Principles and Methods of Immunotoxicology, Volume 1

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Immunotoxicology of Drugs and Chemicals: an Experimental and Clinical Approach

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Immunotoxicology of Drugs and Chemicals: an Experimental and Clinical Approach Volume I: Principles and Methods of Immunotoxicology J. DESCOTES Lyon Poison Center, Pharmacovigilance Unit, Lyon, France

2004

Amsterdam – Boston – Heidelberg – London – New York – Oxford – Paris San Diego – San Francisco – Singapore – Sydney – Tokyo

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First edition 1986 Second edition 1988 Third edition 2004 Library of Congress Cataloging in Publication Data A catalog record is available from the Library of Congress. British Library Cataloguing in Publication Data A catalogue record is available from the British Library. ISBN: 0 444 51093 1 1 The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). W

Printed in The Netherlands.

This book is dedicated to my wife, Christiane, my children and in-laws, Je´roˆme and Virginie, Aure´lie and Lionel, for their patience and love


Preface Exposure of the immune system to xenobiotics may result in complex alterations. With few exceptions, a molecular or even cellular basis for these toxic changes has yet to be elucidated. Perhaps one of the most confusing issues confronting the student of immunotoxicology is the identification of what constitutes an immunotoxic response. Those effects which are short acting, reversible and presumably non harmful should not be considered, in the author’s opinion, as immunotoxic. Immunotoxicology is still a young, but rapidly expanding area of toxicology. Since the second edition of this book published in 1988, a continuously increasing amount of data has been generated so that research, review and position papers have been more and more frequently published. As a bridging discipline between immunology and toxicology, immunotoxicology is genuinely multidisciplinary. Researchers from different disciplines have shown interest in this new field, and presented results of their work on immunotoxicological issues from extremely varied perspectives. It is therefore still difficult to grasp unambiguously the full scope of immunotoxicology at the beginning of this century. The aim of this three-volume book is ambitious. On one hand, it is an attempt to cover as extensively and comprehensively as possible the many facets of current immunotoxicology, such as clinical, experimental, mechanistic and regulatory aspects; on the other hand, the purpose is to provide to all those interested in the immunotoxic effects of xenobiotics, namely pharmaceutical drugs and every type of chemical substances, easy access to published data by using a standardized tabulated format. This first volume covers the principles and methods of experimental and clinical immunotoxicology. The history of immunotoxicology is an important aspect as it helps understand current limitations or biases. An obvious consequence is the focus on immunosuppression that has been and is still given in immunotoxicological research and regulatory decisions, even though other aspects, in particular hypersensitivity, are of critical relevance with regards to human health. This volume includes a chapter on the immune system. It is clearly only an overview of key concepts intended to assist toxicologists unfamiliar with immunology to acquire the minimal knowledge necessary to conduct and interpret immunotoxicity studies. Because toxicology is designed to predict and understand the toxic effects of drugs and other chemicals in living beings, this volume includes a comprehensive chapter on adverse effects in relation to immunotoxicity in human beings. Mechanisms of immunotoxicity are not elucidated in most instances; however, the data available on vii

viii

Preface

a small number of immunotoxicants can be helpful to understand the possible mechanisms of immunotoxicity. A major issue in immunotoxicity as well as general toxicology is to define the animal models and assays that can be used to evaluate the potential of new molecular entities for inducing immunotoxicity. This aspect has been divided into the four major types of immunotoxic effects, namely immunosuppression, immunostimulation, hypersensitivity and autoimmunity. Past, present and expected guidelines or regulatory documents are overviewed and discussed. Current strategies are presented taking into account the current status of standardization and validation of animal models and assays, as well as the philosophy or requirement of available guidelines. Clinical immunotoxicology has so far not received much attention despite the urgent need to accumulate relevant information on the immunotoxicity of drugs and other chemicals in human beings. Another aspect of clinical immunotoxicology is the use of specific antibodies to treat acute poisonings. The interactions of immunotoxicants and the developing immune system, i.e. developmental immunotoxicology, are an emerging new area of growing concern. Wildlife is an often overlooked target of immunotoxicants. New methods are also considered as rapid technological progress is likely to change the face of immunotoxicity evaluation in the few coming years. Finally, a classification of immunotoxicants is proposed. This first volume is therefore an attempt to cover all general aspects of immunotoxicology. It is obvious this area of toxicology has many more facets than often thought. It is hoped that readers will find both a clarification of current concepts and methods, and new avenues of research.

JACQUES DESCOTES Lyon, St. Jean d’Avelanne January 14, 2004

Contents Preface

vii

Chapter 1 Definition, history, and scope of immunotoxicology Definition of immunotoxicology A short history of immunotoxicology First phase: the ‘prehistorical’ phase Second phase: the birth of immunotoxicology Third phase: the scope of immunotoxicology widens Fourth phase: immunotoxicology comes of age Fifth phase: the future of immunotoxicology The scope of immunotoxicology Immunosuppression Immunostimulation Hypersensitivity Autoimmunity References

2 3 4 5 6 7 8 9 9 11 12 13 13

Chapter 2 The immune system: a toxicologist’s overview Introduction The lymphoid organs Central lymphoid organs Peripheral lymphoid organs Immunocompetent cells Lymphocytes Phagocytes Other immunocompetent cells Innate (natural) immunity Components of innate immunity Recognition of pathogen-specific molecular patterns Inflammation Adaptive (specific) immunity Cell –cell interactions Humoral immunity Cellular immunity

19 21 21 23 24 25 27 28 31 31 35 35 36 36 37 39 ix

x External influences on the immune system Psychoneuroimmunology Interactions between the nervous, endocrine and immune systems Age and immunocompetence Implications for immunotoxicity evaluation Conclusion References

Contents 45 45 46 48 48 49 49

Chapter 3 Health consequences of immunotoxic effects Introduction Immunosuppression Impaired resistance against microbial pathogens Immunosuppression and malignancies Other adverse effects related to immunosuppression Immunosuppression vs. immunodepression Immunostimulation Flu-like reactions Increased incidence of autoimmune diseases Hypersensitivity reactions to varied allergens Inhibition of drug-metabolizing enzymes Other complications associated with immunostimulation Hypersensitivity Epidemiology Clinical signs and symptoms Diagnosis Pseudoallergy Autoimmunity Incidence of autoimmune diseases and autoimmune reactions Systemic autoimmune reactions Organ-specific autoimmune reactions Isolated autoantibodies Conclusion References

55 55 56 59 63 64 66 66 67 67 68 70 70 71 71 83 87 88 88 88 91 96 97 97

Chapter 4 Mechanisms of immunotoxic effects Introduction Mechanisms of immunosuppression Introduction

127 128 128

Contents Nonspecific mechanisms Antigen-specific mechanisms Mechanisms of immunostimulation Introduction Flu-like reactions and acute cytokine syndromes Autoimmune diseases and immunostimulation Inhibition of drug-metabolizing enzymes Mechanisms of hypersensitivity reactions Introduction Immune-mediated hypersensitivity reactions Pseudoallergic hypersensitivity reactions Mechanisms of autoimmune reactions Pathophysiological hypotheses Predisposing factors Structure – immunotoxicity relationships Conclusion References

xi 129 130 131 131 131 132 132 133 133 134 143 145 146 148 149 149 149

Chapter 5 Nonclinical evaluation of unexpected immunosuppression Introduction Histopathology Conventional histology examination Histology of lymphoid organs Hematology and clinical chemistry Blood cell counts Bone marrow toxicity Lymphocyte subset analysis Serum immunoglobulins Measurement of humoral immunity General considerations The PFC assay ELISA ELISPOT Measurement of cellular immunity In vivo animal models Lymphocyte proliferation assays T lymphocyte cytotoxicity assays Cytokine assays Measurement of innate immunity Phagocytosis

163 163 164 165 170 170 171 171 172 173 173 174 176 177 177 177 179 180 181 182 182

xii Chemotaxis NK cell activity Complement Host resistance models Experimental infection models Implanted tumor models Conclusion References

Contents 184 184 186 186 186 189 189 189

Chapter 6 Nonclinical evaluation of unexpected immunostimulation Introduction Histopathology Hematology and clinical chemistry Immune function assays Cytokine release assays Host resistance models Experimental infection models Animal models of autoimmune diseases Conclusion References

205 205 206 206 207 207 208 208 212 213

Chapter 7 Nonclinical evaluation of the potential for hypersensitivity Introduction Prediction of immune-mediated hypersensitivity Prediction of IgE-mediated hypersensitivity reactions Prediction of T lymphocyte-mediated hypersensivity reactions Pseudoallergy Conclusion References

219 220 220 224 232 233 233

Chapter 8 Nonclinical evaluation of the potential for autoimmunity Introduction Systemic autoimmune reactions Animal models of systemic autoimmune reactions The popliteal lymph node assay (PLNA) Organ-specific autoimmune reactions

243 243 243 244 250

Contents

xiii

Conclusion References

251 251

Chapter 9 Regulatory aspects of immunotoxicity evaluation Introduction Early immunotoxicity regulations First EPA guideline on pesticide immunotoxicity Commission of the European Communities Japanese drug antigenicity guideline FDA Red Book II Recent immunotoxicity guidelines OECD Guideline 407 Second EPA guideline on pesticide immunotoxicity ICH guideline on the preclinical safety evaluation of biotechnologyderived pharmaceuticals EMEA concept paper on immunotoxicity FDA CDRH immunotoxicity testing guidance CPMP note for guidance on repeated dose toxicity FDA guidance on immunotoxicology evaluation of investigational new drugs Conclusion References

257 257 258 259 260 260 260 261 261 262 262 262 263 264 264 266

Chapter 10 Nonclinical strategies of immunotoxicity evaluation and risk assessment Introduction Good laboratory practices in immunotoxicology Impact of animal-related factors on immunity Standardization of reagents and assays Strategies for the prediction of immunosuppression General considerations Selection of assays Strategies for the prediction of immunostimulation Strategies for the prediction of hypersensitivity Strategies for the prediction of autoimmune reactions Risk assessment Definitions and concept Immunotoxicity risk assessment Conclusion References

269 269 270 272 272 273 277 280 280 281 281 281 282 284 284

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Contents

Chapter 11 Clinical immunotoxicology Introduction Objectives of clinical immunotoxicology Identification of immunotoxicants in human beings Surveillance of at-risk humans exposed to immunotoxicants Validation of nonclinical immunotoxicity assays and animal models Clinical assessment of immunotoxicity Clinical immunology end-points to assess humoral immunity Clinical immunology end-points to assess cellular immunity Clinical immunology end-points to assess nonspecific immunity Biomarkers of immunotoxicity Immune end-points adopted by the ATSDR Recommendations of the US National Research Council Environmental epidemiology studies Experimental vs. observational epidemiological methods Use of epidemiological studies in immunotoxicity assessment Disease clusters and sentinel diseases Disease clusters Sentinel events Conclusion References

295 295 296 297 297 298 298 298 300 300 301 301 302 302 302 303 304 304 305 305

Chapter 12 Immunotherapy of human poisonings Introduction Experimental studies Medicinal products Industrial and environmental chemicals Clinical studies Immunotherapy of envenomations Digitalis Tricyclic antidepressants Colchicine Vaccines for the treatment of drug dependence Mechanism of specific antibodies efficacy Conclusion References

311 312 312 313 315 315 316 317 318 318 320 321 321

Contents

xv

Chapter 13 Chemical immunoteratology and developmental immunotoxicology Introduction Immunity in newborns Chemical teratogens and the immune system Morphological changes in lymphoid organs induced by teratogens Reproduction toxicity assessment of immunopharmaceuticals Nonclinical assessment of developmental immunotoxicity Developmental immunotoxicants Drugs as developmental immunotoxicants Chemicals as developmental immunotoxicants Conclusion References

327 328 329 329 329 330 331 331 337 343 344

Chapter 14 Immunotoxicity and wildlife Introduction Immunotoxicity in fish Fish immune system and diseases Immunotoxicity studies in fish Immunotoxicity of chemicals in fish Immunotoxicology in marine mammals Seals Dolphins Whales Immunotoxicity in birds Conclusion References

355 355 356 356 357 358 358 358 359 359 359 359

Chapter 15 New methods in immunotoxicology Introduction Enhanced pathology Immunohistochemistry Computerized processing and image analysis In situ hybridization In situ polymerase chain reaction In vitro immunotoxicology General considerations

363 363 364 364 365 365 365 366

xvi In vitro assays for the prediction of unexpected immunosuppression In vitro assays for the prediction of unexpected immunostimulation In vitro assays for the prediction of contact hypersensitivity In silico immunotoxicology Molecular immunotoxicology Microarray analysis New animal models Immunodeficient mouse models Genetically modified animals Computational immunotoxicology Conclusion References

Contents 367 368 368 369 369 369 370 370 371 371 372 372

Chapter 16 Classification of immunotoxicants Introduction Selected criteria Potential consequences of exposure to immunotoxicants Reliability and magnitude of available data Proposed classification Group 1: unclassifiable xenobiotics Group 2: possibly immunotoxic xenobiotics Group 3: probably immunotoxic xenobiotics Group 4: certainly immunotoxic xenobiotics Group 5: nonimmunotoxic xenobiotics Examples Discussion References

379 379 380 381 382 382 382 382 382 382 383 383 385

List of Abbreviations

387

Index

389

Chapter 1

Definition, history, and scope of immunotoxicology

An immense variety of medicinal products as well as chemical substances from either household, nutritional, industrial, or environmental sources—for review see Refs. [1,2]— have been suspected or shown to cause histological changes of the lymphoid organs and qualitative or quantitative functional disturbances of the immune system. In laboratory animals, decreases in the weight of lymphoid organs, especially the thymus, were often associated with parallel alterations in the histological architecture. Impairment of functional immune responses and diminished resistance towards experimental infections or implanted tumors has also been frequently reported. Data are accumulating that similar effects occur in wildlife animal species [3,4] and to a much lesser extent in domestic animals [5]. More recently, efforts focused on determining whether drug treatments and chemical exposures can induce immunotoxic changes with significant adverse effects to the health of human beings. Biomarkers of immunotoxicity have been tentatively identified and their use proposed for inclusion in epidemiological studies [6,7]. In the 1980s, several national and international programs conducted under the auspices of either the US National Toxicology Program (NTP) [8], the International Programme on Chemical Safety (IPCS/WHO) jointly with the European Union [9], or the German Drug Agency (BGA/BfArM) [10], to name but a few, in association with private companies from both the pharmaceutical and the chemical industry, contributed to standardizing experimental models and assays, and to validating strategies applicable to the nonclinical immunotoxicity evaluation of new drugs and chemicals. After many hesitant years, several regulatory bodies, such as the Organization for Economic Cooperation and Development (OECD), the US Environmental Protection Agency (EPA), the European Medicines Evaluation Agency (EMEA), and lately the US Food and Drug Administration (FDA), released guidelines, guidance or points to consider concerning the immunotoxicity evaluation of new drugs or chemicals (see Chapter 10 of this volume). Refinements of, or additions to these guidelines, e.g. from the Japanese Ministry of Health, Labor and Welfare, or the International Conference on Harmonization (ICH), were expected at the time of writing. 1

2

Immunotoxicology of drugs and chemicals

During the past 25 years, extensive research efforts have thus enabled to delineate more accurately the scope of immunotoxicology and to make significant progress in the understanding of the immunotoxic effects induced by drugs and chemicals, and in establishing nonclinical procedures that can be considered for use to assess the potential of new drugs and chemicals to produce immunotoxicity.

DEFINITION OF IMMUNOTOXICOLOGY Immunotoxicology was once defined as the “science of poisons to the immune system” [11]. Although, the foundations of immunotoxicology were primarily toxicology and immunology [12,13], other areas of the biomedical sciences, such as occupational medicine and environmental health [14], clinical and internal medicine [15 –17], allergology [18], molecular and cellular biology [19], or risk assessment [20,21], are also critically contributing to our improved understanding of the adverse effects of immunotoxicants, and to the development of new concepts and techniques applicable to the prediction and investigation of the immunotoxic effects of drugs and chemicals. The International Seminar on the Immunological System as a Target for Toxic Damage held in Luxemburg under the auspices of the Commission of the European Community (CEC) and IPCS played a seminal role in the early development of immunotoxicology. During that seminar, the following definition of immunotoxicology was proposed: “[immunotoxicology can be defined as] the discipline of toxicology which studies the interactions of xenobiotics with the immune system resulting in adverse effects” [22]. Although, this definition was coined in 1984, it is still valid today. Of extreme importance is the fact that this early definition distinguished two distinct and absolutely essential steps to ensure that immunotoxicity is reliably evaluated, namely: (1) the identification of the potential of drugs and chemicals for inducing toxic effects on the immune system, essentially in laboratory animals (i.e. immunotoxic hazards), and (2) the evaluation of related adverse clinical consequences (i.e. immunotoxic risks), based either directly on the results of human studies, or indirectly on risk assessment procedures. This definition is in full agreement with the definition subsequently proposed by the US Office of Technology Assessment [23]: “[immunotoxicity is] an adverse or inappropriate change in the structure and function of the immune system after exposure to a foreign substance. Adverse effects can be manifest as immunosuppression, hypersensitivity, or autoimmunity.” As is clearly emphasized in the definitions above, immunotoxicology is not restricted to the mere identification of histological changes in lymphoid organs or functional disturbances of immunocompetence in laboratory animals or human beings, whatever their magnitude, or else to the fundamental investigation of possibly underlying mechanisms, as so many investigators often seem to believe. Although, it is beyond doubt that both aspects are absolutely essential and should be carefully considered, the ultimate goal of immunotoxicology (as that of toxicology in general) is to generate data that can primarily be used to predict as reliably as possible the adverse clinical consequences that can be expected to occur in living beings exposed to the identified immunotoxicants, and as the last resort to decide which industrial or regulatory steps are required to avoid for these clinical consequences to develop.


3

A SHORT HISTORY OF IMMUNOTOXICOLOGY Even today in the very first years of the 21st century, immunotoxicology remains a relatively young area of toxicology [24,25]. The first symposium ever devoted entirely to immunotoxicology was seemingly held in Lyon (France) on 24th October 1974 [26]. This symposium only focused on the immune-mediated adverse effects of drugs, which is quite ironic as ‘drug allergies’ have long been totally overlooked by most immunotoxicologists. Surprisingly, 5 years elapsed before the word immunotoxicology was again included in the title of a scientific meeting. The word immunotoxicology was indeed used for the very first time in the international scientific literature in a special issue of the journal Drug and Chemical Toxicology published in 1979 to describe what was then called the ‘immunotoxicology phenomenon’ [27]. In 1983, Davies [13] proclaimed the birth of immunotoxicology. Twenty years later, it is useful to overview the short history of immunotoxicology as the description of the five phases that can be identified provides a better understanding of what immunotoxicologists know and do not know today, and as importantly, what they believe and do not believe in. Important Dates in the History of Immunotoxicology 1966 1968 1973 1974 1976 1977 1979

1980 1981 1982

1983 1984 1986

First review of infectious complications associated with immunosuppressive drugs Outbreak of PCB-induced Yusho disease in Japan Accidental contamination of cattle feed by PBBs in Michigan associated with immune disturbances in exposed rural populations First meeting ever devoted to immunotoxicology (Lyon, France) Autoimmune-like adverse reactions result in practolol withdrawal from the UK market First literature survey on immunosuppression as related to toxicology The word ‘immunotoxicology’, is used for the first time in the scientific literature Introduction of the concept of tiered protocols in immunotoxicity evaluation First epidemiological study showing a link between lymphoma and immunosuppression in kidney transplant patients The Spanish toxic oil syndrome, a supposedly autoimmune disease of as yet unknown origin, affects 20,000 people and kills 1500 The US NTP initiates an interlaboratory immunotoxicity validation study First International Seminar on Immunotoxicology (University of Surrey, UK) First US EPA guideline for the immunotoxicity evaluation of pesticides CEC recommendation to evaluate the immunotoxicity potential of medicinal products International Seminar on the Immunological System as a Target for Organ Toxicity (Luxemburg) CEC and IPCS initiate the ICICIS interlaboratory validation study

4 1988 1989

1995 1997 2000 2002

Immunotoxicology of drugs and chemicals First report from the NTP interlaboratory validation study International Workshop on the Immunotoxicity of Metals and Immunotoxicology (Hanover, Germany) First book ever devoted to autoimmunity and toxicology L -trytophan induces over 1500 cases of fasciitis-eosinophilia in the US The updated OECD guideline 407 requires systematic histological examination of the main lymphoid organs in 28-day rat toxicity studies The ICH guideline on biotechnology-derived products emphasizes the need to consider immunotoxicity as a critical safety issue The EMEA releases an updated guidance on repeated dose toxicity requiring a limited set of immunotoxicity endpoints The FDA CDER releases a guidance on the immunotoxicology evaluation of investigational new drugs

FIRST PHASE: THE ‘PREHISTORICAL’ PHASE The interest of researchers in the possible adverse effects of toxicants on the immune responses is not as new as is often thought. Examples of very early investigations in the field of what was obviously not known as immunotoxicology at the time of publication, include the effects of ethanol on the resistance of rabbits towards an experimental streptococcal infection [28], the effects of the anesthetics ether, chloroform or chloral hydrate on various experimental infections [29], the effects of ether on phagocytosis [30], or those of sodium salicylate on humoral immunity [31]. The origin of immunotoxicology can be traced back to the introduction of potent immunosuppressive drugs into the clinical setting in the mid-1960s [32]. Although, the concept of immunotoxicology was still to be invented, the adverse clinical effects of immunosuppressive drugs directly related to their (immuno)-pharmacologic activities in humans, in particular, infectious complications were described shortly thereafter [33] and received much attention subsequently. This historical context explains why immunosuppression has so consistently been and is still so often used, even today, as a synonym of immunotoxicity, or was and is still being considered by many investigators as well as regulators as the main cause of concern in relation to immunotoxicity associated with drugs and chemicals. Unsurprisingly, the first international literature survey on immunotoxicology published by Jeff Vos in 1977 focused on immunosuppression as related to toxicology [34]. The observation that immunosuppressive drugs can cause severe and sometimes potentially life-threatening infectious complications led many researchers mainly from the immunological field to investigate the possible effects of drugs and chemicals on the immune responses in the 1960s and 1970s. However, most of these early experimental studies were usually conducted without a minimal understanding that relevant conditions of exposure or adequately selected doses are essential to generate (immuno)-toxicological results that can be soundly extrapolated from animal to man. Therefore, most of these data should only be used, if at all, with great caution. Some studies were also initiated largely for promotional purposes, as exemplified by reports on the supposed influence, either positive or negative, of antimicrobials on immune responsiveness [35].


5

Whatever its obvious limitations, this prehistorical phase of immunotoxicology set the stage to long-lasting effect. Efforts must be paid, even today, to expand the scope and the relevance of immunotoxicological evaluation to better assure that all key issues related to possible immunotoxic effects of drugs and chemicals, and not only immunosuppression, are adequately dealt with.

SECOND PHASE: THE BIRTH OF IMMUNOTOXICOLOGY The year 1979 was an important year for immunotoxicology. That year, the word immunotoxicology was used for the first time in the international literature [36] to summarize the new concepts and findings presented at the Annals of the New York Academy of Sciences Satellite Meeting on immune abnormality and shortly thereafter at the Gordon Research Conference on Drug Safety. That year was also a major turning point with the recommended introduction of fundamental toxicological concepts, such as the selection of relevant animal species, doses, route and duration of exposure, in the nonclinical immunotoxicity evaluation of drugs and chemicals. To some extent, the year 1979 can also be heralded as the first attempt to shift immunotoxicology from immunology to toxicology, and this shift slowly, but inescapably resulted in driving immunotoxicology to a fully recognized area of toxicology as it is today. Jack Dean and co-workers introduced the concept of tiered protocols that same year [37]. Because the immune response involves such a variety of effector and regulatory mechanisms, these authors logically claimed that no single assay could be expected to identify the immunotoxic potential of drugs and chemicals, hence the recommended use of a battery of assays. This claim has never been questioned and is still fully accepted today. To achieve optimal cost/effectiveness, a stepwise approach was proposed. The first step (screening phase ¼ tier I) included a matrix of in vitro and in vivo assays to be used initially for screening purpose. Depending on the results of the first tier, a list of additional assays was suggested for mechanistic purpose (mechanism phase ¼ tier II). Even though the list of assays recommended for inclusion in tier I and tier II at that time has evolved so markedly over the years that the current strategies of immunotoxicity evaluation bear few similarities with those of these early times (see Chapter 10 of this volume), the concept of a stepwise evaluation of the immunotoxic potential of new chemical entities is still widely used and considered a hallmark of nonclinical immunotoxicity evaluation. This phase of immunotoxicology also proved essential as it greatly helped rationalize the nonclinical immunotoxicity evaluation of drugs and other chemicals. Nevertheless, immunotoxicity remained largely synonymous with immunosuppression at that time despite significant methodological progress [38]. The immunotoxic potential of many substances was evaluated during this period. In 1982, the NTP initiated the most important interlaboratory validation program ever [39], which was conducted by Mike Luster and coworkers in the B6C3F1 mouse. Drugs and chemicals included for study in this program were primarily compounds with known or suspected immunosuppressive and/or carcinogenic activity, such as benzidine, biphenyls, chlordane, cyclophosphamide, dioxin, diethylstilbestrol, dimethylbenz[a ]anthracene, ethylcarbamate, formaldehyde, hexachlorobenzene, methylcarbamate, methylisocyanate, organotins, or pentachlorophenol [40].

6


Other interlaboratory validation programs were later conducted, including the Fisher 344 cyclosporine validation study [41], the modified 28-day rat study [10] and the ICICIS rat validation study [9]. Since these programs investigated a very limited number of compounds that were again markedly immunosuppressive, primarily cyclosporine and azathioprine, they did not prove very useful for validation purposes. Nevertheless, they were instrumental in setting the stage for the further refinement of evaluation strategies, essentially by stressing the need for careful standardization and strict selection of animal models and immune assays to be used in nonclinical immunotoxicity evaluation. They also resulted in shifting from the mouse, the animal species immunologists preferred, to the rat, the preferred animal species of toxicologists. In addition to these seminal validation efforts, a number of major symposia contributed to the international development of immunotoxicology as a new discipline of toxicology: among these, the Annals of the New York Academy of Sciences Satellite Meeting on Immune Abnormality and Gordon Research Conference on Drug Safety which were both held in 1979; the Workshop on Immunological Aspects of Toxicology organized in Chicago by the US Pharmaceutical Manufacturers Association Foundation in 1981 [42]; the Symposium on the Immune System as a Target Organ of Toxicity held in Research Triangle Park (North Carolina) in 1982 [43]; the First International Symposium on Immunotoxicology convened at the University of Surrey (UK) in 1982 [44]; and the International Seminar on the Immunological System as a Target for Toxic Damage in Luxemburg in 1984 [22]. The proceedings of these symposia defined the foundations of immunotoxicology and established the pioneering concepts that were to be used by most immunotoxicologists during the next decade and beyond. THIRD PHASE: THE SCOPE OF IMMUNOTOXICOLOGY WIDENS After years of efforts entirely devoted to immunosuppression, the scope of immunotoxicology began to expand, thanks largely to European researchers. An illustration of this new trend is the International Workshop on the Immunotoxicity of Metals and Immunotoxicology held in Hanover (Germany) in 1989 [45]. Although it is, even today, debatable to what extent autoimmune diseases or reactions caused by immunotoxicants are a major health problem, the first new area that drew the attention of European immunotoxicologists was autoimmunity [46] (see Chapter 9 of this volume). Several drugs were indeed withdrawn from the market because of demonstrated or alleged autoimmune adverse effects [47]. The beta-blocking drug practolol, a wellestablished cause of many cases of the oculo-mucocutaneous syndrome [48], was the first drug to be withdrawn. Later on, Guillain – Barre´ syndromes induced by the antidepressant zimeldine [49], or autoimmune hemolytic anemias due to nomifensine [50], another antidepressant drug, showed that medicinal drugs can indeed cause severe autoimmune reactions. In addition, the toxic oil syndrome that severely affected thousands of individuals in Spain [51] and the fasciitis eosinophilia syndrome caused by tryptophan [52] were other pungent examples that autoimmunity induced by immunotoxicants, although probably rare, can nevertheless result in significant morbidity and mortality [53]. Another major new area of interest for immunotoxicologists was drug and chemical allergies (see Chapter 8 of this volume). For many years, drug and chemical allergies


7

or hypersensitivities had been claimed to be beyond reach of experimental testing and toxicological prediction, despite the fact that contact sensitization assays in the guinea-pig had long been widely used [54,55]. However, in the first edition of Toxicology, The Basic Science of Poisons, nowadays the absolute reference book for toxicologists worldwide, John Doull was reluctant to support the view that sensitization reactions were not to be considered as true toxic reactions [56]. The repeated market withdrawal of medicinal drugs that had been reported to cause ‘allergic’ reactions, such as acute pseudo-allergic reactions due to the intravenous anesthetic alfadione [57], bullous toxidermias caused by the nonsteroidal anti-inflammatory drug (NSAID) isoxicam [58], anaphylaxis caused by the NSAID zomepirac [59], or the minor analgesic glafenin [60] increasingly led to consider drug allergies as a cause of major health concern. Today, the prediction of the allergic potential of medicinal drugs in the course of nonclinical safety evaluation is unanimously considered a top priority [61], but much remains to be done in order to design, standardize, and validate the experimental models assuring an adequate level of prediction [62]. FOURTH PHASE: IMMUNOTOXICOLOGY COMES OF AGE After more than 20 years of recognized existence, immunotoxicology has come of age. Major toxicological societies, such as the (US) Society of Toxicology [63], the European Societies of Toxicology (Eurotox) [64], and the British Toxicology Society [65] have established specialty sections entirely devoted to promoting immunotoxicology. These societies regularly organize scientific sessions on immunotoxicology during their annual meetings and circulate focused information to members who expressed their interest in the field. A number of organizations including the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) [66], the International Life Science Institute (ILSI) [67], or the International Commission on Occupational Health (ICOH) [68] have also established working groups or committees to address timely issues in immunotoxicology. Finally, the Foundation in Immunotoxicology and the Summerschool in Immunotoxicology [69] are non profit associations that used to convene meetings entirely devoted to immunotoxicology each year. Although all major journals of toxicology do publish original papers as well as reviews on immunotoxicology, three journals have specifically included immunotoxicology in their editorial scope, namely Academic Press’ International Immunopharmacology (formerly International Journal of Immunopharmacology) [70], Marcel Dekker’s Immunopharmacology and Immunotoxicology [71], and Elsevier’s Toxicology [72]. Quite a few books have been published to review the concepts and data available at the time of publication [1,2, 15 –17,21,22,27,38,44 – 46,73 –80]. In the 1990s, the scope of immunotoxicology further expanded to include three new areas, namely clinical immunotoxicology, immunotoxicity risk assessment, and immunotoxicology in wildlife species. Emphasis indeed began to be put on the human (clinical) aspects of immunotoxicity [15 – 17] (see Chapter 12 of this volume). Panels of experts identified those markers that are more likely to be useful to evidence adverse effects on the immune system in selected groups of the population exposed to occupational and/or environmental chemicals [81,82]. Less attention, if any, has so far been paid to defining

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endpoints potentially useful in the clinical trials of pharmaceuticals [83]. The recent release of guidance on the nonclinical immunotoxicity evaluation of pharmaceuticals as well as other chemicals (see Chapter 10 of this volume) is, however, likely to change the situation rapidly as nonclinical data cannot be expected to achieve a full evaluation of the immunotoxic potential of new chemical entities. The use of immunological endpoints in clinical trials and epidemiological studies will, therefore, be required to rule out or confirm whether and to what extent immune changes seen in laboratory animals are also likely to be noted in human beings. Another breakthrough in the field of immunotoxicology was the recent introduction of risk assessment [20,21] (see Chapter 13 of this volume). As suggested by the definition of immunotoxicology proposed at the Luxemburg seminar [22], immunotoxicologists must not only determine whether drugs and chemicals can provoke immune changes (i.e. immunotoxic hazards), but also assess the potential risks associated with these changes depending on the actual or expected conditions of exposure. It is beyond doubt that much remains to be done to attain a reasonably reliable assessment of immunotoxic risks, especially when medicinal products are considered. Efforts should be made to establishing conclusive correlations between changes in the histology of lymphoid organs and the immune functions of laboratory animals, and possible adverse clinical consequences in human beings. At the present time, data are indeed extremely few and can hardly be used for stringent regulatory purposes, except on occasions when the precautionary principle is considered a priority. A relatively recent interest of immunotoxicologists is the effects of immunotoxicants on wildlife species [4,84] (see Chapter 16 of this volume). Fish are often considered valuable sentinel species to assess the immunotoxic effects of environmental chemicals [3,85 –87]. The impact of immunotoxicants on wildlife has also been illustrated by studies in seals [88] and whales [89]. In contrast, the study of immunotoxicants in domestic animals is still in its infancy [5]. Finally, the applicability of in vitro methods has been an area of research in the recent years [19, 90, 92] (see Chapter 17 of this volume). However, only limited data have been provided to establish whether in vitro endpoints are actually helpful or whether other endpoints should be proposed for use. In addition, new techniques involving new animal models, such as genetically modified animals [93], or computer simulation [94], are increasingly considered for use. FIFTH PHASE: THE FUTURE OF IMMUNOTOXICOLOGY Because regulatory aspects are so critical in toxicology, a significant step in the short history of immunotoxicology is the recent release of several regulatory documents devoted to the nonclinical immunotoxicity evaluation of drugs and chemicals, as discussed later in this volume (see Chapter 10). After many years of debate and hesitation, these guidelines are expected to change dramatically the scope and practice of immunotoxicology in the next few years. Indeed, private companies will have to devote significant efforts to establish adequate resources and design in-house strategies to evaluate the immunotoxic potential of new chemical entities [95]. In doing so, a wealth of data will be generated, which will raise many new


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questions and dilemmas that will have to be addressed jointly by researchers from the industry, academia, and regulatory agencies. There is no doubt this will have a major impact on the development and scope of immunotoxicology. With the introduction of new techniques and the generation of data concerning an ever-increasing number of new chemical entities, the practice of immunotoxicology can be expected to change markedly in the next few years.

THE SCOPE OF IMMUNOTOXICOLOGY Typically, immunotoxic effects used to be classified into two areas, namely immunosuppression and immunostimulation, the latter encompassing allergy and autoimmunity. This classification is no longer accepted. The introduction of potent immunostimulating (‘immunoactivating’) substances, such as the recombinant cytokines, showed that therapeutic agents can induce clinical adverse effects with no direct pathophysiological links to allergy and autoimmunity [96]. Flu-like reactions (also called acute cytokine syndromes when severe) and the inhibition of hepatic drug metabolism pathways are examples of such adverse effects. More frequent autoimmune diseases and hypersensitivity reactions to unrelated allergens have also been described [97]. Importantly, autoimmune reactions induced by few drugs and still fewer chemicals are markedly different from autoimmune diseases that are more frequently noticed in patients treated with certain therapeutic cytokines, such as rIL-2 and the interferons-alpha [98]. In general, only one type of autoimmune reaction is produced by a given drug, whereas the more frequent autoimmune diseases described with immunostimulating drugs are extremely varied. The difference between the increased incidence of hypersensitivity reactions to unrelated allergens in patients treated with immunostimulating drugs, and allergic reactions directed against the drug or chemical a person is being treated with, or exposed to, is obvious. Finally, even though similar mechanisms are involved in certain fundamental aspects of allergy and autoimmunity [99], it is more logical to address allergy and autoimmunity separately, especially because the majority of predictive nonclinical and clinical models and assays are widely different. Nowadays, it is clear that the scope of immunotoxicology definitely consists of four distinct areas, namely immunosuppression, immunostimulation, hypersensitivity, and autoimmunity. Each area requires different modalities of nonclinical and clinical evaluation to identify the corresponding immunotoxic effects involved, to understand the underlying mechanism(s), and to predict the risk of possible adverse consequences. IMMUNOSUPPRESSION As briefly overviewed above, immunosuppression has been and is still a major area of immunotoxicology. Because so many efforts have been devoted to identifying those drugs and chemicals that might have the capacity of decreasing one or several aspects of immune responsiveness, most of the currently available data on the immunotoxicity of drugs and chemicals relate to immunosuppression. However, this should not lead to conclude that immunosuppression is the one and only aspect of immunotoxicology to be dealt with. So far,

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no pharmaceutical products have ever been withdrawn from the market because of unexpected immunosuppressive effects, although the adverse consequences of the immunosuppressive effects of the anti-TNF drug infliximab, a monoclonal antibody used to treat rheumatoid arthritis, were openly recognized only after marketing of the drug [100]. As many unrelated causes or biases can be suspected for contributing to the possibly unnoticed immunosuppressive adverse effects of pharmaceutical drugs, it is important that immunotoxicologists from the nonpharmaceutical field as well as those from regulatory drug agencies are fully aware that immunosuppression is in no way the only relevant aspect of immunotoxicity. Direct immunotoxicity. Data in laboratory animals and human beings show that the immune response, as any other physiological responses, can be reduced or augmented by drug treatments or chemical exposures. Because the dose – response relationship was close to that seen in other areas of toxicity, the term ‘direct immunotoxicity’ has often been used [101,102] to refer to quantitative changes of qualitatively normal immune responses, such as antibody responses and delayed-type hypersensitivity reactions to defined antigens, or mitogen-induced lymphocyte proliferation. Depending on whether the observed changes consist of decreases or increases in immune responses, the words immunosuppression and immunostimulation are used, respectively. Until the recent development of potent immunoactivating substances, such as the recombinant cytokines used for therapeutic purposes, immunostimulation was not at all considered a key aspect of immunotoxicology, explaining why both immunosuppression and immunostimulation were sometimes grouped together under the general heading ‘direct immunotoxicity,’ and why in fact immunosuppression has been a major area of interest for many immunotoxicologists in total contrast to immunostimulation [34,103]. It is obvious that the adverse clinical consequences of immunosuppression and immunostimulation are so totally different that a clear distinction between immunosuppression and immunostimulation is essential and that distinct strategies for drug and chemical nonclinical and clinical safety evaluation have to be utilized (see Chapters 6 and 7 of this volume). Immunosuppression vs. immunodepression. The word immunosuppression, when used whatever the magnitude of the decrease in the immune responses, may be misleading. Strictly speaking, immunosuppression should only refer to a total abrogation of the immune responsiveness. English dictionaries unequivocally differentiate the verb to suppress, which is defined “to prevent something from being known or seen,” from the verb to depress, which is defined “to press, push, or pull something down.” The inhibition of immune-mediated organ graft rejection by cyclosporine or tacrolimus, for instance, clearly refers to an immunosuppressive effect, whereas a 30% decrease in antibody response to a given antigen associated with chemical exposure is merely an immunodepressive effect. Although, immunotoxic risk assessors primarily focusing on environmental chemicals may not adhere to this distinction, immunosuppression and immunodepression cover markedly different situations, especially as far as pharmaceutical products are concerned (and it remains to be demonstrated whether this does not also apply to nonpharmaceutical chemicals). As discussed later in this volume (see Chapter 3), the distinction between


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immunosuppression and immunodepression is critical in several aspects: (1) the adverse clinical consequences resulting from an immunosuppressive or immunodepressive effect, although qualitatively similar in most instances, are different with respect to their magnitude, incidence, and severity; (2) the modalities of assessing the risk of immunodepression and immunosuppression differ due to the functional reserve of the immune system. Even though nobody actually knows how to measure the magnitude of the functional reserve, this concept is strongly supported by the clinical experience alluded to above; and (3) immunodepressive effects used to be interpreted using quite different criteria from immunosuppressive effects in the risk/benefit analysis of medicinal drugs. A large number of marketed drugs have indeed been shown to induce mild to moderate (i.e. immunodepressive) effects with no demonstrated major clinical consequences for the health of treated human beings [1,2]. Therefore, when a certain level of immune responsiveness can still be measured in living beings exposed to an immunotoxicant, either drug or chemical, the word immunodepression is recommended for use instead of immunosuppression. To stress the distinction between immunosuppression and immunodepression, the words underimmunosuppression and overimmunosuppression have sometimes been used [104]. IMMUNOSTIMULATION Immunostimulation refers to an increase in immune responsiveness. Probably because the focus of most immunotoxicologists and the design of the assays they used was primarily immunosuppression, very few drugs and chemicals have been shown to exert unexpected (adverse) immunostimulating effects. In any case, recent clinical data demonstrate immunostimulation is one of the key areas of immunotoxicology. The use and definition of the words immunostimulation and immunomodulation have also been a matter of debate. Presumably because early experimental findings suggested that potent immunosuppressive drugs, such as cyclophosphamide [105] and cyclosporine [106], might increase immune responses, particularly delayed-type hypersensitivity, depending on the dose regimen with respect to the time of antigen injection, the word immunomodulation has often been used. Despite (obviously premature) claims that such experimental findings might be associated with some therapeutic benefit in human patients [107], which subsequently proved untrue, the concept of immunomodulating drugs has been widely used. It has also been often claimed that it was impossible to increase a normal immune response. The word immunorestoration (immunorestoring drugs) has thus also been used, as it was suggested that only impaired immune responses could be increased by pharmacotherapeutic agents. In fact, the few proposed immunorestoring drugs, such as imuthiol [108], proved unsuccessful in the clinical setting. The clinical development of recombinant cytokines showed that the immune response, like any other physiological responses, can be increased as a consequence of pharmacological manipulation. Because the adverse clinical consequences of the administration of therapeutic recombinant cytokines are markedly more severe than those seen after the administration of less potent substances, such as microbial extracts [109], the distinction between immunoactivation and immunostimulation can also be made.

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The question remains whether most assays and strategies that are typically used to predict the unexpected immunosuppressive potential of drugs and chemicals are applicable to the prediction of unexpected immunostimulating activities, as discussed in Chapter 7 of this volume. In fact, data on the use of strategies to evaluate possibly immunosuppressive drugs for the nonclinical immunotoxicity evaluation of immunostimulating drugs have seldom been published [110]. Novel assays or conventional assays using different experimental conditions should probably be selected to generate data helpful to predict the potential for inducing clinical adverse effects in man, but adverse effects linked to immunosuppression and immunostimulation are different. With the expanding introduction of pharmaceutical products intended to stimulate or activate components of the immune system, the need for animal models and assays specifically designed to address this issue is likely to become unavoidable. HYPERSENSITIVITY Hypersensitivity reactions to drugs and chemicals are considered relatively common. In fact no reliable epidemiological data are available on the actual incidence of hypersensitivity reactions to medicinal drugs. A widely accepted estimate is that hypersensitivity accounts for approximately 10% of all reported adverse drug reactions [111,112]. Hypersensitivity reactions to medicinal drugs are also a major issue for the pharmaceutical industry as there has been a number of examples of marketed drugs either withdrawn or halted at a very late stage of clinical development due to the occurrence of hypersensitivity reactions judged either too severe or too frequent compared to the expected therapeutic benefit. Hypersensitivity to occupational and environmental chemicals is also often claimed to be more and more frequent [18,113], although well-documented data on the incidence of these adverse reactions are rare and often poorly reliable when available. A major currently associated concern is the suspected, but as yet unproven increase of asthma and asthma-like diseases in the general population [114]. Fortunately though, hypersensitivity reactions are seldom severe or even life-threatening. The majority of these reactions are actually mild to moderate, and self-limiting. Extremely severe reactions, such as anaphylactic shocks, agranulocytosis, and toxidermias, do nevertheless develop. The clinical manifestations of drug and chemical hypersensitivity are manifold: the skin and the lung are the most commonly affected target organs, but the liver and the kidney can also be involved as well as other target organs, such as the blood cells. A major difficulty when addressing the issue of drug and chemical hypersensitivity is that so little is known of the underlying mechanisms. Only a few mechanisms have been fully elucidated. Examples are T lymphocyte-mediated contact hypersensitivity or IgE-mediated anaphylaxis. Despite continuing and ever-increasing efforts, the prediction of drug and chemical hypersensitivity reactions is only possible to a very limited extent in laboratory animals. In fact, the mechanism of most hypersensitivity reactions is not known, and unsurprisingly their prediction in animals [62] or their diagnosis in human beings [111] is always difficult, not to say impossible in many instances. Progress in this area can be expected when a better understanding of the underlying mechanisms is gained. Fortunately, more and more immunotoxicologists from the industry and academia are becoming aware that drug and chemical allergies do represent a major health issue.


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Another difficulty with hypersensitivity is that either immune-mediated or nonimmunemediated mechanisms can be involved. The word pseudoallergy has been coined to refer to those clinical adverse reactions due to nonimmune-mediated mechanisms, but mimic immune-mediated allergic reactions, namely those reactions that involve specific immunological recognition and memory [115]. Because of frequent uncertainties related to the mechanisms involved in immune allergic reactions and the possible confusion between immuno-allergy and pseudo-allergy, the word hypersensitivity is often used. Recently, the European Academy of Allergology and Clinical Immunology proposed to refer to the generic term hypersensitivity to describe reproducible symptoms initiated by exposure to a defined stimulus at a dose tolerated by normal human beings, and to divide hypersensitivity into immunological and nonimmunological hypersensitivity depending whether immune-mediated mechanisms are suspected or documented to be involved [116]. AUTOIMMUNITY Although autoimmune diseases are frequent diseases in the general population, autoimmune reactions caused by medicinal drugs seem to be relatively rare events [117]. From an immunotoxicological perspective, it is absolutely essential to differentiate autoimmune reactions from autoimmune diseases. Autoimmune diseases have been reported to be more frequent in patients treated with certain therapeutic recombinant cytokines, such as rIL-2 and the interferons-a [98]. There is a recent and growing concern that industrial and environmental chemicals may be ignored causes of autoimmune diseases [118]. Whatever the cause either a drug or a chemical, the issue is whether a supposedly immunotoxic exposure can result in more frequent autoimmune diseases that are totally similar, both clinically and biologically, to spontaneous autoimmune diseases. In general, as suggested by the interferons-a, any kind of autoimmune diseases can be described as potentially more frequent in treated or exposed human beings. The concept of autoimmune reactions is different in that it refers to certain autoimmune processes that are typically associated with only few drug or chemical exposures. For example, the antihypertensive drug a-methyldopa has only been shown to induce autoimmune hemolytic anemia. The mechanisms involved are unknown, whatever the type of autoimmune reaction or disease. In fact, the fundamental mechanisms of autoimmunity in general are unknown. Therefore, progress in this field is likely to be very slow and current procedures to test for drug and chemical-induced autoimmunity in laboratory animals are inadequate [119]. Autoimmunity is nevertheless an important area of immunotoxicology, especially because autoimmune diseases are frequent diseases in the general population and some experimental data suggested a possible link between chemical exposures and autoimmunity.

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[87] Zelikoff JT, Raymond A, Carlson Y et al. (2000) Biomarkers of immunotoxicity in fish: from the lab to the ocean. Toxicol Lett, 112/113, 325– 332. [88] Ross P, De Swart R, Addison R, Van Loveren H, Vos J, Osterhaus A (1996) Contaminant-induced immunotoxicity in harbor seals: wildlife at risk? Toxicology, 112, 157– 169. [89] Fournier M, De´gas V, Colborn T, Omara FO, Denizeau F, Potworowski EF, Brousseau P (2000) Immunosuppression in mice fed on diets containing beluga whale blubber from the St Lawrence estuary and the Arctic populations. Toxicol Lett, 112/113, 311– 317. [90] Sundwall A, Andersson B, Balls M et al. (1994) Immunotoxicology and in vitro possibilities. Toxicol in vitro, 8, 1067– 1074. [91] Reference deleted at proof stage. [92] Pallardy M, Lebrec H, Kerdine S et al. (2000) In vitro immunotoxicology. In: Gad SC (Ed) In vitro Toxicology, 2nd edition, pp 347– 364. Taylor & Francis, New York. [93] Lovik M (1997) Mutant and transgenic mice in immunotoxicology: an introduction. Toxicology, 119, 65 – 76. [94] Karol MH, Graham C, Gealy R, Macina OT, Sussman N, Rosenkranz HS (1996) Structure – activity relationships and computer-assisted analysis of respiratory sensitization potential. Toxicol Lett, 86, 187– 191. [95] Descotes J (1998) Regulating immunotoxicology: issues and needs. Arch Toxicol, Suppl. 20, 293– 299. [96] Vial T, Descotes J (1995) Immune-mediated side-effects of cytokines in man. Toxicology, 105, 31 – 57. [97] Vial T, Chevrel G, Descotes J (2000) Drugs acting on the immune system. In: Dukes MNG, Aronson JK (Eds) Meyler’s Side-Effects of Drugs, 14th edition, pp 1246– 1337. [98] Miossec P (1997) Cytokine-induced autoimmune disorders. Drug Safety, 17, 93 – 104. [99] Griem P, Wulferink M, Sachs B, Gonza´lez JB, Gleichmann E (1998) Allergic and autoimmune reactions to xenobiotics: how do they arise? Immunol Today, 19, 133– 141. [100] De Rosa FG, Bonora S, Di Perri G (2002) Tuberculosis and treatment with infliximab. N Engl J Med, 346, 623– 626. [101] Descotes J, Choquet-Kastylevsky G, Van Ganse E, Vial T (2000) Responses of the immune system to injury. Toxicol Pathol, 28, 479– 481. [102] Van Loveren H, De Jong WH, Vandebriel RJ, Vos JG, Garssen J (1998) Risk assessment and immunotoxicology. Toxicol Lett, 102–103, 261– 265. [103] CPMP (2000) Note for guidance on repeated dose toxicity testing. CPMP/SWP/1042/99. Document available at http://www.health.gov.au/tga/docs/pdf/euguide/swp/ 104299en.pdf. [104] Opelz G, Dohler B (2001) Cyclosporine and long-term kidney graft survival. Transplantation, 72, 1267– 1273. [105] Askenase PW, Hayden BJ, Gershon RK (1975) Augmentation of delayed-type hypersensitivity by doses of cyclophosphamide which do not affect antibody responses. J Exp Med, 141, 697– 702. [106] Thomson AW, Moon DK, Inoue Y, Geczy CL, Nelson DS (1983) Modification of delayed-type hypersensitivity reactions to ovalbumin in cyclosporin A-treated guinea-pigs. Immunology, 48, 301– 308. [107] Spreafico F (1985) Problems and challenges in the use of immunomodulating agents. A general introduction. Int Arch Allergy Appl Immunol, 76(Suppl. 1), 108– 118. [108] Renoux G (1986) Characterization of immunotherapeutic agents: the example of imuthiol. Methods Find Exp Clin Pharmacol, 8, 45 – 50. [109] Descotes J (1985) Adverse consequences of chemical immunomodulation. Clin Res Pract Drug Regul Affairs, 3, 45 – 52. [110] Weston BJ, Henderson DC, Seal S, Spackman V, Robertson F, Dewdney JM (1991) Stimulatory effects of FK156 in a panel of tests designed to detect changes in immune function. Int J Immunopharmac, 13, 117– 124. [111] Bigby M (2001) Rates of cutaneous reactions to drugs. Arch Dermatol, 137, 765– 770.

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[112] Demoly P, Bousquet J (2001) Epidemiology of drug allergy. Curr Opin Allergy Clin Immunol, 1, 305– 310. [113] Vos JG, Younes M, Smith E (1995) Allergic Hypersensitivities Induced by Chemicals. CRC Press, Boca Raton. [114] Wieringa MH, Vermeire PA, Brunekreef B, Weyler JJ (2001) Increased occurrence of asthma and allergy: critical appraisal of studies using allergic sensitization, bronchial hyperresponsiveness and lung function measurements. Clin Exp Allergy, 31, 1553– 1563. [115] Manning ME, Stevenson DD (1991) Pseudo-allergic drug reactions. Aspirin, nonsteroidal antiinflammatory drugs, dye additives and preservatives. Immunol Allergy Clin N Am, 11, 659–678. [116] Johnson SGO, Hourihane JO, Bousquet J, Bruijnzeel-Koomen C, Dreborg S, Haahtela T, Kowalski ML, Mygind N, Ring J, Van Cauwenberge P, van Hage-Hamsten M, Wuthrich B, EAACI (the European Academy of Allergology and Clinical Immunology) nomenclature task force (2001) A revised nomenclature for allergy: an EAACI position statement from the EAACI nomenclature task force. Allergy, 56, 813– 824. [117] Vial T, Nicolas B, Descotes J (1994) Drug-induced auto-immunity: experience of the French Pharmacovigilance system. Toxicology, 119, 23 – 27. [118] Heimer H (1999) Environment and auto-immunity. Environ Health Perspect, 107, A504 –A509. [119] Descotes J (2000) Auto-immunity and toxicity testing. Toxicol Lett, 112/113, 461– 465.

Chapter 2

The immune system: a toxicologist’s overview

There is a long-established tradition that review papers, books or symposia devoted to immunotoxicology include an introductory overview of the immune system. There is no doubt that toxicologists when they are not familiar with immunology can benefit from such overviews. Nevertheless, the question can be asked why immunologists who are seldom familiar with toxicology might not benefit from an overview of toxicology since immunotoxicology is definitely a discipline intended to bridge the gap between immunologists and toxicologists. To avoid fruitless controversy, it will simply be considered that ample evidence is available to demonstrate that immunotoxicology is primarily an area of toxicology. As it is beyond the scope of this chapter to provide a comprehensive coverage of immunology, the focus will be on essential concepts that are of particular relevance for the design of immunotoxicity studies and the interpretation of results. Quite a few excellent textbooks are available for those readers willing to access to a more in-depth and specialized coverage of immunology and the immune system.

INTRODUCTION The immune system is a complex network of cells and molecules sharing a common goal: the detection of self from non-self to defend the integrity of the body [1]. Molecules that can bind to receptors on lymphocytes are antigens which range from small to highly complex molecules. Antigens that elicit immune responses are termed immunogens. Large antigens, such as proteins, can be directly immunogenic. In contrast, smaller antigens called haptens must first be bound to carrier macromolecules to induce a specific immune response. The primary function of the immune system is the protection against invading microbial pathogens including bacteria, viruses, fungi, yeasts, and tumors. This role of the immune system is crucial to survival. In order to defend the host, the immune system either simultaneously or independently utilizes a wide variety of mechanisms which are either nonspecific because they lack immunological memory (innate or natural immunity) or specific (adaptive or acquired immunity). Nonspecific defenses include physical barriers, such as the skin and the mucosa of the respiratory, gastro-intestinal and uro-genital tracts. 19

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When these barriers are breached, an array of cells, primarily the white blood cells (leukocytes) and various serum factors, such as the complement cascade and the interferons (IFN), are activated to fight invading pathogens. Specific immune responses include humoral immunity resulting in the production of antigen-specific antibodies by plasma cells derived from B lymphocytes, and cellular immunity involving T lymphocytes. Upon a first encounter with the invading pathogen, 7 –10 days are generally required for an effective specific immune response to be mounted. The term specific refers to the recognition of foreign structures (antigens) by antigen-recognition molecules, either antibodies or T cell receptors (TCR), which bind to small portions of the antigen—the antigenic determinants or epitopes. A critical feature of specific immune responses is immunological memory. Upon a subsequent encounter with the same antigen, the immune response is both faster and greater. Recent data indicate, however, that innate immunity can also be instrumental in mounting specific immune responses, particularly via the recognition of molecular patterns on certain classes of microbial pathogens via toll-like receptors (TLRs). Closely inter-twined processes assure coordinated immune responses. Specialized (immunocompetent) cells, including leukocytes and accessory cells, are involved in immune responses. The renewal, activation and differentiation of these cells are required to achieve a normal level of immunocompetence and they are under the control of many mechanisms with either redundant or conflicting outcome. Redundancy is indeed an essential feature of the immune system. Thus, the adverse clinical consequences of morphological changes as well as functional immune alterations caused by drug treatments or chemical exposures are often difficult to anticipate because of compensatory mechanisms. Redundancy contributes to the functional reserve capacity of the immune system. There is no widely accepted definition of the functional reserve capacity. However, a large body of evidence shows that it is still possible to mount apparently normal immune responsiveness in the presence of mild to moderate impairment in one or several immune functions. Depletion in immune cells, dysregulation of immune mechanisms or functional defects are expected to facilitate pathological events characterized by disturbances of recognition mechanisms distinguishing the self from the non-self, thus resulting in an increased susceptibility of the host toward infectious pathogens and neoplasia. It is noteworthy that although the primary goal of the immune system is protection of the host, immune responses are not always beneficial. Thus, immune responses against selfconstituents of the host can result in a variety of autoimmune diseases, either organ-specific or systemic, and inadvertent immunological reactivity against obviously innocent antigens, e.g. penicillin or pollens, is the cause of hypersensitivity reactions that can be lifethreatening. Finally, excessive activation of normal immune responses has been shown to be potentially harmful as evidenced by the adverse clinical effects of potent immunoactivating drugs, e.g. recombinant cytokines, or an excessive stimulation of the immune system by infectious agents. An overview of mechanisms involved in the immune-mediated adverse effects of immunotoxicants can be found in Chapter 4 of this volume. From the viewpoint of a toxicologist, a major limitation of current research and teaching in immunology is that a global approach of the immune system and immune responsiveness is seldom, if ever proposed, an opinion that is shared by few, but prominent immunologists [2]. Because of the redundancy of the immune responses, the role of the functional capacity reserve of the immune system, and significant interactions between the immune system


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and other physiological systems of the body, such as the nervous and endocrine systems, the interpretation of immune alterations caused by possible immunotoxicants in nonclinical as well as clinical studies should always be carefully based on a global approach.

THE LYMPHOID ORGANS The immune system consists of various compartments interconnected by the blood and the lymphatic vessels. Although immunocompetent cells can be found in the blood and in almost all organs or tissues of the body, they tend to concentrate into specific anatomical structures, the lymphoid organs. A few organs, such as the eyes, testis and the brain, do not have lymphoid cells and are said to be immunologically privileged. Lymphoid organs contain a large number of major immunocompetent cells, the lymphocytes which closely interact with nonlymphoid cells. Interactions between lymphocytes and nonlymphoid cells are essential for the maturation of lymphocytes as well as the initiation and continuation of immune responses. Typically, lymphoid organs are divided into central (or primary) and peripheral (or secondary) lymphoid organs because they play distinct roles in the production and the maturation of immunocompetent cells, as well as the mounting of immune responses. CENTRAL LYMPHOID ORGANS Central lymphoid organs are lympho-epithelial structures which develop early during organogenesis independently of antigenic stimulations. They assure the production and/or the maturation of lymphocytes. The bone marrow—the major hematopoietic organ in mammals including man—is the equivalent of the bursa of Fabricius in birds. All blood cell types at different stages of maturation and their progenitors (except mature T lymphocytes) can be found in the medullary cavity of bones. Hematopoiesis takes place within the extra-vascular spaces of the bone marrow sinuses. The stroma is made up of various cell types including reticular cells and adipocytes which interdigitate with the single layer of endothelial cells lining sinuses. In normal conditions, senescent blood cells are continuously replaced by the bone marrow. All blood cells derive from a common multipotent progenitor cell. The stroma plays a major role in hematopoiesis by providing a matrix where populations of progenitor cells are produced, but also by releasing soluble factors that regulate hematopoiesis. Among factors that are produced by stroma or accessory cells, cytokines and the growth factors interleukin-3 (IL-3), macrophage-colony stimulating factor (M-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF) exert such a critical influence that recombinant factors have been produced for therapeutic use when hematopoiesis is failing or damaged. Multipotent progenitor cells give rise to distinct lymphoid and myeloid progenitor cells that follow complex differentiation pathways ending in a variety of cell types which are found in the blood and/or tissues (Fig. 1). In addition, B lymphocytes mature in the bone marrow, hence its role as a central lymphoid organ. The B cell lineage is derived from lymphoid progenitor cells that

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Fig. 1. Cells of the immune system and their differentiation pathways. All cells of the immune system derive from a common bone marrow multipotent progenitor cell. Progressive differentiation and maturation leads to the myeloid and lymphoid lineages, and finally in well-characterized cells.


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differentiate from the multipotent progenitor cells. Several stages in B lymphocyte development have been identified, namely the pro-B cells which are characterized by the appearance of specific surface markers, the pre-B cells and immature B cells corresponding to variable expression of components of the B cell receptor, the mature B cells which express surface IgM and IgG, the plasma cells that secrete antibodies and the memory B cells. During the transition from immature to mature B lymphocytes, the process of negative selection removes immature B cells bearing B cell receptors recognizing selfantigens. Alternatively, inactivation of these cells results in the induction of tolerance. The thymus is considered as the main central lymphoid organ because T lymphocytes— the key cells of the immunological orchestra—mature within the thymus. The different stages of T lymphocyte maturation are identified from the molecules expressed on their surface. Bone-marrow-derived thymocytes which enter the subcapsular zone are triple negative (TN) cells as they express none of the three major T lymphocyte markers CD3, CD4 and CD8. Thereafter, gene rearrangements leading to expression of the TCR and commitment to a,b or g,d lineages begin to occur. The next step is the double-positive (DP) thymocytes expressing both the CD4 and CD8 surface markers. They are mainly found in the cortex. Then, DP thymocytes migrate to the medulla and become singlepositive T lymphocytes, namely CD4þCD82 or CD42CD8þ T lymphocytes. Singlepositive thymocytes mature further and leave the thymus via the blood vessels to populate organs and the T lymphocyte areas of lymphoid tissues. The epithelial micro-environment and thymic hormones regulate the generation of mature T lymphocytes from immature thymocytes. More than 95% of immature DP thymocytes die within the cortex by apoptosis. Immature thymic T cells express an immense variety of TCRs and only those cells expressing TCRs that recognize MHC class I or class II molecules and associated self-peptides with moderate affinity escape from apoptosis and continue to differentiate into CD8þ or CD4þ T lymphocytes, respectively. This mechanism—positive selection—generates MHC-restricted T lymphocytes. Remaining auto-reactive T lymphocytes that react with self-antigens and may give rise to autoimmune diseases tend to be further eliminated by negative selection. PERIPHERAL LYMPHOID ORGANS Antigens and immunocompetent cells come into contact in the peripheral lymphoid organs because of their rich blood and lymphatic vascularization. These organs develop late during gestation and reach optimal development only after repeated antigenic stimulations. They contain small clusters of B lymphocytes—the lymphoid follicles—that are divided into primary follicles prior to antigenic stimulation and secondary follicles when containing a germinative center where memory B lymphocytes and precursors of plasma cells are generated following antigenic stimulation. CD4þ T lymphocytes and macrophages are also present. The spleen acts primarily as a filter in the blood. It is the site of immune responses against blood-borne antigens, especially opsonized microbes. It contains two main compartments: the red pulp and the white pulp. The red pulp is the most prominent compartment with

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macrophages, lymphocytes and plasma cells. Macrophages clear aged red blood cells, cell debris and nonopsonized foreign particles. This function is of critical importance for the removal of intra-vascular pathogens before antibody formation. In mice, and in rats to a lesser extent, the red pulp is also the site of extra-medullary hematopoiesis. Importantly, the marked differences in spleen cell functions between rodents and man have been suggested as a cause to reconsider the use of spleen cells for immunotoxicity evaluation [3]. About a quarter of all lymphocytes of the body are located in the white pulp. It consists of periarteriolar lymphoid sheaths—the thymo-dependent area—with many T lymphocytes and adjacent follicles containing B lymphocytes. The white pulp of the spleen plays a major role in immune responses to blood-borne antigens. The marginal zone located at the border of the red pulp and white pulp is the site of B lymphocyte memory and generates humoral responses to T-independent antigens, such as the polysaccharides of encapsulated bacteria. Lymph nodes are usually grouped at sites connecting blood and lymph vessels to assure direct contact between antigens and immunocompetent cells. The general structure of the lymph nodes is made up of three main areas: the cortical, paracortical and medullary areas. Antigens enter lymph nodes by the afferent lymphatic vessels and are processed by macrophages in the medulla. In the paracortex, free or processed antigens are presented to T lymphocytes by interdigitating cells and macrophages. The cortical area is the site where B lymphocytes encounter antigens. The medulla is the primary site of effector reactions involving macrophages, granulocytes and plasma cells. Circulating lymphocytes enter lymph nodes via the paracortical high endothelial venules. Mucosa-associated lymphoid tissue (MALT) is found underneath the secretory epithelium of the gut, the broncho-pulmonary tract (bronchi-associated lymphoid tissue or BALT) and the upper airways (nasal-associated lymphoid tissue or NALT), and tonsils and adenoids forming the ring of Waldeyer. The gut-associated lymphoid tissue (GALT) includes the Peyer’s patches in the duodenum and jejunum, and the appendix of the large intestine. Plasma cells in the lamina propria of the intestine secrete IgA as dimers or larger polymers after incorporation of the J chain and association with the secretory component, a transmembrane epithelial glycoprotein. In addition to IgA-producing plasma cells and intra-epithelial T lymphocytes (of which < 50% are g,d TCR-bearing CD8þ T lymphocytes), MALT contains a particular type of epithelial cells—the M cells—which take up inhaled or ingested antigens by pinocytosis. Because of its role in mucosal immunity, MALT is considered as a first line of defense even though its relevance to immunotoxicity is not fully understood [4].

IMMUNOCOMPETENT CELLS Cells involved in either innate or adaptive immunity originate in the bone marrow from multipotent hematopoietic stem cells and subsequently mature along different pathways. The lymphoid cell lineage gives rise to lymphocytes, and the myeloid cell lineage to neutrophils, basophils and eosinophils, monocytes and macrophages, dendritic cells (DCs), and mast cells (Fig. 1). Leukocytes express a more or less specific pattern of surface


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markers. The name CD—for cluster of differentiation—is universally accepted and to date, over 200 CD have been designated by ad hoc expert groups based on laboratory findings using specific antibodies to identify CD relevant to cell differentiation and function [5]. LYMPHOCYTES Lymphocytes are the primary cells involved in adaptive immunity. They are ovoid cells about 8 –12 mm in diameter. The three major populations of lymphocytes include T lymphocytes, B lymphocytes and natural killer (NK) cells. Although lymphocytes differ in size and morphology, they display no definite cytological or even ultrastructural characteristics, hence the use of cell-surface markers to distinguish between lymphocyte populations. Lymphocytes constitute 10% of circulating blood cells in pigs, 20 –40% in humans and dogs, but the percentage of lymphocytes is greater in monkeys (40 – 65%), mice (50 –70%) and rats (. 80%). In addition, there are marked inter-individual variations related to the age, sex and strain of animals, and even in specific-pathogen-free (SPF) animals [3]. The use of appropriate controls and the comparison to historical background values are therefore absolutely essential. T lymphocytes. Approximately 55 – 70% of circulating lymphocytes are T lymphocytes in man as well as rats, dogs, pigs and monkeys. All T lymphocytes express the TCR, a heterodimer consisting of one a and one b, or one g and one d chains. Over 90 –95% of T lymphocytes bear the a,b TCR, except in pigs where the percentage of g,d TCRbearing T lymphocytes is seemingly much higher. The function of g,d TCR-bearing T lymphocytes is not fully elucidated. Following maturation in the thymus, lymphocytes circulate in the blood and home to T dependent areas in the peripheral lymphoid organs. CD2 and CD3 are surface markers expressed by all T lymphocytes. T lymphocytes are subdivided into two distinct populations, the CD4þ T lymphocytes whose main function is to help other cells to mount immune responses and the CD8þ T lymphocytes which are cytotoxic and kill target cells. CD41 (helper) T lymphocytes consist of approximately two-thirds of all T lymphocytes in man, rats and dogs, but only one half in monkeys. Although all CD4þ T lymphocytes can release IL-3, GM-CSF and TNF-a, they are further divided into TH1 and TH2 lymphocytes which differ by the distinct profile of cytokines they secrete, primarily IL-2 and INF-g for TH1 lymphocytes, and IL-4, IL-5 and IL-10 for TH2 lymphocytes. CD4þ T lymphocytes recognize antigens presented by APCs in close association with MHC class II molecules. The dichotomy between TH1 and TH2 lymphocytes is not a pre-existing feature and instead, TH0 lymphocytes are presumably polarized to mount TH1 or TH2 immune responses by illunderstood mechanisms under the combined action of genetic and environmental factors. TH1 lymphocytes primarily activate the microbicidal function of macrophages by the release of IFN-g, whereas TH2 lymphocytes primarily release IL-4 and IL-5 that stimulate B lymphocytes and promote the secretion of IgE. CD81 (cytotoxic/effector) T lymphocytes recognize antigens presented by APCs in close association with MHC class I molecules. CD8þ T lymphocytes can be activated by encountering antigens presented by APCs or other cells. In any event, activation of CD8þ T lymphocytes requires a second signal derived either from co-stimulatory molecules when

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the antigen is presented by APCs or from cytokines released by CD4þ T lymphocytes. Activated CD8þ T lymphocytes thus differentiate into cytotoxic lymphocytes that lyse target cells. Similar patterns of cytokine secretion have been shown for CD8þ T lymphocytes that can also be subdivided into TC1 and TC2 lymphocytes. However, the respective role of TC1 and TC2 lymphocytes is not as clearly elucidated as that of TH1 and TH2 lymphocytes. B lymphocytes. Approximately 10 – 20% of peripheral blood lymphocytes are B lymphocytes in man and most conventional laboratory animal species. They first develop in the fetal liver, then in the bone marrow. They can be identified by the presence of the surface immunoglobulins IgM, IgD, IgG, IgA or IgE, and various differentiation cell markers including CD19, CD22, CD23 and CD37. Following antigen encounter by APCs, naive B lymphocytes proliferate and this event initiates the formation of germinal centers within the spleen and the lymph nodes. As a general rule, B lymphocytes cannot be activated by the antigen alone. A second accessory signal is required that is delivered by activated T lymphocytes. Antigens that require T– B lymphocyte collaboration to initiate an immune response are thymus-dependent antigens. A few antigens, such as bacterial polysaccharides or dextran, do not require T – B collaboration and are thymus-independent antigens. At the final stage of maturation, B lymphocytes are differentiated into antibodysecreting plasma cells. Memory lymphocytes. It has been estimated that lymphocytes are capable of producing about 1015 different antibodies and a similar number of TCR. The whole range of antigenbinding specificities is called the antigenic repertoire. The diversity of the antigenic repertoire is achieved by extensive rearrangements of less than 400 genes. There are normally no more than a few thousand lymphocytes specific for a given antigen. For the immune system to mount more vigorous and effective responses on re-exposure to previously encountered antigens, memory T and B lymphocytes persist in the body in a resting state for many years and even the whole of life, but there are also examples where protection afforded by memory lymphocytes is short-lived [6]. Although it has long been difficult to distinguish memory lymphocytes from other lymphocytes, there is now a general agreement that memory lymphocytes have a different cell phenotype. Thus, memory T lymphocytes no longer express CD62 (selectin L), but overexpress CD44 and particular isoforms of CD45. It is thought that some activated B lymphocytes can undergo genetic changes to create more effective antibodies. On subsequent exposure to the antigen, memory B lymphocytes respond to mount a more rapid and higher affinity antibody response, while memory T lymphocytes have less stringent requirement for co-stimulatory molecules and MHC molecules ligation to be activated. Natural killer (NK) cells have morphological features resembling those of large granular lymphocytes. Human NK cells are typically CD32 CD56þ CD16þ cells and comprise approximately 15% of all leukocytes. This percentage, however, is only 5% in rats. Importantly, murine NK cells do not express a homolog of CD56, and CD56 is not a suitable marker of NK cells in monkeys. Although most NK cells are non-T cells, a subset of T lymphocytes—the NKT cells—express characteristic markers of NK cells along with


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a TCR. The function of NKT cells is not well understood. The prime role of NK cells is to directly kill target cells, such as cells infected by viruses or tumor cells without prior sensitization through the release of cytotoxic mediators, e.g. perforin and granzymes that are stored in cytoplasmic granules. NK cells are able to recognize and kill cells that either is coated with IgG resulting in ADCC (antibody-dependent cellular cytotoxicity) or express lower than normal levels of MHC. Activation of NK cell cytotoxicity is thought to be the result of a balance between killer-activating and inhibiting receptors that recognize HLA molecules. The killer inhibiting receptors (KIR) primarily recognize the MHC class I molecules HLA-A, -B and -C. All nucleated normal cells express MHC class I molecules on their surface, but cells infected by some viruses and tumor cells lose this ability. Thus, inhibitory signals from KIRs are lacking and NK cells can kill the abnormal target cells. The contribution of NK cells to the host’s defense against viruses is demonstrated by the increased susceptibility of rodents and humans with low NK cell cytotoxic activity to a variety of viral infections [7 – 9] and cancers [10,11]. PHAGOCYTES Phagocytosis is the process by which certain cells can ingest particular ligands whose size exceeds 1 mm. It is the main mechanism of nonspecific host defenses to eradicate microbial pathogens and foreign bodies. Although a variety of cells exert phagocytic capacity, the most effective phagocytic cells are polynuclear neutrophils and the mononuclear phagocytes monocytes/macrophages. Phagocytes move close to their targets under the influence of chemotactic substances (chemokines), which adhere to their surface either nonspecifically (via lectins) or following opsonization, the main opsonins being IgG and C3b. Phagocytes engulf their prey within phagosomes where they release catalytic enzymes, such as lysozyme, cationic proteins, proteases and peroxidase, and free radicals, e.g. oxygen and nitrogen reactive species. Neutrophils are small cells that constitute approximately one half of the circulating white blood cells in most species [12] and over 95% of polynuclear blood cells. However, only 5 –35% of rat leukocytes are neutrophils. Their number is equilibrated between the pool of circulating and marginated neutrophils. Marginated neutrophils loosely adhere to the endothelial wall and can be rapidly released to the circulation. Neutrophils are characterized by a multilobed nucleus and the presence of two types of cytoplasmic granules: the primary (azurophil) granules that contain lytic enzymes, such as lysozyme, myeloperoxidase-derived substances and cationic proteins, and the secondary (specific) granules that contain enzymes, such as lactoferrin and collagenases. Each day, a normal adult releases 1011 neutrophils from the bone marrow to the circulation where their half-life is in the range of 4– 8 h. Because of this rapid turnover, neutrophils are particularly susceptible to toxic or immune-mediated insults. The primary function of neutrophils is phagocytosis. They migrate to the site of infection and/or inflammation through the wall of microvessels (diapedesis) in response to a chemoattractant signal (chemotaxis). Neutrophil accumulation is controlled by G-CSF, GM-CSF and IL-3. The first stage of phagocytosis is engulfment. This is a two-step process including recognition and internalization. In some instances, recognition is

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due to the direct binding of particles on neutrophils, e.g. LPS. Normally, recognition of microorganisms involves prior opsonization due to binding to plasma proteins that are specific (immunoglobulins) or not (complement components). Engulfed microorganisms are killed intracellularly and this process initiates the oxidative respiratory burst with z the subsequent release of superoxide anion (O2 2 ), hydroxyl radical ( OH) and hydrogen peroxide (H2O2). The pivotal role of neutrophils in host defenses against pathogens is illustrated by the development of severe, often life-threatening infections in patients with defects in neutrophil functions, such as the leukocyte adhesion deficiency, chronic granulomatous diseases or neutropenia of various origins [13]. In addition, the normal functions of neutrophils can result in adverse consequences including tissue damage as evidenced by reperfusion injury following thrombolysis in the acute phase of myocardial or cerebral infarction, vasculitis either systemic, such as Wegener’s syndrome, or localized and then typically drug-induced, and the acute respiratory distress syndrome (ARDS). Owing to their metabolizing capacities, neutrophils oxidize a number of xenobiotics and the metabolites thus generated can cause hypersensitivity and autoimmune-like reactions [14]. Monocytes/macrophages. Monocytes that usually represent 5% of circulating leukocytes are larger cells than lymphocytes with an irregular nucleus and numerous cytoplasmic granules and vacuoles. Bone marrow precursors expressing CD34 differentiate into monocytes under the influence of GM-CSF and M-CSF. The life span of monocytes in the circulation is between 8 and 70 h as they migrate to tissues where they mature and differentiate into macrophages. The vast majority of macrophages are found in tissues. However, intra-vascular macrophages fixed to the capillary bed of the lungs are present in humans and pigs, but not in rodents (except cirrhotic rats) and dogs. Their in situ activation can result in lung injury [15]. There is a wide morphological and functional polymorphism of macrophages according to their tissue location, e.g. Ku¨pffer cells in the liver, alveolar and peritoneal macrophages as well as species differences [3]. In contrast to neutrophils, monocytes and macrophages also play the role of APCs and thus participate in adaptive immune responses. However, antigens taken up by macrophages are rapidly degraded because macrophages are avidly phagocytic, so macrophages do not play a major role in antigen presentation. When activated, monocytes and macrophages release cytokines, such as IL-1, IL-6, and TNF-a, and this can result in target-organ toxicity, e.g. hepatotoxicity [16]. OTHER IMMUNOCOMPETENT CELLS In addition to the cells already mentioned, a number of other cell types are involved in nonspecific as well as specific immune responses. Dendritic cells (DCs) are mobile and irregularly shaped cells that actively produce a variety of cell processes [17,18]. Circulating DC precursors arise from bone marrow stem cells expressing CD34, which in turn give rise to immature DCs that home to tissues under the influence of various chemokines. Resident DCs are a heterogeneous and complex system of multiple subsets with distinct biological functions. For instance, interdigitating


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DCs and Langerhans cells are located at sites that interfere with the external environment, such as the skin and the mucosa surfaces, e.g. in the lungs and the gut. Other DC subsets are found in lymphoid and connective tissues or in the afferent lymph. Immature DCs are components of the innate immune system. Being scattered throughout the body, they can determine whether a host response is needed and if so, what kind of response is needed. Because they have a low phagocytic capacity, they degrade antigens by endocytosis to small peptides that can be presented to T lymphocytes. DCs also receive the so-called danger signals via specific receptors that are activated by endogenous substances, such as the heat-shock proteins, CD40L, and the cytokines TNF-a and IL-1, as well as exogenous substances. As discussed later in this Chapter, a number of microorganisms display pathogen-specific molecular patterns which bind to TLRs on the cell surface of immature DCs. Depending on the presence or magnitude of these signals, DCs mature, then attract and cluster with T lymphocytes to present antigenic peptides to those rare CD4þ T lymphocytes that express a TCR recognizing the MHC class II-peptide complexes at the surface of DCs in the presence of co-stimulatory molecules, such as CD11a, CD40, CD54, CD80 and CD86. DCs can present antigens to CD8þ T lymphocytes after proteosomal degradation and binding to MHC class I molecules. Mature DCs play a major role in the regulation of immune responses. They direct antigen-specific T lymphocytes either to die or to become anergic, memory or effector T lymphocytes. Inadequate DC –T lymphocyte interactions due to poor fit of the TCR for MHC class II-peptide complexes or lack of co-stimulation lead to T lymphocyte anergy or apoptosis. DCs are also thought to contribute to memory T lymphocyte persistence, but the mechanism involved is not fully elucidated. Finally, DCs trigger T lymphocyte proliferation. Mature DCs release IL-12 for a period of time less than 12 h, thus skewing the immune response to a TH1 response. In the absence of IL-12 release, they stimulate T lymphocytes toward a TH2 response. Finally, mature DCs regulate immune effector mechanisms, such as antigen-specific CD8þ T cell cytotoxicity and NK cell activity. Polynuclear leukocytes. In addition to neutrophils, polynuclear leukocytes include eosinophils and basophils. Eosinophils account for 1– 3% of peripheral blood leukocytes in normal healthy humans with an upper limit of 350 cells/mm3. Similar values are normally found in most conventional animal species. They can be distinguished morphologically from other granulocytes by their bilobed nucleus and the presence of specific and primary granules. Specific granules store cationic proteins, such as the major basic protein (MBP), eosinophil cationic protein, eosinophil-derived neurotoxin and eosinophil peroxidase. Primary granules are only found at an early stage of development. Eosinophils express a variety of cytokine, chemokine and complement receptors, but because there is no eosinophilspecific surface marker, the use of eosin-like dyes is the most common detection method. Eosinophils are produced in the bone marrow from multipotent stem cells and IL-5 plays a pivotal role in this process. Overproduction of IL-5 in transgenic mice results in eosinophilia [19]. In contrast, IL-5 KO mice present with marked eosinophil depletion following antigenic challenge. Once released from the bone marrow, eosinophils circulate in the blood where their half-life is in the range of 8 – 18 h. Then, they distribute to tissues, especially the lungs and the gut.

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The mechanism of eosinophil activation is not fully elucidated. Activated eosinophils release pro-inflammatory mediators including the cationic proteins stored in specific granules, newly synthesized eicosanoids and cytokines. MBP accounts for 50% of all stored cationic proteins and is directly toxic to the lung epithelial cells with resulting bronchial hyper-responsiveness. The eosinophil-derived neurotoxin is toxic to myelinated neurons. Eosinophil peroxidase is partially homologous to neutrophil myeloperoxidase. Eosinophils are a major source of cysteinyl-leukotrienes, but release relatively low amounts of IL-1, IL-3, IL-4, IL-5, IL-8 and TNF-a. CD69 is a marker of eosinophil activation. Increased numbers of eosinophils in the blood are found in allergic diseases, such as asthma, rhinitis, atopic dermatitis and chemical allergies, in inflammatory disorders and some malignancies, and in helminth infections [20]. While studies in IL-15 KO mice do not support a major role of eosinophils in clearing helminths, eosinophils have pro-inflammatory effects in allergic diseases and asthma. Basophils. Less than 1% of circulating leukocytes are basophils. Although they share many similarities with mast cells, basophils are believed to represent a distinct cell lineage [21]. Basophils exhibit a segmented nucleus and are stained by basic dyes such as toluidine blue. They derive from bone marrow progenitor cells, mature in the bone marrow under the influence of IL-3, and then circulate in the blood with a lifespan of several days. Like mast cells, they express the high affinity receptor for IgE (Fc1RI). Activation of basophils is induced by cross-linking of Fc1RI (as seen in IgE-mediated hypersensitivity reactions) or by the complement components C3a and C5a via specific receptors resulting in pseudoallergic reactions. CC chemokines, N-formyl-methionylleucyl-phenylalanine (FMLP), IL-3, IL-5, GM-CSF and other endogenous substances can prime basophils for maximal activation. Activated basophils abruptly release preformed mediators stored in cytoplasmic granules including histamine, MBP and tryptase. CD63 is a marker of basophil activation. They also release newly formed mediators, such as leukotriene LTC4, and cytokines (IL-4, IL-13). The physiological role of basophils is not known. Mast cells are long-lived cells found in many sites, preferentially near blood vessels and under epithelial, serous and synovial membranes in the skin, the lung and the digestive tract. A characteristic feature is the presence of dense cytoplasmic granules that are stained by toluidine blue. Mast cells derive from bone marrow progenitor cells. They do not circulate in blood. They express the high affinity IgE receptor (Fc1RI), the C3a and C5a receptors and a variety of other receptors on their surface. Mast cells have been divided into subsets according to their contents in preformed mediators [22]. The main preformed mediators are histamine, the neutral proteases tryptase, chymase, carboxypeptidase and cathepsin D, and the proteoglycans heparin and chondroitin sulfate E. Like basophils, mast cells are activated by cross-linking of Fc1RI or by the complement components C3a and C5a (anaphylatoxins). This results in the release of granule contents within minutes. Subsequently, newly synthesized mediators including the eicosanoids prostaglandin D2 and leukotriene LTC4, and cytokines (predominantly TNF-a) are released into the external environment. This cascade of events results in immediate and late-phase hypersensitivity reactions. Pathologic excess of mast cells leads to mastocytosis that is characterized by fixed


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pigmented skin lesions, unexplained flushing and anaphylaxis, and elevated serum tryptase levels [23].

INNATE (NATURAL) IMMUNITY The immune system is traditionally divided into innate and adaptive immunity. The main difference lies in the specificity of the mechanisms involved. Innate immunity which appeared before adaptive immunity during evolution is the first line of defense of the body against invading pathogens [24,25]. It is made up of a number of components including epithelial cells, soluble factors and circulating cells. COMPONENTS OF INNATE IMMUNITY Epithelial defenses. The surface of the body is protected by epithelial barriers. Epithelial cells from the skin, respiratory, gastro-intestinal and uro-genital tracts defend the body from invading pathogens. Epithelial defense mechanisms are varied. They include mechanical mechanisms, e.g. flow of air or mucus over the epithelial surface which can entrap invading bacteria; chemical, e.g. secretion of acid or hydrolytic enzymes (such as lysozyme), or antimicrobial peptides; and microbiological mechanisms, e.g. the competing role of the normal gut flora. Thus, neonates whose gastric pH is less acidic than in adults develop more frequent intestinal infections. As soon as an invading pathogen breaches an epithelial barrier, other mechanisms of innate immunity are brought into play. Soluble factors. A wide variety of soluble factors, including antimicrobial peptides, the complement system, acute-phase proteins, chemokines and pro-inflammatory cytokines are involved in innate immunity. Antimicrobial peptides are polypeptides less than 100 aminoacids divided into two main families: the defensins and the cathelicidins [26]. Defensins are widely represented among vertebrate species. There are two main sub-families, the a and b-defensins with markedly different structure. A third sub-family, the u-defensins, has recently been identified in rhesus monkeys. Defensins are abundant in cells and tissues that are primarily involved in innate immunity. The highest concentrations are found in the granules of leukocytes. However, there are species-differences. For example, mice in contrast to rats have no leukocyte defensins. Defensins are stored in the primary (azurophil) granules of neutrophils. During phagocytosis, defensin-rich granules fuse with phagocytic vacuoles so that microorganisms are exposed to high concentrations of defensins. Most defensins show antimicrobial activity against bacteria, fungi and enveloped viruses by inducing permeabilization of target membranes. The physiological role of defensins is supported by the findings that defensins are inhibited by the high salt content in the airways of patients with cystic fibrosis who develop more frequent respiratory infections. Cathelicidins have so far only been detected in mammals. In humans and in mice, there is only one cathelicidin, whereas in other animal species, such as pigs, many cathelicidins have been identified. Although much less is known of cathelicidins than defensins,

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the finding that cathelicidin genetically deprived mice develop more severe infections lends support to the role of these polypeptides in innate immunity. Complement. This term refers to more than 30 chemically and immunologically distinct proteins in man, hence the recommended use of the term complement system. Several proteins are proteases that are normally present in the circulation and some tissues in the form of functionally inactive molecules. The complement system functions as an enzymatic cascade: inactive proteases are cleaved to form activated proteins with the capacity to cleave downstream proteins. There are marked inter-species differences that must be carefully taken into account when investigating the influence of immunotoxicants on complement activation and function in conventional laboratory animal species. The complement system serves important physiological functions, which has long been shown by the development of various pathological conditions in patients with primary or secondary complements deficiencies [27,28]. Complement activation pathways. Three different pathways of complement activation have been described (Fig. 2). The classical pathway. Activation of the classical pathway is initiated by specific binding of the component C1q to the Fc region of antibodies present in immune complexes or on

Fig. 2. Activation pathways of the complement cascade. Activation of the complement cascades results in the production of either the classical or alternate C3 convertases which cleave C4 to produce the classical and alternate C5 convertases. Thereafter, C5 is cleaved into C5a and C5b which subsequently binds to C6, C7, C8 and C9 to produce the membrane-attack complex with marked cytolytic activity. The activation by-products C3a, C4a and C5a are named anaphylatoxins owing to their strong vasoactive and mediator-releasing properties.


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the surface of pathogens. C1q cannot be directly activated by free antibodies, but only by antibodies bound to multiple sites as on the surface of pathogens. IgM, then IgG1 and IgG3 are the most effective antibodies at activating the complement. C1q is activated after binding to the Fc region of antibodies and in turn activates the normally inactive serineproteases C1r and C1s. C1s interacts with C4 to form C4a and C4b, then with C2 which is cleaved into C2a and C2b. Both C4b and C2b form the complex C4bC2b which is the classical C3 convertase. The extent of complement activation by the classical pathway is under the control of the C1 inhibitor which dissociates C1r and C1s from C1q, and inactivates the spontaneous low activation of C1q. Congenital defects in C1 inhibitor result in hereditary angioneurotic edema. Other regulatory proteins include the C4-binding protein that controls activation of the classical C3 convertase. The alternate pathway. Activation of this pathway that was subsequently described— hence the term alternate pathway—is initiated by the spontaneous cleavage of C3, the most abundant complement protein in the plasma, to C3(H2O) which binds to factor B. Factor B is then cleaved to Bb by factor D. C3(H2O), Bb is a soluble convertase which cleaves C3 into C3a and C3b. A fraction of formed C3b binds to factor B which is then cleaved to Ba and Bb by factor D. C3bBb is the alternate C3 convertase. Regulatory proteins of the alternate pathway are found either in the plasma or on cell membranes. Complement receptor 1 (CR1) and the decay-accelerating factor (DAF or CD55) compete with factor B to prevent its binding to C3b. Factor I together with CR1 and the membrane co-factor of proteolysis (MCP or CD46) cleave C3b to the inactive protein iC3b. Finally, factor H binds to C3b and prevent the formation of C3bBb by competing with Bb. The lectin-mediated pathway. Activation of this third pathway is initiated by a protein very similar to C1q, the mannan-binding protein, which binds specifically to mannose and other sugars on the surface of pathogens. In turn, the bound protein forms a complex with two proteases very similar to C1r and C1s, MASP1 and MASP2, which are activated to cleave C4 and C2 with the resulting formation of the C4bC2b convertase. Effector functions. The activation of the complement cascade serves several important functions. Opsonization. Whatever the activation pathway, complement convertases cleave C3 to C3a and C3b. C3b binds covalently to the surface of pathogens or is hydrolyzed. Five receptors from CR1 to CR5 have been identified. The role of CR1 (CD35) is elucidated. Phagocytes bind C3b via CR1. Large amounts of C3b can be deposited on the surface of pathogens. C3b-bound pathogens are more easily taken up and destroyed by phagocytes. This facilitating mechanism is called opsonization. Mast cell and basophil activation. The small fragments released during activation of the complement cascade exert biological activities. Of these, C3a, C4a and C5a—the anaphylatoxins [29]—cause the release of inflammatory mediators by mast cells and basophils. This mechanism is involved in pseudoallergic reactions. Complement-induced lysis. Although formation of C3b is the major consequence of complement activation, the next step is the binding of the convertases C4bC2b or C3b, Bb to C3b to produce the C5 convertase which cleaves C5 to C5a and C5b. C5b initiates the assembly of the membrane-attack complex. In succession, C5b binds to C6, then C5bC6 to C7, and finally C5bC6C7 to C8, which results in the polymerization of C9. The membraneattack complex forms transmembrane pores leading to perturbation of membrane integrity

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and destruction of the pathogen. The role of the membrane-attack complex in host defense is limited, but deficiency in any of the C5 –C9 proteins results in severe Neisseria infections. This complex is also involved in the immunoallergic destruction of blood cells. Immune complex removal. Activation of the complement system enhances the removal of circulating immune complexes that cannot be engulfed by phagocytes. The number of IgG molecules in immune complexes is sufficient to bind and activate C1q, then C4 and C3. Immune complexes tagged with C4b and C3b are bound to CR1 on the surface of erythrocytes so that macrophages in the spleen and the liver can degrade immune complexes. If circulating immune complexes are not removed, they tend to enlarge and precipitate at the basement membrane of small blood vessels, especially in the kidney glomeruli by binding to CR1 expressed by podocytes. Deposition of immune complexes is a characteristic feature of systemic lupus erythematosus and deficiencies in the early components of the complement cascade. Acute-phase proteins are molecules whose concentrations increase by at least 25% in response to inflammation [30]. In addition to several complement proteins (e.g. C3, C4, C9, Factor B), they include C-reactive protein, serum amyloid A, fibronectin, antiproteases, such as a1-protease inhibitor or a1-antichymotrypsin, coagulation proteins, e.g. fibrinogen, plasminogen and urokinase. Following an infection, various types of trauma or stress, e.g. surgery, the plasma concentrations of acute-phase proteins increase. These changes are primarily associated with fever, but other signs and symptoms, such as behavioral changes including anorexia and somnolence, stimulation of the adrenal cortex, anemia, are typically seen. The protective role of acute-phase proteins is still hypothetical, but current evidence supports their beneficial role in infection, wound healing and adaptation to noxious stimuli. C-reactive protein has been shown to bind phosphocholines on foreign pathogens or damaged cells and thus activate the complement classical pathway. Haptoglobin, hemopexin or a1-antichymotrypsin protect against reactive oxygen species. Clinically, the most widely used indicator of an acute-phase response is C-reactive protein plasma concentration, which tends to replace erythrocyte sedimentation rate. Most normal subjects have C-reactive protein plasma concentrations below 2 mg/l, although normal or elevated plasma concentrations do not consistently correlate with health status. Chemokines are small cytokines which facilitate the directed migration (chemotaxis) of many cells including lymphocytes, monocytes, neutrophils and eosinophils [31,32]. To date, nearly 50 different chemokines have been identified. Depending on the configuration of cysteine residues near the NH2-terminal end, chemokines are classified into four major families, the CC, CXC, C and CX3C chemokines. Over 20 distinct receptors are known. Due to the diversity of chemokines and chemokine receptors, much remains to be done to understand fully their individual function and role. Recent data suggest that chemotaxis may not be their sole function. Chemokines are divided into two broad categories: inducible chemokines that recruit leukocytes in response to stress, and constitutive chemokines that are involved in the background trafficking of leukocytes and the architecture of secondary lymphoid organs. Chemokines play a major role in cell migration, stimulate leukocyte degranulation and promote angiogenesis. Chemokines and chemokine receptors are primarily involved in the extravasation of leukocytes from the blood into lymph nodes and sites of inflammation (diapedesis). The selectins and integrins are the major chemokines involved in this first step. Subsequently, leukocytes


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migrate within secondary lymphoid organs and tissues (chemotaxis). Chemokines have also been shown to play a significant role in a variety of pathological disorders, including cardiovascular diseases, allergy, transplantation, neuro-inflammation, cancer and HIVassociated disease [33]. Pro-inflammatory cytokines. In addition to their role in adaptive immunity, cytokines also directly defend the body against infectious invaders [34]. The interferons alpha (IFN-a) and beta (INF-b) are produced by virus-infected cells. They inhibit the replication of viruses and bind to a receptor expressed on virus-infected and surrounding cells. In addition, they enhance antiviral defenses and NK cell activity. Activated macrophages release the major pro-inflammatory cytokines IL-1, IL-6 and TNF-a, which induce fever and increased production of acute-phase proteins. RECOGNITION OF PATHOGEN-SPECIFIC MOLECULAR PATTERNS Although innate immunity has long been considered to be fully distinct from adaptive immunity, recent evidence showed that it is also involved in the facilitation of mounting specific immune responses. A remarkable feature of the innate immune system is its capacity to recognize a number of pathogens. When microbes breach the epithelial barrier, receptors, especially the TLRs, recognize molecular patterns that are characteristic of particular classes of microorganisms, such as the LPS of gram-negative bacteria, doublestranded RNAs of viruses, or mannan of fungi [35,36]. These molecular patterns are not found on self-tissues. At least 10 receptors from TLR-1 to TLR-10 have been identified in man. They are mainly expressed by cells involved in the first line of the host’s defense, such as macrophages, neutrophils, and epithelial or endothelial cells. Recognition of pathogen-associated molecular patterns results in the activation of signaling pathways to drive and control specific adaptive immune responses. TLRs have been suggested to be involved in sepsis, chronic inflammation or allergy. Recently, children suffering from recurrent pyogenic bacterial infections have been shown to bear mutations on certain TLRs [37]. TLRs are increasingly considered as potential drug targets to treat a variety of pathological conditions and the possible negative influence of toxicants on TLRs should not be overlooked. INFLAMMATION Inflammation is the cause of a variety of clinical signs and symptoms. This is a primary mechanism of the host’s defense against many aggressions produced by physical agents including excessively high or low temperature and irradiation, trauma (e.g. cuts or bruise), chemical agents, microbial pathogens and various immune reactions. Inflammation is primarily characterized by the movement of leukocytes and serum proteins from the blood to the extra-vascular compartment. The local release of vasoactive substances increases the blood flow to the inflamed area with a loss of plasma proteins and fluid due to increased microvascular permeability. The up-regulated expression of adhesion molecules on endothelial cells and the release of chemotactic factors (chemokines) result in the facilitated adherence of leukocytes on endothelial cells and their further migration to the site of inflammation where they can play their phagocytic role.

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A number of soluble mediators are released immediately or secondarily depending on the causative agent, and whether the inflammatory response is acute or chronic. These mediators include a variety of free radicals, such as oxygen and nitrogen reactive species that induce direct tissue and cell injury or promote chain reactions, such as lipid peroxidation resulting in further tissue damage. Membrane lipids are the source of mediators exerting potent biological effects, such as PAF-acether, the prostaglandins and leukotrienes. Tissue injury is associated with the release of a variety of proteases that induce further tissue damage. Activation of the complement cascade is often seen and either the final membrane-attack complex or activation by-products, such as C3a and C5a, contribute to the inflammatory reaction. Other potentially important soluble mediators include the kinins, pro-inflammatory cytokines and acute-phase proteins. The clinical signs and symptoms that are characteristic of inflammation include fever, local pain, and redness at the site of inflammation. Asthenia and sleep disorders can also occur. Fever is induced by the production of IL-1, TNF-a and IL-6. It is generally thought that IL-6 is transported into the brain to reach the preoptic-anterior hypothalamic area and trigger the synthesis of PGE2 which is the major pyrogenic factor. Pain is mainly caused by the interactions of bradykinin with sensory neurons that induce the subsequent release of various mediators, such as substance P and capsaicin. Phagocytic cells tend to remove cellular debris and injured cells during the acute phase of the inflammatory reaction. The cytokines and other soluble mediators that they release promote the mobilization and proliferation of fibroblasts involved in wound healing and whose activity is controlled by many factors including TNF-a, IL-4 and growth factors. However, phagocytosis may cease to be beneficial and lead to chronic inflammation.

ADAPTIVE (SPECIFIC) IMMUNITY Adaptive immunity comprises a variety of responses which, in contrast to innate immunity, are antigen-specific, that is to say they involve the exquisite capacity of the immune system to identify non-self from self-constituents and memorize this information for accelerated responsiveness after subsequent encounter with the antigen. The immense antigenic repertoire is thought to enable the immune system to recognize every non-self antigen. However, only a very small number of immunocompetent cells share the same antigenic specificities so that amplification of immune responses involving primed immunocompetent cells is absolutely essential. Specific immune responses are subdivided into humoral and cellular immune responses and both involved complex cell – cell interactions. CELL –CELL INTERACTIONS Intercellular communications are essential in the mounting of either humoral or cellular immune responses. Antigen-specific immune responses are the results of complex cell – cell interactions including the processing, then the presentation of the antigen to T lymphocytes under the strict genetic control of either MHC class I or II molecules. For optimal T cell activation, 2 signals are required: a TCR signal upon binding to the peptide – MHC on an antigen-presenting cell (APC) and an antigen-independent signal involving


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co-stimulatory molecules. If both signals are provided, T lymphocytes can proliferate and secrete cytokines. In the absence of co-stimulatory signals, T lymphocytes become anergic or apoptotic. Antigen presentation. Unlike surface Ig of B lymphocytes, which can recognize a wide range of antigens in their native form, TCRs can recognize an antigen only in the form of a peptide bound to an MHC molecule at the surface of APCs. Thus, antigens must first be processed into small peptides to be recognized. MHC molecules are membrane glycoproteins. There are two classes of MHC molecules, which each contains three types of molecules, namely the MHC class I molecules HLA-A, -B and -C, and the MHC class II molecules HLA-DP, -DQ, and -DR. Small peptides of 8 –10 aminoacid length are formed by the degradation of large cellular or viral proteins in the cytosol, then delivered to the endoplasmic reticulum where they can bind to MHC class I molecules. Exogenous antigens bind to surface receptors and are taken up by cells via phagocytosis and endocytosis where they are degraded in lysozomes to form larger peptides that are associated with MHC class II molecules. Superantigens are bacterial or viral proteins, which bind to MHC class II molecules. They can stimulate 5– 20% of T lymphocytes and have been involved in the pathogenesis of various diseases, such as septic shock, food poisonings and autoimmune diseases (e.g. diabetes and Crohn’s disease). Once a peptide – MHC complex appears on the cell surface it can be recognized by the corresponding TCR. Almost all cell types with the notable exception of erythrocytes express MHC class I molecules, but none simultaneously expresses both class I and class II molecules. MHC class I molecules present antigens of cellular origin to CD8þ T lymphocytes, whereas MHC class II molecules present antigens of extracellular origin to CD4þ T lymphocytes. Co-stimulatory molecules. When a naive T lymphocyte encounters a peptide –MHC complex, a signal is delivered through the TCR, but this signal is insufficient to trigger activation [38]. A second co-stimulatory signal is required. CD28 is the prototypic T lymphocyte co-stimulatory molecule which binds to the B7 molecules on APCs. CD28 ligation enhances the magnitude and duration of T lymphocyte responses. Other major components of the immunological synapse include CD40L CD40, CD2 LFA-3 (CD48, CD58), and LFA-1 ICAM-1 (Fig. 3). HUMORAL IMMUNITY After contact with an antigen, the immune system can produce antigen-specific antibodies. Antibodies. Antibodies are heterodimeric glycoproteins containing 3 –13% of carbohydrates, the immunoglobulins (Ig). There are five classes of Ig in humans and most mammals, namely IgG, IgM, IgA, IgE and IgD. IgG are further divided into four subclasses, IgG1 – 4, and IgA into 2 subclasses, IgA1 – 2. In rodents, IgG include the subclasses IgG1, IgG2a, IgG2b and IgG2c, but no IgA subclasses. Immunoglobulins consist of two heavy (H) and two light (L) chains linked by disulfide bridges of varying numbers and locations according to the Ig class or subclass. Disulfide bridges are critical to determine the tertiary structure of the immunoglobulins. Both chains contain variable (V) and constant (C) regions

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Fig. 3. The immunological synapse. The presentation of the antigen with a MHC molecule is not a sufficient signal to induce T lymphocyte activation. A second co-stimulatory signal is required involving interactions between C40L-CD40, CD2-LFA-3 (CD48 in mice, CD58 in man), CD28-B7 (CD80, CD86), and the production of cytokines including IFN-g, GM-CSF, IL-4, by T lymphocytes and IL-1, IL-6, Il-12, IL-15 and TNF-a by APCs.

that differ due to the variability of the aminoacid sequence. In addition, disulfide bridges define domains (VH, VL, C1L, C1H, C2H and C3H) in the aminoacid sequence (Fig. 4). The V regions are divided into framework and complementarity determining regions. The latter display the greatest aminoacid variability and act as antigenic determinants (idiotopes). Multiple independent idiotopes in a single immunoglobulin molecule are called idiotypes. Heavy chains differ according to the Ig class: g,m,a,1 or d chains, but there are only two types of light chains, namely k and l, whatever the Ig class. The N-terminal end of the chain is quite different between chains; this is the variable end which supports the antibody specificity. By contrast, the C-terminal end is much more consistent among Ig of the same class or subclass. The antibody site is formed by the variable domains of the light and heavy chains. Each Ig molecule can be divided into 2 Fab fragments, which bind the antibody and one Fc fragment, which is involved in various biological functions, such as complement activation, opsonization and placental transfer. Primary humoral responses. B lymphocytes encounter antigens in peripheral lymphoid organs. There are genes coding for constant ends and genes coding for variable ends of Ig that are located on different chromosomes. Thus, an extremely large number of Ig can be formed following gene rearrangements. Antibody production requires that B lymphocytes expressing antigen-specific membrane Ig are activated, which results in an increased number of antigen-responding cells (clonal expansion). While they proliferate, B lymphocytes are transformed into lymphoblasts that subsequently give rise to long-living memory B lymphocytes and antibody-secreting plasma cells.


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Fig. 4. Schematic representation of the structure of immunoglobulins. Antibodies are immunoglobulins of which five classes IgG, IgM, IgA, IgD and IgE have been identified. They consists of a heavy (H) and a light (L) chain. Disulfide bridges determine the tertiary structure of the molecule define variable (V) and constant (C) domains with more or less marked variability in aminoacid sequence in each chain. The V domains act as idiotopes which are complementary to specific antibody.

After a first encounter with the antigen (primary response), antibody production follows three phases (Fig. 5). The latency period usually lasts 3– 4 days before the first antibodies are detectable, but the duration of the latency period depends on the route of antigen administration, the dose and the nature of the antigen. Antibody titers subsequently grow exponentially and finally decrease either totally or down to a residual level. During a primary response, the first detected antibodies typically are IgM followed by IgG. Secondary humoral responses. The persistence of humoral immune memory is seemingly not due to chronic restimulation by depots of the specific antigen on the surface of follicular DCs as previously thought, but it is more likely due to intrinsic changes in the physiology and lifespan of B lymphocytes. Memory B lymphocytes are generated in germinal centers to become either memory cells in the bone marrow that maintain elevated serum antibody levels, or small resting B lymphocytes of the blood and peripheral lymphoid tissues. After a second encounter with the antigen (secondary response), antibody production is earlier and more marked. Antibodies are primarily IgG of a markedly higher affinity for the antigen (Fig. 5). CELLULAR IMMUNITY Immune responses can involve strictly cellular mechanisms without the production of antigen-specific antibodies. They are mediated by T lymphocytes either CD4þ or CD8þ T lymphocytes. CD4þ T lymphocytes are mainly cytokine-secreting lymphocytes that trigger and control immune responses, whereas CD8þ T lymphocytes are involved in T lymphocyte-mediated cytotoxicity to kill target cells. Cytokines are regulatory polypeptides released by leukocytes and other cells. They play a pivotal role in many immune responses including inflammation or specific immunity by

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Fig. 5. Primary and secondary antibody responses. In primary antibody responses (first encounter with the antigen), IgM are detectable after 3–4 days followed by IgG. In secondary antibody responses, higher affinity IgG are predominantly found and antibody titers are much greater.

providing exquisitely efficient cell –cell communication. In 2000, over 150 cytokines had been identified and cloned [39], and this number is still growing [40]. Cytokines include interleukins, chemokines, growth factors and many other endogenous substances. The cytokine network is extremely complex as cytokines mediate and regulate the metabolic function, differentiation and proliferation of immune cells as well as other cells via specific receptors. Not only one given cytokine usually has an impact on a variety of target cell populations—the so-called pleiotropic effects of cytokines—but distinct cytokines can also exert similar effects on the same targets and thus, contribute to the redundancy of immune responses. A major difficulty in our current understanding of the respective role of individual cytokines is that most data were obtained using in vitro assays or very particular experimental conditions. In addition few experiments have been conducted to provide information on the effects of combined cytokines. It is absolutely essential to keep in mind these limitations either when including cytokine assays in nonclinical as well as clinical


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studies, or when interpreting results obtained when using these assays [41]. It is beyond the scope of this chapter to provide a comprehensive and detailed review of all known cytokines. The focus will be on those cytokines that are of major interest for immunotoxicity assessment. Interferons (IFN) include three families that were initially identified from their respective antiviral activity. The IFNs-a and -b (or type-1 IFNs) have significant antiviral activity, whereas IFN-g primarily exerts immunomodulatory properties. IFN-a. At least 14 different genes code for structurally distinct forms of IFN-a. They are produced ubiquitously by monocytes, macrophages, B lymphocytes and NK cells. They act via the IFN-a/b receptor. The antiviral activity of IFN-a is due to the inhibition of virus replication within virus-infected cells and the stimulation of cytotoxicity by NK cells and T lymphocytes. In addition, IFN-a up-regulates the expression of MHC class I molecules and down-regulates the expression of MHC class II molecules. The immunoregulatory effects of IFN-a are increasingly recognized. Recombinant IFNs-a are widely used in the treatment of viral diseases, particularly chronic hepatitis C and in several malignancies because of their antiproliferative effects. IFN-b. The structure and effects of IFN-b are closely similar to those of IFN-a. There is only one form of IFN-b and its effects are mediated by the IFN-a/b receptor. Recombinant IFN-b has been approved for the treatment of multiple sclerosis. IFN-g is essentially produced by T lymphocytes and NK cells. It exerts limited antiviral activity, but is central in determining the effectiveness of immune responses against microbial pathogens. IFN-g enhances antigen presentation and cytokine production by monocytes, and promotes the intracellular killing of microbial pathogens. It stimulates NK cells and neutrophils. IFN-g is the most important cytokine in cellular immunity. IFN-g increases the expression of constitutive MHC class I molecules and induces the expression of MHC class II molecules on cells that normally do not produce them. It promotes TH2 responses and is also an inhibitor of allergic (TH1) responses mediated by IL-4. Interleukins are mainly released by T lymphocytes. They have been numbered in the order of their discovery. To date, 30 interleukins have been identified. The major sources and biological effects of interleukins are summarized in Table 1. IL-1. The IL-1 family includes IL-1a and IL-1b, the IL-1 receptor antagonist (IL-1ra) and IL-18. IL-1a and IL-1b share similar structures and biological properties. Both are synthesized as pro-IL-1 proteins, but only pro-IL-1a is active and can bind to the two IL-1R. IL-1 is released by many cells including primarily monocytes and macrophages, but also endothelial cells, keratinocytes, astrocytes, and many other cells. Many stimuli, such as LPS of gram-negative bacteria, cytokines, microorganisms and antigens induce IL-1 production. IL-1 plays a crucial role in the development of specific responses to infectious agents and in inflammation. Its major effect is the activation of T lymphocytes by enhancing IL-2 production and IL-2R expression. In addition, IL-1 increases B lymphocyte proliferation and antibody synthesis. IL-1 exerts effects at a distance from the site of release, such as the central nervous system to cause fever, anorexia and consciousness disturbances, and the liver to stimulate the synthesis of acute-phase proteins. IL-1 shares many biological effects of TNF-a, but the latter has no direct effect on lymphocyte

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Table 1.Major sources and biological effects of the interleukins Major sources

Major effects

IL-2

Activated macrophages, NK cells, endothelial cells, B cells, fibroblasts, astrocytes T cells

IL-3 IL-4

T cells TH2 cells, mast cells

IL-5

TH2 cells

IL-6

B and T cells, fibroblasts, macrophages, endothelial cells

IL-7

IL-9

Bone marrow and thymus stroma cells, splenocytes Macrophages, endothelial cells and many cells TH2 cells

Activation of T lymphocytes and APCs, production of acute phase proteins, induction of IL-2 production Stimulation of T and B cells, macrophages, NK cells Growth factor of bone marrow precursor cells Secretion of IgG4 and IgE by B cells, inhibition of TH1 cytokine production Growth factor of eosinophils, proliferation and differentiation of B cells B and T cell proliferation, induction of acutephase reaction, synergistic effects with IL-1 or TNF-a Proliferation and differentiation of thymocytes, B and T cells Chemotactic factor of neutrophils

IL-10

TH2 cells

IL-11 IL-12

Fibroblasts, bone marrow stroma cells Macrophages, B cells

IL-13

T cells

IL-14

B and T cells

IL-15

Epithelial cells, monocytes

IL-16 IL-17 IL-18 IL-19 IL-20 IL-21

CD8þ T cells Activated memory T cells Ku¨pffer cells, osteoclasts, keratinocytes Monocytes Keratinocytes T lymphocytes

IL-22 IL-23 IL-24 IL-25

T lymphocytes, mast cells Macrophages, dendritic cells T lymphocytes, NK cells TH2 lymphocytes

IL-1 ab

IL-8

Stimulation of T cells and mast cells, inhibition of T cell apoptosis Activation and proliferation of B cells, thymocytes and mast cells, inhibition of IFN-g production Growth factor of bone marrow precursor cells Stimulation of IFN-g production, activation and differentiation of TH1 cells, cytotoxicity Proliferation and differentiation of B cells, secretion of IgE Proliferation of activated B cells, inhibition of Ig synthesis Growth factor of T cells, memory T cells, NK cells Chemoattractant for CD4þ T cells Stimulation of T cell proliferation Induction of IFN-g and IL-4 production Apparently similar to IL-10 Apparently similar to IL-10 Maturation and proliferation of B lymphocytes and NK cells Apparently similar to IL-10 Apparently similar to IL-12 Apparently similar to IL-10 Increased IgE production and eosinophilia

proliferation. IL-1ra is thought to modulate the effects of IL-1 and particularly to protect against IL-1 deleterious effects. IL-2 is produced almost exclusively by T lymphocytes when activated by the antigen and co-stimulatory signals. IL-2 augments the expression of its own high-affinity receptors (IL-2R) on other T lymphocytes, which in turn release various cytokines that activate immunocompetent cells. The proliferative effect of IL-2 is crucial because it produces large


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numbers of activated cells from rare antigen-specific naive T lymphocytes. IL-2 is an essential factor in the magnitude of T and NK cells responses, the induction of IFN-g production, the activation of B lymphocytes and macrophages. The importance of IL-2 is reflected in the potent immunosuppressive effects of cyclosporine, tacrolimus and sirolimus which inhibit IL-2 production by different mechanisms. Recombinant human IL-2 is currently approved for the treatment of cutaneous T cell lymphoma, hemangioendothelioma, metastasic melanoma and renal cell carcinoma. Clinical trials are under way in HIV patients or for the treatment of other tumors. IL-3 is produced by T lymphocytes, eosinophils and mast cells. It is a growth factor for most bone marrow precursors of immunocompetent cells. IL-4 is mainly produced by CD4þ TH2 lymphocytes, but also eosinophils and basophils. In eosinophils and mast cells, IL-4 is stored in granules and can be released immediately. Overall, IL-4 promotes allergic responses and inhibits cellular immunity [42]. It assures the differentiation of CD4þ T lymphocytes into TH2 lymphocytes that produce IL-4 and IL5. It enhances the antigen-presenting capacity of B lymphocytes and favors the isotype switch from IgM to IgE. IL-5 is the main regulator of eosinophil production, maturation and activation [43]. It contributes to immune defenses against helminths and infectious agents of the digestive and respiratory tracts. Increased eosinophil number and activation caused by elevated IL-5 production are associated with asthma or hypereosinophilic syndromes. Anti-IL-5 antagonists, such as mepolizumab [44] or imatinib mesilate [45], are being considered as possible treatment of inflammatory and allergic diseases. IL-6 is mainly produced by monocytes and macrophages, but also by B and T lymphocytes, keratinocytes, endothelial cells and many other cell types. IL-6 production is triggered by varied stimuli including LPS, TNF-a, IL-1, and IL-2. IL-6 is involved in the differentiation of B lymphocytes to antibody-secreting plasma cells, and the activation and differentiation of T lymphocytes. IL-6 has marked pro-inflammatory properties and is thought to be the most important inducer of acute-phase proteins production by hepatocytes. However, IL-6 also exerts anti-inflammatory effects by inhibiting IL-1 and TNF-a production and stimulating IL-1ra synthesis. IL-9 was first described as a mast cell-growth factor. It is produced by eosinophils and TH2 lymphocytes. It has important effects in allergic inflammation and on antigen-activated T lymphocytes [46]. IL-10 is a potent anti-inflammatory and immunosuppressive cytokine produced by many cell types including monocytes and B lymphocytes, and also macrophages, T lymphocytes, mast cells and keratinocytes [47]. It inhibits the synthesis of many chemokines as well as cytokines including IL-1, IL-6, IL-8, IL-12 and TNF-a produced by monocytes and macrophages, IL-2 and IFN-g produced by TH1 lymphocytes, IL-4 and IL-5 produced by TH2 lymphocytes. In contrast, IL-10 stimulates B lymphocytes and antibody secretion. Elevated IL-10 levels are associated with decreased resistance to infection due to the inhibition of inflammatory responses induced by microbial pathogens. Based on experimental results showing a protective effect of IL-10 in animal models of autoimmune diseases, recombinant human IL-10 is being considered for the treatment of various immune diseases, such as rheumatoid arthritis, type I diabetes mellitus, lupus erythematosus or psoriasis [48].

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IL-11 contributes to the production of lymphoid cells, red blood cells, platelets and mast cells [49]. It is also a stimulating factor for the synthesis of acute-phase proteins and for fibroblasts. IL-12 is a heterodimer primarily produced by monocytes and macrophages, but also neutrophils, B lymphocytes, mast cells, Langerhans cells and DCs [50]. IL-12 acts via the high-affinity receptor IL-12R. IL-12 primarily triggers activation, proliferation and cytokine production of NK cells. The production of IL-12 is induced by products from microorganisms, such as bacteria, parasites, fungi, double-stranded RNAs and CpG-containing nucleotides, and by various cytokines including IFN-g, IL-4 and IL-13. In contrast, IL-10 is a potent inhibitor of IL-12 production. IL-12 is therefore essential in the interactions between innate and adaptive immunity, but its most striking role is the regulation of the TH1/TH2 balance. IL-12 promotes and sustains TH1 responses with a resulting marked increase in IFN-g production. Thus, IL-12 plays a critical role in promoting resistance against intra-cellular infectious pathogens, such as Leishmania, Toxoplasma gondii, Listeria monocytogenes, and Cryptococcus neoformans, as well as tumors, as shown in many experimental models [51]. IL-13 shares many biological effects of IL-4 [52]. IL-15 effects on T lymphocytes are similar to those of IL-2, but IL-15 is not produced by activated T lymphocytes [53]. The major sources of IL-15 are monocytes, macrophages, epithelial cells and fibroblasts. As IL-15 is involved in the activation and differentiation of T lymphocytes and NK cells, it offers monocytes and macrophages a mean to control T lymphocyte and NK cell function. TNF-a. Tumor-necrosis factor (TNF-a) was first described in 1975 [54], although possible TNF-a related effects were fortuitously described in the late 19th century in inoperable surgical patients [55]. TNF-a is produced by monocytes, macrophages, activated lymphocytes, NK cells, endothelial cells and mast cells. LPS acting through TLRs is the most potent inducer of TNF-a by monocytes. The effects of TNF-a and the closely related lymphotoxin-a (formerly referred to as TNF-b) are mediated by two distinct receptors, namely TNF-R1 (p55) and TNF-R2 (p75). TNF-R1 is expressed constitutively on the surface of most cells, whereas TNF-R2 is found mainly on leukocytes and endothelial cells. The majority of TNF-a effects are thought to be mediated via TNFR1. The intracellular part of TNF-R1 contains a death domain. When TNF-a binds to TNF-R1, a number of accessory proteins are recruited and this results in the activation of the caspase pathway of apoptosis. However, caspase-independent pathways are also involved in the pro-apoptotic activity of TNF-a. TNF-a induces antitumor activity by a direct cytotoxic effect and by stimulation of antitumor immune responses. It interacts with endothelial cells to produce adhesion molecules which facilitate the migration of phagocytes to inflammatory foci. It is also a potent activator of neutrophil functions. TNFa causes cachexia in patients with severe infection or cancer, and is the main mediator of septic shock. Transforming growth factor-b (TGF-b) represents a family of peptides that regulate cell growth [56]. It is released as an inactive precursor that has to be cleaved to become active. It is inhibitory for B lymphocytes, and both CD4þ and CD8þ T lymphocytes.


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The production of TGF-b in GALT stimulates IgA synthesis and is critical for the maintenance of nonresponsiveness to gut pathogens and food allergens.

EXTERNAL INFLUENCES ON THE IMMUNE SYSTEM There are an extremely vast number of studies demonstrating the relevance and potency of external factors on immune responses. These include the central and autonomic nervous system, the neuroendocrine system, nutrition, age. Although it is beyond the scope of this chapter to provide a comprehensive review of the available data, emphasis will be given on aspects that are more likely to impact on the results of immunotoxicity evaluation studies. There is a growing body of scientific evidence lending support to early speculations [57]. Nevertheless, much remains to be done to determine all those changes in other biological systems that bear relevance on the interpretation of results from immunotoxicity studies. PSYCHONEUROIMMUNOLOGY The term psychoneuroimmunology was coined [58] as concern grew that psychological factors and the nervous system might influence immune responses as well as the outcome of a variety of diseases. Psychosocial factors and immunity. Psychosocial factors that have been shown to alter immune responses include psychosocial processes and health behaviors [59]. Psychosocial processes or responses to life events and stressors encompass mental health and mood factors, personality characteristics, social relationships. Health behaviors, such as smoking, exercise and nutrition, often have a direct impact on immune responses, but can also act indirectly via psychological factors. Interestingly, the described immune changes were considered of sufficient relevance to have implications for the treatment of patients with psychiatric disorders [60]. Thus, intended modulation of immune responses is a potential avenue of research for novel therapeutic modalities, whereas the immunotoxic effects of psychiatric drugs should be cautiously evaluated. Stress and immunity. Immune responses are unequivocally altered by acute and chronic stress [61 – 64]. Reduced humoral and cellular immunity in relation to examination stress was shown in medical students [65 – 67]. Another widely studied cause of stress is bereavement. For instance, a significant decrease in mitogen-induced proliferation was observed in widowers [68,69] and a 50% reduction in NK cell activity in women who have recently become bereaved [70]. Impairment of immunocompetence has been reported in unemployed persons [71]. Stressful working conditions can also lead to reduced immune responsiveness as observed in Japanese taxi drivers [72] or stressed nurses [73]. Depression. Although depression is often a factor concomitant to stress or social disruption, it was found to be associated with impaired lymphocyte functions, but other changes including reduced leukocyte numbers, higher CD4þ/CD8þ ratio, increased autoantibody titers and increased IL-1 and IL-6 production, were inconsistent [74 – 77]. Overall, changes were more marked with increasing age and in patients with major depression.

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Health behaviors and immunity Nutrition and immunity. Overall, malnutrition is a major cause of immunodepression [78 – 81]. Insufficient or inadequate food intake is unfortunately very frequent and associated with impairment of immune functions. The thymus is a target in malnutrition [82]. Experimental studies in mice [83,84] showed that inadequate protein intake causes reduction in cellular immunity or immune cell counts. A long-recognized consequence of nutrition deficiencies is the greater severity and possibly the greater incidence of infections [85]. Smoking and drinking. The immunotoxic effects of cigarette smoking and alcohol drinking will be reviewed in Chapter 5 of volume III. There is unequivocal experimental and clinical evidence that both addictive although licit behaviors have a negative impact on immunity. The psychological context of these behaviors may also play an indirect role. Sport, strenuous exercise and immunity. Despite the widely shared view that sport and exercise are good to human health—and actually they are in many instances—there is growing evidence that strenuous exercise or sport practices result in impairment of immune responses. Although high-performance athletes are generally not immunodeficient, they usually present with changes in various immunological parameters including impaired neutrophil functions, reduced serum Ig levels, increased NK cell activity and lower lymphocyte counts [86,87]. Overall, innate immunity tends to be more markedly affected than adaptive immunity [88]. There is an associated increase in the risk of infections [89, 90]. Most of the changes described in athletes have been reproduced in laboratory animals. The actual consequences of sport on immunity are, however, unclear as changes in athletes may differ depending on the time when measurements are made, e.g. at rest or immediately after a strenuous exercise, such as marathon running. In addition, the types of sport, the intensity of training, nutrition are important factors to be taken into consideration. Surgery. In addition to the immunotoxic effects of anesthetics drugs (see Chapter 3 of volume II), surgery itself is a stressful situation with adverse consequences on immune responses. The various components of surgical stress include psychological factors, acute and chronic inflammation due to tissue section and wound healing, malnutrition and the underlying disease. Depression in cellular immunity evidenced by mitogen-induced lymphocyte proliferative responses or skin testing to recall antigens was seen following surgery and lasted for 1 –3 weeks [91 – 93]. The chronic fatigue syndrome [94,95] is a still unexplained condition characterized by disabling physical and mental fatigue that lasts for at least 6 months. Many authors have suggested the involvement of the immune system. In particular, low NK cell activity was often noted. Other frequently reported anomalies include low T lymphocyte numbers and inconsistent slight T lymphocyte activation. Usually, serum immunoglobulin and cytokine levels are normal with the exception of increased serum TNF-a levels. INTERACTIONS BETWEEN THE NERVOUS, ENDOCRINE AND IMMUNE SYSTEMS There is a huge amount of data demonstrating the intricate interferences on the nervous, endocrine and immune systems, each system having the ability to influence the function


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of the other two systems. Some examples have already been mentioned. Because the literature available is so immense, no comprehensive coverage of this crucial area can be proposed in this chapter. The focus will be therefore on three major components involved in these interferences, namely neurotransmitters, cytokines and hormones. Neurotransmitters and immune responses. The primary and secondary lymphoid organs are innervated with neurotransmitter-containing fibers, closely associated with B lymphocytes, T lymphocytes, mast cells and other important immune cells. Four criteria have been defined to evidence the influence of a given neurotransmitter on immune responses: (i) there must be an association between lymphoid organs and specific nerve fibers; (ii) the neurotransmitter must be available to immunocompetent cells; (iii) target cells must express the appropriate receptors; and (iv) the immunoregulatory properties of the neurotransmitter must be clearly identified. Interestingly, the effects of a given neurotransmitter can differ depending on the status of the cell, namely a resting cell or an antigen-activated cell. Thymus, spleen, lymph nodes and MALT have an extensive sympathetic innervation and many types of receptors have been demonstrated on immunocompetent cells including receptors of the catecholamines, noradrenaline, adrenaline, dopamine and serotonin [96 –100]. Under stimulation, catecholamines are released from sympathetic nerve terminals in these organs and in binding to their receptors can affect lymphocyte circulation and proliferation, and modulate cytokine production. However, despite the existence of extensive sympathetic innervation in lymphoid organs, our knowledge of the resulting consequences is extremely modest. Neuropeptides are naturally occurring peptides that are produced and secreted by nerve endings and some of these neuropeptides, such as substance P and neuropeptide Y, have been shown to induce the secretion of cytokines by T lymphocytes. Opioids are also potent immunomodulating substances (see Chapter 3 of volume II). Cytokines and the neuroendocrine system. There is a growing body of evidence that cytokines not only serve as messengers between immunocompetent cells, but also exert physiological effects on nonimmune tissues, including the nervous and endocrine systems [101,102]. Measurement of cytokines in toxicity studies should therefore not be limited to immunotoxicity assessment [41] as they are involved in a variety of cell and tissue injuries [103]. The impact of cytokines on the nervous and endocrine systems is further supported by adverse effects reported in patients treated with recombinant cytokines, such as the IFNs-a [104]. The pro-inflammatory cytokines IL-1, TNF-a, and IL-6 act on the brain and the periphery to induce nonspecific clinical signs and symptoms of infections and inflammation [105]. They also exert marked stimulatory influences on the hypothalamo-pituitary-adreno-cortical axis and provoke the release of glucocorticoids resulting in feedback responses [106]. There is evidence that IL-1 and TNF-a promote sleep via interactions with the growth hormone-releasing hormone (GHRH) and corticotropin-releasing hormone (CRH) [107], whereas IL-6 and IFN-a, at least in humans, acutely disturb sleep [108]. The role of cytokines in the promotion of fever is discussed in Chapter 4 of this volume. In addition, TH1 and TH2 cytokines are produced in the brain, but their role are not clearly elucidated [109].

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Endocrine hormones and the immune system. Hormones exert marked immunomodulatory properties. Differences in the hormonal status are related with the varying incidence of immunopathological conditions, such as autoimmune diseases that are markedly more frequent in women. The impact of hormones and antihormones on immunity is addressed in Chapter 6 of volume II. AGE AND IMMUNOCOMPETENCE The level of immunocompetence depends on the age of the host. Immunocompetence in newborns and infants. Immaturity of the immune system and a different susceptibility to various immunological stimuli or factors has been shown in very young animals as well as newborns and infants (see Chapter 15 of this volume). Immunosenescence. Following the pioneering work of Walford [110], the role of agingrelated changes in immune functions has been clearly established to contribute to a wide range of diseases that are more frequently seen in elderly people [111]. Infectious diseases are a major cause of morbidity and morality in the elderly. Immune functions decline with age [112 – 114]. Although both humoral and cellular immune responses are impaired, cellular immunity is more profoundly affected. This is primarily due to the physiological involution of the thymus that starts at puberty and is nearly complete at the age of 60 in man. The most common findings include a reduction in CD3þ, CD4þ and CD8þ T lymphocyte numbers, and naive CD45þ T lymphocytes, depressed T lymphocyte proliferative responses, alterations in signal transduction due to loss of the co-stimulatory molecule CD28 by T lymphocytes, and a shift in TH1 to TH2 cytokine production with decreased interferon-g vs. increased IL-4 production. In addition, there is a decrease in total B lymphocyte numbers and an increase in serum IgG and IgA levels. Specific antibody responses are less protective: elderly people have a limited ability to mount primary immune responses whereas responses to recall antigens are generally conserved. The phagocytic potency of both neutrophils and macrophages, NK cell activity are decreased in elderly people. A correlation has been suggested between low NK cell activity and increased susceptibility to infections [9]. In addition, low NK cell activity could contribute to the greater incidence of cancer. Serum autoantibody levels tend to be higher, but the incidence of autoimmune diseases does not increase with age. IMPLICATIONS FOR IMMUNOTOXICITY EVALUATION The interactions between the immune system and either the central nervous system or the endocrine system, have not so far received all the attention they deserve [116]. There is, however, increasing awareness that xenobiotics can modulate immune responses both directly and indirectly. Glucocorticosteroids are potent immunosuppressive drugs in laboratory animals and treated patients. Interestingly, a number of xenobiotics act as chemical stressors and there is a correlation between corticosterone levels and immunosuppression [117]. Finally, altered nervous responses can induce changes in immune function and conversely abnormal immune responses can induce various pathological


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conditions involving the nervous system [118]. Eventhough the exact consequences of interactions between the immune system and the neuroendocrine system remain to be fully elucidated, the available evidence supports the recommendation that immune changes induced by drugs and other chemicals in the course of nonclinical as well clinical studies should be carefully evaluated and integrated in a global approach encompassing the suspected mechanisms of immunotoxicity and the impact on other systems that may indirectly affect immune responses. This seems particularly relevant for those drugs and chemicals that have known effects on the nervous and endocrine systems.

CONCLUSION The immune system is an extremely complex array of cells and mediators that interact to mount immune responses, which can be either beneficial or detrimental. Because of this complexity, it is recommended that immunotoxicity evaluation is not based on trivial or isolated end-points that cannot reflect the whole spectrum of mechanisms possibly involved. In any case, one single change cannot be considered as equal to immunotoxicity, but instead should be included in a wider perspective. Establishing the relevance of changes in the morphology of the lymphoid tissue or immune function may therefore require the use of global—host resistance—models.

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[62] O’Leary A (1990) Stress, emotion, and human immune function. Psychol Bull, 108, 363– 682. [63] Stone AA, Bovbjerg DH (1994) Stress and humoral immunity: a review of human studies. Adv Neuroimmunol, 4, 49 – 56. [64] Kort WJ (1994) The effect of chronic stress on the immune response. Adv Neuroimmunol, 4, 1 –11. [65] Dorian BJ, Keystone E, Garfinkel PE, Brown GM (1982) Aberrations in lymphocyte subpopulations and functions during psychological stress. Clin Exp Immunol, 50, 132– 138. [66] Kiecolt-Glaser JK, Glaser R, Strain EC, Stout JC, Tarr KL, Holliday JE, Speicher CE (1985) Modulation of cellular immunity in medical students. J Behav Med, 9, 5 – 21. [67] Workman EA, La Via MF (1987) T-lymphocyte polyclonal proliferation: effects of stress and stress response style on medical students taking national board examinations. Clin Immunol Immunopathol, 43, 308– 313. [68] Bartrop RW, Luckhurst E, Lazarus L, Kiloh LG, Penny R (1977) Depressed lymphocyte function after bereavement. Lancet, I, 834– 836. [69] Schleifer SJ, Keller SE, Camerino M, Thornton JC, Stein M (1983) Suppression of lymphocyte stimulation following bereavement. J Am Med Assoc, 250, 374– 377. [70] Irwin M, Daniels M, Smith TL, Bloom E, Weiner H (1987) Impaired NK cell activity during bereavement. Brain Behav Immun, 1, 98 – 104. [71] Arnetz BB, Wasserman J, Petrini B, Brenner SO, Levi L, Eneroth P, Salovaara H, Hjelm R, Salovaara L, Theorell T et al. (1987) Immune function in unemployed women. Psychosom Med, 49, 3 – 12. [72] Nakano Y, Nakamura S, Hirata M, Harada K, Ando K, Tabuchi T, Matunaga I, Oda H (1998) Immune function and lifestyle of taxi drivers in Japan. Ind Health, 36, 32– 39. [73] De Guch V, Fischler B, Demanet C (1999) Immune dysfunction associated with professional stress in nurses. Psychiatr Res, 85, 105– 111. [74] Schleifer SJ, Keller SE, Meyerson A, Raskin MJ, Davis KL, Stein M (1984) Lymphocyte function in major depressive disorder. Arch Gen Psychiatr, 41, 484– 486. [75] Ravindran AV, Griffiths J, Merali Z, Anisman H (1995) Lymphocyte subsets associated with major depression and dysthymia: modification by antidepressant treatment. Psychosom Med, 57, 555– 563. [76] Andreoli AV, Keller SE, Rabaeus M, Marin P, Bartlett JA, Taban C (1993) Depression and immunity: age, severity and clinical course. Brain Behav Immun, 7, 279– 292. [77] Maes M (1995) Evidence for an immune response in major depression: a review and hypothesis. Prog Neuro-psychopharmacol Biol Psychiatr, 9, 11 – 38. [78] Brussow H, Sidoti J, Dirren H, Freire WB (1995) Effect of malnutrition in Ecuadorian children on titers of serum antibodies to various microbial antigens. Clin Diagn Lab Immunol, 2, 62 –68. [79] Zaman K, Baqui AH, Yunus M, Sack RB, Chowdhury HR, Black RE (1997) Malnutrition, cellmediated immune deficiency and acute upper respiratory infections in rural Bangladeshi children. Acta Paediatr, 86, 923– 927. [80] Grimble RF (2001) Nutritional modulation of immune function. Proc Nutr Soc, 60, 389–397. [81] Calder PC (2003) Immunonutrition. Br Med J, 327, 117– 118. [82] Savino W (2002) The thymus gland is a target in malnutrition. Eur J Clin Nutr, 56(Suppl. 3), 46 – 49. [83] Bounous G, Kongshav N (1982) Influence of dietary proteins on the immune system of mice. J Nutr, 112, 1747– 1755. [84] Nimmanwudipong T, Cheadle WG, Appel SH, Polk HC (1992) Effect of protein malnutrition and immunomodulation on immune cell populations. J Surg Res, 52, 233– 238. [85] Gordon JE, Ingalls TH, Scrimshaw NS, Taylor CE, Gordon JE (1988) Interactions of nutrition and infection. Nutrition, 4, 13– 50. [86] Sharp NCC, Koutedakis Y (1992) Sport and the overtraining syndrome: immunological aspects. Br Med J, 48, 518– 533.


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[87] MacKinnon LT (2000) Special feature for the Olympics: effects of exercise on the immune system: overtraining effects on immunity and performance in athletes. Immunol Cell Biol, 78, 502– 509. [88] Nieman DC, Pedersen BK (1999) Exercise and immune function. Recent developments. Sports Med, 27, 73 – 80. [89] Nieman DC (1998) Exercise and resistance to infection. Can J Physiol Pharmacol, 76, 573– 580. [90] Nieman DC (2000) Special feature for the Olympics: effects of exercise on the immune system: exercise effects on systemic immunity. Immunol Cell Biol, 78, 496– 501. [91] Riddle PR, Berenbaum MC (1967) Postoperative depression of the lymphocyte response to phytohaemagglutinin. Lancet, i, 746– 748. [92] Slade MS, Simmons RL, Yunis E, Greenberg LJ (1975) Immunodepression after major surgery in normal patients. Surgery, 78, 363– 372. [93] Hamid J, Bancewicz J, Brown R, Ward C, Irving MH, Ford WL (1984) The significance of changes in blood lymphocyte populations following surgical operations. Clin Exp Immunol, 56, 49 – 57. [94] Natelson BH, Haghighi MH, Ponzio NM (2002) Evidence for the presence of immune dysfunction in chronic fatigue syndrome. Clin Diagn Lab Immunol, 9, 747– 752. [95] Lyall M, Peakman M, Wessely S (2003) A systematic review and critical evaluation of the immunology of chronic fatigue syndrome. J Psychosom Res, 55, 79 – 90. [96] Schorr EC, Arnason BGW (1999) Interactions between the sympathetic nervous system and the immune system. Brain Behav Immun, 13, 271– 278. [97] Eskandari F, Sternberg EM (2002) Neural-immune interactions in health and disease. Ann NY Acad Sci, 966, 20 – 27. [98] Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES (2000) The sympathetic nerve—an integrative interface between two supersystems: the brain and the immune system. Pharmacol Rev, 52, 595– 638. [99] Sanders WM, Straub RH (2002) Norepinephrine, the b-adrenergic receptor, and immunity. Brain Behav Immun, 16, 290– 332. [100] Madden KS (2003) Catecholamines, sympathetic innervation and immunity. Int Immunopharmacol, 17(Suppl.), 17 – 20. [101] Larsen SJ, Dunn AJ (2001) Behavioral effects of the cytokines. Brain Behav Immun, 15, 371– 387. [102] Haddad JJ, Saade´ NE, Safieh-Garabedian B (2002) Cytokines and neuro-immune-endocrine interactions: a role for the hypothalamic – pituitary– adrenal revolving axis. J Neuroimmunol, 133, 1 – 19. [103] Foster JR (2001) The functions of cytokines and their uses in toxicology. Int J Exp Pathol, 82, 171– 192. [104] Vial T, Choquet-Kastylevsky G, Liautard C, Descotes J (2000) Endocrine and neurological adverse effects of the therapeutic interferons. Toxicology, 142, 161– 172. [105] Wang X, Feuerstein GZ (2000) Role of immune and inflammatory mediators in CNS injury. Drug News Perspect, 13, 133– 140. [106] John CD, Buckingham JC (2003) Cytokines: regulation of the hypothalamo-pituitaryadrenocortical axis. Curr Opin Pharmacol, 3, 78– 84. [107] Krueger JM, Majde JA (2003) Humoral links between sleep and the immune system: research issues. Ann NY Acad Sci, 992, 9 – 20. [108] Marshall L, Born J (2002) Brain– immune interactions in sleep. Int Rev Neurobiol, 52, 93 – 131. [109] Sredni-Kenigsbuch D (2002) TH1/TH2 cytokines in the central nervous system. Int J Neurosci, 112, 665– 703. [110] Walford RL (1969) Immunological Theory of Aging. Munskgaard, Copenhagen. [111] Effros RB (1993) Immunosenescence-related diseases in the elderly. Immunol Allergy Clin North Am, 13, 695– 712.

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[112] Gruebeck-Loebenstein B (1997) Changes in the aging immune system. Biologicals, 25, 205– 208. [113] Ginaldi L, Loreto MF, Corsi MP, Modesti M, De Martinis M (2001) Immunosenescence and infectious diseases. Microb Infect, 3, 851– 857. [114] Lloberas J, Celada A (2002) Effect of aging on macrophage function. Exp Gerontol, 37, 1323– 1329. [115] Reference omitted at proof stage. [116] Descotes J (2000) Integrating immunotoxicity with effects on other biological systems in preclinical safety evaluation: a perspective. Toxicology, 142, 157– 160. [117] Pruett SB, Ensley DK, Crittenden PL (1993) The role of chemical-induced stress responses in immunosuppression: a review of quantitative and cause –effect relationships between chemicalinduced stress responses and immunosuppression. J Toxicol Environ Health, 39, 163– 192. [118] Lawrence DA, Kim D (2000) Central/peripheral nervous system and immune responses. Toxicology, 142, 189– 201.

Chapter 3

Health consequences of immunotoxic effects

INTRODUCTION The continuum model [1] based on the assumption that normal immune responses are, upon chemical exposure, either inhibited (immunosuppression) or enhanced (immunostimulation), the latter situation resulting in hypersensitivity or autoimmunity, did not stand the test of time and clinical experience, even though it had long been recognized that the immunemediated adverse effects of drugs did not fit to this oversimplified vision of immunotoxicity [2,3]. As described later in this chapter, specific adverse effects in relation to immunostimulation have indeed been reported, in particular following the clinical use of therapeutic recombinant cytokines. In addition, a number of drugs and other chemicals that may cause immune-mediated hypersensitivity reactions have never been shown to stimulate normal immune responses. Penicillin is an illustrative example in this regard. Finally, although a variety of autoimmune diseases develop more frequently in patients treated with certain, but not all therapeutic cytokines, a few medicinal drugs have been shown to induce a limited characteristic set of autoimmune reactions. In fact, clinically significant immunotoxic effects are best categorized into the following four situations, namely immunosuppression, immunostimulation, hypersensitivity and autoimmunity. Each of these four categories is associated with markedly different adverse effects.

IMMUNOSUPPRESSION Due to the long clinical experience gained with the use of immunosuppressive drugs [4], particularly in organ transplant patients and the continuing, if not sole interest of immunotoxicologists in immunosuppression during the 20 first years of the history of immunotoxicology, the clinical adverse effects of immunosuppression are very well recognized. Two major types of effects have long been identified [5], namely impaired resistance of the host against various microbial pathogens leading to infectious complications, and more frequent malignancies, e.g. lymphomas. A question that has so far been paid relatively limited, if any attention is whether and if so, to what extent, mild to moderate impairment of the immune responsiveness 55

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(namely immunodepression) has the potential to induce similar adverse clinical effects. This highly critical issue from a risk assessment perspective will be addressed later in this chapter. IMPAIRED RESISTANCE AGAINST MICROBIAL PATHOGENS Primary (congenital) or secondary (acquired) immune defects are associated with microbial infections that are both more frequent and more severe, and sometimes atypical as exemplified by opportunistic infections that normally do not develop in immunocompetent individuals. Therefore, impaired resistance against microbial pathogens is a characteristic feature of immunosuppression. The causative immune defect(s) may affect either nonspecific host defense mechanisms (innate immunity), specific immune mechanisms (adaptive immunity) or both (see Chapter 4 of this volume). Relevance of impaired microbial resistance induced by xenobiotics. Illustrative examples from general medical practice include infants born with a severe immune defect [6,7] as well as cancer and AIDS patients, all of whom develop frequent, severe and potentially lethal infections [8]. Similar examples have been reported in the area of toxicology [9]; some of them will be briefly overviewed. Corticosteroids. The adverse influence of corticosteroid hormones on host defenses has long been recognized [10,11] and infectious complications have been often described in patients treated with corticosteroids [12,13]. Corticosteroids primarily suppress T lymphocyte functions and phagocytosis. Bacterial infections in treated patients are often due to staphylococci and gram-negative bacteria [14], but infections caused by intracellular pathogens, such as mycobacteria [15,16], have also been reported. The risk of disseminated viral infections is high and, for instance, leads to malignant varicella in asthmatic children on long-term corticosteroid therapy [17 – 21]. Pneumocystis carinii infections are the most common parasitic infections. It is noteworthy to mention that nevertheless corticosteroids can prove useful as adjunct therapy of severe infections [22]. Immunosuppressive drugs and chemotherapy. Another typical example is the use of immunosuppressive drugs that are major causes of morbidity and mortality in the posttransplantation period whatever the immunosuppressive regimen [23 – 28]. For instance, the analysis of 102 medical and autopsy records showed that infections were the cause of death in 70% of transplant patients [29]. Bacteria (50%) or fungi (29%) were the most common pathogens. Infectious complications have been reported in transplant patients treated with azathioprine [30,31], cyclosporine [32 – 34], tacrolimus [35] or mycophenolate mofetil [36,37]. Most cytotoxic anticancer drugs exert immunosuppressive properties in addition to marked myelotoxicity and infectious complications are common in cancer patients undergoing chemotherapy [38 – 41]. Environmental and industrial chemicals. The possible occurrence of infectious diseases has not been extensively studied in humans exposed to occupational or environmental chemicals that proved to be immunosuppressive in animal studies. However, there is a seemingly similar trend toward more frequent, even though often clinically unremarkable infections in exposed human beings. Biphenyls. In October 1968, an outbreak of unusual clinical signs and symptoms was reported in the Fukuoka prefecture in Kyushu (Japan). Contamination of a commercial


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brand of rice oil by a mixture of polychlorinated biphenyls (PCBs) was subsequently identified as the cause of what is since then referred to as the Yusho disease [42]. Although general nonspecific clinical signs, skin manifestations, e.g. chloroacne, endocrine, neurological and teratogenic disorders were the most consistent findings, respiratory infections were seen in some patients and were correlated with decreased serum Ig levels and decreased lymphocyte responsiveness to PHA [43]. An outbreak due to PCB contaminants in rice oil was also described in Taiwan. Patients with the Yu-Cheng—the Chinese word for Yusho—disease also developed more frequent respiratory infections associated with decreased cutaneous responses to recall antigens [44]. These clinical findings are consistent with the results of studies in wildlife species showing that exposure to PCBs is associated with immunosuppression and more frequent spontaneous infections [45,46]. Heavy metals. There is a huge amount of evidence that a number of heavy metals cause immunosuppression resulting in impaired host resistance to infections in laboratory animals [47]. Data in humans are so scarce that no conclusion can be made to what extent, if any animal data extrapolate to humans. For example, no increased incidence of malaria was evidenced in Brazilian gold miners heavily exposed to mercury [48] and workers exposed to mercury vapors had slight and inconsistent immune changes, but no increased incidence of infections [49]. In contrast, children exposed to lead were suspected to develop more frequent infections [50]. Pesticides. Despite claims that the potential for pesticide-induced immunosuppression is a threat to human health [51], rare clinical data are supportive [52]. Workers exposed to various organophosphorus insecticides had impaired neutrophil chemotaxis associated with a significantly higher incidence of respiratory infections [53]. Children exposed to DDE, hexachlorobenzene and PCBs were found to be at a greater risk of developing otitis media, whereas the overall risk of infections was not increased [54], which confirmed previous findings in Inuit infants exposed to organochlorine pesticides who had more frequent otitis media [55]. In fact, much remains to be done to ascertain the effects of pesticides on the immune system of exposed human beings [56,57]. Physical agents. One very interesting finding is that UV exposure, which has been shown to exert immunosuppressive effects in a variety of in vitro and in vivo models is associated with more frequent infections in man [58]. Most commonly involved microbial pathogens. Although, no single pathogen is specifically involved in infectious complications associated with immunosuppression, some are more frequently found in immunocompromised subjects exposed to immunosuppressive agents. Importantly, infections in immunocompromised patients can be caused by several microbial species simultaneously. Bacterial infections are preferentially due to staphylococci, e.g. S. aureus and S. epidermidis, streptococci, e.g. S. pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Haemophilus influenzae, nocardia, and mycobacteria [59]. L. monocytogenes is an intracellular pathogen that preferentially occurs in immunocompromised patients [60 – 62]. Meningitis is the most common complication, but bacteremia, brain abscess and meningo-encephalitis [63], or pneumonia may also develop. Nocardiosis primarily affects the lung, but dissemination to the skin, the central

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nervous system, and the skeletal system is also common, particularly in immunocompromised patients [64]. Approximately 20% of all nocardial infections have been reported in organ transplant patients [65].The incidence of tuberculosis in solid-organ transplant patients is greater than in the general population [66 –68]. Apart from tuberculosis, mycobacterial infections involve atypical mycobacteria, such as M. kansasii, M. chelonei, and M. haemophilum. Viral infections. Herpes viruses, the cytomegalovirus and Epstein – Barr virus (EBV) are the most common viruses involved in viral infections in immunocompromised patients [69]. Herpes simplex virus infections are frequent in organ transplant patients, particularly infections to human herpesvirus 6 and 7 [70,71]. They are often due to reactivation of a dormant disease. Clinical symptoms include fever, skin rash, pneumonia, bone marrow suppression and encephalitis. Varicella-zoster virus infections are often atypical and more severe in immunocompromised patients [72]. Cytomegalovirus is one of the most significant pathogens in organ transplant patients [73 – 75]. Clinically, the disease can range from asymptomatic viral shedding to lifethreatening disseminated disease. Epstein –Barr virus (EBV) causes asymptomatic elevation in serum antibody titers and more rarely disseminated lymphoproliferative disorders. Infections of humans with EBV [76] usually occur by contact with oral secretions. The site of persistence of EBV within the body is resting B lymphocytes. Although EBV infections induce both humoral and cellular immune responses, the cellular responses are more important: NK cells and cytotoxic T lymphocytes control proliferating EBV-infected B lymphocytes. Clinically, the most common human EBV infection is infectious mononucleosis, the symptoms of which are attributed to the proliferation and activation of T lymphocytes in response to infection. A variety of cancers are associated with EBV, including nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s disease and various lymphoproliferative disorders. Importantly, EBV is associated with lymphoproliferative diseases in patients with either primary (congenital) or secondary (acquired) immune deficiency. These patients have impaired T lymphocyte functions, which are unable to control the proliferation of EBV-infected B lymphocyte. Other viruses include the hepatitis B, C and delta viruses, the respiratory syncytial, influenza and parainfluenza viruses. As already mentioned viral infections, including measles [77] can be particularly severe in immunosuppressed patients. Fungal infections are a major cause of morbidity and mortality in immunocompromised patients [78 –82]. For instance, current estimates suggest that they may develop in 5 –40% of organ transplant patients. Infections due to Candida, Aspergillus, P. carinii and Cryptococcus are the most frequent fungal infections. The most common site of Candida infections is the oral mucosa, but skin, lung and disseminated infections also occur. Aspergillus infections predominate in the lung and the digestive tract, but disseminated infections can occur [83,84]. P. carinii pneumonia is one of the most common opportunistic infection with variable clinical and radiological presentation [85]. Cryptococcosis, caused by Cryptococcus neoformans, can occur in any organ of the body, although meningitis is the most frequent form.


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Parasitic infections in immunosuppressed patients are mainly caused by Toxoplasma gondii, Strongyloides stercoralis and cryptosporidae in most instances [86,87]. Most common sites of infections in immunocompromised patients Infections of the respiratory tract are the most frequent infections of immunocompromised patients [88 –93]. In organ transplant patients, the incidence of infections of the respiratory tract is in the range 10 –50% depending on the transplanted organ. As the respiratory tract is the major route of entry for microbial pathogens, infections of the upper airways and the lung are the most common infections. Bacteria, including S. pneumoniae, P. aeruginosa and S. aureus are the main causes of infections of the respiratory tract. Cytomegalovirus infections are often silent, but detectable in up to 90% of renal transplant patients. Other pathogens, such as the respiratory syncytial virus, influenza virus, adenoviruses and fungi including Candida, Nocardia, Aspergillus species are less common. Infections of the digestive tract. The digestive tract is the first line of defense against oral pathogens. Any dysfunction of specific and nonspecific defense mechanisms can result in more frequent and/or more severe gastro-intestinal infections with often atypical clinical features and prolonged or recurring outcome [94 – 96]. Even though respiratory infections are more frequent, gastro-intestinal infections often presenting as chronic diarrhea may predominate in some patients. Oesophagitis, colonic and anorectal infections have also been described [97 –99]. Associated risk factors, such as atrophy of the gastric mucosa, increased gastric acid secretion, or alterations of the intestinal flora, contribute to the development of gastro-intestinal infections. Unexpectedly, selective IgA deficiency is seldom associated with gastro-intestinal infections, even though IgA are the predominant Ig class in digestive secretions, which underscores our poor current understanding of the role of mucosal immunity in the host’s defenses. The pathogens which are more often involved in gastro-intestinal infections include C. albicans, salmonella, Aeromonas, rotaviruses, adenoviruses, herpesviruses and the cytomegalovirus. Other infection sites. Beside broncho-pulmonary and gastro-intestinal infections, which are by far the most frequent infections in immunocompromised patients, any other site of the body can be affected. These infectious complications are often atypical. Infections of the central nervous system, such as brain abscess and encephalitis [100,101], or severe and recurring infections of the skin are among the most severe opportunistic infections. For example, cutaneous cryptococcosis [102] and blastomycosis [103]—a disease caused by the fungus Blastomyces dermatitidis—are more severe in immunocompromised patients with multiple organ and central nervous system involvement. In contrast, urinary infections are relatively uncommon. Finally, infectious complications may remain silent, or present as isolated fever. IMMUNOSUPPRESSION AND MALIGNANCIES There is large body of evidence that immunocompromised patients are at a greater risk of developing malignancies. The number of published studies is so large that only a selection can be included in the reference list [104 – 106]. More frequent malignancies have been

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described in patients with various congenital immune defects [107 – 109] or the HIV immunodeficiency syndrome [110 – 113]. Interestingly, the majority of malignancies reported in immunocompromised patients are relatively rare in the general population. They are often associated with (latent) viral infection. Thus, Kaposi’s sarcoma is the commonest malignancy in AIDS patients and a link between Kaposi’s sarcoma and human herpesvirus 8 has been evidenced [114]. Lymphomas are another significant cause of mortality in AIDS patients and a high percentage of AIDS-associated lymphomas are B cell lymphomas involving EBV [115,116]. Human papilloma virus infections are primarily associated with cervical cancers, but also with vulval/vaginal, skin and conjunctival cancers, as well as a variety of oral cancers, including tonsillar, laryngeal and esophageal carcinomas [117,118]. Unsurprisingly, organ transplant patients treated with long-term immunosuppressive regimens also develop more frequent malignancies [119 –121]. In fact, the role of immunosuppression was slowly established based on two sets of data, namely second cancers following successful chemotherapy and epidemiological data in organ transplant patients, in particular kidney transplant patients. Second cancers are de novo cancers that develop following chemotherapy and are unrelated to the first cancer. Approximately 1% of patients have been reported to develop a second cancer within 10 years after completion of chemotherapy, and 3% within 20 years [122]. Acute leukemias, non-Hodgkin’s lymphomas, and carcinomas of the skin, lung, breast, colon or pancreas, are the most common second cancers [123 – 128]. Several factors are likely to play a role in the development of de novo second cancers. As most anticancer drugs are genotoxic, the involvement of a direct carcinogenic effect can be suspected, but owing to the long delay required for carcinogenicity to result in clinically overt tumors, only those second cancers, such as solid tumors that develop slowly and whose incidence rises with time since first treatment can be considered as a consequence of the carcinogenic effect of chemotherapy. Another factor is the role of individual predisposition. Patients who develop de novo second cancers may be more prone to developing cancer than patients with no prior history of cancer. Despite significant progress in our understanding of the genetic and immunological factors predisposing to cancer, much remains to be done, however, to demonstrate such a predisposition. Finally, as most chemotherapeutic agents are immunosuppressive, the role of immunosuppression can be proposed to result in the lymphoproliferative disorders that develop within the few months or years after completion of chemotherapy. Organ transplant patients. Confirmation of the role of immunosuppression resulting in more frequent malignancies primarily derived from epidemiological studies in organ transplant patients. Lymphoproliferative disorders. Many retrospective and prospective studies showed that lymphoproliferative disorders are, indeed, more frequent in renal transplant patients. Although the role of renal insufficiency has sometimes been suspected [129,130], immunosuppression was confirmed as a key factor. The most comprehensive data are those of the Cincinnati Transplant Tumor Registry [131]. Early [132,133] and many subsequent studies [134,135] gave consistent results. For instance, there were 0.65 malignancies for


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each 100 cumulative patient-years of immunosuppression in a 20-year follow-up study of 794 renal transplant patients who all received azathioprine plus prednisone [136]. Variable results have been reported reflecting presumably differences in the population of organ transplant patients under scrutiny, the length and selected criteria of follow-up, the immunosuppressive drug regimen, the method for the calculation of the incidence rate as well geographical differences [121]. However, there is a consistent trend among published studies. One recent estimate is that 1% of renal transplant patients develop lymphoproliferative disorders [137], that is to say 30- to 50-fold greater than in the general population. With the rapid development of other types of organ, e.g. heart [138,139] and liver [140,141] transplantation, similar findings have been published. Other malignancies. Although, lymphoproliferative disorders received more attention, a number of malignancies have also been described in organ transplant patients. Some of these are up to 100-fold more frequent than in the general population [121]. The most common malignancies affecting organ transplant recipients are cancers of the skin and lips [142 – 146]. For instance, skin cancers have been observed in 45% of kidney transplant patients from Queensland (Australia) after 11 years of immunosuppression [147]. A cumulative risk of 43.8% was similarly found in Spanish heart transplant recipients after a 7-year follow-up [142]. In the Cincinnati Transplant Tumor Registry [131], 38% of all cancers were cancers of the skin and lips, more often squamous cell carcinomas, that preferentially developed in sun-exposed areas, a finding consistent with the immunosuppressive effect of UV exposure [58]. The incidence of skin cancers increased with the length of follow-up after transplantation and was higher in younger patients. Only 16% of recorded cancers were non-Hodgkin’s lymphomas. Other reported cancers include renal, uterine, anogenital and hepatocellular carcinomas, and sarcomas of the soft tissues and visceral organs, but no overall increase in the most common cancers was seen in organ transplant patients as compared to the general population. In contrast, Kaposi’s sarcoma which accounts for 1– 6% of all cancers in transplant patients (4% of all cancers in the Cincinnati Transplant Tumor Registry) is 400 –500 times more frequent than in the general population [131]. Contributing risk factors. As mentioned above, variable results have been published despite relatively consistent trends. Thus, the role of contributing factors deserves clarification. Differences related to the type of organ transplantation have already been addressed. Role of the immunosuppressive regimen. Overall, malignancies are more frequent irrespective of the immunosuppressive drugs [149]. Lymphoproliferative disorders have been described in organ transplant patients treated with azathioprine [150,151], cyclosporine [152 – 154], tacrolimus [155 –157], sirolimus [158,159], mycophenolate mofetil [160,161] or the monoclonal antibody OKT3 [162,163]. Although, skin cancers were initially thought to be more frequent in azathioprine-treated patients, a similar incidence of skin cancers was later reported with other immunosuppressive drugs [131]. Conflicting results, however, have been published regarding the respective incidence of lymphoproliferative disorders associated with cyclosporine and tacrolimus [164,165] and the time to onset of lymphoproliferative disorders was shorter with cyclosporine or sirolimus than with azathioprine [166,167]. Finally, the most important factor concerning the role of the immunosuppressive regimen is the magnitude of the immunosuppression

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achieved, since patients submitted to more aggressive immunosuppressive regimens develop more frequent malignancies [168 – 170]. For example, the incidence of cyclosporine-associated skin cancers was found to be dose dependent [171]. Role of latent viral infections. Another important contributing factor is the co-existence of latent viral infections as already mentioned. There is a large body of evidence showing that lymphoproliferative disorders and other malignancies associated with immunosuppression in organ transplant patients as well as patients with other causes of immunosuppression, e.g. HIV, are triggered or at least facilitated by viral infections. EBV is the most common virus detected in organ transplant patients with lymphoproliferative disorders [172 – 177]. EBV-associated lymphomas are mainly B lymphomas, but T lymphomas have also been reported [178]. After a primary infection that is usually asymptomatic or can also manifest as infectious mononucleosis, normally EBV harmlessly resides in B lymphocytes for the whole life of the host. However, EBV can promote post-transplantation lymphoproliferative disorders probably because the proliferation of EBV-infected B lymphocytes is no longer controlled by T lymphocytes. The second-most frequent viral infections that are associated with malignancies in organ transplant patients are human herpesvirus 8 (HHV8) infections. Patients seropositive for HHV8 have a greater risk of Kaposi’s sarcoma [179 – 181]. However, many people infected by HHV8 never develop disease. As already mentioned, the incidence of Kaposi’s sarcoma in organ transplant patients is much greater than in the general population and the role of impaired immune function in the development of Kaposi’s sarcoma is likely even though ill-established contributing factors are presumably also involved. The contributing role of human papilloma viruses in skin cancers of transplant patients is a matter of growing concern [182 –184]. Malignancies in other groups of immunocompromised patients. Although, malignancies were first described in profoundly immunocompromised patients, noncancerous and nontransplant patients treated with immunosuppressive drugs develop similar, but less frequent malignancies [185]. These data can serve as the basis for the distinction between immunosuppression and immunodepression as later discussed in this chapter. Epidemiological studies showed that lymphoproliferative disorders are more frequent in patients with an autoimmune disease than in the general population, despite conflicting published results [121]. The relative risk for lymphoproliferative disorders has been estimated to be from 2.5- to 10-fold that of the general population [186,187]. In fact, a major bias is the seemingly increased incidence of lymphoproliferative disorders in these patients whether or not they have been treated with immunosuppressive drugs or not [188,189]. In contrast to the available data in organ transplant patients in whom the magnitude of immunosuppression, but not the immunosuppressive drugs used is a probable risk factor, the risk of malignancies in patients with an autoimmune disease that are treated with lowdose immunosuppressive drugs tends to vary between therapeutic regimens. Thus, in contrast to early findings, the results of the most recent epidemiological studies support the conclusion that azathioprine carries a low risk of malignancies in patients with multiple sclerosis, rheumatoid arthritis or inflammatory bowel disease [190 –193]. In addition to bladder cancers, low-dose cyclophosphamide has been reported to cause an increased


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incidence of lymphomas and skin cancers, but the increase was depending on the treated autoimmune disease [194,195]. Although, the available data are still limited regarding the risk of malignancies associated with cyclosporine in nontransplant patients, a low and inconsistent increase in lymphomas and skin cancers has been reported [196 –198]. Very few data are available with the anti-TNF-a drug infliximab, but an increased risk of lymphomas is also suspected [199]. In fact, the vast majority of data on the association between malignancies and lowdose immunosuppressive drugs in nontransplant patients relates to methotrexate. Lowdose methotrexate therapy has indeed been and still is widely used in the treatment of autoimmune diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease and systemic lupus erythematosus, despite the recent introduction of anti-TNF-a drugs. Although in vitro assays suggested that methotrexate could be genotoxic, the consistent absence of second solid tumors in patients treated with methotrexate for long periods of time did not confirm these early findings [196,197]. Despite the sometimes conflicting conclusions of published studies, many case reports of methotrexateassociated lymphomas are available in the medical literature [200 –210]. The following findings argue for the causative role of low-dose methotrexate in the development of lymphomas: (i) the clinical features of methotrexate-associated lymphomas are very similar to those of lymphomas associated with other immunosuppressive drugs; (ii) the discontinuation of methotrexate usually resulted in the spontaneous remission of lymphoproliferative disorders [207 – 209]; and (iii) EBV infection was found in one-half of rheumatoid patients with methotrexate-associated lymphomas, whereas most lymphomas in patients without a previous exposure to methotrexate were not associated with EBV infection and had a clinical presentation closer to lymphomas seen in the general population [211]. OTHER ADVERSE EFFECTS RELATED TO IMMUNOSUPPRESSION Beside infectious complications and virus-induced malignancies, few immunosuppressionrelated adverse effects were shown to be associated with exposure to drugs and other chemicals. Drug-induced IgA deficiency. Selective IgA deficiency is the most commonly diagnosed primary immune deficiency in children and adults [212 –214]. Indeed, it has been estimated that approximately 1 in 500 –700 human beings are affected. Although, IgA deficiency was suggested to be associated with a variety of upper respiratory, gastrointestinal, allergic and autoimmune diseases, most people with IgA deficiency actually do not suffer from any clinically significant illness. Acquired IgA deficiency is primarily caused by viruses including the rubella virus and EBV, and drugs. It was formerly described in epileptic patients treated with the anticonvulsant drugs diphenylhydantoin [215 – 218], carbamazepine [219] and sodium valproate [220], and in rare patients treated with the antirheumatismal drug sulfasalazine [221,222], the NSAID fenclofenac [223], and anticancer chemotherapy [224]. Although, a causal relationship was suggested, the lack of matched controls or prospective studies precludes any firm conclusion.

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Selective lymphopenias. Following the discovery that CD4þ T lymphocyte numbers decrease with the progression of the disease in HIV seropositive individuals and are one of the most sensitive parameters for the follow-up and prognosis of affected patients, measurement of the CD4þ and CD8þ T lymphocytes is often routinely included in the nonclinical immunotoxicity evaluation of new molecular entities. Regulatory bodies, such as the European Medicines Evaluation Agency [225] and the US Food and Drug Administration [226] published diverging recommendations regarding the predictive value of this end-point as a first-line screen for nonclinical immunotoxicity evaluation. It is indeed critical to bear in mind that measuring either CD4þ or CD8þ T lymphocytes does not provide any clue on immune function in human beings exposed to drugs and other chemicals. Lymphopenia is relatively common, often reversible and associated with acute illnesses, such as sepsis and cancer [227]. Profound depletion in CD4þ T lymphocytes is associated with opportunistic infections, whereas the clinical consequences of mild to moderate decreases even though statistically significant are not known. The effects of depletion in CD8þ T lymphocytes are poorly documented, but as CD8þ T lymphocytes play a crucial role in antiviral defenses and various immunopathological conditions, such as autoimmune diseases, depletion in CD8þ T lymphocytes can be suspected to cause adverse consequences. Overall, there is limited, if no evidence at all that decreases in either CD4þ or CD8þ T lymphocyte counts do result in immunotoxic effects. IMMUNOSUPPRESSION VS. IMMUNODEPRESSION Definitions. The terms immunosuppression and immunodepression are often used interchangeably. However, this can prove misleading, at least as far as medicinal products are concerned. Strictly speaking, immunosuppression means total inhibition or abrogation of immune responsiveness, whereas immunodepression refers to a mere decrease in immune responsiveness that can still be associated, whatever its magnitude, with a certain degree of immune response. In order to avoid confusion or misinterpretation, immunosuppression and immunodepression should not be used whatever the degree of immune response impairment that is evidenced. The term overimmunosuppression has been proposed to refer to the profound immunosuppression associated with infectious and tumorigenic complications in organ transplant patients [228] and this further illustrates the difference that certainly does exist between immunosuppression and immunodepression. Even though such a distinction may not be considered relevant for risk assessors who extrapolate animal data on industrial and environmental chemicals to man, it is important to bear in mind that the situation is markedly different for medicinal products where the focus is primarily on risk/benefit instead of risk assessment. Depending on the expected therapeutic benefit, the magnitude of which depends on the severity of the disease to be treated and whether other effective therapeutic modalities are available, that a new pharmaceutical product is suspected to induce either immunosuppression or immunodepression impacts on the conclusions of risk/benefit analysis. Adverse consequences of immunodepression. A major issue when distinguishing immunodepression from immunosuppression is whether the ensuing clinical adverse effects


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are qualitatively and quantitatively similar or not. The clinical data overviewed in previous sections of this chapter unambiguously support the view that exposure to drugs and other chemicals known to abrogate immune responses can result in infectious complications and virus-induced malignancies. There are clinical data suggesting that immunodepression can induce similar, although less frequent and less severe adverse effects. Infectious complications. Patients treated with low-dose methotrexate develop infections, such as P. carinii and Nocardia pneumonias, cryptococcosis and histoplasmosis [229 – 235]. More recently, the negative impact of immunodepression on the resistance against infectious pathogens was further demonstrated by the increased risk of developing infections, in particular tuberculosis, in patients treated with immunodepressive antiTNF-a drugs [236,237]. Already, mentioned findings in relation to the Yusho [43] or YuCheng [44] disease, or UV exposure [58] are also supportive of the conclusion that immunodepressive exposures can lead to more frequent infections. However, immunodepression-related infections are often clinically unremarkable even though opportunistic infections have sometimes, but rarely been reported. Importantly, a causal relationship between unremarkable infections and drug or chemical exposures can only be demonstrated by epidemiological studies [238,239]. Malignancies. Rheumatoid arthritis patients treated with low-dose immunosuppressive (i.e. immunodepressive) drugs have an increased risk of death from malignancy (RR ¼ 4.2 vs. 1.1) when compared to matched patients who never received immunosuppressants [240]. Lymphomas have been reported in rheumatoid patients treated with low-dose immunosuppressive drugs (see above). In contrast, no human data suggest that lymphomas could be more frequent in human beings following immunodepressive occupational or environmental exposure. Threshold between immunodepression and immunosuppression. A major and unsolved issue is the determination of those quantitative changes in immune functions that can result in clinically significant adverse effects. Based on the large experience gained from animal studies, the conclusion can be made that a linear dose – response relationship does exist and that increasing the dose of immunosuppressive drugs from low (immunodepression) to high (immunosuppression) results in more frequent and more severe infections reflecting an increasing impairment of resistance toward infectious pathogens. There are, however, no data to determine the immunodepression/immunosuppression threshold above which clinical adverse effects are likely to occur [241]. Correlations have been shown between changes in immune functions and host resistance in laboratory animals [242], but similar data are lacking in man with the sole exception of UV exposure [58]. The parallelogram paradigm that is often used to compare in vitro and in vivo animal data with in vitro human data in order to predict possible in vivo effects in humans [243] can only serve as a gross approximation. A major difficulty that has yet to be overcome is the impact of the functional reserve capacity of the immune system [244 –248]. Thus, a mild to moderate impairment in one immune function may not result in any observable adverse effect. The current lack of human data precludes any documented extrapolation. In addition, redundancy is a key characteristic of the immune system. When one arm of the immune system is impaired, compensatory effector mechanisms are triggered and no immunotoxic effect can be seen.

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One single change in the immune system, therefore, may not result in overt immunotoxicity and a global assessment of general toxicity and immunotoxicity data is absolutely required.

IMMUNOSTIMULATION The term immunomodulation [249 –251] has long been preferred to immunostimulation. The introduction of recombinant therapeutic cytokines into the clinical setting demonstrated, however, that immunological responses can be increased (or stimulated) as any other physiological responses of the body. Other terms, such as immunorestoration [252] or immunoenhancement [253], have also been used, but these purely semantic differences only reflected limitations in our understanding of the potential for pharmacological immunostimulation. Because immunosuppression has been the primary concern of immunotoxicologists during the past 20 –30 years and also because potent immunostimulatory drugs, such as recombinant IL-2 and the IFNs have only been relatively recently introduced into the clinical setting, the clinical adverse effects related to immunostimulation in human beings have not yet been extensively investigated. However, with the steadily increasing number of patients treated with these drugs, the clinical experience is growing rapidly [254,255]. In fact, the most recent findings largely confirmed early isolated case reports of adverse effects associated with medicinal products proposed for use as immunostimulants, such as levamisole, Corynebacterium parvum and thymic hormones [256]. FLU-LIKE REACTIONS Hyperthermic reactions (. 38– 38.5 8C) with chills, arthralgias and malaise have been described shortly after the introduction of medicinal products suspected to exert immunostimulating properties, such as glycan derivatives, C. parvum, BCG, levamisole or early poorly purified interferon preparations [256]. Because of their clinical features, these reactions are generally referred to as flu-like reactions. Clinically similar reactions are observed following the administration of vaccines [257], particularly recall injections in children. Whatever the cause, they are often easily prevented or controlled by the administration of antipyretics and minor NSAIDs. Similar, but typically more severe and sometimes treatment-limiting reactions have been reported in patients treated with natural [258] and recombinant IFNs, including IFN-a [259], IFN-b [260] and IFN-g [261], or with recombinant IL-1 [262], IL-2 [263], IL-10 [264], TNF-a [265] as well as monoclonal antibodies [266,267]. In these patients, hyperthermia can reach 40 8C or even more, and is associated with severe symptoms, such as diarrhea, vomiting, chest pain, hypotension possibly leading to cardiovascular collapse or cardiac ischemia, and neurological disorders, such as tremor, confusion, obnubilation and seizures. Slower administration, lower doses, and treatment with NSAIDS, such as ibuprofen [268] or indomethacin [269], or corticosteroids [270] were shown to reduce the incidence and/or the severity of the reaction. The terms acute cytokine syndrome and cytokine release syndrome tend to be used instead of flu-like reactions to describe severe adverse events.


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INCREASED INCIDENCE OF AUTOIMMUNE DISEASES Shortly after their introduction into the clinical setting, immunostimulatory drugs were found to exacerbate or facilitate a variety of underlying diseases in treated patients, namely dormant diseases, autoimmune diseases and immunoallergic reactions [256]. Early and somewhat speculative reports were later largely confirmed following the introduction of therapeutic recombinant cytokines. When early immunostimulatory drugs were introduced into the clinical setting, they were often empirically administered to patients with varied immunopathological conditions. For instance, patients treated with levamisole, a prototypic immunostimulatory drug in the 1970s, developed skin allergies [271,272], immune complex vasculitis [272,274,275], exacerbation of rheumatoid arthritis [276] or Crohn’s disease [277], but seemingly none developed autoimmunity. Similar, although less frequent adverse reactions were reported with other immunostimulatory drugs, such as thymic hormones, BCG or microbial extracts. More recent data confirmed early findings. Thus, recombinant IFN-a and IFN-b were reported to exacerbate psoriasis [278 – 280], lichen planus [281] or sarcoidosis [282,283]. In fact, one of the most frequent immune-mediated adverse effects of therapeutic recombinant cytokines is a marked increase in the incidence of autoimmune diseases [284 – 286]. As more extensively detailed in Chapter 10 of volume II, an unexpectedly large number of patients treated with rIL-2, or the rIFNs-a, -b and even IFN-g have been reported to develop extremely varied autoimmune diseases including autoimmune thyroiditis, thrombocytopenia, hemolytic anemia and hepatitis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, myasthenia gravis, multiple sclerosis, and Sjo¨gren’s syndrome. Autoimmune thyroiditis is by far the most common finding. IFN treatments were associated with thyroid diseases, including hypothyroidism, hyperthyroidism and biphasic thyroiditis, in 4– 14% of patients and the majority of patients had serum antithyroid thyroid microsomal and antithyroglobulin autoantibodies [287]. Thyroid disease is seemingly more common in patients treated with IL-2. Thus, up to 35% of rIL-2 treated patients with melanoma or renal cancer developed hypothyroidism and antithyroid autoantibodies [288]. The role of chemical exposures in the development of autoimmune diseases is a matter of growing concern [289 – 293]. Although, an epidemiological link was sometimes suggested between given chemical exposures, such as pesticides, and the occurrence of more frequent autoimmune diseases, no mechanistic clues support a causal relationship and in any event, the suspected chemicals are not known to exert immunostimulatory properties. A possible exception is hexachlorobenzene, which was repeatedly shown to induce stimulatory effects [294,295] as well as autoimmunity [296,297] in rats. A causal link has, however, been disputed [298]. HYPERSENSITIVITY REACTIONS TO VARIED ALLERGENS Another type of adverse effects associated with immunostimulatory drugs is an increased incidence of hypersensitivity reactions to varied allergens. Surprisingly, this issue has very rarely been addressed. Post-marketing surveillance data on immunostimulatory drugs are scarce, but exacerbation of asthma, eczema and rhinitis was reported shortly after initiation

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of treatment [299,300]. Elevated serum IgE levels and skin eruptions were more frequent in rheumatoid patients treated with levamisole than untreated controls [301]. Skin eruptions and systemic hypersensitivity reactions to a variety of drugs have been reported in patients concomitantly treated with recombinant cytokines including rIL-2 [302 – 305] and the IFNa and -b [306 – 308]. No differences in total serum IgE levels, however, were noted when comparing chronic hepatitis C patients treated with interferon-a to matched controls [309]. The most compelling evidence that treatment with immunostimulatory drugs may be associated with more frequent hypersensitivity to varied allergens is the increased incidence of adverse reactions to radiological contrast media in patients treated with rIL-2. Several retrospective or prospective studies [310 – 312] confirmed isolated case reports [313 – 315]. These reactions consisted of rash, pruritus, edema, fever, nausea, vomiting, hypotension and oliguria, and usually developed within 1 – 4 h after injection of contrast media. Delayed reactions are also possible. INHIBITION OF DRUG-METABOLIZING ENZYMES In both animals and man, CYP450-dependent metabolizing enzymes are depressed following the administration of interferon or interferon-inducing agents [316 –318]. It has long been recognized that infectious diseases, particularly viral infections, and inflammation exert similar effects [319 – 321]. Further to findings in rats treated with the interferon inducer tilorone [322], the majority of early medicinal products with immunostimulating properties, including poly (rI:rC) and a wide variety of bacterial extracts [323 –328] have been shown to inhibit hepatic drug metabolizing enzymes, either in vitro or in laboratory animals. Cytokines have been shown to depress hepatic microsomal CYP450-mediated metabolism after in vitro or in vivo administration to rodents and in a few instances in man as well. rIL-1a caused a significant reduction in the amount of hepatic CYP450 and CYP reductase activity in rats [329]. There was also a significant reduction in CYP1A2, 2C11, 2D, 2E1 after 24 h post-administration and decreased aniline-hydroxilase, benzphetamine N-demethylase and 7-ethoxycoumarin-O-deethylase activities. This inhibitory effect resulted in diminished hepatotoxicity of carbon tetrachloride and paracetamol in IL-1a treated rats [330]. The effects of IL-1b seem more complex as CYP2C6, 2C13, 2E1, and 3A2 were suppressed in rats, in contrast to CYP2A, 2B1/2, 2C11, and 4A1 [331], whereas other authors found a marked decreased in CYP2C11 and 3A1 [332]. Destruction of CYP450 monooxygenases by free radicals, and downregulation or decreased transcription of CYP450 enzymes have been suggested to be involved. rIL-2. The total CYP450 content of cancer patients treated with high-dose rIL-2 was significantly decreased by 34% with reductions in CYP1A2, CYP2C, CYP2E1 and CYP3A4 of 37, 45, 60 and 39%. In addition, the activity of methoxyresorufin-Odemethylase and erythromycin-N-demethylase was reduced by 62 and 50%, respectively [333]. The effect was clearly dose dependent as patients treated with 3– 6 £ 1026 IL-2 units/m2 instead of 9 –12 £ 1026 IL-2 units/m2 exhibited no changes. These findings confirm previous results in rats [334].


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IL-4. In contrast, IL-4 was reported to up-regulate CYP2E1 in primary cultures of human hepatocytes [335]. rIL-10. Subcutaneous injections of 8 mg/kg/day of human nonglycosylated rIL-10 to 12 human volunteers for 6 consecutive days induced a modest, but significant decrease compared to placebo in CYP3A activity as assessed from midazolam clearance, whereas no changes were seen in CYP1A2, CYP2C9 or CYP2D6 activities [336]. Interferons. rIFN-a was found to influence negatively the pharmacokinetics of theophylline [337,338], antipyrine [339] although inconsistently [340,341], erythromycin [342] and hexobarbital [343] in healthy volunteers as well as in patients treated with therapeutic doses. The subcutaneous administration of a therapeutic dose of IFN-a was shown to decrease cyclophosphamide clearance by 63% with a concomitant 137% increase in half-life. Interestingly, the formation of the cytotoxic metabolite 4-hydroxy-cyclophosphamide was markedly reduced [344]. In contrast, the pharmacokinetics of 5-fluorouracil was not altered by IFN-a2a treatment [345]. Vaccines and drug-metabolizing enzymes. Conflicting results have been observed with influenza and tetanus vaccines [346]. However, it is noteworthy that most studies suggesting possible interactions with a variety of drugs were usually published years before studies showing no effects. Therefore, the role of different formulations can be suspected to account for these discrepancies. A dose-dependent effect is also possible as mice injected with a human dose of whooping cough, tetanus, rubella, or poliomyelitis vaccine consistently had prolonged pentobarbital sleeping time on day 4 post-administration [347]. Bacillus Calmette –Gue´rin was consistently shown to inhibit hepatic drug-metabolizing enzymes in rats [348,349]. Interestingly, 3 of 11 cancer patients who received intradermal BCG as immunotherapy had decreased hepatic N-demethylation of aminopyrine assessed by the aminopyrine breath test [350]. Two weeks following BCG vaccination, the mean serum half-life of theophylline was significantly increased in 20 healthy female volunteers [351]. In contrast, no effect of BCG was noted on dacarbazine metabolism in cancer patients [352]. Influenza vaccines have been extensively studied and published results are markedly conflicting. Influenza vaccination was found to significantly augment the half-life of theophylline in three patients and four healthy volunteers. Importantly, the elevation of plasma theophylline concentrations was associated with clinical signs of theophylline toxicity [353,354]. Theophylline oxidation was slightly decreased 1 day, but not 7 days after influenza vaccination, whereas chlordiazepoxide oxidation was not affected [355]. No changes in plasma theophylline concentrations were found in 34 outpatients with chronic obstructive pulmonary disease 1 – 14 days after influenza vaccination [356] or in 8 asthmatic children, 52 h after influenza vaccination [357]. A significant increase in the mean serum concentrations of phenytoin, phenobarbital and carbamazepine was found between 7 and 14 days after influenza vaccination when compared to baseline values [358], but no changes in the serum concentrations of clozapine and its main metabolites Ndesmethylclozapine and clozapine-N-oxide were evidenced in 14 patients with schizophrenia when measured 2 days before and 2, 4, 7 and 14 days after influenza vaccination [359]. No effects were similarly seen in the pharmacokinetics of antipyrine, paracetamol, alprazolam or lorazepam of healthy volunteers when compared before, and 7 and 14 days

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after influenza vaccination [360]. In 12 patients on long-term stable warfarin treatment, influenza vaccination induced an asymptomatic increase in prothrombin time which was maximal on day 14 post-vaccination [361]. Similar findings were obtained in 8 patients on long-term warfarin treatment, but not in healthy subjects [362]. In contrast, no interaction between warfarin or theophylline and influenza vaccination was observed in 13 elderly persons [363] and the prothrombin time of 21 male patients on long-term warfarin treatment remained unaffected by influenza vaccination for up to 3 months [364]. The effects of acenocoumarol, another oral anticoagulant, measured on the International Normalized Ratio (INR) were not enhanced by influenza vaccination in 43 patients [365]. Finally, influenza vaccination did not alter CYP2E1 activity measured by the clearance of chlorzoxazone 1– 2 weeks post-vaccination [366] nor CYP3A4 activity measured by the erythromycin breath test in 15 healthy volunteers [367]. Tetanus vaccines. In mice, a human dose of tetanus toxoid induced a 1.6 to 1.8-fold increase in hexobarbital sleeping time used as a measurement of drug-metabolizing enzymes activity [368]. A significant increase in the half-life of antipyrine was seen in human volunteers when compared before and 4 days after tetanus vaccination [369]. OTHER COMPLICATIONS ASSOCIATED WITH IMMUNOSTIMULATION Increased organ graft rejection is at least a theoretical complication of unexpected immunostimulation. Suspicion arose that treatment with cimetidine, although a mildly immunostimulatory drug, might result in increased rejection in kidney transplants [370 – 372]. Recombinant IFNs-a are commonly used to treat chronic hepatitis C in organ transplant patients and no case of graft rejection was seemingly reported. Immunosuppressive drugs, such as cyclophosphamide or cyclosporine, have been shown to enhance antigen-specific immune responses, depending on the dose and time of administration in relation to antigen injection [373]. In contrast, immunostimulatory drugs, such as levamisole, were found immunosuppressive in particular conditions [374]. Based on these early experimental works, the term immunomodulating drug was preferred to immunostimulatory drug, although no clinical benefit was actually ever evidenced. However, the risk of immunosuppression in patients treated with immunostimulatory drugs or the opposite seems totally marginal based on the available clinical evidence.

HYPERSENSITIVITY Hypersensitivity reactions are by far the most frequently reported immunotoxic effects of drugs and other chemicals in human beings. These reactions have been a major cause of drug withdrawal from the market in the past few decades. In most instances, the causative drug is administered to patients for therapeutic purposes. There is also a small risk that residues of veterinary drugs may elicit hypersensitivity reactions after consumption of food of animal origin [375]. However, few case reports have been published in the literature and most of these reports involved penicillin as the suspected causative drug [376,377]. Nearly every chemical substance in our domestic, occupational or natural environment can induce hypersensitivity reactions.


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EPIDEMIOLOGY Although, hypersensitivity is widely held as a major cause of clinical adverse events, there is surprisingly few data on the incidence of hypersensitivity reactions in the general population or selected groups of the population. Indeed, most publications consist of case reports and epidemiological data are relatively rare. Under-reporting is an obvious major bias [378]. Whatever the nature, origin and severity of the reaction under scrutiny, scarce or elusive incidence data are available. It is noteworthy that this lack of reliable data is a major difficulty when implementing appropriate prevention or eviction measures that often are the sole effective treatment, but in contrast paves the way to the irrational promotion of excessive and scientifically unfounded views. Severe anaphylactic or anaphylactoid reactions have been suggested to develop in about 1 in 5000 exposures to antibiotics or radiocontrast media [379], or in about 1 in 10,000 drug treatments [378]. Porter and Jick [380] recorded 12 out of 32,812 hospitalized patients with drug-induced anaphylaxis, i.e. 0.04%. In a survey of 90,910 hospital admissions between 1997 and 1999, Thong et al. [381] identified 210 cases with a documented IgE-dependent or T cell mediated drug-induced hypersensitivity reactions. Based on their review of the literature, Demoly and Bousquet [382] concluded that approximately one-third of all druginduced adverse reactions consists of hypersensitivity reactions, either immunoallergic or pseudoallergic. Lower estimates, e.g. 6– 10% of immunoallergic reactions among all druginduced adverse events [383] have also been proposed. Differences in the definition and selection of diagnosis criteria probably account for diverging results [384]. The same lack of reliable data precludes any sound estimate of the role of occupational exposure in hypersensitivity reactions. The skin is the first site of hypersensitivity reactions at the workplace followed by the lung [385]. Allergic contact dermatitis—the most frequent hypersensitivity reaction caused by xenobiotics—was said to affect 0.5 –1.9 per 1000 workers per year [386]. Around 12% of all allergic contact dermatitis were deemed to be occupationally related [387]. In the USA, the incidence of occupational asthma is in the range 29 –710 cases per million workers per year and 10– 25% of all asthmas were suggested to be work related [388]. Food allergies and food intolerance are also frequent. They have been suggested to affect up to 6% of infants younger than 3 years of age [389]. The environment, including indoor air pollutants [390], is a contributing factor to the development of hypersensitivity reactions and medical allergies, but its impact still remains to be quantified [391]. CLINICAL SIGNS AND SYMPTOMS Although hypersensitivity reactions can affect nearly every organ or tissue of the body, one organ or tissue is often a predominant target. The following description of the clinical signs and symptoms of hypersensitivity reactions is based on this premise. There are examples, however, of hypersensitivity reactions that consist of a variety of clinical signs and symptoms with no overt predominant target, such as the so-called multiple chemical sensitivity syndromes. Despite the ongoing controversy whether this is a real disease or not, a number of reports described patients who became polysymptomatic when exposed to low levels of chemicals [392 – 394]. Characteristic features of the

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syndrome is the involvement of more than one organ, the lack of specificity of clinical symptoms and signs which tend to be chronic, their recurrence when the patient is reexposed, the role of multiple unrelated chemical substances and improved clinical condition upon avoidance of the suspected exposure(s). A major difficulty is that no clear definition of the disease is widely accepted [395]. The most common complaints include respiratory symptoms, headache, fatigue, flu-like symptoms, depressive mood, ear, eye, throat and nose irritation, gastrointestinal disorders, muscle and joint pain. Last but not least, the underlying mechanism is not elucidated [396]. Due to the lack of conventional dose – response relationship as patients who develop clinical symptoms and signs are exposed to low and even very low doses in contrast to those exposed to higher doses that develop overt toxicity, the syndrome is often denied a toxic origin. Various theories have been proposed, including an abnormal immunological reaction. Further research is warranted to ascertain whether this syndrome is not merely psychosomatic and if so, what the underlying mechanism actually is. Cutaneous manifestations. Skin reactions are the most frequent immune-mediated adverse reactions to drugs and other chemicals [397 –399]. While relatively rare reactions involve specific IgE antibodies, e.g. urticaria, or circulating immune complexes, e.g. vasculitis, the majority of immune-mediated cutaneous manifestations caused by drugs and chemicals are presumably due to T cell-mediated mechanisms. The clinical presentation is nevertheless extremely varied. Severe toxidermias, such as erythema multiforme (EM), Stevens – Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), presumably combine both toxic and immune-mediated mechanisms. Whatever the clinical manifestation, it is absolutely essential to obtain a detailed medical history and a clinical description as accurate and comprehensive as possible to avoid imprecise diagnosis that is so frequent in the medical literature including even top-rank journals. It is also essential to keep in mind that the diagnosis is usually not based on a standardized step-by-step process, which does not mean, however, that the selection of assays should be made by mere guess! Angioedema was first described by Von Quincke in 1882, hence the frequently use of the term Quincke’s edema. In angioedema, there is an abruptly developing congestion of the mucous membranes that typically involves the eyes, mouth and throat [400]. Swelling of the lips and tongue may impair swallowing or respiration, the latter being also impaired by swelling of the larynx, epiglottis and surrounding tissue. Angioedema can be isolated without systemic involvement [401] or associated with various clinical signs and symptoms including urticaria, breathlessness and cough, or even systemic anaphylaxis. Indeed, the majority of angioedema is due to IgE-mediated mechanisms. Idiopathic nonallergic angioedema is a rare condition primarily linked to a congenital deficiency in complement C1 inhibitor. Nonallergic angioedema is a well-known adverse effect of angiotensinconverting enzyme (ACE) inhibiting drugs and the suspected mechanism is an inhibition of bradykinin degradation [402,403]. It is also a common clinical feature of NSAID intolerance [401]. Bullous eruptions. Drug eruptions may mimic spontaneous bullous dermatoses. Bullae are noticed in the context of severe and possibly life-threatening drug-induced reactions, such as SJS and TEN (see below).


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Contact dermatitis is a frequent complication of occupational exposures [404,405]. It accounts for at least 90% of work-related cutaneous disorders and 10% of all occupational diseases [406]. Contact dermatitis is also provoked by fragrances and cosmetics [407,408], botanical extracts [409], clothes and shoes, and topical drugs [410]. Allergic contact dermatitis is a T lymphocyte-mediated reaction, whereas irritant contact dermatitis is caused by nonantigen-specific mechanisms. Acute allergic contact dermatitis is characterized by pruritic vesicles on an erythematosus background. Long-standing lesions evolve toward skin thickening. Because elimination of the contact allergen is the best treatment, a careful clinical history and patch testing are essential for the identification of the offending chemical(s). Cross-reacting substances should also be listed. Patch testing is a procedure by which well-characterized contact allergens are applied to small areas of the skin under occlusion in standardized conditions. The patches are removed after 48 h and the skin response is quantified using a scoring system. Although patch testing allows accurate diagnosis in a number of patients, false positive responses are frequent. Drug hypersensitivity syndrome. Although most drug-induced toxidermias are hypersensitivity reactions, drug hypersensitivity syndrome (DHS) is a term used to describe reactions that are typically characterized by a severe skin eruption, variable visceral involvement and eosinophilia [411]. The acronym DRESS which stands for Drug Rash with Eosinophilia and Systemic Symptoms is often preferred to avoid confusion as most drug-induced cutaneous eruptions can be considered as hypersensitivity reactions [412 – 414]. The incidence of this syndrome is not known. One estimate is that one in 10,000 patients treated with anticonvulsants may be affected. Usually, clinical signs and symptoms develop after 2 –6 weeks of treatment. Fever and rash are the most frequent features. Initially, the rash is often a morbilliform eruption that subsequently is indurated with facial swelling and exfoliative dermatitis. Purpuric lesions and a few bullae are common. Mucous membrane lesions are possible without necrolysis. Lymphadenopathy, arthritis or hepatitis is seen in approximately 50% of patients. Interstitial nephritis is less frequent. Heart, lung, thyroid and brain involvement is possible, but relatively rare. Hypereosinophilia is noted in 90% of cases and mononucleosis-like lymphocytosis can also be seen. Because one organ is often predominantly involved, DHS is frequently misdiagnosed. The prognosis depends on the visceral involvement. Within a few weeks after discontinuation of the causative drug, full recovery is the expected outcome, but in case of associated hepatitis, the mortality rate may be as high as 8%. The use of corticosteroids to treat DHS is widely recommended despite the lack of scientific evidence. DHS was first described as the anticonvulsant hypersensitivity syndrome [415]. In fact, most early reports of lupus or pseudolymphoma [416] due to anticonvulsants were more probably DHS. Although anticonvulsants are the primary cause of DHS [417], the role of other drugs, including sulphonamides and sulphones, minocycline and allopurinol, is well established. The mechanism of DHS is not known. Experimental data suggest the involvement of specific T lymphocyte responses [418]. Interestingly, the involvement of CD8þ T lymphocytes was demonstrated in the popliteal lymph node assay [419] and in humans as well [420]. The contribution of a latent human herpesvirus 6 infection has been suggested [421,422]. The role of metabolites derived from the offending drug is widely accepted [423], but a direct interaction with T lymphocytes is also possible [424].

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The production of IL-5 and eotaxin [425,426] is observed when marked hypereosinophilia is present. Erythema multiforme (EM) is a usually benign erythematous eruption predominantly found on the extremities. The main causes are herpetic and mycoplasma infections, but drugs are seemingly often involved. The distinction between minor and major EM on the one hand, and major EM and Steven –Johnson syndrome on the other hand is often difficult [427]. When skin biopsies are performed, they show a lymphocytic infiltrate of the dermoepidermal junction. Erythema nodosum consists of inflammatory, tender, erythematous, nodular lesions, usually symmetrical and located on the shins, ankles and knees. It is typically associated with a wide variety of diseases. Sweet’s syndrome is characterized by an acute onset of nonpruritic, painful reddish nodules on the head and neck, chest and/or the upper limbs accompanied by fever and inconsistently extra-cutaneous manifestations including mucosal, musculoskeletal, hematological, pulmonary, hepatic and renal manifestations. Histopathological examination shows a diffuse dermal neutrophilic infiltrate. The pathogenesis is still not understood. Drugs are thought to account for less than 5% of cases of which one half was associated with rGM –CSF therapy [428]. Fixed drug eruptions. This is a bizarre reaction in that the eruption only develops and recurs at a single site [429]. They are relatively frequent and estimates ranked fixed drug eruptions as the second- or third-most frequent drug eruptions. The reaction is characterized by round or oval, erythematous patches with dusky red center on any skin or mucosal surface, with a predilection for the lips and genitalia. The skin lesion can be associated with a burning sensation, and fever, nausea or malaise. Usually, a fixed drug eruption occurs within hours and resolve spontaneously in a few weeks without scarring. The offending drugs are most often sulphonamides, barbiturates, or tetracycline derivatives. The mechanism is unknown. T CD8þ lymphocytes and activated keratinocytes are been suggested to play a key role [430]. Lichenoid eruptions. While lichen planus is a relatively common skin disorder of unknown etiology, Lichenoid eruptions are relatively rare, but can be caused by a variety of drugs including arsenicals, antimalarials, gold salts, a-methyldopa, NSAIDs [431]. The eruption consists of small, shiny, polygonal papules of variable location. Mucosal involvement is possible. It is extremely difficult to differentiate lichen planus from a druginduced lichenoid eruption. Photosensitivity. Sunlight is the cause of or contributing factor in skin diseases. Phototoxic and photoallergic reactions share similar clinical and histological appearance [432]. The most common reactions are phototoxic [433]. As regards drug and chemical exposures, phototoxic reactions are typically dose dependent and due to reactive metabolites produced by radiations in the UVA (315 – 430 nm) or visible regions that cause damages to cellular components within the skin. Theoretically, any person who receives a sufficiently high dose of a phototoxic chemical can develop an adverse reaction that typically manifest as an exaggerated sunburn response limited to sun-exposed areas and resolves shortly after cessation of the causative agent. Photoallergy occurs less frequently. It is triggered by levels of sunlight that are normally harmless. Photoallergic reactions are considered to be a special type of cell-mediated


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hypersensitivity. UV radiation is thought to induce the conversion of the parent molecule to metabolites that either play the role of haptens that directly bind to carrier macromolecules, or are activated to an unstable excited state that reverts to its ground state by releasing energy facilitating conjugation to a carrier macromolecule. Photoallergic reactions normally develop after 5 –10 days of treatment and sunlight exposure. The reaction recurs within 24 –48 h after a subsequent exposure to both the drug and sunlight. Clinically, the reaction is characterized by pruritic or eczematous skin eruption that can spread beyond the area of the skin exposed to sunlight. Histological findings are similar to those seen in contact dermatitis. Photopatch tests can be used to confirm the diagnosis. Drugs are responsible for the vast majority of photoallergic reactions including sulphonamides, quinolones, thiazides, phenothiazines and NSAIDs [434 – 436]. Purpura is defined as the presence of purplish discolorations in the skin produced by small bleeding vessels near the surface of the skin or the mucous membranes. Small purpura spots are called petechiae. Purpura may be associated with normal platelet counts, as in vasculitis, or decreased platelet counts, as in immunoallergic or autoimmune thrombocytopenias. Pustolosis. Acute generalized exanthematous pustulosis is a rare severe toxidermia [437] that is most often caused by a variety of drug treatments [438]. It is characterized by the acute onset of a diffuse edematous erythema rapidly progressing from the face to the trunk with numerous pin-head sterile pustules. Mucous membrane lesions are rare, but fever is nearly constant. Organ involvement, such as hepatitis, is rare. Hypereosinophilia is inconsistently seen. Due to the very short delay between the initiation of drug exposure and the onset of symptoms, it is logical to suspect the role of an underlying viral infection. Closely related generalized drug-induced pustular eruptions include generalized pustular rash and toxic pustuloderma. Rash. This is the most common skin eruption induced by drugs and other chemicals. Typically, morbilliform or exanthematous rash begins on the trunk and extends to cover a larger area of the body. Morbilliform rash is the most frequent and could be occurring in up to 2% of treated patients. The onset varies within 1 – 2 weeks after the start of treatment. A T cell-mediated cytotoxic mechanism is most likely involved [439]. Drugs can form labile, covalent complexes with MHC class II antigens, which are sufficient to stimulate T lymphocytes that bear the TCR for the drug. An additional danger signal is, however, required. This signal can be provided by APCs that enhance the activation of T lymphocytes. A viral infection can serve as a trigger of APC activation. The release of pro-inflammatory cytokines results in the destruction of keratinocytes. The amidopenicillins, e.g. ampicillin and clamoxicillin, are unique as a morbilliform rash develops in approximately 10% of patients and even many more if the patient has a concurrent infection, such as infectious mononucleosis [440]. The mechanism is not known, but the increased production of IL-5 in response to viral infection might upregulate the expression of MHC class II antigens on keratinocytes. The rash may clear spontaneously even though the drug is not discontinued. In most patients with a history of ampicillin rash, readministration does not lead to relapse, which further supports the role of an associated, even though not apparent viral infection. However, drug-specific lymphocyte reactivity was recently been shown in patients who developed amoxicillin rash in the course of infectious mononucleosis [441].

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Stevens– Johnson syndrome and toxic epidermal necrolysis are the most severe, potentially life-threatening, cutaneous complications of drug treatments [442,443]. It is still controversial whether SJS is a minor form of TEN or a distinct disease. The confusion is fuelled by the presence of many similar clinical signs and symptoms despite differences in their magnitude and severity. In addition, the causative mechanism is not fully elucidated. Therefore, SJS and TEN are often included in the wide spectrum of EM diseases. Lyell described the four first patients with TEN and the term Lyell’s syndrome is sometimes used alternatively. Typically, the first clinical symptoms appear within 7 –21 days after the initiation of treatment. Both SJS and TEN are initially characterized by a flu-like reaction of variable severity and mucous membrane involvement. A painful erythema is seen on the face and the trunk. Skin lesions extend within 2 – 3 days with purpuric macules and bullae leading to erosion of the epidermis that typically covers less than 10% of the total surface of the body in SJS and more than 30% in TEN. Purpuric macules are widespread in TEN. Painful erosions of the mucous membranes affect the oropharynx, eyes, genitalia and anus resulting in dysphagia, conjunctivitis, keratitis, diarrhoea and respiratory distress. Hematological anomalies including anemia, neutropenia and lymphopenia are frequent, whereas liver and renal involvements are characteristic of the most severe cases. The diagnosis is confirmed by histological examination of the skin. The prognosis and management of these toxidermias are similar to severely burnt patients. The mortality rate is about 5% for SJS and 30% for TEN. Sepsis is the main cause of death. SJS and TEN affect approximately 0.5– 2 in 1 million persons per year [444 –447]. A fairly large percentage develops in relation to drug treatments, but they also occur in about 3% of bone-marrow transplant patients and more rarely in patients with systemic lupus erythematosus or infection with Mycoplasma pneumoniae. Even though the cause is probably multifactorial, a number of drugs have been shown to be involved. Only 5% of SJS occur in the absence of any drug treatment, whereas drugs are considered to be involved in approximately 50% of TEN. The incidence of drug-induced TEN was shown to be 2.7 times higher in the elderly than in younger patients with a fatal outcome twice as frequent. However, the suspected drugs were similar in both groups [448]. A possible interaction with the HIV virus is suggested by the greater incidence in seropositive patients, i.e. up to 1 in 1000 patients. Epidemiological studies identified which drugs are the most frequently involved [449,450]. Sulphonamides are the leading cause with a relative risk of 172. Trimethoprim accounts for 2/3 of cases involving sulphonamides. Various classes of antimicrobials including the penicillins, cephalosporins, quinolones, tetracyclines and imidazole antifungal drugs have a relative risk between 5 and 25. Antiepileptic drugs are also very commonly involved, particularly carbamazepine, phenobarbital, diphenylhydantoin and valproic acid. Finally, the NSAIDs diclofenac and oxyphenylbutazone, and allopurinol are often associated with either pathological condition. The pathogenic mechanism of SJS and TEN is not fully elucidated, but a number of findings over the last years have supported the theory of an intervention of the immune system. A relation with some HLA phenotypes [451] and the predominant infiltration of CD8þ T lymphocytes in immunohistological examinations of skin biopsy specimens [452,453] have been evidenced. Although most publications propose the involvement of


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a cell-mediated reaction, the link remains to be made between severe skin reactions and effector immune mechanisms. Urticaria is estimated as the second-most frequent drug-induced skin reaction. However, infections account for the majority of urticaria in children, so that differentiating the role of infection and antibiotics is often tricky [454]. Clinically, pink wheals on the skin and pruritus are hallmarks of urticaria. No clinical signs or histological findings can distinguish drug-induced urticaria from other causes including cold, heat, and sunlight or food intolerance. Single lesions typically resolve within 24 h, but new ones may arise. Chronic urticaria is characterized by the persistence of lesions beyond 6 weeks. Angioedema is the corresponding tissue reaction of urticaria. It is characterized by deep swelling, most frequently of the lips and eyes, and may last several days. Acute druginduced urticaria can be due to specific IgE as a manifestation of immediate hypersensitivity (anaphylaxis). However, no more than 10% of urticaria have an immunoallergic origin. Other immune-mediated mechanisms include IgG-mediated reactions and deposition of circulating immune complexes (serum sickness-like diseases) leading to rare urticarial vasculitis preferentially associated with pain instead of pruritus [455]. Vasculitis is an inflammatory reaction of blood vessel walls associated with necrosis. Infections, particularly group A streptococci and hepatitis B virus infections are wellrecognized causes of vasculitis [456]. Drugs have been estimated to account for approximately 10% of all vasculitides [457]. Nearly all classes of drugs have been reported to cause vasculitis. That environmental factors, such as solvents, pesticides and silica may provoke vasculitis was recently suggested [458]. The clinical presentation of drug-induced vasculitis is not different from that of idiopathic vasculitis. The skin is by far the most frequently affected organ. Typically, vasculitis presents as purpura. It is, however, important to recall that all purpuric lesions are not associated with vasculitis and that not all vasculitides are associated with purpura. The histological examination of a skin biopsy may be necessary to confirm the diagnosis of leukocytoclastic vasculitis. Nonskin manifestations of drug-induced vasculitides include arthralgias and arthritis, and more frequently glomerulonephritis with an incidence of up to 40%. Hepatic involvement is also often described. Eosinophilia is more common in systemic than cutaneous vasculitis. In many cases, drug-induced vasculitis resolves within weeks after discontinuation of the offending drug. The measurement of TNF-a and IL-10, but not IL-6 and IL-2R serum levels in patients with hypersensitivity vasculitis was suggested to be a useful diagnosis tool [459]. The generally accepted mechanism is the deposition of circulating immune complexes in skin small vessels [460] resulting in the activation of the complement cascade, the release of vasoactive mediators, the infiltration of neutrophils in the perivascular space and the secretion of cytolytic proteins. When immune complexes have a small antigen excess, they are soluble and cause either vasculitis or serum sickness. When there is a strong antibody excess as in patients hyperimmunized by previous injections of a vaccine, the immune complexes are poorly soluble and induce a localized tissue necrosis (Arthus-type phenomenon). Hematological manifestations. Immunoallergic cytopenias mostly manifest as destruction of one or several blood cell lines due to an immune-mediated mechanism restricted to allergic patients. Even though drugs are often considered the most likely cause

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of immunoallergic cytopenias, the incidence is low and probably not more than 1 in 100,000 –300,000 treated patients [461,462]. In most instances, they are acute phenomena with clinical signs and symptoms related to the cytopenia developing in few hours or days after the offending exposure, a rapid recovery provided the initial manifestations are not too severe, and finally a high probability of reproducing the adverse event upon re-exposure. Agranulocytosis is characterized by the absence of, or a marked decrease (neutropenia) in neutrophils in the peripheral blood. The overall incidence of agranulocytosis has been estimated in the range 1.6 –3.4/year/million inhabitants [463]. Agranulocytosis is more frequent in female and elderly patients, and drug treatments are considered a leading cause. Patients are either asymptomatic and agranulocytosis is diagnosed after a routine blood examination, or develop signs of infection, in particular sore throat. In the latter case, neutropenia is often below 100 per mm3. Infections of the upper airways and pneumonitis are the most frequent complications to be expected. A bone marrow biopsy can show normal or increased neutrophil precursors consistent with immune destruction or complete absence of the myeloid cell line. A direct toxic effect and an immune-mediated reaction are the two main mechanisms of drug-induced agranulocytosis. Drug-dependent neutrophil antibodies have been detected in some patients [464]. A large number of drugs have been associated with immunoallergic agranulocytosis, but a higher risk is linked to treatments with antithyroid drugs, phenothiazine derivatives, quinine, NSAIDs and anticonvulsants. Treatment with hematopoietic grow-factors is used in an attempt to speed recovery [465]. Hemolytic anemias caused by xenobiotics are primarily due either to direct or antibodymediated toxicity to the membrane of erythrocytes [466,467]. Depending whether the involved antibodies are autoantibodies or not, immune-mediated hemolytic anemias are either autoimmune or immunoallergic [468]. However, the distinction may be difficult to make as both autoantibodies and drug-dependent antibodies can be provoked by the same drug and even in the same patient as was described in nomifensine-treated patients [469]. Typically, immunoallergic hemolytic anemias are related to IgM or IgG antibodies that fix complement to the cell membrane of erythrocytes. One single attached IgM is required to activate the first component (C1) of the classical activation pathway of the complement cascade, whereas two closely bound IgG are required to induce activation of C1. Immunoallergic hemolytic anemias are usually acute or subacute, and hemolysis is typically intravascular. There is a variable fall in hemoglobin levels, hemoglobinemia and hemoglobinuria. Clinical symptoms usually develop within hours following drug intake and include abdominal and dorsal pains, headache, malaise, fever, nausea and vomiting. Shock and acute renal failure are noted in 30– 50% of acute anemias. Hemolysis may be less brutal and predominantly extravascular. In rare instances, a cutaneous rash, oedema or other hematological manifestations can be seen. Irrespective of the clinical expression, full recovery is normally very rapidly obtained after discontinuation of the offending drug and supportive measures when required. The diagnosis of immunoallergic hemolytic anemias typically relies on the presence of hemolysis and a positive direct Coombs’ test. The direct Coomb’s test or direct antiglobulin test (DAT) is used to determine whether erythrocytes are coated with antibodies. However, the involvement of varied mechanisms or a short delay since acute hemolysis can result in false positive direct Coombs’s test [470]. Two main mechanisms are involved in drug-induced immunoallergic hemolytic anemias. In the first situation, the causative drug is


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nonspecifically bound to erythrocytes and reacts with circulating drug-specific antibodies. The best examples are hemolytic anemias induced by the beta-lactam antibiotics, especially third-generation cephalosporins [471,472]. One consistent feature is the exquisite specificity of formed antibodies for the causative drug or its metabolites. Specificity can even be so extreme as to differentiate between enantiomers of the same molecule as evidenced by anti-nomifensine antibodies [473]. The diagnosis is based on the clinical course of events and the direct Coombs’ test following incubation of normal erythrocytes, high concentrations of the suspected drug and the patient’s serum [474,475]. In the second situation, the drug binds to a plasma protein to form an antigenic complex. The consequence is the production of IgM against the drug – protein complex that can be passively fixed to erythrocytes. The reintroduction of even a small amount of the drug triggers an antigen – antibody reaction leading to intravascular hemolysis by activation of the complement system [476,477]. The immune reaction can be simulated in vitro after incubation of normal erythrocytes, the patient’s serum and low concentrations of the suspected drug. Thrombocytopenia. Drug-induced immunoallergic thrombocytopenias are rather uncommon with the notable exception of heparin [478] and there are extremely few case reports of thrombocytopenia associated with chemical exposure [479]. Immune mechanisms similar to those resulting in erythrocyte destruction cause thrombocytopenia. Most patients with immune thrombocytopenia have detectable antibodies against platelet glycoproteins, especially the glycoproteins Ib/IX and IIb/IIIa. Due to the presence of immune complexes consisting of the drug and antibodies, platelets are destroyed by complement or taken up by macrophages. Clinically, thrombocytopenia leads to bleeding, such as petechiae and ecchymoses when platelet counts are less than 10 –30,000 per mm3. Bone marrow examination typically shows a normal or increased number of megacaryocytes. Antibodies to clotting factors. Hemophiliac patients require substitutive therapy with factor VIII or IX. Although, antibodies can spontaneously arise, substitutive therapy with either purified factors from blood donors or recombinant factors can produce antibodies in up to 15 – 20% patients and they are sometimes neutralizing [480]. This is an unrewarding illustration of the current difficulties for predicting the immunogenicity of therapeutic proteins in man [481]. Cardiac and vascular manifestations. As many other organs the heart and vessels are susceptible to immune-mediated injury [482]. Cardiac diseases, such as myocardial infarction, cardiomyopathy and myocarditis are occasionally associated with deposition of antibodies within the myocardium or circulating cardiac autoantibodies [483]. Lymphocyte and macrophages infiltration is often noted in chronic inflammatory heart disease. Myocardial inflammation may be associated with irreversible or reversible immune injury. IL-1, IL-6 and TNF-a released by macrophages contribute to cardiac dysfunction [484]. Cytotoxic lymphocytes are also involved in many cardiac diseases by releasing perforin and granzymes that are cytotoxic to cardiac myocytes, and apoptosis via the Fas – FasL system [485]. There is a growing body of evidence that macrophages and T lymphocytes are major components of atherosclerotic lesions, thus opening new avenues of research for better drugs to treat atherosclerosis [486].

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Anaphylactic shock is one of the most severe hypersensitivity reactions [487 – 490]. The risk of death has been estimated to be about 1%. Drugs, food and insect stings are the most common causes of anaphylactic shock. Typically, anaphylaxis unexpectedly develops within 15 –20 min after the last contact with the allergen and almost always within the first 2 h. The rapidity of onset depends on the route of entry, the dose or concentration of the allergen, the patient’s sensitivity. Although the clinical picture may vary, patients usually first complain of intense itching, and then often develop urticaria and angioedema. Tachycardia, hypotension rapidly progressing to cardiovascular collapse and possibly shock in the most severe cases, marked respiratory difficulties with cyanosis are the hallmarks of systemic anaphylactic reactions. Up to 20% of patients may experience biphasic or even delayed anaphylaxis with persistence of symptoms for 6– 8 h after an initial reaction. The clinical signs and symptoms of systemic anaphylaxis reflect the potent biological effects of mediators that are released either abruptly (e.g. histamine) or secondarily (e.g. leukotrienes). There is a marked increased in vascular permeability that can result in the rapid transfer of 50% of the intravascular fluid to the extravascular space. In addition, there is an intense constriction of smooth muscles in bronchi and coronary arteries. In humans, the heart and the lung are the primary target organs. Conduction defects, rhythm disturbances and acute myocardial ischemia can develop in the course of anaphylactic shock, even in patients with normal coronary arteries [491]. Involved organs differ among species. Thus, in the guinea pig there is marked constriction of bronchial smooth muscles leading to bronchospasm, hypoxia and death. In the rabbit, anaphylaxis produces vasoconstriction of the pulmonary artery and right ventricular failure. In the dog, the primary target organ is the venous system of the liver resulting in severe hepatic congestion. Anaphylactic shock is a major emergency. The initial measures tend to assure and maintain normal respiratory and circulatory functions. The key treatment is epinephrine (adrenaline) injected subcutaneously or intramuscularly. In refractory patients, the intravenous route can be used. Patients treated with b-adrenergic receptors antagonists (b-blockers) are at a greater risk of developing more severe anaphylactic shock as the administration of epinephrine may be ineffective [492]. Antihistamine drugs have a minor, if any role in the treatment of anaphylactic shock, but can be useful to prevent anaphylaxis in at-risk patients [493]. Vasculitis is primarily the consequence of an immune-mediated injury of small vessels limited to the skin, i.e. cutaneous vasculitis, or spreading to various organs, in particular the kidneys, i.e. systemic vasculitis (see page 77). Hepatic and gastrointestinal manifestations Hepatitis induced by drugs are infrequent and generally considered as idiosyncratic reactions in most instances. Drug-induced immune-mediated hepatitis is the result of an unpredictable adverse immune response against the liver with cytolytic, cholestatic or mixed clinical features [494,495]. In several clearly documented instances, a metabolite can form adducts with specific constituents of hepatocytes. An immune response can be mounted against these neoantigens, which results in drug-induced autoimmune hepatitis. In fact, the causative mechanism is not known in most cases although the association of liver


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injury with fever, rash, and sometimes hypereosinophilia is suggestive of an immunoallergic reaction. Immunoallergic hepatitis typically develops within 1– 8 weeks after starting drug treatment, but long latency up to 12 months is possible. Importantly, re-introduction of the offending drug usually leads to relapsing hepatitis with a shorter onset. Eosinophilic gastroenteritis. Infiltration of eosinophils into layers of the gastrointestinal tract is associated with food allergy and in very few cases with drug or toxic exposure [496]. Renal manifestations Acute interstitial nephritis. Drug treatments are the leading cause of acute interstitial nephritis, which accounts for 1 –3% of all cases of acute renal failure [497]. The typical clinical presentation is nonoliguric renal dysfunction associated with fever, rash and/or eosinophilia in up to 50% of patients. Proteinuria is always present and the differential diagnosis from glomerulonephritis is possible by renal biopsy only. Drug-induced acute interstitial nephritis is widely considered to be a hypersensitivity reaction, the exact mechanism of which remains, however, to be elucidated. Humoral immune reactions occur within minutes to hours after drug administration and usually involve the parent drug or its metabolites acting as haptens. Cell-mediated immune mechanisms, such as delayed hypersensitivity involving CD4þ T lymphocytes or cytotoxicity due to CD8þ T lymphocytes, are presumably more frequent based on the observed histological changes and the lack of detectable immune complexes in the majority of patients. Glomerulonephritis. IgA nephropathy is the most common form of glomerulonephritis with an incidence between 15 and 40 new cases per million each year [498]. In the early stages of the disease, many patients have no symptoms. Microscopic hematuria and proteinuria may be detected for years and overt signs, such as macroscopic hematuria, loin pain and azotemia often develop after an infection of the urinary or respiratory tract. The course of the disease is highly variable, either totally benign or rapidly progressing toward renal failure. A definitive diagnosis can only be made by renal biopsy demonstrating focal or diffuse expansion of mesangial regions. Immunofluorescence and immunoperoxidase studies show a predominant deposition of IgA within the mesangial regions of glomeruli. A variety of other immunoglobulins and complement components are frequently codistributed with IgA. The cause of primary IgA nephropathy is not known. Increased production of IgA is not sufficient to produce IgA nephropathy. Recent studies have shown that abnormalities of the IgA molecules could play a role. No infectious or environmental causes have been evidenced. Respiratory and pulmonary manifestations Asthma is a pathological condition that is estimated to affect 5 – 10% of the adult population. Although drugs can induce or precipitate asthma, the underlying mechanism is rarely immune mediated, except in the context of a systemic hypersensitivity reaction to the drug [499]. In contrast, occupational [500 – 502] and environmental [503] causes of asthma are prominent. Thus, 5– 15% of adult-onset asthma have been attributed to occupational exposures [504] and more than 250 agents have been adequately documented as causing immunologic occupational asthma [505]. Occupational asthma is caused by sensitizing chemicals in over 90% of cases. There is also a growing concern that environmental

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pollutants, either outdoor [506,507] or indoor [508], do contribute to more frequent asthma, especially in children. Although, asthma was described in the ancient times, the pathogenesis has been only slowly elucidated. The French physician Trousseau described the association of asthma with immune disorders in the 19th century. Today, the pivotal role of chronic inflammation of the airways is well established and this led to shifting from the bronchodilatating b2agonist drugs to corticosteroids as first-line therapy. Inflammation of the airways mucosa occurs as a result of direct cell injury, activation of the complement cascade, release of proinflammatory mediators, or activation of the immune system. Thus, airways hyperreactivity can be provoked by not only physical factors, such as cold or exercise, by chemical irritants beyond a certain concentration threshold, but also by encounter with a given antigen in previously sensitized persons. In this latter situation, asthma is an IgEmediated hypersensitivity reaction involving antigen-specific mast cell activation. Whatever the mechanism, asthma reflects bronchospasm. Typically asthma consists of acute episodes of coughing, wheezing, dyspnoea and chest tightness of variable severity between patients. Inconsistently associated clinical signs include conjunctival, nasal and pharyngeal adverse effects. Chest radiographs and computed tomography are usually not informative. A diagnosis of asthma is based on the history, clinical examination and conventional spirometry. Airways hyper-reactivity can be evidenced by bronchial challenge testing. Standardized protocols using histamine or methacholine are used to show nonspecific hyper-reactivity measured as a dose-dependent increase in airways resistance. Specific hyper-reactivity can also be measured using suspected chemical or particulate antigens. A positive prick test supports the diagnosis if it is associated with appropriate pulmonary functional changes. Bronchoalveolar lavage (BAL) is utilized mainly as a research tool. Hypersensitivity pneumonitis is a group of immunologically mediated lung diseases typically caused by the inhalation of environmental agents in susceptible individuals [509]. Organic dusts from vegetable or animal products are the main causes of hypersensitivity, such as the farmer’s lung. The inhalation of small-molecular-weight chemicals can also be involved. The diagnosis is based on the history of exposure with resulting clinical respiratory symptoms and a constellation of radiological, laboratory, and histological findings [510]. Bronchiolitis, interstitial lymphocytic infiltration and granulomas are the main changes seen at lung biopsy. Occupational exposure to beryllium can result in a spectrum of pulmonary diseases including chronic beryllium disease or berylliosis which is an immune-mediated systemic disorder, as reviewed in Chapter 1 of volume III. Drugs can cause various types of pneumonitis including acute interstitial pneumonia, eosinophilic pneumonia and hypersensitivity pneumonitis [499,511,512]. Clinical signs and symptoms, including fever, cough, eosinophilia, and elevated serum IgE levels vary with the offending drug and this accounts for the variable terminology in use. BAL [513], histological examination of lung biopsies [514] and immunological assays to evidence a drug-specific immune response [515] are useful tools to establish a diagnosis. Pulmonary fibrotic diseases. Interstitial lung diseases are a diverse group of diseases characterized by chronic inflammation and progressive fibrosis of the lung interstitium [516]. A number of drugs and other chemicals can induce lung injury by a variety of


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mechanisms. Fibrosis is a pathologic process that results in the overproduction of extracellular matrix by the connective tissue as a response to tissue damage. The pathogenesis is not fully elucidated, but an immune mechanism, including hypersensitivity may be involved in some cases. Major profibrotic agents are TH2 CD4þ lymphocytes, CD40 –CD40L interaction, IL-4, and TGF-b. The main antifibrotic agent is IFN-g. Fibrosis is considered an irreversible process. Drug fever may also be a manifestation of an immune reaction to a drug [517,518]. If the offending drug is stopped, fever generally recedes within 24 – 48 h. There is no characteristic clinical pattern or biological abnormality. The onset of fever is variable, but usually within the 21 first days of treatment. The mechanism is not known, but the role of the pro-inflammatory cytokines IL-1, IL-6 and TNF-a that induce the release of pyrogenic factors, such as PGE2, is suspected. DIAGNOSIS As previously overviewed, hypersensitivity reactions are primarily immune-mediated adverse drug reactions that exhibits specificity and recurrence on re-exposure to the offending agent. The diagnosis is limited by a number of factors and is therefore still a major challenge [519 – 521]. In most instances, the exact epitope causing the reaction is unknown, the immunological mechanism is unclear, and the presence of immunological recognition is not predictive of a clinical reaction. Finally rechallenge, the gold standard for diagnosis, is a complicated and sometimes dangerous endeavor. During the past few decades, allergologists and clinical immunologists developed and used a variety of in vivo and in vitro tests. Recommendations regarding the preferred tests, however, were often based more on the views of opinion leaders than on scientific evidence. Thus, skin testing, the lymphocyte transformation test (LTT) and human basophil degranulation test were alternatively widely promoted, then strongly rejected and are nowadays progressively reintroduced in clinical practice. Importantly, assuming which mechanism may be involved is an essential step for the selection of adequate diagnosis tests. This assumption is difficult to make because not all mechanisms have been elucidated while diagnosis tests also serve to provide clues regarding the underlying mechanisms. This uncomfortable situation is a hallmark of the diagnosis of hypersensitivity reactions. A careful medical examination is absolutely essential as the time course of the reaction in relation to drug treatment or chemical exposure, and the observed clinical signs and symptoms may allow suspecting the likeliest mechanism. Thus, tests are selected, wherever possible, that can be or should not be used in a given patient. For instance, it is obviously illogical to use tests that explore IgE-mediated hypersensitivity reactions when the clinical signs and symptoms do not lend support to the possible involvement of such a reaction in a given patient. Standard biological end-points. Standard biological end-points can be changed following a hypersensitivity reaction, but the observed changes are rarely useful diagnostic tools. Eosinophilia. In normal subjects, eosinophils account for only a small percentage of leukocytes (1 – 3%). However, in certain disorders, such as parasitic infections, cancer and

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allergic diseases, a marked accumulation of eosinophils may occur. Eosinophilia—in fact hypereosinophilia—corresponds to eosinophil counts higher than the normal limit of 350 eosinophils per ml [522]. Eosinophilia is considered moderate above 1500 and severe above 5000. Importantly, the interpretation of increased eosinophil counts must be based on associated clinical symptoms. Eosinophilia is primarily associated with parasitic infections as eosinophils participate in the host’s defense against helminths. In addition, allergic reactions are typically associated with the release of eosinophil-directed cytokines, such as IL-4, IL-5 and IL-13, and eosinophilia is commonly seen in hypersensitivity reactions [523], including various skin eruptions, the DHS, immunoallergic hepatitis or eosinophilic pneumonitis. Eosinophils contribute to allergic reactions by releasing inflammatory mediators, such as the eosinophil cationic protein, eosinophilderived neurotoxin and eosinophil peroxidase. A number of drugs inhibit the production or action of neutrophils. Of these, glucocorticosteroids are by far the most potent drugs [524]. Leukocytosis. Because T lymphocytes play such a crucial role in specific immune responses, it is not surprising that elevated leukocyte and T lymphocyte counts can be observed in patients with a recent history of hypersensitivity reactions [525]. However, lymphopenia has been described in patients with TEN [526]. Total IgE serum levels. Although IgE play a crucial role in immune-mediated hypersensitivity reactions, the measurement of total IgE serum levels is not helpful for diagnostic purposes. Increased serum levels are inconsistently seen [527] and more importantly do not provide clues on the causative agent. In fact, baseline elevated total IgE levels might be used to predict patients that may be at higher risk of developing hypersensitivity reactions [528], although the predisposing role of atopy, especially regarding drug-induced hypersensitivity reactions is debated (see Chapter 4 of this volume). In vitro tests. Quite a few in vitro tests have been proposed for the diagnosis of hypersensitivity reactions. Several tests can be used for the diagnosis of immediate-type reactions and others for non immediate-type reactions. In vitro tests and immediate-type hypersensitivity reactions. Because the same mediators are often involved in immune-mediated and nonimmune-mediated (pseudoallergic) hypersensitivity reactions, most of the following tests can be used in either situation. Histamine release test. In the early 1940s, histamine release in vitro from basophils isolated from the peripheral blood of sensitized patients was shown following challenge with the antigen. Briefly, basophils or more frequently peripheral leukocytes of a patient with a history of immediate-type hypersensitivity reaction are incubated in standardized conditions with graded concentrations of the suspected offending agent. Histamine is measured in the supernatant. Radioimmunoassay [529] and immunoenzymoassay [530] of histamine markedly improved the sensitivity of the assay. Although, the value of this test was demonstrated by many authors [531 –535], others did not concur with this conclusion [536]. Human basophil degranulation test. This test is based on the same concept that the histamine release test. Here, basophil degranulation is evaluated not from the amount of released histamine, but on basophil counts before and after a standardized incubation of basophils isolated from the peripheral blood of a sensitized patient, with graded concentrations of the suspected allergens [537]. Formerly, rabbit basophils were used in


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the indirect basophil degranulation Shelley’s test [538]. The granules of human basophils in which preformed mediators are stored can be stained by toluidine blue. When basophils degranulate, they are not longer stained and basophils can thus be counted before and after incubation with the suspected allergen. Although, initial studies gave interesting results [539 – 542], it ceased to be recommended without extensive validation [543], in part because it was a time-consuming assay. In the past years, interest in basophil degranulation or activation was rekindled by the introduction of flow cytometry techniques. Activated human basophils were shown to express the surface marker CD63 [544] and CD45 [545]. The double-labeling of basophils with anti-IgE and anti-CD63 antibodies is the most commonly used technique at the present time [546 – 550]. Even though other markers are or may be used, and full validation of basophil activation measured by flow cytometry is still necessary, the available results suggest this is a sensitive test and a good correlation with skin tests has been shown in several groups of sensitized patients [551]. Leukotriene release test. Cysteinyl- or sulfidoleukotrienes are potent mediators liberated by mast cells, but not basophils by IgE- as well as non-IgE-mediated mechanisms. The use of in vitro leukotriene release has logically been evaluated as a possible tool for the diagnosis of immediate-type hypersensitivity reaction. The release of cysteinylleukotrienes from whole blood leukocytes can be measured by the cellular antigen stimulation test (CAST), which is based on detection of LTC4, LTD4 and LTE4 by a monoclonal antibody. When compared to histamine release test, CAST did not prove superior [552]. However, this assay was found to be a reliable tool for the diagnosis of intolerance to NSAIDs [553,554]. Tryptase assay. Tryptase is a neutral serine endoprotease stored in the secretory granules of all mast cells and basophils where it is stabilized by ionic association with heparin. Normal individuals have undetectable serum or plasma tryptase levels (, 1 ng/ml). Abnormally greater levels are seen in systemic anaphylaxis and the measurement of tryptase levels using various immunoassays was proposed as a diagnostic tool [555]. Elevated tryptase levels are also seen in 50% of patients with mastocytosis. A possibly interesting finding is that the tryptase content of mast cells is much higher than the content of basophils, suggesting that elevated tryptase levels can be used as indicators of mast cell activation. Specific IgE measurement. If, as already mentioned, the measurement of total IgE serum levels is of limited, if any value in the diagnosis of immediate-type hypersensitivity reactions, the measurement of specific IgE is a first-line diagnostic tool [556 –560]. Allergen-specific IgE antibody can be detected in the blood using a radioallergosorbent test (RAST). Unfortunately, a limited panel of reagents is available, particularly as regards low-molecular-weight haptens, but the development of new techniques, such as ELISA, is likely to improve the availability of reagents. In vitro tests and non immediate hypersensitivity reactions Lymphocyte transformation test (LTT). The LTT was proposed as an in vitro diagnostic tool for human drug allergies nearly 40 years ago [561]. The early method based on the cytological aspects of lymphoblasts was subsequently refined by the use of tritiated thymidine incorporation. After a long period of debate and nearly total neglect [562], it is

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again considered as a possibly useful tool [563]. In any case, the diagnostic value of the LTT has been demonstrated in various drug-induced hypersensitivity reactions including skin eruptions [441,564,566], immunoallergic hepatitis [567,568] and the DHS [569]. The LTT has major drawbacks. An optimal method, expensive equipment and technical skill are required. A major concern is the frequency of false negative results, which may be related to the involvement of metabolites to trigger lymphocyte proliferation. Activation of specific T cells either by direct binding to MHC molecules or to peptides present in the antigen-presenting groove has been suggested, and this could explain why nonreactive penicillin species, such as poly-L -lysine/penicillin conjugate, do not cause activation of specific T lymphocytes in patients allergic to penicillin, in contrast to reactive species, such as phenoxymethylpenicillin [570]. Co-incubation of the drug with human liver microsomes has been reported to result in drug-specific T lymphocyte reactivity [571]. Macrophage migration inhibition test. The macrophage migration inhibition factor (MIF) is a lymphokine released from sensitized T lymphocytes in the presence of the appropriate antigen. In the past, the MIF test [572,573] was suggested as a useful in vitro test in the diagnosis of hypersensitivity reactions [574 – 576], but is nowadays rarely used. Skin tests. Skin tests include patch tests, prick test and intradermal tests. Epidermal patch tests are a safe, objective and practical method for the diagnosis of allergic contact dermatitis provided the patients are carefully selected [577,578]. It is generally thought that 20 –30 allergens in routine screening tests can identify 50 – 70% of clinically relevant contact allergens. They are also increasingly used for the diagnosis of drug eruptions, especially as T lymphocyte-mediated reactions are thought to be commonly involved [579, 580]. In the prick test [581,582], a drop of the suspected allergen is layered on the surface of the skin and penetration into the epidermis is achieved by a puncture through the drop. Immediate reactions, i.e. wheal and erythema, are read at 15 – 20 min. As several allergens are generally tested simultaneously, punctures should be sufficiently distant to avoid overlapping erythema. A positive control, e.g. histamine, and a negative control, i.e. saline should be used. A number of factors are important to take into account including poor technique, volume, vehicle and prior treatment with antihistamines. In the intradermal test, the allergen is injected intracutaneously. The wheal and flare reaction is also read at 15– 20 min. A subcutaneous injection can lead to false-negative response. The concentration is essential and a 1000-fold higher concentration is normally required compared to prick test. There is a growing consensus that skin tests can be useful in the diagnosis of druginduced eruptions [583 – 586] provided a strict protocol is adhered to [587]. Nevertheless, patch tests were found as an ineffective diagnostic tool in patients with SJS or TEN [588]. The benefit of skin testing in the diagnosis of food and environmental chemical allergies is controversial [589,590], possibly reflecting a much greater variety of (nonimmunologic) mechanisms. Photopatch tests have been shown to be useful in the diagnosis of photosensitivity reactions [591]. Adverse reactions associated with skin testing have been a matter of concern. According to Lockey [592], more than 70 deaths have been reported after skin testing between 1895 and 1964. However, the majority of these deaths were due to antigens, such as horse serum-derived antitoxins that are no longer in use, and only 10 deaths were due to allergens still in u today. In fact, skin tests are generally considered safe. Lin et al.


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[593] reported two cases of systemic reactions in a series of 10,400 tested patients, and Reid et al. [594] reported no fatalities in a questionnaire survey from 1985 to 1989. In a retrospective analysis of 497,656 skin tests performed on 18,311 patients at the Mayo Clinic from 1992 to 1997, only six systemic reactions were identified, corresponding to an overall rate of 33 systemic reactions per 100,000 tests [595]. All six patients had asthma and fully recovered within 1 h. Provocation tests. Positive rechallenge, namely re-introduction of an adverse effect after re-administration of the suspected drug or chemical, is often considered the gold standard to establish the causal relationship between drug or chemical exposure and the adverse effect under scrutiny. A major issue with provocation tests in patients with a history of immune-mediated adverse reaction is the possibility to induce a severe, even more severe, life-threatening adverse event. Therefore, provocation tests can only be recommended in strictly selected patients, such as patients who absolutely require subsequent administration of the suspected drug (e.g. AIDS patients with a history of adverse reaction to sulfamethoxazole). Interestingly, several studies showed that provocation tests may not be as useful as formerly claimed since few positive responses are actually seen [596–598]. However, provocation tests are routinely performed to diagnose food intolerance, probably because the mechanism although not clearly elucidated is not immune-mediated in most instances [599]. PSEUDOALLERGY The first documented individual who developed an anaphylactic reaction was Pharaoh Menes who died from a wasp sting in 2640 BC. Many years later, in 1902, Portier and Richet coined the term anaphylaxis to describe acute reactions developing in dogs after repeated injections of the sea anemon toxin [600]. Subsequently, anaphylaxis was found to be caused by the IgE-dependent release of chemical mediators. However, adverse reactions that are clinically almost indistinguishable and do not involve IgE can develop following the first contact to the offending agent. The term anaphylactoid is often used to describe these IgE-independent reactions [601,602]. Anaphylactoid reactions have been described during anesthesia and many intravenous anesthetics have been shown to be involved [603]. NSAIDs [604] and radiocontrast media [605] are also frequently incriminated. Because anaphylactoid is a vague term, pseudoallergy is preferred [606]. Whatever the mechanism involved (see Chapter 4 of this volume), anaphylactoid reactions are clinically characterized by clinical signs and symptoms that mimic more or less closely a histaminic reaction with namely flush, redness of the skin, headache, cough and abdominal pain [607]. Flush and redness of the skin may be so prominent that the term red man syndrome has been coined to depict pseudoallergic reactions to vancomycin [608]. Typically, cardiovascular changes are not reminiscent of shock as the heart rate is only moderately accelerated while the blood pressure is unchanged, minimally decreased or even slightly increased. Interestingly, a correlation between plasma histamine levels and symptoms was demonstrated in human volunteers submitted to sequential infusion of histamine [609]. Tachycardia was the first symptom to develop, then flush and headache.

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AUTOIMMUNITY Autoimmunity is the 4th category of adverse effects related to immunotoxic exposures [610 –613]. With the introduction of potent immunostimulatory drugs, such as the therapeutic cytokines, autoimmunity cannot be considered only as a mere consequence of immunostimulation-like allergy, even though it is increasingly apparent that common mechanisms can seemingly lead to immune responses potentially resulting in allergy or autoimmunity [614]. INCIDENCE OF AUTOIMMUNE DISEASES AND AUTOIMMUNE REACTIONS Overall, autoimmunity is largely a mystery. Autoimmune diseases are relatively common in the general population, although estimates vary widely, e.g. from 5 to 20% in the US [615 – 617]. Jacobson et al. [618] studied the incidence of 24 well-defined autoimmune diseases based on the results of a literature search from 1965 to 1996 and concluded that over 1 million new cases develop every 5 years in the USA. It is generally accepted that 10 million Americans suffer from an autoimmune disease. This discrepancy in the estimated incidence of autoimmune diseases reflects the lack of consensus surrounding their definition. Autoimmune diseases are indeed clinically very diverse and in many instances, the diagnosis is based on the presence of several among a predefined set of symptoms and clinical signs. SYSTEMIC AUTOIMMUNE REACTIONS Lupus syndromes. Systemic lupus erythematosus (SLE) is estimated to affect 2 – 10 in 10,000 individuals; it is more frequent in young adult women, and black Americans [619]. The causes of SLE are not known, but an interplay of endocrine, genetic and environmental factors is widely accepted to be involved. This complex interaction of factors results in the loss of self-tolerance leading to the polyclonal activation of B lymphocytes and the production of autoantibodies. The role of genetic factors is demonstrated by twin studies, the more frequent association with certain HLA haplotypes, such as A1B8 DR3, or congenital deficiencies in complement components, such as C2. In addition, murine models of SLE (e.g. NZB/NZW, MRL lpr/lpr and BXB mice) support the role of genetic predisposition. The diagnosis of SLE is based on the concomitant presence of four criteria among the following [620]: rash, discoid lupus, photosensitivity, mouth ulcerations, arthritis, pleural or pericardial effusion, renal involvement (proteinuria), convulsions or psychosis, hematological disorders (hemolytic anemia, thrombocytopenia, lymphopenia and/or leucopenia), immunological changes (LE cells, anti-dsDNA or anti-Sm antibodies), and antinuclear antibodies. The natural history of the disease is unpredictable and highly variable. The prognosis depends on the evolution speed of the disease. The lupus syndrome or pseudolupus is the most frequent drug-induced autoimmune reaction [621 – 623]. Since the first description of lupus syndrome associated with sulfadiazine [624], the list of incriminated drugs markedly increased [625]. In the recent years, minocycline appeared as a leading cause of lupus syndrome [626]. Hydralazine and procainamide are, by far, the most frequent causes of drug-induced lupus syndromes [627].


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Autoantibodies have been detected in the sera of up to 25% of patients treated with hydralazine and of 50% of patients treated with procainamide. A relation was found between the prevalence of autoantibodies, the dose and/or duration of treatment, and the slow acetylator phenotype. However, no clinical signs were associated with autoantibodies in the majority of patients. Other drugs seldom reported to induce lupus syndromes include the anti-epileptic drugs trimethadione, ethosuximide, diphenylhydantoin and carbamazepine [628,629], most b-blockers, in particular acebutolol [630], chlorpromazine [631] and isoniazid [632]. Lupus syndromes induced by industrial and environmental chemicals have been quite exceptionally described. There are isolated case reports of lupus syndromes associated with thallium [633], hydrazine [634], hair dyes [635] and trichloroethylene [636]. Clinically, drug-induced lupus syndromes bear few similarities with SLE. Therefore, the use of the term drug-induced SLE must be strictly prohibited. In contrast to SLE where there is a frank female predominance, lupus syndromes are as frequent in men as in women. The number of clinical signs and symptoms concurrently seen in drug-induced lupus syndromes tends to be lower. The most typical clinical signs include arthritis in over 80% of patients, fever, weight loss, and muscular weakness with myalgias. Cutaneous manifestations are often limited to uncharacteristic erythematous eruptions. Renal involvement is inconsistent and usually mild when present. However, renal failure secondary to glomerulonephritis induced by immune complex deposition has been described in lupus syndromes associated with procainamide [637]. No neurological signs are usually noted, whereas neurolupus is a characteristic feature of severe SLE. One major distinction is the high incidence of pleural and/or pericardial effusions in the lupus syndrome. Pleural effusion is seen in up to 40% of patients. Pericardial effusion is somewhat less frequent, but can result in cardiac tamponade. No biological signs are typical of a drug-induced lupus syndrome. Anemia, leucopenia and thrombocytopenia are present in about one-third of patients as well as a positive Coomb’s test. In contrast to SLE, serum complement levels are normal. Antinuclear antibodies are universally detected, whereas antihistone antibodies are found in only 50– 85% of patients with the lupus syndrome. Interestingly, anti-ds (double-stranded) DNA antibodies, namely native DNA antibodies are found in 50– 70% of patients with SLE, but in less than 5% of patients with the lupus syndrome. In contrast, antibodies to denatured DNA are relatively common in patients with the lupus syndrome. Attempts to identify particular autoantibodies as markers of drug-induced lupus syndromes have so far been unsuccessful. The antiphospholipid syndrome combines antiphospholipid antibodies, a group of autoantibodies which include lupus anticoagulant, and a syndrome of hypercoagulability [638]. Antiphospholipid antibodies can arise without associated clinical signs of either SLE or lupus syndrome. The most common cause of drug-induced antiphospholipid antibodies is chlorpromazine. In contrast to SLE, lupus syndromes have a favorable outcome after cessation of the offending drug. The majority of clinical signs and symptoms disappear within a few weeks, except for pleural/pericardial effusions which may persist for several months. Scleroderma-like reactions. Scleroderma or systemic sclerosis is a relatively rare disease characterized by a more or less diffuse infiltration of the dermis and viscera by collagen

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with vascular abnormalities including vasospasm and microvascular occlusion [639 –641]. The disease can be limited to a few zones of the dermis or diffuse with systemic complications affecting the joints and the musculo-skeletal system (arthritis, myalgias), the gut (eosophageal and intestinal dysmotility), the broncho-pulmonary tract (pulmonary fibrosis), the heart (arrhythmias, ischemia and congestive heart failure) and the kidneys (‘renal crisis’ with hypertension, microangiopathy and renal insufficiency). Thickening of the skin is the hallmark of limited and diffuse diseases. It is initially limited to the face and the fingers and can progress to the arms, trunk, back, abdomen and legs. Raynaud’s phenomenon is present in < 90% of patients. The pathogenesis of scleroderma is not elucidated. There is an overproduction of collagen by fibroblasts due to abnormal interactions between fibroblasts and endothelial cells, lymphocytes and monocytes resulting in excessive release of various cytokines and growth factors, such as IL-6, IL-8 and TGF-b [642]. T lymphocytes are thought to play a pivotal role even though T cell functions are normal in patients with scleroderma. As autoantibodies are present in the majority of patients with scleroderma, an autoimmune process has been suggested to occur. The anticentromere, anti-topoisomerase 1 and antiRNA polymerase III autoantibodies are indeed disease-specific and mutually exclusive, but inconsistently present. Other types of autoantibodies, such as antinuclear antibodies, are also commonly found. Evidence that autoantibodies cause tissue damage is extremely limited. A number of patients present with atypical clinical manifestations, which only partly mimic the disease. The term scleroderma-like reactions is used to characterize these related disorders [643] that include eosinophilic fasciitis, localized forms of scleroderma, the eosinophilia – myalgia syndrome (EMS) and pseudoscleroderma. Although drugs and occupational or environmental factors are often thought to be involved, there is clinical evidence that such pathological conditions can arise despite no known or suspected toxic exposure. Only few drugs have been reported to induce scleroderma-like diseases. The most severe was the so-called oculo-mucocutaneous syndrome induced by the beta-blocker practolol. This syndrome included kerato-conjunctivitis, lesions of the conjunctivae with loss of sight, psoriasis-like eruption, and pleural and/or pericardial effusion. An immunotoxic mechanism was suspected [644,645], but never conclusively demonstrated. Practolol was withdrawn from the market in 1975. Other pharmaceutical products that were reported to induce scleroderma-like reactions include the anticancer drugs bleomycin and docetaxel, the analgesics pentazocine, morphine and ketomebidone, and the ergot derivative methysergide [646]. Overall, the clinical features are often atypical and the mechanism is not known. Various occupational exposures have been tentatively linked to the development of scleroderma and scleroderma-like reactions [647 – 650]. Silica is the leading occupational cause of scleroderma and epidemiological studies showed that workers heavily exposed to silica are at a significantly greater risk of developing scleroderma [651,652]. The disease in silica-exposed workers is usually similar to the spontaneous disease, but visceral involvement is typically delayed by a decade or more [653]. The frequent association of silicosis with scleroderma is sometimes referred to as the Erasmus syndrome. These findings led to the assumption that silicone gel-filled implants used for mammary


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augmentation or reconstruction could be the cause of connective tissue diseases (for review, see Chapter 11 of volume II). The available evidence so far, however, does not support a causal relationship [654]. Vinyl chloride is another main occupational cause of scleroderma-like reactions [655,656]. The syndrome consists of thickening of the skin, Raynaud’s phenomenon, acro-osteolysis and visceral involvement. Other occupational exposures shown or suspected to induce scleroderma-like reactions include solvents, particularly organic solvents such as trichloroethylene [657 –661], although the increase in relative risk compared with the general population was usually small. Finally, reports of scleroderma-like reactions have been published in workers exposed to epoxy resins and formaldehyde. Two major epidemics of scleroderma-like reactions developed in the last two decades. In 1981, an oil fraudulently sold as olive oil affected at least 20,000 Spaniards of whom approximately 300 died within the next two months. The so-called Spanish Toxic Oil Syndrome (OTS) was characterized by intense incapacitating myalgias, marked eosinophilia and pulmonary infiltrates [662]. Affected patients later on developed various pathological conditions including scleroderma-like skin changes. In 1989, a syndrome with multiple clinical presentations and variable severity, later called the EMS was described in certain users of preparations containing L -tryptophan [663]. It mainly consisted of eosinophilia, severe myalgias, sclerodermatous skin changes, fasciitis and occasionally myopathy and neuropathy. EMS was shown to be caused by contaminants of L -tryptophan arising during the manufacturing process in one Japanese company [664]. A number of patients with either the OTS or EMS had similar clinical features and histological findings. Although, the underlying mechanisms have not yet been conclusively elucidated, an autoimmune process is considered to play a key role (for review, see Chapter 5 of Volume III). ORGAN-SPECIFIC AUTOIMMUNE REACTIONS Organ-specific autoimmune reactions induced by xenobiotics are characterized by a homogeneous antibody response against a unique target (resulting in the presence of predominant type of autoantibody in the sera of affected patients) and by clinical symptoms closely mimicking those found in the corresponding spontaneous autoimmune disease. Depending on the specific target involved, a variety of organ-specific autoimmune reactions have been described. Cardiac autoimmune reactions. Myocarditis is an insidious inflammation of the heart muscle that may result in sudden death in asymptomatic persons [665]. Although the underlying mechanism is not elucidated, there is a growing awareness that myocarditis may involve an intricate pattern of causative and predisposing factors. That an infectious– immune mechanism is possibly involved is supported by experimental studies, which showed the role of viral infections in the development of myocarditis in several animal models [666], and by several findings in patients with idiopathic dilated cardiomyopathy including an imbalance between CD4þ and CD8þ T lymphocytes, and a variety of circulating autoantibodies. It is also worth mentioning the cross-reactivity existing between antibodies that react with streptococcus protein M and human cardiac myocytes, with the resulting pancarditis seen in acute rheumatic fever.

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CNS-specific autoimmune reactions Guillain– Barre´ syndrome is an acute or subacute polyneuropathy that can occur after minor infections, surgery or without known precipitating factors [667]. The syndrome usually presents with progressive lower extremity weakness and can lead to autonomic dysfunction. Guillain –Barre´ syndrome is potentially fatal and the mortality rate is 3 –5%. Severe residual disabilities are noted in 10% of patients. A still hypothetical link between Guillain– Barre´ syndrome and vaccination has been suggested [668]. Multiple sclerosis is a multifocal demyelinating disease of the central nervous system that may take a relapsing or progressive course, and is typically more frequent in young adult white women [669]. Early symptoms consist of paresthesias, gait disorders, visual loss and diplopia. Relapses evolve on days and resolve gradually. Patients may eventually develop progression of the disease with increasing disabilities usually due to ataxia, fatigue, gait or cognitive dysfunctions. The diagnosis is based on clinical symptoms and confirmatory findings on magnetic resonance imaging, electrophysiologic measurement and cerebro-spinal fluid measurement. Multiple sclerosis is characterized by perivascular cuffing of CD4þ T lymphocytes and myelin destruction. Vaccines, in particular hepatitis B vaccine, have been suggested to induce or facilitate the development of multiple sclerosis, but so far no confirmative epidemiological evidence has been published [670]. Myasthenia is characterized by a loss of muscular strength due to impaired neuromuscular transmission [671]. There is a predilection for certain cranial nerves and virtually all patients complain of ocular symptoms, in particular ptosis and diplopia. Weakness of the oropharyngeal muscles produces difficulties for chewing, swallowing, speaking or breathing. Overall, 80– 90% of patients with generalized myasthenia gravis have IgG antibodies directed against the nicotinic receptors of acetylcholine in the neuromuscular motor plates. There is clear evidence that these autoantibodies are the pathogenic agent in myasthenia gravis even though there is no correlation between serum antibody titers and the clinical severity of the disease. The mechanism leading to the production of these autoantibodies is not elucidated. A few drugs have been shown to induce a pathological condition that is very close clinically and biologically to spontaneous myasthenia gravis. This condition must be differentiated from drug-induced myasthenialike syndromes due to a pharmacodynamic interference between the causative drug and acetylcholine. Penicillamine is by far the most frequent cause of drug-induced myasthenia [672]. The clinical and biological features are so close to those of spontaneous myasthenia that the causal relationship is only based on the slow improvement of clinical symptoms and decrease in serum autoantibodies that do not exist in spontaneous myasthenia. A few other pharmaceutical drugs have been reported to induce myasthenia including the antirheumatismal drugs tiopronine and pyritinol, and the anti-epileptic drug trimethadione. Whatever the causative drug, the underlying mechanism is not known. Endocrine-specific autoimmune reactions. Autoimmune reactions involving endocrine organs are among the most frequent organ-specific autoimmune reactions induced by drugs and chemicals. Insulin-dependent diabetes mellitus (IDDM or type I diabetes) is an autoimmune disease characterized by monocytic and lymphocytic infiltration of insulin-producing pancreatic b cells. This leads to progressive insulin deficiency and clinical hypoglycemia,


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which in turn causes severe metabolic disturbances and medical complications, including retinopathy, renal failure, neuropathy and stroke [673]. The pathogenesis of IDDM is extremely complex and the underlying mechanisms are not fully elucidated. There is evidence that IDDM is genetically controlled even though studies in identical twins found a concordance of only 30 – 50%. The incidence of IDDM is growing worldwide [674] and the role of external factors, such as infection, diet, drug treatment and environmental chemical exposure is suggested by the results of epidemiological studies and animal experiments. No firm conclusion, however, can be drawn from the available data [675]. None of the xenobiotics shown to induce IDDM in animals or man, except streptozotocin is suspected to be diabetogenic via an immune-mediated effect. Thyroid-specific autoimmune reactions. Thyroiditis is one of the most common immune disorders of the endocrine glands [676]. It is due to a specific autoimmune response involving T cells and autoantibodies. Thyroperoxidase, the enzyme involved in the synthesis of thyroid hormones, is the major autoantigen, but thyroglobulin, the precursor of thyroid hormones, or the thyrotropin receptor can be the target of the autoimmune process. The destruction of thyroid follicular cells typically leads to chronic atrophy of the thyroid gland associated with antithyroid autoantibodies and hypothyroidism. Most often, autoimmune thyroiditis presents as a slowly progressing atrophy of the thyroid gland. Less commonly, the clinical aspect is the same as initially described by Hashimoto in 1912, with a hypertrophy of the thyroid gland. It is still unclear whether the initial triggering event is to be found in the abnormal expression of MHC class II molecules on thyroid cells, which thus become fully active antigen-presenting cells, or in a dysfunction of T helper cells. As already mentioned, treatments with rIL-2 and the IFNs are associated with a higher incidence of autoimmune thyroiditis. Gastrointestinal and hepatobiliary specific autoimmune diseases. The importance of the mucosal immune system in the gastrointestinal tract is reflected by the high prevalence of gastrointestinal infections in immunocompromised patients. Inflammatory bowel diseases. Ulcerative colitis and Crohn’s disease are collectively referred to as inflammatory bowel diseases. These are chronic and spontaneously relapsing disorders of unknown causes. Typical symptoms occur as a result of mucosal injury and consist of bloody diarrhea, abdominal pain and constitutional symptoms. Isolated case reports have been published, e.g. with the NSAID diclofenac [677], but it remains to be confirmed whether they actually are true inflammatory bowel diseases. Liver-specific autoimmune reactions. Autoimmune hepatitis [678,679] occurs primarily in young-to-middle-aged women. More than 80% of patients have hypergammaglobulinemia. The presence of autoantibodies, such as antinuclear antibodies, antibodies against smooth muscle and liver – kidney microsomal antibodies, is common, even though their diagnostic value is debated. A liver biopsy is often essential to confirm the diagnosis. Several drugs have been reported to cause hepatitis associated with the presence of highly specific autoantibodies in the sera of affected patients [680]. The mechanism has been clearly elucidated in a few instances: overall, the induction of the specific immune response is linked to structural changes in hepatocyte constituents, such as the cytochromes P450 following the biotransformation of the offending drug into metabolites that selectively bind to these hepatocytic constituents [681,682]. It is noteworthy that approximately

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2% of patients with chronic hepatitis C have anti-CYP2A6 autoantibodies, whereas up to 14% of patients with chronic delta virus hepatitis have antibodies against UDPglucuronosyl-transferases (UGTs). Dihydralazine is an antihypertensive drug closely related to hydralazine that has been in use in a few countries including France and East Germany. Hepatitis characterized by the presence of serum autoantibodies reacting with liver microsomes has been reported in several patients treated with dihydralazine. These autoantibodies were shown to be directed against CYP450 1A2, which is suspected to be involved in the biotransformation of dihydralazine [683]. Halothane is a volatile general anesthetic whose administration is associated with a severe hepatitis in few patients. A mild form of liver injury is much more frequent, but may be due to direct hepatotoxicity and the contribution of factors, such as surgery and hypoxia [684]. The great majority of patients with the most severe form of halothane hepatitis have had halothane anesthesia on several occasions and exhibit rash, arthralgias, eosinophilia and autoantibodies. Halothane is oxidized by CYP450 4E1 to the highly reactive metabolite trifluoroacetylchloride, which either reacts to water to yield trifluoroacetate or binds covalently to liver proteins to produce various trifluoroacetyl adducts in humans and various animal models of halothane hepatitis in rats and rabbits. Interestingly, the major adduct involves CYP450 4E1 [685]. Iproniazid is a monoamine oxidase inhibitor formerly often used to treated depression. Hepatitis is a well-recognized adverse effect of iproniazid [686]. The antimitochondria antiM6 antibody was identified in the sera of patients with iproniazid-induced hepatitis and was shown to specifically recognize liver monoamine oxidase B [687]. Tienilic acid is a thiazide diuretic drug. Over 500 cases of hepatic injury of which at least 25 were fatal have been reported in the 1980s. Another type of autoantibodies, the LKM2 autoantibodies, have been found in the sera of patients with tienilic acid-induced hepatitis, but not in the sera of patients with nontienilic acid-induced hepatitis or nonhepatitic patients treated with tienilic acid [688]. Interestingly, tienilic acid is biotransformed by CYP 2C9 and LKM2 autoantibodies were found to bind to CYP 2C9 in humans suggesting that a reactive metabolite of tienilic acid produced by CYP 2C9 results in structural changes of this isoenzyme and induces an immune-mediated destruction of this isoenzyme [689]. Primary biliary cirrhosis is an autoimmune disease characterized by inflammation of the intrahepatic bile ducts followed by fibrosis and eventually liver failure. Intramitochondrial autoantibodies are frequently detected in the sera of patients, but there is no correlation between measured titers and disease activity. Although the incidence of the disease has been increasing over the past 30 years, very little is known of the mechanisms involved and no toxic cause has so far been incriminated [690]. Hematological autoimmune diseases Autoimmune aplasia. During the first 10 years of recombinant human erythropoietin use, erythropoietin-induced antibodies were a rare complication. In the recent years, there has been an increase in the number of patients developing severe pure red cell anemia due to neutralizing antibodies. Patients present with an absolute resistance to erythropoietin therapy and rapidly develop a very low reticulocyte count. The reason why this complication began developing only after years of therapeutic use is not elucidated [691].


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Autoimmune hemolytic anemias refer to a destruction of erythrocytes by autoantibodies bound to the erythrocyte surface. Autoimmune hemolytic anemias are rare and heterogeneous. The majority of autoimmune hemolytic anemias are spontaneous or secondary to neoplasia, but in a few instances, they are caused by drug treatments, primarily the antihypertensive drug a-methyldopa [692]. Rarely, autoimmune hemolytic anemias are manifested by clinical signs and symptoms of acute anemia. Up to 30% of patients treated with a-methyldopa have a positive Coombs test and autoantibodies against Rhesus erythrocyte antigens. However, hemolysis is patent, as mild anemia with reticulocytosis, nonconjugated hyperbilirubinemia, and diminished serum haptoglobulin, in less than 1% of patients. In very rare patients, anemia is severe and potentially fatal. Anemia usually recovers in the few days following the cessation of the offending drug, but serum autoantibodies can still be found after a few months. Importantly, no antibodies are directed against amethyldopa or its metabolites. Establishing a causal relationship is therefore not easy in most cases, but the time course of events and the recovery after drug treatment cessation are helpful. Even though the mechanism involved in the production of these autoantibodies is unknown, their role in the induction of hemolysis is well established. A small number of medicinal products including chlorpropamide, levodopa, fludarabine, mefenamic acid, nomifensine or procainamide, have been reported to induce autoimmune hemolytic anemias, which nevertheless remain a very rare adverse effect. No occupational or environmental chemicals have so far been suspected to induce autoimmune hemolytic anemias. Autoimmune neutropenias are typically due to a destruction of neutrophils by autoantibodies against the CD16 Fcg RIIIb complex. Although, a number of drugs have been incriminated as causes of neutropenias, it is unclear whether and when an autoimmune mechanism is involved. Autoimmune thrombocytopenias are relatively benign organ-specific autoimmune diseases. The diagnosis is often difficult as the other more frequent causes of thrombocytopenias, including most viral infections, blood transfusion, an immunoallergic reaction (due to drugs or other causes), must be carefully ruled out. Patients with autoimmune thrombocytopenias usually develop autoantibodies that bind to platelet membrane glycoproteins, such as GPIIb –GPIIIa and GPIb– GPIX, resulting in their destruction by phagocytes. The causative role of drugs is debated [693]. Muscle-specific autoimmune reactions Polymyositis is an uncommon disease very rarely induced by medicinal products [694]. It is characterized by muscular deficiency, myalgias and sometimes cutaneous lesions. It is more frequent in rheumatoid patients treated with penicillamine. Macrophagic myofasciitis has only recently been described [695]. The clinical features are not specific and include myalgias, arthralgias, asthenia and fever. Laboratory findings are not specific, but muscle biopsy can confirm the diagnosis by showing infiltrations of CD68þ cells, i.e. macrophages, and a few CD8þ T cells. The causative role of aluminum hydroxide is suggested by the identification of these histological changes in muscles used for vaccine injection, such as the quadriceps muscle in children [696]. In addition, intra-macrophagic crystal deposits have been evidenced in biopsy specimens [697]. Finally, the intramuscular injection of aluminum to rats was shown to reproduce similar lesions. Although, the exact mechanism of macrophagic fasciitis remains to be

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elucidated, chronic immune stimulation by aluminum hydroxide and autoimmunity have been proposed. Skin-specific autoimmune reactions Linear IgA bullous dermatosis is a rare sub-epidermal blistering disorder in which linear deposits of IgA along the basement membrane of the skin are seen on direct immunoflurorescence. There may be deposition of IgG and C3 as well. The disease presents as an asymptomatic or pruritic bullous dermatosis that has a predilection for the trunk, but often involves the limb and acral regions. Mucosal involvement is present in 40% of cases [698]. The reaction usually begins between 24 h and 15 days after commencing drug treatment and there is typically a cessation of new lesion formation 24 –72 h after drug withdrawal. Resolution of all lesions requires 2 – 7 weeks. Of the many drugs reported to induce linear IgA bullous diseases, vancomycin is by far the most frequently involved. The underlying mechanism is not known. The offending drug might cause production of autoantibodies, either by modifying native proteins or altering immune surveillance. Pemphigus is a rare skin disease affecting approximately 1 person per million, but nevertheless the most frequent autoimmune skin disease. Several clinical patterns have been identified, but all are characterized by bullous skin eruptions of varying severity. Histologically, pemphigus is characterized by the destruction of the epidermis (acantholysis) involving autoantibodies against components of the intercellular substance in the deep epidermis including the desmogleins 1 and 3, desmoplakins and envoplakin. A few drugs have been reported to cause pemphigus, e.g. captopril, and the antirheumatismals penicillamine, pyritinol and tiopronine [699,700]. A closely related disease is bullous pemphigoid, which is caused by IgG1 or IgG4 autoantibodies against the BPAG1 and BPAG2 antigens of the dermo-epidermal junction. Other antigens, such as laminin 5 and collagen XVII, have also been shown to be the target of autoantibodies. Bullous pemphigoid is 10 times more frequent than pemphigus in the general population, but is an extremely rare drug-induced adverse effect. The generally accepted hypothesis is that drugs acting as haptens could alter the antigenicity of the lamina propria or attach to other cell sites. The resulting autoantibodies would bind to bullous pemphigoid antigens and activate complement resulting in blister formation. Vasculitis can be associated with antineutrophil cytoplasmic autoantibodies (ANCA) detected by indirect immunofluorescence and ELISA. The clinical manifestations are very similar to those of ANCA-associated idiopathic vasculitis, such as Wegener’s granulomatosis [701]. ANCA are often associated with antibodies to proteinase3 or myeloperoxidase. Hydralazine, propylthiouracil and minocycline are the most commonly incriminated drugs. ISOLATED AUTOANTIBODIES Autoantibodies are the hallmark of autoimmunity as they are normally detected in the sera of patients with autoimmune diseases. However, autoantibodies can also be detected in healthy human subjects [702]. There is a steady increase in the levels of autoantibodies with age. It is known that autoreactive T and B lymphocytes are normally present and that


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natural autoantibodies constitute a large fraction of serum immunoglobulins. The amount of natural autoantibodies does not increase or adapt to antigenic challenge and they are considered as part of innate immunity. The significance of isolated autoantibodies, however, in relation to drug treatment or toxic exposure is unknown. For instance, autoantibodies have been identified in patients treated with a variety of cardiovascular drugs [703] or in workers exposed to silica [704] without overt autoimmune disease. Recently, however, autoantibodies have been detected in the sera of patients many years before they developed SLE [705].

CONCLUSION This review of the immunotoxic effects induced by drugs and other chemicals demonstrate that immunotoxicity can be associated with significant morbidity and mortality. The wide variety of clinical adverse events related to immunotoxicity reflects the pivotal role of the immune system in maintaining homeostasis of the body, but also the existence of close interactions with many organs and physiological systems. Based on the available clinical evidence, it can be unambiguously concluded that a systematic and careful evaluation of the immunotoxic potential of every molecular entity to which human beings are intended or likely to be exposed is absolutely essential [706,707].

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[697] Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, Pellissier JF, Chariot P, Authier FJ (2001) Macrophagic fasciitis lesions assess long-term persistence of vaccinederived aluminum hydroxide in muscle. Brain, 124, 1821– 1831. [698] Kuechle MK, Stegemeir E, Maynard B, Gibson LE, Leiferman KM, Peters MS (1994) Druginduced linear IgA bullous dermatosis: report of six cases and review of the literature. J Am Acad Dermatol, 30, 187 –192. [699] Mutasim DF, Pelc NJ, Anhalt GJ (1993) Drug-induced pemphigus. Dermatol Clin, 11, 463– 471. [700] Brenner S, Bialy-Golan A, Ruocco V (1998) Drug-induced pemphigus. Clin Dermatol, 16, 393– 397. [701] Doyle MK, Cuellar ML (2003) Drug-induced vasculitis. Expert Opin Drug Saf, 2, 401– 409. [702] Tomer Y, Shoenfeld Y (1988) The significance of natural autoantibodies. Immunol Invest, 17, 389– 424. [703] Wilson JD (1980) Antinuclear antibodies and cardiovascular drugs. Drugs, 19, 292– 305. [704] Conrad K, Mehlhorn J (2000) Diagnostic and prognostic relevance of autoantibodies in uranium miners. Int Arch Allergy Immunol, 123, 77 –91. [705] Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, Harley JB (2003) Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med, 349, 1526– 1533. [706] Descotes J (2004) Immunotoxicity guidelines: from animal to man. The viewpoint of an independent expert. Toxicology. In press. [707] Descotes J (2004) Role of immunotoxicology in the safety assessment of drugs. Drug Saf. In press.

Chapter 4

Mechanisms of immunotoxic effects

INTRODUCTION The immunotoxic effects of drugs and other chemicals are extremely varied and as previously described in Chapter 3 of this volume they have been shown to affect almost every organ or tissue of the body. In part because of this wide variety of immunotoxic effects, the underlying mechanisms are not well elucidated, except in a few situations. Over the years, immunotoxic effects have been variably categorized. The continuum model [1] was based on the assumption that normal immune responses can be, upon chemical exposure, either inhibited or enhanced, and immunoenhancement was said to result in hypersensitivity or autoimmunity. Much of the experimental work since the early 1980s until the end of the 20th century strictly adhered to this model [2 –4]. There is, however, a growing awareness that this model does not reflect the whole pattern of possible immunotoxic effects [5] and a recently released immunotoxicity guideline unambiguously confirmed this trend [6]. Today, immunotoxic effects are best divided into immunosuppression, immunostimulation, hypersensitivity and autoimmunity in order to stick to the clinical experience accumulated over the years. Interestingly, another, but only slightly different categorization was recently proposed based mainly on a mechanistic perspective [7]. Here, normal immunity remains the core situation that, upon chemical exposure, may shift either toward hypoimmunity encompassing immunological tolerance and immunosuppression, or toward hyperimmunity including hypersensitivity, autoimmunity and immunostimulation. Although similar immunotoxic mechanisms may impact on the normal immunity to induce a shift toward hyperimmunity and result in either hypersensitivity, autoimmunity or immunostimulation, the three latter categories are sufficiently different both mechanistically and clinically to justify their individualization as different categories of immunotoxic effects. It is beyond doubt that overlapping mechanisms can result in diverse adverse effects, but for the sake of clarity and despite the obvious risk of oversimplification, our current understanding on the mechanisms of immunotoxic effects will be overviewed below based on the perspective of each of these four categories. 127

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MECHANISMS OF IMMUNOSUPPRESSION INTRODUCTION Immunosuppression is associated with more frequent infections and virus-induced malignancies as is widely shown by the clinical experience from patients with congenital or acquired immunodeficiencies, or treated with varied immunosuppressive drugs. Whatever the expected or observed consequences, namely impaired resistance towards microbial pathogens or virus-related malignancies, nonspecific as well as specific mechanisms can be involved exclusively, predominantly or concurrently. Immunosuppression and impaired resistance to infectious pathogens. Immunocompromised patients are at a greater risk of developing more frequent and/or more severe infectious complications. A wide range of mechanisms is involved in defenses of the body against microbial pathogens and this is one reason why the range of clinically significant infectious complications in immunocompromised persons is so varied [8]. Importantly, the nature and severity of infectious complications depend on the type of immune defects, at least to some extent. The clinical data from patients with congenital immune deficiencies are particularly illustrative in that respect [9,10]. This should have an impact on the selection of relevant experimental infections when considered for use as host resistance models in immunotoxicity risk assessment. Appropriate selection of an experimental infection model should therefore be based on the demonstrated adverse influence of the test article on specific components of the immune system and the known immunological mechanisms of the host’s defenses that are involved to fight the selected experimental infection. Thus, selecting of relevant experimental infection model necessarily requires that a minimal knowledge of the immunotoxic effects of the test article is available. Immunosuppression and virus-induced malignancies. Another major adverse effect of immunosuppressive agents is the development of more frequent and typically virusinduced malignancies, including skin cancers and lymphoproliferative disorders. Carcinogens are classified into genotoxic and nongenotoxic (epigenetic) carcinogens [11]. The majority of genotoxic carcinogens that have been tested for their immunotoxic potential proved to be markedly immunosuppressive, but it is as yet unclear whether this correlation is not more than purely coincidental, or the consequence of a biased selection of chemicals for early immunotoxicity intervalidation studies [12]. In fact, immunosuppression should be logically considered as an epigenetic mechanism of cancerogenicity [13] as potent immunosuppressive drugs, which are certainly nongenotoxic, are associated with a greater risk of lymphoproliferative disorders and skin cancers in long-term treated patients. Tumor antigens have long been identified on murine tumor cells, but their presence was evidenced on human tumor cells only relatively recently [14], hence the debate whether the concept of the immunosurveillance of cancer proposed by Paul Ehrlich in the very early days of the last century is valid [15 – 17]. This concept served as a rationale for extensive research efforts to design cancer immunotherapy in the 1960s –1970s, but these efforts


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proved largely unsuccessful. The recent discovery of human tumor antigens fueled the renewed interest in the design of therapeutic vaccines to fight cancer [18,19]. NONSPECIFIC MECHANISMS Epithelial barriers are the first line of defense of the body against infectious pathogens. Xenobiotics that alter the integrity or function of epithelia can facilitate the development of infections as is seen in cancer patients undergoing chemotherapy [20]. Atmospheric pollutants, such as ozone [21,22], sulfuric acid [23], chlorine [24] and nitrogen oxides [25], exert direct toxic effects on the respiratory epithelium that predispose to infections [26,27]. However, they also exert a negative impact on phagocytic cells and cellular immunity. Another example is marijuana smoking [28]. The complement system is a major component of natural (innate) immunity. Congenital [29] or acquired [30] deficiencies in various complement components are associated with potentially severe infections. Although inhibition of the complement cascade can predispose to infections, study of the complement system is very seldom included in the nonclinical immunotoxicity evaluation of xenobiotics. The effects of the anticholinesterase insecticides carbaryl, carbofuran, dichlorvos and paraoxon on complement were studied in human sera in vitro and a concentration-dependent inhibition was noted, except for paraoxon [31]. Chemokines are small molecules that recruit leukocytes, and are involved in leukocyte trafficking and the architecture of secondary lymphoid organs. As their discovery is relatively recent, much remains to be done to clearly identify their relevance in host’s defenses and whether immunotoxicants can exert sufficiently strong negative influences to impair resistance toward infectious pathogens. An inhibition of monkey neutrophil and monocyte chemotaxis induced by the chemokines IL-8 and RANTES was shown following in vitro incubation with the insecticides heptachlor, chlordane or toxaphene [32]. It is noteworthy that chlordane impaired the resistance of rats to Listeria monocytogenes [33] and dichlorovos the resistance of rabbits to Salmonella typhymurium [34], even though other mechanisms may also be involved. In a rat model of pneumonia to Streptococcus pneumoniae, acute alcohol intoxication was associated with increased bacterial burden, decreased survival and inhibited production of several chemokines [35]. Phagocytes. There is a large amount of data showing that xenobiotics can impair the recruitment, activation and microbicidal of the mononuclear phagocytes, i.e. monocytes and macrophages, and polynuclear neutrophils [36,37]. A few studies described possible correlations between impaired phagocyte function and decreased resistance to experimental infections or implanted tumors, including diesel exhaust particles [38], gallium arsenide [39], para-nitrotoluene [40], 2,4-diaminotoluene [41], cyclophosphamide [42,43], cyclosporine [44] and organochlorine insecticides [45]. The rarity of a demonstrated correlation between impaired phagocytic function and increased incidence of infections or malignancies is primarily be due to the uncommon measurement of phagocytic functions in

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nonclinical immunotoxicity evaluation, but may also reflect the relatively limited role of phagocytes in eradicating invading pathogens, which can be readily compensated by specific immune mechanisms, or the lack of a sufficiently strong inhibitory effect of most immunotoxicants on phagocytes. NK cells can destroy virus-infected cells and tumor cells and it is known that impaired NK cell activity is associated with more frequent infections and malignancies. As regards immunotoxicity evaluation, the value of NK cell activity measurement is unclear as few studies evidenced decreased NK cell activity in patients treated with potent immunosuppressive drugs, such as azathioprine and cyclosporine [46,47]. A major concern is indeed that very few drugs and other chemicals consistently inhibited NK cell activity [48]. Morphine derivatives and several metals, e.g. nickel are the most consistently reported inhibitors of NK cell activity [49]. A correlation between decreased NK cell activity in mice and increased susceptibility to experimental cytomegalovirus infection, however, was evidenced [50]. ANTIGEN-SPECIFIC MECHANISMS The importance of antigen-specific mechanisms to fight invading microbial pathogens has been unequivocally demonstrated over the past century with the use of specific immunization to protect against common viral and bacterial infections. Nowadays, vaccines are essential tools to assure a better health status to the general population and the lack of any vaccine to fight given infectious diseases, such as AIDS, is an obvious major issue. Our improved understanding of the mechanisms involved in the successful applications of vaccines showed that antibodies afford the most significant protection against extra-cellular pathogens, whereas CD8þ T lymphocytes are needed to control infections due to intra-cellular pathogens. Humoral immunity. Antibodies are glycoproteins produced by plasma cells derived from B lymphocytes. Variable domains in the N-terminal end of the heavy and light chains constitute the Fab fragment that has two antigen-reactive sites. That humoral immunity can be essential is demonstrated by more frequent infections in patients with congenital defect in humoral immunity. Only a few xenobiotics have been shown to exert exclusive or predominant inhibitory effects on antibody responses [51 – 54]. Cellular immunity. Similarly, cellular immune responses are crucial in immunocompetence and several xenobiotics have been shown to inhibit cellular immunity without markedly altering antibody production [55 – 58]. In most instances, the underlying mechanism has not been clearly elucidated with the exception of immunosuppressive drugs [59]. Thus, azathioprine acts by inhibiting the formation of 5-phosphoribosyl-1pyrophosphate which results in decreased number of T lymphocytes and reduced antibody production. Cyclosporine is a calcineurin inhibitor which blocks IL-2 production. Tacrolimus, a structurally unrelated calcineurin inhibitor, acts through a different site on IL-2 production. Mycophenolate mofetil inhibits T lymphocyte proliferation by inhibiting purine synthesis.


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Thymus atrophy and immunotoxicants. In fact, the vast majority of immunotoxicants induce immunosuppressive effects by altering humoral and cellular immunity simultaneously. The precise mechanism of action is not yet known in most instances. However, the thymus is the target organ of several immunotoxicants including cyclosporine, dioxin and organotins [60,61]. Cyclosporine disrupts the activation of peripheral T lymphocytes, but also blocks the maturation of thymocytes by altering the thymic microenvironment provided by dendritic cells, macrophages and stroma cells in the medulla [62–65]. In contrast, histology of the thymic cortex is not affected by cyclosporine. A similar effect of tacrolimus was reported [66]. Dioxin or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied of halogenated aromatic hydrocarbons. Thymic atrophy primarily involving the cortex is a common finding in rodents even at very low dose exposure [67]. Activation of the arylhydrocarbon receptor (AhR), a ligand-activated transcription factor, by dioxin results in immunosuppression and thymus atrophy and can induce the transcription of an array of genes, including genes involved in cell growth and differentiation, and in apoptosis [68 – 70]. This finding opens new avenues of research for unraveling the molecular mechanism of dioxin-induced immunosuppression. Organotins. Thymic atrophy predominantly affecting the cortex has long been described as a consequence of exposure to organotins. Dioctyltin dichloride [71] and tri-n-butyl-tin oxide [72] produce thymus atrophy at doses that induce no other toxic effects, including immunotoxic effects. Induction of thymocyte apoptosis is a widely accepted mechanism even though other mechanisms may also be involved [73].

MECHANISMS OF IMMUNOSTIMULATION INTRODUCTION Despite many years of hesitation or controversy, it is nowadays well recognized that normal immune responses can be stimulated or activated by drugs and other chemicals. Depending on the baseline level and potency of the immunostimulatory substance, immunostimulation can results in clinically significant adverse effects, the mechanisms of which are at least partly understood. FLU-LIKE REACTIONS AND ACUTE CYTOKINE SYNDROMES Early studies showed that leukocytes stimulated by bacterial products release endogenous pyrogens, later identified as cytokines [74]. Thus, the administration of low doses of LPS to humans resulted in fever, chills and myalgias with associated changes in serum proteins as seen in the acute-phase response of the liver [75]. It is no longer accepted that external pyrogens are involved in flu-like reactions and acute cytokine syndromes. Fever has been related to an increased release of pro-inflammatory, pyrogenic cytokines primarily IL-1b, IL-6 and TNF-a [76]. Cytokines released into the circulation are transported into the brain by still debated mechanisms and reach the preoptic-anterior hypothalamic area where they induce the release of prostaglandins (PG), especially PGE2 [77,78]. However, because increased cytokine levels are usually detected when the febrile response is already ongoing, there have been suggestions that other sources of pyrogenic factors, such as the Ku¨pffer

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cells of the liver may also be involved. Importantly, PGE2 is synthesized by all macrophages including Ku¨pffer cells in response to LPS and blockade of Ku¨pffer cells prevents the release of PGE2 [79]. Another possible mechanism is the activation of the complement cascade as Ku¨pffer cells release PGE2 in response to the C3a anaphylatoxin [80]. Whatever the mechanism actually involved, PGE2 is widely considered as the key pyrogenic factor. In fact, that several different mechanisms can result in PGE2 synthesis could explain why clinically similar reactions develop following the administration of markedly different agents, such as vaccines, microbial extracts used as immunostimulatory drugs, recombinant cytokines and monoclonal antibodies. Interestingly, the release of IL-1 and TNF-a was reported following the inhalation of metal fumes, particularly zinc fumes, suggesting that acute welder reactions involve a similar mechanism, presumably via macrophage activation [81]. AUTOIMMUNE DISEASES AND IMMUNOSTIMULATION Overall, that autoimmune diseases are more frequent in patients treated with recombinant cytokines provides further evidence, if need be, for the pivotal role of cytokines in the pathogenesis of many immunopathological conditions. Because of their pleiotropic effects and redundancy, it is unlikely that one single cytokine can be the key determinant in the occurrence of immune disorders, but it may act as a booster. Cytokines have been shown to play a crucial, albeit complex regulatory role in the control of autoimmunity [82 –84]. Autoimmune diseases result from complex interactions among different T and B lymphocyte subpopulations that target autoantigens on different cell types. TH1 responses are implicated in many autoimmune disorders, whereas TH2 responses via IL-4 have opposing effects, so that autoimmunity is thought to reflect a shift in the subtle balance between TH1 and TH2 responses [85]. Not all autoimmune diseases, e.g. ulcerative colitis and SLE, are strictly TH1 diseases. In fact, IL-2, IFN-a, IFN-b, IFN-g and TNF-a can either enhance or limit autoimmune processes, and although the exact mechanisms leading to either effect are not known, this may explain why the same cytokine can either exert therapeutic efficacy in some patients with autoimmune diseases or induce autoimmune diseases in patients treated with the recombinant form of this cytokine [86]. Among the many hypotheses proposed to account for the observed clinical and immunological changes in patients treated with recombinant cytokines is the overproduction of other cytokines [87]. An abnormal expression of MHC class II molecules induced by IFNg, amplified by IL-1 and TNF-a, is a commonly held hypothesis. Thus, under the influence of IFN-g, thyroid cells express MHC class II molecules and act as antigen-presenting cells with the production of antithyroid autoantibodies as a consequence [88,89]. Importantly, the clinical and biological signs of autoimmune diseases associated with immunostimulating agents are closely similar to those of the corresponding spontaneous diseases in sharp contrast to most drug-induced autoimmune reactions. INHIBITION OF DRUG-METABOLIZING ENZYMES Our understanding of the mechanism involved in the inhibition of drug-metabolizing enzymes following administration of immunostimulatory substances progressed significantly in the past years.


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Infections and inflammation have long been associated with inhibition of drugmetabolizing enzymes in laboratory animals and man [90]. Viral infections were the first infections reported to induce inhibitory effects. Thereafter, a variety of viral, bacterial, fungal and parasitic infections were shown to exert similar effects. The first human report showed decreased theophylline clearance following confirmed infections to influenza and adenoviruses [91]. Similar findings were, for example, noted in patients with herpes simplex virus infection [92] or malaria [93,94]. As regard to inflammation, the activation of Ku¨pffer cells by latex particles inhibited CYP450 [95]. In classical experimental models of inflammation, an inhibition of drug-metabolizing enzymes was also demonstrated [96 –98]. Because IFNs are released to fight viral infections, the effects of IFNs on drugmetabolizing enzymes have been extensively studied. Interestingly, immunostimulatory drugs that increase interferon production down-regulate CYP450. Thus, reduced CYP2C11 mRNA levels have been noted shortly after the administration of the interferon-inducer tilorone [99] and the interferon inducer polyinosinic – polycytidylic acid (poly I:C) was reported to inhibit CYP1A1, CYP1A2, CYP2C11, CYP2E1, CYP3A1 and CYP4A [100 – 104] in the rat. Recombinant human IFNs were shown to inhibit CYP450 in mice [105,106] and similar effects have been obtained with IFN-a, IFN-b and IFN-g. IFNs have been implicated in the down-regulation of most families of CYP450 including CYP1A1, CYP1A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2E1, CYP3A1, CYP3A2 and CYP4A1 [100]. As mentioned in Chapter 3 of this volume, recombinant IFNs exert inhibitory effects on CYP450-induced metabolism at therapeutic doses. The effects of the pro-inflammatory cytokines IL-1, TNF-a and IL-6 have also been extensively studied [107,108]. For instance, IL-1 decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-O-deethylase activities in mice [109]. The administration of recombinant murine TNF-a to Wistar rats was shown to induce pretranslational down-regulation of the microsomal expression of CYP2C11 and 3A2, but had no effects on CYP2C6 and 2A1 [110]. IL-6 was similarly shown to down-regulate CYP450 in the rat [111]. In summary, IFNs, IL-1, IL-6 and TNF-a have been consistently shown to down-regulate the expression of a wide variety of CYP450 isoforms in varied conditions. Interestingly, the underlying mechanism is relatively well understood [112,113]. There is evidence that pretranslational mechanisms are the sites involved in the loss of CYP450 induced by cytokines. The loss of mRNA precedes enzyme loss and the magnitude of the decrease parallels the reduction in enzyme activity. However, the precise molecular target has seldom been directly investigated or remains to be elucidated [114].

MECHANISMS OF HYPERSENSITIVITY REACTIONS INTRODUCTION The clinical manifestations of hypersensitivity reactions as reviewed in Chapter 3 of this volume are extremely varied as these reactions can target nearly every tissue or organ. Hypersensitivity reactions are also characterized by the wide variety of causative

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mechanisms. Broadly, they are divided into immune-mediated and nonimmune-mediated reactions. IMMUNE-MEDIATED HYPERSENSITIVITY REACTIONS A hallmark of immune-mediated hypersensitivity reactions is the exquisite capacity of the immune system to recognize structural elements of non-self molecules or antigens, and to mount exaggerated response against these antigens through the involvement of immunological memory. Thus, in the nonclinical as well as the clinical setting, a diagnosis of immune-mediated hypersensitivity reaction can only be envisioned when a specific immunological mechanism can be reasonably suspected to be involved. Immunogenicity. For any molecular entity to induce an immune-mediated hypersensitivity reaction, a prerequisite is that sensitization of the host has taken place prior to the eliciting contact on the occasion of a prior sensitizing contact. Prior sensitizing contact. It is important to bear in mind that it is normally impossible to demonstrate whether prior contact(s) actually sensitized the treated or exposed host. Therefore, the assumption has to be made that any prior contact may have been sensitizing. In fact, the development of a clinical reaction following a subsequent challenge to the same antigen is the only situation to suspect the involvement of an immune-mediated mechanism. Several aspects, however, are important to consider. The easiness of identifying a prior and supposedly sensitizing contact is variable. In laboratory animals exposed to repeated dose administrations, a prior contact is obvious. In human beings, the situation may be not so obvious and is often totally different whether the causative agent is a drug or a chemical. The identification of a prior contact is straightforward when a patient who developed an adverse reaction is being treated with the suspected drug. A minimum of 5 –7 days of treatment is generally deemed necessary to result in sensitization. Typically drug-induced immune-mediated hypersensitivity reactions develop within the first month of treatment, even though a longer or even much longer period of time is possible. Similarly, no difficulty is expected in patients who have been previously treated with the same drug, even though they did not experience adverse effects induced by the suspected drug, simply because a sensitizing contact often does not result in a clinically overt reaction as already mentioned. In these patients, an immune-mediated hypersensitivity reaction can develop within minutes to several days depending on the route of administration and the underlying mechanism. The identification of a prior contact may be far less easy. This can occur when the prior contact is unknown or overlooked. For example, anaphylaxis has been reported after ingestion of a soft drink containing penicillin [115]. Sensitization can also be due to a closely related molecule and the clinical reaction reflects cross-allergenicity. As discussed below, cross-reactivity is not as common as is often thought and usually limited to a few families of closely related molecules. In general, the identification of a prior contact is difficult, if at all possible for most environmental and occupational chemicals. Although a prior, supposedly sensitizing contact is an absolute prerequisite to develop an immune-mediated hypersensitivity reaction, it should be underscored that a clinical reaction often does not develop following a subsequent contact. A typical example is the


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analgesic drug glafenin which was withdrawn from the market because it had been incriminated in anaphylactic shocks [116]. One of the main uses of glafenin was the treatment of mild and frequent pain, such as headaches. Therefore, affected subjects generally had a long and uneventful history of multiple glafenin intakes prior to developing anaphylactic shock for reasons that have never been elucidated [117]. Sensitization. If a prior sensitization contact is absolutely required, then the drug or chemical to which a laboratory animal or human subject is exposed should be sensitizing. Broadly speaking, a molecule can be sensitizing when the two following criteria are fulfilled. First of all, the molecule must be a foreign molecule for the immune system to mount a specific response. The majority of drugs and other chemicals with the exception, to some extent, of medicinal products of human origin, such as biotechnologically derived human insulin or growth hormone, are foreign molecules. Only foreign molecules large enough can be sensitizing. No minimal requirement has been fully documented. A molecular weight of at least 1000 has been proposed for a foreign molecule to be antigenic [118]. In fact, the minimal requirement is probably greater, maybe above 5000. Foreign macromolecules including proteins, glycoproteins, polypeptides, polysaccharides and microbial extracts can be direct immunogens. Important factors affecting the immunogenicity of a macromolecule include the antigenic foreignness, structural complexity and molecular size, degree of polymerization and biodegradability. The vast majority of xenobiotics, including drugs and other chemicals, are too small— usually below 500—to be direct immunogens. To induce sensitization, they must play the role of haptens [119]. Haptens according to the definition proposed by Landsteiner [120] are small molecules that strongly, but not necessarily covalently, bind to carrier macromolecules, mostly proteins. The hapten – carrier complex can trigger the immune system to mount a specific response against the hapten. Importantly, binding to plasma proteins, primarily albumin is normally not strong enough to produce immunogenic complexes. It appears that the overall stability of the hapten –carrier complex is a more critical factor than any properties of the chemical bond. For example, the benzodiazepine derivative diazepam is 80% bound to plasma albumin, but hypersensitivity reactions have extremely rarely, if ever been reported in treated human subjects. Binding to plasma albumin can be due to covalent linking and even though the percentage of a drug that is covalently bound to plasma albumin may be far less than in the previous situation, immunemediated hypersensitivity reactions, such as anaphylactic shock, may ensue as was shown with the NSAIDs tolmetin and zomepirac [121]. Role of metabolites. Sufficient chemical reactivity is absolutely required for lowmolecular-weight xenobiotics to form immunogenic complexes with carrier proteins or macromolecules. This requirement is fulfilled either inadvertently or purposely by many environmental or industrial chemicals, such as aldehydes [122] and diisocyanates [123]. In contrast, medicinal products have limited, if no chemical reactivity. Importantly, this should be kept in mind when interpreting results obtained in animal models for the prediction of respiratory and contact skin sensitizers as they are unlikely to be valid for medicinal products that are devoid of intrinsic chemical reactivity. To take into account the fact that most molecules intended for therapeutic use are devoid of any significant chemical reactivity, a widely held assumption is that metabolites instead of the parent molecule are actually involved in hapten formation [124 –128]. Because metabolizing processes indeed

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often lead to reactive intermediate metabolites, it is tempting to believe that metabolites play the role of haptens. The most recently published examples of the documented role of metabolites in immune-mediated hypersensitivity reactions include carbamazepine-10,11epoxide in cutaneous eruptions [129], several metabolites of metamizole in immunoallergic cholestatic hepatitis [130], the hydroxylamine and nitroso metabolites of sulfamethoxazole in severe toxidermias [131], and the glucuronides of etodolac and 6-hydroxy-etodolac in immunoallergic anemias [132]. A major limitation to the demonstration of the role of metabolites in immune-mediated hypersensitivity reactions is their short half-life as they are most often highly reactive intermediates that are very rapidly metabolized into more stable metabolites. However, the use of in vitro systems, such as the LTT, can prove useful when the suspected metabolite is not available. In turn, if the role of intermediate metabolites in hapten formation is accepted, the immune system must be expected to discriminate between extremely close chemical structures, such as metabolites and the corresponding parent molecule. The currently proposed in vitro tests for the diagnosis of drug and chemical allergy are likely to generate false negative results when the parent molecule and not the involved metabolite are tested. Cross-allergies are clinical manifestations involving the recognition of closely related molecular structures. It is important to note that because human subjects mount their own specific response toward a given antigen or allergen, cross-reacting antigens may not always induce cross-sensitization. In addition, the presence of homologies in the structure of two molecules may not result in cross-reactivity and finally, cross-reactivity may ensue despite the lack of apparent homology. This is especially true for proteins and macromolecules where conformational instead of sequential epitopes are involved in sensitization. Conformational, in contrast to sequential epitopes consist of bits of a molecule, e.g. several amino-acids that are closely linked when the 3D conformation of the molecules is considered, but may be distant in the linear sequence of the molecule. It is often difficult to ascertain that cross-reactivity is actually involved. Typical examples of cross-allergies can be found among grass [133,134] and tree [135] pollens, house-dust mite [136] and insect [137] allergens, food allergens [138], as well as between each of these categories of allergens [139]. As far as drugs and other chemicals are concerned, the b-lactam antibiotics and the ‘para-amino’ chemicals are the most illustrative. Penicillin derivatives are produced from the cephalosporium mold. Early cephalosporin antibiotics contained traced amounts of penicillin and this led to overestimate the incidence of cross-allergies between penicillins and cephalosporins. Based on the review of the literature, only 8.1% of patients with a history of penicillin allergy had a reaction after treatment with cephaloridine, cephalexin, cephalothin, cefazolin, or cefamandole, as compared to 1.9% of patient without such a history [140]. Even though published studies suggest that an allergic reaction to cephalosporins in patients with a history of allergy to penicillin may overall be up to 8 times more frequent than in patients without a history of allergy to penicillin, it is essential to consider closely in a given patient the type of cephalosporin he (or she) will be given. Penicillin skin testing is useful to exclude sensitization involving the b-lactam ring [141]. Probably due to differences in the side chains attached to the b-lactam ring, cephalosporins are far less likely to induce crossallergies in patients with a history of penicillin allergy [142 – 145]. Nevertheless,


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the possibility for b-lactam cross-reactivity should not be overlooked [146 – 148]. Importantly, there is no cross-reactivity between mono-bactams or carbapenems with beta-lactams [149]. Last but not least, it is important to emphasize the unreliable history of hypersensitivity reactions to b-lactam antibiotics in many patients. Thus, when a welldocumented diagnosis, primarily based on the clinical manifestations and the results of skin testing [150] is not available, the decision whether a patient can be given a penicillin or cephalosporin is only subjective, and therefore can be risky. This is also a major reason why results of studies dealing with cross-sensitization among b-lactam antibiotics are so conflicting. Para-amino chemicals. A variety of drugs and chemicals, including para-phenylenediamine, diaminodiphenylmethane, para-aminophenol, benzocaine, sulfanilamide have a para-di-substituted structure. Cross-allergies have been described although the mechanism leading to the formation of common haptens is not known [151]. Risk factors. Whatever the immunogenic (or sensitizing) potential of a given xenobiotic, not all individuals obviously become sensitized upon exposure, so that the role of contributing factors should be considered. For instance, less than 1– 2% of treated patients are expected to develop an immune-mediated reaction to penicillin G. Risk factors are related either to the patient or to the substance. Risk factors related to the patient Age. Young adults generally develop more frequent immunoallergic reactions to drugs and other chemicals for reasons that remain unclear. In an epidemiologic study of severe anaphylactic and anaphylactoid reactions among hospitalized patients, the number of reactions was 927 per 1 million in patients less than 20 years of age, 221– 276 in patients between 20 and 59 years of age, and only 154 in patients over 60 [152]. Overall, hypersensitivity reactions are as common in children as in adults [153]. However, certain drug-induced reactions are more frequent in children. Thus, a 3-fold incidence of lamotrigine-induced severe cutaneous eruptions, has been reported in children as compared to adults [154] and the majority of reported serum sickness-like diseases associated with cefaclor developed in children [155]. Allergic contact dermatitis is uncommon in the first years of life (, 3– 4 years), then tends to be more frequent to reach an incidence similar to that in adults. In contrast, the overall incidence does not seem to be markedly different between young and older adults. Contact hypersensitivity responses in the Mouse Ear Swelling Test were shown to be weaker in mice less than 5 weeks of age or more than 13 weeks of age [156]. In contrast, food and respiratory allergies are more frequent in young children. Gender. Despite somewhat conflicting data [157 –159], young girls and women seem to be only at a slightly higher rate of developing hypersensitivity reactions to drugs than boys and men so that the role of gender does not seem essential. The female/male ratio tends to increase with age possibly reflecting greater drug consumption in women. It is unclear whether the hormonal status accounts for this slight difference. Atopy. The term atopy was introduced by Coca and Cooke in 1923 to describe the familial predisposition to abnormal reaction after exposure to common allergens. Today, atopy is understood as a pathological condition characterized by excessive production of IgE associated or not with one or several diseases, such as reaginic asthma, hay fever and

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constitutional dermatitis [160]. The incidence of atopy was variably estimated depending on the criteria used, namely the presence of either clinical symptoms or specific IgE to common allergens demonstrated by skin tests or in vitro (elevated serum IgE levels). Due to variations in the definition of atopy, conflicting results have been published regarding the predisposing role of atopy in immune-mediated adverse drug reactions, but the most recent data support the role of atopy as a risk factor for drug-induced hypersensitivity reactions [161,162]. However, the link is seemingly more consistent with non immune-mediated hypersensitivity to NSAIDs [163 –165]. In contrast, atopy was not found to predispose to hypersensitivity to vaccines [166]. Interestingly, chemical exposures, such as maternal smoking have been shown to increase the risk of atopy [167]. Individual (genetic) predisposition. Epidemiological studies, especially multigeneration family and twin studies have demonstrated a strong genetic component in a number of allergic diseases, most notably those involving an IgE-mediated mechanism [168]. However, these studies focused on clearly immune-mediated allergies. As it is increasingly clear that IgE-mediated reactions induced by drugs and chemicals are relatively uncommon or relatively limited to a few chemical classes of compounds, such as the beta-lactam antibiotics, there is no surprise that very limited, if any evidence supports the view that drug and chemical allergies involve a genetic predisposition. However, this does not exclude the possibility that such a genetic predisposition might be strongly involved in specific cases. Clearly, all individuals are not equal regarding the risk of sensitization to drugs and other chemicals: in the same conditions of treatment, only a fraction of patients develop an immunoallergic reaction to penicillin G. However, no association between HLA determinants and penicillin allergy [169] or drug-induced cutaneous eruptions [170] was evidenced. The search for genes predisposing to asthma and atopy is under way and significant progress has been made. Importantly, as genetic factors are essentially predisposing factors, this suggests the role of interactions between genetic and environmental factors [171]. Biotransformation/pharmacokinetics. As already mentioned, the parent molecule is thought to be metabolized to a reactive metabolite that is sensitizing. Therefore, the role of metabolic polymorphisms should be considered as a risk factor. An interesting example in this respect is the suspected role of certain drug-metabolizing phenotypes and genotypes in AIDS patients with hypersensitivity reactions to sulfonamides. Sulfonamides, in particular co-trimoxazole, are known to induce a high rate of severe cutaneous reactions in AIDS and the role of metabolites is clearly established [131]. A slow acetylator phenotype and genotype, and a glutathione S-transferase M1 null genotype were found to be risk factors for such reactions in AIDS patients [172 –176]. However, the most recent data tend to contradict the role of slow acetylator phenotype and genotype as a major risk factor [177 – 179]. As diverging results have often been reported by the same groups of researchers, the possibility that co-factors are involved cannot be discarded. Environment. The role of the global and occupational environment in the development of hypersensitivity reactions is obvious [180]. There is substantial evidence that the incidence of respiratory allergies is increasing in industrialized countries [181], even though the causative factors are not yet fully established and in fact a combination of factors is likely [182]. Air pollution has clearly been identified as a causative factor in asthma. Beside environmental gases, such as ozone, sulfur dioxide and nitrogen oxides [183 –185],


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particles, such as diesel exhaust particles augment allergic immune responses [186]. Indoor air pollution, including passive smoking, should also be considered [187]. The socio-economic situation should finally be considered and the more frequent reporting of allergic and especially asthma symptoms in the upper class might simply reflect a better awareness of this pathological condition than in lower classes [188]. Risk factors related to the substance Chemical structure is another critical, but still ill-understood factor of immunogenicity despite current extensive efforts to search for components of the chemical structure which are more likely to be involved [189 – 194]. More information is obviously needed before it can be possible to identify which components of the chemical structure are specifically involved in the sensitizing potential of xenobiotics. Route of exposure. Although every route of administration or exposure can result in sensitization, there are routes such as topical applications that have a greater potential for sensitization as exemplified by allergic contact dermatitis to drugs administered by transdermal devices [195]. The oral route is typically associated with the induction of tolerance, but sensitization can nevertheless occur. The mechanisms resulting in the breakdown of oral tolerance are not known. When the intravenous route is used, reactions tend to be more severe. Exposure regimen. Intermittent exposures markedly facilitate sensitization and the development of immunoallergic reactions. Thus, immune-mediated tubulo-interstitial nephritis was more frequent after intermittent rifampicin treatment [196]. Mechanisms of immune-mediated hypersensitivity reactions Pathophysiological classifications. In the early 1960s, Gell and Coombs proposed a classification of immunoallergic reactions [197] which is still often used [198] and even unfortunately promoted by some regulatory authorities, such as the US FDA [199]. It should be strongly underscored that it is often useless and misleading as the mechanisms of immune-mediated hypersensitivity reactions are not understood and clearly cannot be classified according to the Gell and Coombs classification [200 – 202]. In further support to this view, delayed-type hypersensitivity reactions—the type-IV reactions according to Gell and Coombs—have been proposed to be divided into four distinct sub-categories, namely type IV-a reactions involving a TH1 response mediated by IFN-g; type IV-b reactions involving a TH2 response mediated by IL-5; type IV-c or cytotoxic reactions; and type IV-d reactions leading to neutrophilic inflammation [203]. Similarly, an overlap between the different mechanisms involved in immune-mediated hemolytic anemias has been underscored [204]. Despite obvious limitations, the Gell and Coombs classification can serve as an introduction to the pathophysiology of hypersensitivity reactions (Fig. 1). Importantly, it must be borne in mind that not all mechanisms are included in this classification, that several mechanisms can be involved concomitantly by one given sensitizing molecule or that different mechanisms can be involved in different patients exposed to a given molecule and presenting with clinically similar reactions. Type I reactions are anaphylactic or immediate hypersensitivity reactions. Antigens that trigger type I reactions are allergens present in the environment as low-molecular-weight particles that are inhaled, eaten or administered as in the case of drugs. Antigens induce the production of a particular class of (reaginic) antibodies. In man, reaginic antibodies are IgE

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Fig. 1. Gell and Coombs classification of drug-induced immunoallergic reactions.

and to a lesser extent IgG4. Depending on the animal species, other classes of Ig can be involved, such as IgE and IgG1 in mice, and mainly IgG1 in guinea pigs. Upon contact with an allergen, B lymphocytes switch to IgE production when they are co-stimulated by IL-4. IgE bind to high affinity receptors (Fc1RI or CD64) on the membrane surface of mast cells in tissues and basophils in the peripheral blood. After a subsequent, but not necessarily the next contact, the reaction between a divalent allergen and specific IgE antibodies triggers the degranulation of mast cells and basophils. This results in the immediate release of preformed mediators that are stored in cytoplasmic granules. Preformed mediators include histamine, the neutral proteases tryptase, chymase, carboxypeptidase and cathepsin D, and the proteoglycans heparin and chondroitin sulfate E. Since its discovery by Dale and Laidlaw in 1911, histamine has been widely considered as the major mediator involved in IgE-mediated hypersensitivity reactions, although the marked biological properties of other preformed mediators should not be overlooked. Histamine is formed by the decarboxylation of histidine. After release, it diffuses very rapidly into the surrounding tissues with a plasma half-life in the range of 5 – 10 min or even less. Histamine is metabolized by histamine-N-methyltransferase and diamine-oxidase (histaminase). Only 2– 3% of histamine is excreted unchanged in the


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urine. Histamine causes vasodilatation, increased capillary permeability, and contraction of smooth muscles in the bronchi and intestine, and enhanced mucus production that are normally of short duration due to its short half-life. Tryptase is a serine protease relatively specific of mast cells, which seems to have very limited local effects. Chymase is another protease that cleaves angiotensin I to angiotensin II and accounts for the effects of immediate hypersensitivity reactions on the coronary vasculature. The IgE-allergen reaction induces the subsequent de novo synthesis of mediators from membrane phospholipids [205]. The release of arachidonic acid from phospholipids is controlled by the enzyme phospholipase A2 and leads to the eicosanoid derivatives PG and leukotrienes (LT). PG are synthesized by cyclooxygenases of which two distinct forms have been identified, the COX-1, which is constitutive and present in the majority of tissues, and the COX-2 which is inducible over a period of hours in inflammatory cells. The cyclooxygenases lead to the synthesis of the prostaglandin PGH2 which is further processed to individual PG. PGD2 and thromboxane A2 are particularly relevant to immunoallergic reactions, as they are potent bronchoconstrictors. The slow-reacting substance of anaphylaxis (S-RSA) first described in the 1930s was later shown to consist of a mixture of the arachidonic acid 5-lipooxygenase metabolites LTC4, LTD4 and LTE4, or cysteinylleukotrienes (Cys-LTs). Two cys-LT receptors Cys-LT1 and cys-LT2 have so far been identified. The pathogenic role of cys-LTs in immediate hypersensitivity reactions is underscored by the finding that the anti-cys-LT1 antagonists montelukast, pranlukast and zafirlukast are effective drugs in the treatment of asthma and other allergic diseases [206]. The clinical manifestations of type I reactions reflect the biological activities of the released mediators. Type II reactions are cytotoxic reactions due to the binding of IgM and IgG to the surface of cells [207]. Typically, type II reactions consist of acute hematological reactions. When the sensitizing chemical bound to the surface of blood cells encounters circulating antibodies, the resulting activation of the complement system provokes the destruction of the blood cells, as is seen in immunoallergic hemolytic anemia. A similar mechanism is involved in some immunoallergic thrombocytopenia [208]. There are other mechanisms involving cytotoxic antibodies, which result in hypersensitivity reactions. Thus, antibodyopsonized erythrocytes can be destroyed by complement activation upon encounter with the antigen. The antibody can be an autoantibody directed against a self-constituent of the body as in autoimmune thyroiditis where the antibody binds to the thyroid-stimulating hormone receptor [209] or in Goodpasture’s syndrome where the autoantibody damages the basement membrane of lungs and kidneys [210]. Type III reactions are immune complex-mediated reactions. In the presence of antigens in greater quantity in the serum than IgM or IgG antibodies, circulating immune complexes can be formed. The size of the immune complexes determines the sites of tissue deposition and the resulting injury. Typically, immune complexes deposit in capillary vessels and activate the complement system, platelets, macrophages and neutrophils through their Fc receptors. Normally, activation of the complement system helps to clear immune complexes, but anaphylatoxins are formed that increase vascular permeability and are chemotactic for leukocytes. Activated cells release a variety of mediators and free radicals, which damage the endothelial cells. If the antigen is present predominantly at one site, immune complexes cause localized damage as in the Arthus reaction [211]. Immune

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complex reactions are one mechanism of cutaneous vasculitis [212]. When the immune complexes are present in the circulation, they may cause generalized vasculitis. The historical clinical presentation is serum sickness with fever, arthralgias, cutaneous eruption and proteinuria that are noted within 9– 11 days after the injection of heterologous serum or monoclonal antibodies [213]. A typical finding is deposition of immune complexes in the glomerular basement membrane which can be evidenced by immunofluorescence. Although the term serum sickness is still often used to describe hypersensitivity reactions induced by low-molecular-weight drug, there is a consistent lack of detectable immune complexes, either circulating in the blood or deposited in the glomeruli. The term serum sickness-like disease is therefore recommended to avoid confusion. Type IV reactions are delayed hypersensitivity reactions. Typically, delayed hypersensitivity reactions include allergic contact dermatitis and photoallergy which can both be easily reproduced in humans with the use of skin tests. Contact dermatitis can be either nonimmune-mediated (irritant contact dermatitis) or immune-mediated (allergic contact dermatitis) [214,215]. Both conditions are sometimes clinically inseparable. Irritant contact dermatitis may be complicated by a superimposed allergic contact dermatitis. Allergic contact dermatitis is characterized by the infiltration of T lymphocytes into the dermis and epidermis. It is caused by skin contact with a chemical that triggers an immunological response leading to inflammatory skin lesions. After penetrating into the skin, low-molecular-weight molecules or their metabolites can play the role of hapten that binds or complexes with cell surface or structural proteins on various cells, including Langerhans cells and keratinocytes. Langerhans cells process and present the antigen to T lymphocytes, which leads to the clonal proliferation of sensitized lymphocytes and to a clinically patent inflammatory reaction. Acute allergic contact dermatitis usually develops as an erythematous, vesicular, edematous eruption at the sites of skin contact with the sensitizing substance. Following repeated exposure, chronic contact dermatitis will develop with erythematous, scalling and thickened skin lesions. As already mentioned, Type IV reactions have been proposed to be divided into four distinct sub-categories [216], which lends further support to the inadequacy of the Gell and Coombs classification. Type IV-a reactions involve a TH1 response and closely corresponds to the classical type V reactions as defined by Gell and Coombs. Type IV-b reactions involve a TH2 response in which IL-5 is suspected to play a key role as in drug hypersensitivity syndromes and drug-induced exanthemas [217]. Type IV-c are caused by cytotoxic T lymphocytes [218] and type IV-d reactions involving IL-8 lead to neutrophilic inflammation [219]. However, it remains to be established whether this classification into four subgroups of type IV reactions is applicable to non cutaneous drug-induced reactions involving T lymphocyte mediated mechanisms. Interestingly, the labile, nonconvalent binding of drugs to the MHC – peptide complex has been proposed as another mechanism of immunogenicity for small-molecular-weight molecules that have structural features allowing them to interact with certain T cell receptors directly [220,221]. Thus, a number of T lymphocyte clones have been identified that can interact with allopurinol, carbamazepine, lamotrigine, lidocaine, mepivacaine or sulfamethoxazole. These were mainly CD4þ T lymphocytes that recognized the drug in combination with MHC class II molecules. Interestingly, these clones were directed against the parent molecule, but not a metabolite. This whole set of findings suggests that the hapten


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theory, although probably still valid in many instances, is not the only mechanism for a small-molecular-weight molecule to induce immune-mediated hypersensitivity reactions. PSEUDOALLERGIC HYPERSENSITIVITY REACTIONS Definition. Although immunoallergic reactions induced by drugs and chemicals are considered relatively common, an immune-mediated mechanism does not account for all hypersensitivity reactions. Because consistent clinical manifestations mimicking true immunoallergic reactions, especially IgE-mediated reactions, have been reproducibly described in patients exposed for the first time to the same offending chemical, the term pseudoallergy was coined to describe these adverse reactions that can develop despite the carefully documented lack of any previous contact. This new concept was heralded by a compilation of original contributions published in 1980 [222]. In a recent revision of its nomenclature for allergy, the European Academy for Allergy and Clinical Immunology recommended using the term nonallergic hypersensitivity instead of pseudoallergy [223], but this view has been criticized essentially because nonallergic hypersensitivity would not mean much to most lay people [224]. In fact, the main issue is to unanimously agree on a definition of what is a nonallergic hypersensitivity or pseudoallergy reaction, and to stick to this definition in the scientific literature in order to avoid confusing statement and case reports. Based on the available scientific and clinical evidence, several points can probably be easily accepted. The first point is that adverse events mimicking anaphylactic reactions can develop in patients after a first contact. The second point is that the same vasoacting pro-inflammatory mediators can be released to cause either nonallergic hypersensitivity or pseudoallergic reactions. The third point that is open to debate and a source of widespread confusion is the difference to be made between idiosyncrasy and pseudoallergy. For the sake of clarity, it is recommended to exclude those adverse reactions that only develop in patients with a predisposing pharmacogenetic defect. Thus, acute hemolysis in patients with a congenital deficiency in the enzyme glucose-6-phosphatase dehydrogenase following the administration of NSAIDs or antimalaria drugs [225] is an idiosyncratic, but not a pseudoallergic reaction. The delineation between idiosyncratic and pseudoallergic reactions may be trickier when an idiosyncratic reaction leads to immunological injury without the overt involvement of reactive metabolites [226]. In addition, a number of drug-induced adverse reactions have been considered as pseudoallergic, even though neither the involvement of a genetic predisposing trait nor the release of mediators have been shown or even suspected. One typical example is the Hoigne´’s syndrome, in fact first described by Batchelor in 1951 [227]. This syndrome is characterized by an acute feeling of thoracic tightness and malaise with hypotension following the injection of the slow-release formulation of procaine – penicillin G suspected to form procaine microcristal aggregates resulting in pulmonary micro-emboli [228]. With an incidence of 1 out of 1000 injections, this syndrome was often erroneously considered as an allergic reaction to penicillin G and it is likely that many elderly people are considered to be allergic to penicillin because they develop the Hoigne´’s syndrome in their childhood when treated with procaine – penicillin G to prevent the cardiac complications of rheumatic fever. Similarly, true immunoallergic systemic reactions to local anesthetics are considered

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to be rare [229]. In fact, shocks associated with loco-regional anesthesia are more likely to be either vagal reactions induced by the combination of pain and fear, or systemic reactions due to the inadvertent intra-arterial injection of the local anesthetic [230]. Mechanisms of pseudoallergic reactions. Although pseudoallergic have been known for more than 20 years, limited progress has been made in the recent years regarding our understating of the mechanisms involved [231,232]. Direct histamine release. Histamine, which is stored in mast cells and basophils, can be released by exocytosis independently of any IgE-mediated mechanism. A cytotoxic or osmotic effect may be involved. Major histamine-releasing compounds include morphine and several morphine derivatives [233], general intravenous anesthetics [234,235], and vancomycin [236]. However, it is difficult to predict the clinical consequences of administering a compound with histamine-releasing properties demonstrated in human healthy volunteers or in vitro. The measurement of serum histamine is seldom conclusive due to the very short half-life of histamine, so that the role of direct histamine release in anaphylactoid reactions associated with general anesthesia although frequently claimed in the past [237], remains speculative. Importantly, pseudoallergic reactions due to direct histamine release only grossly mimic IgE-mediated reactions as clinical manifestations only reflect the short-lived biological effects of histamine. Activation of the complement system. The complement is a complex system, the activation of which is under the control of regulatory proteins. C3a and C5a are potent anaphylatoxins that are released during complement activation and interact with specific receptors. They exert a variety of effects including leukocyte chemotaxis, increased vascular permeability, contraction of bronchial smooth muscle, histamine release, generation of LT, and IL-1 production. This variety of effects explains the close similarities between acute reactions involving direct activation of the complement system and IgE-mediated reactions. Complement activation can be caused by immunological as well as nonimmunological stimuli, among which Cremophor ELw and hydrosoluble radiological contrast media are the most commonly cited medicinal products. Cremophor ELw (polyoxyethylated castor oil) is a pharmaceutical solvent used to dissolve poorly soluble drugs [238]. It is a complex mixture of amphiphilic lipid molecules that form micelles in water, which activates complement either directly or after transformation. Acute pseudoallergic reactions due to Cremophor ELw have been recorded with intravenous formulations of diazepam [239], vitamin K1 [240], alfadione [241] and more recently, cyclosporine [242] and paclitaxel [243]. Histamine release was shown in dogs after injection of Cremophor ELw and several derivatives [244]. When Cremophor ELw is involved as in an intravenous formulation, no risk can be expected when using an oral formulation of the same drug without Cremophor EL, as exemplified by cyclosporine [245]. Hydrosoluble radiological contrast media induce clinically similar adverse reactions [246 – 248]. Such reactions are relatively common, but usually mild to moderate with only one death in approximately 40,000 radiological examinations. The introduction of lowosmolality agents has caused an overall reduction in the number of nonfatal contrast reactions. An immunoallergic mechanism is very unlikely to be involved, despite the concept of ‘iodine allergy’ which is common in the public. Complement activation by


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either the classical or alternate pathway was evidenced, but other biological systems, such as the kallikrein-kinin-factor XII system and the coagulation cascade also play a role [249]. More recently, the role of complement activation in severe first-dose adverse effects was suggested to be involved in patients treated with the murine anti-CD3 monoclonal antibody OKT-3 [250,251]. A similar mechanism was demonstrated with the chimeric anti-CD20 monoclonal antibody rituximab [252]. An increase in C3b/c and C4b/c was observed 30 min after onset of the infusion in five patients. In addition, increased levels of TNF-a followed by IL-6 and IL-8 were also noted. Prostaglandins and leukotrienes. NSAIDs and aspirin in particular can induce acute intolerance reactions, mainly precipitations of asthma attacks noted in 5– 10% of asthmatics [253]. Intolerance reactions typically develop within 1 h after aspirin ingestion as an acute asthma attack, often associated with rhinorrhea and conjunctival irritation [254 – 256]. A certain diagnosis is based on provocation tests only, either oral, inhaled or nasal tests. Urticaria with or without concomitant angioedema is less frequent in patients intolerant to aspirin. One single individual can develop stereotyped reactions following ingestion of different NSAIDs [257]. As chemical structures are too dissimilar, crossallergenicity cannot be involved and an impact on the release of arachidonic derivatives is far more likely. NSAIDs inhibit the enzyme cyclooxygenase (COX), of which two isoforms, namely COX-1 and COX-2, are known. Aspirin and the majority of NSAIDs are much more potent inhibitors of COX-1 than COX-2. Any NSAID with marked COX-1 inhibiting activity can precipitate asthma attacks in contrast to those NSAIDs, such as nimesulide, with preferentially anti-COX-2 activity. Importantly, COX inhibitors devoid of anti-inflammatory effects in humans, such as paracetamol, can nevertheless induce acute intolerance reactions. In contrast, COX-2 inhibitors appear to be safe in patients with a history of aspirin intolerance [258]. There is no firm evidence that COX inhibition results in increased availability of arachidonic acid and/or leukotriene release. It is only possible that inhibition of COX is associated with the overproduction of Cys-LTs that are important mediators of asthma [259]. Why a given patient will develop aspirin intolerance remains elusive. The facilitating role of chronic inflammation, persistent viral infection, or genetic predisposition has been suggested. Inhibition of kinin degradation. Kinins consist of a cascade system resulting in the production of bradykinin from kininogen, which increases vascular permeability, contracts smooth muscles and elicits pain. Bradykinin is degraded by carboxypeptidase N and angiotensin-converting enzyme [260]. Angioedema that is bradykinin-dependent can result from the use of angiotensin-converting enzyme inhibitors [261]. MECHANISMS OF AUTOIMMUNE REACTIONS The mechanisms involved in autoimmunity as well as autoimmune reactions induced by drugs and chemicals are not elucidated [262]. This is the reason why the prediction of these reactions, either systemic or organ-specific, is nearly impossible at the present time. Many theories have been proposed, but very few stood the test of time. The role of contributing or predisposing factors, however, is more clearly established.

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PATHOPHYSIOLOGICAL HYPOTHESES A number of hypotheses have been proposed over the years, but none has stood the test of time. In fact, research on drug-induced autoimmunity is not as active as it was in the 1970s –1980s. It is therefore unsure that certain hypotheses that have been tested with the experimental tools available at the time could not merit to be rekindled. Immune dysregulation. One of the earliest hypothesis was the depression of suppressor T lymphocyte functions (when suppressor T lymphocytes were considered pivotal components of the immunological orchestra) resulting in the polyclonal activation of B lymphocytes and the abnormal production of autoantibodies [263]. However, decreases as well as increases in suppressor T cell activity were later evidenced in patients with autoimmune hemolytic anemia induced by a-methyldopa. Although this hypothesis can no longer be advocated, it could be interesting to investigate whether the cytokine profile of patients with a-methyldopa-induced autoimmune hemolytic anemia is more Th1 or Th2like, which seemingly has so far not been considered. Penicillamine exerts a variety of immunopharmacological properties [264], the exact relevance of which is at best debatable with regard to its therapeutic activity, and it is still impossible to explain why it can induce organ-specific autoimmune reactions in treated patients. Based on the available evidence, the immunomodulation hypothesis cannot account for the reported drug-induced autoimmune reactions and in particular the specificity of autoantibodies. Alterations of cellular constituents. That xenobiotics or their metabolites can bind to cellular constituents with the ensuing formation of neo-autoantigens has long been suspected as a possible mechanism involved in autoimmune reactions. Several targets have been more extensively considered. Xenobiotics-metabolizing enzymes. Most xenobiotics are metabolized into compounds (metabolites) that are more easily eliminated from the body. In some cases, the metabolites produced are either directly or indirectly toxic after binding to a protein. The cytochromes P450 (CYP450) play a pivotal role in the metabolism of many drugs and chemicals, and several CYP450 have been shown to be the target of the reactive metabolite(s) they produce (see Chapter 3 of this volume). Interaction with sulfhydryl groups. The role of sulfhydryls groups in organ-specific autoimmune reactions induced by several drugs products has been suggested, but failed to be formally substantiated [265]. Sulfhydryl groups are present in most proteins and enzymes and are critical to normal cellular functions. Hematopoietic cells are particularly sensitive to SH-reactive compounds. However, penicillamine, thiopronine [266] and the preservative sodium ethylmercuryl thiosalicylate [267] which all contain a thiol group have been described to induce organ-specific autoimmune reactions. Experimental results in Brown– Norway rats with aurothiopropanolsulfonate do not support the role of thio group in the induction of autoimmunity [268]. Interactions with nuclear macromolecules. Hydralazine can indeed bind to DNA and nucleoproteins [269], but the concentrations required are much too high to support a specific binding. In addition, the autoantibodies detected in the sera of patients with the drug-induced lupus syndrome never reacted with the incriminated drug or one of its


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recognized metabolites. No one could ever reproduce the human disease in laboratory animals after the binding of hydralazine or procainamide to various nuclear macromolecules. T lymphocytes from subjects with SLE frequently have reduced DNA methyltransferase activity and hypomethylated DNA. Even though the mechanism is unclear, hydralazine and procainamide have been shown to inhibit methylation of T lymphocyte DNA [270]. Interestingly, N-acetylprocainamide, a procainamide derivative that does not induce lupus, is a 100-fold less potent DNA methylation inhibitor than procainamide [271]. Activation of dormant autoimmune abnormalities. The activation of dormant autoimmune abnormalities [272] is no longer accepted as an attractive hypothesis to account for drug-induced autoimmune reactions. Interestingly, the phenothiazine derivative alimemazine was found to induce overexpression of MHC class II molecules on thyroid epithelial cells and increased presentation of thyroid antigens [273]. Pseudo-GvH reactions. An interesting hypothesis regarding the mechanism of druginduced systemic autoimmune diseases is the concept of pseudo-GvH reactions. The administration of histo-incompatible lymphocytes to a host can induce a potentially lethal, graft-vs.-host (GvH) reaction [274]. The clinical symptoms observed after bone marrow graft, include hyperpyrexia, cutaneous eruptions, arthralgias, lung infiltrates and adenopathies. Remarkably, these clinical symptoms are similar to those observed in patients with drug-induced systemic autoimmune reactions, such as the lupus syndrome or scleroderma-like disease. Thanks to the early work of Helga Gleichmann [275], the hypothesis of a pseudo-GvH reaction came to light. Mice injected into one footpad with the antiepileptic drug diphenylhydantoin, the adverse effects of which mimic a GvH reaction, had a significant increase in political lymph node weight evidenced on day 7. This was the starting point of the development of the political lymph node assay (see Chapter 8 of this volume). The mechanism of pseudo-GvH reactions is not clearly understood [276]. An interference with the molecular mechanisms involved in antigen presentation by APCs to CD4þ T lymphocytes is the most attractive hypothesis. The overactivation of CD4þ T lymphocytes could result in an abnormal polyclonal activation of B lymphocytes with the production of autoantibodies or the proliferation of previously dormant clones of autoreactive lymphocytes. It is noteworthy that this hypothesis is reminiscent of the proposed mechanism for the direct recognition of certain low-molecular-weight molecules by T lymphocytes [220]. Molecular mimicry received much attention recently. In addition to animal models [277], several examples in humans underscore the potential importance of this mechanism [278,279]. Thus, rheumatic fever is an autoimmune response triggered by b-hemolytic streptococcus infection with cross-reactivity between streptococcal and cardiac myosin. In the Guillain– Barre´ syndrome, antibody cross-reactivity has been demonstrated between human gangliosides and lipopolysaccharides from C. jejuni. In type I insulin-dependent diabetes mellitus, T cells recognize a peptide from glutamic acid decarboxylase and a highly homologous peptide from B4 coxsackie virus. In multiple sclerosis, T cells react with the myelin basic protein and peptides from Epstein – Barr virus, influenza virus type

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A and human papilloma virus. Molecular mimicry has been primarily demonstrated for microbial antigens and it remains to be determined whether drugs, and not only vaccines can induce the production of antigens with the capacity to induce autoimmunity by molecular mimicry [280]. PREDISPOSING FACTORS Although the mechanisms of autoimmune reactions induced by drugs and other chemicals are not known, the role of contributing or predisposing factors is suspected from the data available on spontaneous autoimmune diseases. Genetic susceptibility. Epidemiological studies demonstrated that genetic factors are crucial in autoimmune diseases. The rate of concordance for rheumatoid arthritis, systemic lupus erythematosus, type-I insulin-dependent diabetes mellitus or multiple sclerosis, is at least four times higher in monozygotic twins than in dizygotic twins [281]. Family studies have also shown an increased risk of autoimmune disease in relatives of patients with a systemic autoimmune disease. Unfortunately, only a few autoimmune diseases are due to mutations in a single gene so that most autoimmune diseases involve multigenic susceptibility. A number of autoimmune diseases are linked to a particular class I or class II HLA molecules [282], but this association required further linkage with other genes. HLA alleles, such as HLA-DQB1p0602, have been shown to be protective, but the association between HLA alleles and disease may widely vary among different ethnic groups. Finally, recent studies suggested the involvement of MHC and non-MHC genes. Although the role of genetic susceptibility in autoimmune diseases is accepted, further research is needed to elucidate the role if any, of genetic susceptibility in autoimmune reactions. However, there is experimental evidence that genetic susceptibility is also crucial in autoimmune reactions. For instance, mercuric chloride was clearly shown to induce autoimmune glomerulonephritis in Brown– Norway rats, but not in other inbred or outbred strains [283]. Infectious agents can trigger autoimmunity by three distinct mechanisms at least, namely molecular mimicry, polyclonal activation and the release of sequestered antigens. Therefore, the predisposing role of infections is an area of vivid research. Hormonal status. Many epidemiological studies showed that women are at a greater incidence of developing autoimmune diseases. However, the female predominance varies depending on the type of autoimmune disease [284]. Thus, 85% of patients with autoimmune thyroiditis, scleroderma and SLE are women, but only 60% of patients with SLE or rheumatoid arthritis. There is a relatively equal risk among boys and girls as regards early onset autoimmune diabetes. The female predominance is confirmed by models of autoimmunity-prone mice, such as NZB £ NZW F1, NOD and MLR lpr/lpr mice in which females develop more frequent and more severe spontaneous disease. Female sex hormones have long been shown to interfere with immune regulation [285,286]. Age. Autoimmune diseases can occur at any age and there are age differences among diseases [284].


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Chemical exposure. The role of drug treatments and chemical exposures in the development of autoimmune diseases is controversial. Autoimmunity induced by immunotoxicants has been claimed to be a major cause of concern, but well-documented case reports of autoimmune reactions induced by drugs and other chemicals are relatively few [287]. This might reflect underreporting of these adverse events. A survey of the French Pharmacovigilance database showed that autoimmune reactions accounted for less than 1% of all recorded drug-induced adverse effects [288]. That autoimmunity, even though a rare immunotoxic event, can be a significant cause of morbidity and mortality is nevertheless unambiguously exemplified by the Spanish toxic oil syndrome [289] and tryptophaninduced eosinophilia –myalgia [290] which affected thousands of patients.

STRUCTURE –IMMUNOTOXICITY RELATIONSHIPS A structure – activity relationship (SAR) is an association between a structural feature of a drug or chemical and a biological effect, either beneficial or toxic (structure – toxicity relationship). Substructures can serve as structural alerts for potential toxicity (toxicophore). In contrast, a quantitative structure –activity relationship (QSAR) is a mathematical relationship between a quantitative measure defining one characteristic of the chemical structure and a quantitative measure of a biological property. Therefore, the distinction between SAR and QSAR should not be misunderstood. A QSAR provides a means of extrapolating from a physicochemical property or any other measure of chemical structure to the end-point of interest. Although QSARs have been developed for the extrapolation of in vivo end-points, they are probably more adapted to the extrapolation of in vitro end-points. So far, only limited data are available regarding the SAR of immunotoxicants. Most efforts have actually been paid to sensitizing compounds, either contact sensitizers [291 –294] or respiratory allergens [295,296].

CONCLUSION Over the past 30 years, immunotoxicologists have focused most of their efforts on developing animal models and assay that can be reliable to predict immunotoxicity in nonclinical studies. Despite extensive research to understand the mechanisms involved in the immunotoxicity of a few drugs and chemicals that will be addressed in volumes II and III, relatively limited knowledge is available on the mechanisms of immunotoxic effects in general.

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Chapter 5

Nonclinical evaluation of unexpected immunosuppression

INTRODUCTION As immunosuppression has been the main, if not the only focus of immunotoxicologists during 25 years, a number of animal models and assays have been designed and utilized to predict the potential of drugs and other chemicals for suppressing immune responses. Although the majority of these animal models and assays has not been optimally standardized or fully validated from the view point of regulatory safety evaluation, it can nevertheless be claimed that drugs and chemicals that unexpectedly exert potent immunosuppressive properties can be reliably detected by performing state-of-the-art nonclinical studies. HISTOPATHOLOGY Histology is consistently included in conventional nonclinical toxicity studies. Histological alterations are expressed as quantitative and/or qualitative changes of the normal structure of target organs of toxicity. Structural changes are normally associated with modifications in the function of the affected organ(s), whereas it is not established whether modifications in immune functions consistently correlate with structural changes. A further difficulty is that the immune system is not a typical target organ of toxicity since immunocompetent cells are dispersed throughout the body even though they tend to form cell aggregates that are identified as lymphoid organs. Examination of the histology of lymphoid organs has been proposed as a straightforward and cost-effective approach to identify potentially immunosuppressive compounds [1 –7]. A standard histological examination of the major lymphoid organs including the thymus, spleen, bone marrow, the main lymph nodes and Peyer’s patches, is nowadays considered as an early indicator of possible immunosuppression. It is therefore included in all currently applicable guidelines relative to the nonclinical evaluation of unexpected immunosuppression (see Chapter 9 of this volume). Potent immunosuppressive xenobiotics have indeed been shown to produce frank histological changes in lymphoid organs that are easily detected, whereas less potent 163

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compounds may induce only slight morphological changes, if at all. As histological diagnosis often represents a subjective judgment on the nature and the expected consequence of a specific lesion, a central question is to what extent histopathology is helpful in the nonclinical immunotoxicity evaluation of drugs and chemicals. Pathologists can identify alterations in the architecture of lymphoid organs and changes in the distribution of immunocompetent cell populations that are not easily discernible using immune function tests. This led eminent pathologists to claim that routine histological examination of the lymphoid organs during general nonclinical toxicity studies is sufficient to detect unexpectedly immunosuppressive xenobiotics [1,3 – 5]. However, even potent immunosuppressive drugs, such as azathioprine and cyclosporine, did not induce consistent histological changes in certain rodent studies [8]. Logically, there is a growing consensus that immune function tests are essential and necessary additions to the histological examination of the lymphoid organs [9]. CONVENTIONAL HISTOLOGY EXAMINATION State-of-the-art preclinical immunotoxicity evaluation normally includes measurement of the weight and standard histological examination of the main lymphoid organs. Tissue sampling, weighting and staining techniques [2,3,10,11]. Lymphoid organs must be carefully dissected due to possible adherence to nonlymphoid fatty and connective tissues and weighted unfixed for calculation of weight changes relative to total body weight. Although lymphoid organ weights have often been used as an index of immunotoxicity, the weights vary considerably so that statistical significance between controls and treated animals is rarely attained. In addition, changes in the weight of lymphoid organs are of variable immunotoxicological relevance. Whereas decreases in the relative weight of the thymus often characterize a potential for immunosuppression, but may be difficult to ascertain due to normal thymic involution in aged animals, decreases in spleen weight must be clearly dose-dependent to be of possible relevance [12]. Normally, lymph nodes are very small, but they very rapidly increase in size after antigen stimulation to return to their original size after termination of the reaction. Thus, as activated and resting lymph nodes should be examined concomitantly, lymph nodes close to and remote from the site of entry are examined. A selection of lymph nodes is made depending on the site of entry. For example, mandibular and mesenteric lymph nodes are preferred when the oral route is used. The axillary or popliteal lymph nodes are generally recommended for use as reflecting the resting state. Changes in lymph node weight, however, are rarely useful due to marked inter-individual anatomical variability. The bone marrow is usually collected from the sternum or the femur and processed to bone marrow smears stained with conventional dyes, such as MayGru¨nwald –Giemsa. It is unknown whether the bone marrow from these two sites is equally sensitive to immunotoxicants. Cell suspensions can be prepared for cytological or flow cytometry analysis [13 – 16]. Finally, mucosa-associated lymphoid tissue (MALT) is obtained using varied procedures. The gut-associated lymphoid tissue (GALT) is typically obtained from rolled intestinal segments, the so-called ‘Swiss rolls’ that are either fixed or frozen. A lack of effects on Peyer’s patches can be due to the selected Peyer’s patches for microscopic examination. The bronchi-associated lymphoid tissue


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(BALT) is best examined from lung sections. In inhalation studies, the nasal-associated lymphoid tissue (NALT) can also be examined. Finally, it is essential to include sampling of blood and selected nonlymphoid organs, such as skin, lung, kidney and liver for a comprehensive histological examination. The tissues and organs examined for standard histology are fixed as quickly as possible after necropsy. Several fixatives can be used for conventional and immunochemistry staining. Formalin fixation, paraffin embedding and staining with hematoxylin and eosin are the recommended procedures to identify changes in the architecture of lymphoid tissues and the morphology of most immunocompetent cells. Electron microscopy requires special equipment and glutaraldehyde/paraformaldehyde fixatives to preserve ultrastructural details. An overview of emerging histological techniques is available in Chapter 17 of this volume. HISTOLOGY OF LYMPHOID ORGANS Hematological and clinical chemistry data as well as results of necropsy are essential for an in-depth evaluation of histological changes. Because the immune system is characterized by a complex interplay of immune cells and soluble factors, the histology of lymphoid organs is very variable. Although changes related to necrosis and fibrosis or inflammation are easily recognized, the use of a precise nomenclature is absolutely essential. Quantitative changes in cell density, germinal center development or high endothelial veinules are less easily recognized, especially when mild [11]. Bone marrow Normal morphology. The bone marrow is a large and complex organ that is distributed throughout the cavities of the skeleton. At birth, hematopoietic tissue occupies all medullary space, but is progressively limited to the proximal epiphyses of long bones, central skeleton and skull. Fat constitutes about 70 –80% of the bone marrow in rodents, but only 50% in larger animals. Depending on the species, the bone marrow constitutes 1 –5% of the body mass in adults. The bone marrow can be divided into vascular and extravascular compartments. Hematopoiesis takes place in the convoluted extra-vascular spaces of bone marrow sinuses, which are lined by a single layer of endothelial cells interdigitating with various cell types including reticular and dendritic cells. Niches are thus formed in the extra-vascular spaces where hematopoietic and myelopoietic cells can be generated. As described in Chapter 2 of this volume, hematopoietic cells arise from a common multipotent progenitor cell. Maturation and differentiation of hematopoietic cells are controlled by stromal fibroblasts and growth factors among which IL-3, G-CSF, GM-CSF and M-CSF play a crucial role. For cells in continuous proliferation, a cell cycle takes approximately 24 h. Bone marrow evaluation from sections of femur or sternum is typically based on the cytological examination of bone marrow smears to measure cellularity of the erythroid, myeloid, lymphoid and megakaryocytic lines, maturation of cells within cell lines, and ratio of myeloid to nucleated erythroid cells. Interpretation of the results requires a peripheral blood cell count. In most species, the bone marrow myeloid:erythroid ratio has a slight myeloid predominance. In general, histological examination by differential cell counts in bone marrow smears is required when there is altered cellular maturation.

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Changes in the bone marrow induced by immunotoxicants. Using flow cytometry, Burchiel et al. [17] found a shift to immature cell types in bone marrow obtained from mice 5 days after treatment with cadmium or lead and this was associated with an increase in the number of progenitor cells. Suppression of humoral and cellular immunity was also noted. In mice exposed to graded doses of nickel sulfate via the drinking water for 180 days, the primary toxic effects were expressed in the myeloid system with a dose-related decrease in bone marrow cellularity, and granulocyte-macrophage and pluripotent stem-cell proliferative responses [18]. However, the predictability of changes in the bone marrow as reliable, specific indicators of immunosuppression is questionable. Thus, azathioprine—a cytotoxic immunosuppressive drug—induced a dose-dependent decrease in the numbers of nucleated leukocytes in the bone marrow of rats in contrast to the noncytotoxic immunosuppressive drug cyclosporine [19]. That the investigative oral anticancer drug CI-994 induced parallel decreases in bone marrow cellularity and cellular immunity, is consistent with these findings [20]. Based on the limited published data, histological examination of the bone marrow is more likely to serve as a tool to detect drugs and other chemicals that are myelotoxic and therefore should be considered for further extensive immunotoxicological evaluation. The thymus Normal morphology. The thymus is located in the anterosuperior mediastinum, except in guinea pig where it is located in the neck. It consists of two independent lobes attached by connective tissue. Each lobe is divided into lobules—the basic thymic functional units— that are enclosed by a fibrous capsule. Lobules are made up of three main areas (Fig. 1): the subcapsular zone which contains the earliest progenitor cells; a peripheral area—the cortical area—which is packed with immature thymocytes undergoing selection; and a central area—the medulla—which is less densely populated by mature lymphocytes ready to leave the thymus. Hassals’s corpuscles, found only in the medulla, appear to be degenerating epithelial cells. The thymus architecture is grossly similar among species [21]. Blood vessels and closely related nerves enter the lobules at the corticomedullary junction and plunge into the cortex. The normal thymus develops early in fetal life. At birth, the full peripheral immunologic repertoire is established. Marked physiological changes occur in the thymus during life. The size of the thymus increases considerably after birth due to postnatal antigenic stimulation, peaks at puberty and involutes in early adulthood. Most of the parenchyma, in particular the cortical lymphoid tissue, is progressively replaced by adipose tissue so that only sparse lymphoid islets are seen in elderly humans and aged animals. Thymus atrophy is associated with progressively impaired immune responses and an increased risk of infectious diseases in older subjects [22 – 24]. The reason for aging-associated thymus atrophy is not known. Involution of the thymus has also been observed in a number of situations. The sensitivity of the thymus to stress has long been recognized and an increased production of corticosteroid hormones is the hallmark, if not the only mechanism of thymic involution in response to stress [25 – 28]. Acute involution is seen in the first few days after various stresses and injuries. It is essentially due to massive apoptosis of thymocytes. Not all thymocytes, however, are affected: the CD4þCD8þ cortical thymocytes are much more


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Fig. 1. Schematic representation of a thymic lobule.

sensitive than medullary thymocytes. Thymic regeneration starts very rapidly with rapid proliferation of large thymocytes in the outer cortex. The size of the cortex gradually increases with a considerable reduction in size of the medulla. Thereafter, the volume of both the cortex and medulla increases until the thymus has reached its former status. A transient hypertrophy is sometimes seen. Stress has varied causes including malnutrition (i.e. decreased food consumption) which often reflects the systemic toxicity of the test article and can result in thymus atrophy [29,30]. The conditions of the breeding, housing and handling of animals are possible causes of thymic involution [2,31]. Histological changes of the thymus have been described following exposure to potent immunosuppressants [32]. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD) causes thymic atrophy in mice and rats even at very low doses. The thymic epithelium is the primary target of TCDD presumably via the Ah receptor [33]. At higher levels of exposure,

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apoptosis of lymphocytes is also seen. Organotins are another group of immunotoxicants which induce thymic atrophy [34]. Di- and tri-alkyl substituted derivatives act more specifically on the thymus. As the tri-alkyl derivatives are rapidly transformed to their di-alkyl homologues, the latter are considered the primary cause of thymic atrophy [35]. Immunosuppressive drugs, such as cyclosporine [36 – 39], tacrolimus [40 – 42] and sirolimus [43] induced thymic atrophy in rodents. Other examples of chemicals toxic to the thymus include the food additive caramel color [44], estradiol [45], diethylstilbestrol [46], the peroxisome proliferator di(2-ethylhexyl)-phthalate (DEHP) [47], the herbicide propanil [48], the insecticide deltamethrin [49] and 2-hexane-dione, the toxic metabolite of the solvent 2,5-hexane [50]. The spleen Normal morphology. The spleen is surrounded by a capsule of dense connective tissue that form branched trabeculae extending into the parenchyma and contains two main compartments: the red pulp and the white pulp. The red pulp is the most prominent compartment with large, irregular, thin-walled blood vessels—the splenic sinusoids—that are interposed between sheets of reticular connective tissue, the splenic cords of Billroth containing many macrophages. In mice and in rats to a lesser extent, the red pulp is also the site of extra-medullary hematopoiesis. The white pulp consists of a central arteriole surrounded by periarteriolar lymphoid sheaths with many T cells—the thymodependent area—and adjacent follicles containing B cells. The marginal zone, which is easily identified in rats, but less easily in mice, and is absent in humans, is located at the border of the red and white pulps. Histological changes of the spleen and immunosuppressants. Although spleen weight and histology are routinely examined in conventional toxicity studies, examples of significant changes are very few. Spleen weight is a relatively insensitive correlate of immunosuppression [51]. In most instances, spleen atrophy, especially in T cell areas, is associated with thymus atrophy [7]. Decreased cellularity and germinal center development in splenic follicular areas are sensitive parameters. The lymph nodes Normal morphology. Lymph nodes have a bean-shaped structure (Fig. 2). They are usually grouped at sites connecting blood and lymph vessels. The anatomy and architecture of lymph nodes are variable. Rodent species typically have fewer lymph nodes than larger animal species or man, but the general architecture of lymph nodes is to some extent consistent among species despite inter-individual differences and the role of pathogen-free conditions of breeding [21]. Overall, the general structure of lymph nodes comprises three main areas: the cortical, paracortical and medullary areas. Antigens typically enter lymph nodes by the afferent lymphatic vessels, which end in the subcapsular area and finally the medulla. In the paracortex, free or processed antigens are presented to T lymphocytes by interdigitating cells and macrophages. The cortical area is the site where B lymphocytes encounter antigens. There are two types of follicles in the cortical area: the primary follicles, which are aggregates of small resting B lymphocytes and the secondary follicles which contain B lymphoblasts within germinal centers. Resting or unstimulated lymph nodes have


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Fig. 2. Schematic representation of a lymph node.

a small size and store resting B and T lymphocytes. Upon antigen stimulation, the size of lymph nodes increases very rapidly and they show signs of hyperactivity with welldeveloped germinal centers. Histological changes of the lymph nodes induced by immunosuppressants are generally similar and concomitant to changes observed in other lymphoid organs, such as the thymus and the spleen [7,11]. Other lymphoid organs. Because epithelial surfaces are exposed to many environmental antigens, local effector immune cells protect the host. MALT is present in many sites. Peyer’s patches are lymphoid tissue beneath the secretory epithelium of the duodenum and jejunum, i.e. GALT. Other MALT includes the appendix in the large intestine, and lymphoid tissue along the bronchi (BALT) and in the upper airways where the tonsils and the intermediate lymphoid tissue form the ring of Waldeyer. The organization of MALT is similar to that of lymph nodes. A common characteristic of MALT is that IgA is the predominant antibody isotype produced by mucosal B lymphocytes. Although histological examination of the Peyer’s patches is required by most, if not all recent guidelines, relevant end-points still need to be defined [52]. In fact, very few examples of histological changes in Peyer’s patches have been reported. Oral treatment with 10 mg/kg/day of cyclosporine by gavage caused lymphocyte depletion of Peyer’s patches followed by progressive replacement of the lymphoid tissue by cystic and glandular

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epithelial structures in male Fischer 344 rats [53]. In mice, a single intravenous dose of cyclophosphamide (200 mg/kg) induced a marked cellular depletion in Peyer’s patches, which started to recover after 9 days post-injection [54]. A single oral dose of 10 mg/kg of T-2 toxin induced a sharp decrease in the numbers of CD3þ, CD 19þ, CD4þ and CD8þ lymphocytes of mouse Peyer’s patches [55]. The oral administration of 7,12-dimethylbenz[a]anthracene (DMBA) to B6C3F1 mice did not affect the number of lymphocytes in the Peyer’s patches, but inhibited their proliferative response to PHA and LPS [56].

HEMATOLOGY AND CLINICAL CHEMISTRY Clinical chemistry and hematology are included in all conventional toxicity studies. Although most measured end-points are not directly relevant to the prediction of direct immunotoxicity, some warnings can be identified. In addition, specific techniques, such as lymphocyte subset analysis, are commonly utilized in an attempt to enhance predictability. BLOOD CELL COUNTS A number of hematological end-points are typically measured during conventional toxicity testing [57,58]. They include total and differential leukocyte counts in addition to red blood cell end-points. Differential leukocyte counts enumerate neutrophils, lymphocytes, monocytes, eosinophils and basophils in the peripheral blood. It is essential to evaluate the actual numbers of cells/ml and not to interpret relative or percent counts. In general, statistically significant, dose-related changes of 10 –20% from baseline values are considered relevant. However, baseline values normally vary widely and it will be all the more difficult to show a treatment-related effect as the number of tested animals is smaller. Changes in differential leukocyte counts, such as leucopenia, lymphopenia, neutropenia, and eosinophilia, can be suggestive of an immunotoxic effect. However, leukocytosis can be seen in excited or frightened animals and the cell type responsible for the observed increase varies with the animal species: neutrophilia is more frequent in dogs and lymphocytosis more frequent in rats. No specific cell type increase is seen in monkeys. Obviously, this artifact can lead to misinterpretation of results, especially when few animals per group are used, and should be avoided by careful handling of the animals. Neutrophilia is also secondary to inflammatory responses caused by the technical procedure, such as catheter implantation or repeated injections. In the opposite, stressed animals may develop neutropenia, lymphopenia and eosinopenia due to the release of endogenous corticosteroid hormones. However, this situation is most commonly associated with overt toxicity. Neutropenia is usually caused by direct toxicity of the test article and does not reflect a specific immunotoxic effect. In contrast, lymphopenia, especially in rats where the number of lymphocytes is physiologically higher, can be due to immunotoxicity. This is, however, only a very rough end-point and interpretation is best based on the analysis of lymphocyte subsets (see below). Finally, effects on basophil and eosinophil counts are rarely seen during toxicology studies as absolute counts for these cells are very low.


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BONE MARROW TOXICITY Because of their rapid turnover, hematopoeitic cells from the bone marrow are exquisitely sensitive to the cytotoxic effects of anticancer drugs. Bone marrow toxicity is typically evidenced by colony-forming cell assays. Initially, the spleen colony-forming unit assay was considered as a reliable measure of multipotent progenitor cells, which later proved to be inexact. A number of in vitro assays have been designed that can investigate the effects of drugs and other chemicals on virtually all specific progenitor cell populations including CFU-E (erythroid), CFU-GM (granulocytic-macrophage), CFU-MK (megakaryocytic) clonogenic assays [59]. Techniques have been designed for performing these assays in rodents, dogs and monkeys as well as human beings. Despite its crucial role in immunity, the bone marrow has often been ignored as a target organ of immunotoxicity [60,61] and the evidence that toxicity to bone marrow hematopoiesis can result in immunotoxicity is largely indirect or limited to potent myelotoxic drugs. LYMPHOCYTE SUBSET ANALYSIS Nowadays, the use of flow cytometry is widespread to analyze lymphocyte subsets (Fig. 3). Flow cytometry was simultaneously developed in the 1960s. This is a powerful technique for analyzing large populations of single cells usually 10,000 cells or more within a short period of time [62]. The mechanism involves passing individual cells in a fluid stream to position the cells through the path of a laser beam. The light that is emitted is gathered by detectors and amplified by photomultiplier tubes. The light scattered from each cell can produce information related to cell size and density.

Fig. 3. Schematic representation of lymphocyte subset analysis by flow cytometry. Lymphocytes from spleen or peripheral blood are labeling with specific antibodies labeled with a fluorochromes, then passed through a laser beam. In general, cells are gated using two-parameter dot plot of cell size (forward-angle light scatter) vs. granularity (side-angle light scatter).

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Forward-angle (0.5 –28) light scatter is an approximate indicator of cell size and is commonly used to distinguish viable from dead cells which exhibit lower forward-angle light scatter. Increased forward-angle light scatter may be seen with activated cells. Sideangle light scatter (908) generally correlates with the granularity of the cell and an increased side-angle light scatter can be used to measure phagocytosis. A combination of forward-angle and side-angle scatter allows recognizing subsets of cells in complex cell populations. In addition, the power of flow cytometry can be further enhanced by the use of fluorochromes (or fluorescent dyes), which interact with cells. Cells can be labeled with fluorochromes specific for selected structures, e.g. cell-surface markers (CDs). There is a wide choice of commercially available fluorochromes and the selection is based on the parameter to be measured. Modern cytometers can measure several fluorescence parameters simultaneously to perform multiparameter analysis. The resulting fluorescence from the cells can be recorded cell by cell and quantified. In flow cytometry analysis of lymphocyte subsets, lymphocytes are treated with a panel of monoclonal antibodies covalently bound to fluorochromes. Typically, lymphocyte subset analysis in nonclinical immunotoxicity evaluation enumerates B lymphocytes, total T lymphocytes, CD4þ T lymphocytes and CD8þ T lymphocytes. Techniques have been developed to perform lymphocyte subset analysis in all conventional species including mice [63,64], rats [65,66], dogs [67], pigs [68,69], marmosets [70,71], and cynomolgus as well as rhesus monkeys [72 –76]. However, the use of this assay in larger animal species is often limited by the availability of specific or suitable reagents. Cross-reactivity must be carefully assessed when reagents from a different species are to be used. In rodents, splenocytes are commonly used for lymphocyte subset analysis, but recent findings suggest that peripheral blood lymphocytes may be more sensitive [77]. Although lymphocyte subset analysis in B6C3F1 mice has been shown to be a good predictor of immunosuppression [78], the predictive value of this assay was recently re-examined and the conclusion of a panel of experts was that immunophenotyping has not been sufficiently validated for inclusion as a routine assay in nonclinical immunotoxicity evaluation [79]. This conclusion is further supported by the finding that cyclosporine, one of the most potent immunosuppressive drugs available clinically, does not alter immunophenotypes in spleen or thymus of treated animals [80]. In fact, flow cytometry applications should not be restricted to the traditional analysis of a limited panel of lymphocyte subsets [81]. Other potentially important cells, such as monocytes (CD14þ) or natural killer (NK) cells (CD56þ), can be included even though it is not known whether changes in NK cell numbers correlate with changes in NK cell activity. Another avenue of research is the use of activation surface markers, such as CD25, CD62L, CD44 or CD71 that may, theoretically, prove more sensitive predictors of immunotoxicity. However, rare results comparing lymphocyte proliferation and the expression of activation surface markers suggested different sensitivities that deserve careful validation [82]. Finally, flow cytometry can be used for mechanistic purposes, including apoptosis [83]. SERUM IMMUNOGLOBULINS Serum immunoglobulins include IgG, IgM, IgA and IgE. All classes of immunoglobulins can be assayed using ELISA in all conventional animal species [84 – 87]. Another


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technique is radial immunodiffusion, but as total IgE levels are physiologically extremely low, this technique is not applicable. Although this is a simple and relatively inexpensive assay, its predictive value is very limited. In most instances, changes in serum immunoglobulins are not associated with consistent changes in other parameters; mainly functional parameters. Therefore, extremely limited information can be expected from the results obtained.

MEASUREMENT OF HUMORAL IMMUNITY Following the introduction of foreign macromolecules or haptens complexed to carrier proteins, B lymphocytes differentiate and multiply, some of them becoming plasma cells to produce antibodies. Humoral immunity refers to the production of antigen-specific antibodies. In addition to measuring serum immunoglobulin levels, humoral immunity is primarily assessed by specific antibody responses toward a given antigen. The advantage of specific antibody responses as compared to serum immunoglobulin levels is that humoral immunity is explored in conditions that mimic those of a single antigenic stimulation. A limitation to the use of a specific antibody response assay is the claimed requirement for additional animals in toxicity studies as supposed changes caused by antigenic stimulation are not accepted. In fact, this view, although still held by many, is based on purely theoretical grounds and when considering published data there is no indication that this might be true [37,88 – 90]. Typically, humoral immunity can be assessed either by determining antigen-specific antibody-producing spleen cells or by measuring antigen-specific antibody levels in the sera of exposed animals [91]. These two approaches measure humoral immunity from a markedly different perspective, which may result in variable sensitivity. Very few comparative studies have been performed [92 – 96] and the selection of either procedure remains subjective or based on the prior experience of the investigator. The determination of antigen-specific antibody-producing cells, as is done with the plaque-forming cell (PFC) assay, has long been the preferred approach. This is the best validated animal model to predict for the potential of immunosuppression associated with drug treatment and chemical exposure. However, ELISA are more and more frequently used. GENERAL CONSIDERATIONS Exploring specific antibody response requires that a given antigen is injected to the animals included in the study. Administration of the antigen is usually performed either a few days before, or at the end or immediately after the completion of drug treatment or chemical exposure. Due to the half-life of immunoglobulins, the duration of exposure should be at least 21 days and more preferably 28 days in rodents. It is noteworthy that seemingly no study ever showed a different effect after a shorter or longer duration of a given chemical exposure on humoral immunity. Depending on whether the contact with the antigen is a first or subsequent contact, the antibody responses are markedly different. The kinetics of antibody production is

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accelerated in secondary responses as compared to primary responses. The class of antibodies produced is also different: IgM are initially and predominantly produced in primary responses, whereas IgG are the main antibodies found in secondary responses. The magnitude of secondary responses is greater than in primary responses, as is the affinity of antibodies. Nevertheless, only the primary antibody response was routinely explored, at least until very recently. It is important to bear in mind, especially for the purpose of immunotoxicity risk assessment, that secondary antibody responses are generally less sensitive to immunotoxicants and that antibody responses mounted by human beings, except infants and small children, are preferentially secondary responses. Antibody responses require the maturation of B cells in plasma cells to produce antibodies. Most, but not all, antibody responses are under the control of T lymphocytes. Two types of antigens—the T dependent and T independent antigens—can be used to assess humoral immunity. Antibody responses to T dependent antigens are considered more relevant for nonclinical immunotoxicity evaluation as T dependent antigens are by far the most commonly encountered antigens. A large variety of T dependent antigens can be used including SRBC [97,98], tetanus toxoid [99 –102], BSA [103,104], ovalbumin [105,106] and KLH. SRBC have long been used to measure humoral immunity. The Fischer 344 rat was suggested to respond less strongly to antigens on SRBC, which could explain why some authors recommended KLH. That SRBC are not standardized antigens and that not all conventional species can be readily immunized with SRBC are major drawbacks. Ovalbumin can induce IgM and IgG as well as IgE antibody response depending on the experimental protocol used [107]. In addition, ovalbumin as well as SRBC and KLH depending on the dose and route of administration can induce either humoral or cellular immune response. T-independent antigens [108], such as DNP-Ficoll, TNP-Escherichia coli LPS, polyvinylpyrrolidone and flagellin, can sometimes be used in mechanistic studies to identify immunotoxicants which specifically interfere with B lymphocyte function [109]. Typically, antibody levels are measured 5– 10 days after injection of the antigen. Older techniques, such as hemagglutination, complement lysis or antibody precipitation, are no longer used. The reference assay is the PFC assay which, however, tend to be replaced by ELISA and to a lesser extent ELISPOT. THE PFC ASSAY The PFC assay is one of the hallmarks of nonclinical immunotoxicity evaluation (Fig. 4). It measures the production of specific antibodies by antibody-producing cells following a primary or secondary immunization. Direct (IgM) or indirect (IgG) PFCs can be measured [110]. The most widely used antigen is SRBC, but other antigens, such as KLH or tetanus toxoid, can also be used after coupling to SRBC using tannic acid, chromic chloride or carbodiimide. However, considerable variations have been shown depending on the antigen, the experimental protocol and the strain [111], so that only SRBC are used in the direct PFC assay for nonclinical immunotoxicity evaluation purposes. Due to possible variations in the antigenicity of SRBC, it is recommended to use the same commercial source despite possible batch-to-batch variability. SRBC can be stored at þ 4 8C for several weeks.


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Fig. 4. Schematic representation of the direct plaque-forming cell (PFC) assay. Mice or rats are given an intravenous injection of SRBC on day 0. The animals are sacrificed on day 5. The spleen is removed and a suspension of spleen cells is prepared, and then incubated with SRBC and guinea pig complement in Cunningham’s chambers. Typically, after 2–3 h of incubation, the number of yellowish plages each corresponding to an antibody producing cell is counted and the result is given as the number of plaque-forming cells per spleen or 105 –106 splenocytes.

In general, 1 – 5 £ 108 SRBC are injected via the intravenous route to mice or rats, but the intraperitoneal route can also be used. The animals are sacrificed after 4– 5 days (direct PFC assay). For the indirect PFC assay, the sacrifice is on days 7– 10. The spleen is removed and a suspension of splenocytes is incubated in the presence of SRBC and guinea pig complement for 3 h either in agarose according to Jerne’s technique [112], or more often over a slide according to Cunningham’s technique [113]. At the end of the 2 –3 h incubation period, pale areas corresponding to antibody-producing cells can be seen that reflect the lysis of red blood cells around these cells. The number of plaques is counted with the naked eye or at a very small magnification. It is usually given for 105 or 106 splenocytes. IgG response can be measured if an anti-IgG serum is added during the incubation period (indirect PFC assay). The indirect PFC assay is extremely difficult to perform in the rat. The PFC assay has sometimes been performed in guinea pigs [114 –116], hamsters [117], dogs [118], marmosets [119] and pigs [120]. However, the use of the PFC assay in these species is either ethically or scientifically debatable. As already mentioned, the direct PFC assay has long been considered as the main assay to be routinely used for nonclinical immunotoxicity evaluation. In the US NTP interlaboratory validation program, the direct PFC assay was found to be the most sensitive and reliable predictor of immunosuppression [78] and decreases in the PFC assay have been noted despite the lack of histological changes in lymphoid organs [9]. However, the direct PFC has several limitations and drawbacks. As already mentioned, SRBC are not properly standardized antigens and this may result in marked inter- as well as intra-laboratory variability. Cell counting is not automated and results may vary depending on the technical skill and performance of the investigator. The direct PFC assay is time-consuming and must

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be performed shortly after the sacrifice of the animals. Appropriate human resources are therefore required to do a lot of assays in a short period of time. ELISA For the reasons discussed above, enzyme-linked immunoadsorbent assays (ELISA) are increasingly the preferred technique to measure humoral immunity in nonclinical immunotoxicity evaluation. Indeed, a number of samples can be measured by ELISA in a relatively short period of time. The technique is automated and can be properly standardized. Samples can be stored at 4 8C or frozen for subsequent assay. Importantly, ELISA can be used to measure antibody levels on several occasions in the same animal, e.g. before and after termination of exposure. ELISA can measure very low concentrations of specific antibodies and can be designed to detect any class of antibodies. A current limitation, however, is the availability and the quality of reagents. An ELISA is a five-step procedure. The first step involves coating of the microtiter plate wells with antigen. The second step includes the blocking of all unbound sites to prevent false-positive results. The third step is the addition of antibody to the wells. The fourth step is the addition of anti-IgG conjugated to an enzyme. The fifth step involves the reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. The antigen is bound noncovalently to a solid phase, e.g. wells of microtiter plates and after addition of the serum containing specific antibodies, these antibodies bind to the immobilized antigen. Washing is required to remove all remaining nonspecific serum components. The amount of antibody that is specifically bound to the immobilized antigen is measured by adding an enzyme-labeled anti-Ig. Various enzymes, primarily horseradish peroxidase and alkaline phosphatase are used. When the enzyme substrate is added to the wells, the enzymatic reaction is triggered resulting in degradation of the substrate, the extent of which is estimated from the color intensity of the substrate solution measured by a spectrophotometer. In competition ELISA, a soluble antigen is added to a limited amount of antibody. The mixture is placed in wells covered by the antigen. The amount of antibody covered with the antigen during the second step is proportional to the amount of complexes formed during the first step and hence the concentration of serum antibody. Results are variably expressed, e.g. absorbance values, optical density ratios (by comparing optical density of the test sample with reference negative samples), or comparison to a standard curve. Since the discovery of ELISA, many modifications have been introduced. All ELISA are complex systems and a careful selection of the technique, adequate reagents and equipment is crucial to ensure the quality, reproducibility, sensitivity and accuracy of results. In the context of nonclinical immunotoxicity evaluation, ELISA have first been used to measure antibody responses to SRBC [121 – 123]. Recently, other antigens were considered for use of which KLH is currently the most popular antigen [96,124 –126]. Another possible candidate antigen is tetanus toxoid [97,127]. To date, it is too early to make any recommendation on the most appropriate antigen to be selected. Experimental protocols have to be standardized and carefully validated in interlaboratory studies. As immunological responses vary depending on the antigen, differences in sensitivity can be expected and decision on the most appropriate antigen can only be made when adequate data are available.


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ELISPOT The enzyme-linked immunospot assay (ELISPOT) is based on a principle similar to ELISA [128 – 131]. It includes three steps. First, the antigen is bound to a solid phase as in ELISA. Then, antibody-producing cells are added to the wells and allowed to incubate. During the incubation period, the cells can produce antibodies that bind the immobilized antigen. Finally, the cells are washed and an enzyme-labeled anti-Ig is added. Alkaline phosphatase and horseradish peroxidase are the most commonly used enzymes. A substrate is added and where the antibody is bound to the antigen, spots are formed that correspond to antibodyforming cells. Antibodies specific for different heavy chains can be used to measure IgG, IgM, IgA or IgE antibody-forming cells. Results using ELISPOT in the context of nonclinical immunotoxicity evaluation have very rarely been published [132].

MEASUREMENT OF CELLULAR IMMUNITY Cell-mediated immune responses are mediated by T lymphocytes. The main responses involving cellular immunity are delayed-type hypersensitivity, organ transplant rejection, tumor immunity and resistance against infectious pathogens. Assays to measure cellular immunity in nonclinical immunotoxicity evaluation can be divided into in vivo animal models and in vitro assays. IN VIVO ANIMAL MODELS General considerations. Two types of models can be used, either the classical DTH or the contact hypersensitivity model. Despite species differences that impact on the end-points being measured, these models are easy to perform and offer the advantage of measuring cellular immunity in vivo and thus of taking into account the possible influence of other physiological systems, such as the nervous and endocrine systems whose functions may be negatively affected by drugs and other chemicals, which in turn may result in indirect immunotoxicity. In vivo animal models to measure cellular immunity are very close to models used for predicting the potential of drugs and other chemicals to induce cell-mediated hypersensitivity reactions. Here, potent immunogens or contact sensitizers are used to induce a specific cellular immune response that immunosuppressive drugs and chemicals can inhibit. Whatever the selected model, the experimental protocol always comprises three consecutive phases. The first phase is the sensitization or induction phase during which the animals are given one or several administrations of the reference immunogen or contact sensitizer. The second phase is the rest period of variable duration, which is necessary for the immune system to mount the expected cellular immune response. The third phase is the eliciting or challenge phase where the animals are administered the same reference immunogen or contact sensitizer. Overall, there are extremely few, if any objective data to substantiate the recommended variations among experimental protocols regarding the selected immunogen or contact sensitizer [133], the dose and number of injections and the route of administration during

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the sensitization phase, the duration of the rest period, the dose and the route of administration during the induction phase. Although some efforts have been paid to the standardization of DTH as well as contact hypersensitivity models, there are many variations between models and species. In fact, technical skill and acquired experience are essential so that results obtained with potent immunosuppressive compounds are generally similar despite obvious variations in the experimental protocol. It is, however, unclear to what extent variations in the experimental protocols can influence the measured effects of less potent immunosuppressive, i.e. immunodepressive chemicals. DTH models are the most commonly used in vivo models to assess cellular immunity in preclinical immunotoxicity evaluation. Although initially considered as less sensitive than lymphocyte proliferation assays [134], DTH models were later shown to be as sensitive and to generate reproducible results [78]. Experimental protocols Antigen selection. A wide variety of T-dependent antigens can be used. In the context of nonclinical immunotoxicity evaluation, the preferred antigens are SRBC [135 –139], KLH [140 – 142], BSA [143,144], ovalbumin [145,146] and tetanus toxoid [147]. SRBC have long been the most commonly used antigen in rodents. In larger animal species, such as dogs and monkeys, KLH and tetanus toxoid are potent inducers of a DTH response. A DTH-like response can be elicited by the intradermal injection of PHA [148]. There is an induration of the skin at the site of injection and histological changes are those of a DTH reaction. Route and site of antigen administration. In the sensitization phase, the antigen is typically injected by the subcutaneous or intradermal route. In rodents, the antigen is injected into the back, the basis of the tail or the footpad for sensitization. The elicitation is induced in another site, typically the footpad by the subcutaneous or intradermal route. In larger animal species, sensitization is obtained by subcutaneous or intradermal injections in one site, and elicitation in another site. Duration of the rest period is most often in the range of 7 –14 days. Longer durations, e.g. 21 days, have also been used. There is no conclusive evidence whether any duration is optimal. The dose used for elicitation is also variable. Measured end-points. In rodents, the reaction is typically measured from the induced increase in hind-footpad thickness [149 –154]. The measured increase is proportional to the magnitude of the DTH response. Footpad thickness is measured immediately prior to the eliciting injection of the antigen into the hind-footpad, then after 24 and/or 48 h, sometimes later although the reaction normally peaks at 48 h. Various techniques can be used to measure footpad thickness. The use of a dial caliper is a simple and straightforward technique [155,156]. More sophisticated methods, such as 125I iodine diffusion to measure increased capillary permeability and edema due to the DTH reaction [157 – 159], footpad weight [160] or footpad dye accumulation [161] have been proposed, but they are timeconsuming and did not prove their superiority [162]. In higher animal species, such as dogs [163,164] and monkeys [147,165], DTH is assessed after the intradermal administration of the antigen to induce a skin reaction similar to that seen in human skin testing. The magnitude of the reaction is measured semiquantitatively from the size of the erythema and the presence of edema.


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Contact hypersensitivity models. In contact hypersensitivity models, the antigen— typically a hapten—is taken and processed by Langerhans cells to be presented to T CD4þ lymphocytes. These models are primarily used in rodents and guinea pigs, although dogs have, but rarely been used [151]. The protocol is similar to protocols used for contact sensitization assays to test the potential for skin sensitization (see Chapter 7 of this volume). Sensitization is performed topically on the shaved abdomen or inter-scapular area, and elicitation topically on the shaved abdomen (guinea pig, mouse) or the ear (mouse). Rats are seldom used as contact sensitization is difficult to obtain in this species. In guinea pigs, the response is measured semi-quantitatively from the intensity of the erythema and the presence or lack of edema. In mice, ear thickness measurement is the preferred method. Ear thickness is measured with a dial caliper immediately prior to topical elicitation on the ear and again 24 –48 h later. A radioisotopic assay was also proposed [166]. Strong contact skin sensitizers, such as picryl chloride [167], oxazolone [168,169] and DNFB [170,171] in the mouse, and DNCB [172 –174] and picryl chloride in the guinea pig, are used to induce a reaction, the intensity of which can be decreased in animals treated by drugs or exposed to chemicals. Other in vivo assays of cell-mediated immunity. Other in vivo assays have sometimes been used to investigate the influence of drugs and chemicals on cellular immune responses. Because they are time-consuming and require adequate technical skill, skin grafts have exceptionally been used in the context of nonclinical immunotoxicity evaluation. They are obviously more suited to pharmacological investigations [175]. Graft-vs.-host (GvH) disease results from the injection of histo-incompatible immune cells to an immature, immunocompromised or tolerant host: the donor’s cells react with the host’s antigens, whereas the host is unable to mount an immune response against the donor’s cells. As for skin graft models, experimental GvH models are not suitable for nonclinical immunotoxicity evaluation [176]. LYMPHOCYTE PROLIFERATION ASSAYS Mitogen-induced proliferation assay. These assays take advantage of the capacity of cultured lymphocytes to proliferate (Fig. 5). In vitro proliferation is a long recognized property of T lymphocytes [177] and it has been shown to be a good correlate of cellular immunity. At the end of the drug treatment or chemical exposure, animals are killed to collect spleen lymphocytes. It is also possible to collect lymphocytes from peripheral blood, which allows repeated assays in the same animal. Lymphocytes can also be collected from lymph nodes. Lymphocytes are cultured for a varying period of time (often 72 h). The addition of various mitogens induces the proliferation of T lymphocytes. Although PHA has long been the preferred mitogen to induce T lymphocyte proliferation [178], con-A is more often used nowadays. PWM, which stimulates both B and T lymphocytes, is not longer commonly utilized. Four to 24 h prior to the end of the culture, tritiated thymidine is added to the culture medium. When lymphocytes proliferate, they incorporate tritiated thymidine into DNA, so that the amount of incorporated radioactivity in cultured lymphocytes is a correlate of lymphocyte proliferation and hence of cellular immunity. Other methods have been proposed to quantify lymphocyte proliferation. These are essentially

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Fig. 5. Schematic representation of lymphocyte proliferation assay. Lymphocytes from the spleen or peripheral blood are cultured for 72 h in the presence of mitogens, e.g. con-A (a T lymphocyte mitogen) or LPS (a B lymphocyte mitogen). Twenty-four hours prior to the end of the culture, tritiated thymidine is added. Lymphocytes are harvested and the incorporated radioactivity is counted using a liquid scintillation analyzer. The amount of incorporated radioactivity correlates with the magnitude of cellular immune response.

colorimetric methods using MTT [179 – 181] or XTT [182]. BrdU is incorporated into DNA as thymidine [183] and the use of monoclonal antibodies allow measuring lymphocyte proliferation by flow cytometry [184] or ELISA [185]. Comparison of BrdU with thymidine incorporation gave similar results [186]. However, an unsolved issue related to the use of these methods is that proliferation indices are around 10 times less than when thymidine incorporation is used. It remains therefore to be established that smaller proliferation indices are not associated with less sensitivity of the assay for use in nonclinical immunotoxicity evaluation. Allo-antigen-induced proliferation assay. In this assay, the animals are sensitized in vivo by an antigen, such as tuberculin, and lymphocyte proliferation is induced in vitro by the addition of the mycobacterial extract PPD. Another allo-antigen-induced proliferative response of T lymphocytes can be triggered by the use of allogeneic cells. The mixed lymphocyte response assay has been very popular in nonclinical immunotoxicity evaluation [187 – 191]. In this assay, a responder cell suspension derived from the single spleen of exposed animals is prepared and incubated in the presence of stimulator pooled splenocytes from control histo-incompatible animals after inactivation by treatment with mitomycin C. Eighteen hours before termination of the culture tritiated thymidine is added for incorporation into proliferating lymphocytes and the incorporated radioactivity is measured using a liquid scintillation counter. T LYMPHOCYTE CYTOTOXICITY ASSAYS Cytotoxic T lymphocytes (CTL) are T CD8þ lymphocytes that are specifically cytotoxic to target cells. Although CTL can directly destroy their targets, CTL lysis is a major histocompatibility complex (MHC)-restricted process requiring prior sensitization for T lymphocytes to proliferate and differentiate into effector cytotoxic cells via the sequential


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influence of several cytokines. The in vitro induction of CTL from the splenocytes of treated animals is a widely used model to evaluate cellular immunity [192 – 195]. Typically, splenocytes of treated and control animals are co-cultured for five days with target cells inactivated by mitomycin C. The selected target cell line must be histoincompatible for the strain used. Commonly used target cells are P815 mastocytoma cells in mice and Fu-G1 tumor cells in rats. Four hours prior to the end of the culture, the splenocytes are washed and added to fresh target cells labeled with 51chromium. Cytotoxicity is measured from the amount of radioactivity released into the supernatant after a 4 h incubation period. Cell-mediated cytotoxicity can also be measured by flow cytometry with a seemingly similar sensitivity [196,197]. CYTOKINE ASSAYS Cytokines are involved in many innate and specific immune responses including humoral and cellular responses. Cytokine production or activity can be affected by direct toxicity to cytokine-producing cells, inhibition of cytokine production, induction of immunosuppressive factors, alterations in cellular homeostasis, and presumably many other still ill-defined mechanisms. In the past decade, a number of assays have been described to measure cytokines in body fluids or tissues [198,199]. Very low concentrations of cytokines (, 10 pg/ml) are associated with pleiotropic effects on numerous target cells. Identifying their exact role is a complex task. Cytokines can be found under multiple molecular forms, such as monomers/polymers, glycosylated derivatives, precursors, and degradation products with different assay behaviors. In addition, a number of cytokine inhibitors have been described. As the half-life of cytokines is very short (, 10 min in most instances), the conditions of sampling and storage of biological fluids for cytokine measurements are of crucial importance to avoid degradation of cytokines during storage, and cellular production or release of cytokines after sampling. For immunoassays, sterile sampling on EDTA, but not heparin is recommended. Sera must be decanted very rapidly and frozen at 2 80 8C. Cytokines can be measured by bioassays or immunoassays. Bioassays measure cytokine levels in a biological system, usually a cell line, and the levels are calculated from the comparison to a standard dose – response curve obtained with known graded concentrations of the cytokine. Major drawbacks of bioassays are their lack of specificity and sensitivity. They are also time-consuming. Immunoassays, either radioimmunoassays or ELISA, are much more specific and sensitive, but they are based to the binding of antibodies to the cytokine considered as an antigen irrespective of its functional role. The detection limit is usually around 5 pg/ml. Many commercial kits are available, but they have not been properly standardized so that results obtained with one kit may not be reproducible with another kit. Until recently, cytokines were measured in the supernatants of cells stimulated by mitogens, such as Con-A, or antiCD3 antibodies to produce measurable levels of cytokines. A major issue is that subtle changes in cytokine levels cannot be detected. In addition, it is unsure that artificially activated cells do react as in normal conditions. Recent techniques have been developed that allow for measuring cytokine levels in whole blood or intracellular cytokines. Cytokines can also be measured by immunofluorescence

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techniques or by flow cytometry. The newest techniques will be considered in Chapter 16 of this volume. Among critical questions that will have to be addressed carefully to assure the reliability of results in nonclinical immunotoxicity evaluation are which cytokines should be measured and what is measured [200,201]. The pro-inflammatory cytokines IL-1, TNF-a and IL-6, and the main TH1/TH2 cytokines, including IL-2 and IFN-g, and Il-4 and IL-10 respectively are the logical first candidates for assay. The pro-inflammatory cytokines are involved in numerous nonspecific as well as specific immune responses, and changes in the levels of these cytokines may be expected to correlate with immunotoxic effects. In addition, due to the current focus on the TH1/TH2 paradigm, measurement of TH1 and TH2 cytokines is suspected to provide crucial information. Needless to say that extensive effort will have to be paid to identifying which cytokines must be measured in given situations and to conduct validation studies that are absolutely required for the interpretation of assay results.

MEASUREMENT OF INNATE IMMUNITY Nonspecific (innate) defense mechanisms play a major role in the protection of the host against pathogens. A number of parameters, such as serum levels of enzymes and proteins including C-reactive protein, lysozyme, ceruloplasmin, alpha-2-macroglobulin, and a1antitrypsin, have sometimes been measured in an attempt to follow up groups of the population, in particular workers with occupational exposure to known or suspected immunotoxicants [202,203]. To date the predictive value of these parameters is not known and they are not recommended for the nonclinical as well as the clinical immunotoxicity evaluation of drugs and chemicals. In contrast, cells involved in innate immunity, i.e. polymorphonuclear neutrophils, monocytes and macrophages, are of primary interest, at least from an immunotoxicological perspective. In addition, there is much emphasis on NK cell activity in nonclinical immunotoxicity evaluation. PHAGOCYTOSIS The role of phagocytosis is primarily the removal of microorganisms, foreign bodies, but also the elimination of dead or injured cells. Even though alterations in phagocyte functions are an important issue, this aspect of immunotoxicity has so far not received much attention. There are several explanations to this situation. Because phagocytosis is a complex process, a fairly large number of in vivo and in vitro techniques have been proposed to investigate the influence of immunotoxicants, but most assays are only helpful to investigate highly specific aspects of the whole phagocytic process. The clinical consequences of observed changes in selected phagocyte functions are often unclear and are therefore more suited to mechanistic studies than nonclinical immunotoxicity evaluation. As only very few global assays are available, relevant information must be obtained by combining several assays. Finally, the vast majority of available assays have not been properly standardized and validated.


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Clearance assays are in vivo assays based on the measured elimination of a substance by phagocytic cells. One of the earliest assays is the carbon clearance assay in which mice or rats receive an intravenous injection of Indian ink and are blood sampled at different time points to calculate the clearance of colloidal carbon [204,205]. The Listeria monocytogenes clearance assay is currently the standard assay [206 –208]. Rats are injected intravenously with a suspension of L. monocytogenes, and then killed at day 1 or 2 post-injection. Their spleen is removed and serial dilutions are plated to determine the number of colony-forming units. In order to test lung macrophage function, L. monocytogenes can also be inoculated intratracheally. Rats are sacrificed sequentially after the inoculation and the number of viable bacteria is counted in the lungs [209,210]. Ingestion assays. Phagocytic cells engulf microbial pathogens to exert their microbicidal activity. Ingestion assays are based on the counting the number of ingested pathogens per neutrophil or macrophage [211,212]. These assays are time-consuming, cannot be automated and are therefore open to many causes of inter-assay variability due to the technical skill of the investigator and the selected experimental conditions. They are not considered as reliable tools to measure phagocytosis [213]. Metabolic assays. During phagocytosis, phagocytic cells are stimulated. The respiratory burst is a series of metabolic changes which leads to increased oxygen uptake, increased oxidation of glucose uptake via the hexose monophosphate shunt, increased production of hydrogen peroxide, production of the superoxide anion (O2 2 ), generation of photons of light and increased reduction of tetrazolium dyes. Metabolic assays of phagocytosis focus on one of these changes [214]. Superoxide production is measured from cytochrome c reduction by a spectrophotometer [215]. Although this assay is straightforward, it requires technical skill and has been rarely used in nonclinical immunotoxicity evaluation [216]. Chemiluminescence assay. Chemiluminescence is the phenomenon of light emission by stimulated cells. It is the result of the respiratory burst in the early stages of the phagocytosis of microorganisms and other foreign particles [217]. Because the quantity of light emitted as chemiluminescence is extremely low, the use of amplifying agents, such as luminol, is essential. In the presence of reactive substances produced during the respiratory burst, such as hydrogen peroxide and superoxide anion, each molecule of luminol is converted to aminophtalic acid with the release of a photon of light. Photons of light emitted by luminol are easily measured by a chemiluminometer and only nanomole quantities of luminol that are non toxic to cells, are required to amplify the chemiluminescence response to a satisfying level. There are several limitations to the use of chemiluminescence assays. They are time-consuming and require both technical skill and expensive equipment. Although cells known to emit chemiluminescence during the respiratory burst are primarily phagocytic cells, i.e. neutrophils, monocytes and macrophages, other cell types, such as platelets and eosinophils, can generate a significant amount of chemiluminescence. However, chemiluminescence assay on whole blood is possible. Because the intensity of a chemiluminescence response is dependent upon the number of cells, the cell type (neutrophils produce a greater yield of chemiluminescence than macrophages) and the stimulating agent (particulate agents, such as zymosan and latex

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beads, produce a greater stimulation than mitogens), standardization of the experimental conditions is crucial. Interestingly, similar results have been obtained in man, rats, monkeys and dogs [218]. There is a large body of data showing that chemiluminescence assays are useful to evaluate the effects of drugs, in particular antimicrobials on phagocytosis [219 – 221]. Flow cytometry is a rapidly expending technique for the evaluation of phagocytosis [222]. Fluorescein isothiocyanate is widely used to label bacteria yeast and zymosan particles. As already mentioned, discrimination between lymphocytes and polymorphonuclear cells, essentially neutrophils can be achieved by a combination of forward-angle light scatter (size) and side-angle light scatter (granularity). Nonlymphocytes are gated to forward-angle light scatter and analyzed for associated fluorescence related to bacteria, fungi, or beads labeled with a fluorescent dye. The proportion of phagocytozing cells is defined from the percentage of gated cells with target fluorescence. In addition, the oxidative burst can be measured on the conversion of nonfluorescent substrates into fluorescent products. The intensity of the oxidative burst is calculated from the percentage of nonlymphocytes with fluorescence. Flow cytometry is now routinely used to measure phagocytosis in humans. There are differences in the kinetics of phagocytosis response between chemiluminescence and flow cytometry assays [223]. Although flow cytometry measurement of phagocytosis is possible in rats, dogs and monkeys, published results are still rare [224]. CHEMOTAXIS The importance of leukocyte motility and chemotaxis in innate immunity is demonstrated by clinical reports of infectious complications in patients with congenital disorders of phagocyte motility [225], but there are few studies on the adverse effects of immunotoxicants on neutrophil chemotaxis [226 –229]. Various techniques have been proposed [230]. The Boyden’s technique [231] is based on the progression of leukocytes through a polycarbonate or nitrocellulosis filter covered with a chemoattractant substance. After several hours of incubation with the chemoattractant, for instance FMLP, the distance traveled by leukocytes is measured reflecting the magnitude of the chemotactic response. The agarose technique is a simple technique [232]. Leukocyte migration is measured in wells of 2 –3 mm in diameter dug in agarose. Wells contain the leukocyte suspension, the chemoattractant or a control substance, and chemotaxis is measured by comparing the distance traveled by leukocytes toward the well containing the chemoattractant and the well containing the control substance. NK CELL ACTIVITY NK cells are essential in the host’s defense against viral infections and neoplasias. NK cell activity is typically measured by the 51chromium release assay [233 – 235] (Fig. 6). Depending on the species, different target cells are used, for instance YAK-1 murine lymphoma cells in rodents and K562 human myeloma cells in monkeys. Splenocytes or peripheral blood mononuclear cells are incubated for 4 h with labeled target cells at 37 8C and different effector:target cell ratios are used, e.g. 200:1, 100:1, 50:1 or 25:1.


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Fig. 6. Schematic representation of the 51chromium release assay. Target cells are labeled with 51chromium and incubated for 4 h with effector cells from spleen or peripheral blood. Killed target cells release 51chromium in the supernatant. Radioactivity is measured by liquid scintillation counter and the amount of radioactivity is correlated to NK cell activity.

After incubation, the radioactivity of the supernatant is measured using a liquid scintillation counter. NK cell activity is obtained by comparing the radioactivity released by target cells in the presence of effector cells with the maximal radioactivity released by target cells of the presence of HCl or Triton X 100. Spontaneous release is also taken into account. Comparison of the percent lysis at a single effector:target ratio does not provide a quantitative assessment of potency between groups, but it is common practice to use several ratios. Flow cytometry [236] was recently introduced as an alternative to 51chromium release assay in nonclinical immunotoxicity evaluation [237,238]. The assay is based on target cell labeling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNAlabeling with propidium iodide for the identification of dead target cells. The results are expressed as the percentage of dead targets on a cell-to-cell basis. The technique can be used in rats and monkeys, and preliminary results show similar sensitivity of flow cytometry with 51chromium release assay [239,240]. Interestingly, NK cell activity from peripheral blood was seemingly more sensitive to toxic insult than when measured in splenocytes, and this supports other similar findings [77]. The main advantage of this assay is to avoid the use of radioisotopes. In addition, it is possible to combine NK cell activity with NK cell counting in the same animal, and when peripheral blood is used measurement of NK cell activity is possible on several occasions in the same animal. A major issue with NK cell activity, however, is that a limited amount of data is available on drugs and chemicals that actually exert a reproducible influence. For instance, cyclosporine was inconsistently shown to impair NK cell activity in many immunotoxicity validation studies. In humans, impairment of NK cell activity was found either following in vitro exposure [241] or in renal transplant patients [242]. Surprisingly, cyclosporine was also shown to increase NK cell activity in rats [5]. In fact, morphine derivatives and a few environmental chemicals, such as nickel, reproducibly inhibited NK cell activity in rodents. This is in disagreement with the results of the interlaboratory validation study performed

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under the auspices of the U.S. National Toxicology Program showing that NK cell activity is a reliable end-point [78]. At the present time, the validity of the NK cell assay in nonclinical immunotoxicity evaluation is a matter of debate among regulatory agencies. Thus, the EMEA recommends this assay as a possible first-line assay if combined with lymphocyte subset analysis [243], whereas the US FDA considers this assay only as a second-line assay [244]. COMPLEMENT The complement system is an important and complex constituent of innate immunity. Extremely few immunotoxicants have been investigated for adverse effects on activation of the complement system [245,246]. Dioxin [247] and 2,4-diaminotoluene [248] are rare examples of immunotoxicants which have been shown to be inhibit complement activation. Usually, exploration of the complement system when it is included in nonclinical immunotoxicity evaluation is limited to measuring CH50 and sometimes C3 and C4 complement serum levels. It must be emphasized that these end-points are neither sensitive nor comprehensive enough to provide an in-depth analysis of the potential adverse effects of drugs and other chemicals on the complement system. A comprehensive study performed in a laboratory specialized in the complement system is therefore highly recommended when the complement system is suspected to be a target of immunotoxicity.

HOST RESISTANCE MODELS A major issue in preclinical immunotoxicity evaluation is to determine whether the observed changes in the histology of lymphoid organs and/or immune function responses are actually indicative of immunotoxicity. As changes are often mild to moderate, or inconsistent among measured end-points, it is not easy to reach a definitive conclusion in many instances. The immune system utilizes a very complex interplay of nonspecific (innate) and specific (adaptive) defense mechanisms that are often redundant and intricate in order to protect the body against microbial invaders and malignancies. The use of host resistance models is therefore recommended to confirm whether the observed changes parallel to impairment in resistance against either experimental infections or implanted tumors [249,250]. The status of host resistance models varied over the years. In the 1980s, they were considered as pivotal elements for nonclinical immunotoxicity evaluation. After several studies showed correlations between immune function changes and resistance to infection, the use of host resistance models declined and they are underutilized [251]. Although animal welfare issues should be carefully considered in the selection and appropriateness of host resistance models, the utilization of host resistance models is often unavoidable to carefully assess the immunosuppressive potential of drugs and other chemicals. EXPERIMENTAL INFECTION MODELS General considerations. Before selecting an experimental infectious model, several issues must be carefully addressed.


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Pathogenicity of the selected infectious agent must be close to that of pathogens causing diseases in man. Although a number of marginal infectious models have been proposed in the past, it is unclear to what extent, if any, such models are relevant to man. In addition, it is essential for immunotoxicity risk assessment that the defense mechanisms involved in the selected animal species are similar to those in man. Virulence of the infectious agent is another important issue. It is essential to select infectious agents that can be used in experimental conditions ensuring an adequate level of safety and compliance to good laboratory practices. Highly virulent pathogens expose to a significant risk of biohazards and require appropriate safety measures, such as personnel protective devices and containment (class I– III) equipment. Immunological mechanisms involved in the infectious model should match with the immunotoxicity profile of the tested compound. Thus, experimental infections involving humoral mechanisms of defense, such as Streptococcus pneumoniae and Klebsiella pneumoniae [252] infections are not relevant to assess compounds that have been shown to affect primarily cellular immunity. Similarly, compounds known to impair humoral immune responses should not be tested on models involving cellular immunity, such as L. monocytogenes infections. However, as many immunotoxicants impair varied mechanisms and several mechanisms of defense against microbial pathogens are involved, selecting an appropriate infectious model may not be straightforward. A possibility is the use of several models, but the cost-effectiveness of this approach is unclear. Viral infections. Viral infections are particularly interesting because they are a major cause of morbidity and mortality in man. The outcome of the infection depends on the virulence, the susceptibility of the target organ and the host immune competence. A variety of models have been developed and some of these proposed for nonclinical immunotoxicity evaluation [253]. Two models deserve particular attention. Herpes virus infections. Infections due to the herpes virus family include herpes simplex virus (HSV) and cytomegalovirus. In fact, they have been rarely used in nonclinical immunotoxicity evaluation [254,255]. Most murine strains are not susceptible to HSV-1. The pathogenicity of HSV-2 depends on the age of the animal and the route of inoculation. Cellular immunity plays a pivotal role against HSV infection and macrophages are also seemingly essential [256] which could explain the relative susceptibility of very young compared to adult animals. NK cell activity was shown to play a major role in the defense against cytomegalovirus [257]. Influenza virus infections are easily accessible due to the availability of human isolates and the well-characterized mechanisms of pathogenesis. Influenza virus infections can be used in both mice and rats [258,259]. However, nasal administration induces mortality in mice, but not in rats. Quantification of infectious viral titer in the lungs of inoculated animals can also be used. Infections to influenza virus are helpful to assess the global consequences of depressed humoral immunity by drug treatment and chemical exposure. Bacterial infections. Many bacterial infectious models have also been developed, but few are still in use in the context of nonclinical immunotoxicity evaluation. L. monocytogenes infection is a prototypic infectious model [260,261]. The main advantage is the possible use of either rats or mice. Pathogens are cultured and a suspension

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of viable listeria is quantified by turbidimetry and injected intravenously. The majority of injected listeria are removed and destroyed by liver macrophages, whereas surviving listeria multiply in the liver. In normal animals, the infection spontaneously stops within 5 –6 days. This model is particularly interesting to investigate cell-mediated immunity and macrophage functions. The final end-point is either the death of infected animals, or the number of viable pathogens in the spleen and liver of inoculated animals, measured from the clearance of pathogens and/or organ bacterial colony counts. Streptococcal infections are another type of well-characterized infection models [262]. S. pneumoniae and S. zooepidemicus are the two widely species utilized by immunotoxicologists. The complement and humoral immunity have been shown to play a major role in the defense against S. pneumoniae. Fresh inoculates of cultured bacteria are injected intravenously to mice. Mortality induced by inoculates of graded concentrations is noted shortly after injection. Humoral immunity is the major line of defense against S. zooepidemicus and the infection develops more slowly. Other bacterial infections include infections to Escherichia coli, Salmonella typhimurium, Staphylococcus epidermidis, and K. pneumoniae. Most are performed in mice. Parasitic infections. The main advantage of parasitic infections is the more consistent virulence of parasites as compared to bacterial and viral pathogens, and also the possibility to use end-points other than lethality. Plasmodium infections. Infections to Plasmodium berghei and P. yoelii are very similar to the human diseases. Mice and certain strains of rats can be given an intraperitoneal injection of the pathogens. Host resistance depends on specific antibodies. Evaluation criteria include mortality, erythrocyte count and hemoglobin level [263]. Toxoplasma gondii infections are used in mice [264]. The injection of a small ð, 100Þ number of parasites induces a progressively lethal disease. Because virulence is well preserved, results are reproducible and this is an adequate tool to investigate host resistance following medicinal or chemical exposure. Trichinella spiralis infections have been utilized for many years [265,266] and tend to be increasingly used in the context of preclinical immunotoxicity evaluation. The impact of medicinal products, such as ketotifen [267], recombinant IL-3 [268] and salmeterol [269], as well as xenobiotics of various origins including tributyltin oxide [270], zinc [271], diethylstilbestrol [272,273], dioxin [274 – 276], ethanol [277 – 279] and Lactobacillus casei [280] has been investigated using this model. T. spiralis infection can be produced mice and rats [281,282], although hamsters [283,284], guinea pigs [285] and mini-pigs [286] have also been used. Species-dependent differences have been identified. Thus, T. spiralis infected rats proved to be much more susceptible to a single dose of cyclophosphamide than mice [287]. Cellular immunity plays a pivotal role in this response, whereas specific antibodies enhance the elimination of the parasite. The oral administration of larvae as a suspension induces an infection manifesting after one week with worms in the gut of 50– 70% of animals. Animals are killed after 14 days and adult worms are counted. Typically, immunosuppressed animals have a much higher number of encysted larvae than normal animals. Other end-points, such as specific serum IgE levels can be measured as correlate to anti-infectious resistance.


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IMPLANTED TUMOR MODELS The immune system undoubtedly plays a critical role in limiting the onset and development of neoplasias, whatever the mechanisms involved. The exploration of antitumoral resistance has therefore been considered an important step in preclinical immunotoxicity evaluation. Due to their low incidence, spontaneous tumors cannot be used to evaluate antitumoral resistance, and this is true even for spontaneous neoplasias with a greater incidence, such as leukemias in Fischer 344 rats. A number of implanted tumor models have been proposed including fibrosarcoma PYB6, sarcoma 1412, melanoma B16 or B16F10, tumoral ascitis EL-4, Lewis carcinoma in C57Bl/6 mice; tumoural ascitis L1210 or P388 in DBA/2 mice; MKSA (virus SV-40), TKL5, ascitis MOPC-104 in Balb/c mice; and C58NT or mammary adenoma MADB106 in rats. All tumor models include several steps: (i) the injection of tumor cells as a suspension; (ii) a rest period to allow progression of the tumor; (iii) the onset of a palpable tumor; and (iv) death of the animal. The selection of the tumor model depends on the species and strain, as the use of a syngenic tumor is required to avoid rejection of the tumor by specific immune mechanisms. Tumor cells are obtained from solid tumors, cultured neoplastic cell lines, or ascites, and injected either via the intradermal, subcutaneous, intratracheal or intramuscular route, or via the intraperitoneal route to induce ascites. The injection of a small volume, which induces tumors in 10 –30% of the animals, is preferred when an immunosuppressive chemical is investigated. By contrast, a larger volume inducing tumors in 80 – 90% of animals is recommended for potentially immunostimulatory chemicals. Evaluation criteria include the incidence of tumors, or animals with one or more tumors, the delay between inoculation of tumor cells and the identification of palpable tumors, the time of tumor growth, the number of nodules, the death rate, or the delay to death. All these criteria have been more or less adequately standardized and validated. CONCLUSION The panel of immunological end-points, animal models and assays that can be used to predict the potential for a drug or chemical to induce unexpected immunosuppression is extremely large. Some have been correctly standardized and validated, but the majority awaits confirmation of their predictability for nonclinical immunotoxicity evaluation. In addition, because of the extreme variety of animal models and assays, a step by step approach is recommended to afford cost-effective evaluation as will be discussed in Chapter 10 of this volume. The evolution of concepts and the acquisition of data changed the most appropriate approach markedly over the years.

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[255] Selgrade MJ, Daniels MJ, Burleson GR, Lauer LD, Dean JH (1988) Effects of 7,12dimethylbenz[a] anthracene, benzo[a]pyrene and cyclosporin A on murine cytomegalovirus infection: studies of resistance mechanisms. Int J Immunopharmacol, 10, 811– 818. [256] Mogensen SC (1984) Host defences in mice against infections with herpes simplex virus. Microbiol Sci, 1, 127– 130. [257] Selgrade MK, Daniels MJ, Dean JH (1992) Correlation between chemical suppression of natural killer cell activity in mice and susceptibility to cytomegalovirus: rationale for applying murine cytomegalovirus as a host resistance model and for interpreting immunotoxicity testing in terms of risk of disease. J Toxicol Environ Health, 37, 123– 137. [258] Burleson GR, Lebrec H, Yang YG, Ibanes JD, Pennington KN, Birnbaum LS (1996) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on influenza virus host resistance in mice. Fundam Appl Toxicol, 29, 40 –47. [259] Lebrec H, Burleson GR (1994) Influenza virus host resistance models in mice and rats: utilization for immune function assessment and immunotoxicology. Toxicology, 91, 179–188. [260] Bradley SG (1995) Listeria host resistance model. In: Burleson GR, Dean JH, Munson AE (Eds) Methods in Immunotoxicology, volume 2, pp 169– 179. Wiley-Liss, New York. [261] Meade BJ, Hayes BB, Klykken PC (1998) Development and validation of a Listeria monocytogenes host resistance model in female Fisher 344 rats. Toxicol Methods, 8, 45 – 57. [262] Bradley SG (1995) Streptococcus host resistance model. In: Burleson GR, Dean JH, Munson AE (Eds) Methods in Immunotoxicology, volume 2, pp 159– 168. Wiley-Liss, New York. [263] Luebke RW (1995) Assessment of host resistance to infection with rodent malaria. In: Burleson GR, Dean JH, Munson AE (Eds) Methods in Immunotoxicology, volume 2, pp 221– 242. WileyLiss, New York. [264] Descotes J, Brouland JP, Tedone R, Verdier F (1991) Experimental toxoplasmosis in Swiss mice for assessing host resistance. Toxicologist, 11, 207 (abstract). [265] Larsch JE, Kent DE (1949) The effect of alcohol on natural and acquired immunity of mice to infection with Trichinella spiralis. J Parasitol, 35, 45 – 53. [266] Cocker CM (1955) Effects of cortisone on Trichinella spiralis on non immunized mice. J Parasitol, 41, 498– 504. [267] Doligalska M (2000) Immune response to Trichinella spiralis larvae after treatment with the anti-allergic compound ketotifen. Parasitol Res, 86, 232– 238. [268] Korenaga M, Watanabe N, Abe T, Hashiguchi Y (1996) Acceleration of IgE responses by treatment with recombinant interleukin-3 prior to infection with Trichinella spiralis in mice. Immunology, 87, 642– 646. [269] De Waal EJ, De Jong WH, Van der Stappen AJ, Verlaan B, Van der Loveren H (1999) Effects of salmeterol on host resistance to Trichinella spiralis in rats. Int J Immunopharmacol, 21, 523– 529. [270] Vos JG, De Klerk A, Kranjnc EI, Van Loveren H, Rozing J (1990) Immunotoxicity of bis-tri-nbutyltin)-oxide in the rat: effects on thymus-dependent immunity and nonspecific resistance following long-term exposure in young versus aged rats. Toxicol Appl Pharmacol, 105, 144– 155. [271] Fenwicj PK, Aggett PJ, Macdonald D, Hubert C, Wakelin D (1990) Zinc deficiency and zinc repletion: effect on the response of rats to infection with Trichinella infection. Am J Clin Nutr, 52, 166– 172. [272] Fugmann RA, Aranyi C, Barbera PW, Bradof JN, Gibbons RD, Fenters JD (1983) The effect of diethylstilbestrol as measured by host resistance and tumor susceptibility assays in mice. J Toxicol Environ Health, 11, 827– 841. [273] Luebke RW, Luster MI, Dean JH, Hayes HT (1984) Altered host resistance to Trichinella spiralis infection following subchronic exposure to diethylstilbestrol. Int J Immunopharmacol, 6, 609– 617. [274] Luebke RW, Copeland CB, Diliberto JJ, Akubue PI, Andrews DL, Riddle MM, Williams WC, Birnbaum LS (1994) Assessment of host resistance to Trichinella spiralis in mice following preinfection exposure to 2,3,7,8-TCDD. Toxicol Appl Pharmacol, 125, 7 – 16.


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[275] Luebke RW, Copeland CB, Andrews DL (1995) Host resistance to Trichinella spiralis infection in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Fundam Appl Toxicol, 24, 285– 289. [276] Luebke RW, Copeland CB, Andrews DL (1999) Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology, 136, 15 – 26. [277] Steven WM, Kumar SN, Stewart GL, Seelig LL (1990) The effects of ethanol consumption on the expression of immunity to Trichinella spiralis in rats. Alcohol Clin Exp Res, 14, 87 – 91. [278] Seelig LL, Steven WM, Stewart GL (1996) Effects of maternal ethanol consumption on the subsequent development of immunity to Trichinella spiralis in rat neonates. Alcohol Clin Exp Res, 20, 514– 522. [279] Seelig LL, Steven WM, Stewart GL (1999) Second generation effects of maternal ethanol consumption on immunity to Trichinella spiralis in female rats. Alcohol, 34, 520–528. [280] Bautista-Garfias CR, Ixta O, Orduna M, Martinez F, Aguilar B, Cortes A (1999) Enhancement of resistance in mice treated with Lactobacillus casei: effect on Trichinella spiralis infection. Vet Parasitol, 80, 251– 260. [281] Dessein AJ, Parker WL, James SL, David JR (1981) IgE antibody and resistance to infection. I. Selective suppression of the IgE antibody response in rats diminishes the resistance and the eosinophil response to Trichinella spiralis infection. J Exp Med, 153, 423– 436. [282] Van Loveren H, Luebke RW, Vos JG (1995) Assessment of immunotoxicity with the parasitic infection model Trichinella spiralis. In: Burleson GR, Dean JH, Munson AE (Eds) Methods in Immunotoxicology, volume 2, pp 243– 271. Wiley-Liss, New York. [283] Ritterson AL (1968) Effect of immunosuppressive drugs (6-mercaptopurine and methotrexate) on resistance of Chinese hamsters to the tissue phase of Trichinella spiralis. J Infect Dis, 118, 365– 369. [284] Behnke JM, Dehlawi MS, Rose R, Spyropoulos PN, Wakelin D (1994) The response of hamsters to primary and secondary infection with Trichinella spiralis and to vaccination with parasite antigens. J Helminthol, 68, 287–294. [285] Dzik JM, Zielinski Z, Golos B, Jagielska E, Wranicz M, Walajtys-Rode E (2002) Effect of cyclosporin A on immunological response in lungs of guinea pigs infected with Trichinella spiralis. Acta Biochim Pol, 49, 233– 247. [286] Ivanoska D, Cuperlovic K, Lunney JK (1990) Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs. Res Vet Sci, 49, 92– 97. [287] Luebke RW, Copeland CB, Andrews DL, Riddle MM, Smialowicz RJ (1992) Host resistance to Trichinella spiralis in rats and mice: species-dependent effects of cyclophosphamide exposure. Toxicology, 73, 305– 321.


Chapter 6

Nonclinical evaluation of unexpected immunostimulation

INTRODUCTION For the time being, only a very limited amount of data is available regarding the nonclinical evaluation of unexpected immunostimulation because the primary focus of immunotoxicologists was on immunosuppression and active immunostimulatory drugs were only relatively recently introduced into the clinical setting. Very few drugs and other chemicals have been shown to exert significant immunostimulatory properties in animal models and assays, but the clinical relevance of these findings remains to be fully demonstrated in most instances. Among drugs and other chemicals shown to exert immunostimulatory effects, the antiH2 histamine antagonist cimetidine [1], recombinant IL-2 [2], phosphorothioate oligonucleotides [3], vaccine immunoadjuvants [4] and hexachlorobenzene [5,6] are main reference compounds. Due to the very limited available data, it is important to bear in mind that drawing general conclusions from results obtained during nonclinical immunotoxicity studies of unexpected immunostimulatory drugs and other chemicals is not possible or, at least, cannot be based on science-based evidence. Thus, the nonclinical safety evaluation of immunostimulatory drugs as well as that of xenobiotics with unexpected immunostimulatory properties still poses many problems to toxicologists and regulators [7,8].

HISTOPATHOLOGY As is common practice for the detection of unexpectedly immunosuppressive drugs and other chemicals, the histological examination of lymphoid organs should be included in repeated dose toxicity studies in order to detect unexpected immunostimulation. Technical aspects and the interpretation of results pose similar difficulties to those addressed in Chapter 5 of this volume. In addition, limited information is available on which histological changes in lymph organs are more likely to be seen in association with immunostimulation. 205

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Markers of immunostimulation [9 – 12] typically consist of increased spleen weight that may not be associated with any detectable histological changes, and hyperplasia of the bone marrow, red and white pulps of the spleen and lymph nodes with mononuclear cell infiltration and more numerous germinal centers. Mononuclear cell infiltration can be noted in liver, lungs and adrenal glands. Extra-medullary, particularly splenic hematopoiesis has been observed in mice [13]. Thymus histology does not seem to be significantly affected. Hexachlorobenzene increased the number of intraepithelial CD8þ T lymphocytes in the epithelium and lamina propria of the small intestines of Lewis rats [14].

HEMATOLOGY AND CLINICAL CHEMISTRY Mild to moderate hyperleukocytosis has been reported as well as lymphocytosis. Increases in counts of lymphocytes, monocytes, neutrophils, basophils and eosinophils in the peripheral blood have been described in hexachlorobenzene-treated rats [15] as well as rIL2 treated rats [10,11] and dogs [16]. Depending on the mechanism of action, acute-phase response [17] related to immune activation may be reflected in decreased albumin and total protein serum levels. A slight increase in liver weight, liver enzyme serum levels, and bilirubin and fibrinogen levels is a possible finding. Recombinant hematopoeitic growth factors that modulate immune responses increase the proliferation and maturation of bone marrow cell lines, but this is an expected consequence of their biological effects. Increased serum Ig levels have been described [18,19]. Increased B lymphocyte [20] and decreased CD8þ T lymphocyte [21] numbers have been shown after cimetidine treatment. Hexachlorobenzene did not change the numbers of B and T lymphocytes in lymph nodes, but markedly increased the numbers of splenic B and CD4þ T lymphocytes [10,22].

IMMUNE FUNCTION ASSAYS Immune function assays intended to detect unexpected immunosuppressive properties of drugs and other chemicals (see Chapter 5 of this volume) have been, but rarely used to predict unexpected immunostimulation. At least from a theoretical point of view, any assay that measures nonspecific as well as specific immune function can show enhanced responsiveness. A crucial question, however, is whether the same experimental protocol of a given assay can detect immunostimulatory as reliably as immunosuppressive properties. Because they have been primarily designed to detect unexpected immunosuppression, they typically include administration of an optimal dose of an antigen to mount either a humoral or cellular immune response. Similarly, lymphocyte proliferation assays use optimal mitogen concentrations. In either situation, the aim is to mount an immune response strong enough to be readily inhibited by immunosuppressive drugs and chemicals. The question remains whether the use of optimal doses of an antigen or optimal mitogen concentrations is an adequate way to detect unexpected immunostimulation. The use of suboptimal doses of an antigen or suboptimal concentrations of a mitogen resulting in a weaker immune response may be more suitable, but seemingly this issue has not been addressed.


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Only limited data are available to show that current models genuinely designed to detect unexpected immunosuppression can also demonstrate unexpected immunostimulatory effects. Since cimetidine and immunostimulatory oligonucleotides were the most frequently used immunostimulatory compounds in these assays, no general conclusion can, therefore, be made. In addition, the lack of demonstrated or even suspected correlation between animal data demonstrating the immunostimulatory effects of cimetidine and human data should be underscored. Any way, enhanced responses have been shown using immune function assays primarily designed to detect unexpected immunosuppression. Examples of published data include the following. The direct PFC assay is a universally used animal model to measure T-dependent antibody response. Interestingly, cimetidine [23], cinnarizine [24], phosphodiesterase inhibitors [25], iodoxuridine [26] or medroxyprogesterone acetate [27] were shown to enhance specific responses of mice in this assay. Similarly, increased antitetanus toxoid antibody levels were in rats treated with an immunostimulatory oligonucleotide [28]. Enhancement of PFC response to T-independent antigens was also reported [29]. As regards cellular immunity evaluation, augmented delayed-type hypersensitivity response to SRBC [27,30] or L. monocytogenes [31 – 33], and mitogen-stimulated lymphocyte proliferation [34] have been reported. Finally, as far as innate immunity is concerned, both decreased production of superoxide anion by human neutrophils [35] and increased NK cell activity [36] have been observed. Cimetidine has an inhibitory effect on skin graft survival in mice [37]. Increased resistance to a T. spiralis [38] or L. monocytogenes [39] experimental infection by immunostimulatory substances is possible.

CYTOKINE RELEASE ASSAYS Flu-like reactions are adverse effects of immunostimulatory drugs and chemicals, the severity and incidence of which depends on their ability to release various cytokines including IL-1b, IL-6 and TNF-a. As discussed in Chapter 4 of this volume, macrophage activation is a central mechanism in the abrupt release of pro-inflammatory cytokines. In vitro screening systems have been established to predict the cytokine-releasing potency of immunostimulatory drugs. One such system is based on the murine macrophagelike cell line RAW 264 activated with LPS to produce IL-1b, IL-6 and TNF-a [40]. Similar results were obtained with the human monocyte-like histocytic lymphoma U-937 cells [41]. The capacity of the recombinant cytokines and growth factors rhGM-CSF, rhIL-2, rhIL-3, rhIFN-g and rhG-CSF to cause TNF-a release from peripheral blood mononuclear cells in vitro was also shown to correlate with their potential for inducing acute cytokine syndrome [42], thus confirming previous results with monoclonal antibodies [43].

HOST RESISTANCE MODELS Host resistance models are as essential for immunotoxicity risk assessment of unexpectedly immunostimulatory drug and chemicals as they are for unexpected immunosuppressive substances. As regards unexpected immunosuppression, host resistance models, namely

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experimental infection and implanted tumor models are demonstrated correlates of adverse events observed in the clinic including infectious complications and virus-induced malignancies. The clinical experience shows that adverse effects associated with immunostimulation are markedly different from those induced by immunosuppression. For host resistance models to mimic the clinical situation, different host resistance models should, therefore, logically be used. EXPERIMENTAL INFECTION MODELS Experimental infection models have widely been used to investigate the immunosuppressive potential of drugs and other chemicals (see Chapter 5 of this volume). Nevertheless, these models have also been suggested to be helpful for assessing unexpected immunostimulation. In the latter situation, the design of experimental infection models should be different. When the aim is to detect impaired resistance of the host, microbial challenges are used that are likely to kill only a few animals as one of the major end-points measured in experimental infection models is mortality. Thus, increased mortality can be demonstrated following immunosuppressive exposure. When the aim is to detect enhanced resistance, a higher level of microbial challenges expected to kill more animals is recommended so that a decrease in mortality can be supposedly evidenced following immunostimulatory treatment or exposure [44]. Whatever the logic of this approach might be, no published data are available to substantiate it is useful in the context of immunotoxicity risk assessment. Experimental infection models for the immunotoxicity evaluation of immunostimulatory drugs and other chemicals have, however, been proposed that follow the same design as models used for the evaluation of immunosuppressive agents. One example is Candida albicans infection in CBA/J mice [45]. Nonspecific immunity was assessed by C. albicans colony-forming unit (CFU) count from the spleen at 2 h (uptake) and or 22 h (clearance) following intravenous inoculation. Cellular immunity was measured from CFU count on day 6 post-inoculation via the intramuscular route and humoral immunity by anti-Candida antibody titers following multiple subcutaneous injections. In these experimental conditions, the immunosuppressive drugs dexamethasone and cyclosporine decreased host resistance, whereas the immunostimulating drugs liposomal muramyl-tripeptide phosphatidyl-ethanolamine and SK&F 105,685 enhanced host resistance. ANIMAL MODELS OF AUTOIMMUNE DISEASES More frequent autoimmune diseases are typically seen in patients treated with immunostimulatory drugs, such as the recombinant cytokines IL-2 and interferons-a (see Chapter 3 of this volume). Autoimmunity has also been found in animals treated with hexachlorobenzene [46]. It is, therefore, logical to use animal models of autoimmunity to test whether treatment or exposure with a known or suspected immunostimulatory substance may trigger, accelerate or aggravate autoimmunity. A large number of animal models have been developed primarily in an attempt to investigate the pathogenesis of spontaneous autoimmune diseases [47]. These models have also been widely used by pharmacologists to investigate the potential efficacy of new drugs


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intended to treat these diseases [48]. However, none of these models has been adequately standardized and validated from a toxicological point of view in order to be recommended for nonclinical immunotoxicity evaluation at the present time, except as second-line or mechanistic studies. Overall, autoimmune animal models can be divided into two main categories: autoimmunity-prone animal models and experimental autoimmune diseases. Autoimmunity-prone rodent models. Several mouse and rat strains have been shown to develop frequent autoimmune diseases. In most instances, these are genetically prone species, which inherited a genetic predisposition that arose spontaneously. The possibility for modifying the genetic background of rodent species to facilitate autoimmunity is also being extensively pursued. Genetically modified animals, e.g. transgenic or knockout animals, have rarely been used within the context of immunotoxicity evaluation as discussed in Chapter 16 of this volume. In this chapter, only those animal models that result from spontaneous mutations will be described. A major limitation to the utilization of autoimmunity-prone rodent models is the inconsistent effects of drugs and other chemicals among laboratories. In addition, biological and clinical changes suggestive of autoimmunity usually develop after several months of evolution and not all animals develop overt disease. These are obvious major drawbacks to be circumvented for autoimmunity-prone rodent models before they can be accepted as valuable tools during nonclinical immunotoxicity evaluation. In an attempt to both shorten the spontaneous onset and evolution of the disease, and increase the reproducibility of results, pre- or concomitant treatment with known experimental autoimmunogens, such as mercuric chloride has been used, but preliminary results [49,50] await confirmation. Mouse models New Zealand Black (NZB) and (NZB 3 NZW)F1 hybrid mice spontaneously develop an autoimmune syndrome similar to systemic lupus erythematosus (SLE). NZB mice have high serum levels of anti-erythrocyte antibodies and most die of hemolytic anemia. Female (NZB £ NZW)F1 mice develop antiDNA autoantibodies as early as 2 months of age, immune complex glomerulonephritis, proteinuria, and have a shorter life expectancy [51 –53]. Glomerulonephritis is the major cause of death. Immunofluorescence studies can show deposits of IgG and C3 in the mesangium from 5 months of age. Severe cortical thymic atrophy is a consistent finding, whereas spleen and lymph node hyperplasia is of variable intensity. The (NZB £ NZW)F1 mouse model has long been used to screen for pharmaceutical drugs that might lessen the severity or the incidence of autoimmune diseases in treated human beings, but limited data demonstrate an adverse influence of immunostimulatory drugs and other chemicals on the course of the spontaneous disease in (NZB £ NZW)F1 mice. Thus, treatment with 100 mg/kg/day of the interferon-inducer tilorone markedly accelerated the disease in young, but not old (NZB £ NZW)F1 mice [54], and this was to some extent confirmed by repeated administrations of interferon [55 – 57]. Interestingly, exacerbation of the spontaneous disease was shown following cadmium exposure via the drinking water [58] or intranasal exposure to silica dust [59], whereas neither silicone gel [60] nor rIL-2 [61] exerted significant effects. Most importantly, that mercuric chloride [62] and lead [63] induced acceleration of the lupus disease in (NZB £ NZW)F1 mice does not support the predictability of this model for nonclinical

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immunotoxicity evaluation as neither mercuric chloride nor lead have been conclusively shown to facilitate autoimmunity in exposed human beings despite claims that lupus-prone mouse models are reliable tools to evidence environmentally related acceleration of spontaneous autoimmune diseases [64]. MRL-lpr/lpr mice were developed at the Jackson laboratories. Further inbreeding led to separate two different lines, the MRL-lpr/lpr—lpr denoting lymphoproliferation—and MRL/n lacking the lpr gene. MRL-lpr/lpr mice are characterized by massive lymphoproliferation and a high incidence of rheumatoid factor sometimes associated with polyarthritis, SLE autoantibodies, such as anti-Sm autoantibodies that are characteristic of MRL-lpr/lpr mice, and circulating immune complexes [65]. Typically, mice live less than 6 months. Immune glomerulonephritis, degenerative lesions of the coronary arteries and myocardium and massive generalized lymphadenopathy affecting the cervical, axillary and mesenteric lymph nodes as early as 8 weeks of age are typical findings. MRL-lpr/lp mice have rarely been used for nonclinical immunotoxicity evaluation purpose. Mice were exposed to 0, 500, 1000 or 2000 ppm trichloroethylene by inhalation for 4 h a day, 6 days a week, for 8 weeks. Changes in B and T cell functions were seen at the higher dose level [66] and TH1 activation was evidenced [67]. Implantation of silicone induced increases in autoantibody levels, but had not influence on the spontaneous course of the disease [68]. NOD mice. The nonobese diabetic (NOD) mouse is an inbred mouse strain initially developed at the Shianogi Laboratories in Japan [69]. Clinical features in the NOD mice are similar to human type-I insulin-dependent diabetes mellitus (IDDM). Between 90 and 120 days of age, there is an abrupt, but in fact more often progressive onset of hyperglycemia, glycosuria, ketonuria, polydipsia, polyuria and polyphagia [70]. Female mice become permanently hyperglycemic at an earlier age than males (3 – 6 vs. 5 – 8 months). Histological examination shows insulitis consisting of an infiltration of leukocytes, mainly B and T lymphocytes into pancreatic b-islets. The ensuing inflammatory reaction is associated with loss of b-cells. The development of diabetes in NOD mice depends on a variety of contributing factors so that the incidence is generally not more than 80– 100% in females and 30– 50% in males. Clearly identified contributing factors include infections (viral and bacterial infections decrease the incidence of diabetes), diet and housing conditions (crowding and frequent handling increase the incidence of diabetes) [71].The easiest way to detect the onset of diabetes is the measurement of blood glucose levels, but as hyperglycemia results in glucose secretion in the urine, glycosuria is often used to avoid frequent bleeding when monitoring the progression of the disease. Mice normally die within 5 – 7 weeks after blood glucose levels have peaked. There is evidence that T lymphocytes and macrophages, but not antibodies play a critical role in the pathogenesis of the disease. Several self-antigens have been suggested to be involved, but their role is not clearly elucidated. The incidence and course of the disease have been shown to be altered by immunomodulating agents. The interferon inducer poly (I:C) [72], Freund’s complete adjuvant [73,74] and Bacillus Calmette –Gue´rin [75,76], which all induce a TH1 response reduced the incidence or severity of diabetes in treated NOD mice. Results of vaccine administration to NOD mice are conflicting. Thus, Classen [77] found that pertussis vaccine when administered at 8 weeks of age increased the incidence of diabetes, whereas


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multivalent vaccines including pertussis had no effects [78]. Because NOD mice develop spontaneous autoimmune lesions to other targets including the salivary and thyroid glands testis with hemolytic anemia, anti-nuclear autoantibody production and immune complex-mediated glomerulonephritis, the suggestion was made that NOD mice could also be a useful animal model of SLE [79]. Interestingly, a single injection of Bacillus Calmette –Gue´rin prevented diabetes in NOD mice, but precipitated a syndrome similar to SLE [80]. (SWR 3 SJL) F1 mice. SWR and SJL mice do not spontaneously develop autoimmune disease, but the hybrid (SWR £ SJL)F1 mice were found to produce autoantibodies as early as 10 weeks of age with a peak at 40 weeks [81]. At this age, only 10% of males had detectable autoantibodies as compared to 70% of females and approximately 30% of these female mice developed proteinuria due to glomerulonephritis caused by immune complexes deposition. This mouse model has seemingly never been used in nonclinical immunotoxicity evaluation. B 3 SB mice spontaneously develop an autoimmune syndrome with features of SLE that affects males much earlier than females [82]. Mercury was shown to accelerate the spontaneous autoimmune disease in both male and female mice [83]. Rat models. Fewer autoimmunity-prone rat models have been described. As regards autoimmunity and drug or chemical exposure, the Brown– Norway (BN) rat is by far the most commonly used rat strain, but the validity of BN rat models in nonclinical immunotoxicity evaluation is still far from being established. BB—for Bio-Breeding—rats are a classical model of autoimmune insulin-dependent diabetes mellitus. Overall, 60 –80% of male and female BB rats develop diabetes between 60 and 120 years of age. Low doses of the interferon inducer poly (I:C) was shown to exert protective [84] or facilitating [85,86] effects. Brown – Norway (BN) rats have long been used as an experimental model of autoimmunity [87,88]. The administration of mercuric chloride induces a strong polyclonal activation of B lymphocytes resulting in a massive and rapid increase in IgE levels probably due to a dysregulation in various cytokines, especially interferon-g [89]. The increase in antibody levels to both self and nonself antigens results in the formation of immune complexes, leading to glomerulonephritis [90]. However, the validity of the BN rat as a predictive model for autoimmunity can be questioned since no conclusive case report of autoimmunity in humans exposed to mercury has been published suggesting that autoimmunity induced by mercuric chloride in the BN rat is both speciesand strain-specific. In addition, other chemicals known or suspected to induce autoimmunity in humans failed to induce an autoimmune response in the BN rat. Thus, negative results have been obtained with trichloroethylene and silicone gel following 6 weeks of exposure, whereas mercuric chloride and D -penicillamine induced an increase in serum IgE levels, antibody titers to dinitrophenol-HSA and double-stranded DNA autoantibodies [88]. Similarly, BN, but neither Lewis nor Sprague– Dawley rats treated with 20 –50 mg daily of oral D -penicillamine developed systemic toxicity associated with circulating antinuclear antibodies and immune complexes after 3 –4 weeks of treatment. The administration of 20 mg of oral captopril induced no toxicity or immunological changes [91].

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Animal models of experimental autoimmune diseases. A variety of pathological disorders more or less close to autoimmune disorders can be induced in animals. These models have been extensively used for research of the involved pathophysiological mechanisms and candidate drugs that could alleviate the clinical signs and symptoms of the corresponding human disease. Although, the use of experimental autoimmune disease models is as attractive as the use of autoimmunity-prone animal models for nonclinical immunotoxicity evaluation, they have been very rarely used in this context. Experimental arthritis models have long being used in pharmacological research and in the development of drugs to treat rheumatoid arthritis. They are relatively easy to perform and share a number of features similar to those seen in human diseases [92]. Of these models, adjuvant arthritis induced by subcutaneous injections of Freund’s complete adjuvant and collagen type II arthritis in rats or mice are the most commonly used models. Wide variations in the frequency and severity of lesions are observed depending on the strain used. Various mycobacterial preparations can be used, but the epitope involved in the reaction has been identified. The progress and severity of the disease is evaluated by physical examination including footpad swelling and deformity of the joints, histological examination of the joints which can show early subtle changes, and imaging techniques from standard radiological techniques to magnetic resonance imaging. Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system used as a model of multiple sclerosis [93]. EAE is induced by immunization of mice or rats with myelin proteins. The clinical manifestations are loss of tail tonus and forelimb paralysis that can be easily quantified. The interferoninducer tilorone was shown to suppress EAE [94]. Experimental autoimmune uveoretinitis (EAU) can be induced in rats by immunization with retinal photoreceptor-specific antigens [95]. This is a predominantly T cell dependent inflammatory disease of the eye. Injection of the S-antigen into the footpad of female Lewis and PVG rats induces dilation of the iris, increased vascular permeability measured by fluorescein angiography, and histological alterations of the uvea. Recombinant IL-4 which preferentially promotes a TH2 response exacerbated EAU in Lewis rats, whereas an anti-IL-4 antibody attenuated the increased severity of the disease [96]. Dihydrotestosterone decreased, whereas b-estriol slightly increased the progression of the disease [97]. This model is primarily used in pharmacological research to investigate immunomodulatory drugs [98 –101] and has seemingly never been applied to nonclinical immunotoxicity evaluation. Interestingly, Methimazole, an antithyroid drug, known to inhibit the development of autoimmune diseases in animals, was shown to inhibit induction of the disease and the associated antigen-specific lymphocyte proliferation and cytokine production in the draining lymph nodes [102]. CONCLUSION Immunostimulation has long been considered only as a useless or adverse effect of drugs and other chemicals. Indeed, therapeutic benefits from immunostimulation could not be evidenced until recently and misleadingly, immunostimulation was thought to lead to hypersensitivity or autoimmune reactions. Recent progress has shown that immunostimu-


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lation holds therapeutic promises. Therefore, drugs with intended immunostimulatory properties are likely to be increasingly considered for use, and the resulting advances in the nonclinical evaluation of intended immunostimulation, the nonclinical evaluation of unexpected immunostimulation is likely to improve in the next few years.

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Chapter 7

Nonclinical evaluation of the potential for hypersensitivity

INTRODUCTION Hypersensitivity reactions caused by drugs and other chemicals are relatively frequent even though their actual incidence is not precisely known (see Chapter 3 of this volume). In any case, these adverse events lead to significant morbidity and even mortality. In addition to their major health consequences, they may also have negative impact on the industry since a number of compounds including drugs and other chemicals had to be withdrawn from the market or their use strictly limited. Although hypersensitivity reactions are undoubtedly the most frequent immunotoxic effects, limited efforts have been paid to their prediction by nonclinical studies until recently. In fact, they have long been considered unpredictable despite the routine use of contact sensitization animal models since the mid-1940s [1]. Even though predicting the potential of a new molecular entity for inducing hypersensitivity reactions is still difficult, if not at all impossible depending on the chemical and the type of reaction being considered [2 – 6], the a priori unpredictability of animal models is no longer accepted [7]. At least, three characteristics can explain the current difficulties of the nonclinical prediction of hypersensitivity reactions. Firstly, underlying mechanisms are not elucidated in many instances and they are probably often intricate. The lack of a minimal understanding of the mechanism involved, however, is a prerequisite for the design of relevant predictive animal models. The second major characteristic of hypersensitivity reactions is the role of metabolites. Patients exposed to a given drug or a chemical often develop hypersensitivity to a metabolite which is usually highly reactive and thus shortlived, instead of the parent molecule to which they are exposed. Despite significant progress in the identification of causative metabolites and involved metabolic pathways, there are still few instances where hypersensitivity reactions observed in man can be reproduced in animal models. The third and presumably most important characteristic of hypersensitivity reactions from the perspective of nonclinical immunotoxicity evaluation is their low and actually very low incidence which is typically less and often far less than 1% of treated or exposed persons. This reflects a complex interplay of contributing or predisposing factors, 219

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such as the immunogenicity of causative molecular entity, the route and magnitude of the exposure, the variable biotransformation and disposition of chemicals related to individual pharmacogenetic traits, and the intrinsic immunological susceptibility. In the setting of immunotoxicity evaluation and more broadly of nonclinical toxicity evaluation, the use of optimized assays is required to allow for a reliable prediction in a cost-effective way using a number of animals as limited as possible. Optimization is only conceivable when the underlying mechanism is sufficiently well elucidated and not excessively complex. So far, optimization has only proved possible with contact sensitization assays. Optimized procedures are well suited to hazard identification and it remains to be ascertained whether they are reliably applicable to risk assessment. As hypersensitivity reactions involve either immune-mediated or nonimmune-mediated mechanisms, predictive animal models and assays will be described according to this widely accepted distinction.

PREDICTION OF IMMUNE-MEDIATED HYPERSENSITIVITY Immune-mediated hypersensitivity reactions are caused by antigen-specific mechanisms involving either antibodies or T lymphocytes (see Chapter 4 of this volume). As the mechanisms of anaphylaxis and delayed-type contact hypersensitivity are relatively well elucidated, most available animal models and assays deal with these two aspects of immune-mediated hypersensitivity reactions. As regards other mechanisms, attempts have been or are being made, but they are often based on elusive concepts or empirical approaches, and therefore rarely proved useful for nonclinical immunotoxicity evaluation. PREDICTION OF IgE-MEDIATED HYPERSENSITIVITY REACTIONS Anaphylaxis is the term recommended to describe IgE-mediated reactions that typically develop within minutes or a few hours depending on the dose and route of administration following reintroduction of the allergen to which the immune system has been sensitized by prior contact(s) [8]. The induction of specific IgE is therefore a critical step whatever the experimental model used. Because small-molecular-weight substances are not directly immunogenic due their limited size, they play the role of haptens by binding to carrier macromolecules to be sensitizing and induce anaphylactic reactions. The chemical reactivity of the test substance is therefore a critical aspect. Although many industrial, household and environmental chemicals are endowed with sufficient chemical reactivity to act as haptens, nearly all pharmaceutical products have very limited, if no chemical reactivity at all. A metabolic step is thus necessary to generate reactive metabolites, which can play the role of haptens. Up to now, no standardized and validated modalities are available to generate reproducibly such reactive metabolites in laboratory animals despite many attempts. The frequent marked difference in chemical reactivity between drugs and other chemicals should always been borne in mind when attempting to apply to the prediction of drug allergenicity, animal models which have been only validated with other chemicals.


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Systemic anaphylaxis models. There is quite a long experience with the use of systemic anaphylaxis models in guinea pigs to predict the potential of drugs and to a much lesser extent of other chemicals for inducing systemic anaphylaxis [9,10]. These models have also often been used for the control of batch-to-batch consistency. Mice and rats are less commonly utilized. Genetically mast-deficient WBB6F1 mice have been proposed [11], but most of the available data relate to fundamental research so that the validity of this model for nonclinical immunotoxicity evaluation is not known. Differences in susceptibility to sensitization have been shown between guinea pig strains. Dunkin – Hartley guinea pigs produce the highest IgE titers [12], whereas strain two guinea pigs are relatively insensitive to the adverse effects of released mediators during anaphylactic reactions [13]. Female Dunkin – Hartley guinea pigs are more susceptible than males [14]. Although macromolecules, e.g. foreign proteins can be directly immunogenic and induce a specific IgE or IgG1 response, most small-molecular-weight chemicals require coupling to a carrier to play the role of hapten and induce IgE-mediated reactions. The site and degree of conjugation play a critical role [15] and to date, no widely accepted coupling method is available to assure optimal immunogenicity. Depending on the system used for chemical coupling, antibodies can be raised against the carrier and not against the hapten, or IgG and IgM may be raised instead of the expected IgE. Several approaches, however, proved successful, but it is not known to what extent they can be used routinely. For example, specific IgE antibodies against various b-lactam derivatives have been raised in guinea pigs [16,17] and mice [18] following immunization with Ascaris conjugates mixed with aluminum hydroxide or Bordetella pertussis. Polylysine conjugates have also been used successfully [19]. The use of mixtures of the test article with serum albumin is likely to give positive results quite inconsistently. Albumin, either bovine or human serum albumin, is frequently used as a control due to its well-established allergenicity. In order to enhance sensitization, adjuvants are often injected with the test article, either as a mixture or at different sites. It is important to bear in mind that complete Freund’s adjuvant (CFA) preferentially induces a TH1 response and aluminum hydroxide a TH2 response [20 –22]. The use of CFA is therefore illogical to induce IgE responses [23]. Typically, systemic anaphylaxis models comprise three phases. During the first phase—the elicitation or sensitization phase—guinea pigs are given several injections of the test article alone or conjugated to a carrier via the subcutaneous or intradermal route. The intramuscular route is seldom used. There is so far no rationale for selecting an optimal number of injections. However, two injections at a one-week interval are sufficient to induce reproducible sensitization to ovalbumin. Similarly, there is no rationale for selecting the optimally sensitizing dose and dose selection is often empirical. To avoid false negative results, the use of several doses may be preferable [24]. The duration of the second phase—the rest phase—is variable, usually in the range of 7 –21 days. However, it does not seem necessary to prolong the rest period beyond 7 days. The third phase is the eliciting or challenge phase. Guinea pigs are given an intravenous dose of the test article and sensitized animals develop an acute reaction of variable intensity. An intradermal injection is sometimes used to mimic the clinical situation. Because the lung is the primary target of anaphylaxis in guinea pigs, respiratory disorders are prominent. They typically include nasal discharge, increased respiratory rate, difficulties in breathing and cyanosis. In severe reactions, markedly decreased respiratory rate and convulsions may be seen resulting in death of the animal within

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10 –30 minutes. In mild to moderate reactions, all clinical signs normally resolve within 30 minutes. The magnitude and delay of onset of clinical manifestations can be graded. Although systemic anaphylaxis guinea pig models have been routinely used for over 30 years in the pharmaceutical industry, few efforts have been paid to the standardization and validation of these models. It was, however, possible to show reproducible results with ovalbumin in an intra-laboratory validation study [25] and there was a good correlation between results in guinea pigs and the clinical experience using a panel of therapeutic macromolecules including microbial extracts used as immunostimulatory drugs and vaccines [26,27]. However, a major limitation is that macromolecules of human origin or humanized molecules, e.g. rIL-2, induce false-positive reactions in this model. These findings may explain why the use of these models is not recommended by the ICH guideline for the pre-clinical safety evaluation of biotechnology-derived pharmaceuticals [28]. There are numerous data published mainly by Japanese authors [29 – 33] suggesting the predictive value of systemic anaphylaxis guinea pig models. It is noteworthy, however, that negative results have been obtained with nonconjugated low-molecular-weight compounds [9], so that the lack of antigenicity demonstrated for low-molecular-weight drugs in this assay is of debatable relevance, with the possible exception of b-lactam antibiotics. Passive cutaneous anaphylaxis. (PCA) is a model developed in the early 1950s [34 –36]. Guinea pigs or mice are sensitized as described for systemic anaphylaxis models. After several supposedly sensitizing injections, blood samples of the animals are withdrawn and the sera injected intradermally to naı¨ve animals, e.g. guinea pigs, mice or rats. Rats are not used for the sensitization phase as they usually do not mount sufficient antibody response. Thereafter, the animals are injected intravenously with the test article mixed in a dye, such as blue Evans, and when specific antibodies are present in the dermis, the antigen – antibody reaction induces activation of mast cells and the release of vasoacting mediators. The local increase in capillary permeability results in extravasation of the dye in the tissues surrounding the injection site so that a blue spot is formed. The intensity of the reaction is measured from the size of the spot. In the reversed PCA model, the test article is injected intradermally and the sera of sensitized animals is injected intravenously [37], but the sensitivity is 100 –200-fold less than the direct PCA model [38]. Overall, data from the literature tend to show similar results when using systemic anaphylaxis or PCA models, but no studies seemingly attempted to compare the relative sensitivity of both models to predict the allergenicity of drugs and other chemicals. Models of food allergy. Food allergies are relatively frequent even though the mechanism involved in a given individual with a history of food allergy is often not elucidated. Either IgE- or non-IgE-mediated mechanisms may be involved [39]. Uncertainties regarding the underlying mechanism lead to consider oral provocation tests as useful diagnosis tools in humans [40] in contrast to hypersensitivity reactions caused by drugs and other chemicals. Anyway, IgE-mediated food allergies do exist [41] and concern related to food allergies was recently fueled by the introduction of genetically modified foods [42]. A number of animal models, including mouse, rat and dog models are under development for the prediction of food protein allergenicity [43]. A more detailed description of available models can be found in Chapter 6 of Volume III.


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So far, intraperitoneal administrations of food proteins to Balb/c mice could identify the allergenic potential of proteins when comparing ovalbumin to food proteins known to induce food allergies, such as peanut proteins, or not, such as potato extracts [44,45], but their usefulness for nonclinical immunotoxicity evaluation is not established. In fact, they may be more suitable for the assessment of the intrinsic allergenic potential of food proteins. Models of food allergy in rats have been developed in BN rats as BN rats proved much more sensitive than outbred rat strains [46,47]. Daily gavage with several food proteins including cow’s milk, hen’s egg white and peanut proteins, without the use of adjuvants, resulted in significant antigen-specific IgE responses. Interestingly, local and systemic immunemediated effects, such as increased gastro-intestinal permeability, decreased breathing frequency and blood pressure, could be observed in sensitized animals after oral challenge. Validation of this promising model with weak food allergens and nonallergens is awaited to confirm its validity for nonclinical immunotoxicity evaluation. There are also recent data suggesting that the dog [48], in particular the atopic dog [49] is a suitable species for the prediction of food allergens. In general, dogs are sensitized by repeated subcutaneous injections of the allergen and challenged orally. However, available data in the dog are still very limited and further studies are needed to determine its validity as a predictive model. Models of respiratory allergy. Animal models are extensively used to detect respiratory allergens [50], but in contrast to systemic anaphylaxis where the guinea pig is often the selected species, models of respiratory allergy have been developed in mice [51,52], guinea pigs [53,54] as well as rats [55]. A number of experimental protocols have been proposed, but none have so far been fully standardized and validated. Sensitization is produced by subcutaneous administration [56], inhalation [57,58], intranasal [59,60] or intratracheal instillation [61,62] daily for several consecutive days or once weekly for several weeks. Depending on the species or the model, the reaction can be assessed from the severity of respiratory disorders, essentially changes in respiratory rate, after a challenge administration by the respiratory route [63], but more often from the induction of specific IgE or IgG1 antibodies. Serum IgE are increasingly used indicators of respiratory allergenicity as discussed below. However, the route of administration and the dose may be critical factors for the induction of sensitization [64,65]. If macromolecules, such as proteins can be directly immunogenic, low-molecular-weight chemicals must bind to larger carrier molecules. Serum albumin is often used as a carrier owing to the presence of nucleophilic side-chains to react with electrophilic haptens. Although bovine or human serum albumin are sometimes used, it is more logical to use serum albumin from the species being tested, e.g. guinea pig serum albumin [66]. There is so far extremely few, if any data regarding the use of these models with small-molecular-weight pharmaceutical drugs. Serum specific IgE responses. Although guinea pigs produce more specific IgG1 than IgE when sensitized to an allergen, the detection of specific IgE is an assay used for the evaluation of the allergenicity of drugs [67,68]. Sensitization is usually induced using a protocol similar to systemic anaphylaxis or PCA models. Interestingly, ELISA can detect lower levels of specific IgE than PCA. Specific IgE can similarly been produced in mice [18]. It is noteworthy that most published results were obtained with penicillins and

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cephalosporins conjugated to various proteins, but it is unsure that such results can readily be extrapolated to other classes of drugs that have less chemical reactivity. Although specific IgE can be produced in mice sensitized by the respiratory route, an interesting finding was that elevated total serum IgE levels can also be observed following intraperitoneal injections of proteins inducing respiratory allergy, but not BSA [70]. Subsequently, topical applications of the respiratory allergen trimellitic anhydride (TMA) were shown to produce increased serum IgE levels in contrast to the contact sensitizer dinitrochlorobenzene (DNCB). These findings served as a basis to propose the mouse IgE test for the identification of respiratory allergens [71,72]. An inter-laboratory study involving five laboratories found reproducible and confirmative results with TMA and DNCB, and in addition showed that Balb/c mice are more sensitive than C56Bl/6 mice [73]. In BN rats, TMA similarly induced elevated serum IgE levels although the increase was notably less after inhalation than dermal exposure [74]. However, toluene diisocyanate, hexamethylene diisocyanate and hexahydrophthalic anhydride induced no increased in total serum IgE levels in BN rats when compared to TMA, but IL-4 and IL-10 mRNA expression was markedly increased in mice topically exposed to these respiratory allergens [75]. These results led to suggest that the mouse IgE test may be not sensitive enough to identify respiratory allergens, but the conclusion that cytokine profiling is a more suitable model awaits confirmation with a more extensive panel of chemicals and inter-laboratory validation. PREDICTION OF T LYMPHOCYTE-MEDIATED HYPERSENSIVITY REACTIONS There is a growing awareness that T lymphocyte-mediated mechanisms play a major role in a variety of hypersensitivity reactions. The most frequent T lymphocyte-mediated hypersensivity reaction that is reliably predictable using animal models is allergic contact dermatitis. Limited attempts have been made to predict other types of T lymphocyte mediated reactions. Contact sensitization Introduction. The use of patch test to assess contact allergy in man was introduced by Jadasshon in 1895 [76]. Following the pioneer work of Landsteiner on chemical sensitization [77], the guinea pig was selected as the reference animal species to design test methods for the prediction of contact sensitization. In 1944, Draize et al. [78] proposed the first experimental protocol, which was progressively refined. Many guinea pig methods were developed thereafter and alternative mouse models began to be proposed after 40 years of guinea pig utilization. Even though mouse models, in particular the local lymph node assay (LLNA), are increasingly popular, guinea pig models still offer the advantages of a very long experience and extensive validation enabling correlations between animal and human results. Guinea pig contact sensitization tests. At least seven guinea pig tests have been accepted by regulatory authorities at some time point [79], but other experimental procedures or modifications of main procedures have been proposed [80]. Although these methods were primarily intended for labeling chemicals, no general agreement has ever been reached regarding the conduct of these tests neither their relative sensitivity [81,82]. Different guinea pig strains have been used, but results were reportedly similar [83]. No sex


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differences were noted either [84]. Overall, the technical skill and in-house historical data seem to be more important than the selected experimental procedure. However, adjuvant-free methods are generally considered less sensitive [85 –87], whereas adjuvant methods have sometimes been claimed to be too sensitive or to induce false-positive responses [88]. A major and largely unsolved issue is the detection of weak human contact sensitizers [89]. As for inducing any delayed-type hypersensitivity (DTH) reaction, the experimental protocol includes an induction phase during which guinea pigs receive epidermal or intradermal applications of the test article. The use of a mildly to moderately irritating concentration is necessary to assure optimal sensitization. When the test article is absolutely nonirritating, a slightly irritating solvent/vehicle can be used. After a rest period of 10– 21 days, sensitization is assessed after one topical challenge (eliciting) application of a nonirritating concentration of the test article. That the concentration used for elicitation is nonirritating is absolutely essential, but may not be possible in all instances. The determination of the mildly irritating and nonirritating concentrations of the test article is performed in a preliminary study prior to the main study. The evaluation of the epidermal challenge is typically made by visual scoring of the erythema and edema usually including four grades, namely 0 ¼ no visible change; 1 ¼ discrete or patchy erythema; 2 ¼ moderate and confluent erythema; 3 ¼ intense erythema and edema. Histological examination of the skin may prove helpful when ambiguous changes are present, especially when the eliciting concentration retains a certain level of primary irritancy. In general, positive reference contact sensitizers are not included in the experimental protocol, but it is recommended to perform the selected assay every 6 months with 2 positive controls to assure the consistency of results. The selection of positive controls has been a matter of continuing debate over the years, and no consensus emerged among expert groups and regulatory bodies. The Draize test was primarily intended to detect potent skin sensitizers and was the first test included in a guideline [78]. The induction and elicitation are performed by the intradermal route using the same concentration. To improve the sensitivity of the test, various modifications have been proposed including a 2.5-fold greater concentration for sensitization (induction) than challenge (elicitation), and simultaneous intradermal and epicutaneous administrations on different sites for both induction and challenge [90]. Because negative results have been obtained with known human contact sensitizers, other methods have been developed. Buehler test. The occlusive patch test, subsequently named the Buehler test, was originally proposed in 1965 [91]. Typically, the experimental protocol involves 20 tested animals and 10 or more control animals, and consists of an occluded topical patch technique for the induction and elicitation of contact sensitization. Induction patches are applied to the same shaved site on the dorsal surface of the animals for 6 h and one patch is applied once a week for 3 –9 consecutive hours. The induction site is approximately 8 cm2 on the animal’s left shoulder clipped 24 h before, and a 4 cm2occlusive patch is maintained for 6 h. A modestly irritating concentration of the test article or the anticipated (end-use) concentration for human exposure is used. After a rest period of 10 –14 days, the animals receive a 6-h challenge patch with a nonirritating concentration of the test article applied to a naı¨ve clipped skin site. Reactions are graded for erythema 24 and 48 h after patch removal using a 0– 3 scale. A test chemical is

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considered positive if at least 15% of animals have a positive response. Although the Buehler test was criticized because several human sensitizers were negative, validation studies showed a relatively good correlation between results in the Buehler test and clinical data [92,93]. One of the main advantages of the Buehler test is the possibility to test the final concentration or the whole formulation that will be used commercially. Maximization test. The guinea pig maximization test (GPMT) initially proposed by Magnusson and Kligman [94,95] has long been the most widely used technique in Europe [96]. It is considered as the most sensitive, but to such an extent that the original classification system of results did not include nonsensitizers (Fig. 1). The induction consists of two phases with paired intradermal injections of 0.1 ml into the clipped and shaved shoulder of CFA, the test article in a suitable vehicle and a mixture of the test article dissolved or suspended in CFA. On day 7, the induction is boosted by an occlusive patch applied for 48 h on the shoulder clipped 24 h before. A mildly irritating concentration of the test article is recommended for induction. The suitable concentration is determined in

Fig. 1. The Magnusson and Kligman (maximization) test. The induction consists of paired intradermal injections of 0.1 ml of the test article, CFA, and a 1:1 mixture of the test article in CFA into the clipped and shaved shoulder region. On day 7, induction is boosted by the topical application of a mildly irritating concentration of the test article on the shoulder of the animals using a 48-h occlusive patch. On day 21, challenge is performed by topical application of a nonirritating concentration of the test article using a 24-h occlusive patch. The reaction is read 24 and 48 h after removal of the occlusive patch and scored according to a 0–3 grade scale.


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a preliminary study. With nonirritating test articles, pretreatment of the clipped shoulder area by 10% serum lauryl sulfate is recommended on day 6. Challenge is performed on day 21. A 4 cm2 skin site on the left flank of the abdomen is shaved and a nonirritating concentration of the test article in suitable vehicle is applied using a 24-h occlusive patch. The challenge site is observed 24 and 48 h after removal of the occlusive patch. The GPMT is a very sensitive test for the contact allergenicity testing of topical drugs and other chemicals as shown by comparisons with other guinea pig tests described in this section [85, 97,98]. Initially, the ranking of contact sensitizers was based on a 5-grade scale (Table 1). Other guinea pig tests CFA test. The CFA test uses 10 –20 tested animals and 10 controls. The induction is performed by the intradermal injection of a 1:1 mixture of CFA and the test article at a concentration not exceeding 5% into the suprascapular region on days 0, 4 and 8. The challenge consists of epicutaneous applications of several concentrations of the test article on 2 cm2 areas of the clipped flank skin. This is an easy test to perform and results are similar to the Magnusson and Kligman test [99]. Optimization test. The experimental protocol consists of 10 intracutaneous applications of a 0.1% solution or suspension of the test article every other day [100,101]. During the second and third weeks, the test article is incorporated into CFA at the same concentration. The animals are challenged intradermally 14 days after the last application, then epicutaneously after a further rest period of 10 days. Most of the allergens producing allergic contact dermatitis in 1% of exposed humans can be detected with this test and there is a good correlation with the Magnusson and Kligman test [102]. Split adjuvant test. This test was designed to allow for epidermal exposure of formulations to be used as a natural route [103]. On day 0, the induction site is exposed to dry ice for 5 s prior to the application of the test article on a 2 cm2 area of previously shaved skin in the suprascapular region. The area is kept under occlusion for 48 h until the next application. A total of four applications is used. Two intradermal injections of 0.1 ml of CFA are administered immediately before the fourth application on each side of the application site. On day 21, the challenge consists of an open or occlusive application of the test article and the reaction is read after 24 –72 h. This test has not received wide acceptance and the available database is relatively limited. Open epicutaneous test. This test consists of the epicutaneous application of the test article on the intact skin of the flank [99]. Daily applications for 5 – 7 days are used for the induction, then 3:1 progressive dilutions up to a total of 20 applications in general. On days 21 and 29, the animals are challenged on the contralateral clipped side of the flank Table 1. Contact allergenicity ranking according to Ref. [94] Sensitization rate (%)

Grade

Classification

0–8 9–28 29–64 65–80 81–100

I II III IV V

Weak Mild Moderate Strong Extreme

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with the test article or a nonirritating concentration. The reactions are read at 24, 48 and 72 h. A major advantage of this test is that there are few limitations due to the physicochemical properties of the test article. In addition, assessment of dose responses is possible. Epicutaneous maximization test. This test was developed to allow for the assessment of the allergenicity potential of end-use products [104]. A preliminary study is conducted to determine the maximal nonirritating concentration of the test article by a single topical application using a 48-h occlusive patch. The induction consists of an intradermal injection of 0.1 ml of CFA together with a topical application of the test article using a 48-h occlusive patch on the shaved suprascapular region. On days 2, 4, 7, 9, 11 and 14, the test article is applied topically using a 48-h occlusive patch. On day 28, the challenge consists of the topical application of a nonirritating concentration of the test article on a naı¨ve skin site using a 48-h occlusive patch. The reaction is read at 24, 48 and 72 h. Mouse contact sensitization assays. In contrast to guinea pig assays, which have been routinely used for many years, mouse assays have been developed rather recently. In fact, mice were considered unable to mount DTH response until Crowle [105 – 107] showed that DTH as well as contact hypersensitivity can be elicited in the mouse. Nearly 10 more years were needed for ear swelling to be introduced as an endpoint to measure contact hypersensivity [108]. Initially, ear thickness was measured with a micrometer [109], but nowadays dial calipers are preferred due to improved accuracy of the technique. The mean variation in measured ear thickness of normal mice is less than 5% [110,111]. Quantitative techniques using 125I-iododeoxyuridine incorporation in the ear [112] or wet ear weight measurement [113], in addition to requiring technical skill or expensive equipment, either did not prove more sensitive [114 –116] or even less [117], and thus never gained wide acceptance. Mouse ear swelling test (MEST). Within a few years, three groups proposed slightly different experimental approaches to identify contact sensitizers in mice based on ear swelling measurement. Early findings. The first set of data showed that mice pre-fed with vitamin A developed a significant ear swelling after topical application of 0.03% oxazolone, a prototypic contact sensitizer in mice which is normally not sensitizing at so low concentrations [118]. These early findings were subsequently confirmed using a panel of 12 fragrances [119]. Interestingly, the enhancing effects of vitamin A on the contact sensitizing potential of chemicals were demonstrated in the MEST [111] and LLNA [120]. In the mouse ear sensitization assay [121], mice received a topical application of the test article on both sides of the right ear on days 1, 4, 6, 8, 11, 13 and 15. In addition, they were injected subcutaneously with 0.05 ml of CFA in the scapular area on day 1. They were challenged by a topical application on both sides of the left ear on day 27. Left ear thickness was measured with a dial caliper immediately prior to the challenge application, then 24 and 48 h later. Mean left ear thicknesses prior to and following challenge were compared statistically. The results obtained with 18 known sensitizers and 10 non-sensitizers showed that the ability of this assay to detect strong as well as moderate contact sensitizers. No further studies were published to confirm the suitability of this protocol as a predictor of contact sensitization.


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MEST. The MEST was developed in an attempt to offer a standardized and validated procedure to investigate the contact sensitizing potential of chemical substances in mice as an alternative to guinea pig assays [122]. The maximal concentrations inducing minimal irritation on the abdomen and no irritation on the ear are determined during a preliminary study. Initially, the MEST design incorporated two intradermal injections of 0.05 ml of CFA into the abdomen immediately prior to the first topical application of the test article. Mice are topically dosed with 100 ml of a minimally irritating concentration of the test article or with the vehicle at the center of a previously shaved and tape-stripped abdominal area for four consecutive days. Seven days after the last topical application, 20 –40 ml of the test article are applied to the left ear and 20 –40 ml of the vehicle to the right ear. Mice are lightly anesthetized at 24 and 48 h post-challenge and the thickness of both ears is measured with a micrometer. This protocol was validated with 72 substances. The concentrations used are selected to avoid irritation. Among 49 known positive compounds, only one, vanillin, a mild sensitizer in both guinea pigs and man, was found negative. All 23 nonsensitizers were negative. When comparing several strains of mice, CF-1 and Balb/c mice proved superior in response, but no clear differences between males and females were seen. Mice less than 5 weeks of age or more than 13 weeks of age gave weaker responses. The initial MEST protocol was subsequently refined [123]. As indicated above, mice are given a diet supplemented with 250 IU/g vitamin A acetate starting 21 days before and maintained throughout the experiment had a near doubling of ear swelling compared to nonpretreated controls [111]. Topical applications on the back were also found to be more suitable as detection of weak contact sensitizers, such as eugenol and paraphenylenediamine was possible without CFA following topical application on the back for three consecutive days [124]. Despite probably premature expectations that MEST could serve as an alternative to guinea pig assays [125,126], results from various laboratories showed that MEST was not reliable to detect weak to moderate sensitizers [127], even when 125I-iododeoxyuridine incorporation was used [128], and that it is a predictor of contact sensitizers not as good as the GPMT [129]. Despite conflicting results of comparisons between the MEST and the LLNA, since the MEST was found to be either a more [130] or less [131] or equally [132] sensitive predictor of contact sensitization, the LLNA was rapidly adopted by a number of laboratories and today the MEST is rarely, if ever used. Local lymph node assay (LLNA). The LLNA is nowadays widely accepted by regulatory authorities for the assessment of the contact sensitizing potential of chemicals [133 –135], but also drugs [136]. The validation of toxicity tests is a key issue and the process which ended in the regulatory acceptability of the LLNA can serve as a good example [137]. Pioneering work on the LLNA as a potential assay for the identification of contact sensitizers was published in 1986 by Kimber et al. [138]. The originality of the concept was to focus on changes induced in the draining lymph nodes instead of the skin response elicited in animals previously sensitized as in guinea pig contact sensitization assays. The stimulation of lymph node cells turned out to be the most sensitive marker of skin sensitization [139] and initially proliferative responses were measured ex vivo in cultured lymph node cells. The next step was the use of in situ measurement of proliferative responses following the intravenous injection of 3H-methyl-thymidine [140]. The experimental protocol was evaluated, improved and validated following a number

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of laboratory [141] and inter-laboratory [142 – 147] studies. Results with known contact sensitizers and nonsensitizers were also compared using the LLNA and widely used guinea pig tests [148 – 150] and finally compared to human data [151,152]. The basic principle underlying the LLNA is that contact sensitizers can induce the proliferation of lymphocytes in the draining lymph node, i.e. the auricular lymph node, after topical applications on the ear (Fig. 2). Lymphocyte proliferation is measured as a function of 3H-thymidine incorporation into the DNA of dividing lymphocytes. Proliferation is generally proportional to the dose applied. Typically, young adult female CBA/Ca or CBA/J mice are used. Other strains, such as Balb/c, DBA/2 and B6C3F1 mice, in contrast to C57BL/6 and SJL/J mice gave similar results [153]. Males can also be used, but much less data is available in males than females. Solid test articles should be dissolved in appropriate solvents or vehicles, and diluted if necessary [154]. The recommended solvents/vehicles are acetone/olive oil [4:1], N,Ndimethyl-formamide, methyl-ethyl-ketone, propylene glycol and dimethylsulfoxide. Negative, i.e. solvent/vehicle, and positive controls should be used. Hexyl cinnamic aldehyde and mercaptobenzothiazole are usually the preferred positive controls. The maximum concentration tested should the highest achievable concentration that induces neither overt systemic toxicity nor excessive local irritation. On days 1, 2 and 3, each mouse is applied 25 ml/ear of the appropriate dilution of the test article to the dorsum of both ears. On day 6, mice are injected with 250 ml of sterile PBS containing 20 mCi of 3 H-methyl-thymidine or 2 mCi of 125I-iododeoxyuridine and 1025 M fluorodeoxyuridine via the tail vein. Five hours later, the auricular lymph node of each ear is excised and pooled in PBS for each animal. A single cell suspension of lymph node cells is prepared for each animal. A stimulation index . 3 is considered a positive response.

Fig. 2. Standard PLNA protocol. Mice receive a topical application on the ear on days 0, 1 and 3. On day 5, they are administered 20 mCi of 3H-methyl-thymidine intravenously and the auricular lymph node of each ear is excised 5 h post-injection. Radioactivity of a single lymph node cell suspension per animal is measured using a liquid scintillation counter and a stimulation index . 3 is considered as a positive response.


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Various modifications of the original protocol have been proposed. A 3-week pretreatment with vitamin A was expectedly shown to enhance responses to several weak contact sensitizers, but this addition to the original protocol is rarely if ever used [121]. To avoid the use of radioisotopes, attempts were made to measure proliferation by immunohistochemistry [155], IL-2 production [156] or BrdU incorporation [157,158]. As primary irritants can induce false-positive response, a two-step strategy was proposed including a primary irritancy test prior to the LLNA [159]. Interestingly, the percentage of B220þ B lymphocytes in lymph nodes measured by flow cytometry was increased by contact sensitizers, but not by primary irritants [160]. In order to improve the sensitivity of the LLNA, the value of other endpoints has been tested including IL-6 [161], IL-12 and IFN-g [162] production, but neither endpoint proves more sensitive. Finally and in keeping with findings already mentioned, a different cytokine secretion pattern was observed in the LLNA with contact sensitizers and respiratory allergens. Thus, the contact sensitizers DNCB and formaldehyde induced the secretion of the TH1 cytokine IFN-g, whereas the respiratory allergens TMA and diphenylmethane diisocyanate induced the secretion of TH2 cytokines IL-4 and IL-10 [163]. These results obtained with potent allergens, however, await confirmation with a panel of weak to strong allergens. Contact sensitization models in other animal species. Rare published results suggest that the hamster is an acceptable species for the evaluation of contact sensitizers [164], including the LLNA [165]. Neither the rat nor the dog has been used to predict for the potential of allergic contact dermatitis induced by chemicals. The use of the LLNA in marmosets [166] is questionable both from an ethical and cost-effectiveness perspective. Prediction of photosensitivity reactions. In contrast to phototoxicity, photoallergy is a rare complication of drugs and other chemicals. Mice were photosensitized to intraperitoneal administration of sulfanilamide or chlorpromazine followed by UVB and UVA irradiation of shaved flank skin on two consecutive days. All animals were challenged on day 5 with an intraperitoneal injection of the drug followed by UVA irradiation of one ear. Mice that had been previously immunized developed ear swelling and erythema 24 h after challenge [167]. In another experimental protocol, cyclophosphamide-pretreated Balb/c mice received topical applications on the dorsal skin surface of the ear on 3 consecutive days and challenged on the ears 5 days after the last induction. For each induction and challenge treatment, mice were irradiated with UVA and UVB radiation 30 min –1 h after each topical application. Nine human photoallergens were successfully detected using this model [168]. Guinea pig models can also be used [169,170]. The experimental protocol generally consists of several topical applications of the test article using occlusive patch or not, intradermal injection of CFA or not and consistently associated with UVA alone or UVA þ UVB irradiation, then challenge with one or two topical applications followed by irradiation after a rest period of variable duration. Despite variations among experimental protocols, strong human photoallergens are successfully detected, whereas drugs associated with a very low incidence of photoallergic reactions are not. Other predictive animal models of T lymphocyte mediated hypersensitivity reactions. Because T lymphocytes are increasingly suspected to be involved in a number of

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immune-mediated hypersensitivity reactions, it is tempting to use available DTH or contact sensitization models to predict the T lymphocyte mediated allergenicity of drugs and other chemicals. Thus, a subcutaneous injection of benzyl-penicillin conjugated to human serum albumin (HSA) and CFA sensitized mice as evidence by footpad swelling 24 h after a challenge injection of the conjugate into the footpad [171]. Similar findings were obtained with unconjugated cephalothin after several biweekly subcutaneous injections [172]. More recently, footpad swelling induced by sulfamethoxazole mixed with S9 mix was obtained in mice previously sensitized by subcutaneous injections in the back, showing the role of metabolites in the induction of sulfamethoxazole DTH [173]. As guinea pig contact sensitization assays have long been used, there is a huge database on drugs and other chemicals that are commercially or environmentally available. It is therefore possible to compare the results obtained in these assays with the clinical experience in order to determine whether contact sensitization assays could also serve as predictors of T lymphocyte mediated reactions other than allergic contact dermatitis. The published GPMT results on seventy drugs and other chemicals were compared to clinical reports. A close correlation was found with 43 substances and a relatively good one with 16 substances. Conflicting results were found with 11 substances only [174]. Similarly, Hattori et al. [68] concluded that the GPMT is the best predictors of systemic DTH induced by 8 b-lactam derivatives. Interestingly, the sensitizing potential of 6 pharmaceutical drugs was tested in the GPMT and there was a correlation between guinea pig results and their respective incidence of systemic hypersensitivity reactions in humans. In addition, the inhibition of the migration of peritoneal macrophages from sensitized guinea pigs following in vitro incubation paralleled the GPMT results and even showed a better sensitivity [175]. However, a recent study showed that among 83 drugs reported to the US FDA for inducing systemic hypersensitivity reactions in humans, only 14 had a positive guinea pig test [176]. Therefore, whether guinea pig tests can predict the potential of drugs for inducing systemic hypersensitivity is at best an open question. The popliteal lymph node assay (PLNA) was initially developed as a tool to predict systemic autoimmune reactions induced by drugs and other chemicals. Nowadays, it is also tentatively used as a potential tool to predict the immunogenicity of small molecules as discussed in Chapter 8 of this volume.

PSEUDOALLERGY Because the definition of pseudoallergy is not consistent among authors and the role of pseudoallergic mechanisms in immune-mediated adverse effects often overlooked or not elucidated in many instances, possibilities for predicting the potential of new chemical entities to induce pseudoallergic reactions are limited in animal models. In fact, in vitro assays using human cells may be more valuable and the experimental protocols are similar to those used for the diagnosis of clinical reactions. Thus, histamine release [177], basophil degranulation [178] or complement activation [179] can be tested in vitro with human cells or peripheral blood using increasing concentrations of the test article. Histamine release by nonimmunological mechanisms is associated with clinical signs and symptoms mimicking IgE-mediated anaphylactic reactions. Administration to dogs


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that are particularly sensitive to nonspecific degranulation of mast cells and basophils can be a useful tool to evidence the histamine-releasing properties of a test substance. The clinical observation of animals together with the measurement of plasma histamine levels in conditions of administration mimicking the clinical conditions of use proved useful in several instances [180 – 184]. Activation of peripheral blood leukocytes by compound 48/80 has been evidenced by chemiluminescence measurement from blood samples in rats and dogs [185]. The increase in chemiluminescence three times greater in dogs than in rats was considered as suggestive of an anaphylactoid reaction. Activation of dog and rat mast cells can be measured by flow cytometry [186] following in vivo administration or from the histamine released into the supernatant following incubation with the test article [187 – 191]. Although these assays have been primarily used to test for antiallergic properties of drug candidates, there is sufficient evidence that activation of either dog or rat mast cells following in vivo or in vitro exposure is a reliable predictor of drug-induced pseudoallergic reactions.

CONCLUSION In contrast to the high incidence of hypersensitivity reactions in man, few animal models and assays are available to predict the potential for inducing hypersensitivity in nonclinical immunotoxicity evaluation. This situation is obvious as far as low-molecular-weight chemicals are considered with the exception of contact sensitizers or highly reactive respiratory allergens. The prediction of the immunogenicity of therapeutic proteins is also extremely difficult as immunogenicity in man cannot be straightforwardly predicted in conventional animal species [192]. In rare instances, the repeated weekly administration of therapeutic proteins in CFA proved weakly immunogenic in nonhuman primates and these results correlated with the subsequent clinical experience.

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[124] Garrigue JL, Nicolas JF, Fraginals R, Benezra C, Bour H, Schmitt D (1994) Optimization of the mouse ear swelling test for in vivo and in vitro studies of weak contact sensitizers. Contact Dermatitis, 30, 231– 237. [125] Thorne PS, Hillebrand JA, Lewis GR, Karol MH (1987) Contact sensitivity by diisocyanates: potencies and cross-reactivities. Toxicol Appl Pharmacol, 87, 155– 165. [126] Thorne PS, Hawk C, Kaliszewski SD, Guiney PD (1991) The noninvasive mouse ear swelling assay. II. Testing the contact sensitizing potency of fragrances. Fundam Appl Toxicol, 17, 807– 820. [127] Dunn BJ, Rusch GM, Siglin JC, Blaszcak DL (1990) Variability of a mouse ear swelling test (MEST) in predicting weak and moderate contact sensitization. Fundam Appl Toxicol, 15, 242– 248. [128] Cornacoff JB, House RV, Dean JH (1988) Comparison of a radioisotopic incorporation method and the mouse ear swelling test (MEST) for contact sensitivity to weak sensitizers. Fundam Appl Toxicol, 10, 40 – 44. [129] Viluksela M, Haasio K, Mannisto PT (1990) Studies on the contact sensitizing activity of dithranol (anthralin) and 10-butyryl dithranol (butantrone). Contact Dermatitis, 23, 103– 110. [130] Hayes BB, Gerber PC, Griffey SS, Meade BJ (1998) Contact hypersensitivity to dicyclohexylcarbodiimide and diisopropylcarbodiimide in female B6C3F1 mice. Drug Chem Toxicol, 21, 195– 206. [131] Hayes BB, Meade BJ (1999) Contact sensitivity to selected acrylate compounds in B6C3F1 mice: relative potency, cross reactivity, and comparison of test methods. Drug Chem Toxicol, 22, 491– 506. [132] Sailstad DM, Tepper JS, Doerfler DL, Qasim M, Selgrade MK (1994) Evaluation of an azo and two anthraquinone dyes for allergic potential. Fundam Appl Toxicol, 23, 569– 577. [133] ICCVAM (2001) Murine local lymph node assay (LLNA). Document available at http:// iccvam. niehs.nih.gov/methods/llnadocs/LLNAProt.pdf. [134] OECD (2002) Skin sensitization: local lymph node assay. OECD Guideline 429. Document available at http://www.oecd.org. [135] US Environmental Protection Agency (2003) Skin sensitization. Document available at http:// www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/ 870-2600.pdf. [136] FDA (2002) Guidance for industry: immunotoxicology evaluation of investigational new drugs. Document available at http://www.fda.gov/cder/guidance/4945fnl.pdf. [137] Kimber I, Dearman RJ, Basketter DA, Ryan CA, Gerberick GF (2002) The local lymph node assay: past, present and future. Contact Dermatitis, 47, 315– 328. [138] Kimber I, Mitchell JA, Griffin AC (1986) Development of a murine local lymph node assay for the determination of sensitization potential. Food Chem Toxicol, 24, 585– 586. [139] Kimber I, Dearman RJ (1991) Investigation of lymph node cell proliferation as a possible immunological correlate of contact sensitizing potential. Food Chem Toxicol, 29, 125–129. [140] Kimber I, Hilton J, Weisenberger C (1989) The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis, 21, 215– 220. [141] Kimber I, Weisenberger C (1989) A murine local lymph node assay for the identification of contact allergens. Assay development and results of an initial validation study. Arch Toxicol, 63, 274– 282. [142] Kimber I, Hilton J, Botham PA, Basketter DA, Scholes EW, Miller K, Robbins MC, Harrison PT, Gray TJ, Waite SJ (1991) The murine local lymph node assay: results of an inter-laboratory trial. Toxicol Lett, 55, 203– 213. [143] Basketter DA, Scholes EW, Kimber I, et al. (1991) Interlaboratory evaluation of the local lymph node assay with 25 chemicals and comparison with guinea pig test data. Toxicol Meth, 1, 30 – 43.

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[144] Scholes EW, Basketter DA, Sarll AE, Kimber I, Evans CD, Miller K, Robbins MC, Harrison PT, Waite SJ (1992) The local lymph node assay: results of a final inter-laboratory validation under field conditions. J Appl Toxicol, 12, 217– 222. [145] Kimber I, Hilton J, Dearman RJ, Gerberick GF, Ryan CA, Basketter DA, Scholes EW, Ladics GS, Loveless SE, House RV, et al. (1995) An international evaluation of the murine local lymph node assay and comparison of modified procedures. Toxicology, 103, 63 – 73. [146] Loveless SE, Ladics GS, Gerberick GF, Ryan CA, Basketter DA, Scholes EW, House RV, Hilton J, Dearman RJ, Kimber I (1996) Further evaluation of the local lymph node assay in the final phase of an international collaborative trial. Toxicology, 108, 141– 152. [147] Kimber I, Hilton J, Dearman RJ, Gerberick GF, Ryan CA, Basketter DA, Lea L, House RV, Ladics GS, Loveless SE, Hastings KL (1998) Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation. J Toxicol Environ Health A, 53, 563– 579. [148] Kimber I, Hilton J, Botham PA (1990) Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs. J Appl Toxicol, 10, 173– 180. [149] Basketter DA, Scholes EW (1992) Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens. Food Chem Toxicol, 30, 65–69. [150] Basketter DA, Selbie E, Scholes EW, Lees D, Kimber I, Botham PA (1993) Results with OECD recommended positive control sensitizers in the maximization. Buehler and local lymph node assays. Food Chem Toxicol, 31, 63 – 67. [151] Basketter DA, Scholes EW, Kimber I (1994) The performance of the local lymph node assay with chemicals identified as contact allergens in the human maximization test. Food Chem Toxicol, 32, 543– 547. [152] Ryan CA, Gerberick GF, Cruse LW, Basketter DA, Lea L, Blaikie L, Dearman RJ, Warbrick EV, Kimber I (2000) Activity of human contact allergens in the murine local lymph node assay. Contact Dermatitis, 43, 95 –102. [153] Woolhiser MR, Munson AE, Meade BJ (2000) Comparison of mouse strains using the local lymph node assay. Toxicology, 146, 221–227. [154] Basketter DA, Gerberick GF, Kimber I (2001) Skin sensitisation, vehicle effects and the local lymph node assay. Food Chem Toxicol, 39, 621– 627. [155] Boussiquet-Leroux C, Durand-Cavagna G, Herlin K, Holder D (1995) Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay. J Appl Toxicol, 15, 465–475. [156] Hatao M, Hariya T, Katsumura Y, Kato S (1995) A modification of the local lymph node assay for contact allergenicity screening: measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay. Toxicology, 98, 15 – 22. [157] Takeyoshi M, Yamasaki K, Yakabe Y, Takatsuki M, Kimber I (2001) Development of nonradio isotopic endpoint of murine local lymph node assay based on 5-bromo-20 -deoxyuridine (BrdU) incorporation. Toxicol Lett, 119, 203– 208. [158] Suda A, Yamashita M, Tabei M, Taguchi K, Vohr HW, Tsutsui N, Suzuki R, Kikuchi K, Sakaguchi K, Mochizuki K, Nakamura K (2002) Local lymph node assay with nonradioisotope alternative endpoints. J Toxicol Sci, 27, 205– 218. [159] Ulrich P, Streich J, Suter W (2001) Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals. Arch Toxicol, 74, 733– 744. [160] Gerberick GF, Cruse LW, Ryan CA, Hulette BC, Chaney JG, Skinner RA, Dearman RJ, Kimber I (2002) Use of a B cell marker (B220) to discriminate between allergens and irritants in the local lymph node assay. Toxicol Sci, 68, 420– 428. [161] Dearman RJ, Scholes EW, Ramdin LS, Basketter DA, Kimber I (1994) The local lymph node assay: an interlaboratory evaluation of interleukin 6 (IL-6) production by draining lymph node cells. J Appl Toxicol, 14, 287– 291.


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[184] Furuhata K, Hayakawa H, Soumi K, Arai H, Watanabe Y, Narita H (1998) Histamine-releasing properties of T-3762, a novel fluoroquinolone antimicrobial agent in intravenous use. I. Effects of doses and infusion rate on blood pressure, heart rate and plasma histamine concentration. Biol Pharm Bull, 21, 456– 460. [185] Wilhelms O-H, Lipponer L, Seilnacht W (1990) Chemilumiscence (CL) in blood samples reflects leukocyte activation (LA) during anaphylactoid reactions in dogs. J Biolumin Chemilumin, 5, 197– 202. [186] Nakagawa T, Stadler BM, De Weck AL (1980) Determination of rat mast cells by flowcytometry. J Immunol Methods, 33, 87 – 92. [187] Ennis M, Lorenz W, Kapp B, Luben L, Schmal A (1985) Comparison of the histaminereleasing activity of cremophor E1 and some of its derivatives in two experimental models: the in vivo anaesthetized dog and in vitro rat peritoneal mast cells. Agents Actions, 16, 265– 268. [188] Williams PD, Laska DA, Shetler TJ et al. (1991) Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1). Agents Actions, 32, 217– 223. [189] Decorti G, Bartoli Klugmann F, Candussio L, Baldini L (1996) Effect of paclitaxel and Cremophor EL on mast cell histamine secretion and their interaction with adriamycin. Anticancer Res, 16, 317– 320. [190] Sugimoto Y, Iba Y, Utsugi K, Kamei C (2000) Influences of everninomicin, vancomycin and teicoplanin on chemical mediator release from rat peritoneal mast cells. Jpn J Pharmacol, 83, 300– 305. [191] Mori K, Maru C, Takasuna K (2000) Characterization of histamine release induced by fluoroquinolone antibacterial agents in-vivo and in-vitro. J Pharm Pharmacol, 52, 577– 584. [192] Wierda D, Smith HW, Zwickl CM (2001) Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology, 158, 71 – 74.

Chapter 8

Nonclinical evaluation of the potential for autoimmunity

INTRODUCTION Major difficulties for the nonclinical evaluation of the potential of xenobiotics to induce autoimmune reactions are the near total lack of understanding of the underlying mechanisms and the widely accepted role of individual predisposition factors as well viral and environmental causes. Interestingly, recent findings evidenced genetic elements in mouse and rat models of autoimmune diseases common to a number of human diseases [1]. To date, predicting the risk for autoimmune reactions induced by new drugs and other chemicals during nonclinical immunotoxicity evaluation is clearly an impossible task [2]. Research efforts are needed to gain a better understanding of the mechanisms involved and the role of contributing factors in order to design relevant and predictive animal models and assays. As described in Chapter 3 of this volume, autoimmune reactions as well as autoimmune diseases can be divided into systemic and organ-specific reactions.

SYSTEMIC AUTOIMMUNE REACTIONS Typically, systemic autoimmune reactions are characterized by clinical signs and symptoms that more or less mimic systemic autoimmune diseases, and also by heterogeneous auto-antibody responses, including antinuclear, anti-histone, anti-DNA and other autoantibodies. ANIMAL MODELS OF SYSTEMIC AUTOIMMUNE REACTIONS Early studies showed the possibility to induce the production of various autoantibodies in mice, rats or dogs treated with drugs and other chemicals including procainamide, isoniazid, hydralazine, penicillamine, gold salts or mercury [3 –8]. For example, Ten Veen and Feltkamp [9] treated Balb/c, C57Bl/6 and A/JAX mice daily via the drinking water for 243

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up to 8 months with hydralazine, isoniazid, procainamide, a-methyldopa, chlorthalidone or saline. Although, the five tested drugs have been reported to induce more or less frequent autoimmune reactions in humans, a slight increase in antinuclear antibodies was only evidenced in mice treated with hydralazine and procainamide. However, these early findings were obtained in experimental conditions that did not mimic the conditions of human treatment and exposure, or failed to be reproduced by others. In fact, mercuric chloride is the only chemical that consistently and reproducibly induced systemic autoimmune reactions in few strains of mice and rats [10,11]. It is, however, important to bear in mind that human exposure to mercury is not associated with autoimmunity [12 –15]. Because autoantibodies are the hallmark of autoimmunity in humans with autoimmune diseases, it is logical to search for autoantibodies in the sera of animals in conventional toxicity testing [16]. Extremely few studies have been conducted and as indicated above they gave either negative or conflicting results so that the search for autoantibodies in conventional toxicity studies cannot be recommended. Recently, histological findings suggestive of an autoimmune process have been described [17]. The histological hallmark of autoimmunity in laboratory animals is inflammation. Although, mononuclear cell infiltration is commonly seen in autoimmune diseases, it is also observed in other pathological conditions and is not a reliable histological indicator of autoimmunity. However, a predominantly CD4þ T lymphocyte infiltrate may be a better indicator. Autoimmune diseases are frequently associated with thymic hyperplasia, but thymus atrophy is also possible as in NZB £ NZW F1 mice. Follicular hyperplasia in the spleen and lymph nodes may be an early indicator of autoimmunity. Because genetic predisposition is thought to play a key role in autoimmunity, the use of autoimmunity-prone animal species is often considered for the nonclinical prediction of the potential of drugs and other chemicals to induce autoimmune reactions. The BN rat has been widely used for fundamental research on the mechanisms of autoimmunity, especially following treatment with mercuric chloride [18]. Recently, the usefulness of this model to predict the potential of drugs and other chemicals was suggested [19], but confirmatory results are absolutely needed before any conclusion can be drawn. Similarly, the usefulness of autoimmunity-prone mouse strains, such as NZB £ NZW and BXSB [20] mice is not established. THE POPLITEAL LYMPH NODE ASSAY (PLNA) To date, the PLNA is the only assay that can be proposed as a potential useful predictor of systemic autoimmune reactions induced by drugs and other chemicals. The PLNA was developed for use in nonclinical immunotoxicity evaluation after the seminal work of Helga Gleichmann [21] showing that injection of the anticonvulsant diphenylhydantoin into the footpad of mice induced a significant weight increase of the treated popliteal lymph node as compared to the contralateral control lymph node. Due to clinical similarities between the anticonvulsant hypersensitivity syndrome, and GvH diseases, the hypothesis was made that clinical adverse effects associated with diphenylhydantoin might reflect a pseudo-GvH reaction [22]. Ford et al. [23] had indeed previously shown that injection of histo-incompatible lymphoid cells into the footpad of rats induced an enlargement of the popliteal lymph node. Thereafter, the PLNA was proposed as a tool to predict systemic


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autoimmune diseases [24 – 27]. Several different experimental protocols were designed for toxicity evaluation or mechanistic purposes, and this resulted in proposing the PLNA as well for the identification of sensitizing drugs and chemicals supposedly acting via T lymphocyte-mediated mechanisms [28 –31]. This latter aspect will be dealt with in this chapter for the sake of clarity, but the PLNA could also be considered as a potential tool for the prediction of T lymphocyte-mediated hypersensitivity reactions as indicated in Chapter 7 of this volume. The direct PLNA is the original protocol for performing the PLNA [32]. This is a rapid, simple and inexpensive assay that can be performed in mice [33] and rats [34]. Standard protocol. Typically, the test article is injected subcutaneously via a hypodermic needle into one footpad in the heel to toe direction, and the same volume of the vehicle into the control-lateral footpad (Fig. 1). After a rest period of 7 days, both popliteal lymph nodes are removed and smoothly dissected. Dissection of lymph nodes is trickier in mice than in rats due to the smaller size of popliteal lymph nodes. There is no anatomical variation in the localization or number of popliteal lymph nodes. Both lymph nodes are immediately weighted and the weight index is calculated as the ratio of treated:control lymph node weight. There is no consensus on the definition of positive PLNA response. A positive response can be defined as a weight index above the upper limit of the confidence ðp , 0:05Þ interval. The accumulated experience shows that a weight index . 2 can be considered as a positive response. Indices . 10 are rarely observed. Rats or mice received a subcutaneous injection of the test article into one hind footpad and the vehicle in the contralateral footpad. On day 7, the animals are sacrificed and both popliteal lymph nodes are removed, dissected and immediately weighted. The ratio of treated:control lymph node weight (weight index) is calculated. A weight index . 2 is usually considered as a positive response.

Fig. 1. Schematic representation of the direct PLNAs.

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Technical issues. A number of technical issue must be considered. The route of administration should be carefully subcutaneous as an intradermal injection gives either a negative response or at least, a lower weight index than the subcutaneous route. The volume of administration is usually 50 ml in mice and rats. Seemingly, no study addressed the possible variations in response depending on the volume injected. It is, however, logical to assume that larger volumes may cause inflammation and false positive responses. The selection of the vehicle is a critical issue. Saline is the first-choice vehicle. However, poor solubility is a common problem. 20% DMSO has often been used and seems suitable. In contrast, acetone, methyl-ethyl-ketone and pure ethanol [35] induce falsepositive responses due to primary irritation and their utilization is not recommended. The use of 25% ethanol is possible. Other vehicles, such as corn oil, paraffin oil, and 1,2 propylene-glycol, are not recommended as they cause false-positive responses due to either primary irritation or excessive viscosity [34]. Dose selection. Until recently, the selected dose was arbitrarily 1 mg in mice and 5 mg in rats. However, increasing the dose has been shown to produce positive responses with procainamide [36], but systemic toxicity was described after administration of greater doses of mercuric chloride [37]. It seems logical to use several dose levels. However, preliminary results suggest that the dose – response relationship is relatively elusive [38]. Thus, no dose – response relationship could be evidenced with diphenylhydantoin, streptozotocin, ofloxacin, phenobarbital or metformin in mice. The selection of the animal species is not an issue as similar results have been obtained in mice and rats. In contrast, the selection of the strain has been a matter of debate even though a limited amount of data is available on strain comparison. An early study showed that Balb/c and C57B1/10 mice gave stronger responses than DBA/2 mice [33]. In the rat, early findings led to the conclusion that BN rats were more suitable than Sprague– Dawley rats [39], but this was not confirmed by a subsequent study showing that Sprague– Dawley, Wistar, Wistar– Furth, Lewis, Fisher 344 and BN rats had similar responses to barbital, diphenylhydantoin, isoniazid, procainamide and streptozotocin [40]. Kinetics of the response. The optimal duration of the rest period between subcutaneous injection of the test article and removal of popliteal lymph nodes has not been investigated. Typically, the selected duration is 7 days. However, kinetic studies showed a stronger response with zimeldine [41] and streptozotocin [42] when popliteal lymph nodes are removed after 9– 11 days. As the possibility remains that the kinetics of PLNA responses may vary depending on the test article, it is logical to recommend using several time points for popliteal lymph node removal. Other measured end-points. The weight index is generally considered as the gold standard in the direct PLNA. However, other end-points have been used and some proved more sensitive. Cellularity index Popliteal lymph nodes when weighted are crushed against a stainless grid, the cells carefully recovered, then centrifuged and resuspended. Cells are counted either microscopically or by flow cytometry. The cellularity index is calculated from the ratio of treated:control lymph node cellularity. The cellularity index is consistently


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greater than the weight index and allows improved accuracy when measuring the PLNA response [43]. As for the weight index, there is no definition of a positive response assessed from the cellularity index, but the experience shows that a cellularity index . 5 can be considered as a positive response. Indices . 50 have sometimes been reportedly, especially with streptozotocin. Tritiated thymidine incorporation. Tritiated thymidine incorporation has rarely been used [44,45]. As cellularity index and still more so, weight index may be considered to lack sufficient accuracy, the use of tritiated thymidine incorporation was recently evaluated as a surrogate end-point [38]. The technique is closely similar to that used in the local lymph node assay (LLNA) as described in Chapter 7 of this volume. On day 7, all animals receive an intravenous injection of 250 ml of PBS containing 20 mCi of [3H] methyl-thymidine. Five hours after the injection of thymidine, the mice are sacrificed and the draining popliteal lymph nodes excised. A single-cell suspension is prepared from each lymph node and thymidine incorporation is measured by liquid scintillation counting. No significant differences between the weight index and thymidine incorporation were evidenced and tritiated thymidine incorporation does not seem to be a useful add-on to the standard protocol of the direct PLNA. Lymphocyte subset analysis. Changes in lymphocyte subsets have seldom being studied in the direct PLNA. A global increase in total T lymphocytes, CD4þ T lymphocytes, CD8þ T lymphocytes, and B lymphocytes has been described in mice [42] and rats [46]. Recently, streptozotocin was shown to induce an early fall in CD4þ T lymphocytes which reversed on day 6, while there was an increase onward that was still present on day 12. CD8þ T lymphocytes followed an opposite pattern. In addition, there was a transient early increase in CD25þ and CD69þ T lymphocytes suggesting the possible utility of analyzing surface markers of activation [47]. In contrast, mercuric chloride induced slight changes and hydralazine a small reduction in CD4þ T lymphocytes. Histological examination. Standard examination of popliteal lymph nodes shows changes typically reminiscent of a GvH reaction in positive responses. There is a more or less pronounced blurring of the lymph node architecture resulting from an intermingling of the cortical and paracortical areas, numerous immunoblasts in paracortical areas, highgrade maturation of germinal centers and numerous plasma cells in medullary areas [48, 49]. Immunochemistry has rarely been used and no consistent findings have been obtained. IgG production has rarely been measured in the direct PLNA [50], although it can provide functional evidence of B-cell activation. This end-point has been suggested to be more sensitive than either weight or cellularity indices. Cytokine fingerprinting. Due to the pivotal role of cytokines in immune responses, the production of cytokines is an attractive end-point to improve the sensitivity of the direct PLNA. Streptozotocin induced a dramatic increase in IFN-g mRNA production measured by RT-PCR which correlated with an increase in weight and cellularity indices, whereas no changes in the production of IL-4 mRNA was seen [51]. Subsequently, streptozotocin was shown to induce a marked increased in the production of IL-6, IFN-g, TNF-a, IL-1a, IL1b, IL-2R and IL-12 mRNAs, whereas neither ethanol nor acetone induced changes in the production of IL-6 and IFN-g mRNAs [52]. In addition, increases in the production of TNFa, IL-1a, IL-1b, IL-2R and IL-12 mRNAs were 2 –3 times lower with acetone or ethanol than with streptozotocin. These findings suggest that measuring cytokine production could

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be helpful to rule out positive responses due to primary irritation. The effect of streptozotocin on IL-2 and IFN-g production was confirmed by Tuschl et al. [47] who measured the intracellular levels of these two cytokines by flow cytometry. However, the measurement of cytokine production in the direct PLNA was recently questioned. Diclofenac was shown to induce a slight increase in the production of the TH1 cytokines IL-2, IL-12 and IFN-g, and the proinflammatory cytokines IL-6, TNF-a and MCP-1 by popliteal lymph node cells, but no changes in the production of the TH2 cytokines IL-4 and IL-5 [53]. In addition, neither diphenylhydantoin nor hydralazine nor minocycline nor imipramine induced changes in the production of either TH1 or TH2 cytokines. Results. Overall, results on more than 130 compounds in the PLNA have been published. Interestingly, there was a nice parallelism between human data and experimental results in some instances. One inter-laboratory study involving three laboratories showed consistent blind results in the direct PLNA in Wistar rats with chlorpromazine, zimeldine, hydrazine and streptozotocin, which have been reported to cause systemic autoimmune reactions in humans, and barbital which has not [54]. However, (false) positive responses have been reported with drugs and other chemicals that never induced systemic autoimmune reactions in man and (false) negative reactions as well. A major cause of false positive responses in the direct PLNA is sensitization or immunogenicity. Thus, strong positive responses have been obtained in the direct PLNA with DNCB and picryl chloride [48,49]. However, weak to moderate contact sensitizers are negative [33]. The injection of SRBC also induced a strong positive response. Contact sensitizers can easily be detected in guinea pig or mouse assays (see Chapter 7 of this volume). In addition, conventional histological examination of popliteal lymph nodes shows changes that are markedly dissimilar, in particular a conserved architecture of the lymph node and numerous germinal centers [49]. The second cause of false positive responses is nonspecific inflammation due to the primary irritancy of the test article. As already mentioned, this can explain false positive responses induced by acetone or pure ethanol. The false positive response to imipramine has been demonstrated to involve a primary irritant effect [55] and is not due to a hypothetical modulation of the immune response via serotonin pathways [50]. It is also possible that false positive responses to phenylbutazone [33] were due to nonspecific irritation. Unfortunately, histological examination of the popliteal lymph nodes provides no clues for the identification of these false positive responses due to primary irritation. It is, however, possible to rule out these false positive responses by depleting mice in CD8þ T lymphocytes prior to performing the PLNA or by performing fingerprinting of cytokines in the popliteal lymph nodes [52]. Depletion of mice in CD8þ T lymphocytes using an anti-CD8 monoclonal antibody did not suppress the false positive responses to acetone and ethanol as compared to nondepleted mice, whereas the positive response to streptozotocin was completely inhibited. When comparing the profile of released cytokines, marked differences were seen between mice treated with either acetone or ethanol, and streptozotocin as already mentioned. When comparing the kinetics of the primary and secondary PLNA responses, Suda et al. [56] showed that primary irritants


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did not induce an increased secondary response. Finally, quartz crystals have been shown to induce false-positive responses, which were presumably due to the persistence of the crystals resulting in prolonged stimulation of macrophages and enhanced cytokine production [57]. False negative results have been obtained with procainamide and isoniazid. They are probably due to the involvement of metabolites instead of the parent molecules. In vitro pre-incubation with S-9 mix as shown with procainamide [58] and isoniazid [59], pretreatment with an enzyme-inducing agent, such as b-naphthoflavone as shown with isoniazid [59], or by using higher doses as shown with procainamide [36] can rule out false negative responses. The secondary PLNA was designed since a positive response in the direct PLNA does not demonstrate whether the reaction is antigen specific. To test whether T lymphocytes have been sensitized by the test article, the capacity of mounting a secondary response must be investigated. This can be done either by performing a direct PLNA and challenging the same animals after a rest period (4 –6 weeks) sufficient to allow for total resolution of the primary PLNA response, into the same footpad, but with a lower dose of the test article [60]. Animals can also be primed by a different route, either intranasally [61], orally [62] or intravenously [63]. Thereafter, primed animals are challenged into the footpad using a dose of the test article insufficient to induce a primary positive response. In the secondary PLNA, mice are injected with the test article either subcutaneously into one hind footpad as in the direct PLNA, or via other routes, e.g. intramuscularly or orally. The animals are subsequently challenged with a sub-optimal nonsensitizing dose into the footpad. Weight and/or cellularity indices are calculated as previously described 4– 6 days after challenge. The adoptive transfer PLNA is a more elegant, but time-consuming technique. In this test, T lymphocytes from a previously exposed donor are transferred to a syngeneic recipient. The donor animals can be exposed to the test article under conditions that mimic the human exposure. Thereafter, T cells of the donor are injected subcutaneously into the hind footpad of an unexposed recipient and the next day, the recipient is injected subcutaneously at the same site a suboptimal dose of the test article. Using this technique in mice, it was possible to show specific T lymphocyte responses to mercuric chloride [64], and to metabolites of gold [65] and procainamide [66]. Unfortunately, all these results have been obtained in one single laboratory and have never been reproduced elsewhere. Their applicability to immunotoxicological evaluation is unknown. The modified PLNA. The most recently introduced variation of the PLNA is the modified PLNA [67]. The local specific antibody response to the reporter antigens TNP-Ficoll or TNP-ovalbumin elicited by the test article is used to characterize its immunostimulatory potency. The test article is injected into the footpad together with 10 mg/mouse of TNPFicoll or TNP-ovalbumin. The specific antibody response to these antigens is measured on day 7 by ELISPOT. Because TNP-Ficoll is a T-independent antigen and TNP-ovalbumin, a T-dependent antigen, the type of response can discriminate compounds that induce

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inflammation, i.e. IgG response to TNP-ovalbumin, but not to TNP-Ficoll, from those inducing a T-lymphocyte mediated reaction, i.e. IgG response to both TNP-ovalbumin and TNP-Ficoll. As there is a 10– 1000 increase in cells producing anti-TNF-Ficoll IgG vs. only a 20-fold increase in cells producing anti-TNF-ovalbumin IgG, the potential for discriminating between sensitizing and nonsensitizing compounds is high as is the possibility, at least theoretically, to rank compounds according to their immunostimulatory potency. The measurement of IgG1, and IgG2a and IgE can also differentiate TH2 and TH1 responses, respectively. Although data using this sophisticated experimental protocol are still extremely limited, streptozocin was shown to stimulate an Ig2a response and mercuric chloride an IgG1 response [68]. Although, the modified PLNA still requires extensive validation, the few available results suggest it could be more suitable to identify sensitizing drugs that have been reported to induce anaphylaxis in man. Thus, diclofenac, zomepirac and glafenin gave inconsistent results in the direct PLNA, but induced a marked anti-TNP-ovalbumin IgE response in the modified PLNA [69]. In contrast, the modified PLNA may not be appropriate to detect the potential for inducing autoimmune reactions as neither hydralazine nor isoniazid nor a-methyldopa induced positive responses [70]. Perspectives. A number of studies have been devoted to the PLNA, either the direct PLNA or variations of the standard protocol. Many drugs and other chemicals have been tested and expected results based on the clinical experience have often, even though inconsistently been reported. However, the diversity of protocols and the lack of understanding of the mechanisms involved in PLNA response are major limitations to the routine use of the PLNA in nonclinical immunotoxicity evaluation. At the present time, it remains to be seen whether refinements of the direct PLNA could improve the identification of drugs and other chemicals that induce systemic autoimmune reactions, whereas validation of the modified PLNA is needed to confirm its predictive value for the identification of drugs and other chemicals that induce anaphylactic reactions.

ORGAN-SPECIFIC AUTOIMMUNE REACTIONS No animal models or assays are available to predict organ-specific reactions induced by drugs and other chemicals because the mechanisms are not known. It is also noteworthy that these reactions are extremely rare, which suggests either a specific mechanism, e.g. the formation of reactive metabolite in a few patients, or the role of contributing as well as predisposing factors that cannot predicted. In fact, these two hypotheses are not mutually exclusive. An illustrative example of the current difficulty, if not impossibility to predict the potential for inducing organ-specific autoimmune reactions is autoimmune hemolytic anemia induced by the antihypertensive drug a-methyldopa. The majority of published drug-induced autoimmune hemolytic anemias are due to a-methyldopa (see Chapter 4 of volume II). Although, the dog is the only conventional animal species that spontaneously develops autoimmune hemolytic anemias similar to those seen in man [71], there is seemingly no published report of autoimmune hemolytic anemia caused by drugs and


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other chemicals in the dog. Hemolytic anemia is a typical manifestation in autoimmunity-prone NZB mice. Unexpectedly, NZB mice when treated with 60 mg/ kg/day of a-methyldopa had a slight increase in their hematocrit as well as a slower elevation in anti-erythrocyte antibody levels at 66 weeks of age as compared to controls [72]. In contrast, combined treatment with the structurally related drugs carbidopa and levodopa induced a decrease in the hematocrit of NZB mice after 20 weeks of age that became significant at 66 weeks, and the peak anti-erythrocyte antibody levels in treated animals was reached 8 weeks earlier than in the controls. Neither drug accelerated the course of the spontaneous disease. It is noteworthy that levodopa, however, induced autoimmune hemolytic anemia extremely rarely in treated patients [73]. In addition, treatment with a-methyldopa consistently failed to induce autoimmune hemolytic anemia or related immunohematological changes in conventional mice [5,74,75]. Interestingly, Owens et al. [76] tested the potential for a-methyldopa to induce autoimmune hemolytic anemia in chimpanzees. Four animals were injected with membranes of their own erythrocytes incubated with a-methyldopa plus diethyl-dithiocarbamate, 3 times a week for 6 consecutive weeks. Two had a moderately positive direct Coombs test, which was then considered as the indication that oxidized metabolites of a-methyldopa might be involved in the generation of anti-erythrocyte autoantibodies. The same experimental protocol, however, was unsuccessful in rabbits.

CONCLUSION The nonclinical prediction of the potential of drugs and other chemicals to induce autoimmune reactions is beyond reach at the present time. No animal model or assay can be recommended. Research efforts are needed to gain a better understanding of the mechanisms involved to design models that may prove good predictors in the long term.

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Chapter 9

Regulatory aspects of immunotoxicity evaluation

INTRODUCTION Despite the growing awareness that immunotoxicity is an important issue regarding the safety of drugs and other chemicals, regulatory authorities have often been hesitant until relatively recently, to release guidelines or guidances dealing with nonclinical immunotoxicity assessment. Regulatory issues, however, have been underscored quite a long time ago [1]. Over the years, a great number of authors, institutions or societies from industry or academia, however, published recommendations or position papers reflecting what were considered the state-of-the-art strategies for nonclinical immunotoxicity evaluation at the time [2 – 21]. So far, the regulatory process regarding immunotoxicity assessment has been a long and winding road [22 –30]. In fact, the first regulations regarding the nonclinical immunotoxicity evaluation of drugs and other chemicals have been published more than 30 years ago. Because they were either too vague or premature, they have never been implemented by the regulatory authorities who released them. Then, more than a decade elapsed before new regulations emerged, but they either applied to a very restricted type of compounds, such as biotechnology-derived pharmaceuticals [31], or were overcautious, such as OECD guideline 407 [32]. Nevertheless, they heralded a continuing effort to define nonclinical procedures to assure cost effectively that new molecular entities are not unduly immunotoxic in treated or exposed human subjects. Subsequently, guidelines have been published on the nonclinical immunotoxicity evaluation of pesticides [33], medical devices [34] and pharmaceutical products [35,36]. As regards the latter, the decision by the ICH steering committee that sufficient scientific evidence is available for immunotoxicity to enter the harmonization process of human pharmaceuticals as topic S8 [37] with a preset deadline in 2005 is a highly significant step in the ongoing immunotoxicity regulatory process.

EARLY IMMUNOTOXICITY REGULATIONS Although major immunotoxicity guidelines have only been published in the late 1990s and later, a few regulatory authorities published guidelines more than 20 years ago. 257

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FIRST EPA GUIDELINE ON PESTICIDE IMMUNOTOXICITY In the early 1980s, the US National Toxicology Program (NTP) funded a far-reaching program in an attempt to define, rationalize, standardize and validate animal models to be used for the nonclinical prediction of the unexpected immunosuppressive effects of xenobiotics. The selected protocol [38] was based on the previously proposed concept of a two-tier protocol in order to take into account the well-recognized complexity of the immune response and offer a cost-effective approach [39]. B6C3F1 mice were selected because the use of a hybrid strain—B6C3F1 mice are bred from C57Bl/6N female and DNA/2N male mice—was felt to make immunological assays easier to be performed than in an outbred strain due to genetic variability. Over 50 compounds were tested in several laboratories. The most significant conclusions [40,41] to be drawn from this study were that reasonably reproducible results can be achieved using standardized animal models and assays and quantifiable correlations between functional immune assays and host resistance models could be identified. In 1982, the Office of Pesticide Programs of the U.S. Environmental Protection Agency issued a guideline for the immunotoxicity evaluation of pesticides (Subdivision M Pesticide Assessment Guidelines) requiring that any new pesticide be evaluated according to the following tier-1 and tier-2 assay procedure summarized in Table 1. Needless to say that this guideline was extremely premature as the recommended animal models and assays had been neither standardized nor validated at the time. Indeed, the final results of the NTP inter-laboratory validation study would be available only 10 years after the guideline release. Modifications of this guideline therefore were soon a necessity as implementation of this guideline proved impossible. In 1988, the US National Agricultural Chemicals Association proposed to include gross immunotoxicity end-points into 90-day conventional rat studies (tier-1) and to perform immune function tests in specific 28-day rat studies (tier-2) only when deemed necessary. The final decision would be based on the results of host resistance assays, e.g. experimental infections (tier-3). That same year, the EPA proposed a revision of the initial guideline [22]. Although rodents remained the preferred species, other species were accepted. The sensitivity of each selected assay would have to be confirmed in positive control animals. At least, three dose levels not inducing general toxicity would have to be selected for 30 days of exposure. Assays were still divided into two tiers. The first tier would include total serum Ig levels or plaque-forming cell (PFC) assay to assess humoral immunity, MLR, DTH response or T lymphocyte cytoxicity to assess cellular immunity, and natural killer (NK) cell activity or macrophage functions to assess innate immunity. Tier-2 would have to be performed only when positive or not interpretable results are obtained in tier-1, or when additional data, such as a chemical structure or mechanism close to that of a known immunotoxicant are present. Assays to be included in tier-2 were expected to evaluate the time course of recovery from immunotoxicity and the resistance to experimental infections. The inclusion of additional tests, such as serum complement levels, T-independent antibody response, or lymphocyte subset analysis, was recommended as needed. This draft guideline was never finalized as such, but nevertheless served as a basis for the final guideline to be published in 1996 (see below).


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Table 1. Tier-1 and tier-2 assays to be included in the nonclinical immunotoxicity assessment of pesticides according to 1982 US EPA guideline Tier I Immunopathology Hematology (blood cell counts) Total, spleen, thymus and liver weight Spleen cellularity Histology of the spleen, thymus and lymph nodes Humoral immunity Direct plaque-forming cell (PFC) assay or primary antibody response to antigen Cell-mediated immunity Mitogen-induced lymphocyte proliferation or mixed leukocyte reaction Nonspecific immunity NK cell activity Tier II Immunopathology Enumeration of B and T cells Humoral immunity Secondary antibody response to antigen Cell-mediated immunity T lymphocyte cytotoxicity assay Delayed-type hypersensitivity response Nonspecific immunity Enumeration and phagocytic activity of peritoneal macrophages Resistance assays Implantation of syngeneic tumour cells (PYB6 fibrosarcoma, B6F10 melanoma) Bacterial infections (Listeria monocytogenes, Streptococci) Viral infections (Influenza) Parasitic infections (Plasmodium yoleii)

COMMISSION OF THE EUROPEAN COMMUNITIES The Council (subsequently named Commission) of the European Communities was the first regulatory authority to issue a recommendation emphasizing the need for a systematic immunotoxicological evaluation of new medicinal products [42]. This recommendation— not a guideline—stated that the interferences of medicinal products with the immune system should be considered even when not expected from the intended therapeutic use of these products as they may result in potentially severe adverse reactions including infectious complications and neoplasias. Emphasis was given to the histological examination of lymphoid organs. If changes were noted, additional assays would have to be performed, but no recommendations on relevant assays were provided. Despite obvious limitations, this recommendation was the first to be issued regarding the nonclinical immunotoxicity evaluation of new medicinal products. It is still unclear why it had no impact whatsoever on the nonclinical safety assessment of new medicinal products for nearly 20 years.

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JAPANESE DRUG ANTIGENICITY GUIDELINE An unofficial guideline emitted by the Japanese Ministry of Health, Labour and Welfare in 1990 required antigenicity testing for every new medicinal product using the raw material and a protein-conjugate in three different assays [43], namely systemic anaphylaxis in guinea pigs with two sensitizing oral or transcutaneous injections at one-week intervals, and intravenous or intradermal challenge after 10 days; passive cutaneous anaphylaxis reaction in guinea pigs or rabbits; and indirect hemagglutination in mice with two subcutaneous injections of sensitized rabbit serum at 2 – 4 week intervals. The recommended tests were not validated. They are still neither standardized nor validated, as discussed in Chapter 7 of this volume. Although this guideline has never been published officially by the Japanese authorities, the pharmaceutical industry very often follows this unofficial requirement despite a total lack of demonstrated relevance. FDA RED BOOK II In consideration of the need for defining immunotoxicity with regard to regulated products of the US FDA, an intra-agency taskforce was convened to arrive at a definition of immunotoxicity. Pursuant to these efforts, immunotoxicity testing guidelines in draft form [23,28] were proposed to be included in the second edition of Toxicological Principles for the Safety Assessment of Direct Food and Color Additives Used in Food (referred to as the Red Book). Initially, two levels of immunotoxicity testing were defined, namely level I, which did not require injection of an antigen to animals, so that selected end-points could be measured in the same animals as those used in standard toxicity studies, and level II, which was defined as functional testing and required a concurrent satellite group of animals. Primary indicators of immunotoxicity included total and differential white blood cell counts, clinical chemistry end-points, lymphoid organ weights and standard histological examination of the spleen, thymus, lymph nodes and Peyer’s patches. Expanded level I serum chemistry screen might include quantification of serum immunoglobulin and autoantibody levels, NK cell and macrophage functional activity, assessment of hemolytic complement and enhanced histopathology based on the immunostaining of lymphocytes in tissues. Level II testing was suggested to include DTH responses and humoral responses to both T-dependent and T-independent antigens. In fact, a final version has not yet been officially published, but interestingly additional approaches including hypersensitivity reactions or the allergenic potential of new foods and food constituents are being considered.

RECENT IMMUNOTOXICITY GUIDELINES Although there is wide belief that immunotoxicity guidelines are only a recent trend among regulatory authorities, several guidelines or guidances have been published over the past decades. These guidelines will be considered chronologically according to their date of publication.


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OECD GUIDELINE 407 The Organization for Economic Development and Cooperation (OECD) released a revised version of guideline 407 adopted on 27 July 1995 [32]. This guideline was aimed at defining the required protocol for use in 28-day conventional toxicity rat studies, which are the minimal repeated dose toxicity evaluation for industrial chemicals. Two major aspects of the revision were the inclusion of neurobehavioral and immunotoxicity end-points. As regards immunotoxicity end-points and after a long vivid debate, the final decision was to restrict the required immunotoxicity assessment to a standard histological examination of the lymphoid organs in the control and high dose groups, including the thymus, spleen, lymph nodes (with one lymph node covering the route of administration and one distant lymph node to cover systemic effects), and the Peyer’s patches. Although the systematic inclusion of standard histological examination of lymphoid organs was a significant progress, it is unfortunate that no immune function assays were deemed necessary. Despite claims that standard histology examination is a sensitive predictor of unexpected immunosuppression [44 –47], there are instances where decreased PFC responses can be seen without any remarkable histological changes [48]. SECOND EPA GUIDELINE ON PESTICIDE IMMUNOTOXICITY After 15 years of debate, the immunotoxicity testing guideline for pesticides under the EPA’s Toxic Substances Contract Act (TSCA) was recently updated in final form [33]. Although allergy and autoimmunity are included in the scope of immunotoxicity, this guideline is restricted to immunosuppression. Interestingly, emphasis is placed on immune function, even though it is clearly stated that the proposed assays do not represent a comprehensive assessment of immune function. Relevant information on histological changes in lymphoid organs is considered to be available from the results of conventional toxicity testing. Mice and rats are the recommended species, but one species can be used, unless no adequate ADME data are available. At least, eight animals of each sex per group should be used, unless one sex is known to be more sensitive to the test article. At least, three dose levels should be used for dose –response relationship and the determination of a no-observed immunotoxic effect level. Ideally, the lowest dose should not induce immunotoxic effects and the highest dose should produce measurable signs of moderate general toxicity, such as a 10% loss in body weight, but neither stress, nor malnutrition nor fatalities. The primarily recommended function assay is the PFC assay to be performed in animals dosed for 28 days. However, immunoglobulin quantification by ELISA is an accepted alternative. In the event that a significant suppression of PFC response is observed, lymphocyte subset analysis using flow cytometry and NK cell activity should be considered. Finally, a positive control group of eight animals at least, treated with cyclophosphamide should be used. It is unclear why should immunotoxicity studies include a positive control group as conventional repeated dose administration studies should not. This guideline reflects two major changes in the regulatory approach of nonclinical immunotoxicity evaluation. At least one immune function assay is required and histological examination of the lymphoid organs is no longer considered as a sufficiently sensitive

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predictor of unexpected immunosuppression. The strict tiered approach is no longer considered a hallmark of immunotoxicity evaluation and antibody response to a T-dependent antigen can serve as an initial screen. ICH GUIDELINE ON THE PRECLINICAL SAFETY EVALUATION OF BIOTECHNOLOGY-DERIVED PHARMACEUTICALS The ICH guideline S6 on the preclinical safety evaluation of biotechnology-derived pharmaceuticals was adopted in 1997 [31]. This is the first guideline putting great emphasis on the nonclinical immunotoxicity evaluation of pharmaceutical products, even though only biotechnology-derived products are concerned. Most biotechnology-derived products are large molecules that can be immunogenic due to subtle changes as compared to their natural human counterpart because of a different 3D conformation or glycosylation for instance. They are normally immunogenic in animals, which can preclude the conduct of standard toxicity studies in conventional animal species, but the production of specific antibodies in animals is not predictive of their immunogenicity in man. The use of systemic anaphylaxis guinea pigs is not recommended, a claim that is only acceptable when humanized products are tested. For the routine evaluation of unexpected immunosuppression, a classical tier-testing approach is not recommended either, and it is noteworthy that ICH guideline S6 is in keeping with the second US EPA guideline for pesticide immunotoxicity on this aspect. A major limitation of this guideline, however, is that an extremely wide variety of products is considered and as safety issues are obviously different depending on the particular type of products being tested, recommendations are only broadly general. EMEA CONCEPT PAPER ON IMMUNOTOXICITY In 1998, the Committee for Proprietary Medicinal Products (CPMP) of the European Agency for the Evaluation of Medicinal Products (EMEA) released a concept paper on immunotoxicity [49]. This short concept paper is important as for the first time, the focus is no longer only on immunosuppression as it used to be in most previous immunotoxicity guidelines, but put clear emphasis on other aspects of drug-induced immunotoxicity including autoimmunity and hypersensitivity. FDA CDRH IMMUNOTOXICITY TESTING GUIDANCE The FDA Center for Devices and Radiological Health (CDRH) published this guidance in 1999 [34]. The framework of this guidance was to propose the best tests based on the knowledge at the time of publication to predict any adverse effect that may occur on the structure or function of the immune system and results in immunosuppression, inflammation, hypersensitivity or autoimmunity in relation to the use of medical devices. The guideline consists of a flow chart used for determining whether immunotoxicity testing is needed and for providing a rationale for the selection of relevant tests.


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Briefly, immunotoxicity testing is required when the device contacts the body and contains potentially immunotoxic material, the immunotoxic potential of which has not been previously established. Possible tests are divided into critical and noncritical tests. Histopathology is considered as a critical test for inflammation and autoimmunity, but a noncritical test for hypersensitivity and immunostimulation, which is in strong disagreement with the available experience [50]. Humoral immunity assays are judged critical, except for inflammation. Cellular immunity assays are judged critical in all situations, including inflammation. NK cell activity is judged critical for immunosuppression, which is in contradiction with the available experience [51]. Macrophage function assays are judged critical for inflammation and immunosuppression, and host resistance assays only for immunosuppression. A list of the main assays to be considered is provided. CPMP NOTE FOR GUIDANCE ON REPEATED DOSE TOXICITY In 2000, the CPMP released a note for guidance on repeated dose toxicity, which in fact is essentially focused on immunotoxicity [35]. That the title of this note for guidance does not explicitly refer to immunotoxicity may reflect a certain degree of disagreement or lack of frank consensus among state member representatives of the CPMP. Anyway, this note for guidance is exclusively considering immunotoxicity from the viewpoint of immunosuppression, which is somewhat unexpected and untimely. That immunotoxic effects encompass immunosuppression, immunoenhancement, hypersensitivity and autoimmunity is only very briefly mentioned. The recommended approach is extremely conventional. Nonclinical immunotoxicity evaluation should be preferably incorporated in a 28-day rodent study. A longer period of exposure is possible as well as the selection of a nonrodent species, but this must be justified. Immunotoxicological end-points are divided into initial screen and extended studies, a division clearly reminiscent of the classical tiered approach. The initial screen consists of hematology, bone marrow cellularity and histological examination of the main lymphoid organs, and finally assessment of immune function. To assess immune function, it is possible to combine NK cell activity measurement and analysis of lymphocyte subsets or, as an alternative, to perform a primary antibody response assay to a T-dependent antigen. It is noteworthy that neither NK cell activity measurement [51] nor lymphocyte subset analysis [52] are considered as sensitive predictors of immunosuppression. Depending on initial results, extended studies should be performed including DTH responses, mitogen- or antigen-induced lymphocyte proliferation, macrophage function, primary antibody response to a T-independent antigen and experimental infections. This note for guidance offers negative and positive aspects. The major positive aspect is that nonclinical immunotoxicity assessment including assessment of immune function is systematically required for the marketing approval of every new drug. It has already been mentioned that histological examination is not a sufficient predictor of immunosuppression and the required assessment of immune function is clearly welcome. The major negative aspect is the conservative approach that has not yet properly been validated for medicinal drugs as it was for chemicals.

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FDA GUIDANCE ON IMMUNOTOXICOLOGY EVALUATION OF INVESTIGATIONAL NEW DRUGS The most recently guideline on immunotoxicity evaluation at the time of writing is the FDA Guidance for industry published by the FDA Center for Drug Evaluation and Research (CDER) [36]. This is a long, comprehensive guideline including many key scientific references. The scope of immunotoxicology is nicely detailed, but surprisingly divided into five categories, namely immunosuppression, adverse immunostimulation, immunogenicity, hypersensitivity and autoimmunity. It is unclear why only immunostimulation is considered as an adverse effect and not immunosuppression as well. The distinction between immunogenicity and hypersensitivity is unfounded, even though the prediction of the immunogenicity of therapeutic proteins and hypersensitivity reactions induced by small-molecular-weight drugs is not based on the same strategy. Unfortunately, hypersensitivity reactions are still classified into type-1 to type-4 reactions despite growing evidence that such a classification does not stand the test of science as discussed in Chapter 4 of this volume. Interestingly, this guideline proposes a critical state-of-the-art review of the current status of animal models and assays that can be considered for inclusion in nonclinical immunotoxicity evaluation. Finally, a flowchart is designed to guide in the selection of recommended testing. As regards immunosuppression, a major criticism is that no immune function assay is required on a systematic basis. In sharp contrast to the second EPA guideline on pesticide immunotoxicity [33] and the CPMP guideline on repeated dose toxicity study [35], the histological examination of lymphoid organs combined with standard hematology and clinical chemistry parameters, and clinical examination is considered a sufficiently sensitive predictor of unexpected immunosuppression. The imperfect predictive value of histology has already been mentioned. Standard hematology and clinical biochemistry parameters are unlikely to be markedly modified when the tested drug is not cytotoxic. Finally, the chance of observing clinical changes suggestive of immunosuppression is very small in laboratory animals under GLP conditions. However, assessment of immune function is required when the tested drug is intended to be used as an immunosuppressant, in AIDS patients, in pregnant women and when there is evidence that the drugs or its metabolites accumulate in lymphoid tissues.

CONCLUSION Although regulatory aspects have long been a matter of concern and debate, only limited and contradictory guidelines are available, as tentatively shown in this chapter. Several issues and needs can be identified as regards regulatory immunotoxicology [53]. Before requiring for nonclinical immunotoxicity testing, it must be clearly established which hazards related to immunotoxic effects have been identified, what are their incidences and their severity. As reviewed in Chapter 3 of this volume, there is no doubt that, overall, immunotoxic effects are relatively common and associated with significant morbidity and mortality. There is evidence that patients treated with immunosuppressive


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drugs develop more frequent and severe infections, and more frequent virus-related malignancies. The evidence in patients exposed to industrial or environmental chemicals is less strong, but consistent. Hypersensitivity reactions are the most frequent immunotoxic effects and some are life-threatening. Autoimmune reactions, although probably rare, can result in massive epidemics with significant mortality as demonstrated by the Spanish toxic oil syndrome. Another issue is the acceptability of risk. Although there is no widely accepted definition of what an acceptable toxic risk is, immunotoxic effects can result in severe complications so that many immunotoxic effects can be considered as unacceptable risks. The next issue is whether reasonably adequate methods are available to assess immunotoxic hazards which may result in unacceptable risks. Extensive efforts have been paid to design, standardize, and validate animal models and assays useful for the nonclinical prediction of immunotoxic hazards. Not all immunotoxic effects can be equally predicted, but some can be. Unmet needs explain why adequately standardized and validated methods are not available to evaluate every potential immunotoxic effect. It is essential to bear in mind that immunotoxicity is not equal to immunosuppression. Despite the fact that hypersensitivity is the major immunotoxic risk, immunosuppression was the primary focus of immunotoxicological investigations and current guidelines unfortunately reflect this focus. However, hypersensitivity and autoimmunity are increasingly areas of research. Although science-oriented studies are absolutely essential to gain a better understanding of underlying mechanisms, which is a prerequisite for designing more reliable animal models and assays, combined efforts in the industry and academia should be paid to standardize, and validate or invalidate available experimental protocols applicable to regulatory nonclinical immunotoxicity evaluation. Validation requires that a large panel of known immunotoxic and nonimmunotoxic compounds are tested blindly using the same experimental protocol in several laboratories. It is essential that other compounds than potent immunotoxicants, such as the immunosuppressants cyclosporine or cyclophosphamide, the chemical sensitizers DNCB or TMA, or mercuric chloride are included in validation studies. It is also important to bear in mind that results obtained with industrial and environmental chemicals may not necessarily be extrapolated to medicinal products. Finally, clinical immunotoxicology is a major unmet need. The lack of human data precludes the comparison of immunotoxic effects obtained in conventional animal species. Immune end-points should be selected that can be measured in laboratory animals, and not only rodents, as well as in man. When animal and human data are available, it becomes possible to extrapolate animal data to man, and in turn to validate animal data by comparison to human data. Recently released regulations in immunotoxicology will have and already have had a major impact on the development of immunotoxicology. To meet regulatory requirement, laboratories throughout the world will have to test a wealth of new compounds under development and compare these results with those obtained with older compounds. Experience will be gained everywhere in the best way to conduct immunotoxicity studies and more importantly, new methods and concepts will undoubtedly have to be imagined to improve the interpretation of results and the predictability of immunotoxicity evaluation.

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References [1] Descotes J (1986) Immunotoxicology: health aspects and regulatory issues. Trends Pharm Sci, 7, 1 –4. [2] Dean JH, Padarathsingh ML, Jerrells TR, Keys L, Northing JW (1979) Assessment of immunobiological effects induced by chemicals, drugs or food additives. II. Studies with cyclophosphamide. Drug Chem Toxicol, 2, 133– 153. [3] Norbury KC (1982) Methods currently used in the pharmaceutical industry for evaluating immunotoxic effects. Pharmacol Rev, 34, 131– 136. [4] Dean JH, Vos JG (1986) An introduction to immunotoxicology assessment. In: Descotes J (Ed) Immunotoxicology of Drugs and Chemicals, 1st edition, pp 3 – 18. Elsevier, Amsterdam. [5] ECETOC (1987) Identification of Immunotoxic Effects of Chemicals and Assessment of their Relevance to Man. Monograph No. 10. European Chemical Industry Ecology and Toxicology Centre, Brussels. [6] Trizio D, Basketter DA, Botham PA, Graepel PH, Lambre C, Magda SJ, Pal TM, Riley AJ, Ronneberger H, Van Sittert NJ, et al. (1988) Identification of immunotoxic effects of chemicals and assessment of their relevance to man. Food Chem Toxicol, 26, 527–539. [7] Luster MI, Germolec DR, Rosenthal GJ (1990) Immunotoxicology: review of current status. Ann Allergy, 64, 427– 432. [8] Burrel R, Flaherty DK, Sauers LJ (1992) Regulatory immunotoxicology. In: Burrel R, Flaherty DK, Sauers LJ (Eds) Toxicology of the Immune System. A Human Approach, pp 293– 309. Van Rostrand-Reinhold, New York. [9] Descotes J, Vial T, Verdier F (1993) The how, why and when of immunological testing. Comp Haematol Int, 3, 63 –66. [10] ECETOC (1994) Immunotoxicity: Hazard Identification and Risk Characterisation. Monograph No. 21. European Chemical Industry Ecology and Toxicology Centre, Brussels. [11] Immunotoxicology Technical Committee (1995) Immunotoxicity Testing and Risk Assessment: summary of a 1994 workshop. ILSI Health and Environmental Sciences Institute. Food Chem Toxicol, 33, 887– 894. [12] Vandebriel RJ, Garssen J, Van Loveren H (1995) Methods in immunotoxicology. Methods Neurosci, 24, 151– 169. [13] Banerjee BD, Koner BC, Ray A (1996) Immunotoxicity of pesticides: perspectives and trends. Indian J Exp Biol, 34, 723–733. [14] Dean JH (1997) Issues with introducing new immunotoxicology methods into the safety assessment of pharmaceuticals. Toxicology, 119, 95 – 101. [15] Van der Laan WJ, Van Loveren H, Vos JH, Dean JH, Hasting KL (1997) Immunotoxicity of pharmaceuticals: current knowledge, testing strategies, risk evaluation, and consequences for human health. Drug Info J, 31, 1301– 1305. [16] Kimber I (1998) Regulatory immunotoxicology—the scientist’s point of view: an introduction. Arch Toxicol, 20, 271– 274. [17] Dean JH, Hincks JR, Remandet B (1998) Immunotoxicology assessment in the pharmaceutical industry. Toxicol Lett, 102– 103, 247– 255. [18] Anderson JM, Langone JJ (1999) Issues and perspectives on the biocompatibility and immunotoxicity evaluation of implanted controlled release systems. J Control Release, 57, 107– 113. [19] Harleman JH (2000) Approaches to the identification and recording of findings in the lymphoreticular organs indicative for immunotoxicity in regulatory type toxicity studies. Toxicology, 142, 213– 219. [20] Putman E, Van Loveren H, Bode G, Dean JH, Hastings K, Nakamura K, Verdier F, Van der Laan JW (2002) Assessment of the immunotoxic potential of human pharmaceuticals: a workshop report. Drug Info J, 36, 417– 427. [21] Luster MI, Dean JH, Germolec DR (2003) Consensus workshop on methods to evaluate developmental immunotoxicity. Environ Health Perspect, 111, 579–583.


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Immunotoxicology of drugs and chemicals assessment of, chemicals identified as immunosuppressive in routine toxicology. Food Chem Toxicol, 33, 239– 243. Gopinath C (1996) Pathology of toxic effects on the immune system. Inflamm Res, 2, 74 – 78. Crevel RW, Buckley P, Robinson JA, Sanders IJ (1997) Immunotoxicological assessment of cyclosporin A by conventional pathological techniques and immune function testing in the rat. Hum Exp Toxicol, 16, 79 – 88. Germolec DR, Nyska A, Kashon M, Kuper CF, Portier C, Kommineni C, Johnson KA, Luster MI (2004) Extended histopathology in immunotoxicity testing: interlaboratory validation studies. Toxicol Sci, 78, 107– 115. EMEA (1998) Concept paper on immunotoxicity, CPMP/SWP/160/98, 26 February 1998, London. Kuper CF (2004) Issues in the histological examination of lymphoid organs. Toxicology. In press. Cederbrant K, Marcusson-Stahl M, Condevaux F, Descotes J (2003) NK-cell activity in immunotoxicity drug evaluation. Toxicology, 185, 241– 250. Immunotoxicology Technical Committee (2001) Application of flow cytometry to immunotoxicity testing: summary of a workshop. Toxicology, 163, 39 –48. Descotes J (1998) Regulating immunotoxicity evaluation: issues and needs. Arch Toxicol, 20(Suppl.), 293– 299.

Chapter 10

Nonclinical strategies of immunotoxicity evaluation and risk assessment

INTRODUCTION The nonclinical evaluation of the toxicity as well as the immunotoxicity of drugs and other chemicals must take notice of a number of important, but sometimes conflicting considerations. The four following aspects are deemed essential. First of all, this evaluation should aim at identifying the hazards in relation to the test article to the health of living beings and to the environment; secondly, this evaluation should provide relevant information for use in the risk assessment process for determined conditions of exposure; thirdly, this evaluation should be based on state-of-the-art scientific concepts at the time of evaluating; fourthly, this evaluation should be cost-effective to ensure that optimal safety is assured at a manageable cost not only for the manufacturers, but for the consumers who will buy and use the product. Despite continuing efforts over the last two decades, the design of adequate nonclinical strategies for immunotoxicity evaluation is still in its infancy. A lot of work, however, has been done as regards immunosuppression, but the three other aspects of immunotoxicity, namely immunostimulation, hypersensitivity and autoimmunity have long been neglected. Much debate is still ongoing on the optimal nonclinical strategy for predicting unexpected immunosuppression. A number of inter-laboratory studies have been conducted to determine the relevance of animal models and assays. In fact, most of these studies focused on immunosuppression and very limited data are available regarding the other three aspects of immunotoxicity. This chapter is an attempt to summarize the available information in order to propose possible strategies including the four aspects of immunotoxicity based on the author’s personal experience. Importantly, the final strategy to be selected not only always depends on the nature of the drug or chemical being tested as well as the expected conditions of human treatment or exposure, but it should also take into account specific regulatory requirements, when available or applicable. GOOD LABORATORY PRACTICES IN IMMUNOTOXICOLOGY In the 1970s, the identification of frauds in preclinical laboratory studies submitted to the US FDA was an incentive to the definition of Good Laboratory Practices (GLP), which 269

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described requirements for testing facility management, data tracking, test article characterization and handling, protocol development, data acquisition, report preparation and keeping [1]. Thereafter, similar regulations have been published throughout the world and despite slight differences between national and/or international regulatory authorities, they all adhere to the same concepts [2]. Surprisingly, the application of GLP rules to preclinical immunotoxicity evaluation has seldom been addressed [3]. There are no specific requirements for immunotoxicity studies regarding animal records, handling and housing, monitoring, maintenance of laboratory equipment, data acquisition and storage, or personnel training, as compared to conventional toxicity studies. However, the impact of several factors can be highlighted. IMPACT OF ANIMAL-RELATED FACTORS ON IMMUNITY Nonclinical immunotoxicity evaluation studies normally adhere to GLP rules, especially when performed for regulatory purposes. The animal characteristics, the conditions of housing and handling are assumed to be strictly identical. However, some inadvertent or intended changes may ensue and these changes may impact on the immune system of laboratory animals. When changes are purposely made, it is important to confirm that the immune responses of animals are not affected and if they are, to measure the impact of these changes in immune responsiveness on the immunotoxic potential of known immunotoxicants. Animal source. The influence of the commercial sources of animals used in immunotoxicity studies has rarely been addressed. Kajioka et al. [4] found marked differences in the spleen weight, T lymphocyte numbers and T lymphocyte proliferative response to mitogens of C56Bl/6 mice from two different commercial sources. Animal shipping. The influence of transport conditions on immune functions has been underscored [5]. Thus, mice shipped either by truck or plane had significantly increased serum corticosterone levels with impaired humoral and cellular immunity. However, these changes were of short duration as normal values were found after 48 h post-shipping. Housing conditions. The environment of laboratory animals is extremely complex and involves many factors that must be kept constant as much as possible. These factors include light, noise, humidity, temperature, bedding, cage size and many others. Variations in housing conditions can impact on immunocompetence even though the inconsistency of reported findings is a difficulty in making any firm conclusion. More attention, however, should be paid to the possible influence of changes in housing conditions that are expected to be recommended by regulatory bodies in the very near future as they may impact on the results of immunotoxicity studies. Mice housed one per cage had stronger humoral response to SRBC than mice housed five per cage [6], whereas no differences in humoral response were evidenced in rats housed either one or five per cage [7]. Mice switched from five to one per cage had decreased humoral responses to SRBC and this decrease was inhibited by diazepam [8]. A stronger resistance to experimental Candida albicans infection was noted in mice housed one per


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cage [9] and this was linked to an increased phagocytosis of C. albicans organisms by macrophages in these animals [10]. No differences in NK cell activity were found among individually- and group-housed mice, or in mice housed individually for 4 weeks and switched to group housing [11]. Finally, the production of IL-4, but not IL-2 by splenocytes was increased in individually housed Balb/c mice as compared to group-housed mice, whereas the production of IL-2, but not IL-4 was increased in individually house C57Bl/6 mice as compared to group-housed mice [12]. Neither switching from one animal to five per cage nor from five animals to one had any influence. Conflicting results have also been published about the duration of immune changes following different conditions of housing. Thus, mice housed one or five per cage had similar humoral response to SRBC and resistance to experimental C. albicans infection after 3 weeks [9]. The role of sex was suggested in mice grouped six per cage as males had significantly lower proliferative responses than age-matched females, and individually housed males exhibited responses similar to those of grouped females [13]. Rabbits. No significant differences in antibody production and DTH responses were seen in young female New Zealand White (NZW) rabbits either individually or group-housed [14]. Turner et al. [15] compared NZW or Dutch (DXL) group-housed rabbits. Although anti-HSA antibody responses were similar, the anti-SRBC responses were significantly lower in DXL rabbits. The lymphoproliferative responses to PHA and Con-A as well as the phagocytic activity assessed by chemiluminescence responses to zymosan and latex particles was not different between NZW and DXL rabbits. Dogs. No changes in antibody response to KLH, total leukocyte counts, lymphocyte subsets and plasma cortisol levels were noted in dogs housed individually or two per cage [16]. Monkeys housed one per cage had significantly increased serum corticosterone levels and decreased T lymphocyte counts, and these changes were still present 11 weeks after removal from their social group [17]. In keeping with these results, Schapiro et al. [18] found significantly decreased CD4þ T lymphocytes in singly housed monkeys as compared to monkeys housed two or four per cage. Although, lymphocyte proliferative responses did not differ among groups, the production of interferon-g and IL-10 was markedly decreased in singly caged animals. Environmental enrichment is a current trend in regulatory toxicity studies and strict requirement are expected to be published in the near future. Several authors showed, however, immune changes in relation to enrichment, the consequences of which deserve close scrutiny as they may influence the results of nonclinical immunotoxicity studies. Decreased lymphoproliferative responses to Con-A and reduced T lymphocyte numbers were found in mice after enrichment with in-cage treading mills and various objects as compared to nonenriched mice [19]. However, enrichment-housed mice were less sensitive to immune changes induced by acute stress. Another study in male CFLP mice showed that enrichment with shelves and nest boxes was associated with decreased antibody response and resistance to Babesia microti infection [20]. In rhesus monkeys, various environmental enrichment procedures, such as toys, foraging devices and video-tapes, did not produce changes in immunocompetence, but there was a trend toward enhanced cellular immunity in enriched as compared to nonenriched animals [21]. Finally, mice housed on wood

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bedding had an increased number of Peyer’s patches and an augmented production of total IgA as compared to mice on cotton bedding [22]. Handling stress is associated with a certain degree of immunosuppression as evidenced by decreased PFC responses in stressfully handled rats [23]. Similarly, stressful handling was shown to induce decreased antibody response to KLH and ConA-induced lymphocyte proliferation in mice, but no consistent changes in lymphocyte subsets [24]. Rectal temperature measurement in mice used as handling stress was associated with significantly increased release of IL-1a, IL-1b, IL-6, TNF-a, IFN-a, but not IL-2 by peripheral blood lymphocytes stimulated by LPS [25]. Thus, appropriate experimental procedures should be implemented to avoid any stressful conditions likely to result in immune changes that might be misinterpreted as being induced by the test article. STANDARDIZATION OF REAGENTS AND ASSAYS The poor standardization of reagents and assays is another critical and as yet unsolved issue. Most immunological reagents have not been carefully standardized as other reagents used in nonclinical toxicity studies. The careful labeling of reagents, the identification of commercial sources, the consistent conditions of storage and handling are not sufficient to assure reproducible quality. SRBC are a typical example. Although, they are widely used to test humoral immunity in rodents, there is a marked variability of results that may be at least partly due to the use of different animals to produce red blood cells, but also to the delay between blood samplings in the same animal. This led the ground to the current trend to shift from SRBC to KLH as the preferred T-dependent antigen for use in assessment of humoral immunity. Although, the commercial offering for immunological reagents is rapidly expanding, there is still a lack of reagents for use in several animal species, in particular monkeys and dogs. To overcome this difficulty, it is common practice to use human reagents, especially antibodies that have been shown to cross-react with monkeys [26 –28]. However, crossreactivity does not necessarily mean that the same functions or immune cell types are actually being investigated as exemplified by NK cells in the Rhesus monkey where the CD56 marker is not specific of NK cells as in humans [29]. Standard operating procedures must be strictly adhered to by authorized personnel, but the lack of inter-laboratory standardization can nevertheless result in poorly reproducible results that cannot be merely explained by the intrinsic inter-individual variability of immune responses. It is important to bear in mind that very few, if any inter-laboratory validation studies used exactly the same procedure. Thus, future studies should pay particular attention to a strict adherence to every, even minimal aspect of the experimental protocol to avoid undue variability of results and inadequate conclusions.

STRATEGIES FOR THE PREDICTION OF IMMUNOSUPPRESSION Immunosuppressive drugs were the first therapeutic agents exerting marked effects on the immune system to be introduced into the clinical setting. Shortly thereafter, adverse effects


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associated with immunosuppression, in particular infectious complications, were described. Immunosuppression resulting from industrial or environmental chemical exposures has most often been the primary focus of immunotoxicologists, and it remains to be established that results from studies with industrial and environmental chemicals concur with results from studies with medicinal products. Although, immunostimulation, hypersensitivity, and autoimmunity are important aspects of growing concern, immunosuppression remains the main focus of nonclinical immunotoxicity evaluation. Therefore, unexpected immunosuppression is the only aspect of immunotoxicity caused by drugs and other chemicals which can be reasonably predicted today, at least to some extent. GENERAL CONSIDERATIONS Before defining the optimal strategy for the identification of unexpected immunosuppression induced by drugs and other chemicals, a number of factors should be considered. Selection of species. The selection of species for nonclinical immunotoxicity evaluation has been a matter of debate, largely because this selection had to take into consideration two critical, but often conflicting issues. It is indeed essential that a comprehensive knowledge of the immune system and immunopathology in the selected species is available together with specific immunological reagents. It is also important to bear in mind that immunotoxicity is only one aspect of the global safety evaluation of drugs and other chemicals. The observed immunotoxic effects should always be compared and interpreted in the light of all other toxic effects observed in similar conditions of exposure and metabolism. Mouse. The mouse has long been the preferred species for nonclinical immunotoxicity evaluation. The first and still most comprehensive inter-laboratory immunotoxicity validation study conducted under the auspices of the US National Program of Toxicology (NTP) used the B6C3F1 mouse [30] as the use of a hybrid strain—B6C3F1 mice are bred from C57Bl/6N female and DNA/2N male mice—was at that time expected to decrease inter-animal variability. In fact, no greater inter-animal variability was evidenced in outbred mice, such as the CD-1 mice [31]. An enormous amount of information on the mouse immune system and a nearly endless variety of murine immunological reagents are available. As mice have limited applications during conventional toxicity testing compared to other species, the interpretation of immunotoxicity changes in mice in relation to the general toxic effects of the test article may not be possible. The use of mice for nonclinical immunotoxicity evaluation is therefore limited, especially in the context of drug safety evaluation. However, the largest immunotoxicity database was compiled from mouse studies [32,33]. Most commonly recommended immune function assays [34 – 36] as well as host resistance assays [37,38], including experimental infections and implanted tumor models, have indeed been initially used in mice, and subsequently adapted whenever possible to other animal species. Rat. Limited information on the rat immune system and few immunological reagents have long restricted the selection of the rat for use in nonclinical immunotoxicity evaluation. As the rat is the first-line species for conventional toxicity studies,

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the comparison of immunotoxic effects with general toxicity in similar conditions of exposure is nearly always possible. Immunotoxicity studies have been increasingly performed in the rat, especially after the rat was shown to be an adequate species for immunotoxicity evaluation [39 – 41]. In turn, this expanded our knowledge on the rat immune system and increased the availability of rat-specific commercial reagents. Although, the rat is the most commonly used or recommended species for nonclinical immunotoxicity evaluation, differences in immunotoxic effects have been reported between mice and rats [42 – 44]. These differences may be due to metabolism of the test article, but the mechanism is not always elucidated. In any case, this highlights the need for a careful selection of species. Alternative species should always be considered. A matter of debate was whether an inbred or outbred strain of rat should be used. When the rat began to be widely considered as a possible species for immunotoxicity evaluation, the Fischer F344 rat was used by many investigators and is still widely used [44 –52]. However, several intra- and inter-laboratory validation studies showed that outbred strains of rats, such as Sprague– Dawley or Wistar rats, can also be used with acceptable interanimal variability [53 – 57]. Today, the rat is definitely the species of choice for nonclinical immunotoxicity evaluation, which does not exclude the possibility to conduct immunotoxicity studies in other animal species. Other species may indeed be more relevant depending on the origin, mechanism of action or metabolism of the test article. There is a large amount of data on histological changes in lymphoid organs of rats exposed to immunotoxicants [58 –61]. Immunophenotyping of lymphocytes subsets by flow cytometry can routinely be included in conventional toxicity as well as immunotoxicity repeated dose studies [62,63]. Validated immune function assays for use in rat immunotoxicity studies include humoral function assays, such as the direct PFC assay [64], ELISA to measure anti-SRBC [65,66], anti-KLH [67,68] and anti-tetanus toxoid [69] antibodies, and to a lesser extent ELISPOT [70]; cellular immune function assays, such as DTH response [71] and mitogen- or alloantigeninduced lymphocyte proliferation; and nonspecific immune function assays, such as NK cell activity [72] and phagocytosis [73]. As compared to mice, only a limited number of experimental infection models have been extensively studied, in particular T. spiralis [74], L. monocytogenes [75] and influenza [76] infections, but they were shown to be easily applicable and they are frequently utilized. Dog. Surprisingly, extremely few immunotoxicity studies have been conducted in the dog. The dog is an overlooked species [77], although a lot of information on the dog immune system is available [78] and the dog is the first nonrodent species for use in conventional repeated dose toxicity testing. Seemingly, there are no published data on histological changes of the dog lymphoid organs following exposure to immunotoxicants. The dog can be selected for nonclinical immunotoxicity evaluation as the main immunotoxicity end-points can be measured in the dog, such as lymphocyte subsets [79], antibody responses to T-dependent antigens [80], mitogen-induced lymphocyte proliferation [81,82], NK cell activity [83] and phagocytosis. Minipig. Although, introduced in nonclinical safety assessment approximately 2 decades ago [84], the minipig was only recently considered for use as an animal species for nonclinical immunotoxicity evaluation. The available data are, therefore, extremely limited. There is, however, evidence that minipigs can mount humoral responses to conventional


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antigens, such as SRBC [85 – 87]. Cyclophosphamide was shown to induce the expected changes in the histology of the thymus, spleen and submandibular lymph nodes, and marked decreases in neutrophils, lymphocytes and T lymphocytes [87]. Lymphocyte subsets can be analyzed and T lymphocyte proliferation measured by flow cytometry [88]. The 51Cr release assay can be used to measure NK cell activity [89]. Nonhuman primates are increasingly utilized for the nonclinical safety evaluation of medicinal products, particularly biotechnology-derived products and immunostimulatory drugs [90,91], while the immunotoxic potential of few chemicals has been investigated in nonhuman primate models [92 – 97]. Macaques, i.e. rhesus and cynomolgus, are more commonly used than marmosets. A model of delayed-type hypersensitivity was proposed in the cynomolgus monkey [98]. This model is based on the use of recall antigens, but no extensive database is available to confirm that the selected antigens are actually recall antigens in the monkey. To overcome this difficulty, monkeys can be sensitized and later challenged with the selected antigens. As delayed-type hypersensitivity using a single antigen, such as tetanus toxoid [99], is common practice, the need to use recall antigens is unclear. Lymphocyte subset analysis can be easily performed in cynomolgus [100] or rhesus monkeys [101], and marmosets [102], but due to lack of specific reagents, crossreacting human reagents often have to be used [26,103]. Phagocytosis can be measured by flow cytometry [80] and NK cell activity by the 51Cr release assay [104]. Selection of doses. As in any toxicity testing, the selection of doses is a critical step. Ideally, one selected dose of the tested chemical at least should induce histological or functional changes in animals for extrapolation to man and risk assessment. In fact, the selection of doses for the identification of unexpected immunosuppression is difficult. High doses that are more likely to induce statistically significant impairment of immune function or changes in the histology or lymphoid organs may also induce overt toxicity. Overt toxicity is associated with stress resulting in increased secretion of corticosterone. Correlations are often noted between corticosterone levels and immunosuppression in stressful situations [105,106]. In addition, overt toxicity is typically associated with reduced food intake and malnutrition is another well-recognized cause of immunosuppression. Increased corticosterone levels and malnutrition are associated with thymus atrophy [107 – 109]. Although, changes in the histology of lymphoid organs induced by stressrelated general toxicity during conventional repeated-dose toxicity studies have been claimed to be distinguishable from histological changes related to direct immunotoxicity, the selection of a dose-producing overt toxicity is not recommended. Indirect immunotoxicity as a consequence of general toxicity can indeed result in impaired immune function. It is also important to bear in mind that the objective of nonclinical immunotoxicity studies is the identification of direct immunotoxicants and not the identification of indirect immunotoxic effects related to general toxicity. There is a general agreement that the selected high dose should be not to produce a decrease in weight gain more than 10% as compared to controls. Dose –response relationships in immunosuppression are generally thought to be linear with a threshold [110]. In fact, there is some evidence that dose responses may not always be linear, but follow a hockey cross-type curve. Thus, a high dose of a given compound may cause immunosuppression, an intermediate dose may induce no effect and a low dose may

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be associated with immunostimulatory effect [111 –113]. These findings are in keeping with the concept of hormesis [114,115]. The selection of appropriate dose levels is, therefore, critical for the purpose of hazard identification and risk assessment. When a dose level much higher than the equivalent of the therapeutic dose is selected and even though no overt toxicity is induced, unexpected immunosuppression may be evidenced. This finding may be of no practical relevance for safety evaluation if a low dose close to the therapeutic dose is associated with immunostimulation. The reasoning based on the comparison to the therapeutic dose is also applicable to occupational and environmental exposure. Therefore, the selection of an exceedingly high dose is always inappropriate either because overt toxicity may hide direct immunotoxic effects or because the observed immunotoxic effects may not be extrapolated to man. In contrast, a low dose as reasonably low as possible should also be selected to assure the dose response curve of the immunotoxic effects of the test article is known. Exposure duration. It is common practice to treat or expose rodents for 28 consecutive days as shorter periods of exposure, e.g. 14 days have been shown to produce unexpectedly negative results [32]. The selected period of exposure should be long enough to show alterations in the synthesis of antibodies. There are no data whether long-term studies are helpful to identify immunotoxic effects or changes that could not be detected in short-term studies, or alternatively whether immunotoxic effects evidenced in short-term studies tend to alleviate in long-term studies. Comparative studies, however, have seemingly never been conducted. To date, there is no reason to avoid the inclusion of immunological end-points in long-term conventional toxicity studies or to perform immunotoxicity studies longer than 28 days, particularly in larger animal species, such as the dog and monkey. Route of exposure. The widely accepted rules of safety evaluation should apply and the route(s) of exposure in immunotoxicity evaluation should be the route(s) of expected exposure in humans. The oral route poses no particular difficulties as most immunotoxicity animal studies have been performed following oral exposure. In contrast, other routes, such as inhalation [116 –120], have rarely been used. Due to the lack of historical background data, the interpretation of results may be difficult. Thus, differences in immunotoxic effects have been shown depending on the route of exposure [121,122]. Conventional vs. focused immunotoxicity studies. Nonclinical immunotoxicity evaluation can be done either during conventional toxicity studies or in focused immunotoxicity studies. In conventional toxicity studies, satellite groups of animals are generally used for immune function assays because of the widely held, although not substantiated belief that the immunization of animals may induce inadvertent changes in the toxic profile of the test article. The FDA guidance on the immunotoxicology evaluation of investigational new drugs unfortunately supports this belief [123]. Several studies have indeed shown that the immunization of animals is acceptable [64,124,125] and so far no published studies have shown it is not. The simultaneous immunization with different antigens in the same animal was even proposed [126]. When satellite groups of animals are used for immunotoxicity evaluation in a conventional repeated dose toxicity study, the selected doses may not be appropriate as


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the high dose is normally aimed at inducing overt toxicity to identify target organs. If immune function assays are performed with only the two lower doses, a full immunotoxicity evaluation may not be possible. Therefore, focused immunotoxicity studies are recommended. However, they should not include a single immune function assay, but also a histological examination of the lymphoid organs and the measurement of standard hematology and clinical chemistry parameters. Three doses should preferably be used, of which one or two have already been tested in conventional toxicity studies in the same species. Thus, a global interpretation is possible taking into account the results of the immune function assay, and the changes in histology of the lymphoid organs, and standard hematology and clinical chemistry, compared to the toxicity profile of the test article in the same species. Timing of nonclinical immunotoxicity studies in drug development. Available immunotoxicity guidelines do not give any indication on the optimal timing for conducting nonclinical immunotoxicity studies during drug development. An emerging consensus in the pharmaceutical industry, is that such studies should be performed prior to or during phase II clinical trials [127]. From a medical perspective, this timing is inadequate. As the immune system is a significant target organ of toxicity, it is debatable if not unethical to expose human volunteers or patients to a drug that can be immunotoxic since conventional repeated dose toxicity testing cannot reliably predict for this risk. Furthermore, it is essential that immunological end-points are included in clinical trials for systematic further immunotoxicity evaluation. For bridging animal and human studies, it is logical to select end-points that have already been investigated in nonclinical studies. This is impossible if nonclinical immunotoxicity studies are conducted at a late stage of drug development. As immunotoxicity is a significant cause of morbidity and mortality, it would instead seem logical to screen for the immunotoxicity potential of new molecular entity at a very early stage. The inclusion of immune function assays in safety pharmacology studies, which are normally performed prior to conducting clinical trials is worth considering. The direct PFC assay or DTH response in mice which enable the detection of unexpected immunosuppression rapidly and cost-effectively are typical examples of simple assays that can be used as a very early screen [128]. SELECTION OF ASSAYS As already mentioned, quite a few assays have been standardized and validated in interlaboratory studies. Validation in mice is quite extensive and a fairly large number of compounds have been tested [32,33]. However, only one large inter-laboratory validation study has been conducted in mice and few medicinal products have been tested. In contrast, several inter-laboratory validation studies have been conducted in rats, but they compared results obtained only with cyclosporine or azathioprine [43,44,48,53,54,56]. No validation studies have ever been conducted in nonrodent species. Following the pioneering work of American authors in the late 1970s [129], the concept of tiered protocols was introduced into nonclinical immunotoxicity evaluation and has since then been widely utilized. Originally, a 2-tier approach was proposed including the tier I (screening phase), which consisted of blood cell counts, weight and histology of

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lymphoid organs, the direct PFC assay, lymphocyte proliferation response and NK cell activity. Depending on the results of the first tier, a list of additional assays was suggested for mechanistic purpose (tier II) including enumeration of B and T lymphocytes, antibody response to a T-independent antigen, DTH response, T lymphocyte cytotoxicity, macrophage function and host resistance assays. Over the years with the refinement or introduction of new techniques, the contents of tiered protocols markedly evolved to be reduced to a cost-effective minimum battery of end-points, which have proven sensitivity to detect the potential of new drugs and other chemicals to induce unexpected immunosuppression. Although, the term of tiered protocols is less frequently used, the general stepwise concept for nonclinical immunotoxicity evaluation is still valid. First-line assays are intended to be used as a first screen to identify the potential of drugs and other chemicals to exert unexpected immunosuppressive effects. As compared to the original tier-I, fewer assays are nowadays considered useful as first-line assays. Histological examination of the lymphoid organs. The histological examination of lymphoid organs is unambiguously considered as an essential component of nonclinical immunotoxicity studies intended to detect unexpected immunosuppression. As a matter of fact, it is required by all recent immunotoxicity guidelines since the adoption of OECD Guideline 407 in 1995 [130]. The thymus, spleen, lymph nodes and Peyer’s patches should be examined using standard histological techniques. The examination should be carefully conducted to avoid misinterpretation [131,132]. Quantitative microscopic and immunohistochemical techniques are being considered as add-ons to conventional examination [133,134], but so far they have not been conclusively demonstrated to significantly enhance sensitivity although they can be useful add-ons in particular circumstances. Although focused immunotoxicity evaluation is often restricted to one animal species, it is recommended that a histological examination of lymphoid organs be included in every conventional repeated dose toxicity study whatever the species or the duration of the study. As marked differences in the immune system exist across species, histological examination of the lymphoid organs in several species [135] is likely to enhance the detection of unexpected immunosuppression, to allow for comparisons among species and to identify possible species-specific effects. Standard hematology and clinical chemistry are essential components of nonclinical immunotoxicity evaluation as changes, such as reduction in total or specific blood cell counts may serve as a predictor for possible immunotoxicity. Results of standard hematology and clinical chemistry are also useful to guide the interpretation of histological changes. T-dependent antibody response. Histological examination of the lymphoid organs has been considered as a sensitive predictor of unexpected immunosuppression by several authors [59,136– 139] and this view is supported by the recent FDA guideline on the immunotoxicology evaluation of investigational new drugs [123]. However, the recent findings that decreased PFC responses may not be associated with histological changes [140] challenge this conservative view. Therefore, a T-dependent antibody response assay is strongly recommended as a systematic first-line assay for the identification of unexpected immunosuppressive drugs and chemicals.


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The direct PFC assay has long been the reference assay following the demonstration by the NTP inter-laboratory validation study that it was the most sensitive immune function assay [32]. ELISA has been proposed as an alternative to measure primary antibody responses to SRBC, but since SRBC are standardized antigens, other T-dependent antigens, in particular KLH [67, 68, 141], are increasingly used. A major advantage of KLH is that it can be used in nonrodent species. ELISA are less time-consuming, can be automated and samples can be frozen for subsequent assay. Although, ELISA may become more and more popular, there is a need to standardize experimental protocols and to perform validation studies to determine the sensitivity of the results for the identification of unexpected immunosuppression and risk assessment. Second-line assays are primarily recommended when first-line assays suggest an unexpected immunosuppressive potential of the test article and the selection of assays will depend on the type and magnitude of changes observed in first-line assays. In addition, second-line assays may be necessary when it bears structural or biological similarities with a known immunosuppressive drug or chemical. An extensive nonclinical immunotoxicity evaluation including second-line assays may also be required when the test article is an investigational new drug intended for use as an immunosuppressive drug, or in AIDS patients or pregnant women, or finally when there is suggestion that it accumulates in lymphoid tissues [123]. A description of the main assays to be considered is available in Chapter 5 of this volume. It is, however, important to keep in mind that none of these assays has been extensively validated and that the selection should be based on a case by case approach. Lymphocyte subset analysis is often included in conventional repeated dose toxicity studies as no satellite groups of animals are necessary. It is important to bear in mind that the predictive value of lymphocyte immunophenotyping is not known. Recently a panel of experts reached the conclusion that validation is insufficient to recommend its inclusion in routine immunotoxicity studies [144]. Although, the CPMP considers this assay as an acceptable first-line assay if combined with NK cell activity measurement [145], it is not recommended to rely on lymphocyte subset analysis as a sensitive predictor of unexpected immunosuppression. Serum immunoglobulin levels are another end-point frequently included in conventional repeated dose toxicity studies as this is an easy and inexpensive assay. However, the predictive value of this assay is extremely limited. Cellular immunity assays, either DTH response or mitogen- and alloantigen-induced lymphocyte proliferation can be used. There is a general agreement that either assay is equally sensitive. The selection will, therefore, be based on in-house expertise and historical data. Phagocyte functions. A wide variety of assays can be used to assess phagocyte functions, but they are rarely included in nonclinical immunotoxicity evaluation. Flow cytometry measurement of phagocytosis and oxidative burst can easily be performed in most conventional animal species including rats, monkeys and dogs. NK cell activity is sometimes considered as a first-line assay [145]. In fact, very rare medicinal products reproducibly decreased NK cell activity and the predictive value of this assay is not known.

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STRATEGIES FOR THE PREDICTION OF IMMUNOSTIMULATION Due the very limited published data, no general strategy can be proposed for routine use in nonclinical immunotoxicity evaluation. Few systematic studies have been published, but unexpected immunostimulation seems to be very infrequent and, in any case, much less frequent than unexpected immunosuppression [57]. Immunostimulation corresponds to the enhancement of otherwise normal immune responses. There are reports that the histological examination of the lymphoid organs can detect changes suggestive of immunostimulation, such as hyperplasia of the bone marrow, red and white pulps of the spleen and lymph nodes with mononuclear cell infiltration and more numerous germinal centers [146,147]. Splenomegaly may also be observed. Animal models and assays that are being recommended for the identification of unexpected immunosuppression have rarely been used [148]. One partly unsolved question is whether assays that have been designed to detect immunosuppression can also serve as predictors of unexpected immunostimulation. In the former situation, a strong antigen-specific or mitogen-induced response is likely to be more susceptible to immunosuppressive effects. Few published results tend to demonstrate that using the same experimental protocol, unexpected immunostimulation can be evidenced. Thus, enhancement of PFC responses or anti-tetanus antibody production [149,150] as well as DTH responses [151,152] has been reported with several immunostimulatory drugs. However, it must be kept in mind that no inter-laboratory studies have ever been conducted to validate these animal models and assays for the identification of unexpected immunosuppression. As reviewed in Chapter 6 of this volume, host resistance models should focus on the known adverse consequences of immunostimulation. No model has properly validated and the validity of autoimmunity-prone rodent species is unknown. Finally, in vitro cytokine release assays have been shown to be sensitive predictors for the risk of flu-like reactions [153], the validity of autoimmunity-prone rodent species for predicting the risk of autoimmune reactions is not known.

STRATEGIES FOR THE PREDICTION OF HYPERSENSITIVITY The prediction of the sensitizing potential of most low-molecular-weight drugs and other chemicals is nearly impossible to date [154]. Exceptions may be highly reactive respiratory industrial or environmental chemicals although no comprehensive interlaboratory validation studies of available animal models and assays have so far been conducted. Models for the identification of allergenicity of proteins, either therapeutic [155] or food [156] proteins are still under development. However, most contact sensitizers can reliably be detected using either guinea pig or mouse (LLNA) assays as described in Chapter 7 of this volume. A number of experimental approaches are under development to predict the risk of systemic hypersensitivity reactions involving either IgE or T lymphocytes. None of these approaches has been properly validated and their use should be restricted to very specific situations, especially for mechanistic purpose.


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Similarly, no general strategy can be proposed for the prediction of pseudoallergic reactions. Selection of relevant assays will depend on the chemical structure and nature of the test article, the route of administration (intravenous), the solvent used and the results of conventional repeated dose toxicity studies.

STRATEGIES FOR THE PREDICTION OF AUTOIMMUNE REACTIONS Systemic as well as autoimmune reactions cannot be predicted for the time being. Although, many studies have been devoted to validating and refining the PLNA, it is still not known to what extent, if any it can prove useful to identify drugs and other chemicals that can induce systemic autoimmune reactions and possibly certain types of hypersensitivity reactions as well. More research is needed before it can be recommended for routine evaluation. The search of autoantibodies in the sera of animals during conventional repeated dose toxicity studies is usually negative, whatever the test article.

RISK ASSESSMENT Risk assessment is a systematic scientific characterization of potential adverse health effects resulting from human exposure to hazardous agents or situations [157]. DEFINITIONS AND CONCEPT Hazard and risk. Although the terms hazard and risk are often used interchangeably, their definition is different. Hazard refers to the intrinsic toxic properties of a substance. Risk is the likelihood for these toxic properties to result in adverse health effects in an individual or in a population following particular conditions of exposure. In contrast to hazard that can be objectively determined provided adequate methodologies are selected and used, uncertainty is a major and unavoidable feature of risk. Risk assessment process. Risk assessment is a process including four steps. Hazard identification is the first step of the risk assessment process. In toxicology, hazard equals toxicity. The data usually derive from animal studies, structure – activity relationships, in vitro studies and rarely from epidemiological or population-based studies in humans. The quantitative nature of the relationship between the dose and the toxic response is considered in the second step or dose –response assessment. When a threshold is assumed to exist beyond which toxicity develops, the assumption can be made that no toxicity will occur below the threshold. Major difficulties are the extrapolation from animals to man and from high to low doses. Doses used in animal toxicity studies are arbitrarily selected and the relative sensitivity of animal species and man to a given toxicant is generally unknown, especially as few human data are available. To overcome this difficulty, a no-effect dose level (NOEL) is determined during animal toxicity studies and uncertainty factors are applied to take into account intra-species and inter-species variability. As there are many

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causes of variations in the determination of the NOEL, the benchmark dose approach is increasingly used. It is a mathematically derived model that yields a statistical lower confidence level on the dose producing a predetermined level of toxic effects. Uncertainty factors can also be applied to this approach. The third step—exposure assessment—attempts to determine the duration, magnitude, frequency and routes of exposures as well as the number and characteristics of exposed subjects, e.g. at risk subjects, age, gender, underlying pathological conditions. This is the most neglected aspect of the risk assessment process. Risk characterization is the final step of the process during which the incidence of toxic effects is estimated in a given population. The reliability and acceptability of the estimate depend on the quality of the whole process and on the identification of uncertainties. This is fundamentally a science-based judgment, but since the final results of the risk assessment process are aimed at determining adequate measures to control or remove the risk (risk management), the perception of risk should also be taken into consideration. IMMUNOTOXICITY RISK ASSESSMENT No studies specifically focused on issues related to immunotoxicity risk assessment. This is, however, an area of slowly growing interest [158 –164]. General considerations. In general, immunotoxicity studies were designed for hazard identification and there is an immense database of results demonstrating the immunotoxic effects of drugs and other chemicals as reviewed in volumes II and III. However, a significant number of studies have been conducted in experimental conditions that preclude straightforward extrapolation from animal to man. A threshold for immunosuppression [110] and contact sensitization [165] has been evidenced and quantitative risk assessment is therefore possible [166]. Immunotoxicity risk assessment was often proposed as the mere extrapolation of animal data whether or not they have demonstrated relevance for the prediction of unexpected immunosuppression. Therefore, the question of the extrapolation of animal results to man has been repeatedly raised [157,167– 169], but no definitive and widely accepted answer has so far been provided. The parallelogram concept in which comparisons between results of in vivo animal models and in vitro assays are compared with results of human in vitro assays is an attempt to predict in vivo human effects (Fig. 1). Although, this approach is often referred to, its

Fig. 1. The parallelogram approach in risk assessment.


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validity for risk assessment as never been critically assessed because human data, both in vivo and in vitro, are often lacking. Critical factors in immunotoxicity risk assessment. During the process of risk assessment, immunotoxicologists are faced with several major challenges. Dose –response. Dose –response relationships of immunotoxic effects are often atypical. As mentioned above, a hockey cross-type curve may be seen with drugs and other chemicals that are immunosuppressive at high doses, but immunostimulatory at lower doses. Depending on the expected level of human exposure, adverse health consequences may be totally different. For the purpose of risk assessment, the selection of doses for use in nonclinical immunotoxicity studies should not disregard the expected level of human exposure. Criteria selected for follow-up of exposed groups of the general population should also take into account the expected consequences to be seen in realistic conditions of exposure based on relevant experimental evidence. Thus, although metals are widely considered as immunosuppressive chemicals, the clinical human data evidence either no effects or immunostimulation. Dose– response curves are also often atypical as far as hypersensitivity reactions are concerned. Although, a linear dose – response with a threshold is increasingly accepted for contact sensitizers [167], a bell-shaped curve is probably a general rule with most antigen-specific reactions. This unusual dose – response curve should have an influence of the selection of doses as only a narrow range of doses or concentrations is likely to induce antigen-specific hypersensitivity, whereas lower as well as higher doses or concentrations may be associated with false negative responses. Redundancy refers to the capacity of the immune system to trigger compensatory mechanisms when one effector mechanism is inhibited. Thus, changes in one single immune function assay or subtle histological changes should not be considered as sufficient evidence that the test article is immunotoxic. The logical consequences are that more than one end-point should be used to predict the potential for immunotoxicity and that a global evaluation of all the results of immunotoxicity as well as general toxicity studies is essential to avoid misleading conclusions. Functional reserve capacity. Because of the redundancy of many effector mechanisms and the pleiotropic effects of many immunocompetent cells and soluble factors, such as cytokines, there is a wide functional reserve functional capacity of the immune system. This is a widely accepted but very general concept. To date, no sufficient database enables to correlate mild to moderate changes in animal or human immune functions with clinical adverse health effects. Genetic factors are thought to play a key role not only in the metabolic capacities of the host, but also in the nature and magnitude of immune responses. The intrinsic inter-individual variability of immune responses is the reason why the variability of immunotoxicity results is usually greater than that of conventional repeated dose toxicity studies. It is, therefore, advisable that the number of animals be sufficient to keep the power of statistical analysis at an acceptable level. The comparison of results in treated animals to those of study controls as well as historical controls is necessary as the percentage of responder and nonresponder animals in any group, including the control group cannot be predicted. As genetic predisposition is known to play an important role in hypersensitivity

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and autoimmune reactions, this should be an incentive to the development of genetically prone or genetically modified animal models. Interspecies differences are not a specific characteristic of the immune system [35]. However, they do exist and should be taken into account for the selection of animal species and the interpretation of results. Therefore, rodent studies cannot be expected to provide reliable identification of immunotoxicants and studies in nonrodent species and man are critical to confirm initial results obtained in rodents. To improve the applicability of immunological end-points measurements in most conventional repeated dose toxicity studies, there is a need to design, standardize and validate noninvasive or ex vivo assays that can be performed without disturbing the normal design of these studies. It is logical that certain immunological end-points can be measured in rodent, nonrodent and clinical studies. As regards unexpected immunosuppression, a T-dependent antibody response assay is often considered as a sensitive initial immune function screen. SRBC can be only used in rodents and KLH is a logical alternative antigen. However, even though KLH has sometimes being used as an human antigen [171], sensitization to KLH for measuring either humoral or cellular immunity cannot be recommended in human clinical trials as there is a recognized greater risk of hypersensitivity reactions to sea foods and other allergens in KLH- or hemocyanin-sensitized subjects [172 –175]. Cytokine assays when properly standardized and validated may prove useful end-points in the future.

CONCLUSION Although a large number of animal models and assays have been used to study the immunotoxic potential of drugs and other chemicals, very few have been properly standardized and validated. Therefore, the selection of optimal strategies for the identification of immunotoxicity, especially for regulatory purposes is always difficult and even impossible as regards most hypersensitivity reactions, and autoimmune reactions. Combined efforts in the industry and academia should therefore be paid to defining which animal models and assays are the most valid for nonclinical immunotoxicity evaluation and to designing more sensitive predictive models.

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[147] Cesario TC, Vaziri ND, Ulich TR, Khamiseh G, Oveisi F, Rahimzadeh M, Yousefi S, Pandian MR (1991) Functional, biochemical, and histopathologic consequences of high-dose interleukin-2 administration in rats. J Lab Clin Med, 118, 81 – 88. [148] Weston BJ, Henderson DC, Seal S, Spackman V, Robertson F, Dewdney JM (1991) Stimulatory effects of FK156 in a panel of tests designed to detect changes in immune function. Int J Immunopharmacol, 13, 117– 124. [149] Cleveland RP, Kumar A (1987) Enhancement of anti-sheep erythrocyte plaque-forming cell levels by cimetidine in vivo. Immunopharmacology, 14, 145– 150. [150] Eastcott JW, Holmberg CJ, Dewhirst FE, Esch TR, Smith DJ, Taubman MA (2001) Oligonucleotide containing CpG motifs enhances immune response to mucosally or systemically administered tetanus toxoid. Vaccine, 19, 1636 –1642. [151] Descotes J, Tedone R, Evreux JC (1983) Effects of cimetidine and ranitidine on delayed-type hypersensitivity. Immunopharmacology, 6, 31 – 35. [152] Yoshikai Y, Miake S, Koga T, Watanabe Y, Nomoto K (1984) Effect of Bacillus Calmette– Guerin infection on delayed footpad reaction to Listeria monocytogenes. Cell Immunol, 83, 404– 413. [153] Oshiro Y, Morris DL (1997) TNF-alpha release from human peripheral blood mononuclear cells to predict the proinflammatory activity of cytokines and growth factors. J Pharmacol Toxicol Methods, 37, 55 – 59. [154] Choquet-Kastylevsky G, Descotes J (1998) Value of animal models for predicting hypersensitivity reactions to medicinal products. Toxicology, 129, 27 – 35. [155] Wierda D, Smith HW, Zwickl CM (2001) Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology, 158, 71 – 74. [156] Atherton KT, Dearman RJ, Kimber I (2002) Protein allergenicity in mice: a potential approach for hazard identification. Ann NY Acad Sci, 964, 163– 171. [157] Faustman EM, Omenn GS (2001) Risk assessment. In: Klaasen DC (Ed) Casarett’s and Doull’s Toxicology. The Basic Science of Poisons, 6th edition, pp 83 – 104. McGraw-Hill, New York. [158] Luster MI, Pait DG, Portier C, Rosenthal GJ, Germolec DR, Comment CE, Munson AE, White K, Pollock P (1992) Qualitative and quantitative experimental models to aid in risk assessment for immunotoxicology. Toxicol Lett, 64/65, 71 – 78. [159] Luster MI, Portier C, Pait DG, Germolec DR (1994) Use of animal studies in risk assessment for immunotoxicology. Toxicology, 92, 229– 243. [160] Selgrade MK, Cooper KD, Devlin RB, van Loveren H, Biagini RE, Luster MI (1995) Immunotoxicity—bridging the gap between animal research and human health effects. Fundam Appl Toxicol, 24, 13 –21. [161] Van Loveren H, De Jong WH, Vandebriel RJ, Vos JG, Garssen J (1998) Risk assessment and immunotoxicology. Toxicol Lett, 102/103, 261– 265. [162] Selgrade MK (1999) Use of immunotoxicity data in health risk assessments: uncertainties and research to improve the process. Toxicology, 133, 59 – 72. [163] Thomas PT (1998) Immunotoxicology: hazard identification and risk assessment. Nutr Rev, 56(Suppl.), 131– 134. [164] Descotes J (2003) From clinical to human toxicology: linking animal research and risk assessment in man. Toxicol Lett, 140/141, 3 – 10. [165] Boukhman MP, Maibach HI (2001) Thresholds in contact sensitization: immunologic mechanisms and experimental evidence in humans—an overview. Food Chem Toxicol, 39, 1125– 1134. [166] Felter SP, Ryan CA, Basketter DA, Gilmour NJ, Gerberick GF (2003) Application of the risk assessment paradigm to the induction of allergic contact dermatitis. Regul Toxicol Pharmacol, 37, 1 – 10. [167] Van Loveren H, Steerenberg PA, Vos JG (1995) Early detection of immunotoxicity: from animal studies to human biomonitoring. Toxicol Lett, 77, 73 – 80. [168] Vos JG, Van Loveren H (1995) Markers for immunotoxic effects in rodents and man. Toxicol Lett, 82/83, 385– 394.


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Chapter 11

Clinical immunotoxicology

INTRODUCTION Immunotoxicology is definitely an area of toxicology. Experimental research is generally a more developed aspect of toxicology despite the obvious, although frequently overlooked need to link animal research to human toxicity since toxicological research is genuinely not intended to enable the identification and understanding of the toxic effects of xenobiotics in rodents, but clearly to assure an optimal level of safety to exposed human beings [1]. Thus, there is a need to promote clinical research and to use more efficiently the existing human databases [2]. Accordingly, immunotoxicologists primarily focused their efforts on unraveling the fundamental mechanisms of immunotoxic effects and on designing new animal models and assays applicable to the nonclinical immunotoxicity evaluation of drugs and other chemicals. As described in volumes II and III, there is a wealth of experimental data describing the immunotoxic effects of drugs and other chemicals as well as their mechanisms of action, essentially in rodent systems. However, very little information is available on the immunotoxic effects of most xenobiotics in exposed humans. Over the years, the clinical aspects of immunotoxicity are nevertheless a matter of growing interest and investigation [3 – 12].

OBJECTIVES OF CLINICAL IMMUNOTOXICOLOGY Clinical immunotoxicology remains a relatively neglected area of immunotoxicology as compared to other areas that focus much more attention and attract more research and funding. Anyway, clinical immunotoxicology should become an increasingly important area of immunotoxicology as our current ignorance of immunotoxic effects in man is a major hurdle to the development and acceptability of immunotoxicity evaluation and more importantly, may represent a threat to public health by allowing the commercial use of or the exposure to potentially immunotoxic drugs and other chemicals that have not been properly tested in nonclinical immunotoxicity studies and subsequently in human studies. 295

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Relatively few comprehensive human immunotoxicity studies have so far been conducted [13 – 25], whereas a large number of different immunotoxic effects have been described in humans as reviewed in Chapter 3 of this volume. At least three major objectives of clinical immunotoxicology can be proposed. IDENTIFICATION OF IMMUNOTOXICANTS IN HUMAN BEINGS In contrast to the extremely large database on immunotoxic effects that has been accumulated primarily in rodents over the years, the currently available database on immunotoxic effects in human subjects is so small that the immunotoxic effects of many xenobiotics which have been extensively studied in laboratory animals are totally unknown in humans. There is a long and thus increasingly urgent need to conduct clinical immunotoxicity studies to construct a decent and needed database of immunotoxic effects in man. Various clinical studies of relevance for immunotoxicity evaluation can be conducted. Clinical trials of drugs. Guidelines requiring that the immunotoxic potential of drug candidates be assessed during nonclinical studies have recently been released [26,27]. Although, immunotoxicity assessment is to date primarily based on rodent studies, the primary aim of nonclinical toxicity studies is to identify safety issues for subsequent evaluation during clinical trials. It can, therefore, be suspected that inclusion of immunological end-points will be more and more often necessary to confirm or rule out immunotoxic effects demonstrated or suggested during nonclinical studies. Unfortunately, scarce attention has so far been paid to the role of clinical trials in immunotoxicity evaluation and the optimal protocol to be used depending on the type of drug candidate [28,29]. Immunological end-points in clinical trials may consist of immune function assays similar to those used in rodent and nonrodent studies for the identification of unexpected immunosuppression or immunostimulation. For bridging purpose of nonclinical with clinical findings, the use of some immune function assays that can be performed in rodent and nonrodent animals as well as in man is an attractive solution. For example, as T-dependent antibody responses are widely considered as the more sensitive screen for unexpected immunosuppression, the selection of the same antigen throughout nonclinical and clinical immunotoxicity evaluation may generate results that can be more easily interpreted. SRBC are not applicable to man. KLH can be used in all mammalian species including man, but KLH sensitization has been reported to induce sea food allergies as well as possible allergies to common allergens, such as dust mites [30 – 33]. Tetanus toxoid may therefore be a more adequate T-dependent antigen. Clinical observations reflecting adverse events developing in treated vs. control patients are important to consider carefully during clinical trials. Infections are the most frequent complications of unexpected or intended immunosuppressive drugs. Although opportunistic infections are easily diagnosed, a mild to moderate immunosuppressive drug may induce more frequent, but clinically and microbiologically unremarkable infections. Therefore, the statistical comparison of the incidence of carefully diagnosed infections may be the only way to demonstrate whether impaired immune function or histological changes


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in the lymphoid organs of rodents are associated with clinically relevant consequences in treated patients or not. The clinical protocol should, therefore, include a detailed flowchart of clinical, biological and microbiological end-points to be documented to avoid vague retrospective diagnosis. The same approach applies to hypersensitivity and autoimmune reactions, even though nonclinical repeated dose toxicity studies are unlikely to serve as reliable predictors.

Epidemiological studies. Epidemiological and population-based studies are similarly urgently needed as discussed later. They may focus on predefined immune function changes or immune-related diseases, and search for possible correlations with chemical exposures, or alternatively try to identify causal links between exposure to a given immunotoxicant and functional or clinical immune-mediated adverse effects.

SURVEILLANCE OF AT-RISK HUMANS EXPOSED TO IMMUNOTOXICANTS There are plenty of experimental studies that suggest that the incidence and the severity of immunotoxic effects may depend on various contributing or predisposing factors [34]. Age is the most unequivocally contributing factor, as the functioning of the immune system is not optimal in either young [35 – 37] or old [38 –42] subjects, and this can result in more pronounced immunotoxic effects. Genetic predisposition, pre-existing immune status or associated illness may also be significant predisposing factors. As the exact role of these factors is not known in human subjects exposed to immunotoxicants, there is a need to substantiate or rule out the assumption that immunotoxic effects may be altered when supposedly predisposing factors are present. When at-risk groups of the population have been unambiguously identified, an immunological follow-up may be an early detector of immunotoxicity as suggested with beryllium exposure and the risk of chronic beryllium disease in occupationally exposed workers [43].

VALIDATION OF NONCLINICAL IMMUNOTOXICITY ASSAYS AND ANIMAL MODELS The results of nearly all currently recommended animal models and assays for the nonclinical immunotoxicity evaluation of drugs and other chemicals have not been adequately compared to human results obtained using similar assays in relevant conditions of exposure. Clinical trials and epidemiological studies including immunological endpoints and clinical observations of immune-mediated pathological conditions are necessary for the validation of animal models and data. Extrapolation from animal to man can only be expected to bear some relevance when a sufficient number of reference immunotoxicants have been tested in animals and man. Human data are needed to validate selected immunological assays and end-points for use in the nonclinical immunotoxicity evaluation of new molecular entities.

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CLINICAL ASSESSMENT OF IMMUNOTOXICITY Over the years, clinical immunologists designed and used a wide variety of assays for the assessment of immune function in human beings. Although these assays were not intended for use in immunotoxicity assessment, but more often for the assessment of humans with suspected immune deficiencies, they may be considered for inclusion in clinical trials or epidemiological studies [44 –48]. Many assays are also used in nonclinical immunotoxicity evaluation studies. A description of these assays can be found in Chapters 5– 8 of this volume and only specific aspects in relation to their use in human beings will be addressed in this chapter. CLINICAL IMMUNOLOGY END-POINTS TO ASSESS HUMORAL IMMUNITY Antibody responses to a T-dependent antigen are increasingly considered as the best early predictor of unexpected immunosuppression. Although primary immune deficiencies involving humoral immunity are typically associated with slightly decreased lymphocyte numbers as there are normally fewer B lymphocytes than T lymphocytes in the peripheral blood, and lower serum immunoglobulin levels, both parameters are unlikely to be sensitive predictors of immunosuppression due to treatment or exposure to drugs and other chemicals. B lymphocytes can be counted by flow cytometry. Using specific B lymphocytespecific markers, CD19þ and CD20þ normally account for 4 and 10% of lymphocytes in the peripheral blood, respectively. The use of vaccine antigens is a theoretically interesting, but as yet nonvalidated alternative [49]. Tetanus toxoid has been used in rodent [50 – 55] and nonrodent [56,57] studies. Booster immunization of normal individuals with tetanus toxoid produces significant levels of plasma anti-tetanus toxoid IgM and IgG that can be detected by ELISA [58]. This method is commonly used in clinical immunology to investigate patients with a suspected diagnosis of innate or acquired immune deficiency [59 – 64]. Influenza virus has been used for the induction of experimental infections in rodents, but rarely for the assessment of humoral immunity. Burns et al. [65] investigated the utility of influenza vaccination as a tool to assess humoral immunity in occupationally exposed employees. As only 50% of the tested subjects developed a significant primary antibody response measured by ELISA, their conclusion was that influenza vaccination is not recommended to assess humoral immunity in human subjects. B lymphocyte function can be evaluated in vitro in lymphocyte proliferation assays using mitogens. PWM requires the presence of T lymphocytes to induce the proliferation of B lymphocytes. Other mitogens, such as LPS, induce a specific B lymphocyte proliferative response. CLINICAL IMMUNOLOGY END-POINTS TO ASSESS CELLULAR IMMUNITY Cellular immunity can be assessed from the enumeration or function of T lymphocytes. Overall, in vitro or in vivo assays can be used to measure cellular immunity. In vivo assays offer the advantage of not requiring laboratory facilities, but they are often poorly standardized. Both in vitro and in vivo assays are characterized by wide variations among


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normal individuals. Conflicting conclusions have been made when comparing in vitro and in vivo assays [66 – 68]. Lymphocyte subset analysis. Measurement of total T lymphocytes and subsets can be easily performed by flow cytometry. Typically, human T lymphocytes are analyzed using the CD3, CD4 and CD8 cell surface markers. The use of activation surface markers, such as CD25, CD40, CD69 or CD71 is expected to provide useful information, but there is still limited consensus as regards the selection of the most sensitive activation surface markers and the significance of observed changes in the expression of these markers. Quantification of NK cells and monocytes is also possible. In vitro assays require specialized equipment and technical skill, and viable cells must be rapidly delivered to the laboratory performing these assays, which can be a limitation to the use of these assays when treated or exposed human beings are remote from the laboratory performing these assays. Lymphocyte proliferation assays have long been used to measure cellular immune responses in humans. The mitogens PHA and Con-A have long been used as triggers of T lymphocyte proliferation, but the preferred technique nowadays is anti-CD3 Mabs, which are directed against the TCR. Recall antigens, such as tetanus toxoid or candida, and irradiated allogeneic lymphocytes can also be used. Typically, T lymphocytes are cultured for 3 –7 days depending on the stimulus and 3H-thymidine is added to the culture for quantification of cellular proliferation. Normal values have wide intra- and inter-laboratory variations. Most often, results are reported as median stimulation index (SI), which is the ratio of cpm in stimulated vs. unstimulated cultured lymphocytes. Normal SI is . 5 for antigen-stimulated responses and . 100 for mitogen-stimulated responses. BrdU incorporation has been proposed as an alternative to 3H-thymidine to avoid the use of radioactive isotopes, but SI are usually markedly smaller with SI in the range 7– 8 for mitogen responses. A critical issue is the shipping conditions and time when blood must be transported to a distant laboratory. In general, overnight delay can lead to loss of responsiveness. Because of the wide distribution of normal values, it may be difficult to evidence statistically significant results. T lymphocyte cytotoxicity assays examine the ability of T lymphocytes to kill allogeneic targets. One aliquot of T lymphocytes from the donor are sensitized with irradiated cells from an unrelated donor and a second aliquot of T lymphocytes from the same donor are labeled by 51Cr. The sensitized and labeled T lymphocytes are incubated using varying effector to target cell ratios. The amount of 51Cr released into the medium correlates with the cytolytic activity of effector cells. Cytotoxicity can also be measured by flow cytometry using target cells labeled with a specific dye. Measurement of cytokine release is increasingly used for the qualitative assessment of the nature of immune responses, e.g. TH1 or TH2 responses. Cytokine secretion can be measured after mitogen stimulation of lymphocytes in whole blood or from cultured lymphocytes. Recent techniques suggest that functional evaluation from the measurement of released cytokines can also be possible [69,70].

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In vivo assays. DTH skin tests are prototype assays for the in vivo assessment of cellular immunity. This is a simple and straightforward method. Typically, one or several antigens are injected intradermally or pressed into the skin with a pronged applicator. Intradermal injections are however preferred, as antigen delivery is more consistent and reliable. The site is observed for erythema and induration after 24 and 48 h. When properly performed, DTH skin tests correlate with other measures of T lymphocyte function. Recall antigens are antigens that the tested subject is suspected or known to have previously encountered. Because positive DTH responses to even well-selected and standardized recall antigen are not seen in more than 60– 70% of the normal population, a panel of recall antigens is used including candidin, mumps, coccidioidin, trichophyton, tuberculin, streptokinase – streptodornase antigens, diphteria or tetanus toxoid. The MultiTestw is a standardized device that has been widely used, but not so much today [71 – 73]. A limitation to the use of recall antigens is that historical data from closely comparable subjects must be available to interpret results in the tested subjects [74 –83]. Inconstant differences in DTH responses to standardized recall antigens have indeed been shown depending on age, gender, race, smoking or income [84 – 86]. To some extent, antigens can be selected from the history of prior exposure by immunization, e.g. tetanus [87 – 89] and mumps [90,91], or environment, e.g. candidin [92] and tuberculin [93]. DNCB can be used without prior sensitization [94], but is no longer recommended owing to the risk of severe cutaneous reactions. Similarly, skin tests to KLH have also been used [95], but immunization to KLH may expose to the risk of shellfish allergies as already mentioned and anti-KLH antibodies have been detected in the sera of normal nonsensitized individuals [96]. DTH skin tests are commonly used to evaluate cellular immunity in human beings, but rarely in the context of immunotoxicity evaluation. Examples of studies where DTH skin testing has been used to evaluate the influence of drugs and other chemicals on cellular immunity include acute injections of thiopentone and propofol [97], inhalation of the corticosteroid fluticasone [98] in healthy volunteers, acute and chronic alcohol intake [99] and chronic exposure to dioxin [100]. CLINICAL IMMUNOLOGY END-POINTS TO ASSESS NONSPECIFIC IMMUNITY Flow cytometry techniques are available to measure NK cell activity [101,102], phagocytosis or oxidative burst [103,104] from peripheral blood of treated humans or after in vitro exposure. Other functional assays of monocytes and neutrophils include chemotaxis.

BIOMARKERS OF IMMUNOTOXICITY Biomarkers are essential tools that enable toxicologists to measure the exposure to hazardous chemicals, the magnitude of toxic responses and/or to predict the likelihood of these responses [105]. Three possibly overlapping categories of biomarkers have be defined, namely biomarkers of exposure, biomarkers of toxicity and biomarkers of susceptibility. Biomarkers may be simple or complex, specific to mammals or other species, and used in in vitro or in vivo systems. During the past decades, clinical


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immunologists devoted extensive efforts to design assays helpful for the laboratory diagnosis of immune diseases in patients with primary or secondary immune deficiencies, or a history of hypersensitivity reaction or autoimmune disease. Only limited efforts have paid to transferring these assays to immunotoxicity assessment [106]. Because many end-points have been proposed and most of these end-points have initially been developed to specifically address clinical immunology issues, a selection of relevant end-points was needed to provide a cost-effective list of biomarkers to be included in field or epidemiological studies. Among the several panels of experts that were convened to select immune end-points as biomarkers of immunotoxicity, the conclusions of two panels deserve particular attention. IMMUNE END-POINTS ADOPTED BY THE ATSDR The panel of experts convened by the Agency for Toxic Substances and Disease Registry (ATSDR) recommended that available immune tests are divided into three categories or levels [107], namely basic tests (level 1), focused tests (level 2) and research tests (level 3). Basic tests (level 1) were selected because they can be easily performed and are relevant to the detection and diagnosis of immune deficiency, hypersensitivity and autoimmunity. They include serum levels of antinuclear antibodies, C reactive protein, IgG, IgM and IgA, and total proteins, total white blood cell count, total lymphocyte and eosinophil counts, CD4þ and CD8þ lymphocyte counts. Focused tests (level 2) were selected for the follow-up of abnormal basic test results. They are intended for use in specific patients or groups of patients. Recommended tests focusing on immune deficiency include measurement of serum antibody levels to a given antigen, such as a vaccine, and serum isohemagglutinins, complement CH50 assay, granulocyte tetrazolium dye reduction assay, mitogen-induced lymphocyte proliferation assay and skin tests. Recommended tests focusing on hypersensitivity include total and specific IgE serum levels, beryllium lymphocyte transformation test, leukocyte histamine release assay and skin tests. Finally, recommended tests focusing on autoimmunity include serum levels of antithyroglobulin, antimitochondrial, antiphospholipid, and anti-smooth muscle antibodies and rheumatoid factor. Research tests (level 3). All other tests are considered as research tests (level 3) and are not recommended for use in epidemiological immunotoxicity studies. RECOMMENDATIONS OF THE US NATIONAL RESEARCH COUNCIL The Subcommittee of the US National Research Council reviewed the status of biomarkers of immunotoxicity [108]. Recommendations were that “clinical studies in humans are needed to determine the relationship between chemical exposures and immune-mediated diseases.”

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Emphasis was placed on the use of sensitive and validated assays and standardized case definitions. The need for methods to extrapolate animal data to human beings more accurately was stressed, as was the need for better quantification of exposure, improved identification of confounding and risk factors. That the recommendations of this panel of experts focused more on research needs than on possible modalities of use of biomarkers of immunotoxicity is not surprising, as available biomarkers have obviously been poorly relevant and helpful, if at all.

ENVIRONMENTAL EPIDEMIOLOGY STUDIES Ideally, the identification of immunotoxicants in human beings should be based on epidemiological studies. Environmental epidemiology can be defined as “the study of environmental factors that influence the distribution and determinants of disease in human populations” [109]. Clearly, immunotoxicants are environmental factors that can potentially result in human diseases. EXPERIMENTAL VS. OBSERVATIONAL EPIDEMIOLOGICAL METHODS Various methods have been designed to find associations between exposure and disease: they are divided into experimental and observational methods. Experimental methods include clinical trials which use patients as subjects, in particular to evaluate the efficacy of novel medicinal products, field trials which use healthy patients in a controlled environmental situation, for instance to evaluate the preventive value of a new modality of nutritional supplementation in a fraction of the population, and community trials, for instance to evaluate either the efficacy or safety of new processes, such as the fluorination of drinking water. These methods are also often called intervention studies in the sense that the investigators typically change the conditions of exposure. Observational methods include descriptive, analytical and cross-sectional studies. Descriptive studies are confined to describing a population of interest with regard to potential health problems. Because they are unable to establish causality between exposure and disease, these fishing expeditions may identify purely random, statistically significant events, sometimes leading to erroneous or misleading conclusions. Analytical studies are the core epidemiological studies, including longitudinal studies, e.g. cohort and casecontrol studies, and cross-sectional studies. When properly designed, these methods are critical to establish causality between exposure and disease. USE OF EPIDEMIOLOGICAL STUDIES IN IMMUNOTOXICITY ASSESSMENT Despite enormous methodological efforts and scientific achievements in the past decades, epidemiology has its own limits [110]. When immunotoxicity assessment


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is the focus of epidemiological studies, a number of specific difficulties must be overcome [111,112]. As already mentioned, most available assays that can be used for assessing immune function have neither been adequately standardized nor extensively validated. The functional reserve capacity of the immune system is so poorly defined that it remains to be established whether recorded changes of a given end-point, for example a 20 –30% decrease in antibody response against a selected antigen, are truly indicative of immunotoxicity. The appropriate selection of controls is essential to avoid factors which may interfere with immunocompetence. Many factors have been identified included age, gender, nutritional status, smoking or underlying illnesses. As so many confounding factors have to be taken into account, the size of the exposed and the control populations in epidemiological immunotoxicity studies have often to be much larger than in other epidemiological studies, and this may result in additional cost that may prove prohibitive. Chemical exposure must be documented as accurately as possible. In most instances, exposure should be sufficiently high to be associated with detectable immunological changes, as most current clinical immunology assays have limited sensitivity. Importantly, as the level of environmental or occupational exposures is steadily decreasing in many areas of the world, epidemiological immunotoxicity studies might well result in falsely negative results. In addition, the level of exposure must be carefully considered for the interpretation of results as low levels of exposure may be associated with unexpected immunostimulation and high levels of exposure to the same chemical with unexpected immunosuppression. Sample acquisition at sites geographically distant from the investigator’s laboratory is a major difficulty to be overcome when using available assays. The availability and/or implementation of assays is often more critical in the selection of biomarkers than scientific considerations. Finally, questionnaires are a prime tool in epidemiological studies. A number of organizations have established questionnaires that have been widely used. Questionnaires focus on defining exposure, identifying health effects and detecting confounding or predisposing factors. However, none of these questionnaires have been designed to address specific issues in relation with exposures to immunotoxic agents. In fact, very few epidemiological studies have been conducted to assess the adverse consequences of exposures to suspected or known immunosuppressive agents in man. Other epidemiological studies focused on selected health effects and searched for a correlation with immunotoxic exposures.

DISEASE CLUSTERS AND SENTINEL DISEASES Environmental epidemiological studies are typically designed to identify greater rates of diseases in relation to certain exposures. In some instances, the distribution of diseases is not uniform in a given population and the question arises to know whether case occurrence within a certain location is increased over expected number: cases seemingly grouped within a certain location are called disease clusters [113].

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DISEASE CLUSTERS Immunotoxicity has rarely been evoked as a cause of disease cluster. The subsequently unconfirmed finding that CD4þ T lymphocytes were decreased in a small group of women residing in households with groundwater contaminated by the pesticide aldicarb [114], can be considered as an example of an immunotoxicity cluster. In fact, numbers are usually very small so that it is very difficult to demonstrate increased occurrence rates. Cluster studies have essentially been devoted to certain types of cancers, such as leukemias [115], and have often been a matter of excessive hype by the media and the public. SENTINEL EVENTS General considerations. Because of the limitations in available biomarkers of immunotoxicity, the surveillance of sentinel events was suggested to be another potentially useful approach to identify human immunotoxicants [109,112,116]. The concept of sentinel events has already been used in other areas of medicine, such as infectious diseases, Parkinson’s disease, cerebro-vascular accidents or suicide, as well as in the fields of occupational toxicology, veterinary toxicology, or mutagenicity [117]. From a toxicological perspective, a sentinel event is an adverse event whose underlying pathophysiological mechanism is sufficiently well understood to indicate or, at least, strongly suggest that a chemically-mediated injury to a given target organ or function has occurred, and that a causal relationship between the toxic injury and a documented chemical exposure might exist. Relatively rare events should be preferably selected as sentinel events to avoid excessive background noise. A characteristic of sentinel programs is that they are based on spontaneous case reporting and therefore rely on less stringent and expensive procedures than epidemiological studies. The advantage of this approach is that a wider fraction of the general population can be included in such programs, but their major limitation is that incidence rates cannot be calculated. Changes in reported sentinel event rates may, however, be suggestive of a causal link, which will have to be confirmed by appropriate epidemiological studies. Another advantage of sentinel programs is the possibility to use medical records from general practitioners and clinicians. Immunotoxicity sentinel events. There is a variety of pathological conditions that may be considered as sentinel events suggestive of immunotoxicity. Opportunistic infections that normally do not develop in nonimmunocomprised patients and non-Hodgkin’s lymphomas can be considered as sentinel events when other frequent causes such as cancer or AIDS are carefully ruled out. Autoimmune diseases and autoimmune reactions are other typical examples, even though for unknown reasons, general practitioners and clinicians do not seem to search for causal link with chemical exposure. Severe hypersensitivity reactions, such as toxidermias and anaphylaxis are other possible sentinel events to be investigated. It is important to bear in mind that only relatively rare and clinically suggestive pathological conditions can be considered as possible sentinel events. A sentinel program was tentatively set up in France in the 1990s [118]. The following autoimmune diseases were selected as sentinel events, namely SLE, myasthenia,


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pemphigus and thyroiditis, Gougerot – Sjo¨gren syndrome, rheumatoid arthritis, Sharp’s syndrome, dermatopolymyositis and sclerodermia. Collaborating physicians were provided with specific reporting forms to obtain detailed information on the patient’s medical history, past drug and chemical exposures, and clinical and biological diagnostic criteria of the disease. Although, a possible causal link between silica exposure and scleroderma was suggested in dental prosthesis workers, the program was stopped by lack of funding.

CONCLUSION As can be seen from this overview, clinical immunotoxicology is a largely neglected area of immunotoxicology. Efforts in the 1990s have not been pursued and today, studies dealing with clinical immunotoxicity issues are rarely conducted. Nevertheless, the need for human immunotoxicity data is still present and it can be expected that the implementation of recent guidelines for nonclinical immunotoxicity evaluation will serve as a renewed impetus for the development of this critical aspect of immunotoxicity.

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Chapter 12

Immunotherapy of human poisonings

INTRODUCTION Although the introduction of modern methods of resuscitation dramatically changed the prognosis of acute poisonings with, for instance, a reduction from approximately 30 to 1% in the death rate of barbiturate-induced suicidal ingestions [1], the search for new antidotes is still a central issue for clinical toxicologists [2]. Antidotes are medications, which are given to counteract the action of certain poisons or toxins by specifically neutralizing toxic effects, preventing the absorption of the poison, or producing opposite effects of the poison. The idea of using the serum from immunized animals to treat human poisonings dates back to the end of the 19th century. The French microbiologist Albert Calmette—the co-developer of Bacillus Calmette –Guerin (BCG) as a vaccine against tuberculosis—studied the venom of the cobra snake after founding the Pasteur Institute of Saigon (today Hochiminhville). In 1891, he identified the neurotoxin of the cobra venom and taking advantage of his experience at the Pasteur Institute in Paris, he succeeded in attenuating the neurotoxin by multiple exposures to calcium hypochloride, thus making possible the immunization of rabbits and larger animals [3]. Initially, antibodies were generally produced by repeated injections of complexes of the toxic substance covalently coupled to a carrier protein. Rabbits were the preferred species. As the parent molecule could not be covalently bound to carriers in most instances, a major issue was to ensure that antibodies against even slightly modified chemical derivatives cross-react with the parent molecule. Antibodies raised by this procedure include antibodies against the hapten, i.e. the toxic substance, and antibodies against the carrier. Purification is therefore a critical step to remove antibodies that are not directed against the toxic substance. With the development of modern techniques enabling to produce large amounts of high affinity antibodies and antibody fragments for immunoassays, the intravascular administration of exogenous, specific antibodies and antibody fragments could be considered as a possible modality for the management of acute life-threatening human poisonings [4]. As is well known today, specific antibodies can inhibit the physiological or pharmacological effects of therapeutic peptides and proteins. Similar consequences can be achieved when small molecules are tightly bound to macromolecular carriers to raise specific antibodies [5]. 311

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Attempts to produce antibodies to neutralize small biologically active molecules in animal models have resulted in the first successful treatment of human poisoning in the mid 1970s: Smith et al. [6] reported the first potentially lethal digitalis poisoning recovering uneventfully after the administration of digoxin-specific antibodies. Thereafter, the therapeutic use of digoxin-specific antibodies was extensively studied and they have been approved in many countries as first-line therapy for advanced digitalis poisonings [7]. Besides the wide use of specific immunotherapy to treat human envenomations by animal stings and bites [8], other successful, but unfortunately limited attempts to treat acute human poisonings include tricyclic antidepressants [9] and colchicine [10].

EXPERIMENTAL STUDIES A number of experimental studies have tested whether specific sera or antibodies can protect against the toxic effects of miscellaneous drugs and other chemicals. In most instances, however, they have been limited to animal or even in vitro studies. MEDICINAL PRODUCTS Adriamycin. A major adverse effect of adriamycin is dose-dependent irreversible cardiotoxicity. Adriamycin-specific rabbit antibodies administered to mice were shown to block adriamycin-induced inhibition of myocardial Naþ –Kþ ATPase, reduce adriamycin concentrations in myocardial tissue and increase survival [11]. These findings have not seemingly been confirmed or expanded. Ajmaline, a purified alkaloid isolated from Rauwolfia serpentina, was introduced into the clinic for the treatment of cardiac arrhythmias in 1958. Despite the use of state-of-the-art resuscitation techniques, 15% of acute ajmaline poisonings were fatal. The possibility that specific antibodies can improve the prognosis of ajmaline-induced acute poisonings was therefore considered. Rabbits were immunized by repeated intradermal injections of the 17-hemisuccinate ester of ajmaline-21-acetate coupled to BSA. A statistically significant protection assessed from the delay for the QRS interval on standard ECGs to increase by 300% and the time to death was evidenced in immunized rabbits as compared to controls when ajmaline hydrochloride was continuously injected as a slow intravenous infusion [12]. In the same experimental conditions, short-term intravenous administration of serum from actively sensitized rabbits, either to naive intoxicated rabbits or mice, had a protective effect greater than sodium lactate, the reference treatment for ajmaline-induced rhythm disturbances [13]. However, no further experiments were conducted as ajmaline was withdrawn from the market by the manufacturer. Aspirin. Butler et al. [14] showed that antibodies from rabbits immunized by aspirin coupled to horse serum globulin inhibited the antipyretic effects of aspirin.


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Morphine. In an attempt to evidence the possible role of an immune-mediated mechanism of tolerance to narcotic analgesics, Berkowitz and Spector [15] showed that active immunization of mice by 3-carboxymethylmorphine coupled to BSA had serum concentrations of [3H]dihydromorphine 30 –50 times higher than nonimmunized mice and a prolonged half-life. However, the typical pharmacological effects of morphine, such as lethality, analgesia and decreased induction of writhing by p-phenylquinone, were only marginally affected. In contrast, antibodies raised in rabbits by repeated administrations of 6-succinyl-morphine conjugated to BSA antagonized the electrically induced contractions of isolated guinea pig ileum induced by morphine in a concentration-dependent manner [16]. Rabbits actively immunized with 6-succinyl-morphine conjugated to BSA had delayed clearance of morphine from the circulation [17]. Vinca alkaloids. The inadvertent administration of anticancer overdose occurs rarely, but may have serious consequences [18]. The use of the antibody 27.8.1A was investigated to reduce the toxicity of vinca alkaloids [19]: in vitro, this antibody reversed the cytotoxicity of a vinca derivative on a human acute lymphoblastic leukemia cell line, whereas nude mice treated with the antibody survived toxic doses of the vinca derivative. INDUSTRIAL AND ENVIRONMENTAL CHEMICALS Amanita phalloides. are markedly toxic mushrooms causing a significant number of lethal poisonings. Acute poisonings are typically characterized by severe hepatotoxicity which, in humans, is mainly due to amatoxins [20]. Attempts to treat Amanita phalloids poisonings date back to the early 20th century when Dujarric de la Riviere [21] immunized horses with extracts of the mushroom. However, the protective effects of this antiserum have never been clearly demonstrated. Over the years, various antibodies have been produced in an attempt to develop a reliable assay for use as a diagnostic tool in human poisonings. Surprisingly, the administration of specific rabbit IgG to mice intoxicated with a-amanitin induced a 2-fold increase in the toxic effects of a-amanitin [22]. Similar findings were obtained by the same authors when using Fab fragments from a monoclonal antibody against a-amanitin that induced in mice a 50-fold increase in toxicity mainly due to unexplained nephrotoxicity [23]. Arsenic. Polyclonal specific antibodies were raised in rabbits sensitized with biweekly intravenous injections of an arsenix – ox – globulin complex. Mice received an intraperitoneal dose of para-amino-phenyl-arsenic acid, phenol-p-azo-phenyl-arsenic acid, or resorcinol-tris-azo-phenyl-p-arsenic acid, followed by a subcutaneous injection of different amounts of serum from immunized rabbits. Despite variable results depending on the arsenic derivative used, protection was achieved by serum administration [24]. Dioxin and biphenyls. Mice passively immunized with rabbit antiserum against 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) coupled to HSA were neither protected nor cured

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from the lethal effects of a single oral dose of 250 mg/kg TCDD [25]. In contrast, the passive immunization of mice with rabbit antiserum against 4-monochloro-biphenyl or 3,4,30 ,40 tetra-chlorobiphenyl increased the plasma concentrations of either biphenyl suggesting that antibodies may be used to remove chlorinated biphenyls from tissue stores [26]. Organophosphorus compounds. are extremely toxic chemicals initially synthesized for warfare purposes. After World War II, a number of less toxic derivatives were marketed for use as insecticides. Although atropine and oximes are the most widely used antidotes to treat human poisonings with organophosphorus compounds, several researchers tested the possible protective effects of specific antibodies in laboratory animals. Paraoxon. is the metabolite responsible for the toxicity of the organophosphorus insecticide parathion. Polyclonal antibodies raised in rabbits were protective after the injection of 4 LD50 [27] or 2 LD95 [28] of paraoxon. The protective effects of antiparaoxon antibodies were also shown in mice pre-injected with the serum of sensitized rabbits [29]. Soman. Monoclonal antibodies raised in sensitized rabbits were shown to react in vitro with soman in a competitive inhibition enzyme immunoassay using acetylcholinesterase. In addition, these antibodies increased 2-fold the LD70 or LD90 of soman in mice [30]. VX (ethyl S-2-diisopropylaminoethyl methylphosphonothioate) is one of the most potent organophosphorus derivatives for warfare purposes. VX coupled to tetanus toxoid or BSA was used to raise specific antibodies by repeated administrations to rabbits [31]. Protection by active immunization was demonstrated by the intramuscular injection of one LD95 of VX to 10 immunized rabbits of which only one died. When rabbit anti-VX serum was injected intravenously immediately before one LD95 of VX to mice, total protection was achieved for 1– 24 h depending on the dose of anti-VX serum. Finally, the inhibition of Torpedo acetylcholinesterase by VX was reversed dose dependently by the addition of antiVX serum in contrast to the inhibition by the related organophosphorus compounds soman and sarin. Paraquat is a nonselective contact herbicide that is highly toxic in humans. Accidental or suicidal ingestions are associated with a high percentage of mortality, primarily due to the development of progressive and irreversible lung fibrosis. Up to now, none of the many therapeutic modalities that have been proposed has succeeded in improving the prognosis of acute paraquat poisonings. Mortality in rats passively immunized by a paraquat derivative covalently bound to BSA and in non-immunized rats when given a lethal dose of paraquat was not different, but a slight increase in survival time was noted in immunized rats [32]. The efficacy of specific antiparaquat F(ab0 )2 antibodies raised in immunized rabbits was tested in mice. Although the paraquat plasma concentrations in mice pretreated with F(ab0 )2 antibodies were significantly increased compared to control mice given the same intravenous sublethal dose of 8 mg/kg of paraquat, no difference in mortality rate or delay of survival was seen [33]. That antibodies are unlikely to protect against paraquat toxicity was confirmed by the lack of protection afforded by the administration of a specific monoclonal antibody to mice given a sublethal intraperitoneal dose of paraquat [34]. Similarly, the injection of one of three different anti-paraquat monoclonal antibodies to rats increased plasma paraquat


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concentrations, but did not affect urinary elimination [35]. However, in vivo and in vitro results are conflicting. Thus, anti-paraquat sheep polyclonal and murine monoclonal antibodies were able to prevent paraquat accumulation into rat lung slices [36], while antiparaquat Fab fragments and monoclonal antibody significantly inhibited paraquat uptake in vitro by suspensions of rat alveolar type II pneumocytes and this was associated with a 50 – 80% reduction in paraquat cytotoxicity [37]. Ricin, the toxic lectin from the castor bean (Ricinus communis) is one of the most potently toxic substances with an intravenous LD50 of 2.7 mg/kg in mice. Antibodies raised against either the A or B chain of the toxin induced protection of mice against a lethal subcutaneous dose of ricin when injected within 10 h after ricin injection. However, only partial protection could be achieved when ricin was injected intravenously [38]. These early findings were confirmed by the recent use of ricin vaccines administered intranasally [39], intramuscularly [40] or orally [41], which were shown to afford protection in mice intoxicated by aerosolized ricin. Strychnine. Mononitrostrychnine and dinitrostrychnine were coupled to horse serum proteins and the complexes were injected three times weekly for 4 weeks to rabbits. Although the reactivity of raised antibodies with strychnine was clearly demonstrated, all mice injected with 0.05 mg of strychnine nitrate intraperitoneally died shortly after the injection despite the injection of 1 ml of serum from sensitized rabbits, 15 min to 96 h prior to strychnine injection [42].

CLINICAL STUDIES In humans, the use of immunotherapy has so far been limited to envenomations by animal bites and stings, and acute poisonings by digitalis derivatives, tricyclic antidepressants and colchicine. As the clinical application of specific antibodies was based on extensive experimental studies, a summary of animal and in vitro findings will be presented before the description of clinical results. Finally, an emerging field is the development of therapeutic vaccines for drug dependence. IMMUNOTHERAPY OF ENVENOMATIONS Envenomations caused by animal bites and stings represent a public health problem around the world despite variable importance depending on the region being considered. It has been estimated that at least 5 million cases of snakebites occur each year with a total death toll ranging from 50,000 to 100,000 [43]. Spider bites and scorpion stings are another major cause of concern. Finally, envenomations can also due to fish, molluscs and insects. There is a bewildering variety of animal venoms, most of them being proteins and polypeptides. They exert many toxicological effects, including neurotoxicity, myotoxicity, proinflammatory activity and coagulation disturbances.

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The parenteral administration of antibodies and antibody fragments have become the mainstay in the therapy of human envenomations [44]. Such antibody preparations or antivenoms are produced from the plasma of animals, mainly horses and sheep, immunized with the venom of interest. Thus, currently used antivenoms are made of polyclonal antibodies of heterologous origin. Although monoclonal antibodies have been produced and proved to neutralize the venom toxins of arthropods and snakes, they are not yet available for clinical use. Heterologous sera tend to be replaced by purified IgG antivenoms or F(ab0 )2 and Fab fragments prepared by pepsin or papain digestion, respectively. A number of clinical studies compared the distribution of these three types of antivenoms following intravenous injection. Fab and F(ab0 )2 fragments were found to have a larger volume of distribution, whereas Fab fragments had a shorter half-life [45,46]. Depending on the antivenoms, neutralization can be obtained against venoms from closely related animal species or venoms from a given species only [47]. A number of experimental studies showed that antibodies or their fragments can block the molecular regions of the venom toxins that are involved in the development of toxic effects. To date, a number of individual antivenoms are available for the management of human envenomations. As a detailed review of these antivenoms is beyond the scope of this chapter, interested readers are referred to comprehensive sources of information in this area [48,49]. DIGITALIS Digitalis, and in particular digoxin, are still often prescribed for the treatment of congestive heart failure. Studies have shown that mortality ranged from 3 to 21% of hospitalized digoxin-intoxicated patients. The advent of digoxin-specific antibody fragments for the immunotherapy of treatment overdosage or acute poisoning has dramatically changed the management and prognosis of digitalis poisonings. Experimental studies. Digoxin-specific antibodies were raised by coupling the D ring hydroxyl groups of 5 –7 molecules of digoxin to BSA in mice, rabbits and sheep. To avoid immunogenicity, slow distribution into interstitial spaces and the lack of excretion by the kidney, whole antibodies were fragmented [50]. Subsequently, monoclonal antibodies were produced [51 –54]. Today, two commercial preparations of Fab fragments are available. Early in vitro studies showed a good specificity and high affinity of antibodies [55] that could reverse the effects of digoxin in various systems including guinea pig atrial strips [56] or rubidium transmembrane transport [57]. Quite a few experimental studies using various antibody preparations consistently showed that digoxin-specific antibodies can reverse or prevent the effects of toxic and even lethal doses of digoxin in rabbits [58], dogs [59,60], cats [61] and guinea pigs [62]. Depending on the experimental conditions, digoxin-specific antibodies prevented or reversed malignant ventricular arrhythmias, or a significantly higher dose of digoxin was needed to produce the expected toxic effects. The administration of digoxin-specific antibodies was shown to induce elevated digoxin serum levels, accelerated clearance and reduced tissue concentrations in mice [63], dogs [64], rats [65, 66], rabbits and baboons [67]. Protection against digitalis glycosides in toad venom was evidenced in mice [68].


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Clinical studies. In 1976, the first report of the successful use of digoxin-specific antibodies in human poisoning was reported [5]. Subsequently, the efficacy of specific antibodies was demonstrated in large series of intoxicated patients [69 – 72]. Interestingly, digoxin-specific antibodies were also shown to be effective in patients intoxicated with other digitalis glycosides, such as lanatoside C [73], digitoxin [74], oleander glycosides [75,76] and foxglove extracts [77]. Fab fragments are normally administered in 30 min infusions unless emergency circumstances dictate more rapid administration. The efficacy of Fab fragment has been reported in children [78] and patients with renal failure [79]. Although the efficacy of digoxin-specific antibodies is well established, usual aggressive therapeutic measures are nevertheless necessary in severe digitalis poisonings. Allergic reactions are uncommon, usually limited to rash or pruritic eruption. So far, neither anaphylactoid reaction nor serum sickness has been observed.

TRICYCLIC ANTIDEPRESSANTS Tricyclic antidepressants are one of the leading causes of mortality from intentional drug overdoses, primarily because of their marked cardiotoxicity. Based on the clinical experience gained with antidigoxin antibodies, the potential benefit of specific immunotherapy in acute poisonings with tricyclic antidepressants has been extensively investigated. Experimental studies. Initially, that specific antibodies can afford protection from the toxicity of desmethyl-imipramine, or desipramine, the active metabolite of imipramine, was demonstrated by the intra-arterial injection of 2 mg of [3H]-desipramine to rabbits followed by 1 g of anti-desipramine Fab fragments prepared from sheep IgG [80]. Treatment with Fab induced an increase in serum desipramine levels and in unmetabolized desipramine in the urine. These findings were later confirmed in rats using an IgG1 antidesipramine monoclonal antibody [81]. Rats received 1 mg of desipramine or imipramine, followed by graded doses of specific antibody by the intravenous route which resulted in a dose-related increase in the serum concentrations of desipramine or imipramine. The efficacy of various specific antibody fragments was later demonstrated in rats administered a toxic dose of desipramine. The toxicity of desipramine in rats as in humans is characterized by hypotension and prolongation of the QRS interval on the ECG. Rats were given 2 g/kg of anti-desipramine F(ab0 )2 fragments intravenously over 10 min starting 15 min after a toxic dose of desipramine. This resulted in a rapid reduction of QRS prolongation which lasted for 45 min at least [82]. Pretreatment with an antidesipramine monoclonal antibody was also shown to protect rats from the cardiotoxic effects of desipramine as a reduction of 30 –50% of the dose of desipramine was needed to induce the same effect on QRS duration in nonpretreated rats [83]. In a subsequent study [84], rats were given 30 mg/kg of desipramine intraperitoneally followed by 1 g of anti-desipramine Fab0 fragments. This resulted in a rapid 15-fold in desipramine serum concentrations, demonstrating the redistribution of desipramine out of tissues and this was

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associated with a reduction in QRS prolongation. As the production of monoclonal antibodies was time-consuming and expensive at that time, the effects of ovine polyclonal Fab fragments were tested in similar conditions and protective effects were demonstrated [85]. The same Fab fragments were evaluated in different experimental conditions. Rats were infused with desipramine until death and survival time was increased by 58% following the administration of 2 g/kg of ovine anti-desipramine polyclonal Fab fragments [86]. In another study, rats were infused with desipramine until a 300% in QRS duration was obtained and then graded doses of specific Fab fragments were injected. There was a dose-dependent relationship, as the animals given the highest dose showed the greatest improvement [87]. Recombinant desipramine-specific single chain Fv fragments (B9-sFv) were also tested and the marked increase in the serum concentrations of [3H]-desipramine induced by B9-sFv indicated that even very small fragments can bind to desipramine and its redistribution out of tissues [88]. Interestingly, nortriptyline-specific antibodies were also shown to increase amytriptyline serum levels and reduce toxicity when injected to nai€ve rabbits [89]. Clinical efficacy. Heard et al. [90] treated a 48-year-old man who had ingested 1.5 g of amitryptiline in association with several other drugs. As his condition did not improve despite gastric lavage and symptomatic therapeutic measures, he was given a total intravenous dose of 14 g of ovine Fab fragments over 4 h. Electrocardiographic changes normalized rapidly and the patient recovered quickly. Seemingly, this is the only case report of the clinical application of tricyclic antidepressant-specific antibodies in human acute poisoning. COLCHICINE Colchicine is a drug used in the treatment of gout. Although relatively uncommon, colchicine poisoning may produce life-threatening systemic effects and treatment requires early recognition and supportive care. Anti-colchicine antibodies raised in rabbits were shown to protect naive rabbits from acute colchicine intoxication [91]. There was a dosedependent protective effect of specific antibodies and increased colchicine serum levels in treated rabbits. Similar findings were obtained in mice treated with goat colchicine-specific antibodies [92,93]. Goat Fab fragments also exerted protective effects in a murine model of severe acute colchicine poisoning [94,95]. So far, only one patient with severe colchicine was successfully treated with colchicine-specific Fab fragments [10]. VACCINES FOR THE TREATMENT OF DRUG DEPENDENCE The use of therapeutic vaccines is a novel modality for the therapy of drug addictions [96, 97]. Although the efficacy of this approach remains to be established, several candidate vaccines have been tested in laboratory animals. The available experimental data demonstrate that immunization of animals is associated with reduced effects and selfadministration of the addicting drug. Although it is as yet not known whether immunization


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produced similar effects in human addicts, therapeutic vaccines are considered as potential helpful adjuncts to current modalities of therapy. At the time of writing, several cocaine vaccines were being tested in phase II trials. Cocaine. The first attempt to develop an anti-cocaine vaccine was published in 1992 [98]. Fisher rats immunized twice with cocaine conjugated to KLH had a significantly reduced analgesic response to cocaine. Later, mice actively immunized against a cocaine conjugate had a significant reduction in cocaine brain concentrations that could be evidenced 30 s after an intravenous injection of cocaine [99] and similar findings were obtained with an anti-idiotypic cocaine vaccine [100]. Using a cocaine – KLH conjugate, Carrera et al. [101] showed that rats immunized by three injections of the conjugate selfadministered cocaine significantly less than controls and that a 8-fold increase in cocaine dose was needed to produce similar effects than in controls. In rats, the injection of the anti-cocaine Mab MO240 decreased cocaine self-administration and the effect was related to serum anti-cocaine antibody levels [102]. Similar findings were obtained with another candidate vaccine IPC-101 [103]. Pursuing with their development of anti-cocaine Mabs that could be better vaccine candidates for clinical use, Carrera et al. [104] found that the Mab GNC92H2 reduced the effects of cocaine on locomotor activity in rats to an extent similar that seen with a cocaine –KLH conjugate. A phase I clinical trial of a therapeutic cocaine vaccine showed a significant increase in anti-cocaine antibodies that peaked at 3 months and declined to baseline by 1 year. The vaccine was well tolerated and a dosedependent antibody response was obtained. Preliminary data of a phase IIa clinical trial confirmed these early results and interestingly 4 of the 9 patients abstained from cocaine during the 12-week trial [105]. Nicotine. It is beyond doubt that nicotine addiction is a major health problem worldwide. Although many therapeutic approaches are being used to help people refrain from smoking, none has proved optimally effective. The search for nicotine therapeutic vaccines is ongoing and the available results to date are promising enough for candidate vaccines to enter clinical trials [106]. Pentel et al. [107] showed that injection of specific IgG from rats immunized with nicotine conjugated to Pseudomonas aeruginosa exoprotein A dose dependently reduced nicotine brain concentrations and the increase in blood pressure induced by nicotine. Using the same conjugate, it was possible to demonstrate that antibodies to nicotine in rats can be raised despite continuation of nicotine exposure and that immunized rats had a 40– 60% reduction in nicotine brain concentrations [108]. Immunization of rats with three doses of the same conjugate over 6 weeks significantly reduced the incidence of seizure induced by nicotine [109]. Following immunization with the nicotine conjugate IP-18-KLH, rats developed plasma antibodies that are highly specific for nicotine and had reduced dopamine release in nucleus accumbens, a biochemical correlate of nicotine effects [110]. In addition, rats with the higher titers of antibodies against nicotine did not reinstate nicotine self-administration behavior when they are exposed to nicotine [111]. Rats immunized with another peptide – nicotine vaccine

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had attenuated behavioral effects despite repeated high exposure to nicotine [112]. Interestingly, the administration of a dose of nicotine equal to twice the binding capacity of nicotine in immunized rats resulted in decreased nicotine concentrations in muscle, testis, spleen, liver, heart, kidney and brain, suggesting that antibodies not only neutralize nicotine, but also produce the redistribution of nicotine [113]. Phencyclidine. is another widely abused substance. Experimental works suggest that immunization against phencyclidine could also be considered as a potential therapeutic approach. The administration of high-affinity anti-phencyclidine Fab fragments induced a 17 –56-fold increase in serum phencyclidine concentrations in dogs [114] and significant increase of serum phencyclidine concentrations and decrease in the brain of rats [115]. The administration of these fragments was later shown to reverse the behavioral effects of phencyclidine and closely related analogs in rats [116]. A single administration of IgG, but not Fab fragments, significantly decreased brain phencyclidine concentrations for up to 27 days [117] and protected rats against the behavioral effects of phencyclidine for at least 2 weeks [118]. A protective effect was obtained when a dose as low as 1/100th the molar equivalent of the phencyclidine body burden was used [119].

MECHANISM OF SPECIFIC ANTIBODIES EFFICACY Schematically, one molecule of a specific antibody reacts with one molecule of a hapten. From a theoretical point of view, specific antibodies can neutralize the biological effects of toxic substances by different mechanisms including the binding of the toxicant to specific antibodies within the blood, creating complexes that are eliminated by the kidney and avoiding toxicity in target organs, the binding of the toxicant to specific antibodies in the extra-cellular fluid creating a concentration gradient promoting the egress of the toxicant from cells, a direct interaction with the toxicant and specific antibodies at the cell membrane level resulting in the inactivation and/or dissociation of the toxicant and finally the entry of the antibodies into the cells to bind the toxicant intracellularly. The intravascular administration of high affinity antibodies or antibody fragments results in the binding of the hapten. The binding leads to a reduction in the plasma concentration of the free hapten. Since the free concentration of the hapten is assumed to be equal throughout the body water, the free hapten returns from tissue spaces into the circulation to re-establish the previous equilibrium. This, in turn, causes the dissociation of the hapten from tissue binding sites provided the relative affinity of the hapten for specific antibodies or antibody fragments is much higher than for tissue binding sites. If a sufficient quantity of antibodies or fragments is administered, a substantial depletion of the toxic substance can be achieved. The impact of antibody administration on the pharmacokinetics of a toxic substance depends on the initial free fraction of the substance in the plasma, the mode of elimination from the body and the binding capacity of antibodies and antibody fragments [120].


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CONCLUSION Specific antibodies have unequivocally been shown to save the life of acutely poisoned human patients [121]. However, their clinical use is still restricted to digitalis poisonings and snake envenomations. Research in this area which was very active in the 1980s and 1990s has markedly slowed with the notable exception of candidate vaccines for the treatment of drug addiction. Relatively few patients are expected to benefit from the administration of specific antibodies, either because conventional resuscitation measures are generally appropriate for the management of these patients as is the case for tricyclic antidepressants, or because human poisonings which could benefit from the use of specific antibodies, e.g. colchicine poisonings are extremely rare. Finally, experimental research has suggested potential efficacy for only few specific antibodies.

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[83] Brunn GJ, Keyler DE, Pond SM, Pentel PR (1992) Reversal of desipramine toxicity in rats using drug-specific antibody Fab0 fragment: effects on hypotension and interaction with sodium bicarbonate. J Pharmacol Exp Ther, 260, 1392– 1399. [84] Pentel PR, Ross CA, Landon J, Sidki A, Shelver WL, Keyler DE (1994) Reversal of desipramine toxicity in rats with polyclonal drug-specific antibody Fab fragments. J Lab Clin Med, 123, 387– 393. [85] Pentel PR, Scarlett W, Ross CA, Landon J, Sidki A, Keyler DE (1995) Reduction of desipramine cardiotoxicity and prolongation of survival in rats with the use of polyclonal drugspecific antibody Fab fragments. Ann Emerg Med, 26, 334– 341. [86] Hursting MJ, Opheim KE, Raisys VA, Kenny MA, Metzger G (1989) Tricyclic antidepressantspecific Fab fragments alter the distribution and elimination of desipramine in the rabbit: a model for overdose treatment. J Toxicol Clin Toxicol, 27, 53 – 66. [87] Dart RC, Sidki A, Sullivan JB, Egen NB, Garcia RA (1996) Ovine desipramine antibody fragments reverse desipramine cardiovascular toxicity in the rat. Ann Emerg Med, 27, 309– 315. [88] Shelver WL, Keyler DE, Lin G, Murtaugh MP, Flickinger MC, Ross CA, Pentel PR (1996) Effects of recombinant drug-specific single chain antibody Fv fragment on [3H]-desipramine distribution in rats. Biochem Pharmacol, 51, 531– 537. [89] Sabouraud A, Denis H, Urtizberea M, Chappey O, Scherrmann JM (1990) The effect of nortriptyline-specific active immunization on amitriptyline toxicity and disposition in the rabbit. Toxicology, 62, 349– 360. [90] Heard K, O’Malley GF, Dart RC (1999) Treatment of amitriptyline poisoning with ovine antibody to tricyclic antidepressants. Lancet, 354, 1614– 1615. [91] Scherrmann JM, Urtizberea M, Pierson P, Terrien N (1989) The effect of colchicine-specific active immunization on colchicine toxicity and disposition in the rabbit. Toxicology, 56, 213– 222. [92] Terrien N, Urtizberea M, Scherrmann JM (1989) Influence of goat colchicine specific antibodies on murine colchicine disposition. Toxicology, 59, 11 –22. [93] Terrien N, Urtizberea M, Scherrmann JM (1990) Reversal of advanced colchicine toxicity in mice with goat colchicine-specific antibodies. Toxicol Appl Pharmacol, 104, 504–510. [94] Sabouraud A, Urtizberea M, Grandgeorge M, Gattel P, Makula ME, Scherrmann JM (1991) Dose-dependent reversal of acute murine colchicine poisoning by goat colchicine-specific Fab fragments. Toxicology, 68, 121– 132. [95] Urtizberea M, Sabouraud A, Cano N, Grandgeorge M, Rouzioux JM, Scherrmann JM (1991) Reversal of murine colchicine toxicity by colchicine-specific Fab fragments. Toxicol Lett, 58, 193– 198. [96] Bunce CJ, Loudon PT, Akers C, Dobson J, Wood DM (2003) Development of vaccines to help treat drug dependence. Curr Opin Mol Ther, 5, 58 –63. [97] Kosten TR, Biegel D (2002) Therapeutic vaccines for substance dependence. Expert Rev Vaccines, 1, 363– 371. [98] Bagasra O, Forman LJ, Howeedy A, Whittle P (1992) A potential vaccine for cocaine abuse prophylaxis. Immunopharmacology, 23, 173– 179. [99] Fox BS, Kantak KM, Edwards MA, Black KM, Bollinger BK, Botka AJ, French TL, Thompson TL, Schad VC, Greenstein JL, Gefter ML, Exley MA, Swain PA, Briner TJ (1996) Efficacy of a therapeutic cocaine vaccine in rodent models. Nature Med, 2, 1129– 1132. [100] Schabacker DS, Kirschbaum KS, Segre M (2000) Exploring the feasibility of an anti-idiotypic cocaine vaccine: analysis of the specificity of anticocaine antibodies (Ab1) capable of inducing Ab2beta anti-idiotypic antibodies. Immunology, 100, 48 – 56. [101] Carrera MR, Ashley JA, Zhou B, Wirsching P, Koob GF, Janda KD (2000) Cocaine vaccines: antibody protection against relapse in a rat model. Proc Natl Acad Sci USA, 97, 6202– 6206. [102] Kantak KM, Collins SL, Lipman EG, Bond J, Giovanoni K, Fox BS (2000) Evaluation of anticocaine antibodies and a cocaine vaccine in a rat self-administration model. Psychopharmacology, 148, 251– 262.

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Chapter 13

Chemical immunoteratology and developmental immunotoxicology

INTRODUCTION As in many areas of biology and immunology, our understanding of developmental and reproductive immunology progressed rapidly over the years. In parallel, the abnormal embryonic development of organs and organ systems has been the focus of extensive research for several decades and reproductive toxicity testing is routinely performed as an obligatory component of the safety evaluation of many drugs and other chemicals. The adverse consequences of chemical exposure on reproduction are extremely varied. Regulatory toxicity tests have been divided into three segments to detect effects that may negatively impact on the fertility and reproductive function of mating parents, the normal anatomical development of organs and tissues in the embryo, and the physiological function of organs and systems later in the fetus and after birth until weanling. There is evidence that immunological factors can lead to infertility and play a crucial role in the failure or success of pregnancy [1]. The term immunoteratology has long been coined to address abnormal immune responses that may occur between the fetus and the mother [2]. Chemical immunoteratology is proposed to address the induction of anatomical malformations of the lymphoid organs by exposure to drugs and other chemicals, and also the modulation of malformations in the lymphoid and nonlymphoid organs by chemical exposures influencing immune responses. In fact, the finding that in utero chemical exposures can result in functional changes evidenced at a later stage of life gave an impetus to a rapidly expanding new area of immunotoxicology, namely developmental immunotoxicology. Although, some of the earliest works in immunotoxicology focused on the effects of developmental exposure to lead [3,4] and dioxin [5], alterations in the immune responsiveness following in utero exposure to medicinal products as well industrial and environmental toxicants became an issue of concern only recently [6 – 12]. Immune responses play an important role in various reproductive processes. The question of why the semi-allogeneic fetus is not rejected by the mother remains unresolved [13]. The fetus can be regarded as a semi-allogeneic graft within the uterus, which has to be 327

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tolerated over the whole gestation [14]. Mammalian species use a variety of strategies to avoid maternal immune attack and rejection of the fetus [15]. In this setting, the placenta plays a central role both as a physiological barrier and as a zone of contact and exchanges between the mother and the fetus. As the maternal immune system is aware of the presence of the fetus, tolerance must be present to avoid rejection of the fetus [16]. The involvement of many mechanisms has been hypothesized, but their respective role has yet not been clearly elucidated. Several hormones, such as progesterone and growth hormone, because they modulate the immune responses, may interfere. As fetal antigens and whole cells can pass through the placenta, they are suspected to induce peripheral tolerance. In addition, local tolerance can be induced by components of the placenta, such as trophoblasts, which do not express the class I MHC antigens HLA-A and HLA-B. There is a shift in the TH1/TH2 balance in favor of TH2 responses during pregnancy. Cytokines play a pivotal role in the early stages of pregnancy [17] and the pro-inflammatory cytokines IL-1, TNF-a and IFN-g have been suspected of causing pregnancy loss and miscarriage [18]. The TH1 cytokines TNF-a and IFN-g affect many reproductive processes [19]. Autoantibodies to cardiolipin and possibly phosphatidylserine are the immunological factors that have been most clearly associated with recurrent abortion, even though the antiphospholipid syndrome occurs in less than in 5% of women with recurrent pregnancy losses [20].

IMMUNITY IN NEWBORNS In mammals, the immune system undergoes dynamic changes during the early stages of development. These changes include the sequential formation and expansion of multipotent hematopoietic stem cells, the expansion of lineage-committed stem cells, the colonization of post-natal lymphopoietic compartments and maturation to immunocompetence. Establishment of a normal immune system requires a sequence of carefully timed and co-ordinated developmental events that begin early in embryonic/fetal life and continue through the early post-natal period [21]. Fetal liver hematopoiesis is first detectable at day 10 of gestation in mice and at about 6 weeks of gestation in humans. The mouse spleen contributes to hematopoiesis from day 15 of gestation until several weeks after birth. In mice, the spleen never completely loses hematopoietic function, but hematopoiesis has ceased in the human spleen by the time of birth. Liver multipotent stem cells start migrating to the bone marrow by gestational days 17– 18 in mice and by week 20 in humans. Thymic colonization occurs in the latter half of gestation in mice, whereas thymic colonization and early T lymphocyte development occur in the first trimester of fetal development in humans. Immunity is deficient in newborns [22 – 24]. Every component of the immune system is affected. Although the number of phagocytic cells is normal at birth, chemotaxis and phagocytosis are impaired. The number of NK cells is low and even lower in pre-term infants. Complement levels are below adult levels. At birth, the number of B lymphocytes is normal or even higher than in adulthood, but their ability to proliferate is decreased. Serum IgM and IgA levels are very low, whereas IgG are mainly of maternal origin. Nevertheless, newborns have the capacity of mounting IgG, IgM and IgA responses even though the production of specific antibodies is decreased until 5 –6 months of age in human babies.


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The number of T lymphocytes is normal at birth and the CD4þ:CD8þ ratio similar to that of adults. It is unclear whether the diversity of the T lymphocyte repertoire is lower at birth than in adulthood. There is a general consensus that newborns preferentially mount TH2 responses, which might be attributed to the cytokine milieu in the maternal – fetal interface, and high levels of progesterone. The polarized TH2 response is associated with a relative deficiency in TH1 and cytotoxic T lymphocyte responses. APCs and in particular dendritic cells have a diminished capacity to express CD40 and CD86, which results in reduced signaling to T lymphocytes.

CHEMICAL TERATOGENS AND THE IMMUNE SYSTEM MORPHOLOGICAL CHANGES IN LYMPHOID ORGANS INDUCED BY TERATOGENS In contrast to developmental immunotoxicology, this aspect of chemical immunoteratology has so far not received much attention. Abnormal thymus development has been described following in utero exposure to several drugs and other chemicals. It has been described in hamsters exposed to retinoic acid [25] and thymic hypoplasia has been reported at least once in a newborn exposed to isotretinoin in utero [26]. Abnormal development of the thymus was also described in rats exposed to the antiviral drug acyclovir [27]. Although cyclosporine, tacrolimus and sirolimus can induce thymic atrophy in adult rodents, no thymic anomalies have seemingly been reported following in utero exposure. Oral gavage of pregnant C57BL/6J mice with N,N-bis(dichloroacetyl)-1,8-octamethylene diamine on day 10 of gestation induced cortical hypoplasia of the thymus in the offspring at 36 days post-natally. This change was associated with diffusely distributed white pulp of the spleen [28]. REPRODUCTION TOXICITY ASSESSMENT OF IMMUNOPHARMACEUTICALS The term refers to medicinal products that are intended to treat (or prevent) diseases via direct effects on the immune system. The major immunotherapeutics are immunosuppressive and immunostimulatory drugs, therapeutic cytokines and anti-cytokine antibodies, monoclonal antibodies and vaccines. Although these products are increasingly used and some have been in use for many years, few results of teratogenicity studies are available in the literature, with the exception of cytotoxic immunosuppressive drugs, such as azathioprine, cyclophosphamide, or methotrexate. Cyclosporine which was shown to induce malformations to cultured embryos by inhibition of the arachidonic acid pathway [29], has not been shown to induce malformations in humans [30]. IFN-g has been suspected to exert teratogenic effects in mice, but these findings were not confirmed in other species [31]. As immunopharmaceuticals do not appear to present any particular risk on reproduction, there is no obvious reason to deviate from usual reproduction toxicity strategies [32,33].

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However, protection of the embryo against the teratogenic effects of cyclophosphamide, dioxin, methyl-nitrosourea, urethane and valproic acid was observed in mice following nonspecific stimulation of the maternal immune system by Bacillus Calmette – Gue´rin, a TH1 known immunostimulating agent [34]. These results are in keeping with previous findings [35,36]. Allergic diseases, such as asthma in childhood, have been suggested to start in utero [37] and the role of faulty TH1 cell control is a likely mechanism [38]. Overall, these data suggest that the maternal immune system has an influence on reproduction, the consequences of which warrant further clarification. Careful attention should be paid to the possible adverse influence of compounds inducing a shift in TH1/TH2 balance. This lends further support to the current trend leading to consider the need for reproduction toxicity studies of vaccines [39]. NONCLINICAL ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY The majority of immune functions appear before the end of the first trimester of pregnancy in humans. In contrast, some functions appear only after birth in rodents [40]. Differences in placental structure between humans and laboratory animals are also likely to play a major, if not fully clarified, role. Selection of animal models for nonclinical developmental immunotoxicology Mouse. The mouse has been extensively used for demonstrating post-natal immune deficits following pre- and perinatal exposure to immunotoxicants [41 – 53]. Assays utilized have historically been those developed for adult mice [54 –56], but their validity in the context of developmental immunotoxicity studies remains to be fully established [57]. Rat. The rat is the preferred animal species for nonclinical immunotoxicity evaluation and, therefore, logically considered for use in developmental immunotoxicity studies [58,59]. A number of studies have been conducted in rats exposed pre- or perinatally [2,3, 27,60 –73]. The PFC assay was shown to be possible in rats from 2 weeks of age [74]. Dog. Although, information available on the ontogeny of the immune system in the dog is limited, the dog presents more similarities to the human immune system than rodents, which should make the dog a more relevant model for developmental immunotoxicity assessment than mice or rats [75]. A major limitation to the use of the dog model is that nearly no immunotoxicity data are available and that assays have to be standardized and validated. Pig. Similarly, the pig could be an interesting model for developmental immunotoxicity assessment as a lot of information is available on the ontogeny of the pig immune system [76]. However, very few immunotoxicity data are available in this species. Monkey. Although monkeys are well suited for the nonclinical safety evaluation of drugs, limited information is available of their suitability for use as a valuable model for developmental immunotoxicity assessment [77,78]. Assessment strategies. Although, developmental immunotoxicity assessment is considered an important issue [9 –12] and children, a sub-population of increased susceptibility to immunotoxic insult [79], there are currently no validated methods for evaluating the effects of a chemical on the developing immune system. A key question is to determine


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the most appropriate species and strains to model the developing human immune system. Critical windows of exposure have been tentatively determined. There is uncertainty about how to apply standard animal models and assays to a developing animal. A frequent confusion is between prenatal and perinatal exposure. For prenatal exposure, conventional young adult animals may be appropriate as a number of studies showed that prenatal exposure was associated with persistent changes in immune functions measured in adult animals. For assessing the short-term consequences of pre- as well as perinatal exposure, very young animals have to be used, which may pose technical problems.

DEVELOPMENTAL IMMUNOTOXICANTS Organogenesis of the immune system occurs during the prenatal and, to a lesser extent, early post-natal periods of mammalian development. Therefore, high sensitivity to immunotoxicants that cross the placenta or enter the neonate via lactation is noted during the perinatal period. Post-natal immunotoxic consequences from chemical exposures during the establishment of the immune organs may be both more severe and more persistent than those that occur in at similar level of exposure as evidenced by a number of rodent studies. It is not surprising that later in development when the T and B lymphocyte repertoires are well established, the risk of permanent immunotoxicity might differ from that induced by prenatal or perinatal exposures. Due to the ever-increasing population of patients treated with immunosuppressive drugs, the chance that women of child-bearing age become pregnant while on immunosuppressive treatment is also increasing [80 – 85]. The available data are reassuring in that no excess of malformations was seen in the offspring of immunosuppressant-treated mothers whatever the treatment regimen [30,86] and in particular, no malformations of the lymphoid organs has seemingly been reported. In contrast, information on the immune status of babies exposed in utero or via breast feeding to immunosuppressive drugs is extremely scarce so that no firm conclusion can be made at the present time. Takabashi et al. [87] found severe B lymphocyte depletion, but no changes in the percentage of CD2þ, CD4þ and CD8þ T lymphocytes in the cord blood and peripheral blood at 1 month of age of 5 normal newborns from renal transplant mothers taking azathioprine, cyclosporine and methylprednisolone during their pregnancy. B lymphocyte counts returned to or near baseline values at 3 months of age. One recent isolated report described various autoimmune disorders in a 23-year-old woman born to a renal transplant mother treated with azathioprine and prednisone throughout pregnancy [88]. Although disturbances of organ functions have been recognized as a possible consequence of prenatal exposure to drugs and chemicals, the developmental immunotoxicological impact of only very few drugs and chemicals has been investigated in animals. As no human data are available in the majority of cases, the relevance or animal data is not known. DRUGS AS DEVELOPMENTAL IMMUNOTOXICANTS Acyclovir. This antiviral agent has been shown to induce congenital abnormalities in rats [89]. In the male and female offspring of Wistar rats given either 100 or 300 mg/kg of

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acyclovir subcutaneously at day 10 of gestation, the thymus weight was significantly reduced, whereas the spleen weight was increased, but only at the higher dose [90]. There was an inconsistent increase in IgM and IgG responses to the parasite Trichinella spiralis depending on the sex and age of the animals. The most consistent finding was an increase in IgE responses of animals exposed to the lower dose of acyclovir. Male offspring of dams exposed during gestation had impaired resistance to T. spiralis at 6 weeks of age evidenced by lower IgG, IgE and IgA antibody response to Trichinella antigens and a higher number of muscle larvae in the tongue muscle. Ampicillin. The offspring of mice treated with ampicillin during gestation had lowered cellular immune response, a decrease in T lymphocytes in lymph nodes, and an increase in humoral immune response [91]. This latter finding was confirmed by another study showing that mice administered 21 mg/kg/day of a mixture of ampicillin and cloxacillin (2:1) intraperitoneally from day 11 to day 15 of gestation had significantly higher plaque-forming cell responses [92]. Treatment of mice during the last week of gestation induced decreased relative thymus, spleen and lymph nodes weight, but no associated histological changes, and lymphocytosis in the offspring at 30 days of age [93]. Azathioprine. The influence of azathioprine is seemingly more pronounced, although it is often difficult to differentiate the effects due to azathioprine from those due to glucocorticosteroids in combination immunosuppressive regimens. Thymic hypoplasia, lymphopenia and transient decreases in serum IgG, IgM and IgA levels have been reported [94 – 96]. These anomalies were usually transient and reversed before the first year of age. They do not seem to develop when low doses of azathioprine are used [87]. Although, children exposed to azathioprine were suggested to be prone to myelosuppression and pancytopenia, humoral and cellular immunity between 1 and 6 years of age were in the normal range, even though azathioprine had seemingly a greater impact than cyclosporine [97]. Overall, these results lend support to the recommendation that children exposed to azathioprine in utero are monitored for immunological changes. Benzodiazepines. Diazepam has been one of the most commonly prescribed tranquilizers. A number of experimental studies show that in utero exposure to diazepam may adversely affect the immune response of offspring [98]. Subcutaneous injections of 1.25 mg/kg of diazepam from day 14 to day 20 of gestation in rats induced a 50% decrease in lymphocyte proliferative responses induced by con-A or a mixed lymphocyte culture, and this was noted for up to 2 months after birth [99]. The same treatment conditions induced a significant decrease in IL-2 release, but no changes in IL-1 release in pre-weanling rats. However, IL-1 release was significantly decreased in post-weanling rats until day 83, whereas IL-2 release returned to the normal range after day 60. The percentage of IL-2R (CD25þ) T-cells was not affected at any time point [100]. In other experiments using the same treatment conditions, IL-6 release by mitogen-stimulated splenocytes or macrophages [101] and TNF-a release by mitogen-stimulated splenocytes [102] were significantly decreased until 8 weeks of age. Interestingly, diazepam and a close derivative clonazepam were found to have higher affinity for peripheral benzodiazepine receptors expressed in the thymus and spleen of in utero treated pups than normal adult mice [103].


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Livezey et al. [104] described a higher incidence of uterine, lung and skin infections, and tumors associated with a lower number of plasma cells and a higher number of white blood cells in the offspring of female rats exposed to 6 mg/kg/day between day 15 and 20 of the gestation. In addition, a single subcutaneous dose of 10 mg/kg on the 7th post-natal day, or 3 doses of 5 mg/kg/day between the 5th and 7th post-natal day induced statistically significant decreases (range 20 –50%) in serum antibody titers to sheep erythrocytes and ovalbumin, and DTH to BSA and lymphocyte proliferative response to con-A when measured in rats at the age of 6 and 12 months [105]. Subcutaneous injections of 1.25 mg/kg/day diazepam from day 14 to day 20 of gestation in rats induced impaired resistance to an experimental T. spiralis infection as evidenced by a decrease in the number of larvae and an increase in Trichinella-specific serum IgG and IgA levels [62]. Subcutaneous injections of 1 or 1.5 mg/kg/day diazepam from day 9 to day 15 of gestation in hamsters induced impaired resistance of the offspring toward an experimental Mycobacterium bovis infection at 3– 4 months of age [106]. Corticosteroids. Although corticosteroids are often used in pregnant women, they have been claimed to exert no long-lasting effects on the immune function of the offspring [107]. However, several studies in either humans or laboratory animals somewhat contradict this conclusion. Betamethasone. Offspring of rats that were given 0.2 mg/kg of betamethasone on days 19 and 20 of gestation had decreased lymphocyte proliferative response to PHA and IL-2 6 days, but not 12 days after birth [108]. Newborns from mothers treated with betamethasone had enhanced NK cell activity and decreased lymphoproliferative responses measured in the cord blood [109]. In another study, decreased CD4þ T lymphocyte numbers and impaired neutrophil functions were found in preterm infants whose mothers had received betamethasone for the prevention of the respiratory distress syndrome [110]. In addition, 26 of 28 babies born from mothers treated with either betamethasone or dexamethasone for 3 consecutive days during the 29 –36 weeks of gestation had normal serum Ig levels [111]. Similarly, 10 preterm infants had normal IgM levels at birth after maternal treatment with 12 mg of betamethasone 1– 5 days prior to delivery [112]. Dexamethasone. An abrupt rise in leukocyte count involving neutrophils and neutrophil precursors was reported in 5 infants born from mothers treated with dexamethasone until 24 h before delivery in some of them [113]. Interestingly, similar findings have been reported in sheep [114]. Cellular immunity evaluated by lymphocyte proliferation induced by PHA or PWN was not affected in 6 premature infants whose mothers were treated by 4 –12 mg dexamethasone intramuscularly for 3 consecutive days prior to delivery [115]. Decreased humoral and cellular immunity was observed in rats at 7 months of age after a single injection of 1 mg/kg dexamethasone at 7 months of gestational age [116]. The intraperitoneal injection of 400 mg dexamethasone/female rats on days 17 and 19 of pregnancy induced an increase in CD4þ lymphocytes in the thymus, but not in the spleen on days 7 and 20 post-birth [117], which may be due to a delayed expression of glucocorticosteroid receptors as suggested by results in a transgenic mouse model [118]. Lymphocyte proliferation to Con-A was decreased in monkeys at 1– 1.5 years of age after 2 days of exposure for 2 days, 1 month before delivery [119], and confirmed the decreased proliferative capacity of lymphocytes in mixed lymphocyte reaction in monkeys exposed

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to dexamethasone for 2 days at 4.5 months after conception [120]. The most recent study compared the effects of 0, 0.0625, 0.125, 0.25, and 0.5 mg/kg/day dexamethasone-21 phosphate administered subcutaneously at days 6 – 21 of gestation [121]. Marked hypoplasia of thymic medulla was observed at 0.125 mg/kg in young and adult animals. DTH to KLH was depressed at 5 weeks of age whatever the dose, but only in the high dose group at 13 weeks of age. There was an increase in anti-KLH IgG antibody levels at 0.125 mg/kg and a decrease in total IgE levels. No consistent changes were seen in cell surface markers analyzed by flow cytometry. Macrophage function was only marginally affected. Prednisone is often included in immunosuppressive therapeutic regimens as an adjunct to first-line immunosuppressants, or used alone for the therapy of auto-immune diseases. Two babies exposed to prednisone and azathioprine had thymic hypoplasia without signs of immunodeficiency at birth, and decreased lymphocyte counts during a 2-year follow-up [122]. Another baby born to a mother with lupus erythematosus treated with prednisone had symptomatic cytomegalovirus infection at birth [123]. In contrast, the offspring of seven mothers who received prednisone and azathioprine during the whole pregnancy for kidney transplantation had normal cord blood immunoglobulin concentrations [124]. Cyclophosphamide. Mice were injected daily either intravenously with 5 mg/g of body weight on day 12 of gestation, or 1 mg/g of body weight from day 9 to day 12 or from day 14 to day 17 of gestation, intraperitoneally with 1, 2.5, or 5 mg/g of body weight on day 12 of gestation, or 5, 10, or 20 mg/g of body weight on day 17 of gestation [125]. No consistent enhancement or suppression of cellular or humoral immune function was observed in offspring of treated dams. Increases in B lymphocyte proliferative response to LPS in 5-week-old animals and plaque-forming cell responses in 8-week-old offspring exposed to cyclophosphamide at 20 mg/g on day 17 of gestation were the only findings. A single dose of 23.5 mg/kg cyclophosphamide at the 16th day of gestation in mice has been reported to induce decreased lymphocyte proliferative responses to LPS, but not to con-A or PHA in the pups that had resolved at 16 weeks of age, and transient increase in both delayed-type hypersensitivity and peritoneal macrophage activity [126]. In contrast, a single intraperitoneal administration of up to 30 mg/kg of cyclophosphamide to pregnant rats at day 11 or 15 of the gestation induced neither changes in the histology of the thymus and spleen of the fetuses, nor in lymphocyte subsets and resistance to an experimental T. spiralis infection of pups up to 6 weeks of age, whereas dosedependent gross malformations were seen [61]. Cyclosporine. Only rare animal studies are available. Pups born to female rats given 15 mg/kg/day cyclosporine from coitus to day 21 of gestation had transient histological changes in the thymus, spleen and bone marrow [127]. The daily subcutaneous administration to mice of 15 or 25 mg/kg/day of cyclosporine for 20 days after birth (corresponding to the lactation period) induced a significant decrease in thymus weight and cellularity in the pups with a nearly complete loss of the cortical area of the thymus. Thymocyte proliferative responses to con-A plus IL-2 were decreased in both the mothers and pups, whereas splenocyte proliferative responses were only noted in the pups. All changes disappeared within 30 days post-weaning [128]. The relatively slight effect of in utero


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exposure to cyclosporine on the immune status of pups is probably related to a poor placenta crossing in rodents. Limited information is available on the possible adverse effects of cyclosporine treatment of pregnant women on their offspring. Importantly, accumulation of cyclosporine in the embryo was observed in the first trimester of pregnancy [129]. Infants born to mothers treated by cyclosporine during pregnancy show a delay in T and B cell development and maturation [130]. The 1 year follow-up of six infants evidenced a decrease in total, CD4þ and CD8þ T lymphocytes, which rapidly normalized after birth, T lymphocyte activation markers below normal during the whole period, low B lymphocyte counts and subnormal serum IgG1 and IgG3 concentrations up to six months after birth, but normal numbers of NK cells [131]. A decrease in B lymphocyte counts was also noted during the first 2 years of life in one infant [132]. B lymphocyte depletion was particularly severe at birth when cyclosporine was combined with prednisone and azathioprine [87]. Minimal effects on T cell development were noted in seven children [97], or on lymphocyte counts and lymphocyte subset analysis in three neonates exposed to cyclosporine in utero [133,134]. With the growing number of babies born to mothers treated with cyclosporine during pregnancy, a better evaluation for safety is expected. Although cyclosporine is often used in the treatment of auto-immune diseases, it can paradoxically induce autoimmunity in laboratory animals. Autoantibodies have been detected in 11 of 50 offspring born to pregnant mice treated with cyclosporine [135]. However, at the present time, whether in utero exposure to cyclosporine can predispose human beings to autoimmunity is not known. Diethylstilbestrol (DES). It has been estimated that up to 4 million human female fetuses worldwide have been exposed to the estrogen derivative (DES) in the 1940s –1970s for the mistaken use as a preventive measure against miscarriages and preterm births. DES has been shown to induce a variety of cancers, most notably adenocarcinomas of the vagina as well as morphological and functional anomalies in the reproductive tract of exposed humans of either sex [136]. The question whether in utero exposure to DES is associated with changes in immune function or immune-mediated diseases in human adults of either sex is still a matter of debate and conflicting results have been reported [137,138]. Enhanced T lymphocyte proliferative responses to PHA were evidenced in 8 adult DES-exposed women compared to 8 matched nonexposed controls [139]. Similarly, lymphocyte proliferation was found to be increased 3-fold in response to Con-A, 2-fold to IL-2, and by 50% to PHA in 13 women exposed to DES in utero when compared to 13 matched unexposed women [140]. The child of a DES-treated mother developed acute lymphoblastic leukemia, but obviously no causal relationship can be established [141]. When comparing 179 women exposed to DES in utero with 123 controls using a questionnaire for immune-related diseases, Vingerhoets et al. [142] found more allergies, autoimmune diseases and infections in exposed women, but the increase was only statistically significant for measles and urinary infections probably due to the small number of surveyed women. Another study comparing 253 male and 296 female patients exposed to DES in utero with 241 male and 246 female nonexposed controls found a similarly increased incidence of allergies, infections and autoimmune diseases in both groups [143].

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Although DES is a recognized immunotoxicant in laboratory animals [144,145], perinatal immunotoxicity studies gave rather diverging results. The daily subcutaneous injection of 5 mg per mouse of DES for 5 consecutive days starting within 24 h after birth induced a reduction in natural killer cell activity [146] and antibody response to the T-independent antigen LPS and to sheep erythrocytes [147] in female Balb/c mice. At 4 months of age, Balb/c mice treated with one daily subcutaneous injection of 1 mg DES for 5 consecutive days immediately after birth had significantly reduction in lymphocyte proliferative response to PHA [148]. In contrast, the administration of up to 60 mg/kg of DES to pregnant rats by gavage at days 0, 6, 11, 16 and 21 of gestation, however, induced no changes in the histology of the main lymphoid organs or antibody responses to SRBC in pups at 4 weeks of age [61]. Dimercaptosuccinic acid (DMSA) is used as a chelating agent in the oral treatment of lead poisoning. Fisher 344 rats were given 30 or 60 mg/kg/day of DMSA orally from day 6 to day 21 of gestation [67]. No significant differences were noted either for litter size, offspring sex ratio, growth rate, hematocrit and total leukocyte count, or lymphocyte subset analysis, IgG, IgG1 and IgG2a responses to KLH. DTH to KLH was mildly, but significantly suppressed at the higher dose of DMSA. No changes in IFN-g, IL-2 or IL-4 production by ConA-stimulated splenocytes were seen. Diphenylhydantoin is an anticonvulsant drug that is teratogenic in rodents. In humans, it can induce the so-called fetal hydantoin syndrome, but no malformations of the lymphoid organs have been included in this syndrome. Limited data suggest that it might be a developmental immunotoxicant. C3H mice were given 25 mg/kg phenytoin twice a day by gavage during the whole gestation. Pups sacrificed at day 5 after birth had reduced thymic cortex and enlarged white pulp of the spleen, whereas no similar changes were seen in the mothers [149]. Balb/c mice were given 0, 20, 40, or 60 mg/kg/day of phenytoin by gavage from day 9 to day 18 of gestation. A dose-dependent decrease in humoral immunity assessed from serum antibody levels to type III pneumococcal polysaccharide, was observed in offspring at 25 days, but not at 15 weeks of age. Cell-mediated immunity assessed by contact sensitivity to oxazolone was not affected by prenatal phenytoin exposure [150]. Promethazine is an H1 histamine receptor antagonist formerly widely used in the treatment of allergic disorders. Early studies suggested that promethazine exert immunosuppressive effects (see Chapter 7 of volume II). A profound decrease in B and T lymphocyte numbers at birth was found in the cord blood of 15 babies born from mothers with erythroblastosis treated by promethazine [151]. At 6 weeks of age, B and T cell numbers had returned to normal value. In addition, normal B and T lymphocyte numbers were found in 5 of these women. A similar decrease in B and T cell numbers was also reported in 3 newborns [152]. However, the role of promethazine is debated. Thalidomide is a very potent teratogen first marketed in Germany in 1956. Due to its anxiolytic and anti-emetic activities, it was widely used in the early stage of pregnancy. In 1961, the causative role of thalidomide in thousands of cases of limb gross malformations,


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particularly phocomelia, was suspected and it was withdrawn from the market. In the recent years, thalidomide was rediscovered as a potentially useful drug in several severe skin diseases. Decreased expression of the integrins CD11a, CD11b, CD18, CD49 and CD61 as well as the adhesion receptors CD54, CD62L and CD71 was evidenced on limb bud cells of rats, but not of monkeys, suggesting a possible common mechanism for thalidomideinduced teratogenicity and immunomodulatory effects [153]. CHEMICALS AS DEVELOPMENTAL IMMUNOTOXICANTS The immunotoxic effects of pre-or perinatal exposure to a number of chemicals have been studied. However, the majority of available data derives from animal studies and their relevance to man is largely unknown. Pesticides. There is relatively limited evidence that pesticides exert immunotoxic effects. The available data are restricted to a few compounds or groups of compounds (see Chapter 2 of volume III). Of these, the immunotoxic effects of selected compounds have been investigated following pre- or perinatal exposure. Chlordane. Although immune changes have been shown in adult animals exposed to this organochlorine cyclodiene insecticide, the available data suggest more pronounced effects on the developing than mature immune system. A number of animal studies, particularly in Balb/c mice, investigated the effects of chlordane on immune functions after prenatal exposure, but no human data is available. Pregnant Balb/c mice were given daily 0, 4 or 16 mg/kg/day of technical chlordane mixed in food from the start of gestation until day 19. Contact hypersensitivity to oxazolone was depressed at 100, but not 30 days of age. Lymphocyte proliferative responses were either unchanged when induced by the mitogens PHA or LPS, enhanced by con-A, or depressed in a mixed lymphocyte reaction [154]. In contrast, oral exposure to 0.12, 2, 4 or 8 mg/kg/day of technical chlordane from the start of gestation until day 19 enhanced the primary and secondary antibody response to influenza type A virus in female Balb/c mice, but not in males, and slightly enhanced survival to this experimental infection at day 38 [155]. The oral exposure of Balb/c mice to 4 or 8 mg/kg/day of analytical grade chlordane from day 1 to day 18 of gestation caused a significant decrease in the NK cell activity of the offspring at 200, but not 100 days of age, whereas T lymphocyte cytotoxicity was not affected [156]. In another study, the offspring of Balb/c mice exposed orally to 0 or 8 mg/kg/day of chlordane from day 1 to day 18 of gestation, had delayed and/or impaired tumoricidal activity, including hydrogen peroxide and nitric oxide release by macrophages in vitro, and cytolytic activity against P815 cells, at 100 –125 days of age [157]. Resistance toward an experimental influenza infection was enhanced. No direct or indirect changes in T lymphocyte functions were evidenced, but a marked depression of the tumoricidal activity of macrophages was reported [158]. Cypermethrin. Pyrethroid insecticides are widely used for agricultural and domestic applications due to their potency and low toxicity to mammals. Although their effects on the immune system have not been extensively investigated, a few studies suggest that the type II synthetic pyrethroid cypermethrin can exert immunotoxic effects following in utero exposure. Female Wistar rats were exposed to 50 mg/kg cypermethrin or corn oil orally

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from day 7 to day 16 of the gestation and showed no evidence of toxicity. A significant [20%] increase in the percentage of double positive CD4þ/CD8þ thymocytes, and a marked decrease in CD4þ and CD8þ T lymphocytes were observed in the offspring between 15 and 90 days after birth and gradually recovered from 2 50% to 2 15% during this period of time [159]. In addition, the proliferative responses of thymocytes to recombinant human IL-2 (rhIL-2) or con-A was inhibited as was con-A-induced release of IL-2 by thymocytes. In the same experimental conditions, there was an increase in total, CD4þ and CD8þ T lymphocyte numbers in the peripheral blood, but a decrease in the spleen. Similarly, the proliferative responses to con-A or rhIL-2 were enhanced in T lymphocytes of the peripheral blood, but decreased in lymphocytes from the spleen. Interestingly, enhanced proliferative responses of lymphocytes of the peripheral blood that were still noted at day 90 after birth paralleled the increase in plasma noradrenaline but not adrenaline concentrations [160]. As type II synthetic pyrethroids are known to release noradrenaline and adrenaline, the assumption was made that noradrenaline plays a role in the modulation of T lymphocytes functions by cypermethrin. Finally, a significant enhancement of NK cell activity and antibody-dependent cellular cytoxicity (ADCC) in the peripheral blood, but a decrease in the spleen was observed in pups from day 60 to day 120 after birth [161]. That prenatal exposure to cypermethrin was the cause of these changes is suggested by the lack of effects in exposed pregnant dams. Dichlorophenoxyacetic acid. The n-butylester of 2,4-dichlorophenoxyacetic acid (2,4-D) was administered orally to mice on day 11 of gestation. The PFC response was not altered by exposure during gestation. The proliferative responses of lymphocytes induced by con-A and LPS were slightly reduced in the group exposed to 200 mg/kg [162]. The effect of 2,4-D was evaluated on urethan-induced pulmonary adenoma formation in female offspring at 19 weeks after birth after exposure to 0 – 650 mg/kg 2,4D on days 6 – 16 of gestation in drinking water. 2,4-D in utero exposure did not affect the number of tumors, but reduced the mean tumor diameter in the highest dose group [163]. In another study using the same experimental exposure, the effects of 2,4-D were evaluated in offspring 7 weeks after birth. Con-A induced-lymphocyte proliferation was observed in the high dose group. There was also an increase in B lymphocyte numbers and a reduction of T cytotoxic lymphocytes [51]. Heptachlor. Pregnant Sprague –Dawley rats were given 0, 30, 300, or 3000 mg/kg/day of heptachlor from gestation day 12 to post-natal day 7. The pups were subsequently dosed until post-natal day 42. There was no toxicity on dams or pups. Lymphoid organ weights, NK cell activity, and mitogen- and alloantigen-induced lymphoproliferative responses were normal as compared to controls at 8 weeks of age. DTH or contact hypersensitivity responses were not affected at 10 or 17 weeks of age, respectively. Primary and secondary humoral immune responses to SRBC were dose-dependently suppressed in males, but not females, and this effect persisted for up to 20 weeks [69]. Hexachlorobenzene (HCH) is one of the rare environmental chemicals to exert immunostimulatory effects in the rat, although not in the mouse. A few studies suggest that the developing immune system is more vulnerable in rats as well as mice. Vos et al. [164] performed a fairly comprehensive pre- and post-natal study in rats given 50 or 150 mg/kg/ day of HCH continuously from day 1 –3 of gestation to 5 weeks after birth. They observed


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an increased number of high-endothelial venules in the paracortex of mesenteric and popliteal lymph nodes, and focal accumulation of alveolar macrophages, but no effects on serum total IgM and IgG levels and lymphocyte counts. At both dose levels, the resistance to L. monocytogenes infection was decreased, the rejection of skin tail grafts was delayed and the mitogen-induced proliferative responses of splenocytes and thymocytes were suppressed. In contrast, IgG response to tetanus toxoid and IgE response to T. spiralis antigen were markedly increased, whereas the IgM response to the T-independent antigen LPS was unchanged. No effects were noted on passive acute anaphylaxis to T. spiralis or the clearance of carbon particles and L. monocytogenes. As general toxicity and immunotoxic effects were noted at higher doses in adult rats, these authors concluded that the immune system is a target organ of toxicity of HCB and that the developing immune system is particularly vulnerable. The same group confirmed these results using the same experimental conditions with lower exposure levels, namely 4 and 20 mg/kg [165]. NK cell activity was not affected. In contrast, Balb/c mice exposed throughout gestation to 0.5 or 5 mg/kg/day of HCH had a significant suppression of contact hypersensitivity to oxazolone at 45 days of age. Lymphocyte proliferation was inhibited in a mixed lymphocyte response, but was unaffected when stimulated by mitogens. T lymphocyte counts were increased and B lymphocyte counts decreased in the spleen. Finally, no effects were evidenced on the PFC assay [166]. Hexachlorocyclohexane. DTH response to SRBC was significantly higher in pups of Swiss mice exposed to 10 mg/kg throughout the gestation, whereas it was significantly impaired in pups of dams exposed to 100 mg/kg. Lymphocyte proliferative responses to con-A and LPS were 2-fold and 8-fold higher in the low-dose exposed pups as compared to controls, respectively, but were unchanged in the high-dose group. Similarly, PFC assay showed a 2-fold higher response in pups exposed to 10 mg/kg, but not in pups exposed to 100 mg/kg [167]. Tebuconazole, a triazole fungicide, was administered daily to Sprague– Dawley dams by oral gavage (0, 6, 20, or 60 mg/kg) from gestational day 14 to post-natal day 7. Then, pups were exposed daily to the same dose levels until post-natal day 42. No significant changes in immune function were evidenced [70]. Environmental and industrial chemicals Benzo[a]pyrene. Exposure of mice to a single small dose of benzo[a ]pyrene in utero led to a severe and persisting suppression of PFC response with subnormal cellular immunity shortly after birth, and an increased incidence of tumors following X-ray irradiation [168,169]. Abnormal changes in developing T lymphocytes have repeatedly been reported [170,171]. Biphenyls. Swiss –Webster mice were fed 0, 10, 100, 250, or 1000 ppm of Aroclor 1254 for 12 weeks [172]. Exposure continued during the whole of gestation and lactation. At 8 weeks of age, the only statistically significant effect was a 20% decrease in serum antibody titers to BSA at the highest, but not at all other exposure levels. No effects were evidenced on the contact hypersensitivity response to oxazolone and the phagocytic activity of peritoneal macrophages. In another study, adult female mice received 3 subcutaneous injections per week of 50 mg/kg Aroclor 1242, Aroclor 1254 or corn oil for 2 weeks and then mated with nontreated males. First-litter pups were sacrificed at 7 or 28 days of age.

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Splenic IL-2 production was decreased in 7-day-old pups exposed to Aroclor 1242 or Aroclor 1254. Increased spleen weight was noted in Aroclor 1254-exposed pups only, whereas 28-day-old pups had a significant decrease in thymus weight, a decrease in CD4þ T lymphocyte and B lymphocyte counts [52]. Carbon monoxide. Wistar female rats were exposed to 75 or 150 ppm of carbon monoxide from day 0 to day 20 of pregnancy. Splenic macrophage phagocytosis of C. albicans was significantly decreased in 15-day-old male pups prenatally exposed to 75 and 150 ppm. Prenatal CO (150 ppm) significantly decreased splenic macrophage O22 release in both groups. No alterations were observed in 60-day-old rats [60]. 2,4-Dichlorophenol is a drinking and waste-water contaminant formed following the chlorination of water for disinfection and deodorization. Rats were exposed to 0, 3, 30, or 300 ppm in drinking water in utero or for 12 week post-natally following in utero exposure. Rats that were exposed in utero and post-natally to 300 ppm had significantly increased spleen weights, enhanced humoral immunity and depressed cellular immunity. In contrast, at 6 weeks of age, offspring of dams had normal immune functions [173]. Dioxin (TCDD) is by far the environmental immunotoxicant that has been the most comprehensively investigated in utero or perinatally [46,63,64,68,174,175]. When administered during the maturational development of the immune system, TCDD causes more persistent immunosuppression than in adult life. Thymic atrophy in neonates occurs at low-level of exposure; although less than 0.5% of TCDD crosses the mouse placenta single-dose or subacute exposure of pregnant mice to 1 mg/kg causes a highly significant involution of the fetal thymus and inhibition of thymocyte differentiation. The inhibition of T lymphocyte proliferative responses lasts 8 –10 weeks after birth. Delayed skin graft rejection and suppressed DTH responses have also been reported. In addition, perinatal TCDD exposure increases susceptibility to challenge with various infectious agents or syngeneic tumor cells. However, no relationship between pre- and post-natal PCB/dioxin exposure and upper or lower respiratory tract symptoms or humoral antibody production was evidenced in Dutch infants exposed to biphenyls and dioxin from birth to 18 months of age [176]. In a subsequent study on a subgroup of 85 children, the same authors found an increased number of total lymphocytes, T lymphocytes, CD4þ and CD8þ T lymphocytes and activated T lymphocytes associated with lower antibody levels to mumps and measles at preschool age. They also found a higher prevalence of recurrent middle-ear infections and chicken pox and a lower prevalence of allergic reactions [178]. These conflicting results obviously await confirmation and clarification to draw definitive conclusions. Lead is of particular toxicological concern during early development. There is evidence that lead can interfere with immune responses. Several studies have shown that in utero lead exposure can exert immunotoxic effects [3,4]. Rats exposed in utero and for 4 weeks post-natally were present with various changes in the humoral and cell-mediated immune response. Swiss – Webster mice were fed 0, 10, 100, 250 or 1000 ppm of lead, as lead acetate. They were then bred and exposure continued during the whole of gestation and lactation. At 8 weeks of age, several immune parameters were analyzed in the offspring. No effects were evidenced on the contact hypersensitivity response to oxazolone, on serum antibody titers to BSA and the phagocytic activity of peritoneal macrophages [172]. Female Fisher 344 rats were exposed to 0, 100, 250 or 500 ppm lead in the drinking water


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and housed with male rats. Exposure was continued until parturition. Immune function was investigated in female offspring at 13 weeks of age. No statistically significant changes in leukocyte subpopulations, NK cell activity, KLH-specific IgG titers, or IL-2 production by ConA-stimulated splenocytes were noted whatever the level of exposure. A significant 2-fold increase in total serum IgE levels was the only change seen in 100 ppm exposed animals. The offspring of 250 ppm exposed dams had an elevated in vitro production of TNF-a by cultured splenocytes and a small, but statistically significant decrease in DTH response to KLH. Finally, the offspring of 500 ppm dams had a significantly decreased IFN-g production by Con-A stimulated splenocytes [65]. Although the relevance of these changes is unclear, they nevertheless indicate that in utero lead exposure may induce persistent changes in some aspects of the immune response. When comparing male and female Fisher 344 rats at 5 and 13 weeks, after exposure to 50, 100, or 250 ppm lead ad libitum throughout gestation, more profound immunotoxicity was found in females than males. Interestingly, some changes were seen only in adult animals and most changes were persistent [179]. Methylmercury. Balb/c mice were exposed to 0, 0.5 or 5 mg Hg/kg as methylmercury in the diet. Dams were exposed for 10 weeks prior to mating, during gestation and lactation, and pups were directly exposed to mercury until day 15 of lactation. Lymphoid organs were unaffected by exposure. Offspring from the high-dose group had increased numbers of splenocytes and thymocytes. There was a significant decrease in the number of CD4þ T lymphocytes at 10, but not at 22 days of age. In addition, CD8þ T lymphocyte numbers were increased in the high-dose group at day 22 and 50 post-partum. The proliferative response of splenocytes to LPS and the primary antibody response to the viral antigen PR8 were increased in offspring from the high-dose group. No changes in T lymphocyte proliferation induced by con-A or NK cell activity was evidenced [49]. Ochratoxin A is an immunosuppressive myocotoxin. Sprague –Dawley dams were given a single dose of 0, 10, 50 or 250 mg/kg on day 11 of lactation. The con-A induced proliferative response of splenocytes was significantly increased following 10 or 50 mg/kg and that of thymocytes after 50 mg/kg. In a long-term study, dams received 50 mg/kg orally 5 days a week, 2 weeks before mating, during gestation and then 7 days a week until weaning. Suppressed lymphoproliferative responses to LPS and Con A were observed on day 14 of lactation. At 13 weeks, the response to LPS was still impaired. The primary antibody response to the viral antigen PR8 was also lower in prenatally exposed pups [180]. The effects of maternal exposure to a single dose of ochratoxin A during gestation or lactation were also studied in Balb/c mice. A single 500 mg/kg dose on day 16 of gestation resulted in decreased mitogen-induced proliferation of splenic and thymic lymphocytes in the pups at 15 days of age. There was a lower percentage of mature CD4þ lymphocytes. In contrast, a single dose on day 10 post-partum significantly increased mitogen-induced proliferative responses of lymphocytes in the offspring 3 days after the exposure in the highest dose group [48]. Phenol was administered to Sprague –Dawley rats in the drinking water at the concentrations of 0, 200, 1000, or 5000 ppm, for 10 weeks prior to mating, during mating, gestation, lactation, and until sacrifice. No changes in either thymus and spleen histology, or the plaque-forming cell assay were evidenced in the offspring [181].

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Tin. Pregnant and/or lactating Fischer 344 rats were exposed prenatally, both pre- and post-natally, or post-natally to dioctyltin chloride by oral gavage of pregnant and/or lactating females. Treatment on days 10 –20 of gestation, or days 11 –20 of gestation and days 2 –11 of age, or days 2 –13 of age resulted in no alterations in offspring immune responses. Oral dosing beginning at 3 days of age and then 3 times per week up to 24 days of age resulted in significant suppression of mitogen-induced T lymphocyte proliferative responses in 10-week-old rats. Lymphoproliferative responses returned to control levels by 12 weeks of age [182]. 2,4,6-Tribromophenol (TBP) is a chemical intermediate in the manufacturing of pharmaceuticals, dyes, plastics and pesticides. Female Wistar rats were exposed to 0, 0.03, 0.1, 0.3 or 1 mg/m3 TBP 24 h/day by whole body inhalation from day 1 to day 21 of gestation. No effect on phagocytosis was noted [183]. Urethan (methylcarbamate) is a potent carcinogen with immunosuppressive properties. Female C57Bl/6 mice were injected daily via the subcutaneous route with 0.05 or 0.1 mg/g of body weight from day 7 to day 17 of gestation. Although pups from exposed dams had a normal NK cell activity, they had elevated white blood well counts, increased spleen and thymus weight, markedly decreased lymphoproliferative responses to mitogens as well as PFC response [41]. Substances of abuse Alcohol is a known teratogen and immunoteratogen [184 – 195]. The fetal alcohol syndrome was first described in 1968 by French authors [196], but their findings were confirmed only 5 years later [197]. The syndrome is characterized by low birth weight, various craniofacial and skeletal anomalies as well as cardiac and brain defects. A number of animal and human studies linked prenatal alcohol exposure to changes in immune competence [198]. Children with the fetal alcohol syndrome develop more frequent infections [199 – 203] and cellular immunity is seemingly the primary target of in utero alcohol exposure. Defects in B and T cell functions were evidenced in children with fetal alcohol syndrome as well as recurrent or more severe infections. There is a large body of evidence that in utero exposure to alcohol exerts a negative influence on the thymus. Several cases of DiGeorge syndrome associated with maternal alcoholism have been reported [204] and similar findings could be reproduced in mice [205, 206]. This is a congenital immuno-deficiency syndrome characterized by abnormal facies, congenital heart disease, hypocalcemia, increased susceptibility to infections and the absence of thymus. Significantly reduced thymus weight was noted in 18-day-old fetuses of C57Bl/6 mice fed a liquid diet containing 25% ethanol-derived calories [207]. In addition, there was a reduced proliferative response of fetal thymocytes stimulated by Con-A. An immaturity of thymocytes with blurring of the histological distinction between the cortex and medulla was found using the same conditions of exposure [208]. In utero exposed offspring of mice aged 5 – 8 months had inhibition of B and T lymphocyte mitogen responses, but NK cell activity was unchanged [209]. A decrease in serum antibody titers to tetanus toxoid without changes in T lymphocyte proliferation and monocyte phagocytosis was found in nonhuman primates exposed weekly during pregnancy [210]. Cocaine. Pregnant Sprague –Dawley rats and C57BL/6 mice were injected subcutaneously twice a day from day 5 of gestation until the day before parturition with 10


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or 20 mg/kg/day in rats and 10 mg/kg/day in mice. At 8 weeks of age, only the female rat offspring of the lower dose group had a statistically significant decrease in the PFC assay. No effect on the DTH response to KLH was noted. There was no difference in lymphocyte subsets, except for an increase in CD8þ T lymphocytes in males. No effects were seen in the mouse offspring at 4 weeks of age [50]. Interestingly, peripheral blood mononuclear cells isolated from the cord blood of newborns from cocaine-addicted mothers showed decreased responses to PHA and phorbol 12-myristate 13-acetate. IL 1 and IL 2 concentrations were suppressed in the cocaineexposed fetal serum [211]. Tobacco smoking. The relationship between maternal smoking and low birth weight was first described in 1957. Subsequently, the risk of congenital anomalies and fetal death was demonstrated [212]. There is a growing body of evidence that maternal smoking is associated with immunological adverse effects on the offspring. In a study of 223 babies, Devereux et al. [213] found a correlation between maternal smoking and increased in vitro proliferative responses of cord blood lymphocytes to PPD, timothy grass pollen extract and house dust mite extract, suggesting that maternal smoking is associated with a greater risk of atopy. Similarly, children who had been exposed to tobacco smoke prenatally had a higher incidence of food allergies, but no more frequent sensitization to respiratory allergens [214]. However, inconsistent results have been published regarding the risk of higher IgE levels at birth or sensitization in childhood following exposure to tobacco in utero [215]. The comparison of 163 mothers who smoked during pregnancy with 130 mothers who did not smoke showed a significant increase in IgG, IgM and IgA levels in the cord blood of newborns from mothers who smoke [216]. Cord blood mononuclear cells from babies exposed to maternal smoking in utero produced more IL-13 when stimulated by house dust mite extract or ovalbumin [217]. A similar, but not statistically significant trend was observed for IL-5, IL-6 and IL-9. Finally, newborns from smoking mothers had reduced numbers of neutrophils [218]. Thousands of different chemicals have been identified in tobacco smoke many of which have toxic effects, including carcinogenic and immunosuppressive effects [219]. Nicotine is responsible for tobacco addiction. There is evidence that nicotine is immunosuppressive in adult humans and rodents. The subcutaneous injection of 6 mg/kg/day of nicotine from gestation days 10 to 20 to pregnant rats induced impaired lymphoproliferative responses of splenocytes to Con-A and LPS from days 9 to 86 in the offspring [220].

CONCLUSION There is a large and still growing body of evidence demonstrating that exposure to drugs and other chemicals pre- or perinatally may result in significant changes of immune responses. Importantly, the developing immune system may be more susceptible to immunotoxic injury than the immune system of adults. Developmental immunotoxicity is therefore a major concern. However, the available data underscore the need for improving our understanding of the mechanisms involved and for defining the animal models and assays, as well as the strategies that should be applied to enhance the prediction of developmental immunotoxic hazards and risks.

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Chapter 14

Immunotoxicity and wildlife

INTRODUCTION Laboratory studies have shown that environmental contaminants can suppress immune function and increase susceptibility to infectious diseases. Often these chemicals act at low doses and cause persistent effects, especially following pre- or perinatal exposure. These studies raise concerns about the potential immunotoxic impacts on wildlife and humans. Because fish are exposed to pollutants present in contaminated water, they can be used as sentinel animals or biomarkers of immunotoxicity [1]. The epidemic which killed thousands of seals from the Northern Sea was another important impetus to immunotoxicity studies in wildlife [2]. In contrast, limited information is available on the immunotoxicity of veterinary medicinal drugs and environmental chemicals on domestic animals. Overall, the rarely reported effects in domestic animals are similar to those noted in either laboratory animals or humans [3,4]. As immunotoxicity assessment in wildlife species is a broad specialized area of immunotoxicology, this chapter will only give a brief overview of the available data.

IMMUNOTOXICITY IN FISH Immune defense mechanisms of fish have not been investigated as extensively as in mammals, but they appear to share a number of structural and functional similarities. Immune cells of fish include B and T lymphocytes, nonspecific cytotoxic cells (proposed as equivalent to mammal NK cells) and phagocytic cells. Because of these similarities, immune responses of fish are increasingly considered as models for immunotoxicological evaluation. Because of the development of aquaculture, concern arose regarding the economic impact of diseases in fish, and this in turn served as a basis for the development of research in fish immunology and microbiology [5 – 7]. In addition, the impact of water contamination by chemical pollutants on fish health status is an expanding research area. 355

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FISH IMMUNE SYSTEM AND DISEASES The functions of the immune system in fish are similar to those in mammals in many aspects [5]. Over 20,000 species are known, however, and there are inter-species differences. The fish immune system is formed of nonspecific and specific mechanisms, the latter including humoral and cellular immune responses. Thus, fish can produce antigenspecific antibodies, mount DTH responses or reject allografts. Fish lymphocytes proliferate in response to mitogens and release cytokines, while granulocytes and macrophages can phagocytose foreign pathogens. Neither lymph nodes nor bone marrow is found in fish, but an equivalent of the hematopoietic tissue is located in several areas of the body, such as the spleen, thymus, head and kidney. Fish have circulating white blood cells, which are functionally and morphologically similar to those of mammals. The most common diseases in fish related to pollution are skin diseases, such as lymphocystis, papillomas, fin rot and skin ulcers [5,8,9]. These diseases can be easily identified and because they are caused by bacteria or viruses, the role of the immune system is obvious. Liver tumors have also been suggested to correlate with water pollution. Nevertheless, no conclusive correlations between these diseases and environmental pollution have so far been obtained. Although laboratory studies are helpful to identify the immunotoxic effects of pesticides, heavy metals, chlorinated hydrocarbons present, for instance, in harbor sediment, sewage sludge, or pulp mill effluent, it is far more difficult to delineate the role of immunotoxic chemicals in field conditions, as other factors, such as migratory patterns, temperature, and stress, may interfere with the development of the disease [10 – 12].

IMMUNOTOXICITY STUDIES IN FISH Many screening and functional immunotoxicological techniques routinely used in mammals have been rather successfully applied to the study of fish immunology and immunotoxicology [13 –15]. However, most of these techniques are only available in research laboratories, which is a major limitation to their use in field studies. Immunosuppression is the primary focus of immunotoxicity studies in fish. In addition to the economical impact on aquaculture of such deleterious exposures, adverse effects on the health status of fish can be indicative of possible consequences on human health [1,10,12]. Fish meet several criteria for use as biomarkers of immunotoxicity. Fish are sensitive to environmental immunotoxic exposures. Immunological assays in fish give reproducible and validated changes, and the observed changes are dose-dependent. A wide range of fish species can be used and the selection is primarily based on the investigator’s experience and the laboratory’s background. The preferred species are trout [16], carp (Cyprinus carpio) [17] and flounder [18] because they are more easily manageable due to their size, which enables blood and tissue sampling. Smaller species, e.g. guppies, have the advantages of easy husbandry and lowered cost, but blood and tissue samplings are difficult. White blood cell counts and lymphocyte subset analysis can easily be performed in a blood sample drawn from a live fish. In fish of adequate size, the spleen can be easily


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removed and weighted, but the lymphoid tissue is poorly developed in the spleen of most fish species. Thymus weight is seldom used in fish immunotoxicology because of its complex localization in most fish species. Thymus morphology could be more useful, and thymus atrophy could be evidenced in several, but not all fish species, after exposure to tributyltin oxide [19]. Melanomacrophage centers are widely distributed throughout the body of fish. They are formed of clusters of macrophages, the function of which is not yet fully understood. They have been suggested to be primitive analogs of lymphoid follicles, and tend to increase with age and after stress. Macrophage function tests, such as chemiluminescence, chemotaxis and phagocytosis, are considered as useful indicators of immunotoxicity. Fish macrophages indeed share most morphological and functional features of mammalian macrophages, and a number of studies on the impact of environmental chemicals on macrophage and phagocyte functions have been published [20 –26]. NK cell activity can be measured by flow cytometry [27]. Humoral immunity can be assessed by the determination of total circulating immunoglobulin levels or the specific antibody response to standard antigens [28 – 30]. Agglutination and ELISA are the most commonly used techniques. The plaque-forming cell (PFC) assay is less commonly used. However, a similar pattern of inhibition on antibody production was shown using this assay when comparing 14 immunotoxicants in tilapia (Oreochromis niloticus) and rodents [31]. LPS can induce the proliferation of fish B lymphocytes. Cellular immunity in fish is quite similar to cellular immunity in mammals. Evaluation of cellular immunity can be best performed using mitogen-induced lymphocyte proliferation assays [32,33]. IMMUNOTOXICITY OF CHEMICALS IN FISH Fish are exposed to an enormous variety of chemicals due to discharges to rivers and lakes, marine dumping and atmospheric fallout. A fairly large number of environmental chemicals have been tested for their immunotoxic potential in fish. Most metals have been shown to exert immunotoxic properties in fish [20]. Lead and cadmium have been shown to be immunosuppressive with reduced antibody response as seen in mammals, even though a few experimental data found immunostimulatory properties. The ‘biphasic’ effects of heavy metals depending on the level of exposure and the time of exposure with respect to antigen injection previously discussed in this volume was also evidenced to occur in fish. Impaired cellular immunity was reported following cadmium exposure, but inconsistently. Cadmium was also shown to impair nonspecific immune responses, such as trout macrophage phagocytosis [34]. Manganese, a trace element necessary for life in mammals and fish, was shown to be highly toxic in fish, nevertheless consistently increased NK cell activity, lymphocyte proliferation and macrophage functions. The immunotoxic effects of nickel have seldom been investigated in fish, but nickel proved to suppress humoral immunity and macrophage functions. Pesticides are widely used and water contamination by pesticides is widespread. The organochlorine insecticides are major pollutants of aquatic ecosystems because of their persistence. No immunotoxic effect of lindane, except hypocellularity of the spleen was reported [35]. Several organophosphate insecticides, such as ediphos [36], malathion [13],

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dichorvos and trichlorphon [37], and the herbicides glyphosate [36] and atrazine [38] were shown to be immunosuppressive. Contamination of rivers, lakes and seas by halogenated aromatic hydrocarbons is also widespread. The humoral and innate immunity of fish is depressed by biphenyls [39,40] whereas 7,12-dimethylbenz[a]anthracene (DMBA) affects only innate immunity. IMMUNOTOXICOLOGY IN MARINE MAMMALS Marine mammals including seals, dolphins, and whales, are exposed to contaminants and in the recent years, have been shown to be sensitive targets of immunotoxicants. SEALS In 1988, an epizootic resulted in the deaths of approximately 20,000 harbor seals (Phoca vitulina) and several hundred gray seals living in seas of Northern Europe. Affected animals presented with fever, cutaneous lesions, gastro-intestinal dysfunction, neurological disorders and respiratory distress. The causative agent was a previously unidentified morbillivirus, named phocid distemper virus-1 or PDV-1. However, the epidemic was so severe that contributing factors to the observed morbidity and mortality were suspected to be involved. In order to test the hypothesis that the contributing role of polyhalogenated aromatic hydrocarbons, including polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans might explain the unusual severity of the epidemic, two groups of captive harbor seals were fed herrings from the heavily polluted Baltic Sea or from the relatively uncontaminated Atlantic Ocean over 2.5 years. T lymphocyte proliferative responses induced by mitogens and mixed lymphocyte culture were significantly reduced in seals fed Baltic herring. Similarly, allo-antigenic lymphoproliferative responses to tetanus toxoid and rabies virus antigen were suppressed. Significant suppression was noted after 2 years of exposure. In contrast, humoral immunity was not affected [41]. Female adult rats were fed for a period of four-and-a-half months—a freeze-dried diet made from herrings caught either in the heavily contaminated Baltic Sea or in uncontaminated areas of the Atlantic Ocean. Although no differences in lymphocyte subsets, mitogeninduced lymphoproliferative responses and NK cell activity were seen, cytomegalovirus titers in salivary glands after experimental infestation were higher in rats fed herrings from the Baltic Sea [42]. Finally, pregnant rats were exposed daily via the oral route to oil extracted from herring caught either in the Baltic Sea or the Atlantic Ocean. Reduced thymus weight, decreased lymphocyte proliferative responses to T-dependent mitogens and impaired NK cell activity at 11, 25, 46 or 59 days of age were found only in offspring of rats exposed to contaminated herring [43]. A similar, but more marked pattern of immunotoxic effects was seen in offspring of rats exposed to uncontaminated herring spiked with TCDD. DOLPHINS Similarly, dolphins from the Atlantic Ocean or the Mediterranean Sea have been shown to be exposed to high levels of chemical contaminants in water. Although studies on the immune status of dolphins in relation to environmental contamination have seemingly


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not been performed, the immunotoxic effects of contaminants are likely to play a role in the epizootic which killed many dolphins from the Mediterranean Sea [44]. WHALES The effects of in vitro exposure to different concentrations of PCB 138, 153, 180, and 169, and 2 DDT metabolites ( p,p0 -DDT and p,p0 -DDE) were evaluated on peripheral blood leukocytes and splenocytes from beluga whales. PCB 138 and p,p0 -DDT, but not PCB 153, 180, 169, and p,p0 -DDE, reduced significantly the proliferative response of beluga splenocytes stimulated by PHA. No effect on phagocytosis was observed [45]. These findings confirmed the immunocompromised status of beluga whales of the St Lawrence estuary [46]. Indeed, 44% of beluga whales had opportunistic bacteria in significant concentrations in at least two organs. Ten of 45 necropsied whales had malignant neoplasia and 15 had pneumonia. IMMUNOTOXICITY IN BIRDS There have been few immunotoxicological investigations of other free-living wildlife, especially birds. However, birds are another group of sentinel animals for environmentally induced toxic effects including immunotoxicity. Polychlorinated biphenyls were shown to induce marked immunotoxic effects in avian species. Young Caspian terns from Lake Huron had a marked reduction in lymphoproliferative response to PHA that negatively correlated with PCB concentrations in their eggs and plasma [47]. American kestrels were fed a diet containing 7 mg/kg/day of a mixture of Aroclor 1248:1254:1260 over approximately one year. Males, but not females had increased leukocyte numbers related to lymphocytosis and a greater skin response to PHA. However, the F1-generation birds did not show any effects [48]. These results are in disagreement with the increased antibody response to DNP-KLH in females and decreased responses in males observed by the same authors using the same conditions of exposure. In addition, F1-generation birds had a much lower antibody response than nonexposed controls [49]. Mallards exposed to 0– 500 mg/kg of Aroclor 1254 twice a week for 5 weeks did not show any adverse effect on their antibody response to SRBC, NK cell activity or PHA-induced lymphoproliferation [50]. CONCLUSION The available data demonstrate that wildlife species are potential targets of immunotoxicants. In addition, studies of wildlife species immune responses can serve to predict risk of populations exposed to chemical contaminants. Recently, invertebrates have also been considered for use [51,52].

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[2] Van Loveren H, Ross PS, Osterhaus AD, Vos JG (2000) Contaminant-induced immunosuppression and mass mortalities among harbor seals. Toxicol Lett, 112/113, 319– 324. [3] Damstra T, Jurgelski W, Posner HS, Vouk VB, Bernheim NJ, Guthrie J, Luster MI, Falk HL (1982) Toxicity of polybrominated biphenyls (PBBs) in domestic and laboratory animals. Environ Health Perspect, 44, 175– 188. [4] Black RD, McVey DS, Oehme FW (1992) Immunotoxicity in the bovine animal: a review. Vet Human Toxicol, 34, 438–442. [5] Van Muiswinkel WB, Anderson DP, Lamers CHJ et al. (1985) Fish immunology and fish health. In: Manning MJ, Tatmer MF (Eds) Fish Immunology, pp 316– 333. Academic Press, London. [6] Anderson DP, Zeean MG (1995) Immunotoxicology in fish. In: Rand G (Ed) Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment, pp 371–404. Taylor & Francis, Washington, DC. [7] Zelikoff JT (1994) Fish immunotoxicology. In: Dean JH, Luster MI, Munson AE, Kimber I (Eds) Immunopharmacology and Immunotoxicology, 2nd edition, pp 71 – 95. Raven Press, New York. [8] Vos JG, Van Loveren H, Wester PW, Vethaak AD (1988) The effects of environmental pollutants on the immune system. Eur Environ Res, 2, 2 – 7. [9] Vethaak AD, Jol JG, Meijboom A, Eggens ML, Rheinallt T, Wester PW, van de Zande T, Bergman A, Dankers N, Ariese F, Baan RA, Everts JM, Opperhuizen A, Marquenie JM (1996) Skin and liver diseases induced in flounder (Platichthys flesus) after long-term exposure to contaminated sediments in large-scale mesocosms. Environ Health Perspect, 104, 1218– 1229. [10] Wester PW, Vethaak AD, Van Muiswinkel WB (1994) Fish as biomarkers in immunotoxicology. Toxicology, 86, 213– 232. [11] Arkoosh MR, Casillas E, Clemons E et al. (1998) Effect of pollution on fish diseases: potential impacts on salmonid populations. J Aquat Anim Health, 10, 182– 190. [12] Zelikoff JT (1998) Biomarkers of immunotoxicity in fish and other non-mammalian sentinel species: predictive value for mammals. Toxicology, 129, 63 – 71. [13] Beaman JR, Finch R, Gardner H, Hoffmann F, Rosencrance A, Zelikoff JT (1999) Mammalian immunoassays for predicting the toxicity of malathion in a laboratory fish model. J Toxicol Environ Health A, 56, 523– 542. [14] Lunden T, Bylund G (2002) Effect of sulphadiazine and trimethoprim on the immune response of rainbow trout (Oncorhynchus mykiss). Vet Immunol Immunopathol, 85, 99 – 108. [15] Kollner B, Wasserrab B, Kotterba G, Fischer U (2002) Evaluation of immune functions of rainbow trout (Oncorhynchus mykiss)—how can environmental influences be detected? Toxicol Lett, 131, 83 – 95. [16] Miller GG, Sweet LI, Adams JV, Omann GM, Passino-Reader DR, Meier PG (2002) In vitro toxicity and interactions of environmental contaminants (Arochlor 1254 and mercury) and immunomodulatory agents (lipopolysaccharide and cortisol) on thymocytes from lake trout (Salvelinus namaycush). Fish Shellfish Immunol, 13, 11 –26. [17] Betoulle S, Etienne JC, Vernet G (2002) Acute immunotoxicity of gallium to carp (Cyprinus carpio L.). Bull Environ Contam Toxicol, 68, 817– 823. [18] Mondon JA, Duda S, Nowak BF (2000) Immune response of greenback flounder Rhombosolea tapirina after exposure to contaminated marine sediment and diet. Mar Environ Res, 50, 443– 450. [19] Scott AL, Rogers WA, Klesius PH (1985) Chemiluminescence by peripheral blood phagocytes from channel catfish: function of opsonin and temperature. Dev Comp Immunol, 9, 241– 250. [20] Zelikoff JT (1993) Metal pollution-induced immunomodulation in fish. Annu Rev Fish Dis, 2, 305– 325. [21] Zelikoff JT, Enane NA, Bowser D, Squibb KS, Frenkel K (1991) Development of fish peritoneal macrophages as a model for higher vertebrates in immunotoxicological studies. I. Characterization of trout macrophage morphological, functional, and biochemical properties. Fundam Appl Toxicol, 16, 576 –589. [22] Enane NA, Frenkel K, O’Connor JM, Squibb KS, Zelikoff JT (1993) Biological markers of macrophage activation: applications for fish phagocytes. Immunology, 80, 68 – 72.


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[23] Bowser DH, Frenkel K, Zelikoff JT (1994) Effects of in vitro nickel exposure on the macrophage-mediated immune functions of rainbow trout (Oncorhynchus mykiss). Bull Environ Contam Toxicol, 52, 367– 373. [24] Betoulle S, Duchiron C, Deschaux P (2000) Lindane increases in vitro respiratory burst activity and intracellular calcium levels in rainbow trout (Oncorhynchus mykiss) head kidney phagocytes. Aquat Toxicol, 48, 211– 221. [25] Palchaudhuri S, Raymond A, Carlson EA, Li Y, Zelikoff JT (2001) Cytotoxic and cytoprotective effects of selenium on bluegill sunfish (Lepomis macrochirus) phagocytic cells in vitro. Bull Environ Contam Toxicol, 67, 672– 679. [26] Clerton P, Troutaud D, Verlhac V, Gabaudan J, Deschaux P (2001) Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes. Fish Shellfish Immunol, 11, 1 – 13. [27] Oumouna M, Jaso-Friedmann L, Evans DL (2001) Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells. Cytometry, 45, 259– 266. [28] Le Morvan C, Deschaux P, Troutaud D (1996) Effects and mechanisms of environmental temperature on carp (Cyprinus carpio) anti-DNP antibody response and non-specific cytotoxic cell activity: a kinetic study. Dev Comp Immunol, 20, 331– 340. [29] Alcorn SW, Pascho RJ (2002) Antibody responses by chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss) to various protein antigens. Fish Shellfish Immunol, 13, 327– 333. [30] Irie T, Watarai S, Kodama H (2003) Humoral immune response of carp (Cyprinus carpio) induced by oral immunization with liposome-entrapped antigen. Dev Comp Immunol, 27, 413– 421. [31] Smith DA, Schurig GG, Smith SA, Holladay SD (1999) Tilapia (Oreochromis niloticus) and rodents exhibit similar patterns of inhibited antibody production following exposure to immunotoxic chemicals. Vet Hum Toxicol, 41, 368– 373. [32] Siwicki A, Dobosz S, Goryczko K, Trapkowska S, Pozet F (2003) Cell-mediated immunity in yellow forms of rainbow trout. Pol J Vet Sci, 6, 49 – 50. [33] Reynaud S, Duchiron C, Deschaux P (2003) 3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L.). Aquat Toxicol, 63, 319– 331. [34] Zelikoff JT, Bowser D, Squibb KS, Frenkel K (1995) Immunotoxicity of low level cadmium exposure in fish: an alternative animal model for immunotoxicological studies. J Toxicol Environ Health, 45, 235– 248. [35] Hart LJ, Smith SA, Smith BJ, Robertson J, Holladay SD (1997) Exposure of tilapian fish to the pesticide lindane results in hypocellularity of the primary hematopoietic organ (pronephros) and the spleen without altering activity of phagocytic cells in these organs. Toxicology, 118, 211– 221. [36] el-Gendy KS, Aly NM, el-Sebae AH (1998) Effects of edifenphos and glyphosate on the immune response and protein biosynthesis of bolti fish (Tilapia nilotica). J Environ Sci Health B, 33, 135– 149. [37] Dunier M, Siwicki AK, Demael A (1991) Effects of organophosphorus insecticides: effects of trichlorfon and dichlorvos on the immune response of carp (Cyprinus carpio). III. In vitro effects on lymphocyte proliferation and phagocytosis and in vivo effects on humoral response. Ecotoxicol Environ Saf, 22, 79 – 87. [38] Rymuszka A, Siwicki AK (2003) Immunomodulatory activity of the dimerized lysozyme KLP602 after earlier suppression by atrazine in carp (Cyprinus carpio L.). Pol J Vet Sci, 6(Suppl.), 43 –46. [39] Arkoosh MR, Clemons E, Myers M, Casillas E (1994) Suppression of B-cell mediated immunity in juvenile chinook salmon (Oncorhynchus tshawytscha) after exposure to either a polycyclic aromatic hydrocarbon or to polychlorinated biphenyls. Immunopharmacol Immunotoxicol, 16, 293– 314.

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[40] Regala RP, Rice CD, Schwedler TE, Dorociak IR (2001) The effects of tributyltin (TBT) and 3, 30 ,4,40 ,5-pentachlorobiphenyl (PCB-126) mixtures on antibody responses and phagocyte oxidative burst activity in channel catfish, Ictalurus punctatus. Arch Environ Contam Toxicol, 40, 386– 391. [41] De Swart RL, Ross PS, Timmerman HH, Vos HW, Reijnders PJ, Vos JG, Osterhaus AD (1995) Impaired cellular immune response in harbour seals (Phoca vitulina) feeding on environmentally contaminated herring. Clin Exp Immunol, 101, 480– 486. [42] Ross PS, Van Loveren H, De Swart RL, van der Vliet H, de Klerk A, Timmerman HH, van Binnendijk R, Brouwer A, Vos JG, Osterhaus AD (1996) Host resistance to rat cytomegalovirus (RCMV) and immune function in adult PVG rats fed herring from the contaminated Baltic Sea. Arch Toxicol, 70, 661– 671. [43] Ross PS, De Swart RL, Van der Vliet H, Willemsen L, de Klerk A, van Amerongen G, Groen J, Brouwer A, Schipholt I, Morse DC, van Loveren H, Osterhaus AD, Vos JG (1997) Impaired cellular immune response in rats exposed perinatally to Baltic sea herring oil or 2,3,7,8-TCCD. Arch Toxicol, 71, 563– 574. [44] Borrell A, Aguilar A, Corsolini S, Focardi S (1996) Evaluation of toxicity and sex-related variation of PCB levels in Mediterranean striped dolphins affected by an epizootic. Chemosphere, 32, 2359– 2369. [45] De Guise S, Martineau D, Beland P, Fournier M (1998) Effects of in vitro exposure of beluga whale leukocytes to selected organochlorines. J Toxicol Environ Health A, 55, 479– 493. [46] Martineau D, De Guise S, Fournier M, Shugart L, Girard C, Lagace A, Beland P (1994) Pathology and toxicology of beluga whales from the St Lawrence Estuary, Quebec, Canada. Past, present and future. Sci Total Environ, 154, 201– 215. [47] Grasman KA, Fox GA (2001) Associations between altered immune function and organochlorine contamination in young Caspian terns (Sterna caspia) from Lake Huron, 1997– 1999. Ecotoxicology, 10, 101– 114. [48] Smits JE, Fernie KJ, Bortolotti GR, Marchant TA (2002) Thyroid hormone suppression and cellmediated immunomodulation in American kestrels (Falco sparverius) exposed to PCBs. Arch Environ Contam Toxicol, 43, 338– 344. [49] Smits JE, Bortolotti GR (2001) Antibody-mediated immunotoxicity in American kestrels (Falco sparverius) exposed to polychlorinated biphenyls. J Toxicol Environ Health A, 62, 217– 226. [50] Fowles JR, Fairbrother A, Trust KA, Kerkvliet NI (1997) Effects of Aroclor 1254 on the thyroid gland, immune function, and hepatic cytochrome P450 activity in mallards. Environ Res, 75, 119– 129. [51] Bunn KE, Thompson HM, Tarrant KA (1996) Effects of agrochemicals on the immune systems of earthworms. Bull Environ Contam Toxicol, 57, 632– 639. [52] Galloway TS, Depledge MH (2001) Immunotoxicity in invertebrates: measurement and ecotoxicological relevance. Ecotoxicology, 10, 5 –23.

Chapter 15

New methods in immunotoxicology

INTRODUCTION It is important to keep in mind that initially immunotoxicology benefited from the input of immunologists or cellular biologists [1,2], but that, progressively, it became a fully recognized area of toxicology [3 –5]. Most animal models and assays that have been and are still used today for nonclinical as well as clinical immunotoxicity assessment derive more or less directly from immunological research and practices [6]. However, the growing numbers of studies, especially those that are nowadays required for regulatory purposes, tend to generate results that cannot be easily, if at all addressed by ‘first-generation’ immunotoxicological assays. Biological technologies have achieved tremendous progress in the past years and the introduction of these new techniques is likely to change our understanding of immunotoxic effects as well as the practice of immunotoxicity assessment in the next few years. The variety and sophistication of the most recent biological tools applicable to immunotoxicology are such that this chapter cannot provide an in-depth and accurate coverage of all possible tools and their applications. In addition, available new tools are improved or proposed almost daily. Modestly, the aim is providing a general description of the potential utility and current limitations of new tools for nonclinical as well as clinical immunotoxicity evaluation.

ENHANCED PATHOLOGY The term enhanced pathology was coined in the mid-1990s when conventional histological examination of the lymphoid organs of rodents proved to lack the required sensitivity to predict the potential of drugs and other chemicals to induce unexpected immunotoxicity [7]. A number of new techniques have been introduced in toxicological pathology and are progressively applied to immunotoxicity evaluation. Emphasis has also since then been placed on the required technique and skill to perform optimal conventional histological examination of lymphoid organs [8,9]. Nevertheless, new techniques are being considered. 363

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IMMUNOHISTOCHEMISTRY General considerations. Immunohistochemistry is the immunological detection of proteins in tissue samples using specific polyclonal or monoclonal antibodies. Primary antibodies are used to bind specific epitopes and a secondary marker, such as fluorochromeconjugated secondary antibody or streptavidin –biotin labels is necessary to visualize specific binding of the primary antibody. The selection of the fluorochrome depends on the tissue being examined, the technique used and the need for double-labeling. Nonfluorescent markers, such as immunoperoxidase and immunogold conjugates are suitable for brightfield microscopy and offer increased stability. Immunohistochemistry provides means to evaluate characteristics of immune cells within tissue sections [10,11]. A major limitation to immunohistochemistry is that it cannot be used for quantitative measurement, except when cell proliferation is being studied. Another significant limitation is the availability of antibodies specific for the target molecule. When antibodies are available, and many antibodies directed against the same molecule are commercially available, a careful selection based on prevalidation studies is absolutely required. When no antibodies are available, cross-reactivity among species is possible, but must be carefully checked. Target molecules must be immobilized to avoid diffusion in tissues. The classical procedure with formalin-fixed, paraffin-embedded tissue may not be appropriate as antigens may be lost by cross-linking. The preferred tissue preparation is freezing following mild fixation, e.g. with acetone, which allows optimal conditions for most antibodies. Immunohistochemistry in immunotoxicity assessment. Immunohistochemistry can be used to detect changes in cell-surface markers of immune cells in response to inflammation or immunomodulation [12 – 17]. Importantly, changes in the number or distribution of cellsurface markers may be evidenced in the absence of apparent changes in lymphoid tissue. In addition, toxic changes, such as apoptosis, can easily be detected [18]. Thus, immunohistochemistry of lymphoid organs is increasingly considered for inclusion in routine immunotoxicity assessment. However, changes observed using immunohistochemistry provide no information on cellular function. In addition, several immune cells may express the same cell-surface marker and this may result in misinterpretation of the observed changes. Therefore, validation using a large panel of known immunotoxic and nonimmunotoxic compounds is needed to compare the relative sensitivity of conventional histology and immunohistochemistry. An interesting application of immunohistochemistry is the identification of cross-reactivity between therapeutic Mabs and human tissues in order to rule out the risk of toxic effects related to inadvertent binding on nontargeted tissues [19]. COMPUTERIZED PROCESSING AND IMAGE ANALYSIS Conventional histological examination of the lymphoid organs can only provide a semiquantitative assessment of morphological changes. With the introduction of new techniques, such as immunofluorescence, there is a need to provide a quantitative assessment of observed changes [20]. Quantitative analysis can be performed electronically


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utilizing image analysis and stereological techniques to provide numerical values. Morphometry is the quantitative description of a structure, such as the thymus [21]. Confocal microscopy combines the power of computer technology with the precision of scanning laser beam [22]. Confocal microscopes offer the advantages of illuminating a single point instead of broad regions of a specimen. IN SITU HYBRIDIZATION In situ hybridization is based on the principle that labeled sequences of nucleic acids (probes) can specifically hybridize with cellular mRNA or DNA (gene) in tissues. Various probes can be used, either single or double-stranded DNA, RNA or oligonucleotides. RNA probes have the advantage of higher sensitivity, but they are labile and require technical skill. Labeling is achieved by radio-isotopic or nonradio-isotopic methods. 35S- and 33Plabeled probes are the most frequently used radiolabeled probes. Nonisotopic probes are labeled with fluorochromes or the enzymes horseradish peroxidase and alkaline phosphatase. Although in situ hybridization has essentially been used for discovery, it is considered for the study of gene expression following drug administration, although no data have seemingly been published in the area of immunotoxicology. As in situ hybridization is a powerful tool for studying gene expression and distribution at the cellular level, it is often used to localize the vector of gene therapy products and to determine whether observed lesions are related to the presence of transferred DNA [23,24]. IN SITU POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) is a powerful method that is capable of quantifying even minute quantities of DNA. Thus, in situ PCR is capable to detect low-expression mRNA and is a more sensitive tool than in situ hybridization. However, its exquisite sensitivity makes it a tool more useful for discovery than toxicology. So far, the relevance of subtle changes detected by this technique is not known [25]. In addition, sophisticated technical skill and equipment are required, which may be major limitations for extensive use in immunotoxicity assessment, unless future studies indicate circumstances where it is a helpful add-on to the prediction of immunotoxic effects.

IN VITRO IMMUNOTOXICOLOGY In 1959, Russel and Burch [26] defined the 3Rs concept, which stands for Refinement, Reduction and Replacement, in an attempt thereafter widely supported to restrict, if not abolish the use of animals in the safety assessment of xenobiotics. Not unwittingly, Gad proposed another definition of the 3Rs, namely Relevance, Reliability and Reproducibility, based on a toxicologist’s perspective, or the use of a fourth R, namely Responsibility, as despite the commitment of toxicologists to Russel and Burch’s 3Rs, toxicologists must ensure an optimal safety of the drugs and chemicals to which human beings are to be

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exposed [27]. Although it is not within the scope of this book to contribute to this debate, data derived from in vitro immunotoxicity studies illustrate the difficulties of reconciling these approaches of the 3Rs. GENERAL CONSIDERATIONS Strictly speaking, in vitro immunotoxicology plays a very limited role in immunotoxicity evaluation despite recent incentives to promote research in this area [28]. Few assays have indeed been specifically developed for the in vitro immunotoxicity screening of drugs and other chemicals, even though more assays are being considered for mechanistic purposes. In fact, most in vitro assays that are commonly used are ex vivo assays, where blood samples are drawn from animals before and at the end of exposure to measure selected endpoints. Routine ex vivo immunotoxicity assays include lymphocyte proliferation assays, and measurement of NK cell activity and phagocytosis (see Chapter 5 of this volume). Advantages and limitations of in vitro immunotoxicity assays compared to in vivo assays. Despite the growing willingness of many people to restrict, if not ban the use of animals for the safety evaluation of new molecular entities prior to commercial use, there is a need to carefully assess the advantages and limitations of in vitro assays as compared to conventional animal models. Advantages. Overall, major advantages of in vitro assays include the reduction of animal use, the requirement of only tiny amounts of tested chemicals, which might be crucial at a very early stage of development when production scale is limited, and finally the possibility to compare effects on different cellular or organ systems in the same species or across species. In addition, in vitro assays when carefully validated are cost-effective and can be used to test a large number of new molecular entities in a relatively short period of time as compared to in vivo models. As far as immunotoxicity assessment is concerned, additional advantages include the possibility of utilizing a much wider range of concentrations much wider than in in vivo testing. Immunotoxic effects are characterized by variable dose – response relationships depending on the tested article and the type of immunotoxic effects being investigated. Therefore, the use of a wide range of concentrations can provide useful clues to delineate specific dose –response relationships, which may prove essential for sound immunotoxicity risk assessment. In vitro assays have been suggested to improve the integration of immunotoxicology in drug discovery and development [29], and this probably applies to any type of new chemical entity. In this context, expected benefits from in vitro immunotoxicity assays are either the early detection of the potential for immunotoxicity, or the quick confirmation and understanding of observations in conventional in vivo toxicity animal testing. Limitations. In vitro assays for immunotoxicity assessment, however, have several major limitations as well, which must be overcome for these assays to have their share of reliable immunotoxicity prediction and safety assessment. One limitation is the role of metabolism in many immunotoxic effects. Therefore, in vitro assays including metabolizing systems are absolutely essential. In that respect, it is unsure whether widely used


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procedures, such as preincubation with liver microsomal fractions from rodents exposed to enzyme inducers, e.g. S9-mix, is a reliable method as the few published results are conflicting [30 –32]. No inter-laboratory validation studies have so far been conducted. Other results suggest that co-culture of splenocytes or lymphocytes with hepatocytes is a better alternative [33 – 36]. In fact, the role of metabolites has more often been shown in in vitro assays when fully identified and commercially available metabolites were available. Another major limitation of in vitro immunotoxicity assays is that they cannot take into account the critically significant interactions that occur between the immune system and the nervous or endocrine systems. To date, relatively few studies have been conducted to compare results of in vitro assays with those obtained in vivo with the same test article. Inconsistencies have been reported. Thus, rhodamine B enhanced PFC responses and mitogen-induced lymphoproliferation in mice, but had suppressive effects in vitro [37]. 3-Acetyldeoxynivalenol exerted no effect in mice, but antibody response to SRBC was increased and lymphocyte proliferation to DNP-Ficoll or LPS was dose dependently suppressed in vitro [38]. Viora et al. [39] compared the effects of clindamycin, piritrexim, and pyrimethamine on mitogen- and IL-2-induced lymphocyte proliferation and NK cell activity following in vivo treatment in rats and in vitro incubation with human PMNC. The three drugs induced a dose-dependent decrease in lymphocyte proliferation in both systems, but NK cell activity was only reduced in vitro at high concentration levels. Culturing immunocompetent cells pose particular problems [40]. To mount an antigenspecific immune response in vitro, it is absolutely required that at least two populations of cells are present, namely APCs and lymphoid cells. The spleen is the main source of lymphoid cells in rodents. It can be easily removed from the surrounding connective tissue. After gentle crushing or teasing, a mouse spleen normally yields 5 – 10 £ 107 lymphocytes. In rats, the cell yield is approximately 10 times larger. Lymph nodes can also be used and the yield of lymphoid cells is 3 – 5 £ 107 =mouse and 1 – 2 £ 108 =rat: Peripheral blood is obtained by cardiac puncture or from the retro-orbital plexus in mice and rats. Blood can also be withdrawn from a tail or sublingual vein in the rat. In larger animals and humans, blood is withdrawn from a large vein, such as the jugular, femoral, brachial or saphenous vein depending on the species. Citrate or heparin should be avoided because of the risk of leukocyte clumping. The use of EDTA is recommended. Purification of PBMC up to 1000 can be achieved by centrifugation using a density-gradient procedure. Thus, approximately 70% of PBMC consist of T lymphocytes. Short-time manipulations of cells can be performed at room temperature. Otherwise, cells are cultured at 37 8C with 5% CO2 in an incubator. Depending on the assay, various culture media and buffers can be used. IN VITRO ASSAYS FOR THE PREDICTION OF UNEXPECTED IMMUNOSUPPRESSION Regulatory authorities tend to require that toxicity testing, including immunotoxicity, should be performed on every new xenobiotic before human exposure [41]. As the number of new chemicals produced each year is enormous, there is an obvious shortage of resources to perform safety assessment based on animal toxicity studies, let alone the unacceptable

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use of many more animals to achieve this goal. Thus, there is an urgent need to design, standardize and validate in vitro assays that can be used as a screen. Despite continuing research effort, such as the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in the USA [42] or the European Centre for the Validation of Alternative Methods (ECVAM) [43], in vitro assays that can be used as a screen for immunotoxicity assessment are very few. The Mishell – Dutton assay [44] is the first in vitro assay that has been proposed to measure immune function. In this assay, mouse spleen cells are cultured with SRBC generally for one day and direct PFC response is measured using a procedure very similar to the in vivo PFC assay. Inhibition of in vitro PFC responses has been demonstrated with known immunosuppressants, including cyclosporine [45], methotrexate [46], cyclophosphamide [30], benzopyrene [47] and dioxin [48]. Applications of the Mishell –Dutton, however, gave somewhat inconsistent results when compared to in vivo findings [49 –51]. Fautz and Miltenburger [52] proposed a multiple end-point system using suspensions of thioglycollate-elicited peritoneal macrophages and erythrocyte-free splenocytes obtained from C57B1 female mice. Immune functions, namely antibody-dependent phagocytosis of SRBC, LPS-induced activation of macrophages, NK cell activity, lymphoproliferative response to B and T lymphocyte mitogens, and in vitro immunoglobulin synthesis by B lymphocytes, were studied in parallel with aliquots derived from the same cell suspension. When applicable, tested chemicals were activated with S-9 mix prior to incubation of cells. Results obtained with tributyltin oxide, 7,12-dimethyl-benzathracene, lead acetate, cyclophosphamide and dexamethasone were in accordance with in vivo data from the literature. However, this approach has seemingly not been further tested by others. A number of immune function assays that are included in animal studies are ex vivo assays so that they can easily be performed using exclusively in vitro conditions [53,54]. In vitro cytokine release (IL-1b and IL-4) using human whole-blood following stimulation by LPS or staphylococcal enterotoxin B was suggested to predict unexpected immunosuppression as well as immunostimulation [55]. As already mentioned, no extensive inter-laboratory studies have been performed to standardize experimental protocols, in particular the selection of relevant concentrations, and compare results in vivo and in vitro to determine the relative sensitivity and reliability of both approaches for immunotoxicity assessment. IN VITRO ASSAYS FOR THE PREDICTION OF UNEXPECTED IMMUNOSTIMULATION With exception of in vitro cytokine release assays that have been shown to be good predictors of acute cytokine syndrome [56] and possibly immunostimulation [55], no in vitro assay has been considered for use as predictors of expected immunostimulation. However, enhanced PFC responses were shown using the Mishell – Dutton assay [57]. IN VITRO ASSAYS FOR THE PREDICTION OF CONTACT HYPERSENSITIVITY No in vitro assays are available to predict the risk of immune-mediated hypersensitivity reactions. Several in vitro assays can be used to predict pseudoallergic reactions as discussed in Chapter 7 of this volume.


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During the past two decades, studies on the sensitizing potential of xenobiotics have been based on in vivo animal models and research is ongoing to design in vitro tests predictive of contact sensitization. However, none has so far been validated despite various attempts [58]. Early attempts focused on hapten-induced cytokine responses of cultured keratinocytes, alterations in the expression of adhesion molecules on keratinocytes and epidermal Langerhans cells, and hapten-mediated T lymphocyte activation by Langerhans cells [59 –62]. With the recent discovery that dendritic cells play a crucial role in contact sensitization [63]. In fact, contact sensitization is the result of a complex interplay of events involving interactions between several cellular populations. The current trend is therefore to develop models using combinations of regulatory and effector cells [64,65] or artificially reconstituted skin.

IN SILICO IMMUNOTOXICOLOGY The extraordinary advances in molecular biology techniques led to recommend the introduction of molecular end-points in immunotoxicology [66]. Beside genetically modified animals as discussed later in this chapter, molecular immunotoxicology testing essentially focused on the mRNA expression of cytokines. However, a lot of research efforts need to be paid to the refinement, standardization and validation of these tests before they can come out from the area of fundamental research to fit to the specific requirements of (regulatory) immunotoxicity assessment. Toxicogenomics is another novel area of toxicology expected to bring new avenues of research and to contribute to risk assessment. With the use of innovative technologies, such as microarrays [67] and toxicogenomics [68], a wealth of new biological end-points measured simultaneously, either in vitro or in vivo, can be expected to evidence alterations of gene expression as early warnings of toxicological events. Limited attempts have been made to use these approaches as a predicting tool in immunotoxicology [69]. MOLECULAR IMMUNOTOXICOLOGY As cytokines play a key role in the immune system, molecular immunotoxicology is focusing on the analysis of cytokine expression. Beside the measurement of cytokine levels in blood or in the supernatant of cultured cells typically by ELISA [55,70], cytokines can be analyzed at the mRNA level. Reverse transcription-polymerase chain reaction (RT-PCR) is the preferred technique due to enormous sensitivity, but it requires sophisticated technical skill and dedicated equipment [71,72]. To date analysis of cytokine mRNA expression has largely been restricted to mechanistic studies [73 –80] as correlations with changes in immune function are unclear [81,82]. MICROARRAY ANALYSIS Microarray analysis can be defined as the simultaneous measurement of the mRNA abundance of thousands of genes in a given sample by means of hybridization. The first

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successful attempts to classify compounds on the basis of expression profiles have been reported [83]. To establish patterns of toxicity and classes of toxic compounds, databases consisting of expression profiles with correlations to histological and functional changes are required. So far, immunotoxicity has not been included in these initiatives [84].

NEW ANIMAL MODELS Recent dramatic progress in the development of new animal models for use in various areas of biology, including immunology and toxicology, opened interesting avenues of research. These new animal models that could prove useful in immunotoxicology, include SCID mice, and genetically modified, e.g. knock-out and transgenic, animals. IMMUNODEFICIENT MOUSE MODELS SCID mice. Severe combined immune deficiency (SCID) is a rare congenital disease syndrome that results in loss of B and T cell immunity. It was first recognized in human infants in the mid-1950s. Babies present with recurring infections and general failure to thrive. They usually die within one or two years after birth unless they have successfully engrafted with normal bone marrow. In 1983, an autosomal recessive mutation on chromosome 16 was reported in Balb/c C.B-17 mice [85]. Homozygous SCID mice have a profound lymphopenia affecting both B and T cells. Some mice, however, develop a certain degree of B and T lymphocyte function and serum immunoglobulin levels can be detected in a percentage of mice that is increasing with age. Therefore, SCID mice must be screened for immunological leakiness before use. Typically, the size of their thymus is only 1–10% of the normal thymus and their Peyer’s patches are hypoplastic and contain few lymphoid cells. They are unable to mount immune responses directed against T-dependent as well as T-independent antigens and are highly susceptible to infections. NK cell activity and the bone marrow, however, are normal. Due to their immune defect, SCID mice must be kept in a protected environment to avoid contact with microbial pathogens. In a pathogen-free environment, SCID mice normally survive for 1–2 years, but 10% develop a T cell lymphoma [86]. A major interest in the use of SCID mice for immunotoxicity studies is the possibility to graft human tumors and liver, thyroid or skin tissues as well as human immunocompetent cells [87]. SCID mice can be reconstituted with congenic, allogeneic or xenogeneic cells, including mouse, rat, bovine, equine and human cells. Genetically manipulated cells can also be used for reconstitution. After reconstitution, SCID mice can mount normal or nearly normal immune responses. Two SCID mouse models have been used by immunotoxicologists, but limited experience is available with either model so that their utility for immunotoxicity assessment in not known. The hu-PBL-SCID mouse is reconstituted with human peripheral blood mononuclear cells injected intraperitoneally. The reconstitution lasts several months. However, when EBV-positive donors are used, the reconstituted mice can develop aggressive B lymphomas. The injection of an excessive number of reconstituting cells can also result in a graft-vs.-host disease. Hu-PBL-SCID mice have been used to investigate the immunosuppressive effects of dioxin and cyclosporine [88], cyclophosphamide and


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cyclosporine [89], and allopurinol [90], or the adjuvant effects of diesel exhaust particles on sensitization to birch pollen [91]. SCID-hu mice. Reconstitution can also be achieved by the transplantation of fragments from fetal human lymphoid organs, such as liver, thymus or bone marrow, under the kidney capsule of SCID mice. Mice reconstituted according this procedure, namely SCID-hu mice [92], have been widely used for the study of hematopoiesis and lymphopoiesis, and the pathophysiology of certain infections, e.g. HIV infections. Their use for studies on the human immune responses is limited because the generation of normal immune responses is difficult to achieve. However, SCID-hu can be useful tools for studies of immunotoxic effects specifically targeting lymphoid tissues, such as the thymus [93,94]. Alternatively, SCID-ra mice can be used following implantation of rat fetal or post-natal thymus and liver explants under the renal capsule of SCID mice resulting in thymus grafts with a histological appearance virtually identical to that of normal rat thymus [93]. The beige/nude/xid (BNX) mouse is another immunodeficient mouse model with a triple mutation resulting in reduced numbers of natural and lymphokine-activated killer cells, and impaired B and T lymphocyte functions. However, reconstitution is much more difficult to achieve in BNX than SCID mice [87]. Despite expectations that immunodeficient mouse models could prove useful tools for immunotoxicity assessment [95], limitations including marked inter-individual variability, inconsistent reconstitution and strict housing requirement are such that no further studies using either hu-PBL-SCID or SCID-hu mice have been published over the past years. GENETICALLY MODIFIED ANIMALS A major achievement in biological research is the development of animals with new functional genes added to their genome, and animals with altered or inactivated genes (transgenic, knock-out and knock-in animals). The variety of genetically modified animals is growing daily. In the field of toxicology, there is particular interest in genetically modified animals to increase their sensitivity to screen for toxic effects or to investigate mechanisms [96]. A number of cytokine knock-out mice have been developed. Their use in mechanistic studies is being investigated in order to show their superiority over conventional animals for immunotoxicity evaluation [97–99]. Modulation of TH1 cytokine gene expression may affect cell-mediated immune responses, whereas overexpression of TH2 cytokines enhances IgEmediated responses and deletion of these cytokines attenuates and/or prevents allergic responses. Despite expectations that genetically modified rodents may prove useful tools in the future for immunotoxicity assessment [100], rare studies have been published and all dealt with nonclinical assessment of biotechnology-derived pharmaceuticals [101–103].

COMPUTATIONAL IMMUNOTOXICOLOGY Computational programs (expert systems) are being developed as an aid to predict the potential of new molecular entities to induce toxicity by the identification of structural alerts or toxicophores [104].

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Structure –activity relationship (SAR) models have been reported for respiratory and contact sensitizers. Several models have accurately predicted strong sensitizers by identifying electrophiles, nucleophiles, proelectrophiles, and pronucleophiles, but had moderate success in identifying weak sensitizers, such as the CASE/MultiCASE system [105] or to a lesser extent DEREK expert system which includes 59 structural alerts [106, 107]. A model for respiratory chemical sensitizers has been described [108]. The database consists of chemicals identified from human case reports. The model has identified chemical fragments associated with activity and is expected to enable predictions of the respiratory sensitizing capacity of chemicals. A number of other computational SAR programs have been developed including TOPKAT, HazardExpert, OncoLogic and COMPACT [109], but none of these programs included immunotoxicity data. Beside SAR, expert systems could also be used as an aid to determine step by step the optimal strategy for the safety assessment of drugs and other chemicals. They use a knowledge base, which is organized in rules simulating human reasoning. After processing the new data provided by the investigator, the inference engine offer conclusions in a userfriendly manner. Although this type of expert systems is increasingly used in many areas, including clinical toxicology [110], limited attention has been paid to their application in toxicity evaluation.

CONCLUSION New tools are emerging for application in biomedical sciences. Because of their sophistication, they required technical skill and dedicated equipment. They potentially offer powerful ways to improve the early detection of toxic effects. However, because of this sophistication and extreme sensitivity, the data they generate are still not straightforwardly interpretable in most instances. They are being increasingly used in drug discovery and general toxicology, but have only been timidly explored for immunotoxicity assessment. At the time of writing, it is nearly impossible to predict which tools will prove helpful add-ons to current animal models and assays for the identification of immunotoxicants. However, the data they will generate are expected to open new avenues of research and to end in more reliable immunotoxicity risk assessment.

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Chapter 16

Classification of immunotoxicants

INTRODUCTION Classifications of xenobiotics based on their effects on target organs of toxicity have been proposed for carcinogenic and mutagenic [1,2], teratogenic [3] or contact sensitizing [4,5] chemicals. One of the declarations made during the Conference on Environment and Development in Rio de Janeiro (1992) was the need to generate a globally harmonized hazard classification of chemicals on which a compatible labeling system could be based to create a common international framework to facilitate communication on hazards and the trade of chemicals whose hazards have been carefully evaluated. The Organization for Economic and Co-operation and Development (OECD) and several United Nations agencies are among the international institutions involved in this harmonized classification process. There is a large and still growing amount of data on the possible or demonstrated adverse effects of drugs and other chemicals on the immune system. The majority of these data, however, was obtained in laboratory animals or in vitro assays and thus, the immunotoxicity of most drugs and chemicals in human beings is still not well known, if at all. Nevertheless significant efforts have been paid to the design of methods for the assessment of immunotoxic hazards based on animal studies [6,7] or the identification of human biomarkers of immunotoxicity for inclusion in field studies [8,9]. With the sole exception of contact sensitizers [4,5], no classification of immunotoxic xenobiotics is available. This chapter is an attempt to propose a classification scheme that merely reflects the author’s personal thoughts. Even though any classification process must be based on the input from experts and institutions of varied origin, this proposed classification may hopefully serve as an impetus for further definition of data and concepts useful to the establishment of such a needed classification.

SELECTED CRITERIA The main purpose of any classification is to systematically identify hazardous chemicals. Chemical hazards are conventionally divided into physicochemical, toxicological and 379

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ecotoxicological hazards. As regards immunotoxic effects, the main hazards are toxicological, i.e. health hazards, even though there is a growing amount of data showing that chemicals can also adversely affect the immune system of wild-life species [10,11]. POTENTIAL CONSEQUENCES OF EXPOSURE TO IMMUNOTOXICANTS A prerequisite to any classification of dangerous chemicals is to characterize the potential health consequences of exposure to these chemicals. Four broad categories of immunotoxic effects, i.e. immunosuppression, immunostimulation, hypersensitivity and autoimmunity, have been identified. It is noteworthy that the clinical or immunological presentation of immunotoxic effects may differ depending whether they are observed in animals or in man. As xenobiotics can induce one or several different categories of immunotoxic effects, the classification is based on the total weight of evidence. When a drug or chemical is known or suspected to induce several categories of immunotoxic effects, the most reliable data available serve for evidence determination. When only one category of immunotoxic effects is known or suspected to occur, evidence determination is based on the data related to these immunotoxic effects. Immunosuppression is primarily expected to induce histological changes in the lymphoid organs of exposed animals, to decrease immune functions in either animals or humans or both, and to impair the resistance of laboratory animals toward experimental infections. In man, infections, especially opportunistic infections, and virus-induced cancers, such as skin cancers and B lymphomas, are the major adverse effects reportedly associated with immunosuppressive drugs and other chemicals. In addition, decreases in immune function have also been described in in vitro assays using animal or human cells. Immunostimulation can be associated with histological changes in the lymphoid organs of animals, enhanced immune responses in both animals and humans, and acceleration of spontaneous autoimmune diseases in genetically prone animals or experimental autoimmune diseases. In humans, immunostimulation may cause flu-like reactions of variable severity, more frequent autoimmune diseases and hypersensitivity reactions to varied allergens, and inhibition of drug-metabolizing enzymes. Enhancement or activation of immune responses can be induced using in vitro systems. Hypersensitivity. In animals, drugs and other chemicals can induce immune-mediated hypersensitivity reactions, such as IgE-mediated anaphylaxis, respiratory allergy or contact sensitization, and nonimmune-mediated hypersensitivity reactions. Similar reactions can be seen in humans, but their incidence is far higher in man than in animals due to the limited availability of animal models. In vitro assays are almost restricted to nonimmune-mediated hypersensitivity reactions. Autoimmunity. In animals as well as humans, drugs and other chemicals can result in the production of autoantibodies or autoimmune reactions. However, because mechanisms are not elucidated and predisposing factors play a crucial role, there are rare data in animals and they are often the consequence of exposure to a limited number of


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drugs and chemicals in genetically predisposed animal strains. In addition, no in vitro assay is available. RELIABILITY AND MAGNITUDE OF AVAILABLE DATA Immunotoxic drugs and chemicals are ranked according to the magnitude and the reliability of their reported effects. Ideally, the highest degree of plausibility is associated with immunotoxic effects described in a significant number of exposed humans. The next lower degree of plausibility consists of immunotoxic effects that have been seldom observed in humans, but for which there is a large and consistent database in animals, followed by those immunotoxic effects that have only been evidenced in animal studies. Finally, there are drugs and other chemicals for which no data are available, either in animals or in man. Although the incidence and severity of immunotoxic effects may differ depending on the category of effects being considered, any drug or chemical known or suspected to induce at least one category of immunotoxic effects is labeled as immunotoxic. It is logical to separate mildly from moderately or markedly immunotoxic substances. Even though the available data tend to make judgments excessively subjective, this should be considered as an incentive for additional validation studies and research work, and certainly not as a cause for rejecting attempts to classify immunotoxic xenobiotics. In fact, the degree of subjectivity depends on the category of immunotoxic effects being considered. Thus, markedly immunosuppressive xenobiotics induce severe infections and more frequent skin cancers. The difference between mildly and moderately immunosuppressive xenobiotics is more clearly subjective. Moderately immunosuppressive xenobiotics can be proposed to include those xenobiotics whose exposure is associated with clinically unremarkable infections. Mildly immunosuppressive xenobiotics are those that induce decreases in immune functions or changes in the histology of lymphoid organs with no associated increases in clinical infections or significant impairment of resistance to experimental infections. Markedly immunostimulatory xenobiotics are those that cause acute cytokine syndrome and a significant increase in the incidence of autoimmune diseases or hypersensitivity reactions to varied allergens. Moderately immunostimulatory xenobiotics are those that cause less severe flu-like reactions and isolated case reports of autoimmune diseases or hypersensitivity reactions. Mildly immunostimulatory xenobiotics are those which only cause enhancement of immune functions or histological changes in lymphoid organs. The variable severity of clinical hypersensitivity reactions as well as autoimmune reactions induced by xenobiotics will serve as a basis to differentiate between markedly, moderately and mildly immunotoxic xenobiotics. Severe reactions are characteristic of markedly immunotoxic xenobiotics, while moderately severe reactions are characteristic of moderately immunotoxic xenobiotics and benign reactions characteristic of mildly immunotoxic xenobiotics. Due to the complexity, the redundancy and the functional reserve of the immune system, it may be difficult to reach a generally agreed conclusion. Both negative and positive results, including structure/activity relationships when known, in vitro assays when available, relevant animal studies, clinical case reports and epidemiological studies will be used.

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PROPOSED CLASSIFICATION GROUP 1: UNCLASSIFIABLE XENOBIOTICS Group-1 xenobiotics cannot be classified with respect to immunotoxic hazard as no or overtly inadequate data are available. Usually, these data have been obtained either in irrelevant conditions of exposure, in particular excessively high-level exposures, or using outdated or inappropriate experimental procedures. Unfortunately, the majority of drugs and other chemicals in commercial use to date are classified in this category, which underscores the obvious need for additional, relevant immunotoxicity studies to be conducted in both animals and man. GROUP 2: POSSIBLY IMMUNOTOXIC XENOBIOTICS Although limited data are available, xenobiotics that fall into this category can be suspected to be associated with immunotoxic hazard, at least from a theoretical perspective. Because of structural similarities with known immunotoxicants or in their mechanism of action, immunotoxic effects may develop in treated or exposed human beings. Structure –activity relationships, for example, were identified for a number of drugs including the beta-lactam antibiotics and neuromuscular blocking drugs that have been reported to induce anaphylactic reactions in humans [12]. This can serve as a basis for the allergenic evaluation of new drugs as part of their safety assessment. Similarly, beta-blocking drugs have been reported to induce systemic autoimmune reactions. Even though the majority of beta-blockers were only involved in isolated case reports of lupus syndrome, acebutolol was associated with a significant incidence of autoantibodies [14] and lupus syndromes, whereas practolol induced severe oculo-mucocutaneous syndromes [15] and was withdrawn from the market. Although the underlying mechanism is not elucidated, any new beta-blocker should be suspected to be immunotoxic. The weight of evidence will unavoidably often be judged subjectively. GROUP 3: PROBABLY IMMUNOTOXIC XENOBIOTICS These are xenobiotics for which a sufficient amount of relevant animal data is available to suspect they represent a probably immunotoxicity hazard. Group 3 might be further subdivided into 3A when only animal data are available and 3B when some, but limited human data complement animal evidence. GROUP 4: CERTAINLY IMMUNOTOXIC XENOBIOTICS These are xenobiotics that have been conclusively documented to induce immunotoxic effects in humans, whatever the degree of animal evidence. GROUP 5: NONIMMUNOTOXIC XENOBIOTICS Nonimmunotoxic xenobiotics are defined as xenobiotics that never caused adverse effects on the immune system despite well-documented high levels of exposure in a large number


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of human beings. Needless to say, those extremely few xenobiotics can be categorized as 5 due to the limited database that is currently available. EXAMPLES To illustrate how this classification can be applied, three examples of immunotoxic drugs and chemicals are presented in Tables 1 and 2. The weight of evidence is based on data that are detailed in volume II for pharmaceuticals drugs and volume III for other chemicals. In the following tables, only a brief summary of known effects or lack of it is included. The proposed judgment is based on the weight of evidence at the time of writing. DISCUSSION Classifications have been proposed and used for compounds with varied toxic potentials, such as carcinogens, teratogens or environmental toxicants. By and large, these classifications proved helpful to assess the toxicity of xenobiotics, particularly from a regulatory perspective, but also to set research priorities. In addition to this, the proposed classification for immunotoxicants can serve as a basis to evaluate the available evidence for immunotoxicity to humans and to identify where relevant data are lacking. Obviously, classifications such as

Table 1. Proposed judgment on the immunotoxic potential of selected drugs Weight of evidence

Proposed judgment

Ampicillin

Immunosuppression Inadequate animal data No human data Immunostimulation: no data Hypersensitivity Frequent clinical reactions Relevant animal data Autoimmunity: no data

Certainly immunotoxic (Group 4)

Cyclosporine

Immunosuppression Infections and malignancies in man Consistent animal data Immunostimulation: no data Hypersensitivity: no data Autoimmunity: only scare animal data

Certainly immunotoxic (Group 4)

Diazepam

Immunosuppression Animal data only at high doses Developmental immunotoxicant No human data Immunostimulation: no data Hypersensitivity: no data Autoimmunity: no data

Possibly immunotoxic (Group 2)

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Table 2. Proposed judgment on the immunotoxic potential of selected chemicals Weight of evidence

Proposed judgment

Dioxin

Immunosuppression Consistent animal data Inconsistent human data Immunostimulation: no data Hypersensitivity: no data Autoimmunity: no data

Possibly immunotoxic (Group 2) or Probably immunotoxic (Group 3A)

Hexachlorobenzene

Immunosuppression: no data Immunostimulation Relevant and consistent animal data No human data Hypersensitivity: no data Autoimmunity Consistent animal data No human data

Possibly immunotoxic (Group 2)

Mercuric chloride

Immunosuppression Positive animal data at high doses No human data Immunostimulation: no data Hypersensitivity: no data Autoimmunity Positive data in selected strains No human data

Unclassifiable (Group 1)

the one which is proposed here, require that a large consensus can be achieved among both the scientific and the regulatory communities, to be operationally helpful. When a classification of immunotoxicants is used, the following questions have to be carefully addressed, and this is another expected beneficial outcome †

†

†

Is immunotoxic hazard based on merely qualitative or quantitative alterations of the immune response, or should one need correlations with adverse clinical effects in human beings? Is a change in one single immune parameter sufficient evidence to classify a chemical as immunotoxic? Alternatively, is a consistent pattern of changes required to reach this conclusion and if so, what types of pattern are expected? Should observed changes be morphologic or functional or both?

There is no doubt that a comprehensive analysis of validated data using a panel of chemical families as large and varied as possible is absolutely necessary to identify the usefulness and weaknesses of this classification. Although, any classification has drawbacks as well as merits, it is hoped that the proposed classification can at least serve as a useful starting point.


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References [1] Kramer PGN, Roelfzema H (1992) Classification of chemicals for carcinogenic and mutagenic properties. Toxicol Lett, 64/65, 173– 182. [2] Dearfield KL, Cimino MC, McCarroll MI, Valcovic LR (2002) Genotoxicity risk assessment: a proposed classification strategy. Mutat Res, 521, 121– 135. [3] Sullivan FM (1992) The European Community classification of chemicals for reproductive toxicity. Toxicol Lett, 64/65, 183– 189. [4] Botham PA (1992) Classification of chemicals as sensitizers based on new test methods. Toxicol Lett, 64/65, 165– 171. [5] ECETOC (2003) Contact sensitization: classification according to potency. Technical Report no. 87. Brussels. [6] Luster MI, Selgrade MJ, Germolec DR, Burleson FG, Kayam F, Comment CE, Wilmer JL (1994) Experimental studies in immunosuppression: approaches and application in risk assessment. In: Dean JH, Luster MI, Munson AE, Kimber I (Eds) Immunotoxicology and Immunopharmacology, 2nd edition, pp 51 –70. Raven Press, New York. [7] Selgrade MJ (1999) Use of immunotoxicity data in health risk assessment: uncertainties and research to improve the process. Toxicology, 133, 59 – 72. [8] National Research Council, (1992) Biologic Markers in Immunotoxicology. National Academy Press, Washington, DC. [9] Straight JM, Kipen HM, Vogt RF, Amler RW (1994) Immune Function Test Batteries for Use in Environmental Field Studies. Agency for Toxic Substances and Disease Registry, Atlanta. [10] Zelikoff JT, Raymond A, Carlson Y, Li Y, Beaman JR, Anderson M (2000) Biomarkers of immunotoxicity in fish: from the lab to the ocean. Toxicol Lett, 112/113, 325– 332. [11] Galloway TS, Depledge MH (2001) Immunotoxicity in invertebrates: measurement and ecotoxicological relevance. Ecotoxicology, 10, 5 –23. [12] Baldo BA, Pham NH (1994) Structure –activity studies on drug-induced anaphylactic reactions. Chem Res Toxicol, 7, 705– 721. [13] Baldo BA, Pham NH, Zhao Z (2001) Chemistry of drug allergenicity. Curr Opin Allergy Clin Immunol, 1, 327– 335. [14] Booth RJ, Bullock JY, Wilson JD (1980) Antinuclear antibodies in patients on acebutolol. Br J Clin Pharmacol, 9, 515– 517. [15] Behan PO, Behan WM, Zacharias FJ, Nicholls JT (1976) Immunological abnormalities in patients who had the oculomucocutaneous syndrome associated with practolol therapy. Lancet, ii, 984–987.


LIST OF ABBREVIATIONS ADCC BALT BSA CD CFA CFU ConA DC DES DNCB DNFB DTH ECG EMEA EPA FDA FMLP G-CSF GALT GM-CSF GPMT HSA HCB HGG IDDM IFN IL KIRs KLH KO mice LLNA LTT Mab M-CSF MALT MBP MEST MHC MLR NALT NK NTP

Antibody-dependent cellular cytoxicity Bronchi-associated lymphoid tissue Bovine serum albumin Cluster of differentiation Complete Feund’s adjuvant Colony-forming unit Concanavalin-A Dendritic cells Diethylstilbestrol Dinitrochlorobenzene Dinitrofluorobenzene Delayed-type hypersensitivity Electrocardiogram European Medicines Evaluation Agency (US) Environmental protection Agency (US) Food and Drug Agency N-formyl-methionyl-leucyl-phenylalanine Granulocyte colony-stimulating factor Gut-associated lymphoid tissue Granulocyte/macrophage colony-stimulating factor Guinea-pig maximization test Human serum albumin Hexachlorobenzene Human gamma-globulin Insulin-dependent diabetes mellitus Interferon Interleukin Killer inhibiting receptor Keyhole limpet hemocyanin Knock-out mice Local lymph node assay Lymphocyte transformation test Monoclonal antibody Macrophage colony stimulating factor Mucosa-associated lymphoid tissue Major basic protein Mouse eat swelling test Major histocompatibility complex Mixed lymphocyte reaction Nasal-associated lymphoid tissue Natural killer (US) National Toxicology Program 387

388 PBS PHA PBMC RT-PCR SLE SRBC TCR TGF-b

Immunotoxicology of drugs and chemicals Phosphate-buffered saline Phytohemagglutinin Peripheral blood mononuclear cells Reverse transcriptase polymerase chain reaction Systemic lupus erythematosus Sheep red blood cells T cell receptor Transforming growth factor-b

Index A a-Methyldopa 74, 95, 146, 244, 250 ACE inhibitors 72 Acebutolol 89 Acetone 246– 248 Acute-phase proteins 34, 41, 206 Acute cytokine syndrome 66, 131 Acyclovir 329, 331 ADCC 27 Adrenaline 47, 338 Adriamycin 312 Age and immunity 48, 166, 297 Agranulocytosis 78 Ajmaline 312 Alcohol see ethanol Aldehydes 135 Aldicarb 304 Alfadione 7, 144 Alimemazine 147 Allopurinol 73, 76, 142, 370 Aluminum hydroxide 96 Amanita phalloides 313 Ampicillin 75, 332, 383 Amitryptiline 318 Anaphylactoid reactions 87 Anaphylatoxins 30, 141, 144 Anaphylaxis 71, 80, 87, 139, 220 Angioedema 33, 72, 77 Animal handling/housing 270– 272 Antibiotics 71 Anticancer drugs 56, 60, 171 Antiepileptic drugs 73, 76, 78 Antigen-presenting cells 28, 37 Antigens 26, 174 Antimicrobial peptides 31 Antiphospholipid syndrome 89 Antiproteases 34 Antithyroid drugs 78 AntiTNF-a drugs 10, 63, 65

Arsenic 313 Arthus 77, 141 Aspergillosis 58 Aspirin 145, 312 Asthma 71, 81, 82 Atherosclerosis 80 Atopy 137 Autoantibodies 97, 243, 244, 281 Autoimmune diseases 67, 88, 145, 243 Animal models 209, 212, 244, 281 Autoimmunity 243 Azathioprine 56, 61, 130, 164, 166, 331, 332 Azurophil granules 27, 31 B Bacterial infections 57 BALT 165, 169 Barbital 246 Barbiturates 74 Basophils 30, 33 Basophil degranulation test 84 BCG 66, 67, 69, 210, 330 Beige mice 371 Benzo(a)pyrene 339, 368 Benzodiazepines 332 Benzyl-penicillin 232 Beryllium 82, 297 Betamethasone 333 Biomarkers of immunotoxicity 300 Biphenyls 56, 339, 358 Birds 359 Bleomycin 90 Blood cell counts 170 Bone marrow 21, 164, 165, 171, 206 Bovine serum albumin 174, 178 Bradykinin 36 BrdU 180, 299 Buehler test 225 389

390 Bullous pemphigoid 96 Bursa of Fabricius 21 C C3a 30, 36 C3b 27 C5a 30, 36 C-Reactive protein 34, 182 Cadmium 166, 209, 357 Cancer see Malignancies Candida albicans 58, 208 Capsaicin 36 Captopril 96, 211 Caramel color 168 Carbamazepine 63, 76, 89, 142 Carbaryl 129 Carbofuran 129 Carbon clearance assay 182 Carbon monoxide 340 Carboxypeptidase 30, 140 Cathecolamines 47 Cathelicidins 31 Cathepsin D 30, 140 Cationic proteins 27 CD2 25 CD3 25, 26 CD4 25, 172 CD8 25, 172 CD11 29 CD14 172 CD16 26 CD19 26, 298 CD20 298 CD22 26 CD23 26 CD25 172, 299 CD28 37 CD34 28 CD37 26 CD40 29, 299 CD40L 29 CD44 26, 172 CD45 26, 85 CD46 33

Index CD54 29 CD55 33 CD56 26, 172, 272 CD62 26, 172 CD63 30, 85 CD64 140 CD69 30, 299 CD71 172, 299 CD80 29 CD86 29 Cefaclor 137 Cellular immunity 39, 130, 177, 188, 298 Cephalosporins 76, 79, 136, 224 Cephalothin 232 Ceruloplasmin 182 Chemiluminescence assay 183 Chemokines 30, 34, 129 Chemotaxis 27, 34, 184 Chemotherapy 56, 60, 63 Chimpanzees 251 Chlordane 129, 337 Chloral hydrate 4 Chlorine 129 Chloroform 4 Chlorpromazine 89, 90, 231, 248 Chlorpropamide 95 Chlorthalidone 244 Chromium release assay 180, 184 Chronic fatigue syndrome 46 Chymase 30, 140 Cimetidine 205– 207 Cinnarizine 207 Clamoxicillin 75 Clinical immunotoxicology 295 Clonazepam 332 Cloxacillin 332 Cocaine 317, 342 Colchicine 316 Collagen-induced arthritis 212 Collagenase 27 Complement 32, 36, 72, 88, 129, 132, 141, 144, 186 Concanavalin-A 179, 181 Contact dermatitis 71, 73, 142 Contact hypersensitivity models 179, 224

Index Coombs’ test 78, 79, 89, 251 Corticosteroids 48, 56, 333 Corticosterone 48, 166, 275 Corynebacterium parvum 66 Co-trimoxazole 138 Cremophor El8 144 Cross-allergy 136 Cryptococcus 58 Cyclophosphamide 62, 70, 129, 170, 231, 275, 334, 368, 370 Cyclosporine 43, 56, 61, 70, 129, 130, 144, 164, 166, 168, 169, 172, 185, 329, 331, 334, 368, 370, 383 Cypermethrin 337 Cytochrome P450 68, 133 Cytomegalovirus 58, 59, 130, 187 Cytokine release syndrome 66 Cytokines 35, 39, 47, 68, 132, 181, 247 Cytopenias 77 Cytotoxicity assays 180, 299 D DDT 57, 359 Defensins 31 DEHP 168 Delayed hypersensitivity 142, 178, 207, 279, 280, 300 Deltamethrin 168 Dendritic cells 28 Depression and immunity 45 DES 168, 188, 335 Desipramine 317 Developmental immunotoxicology 327 Dexamethasone 333, 368 Dextran 26 Diabetes 93, 147, 210 2,4-Diaminotoluene 129 Diapedesis 27, 34 Diazepam 144, 270, 332, 383 Dichlorophenol 340 Dichlorophenoxyacetic acid 338 Dichlorvos 129 Diclofenac 76, 94, 248, 250 Diesel exhaust particles 129, 139, 370

391 Diethylstilbestrol see DES Digitalis 316 Digoxin 316 Dihydralazine 94 Dihydrotestosterone 212 Diisocyanates 135 Dimercaptosuccinic acid 336 7,12-Dimethylbenzathracene 368 Dioctyltin dichloride 131, 342 Dioxin 131, 167, 188, 300, 313, 327, 340, 358, 368, 370, 393 Diphenylhydantoin 63, 76, 89, 147, 244, 246, 336 Direct immunotoxicity 10 DMBA 170 DNCB 179, 224, 231, 248, 300 DNFB 179 DNP-Ficoll 174 Docetaxel 90 Dog 25, 80, 170, 178, 223, 250, 271, 274, 330 Dolphins 358 Dopamine 47 Dose response relationships 283 Draize test 225 DRESS 73 Drug hypersensitivity syndrome see DRESS E ELISA 176, 279 ELISPOT 177, 249 Epstein –Barr virus 58, 60, 62, 63, 147 Endocytosis 29 Eosinophils 29, 84 Epicutaneous maximization test 228 Epidemiological studies 65, 302 Epoxy resins 91 Erasmus syndrome 91 Erythema multiforme 74 Erythema nodosum 74 Erythropoeitin 95 Ethanol 4, 46, 188, 246 – 248, 300, 342 Ether 4

392 Ethosuximide 89 Expert systems 371 F Farmer’s lung 82 Fenclofenac 63 Fetal alcohol syndrome 342 Fever 34, 36, 41, 131 Fibronectin 34 Fish 355 Fixed drug eruptions 74 Flagellin 174 Flu-like reactions 66, 131, 207 Fludarabine 95 Fluticasone 300 Food allergy/intolerance 71, 222 Formaldehyde 91 Free radicals 27, 36 Freund’s complete adjuvant 210, 212 Functional reserve capacity 20, 65, 283 Fungal infections 58 G G-CSF 21, 27, 165, 207 Gallium arsenide 129 GALT 169 Gastro-intestinal infections 59 Gell and Coombs classification 139, 142 Glafenin 7, 135 Glomerulonephritis 81 Glycans 66 GM-CSF 21, 25, 27, 28, 30, 74, 165, 207 Gold 243, 249 Good Laboratory Practices 269 Goodpasture’s syndrome 141 Guidelines 257 Guillain– Barre´ syndrome 92, 147 Guinea-pigs 37, 80, 140, 179, 221, 224 H Hair dyes 89 Halothane 94

Index Hamster 231 Hapten 135, 142, 220 Haptoglobin 34 Heat-shock proteins 29 Hemolytic anemias 78, 95, 141 Hemopexin 34 Heparin 140 Hepatitis 80, 94 Heptachlor 129, 338 Herpesvirus 58, 62, 187 Hexachlorobenzene 57, 67, 205, 206, 338, 393 Hexachlorocyclohexane 339 Hexahydrophthalic anhydride 224 Hexamethylene diisocyanate 224 2,5-Hexane 168 Histamine 30, 140 Histamine release test 84, 144 Histology 163, 205, 278, 363 Bone marrow 164, 165, 280 Lymph nodes 164, 168, 280 MALT 164, 169 Peyer’s patches 164, 169 Spleen 164, 168, 280 Thymus 164, 166 HLA 148 Hoigne´’s syndrome 143 Hormones 48, 148 Host resistance models 186, 207, 280 Humoral immunity 37, 130, 173 , 298 Hydralazine 89, 97, 146, 243, 247, 250 Hypereosinophilia 73, 81, 82, 84 Hypersensitivity 70, 280 Hypersensitivity pneumonitis 82 Hypersensitivity reactions 68, 133, 219 I ICICIS 1, 3 Idiosyncrasy 143 IgA 37, 169 IgA deficiency 59, 63 IgA linear bullous dermatosis 96 IgA nephropathy 81 IgD 37

Index IgE 25, 37, 140, 250 Specific IgE 85, 220, 223 Total IgE 84, 173 IgG 37, 141 IgG1 140, 221, 223, 250 IgG2 37, 250 IgG4 140 IgM 37, 141 IL-1 28– 30, 35, 41, 47, 66, 68, 131, 133, 207, 328 IL-1Ra 41 IL-2 25, 42, 66 – 68, 205 – 207, 209, 222 IL-3 21, 25, 27, 30, 43, 165, 188, 207 IL-4 25, 30, 43, 69, 212 IL-5 25, 29, 30, 142 IL-6 28, 36, 43, 47, 131, 133, 207 IL-8 30, 142 IL-9 43 IL-10 25, 43, 66, 68 IL-11 44 IL-12 29, 44 IL-13 44 IL-15 44 IL-18 41 Imipramine 248, 317 Immune complexes 34, 141 Immunodepression 56, 64, 178 Immunogenicity 134 Immunoglobulins 37, 172, 279 Immunohistochemistry 364 Immunomodulation 66 Immunostimulation 66, 205, 280 Immunosuppression 4, 9, 55, 56, 59, 128, 164, 168, 272, 282, 367 Immunoteratology 327 Immunotherapy of poisonings 312 Adriamycin 312 Ajmaline 312 Amanita phalloides 312 Arsenic 313 Aspirin 312 Colchicine 318 Cocaine 319 Digitalis 316

393 Digoxin 316 Dioxin 313 Morphine 312 Nicotine 319 Paraquat 314 Parathion 314 Phencyclidine 320 Ricin 315 Soman 314 Snake envenomations 314 Strychnine 315 Tricyclic antidepressants 317 Vinca alkaloids 312 VX 314 Implanted tumors 189 In silico immunotoxicology 369 In situ hybridization 365 In vitro immunotoxicology 363 Indoor air 71, 82 Infections 56, 128 Bacterial infections 57, 187 Experimental infections 186, 208 Fungal infections 58 Gastro-intestinal infections 59 Parasitic infections 59, 188 Respiratory infections 59 Viral infections 58, 60, 62, 187 Inflammation 35 Infliximab 10, 63 Influenza vaccines 69 Influenza virus 187 Insecticides 129 Integrins 34 Interferons Interferon-a 35, 41, 66, 67, 69, 70, 133 Interferon-b 35, 41, 66, 67, 133 Interferon-g 25, 41, 43, 66, 67, 133, 207, 328, 329 Interstitial nephritis 81 Iodoxuridine 207 Iproniazid 94 Isoniazid 89, 243, 246, 249, 250 Isotretinoin 329 Isoxicam 7

394

Index

K

M

Kaposi’s sarcoma 60, 62 Ketotifen 188 Keyhole limpet hemocyanin 174, 176, 178, 279, 296, 300 Kinins 36, 145 Ku¨pffer cells 28

M-CSF 21, 28, 165 Macrophages 28, 132 Macrophagic fasciitis 96 Magnusson and Kligman test 227 Marijuana 129 Major basic protein 29, 30 Malignancies 59, 128 Malnutrition 167 MALT 24, 47, 164 Manganese 357 Marijuana 275 Marmoset 231 Mast cells 30, 33 Mastocytosis 30, 85 Maximization test 226 Medical devices 257, 262 Medroxyprogesterone acetate 207 Mefenamic acid 95 Memory lymphocytes 26, 39 Mepivacaine 142 Mercury 57, 209, 211, 243, 247, 249, 393 MEST 228 Metals 57, 357 Metformin 246 Methimazole 212 Methotrexate 63, 65 Methylmercury 341 Methylprednisolone 331 Methysergide 91 MHC 142, 148 MHC Class I molecules 25, 27, 29, 37, 41 MHC Class II molecules 29, 37, 41, 142 Minocycline 73, 89, 97, 248 Mishell –Dutton assay 368 Mixed lymphocyte response 180 Molecular mimicry 147 Monkey 25, 26, 170, 178, 271, 275, 330 Monoclonal antibodies 66 Monocytes 28 Morphine 90, 130, 144, 185, 312 Mouse 26, 31, 140, 270, 273, 330 Mouse IgE test 224

L L -Tryptophan 3, 6, 91 Lactoferrin 27 Lamotrigine 137, 142 Langerhans cells 29, 142 Lead 57, 166, 209, 327, 340, 368 Leukocytosis 84, 170, 206 Leukotrienes 30, 36, 85, 141 Levamisole 66 –68, 70 Levodopa 95, 251 Lichenoid eruptions 74 Lidocaine 142 Listeria monocytogenes 182, 187, 207 LLNA 229 Local anesthetics 143 LPS 35, 44, 174, 298 LTC4 30, 141 LTD4 141 LTE4 141 LTT 85, 136 Lupus 88 Lymph nodes 24, 47, 164, 168, 206, 244 Lymphocyte proliferation 45, 179, 279, 298, 299 Lymphocyte subset analysis 64, 171, 279, 299 Lymphocyte transformation test see LTT Lymphocytes 21, 23, 25, 26 Lymphocytosis 170 Lymphoid organs 21, 23, 280 Lymphomas 58, 60 Lymphopenia 64, 84, 170 Lymphotoxin-a 44 Lysozyme 27, 31, 182

Index MRL mice 210 MTT 180 Multiple chemical sensitivity 71 Multiple sclerosis 92, 147 Mycophenolate mofetil 56, 61, 13 Myeloperoxydase 30 Myelotoxicity 56 Myocarditis 79, 92 Myasthenia 92 N N-Acetylprocainamide 147 NALT 165 Natural Killer cells see NK cells Neonate and immunity 48 Neuropeptide Y 47 Neuroendocrine system 47 Neurotransmitters and immunity 47 Neutropenia 78, 95, 170 Neutrophilia 170 Neutrophils 27 Newborns 328 Nickel 130, 166, 185, 357 Nicotine 319, 343 Nimesulide 145 Nitrogen oxides 129, 138 NK cells 26, 35, 45, 46, 130, 184, 187, 207, 279, 300 Nocardiosis 58 NOD mice 210 Nomifensine 6, 78, 79, 95 Noradrenaline 47 NSAIDs 72, 74, 75, 78, 85, 138, 143, 145 Nutrition and immunity 46 NZB £ NZW (F1) mice 209, 244, 251 O Ochratoxin 341 Ofloxacin 246 OKT-3 61, 145 Oligonucleotides 205, 207 Open epidermal test 227 Opioids 47

395 Opportunistic infections 64 Opsonization 27, 33 Optimization test 227 Organochlorine insecticides 57, 129 Organophosphorus compounds 314 Organophosphorus insecticides 57 Organotins 131, 168 Ovalbumin 174, 178, 221, 223 Overimmunosuppression 11, 64 Oxazolone 179, 228 Oxyphenylbutazone 76 Ozone 129, 138 P Paclitaxel 144 PAF-acether 36 Papilloma virus 60 Paracetamol 145 Parallelogram paradigm 65 Paraoxon 129, 314 Paraquat 314 Parasitic infections 59 Patch tests 73, 86, 224 PCBs 56, 57, 314, 359 Pemphigus 96 Penicillamine 92, 96, 146, 211, 243 Penicillin 70, 76, 86, 134, 136, 138, 143, 223 Pentazocin 90 Pesticides 57, 129, 257, 337, 357 Peyer’s patches 164, 169 Phagocytes 27, 129 Phagocytosis 27, 172, 182, 279, 300 Phencyclidine 320 Phenobarbital 76 Phenol 341 Phenothiazines 75, 78 Phenylbutazone 248 Phosphodiesterase inhibitors 207 Photoallergy 74 Photosensitivity 74 Phototoxicity 74 Phytohemagglutinin 178, 179 Picryl chloride 179, 248

396 Pig 25, 274, 330 Plaque Forming Cell (PFC) assay 174, 207, 278, 280 Pleiotropy 40 Pneumocystis carinii 56 Pokeweed mitogen 179, 298 Poly-(I:C) 68, 133, 210, 211 Polybrominated biphenyls see PBBs Polychlorinated biphenyls see PCBs Polymyositis 96 Polynuclear leukocytes 29 Polysaccharides 26 Polyvinylpyrrolidone 174 Popliteal lymph node assay 147, 244 Practolol 6, 90 Prednisolone 331 Prednisone 61, 334 Prick tests 86 Primary granules 27, 31 Procainamide 89, 95, 147, 243, 246, 249 Promethazine 336 Propanil 168 Propofol 300 Propylene glycol 246 Propythiouracil 97 Prostaglandins 30, 36, 131, 141 Proteases 27, 36 Provocation tests 87, 145, 222 Pseudo-GvH 147 Pseudoallergy 87, 143, 281 Psychoneuroimmunology 45 Purpura 75 Pustolosis 75 Pyritinol 93, 96 Q Quinine 78 Quinolones 75, 76 R Rabbit 80, 271 Radiocontrast media 68, 71, 144

Index Rash 75 RAST 85 Rat 25 – 27, 31, 37, 170, 273, 330 Redundancy 20, 283 Red man syndrome 88 Regulatory immunotoxicology 257 Respiratory allergy 223 Respiratory infections 59 Retinoic acid 329 Ricin 315 Rifampicin 139 Ring of Waldeyer 24, 169 Risk assessment 269, 281 Rituximab 145 S Salmeterol 188 Scid mice 370 Scleroderma 90 Seals 308 Second cancers 60 Secondary granules 27 Selectins 26, 34 Sentinel diseases 303 Serotonin 47 Serum amyloid A 34 Serum sickness 142 Shelley’s test 85 Silica 91, 97, 209, 305 Silicone gel 91, 209 –211 Sirolimus 43, 61, 168, 329 Skin cancers 61 Skin graft models 179, 207 Skin tests 86 Smoking 46, 138, 343 Sodium salicylate 4 Sodium valproate 63 Solvents 91 Soman 314 Spanish toxic oil syndrome 3, 6, 91 Specific immunity 36 Spleen 23, 47, 164, 168, 206, 244 Split adjuvant test 227

Index Sport and immunity 46 SRBC 174, 176, 178, 207, 248, 272, 279, 296 Streptozotocin 246– 249 Stress and immunity 45, 46, 166, 275 Stevens– Johnson syndrome 76 Structure – immunotoxicity relationships 149 Substance P 36, 47 Sulfasalazine 63 Sulhydryl groups 146 Sulfadiazine 89 Sulfamethoxazole 142, 232 Sulfanilamide 231 Sulfur dioxide 138 Sulfuric acid 129 Sulphonamides 73 – 76, 138 Sulphones 73 Surgery and immunity 46 Sweet’s syndrome 74 T T lymphocytes 25, 26, 42, 43 T-2 toxin 170 Tacrolimus 43, 56, 61, 131, 168, 329 TBTO 131, 168, 188, 357, 368 TCDD see Dioxin Tebuconazole 339 Tetracyclines 74, 76 Tetanus toxoid 174, 176, 178, 207, 296, 298 Tetanus vaccines 70 TGF-b 44 TH1/TH2 responses 25, 41, 44, 132, 250, 328, 329 Thiazides 75 Thalidomide 336 Thallium 89 Thiopentone 300 Thrombocytopenias 79, 95, 141 Thymic hormones 67 Thymus 23, 47, 164, 166 Thyroiditis 67, 93

397 Tienilic acid 94 Tiered testing 258, 277 Tilorone 133, 209, 212 Tiopronine 93, 96, 146 TNF-a 25, 28– 30, 35, 44, 47, 66, 131, 133, 207 TNF-b 44 TNP-Ficoll 174 Toll-like receptors 29, 35 Tolmetin 135 Toluene diisocyanate 224 Toluidine blue 30 Toxaphene 129 Toxic epidermal necrolysis 76 Transgenic mice 371 Tri-n-butyl tin oxide see TBTO 2, 4, 6-Tribromophenol 342 Trichinella spiralis 188, 207, 332 Trichloroethylene 89, 91, 210, 211 Tricyclic antidepressants 317 Trimellitic anhydride 224, 231 Trimethadione 89, 93 Trimethoprim 76 Tryptase 30, 85, 140 Tuberculin 180 Tuberculosis 58 U Urethan 342 Urticaria 77 UV 57, 65 V Vaccines 66, 69, 92, 96, 138, 211, 222 Valproic acid 76 Vancomycin 88, 144 Vasculitis 77, 97, 142 Viral infections 58 Vitamin K1 144 Vinyl chloride 91 VX 312

398

Index

W

Y

Welder’s fumes 132

Yu-Chen disease 3, 57 Yusho disease 3, 57

X

Z

XTT 180 Whales 359 Wildlife 355

Zimeldine 6, 57, 246, 248 Zinc 132, 188 Zomepirac 7, 135

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