammatory response. If one transfers this concept to drug hypersensitivity, one has to differentiate between contact dermatitis and generalized exanthema. In contact dermatitis, the hapten penetrates the barrier from outside, forms hapten–carrier complexes,

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Table 94.4 Tolerance mechanisms to drugs. Nonrecognition Even if drugs bind to immune receptors, the interaction (affinity, surface contact) is too weak to elicit a significant reaction Lack of danger signals and preactivation Hapten–carrier complexes do not sufficiently stimulate innate immunity, which is necessary to develop a primary immune response. Or, in the case of the p-i concept, stimulation of the T-cell receptor by the drug is not sufficient to induce cytokine production and proliferation, as the T cell is not sufficiently preactivated to react to this signal Regulatory T cells Treg as well as IL-10/TGF-b-producing Tr1 cells may be insufficiently active in some patients, making them prone to react to small chemical compounds. These patients may have multiple drug allergies and may also suffer from autoimmunity The liver as a tolerogenic organ The generation of reactive metabolites in the liver may induce tolerance to the drug, which might even prevent the development of an immune reaction to the drug in the periphery

and activates dendritic/Langerhans cells. They migrate to the local lymph node, where a hapten-specific T-cell immune response is generated. The effector T cells [CCR6 and cutaneous lymphocyte antigen (CLA) positive] leave the lymph nodes and subsequently localize in the affected skin area. However, in systemic drug hypersensitivity reactions, the hapten concept does not appear to account for all the features of the disorder, whereas the p-i concept does. One has to be aware that drugs are distributed ubiquitously throughout the body, and are not concentrated in certain lymphatic areas like protein antigens. According to the p-i concept, drugs may stimulate T cells throughout the body, if they fit into some TCR and if the T cells are ready to react (effector T cells, with a low threshold of activation). In addition, interaction with MHC molecules (antigen-presenting cells) is needed to make this drug–TCR interaction functional. Such conditions are preferentially found in the skin with the close network of T cells and dendritic cells, and to a certain degree in lymph nodes. Orally applied drugs can be found in the skin soon after administration. There they may interact with the αβ TCR of the resident CCR8+ sentinel T cells, which have a low threshold of activation. On interaction with the MHC molecules on Langerhans and dendritic cells, these T cells may release IFN-γ, TNF-α, and eventually CXCL8. This T-cell stimulation in the skin would result in an initial activation of keratinocytes, which release inter alia CCL20, the ligand for CCR6. Activation of endothelial cells by these proinflammatory cytokines leads to upregulation of adhesion molecules. Circulating effector T cells (CCR6+) are thus stimulated and recruited to the skin. To what extent this drug stimulation of T cells in the skin leads to dendritic/Langerhans cell migration is unclear. Most likely effector T cells (CCR6+) are similarly stimulated in the lymph nodes. Thereby, a concomitant massive stimulation of the immune system in the lymph nodes by herpesviruses for example would enhance the production

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of effector T cells and the readiness of T cells to react to drugs. Such a scenario would explain the following: • the preferential involvement of the skin in drug hypersensitivity reactions; • the rather diffuse, generalized involvement of the skin in drug hypersensitivity, due to the ubiquitous distribution of the drug in the skin; • it is unnecessary to postulate drug metabolism or creation of danger signals due to drug metabolism, which mostly occurs in the liver and cannot be explained by skin involvement; • generalized viral infections are cofactors for drug allergies. Depending on the type of T-cell stimulation, different forms of exanthems will develop (Fig. 94.2).

Clinical features and pathophysiology of cutaneous reactions The most frequent manifestations of drug allergies are delayed cutaneous reactions, so-called “rashes.” These comprise a broad spectrum of clinical and distinct histopathologic features which appear 6 hours to 10 days after drug intake (Figs 94.5 and 94.6). In all forms of delayed drug-induced reactions like exanthems, nephritis and probably hepatitis, cytotoxic mechanisms seem to play an important role. The clinical picture is determined by the strength of cytotoxicity (number of drugspecific cytotoxic T cells and tissue destruction), which is also related to the proportion of cytotoxic CD8 versus cytotoxic CD4 cells, as cytotoxic CD8 cells have a broader target cell repertoire, as well as the type of associated effector mechanism (monocyte or activation of eosinophils and neutrophilic granulocytes).

Maculopapular exanthem Maculopapular exanthem (MPE) is the most frequent drug hypersensitivity reaction, affecting 2–8% of hospitalized pati-

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Effector response Th1

IV a Without drug

LC/DC

Plus drug T cell

T CCR8+

Th2 2

LC

1

IV b TNFa IFN-g CXCL8

Drug

KC

MC KC KC

KC

Eos.

KC

Perforin CCL20 gradient

T CCR8+

IV c

4

CCL20

CTL Granzyme B

T cell CCR6+

Drug 3 Dermis

PMN

Drug

e E-selectin

T CCR8+ EC

3

Blood

CXCL8 GM-CSF

IV d

T cell CCR6+

EC

IL-4 T cell IL-5 CCR6+

KC

CCL1

CCL1

IFN-g T cell CCR6+

CCR8+

CCL1

Epidermis

MC

MØ

T CCR6+

5 EC ICAM T cells: CLA+ CCR6+

Memory CCR6+ 6

EC T cell

DC

Lymph node Fig. 94.5 Schematic representation of critical events involved in the frequent manifestation of drug hypersensitivity in the skin based on the p-i concept. Skin resident CCR8+, CD4+, TCRab+ sentinel T cells are rather easily stimulated by the drug directly (p-i mechanism), as the drug is present in sufficient amount in the tissue and the sentinel cells ready to react. This drug induced activation of the local T cell is facilitated by the close contact of T cells to DC/LC in the skin. This initial T cell stimulation provides signals to local

epidermal and endothelial cells and thus sets an alarm signal. Simultaneously, T cells with a low threshold may be activated by the drug in the lymph nodes, where they expand and them emigrate. The local CCR8+, but mainly the recruited T cells (CCR6+) would evoke an generalized inflammatory skin response, as it is typical for exanthems. The cytokine pattern released by the T cells leads to different forms of the exanthema, which can be subclassified into IVa, IVb, IVc and IVd type reactions. (See CD-ROM for color version.)

ents, especially after treatment with β-lactams, sulfamethoxazole, quinolones, diuretics, and many more (Bigby et al. 1986; Hunziker et al. 1997). It appears mainly 8–11 days after start of treatment, sometimes even 1–2 days after stopping treatment (Fig. 94.6a). In previously sensitized individuals it may appear on the first day. It is clearly a dose-dependent reaction. A study investigating exanthem after treatment with gemifloxacin (a quinolone) showed that females of childbearing age had a higher risk of exanthem, suggesting an influence of estrogens on the clinical manifestation (Schmid et al. 2006). Most MPEs, particularly if caused by β-lactams or gemifloxacin, are rather mild diseases, and treatment with an emollient cream, and possibly topical corticosteroids, or systemic antihistamines for the pruritus is sufficient. Some patients can be treated without aggravation. The exanthem often heals with desquamation within 2–10 days after halt-

ing the incriminated drug. That SJS/TEN develops from an MPE is rather unlikely, as the cells involved are different. On the other hand, some drugs may induce a mixed CD4/CD8 cell activation (Table 94.5). In such cases, prolonged treatment may lead to confluence of the papules, the patient may complain about malaise and fever, and liver function tests indicate hepatitis (alanine aminotransferase/aspartate aminotransferase more than threefold increased). In the blood an eosinophilia (> 0.5 × 109/L) and activated CD8 cells are found (Hari et al. 2001). This illustrates that even “mild” drug hypersensitivity reactions are systemic diseases, and that cutaneous manifestations may only be the tip of the iceberg.

Immunohistology Immunohistology revealed drug-specific CD4 cell infiltration (CLA+CCR6+) in perivascular areas of the dermis (Hari

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45

Number of patients

40 35 30 25 20 15 10 5 0 1 (a)

2

3

4

5

6

7

(b)

8

9

10

11

12

13

14

15

16

17

Days

Fig. 94.6 (a) Typical maculopapular exanthem with partly confluent maculae. (b) Appearance of maculopapular exanthem in gemifloxacintreated healthy volunteers. The biggest study ever to investigate druginduced side effects was performed with gemifloxacin, a quinolone. Initial data revealed that rashes appeared more commonly in women under 40 years of age and in those patients submitted to longer treatment (> 7 days). A prospective study was performed in 987

healthy women aged 18–40 years: 790 (80%) were treated with gemifloxacin for 10 days while the remaining 197 (20%) received ciprofloxacin for 10 days. An exanthem appeared in 31.7% (n = 260) of the gemifloxacin treated women, with a clear peak around day 8–10 after treatment onset. Aminopenicillin-induced exanthems have a similar time course. (From FDA hearing on gemifloxacin, reviewed in Schmid, Campi & Pichler 2006). (See CD-ROM for color version.)

et al. 2001; Pichler 2003). Some T cells progress into the dermoepidermal junction zone and epidermis, where they are reactivated (they express MHC class II and CD25+) and kill keratinocytes in a contact-dependent way by releasing perforin/granzymeB. Some keratinocytes undergo hydropic degeneration, but this apoptosis is not as extensive as with CD8+ T cell-mediated killing (Table 94.4). In addition, the immigrating CD4+ T cells exhibit a heterogeneous cytokine profile, including type 1 (IFN-γ) and type 2 (IL-4, IL-5) cytokines, suggesting that both Th1 and Th2 cells infiltrate the

skin. The cytokine IL-5 is also detectable in the serum. A tissue and blood eosinophilia can be found (Pichler et al. 1997; Hari et al. 1999, 2001). The recruitment of eosinophils is also enhanced by the expression of the chemokines eotaxin and RANTES in MPE lesions (Table 94.5).

Acute generalized exanthematous pustulosis AGEP is a rare disease (about 1 in 100 000 treatments) with an estimated incidence equal to severe bullous skin diseases (SJS and TEN combined) (Roujeau et al. 1991; Britschgi et al.

Table 94.5 Immunologic findings in drug-induced exanthem. Drug-specific T cells are found in the blood, in affected skin and in positive patch tests Drug-specific T cells show a high frequency for many years after the reaction (1 in 250 to 1 in 3000 CD4+ T cells react with the drug) Drug-specific CD4 and CD8 cells can kill in a drug-dependent manner. CD4-mediated killing is perforin/granzymeB dependent and responsible for focal hydropic degeneration of keratinocytes in maculopapular exanthem The clinical picture of the exanthem is determined by the cytokine released by the T cells infiltrating the skin: secretion of IFN-g leads to macrophage activation; secretion of IL-5 leads to eosinophil activation; secretion of CXCL-8 and GM-CSF leads to neutrophil activation and recruitment A large number of drug-specific CD8+ T cells causes more severe bullous skin diseases, probably because all cells are targets for CD8+-mediated cytotoxicity CD8-mediated severe reactions to carbamazepine, allopurinol, and abacavir show a striking HLA-B* association, which is different with different drugs

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Table 94.6 Drugs eliciting severe cutaneous or systemic reactions (list incomplete). Acute generalized exanthematous pustulosis Aminopenicillins* Cephalosporins Pristinamycin Celecoxib Quinolone Diltiazem Terbinafine Macrolides Stevens–Johnson syndrome and toxic epidermal necrolysis Nevirapine* Allopurinol*† Phenytoin* Carbamazepine*† Lamotrigine Co-trimoxazole Barbiturate NSAIDs (oxicams) Drug-induced hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms‡ Carbamazepine*† Phenytoin* Lamotrigine* Minocycline Allopurinol† Dapsone Sulfasalazine Co-trimoxazole Abacavir† * Most frequent elicitors. † Type of reaction might be determined by the presence of a certain HLA-B phenotype. ‡ Abacavir- and lamotrigine-induced systemic reactions often lack eosinophilia; abacavir preferentially affects the respiratory and gastrointestinal tracts; minocycline induces lymphadenopathy more frequently. Compare Peyriere et al. (2006).

2001). It is caused by drugs in over 90% of cases (Table 94.6). Its clinical hallmark is the presence of myriads of disseminated, sterile pustules in the skin (Fig. 94.7), which appear rather rapidly, often after 3–5 days after starting treatment. Patients have fever and massive leukocytosis in the blood, sometimes with eosinophilia, but no involvement of mucosa. Epicutaneous patch-test reactions can cause a similar pustular reaction locally (Fig. 94.7). Immunohistology of the acute lesion reveals subcorneal or intraepidermal pustules, which are filled with neutrophilic granulocytes and are surrounded by activated HLA-DRexpressing CD4+ and CD8+ T cells. Keratinocytes show elevated expression of the neutrophil attracting chemokine IL-8 (CXCL8), and even the T cells migrating into the epidermis

Drug Hypersensitivity

express CXCL8 and GM-CSF. Analysis of sequential patchtest reactions at 48–96 hours suggests that drug-specific cytotoxic T cells emigrate first, causing formation of vesicles by killing keratinocytes. Subsequently T cells and keratinocytes release CXCL8, which recruits granulocytes into the vesicles, which are then transformed to pustules (Britschgi et al. 2001). Some pustules coalesce together and may form bullae. Mortality is about 2–4%, and occurs particularly in older people. Healing occurs within 5 days after stopping the drug. This disease and the underlying T-cell reaction seems to be a model for sterile neutrophilic inflammations (type IVd) like pustular psoriasis and Behçet disease (Keller et al. 2005).

Bullous exanthem, Stevens–Johnson syndrome, and toxic epidermal necrolysis The most severe forms of drug-induced skin reactions accompany formation of bullae. The most severe bullous skin diseases are SJS and TEN. TEN and SJS are rare (incidence 1 in 1 million for TEN, about 1 in 100 000 for SJS). Nowadays, SJS is considered to be the milder and TEN the more severe form of the same disease (SJS < 10%, TEN > 30% skin detachment). They are graded according to SCORTEN, whereby age, underlying disease, and amount of maximal skin detachment are the most important prognostic factors (Roujeau & Stern 1994). According to the European study group of severe cutaneous drug reactions, SJS has a mortality of about 13% and TEN of about 39%. The intermediate form with 10–30% skin detachment is called SJS/TEN overlap syndrome and has a mortality of about 21%. SJS/TEN are clearly different from erythema multiforme exudativum, which is mainly caused by viral infections (Roujeau & Stern 1994), is often recurrent, and affects younger persons (mean age 24 years). In about 6%, no drug treatment was given the week before SJS/TEN started and an infectious origin (Mycoplasma pneumoniae, Klebsiella pneumoniae) is suspected. It can also be due to graft-versus-host disease. Most reactions start within the first 8 weeks of treatment (mean of first symptoms at about 17 days), with some differences according to the causative drug (e.g., with sulfanilamide antibiotics it may appear late). The disease may develop quite rapidly. Initially a macular purple-red exanthem can often be observed, which can become painful, an ominous sign. Within 12–24 hours bullae might already be seen, and the Nikolsky sign is positive. Stopping drug treatment at this time may not prevent SJS, but might prevent an even more severe reaction (TEN). Mucous membranes (mouth, genitalia) are involved with blister formation, and a purulent keratoconjunctivitis with formation of synechiae may result in permanent eye damage. The main causes for SJS/TEN are drugs (Table 94.6), which might differ in frequency in various regions due to genetic and ethnic background. The most important risk factor is HIV infection (low CD4, high CD8 counts), but renal disease and active systemic autoimmune diseases like systemic lupus

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erythematosus, Still syndrome, Sjögren disease and rheumatoid arthritis are also associated with immune stimulation. TEN has to be differentiated from staphylococcal scalded skin syndrome (SSSS), which appears mainly in children under 5 years old, but occasionally also in adults. It is caused by toxigenic strains of Staphylococcus aureus, which secrete two exotoxins, epidermolytic toxin A and epidermolytic toxin B. These toxins cause intraepidermal splitting by specific cleavage of desmoglein 1, a desmosomal cadherin protein that mediates cell–cell adhesion of keratinocytes. In histologic examination a subcorneal split is consistent with SSSS (Stanley & Amagai 2006). In TEN many dead keratinocytes are found, but cell infiltration is only scarcely seen. However, the bullae may be filled by cytotoxic CD8+ T cells, expressing CD56 and αβ TCRs, which kill via perforin/granzymeB but not via the Fas-mediated pathway at this stage of the disease (Nassif et al. 2002). On the other hand, the massive cell death of keratinocytes is hard to reconcile with a cell contactdependent killing process. It has been proposed that the apoptosis of keratinocytes is due to FasL, a soluble molecule of the TNF family, which binds to keratinocytes via Fas and functions as a so-called death receptor (Viard et al. 1998). Since blocking anti-Fas antibodies are found in immunoglobulin preparations, it has been proposed to treat patients with TEN with immunoglobulins. However, the efficiency of this treatment is controversial, and the content of such antibodies in immunglobulin preparations highly variable (Bachot & Roujeau 2003).

Pharmacogenetic associations Extensive research has not revealed a pharmacogenetic predisposition or low glutathione levels in affected persons. How-

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Fig. 94.7 (a) Pustular drug eruption (acute generalized exanthematous pustulosis): disseminated pustules cover the skin; note that the pustules are intraepidermal, nonfollicular (histology). (b) A patch test reaction leading to a pustular reaction. For details see text. (See CD-ROM for color version.)

ever, recent data have shown striking HLA associations of severe CD8-mediated drug hypersensitivity reactions (Mallal et al. 2002; Chung et al. 2004; Hung et al. 2005). Three factors play a role: (i) the type of drug; (ii) the type of reaction, as it was found for cytotoxic CD8 reactions only; and (iii) possibly specific populations, as some associations were exclusively found in Han Chinese. The HLA-B allele, which is the most polymorphic HLA allele, seems to be involved. In carbamazepineinduced SJS/TEN it is HLA-B*1502, for phenytoin HLAB*5601, and for abacavir HLA-B*5701 together with HSP70. It seems that in these CD8+ T-cell reactions, a certain HLA-B allele favors the presentation of certain peptides capable of optimally presenting the drug acting as hapten. Alternatively, certain MHC class B alleles might supplement the T-cell stimulation via these drugs (p-i mechanism) better than others, while absence of this allele renders the T cell insufficiently responsive to the drugs. The extremely high association of certain HLA-B alleles and hypersensitivity reactions to certain drugs may be used to prevent such side effects in the future. HLA typing may identify patients at risk, and their exclusion from treatment reduces the incidence of side effects (Rauch et al. 2006).

Systemic drug reactions: severe drug hypersensitivity syndromes Some drugs are known to cause a severe systemic disease with fever, lymph-node swelling, massive hepatitis, and various forms of exanthems (Table 94.6). Occasionally patients develop colitis, pancreatitis, or interstitial lung disease (Knowles et al. 1999). Over 70% of these cases have a marked eosinophilia (often > 1 × 109/L), and activated lymphocytes are often found in the circulation, similar to acute HIV or

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generalized herpesvirus infections. This syndrome has many names, but the most frequently used are drug (induced) hypersensitivity syndrome (DHS or DiHS) or drug rash with eosinophilia and systemic symptoms (DRESS). Importantly, the symptoms can start up to 12 weeks after start of treatment, often after increasing the dose, and may also persist and recur for many weeks after cessation of drug treatment. The clinical picture resembles a generalized viral infection, e.g., an acute Epstein–Barr virus infection, but is distinguished by the prominent eosinophilia. Many patients have facial swelling, and some have signs of a capillary leak syndrome, namely enhanced vascular permeability of small vessels with generalized edema, also affecting the lungs, similar to patients with a cytokine release syndrome. Indeed, various cytokines are massively increased in the serum of these patients, not as high but still similar to those caused by TGN1412 (a peculiar anti-CD28 monoclonal antibody able to stimulate T cells to cytokine production and proliferation). The reaction to TGN1412 has certain clinical similarities to DRESS, and supports the idea that certain drugs can act like superagonists or superantigens (Pichler 2002; Suntharalingam et al. 2006). There are certain differences in the clinical pictures depending on the drug, e.g., abacavir-induced reactions lack eosinophilia and hepatitis but exhibit mainly gastrointestinal and respiratory symptoms; lamotrigine does rather rarely cause eosinophilia; and allopurinol-induced DRESS often shows kidney involvement (Peyriere et al. 2006). As the clinical picture is quite dramatic and the disease tends to persist despite stopping the drug, many patients are not diagnosed timely and correctly. However, any doctor using anticonvulsants should be aware of this syndrome, as it might occur in 1 in 3000 patients. The mortality is about 10%, and some patients only survive after an acute liver transplantation. This disease is most likely causing more deaths than anaphylaxis (Clarkson & Choonara 2002). Patients with DiHS/DRESS have many activated T cells in the circulation. These drug-specific T cells are stimulated by the parent compound (p-i concept) and they secrete large amounts of IL-5 and IFN-γ (Naisbitt et al. 2003). A peculiar feature of this syndrome is its long-lasting clinical course despite withdrawal of the causative drug (Chung et al. 2004; Peyriere et al. 2006). There may also be a persistent intolerance to other, chemically distinct drugs, leading to flare-up reactions to a rather innocuous drug (e.g., acetaminophen) weeks after stopping the initial drug therapy, further adding to the confusion (see below). Treatment often includes high doses of corticosteroids, particularly if hepatitis is severe. Recently, it has been shown that in many patients with this syndrome, human herpesvirus 6 and/or cytomegalovirus and/or Epstein–Barr virus DNA can be detected in serum during the third or fourth week of the disease (but not before), followed by an increase in antibodies to these herpesviruses (Hashimoto et al. 2003). Thus, similar to HIV, where T-cell activation can also enhance virus production, drug-induced

Drug Hypersensitivity

massive immune stimulation may reactivate these latent lymphotropic herpesviruses, which subsequently replicate and possibly contribute to the chronic course and persistent drug intolerance in affected patients. While in DRESS/DiHS the exanthema might help diagnosis of a drug hypersensitivity reaction, there are other drug hypersensitivity reactions without exanthemas. Many drugs can induce an isolated hepatitis and some (penicillins, proton pump inhibitors, quinolones, disulfiram) an (interstitial) nephritis. Rarer are interstitial lung diseases (Furadantin), pancreatitis, isolated fever, or eosinophilia as the only symptom of a drug allergy. In drug-induced interstitial nephritis, eosinophils can sometimes be detected in the urine (even in the absence of eosinophilia in the blood).

Multiple drug hypersensitivity syndrome The term “multiple drug hypersensitivity” is used for different forms of side effects to various drugs. Some use it to characterize patients with multiple drug intolerance (“pseudoallergy” to various NSAIDs, etc.), while others reserve this term for well-documented, repeated and clearly immune-mediated reactions to structurally unrelated drugs (Sullivan 1989). Cross-reactivity due to structural similarity is not included. In this author’s experience, about 10% of patients with well-documented drug hypersensitivity (skin and/or lymphocyte transformation test positive) have multiple drug allergies (Gex-Collet et al. 2005). For example, a patient reacted to injected lidocaine with massive angioedema; years later the same patient developed a contact allergy to corticosteroids. Alternatively, a patient reacts to amoxicillin, phenytoin, and sulfamethoxazole within a few months, but with different symptoms (MPE, DiHS/DRESS, erythroderma). Most patients have had rather severe reactions to at least one drug. An IgE-mediated reaction might be followed by a T cellmediated reaction. The reason for this accumulation of drug hypersensitivity in one individual is unknown. One explanation might be a deficient tolerance mechanism against small chemical compounds/xenobiotics (see Table 94.3). An immune reaction to a drug, be it via hapten or p-i mechanism, can be seen as a failure of tolerance, and the same patient might not only be prone to develop other drug allergies as well but also autoimmunity. Preliminary data indeed suggest that previous drug allergy might be a risk factor for the development of a delayed hypersensitivity reaction to contrast media.

Flare-up reactions Multiple drug hypersensitivity should be differentiated from flare-up reactions. In patients with systemic drug allergies like severe MPE or DRESS/DiHS, the T-cell immune system is massively activated, similar to acute viral infections. As in the latter, these patients seem to have a lower threshold of reaction to a new drug and might show a flare-up of their rash to a new drug, which is often an antibiotic. The patient is then labeled as allergic to the first and second antibiotic, but

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testing after remission might reveal only sensitization to the first antibiotic, and the second is well tolerated in provocation testing (personal observation). The T-cell reactivity to the second antibiotic would require a massive T-cell stimulation, which is absent under normal conditions.

T-cell activation and symptoms The clinical observation of a flare-up reaction and the previously mentioned cofactors for drug hypersensitivity (viral infections, etc.) suggest that the efficient stimulation of T cells by a drug is the sum of drug–TCR affinity and readiness of the cell to react. Many TCRs have a key ability to interact with drugs, probably with different affinities. If the immune system is quiescent, only drug–TCR interactions of high affinity may be able to stimulate T cells sufficiently to cause T-cell expansion. If sufficient cells react, symptoms might arise. But even such a high-affinity interaction may remain unnoticed if too few cells are stimulated. If the immune system is activated and the readiness to react increased (lower threshold), even relatively low affinity drug–TCR interactions may suffice to activate many T cells and symptoms arise. After the cofactor is eliminated, the low-affinity binding drug remains negative in skin tests and is later well tolerated, if the costimulatory conditions are no longer present. This hypothesis might also explain the transient nature of many exanthems caused by antibiotics in childhood, as the viral cofactors are missed during testing and provocation.

Diagnosis of drug hypersensitivity The diagnosis of drug hypersensitivity addresses three questions: is it a drug hypersensitivity, which mechanism might be involved and which drug has caused it? The symptoms can be extremely heterogeneous and therefore in a patient with “bizarre” symptoms a drug hypersensitivity reaction should be included in the differential diagnosis. On the other hand, some of the above-described skin exanthems are rather typical for drug hypersensitivity and easy to recognize.

Clinical diagnosis A drug hypersensitivity is often suspected if a “rash” appears. Differential diagnosis of this “rash” includes viral exanthems, occasionally other infections, food allergy, and graft-versus-host reaction. A strong itch, an eosinophilia, and the fact that a new drug was recently administered speak for an allergic reaction. Regretfully, many drug hypersensitivity reactions are not well documented, although (or because?) they are iatrogenic diseases. It is extremely important to better educate the treating physician to document a suspected drug hypersensitivity, since the clinical symptoms and its relationship to drug intake are the most important parameters when advising the patient about future use of the drug. Many so-called drug allergies (e.g., diarrhea, stomach ache, headache) to penicillin

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preparations are actually not allergies, and this important class of antibiotics is withheld without reason. It is important to document the severity of the presumed drug hypersensitivity, as this might be decisive in giving the drug again. Special attention should be paid to type and extent of skin symptoms, the involvement of mucosal areas, lymph node enlargement, fever, and some subjective symptoms like malaise, as this could indicate involvement of internal organs. A painful skin and a positive Nikolsky sign might herald a severe bullous skin reaction, which can develop within hours. One should be aware of danger signs (Table 94.7) and the most important elicitors of SJS/TEN and DRESS/DiHS should be known. Some patients have no skin involvement but an isolated drug allergic hepatitis or interstitial nephritis, which might be associated with some rather nonspecific symptoms and be diagnosed only by laboratory investigations. Different mechanisms can lead to drug hypersensitivity symptoms. They should be differentiated, as they need distinct diagnostic steps and may have a different prognosis. Symptoms, together with the time course, usually help to discriminate IgE-mediated from delayed reactions. Immediate reactions start less than 1 hour after drug intake and are usually IgE-mediated (or due to drug-induced mast cell release). Occasionally, after oral drug uptake, IgE-mediated reactions may appear later than 1 hour. Typical symptoms are urticaria and anaphylaxis. Delayed reactions start 6–12 hours after drug intake and are mostly non-IgE-mediated. They represent T cell-orchestrated inflammation or IgG-mediated reactions. In highly sensitized individuals symptoms can arise within 2–4 hours after drug intake: the more drug-specific precursor T cells are present, the more rapidly symptoms may appear. Note that the superagonist anti-CD28 antibody TNG1412, which stimulates almost all T cells via CD28 and which was given to six volunteers in a phase 1 study, started to elicit the catastrophic symptoms after only 60 min, meaning that some T cell-related symptoms, if elicited by high cytokine levels, can appear very rapidly (Suntharalingam et al. 2006). A patient with DRESS/DiHS may therefore react with systemic symptoms within hours to reexposure (or even only to a patch test; personal observation). Last but not least, experience with the drug is important: it is often documented in books or on the drug data sheet (Zuercher & Krebs 1992), e.g., vancomycin can cause basophil degranulation after rapid intravenous injections (“red man” syndrome), which is due to a direct stimulating effect of vancomycin on basophils and can be avoided by slower injections and using lower doses. In perioperative anaphylaxis, one knows that neuromuscular blocking agents account for 60% of anaphylactic reactions, which often start rapidly after onset of the anesthesia. Latex, antibiotics, and sometimes dyes (e.g., methylene blue) for demonstrating lymphatic vessels and lymph nodes are the major causes of the remaining reactions. Their symptoms

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Table 94.7 Clinical and laboratory investigations and danger signs* in drug-induced exanthem. Clinical Extent and type of exanthem (infiltration, bullae, pustules) Pain of skin, Nikolsky sign Involvement of mucous membranes Systemic symptoms (malaise, fever) Lymphadenopathy Hepatosplenomegaly Laboratory Eosinophilia (> 1 × 109/L†); atypical (activated) lymphocytes in the circulation (> 2%†) CRP elevation ALAT/ASAT increase more than threefold† (particularly if together with bilirubin twice upper limit of normal†) Additional investigations depend on clinical signs: liver, kidney, lung, pancreas involvement (urine analysis, creatinine) * Danger signs in italic. † Cutoff values of the laboratory parameters are estimates, and are not based on prospective studies; severe reactions can also develop in the absence of these signs. ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; CRP, C-reactive protein.

often start later. Skin testing is well established in these reactions (Hashimoto et al. 2003). Laboratory investigations can also help to determine the severity of the reaction. In more severe acute reactions, tryptase levels should be determined, optimally 2–4 hours after the peak of the reaction to confirm mast cell involvement. The analysis should be repeated later (> 2 days) to rule out a constitutively elevated tryptase level (e.g., mastocytosis). Such measurements are particularly important if anaphylaxis is suspected during anesthesia, where the sole sign of anaphylaxis might be cardiac arrest without skin symptoms. In delayed reactions a differential blood count may reveal the presence of activated lymphocytes and an eosinophilia, which is a rather common sign for drug hypersensitivity (observed in up to 40% of patients with MPE and in 30% of those with AGEP together with leukocytosis) and in more than 70% in DiHS/DRESS (Rauch et al. 2006). Measurements of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase should be done in patients who complain about malaise, in those with extensive skin involvement, or if drugs known to cause DiHS/DRESS or hepatitis/cholestasis are involved. A transient mild hepatitis is not rare and seems to occur in about 25% of patients with more severe MPE (Hari et al. 2001). Depending on the symptoms, other tests may also be indicated (creatinine, urinary analysis, etc.). C-reactive protein may either be elevated (e.g., in drug-induced interstitial lung or kidney diseases) or normal, even in severe hypersensitivity reactions like DiHS/DRESS.

Identifying the culprit drug Again, the history is the most important factor for identifying

the relevant drug, and it is noteworthy that no test exists that can overrule a convincing history. Moreover, drug hypersensitivity is not one disease, but many different diseases, relying on different pathomechanisms. A single test procedure most likely covers only a part of the possible reactions, and a negative test can rarely exclude a drug hypersensitivity. Important questions include the following. • What drugs have been taken and since when? • Was the dose increased? • Are there comedications and do they possibly interfere with drug metabolism? • How were the drugs tolerated previously? • Have other drugs been tolerated that are known to cause similar effects? • What was the underlying disease? • Have similar reactions occurred previously with or without drugs? Books listing side effects, websites (e.g., http:\\www. pneumotox.com), and drug companies can provide information about known side effects of a drug. In many instances, the history and these sources may allow a rather conclusive allocation of symptoms and drug intake. However, many patients have taken multiple drugs and history alone may be insufficient. In these cases further tests can be justified, which are however often not well validated. The in vivo (skin test) and in vitro tests for drug allergy diagnosis are difficult to standardize and to validate. Although drug allergy is common, it is rather rare for an individual drug; a single drug might elicit different types of symptoms, requiring different tests; provocation tests, which would prove the allergy and the sensitivity of the test, are often not performed for ethical reasons; and for some tests not serum

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but living cells are needed, which thus require a great logistic effort. Despite these obstacles, many groups perform tests, but three rules are important: 1 the test is solely supplementing the history; 2 the sensitivity of the tests is generally low and thus a positive test is more meaningful than a negative test, which cannot rule out a drug allergy; 3 combining different tests might increase the sensitivity.

reactions (Aberer et al. 2003). Moreover, delayed reactions are difficult to prove or disprove by provocation: cofactors are absent during provocation, the dose to elicit symptoms will often not be reached, and patients may experience the symptoms at home. Thus one might be able to exclude immediate reactions to the dose provoked, but treatment with higher doses might nevertheless lead to reappearance of symptoms.

In vitro tests In vivo tests For immediate reactions both skin-prick and intradermal tests are available. There are protocols on the concentrations recommended, e.g., for neuromuscular blocking agents (Mertes et al. 2005). The most common elicitor of drug reactions are the β-lactam antibiotics, in particular amoxicillin. Tests have been available for many years and have been widely used. To form repetitive determinants able to cross-link two FcεRbound IgE molecules, the drug is coupled to polylysines, either by opening the penicillin ring and forming penicilloyl carriers (see Fig. 94.1a), or by binding via the thiol structure (minor components, MDM-PLL). In addition, the eliciting drug (e.g., amoxicillin) should also be evaluated; it is frequently positive as well, if the polylysine-coupled tests show a reaction. The sensitivity of these tests is controversial: older studies from the USA postulated a sensitivity of over 95%, whereas recent studies from Spain found a sensitivity of only 70% (Torres et al. 2003). Other drugs, in particular antibiotics, pyrazolones, contrast media, and many more can also be tested by skin-prick or intradermal tests. An extensive literature exists for test procedure and test concentrations (Brockow et al. 2002; Empedrad et al. 2003). Some drugs might cause false-positive reactions (e.g., to quinolones). Therefore, if a test is not validated, control individuals have to be tested as well to exclude an irritative effect. Intradermal tests should be read after 24 hours and again possibly after 48 hours, to detect delayed reactions. Intradermal tests to amoxicillin appear to be slightly more sensitive than patch tests for detecting delayed reactions, but are also more frequently false positive. In Europe it is common to perform patch tests for delayed reactions (as for contact dermatitis) (Barbaud et al. 2001). Drugs are usually dissolved in petrolatum or phosphatebuffered saline (5–20%). The overall sensitivity is considered to be about 50% (Barbaud et al. 2001) and depends on the disease (often negative in macular reactions and delayed urticarial exanthem, but valuable in severe MPE, DiHS/DRESS and AGEP). It is also a reliable test for abacavir hypersensitivity, even if only hepatitis occurs. Provocation tests are considered to be the gold standard for diagnosis (Messaad et al. 2004). Indeed, they are useful for immediate reactions, but the intention of a provocation test is mostly to show that no reaction occurs or that an alternative drug is tolerated. One should be aware of its limitations and contraindications, and it is not indicated in very severe

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In vitro tests for immediate reactions include the determination of specific IgE to a certain drug. However, the repertoire of drugs to be tested is very limited (benzylpenicillin, amoxicillin, suxamethonium, sulfamethoxazole) and the sensitivity of commercial assays to detect drug-specific IgE appears to be rather low and coupling to a carrier molecule may block the antigenic site of the drug (Schmid et al. 2006). Moreover, circulating drug-specific IgE has a relatively short half-life (often only 1 year). On the other hand, some published “homemade” tests have repeatedly proven helpful in the diagnosis and discrimination of pseudoallergic reactions (Pichler 2003; Manfredi et al. 2004). An interesting alternative to these rather classical solidphase ELISA type assays may be basophil degranulation/ activation tests (upregulation of CD63 or CD203c). These have been proposed to be usable for drug hypersensitivity diagnosis, with a sensitivity in IgE-mediated reactions of about 40% and in “pseudoallergic” reactions to NSAIDs of about 30–60% if five distinct NSAIDs were tested (Sanz et al. 2005; Kleine-Tebbe et al. 2006). The lymphocyte transformation (proliferation/activation) test (LTT) relies on the activation and expansion of T cells in cell culture supplemented by the drug (Pichler & Tilch 2004). Reactivity can be measured by 3H-thymidine incorporation after 5–6 days of culture, but also by other means (e.g., ELISPOT, CSFE staining, CD69 upregulation) (Beeler et al. 2006). It is a cumbersome test, but a clearly positive value is very useful. The sensitivity of the LTT depends on the pathomechanism of the drug hypersensitivity but seem to be more than 90% for DiHS/DRESS, but low in cytotoxic reactions like SJS/TEN. It is, like the patch test, positive in drugs sensitizations occurring via the hapten mechanism but also via the p-i mechanism. The identification of the relevant drug in IgG-mediated reactions like vasculitis or blood cell dyscrasias is difficult. Skin tests, including intradermal tests with delayed reading at 6 and 24 hours, are often negative; LTT is also negative. In hemolytic anaemia, the Coombs test in the presence of the drug might be helpful.

Therapeutic aspects The therapy of drug hypersensitivity diseases is dependent on the symptoms. It can range from the usual treatment of

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anaphylaxis to acute liver transplantation. In exanthema, H1 histamine antagonists and emollient creams are used to reduce the itch. Oral prednisone is used in MPE/DRESS if symptoms are severe or internal organs are affected. On the other hand, the use of prednisone is questionable in SJS and TEN, and there are well-documented cases where TEN appeared with 50 mg prednisone, suggesting that the pathomechanism underlying TEN is quite resistant to corticosteroid treatment. Stopping the possible culprit drugs and avoiding them until clarity is achieved should be possible in most circumstances and might reduce the development of more severe symptoms. In DRESS/DiHS, the use of other xenobiotics should be reduced to a minimum, as flare-up reactions can be provoked. In some milder skin reactions like nonbullous exanthem due to sulfamethoxazole in HIV-positive patients, experience has shown that continuation of treatment might be possible. However, this is not generally advisable. Reintro-

Drug Hypersensitivity

ducing the drug after stopping it might actually precipitate more severe symptoms. An important problem in clinical practice is cross-reactivity. Some general rules are given in Table 94.8; in case of doubt it is advisable to start using graded drug challenges (see below).

Desensitization Under certain circumstances the drug causing allergic or “pseudoallergic” side effects is essential for treatment. This might be the case for penicillin when treating syphilis during pregnancy or the use of certain cytostatic drugs (e.g., cisplatin) in cancer treatments. Most protocols refer to hypersensitivity reactions involving mast cell degranulation, and the procedure is best established for penicillin and NSAID desensitizations (Solensky 2004). It can be performed using oral or, as for cytostatic drugs, by intravenous administration (Table 94.9) (Sullivan 1993).

Table 94.8 Cross-reactivity. In NSAID-induced “pseudoallergy”, cross-reactivity seems to be due to pharmacologic action (strength of cyclooxygenase 1 inhibition) and not the structure Risk of cross-reactivity depends on the type of reaction: IgE-mediated reactions have a higher degree of cross-reactivity than T-cell reactions, probably because antibodies can recognize small molecular components, while T cells tend to recognize the complete structure Cross-reactivity of T cell and antibodies is common within the same class of drugs (penicillins, quinolones, pyrazolones, cephalosporins) Penicillin–cephalosporin cross-reactivity: no problem in T-cell-mediated reactions (maculopapular exanthem); in IgE-mediated reactions, cross-reactivity might occur in about 4% of penicillin skin test-positive patients with first and second-generation cephalosporins. The chance of cross-reactivity is very low if the second drug is negative in skin tests Sulfonamide cross-reactivity: there is extensive cross-reactivity between sulfonamide-containing antibacterials (sulfanilamides), which mainly cause sensitization, but not with other drugs containing a sulfonamide (furosemide, celecoxib, glibencamide)

Table 94.9 Penicillin oral desensitization protocol. (From Sullivan 1993, with permission.)

Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Step 14

Penicillin (mg/mL)

Amount (mL)

Dose (mg)

Cumulative dose (mg)

0.5 0.5 0.5 0.5 0.5 0.5 0.5 5 5 5 50 50 50 50

0.1 0.2 0.4 0.8 1.6 3.2 6.4 1.2 2.4 5 1 2 4 8

0.05 0.1 0.2 0.4 0.8 1.6 3.2 6 12 25 50 100 200 400

0.05 0.15 0.35 0.75 1.55 3.15 6.35 12.35 24.35 49.35 100 200 400 800

Interval between doses is 15 min. At end of the updosing protocol, observe patient for 30 min, then give full therapeutic dose by the desired route.

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The starting dose for desensitization is determined by the dose the patient tolerated in skin testing. This dose generally translates to 1 in 10 000 of the therapeutic dose. Doubling doses are administered every 15 min until the full dose is reached. Once desensitization is completed, the patient can receive the full therapeutic course of penicillin via the desired route. If treatment is discontinued for more than 48 hours, the patient is again at risk of anaphylaxis. A new desensitization has to be started. There are some protocols for desensitization in delayed reactions (e.g., to allopurinol) (Fam et al. 2001). They need a very slow dose increment lasting for weeks and do not always work (personal experience). The mechanism of desensitization in immediate or delayed drug hypersensitivity is not understood. In dubious skin test-negative cases, where one actually expects no allergy, graded drug challenges under careful supervision may be used, starting with about 1 in 100 of the dose, and increasing the dose stepwise (double or triple) every 30– 60 min. The full dose might be achieved in one day and can be continued during the next few days. Tolerance may indicate that no drug hypersensitivity was present, and thus the next treatment may be tolerated without graded drug challenge.

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Hypersensitivity to Aspirin and other NSAIDs Andrzej Szczeklik, Ewa Ni-ankowska-Mogilnicka and Marek Sanak

Summary Aspirin was introduced into medicine over a century ago and has become the most popular drug in the world. Its use has further increased over the last decades when it was shown to reduce the risk of myocardial infarction and stroke. Most people tolerate aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) well, but patients with asthma or urticaria are an exception. In several of them NSAIDs aggravate the disease. These are the most common clinical presentations of aspirin hypersensitivity, i.e. aspirin-induced asthma and aspirin-induced urticaria. Aspirin and other NSAIDs precipitate asthmatic attacks in up to 10% of asthmatics. In fact, by doing so, they uncover a specific clinical syndrome, characterized by aggressive and continuous inflammatory disease of the airway. This syndrome develops according to a characteristic sequence of symptoms, and progresses from upper to lower respiratory tract. It is more frequent in women than men, beginning in adulthood on average at the age of 30 years. Rhinorhea and nasal congestion are usually first symptoms, subsequently complicated by nasal polyposis. Asthma and aspirin hypersensitivity develop a few years later. Asthma, usually of severe type, is characterized by blood and nasal eosinophilia, and runs a protracted course despite avoidance of analgesics. Almost half of the patients require systemic corticosteroids to control their sinusitis and asthma. At the biochemical level, the characteristic feature is profound alteration in eicosanoid biosythesis and metabolism, best exemplified by overproduction of cysteinyl leukotrienes. Acute reactions of hypersensitivity are elicited via cyclooxygenase 1 inhibition by NSAIDs. Aspirin and other NSAIDs can induce or exacerbate skin eruptions in up to 40% of patients with chronic idiopathic urticaria. The attacks are related to inhibition of cyclooxygenase 1. Patients who react to aspirin with skin rash share a similar eicosanoid profile with aspirin-induced asthmatics, although the eicosanoid alterations are less pronounced. Similarly to aspirininduced asthma, mastocytes appears to be the main cellular source of the participating inflammatory mediators.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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In some patients drug-specific immune-mediated allergic reactions to NSAIDs occur independent of cyclooxygenase inhibition. These present as generalized pruritis, erythema, urticaria, angioedema, rhinorhea, asthma, and anaphylactic shock. Allergy to pyrazolone drugs, especially metamizole and aminophenazone, are relatively common and life-threatening. In patients with allergy to individual NSAIDs, skin tests are often positive and specific IgE can usually be demonstrated in specialized laboratories.

Classification Aspirin can precipitate or aggravate symptoms in about 10% of asthmatics as well as a lesser percentage of patients with chronic urticaria. These hypersensitivity reactions are the markers of two distinct clinical syndromes, which develop according to characteristic patterns and follow a specific clinical course. The first, called aspirin-induced asthma (AIA), is characterized by chronic eosinophilic rhinosinusitis with nasal polyposis and persistent, often severe, asthma. The second is marked by aspirin-triggered urticaria/angioedema. These acute nonallergic hypersensitivity reactions to aspirin are elicited via inhibition of cyclooxygenase (COX)-1 by nonsteroidal antiinflammatory drugs (NSAIDs). The two syndromes, cared for by different specialists, are characterized at the biochemical level by profound alterations in eicosanoid biosynthesis, best exemplified by overproduction of cysteinyl leukotrienes. They should be clearly differentiated from other adverse reactions to NSAIDs with allergic background.

History Aspirin was introduced into medicine in 1899, and already three years later it was implicated as the cause of an anaphylactic reaction. Hirschberg (1902) described the first case of a transient acute angioedema/urticaria that occurred shortly after ingestion of aspirin. The symptom of acute bronchospasm was first reported in 1919 and a year later the first death from asphyxia due to aspirin was described. The association

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of aspirin sensitivity, asthma and nasal polyps was reported by Widal et al. (1922), and the syndrome was popularized by Samter and Beers (1968), who presented a description of its clinical course. In the 1970s the link between precipitation of asthmatic attacks and inhibition of COX by aspirin and other NSAIDs was described (Szczeklik et al. 1975). In the following three decades other alterations in arachidonic acid metabolism have been discovered and found recently to be common also in aspirin-induced angioedema/urticaria. Adverse reactions to aspirin and NSAIDs may have different clinical symptoms and different pathogenesis. The two most common presentations of aspirin hypersensitivity are bronchial asthma and urticaria/angioedema.

Aspirin-induced asthma Definition This term refers to a distinct clinical syndrome of intractable inflammation in both the upper and lower respiratory tract, characterized by chronic eosinophilic rhinosinusitis with nasal polyposis and asthma. Aspirin and other NSAIDs that inhibit COX-1 exacerbate this condition. At the biochemical level profound alterations in arachidonic acid metabolism are characteristic. The disease runs a protracted course, even if COX-1 inhibitors are avoided, and at least half of the patients require systemic corticosteroids to control their rhinosinusitis and asthma. Since exposure to aspirin does not initiate the underlying inflammatory disease, this syndrome is increasingly referred to as aspirin-exacerbated respiratory disease (AERD).

Prevalence In the general population the prevalence of aspirin hypersensitivity ranges from 0.6 to 2.5% (Hedman et al. 1999; Vally et al. 2002; Kasper et al. 2003; Gomes et al. 2004). In asthmatic subjects three large questionnaire surveys, carried out in Finland, Poland and Australia, reported AIA in 8.8, 11 and 4.3% of patients, respectively (Hedman et al. 1999; Vally et al. 2002; Kasper et al. 2003). Many asthmatics may not be aware of their hypersensitivity because they have not ingested aspirin or other NSAIDs (mostly acetominophen users). Analgesic ingestion frequently occurs coincidentally with asthma exacerbation, often in the absence of a direct cause-and-effect relationship. Underdiagnosis may also be caused by the lack of routine diagnostic aspirin challenges. Jenkins et al. (2004) carried out a metaanalysis of 15 studies, using oral aspirin challenges to diagnose AIA, and found that the combined prevalence of AIA reached 21% (CI 14–29%). In populations of patients with chronic hyperplastic eosinophilic sinusitis (CHES) and nasal polyposis, the prevalence of aspirin hypersensitivity is even more elevated, reaching 30–40% (Stevenson & Szczeklik 2006; Szczeklik & Sanak 2006; Wenzel 2006). AIA is very rare in preschool children, somewhat more frequent in teenagers; in asthmatic children, as confirmed by

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oral aspirin challenge, it may reach 5% (Jenkins et al. 2004). Bronchospastic reactions to ibuprofen were found in 2% of children exposed to oral challenge with this drug (Debley et al. 2005). More females than males acquire this disease, females outnumbering men by a ratio of 2 : 1. No racial or ethnic predilection for AIA has been identified. In 1–6% of cases there is a family history of aspirin hypersensitivity (Berges-Gimeno et al. 2002; Szczeklik et al. 2000).

Pathogenesis Aspirin hypersensitivity remains a disease of unknown etiology. It is an acquired condition with a low prevalence during childhood. A slow onset and manifestations delayed to the third or fourth decade of life led to hypotheses of transmissible or autoimmune pathogenesis of aspirin hypersensitivity. Familial aggregation of the disease is rare. Consistent with the acquired syndrome, in rare instances aspirin hypersensitivity is self-limiting and subsides after several years. Mostly, however, the disease affects the patient for life.

AIA and its relation to innate antiviral responses A characteristic slow onset reminiscent of a protracted viral infection of the respiratory tract raised the suspicion of a viral origin of the disease (Szczeklik 1988). As the initial symptoms are similar to a common cold, which tends to persist and progress, rhinoviruses were the suspect pathogens. Human rhinovirus (HRV) is a leading cause of acute infections of the upper respiratory tract. It was only recently, however, that rhinoviral infection was demonstrated to affect the lower airways. Using a sensitive in situ reverse-transcriptase polymerase chain reaction (RT-PCR) method, rhinovirus was detected in bronchial biopsies of 73% of stable asthmatics (N = 30) free for at least 3 weeks of clinical signs of respiratory infection, as compared to 22% controls. All aspirin-sensitive patients (N = 7) had rhinoviral RNA (Woj et al. 2006, 2008). Delayed clearance of the virus is related to a deficiency in antiviral response and seems a characteristic feature of asthmatic bronchial epithelium. In cell cultures Peng et al. (2006) demonstrated that experimental HRV14 infection is not accompanied by an adequate interferon (IFN)-β response and that HRV14 quite specifically inhibits interferon regulatory factor-3, a transcription factor required to induce type I interferon and orchestrate the antiviral response of the epithelial cell. This effect, though specific to HRV14, had broader consequences, because infected cells did not react to a double-stranded RNA, which normally elicits similar antiviral responses mediated by the transcription factor NF-κB and by interferons. Thus, it remains possible that HRV is a trojan virus, disrupting antiviral defense mechanisms of respiratory epithelia. A permissive host phenotype for HRV was recently characterized ex vivo by another research group. Firstly, Wark et al. (2005) found that HRV infection of primary bronchial epithelia in vitro results in dimished production of IFN-β as compared with control cells. Such a defect resulted in

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increased replication and depressed apoptosis of HRV16 in the cell cultures of asthmatics. Contoli et al. (2006) extended this observation to the novel family of IFN-λ, deficiency of which was demonstrated in HRV16-infected bronchial cells and pulmonary macrophages and inversely correlated with pulmonary lung function of experimentally inoculated asthmatics. However, this deficit was not specific for HRV infection and was detectable also on stimulation by bacterial enterotoxin. In summary, it can be generalized that AIA possibly affects asthmatic subjects due to an incompetent antiviral response; HRV appears to be a model pathogen.

Genetic susceptibility to aspirin hypersensitivity: etiologic link A number of approaches attempting to elucidate the pathogenesis of aspirin hypersensitivity have sought a genetic susceptibility. This seemed a paradoxical quest, considering the acquired character of the disease. However, susceptibility to the disease can manifest only following an appropriate triggering event, which could be a persistent pathogen infection. Studies on the genetic variability of human leukocyte antigen (HLA) class II revealed that in many inflammatory diseases, e.g., type I diabetes, rheumathoid arthritis or inflammatory bowel disease, genetic associations with particular antigens increase the risk of morbidity. In aspirin hypersensitivity such an association has been found in two ethnically distant populations of Slavs and Asians: the relatively common HLA-DRB1*0301 allele was found in aspirin-hypersensitive Polish patients by Dekker et al. (1997) and in aspirin-hypersensitive Koreans by Choi et al. (2004a). The immunologic restriction of the HLA class II system can lead to incompetent recognition of a pathogen and persistence of viral infection. It

is interesting that a similar finding of genetic association was reported in patients with aspirin-sensitive urticaria by Kim et al. (2005a) for the HLA-DRB1*1302 allele. Because there is unlikely to be any relation between aspirin-induced urticaria and HRV, this association is either a marker for an autoimmune response or suggests involvement of another pathogen in skin manifestations of aspirin hypersensitivity.

Effector pathways of aspirin hypersensitivity Prostaglandins A nonallergic mechanism underlying precipitation of asthmatic attacks by aspirin in hypersensitive patients was proposed over 30 years ago (Szczeklik et al. 1975). It was founded on pharmacologic inhibition of COX and explained the crossreactivity between NSAIDs with different chemical structures (Fig. 95.1). This cyclooxygenase theory was confirmed by several studies (Szczeklik 1990) and was further refined following discovery of the second COX isoenzyme, COX-2. Lack of drug tolerance in susceptible patients correlates with its activity against the noninducible cyclooxygense, COX-1, which in contrast to COX-2 has a rather constitutive expression (Szczeklik & Sanak 2002). Because inhibition of COX-1 (and COX-1 gene derived alternate splicing variant COX-3) is one of the most common pharmacologic interventions, it required explanation why only a fraction of individuals suffer from hypersensitivity reactions. Currently, no genetic variants of the COX-1 or COX-2 gene are known to associate with aspirin hypersensitivity. Despite many studies, no plausible explanation has been proposed for the unusual sensitivity of COX-1 to pharmacologic inhibition in the disease. Using mRNA and protein analysis, only the genetic expression of COX-2 was decreased in nasal polyps and

Aspirin NSAIDs

LTC4 LTD4 PGD2

1968

COX-1

pPGE2

Fig. 95.1 Symptoms of aspirin hypersensitivity are triggered by inhibition of cyclooxygenase (COX)-1. This results in decrease of PGE2 level below the threshold required to inhibit activation of inflammatory cells. Release of mast cell mediators (LTC4, LTD4, PGD2) and activation of cysteinyl biosynthesis in eosinophils mediates the symptoms. A role for persistent viral infection in perpetuating inflammation of respiratory mucosa has been suggested.

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bronchial fibroblasts (Kowalski et al. 2000; Picado et al. 2000; Pierzchalska et al. 2003). In contrast, no difference in COX-1 levels were found in relation to aspirin hypersentivity. These results should be interpreted with caution, because of the substantial inducibility of COX-2 enzyme and its relatively high expression in the airway epithelia. Some in vitro studies, based on peripheral blood cell preparations, also pointed to other peculiarities of arachidonic acid metabolism. Leukocytes from hypersensitive patients exposed to aspirin or other NSAIDs release more 15-hydroxyeicosatetraenoic acid (15-HETE). The relevance of this finding to the pathogenesis is unknown, although this phenomenon has been proposed for in vitro diagnosis (Kowalski et al. 2003, 2005). It is known that COX-1 products can contain a small amount of 15-HETE but the fraction of this metabolite is not affected by pharmacologic inhibition of the enzyme. Accumulation of 15-HETE is due to 15-lipoxygenase activity, an enzyme present in eosinophils and basophils, which pro bably becomes activated during experimental inhibition of COX-1. In most cells, except blood platelets, the main product of COX-1 and COX-2 is prostaglandin (PG)E2. A special role for PGE2 in the pathogenesis of AIA has been suggested (Szczeklik 1995). Peripheral blood mononuclear cells of some patients with AIA undersynthesize PGE2 at baseline (Schaefer et al. 1999). This places patients with AIA at the disadvantage of lacking sufficient concentrations of cellular or transcellular PGE2 to stabilize mast cells and slow synthesis of cysteinyl leukotrienes (Stevenson & Szczeklik 2006). The biological action of PGE2 is dependent on cellular receptors, which differ in signal transduction mechanism. The type 1 receptor for PGE2 is coupled to phospholipase C and inositol trisphosphate/ diacylglycerol mediators. Type 2 and 4 receptors (EP2 and EP4) stimulate adenylate cyclase leading to increased cyclic adenosine monophosphate (AMP), while the type 3 receptor causes inhibition of adenylate cyclase. The EP2 receptor is present on many inflammatory cells, including macrophages, mast cells, eosinophils, and T lymphocytes (Ying et al. 2006). It is regarded as a prostanoid inhibitory receptor and can inhibit release of cysteinyl leukotrienes from activated mast cells (Celik et al. 2001) (Fig. 95.2). Interesting interactions of PGE2 with 15-HETE production in vitro suggest an additional link between inhibition of COX-1 and release of cysteinyl leukotrienes in subjects with aspirin hypersensitivity. A nonspecific agonist of EP2/EP3 receptors for PGE2, misoprostol, caused inhibition of 15-HETE formation at low concentrations but was ineffective at higher concentrations (Kowalski et al. 2003). It seemed to restore the profile of 15-HETE production typical of aspirin-tolerant control subjects within the range of concentration required for stimulation of EP2 receptors. The EP2 receptor mediates some of the effects of PGE2 by activation of adenylate cyclase and increase in cellular cyclic AMP. In a large genomic study, EP2 gene polymorphism,

Hypersensitivity to Aspirin and other NSAIDs

Arachidonic acid 15-LO

15S-HETE 5-LO

COX-2

5-LO

5-HPETE 15-LO

+ ASA

15R-HETE 12-LO

5-LO

5S,6S,15* epoxytetraene

HO

OH

O OH

15 H OH

Lipoxin A4

Fig. 95.2 A simplified scheme of transcellular biosynthesis of lipoxins. Lipoxygenases of arachidonic acid have different expression patterns: 5lipoxygenase (5-LO) in neutrophils, basophils, eosinophils and mast cells, 15lipoxygenase (15-LO) in eosinophils, airway epithelial cells and monocytes, 12-lipoxygenase (12-LO) in blood platelets. Cyclooxygenase (COX)-2 is inducible in monocytes and endothelial and epithelial cells and contributes to lipoxin biosynthesis only if inhibited by aspirin. An isomer of lipoxin (LX)A4, LXB4, is also generated by hydration of an epoxytetraene intermediate and differs from LXA4 by the presence of hydroxyl at carbons 5, 14 and 15 rather than 5, 6 and 15. Aspirin-specific lipoxins are epimers, their hydroxyl at carbon 15 is on the same side of the arachidonate molecule as the hydroxyl at carbon 5.

located within a distant regulatory region of the gene PTGER2, was associated with aspirin hypersensitivity in asthmatic patients (Jinnai et al. 2004). EP2 receptor signaling alterations would be difficult to prove functionally in aspirinhypersensitive patients, as this receptor frequently colocalizes with the EP3 receptor, which has the opposite effects on cyclic AMP. However, it was shown by immunochemistry that infiltrating inflammatory cells in aspirin-sensitive rhinosinusitis had diminished expression of EP2 receptors (Ying et al. 2006).

Leukotrienes Cysteinyl leukotrienes originate from arachidonic acid specifically oxidized by 5-lipoxygenase, an enzyme abundant in granulocytes (Wenzel 2003). However, only limited types of cells can express leukotriene (LT)C4 synthase, a key enzyme controlling production of cysteinyl leukotrienes. AIA is marked by increased production of cysteinyl leukotrienes (Fig. 95.3). LTE4 baseline levels in urine are distinctly elevated and they rise further following aspirin challenge (Shuaib Nasser & Lee 1998). They increase also in nasal exudates, induced sputum, bronchoalveolar lavage fluid, and exhaled air condensates following exposure to aspirin in a time-dependent manner (Sgadek & Szczeklik 1993; Szczeklik et al. 1996; Sanak & Sampson 1999; Obase et al. 2002; iwierczykska et al. 2003; Sanak et al. 2004). In aspirin-sensitive asthmatics, the cells producing cysteinyl leukotrienes were identified as eosinophils

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LTE4 pg/mg creatinine

10 000

1000

100

10

0 Controls

ATA log HPLC-MS

AIA log ELISA

Fig. 95.3 Urinary excretion of leukotriene (LT)E4 in aspirin-hypersensitive and aspirin-tolerant asthmatic patients versus healthy controls. LTE4 was measured by two methods: enzyme-linked immunoassay (ELISA) and high-performance liquid chromatography–mass spectrometry (HPLC-MS) and standardized for urinary creatinine. Correlation between the methods was significant (P < 0.001, R2 0.45). At 100 pg/mg creatinine threshold, HPLC-MS had 31% sensitivity and 95% specificity for detecting aspirin intolerance among asthmatic patients.

and mast cells. A distinct feature of bronchial biopsies in AIA patients is increased expression of LTC4 synthase; protein immunoreactivity also negatively correlated with tolerance to aspirin measured as a provocative dose (Cowburn et al. 1998). A regulatory region of the gene coding for LTC4 synthase has a common single nucleotide polymorphism (A444C). The variant allele −444C was demonstrated to cause increased expression of the enzyme and/or cysteinyl leukotriene overproduction, although this association is not limited to aspirin hypersensitivity but characterizes severe asthma in general (Sanak et al. 2000a). Several other polymorphisms have been studied in aspirin hypersensitivity (Kim et al. 2005b,c, 2006; Park et al. 2005), although most of these findings await replication in other populations. Eosinophils and mast cells closely cooperate as a functional unit during allergic inflammation (Piliponsky et al. 2001). In aspirin hypersensitivity this common inflammatory mechanism operates separately from allergic sensitization (Mita et al. 2001). Because cross-reactivity between drugs can be predicted by COX-1 inhibitory potency rather than chemical structure, any specific immunologic sensitization is unlikely. In the following sequence of events mast cells specifically produce PGD2, as evidenced by rise in the stable metabolite 9α,11β-PGF2 in plasma and urine (Bochenek et al. 2003). Tryptase, another marker of mast cell activation, also increases following aspirin challenge (Sgadek & Szczeklik 1993). Activation of complement may contribute to release of these mediators (Lee et al. 2006). In contrast to the IgE-mediated immediate reaction, aspirin hypersensitivity is accompanied by much more profound alterations in metabolism of eicosanoids. Baseline production of cysteinyl leukotrienes is

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increased to the point where asthmatic patients with aspirin hypersensitivity differ significantly from aspirin-tolerant asthmatics, also during a symptom-free period (Sanak et al. 2004). A steady state of cysteinyl leukotriene overproduction does not necessarily indicate alterations in mast cell–eosinophil activation. Following surgical intervention to reduce hyperplastic rhinosinusitis, the level of cysteinyl leukotriene metabolites in urine diminished (Higashi et al. 2004). There are two subtypes of cysteinyl leukotriene receptors. The CysLT1 receptor is the predominant one. It is expressed in the respiratory tract, on infiltrating inflammatory cells including eosinophils, mast cells, lymphocytes, macrophages, and neutrophils, bronchial smooth muscle, and epithelia. Recently, upregulation of CysLT1 was described in allergic inflammation but enhanced expression of this receptor has been detected quite specifically in aspirin-hypersensitive patients (Sousa et al. 2002). Increased expression of CysLT2 has also been described by Corrigan et al. (2005) on virtually the same infiltrating inflammatory cells. It is characteristic of chronic rhinosinusistis by its presence on epithelial and glandular cells of the respiratory epithelium, but does not differ with status of aspirin sensitivity. Currently, the only pharmacologic target is CysLT1, which is antagonized by montelukast, pranlukast and zafirlukast. It is interesting that CysLT1 receptor-positive cells decrease following intranasal desensitization with lysyl-aspirin (Sousa et al. 2002). However, involvement of CysLT1 in chronic rhinosinusitis seems secondary rather than the initial mechanism. Chao et al. (2006) described high immunoreactivity for this receptor in nasal polyps. Both stromal cells and epithelia of the polyps expressed CysLT1 and no clear difference between aspirin-sensitive and aspirin-tolerant patients was found.

Lipoxins Lipoxins are transcellular products of the enzymatic oxidation of arachidonic acid. Only some cells, like eosinophils, express the two distinct lipoxygenases, e.g., 5-lipoxygense and 15lipoxygenase, required to produce lipoxins. Inhibition of COX-1 specifically by aspirin can trigger production of lipoxin (LX)A4, which is even more potent because of altered stereochemistry of the reaction. Patients with aspirin hypersensitivity have a deficiency in the capacity for lipoxin biosynthesis. This phenomenon was observed mostly in vitro following activation of peripheral blood granulocyte or eosinophil preparations (Sanak et al. 2000b; Kowalski et al. 2003; Perez-Novo et al. 2005). Lipoxins are unique representatives of antiinflammatory eicosanoids and have been proven to diminish inflammation in an animal model of asthma by inhibition of granulocyte transmigration to the inflammation site. Mast cells and eosinophils are sources of arachidonic acid mediators, of which cysteinyl leukotrienes are the hallmark of AIA. It has to be emphasized that these effector mechanisms are also common in allergic reactions and the difference is rather a quantitative one. For example, increased produc-

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tion of cysteinyl leukotrienes and diminished biosynthesis of lipoxins have been reported in severe asthma (Levy et al. 2005). Upregulation of the lipoxygenase pathway due to genetic variability only partially contributes to aspirin hypersensitivity, as promoter variants of the 5-lipoxygensae gene (ALOX5) and LTC4 synthase (LTC4S) associate with severe asthma in general (Choi et al. 2004b). Common mechanisms of inflammation are the reason for difficulties in delineatiing between aspirin hypersensitivity manifested by airway disease and other types of asthma. Thus, identification of a triggering mechanism for aspirin hyperensitivity, i.e., inhibition of COX-1 by a drug, remains the most searched for characteristic of the disease.

Natural history and presentation AIA is usually preceded by chronic rhinitis/rhinosinusitis. According to the two large studies involving patients from Europe (Szczeklik et al. 2000) and America (Berges-Gimeno et al. 2002), the first nasal symptoms appeared at the age of 29 and 34 years, respectively. The presentation was mainly as watery rhinitis and nasal congestion and many patients described an upper respiratory tract infection (common cold). Perhaps a viral infection may initiate the vicious circle of inflammatory events leading to AIA in genetically susceptible subjects (Szczeklik 1988). Asthma is diagnosed usually 2–3 years later and at about the same time the first unexpected adverse clinical reaction precipitated by aspirin or other NSAID appears in patients who previously tolerated these drugs very well. The “classic” adverse reactions to aspirin ingestion include bronchospasm of different severity, usually accompanied by profuse rhinorhea, nasal congestion, sneezing and itching, ocular “injection,” tearing, and rarely periorbital swelling. Some patients experience skin rush and erythrema of the head and neck. The severity of adverse reactions to aspirin or other NSAID may range from isolated rhinitis to life-threatening anaphylactic reactions. Rare patients following aspirin ingestion manifest nausea, stomach cramps, or myocardial ischemia (Szczeklik et al. 2002). AIA is usually a severe type of asthma and runs a protracted course despite complete avoidance of aspirin and other NSAIDs. Up to 50% of patients require systemic corticosteroids to control their asthma. In a recent Japanese study, patients with multiple exacerbations of asthma more frequently had hypersensitivity to aspirin and reported more asthma-related hospitalizations (Koga et al. 2006). Aspirin hypersensitivity was also strongly associated with near-fatal asthma (Yoshimine et al. 2005). Positive skin-prick tests to at least one aeroallergen were documented in 34–64% of patients with AIA (Bochenek et al. 1996; Berges-Gimeno et al. 2002). Marked blood and sputum eosinophilia are common. The majority of patients suffer from CHES and polyposis usually filling all the sinuses and finally destoying bone structures. Loss of smell indicates the development of nasal polyposis and normal olfaction corre-

Hypersensitivity to Aspirin and other NSAIDs

lates negatively with AIA diagnosis (Stevenson et al. 2003). Symptoms from the upper respiratory tract are often exacerbated by sinus infections, on average five to six episodes each year. Aspirin-sensitive nasal polyps are characterized by rapid regrowth, resulting in multiple sinus surgery (Berges-Gimeno et al. 2002). Sinus involvement, as evidenced by computed tomography, is of higher magnitude in AIA patients compared with patients who tolerate aspirin well (Kordek et al. 2000; Mascia et al. 2005).

Cross-reactions with aspirin and NSAIDs Typically, patients with AIA are also hypersensitive to all NSAIDs that preferentially inhibit COX-1 (Szczeklik et al. 1975, 1977; Mathison & Stevenson 1979). The most common analgesics provoking hypersensitivity reactions in the USA are aspirin (80%) and ibuprofen (41%) (Berges-Gimeno et al. 2002). Acetaminophen is a weak inhibitor of COX-1, and in doses not exceeding 500–1000 mg is regarded as a relatively safe therapeutic alternative in most patients with AIA. According to Jenkins et al. (2004) less than 2% of asthmatics are sensitive to both aspirin and acetaminophen. Several studies with NSAIDs that preferentially inhibit COX-2 have been were carried out over the last few years. Meloxicam and nimesulide are usually well tolerated by patients with AIA when given at low doses. However, higher doses could elicit hypersensitive reactions in some AIA patients (Asero 2000; Quaratino et al. 2000; Bavbek et al. 2004, 2006). Highly selective COX-2 inhibitors (rofecoxib, celecoxib or the less popular valdecoxib, etoricoxib, parecoxib and lumiracoxib) were found to be well tolerated in a series of placebo-controlled clinical trials (Yoshida et al. 2000; Stevenson & Simon 2001; Szczeklik et al. 2001a; Woessner et al. 2002; Gyllfors et al. 2003; El Miedany et al. 2006; Viola et al. 2006). However, rofecoxib and valdecoxib have been withdrawn from the market because of concerns about increased incidence of cardiovascular complications. Some patients who are extremely sensitive to aspirin may develop hypersensitive reactions to celecoxib and other coxibs (Levy & Fink 2001; Murr et al. 2003; Passero 2003; Bavbek et al. 2004; Baldassarre et al. 2006; Mastalerz et al. 2006a; Morais-Almeida et al. 2006). In individual cases these unusual reactions may be partially IgE-mediated (see below).

Diagnosis Establishing a diagnosis of aspirin hypersensitivity is of utmost importance. It provides the patient with a comprehensive list of common drugs that must be avoided because of the high risk of life-threatening reactions (Table 95.1) and indicates which NSAIDs can be taken safely. Although a history of adverse reactions, as well as a typical clinical presentation of this syndrome, may raise suspicion of aspirin hypersensitivity, the diagnosis of AIA can be definitely established only through aspirin challenge. There is no reliable in vitro test, though the search for one continues (Gamboa et al. 2004;

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Kowalski et al. 2005). There are four types of provocation challenge with aspirin: oral, inhalation (bronchial), nasal, and intravenous (Dahlen & Zetterstrom 1990; Milewski et al. 1998; Nizankowska et al. 2000; Melillo et al. 2001; AlonsoLlamazares et al. 2002; Stevenson et al. 2003; Mita et al. 2004; Nizankowska-Mogilnicka et al. 2007). Oral, inhalation and intravenous aspirin challenges have to be carried out in a hospital under the direct supervision of a physician and technicians skilled in performing these tests. Emergency resuscitative equipment should be readily available. Patients should have an intravenous line and asthmatics must be in a stable clinical condition. Baseline forced expiratory volume in 1 s (FEV1) should be at least 70% of the predicted value. Aspirin challenge should be always preceded by a placebo challenge. The oral challenge test with aspirin was introduced into clinical practice in the early 1970s (Szczeklik et al. 1975, 1977). The oral route mimics natural exposure and the challenge procedure does not require special equipment, except FEV1 measurement. There exist various protocols of oral challenges. The EAACI/GA2LEN guidelines use four exponentially increasing doses of aspirin (27, 44, 117, and 312 mg) at 1.5–2 hour intervals until a cumulative dose of 500 mg is reached (Fig. 95.4). If a patient gives a history of a severe hypersensitivity reaction (profound dyspnea and/or anaphylactic shock) the test is commenced with 10 mg of aspirin and the next dose of 17 mg is administered 1.5–2 hours later, i.e., the 27-mg dose is divided into two doses for safety reasons (Fig. 95.4). If a patient with a very strong suspicion of aspirin hypersensitivity shows no reaction after the final dose of 312 mg aspirin (i.e., after administration of a cumulative dose

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900 Cumulative dose of aspirin (mg)

Diclofenac (Voltaren, Cataflam) Diflunisal (Dolbid) Etodolac (Lodine) Fenoprofen (Nalfon) Flurbiprofen (Ansaid) Ibuprofen (Motrin, Rufen, Advil) Indomethacin (Indocin) Ketoprofen (Orudis, Oruval) Ketorolac (Toradol) Meclofenamate (Meclomen) Mefenamic acid (Ponstel) Metamizole (Pyralginum) Nabumetone (Relafen) Naproxen sodium (Anaprox, Aleve) Naproxen (Naprosyn) Oxaprozin (Daypro) Piroxicam (Feldene) Sulindac (Clinoril) Tolmetin (Tolectin)

500 mg

1000

Table 95.1 Nonsteroidal antiinflammatory drugs that inhibit cyclooxygenase 1 and which invariably elicit reactions in patients with aspirin-induced asthma. (Brand names in parentheses)

800 700 600 312 mg

500 400

117 mg

300 200 100

27 mg (10 mg)* (17 mg)*

44 mg

0 0

1.5

3

4.5

6

7.5

Time (h) Fig. 95.4 Oral aspirin challenge flowchart (for explanation see text).

of 500 mg), another capsule containing 500 mg of aspirin may be administered 1.5–2 hours following the preceding dose; the cumulative dose in this case will be equivalent to 1000 mg of aspirin (Fig. 95.4). FEV1 is measured before each consecutive dose of aspirin and subsequently every 30 min, i.e., at 30, 60 and 90 (120) min thereafter. The challenge is interrupted if a decrease in FEV1 of 20% or more of baseline occurs (a positive reaction), or when the maximum cumulative dose of aspirin (1000 mg) is reached without a fall in FEV1 of 20% or more and the symptoms of aspirin hypersensitivity do not appear (a negative reaction). The test could also be regarded as positive when severe extrabronchial symptoms (e.g., very severe nasal congestion, profound rhinorrhea) of aspirin hypersensitivity appear. In inhalation (bronchial) challenge lysine-aspirin (soluble form of aspirin) is administered by a dosimeter-controlled jet-nebulizer; increasing doses of lysine-aspirin are used up to the cumulative dose of 188 mg of aspirin (NizankowskaMogilnicka et al. 2007). This challenge, more frequently used in Europe, is safer and faster to carry out than the oral test, although it is slightly less sensitive (Dahlen & Zetterstrom 1990; Nizankowska et al. 2000). Treatment of adverse respiratory reactions to oral and bronchial aspirin challenges has been reviewed in detail elsewhere (Stevenson et al. 2003; Nizankowska-Mogilnicka et al. 2007). Nasal aspirin challenge with lysine-aspirin (Milewski et al. 1998; Alonso-Llamazares et al. 2002) or rarely ketorolac solutions (White & Stevenson 2006) are less popular than the oral or inhalation tests. Evaluation of nasal responses following nasal instillation of 16 mg of aspirin (as lysine-aspirin solution) is based on nasal symptom scores and rhinomanometry or acoustic rhinometry or peak nasal inspiratory flow. Unlike oral challenge, the nasal test does not produce systemic reactions. It is recommended particularly for patients

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with predominantly nasal symptoms and for those in whom oral or inhalation tests are contraindicated because of asthma severity. As the negative predictive value is lower than in the other two tests, a negative nasal challenge should be followed, whenever possible, by the oral or inhalation test. However, patients with septal perforation or important nasal blockade due to nasal polyposis are not suitable candidates for nasal provocation testing assessed by rhinomanometry (Milewski et al. 1998). Intravenous tests with lysine-aspirin (administration of increasing doses of aspirin every 30 min: 12.5, 25, 50, 100, 200 mg) are used preferentially in Japan (Mita et al. 2004).

Prevention and treatment Aspirin and NSAIDs which inhibit COX-1 are popular overthe-counter drugs, so patients with asthma should be alerted by their physicians and pharmacists to the possibility of an adverse reaction. Once diagnosed with AIA, patients must not use aspirin or any other NSAID inhibiting COX-1; education is of utmost importance. Patients should receive a list of drugs contraindicated and a list of drugs that are well tolerated (Table 95.1). Acetaminophen, coxibs and codeine are optimal preferential choices for acute pain. Coxibs are generally well tolerated by AIA patients, although the small degree of residual COX-1 inhibition displayed by these compounds may be enough to trigger hypersensitive reactions when used at high doses (Woessner 2003). Therefore, when administering acetaminophen and coxibs it is safer to administer the first dose in a physician’s office. In general, treatment of AIA should follow international guidelines (GINA 2006). However, this type of asthma is often severe and patients require complex therapy to control their symptoms; frequently high doses of inhaled corticosteroids and oral corticosteroids are necessary. In the AIANE (European Network on Aspirin-Induced Asthma) study more than 50% of AIA patients were on chronic oral and inhaled corticotherapy (Nizankowska et al. 1998; Szczeklik et al. 2000). In the USA systemic corticosteroids were used as short courses in 134 patients (45%), and on a daily basis in 95 (32%) patients (Berges-Gimeno et al. 2002). The discovery of the antileukotrienes, such as montelukast, zafirlukast and pranlukast (CysLT1 receptor inhibitors) and zileuton (5-lipoxygenase inhibitor), has provided new opportunities for management of aspirin-induced asthma and rhinosinusitis. In the Polish–Swedish double-blind placebocontrolled study, the clinical efficacy of zileuton as an add-on treatment was well documented (Dahlen et al. 1998). Similarly, montelukast was also shown useful in controlling AIA; it led to an improvement of asthma symptoms, an increase in morning FEV1 and peak expiratory flow values, and less asthma exacerbations (Dahlen et al. 2002). It also improved nasal function (Micheletto et al. 2004). Contrary to expectations, aspirin-sensitive asthmatics do not seem to experience greater benefit from leukotriene receptor antagonists com-

Hypersensitivity to Aspirin and other NSAIDs

pared with aspirin-tolerant asthmatics. Treatment success was significantly better in the carriers of the variant C allele of LTC4S (Sampson et al. 2000; Szczeklik et al. 2001b; Asano et al. 2002; Mastalerz et al. 2002) and HLA-DPB1*0301 marker (Park et al. 2004). In atopic AIA patients allergen avoidance, immunotherapy, and anti-IgE treatment should be strongly considered. Aspirin-sensitive eosinophilic rhinosinusitis is particularly refractory and difficult to treat. All patients should use longterm high-dose intranasal corticosteroids for reducing inflammation and retarding the formation of nasal polyps (Aukema et al. 2005). During acute bacterial sinus infections, extended courses of broad-spectrum antibiotics are frequently needed. Some patients often require 1–3 week bursts of systemic corticosteroids to control their symptoms (“medical polypectomy”). This treatment results in shrinking of the nasal polyps. Oral and nasal decongestants and antihistamines may give additional relief. In many patients local medical management of nasal polyposis fails, and nasal patency can be restored only through surgery. Simple polypectomy results in scarring that makes inevitable revisional procedures more difficult (Hosemann 2000). More extensive procedures, such as functional endoscopic sinus surgery, must be carried out (“pansinus surgery”) (Jankowski et al. 1997). The subjective success rate for nasal symptoms after pansinus surgery reaches 80% but generally the benefits of this procedure are short-lived, since the polyps almost always recur. However, a retrospective analysis published recently showed long-term postoperative improvement of asthma in 94% of patients subjected to sinus surgery (Loehrl et al. 2006). In a first randomized prospective study comparing surgical versus medical therapy of chronic rhinosinusitis and concomitant asthma, overall asthma control was better maintained after medical therapy as evidenced by an increase in FEV1 and decrease in exhaled nitric oxide (Ragab et al. 2006). One of the treatment options is chronic desensitization to aspirin. AIA patients can be desensitized to acetylsalicylic acid (ASA) or NSAID using a protocol that requires 1–3 days of inpatient treatment and continuous daily ASA ingestion (600–1200 mg) to maintain the desensitized state (Stevenson 2003). Desensitization is utilized in some centers for treatment of refractory AIA patients with predominant symptoms of chronic sinusitis and recurrent nasal polyps requiring repeated polypectomies (Stevenson 2003; Stevenson & Simon 2006). It should also be considered in AIA patients controlled only with unacceptably high doses of systemic corticosteroids and in patients who require NSAIDs for treatment of other diseases, particularly ischemic heart disease (Gollapudi et al. 2004; Silberman et al. 2005). The detailed data concerning aspirin desensitization have been reviewed elsewhere (Stevenson 2003; Stevenson & Simon 2006). More recently, in a controlled trial the clinical effectiveness of topical lysine-aspirin (nasal administration) was assessed

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in 11 patients with aspirin-sensitive nasal polyposis. Multivariate analysis did not reveal any significant clinical benefit of this procedure as compared to placebo (Parikh & Scadding 2005).

also involve trunk and extremities. Standardized scales have been developed to assess the intensity of skin eruptions (Zembowicz et al. 2003) (Fig. 95.5).

Histopathology

Urticaria/angioedema Definition and prevalence Aspirin and other NSAIDs can induce or exacerbate skin eruptions in some patients with chronic idiopathic urticaria. This particular kind of urticaria is called aspirin-induced urticaria and its mechanism is related to inhibition of COX-1. Incidence has been reported to be as high as 20%, but may not exceed 5% (A. Kaplan, personal communication, 2007); properly controlled studies on this are missing.

Clinical presentation The reaction may occur in just 15 min or up to 24 hours following aspirin ingestion, but on average it develops within 1–4 hours. Most cases settle in a few hours, but in severe reactions bouts of multiform skin eruptions covering most of the body may continue for 10 days after aspirin ingestion. The clinical morphology of the lesions comprises angioedema, transient wheals, and confluent evanescent pale erythematous macules. Occasionally, patients develop lesions described as annular, gyrate and figurate erythematous plaques, sometimes with raised borders. The skin rashes with pleomorphic clinical morphology last longer (3–10 days) and are most prominent over the head and neck region, but may

The histologic spectrum of cutaneous reactions to aspirin in chronic idiopathic urticaria was reported by Zembowicz et al. (2004). Aspirin in up to 500-mg doses induced a restricted range of histologic responses, with a classic pattern of urticarial tissue reaction occurring in 12 of 16 cases. Histologic criteria of urticaria included the presence of dermal edema, lymphatic dilatation, a perivascular mononuclear infiltrate, and interstitial eosinophils and neutrophils. Mast cells around blood vessels and in the interstitial space were identified in all cases. There was no evidence of leukocytoclasis, endothelial swelling, red blood cell extravasation, or fibrin deposition to suggest vasculitis. Of the 16 biopsies, two showed an interstitial fibrohistocytic (granuloma/annular-like) reaction pattern. Polymorphism of histologic patterns induced by aspirin suggests that in addition to the drug-specific mechanisms triggering eruptions, individual host factors also play a role in determining the ultimate histologic phenotype of a drug response.

Mechanisms Patients with chronic idiopathic urticaria who react to aspirin challenge with a skin rash share a similar eicosanoid profile with aspirin-induced asthmatics (Mastalerz et al. 2004). In fact, about 10% of challenged individuals also developed airway manifestations, e.g., nasal congestion and less frequently dyspnea. When contrasted with aspirin-tolerant chronic urticaria patients, hypersensitivity to aspirin correlated with increased LTE4 excretion in urine at baseline and a few hours following appearance of symptoms after provocation tests. Likewise, the PGD2 metabolite increased in plasma but within a shorter time course. The extent of the eruption following the challenge correlated with urinary LTE4. These biochemical markers are not absolutely specific for aspirin hypersensitivity because aspirin-tolerant patients with chronic urticaria also produce greater amounts of LTE4 than healthy controls, and aspirin challenge in these patients provokes slow release of PGD2 in plasma (Mastalerz et al., 2004). Aspirininduced urticaria aggregates in families inheriting the LTC4 synthase allelic variant. Segregation of aspirin sensitivity in these families does not follow a clear Mendelian pattern (Mastalerz et al. 2006b).

Diagnosis History suggests the diagnosis, which however can be established only by oral challenge tests. Such standardized tests have been recently described (Nizankowska-Mogilnicka 2007). Fig. 95.5 Multiform skin eruptions fluctuating during the 3 days after a severe bronchospastic reaction to 150 mg aspirin in a 30-year-old woman suffering from rhinosinusitis and asthma. (See CD-ROM for color version.)

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Prevention and treatment Aspirin and all drugs that inhibit COX-1 should be avoided in patients who have already had adverse reactions. Others

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should pay attention to whether NSAIDs affect their lives. Coxibs are usually well tolerated, although sporadic adverse reactions have ben reported (Levy & Fink 2001; Murr et al. 2003; Passero 2003; Bavbek et al. 2004; Baldassarre et al. 2006; Mastalerz et al. 2006a; Morais-Almeida et al. 2006). In treatment of the reactions, antihistamines are usually sufficient but in more severe cases epinephrine and corticosteroids may be warranted.

Allergic reactions to NSAIDs Although pharmacologic inhibition of COX-1 with overproduction of cysteinyl leukotrienes represents the most frequent pathomechanism of the adverse reactions to aspirin and other NSAIDs, in a subset of subjects the reactions could have an allergic or pseudoallergic background. According to Stevenson et al. (2001, 2003) these reactions can be classified into the following types: (i) single NSAID-induced urticaria/ angioedema in otherwise normal subjects; (ii) single NSAIDinduced anaphylaxis and anaphylactoid reactions; (iii) aseptic meningitis caused by a specific NSAID; and (iv) hypersensitivity pneumonitis caused by a specific NSAID. These reactions are usually drug specific, independent of COX-1 inhibition, and probably in the majority of cases immune mediated. In the first two types of reaction, cross-reactivity between aspirin and NSAID does not occur. Only in some studies does atopy appear to be a risk factor for single NSAID-induced urticarial and anaphylactic reactions. Usually urticaria/ angioedema develops on reexposure to NSAIDs (after prior sensitization). NSAIDs, like many drugs, can function as drug haptens to induce immune sensitization, followed by probable IgE-mediated reactions involving mast cell and basophil activation. The average relative risk for these allergic reactions to one NSAID varies from 0.1 to 3.6% of subjects who take NSAIDs intermittently or chronically for acute pain, depending on the population studied. Most drug-induced anaphylactic episodes are attributed to aspirin, diclofenac, naproxen, and ibuprofen, but almost any other NSAID could induce anaphylactic reactions (e.g., fenoprofen, piroxicam, indomethacin, acetaminophen) (Van Diem & Grilliat 1990; Quiralte et al. 1997; Van Puijenbroek et al. 2002). Such patients can be challenged with structurally different NSAIDs without adverse effects.

Pyrazolone derivatives Pyrazolone derivatives are analgesic substances known for a long time. The use of antipyrine (phenazone) and aminopyrine was sharply curtailed after their bone marrow toxicity was reported. However, others (phenylbutazone, metamizole, sulfinpyrazone and propyphenazone) are widely used and can be obtained without prescription in many countries. They are not infrequently a cause of adverse reactions, ranging from urticaria, angioedema or asthma to anaphylactic shock.

Hypersensitivity to Aspirin and other NSAIDs

Based on their mechanism, these reactions can be clearly separated into two groups (Czerniawska-Mysik & Szczeklik 1981; Szczeklik et al. 1997). In the first group (which corresponds to AIA): 1 metamizole, aminophenazone, phenylbutazone and sulfinpyrazone as well as several other COX-1 inhibitors including aspirin precipitate bronchoconstruction; 2 skin test with pyrazolone drugs are virtually negative; 3 all patients have chronic rhinosinusitis and/or asthma. In the second group, the following may be observed. 1 Reactions are of an allergic type, most likely IgE-mediated (Himly et al. 2003) and can be life-threatening. They are limited to a single pyrazolone drug or two drugs that are chemically closely related (e.g., metamizole and aminophenazone). This strict clinical specificity is corroborated by results in experimental animals (Schneider et al. 1987). 2 Skin tests with the incriminated drug are positive. 3 Other pyrazolones (e.g., phenylbutazone or sulfinpyrazone in cases of allergy to metamizol), aspirin, and other COX-1 inhibitors can be taken with impunity. 4 Chronic bronchial asthma is present in only about onequarter of patients. These allergic reactions may have a genetic predisposition (Kowalski et al. 1998). Azapropazone, a benzotriazone misclassified originally as a pyrazolone, rarely if ever causes the above pseudoallergic or allergic reactions (Szczeklik et al. 1989). Very rarely a patient may develop aseptic meningitis after ingestion of a specific NSAID (ibuprofen, sulindac, tolmetin and naproxen) (Stevenson et al. 2003). Cross-reaction with NSAIDs does not occur and aspirin has not been reported to cause aseptic meningitis. Some NSAIDs can cause hypersensitivity pneumonitis; in these cases disappearance of pulmonary infiltrates spontaneously or following corticosteroid therapy has been reported (Stevenson et al. 2003).

Other types of adverse reaction to ASA/NSAIDs NSAIDs can induce a number of other adverse reactions including bleeding disorders, anemia, thrombocytopenia, erythema nodosum, erythema multiforme, fixed drug eruptions, toxic epidermal necrolysis, Stevens–Johnson syndrome, leukocytoclastic vasculitis as well as hepatotoxicity, interstitial nephritis and, of course, the well-known gastric cytotoxicity (Stevenson et al. 2001; Stevenson et al. 2003).

References Alonso-Llamazares, A., Martinez-Cocera, C., Dominguez-Ortega, J. et al. (2002) Nasal provocation test (NPT) with aspirin: a sensitive and safe method to diagnose aspirin-induced asthma. Allergy 57, 632–5. Asano, K., Shiomi, T., Hasegawa, N. et al. (2002) Leukotriene C4 synthase gene A(-444)C polymorphism and clinical response to an LT(1) antagonist, pranlukast, in Japanese patients with moderate asthma. Pharmacogenetics 12, 565–70.

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eicosanoid profile of the hypersensitivtiy reactions triggered by aspirin and celecoxib in a patient with sinusitis, asthma and urticaria. J Allergy Clin Immunol 118, 957–8. Mastalerz, L., Setkowicz, M., Sanak, M. et al. (2006b) Familial aggregation of aspirin-induced urticaria and leukotriene C synthase allelic variant. Br J Dermatol 154, 256–60. Mathison, D.A. & Stevenson, D.D. (1979) Hypersensitivity to nonsteroidal anti-inflammatory drugs: indications and methods for oral challenge. J Allergy Clin Immunol 64, 669–74. Melillo, G., Balzano, G., Blanco, S. et al. (2001) Report of the INTERASMA Working Group on Standardization of Inhalation Provocation Tests in Aspirin-induced Asthma: oral and inhalation provocation tests for the diagnosis of aspirin-induced asthma. Allergy 56, 899–911. Micheletto, C., Tognella, S., Visconti, M. et al. (2004) Montelukast 10 mg improves nasal function and nasal response to aspirin in ASA-sensitive asthmatics: a controlled study vs placebo. Allergy 59, 289–94. Milewski, M., Mastalerz, L., Nizankowska, E. & Szczeklik, A. (1998) Nasal provocation test with lysine-aspirin for diagnosis of aspirinsensitive asthma. J Allergy Clin Immunol 101, 581–6. Mita, H., Endoh, S., Kudoh, M. & Akiyama, K. (2001) Possible involvement of mast-cell activation in aspirin provocation of aspirin-induced asthma. Allergy 56, 1061–7. Mita, H., Higashi, N., Taniguchi, M. et al. (2004) Increase in urinary leukotriene B4 glucuronide concentration in patients with aspirinintolerant asthma after intravenous aspirin challenge. Clin Exp Allergy 34, 1262–9. Morais-Almeida, M., Marinho, S., Rosa, S. et al. (2006) Multiple drug intolerance including etoricoxib. Allergy 61, 144–5. Murr, D., Bocquet, H., Lelouet, H. et al. (2003) Adverse cutaneous reaction to celecoxib: 6 cases. Ann Dermatol Venereol 130, 519–21. Nizankowska, E., Duplaga, M., Bochenek, G. & Szczeklik, A. on behalf of the AIANE project (1998) Clinical course of aspirin-induced asthma, results of AIANE. In: Szczeklik, A., Gryglewski, R., Vane, J., et al., eds. Eicosanoids, Aspirin and Asthma. Marcel Dekker, New York, pp. 451–72. Nizankowska, E., Bestynska-Krypel, A., Cmiel, A. & Szczeklik, A. (2000) Oral and bronchial provocation tests with aspirin for diagnosis of aspirin-induced asthma. Eur Respir J 15, 863–9. Nizankowska-Mogilnicka, E., Bochenek, G., Mastalerz, L. et al. (2007) EAACI/GA2LEN guideline: aspirin provocation tests for diagnosis of aspirin hypersensitivity. Allergy 62, 1111–8. Obase, Y., Shimoda, T., Tomari, S.Y. et al. (2002) Effects of pranlukast on chemical mediators in induce sputum on provocation tests in atopic and aspirin-intolerant asthmatic patients. Chest 121, 143– 50. Parikh, A.A. & Scadding, G.K. (2005) Intranasal lysine-aspirin in aspirin-sensitive nasal polyposis: a controlled trial. Laryngoscope 115, 1385–90. Park, H.E., Kim, S.H., Sampson, A. et al. (2004) The HLA-DPB1*0301 marker might predict the requirement for leukotriene receptor antagonist in patients with aspirin-intolerant asthma. J Allergy Clin Immunol 114, 688–9. Park, J.S., Chang, H.S., Park, C.S. et al. (2005) Association analysis of cysteinyl-leukotriene receptor 2 (CYSLTR2) polymorphism with aspirin-intolerant asthma. Pharmacogenet Genomics 20, 232–6. Passero, M. (2003) Cyclo-oxygenase-2 inhibitors in aspirin sensitive asthma. Chest 123, 2155–6.

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Anaphylaxis and Allergy to Food and Drugs

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Insect Sting Allergy Ulrich R. Müller

Summary

Introduction

Insect stings may cause generalized allergic reactions which can even be fatal. Social Hymenoptera like honeybees, vespids, and ants are the species most often responsible for such reactions. They sting in defense and inject venom into the skin of the victim using their venom apparatus, situated at the end of the abdomen. The venom contains biogenic amines, peptides and proteins, mostly enzymes, that are cytotoxic and cause local pain and inflammation. Proteins may also induce sensitization, causing allergic reactions to later stings. Very rarely other insect species, like horseflies, mosquitoes, fleas or bed bugs, which bite humans to suck blood, may also provoke generalized allergic reactions due to sensitization to proteins from the salivary glands of these insects. The generalized allergic reactions are most often IgEmediated, and consist of cutaneous symptoms like urticaria or angioedema, dyspnea due to bronchial asthma or laryngeal edema, and anaphylactic shock. Involvement of the respiratory and cardiovascular tract is responsible for the occasional fatal reactions, which occur with an incidence of 0.1–0.5 per million population each year in Europe and the USA. The diagnosis of insect sting allergy is based on the clinical history and the detection of venom-specific IgE antibodies by skin tests or in serum by immunoassays. Emergency treatment of allergic reactions includes shock positioning, application of intramuscular epinephrine, volume substitution, as well as intravenous antihistamines and corticosteroids. Cardiopulmonary resuscitation may be necessary in severe cases. All patients with severe reactions should therefore be hospitalized, treated and supervised until completely recovered. Venom immunotherapy over 3–5 years has been proved to be effective while providing complete protection from further sting reactions in 80 to over 95% of patients. No suitable extracts for diagnosis and treatment of the rare generalized allergic reactions to stings by bloodsucking insects are commercially available.

Insects may induce allergies in humans in various different ways. The decayed bodies of insects or their feces can cause inhalation allergies such as bronchial asthma or allergic rhinitis and stings or bites by insects may induce local cutaneous or systemic allergic reactions including life-threatening anaphylaxis. Insect sting-related allergies are the topic of this chapter. Stings by insects may have different goals. Hymenoptera sting humans to defend themselves and while stinging inject venoms containing toxic substances. Besides harming effects due to toxicity, venom proteins may also lead to allergic sensitization. Other insects like mosquitoes, horseflies, midges, fleas, or bed bugs sting, or rather bite, to nourish themselves. While sucking blood they release protein enzymes that may also induce sensitization of their victims. Ticks and spiders also inflict stings on humans; however, because arthropods are not insects, they will not be considered in this chapter. With regard to the difference between stings for defense and bites for nourishment, we will use the term “sting” for Hymenopterans which defend themselves against humans by stinging, and “bite” for all other insects which nourish themselves by attacking humans. With regard to allergies, stings by insects of the order Hymenoptera (bees, wasps and ants) are much more important than bites by mosquitos, horseflies and other insects. Stings by Hymenoptera can induce acute systemic allergic reactions that kill several hundred patients in Europe and America every year, while systemic allergic reactions to bites are very rare. Infections are only transmitted by blood-sucking insects.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Taxonomy and entomologic aspects With regard to taxonomy, we follow the Chinery classification (Chinery 1979). Insects that inject venom while stinging and which thus induce sensitization to venom proteins all belong to the order Hymenoptera, almost exclusively to the families Apidae, Vespidae, Formicidae and Myrmicidae of the suborder Aculeata (Fig. 96.1). Other Aculeata may induce

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Order

Hymenoptera

Suborder

Apocrita

Legion

Aculeata

Superfamily

Apoidea

Vespoidea

Family

Apidae

Vespidae

Subfamily

Insect Sting Allergy

Scolioidea

Apinae

Bombinae

Vespinae

Polistinae

Genus

Apis

Bombus

Vespula

Dolichovespula

Vespa

Polistes

Myrmecia

Species

Apis mellifera & others

Bombus pratorum & others

Vespula germanica vulgaris maculifrons & others

D. media D. saxonica D. maculata D. arenaria D. sylvestris

V. crabro V. orientalis

P. dominulus P. gallicus P. exclamans P. fuscatus

M. pilosula & others

Myrmicidae

Formicidae

Myrmicinae

Formicinae

Solenopsis Pogonomyrmex Formica

S. invicta & others

P. rugosus & others

F. rufa & others

Fig. 96.1 Taxonomy of Hymenoptera.

painful stings but repeated stings by one species are so unlikely that allergy to them is practically unheard of.

Apidae The honeybee (Apis mellifera) (Fig. 96.2a) is doubtless the most important species of this family in causing allergies. Its ability to produce honey and to pollinate fruit trees and many vegetables has led to its domestication and worldwide distribution. One beehive may consist of several tens of thousands of working bees. Stings occur either in the vicinity of beehives or when the insect feels threatened by human occupations such as cutting flowers or walking barefoot in the grass. Since the whole beehive survives during winter, stings may not only occur in spring and summer, but occasionally also on warm winter days. Honeybees are brown and moderately hairy. When stinging, in contrast to other Hymenoptera, they usually lose their barbed stinger and die within a few days. The Africanized honeybee resulted from cross-breeding of African with South American honeybees and is more aggressive than native American and European bees (McKenna 2003). It may attack in swarms in the vicinity of the beehive and thus cause fatalities from multiple stings. It has spread from South America to Mexico and southern USA, but will probably not get farther north because it cannot survive winter in a cold climate. Its venom is very similar to the venom of native American and European honeybees. The bumble-bees (Bombus terrestris, B. agrorum and others) are increasingly used as pollinators in greenhouses because they have a longer proboscis than honeybees and pollinate at

lower temperatures. They may occasionaly cause allergic sting reactions in greenhouse workers (de Groot 2006). Bumblebees are larger than honeybees, more hairy, and most species have distinct yellow or white bands on their abdomen (Fig. 96.2b). Their small nests are usually built in the ground. Stings outside greenhouses are extremely rare. Bumble-bees do not usually loose their stinger when stinging.

Vespidae The vespids are divided into the subfamilies Vespinae and Polistinae, which differ morphologically at the junction from thorax to abdomen (Fig. 96.2c,d). The abdomen becomes thicker rapidly after the waist in the Vespinae, but only gradually in the Polistinae. Vespids are almost hairless, and in most species their abdomen is striped black and yellow. While stinging, vespids do not usually lose their stinger. Only the queen survives winter, starts building a new nest in spring and establishs a new population. Larger numbers of worker vespids develop only in summer and most stings therefore occur in summer and fall. The subfamily Vespinae contains the three genera Vespula, Dolichovespula, and Vespa (Chinery 1979; Müller 1990; Bilò et al. 2005). Vespula germanica and V. vulgaris are the most common species in Europe and V. maculifrons and V. germanica the most common species in the USA; Vespula spp. are called wasps in Europe and yellow jackets in the USA. They breed in the ground, in attics or shelters and their colonies may consist of several thousand workers. The genus Vespula is by far the most aggressive of Vespidae. Humans are stung not only

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(a)

(b)

(c)

(d)

Fig. 96.2 Stinging social Hymenoptera: (a) Apis mellifera, the honeybee; (b) Bombus pratorum, the bumble-bee; (c) Vespula germanica, the common wasp (yellow jacket in USA); (d) Polistes gallicus, the European field wasp; (e) Vespa crabro, the European hornet. (See CD-ROM for color version.)

(e)

near the nests but more often when eating outdoors, under orchards with fallen fruits, or near open waste-bins. Most species of Dolichovespula (D. media, D. sylvestris, D. saxonica in Europe; D. maculata, D. arenaria in the USA) look very similar to Vespula with black and yellow stripes on the abdomen and

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are only slightly larger. They can be distinguished from Vespula by the larger distance between the eyes and the mandibles. Only D. maculata, the bald-faced hornet in the USA, is easy to distinguish from other vespids by its mostly black abdomen. Dolichovespula build their nests hanging from tree branches or

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Table 96.1 Popular names for common vespids in Europe and the USA.

Vespula spp. (Fig. 96.2c) Dolichovespula Vespa crabro Polistes (Fig. 96.2d)

Europe

USA

Wasp Wasp Hornet Field wasp

Yellow jacket Hornet European hornet Wasp

under the roof of houses and their colonies are much smaller than those of Vespula, usually not exceeding a few hundred workers. Dolichovespula do not go after human food and therefore stings occur most often in the vicinity of their nests. The genus Vespa (V. crabro, the European hornet; V. orientalis, the Oriental hornet) is easy to distinguish from other vespids by its much larger size (Fig. 96.2e). The European hornet was introduced to America during the 19th century. The Oriental hornet is present in south-eastern Europe, Asia and Africa. Hornet stings are rare and occur almost exclusively in the vicinity of nests, which are usually built in hollow tree trunks or bird nest-boxes. Polistinae (P. annularis, P. exclamans and P. fuscatus in the USA; P. dominulus, P. gallicus and P. nympha in Europe) live mostly in the southern USA and in the Mediterranean area of Europe, where cases of Polistes sting anaphylaxis are frequently reported. However, small colonies may be observed all over Europe except for the British Isles. Their small nests consist of only one womb and are built in trees or under roofs. Much confusion is created by the fact that common English names for Vespula, Dolichovespula and Polistes species differ in Europe and the USA (Table 96.1).

Ants (Myrmicinae, Formicinae) In South and Central America and the southern states of the USA the fire ants (Solenopsis invicta, S. richteri) are responsible for many systemic allergic sting reactions (Reichmuth & Lockey 2003). Fire ants build their mounds in yards, playgrounds and fields and multiple stings may be inflicted on humans in these areas. Occasionally, allergic sting reactions have been described to Pogonomyrmex, the harvester ant in the USA, extremely rarely also to the red ant, Formica rufa in Europe (Seebach et al. 2000). In contrast, species of Myrmicinae, especially Myrmecia pilosula, the jack-jumper ant, are an important cause of allergic sting reactions in southern Australia (Brown et al. 2003) and especially Tasmania. Ants do not usually lose their stinger while stinging.

Allergens in Hymenoptera venoms (see also Chapter 50) Low-molecular-weight substances like biogenic amines, phospholipids, amino acids, and carbohydrates are found in

Insect Sting Allergy

all Hymenoptera venoms. Local toxicity of Solenopsis invicta venom is mainly due to dialkyl piperidines. All Hymenoptera venoms contain low-molecular-weight peptides like mellitin, apamin or kinins. The peptides contribute to the toxic effect induced by a sting. They usually do not induce IgE antibody formation and are therefore probably irrelevant with regard to allergies. Specific IgE antibodies to melittin are occasionally observed in humans. Moreover, melittin was shown to have an adjuvant effect for sensitization in animal studies (Kind et al. 1981). Most of the allergens of the important stinging Hymenoptera are glycoproteins of 10–50 kDa. Major allergens in bee venom are phospholipase A2, hyaluronidase and acid phosphatase, in vespid venoms antigen 5, phospholipase A1 and hyaluronidase. A number of minor allergens have been described in both bee and vespid venoms. Ant venom from Solenopsis invicta contains a 37-kDa and a 24-kDa allergen with some sequence homology to phospholipase A1 and antigen 5 from vespid venom. Today sequences of most major venom allergens are known. Several of them have also been expressed in recombinant refolded form and shown to be comparable in allergenic activity and biological function, like enzyme activity, to their natural counterparts (King et al. 1996; Müller 2002; Grunwald et al. 2006). The amount of venom injected during a sting varies from species to species and also within one species, especially with vespids. Bees release 50–140 μg protein per sting, vespids much less (2–17 μg) (Hoffmann & Jacobson 1984; Schumacher et al. 1994). Commercially available venoms of the honeybee are collected by electrostimulation (Benton et al. 1963), while those of vespids are usually obtained by venom sac extraction and thus may contain some body proteins derived from the venom sacs as impurities (Guralnick & Benton 2003). No venom preparations of Formicidae or Myrmicidae are so far commercially available for diagnosis or treatment of ant sting allergy.

Pathogenesis and clinical presentation of Hymenoptera venom allergy The clinical symptoms of allergic reactions to Hymenoptera stings are broad. Besides large local reactions at the sting site, immediate IgE-mediated allergic reactions like urticaria, angioedema, asthma, and anaphylactic shock are most frequently observed. Occasionally, allergic reactions are delayed in their appearance, e.g., in serum sickness-like syndromes or in genealized vasculitis, which are probably not IgE-mediated but mostly due to IgG antibodies or cellular immunologic mechanisms. Rarely nonimmunologic mediator release, e.g., direct complement activation, may play a role. Massive activation of the bradykinin cascade has been noted during anaphylaxis based on cleavage of high-molecular-weight kininogen (Smith et al. 1980). Following multiple stings generalized

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symptoms are due to venom toxicity. The clinical presentation is classified into normal, large local, systemic allergic, unusual, and systemic toxic reactions (Müller 1990; Bilò et al. 2005).

Normal local reactions The normal local reaction to a Hymenoptera sting in a nonallergic subject consists of painful, erythematous, sometimes itchy, local wheal and flare reactions, followed by swelling of up to 5–10 cm in diameter. Usually, local symptoms resolve within a few hours, by definition within 24 hours. The local reaction is due to the effect of biogenic amines, the cytotoxicity of low-molecular-weight peptides like melittin, kinins or mast cell-degranulating peptides, and the membrane toxicity of enzymes like phospholipases. The first local symptom to stings by Solenopsis, the American fire ant, is a wheal and flare reaction. A vesicular lesion then develops, finally leading to pustulous local necrosis, which only heals after 1–2 weeks (Reichmuth & Lockey 2003). While the initial wheal and flare reaction is due to histamine release from mast cells, local necrosis is the result of the highly toxic dialkyl piperidines of Solenopsis venom.

Large local reaction We define a large local reaction (LLR) as a swelling around the sting site exceeding a diameter of 10 cm, which develops minutes to hours after the sting, and lasts more than 24 hours (Müller 1990). LLR can be very disturbing, especially when they last for days or even weeks and involve a whole limb, eyelids or lips. They may even be life-threatening if developing after a sting in the oral cavity. Sometimes they are accompanied by lymphadenopathy or lymphangitis. They can also be associated with nonspecific systemic inflammatory symptoms, like malaise, fever, shivering, or headache. However, the development of local infection, abscess or phlegmon at the sting site is unlikely because of the bacteriostatic effect of Hymenoptera venoms. Nevertheless, scratching after stings, especially by the American fire ant Solenopsis or bites from blood-sucking insects such as midges, can entail a skin infection. Pathogenetically, LLRs involve both immediate and delayedtype allergic responses. In the majority of these patients immediate-type skin tests with the respective venoms are positive and venom-specific serum IgE antibodies are present. Often, positive lymphocyte transformation tests suggest involvement of a cellular immune response (Case et al. 1981; Müller 1990).

Systemic allergic reaction Systemic anaphylactic reactions are usually IgE-mediated. In the skin pruritus, urticaria, flush and angioedema are most frequently observed. Gastrointestinal symptoms may consist of abdominal cramps, dysphagia, vomiting or diarrhea, due to mucosal edema and spasms of smooth muscles of the intestine. Women sometimes complain of heavy lower abdominal

1984

cramps, which may even be accompanied by vaginal bleeding, suggesting spasms of the uterine muscles and mucosal alterations as their origin. In the respiratory tract laryngeal edema accompanied by hoarseness and stridor, bronchial obstruction with wheezing due to bronchospasm, mucosal edema and secretion, or occasionally even pulmonary edema, due to either increased vascular permeability or left ventricular failure, may lead to dyspnea, wheezing and respiratory distress. Cardiovascular symptoms consist of arterial hypotension, arrhythmias (ventricular or atrial tachycardia, ventricular or atrial fibrillation or atrioventricular block have been described), collapse and shock with loss of consciousness, often incontinence, and occasionally epileptiform seizures due to cerebral ischemia. A number of various pathogenetic factors may lead to anaphylactic shock. Increased vascular permeability, vasodilatation, and fluid loss by vomiting, diarrhea or bleeding during disseminated intravascular coagulation leads to hemoconcentration and hypovolemia. Bronchial obstruction and pulmonary edema induce respiratory distress with hypoxia and acidosis, and direct cardiotoxicity of mediators released from mast cells and basophils (histamine, leukotrienes or prostaglandins) may be responsible for arrhythmias, coronary spasms and reduced contractility of cardiac muscle. In elderly patients with preexisting cardiovascular or cerebrovascular disease, anaphylactic shock may induce myocardial or cerebrovascular infarction (Müller 2007). The most often used classification of systemic allergic reactions is the one proposed by Mueller (1966) (Table 96.2). Usually symptoms appear within minutes to 1 hour after the sting and the patient recovers within a few hours. Rarely a protracted course over more than a day or a biphasic course have been described (Reisman 1987).

Unusual reactions Unusual sting reactions are reported by less than 5% of patients and may appear after hours to days. More than half of them are accompanied by immediate large local or systemic reactions (Müller 1990; Bilò et al. 2005; Reisman 2005). NonIgE-mediated, immune complex, or cellular immunologic

Table 96.2 Classification of generalized allergic Hymenoptera sting reactions. (From Müller 1990, with permission.) Grade I: generalized urticaria, itching, malaise, anxiety Grade II: any of the above plus two or more of the following: angioedema, chest constriction, nausea, vomiting, diarrhea, abdominal pain, dizziness Grade III: any of the above plus two or more of the following: dyspnea, wheezing, stridor, dysarthria, hoarseness, weakness, confusion, feeling of impending disaster Grade IV: any of the above plus two or more of the following: fall in blood pressure, collapse, loss of consciousness, incontinence, cyanosis

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mechanisms may play a role, but involvement of late-phase IgE-mediated mechanisms has been suggested (Lichtenstein & Golden 1983). Serum sickness-like syndromes with fever, arthralgias, exanthema and lymphadenopathy are rather well documented. Less frequently, symptoms of the nervous system (peripheral neuropathy, polyradiculomyelitis, acute disseminated encephalomyelitis, multiple sclerosis) have been suggested to be due to, or elicited by, previous Hymenoptera stings. In rare instances symptoms associated with the kidneys (glomerulonephritis, interstitial nephritis) or blood and blood vessels (hemolytic anemia, thrombocytopenia, Henoch–Schönlein syndrome and other forms of vasculitis) have been reported following Hymenoptera stings. The causal relation to the sting event often remains uncertain, especially in generalized neurologic syndromes, kidney diseases, or blood dyscrasias.

Systemic toxic reactions Toxic reactions are dose dependent. A clinically significant toxic effect must be considered only after multiple, usually 50 to several hundred stings (Müller 1990; Bilò et al. 2005). They are mostly due to the cytotoxic effects of venom components such as peptides or enzymatically active venom proteins. The main toxic effects develop within hours to days and consist of rhabdomyolysis and intravascular hemolysis, leading to acute renal failure with tubular necrosis. Myocardial damage, hepatic dysfunction, coagulation disorders, and brain edema and/or necrosis may also occur. The number of stings that cause a fatal reaction varies between 200 and over 1000. In small children, however, less than 50 stings may be lethal. In most cases, death occurs only after several days, usually due to kidney failure, blood clotting disturbances, or necrosis of the brain.

Quality-of-life aspects An anaphylactic reaction after a Hymenoptera sting is a very traumatic event for many patients and often also for their families. It can result in a dramatic change in lifestyle, where some of these patients are frightened to go outdoors during the flying season of Hymenoptera. This can have deep effects on emotional functioning and social, in some cases also professional, activites. Recently a disease-specific questionnaire was designed and validated for assessing health-related quality of life in patients with anaphylactic reactions to Vespula stings (Oude Elberink & Dubois 2003). It documented a significantly reduced quality of life as a consequence of the emotional distress associated with the allergy.

Epidemiologic aspects Prevalence of allergy to Hymenoptera stings Cumulative lifetime sting rates of 61–95% have been reported in adult individuals in Israel (Graif et al. 2006). Of course this increases with age and may vary considerably in different

Insect Sting Allergy

regions of the world. Hymenoptera sting allergy may occur at any age. In general, based on more frequent outdoor activities, men are more frequently stung than women, children more often than adults. In children a prevalence of 0.4– 0.8% was reported in American studies (Settipane & Boyd 1970). The cumulative lifetime prevalence of LLR in adults ranges from 2 to 26%, and that of systemic Hymenoptera sting reactions from 0.3 to 7% (Antonicelli et al. 2002). In highly exposed beekeepers it varies between 14 and 43% (Müller 2005b).

Risk factors for Hymenoptera allergy The risk of developing a sting allergy increases with the number of stings. It is especially high if two stings occur within a short period of a few weeks to 2 months (Settipane & Boyd 1970). However, beekeepers stung less than 15 times a year have a much higher risk of systemic allergic reactions than those stung more than 50 times, and beekeepers with more than 200 stings report no systemic allergic reactions at all (Müller 2005). Allergic sting reactions in children tend to be less severe than in adults. The prevalence of allergic sting reactions is higher in men than in women in most studies. This excess is particularly noticeable in the death statistics. It is assumed that the higher exposure of men, who more often work outdoors, is responsible for this difference (Müller 1990). In large urban areas in Europe, where Vespula is responsible for more than 80% of allergic sting reactions, the prevalence of such reactions is higher in women, probably because most stings occur in relation to food preparation or eating outdoors. Especially in elderly patients with preexisting cardiovascular diseases, treatment with beta-blocking drugs or angiotensinconverting enzyme (ACE) inhibitors (Tunon-De-Lara et al. 1992; Müller & Haeberli 2005) is associated with more severe or even fatal sting reactions. However, beta-blockers do not increase the overall risk for systemic sting reactions. Atopy is not more frequent in Hymenoptera sting-allergic patients than in the whole population. However, in an atopic patient with Hymenoptera venom allergy, systemic reactions may be more severe and affect the respiratory tract more frequently (Müller 1990; Bilò et al. 2005). Interestingly, about 50% of venom-allergic beekeepers are atopic compared with 20–30% of the whole European population. It is suggested that this is due to sensitization of atopic beekeepers at least partly over the mucous membranes by inhalation of venomcontaining dust during work in the beehive (Müller 2005). Mastocytosis (Fricker et al. 1997; Oude Elberink et al. 1997) and elevated baseline serum tryptase levels, reflecting increased whole-body mast cell load (Ludolph-Hauser et al. 2001; Haeberli et al. 2003), have been reported to be associated with an increased risk of developing severe or even fatal cardiovascular reactions, especially in vespid venom-allergic patients.

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Mortality due to Hymenoptera stings The incidence of mortality from Hymenoptera stings varies from 0.1 to 0.5 deaths per million population per year (Sasvary & Müller 1994). The annual rate of fatal Hymenoptera sting reactions of about 150 in Europe and 40 in the USA is probably underestimated. Autopsy studies in people dying outdoors for unknown reasons have revealed elevated serum IgE antibodies to Hymenoptera venoms in up to 20% (Schwartz et al. 1988). The vast majority of fatal sting reactions occur in adults older than 45 years of age (Sasvary & Müller 1994; Bilò et al. 2005). Additional risk factors include a positive history of sting allergy, male gender, stings in the head or neck, and mastocytosis (Oude Elberink et al. 1997). Autopsies have documented preexisting cardiovascular disease in most cases (Sasvary & Müller 1994). Approximately half of the deaths occur in subjects with no known prior history of allergic sting reactions, a figure which again is probably underestimated, because minor systemic sting reactions may not have been documented in the environment of the victim. Annual deaths rates in Switzerland during 1961–2004 ranged from zero to eight fatalities with an average of three per year (Sasvary & Müller 1994).

Natural history of Hymenoptera sting allergy In prospective clinical studies the risk of developing a systemic allergic reaction after an LLR is 5–10%, after a mild allergic reaction 15–30%, and after a severe allergic reaction 50–75% (Ruëff et al. 1996; Müller 1990). A short interval since the last sting (2 weeks to a few months) increases the risk (Antonicelli et al. 2002). With a longer interval since the last sting reaction the risk gradually decreases, but occasional reactions have been observed after an interval of more than 20 years. The degree of severity of an index sting reaction is an important factor determining the risk at reexposure. Children are at a lower resting risk than adults (Golden 2006). The resting risk is definitely lower in Vespula-allergic than in bee venomallergic patients, probably because of the much smaller and less constant amount of venom applied, since vespids do not lose the stinger when stinging. Depending on whether they have already stung other people, mammals or insects, the amount of venom available in their venom sac may vary considerably (Müller et al. 1992). Occasional patients claim that they reacted systemically to the first sting they ever experienced. In such cases, however, it is difficult to exclude stings during early childhood which may not be remembered. An alternative explanation may be that reactions in these patients are due to cross-reactivities between plant and venom allergens, such as the well-known cross-reacting carbohydrate determinants (Hemmer et al. 2001).

Epidemiologic aspects of allergic reactions to ant stings In fire ant endemic areas of the USA nearly 50% of the inhabitants are stung every year (Reichmuth & Lockey 2003).

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Most report LLRs, while up to 1% of patients develop anaphylaxis, and even fatal reactions have been reported. In Australia more than 90% of ant venom anaphylaxis is caused by Myrmecia pilosula. Age over 35 years, annual sting exposure rate, and allergy to bee venom were predictors for more severe reactions. Deaths have also been reported (Brown 2005).

Diagnosis Case history A carefully taken case history is the basis of any diagnosis of Hymenoptera sting allergy. This includes date, number and circumstances of sting reactions (e.g., environment, activities), interval to onset of symptoms, type and severity of symptoms, emergency treatment, sting site, retained or removed stinger, risk factors for a particularly severe reaction (e.g., comorbidity, drugs), tolerated stings between the first and last or after the last systemic reaction, and other allergies (Müller 1990; Bilò et al. 2005). For further exploration and treatment, the physician must try to find the answer to the following questions. • Which insect was responsible? In nearly all cases patients realize the connection between the sting from the insect and the reaction experienced afterwards, but they are often unable to identify the responsible insect. Only beekeepers, farmers, gardeners or family members can normally identify the species of insect correctly, but other further questions may be helpful. • Did the stinger remain in the skin as is usually the case with honeybees? • At what season of the year did the sting occur? Bee stings may occur from early spring to fall, while Vespula stings mostly occur from late summer to fall. • Where did the sting occur? If picknicking or in an orchard with fallen fruits, a vespid would be the more likely offender; if walking barefoot in the grass or cutting flowers, a bee was probably responsible. • What did the insect look like? Is the patient able to identify it on a color photograph? (See Fig. 96.2a–e.) • Was it an allergic or a nonallergic reaction? A painful sting by a bee or vespid may provoke symptoms like hyperventilation, paresthesias, dizziness, or even vasovagal collapse. On the other hand, symptoms like urticaria, angioedema, red itchy eyes, sneezing, wheezing or stridor definitely point to an IgE-mediated allergic reaction. If possible, the emergency physician should be contacted to obtain an objective description of symptoms. • How great is the risk for and from further stings? This risk is dependent on the exposure and the severity of previous reactions (Müller 1990; Bilò et al. 2005). The likelihood of being stung again may be estimated from the number of stings received already, from the area where the individual lives (urban or rural), and lifestyle (job, hobbies). Patients

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with previous severe reactions, including definite cardiovascular and respiratory symptoms, are at increased risk because symptoms tend to repeat with subsequent stings. Preexisting cardiovascular disease and intake of drugs like beta-blockers or ACE inhibitors (Tunon-De-Lara et al. 1992; Müller & Haeberli 2005) may favor especially severe reactions. In individuals with only an LLR, no further diagnostic tests are usually recommended (Bilò et al. 2005). Besides taking a careful history, basic diagnostic tests such as skin tests and estimation of venom-specific serum IgE antibodies (sIgE) should be performed in all patients with a systemic reaction. They will be able to distinguish between an allergic and a nonallergic reaction, identify the responsible insect, and decide about further management. In special situations additional tests such as basophil histamine or leukotriene release, basophil activation test, or estimation of venom-specific IgG antibodies may also be of value.

Insect Sting Allergy

Immediately after a sting, sIgE may be low or even undetectable. It usually increases within days or weeks after a sting reaction. If no sIgE is detectable, the test should be repeated after 2–4 weeks (Bilò et al. 2005). In the presence of double positivity to bee and vespid venoms, a strong increase in sIgE to a particular insect venom after the sting reaction may help in identifying the responsible insect.

Sensitivity and specificity of skin tests and sIgE

It is recommended that skin tests are not performed before 3 weeks after a sting reaction in order to avoid false-negative results during the refractory period. Skin tests involve either intradermal or skin-prick end-point titration (Bilò et al. 2005). For the intradermal test, 0.02 mL of venom solution is injected in increasing concentrations (from 0.00001 to 1 μg/mL) into the volar surface of the forearm. The test is considered positive if a wheal of at least 5 mm in diameter with surrounding erythema results after 20 min at 0.1 μg/mL or less, and doubtful if it only occurs at 1 μg/mL. At even higher concentrations nonspecific toxic skin reactions are elicited. For the skin-prick test, concentrations of 0.01–300 μg/mL are used. However, even at 300 μg/mL the sensitivity of the prick test is clearly lower than that of the intradermal test at 1 μg/mL. We therefore prefer the intradermal test. If skinprick tests are negative in the presence of a strong history of systemic allergic sting reaction, this result should be controlled by intradermal tests.

Sensitivity of both these tests is over 90% in patients with a history of systemic reactions within the last year (Müller 1990). With an increasing interval from the sting, the percentage of patients with positive tests decreases slowly. However, even at an interval of several years tests remain positive in the majority of patients, in some even after decades, with large interindividual variation. As a rule the intradermal test remains positive longer than sIgE. Unfortunately, both skin tests and sIgE are not reliable predictive tests with regard to a future systemic reactions of untreated patients or protection in patients on venom immunotherapy (VIT) (Müller et al. 1989; van der Linden et al. 1994). Despite a typical severe reaction to stings, a few patients have no detectable sIgE and negative skin tests (Golden et al. 2001). This may be due to insufficient sensitivity of available tests or to a long interval between the severe reaction and testing, with spontaneous decrease in sensitization. On the other hand, non-IgE-mediated immunologic or nonimmunologic mechanisms of mediator release, e.g., in systemic mastocytosis (Fricker et al. 1997), may be responsible in rare cases. Specificity may cause problems. About 10–20% of individuals without a history of systemic reactions have positive diagnostic tests to Hymenoptera venoms. Although sensitization following a previous sting is difficult to exclude, positivity may also be due to cross-reactions (see below). For ant allergy, only whole-body extracts are commercially available. Their diagnostic sensitivity and specificity are not well documented.

Venom-specific serum IgE antibodies

Cross-reactivity

A number of different in vitro immunoassays for the detection of sIgE have been derived from the original RAST (radioallergosorbent test) and are commercially available. In most of these the allergen is fixed to an immunosorbent, then incubated with the patient’s serum and fixed specific IgE antibodies are then detected by radiolabeled or enzymelabeled anti-IgE antibody (Müller 1990). In newer tests the anti-IgE is fixed to the immunosorbent, then incubated with the patient’s serum. The fixed allergen-specific IgE antibodies are detected by adding labeled allergen. In this second approach interference with abundant allergen-specific IgG antibody is avoided and the producers claim that it allows for a better consideration of antibody affinity, resulting in higher specificity (Petersen et al. 2004).

Cross-reactivity between venom allergens is strong within a genus, e.g., between Vespula, Dolichovespula and Vespa, but only limited between Vespinae and Polistinae and also between honeybees and bumble-bees. There is little cross-reactivity between bee and vespid venom on a protein basis, mainly due to about 50% sequence identity between hyaluronidases of the two families. Nevertheless, in diagnostic skin and sIgE tests, double positivity to both venoms is observed in more than 50% of patients tested within a year after a systemic reaction (Müller et al. 2006), which may reflect true double sensitization or cross-reactivity. Besides the partial sequence homology of hyaluronidase, carbohydrate-containing epitopes are important. Cross-reacting carbohydrate determinants (CCDs) are present in many major Hymenoptera

Skin tests

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venom allergens such as hyaluronidase, acid phosphatase, and phospholipase A2, but also in many plant proteins, e.g., in pollen of rape or bromelain. CCDs are certainly responsible for part of the double positivity in diagnostic tests of bee and vespid venoms. They may also explain some of the positive tests in individuals with no history of a systemic reaction and in individuals with systemic reactions who claim that they have never been stung. The CCDs are considered to be of no clinical relevance (Hemmer et al. 2001), although this has not been proven by sting challenges in history-negative patients with sIgE to CCDs. The RAST-inhibition test with venoms and CCDs is helpful in distinguishing between true double sensitization and cross-reactivity (Hemmer et al. 2001; Jappe et al. 2006), but is not always conclusive. Estimation of speciesspecific nonglycosylated major venom allergens of both species appears to be helpful in this situation but tests are not yet commercially available (Müller et al. 2006).

Cellular tests When skin tests and sIgE to venoms of the locally important insects are negative in patients with a history of a systemic reaction, cellular tests may be helpful for demonstrating sensitization (Bilò et al. 2005). 1 In the basophil histamine release test, peripheral blood leukocytes are incubated with venom allergens. The reaction with cell-bound IgE antibodies leads to histamine release. 2 In the cellular antigen stimulation test (CAST), leukocytes of patients are pre-stimulated with interleukin (IL)-3 and then exposed to venom allergens. The released sulfidoleukotrienes are determined by ELISA. 3 The basophil activation test is based on the flow cytometric demonstration of an altered membrane phenotype of basophils stimulated by IL-3 and allergen exposure. At present the most commonly used expression marker is CD63 (Ebo et al. 2006). All these tests have an apparently high sensitivity. However, in a significant percentage of patients the cells do not react even to the positive control, anti-IgE, and the test can thus not be evaluated. Moreover, cellular tests are expensive and logistically more complicated than skin tests or sIgE. Data on their predictive value in relation to a sting challenge in patients on VIT are disappointing (Erdmann et al. 2004).

Serum tryptase The commercially available fluorescence immunoassay measures total tryptase. The α-tryptase is an enzymatically inactive monomer and is secreted continuously. It reflects the whole body mast-cell load. Elevated values are seen in cutaneous and systemic mastocytosis. β-Tryptase is the enzymatically active tetramer released during mast-cell activation. An elevated serum tryptase level within 30 min and 4 hours after a systemic reaction of questionable pathogenesis is thus an indicator of anaphylaxis. In the presence of only a slight elevation, comparison with a baseline serum level obtained at least a few days after the reaction may be helpful. Because of the association between an elevated baseline serum tryptase level (> 11.4 μg/L) and especially severe, sometimes sIgE negative, systemic sting reactions and also cutaneous or systemic mastocytosis (Oude Elberink et al. 1997; LudolphHauser et al. 2001; Haeberli et al. 2003), this enzyme should be determined in all patients with a history of a systemic reaction.

Sting challenge tests It is well known from studies on VIT that although patients tolerate an injected maintenance venom dose of 100 μg, corresponding to one or even several stings, they may react to the next sting by a live insect. Therefore a sting challenge under well-supervised clinical conditions is helpful in evaluating the efficacy of VIT (Ruëff et al. 1996). Sting challenges have also been used in untreated patients with a history of systemic reactions and these studies have shown that only some of these patients will react again (see Natural history) (van der Linden et al. 1994). However, a tolerated sting challenge does not definitely exclude a potential reaction to future stings. In a study with repeated sting challenges in adult history-positive patients, 21% reacted only to the second challenge, some of them severely (Franken et al. 1994). A well-tolerated sting challenge in an untreated patient may boost a preexisting sensitization. Because of these observations we do not recommend sting challenge as a diagnostic test in untreated patients, either before or after VIT. However, it is a useful test during VIT, especially in highly exposed patients.

Allergen-specific IgG The presence of specific IgG, primarily IgG4, reflects exposure to the respective venom. Extremely high levels are observed in heavily exposed beekeepers (Müller 2005). Venom-specific IgG increases after a sting, irrespective of the presence or absence of an allergic sting reaction. This increase, as for sIgE, may be helpful in identifying the responsible insect in cases of double positivity. VIT induces a rise in sIgG. However, there is no close correlation between the concentration of sIgG or the sIgE/sIgG ratio and protection induced by immunotherapy as indicated by a well-tolerated sting challenge (Müller et al. 1989).

1988

Prevention and treatment Prevention All patients with a history of systemic reaction must be instructed in detail with regard to avoidance of future stings. Knowledge of the behavior and ecology of the various Hymenoptera is the basis of a series of recommendations that can reduce the risk of a field resting very considerably (Table 96.3). Patients must be aware that Hymenoptera sting only in selfdefense when feeling threatened or in defense of their colony in the vicinity of their nests (Bonifazi et al. 2005).

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Table 96.3 Prevention measures: avoid activities with high risk of being stung by Hymenoptera. Activity

Expected hymenoptera

Outdoor eating and drinking Under trees with fallen fruit Walking barefoot in the lawn Gardening, cutting flowers Staying close to beehive, especially during honey collection Near vespid nests in trees, attic, windows

Vespula Vespula Honeybee, Vespula Honeybee Honeybee Various vespids

Treatment of large local reactions Oral antihistamines and cooling of the sting site (e.g., with ice cubes) reduces local swelling, pain, and itch. Immobilization of the involved area if possible and antiphlogistic ointments or topical corticosteroids may diminish the local inflammatory process. In cases of severe swellings, oral corticosteroids together with antihistamines over several days are recommended (Müller 1990; Bonifazi et al. 2005).

Systemic allergic reactions Sympathomimetics, antihistamines and corticosteroids are the most effective drugs for symptomatic treatment of systemic reactions. All patients with systemic reactions have to be medically observed until the symptoms resolve and the blood pressure is stable (Table 96.4). Mild and only cutaneous reactions may be treated orally with rapidly acting antihistamines (cetirizine, fexofenadine) alone. In the presence of respiratory or cardiovascular symp-

toms, intramuscular epinephrine must be given immediately, intravenous access established, and antihistamines (e.g., clemastine, diphenhydramine) and corticosteroids (e.g., methylprednisolone) given intravenously. All patients with systemic allergic reactions of grade I and II must be treated and supervised until symptoms definitely recede, usually for at least 1–2 hours. All patients with severe respiratory and cardiovascular symptoms should be hospitalized and supervised until complete recovery. Patients with cardiovascular symptoms have to be treated and transported in the supine position and intravenous volume substitution is indicated. Further investigations by an allergist with regard to prevention measures, prescription of emergency self-medication, and VIT are indicated in every patient with systemic allergic sting reactions (Bonifazi et al. 2005).

Emergency medication kit (Table 96.5) All patients with a history of a systemic reaction should carry

Table 96.4 Emergency treatment of generalized allergic reactions Symptoms

Medication

Other measures/comments

Mild urticaria, angioedema

Antihistamines (oral or parenteral)

Check blood pressure, pulse, respiration Observation for 1–2 hours

Severe urticaria, angioedema

Antihistamines i.v. Corticosteroids i.v. (e.g., prednisolone 0.5–1.0 mg/kg) Epinephrine: adults 0.3 mg i.m., children 0.01 mg/kg i.m.

Check blood pressure, pulse, respiration Observation until symptoms disappear completely

Laryngeal edema

Epinephrine (inhalation or i.m.) Antihistamines and corticosteroids i.v.

Oxygen supply If necessary intubation or tracheotomy, ventilation Hospitalization

Bronchial obstruction

b2 Agonists (inhaled) Epinephrine i.m. Corticosteroids i.v.

Oxygen supply If necessary intubation, ventilation Hospitalization

Anaphylactic shock

Epinephrine (if necessary repeated after 10 min): adults 0.3–0.5 mg i.m., children 0.01 mg/kg i.m. Volume replacement Antihistamines and corticosteroids i.v. Epinephrine, dopamine or norepinephrine continuous infusion Glucagon 0.1 mg/kg i.v.

Place in supine position, oxygen supply Hospitalization for 24 hours advisable (risk of biphasic reaction) In cases of protracted hypotension or shock In patients on beta-blockers

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Table 96.5 Emergency kit for self-medication.

Table 96.6 Treatment protocols for venom immunotherapy.

Prescribed emergency drugs Epinephrine autoinjector (e.g., EpiPen) Antihistamine with rapid action (e.g., cetirizine two 5-mg tablets or fexofenadine two 180-mg tablets) Corticosteroid (e.g., prednisolone two 50-mg tablets)

Conventional

Procedure if restung Take the four emergency tablets immediately Prepare EpiPen for injection If any systemic allergic symptoms arise: apply EpiPen immediately in the lateral thigh and seek medical care

an emergency kit for self-administration during the flying season of Hymenoptera. After a sting, the stinger, if still in the skin, must be removed as fast as possible. Patients have to take both their antihistamine and corticosteroid tablets immediately, irrespective of any symptom. If systemic symptoms such as urticaria, dyspnea, generalized weakness or dizziness occur, the EpiPen autoinjector (0.3 mg epinephrine) should be administered immediately intramuscularly in the lateral thigh. In children under 30 kg body weight, the Epipen Jr (0.15 mg epinephrine) and half the dose of antihistamines and corticosteroids should be used, and parents and teachers should also be instructed about the use of the emergency kit. If any systemic allergic symptoms occur, medical care must be sought immediately.

Venom immunotherapy (Bonifazi et al. 2005) Indications VIT is indicated in children and adults with a history of severe systemic reactions (grade III–IV) if sensitization to the relevant venom is demonstrated either by skin test and/or serum sIgE. Large local reactions are not usually an indication for VIT, but may be so in heavily exposed patients such as farmers or gardeners with repeated extensive and long-lasting reactions. Unusual reactions do not qualify for VIT. VIT is also recommended in patients with repeated mild reactions that are not life-threatening and who are at high risk of reexposure, such as beekeepers or their family members. Concomitant cardiovascular disease, mastocytosis, or strongly impaired quality of life due to venom allergy are also indications for VIT in patients with sting reactions that are not life threatening (Oude Elberink & Dubois 2003). Contraindications for VIT are the same as for immunotherapy with other allergens. Treatment with beta-blockers or ACE inhibitors (Tunon-De-Lara et al. 1992; Müller & Haeberli 2005) is a relative contraindication and these drugs should preferably be replaced by other substances. In all patients with coronary heart disease, arrhythmias or cardiac failure, where the life-prolonging effect of these drugs is well documented, the situation should be evaluated carefully together with the family practitioner and a cardiologist.

1990

Week

Amount of venom (mg)

1 2 3 4 5 6 7 8 9 10 11 12

0.01 0.1 1 2 4 8 10 20 40 60 80 100

Ultra-rush

Day

Minutes

Amount of venom (mg)

1

0 30 60 90 150 210

0.1 1 10 20 30 50

8

0 30

50 50

21 49

0 0

100 100

Afterwards 100 mg monthly for 1 year, then every 6 weeks from year 2 to 5.

Dosage and treatment regimens VIT may be initiated by a conventional or an ultra-rush protocol (Table 96.6). The recommended starting dose is usually 0.1 μg and the maintenance dose 100 μg of the venom, applied subcutaneously in the lateral upper arm, both in children and adults. The maintenance dose of 100 μg of venom protein is equivalent to approximately two bee stings and to several vespid stings. A higher dose (200 μg) is recommended when systemic reactions occur after reexposure by a field sting or a sting challenge during VIT. In highly exposed subjects such as beekeepers 200 μg as maintenance dose is advised in the first place because several stings may often occur together. The injection interval for maintenance VIT is 4 weeks for the first year. Afterwards intervals may be extended to 6 weeks if injections are well tolerated.

Adverse reactions to VIT The overall incidence of systemic adverse reactions to VIT varies between 5 and 40%. VIT with bee venom causes more side effects than with Vespula venom (Golden 2006). Ultrarush is associated with a somewhat higher risk of side effects than conventional protocols (Birnbaum et al. 2000). Most systemic side effects are mild; approximately one-third will require medical treatment. In several controlled studies (Müller et al. 2001) pretreatment with antihistamines reduced large local and other cutaneous reactions such as urticaria, while the much rarer severe systemic reactions were not reduced significantly, maybe owing to the low frequency of such side effects (type 2 error). Based on these observations many authors recommend giving antihistamines 2 hours before the injection in the up-dosing phase of VIT until the maintenance dose has been well tolerated repeatedly.

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Efficacy of VIT In addition to three prospective controlled trials (Hunt et al. 1978; Müller et al. 1979; Brown et al. 2003), the efficacy of VIT has been shown in a number of uncontrolled prospective studies that observed well-tolerated sting challenges during VIT (Ruëff et al. 1996). Treatment with bee venom results in full protection, as indicated by the complete absence of systemic allergic symptoms, in 80–90%, and with Vespula venoms in 95–100% of patients. On the other hand, 60– 75% of patients in the placebo or whole-body extract-treated groups of controlled studies developed systemic reactions again when restung by the culprit insect. In patients with Solenopsis allergy, the efficacy of immunotherapy with commercially available Solenopsis whole-body extract is not documented in controlled studies or by sting challenge tests during VIT. However, excellent results, comparable to those of VIT with Vespula venom, were obtained in the Australian double-blind placebo-controlled study with venom of Myrmecia pilosula (Brown et al. 2003).

Mechanisms of VIT Numerous publications have shown that VIT influences both humoral and cellular immune response in a very significant way. During VIT both skin sensitivity and sIgE initially increase somewhat and then decrease slowly, but are often still present when the patient is already well protected. The decrease in these markers of IgE-mediated allergy can thus not be the only reason for protection (Müller 1990; Bilò et al. 2005). In highly exposed beekeepers and in patients on VIT, sIgG is strongly increased and a protective effect of bee venomspecific IgG antibodies has been documented both in vitro by inhibition of histamine release from basophils, and in vivo, where passive immunotherapy with beekeeper gammaglobulin was able to increase the tolerated venom dose during VIT very significantly (Müller 1990). Competitive inhibition of allergen binding to effector cell-fixed sIgE seems a plausible mechanism for this effect. However, neither the serum level of sIgG nor the quotient of sIgG/sIgE are closely related to the protective effect of VIT (Müller et al. 1989). Ultra-rush VIT significantly decreases release of both histamine and leukotrienes from basophil cells early during VIT (Jutel et al. 1996) and thus may be responsible for early protection, when sIgG is not yet increased. Order

Profound alterations in the cellular immune response have also been described during VIT and are now thought to be the basic mechanism of protection induced by this treatment: During the first weeks of VIT the previously Th2-shifted cellular immune response, as indicated by proliferation and secretion of IL-4, IL-5 and IL-13 following in vitro allergen stimulation, is strongly decreased (Jutel et al. 1995), while regulatory T cells appear, which by secretion of IL-10 dampen Th2 cells and as a consequence also the IgE-mediated immune reponse (Blaser & Akdis 2004).

Duration of VIT Treatment for life may be the safest recommendation. However, after some years of VIT the majority of allergic individuals may lose motivation. Studies which addressed the protection rate 1–7 years after discontinuation of VIT of at least 3 years’ duration showed persisting protection in over 80% of both adults and children. Long-term protection after stopping seems to be superior in patients who were treated for 5 years rather than for only 3 years (Lerch & Müller 1998; Golden 2001). In most allergy centers, VIT for at least 5 years is recommended today. Even longer treatment should be considered in all high-risk patients such as those with very severe systemic sting reactions, preexisting cardiovascular or pulmonary disease, systemic allergic reactions to VIT or stings during VIT, and for subjects with elevated basal serum tryptase levels. Lifelong VIT is advised for patients with cutaneous or systemic mastocytosis. Three of four cases of fatal sting reactions after stopping VIT have been described in patients with systemic mastocytosis.

Allergic reactions to biting insects Biting insects may cause local and very rarely systemic allergic reactions that are due to sensitization to salivary proteins introduced during the process of blood sucking. The responsible insects belong to the orders Diptera, Hemiptera and Siphonaptera (Hoffman 2003) (Fig. 96.3).

Clinical symptoms Local reactions to insect bites may be of the immediate wheal and flare type, or delayed with pruritic erythema and papules developing after 12–24 hours and lasting for days to weeks, Diptera

Hemiptera

Siphonaptera

Family

Culicidae

Simuliidae

Tabanidae etc

Triatomidae

Cimicidae

Pulicidae

Species

Mosquito

Blackfly

Horsefly, deer fly

Kissing bug

Bed bug

Human flea

Aedes, Culex, Anopheles

Simulium

Tabanus

Triatoma protracta

Cimex lectularius

Pulex irritans

e.g., Fig. 96.3 Biting insects that may cause allergic reactions.

Insect Sting Allergy

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or with features of both. In delayed and combined types of reactions, vesicular, bullous or even necrotic lesions may develop. Systemic reactions are very much rarer than is the case with Hymenoptera stings. However, they have occasionally been described, especially following bites by horseflies (Tabanus spp.) and the kissing bug (Triatoma protracta) (Hoffman 2003).

Allergens The proteins in salivary glands have either digestive function (amylases, esterases) or hemostatic function (e.g., factor Xa inhibition). Numerous IgE-binding proteins with molecular masses of 15– 81 kDa have been described, especially in the saliva of mosquitos, but also of horseflies and kissing bugs and some have even been cloned (Simons & Peng 2003).

Prevention and treatment Application of insect repellents and prophylactic intake of antihistamines have a preventive effect for outdoor exposure (Simons & Peng 2003). Screens on windows and doors and mosquito nets over the bed are effective in homes. Bedbug infestation should be eliminated by appropriate pesticides, and infestation of pet animals by fleas eliminated in cooperation with a veterinarian. Local reactions may be treated with topical steroids or oral antihistamines. Immunotherapy for systemic allergic reactions is controversial, because only whole-body extracts are commercially available for diagnosis and treatment. Since systemic reactions are so rare and salivary extracts so difficult to prepare, it seems unlikely that salivary extracts or recombinant salivary proteins will ever be commercially available for diagnosis and immunotherapy.

Perspectives A number of promising perspectives are based on the availability of recombinant venom allergens for both diagnosis and immunotherapy (King et al. 1996; Müller 2002; Grunwald et al. 2006). By using recombinant cocktails with all major allergens for diagnosis instead of the whole venom, the specificity of these tests could be improved considerably. In the frequently observed double positivity of diagnostic tests with honeybee and Vespula venom, recombinant species-specific nonglycosylated major allergens from honeybee and Vespula venom should allow reliable differentiation between true double sensitization and cross-reactivity, which is important for the choice of venoms for immunotherapy (Müller et al. 2006). Both the efficacy and safety of VIT with vespid venoms are excellent, but in bee venom-allergic patients VIT is associated with significantly more side effects and is also somewhat less effective. Therefore, new approaches to VIT for bee venom allergy are especially sought. Various possibilities for reducing

1992

allergenicity while preserving immunogenicity of allergenic proteins have been proposed and studied experimentally, such as point-mutated recombinant allergens, polymers, Tcell epitope peptides of bee venom allergens (Reunala et al. 1991; Müller et al. 1998), or gene recombination of major allergens (Akdis et al. 1996; Karamloo et al. 2005). With such preparations it should be possible to reduce the side effects of VIT while increasing the maintenance dose and thus the efficacy of the treatment.

References Akdis, C.A., Akdis, M., Blesken, T. et al. (1996) Epitope specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL15. J Clin Invest 98, 1667–83. Antonicelli, A., Bilo, M.B. & Bonifazi, F. (2002) Epidemiology of Hymenoptera allergy. Curr Opin Allergy Clin Immunol 2, 341–6. Benton, A.W., Morse, R.A. & Stewart, J.D. (1963) Venom collection from honey bees. Science 142, 228–9. Bilò, M.B., Ruëff, F., Mosbech, H. et al. (2005) Diagnosis of Hymenoptera venom allergy. Allergy 60, 1339–49. Birnbaum, J., Vervloet, D., Ramadour, M. & Magnan, A. (2000) Hymenoptera ultra rapid venom immunotherapy (ultra-RVIT) (210 minutes): safety and risk factors. J Allergy Clin Immunol 105, S60. Blaser, K. & Akdis, C.A. (2004) Interleukin-10, T regulatory cells and specific allergy treatment. Clin Exp Allergy 34, 328–31. Bonifazi, F., Jutel, M., Bilo, M.B. et al. (2005) Prevention and treatment of Hymenoptera venom allergy. Allergy 60, 1459–70. Brown, S.G. (2005) Cardiovascular aspects of anaphylaxis: implications for treatment and diagnosis. Curr Opin Allergy Clin Immunol 5, 359– 64. Brown, S.G.A., Wiese, M.D., Blackmann, K.E. & Heddle, R.J. (2003) Ant venom immunotherapy: a double blind, placebo-controlled crossover trial. Lancet 361, 1001–6. Case, R.L., Altman, L.C. & VanArsdel, P.P. (1981) Role of cell-mediated immunity in Hymenoptera allergy. J Allergy Clin Immunol 68, 399– 405. Chinery, M. (1979) Insekten Mitteleuropas. Verlag Paul Parey, Hamburg. de Groot, H. (2006) Allergy to bumblebees. Curr Opin Allergy Clin Immunol 6, 294–297. Ebo, D.G., Sainte-Laudy, J., Bridts, C.H. et al. (2006) Flow-assisted allergy diagnosis: current applications and future perspectives. Allergy 61, 1028–39. Erdmann, S., Sachs, B., Kwiecien, R., Moll-Slowody, S., Sauer, I. & Merk, H. (2004) The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy. Allergy 59, 1102–9. Franken, H.H., Dubois, A.E.J., Minkema, H.J., van der Heide, S. & de Monchy, J.G.R. (1994) Lack of reproducibility of a single negative sting challenge response in the assessment of anaphylactic risk in patients with suspected yellow jacket hypersensitivity. J Allergy Clin Immunol 93, 431–6. Fricker, M., Helbling, A., Schwartz, L. & Müller, U. (1997) Hymenoptera sting anaphylaxis and urticaria pigmentosa: clinical findings and results of venom immunotherapy in ten patients. J Allergy Clin Immunol 100, 11–15.

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Golden, D.B.K. (2001) Discontinuing venom immunotherapy. Curr Opin Allergy Clin Immunol 1, 353– 6. Golden, D.B.K. (2006) Insect allergy in children. Curr Opin Allergy Clin Immunol 6, 289–93. Golden, D.B.K., Kagey-Sobotka, A., Norman, P.S. et al. (2001) Insect sting allergy with negative venom skin test responses. J Allergy Clin Immunol 107, 897–901. Graif, Y., Romano-Zelekha, O., Livne, I., Green, M.S. & Shohat, T. (2006) Allergic reactions to insect stings: Results from a national survey of 10 000 junior high school children in Israel. J Allergy Clin Immunol 117, 1435–9. Grunwald, T., Bockisch, B., Spillner, E. et al. (2006) Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3). J Allergy Clin Immunol 117, 848–54. Guralnick, M.W. & Benton, A.W. (2003) Entomological aspects of insect sting allergy. In: Levine, M.I. & Lockey, R.F., eds. Monograph on Insect Allergy, 4th edn. Dave Lambert Associates, Pittsburgh, pp. 11–25. Haeberli, G., Brönnimann, M., Hunziker, Th. & Müller, U. (2003) Elevated basal serum tryptase and hymenoptera venom allergy: relation to severity of sting reactions and to safety and efficacy of venom immunotherapy. Clin Exp Allergy 33, 1216–20. Hemmer, W., Focke, M., Kolarich, D. et al. (2001) Antibody binding to venom carbohydrates is a frequent cause for double positivity to honey bee and yellow jacket venom in patients with stinging insect allergy. J Allergy Clin Immunol 108, 1045–52. Hoffman, D.R. (2003) Allergic reactions to biting insects. In: Levine, M.I. & Lockey, R.F., eds. Monograph on Insect Allergy, 4th edn. Dave Lambert Associates, Pittsburgh, pp. 161–74. Hoffmann, D.R. & Jacobson, R.S. (1984) Allergens in Hymenoptera venoms. XII: How much protein is in a sting? Ann Allergy 52, 276–78. Hunt, K.J., Valentine, M.D., Sobotka, A.K., Benton, A.W., Amodio, F.J. & Lichtenstein, L.M. (1978) A controlled trial of immunotherapy in insect hypersensitivity. N Engl J Med 299, 157–61. Jappe, U., Raulf-Heimsoth, M., Hoffmann, M., Burow, G., HübschMüller, C. & Enk, A. (2006) In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 61, 1220–29. Jutel, M., Pichler, W.J., Skrbic, D., Urwyler, A., Dahinden, C. & Müller, U.R. (1995) Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures. J Immunol 154, 4187–94. Jutel, M., Müller, U.R., Fricker, M., Rihs, S., Pichler, W.J. & Dahinden, C. (1996) Influence of bee venom immunotherapy on degranulation and leukotriene generation in human blood basophils. Clin Exp Allergy 26, 1112–18. Karamloo, F., Schmid-Grendelmeier, P., Kussebi, F. et al. (2005) Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes. Eur J Immunol 35, 3268–76. Kind, L.S., Ramaika, C. & Allaway, E. (1981) Antigenic, adjuvant and permeability enhancing properties of Melittin in mice. Allergy 36, 155– 60. King, T.P., Lu, G., Gonzalez, M. et al. (1996) Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with hornet and wasp homologs and possible implications for clinical allergy. J Allergy Clin Immunol 98, 588– 600.

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Lerch, E. & Müller, U.R. (1998) Long-term protection after stopping venom immunotherapy: results of re-stings in 200 patients. J Allergy Clin Immunol 101, 606–12. Lichtenstein, L.M. & Golden, D.B. K. (1983) Postscript to bee stings: delayed “serum sickness”. Hosp Pract 18, 36–46. Ludolph-Hauser, D., Ruëff, F., Fries, C., Schöpf, P. & Przybille, B. (2001) Constitutively raised serum concentration of mast cell tryptase and severe anaphylactic reactions to Hymenoptera stings. Lancet 357, 361–2. McKenna, W.R. (2003) Africanized honey bees. In: Levine, M.I. & Lockey, R.F., eds. Monograph on Insect Allergy, 4th edn. Dave Lambert Associates, Pittsburgh, pp. 27–35. Mueller, H.L. (1966) Diagnosis and treatment of insect sensitivity. Asthma Res 3, 331–3. Müller, U. (1990) Insect Sting Allergy. Gustav Fischer Verlag, Stuttgart. Müller, U. (2002) Recombinant Hymenoptera venom allergens. Allergy 57, 570–6. Müller, U.R. (2005a) Cardiovascular diseases and allergy. Allergo J 14, 569–74. Müller, U.R. (2005b) Bee venom allergy in beekeepers and their family members. Curr Opin Allergy Clin Immunol 5, 343–7. Müller, U. & Haeberli, G. (2005) Use of beta-blockers during immunotherapy for Hymenoptera venom allergy. J Allergy Clin Immunol 115, 606–10. Müller, U., Thurnheer, U., Patrizzi, R., Spiess, J. & Hoigné, R. (1979) Immunotherapy in bee sting hypersensitivity. Bee venom versus wholebody extract. Allergy 34, 369–78. Müller, U., Helbling, A. & Bischof, M. (1989) Predictive value of venom-specific IgE, IgG and IgG subclass antibodies in patients on immunotherapy with honey bee venom. Allergy 44, 412–18. Müller, U., Helbling, A. & Berchtold, E. (1992) Immunotherapy with honeybee venom and yellow jacket venom is different regarding efficacy and safety. J Allergy Clin Immunol 89, 529–35. Müller, U., Akdis, C.A., Fricker, M. et al. (1998) Successfull immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol 101, 747–54. Müller, U., Hari, Y. & Berchtold, E. (2001) Premedication with antihistamines may enhance efficacy of specific-allergen immunotherapy. J Allergy Clin Immunol 107, 81–6. Müller, U., Johansen, N., Petersen, A., Haeberli, G. & FrombergNielsen, J. (2006) Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of species specific major allergens Api m1 and Ves v5. Collegium Intern Allergol 26, 45–6. Oude Elberink, J.N. & Dubois, A.E.J. (2003) Quality of life in insect venom allergic patients. Curr Opin Allergy Clin Immunol 3, 287–93. Oude Elberink, J.N.G., de Monchy, J.G.R., Kors, J.W., van Doormaal, J.J. & Dubois, A.E.J. (1997) Fatal anaphylaxis after a yellow jacket sting, despite venom immunotherapy, in two patients with mastocytosis. J Allergy Clin Immunol 99, 153–4. Petersen, B.R., Gudmann, P., Milvang-Gronager, P. et al. (2004) Performance evaluation of a specific IgE assay developed for the ADVIA centaur® imunoassay system. Clin Biochem 37, 882–92. Reichmuth, D.A. & Lockey, R.F. (2003) Clinical aspects of ant allergy. In: Levine, M.I. & Lockey, R.F., eds. Monograph on Insect Allergy, 4th edn. Dave Lambert Associates, Pittsburgh, pp. 133–51. Reisman, R.E. (1987) Late onset reactions following venom immunotherapy (VIT) and venom skin tests. J Allergy Clin Immunol 79, 232.

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Reisman, R.E. (2005) Unusual reactions to insect stings. Curr Opin Allergy Clin Immunol 5, 355– 8. Reunala, T., Lapallainen, P., Brummer-Korvenkontio, H., Coulie, P. & Palasuo, T. (1991) Cutaneous reactivity to mosquito bites. Effects of cetirizine and development of anti-mosquito antibodies. Clin Exp Allergy 21, 617–24. Ruëff, F., Przybilla, B., Müller, U. et al. (1966) The sting challenge test in Hymenoptera venom allergy. Allergy 51, 216–25. Sasvary, T. & Müller, U. (1994) Fatalities from insect stings in Switzerland 1978–87. Schweiz Med Wochenschr 124, 1887–94. Schumacher, M.J., Tveten, M.S. & Egen, N.B. (1994) Rate and quality of delivery of venom from honeybee stings. J Allergy Clin Immunol 93, 831–5. Schwartz, H.J., Sutheimer, C., Gauerke, M.B. & Yunginger, J.W. (1988) Hymenoptera venom-specific IgE antibodies in post-mortem sera from victims of sudden, unexpected death. Clin Allergy 18, 461–8. Seebach, J.D., Bucher, Ch., Anliker, M., Schmid-Grendelmeier, P. &

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Wüthrich, B. (2000) Ant venoms: a rare cause of allergic reations in Switzerland. Schweiz Med Wochenschr 130, 1805–13. Settipane, G.A. & Boyd, G.K. (1970) Prevalence of bee sting allergy in 4992 boy scouts. Acta Allergol 25, 286–91. Simons, F.E.R. & Peng, Z. (2003) Mosquito allergy. In: Levine, M.I. & Lockey, R.F., eds. Monograph on Insect Allergy, 4th edn. Dave Lambert Associates, Pittsburgh, pp. 175–203. Smith, P.I., Kagey-Sobotka, A., Blecker, E., Traystman, R., Kaplan, A.P. & Gralnick, H. (1980) Physiologic manifestations of human anaphylaxis. J Clin Invest 66, 1072–80. Tunon-De-Lara, J.M., Villanueva, P., Marcos, M. & Taytard, A. (1992) ACE inhibitors and anaphylactoid reactions during venom immunotherapy. Lancet 940, 908. van der Linden, P.W.G., Hack, C.E., Struyvenberg, A. & van der Zwan, J.K. (1994) Clinical aspects of allergic disease. Insect-sting challenge in 324 subjects with a previous anaphylactic reaction: current criteria for insect-venom hypersensitivity do not predict the occurrence and the severity of anaphylaxis. J Allergy Clin Immunol 94, 151–9.

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Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Prevention of Allergic Disease Susan L. Prescott and Bengt Björkstén

Summary

Introduction

It is now well established that there is a pronounced global increase in allergic diseases, particularly in affluent societies. It would obviously be preferable to prevent the development of disease, rather than limiting efforts to treatment of already established disease manifestations. Allergic diseases are often evident within the first months of life. This highlights the need to investigate the role of very early life events and exposure that possibly leads to the onset of the allergic march and then to develop strategies for prevention early in life. Currently, our capacity to prevent allergic disease is constrained by limited understanding of disease pathogenesis and etiologic factors, particularly of the early exposures responsible for the recent increase in allergic disease. There is also an inability to accurately identify atopic individuals before sensitization occurs. While there has been considerable success in managing established disease, strategies to prevent the development of these processes have been largely disappointing, including efforts to reduce allergen exposure early in life. In infants with a family history of allergy and who for some reason are not breast-fed, extensively hydrolyzed infant formulas reduce the incidence of food allergy and eczema during infancy, although they do not prevent respiratory allergies later in life. While exposure to tobacco smoke is a risk factor for wheezing in infancy, it does not appear to cause allergic disease. Similarly, breast-feeding protects against infant wheezing, but any allergypreventive effect is small. Vaccinations are neither protective nor risk factors in relation to allergy development. Controlled studies with probiotic lactobacilli suggest a modest eczema-preventive effect, at least in studies in which the bacteria were given both to mothers during pregnancy and then to their newborn babies for several months. Several ongoing studies in which the gut microflora early in life is modified by prebiotics and probiotics will show if such procedures have any sustained allergypreventive effects. Future intervention studies may also reveal if other dietary interventions, e.g., fish oil, vitamins or other dietary supplements, will have an impact. Also, the potential to prevent respiratory allergy, including asthma, by allergen vaccines is currently being studied.

Most current therapeutic strategies for allergic disorders are directed at controlling symptoms rather than reversing the underlying processes. It is increasingly urgent to define the causal pathways leading to disease and to develop strategies to avert the development of these diseases. As allergic diseases are very common, strategies that would reduce the risk or the severity of disease even slightly could have a major impact in a global context. Allergic diseases are often evident within the first months of life. This highlights the need to further investigate the role of very early events possibly leading to the onset of the allergic march and to develop strategies for prevention early in life. By “allergy prevention” we mean measures taken that would reduce the incidence of manifest allergic disease. Temporary sensitization, as demonstrated by allergen-specific IgE antibodies, is common in infancy and early childhood and is part of the normal development of immunity during the first years of life. This review does not focus on areas of secondary and tertiary prevention as these denote treatments that aim to avoid progression of disease and airway remodeling. Until recently, most of the interest has focused on identifying risk factors, i.e., environmental factors, that would increase the likelihood for sensitization. Thus, excessive exposure to indoor allergens, particularly house-dust mite (HDM), tobacco smoke and poor indoor ventilation, have all been suggested as major risk factors. As a consequence, prevention has focused on reducing exposure to these putative risk factors. However, none of the suggested risk factors can more than marginally explain the large regional differences in allergy prevalence, and the results of intervention studies have largely been disappointing. Over the past few years, novel discoveries related to the maturation of the immune system have resulted in an interest in identifying factors that protect against the development of manifest allergic disease, rather than searching for risk factors. An understanding of the development of immune responses to allergens is a prerequisite for understanding why the prevalence of allergy is increasing in developed countries and for the implementation of primary prevention.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Table 97.1 World Health Organization categories of evidence and strength of recommendations. Categories of evidence Ia Evidence from metaanalysis of randomized controlled trials Ib Evidence from at least one randomized controlled trial IIa Evidence from at least one controlled study without randomization IIb Evidence from at least one other type of quasi-experimental study III Evidence from nonexperimental descriptive studies, such as comparative studies, correlation studies and case–control studies IV Expert opinion of the PAA working group Strength of recommendations A Directly based on category I evidence B Directly based on category II evidence or extrapolated recommendation from category I evidence C Directly based on category III evidence or extrapolated recommendation from category II evidence D Directly based on category IV evidence or extrapolated recommendation from category III evidence

The strength of evidence for recommendations in clinical practice varies. The World Health Organization (WHO) have suggested Categories of Evidence (Table 97.1). These categories of evidence are more applicable to therapeutic interventions than to measures and recommendations based on epidemiologic and basic research studies, where randomization or blinding may not be possible (such as with breast-feeding). Thus, a recommendation categorized B cannot always be interpreted as a lesser recommendation than one coded A, as this may reflect the types of studies possible in generating an evidence base. Despite these limitations, we have assessed various preventive measures according to the WHO Categories of Evidence.

feeding and the harm of tobacco smoking. However, even seemingly innocent advice may have negative consequences, e.g., guilt feelings that not enough was done if the child develops disease. Advice about cleaning procedures may be taken so far that they interfere with daily life, “good” ventilation may come into conflict with energy conservation, and “no air pollution” may prevent a family from painting the house. Advice on pet avoidance may profoundly affect a family with a loved pet, visits to grandparents who keep pets, and may even force people to move from a farm. Thus, the consequences of advising preventive measures should always be considered, including how advice may be interpreted by those receiving it.

Ethical aspects

Identifying target populations for allergy prevention

Preventive measures that are recommended should be based on scientific evidence that is evaluated as strictly as that for medical treatment, as even seemingly innocent advice may have a profound impact on a family. The WHO has defined certain principles for decisions on preventive measures. First, the disease should be common and have potentially serious consequences. Second, the causes should be known and measures should be effective, safe and acceptable. There should also be resources for implementing the measures. Finally, the health economic consequences of the measures should be known. The ethical aspects regarding prevention are basically the same as for other medical therapies and interventions. They are based on four major principles: (i) do no harm, (ii) respect the autonomy of the individual to make his or her own decisions, (iii) only implement effective measures, and (iv) do not discriminate and distribute resources well. It could be argued that it would never be harmful to give healthpromoting advice regarding for example the value of breast-

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The reasons for identifying people with an increased risk for a disease should be clearly defined. The first obvious prerequisite is that it should be possible to prevent the disease, or at least significantly modify the progress of it. To merely predict a potentially severe disease without offering efficient preventive measures is unethical. Not only should it be possible to prevent a disease, the necessary resources for implementing the measures should also be available. Furthermore, the social and economic costs of preventive measures should clearly outweigh those of the disease to be prevented. Finally, accurate predictive markers should be available and they should be cost-effective. No diagnostic test or procedure is perfect in the sense that every individual who will develop a disease can be identified by the test and at the same time not a single person who will not develop disease will show a falsely-positive result. The clinical value therefore depends on sensitivity, specificity, negative and positive predictive value, and efficiency (Fig. 97.1).

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Test Allergy: No allergy:

Positive TP FP

Negative FN TN

Sensitivity:

TP TP + FN

Positive predictive value:

TP TP + FP

Specificity:

TN TN + FP

Negative predictive value:

TN TN + FN

Efficiency:

TP + TN TP + FP + TN + FN

Fig. 97.1 Summary of definitions employed to assess the clinical usefulness of a test.

Sensitivity is defined as the proportion of true positive outcomes among those who will develop disease, while specificity denotes the proportion of true negative outcomes among those who will not develop disease. The positive predictive value indicates the individuals who will develop disease as a percentage of all test-positive individuals. Negative predictive value is a measure of individuals without disease of all test-negative persons. Finally, the most important indicator of the clinical value of a predictive test or other indicator is efficiency. This is defined as the percentage of correctly classified individuals of all tested. In the following sections various indicators used for the prediction of allergic disease will be discussed.

Genetic markers to predict allergy risk The clear familial association of asthma and allergic disease suggests that genetic factors are important in the pathogenesis, although no reliable genetic markers for IgE sensitization or specific allergic diseases have been identified. Children born into atopic families are more likely to develop allergic diseases (50–80% risk) compared to those with no family history atopy (20%). The risk appears to be higher if both parents are allergic (60– 80%) as opposed to only one parent. The risk is also higher if the mother (as compared to the father) has allergic disease (Zeiger et al. 1992; Kjellman 1998). A number of candidate genes have been defined, but none of them have been shown to accurately predict disease (reviewed recently in Halapi & Hakonarson 2004). It is likely that different genes may be operative in different populations and different individuals. None of these has yet been confirmed to play a role in pathogenesis and at this stage genetic studies are not of any practical predictive value. Specific allergic conditions such as asthma and atopic dermatitis are strongly associated with allergic sensitization (the predisposition to produce allergen-specific IgE). However, sensitization and development of overt disease are likely to be regulated by different processes (and therefore different genes) (Lau et al. 2000). The precise relationship between sensitization and development of disease is still poorly understood. Genetic factors are also critical in understanding gene– environment interactions. The recent increase in allergic

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diseases suggests that environmental changes are responsible for unmasking genetic predisposition. Functional genetic polymorphisms appear to result in differences in the vulnerability to environmental factors such as microbial exposure (Eder et al. 2004) or tobacco smoke (Colilla et al. 2003; Meyers et al. 2005) underscoring the likelihood of complex multidirectional gene–environment interactions. This area is still poorly understood, but it is possible that in the future environmental modification may be targeted to individuals with particular genotypes.

Biological markers Determination of cord blood IgE is the most extensively evaluated laboratory test employed in allergy prediction so far, and in the early 1990s routine screening in delivery wards was considered. The most extensive study was done by Croner and Kjellman (1986), who followed 1700 consecutively born infants over an 11-year period. An elevated cord blood IgE over 0.9 kU/L was strongly associated with allergy development, particularly asthma and multiple allergies, but the positive predictive value was only 67% and sensitivity 26%. Furthermore, the severity of future disease could not be predicted by the test. Several subsequent studies have confirmed the limited clinical usefulness of IgE determinations for allergy prediction. There is accumulating evidence of early presymptomatic differences between atopic and nonatopic individuals at birth (Rinas et al. 1993; Tang et al. 1994; Warner et al. 1994; Martinez et al. 1995a; Liao et al. 1996; Prescott et al. 1998a,b; Upham et al. 1999; Macaubas et al. 2003). These differences seem to affect a number of different markers of immune activity at birth, including the magnitude (Piastra et al. 1994; Warner et al. 1994; Miles et al. 1996) and pattern (Rinas et al. 1993; Tang et al. 1994; Warner et al. 1994; Martinez et al. 1995a; Liao et al. 1996; Prescott et al. 1998b) of cellular responses in vitro, circulating neonatal levels of cytokines (Macaubas et al. 2003) or cytokine-producing cells (Spinozzi et al. 2001), and activity of progenitors that give rise to proallergic inflammatory cells (eosinophil progenitors) (Upham et al. 1999). Proteins involved in the activation of antigenpresenting cells (APCs) and pro-Th1 signaling (such as sCD14) have also been detected in amniotic fluid and are reduced in those who go on to develop atopic dermatitis (Jones et al. 2002). Börres and Björkstén (2004) prospectively studied the predictive value of blood and nasal eosinophil counts and serum interleukin (IL)-4 levels in infancy with regard to development of allergic disease up to 6 years of age. Although eosinophilia and elevated IL-4 during the first year of life were both significantly related to later developing allergic disease, the diagnostic efficiency was low and the tests too complex for routine use. Other parameters that have been with regard to allergy prediction early in life include leukocyte phosphodiesterase levels (Odelram et al. 1994), skin dryness and sensitivity to

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histamine, erythema toxicum, and neonatal eosinophil, leukocyte differential and platelet counts. None of them had an efficiency exceeding 58% (Odelram et al. 1995). Thus none of the biological markers tested so far (either alone or in combination) is of sufficient sensitivity or specificity to be used in clinical practice.

Targeting children with early disease: preventing disease progression Another target population for allergy prevention is children with very early disease (such as food allergy or atopic dermatitis), in order to prevent the progression to more persistent forms of disease (such as asthma and allergic rhinitis). Approaches to “secondary” prevention are discussed further below. Although children with these early forms of disease have a well-recognized risk of aeroallergen-associated disease, the natural disease history is extremely variable between individuals, and many early symptoms are nonspecific. Identifying children with an early atopic phenotype will become more relevant when effective immunomodulatory strategies are available (see section Prevention of disease progression in children with early sensitization or disease).

Summary Genetic factors are likely to be important in disease pathogenesis, but no genetic markers are currently documented in allergy prevention. There are no predictive genetic markers for the type or severity of future allergic disease. A family history of allergy and asthma and several laboratory tests including cord-blood IgE determinations are associated with an increased risk of allergic disease. The specificity and particularly sensitivity are both too low for routine screening purposes.

Prevention strategies in pregnancy The frequent appearance of allergic symptoms in the first months of life suggests that disease pathways are initiated very early in life, possibly even before birth. This has led to interest in the role of environmental exposures in pregnancy. Although there is growing evidence that maternal exposures, including microbial products (Ege et al. 2006), smoking (Noakes et al. 2006) and dietary factors (Dunstan et al. 2003), can influence infant immune development, experience of allergy prevention strategies are still limited in pregnancy. Most intervention studies for allergy prevention have involved interventions in both the antenatal and postnatal periods (Brunekreef et al. 2002; Woodcock et al. 2004; Chan-Yeung et al. 2005), making it difficult to determine the isolated effect of interventions in pregnancy. One ongoing Dutch prevention study (PREVASC) has been designed to also allow comparison of early versus prolonged interventions (Kuiper et al. 2005), although the results are not yet known.

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Allergen exposure during pregnancy While there is growing evidence that the fetus is exposed to allergens, this may be physiologic and not a risk factor for allergic disease. So far, strategies to avoid or reduce allergen exposure in pregnancy have been disappointing in reducing disease. The avoidance of multiple potential food allergens in pregnancy has not been shown to reduce the risk of allergic disease in randomized, double-blind, controlled trials (FälthMagnusson & Kjellman 1987, 1992; Kramer 2000a). Current consensus is that this practice should be discouraged because of potential nutritional compromise to the mother and fetus. Furthermore, it was observed in one of the studies (FälthMagnusson & Kjellman 1992) that all children with an allergy to egg extending beyond the age of 4 years belonged to the intervention group where the mothers avoided eating eggs during the last trimester of pregnancy. In the light of animal studies on tolerance induction by intrauterine antigen exposure (Jarrett & Hall 1984), the observations warrants further caution with dietary intervention. The opposite of avoidance, i.e., high intake of allergenic foods like cow’s milk and egg, has also been tried in an effort to induce tolerance (Lilja et al. 1989). This had no impact on subsequent development of allergic disease up to the age of 18 months. A number of large-scale studies are in progress to assess the role of early inhalant allergen exposure, using strategies that have been shown to effectively reduce HDM exposure (Brunekreef et al. 2002; Simpson et al. 2003). Several of these initiated HDM reduction in pregnancy and continuing through the first year of life, including the Manchester Asthma and Allergy Study (MAAS) (Woodcock et al. 2004), the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study based in the Netherlands (Brunekreef et al. 2002; Koopman et al. 2002), and the Childhood Asthma Prevention Study (CAPS) in Australia. In the Manchester study, HDM reduction was associated with significantly better lung function at 3 years of age (Woodcock et al. 2004). However, the trend for lower respiratory symptoms in the intervention group did not reach statistical significance. The authors also observed a paradoxical increase in the risk of sensitization in the active group (Woodcock et al. 2004). The Dutch PIAMA study has so far also failed to show any reduction in asthma, respiratory symptoms (with the exception of nocturnal cough in the second year of life) (Koopman et al. 2002), or sensitization. The recently published 5-year outcomes of the Australian CAPS also indicate that HDM reduction strategies did not have any effect on the prevalence of wheeze, asthma or allergen sensitization despite a 61% reduction in mite levels (Marks et al. 2006). If anything the rates of atopic dermatitis were higher in the HDM reduction group (P = 0.06).

Summary There is no evidence of a protective effect of dietary avoid-

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ance in pregnancy. Aeroallergen avoidance in pregnancy has not been shown to reduce allergic sensitization (and may even increase the risk) or the development of asthma.

Recommendations • Food avoidance in pregnancy is not recommended (strength of recommendation A). • Strategies for reduction of HDM in pregnancy are not associated with less allergy development and could even be associated with an increased risk (strength of recommendation A).

Maternal smoking in pregnancy The adverse effects of maternal smoking in pregnancy on infant lung development are well recognized (Brown et al. 1995; Stick et al. 1996; Hoo et al. 1998). There are also strong associations between maternal smoking in pregnancy and reduced lung function in later childhood (Gilliland et al. 2000, 2003). The adverse effect of antenatal smoke exposure on lung function was much greater than subsequent postnatal effects (Gilliland et al. 2003). More recent studies also suggest that maternal smoking could have additional immune effects, which could contribute to allergic risk (Noakes et al. 2003, 2006).

Summary Maternal smoking in pregnancy has adverse effects on lung development and may also have effects on immune development. This common and avoidable exposure has wellestablished adverse effects on many other developing systems. Recommendations • Pregnant women should be advised not to smoke in pregnancy (strength of recommendation A).

Role of immunomodulatory dietary nutrients in pregnancy There has been growing interest in potentially proinflammatory changes in Western diets, including the specific role of dietary components with recognized immunomodulatory effects such as antioxidants and polyunsaturated fatty acids (PUFA) (Black & Sharpe 1997; Weiss 1997). As discussed in recent comprehensive reviews (Devereux & Seaton 2005; Devereux 2006), potential effects on immune function could be greatest in early life including fetal life, when systems and responses are developing. Maternal dietary antioxidant intake (vitamin E) has been associated with neonatal immune responses to allergens (Devereux et al. 2002), justifying further studies of the effects of antioxidants on early immune function. To our knowledge, there has only been one intervention study in pregnancy to examine the effects of dietary nutrients on immune function. This study demonstrated that maternal n-3 PUFA (fish oil) supplementation had effects on neonatal immune function (Dunstan et al. 2003). Although this study

Prevention of Allergic Disease

was not designed to examine clinical effects, it is worth noting that infants in the fish oil group were consistently less likely to develop clinical features including food allergy, recurrent wheeze, persistent cough, diagnosed asthma, angioedema, or anaphylaxis compared with the control group (Dunstan et al. 2003). Children in the fish oil group were also three times less likely to have a positive skin-prick test to egg at 1 year of age [odds ratio (OR) 0.34, 95% CI 0.11–1.02; P = 0.055]. Further larger studies already underway in a number of centers to examine the suggested benefits of n-3 PUFA, and their role in allergy prevention. The effects of fish oil supplementation in the postnatal period are discussed further below.

Summary There is preliminary evidence that dietary composition can influence immune function and allergic symptoms in early life. Further research is required to confirm any potential benefits of dietary supplements in pregnancy and to determine if there are risks associated with the use of vitamin supplements. Recommendations None.

Prevention strategies in infancy Breast-feeding There is consensus that breast-feeding has multiple health benefits and should be encouraged. This is particularly true in developing countries, where protection against infections may be a matter of life or death. Human milk affects host defense and immunity of the infant in several ways (reviewed by Cleary 2004; Labbok et al. 2004; Walker 2004). It provides passive protection against infections, through numerous components of innate immunity and IgA antibodies, but it also provides the baby with components that enhance the development of the immune system. As the content of food allergens, such as egg and cow’s milk protein, is much lower than when directly given to the baby, it is not surprising that the potential allergy-preventive effects of breast-feeding have raised major interest among pediatricians. However, the relationship between breast-feeding and subsequent development of allergy is complex.

Breast-feeding and subsequent asthma and allergy Short duration of breast-feeding has been associated with an increased incidence of allergic disease since the 1960s (reviewed by Björkstén 1983), but although there have been over 40 studies examining the relationship between breast-feeding and the development of allergy, results remain conflicting. There are inherent limitations in studies of this nature (confounding factors, recruitment and reporting biases, perceptions modifying feeding practices, inability to randomize and blind).

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Importantly, many studies do not make the distinction between “exclusive” breast-feeding and “any” breast-feeding. Furthermore, “allergy” and “allergic disease” are often not clearly defined. For example, wheezing in infancy is rarely caused by an allergy but is a consequence of a viral respiratory disease and usually not associated with asthma later in life. As breast-feeding protects against infections, it is not surprising that most authors agree that breast-feeding is associated with less infant wheezing. Many studies have shown a weak protective effect on early symptoms of allergic disease (reviewed by Foucard 2000), including atopic dermatitis (Lucas et al. 1990; Schoetzau et al. 2002) and early wheezing (Burr et al. 1993; Rylander et al. 1993; Martinez 1995; Wright et al. 1995; Wilson et al. 1998; Oddy et al. 1999). A systematic review (Gdalevich et al. 2001) found that exclusive breast-feeding in the first months of life was also associated with reduced rates of subsequent asthma (OR 0.70, 95% CI 0.60– 0.81). This included 12 prospective studies (8183 infants) and found that the protective effect was greater in studies of children with an increased risk of allergy (OR 0.52, CI 0.35–0.79). Others showed that although breast-feeding protected from early transient wheeze, there was the opposite relationship with persistent wheeze (subsequent asthma) (Rusconi et al. 1999; Wright et al. 2001). It has been proposed that protection from “transient wheeze” may be due to protection from infection but this does not explain all of the effect (Sly & Holt 2002). Other studies have challenged the protective effects of breast-feeding (reviewed by Sly & Holt 2002). However, a recent review examined over 4000 articles relating to breast-feeding and allergic disease and concluded that breast-feeding in the first 4 months of life reduced the risk of wheezing and asthma (van Odijk et al. 2003). The effect of breast-feeding on sensitization and atopy is less clear. A number of studies have suggested that breastfeeding also reduces the subsequent development of atopy in later childhood (Saarinen & Kajosaari 1995; Oddy et al. 1999). In a large prospectively followed cohort of 2187 Australian children (enrolled before birth) the risk of developing a positive skin-prick test reaction to common aeroallergens at 6 years of age was increased if exclusive breast-feeding was stopped (other milk was introduced) before 4 months (OR 1.30, 95% CI 1.01–1.62) (Oddy et al. 1999). Other studies have shown no long-term benefits or even increased atopy at 6 years (Kaplan & Mascie-Taylor 1985; Rusconi et al. 1999; Wright et al. 2001; Bergmann et al. 2002), including a New Zealand study (Sears et al. 2002) which found that breast-fed children (N = 504) were more likely to develop allergic disease and sensitization at 13 years than if they were formula fed (N = 533), although significant methodologic concerns have been raised (Kemp & Kakakios 2004).

Summary The relationship between breast-feeding, asthma and allergy

2002

development is unclear, and may remain difficult to ascertain because of difficulties conducting randomized controlled trials in this area. Exclusive breast-feeding for the first 4–6 months is associated with reduced rates of wheezing in the first years of life although this effect is modest. The effects of breast-feeding on the development of allergic disease in later childhood are uncertain. There appear to be different effects of breast-feeding on allergic disease as compared to atopy per se.

Recommendations • Breast-feeding is recommended because of a wide number of clearly beneficial effects, particularly in developing countries (strength of recommendation A). • Any protection against allergic disease is small and some studies have raised concerns that breast-feeding may actually increase the risk of disease. Breast-feeding is therefore not particularly recommended in allergy prevention (strength of recommendation C).

Role of maternal allergen avoidance during lactation Although some studies have suggested that maternal avoidance of potential food allergens (milk, egg, and fish) while breast-feeding may reduce the risk of atopic eczema in the first years of life (Businco et al. 1983; Chandra et al. 1986; Chandra 1997), the validity of several of the key papers (Chandra et al. 1986; Chandra 1997) have been questioned, casting serious doubts over the data (White 2004). Other studies have failed to show a protective effect of maternal dietary restrictions (Lilja et al. 1989; Sigurs et al. 1992; Hattevig et al. 1999). Thus, while the previous Cochrane review suggested some benefits of dietary restriction on early atopic eczema, this is now difficult to interpret and highlights the need for further research in this area (Kramer 2000b).

Summary There is no convincing evidence to support a protective effect of allergen avoidance during lactation. Recommendations • Maternal dietary restrictions during breast-feeding are not recommended for disease prevention (strength of recommendation A).

Role of infant feeding Infant formulas With the rising rates of allergy there has been concern that early exposure to allergenic food proteins, such as cow’s milk, could increase the risk of allergic disease. It was thought that by preventing food allergy and infant eczema, continuation of the the “atopic march” toward respiratory allergic disease could also be prevented. It is now clear, however, that complete avoidance of a certain allergen, such as cow’s milk or

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eggs, can only be expected to prevent sensitization and manifest allergy to that allergen. In order to avoid allergy to cow’s milk, soy formulas have been tried but they are as allergenic as regular cow’s milk formulas (Businco et al. 1992; Burr et al. 1993; Chandra et al. 1989; Chandra & Hamed 1991). This also seems to be the case for infant formulas based on milk from other animals than cows, e.g., goat’s formula. This was the basis for developing alternatives with reduced allergenicity, such as extensively hydrolyzed formulas. It has become largely accepted that these hydrolyzed formulas are the best alternatives if breast-feeding is not possible, but there is no evidence that these are superior to exclusive breastfeeding in preventing allergy (Osborn & Sinn 2003). This metaanalysis found a significant reduction in allergy incidence in infancy [typical relative risk (RR) 0.63, 95% CI 0.47, 0.85; RD −0.15, 95% CI −0.25, −0.06], although this has had to be withdrawn because of the inclusion of a study which is questioned (Chandra et al. 1989). Despite this, a number of other studies suggest that the use of extensively hydrolyzed formulas in combination with avoidance of cow’s milk proteins and solid foods during the first 4 months of life in high-risk infants are associated with reduced cumulative incidence of atopic eczema and food allergy, especially cow’s milk allergy in the preschool years (Arshad & Hide 1992; Halken et al. 1992; Mallet & Henocq 1992; Zeiger et al. 1992; Halken et al. 1993; Hide 1994; Zeiger 1995). Partially hydrolyzed formulas (with moderately reduced allergenicity) have also been reported to have an allergypreventive effect in randomized prospective studies of highrisk infants (Vandenplas et al. 1988, 1992, 1995; Chan et al. 2002). Recent data from studies comparing the allergypreventive effect of various hydrolyzed formulas suggest that extensively hydrolyzed formulas have a greater protective effect compared with partially hydrolyzed formulas in some studies (Oldaeus et al. 1997; Halken et al. 2000) but not all (Nentwich et al. 2001). Although the recent metaanalysis by Osborn and Sinn (2003) found no difference between the effects of extensive and partially hydrolyzed formulas, this will need to be reevaluated following the exclusion of particular studies (Chandra et al. 1989; Chandra & Hamed 1991).

Summary There is no evidence that soy formulas are less allergenic than normal cow’s milk formulas. Extensively hydrolyzed formulas appear to have a significant protective effect on early manifestations of allergic disease, notably eczema and cow’s milk allergy but not on respiratory allergies appearing later in life. Partially hydrolyzed formulas also appear to have a (probably weaker) protective effect. Further longer-term studies are needed. Because of great variations in study design and diagnostic criteria, the relative efficacy of the different formulas tested in different studies cannot be compared directly.

Prevention of Allergic Disease

Recommendations • If breast-feeding is not possible, an extensively hydrolyzed formula is recommended (rather than conventional cow’s milk-based formulas) in infants with a family history of allergy in order to prevent food allergy and eczema in early childhood (strength of recommendation B; the metanalyses need to be revised after excluding studies that are now suspected of being unreliable). • If an extensively hydrolyzed formula is not accessible, a partially hydrolyzed formula may be considered, but more data are needed (strength of recommendation B). However, these formulas have not been shown to prevent respiratory allergies.

Role of weaning foods Allergens were among the first candidate factors to be investigated as causes of the increasing prevalence of allergy, but there has been no clear evidence that allergen exposure has changed significantly during the “allergy epidemic.” The guiding principle behind allergen avoidance strategies is the hypothesis that reducing allergen levels may reduce the risk of allergen sensitization and hence the risk of allergic disease. However, despite the clear association between sensitization and the development of allergic disease, the processes leading to sensitization appear to be independent of those leading to disease. In many cases, allergen avoidance strategies have been ineffective in reducing allergic sensitization, and some studies even suggest unexpected paradoxical effects (as discussed below). Moreover, new allergy prevention studies are focusing on the potential role of controlled allergen administration (immunotherapy) rather than allergen avoidance (also discussed further below). Cumulatively, the literature suggests that delaying the introduction of solid foods may reduce or delay the onset of infantile atopic dermatitis and food allergies (Kajosaari & Saarinen 1983; Zeiger et al. 1989, 1992; Fälth-Magnusson & Kjellman 1992; Zeiger 1995; Zeiger & Heller 1995). However, these effects are modest, and any long-term benefits are not certain. A 2002 Cochrane metaanalysis (Ram et al. 2002) reviewed six randomized control trials that examined the effects of dietary restriction (in combination with hydrolyzed formulas) in infants at high risk of allergy. Although the analysis concluded that these interventions were associated with reduced risk (RR 0.4, 95% CI 0.19–0.85) of wheezing at 1 year of age, there are no data to confirm that this has any long-term benefits in reducing asthma, particularly in view of the heterogeneous etiology of early infant wheeze. Another recent study (Morgan et al. 2004) also suggested that preterm infants are also significantly more likely to develop atopic eczema (OR 3.49) if solid foods were introduced before 17 weeks of age. The long-term effects were not examined. One study has suggested that delaying solids (by parental choice) does not reduce asthma or eczema at 5 years (Zutavern et al. 2004). However, these data were collected retrospectively

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and could have been confounded by reverse causality (the parents chose to delay solids because they perceived the child to have signs of allergy). Preliminary results of multicenter interventions to reduce allergy (including delayed introduction of solids, breast-feeding, or hydrolyzed formulas and HDM reduction strategies) have also shown disappointing results (Tauber et al. 2002). The duration of dietary restrictions examined in different studies varies. All studies showing a preventive effect on eczema and food allergy have involved exclusive breastfeeding or use of extensively hydrolyzed formula and avoidance of cow’s milk and solid foods for 4– 6 months. There is currently no evidence that dietary restrictions for longer periods have additional benefits. In children with existing sensitizations it has been common clinical practice to recommend avoidance of potentially allergenic foods such as egg and milk until 12 months of age, and peanuts, nuts and shellfish until after 2–4 years of age (although these time-frames are arbitrary and variable). This practice is based on theoretical concepts that it may be best to avoid these foods until the “immune system is more mature.” There is little evidence to substantiate this. However, the rationale behind this nonevidence-based “reasonable” advice has been that there is no evidence that avoiding these foods (peanuts, nuts, shellfish) during early life is harmful. However, there has been growing concern that avoidance of allergens may interrupt the development of normal oral tolerance, which appears to be driven by early and repeated food allergen exposure. This is the basis for new studies that propose to examine whether the early administration of foods (such as peanut allergens) has a role in reducing the risk of subsequent peanut allergy. The results are awaited with interest.

Summary There appears to be a transient benefit from delaying introduction of solids for 4– 6 months in infants with a family history of allergy (also reviewed by Gore & Custovic 2004) in that the incidence of eczema and food allergy is slightly reduced. So far, any long-term benefits in prevention of allergic disease have not been shown. It should be remembered, however, that avoidance of proteinaceous food with high nutritional value may be associated with malnutrition and suboptimal growth and that any dietary restrictions might interfere with family life. Recommendations • Complementary foods (including normal cow’s milk formulas) may be delayed for up to 4– 6 months in infants with a distinct family history of allergy and/or elevated cord blood IgE levels. (strength of recommendation C). The metaanalysis by Ram et al. (2002) did not look at this intervention in isolation.

2004

• There is no evidence for a preventive effect of diet after the age of 6 months (strength of recommendation B).

Role of inhalant allergen exposure Role of HDM avoidance Stringent environmental control measures can dramatically reduce HDM levels (Simpson et al. 2003; Gore & Custovic 2004), and even less stringent measures (mite covers for bedding and washing instructions) significantly reduce HDM levels (Koopman et al. 2002). Many groups have shown a dose relationship between HDM levels in the home and sensitization to HDM (Lau et al. 1989; Wahn et al. 1997; PlattsMills et al. 2000). However, while sensitization is a strong risk factor for persistent asthma, wheeze and bronchial hyperactivity (Lau et al. 2002; Ponsonby et al. 2002), the relationship between early allergen exposure and the development of clinical symptoms has been much harder to confirm. The hypothesis that allergen avoidance early in life would prevent asthma is based on two independent observations, namely that exposure to high levels of inhaled allergen is associated with increased likelihood of sensitization and that asthmatic children were often sensitized in early childhood. However, no studies have confirmed that the two observations are related to each other. On the contrary, exposure to allergens may facilitate tolerance after a transient period of sensitization. A detailed cohort study failed to demonstrate a relationship between early indoor allergen exposure and the prevalence of asthma, suggesting that the induction of specific IgE responses and the development of childhood asthma are determined by independent events (Lau et al. 2000). As indicated previously (in section Allergen exposure during pregnancy), a number of prevention studies have initiated HDM avoidance strategies in late gestation, either as a single strategy such as in MAAS (Simpson et al. 2003; Woodcock et al. 2004), PIAMA (Brunekreef et al. 2002; Koopman et al. 2002) and CAPS (Mihrshahi et al. 2003; Peat et al. 2004; Marks et al. 2006), or as part of a multifaceted intervention program such as the Canadian Asthma Primary Prevention Study (Chan-Yeung et al. 2000, 2005; Becker et al. 2004). Others have initiated HDM reduction in the immediate postnatal period, again either as an isolated strategy such as the Study of Prevention of Allergy in Children of Europe (SPACE) (Halmerbauer et al. 2002; Tauber et al. 2002; Horak et al. 2004) or as part of a combined approach with food allergen avoidance such as the Isle of Wight Prevention study (Arshad et al. 1992, 2003; Hide 1994). The Manchester study demonstrated that a significant reduction in HDM levels in pregnancy and the postnatal period was associated with less respiratory symptoms at 1 year of age (Custovic et al. 2001) and better lung function at 3 (Woodcock et al. 2004). However, there was a significantly increased risk of sensitization (RR 1.61, 95% CI 1.02–2.55; P = 0.04) (Woodcock et al. 2004) as noted previously. These

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differential effects of HDM reduction on sensitization and disease suggest that IgE responses develop by independent processes to those which lead to asthma. There has also been speculation that these measures may have also reduced potentially protective endotoxin levels (as discussed below). The 7-year outcomes of the multifaceted Canadian study have recently been published, suggesting that a combined approach (using avoidance of HDM, pets, and cigarette smoking in combination with breast-feeding and delayed weaning) may reduce the risk of asthma (adjusted OR 0.39, 95% CI 0.22– 0.71), although there was no effect on bronchial hyperresponsiveness, sensitization, or other allergic disease (allergic rhinitis or atopic dermatitis) (Chan-Yeung et al. 2005). However, most other large intervention studies have not shown consistent effects on lung function, respiratory symptoms, or sensitization. In the Australian prevention study (CAPS), HDM avoidance had no significant effects on respiratory symptoms or sensitization at 18 months (Mihrshahi et al. 2003), 3 years (Peat et al. 2004), or 5 years (Marks et al. 2006). Although the PIAMA study showed a reduction in nocturnal cough in the second year of life (adjusted OR 0.65, 95% CI 0.4–1.0), HDM reduction had no other effects on respiratory disease or sensitization (Brunekreef et al. 2002; Koopman et al. 2002). Similarly, although the multicenter SPACE study reported a reduction in allergen sensitization at 1 year (Halmerbauer et al. 2002), there were no effects at 2 years of age (Horak et al. 2004) and no effects on lung function in a subgroup analysis. These studies require more definitive follow-up analysis. HDM reduction interventions in older children (5–7 years) have also been shown to reduce the rate of new sensitization to HDM after 12 months (Arshad et al. 2002), although more studies are needed. There is some evidence that some forms of bedding (such as feather bedding) may reduce the risk of HDM sensitization (Ponsonby et al. 2002), but more studies are needed. Earlier studies suggested that HDM avoidance may delay rather than prevent the development of asthma (Arshad et al. 1992; Hide et al. 1996), highlighting the need for long-term follow-up in these studies. At this stage there is not a strong case for HDM allergen avoidance in the prevention of allergic disease. However, this may have a future place in multifaceted prevention programs. Extensive house cleaning puts strain on families for a long period of time and should therefore only be considered in highly motivated families.

Summary The pattern of specific allergen reactivity reflects exposure patterns, but there is no evidence that allergens are directly responsible for the underlying propensity for allergic immune responses. Reducing allergen exposure is therefore unlikely to modify the allergic propensity as suggested by recent trials. Reduction in HDM levels may have a marginal benefit on

Prevention of Allergic Disease

lung function in early childhood, but the effects on sensitization remain controversial.

Recommendations • There is some evidence that HDM avoidance may have a limited benefit on early symptoms and lung function. In those patients with established disease and sensitivity, HDM avoidance has been shown to be of help (strength of recommendation C). • Clinical studies do not support the proposal that HDM avoidance significantly reduces the risk of allergic disease (strength of recommendation B).

Exposure to pet allergens As for HDM, the relationship between pet sensitization and respiratory allergic diseases is not clear and likely to be regulated by different processes. While it is known that high levels of cat allergen in house dust are associated with an increased incidence of sensitization in early life (Lau et al. 1989), there are no studies showing that low levels are associated with less allergic disease. On the contrary, a growing number of studies are challenging the traditional approach of reducing pet exposure in early life to prevent sensitization (and/or disease). Hesselmar et al. (1999) reported that pet exposure in the first year of life was associated with a lower prevalence of sensitization, asthma, and allergic rhinitis in later childhood. It is also of note that in a recent North American cohort, Ownby et al. (2002) reported that having pets (two or more cats or dogs) in the household during the first year of life was associated with not only less sensitization to pet allergens at 6 years of age but also less sensitization to other allergens, including molds and pollens. This is also consistent with findings of a Dutch study (Anyo et al. 2002). The relationship between pet allergen exposure and sensitization appears to be “bell shaped” rather than linear (PlattsMills et al. 2002; Cullinan et al. 2004), such that sensitization is less likely at both very low levels and at very high levels (which may induce tolerance). Many pet-exposed children who do not develop IgE responses have IgG4 antibody responses to pets (Platts-Mills 2002) which are believed to be the result of type 2 cytokine-driven immunoglobulin isotype switching. The potential mechanisms are not yet understood and the significance for the development of allergic disease is unknown. The relevance of these immunologic observations for disease is not known. Thus, there is accumulating evidence that recommendations for pet allergen avoidance in the first year of life to prevent allergic disease are not justified, particularly as in some communities 75% of cat-allergic individuals have never lived in a house with a cat (Rönmark et al. 1998). Further studies are also required to determine the mechanisms of this emerging protective relationship, which may be related to higher levels of bacterial endotoxin (von Mutius et al. 2000)

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(see below) in the presence of cats, dogs and cockroaches in the home (Heinrich et al. 2001). Endotoxin exposure has been proposed as an explanation for the protective relationship between early animal exposure and allergic sensitization that has been observed in European farming communities (Horak et al. 2002). This has added to speculation that this may be related to the immunostimulant effects of bacterial endotoxin (von Mutius et al. 2000). This could also account for the protective effects of domestic pets in some studies. These observations have provided ongoing support for the “hygiene hypothesis” that rising rates of allergic disease are due to increasingly “clean” environments, which fail to provide adequate stimulation of type 1 responses (and regulatory immune responses) for suppression of type 2 allergic responses (von Mutius 2001).

Summary There is no consistent evidence that either exposure to, or avoidance of, pet allergens has a protective effect against development of allergic disease. The potential compounding effects of endotoxin are not clear. Recommendations • In patients with established allergic disease and sensitization to pet allergen, pet removal may be of some benefit (strength of recommendation C). • In infants without apparent allergic disease, exposure to household pets does not seem to increase the risk of subsequent allergic disease, and may even have a protective effect in some situations (strength of recommendation B).

Role of microbial exposure Although infectious agents have a clear role in triggering established allergic diseases (such as asthma and atopic dermatitis), their role in the development of allergic disease remains controversial. The effects of microorganisms are likely to vary with the timing of exposure and the nature of the organism.

Bacteria Bacteria are the most powerful immunostimulants in the normal environment, activating the immune system via a range of pattern-recognition receptors (Toll-like receptors, TLRs). Although TLRs are found principally on cells of the innate immune system (including granulocytes, monocytes, and natural killer cells), they are also present on cells involved in programming and regulating “adaptive” immune responses (such as APCs and regulatory T cells). It has been proposed that early microbial activation of both APCs and regulatory T cells may promote Th1 maturation and play an important role in reducing the risk of Th2-mediated allergic responses (Holt et al. 1999; Wills-Karp et al. 2001). This is supported by animal studies demonstrating that bacterial lipo-

2006

polysacharide (LPS) endotoxin exposure can prevent allergic sensitization if given before allergic responses are established (Tulic et al. 2000; Blumer et al. 2005). These effects may be of greater significance in genetically susceptible individuals who appear to have weaker Th1 responses in the perinatal period (Holt et al. 1992). Genetic studies also support a role for CD14/LPS (Baldini et al. 1999) and TLR (Eder et al. 2004) pathways in the development of allergic disease. Studies in germ-free animals confirm that a microbial gut flora is essential for the development of oral tolerance and for the induction of normal immune regulation (Sudo et al. 1997). The controversy regarding the role of gut bacteria in allergy development thus lies in the clinical consequences of these findings and not so much the way they affect the immune system. Studies investigating the relationship between early childhood infection and risk of atopy have been inconsistent or difficult to interpret (Ponsonby et al. 1999; Wickens et al. 1999a). The immunologic effects of microbial agents differ with the type of infectious agent and the site of infection (Matricardi et al. 2000). Differences are also seen in the responses to vaccine antigens compared to the wild-type infections they prevent. Furthermore, nonpathogenic colonizing organisms are also likely to play a central role in immune development (Bottcher et al. 2000; Björkstén 2004). A recent large Danish national cohort study involving 24 341 mother– child pairs (Benn et al. 2004) found that early infections did not protect against atopic dermatitis. However, the study observed that other environmental factors, sometimes taken for indirect markers of microbial exposure (such as early daycare attendance, having three or more siblings, farm residence, and pet keeping), were protective. It is possible though that these protective factors are due to other than microbial exposure. For example, the inverse relationship between number of older siblings and allergy risk may be due to altered maternal immunity as a consequence of repeated pregnancies and exposure to animals could possibly be explained by high-zone tolerance induction. This highlights the emerging concept that overall “microbial burden” rather than specific infections may be more relevant in early life (Martinez 2001). Thus, while there is accumulating epidemiologic and experimental evidence to support the notion that exposure to microbes and microbial products early in life facilitates the development of immune regulation and thus prevent allergic disease, there is no definitive proof and it remains an area of debate (Platts-Mills et al. 2001; von Mutius 2001).

Summary There is no clear relationship between early childhood infection (or the use of antibiotics) and the risk of allergy. Interactions between microbial agents and the immune system are likely to depend on the type of infectious agent, the site of infection, the severity of infection and host response, and the genetic

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predisposition of the host. Overall “microbial burden” rather than specific infections may be more relevant in early life.

Recommendations None.

Recommendations None.

Role of antibiotics

Viral respiratory tract infections The relationship between early respiratory tract infections and allergic airway disease is complex. It is now well established that virus-associated “infant wheezers” are a heterogeneous group (Martinez et al. 1995b) and only a proportion will ultimately develop asthma and allergic diseases. Viral infections have been clearly identified as asthma triggers in children with established disease, and respiratory syncytial virus (RSV) infection in infancy has also long been regarded as a risk factor for subsequent asthma, at least in the first 6 years of life (Stein et al. 1999). This may be partly because of the Th2-trophic (proallergic) properties of this and other respiratory viruses (Sigurs et al. 1995), but may also be an indirect consequence of the delayed capacity to mount Th1 interferon (IFN)-γ responses in the early postnatal period (Rowe et al. 2001). In this context, Oddy et al. (2002) recently demonstrated that predisposition to wheezing lower respiratory infection in the first year of life is a strong risk factor for asthma at 6 years of age in both nonatopic (OR 4.1; P < 0.0005) and atopic (OR 9.0; P < 0.0005) children. The findings strongly suggested that significant infection-induced airway inflammation during the early period of postnatal lung growth and development can have profound long-term effects that may be more marked than similar insults at later ages (Holt & Sly 2002; Holt et al. 2004). Moreover, susceptibility to RSV infection and its subsequent spread to the lower respiratory tract in children at high risk of atopy appears directly related to their diminished capacity to mount Th1polarized immune responses during the early postnatal period (Holt & Sly 2002; Kimura et al. 2002). The notion that infection can serve only as a priming factor for subsequent allergic inflammation is at odds with several epidemiologic observations that, under some circumstances, infections appear to protect from allergic disease (Gerrard et al. 1976; Strachan 1989; Bråbäck et al. 1994; Martinez 1994, 1995). These studies support an alternative hypothesis that early infections may accelerate the maturation of local immune networks promoting Th1 defense responses which may override the Th2 default response in immature infants.

Summary Variations in the consequences of viral infections may be due to differences in the timing of exposure, the nature of the infectious agent, the location of the infection (upper or lower airway), as well as the genetic susceptibility of the individual. While viruses play a clear role as triggers for asthmatic symptoms in established disease, their role in disease pathogenesis remains poorly understood.

Prevention of Allergic Disease

The growing awareness of the potential importance of early microbial exposure for early immune development has prompted speculation about the role of antibiotics and other antimicrobials in the first year of life. A number of studies have suggested a possible link between antibacterial exposure in infancy and an increased risk of subsequent allergic disease (Wickens et al. 1999b; Droste et al. 2000), although other studies have failed to find any association (Ponsonby et al. 1999; Celedon et al. 2002). In a retrospective analysis, Farooqi and Hopkin (1998) investigated the putative relationship between childhood infections and atopic diseases in over 1900 children and young adults aged 12–21 years. Treatment with antibiotics in the first 2 years of life was found to be significantly associated with subsequent atopic disease (OR 2.07, 95% CI 1.64–2.60). The association was even stronger for asthma (OR 3.19, 95% CI 2.43–4.18). The study is important, as the authors analyzed the effects of both different types of antibiotics given during the first 2 years of life and the indications for treatment. Thus, the relationship was not limited to treatment of upper and lower respiratory tract infections, but was also noted for urinary tract infections for example. Importantly, the association was more marked for treatment with broadspectrum antibiotics, while no such trend was observed for infections treated with penicillin. Thus, treatment with broadspectrum antibiotics after the age of 2 years did not seem to be associated with any particularly increased risk for atopic disease. Several authors have subsequently assessed the possibility that antibiotics may be a risk factor for the development of asthma and other allergies and the results are slightly conflicting. In a study from New Zealand, antibiotic use during the first year of life was significantly associated with having a history of asthma in 5–10 year old children (OR 4.05, 95% CI 1.55–10.59) (Wickens et al. 1999b). In contrast, the use of antibiotics after the first year was only associated with a slightly increased risk for asthma, which did not reach statistical significance (OR 1.64, 95% CI 0.60–4.46). A more recent multicenter cross-sectional study of 6630 children reported that antibiotic use during the first year of life increased the risks of rhinoconjunctivitis, asthma, and atopic eczema (Flöistrup et al. 2006). Other large-scale studies include a UK study of children (N = 29 238) registered through a general practice research database. They found that receiving more than four courses of antibiotics in the first year of life was associated with an increased risk of asthma, eczema, and hay fever, but these associations were reduced (below significance in most cases) after adjusting for differences in consulting behavior (McKeever et al. 2002). However, these associations are not supported by all studies. Bremner et al. (2003) conducted a nested case–control study derived from a

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database containing 116 493 children continuously registered from birth to at least 5 years of age, of whom 7098 developed allergic rhinitis after the age of 2 years. The findings suggested that antibacterial exposure in early infancy did not have an important effect on subsequent hay fever risk. However, ordinary penicillin and broad-spectrum antibiotics were not analyzed separately. There have also been reported links between the use of antibiotics before birth in pregnancy and a higher risk of subsequent asthma and allergic disease. In a population-based cohort comprising 3000 children and their mothers it was observed that use of antibiotics in pregnancy was associated with a modest increase in asthma, as defined by usage of antiasthmatic medication during the fifth year of life in the children (OR 1.7, 95% CI 1.1–2.6) (Benn et al. 2002). There was also a similar, independent association between usage of antiasthmatic medication by the children and the presence of staphylococci in the maternal vaginal flora during pregnancy (OR 2.2, 95% CI 1.4–3.4). These bacteria are not part of the normal vaginal flora, which is normally dominated by lactic acid bacteria. The findings are interesting, as the vaginal flora is the first microbial environment that the infant comes in contact with during the birth process and thus has an impact on postnatal colonization. A larger study (N = 24 690) of children registered through the same UK general practice research database found that exposure to antibiotics in utero was associated with an increased risk of asthma in a doserelated manner (McKeever et al. 2002). Similar associations were noted for eczema and hay fever. However, it is difficult to assess potential separate effects of infectious exposures and the antibiotics used to treat these disorders.

Summary There is evidence that use of broad-spectrum antibiotics but not penicillin in the first year of life is associated with an increased risk of allergic disease, although the data are conflicting. Recommendations • The indication for prescribing antibiotics should be carefully assessed for many reasons (to avoid side effects and the emergence of antibiotic-resistant organisms). If antibiotics are clearly indicated clinically, they may be prescribed without concern that they may possibly increase the risk of allergic disease (strength of recommendation B).

Role of childcare attendance Regular daycare attendance is associated with more frequent and severe respiratory tract infections (Wald et al. 1988) and asthma exacerbations in children with established disease (Connett et al. 1994). Daycare attendance is a crude (and problematic) marker of early-life microbial exposure and there is no strong evidence for a role of daycare attendance in allergic disease development or prevention. The first study to examine

2008

this relationship (in the early 1980s) actually explored the earlier alternate hypothesis that infections acquired in daycare might increase the risk of allergic sensitization, but found no relationship (Backman et al. 1984). Subsequently, some studies suggest a protective effect of attending daycare (Kramer et al. 1999; Benn et al. 2004), while others show no benefit (Strachan et al. 1997; Svanes et al. 1999).

Summary Children attending daycare (or having other regular contact with groups of other small children) are more likely to develop upper respiratory tract infections, including otitis media, but there is no definitive evidence that this protects from, or would contribute to, the risk of allergic disease. Recommendations • Parents can be informed that their children are more likely to develop upper respiratory tract infections if they attend daycare, but that there is no clear relationship with the development of allergic disease (strength of recommendation B).

Role of early gut colonization, probiotics and prebiotics Probiotic intestinal flora are arguably the most abundant source of early immune stimulation, and contribute significantly to “microbial burden” in early life. There is good evidence from germ-free animal models that bacterial gut colonization is essential for maturation of immune function and induction of oral tolerance (Sudo et al. 1997). A number of studies have suggested differences in colonization patterns of infants who go on to develop allergic disease (Björkstén et al. 1999, 2001; Bottcher et al. 2000; Kalliomäki et al. 2001a; as reviewed by Björkstén 2004). These differences were already apparent at 1 week of age, suggesting that early colonization can influence subsequent patterns of immune development. It has been logical to explore the benefits of probiotics earlier when immune responses are still developing, and there are now a number of studies addressing the role or probiotics in primary allergy prevention. In animal (mouse) models of food anaphylaxis, the intraperitoneal administration of a Lactobacillus strain (Shida et al. 2002) induced Th1-trophic IL-12 responses and suppressed the development of IgE responses and anaphylaxis. The first human study to assess the role of probiotics in this context administered probiotics to mothers (in pregnancy) and to infants in the first 6 months of life. This was reported to reduce the incidence of eczema at 2 years by around 50% (Kalliomäki et al. 2001b). Although this effect was still evident at 4 years, there was no reduction in respiratory allergy, IgE levels, or allergic sensitization (Kalliomäki et al. 2003). Effects on underlying immune response were not reported and a number of methodologic concerns have been raised about the study (Matricardi 2002). There are now at least six other studies (in Australia, Finland, New Zealand, Singapore, Sweden and the UK) examin-

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ing the effects of various probiotic strains for allergy prevention using direct infant supplementation. Some are still in progress. In the Australian study, high-risk infants (with maternal allergic disease) were randomized to receive 3 × 109 Lactobacillus acidophilus (LAVRI 01) or placebo daily for the first 6 months of life (Taylor et al. 2007). There was no evidence that the probiotic reduced the risk of atopic dermatitis or food allergy at 12 months of age. Rather, the rates of allergic sensitization were significantly higher in the probiotic group (P = 0.03) and the prevalence of skin-prick test-positive atopic dermatitis was also higher in this group (P = 0.042). In the Swedish study, comprising 230 infants and their mothers, which used a strain of Lactobacillus reuteri, there was also no reduction in allergic disease or sensitization (although the rate of skin-prick test-positive atopic dermatitis was lower in the probiotic group) (Abrahamsson et al. 2007). In this study, pregnant women expecting a child with a family history of allergy were given 109 bacteria or placebo daily for 4 weeks before delivery; the babies then received the same product daily for 1 year. Follow-up was done at 3, 6 and 12 months and at 2 years. Probiotic treatment did not reduce the incidence of eczema or other allergic manifestations. However, there were less infants with atopic eczema (dermatitis and a positive skin-prick test) at 2 years in the infants who had been treated with probiotics. In another more recent Finnish study, 1223 pregnant women carrying children with a family history of allergy received a probiotic preparation or a placebo for 2–4 weeks before delivery (Kukkonen et al. 2006). Their infants received the same probiotics plus galacto-oligosaccharides (N = 461) or a placebo (N = 464) for 6 months. Probiotic treatment showed no effect on the incidence of all allergic diseases by age 2 years but reduced the incidence of eczema, especially atopic eczema. To our knowledge at least two of the other probiotic trials failed to show any preventive effect of probiotics on allergic disease and the publication of these data is awaited with interest. However, it is interesting to note that in the studies in which probiotics seemed to reduce some forms of eczema, the product was given to the mothers during pregnancy as well as to the infants (Kalliomäki et al. 2001b; Kukkonen et al. 2006; Abrahamsson et al. 2007). As it appears increasingly unlikely that supplementation with a single probiotic strain will be sufficient to overcome the high environmental pressure to develop allergic disease, there has been a shift in interest to dietary substrates that could potentially have a more global effect on gut flora, namely prebiotics. Prebiotics are nondigestible but fermentable oligosaccharides (food starches) that specifically stimulate the growth of Bifidobacterium and Lactobacillus species. Altering the intake of foods containing these products can directly influence the composition and activity of intestinal microflora. This could explain some of the protective effects of grains and cereals that have been seen in

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epidemiologic studies (Ellwood et al. 2001). At this stage there are still very few data to directly confirm the immunologic or therapeutic effects of prebiotic supplements, although one recent study has reported encouraging results (Moro et al. 2006).

Summary There is evidence that differences in fecal flora may be associated with allergic disease. However, supplementation with a single species may not be sufficient to reduce the risk of disease. Recommendations None.

Role of other bacterial products The potential for other bacterial products to promote Th1 immunity (and immunoregulatory pathways) have made these logical agents for allergy prevention. However, with the exception of a German study looking at the potential protective effect of endotoxin (study still in progress), these are not yet in clinical trials. As potent Th1 immunostimulants, mycobacterial antigens have been considered as vaccine candidates for both prevention and treatment of allergic disease (reviewed by Beasley et al. 2002). Mycobacteria have also been associated with improved pulmonary function in sensitized animals (Hopfenspirger & Agrawal 2002) and reduced airway inflammation (Tsai et al. 2002), eosinophilia (Major et al. 2002) and expression of adhesion molecules (Yang et al. 2002). Although the mechanisms are unclear, there is some evidence (again in mice) that mycobacteria give rise to allergen-specific regulatory cells that mediate a reduction in airway inflammation through the production of transforming growth factor (TGF)β and IL-10 (Zuany-Amorim et al. 2002). In humans, BCG vaccination has also been associated with reduced total and specific IgE in allergic individuals (Barlan et al. 2002; Cavallo et al. 2002; Ota et al. 2002) and improved lung function in asthmatics (Choi & Koh 2002). Intradermal administration of mycobacteria has previously been used therapeutically in children with existing atopic dermatis with some success (Arkwright & David 2001). In newborns, BCG and can influence immune responses to other antigens (vaccine antigens), promoting both Th1 and Th2 responses (Ota et al. 2002). However, in humans mycobacterial antigens have not yet shown any benefits in reducing the risk of disease (Strannegård et al. 1998). Other potential neonatal vaccination strategies for allergy prevention include the use of CpG oligodeoxynucleotides or plasmid DNA. Although there has been some success with these approaches in animals (Nguyen et al. 2001), there are still concerns about negative side effects particularly when vaccinating young children. This is likely to be a major factor delaying human trials.

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Summary There is good evidence from animal studies that bacterial products have immunomodulatory effects. Preliminary studies also suggest that these products may modify disease expression. Their role in allergy prevention remains to be confirmed. Further information is also needed about the effects (and safety) of other bacterial adjuvants. Recommendations None.

Role of postnatal pollutant exposure There is a clear association between parental smoking and wheezing illness in early childhood (Martinez et al. 1992, 1995b; Halken et al. 1995). The relationship between cigarette smoke exposure and atopy is less clear. While some studies have reported associations with increased risk of atopy (Weiss et al. 1985; Ronchetti et al. 1990; Wjst et al. 1994; Kulig et al. 1999), a large prospective study of asthma and wheezing in childhood found that although maternal smoking was associated with wheezing in the first 3 years of life, this was not associated with asthma and allergies at 6 years (Martinez et al. 1995b). Furthermore, in a metaanalysis of published studies on the relationship between exposure to tobacco smoke and allergy development in children, Strachan (1998) came to similar conclusions. The role of other indoor pollutants is also poorly understood. It is well established that air pollution indoors and outdoors may increase hyperreactivity and trigger symptoms in asthmatic children and adults. The use of home gas appliances has also been associated with an increased risk of HDM sensitization and subsequent respiratory symptoms (Ponsonby et al. 2001), but this needs to be confirmed, as other studies have not shown indoor pollutants to cause allergic disease.

Summary As with antenatal exposure (see above), parental smoking in the postnatal period is associated with increased respiratory tract symptoms and disease, but probably not with allergic disease. Recommendations • Parents should be advised not to smoke (strength of recommendation A). • Children should not be exposed to cigarette smoke in confined spaces (strength of recommendation A).

Dietary interventions The role of dietary nutrients has been examined in the postnatal period (see section on role in pregnancy). The Australian CAPS study (Mihrshahi et al. 2003; Peat et al. 2004; Marks et al. 2006) also included a dietary intervention. They gave n-3 PUFA fish oil supplements from around

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6 months of age. At 18 months of age they reported a 9.8% absolute reduction (95% CI 1.5–18.1; P = 0.02) in the prevalence of wheezing, but no effects on total serum IgE, sensitization to foods, or atopic dermatitis (Mihrshahi et al. 2003). At 3 years of age there was no longer any difference in wheezing, but there was a significant 10.0% (95% CI 3.7– 16.4) reduction in the prevalence of cough in atopic children in the active diet group (P = 0.003) but no significant effect in the nonatopic children (Peat et al. 2004). There was a 7.2% (95% CI 10.11–14.3) reduction in sensitization to HDM in the active allergen avoidance group (P = 0.05). However, by 5 years of age, there was no evidence of any difference in the prevalence of asthma, wheezing, atopic dermatitis, or sensitization between the groups (Marks et al. 2006). The role of other vitamins and dietary supplements is not known at this stage. One recent population-based study of over 8000 patients in the USA found that multivitamin supplements in early infancy (first 6 months of life) was associated with an increased risk of asthma (OR 1.27, 95% CI 1.04–1.56), although this was only seen in African American infants (Milner et al. 2004). Food allergies were also more common with vitamin supplementation (OR 1.63, 95% CI 1.21–2.20), but only in infants who were exclusively formula fed (Milner et al. 2004). Further studies are needed.

Summary Although there have been epidemiologic associations between dietary factors including n-3 PUFA (Peat et al. 1992), there have been few intervention studies to address the effects of supplementation. The only study to do this so far has not shown any long-term benefits. Recommendations None.

Prevention of disease progression in children with early sensitization or disease Existing strategies to prevent allergies are relatively ineffective and a significant proportion of children with a family history of allergy will still develop disease. If these children (discussed above in section on targeting children with early disease) can be identified when they have early disease, future strategies may also provide avenues of reducing the risk of new sensitizations and progression to other persistent forms of disease (in the “atopic march”) and/or reducing the severity of disease. A number of recent observations justify further investigation in this area of “secondary” prevention. Möller et al. (2002) observed that children who are treated with specific (pollen) immunotherapy for allergic rhinitis are significantly less likely to develop asthma than those who do not receive active treatment. Immunotherapy can also reduce the devel-

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opment of new sensitizations in patients sensitized to a single aeroallergen (HDM) (Pajno et al. 2001). Other interventions, such as the use of antihistamines in children with early disease, are also being investigated because of some preliminary evidence that this may modify disease progression (Warner 2001). In this study infants with atopic dermatitis were treated with cetirizine between 1 and 2 years of age. A subgroup analysis showed that children with aeroallergen sensitivity who were treated with cetirizine were significantly less likely to have asthma compared with those treated with placebo over the 18 months of treatment. As this was a post hoc analysis, no conclusions can be drawn from the study. The latest approach in this area is the use of sublingual immunotherapy (SLIT) in the prevention of aeroallergen sensitization in children with food allergy. This study commenced in 2006 (as discussed in the section below on future directions) and the results are awaited with great interest.

Summary There is preliminary evidence that early immune interventions in allergic children could modify the development of new allergic diseases (level of evidence 1b). Recommendations • Allergy vaccination may be considered in children with allergic rhinoconjunctivitis as a measure to prevent the development of asthma (strength of recommendation B).

Overview of current preventive strategies Table 97.2 provides a summary of current proposed recommendations based on the current evidence (as of 2006). The conclusion of this summary is that there are currently no specific allergy-preventive measures that would meet the same stringent criteria as those required for other medical therapies and interventions. Most previous approaches to allergy prevention were based on avoiding candidate factors that could be implicated in allergic disease, including allergens and environmental pollutants. These have had limited success as summarized above. More recent strategies include the use of immunomodulatory factors such as probiotics, prebiotics, and dietary nutrients (such as n-3 PUFA), although data are still limited and there is still insufficient evidence to make specific recommendations. There are also several studies (not yet completed) that are examining whether early allergen administration (rather than avoidance) may have a role in promoting the development of tolerance. These include the early administration of peanut allergen or inhalant allergens before any evidence of sensitization, with the aim of inducing immune tolerance. While these areas hold promise, it will be many years before the results of current studies are known.

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General discussion How do current strategies meet our objectives? Until the late 1970s textbooks in pediatrics or allergology did not discuss allergy prevention, or only mentioned it in passing. The increasing awareness of environmental pollution and the continuing increase in the prevalence of allergies brought public attention to environmental factors that could explain the increase. Soon the medical community became interested in the potential to prevent allergic disease by modifying the environment. Air pollution and allergen exposure were the two obvious suspects. The lesson learnt from 20 years of epidemiologic analyses, observational studies and recent intervention studies show that strategies for allergy prevention have not been successful so far. The avoidance of allergenic foods during the first 4–6 months of age is the only advice with a confirmed allergy-preventive effect, i.e., less infant eczema and food allergy, that has been shown in several intervention studies. Other advice with general health-promoting effects include breast-feeding and avoidance of exposure to tobacco smoke. Despite the lack of evidence of efficacy, advice is often given to parents. This is to some extent understandable, as families with allergic family members are often desperate for advice and medical staff often feel obliged to “say something.” It can also be argued that certain advice, such as avoidance of tobacco smoke and the value of clean indoor air and breast-feeding, is good in general health terms. This reasoning raises ethical concerns, however, as even seemingly innocent advice may have social, economic, and psychological consequences for the family. Recent studies indicate that some advice may possibly even increase the risk for allergy development. For many years families with a history of allergy expecting a baby were strongly advised against keeping furred pets. Avoiding pets is strongly indicated as part of the treatment of asthmatic pet-allergic patients and it was assumed that it would also be beneficial as a preventive measure. In recent years, focus has switched from researching environmental risk factors toward an interest in factors that could induce and maintain tolerance to allergens. Thus, current research is more directed toward an understanding of how immune regulation develops and how tolerance could be induced early in life.

Future directions The use of allergen vaccines/preventive immunotherapy has been long proposed as one method of primary prevention (Holt 1994). Potential strategies involve utilizing and enhancing the natural processes, which in most cases efficiently terminate IgE responses to allergens in infancy. Accordingly, vaccines for primary prevention would need to be administered in early infancy, when immune responses are still “plastic” and not “committed.” In murine systems neonatal

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Table 97.2 Summary of recommendations for allergy prevention. Measure

Conclusion of studies

Advice to parents

Identifying infants at risk of allergic disease

A family history of allergy and asthma may identify children at increased risk of allergic disease. The sensitivity is low

This is usually of no practical consequence

Allergen avoidance in pregnancy

Dietary restrictions in pregnancy are not recommended Aeroallergen avoidance in pregnancy has not been shown to reduce allergic disease, and is not recommended

A normal balanced diet is optimal for the baby. No need to remove any pets

Breast-feeding

Results are controversial but any preventive effect is small

Breast-feeding has many advantages, including less infections but does not prevent allergy

Infant formulas

Extensively hydrolyzed formulas reduce the incidence of food allergy and eczema (but not respiratory allergies) compared with standard formulas Soy and other (e.g., goat’s milk) formulas are not superior to regular formula for the reduction of food allergy risk

If you are very concerned about allergy and are not breast-feeding, then a formula with extensively hydrolyzed proteins may be considered

Infant diet

Complementary foods (including normal cow’s milk formulas) may be delayed for 4–6 months in infants with a family history of allergic disease There is no evidence that an elimination diet after the age of 6 months provides a protective effect Peanut, nut, and shellfish avoidance in infants, even those with a family history of allergy, during the first years of life is based on theoretical concepts rather than clinical studies

No particular measures required

House-dust mite (HDM) exposure

Reducing HDM levels does not reduce the risk of allergic disease

No particular measures required

Pet exposure

Pet exposure in early infancy does not increase the risk of allergy, and may even have a protective effect in some situations Children with confirmed allergy to pets should avoid exposure

No need to remove any pets unless your child has developed a confirmed allergy giving clinical symptoms

Smoking and other irritants

Pregnant women should be advised not to smoke in pregnancy Parents should be advised not to smoke

Smoking during pregnancy and smoke exposure of your baby are associated with many negative outcomes, including lung health

Role of microbial agents

The role of early childhood exposure to microbial products and infections in allergy development is unclear

Neither excessive cleaning of your home nor exposure to dirt are recommended

Dietary nutrients

Potential benefits of dietary supplements (such as n-3 PUFA) in allergy prevention is unconfirmed Potential risks of dietary supplements (such as vitamins) for allergy development is unconfirmed

Your baby should have a normal balanced diet that will secure his/her growth

Vaccinations

Vaccines are neither protective nor risk factors in relation to allergy development

Your baby should participate in the vaccination program as recommended in your country

Prebiotics and probiotics

Probiotic strains of Lactobacillus have in some, but not all, studies modestly reduced the incidence of some forms of eczema, when given to mothers before birth and then to the infants

Probiotic products have not been documented for allergy prevention. As a modest effect against infant eczema has been observed in some studies and they appear to be safe, it is your decision

administration of allergen can inhibit the development of Th2-type airways disease, but the dose and delivery method appear crucial (Hogan et al. 1998). The enteric mucosal immune system plays an extremely efficient and pivotal role in the development of tolerance.

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Repeated exposure to allergen through the gastrointestinal tract during this period of life leads to the development of tolerance, even in highly atopic individuals (reviewed by Holt 1994). It is proposed that exposure to aeroallergens through this route may promote local (IgA) immune responses, which

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promote persistent systemic tolerance and prevent the emergence of pathogenic Th2-responsive memory T cells. A number of studies are currently addressing the effects of these strategies in humans, including the effects of intranasal and sublingual administration of allergen that may theoretically have similar benefits. There is preliminary evidence that SLIT may reduce the risk of asthma developing in children with allergic rhinitis (Novembre et al. 2004). Novel studies are now planned to examine the effects of SLIT for prevention of aeroallergen sensitization. In this proposed randomized controlled trial 200 children (aged 18 months to 3 years and at high risk of developing allergic respiratory disease) will receive inhalant allergen SLIT (HDM, cat and timothy grass) or a placebo for 12 months. These children will already have evidence of allergic disease (food allergy or atopic dermatitis) but no sensitization to inhalants. The children will be monitored for 3 years, aiming to detect a 50% reduction in IgE and allergic Th2 responses to allergens, as well as a 50% reduction in asthma (http://www.immunetolerance.org/research/ allergy/trials/holt.html). The results are awaited with great interest.

Concluding comments There is a growing need to reduce the mounting personal, social and economic cost of allergic disease. While there has been some success in managing established disease, strategies to prevent the development of these processes will be of greater value in the long term. Currently, our capacity to prevent allergic disease is constrained by limited understanding of disease pathogenesis and etiologic factors, particularly of the early exposures responsible for the recent increase in allergic disease. There is also an inability to accurately identify atopic individuals before sensitization occurs. All these areas need to be investigated more fully in order to determine how tolerance mechanisms can be promoted without adverse effects.

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Prevalence of Atopic Disorders in a Developing World: Pitfalls and Opportunities Maria Yazdanbakhsh, Taniawati Supali and Laura C. Rodrigues

Summary Most studies of atopic disorders have been conducted in the so-called Western countries using tools developed in these regions. There is increasing interest in a more global view of allergies, which would enable us to estimate the burden of these disorders in developing countries and to identify risk and protective factors associated with the development of allergies in a wider geographical area. This chapter highlights how extending epidemiologic and mechanistic studies to Asia, Africa and Latin America would benefit not only health planners but also those interested in developing effective and novel control measures against allergic disorders.

Introduction Many studies have documented that the prevalence of atopy has been increasing in industrialized developed countries, leading to an increase in asthma, rhinitis and eczema (Eder et al. 2006). In fact, the prevalence of these allergic disorders is increasing among affluent people not only in developed but also in developing countries (Weinberg 2000; von Hertzen & Haahtela 2004). The geographic variation in the prevalence of allergic diseases suggests that a rural lifestyle confers protection against allergic disorders, whereas urbanization appears to be an important risk factor for diseases such as asthma and atopic dermatitis. In rural Ethiopia (Yemaneberhan et al. 1997, 2004), South Africa (Steinman et al. 2003), India (Vedanthan et al. 2006), Kenya (Ng’ang’a et al. 1998), Gambia (Nyan et al. 2001) and also among traditional rural farmers in parts of Europe (Germany, eastern Europe), there is little allergy compared with major urban centers (Von Ehrenstein et al. 2000). Understanding the cause of the change in prevalence of allergies may help us understand the risk factors, not only at the environmental but also at the mechanistic level, that

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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are responsible for the so-called “allergic march” worldwide (Wahn 2000). This knowledge could be applied to stop, or reverse, the worldwide allergy epidemic. This chapter focuses on one of the environmental factors, infections, that may have played an important role in keeping the prevalence of allergic disorders low. Infections covered will be those that are highly prevalent in developing countries. The associations between such infections and prevalence of allergic disorders is discussed at the epidemiologic level but also at the level of the immune system to emphasize the importance of studying allergies in areas where there are strong dynamic changes in lifestyle and environment.

International studies of asthma and other atopic disorders: geographic variation Most studies published on the prevalence of allergies are based on data collected via questionnaires on disease symptoms using standardized documents and methodologies developed by ISAAC (International Study of Asthma and Allergies in Childhood; http://isaac.auckland.ac.nz) or by ECRHS (European Community Respiratory Health Survey). A few of these studies have also included objective measurements of allergy, such as skin-prick test (SPT) to allergens or exercise-induced bronchospasm (EIB). Several large studies involving many centers in diverse geographic locations have reported great variation in the prevalence of atopic disorders. ECRHS has estimated the prevalence of asthma and atopy in 140 000 adults in many centers worldwide with particular focus on European, North American, Australian and New Zealand populations. It reported higher prevalence of allergic disorders in Englishspeaking countries but lower prevalence in Mediterranean and eastern European countries (Janson et al. 2001). ISAAC has set out to examine the international prevalence patterns of asthma, allergic rhinoconjunctivitis, and eczema in children aged 6–8 years and 12–14 years. Phase I of ISAAC was conducted in 156 centers in 56 countries using a standard questionnaire which collected data from 700 000 children. Of the participating countries, six in Africa, 18 in Asia (including the Middle East), and nine in Latin America were

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included. The results indicated that the highest prevalence of symptoms of asthma (wheeze in past 12 months) was found in countries such as the UK (18.4%), New Zealand (29.7%), Ireland (33%), and the USA (22.9%), and lowest among others in Indonesia (2.1%), Albania (2.6%), or China (average 5%) (ISAAC 1998). On the whole, it appeared that most developing countries tended to have lower prevalence of symptoms of allergic disorders compared with countries with full economic development. However, it was also noted that there was great variation in the reported prevalences in developing country groups. For example, in some centers in Latin America, a high prevalence of allergies was reported (Mallol et al. 2000) whereas in most Asian centers this tended to be low (Asher et al. 2006). Highlighting the situation within one developing country, such as India, it appeared that the prevalence of asthma symptoms reported varied between 2 and 20% depending on geographic area in the country (ISAAC 1998). Ecologic analysis of ISAAC phase I data has indicated that in addition to affluence (Stewart et al. 2001) and dietary factors (Weiland et al. 1999), infections (von Mutius et al. 2000) might play a role in the variations recorded. In phase II of ISAAC, a more in-depth investigation of the causes of allergies, was instigated in 30 centers within 22 countries. Here, in addition to questionnaires, objective measurements of atopy such as SPT and IgE measurements were included in the surveys (Weiland et al. 2004). In this phase of ISAAC (http://isaac.auckland.ac.nz/Phasetwo/Phs2Frame .html), very few centers in developing countries could participate (total of seven non-European countries). There are as yet very few data published from this phase II study. In phase III of ISAAC was designed to study the changes over time in prevalence of asthma, allergic rhinoconjunctivitis, and eczema using the standard ISAAC questionnaire. The study results have been published to show the time trends for the symptoms of these disorders in 6–7-year-old children (N = 193 404) in 66 centers within 37 countries and in 13–14-year-old children (N = 304 679) in 106 centers in 56 countries. The average time between phase I and phase III was 7 years. A wealth of data was generated from many developing country centers (Asher et al. 2006). The data show not only increases but also decreases in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema. The data indicate that centers where prevalence of the three disorders was high in the phase I study tend to show either no change or a decrease, whereas those with low prevalences are more likely to show an increase in allergy symptoms. The only centers that showed an increase in all three symptoms were in Asia-Pacific and India. This was followed by centers in eastern Euorope, Africa and Asia, which tended to report more often an increase than a decrease in a symptom over the study period. The strengths of the ISAAC study are the sheer numbers of children that participate and the use of the same questionnaire to enable comparisons. It has provided valuable data for health officials in many countries,

which can use the data to plan their health services accordingly. However, the limitations of this approach need to be considered when the data are used to investigate risk factors and the mechanisms underlying the change in prevalence of asthma. Some of the limitations include the overrepresentation of urban centers (particularly from developing countries) and the use of a questionnaire of reported symptoms in the absence of “objective” measurements of the diseases studied. These issues are discussed later in this chapter. Affiliated to ISAAC studies or outside it, there are numerous data generated from surveys in developing countries where the prevalence of allergic diseases has been estimated. For example, during ISAAC phase III study in Africa, in addition to the six countries already involved since ISAAC I, another 10 countries estimated the prevalence of symptoms of allergic disorders using the questionnaire. These centers could not be included in the time trend analysis but nevertheless provided interesting data on the point prevalence of asthma, allergic rhinoconjunctivitus, and eczema in areas where very little information on these disorders has been available (Ait-Khaled et al. 2007). Again these studies have shown marked variation not only between countries but also between centers in the same country. Current wheeze varied between 5.7% in Yaounde in Cameroon and 21.5% in Réunion Island (Ait-Khaled et al. 2007); within Morocco, rates varied from 6.4% in Marrakech to 16% in Casablanca (Bouayad et al. 2006).

Differences in prevalence of allergic diseases in rural and urban communities The prevalence of atopy and atopic diseases has been reported to vary between urban and rural populations. Within Europe, the prevalence of these disorders tends to be lower in rural areas with traditional lifestyles (Priftanji et al. 2001), in children raised on traditional farms (Riedler et al. 2001), and in Scotland (Iversen et al. 2005). The difference in prevalence of allergic disorders in rural and urban areas has often been reported for developing countries. Higher prevalences of atopic disorders have been documented in urban centers of South Africa, not only more than 25 years ago (Van Niekerk et al. 1979) but also more recently (Steinman et al. 2003). Similarly, recent studies in Ethiopia (Yemaneberhan et al. 1997), Ghana (Addo-Yobo et al. 2007), India (Vedanthan et al. 2006), Gambia (Nyan et al. 2001), Kenya (Ng’ang’a et al. 1998; Perzanowski et al. 2002), and China (Wong et al. 2004) have confirmed that higher prevalences are found in more urbanized areas compared with more rural ones. However, although most studies have shown higher prevalences of symptoms of allergic diseases or EIB with an increasing rural to urban gradient, the distribution of SPT results have shown a less clear pattern. For example, in Ethiopia, the prevalence of SPT to house-dust mite and cockroach was significantly

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higher in rural than in urban areas even though the prevalence of wheeze was lower in the rural communities (Scrivener et al. 2001). In addition, recent results gathered in Indonesia (T. Supali et al., unpublished data) have indicated that there might be no difference in prevalences of SPT to certain aeroallergens and foods in rural areas in Indonesia compared with more urbanized centers. A neglected issue is the lack of any standardized classification of what is “urban” and what is “rural” in an increasingly urbanizing world particularly in developing countries. The definitions can vary from population density to the level of infrastructure available (Fotso 2006). It is essential to record in detail the characteristics of areas under study to be able to identify the “factors” present in rural areas that may lead to the lower prevalence of allergic diseases.

Atopy and allergic diseases are not the same Atopy is usually diagnosed as either high levels of allergenspecific IgE or skin-test positivity to allergens, but they are not always perfectly correlated, neither in Europe (Annus et al. 2001) nor in developing countries (van Den Biggelaar et al. 2001). The relationship between diagnosed atopy and diagnosed allergic disease is also not simple. A negative SPT may result not from absence of atopy but from the use of wrong allergens for a particular study site. Moreover, a very

Not allergic Allergens Early exposures (permanent effect)

Early exposure to infections leading to immune programming with strong regulation

Allergens

High allergen-specific IgE

Positive skin test to allergen

Asthma

Current exposures (removable effect)

Current exposure to infections leading to downregulation

Atopic diseases Eczema Rhinitis

Fig. 98.1 Proposed modifying effect of environmental factors, such as infections, on the sequence of events that follows allergen exposure and the development of atopy and atopic disease. Infections modify the association between levels of allergen-specific IgE and skin-test positivity, and between skin-test positivity and clinical disease. There are two patterns: infection during early programming leads to a long-lasting effect that remains after removal of helminths; and current infection leads to an effect that is eliminated with removal of the infection. (See CD-ROM for color version.)

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important issue is that atopy is not always accompanied by asthma or eczema, and not all asthma and eczema is atopic. It is likely that the proportions of atopic subjects that develop clinical disease varies, and a change in this proportion may well have contributed to the allergic march (Fig. 98.1). In Western countries, the SPT has a relatively high predictive value for clinical allergy, and the proportion of asthma cases in children in industrialized countries with a positive SPT ranges from 30 to 57% (Pearce et al. 1999), whereas in Indonesia this proportion ranges up to 15% (S. Wahyuni et al., unpublished data). It is clear that allergen induction of mast cell degranulation (leading to a positive SPT result) does not always lead to overt inflammation in affected tissues (e.g., in the lungs). Whether this is related to a local tissue physiologic or immunologic change leading to a different threshold of reactivity to inflammatory signals, in populations that are often exposed to infectious agents, is not yet known. Further research on the factors regulating the relationship between allergic sensitization and clinical manifestation (Fig. 98.1) is much needed.

Tools used to assess prevalence of atopic disorders and of atopy and their drawbacks in international studies Different tools are used to identify atopy (levels of IgE to allergen, SPT to allergen) and allergic diseases (questionnaires, clinical investigations). Below we review some of the instruments used in international epidemiologic studies to identify both atopy and allergic diseases.

Questionnaires It is indisputable that simple tools are needed to estimate the prevalence of allergies in large epidemiologic studies, and questionnaires are the method of choice. The most widely used standardized questionnaire for allergy symptoms originates from ISAAC. Much effort has gone into standardization of the ISAAC questionnaire, which has been validated in a number of different ethnic and cultural settings and reported to be adequate for distinguishing cases from noncases (BraunFahrlander et al. 1997; Gibson et al. 2000; Chan et al. 2001; Vanna et al. 2001; Haileamlak et al. 2005). Although the questionnaire used is the same across the ISAAC studies, in the absence of an objective measure its validity is likely to be influenced by issues such as culturally sensitive interpretation of the questions, awareness of disease, and symptom management. Definitions of symptoms can be problematic across language and cultural barriers, for example there is no word for wheeze in the German language. Many more of the 56 countries included in ISAAC surveys would be expected not to have the “right” words to describe many of the symptoms of atopic disorders. Indeed, perception of wheeze is highly variable and dependent on, among other factors, the

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level of education and access to health systems (Crane et al. 2003). Given that questionnaires are highly sensitive to socioeconomic and cultural variation, their use in developing countries would be particularly problematic as these countries often exhibit great variation in socioeconomic status and also enormous variation in ethnic groups and therefore local cultures and languages. Questions asking about “ever wheeze” or “wheeze in the past 12 months” or “doctor-diagnosed asthma” as well as bronchial hyperreactivity after a 6-min run have given different prevalences of asthma in schoolchildren in Indonesia, with limited sensitivity and predictive values (S. Wahyuni, unpublished data). Overall, the important question of how we should define asthma in epidemiologic studies is still awaiting a unified answer (Crane et al. 2003), bearing in mind that severe asthma is more likely to be diagnosed than mild disease. Moreover, use of medication might influence the accurate capture of asthma via questionnaires in two ways: it might mask the presence of disease but it may also allow detection of the disease if data can be acquired on drug intake. An extra worry in developing countries is that wheezing can be caused by viral and bacterial infections of the respiratory tract rather than by asthma. The relative proportion of wheezing in the last 12 months attributable to asthma may therefore vary geographically, and be smaller in developing countries, where infections are rampant. Efforts to improve the performance of questionnaires has resulted in the development of a video questionnaire, where symptoms of asthma are visually presented in an attempt to overcome the language barrier (Shaw et al. 1995). In some African centers, the prevalence of symptoms of asthma acquired using the standardized ISAAC questionnaire and the video questionnaire was compared (Ait-Khaled et al. 2007). Not surprisingly, less variation was found in the symptoms of asthma between different centers using the video questionnaire compared with the standard questionnaire (Ait-Khaled et al. 2007). It might seem trivial, but it is important to use individuals of different race and color in the videos to depict different aspects of asthma symptoms. The other important symptoms often studied are allergic rhinoconjunctivitus and eczema. Their diagnosis is even more problematic using questionnaires in developing countries. In a study carried out in Ethiopia to establish the prevalence of eczema in children, it was concluded that neither the ISAAC questionnaire nor the Hanifin and Rajka (UK) diagnostic criteria perform adequately in identifying cases (Haileamlak et al. 2005). The complication of differential diagnosis applies to both allergic rhinoconjunctivitus and eczema. Fungal infections and scabies, both frequent in many developing countries, can cause itchy rash and may therefore be mistaken for eczema. Equally frequently, bacterial and viral infections lead to a runny nose which could therefore be mistaken for rhinitis symptoms. Fortunately, in many developing countries, medical staff are highly skilled at recognizing infectious diseases and it would therefore be important to ensure

that consultation with such skilled medical teams takes place when designing locally adapted questionnaires. Given the problems with the use of questionnaires, it is important to consider using more “objective” parameters associated with allergic disorders. However, it has to be realized that in developing countries, with limited resources, the cost of such tests is an important consideration.

Lung function tests Additional diagnostic methods for diagnosing asthma, such as measurement of peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV1), forced vital capacity (FCV), and EIB have been used in several epidemiologic studies in Africa (Addo-Yobo et al. 2002; Calvert & Burney 2005). Many of these studies follow protocols and guidelines established by the European Respiratory Society or the American Thoracic Society. However, there is a need to determine whether reference values of healthy populations from affluent industrialized countries are valid for other ethnic groups and/or different socioeconomic backgrounds (McKenzie et al. 2002). More complicated and time-consuming are tests that use challenges such as methacholine, histamine, isotonic saline, or even allergen. These tests have seldom been used in largescale studies in developing countries (Steinman et al. 2003).

Tools for the diagnosis of atopy The presence of IgE antibodies to allergens is a strong predictor of allergic disorders in Western countries. The presence of allergen-specific IgE antibodies is commonly referred to as allergen sensitization and can be assessed by direct measurement or by SPT.

Skin-prick test The SPT for allergic sensitization consists of topical application of one (or more commonly many) allergens followed by a small subcutaneous incision that allows the allergen to reach any allergen-specific IgE bound to Fcε receptors on mast cells in the skin. Triggering of mast cell degranulation and histamine release leads to a wheal and flare reaction in the skin. However, if allergen-specific IgE antibodies are not represented among the IgE bound to surface of mast cells, there will be no skin reaction. Most standardized tests measure the average of the two largest perpendicular diameters of the wheal and if the diameter is 3 mm or more, the test is considered positive (Carr 2006) after considering the positive reaction to histamine and the negative reaction to the diluent. There are two relevant methodologic issues in the interpretation of atopy surveys in different geographical settings: the choice of allergens and the choice of a cutoff point. It is likely that the relative importance of different allergens varies geographically. It is conceivable that only a fraction of relevant allergens are used in SPTs in developing countries where little information is available on local allergens of importance. Thus it is essential to be aware of the importance of

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identifying local allergens and using them in international studies. For example, many of the studies mentioned above (Kenya, Ghana, India) that used SPT in rural/urban comparisons and which showed higher prevalences in urban centers did not include cockroach in their panel of allergens; in contrast, in areas where cockroach was used, for example Ethiopia, different patterns of SPT results between rural and urban areas were seen. The second and more complex methodologic issue is the choice of cutoff value for SPT positivity and whether this should vary geographically. The current value (3 mm) was determined from studies in Western populations. The question of what should be the best choice for a cutoff point for developing countries is not simple. If there are factors that influence the relationship between allergen-specific IgE, SPT positivity, and clinical manifestation, would the “right” cutoff point be based on a certain IgE level or on the proportion of children that develop allergic disease? A report from South Africa suggested that for black skin the cutoff point for a positive SPT should be 5 mm (Steinman et al. 2003). No data were presented to justify or substantiate this statement. Therefore a discussion is needed on this issue of SPT as a diagnostic marker for atopy in developing countries. Finally, an additional point to consider carefully is the variation in SPT values that can result from the methods used by individual skin-prick testers. It is therefore important, particularly for epidemiologic studies, that considerable attention is paid to standardized training of the individuals who will be performing the tests to ensure uniformity.

IgE antibodies: in vitro measurements Another method of assessing the presence of allergen-specific IgE is the laboratory measurement of allergen-specific IgE by either radioallergosorbent test (RAST) or the increasingly used commercial Immuno-CAP method (Bousquet et al. 1990). The diagnostic value of determining levels of IgE in developing countries is complicated by the fact that in many areas of the developing world, particularly in rural areas, parasitic helminth infections are highly prevalent. These infections are the strongest natural stimuli for IgE production (Maizels & Yazdanbakhsh 2003). Therefore total IgE levels can be extremely high in populations residing in areas where parasitic infections are prevalent (Fig. 98.2). Moreover, it is possible that due to cross-reactivity between environmental allergens and parasitic antigens, high levels of allergen-specific IgE are found. One of the concerns about the use of serum IgE testing as a diagnostic tool is that allergen-specific IgE in parasitized children is often not accompanied by a positive SPT (van den Biggelaar et al. 2001). This is in contrast to the findings in Western countries where positive IgE to housedust mite could lead to 20-fold higher risk of having a positive SPT compared with sixfold in children in Gabon (J. van der Zee, A. van den Biggelaar unpublished results). The question of why allergen-specific IgE is detected but no skin

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100 Normal % of the population

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Helminth infections 50 Rhinitis Asthma 0

10

Atopic dermatitis

1000 100 Total IgE (IU/mL)

10 000

Fig. 98.2 Levels of total IgE measured in serum samples. In Western countries, nonallergic individuals have normal total IgE levels of up to 100 IU/mL. These values are higher in patients with rhinitis, asthma, and atopic dermatitis. In areas where helminth infections are endemic, the total IgE level in the population is high irrespective of whether individuals have any allergic disorders. (See CD-ROM for color version.)

reactivity to an allergen is found in helminth-infected subjects has yet to be answered with certainty.

Relationship between IgE levels and SPT in developing countries The discrepancy between the presence of specific IgE and a positive SPT may result from the difference in biological activity of the IgE antibodies. The biological activity of IgE can be assessed by the in vitro basophil histamine release assay (van Ree et al. 2006). This assay can determine what concentration of allergen is needed to induce histamine release from basophils, which have IgE from a sensitized subject bound on their Fcε receptors. Preliminary experiments have demonstrated that histamine release from basophils bearing allergen-specific IgE of children with chronic helminth infections is only observed at extremely high allergen concentrations compared with when basophils bear allergen-specific IgE of subjects from Europe (Ronald van Ree, unpublished data). This suggests that the anti-allergen IgE in helminth-infected subjects may have poor avidity and therefore would need much higher concentrations of allergen before being triggered to start the cascade of allergic inflammation (Fig. 98.3a). The lower avidity of the IgE might be due to the fact that the IgE in helminth-infected individuals represents cross-reactive antibodies to allergen- and parasite-derived molecules and, in analogy with cross-reactive IgE antibodies to tree pollen and food allergens, has little functional significance (van Ree 2004). The molecular events within B cells that lead to lowaffinity antibodies under situations of cross-reactive epitopes or under conditions of high microbial/allergen exposure (Aalberse & Platts-Mills 2004) have yet to be fully defined. Another reason why despite the presence of IgE, often no skin reactivity to allergen is found in helminth-infected children is that suppressor molecules interfere with the allergen-

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Allergen

Mast cell degranulation Histamine, leukotrienes, prostaglandins

B cell with low-affinity allergen-specific IgE B cell with high-affinity allergen-specific IgE

INFLAMMATION

Epithelial/parenchymal cells

Mucus

Goblet cell Smooth muscle cell Allergen

Smooth muscle cell contraction

(a)

Allergen

Mast cell degranulation Histamine, leukotrienes, prostaglandins

Nonspecific IgE Allergen-specific IgE Epithelial/parenchymal cells

INFLAMMATION

Goblet cell

Mucus

Smooth muscle cell Allergen (b)

Smooth muscle cell contraction

Regulatory APC IL-10

Allergen

Regulatory Breg B cell Regulatory T cell

Treg

Immunosuppressory molecule such as IL-10 Allergen-specific IgE Epithelial/parenchymal cells

Histamine, leukotrienes, prostaglandins

INFLAMMATION Mucus

Smooth muscle cell Allergen

Exposure to microorganisms and parasites: risk or protective factor? During the course of evolution humans have constantly been exposed to a wide variety of microorganisms and parasites, ranging from harmless to life-threatening ones. Our immune system has evolved to counteract dangerous infections but to tolerate or even to depend on harmless ones. For example, the gut microflora is essential for the normal development of the immune system, as shown in studies of animals raised under germ-free conditions (Ostman et al. 2006). Elegant studies of the interaction of the immune system with molecular patterns present on bacteria in the gut have shown that bacterial polysaccharide, for example, is essential for the development of a fully functional adaptive immune system (Mazmanian et al. 2005). In line with this, there has been

Mast cell degranulation

Goblet cell

(c)

induced histamine release from tissue mast cells. As early as the 1970s it was suggested that high levels of total IgE in helminth-infected subjects may saturate the Fcε receptors on mast cells and thus compete with allergen-specific IgE (Fig. 98.3b). Given the extremely high levels of IgE found in helminth-infected subjects, this sounds plausible and although there is some evidence for this (Larrick et al. 1983) recent studies during trials with anti-IgE have shown that Fcε receptors on mast cells and basophils are upregulated and downregulated with changing levels of serum IgE (MacGlashan et al. 1997) and therefore it might be difficult to saturate these receptors even when high levels of total IgE are present in serum (Mitre et al. 2005). The other mechanisms whereby mast cell degranulation might be inhibited is via the activity of suppressor molecules such as interleukin (IL)-10. The inhibitory cytokine IL-10 is upregulated during chronic helminth infections and has been shown to be able to inhibit basophil degranulation (Fig. 98.3c) (Royer et al. 2001). The question is whether high levels of IL-10 are indeed present in tissues where mast cells reside, how they are induced, and what is the source of this IL-10. These are issues that need to be addressed before this model is accepted.

Smooth muscle cell contraction

Fig 98.3 Proposed mechanisms whereby Th2 responses do not lead to allergic disease. (a) When the affinity of IgE for allergen is low, the binding of allergen to IgE antibody does not lead to mast cell degranulation. It is possible that during helminth infections, low-affinity IgE antibodies to allergens are generated, explaining the absence of skin-prick test responses to an allergen to which IgE antibodies are found. (b) The presence of high levels of nonspecific total IgE could compete with allergen-specific IgE to bind to Fce receptors present on mast cells. (c) The presence of a strong regulatory network, i.e., regulatory antigen-presenting cells (APC), regulatory B cells or regulatory T cells, can lead to the production of suppressor molecules such as IL-10, which prevents mast cell degranulation or inflammation in the target tissue. (See CD-ROM for color version.)

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much interest in, and controversy over, the relationship between infections and allergies.

Population studies The hygiene hypothesis was initially proposed by David Strachan when he observed that in a British cohort study allergic rhinitis was more prevalent among first-borns and less frequent in children from large families or children from families with lower socioeconomic status (Strachan 1989). He suggested that frequent exchange of childhood infections among siblings in large and less affluent families was responsible for the lower incidence of allergic diseases. Numerous studies have examined the relationship between infections and allergic disorders using approximations to measure infections. Here this chapter will only consider a few that have used markers of infections or direct measurement of infectious agents to assess the relationship between infections and allergies. Antibodies to hepatitis A, Toxoplasma gondii or Helicobacter pylori (Matricardi et al. 1997) as well as cellular responses to mycobacterial antigens (Shirakawa et al. 1997) were shown to be inversely associated with prevalence of asthma or SPT positivity. The only studies that have related the actual presence of infections to allergic disorders are those measuring viral or parasitic infections in the cohorts studied. Whereas respiratory viral infections have been shown to be associated with exacerbation of asthma (Mallia & Johnston 2006), parasitic helminth infections have often been shown to be negatively associated with allergic disorders (Yazdanbakhsh et al. 2002). However, this is thought to depend on the intensity and chronicity of these infections (Yazdanbakhsh et al. 2001) as a number of studies have indicated that helminth infections increase the risk of allergic sensitization, atopy and symptoms (Palmer et al. 2002; Daschner et al. 2005; Hunninghake et al. 2007). In a recent metaanalysis of published data on the relationship between intestinal helminth infections (Ascaris, Trichuris, and hookworms) and allergies, no consistent role for nematode infections in preventing allergic asthma was found, although hookworm infections did seem to show some protective effect (Leonardi-Bee et al. 2006). In the metaanalysis, all studies were included that reported parasite infection in at least 1% of the study population. This led to enormous variation in helminth intensities, in dominant species of helminths involved, and in the populations studied in terms of genetics and environmental exposures. It is known that in some endemic areas close to 100% of the inhabitants can be infected, representing intense and chronic helminth infections, while in others helminth infections can occur sporadically and in a small proportion of the population and in low intensities. Moreover, there might be variation in the ability of different species of helminth to influence allergic reactions. Ascaris and Trichuris infections have largely an intestinal passage/residence; hookworms have an interstitial migratory phase, whereas filarial nematodes and schistosomal trematodes cause systemic infections.

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It is possible that depending on the tissues inhabited by a parasitic worm, the effect on the immune system and allergic reactivities would vary. In this respect, several studies examining the relationship between schistosome infections and allergies have been consistent in showing a suppressive role for this infection on allergic disorders (Araujo et al. 2000; van den Biggelaar et al. 2000; Medeiros et al. 2003). Short-term application of anthelminthic drugs (12 months) in Ecuador (Cooper et al. 2006) did not change the prevalence or atopy nor of clinical signs of allergy (wheeze) in comparison to the untreated group. However, long-term anthelminthic treatment in children in Venezuela (> 22 months) (Lynch et al. 1993) or Gabon (> 30 months) (van Den Biggelaar et al. 2004) resulted in increased SPT reactivity to housedust mite, supporting a direct effect of helminth infections on atopy. Viral infections can often be detected by molecular techniques whereas helminth infections, in addition to molecular methods, can also be detected by the presence of circulating antigens, eggs/larvae in feces or urine, and larvae in peripheral blood. The method of direct detection of viruses or parasites is superior to serologic markers of infections that are thought to indicate exposure. The problems of cross-reactivity and diminishing serologic or cellular responses to an infection would complicate the interpretation of how infections may be associated with allergies. It is also important to note that the presence of a current viral or parasitic infection does not provide the investigator with a history of exposure to infections. With increasing evidence that early infections may shape the developing immune system in infants, determining current infection might not capture the situation earlier in life. Needless to say, large prospective studies where infections are measured and can be related to sensitization and clinical allergies are needed. In addition, to prove that infections and not confounding factors are associated with allergies, double-blind placebo-controlled intervention studies would have to be carried out. Such studies need to be considered carefully, taking into account ethical considerations that would be expected to be widely different depending on which infection one is dealing with.

Animal models To study the causal relationship between helminth infections and the development of allergic diseases, several groups have developed combined murine models of infection and asthma. Studies with rodent nematodes, such as Heligmosomoides polygyrus, have demonstrated that infection leads to strongly reduced ovalbumin-driven eosinophilic airway inflammation (Wilson et al. 2005; Kitagaki et al. 2006). In another study, immune responses to food allergens (IgE and IL-13 production) were downmodulated by infection with H. polygyrus (Bashir et al. 2002). A similar inhibition of allergic eye disease or lung inflammation (McConchie et al. 2006) and airway hyperresponsiveness (Wohlleben et al. 2004) was shown by

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either Ascaris suum eggs, Ascaris extract implants, or by infection with Nippostrongylus brasiliensis. However, there are also a number of studies showing no effect or a selective effect on a few immunologic markers, such as T-cell cytokine responses or allergen-specific IgE production, but no decrease in eosinophilic airway inflammation during infection with Strongyloides venezuelensis or S. stercoralis (Negrao-Correa et al. 2003). Some studies even find an exacerbation of allergic disease. For example, early stages of A. suum potentiated airway inflammation (Enobe et al. 2006) and infection with Trichinella spiralis increased anaphylaxis in mice (Strait et al. 2003). The observations that different parasitic helminths induce opposing effects in mouse models may suggest variation in the immune-modulating capacity of the distinct species or variations in intensity and chronicity of the different infection studied. Recent studies in animal models of schistosome infection, where the acute and chronic phases can be distinguished and studied in isolation, have shown that whereas acute infections have no effect or even a slight exacerbating effect on airway hyperresponsiveness, chronic infections are associated with decreased airway inflammation following ovalbumin sensitization (Smits et al. 2007).

Cellular immunologic profiles associated with allergic disorders in developing countries In Western countries where the increase in allergic disorders has been observed, the hypothesis was put forward that allergic responses might result from faulty maturation and polarization of the immune system. It was presumed that the decrease in exposure to bacterial and viral infections might lead to a slower development of Th1 adaptive responses, allowing proallergic Th2 responses to develop unhindered (Cookson & Moffatt 1997). The birth cohort studies in Australia supported the notion that a slow developing Th1 response could indeed be responsible for increased susceptibility to allergic disorders (Prescott et al. 1999). The distorted balance between Th1 and Th2 responses becomes interesting when considering developing countries and in particular the rural areas in these countries where infections leading to Th2-type responses are highly prevalent. The parasitic helminths are the strongest natural stimuli for the development of Th2 responses, characterized by IL-4, IL-5 and IL-13 secretion from T cells and resultant increased IgE antibodies and eosinophils in peripheral blood (Maizels & Yazdanbakhsh 2003). Importantly, these infections when present during pregnancy of an infected mother can affect the immune response of the fetus. Increased Th2 responses have been measured in neonates born to mothers with parasitic helminths (Malhotra et al. 1999). However, formal studies are needed to establish the relative development of Th1 and Th2 responses in infancy and early childhood in

areas where not only helminth infections but also other infections are highly prevalent. These are areas of particular interest because of their low prevalence of allergic disorders. As mentioned above, studies carried out in areas where helminth infections are highly prevalent have indicated that such Th2-inducing infections might actually reduce the risk of developing atopy or allergic diseases (Yazdanbakhsh et al. 2002; Cooper et al. 2003; Flohr et al. 2006). The question of how a Th2-inducing infection might be associated with decreased risk of atopy has not received a definitive answer. As discussed earlier, the functional capacity of the IgE generated to house-dust mite, for example, during such infections might be different such that no mast cell degranulation occurs when these antibodies present on mast cells encounter an allergen. Another possibility is that one of the important properties of helminth infections, namely their capacity to induce regulatory responses (Taylor et al. 2005), is responsible for suppression of the effector phase of the allergic response (Yazdanbakhsh et al. 2002; Wilson et al. 2005). In a study carried out in Gabonese schoolchildren, it has been shown that children with an increased parasite-induced IL-10 production capacity had a decreased risk of being atopic to house-dust mite (van den Biggelaar et al. 2000). This increased parasiteinduced IL-10 production capacity did not have any effect on the levels of house-dust mite-specific IgE. Although human studies are complicated by confounders that cannot be fully controlled, animal models have recently confirmed the protective role of regulatory immune responses in allergic airway inflammation (Wilson et al. 2005; Smits et al. 2007). Little is known about the emerging concept that regulatory immune cells might be an important component of the so-called “maturing immune system.” Thus not only Th1 or Th2 but also regulatory T cells might need to mature as part of a well-balanced adaptive immune system (Wing et al. 2005). Such cells will then ensure that there is no overshoot of Th1 or Th2 responses during childhood (Fig. 98.4). Thus any inflammation resulting from an insult or a pathogen would be kept under control and would not lead to tissue damage or organ failure otherwise expected to result from an uncontrolled polarized immune response. Much has yet to be elucidated on the mechanism whereby regulatory responses control allergic inflammation. The question of antigen specificity has not yet been answered. In some reports where regulatory T cells were shown to play an important role in controlling allergies, either during immunotherapy or during seasonal exposure to allergy, antigen specificity of the regulatory cells was suggested (Akdis & Akdis 2007). However, more extensive studies are needed, not only at the immunoepidemiologic but also at the mechanistic level. If intense infections protect against allergies, do they do so against one allergen or against several? Following on from this, are the regulatory responses allergen specific or represent a bystander effect? The answers need to be sought, as the specificity of a suppressor mechanism is essential to

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Th1

Th2 B reg

Treg Treg

(a)

Th1

Th2 Th2 Th2

Th1 B reg

Treg Treg Treg

Th2 Th2 Th2

(b)

enable the development of therapeutics with few undesired effects on the general immune system.

Concluding remarks The study of allergy and asthma in Africa, Asia and Latin America is of great importance as the increasing westernization of lifestyle, particularly in urban areas, is leading to an increased burden of allergic disorders. The questions regarding the steps involved in the development of full-blown allergic disease and the modifiers of each step need to be addressed (Fig. 98.1). Equally important is to understand the modifiers that affect early life events permanently and those factors that are currently modifying events and which may be eliminated (Fig. 98.1). These studies need tools that can help generate accurate data. These tools can then be utilized to understand risk and protective factors which, at the epidemiologic and individual level, should help measures to control allergic disorders effectively. At the mechanistic level, understanding the physiologic and immunologic pathways that prevent the development of allergies (Figs 98.3 and 98.4) will help the field devise therapeutics to prevent new epidemics.

References Aalberse, R.C. & Platts-Mills, T.A. (2004) How do we avoid developing allergy: modifications of the TH2 response from a B-cell perspective. J Allergy Clin Immunol 113, 983– 6. Addo-Yobo, E.O., Custovic, A., Taggart, S.C., Asafo-Agyei, A.P. & Woodcock, A. (2002) Seasonal variability in exercise test responses in Ghana. Pediatr Allergy Immunol 13, 303– 6. Addo-Yobo, E.O., Woodcock, A., Allotey, A., Baffoe-Bonnie, B., Strachan, D. & Custovic, A. (2007) Exercise-induced bronchospasm and atopy in Ghana: two surveys ten years apart. PLoS Med 4, e70.

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Treg

(c)

Fig. 98.4 Proposed schematic representation of the balance between Th1 and Th2 and the regulatory network that results in (a) no inflammation, (b) no allergic disease in the face of a Th2-skewed response, and (c) Th2-skewed response and allergic disease. (See CD-ROM for color version.)

Ait-Khaled, N., Odhiambo, J., Pearce, N. et al. (2007) Prevalence of symptoms of asthma, rhinitis and eczema in 13- to 14-year-old children in Africa: the International Study of Asthma and Allergies in Childhood Phase III. Allergy 62, 247–58. Akdis, M. & Akdis, C.A. (2007) Mechanisms of allergen-specific immunotherapy. J Allergy Clin Immunol 119, 780–91. Annus, T., Bjorksten, B., Mai, X.M. et al. (2001) Wheezing in relation to atopy and environmental factors in Estonian and Swedish schoolchildren. Clin Exp Allergy 31, 1846–53. Araujo, M.I., Lopes, A.A., Medeiros, M. et al. (2000) Inverse association between skin response to aeroallergens and Schistosoma mansoni infection. Int Arch Allergy Immunol 123, 145–8. Asher, M.I., Montefort, S., Bjorksten, B. et al. (2006) Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys. Lancet 368, 733– 43. Bashir, M.E., Andersen, P., Fuss, I.J., Shi, H.N. & Nagler-Anderson, C. (2002) An enteric helminth infection protects against an allergic response to dietary antigen. J Immunol 169, 3284–92. Bouayad, Z., Aichane, A., Afif, A. et al. (2006) Prevalence and trend of self-reported asthma and other allergic disease symptoms in Morocco: ISAAC phase I and III. Int J Tuberc Lung Dis 10, 371–7. Bousquet, J., Chanez, P., Chanal, I. & Michel, F.B. (1990) Comparison between RAST and Pharmacia CAP system: a new automated specific IgE assay. J Allergy Clin Immunol 85, 1039–43. Braun-Fahrlander, C., Wuthrich, B., Gassner, M. et al. (1997) Validation of a rhinitis symptom questionnaire (ISAAC core questions) in a population of Swiss school children visiting the school health services. SCARPOL-team. Swiss Study on Childhood Allergy and Respiratory Symptom with respect to Air Pollution and Climate. International Study of Asthma and Allergies in Childhood. Pediatr Allergy Immunol 8, 75– 82. Calvert, J. & Burney, P. (2005) Effect of body mass on exerciseinduced bronchospasm and atopy in African children. J Allergy Clin Immunol 116, 773–9. Carr, W.W. (2006) Improvements in skin-testing technique. Allergy Asthma Proc 27, 100–3. Chan, H.H., Pei, A., Van Krevel, C., Wong, G.W. & Lai, C.K. (2001) Validation of the Chinese translated version of ISAAC core questions for atopic eczema. Clin Exp Allergy 31, 903–7.

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(1993) Effect of anthelmintic treatment on the allergic reactivity of children in a tropical slum. J Allergy Clin Immunol 92, 404–11. McConchie, B.W., Norris, H.H., Bundoc, V.G. et al. (2006) Ascaris suum-derived products suppress mucosal allergic inflammation in an interleukin-10-independent manner via interference with dendritic cell function. Infect Immun 74, 6632–41. MacGlashan, D.W. Jr, Bochner, B.S., Adelman, D.C., Jardieu, P.M., Togias, A. & Lichtenstein, L.M. (1997) Serum IgE level drives basophil and mast cell IgE receptor display. Int Arch Allergy Immunol 113, 45–7. McKenzie, S.A., Chan, E., Dundas, I. et al. (2002) Airway resistance measured by the interrupter technique: normative data for 2–10 year olds of three ethnicities. Arch Dis Child 87, 248–51. Maizels, R.M. & Yazdanbakhsh, M. (2003) Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol 3, 733– 44. Malhotra, I., Mungai, P., Wamachi, A. et al. (1999) Helminth- and Bacillus Calmette-Guerin-induced immunity in children sensitized in utero to filariasis and schistosomiasis. J Immunol 162, 6843–8. Mallia, P. & Johnston, S.L. (2006) How viral infections cause exacerbation of airway diseases. Chest 130, 1203–10. Mallol, J., Sole, D., Asher, I., Clayton, T., Stein, R. & Soto-Quiroz, M. (2000) Prevalence of asthma symptoms in Latin America: the International Study of Asthma and Allergies in Childhood (ISAAC). Pediatr Pulmonol 30, 439–44. Matricardi, P.M., Rosmini, F., Ferrigno, L. et al. (1997) Cross sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis A virus. BMJ 314, 999– 1003. Mazmanian, S.K., Liu, C.H., Tzianabos, A.O. & Kasper, D.L. (2005) An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–18. Medeiros, M. Jr, Figueiredo, J.P., Almeida, M.C. et al. (2003) Schistosoma mansoni infection is associated with a reduced course of asthma. J Allergy Clin Immunol 111, 947–51. Mitre, E., Norwood, S. & Nutman, T.B. (2005) Saturation of immunoglobulin E (IgE) binding sites by polyclonal IgE does not explain the protective effect of helminth infections against atopy. Infect Immun 73, 4106–11. Negrao-Correa, D., Silveira, M.R., Borges, C.M., Souza, D.G. & Teixeira, M.M. (2003) Changes in pulmonary function and parasite burden in rats infected with Strongyloides venezuelensis concomitant with induction of allergic airway inflammation. Infect Immun 71, 2607–14. Ng’ang’a, L.W., Odhiambo, J.A., Mungai, M.W. et al. (1998) Prevalence of exercise induced bronchospasm in Kenyan school children: an urban-rural comparison. Thorax 53, 919–26. Nyan, O.A., Walraven, G.E., Banya, W.A. et al. (2001) Atopy, intestinal helminth infection and total serum IgE in rural and urban adult Gambian communities. Clin Exp Allergy 31, 1672–8. Ostman, S., Rask, C., Wold, A.E., Hultkrantz, S. & Telemo, E. (2006) Impaired regulatory T cell function in germ-free mice. Eur J Immunol 36, 2336–46. Palmer, L.J., Celedon, J.C., Weiss, S.T., Wang, B., Fang, Z. & Xu, X. (2002) Ascaris lumbricoides infection is associated with increased risk of childhood asthma and atopy in rural China. Am J Respir Crit Care Med 165, 1489–93. Pearce, N., Pekkanen, J. & Beasley, R. (1999) How much asthma is really attributable to atopy? Thorax 54, 268–72.

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PART 14

Prevention of Allergic Disease

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Note: page numbers in italics refer to figures; page numbers in bold refer to tables. 2282del4 gene, mutations, 1228 AA see atopic asthma (AA) AAC see acute allergic conjunctivitis (AAC) AAD (adaptive aerosol delivery), 778 17-AAG (17-allylamino-17demethoxygeldanamycin), in mastocytosis treatment, 923 AAHR (asymptomatic airway hyperresponsiveness), 1568 ABC (atopic blepharoconjunctivitis), 243–4 ABCB1 gene, 740 ABCC2, 735 abciximab, 1951 ablation, airway smooth muscle, 885–6 ABPA see allergic bronchopulmonary aspergillosis (ABPA) Absidia corymbifera (fungus), 1761 Acarus siro (storage mite), 1101 ACD see allergic contact dermatitis (ACD) ACE see angiotensin-converting enzyme (ACE) ACE (angiotensin-converting enzyme) inhibitors, 1867–8 acebutolol, 679 acetylcholine, 784, 824 N-acetylglucosamine residues, 1126 ACGIH (American Conference of Governmental Industrial Hygienists), 1017–61 acid anhydrides, 1017 acidic C-serum protein, 1168–9 acidic mammalian chitinase (AMC), 387 acid phosphatase, 1123–4 Acinetobacter spp. (bacteria), 1760 ACQ (Asthma Control Questionnaire), 1575 acquired C1 inhibitor deficiency, 1866 acrivastine, 554, 555 ACT (Asthma Control Test), 1575 ACTH see corticotropin α-actin, 877, 878–9 Actinaria spp. (sea anemones), 3–4 actinidin, 1153 actinomycetes, 963 abundance, 964 spores, 964 thermophilic, 1757 activating receptors, blockade, 328

activation-induced cytidine deaminase (AID), 119, 126, 127, 129, 130–1, 133, 134 activator protein-1 (AP-1), 72, 717 active immunization models, 1204–8 allergen challenge issues, 1205–7 administration route, 1207 dose, 1206 genetic background, 1206–7 allergen selection, 1204–5 fungal allergens, 1205 protein allergens, 1204–5 chronic injury mechanisms, 1208 protocols, 1204 remodeling changes, persistence, 1207–8 acute allergic conjunctivitis (AAC), 1491 clinical features, 1491 definition, 1387 pathogenetic mechanisms, 1491 treatment, 1491 acute asthma, antileukotrienes in, 704–5 acute bronchospasm, 1966 acute ethmoiditis, orbital complications, 1474 acute exacerbations in chronic obstructive pulmonary disease adrenergic agents, 688 anticholinergic agents, 688–9 acute generalized exanthematous pustulosis (AGEP), 1953, 1956–7 acute nonviral (bacterial) rhinosinusitis, definition, 1393 acute respiratory distress syndrome (ARDS), 311, 423 acute rhinosinusitis (ARS) antibiotics, indications, 1473 clinical features, 1472–3 comorbidities, 1472 definition, 1393 diagnosis, 1472, 1473 differential diagnosis, 1473 disease course, 1474 epidemiology, 1469–70 etiology, 1470 future trends, 1474–5 genetics, 1470 management, 1473–4 mechanisms, 1470 pathogenesis, 1470 pathology, 1470–2 prevalence, 1469–70 prognosis, 1474

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

surgery, 1474 treatment, 1473–4 acute severe asthma, systemic glucocorticoids in, 726 acute urticaria, 1929 acute viral infections and exacerbations, 1247 infants, 1245 acute viral rhinosinusitis, definition, 1393 AD see atopic dermatitis (AD) ADAM 10, 111 ADAM 17, 387 ADAM33 gene, 40, 387, 1229, 1717 in airway remodeling, 1619 polymorphisms, 1207 ADAM family, 387 adapter proteins, 207 adaptive aerosol delivery (AAD), 778 adaptive immune responses development, 436 epithelium in, 385–7 mediation, 197 adaptive immune system, interactions, with innate immune system, 193 adaptive immunity, 119 adenoid hypertrophy, with allergic rhinitis, 1388–9 adenosine, 786, 833 adenosine antagonists, 1718–20 adenosine monophosphate (AMP), 236, 698 bronchial hyperresponsiveness, 790 bronchoconstriction, 787–8 adenosine receptors, 786, 1719–20 adenosine triphosphate (ATP), 851 inhalation, 833 adenylate cyclase, activators, 328–9 adherens, 369 adherens junctions (AJs), 858–9, 860, 864–5, 868 adhesion molecule blockade, 1723 adhesion molecules, 264, 337, 381–2, 1304–6 in allergic disease, 345–53 and chemokines, 472–3 expression, 345–6 soluble, 346 see also intercellular adhesion molecule-1 (ICAM-1); junctional adhesion molecules (JAMs); platelet endothelial cell adhesion molecule-1 (PECAM-1); vascular cell adhesion molecule-1 (VCAM-1)

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adhesion receptor antagonists, in allergic disease treatment, 353 adhesion receptors function, in allergic inflammation, 352– 3 in vivo models, 352– 3 roles, 1455 adjuvants, 1720–1 adolescents, atopic dermatitis, 1816 ADRB3, sequencing, 672 adrenaline see epinephrine adrenal suppression, and inhaled glucocorticoids, 723– 4 adrenergic agents in chronic obstructive pulmonary disease, acute exacerbations, 688 discovery, 683 adrenergic agonists, 668– 82 administration, 668, 677 β2-adrenergic agonists, 405 as bronchodilators, 668 adrenergic antagonists, 668– 82 adrenergic receptors early studies, 668 ligands as, 668 β2-adrenergic receptors, interactions, with glucocorticoids, 717–18 adrenergic urticaria, 1864 adrenoceptor agonists, mechanisms, 672 α-adrenoceptor agonists, 680 β-adrenoceptor agonists, 672– 9 adverse effects, 677– 9 cardiac, 678 metabolic, 679 aerosol administration, 677 affinity, 674– 5 desensitization, 677– 8 efficacy, 674–5 and hypokalemia, 679 and hypoxemia, 679 metabolism, 672– 4 oral administration, 677 parenteral administration, 677 pharmacogenetics, 679 pharmacokinetics, 677 physiologic effects, 675–7 cardiovascular, 677 selectivity, 674–5 structure, 672–4 and tremor, 678 β2-adrenoceptor agonists, 796 in asthma treatment, 1712 effects on airway smooth muscle, 885 limiting factors, 885– 6 α-adrenoceptor antagonists, 680 β-adrenoceptor antagonists, 679– 80 β adrenoceptor-associated kinase (βARK), 678 adrenoceptors biology, 670– 2 localization, 669–70 molecular structure, 670–2 tissue distribution, 669–70 α adrenoceptors, 670 subtypes, 671

β adrenoceptors, 669–70 mRNA, 671 adrenocorticotropic hormone (ACTH) see corticotropin adult asthma, anticholinergic agents in, 689–90 adults atopic dermatitis, 1816 atopic eczema, 1816 pet sensitization studies, 1012 tracheobronchial airways, species differences, 1188–92 see also elderly AECs (airway epithelial cells), 180, 181 Aedes aegypti (mosquito), 1139 AEG12, 1138–9 aeroallergens, plant-derived, and air pollution, 1270–2 AeroEclipse, 772 aerosol delivery adaptive, 778 and mechanical ventilation, 780 to nasal mucosa, 780–1 pediatric, 779–80 aerosol delivery systems, 768–82 applications, in allergic diseases, 778–81 assessment, 771–3 developments, 778 see also spacers aerosols aerodynamics, 769–71 characteristics, 768–9 definition, 768–9 generation, 776–8 hyperosmolar, 814–15 inhaled mass, 771 interfaces, 771 lung deposition, 772–3 and breathing patterns, 769–70 deep breathing, 770 and expiration, 770 mechanisms, 769 optimization, 769–71 slow breathing, 770 penetration, beyond oropharynx, 769 production, 785 afferent innervation see sensory innervation A-fibers, 826 Africanized honeybees, 1981 AFS (allergic fungal rhinosinusitis), development, 1464 age and asthma, 1243 and atopic dermatitis, 1259–60, 1261 and skin tests, 1339 Agelaia pallipes pallipes (wasp), 1124 AGEP (acute generalized exanthematous pustulosis), 1953, 1956–7 agonists, 14 β agonists in asthma treatment, 1332 in clinical use, 673 in combination drugs, 686 and late-phase allergic reactions, 531 structure, 673

β2 agonists, 717–18, 814 adult studies, 719–20 adverse effects, 1651 in chronic asthma treatment, 1650–1 clinical use, 1651 efficacy, 722 mechanisms, 1651 pediatric studies, 720 in persistent asthma, 703–4 safety, 1651 tolerance, 1651 ultra-long acting, 1717 vs. antileukotrienes, 700–2 see also long-acting β2 agonists (LABAs) agricultural environments, fungal spores, 972 AHR see airway hyperresponsiveness (AHR) AIA see aspirin-induced asthma (AIA) AIANE (European Network on Aspirin-Induced Asthma), 1973 AICs (allergen immunostimulatory conjugates), 1561–2 AID see activation-induced cytidine deaminase (AID) AIR (Asthma Insights and Realities), surveys, 1661 airborne allergen-carrying particles, and air pollution, 1270–1 airborne allergens, in workplace, 1017–122 airborne irritants, in workplace, 1017–122 air conditioning, contaminated, 1760 air-conducting segments, cell types, 1215–17 AIRE gene deficiency, 50 mutations, 50 airflow limitation and bronchial hyperresponsiveness, 783 drug-induced, 791 measurement, asthma, 1579–80 reversibility glucocorticoids, 1581 rapid-acting bronchodilators, 1580 use of term, 784 airflow obstruction assessment, 750–4 monitoring methods, 752 persistent, with chronic asthma, 755–6 air inhalation, in exercise-induced bronchoconstriction, 816 air particulate matter and immunoglobulin E production, 144–5 age effects, 144 particle size effects, 144 air pollution aeroallergens, plant-derived, 1270–2 and airborne allergen-carrying particles, 1270–1 and allergic respiratory diseases, 1266–7 and asthma, 1250 and asthma exacerbations, 1267 and atopic diseases, 1250 and bronchial asthma, 1267 and climate change, 1273–4 epithelial injury, 1616 and plants, 1273–4 see also indoor air pollution; outdoor air pollution Air Pollution and Health: a European Approach (APHEA), 1250

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air quality effects, 1279– 80 legislation, 1279 see also indoor air quality Air Quality Framework Directive (EU), 1279 air trapping, assessment, 754– 5 airway caliber, 786 and bronchial hyperresponsiveness, 787 airway challenge, late-phase allergic reactions, 525 airway diseases antihistamine therapy, 559 see also allergic airway diseases; occupational airway diseases airway dysfunction, and eosinophilic airway inflammation, 1373– 5 airway epithelial cells (AECs), 180, 181 airway epithelial dysfunction, mast cells and, 240 airway epithelium, 366– 97 anatomy, 366– 71 in asthma, 1614–19 cell types, 371 pathophysiology, 372– 87 physiology, 366–71 airway function and airway vascularity, 400–1 and submucosal swelling, 401 tests, 750–4 airway growth, 1192– 3 exposure responses, 1195– 6 airway hyperresponsiveness (AHR), 62, 63, 64, 277– 8, 279 allergen-induced, 1206 in allergic rhinitis, 840–1 animal models, 1202 in asthma, 749, 812, 840 –1, 1608 asymptomatic, 1568 cold air, 802 development, 65, 484, 694 late-phase, in dogs, 801–2 and leukotrienes, 582– 3 measurement, 1568, 1580–1, 1717 mechanisms, 832– 3 neurotrophins in, 503–5 reversal, 716 substance P-induced, 530–1 and T cells, 536–7 treatment, 840, 841 see also bronchial hyperresponsiveness (BHR) airway hypersecretion in allergic rhinitis, pathophysiology, 844–5 in asthma, pathophysiology, 842– 4 new drugs for, 847–52 schematics, 847 treatment, 840–1, 846 airway immune system, exposure responses, 1197– 9 airway inflammation and airway remodeling, 1617–18, 1619–20 in asthma pathogenesis, 1608– 9 and bronchial hyperresponsiveness, 783, 787–8 and exercise-induced bronchoconstriction, 809 late-phase, in dogs, 800–1 markers, eosinophils, 809

and mast cells, 1374, 1375 measurement, 1368–72 mediators, 512 neuromodulation in, 828–31 neurotrophins in, 502–3 repetitive hyperventilation-induced, 802 systemic signaling, 1410 variable, in asthma, 1623 see also eosinophilic airway inflammation airway inflammation assessment exhaled nitric oxide, 1371–2 methodology, 1371 future trends, 1377 ideal monitoring tool, 1377 induced sputum, 1368–71 methodology, 1368–9 airway inflammation monitoring clinical roles, 1375–7 in asthma monitoring, 1376–7 in diagnosis, 1375–6 airway innervation, 512–13 anatomy, 512 see also human respiratory sensory innervation airway mucus, 841–2 production, 842 airway nerve fibers, activation, 511–12 airway nerves calcitonin gene-related peptide in, 531 substance P in, 513–14 airway obstruction late-phase, in animals, 799–802 repetitive hyperventilation-induced, in dogs, 802 airway remodeling, 64–5, 194–5, 413 active immunization models, 1204–8 and airway inflammation, 1617–18, 1619–20 in asthma, 1202, 1203, 1619–20, 1623, 1632–3 mechanisms, 1202–3 neurotrophins in, 503–5 occurrence, 1595 repetitive hyperventilation-induced, 802–3 small animal models, 1202–13 transgenic models, 1208–10 treatment, 1724 use of term, 1202 airway responses to deep inspiration, 757 to helium–oxygen mixtures, 756–7 airways allergen recognition, 1609 antimicrobials, 373–6 asthmatic patients, 1632, 1633 autonomic control of, 683–4 blood flow, 399–400 cells types, 366–8 cooling, 809 dehydration, 809 dendritic cells, 31 extrathoracic, 762–3 and immune development, 31 inflammation, 194 irreversible changes, prevention, 721 neural inflammation, 530–1 neurophysiology, 823–8

pollutant effects, 376 small, 757–8 T cells in, and asthma, 70–1 visualization, 757 see also lower airways; tracheobronchial airways; upper airways airways conductance (Gaw), measurement, 785 airway secretions, and leukotrienes, 583 airway sensitizers, listed regulatory, 1017–61 airway size, computed tomography studies, 758 airway smooth muscle (ASM), 874–91 ablation, 885–6 allergic roles, in asthma, 880–1 anatomy, 874–5 antiasthma drug effects on, 885–6 and asthma, 759–60 calcium influx, 878–9 chemokine release, 350 contractile elements, 875–6 contractile roles, in asthma, 878–80 cytoskeletal elements, 875–6 development, 1193 early studies, 874 exposure responses, 1197, 1199 factor expression, 881 and fibrosis mediation, 419 functions, 874 in healthy lung, 877 immunomodulatory roles, in asthma, 881–3 integrin-dependent interactions, 883 localization, 874–5 in lung diseases, 877–85 and lung volume change, 759–62 mast cell infiltration, 237–8 and mast cells, 1296 adhesion, 238 mediation effects, 239–40 putative interactions, 238–40 recruitment, 238 mechanical plasticity, in asthma, 878–80 myogenesis, 876–7 ontogeny, 876–7 plasticity, 1638–9 and platelet-activating factor, 606 proliferative roles, in asthma, 883–5 ultrastructure, 875–6 wall content decrease, in asthma, 886–7 airways resistance (Raw), 749, 752–3 exposure responses, 1195 measurement, 785 sites of increased, 756–7 airway submucosal glands, mast cell microlocalization in, 241 airway surface liquid (ASL) hyperosmolarity, 809 osmolarity, roles, 797 airway vascularity in asthma, 398–411 effects on airway caliber, 400–1 airway vasculature, exposure responses, 1197 airway vessels see bronchial vessels airway wall nerves, in asthma, 1641 AJs (adherens junctions), 858–9, 860, 864–5, 868

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AKC see atopic keratoconjunctivitis (AKC) AL-438, 1718 Albert, Prince of Monaco (1848–1922), 3– 4 albumins, 1151, 1152 transport, 860 see also bovine serum albumin (BSA); ovalbumin (OVA) albuterol, 686 alder pollen, 954 sensitization, 954 alemtuzumab, 742 Alexander, Harry L. (1887–1969), 784 algal blooms, and asthma, 1585 algorithms, for allergen sensitization, 1148 alkaloids, 683 atropine-like, 684– 5 allelic exclusion, 122, 125, 126, 131–2 mechanisms, 130 allergen avoidance, 1330–1, 1433– 4, 1598– 9, 1601–2 and childcare attendance, 2008– 9 chronic asthma, 1654 during lactation, 2002 food allergy, 1924, 1935 in occupational asthma, 1704–5 allergen-derived T-cell peptides, and late-phase allergic reactions, 533– 7 allergen exposure, 1248– 9 and atopic diseases, 1261–3 and disease, 1248– 9 inhalant, 2000, 2004– 6 mechanisms, 2022 allergen extracts, 897–8, 928– 41 aqueous, 1527– 8 commercial, 1527– 8 incubation, 932 mixtures, 1528 skin tests, 1339 allergen genes, cloning, 1556 allergen hybrids, for allergen-specific immunotherapy, 1558– 9 allergenicity, and immunoglobulin E, 915 allergen immunostimulatory conjugates (AICs), 1561–2 allergen immunotherapy see allergen-specific immunotherapy (SIT) allergen injection immunotherapy, 1510–21 adverse effects prevention, 1532–7 systemic reactions, 1530 treatment, 1537 algorithms, 1537– 9 allergen products, 1527– 8 annotations, 1537– 9 contraindications, 1525 absolute, 1525 relative, 1525 disease-modifying capacity, 1525– 6 dosage, 1530–2 during allergen season, 1532 maintenance, 1531 dose–efficacy studies, 1528– 9 dose-increase regimens, 1528 dose modifications, 1531–2 dose–response relationships, 1528– 9

effectiveness, 1511 and effector cell recruitment, 1513 effects on T-cell responses, 1515–17 and eosinophil recruitment, 1513 guidelines, 1522 and immunoglobulin responses, 1513–15 indications, 1523–5 patient profiling, 1525 monitoring, 1532 forms, 1533–6 novel strategies, 1517–18 organization of, 1526–7 patients, 1527 diagnostic procedures, 1527 information, 1527 observation, 1527 practice, 1522–42 preventative capacity, 1525–6 rescue facilities, 1526–7 risk–benefit assessments, 1524 risk factors, 1529 safety issues, 1529–30 systemic reactions, frequency, 1530 treatment strategies, 1523 allergen M, 1151 allergen molecules purified natural, 897–8 recombinant, 897–8 allergen products for allergen injection immunotherapy, 1527–8 composition, 934–5 allergen proteins, genetically modified, 1517 allergens antibody binding, 914–15 and asthma, 142–3, 984, 1570–1 and atopic dermatitis, 1819 biochemical characterization, 904–5 biochemistry, 895–912 biological functions, 903–4 classification, 895, 899–901, 1834 clinical characterization, 904–5 cross-reactions, 1847 definition, 1280 and exercise-induced bronchoconstriction, 812–13 fungi as, 963–83 host responses to, 913–27 immunoassays, 930–4 and immunoglobulin E production, 142–4 climate change, 143–4 dose exposure, 143 ecological environment change, 144 geographical spread, 143–4 indoor vs. outdoor exposure, 142–3 level changes, 143–4 immunologic characterization, 904–5 and indoor air pollution, 1280 inhalation tests, 1583 interactions with diesel exhaust, 145–6 isoforms, 900 and late asthmatic reactions, 527, 528 and late-phase allergic reactions, 524 marker, 901 minor, 899–900, 915

natural, 900 nomenclature, 899–901 occupational asthma induction, 1018– 60 occurrence, 142 origin of term, 5 and pregnancy, 142 recognition, in airways, 1609 sensitization, 1248 algorithms, 1148 hierarchies, 901 prevalence, 166 risk, 142 β-sheet structure, 903 sources, 142, 897–8, 899–901, 902, 913–14, 928 structural characterization, 903–4 triggers, algorithms, 1148 types of, 1280 use of term, 895 see also animal allergens; dust mite allergens; environmental allergens; food allergens; fungal allergens; indoor allergens; insect allergens; major allergens; mammalian allergens; pet allergens; plant allergens; recombinant allergens; respiratory allergens; superallergens allergen-specific antibody responses, 92–3 allergen-specific immunotherapy (SIT) allergen hybrids for, 1558–9 for allergic rhinitis, 1439–40 current status, 1176–7 cytosinephosphoguanosine oligonucleotides in, 1561–2 definition, 1522, 1543 effectiveness, 1555 future trends, 1177–8 hypoallergenic recombinant allergens for, 1556–8 in latex allergy, 1176–8 mechanisms, 90–6, 1333 novel approaches, 1555–64 principles, 1332 procedures, 1332–3, 1555 recombinant allergens for, 1555–6 T-cell peptide epitopes in, 1559–61 allergen-specific vaccines, for immunotherapy, 94–6, 1439–40 allergen standardization, 928–41 biological vs. major allergen measurements, 929 future trends, 936–7 historical background, 928–9 without antibodies, 936–7 allergic airway diseases and indoor air pollution, 1280 nerves in, 831–5 neurogenic inflammation in, 495 and outdoor air pollution, 1266–78 allergic airway inflammation, animal models, 502–3 allergic airways, basophil recruitment to, 325–6 allergic alveolitis, 1282, 1324 allergic angiogenesis, basophils in, 327–8 allergic Aspergillus sinusitis, 1747–8

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allergic asthma characterization, 1667 immunotherapy, 990 inflammatory cascade, 1610 in vitro tests, 1583 in vivo tests, 1582– 3 neurotrophins in, 502 prevalence, 634 primate models, 1187– 201 skin tests, 1582– 3 use of term, 1214 see also persistent severe allergic asthma allergic bronchopulmonary aspergillosis (ABPA), 1743–56 and asthma, 1746– 7 biology, 1749 clinical characteristics, 1748– 9 and cystic fibrosis, 1747 definition, 1743 diagnosis, 1746– 8, 1792 diagnostic criteria, 1792 exacerbations, 1750 genetic factors, 1745 issues, 1753 onset, 1748 pathogenesis, 1744 pathology, 1746 pathophysiology, 1744– 6, 1792 prevalence, 1791 pulmonary function tests, 1740– 51 radiology, 1749– 51 respiratory function tests, 1751 stages, 1748– 9 symptoms, 1792 tissue damage, 1746 treatment, 1751–3, 1792– 3 allergic bronchopulmonary fungal disease, 1585 allergic bronchopulmonary mycosis, 1791–3 allergic cells, signal transduction, 203–13 allergic conjunctivitis animal models, 1486– 7 classification, 1387 mast cells in, 243– 4 sublingual immunotherapy, clinical efficacy, 1547 symptoms, 1432 see also acute allergic conjunctivitis (AAC); perennial allergic conjunctivitis (PAC); seasonal allergic conjunctivitis (SAC) allergic contact dermatitis (ACD), 1831 cellular mechanisms, 1833 chemical issues, 1834 clinical diagnostic tests, 1844–8 clinical features, 1838– 42 clinical history, 1843– 4 diagnosis, 1843– 8 eyelids, 1841 face, 1840–1 hands, 1839, 1840 histopathology, 1834–5 patch tests, 1832, 1834–5, 1844– 8 patterns, 1841–2 phases, 1833 photopatch tests, 1848 types of, 1839– 42

allergic diseases aerosol delivery systems, 778–81 and atopy compared, 2022 basophils in, 320–36 and bradykinin pathways, 451–70 cellular immunologic profiles, developing countries, 2027–8 characterization, 1290 contiguous immunity in, 195–6 cytokines in, 1297–303 dendritic cells as drug targets in, 179 desensitization, 1332–3 diagnosis, 1319–80 practice, 1321–34 principles, 1321–34 specific clinical conditions, 1323–8 tests, 1323 drug therapy, 1331–2 early origins, 23–4 environmental factors, 24, 31–9 eosinophils in, 1293–5 epidemics, 1290 and epithelial cells, 1297 etiology, studies, 1354–5 and helminth infections, 154 hyposensitization, 1332–3 and immunoglobulin E, 1292–3 immunoglobulin E in, 141–2, 634 and immunoglobulin E receptors, 1292–3 immunotherapy, 1332–3 incidence, 1290–1 inflammatory cells in, 1293–7 and innate immune system, 193–6 innate immunity in, 187–202 mast cells in, 231–45 molecular immunopathology, 1290–317 molecular techniques, 1291–2 neurotrophins in, 502–8 and neutrophils, 1297 neutrophils in, 312–13 and particulate matter, 144–5 patient perceptions, 1524 perinatal risk factors, 37–9 and pets, 35, 1282 phenotypes, 194 prevalence, 23, 634 rural vs. urban areas, 2021–2 prevention developing countries, 1995–2030 in fetus and newborn, 1997–2019 remodeling in, 1308–9 sensitization, prevalence, 166 superallergens in, 326–7 T cells in, 1295–6 transcription factors in, 1306–8 treatment, 1330–3 adhesion receptor antagonists, 353 use of term, 6 see also hay fever; ocular allergic diseases; respiratory allergic diseases allergic eosinophilic gastroenteritis, 1927–8 allergic fungal rhinosinusitis (AFS), development, 1464 allergic inflammation adhesion receptor function, 352–3

and barrier cells, 188 and basophils, 1296–7 chemokine receptors in, 480–6 chemokines in, 480–6, 1303–4 cytokines in, 48–82 histamine in, 553, 554 leukocyte adhesion in, 337–65 and mast cells, 230–1, 1296 mechanisms, 895 neuropeptides in, 511–23 and prostaglandin E2, 594– 6 and regulatory T cells, 1296 T cells in, 48–82 allergic march, use of term, 896, 2020 allergic patients assessment, 1322–3 disease perceptions, 1524 history, 1322–3 physical examination, 1322–3 allergic reactions emergency treatment, 1989 mechanisms, 1346, 1347 see also early-phase allergic reactions (EPRs); late-phase allergic reactions (LPRs) allergic respiratory diseases see respiratory allergic diseases allergic responses development prevention, 83 and immunotherapy, 1511–12 late, 1511–12 allergic rhinitis (AR), 1383–401 with adenoid hypertrophy, 1388–9 airway hyperresponsiveness in, 840–1 airway hypersecretion in, pathophysiology, 844–5 allergen avoidance, 1433–4 allergen-specific immunotherapy, 1439–40 antileukotrienes in, 705–6 with asthma, 1386–7, 1430, 1431 antileukotrienes in, 706 treatment, 1443–5 autonomic nervous system in, 823–39 basophils in, 1418 cellular bases, 1409–18 cellular events, 1409 classification, 1383, 1430 ARIA, 1386 clinical history, 1432 comorbidities, 1383, 1386–9, 1430, 1431 treatment guidelines, 1441–5 complications, 1386–9, 1431 with conjunctivitis, 1387 costs of, 1430 definitions, 1384–5 clinical, 1384 epidemiological, 1384–5 dendritic cells in, 179 development, 1407–9 diagnosis, 1431–3 diagnostic tests, 1431 differential diagnosis, 1383, 1384 disabling effects, 1524 drug therapy, 1434–9 administration routes, 1436 intranasal administration, 1436

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allergic rhinitis (AR) (Cont’d ) ear, nose and throat examination, 1432 in elderly, 1446 environmental control, 1433– 4 eosinophils in, 1417–18 epidemiology, questionnaires, 1384– 5 genome screens for, 1227 intermittent, 1385 issues, 1445– 6 and kinins, 464 leukocyte infiltration, 1417–18 luminal cell recruitment, 1413–17 management, 1430–53 education, 1446 in pharmacies, 1445 recommendations, 1443 mast cells in, 243, 1296, 1410–12 mediators, 1418–20 mucociliary clearance in, 845– 6 mucus in, 840–56 with nasal polyps, 1388 nasal symptoms, 1403 neurotrophins in, 505– 6 omalizumab treatment, 1680 with otis media with effusion, 1389 over-the-counter management, 1445 pathophysiology, 1402–29 pediatric issues, 1445– 6 perennial, 243, 1385 persistent, 1385 and pregnancy, 1446 prevalence, 1383, 1430 receptors, 1418–20 with rhinosinusitis, 1387– 8 seasonal, 243, 1384 sensory nervous system in, 823–39 severity assessment, 1385– 6, 1431–3 objective, 1386 subcutaneous immunotherapy, 1439– 40, 1526 sublingual immunotherapy, 1440 clinical efficacy, 1545–7 symptoms, 243, 1402, 1430, 1432 classical, 1385– 6 expression, 1418– 20 social, 1386 systemic cell signaling, 1420–1 T cells in, 1412–13 therapeutic implications, 1421 tissue recruitment, 1413–17 treatment, 506, 1430–53 evidence-based, 1441–3 issues, 1524–5 treatment guidelines, 1441–5 development, 1441–2 triggers, 1383 with tubal dysfunction, 1389 Allergic Rhinitis and its Impact in Asthma (ARIA) initiative, 1240, 1544, 1573 classification, 1385, 1386 guidelines, 1387, 1441–2, 1443, 1444 allergic rhinoconjunctivitis allergen injection immunotherapy indications, 1523–5 treatment strategies, 1523

antihistamine therapy, 557–9 diagnosis, 1335 and latex allergy, 1173 prevalence, 23 seasonal, 1260 allergic sensitization definition, 166 dendritic cells in, 176–7 and house-dust mites, 1281–2 and pets, 1002–12 risk factors, 1944, 1997 use of term, 896 see also immunoglobulin E (IgE) sensitization; pet sensitization allergic skin inflammation, neurotrophins in, 507 allergology, field of study, 3 allergy and asthma, 1325 and breastfeeding, 2001–2 children, 1330 concepts, 3–22 conditions, 1322 corruption of term, 5–6 development, 23–47 early studies, 3 environmental factors, 24, 31–9 etiology, 634 fungi-induced, 965 genetics, 24, 39–40 history, 3–22 and hypersensitivity, 5–6 immunology, 23–30 incidence, 634 mimics, 1322 and nasal polyps, 1461–2 nomenclature, 1921 origin of term, 4–5, 895 pediatric, 1333 and pediatric asthma, 1597–9 prophylaxis, 906–7 regulatory T cells in, 83–102 roles, 1328–30 in nonspecific/polysymptomatic illness, 1330 and skin, 1326–7, 1811–93 specialization in, 1321 tolerogenic mechanisms in, 83–102 type I, 1947–9 type II, 1950–2 type III, 1357–8, 1950 use of term, 634 see also biting insect allergy; food allergy; fruit allergy; hygiene hypothesis; insect sting allergy; latex allergy; ocular allergy; pollen allergy; pseudoallergy; unconventional allergy; vegetable allergy allergy awareness, healthcare workers, 1321–2 allergy diagnosis, basophil simulation tests, 1359–64 allergy march, 1259–65 use of term, 1259 allergy prevention and breastfeeding, 2001–2 children, 2000 in developing countries, 1995–2030

ethical issues, 1998 future trends, 2011–13 infants, 2001–10 issues, 2011 in pregnancy, 2000–1 recommendations, 2012 strategies, 2011 target populations, 1998–2000 use of term, 1997 allergy pyramid, 40 allergy risk, 1944, 1997 biomarkers, 1999–2000 genetic markers, 1999 allergy tests in vitro, 1583 in vivo, 1582–3 laboratory, 1346–67 see also basophil simulation tests; inhalation tests; puncture tests; radioallergosorbent test (RAST); skin tests allergy treatment, 906–7, 1330– 3 chemokine blockade, 485–6 drug therapy, 1331–2 future trends, 1727 new drugs, 1712–39 allodyspneic effects, 833 allotussive effects, 833 17-allylamino-17-demethoxygeldanamycin (17-AAG), in mastocytosis treatment, 1891 almonds, allergens, 1152 Aln g 1, 954 Alnus spp. (alders), 954 see also alder pollen ALOX5 gene, 811, 1971 Alt a 1, 929, 973, 975 Alternaria spp. (fungi), 929, 975 abundance, 967 allergens, 965, 973, 1061 seasons, 967 spores, 970 Alternaria alternata (fungus), 965, 973 enolases, 1170 alternative pathway, 438, 441, 444 –8 alternative (unconventional) allergy, diagnosis, 1333–4 altrakincept, 73 aluminum smelting, emissions, 1101 alveolar development, 1194 alveolar epithelium, 370 alveolar function, assessment, 755 alveolar macrophages (AMs), 167 and fibrosis mediation, 420 functions, 180, 181 homeostasis, 180–1 origins, 179–80 roles, 188 ALX receptor (ALXR), 601–2 Amaranthus spp. (pigweeds), 957 Amb a 1, 914, 920, 1518, 1561, 1562 Amb a 2, 920 Amb a 5, 920 Ambrosia spp. (ragweeds), 943, 958, 959 see also ragweed pollen Ambrosia artemisiifolia (common ragweed), 958

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Ambrosia trifida (giant ragweed), 958 AMC (acidic mammalian chitinase), 387 American Academy of Allergy, Asthma and Immunology, 949 guidelines, 1522 American Academy of Pediatrics, 1905 American College of Allergy, Asthma and Immunology, guidelines, 1522 American Conference of Governmental Industrial Hygienists (ACGIH), 1017– 61 American Thoracic Society (ATS), 1371, 1575 occupational asthma studies, 1689– 90 amino acids, modification, 203– 4 aminophylline, in anaphylaxis treatment, 1915 aminopyrine, 1975 ammonium hexachloroplatinate, 1703 AMP see adenosine monophosphate (AMP) AMs see alveolar macrophages (AMs) α-amylase, 1703 anaphylactic mediators, 8– 9 anaphylactic response, components, 1900–1 anaphylactic shock, 1948– 9 use of term, 1949 see also anaphylaxis anaphylatoxin receptors, 376, 447 anaphylatoxins, 324, 1906 anaphylaxis, 6, 1897– 920, 1948–50 acute attacks, management, 1914–16 agents, 1900 airway reactions, 1914–15 antihistamine therapy, 559– 60 biochemical features, 1899– 910 and bradykinin, 465 cardiovascular reactions, 1915–16 classification, 1901 clinical features, 1899– 910 cutaneous reactions, 1914–15 definition, 1330, 1899 desensitization long-term, 1913–14 short-term, 1913 diagnosis, 1330 diagnostic criteria, 1899 differential diagnosis, 1910–11 discovery, 3– 4 drug-induced, 1901–2 early studies, 567 epidemiology, 1899 etiology, 1899– 908 exercise-induced, 1863– 4, 1907– 8 and food allergy, 1328, 1903–5 food-induced, 1903–5, 1930–1, 1935 gastrointestinal, 1927 historical background, 3– 4, 1897– 8 chronology, 1898 and Hymenoptera venom, 1898, 1902– 3, 1905, 1984 idiopathic, 1908 immunoglobulin-E-mediated, 1901– 6 immunotherapy, 1913–14 and insect venoms, 1902–3, 1914 intraoperative, 1165 and latex, 1898, 1903 and latex allergy, 1173, 1903 management, 1911–16

acute, 1422 kits, 1176 principles, 1911–13 and mast cells, 8 mast cells in, 244 mechanisms, 1900 occupational, 1326 origin of term, 1897 pathogenesis, 1908–9 and penicillin, 1898, 1901 pharmacologic pretreatment, 1913 postcoital, 1906 prevention, 1911, 1913–16 prognosis, 1916 recovery, 1916 risk factors, 1529 therapeutic-protein-induced, 1905 treatment, 1911–16 triggers, 1330 novel, 1905–6 unknown mechanisms, 1907–8 use of term, 5 venom, 1511 see also non-IgE-mediated anaphylactoid reactions anaphylaxis hypothesis, 1905 Anaplasma phagocytophila (bacterium), 311 ANCAs (antineutrophil cytoplasmic antibodies), 1786, 1787 Ancient Egypt, anaphylactic shocks, 3 Ancylostoma braziliense (dog hookworm), 1791 Ancylostoma duodenale (hookworm), 1791 infestations, 1247 anemophilous plants, 943 anesthetic agents, allergic reactions to, 1326 ANG12, 1138–9 angioblasts, and stem cells, 404 angioedema, 20, 1326, 1853–77, 1929 after direct mast cell degranulation, 1865 and arachidonic acid metabolism abnormalities, 1865 blood product reactions, 1864 bradykinin in, 1859 cellular infiltrate in, 1857–8 characterization, 1853 chronic idiopathic, 1865 diagnostic approaches, 1859–60 epidemiology, 1854 hereditary, 1866–7, 1910 IgE-dependent, 1860–1 IgE receptor-dependent, 1860–1 and infections, 1865 laboratory findings, 1868–9 mast cells in, 1854–5 pathogenesis, 1854–9 physical, 1861–4 prevalence, 1854 specific antigen sensitivity, 1860–1 testing procedures, 1869 treatment, 1869–72 vibratory, 1862 see also aspirin-induced urticaria/angioedema; urticaria angiogenesis in asthma, 399

drivers of, 402–3 mast cells in, 228–9 mechanisms, 402 prepared fields, 404 and vasodilation, 401–2 Angiostrongylus cantonensis (nematode), 273 Angiostrongylus spp. (nematodes), 273 angiotensin-converting enzyme (ACE), 452, 462, 515–16 inhibition, kinins and, 464 roles, 463 angiotensin-converting enzyme (ACE) inhibitors, 1867–8 animal allergens, 997–1016 aerodynamics, 998–9 distribution, in homes, 998–9 and extrinsic allergic alveolitis, 1762 and perennial rhinitis, 1324 studies, 997 see also pet allergens animal confinement facilities, occupational airway diseases, 1061 animal models age effects, 1219 airway hyperresponsiveness, 1202 allergic airway inflammation, 502–3 allergic conjunctivitis, 1486–7 animal supplier issues, 1219 asthma, 54–5, 65, 1185–222 eosinophils in, 276–7 issues, 1214–22 mast cells in, 241 bronchi, 1217 endotoxins, 150 exercise-induced bronchoconstriction, 794–807 food allergens, 1158 helminth infections, 154 hyperventilation-induced bronchoconstriction, 794–807 infections, 2026–7 innervation in, 1216–17 mucociliary clearance, 1216 precautions, 1220 primates, 1187–201 respiratory tract, species differences, 1215–19 strain effects, 1219 tobacco smoke effects, 148–9 whole-lung, 794, 795 see also mouse models; small animal models animal proteins, 1102 animals laboratory, 1701–2 late-phase airway obstruction, 799–802 proximity to, and hygiene hypothesis, 1246 see also pets annatto, 1926 anogenital area, lichenification, 1817 Anopheles gambiae (mosquito), 1138–9 anterior neck folds, 1818 anthropometric measurements, and allergic diseases, 38 anthroposophic lifestyle, and hygiene hypothesis, 1246 antiallergens, immunoglobulin E responses, spectral studies, 915–17

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antiasthma drugs and bronchial hyperresponsiveness, 788– 9 evaluation, 788 antibiotics and allergic diseases, 35– 6 and anaphylaxis, 1901–2 asthma induction, 2007– 8 in atopic dermatitis treatment, 1826 early exposure to, 35 extensive use of, 1147 and infants, 2007– 8 macrolide, 851–2 antibodies, 741–2 binding, 1351–3 to allergens, 914–15 characteristics, 438 function, 119– 20, 121–2 polyclonal, 741 specificity, 119– 20 structure, 121–2 see also monoclonal antibodies antibody-mediated drug hypersensitivity reactions, 1947–52 classification, 1948 type I, 1947– 9 antibody repertoire, 119–20 anti-CCR3, in nasal polyposis treatment, 1469 anticholinergic agents, 824 in adult asthma, 689– 90 adverse effects, 690, 1652 for airway hypersecretion, 848 in allergic rhinitis treatment, 1439 in asthma treatment, 1332 as bronchodilators, 683 in chronic asthma treatment, 1651–2 in chronic obstructive pulmonary disease acute exacerbations, 688– 9 stable, 686– 8 clinical uses, 686– 90 dose–response relationships, 686 pharmacology, 684–5 rationale for use of, 683–4 see also cholinergic antagonists anti-eosinophil therapeutics, 280–1 anti-eotaxin, in nasal polyposis treatment, 1469 antifungal drugs, in allergic bronchopulmonary aspergillosis treatment, 1752–3 antigen 5, 1124–5, 1126 antigen–antibody complexes, 436, 438 antigen avoidance, extrinsic allergic alveolitis, 1771–2 antigen-binding test, 1359 antigenicity, use of term, 1944 antigen presentation, 168–70, 267– 9 endogenous, on major histocompatibility complex class I to CD8+ T cells, 169 exogenous on major histocompatibility complex class I to CD8+ T cells, 169–70 on major histocompatibility complex class II, to CD4+ T cells, 168– 9 and mast cells, 230 and rhinitis, 1407– 9

antigen-presenting cells (APCs), 472, 595–6 activation, 1999 and late-phase allergic reactions, 529 antigen processing, in dendritic cells, 168 antigens diagnostic, 979 in immune complex disease, 438 antigen uptake, mechanisms, 167–8 antihistamines, 551–65, 705–6, 849–50 adverse effects, 560–2 in allergic rhinitis treatment, 1436–8 in anaphylaxis treatment, 1911 in atopic dermatitis treatment, 1826 in atopic keratoconjunctivitis treatment, 1499–500 clinical pharmacology, 554–60 in hay fever treatment, 1331 and late-phase allergic reactions, 531–2 pharmacodynamics, 555–7 pharmacokinetics, 554–5 in seasonal allergic conjunctivitis treatment, 1489, 1490 in urticaria treatment, 1869–72 H1-antihistamines efficacy, 557 first-generation adverse effects, 560–1 efficacy, 557, 559 in elderly, 562 pharmacodynamics, 555 pharmacokinetics, 554 therapeutic index, 560 future trends, 562 in infants, 561–2 in lactation, 562 in mastocytosis treatment, 1889, 1890 oral, 1436–7 properties, 1437 in pregnancy, 562 second-generation adverse effects, 561 clinical pharmacology, 557–60 dosing, 557 efficacy, 557 pharmacodynamics, 555–7 pharmacokinetics, 554–5 topical, 1437– 8 anti-IgE in allergic rhinitis treatment, 1440–1 in asthma treatment, 1712, 1720 in chronic asthma treatment, 1653–4 development, 1667–8 and late-phase allergic reactions, 532–3 in persistent severe allergic asthma, 1661–86 see also omalizumab anti-IgE receptor antibody binding assays, 1856 functional assays, 1855–6 anti-IL-5 see anti-interleukin-5 (anti-IL-5) antiinflammatory agents in chronic urticaria treatment, 1871–2 in exercise-induced bronchoconstriction, 812 antiinflammatory cytokines, 1723, 1764 anti-interleukin-5 (anti-IL-5), 1373 in hypereosinophilic syndrome treatment, 1507

antileukotrienes, 694–714, 849–50, 1973 in acute asthma, 704–5 as add-on therapy, 722 administration, oral vs. inhaled, 697 adverse effects, 697 in allergic rhinitis, 705–6 in allergic rhinitis treatment, 1438–9 applications, 706–8 in aspirin-induced asthma, 706–7 asthma monotherapy, 697–9 in chronic asthma treatment, 1653 in concomitant asthma and allergic rhinitis, 706 drug development, 696 efficacy, 708–9 in exercise-induced asthma, 707–8 indications, 697 and inhaled corticosteroids, in asthma, 699–704 pathophysiology, 695–6 prescribing, 696–7 responsiveness, pharmacogenetic determinants, 708 vs. β2 agonists, 700–2 antimetabolites, 737 antimicrobials, secreted, 373–6 antimuscarinic agents, pharmacodynamics, 685 antineutrophil cytoplasmic antibodies (ANCAs), 1786, 1787 antineutrophil strategies, 313 antioxidants, 852, 2001 antiproliferative agents, 733–7 antipyrine, 1975 antisense oligodeoxynucleotides (AS-ODNs), 1725–6 structure, 1726 antiserum, 4 antitoxins, 4–5, 12 ants, 1983 insect sting allergy, 1983 see also fire ants ant stings, allergic reactions, epidemiology, 1986 ant venom allergens, 1125 AP-1 (activator protein-1), 72, 717 APCs see antigen-presenting cells (APCs) APHEA (Air Pollution and Health: a European Approach), 1250 apical intercellular adhesion complex, 369 Apidae (bees), 1123, 1902 see also honeybees Api g 1, 1150 Api m 1, 95, 1558, 1559, 1560–1 Api m 2, 95, 1559 Api m 3, 95, 1559 Api m 6, 1124 Apis cerana (eastern honeybee), 1124 Apis dorsata (giant honeybee), 1124 Apis mellifera (honeybee), 1902, 1981 venom, 95, 1123–4 aplastic anemia, 439 apoptosis, 20, 49–50 neutrophils, 307–8 signaling pathways, 308–10 spontaneous, 308 apovitelenins, 1151 apples, allergens, 1153 APRIL (a proliferation-inducing ligand), 128, 132

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a proliferation-inducing ligand (APRIL), 128, 132 aprotinin, 868 aquagenic pruritus, 1864 aquagenic urticaria, 1864 AR see allergic rhinitis (AR) arachidonic acid, 37 products, 1410, 1411 arachidonic acid metabolism abnormalities, 1865 modulation, 1907 Ara h 1, 914, 915, 916–17, 1149, 1151, 1154, 1155 reactivity, 1158 Ara h 2, 914, 915, 916–17, 1151, 1154, 1156 Ara h 3, 914, 916–17, 1154 Ara h 4, 914, 916–17 Ara h 6, 914, 915 Ara h 8, 1153 ARDS (acute respiratory distress syndrome), 311, 423 area under the concentration–time curve (AUC), 732 ARF, 205 ARIA initiative see Allergic Rhinitis and its Impact in Asthma (ARIA) initiative arofylline, structure, 644 arrestins, 479 ARS see acute rhinosinusitis (ARS) arsenic, 1285 Artemisia spp. (wormwoods and mugworts), 958 pollen, 960 arthritis, 437 Arthus, Maurice (1862–1945), 4–5 Arthus reaction, 18, 439 Art v 1, 921 asbestos, 1283 Ascaris spp. (nematodes), infections, 2026 Ascaris lumbricoides (nematode), 154 infection, 1247, 1790 tropomyosins, 1137 Ascaris suum (nematode), 2027 infections, 1585 Ascaris pneumonia definition, 1790 symptoms, 1790 treatment, 1790 ascospores, 965 ASGPR (asialoglycoprotein receptor), 109, 110–11 asialoglycoprotein receptor (ASGPR), 109, 110–11 ASL see airway surface liquid (ASL) ASM see airway smooth muscle (ASM) AS-ODNs see antisense oligodeoxynucleotides (AS-ODNs) Asp303, 1138 asparagine residues, 1126 aspartic protease, 1131 Aspergillus spp. (molds), 230, 974, 1282, 1570–1, 1743– 4 abundance, 967 allergens, 974, 1061, 1205 infections, 1743, 1791 occurrence, 971, 972–3, 977– 8 seasons, 967 spores, 1658 Aspergillus flavus (mold), 1464

Aspergillus fumigatus (mold), 376, 485, 1170, 1464, 1743–4 allergens, 974, 1205, 1749 characteristics, 1745 and eosinophilic esophagitis, 1925 infections, 1745 skin tests, 1342 spores, 481, 1658 Aspergillus oryzae (mold), 1761 α-amylase, 1703 Aspergillus bronchitis, in cystic fibrosis, 1748 Asp f 1, 974, 1749 Asp f 2, 1749 Asp f 3, 974, 1749 Asp f 4, 1749 Asp f 5, 974, 1749 Asp f 6, 974 Asp f 11, 974 Asp f 22, 1749 asphyxia, 1949, 1966 aspirin activity, and cyclooxygenases, 590–1 and anaphylaxis, 1907 and angioedema, 1865 asthma induction, 236–7 cross-reactions, 1971 nasal challenge, 1972–3 oral challenge test, 1972 and urticaria, 1865 aspirin hypersensitivity, 1326, 1966–79 diagnosis, 1971–3 effector pathways, 1968–71 genetic susceptibility, 1968 aspirin-induced asthma (AIA), 236–7, 586, 1967–73 antileukotrienes in, 706–7 characterization, 1966 definition, 1967 diagnosis, 1971–3 disease course, 1971 historical background, 1966–7 and innate antiviral responses, 1967–8 management, 1657 and nasal polyposis, 1573 natural history, 1971 pathogenesis, 1967–71 presentation, 1971 prevalence, 1967 prevention, 1973 treatment, 1973 and variant C allele, 708 aspirin-induced urticaria/angioedema, 1974–5 characterization, 1966 clinical presentation, 1974 definition, 1974 diagnosis, 1974 histopathology, 1974 historical background, 1966–7 mechanisms, 1974 prevalence, 1974 prevention, 1974–5 treatment, 1974–5 aspirin-sensitive respiratory disease (ASRD), 590–1, 603 aspirin-sensitive rhinitis, 513

aspirin-sensitive rhinosinusitis (ASRS), 1462 aspirin sensitivity, and nasal polyposis, 1459, 1462–3 ASRD (aspirin-sensitive respiratory disease), 590–1, 603 ASRS (aspirin-sensitive rhinosinusitis), 1462 astemizole, 1437 asthma, 1565–739 acute, 704–5 acute severe, 726 adult, 689–90 aerosol delivery systems, 778–9 airflow limitation measurement, 1579–80 reversibility, 1580, 1581 and air pollution, 1250 airway epithelium in, 1614–19 airway hyperresponsiveness in, 749, 812, 840–1, 1608 airway hypersecretion in, pathophysiology, 842–4 airway inflammation monitoring, 1376–7 variable, 1623 airway remodeling in, 1202, 1203, 1619–20, 1623, 1632–3 and airway smooth muscle, 759–60 airway smooth muscle in allergic roles, 880–1 contractile roles, 878–80 mechanical plasticity, 878–80 proliferative roles, 883–5 wall content decrease, 886–7 airway vascularity in, 398–411 airway wall nerves in, 1641 allergen-induced, 585 allergen injection immunotherapy indications, 1523–5 treatment strategies, 1523 allergens, 142–3, 984, 1570–1 and allergic bronchopulmonary aspergillosis, 1746–7 with allergic rhinitis, 1386–7, 1430, 1431 antileukotrienes in, 706 treatment, 1443–5 and allergy, 1325 alveolar macrophages in, 181 ancillary tests, 1583–4 angiogenesis in, 399 animal models, 54–5, 65, 1185–222 issues, 1214–22 antibiotic-induced, 2007–8 anticholinergic agents in, 689–701 antileukotriene monotherapy, 697–9 autonomic nervous system in, 823–39 basophils in, 1613 biomarkers, 1716–17 and bradykinin, 464–5 and breastfeeding, 1572, 2001–2 bronchial blood flow in, 763 bronchial hyperresponsiveness in, 787–8 bronchial smooth-muscle mass increase, 1637–9 hyperplasia vs. hypertrophy, 1638 muscle plasticity, 1638–9 bronchial vasculature in, 1639

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asthma (Cont’d ) CD8 in, 71–2 changes in clinical remission, 756 characterization, 1214 children, 1591– 607 chronic allergic, 233– 4 and chronic obstructive pulmonary disease, 1574–5, 1656 and Churg–Strauss syndrome, 1785 classification, 1568–70 clinical, 584–5 clinical features, 1573– 6 clinical phenotypes, 1573– 6 and cockroach allergens, 1131– 45, 1571 comorbidities, 1430, 1431 costs, 23, 1593 cough variant, 1573– 4 and cyclooxygenases, 589– 90 cytokines in, 48– 82 definition, 1240, 1567– 8 dendritic cells in, 177, 1641 diagnosis, 1325, 1335, 1433, 1576– 82 and diet, 1249, 1572 differential diagnosis, 790, 1581, 1584–5 difficult, 1575 disease course, 1593– 4 distal lung in, pathology, 1641–2 drug-induced, 236, 1390, 1572 and drugs, 1572 and dust mites, 984– 96 exposure to, 988 early studies, 9–14, 831–2 economic burden, 1593, 1661 elderly, 1657 and endotoxins, 34 and environmental tobacco smoke, 149, 1571 and eosinophilia, 984 eosinophilic, 1621, 1794–5 eosinophils in, 276–9, 984, 1611–13 animal models, 276–7 epidemics, 1225 epidemiology, 1239–58 and epithelial cells, 418 epithelial changes, 1634–5 etiology, 1325, 1569 exacerbations, 279, 1575– 6, 1577 and acute viral infections, 1247 and air pollution, 1267 management, 1578, 1653 exhaled breath condensate, 1584 experimental allergen-induced, 231–2 early reaction, 231–2 late reaction, 232 extrathoracic airways in, 762–3 and fibrosis, 412–13 food-induced, 1930 fractional exhaled nitric oxide, 1584 fungus-induced, 965 future research, 1623– 4 and gastroesophageal reflux, 1573 and gender, 1243– 4, 1570 genes, 1229 genetics, 1206– 7, 1225– 38, 1247– 8, 1260, 1570, 1717 novel mechanisms, 1623

genome screens for, 1226–7 genome-wide association studies, 1227–8 geographic variations, 2020–1 and hay fever, 12 heterogeneity, 1620–2, 1623 histopathology, 1634–9 hospital admission rates, 1243 and house-dust mite allergens, 142, 1570–1 animal models, 1194 hypervascularity in, 399 immune cells in, 1219–20 immune response, 1609–10 and immunoglobulin E, 141, 1239 and immunoglobulin E sensitization, 1240–1 and indoor air pollution, 1280, 1572, 2010 induced sputum, 1583–4 infants, 1591–607 and infections, 1571 inflammation in, 1639–41 inhalation tests, 1583 intrinsic, 12 irritant-induced, 1571 leukotrienes in, 584–6 and lipoxins, 602–4 lung appearance at postmortem, 1633–4 lung function tests, 750–6, 1580–2, 2023 macrophages in, 1613 and major histocompatibility complex, 1227 mast cells in, 231–43, 1296, 1610–11, 1640 animal models, 241 evidence, 231–7 medical history, 1577–9 monocytes in, 1613 mortality, 1593, 1661 and mouse allergen, 142–3 mucociliary clearance in, 845 mucus in, 840–56 and nasal polyposis, 1573 and nasal polyps, 1461 natural exacerbations, and eosinophils, 279 natural history of, 1593–4 natural killer T cells, 71–2 nerve growth factor in, 513–14, 1620 nerves in, 831–4 neural remodeling in, 1620 neurotrophins in, 1620 neutrophils in, 313 new cytokines in, 65–70 nocebos, 834 nocturnal, 833–4 nonatopic, 63 noneosinophilic, 64, 1621 and nutrition, 1249 and obesity, 1570 occupational agent-induced, 1061, 1102 and occupational sensitizers, 1571 and outdoor air pollution, 1572 pathogenesis, 1608–31 airway inflammation, 1608–9 cytokines in, 62–74 hypothesis, 63 T cells in, 62–74 pathology, 1632–49 pathophysiology, 1325

and eosinophilic airway inflammation, 1374–5 paucigranulocytic, 1621 persistence, and atopy, 1618–19 persistent, β2 agonists in, 703–4 and pets, 998, 1001, 1002–12 phenotypes, 63–4, 276 and phosphodiesterase 4 inhibitors, 643 physical examination, 1579 physiology, 749–67 placebos, 834 and plant allergens, urban areas, 1272–3 and platelet-activating factor, 606–8 and pregnancy, 1657 premenstrual, 1574 prevalence, 23, 1225, 1239, 1661 racial and ethnic differences, 1244 regional differences, 992–3 prostaglandin E2 in, 598–9 prostanoids in, 591–600 regulatory T cells in, 83–102 remodeling, 277–8 and respiratory infections, 1572–3 respiratory tract mucosa, 136 reticular basement membrane in, 1635–7 and rhinitis, 1573 risk factors, 29, 1570–3 age, 1243 air pollution, 1572 allergens, 1570–1 contributing, 1572–3 diet, 1572 drugs, 1572 environmental, 1570–2 evaluation, 1582–4 family structure, 1244–5 gastroesophageal reflux, 1573 gender, 1243–4, 1570 genetic, 1570 host, 1570 infections, 1571 medical history, 1582 nasal polyposis, 1573 obesity, 1570 occupational sensitizers, 1571 personal, 1243–5 race, 1244 respiratory infections, 1572–3 rhinitis, 1573 sinusitis, 1573 socioeconomic status, 1244 tobacco smoke, 1571 sensory nervous system in, 823–39 severe, 64 severity, 1569 classification, 1662–3 and sinusitis, 1472, 1573 small airways in, 757–8 and smoking, 1574–5, 1657–8 and soybean dust, 1272–3 spirometry, 1579–80 steroid-resistant, 718 subepithelial matrix deposition, 1635–7 sublingual immunotherapy, clinical efficacy, 1545–7

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subphenotypes, categorization, 1622 susceptibility genes, 1229, 1230, 1623, 1624 symptoms, 1325, 1573 T cells in, 48– 82 and airways, 70–1 γδ T cells in, 71–2 T helper 2 cells in, 62–3, 70–1, 1609–10 therapeutic targets, novel, 241–3 tissue remodeling, 398 and tobacco smoke, 148, 1571, 1621 tolerogenic mechanisms in, 83–102 toluene diisocyanate, 235– 6 and transforming growth factor-β, 88, 1636–7 triggers, 10–11, 829, 984, 1325 and tuberculin sensitivity, 1264 types of, 63– 4 upper airways in, pathology, 1642– 3 use of term, 1214 vascular remodeling in, 1620 vasodilator hypothesis, 668– 9 and viral infection, 26 virus-induced, 235 western red cedar, 235– 6 wheezy breathlessness, 277, 763 see also aspirin-induced asthma (AIA); atopic asthma (AA); brittle asthma; bronchial asthma; chronic asthma; exercise-induced asthma (EIA); late asthmatic reactions (LARs); near-fatal asthma (NFA); nonallergic asthma; occupational asthma; pediatric asthma; refractory asthma; thunderstorm asthma asthma control, 1569–70, 1655 levels, 1652, 1663 measurement, 1575 Asthma Control Questionnaire (ACQ), 1575 Asthma Control Scoring System, 1575 Asthma Control Test (ACT), 1575 asthma genes, 40 Asthma Insights and Realities (AIR), surveys, 1661 asthma management goals, 1368 guidelines, 1567– 8 Asthma Therapy Assessment Questionnaire (ATAQ), 1575 asthmatic airways cross-sections, 1608 diagnostic criteria, 832 mast cell microlocalization, 237– 41 asthmatic responses bronchial vessels in, 398, 399 cellular infiltration, 398 asthma treatment, 72– 4, 1330– 3, 1565–739 current, 1713 drug therapy, 1331–2 future trends, 1727 new drugs, 1712–39 goals, 1650 inhaled corticosteroids, 1332, 1370, 1712, 1713 and antileukotrienes, 699–704 inhaled glucocorticoids, 721–2 new approaches, rationale, 1713–14 novel targets, 1713, 1714

asymptomatic airway hyperresponsiveness (AAHR) differential diagnosis, 1568 risk factors, 1568 ATAQ (Asthma Therapy Assessment Questionnaire), 1575 atenolol, 679 atopic asthma (AA), 63, 179 children, 1594 atopic blepharoconjunctivitis (ABC), 243–4 atopic conjunctivitis definition, 1387 see also atopic keratoconjunctivitis (AKC) atopic dermatitis (AD), 26, 1813–30 acquired immunity, 1823 adolescents, 1816 adults, 1816 and age, 1259–60, 1261 and allergens, 1819 antihistamine therapy, 559 as autoimmune disease, 1824–5 and bacterial infections, 1820 chemokines in, 1823–4 children, 1816 clinical features, 1814–18, 1929–30 complications, 1820–1 cytokines in, 1823–4 definition, 1813 dendritic cells in, 178–9, 1824 diagnosis, 1326–7 diagnostic criteria, 1818–19 differential diagnosis, 1815, 1821 disease course, 1813, 1814–18 education, 1827 and food allergy, 1819 future trends, 1827 genetics, 1225–38, 1260, 1821–2 genome screens for, 1227 and growth delay, 1821 histology, 1819 and hygiene hypothesis, 1822 immunologic mechanisms, 1823–4 infants, 1815 innate immunity, 1823 itch triggers, 1815 latex-induced, 1165 mast cells in, 244–5 and microbial agents, 1819–20, 1824 minimal variants, 1817 neurotrophins in, 506–7 ocular complications, 1499 pathogenesis, 1821–4 prevalence, 1225, 1259–60, 1813, 1814 prognosis, 1260, 1262 psychological factors, 1822 and quality of life, 1820–1 and skin barrier dysfunction, 1822 and stress, 1819 T cells in, 1823 treatment, 1825–7 systemic, 1826–7 topical, 1825–6 trigger factors, 1819–20 and viral infections, 1820 see also atopic eczema; eczema

atopic diseases and air pollution, 1250 and allergen exposure, 1261–3 and diet, 1249 and domestic environment, 1261–3 epidemiology, 1239–58 geographic variations, 2020–1 hereditary factors, 1260 and immune system development, 1259 and lifestyle, 1260–1 and microbial infection, 1232–3 natural history of, 1259–60 nongenetic factors, 1260–4 parental, 1821 and pollutants, 1263 prevalence, 1165– 6, 1264 assessment tools, 2022–5 developing countries, 2020–30 protection against, and microbial exposure, 1233 questionnaires, 2022 –3 and tobacco smoke, 1263 atopic eczema adults, 1816 children, 1598 development, 30 diagnosis, 1326–7 differential diagnosis, 1821 infants, 1815 lichenification, 1817 neonatal protection, 30 symptoms, 30 triggers, 1327 use of term, 1813 see also atopic dermatitis (AD) atopic keratoconjunctivitis (AKC), 243–4, 1482, 1497–500 clinical features, 1497–9 histology, 1499 pathogenesis, 1487 pathogenetic mechanisms, 1499 treatment, 1499–500 atopic stigmata, 1817–18 atopy, 1813–14 and allergic diseases compared, 2022 and asthma persistence, 1618–19 and breastfeeding, 2002 definition, 1239–40 development, 23–47 diagnosis, 2023–5 epidemiology, 1239–58 and latex allergy, 1172 maternal, 37–8 occupational asthma, 1691–2 origin of term, 13 prevalence assessment tools, 2022–5 developed countries, 2020 questionnaires, 2022–3 skin-pricking tests, 2023–4 use of term, 1225, 1813–14 ATP see adenosine triphosphate (ATP) Atriplex spp. (saltbushes), 957 Atropa belladonna (deadly nightshade), atropine, 683

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atrophic rhinitis, etiology, 1391–2 atropine, 683, 684–5 adverse effects, 690 atropine sulfate, structure, 685 Atrovent see ipratropium bromide ATS see American Thoracic Society (ATS) AUC (area under the concentration–time curve), 732 Austen, K. Frank (1928– ), 9, 10 Australia, thunderstorm asthma, 1271–2 autoallergic, use of term, 6 autoimmune, use of term, 6 autoimmune diseases atopic dermatitis as, 1824–5 prevalence, 23 autoimmune lymphoproliferative syndrome, 49–50 autoimmunity, and chronic urticaria, 1855 autonomic innervation lower airways, 827 parasympathetic, 824, 827 sympathetic, 824–5, 827 upper airways, 824–5 autonomic nerves, modulation, 830 autonomic nervous system in allergic rhinitis, 823–39 in asthma, 823– 39 components, 512 AVE5883, 531 AWD-12-281, structure, 644 axon reflex, 495 mechanisms, 825 azathioprine, 733–5 adverse effects, 734–5 in atopic dermatitis treatment, 1826–7 drug interactions, 734–5 indications, 734 mechanisms, 734 metabolic pathway, 734 monitoring methods, 734 pharmacogenetics, 733– 4 pharmacokinetics, 733 structure, 733 azelastine, 557, 1437– 8 in perennial allergic conjunctivitis treatment, 1492–3 aztreonam, 1902 azurocidin, 868 azurophil granules, 297, 305 inhibition, 311 bacillus Calmette-Guérin (BCG), 1246–7, 2009 Bacillus cereus (bacterium), 1760 Bacillus subtilis (bacterium), 1760 bacteria abundance, 964 exposure, infants, 2006–7 and extrinsic allergic alveolitis, 1758– 60 and indoor air pollution, 1282–3 particles, 963 bacterial immunoglobulin superallergens, 327 bacterial infections and atopic dermatitis, 1820 in chronic rhinosinusitis, 1454–5 mast cells and, 230

bacterial products, and immunity, 2009–10 BAFF (B-cell activating factor), 128, 132, 386–7 bagassosis, 1758–60 bakers, occupational asthma, 1700, 1702 baking industry enzymes, 1703 occupational airway diseases, 1061 BAL see bronchoalveolar lavage (BAL) BALB/c animal models, 1158, 1206, 1207 particle deposition, 1219 BALT (bronchial-associated lymphoid tissue), 385 bambuterol, 674 bananas, allergens, 1150 barium enema catheters, 1165 βARK (β adrenoceptor-associated kinase), 678 barrier cells and allergic inflammation, 188 and innate immunity, 188 basal epithelial cells, 370 roles, 1635 base excision repair (BER), 127 basement membrane, 371, 415 see also reticular basement membrane (RBM) basement membrane zone (BMZ) development, 1193 exposure responses, 1196–7 thickening, 1194 BASH (B-cell linker protein), 120, 207 basidiomycetes, allergens, 974–5 basidiospores, 965, 971, 974, 975 basogranulin, 322–3 basophil activation analysis, read-outs, 1362 flow cytometric analysis, 1360–1 tests, 1175 basophil adhesion, 349 basophil migration, 349 basophil responses, heterogeneity, 1359–60 basophils, 8, 295 and allergic inflammation, 1296–7 in allergic rhinitis, 1418 in asthma, 1613 biological properties, 320–36 in bronchial asthma, 325 in chronic urticaria, 1858 degranulation, 896–7 functions, 324 and mast cells compared, 320 mediator release, 320–2 mediators, 322–3 morphology, 322 origin, 322 pharmacologic modulation, 328–30 reactivity, 1359–60 recruitment to allergic airways, 325–6 by chemokines, 483 releasibility, 1858 roles in allergic angiogenesis, 327–8 in allergic diseases, 320–36 sensitivity, 1359–60 surface markers, 323–5 ultrastructure, 322

basophil simulation tests allergen selection, 1361 for allergy diagnosis, 1359–64 challenge concentration, 1361 clinical results, 1362–3 experimental issues, 1361 indications, 1363–4 interpretation, 1364 methods CD63-based, 1362 CD203c-based, 1362 mediator-based, 1362 negative control, 1361 performance, 1364 positive control, 1361 read-outs, 1362 signal enhancement, 1361 technical issues, 1361 BAY 60-7550, 641 structure, 641 4-1BB, 54 B-cell activating factor (BAFF), 128, 132, 386–7 B-cell linker protein (BLNK), 120, 207 B-cell maturation antigen (BCMA), 128 B-cell receptor (BCR) complex, 108 B cells, 28–9, 1292 antigen recognition, 49 development, 120 and diesel exhaust, 146 differentiation, 120 into plasma cells, 134 expression, 48–9, 386 interactions, 193 response amplification, 1746 BCG (bacillus Calmette-Guérin), 1246–7, 2009 Bcl-2, 308–10 BCMA (B-cell maturation antigen), 128 BCR (B-cell receptor) complex, 108 BDNF see brain-derived neurotrophic factor (BDNF) BDP see beclomethasone dipropionate (BDP) Becker, Elmer L., 5 beclomethasone, 699 beclomethasone dipropionate (BDP), 405, 718–19 adverse effects, 724 efficacy, 721 in pediatric asthma treatment, 1602–3 structure, 718 bees, 1123, 1902 see also bumble bees; honeybees bee venom allergens, 914–15, 917, 1123–4, 1326, 1558, 1559, 1902 bee venom allergy, T-cell peptide epitopes in, 1560–1 bee venom immunotherapy, 1516 Bennich, Hans (1930– ), 7, 8, 1346, 1665–6 benzalkonium chloride, 1390 benzoates, 1328 benzylpenicillin, 1945 Beraldo, Wilson Teixeira (1917– 98), 9 BER (base excision repair), 127 Bergstrom, Sune (1916–2004), 567

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beta-blockers, 1529 cardioselective, 1572 Betula spp. (birch trees), 944, 954–5 see also birch pollen Betula pendula (silver birch), 954–5 Betula verrucosa (white birch), allergens, 899 Betulaceae (birch family), 954 bet v 1, 95, 168, 899, 901, 913, 916, 920, 930, 954, 1152 and air pollution, 945 cross-reactivity, 1149–50, 1355 extracts, 1349 mutations, 1556 processing, 1156 bet v 2, 916 bet v 4, 916 bet v 7, 916 bet v 11, crystal structure, 1556–7 BGs (Birbeck granules), 167 BH3 domain, 309–10 BHR see bronchial hyperresponsiveness (BHR) Bifidobacterium spp. (bacteria), 2009 Bifidobacterium infantis (bacterium), 36 ‘Big Eight’, 1147 biglycan, 414 bimosiamose, 1723 ε-binding protein (galactin-3), 113 biological markers see biomarkers biologics, in atopic dermatitis treatment, 1827 biomarkers allergy risk, 1999– 2000 for chronic obstructive pulmonary disease, 1717 in exhaled breath, 1716–17 in sputum, 1716–17 Birbeck granules (BGs), 167 birch pollen, 897, 899, 913, 954– 5, 1150, 1178 extracts, 1349, 1355 proinflammatory substances, 945– 6 seasons, 943, 955 severity, annual variations, 955 tyrosine components, 946 birch pollen allergen, 95, 901 birds, and allergic diseases, 1324 birth order, and allergic diseases, 32 bisoprolol, 679 biting insect allergy, 1991–2 allergens, 1992 prevention, 1992 symptoms, 1991–2 treatment, 1992 Bixa orellana (lipstick plant), 1926 Blackley, Charles H. (1820–1900), 12, 13, 524, 945, 1592 Bla g 1, 1131–2, 1134, 1135 antibodies, 1136 exposure, 1132, 1133, 1134 functions, 1132 prevalence, 1139 reduction, 1139 Bla g 2, 914, 916, 922, 1131–2 antibodies, 1136 cloning, 1134 exposure, 1132, 1133, 1134 prevalence, 1135

reduction, 1139 structure, 1132 crystal, 1137 tertiary, 1137–8 Bla g 4, 1131, 1134, 1137 binding, 918 functions, 1132 reproductive function, 1139 structure, tertiary, 1137 Bla g 5, 914, 916, 1131, 1134 binding, 918, 922 prevalence, 1135 Bla g 6, 1134 homologs, 1137 structure, tertiary, 1137 Bla g 7, 922, 1131, 1134 binding, 918 homologs, 1137 Bla g 8, 1134 Blattella germanica (German cockroach) allergens, 1131–2, 1134 antibodies, 1136 antigen sensitization, 1132, 1135 occurrence, 1134 bleaches, 1101 BLIMP1 (B-lymphocyte maturation protein 1), 121, 134, 136 BLNK (B-cell linker protein), 120, 207 Bloch, B., 1832 blocking antibody discovery, 13–14 model, 1514–15 Blomia tropicalis (house-dust mite), 916 blood flow, airways, 399–400 blood mast cells see basophils blood pressure, regulatory mechanisms, 463 blood tests, 1322 BLT1, 566, 576–7 function, 577 structure, 577 BLT2, 566, 577 B-lymphocyte maturation protein 1 (BLIMP1), 121, 134, 136 B lymphocytes see B cells BMMCs (bone marrow-derived mast cells), 219 BMPs (bone morphogenetic proteins), 1636–7 BMT (bone marrow transplantation), in mastocytosis treatment, 1890 BMZ see basement membrane zone (BMZ) Bombus agrorum (bumble bee), 1981 Bombus pennsylvanicus (bumble bee), 1124 Bombus terrestris (bumble bee), 1981 bone marrow dendritic cells, 167 eosinophil egress from, 346–7 eosinophilia, 1803 neutrophils, 295–7 bone marrow aspirate, 1886 mastocytosis, 1884–5 bone marrow biopsies, mastocytosis, 1884–5 bone marrow-derived mast cells (BMMCs), 219 bone marrow transplantation (BMT), in mastocytosis treatment, 1890

bone metabolism, and inhaled glucocorticoids, 724 bone morphogenetic proteins (BMPs), 1636–7 Bonnevie, P., 1832 Bos d 2, 1137 Bos d 5, 1137 Bostock, John (1773–1846), 12, 13 bovine serum albumin (BSA), 1150 radiolabeled, 436–7 bowel flora, and hygiene hypothesis, 1245–6 BR see bronchial reactivity (BR) bradykinin, 1906 accumulation, 1867–8 and anaphylaxis, 465 assembly, on cell surfaces, 454–8 and asthma, 464–5 contact activation, 452–4 discovery, 9 formation, 20, 465 pathways, 451–2 inactivation, 461–2 roles in angioedema, 1859 in diseases, 462–5 bradykinin pathways, and allergic diseases, 451–70 brain-derived neurotrophic factor (BDNF), 494, 1620 binding, 497 discovery, 496 expression, 498 levels, 502, 503, 507 receptors, 500 signaling, 505 synthesis, 499, 501 Bra r 1, 1557 Brassica spp. (cabbages/mustards), allergens, 1557 Brassica napus (oilseed rape), pollen, 943 breastfeeding, 1330 and allergic diseases, 38–9 and allergy, 2001–2 and allergy prevention, 2001–2 and asthma, 1572, 2001–2 and atopy, 2002 breast milk, in ovalbumin, 1154 breathing patterns, 828 and aerosol deposition, 769–70 breathlessness, nerves in, 833 breeds and cat allergens, 998 and dog allergens, 998 Britannicus (41–55), 3 British Committee on the Safety of Medicines, 1526 British Society for Allergy and Clinical Immunology, 1914 brittle asthma, 1574 management, 1657 type 1, 1574 type 2, 1574, 1657 use of term, 1574 brochial thermoplasty, 762 Brocklehurst, Walter, 567 bromelain, 1148, 1703

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bronchi animal models, 1217 branching patterns, 1215, 1216 dichotomous, 1215, 1216 monopodial, 1215, 1216 canine, 800 bronchial arteries, species differences, 1217–18 bronchial-associated lymphoid tissue (BALT), 385 bronchial asthma and air pollution, 1267 basophils in, 325 diagnostic criteria, 783 epidemics, 1274 leukotrienes in, 581–2 bronchial blood flow, in asthma, 763 bronchial circulation, 398 see also airway vascularity bronchial congestion, 1639 bronchial eosinophilia, 1925 bronchial hyperresponsiveness (BHR), 235, 237– 8, 242, 783– 93 and airflow limitation, 783 and airway caliber, 787 and airway inflammation, 783, 787– 8 and antiasthma drugs, 788– 9 in asthma, 787– 8 asymptomatic, 788 definition, 784 development, 239, 240 direct, 788– 9 etiology, 986 historical background, 784 indirect, 788– 9 measurement, 783, 785 –6 airway response, 785– 6 stimulus delivery methods, 785 mechanisms, 786–7 occurrence, 783 and pediatric asthma, 1597 quantification, 786 receptors, 786–7 reversibility, 984 stimuli direct, 784, 786, 789– 91 indirect, 784, 786–7, 789– 91 pharmacologic, 790 physical, 790–1 physicochemical, 790–1 see also airway hyperresponsiveness (AHR) bronchial provocation testing, 786 bronchial reactivity (BR) predictors, 1240 standardized tests, 1240 bronchial smooth-muscle mass, increases, 1637– 9 bronchial symptoms, without asthma, 1389 bronchial vasculature in asthma, 1639 development, 1194 bronchial vessels characteristics, 398– 9 and immune response, 403– 4 leakage, 404–5 pharmacological responsiveness, 405 roles, in asthmatic responses, 398, 399

bronchoalveolar lavage (BAL), 231, 232–4, 235, 236, 237, 242 in eosinophilic pneumonia diagnosis, 1781 eosinophils in, 1195, 1268 epithelial cells in, 798 extrinsic allergic alveolitis, 1768 in idiopathic acute eosinophilic pneumonia diagnosis, 1784–5 in idiopathic chronic eosinophilic pneumonia diagnosis, 1783 inflammatory cells in, 788 mediators in, 798 T cells in, 1568 bronchoalveolar lavage fluid (BALF), 502, 503 bronchocentric granulomatosis, 1793 diagnosis, 1793 disease progress, 1793 symptoms, 1793 treatment, 1793 bronchoconstriction cellular pathways, 784 drug-induced, 784, 1669 see also exercise-induced bronchoconstriction (EIB); hyperventilation-induced bronchoconstriction (HIB) bronchoconstrictor response, expression, 813–14 bronchoconstrictor stimuli, classification, 1581 bronchodilation, 464 bronchodilators, 668 adverse effects, 1717–18 anticholinergic agents as, 683 in chronic asthma treatment, 1650–2 in combination drugs, 686 developments, 1717 new, 1717–18 rapid-acting, 1580 responses, 677 bronchomotor tone, cholinergic, 684 bronchopulmonary aspergillosis, 1658 bronchorestriction, 464 bronchospasm, acute, 1966 bronchospastic stimuli, protection against, 686 bronchovascular leakage, 796–7 Brown-Norway rats, 1219 Brugia malayi (nematode), 1789 BSA see bovine serum albumin (BSA) BTEX compounds, 1284 bucindolol, 680 budesonide, 699, 700, 718–20 adverse effects, 724 efficacy, 721 structure, 718 building materials, fungal particles from, 967–9 Building Research Establishment (UK), studies, 1282 bullous exanthem, 1957–8 Bullowa, J. G. M., 669 bumble bees, 1981 venom, 1124 Burkard volumetric traps, 946 C1, activation control, 442, 444–5 C1 inhibitor, 444–5 C1 inhibitor deficiency, 462–3 acquired, 1866–7

C2, activation control, 443, 445 C3, 437, 438, 440 activation control, 445–7 functions, 444 structure, 444 C3a, receptors, 447 C3b, 437 C4, 440 activation control, 442–3, 445 C4a, receptors, 447 C5, binding, 443 C5a, receptors, 447 C5m binding, 443 C6, binding, 443 C7, binding, 443 C8, binding, 443 C9, binding, 443 Cadham, F. T., 963 cadherin cell–cell adhesion molecules, 858–9 cadherins, 369, 858–9, 864 Caelius Aurelianus (fl. 5th century), 1591 Caenorhabditis elegans (nematode), 860 calcineurin (Cn), 329, 1825 calcineurin inhibitors, 732, 737 adverse effects, 738 mechanisms, 737–8 oral bioavailability, 738 structure, 739 topical, 1825 toxicity, 738 calcitonin gene-related peptide (CGRP), 385, 495, 511, 513, 516–18, 849 in airway nerves, 531 clinical applications, 518 and late-phase allergic reactions, 534–6 localization, 1406 metabolism, 518 pulmonary effects, 517–18 receptors, 517 calcium, cytostolic, 878–9 calcium-activated chloride channel inhibitors, 851 calcium-independent phospholipase A2 (iPLA2), 568, 569 calcium ions, and vascular permeability, 862–3 calmodulin (CaM), 329 calponin, 877 CaM (calmodulin), 329 cAMP see cyclic adenosine 3′,5′-monophosphate (cAMP) Campath-1H (alemtuzumab), 742 CAMs (complementary/alternative medicines), allergic rhinitis treatment, 1441 Canadian Asthma Primary Prevention Study, 2004, 2005 Candida spp. (fungi) allergens, 1205 infection, 776–7 Candida albicans (fungus), 1761 Candida guiliermondii (fungus), 1761 Can f 1, 1137, 1353 concentration in dust, 1000 distribution, 999 exposure to, 1001 structure, 998 Can f 2, 1137

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canine bronchi, 800 CAP assay, 915, 1126 inhibition, 933 CAP-FEIA, 1901 capillary zone electrophoresis (CZE), 936–7 capsaicin, 1391 CAPS (Childhood Asthma Prevention Study), 2000, 2004, 2005, 2010 capsin-recruitment domain (CARD), 379, 380 Car b 1, 954 carbon dioxide, exposure, 143– 4 carbon monoxide, and indoor air pollution, 1285 carboplatin, 1905 carcinoid tumors, differential diagnosis, 1585 CARD4 (caspase recruitment domain protein 4), 40 CARD (capsin-recruitment domain), 379, 380 Cardif, 192 cardiopulmonary resuscitation (CPR), for acute anaphylaxis, 1914 cardiovascular events, and chronic obstructive pulmonary disease, 690 carmine, 1926 carmoterol, 1717 cascade impactors, 771 CAS (Chemical Abstracts Service), occupational agents, 1061 α-casein, 917 β-casein, 917 casein family, 914, 917 caseins, 1150 in gloves, 1166 cashew nuts, allergens, 1152 caspase recruitment domain protein 4 (CARD4), 40 Castanea spp. (chestnuts), 955– 6 see also chestnut pollen CAST (cellular antigen stimulation test), 1988 cat allergens, 95, 916, 921, 998 aerodynamics, 998– 9 airborne levels, 1000 and asthma, 142 breed-specific, 998 exposure, 1136 occurrence, 1013 sensitization, 1001, 1002–3, 1012 sources, 1282 tolerance, 992 cat allergy sensitization, 1559– 60 T-cell peptide epitopes in, 1559– 60 cataracts, and inhaled glucocorticoids, 724 catecholamines, 824– 5 structure, 672, 673 use of term, 672 α-catenin, 858–9 β-catenin, 858– 9 γ-catenin, 858–9 catenins, 369, 858– 9 cathelicidins, 374–5 cats and asthma, 998 ownership, 1012 as pets, 997–8 washing, 1002

caveolae, 860–1, 862 caveolin-1, 861 caveolin, 860–1 CBMCs (cord blood-derived mast cells), 219 CC chemokines, 473–4, 479 CC (closing capacity), 754 CCDs (cross-reacting carbohydrate determinants), 1355, 1356, 1987–8 CCL2, 1304 CCL3, 1413, 1416 neutralization, 483 CCL5, 1304, 1416 CCL7, 1416 CCL8, 1416 CCL11, 1304, 1416 deficiency, 484 synthesis, 472–3 CCL13, 1416 CCL17 see thymus activation-regulated chemokine (TARC) CCL17, see also thymus activation-regulated chemokine (TARC) CCL19, 1609, 1611 expression, 1639 CCL20, 1954 CCL21, 1609 expression, 1639 CCL22, 481–2 CCR2, 1723 CCR3, 71, 1415, 1416 antagonism, 483, 1612 expression, 483, 484, 485, 920, 1304, 1723 future applications, 1654 see also anti-CCR3 CCR4, 29 expression, 30, 57, 71, 481, 920 inhibitors, 1610 CCR5, 530, 1612 CCR6, 1954 CCR7, 56, 1609, 1611 expression, 1639 CCR8, 71, 530 expression, 481, 920 CCR9, blockade, 482 CCR10, 29, 530 expression, 30 CD1d, 55–6 CD3+, 1296, 1641–2 CD4, 19, 49, 1409 expression, 1295 CD4+, 20, 48, 49, 50, 56, 1295 allergen-activated, 528–9 antigen presentation, 168–9 increase, 1642 inhibition, 64–5 in perennial rhinitis, 1412 phenotypes, 56–7 trafficking, 270–2 CD4+CD25+Foxp3+ Treg, naturally occurring, 85–7 CD4+CD25+ regulatory T cells, 27, 87, 92, 1220, 1515 identification, 1296 CD8, 49 in asthma, 71–2

CD8+, 49, 50, 1295, 1296 antigen presentation, 169–70 CD11c, 1409 CD14, 38, 39–40, 1231 expression, 1230 soluble, 38 CD14 gene, 149, 151, 1234 CD21, 112 binding, 110 discovery, 104, 121 expression, 104 roles, 104 CD23, 109–11, 1514 binding, 1720 sites, 107–8 counterreceptors, 112–13 functional diversity, 104 functions, 122–3 membrane-bound, 103, 110–11 roles, 103–4 in food allergy, 1148 in immunoglobulin E homeostasis, 134–5 structure, 110 see also soluble CD23 (sCD23) CD25+ Treg inhibitory mechanisms, 86–7 naturally occurring, markers, 86 CD28, 50, 55, 1409 signaling, 1609 superagonists, 1715 CD30, 54 CD34, 339 expression, 404 precursors, 1611, 1612 CD34+, 1410, 1879 CD35 occurrence, 446 roles, 446, 447 CD40, 1408 CD40L, 54, 132, 1408 CD44, 882 CD45, surface markers, 404 CD45RA, 56 CD45RO, 56 CD45+, 1642 CD59, 447–8 CD63, 1360–1 in basophil simulation tests, 1362 CD80, 54, 1408, 1609 CD86, 50, 54, 1409, 1609 CD99, 337 function, 344 structure, 344 CD152, 50 CD203c, 325, 1360–1 in basophil simulation tests, 1362 cDNA, 1291–2 allergen-encoding, 898–9, 903 CDSN gene, 1229 cedar pollinosis, 1250 ceftazidime, 1902 celery–mugwort-spice syndrome, 1153 celiac disease, diagnosis, 1322, 1329 cell-surface structures, 265–6, 267 cellular adhesion molecules (CAMs), 300–1

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cellular antigen stimulation test (CAST), 1988 cellular immunologic profiles, allergic diseases, developing countries, 2027– 8 cellular infiltrate in angioedema, 1857– 8 in urticaria, 1857– 8 cellular infiltration, 398 cellular tests, insect sting allergy, 1988 central nervous system (CNS) antihistamine and, 560 histamine expression, 551 modulation in, 829–30 cephalosporins, 1902 Cephalosporium acremonium (fungus), 978 cetirizine, 554, 556–7, 561–2, 1437, 1439, 1869 CF see cystic fibrosis (CF) CFCs (chlorofluorocarbons), 768 C-fibers, 826–7 activation, 829, 830 sensitization, 830 CFTR see cystic fibrosis transmembrane conductance regulator (CFTR) cGMP (cyclic guanosine monophosphate), 863 CGRP see calcitonin gene-related peptide (CGRP) Chaetomium spp. (fungi), 963 occurrence, 978 challenge testing, for latex allergy, 1176 Charcot, Jean Martin (1825– 93), 11, 12 Charcot–Leyden crystals (CLCs), 11, 259, 984, 1464, 1495 in asthma, 1634 cheese, allergy to, 2 cheilitis, 1817 Chemical Abstracts Service (CAS), occupational agents, 1061 chemical sensitizers, inhalation tests, 1583 chemiluminescence technology, 1323 chemokine antagonists, 485– 6 chemokine function, modulation, 479 chemokine receptor antagonists, 328, 1723 future trends, 1654 chemokine receptor expression, regulatory mechanisms, 29–30 chemokine receptors, 264, 474– 9, 1209 activation, 475–6 in allergic inflammation, 480– 6 and downstream signaling, 476– 8 expression, 920, 1712 regulatory mechanisms, 478– 9 G-protein coupling, 476– 8 ligands, 477 proteolytic processing, 479 chemokines, 29–30, 337– 8, 471– 93, 920 activation, 1232 and adhesion molecules, 472–3 in airway epithelium, 382–5 in allergic inflammation, 480– 6, 1303– 4 in atopic dermatitis, 1823– 4 basophil recruitment, 483 binding, structural determinants, 475– 6 blockade, in allergy treatment, 485– 6 classification, 473– 4 and cytokines, 472– 3 dendritic cell recruitment, 480 and diseases, 472

eosinophil recruitment, 483–5 expression, 323 interactions, with glycosaminoglycans, 474 and late-phase allergic reactions, 530 mast cell derived, 224 mast cell recruitment, 482–3 nomenclature, 474, 475 roles, 472, 1455 scavenging, 479 structure, 473 T cell recruitment, 480–2 chemotactic cytokines see chemokines chemotactic factor receptors, 264 chemotaxis, 471 Chenopodiaceae (goosefoot family), 957 chenopod pollen, 957–8 chestnut pollen, 955–6 seasons, 956 chest radiology, extrinsic allergic alveolitis, 1766–7 chest X-rays, 1749–50 childcare attendance, and allergen avoidance, 2008–9 childhood asthma see pediatric asthma Childhood Asthma Prevention Study (CAPS), 2000, 2004, 2005, 2010 children aerosol delivery, 779–80 allergen avoidance, 1598–9, 1601–2 allergy, 1330 allergy prevention, 2000 asthma, 1591–607 atopic asthma, 1594 atopic dermatitis, 1816 atopic eczema, 1598 diet, 1330 early sensitization, disease progression prevention, 2010–11 food allergen avoidance, 2004 high-risk, pet sensitization, 1003 insect sting allergy, 1985 see also infants; pediatric asthma Chinery classification, 1980–1 Chinese restaurant syndrome, 1328 chitin, 387 chitinases, 1153, 1166, 1170 chitin-binding proteins, 1169 Chlamydophila pneumoniae (bacterium), 1233, 1573, 1621 infection, 1655 chloride channels, regulatory mechanisms, 1618 chlorofluorocarbons (CFCs), 768 chlorpromazine, 14 cholinergic antagonists, 683–93 applications, 683 see also anticholinergic agents cholinergic urticaria, 1862–3 and cold urticaria, 1863 chromatin, 129–30 structure, 131 chromosomes 1q, 1615, 1616 1q13, 1615 1q21, 1227, 1228, 1230 2q14, 1229

3q21, 1229, 1821 5q31–33, 1804, 1821–2 5q34, 1234 7p14, 1229 10q23, 1229 11q13, 1234 13q14, 1229 16p, 1229 16q, 1821 17q11, 1822 17q25, 1227, 1229, 1821 18q11–12, 1227 20p, 1229, 1821 20q12, 1231 chronic allergic asthma, mast cells and, 233–4 chronic angioedema, treatment, 1870–1 chronic asthma allergen avoidance, 1654 alternative therapies, 1654 controller therapies, 1652–4 epithelium in, 1615 future therapies, 1654 goblet cells in, 1618 immunotherapy, 1654 management, 1650–60 education, 1656 methods, 1654–6 special issues, 1657–8 step-down, 1655–6 with persistent airflow obstruction, 755–6 stepwise pharmacologic therapy, 1654–5 surgery, 1658 treatment bronchodilators, 1650–2 new drugs, 1724 steroid-sparing strategies, 1653 chronic bronchitis, 1637 see also chronic obstructive pulmonary disease (COPD) chronic idiopathic angioedema, 1865 chronic idiopathic urticaria, 1865 chronic injury, mechanisms, 1208 chronic obstructive pulmonary disease (COPD) acute exacerbations, anticholinergic agents in, 688–9 airway wall remodeling, 413 and asthma, 1574–5, 1656 biomarkers, 1717 and bronchial hyperresponsiveness, 783 and cardiovascular events, 690 differential diagnosis, 790, 1581, 1584–5, 1656 etiology, 413 lung function decline, reduction in rate of, 1371 mast cells in, 790 myofibroblasts, 417 and phosphodiesterase 4 inhibitors, 643 and sinusitis, 1472 and smoking, 1574–5 sputum eosinophil count, 1373, 1377 stable, anticholinergic agents in, 686–8 chronic rhinosinusitis (CRS), 1386, 1389, 1392, 1394 bacterial infections in, 1454–5 clinical features, 1472–3 comorbidities, 1472

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definition, 1393 differential diagnosis, 1473 disease course, 1474 epidemiology, 1469– 70 etiology, 1470 future trends, 1474–5 genetics, 1470 macrolide therapy, 1475 management, 1473– 4 mechanisms, 1470 molds in, 1388 pathogenesis, 1470 pathology, 1470–2 prevalence, 1388, 1469– 70 prognosis, 1474 studies, 1464 symptoms, 1393 treatment, 1473– 4 use of term, 1454 vicious circle, 1471 see also nasal polyposis chronic rhinosinusitis with nasal polyps see nasal polyps chronic urticaria, 1326 and autoimmunity, 1855 basophils in, 1858 incidence, 1855 treatment, 1870–2 Churg–Strauss syndrome (CSS), 697, 1459, 1585, 1785– 7 and asthma, 1785 definition, 1785 diagnosis, 1785–7 prevalence, 1785 prognosis, 1787 and rhinitis, 1786 risk factors, 1787 symptoms, 1785 treatment, 1787 chymase, 239– 40, 330 CIBA Foundation, Guest Symposium (1959), 1240 ciclesonide, 719, 1712 ciclosporin A see cyclosporin A (CsA) cigarette smoking see smoking ciliated cells, 1618 ciliated epithelial cells, 368– 9 ciliated epithelium, structure, 368– 9 cilomilast, 1712–13 adverse effects, 1724 efficacy, 1724 structure, 644 cimetidine, in anaphylaxis treatment, 1915 cisplatin, 1905 Cit s 1, 1153 c-kit activation, 218 mutations, 1886, 1887– 8 CLA see cutaneous lymphocyte antigen (CLA) Cladosporium spp. (fungi), 1282 abundance, 967 allergens, 973 occurrence, 978 seasons, 966–7

Cladosporium herbarum (mold), enolases, 1170 cladribine, in mastocytosis treatment, 1890 Clara cells, 370, 1191–2, 1618 species differences, 1192 Clarkson disease, 868 classical pathway, 438, 441, 442–3 class II-associated invariant chain peptide (CLIP), 168–9 class-switch recombination (CSR), 119, 120, 121, 126, 127–9 regulation, to immunoglobulin E, 132–4 claudin family, 859–60 CLC protein, 260–2 CLCs see Charcot–Leyden crystals (CLCs) Clean Air Acts (1956; 1968) (UK), 1279 cleaning agents, and occupational airway diseases, 1101 clenbuterol, 673–4 climate change and air pollution, 1273–4 and allergen levels, 143–4 and pollen allergy, 1274 and pollinosis, 1273–4 clinical studies, safety, 1714–15 CLIP (class II-associated invariant chain peptide), 168–9 clonidine, 680 closing capacity (CC), 754 closing volume (CV), 754 Clostridium botulinum (bacterium), neurotoxins, 850 Clostridium difficile (bacterium), 36 CLPR see cutaneous late-phase reaction (CLPR) CM see cutaneous mastocytosis (CM) c-Maf, 1306 Cn (calcineurin), 329, 1825 CNS see central nervous system (CNS) coagulation pathway-associated proteases, fibrosis mediation, 423–4 coagulation pathways, activation, 1906 coarse particles, definition, 1281 cobra venom factor, 437 Coca, Arthur Fernandez (1875–1959), 5, 13 cocaine sniffing, 1390 cockroach (CR) allergens, 914, 916, 918, 1123 in active immunization models, 1205 aerodynamics, 1000–1 and allergic diseases, epidemiology, 1132–4 and asthma, 142, 1131–45, 1571 hospitalization rates, 1133 characteristics, 985 dose–response relationships, 992 early studies, 1131–2 environmental exposure, 1131–45 exposure, 1132, 1133–4 functions, 1135 biological, 1138–9 global, 1134 immune responses, 1135–7 immunoglobulin E reactivity, 1135–7 insecticide effects, 1139–40 molecular structure, 1137 nomenclature, 1135 regional differences, 1134

remediation procedures, clinical trials, 1139–40 repertoire, 1134–9, 1141–2 research, 1132 sensitization, 1132 prevalence, 1132–3 structure, 1132 Codex Alimentarius Commission, 1147 codfish, allergens, 1151 codon 816, c-kit mutation, 1887 cold air, and exercise-induced bronchoconstriction, 809, 812, 813 cold urticaria, 1862, 1911 and cholinergic urticaria, 1863 collagen in extracellular matrix, 413–14 structure, 413 collagen IV, in bronchial vessels, 399 collectins, 375, 1231 colophony applications, 1703 and occupational asthma, 1703 combination drugs, 686 combination inhalers, in chronic asthma treatment, 1655 Committee on the Safety of Medicines (CSM) (UK), 1913–14 common cold (acute viral rhinosinusitis), 1393 COMPASS trial, 1713 competitive allergen assays, 933 complement activation pathways, 441 alternative, 438, 441, 444–8, 1906 classical, 438, 441, 442–3 lectin, 438, 441, 443–4 in afferent limb of immune response, 448 deficiencies, 448–9 early studies, 441 functions inflammatory peptide generation, 442 lytic, 441 particle opsonization, 441–2 overview, 441–2 roles, 1856 in disease, 436–50 in host defense, 436–50 in immune complex clearance, 437 complementarity determining regions (CDRs), 122 complementary/alternative medicines (CAMs), allergic rhinitis treatment, 1441 complement cascade, late steps, control, 447–8 complement components, roles, in airway diseases, 376 complement control molecules, 444–7 complement proteins, 442 complement receptors, 264, 265, 444–7 complex platinum salts, and occupational asthma, 1703 complex traits, 1225 Compositae (daisy family), and dermatitis, 1841 compound allergies, use of term, 1847–8

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computed tomography (CT), 749 airway visualization, 758 see also high-resolution computed tomography (HRCT); positron emission tomography (PET); single photon emission computed tomography (SPECT) confectioneries, occupational airway diseases, 1061 congeners, 1328 conglycinins, 1159 conjunctiva anatomy, 1484 – 5 surface markings, 1484 conjunctival cytology, 1503 conjunctival immune tissue, anatomy, 1485 conjunctivitis with allergic rhinitis, 1387 atopic, 1387 use of term, 1387 see also allergic conjunctivitis; atopic conjunctivitis; contact lens conjunctivitis; giant papillary conjunctivitis (GPC); rhinoconjunctivitis; vernal conjunctivitis (VC); vernal keratoconjunctivitis (VKC) connective tissue, inhaled glucocorticoid effects, 724 connective tissue disease idiopathic pulmonary fibrosis in, 1795 interstitial pneumonia in, 1795 connective tissue mast cells (CTMCs), 220 contact activation bradykinin, 452– 4 inhibition, 460–1 contact allergens, fungi, 965 contact dermatitis, 1831–52 advice, 1848– 9 cellular mechanisms, 1833– 4 chemical issues, 1834 classification, 1831 clinical features, 1835– 43 counseling, 1848– 9 definition, 1831 diagnosis, 1322, 1327 epidemiology, 1832– 3 feet, 1841– 2 food-induced, 1930 histopathology, 1834– 5 historical background, 1831– 2 legislative changes, 1849 lips, 1842 management, 1848– 9 mucosal, 1842– 3 patch testing, 1323 treatment, clinical, 1849 triggers, 1327 see also allergic contact dermatitis (ACD); irritant contact dermatitis (ICD); photocontact dermatitis; protein contact dermatitis contact lens conjunctivitis, definition, 1387 contact urticaria, 1842, 1864 contiguous immunity, in allergic diseases, 195– 6 controller therapies, chronic asthma, 1652– 4 Cooke, Robert Anderson (1880–1960), 13, 14, 928, 1513 Coombs and Gell classification, 14– 20

Coombs, Robin Amos (1921–2006), 5–6, 14–20 COPD see chronic obstructive pulmonary disease (COPD) Cor a 1, 954 Cor a 1.04, 1150 Cor a 8, 930 cord blood, immunoglobulin E, 1999 cord blood-derived mast cells (CBMCs), 219 Corey, E. J., 9 cornea anatomy, 1485 histology, 1485 corneodesmosin, 1215 corticosteroid resistance, 726–7, 1373, 1376 corticosteroid-resistant asthma, management, 1656–7 corticosteroid responsiveness, 1373, 1375–6 corticosteroids, 405, 1373 in allergic bronchopulmonary aspergillosis treatment, 1792–3 in allergic rhinitis treatment, 506, 1437–8 in angioedema treatment, 1869–70 in asthma treatment, 1712 in bronchocentric granulomatosis treatment, 1793 in Churg–Strauss syndrome treatment, 1787 in hay fever treatment, 1331 in hypereosinophilic syndrome treatment, 1789, 1807 in idiopathic acute eosinophilic pneumonia treatment, 1785 in idiopathic chronic eosinophilic pneumonia treatment, 1783–4 and late-phase allergic reactions, 532 in mastocytosis treatment, 1890 new, 1718 reduction studies, 1372 in seasonal allergic conjunctivitis treatment, 1490 in urticaria treatment, 1869–70 see also glucocorticoids; inhaled corticosteroids (ICSs); oral corticosteroids; systemic corticosteroids; topical corticosteroids corticotropin reduction, 723 synthesis, 831–2 corticotropin-releasing factor (CRF), 1406 cough, 828 nerves in, 833 neuropeptides and, 519–20 questionnaires, 1590 sputum eosinophil count, 1373 without asthma, 1389 cough variant asthma, 1573–4 cow allergens, 1061 cow’s milk allergens, 1150–1, 1922 lactoglobulin, 1148 malabsorption, 1329 sensitization, 1905, 2002–3 COX-1 see cyclooxygenase-1 (COX-1) COX-2 see cyclooxygenase-2 (COX-2) COX (cyclooxygenase) pathway, 566, 567, 695 coxibs, 1974–5 CP96345, 516

CP99994, 516, 520 CpG see cytosinephosphoguanosine (CpG) cPLA2α (cytosolic phospholipase A2α), 568 CPR (cardiopulmonary resuscitation), for acute anaphylaxis, 1914 CR2 see CD21 CRACM1, 225 C-reactive protein (CRP), 1231 cresols, 1166 CRF (corticotropin-releasing factor), 1406 CRHT2 receptors, 592 critical period, concept of, 830–1 Crohn disease, 312, 322, 1229 diagnosis, 1329–30 leukotrienes in, 587 cromoglycate, 1439, 1443 cromones in allergic rhinitis treatment, 1439 in chronic asthma treatment, 1653 cross-reacting carbohydrate determinants (CCDs), 1355, 1356, 1987–8 croton oil, 1835 CRP (C-reactive protein), 1231 CRS see chronic rhinosinusitis (CRS) CRTH2, 57, 1420, 1721–2 CRTH2+CD4+, 57 cryoglobulinemia, 438 Cryptococcus albidus (fungus), 1761 cryptogenic fibrosing alveolitis, leukotrienes in, 587 Cryptomeria japonica (Japanese cedar), pollen, 144, 956 CsA see cyclosporin A (CsA) CSM (Committee on the Safety of Medicines) (UK), 1913–14 CSR see class-switch recombination (CSR) CT see computed tomography (CT) CTACK, 29 CTLA-4 (cytotoxic T-lymphocyte antigen-4), 26, 58, 86 CTLs (cytotoxic T lymphocytes), 169 CTMCs (connective tissue mast cells), 220 cupin superfamily, 1148, 1149, 1150 Cupressaceae (cypress family), 956 Cupressus spp. (cypresses), 956 see also cypress pollen Cupressus alba, 956 Cupressus arizonica (Arizona cypress), 956 Cupressus glaucophylla (white cypress), 956 Cupressus lawsoniana (Lawson’s cypress), 956 Cupressus sempervirens (Mediterranean cypress), 956 Curschmann spirals, 1634 Curvularia spp. (fungi), abundance, 967 cutaneous challenge, late-phase allergic reactions, 526 cutaneous food-allergic reactions, 1929–30 cutaneous late-phase reaction (CLPR), 524–5, 526, 528 allergen-induced, 532 cutaneous lymphocyte antigen (CLA), 29, 530 expression, 30, 350 cutaneous mastocytosis (CM), 1880 diffuse, 1880 infants, 1891

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skin biopsies, 1884 symptoms, 1881–3 see also maculopapular cutaneous mastocytosis cutaneous reactions, pathophysiology, 1954– 8 CV (closing volume), 754 CX3C chemokines, 473– 4 CX3CL1, 473– 4 CXC chemokines, 473– 4, 528 CXCL4, 474 CXCL8, 1413, 1611, 1953, 1957 CXCL10, 1611 CXCL12, 480–1 CXCR2, 1416, 1611 CXCR3, 530, 1611 CXCR3 ligands, 481 CXCR4, 1409, 1416, 1609 expression, 299, 480–1, 484 CXCR12, 1409, 1609 cyclic adenosine 3′,5′-monophosphate (cAMP), 329, 471, 642, 672 degradation, 634 in endothelial cells, 865– 6 hydrolysis, 635, 636 inactivation, 635 intracellular, 885 cyclic guanosine monophosphate (cGMP), 863 cyclic guanosine 3′,5′-monophosphate (gAMP) degradation, 634, 642–3 hydrolysis, 635, 636 inactivation, 635 cyclic nucleotide phosphodiesterases function, 635 properties, 635– 7 selective inhibitors, 637 cyclic peptides, 519 cyclic purine phosphodiesterases, structure, 636 cycling, exercise monitoring, 813 cyclooxygenase-1 (COX-1), 380, 566–7, 1968– 9 inhibition, 1390, 1966, 1967, 1969, 1971, 1973 inhibitors, 1972 roles, 589– 90 cyclooxygenase-2 (COX-2), 236–7, 380, 566–7, 1968– 9 inhibition, 1390, 1971 roles, 589– 90 cyclooxygenase (COX) pathway, 566, 567, 695 cyclooxygenases and aspirin activity, 590–1 and asthma, 589– 90 blocking, 800 roles, 589 cyclophilin (CyP), 329 cyclophosphamide, 736– 7 adverse reactions, 737 indications, 737 mechanisms, 737 monitoring methods, 737 pharmacogenetics, 737 pharmacokinetics, 736–7 structure, 736 cyclosporin A (CsA), 72–3, 329, 738– 9 adverse effects, 739 in atopic dermatitis treatment, 1826 drug interactions, 739 indications, 739

and late-phase allergic reactions, 532 monitoring methods, 739 pharmacogenetics, 739 pharmacokinetics, 738–9 structure, 738, 739 topical, in vernal keratoconjunctivitis treatment, 1497 Cynodon dactylon (Bermuda grass), 1354 CYP1A1, 652 CYP1A2, 652 CyP (cyclophilin), 329 cypress pollen, 956 cross-reactivity, 956 seasons, 956 cyproheptadine, 560 Cys (cysteinyl), 329 CysLT1, 578–9 expression, 579 function, 579 gene, 578–9 receptors, 566, 1970 identification, 578 CysLT2 expression, 580 function, 580 gene, 580 receptors, 566, 580–1, 1970 identification, 579–80 transgenic mice, 580 CYSLT2 gene, polymorphisms, 580 CYSLTR2 gene, 580 Cysltr2-null mice, 580 CysLTs see cysteinyl leukotrienes (CysLTs) cysteinyl (Cys), receptor antagonists, 329 cysteinyl leukotriene receptor antagonists, 849–50 cysteinyl leukotriene receptors, 577–81 types of, 1970 cysteinyl leukotrienes (CysLTs), 566, 575 biological activity, 575, 694 biosynthesis, 695, 708 effects, 695–6 and exercise-induced bronchoconstriction, 810–11 roles, 695 cystic fibrosis (CF) and allergic bronchopulmonary aspergillosis, 1747 Aspergillus bronchitis in, 1748 differential diagnosis, 1585 mortality, 1463 and nasal polyposis, 1394 and nasal polyps, 1463 prevalence, 1463 cystic fibrosis transmembrane conductance regulator (CFTR), 1463 mutations, 1745, 1791 cytokeratin 1, 456–7 cytokine antagonists, 850 cytokine assays, 1175 cytokine inhibitors, 1722–3 cytokine receptors, 264–5 cytokines, 24–5, 26, 29, 337–8 in airway epithelium, 382, 383 in airway smooth muscle, 881–2

in allergic diseases, 1297–303 in allergic inflammation, 48–82 in asthma, 48–82 in atopic dermatitis, 1823–4 and chemokines, 472–3 eosinophil-generated, 260, 261 expression, 1208, 1209, 1291 immunolocalization, 1291 and late-phase allergic reactions, 529–30 mast cell derived, 223 new, in asthma, 65–70 in ocular allergy, 1487 roles, 1455 in asthma pathogenesis, 62–74 see also antiinflammatory cytokines; chemokines cytopenias, and systemic mastocytosis, 1883 Cytophaga spp. (bacteria), 1760 cytosinephosphoguanosine (CpG), 1726 motifs, 1561 oligodinucleotides, in allergen-specific immunotherapy, 1561–2 cytosolic phospholipase A2α (cPLA2α), 568 cytotoxicity, mechanisms, 1953 cytotoxic T-lymphocyte antigen-4 (CTLA-4), 26, 58, 86 cytotoxic T lymphocytes (CTLs), 169 CZE (capillary zone electrophoresis), 936–7 D6, chemokine scavenging, 479 Dactylis glomerata (cocksfoot), pollen, 943 daily living, and persistent severe allergic asthma, 1662 Dale, Sir Henry H. (1875–1968), 8, 9, 668, 680 damage-associated molecular patterns (DAMPs), 170, 171–2 DAMPs (damage-associated molecular patterns), 170, 171–2 dander, 1282, 1349, 1570–1 DARC (Duffy antigen receptor complex), chemokine scavenging, 479 Datura stramonium (stramonium), atropine, 683 DBPCFCs (double-blind, placebo-controlled food challenges), 1150, 1323 DCs see dendritic cells (DCs) decongestants in allergic rhinitis treatment, 1439 in hay fever treatment, 1331 decorin, 414 deep inspiration (DI), 749 airway responses, 757 effects, 760–2 prohibition, 760–2 defensins, 375, 1231 α defensins, 375 β defensins, 375 Dekker, H., 984 delivery mode, and allergic diseases, 37 dendritic cells (DCs), 24–5, 1408–9, 1721–2 activation, 57, 58, 170–1 airways, 31 allergen processing, 1609 in allergic diseases, 596 in allergic rhinitis, 179 in allergic sensitization, 176–7 antigen-presenting, 166–86

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dendritic cells (DCs) (Cont’d ) antigen processing, 168 antigen uptake, 167– 8 in asthma, 177, 1641 in atopic dermatitis, 178– 9, 1824 as drug targets, in allergic diseases, 179 expression, 24 heterogeneity, 166–7 and immune response, 170– 6 importance of, 166 in lung, 188 regulatory T cell control, 176 migration, 170 to draining lymph nodes, 171–2 morphology, 167 natural killer, 167 and neurotrophins, 499 origins, 167 polarization by, 173– 5 recruitment, via chemokines, 480 renewal, 166–7 roles, 596 subtypes, 166–7 T-cell activation, 172–3 terminology, 166–7 tolerance induction by, 175– 6 see also myeloid dendritic cells (mDCs); plasmacytoid dendritic cells (pDCs) Dennie-Morgan lines, 1818 deposition, 772–3 and dose–response characteristics, 775 deposition fraction (DF), 769 DEPs see diesel exhaust particles (DEPs) Der f 1, 930 assays, 987 Der f 2, 930 dermatitis eyelid, 1817 palmar, 1817 plantar, 1817 use of term, 1831 see also atopic dermatitis (AD); contact dermatitis dermatitis herpetiformis, 1930 dermatographism, 1861 Dermatophagoides farinae (American house-dust mite), 231, 1281–2 allergens, 916, 920–1, 1205 Dermatophagoides pteronyssinus (European housedust mite), 231, 984, 1281–2, 1342 allergens, 369, 916, 920–1, 985– 6, 1228 cultures, 986, 987 omalizumab studies, 1669 Der p 1, 93, 111, 143, 168, 176–7, 369, 930 and allergic sensitization, 1281–2 assays, 987 binding, 916, 918 epitopes, 990 as protease, 903, 1228 purification, 985– 6 toxicity, 1232 Der p 2, 929, 930, 934, 986, 1353, 1358 binding, 916, 918 epitopes, 990 as protease, 1228 toxicity, 1232

Der p 3, 986 binding, 918 Der p 4, 916, 986 binding, 918 Der p 5, 916 binding, 918 Der p 7, 916 binding, 918 Der p 8, binding, 918 Der p 10, binding, 918 Der p 20, binding, 918 desensitization β-adrenoceptor agonists, 677–8 allergic diseases, 1332–3 anaphylaxis, 1913–14 drug hypersensitivity, 1962–3 systemic, 1543 desloratadine, 329, 554, 555, 557, 561–2, 1869 desmosomes, 369 detergents, 1101, 1703 developed countries, atopy, 2020 developing countries allergic disease prevention, 1995–2030 allergic diseases, cellular immunologic profiles, 2027–8 allergy prevention, 1995–2030 atopic diseases, prevalence, 2020–30 immunoglobulin E levels, 2024–5 rhinitis management, 1445 DF (deposition fraction), 769 DI see deep inspiration (DI) dialysis membranes, and anaphylaxis, 1906 diapedesis see transendothelial migration (TEM) Dictyostelium discoideum (slime mold), chemotaxis, 471 Didymella spp. (fungi), seasons, 967 Didymella exitialis (fungus) and asthma, 965 periodicity, 967 diesel exhaust exposure, 1270 and immunoglobulin E production, 145–6 direct effects, 146 exacerbation of inflammation, 145 indirect effects, 146 interactions with allergens, 145–6 and respiratory diseases, 145 diesel exhaust particles (DEPs), 147, 148, 945 composition, 1269 and immunoglobulin E production, 145–6 urban areas, 1269–70 diet and allergic diseases, 36–7 and asthma, 1249, 1572 and atopic diseases, 1249 children, 1330 see also food; nutrition dietary interventions, infants, 2010 dietary protein-induced enteropathy, 1929 diethylcarbamazine, 1790 di-2-ethylhexyl adipate, 1101 diet restriction, in atopic dermatitis treatment, 1827 difficult asthma, use of term, 1575 diffuse cutaneous mastocytosis, 1880

digestibility, and food allergy, 1155 DiHS (drug-induced hypersensitivity syndrome), 1958, 1961, 1962 diisocyanate asthma, use of term, 1214 diisocyanates, and occupational asthma, 1692, 1701 dimenhydrinate, 560 dipeptidyl peptidase IV, 518 diphenhydramine, 560, 1437, 1869 diphtheria, treatment, 436 Dirofilaria immitis (dog parasite), 1791 disease genes, finding, 1225–6 diseases and allergen exposure, 1248–9 bradykinin in, 462–5 and chemokines, 472 complement in, 436–50 dust mite exposure in, 991 eosinophils in, 272–80 immune complexes in, 436–50 impacts on innate immunity, 196 leukotriene release, 583–8 neutrophils in, 311–13 progression prevention, children with early sensitization, 2010–11 see also airway diseases; allergic diseases; atopic diseases; autoimmune diseases; intestinal diseases; lung diseases distal lung in asthma early studies, 1641 pathology, 1641–2 DLG5 gene, 1229 DNA, allergen-encoding, 898– 9 DNA microarrays, 1292 DNA replication, 131–2 DNA viruses, 380 DNK333, 516, 531 dog allergens, 998 aerodynamics, 998–9 airborne levels, 1000 breed-specific, 998 occurrence, 1013 sensitization, 1001, 1002–3, 1012 dogs late-phase airway hyperresponsiveness, 801–2 late-phase airway inflammation, 800–1 as pets, 997 repetitive hyperventilation-induced airway obstruction, 802 Dolichovespula spp. (yellowjackets), 1982–3, 1987 Dolichovespula maculata (white-faced hornet), 1124–5, 1982 Dolichovespula media (median wasp), 1982 Dolichovespula saxonica (Saxon wasp), 1982 Dolichovespula sylvestris (tree wasp), 1982 domestic environment, and atopic diseases, 1261–3 domestic pets, and allergic diseases, 35 dopamine, 672 in insect venoms, 1127 dose, optimal, 930 dose–response relationships allergen injection immunotherapy, 1528–9 anticholinergic agents, 686

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cockroach allergens, 992 and deposition, 775 dust mite allergens, 992 environmental allergens, 1259 histamine, 785 inhaled corticosteroids, 695 inhaled glucocorticoids, 720–1 ipratropium, 686 mammalian allergens, 992 methacholine, 785 double-blind, placebo-controlled food challenges (DBPCFCs), 1150, 1323 double-stranded RNA (dsRNA), 379– 80 downstream events, 537 downstream signaling, chemokine receptors and, 476– 8 doxylamine, 560 DPIs (dry powder inhalers), 768, 777– 8 DPP10 gene, 1229, 1232, 1234, 1717 DP receptor, 592 activation, 330 DRESS (drug rash with eosinophilia and systemic symptoms), 1958, 1959, 1961, 1962 Drosophila spp. (fruit flies), 189 FLJ 14466, 225 Drosophila melanogaster (fruit fly), dunce gene, 643 drug administration, 668, 677, 697 allergic rhinitis, 1436 intranasal advantages, 1436 disadvantages, 1436 drug allergy, diagnosis, 1326 drug compliance, issues, 1524 drug hypersensitivity, 1901, 1943– 65 classification, 1943, 1947–59 desensitization, 1962– 3 diagnosis, 1959– 62 clinical, 1959– 61 disease induction, 1944 drug identification, 1961–2 in vitro tests, 1962 in vivo tests, 1961–2 multiple, 1959 nonimmune-mediated, 1949 preferential skin involvement, 1953– 8 sensitization risk, 1944 treatment, 1962–3 see also antibody-mediated drug hypersensitivity reactions; multiple drug hypersensitivity syndrome; T-cell-mediated delayed drug hypersensitivity reactions drug-induced adverse reactions classification, 1943 type A, 1943 type B, 1943 drug-induced eosinophilic pneumonia, 1794 diagnosis, 1793 symptoms, 1793 drug-induced exanthems, 1959– 60 drug-induced hypersensitivity syndrome (DiHS), 1958, 1961, 1962 drug-induced rhinitis, 1439 etiology, 1390 drug manufacturing plants, occupational airway diseases, 1101

drug rash with eosinophilia and systemic symptoms (DRESS), 1958, 1959, 1961, 1962 drugs and anaphylaxis, 1901–2 antifungal, 1752–3 and asthma, 1572 Category A, 562 Category B, 562 Category C, 562 combination, 686 cross-reactivity, 1962, 1963 eosinophilic pneumonia induction, 1793–4 and extrinsic allergic alveolitis, 1762 herbal, 683, 1441 immune recognition of, 1944–6 inhalation tests, 1583 and late-phase allergic reactions, 531–3 new, 634–6 in allergy treatment, 1712–39 in asthma treatment, 1712–39 and skin tests, 1340 tolerance mechanisms, 1954 see also antiasthma drugs; antibiotics; anticholinergic agents; antihistamines; antiinflammatory agents; antileukotrienes; immunosuppressive drugs; nonsteroidal antiinflammatory drugs (NSAIDs); translational medicine drug targets, dendritic cells as, 179 drug therapy allergic rhinitis, 1434–9 allergy treatment, 1331–2 asthma, 1331–2 hay fever, 1331 dry air early responses, hyperventilation, 795–7 eucapnic voluntary hyperpnea, 814 and exercise-induced bronchoconstriction, 809 dry powder inhalers (DPIs), 768, 777–8 dsRNA (double-stranded RNA), 379–80 Duffy antigen receptor complex (DARC), chemokine scavenging, 479 Dunbar, William Philipps (1863–1922), 12 dunce gene, 643 durum wheat, allergens, 1101 dust mite allergens, 984 aerodynamics, 1000–1 allergic responses to, 990–1 avoidance, 988–9, 1598–9 issues, 992 characteristics, 985 dose–response relationships, 992 and dust mite fecal particles, 985–91 exposure measurement, 986–7 inhalation, 986 dust mite exposure and asthma, 988 avoidance, 1331 and dust mite sensitivity, 987–8 isotype diversity, 988 reduction strategies, 989 and rhinovirus, 991–2 roles, in diseases, 991 dust mite fecal particles, and dust mite allergens, 985–91

dust mites and asthma, 984–96 early studies, 984–5 immunoglobulin E antibodies, 990 see also house-dust mites (HDMs) dust mite sensitivity allergen-specific treatment, 988–90, 992 and asthma, 987 and dust mite exposure, 987–8 epidemiology, 984, 987–8, 992 immunotherapy, 985–6, 990 future trends, 990–1 regional differences, 987, 992 dust reservoirs, animal allergens in, 998 dyshidrotic eczema, 1817 dysphonia, 1653 dyspnea, nerves in, 833 EAA see extrinsic allergic alveolitis (EAA) EAACI see European Academy of Allergology and Clinical Immunology (EAACI) EAAs (excitatory amino acids), 830 EAN (European Aeroallergen Network), 947 EAR (early asthmatic reaction), 231–2 early asthmatic reaction (EAR), 231–2 early nodule-specific protein, 1170 early-phase allergic reactions (EPRs), 531 priming, 527 progression, 526–7 ear, nose and throat (ENT) examination, allergic rhinitis, 1432 EB see eosinophilic bronchitis (EB) EBC see exhaled breath condensate (EBC) EBV receptor see CD21 ECM see extracellular matrix (ECM) EC-MLCK, 862 ecological environment change, and allergen levels, 144 ECP see eosinophil cationic protein (ECP) ECRHS see European Community Respiratory Health Survey (ECRHS) eczema definition, 1240 discoid, 1840 dyshidrotic, 1817 and eosinophilia, 29 hands, 1840 and immunoglobulin E, 1239 and immunoglobulin E sensitization, 1240–1 lichenified, 1839 mast cells in, 244– 5 neck, 1841 nipple, 1817 numular, 1817 prevalence, 23, 1239, 1244 use of term, 1831 vesicular, 1838 see also atopic dermatitis (AD); atopic eczema; infantile eczema; nonatopic eczema eczema herpeticum, 1820 EDC (epidermal differentiation complex), 1228–9 edema, 1639 and phosphodiesterase inhibitors, 650–1 edema formation, 398, 857 EDN see eosinophil-derived neurotoxin (EDN)

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EE see eosinophilic esophagitis (EE) EECDRG (European Environmental and Contact Dermatitis Research Group), 1832 efalizumab, in atopic dermatitis treatment, 1827 effector cells recruitment, and allergen injection immunotherapy, 1513 suppression, 93– 4 use of term, 1880 see also mast cells efferent innervation see autonomic innervation efficiency, definition, 1999 EGF (epidermal growth factor), 205, 1617 EGFRs see epidermal growth factor receptors (EGFRs) egg allergens, 914, 917, 1151 exposure, 1154 early, 1159 eggs allergic reactions, 1147, 1922 digestion, 1156 lysozyme, 1148 EGIDs (eosinophil-associated gastrointestinal diseases), 279– 80 EHNA see erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) Ehrlich, Paul (1854–1915), 7– 8, 320 EIA see exercise-induced anaphylaxis (EIA); exercise-induced asthma (EIA) EIB see exercise-induced bronchoconstriction (EIB) eicosanoid mediators, 799 eicosanoids biosynthesis, 573 definition, 566 roles, 567 elafin, 373– 4, 1231 elastin, in extracellular matrix, 414 elderly allergic rhinitis, 1446 H1-antihistamines in, 562 asthma, 1657 insect sting allergy, 1985 rhinitis, 1391 electromagnetic fields (EMFs), and indoor air pollution, 1286–7 electrospray ionization time of flight mass spectrometry (ESI-TOF MS), 937 ELISA see enzyme-linked immunosorbent assay (ELISA) emedastine, 557 in seasonal allergic conjunctivitis treatment, 1490 emergency medication kits insect sting allergy, 1989– 90 self-medication, 1990 EMIT (enzyme-multiplied immunoassay technique), 736 emotions, and rhinitis, 1391 EMPD (extra-cellular membrane-proximal domain), 108 emphysema airway wall remodeling, 413 see also chronic obstructive pulmonary disease (COPD)

EMT see epithelial-to-mesenchymal transition (EMT) EMTU see epithelial–mesenchymal trophic unit (EMTU) ENDA (European Network For Drug Allergy), 1901 endobronchial catheter studies, 756 endocytosis, and antigen uptake, 167 endo-β-1,3-glucanase, 1150 endoplasmic reticulum (ER), roles, 298 endothelia, cultured, and intact microvessels compared, 866–7 endothelial adhesion molecules, expression, 345–6 endothelial barrier function, agents enhancing, 865–6 endothelial cells roles, in kinin cascade activation, 458–60 see also human umbilical vein endothelial cells (HUVECs) endothelial contractility, 862–3 and Rho subfamily, 863– 4 endothelial hyperpermeability leukocyte-mediated, 867–8 mitogen-activated protein kinases in, 865 endothelial nitric oxide synthase (eNOS), 860, 861, 862–3 endothelial selective adhesion molecules (ESAMs), 301, 860 endothelium, resting, pores in, 860–1 endotoxins and allergic diseases, 34–5 animal models, 150 benefits vs. detriments, 149–50 exposure, 1264 and immunoglobulin E production, 149–52 mechanisms, 151–2 receptor expression, 150–1 occurrence, 1282–3 ENFUMOSA (European Network For Understanding Mechanisms Of Severe Asthma), 1667 eNO (expired nitric oxide), 809 enolase, 1170 eNOS (endothelial nitric oxide synthase), 860, 861, 862–3 ENT (ear, nose and throat) examination, allergic rhinitis, 1432 enteric infections, and hygiene hypothesis, 1245 enterotoxins, 1720–1, 1824 roles, 1464–5 entomophilous plants, 943 environmental allergens dose–response relationships, 1259 sensitization, 1259, 1261 see also indoor allergens; outdoor allergens environmental factors allergic diseases, 24, 31–9 in immunoglobulin E production, 141–65 environmental postnatal airways disease, experimental models, 1194–5 environmental tobacco smoke (ETS), 148, 1286 and asthma, 149, 1571 constituents, 1281 effects, animal models, 148–9

emissions, 1281 and indoor air pollution, 1281 enzyme inhibitors, in airway epithelium, 387 enzyme-linked immunosorbent assay (ELISA), 932, 947, 1152, 1349 applications, 1323 competitive, 933 immunotherapy monitoring, 1359 inhibition, 933 sandwich, 932–3 enzyme-multiplied immunoassay technique (EMIT), 736 enzymes in airway epithelium, 387 in baking industry, 1703 and occupational asthma, 1703–4 eos47 gene, 269 eosin, 8 eosinophil adhesion, 346–9 eosinophil-associated disease, 1741–809 eosinophil-associated gastrointestinal diseases (EGIDs), 279–80 eosinophil cationic protein (ECP), 29, 259–60, 1293, 1370, 1414–15, 1460 functions, 259 in idiopathic chronic eosinophilic pneumonia, 1783 serum concentrations, 1372 eosinophil-derived neurotoxin (EDN), 259, 260, 1417 in idiopathic chronic eosinophilic pneumonia, 1783 eosinophil development, 269–70 eosinophil granule proteins, 258–64 eosinophil hematopoietin, 1805–6 eosinophilia, 1293, 1302 and asthma, 984 in bone marrow, 1803 bronchial, 1925 and eczema, 29 esophageal, 1925 familiar, 1804 and hypereosinophilic syndromes, 1802–3, 1804 markers, 29 see also pulmonary eosinophilia; tropical eosinophilia eosinophilic airway inflammation and absence of asthma, 1373– 4 and airway dysfunction, 1373–5 and asthma pathophysiology, 1374–5 prevalence, 1374 eosinophilic asthma, 1621, 1794–5 eosinophilic bronchitis (EB), 237–8, 1795 diagnosis, 1373–4 eosinophilic cationic protein (ECP), 1495 eosinophilic esophagitis (EE), 1928 pathogenesis, 1925 eosinophilic gastroenteropathies, 1927 eosinophilic lung diseases, 1585 definition, 1779 of determined cause, 1789–95 of undetermined cause, 1782–9 eosinophilic pneumonias, 1779–80 after radiation therapy, 1794

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classification, 1779– 80 definition, 1779 diagnosis, 1780–2 of infectious origin, 1789– 91 of parasitic origin, 1789– 91 pathology, 1780 toxic-agent-induced, 1794 toxocariasis-induced, 1790 see also drug-induced eosinophilic pneumonia; idiopathic eosinophilic pneumonias eosinophilic rhinitis, etiology, 1391 eosinophil migration, 346– 9 through vascular endothelium, 347– 8 in tissue, 348– 9 eosinophilopoiesis, 346–7 eosinophil peroxidase (EPO), 258– 9, 260, 1293, 1414–15 eosinophil protein X (EPX), 29 eosinophils, 29, 73, 295 in allergic diseases, 1293–5 in allergic rhinitis, 1417–18 apoptosis, 1669 in asthma, 276– 9, 984, 1611–13 animal models, 276–7 biological properties, 258– 94 in bronchoalveolar lavage, 1195, 1268 discovery, 8 and fibrosis mediation, 420–1 and gastrointestinal diseases, 279– 80 and immune regulation, 266– 9 in infection, 273– 6 and late asthmatic reaction, 278 and late-phase allergic reactions, 527– 8 as markers of airway inflammation, 809 maturation, 1712 in nasal polyposis, 1461 and neurotrophins, 500 origin, 1803 recruitment and allergen injection immunotherapy, 1513 by chemokines, 483–5 roles, 258 in diseases, 272– 80 in health, 280–1 signal transduction, 210–11 SNARE isoform expression, 262– 4 in sputum, 787, 1600, 1716–17 thymic, 266–7 eosinophil surface markers, 264– 6, 267 eosinophil trafficking, 270–2 chemokine regulation, 270–2 eotaxins, 271–2, 484, 573, 920, 1303, 1415 expression, 1460 production, 1303– 4 receptors, 1416 Ephedra equisetina (ma-huang), 680 ephedrine, 680, 1439, 1441 structure, 673 Epidemiological Study of Xolair Evaluating Clinical Effectiveness and Long-Term Safety in Patients with Moderate to Severe Asthma (EXCELS), 1670, 1675 epidermal differentiation complex (EDC), 1228–9 epidermal growth factor (EGF), 205, 1617

epidermal growth factor receptors (EGFRs), 851, 1617 ligands, 1618 upregulation, 1614 epidermis, innate immune system, 1823 EPIDERM project (UK), 1832 EPI (European Pollen Information), 947–9 epinastine, 557, 1489 epinephrine, 668, 669, 672, 680 administration, 677 in anaphylaxis treatment, 1911, 1912–13, 1915–16, 1935 in hypotension treatment, 1890 structure, 673 epithelial cells, 367 adhesion, 369 airway, 180, 181 and allergic diseases, 1297 and asthma, 418 in bronchoalveolar lavage, 798 ciliated, 368–9 extracellular matrix production, 418–19 models, 371–2 roles, in remodeling, 419 epithelial changes, asthma, 1634–5 epithelial differentiation, 1193 epithelial diseases, genes, 1229 epithelial Duox, 376 epithelial immunity barrier defenses, 1230–1 danger recognition, 1231–2 danger responses, 1232 mechanisms, 1229–32 epithelial injury, 1614 air pollution, 1616 repair, 1617 epithelial innervation, 1194 exposure responses, 1197 epithelial–mesenchymal trophic unit (EMTU), 1187 concept of, 1188–9 structure, 1188 epithelial receptors, for sensing microbes, 377 epithelial-to-mesenchymal transition (EMT), 417, 423 mechanisms, 418–19 epithelium in chronic asthma, 1615 olfactory, 371 roles in adaptive immune responses, 385–7 in inflammation, 380–5 in innate immunity, 373–80 see also airway epithelium epitope–major histocompatibility complex specificity, 49 EPO (eosinophil peroxidase), 258–9, 260, 1293, 1414–15 EP receptors, 597–8 Epstein–Barr virus (EBV) receptor see CD21 EPX see eosinophil-derived neurotoxin (EDN); eosinophil peroxidase (EPO) EPX (eosinophil protein X), 29 Equ c 1, 1137 Equ c 2, 1137

ER (endoplasmic reticulum), roles, 298 ERK (extracellular signal-regulated kinase), 884 ERK pathway, 210 ERS (European Respiratory Society), 1371, 1575 Erysiphe spp. (powdery mildew), seasons, 966 erythrocytes, clearance mechanisms, 440 erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), 641 structure, 641 ESAMs (endothelial selective adhesion molecules), 301, 860 ESCD (European Society of Contact Dermatitis), 1832 Escherichia coli (bacterium), 312 allergens, 1135 BLT1, 577 enterotoxins, 1720–1 K1, 446 ovomucoid, 1150 rBet v 1 production, 1556, 1557 Escherich, Theodor (1857–1911), 4 ESI-TOF MS (electrospray ionization time of flight mass spectrometry), 937 ESL-1, 340 esmolol, 679 esophageal eosinophilia, 1925 ESSCA (European Surveillance System of Contact Allergies), 1832 etanercept, 73 ethylene oxide, and anaphylaxis, 1906 ETOPA study, 1670, 1673, 1675, 1679, 1681 ETS see environmental tobacco smoke (ETS) EtxB, 1720–1 EU see European Union (EU) eucapnic voluntary hyperpnea (EVH), 813 with dry air, 814 European Academy of Allergology and Clinical Immunology (EAACI), 1239, 1337, 1544 allergy nomenclature, 1921 guidelines, 1439, 1441, 1522, 1523, 1529, 1530 anaphylaxis, 1901 European Aeroallergen Network (EAN), 947 European Commission (EC) Scientific Committee on Health and Environmental Risks (SCHER), 1280 Strategy on Environment and Health, 1279 European Community Respiratory Health Survey (ECRHS), 1240, 1384, 1387, 1922 allergy prevalence studies, 2020 geographic variations, 1241–2 II, 1248, 1249 time trends, 1242 European Environmental and Contact Dermatitis Research Group (EECDRG), 1832 European Fighting for Breath survey, 1662 European Network on Aspirin-Induced Asthma (AIANE), 1973 European Network For Drug Allergy (ENDA), 1901 European Network For Understanding Mechanisms Of Severe Asthma (ENFUMOSA), studies, 1667 European Pollen Information (EPI), 947–9 European Respiratory Society (ERS), 1371, 1575

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European Society of Contact Dermatitis (ESCD), 1832 European Surveillance System of Contact Allergies (ESSCA), 1832 European Union (EU) air quality legislation, 1279 directives, 1061, 1102, 1849 Sixth Environment Action Programme, 1279 European Union Respiratory Health Survey see European Community Respiratory Health Survey (ECRHS) Eurotium spp. (fungi), occurrence, 971, 978 Eurotium amstelodami (fungus), 1761 everolimus adverse effects, 741 discovery, 740 drug interactions, 741 indications, 741 mechanisms, 741 monitoring methods, 741 pharmacogenetics, 740–1 pharmacokinetics, 740 structure, 740 EVH see eucapnic voluntary hyperpnea (EVH) evidence-based treatment allergic rhinitis, 1441–3 availability, 1442–3 exacerbations asthma, 279, 1575– 6, 1577 and acute viral infections, 1247 management, 1578, 1653 of inflammation, 145 see also acute exacerbations exanthems, drug-induced, 1959– 60 EXCELS (Epidemiological Study of Xolair Evaluating Clinical Effectiveness and LongTerm Safety in Patients with Moderate to Severe Asthma), 1670, 1675 excitatory amino acids (EAAs), 830 exercise, and anaphylaxis, 1863– 4, 1907– 8 exercise hyperventilation syndrome, 808 exercise-induced anaphylaxis (EIA), 1907– 8 food-dependent, 1908 exercise-induced asthma (EIA), 236, 585– 6 antileukotrienes in, 707– 8 use of term, 808 exercise-induced bronchoconstriction (EIB), 790, 791 and airway inflammation, 809 and allergens, 812–13 animal models, 794– 807 diagnosis, 813–14 surrogate challenges, 814–15 differential diagnosis, 795– 6, 808 human models, 808–22 selection criteria, 815–16 and hyperosmolar aerosols, 814–15 identification, with pharmacologic agents, 815–16 mechanisms, 809 and mediators, 809–12 and physical training, 817 prevalence, 812–13 prevention, 816–17 nonpharmacologic methods, 816–17

stimuli, 809 dry air, 809 use of term, 808 see also hyperventilation-induced bronchoconstriction (HIB) exercise monitoring, cycling, 813 exercise tolerance, ozone effects, 1268 exertional asthma, use of term, 1214 exhaled air, inflammation markers, 1368–80 exhaled breath, biomarkers in, 1716–17 exhaled breath condensate (EBC), 1372 analytical issues, 1717 asthma, 1584 hydrogen peroxide, 1717 markers, 1372 exhaled nitric oxide airway inflammation assessment, 1371–2 as biomarker, 1716–17 fractional, 1584 measurement, 1600 characteristics, 1371–2 portable analyzers, 1371 β-expansin, 1154 expired nitric oxide (eNO), 809 extracellular matrix (ECM) altered turnover, 413 breakdown, 402 components, 413–15 and extrinsic allergic alveolitis, 1764 and fibroblasts, 412–35 extracellular matrix-producing cells, 415–19 extracellular membrane-proximal domain (EMPD), 108 extracellular signal-regulated kinase (ERK), 884 extrathoracic airways, in asthma, 762–3 extrinsic allergic alveolitis (EAA), 18, 1757–78 acute, 1764–5, 1766, 1768–9, 1770 and animal allergens, 1762 antibodies, 1764 antigen challenge, 1769 antigens, 1759–60 avoidance, 1771–2 bronchoalveolar lavage, 1768 chest radiology, 1766–7 chronic, 1765, 1767, 1769, 1770 classification, 1757 clinical features, 1764–5 definition, 1757 diagnosis, 1765–9 differential diagnosis, 1769–71 disease course, 1772 and drugs, 1762 early studies, 1757–8 epidemiology, 1757–8 etiology, 1758–62 future research, 1773 genetics, 1758 histopathology, 1768–9 and humidifier fever, 1771 immunopathogenesis, 1762–4 infections, 1769 and inflammatory cells, 1762–3 laboratory studies, 1766 and low-molecular-weight compounds, 1762 and lung diseases, 1771

management, 1771–2 mediators, 1763–4 and microbial antigens, 1758– 62 modulating factors, 1764 and organic dust toxic syndromes, 1771 pharmacotherapy, 1772 prevalence, 1758 prevention, 1772–3 prognosis, 1772 pulmonary function tests, 1768 radiological features, 1766–8 and sick building syndrome, 1771 and smoking, 1764 subacute, 1765, 1769 treatment, 1771–2 and viral infections, 1764 eye, outer, 1482–5 eye deposition, 774 eyelid dermatitis, 1817 eyelids allergic contact dermatitis, 1841 anatomy, 1482–4 FAAN (Food Allergy and Anaphylaxis Network) (US), 1899 face, allergic contact dermatitis, 1840–1 facemasks, 774 FACET (Formoterol and Corticosteriods Enabling Therapy), 1713 facial deposition, 774 facilitated antigen presentation (FAP), 1562 factor XII, 452 activation, 1906 binding, to human umbilical vein endothelial cells, 456 PK–HK complex activation, 458–60 FADD (FAS-associated via death domain), 380 Fagus spp. (beech trees), 944 FALCPA (Food Allergen Labeling and Consumer Protection Act) (2006) (US), 1147 fall rhinitis, 1323 familiar eosinophilia, use of term, 1804 family size, and allergic diseases, 32 family structure, and asthma, 1244–5 FAP (facilitated antigen presentation), 1562 farm environments and allergic diseases, 33–4 childhood benefits, 33–4, 149 farmer’s lung see extrinsic allergic alveolitis (EAA) farming, and hygiene hypothesis, 1246 FARRP (Food Allergy Research and Resource Program), 902–3 Fas expression, 49–50 mutations, 49–50 FAS-associated via death domain (FADD), 380 fatty acids, 1249 Fcε2-4, crystal structure, 106 Fcε3-4, crystal structure, 105, 106 Fcε3-4/FcεRIα complex, crystal structure, 109 Fcε antibodies, FcεRIα, 14–18 FCER1B gene, 1229 FcεRI, 103, 108–9, 227, 235, 482–3 in airway smooth muscle, 881 binding, 320–2, 1514

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binding sites, 107 detection, 1298 expression, 1297 receptors, 1667 roles, 104 see also immunoglobulin E–FcεRI interaction FcεRI+ activation, 326–7 receptor antagonist cell-derived mediators, 329–30 targeting, 328 FcεRII see CD23 FcεRIβ gene, 1234 FcγR, roles, 439 FcγRI, 439 FcγRIIA, 439 FcγRIIB, 440 FcγRIII, 439, 440 FcγRIV, 439 Fc receptors, 441, 533 feet, contact dermatitis, 1841–2 Fel d 1, 538, 914, 916, 919, 921, 922, 929, 990–1 activity, 1232 airborne, 999, 1000 exposure, 1001, 1003, 1012, 1136 extracts, 1349 impacts, 998 levels, 999 and cat washing, 1002 particle size distribution, 999 sensitization, 1559– 60 structure, 998 studies, 1355 Fel d 2, 916 Fel d 3, 916 Fel d 4, 916, 1137 Feldberg, W., 567 Felis domesticus (domestic cat), allergens, 998 FENO see exhaled nitric oxide fenoterol, 674, 675 structure, 673 Ferdnand Widal triad, 1394 FESS (functional endoscopic sinus surgery), 1468 fetal growth, and allergic diseases, 38 fetus, allergic disease prevention, 1997–2019 FEV1 see forced expiratory volume in 1 second (FEV1) fexofenadine, 554, 555, 556–7, 561, 1869 FGF-1 (fibroblast growth factor-1), 1617 FGF-2 (fibroblast growth factor-2), 1617 fibers, and indoor air pollution, 1283 fibrinolysis, roles, 463 fibroblast growth factor-1 (FGF-1), 1617 fibroblast growth factor-2 (FGF-2), 1617 fibroblasts, 1308–9 activation, 416 and extracellular matrix, 412–35 extracellular matrix production, 415–17 see also myofibroblasts fibrocytes, extracellular matrix production, 417–18 fibrosis and asthma, 412–13

and lung diseases, 413 mediators, 422–5 indirect, 419–22 targeting, 425 see also cystic fibrosis (CF); idiopathic pulmonary fibrosis (IPF) Ficus benjamina (weeping fig), allergens, 1286, 1703 Ficus–fruit syndrome see latex–fruit syndrome filaggrin (FLG), 1228 encoding, 1615 fine particle fraction (FPF), 769 fine particles, definition, 1281 FIP1L1-PDGFRA fusion gene, 1885, 1888 fire ants, 1983 venom, 1125 fish allergens, 1151, 1152 allergic reactions, 1147 see also shellfish fish poisoning, scombroid, 1921 FK-224, 516, 531 FLAP (5-lipoxygenase-activating protein), 570–1, 594 Fleming, Sir Alexander (1881–1955), 373 FLG see filaggrin (FLG) FLJ 14466, 225 floricultures, occupational airway diseases, 1061 flour allergens, 1102, 1155 flow cytometric analysis, 1886 of basophil activation, 1360–1 diagnostic performance, 1363 isolated basophils, 1361 whole blood cells, 1361 flowers, allergens, 1061 flow–volume tests, 753–4, 1582 Floyer, John (1649–1734), 10, 11 Treatise of the Asthma (1698), 1591 flunisolide, structure, 718 fluorescence resonance energy transfer (FRET), 105 fluticasone propionate (FP), 405, 695, 699, 718–19, 1438 adverse effects, 724 efficacy, 721 structure, 718 fMLP see N-formyl-methionyl-leucylphenylalanine (fMLP) follicular dendritic cells (FDCs), 120 food and anaphylaxis, 1903–5 tolerance, 1925 weaning, 2003–4 see also diet; nutrition food additives, 1929–30 allergic reactions to, 1328, 1926 Food Allergen Labeling and Consumer Protection Act (FALCPA) (2006) (US), 1147 food allergens, 902–3, 914, 918, 1146–63, 1929–30 animal models, 1158 avoidance children, 2004 in pregnancy, 2000

characterization, 1147 class I, 1925 classification, 1148 class II, 1925 cross-reactivity, 1155, 1929–30 databases, 1158 definition, 1146 epitopes, 1149–50 exposure, 1153–4 future research, 1159 genetically modified, 1156–8 mechanisms, 1147 respiratory, 1155 sequences, 1158 structure, 1148–50 threshold doses, 1154–5 food allergy, 1921–42 allergen avoidance, 1924, 1935 and anaphylaxis, 1328, 1903–5 and atopic dermatitis, 1819 childhood, 1147 classification, 1921–2 clinical features, 1926–31 clinical investigations, 1931–4 definition, 1921–2 diagnosis, 1327–8 differential diagnosis, 1934 and digestibility, 1155 disease course, 1936 epidemiology, 1922–3 etiology, 1146–7, 1923–4 and food processing, 1155–6 future therapies, 1935–6 future trends, 1936–7 and gastrointestinal diseases, 1929 genetics, 1923 and latex allergy, 1173–4 management, 1935–6 mechanisms, 1147–8, 1923–4 pathogenesis, 1924–6 pathology, 1926–31 perception, prevalence, 1152 prevalence, 1922 prevention, 1936 prognosis, 1936 treatment, 1935–6 use of term, 1921 see also fruit allergy Food Allergy and Anaphylaxis Network (FAAN) (US), 1899 Food Allergy Research and Resource Program (Farrp), 902–3 food avoidance education, 1935 rates, 1922 food hypersensitivities use of term, 1921 see also food intolerance food-induced contact dermatitis, 1930 food-induced rhinitis, etiology, 1391 food intolerance and intestinal diseases, 1329 nonallergic forms of, 1328 use of term, 1327, 1921 food poisoning, 1328

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food processing and food allergy, 1155– 6 fungal spores in, 972– 3 food protein-induced enterocolitis syndrome (FPIES), 1928– 9 infants, 1924 food proteins adverse reactions, 1924 allergenic, 1146, 1147 avoidance education, 1935 food sensitivity, use of term, 1327 forced expiration tests, 750– 2 forced expiratory spirograms, 750–1 forced expiratory volume in 1 second (FEV1), 516, 519, 525, 585, 586, 751 anticholinergic agent studies, 686– 7 applications, 2023 in asthma management, 749 in asthmatic airway diagnosis, 832 changes, 775 decline, 1574 drug-improved, 674 in exercise-induced bronchoconstriction, 812, 813–14 histamine effects, 809–10 measurement, 808 in occupational asthma studies, 1696– 7 PC20, 786 PD20, 786 PDE4 inhibitor studies, 651–2 and placebos, 834 in spirometry, 1579– 80 tests, 785– 6 forced oscillation technique (FOT), 753 forced vital capacity (FVC), 652, 687, 751, 754, 2023 forkhead winged transcription factor-3 (Foxp3), 58, 86, 922 formaldehyde, and indoor air pollution, 1283– 4 Formica rufa (red ant), 1983 Formicidae (ants), 1123 Formicinae (ants), insect sting allergy, 1983 formoterol, 674, 675, 1717 administration, 677 in combination drugs, 686 in exercise-induced bronchoconstriction, 816 structure, 673 Formoterol and Corticosteroids Enabling Therapy (FACET), 1713 N-formyl-methionyl-leucyl-phenylalanine (fMLP), 302, 324 receptors, 325 fossil fuels, pollutants, 144 FOT (forced oscillation technique), 753 Foxp3 (forkhead winged transcription factor-3), 58, 86, 922 FP see fluticasone propionate (FP) FPF (fine particle fraction), 769 FPIES see food protein-induced enterocolitis syndrome (FPIES) fractalkine, 350 Fraxinus exelsior (ash), 957 FRC (functional residual capacity), 752, 755 Freeman, John (1877–1962), 12–13

FRET (fluorescence resonance energy transfer), 105 Frey–Wyssling particles, 1167 FR receptors, 592–3 fruit allergy, 1926 and latex allergy, 1172, 1173–4 prevalence, 1922–3 fruit–latex syndrome see latex–fruit syndrome fruits allergens, 1153 cross-reactivity, 1153 fucose, 1126 functional endoscopic sinus surgery (FESS), 1468 functional fucosyltransferase 2 (FUT2), 373 functional residual capacity (FRC), 752, 755 fungal allergens, 973–5, 1205 recognition, 975 release, 964, 969 fungal cells, structure, 964 fungal particles, from building materials, 967–9 fungal sinusitis, 1463–4 see also allergic fungal rhinosinusitis (AFS) fungal spores abundance, 963–4 circadian periodicity, 966 dispersal, 965 future research, 979 geographic distribution, 967 local sources, 967 occupational exposure, 979 outdoors, 969 occurrence agricultural environments, 972 food processing, 972–3 indoors, 970–3 mushroom farms, 972 nonindustrial environments, 971–2 outdoors, 965–9 residential environments, 971–2, 979 release mechanisms, 966 sampling equipment, 979 seasons, 966–7, 968 fungal studies, Hurricane Katrina, 976–9 fungi agents, 964–5 as allergens, 963–83 allergy induction, 965 classification, 964 contact allergens, 965 early studies, 963 and extrinsic allergic alveolitis, 1761–2 and indoor air pollution, 1282–3 nonallergic factors, 976 particles, 963 respiratory allergens, 965 sensitization, 965 sexual reproduction, 964 see also fungal spores; molds furosemide, inhalation, 833 Fusarium spp. (fungi), seasons, 967 fusion proteins, 742–3 FUT2 (functional fucosyltransferase 2), 373 FVC (forced vital capacity), 652, 687, 751, 754, 2023

Gad c 1, 917, 1151 GAF domains, 637 GAGs see glycosaminoglycans (GAGs) GAIN (Genetics and Asthma International Network), studies, 1134 α-galactosylceramide (α-GalCer), 56 α-GalCer (α-galactosylceramide), 56 galectin-3, 113 GALT (gut-associated lymphoid tissue), 1147 gAMP see cyclic guanosine 3′,5′-monophosphate (gAMP) Ganoderma spp. (fungi), 974 garden cities, 1279 gases, and immunoglobulin E production, 146 gas-exchange reactions, lung, 1218–19 Gastão Rosenfeld (1912–90), 9 gas trapping, computed tomography studies, 758 gastroesophageal reflux (GOR) and asthma, 1573 management, 1573 treatment, 1602 gastrointestinal anaphylaxis, 1927 gastrointestinal diseases eosinophil-associated, 279–80 and food allergy, 1929 gastrointestinal flora, 30–1 and allergic diseases, 36 gastrointestinal food-allergic disorders, 1926–9 gastrointestinal tract functions, 1147 and immune development, 30–1 GATA-1, expression, 269–70 GATA-3, 51, 52 in allergic diseases, 1306, 1308 Gaw (airways conductance), 785 Gay, Leslie N., 831 gC1qR, 455, 456–7, 458 GCP (Good Clinical Practice), 1714–15 GEFs (guanine nucleotide exchange factors), 863, 864, 865–6 gelatin, 1926 Gell, Philip George Houthem (1914–2001), 5–6, 14–20 Gell–Coombs classification, 14–20 GenBank, nomenclature, 637–8 gender and asthma, 1243–4, 1570 and skin tests, 1339 gene expression control, in immune system, 129–31 principles, 129 genes asthma, 1229 disease, 1225–6 epithelial diseases, 1229 tissue-specific, 40 see also immunoglobulin genes; susceptibility genes gene therapy, 1726–7 genetically modified allergens, 1156–8 genetic markers, allergy risk, 1999 genetics acute rhinosinusitis, 1470 allergy, 24, 39–40

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asthma, 1206–7, 1225–38, 1247– 8, 1260, 1570, 1717 novel mechanisms, 1623 atopic dermatitis, 1225–38, 1260, 1821–2 chronic rhinosinusitis, 1470 extrinsic allergic alveolitis, 1758 food allergy, 1923 hypereosinophilic syndromes, 1804 immunoglobulin E production, 141–2 pollutants and, 147– 8 tobacco smoke and, 149 immunoglobulin E sensitization, 1247– 8 nasal polyposis, 1459– 60 Genetics and Asthma International Network (GAIN), studies, 1134 gene transcription, glucocorticoid effects, 715 genome screens for allergic rhinitis, 1227 for asthma, 1226–7 for atopic dermatitis, 1227 genome-wide association (GWA) studies, 1226 asthma, 1227– 8 Germany, atopic diseases, 1260–1, 1264 germinal center reactions, 120 giant papillary conjunctivitis (GPC), 1500– 2 clinical features, 1501 etiology, 1487 histology, 1501 pathogenetic issues, 1501–2 treatment, 1502 GINA see Global Initiative for Asthma (GINA) glass hypothesis, 879– 80 gliadins, 1154 Global Initiative for Asthma (GINA), 1567– 8 guidelines, 1662, 1663–5, 1712 severity classification, 1568 glomerulonephritis, 437 glottis, in asthma, 762–3 gloves (latex) see latex gloves β-1,3-glucanase, 1166, 1167 glucans, 964 glucocorticoid receptors (GRs), 716–17, 885, 1713 glucocorticoid resistance, 726– 7 molecular mechanisms, 726– 7 reversal, 727 therapy, 727 glucocorticoid response elements (GREs), 716–17 glucocorticoids, 72, 329, 669, 715– 31 for airway hypersecretion, 847– 8 in allergic rhinitis treatment, 1437– 8, 1443 in asthma, 1572, 1574, 1581 chemical structure, 718 effects cellular, 715–16 on gene transcription, 715 on inflammation, 717 effects on airway smooth muscle, 885 limiting factors, 885– 6 effects on nasal polyps, 1461 interactions, with β2-adrenergic receptors, 717–18 intranasal, 1438 properties, 1438 safety, 1443– 4 mechanisms, 715–18

pharmacokinetics, 718–19 topical, 1825 see also inhaled glucocorticoids; systemic glucocorticoids glucocorticosteroids see glucocorticoids glutathione transferase, 1131 glutathione S-transferase (GST), 148 antibodies, 975 gluten, 1151 GlyCAM-1 (glycosylated cell adhesion molecule1), 339 glycoallergens, 1153 glycoproteins, in insect allergens, 1126 glycosaminoglycans (GAGs), 414, 485 interactions, with chemokines, 474 glycosylated cell adhesion molecule-1 (GlyCAM1), 339 Gly m Bd 28K, 1156 Gly m Bd 30K, 1148, 1151, 1158 GM-CSF see granulocyte–macrophage colonystimulating factor (GM-CSF) GMFA, 1156 goat’s milk, allergy to, 3 goblet cells, 369–70, 848 apoptosis induction, 851 in chronic asthma, 1618 hyperplasia, 1637 Goldblatt, M. W., 567 Golgi apparatus, neutrophils, 298 Good Clinical Practice (GCP), 1714–15 GPC see giant papillary conjunctivitis (GPC) GPCRs see G protein-coupled receptors (GPCRs) GPRA gene, 1229, 1234 G protein-coupled receptors (GPCRs), 204, 471, 472, 475–6, 1723 see also chemokine receptors G-protein coupling, chemokine receptors, 476–8 G proteins, 302, 671–2 heterotrimeric, 204–5 signal transduction, 204–5 small, 205 grain dust, and occupational airway diseases, 1101 grain fever, 1101 Gramineae (grass family), 950 granulocyte–macrophage colony-stimulating factor (GM-CSF), 94, 170–1, 177, 264–5, 528, 1418 tissue densities, 1460 granulopoiesis, neutrophils, 295, 298 grass allergens, 897, 916, 918 hybrid, 1558–9 grasses distribution, 950 and hay fever, 1324 wind pollination, 950 grass pollen, 950–3 counts, 953–4 cross-reactivity, 950 daily variations, 951–3 diurnal variations, 951–3 grains, 950–1 granule release, 945 pollen blocker creams, 1441 rupture, 945 seasons, 944, 951, 952–3

severity, 951 timing, 951 subgroups, 950 grass pollen allergens, 913–14, 915, 1359, 1364, 1557–8 recombinant, 1555–6 grass pollen allergy, 95, 1385 grass pollen immunotherapy, 1512 sublingual, 1712, 1720 Grave’s disease, 14 greenhouses, occupational airway diseases, 1061–101 growth, and inhaled glucocorticoids, 724–5 growth delay, and atopic dermatitis, 1821 growth factors, expression, 1208, 1209 GRs (glucocorticoid receptors), 716–17, 885, 1713 GSK 685 698, 1712 GSK-159797, 1717 GST see glutathione S-transferase (GST) GSTM1 gene, 148, 149 GSTP1 gene, 148, 149 GTPases (guanosine triphosphatases), 863–4 GTP (guanosine triphosphate), 863–4 guanine nucleotide exchange factors (GEFs), 863, 864, 865–6 guanosine triphosphatases (GTPases), 863–4 guanosine triphosphate (GTP), 863–4 gustatory rhinitis, 1391, 1921 gut, immune system, 30–1 gut-associated lymphoid tissue (GALT), 1147 gut colonization, infants, 2008–9 GWA studies see genome-wide association (GWA) studies Haemophilus influenzae (bacterium), 1463, 1470 Hageman factor see factor XII hairdressing salons, occupational airway diseases, 1101 Hamburger, Franz (1874–1954), 5 hands allergic contact dermatitis, 1839, 1840 eczema, 1840 HapMap project, 1226 hapten concept, 1944–6 haptens, 1901 Harmonia axyridis (Asian lady beetle), allergens, 144 hay fever, 943, 1219, 1225 and asthma, 12 definition, 1240 diagnosis, 1323–4 drug therapy, 1331 early studies, 9–14 etiology, 1323, 1324 and immunoglobulin E, 1239 and immunoglobulin E sensitization, 1240–1 and pollen, 12 prevalence, 945, 1239, 1323–4 see also histamine hazelnuts allergens, 1152 threshold doses, 1155 lipid transfer protein, 930 hCLCA1, 851

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HCP-1 (high affinity cAMP-specific phosphodiesterase 1), 646 HCWs see healthcare workers (HCWs) HDAC2 (histone deacetylase 2), 717, 726– 7 HDI (hexamethylene diisocyanate), 1699 HDMAs see house-dust mite allergens (HDMAs) HDMs see house-dust mites (HDMs) headaches, food-induced, 1328 health eosinophils in, 280–1 mast cells in, 228–31 neutrophils in, 313 healthcare workers (HCWs) allergy awareness, 1321–2 and latex allergy, 1165, 1166, 1168, 1171–2, 1703 health issues, indoor air pollution, 1286–7 heart, adrenoceptors, 669 heating, ventilation and air-conditioning (HVAC) systems, allergens, 972 heavy metals, 1285 Heiner syndrome, 1930 Hel as 1, 1137 helicase family, 379– 80 helicases, 380 Helicobacter pylori (bacterium), 1245, 2026 infection, 1822 Heligmosomoides polygyrus (nematode), 154, 2026– 7 helium–oxygen mixtures, airway responses, 756–7 helminth infections, 2026–7 and allergic diseases, 154 animal models, 154 helminths, 36 hemidesmosomes, 369 hemolytic anemia, 14, 1950–1 hemopoietic stem cells (HSCs), 404 Henoch–Schönlein purpura, 438 hen’s eggs, sensitization to, 29 HEPA (high-efficiency particulate air), filters, 1001–2, 1433– 4 hepatitis A, 152, 1245, 2026 hepatitis C, 438 hepatomegaly, and systemic mastocytosis, 1883 herbal medicines, 683, 1441 hereditary angioedema, 1866– 7 hereditary factors atopic diseases, 1260 see also genetics Hertoghe’s sign, 1818 HESs see hypereosinophilic syndromes (HESs) 5-HETE see 5S-hydroxyicosatetraenoic acid (5-HETE) Hev b 1, 1164, 1167, 1178 Hev b 2, 1167 Hev b 3, 1164, 1167– 8, 1178 Hev b 4, 1168 Hev b 5, 1164, 1168– 9, 1174, 1175, 1178 in latex gloves, 1168, 1169, 1171, 1172 T-cell mapping, 1169

Hev b 6, 1164, 1169, 1178 Hev b 6.01, 1169, 1175, 1558 Hev b 6.02, 1169, 1171, 1178 cross-reactivity, 1170 in latex gloves, 1170 mutations, 1558 Hev b 6.03, 1169 Hev b 7, 1169–70 Hev b 7.01, 1170 Hev b 7.02, 1170 Hev b 8, 1170 Hev b 9, 1170 Hev b 10, 1170 Hev b 11, 1170 Hev b 12, 1170 Hev b 13, 1164, 1170, 1171, 1178 Hevea brasiliensis (rubber tree) latex extraction, 1166, 1702 proteins, 1164, 1166, 1173 hevein, 1153, 1166 hevein precursor, 1169 hexamethonium, inhalation, 1715 hexamethylene diisocyanate (HDI), 1699 HFAs (hydrofluoroalkanes), as propellants, 776 HIB see hyperventilation-induced bronchoconstriction (HIB) high affinity cAMP-specific phosphodiesterase 1 (HCP-1), 646 high-efficiency particulate air (HEPA), filters, 1001–2, 1433–4 high mobility group box 1 (HMGB1) protein, 170 high-molecular-weight kininogen (HK), 452 binding, to human umbilical vein endothelial cells, 454–6 in contact activation, 454 structure, 453 high-performance liquid chromatography (HPLC), 936–7 high-resolution computed tomography (HRCT), 754–5, 758, 759 extrinsic allergic alveolitis studies, 1767–8 high-volume samplers (HVSs), 947 Hippocrates (c.450–c.370 BC), 3, 1591 Hirschberg, V. G. S., 1966 Hirst spore traps, 946 histamine, 8–9, 322–3, 529, 833 and airflow limitation, 791 in allergic inflammation, 553, 554 biological effects, 551 control, 1338 dose–response relationships, 785 and exercise-induced bronchoconstriction, 809–11 exposure responses, 1195 expression, 551 hyperresponsivity, 784 and immunomodulation, 553–4 inhalation challenges, 1581 inhibition, 329 in insect venoms, 1127 mast cell mediation, 1908 see also antihistamines histamine antagonists, 1718–20

histamine receptors, 551, 552, 553 H1, 551, 552, 554, 1419 activity, 553–4 antagonists, 849 two-state model, 553 H2, 551, 552, 554, 1419 activity, 553–4 H3, 551, 552, 554, 1419 activity, 553 antagonists, 1712 H4, 551, 552, 554, 1419 antagonists, 1712 histamine receptor type 2 (HR2), in peripheral tolerance, 94 histone deacetylase 2 (HDAC2), 717, 726–7 histones, 129 HK see high-molecular-weight kininogen (HK) HLA (human leukocyte antigen), 1485, 1692 HLA class II genes, 1923 HLA-DR, 1485 HLA-G gene, 1717 HLMCs (human lung mast cells), 226, 227, 228 HMC-1 cells, 1887 HMGB1 (high mobility group box 1) protein, 170 homeostasis, alveolar macrophages, 180–1 homopoiesis, 404 honeybees, 1124, 1902 Africanized, 1981 venom, 95 hormonal rhinitis, etiology, 1391 horseradish peroxidase, 1126 horse serum, 4–5 toxicity, 436 hospital admission rates, asthma, 1243 hospitalization, persistent severe allergic asthma, 1661–2 host defense complement in, 436–50 immune complexes in, 436–50 host responses, to allergens, 913–27 house dust radioallergosorbent tests, 1354 skin tests, 984 house-dust mite allergens (HDMAs), 143, 903, 916, 1187 in active immunization models, 1205 and asthma, 142, 1570–1 animal models, 1194 exposure responses, 1197–9 quantity measurement, 1324 sensitization, 2005 house-dust mites (HDMs), 111, 144, 914, 1228 avoidance, 2000, 2004–5 Blomia tropicalis, 916 and indoor air pollution, 1281–2 omalizumab studies, 1669 and perennial rhinitis, 1324 see also Dermatophagoides farinae (American house-dust mite); Dermatophagoides pteronyssinus (European house-dust mite) HPA (hypothalamic–pituitary–adrenal) axis, suppression, 723–4 HPLC (high-performance liquid chromatography), 936–7 HR2 (histamine receptor type 2), 94

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HRCT see high-resolution computed tomography (HRCT) hRSV infection, 274 –5 HRV14 infection, 1967 HRVs see human rhinoviruses (HRVs) Human Genome Project, 1226 human leukocyte antigen (HLA), 1485, 1692 human lung mast cells (HLMCs), 226, 227, 228 human models, exercise-induced bronchoconstriction, 808–22 human respiratory sensory innervation, 513–14 characteristics, 513 human rhinoviruses (HRVs), 1233 infections, 1967– 8 humans late-phase allergic reactions, 524– 48 repetitive airway exposure, to cold air, 802–3 human umbilical vein endothelial cells (HUVECs), 859, 864, 866–7, 868 binding, 457–8 to factor XII, 456 to high-molecular-weight kininogen, 454– 6 cell membranes, interactions, 456–7 Humicola fuscoatra (fungus), 1761 humidifier fever, 972 and extrinsic allergic alveolitis, 1771 humoral factors, 187– 8 Hurricane Katrina, aftermath, fungal studies, 976– 9 HUVECs see human umbilical vein endothelial cells (HUVECs) HVAC (heating, ventilation and air-conditioning) systems, allergens, 972 HVSs (high-volume samplers), 947 hyaluronidase, 1123– 4, 1126 homologs, 1125 roles, 1127– 8 structure, 1125 hybridization signals, detection, 1291 hydrocortisone, 719 structure, 718 hydrofluoroalkanes (HFAs), as propellants, 776 hydrogen chloride, 1101 hydrogen peroxide, exhaled breath condensate, 1717 hydroxycarbamide, 1807 15-hydroxyicosatetraenoic acid, release, 811 5S-hydroxyicosatetraenoic acid (5-HETE), 569, 577 biosynthesis, 1969 hydroxyzine, 554, 560, 1869 hygiene hypothesis, 32– 6, 149, 1245– 7, 1286, 1720, 2006 and atopic dermatitis, 1822 and latex allergy, 1165– 6 origins, 1244, 2026 Hymenoptera (sawflies, ants, bees, and wasps), 1123, 1124 classification, 1902 stings, 1980 mortality, 1986 reaction classification, 1984 taxonomy, 1981 Hymenoptera sting allergy see Hymenoptera venom allergy

Hymenoptera venom allergens, 1983 and anaphylaxis, 1898, 1902–3, 1905, 1984 local reactions large, 1984 normal, 1984 systemic reactions, 1984 toxic, 1985 unusual reactions, 1984–5 Hymenoptera venom allergy allergen injection immunotherapy contraindications, 1525 indications, 1525 atopy, 1985 clinical presentation, 1983–5 natural history, 1986 pathogenesis, 1983–5 prevalence, 1985 quality of life issues, 1985 risk factors, 1985 hyperactivity, 1328–9 hypercapnia, 1576 hypereosinophilic syndromes (HESs), 280, 1788–9, 1802–9 classification, 1802–3 clinical features, 1804–5 definition, 1788, 1802–3 diagnostic criteria, 1803 differential diagnosis, 1805–7 epidemiology, 1803 etiology, 1788, 1804 genetics, 1804 management, 1807–8 pathogenesis, 1804 physiology, 1803 prevalence, 1789 prognosis, 1789, 1808 symptoms, 1789 treatment, 1789, 1807–8 variants, 1788 see also lymphocytic variant hypereosinophilic syndrome; myeloproliferative variant hypereosinophilic syndrome hyperinflation, assessment, 754–5 hyperkinesis, 1328–9 hyperosmolar aerosols, and exercise-induced bronchoconstriction, 814–15 hyperpermeability, in microvessels, 861–2 hyperplasia, 1638 hyperpnea, 796, 797, 799, 801–2 see also eucapnic voluntary hyperpnea (EVH) hyperreflectoric rhinitis, 513 hypersensitivity pneumonitis see extrinsic allergic alveolitis (EAA) hypersensitivity reactions and allergy, 5–6 to aspirin, 1966–79 classification, 1966 concepts, 3–22 Coombs and Gell classification, 14–20 early studies, 4 history, 3–22 landmark discoveries, 15–18 to nonsteroidal antiinflammatory drugs, 1966–79

type I, 14, 19 in vernal keratoconjunctivitis, 1487 type II, 14–18, 19 type IIa, 18 type IIb, 18 type III, 18–19 immunoglobulin G in, 1357–8 type IV, 19–20 type IV Th1, 20 type IV Th2, 20 see also drug hypersensitivity hypertonicity, 797, 798–9 hypertonic saline challenge, 790–1 hypertrophy, 1638 adenoid, 1388–9 hypervascularity, in asthma, 399 hyperventilation, dry air, early responses, 795–7 hyperventilation-induced airway injury, 798–9 hyperventilation-induced bronchoconstriction (HIB) animal models, 794–807 development, 795 differential diagnosis, 795–6 mammals, 794, 795 mechanisms, 796–7 modulation, 796 see also exercise-induced bronchoconstriction (EIB) hyperventilation-induced mediator production, 799 hyperventilation-induced mediator release, 799 hypoallergenic recombinant allergens, for allergenspecific immunotherapy, 1556–8 hypoallergens, future trends, 937 hypokalemia, and β-adrenoceptor agonists, 679 hyposensitization, allergic diseases, 1332–3 hyposmia, 1466–7 hypotension, treatment, 1890 hypothalamic–pituitary–adrenal (HPA) axis, suppression, 723–4 hypoxemia, 1576 and β-adrenoceptor agonists, 679 Hypsizigus marmoreus (mushroom), 1761 IAEP see idiopathic acute eosinophilic pneumonia (IAEP) IASP (International Allergen Standards Program), 928–9 IBD see inflammatory bowel disease (IBD) IBS (irritable bowel syndrome), diagnosis, 1329 ibudilast, structure, 644 IC-485, structure, 644 ICAM-1 see intercellular adhesion molecule-1 (ICAM-1) ICAMs (intercellular adhesion molecules), 342–3 ICAMs (intracellular adhesion molecules), 1304–5 ICAS (Inner City Asthma Study) (USA), 1133, 1139–40 ICC (immunocytochemistry), 1291 ICD see irritant contact dermatitis (ICD) ICDRG (International Contact Dermatitis Research Group), 1832 ICEP see idiopathic chronic eosinophilic pneumonia (ICEP)

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ichthyosis vulgaris, genetics, 1228 ICOS, 54–5 ligands, 1609 ICSs see inhaled corticosteroids (ICSs) IDEC (inflammatory dendritic epidermal cells), 1824 IDEX syndrome, 58 idiopathic acute eosinophilic pneumonia (IAEP), 1784–5 diagnosis, 1781, 1784–5 diagnostic criteria, 1784 onset, 1784 pathology, 1780 spontaneous recovery, 1785 symptoms, 1784 treatment, 1785 idiopathic anaphylaxis, 1908 idiopathic chronic eosinophilic pneumonia (ICEP), 1782– 4 diagnosis, 1782–3 onset, 1782 pathology, 1780 spontaneous resolution, 1783 symptoms, 1782 treatment, 1782–3 idiopathic eosinophilic pneumonias, 1782–5 classification, 1782 see also idiopathic acute eosinophilic pneumonia (IAEP); idiopathic chronic eosinophilic pneumonia (ICEP) idiopathic pulmonary fibrosis (IPF) in connective tissue disease, 1795 and epithelial-to-mesenchymal transition, 417–18 incidence, 413 myofibroblasts, 417 symptoms, 413 idiopathic rhinitis, 1392 see also vasomotor rhinitis IDO (indoleamine 2,3-dioxygenase), 176 IFN-α see interferon-α (IFN-α) IFN-β (interferon-β), production, 1616 IFN-γ see interferon-γ (IFN-γ) IgA (immunoglobulin A) antibodies, 918 IgE see immunoglobulin E (IgE) IgE assays see immunoglobulin E (IgE) assays IgE-Fc binding sites, 107 crystal structure, 105–7 IgE (immunoglobulin E) mediation, anaphylaxis, 1901– 6 IgE-mediated diseases, omalizumab treatment in, 1680 IgE receptors see immunoglobulin E (IgE) receptors IgE sensitization see immunoglobulin E (IgE) sensitization IgE testing see immunoglobulin E (IgE) testing IGFs (insulin-like growth factors), 325 IgG see immunoglobulin G (IgG) IgG1 (immunoglobulin G1), induction, 1557 IgG44 see immunoglobulin G4 (IgG4) IgG (immunoglobulin G ) antibodies, 917–18 IgG (immunoglobulin G) testing, 1357– 9

IgG-mediated reactions see immunoglobulin G (IgG)-mediated reactions IgM see immunoglobulin M (IgM) IgND (immunoglobulin ND), in allergic asthma, 1665–6 IHRs (in-house references), 929 IKKi, 380 IL-1 (interleukin-1), 1418 IL-2 see interleukin-2 (IL-2) IL-3 see interleukin-3 (IL-3) IL-4 see interleukin-4 (IL-4) IL-5 see interleukin-5 (IL-5) IL-6 see interleukin-6 (IL-6) IL-8 see interleukin-8 (IL-8) IL-9 see interleukin-9 (IL-9) IL-10 see interleukin-10 (IL-10) IL-11 see interleukin-11 (IL-11) IL-12 see interleukin-12 (IL-12) IL-13 see interleukin-13 (IL-13) IL-14 (interleukin-14), fibrosis mediation, 422–3 IL-15 see interleukin-15 (IL-15) IL-16 see interleukin-16 (IL-16) IL-17 see interleukin-17 (IL-17) IL-17A (interleukin-17A), 1302 IL-18 see interleukin-18 (IL-18) IL-19 (interleukin-19), roles, 69 IL-21 (interleukin-21), 1723 IL-23 (interleukin-23), 1723 IL-25 see interleukin-25 (IL-25) IL-33 (interleukin-33), and allergic diseases, 1302–3 imatinib mesylate, in mastocytosis treatment, 1890–1 Immulite test, 1350, 1353 immune cells, in asthma, 1219–20 immune complex clearance, complement in, 437 immune complex disease antigens in, 438 studies, 438–9 immune complexes, 18, 1906 deposition, 1952 roles in disease, 436–50 in host defense, 436–50 studies, 440 immune development, tissue-specific issues, 30–1 immune deviation, and immunotherapy, 1515, 1517–18 immune regulation, and eosinophils, 266–9 immune response afferent limb, complement in, 438 asthma, 1609–10 bronchial vessels and, 403–4 dendritic cells and, 170–6 innate, 39–40 see also adaptive immune responses immune system adaptive, 193 airway, 1197–9 development, and atopic diseases, 1259 early studies, 436–7 gene expression control, 129–31 interactions, with mast cells, 230–1 maturing, 2027 molecular biology, 1214–15

mucosal, 1193–4 and neurotrophins, 498–501 see also innate immune system immunity adaptive, 119 and bacterial products, 2009–10 and serotherapy, 4 see also epithelial immunity; innate immunity immunization models protocols, 1204 see also active immunization models immunoassays allergens, 930–4, 1132 major allergens, 934–6 immunocytochemistry (ICC), 1291 immunogenetics, 1225–9 immunogenicity, use of term, 1944 immunoglobulin A (IgA) antibodies, 918 immunoglobulin E (IgE), 28, 103–18, 990 and allergenicity, 915 allergen recognition, 895–6 allergen-specific, 386, 1126, 1239–40 clinical utility, 1356 and parasites, 154 testing, 1349–57 and allergic diseases, 1292–3 in allergic sensitization, 166 antiallergen responses, spectral studies, 915–17 antibody responses, 992 and asthma, 141, 1239 binding, 320–2, 897, 913–14, 915–17, 1153, 1666 class-switch recombination regulation to, 132–4 cockroach allergen antibodies, prevalence, 1136 cord blood, 1999 cross-reactivity, 1354–6 crystal structure, 105 discovery, 6–7, 21, 103, 1346, 1665–6 and eczema, 1239 epitope mapping, 1150 epitopes, 903–4, 1557 expression, 132–6 in food allergy, 1146–7, 1923–4 food-specific, 1147 function, 122–3 and hay fever, 1239 homeostasis, 134–5 immunogenicity, 1148–9 in vitro measurement, 2024 levels, developing countries, 2024–5 and mast cell activation, 222–7 memory responses, 896 molecular structure, 104–8 in nasal polyps, 1461–2 network, 104, 113–14 in occupational asthma, 1694 origin of term, 103 phosphodiesterase inhibitor mediation, 649–50 as radioallergosorbent test calibration tool, 1354 reactivity, 904 to cockroach allergens, 1135–7 regulatory mechanisms, 132–6 in allergy target organs, 135–6 and rhinitis, 1407–9

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roles, 103 in allergic diseases, 141–2, 634 schematics, 1666 serum-specific, 92 structure, 122– 3, 1666 synthesis, 28, 945, 1280 therapeutic prospects, 113–14 total serum concentrations, 1259, 1260 see also serum immunoglobulin E (sIgE) immunoglobulin E (IgE) assays in vitro latex-specific, 1174–5 in vivo latex-specific, 1175– 6 immunoglobulin E (IgE) mediation, anaphylaxis, 1901– 6 immunoglobulin E (IgE) production, 39 and allergens, 142– 4 and endotoxins, 149–52 environmental factors, 141– 65 genetics, 141–2 and parasites, 154–5 and pollutants, 144– 8 and tobacco smoke, 148– 9 and viruses, 152– 4 immunoglobulin E (IgE) receptors, 103–18, 439 and allergic diseases, 1292–3 discovery, 14, 103 immunoglobulin E (IgE) sensitization, 1239– 40 and asthma, 1240–1 and eczema, 1240–1 genetics, 1247– 8 geographic variation, 1241–2 and hay fever, 1240–1 and hygiene hypothesis, 1245–7 personal risk factors, 1243–5 time trends, 1242–3 immunoglobulin E (IgE) testing in vitro, 1347–57 total, 1347– 8 clinical utility, 1348 immunoglobulin E-directed therapy, 1720 immunoglobulin E–FcεRI interaction, 107– 8, 109 inhibition, 112 immunoglobulin G (IgG), 28– 9, 92 allergen-specific, 1988 testing, 1359 antagonism, 1469 binding, 917–18 epitope mapping, 1150 Fc fragment, 439 Fc receptors, 441, 533 IgG1, 50–1 IgG3, 50–1 reactivity, 904 roles, 438 studies, 440 in type III hypersensitivity, 1357– 8 immunoglobulin G (IgG) antibodies, 917–18 immunoglobulin G (IgG)-mediated reactions, 1949 type II, 1950–2 type III, 1952 immunoglobulin G (IgG) testing, 1357– 9 immunoglobulin G1 (IgG1), induction, 1557 immunoglobulin G4 (IgG4), 1514 in diseases, 1358– 9

induction, 1557 roles, 1358 immunoglobulin genes hypermutation, 129–31 organization, 123–32 immunoglobulin loci, 123–4 immunoglobulin M (IgM) roles, 438 studies, 440 immunoglobulin ND (IgND), in allergic asthma, 1665–6 immunoglobulin receptors, 265 immunoglobulins function, 342–5 responses, and allergen injection immunotherapy, 1513–15 in seasonal allergic conjunctivitis, 1488 structure, 342–5 immunolocalization, cytokines, 1291 immunologic disease, murine models, 439 immunologic tests, occupational asthma, 1701 immunology, of allergy, 23–30 immunomodulating drugs, 732–46 see also immunosuppressive drugs immunomodulation, and histamine, 553–4 immunomodulatory dietary nutrients, in pregnancy, 2001 immunomodulatory therapy, rhinosinusitis, 1475 immunophilin ligands, 329 immunoreceptor tyrosine-based activation motifs (ITAMs), 108, 207–8, 222 immunoreceptor tyrosine-based inhibitory motif (ITIM), 207–8, 210, 281, 439 immunoreceptor tyrosine-based switch motif (ITSM), 207–8 immunostimulatory sequences (ISS), 1561 immunosuppressants, topical, 1825 immunosuppression generic complications, 733 in nasal polyposis treatment, 1469 and pregnancy, 743 immunosuppressive drugs, 733–43 targets, 732, 733 immunotherapy administration routes, 1543 allergen specificity, 90 allergen-specific vaccines, 94–6, 1439–40 allergic asthma, 990 allergic diseases, 1332–3 and allergic responses, 1511–12 anaphylaxis, 1913–14 in atopic dermatitis treatment, 1827 chronic asthma, 1654 dust mite sensitivity, 985–6, 990 future trends, 990–1 effectiveness, 1511 effects on T cells, 1510 and immune deviation, 1515, 1517–18 and interleukin-10, 1515–17 and late-phase allergic reactions, 533 major allergens in, 930 monitoring, 1359 novel strategies, 1517–18, 1720–1 organization of, 1526–7

and regulatory T cells, 1515–17 regulatory T cells in, 93–4 rush, 1528 seasonal allergic conjunctivitis, 1490 successful, 1522–3 see also allergen injection immunotherapy; allergen-specific immunotherapy (SIT); grass pollen immunotherapy; subcutaneous immunotherapy (SCIT); sublingual immunotherapy (SLIT); venom immunotherapy (VIT) indacaterol, 674, 1717 indoleamine 2,3-dioxygenase (IDO), 176 indolent systemic mastocytosis (ISM), 1880 symptoms, 1881, 1883 treatment, 1890 indoor air pollution, 1279–89 and allergens, 1280 and allergic airway diseases, 1280 and asthma, 1280, 1572, 2010 and bacteria, 1282–3 and carbon monoxide, 1285 contaminants, 1285–6 and electromagnetic fields, 1286–7 and environmental tobacco smoke (ETS), 1281 and fibers, 1283 and formaldehyde, 1283–4 and fungi, 1282–3 health issues, 1286–7 and house-dust mites, 1281–2 mitigation, 1286–7 and nanoparticles, 1281 and nitrogen dioxide, 1284–5 and organic compounds, 1280 and ozone, 1285 and particles, 1281 and pet allergens, 1281–2 and plasticizers, 1284 significance of, 1279–80 and viruses, 1282–3 and volatile organic compounds, 1284 indoor air quality, 1280–6 factors affecting, 1280 guidelines, 1286 indoor allergens characteristics, 985 immunoglobulin E production, 142–3 nomenclature, 985 indoor environments, fungal spores, 970–3 induced sputum airway inflammation assessment, 1368–71 asthma, 1583–4 cell counts, 1371 cell types, 1370 inflammation markers, 1368–80 limitations, 1377 measurement characteristics, 1370–1 molecular markers, 1370 see also sputum induction inducible nitric oxide synthase (iNOS), synthesis, 1297 infant formulas and allergy prevention, 2002–3 hydrolyzed, 2003

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infantile eczema prevalence, 1225 see also atopic dermatitis (AD) infantile food protein-induced proctocolitis, 1928 infants acute viral infections, 1245 aerosol delivery, 779– 80 allergy prevention, 2001–10 and antibiotics, 2007– 8 H1-antihistamines in, 561–2 asthma, 1591– 607 atopic dermatitis, 1815 atopic eczema, 1815 bacterial exposure, 2006–7 cutaneous mastocytosis, 1891 dietary interventions, 2010 early gut colonization, 2008– 9 feeding, 2002– 4 food protein-induced enterocolitis syndrome, 1924 inhalant allergen exposure, 2000, 2004– 6 microbial burden, 2008 microbial exposure, 2006–10 pet allergen exposure, 2005– 6 and smoking mothers, 1571 viral respiratory tract infections, 2007 wheezing, 1594, 1618–19, 2007 see also pediatric asthma infant wheezing, 1594 infections and angioedema, 1865 animal models, 2026– 7 and asthma, 1571 early exposure to, 1263– 4 early life, and allergic diseases, 33 enteric, 1245 eosinophils in, 273– 6 in extrinsic allergic alveolitis, 1769 and hygiene hypothesis, 1245– 7 population studies, 2026 and urticaria, 1865 see also bacterial infections; helminth infections; parasitic infections; respiratory infections; viral infections infectious rhinitis see rhinosinusitis inflammation in asthma, 1639– 41 epithelium in, 380– 5 and mast cells, 229 neutrophilic, 1640–1 neutrophil recruitment, 299–303 switching off, glucocorticoid-induced, 717 see also airway inflammation; allergic inflammation; neurogenic inflammation inflammation markers in exhaled air, measurement, 1368– 80 in induced sputum, measurement, 1368– 80 inflammatory bowel disease (IBD), 1225 susceptibility genes, 1229 inflammatory cascade, allergic asthma, 1610 inflammatory cells β-adrenoceptor agonist effects, 676 in allergic diseases, 1293– 7 in bronchoalveolar lavage, 788 and extrinsic allergic alveolitis, 1762– 3

interactions, with neurons, 512 and platelet-activating factor, 606–8 signal transduction, 203–13 inflammatory conditions, alveolar macrophages in, 181 inflammatory dendritic epidermal cells (IDEC), 1824 inflammatory genes, suppression, by inhaled glucocorticoids, 716 inflammatory mediator antagonists, 849–50 inflammatory peptides, generation, 442 inflammatory responses loss of Treg suppression, 87 regulatory T cells in, 93–4 infliximab, in atopic dermatitis treatment, 1827 influenza A virus, 153 inhalant allergen exposure, infants, 2000, 2004–6 inhalation tests, 1323 asthma, 1583 developments, 1699 in occupational asthma, 1697–701 procedures, 1699–700 see also lung function tests; pulmonary function tests inhaled allergen challenge, 1715 inhaled corticosteroids (ICSs), 768, 778–9, 1714–15 adverse effects, 1653 and antileukotrienes, in asthma, 699–704 in asthma treatment, 1332, 1370, 1712, 1713 in chronic asthma treatment, 1652–3, 1655 clinical use, 1652–3 dose–response relationships, 695 in exercise-induced bronchoconstriction, 816 mechanisms, 694 new, 1718 in pediatric asthma treatment, 1602–4 in persistent severe allergic asthma treatment, 1665 inhaled glucocorticoids, 718–25 add-on therapy, 722 and adrenal suppression, 723–4 adult studies, 719–20 adverse effects, 722–5 local, 722–3 systemic, 723–5 in asthma, 721–2 and bone metabolism, 724 and cataracts, 724 comparisons, 721 connective tissue effects, 724 dose–response relationships, 720–1 efficacy, 721–2 and growth, 724–5 high-dose, 720 inflammatory gene suppression, 716 irreversible airway change prevention, 721 metabolic effects, 725 mortality reduction, 721 pediatric studies, 720 and pregnancy, 725 psychiatric effects, 725

inhaled mass aerosols, 771 measurement, 773–4 pediatric in vitro models, 773–4 inhaled medications, in pediatric asthma, 1603–4 inhaler devices, 1655 pressurized metered-dose, 776–7 see also nebulizers inhibitory receptors, 266 in-house references (IHRs), 929 injection immunotherapy see allergen injection immunotherapy innate antiviral responses, and aspirin-induced asthma, 1967–8 innate immune system activation, 192–3 and allergic diseases, 193–6 components, 187–9 in disease initiation, 194 in disease perpetuation, 194–6 interactions, with adaptive immune system, 193 pathogen recognition, 189–92 target choice, 196–7 therapeutic exploitation of, 196–7 innate immunity, 119 in allergic diseases, 187–202 and barrier cells, 188 disease impacts on, 196 epithelium in, 373–80 mechanisms, 436 Inner City Asthma Study (ICAS) (USA), 1133 innervation, 1194 in animal models, 1216–17 see also autonomic innervation; epithelial innervation; parasympathetic innervation; sensory innervation; sympathetic innervation INNOVATE study, 1670, 1672–3, 1674, 1675, 1678, 1681 iNOS (inducible nitric oxide synthase), synthesis, 1297 inositol 1,4,5-trisphosphate (IP3), 672, 879 insect allergens, 902–3 see also cockroach (CR) allergens; dust mite allergens insect bites definition, 1980 see also biting insect allergy insects, pollination, 943 insect sting allergy, 1326, 1980–94 case histories, 1986–7 cellular tests, 1988 children, 1985 diagnosis, 1986–8 elderly, 1985 emergency medication kits, 1989–90 entomologic issues, 1980–3 epidemiology, 1985–6 future trends, 1992 prevalence, 1985 prevention, 1988–9 skin tests, 1987 taxonomy, 1980–3 treatment, 1989–91

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large local reactions, 1989 systemic reactions, 1989 venom immunotherapy, 1990–1 see also Hymenoptera venom allergy insect stings definition, 1980 goals, 1980 insect venom allergens, 1123– 30, 1902– 3 biochemical data, 1123–5 cross-reactivity, 1987– 8 recombinant, 1126–7 see also bee venom allergens; stinging insect venom allergens insect venoms allergenicity, 1127– 8 and anaphylaxis, 1902–3, 1914 low-molecular-weight components, biochemical data, 1127 sequenced, 1126 in situ hybridization (ISH), 1291 inspiratory stridor, 808 insulin-like growth factors (IGFs), 325 intact microvessels, and cultured endothelia compared, 866– 7 integrin receptors, 104, 112–13 integrins, 369 function, 340–1 regulatory mechanisms, 342 structure, 340–1 see also leukocyte integrins integrins αVβ3, 342 intercellular adhesion, loss of, 864– 5 intercellular adhesion molecule-1 (ICAM-1), 301, 341, 868, 882, 1413 binding, 351 expression, 345– 6, 382, 1460–1 function, 342–3 structure, 342–3 intercellular adhesion molecules (ICAMs), 342–3 interfaces, aerosols, 771 interference RNA (RNAi), 1725– 6 interferon-α (IFN-α) in hypereosinophilic syndrome treatment, 1807 in mastocytosis treatment, 1890 interferon-β (IFN-β), production, 1616 interferon-γ (IFN-γ), 19, 529 encoding, 1515 production, 26, 50–1, 1616 reduction, 1147 interferon-producing cells see plasmacytoid dendritic cells (pDCs) interferon regulatory factor-4 (IRF4), 120 interferons (IFNs), type 1, 380 interleukin-1 (IL-1), 1418 interleukin-2 (IL-2), 19, 50–1, 1421 interleukin-2 receptors, antibodies, 742 interleukin-3 (IL-3), 264–5, 528, 990, 1418, 1988 in basophil simulation tests, 1361 expression, 1804 tissue densities, 1460 interleukin-4 (IL-4), 51, 528, 529, 538, 990, 1209, 1408 activity, 1722 and allergic diseases, 1297– 9 in asthma, 1639– 40

and fibrosis, 420, 421 and food allergy, 1147 immunoreactivity, 1412 macrophage stimulation, 420 secretion, 323, 801, 1610 tissue densities, 1460 interleukin-4 antagonists, in nasal polyposis treatment, 1469 interleukin-4 receptors, 73, 1822 soluble, 1712 interleukin-5 (IL-5), 51, 264–5, 346–7, 528, 529, 538, 1209, 1417–18, 1421 activity, 1722 and allergic diseases, 1299 in asthma, 1639–40 blockade, 1612 in eosinophil maturation, 1712 expression, 1804 production, 990, 1806 roles, 1805 tissue densities, 1460 see also anti-interleukin-5 (anti-IL-5) interleukin-5 antagonists, in nasal polyposis treatment, 1468–9 interleukin-6 (IL-6), 529, 1408 roles, 68 interleukin-8 (IL-8), 528 expression, 1303 roles, 68 upregulation, 1822 interleukin-9 (IL-9), 51, 65–8, 528 and allergic diseases, 1299–300 expression, 1209 induction, 1722 interleukin-10 (IL-10), 26, 88–9, 93, 530, 1723 expression, 1209, 1515–16 immunologic properties, 85 and immunotherapy, 1515–17 production, 89–90, 921–2, 1296 recombinant, 73 response initiation, 1517 roles, 69, 482 interleukin-11 (IL-11) expression, 1209 roles, 68 interleukin-12 (IL-12), 24, 25, 26, 39–40, 51, 1412–13 mRNA, 1515 recombinant, 73–4 regulatory mechanisms, 1723 interleukin-13 (IL-13), 26, 51, 64, 68, 323, 528, 538, 1209, 1408 activity, 1722 and allergic diseases, 1300–1 in asthma treatment, 1712 and fibrosis, 420, 421 fibrosis mediation, 422–3 and latex allergy, 1171 macrophage stimulation, 420 polymorphisms, 1229 roles, 73, 1618 secretion, 1610 upregulation, 1640 interleukin-13 antagonists, in nasal polyposis treatment, 1469

interleukin-13 receptors, 73 interleukin-14 (IL-14), fibrosis mediation, 422–3 interleukin-15 (IL-15), 64 blocking, 1722 interleukin-16 (IL-16) and allergic diseases, 1301–2 release, 801 roles, 68 interleukin-17 (IL-17) and allergic diseases, 1302 family, 52–3 production, 1722–3 roles, 68 interleukin-17A (IL-17A), 1302 interleukin-18 (IL-18) and latex allergy, 1171 promoter variants, 1723 roles, 69 interleukin-19 (IL-19), roles, 69 interleukin-21 (IL-21), 1723 interleukin-23 (IL-23), 1723 interleukin-25 (IL-25), 53 and allergic diseases, 1302 interleukin-33 (IL-33), and allergic diseases, 1302–3 intermittent rhinitis, 1402–3 definition, 1403 International Allergen Standards Program (IASP), 928–9 International Consensus for Rhinitis, guidelines, 1441 International Contact Dermatitis Research Group (ICDRG), 1832 International Eosinophilic Society, 1802 Hypereosinophilic Syndromes Working Group, 1804 International Life Sciences Institute, 1158 International Study of Asthma and Allergies in Childhood (ISAAC), 23, 1387 allergy prevalence studies, 2020–1 geographic variations, 1241–2 pediatric asthma studies, 1592 questionnaires, 1240, 2022–3 time trends, 1242–3 International Union Against Tuberculosis and Lung Disease (IUATLD) Asthma Guide, 1445 Bronchial Symptoms Questionnaire, 1240 International Union of Immunological Societies (IUIS), 899, 913, 1167 Allergen Standardization Subcommittee, 928–9 Committee in Standardization, 928 interstitial pneumonia, in connective tissue disease, 1795 intestinal diseases diagnosis, 1329–30 and food intolerance, 1329 see also gastrointestinal diseases intracellular adhesion molecules (ICAMs), 1304–5 intracellular molecules, signal transduction, 204 intradermal tests, 1323 comparisons, 1337 grading, 1338 methods, 1336–7

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intrauterine development, and allergen-specific T-cell memory initiation, 25– 6 intrinsic atopic dermatitis see nonatopic eczema intrinsic sensitivity, 1360 IP3 (inositol 1,4,5-trisphosphate), 672, 879 IPC-1, efficacy, 1560 IPC-2, efficacy, 1560 IPCs see plasmacytoid dendritic cells (pDCs) IPF see idiopathic pulmonary fibrosis (IPF) iPLA2 (calcium-independent phospholipase A2), 568, 569 ipratropium, 832 adverse effects, 690 in asthma treatment, 689– 90 in combination drugs, 686 development, 683 dissociation half-life, 685 dose–response relationships, 686 studies, 687 ipratropium bromide, 685, 1332, 1391, 1439 IPS-1 (Cardif), 192 IRAK-4, 194, 207 IRF4 (interferon regulatory factor-4), 120 irritable bowel syndrome (IBS), diagnosis, 1329 irritant contact dermatitis (ICD), 1831 acute, 1837 cellular mechanisms, 1833– 4 chemical issues, 1834 chronic, 1837– 8 clinical features, 1836– 8 cumulative, 1838 high-risk occupations, 1836 histopathology, 1835 occupational irritants, 1836 responses, 1836 irritant-induced asthma, 1571 irritants occupational asthma induction, 1062–100 occupational rhinitis induction, 1102, 1103 see also airborne irritants; respiratory irritants ISAAC see International Study of Asthma and Allergies in Childhood (ISAAC) ISH (in situ hybridization), 1291 Ishizaka, Kinshige (1925– ), 6–7, 928, 1346, 1665– 6 Ishizaka, Teruko (1926– ), 6–7, 928, 1346, 1665– 6 ISM see indolent systemic mastocytosis (ISM) isocyanates, 1017, 1102, 1762 isoenzyme-selective phosphodiesterase inhibitors, 634– 67 isoprenaline, structure, 673 isoproterenol, 672, 675 isothiazolinines, 1286 ISS (immunostimulatory sequences), 1561 ITAMs (immunoreceptor tyrosine-based activation motifs), 108, 207– 8, 222 ITIM (immunoreceptor tyrosine-based inhibitory motif), 207– 8, 210, 281, 439 itraconazole, 1752 in allergic bronchopulmonary aspergillosis treatment, 1792–3 ITSM (immunoreceptor tyrosine-based switch motif), 207–8

IUATLD see International Union Against Tuberculosis and Lung Disease (IUATLD) IUIS see International Union of Immunological Societies (IUIS) Jadassohn, Josef (1863–1936), 1832 JAKs (Janus-activated kinases), 132–3, 134, 884–5 Jak–STAT pathway, 209 JAMs see junctional adhesion molecules (JAMs) Janus-activated kinases (JAKs), 132–3, 134, 884–5 Janus family kinases, 60–1, 206 Johansson, Gunnar (1938– ), 7, 8, 928, 1346 Journal of Allergy, 5 Journal of Allergy and Clinical Immunology, 1147 Journal of Experimental Medicine, 65 Journal of Hypersensitivity and Hypersensitivity Disorders, 5 Jug r2, 1155 junctional adhesion molecules (JAMs), 301–2, 337, 860, 866 function, 344 JAM-A, 344, 866 JAM-B, 344 JAM-C, 344, 866 structure, 344 Juniperus spp. ( junipers), 956 Juniperus communis (common juniper), 956 Juniperus sabinoides (mountain cedar), 956 kallikrein, 463, 1868 plasma, 452 tissue, 451 see also prekallikrein (PK) Kaplan, D. M., 669 Kartagener syndrome, 1459 Kellaway, Charles H., 9, 10, 567 keratinocytes, 29, 1229, 1230 keratosis pilaris, 1817 ketorolac, 1439 ketotifen, 1489 ketotifen fumarate, in seasonal allergic conjunctivitis treatment, 1490 keyhole limpet hemocyanin (KLH), 146 kinase inhibition, 1724–5 kinase inhibitors, development, 1713 kininase I, 1859 kininase II, 1859 kinin cascade activation, 458–60 by cell surface binding, 458 kinin receptors B1, 1419–20 B2, 1419–20 kinins and allergic rhinitis, 464 and angiotensin-converting enzyme inhibition, 464 degradation, 452 production, 452 roles, 451 Kit, 1887 mutations, 1888 structure, 1879

Kiwellin, 1153 kiwi fruit, allergens, 1153 Klebsiella ozaenae (bacterium), 1760 infection, 1391 KLH (keyhole limpet hemocyanin), 146 Kochia scoparia (burning bush), 957 KPNA3 gene, 1229 KS505a, 638 Kurzok, Raphael (b.1895), 567 Küstner, Heinz (1897–1963), 6, 7, 103, 1346, 1665 LABAs see long-acting β2 agonists (LABAs) laboratory animals, and occupational asthma, 1701–2 α-lactalbumin, 917, 1150 β-lactam antibiotics, 1901 lactase deficiency, 1921 lactation allergen avoidance during, 2002 H1-antihistamines in, 562 Lactobacillus spp. (bacteria), 2008 Lactobacillus acidophilus (bacterium), 2009 Lactobacillus reuteri (bacterium), 2009 lactoferrin, 373 lactoglobulin, 1148 α-lactoglobulin, 1154 β-lactoglobulin, 917, 1150 lactoglobulins, 1150 lactose intolerance, 1921 LAD see leukocyte adhesion deficiency (LAD) LAG-3 (lymphocyte activation gene-3), 86 LAL see low ammoniated latex (LAL) LA (lung attenuation), 758 laminins, 369, 876, 877 in extracellular matrix, 414 Langerhans cell granulomatosis, 1795 Langerhans cells (LCs), 166–7 Langley, J. N., 668 large local reactions (LLRs), 1986 definition, 1984 treatment, 1989 LARs see late asthmatic reactions (LARs) larva migrans syndrome, 1790 laryngeal edema, 1910 late asthmatic reactions (LARs), 231, 232, 525, 538 allergen-induced, 527, 528 development, 524 drug-induced, 531 and eosinophils, 278 inhibition, 532–3 occupational agent-induced, 527 peptide-induced, 533–4, 536, 537 late-phase allergic reactions (LPRs), 650 airway challenge, 525 and allergen-derived T-cell peptides, 533–7 characteristics, 524 cutaneous challenge, 526 and drugs, 531–3 early studies, 524 elicitation mechanisms, 525–6 in humans, 524–48 hypothesis, 538, 539 immunology, 527–30

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nasal challenge, 525– 6 priming, 527 progression, 526–7 see also cutaneous late-phase reaction (CLPR) latex and anaphylaxis, 1898, 1903 applications, 1164 and occupational asthma, 1702–3 see also low ammoniated latex (LAL); natural rubber latex (NRL); nonammoniated latex (NAL) latex allergens, 1167– 9 avoidance, 1176 cross-reactivity, 1164 identification, 1164, 1167 mutations, 1558 latex allergy, 1164– 84 allergen avoidance, 1176 and allergic rhinoconjunctivitis, 1173 and anaphylaxis, 1173, 1903 and atopy, 1172 challenge testing, 1176 clinical features, 1173– 4 comorbidities, 1173 diagnosis, 1174– 6 early studies, 1164–5, 1702 epidemics, 1165– 6 epidemiology, 1170–2 and food allergy, 1173– 4 and fruit allergy, 1172, 1173– 4 future research, 1178– 9 historical background, 1164–5 and hygiene hypothesis, 1165– 6 incidence studies, 1171 management, 1174–8 and occupational asthma, 1173 prevalence studies, 1171, 1172 recommendations, 1179 risk factors, 1170–1, 1172 genetic, 1171 occupational, 1171–2 specific immunotherapy, 1176– 8 subcutaneous immunotherapy, 1176–7 sublingual immunotherapy, 1176– 7 latex–fruit syndrome, 1150, 1156, 1170–1, 1173– 4, 1903 allergens, 1153 latex gloves, 1164, 1165 allergy studies, 1171–2 latex allergens, 1168, 1169, 1170, 1171, 1172 manufacture, 1166–7 latex proteins, aerosol transmission, 1165 lauryl dimethyl benzyl ammonium chloride, 1101 LCs (Langerhans cells), 166–7 lectin, C-type superfamily, 104 lectin domain, 109 trimerization, 110 lectin pathway, 438, 441, 443– 4 Legionella spp. (bacteria), 1283 Legionnaires’ disease, 1283 legumins, 1152 Leishmania spp. (trypanosome protozoa), 312 LEKTI gene, 1228 Len c 1, 1149 Leonardo da Vinci (1452–1519), 398

LEP gene, 1228 Lepidoglyphys destructor (storage mite), 1101 Leucogyrophana pinastri (fungus), 1761 Leucophaea maderae (Madeira cockroach), 1138 leukocyte adhesion, 346–52 in allergic inflammation, 337–65 mechanisms, 337–8 leukocyte adhesion deficiency (LAD), 298, 300 I, 342 II, 339 III, 342 leukocyte adhesion receptors function, 338–45 structure, 338–45 leukocyte immunoglobulin-like receptors (LIRs), 324 leukocyte integrins expression, 341–2 αEβ7, 341–2 function, 341–2 αLβ2, 341 αMβ2, 341 structure, 341–2 leukocyte migration, 337, 346–52 leukocyte populations recruited, 188–9 resident, 188 leukocytes endothelial hyperpermeability mediation, 867–8 infiltration, in allergic rhinitis, 1417–18 see also polymorphonuclear leukocytes (PMNs) leukotriene A4 (LTA4), 566, 571–2, 695 urinary excretion, 1970 leukotriene B4 (LTB4), 302, 566, 571–2, 695–6 binding, 577 receptors, 576–7 leukotriene C4 (LTC4), 9, 323, 529, 566, 571–2, 695 and airway secretions, 583 biological activity, 575 in bronchial asthma, 581–2 excretion, 1974 receptors, 578 leukotriene C4 synthase, 708 expression, 1969–70 leukotriene D4 (LTD4), 9, 566, 567, 571–2, 650, 695 and airway responsiveness, 583 and airway secretions, 583 in asthma, 1611 biological activity, 575 in bronchial asthma, 581–2 receptors, 578 leukotriene E4 (LTE4), 9, 529, 566, 567, 571–2, 695 and airway secretions, 583 in asthma, 584–7 biological activity, 575 in bronchial asthma, 581–2 in myocardial infarction, 587–8 receptors, 578 urinary excretion, 810 leukotriene modifiers, efficacy, 1675

leukotriene receptor antagonists (LTRAs), 405, 696, 1718 adverse affects, 697 in allergic rhinitis, 705–6 in asthma, 700–2 in asthma monotherapy, 698–9 efficacy, 708–9 in exercise-induced bronchoconstriction, 816 and late-phase allergic reactions, 532 in pediatric asthma treatment, 1602, 1603 studies, 708 leukotriene receptors, 265, 566, 576–81 in asthma, 586–7 leukotrienes, 171, 538, 569–88, 1420, 1719 and airway hyperresponsiveness (AHR), 582–3 and airway secretions, 583 and aspirin hypersensitivity, 1969–70 in asthma, 584–6 biological activity, 574–5 biosynthesis, 569–74 regulatory mechanisms, 572–3 sites of, 573–4 transcellular, 572 in bronchial asthma, 581–2 cellular sources, 570–2 discovery, 567 in diseases, 587–8 elimination, 572 and exercise-induced bronchoconstriction, 809–10 metabolism, 572 release in biological fluids, 584 in diseases, 583–8 roles, 566 biological, 586–7 structure, 570 see also antileukotrienes; cysteinyl leukotrienes (CysLTs) levocabastine, 557 in perennial allergic conjunctivitis treatment, 1492 in seasonal allergic conjunctivitis treatment, 1489–90 levocetirizine, 554, 555, 557, 560 LFA-1 (lymphocyte function-associated antigen1), 353, 1763 lichenification in allergic contact dermatitis, 1838 of anogenital area, 1817 atopic eczema, 1817 lidocaine, inhalation, 833 Lieb, Charles, 567 life, classification, 964 lifestyle, and atopic diseases, 1260–1 ligands, as adrenergic receptors, 668 light scattering, 771 Ligustrum vulgare (privet), 957 LIM kinase, 865 linoleic acid, 37 lipid mediators, 380–1, 566–633 lipid rafts, 207 lipid transfer protein (LTP), 930, 1170, 1355 lipocalin, 1131 allergens, 1137

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lipopolysaccharides (LPSs), 131, 149, 151, 188, 189– 90, 191, 1518 and airway inflammation, 194 and allergic sensitization, 152 exposure, 150 occurrence, 1264, 1282–3 responses to, 192 lipoxin A4 (LXA4), 567, 600–1 binding, 602 lipoxin B4 (LXB4), 601 lipoxin receptors, 601–2 lipoxins, 567, 600– 4 and aspirin hypersensitivity, 1970–1 and asthma, 602– 4 biosynthesis, 600–1, 1968 elimination, 600–1 immunomodulatory activity, 603 metabolism, 600–1 physiologic actions, 602 5-lipoxygenase (5-LO), 566, 569– 70, 571–3 blocking, 800 coding, 811 5-lipoxygenase-activating protein (FLAP), 570–1, 594 5-lipoxygenase inhibitors, 696 lips, contact dermatitis, 1842 LIRs (leukocyte immunoglobulin-like receptors), 324 LK (low-molecular-weight kininogen), 451 LL-37, 1218 LLRs see large local reactions (LLRs) LMW-PTP (low-molecular-weight protein tyrosine phosphatase), 866 5-LO see 5-lipoxygenase (5-LO) local heat urticaria, 1863 lodoxamide, in seasonal allergic conjunctivitis treatment, 1490 lodoxamide tromethamine, 1496 Löffler syndrome, 1791 etiology, 1791 Lolium perenne (perennial ryegrass) allergens, 935, 943– 4, 951 pollen, 945 Lol p 1, 943– 4, 1359 Lol p 1X, 943– 4 Lol p 5, 929, 1359, 1557– 8 long-acting β2 agonists (LABAs), 702, 762 in asthma treatment, 1712, 1713 in chronic asthma treatment, 1651, 1655 developments, 1717 in exercise-induced bronchoconstriction, 816 issues, 1714–15 in pediatric asthma treatment, 1603 in persistent severe allergic asthma treatment, 1664–5, 1670, 1672, 1673, 1674, 1680, 1681 loratadine, 329, 554, 557, 560, 1437 in allergic rhinitis, 1438– 9 and exercise-induced bronchoconstriction, 811 Loveless, Mary Hewitt (1899–1991), 6, 7, 13–14, 928 low ammoniated latex (LAL), 1175– 6, 1177 sensitivity, 1173 lower airways autonomic innervation, 827

neurogenic inflammation, 828 neurophysiology, 825–8 reflexes, 827–8 autonomic, 827–8 breathing patterns, 828 cough, 828 sensory innervation, 826–7 lower respiratory tract infections (LRTIs), 33 and antibiotics, 35–6 low-molecular-weight compounds, and extrinsic allergic alveolitis, 1762 low-molecular-weight kininogen (LK), 451 low-molecular-weight protein tyrosine phosphatase (LMW-PTP), 866 low-volume samplers (LVSs), 947 LPRs see late-phase allergic reactions (LPRs) LPSs see lipopolysaccharides (LPSs) LRTIs see lower respiratory tract infections (LRTIs) LTA4 see leukotriene A4 (LTA4) LTB4 see leukotriene B4 (LTB4) LTC4S gene, 1971, 1973 LTC4 see leukotriene C4 (LTC4) LTD4 see leukotriene D4 (LTD4) LTE4 see leukotriene E4 (LTE4) LTP (lipid transfer protein), 930, 1170, 1355 LTRAs see leukotriene receptor antagonists (LTRAs) LTT see lymphocyte transformation test (LTT) lumiliximab, 104, 1720 luminal cells, in allergic rhinitis, 1413–17 lung adrenoceptors, 669–70 appearance at postmortem, 1633–4 dendritic cell function, 188 regulatory T cell control, 176 gas-exchange reactions, 1218–19 myeloid dendritic cells, 174 neutrophils in, 301 lung attenuation (LA), 758 lung cells, β-adrenoceptor agonist effects, 675 lung diseases airway smooth muscle in, 877–85 diffuse parenchymal, 1771 eosinophilia-associated, 1795–6 and extrinsic allergic alveolitis, 1771 and fibrosis, 413 textile dust-related interstitial, 1771 see also asthma; chronic obstructive pulmonary disease (COPD); eosinophilic lung diseases; fibrosis lung function tests allergic bronchopulmonary aspergillosis, 1751 asthma, 750–6, 1580–2, 2023 in idiopathic chronic eosinophilic pneumonia, 1783 see also forced expiratory volume in 1 second (FEV1); inhalation tests; maximum expiratory flow–volume (MEFV); peak expiratory flow (PEF); pulmonary function tests; spirometry lung volume changes, and airway smooth muscle, 759–62 measurement, 754–5, 1582 lupine, allergens, 1153 lupus (systemic lupus erythrematosus), 437, 439, 440–1

Lutjanus argentimaculatus (red snapper), allergens, 1152 Lutjanus johnii (golden snapper), allergens, 1152 LVSs (low-volume samplers), 947 LXA4 see lipoxin A4 (LXA4) LXB4 (lipoxin B4), 601 lymph nodes, draining, dendritic cell migration, 171–2 lymphocyte activation gene-3 (LAG-3), 86 lymphocyte function-associated antigen-1 (LFA-1), 353, 1763 lymphocytes migration, 351 and neurotrophins, 499–500 see also B cells; T cells lymphocyte transformation test (LTT) mechanisms, 1962 sensitivity, 1962 lymphocytic infiltrates, lung disorders, 1771 lymphocytic variant hypereosinophilic syndrome, 1806 use of term, 1804 Lyn, activation, 210–11 Lyophyllum aggregatum (mushroom), 1761 lysis, 441 lysozyme, 373, 914, 1148, 1151 M1, 684 M2, 684 M2 receptors, 827 M3, 684, 686 M3 receptors, 827 occurrence, 824 MAAS (Manchester Asthma and Allergy Study) (UK), 1262, 2000, 2004–5 Mac-2 (galectin-3), 113 McABC, 900 McAC, 900 Macaca arctoides (stump-tail macaque), 1187 Macaca fascicularis (cynomolgus macaque), 1187 Macaca fuscata (Japanese macaque), 1187 Macaca mulatta (rhesus monkey), 1187 airway epithelium, exposure responses, 1196 airway growth, 1192–3 exposure responses, 1195–6 airway immune system, exposure responses, 1197–9 airway smooth muscle, 1193 exposure responses, 1197, 1199 airway vasculature, exposure responses, 1197 alveolar development, 1194 basement membrane zone, 1193 exposure responses, 1196–7 bronchial vasculature, 1194 environmental postnatal airways disease, experimental models, 1194–5 epithelial differentiation, 1193 epithelial innervation, 1194 exposure responses, 1197 exposure responses, 1195–9 immunologic, 1195 physiologic, 1195 mucosal immune system, 1193–4 nerve fibers, 1197, 1198 neurons, 1197, 1198

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postnatal development, 1192– 4 tracheobronchial airways architecture, 1189– 90 cellular composition, 1190– 2 macaque monkeys, 1187 macrolide antibiotics, 851–2 macrolide therapy, chronic rhinosinusitis, 1475 macrophage-derived chemokine (MDC), 146, 530, 1301 macrophage inflammatory protein-1α (MIP-1α), 1763– 4 macrophage inflammatory protein-1β (MIP-1β), 530 macrophages antigen-presenting, 166– 86 in asthma, 1613 functions, 179– 80 importance of, 166 and neurotrophins, 499 origin, 179– 80 roles, 188 stimulation, 420 see also alveolar macrophages (AMs) macropinocytosis, and antigen uptake, 167– 8 maculopapular cutaneous mastocytosis, 1880 skin biopsies, 1884 symptoms, 1881 treatment, 1890 maculopapular exanthem (MPE), 1955 magnetic resonance imaging (MRI), 749 airway visualization, 758 Maimonides, Moses (1135–1204), 11, 963 Treatise on Asthma, 9–10 major allergens, 899– 900, 913–14 immunoassays complex interpolation, 935– 6 strengths, 934– 6 weaknesses, 934– 6 in immunotherapy, 930 measurements, 929 major basic protein (MBP), 258, 259, 372, 1293, 1417, 1495 extracellular, 1464 immunostaining, 1294 major histocompatibility complex (MHC), 24, 31, 166 antigens, 48, 122 and asthma, 1227 class I, antigen presentation, 169–70 class II antigen presentation, 168– 9 dendritic cells, 167 Malassezia spp. (yeasts), type I sensitization, 1233 Malassezia furfur (yeast), and atopic dermatitis, 1819– 20 Mal d 1, 1150, 1156, 1355 Mal d 3, 1153 MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) spectrometry, 936–7 malignant neoplasia, and omalizumab, 1677 MALT (mucosal-associated lymphoid tissue), 1485 mammalian allergens characteristics, 985 dose–response relationships, 992

mammals antibody repertoire, 119–20 hyperventilation-induced bronchoconstriction, 794, 795 Manchester Asthma and Allergy Study (MAAS) (UK), 1262, 2000, 2004 –5 manganese superoxide dismutase, 1170 mannitol in exercise-induced asthma, 707–8 in exercise-induced bronchoconstriction, 814–15 mannose, 1126 mannose-binding lectin (MBL), 438, 443, 449 MAPK (mitogen-activated protein kinase) inhibitors, 851 MAPK (mitogen-activated protein kinase) pathway, 205, 208, 338 MAPKs see mitogen-activated protein kinases (MAPKs) MARCKS see myristoylated alanine-rich C-kinase substrate (MARCKS) Marcussen, P. V., 1832 margination, 298–9 masks, in exercise-induced bronchoconstriction, 816–17 MASPs see MBL-associated serine proteases (MASPs) mass median aerodynamic diameter (MMAD), 768, 769, 770, 771–2 mast cell activation and anaphylaxis, 1908–9 immunoglobulin E-dependent, 222–6 monomeric, 227 mechanisms, 222–8 nonimmunologic stimuli, 227–8 mast cell adhesion, 349–50 mast cell-deficient mice, 228 mast cell hyperplasia, 483, 1878, 1879–80 mast cell migration, 349–50 inhibition, in asthma therapy, 242–3 mast cell regulation, 269 mast cells activation, 1611, 1855, 1857 direct, 1907 and airway inflammation, 1374, 1375 in allergic conjunctivitis, 243–4 and allergic inflammation, 230–1, 1296 allergic inflammatory response initiation, 230–1 in allergic rhinitis, 243, 1296, 1410–12 in anaphylaxis, 8, 244 in angioedema, 1854–5 in angiogenesis, 228–9 and antigen presentation, 230 in asthma, 231–43, 1296, 1610–11, 1640 in atopic dermatitis, 244–5 and bacterial infections, 230 and basophils compared, 320 biological properties, 215–57 biology, 220–2, 1879–80 bone marrow-derived, 219 in chronic obstructive pulmonary disease, 790 connective tissue, 220 cord blood-derived, 219 degranulation, 896–7

development, 217–19 discovery, 7–8, 215 in eczema, 244–5 heterogeneity, 219–20 human lung, 226, 227, 228 immunophenotypes, in mastocytosis, 1886 infiltration, of airway smooth muscle, 237–8 and inflammation, 229 interactions, 193 with airway smooth muscle, 238–40 with immune system, 230–1 and late-phase allergic reactions, 527 mediator release, 1611 mediators, 222, 1908 microlocalization, in asthmatic airways, 237–41 morphology, 220–2 atypical, 1886 mucosal, 220 and neurotrophins, 500–1 occurrence, 215 and parasitic infections, 230 recruitment, by chemokines, 482–3 roles, 215, 500 in allergic diseases, 231–45 in health, 228–31 sensitization, 14 signal transduction, 211 synthesis, 1879 and T helper 2 cell differentiation, 230 in urticaria, 244–5, 1854–5 and viral infections, 230 in wound repair, 228–9 see also effector cells; precursor mast cells (pMCs) mast cell stabilizers, and late-phase allergic reactions, 532 mast cell tryptase inhibitors, 850 mastocytosis, 1878–93, 1985 bone marrow aspirate, 1884–5 and cardiovascular problems, 1883 classification, 1880, 1885 diagnosis, 1884–7 diagnostic criteria, 1885–7 major, 1885 minor, 1886–7 differential diagnosis, 1888 epidemiology, 1880 laboratory investigations, 1887–8 mast cells biology, 1879–80 immunophenotypes, 1886 and osteoporosis, 1890 pathology, 1878–9 pediatric, 1891 prevalence, 1880 and respiratory problems, 1883 serum tryptase measurement, 1886–7 skin biopsies, 1884 skin involvement, 1881–3 smoldering, 1880 symptoms, 1878, 1881–4 gastrointestinal, 1883, 1890 generalized, 1883–4 hematologic, 1883 lymphoid, 1883 musculoskeletal, 1883

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mastocytosis (Cont’d ) treatment, 1889– 91 see also cutaneous mastocytosis (CM); systemic mastocytosis (SM) mastoparan, 1127 maternal atopy, and allergic diseases, 37– 8 matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) spectrometry, 936–7 matrix degradation, 424–5 matrix metalloproteinase inhibitors, in nasal polyposis treatment, 1469 matrix metalloproteinases (MMPs), 180–1, 305, 387, 402, 415, 505 in airway smooth muscle, 882–3 chemokine modulation, 479 and fibrosis, 424–5 MMP-3, 1611 MMP-9, 1595– 6, 1611 maturing immune system, concept of, 2027 MAVS (Cardif), 192 maximum expiratory flow–volume (MEFV), 749, 751 curves, 753– 4, 770 helium–oxygen mixtures, 756–7 maximum inspiratory flow–volume (MIFV), 749 curves, 753 MBL see mannose-binding lectin (MBL) MBL-associated serine proteases (MASPs), 443– 4 activation control, 444– 5 MBP see major basic protein (MBP) mCD23 (membrane-bound CD23), 103, 110–11 mCLCA3, 851 mCLCA, 851 MCP-2 (membrane cofactor protein-2), 1415 MCP-3 (membrane cofactor protein-3), 1415 MCP-4 see membrane cofactor protein-4 (MCP-4) MCP (membrane cofactor protein), roles, 446–7 MCPs (monocyte chemotactic proteins), 1303 MDC/CCL22, 71 MDC (macrophage-derived chemokine), 146, 530, 1301 MDC (monocyte-derived chemokine), 920 mDCs see myeloid dendritic cells (mDCs) MDIs see metered-dose inhalers (MDIs) meat wrapping, and occupational airway diseases, 1101 mechanical ventilation, and aerosol delivery, 780 meclizine, 560 mediator antagonists, 1718–20 mediator cells, early studies, 7– 9 mediators allergic rhinitis, 1418–20 anaphylactic, 8 – 9 early studies, 7– 9 eicosanoid, 799 eosinophil-derived, 260–2 and exercise-induced bronchoconstriction, 809–12 inflammatory, 1495 lipid, 380–1, 566– 633 pharmacologic, 529 medication see drugs MEFV see maximum expiratory flow–volume (MEFV) MEL 14, 338

melanophores, 578 melittin, 1124, 1127, 1559 structure, 1125 membrane-bound CD23 (mCD23), 103, 110–11 membrane cofactor protein-2 (MCP-2), 1415 membrane cofactor protein-3 (MCP-3), 1415 membrane cofactor protein-4 (MCP-4), 1303–4, 1415 expression, 1304 immunostaining, 1305 membrane cofactor protein (MCP), roles, 446–7 Menes (fl. 2641 BC), 3, 1897 mepolizumab, 73, 1613 6-mercaptopurine, 733–5 Mercurialis annua (annual mercury), pollen, 960 mesenchymal stem cells, 404 mesothelioma, 1283 metabisulfates, 1328 metamizole, 1975 metaproterenol, 672 Met e 1, 1137 metered-dose inhalers (MDIs), 686, 721–2, 771 use with spacers, 723 see also pressurized metered-dose inhalers (pMDIs) methacholine, 698, 833, 1669 and airflow limitation, 791 bronchial hyperresponsiveness, 783, 784, 787 dose–response relationships, 785 in exercise-induced bronchoconstriction, 815 inhalation challenges, 1581 methotrexate, 72 methoxamine, 680 structure, 673 α-methyldopa, 680 methylxanthines, discovery, 683 metoprolol, 679 MgA, 900–1 MgB, 900–1 MgC, 900–1 MHC see major histocompatibility complex (MHC) mice allergens, 917 gene disruption, 586–7 immunologic disease models, 439 see also mouse models microarray technology, 1292, 1717 microbe-sensing molecules, in airway epithelium, 379–80 microbial agents, and atopic dermatitis, 1819–20, 1824 microbial antigens, and extrinsic allergic alveolitis, 1758–62 microbial burden, infants, 2008 microbial exposure, 32, 33, 34 early, 1263–4 infants, 2006–10 and protection against atopic diseases, 1233 protective factors, 2025–7 risk factors, 2025–7 microbial infection, and atopic diseases, 1232–3 microbial products, 1720–1 microbial volatile organic compounds (MVOCs), 1284

microbicidal proteins, 187 Micrococcus lysodeikticus (bacterium), 373 microhelix complex component, 1168 microvascular leakage, and phosphodiesterase inhibitors, 650–1 microvessels basal permeability, 857–61 hyperpermeability in, 861–2 MIF (migration inhibitory factor), 1721, 1766 MIFV see maximum inspiratory flow–volume (MIFV) MIGET (multiple inert gas elimination technique), 749, 758–9 migration inhibitory factor (MIF), 1721, 1766 milk allergens, 914, 917 allergic reactions, 1147, 1926 breast, 1154 exposure, 1154 goat’s, 3 see also cow’s milk; infant formulas milk proteins in gloves, 1166 malabsorption, 1329 Millar, John (fl. 1769), 1591–2 Mini-AQLQ (Mini Asthma Quality of Life Questionnaire), 1662 Mini Asthma Quality of Life Questionnaire (Mini-AQLQ), 1662 MIP-1α (macrophage inflammatory protein-1α), 1763–4 MIP-1β (macrophage inflammatory protein-1β), 530 MIRR (multichain immune recognition receptor), family, 222 MistyNeb, 772 mitochondria, in neutrophils, 298 mitogen-activated protein kinase (MAPK) inhibitors, 851 mitogen-activated protein kinase (MAPK) pathway, 205, 208, 338 mitogen-activated protein kinases (MAPKs), 147, 208 in endothelial hyperpermeability, 865 mitogenesis, and phosphodiesterase 1, 639 mitogens, and airway smooth muscle growth, 884 mitogillin, 974 mitomycin C, topical, in vernal keratoconjunctivitis treatment, 1497 mizolastine, 329 MLA (monophosphoryl lipid A), 1518 MLCK (myosin light chain kinase), 862, 867–8 MLCP (myosin light chain phosphatase), 864, 879 MMAD (mass median aerodynamic diameter), 768, 769, 770, 771–2 MMCs (mucosal mast cells), 220 MMF see mycophenolate mofetil (MMF) MMPs see matrix metalloproteinases (MMPs) MOAHLFA index, definition, 1832 MOAHL index, 1832 MOHL index, 1832 molds allergens, 1061–101, 1385 see also Aspergillus spp. (molds)

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molecular immunopathology, allergic diseases, 1290–317 molecular techniques, in allergic diseases, 1291–2 mometasone, 1712 mometasone furoate, 1438 monkeys macaque, 1187 see also Macaca mulatta (rhesus monkey) monobactams, 1902 monoclonal antibodies, 741–2 fine specificity, 934 monocyte chemotactic proteins (MCPs), 1303 monocyte-derived chemokine (MDC), 920 monocyte differentiation, and phosphodiesterase 1, 639– 40 monocytes in asthma, 1613 and neurotrophins, 499 mononuclear phagocyte system, 437 studies, 440 monophosphoryl lipid A (MLA), 1518 montelukast, 329, 405, 696, 698– 9, 700 in allergic rhinitis, 705– 6, 1438– 9 in aspirin-induced asthma, 706– 7, 1973 in chronic asthma treatment, 1653 and exercise-induced bronchoconstriction, 810, 811 structure, 696 Moraxella catarrhalis (bacterium), 379, 1470 Moro, Ernst (1874–1951), 5 mouse allergen, and asthma, 142–3 mouse models active immunization, 1204– 8 airway remodeling, 1202–10 criticisms, 1207 issues, 1210 transgenic, 1208–10 MPAG (mycophenolic acid glucuronide), 735– 6 MPA (mycophenolic acid), 735– 6 MPE (maculopapular exanthem), 1955 MPO see myeloperoxidase (MPO) MRI see magnetic resonance imaging (MRI) mRNA β adrenoceptors, 671 allergen-encoding, 898– 9 degradation, 717 detection, 1291 expression, 1460 MUC1, 845 MUC2, 844, 845 MUC5AC, 842, 844, 845, 852 MUC5B, 842, 844 MUC gene expression, 842, 851 suppression, 851 mucins, 370 Muckle–Wells syndrome, 1865 mucoactive agents, 852 mucociliary apparatus, 1457– 8 mucociliary clearance in allergic rhinitis, 845– 6 animal models, 1216 in asthma, 845 use of term, 841 mucolytics, 852

mucosal addressin cell adhesion molecule-1 (MAdCAM-1), 339 function, 344–5 structure, 344–5 mucosal-associated lymphoid tissue (MALT), 1485 mucosal contact dermatitis, 1842–3 mucosal immune system, development, 1193–4 mucosal mast cells (MMCs), 220 mucous cells differentiation, 1193 species differences, 1192 mucus in allergic rhinitis, 840–56 antimicrobial properties, 373 in asthma, 840–56 composition change, 373 see also airway mucus mucus–plasma interactions, 843, 844 mucus-secreting cells, alterations, 1637 mucus secretion, inhibition, 850 mugwort allergen, 921 mugwort–mustard allergy syndrome, 1153 Multicentre Atopy Study (MAS) (Germany), 1262, 1263 multichain immune recognition receptor (MIRR), family, 222 multiple drug hypersensitivity, use of term, 1959 multiple drug hypersensitivity syndrome, 1959 flare-up reactions, 1959 symptoms, 1959 T-cell activation, 1959 multiple inert gas elimination technique (MIGET), 749, 758–9 Munc18B, 850 Munchener Medizinische Wochenschrift, 4 muramyl dipeptide, 191–2 muromonab CD3 (OKT3), 742 Murphy, Robert, 9 muscarinic receptors, 827 subtypes, 684, 824 mushroom farms, fungal spores, 972 Mus m 1, 1137 MVOCs (microbial volatile organic compounds), 1284 Mycelia sterile (fungus), 1282 mycetomas, 1463–4 MYC genes, 120 Mycobacterium spp. (bacteria), 55 Mycobacterium abscessus (bacterium), 1791 Mycobacterium avium (bacterium), 1760 Mycobacterium immunogenum (bacterium), 1760 Mycobacterium tuberculosis (bacterium), 1283 mycophenolate, 735–6 adverse reactions, 736 drug interactions, 736 indications, 736 mechanisms, 736 monitoring methods, 736 pharmacogenetics, 736 pharmacokinetics, 735–6 structure, 735 mycophenolate mofetil (MMF), 735 in atopic dermatitis treatment, 1826 mycophenolic acid glucuronide (MPAG), 735–6 mycophenolic acid (MPA), 735–6

Mycoplasma spp. (bacteria), 55 Mycoplasma pneumoniae (bacterium), 1233, 1573, 1621 infection, 1656 mycotoxins, 976 myeloid dendritic cells (mDCs), 166–7, 169–70 in lung, 174 roles, 176 myeloma-IgND, 7 myeloperoxidase (MPO), 305 secretion, 1297 myeloproliferative variant hypereosinophilic syndrome, 1806–7 differential diagnosis, 1888 use of term, 1806 myocardial infarction, leukotrienes in, 587–8 myocardin, 877 myofibroblasts, 1308–9 extracellular matrix production, 417 sources, 417 myogenesis, airway smooth muscle, 876–7 myosin, 878–9, 1132 activation, 205 myosin light chain kinase (MLCK), 862, 867–8 myosin light chain phosphatase (MLCP), 864, 879 myristoylated alanine-rich C-kinase substrate (MARCKS), 850 inhibitors, 850–1 Myrmecia pilosula ( jumper ant), 1983 venom, 1125 Myrmicinae (ants), 1983 insect sting allergy, 1983 Myrothecium spp. (fungi), occurrence, 978 NAB (National Allergy Bureau) (USA), 949 NAC see nasal allergen challenge (NAC) NACHT, 379 nadolol, 679 NADPH, 377 NADPH oxidase, 297, 298, 304, 311 catalysis, 376 inhibition, 312 neutrophil, 306 NAIP (neuronal apoptosis inhibitory protein), 379 NAL see nonammoniated latex (NAL) NALPs, 379 NANC (nonadrenergic, noncholinergic) neurons, 824, 827 nanoparticles definition, 1281 and indoor air pollution, 1281 NARES see nonallergic rhinitis with eosinophilia syndrome (NARES) nasal airflow sensation, 1433 nasal airways, parasympathetic innervation, 824 nasal allergen challenge (NAC), 1715–16 advantages, 1716 procedures, 1716 nasal cavity, anatomy, 1404, 1456 nasal challenge aspirin, 1972–3 late-phase allergic reactions, 525–6 studies, 1419 nasal cycle, 825 nasal filters, 1441

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nasal hyperactivity, 1403 nasal innervation, 823– 4 nasal mucosa, 136, 1293– 4 aerosol delivery to, 780–1 neural innervation, 1405–7 nasal obstruction, measurement, 1433 nasal polyposis, 1459–69, 1973 and aspirin-induced asthma, 1573 and aspirin sensitivity, 1459, 1462–3 and asthma, 1573 clinical features, 1466 comorbidities, 1459 course, 1468 and cystic fibrosis, 1394 differential diagnosis, 1466 disease course, 1468 eosinophils in, 1461 epidemiology, 1459 etiology, 1394, 1461–5 future research, 1475 future trends, 1468– 9 genetics, 1459– 60 incidence, 1459 investigations, 1466 mechanisms, 1460–1 pathogenesis, 1461–5 pathology, 1465– 6 prevalence, 1459, 1460 prognosis, 1468 symptoms, 1394 therapeutic scheme, 1467 treatment, 1466– 8 future modalities, 1468– 9 nasal polyps, 325, 1393, 1454– 81 with allergic rhinitis, 1388 and allergy, 1461–2 and asthma, 1461 and cystic fibrosis, 1463 recurrence, 1394 use of term, 1454 nasal reflex physiology, 825 nasal resistance, 1433 nasal respiratory mucosa, 823– 4 nasal secretions, sources, 824 nasal symptoms chemical factors, 1391 physical factors, 1391 nasal vasculature, 1404–5 schematic, 1405 natalizumab, 353, 1712 National Allergy Bureau (NAB) (USA), 949 National Ambulatory Medical Care Survey (UK), 1854 National Cooperative Inner City Asthma Study (NCICAS) (USA), 1132, 1133, 1139, 1140 National Health Service (NHS) (UK), allergic diseases, costs, 23 National Heart, Lung, and Blood Institute (NHLBI) (US) Expert Panel Report, 1568 National Asthma Education and Prevention Program Clinical Practice Guidelines, 1194 National Institute of Allergy and Infectious Diseases (NIAID) (US), 1899

National Institute for Clinical Excellence (NICE) (UK), 1603 National Institutes of Health (NIH) (USA), 1132, 1567 Surveillance, Epidemiology, and End Results (SEER) database, 1677 National Study of Lead and Allergens in Housing (NSLAH) (USA), 1132 natural killer (NK) cells, 167, 188 natural killer T (NKT) cells, 20, 55–6, 188 in asthma, 71–2 and chemokines, 482 natural organic compounds, 1102 natural rubber latex (NRL) allergy, 1164 applications, 1164, 1165 chemistry, 1166–7 exposure, 1170–1 fractionation, 1167 manufacture, 1166–7, 1702 see also latex NCICAS (National Cooperative Inner City Asthma Study) (USA), 1132, 1133, 1139, 1140 near-fatal asthma (NFA), 1576 definition, 1574 studies, 1574 nebulizers, 768, 778, 785 developments, 778 Necator americanus (hookworm), 1791 metalloproteases, 479 neck, eczema, 1841 nedocromil, 1443 nedocromil sodium, 1496 in asthma treatment, 1332 in exercise-induced bronchoconstriction, 816 in perennial allergic conjunctivitis treatment, 1492 in seasonal allergic conjunctivitis treatment, 1490 negative expiratory pressure (NEP), 754 negative selection, 50 Neisseria spp. (bacteria), infections, 448 Neisseria gonorrhoeae (bacterium), 448 Neisseria meningitidis (bacterium), 192, 448 nematodes, 154 NEMO, 310 deficiency, 209 NE (neutrophil elastase), 424–5 NEP see negative expiratory pressure (NEP) nerve growth factor (NGF), 269, 494 discovery, 496 interaction, 497 overexpression, 502, 503 receptors, 498 roles, 499–502 in allergic diseases, 502–8 in asthma, 513–14, 1620 synthesis, 499 nerves airway wall, 1641 in allergic airway diseases, 831–5 in asthma, 831–4 autonomic, 830 primary afferent, 829

in rhinitis, 834 sensory-efferent, 849 sympathetic, 825 vagus, 833 in wheezing, 832–3 see also airway nerves nervous system peripheral, 497–8 see also autonomic nervous system; central nervous system (CNS); sensory nervous system Netherton’s disease, genetics, 1228 neural inflammation, 530–1 neural innervation, nasal mucosa, 1405–7 neural remodeling, in asthma, 1620 neurogenic inflammation, 494–6 in allergic airway diseases, 495 concept of, 494, 511 lower airways, 828 mechanisms, 511–12 neuropeptides in, 514–19 upper airways, 825 neurokinin A (NKA), 514–15, 530–1, 786, 788, 825, 849, 1406 neurokinin receptors, activity, 799 neuromediators, 512 neuromodulation, in airway inflammation, 828–31 neuronal apoptosis inhibitory protein (NAIP), 379 neurons cholinergic, 827 interactions, with inflammatory cells, 512 neuropeptides, 385, 824–5 in allergic inflammation, 511–23 and cough, 519–20 immunoregulation, 495 inflammatory, 1406 in neurogenic inflammation, 514–19 roles, 508 neuropeptide tyrosine (NPY), 513, 518 clinical applications, 518 metabolism of, 518 pulmonary effects of, 518 neuropeptide Y (NPY), 825, 1407 neurophysiology, airways, 823–8 neuropilin 1 (NRP1), 328 neurotoxins, 850 neurotransmitter antagonists, 788 neurotransmitter release, inhibition, 849 neurotrophin-3 (NT-3), 494 discovery, 496 expression, 498, 499, 501 interactions, 497 roles, 500 neurotrophin-4 (NT-4), 494 discovery, 496 expression, 499, 501, 507, 508 neurotrophin-5 (NT-5), 494 discovery, 496 neurotrophin receptors, biology, 496–7 neurotrophins, 494–510 in asthma, 1620 biology, 496–7 cellular sources, 497–502

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effects, 497–502 expression, 508 and immune system, 498–501 as neurotrophic factors, 497 pathophysiology, in allergic diseases, 502– 8 and peripheral nervous system, 497– 8 signaling, 496–7 and structural tissue cells, 501–2 synthesis, 508 neutral endopeptidase (NEP), 515–16 inhibitors, 518 neutropenia, drug-induced, 1951–2 neutrophil counts, 1371 neutrophil elastase (NE), matrix degradation, 424–5 neutrophil NADPH oxidase, 306 neutrophil protease inhibitors, 850 neutrophil proteases, roles, 306 neutrophils abundance, 295 activation, 303– 6 adhesion, 300–1 and allergic diseases, 1297 apoptosis, 307– 8 signaling pathways, 308–10 apoptotic, phagocytic removal, 310–11 biological properties, 295–319 bone marrow release, 298 chemotactic factors, 302 chemotaxis, 302–3 deactivation, 307–11 definition, 297 degranulation, 304 – 6 development, 295–7, 298 differentiation, 297 and fibrosis mediation, 421– 2 Golgi apparatus, 298 granule constituents, 304– 5 granule development, 305– 6 granulopoiesis, 295, 298 kinetics, 298– 9 and late-phase allergic reactions, 528 lifespan, 299 maturation, 298 mitochondria in, 298 origin, 298 phagocytosis, 303– 4 plasma membrane, 297 recruitment, to inflamed sites, 299– 303 removal, 307–11 roles in allergic diseases, 312–13 in diseases, 311–13 in health, 313 in inflammatory diseases, 311–12 physiologic, 299 sequestration, 300–1 structure, 297–8 transmigration, 301–2 newborn, allergic disease prevention, 1997– 2019 New Orleans (USA), fungal studies, 976– 9 New Zealand, asthma, racial and ethnic differences, 1244 NFA see near-fatal asthma (NFA) NFAT, phosphorylation, 205

NF-κ-B, 717 activation, 1724 NF-κ-B inhibitors, 727, 1724 NF-κ-B pathway, 208–9 NGF see nerve growth factor (NGF) NHEJ (nonhomologous end-joining) pathway, 125, 129 NHLBI see National Heart, Lung, and Blood Institute (NHLBI) (US) NHS (National Health Service) (UK), allergic diseases, costs, 23 NIAID (National Institute of Allergy and Infectious Diseases) (US), 1899 NICE (National Institute for Clinical Excellence) (UK), 1603 nickel allergy, 1841 Nigrospora spp. (fungi) abundance, 967 seasons, 967 NIH see National Institutes of Health (NIH) (USA) nipple eczema, 1817 Nippostrongylus brasiliensis (nematode), 154, 230, 273, 2027 nitric oxide (NO) exhalation, 377 expired, 809 in hyperventilation-induced bronchoconstriction, 799 measurement, 1466 and vascular permeability, 862–3 see also exhaled nitric oxide nitric oxide synthase (NOS), 377, 1413–14 nitrites, 1328 nitrogen dioxide, 146, 1267 exposure, 1250 and indoor air pollution, 1284–5 sources, 1268–9 urban areas, 1268–9 nitrous oxides, 946 NK1 receptor antagonists, 516, 520, 531 NK1 receptors, 514–15, 530–1, 786, 849 activation, 825 NK2 receptor antagonists, 516, 531 NK2 receptors, 514–15, 530–1, 786, 788, 849 NK3 receptors, 515, 849 NKA see neurokinin A (NKA) NK (natural killer) cells, 167, 188 NKT cells see natural killer T (NKT) cells NLRs see Nod-like receptors (NLRs) NMR (nuclear magnetic resonance) spectroscopy, 104 NO see nitric oxide (NO) nocebos, asthma, 834 NOD2 gene, 1229 NOD/CARD proteins, 1231 Nod-like receptors (NLRs), 189 family, 379 in innate immune system, 191–2 nonadrenergic, noncholinergic (NANC) neurons, 824, 827 nonallergic asthma mast cells and, 234–5 pathogenesis, 1614

nonallergic rhinitis with eosinophilia syndrome (NARES) definition, 1391 etiology, 1391 nonammoniated latex (NAL), 1175–6 applications, 1174 nonatopic asthma, 63 nonatopic eczema symptoms, 1814 use of term, 1814 noncholinergic nonadrenergic innervation see sensory innervation noneczematous contact reactions, 1843 noneosinophilic asthma, 64, 1621 nonhomologous end-joining (NHEJ) pathway, 125, 129 non-IgE-mediated anaphylactoid reactions, 1906–7 arachidonic acid metabolism modulation, 1907 coagulation pathway activation, 1906 complement-mediated, 1906 immune complexes, 1906 mast cell activation, 1907 multiple mechanisms, 1907 sulfite sensitivity, 1907 nonindustrial environments, fungal spores, 971–2 nonreceptor tyrosine kinases (NRTKs), 205–6 nonspecific bronchoconstrictor stimuli, 783, 784 nonspecific interstitial pneumonitis (NSIP), 1769, 1771 nonsteroidal antiinflammatory drugs (NSAIDs), 590–1 adverse reactions, 1975 allergic reactions to, 1975 and anaphylaxis, 1907 and angioedema, 1865 asthma induction, 236, 1390, 1623 cross-reactions, 1971 hypersensitivity reactions to, 1966–79 and nasal polyposis, 1394, 1462–3 rhinitis induction, 1390 in seasonal allergic conjunctivitis treatment, 1490 and urticaria, 1865 Noon, Leonard (1877–1913), 12, 13 noradrenaline (norepinephrine), 669, 672 norepinephrine, 669, 672 normocapnia, 1576 normoxemia, 1576 nose adrenoceptors, 669 anatomy, 1404–7, 1456 in asthma, 762 neural inflammation, 531 physiology, 1404 – 7 NOS (nitric oxide synthase), 377, 1413–14 NPY see neuropeptide tyrosine (NPY) NPY (neuropeptide Y), 825, 1407 NRL see natural rubber latex (NRL) NRP1 (neuropilin 1), 328 NRTKs (nonreceptor tyrosine kinases), 205–6 NSAIDs see nonsteroidal antiinflammatory drugs (NSAIDs) NSIP (nonspecific interstitial pneumonitis), 1769, 1771

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NSLAH (National Study of Lead and Allergens in Housing) (USA), 1132 NT-3 see neurotrophin-3 (NT-3) NT-4 see neurotrophin-4 (NT-4) NT-5 see neurotrophin-5 (NT-5) nuclear magnetic resonance (NMR) spectroscopy, 104 nuclear topology, 131 nucleic acid therapy, 1725– 6 numular eczema, 1817 nutrition and asthma, 1249 see also diet; food OAS see latex–fruit syndrome obesity and allergic diseases, 37 and asthma, 1570 prevalence, 36 occupational agents asthma-inducing, 1061, 1102 future research, 1102 and late asthmatic reactions, 527 rhinitis-inducing, 1101– 2 occupational airway diseases animal confinement facilities, 1061 baking industry, 1061 and cleaning agents, 1101 confectioneries, 1061 drug manufacturing plants, 1101 floricultures, 1061–101 and grain dust, 1101 greenhouses, 1061–101 hairdressing salons, 1101 and meat wrapping, 1101 potrooms, 1101 workplaces, 1061–102 World Trade Center collapse, 1101 occupational asthma, 63, 1687–711 allergen avoidance, 1704–5 allergen-induced, 1018– 60 atopy, 1691–2 bakers, 1700, 1702 and colophony, 1703 and complex platinum salts, 1703 definitions, 1687– 8 determinants, 1690–2 diagnosis, 1325– 6, 1695 and diisocyanates, 1692, 1701 early studies, 10 economic impacts, 1688 and enzymes, 1703– 4 epidemiology, 1688– 93 etiology, 1017–122, 1688 by occupational group, 1688, 1689 examples, 1701– 4 exposure, 1691 to sensitizing agents, 1693– 4 frequency, 1688 fungus-induced, 969, 970 genotype, 1692 identification, 1695–701 historical background, 1695– 6 investigations, 1696–701 immunologic tests, 1701

incidence, 1690 inhalation tests, 1697–701 irritant-induced, 1062–100, 1694 and laboratory animals, 1701–2 and latex, 1702–3 and latex allergy, 1173 management, 1704 mast cells and, 235–6 mechanisms, 1693–4 outcomes, 1692–3 population-based studies, 1689–90 prevalence, 1102 prevention, 1704–5 routine surveillance, 1689 susceptibility, 1102 types of, 1688 use of term, 1687 workplace studies, 1690 work-related airflow limitation, functional tests, 1696–7 Occupational Physicians Reporting Activity (OPRA), 1832 occupational rhinitis, 1017 allergen-induced, 1102 definition, 1101–2 etiology, 1101–2, 1389–90 future research, 1102 irritant-induced, 1102, 1103 occupational-agent-induced, 1101–2 subtypes, 1101–2 occupational sensitizers, and asthma, 1571 OCT1 (octamer-binding protein-1), 120 octamer-binding protein-1 (OCT1), 120 OCTN gene, 1229 ocular allergic diseases atopy incidence, 1488 classification, 1482, 1483 mechanisms, 1485–7 ocular allergy, 1482–509 clinical features, 1502–3 incidence, 1482 laboratory investigations, 1503 pathophysiology, 1485–7 studies, 1502–3 use of term, 1482 ODNs (oligodinucleotides), 1561 oilseed rape, and hay fever, 1324 OKT3 (muromonab CD3), 742 Olea spp. (olive trees), 944 see also olive pollen Olea europaea (olive), 956 cross-reactivity, 957 Ole e 1, 913–14, 929 olfaction, mechanisms, 1457 olfactory disturbances, etiology, 1457 olfactory epithelium, 371 oligodinucleotides (ODNs), 1561 olive pollen, 956–7 cross-reactivity, 957 seasons, 943 severity, 957 olopatadine, 557 omalizumab, 533, 727, 1440–1 adverse effects, 1675–8 in allergic rhinitis treatment, 1680

antiinflammatory mechanisms, 1668–9 in asthma treatment, 1332, 1720 in atopic dermatitis treatment, 1827 in chronic asthma treatment, 1653–4 clearance, 1670 clinical pharmacology, 1669–75 controlled studies, 1670, 1671 development, 1667–8 dose reduction, 1670 dosing tables, 1671 efficacy with coexisting rhinitis, 1675 ETOPA study, 1670, 1673, 1675, 1679, 1681 INNOVATE study, 1670, 1672–3, 1674, 1675, 1678, 1681 in persistent severe allergic asthma treatment, 1670–4, 1681 pooled analysis, 1673–4 studies, 1670–4 future trends, 1680 hypersensitivity, 1677 in IgE-mediated disease treatment, 1680 indications, 1679–80, 1681 and malignant neoplasia, 1677 mechanisms, 1668–9, 1670 and parasitic infections, 1677–8 patient responders, post-treatment identification, 1678 patient selection, 1678–80 based on pretreatment clinical characteristics, 1678 economic issues, 1678–9 in pediatric asthma treatment, 1680 in persistent severe allergic asthma treatment, 1665–81 quality of life studies, 1673–4 safety, 1675–7 and thrombocytopenia, 1678 tolerability, 1675–7 OME (otis media with effusion), with allergic rhinitis, 1389 ONO 6126, structure, 644 ontogeny, airway smooth muscle, 876–7 OP-1 (osteogeneic protein 1), 1637 OPRA (Occupational Physicians Reporting Activity), 1832 oral allergy syndrome (OAS) etiology, 1328 symptoms, 1328 see also latex–fruit syndrome oral candidiasis, 1653 oral challenge test, aspirin, 1972 oral corticosteroids in allergic bronchopulmonary aspergillosis treatment, 1751–2 in atopic dermatitis treatment, 1826 efficacy, 1674–5 oral methoxypsoralen therapy, 1890 oranges, allergens, 1153 orbicules, 945 organic compounds in diesel exhaust particulate, 1269 and indoor air pollution, 1280 organic dust toxic syndromes, and extrinsic allergic alveolitis, 1771

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Index

organizing pneumonia, 1795 organophosphates, 1285 ORMDL3 gene, 1227– 8 oropharyngeal candidiasis, 723, 776–7 Orthoptera (cockroaches), 1123 Osler, Sir William (1849–1919), 11, 1593 osteogeneic protein 1 (OP-1), 1637 osteoporosis, and mastocytosis, 1890 otis media with effusion (OME), with allergic rhinitis, 1389 outdoor air pollution and allergic airway diseases, 1266–78 and asthma, 1572 future research, 1274 rural areas, 1266 traffic-related, 1266, 1267 urban areas, 1266, 1267–70 outdoor allergens, immunoglobulin E production, 142–3 outdoor environments, fungal spores, 965– 9 outer eye, anatomy, 1482–5 OVA see ovalbumin (OVA) ovalbumin (OVA), 29, 144, 150, 174, 914, 1148, 1151 in active immunization models, 1204–5, 1206 aerosolization, 169 in breast milk, 1154 sensitization, 34–5, 153, 1202 tolerance to, 175 over-the-counter management, allergic rhinitis, 1445 ovomucoid, 914, 917, 1150, 1151, 1154 digestion, 1156 ovotransferrin, 914, 1151 OX40, 51, 54, 58, 882 OX40 ligand (OX40L), 51–2, 58, 882 expression, 136 oxidative stress, hierarchical model, 147 oxindole, 641 structure, 641 oxitropium, development, 683 oxitropium bromide, 685 Oxivent (oxitropium bromide), 685 oxymetazoline, 680 ozone, 146, 147, 148, 946, 988 effects on exercise tolerance, 1268 exposure, 1250, 1268 responses, 1195 generation, 1273 and indoor air pollution, 1285 studies, 1268 urban areas, 1267–8 P2Y2 antagonists, 851 P2Y2 receptors, 851 p21-activated kinases (PAKs), 865 activated, 866 p75NTR, 496–7, 504–5 PAC see perennial allergic conjunctivitis (PAC) PACAP (pituitary adenylate cyclase-activating peptide), 518 Paddock, Royce (1889–1969), 784 Paecilomyces spp. (fungi), occurrence, 978 PAF see platelet-activating factor (PAF) PAFR (platelet-activating factor receptor), 607– 9

PAHs see polycyclic aromatic hydrocarbons (PAHs) paired box gene 5 (PAX5), 120, 130, 132, 134 expression, 131 PAKs see p21-activated kinases (PAKs) palmar dermatitis, 1817 palmar hyperlinearity, 1817–18 PALMLTB4, 945–6 PAMPs see pathogen-associated molecular patterns (PAMPs) panallergens, 1153 Paneth cells, 375 papain, 1703 papular urticaria, 1864 PAQLQ (Pediatric Asthma Quality of Life Questionnaire), 1680 PAR2 (protease-activated receptor-2), 1611 parainfluenza virus, 1571 Paramyxoviridae (RNA viruses), 274 paranasal sinuses anatomy, 1456–7 physiology, 1459 parasites and allergen-specific immunoglobulin E, 154 exposure protective factors, 2025–7 risk factors, 2025–7 and hygiene hypothesis, 1247 and immunoglobulin E production, 154–5 regulatory mechanisms, 154–5 parasitic helminth infection, eosinophils in, 273–4 parasitic infections mast cells and, 230 and omalizumab, 1677–8 parasympathetic fibers, 827–8 parasympathetic ganglia, 827 parasympathetic innervation, 512 lower airways, 827 upper airways, 824 parent-of-origin effects, 1233–4 Parietaria spp. (pellitories), pollen, 958, 1272, 1323, 1385 Parietaria judaica (spreading pellitory), pollen, 958 Parietaria officinalis (pellitory of the wall), pollen, 958 paroxysmal nocturnal hemoglobinuria (PNH), 447 PARs see protease-activated receptors (PARs) PARSIFAL (Prevention of Allergy Risk Factors for Sensitization in Farming and Anthroposophic Lifestyle), 1246 Parthenium spp. (feverfews), 960 Parthenium hysterophorus (Santa Maria feverfew), pollen, 960 particle-associated metals, and immunoglobulin E production, 144–5 particle opsonization, 441–2 particles airborne allergen-carrying, 1270–1 bacteria, 963 definitions, 1281 deposition, 1219 fungi, 963 and indoor air pollution, 1281 plant allergens on, 945 see also diesel exhaust particles (DEPs); nanoparticles

particle size effects, 144 measurements, 771–5, 1281 clinical applications, 771 particulate matter (PM), 147 and allergic diseases, 144–5 deposition, 1219 PM1.0, 1281 PM2.5, 976, 1281 PM3.5, 1281 PM10, 1281 urban areas, 1269–70 see also air particulate matter; diesel exhaust particles (DEPs) parvalbumins, 1151, 1152 PAS domains, 637 patatin-like protein, 1169–70 patch tests, 1323, 1961 for allergic contact dermatitis, 1832, 1834–5, 1844 –8 complications, 1847–8 false negative reactions, 1847 false positive reactions, 1847 interpretation issues, 1847–8 reading, 1846–7 repeat, 1848 test allergens, 1845 see also photopatch tests pathogen-associated molecular patterns (PAMPs), 24, 61, 170, 171–2, 1231, 1232 expression, 119, 136 receptors, 379 recognition, 377 pathogens recognition, 303 by innate immune system, 189–92 pathologic conditions, and skin tests, 1339–40 patient compliance, sublingual immunotherapy, 1549 patients allergen injection immunotherapy, 1527 profiling, 1525 sublingual immunotherapy, 1549 see also allergic patients pattern-recognition receptors (PRRs), 119, 136, 192, 1231, 1232 localization, 377 paucigranulocytic asthma, 1621 PAX5 see paired box gene 5 (PAX5) PBMCs see peripheral blood mononuclear cells (PBMCs) PC20, 1560 PCNA (proliferating cell nuclear antigen), 1614 PCR (polymerase chain reaction), 899 PD20, 1560 pDCs see plasmacytoid dendritic cells (pDCs) PDE1 see phosphodiesterase 1 (PDE1) PDE1A gene, 638 PDE1B gene, 638 induction, 640 transcription, 639 PDE1C gene, 638 PDE1 inhibitors see phosphodiesterase 1 (PDE1) inhibitors PDE2 see phosphodiesterase 2 (PDE2)

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PDE2 gene, expression, 641 PDE3 see phosphodiesterase 3 (PDE3) PDE3A, 655 PDE3B, 655 PDE3 gene, expression, 655 PDE3 inhibitors see phosphodiesterase 3 (PDE3) inhibitors PDE4 see phosphodiesterase 4 (PDE4) PDE4 gene, encoding, 643 PDE4 inhibitors see phosphodiesterase 4 (PDE4) inhibitors PDE5 see phosphodiesterase 5 (PDE5) PDE7 see phosphodiesterase 7 (PDE7) PDE7A1, 646–7, 648 PDE7A2, 646–7 PDE7A3, 646–7 PDE7A gene, 646, 648 PDE7B gene, 646 PDE8A gene, 648– 9 PDE8B gene, 648– 9 PDE8 (phosphodiesterase 8), 648– 9 PDE inhibitors see phosphodiesterase (PDE) inhibitors PDE isoenzymes see phosphodiesterase (PDE) isoenzymes PDEs see phosphodiesterases (PDEs) PDGF (platelet-derived growth factor), 1617 PDGFRA, 280–1 PDGFRA gene, 1804 mutations, 1807 peak expiratory flow (PEF), 751–2, 2023 in asthma management, 749 in chronic asthma management, 1654 diurnal variation, 752 modeling, 1581–2 monitoring, 1568 in occupational asthma studies, 1696– 7 tests, 785 peak expiratory flow rate (PEFR), PDE4 inhibitor studies, 651–2 peanut allergy diagnosis, 1904 mortality, 1904 prevalence, 1904, 1922 treatment, 1904–5 peanuts allergens, 914, 916–17, 1151, 1155, 1158 allergic reactions, 1147 and anaphylaxis, 1904–5 digestion, 1148 exposure, 1154 PECAM-1 see platelet endothelial cell adhesion molecule-1 (PECAM-1) pecans, allergens, 1152 pediatric allergy, diagnosis, 1333 pediatric asthma, 1591– 607 and allergy, 1597– 9 anticholinergic agents in, 690 and bronchial hyperresponsiveness, 1597 clinical features, 1600 early studies, 1591–2 economic burden, 1593 epidemiology, 1592– 3 gender differences, 1594 historical background, 1591–2

immunopathology, 1595–6 management, 1601–4 nonpharmacologic, 1601–2 mortality, 1593 natural history of, 1593–4 omalizumab treatment, future trends, 1680 pathology, 1643–4 pharmacotherapy, 1602–4 prevalence, 1592 psychosocial issues education, 1604 management, 1604 and rhinovirus, 1596 studies, 1601 and viral infections, 1596–7 and wheezing, 1600 Pediatric Asthma Quality of Life Questionnaire (PAQLQ), 1680 pediatric in vitro models, inhaled mass, 773–4 pediatric issues, allergic rhinitis, 1445–6 pediatric mastocytosis, 1891 PEEP (positive end-expiratory pressure), 399 PEF see peak expiratory flow (PEF) PEFR (peak expiratory flow rate), PDE4 inhibitor studies, 651–2 Pen a 1, 1137 Pen i 1, 1137 penicillin and anaphylaxis, 1898, 1901 desensitization, 1962, 1963 Penicillium spp. (fungi), 963, 1282 abundance, 967 allergens, 974 occurrence, 971, 972–3, 977–8 seasons, 967 Penicillium brevicompactum (fungus), 1761 mycophenolate, 735 Penicillium camemberti (fungus), 1761 Penicillium frequentans (fungus), 1761 Penicillium olivicolor (fungus), 1761 Penicillium roqueforti (fungus), 1761 pentraxins, 375–6 peptide histidine isoleucine (PHI), 1407 peptide immunotherapy, 1559–60 peptides future trends, 937 and late asthmatic reactions, 533–4, 536, 537 silencing, 49 peptide-specific T cells, and late-phase allergic reactions, 537 Peptostreptococcus magnus (bacterium), 326, 327 Pepys, Jack (1914–96), 18, 21, 1757 Per a 1, 1134 Per a 2, 1134 Per a 3, 1134, 1135 Per a 6, 1134 homologs, 1137 Per a 7, 1134 homologs, 1137 perennial allergic conjunctivitis (PAC), 243–4, 1482, 1491–3 pathogenic issues, 1492 pathology, 1492 pathophysiology, 1487 treatment, 1492–3

perennial rhinitis diagnosis, 1324–5 etiology, 1324 symptoms, 1324 use of term, 1402–3 periarterial space, allergic reactions, 1218 perinatal risk factors, allergic diseases, 37–9 periorbital darkening, 1818 peripheral blood mononuclear cells (PBMCs), 92, 146, 918–20, 921–2, 1518 grass allergen responses, 1558–9 immunotherapy studies, 1515, 1516 proliferation assays, 1175 peripheral nervous system, and neurotrophins, 497–8 peripheral tolerance histamine receptor type 2 in, 94 induction, 94 –6 T cells, 91, 93 Periplaneta americana (American cockroach), 1131–2, 1138 allergens, 1134 antigen sensitization, 1132 occurrence, 1134 Periplaneta fuliginosa (smokybrown cockroach) allergens, 1134 occurrence, 1134 perlecan, 414 peroxisome proliferator-activated receptor (PPAR) antagonists, 1713 peroxisome proliferator-activated receptor-γ (PPAR-γ), 1725 persistent rhinitis, 1402–3 definition, 1403 persistent severe allergic asthma anti-IgE in, 1661–86 targeting rationale, 1666–7 burden, 1661–2 and daily living, 1662 economic impacts, 1662 future trends, 1680 hospitalization, 1661–2 mortality, 1661–2 prevalence, 1661 and quality of life, 1662 treatment omalizumab in, 1665–81 options, 1662–5 steroid-sparing strategies, 1675 pesticides, 1285 PET see positron emission tomography (PET) pet allergens aerodynamics, 998–9, 1000–1 avoidance, 1001–2, 1601 clinical effectiveness, 1002 distribution, 1000–1 in homes, 998–9 in dust reservoirs, 998 exposure, infants, 2005– 6 and indoor air pollution, 1281–2 particle size distribution, 999 in public buildings, 1000 in public transport, 1000 sources, 998 see also cat allergens; dog allergens

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pet ownership, 1001, 1002–12 issues, 997– 8 risk factors, 1002–3 pets and allergic diseases, 35, 1282 and allergic sensitization, 1002–12 and asthma, 998, 1001, 1002–12 see also cats; dogs pet sensitization, 1001 adult studies, 1012 asthma case-control studies, 1012 asthma clinic studies, 1012 birth cohort studies, 1003 and high-risk children, 1003 population-based, 1003 cohort studies, 1003–12 cross-sectional studies, 1003–12 risk factors, 1013 Peyer’s patches, 30 Pezizia domiciliana (mushroom), 1761 Pfam database, 204 PGD2 see prostaglandin D2 (PGD2) PGE2 see prostaglandin E2 (PGE2) PGE, 37 PGF2α (prostaglandin F2α), 592–3 PGG2 (prostaglandin G2), 566 PGH2 (prostaglandin H2), 566, 588– 9 PGI2 (prostaglandin I2), 599– 600 phagocytosis, 304 pharmacodynamics antihistamines, 555–7 definition, 555 pharmacokinetics antihistamines, 554– 5 definition, 554 pharmacologic agents, exercise-induced bronchoconstriction identification, 815–16 pharmacologic mediators, and late-phase allergic reactions, 529 phenacetin, 14 phenazone, 1975 phenols, 1166 phenoxybenzamine, 680 phentolamine, 680 phenylbutazone, 1975 phenylephrine, 1439 structure, 673 phenylpropanolamine, 680, 1439 PHF11 gene, 1229, 1717 Phillips, E. W., 831 PHI (peptide histidine isoleucine), 1407 Phl p 1, 168, 915–16, 918, 1232, 1555– 6, 1557 encoding, 1558 Phl p 2, 915, 918, 1555– 6 encoding, 1558 Phl p 5, 93, 95, 915–16, 918 encoding, 1558 Phl p 5a, 1555– 6 Phl p 5b, 1555– 6 Phl p 6, 1555– 6 encoding, 1558 Phl p 7, 916 Phl p 12, 916 Pholiota nameko (Shiitake mushroom), 1761 phosphatidylcholine, 1154

phosphatidylinositol 3-kinase (PI3K), 302–3, 471 activation, 884 phosphatidylinositol 3,4,5-triphosphate (PIP3), 302–3, 471 phosphodiesterase 1 (PDE1), 638–40 and mitogenesis, 639 and monocyte differentiation, 639–40 therapeutic indications, 639–40 tissue distribution, 638 phosphodiesterase 1 (PDE1) inhibitors, 638, 639 selective, 638 phosphodiesterase 2 (PDE2), 640–1 selective inhibitors, 640–1 therapeutic indications, 641 tissue distribution, 640–1 phosphodiesterase 3 (PDE3), 642 therapeutic indications, 642 tissue distribution, 642 phosphodiesterase 3 (PDE3) inhibitors adverse events, 655 selective, 642 phosphodiesterase 4 (PDE4), 642–5 regulatory mechanisms, 643–5 selective inhibitors, 643 therapeutic indications, 643 tissue distribution, 643 phosphodiesterase 4 (PDE4) inhibitors, 1724 adverse events, 654–5 for airway hypersecretion, 848 clinical pharmacology, 651–2 drug metabolism, 652–4 pharmacokinetics, 652–4 safety, 652–4 selective, 643 tolerability, 652–4 phosphodiesterase 5 (PDE5), 645–6 selective inhibitors, 645 therapeutic implications, 645–6 tissue distribution, 645 phosphodiesterase 7 (PDE7), 646–8 inhibition, adverse events, 655 selective inhibitors, 646–7 therapeutic implications, 647 tissue distribution, 646–7 phosphodiesterase 8 (PDE8), 648–9 phosphodiesterase (PDE) inhibitors, 328–9, 634–5, 1724 adverse events, 654–5 antiinflammatory effects, 649–51 development, 635 and edema, 650–1 hybrid, 649–51 immunoglobulin E-mediated processes, 649–50 and microvascular leakage, 650–1 nonselective, 649–51 proinflammatory cell infiltration, 650 phosphodiesterase (PDE) isoenzymes, 634–5 profiles, 640 phosphodiesterases (PDEs), 635 genetics, 635–6 standardized nomenclature, 637–49 phospholipase A1 (PLA1), 1124, 1125

phospholipase A2 (PLA2), 374, 380, 567, 568–9, 1123–4 isoforms, 323 structure, 1125 phospholipases, 1126 phosvitin, 1151 photocontact dermatitis, 1843 photopatch tests, for allergic contact dermatitis, 1848 phototherapy, in atopic dermatitis treatment, 1827 phthalates, and indoor air pollution, 1284 phthalic anhydride, 1101 Physalia spp. (Portuguese Man-of War jelly fish), 3–4 physical training, and exercise-induced bronchoconstriction, 817 phytocystatin, 1153 phytophotodermatitis, acute, 1840 PI2K–AKT pathway, 209–10, 211 PI3K see phosphatidylinositol 3-kinase (PI3K) PIAMA (Prevention and Incidence of Asthma and Mite Allergy), 2000, 2004, 2005 Pichia pastoris (yeast), allergens, 1135 p-i concept, 1946 picornaviruses, 380 pilocarpine, 784 pimecrolimus, 732, 1825 pindolol, 679 pineapple, bromelain, 1148 PIP3 (phosphatidylinositol 3,4,5-triphosphate), 302–3, 471 pirbuterol, 673–4 Pis s 1, 1149 pituitary adenylate cyclase-activating peptide (PACAP), 518 pityriasis alba, 1817 Pityrosporum spp. see Malassezia spp. (yeasts) Pityrosporum orbiculare see Malassezia furfur (yeast) Pityrosporum ovale see Malassezia furfur (yeast) PK see prekallikrein (PK) PKA (protein kinase A), 885 PKC see protein kinase C (PKC) PK–HK complex, 453, 457–8 factor XII-independent activation, 458–60 PLA1 (phospholipase A1), 1124, 1125 PLA2 see phospholipase A2 (PLA2) placebos, asthma, 834 placental growth factor (PlGF), 324 plant aeroallergens, monitoring, 947 Plantago spp. (plantains), 958 plant allergens and asthma in urban areas, 1272–3 exposure, 1285–6 on particles, 945 see also grass allergens; grass pollen allergens; latex allergens plantar dermatitis, 1817 plantar hyperlinearity, 1817–18 plant defense systems, allergens, 1149 plant proteins, 1102 plants aeroallergens, 1270–2 and air pollution, 1273–4 anemophilous (wind-pollinated), 943

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plants (Cont’d ) entomophilous (insect-pollinated), 943 sexual reproduction, 942–3 plasma cells B-cell differentiation into, 134 differentiation, 120–1 plasmacytoid dendritic cells (pDCs), 166–7, 169–70 roles, 176, 177 plasma exudation, 842–3 mechanisms, 844 plasma kallikrein, 452 plasticizers, and indoor air pollution, 1284 platelet-activating factor (PAF), 604–10 and asthma, 606– 8 biological activity, 606– 8 on airway smooth muscle, 606 on secretions, 606 biosynthesis, 604– 6 biosynthetic pathways, 605 catabolism, 608 cellular release, 608 and inflammatory cells, 606– 8 in ocular allergy, 1488 structure, 604 vascular permeability, 606 platelet-activating factor acetylhydrolase, 569 platelet-activating factor receptor (PAFR), 607– 9 platelet-activating factor receptor antagonists, 609–10 platelet-derived growth factor (PDGF), 1617 platelet endothelial cell adhesion molecule-1 (PECAM-1), 301, 337, 860 function, 343 structure, 343 Platts-Mills, Thomas A. E., 1513–14 Pleurotus ostreatus (tree oyster mushroom), 972, 974–5, 1761 PlGF (placental growth factor), 324 PM see particulate matter (PM) pMDIs see pressurized metered-dose inhalers (pMDIs) PMN (polymorphonuclear) cells, 295 PMNs (polymorphonuclear leukocytes), 569, 570, 574, 575, 576 PNECs (pulmonary neuroendocrine cells), 370–1 pneumococcal disease, invasive, 1233 pneumonia interstitial, 1795 organizing, 1795 see also Ascaris pneumonia; eosinophilic pneumonias Pneumovirinae (RNA viruses), 274 PNH (paroxysmal nocturnal hemoglobinuria), 447 Poa annua (annual bluegrass), 951 Pogonomyrmex spp. (harvester ants), 1983 Pol a 5, 1558 Polistes spp. (paper wasps), 1123, 1902, 1983 Polistinae (paper wasps), 1981, 1983 pollen, 942– 62 abundance, 944– 5 and air pollution, 945– 6 characteristics, 942– 4 dispersal, 944–5 exposure, 142, 144, 1592

grains, 943 and hay fever, 12 information sources, 946–9 proteins on, 945–6 seasons, 942 and thunderstorms, 1271–2 see also grass pollen; tree pollen; weed pollen pollen allergy, 1385 and climate change, 1274 see also grass pollen allergy pollen blocker creams, 1441 pollen counts, 945, 946 applications, 946–7 pollen–food allergy syndrome, 1153, 1156, 1926–7 pollen grains, 1270–1 allergens, 1274 dissemination, 1324 pollen immunotherapy, 1513 pollen monitoring networks, 947–9 pollination insects, 943 mechanisms, 943–4 triggers, 944 and weather patterns, 944–5 see also wind pollination pollinosis, 943, 945, 947, 954, 956 cedar, 1250 and climate change, 1273–4 pollutants and atopic diseases, 1263 effects, on airways, 376 exposure, postnatal, 2010 and immunoglobulin E production, 144–8 genetics, 147–8 mechanisms, 146–7 see also air pollution Polybia scutellaris (wasp), 1124 polyclonal antibodies, 741 polycyclic aromatic hydrocarbons (PAHs), 145, 147, 1281 in diesel exhaust particles, 1270 polymerase chain reaction (PCR), 899, 1291–2 polymorphisms, 1225–6 see also single nucleotide polymorphisms (SNPs) polymorphonuclear leukocytes (PMNs), 569, 570, 574, 575, 576 polymorphonuclear (PMN) cells, 295 polysaccharides, 964 polyunsaturated fatty acids (PUFAs), 2001, 2010 dietary intake, 37 polyvinyl chloride, degradation products, 1101 population studies infections, 2026 pet sensitization, 1003 Portier, Paul J. (1866–1962), 3–4, 6, 1897 positive end-expiratory pressure (PEEP), 399 positivity criteria, 1338 positron emission tomography (PET), 749 airway visualization, 758 postganglionic fibers, 824 potrooms, occupational airway diseases, 1101 poultry, exposure to, 1061 PPAR (peroxisome proliferator-activated receptor) antagonists, 1713

PPAR-γ (peroxisome proliferator-activated receptor-γ), 1725 PR-10, 1153 pranlukast, in aspirin-induced asthma, 1973 Prausnitz, Otto Karl W. (1876–1963), 6, 7, 103, 1346, 1665 prazosin, 680 prebiotics, 2008–9 precursor mast cells (pMCs) binding, 349 migration, 350 predictive values negative, 1999 positive, 1999 prednisolone, 718, 719, 725, 726, 1653 pregnancy and allergens, 142 and allergic rhinitis, 1446 allergy prevention in, 2000–1 H1-antihistamines in, 562 and asthma, 1657 food allergen avoidance, 2000 immunomodulatory dietary nutrients in, 2001 and immunosuppression, 743 and inhaled glucocorticoids, 725 and smoking, 1330, 2001 see also breastfeeding prekallikrein (PK), 452 activation, 458–60 prenyltransferase, 1167 pressure urticaria, 1861 pressurized metered-dose inhalers (pMDIs), 768, 771, 776–7, 779, 780 accessory devices, 776–7 in pediatric asthma treatment, 1603–4 reformulation without chlorofluorocarbons, 777 and spacers, 776–7 PREVASC study, 2000 Prevention of Allergy Risk Factors for Sensitization in Farming and Anthroposophic Lifestyle (PARSIFAL), 1246 Prevention and Incidence of Asthma and Mite Allergy (PIAMA), 2000, 2004, 2005 prick-puncture tests, 1336 primary afferent nerves, modulation, 829 primary positive selection, 49 primate models, allergic asthma, 1187–201 primin, 1841 Primula spp. (primulas), primin, 1841 probiotics, 2008–9 procaterol, 675 profilin, 1170, 1349 profilins, 1152, 1153, 1154, 1155 progenitor cells, 404 prohapten concept, 1944–6 prohevein, 1169 proinflammatory cell infiltration, phosphodiesterase inhibitors, 650 prolamin superfamily, 1148, 1150 proliferating cell nuclear antigen (PCNA), 1614 promethazine, 560 properdin, 444 propranolol, 679 propyphenazone, 1975

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prorenin, 463 prostacyclin (prostaglandin I2), 599– 600 prostaglandin D2 (PGD2), 529 in asthma, 591–2, 1611 biosynthesis, 591 receptor antagonists, 1712 sources, 811 prostaglandin E2 (PGE2), 594– 9, 1969 and allergic inflammation, 594– 6 in asthma, 598– 9 and dyspnea, 833 receptors, 596– 8 sources, 803 prostaglandin F2α (PGF2α), 592–3 prostaglandin G2 (PGG2), 566 prostaglandin H2 (PGH2), 566, 588– 9 prostaglandin I2 (PGI2), 599– 600 prostaglandin receptors, 265, 591 prostaglandins, 171, 529, 566, 588– 600, 1719 and aspirin hypersensitivity, 1968– 9 biosynthesis, 588– 91 cellular sources, 588– 91 early studies, 567 and exercise-induced bronchoconstriction, 809–10 prostanoid antagonists, 1718 prostanoid biosynthetic pathway, 588 prostanoids, 566–7 pharmacologic properties, in asthma, 591– 600 roles, 567 protease-activated receptor-2 (PAR2), 1611 protease-activated receptors (PARs), 387, 424 protease inhibitors, 850 proteases, 1124 roles, 1127– 8 protein A, 326, 327 protein allergens, 1204–5 protein contact dermatitis, 1842 origin of term, 1842 protein Fv, 326 protein kinase A (PKA), 885 protein kinase C (PKC), 860 activation, 204–5 proteins adapter, 207 allergen, 1517 allergenic, 1149 and anaphylaxis, 1905 animal, 1102 bone morphogenetic, 1636–7 classification, 1148 expression, 1229 metabolic, 1149 microbicidal, 187 modification, 203– 4 plant, 1102 on pollen, 945– 6 radiolabeled, 436–7 see also food proteins; G proteins; milk proteins; RAG proteins proteoglycans, in extracellular matrix, 414 proteolytic processing, chemokine receptors, 479 provocation tests, 1323 proximal intrapulmonary airways, species differences, 1192

PRRs see pattern-recognition receptors (PRRs) Pru av 2, 1150 Prunus avium (cherry), proteins, 1150 pruritus aquagenic, 1864 neurotrophins in, 507–8 pseudoallergy, 6, 1949–50 pseudoephedrine, 680, 1439 Pseudomonas aeruginosa (bacterium), 376, 1463 Pseudomonas spp. (bacteria), 1760 PSGL-1 see P-selectin glycoprotein ligand (PSGL-1) psoralen ultraviolet A (PUVA), 1890 psoriasis gene expression studies, 1228–9 leukotrienes in, 587 susceptibility genes, 1229 PSORS2 gene, 1229 PTEN enzyme, 471 PTGD2, 1229 PTGER2 gene, 1969 public buildings, pet allergens in, 1000 public transport, pet allergens in, 1000 PUFAs see polyunsaturated fatty acids (PUFAs) pulmonary embolism, 1585 pulmonary eosinophilia, 1779–801 after transplantation, 1795–6 drug-induced, 1794 tropical, 1790 see also eosinophilic lung diseases pulmonary fibrosis, 1724 pulmonary function tests allergic bronchopulmonary aspergillosis (ABPA), 1740–51 extrinsic allergic alveolitis, 1768 small animals, 1219 see also lung function tests pulmonary neuroendocrine cells (PNECs), 370–1 pulmonary resistance, helium–oxygen mixtures, 757 puncture tests comparisons, 1337 methods, 1335–6 pyrazolone derivatives, allergic reactions to, 1975 pyrilamine, 560, 1489 pyrin domain, 379 QAS see Quidel Allergy Screen (QAS) QoL see quality of life (QoL) quality of life (QoL) and atopic dermatitis, 1820–1 and Hymenoptera venom allergy, 1985 and persistent severe allergic asthma, 1662 quantitative immunoblotting, 933–4 quaternary ammonium compounds, 685 Quercus spp. (oak trees), 944 Quercus suber (cork oak), 1761 questionnaires allergic rhinitis epidemiology, 1384–5 atopic diseases, 2022–3 atopy, 2022–3 cough, 1590 improvements, 2023 Quidel Allergy Screen (QAS) principles, 1351 sensitivity, 1351

R501X gene, mutations, 1228 Rab, 205 rabbits, serum sickness, 437 RAc, 205 roles, 866 race and asthma, 1243–4 and skin tests, 1339 racehorses, as models, 795 racial and ethnic differences, asthma, 1244 Rackemann, Francis (1887–1973), 12 radiation therapy, eosinophilic pneumonia induction, 1794 radioallergosorbent test (RAST), 928, 929, 947, 974, 975, 1349–57 adsorption, 1349 allergosorbent, 1349 antibody binding, 1351–3 applications, 1323 autologous dust, 1354 calibration tools, 1354 chemical binding, 1350 cockroach allergen sensitization studies, 1132 Immulite technology, 1350, 1353 immunotherapy monitoring, 1359 inhibition, 933 and immunoglobulin E cross-reactivity, 1354–6 ligand binding, 1350 multiallergen, 1350–1 results, interpretation, 1356–7 reversed, 1350 reversed immunoglobulin G, 1359 sensitivity, 1353–4 solid phase, 1349 standardization issues, 986–7 use of term, 1349 variable incubation times, 1352 variants, 1350 radiographic contrast media (RCM), sensitivity to, 1913 radiolabeled proteins, 436–7 radiology, allergic bronchopulmonary aspergillosis, 1749–51 radon, 1285 RADS (reactive airways disease syndrome), 1101, 1691, 1696 RAG1 gene, 119 RAG2 gene, 119 RAG proteins, 125, 126 gene expression, 132 ragweed allergy, 1561–2 ragweed immunotherapy, 1513 ragweed pollen, 958 distribution, 959 seasons, 958 ragweed pollen antigens, 1720 rain, effects on fungal spores, 966, 967 Ramazzini, Bernadino (1633–1714), 10, 11, 979 Ramirez, M. A., 6 Ran, 205 randomized controlled trials, sublingual immunotherapy, 1547 ranitidine, in anaphylaxis treatment, 1915

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RANTES see regulated upon activation, normal T-cell expressed, and secreted (RANTES) rapamycin see sirolimus rapidly adapting receptors (RARs), 826–7, 828 RARs (rapidly adapting receptors), 826–7, 828 Ras, 205 rAsp f 4, 1749 rAsp f 6, 1749 RAST see radioallergosorbent test (RAST) Rat n 1, 1137 Raw see airways resistance (Raw) rBet v 1, 901 rBla g 1, 1136, 1556 rBla g 2, 1136 structure, 1138 rBla g 4, 1136 rBla g 5, 1136 rBla g 7, 1136 RBM see reticular basement membrane (RBM) RCBTB1 gene, 1229 RCM (radiographic contrast media), sensitivity to, 1913 reactive airways disease syndrome (RADS), 1101, 1691, 1696 reactive nitrogen species (RNS), 376– 7 reactive oxygen species (ROS), 146–7, 376, 868 reactive upper airways dysfunction syndrome (RUDS), 1102 reagin, 1346 early studies, 6–7 receptor editing (RE), 125– 6 receptor expression, and immunoglobulin E production, 150–1 receptor-operated channels (ROCs), 862 receptor revision (RR), 125– 6 receptors activating, 328 allergic rhinitis, 1418–20 promiscuous, 602 see also adhesion receptors; adrenergic receptors; chemokine receptors; histamine receptors; muscarinic receptors; pattern-recognition receptors (PRRs); Toll-like receptors (TLRs) receptor tyrosine kinases (RTKs), 205 inhibitors, 1713 recombinant allergens for allergen-specific immunotherapy, 1555– 6 biochemistry, 898– 9 clinical use, 905– 7, 1136 future trends, 937 impacts, 900 production, 899 reactivity, 903–4 see also hypoallergenic recombinant allergens recombinant DNA technology, 94–5 refractoriness, 811–12 identification, 817 refractory asthma, 1751 as distinct inflammatory phenotype, 1621–2 management, 1656–7 see also severe refractory asthma (SRA) regional differences asthma prevalence, 992–3 cockroach allergens, 1134 dust mite sensitivity, 987, 992

regulated upon activation, normal T-cell expressed, and secreted (RANTES), 146, 147, 153, 154, 573, 580, 1413–14, 1418, 1460 increase, 1303 levels, 530 and nasal polyposis, 1469 release, 260, 262 synthesis, 274–5 regulatory T cells (Tregs), 24–5, 26–8, 50, 58–9, 1220, 1721–2 adaptive, 59 in allergen-specific immunotherapy, 90–6 and allergic inflammation, 1296 in allergy, 83–102 antigen-specific, 87–90 in asthma, 83–102 and chemokines, 482 generation, 89 identification, 1291 in immunotherapy, 93–4 and immunotherapy, 1515–17 interleukin-10-secreting, 88–9 lung dendritic cell function control, 176 maturation, 26–7 naturally occurring, 85–7 therapeutic applications, 87, 89–90 roles, 59, 1722 suppression, 83, 84, 84, 86–7 loss of, 87 therapeutic applications, 89 Reisseisen, Franz (1773–1828), 874 Reisseisen’s muscles, 874 remodeling in allergic diseases, 1308–9 neural, 1620 tissue, 398 vascular, 1620 see also airway remodeling repetitive airway exposure, cold air, 802–3 replication protein A (RPA), 126 RE (receptor editing), 125–6 RER (rough endoplasmic reticulum), 221, 222–4 residential environments, fungal spores, 971–2, 979 residual oil fly ash (ROFA), 144–5 residual volume (RV), 751, 753, 754–5, 756, 763 resistance nasal, 1433 steroid, 726–7 see also airways resistance (Raw); corticosteroid resistance; glucocorticoid resistance respiration mechanisms, 1457–8 protection, 1457–8 respiratory allergens, 902–3 fungi, 965 respiratory allergic diseases and air pollution, 1266–7 diagnosis, skin tests, 1335–45 epidemiology, 1266–7 immunoglobulin-E-mediated, 1335 management, skin tests, 1335–45 respiratory diseases, and diesel exhaust, 145 respiratory food allergens, 1155

respiratory food hypersensitivity reactions, 1930 respiratory function tests see lung function tests respiratory infections and asthma, 1572–3 see also lower respiratory tract infections (LRTIs) respiratory irritants, 1102 respiratory sensitizers, 1102 respiratory sensory innervation species differences, 513 see also human respiratory sensory innervation respiratory syncytial virus (RSV), 26, 234, 1571 infection, 31, 33, 153, 235, 1283 eosinophils in, 274–6 roles, 152 respiratory syncytial virus (RSV) bronchiolitis, 1594 respiratory tract structure, species differences, 1215–19 viral infections, infants, 2007 respiratory tract mucosa, 136 restriction fragment length polymorphism (RFLP), 1885 retargeted clostridial endopeptidases, 850–1 reticular basement membrane (RBM) in asthma, 1635–7 thickening, 1636, 1643–4 retinoic acid receptor antagonists, 851 reverse-transcriptase polymerase chain reaction (RT-PCR), 1291–2 RFLP (restriction fragment length polymorphism), 1885 rhesus monkey see Macaca mulatta (rhesus monkey) rHev b 1, 1167 rHev b 2, 1167 rHev b 5, 1168, 1174 rHev b 11, 1170 rHev b 12, 1170 rhinitis and antigen presentation, 1407–9 aspirin-sensitive, 513 and asthma, 1573 atrophic, 1391–2 and Churg–Strauss syndrome, 1786 classification, 1383, 1402–3 comorbidities, 1383 definition, 1240, 1383 differential diagnosis, 1383 elderly, 1391 and emotions, 1391 eosinophilic, 1391 etiology, 1324–5, 1389–92 experimental allergen-induced, 243 fall, 1323 food-induced, 1391 gustatory, 1391, 1921 hormonal, 1391 hyperreflectoric, 513 and immunoglobulin E, 1407– 9 management, in developing countries, 1445 nerves in, 834 prevalence, 1244 respiratory tract mucosa, 136 seasonal, 1402–3 severity assessment, 1433 in smokers, 1391

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symptoms, 1383, 1402 toxic, 513 treatment, 1433– 41 guidelines, 1441– 2 omalizumab in, 1675 see also allergic rhinitis (AR); drug-induced rhinitis; idiopathic rhinitis; intermittent rhinitis; nonallergic rhinitis with eosinophilia syndrome (NARES); occupational rhinitis; perennial rhinitis; persistent rhinitis rhinitis medicamentosa, use of term, 1390 rhinoconjunctivitis food-induced, 1930 see also allergic rhinoconjunctivitis Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ), 1441 rhinophototherapy, 1441 rhinorrhea, 1391, 1466–7 rhinosinusitis, 1392–3, 1454– 81 acute nonviral (bacterial) rhinosinusitis, 1393 acute viral, 1393 with allergic rhinitis, 1387– 8 anatomic issues, 1456–7 aspirin-sensitive eosinophilic, 1973 classification, 1454–5 definitions, 1454– 5 clinical, 1392 epidemiologic, 1393 research, 1393 duration, 1392 epidemiology, 1387– 8 etiology, 1389 future research, 1475 immunomodulatory therapy, 1475 physiologic issues, 1457– 9 severity, 1392 use of term, 1383, 1389, 1454 see also acute rhinosinusitis (ARS); chronic rhinosinusitis (CRS); nasal polyps rhinovirus and dust mite exposure, 991–2 and pediatric asthma, 1596 see also human rhinoviruses (HRVs) RHo, 205 RhoA, 880 activation, 879 Rhodotorula rubra (red yeast), 1761 RHo kinase (ROK), 864, 865 Rho subfamily, and endothelial contractility, 863– 4 Richet, Charles Robert (1850–1935), 3– 4, 6, 1897 Rienhoff, W. F., 831 RIG-1, 379– 80 Riley, James F. (1912– 85), 8, 9 rimiterol, 673 administration, 677 rituximab, 742, 1787 rMal d 1, 1156 RNA chain termination, 134 double-stranded, 191 RNAi (interference RNA), 1725– 6 RNA probes, single-stranded, 1291 εRNA transcripts, expression, 1293

RNA viruses, 379–80 RNS (reactive nitrogen species), 376–7 Ro 23-1392, 519 Ro 23-1553, 519 Rocha e Silva, Mauricio Oscar da (1910–83), 9 ROCs (receptor-operated channels), 862 ROFA (residual oil fly ash), 144–5 roflummilast, 1712–13, 1724 adverse effects, 1724 structure, 644 ROK (RHo kinase), 864, 865 Rosaceae (rose family), proteins, 1150 ROS (reactive oxygen species), 146–7, 376, 868 Rotorod system, 946 rough endoplasmic reticulum (RER), 221, 222–4 RPA (replication protein A), 126 rPer a 7, 1136 RQLQ (Rhinoconjunctivitis Quality of Life Questionnaire), 1441 RR (receptor revision), 125–6 RSV see respiratory syncytial virus (RSV) RSV (respiratory syncytial virus) bronchiolitis, 1594 RTKs see receptor tyrosine kinases (RTKs) RT-PCR (reverse-transcriptase polymerase chain reaction), 1291–2 rubber extraction, 1166 see also latex rubber elongation factor, 1167 rubber particles, 1167 rubber products, manufacture, 1166–7 RUDS (reactive upper airways dysfunction syndrome), 1102 Rumex spp. (docks and sorrels), 958–60 Runx3, 480 rupatadine, 555 rural areas allergic diseases, 2021–2 outdoor air pollution, 1266 rush immunotherapy, 1528 RV (residual volume), 751, 753, 754–5, 756, 763 ryanodine receptor (RyR), 879 RyR (ryanodine receptor), 879 S1P (sphongosine 1-phosphate), 866 S100A12 gene, expression, 1229 S100 proteins, 1232 encoding, 1615 SAA (serum amyloid A), 376 SABAs see short-acting β2 agonists (SABAs) SAC see seasonal allergic conjunctivitis (SAC) Saccharomyces cerevisiae (baker’s yeast), 1061 SAEs see Staphylococcus aureus enterotoxins (SAEs) salbutamol, 405, 672 administration, 677 in anaphylaxis treatment, 1915 limitations, 673 structure, 673 saline, in exercise-induced bronchoconstriction, 814–15 saline douches, 1441 salmeterol, 674, 1717 in exercise-induced bronchoconstriction, 816 structure, 673

Salmeterol Multicenter Asthma Research Trial (SMART), 1714 –15 Salmonella spp. (enterobacteria), 311 Salmonella minnesota (enterobacterium), lipopolysaccharides, 1518 Sal s 1, 1152 Salsola kali (Russian thistle), 957 Salsola pestifera, 957 Salter, Henry Hyde (1823–71), 10–11, 12, 668, 831 Samter’s triad, 1326 Samuelsson, Bengt I. (1934– ), 9, 10, 567 sandwich enzyme-linked immunosorbent assay, 932–3 SAPALDIA (Swiss Study on Air Pollution and Lung Diseases), 1385 sarcoidosis, 1795 SARs (slowly adapting receptors), 826–7, 828 SB see spina bifida (SB) scallops, allergens, 1155 SCAR-POL community study, 1407–8 sCD14 (soluble CD14), 38, 39–40 sCD23 see soluble CD23 (sCD23) SCF see stem cell factor (SCF) SCG see sodium cromoglycate (SCG) SCHER (Scientific Committee on Health and Environmental Risks), 1280 Schick, Béla (1877–1967), 4, 5, 436, 439 Schild, H. O., 8–9 Schistosoma haematobium (trematode), 154, 1247, 1791 Schistosoma japonicum (trematode), 154 Schistosoma mansoni (trematode), 154, 273, 1791 school performance, and allergic rhinitis, 1386 SCID (severe combined immunodeficiency), 206, 480 Scientific Committee on Health and Environmental Risks (SCHER), 1280 SCIT see subcutaneous immunotherapy (SCIT) scombroid fish poisoning, 1921 scopolamine, 685 Scott, W. J. M., 831 SDAP (Structural Database of Allergenic Proteins), 902, 1158 SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), 933, 934, 1152 seafood, allergens, 1155 seasonal allergic conjunctivitis (SAC), 243–4, 1482, 1487–90 definition, 1387 immunoglobulins in, 1488 immunotherapy, 1490 management, 1489 mediators, 1488–9 pathology, 1488 pathophysiology, 1487 treatment, 1489–90 seasonal rhinitis, use of term, 1402–3 Secale cereale (rye), allergens, 935 secretory leukocyte proteinase inhibitor (SLPI), 373–4, 1231 secretory phospholipase A2 (sPLA2), 568–9 isoforms, 568 Sedormid, 14

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SEER (Surveillance, Epidemiology, and End Results) database, 1677 E-selectin, 338, 339– 40, 1413 L-selectin, 338, 339– 40 P-selectin, 338 antagonists, 353 P-selectin glycoprotein ligand (PSGL-1), 339– 40 expression, 349 selectin antagonists, 1723 selectin counterstructures function, 339– 40 structure, 339– 40 selectins function, 338– 9 structure, 338– 9 seminal plasma, and anaphylaxis, 1906 sensitivity, definition, 1999 sensitization (allergic) see allergic sensitization sensory-efferent nerves, inhibition, 849 sensory innervation, 512–13 lower airways, 826–7 upper airways, 823– 4 see also respiratory sensory innervation sensory nerve activation, inhibition, 849 sensory nervous system in allergic rhinitis, 823–39 in asthma, 823–39 sensory neuropeptides, 825 Serhan, Charles N., 567 serotherapy, and immunity, 4 serous acini, loss of, 1637 serous cells differentiation, 1193 species differences, 1192 Serpula lacrymans (dry-rot fungus), 971 serum amyloid A (SAA), 376 serum immunoglobulin E (sIgE) sensitivity, 1987 specificity, 1987 venom-specific, 1987 serum sickness, 4, 1910 early studies, 436–7 studies, 438– 9 use of term, 436 serum transfer, early studies, 1346 serum tryptase, 1988 measurement, 1886–7 sesame seeds, allergens, 1152–3 sesquiterpene lactones, 1841 SES (socioeconomic status), and asthma, 1244 SETDB1 gene, 1229 severe combined immunodeficiency (SCID), 206, 480 severe drug hypersensitivity syndromes, 1958– 9 severe refractory asthma (SRA), 1575 use of term, 1575 Sgp200, 339 SH2-containing inositol phosphatase (SHIP), 439 β-sheet structure, allergens, 903 shellfish allergens, 1151–2 allergic reactions, 1147 SHIP (SH2-containing inositol phosphatase), 439 SHM (somatic hypermutation), 119, 120, 121, 122, 126–7

short-acting β2 agonists (SABAs) in chronic asthma treatment, 1651, 1654–5 in exercise-induced bronchoconstriction, 816 short-term exposure limit (STEL), 1102 shrimp, allergens, 1151, 1155 sialomucins, 339 sibling effect, 32 sick building syndrome, and extrinsic allergic alveolitis, 1771 SIDS (sudden infant death syndrome), 1905 sIgE see serum immunoglobulin E (sIgE) signal amplification, 207–8 signal generation, 203–7 signaling, G protein-coupled, 204–5 signaling integration, 208–10 signaling molecules, 497 signaling research, 204 signal termination, 210 signal transducers and activators of transcription (STATs), 209, 210, 211 signal transduction in allergic cells, 203–13 in eosinophils, 210–11 in inflammatory cells, 203–13 inhibition, 328 in mast cells, 211 single gene disorders, 1228 single nucleotide polymorphisms (SNPs), 672, 1226 microarrays, 1292 single photon emission computed tomography (SPECT), 749 airway visualization, 758 sinuses anatomy, 1456–7 CT images, 1458 see also paranasal sinuses sinusitis and asthma, 1472, 1573 and chronic obstructive pulmonary disease, 1472 subgroups, 1455 use of term, 1383 see also rhinosinusitis sirolimus, 73 adverse effects, 741 discovery, 740 drug interactions, 741 indications, 741 mechanisms, 741 monitoring methods, 741 pharmacogenetics, 740–1 pharmacokinetics, 740 structure, 740 sirolimus inhibitors, mammalian targets, 740–1 SIT see allergen-specific immunotherapy (SIT) Sitophilus oryzae (weevil), 1101 SJS (Stevens–Johnson syndrome), 1953, 1957–8 skin allergic responses, 1512 and allergy, 1326–7, 1811–93 and immune development, 30 mastocytosis, 1881–3 neural inflammation, 531 repetitive injury, 1837–8

T-cell-mediated delayed drug hypersensitivity reactions, 1953–4 skin barrier dysfunction, and atopic dermatitis, 1822 skin biopsies, mastocytosis, 1884 skin disorders antihistamine therapy, 559 see also eczema; psoriasis skin irritation, fungi, 965 skin-pricking tests (SPTs), 1170, 1175–6, 1239, 1322, 1349, 1385 atopy, 2023–4 common errors in, 1336 comparisons, 1337 food allergy, 1327–8, 1931–2 and immunoglobulin E levels, 2024–5 methods, 1335–6 in ocular allergy, 1503 precautions, 1583 rural vs. urban areas, 2021–2 skin tests, 915, 1323, 1326 and age, 1339 allergen extracts, 1339 allergic asthma, 1582–3 body areas, 1339 cockroach allergen sensitization studies, 1132 correlation, 1342 diagnostic value, 1342 and drugs, 1340 factors affecting, 1339–40 false-negative, 1340 false-positive, 1340 frequency of, 1339 and gender, 1339 grading, 1337–9 systems, 1338–9 historical background, 1335 house dust, 984 insect sting allergy, 1987 interpretation, 1340–2 measurement, 1337–8 methods, 1335–7 negative control solutions, 1337 number of, 1339 and pathologic conditions, 1339–40 positive, 1340–2 positive control solutions, 1337 positivity criteria, 1338 precautions, 1336 and race, 1339 in respiratory allergic diseases, 1335–45 seasonal variations, 1339 sensitivity, 1987 specificity, 1987 techniques, 1335–9 treatment inhibitory effects, 1341 see also intradermal tests; patch tests; skin-pricking tests (SPTs) SLAM, 54 SLC9A3R1 gene, 1229 SLC12A8 gene, 1229 SLC22A4 gene, 1229 sled dogs, as models, 795 sleep apnea, and allergic rhinitis, 1386 sleep disturbance, and allergic rhinitis, 1386

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SLE (systemic lupus erythrematosus), 437, 439, 440–1 sliding filament hypothesis, 879 SLIT see sublingual immunotherapy (SLIT) slowly adapting receptors (SARs), 826–7, 828 slow-reacting substance of anaphylaxis (SRS-A), 9 early studies, 567 SLP65 (B-cell linker protein), 120, 207 SLPI (secretory leukocyte proteinase inhibitor), 373– 4, 1231 SM see systemic mastocytosis (SM) small airways, in asthma, 757– 8 small animal models airway remodeling in, 1202–13 transgenic, 1208–10 see also mouse models small animals, pulmonary function tests, 1219 small proline-rich proteins (SPRRs), 1228– 9 small rubber particle protein, 1167– 8 SMART (Salmeterol Multicenter Asthma Research Trial), 1714–15 SMART (Symbicort as Maintenance and Reliever Therapy), 1713 SMILE trial, 1713 smogs, 1279 smokers, rhinitis in, 1391 smoking, 1249–50 and asthma, 1574–5, 1657– 8 and chronic obstructive pulmonary disease, 1574–5 and extrinsic allergic alveolitis, 1764 mothers, infants and, 1571 passive, 1249–50 personal, 1249 and pregnancy, 1330, 2001 see also tobacco smoke smoldering mastocytosis, use of term, 1880 SMX-NHOH (sulfamethoxazolehydroxylamine), 1946 SMX-NO (sulfamethoxazolenitroso), 1946 SMX (sulfamethoxazole), 1946 SNARE see soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) SNPs see single nucleotide polymorphisms (SNPs) socioeconomic status (SES), and asthma, 1244 SOCS1, 60 SOCS3, 61 SOCS5, 60–1 SOCS/CIS/JAB gene family, 60–1 SOCS gene, 60–1 SOCS (suppressor of cytokine signaling), 60–1, 210 SOD (superoxide dismutase), 376 sodium cromoglycate (SCG) in asthma treatment, 1332 in chronic asthma treatment, 1653 in exercise-induced bronchoconstriction, 816 in giant papillary conjunctivitis treatment, 1502 in hay fever treatment, 1331 in seasonal allergic conjunctivitis treatment, 1489 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 933, 934, 1152

sodium lauryl sulfate, 1835 sodium metabisulfite, 833 solar urticaria, 1863 Solenopsis spp. (fire ants), stings, 1984 Solenopsis invicta (red imported fire ant), 1983 local toxicity, 1983 venom, 1125 Solenopsis richteri (black imported fire ant), 1983 Sol i 1, 1125 Sol i 2, 1125 Sol i 3, 1125 Sol i 4, 1125 Solidago spp. (goldenrods), pollen, 943 Solis-Cohen, Solomon (1886–1952), 668–9 solitary mastocytoma of skin, 1880 soluble adhesion molecules, 346 soluble CD14 (sCD14), 38, 39–40 soluble CD23 (sCD23), 103, 109, 111 integrin-mediated functions, 112 roles, 104 soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) expression, 306 isoforms, eosinophil expression, 262–4 proteins, 850 somatic hypermutation (SHM), 119, 120, 121, 122, 126–7 somatic recombination, epigenetic control, 129–31 sotalol, 679 Southern California Children’s Health Study, 1250 soybean dust, and asthma, 1272–3 soybeans allergens, 1148, 1151 allergic reactions, 1147 β-conglycinin, 1149 spacers, 719 advantages, 723 and pressurized metered-dose inhalers, 776–7 SPACE (Study of Prevention of Allergy in Children of Europe), 2004 spaglumic acid, 1496 species differences bronchial arteries, 1217–18 respiratory tract, 1215–19 tracheobronchial airways, 1188–92 specific immunotherapy (SIT) see allergen-specific immunotherapy (SIT) SPECT see single photon emission computed tomography (SPECT) spermatophytes, 942–3 SPF rats, 1219 Sphingobacterium spiritivorum (bacterium), 1760 sphongosine 1-phosphate (S1P), 866 spices allergic reactions to, 1926 use of term, 1926 Spieksma, Frits Th. M. (1936– ), 984 spina bifida (SB), and latex allergy, 1164, 1165, 1168 SPINK5 gene, 1229 expression, 1231 mutations, 1234 polymorphisms, 1228

Spiriva see tiotropium bromide spirometry asthma, 1579–80 in chronic asthma management, 1654 see also lung function tests sPLA2 see secretory phospholipase A2 (sPLA2) SPM (suspended particulate matter), 945 Sporobolomyces spp. (fungi), seasons, 966 Sprague–Dawley rats, 1189–90, 1219 SPRR2C gene, expression, 1229 SPRR gene, 1228 SPRRs (small proline-rich proteins), 1228–9 SPTs see skin-pricking tests (SPTs) sputum biomarkers in, 1716–17 eosinophil count, 787, 1600, 1716–17 in chronic obstructive pulmonary disease, 1377 processing, 1369 see also induced sputum sputum induction methodology, 1368–9 protocols, 1369, 1370 see also induced sputum sputum neutrophil counts, reduction, 1371 sputum plugs, 1369 SRA see severe refractory asthma (SRA) Src family kinases, activation mechanisms, 206–7 SRS-A see slow-reacting substance of anaphylaxis (SRS-A) Stachybotrys spp. (fungi), occurrence, 978 Stachybotrys chartarum (fungus), 1282 occurrence, 971 standards, natural vs. recombinant, 935 Staphylococcus aureus (bacterium), 1463 in atopic dermatitis, 1819, 1824 in chronic rhinosinusitis, 1454–5 colonization, 1232–3 in nasal mucosa, 136 protein A, 326, 327 Staphylococcus aureus enterotoxins (SAEs) immune responses, 1465 production, 1462 roles, 1464–5 Starling, Ernest (1866–1927), 857 Starling’s principle, 857–8 START (Steroid Treatment as Regular Therapy) trial, 1602 STAT1, 52 STAT3, 133, 884–5 STAT4, 52, 60 activation, 61 STAT5, 133 STAT6, 60–1, 132, 133 in allergic diseases, 1306–8 STAT proteins, 60 STATs (signal transducers and activators of transcription), 209, 210, 211 STAY trial, 1713 STEAM trial, 1713 STEL (short-term exposure limit), 1102 stem cell factor (SCF), 218–19, 500, 1410, 1411–12, 1879 regulatory mechanisms, 1725

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stem cells, 1726–7 and angioblasts, 404 see also hemopoietic stem cells (HSCs); mesenchymal stem cells STEP trial, 1713 stepwise pharmacologic therapy, chronic asthma, 1654– 5 steroid phobia, 1524 steroid resistance, molecular mechanisms, 726–7 steroid-sparing strategies in chronic asthma treatment, 1653 effects, 1675 Steroid Treatment as Regular Therapy (START) trial, 1602 Stevens–Johnson syndrome (SJS), 1953, 1957– 8 sting challenge tests, 1988 stinging insect allergy see insect sting allergy stinging insect venom allergens, 1123– 30 antigenic cross-reactivity, 1126 Stipa tenacissima (esparto grass), 1761 Strachan, David P., 2026 Streptococcus pneumoniae (bacterium), 1233, 1470 Streptomyces argenteolus (bacterium), KS505a, 638 stress, and atopic dermatitis, 1819 Strongyloides spp. (trematodes), 273 Strongyloides stercoralis (trematode), 154, 2027 infections, 1585, 1790–1 Strongyloides venezuelensis (trematode), 2027 strongyloidiasis etiology, 1790 severe disseminated, 1791 symptoms, 1790–1 structural cells, in allergic rhinitis, 1413–17 Structural Database of Allergenic Proteins (SDAP), 902, 1158 structural tissue cells, and neurotrophins, 501– 2 Study of Prevention of Allergy in Children of Europe (SPACE), 2004 subcutaneous immunotherapy (SCIT) adverse reactions, 1550 algorithm, 1538 allergic rhinitis, 1439– 40, 1526 costs, 1549 development, 1544 dose guidelines induction phase, 1531 maintenance phase, 1531 future trends, 1550 indications, 1440 latex allergy, 1176– 7 mechanisms, 1549–50 and quality of life, 1549 studies, 1177, 1545–7 and sublingual immunotherapy compared, 1547 subepithelial fibrosis, mast cells and, 240 subepithelial matrix deposition, in asthma, 1635–7 sublingual grass pollen immunotherapy, 1712, 1720 sublingual immunotherapy (SLIT), 930, 931, 1543–54 additional effects, 1548 administration modalities, 1548

adverse effects, 1548 allergic rhinitis, 1440 applications, 2011 in atopic dermatitis treatment, 1827 clinical efficacy, 1545–7 clinical safety, 1547–8 postmarketing surveys, 1547–8 randomized controlled trials, 1547 contraindications, 1550–1 developments, 1333 dosage, 1548 efficacy studies, 1543 future research, 2012 historical background, 1543–4 indications, 1545, 1550 latex allergy, 1176–7 mechanisms, 94 patient compliance, 1549 patient issues, 1549 pharmacokinetics, 1549–50 phases, 1544–5 practical issues, 1544–5 studies, 1177 and subcutaneous immunotherapy compared, 1547 sublingual-swallow immunotherapy see sublingual immunotherapy (SLIT) sublobar airway models, 794–5 submucosal swelling, and airway function, 401 substance P, 511, 514–15, 530–1, 788, 825, 849 in airway nerves, 513–14 localization, 1406 roles, 495, 508 suction trapping, 946 sudden infant death syndrome (SIDS), 1905 suicide inhibitor, 444–5 sulfamethoxazole (SMX), 1946 sulfamethoxazolehydroxylamine (SMX-NHOH), 1946 sulfamethoxazolenitroso (SMX-NO), 1946 sulfinpyrazone, 1975 sulfites, 1926 sensitivity, 1907 sulfur dioxide, 146, 148, 1285 sources, 1269 urban areas, 1269 superallergens in allergic diseases, 326–7 bacterial immunoglobulin, 327 endogenous, 326–7 viral immunoglobulin, 326–7 superoxide dismutase (SOD), 376 suppressor of cytokine signaling (SOCS), 60–1, 210 surface markers, basophils, 323–5 surgery acute rhinosinusitis, 1474 chronic asthma, 1658 functional endoscopic sinus, 1468 Surveillance, Epidemiology, and End Results (SEER) database, 1677 susceptibility genes asthma, 1229, 1230, 1623, 1624 concept of, 148 identification, 1225–6

inflammatory bowel disease, 1229 psoriasis, 1229 suspended particulate matter (SPM), 945 Swiss-Prot database, 204 Swiss Study on Air Pollution and Lung Diseases (SAPALDIA), 1385 Swiss Webster mice, 1189–90, 1191 Symbicort as Maintenance and Reliever Therapy (SMART), 1713 sympathetic innervation, 512 lower airways, 827 upper airways, 824–5 see also parasympathetic innervation sympathetic nerves, stimulation, 825 Syringa vulgaris (lilac), 957 systemic cell signaling, in allergic rhinitis, 1420–1 systemic corticosteroids in chronic asthma treatment, 1653 in hay fever treatment, 1331 limitations, 1331 systemic desensitization, 1543 systemic drug reactions, 1958–9 systemic glucocorticoids, 719, 725–6 in acute severe asthma, 726 in allergic rhinitis treatment, 1439 maintenance treatment, 719, 725–6 short courses, 725 systemic lupus erythrematosus (SLE), 437, 439, 440–1 systemic mastocytosis (SM), 1880, 1911 and cytopenias, 1883 diagnostic criteria, 1885–7 and hepatomegaly, 1883 markers, 1887 treatment, 1890 see also indolent systemic mastocytosis (ISM) Tabanus spp. (horseflies), 1992 TACE (tumor necrosis factor-α-converting enzyme), 74 tachykinin antagonists, 516 clinical applications, 516 tachykinin ligands, 514 tachykinin receptor antagonists, 849 tachykinin receptors, 514 tachykinins, 495, 508, 511, 513, 514–16, 788 asthma mediation, 790 binding, 515 localization, 1406 metabolism, 515–16 pulmonary effects, 515 release, 799 TACI (transmembrane activator and calciummodulator and cytophilin ligand interactor), 128 tacrolimus, 329, 732, 739–40 adverse effects, 740 in atopic dermatitis treatment, 1825 drug interactions, 740 indications, 740 monitoring methods, 740 pharmacogenetics, 739–40 pharmacokinetics, 739 structure, 739

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TAFI (thrombin activatable fibrinolysis inhibitor), 462 TANK-binding kinase 1 (TBK1), 380 TAP (transporter associated with antigen processing), 169 TARC (thymus activation-regulated chemokine), 29, 71, 178, 530, 537, 920, 1301, 1413 targeting-by-timing hypothesis, 297 tartrazine, 1328 TBC1269, 353 T-bet, in allergic diseases, 1308 TBM (tidal breathing mode), 778, 779 T-cell adhesion, 350–2 T cell costimulatory molecules, 53–5, 58 T-cell effector responses, 918–20 T-cell epitopes, 49, 920, 990, 1149–50 T-cell function, toll-like receptors in, 191 T-cell hypothesis of asthma, 64 issues, 72– 4 T-cell-mediated delayed drug hypersensitivity reactions, 1952–3 clinical features, 1954– 8 immunohistology, 1955– 6 mechanisms, 1952 pharmacogenetic associations, 1958 preferential skin involvement, 1953– 4 tolerance mechanisms, 1953 type IVa, 1952 type IVb, 1952 type IVc, 1953 type IVd, 1953 T-cell memory, 56– 8 allergen-specific, 25– 6 responses, 896 T-cell migration, 350–2 T-cell peptide epitopes in allergen-specific immunotherapy, 1559– 61 in bee venom allergy, 1560–1 in cat allergy, 1559– 60 T-cell receptor (TCR), 52, 87, 206 T cells, 19– 20, 25– 8 activated, 481–2 activation, by dendritic cells, 172–3 and airway hyperresponsiveness, 536–7 in airways, 70–1 in allergic diseases, 1295– 6 in allergic inflammation, 48– 82 in allergic rhinitis, 1412–13 antigen recognition, 49 antigen-specific, 1721–2 in asthma, 48– 82 in atopic dermatitis, 1823 cytotoxic, 20 dendritic cell regulation, in lung, 176 development, 48– 62 early, 49–50 differentiation, 50– 2, 990, 991 and fibrosis mediation, 420 immunotherapy effects on, 1510 interactions, 193 and late-phase allergic reactions, 528– 9, 536–7 maturation, 1721 naive, 480–1 peripheral tolerance, 91, 93

phenotypes, 49 properties, 48–62 recognition, 920–1 recruitment, by chemokines, 480–2 regulatory responses, 921–2 responses, to allergen injection immunotherapy, 1515–17 roles, 48 in asthma pathogenesis, 62–74 in pathogenesis, 732 see also natural killer T (NKT) cells; regulatory T cells (Tregs) γδ T cells, 55 in asthma, 71–2 TCR (T-cell receptor), 52, 87, 206 TDI see transition dyspnea index (TDI) TDI (toluene diisocyanate) asthma, 235–6 TEAM (Total Exposure and Assessment Methodology), 1284 tears, collection, 1503 telangiectasia macularis eruptiva perstans (TMEP), 1883 treatment, 1890 TEM see transendothelial migration (TEM) TEN (toxic epidermal necrolysis), 1953, 1957–8 tenascin, in extracellular matrix, 414–15 TENOR study, 1662 terbutaline, 672 administration, 677 limitations, 673 structure, 673 terfenadine, 554, 1437 terminal bronchioles, species differences, 1192 tertiary ammonium compounds, 684–5 test allergens, 1845 tetomilast, structure, 644 TGF-β see transforming growth factor-β (TGF-β) TGFβ gene, 1229 TGN 1412, 1715, 1958 Th1 cells see T helper 1 (Th1) cells Th1/Th2 paradigm, 25–6, 27 development, 61–2 Th2 cells see T helper 2 (Th2) cells Th3 cells see T helper 3 (Th3) cells Th17 cells see T helper 17 (Th17) cells thaumatin-like protein (TLP), 1153, 1355 The c 1, 1152 T helper 1 (Th1) cells, 19–20, 24–6, 529–30, 1295 characterization, 52 differentiation, 51 immune responses, 1822 immunoregulatory pathways, 2009 responses, 2027, 2028 early innate modulation, 1232 T helper 2 CD4+ responses, induction, 1745–6 T helper 2 (Th2) cells, 20, 24–6, 529–30, 538, 1295 in allergic sensitization, 166 in asthma, 62–3, 70–1, 1609–10 characterization, 52 differentiation, 51 and mast cells, 230 immune responses, 51, 1822 polarization, 136

responses, 2025, 2027, 2028 early innate modulation, 1232 responsiveness, 1147 T helper 3 (Th3) cells, 88 T helper 17 (Th17) cells, 20, 52–3 discovery, 53 theophylline, 634–67 adverse events, 652–4 in chronic asthma treatment, 1652 clinical pharmacology, 651 clinical use, 1652 drug metabolism, 652–4 glucocorticoid resistance reversal, 727 as nonselective phosphodiesterase inhibitor, 649–51 pharmacokinetics, 652–4 safety, 652–4 tolerability, 652–4 Thermoactinomyces sacchari (bacterium), 1758–60 Thermoactinomyces vulgaris (bacterium), 1760 thiazoles, 1166 thiopurine-S-methyltransferase (TPMT), 733–4 thioredoxins, 1154 Third World see developing countries thiurams, 1166 thrombin, fibrosis mediation, 423–4 thrombin activatable fibrinolysis inhibitor (TAFI), 462 thrombocytopenia, 1951 heparin-induced, 1951 and omalizumab, 1678 thrombocytopenic purpura, 14 thromboxane receptors, 591 thromboxanes, 566, 567, 588–600 biosynthesis, 588–91 cellular sources, 588–91 thrush (oropharyngeal candidiasis), 723, 776–7 Thuja orientalis (Chinese arborvitae), 956 thunderstorm asthma, 945 characteristics, 1272 during pollen season, 1271–2 thymic eosinophils, 266–7 thymic stromal lymphopoietin (TSLP), 51, 58, 171, 175, 177, 379 activity, 1721 allergen mediation, 57 in allergy treatment, 1712 in atopic dermatitis, 1823–4 expression, 50, 52, 61–2, 70, 136 production, 1617 thymus, development, 49 thymus activation-regulated chemokine (TARC), 29, 71, 178, 530, 537, 920, 1301, 1413 thyroid-stimulating hormone (TSH), 14 tidal breathing mode (TBM), 778, 779 tidal flow, 770 tidal inhalation mode (TIM), 778, 780 tight junctions (TJs), 859–60, 864 –5, 868 roles, 860 TIM-1, 60, 152 TIM-2, 60 TIM-3, 60 TIM-4, 60 TIM (tidal inhalation mode), 778, 780

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time-weighted average (TWA), 1102 TIM gene family, 60–1 TIM1 gene, 152 timolol, 679 TIMPs see tissue inhibitors of matrix metalloproteinases (TIMPs) tiotropium adverse effects, 689, 690 in combination drugs, 686 development, 683 dissociation half-life, 685 half-life, 686–7 studies, 687– 9 tiotropium bromide, 685, 1717 structure, 685 tissue eosinophil migration in, 348– 9 recruitment, in allergic rhinitis, 1413–17 tissue damage, in allergic bronchopulmonary aspergillosis, 1746 tissue inhibitors of matrix metalloproteinases (TIMPs), 387, 505 TIMP-1, 1595– 6 tissue kallikrein, 451 tissue remodeling, in asthma, 398 TJs see tight junctions (TJs) TLC (total lung capacity), 750, 751, 753– 4, 755, 756 TLP (thaumatin-like protein), 1153, 1355 TLR2 gene, 150, 1234 polymorphisms, 151 TLR4 gene, 148, 150 expression, 1230 polymorphisms, 151 TLRs see Toll-like receptors (TLRs) T lymphocytes see T cells TMEP see telangiectasia macularis eruptiva perstans (TMEP) TNFA gene, 1229 TNF-α see tumor necrosis factor-α (TNF-α) tobacco smoke allergic responses primary, 148– 9 secondary, 148– 9 and asthma, 148, 1571, 1621 and atopic diseases, 1263 constituents, 148, 1281 drug-metabolizing enzyme induction, 652 effects, animal models, 148– 9 and immunoglobulin E production, 148– 9 genetics, 149 and rhinitis, 1391 see also environmental tobacco smoke (ETS) tobramycin, 1463 tofimilast, structure, 644 tolerogenic mechanisms in allergy, 83–102 in asthma, 83–102 Toll-like receptors (TLRs), 24–5, 86, 143, 150, 170, 174, 266 activity, 1231 in airway epithelium, 377– 8 binding, 986 expression, 191, 192, 193, 1230 initiation, 1617

in innate immune system, 187, 189–91 ligands, 25, 1561 localization, 2006 pathogen recognition, 303 polymorphisms, 40 roles, 191, 1721 signaling, 191, 193, 197 structure, 377–9 types of, 189–90 toluene diisocyanate (TDI), and occupational asthma, 1692, 1694, 1701 toluene diisocyanate (TDI) asthma, 235–6 topical antimicrobial therapy, in atopic dermatitis treatment, 1826 topical calcineurin inhibitors, in atopic dermatitis treatment, 1825 topical corticosteroids adverse effects, 1496–7 in atopic keratoconjunctivitis treatment, 1500 topical glucocorticoids, in atopic dermatitis treatment, 1825 Total Exposure and Assessment Methodology (TEAM), 1284 total lung capacity (TLC), 750, 751, 753–4, 755, 756 total volatile organic compounds (TVOCs), 1284 toxic agents, eosinophilic pneumonia induction, 1794 toxic complex syndrome, 18 toxic epidermal necrolysis (TEN), 1953, 1957–8 toxic reactions, 1921 toxic rhinitis, 513 Toxocara canis (dog roundworm), 1790 toxocariasis, 1585 definition, 1790 eosinophilic pneumonias induction, 1790 Toxoplasma gondii (parasitic protozoon), 1245, 2026 infection, 1822 TPMT (thiopurine-S-methyltransferase), 733–4 trachea, in asthma, 762–3 tracheobronchial airways adults, species differences, 1188–92 architecture, 1189–90 cellular composition, 1190–2 TRAFs (tumor necrosis factor-associated factors), 133 TRAIL (tumor necrosis factor-related apoptosis inducing ligand), 218 transcriptional enhancers, 123–4 transcription factors, 121, 129, 131, 132, 133, 134 in allergic diseases, 1306–8 inhibition, 1724–5 transendothelial migration (TEM), 337 mechanisms, 338 transforming growth factor-β (TGF-β), 26, 50, 530, 876, 1209, 1415–16 and asthma, 88, 1636–7 fibrosis mediation, 422 immunologic properties, 85 isoforms, 69–70 in nasal polyposis, 1461 production, 89–90 transgenic mice, CysLT2, 580 transgenic models, 1208–10

transition dyspnea index (TDI), 687 translational medicine, 1715–17 definition, 1715 transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI), 128 transporter associated with antigen processing (TAP), 169 tree nuts allergens, 1148, 1152 allergic reactions, 1147 tree pollen, 953–7 seasons, 944 see also alder pollen; birch pollen; chestnut pollen; cypress pollen; olive pollen Tregs see regulatory T cells (Tregs) T regulatory cells see regulatory T cells (Tregs) tremor, and β-adrenoceptor agonists, 678 Trethewie, Everton R. (1913–84), 9, 10, 567 TRFK-5, 1421 triamcinolone acetonide, 725–6 structure, 718 Triatoma protracta (kissing bug), 1992 Trichinella spiralis (nematode), 483, 2027 trichinellosis, 1585 Trichophyton rubrum (fungus), 921 Trichosporon spp. (fungi), 1761 Trichuris spp. (whipworms), infections, 2026 triprolidine, 554 Tri r 2, 921 TrkA, 499 expression, 498, 501 TrkB, 497 expression, 499, 501 Trk (tropomyosin-related tyrosine kinase) receptors, 496–7 tropane, 685 tropical eosinophilia, 1789–90 diagnosis, 1789–90 etiology, 1789 stages, 1789 symptoms, 1789 treatment, 1790 tropomyosin-related tyrosine kinase (Trk) receptors, 496–7 tropomyosins, 1131, 1132, 1138, 1152 homologs, 1137 troponin C, 1137 troponins, 1131, 1132 TRPV1, 506 TRPV1 antagonists, 520 TR receptors, 593–4 trypsin, 9 tryptase, 529 and exercise-induced bronchoconstriction, 810–11 α-tryptase, 244, 1908, 1988 β-tryptase, 244, 330, 1908, 1910, 1988 TSH (thyroid-stimulating hormone), 14 TSLC-1 (tumor suppressor in lung cancer-1), 882 TSLP see thymic stromal lymphopoietin (TSLP) tubal dysfunction, with allergic rhinitis, 1389 tuberculin sensitivity and asthma, 1264 and hygiene hypothesis, 1246–7 tuberculosis, 1283

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Tucson Children’s Respiratory Study, 1003 tumor necrosis factor-associated factors (TRAFs), 133 tumor necrosis factor-related apoptosis inducing ligand (TRAIL), 218 tumor necrosis factor-α (TNF-α), 70, 196– 7, 218, 850, 1416 attenuation, 373 blockade, 73 expression, 1622 mast cell-derived, in asthma, 242 roles, 1622 tumor necrosis factor-α-converting enzyme (TACE), 74 tumor necrosis factor-α-directed therapy, 1722 tumor suppressor in lung cancer-1 (TSLC-1), 882 TVOCs (total volatile organic compounds), 1284 TWA (time-weighted average), 1102 TXA2, 593– 4 type 1 diabetes, prevalence, 23 type I allergy, 1947 – 9 type II allergy, 1950– 2 type III allergy, 1950 immunoglobulin G in, 1357– 8 tyramine, 1328 tyrosine kinase inhibitors, 851 tyrosine kinases clinical relevance, 206 signal transduction, 205–7 tyrosines, in pollen, 946 UCN (urocortin), 1406 UIP (usual interstitial pneumonitis), 1769, 1771 ulcerative colitis diagnosis, 1329– 30 leukotrienes in, 587 ultrafines see nanoparticles umbilical cord blood, immunoglobulin E, 28 unconventional allergy, diagnosis, 1333– 4 United Kingdom (UK) allergen injection immunotherapy studies, 1530 allergic diseases, 23, 1321–2 asthma costs, 1593 mortality, 1593 racial and ethnic differences, 1244 hay fever, 1323– 4 pollen counts, 947 smogs, 1279 United States of America (USA) allergen injection immunotherapy studies, 1530 asthma costs, 23 racial and ethnic differences, 1244 cockroach allergens, epidemiology, 1132 hay fever, 1323 pollen counts, 946 pollen monitoring network, 949 United States Environmental Protection Agency (US-EPA), 1284 United States Joint Council of Allergy Asthma and Immunology, 1337 UP see maculopapular cutaneous mastocytosis

u-PAR (urokinase plasminogen activator receptor), 456–7 uPA (urokinase plasminogen activator), 328 upper airways in asthma, pathology, 1642–3 neurogenic inflammation, 825 neurophysiology, 823–5 sensory innervation, 823–4 upper airways diseases, 1383–401 urban areas allergic diseases, 2021–2 asthma, and plant allergens, 1272–3 diesel exhaust particles, 1269–70 nitrogen dioxide, 1268–9 outdoor air pollution, 1266, 1267–70 ozone, 1267–8 particulate matter, 1269–70 sulfur dioxide, 1269 urban climate effect, 1273 uridine triphosphate (UTP), 851 urocortin (UCN), 1406 urokinase plasminogen activator receptor (u-PAR), 456–7 urokinase plasminogen activator (uPA), 328 urticaria, 14, 436, 439, 1853–77 acute, 1929 adrenergic, 1864 after direct mast cell degranulation, 1865 antihistamine therapy, 559 aquagenic, 1864 and arachidonic acid metabolism abnormalities, 1865 blood product reactions, 1864 cellular infiltrate in, 1857–8 characterization, 1853 chronic, 1326 chronic autoimmune, 1861 chronic idiopathic, 1865 clinical entities, 1860–8 contact, 1842, 1864 diagnosis, 1326 diagnostic approaches, 1859–60 epidemiology, 1854 IgE-dependent, 1860–1 IgE receptor-dependent, 1860–1 and infections, 1865 laboratory findings, 1868–9 latex-induced, 1165–6 local heat, 1863 mast cells in, 244–5, 1854–5 papular, 1864 pathogenesis, 1854–9 physical, 1861–4 pressure, 1861 prevalence, 1854 solar, 1863 specific antigen sensitivity, 1860–1 testing procedures, 1869 treatment, 1869–72 and vasculitis, 1868 see also angioedema; aspirin-induced urticaria/angioedema; cholinergic urticaria; cold urticaria urticaria pigmentosa see maculopapular cutaneous mastocytosis

US-EPA (United States Environmental Protection Agency), 1284 Ustilago spp. (smuts), seasons, 966–7 usual interstitial pneumonitis (UIP), 1769, 1771 UTP (uridine triphosphate), 851 vaccinations and allergic diseases, 35 and hygiene hypothesis, 1246–7 vaccines (allergen-specific) see allergen-specific vaccines vagus nerves, electrical stimulation, 833 VAMPs (vesicle-associated membrane proteins), 262–4 Vander Veer, Albert (1841–1929), 13 Vane, John Robert (1927–2004), 567 vanilloid receptor-1 (VR-1), 506, 849 variant C allele, and aspirin-induced asthma, 708 VAS (visual analog scales), 1386, 1433 vascular cell adhesion molecule-1 (VCAM-1), 341–2, 882, 1298–9, 1304–5, 1413 binding, 348, 351 expression, 345–6, 347–8, 1300–1, 1469 function, 343 structure, 343 vascular endothelial growth factor (VEGF), 229, 324, 328, 528, 862, 864 in angiogenesis, 402–3 in asthma, 1620 expression, 1460–1 receptors, 403 regulatory mechanisms, 398 release, 1302 VEGF-A, 325, 327–8, 330 vascular endothelium, eosinophil migration, 347–8 vascular permeability biology, 857–73 and cell signaling, 862–8 increased, 857 loss of, 864–5 mechanisms, 868– 9 platelet-activating factor, 606 vascular permeability factor (VPF), 402–3 vascular remodeling, in asthma, 1620 vasculature, adrenoceptors, 669 vasculitis, 437 and urticaria, 1868 vasoactive intestinal polypeptide (VIP), 512, 518–19, 824, 1407 analogs, 1717–18 clinical applications, 519 metabolism, 518 pulmonary effects of, 518–19 roles, 519 vasoconstrictors, 405 in seasonal allergic conjunctivitis treatment, 1489 vasodilation, and angiogenesis, 401–2 vasodilator hypothesis, 668–9 vasomotor rhinitis use of term, 1392 see also idiopathic rhinitis VC see vernal conjunctivitis (VC)

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VCAM-1 see vascular cell adhesion molecule-1 (VCAM-1) VCD see vocal cord dysfunction (VCD) V(D)J recombination, 119– 20, 124– 5, 130 functions, 125– 6 vegetable allergy, 1926 prevalence, 1922– 3 VEGF see vascular endothelial growth factor (VEGF) venom allergens, 1123 cross-reactivity, 1987– 8 structure, 1125 see also insect venom allergens venom anaphylaxis, allergen injection therapy, 1511 venom immunotherapy (VIT), 1914, 1988 adverse reactions, 1990 contraindications, 1990 dosage, 1990 duration, 1991 efficacy, 1991, 1992 future trends, 1992 indications, 1990 insect sting allergy, 1990–1 mechanisms, 1991 treatment regimens, 1990 venom peptides, 1123 ventilation inhomogeneity, 758– 9 vernal conjunctivitis (VC), 243– 4 definition, 1387 vernal keratoconjunctivitis (VKC), 1482, 1493– 7 clinical features, 1493 histology, 1493– 5 inflammatory mediators, 1495 mechanisms, 1495– 6 pathogenetic issues, 1495 treatment, 1496– 7 medical, 1496–7 surgical, 1497 type I hypersensitivity, 1487 versican, 414 very late antigen-3 (VLA-3), 1854 very late antigen-4 (VLA-4), 1305, 1854 antagonists, 353, 1712, 1723 inhibition, 347– 8 studies, 1723 very late antigen-5 (VLA-5), 1854 vesicle-associated membrane proteins (VAMPs), 262– 4 vesiculovacuolar organelles (VVOs), 860, 861 Vespa spp. (hornets), 1983, 1987 Vespa crabro (European hornet), 1983 Vespa mandarinia (Asian hornet), 1124 Vespa orientalis (Oriental hornet), 1983 Vespidae (wasps), 1123, 1902 insect sting allergy, 1981–3 vespid venom allergens, 1124–5 Vespinae (wasps), 1981 Vespula spp. (hornets, wasps), 1902, 1981–2, 1987 characteristics, 1982– 3 venom, 1992

Vespula germanica (German wasp), 1981 Vespula maculifrons (common wasp), 1981 Vespula vulgaris (common wasp), 1981 venom, 1124, 1125 vessel permeability see vascular permeability vestibular system disorders, antihistamine and, 560 Ves v 5, 921, 1125, 1558 vibratory angioedema, 1862 vicilins, 1152, 1159 VIP see vasoactive intestinal polypeptide (VIP) viral immunoglobulin superallergens, 326–7 viral infections and asthma, 26 and atopic dermatitis, 1820 effects, 152–3 and extrinsic allergic alveolitis, 1764 and hygiene hypothesis, 1245 mast cells and, 230 and pediatric asthma, 1596–7 respiratory tract, infants, 2007 sensors, 192 see also acute viral infections virokinin, 515 viruses asthma induction, 235 and immunoglobulin E production regulatory mechanisms, 152–3 virus-induced mechanisms, 153–4 and indoor air pollution, 1282–3 VISA (Cardif), 192 visceral larva migrans see toxocariasis visual analog scales (VAS), 1386, 1433 VIT see venom immunotherapy (VIT) vitamin E, deficiency, 36 VKC see vernal keratoconjunctivitis (VKC) VLA-3 (very late antigen-3), 1854 VLA-4 see very late antigen-4 (VLA-4) VLA-5 (very late antigen-5), 1854 vocal cord dysfunction (VCD), 763, 814, 1582 differential diagnosis, 808 VOCs see volatile organic compounds (VOCs) volatile organic compounds (VOCs), 1281, 1283 and indoor air pollution, 1284 reduction guidelines, 1286 see also microbial volatile organic compounds (MVOCs); total volatile organic compounds (TVOCs) volumetric trapping, 946 Von Ebner’s glands, 998 von Euler, Ulf (1905–83), 567 von Leyden, Ernst V. (1832–1910), 11, 12 von Pirquet, Clemens (1874–1929), 4, 5, 6, 436, 439, 895 Voorhorst, Reindert (1915–2005), 984 voriconazole, 1752–3 VPF (vascular permeability factor), 402–3 VR-1 (vanilloid receptor-1), 506, 849 vulcanization, 1166 VVOs (vesiculovacuolar organelles), 860, 861

walnuts, allergens, 1152, 1155 WAP (whey acidic protein), 1231 wasps, 1123, 1902, 1981–3 allergens, 921, 1558 wasp stings, and anaphylaxis, 3, 1326, 1902–3 wasp venom allergy, 1902 weaning foods, and allergy prevention, 2003–4 weather patterns, and pollination, 944–5 and spore release, 966 weed pollen, 957–60 see also ragweed pollen weed pollinosis, 1153 weening, early, 1330 Wegener’s granulomatosis, 312 Weibel–Palade bodies, 338 West, Geoffrey B. (1916– ), 8, 9 western red cedar asthma (WRCA), 235–6 wet wrap therapy, in atopic dermatitis treatment, 1825–6 Wharton-Jones, Thomas (1808–91), 8 wheat allergens, 1151 allergic reactions, 1147 exposure, 1154 wheezing early, 1260 infant, 1594, 1618–19 infants, 1594, 2007 nerves in, 832–3 nonatopic persistent, 1594 and pediatric asthma, 1600 prevalence, 1263 transient early, 1594 use of term, 1600 wheezy breathlessness, in asthma, 277, 763 whey acidic protein (WAP), 1231 white dermographism, 1818 WHO see World Health Organization (WHO) whole blood assays, 1716 whole-lung models, 794, 795 WHO/NHLBI Workshop Report, 1433 Willis, Thomas E. (1621–75), 10, 11 wind pollination, 943 grasses, 950 work performance, and allergic rhinitis, 1386 workplaces airborne allergens, 1017–122 airborne irritants, 1017–122 occupational airway diseases, 1061–102 see also occupational agents; occupational asthma World Health Organization (WHO), 899, 930, 1439, 1567 Committee on Biological Standardization, 928 Immunoglobulin Reference Centre, 1666 mastocytosis classification, 1880, 1885 recommendations, 1998 World Trade Center collapse, occupational airway diseases, 1101 wound repair, mast cells in, 228–9 WRCA (western red cedar asthma), 235–6 Wuchereria bancrofti (nematode), 1789

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xanthates, 1166 xanthines, in asthma treatment, 1332 X-box binding protein 1 (XBP1), 120, 134 XBP1 (X-box binding protein 1), 120, 134 Xenopus laevis (African clawed frog) melanophores, 578 oocytes, 579, 608 xerosis, 1817 XI–HK complex, 453, 457– 8 XLAAD (X-linked autoimmune and allergic dysregulation) syndrome, 86

X-linked autoimmune and allergic dysregulation (XLAAD) syndrome, 86 Xolair, 1712 X-ray crystallography, 104 xylometazoline, 680 xylose, 1126 yohimbine, 680 zafirlukast, 696, 704–5 in aspirin-induced asthma, 1973

in chronic asthma treatment, 1653 in exercise-induced asthma, 707 and exercise-induced bronchoconstriction, 810 structure, 696 studies, 698, 699–700 Zea mays (maize), pollen, 943 zeolites, 1283 zileuton, 696, 811 adverse affects, 697

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