Advances in Immunology Volume 13

ADVANCES I N

Immunology

VOLUME 13

CONTRIBUTORS TO THIS VOLUME ELMERL. BECKER

HANSBENNICH BARRYR. BLOOM A. D. M. BRYCESON JOHN

E. HOPPER

S. GUNNAR0. JOHANSSON

ALFREDNISONOFF

J. L. TURK

ADVANCES IN

Immunology EDITED BY

F. J. DIXON, JR.

H E N R Y G. KUNKEL

Division of Experimenfal Pathology Scripps Clinic and Research Foundafion

The Rockefeller Universify N e w York, N e w York

La Jolla, California

V O L U M E 13 1971

ACADEMIC PRESS

New York and London

COPYRIGHT 8 1971, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN A N Y FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION PROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New

York, New York 10003

United Kingdom Edition published b y ACADEMIC PRESS INC. (LONDON) LTD.

24/28 Oval Road, London)NWI IDD

LIBRARY OF CONGRESS CATALOG CARD

NUMBER: 61-17057

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS LIST OF COSTRIBUTORS. PREFACE

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vii ix

CONTENTS OF PREVIOUS VOLUhlES

xi

Structure and Function of Human Immunoglobulin E

HANSBESNICHA N D S. GUSNAR0. JOHANSSON

I. Introduction . . . . . . . . . . . . 11. Isolation and Physicochemical Characteristics of Inimunoglobulin E 111. Properties of Antigenically and Biologically Active Structural Regions of Immunoglobulin E . . . . . . . . IV. Methods for Determination . . . . . . . . . V. Levels of Immunoglobulin E in Healthy Individuals . VI. Levels of Inimunoglobulin E in Disease . . . . . . VII. Detection of Antibody Activity in the Immunoglobulin E Class . VIII. Metabolism . . . . . . . . . . . . IX. Concluding Remarks . . . . . . . . . . References . . . . . . . . . . .

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19 28 29 35 45 49 51 51

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Individual Antigenic Specificity of Immunoglobulins

JOHNE. HOPPERAND ALFRED NISONOFF

I. Introduction . . . . . . . . . . . . 11. Individual Antigenic Specificities in Monoclonal Proteins . . . 111. Individual Antigenic Specificities in Antibody Populations . . IV. Cross-Reactions of Antiidiotypic Sera and Evidence for Identical . . . . . Molecules in Different Individual Animals V. Evidence Based on Idiotypic Specificity for Limited Heterogeneity of Normal Antibody Populations . . . . VI. Persistence and Changes of Antibody Populations during Prolonged Immunization . . . . . . . . . VII. Shared Idiotypic Determinants in IgC and IgM Antibodies of the Same Specificity . . . . . . . . . . VIII. Localization of Individually Specific Antigenic Determinants . IX. Cross-Reactions of Anti-ind Antibodies with Nonspecific Immunoglobulins . . . . . . . . . . . X. Monoclonal Origin of Molecules with Individually Specific Antigenic Determinants . . . . . . . . . XI. Summary . . . . . . . . . . . . References . . . . . . . . . . . .

58 60 63 69

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75 76 81 85 92 94 95 97

In Vitro Approaches to the Mechanism of Cell-Mediated Immune Reactions

BARRYR. BLOOM

I. Introduction . . . . 11. Lymphocyte Transformation

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CONTENTS

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111 Direct Cytotoxicity of Target Cells by Lymphocytes . . . IV . Mediators-Qualitive Basis of the Response . . . . . V. Enumeration of Specifically Sensitized Cells-Quantitative Basis of the Response . . . . . . . . . . VI . Reality Testing-Relationships between in Vitro Results and CellMediated Immunity in Vioo . . . . . . . . VII . Relationships between Cell-Mediated Immunity and Antibody Formation . . . . . . . . . . References . . . . . . . . . . . .

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111 122

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178 193

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Immunological Phenomena in leprosy and Related Diseases

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J . L TURKAND A . D M . BRYCESON

. . . . . . . . . . . I . Introduction . I1. Leprosy . . . . . . . . . . . . . 111. Leishmaniasis . . . . . . . . . . . . IV . Concept of a Host-Determined Spectrum of Clinical Manifestations in Other Chronic Infections in Man . . . . . . . References . . . . . . . . . . . .

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209 210 237

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259 261

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267 270 271 285 286 289 291 298 299 305 307 308

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Nature and Classification of Immediate-Type Allergic Reactions

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ELMERL BECKER

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I Introduction . . . . . . . . . I1 Sensitization . . . . . . . . . 111. Components of the Allergic Reaction . . . . IV . Sites of the Antigen-Antibody Reaction . . . V. Time Course of Allergic Reactions . . . . VI The Terrain . . . . . . . . . VII . Basis and General Description of the Classification VIII Direct Responses (Non-mediator Determined) . . IX . Indirect Responses (Mediator Determined) . . X . Mixing of Categories in Natural Reactions . . XI Pseudoallergic Reactions . . . . . . References . . . . . . . . .

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AUTHOR INDEX .

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SUBJECTINDEX .

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315 333

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ELMERL. BECKER, Department of Pathology, University of Connecticut Schools of Medicine and Dentistry, Farmington, Connecticut (267) HANS BENNICH, The Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden (1) BARRYR. BLOOM, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York (101) A. D. M. BRYCESON, Department of Medicine, Ahmadu Bello University, Zaria, Nigeria (209)

E. HOPPER, Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, Zllinois ( 5 7 )

JOHN

S. GUNNAR0. JOHANSSON, The University Blood Center, University of Uppsala, Uppsala, Sweden (1) ALFREDNISONOFF, Department of Biological Chemistry, University of Zllinois College of Medicine, Chicago, Zllinois (57)

J. L. TURK,Department of Pathology, Royal College of Surgeons of England, Lincoln’s Inn Fields, London, England ( 2 0 s )

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PREFACE It can safely be predicted that the seventies will be the decade of cellular immunology. Already the expansion is cvideiit on all sides and many immunologists, previously involved in the antibody field, are turning to cellular work. To somc cxtent this may be unfortunate because that most basic of problcms, thc mechanism of antibody variability, remains an enigma. Immunologists are still evenly divided on the issue of whether the germ line theory or a typc of somatic model holds the explanation. Possibly the work at thc cellular level might provide the answer. The Advances will participate actively in this timely movement and, as exemplificd by this volumc, will also continue to be involved in the currently lcss popular but no lcss important areas of immunology, The articlc by Drs. John E. Hopper and Alfred Nisonoff concerns that very uscful label of the immunoglobulins, their individual antigenic specificity or idiotypic specificity. The authors have utilized this property in superb fashion to tracc the devclopment of different antibody producing clones of cells in the primary and secondary response. It is abundantly clear that antigens related to thc V regions aiid antibody combining sites are followed in thesc studies. There are few areas where problcms of iiomcnclature are more varied and confusing than in thc field of allergic reactions. Dr. Elmer L. Becker treats this subject from all aspects, ranging from the initiating antigen, through the mediators produced, to the final cell involvement. A very reasonable classification of immediate-type allergic reactions has emerged that takes into account the many different phases of these reactions. One of the exciting chaptcrs in immunology has been the recent dclineatioii of the IgE class of iniiiiuiioglobulins and the demonstration of its significance for atopic allergic disorders. Just as in all other areas of immunoglobulin work, the discovery of a myeloma protein of the IgE typc contributed cnorinously to thc successful evolution of this work. Drs. Hans Bennich and S. G~iiiiiar0. Johansson wcre responsible for this important aspcct and they not only review this field but also present many observations that have not been published elsewhere. Because of thc low concentration of IgE in most scra, its measurement has prescnted a special challenge. Thc ingcnious procedures devcloped by the authors, as me11 as other methods, arc discussed in useful detail. Drs. J. L. Turk and A. D. hl. Bryceson review the various different immunological reactions to the specific organisms in leprosy and ix

X

PREFACE

leishnianiasis. These authors have played a primary role in interpreting these reactions in terms of modern concepts of immunology. Defects in cellular immunity clearly play a major role in special forms of these disorders and many of the principles derived from these studies hold implications for a number of other diseases. Dr. Barry R. Bloom, one of the leaders in the cellular immunity expansion, describes some of the forefronts of this field. The inany mediators involved in lymphocyte reactions are considered in special detail. None of these factors has been isolated in pure form, which will be essential for their eventual understanding. However, an overall picture of the intricacies of cellular iminunity is beginning to emerge which relates the various experimental models to in vivo events. The cooperation and valuable assistance of the publishers in the production of Volume 13 are gratefully acknowledged. H. G . KUNKEL F. J. DIXON July 1971

Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance

M. HA~EK, A. LENGEROVA, AND T. HRABA Immunological Tolerance of Nonliving Antigens

RICHARDT. SMITH Functions of the Complement System

ABRAHAMG. OSLER In Vitro Studies of the Antibody Response

ABRAMB. STAVITSKY Duration of Immunity in Virus Diseases

J. H. HALE Fate and Biological Action of Antigen-Antibody Complexes

WILLIAM 0. WEIGLE Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. GELLAND B. BENACERF~AF The Antigenic Structure of Tumors

P. A. GORER AUTHORINDEX-SUB JECX INDEX Volume 2 Immunologic Specificity and Molecular Structure

FREDKARUSH Heterogeneity of y-Globulins JOHN

L. FAHEY

The Immunological Significance of the Thymus

J. F. A. P. MILLER,A. H. E. MARSHALL, AND R. G. WHITE Cellular Genetics of Immune Responses

G. J. V. NOSSAL Antibody Production by Transferred Cells

CHARLESG. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICK ROWLEY xi

xii

CONTENTS OF PREVIOUS VOLUMES

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOUISBY Embryological Development o f Antigens

REED A. FLICKINCER AUTHOR INDEX-SUB JECT INDEX Volume 3 In Vitro Studies of the Mechanism of Anaphylaxis

K. FRANK AUSTENAND JOHN H. HUMPHREY The Role of Humoral Antibody in the Homograft Reaction

CHANDLER A. STETSON Immune Adherence

D. S. NELSON Reaginic Antibodies

D. R. STANWORTH Nature of Retained Antigen and Its Role in Immune Mechanisms

DANH. CAMPBELL AND JUSTINE S. GARVEY Blood Groups in Animals Other Than Man

W. H. STONEAND M. R. IRWIN Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R.

JENKIN

AUTHOR INDEX-SUB j ~ c INDEX r Volume 4 Ontogeny and Phylogeny o f Adoptive Immunity

ROBERTA. GOODAND BENW. PAPERMASTER Cellular Reactions in Infection

EMANUEL SUTERAND HANSRUEDY RAMSEIER Ultrastructure of Immunologic Processes JOSEPH

D. FELDMAN

Cell W a l l Antigens of Gram-Positive Bacteria

MACLYNMCCARTY AND STEPHEN I. MORSE Structure and Biological Activity of Immunoglobulins SYDNEY COHEN AND

RODNEYR. PORTER

CONTENTS OF PREVIOUS VOLUXZES

Autoantibodies and Disease

H. G. KUKKELAND E. M. TAN Effect of Bacteria and Bacterial Products on Antibody Response

J. MUNOZ AUTHOR INDEX-SUBJECT INDEX Volume 5 Natural Antibodies and the Immune Response

STEPHENV. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAELSELA Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIPY. PATERSON The Immunology of Insulin

C. G. POPE Tissue-Specific Antigens

D. C. DUNONDE AUTHOR INDEX-SUB j ~ c INDEX r Volume 6 Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR. UNAMJEAND FRANK J. DIXON Chemical Suppression of Adaptive Immunity

ANNE. GABRIELSON AND ROBERT A. GOOD Nucleic Acids as Antigens

OTTOJ. PLESCIA AND WERNER BRAWN In Vifro Studies of Immunological Responses of lymphoid Cells

RICHARDW. DUTTON Developmental Aspects of Immunity JAROSL.4V

STERZLAND ARTHURM.

SILVERSTEIN

Anti-antibodies

PHILIPG. H. GELLAND ANDREWS. KELUS Conglutinin and lmmunoconglutinins

P. J. LACHMANN AUTHORINDEX-SUB JECT IXDEX

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Xlll

xiv

CONTENTS OF PREVIOUS VOLUMES

Volume 7 Structure and Biological Properties of Immunoglobulins

SYDNEY COHENAND CESAR MILSTEIN Genetics of Immunoglobulins in the Mouse

MICHAELPOTTER AND ROSELIEBERMAN Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN

B. ZABRISKIE

Lymphocytes and Transplantation Immunity

DARCY B. WILSON AND R. E. BILLINGHAM Human Tissue Transplantation

JOHNP. MERRILL AUTHOR INDEX-SUB J E INDEX ~ Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. MULLER-EBERHARD Regulatory Effect of Antibody on the Immune Response

JONATHAN W. UHRAND GORANMOLLER The Mechanism of Immunological Paralysis

D. W. DRESSER AND N. A. MITCHISON In Vifro Studies of Human Reaginic Allergy

ABRAHAMG. OSLER, LAWRENCE M. LICHTENSTEIN, AND DAVID A. LEVY AUTHOR INDEX-SUB JECT INDEX Volume 9 Secretory Immunoglobulins

THOMAS B. TOMASI, JR.,AND JOHNBIENENSTOCK Immunologic Tissue Injury Mediated by Neutrophilic leukocytes

CHARLES G. COCHRANE The Structure and Function of Monocytes and Macrophages

ZANVIL A. COHN The Immunology and Pathology of NZB Mice

J. B. HOWIEAND B. J. HELYER AUTHOR INDEX-SUB J E INDEX ~

CONTENTS OF PREVIOUS VOLUMES

Volume 10 Cell Selection by Antigen in the Immune Response

GREGORY W. SISKINDAND BARUJBENACERRAF Phylogeny of Immunoglobulins

HOWARD M. GREY Slow Reacting Substance of Anaphylaxis

ROBERTP. ORANGEAND K. FRANK AUSTEN Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, and the Inflammatory Response

OSCARD. RATNOFF Antigens o f Virus-Induced Tumors

KARL HMEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARDAMOS AUTHORINDEX-SUB JECT INDEX Volume 1 1 Electron Microscopy of the Immunoglobulins

N. MICHAELGREEN Genetic Control of Specific Immune Responses

HUGH0. MCDEVITTAND BARUJBENACERRAF The lesions in Cell Membranes Caused by Complement JOHN

H. HUMPHREY AND ROBERTR. DOURMASHKIN

Cytotoxic Effects of lymphoid Cells In Vitro

PETERPERLMANN AND GOFIANHOLM Transfer Factor

H. S. LAWRENCE Immunological Aspects of Malaria Infection

IVORN. BROWN AUTHORINDEX-SUB JECT INDEX Volume 12 The Search for Antibodies with Molecular Uniformity

RICHARDM. KRAUSE Structure and Function of y M Macroglobulins

HENRYMETZGER

xv

XVi

CONTENTS OF PREVIOUS VOLUMES

Transplantation Antigens

R. A. REISFELDAND B. D. KAHAN The Role of Bone Marrow in the Immune Response

NABIHI. ABDOUAND MAXWELLRICHTER Cell Interaction in Antibody Synthesis

D. W. TALMAGE, J. RADOVICH,AND H. HEMMINGSEN The Role of Lysosomes in Immune Responses

GERALDWEISSMANN AND PETERDUKOR Molecular Size and Conformation of Immunoglobulins

KEITH J. DORRINGTON AND CHARLES TANFORD AUTHORINDEX-SUB JECT INDEX

Structure and Function of Human Immunoglobulin E HANS BENNICH AND S . GUNNAR 0 . JOHANSSON The Wallenberg laboratory and The University Blood Center. University o f Uppsala. Uppsala. Sweden

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I Introduction . . . . . . . . . . . I1. Isolation and Physicocheniical Characteristics of Immunoglobulin E . . . . . . . . . . A . Identification of Myeloma Protein N D . . . . . B Isolation . . . . . . . . . . . C . Chemical and Physical Characteristics . . . . . I11. Properties of Antigenically and Biologically Active Structural Regions of Immunoglobulin E . . . . . . . A . Properties of e Chains . . . . . . . . B . Properties of Enzymatic Fragments . . . . . IV . Methods for Determination . . . . . . . . V Levels of Immunoglobulin E in Healthy Individuals . . . A . Levels in Serum . . . . . . . . . B. Levels in Secretions . . . . . . . . . VI . Levels of Immunoglobulin E in Disease . . . . . A . Levels in Serum . . . . . . . . . B Levels in Secretions . . . . . . . . . C . Factors Influencing Immunoglobulin E Level . . . VII . Detection of Antibody Activity in the Immunoglobulin E Class A . Red Cell-Linked Antigen-Antiglobulin Reaction . . B. The Radioallergosorbent Test . . . . . . C. Allergen Antibodies in Serum . . . . . . . VIII . Metabolism . . . . . . . . . . . . . . . . . . . IX Concluding Remarks . References . . . . . . . . . . .

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I. Introduction

Immunoglobulin E ( I @ ) represents a mincjr but distinct class of proteins present in serum of man and higher primates and possibly also in the serum of other species. In healthy individuals. the upper range of concentration is usually below 1 pg./ml. The detection and quantitation of IgE require very sensitive methods. Immunoglobulin E is elevated 4-30 times normal in various diseases. among which atopic disorders and parasitic infestations appear to be the most prominent . Pathological amounts of IgE have also been found in the serum of patients with yE myeloma. The association of certain reaginic antibodies to a new class of im1

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HANS BENNICH AND S. GUNNAR 0. JOHANSSON

munoglobulin was postulated by K. Ishizaka and co-workers ( 1966a,b). The discovery that the myeloma protein ND and its normal counterpart share antigenic characteristics with reaginic antibodies and, in addition, carry skin-fixing structures, which appear similar to those of reagins, opened new possibilities to study the immunological and structural features of immediate hypersensitive reaction. The aim of this paper is to summarize our present knowledge of the biological and structural properties of IgE and its occurrence in various body fluids in health and disease, To this end, particular emphasis has been given to the methodology of identification and quantitation and also to the problem of isolation and characterization of IgE. References to the massive literature on reagins will be made only when found to be relevant for the understanding of a particular problem, and no attempts have been made to portray the long history of reagins, since this has been so masterly done in previous reviews by several authors (see K. Ishizaka and Ishizaka, 1968a; Sehon and Gyenes, 1965; Stanworth, 1963). II. Isolation and Physicochemical Characteristics of Immunoglobulin E

As a result of the obvious difficulties encountered in the isolation of reasonably homogeneous samples of a protein, which, like IgE, represents only a minor serum constituent, the physicochemical characteristics given in this paper will refer mainly to the first described E myeloma protein, ND. However, there is sufficient experimental evidence now available to support the belief that the ND protein has its major biological, immunological, and physicochemical characteristics in common with the IgE present in normal serum (Bennich et al., 1968). OF MYELOMA PROTEIN ND A. IDENTIFICATION

Our first attempt in 1965 to isolate the atypieal myeloma protein ND was done by zone electrophoresis ( Johansson and Bennich, 19674. The M component migrated in the fast y region. The isolated fraction, containing 93% of the M component contaminated mainly with IgG, was used for the first immunization experiments in rabbits and for carbohydrate analysis. The latter indicated that the M-component contained about 10%of total carbohydrate-a result suggesting a possible relationship of ND to IgA or IgM. Gel filtration experiments on serum ND on calibrated columns of Sephadex G-150 gave results in the same direction; indications that the M component distributed within the same elution volume as monomeric or 7 S IgA initiated a direct comparison of a monomeric A myeloma protein and protein ND. Both proteins were isolated from serum by precipitation with sodium sulfate and subsequently purified by recycling chromatography on Sephadex G-150 to

HUMAN IMhlUNOGLOBULIN E

3

eliminate contaminating IgG. The purified A and ND proteins were added to a solution of monomeric normal IgG and thc mixture was analyzed on a calibrated column of Sephadex G-200. The distribution of IgA and protein ND coincided completely as determined by quantitative immunological analysis, and the elution volume was significantly smaller than that of IgG. In contrast to these results, ultracentrifugal analyses indicated a significantly difference for IgA and protein ND, the sedimentation constant values ( so! ) were 6.5 and 7.9, respectively. Molecular weight determinations gave a value of about 139,000 for IgA and 196,000 for protein ND using a partial specific volume of 0.713 for both proteins. By reduction of protein ND with niercaptan followed by dissociation in acid, about 20% of the protein moiety could be rccovered as chains. The remaining 80%constituted a single carbohydratecontaining component with a characteristic electrophoretic mobility in starch gel electrophoresis in acid urea. This major constituent was regarded as representing the heavy chain of an atypical immunoglobulin. The problem of preparing class-specific antisera to IgE( N D ) was not solved until fragments of ND protein were isolated (see Section II1,B). Hereby it also became possible to develop the radioimniunosorbeiit test (RIST) described in Section IV and the radioallergosorbent test (RAST) described in Section VII. By using the RIST, a counterpart to ND was found in nornial serum. The concentration in healthy individuals are usually found to be extremely low as will be further discussed in Section V. However, by chance the serum from one of the apparently healthy blood donors included in the first series of experiments was found to contain a significantly highcr level of IgE( N D ) than the main level of the test group. The donor was subsequently found to have a previously clinically undiagnosed hypersensitivity to dog dander, a finding which initiated a study of the level of I g E ( N D ) in patients suffering from asthma and hay fever, as will be further discussed in Section VI. The significantly higher level of IgE( N D ) found in cases of extrinsic asthma strongly suggested a relation to reaginic antibodies as did the presence of allergen antibodies of IgE class in these patients. In 1966, K. Ishizaka et al. (1966a), from their studies on nntiragweed antibodies in reagin-containing fractions of atopic sera, suggestcd the prcsence of a unique immunoglobulin as a carrier of reaginic activity. The specific activity was found in the y, region by radioinimunoelectrophoresis and the protein was tentativcly designatecl YE-globulin. An exchange of antisera between Denver and Uppsala was made in March 1967 and, by comparatively antigenic analyses of myeloma protein ND and YE-globulin, direct imniunological evidence was obtained that ,lL.

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HANS BENNICH AND S. GUNNAR 0. JOHANSSON

the two proteins are structurally related (Bennich et al., 1969b). Furthermore, these results gave direct support both for the hypothesis of a unique imniunogolobulin as a carrier of reaginic activity and for a relationship between reaginic antibodies and elevated levels of IgE ( ND ) in allergic serum. In addition, a biologicostructural relationship to reaginic antibodies was indicated by a specific inhibition of the Prausnitz-Kiistner reaction given by serum ND and purified protein ND at high dilutions ( Stanworth et aZ., 1967). B. ISOLATION 1. Salt Precipitation The recovery of IgE( ND) and IgG from serum precipitated with sodium and ammonium sulfate as followed by the single radial immunodiffusion technique of Mancini et al. (1965). A quantitative recovery of ND protein was obtained by precipitation with 40% saturated ammonium sulfate ( p H 7 ) or with anhydrous sodium sulfate (18 gm./100 ml. serum) at pH 7.5 and 25°C. These results are in agreement with those reported for reagins by Augustin and Hayward (1960) and by Sehon (1960). At 35% saturated ammonium sulfate, about 75%of the IgG was recovered as compared to 30%for IgE( ND). By adding 16 gm. of sodium sulfate to 100 ml. serum, IgG is quantitatively recovered but only 60%of I g E ( N D ) . The solubility properties of purified E myeloma protein were in close agreement to that of the M component in serum. Isolation of the latter by precipitation with salt was not found to alter its biological, immunological, and physicochemical properties ( Bennich, 1968). 2. Electrophoresis Protein ND migrates in the fast y to slow ,8 region in agar immunoelectrophoresis at p H 8.6. Reagins have been similarly characterized, and K. Ishizaka et a2. (1966a)' described the purified fraction of antiragweed antibodies, yE-globulin, to have yl mobility. Figure 1 illustrates the distribution of IgE relative to the other immunoglobulins in two sera from nonallergic individuals-one lacking IgA and IgD-after starch block electrophoresis for 24 hours at p H 8.6.

3. Chromatography Fractionation of serum ND by column chromatography on diethylaminoethyl ( DEAE )-Sephadex A-50 indicated that the M component distributed as a single peak, which by means of gradient elution (trisHC1 gradient from 0.1 to 1M , pH 8.0) was separated from other

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H U M A N IMMUNOGLOBULIN E

1

I i I 1 I IDi

211011

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I 15 -

5-

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M

-d E

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5

10

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FIG.1. Electrophoretic distribution of immunoglobulins A, D, G, M, and E in two normal sera of which one (right-hand figure; sample G. S . ) was lacking IgA and IgU. Starch block electrophoresis ( 50 X 30 X 1 cni.), barbital buffer ( I = O . l ) , pH 8.6, 3 5 0 4 0 0 V., 100-110 mA., 20-24 hours, 4OC. Immunoglobulins A, D, G, and M were determined by single radial innnunodiffusion (Mancini et al., 1965) and IgE by radioimmunosorbent test. The concentrations of the immunoglobulins in the normal serum (left-hand figure) were A 1.1 mg./ml.; D 0.05 mg./ml.; G 10.5 mg./ml.; X I 0.49 mg./ml.; and E 510 ng./ml. In sample G. S.: A 0.01 mg./ml.; D 0.01 mg./mI.; G 12.8 mg./mI.; M 1.20 mg./mI.; and E 210 ng./rnI.

<

<

immunoglobulins except for fast IgG (Fig. 2 ) . Protein ND was recovered in fractions between 0.1 and 0.2 M concentrations of salt, and the yields from serum were approximately 90%. Similar results were obtained by chromatography of samples of ND protein previously isolated from serum or plasma ND by salt precipitation (Bennich, 1968). Fractionation of serum from healthy individuals performed under essentially the same experimental conditions gave an elution profile of IgE in close agreement with that found to be characteristic for ND protein (Johansson and Bennich, 1967b). Rechromatography of samples of purified protein ND gave no indications that the protein should be regarded as a particularly labile immunoglobulin, which has been suggested to be a characteristic of reagins (see review, Sehon and Gyenes, 1965). However, occasionally, in some serum samples an additional

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HANS BENNICH AND S. GUNNAR 0. JOHANSSON

2

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8 N W

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75

100

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FIG. 2. Fractionation of plasma ND (top) and salt precipitated E myeloma protein ND (bottom) on diethylaminoethyl-Sephadex A-50 (3.2 X 30 cm. ). Fractions of 4 ml. were collected for analysis: absorbancy 280 nm. ( 0 ) ; IgE( ND) (A)and IgG (0) determined by single radial immunodiffusion. (See Fig. 4 for experimental conditions. ) Broken line: tris-HC1 ( M X lo-').

component was found to be eluted with the trailing portion of ND protein. By agar immunoelectrophoresis at pH 8.6 of such contaminated ND fractions, two components having similar immunological characteristics could be resolved. The major component was indistinguishable from the ND protein, whereas the other component, which migrated

7

HUMAN IMMUNOGLOBULIN E

toward the cathode in some respects was similar to F(ab’j2 fragment produced by pepsin (see Section II1,B). Figure 3 shows that an additional IgE-reacting component could be detected also in an allergic serum containing reaginic antibodies to dog dandruff. In view of these findings and the frequently reported observations for a chromatographic heterogeneity of reaginic antibodies, a further characterization of the minor ND component was attempted. By rechromatography of a DEAE fraction of ND protein on carboxymethyl (CM)-Sephadex C-50, the component was eluted with the starting buffer (0.1 M tris-HC1, pH 8.0) followed by a major fraction of fast IgG, while protein ND was adsorbed until a gradient was applied (Bennich, 1970). Fractionation of the first CM peak by gel filtration on

W

n

2 1 (3

25

50

75

400

(25

t50

475

TUBE NUMBER

FIG.3. Fractionation on diethylaminoethyl-Sephadex A-50 ( 3 . 2 X 30 cm. ) of serum (120 ml.) from a patient hypersensitive to dog dandruff. Quantitative determination of immunoglobrllin was made by radioimniunosorbent test (see Section I V ) for IRE, and by single d i a l innnunodiffusion for IgA, D, G , and hl. (See Fig. 4 for experimental conditions. )

8

HANS BENNICH AND S. GUNNAR 0 . JOHANSSON

Sephadex G-200 revealed that the minor component was eluted closely after the void fraction, thus indicating that its size was apparently larger than that of native protein ND. By ultracentrifugal analyses using the sedimentation equilibrium techniques of Yphantis (1964), a molecular weight of about 250,000 (p = 0.715) was obtained for the isolated component, In contrast to the results obtained from gel filtration and ultracentrifugation experiments, starch gel analysis in acid urea buffer indicated that the unidentified protein could dissociate into a smaller component, similar in size to F( ab’)? fragment. Furthermore, by starch gel analysis of the reduced component, it was observed that in addition to light chains and a fragment resembling Fd’, a third fragment was also released. In conclusion, these results suggest that the minor component isolated from plasma ND represents a noncovalently linked dimcr of a fragment of protein ND presumably formcd by an asymmetrical cleavage of the L chains. In this context, it is interesting to note the findings by Girard (1967) of two types of reaginic antibodies in serum from a patient hypersensitive to penicillin, one corresponding to IgM and the other to IgA as regards their elution volumes on Sephadex G-200. A similar polydispersity was subsequently observed for reaginic antibodies to ragweed as reported by K. Ishizaka and Ishizaka (1968a). A careful rcexaniination of high molecular weight reaginic antibodies with regard to their skin-fixing activity would be of interest since the 250,000-molecular weight fragment of ND protein, in contrast to the native protein, was apparently devoid of skin-fixing properties as concluded from its inability to inhibit the passive cutaneous anaphylaxis (PCA) reaction in monkeys (Bennich et al., 1971). It is evident that a combination of criteria such as allergen-binding activity, skin-fixing activity, antigenic characteristics and molecular size distribution must be fulfilled before any statement can be made regardng the presence of different types of reaginic antibodies in a biological specimen. Thus, from this point of vicw, the additional IgE peak shown in Fig. 3 cannot be regarded as an intact reaginic antibody. The procedures used for the isolation of protein ND are summarized in Fig. 4. The conccntration of fast IgG, amounting to 1-2% in IgE( ND) containing DEAE fractions, was decreased by fractionation on Sephadex G-150 ( 3 cycles on a 90-cm. column) to less than 0.18. Preparations of IgE ( ND ) obtained by these procedures were apparently homogeneous as detcrmincd by ininiunoelectrophoresis, starch gel analysis in acid urea buffer, and by ultracentrifugation analysis. The yields from serum wcre approximatcly 80%. The procedures outlined in Fig. 4 can also be applied for the isola-

H U M A N IMMUNOGLOBULIN E

9

S e i u m I M-comp ND, 10-15mg p e r n

18% Na2S04. 25"C,60 min or

40°/o(NH4)2S04,~H7.250C,60m i n I

I

P P t i n 0.1 M T r i s - H C l , p H 8 O : 2 O 0 C

I

DEAE-Sephadex A 5 0 g r a d i e n t 01 t o 1 M Tris-HCl,pH80,20°C

01-02 M

----------+

I

C M - S e p h a d e x C-50 g r a d i e n t 01 t o 1 M T r i s - H C I I PH 8 0 . 2OoC

i

01 M

I

S e p h a d e x G-150 rcc.3cycles 300cm

1 8s

l g E(ND)

4

01-02 M

I

S e p h a d e x G-150

I

1

11 S"lg E"( N D )

1 8s lg EIND)

FIG. 4. Procedure for isolation of IgE (and an 11S fragment) from serum. DEAE-diethylaminoethyl; Chl-carboxymethyl; rcc-recycling chroniatography.

tion of IgE from nonmyeloma serum (Bennich and Johansson, 1971). Since the concentration of IgE in most samples of interest will be in the order of micrograms per milliliter, particular attention should be given to the design and performance of chromatographic experiments. To eliminate one possible source of erroncous results, fractionation of samples collected from different individuals or at different times from one individual should be made on separate ion-exchange columns using standardized experimental conditions. Furthermore, stepwise elution techniques should be avoided and replaced by gradient elution chromatography, whenever the distribution of IgE is the subject of investigation. Finally, evaluation of IgE in different chromatographic fractions should preferably be made by sensitive immunological procedures rather than by detcrnmination of biological activity.

4 . Isolation of ZgE by Means of Zmmunoadsorbents Gyenes and Sehon ( 1960) demonstrated that skin-sensitizing and blocking antibodies to ragweed allergens were effectively removed from serum by absorption with a polystyrene-allergen conjugate. Attempts

10

HANS BENNICH AND S. CUNNAR 0. JOHANSSON

to desorb specifically bound antibodies with a variety of solvents from pH 3 to 11 indicated that skin-sensitizing and blocking activity could be recovered by elution with dilute hydrochloric acid at pH 3; however, the yields were extremely small (less than 2%of the original activity). The use of an IgE-specific adsorbent for isolation of reaginic antibodies to various allergens was recently reported by Ito et al. (1969). Immunoadsorbents were prepared by copolymerization of a rabbit antiserum raised against IgE( PS), using the ethyl chloroformate procedure of Avrameas and Ternynck ( 1967a). Determination of the PrausnitzKustner (P-K) titers in various human reaginic sera, before and after adsorption with such preparations of insolubilized anti-IgE, demonstrated that IgE antibodies to ragweed, grass pollen, house dust, and penicillin allergens were removed from solution. The presence of detectable amounts of IgG, A, M, and D in supernatants was demonstrated by gel diffusion analysis. Attempts to elute reaginic antibodies to grass pollen were also made. Elution of different preparations of the immunoadsorbent, using 2 M sodium chloride pH 7.2, 2 hl magnesium chloride pH 7.5, and 0.2 M glycine-HC1 pH 2.2, yielded a recovery of P-K activity of 0.7, 2, and 8%on the basis of the titer in unabsorbed serum. A simple procedure for chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides has been described by A x h et al. (1967). By this technique both Sephadex and Sepharose can be activated to yield efficient immunoadsorbents for analytical and preparative purposes (Porath et al., 1967). The mechanism for the chemical reaction is not fully elucidated as yet. However, it is assumed that cyanates and iminocarbonic acid esters form the reactive sites of the activated polysaccharide. Specific anti-IgE antibodies have also been coupled to cyanogen bromide-activated Sepharose to obtain an immunoadsorbent suitable for the isolation of IgE from nonmyeloma serum (Bennich, 1969; Bennich and Johansson, 1971). Sepharose, a bead polymerizate of agarose, is commercially available in three concentrations of polymer : types 2B, 4B, and 6B. The results discussed in this paper will refer to studies using Sepharose 2B. The activation of polymer and subsequent coupling with antibody (or antigen) is outlined in Fig. 5. The polysaccharide is activated with cyanogen bromide (2.5250 mg./ml.) at room temperature under strong alkaline conditions (pH 10-11) by addition of a molar solution of sodium hydroxide to constant pH. To get reproducible results as regards the degree of activation, the amount of alkali should be determined rather than the time for the activation process. In the presence of an excess of cyanogen bromide and at a given pH, the degree of activation will become a function of the amount

11

H U M A N IMMUNOGLOBULIN E PRETREATMENT-ACTIVATION-COU 2 0 60g'polymer 2 M Pyrtdtne

4

Water t o pH 7

4

2 M Acetic acid

Ig'polyrner

P L I N G in

activated polymer in

150rnl CNBr 2 5 %

p r o t e i n s o l n ,1 5 rng p e r r n l

N N a O H ( 5 15 m l ) t o

pH6 9

c o n s t p H 10 5+0 2 2OoC

6 t o 4 8 h r s 4'C

I i c e c o l d NaHCO,.O 2 M . 5 ~ 1

N saline t o zero UV

c

4 4 4 4

deactivate unreacted sites check s t a b i l i t y e q u i l i b r a t e pH 7 s t o r e at 4

- 6OC

4 1 0 0 - 400 m g p r o t e i n p e r g' p o l y m e r (4 t o 6 hours)

(1 to 2 hours)

(3 t o 6 d a y s )

'dry weight

FIG. 5. Scheme for cyanogen bromide activation of agarose (Sepharose 2 B ) and the subsequent coupling with protein to obtain an immunoadsorbent.

of sodium hydroxide added. When a given aniount of alkali has been consumed at constant pH, the activation is interrupted by suspending the particles in several volumes of ice-cold sodiuni hydrogen carbonate (0.1M , pH 9 ) . The activated support is then ready for reaction with protein. The activity will decrease slowly upon standing, in particular at low pH. About 60%activity was found to be lost in a preparation kept 5 days in the cold at pH 7.5. Activation and coupling reactions should preferably be made the same day. However, the activation is a rapid proccdure and several batches of activated polynier can be prepared within a few hours. The coupling is simply made by mixing protein with activated support by gentle stirring for 6 to 48 hours in the cold. The optimal conditions may vary slightly depending upon the chemical characteristics of the particular compound to be coupled, but in general the yield will increase the higher the pH. For immunoglobulins, the coupling reaction i s conveniently made in 0.1 A l sodium hydrogen carbonate for 12 to 24 hours in the cold. After this step it is very iniportant that unbound protein is thoroughly removed. In addition unreacted sites on the support must be deactivated. Since the activity decreases upon standing at low pH, presumably by hydrolytic conversion

12

HANS BENNICH AND S . GUNNAR 0. JOHANSSON

of reactive sites into stable nonreactive carbaminic acid esters, deactivation is made by suspending the particles in several volumes of sodium acetate (0.2h1, pH 4) for at least 48 hours in the cold. Furthermore, it is recommended that the inimunoadsorbent be subjected to the same conditions of buffer, pH, temperature, etc., as used for the subsequent elution of specifically absorbed protein. As an example, the isolation of anti-IgE antibodies from sheep serum and isolation of IgE from nonmyeloma serum will be described. Twenty milliliters of a sheep antiserum specific to IgE( ND) was stirred with an Sepharose-IgE( ND) conjugate (precycled with 6 M urea, pH 5; 0.2 A1 tris-HC1, pH 8; and 0.15 hl saline) for 2 hours at 4°C. No antibodies were detected in the supernatant by gel diffusion analysis. After removal of unbound protein, the immunoadsorbent was “dried by suction filtration ( Millipore filter), Elution of bound antibodies was made by resuspending the filter cake in 0.1 M acetic acid (14 ml.) for 2 hours at 4°C. The supernatant (13.5 ml.) and one washing with acetic acid (18 ml.) were collected by gentle suction filtration, immediately neutralized with base, and dialyzed against 0.01 M tris-HC1, pH 8, containing 0.15 M saline. The protein concentration (UV basis) was about 1.6 mg./ml. in combined eluates after dialysis (33 ml.). Gel diffusion analysis revealed a reaction of equivalence at about 0.6 mg. of IgE( ND) per milliliter for eluted antibodies as compared to about 1.2 mg./ml. for unabsorbed antiserum. Starch gel analysis of isolated antibodies at a concentration of about 10 mg./ml. indicated a single band with a mobility similar to that of nonantibody sheep y-globulin included as reference. About 20 mg. of such purified anti-IgE(ND) antibodies were coupled to activated Sepharose (40 ml. bed volume) as described above. The final preparation of Sepharose-anti-IgE ( ND )-antibody conjugate was equally distributed into four small columns (0.9 x 15 c m . ) - o n e was used for control and the others, for isolation of IgE from serum. The capacity for the immunoadsorbent column to remove autologous antigen from solution was studied, as shown in Fig. 6. About 2 mg. of IgE(ND) could be absorbed into the column ( 9 ml.). Desorption of bound protein attempted by elution with glycine-HC1 (0.1 M, pH 3.5) was achieved only in the presence of saline (0.15 M ) . The yield of protcin was about 90%,estimated on UV basis, and about 70%, estimated by immunological analysis. An additional small amount of protein was recovered by further elution with 5 M potassium iodide at pH 7.5 (Avranieas and Ternynck, 196%). The results indicated that the capacity of the immunoadsorbent was not significantly affected by the procedures used for desorption. In addition it was found that elution

13

HUMAN IMhlUNOGLOBULIN E

1

"\

/" 12

24

30 40 E f t l u e n t bed v o l u m e

00

72

FIG. 6. Sorption and desorption characteristics of an agarose-sheep antiIgE( ND) serum conjugate (0.9 X 15 cm.) tested with IgE( ND). Two milliliters of protein ( 1 nig./ml.) in 0.1 M tris-HCI, pH 8.0, was applied at A followed by buffer (bottom). ( B ) Elution with 0.1 M glycine-HCI, pH 3.5; ( C ) elution with 0.2 M NaCI. After washing with 5 h i potassium iodide (trace of protein eluted) and equilibration with buffer, the experiment was repeated as shown (top). ( D ) Elution with 3 . 5 M sodium thiocyanate, pH 6.8. Note the very sharp displacement Unabsorbed protein; ( 0 )eluted protein by desorption; ( 0) effluent obtained. ( 0) pH; ( V ) efluent conductivity; and ( A ) sodium thiocyanate.

with 3.5 M sodium thiocyanate (Dandliker and de Saussure, 1968) at pH 6.8 yielded an amount of recovered IgE( ND) comparable to that obtained by acid dissociation of the complex. The experiences gained from these experiments as regards capacity and desorption conditions were taken into consideration for the isolation of IgE from serum as outlined below. A volume of recalcified reaginic plasma, corresponding to 1 to 2.5 mg. of IgE, was percolated through a column (0.9 X 15 cm.) of im-

14

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

munoadsorbent at a flow rate of 30 ml./hour. An equal volume of absorbed plasma was collected for analysis, after which the column was washed free of protein with saline (10-15 bed volumes). Specifically bound protein was dissociated by elution with sodium thiocyanate (5 bed volumes, in 3.5 M phosphate, pH 6.8) at a flow rate of 15 ml./hour. Fractions ( 1 ml. ) were collected for protein analysis. Protein-containing fractions were pooled, dialyzed (0.1 M tris-HC1, pH 8, 4OC), and finally concentrated by pressure dialysis. The IgE concentration in unabsorbed plasma, absorbed plasma, and in concentrated eluates was determined by RIST as described in Section IV. Absorption-desorption studies of three reaginic sera on separate columns of immunoadsorbent-containing anti-IgE ( ND ) antibodies gave the following results. The average amount of bound IgE in five experiments was about 1400 pg. for sera A and B and about 790 pg. for serum C. However, the average of absolute recovery of protein was 350, 260, and 200 pg. or 27, 19, and 25% of bound protein. An explanation of this apparent inconsistency in results was obtained by examination of the IgE concentration in effluents collected during washing with saline, Between 30 and 50%of the IgE absorbed from serum was found to be eluted before any dissociation was attempted. This indicates that the actual amount of IgE specifically absorbed cannot be estimated on basis of the concentration in serum before and after absorption. Frontal analysis of the three sera as shown in Fig. 7 revealed that the capacity of the anti-IgE( ND ) system to bind nonmyeloma IgE was only about 20%of that found for the autologous protein. However, the recovery of IgE from two columns (sera A and C ) was of the same order of magnitude (70-808) as that found for IgE( ND). For the third column (serum B), the recovery was significantly lower or about 40%. The latter also gave a different concentration profile, indicating a difference in affinity for the immunoadsorbent antibodies. Immunization experiments revealed that none of the preparations were immunologically homogeneous. The presence of trace amounts of proteins other than IgE was detected also in a preparation isolated by acid elution from a 25ml. column of another preparation of Sepharose-anti-IgE ( N D ) , Quantitative analysis for immunoglobulin by single radial diffusion ( Mancini et al., 1965) gave the following results: IgG 25.1; IgA 2.5, IgM 5.6, IgD not determined and IgE 60.5 mg. %. By gel diffusion analysis, only IgG could be detected in addition to IgE. However disappointing these figures might seem, they, nevertheless, illustrate the potential efficiency of immunoadsorbents. Thus, by a single filtration step, it was possible to change the concentration ratio of IgE to IgG from 1:20OO as found in the serum to 2.4:l in the eluate.

HUMAN IMMUNOGLOBULIN E I

I

I

I

15 I

c

0

30

60

90

120

/'

150

Effluent, ml

FIG.7. Frontal analyses of sera A, B, and C. The amount of IgE bound by the inmunoadsorbent was 360 pg. in A, 476 JLg. in B, and 336 p g . in C. Correspondingly relative amount recovered by elution (NaSCN) was about 69% ( A ) , 41% ( B ) , and 79% ( C ) .

C. CHEMICAL AND PHYSICAL CHARACTERISTICS

1. Ultracentrifugal Analysis Several preparations of IgE ( ND ) isolated from samples collected from 1965 to 1967 have been subjected to ultracentrifugal analyses. The protein sedimented as a monomeric component, and for three preparations studied at the same time the sedimentation constant ( s : ~ , ~ ) was 8.20 S (Bennich, 1968). This value is in agreement with that of 7.92 S previously reported for another preparation ( Johansson and Bennich, 1967a). The diffusion constant ( D & , w )was found to be 3.71 x lo-' cm'/second calculated from results of three preparations (Bennich, 1968). The molecular weight of IgE( ND) was determined as 196,000 (Johansson and Bennich, 1967a) from measurements at different protein concentrations by the Archibald method, described by Ehrenberg (1957), and using a partial specific volume of 0.713. When these studies were repeated on three preparations at concentrations of 1.8, 5.4, and 8.8 mg./

16

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

ml., respectively, the molecular weight was calculated as 209,000 ( B = 0.713). However, this value is likely to be much too high, since calculations from sedimentation and diffusion data gave a value of 193,000 for p = 0.713. Furthermore, recent studies of another preparation (Dorrington and Bennich, 1971) has given a molecular weight for IgE(ND) of 188,100 3000 based on sixteen determinations using the sedimentation equilibrium techniques of Yphantis (1964). Though the results of ultracentrifugation studies of reaginic sera appear controversial, sedimentation constants of about 8 S have been reported by several authors (Rockey and Kunkel, 1962; Anderson and Vannier, 1964; K. Ishizaka et ul., 1966b). Ultracentrifugation studies of IgE( ND) in a sucrose gradient and IgE in a reaginic serum also indicated that they have similar sedimentation characteristics in a sucrose gradient ( Bennich and Johansson, 1968). Physicochemical characteristics of IgE ( ND ) and its constituent polypeptide chains are summarized in Table I. TABLE I PHYSICOCHEMICAL CHARACTERISTICS OF IMMUNOGLOBULIN E(ND) AND ITS CONSTITUENT POLYPEPTIDE CHAINS

Immunoglobulin IgE(ND)

c

Chain Chain ND

Molecular weight (method) 196,000 (Archiba1d)a 193,000 (sed. and diff . ) b 188,100 3000 (Yphantisy 72,500 & 2400 (Yphantisp 22, 600d

Sedimentation constant,

Diffusion constant, D2OoBW

7.92" 8.20' -

3.71b

0 SO0.W

c

-

-

-

One preparation; P = 0.713; from Johansson and Bennich (1967). Three preparations; P = 0.713; from Bennich (1968). c One preparation (16 determinations), = 0.713; from Dorrington and Bennich (1971 . .). Calculated on basis of amino acid data, see Table 11. a

2. Amino Acid and Carbohydrate Analysis The amino acid composition of IgE(ND) and its constituent polypeptide chains is given in Table 11. The content of tryptophan was calculated from the tyrosine-tryptophan molar ratio determined by spectrophotometry, as described by Edelhoch (19671, and the value for tyrosine was obtained by amino acid analysis. Carbohydrate analyses were made on samples simultaneously taken for amino acid analysis using classic colorimetric methods. The total carbohydrate content calculated from these determinations was 11.7%.

HUMAN IMMUNOGLOBULIN E

17

TABLE I1 AMINOACID .4ND C.%RIIOHYDR:tTE COMPOSITION O F I M M U N O G M R U L I N E CONSTITUENT POLYPEPTIDE CHAINS~

AND

Native protein

Substance Tryptophan Lysine His tidine Arginine Aspartic acid Th reon ine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine N-Acetylglucosamine* Galactose Mannose Fucose N-Acetylneuraminic acid

Residues per 190,000 gm 37.5 62.3 29.9 68.6 111.2 169.5 186.1 131.5 102.6 104.3 104.9 40.0 107.2 16.9 38.6 102.2 54.0 50.6 36 36 17 4 18

Heavy chain (c) Light chain (A) residues per residues per moles 62,000 gm 22,600 gm

yo

2.5 4.1 2.0 4.5 7.3 11.2 12.3 8.7 6.8 6.9 7.0 2.6 7.1 1.1 2.5 6.7 3.6 3.3

-

-

17.3 19.4 12.5 30.3 43.6 68.8 58.4 47.2 35.4 36.6 35.7 15.2 40.1 7.6 15.8 38.4 16.8 21.4 -

1.9 11.6 2.9 5.1 13.3 19.1 35.1 20.6 14.8 17.2 18.4 4.8 15.1 1.0 4.1 14.1 11.0 5.1 -

-

-

-

-

Data from Bennich (1968).

* Bennich and Clamp (1971). Slightly different results were obtained by gas liquid chromatography of trimethyIsily1 (TMS ) derivatives (Bennich and Clamp, 1971). The total carbohydrate content obtained by this method was 11.%. The extinction coefficient ( E i z i::) for IgE( ND) was 15.33, calculated from the content and molar extinction coefficients (280 nni, pH 7 ) of cysteine, tryptophan, and tyrosine as described by Edelhoch ( 1967).

3. Reduction Amino acid data (sce Table 11) indicate that IgE(ND) contains about forty half-cystinyl residues per molecule of 190,000. Reduction experiments revealed that about 40% or sixteen of these residues were

18

HANS BENNICH AND S . GUNNAR 0. JOHANSSON

available in the native protein for reaction with 2-mercaptoethanol( 2ME) or dithiothreitol (DTT), as determined from the recovery of CM-cysteine following alkylation with iodoacetamide. Using 2-ME (0.1 M final concentration), about 10 moles of CM-cysteine were recovered per mole of protein within less than 30 minutes at 2OoC, which is similar to the results obtained for human IgG included as a control. However, by prolonging the time of reduction or when a higher concentration of mercaptan was used, a significant increase in the recovery of CM-cysteine was observed only by IgE( ND). Experiments performed with D7T revealed that this reagent was 23 times more e5cient than 2-ME at 0.01 M concentration, whereas no significant differences in results were obtained in 0.3 M solutions. These results indicated that the number of disulfide bonds found accessible for reductive cleavage under mild conditions is dependent upon conformational features of the protein and independent of the reducing agent per se. Starch gel analysis of reduced alkylated IgE(ND) is illustrated in Fig. 8. Complete reduction yielded two bands of distinct mobility, indicating the presence of two kinds of polypeptide chains in the IgE( ND), which would be in agreement with the findings for other

FIG. 8. Starch gel analysis of reduced alkylated immunoglobulin E(ND) and human G . IgE(ND): unreduced (l), reduced alkylated in aqueous buffer ( 2 ) , and reduced alkylated in 8 M urea buffer ( 4 ) . IgG: reduced alkylated in aqueous buffer ( 3 ) . Electrophoresis in 8 M urea-formate pH 3. Cathode on top.

HUMAN IhfMUNOGLOBULIN E

19

immunoglobulins. The yields of light chains, recovered by acid gel filtration of completely or partially reduced and alkylated samples, were about 20% (assuming equal extinction coefficients for both components). These results are in close agreement with the yields expected (24%)when assuming two light chains per molecule of protein ND. Isolated light chains were in all respects very similar to a BenceJones protein of Type L isoIated from the serum and urine of patient ND. The amino acid composition of these proteins is given in Table 11. Carbohydrate was detected neither in isolated X chain nor in BenceJones protein ( N D ) . The minimum molecular weight of the light ( A ) chain of IgE( ND) was calculated to be 22,600, which was found to correspond to a polypeptide of 214 amino acid residues of which 5 represent half-cystine and 1 methionine. Amino acid analysis on light chains isolated from two saniples of IgE( ND) containing 9.4 and 16.4 moles CM-cysteine per mole of protein, revealed the presence of 0.91.1 moles of CM-cysteine per mole of 22,600. Since no light chains could be observed to be released from native IgE ( ND ) when dissolved in denaturating media, it can be concluded that a single disulfide bond forms the linkage between a A chain and an c chain. Furthermore, this interchain bond must be readily accessible to reductive cleavage since similar yields (21.3 and 21.5%)of light chains were recovered from the two samples of reduced IgE( ND). The molecular weight of an c chain ND was calculated to be about 72,200. U1tracentrifugation studies in 6 M guanidine of completely reduced, alkylated, €-chain monomers, using the sedimentation techniques of Yphantis (1964), gave a molecular weight of 72,500 2 2400 ( Dorrington and Bennich, 1971). By taking into account the contribution of the carbohydrate moiety, the molecular weight of the 6 polypeptide was estimated to be about 61,000, which is very similar to the size of the 1-1 polypeptide (about 58,000) as calculated from the data given by Miller and Metzger (1965). Ill. Properties of Antigenically and Biologically Active Structural Regions of Immunoglobulin E

A. PROPERTIES OF c CHAINS Studies of the effect of partial reduction and alkylation on the antigenicity of I g E ( N D ) (Bennich, 1968) revealed that cleavage of about five of the eight disulfide bonds accessible for reaction with mercaptan under mild conditions does not significantly alter the antigenic properties, as measured by single radial immunodiffusion (Mancini et al., 1965) and by RIST (see Section I V ) . In accordance with these results

20

HANS BENNICH AND S . GUNNAH 0. JOHANSSON

were the findings that z chains isolated from such reduced alkylated samples of IgE( ND) are precipitated by antisera specific to the FcE fragment to give a reaction of identity with untreated IgE(ND). By cleavage, also of the remaining three disulfide bonds found to be susceptible to reduction in aqueous media at pH 8, the antigenic characteristics of IgE ( ND) become drastically modified. Thus after reduction of about eight disulfide bonds, the decrease in antigenicity-expressed as relative concentration of protein-amounted to about 85%as determined by RIST and about 30% as determined by single radial diffusion. The difference in results reflects principal differences of the quantitative methods used. Since RIST, as it was performed in these experiments, is based on the competitive inhibition between a protein in a sample and an analogous radiolabeled reference protein for their binding to specific antibodies coupled to an insoluble support, alterations of the conformational integrity of antigenic regions in the protein to be determined would preferably favor a reaction between antibody and reference protein. In single radial immunodiffusion, on the other hand, the reaction between antigen and antibody is more straightforward, and, since it is generally allowed to proceed until equilibrium is reached, weakened antigenic structures caused by discrete conformational changes would hardly be detected. In addition to alterations in antigenic properties, reduction of IgE( ND) will also induce changes of its characteristic tissue-binding activity ( Bennich et al., 1969a). From PCA experiments in monkeys, using a grass pollen reaginic serum for sensitization, it was concluded that at least two of the eight accessible disulfide bonds within the C-terminal halfs of the E chains are critical for the tissue-binding activity of IgE(ND) (Stanworth et al., 1970).

B. PROPERTIES OF ENZYMATIC FRAGMENTS 1. Papain Fragments Under Porter’s conditions ( 1959), papain produces a complex mixture of fragments, which will change in composition with time as shown in Fig. 9. Five major fragments were isolated for further studies, and their immunological and physicochemical properties are summarized in Table 111. By 2 hours of digestion, a 7s fragment and light chain fragments ( C X ) were produced in addition to Fc and two kinds of Fab fragments, one containing carbohydrate. By further digestion, there was an increased production only of Fc and CXfragments, with maximum yields obtained after about 3 to 4 hours of digestion. By prolonged digestion (16 hours or longer), all fragments but one are recovered as small inactive glycopeptides and peptides. This apparently resistant

21

HUMAN IMMUNOGLOBULIN E

1.5

I

2 hrs

L

5 x

g 1.0 0 W N

V > 2

4 0.5

m a 0

v)

m

a 25

50

75

100

125

150

175

150

175

FRACTION NUMBER 0 . 6 ml vol.) 2.0

u E

I. 5

la

f 0

%

1.0

> 0 2

a

m

a

0

v)

0.5

m

a

25

50

75

100

125

FRACTION NUMBER 0 . 6 ml wol.)

FIG. 9. Gel filtration on Sephadex G-150 of serial samples of IgE(ND) undergoing papain digestion. Ultraviolet absorption; 280 nm. (line of black circles); hexose (line of open circles). The peaks were identified as IgE ( A ) , 7s fragment ( A ) , Fc (O), Fab, containing carbohydrate ( V ), Fab ( V), Fc" ( @ ) , and A-chain fragment, Ch ( 0).

fragment, designated Fc:, was found to be antigenically related to Fc, as will be discussed further below. Fragment Fc, was defined as the fragment in the digest that does not contain light chains but carries antigenic determinants, characteristic for the IgE class. In immunoelectrophoresis, Fc, occupies a position just anodal relative to intact IgE( ND), and by gel filtration on Sephadex G-150the fragment appears similar in size to that of F( ab') 2 y . Ultracentrifugation analysis indicated that Fc, has a molecular weight of about 98,000

TABLE 111 IMMUNOLOGICAL AND PHYSICOCHEMICAL PROPERTIES OF ENZYMATIC FRAGMENTS OF IMMUNOGLOBULIN E (ND)a ~

~~~~~

Tryptic fragm. Properties Molecular weightb Sedimentation const! ( s : , , ~ Carbohydrate

Papain fragm.

Peptic fragm.

Native IgE (ND)

75

Fc

Fab

Fab

Fc"

CX

7 s

F(ab)2 (t)

190,000

n.d.

95,000

50,000

40,000

38,000

25,500

n.d.

103,000 40,000 140,000 30,000

8.2S

7S

5.1 S

n.d.

n.d.

n.d.

n.d.

7 s

n.d.

6.75

6.75

n.d.

11.7

+

18.5

+

-

20-23

-

+

8

12-14

8.5

2.4

~

Fc"(t)

F(ab')2

pFc

(%)

Antigenic determinants" Epsilon, D.0 DJ D2 Light chain A Inhibitory P-K P-Kk activityd on (<5) P-K or reagin- PCA induced PCA (1-5) (baboons)(pg.)

+ + ++

+

+

+ + ++

F 3

3.

3 v)

-

+ +-

+

+

+

(*)

-

+

+

-

-

-

+-

+ + (*) +

-

+

+ ++

P-K P-KP-K - P-K - P-K (<5) PCA+ PCAPCAPCAPCAP C A k PCA(<2) (>loo) (>loo) (>loo) (>loo) (10-20) (>40)

From Bennich and Johansson (1971); n.d. = not determined. Calculated on basis of r' = 0.713. D.0-idiotypic determinants; D.1 and D.2-determinants in the Fc portion of From Stanworth et al. (1968, 1970).

e

chains.

+-

+ +-

-

-

-

(+)

-

P-K n.d.

PCA(>28)

2

i 9

L(

PCA(>40)

F

HUMAN IMMUNOGLOBULIN E

23

(B = 0.72) and a sedimentation constant (s:,) of 5.1 S. Maximum yields of Fc, (about 30%)were obtained by 3 hours of digestion at p H 7 and 37OC using an enzyme to substrate ratio of 1:120 in the presence of cysteine (10 mM). Since the fragment represents an intermediary product, which gradually is cleaved to produce Fc: and smaller fragments including characteristic glycopeptides, isolated Fc, shows a considerable degree of microheterogeneity. Carbohydrate analysis indicates the presence of 14 to 17%carbohydrate, and starch gel analysis of reduced samples indicates the presence of one major and two minor components, which all three have similar mobilities. According to the definition, Fc, is antigenically indistinguishable from IgE ( ND ) using an antiserum specific to the latter. Furthermore, Fc, is the only fragment produced by papain that inhibits the P-K reaction in human skin (Stanworth et al., 1968) and PCA reaction in monkeys (Stanworth et al., 1970). Fragment Fc” represents an enzymatically resistant portion of the amino terminal part of Fc. It gives strong precipitin reactions with antisera specific for Fe, but is antigenically deficient as compared to the latter fragment; Fc” is a potent immunogen in rabbits. Antiserum to Fc” gives strong precipitin reactions with Fc, and F ( ab’),, distinct but slowly developing reactions with IgE( ND), but no visible reactions with partially reduced, alkylated E chains. The carbohydrate content of Fee" has been estimated to be about 20%and sedimentation equilibrium studies indicate that the fragment has a molecular weight of about 38,000 (7= 0.713; protein concentration = 0.5 mg./ml. ) . In agar immunoelectrophoresis (pH 8.6), Fc; is easily identified by its very fast anodal migration. In addition to Fc and Fc” fragments, two Fab fragments have been identified which differ in carbohydrate content. However, the yields of these fragments are very low due to their rapid breakdown into smaller fragments. The A chain of E myeloma ND appears to be very susceptible to attack by papain. The material in the CA peak (Fig. 9) reacted immunologically like A chain. However, both gel filtration data and starch gel analysis indicated that it constituted the constant portion of A chain; these results are in agreement with those obtained by enzymatic studies on Bence-Jones protein ND ( BerggBrd, 1970). 2. Tryptic Fragments

Trypsin splits IgE( ND) into a 7 s fragment, which by prolonged digestion yields F( ab),( t ) and Fc”(t ) and smaller peptides, but no Fc( t ). The chemical and imniunological properties of tryptic fragments are summarized in Fig. 10 and Table 111. By short-time digestion (less

24

> 0

C

m

n L

0

In

n

a

E l u t i o n v o l u m e , Ve : Vt

B

5

z

0

3

C

a 9 L

0 y1

n

a

1

50

150

250

3 50

Fraction ( 4 m l )

FIG. 10. Top: gel filtration on Sephadex G-150 of a tryptic digest (1 6 hours) of I g E ( N D ) . The peaks were identified as IgE ( A ) , 7 s fragment ( B ) , and F( ab)?(t ) ( C ) . Bottom: chromatography (diethylaminoethyl-Sephadex A-50) of a tryptic digest of IgE( ND). Peaks identified as shown in top figure. Peak D contained in addition to trypsin inhibitor, also Fc”( t).

HUMAN IMMUNOGLOBULIN E

25

than 60 minutes), a 7 S fragment is the main product, which is essentially indistinguishable from the F( ab’), fragment produced by pepsin, However, analysis of reduced samples of 7 s fragment indicates a difference as regards constituent heavy-chain fragments. The tryptic 7 s fragment was found to inhibit PCA reactions in monkey skin. However, these findings must be considered with caution since the inhibitory activity appeared weak on a molar basis and contamination with intact IgE( ND)-less than 5%contamination in purified 7 S preparations would be sufficient-could not be excluded. By further digestion, a smaller but immunologically related fragment is produced. The molecular weight of this fragment, designated F ( ab) ( t ), is about 103,000 ( = 0.713; protein concentration 0.2 mg./ml. ) . The F( ab),( t ) fragment is antigenically deficient as compared to Fc, produced by papain and lacks PCA inhibitory activity. By prolonged digestion a resistant fragment of about 40,000 molecular weight is produced in increasing yields which was designated Fc”(t) since it is similar to papain Fc”. 3. Peptic Fragments

The physiochemical and immunological properties of peptic fragments are shown in Fig. 11 and Table 111. Digestion of IgE(ND) by pepsin at pH 4.5 produces two fragments, both rich in carbohydrate. The inajor fragment, carrying both h antigenic sites and some of the antigenic determinants characteristic for c chains, was assumed to represent F( ab’ ) fragment ( Bennich and Johansson, 1967). However, the yield was surprisingly high-about 75% as calculated on UV basisand the carbohydrate content represented about two-thirds of that of IgE ( ND). The remaining one-third of carbohydrate was recovered together with a minor fragment, designated pFc, which constituted about 15%of the native protein. About 10%of the digest was recovered as small immunologically inactive peptide fragments completely devoid of any carbohydrate. Ultracentrifugal analysis of F( ab’ ) fragment indicated a molecular weight of about 140,000 + 4000 and a sedimentation constant ( ) of 6.7 S . After reduction and alkylation the fragment dissociates in two components, which were isolated by gel filtration in acid buffer. The minor component was identified as X chains and, accordingly, the major, carbohydrate-containing, component was assumed to constitute F d fragment. The latter has a molecular weight of about 45,000 as found by sedimentation equilibrium studies in 6 M guanidine of completely reduced and alkylated fragments ( Dorrington and Bennich, 1971). Partially reduced alkylated F( ab‘)? fragments dissociate at neutral pH to give Fab’ which

I

5

3 > 0 C

m

n L

0 U

n

U

1

E I u t i o n v o l urn 8 , V, : Vt

5

>

0

3

C

m 0 L

0 U

n 4 -I

1

Fraction ( 4 m l )

FIG.11. Top: gel filtration on Sephadex G-150of a peptic digest (20 minutes) of IgE(ND). The peaks were identified as F(ab'): ( A ) and pFc ( B ) . Immunoglobulin E ( N D ) is eluted by a volume of 0.45 V t . Bottom: chromatography (diethylaminoethyl-Sephadex A-50) of a peptic digest ( 8 hours) of IgE(ND). Peaks identified as in top figure.

HUMAN IMMUNOGLOBULIN E

27

indicates that inter-heavy-chain disulfides have cleaved. Fragment F ( ab’) comes off Sephadex G-150 columns in a volume fraction similar to that characteristic for human IgG. By chromatography on DEAESephadex A-50, the fragment is recovered in fractions of 0.2 to 0.3 M tris-HC1, pH S.0 (see Fig. 11).In immunoelectroplioresis (agar pH &6 ), F(ab’), is located on the cathodal side of IgE( N D ) similar to Fab or Fab’ fragments produced by other proteolytic enzymes and similar also to the behavior of 11 S IgE fragments isolated from serum (see Section I1,B). Fragment F( ab’) is precipitated by antiserum specific for Fc ~, it lacks, however, some of the characteristic antigenic sites common to Fc and IgE( ND), but will give a reaction of identity with Fc” [and F c ” ( t ) ] using antiserum specific either for Fc” or for Fc. The cleavage of IgE( ND) by pepsin at pH 4.5 and 37°C is a very rapid process; within less than 15 minutes more than 90%of the substrate is cleaved to yield F( ab’) and pFc fragments and small peptides. The immunological and physicochemical properties of F( ab’ ) do not change by prolonged digestion up to about 24 hours. The pFc fragment, which is enzymatically labile, represents a portion of the carboxy terminal part of the E chains that carries some of the antigenic determinants characteristic of Fc. The molecular weight of pFc fragment was determined to be about 30,000 ( V = 0.713; protein concentration 0.5 mg./ml.) by sedimentation equilibrium experiments. The carbohydrate content is high, about 24%,which corresponds to about one-third of the total carbohydrate of native IgE( ND), By adding the carbohydrate data for F(ab’), calculated on the basis of a molecular weight of 135,000, the sum of carbohydrate residues will be in close agreement with the carbohydrate data for IgE( N D ) (Bennich and Clamp, 1971). Attempts to inhibit PCA reactions with pFc fragments have so far been unsuccessful. In summary, fragmentation studies of IgE ( N D ) have shown that antigenic determinants ( D ) , specific to the c chain and located within the Fc fragment, can be separated in two groups, in the following referred to as D,1 and D,2. Fragments F (ab’)? and Fc share the antigenic determinants, D J , which arc localized within the amino terminal portion of the Fc fragment. This part of thc E chain, which represents a glycopeptide consisting of about 120 amino acid residues, is characterized by a relative resistance to papain, pepsin, or trypsin, and, even by prolongc~ldigestion, a covalently linked dimcr of about 40,000 molecular weight [Fc” or Fc”(t ) J having D,1 specificity can be recovered. Amino acid data and reduction experiments indicate that Fc” represents a “domain” containing four half-cystinyl residues of which two are engaged in interchain bonds. In contrast, structures carrying

28

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

D,2 specificity are readily cleaved to produce inactive fragments. No fragments have been identified to carry only D,2 specificity with the exception of pFc which represents a noncovalently linked glycopeptide from the carboxy terminal part of Fc. Separate regions carrying D,1 and D,2 specificity can be shown to be present also on IgE from normal and atopic sera and from secretions (Bennich and Johansson, 1971). The inhibitory activity of IgE( ND) on reagin-induced PCA in baboons (or P-K in humans) has so far been recovered only in the Fc fragment, which carries both D,1 and D,2 specificity. However, the absence of inhibitory activity in other fragments does not justify speculations on the further localization or nature of the skin-fixing sites of c chains, but rather directs the attention to a weakness of indirect methods such as should be complemented by techniques for a direct study of the nature of binding of IgE and its fragments to cell surfaces. IV. Methods for Determination

The best way to determine IgE is to use immunochemical methods based upon the reaction between IgE and antibodies specific for the TABLE I V COMPARISON OF RADIOIMMUNOCHEMICAL METHODSFOR ESTIMATION OF IMMUNOGLOBULIN E Criteria

RISTa

Sensitivity (IgE estimation) lteproducibili ty Capacity; samples per day and technician Stabilky of reagents Reagents needed, antibody

1 ng./ml. 5-1Oa/, 100

antigen

Results obtained within Special equipment required

0

Catt “sandwich”b 1 ng./ml. 5-1070

100

Very good Very good Very small Very small quantity quantity specific specific Very little, pure Rather much, pure or crude 8-24 hours Scintillation detector; centrifuge for washing

8-24 houm Scintillation detector

RIST-radioimmunosorbent test. From Salmon et al. (1969); Johansson and Bennich (1970). RSRD-radioimmune single radial ditrusion. From Rowe (1969).

RSRD 40 ng./ml. 10-200/, 50

Very good Very small quantity specific Serum with known IgE concentration 5-10 days None

HUMAN IMMUNOGLOBULIN E

29

class-related antigenic determinants of IgE. Gel diffusion methods, such as single radial diffusion in gel (Mancini et al., 1965) are handicapped by low sensitivity; radioimmune assays have proven to be superior. Only a few radioimmune methods have been used for IgE estimation. RIST (Wide and Porath, 1966; Johansson et al., 1968a) and a ''sandwich" modification (Salmon et at., 1969) of the solid phase method described by Catt et al. (Catt and Tregear, 1967; Catt et al., 1967) and the double antibody technique (Gleich et al., 1971) seem to be useful. A two-step modification of the Mancini method was described by Rowe (1969) where the introduction of isotope-labeled antiglobulin antiserum enabled the detection of IgE levels as low as 50-100 ng./ml. In Table IV some criteria, such as sensitivity and precision of the methods, are compared. V. levels of Immunoglobulin E in Healthy Individuals

A.

LEVELSIN SERUM

The dominating immunoglobulin in cord serum is IgG. Only very low concentrations of IgM, about 10%of adult mean level (Franklin and Kunkel, 1958; Johansson and Berg, 1967) can be found. Immunoglobulin A and D are usually below the limit of detection for gel precipitation methods (Johansson and Berg, 1967); IgE can be detected in cord serum with the use of RIST. In a study of samples from healthy newborns, a mean IgE concentration of 36 to 38 ng./ml. was found with a range of 16.0 to 97.5 ng./ml. (Johansson and Bennich, 196%; Johansson, 1968a). This level corresponds to about 15%of the adult level. Since the IgE values did not distribute in a Gaussian manner, before calculations all values were changed to logarithms to the base 10. The confidence limits (mean value +2 S.D.) for the IgE in the cord serum study was 12.9-102 ng./ml. Sera were also collected from 14 anamnestically allergic mothers and from their respective newborns (cord serum). No correlation was found ( T = 0.002) between the IgE concentration of the mothers and their respective newborn (Johansson, 1968a). This seems to indicate that the IgE present in cord serum consists mostly of material synthesized de novo by the fetus. The quantitation of such low IgE concentrations is open to some question due to the technical problems involved. Recent studies seem to indicate that the IgE concentration in cord serum is even lower than that reported. In Table V the IgE concentration in cord serum is compared with the IgE levels in children and adults. The development of the immunoglobulin levels during childhood have been studied by many authors (Claman and Merrill, 1964; Stiehm and Fudenberg, 1966; Collins-Williams et al., 1967; Johansson and Berg,

30

HANS BENNICH AND S. GUNNAR 0 . JOHANSSON

TABLE V SERUMIMMUNOQUJBULIN E LEVELSIN HEALTHY CHILDREN A N D ADULTS. IgE levels (ng./ml.)

Serum Cord serum Children If-44 months 4+-9 months 9 months-3 years 3-5 years 6-10 years 11-15 years Adults (I

Confidence limits (96% interval)

Geometric mean

Range

36. 3

12.9-102

16.0-97.5

60. 6 75.7 114 158 190 246 248

43.5-84.5 24.7-233 29.0-450 45.3-528 55.6-648 71.9-838 61.4-1000

50.0-86.0 24.0-223 49.5-540 62.0-308 63.0-535 54.0-840 66.0-1830

From Johansson (1968a) and Berg and Johansson (1969b).

1967). In Fig. 12 the development patterns for IgG, IgA, IgM, IgD, and IgE are summarized. The striking difference between the slow rise of IgA and the fast increase of IgM is well known. The pattern of IgD in the first year is not yet known but will be easy to investigate by the new highly sensitive methods. In a first study of the IgE development in 50 healthy children, 6 weeks to 5 years of age, a rather slow increase with age was found (Johansson, 1968a). Since the regression

/

mg /ml

mg I00 ml Partus

I

............. gM9' (000 ..................l g M d

4

I

Week of gestation

Months

500

Years

FIG. 12. The development during childhood of the serum concentrations of the five immunoglobulins. Only the mean levels are given. (Data from Johansson and Berg, 1967; Berg, 1969; Berg and Johansson, 1969b; by permission of Kabi, Stockholm. )

HUMAN IMMUNOGLOBULIN E

31

line satisfying all samples gave a level at 6 weeks of age that was somewhat higher than the mean of cord sera, a pattern for IgE similar to that of IgM was proposed. However, further studies (Berg and Johansson, 1969b) of 138 randomly sampled healthy children gave a distribution pattern more like that of IgA. When 10 children were followed with repeated samples during their first year of life (Berg and Johansson, 1969b), a very slow increase of IgE concentrations with age was found. This is in good agreement with IgA but is in sharp contrast to what has been found for IgM (Johansson and Berg, 1967; Berg, 1968). Even in premature newborns, a very fast increase in IgM has been reported (Berg and Johansson, 1967b; Berg, 1969). The difference in IgG and IgM concentrations which was found in boys and girls could not be shown for IgE (Berg and Johansson, 1969b). This is of particular interest since it is well known that of children suffering from asthma, there are twice as many boys as girls. With aging in adults, only small changes in the immunoglobulin concentrations (Johansson et al., 1968b) are seen, and this seems also to be true for IgE (Johansson, 1968a; Johansson et al., 1968b). The difference between men and women which has been reported for IgM and perhaps also for IgD (Johansson et al., 1968b) could not be found with IgE. The decrease of allergic disorders which occurs with increasing age does not seem to be accompanied by a decrease in IgE concentrations. One complication in the studies of the normal distribution of IgE is in the selection of healthy, nonallergic individuals. For practical reasons, we had to select our cases from interviews by a clinical allergologist. No allergological investigation such as skin or provocation tests were made. By this procedure some individuals with subclinical allergy might have been included. Supporting this idea are the findings of a tendency for subnormal IgE levels in patients with asthmatic symptoms but with negative findings on allergological investigation (Johansson, 1967; Berg and Johansson, 1969a). In a study (Berg and Johansson, 1969a) of children with asthma (from Dr. Aas, Olso), it was found that the 28 children with negative findings on clinical investigation had a mean IgE level of 113%of mean for the age. Twenty of these had a positive histamine provocation test, indicating hypersensitive bronchial mucosa which is found in high frequency among allergic individuals. Their mean IgE level was 134%.The other 8 had normal bronchial histamine sensitivity and were, therefore, so far as was possible to evaluate at the time, nonallergic. Their mean IgE level was only 62%. The influence of genetic factors on the IgE Ievels has been studied (Rowe et al., 1971) in a twin population. One hundred and twenty-eight twin pairs were studied, of which fifty-nine were monozygotic (Rowe

32

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

et al., 1968a). For monozygotic men below 20 years of age, a significant intrapair variance was found, This was the only result that indicated a genetic influence on the IgE levels. Obviously the environmental factors are of importance for the development of the IgE concentrations. These results fit very well with the meaning of atopy: an increased tendency, influenced by hereditary factors, to develop disorders based upon immediate-type hypersensitivity reactions. The relation between the concentration of IgE and IgA in an individual was investigated in the twin study. The absence of correlation between IgE and IgA would seem to indicate that there is no firm relationship for the production of these two immunoglobulins.

B. LEVELS IN SECRETIONS The reagin-mediated immunological reactions of asthma and hay fever take place in or in the immediate neighborhood of mucose membranes. It could very well be that the circulating IgE represents an excess of reagin from the affected organ, the lung, or the nose. The information about IgE in secretions is limited (Johansson et d., 1971a). The IgE concentrations in saliva from healthy individuals have been estimated by RIST in a preliminary study (Turner and Johansson, 1971). Very low concentrations were found ranging between 1 to 10 ng./ml. No significant difference in absolute concentrations could be found between whole saliva and parotid secretion. The IgE/IgA (serum comIgA was used as a standard) ratio for whole saliva was 5.7 X pared to 13 x for parotid secretion. The same ratio for serum was 0.1 x The tendency for relatively lower IgE concentrations in the whole saliva might be attributed to addition of gingival pocket secretion which is known to have an immunoglobulin composition very similar to that of serum (Brandtzaeg, 1965). The absolute IgE levels in gingival pocket fluid in healthy adults and patients with parodontiasis seem to be the same as in the serum (Killander et al., 1971). A few samples of nasal washings from healthy individuals have been studied. Very low levels of IgE were found, the absolute concentrations ranging from 3 to 30 ng./ml. The IgE levels have also been studied in bronchial washings from 20 patients. Immunoglobulin E could not be detected in 12 of the unconcentrated secretions. The mean IgE concentrations in the other 8 samples was 5.4 ng./ml. with a range of 1.8 to 12.0 ng./ml. (Deuschl and Johansson, 1970). It is obvious that it is possible to detect IgE in nasal and bronchial secretions of healthy individuals by RIST. Further studies are now in progress to estimate to what proportion the amount of IgE can be attributed to local production. Nasal polyp fluid from allergic patients is known to contain very high

33

HUMAN IMMUNOGLOBULIN E

concentrations of reagins ( Berdal, 1954). Relatively high levels of IgE were also found in individuals without any signs or symptoms of atopic allergy (Donovan et al., 1970). However, since nasal polyps cannot be regarded as a normal status, this condition will be discussed in detail in Section VI,B. The secretion containing the highest immunoglobulin concentrations, and predominately IgA, is colostrum. The IgA concentration can reach a few grams per 100 ml. The IgE levels in colostrum have been studied in the first week of lactation. Very high concentrations were found, in some mothers more than 20 times higher than that of serum. In Fig. 13 is shown the development of the IgE levels in colostrum during the first week of lactation. Two different patterns are shown. In one case the highest levels were found about day 1-2 after delivery and, thereafter, they decreased; in the other case, a steady increase was seen up to day 5-6. When the IgE/IgG ratio in the colostrum was calculated, a very fast rise was found in both patients; the slopes of the two curves were almost parallel. These findings can be explained on the basis of 1oooc

1000

P

LOO0

LOO

2000

200

1000

100

-2 LOO

10

200

10

100

10

* X

0

C

2 -m 0

-

e'

0 PARTUS

1

2

3

4 5 Days

6

7

8

FIG. 13. The development of IgE levels in Iiunian colostrum from two women, ( 0) and ,).( respectively, during the first days of lactation compared with serum IgE levels, and ( @ ) , respectively. Imniunoglobulin E levels are given in absolute concentrations (-) and as a IgE/IgG ratio in colostrum ( - - - - ) .

(a)

34

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

selective transport of serum IgE or by local synthesis. Since there seems to be no correlation between the concentration of IgE in serum and colostrum the hypothesis of local production is likely (Johansson et al., 1971a). Preliminary studies on the molecular size have shown that the major part of clostrum IgE comes off a calibrated Sephadex G-150 column in the same volume as serum IgE (Fig. 14). The immunoglobulin concentration, with special reference to IgE, has been studied in urine from 16 healthy individuals (Turner et al., 1970). The immunoglobulins were simultaneously quantitated in serum and urine, and clearance data were used to estimate the amount of protein locally produced. The results indicate a slight local production of IgG and IgA. A very high clearance of IgE was found, which is thought to represent local production. As much as 100 times more IgE was found than would have been expected from the clearance data. The amounts of IgG and IgA produced locally in percentage of total urinary IgG and IgA varied from 9 to 92%. The same percentage for IgE showed less variation ranging from 62 to 100%.The high urine IgE concentrations can also be explained on the basis of degradation of IgE in the urinary tract. However, so far the only in vitro produced frag-

L

0 u)

n

a

0.20

0.32

0.44

0.56

0.68

E l u t i o n v o t u m e , v,:

0.80

0.92

1.04

vt

FIG. 14. Gel filtration of colostrum ( 6 ml.) from a nonallergic woman on a calibrated column (3.2 X 92 cm.) of Sephadex G-150. Absorbancy, 280 nm. (0); colostral IgE, as determined by radioimmunosorbent distribution of a purified sample of IgE(ND) (17).

(a);

HUMAN IMMUNOGLOBULIN E

35

ments containing E determinants are the F( ab’)? and the Fc fragments, both having a molecular weight larger than that of albumin (Section 111). The lack of correlation with serum lcvels on patients with atopic diseases and various renal disorders also is in favor of a local synthesis. It, therefore, seems very likely that the IgE in urine is produced locally somewhere in the urinary tract. The function of such IgE is yet unknown although some beneficial function is perhaps more probable than a local urinary reagin-mediated disorder. The findings of IgE in several secretions and the data indicating that this is the result of a local production are very interesting. Fluorescent studies using specific antisera to IgE have shown a localization of IgE-staining plasma cells to mucosal membranes of the respiratory and gastrointestinal tracts ( Tada and Ishizaka, 1970). Further studies are of interest to evaluate the importance of local IgE in relation to serum IgE and its role in disease. VI. levels of Immunoglobulin E in Disease

A. LEVELSIN SERUM 1. Atopic Diseases In a preliminary study of patients suffering from asthma and hay fever (Johansson and Bennich, 1967b), some cases with highly increased serum levels were found. The mean IgE value in a group consisting of 31 cases with proven allergy was 1191 ng./ml. with a range of 133 to 5850 ng./ml. These values should be compared with mean IgE of 2,48 ng./ml. in healthy individuals. The highest concentration observed in this study, 5850 ng./ml., is about 20 times higher than the normal mean. In order to investigate the I g E distribution in asthma, a group of adult patients with asthma was analyzed (Johansson, 1967). The study consisted of 38 cases which, according to the allergological investigation, were divided into two groups. The patients were tested by intradermal tests using fifteen commercial allergens including four pollen mixtures and nine animal dandruffs, mold mixture, and house dust. Positive skin reactions were verified by a bronchial provocation test. All patients with positive skin and provocation tests to at least one allergen were brought together in a group called “allergic asthma.” The other group of patients was called “nonallergic asthma.” No difference was found for the concentrations of IgG, IgA, IgM, and IgD between the allergic and nonallergic group. This is in contrast to the findings of Kohler and Farr (1967) who reported high concentrations of IgD in allergic diseases.

36

HANS BENNICH AND S . GUNNAR 0. JOHANSSON

The mean IgE level for the whole study was 828 ng./ml. which is about 3 times the normal mean level. However, the allergic group had a mean of 1589 ng./ml. compared to 275 ng/ml. in the nonallergic group. In the allergic group, 8 of the 16 patients had pathologically high levels (more than 1000 ng./ml.). Immunoglobulin E levels were also studied in children with asthma and hay fever (Berg and Johansson, 1969a). Samples were analyzed from 94 untreated children (70 boys and 24 girls) which all had a positive allergological investigation including provocation tests. As can be seen in Table VI, 13 children had only symptoms of asthma. Their IgE mean level was 563%of normal mean for their age. The 22 children with rhinoconjunctivitis as the predominant symptom had a mean IgE level of 297%. One explanation for this difference could be the more severe and prolonged symptoms that usually occur in asthma. Thus it could be shown that children with perennial symptoms of asthma had a higher mean IgE level (711%)compared to the children with symptoms mainly during pollination season (230%).Of 22 children with hay fever, only 8 had elevated levels, and the mean IgE value for the group was 297%.The influence of allergenic stimulation on the IgE concentrations was clearly shown when the IgE levels were followed in untreated children during a pollination season. The mean level before season was 12652, during season 286%,and after season 242%. TABLE VI IMMUNOGLOBULIN E LEVELSI N CHILDREN WITH ATOPIC DISEASES~

Diagnosis Bronchial asthma Hay fever Bronchial asthma and hay fever Bronchial asthma and atopic eczema Hay fever and atopic eczema Negative allergologic investigation

No. of children

No. of children with high levels*

Mean IgE concentrationc

13 22 2s

10 8 15

563 297 350

S

7

744

4

2

599

7

1

111

Data from Berg and Johansson (1969a). Immuiioglobulin E concentration higher than mean $ 2 S.D. for the age. 111 percent of the predicted arithmetic mean value for healthy children of the same age.

HUMAN IMhfUNOGLOBULIN E

37

Different types of allergens might be more-or-less potent in their ability to stimulate the IgE production. Thus, dust and mold allergens have been regarded as weak allergens by the allergologists, whereas common animal dandruff and pollen allergen have been regarded as strong allergens. In a study (Berg and Johansson, 1969a) of children with asthma (from Dr. Aas, Oslo), it was found that 11 children with positive skin and provocation tests for dust and/or mold had a mean IgE level of only 104%of mean for their age compared to 338% for 42 children allergic to other common allergens. Atopic eczema (prurigo Besnier) is a disease the relationship of which to reagin-mediated disorders has been discussed. It is, therefore, of interest that patients with atopic dermatitis have raised IgE levels. In the study of allergic children (Berg and Johansson, 1969a) it was found that when the children had asthma and eczema or hay fever and eczema, the mean IgE level was 744 and 599%of mean for their age, respectively, compared to 563 and 297%in pure asthma and hay fever, respectively. Clear-cut changes were found when adult patients with pronounced atopic dermatitis were investigated (Juhlin et al., 1969). Studies of 28 patients showed a mean IgE value of 2733 ng./ml. which corresponds to about 11 times the normal mean. As high a value as 31,000 ng./ml. was found in 1 patient who in addition to his eczema also suffered from severe asthma due to animal and pollen hypersensitivity. Five patients (18%)had normal IgE levels, but this number increased with the number of less severe cases included (Ohman and Johansson, 1971). 2. lmmunobgical and lnfectious Diseases The Wiskott-Aldrich syndrome is characterized by increased susceptibility to infections, thrombocytopenia, and eczema ( Wiskott, 1937; Aldrich et al., 1954). Immunoglobulin E concentrations were measured in a study of 6 cases of Wiskott-Aldrich syndrome (Berglund et al., 1968). High levels were found in all 6 patients studied, but there was a remarkable change in the levels with time. Further studies of the children seem to indicate that the IgE level varies with the state of the eczema. When the eczema was pronounced the IgE value was high. Concentrations of IgE have been studied in immunological disorders other than the Wiskott-Aldrich syndrome. No significant changes were found in diseases such as rheumatoid arthritis, systemic lupus erythematosus, and ulcerative colitis. In the latter group, 49 patients were studied (Johansson et al., 1 9 7 1 ~ ) The . mean IgE value for the group was 334 ng./ml., but 3 of the patients had raised levels (1063, 2500, and 4300 ng./ ml. respectively) and 1 patient had a hypogammaglobulinemia

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

38

TABLE VII SERUMIMMUNOGLOBULIN LEVELSIN PATIENTS WITH M COMPONENTS, HYPOQAMMAGLOBULINEMIA, A N D SELECTIVE IMMUNOGLOBULIN A DEFICIENCY Age (years)

Sex

Diagnosis

IgEa

IgGb

IgAb

IgMb

IgDb

65 64 69 74 59 65 63

m f f m m f m

B.H. G.E. S.L.e A.K.1 L.S. B.K. A.K. G.S. J.R. B.H.

6120 5000 280 1410 525 5000 1420 19
20 <1
20 20 10 3180 12 40 40 <1
<1

f m f m f m m m m f

37 79 2600 303 34 37 24 58 59 10 15 37 20 112 65 58 123 145 110

40 40 2770 100 12 130 250 -

25 14 5/12 57 21 25 63 38 30 17

J.Z.9

26

f

140

1320

<1

290

N.T.

K.C.

5

f

82

1350

<1

132

N.T.

M.H.

5

f

71

1567


79

N.T.

K.B.

17

f

53

1867

<1

128

<1

1.E.h

40

f

87

2528

<1

364


M.A.h

33

f

12

140

<1

4

<1

F.E. P.' H.F.i

-

2

m m

M comp., GL M GL YFL M AK M ML M DL MY MY Mff Mff Hypogamma. Hypogamma. Hypogamma. Hypogamma. A-IgA, healthy A-IgA, healthy A-IgA, healthy A-IgA, healthy A-IgA, healthy A-IgA, Atax. telang. A-IgA, Atax. telang. A-IgA, Infect. prone. A-IgA, Infect. prone. A-IgA, SLEk A-IgA, Malabs. A-IgA, Malabs. A-IgA, Atopi c A-IgA, Atopic A-IgA, Atopic

1690 1334


63 214

N.T.

N.T.

N.T.

Patient F.L. V.O. E.W. A.E. E.E. R.E." A.J.c Ded Aid

-

3

-

+

1190 8800 1035

N.T.

<1


<1
-

(1 <1

N.T. <1 7 22.3
2.9

Immunoglobulin E in ng./ml. Immunoglobulins GI A, MI and D in mg./100 ml.; N.T. = not tested. Gamma heavy-chain disease sera kindly provided by Dr. 0. Wager, Helsinki; reported by Wager et al. (1969). Alpha heavy-chain disease sera kindly provided by Dr. M. Seligmann, Paris; reported by Seligmann et al. (1968). * Thymic alymphoplasia receiving 7-globulin therapy (from Berg and Johansson, 0

19678).

HUMAN IMMUNOGLOBULIN E

39

(IgE 20 ng./ml.). The mean IgE level for the other 45 was 188 ng./ml. (range 49/715 ng./ml. ) which is normal. Other kinds of gastrointestinal diseases have also been investigated. Patients suffering from celiac disease (Hobbs et al., 1969) and various types of malabsorption disorders (Bjernulf et al., 1971) were found to have normal or slightly decreased IgE levels. In myeloma and macroglobulinemia the IgE levels vary with the other normal immunoglobulins (Table VII) . Thus, low concentrations of IgA and IgE and sometimes also of IgM were found in sera with a very high M component of Type G. A few cases of M components have been found which had high IgE concentrations. However, these findings could be explained by the fact that the patient was suffering from atopic allergy. Immunoglobulin E concentrations have also been studied in a limited number of patients with hypogammaglobulinemia, some of which are shown in Table VII. Very low IgE levels were found; in a few cases lower than was found in cord serum. However, in no case was the IgE level below the limit of detection of the method used, i.e., 1 ng./ml. A family with two cases of Kartagener syndrome had normal IgE concentrations ( Aas, 1970). Individuals with selective absence of IgA in serum (Rockey et al., 1964) seem to have normal IgE levels (Johansson and Bennich, 1967a; Johansson, 1968b; Johansson et al., 1968a). A few IgA-deficient patients suffering from atopic allergy have been found to have raised IgE levels (Aas, 1970; Fudenberg, 1970). In Table VII the concentration of the five immunoglobulins are given in sera from healthy individuals and patients suffering from various diseases. The mean IgE value is within the normal range. As many as 11 out of the 24 individuals tested had an IgD concentration below 1 ng.1100 ml. This is a higher frequency than has been reported in healthy individuals with normal concentration of IgE (Rowe and Fahey, 1965; Johansson et al., 1968b) but in agreement with an earlier report (Johansson et d., 1968b). Patients suffering from ataxia telangiectasia sometimes lack detectable amounts of IgA (Thieffry et al., 1961; Young et al., 1964). In a study of such patients, Ammann et al. (1969) used the reversed cutaneous anaphylactic reaction (K. Ishizaka and Ishizaka, 196813) to estimate the IgE concentration. Plasma cell leukemia without detectable illcomponent. Kindly provided by Dr. Z. Haddad, Los Angeles. From Bjernulf ct al. (1971). Kindly provided by Dr. H. H. Fudenberg, San Francisco. i Kindly provided by Dr. K. Aas, Oslo. k SLE-systemic lupus erythematosus.

0

40

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

They found that 11 of the 16 patients had a negative skin reaction when guinea pig anti IgE diluted 1:2000 was injected; they interpreted this as IgE deficiency. All patients lacking IgA gave a negative skin reaction to anti-IgE. The few patients we have studied so far have low IgE levels (Table VII). With sera from cases of ataxia telangiectasia,l it was found that the mean IgE value of 8 samples was 56.7 ng./ml. which represents 26.4%of mean for age with a range of 13.7 to 38%. Obviously there is a tendency to low IgE concentrations in patients with ataxia telangiectasia. However, of the 10 patients in which IgE concentrations have been measured, only 3 had an IgE level below the 2 S.D. lower border line for healthy individuals of the same age. In a later study, Cain et al. (1969) could not verify their previous findings of a relation between IgA and IgE deficiency. Infectious diseases have been studied with special reference to the IgE levels. In a group of 12 adults with acute pneumonia, in which 4 of the cases were caused by a mycoplasma infection, no significant changes of the IgE levels were found (Nordbring et al., 1969) during a 50-day follow-up period. However, a tendency was seen to a decline of the levels during hospitalization. No significant changes of IgE were found in patients with hepatitis. In patients with mononucleosis (Nordbring et al., 1971), however, the patients had high IgE levels in the first sample taken at about day 10 of the disease. A significant decrease of their IgE levels during the 50-day period of investigation was found. The IgE pattern is similar to that of IgM but in contrast to that of IgG and IgA (Wollheim, 1968; Nordbring et al., 1971). None of the patients had any allergic disorders such as atopic diseases or drug reactions. 3. IgE Myelomatosis

The first case of E myeloma appeared in July 1965 (Johansson and Bennich, 1967a). The patient ( N D ) , a 50-year-old farmer, had the typical signs and symptoms of myelomatosis: fatigue, back pain, immature plasma cells in the bone marrow smear, high sedimentation rate, and typical M component in serum and urine. More unusual was the high concentration of circulating plasma cells. The case history and some findings are summarized in Fig. 15. The M-component in serum was of type L; the urine component was a Bence-Jones protein of Type L. By reduction it was shown that the serum protein contained both light chains and heavy chains (see Section 11). In September 1968, a second case (PS) of E myeloma was found in Hanover, New Hampshire (U. S. A,) (Ogawa et al., 1969). This E Samples for IgE estimation were kindly made available by Dr. Hong.

SR mm/hr 160. 0--0

Crcatininc

120 80

40 Hb gY. Wbc 15 30 000

fl Sendoxan (g) 3.1 0-0

Plasma cells

10

5 M

1965

1966

1967

1968

FIG. 15. The case history of E myeloma patient (ND). The plasma cells count in peripheral blood (shaded area) shows a peak in relation to the relapses of the disease reaching 150,00O/mm? (96% of total white cell count) in the final stage. The intermittent therapy with Sendoxan did not significantly diminish the M component but apparently caused a partial remission of the disease. When Sendoxan was given continuously in 1968 the paraprotein decreased. The final relapse came during Sendoxan therapy after 3 years of observation. Hb-hemoglobin; W b c w h i t e blood cells; SR-sedimentation rate.

rp P

42

HANS BENNICH AND S . GUNNAR 0. JOHANSSON

myeloma protein PS was also of Type L, and there were other striking similarities in the clinical picture of the two patients, ND and PS. Both had plasma cell leukemia and diffuse osteolytic lesions, a prominent M component of Type L, and Bence-Jones protein in the urine. The simultaneous occurrence of E myeloma and plasma cell leukemia is interesting. Attempts were made to start a long-term tissue culture of the plasma cells from patient ND. Samples were taken from bone marrow, superficial lymph nodes, and peripheral blood at different times in the disease and also at autopsy. Several cell lines were started from the different sources. Most of them produced a monoclonal IgG of Type K after an initial lag phase of a few weeks. However, two of the cell lines, one from peripheral blood taken a few weeks before death and one from bone marrow taken at autopsy showed a different behavior. After a lag phase of 5 months the cells switched into plasma cells, which produced IgE of Type L. Gel diffusion studies showed that the IgE produced in the tissue culture carried the same individual (clone) specific, idiotypic, antigenic determinants in its Fab piece as the intact E myeloma protein ND (Nilsson et al., 1970). 4. Parasitic lnfections

Frequent reports of high incidences of raised serum immunoglobulin levels in Africans compared with Europeans have been published (for review, see Turner and Voller, 1966; Rowe et al., 1968b). It seemed of interest to analyze the situation with special regard to IgE (Johansson The serum samples were collected as a part of the activiet al., 1968~). ties at the Children’s Nutrition Unit in Addis Ababa. Mean IgE values of 2880 and 3520 ng./ml., respectively, were found in different population groups. These concentrations should be compared with a mean of 160 ng./ml. for Swedish children of the same age. Atopic manifestations such as asthma, hay fever, and eczema are said to be rare in Ethiopia and Africa. The most likely explanation for the very high IgE levels thus was parasitic infestations. A group of 25 children with stools positive for Ascaris lumbricoides was compared with a group of 19 children from a children’s home with negative stools. The mean IgE value for the Ascaris-positive group was 4400 ng./ml. (range 240-14 300 ng./ml.) compared with a mean of 860 ng./ml. (range 120-5250 ng./ml.) in the Ascaris-negative group. The increase of serum IgE concentrations in parasitic disorders was further confirmed in a study of visceral larva migrans (Hogarth-Scott et al., 1969). A group of 26 patients with a clinical history suggestive of visceral larva migrans was investigated by an in vitro fluorescent test for antibodies to excretory-secretory

H U M A N IMMUNOGLOBULIN E

43

antigens of Toxocara canis (Hogarth-Scott, 1966), by passive PCA in baboons using saline extract of adult T . canis, and by IgE quantitation. A good correlation was found between the existence of homocytotropic antibody and raised IgE levels. Seven of the 26 patients had detectable concentrations of reagin, and thcir mean IgE value was 2392 ng./ml. compared to 267 ng./ml. for the other patients in the group, Intestinal capillariasis is a new disease in man (Whalen et al., 1969) caused by a species of roundworm, Capillaria philippinensis. The serum concentrations of IgG, IgA, and IgM is normal or low; in contrast, very high levels of IgE were found (Roseuberg et al., 1971). In a group of 20 adult Filipinos, a mean IgE lcvel of 5942 ng./nil. (range 690 to 19,OOO ng./ml.) was found. Three severely ill patients had IgE values over 15,000 ng./ml. A group of 10 apparently healthy adult Filipinos and without any signs of capillariasis had a mean IgE level of 1390 ng./nil. Isolated cases with other parasitic disorders have also been found to have high IgE levels. In a case infected with hookworm (Necator Americanus ) , followcd with repeated examinations during several years (Ball and Barlett, 1969), it was found that very high IgE levels increasing up to 10,000 ng./ml. developed after a lag phase of about a year (Voller et al., 1971). Another case, suffering from ankylostoniiasis ( Reerink-Brongers, 1970), was found to have 140,000 ng./ml. In the Ethiopian study, no individual correlation was found between IgE and eosinophilia. However, a correlation was found between diiferent population groups: high mean IgE value in a group was accompanied with high eosinophile count and low mean IgE value with low eosinophile count. Single nonatopic cases with eosinophilia of unknown origin have been found to have very high IgE levels, i.e., 1 case with 70%eosinophilia had an IgE level of 70,000 ng./ml. In children with atopic diseases, no correlation was found between IgE levels and blood eosinophilia ( Berg and Johansson, 1971). However, these findings can be explained on the basis of rapid changes in eosinophile count compared to more steady IgE levels. Immunoglobulin E levels have also been investigated in sera from patients with malaria and syphilis. Although a limited number of sera from different stages of the diseases have becn analyzed, no evidence has been found to indicate that IgE plays a major role in the immunity of these diseases. Further studies would be of interest.

B. LEVELSIN SECRETIONS The immunological proccsses involved in the immediate-type allergic diseases probably take place mostly in the affected peripheral organ, i.e., mucose membranes and skin. It is, therefore, of interest to estimate the

44

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

IgE distribution in various secretions. However, since many technical problems are involved, such as small volumes and protein, carbohydrate, and lipoprotein composition difFerent from that of serum, very few data are as yet available on IgE concentrations in secretions. The cause of benign mucous nasal polyps is not clear. An allergic etiology has been discussed but in the majority of these patients skin tests to a wide range of allergens are negative (Pepys and Duveen, 1951). However, it has been shown that nasal polyp fluid from allergic individuals may contain a higher titer of reagins than serum (Berdal, 1954). The concentrations of IgE, IgA, IgG, IgM, albumin, and macroglobulin were measured in paired samples of nasal polyp fluid and serum from a group of patients (Donovan et al., 1970). Using the ratio of the concentrations of albumin and macroglobulin in nasal polyp fluid and serum, it is possible to calculate how much of the immunoglobulins could be attributed to local production in the nasal polyp. A considerable degree of local production of IgE was found in almost all the polyps. This was also true for IgG and IgA but less pronounced for IgM. Further studies are now under way to ascertain the importance of local IgE production in other niucosal membrane systems.

C. FACTORS INFLUENCING IMMUNOGLOBULIN E LEVEL From the studies of IgE levels in children with asthma and hay fever (see above), it was apparent that a period of symptoms stimulated the IgE production. It was shown that rapid increase in IgE concentrations occurred in untreated hay fever patients in the beginning of the pollination season; the decrease after the end of the season was slower. In order to study the immediate effect of specific “rush hyposensitization” on the IgE levels, 37 children were followed during the first weeks of therapy (Berg and Johansson, 1969a). All of the children except 3, showed an increase in their IgE concentrations. This increase already was significant after 3 to 4 days; after 14 days the mean increase was more than 50%of the basic value. The IgE concentrations seem to stay high for a considerable time. When a group of nonallergic volunteers was subjected to the same treatment with ragweed allergen extract, no changes in the IgE levels were found (Berg et al., 1971a). Different types of drugs, such as steroids and cytostatics, used in the treatment of allergic diseases might also influence the IgE levels. Patients with atopic eczema having high IgE concentrations were treated with ointments, local steroids, and other symptomatic therapy with good improvement of the clinical state. No change in the IgE levels could be found over a period of a few weeks. Some of the patients were given

HUMAN IMMUNOGLOBULIN E

45

prednisone systemically in rather high dosage during 2 weeks with the same results (Johansson and Juhlin, 1971). No difference could be found in the mean IgE values in a group of adults with asthma receiving steroids and a group not on steroids (Johansson, 1967). Patients with atopic eczema were given azathioprine (150 mg. daily) for 3 to 4 weeks, and no changes in the IgE levels were detectable (Johansson and Juhlin, 1971). It would seem obvious that short-term therapy with steroids and cytostatic agents has rather little influence on IgE concentrations. Disodiuni chromoglycate has been reported to be useful for the treatment of asthma (Altounyan, 1967). In order to evaluate the effect on the IgE levels, 52 adults with asthma were followed before and after treatment (Arner et al., 1971). Most of the patients showed steady levels, and it is likely that the drug as such does not have any influence on the IgE levels. VII. Detection of Antibody Activity in the Immunoglobulin E Class

The association of reaginic antibody activity within the IgE class of immunoglobulins was established by the work of Ishizaka et al. (for review, see K. Ishizaka and Ishizaka, 1968a, 1970). They used 1311-labeled ragweed allergen E to show that only antibodies of the IgE class paralleled the reaginic activity, measured by passive sensitization of normal human skin (the Prausnitz-Kiistner reaction), when allergic sera were fractionated by gel filtration and ion-exchange chromatography ( K. Ishizaka and Ishizaka, 1967). Independently it was shown (Wide et al., 1967) that by the use of the antiglobulin principle (RAST), it was possible to detect IgE antibodies to several different allergens in serum of allergic patients. A. REDCELL-LINKEDANTIGEN-ANTIGLOBULIN REACTION The antiglobulin test for detection of incomplete antibodies was described in 1953 (Coombs et al., 1953). A modified method, the red cell-linked antigen-antiglobulin reaction ( RCLAAR ) , is a sensitive passive hemagglutination procedure suitable for detection of nonprecipitating antibodies. It involves the coupling of allergen to rabbit antibody to human red cells by a photooxidation procedure (Fig. 16). Antibodies to the allergen then attach, and, if they are IgE in character they are agglutinated by IgE antisera. The RCLAAR has been used to measure antibodies to castor bcan allergen in serum of patients living in the neighborhood of a castor bean oil mill (Coombs et al., 1968). It was found that antibodies of IgG and usually also of the IgA and IgM class could be found in sera from almost all exposed individuals. However,

46

HANS BENNICH AND S . CUNNAR 0. JOHANSSON

- -

RAST

RCLAAR

Allrrprn

+

H

+ 4 7

Rabblt cell antibody

Photo-oxidation

Rod

Mood a l l $

Anlibodiom to tho ollrcpm

Rodioadin comploc

Agglutination

FIG.16. Diagrammatic representation of the radioallergosorbent test (RAST) and the red cell-linked antigen-antiglobulin reaction ( RCLAAR)

.

only individuals with allergic symptoms to castor bean had circulating antibodies of the IgE class. Some of these were tested with PCA in baboons with postive results. These sera also had high IgE levels (Johansson and Bennich, 1970). The RCLAAR is a most valuable tool in research. The lack of a suitable coupling principle, however, is a disadvantage. Pollen allergens are not easily coupled by photooxidation and some allergens, i.e., the allergenic substances in Ascaris antigen preparations, seem to be damaged by the coupling reagents (Coombs, 1970).

B. THERADIOALLERGOSORBENT TEST The RAST is an antiglobulin reaction based upon the detection of IgE by lz5I-1abeledimmunoadsorbent purified antibodies specific to IgE, on particles to which allergens have been coupled (Fig. 16). A typical experiment is shown in Fig. 17. A positive control, consisting of serum from an allergic 5atient with high concentration of circulating reagins to the particular allergen to be analyzed, and a negative control, consisting of serum from a healthy individual, are always in-

47

HUMAN IMMUNOGLOBULIN E

C

6696

0 K

5810

S A

7279

1

I'

L J-M7278

7268

W L F K

7245

2 U

7227

A. P.

7225

D E.

7179

1 I

I

Control, positive ( 5 2 7 8 )

-

11-

negative

200

500

1000 cpm

2000

5000

FIG. 17. Radioallergosorbent test estimation of IgE antibodies to birch pollen allergen in serum of atopic children. The blocks represent the amount of radioactivity bound to the respective APC. A serum with high concentration of IgE antibodies to the allergen was used as a positive control, and a serum of a healthy individual with low IgE concentration was used as a negative control.

500

1

200 1 00

50 "0

Lx 20 E

," to 5 2 1/1000 1/4001/200 V80 1/20 118 serum dilutions t

10

I00

1/2

1/1

4 000

Yo0of reference reaginic serum

FIG.18. Quantitation of IgE antibodies by radioallergosorbent test. A serum from an untreated birch pollen sensitive patient (0) was used as reference serum. A good parallelisnl was obtained with a serum containing IgE antibodies to horse dandruff ( 0 )but not with a serum from a patient allergic to dog dandruff subjected to specific hyposensitization for several years ( 0) .

48

HANS BENNICH AND S . GUNNAR 0. JOHANSSON

cluded in the experiment. The amount of radioactivity on the particles is roughly proportional to the amount of reagin in the sample. When allergic sera are analyzed in different dilutions, parallel lines are obtained (Fig. 18). This is also the case when different allergen-polymercomplexes, APCs, are used, Thus, it is possible to use an allergic serum or a serum pool as a 100%reference preparation (Johansson et aZ., 1971f). Particle-coupled IgE has also been used as a standard (Stenius and Wide, 1969). The sensitivity of the method has been compared with the P-K reaction in a fish system, and it was found that the sensitivity of RAST is of the same order as that of the P-K test. A problem in allergological investigations is the present lack of properly standardized allergens. Characterization of a number of commercially available pollen and dandruff allergens using RAST reveals both qualitative and quantitative differences between different products as well as batches of one and the same allergen (Johansson et al., 1971h). One example will be described of an unexpected reaction obtained as a result of the variation in quality of allergens. Using a commercial preparation of cow dandruff, positive reactions in RAST were obtained in 40% of sera from healthy adults and children. The reaction was completely inhibited by cow dandruff allergen (more than 1 mg./ml.), cow milk allergen (0.1 mg./ml.), bovine serum (diluted 1:100), bovine y-globulin (0.01 mg./ml.), or sheep y-globulin (0.01 mg./ml.). No inhibition was obtained with r-globulins from rabbit, pig, horse, or guinea pig (up to 1 mg./ml.). Since the cow dandruff allergen was found to contain bovine serum y-globulin (250 pg./ml.) and the labeled anti-IgE was of sheep origin, the explanation for the “false” RAST reaction is that the sera contain heterophilic antibodies reacting with immunoglobulins from cow and related species (Johansson et al., 1971e). The frequent occurrence of heterophilic antibodies deserves particular attention since they may interfere in several immunological tests. C. ALLERGEN ANTIBODIESIN SERUM In a study of 31 adults with asthma and hay fever (Wide et al., 1967), the result of RAST was compared with that of intradermal provocation tests. In 47 of 140 skin tests the results were positive both in RAST and in the skin, and in 47 the results were negative in both tests, giving an agreement of 68%. Positive skin tests with negative RAST were found in 41 tests and negative skin reactions with positive RAST were found in 5 tests. Better correlation was found when RAST was compared with provocation tests. In 28 tests, both methods were positive, and in 21 tests, both were negative, out of the 51 tests compared which corresponds to a 96%agreement. When RAST was compared with

49

HUMAN IMMUNOGLOBULIN E

independently performed provocation tests in a study of about 100 allergic children slightly different results were obtained (Berg et al., 1971b). An overall correlation of 74% was obtained with no significant differences between bronchial or rhinoconjunctival provocation tests. However, in patients positive to diluted allergen solutions, the correlation increased to about 95% (Table VIII). The RAST can also be used to detect IgE antibodies to penicillin (Juhlin and Wide, 1970), to mite allergens in dust (Stenius and Wide, 1969), and to allergens from several parasites, such as Ascaris lumbricoides, Toxocara canis (Johansson et al., 1971b), NecatoT americanus (Johansson et al., 1971d), and Echinococcus (Johansson et at., 1971g). The reagin titers in sera from untreated children with hay fever sensitive to birch and timothy pollens was followed during pollination season (Berg and Johansson, 1971). All children showed an increase in reagins. The titer increased rapidly, and the mean changed over 400%. In 1 case, an increase from 6 to 100%(relative to a standard reaginic serum) was found to take place within 1 month. But the decrease after the pollination season was slower, the mean decreasing about 308. The level of IgE antibodies has also been followed in the immediate course of “rush desensitization” ( Berg and Johansson, 1970). The mean level was 160% of the initial value after 2 to 4 weeks observation. VIII. Metabolism

The serum immunoglobulin concentrations are the result of synthesis, catabolism, and pattern of distribution. Low serum levels of IgE limit studies of the IgE metabolism to the use of labeled myeloma proTABLE VIII CORRELATION BETWEEN POSITIVE PROVOCATION TESTSA N D RADIO.~LLERGOSORBENT TEST(RAST) I N RELATION TO THE DEGREEOF SENSITIVITY~ Positive provocation tests with allergen dilutionb Any dilution 1 : 10 (w/v) 1 : 100 (w/v) 1 : 1000 (w/v) or more a

No. of tests 283 107 76 54

Agreement with RAST 217 66 70 54

(76.7%) (61.2%) (92.1%) (100%)

Data from Berg et al. (1971b).

* The allergen dilution given represents the highest dilution giving a positive bronchial (in asthma) or rhinoconjunctival (in rhinoconjunctivitis) provocation test. Thus increasing allergen dilution indicates increasing sensitivity.

50

HANS BENNICH AND S. GUNNAR 0. JOHANSSON

tein. That this could introduce systematic errors is indicated by observations that the subclasses of IgG differ in catabolic rates (Spiegelberg et al., 1968; Morrell et al., 1969). The metabolism of IgE and IgG was studied in patient ND using "Y-labeled IgE( ND) and 1311-labeledIgG. The VIP ratio was constant during the whole experiment which indicates that native protein was used in the experiment (Fig. 19). The fractional catabolic rate, calculated as VIP ratio was 15.8%of the intravascular pool per day. The extravascular-intravascular ratio calculated on day 25, was 11.0 (Norberg et al., 1971). However, the extravascular fraction calculated in this way might give too high values if the isotope is excreted through other ways than by the kidneys ( Ahlinder et al., 1968). The halflife for IgE( ND) in patient ND was 4.4days calculated from the plasma curve. Studies of the IgE metabolism in 2 other patients gave a very rapid decrease of intravascular labeled IgE ( ND) and a correspondingly high excretion in the urine. The mean catabolic rate for IgE during 2 weeks in a case of IgG myeloma was 95.8%and in a terminal case of cerebral hemorrhage, 90.4%.Since the decrease was so fast it was not possible to calculate the extravascular distribution on day 25. However, the VIP ratios were in both cases constant during the whole period of observation and at the same high level as found in patient ND. The halflife in the IgG myeloma patient was 0.7 day. The catabolic rate in the homologous studies reported here are in agreement with the results for IgE given by Waldmann (1969). However, the half-life reported by Waldmann was 2.3 days. Immunoglobulin E has some characteristics that might complicate the

---._. -.-._Retained Qse ExtroMxulor

Catabolism "/P

. *.

0

5

10

15

20

lntnrmscular

25

Days

FIG. 19. Turnover studies in E myeloma patient ND using "'I-labeled IgE( N D ).

HUMAN IMMUNOGLOBULIN E

51

metabolic studies performed in the usual way. The high affinity of IgE to some circulating cells and peripheral organs might complicate metabolic studies giving other distribution ratios than those upon which the calculation formula is based. Thus the halflife of persistence of human reagins in human skin investigated by the P-K principle was found to be in the order of 7 to 13 days (Stanworth and Kuhns, 1965; Augustin, 1967). If the major part of IgE is not catabolized in the intravascular space, other methods have to be used for the calculations. In a turnover experiment, about 2 mg. of labeled IgE( ND) is given to the patient which is a very large amount relative to the total body pool of healthy individuals. IX. Concluding Remarks

The nature of the circulating factor underlying the imniediate-type hypersensitivity reaction, first reported by Ramirez, has for the last 50 years been the subject of intense studies. The elaborate immunochemical studies on reaginic antibodies to ragweed allergen E by Ishizaka and Ishizaka and coworkers and the discovery of the atypical myeloma protein ND and its normal counterpart has directed a new approach to the problem. The access of iinniunological reagents to IgE makes it possible to study the role of IgE in the immune system by serological methods. The beneficial function of IgE is still obscure. Persistence of the immunoglobulin class through evolution indicates that it might have some kind of a positive function. Atopic diseases do not seem to be a condition that promotes the survival of the species. Perhaps the solution is to be found in the widespread occurrence of IgE-medicated reactions in parasitic infestations in man and animaIs.

REFERENCES Aas, K. ( 1970). Personal communication. Ahlinder, S., Birke, G., Norberg, R., Olhagen, B., Plantin, L. O., and Reizenstein, P. (1968). Acta Med. Scand. 184, 25. Aldrich, R. A,, Steinberg, A. G., and Campbell, D. C. (1954). Pediatrics 13, 133. Aftounyan, R. E. C. (1967). Acta Allergol. 22, 487. Ammann, A. J., Cain, W. A., Ishizaka, K., Hong, R., and Good, R. A. (1969). N . Engl. J . Med. 277, 250. Anderson, B. R., and Vannier, W. E. (1964). J. E x p . Med. 120, 31. Amer, B., Bertler, A., and Johansson, S. G. 0. ( 1971). Acta Allergol. In press. Augustin, R. ( 1967). I n “Handbook of Experimental Immunology” (D. M. Weir, ed.), p. 1098. Blackwell, Osford. Augustin, R., and Hayward, B. J. (1960). Immunology 3, 45. Avrameas, S., and Ternynck, T. (1967a). J. Biol. Chem. 242, 1651. Avrameas, S., and Ternynck, T. (19671,). Biochem. J. 102, 37c. AxCn, R., Porath, J., and Ernback, S . (1967). Nature ( L o n d o n ) 214, 1302.

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Ball, P. A. J., and Barlett, A. (1969). Trans. Roy. SOC. Trop. Med. Hug. 63, 362. Bennich, H. (1968). Acta Uniu. Upsaliensis 53. Belmich, H. (1969). Int. Symp. Brit. Allergy Soc., 1969. Bennich, H. ( 1970 ). Unpublished observations. Bennich, H., and Clamp, J. R. (1971). In preparation. Bennich, H., and Johansson, S. G. 0. ( 1967). In “Gainma Globulins, Structure and Control of Biosynthesis” ( J . Killander, ed.), p. 199. Almqvist & Wiksell, Stockholm. Bennich, H., and Johansson, S. G . 0. (1968). Unpublished observations. Bennich, H., and Johansson, S. G . 0. (1971). In preparation. Bennich, H., Ishizaka, K., Johansson, S. G. O., Rowe, D. S., Stanworth, D. R., and Terry, W. D. (1968). B t d . W.H.O. 38, 151. Bennich, H., Johansson, S. C . O., and Stanworth, D. R. (1969a). Acta Pathol. Microbiol. Scand. 77, 333. Bennich, H., Ishizaka, K., Ishizaka, T., and Johansson, S. G. 0. (1969b). J . Immunol. 102, 826. Bennich, H., Johansson, S. G . O., and Stanworth, D. R. (1971). In preparation. Berdal, P. (1954). Actu Oto-Laryngol., Suppl. 115, 7 . Berg, T. (1968). Acta Paediat. Scand. 57, 369. Berg, T. (1969). Acta Paediat. Scand. 58, 229. Berg, T., and Johansson, S. G. 0. (1967a). Acfa Paediat. Scand. 56, 430. Berg, T., and Johansson, S. G. 0. (1967b). Acta Paediat. Scand., Suppl. 177, 93. Berg, T., and Johansson, S. G . 0. (1969a). Int. Arch. Allergy Appl. Immunol. 36, 219. Berg, T., and Johansson, S. G . 0. (1969b). Acta Paediat. Scand. 58, 513. Berg, T., and Johansson, S. G . 0. (1970). Acta. Paediat. Scand. 59, Suppl. 206, 85. Berg, T., and Johansson, S. G . 0. (1971). To be published. Berg, T., Foucard, T., and Johansson, S. G. 0. (1971a). To be published. Berg, T., Bennich, H., and Johansson, S. G . 0. (1971b). Int. Arch. Allergy Appl. Immunol. 40, 770. Berggird, I. ( 1970). Personal communication. Berglund, G., Finnstrijin, O., Johansson, S. G . O., and Moller, K. L. (1968). Acta Paediat. Scand. 57, 89. Bjernulf, A., Johansson, S. G . O., and Parrow, A. (1971). Acta Med. Scund. (in press ) . Brandtzaeg, P. (1965). Arch. Oral Biol. 10, 795. Cain, W. A., Ammann, A. J., Hong, R., Ishizaka, K., and Good, R. A. (1969). J . Clin. Inuest. 48, 12A. Catt, K., and Tregear, G . W. (1967). Science 158, 1570. Catt, K., Niall, H. D., and Tregear, G. W. (1967). J . Lab. Clin. Med. 70, 820. Claman, H. N., and Merrill, D. (1964). J . Lab. Clin. Med. 64, 685. Collins-Williams, C., Tkachyk, S. J., Toft, B., and Moscarello, M. (1967). h t . Arch. Allergy Appl. Immunol. 31, 94. Coombs, R. R. A. (1970). Personal conimunica t’ion. Coombs, R. R. A., Howard, A. W., and Mynors, L. S. (1953). Brit. J . Exp. Pathol. 35, 472. Coombs, R. R . A., Hunter, A., Jonas, W. E., Bennich, H., Johansson, S. G. O., and Panzani, R. (1968). Lancet 1, 1115. Dandliker, W. B., and de Saussure, V. A. (1968). Immunochemistry 5, 357. Deuschl, H., and Johansson, S. G. 0. (1970). Unpublished data. Donovan, R., Johansson, S. G. O., Bennich, H., and Soothill, J. F. (1970). Int. Arch. Allergy Appl. Immunol. 37, 154.

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Dorrington, K. J., and Bennich, €1. (1971). In preparation. Edelhoch, H. ( 1967). Biochemi.stry 6, 1948. Ehrenherg, A. (1957). Acfu C/icm. Scand. 11, 1257. Franklin, E. C., and Kunkel, H. C;. ( 1958). J. Lab. C h i . Med. 52, 724. Fudenl,crg, I I. ( 1970). Personal commrinication. Girard, J.-P. ( 1967). I n t . Arch. Allerg!/ Appl. ImnrunoL 32, 294. (ileich, C . J., Avcrbc~k,A . K., and S\\wtllund, H. A. ( 1971 ). J. Lab. C h . Med. 77, 890. Gyenes, L., antl Sehon, A . H. (1960). Can. J. Biochem. Physiol. 38, 1249. Hobbs, J. R., Hepner, G. W., Douglas, A. P., CrabbC., P. A., and Johansson, S. G . 0. (1969). Lancet 2, 649. Hogarth-Scott, H. S. (1966). Immunology 10, 217. Hogarth-Scott, R. S., Johansson, S. G. O., and Bennich, H. (1969). Clin. E x p . Immunol. 5, 619. Ishizaka, K., and Ishizaka, T. (1967). J. Immunol. 99, 1187. Ishizaka, K., and Ishizaka, T. ( 196%). J. Allergy 42, 330. Ishizaka, K., and Ishizaka, T. (196811). J. Irrmrcnol. 100, 554. Ishizaka, K., and Ishizaka, T. (1970). Clin. E x p . Immunol. 6, 25. Ishizaka, K., Ishizaka, T., and Hornbrook, M. M. (196Ga). J. Immunol. 97, 75. Ishizaka, K., Ishizaka, T., and Hornbrook, M. hl. (1966b). J. lmmunol. 97, 840. Ito, K., Wicher, K., and Arbesman, C. E. (1969). J. Immunol. 103, (322. Johansson, S. G. 0. (1967). Lancet 2, 951. Johansson, S. G. 0. (1968a). lilt. Arch. Allergy Appl. Immunol. 34, 1. Johansson, S. G. 0. (196%). Acta Uniu. Upsaliensis 52, 11. Johansson, S. C. 0. (1971). To be published. Johansson, S. G. O., and Bennich, H. (1967a). Immunology 13, 381. Johansson, S. G. O., and Bennich, H. (1967b). In “Gamma Globulins, Structure and Control of Biosynthesis” ( J . Killander, ed.), p. 193. Almqvist & Wiksell, Stockholm. Johansson, S. G. O., and Bennich, H. (1970). Unpublished results. Johansson, S. G . O., and Berg, T. (1967). Acta Paediat. Scand. 56, 572. Johansson, S. G. O., and Berg, T. (1971). To be published. Johansson, S. G . O., and Foucard, T. (1971). To be published. Johansson, S. C;. O., and Jithlin, L. (1971). B r i t . J. Dermutol. In press. Johansson, S. G. O., Bennich, H., and Wide, L. (1968a). Immunology 14, 265. Johansson, S. G. O., Hogman, C. F., and Killnnder, J. (1968b). Acta Pathol. Alicrobiol. Scand. 74, 519. johansson, S. G. O., Mellbin, T., and Vahlqvist, B. ( 1 9 6 8 ~ )Luncet . 1, 1118. Johansson, S. G. O., Bennich, H., and Foucard, T. (1971a). Acta Pathol. Microbiol. Scand. (in press). Johansson, S. G. O., Hogarth-Scott, H. S., and Bennich, H. (1971b). To be published. . be published. Johansson, S. G . O., Bennich, I f . , and Perhnann, P. ( 1 9 7 1 ~ )To Joharisson, S. G. O., Ball, P. A. J., Voller, A., and Bennich, H. (1971d). To be published. Johansson, S. G. O., Bennich, H., Foucard, T., and Lundkvist, U. (1971e). Acta Patliol. Microbial. Scand. (in press). Johansson, S. C . O., Bennich, H., and Berg, T. (1971f). Int. Arch. Allergy Appl. Znimunol. In press. Johansson, S. C,. O., Bennich, II., antl Holdt, C. ( 1971g). To he pul)lished. Johansson, S. C. 0.. Bennich, H., Bcrg, T., and Foucard, T. (1971h). I n preparation. Juhlin, L., and Wide, L. (1970). Personal communication.

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HANS BENNICH AND S. GUNNAR 0. JOHANSSON

Juhlin, L., Johansson, S. G. O., Bennich, H., Hogman, C. F., and Thyresson, N. (1969). Arch. Dermatol. 100, 12. Killander, J., Holmberg, K., Bennich, H., and Johansson, S. G. 0. ( 1971). I n preparation. Kohler, P. F., and Farr, R. S. (1967). J. Allergy 39, 311. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965). lmmunochemistry 2, 235. Miller, F., and Metzger, H. (1965). 1. Biol. Chem. 240, 3325. Morrell, A,, Terry, W., and Waldman, T. (1969). Clin. Res. 17, 356. Nilsson, K., Bennich, H., Johansson, S. G. O., and Pontbn, J. (1970). Clin. Exp. Immunol. 7, 477. Norberg, R., Bennich, H., and Johansson, S. G. 0. ( 1971). In preparation. Nordbring, F., Hogman, C. F., and Johansson, S. G. 0. (1969). Scand. J. Infec. Dis. 1, 99. Nordbring, F., Hogman, C. F., Johansson, S. G. O., and Wahren, B. ( 1971). To be published. Ogawa, M., Kochwa, S., Smith, C., Ishizaka, K., and McIntyre, 0. R. (1969). N . Engl. J . Med. 281, 1217. dhman, S., and Johansson, S. G. 0. (1971). In preparation. Pepys, J., and Duveen, G. W. (1951). lnt. Arch. Allergy Appl. lmmunol. 2, 147. Porath, J., Axbn, R., and Emback, S. (1967). Nature (London) 215, 1491. Porter, R. R. (1959). Biochem. I . 73, 119. Reerink-Brongers, E. E. ( 1970). Personal communication. Rockey, J. H., and Kunkel, H. G. (1962). Proc. SOC. Exp. Biol. Med. 110, 101. Rockey, J. H., Hansson, L. A., Heremans, J. F., and Kunkel, H. G. (1964). J . Lab. Clin. Med. 63, 205. Rosenberg, E. B., Whalen, G. E., Bennich, H., and Johansson, S. G. 0. (1971). N . Engl. I. Med. 283, 1148. Rowe, D. S. (1969). Bull. W. H . 0. 40, 613. Rowe, D. S., and Fahey, J. L. (1965). J. E x p . Med. 121, 185. Rowe, D. S., Boyle, J. A., and Buchanan, W. W. (1968a). Clin. Exp. Immunol. 3, 233. Rowe, D. S., MeGregor, I. A., Smith, S. J., Hall, P., and Williams, K. (1968b). Clin. Exp. Immunol. 3, 63. Rowe, D. S., Bennich, H., and Johansson, S. G. 0. (1971). To be published. Salmon, S. E., Mackey, G., and Fudenberg, H. H. (1969). J. Immunol. 103, 129. Sehon, A. H. ( 1960). In “Mechanisms of Antibody Formation” (M. Holub and L. Jaroskovi, eds.), p. 79. Academic Press, Inc. New York. Sehon, A. H., and Gyenes, L. (1965). In “Immunological Diseases” (M. Samter and H. L. Alexander, eds.), pp. 519-538. Little, Brown, Boston, Massachusetts. Seligmann, M., Danon, F., Hurez, D., Mihaesco, E., and Preudhomme, J.-L. (1968). Science 162, 1396. Spiegelberg, H. L., Fishkin, B. G., and Grey, H. (1968). Fed. Proc. Fed. Amer. SOC. Exp. Bibl. 27, 731. Stanworth, D. R. (1963). Aduan. lmmunol. 3, 181. Stanworth, D. R., and Kuhns, W. J. (1965). Immunology 8, 323. Stanworth, D. R., Humphrey, J. H., Bennich, H., and Johansson, S. G. 0. (1967). Lancet 2, 330.

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Stanworth, D. R., Humphrey, J. H., Bennich, H., and Johansson, S. G. 0. (1968). Lancet 2, 17. Stanworth, D. R., Housley, J., Bennich, H., and Johansson, S. G. 0. (1970). Immunochemistry 7, 321. Stenius, B., and Wide, L. (1969). Lancet 2, 455. Stiehni, E. R., and Fudenberg, H. H. (1966). Pediatrics 37, 715. Tada, T,, and Ishizaka, K. (1970). J. Immunol. 104, 377. Thieffry, S., Arthris, M., Aicardi, J., and Lyon, G. (1961). Reo. Neurol. 105, 390. Turner, M. W., and Johansson, S. G. 0. (1971). To be published. Turner, M. W., and Voller, A. (1966). J. Trop. Med. Hyg. 69, 99. Turner, M. W., Johansson, S. G. O., Barrat, T. M., and Bennich, H. (1970). Int. Arch. Allergy Appl. Immunol. 37, 409. Voller, A., Ball, P. A. J., Bennich, H., and Johansson, S. G. 0. ( 1971). To be published. Wager, O., Rkanen, J. A,, Lindeberg, L., and Makela, V. (1969). Actu Pathol. Microbiol. Scand. 75, 350. Waldmann, T. A. (1969). N . Engl. J. Med. 281, 1170. Whalen, G. E., Rosenberg, E. B., Strickland, G. T., Gutman, R. A., Cross, J. H., and Watten, R. H. (1969). Lancet 1, 13. Wide, L., and Porath, J. (1966). Biochim. Biophys. Actu 130, 257. Wide, L., Bennich, H., and Johansson, S. G. 0. (1967). Lancet 2, 1105. Wiskott, A. ( 1937). Monatsschr. Kinderheilk. 68, 212. Wollheim, F. A. (1968). Scand. J. Haematol. 5, 97. Young, R. R., Austen, K. F., and hloser, H. W. (1964). Medicine (Baltimore) 43, 423. Yphantis, D. A. (1964). Biochemistry 3, 297.

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Individual Antigenic Specificity of Immunoglobulins JOHN E. HOPPER AND ALFRED NISONOFF Deportment o f Medicine, Prifzker School of Medicine, University o f Chicogo ond Deportment of BiologicalChemistry, University o f Illinois College of Medicine, Chicago, Illinois

I. Introduction . . . . . . . . . . . . 11. Individual Antigenic Specificities in Monoclonal Proteins. . . 111. Individual Antigenic Specificities in Antibody Populations . . A. Qualitative Analysis of Individual Antigenic Specificities in Antibodies . . . . . . . . . . . B. Quantitative Investigations of Idiotypic Specificities . . IV. Cross-Reactions of Antiidiotypic Sera and Evidence for Identical Molecules in Different Individual Animals . . . . . . A. Idiotypic Cross-Reactions of Antibodies from Different Donors . . . . . . . . . . B. Unrelatedness of Idiotypic Determinants in Antibodies of . . . Different Specificity from the Same Individual C. Cross-Reactivity among Myeloma Proteins; Presence of Identical Molecules in Different Individuals . . . . . D. Evidence Based on Amino Acid Sequence for the Presence of . Identical Immunoglobulin Molecules in Different Individuals V. Evidence Based on Idiotypic Specificity for Limited Heterogeneity of Normal Antibody Populations . . . . . VI. Persistence and Changes of Antibody Populations during Prolonged Immunization . . . . . . . . . A. Evidence Based on Idiotypic Specificities . . . . . B. Evidence Based on Amino Acid Sequence Analysis for Persistence of Similar or Identical Molecules in Serum . . VII. Shared Idiotypic Determinants in IgC and IgM Antibodies of the Same Specificity . . . . . . . . . . A. Shared Determinants in Anti-Salmonella Antibodies . . . B. Structural Relationship between IgC and IgM Produced by a Clone of Cells . . . . . . . . . . VIII. Localization of Individually Specific Antigenic Determinants . . A. Localization of a Major Idiotypic Determinant to the Region of the Combining Site of an Antibody Molecule . . . . B. Individually Specific Determinants in Isolated Heavy and Light Chains and in Reconstituted Molecules; Determinants . . . . . Dependent on Native Conformation . C. Localization of Individually Specific Determinants of Isolated Light Chains to the Variable Region . . . . . . IX. Cross-Reactions of Anti-ind Antibodies with Nonspecific Immunoglobulins . . . . . . . . . . . 57

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X. Monoclonal Origin of Molecules with Individually Specific Antigenic Determinants

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I. Introduction

Monotypic immunoglobulins, such as myeloma and Bence-Jones proteins, and antibody populations from individual animals possess individually specific antigenic determinants ( Slater et al., 1955; Oudin and Michel, 1963; Kunkel et al., 1963; Gell and Kelus, 1964). For example, one can prepare an antiserum in a rabbit which when appropriately absorbed will react with the human myeloma protein used as immunogen but, with rare exceptions, not with any other myeloma protein or immunoglobulin (Slater et al., 1955). Alternatively, one can immunize a rabbit with antibody from an individual human or rabbit and elicit specific anti-antibodies (Kunkel et al., 1963; Oudin and Michel, 1963; Gell and Kelus, 1964). Oudin (1966) has proposed the term, “idiotypic determinants,” to designate those antigenic determinants in a population of antibody molecules which are not observed in other immunoglobulins of the donor animal nor in antibody directed to the same antigen from other animals of that species. The restriction of idiotypic determinants to antibodies of a single specificity from an individual donor clearly differentiates idiotypy from allotypy, since allotypic determinants are shared by the various antibodies of an individual. As currently defined, idiotypy applies to determinants recognized by an antiserum prepared in the same species, and the term, individually specific antigenic determinant, is generally used with reference to determinants defined by an antiserum prepared in a heterologous species, such as a rabbit antiserum directed to a purified human antibody or myeloma protein ( Kunkel, 1970). There may be no essential difference between idiotypic and individually specific antigenic determinants since each must reflect a unique amino acid sequence, characteristic of a given myeloma protein or antibody, and, therefore present in the variable regions of the polypeptide chains. However, it has not been established whether or not antisera from the homologous or a heterologous species recognize the same determinants. A contribution of the constant region to an individually specific determinant is possible, but individually specific differences among proteins must reflect differences in the variable regions. It may be noted that isotypic and allotypic antigenic determinants similarly reflect particular amino acid sequences, which in many instances have been defined.

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For brevity, we shall frequently use the terms “ind determinants” and “anti-ind antibodies” in referring to individually specific antigenic determinants and their corresponding antibodies. It is noteworthy that antiidiotypic antibodies can be prepared against antibodies from an individual donor and that the latter are almost always heterogeneous. It is apparent, therefore, that an antiidiotypic antiserum may recognize several specificities present in the donor population. The frequent appearance of multiple lines in double-diffusion experiments in agar gel supports this view. In a sense this is also true of a homogeneous myeloma protein, since unrelated determinants niay be present on the same molecule. (This is probably the case since precipitates are sometimes formed in the reactions of Fab fragments of myeloma proteins with anti-ind antisera, indicating multivalency of the antigen; however, as would be expected, only a single line is ordinarily observed in a double-diflcusion experiment. ) The simplest explanation of the existing data on myeloma proteins and antibodies is that an itid or idiotypic determinant is characteristic of a population of immunoglobulin molecules produced by a single clone of cells (Gel1 and Kelus, 1964). This concept has been utilized by a number of investigators in the interpretation of their results. Oudin and Michel (1969a) have pointed out that idiotypy in proteins may be restricted to the immunoglobulins; i.e., that other proteins do not have structures that are different in each individual of a species. However, if all antibody sequences are encoded by germ-line genes, the variations observed could reflect the expression of different genes in different individuals, who might possess essentially the same capabilities. If, on the other hand, antibody diversity is based on somatic mutation the number of potential scquences may be so large that the capacity of an individual to produce a unique antibody molecule may be a valid concept. It is now evident that occasional cross-reactions may be observed among the idiotypic determinants of antibodies of different individuals although so far structural identity has not been established. Also, monoclonal L chains from different mice with multiple myeloma have been shown to have identical amino acid sequences (Appella and Perham, 1967; Weigcrt et al., 1970; Hood et al., 1970), and there is evidence for structural identity of the L chains of two independently derived mouse myeloma proteins ( Potter and Lieberman, 1970) which share ind determinants. The original definitions of idiotype and iiidividually specific antigenic determinant do not specifically consider such cross-reactions. In our estimation, the discovery of cross-reactions or identity among ininiunoglobulins from different donors would not com-

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promise the usefulness of these terms, and the definitions might be qualified accordingly. The existence of idiotypic determinants has proved to be very useful in pursuing a number of lines of investigation relating to the structure and biosynthesis of antibodies. These include the following. ( a ) Strong idiotypic cross-reactions have been observed among certain mouse myeloma proteins which combine with the same antigen. ( b ) Idiotypy has been a useful marker in probing the onset, persistence, and replacement of antibody molecules of a given structure during the course of prolonged immunization. These data have been interpreted as reflecting the persistence and replacement of clones of antibody-forming cells. ( C) Evidence has been adduced for the biosynthesis of IgG and IgM by a single clone of cells, and a relationship between the primary structures of molecules of the two classes produced by a single clone has been proposed. ( d ) The region of the combining site of an antibody molecule has been found to be an important idiotypic determinant. ( e ) The role of heavy and light chains in idiotypy has been explored. ( f ) Evidence has been secured for the existence of homogeneous subpopulations in the heterogeneous antibody of a single specificity from an individual donor. ( g ) The broad range of idiotypic specificities of antibodies directed to a single antigen or hapten has provided additional insight into the great diversity of antibody molecules. In the following sections experimental results will be considered in detail and some of their implications explored. II. Individual Antigenic Specificities in Monoclonal Proteins

Before the discovery of individual antigenic specificities of myeloma proteins, it had been known for some time that such proteins are deficient in antigenic determinants as compared with nonspecific immunoglobulin preparations. This was taken generally as evidence for the abnormality of these monoclonal proteins. The present interpretation would be that a myeloma protein has H and L chains belonging to just one subclass or type and would, therefore, react with only part of the antibodies produced against pooled nonspecific immunoglobulin of the same class. Also, in some of the earlier work, the myeloma proteins studied may have been of the IgA class but were tested against antiserum prepared against a mixture of IgG and IgA. In any event, research on the various antigenic specificities in myeloma and Bence-Jones proteins provided the basis for the eventual elucidation of classes and subclasses of heavy chains and of the light chain types. The experiments of Wuhrmann et al. (1950) suggested the presence of individually specific antigenic determinants in myeloma proteins.

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Injection of whole globulin fractions derived from the sera of patients with multiple myeloma elicited antisera in rabbits which, after absorption with normal globulin, reacted only with the homologous myeloma globulin preparation and not with a variety of other myeloma or normal sera. Unfortunately, these reactions were carried out with globulin fractions, and in only one instance was the antiserum tested against untreated donor serum. Some of these results might, therefore, conceivably have been explained by denaturation of the globulin during precipitation with methanol, thus giving rise to unique determinants. Some early evidence for individual antigenic specificity in macroglobulins from patients with Waldenstrom’s disease was presented by Habich ( 1953). Rabbit antisera were prepared by immunization with the whole serum of the patient. After absorption with normal serum and with serum rich in IgM from another patient, the rabbit antiserum formed precipitates with the serum of the donor but not with those of several other patients. It was not specified whether negative results were observed with all of the panel of sixteen available sera with high concentrations of IgM. The first unequivocal demonstration of individual antigenic specificity was made by Slater et al. (1955), who studied myeloma proteins from 21 patients. The proteins were classified as y- or ,@globulins according to electrophoretic mobility; from their properties these proteins may have been IgG and IgA, respectively. Antisera to individual myeloma proteins gave maximal precipitation at equivalence with the protein used as immunogen; y-myeloma proteins cross-reacted with other y proteins more strongly than with /3-myeloma proteins and vice versa. Absorption experiments showed that each of several myeloma proteins tested possessed individual antigenic specificities. Thus, rabbit antiserum to a given myeloma protein failed to precipitate with any myeloma protein after absorption with the immunogen; however, precipitin reactions with the immunogen persisted after absorption with nonspecific y-globulin or with any of the twenty heterologous myeloma proteins. In the Ouchterlony test, with the homologous and a heterologous protein placed in adjacent cells, the homologous protein formed a spur in the reaction with its unabsorbed antiserum. These results were confirmed by Korngold and Lipari (1956) who prepared antibodies in rabbits against three human myeloma proteins, migrating in the slow y-, fast 7-, and p-globulin regions, respectively. Tests of specificity were carried out by precipitin analysis with twentyfour myeloma globulins. Each of the three imniunogens possessed individually specific determinants. All of the twenty-four proteins had determinants in common with nonspecific y-globulin.

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Korngold and Van Leeuwen (1957a,b) and, at about the same time, Boerma and Mandema (1957) extended these findings to include human macroglobulins isolated from sera of patients with Waldenstrom’s disease. With appropriately absorbed rabbit antisera, it was shown by the Ouchterlony method that each macroglobulin possessed determinants not present in any of the several other macroglobulins tested. Similar investigations were carried out with human Bence-Jones proteins ( L chains) of the kappa and lambda types by Stein et al. ( 1963), who also used rabbit antisera. Appropriate absorptions revealed the presence of antigenic determinants present only in the immunizing antigen and not in any other of the small panel of Bence-Jones proteins tested. Numerous subsequent investigations have amply confirmed these findings. It appears that each myeloma or Bence-Jones protein possesses unique, individually specific antigenic determinants, demonstrable with antisera prepared in a heterologous species and suitably absorbed. Precipitin tests in agar gel have generally been employed. Cross-reactions, which have been observed among certain mouse myeloma proteins, will be considered later. Mehrotra (1960) observed that cold agglutinins of patients with acquired hemolytic anemia possess individual antigenic specificities; by analogy with subsequent findings, these proteins were probably all of the IgM class, although this was not establisbnd. The methods he used were similar to those employed in the investigations already summarized. Absorbed rabbit antisera and a panel of seven cold agglutinins were employed, and individual specificity was found in each protein by the precipitin test. The presence of individual antigenic specificities in 19 S cold agglutinins was confirmed in the studies of Harboe and Deverill ( 1964), who utilized rabbit antisera prepared against cold agglutinins from 6 patients. Individual antigenic specificities were also noted by Williams et al. (1968) in each of ten human cold agglutinins of the IgM class investigated by precipitin techniques with rabbit antisera. In addition, however, they showed that these proteins possessed other antigenic specificities common to cold agglutinins from many individuals but not present in any of the fifty Waldenstrom macroglobulins tested. The common specificities were demonstrated with partially absorbed antisera. Individual antigenic specificity was identified by spur formation in the Ouchterlony test, showing unique determinants on the immunogen not present in other cold agglutinins. Hypotheses put forth to explain these results were: ( a ) the combining site of the cold agglutinin is an antigenic determinant, and, thus, cold agglutinins may have similar

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combining sites, and ( b ) the cold agglutinins belong to a minor subgroup of IgM. Arguing against the first hypothesis was the observation that cross-reactions included cold agglutinins of different blood group antigenic specificities ( I and i ) . It is of interest that one of the crossreacting cold agglutinins had light chains of the lambda type; the remainder were kappa. The findings of Williams et al. (1968) define a unique category of antigenic specificities, present in a group of molecules with similar function-in this case the capacity to agglutinate erythrocytes at low temperature. An apparently related observation was made by Hannestad et al. (1970), who investigated two Ighl monoclonal proteins with different electrophoretic mobilities, isolated from the serum of a single patient. Both proteins reacted specifically with a trinitrophenyl derivative of hemocyanin. The IgM proteins were not identical in specificity, however, since only one of them reacted with a Klebsiella polysaccharide. Each protein had individual, unrelated ind determinants but also held in common other antigenic determinants that were not found in ninety-nine other serums from patients with Waldenstrom’s macroglobulinemia or rheumatoid arthritis. Individually specific antigenic dcterminaiits in monoclonal IgG and IgM have been localized to the Fab fragment produced by proteolysis of these proteins (Grey et al., 1965; Seligmann et al., 1966; Wang et al., 1970a; Solheim et al., 1971). Ill. Individual Antigenic Specificities in Antibody Populations

A. QUALITATIVE ANALYSIS OF INDIVIDUAL ANTIGENIC SPECIFICITIES IN ANTIBODIES A major advance was made in two laboratories when it was discovered that antibody populations frequently possess unique antigenic specificities (Kunkel et al., 1963; Oudin and Michel, 1963). At the same time similar studies were being carried out by Gel1 and Kelus (1964). Oudin and Michel elicited anti-Salmonella t y p h i antibodies in individual rabbits, coated bacteria with these antibodies, and immunized individual recipient rabbits with the antibody-bacteria complexes. After prolonged immunization, antibodies were produced in recipients which reacted, in precipitation or passive cutaneous anaphylaxis tests, with the hyperimmune serum of the donor rabbit but not with the preimmune serum of the donor, nor with hyperimmune (anti-Salmonella) serums of 17 other rabbits. By immunoelectrophorcsis the antigenic reactivity was shown to reside in the 7-globulin fraction of the donor’s serum; this reactivity was almost completeIy removed by absorption of that serum

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FIG. 1. Double-diffusion reactions in agar gel of antiidiotypic antiserum directed to anti-SalmoneZlu antibody from a donor rabbit. The antiidiotypic antiserum was placed in a trough across the lower edge of the figure. A trough comprising three sections (SI, SII, and SV) was present across the upper edge. ( S I ) Preimmune serum from the donor rabbit; ( S I I ) hyperimmune (anti-Salmonella antiserum; ( S V ) serum taken from the same rabbit 11 months later, with no intervening inoculations. [Reprinted, by permission, from Oudin and Michel ( 1963).]

with Salmonella typhi. The reactivity of the donor serum disappeared after 11 months without challenge by Salmonella (Fig. 1). Thus, the reactivity of the donor serum was attributable to the presence of antiSalmonella antibodies and was not observed in anti-Salmonella antibodies of other rabbits. Although the allotypes of the donor and recipient animals were not specified, allotypy did not appear to play a significant role in these experiments since pre- and postimmune donor sera were not reactive with the anti-antibodies. Kunkel et al. (1963) demonstrated the presence of individual antigenic specificities in human immunoglobulins by utilizing rabbit antisera prepared against isolated human antibodies. For example specifically purified, human anti-A substance from an individual donor was used to immunize a rabbit, and the resulting antiserum was absorbed with normal human serum and y-globulin. Two of seven anti-A preparations tested elicited antibodies to individually specific antigenic determinants; the absorbed rabbit antibodies reacted only with the immunogen and not with anti-A antibodies from 7 other individuals, nor with a variety of other antibodies or normal sera. An immunoelectrophoretic pattern demonstrating reactions of absorbed and unabsorbed rabbit antiserum to purified anti-A antibody is shown in Fig. 2. The anti-A antibodies were shown to be 7 s y-globulins and their individually specific antigenic determinants were localized to the Fab or Fab’ fragments. The latter finding is consistent with the fact that Fragment Fc is common to immunoglobulins of a given class; one would, therefore, not expect it to express individual antigenic specificity. Kunkel et al. (1963) also demonstrated individual antigenic specificity in one of two purified human antidextran antibody preparations

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65

FIG.2. Immunoelectrophoretic pattern showing the reaction of a serum containing human anti-A substance with a rabbit antiserum to anti-A antibodies specifically purified from that hunian serum. In each case the human anti-A antiserum was first subjected to electrophoresis. In the lower section the unabsorbed rabbit antiserum was placed in the trough; in the upper section the rabbit antiserum had been absorbed with normal human serum. The arc in the upper section, which has its counterpart also in the lower section, represents the reaction with individually specific antigenic determinants in the human anti-A antibody. [Reprinted, by permission, from Kunkel et al. (1963). Copyright 1963 by the American Association for the Advancement of Science.]

and in one human antilevan antibody. Since the panel of such antibodies available was small, proof of individual antigenic specificity rested on the failure to react with antibody preparations of different specificities from other individuals. Of considerable interest was their finding that two antibodies, anti-A substance and antilevan, isolated from the serum of one individual, possessed unrelated individual antigenic specificities. Investigations with specifically purified human anti-A antibodies were extended by Kunkel et al. (1966). Rabbit antibodies were prepared against a third human anti-A preparation, and a panel of twentyeight anti-A antibodies was used to test the absorbed rabbit antisera. Positive precipitin reactions were noted only with the homologous preparation. Extensive studies of idiotypic specificities in rabbit anti-Proteus vulgaris antibodies have been carried out (Gell and Kelus, 1964; Kelus and Gell, 1968). These investigators immunized rabbits with complexes of proteus with anti-proteus antibody from an individual donor rabbit. The allotypes of the donor and recipient were matched with respect to major specificities to minimize the possibility of eliciting antiallotype antibodies. Rabbits were immunized against anti-Proteus antibodies from 10 different donors and a panel of serums from 60 rabbits hyperimmunized to Proteus was used in testing for cross-reactions, In each instance the reactive antigen in the donor population was shown to be anti-

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Proteus antibody of the IgG class. Each antiidiotypic antiserum formed a precipitate only with anti-Proteus antibody of the donor and not with the donor’s preimmune serum, nor with anti-Proteus antibodies from the large panel of hyperimmunized rabbits, which in some cases included members of the family group of the donor. In each instance absorption of anti-Proteus antibody from a donor’s serum eliminated its reactivity with the antiidiotypic antiserum. No precipitation reactions were observed with the normal sera of 80 relatives and progeny of one of the donors. In 4 cases, autoimmunization of the rabbit was attempted with its own anti-Proteus antibody complexed to bacteria; no antibodies were produced that reacted with antiserum of high titer against Proteus, collected earlier from the same rabbit. The idiotypic determinants of the donor antibody were localized to the Fab fragment (as in the case of human antibodies, discussed above). In this large-scale investigation, Kelus and Gel1 were successful in eliciting antiidiotypic antibodies in nearly every attempt when immunization was sufficiently prolonged. The research we have summarized established the presence of ind or idiotypic determinants in myeloma proteins, macroglobulins, and Bence-Jones proteins as well as in human and rabbit antibodies directed to a variety of antigens. Although not all immunizations were successful in eliciting antiidiotypic antibodies, there appears to be no strong evidence so far against the possibility that essentially all monoclonal proteins and antibody populations possess i i 7 d or idiotypic specificities. The infrequency of idiotypic cross-reactions is consistent with the great heterogeneity of the variable regions of heavy and light chains of immunoglobulin molecules.

B. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC SPECIFICITIES The studies described so far utilized qualitative techniques, principally double diffusion in agar gel. A question of interest is the percentage of molecules in a donor myeloma or antibody population reactive with its anti-iiad or antiidiotypic antiserum. In the case of myeloma proteins, one might expect that this fraction would approach 100%;evidence that this is the case is presented below. However, for antibody populations that are heterogeneous, the answer to this question is not necessarily predictable. 1. Precipitating Antibody Systems Quantitative investigations of the fractions of antibody populations reactive with antiidiotypic antisera were undertaken by using specifically purified rabbit anti-p-azobenzoate antibody as the immunogen ( Daugharty et al., 1969; Hopper et al., 1970). Each recipient rabbit was

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67

matched to the donor with respect to nine allotypic specificities. A high percentage of positive responses was obtained when the donor antibody was polymerized with glutaraldehyde prior to inoculation. Quantitative precipitin tests were carried out with monomeric, Wlabeled, antibenzoate (donor) antibodies. Fractions of donor populations precipitated varied from 2 to 74%. Positive tests were obtained by the Ouchterlony procedure when as little as 2 to 4% of the antigen molecules were precipitable. With thirteen antiidiotypic antisera prepared against purified antibenzoate antibodies from 7 donors, no crossreactions were observed by direct precipitation, using antibenzoate antibodies from a panel of 14 rabbits for the tests. These antibodies comprised all the major allotypic specificities of the donor rabbits. Removal of antibenzoate antibodies from donor sera eliminated the capacity to precipitate with antiidiotypic antisera. A sensitive quantitative test for cross-reactivity, based on coprecipitation of heterologous, 1z51-labeled,antibenzoate antibodies, similarly failed to reveal any cross-reactions. 2. Nonprecipitating Antibody Populations It was found that idiotypic specificities are present on molecules which are not directly precipitable by antiidiotypic antisera ( Hopper et al., 1970). This observation came from experiments based on an indirect method of precipitation, using '"I-labeled F( ab') fragments of the purified donor (rabbit antibenzoate ) antibody, antiidiotypic rabbit antiserum, and goat antirabbit Fc. The latter reagent was used in excess to precipitate all rabbit IgG in the antiidiotypic antiserum as well as complexes of labeled F( ab'), fragments with antiidiotypic antibody, A comparison of results obtained by direct and indirect precipitation is shown in Table I. In nearly all instances a higher percentage of the donor population was precipitable by the indirect method. In some cases substantial amounts of donor antibody fragments were precipitated by antisera that failed to react in the Ouchterlony test with the donor ( antibenzoate ) population. It appears, therefore, that direct precipitation may fail to reveal the presence of some or all molecules bearing idiotypic determinants. The greater sensitivity of the indirect method was attributed to a low antigenic valence of molecules in the donor population. The indirect test should detect even univalent antigens, but at least three antigenic determinants per molecule may be minimal for direct precipitation (Valentine and Green, 1967). Additional evidence for the importance of antigenic valence is the finding of Potter et al. (1966) and Potter and Lieberman (1970) that Fab fragments of mouse myeloma proteins often fail to precipitate with

JOHN E. HOPPER AND ALFRED NISONOFF

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TABLE I COMPARISON OF PEHCENTAGES OF DONORANTIBODY (ANTIBENZOATE) PRECIPITABLE BY ANTIIDIOTYPIC ANTISERABY THE DIRECT AND INDIRECT METHODSO

yo Precipitablef Donor rabbit

Recipient rabbit

AZ5b AZ5c AZll AZld AZld AZ1e A5 A5 A5 A6A A6A A6B A6B A7 A7 V15 V15

RD5 9Y 2x 11 RD8 9N 7A 7c 7D 70 7E 71 7K 7B 7M El E2

Allotype of donor and recipient 1, 3, 4, 7, 21 1, 3, 4, 21 1, 3, 4, 7, 21

Ouchterlony Direct Indirect test precipitation precipitation

+ + +0 0

1, 3, 4, 7, 21

1, 3, 4, 7 1, 4, 7 1, 4, 7, 21 1, 4, 7

+ + + + + +0 0

+ + + +

2 33 7 1 1 22 4 10 11 14 13 4 4 56 41 32 29

23 70 13 34 42 51 29 43 43 58 41 28 32 69 74 35 31

Data compiled from Hopper ct a / . (1970) and MacDonald and Nisonoff (1970). antibodies isolated approximately 2 months after the start of immunization of rabbit AZ5. Antibenzoate antibodies isolated approximately 8 months after the start of immunization of rabbit AZ5. Antibenzoate antibodies isolated approximately 2 months after the start of immunization of rabbit AZ1. Antibenzoate antibodies isolated approximately 8 months after the start of immunization of rabbit AZ1. f 1z51-Labeled,specifically purified, anti-p-azobenzoate antibody was used as antigen in the direct precipitations; lZ5I-labeledF(ab')z fragments were used in the indirect method. 0

* Antibenzoate

anti-ind antibodies which do form precipitates with the intact myeloma protein. Also, Harboe et al. (1969) observed inhibition of the precipitation of monotypic IgM by anti-ind antiserum in the presence of the 7 s subunit of the IgM, which would necessarily have a lower antigenic valence. Increased precipitability by the indirect method has also been observed in quantitative studies of certain rabbit allotypic specificities (Gilman et al., 1964). The fact that Fab fragments sometimes form precipitates with anti-ind antibodies and sometimes inhibit precipitation

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is undoubtedly attributable to variation in the number of ind determinants recognized by different antisera. IV. Cross-Reactions of Antiidiotypic Sera and Evidence for Identical Molecules in Different Individual Animals

A. IDIOTYPIC CROSS-REACTIONS OF ANTIBODIES FROM DIFFERENT DONORS The indirect procedure provided a sensitive means for investigation of idiotypic cross-reactions among antibenzoate antibodies from different donor rabbits (Hopper et al., 1970). Such tests were carried out with unlabeled antibenzoate antibody, or whole serum containing such antibody, in large excess as an inhibitor of the indirect precipitation of IsaIlabeled F( ab' ) fragments by homologous antiidiotypic antiserum. As expected, the unlabeled immunogen was a potent inhibitor in each system. However, in a large number of tests the degree of inhibition by a sixty-fold excess of unlabeled, heterologous, antibenzoate antibodies did not exceed 21%. Significant inhibition (greater than 10%)was noted in 13 of the 138 tests of cross-reactivity. Cross-reactions wcre also investigated, in a smaller number of systems, by testing the capacity of heterologous antiidiotypic antisera to react, by the indirect procedure, with '?'I-labeled F( ab') fragments of antibenzoate antibodies. One strong cross-reaction occurred in these tests. One may conclude that direct precipitation may fail to reveal crossreactions that are detectable by the more sensitive methods of indirect precipitation or inhibition of indirect precipitation. It should be eniphasized, however, that such cross-reactions are in general infrequent and involve a small proportion of the idiotypic antibody population. Cross-reactions have been demonstrated by direct precipitation methods (ring tests or double diffusion in agar gel) by Oudin and Bordenave ( 1971) and Bordenave and Oudin ( 1971) , who prepared rabbit antiidiotypic antisera against anti-Salmonella abortus equi (antiSAE) antibodies from individual donor rabbits. In contrast to the antiSalmonella typhi system (Oudin and Michel, 1963; Oudin and Michel, 19694, discussed earlier, a considerable number of cross-reactions was observed when anti-SAE antisera from individual rabbits were tested against heterologous antiidiotypic antisera. In 132 such tests, utilizing six different antiidiotypic antisera, 23 positive results were obtained by the ring test; 9 of these reactions were also visible in agar gel. Ten of the 22 anti-SAE antisera tested exhibited no cross-reactions. Frequently the homologous idiotypic reaction gave several lines in agar

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gel, none of which could be completely abolished by absorption of the antiidiotypic antiserum with a cross-reacting ( heterologous ) anti-SAE serum, On this basis the authors concluded that the anti-SAE antibodies in cross-reacting antisera were similar but not identical in structure. Since whole sera were used for absorption the amounts were necessarily limited because of dilution effects, so that the presence of small concentrations of identical molecules does not appear to be completely ruled out. Another example of partial idiotypic cross-reaction is provided by the work of Braun and Krause (1!368). Goat antiserum was prepared against a relatively homogeneous preparation of specificially purified antistreptococcal ( Group C ) antibody from an individual rabbit. Exhaustive absorption of the goat antiserum with nonspecific rabbit y-globulin (Fraction 11) eliminated the precipitability of the goat serum with purified antistreptococcal antibodies from several other rabbits, but not with the immunogen. The absorbed goat antiserum did precipitate with antistreptococcal antibody from one other rabbit, but the homologous precipitin line formed in agar gel spurred over the line due to the heterologous reaction. Presumably, further absorption of the goat antiserum with the cross-reacting rabbit antibody would have rendered it specific for idiotypic determinants on the rabbit antistreptococcal antibody used as the immunogen. Very recently, strong ind cross-reactions have been observed among members of the same family of rabbits, selected for breeding on the basis of their capacity to produce antistreptococcal antibodies of limited heterogeneity ( Krause, 1971). A significant number of ind crossreactions was observed among antistreptococcal antibodies from rabbits of a family group. A number of cross-reactions involving ind determinants has also been observed among mouse myeloma proteins. These data will be considered later. B. UNRELATEDNESS OF IDIOTYPIC DETERMINANTS IN ANTIBODIES OF DIFFERENT SPECIFICITYFROM THE SAMEINDIVIDUAL As indicated earlier, Kunkel et al. (1963) observed that anti-A substance and antilevan, specifically purified from the serum of a single human donor, possessed unrelated ind specificities. Similarly, Oudin and Michel (1963) found that a rabbit antiserum directed to idiotypic determinants in rabbit anti-Salmonella antibody failed to react with antipneumococcal antibody from the same donor rabbit. In a subsequent experiment, antibodies from a single donor, specific for Salmonella typhi and Salmonella tranoroa, respectively, were found to have un-

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related idiotypic specificities (Oudin and Michel, 19694. Kelus and Gell (1968) studied this question by immunizing a rabbit with Proteus vulgaris, allowing the titer to disappear, and then injecting human IgG. Antiidiotypic antibodies directed to the anti-proteus did not react with the antiserum, taken subsequently, which contained anti-IgG. Daugharty et al. (1969) and MacDonald and Nisonoff (1970) used rabbit antiidiotypic antisera directed to specifically purified rabbit antibenzoate antibodies. Such antisera reacted with the serum of the donor rabbit, but the reaction was eliminated by absorption of antibenzoate antibodies from the serum. Each donor rabbit had been immunized with bovine y-globulin conjugated with p-azobenzoate groups and possessed a high titer of antibodies against the protein carrier. The failure of antiidiotypic antiserum to react with the donor serum after removal of antibenzoate antibodies indicates that the antibovine y-globulin and antibenzoate do not share idiotypic determinants. Thus, in this experiment different determinants present on the same immunogen molecule, bovine y-globulin-azobenzoate, elicited antibodies with unrelated idiotypic specificities. The available data provide no evidence for idiotypic cross-reactions among antibodies of different specificity from the same individual animal. A related question is whether antibodies from an individual animal, directed to a particular antigen, possess more than one set of idiotypic specificities. In view of the known heterogeneity of antibodies reactive with a single antigen or hapten, one might predict that multiple idiotypic specificities would be present, and there is good evidence that such is the case. Thus, multiple precipitin lines are frequently observed when the donor antibody and antiidiotypic antiserum are allowed to react by double difision in agar gel (Oudin and Michel, 1963, 1969a; Kelus and Gell, 1968). Also, Braun and Krause (1968) and Eichmann et al. ( 1970) were able to isolate subfractions of purified antistreptococcal cellwall antibodies from an individual rabbit by taking advantage of a difference in eIectrophoretic mobiIity. Goat antisera prepared against each subfraction and absorbed with nonspecific rabbit IgG identified different ind determinants in the two populations of antistreptococcal antibodies, although some evidence for partial cross-reactivity was also obtained in one instance. Another approach to this question was made by Hopper et al. (1970), who immunized different recipient rabbits with specifically purified anti-p-azobenzoate antibody from one rabbit. Frequently, antiidiotypic antisera from two recipients reacted with overlapping but nonidentical subfractions of the molecules in the donor ( antibenzoate ) population. Since an indirect method of precipitation was used, only a single antigenic determinant should have sufficed for precipitation; the differences, therefore, cannot be attributed to

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recognition of additional idiotypic determinants on the same molecule by that antiserum which precipitated the larger percentage of donor molecules. The results thus indicate the presence in a rabbit of antibenzoate antibodies bearing different idiotypic specificities. C. CROSS-REACTIVITY AMONG MYELOMA PROTEINS;PRESENCE OF IDENTICAL MOLECULES IN DIFFERENT INDIVIDUALS The data already discussed indicate that idiotypic or ind crossreactions among antibodies of the same specificity from different animals, or among human myeloma proteins, are infrequent. Studies with inbred mice, although still limited in scope at this point, suggest that crossreactions of ind determinants may be more frequent in such animals. Cohn et al. (1969) investigated an IgA myeloma protein from a BALB/c mouse which reacted specifically with pneumococcal C carbohydrate. A plasmacytoma had been induced in the mouse by intraperitoneal injection of mineral oil (Potter and Robertson, 1960). Antibodies to this myeloma protein, prepared in an A/J mouse, precipitated with the immunogen, but with only 1 of 160 other BALB/c myeloma proteins tested. Of great interest was the finding that this cross-reacting IgA protein also combined with pneumococcal C-carbohydrate. These investigators then examined the question as to whether antisera obtained from normal BALB/c mice immunized with pneumococci would react with the same anti-ind antiserum. Positive reactions were obtained in the Ouchterlony test with six of ten such hyperimmune sera. Tests were not carried out to establish whether the myeloma protein used as immunogen gave lines of identity with the hyperimmune anti-C carbohydrate antisera. Thus, the myeloma protein with anti-C activity shares some, although not necessarily all ind determinants with normal antibodies of the same specificity. Implicit in this finding is the possibility that normal antibodies of a given specificity produced in inbred mice will show a much greater frequency of idiotypic cross-reactions than antibodies elicited in humans or outbred rabbits. This work was confirmed and extended by Potter and Lieberman (1970), who isolated six additional IgA myeloma proteins, from BALB/c mice bearing induced tumors, which precipitated with pneumococcal C carbohydrate. Anti-ind antisera were prepared in other strains of inbred mice against all eight of the available myeloma proteins with activity against this antigen. Three of the eight proteins were found to possess individual antigenic specificity unrelated to one another or to any of 120 myeloma proteins tested by precipitin analysis. However, the remaining five myeloma proteins with anti-C activity had ind specifici-

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ties that were indistinguishable from one another, giving lines of identity in the Ouchterlony test with appropriately absorbed antisera against each of the five myeloma proteins. This group included the two crossreacting proteins with anti-C carbohydrate activity discovered by Cohn et al. Chemical investigations were initiated, in collaboration with L. Hood, on two of the cross-reacting myeloma proteins; peptide maps and amino-terminal sequences of their light chains ( K type) were found to be identical but were different from those of light chains of two of the myeloma proteins with anti-C carbohydrate activity which possessed unrelated ind determinants. The possibility was therefore suggested that the proteins with cross-reacting individual antigenic specificities might be structurally identical; data on amino acid sequences in the heavy chains should be of great interest. Potter and Lieberman also observed that each of these proteins reacted, in addition, with phosphorylcholine (cf. Potter and Leon, 1968), which is a component of the pneumococcal cell wall, and also with an extract of Lactobacillus acidophilus. Since the latter organism is prevalent in the gastrointestinal tract of BALB/c mice, it is possible that the cell which underwent malignant transformation belonged to a clone which had been stimulated by the Lactobacillus organism, D. EVIDENCE BASEDON AMINO ACID SEQUENCEFOR OF IDENTICAL IMMUNOGLOBULIN MOLECULESIN DIFFERENT INDIVIDUALS

THE

PRESENCE

The first evidence for the presence of identical molecules in different animals actually was based on analysis of primary structure, rather than antigenic specificity ( Appella and Perham, 1967, 1968), Bence-Jones proteins of the h type, associated with myeloma tumors independently induced in two mice, were indistinguishable by several criteria, including tryptic peptide maps, a sequence of nine amino acids at the COOHterminus, and of ten amino acids at the NH,-terminus (Appella and Perham, 1968). More recently, an essentially complete sequence analysis of these proteins provided further evidence for identity (Appella, 1971). This work was substantially extended by the remarkable findings of Weigert et al. (1970), who determined amino acid sequences of the variable regions (113 residues) of ten mouse h chains. These sequences are essentially complete except for alignment of amino acids in certain peptides. Two of the proteins were derived from the same tumor lines utilized by Appella and Perham. Four of the ten h chains were isolated from IgA myeloma proteins; the remainder were urinary Bence-Jones proteins from mice having serum myeloma proteins of the IgA, IgM, or IgG,, class. Of the ten proteins investigated, six appeared to have

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JOHN E. HOPPER AND ALFRED NISONOFF

identical amino acid sequences, (Glutamine and asparagine were determined as the hydrolyzed products, glutamic acid or aspartic acid, respectively; nevertheless, there were apparently no differences among the six proteins since their corresponding peptides had identical mobilities,) Of the remaining four proteins, two differed by only one residue from the six identical chains, one differed by two residues, and one by three residues. All changes occurred in the three regions of the chain which are highly variable in human K or h chains, near positions 30,50,and 90. Because of their variability or the results of affinity labeling, these regions have been postulated to be near or part of the antibody-combining site. The results of Appella differed slightly from those of Weigert et al. in that the latter investigators found leucine at position 50 of protein RPC-20, where Appella reported isoleucine to be present. These results are strikingly different from those observed with the human L chains studied, where two chains are rarely found to be identical. To account for the data, Weigert et al. postulate first that the number of mouse germ-line genes coding for h chains is smaller than the number coding for K chains. This conjecture is also supported by the low ratio of h to K chains in normal mouse serum. They propose that the sequence differences observed in four of the chains arose as a consequence of somatic mutation; i.e., that only a single germ-line gene encodes the h chains which they investigated (all of which were secreted by experimentally induced tumors ). The small number of somatic mutations, as compared to human chains of a single subgroup of the K or h type, was attributed to the short life-span of the mouse. This carries the implication that each subgroup of human light chains has a corresponding germ-line gene which can undergo somatic mutation. Evidence for the possible presence of identical light chains in two humans was reported by Capra and Kunkel (1970). Two patients with hypergammaglobulinemic purpura had elevated serum levels of an IgG protein of restricted electrophoretic heterogeneity. Each protein had antibody activity against aggregated y-globulin. The first forty NH,-terminal residues and the peptide maps of the light chains of the two proteins were identical, strongly suggesting complete identity of the two chains. Recently, Hood et al. (1970) reported on an extensive study of sequences at the NH,-terminal end of mouse K chains and were able to divide them into at least nine subtypes. Additional investigation was carried out on several of these proteins, and two appeared identical by peptide mapping and partial sequence analysis. These two K chains also possessed identical individual antigenic specificity. As mentioned

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above, another pair of mouse K chains with identical peptide maps and ind specificity had been previously identified by Potter and Lieberman. The complete sequences of the variable regions of these two pairs of K chains have not yet been reported. V. Evidence Based on ldiotypic Specificity for limited Heterogeneity of Normal Antibody Populations

Most if not all myeloina proteins possess individual antigenic specificity. In addition, such specificities are observed in relatively homogeneous rabbit antibody populations, such as those recently studied by Krause, Haber, and co-workers; their work will be summarized below. However, most populations of antibody of a given specificity from an individual animal are known to be quite heterogeneous. The investigations of idiotypic specificities to be considered in this section suggest that this heterogeneity is limited in the sense that homogeneous subpopulations may be present in substantial concentration in specifically purified antibodies from an individual donor. One would anticipate that such homogeneous subfractions might be more prevalent in antisera directed to haptcns, or to antigens with repeating determinants, such as erythrocytes or bacterial cell w ~ I s , than in antiprotein antibody. One type of evidence suggestive of such limited heterogeneity is the small number of bands generally observed in idiotypic precipitin reactions carried out by double diffrrsion in agar gel. Although several bands are sometimes seen, one or two often predominate (Kelus and Gell, 1968; Daugharty et al., 1969; Oudin and Michel, 1969a). Quantitative measurements of percentages of antibenzoate molecules from an individual donor reactive with antiidiotypic antisera from 2 or more recipients led to a similar conclusion (Hopper et al., 1970). Often, two different antisera reacted with the same subpopulation of molecules in the donor antibody preparation. When one antiserum reacted with a larger fraction than the other, the larger subpopulation invariably included the smaller. It was proposed that the reactive fraetion of the donor molecules comprises a discrete number of homogeneous ( i.e., monoclonal) subpopulations The remainder is so heterogeneous that it is noiiimmunogenic in any recipient. That different recipient rabbits often produce antisera reacting with the same donor antibody molecules was shown qualitatively by Kelus and Gell (1968), who utilized the Ouchterlony method. Finally, it scems reasonable that elicitation of antiidiotypic antibody would require the presence of a significant amount of homogeneous antibody in the population of inolecules used as the immunogen.

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JOHN E. HOPPER AND ALFRED NISONOFF

VI. Persistence and Changes of Antibody Populations during Prolonged Immunization

A. EVIDENCE BASEDON IDIOTYPIC SPECIFICITIES Since the half-life of an antibody molecule (Taliaferro and Talmage, 1956; Weigle, 1958) or of most antibody-producing cells (Schooley, 1961; Makinodan and Albright, 1967) is of the order of a few days, one may ask whether antibody molecules isolated at various times during the course of immunization are structurally related. Roholt et al. (1965) have shown that the activity of heavy chains of a rabbit antihapten antibody is not appreciably enhanced upon recombination with light chains isolated from antibody of the same specificity but from another rabbit, whereas a substantial fraction of the original antibody activity is recovered if the autologous light chains are utilized in preparing the recombinant. This general approach was used to provide evidence that structurally related antibody molecules are present in the serum of an immunized rabbit for a long period of time ( MacDonald et al., 1969). Anti-p-azobenzoate antibodies were purified from sera of individual hyperimmunized rabbits at intervals of about 6 months. Recombinants of heavy (or light) chains from the earlier bleeding of a rabbit with light (or heavy) chains from the later bleeding of the same rabbit had specific activities 60-100% as great as the activities of the autologous recombinants. It was proposed that “memory” cells give rise upon subsequent challenge by antigen to antibody molecules identical to those synthesized initially by that cell line; i.e., that a long-lived clone of cells continues to synthesize molecules of a particular structure. The study of idiotypic specificities provides a much more convenient tool for following the persistence of structurally similar or identical antibody molecules during prolonged immunization. A fundamental experimental variable in this type of investigation is the frequency of challenge by antigen, i.e., whether the animal is allowed to rest for a long period of time between inoculations or is repeatedly stimulated. One might, for example, predict that frequent exposure to antigen would tend to stimulate new clones of cells and thus lead to a more rapid replacement of idiotypic specificities than would be observed if the antigen were given infrequently. Nisonoff et al. (1970) and MacDonald and Nisonoff (1970) reported experiments in which the antigen (bovine IgG-p-azobenzoate ) was administered to rabbits at weekly or biweekly intervals. Antiidiotypic antibodies were prepared in rabbits against specifically purified anti-p-azobenzoate antibodies isolated from the

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donor rabbit after 2 or 8 months of immunization. Quantitative measurements of the persistence of idiotypic antibodies were carried out by an indirect precipitation method which utilized 1231-labeledF ( ab’ ) fragments of antibenzoate antibody (the immunogen) and its homologous antiidiotypic antiserum. Unlabelcd antibenzoate antibodies from the same rabbit, isolated at intervals from 2 to 17 months after the start of immunization, were tested as inhibitors. Results obtained with one donor rabbit are shown in Figs. 3 and 4. In Fig. 3 the antiidiotypic antibody was prepared against antibenzoate antibody from the month 2 bleedings. The homologous unlabeled antibody inhibited the indirect precipitation almost completely when present in excess. It is evident that antibodies having the same individual specificities were still present in comparable concentration 2 months later. After month 4, however, there was a rapid transition and new, unrelated individual specificities emerged. Thc transition occurring after month 4 was confirmed with antiidiotypic antibodies prepared against antibenzoate antibodies from the month 8 bleeding (Fig. 4).The antiidiotypic antibodies were not inhibited by antibodies from bleedings taken at months 2, 3, or 4. Idiotypic specificities present at month 8 were first identified at month 5

d

0

2

4

6

8

33

pg UNLABELED COMPETITOR

FIG. 3. Inhibition of binding of labeled F( ab’)z fragments of anti-p-azobenzoate antibodies from rabbit AZ1 to homologous antiidiotypic antiserum. The antibenzoate antibodies \\’ere isolated from sera taken approximately 2 months after the start of iiniiiunization of rabbit AZ1. Competitors are unlabeled, specifically purified antibenzoate antibodies prepared from sera of rabbit AZ1 at various times after the start of immunization; the approximate number of months is indicated by the numeral on each curve. [Reprinted, by permission, from MacDonald and Nisonoff,

(1970).]

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I

I

I

2

4

6

1

8

30

pg UNLABELED COMPETITOR

FIG.4. Inhibition of binding of labeled F(ab’)l fragments of anti-p-benzoate antibodies from rabbit AZ1 to antiidiotypic antiserum prepared in a recipient rabbit. The antibenzoate antibodies were isolated from sera taken approximately 8 months after the start of immunization of rabbit AZ1. Competitors are unlabeled, specifically purified antibenzoate antibodies prepared from sera of rabbit AZ1 at various times after the start of immunization; the approximate number of months is indicated by the numeral on each curve. [Reprinted, by permission, from MacDonald and Nisonoff ( 1970).]

and then persisted through month 17, although gradual changes in the concentration of idiotypic specificities occurred. The most effective inhibitor was the immunogen, i.e., the antibody isolated during month 8. The change in idiotype after month 4 was noted in two other rabbits, similarly immunized, although the transition was more gradual in one of these animals. Prolonged persistence of idiotype for at least a year was observed also in the rabbits which survived for that period of time. Similar results have since been obtained with other schedules of immunization with this protein-hapten conjugate ( Spring and Nisonoff, 1971 ) . Oudin and Michel (1969a), using double diffusion in agar gel, in-

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vestigated the persistence of idiotype in anti-Salmonella antibodies of 4 rabbits. In 1 rabbit, molecules of the same or related idiotype were present over a period of 29 months. The rabbit was allowed to rest for 17 months before the final set of injections and bleedings. In another rabbit they observed a loss of one set of idiotypic specificities but the persistence of another set during the first few weeks of immunization. Idiotypic specificities were found to persist in 2 other rabbits during a period between 2 and 5 weeks after the start of immunization, Persistence of ind specificities has also been demonstrated in antibody populations of limited heterogeneity elicited in certain rabbits by bacterial antigens. Eichmann et aE. (1970) were able to separate from the serum of individual rabbits, components of antistreptococcal antibody having distinct electrophoretic mobilities. Two examples are shown in Fig. 5. Goat antisera were prepared against each of the two major components separated by preparative electrophoresis. Individual antigenic specificity was indicated by formation of a spur in an Ouchterlony test when the immunogen was placed in a well adjacent to nonspecific rabbit IgG, with the goat antiserum in the center well. In an individual rabbit, the antibodies of slow and fast mobility were shown to have different ind specificities. Furthermore, in 2 rabbits, the individual specificity present in a particular electrophoretic component after a primary course of immunization was observed in a component of the same mobility after a secondary course, 7 months later. No immunoglobulin with ind specificity was detected during an interval between courses of immunization when the serum did not contain antistreptococcal antibody. These interesting experiments illustrate another means of following the persistence of molecules of a particular idiotype, namely through their characteristic electrophoretic mobility.

B. EVIDENCE BASEDON AMINO ACID SEQUENCE ANALYSISFOR PERSISTENCE OF SIMILAROR IDENTICAL MOLECULES IN SERUM A third, and perhaps most definitive method for observing persistence is the analysis of amino acid sequence. The idiotype of a molecule must be defined by sequences in the variable portions of its heavy and light chains. Amino acid sequence analysis has been used to observe the persistence of molecules of related or identical structure during repeated immunizations with Type VIII pneumococcal vaccine (Jaton et al., 1971). A rabbit received three l-month courses of immunization with a rest period of 1 month after each course. Antibodies isolated after each course will be referred to as Ab-1, Ab-2, and Ab-3. Specifically purified Ab-2 was homogeneous by the criterion of electrophoresis. Antiind antiserum, specific for this purified antibody, also reacted, although not as strongly, with Ab-1 and Ab-3. Light chains from Ab-2 were

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FIG. 5. Microzone electrophoretic patterns of antisera directed to streptococcal (Group A ) cell walls, before and after absorption with anti-ind antisera. The uppermost pattern is that of immune serum from rabbit A23-61 after secondary immunization. The two major IgC components (bands on the left) were isolated, and goat antiserum was prepared against the slower moving fraction and rendered specific for ind determinants by absorption with nonspecific rabbit y-globulin. The anti-ind antiserum was then insolubilized by polymerization with glutaraldehyde. The antistreptococcal antiserum was absorbed with this polymer; the electrophoretic pattern subsequent to absorption is shown in the second diagram from the top. The lower three patterns show the results of similar experiments with the hyperimmune antistreptococcal antiserum of another rabbit ( R22-79) after secondary immunization. However, in this case, the last pattern was obtained by absorbing the antistreptococcal antiserum with polymerized goat anti-ind antiserum directed to the slow electrophoretic component present in an earlier bleeding of the rabbit. The symbols 1' and 2' refer to primary and secondary immunizations. This experiment shows that the slow and fast components (labeled S and F ) have different ind specificities and that, for rabbit R22-79, the slow component after primary immunization showed some of the ind specificity of the corresponding component observed after the secondary immunization. [Reprinted, by permission, from Eichmann et ol. (1970).]

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analyzed in an automatic sequenator and found to have a unique sequence for the first twenty-one residues, starting from the NH,-terminus. This of course contrasts with nonspecific light chains of the same allotype (b4) which have multiple residues at almost every position (Jaton et al., 1970). The unique sequence observed in the light chains of Ab-2 also predominated in the light chains isolated from Ab-1 and Ab-3; the latter two preparations, however, were not homogeneous by the criterion of sequence since more than one amino acid was found at many positions in the chain. There was good quantitative correlation, among antibodies isolated after each course of immunization, between the percentage of molecules bearing the ind determinants of Ab-2 and the percentage of light chains having the same partial sequence of amino acids as the light chains of Ab-2. Slightly less than half of the light chains of Ab-1 and Ab-3 shared the unique sequence found at the NH,terminus of the light chains of Ab-2. It is of interest that the total serum concentration of molecules bearing the ind specificity of Ab-2 did not actually decrease after the second course of immunization, because the total serum concentration of antipneumococcal antibody increased by a factor of almost 3 during the third course. This increase was, therefore, largely attributable to the initiation of new clones of cells, with the retention of the single major clonk expressed after the second course. From the work described in the preceding sections, it is evident that the study of individually specific determinants provides a sensitive method for detecting the onset and persistence of clones of cells. The product of an individual clone can be observed even in the presence of a large amount of antibody directed to the same antigen but of different idiotype. It is evident that clones of cells may persist for long periods of time and that gradual changes occur under the influence of repeated inoculations of the antigen, which lead to the induction of new clones. How the rate of change is related to the dosage and timing of the administration of antigen is a subject for future study. The analysis of individually specific antigenic determinants represents a convenient substitute for the ultimate test of structural relatedness, namely, the amino acid sequence. In addition, antiidiotypic antisera can detect small subpopulations that would not be observable by sequence analysis. VII. Shared ldiotypic Determinants in IgG and IgM Antibodies

of the Same Specificity

A. SHARED DETERMINANTS IN ANm-Sa~monelihANTIBODIES

Oudin and Michel (1969b) isolated IgG and IgM fractions of antisera prepared against Salmonella typhi in 2 rabbits. In each case the

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IgM and IgG gave lines of identity in agar gel in their reaction with antiidiotypic antiserum directed to the total anti-Salmonella antibody population. This is illustrated in Fig. 6. This important finding strongly suggests that IgG and IgM are synthesized by cells of the same clone, which are probably derived from a single precursor cell. That IgG and IgM may be synthesized by a single clone of cells had been suggested by the investigations of Nossal et al. (1964) with

FIG.6. Double diffusion in agar gel showing shared idiotypic determinants in IgC and IgM and anti-Salmonella antibodies from the same rabbit. In each section a trough cut along the bottom edge contained antiidiotypic antiserum directed to the anti-SaZmoneZZu antibodies. The upper horizontal troughs are divided into two sections. ( A ) Left upper trough, IgM-containing fraction of the donor serum; right, IgC fraction. ( B ) Same as A except that the IgC is diluted 1:5. (The purpose of this part of the experiment is to show that when the IgG is diluted so that its line is as close to the upper trough as that of IgM, the resulting patterns are different in appearance, with that of IgC being more diffuse. This supports the view that the IgM line is close to the trough because of the higher molecular weight of IgM.) In D, the IgC in the left trough is diluted 1:50 and no line appears. Since the maximum contamination of the IgM by IgC had been quantified and shown to be less than 2%,this experiment indicates that the line attributed to IgM in A was not due to contamination by IgC. [Reprinted, by permission, from Oudin and Michel ( 1969b).]

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individual antibody-forming cells. Their data indicated that a single cell was capable of synthesizing both IgG and IgM molecules when tested in uitro approximately 4 days after the start of immunization. Prior to this time, singlc cells manufactured only IgM, and a week or more later, only IgG. Identification as IgM or IgG was based on sensitivity or resistance to mercaptoethanol. The work of Oudin and Michel (1969b) also indicated that there is constant regeneration, within a clone, of cells capable of synthesizing IgM as well as IgG, since both IgG and IgM molecules with the same idiotype were present at widely spaced intervals.

B. STRUCTURAL RELATIONSHIPBETWEEN IgG AND IgM PRODUCED BY A CLONE OF CELLS Investigations of the serum of a single patient with multiple myeloma having two monoclonal proteins, IgG and IgM, provided insight into a possible switching mechanism which permits the biosynthesis of IgG and IgM by a single clone of cells. The patient (Ti1) having two monoclonal serum proteins ( IgG2-K and IgM-K) was studied by Wang et al. (1969). The light chains of the two proteins were isolated and found to be identical by several criteria, including the peptide map, amino acid composition, and optical rotatory dispersion. Upon electrophorcsis in starch gel at p H 3 or 8 the L chains derived from the two proteins had identical electrophoretic mobilities. More recently, a sequence of thirty amino acids starting at the NH,-terminus was shown to be identical in the L chains (Wang and Fudenberg, 1971) . Anti-ind antisera were prepared in rabbits against the isolated monotypic IgG and IgM and rendered specific for individual determinants by appropriate absorptions (Wang et al., 1970a). No cross-reactions were observed in tests with over thirty sera from other patients with multiple myeloma. The anti-incl antiserum to Ti1 IgG gave a line of idcntity in reacting \vith Ti1 IgG and Ighl. The anti-ind antiserum to Ti1 IgM formed two lines in its reaction with Ti1 IgM, one of which showed identity with Ti1 IgG. In quantitative studies, about 90% of F(ab'), or Fab fragments of Ti1 IgG or IgM were precipitable by the antiglobulin technique. Either unlabeled Ti1 IgG or IgM was capable of completely displacing '"Ilabeled F( ab'), fragments of Ti1 IgG or IgM from anti-ind antibodies to Ti1 IgG; t11c IgG was somewhat more effective on a weight basis. In the converse experiment, with labeled Fab fragments from Ti1 IgM, unlabeled IgM completely displaced the fragments from anti-ind antibody directed to Ti1 IgM, but the IgG gave a maximum of 45% displace-

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ment. Thus, Ti1 IgG and IgM share individually specific determinants, but quantitative analysis demonstrated that they are not identical. Of particular interest was the finding that Ti1 L chains did not precipitate with anti-ind antiserum to either Ti1 IgG or IgM and were incapable of inhibiting the reaction of labeled Fab fragments of the Ti1 proteins with anti-ind antisera. A recombinant of Ti1 L chains with Ti1 y chains reacted with the anti-ind antisera, but a recombinant of T i l L chains with H chains from another myeloma protein was completely inactive. These findings suggested that the H chains of the IgG and IgM contribute to the shared individual antigenic specificity. A structural relationship between the H chains of Ti1 IgG and IgM was strongly supported by the results of amino acid sequence analysis. The first twenty-scven residues, starting from the NH,-terminus, are identical in the two proteins ( Wang et al., 1970a). Both H chains have free glutamic acid as the NH,-terminal residue rather than the more frequently encountered pyrollidone carboxylic acid and, thus, belong to the same subgroup of variable regions of H chains (Wang et al., 1970b); the subgroup has been denoted V,,,, by Kohler et al. (1970). The data, although incomplete, strongly suggest that Ti1 IgG and IgM share identical V,, regions ( V = variable; H = heavy chain) as well as identical L chains. The quantitative differences between the IgG and IgM observed in reactions with anti-ind antisera might be due to steric hindrance or to a contribution of the C, region to individually specific deterniinants. Related findings have been reported by Penn et al. ( 1970). Monotypic IgG and IgM from the same patient were shown to share individually specific determinants which were not present on isolated L chains. Recently these findings were extended to several other patients who were found to have double myelomas of various immunoglobulin classes with shared ind dcterminants ( Penn and Kunkel, 1971). To account for the data obtained with Ti1 proteins it was proposed that a clone of malignant cells originally synthesized either Ti1 IgG or IgM and that a switching mechanism occurred in one or more cells which repressed the gene controlling, say, CHp,and simultaneously derepressed the gene controlling Crry. That at least two genes control the biosynthesis of a single L or H chain had been proposed by a number of investigators (Hilschmann and Craig, 1965; Dreyer and Bennett, 1965; Milstein, 1967; Hood and Ein, 1968). The data obtained with the protcins of patient Ti1 provide direct evidence in support of this hypothesis, On the basis of these data and the shared idiotypic determinants in IgG and IgM discovered by Oudin and Michel (1969b), it was further

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p Chain

FIG.7 . Shared amino sequences (shaded areas) in the heavy and light chains of the monotypic IgC and IgM of patient Til, and, hypothetically, in normal IgG and IgM antibodies produced by a single clone of cells. It is assumed that three of four genes controlling the four chain segments are the same for the two classes of molecule.

proposed that a similar switching event occurs during normal immunoglobulin biosynthesis; i.e., that different cells of a single normal clone synthesize IgG and IgM (and perhaps other classes as well) and that the L chains and V, regions of the molecules synthesized within that clone are identical. A corollary of this proposal is that molecules of different classes made by the same clone of cells may have identical antigenbinding sites. The fact that the antigen-binding site appears to be part of a major idiotypic determinant (Brient and Nisonoff, 1970) lends additional weight to this hypothesis. Although the nature of the switching mechanism is obscure, it would appear that it involves only the C,, gene and does not affect the expression of the genes controlling the CL, VL, or VH regions. The structural relationship between Ti1 IgG and IgM and, hypothetically, between normal IgG and IgM from a single clone of cells is illustrated in Fig. 7. Some of these findings were supported by studies carried out at the cellular level. Individual plasma cells derived from the bone marrow of patient Ti1 were shown, with class-specific fluorescent antisera, to synthesize either IgG or IgM, but no individual cells producing both classes were identified ( Wang et al., 1969). In a subsequent study it was found that 100%of plasma cells that were stained by anti-ind antibodies to Ti1 IgG also reacted with anti-ind antibodies prepared against Ti1 IgM (Levin et al., 1971). Nearly all of the bone marrow plasma cells of the patient were stained by the ffuorescent anti-ind reagents. VIII. Localization of Individually Specific Antigenic Determinants

One would expect that idiotypic or ind determinants are largely, if not entirely, determined by variable sequences in immunoglobulin

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molecules. Evidence has already been presented that such determinants are confined to the Fab fragment, which includes all of the variable sequence of each chain as well as invariant regions. Also, investigation of the monotypic proteins of patient Til, already discussed, showed that the VH region contributes to the individual antigenic specificity; Ti1 IgG and IgM, which have different C, regions, share ind specificity which requires participation of the H chain. It should be noted that a possible minor contribution of the constant regions of the molecule to such a determinant cannot a priori be ruled out; an antigenic determinant might include a portion of both a variable and invariant segment. As indicated above a possible participation of the constant region of the H chain was suggested by quantitative differences in the effectiveness of Ti1 IgG and IgM in interacting with anti-ind antibodies. Although the constant region obviously would not be expected in itself to possess individual antigenic specificity, it might contribute through spatial proximity to a variable segment, with the formation of an antigenic determinant comprising a portion of both regions. Other studies relating to the localization of individually specific determinants are summarized in this section. The role of “conformational determinants,” i.e., antigenic determinants dependent upon the interaction of H and L chains, will also be considered. A. LOCALIZATION OF A MAJORIDIOTYPIC DETERMINANT TO THE REGIONOF THE COMBINING SITE OF AN ANTIBODY MOLECULE Quantitative studies of antiidiotypic antibodies directed to specifically purified rabbit anti-p-azobenzoate antibody provided data which indicate that the combining site of the antibenzoate antibody is part of, or close to a significant idiotypic determinant ( Brient and Nisonoff, 1970). The reaction of 1251-labeledF( ab’) fragments of antibenzoate antibody with its antiidiotypic antiserum was significantly inhibited by homologous haptens, i.e, by benzoate derivatives. There was a close correlation between the affinity of a hapten for the antibody and its capacity to prevent the combination of antibenzoate antibody with antiidiotypic antibody. Compounds unrelated to benzoate were not inhibitory. As much as 69% inhibition was observed with the hapten of highest affinity, p- ( p’-hydroxy )phenylazobenzoate, when tested at a concentration of 1.6 X M. This hapten caused significant inhibition of binding of F( ab’)? fragments with antiidiotypic antiserum in each of six systems investigated. Two possible interpretations of these data are that ( a ) the combining site of antibenzoate antibody is part of a major idiotypic determinant and the hapten interferes sterically with the interaction, and ( b ) the

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hapten causes a conformational change in the antibenzoate antibody which results in an alteration of one or more idiotypic determinants in a region removed from the combining site. If the second hypothesis is correct the conformational change must be limited in scope since haptens were shown to have little effect on the reaction of F(ab’), fragments of antibenzoate antibodies with goat and antirabbit Fab, and no detectable effect on the reaction of the same fragments with rabbit antiallotype antisera directed to determinants on either H or L chains. Thus, if conformational changes do occur they are probably restricted to the region of the hapten-binding site of the antibody molecule. Upon variation of the order of addition of hapten and antiidiotypic antiserum, approximately the same results were obtained whether the hapten was added to the F( ab’)2 fragments prior or subsequent to the antiidiotypic antiserum. Thus, the hapten is capable of displacing antiidiotypic antibody. These findings contrasted with those of Kelus and Gel1 (1968), who found that anti-Proteus vulgaris antibodies react with their 11on~ologous antiidiotypic antibodies even when the anti-Proteus is combined with Proteus. This suggested that the active site is not an important idiotypic determinant. Two alternative possibilities are suggested that might reconcile our conclusions. First, since their reactions were carried out with bivalent anti-proteus antibody, one of the combining sites of some of the anti-proteus molecules complexed with bacteria may have been free and available for reaction with antiidiotypic antibody. Also, their antiidiotypic antibody was elicited by immunization with Proteus-antiProteus complexes. It is possible, therefore, that some of the antiidiotypic antibody was specific for the complex; that is, for antibodies in which the combining sites were occupied. This would not apply to the entire antiidiotypic antibody population since it also reacted with anti-Proteus in the absence of bacteria.

B. INDIVIDUALLY SPECIFICDETERMINANTS IN ISOLATED HEAVY AND LIGHT CHAINSAND IN RECONSTITUTED MOLECULES; DETERMINANTS DEPENDENT ON NATIVECONFORMATION In a number of investigations, H and L chains have been isolated and tested for their reactivity with anti-ind antibody directed against the parent immunoglobulin. Details of this work will be described here. Variable results have been obtained, but, with some notable exceptions, the isolated polypeptide chains have reacted less strongly and sometimes not at all with such anti-ind antibodies. When weak reactivity of isolated chains is observed, this might bc attributable either to a change in conformation upon isolation of the chain or to the dependence of

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ind determinants on a structure resulting from the combination of H and L chains. Indeed, in some instances i d determinants were restored only when the homologous H and L chains were allowed to recombine and were not found in heterologous recombinants, consisting of the L (or H ) chain of the immunogen and an H (or L) chain from a different molecule. This indicates the dependence of ind determinants on the integrity of the native structure in such molecules but does not necessarily prove that a determinant comprises amino acids of both the H and L chain. For example, a determinant might be exclusively localized on the L chain, but might only be expressed when the L chain is combined with its homologous H chain. The data generally do not permit a distinction to be made between these two possibilities. Since the combining site of an antibody molecule appears to utilize directly both the H and L chain, it is possible, although not certain, that those idiotypic determinants of an antihapten antibody that are blocked in the presence of small hapten molecules comprise amino acids from both chains (Brient and Nisonoff, 1970). Grey et al. (1965) examined the L and H chains from eleven human IgG myeloma proteins for their capacity to react with rabbit anti-ind antisera directed to each intact protein. ( I n three instances, insolubility of the H chains prevented antigenic analysis.) Proteins with K or A light chains differed markedly in their antigenic properties. Of the five proteins with A chains the individual antigenic specificity was localized only to the L chain in three instances and only to the H chain in the other two proteins. In each case, a line of identity was observed when the isolated polypeptide chain bearing the ind specificity was compared by double diffusion in agar gel with the intact protein; this suggested that all of the specificity was restricted to one or the other chain. (It may be noted, however, that a single determinant requiring the participation of both chains would probably not be sufficient to give a spur, even though it would be duplicated in the two Fab regions. A minimum of three or more determinants is probably required for precipitation.) A finding that is difficult to interpret was the absence of ind determinants from molecules prepared by recombining the homologous L and H chains of each of the five proteins of type A, despite their presence in either the isolated L or H chain. Quite different results were obtained with the six proteins having type K L chains. In no instance was a precipitation reaction observed between isolated L chains and anti-ind antiserum directed to the intact myeloma protein. In two cases the isolated H chains did react, giving a line of partial identity in agar gel with the myeloma protein. In four other cases, recombination of the H and L chains was required for

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restoration of ind determinants. In these investigations there was no obvious correlation between the antigenic properties of the isolated chains and the IgG heavy-chain subgroup to which the myeloma protein belonged. Comparable, although not identical results with respect to L-chain type were obtained by Hurez et al. (1968), who investigated two human myeloma proteins of the IgG class. Rabbit anti-ind antiserum to one of these proteins, which had K chains, failed to precipitate isolated H or L chains in agar gel, but a recombinant of the chains gave a line of identity with the native myeloma protein. In contrast the isolated L chains of the second protein (type A ) did react in agar gel with anti-ind antiserum against the native protein. However, analysis by the Ouchterlony method indicated that the L chains did not possess all of the ind determinants of the myeloma protein. The H chains were not tested. Thus, of six IgG myeloma proteins of the h type investigated so far, ind specificity of the native molecule was found on either the L or H chain in each case; in five instances the antigenically active chains gave a line of identity with the native protein. Somewhat similar data were obtained with two monoclonal IgM proteins, one K and one A, from patients with Waldenstrom’s disease ( Seligmann and Mihaesco, 1967). Rabbit antibodies directed to each macroglobulin, and absorbed so as to render them specific for ind determinants, formed precipitates with the Fab subunit of IgM and also with the 7s subunit prepared by reduction. Isolated L chains of the K protein failed to precipitate with the anti-ind antibodies; a weak reaction was obtained with the H chains. In contrast, L chains of the type protein reacted with anti-ind antibodies to the macroglobulin, although they were antigenically deficient when compared with the Fab fragment. The H chains of this protein showed no reaction. Thus, monotypic IgG and IgM are similar in that L chains of the K type have consistently failed to react with anti-ind antiserum directed to the native protein, whereas, in several instances A light chains reacted. The IgM protein differed in that the A chains gave a reaction of only partial, rather than complete identity with the intact protein. It should be noted that caution is required in interpreting reactivity of H chains with anti-ind antisera when the H chains are isolated by gel filtration of reduced immunoglobulin in acetic or propionic acid, since they have frequently been shown to be contaminated with L chains, The data described so far were secured by qualitative precipitin tests in agar gel. Quantitative studies were carried out to assess the capacity of isolated H and L chains to react with rabbit anti-ind anti-

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bodies directed to a human IgG myeloma protein having L chains of the K type (Wang et al., 1970a). These were mentioned briefly above in discussing the relationship of monotypic IgG and IgM from the same patient. A 200-fold excess of unlabeled L chains failed to displace any 1251-labeledF( ab’)a fragments of the native myeloma protein from the anti-ind antibodies. Recombinants of the L chains with heterologous H chains from three different proteins were similarly inactive. Also recombinants of the H chains of the same protein with various unrelated L chains had virtually no inhibitory activity (Wilson et al., 1971a). A homologous recombinant, comprising H and L chains of the immunogen, reacted strongly with the anti-ind antiserum. On a weight basis it was approximately one-third as active as an inhibitor as the native protein. Similar quantitative studies were carried out with rabbit anti-ind antibodies directed to two other monotypic proteins of the K type, one IgG and one IgM (Wilson et aZ., 1971b). In both cases the L chains of the immunogen or recombinants of the L chains with various heterologous H chains were completely inactive as inhibitors. Although slight activity was observed in recombinants of the homologous H chains with heterologous L chains, this might have been attributable to contamination of the H chains. In both instances the homologous recombinant of H and L chains reacted with the corresponding anti-id antiserum. In summary, the results obtained so far with myeloma proteins indicate that the native structure is essential for the expression of ind determinants on most proteins of the K type, whereas isolated L or H chains of type #i proteins frequently have activity. It should be emphasized that the existing data do not rule out the possible presence of additional determinants on all type #i proteins that require the interaction of H and L chains, since a line of identity in Ouchterlony analysis does not exclude the possible presence of an additional antigenic determinant. The presence of idiotypic determinants on isolated L and H chains of rabbit antibodies was investigated by Bordenave (1971), who used rabbit antiidiotypic antisera directed against rabbit anti-Salmonella abortus equi ( anti-SAE) antibodies. Antiidiotypic antisera were prepared in 3 rabbits against each of two anti-SAE preparations. In each case isolated H chains gave a positive ring test in liquid medium against the antiidiotypic antiserum. The L chains of one donor antibody failed to react with each of the three homologous antiidiotypic antisera. However, a recombinant of the same L chains with nonspecific H chains gave a weak reaction with two of the antisera. In the second system isolated L chains did give a weak reaction with two of the three antiidiotypic antisera, but only after prolonged immunization. Antiidiotypic antisera with strong reactivity against the

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native anti-SAE antibody, but with no activity directed to isolated L chains, appeared early in the course of immunization of each of the 3 recipient rabbits. It thus appears that thc major idiotypic determinants in these rabbit antibodies require the interaction of H and L chains; only weak reactions were obtained with the isolated chains. Tests, by double diffusion, for antigenic similarity between the isolated chains and the native protein were not reported. C. LOCALIZATION OF INDIVIDUALLY SPECIFICDETERMINANTS OF ISOLATED LIGHTCHAINSTO THE VARIABLE REGION

As noted earlier, Bence-Jones proteins or L chains isolated from myeloma proteins have individually specific antigenic determinants (Stein et al., 1963). (These are not necessarily the same as determinants expressed on L chains in the intact immunoglobulin molecule; L chains may acquire a new conformation upon isolation.) It has recently been demonstrated that such determinants are localized in the variable segment of the chain. Molecules comprising only the variable half of the light chain are sometimes found in the urine of patients with multiple myeloma (Cioli and Baglioni, 1966; Solomon et al., 1966; Tan and Epstein, 1967). It is believed that they can be formed by proteolysis of Bence-Jones proteins in the serum or urine (Solomon et al., 1966; Cioli and Baglioni, 1968), although the direct biosynthesis of appreciable quantities of these chain segments has also been reported (Solomon and McLaughlin, 1969; Schubert and Cohn, 1970). Such a fragment was isolated from the urine of a patient by Tan and Epstein (1967) and characterized by its peptide map. The Bence-Jones protein was also purified and used to prepare antibodies in rabbits which, after appropriate absorption, reacted with individually specific determinants of the immunogen. A line of identity was obtained in the reaction of these anti-ind antibodies with the Bence-Jones protein and the fragment comprising the variable region. Thus, all of the individually specific determinants appeared to be represented in the variable segment. This work was extended by Solomon and McLaughlin (1969), who utilized the urine of a patient with multiple myeloma, which contained Bence-Jones protein as well. as fragments corresponding to the VL and C, regions. Anti-ind antibodies to the Bence-Jones protein failed to react with C,, but gave a h e of identity in its reaction with VL and the Bence-Jones protein. The size of the panel of proteins used to demonstrate incl specificity was not indicated. Solomon and McLaughlin also were able to cleave the Bence-Jones protein or L chains isolated from the serum myeloma protein with proteolytic enzymes and in this way to produce artificially the VL and

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C, segments. Enzymes that wcrc effective included trypsin, pepsin, subtilisin, and papain, as well as an enzyme present in normal or pathological urine; the latter enzyme was active only at low pH. Differences in susceptibility to proteolysis were noted among various BenceJones proteins. In each of 3 patients the undegraded Bence-Jones protein gave a line of identity with the V,, fragment in reacting with anti-ind antiserum to the Bence Jones protein; the CL fragment did not react, The capacity of various enzymes to cleave the L chain in the same general region suggested that the VL and CL segments are compact and relatively resistant to proteolysis but are joined by a loosely folded sequence of amino acids which is susceptible to enzymatic attack. It is of interest that the cleavage occurs between the two large disulfidebonded loops present in the VL and CL segments; this suggests that the polypeptide segment within a loop has a compact structure. Upon prolonged digestion the CL region was degraded, whereas the V, segment was relatively resistant; this could account for the more frequent detection of VL, as compared to CL fragments in the urine of patients with multiple myeloma. IX. Cross-Reactions of Anti-ind Antibodies with Nonspecific Immunoglobulins

The data to be summarized in this section suggest that there are two kinds of individually specific determinants in myeloma proteins. The first type cannot be detected in nonspecific immunoglobulin, even with extremely sensitive techniques. The second is apparently present in the nonspecific population; however, it is not certain whether the cross-reacting determinants are identical or only related. The most convincing evidence that certain individually specific determinants of myeloma proteins are absent from nonspecific immunoglobulin was presented by Kunkel (1970). Anti-ind antisera were prepared in monkeys or rabbits and were tested by passive hemagglutination, using erythrocytes to which the immunogen (IgG myeloma protein) was covalently conjugated. In a typical tcst system 8 x 10-G mg./ml. of the homologous myeloma protein significantly inhibited passive hemagglutination whereas 60 mg./ml. of nonspecific human Fraction I1 was not inhibitory; the ratio of the two numbers is approximately 7 x loG.In the twelve systems studied, no inhibitory capacity was detected in pooled nonspecific immunoglobulin. Minimum ratios reported for the inhibitory capacities of the immunogen, as compared to nonspecific IgG, ranged from 6 x 10' to 3 x lo'. In other words, less than one molecule in 3 x 10' of nonspecific IgG, in the later instance, shared the individual antigenic specificity of the myeloma protein.

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The same methods were applied to individually specific antigenic determinants of four Bence-Jones proteins ( Kunkel, 1970). Again, no inhibition of the passive hemagglutination with anti-ind antisera was observed with large concentrations of nonspecific human IgG or L chains isolated from the nonspecific IgG. The ratio of the inhibitory capacity of the homologous Bence-Jones protein to that of nonspecific L chains varied from 7 x 10' to 2 x 1 0 . These are minimum values since no inhibition was observed with the highest concentration of nonspecific L chains tested. Similar results were obtained with a different but less sensitive test system (Wang et al., 1970a), utilizing 0.5 pg. of 1251-labeledF( ab'), fragments of IgG myeloma protein and rabbit anti-id antiserum. Complexes were precipitated by goat antirabbit Fc. Twenty micrograms of unlabeled homoIogous myeloma protein inhibited the reaction almost completely whereas 1200 p g . of nonspecific IgG had no detectable effect on the reaction. More recently, these results were extended by using smaller amounts of the labeled reference fragments. The ratio of the inhibitory capacity of homologous protein to nonspecific IgG exceeded 3 x lo' to 1 (Wilson et al., 1971b). Quite different results were obtained when cross-reactions were investigated by inhibition of specific precipitation. In nearly all systems studied nonspecific IgG inhibited, partially or completely, the in vitro precipitation of IgG myeloma proteins by rabbit anti-id antisera; classic precipitin tests were employed (Grey et al., 1965; Hurez et al., 1968). In a recent investigation both precipitin and binding tests were carried out with the same system (Wilson et al., 1971b). It was shown that nonspecific IgG had no effect on binding of labeled F( ab')2 fragments by anti-ind antiserum but, when present at high concentration, completely inhibited precipitation of the myeloma IgG by anti-ind antiserum. Other myeloma proteins tested had no effect on either precipitation or binding. The following are possible explanations for these apparently conflicting data. ( a ) Precipitation requires the participation of multiple ind determinants, only some of which are present in similar or identical form in nonspecific immunoglobulin. At least one determinant in the myeloma protein must be completely unique to account for the data. ( b ) A high concentration of normal imniunoglobuh nonspecifically inhibits precipitation. This seems rather unlikely since concentrations as low as 20 mg./ml. of nonspecific IgG are inhibitory in some cases; this value does not greatly exceed the concentration in normal serum. The fact that other myeloma proteins failed to inhibit precipitation also argues against this hypothesis. ( c ) A third possibility would invoke the differ-

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ence in conditions of the precipitin and binding tests. The latter were carried out at much lower concentrations of anti-id antibodies. If the determinants shared by the myeloma protein and nonspecific IgG react with antibodies of low affinity, these antibodies might not participate during the tests of binding, but might be active at the higher concentrations used in precipitin tests. X. Monoclonal Origin of Molecules with Individually Specific Antigenic Determinants

It is apparent that substantial concentrations of antibody molecules bearing a given idiotypic specificity are present in the serum of an immunized animal. For example, strong precipitin bands are frequently observed on analysis by the Ouchterlony technique. It was suggested by Gel1 and Kelus (1964) that molecules sharing a given individual specificity may be synthesized by a single clone of antibody-forming cells. Although it is difficult to prove this directly, there is experimental support for the converse hypothesis; namely, that a single clone of cells produces molecules, all of which have the same individual specificity. This applies to myeloma proteins which are almost certainly of monoclonal origin. [The strongest evidence for this is the continued production of identical monoclonal molecules after repeated passages of myeloma tumor cells in mice (Potter and Lieberman, 1967) or in tissue culture ( Cohn et al., 1969; Paraf et al., 1970)l. Recently Matsuoka et al. (1969a,b) showed that each of three lymphocytic cell lines, established in tissue culture from the cells of a patient with myelogenous leukemia, secreted an IgGl protein which possessed individual antigenic specificity. The specificity appeared identical in two of the lines but was different in the third. Each protein reacted with its homologous anti-ind antiserum to form a single band upon double diffusion in agar gel. This observation, together with the fact that molecules of each cell line belong to a single IgG subgroup and have L chains of a single type, are suggestive of molecular homogeneity. It is noteworthy that these apparently homogeneous molecules were secreted only after prolonged maintenance of the cell lines in tissue culture and that the cultured cells were lymphocytes, presumably from nonmalignant clones. An interesting application of individual antigenic specificity was the demonstration that malignant mouse myeloma cells differentiated into two phenotypic variants in tissue culture, one resembling fibroblasts and the other epithelioid cells. The common origin of the two cell types was demonstrated by the fact that each secreted an immunoglobulin with the same individual antigenic specificity as that of the original myeloma protein (Paraf et al., 1970).

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XI. S u m m a r y

The following are some of the major conclusions derived from studies of idiotypic or ind determinants. 1. Antibodies specific for incl or idiotypic determinants can be prepared against myeloma and Bence-Jones proteins and against antibodies directed to bacteria, blood group antigens, carbohydrates, and haptens. The large proportion of successful immunizations suggests that individually specific determinants may be present on virtually all antibodies or monoclonal proteins. 2. Cross-reactions among ind or idiotypic determinants are very rare among human myeloma or Bence-Jones proteins and among antibodies from different rabbits directed to Salmonella typhi or Proteus vulgaris, which have been studied extensively. When tested by direct precipitation methods, idiotypic cross-reactions among rabbit antibenzoate antibodies are similarly infrequent. A more sensitive test, utilizing inhibition of indirect precipitation, reveals occasional weak cross-reactions, Infrequent but definite idiotypic cross-reactions are observed by direct precipitation methods, among antibodics to Salmonella abortus equi from different rabbits. Such cross-reactions are very infrequent among antibodies from outbred rabbits to streptococcal cell walls, but the frequency becomes significant if the antibodies are elicited in rabbits which have been partially inbred on the basis of their ability to produce antistreptococcal antibodics of limited heterogeneity. Randomly d selected myeloma proteins from BALB/c micc do not normally share i determinants, but the frequency of cross-reaction is highly significant if the myeloma proteins are selected according to their capacity to combine with pneuniococcal C carbohydrate. The frequency of cross-reaction would be expected to be high among mouse A chains, in view of their greatly restricted heterogeneity; data so far are largely confined to amino acid sequences. 3. As would be expected, individual antigenic specificities are determined by variable regions of immunoglobulin molecules. They have been localized to Fab fragments and, in the case of isolated light chains, to that half of the chain comprising the variable segment. In at least one instance the contribution of the heavy chains of IgG and IgM to a shared ind specificity has been corrclated with similarity or identity of amino acid sequence in the variable segments of the two heavy chains. The combining site of rabbit antibenzoate antibody is either part of, or close to a major idiotypic determinant. Isolated light chains in general do not react with anti-ind antisera directed to human myeloma proteins of the K type; occasional weak cross-reactions with isolated heavy chains have been noted. Recombina-

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tion of heavy chains with their autologous K chains generally restores ind determinants. In contrast with type K proteins, either the heavy or light chain frequently reacts strongly with anti-ind antiserum prepared against a human myeloma protein of the type. The number of investigations of this kind reported so far is rather limited. Heavy chains of anti-Salmonella antibodies from some rabbits react, although quite weakly, with antiidiotypic antisera to the rabbit antiSalmonella antibody from which the chains were derived. Isolated light chains also react to some extent, but only with antisera obtained after prolonged immunization with the anti-Salmonella antibodies; antiidiotypic antibodies reactive with anti-Salmonella appear much earlier. 4. Anti-Salmonella antibodies of the IgG and IgM classes from individual rabbits were shown to share idiotypic determinants. This provides strong evidence that both classes of molecule can be synthesized by members of a single clone of cells. The structural relationship between IgG and IgM monoclonal proteins from a single patient suggests that molecules of the two classes, synthesized by a given clone of cells, share identical light chains and variable regions of their heavy chains, and differ only with respect to the constant regions of their heavy chains. A genetic switching mechanism was proposed to account for this structural relationship, and the hypothesis was extended to include normal immunoglobulin biosynthesis. Individual plasma cells from the patient with monotypic IgG and IgM synthesized one class of protein or the other, but not both; however, all of his cells producing myeloma protein were stained by fluorescent anti-ind antibody directed to either the IgG or IgM. 5. The concept that a given idiotypic specificity is characteristic of molecules synthesized by a single clone of cells permitted the assessment of the onset, persistence, and replacement of clones of cells through analysis of idiotypic specificities in antibodies present in serum during the course of prolonged immunization. From these studies it is apparent that clones of cells can persist for long periods of time. During repeated inoculation with protein-benzoate conjugates, clones present 1-2 months after the start of immunization are replaced quite rapidly by new clones, which then persist with gradual changes, despite frequent challenges by the antigen. 6. The number of idiotypic antigenic determinants per molecule is frequently quite limited. This is shown by the greater percentage of molecules identified by an indirect ( antiglobulin) precipitation technique, as compared to direct precipitation. ( Indirect precipitation probably requires only a single antigenic determinant. ) Also, Fab fragments of myeloma proteins in some instances inhibit the precipitation of the intact protein with its anti-ind antiserum.

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7. I t would appear the some inn determinants in human myeloma proteins have their countcrpart in nonspecific human IgG, since a large excess of IgG inhibits the precipitation of many myeloma proteins with homologous anti-inn antiserum. It is not certain whether the crossreacting determinants are similar or identical. In several myeloma proteins studied, however, at least one ind determinant was identified that was not present at all in nonspecific IgG. In one case, less than one molecule in 3 x 10: of nonspecific IgG was identical to the human myeloma protein with respect to such determinant ( s ) . The limited available data thus suggest that one type of ind determinant on a myeloma protein molecule may be found in the nonspecific IgG population whereas another type is unique. Inhibition of precipitation could occur even if antibody to one ind determinant (per Fab fragment) is unreactive with nonspecific IgG. 8. When different recipient rabbits are immunized with the same preparation of rabbit antibody molecules, the resulting antisera generally react with the same or overlapping subfractions of the donor antibody population. This suggests that a portion of the donor antibody consists of a discrete number of homogeneous subpopulations, each sufficient in concentration to elicit antiidiotypic antibodies. The remainder of the donor population may be so heterogeneous that it is not immunogenic in any recipient. REFERENCES Appella, E. (1971). Proc. Nut. Acad. Sci. U . S. 68, 590. Appella, E., and Perham, R. N. (1967). Cold Spring Harbor Syrnp. Quant. Biol. 32, 37. Appella, E., and Perham, R. N. ( 1968). J. Afol. Biol. 33, 963. Boerina, F. W., and Mandeina, E. (1957). J. Lab. Clin. Med. 49, 358. Bordenave, G. ( 1971 ). Submitted for publication. Bordenave, G., and Oudin, J. (1971). Submitted for publication. Braun, D. G., and Krause, R. M. (1968). J. E x p . Aged. 128, 969. Brient, B., and Nisonoff, A. (1970). J. E x p . Med. 132, 951. Capra, J. D., and Kunkel, H. G. (1970). Proc. N u t . Acad. Sci. U . S. 67, 87. Cioli, D., and Baglioni, C. (1966). J. Ago!. B i d . 15, 385. Cioli, D., and Baglioni, C. (1968). J. E x p . A4ed. 128, 517. Cohn, hl., Notani, G., and Rice, S. A . (1969). Immunochemistry 6, 111. Daugharty, H., Hopper, J. E., MacDonald, A. B., and Nisonoff, A. (1969). J. E x p . Med. 130, 1047. Dreyer, W. J., and Bennett, J. C. (1965). Proc. Nut. Acad. Sci. U . S. 54, 864. Eichmann, K., Braun, D. G., Feizi, T., and Krause, R. M. (1970). J. E x p . Med. 131, 1169. Gell, P. G. H., and Kelus, A. S. (1964). Nature (London) 201, 687. Gilman, A., Nisonoff, A., and Dray, S . (1964). Iniiwnockernistry 1, 109. Grey, H. M., Mannik, M., and Kunkel, H. C . (1965). J. E x p . Med. 121, 561.

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Habich, von H. (1953). Schweiz. Med. Wocltenschr. 52, 1253. Hannestad, K., Eriksen, J,, Christensen, T., and Harboe, M. (1970). Immunochemistry 7, 899. Harboe, M., and Deverill, J. (1964). Scand. J. Haematol. 1, 223. Harboe, M.,Solheini, B. G., and Deverill, J. (1969). 1. E x p . Med. 129, 1217. Hilschniann, N., and Craig, L. C. (1965). Proc. Nut. Acad. Sci. U. S . 53, 1403. Hood, L. E., and Ein, D. (1968). Natrrre (London) 220, 764. Hood, L. E., Potter, M.,and McKean, D. J. (1970). Science 170, 1207. Hopper, J. E., MacDonald, A. B., and Nisonoff, A. (1970). J. E x p . Med. 131, 41. Hurez, D., Meshaka, G., hlihaesco, C., and Seligniann, M. (1968). J. Immunol. 100, 69. Jaton, J. C., LVaterfield, M. D., Margolies, M. N., and Haber, E. (1970). PTOC.Nut. Acad. Sci. U. S. 66, 959. Jaton, J. C., Waterfield, M. D., Margolies, M. N., Bloch, K. J., and Haber, E. (1971). Biochemistry 10, 1583. Kelus, A. S., and Cell, P. G. H. (1968). J. E x p . Med. 128, 215. Kohler, H., Shimizu, A,, Paul, C., Moore, V., and Putnam, F. (1970). Nature (London) 227, 1318. Korngold, L., and Lipari, R. ( 1956). Cancer (Philadelphia) 9, 183. Korngold, L., and Van Leeuwen, C. ( 1957a). J. E x p . Med. 106, 467. Korngold, L., and Van Leeuwen, C. (1957b). J. E x p . Med. 106, 477. Krause, R. hl. ( 1971 ). Personal communication. Kunkel, H. C. (1970). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 29, 55. Kunkel, H. G., Mannik, hl., and Williams, R. C. (1963). Science 140, 1218. Kunkel, H. G., Killander, J., and Mannik, M. (1966). Acta Med. Scand. 445, 63. Levin, A. S., Fudenberg, H. H., Hopper, J. E., Wilson, S. K., and Nisonoff, A. (1971). Proc. Nut. Acad. Sci. U. S. 68, 169. MacDonald, A. B., and Nisonoff, A. (1970). J. E x p . Med. 131, 583. MacDonald, A. B., Alescio, L., and Nisonoff, A. (1969). Biochemistry 8, 3109. Makinodan, T., and Albright, J. F. (1967). Progr. Allergy 10, 1. Matsuoka, Y., Yagi, Y., hloore, G. E., and Pressman, D. (1969a). J. Immunol. 103, 1176. Matsuoka, Y., Yagi, Y., Moore, G. E., and Pressman, D. (1969b). J. Immunol. 103, 962. hlehrotra, T. N. (1960). Nature (London) 185, 323. Milstein, C. (1967). Nature (London) 216, 330. Nisonoff, A., MacDonald, A. B., Hopper, J. E., and Daugharty, H. (1970). In “Symp. Exp. Approaches to Homogeneous Antibody Populations April, 1969.” Fed. Proc., Fed. Amer. SOC. E x p . Biol. 29, 72. Nossal, C. J. V., Szenberg, A., Ada, C. L., and Austin, C. M. (1964). J. Exp. Med. 119, 485. Oudin, J. (1966). Proc. Roy. SOC.,Ser. B 166, 207. Oudin, J., and Bordenave, G. ( 1971). Nature (London) 231, 86. Oudin, J., and Michel, M. (1963). C. R . Acad. Sci. 257, 805. Oudin, J., and Michel, M. (1969a). 1. E x p . Med. 130, 595. Oudin, J., and Michel, hl. (1969b). J. E x p . Med. 130, 619. Paraf, A., Moyne, M. A., Duplan, J. F., Scherrer, R., Stanislawski, M., Bettane, M., Lelievre, L., Rouze, P., and Dubert, J. M. (1970). Proc. Nut. Acud. Sci. U. S. 67, 983.

INDIVIDUAL ANTIGENIC SPECIFICITY OF IMMUNOGLOBULINS

99

Penn, G. M., and Kunkel, H. G. (1971). Personal communication. Penn, G. M., Kunkel, H. G., and Grey, H. M. (1970). Proc. Soc. Exp. Biol. Med. 135, 660. Potter, M., and Leon, M. A. (1968). Science 162, 369. Potter, M., and Lieberman, R . (1967). Advan. Immunol. 7, 92. Potter, M., and Lieberman, R. (1970). J. E x p . Aled. 132, 737. Potter, M., and Robertson, C. L. (1960). J. Nut. Cancer Inst. 25, 847. Potter, M., Lieberman, R., and Dray, S. (1966). J. Mol. Biol. 16, 334. Roholt, 0. A., Radziniski, G., and Pressman, D. (1965). Science 147, 613. Schooley, J. C. (1961). J. lrnmunol. 86, 331. Schubert, D., and Cohn, M. (1970). J. hlol. Biol. 53, 305. Seligmann, M., and Mihaesco, C. (1967). In “Third Nobel Symposium on Gamma Globulin” ( J . Killander, ed.), p. 169. Interscience, New York. Seligniann, M., Meshaka, G., Hurez, D., and Mihaesco, C. (1966). Zmmunopathol. Int. S y m p . 4th, 1965 p. 229. Slater, R. J., Ward, S. M., and Kunkel, H. G. (1955). J. E x p . Med. 101, 85. Solheini, B. G., Harboe, M., and Deverill, J. (1971). Submitted for publication. Solomon, A., and McLaughlin, C. L. (1969). J. B i d . Chem. 244, 3393. Solomon, A., Killander, J., Grey, H. M., and Kunkel, H . G. (1966). Science 151, 1237. Spring, S . B., and Nisonoff, A. (1971). J. E x p . Med. In press. Stein, S., Nachman, R. L., and Engle, R. L., Jr. (1963). Nature (London) 200, 1180. Taliaferro, W. H., and Talmage, D. W. (1956). 1. Infec. Dis. 99, 21. Tan, M., and Epstein, W. (1967). J. Immunol. 98, 568. Valentine, R. C., and Green, N. M. (1967). I. Mol. Biol. 27, 615. Wang, A. C., and Fudenberg, H. H. (1971). Personal communication. Wang, A. C., Wang, I. Y. F., McCormick, J. N., and Fudenberg, H. H. (1969). lmmunochemistry 6, 451. Wang, A. C., Wilson, S. K., Hopper, J. E., Fudenberg, H. H., and Nisonoff, A. (1970a). PTOC.Nut. Acad. Sci. U . S. 66, 337. Wang, A. C., Pink, J. R. L., Fudenberg, H. H., and Ohms, J. (1970b). Proc. Nut. Acad. Sci. U . S. 66, 657. Weigert, M . G., Cesari, I. M., Yonkovich, S. J., and Cohn, M. (1970). Nature (London) 228, 1045. Weigle, W. 0. (1958). J. Irnmunol. 81, 204. Williams, R. C., Jr., Kunkel, H. G., and Capra, J. D. (1968). Science 161, 379. Wilson, S. K., Nisonoff, A,, and Fudenberg, H. H. (1971a). Unpublished results. Wilson, S. K., Hopper, J. E., Nisonoff, A., and Fudenberg, H. H. (1971b). A n n . N . Y . Acad. Sci. In press. Wuhrmann, F. H., Wunderly, C. H., and Hassig, A. (1950). Brit. J . E x p . Pathol. 31, 507.

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In Vitro Approaches to the Mechanism of Cell-Mediated Immune Reactions

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BARRY R BLOOM1 Deportment o f Microbiology ond Immunology. Albert Einrfein College of Medicine. Bronx. New York

I . Introduction . . . I1. Lymphocyte Transfomiation

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A . Relation of Lymphocyte Transformation to Cell-Mediated . . . . . . . . . . . Immunity B . Cell Cooperation and Lymphocyte Activation . . . . C. Conclusions . . . . . . . . . . . 111. Direct Cytotoxicity of Target Cells by Lymphocytes . . . A . Introduction . . . . . . . . . . . B Cytotoxicity of Target Cells Bearing hlembrane-Associated . . . Antigens Mediated by Sensitized Lymphocytes C . Cytotoxicity of Lymphocytes Stimulated by Soluble . . . . . . . . Antigens or Mitogens . D . Correlation of Direct Lymphocyte Cytotoxicity with Cell. . . . . . . . Mediated Immunity . E . Conclusions . . . . . . . . . . . . . . . IV . Mediators-Qualitative Basis of the Response A . Migration Inhibitory Factor and Factors Affecting . . . . . . . . . . Macrophages B. Lymphotoxin and Growth Inhibitory Factors . . . . C . Blastogenic Factor and Factors Affecting Lymphocytes . . D . Chemotactic Factor . . . . . . . . . E . Interferon . . . . . . . . . . . F. Antibodies . . . . . . . . . . . G . Skin-Reactive Factors . . . . . . . . . V . Enumeration of Specifically Sensitized Cells-Quantitative Basis . . . . . . . . . . of the Response . A . Antibody Formation . . . . . . . . . . . . . . . B. Mixed Lymphocyte Interaction . C . Delayed-Type Hypersensitivity . . . . . . . VI . Reality Testing-Relationships between in Vitro Results and CellMediated Immunity i n Vioo . . . . . . . . A Models . . . . . . . . . . . . B. Histology-Nature and Origin of Cells in Delayed-Type . . . . . . . Hypersensitivity Reactions C . Rejektion of Allografts and Tumors . . . . . .

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'Recipient of U . S . Public Health Service Career Development Award 5K03. Work from the author's laboratory has been supported IJY U . S . Public Health Service Grants AI-07118 and AI.09807 .

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D. Adjuvant Effects . . . . . . E. Cellular Resistance . . . . . . F. Unified Model . . . . . . . VII. Relationships between Cell-Mediated Immunity and Formation . . . . . . . . . A. Effector Cells in Delayed-Type Hypersensitivity B. Cells Involved in Antibody Formation . . References . . . . . . . .

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“Only connect ..... E. M. Forster, “Howard’s End”

I. Introduction

The existence of the delayed-type hypersensitivity or cell-mediated immune response’ has been known since the time of Jenner and Koch, but appreciation of its importance in disease, and perhaps more significantly in health, has only recently become possible. For many years, cell-mediated immunity has been associated with resistance to certain bacterial, mycotic, and viral infections, especially intracellular parasites. Another suggested role for the delayed-type hypersensitivity response, perhaps its primary function and the evolutionary basis for its 300million-year existence, is that of “surveillance,” i.e., the rejection of cells in the body antigenically altered by neoplastic events (Thomas, 1959; Burnet, 1967). Support for these possibilities comes from many experiments but, perhaps, most impressively from the study of those conditions of man in which there is deficiency or suppression of this immune response (Peterson et al., 1966; Balner, 1970). In addition, the cell-mediated immune response serves as the principal obstacle to successful organ and tissue transplantation, as well as being involved in the pathogenesis of a number of autoimmune diseases of man and experimental animals. It has been clear since the work of Chase (1945) that the immunological information for the immune response resides in living lymphoid cells, since they can passively transfer cell-mediated immunity to normal animals. And yet, a detailed understanding of how these cells bring about the reactivity in the recipient remains elusive. Attempts to understand the mechanism of this response by tissue culture methods were pioneered by Rich and Lewis (1932) and, although fraught with considerable difficulties ( Heilman, 1963), have in recent years begun t o fulfil their promise (Lawrence and Landy, 1970). Considerable interest in the in uitro models for delayed-type hypersensitivity as diagThe terms cell-mediated immunity and deluyed-type hypersensitivity will be used interchangeably throughout this article.

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nostic and clinical tools has been generated, and the activities and products of lymphoid cells in vitro will hopefully provide insight into the mechanism of their response in uivo. The focus of this review will be on the study of cell-mediated immunity in vitro, principally in terms of explaining the effector process of the response. Descriptions of many of the in vitro methods used, and discussion of their applications and limitations appears in Bloom and Glade (1971). To organize observations in many species and systems, it has been convenient to make four general assumptions. 1. The diversity of information for cell-mediated immunity is carried by lymphocytes and is of the same order of magnitude and specificity as for circulating antibodies. Yet no assumption about whether the antigen-specific receptor possessed by sensitized cells is an immunoglobulin molecule is made here.'" 2. All cell-mediated immune reactions have a common basis, including the tuberculin-type reaction, contact hypersensitivity, cellular resistance to infection, allograft rejection, and tumor immunity. 3. The increase in responsiveness after a process of sensitization or immunization is consistent with a clonal selection by antigen of reactive cells ( Burnet, 1959). Information for cell-mediated immunity is carried by thymus-dependent lymphocytes (J. F. A. P. Miller et al., 1962; Peterson et al., 1966; J. F. A. P. Miller and Osoba, 1967), but whether their specificity is generated by genes (Jerne, 1971), informational molecules from other cells (Lawrence, 1969), or antigen itself remains unclear. 4. The mechanisms of effector reactions mediated by delayed-type hypersensitivity and humoral antibody are fundamentally different. Explicit discrimination between these two mechanisms must be made wherever possible, not only in vivo but in vitro as well. The most obvious example of the importance of this point would be to confuse the result of an in vitro assay for tumor immunity as indicating a strong cell-mediated immunity, when antibodies of a tumor-enhancing type are perhaps being measured. It must be admitted at the outset that certain limitations have been imposed on this review in consequence of its overall design, which has been to analyze the basic mechanisms of the response as derived from a great number of in vitro studies, to compare the results obtained in vitro with the realities obscrved in vivo, and to explore possible relationships of both to the antibody response. To cover the vast amount of information, it has been neccssary to be arbitrarily selective, rather than truly comprehensive. The antigen receptor problem has recently been well reviewed by Metzger (1970).

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II. Lymphocyte Transformation

Clearly immunological information for the cell-mediated immune response is carried by lymphocytes, and specially the small lymphocyte (Gowans and McGregor, 1965; J. F. A. P. Miller and Osoba, 1967). When relatively pure populations of lymphocytes have been obtained from hypersensitive animals, they effected passive transfer of reactivity to normal recipients (Chase, 1945; Wesslkn, 1952a). Small lymphocytes initiated sensitization in the graft-versus-host reaction ( Gowans et al., 1962; McBride, 1966). Lymphocytes do perfuse the tissues (Hall, 1969), and sensitization is likely to occur in the periphery (Strober and Gowans, 1965), the lymphocytes migrating from a graft or tissue site to the regional lymph node ( Elves, 1970). Moreover, lymphocytes have been shown to carry immunological memory (Gowans and Uhr, 1966). During the induction of graft-versus-host disease ( Pederson and Morris, 1970) or during the sensitization of normal guinea pigs to chemical allergens (Oort and Turk, 1965; Turk, 1967), morphological transformation of small lymphocytes to large pyroninophilic or blast cells has been observed. Thus, in the process of antigenic stimulation, small lymphocytes are transformed, morphologically and functionally (Ling, 1968; Naspitz and Richter, 1968). Studies designed to unravel the mechanism and significance of transformation of lymphocytes by tissue culture techniques followed the observation by Nowell ( 1960) that transformation of normal lymphocytes could be initiated in vitro by phytohemagglutinin (PHA) (Ling, 1968; Naspitz and Richter, 1968). Consequently, the present discussion will focus only on two problems-the mechanism of activation in general and the relationship of blast cell transformation induced by antigens to cellmediated immunity. The fact that lymphocytes can be transformed at will in uitro by specific antigens or many nonspecific agents, such as PHA, concanavalin A ( ConA) , streptolysins, antilymphocyte sera, anti-immunoglobulins, and antiallotype sera, suggests that this system might be useful for studying mechanisms of regulation and control in mammalian cells, and possibly the process of gene activation. Remarkably little is known about gene activation and regulation in mammalian cells, and even the best studies, for example, the induction of tyrosine aminotransferase ( cf. G. M. Tompkins et al., 1969) in liver tumor cells in &To, leave questions about the preexistence of low levels of gene products, the relation of the inducer, a steroid hormone, to the product, and the relation of gene activation in neoplastic cells to normal processes. However, there is absolutely no evidence to indicate that activation of lympho-

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cytes by any agent results in expression of a previously repressed gene or in production of new messenger RNA and the release of products synthesized cle nouo (see Section IV). If one looks at the changes brought about in lymphocytes by activation with nonspecific agents, particularly PHA, which engage most of the cells, the following events are known to occur: ( 1 ) reaction of the inducer at the plasma membrane of the lymphocyte; ( 2 ) increase in turnover (up to 21-fold) in phosphatidyl inositol in the membrane which can be detected within 5 minutes (Fisher and Mueller, 1968); (3) acetylation of histones (Pogo et al., 1966) and binding of acridine orange dye to the deoxyribonucleoprotein ( Killander and Rigler, 1969) within 15 minutes; ( 4 ) increased synthesis of ribonucleic acid (RNA), predominantly polydisperse RNA ( H . L. Cooper, 1969), in 30 minutes to 2 hours; ( 5 ) morphological changes, enlargement of the cell and reorganization of nuclei and nucleoli ( described as transformation of lymphocytes to blast cells), which may begin at about 20 hours, followed by an increase in endocytosis and redistribution of acid hydrolases in lysosomes (Brittinger et al., 1968; R. Hirschhorn et al., 1968); ( 6 ) incorporation of thymidine into deoxyribonucleic acid ( DNA) beginning at about 36 hours for mitogens and at 40 hours for antigens, followed by mitosis, Although the kinetics of division vary with different inducers and species, in no system has synchronous activation been shown (see Ling, 1968; Naspitz and Richter, 1968). Similarly, although there is good evidence that the pyroninophilic cells divide to form small lymphocytes in uiuo (Gowans et al., 1962), there is some question as to the fate of blast cells that arise in uitro. For example, it has been observed in some experiments that blast cells become small lymphocytes but do so without division (Polgar and Kibrick, 1970). Significantly the majority (90%) of peripheral blood lymphocytes which transform with PHA, or in graft-versus-host reactions, are thymus-derived (Davies et d.,1966, 1968). In uitro transformation was believed to be a system in which Iymphocytes differentiated into antibody-producing cells, even though abortively in most cases. It has not been established that blast cells are engaged in significant production of immunoglobulins, although they are able to incorporate amino acids into proteins of all electrophoretic mobilities (Turner and Forbes, 1966). A. RELATIONOF LYMPHOCYTE TRANSFORMATION TO CELLMEDIATED IMMUNITY Perhaps the earliest report of an in uitro test for cell-mediated immunity is that of Achard and Bkrnard (1909) in which it was said

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that morphine had a depressing effect upon the leukocytes of morphine addicts, whereas Old Tuberculin stimulated the activity of tuberculinsensitive leukocytes in vitro. Unfortunately, it was not quite clear in the report precisely what activity of the cells was being followed. Peripheral blood or lymph node lymphocytes from individuals with tuberculin hypersensitivity were found to transform in vitro when cultured in the presence of specific antigens (Schrek, 1963; Pearmain et al., 1963; Marshall and Roberts, 1963). In studies of reactivity of guinea pig lymphocytes from donors with delayed hypersensitivity to tuberculin, ovalbumin, bovine 7-globulin ( BGG) and dinitrophenyl ( DNP)-BGG, transformation was induced in all cases by specific antigen (Oppenheim et al., 1967). In contrast, peripheral lymphocytes from animals immunized to produce only circulating antibodies failed to respond (Mills, 1966). The specificity of reactions of hapten-protein conjugates was found to be primarily conjugate-specific or carrier-specific and not hapten-specific (Mills, 1966; Oppenheim et al., 1967). As little as a 10-minute exposure to purified protcin derivative ( PPD ) or diphtheria toxoid initiated transformation ( Caron, 1967a). A strong argument favoring usefulness of transformation as a correlate in uitro of delayed hypersensitivity was offered by Mills (1966) and by Oppenheim (1968), who emphasized the correlation among hypersensitivity of the donors, carrier specificity, and probable thymic origin of responding lymphocytes. The fate of antigen-transformed cells from human peripheral blood was studied cinemicrographically by Marshall et al. (1969), who observed that only a small number of cells were stimulated to divide initially. However, once stimulated, they divided by clonal proliferation, each daughter cell dividing with remarkably constant generation and doubling times of about 8 to 13 hours. (See Fig, 1.) The question whether lymphocytes are pluripotential, i.e., specific for more than one antigen, will be discussed in Section V. Lymphocytes from individuals sensitized to multiple antigens may be stimulated additively when challenged in vitm with multiple antigen (Caron, 1967b). By using the ingenious technique of allowing cells activated by one antigen, streptokinase-streptodornase (SKSD) to incorporate bromodeoxyuridine (BUDR) and then killing those cells with visible light, Zoschke and Bach (1970) were able to eliminate cells responding to one antigen without affecting the response of lymphocytes to a second, tetanus toxoid. Thus, either lymphocytes have unique specificity for individual antigens, or the number of antigens to which a single cell can respond is quite restricted,

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In most cases lymphocyte transformation in vitro correlates with delayed-type hypersensitivity in vivo, but many individuals possessing cell-mediated immunity also producc circulating antibodies to the same antigen. For example, virtually all adults have antibodics to PPD (Sehon, 1962; Freedman et ul., 196.3). Thc~critical question is whether this correlation is invariant ancl constitutcs sufficient evidencc for inferring cell-mediated immunity is prcscnt. Unfortunately considerablc recent evidence suggcsts that it is not. a. Lymphocytes obtaincd from antibody-producing animals, given a secondary antigenic stimulation in vitro, show a marked increase in incorporation of labeled thymidine into DNA ( Dutton and Pcarce, 1962; Dutton, 1967). Intcrestingly thc specificity of rccognition of the antibody-primed lymphocytes in this in vitro secondary rcsponsc is largely conjugate- or carrier-specific, rathcr than hapten-spccific ( Ovary and Benacerraf, 1963; Dutton and Bulnian, 1966). b. Guinea pigs sensitizc~lto shecp red blood cells (SRBC) in complete Freunds adjuvant devcloped considerable levels of delayed hypersensitivity, whcrcas those immunized with red cells intravenously lacked delayed hypersensitive dcrnial reactivity ( Loewi et al., 1968). When lymphocytes from either group were stimulated in vitro with SRBC a high degree of transformation occurred, indicating that lyniphocytes from nonhypersensitive donors wcre capably of responding. In similar experiments, rabbits were immunized with complete or incomplete adjuvant; delayed hypersensitivity devclopcd only when complete adjuvant was used (Benezra et al., 1970). Nevertheless, lymphocyte transformation occurred in both groups. c. Under appropriate conditions, lymphocytcs from normal nonimmunized humans can clearly be stimulatcd by preformed antigenantibody complexes to transform and incorporate labeled thymidine into DNA ( Bloch-Shtacher et al., 1968). d. Peripheral blood lymphocytes from patients known to have immediate-type hypersensitivity and apparcntly lacking delayed hypersensitivity arc often transfornicd by thc specific allergen. For example, lymphocytes from human donors who arc allergic to pollen antigens have been transformed ( 3 0 4 0 % blast cclls) by pollen antigens, and those from dcsensitizcd or untreated individuals have done so to about the same degree (Zeitz et al., 1965; Girard et al., 1967; Brostoff and Roitt, 1969; Brostoff et ul., 1969). Transformation was also found in patients with immediate-type reactivity to penicillin (Fellner et al., 1967) and to a variety of peptide hormones, alkaloids, local anesthetics, iodides, or antibiotics ( Halpern et al., 1967). But in

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many of the situations, an immediate-type reaction in the skin precluded or obscured existence of an underlying component of delayed hypersensitivity. These results indicated that cells primed for antibody formation, or normal cells in the presence of antibodies, might under appropriatc conditions respond to antigens by transformation. e. Two experiments demonstrated uncorrelated lymphocyte transformation and cell-mediated immunity. In one, patients with chronic mucocutaiieous candidiasis werc found unable to resist their Candidu infection or to give positive delayed-type skin reactions to Candida antigen, although they were capable of giving skin reactions to other antigens such as PPD or SKSD (Chilgren et al., 1967). More striking is the ability of some of these patients’ cells to respond normally in vitro to Canclicla antigens by thymidine incorporation and yet be unable to make one of the putative mediators of hypersensitivity, migration inhibitory factor ( MIF) ( Rocklin et al., 1971; Valdimarsson et al., 1970). At the other extreme, guinea pigs sensitized to a carbohydrate antigen of mycobacteria (Chaparas et al., 1970) or peptides of tobacco mosaic virus (Spitler et al., 1970) demonstrated positive delayed skin reactivity to these antigens, produced MIF or demonstrated inhibition of macro-

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FIG. 1A. FIG. 1. Kinetics of lymphocyte transformation. ( A ) Absolute counts of lymphoblasts in cultures of sensitized human lymphocytes stimulated with tuberculin PPD. No lymphoblasts were encountered at the start of the experiments or after 24 hours of incubation. Counts are displayed on a logarithmic scale. ( B ) Exponential proliferation of single lymphoblasts from a tuberculin-sensitive donor following PPD stimulation. The horizontal line at the left represents the original lyniphoblast; following across toward the right, each division of the line represents a mitotic division of the cell. Generation times are written in hours over the appropriate lines. Those times marked with an asterisk are average times for a group of cells where it was not possible to follow the fate of single cells but where the group itself was discrete and all mitoses could be clearly seen. (From Marshall et al., 1969.)

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FIG.1B.

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phage migration in vitro, and yet were not stimulated in either case to transform or incorporate thymidine. These experiments point to clear dissociation between the response in vioo and lymphocyte transformation in vitro.

B. CELL COOPERATION AND LYMPHOCYTE ACTIVATION As mentioned, it has been impossible to demonstrate synchrony of stimulation by addition of any agent, specific or nonspecific, which induces lymphocyte transformation. Since antigen is added immediately and might be assumed to react instantly with receptors on lymphocytes, it is perplexing that cells undergo thymidine incorporation and division over such an enormous time period (2-9 days for human lymphocytes). One possible explanation for this asynchrony would be that antigens cannot directly stimulate lymphocytes and must either be processed by another cell or be presented to the lymphocyte on the surface of another cell. When macrophages or monocytes were removed from peripheral blood cells by glass bead columns (Cline and Swett, 1968; Hersh and Harris, 1968), transformation by antigens such as PPD, SKSD, and streptolysin 0, was abolished. Reactivity was regained when purified macrophages were restored to the sensitized lymphocytes, with an optimal ratio of about 1 macrophage to 30 lymphocytes. Similar results indicated that the cell required for activation was radiation-resistant (Seeger and Oppenheim, 1970; Schechter and McFarland, 1970), consistent with behavior of monocytes and macrophages. This point was demonstrated nicely by the use of monocytes purified on small cover slips to restore reactivity of monocyte-depleted lymphocytes to antigens (Levis and Robbins, 1970a) and even to PHA (Levis and Robbins, 1970b). It is clear that two types of cells are required for activation of the lymphocytes. The helper cell is a glass-adherent cell, and because of its adherence to glass, likely to be a monocyte, yet some lymphocytes can adhere strongly to glass surfaces as well (Bianco et al., 1970). If, indeed, the cooperative activity depends upon monocytes or macrophages, it is not clear whether the synergism is due to antigen processing, presentation of antigen in proper form, or simply production of nutrients in the medium required for lymphocyte survival. In the case of mixed lymphocyte interaction, once again a glassadherent cell is required for activation of lymphocytes (Cordon, 1968; J. F. Bach et aE., 1970; Alter and Bach, 1970). Highly purified lymphocytes are not stimulated by antigens or in mixed lymphocyte culture. In a potentially important experiment, Alter and Bach (1970) found that the glass-adherent cell could be totally replaced by the supernatant of

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normal, unstimulated, glass-adherent cells, suggesting a conditioned medium factor. Conclusions from this experiment, if confirmed, would contradict the notion that the macrophage is required for processing or presenting antigen to lymphocytes in special form. In the mouse system, for high degree of transformation of sensitized lymphoid cells with PHA and specific antigens, a natural antibody to mouse cells present in normal human serum seems required as well (Adler et al., 1970a,b). The only direct evidence against cell cooperation appears to be the successful transformation of lymphocytes in agar by PHA (Coulson et al., 1968) where agar might be expected to prevent cell interaction. But the possibilities that cells are mobile and can interact, that responding cells occur in clumps, or that macrophages act as feeder cells and condition the medium cannot be excluded. C. CONCLUSIONS Lymphocyte transformation seems to be a consequence of stimulation of sensitized lymphocytes by specific antigens. Although it correlates with delayed-type hypersensitivity, clearly, lymphocyte transformation can occur in individuals lacking delayed hypersensitivity, e.g., undergoing secondary antibody rcsponses or having reaginic sensitization; conversely, it may not occur in individuals known to possess delayed hypersensitivity to certain antigens. Studies of morphological and biochemical changes in lymphocytes do not explain the nature of the lesion or cellular resistance to infection associated with deIayed hypersensitivity reactions in vivo. Therefore, it is necessary to consider the direct and indirect effects of hypersensitive lymphocytes on other cells resulting from stimulation by specific antigens in uitro. I l l . Direct Cytotoxicity of Target Cells by Lymphocytes

A. INTRODUCTION Two medically significant manifestations of cell-mediated immunity, allograft rejection and tumor immunity, are characterized by rapid and specific destruction of massive numbcrs of cells. Evidence indicates that the basic immunological mechanism responsible for this destruction is cellular immunity rather than humoral antibodies ( Medawar, 1959). Since the rejection of allografts and tumors could be passively transferred regularly from sensitized donors to normal recipients with lymphocytes but not with serum, much effort has been devoted to studying

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lymphocytes in vitro to explore the underlying mechanisms and to determine whether lymphocytes from immunized individuals were capable of destroying target cells bearing foreign antigens to which the donors were sensitized. The difficulties involved were first overcome by Govaerts (1960) using thoracic duct cells of a dog which had rejected a kidney graft 4 days previously. These cells destroyed a monolayer of cells obtained from the other kidney of the same donor. Later, Rosenau and Moon (1961) demonstrated that spleen cells of BALB/c mice, which had been immunized against L-cell tissue culture cells ( neoplastic cells derived from the C3H mouse strain), were capable of killing L cells or releasing them from the monolayer. Added complement was not required, and no mouse serum proteins could be detected on the target cells by fluorescent antibody techniques. Although there was a greater cytotoxicity of immune cells, there was a significant cytotoxic effect of nonimmune cells. Wilson ( 1965a,b ) modified experimental conditions to permit more quantitative study using inbred mouse and rat kidney or tumor cells as targets. Normal, nonimmune cells had no detectable cytotoxicity in this system. Immune cells showed no effect on syngeneic target cells, but produced dramatic cytotoxicity in the specific, allogeneic target cells. An approximate first-order relationship between the number of lymphoid cells added and the percentage of target cells surviving was obtained, suggesting single-hit kinetics, i.e., one target cell killed by a single immune cell. Further, 1-2% of the sensitized lymphoid population was estimated to be cytotoxic (Wilson, 1965b)-a figure to which we shall return (Section V ) . Therefore, under appropriate conditions, sensitized lymphocytes were capable of direct target cell cytotoxicity in vitro, and could be analyzed. Extensive literature on the cytotoxic effects of lymphoid cells in vitro has been well reviewed by Perlniann and Holm (1969a), and they indicated at least four different situations in which target cell destruction could be mediated directly by lymphocytes: (a) lymphocytes from sensitized donors may be cytotoxic for target cells with membrane-associated antigens; ( b ) lymphocytes sensitized to soluble antigens or activated by nonspecific mitogens may exert cytotoxicity on target cells antigenically unrelated to the stimulating antigen or mitogen; ( c ) lymphoid cells from normal individuals may be induced to cause cytolysis of target cells by the presence of specific antitarget cell antibodies; and ( d ) normal lymphoid cells may lyse target cells coated with complement components. It would be naive to presume that a cell population so heterogeneous and diverse as lymphocytes exert cytotoxicity, with its potential for selfdestruction, in any but a very complex fashion.

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B. CYTOTOXICITY OF TARGET CELLSBEARINGMEMBRANEASSOCIATEDANTIGENSMEDIATED BY SENSITIZED LYMPHOCYTES 1 . Nature of Antigenic Determinants of Target Cell Cytotoxicity has been observed in a variety of species using lymphoid cells sensitized against transplantation antigens, organ-specific antigens, and tumor antigens, rendering it a most useful model to study allograft rejection, certain autoimmune diseases, and tumor immunity ( Perlmaim and Holm, 1969a). Recent modifications of microassay techniques have opened new possibilities for clinical assessment of immunological reactivity of patients to tumors (Hellstrom et al., 1968a,b; Bubenik et al., 1970b; Takasugi and Klein, 1970; Hellstrom and Hellstrom, 1971). Target cells may be nucleated or nonnucleated. Further, soluble antigens may be attached to target cells, either to erythrocytes (Perlmann et at., 1968, 1970a) or to mouse mastocytoma cell (Henney, 1970a,b) coupled covalently to the target cells by water-soluble carbodiimide. Target cells from different sources bearing the same transplantation antigen vary greatly in susceptibility to cytolysis. Further, the rate and degree of target cell lysis vary considerably with the type of antigen. For example, greater lysis of cells occurs across a major histocompatibility barrier than across a weak histocompatibility barrier or by syngeneic lymphocytes reacting against a tumor-specific antigen (Brondz, 1964; Rosenau and Morton, 1966). In fact, only by the most rigorous immunization schedule was cytotoxicity for tumor antigen achieved in a syngeneic system (Brunner et al., 1970) (see Section VI). The most resistant cells to lymphocyte-mediated cytotoxicity appear to be other lymphocytes or macrophages (Holm, 1967), although destruction of lymphoid cell lines (Steel and Hardy, 1970; Hardy et al., 1970) and macrophages (Brondz, 1964) has been observed. 2. Nature of the Aggressor Cell Lymphocytes obtained from spleen, lymph node, peripheral blood, or thoracic duct have all been found capable of cytotoxic activity after sensitization. Obviously the degree of reactivity depends on the time interval after sensitization and the nature and route of antigenic stimulation. In general, living cells as source of antigen are considerably more immunogenic than killed cells or extracts. There is sonic evidence that more than one kind of lymphocytes may be cytotoxic; one type arising 6-12 days after immunization (Brunner et al., 1970) is resistant to radiation, fluorodeoxyuridine (FUDR) ,

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mitomycin, and actinomycin D (Denham et al., 1970); the other arising later, at about 21 days, is radiation-sensitive and killed by agents affecting DNA. The thymic origin of the cytotoxic lymphocytes in one of the most sensitive systems, i.e., lysis of mouse DBA/2 mastocytoma cells by sensitized C57B1/6 lymphocytes, has recently been established (Cerottini et al., 1970a,b), Lymphocytes from C57B1 mice were sensitized in a graft-versus-host situation by inoculation into irradiated DBA mice. When thymus cells were inoculated, spleen cells, obtained from the recipient after the onset of graft-versus-host sensitization, contained exclusively thymus-derived cells from the donor and were highly cytotoxic for DBA mastocytoma in vitro but did not produce cytotoxic alloantibodies. Conversely, when bone marrow cells were inoculated into the irradiated recipients, only a very low degree of cytotoxicity of the spleen cells (which could be completely eliminated by treating the bone marrow donor with antilymphocyte serum) was found, suggesting that this low level was due to circulating thymus-derived lymphocytes and not to bone marrow-derived cells (Cerottini et al., 1970a). Further studies (Cerottini et al., 1970b; Brunner et al., 1971) have shown that, in the presence of complement, pretreatment of spleen cells with antibody against the '6 antigen (Reif and Allen, 1964; Raff, 1969) which is present only on thymus-derived lymphocytes, completely eliminated direct lymphocyte-target cell cytotoxicity but allowed the production of alloantibody by bone marrow-derived lymphocytes. Conversely, antibodies to p chains of mouse IgM, in the presence of complement, completely blocked the production of alloantibodies but had no inhibitory effect on direct lymphocyte-target cell cytotoxicity. These studies demonstrated that in this system, cytotoxicity was a property of thymusderived lymphocytes and distinct from antibody production. Further, no diminution in cytotoxicity was noted when sensitized spleen cells were first passed over glass bead columns, under conditions in which macrophages would be removed (Brunner et al., 1971), indicating that cytotoxicity can be brought about exclusively by lymphocytes. In this system, cytotoxicity is mediated by thymic-derived lymphocytes directly, yet it is possible that bone marrow-derived lymphocytes or other leukocytes may also be capable of cytotoxicity in other systems (see below).

3. Mechanism of Cytotoxicity a. Contact. In all studies of lymphocyte-mediated cytotoxicity, the earliest morphological event detectable is a clustering and attachment of the lymphocytes to target cells. A barrier to attachment, such as Millipore filter separation, prevents cytotoxicity. Cinemicrographic and electron-microscopic studies ( McFarland and Schechter, 1970; Ax et al.,

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1968) indicate that lymphocytes attach to target cells by means of an organelle or tail, known as the uropod ( Roscnau, 1963) ; McFarland et al., 1966; Able et al., 1970). However, the finding that aggregation of lymphocytes and target cells caused by heterologous antimouse lyniphocytic sera does not result in cytotoxicity indicates that aggregation is a necessary but insufficient condition for this cytoxicity (Mauel et al., 1970). In virtually all studies of direct lymphocyte-target cell interaction in which specific antigens are membrane-bound, attempts to detect a cytotoxic factor in the medium have been unsuccessful (Perlmann and Holm, 1969a; Brunner et al., 1969, 1971; for further discussion, see Sections IV,C and V1,B). b. Kinetics. With the use of the mouse mastocytoma system and W r release to assay cytotoxicity, 708 of label was released from target cells in 1 hour using lymphocytes at 1 O O : l target cell ratio (Brunner et al., 1970). Again, it could be calculated that one lymphocyte was capable of killing more than one target cell. At lower ratios, the rate of target cell destruction was decreased, although at ratios as low as 1:1, 20% of the target cells were lysed within 6 hours. At lymphocyte-target ratios of 100: 1, fewer embryonic fibroblasts or allogeneic spleen cells were lysed than mastocytoma cells, indicating variation in sensitivity of different target cells to the same lymphocytes. c. Znhibition of Cgtotoxicity. All available evidence indicates that lymphocytes niust be alive and capable of metabolic synthesis in order to be cytotoxic. Although aggregation occurs at low temperatures, no destruction of target cells is observed (Wilson, 1965a). Agents that suppress RNA and protein synthesis such as Imuran (Wilson, 1965b), actinomycin D (Brunner et al., 1968b) and Puromycin (Brondz and Sidorova, 1969) block cytotoxicity. Antiinycin A, which suppresses electron transport and inhibits respiration, similarly impairs cytotoxicity ( Perlmann and Holm, 1969b). Additionally, divalent cations are required for cytotoxicity (Mauel et al., 1970) which is reversibly inhibited ethylenediaminetetraacetate ( EDTA) . However, all studies on kinetics indicate that cytotoxicity occurs prior to DNA synthesis. For example, inhibition of DNA synthesis with FUDR, an inhibitor of thymidine synthetase, had no effect on the lytic capacity of sensitized lymphocytes (Mauel et al., 1970). Alloantibodies directed against the transplantation antigens of the target cell blocked cell toxicity ( E . Moller, 1965b; Brunner et al., 1968b), whereas antibodies against the alloantigens of the aggressor cells, curiously, did not block cytotoxicity ( Brunner et al., 1967). Rabbit antiserum to mouse IgG, IgA, or IgM had no inhibitory effect on cytotoxcity in the allogeneic system (Mauel et al., 1970).

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d. Specificity. Generally in allogeneic sensitization, direct lymphocyte-target cell cytotoxicity is specific for cells carrying the immunizing antigens. ( S e e Fig. 2.) For cytotoxicity of mouse cells across the H-2 histocoinpatibility barrier, aggrcssor lymph node cells had to be sensitized to all or almost all of the determinants on target cells (Brondz, 1968). When donors were sensitized to oidy n portion of the antigens, cytotoxicity was not obtained. Synergism by lymphocytes of two donors, each sensitized against part of the H-2 antigens, was not observed. In tumor systems, lymphocytes from mice immunized against noncross-reacting, chemically induced tumors show only tumor-specific cytotoxicity in vitro ( Rosenau and Morton, 1966; Hellstrijm et al., 1968a). Because cytotoxic antibodies against chemical tumors are difficult to obtain, these studies using cell-mediated immunity in vitro helped to establish that the tumors had tumor-specific transplantation antigens.

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FIG. 2. Specificity of lymphocyte-target cell cytotoxicity. Test for nonspecific lysis of syngeneic target cells added to a lytic system composed of sensitized lymphocytes and allogeneic target cells. Sensitized spleen cells obtained by transfer of 50 X 10" C57B1/6 spleen cells into 800 r irradiated DBA/2 mice, and by transfer of 50 X 10" DBA/2 spleen cells into 800 r irradiated C57B1/6 mice. Specific lysis assayed after 3 and 6 hours incubation at lymphocyte target cell ratios of 30:l. Reaction mixtures contained: ( W ) C57Bl anti-DBA/2 lymphocytes, "Cr-labeled DBA/2 mastocytomn cells, and unlabeled C57Bl (EL-4) cells; ( A )DBA/2 antiC57B1 lymphocytes, "Cr-labeled C57B1 ( EL-4) cells, and unlabeled DBA/2 mastocytoina cells; ( 0) C57B1 anti-DBA/2 lymphocytes, unlabeled DBA/2 mastocytoma cells, and "Cr-labeled C57B1 (EL-4) cells; ( A ) DBA/2 anti-C57B1 lymphocytes, unlabeled C57B1 (EL-4) cells, and "Cr-labeled DBA/2 mastocytoma cells. (From Brunncr et al., 1971.)

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From the evidence that lymphocytes sensitized to one tissue antigen do not kill other allogeneic or xenogeneic cells, it can be argued that nonspecific target cells fail to activate the lymphocytes. Hence these experiments do not clearly distinguish the specificity of cytotoxicity from that of activation. Three different systems have resolved this point using a mixed target cell experiment. Sensitized lymphocytes were added to a mixture of specific target cells and an irrelevant cell which was isotopically labeled. The question was, with lymphocytes activated by specific antigen to kill specific target cells, would the bystander labeled cells be destroyed? Using mice spleen lymphocytes presensitized to alloantigens (Cerottini, 1971; Brunner et nl., 1971), normal lymphocytes sensitized in witro in the mixed leukocyte reaction (Canty and Wunderlich, 1970; Hayry and Defendi, 1970b), or rat-mouse xenogeneic sensitization against mouse H-2 antigens ( Ginsburg, 1968; J. R. Cohen and Feldman, 1971), cytotoxicity of only the specific target cell was observed. No label was released from the bystander cells. These experiments indicate an “exquisite” specificity of cytotoxicity and argue against killing of target cells by release of a nonspecific mediator into the medium (see Section VI).

C. CYTOTOXICITY OF LYMPHOCYTES STIMULATEDBY SOLUBLEANTIGENSOR MITOGENS Do lymphocytes activated to undergo blast transformation by mitogens or antigens develop any new functions, e.g., become cytotoxic? Holm et al. (1964) and Holm and Perlmann (1965) initially observed that human lymphocytes stimulated by FHA clustered and aggregated around allogeneic target cells, HeLa, or fetal kidney cells, causing release of radioactive thymidine from prelabeled target cells and destruction of the monolayers. These experiments were confirmed using allogeneic and xenogeneic target cells and lymphocytes from several species (Perlmann and Holm, 1969a). The principle was extended (Holm and Perlmann, 1967) to include other agents that caused activation of lymphocytes, particularly antigens. Specifically, lymphocytes from a tuberculin-positive donor, when stimulated by PPD in oitro, became capable of killing Chang cells in tissue culture. That cytotoxicity was not simply the result of aggregation was indicated by experiments in which streptococcal filtrates, which induced blast transformation in the majority of human lymphocytes but did not cause aggregation, were found to induce cytotoxicity almost as well as PHA (Holm and Perlmann, 1967). In fact, a high dose of PHA induces aggregation and inhibits cytotoxicity indicating that agglutination alone is insufficient for cytotoxicity; further, there are optima1 doses of inducer required for

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activation. Brief pretreatment of the lymphocytes with PHA or staphylococcal filtrate activates irreversible cytotoxicity. However, these substances are known to bind to cells, and it cannot be concluded that once lymphocytes are triggered they no longer require the presence of inducer. The majority (89%) of cells that transform in the peripheral blood with PHA are of thymic origin, as determined by using the T6T6 chromosomal marker in mice ( Davies et al., 1968). Difficulties in determining the initial number of cells responding will be discussed (Section V ) , but whether morphologically transformed and cytotoxic lymphocytes are the same and are exclusively of thymic origin remains unsettled. The early experiments on induction of cytotoxicity by antigens were extended in studies on lymph node cells from rats hypersensitive to PPD, BGG, or albumin ( Ruddle and Waksman, 1968a,b,c). Perhaps most significantly, these studies demonstrated that antigenic differences between the target cell and the aggressor lymphocyte were unnecessary, because lymphocytes from sensitized inbred rats, when activated by specific antigens, became capable of killing syngeneic target cells. Lymphocytes from human donors sensitized to Salmonella became cytotoxic after stimulation i n uitro with Salmonella vaccine (Lundgren et al., 1968a). The kinetics of cytotoxicity by lymphocytes activated with soluble antigens or mitogens are appreciably slower than those following activation by membrane-bound antigens. In fact, in most systems, cytotoxicity is maximal approximately 2 days after stimulation with PHA but 3 days after stimulation with antigens. However, it is clear that maximal cytotoxicity occurs either before DNA synthesis begins, or only very early in DNA synthesis. In all studies, there is a distinct lack of temporal correlation with mitogenesis. Whether the relationship between cytotoxicity and blast cell transformation is invariant was probed by Perlmann et al. (1970b) who were able to activate lymphocytes to transform in approximately equal degree by two mitogens, PHA and Con A. They observed that lymphocytes transformed by Con A were not cytotoxic; whereas, as mentioned earlier, those activated by PHA were highly cytotoxic. By adding Con A to PHA in various proportions, transformation remained approximately constant, but Con A competitively inhibited the cytotoxic reaction. Also, antibodies to human y-globulin were found capable of activating lymphocytes to transformation, but blocked cytotoxic activity induced by PHA (Perlmann and Holm, 1969b). They point out clearly the hazard of drawing inferences about the nature of antigen receptors by inhibition studies using anti-immunoglobulin sera. For example, if the i n vitro cytotoxicity of antigen-stimulated cells were inhibited by anti-immuno-

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globulins, just as transformation is inhibited (Greaves et al., 1969), one would be tempted to infer that the mode of action of the anti-immunoglobulin is to block the antigen receptor. However, when the antiimmunoglobulin antibodies inhibit cytotoxicity for a variety of allogeneic and xenogenic cells produced by PHA-activated lymphocytes ( Perlmann, 1970), the flaws in concluding that the lymphocytes possess receptors for all the target cells involved or that the receptor for PHA is an immunoglobulin become obvious. This is not to say that immunoglobulins could not serve as receptors of sensitized lymphocytes. In terms of mechanism of the response, many attempts to isolate a cytotoxic factor from thc mcdium have been unsuccessful (Perlmann and Holm, 1969b). However, Ruddle and Waksman ( 1968c) presented preliminary evidence for a factor inhibiting growth or attachment of fibroblast target cells after incubation with PPD-stimulated sensitized rat lymphocytes. Related studies of Granger (1970; see Section IV,B) indicated that a toxic factor could be released into the medium under appropriate conditions following activation of normal lymphocytes by PHA or sensitized lymphocytes by antigen. Because of different culture conditions, e.g., the cell density required for optimal lymphotoxin release is much higher than that for direct cytotoxicity and the fact that target celIs do not contact activated lymphocytes where the possibility of destruction of a released cytotoxin or absorption onto membranes are lessened, assessment of the role of such a mediator in this system is difficult. Another possible mechanism is phospholipid release ( Fisher and Mueller, 1968; Ax et al., 1968) and potential membrane effects caused by these dctcrgent-like substanccs. In contradistinction to membrane-bound antigens, in this system once lymphocytes are activated, they become cytotoxic in nonspecific fashion and kill target cells which are antigenically unrelated to the inducing antigen. The fact that lymphocytes activated by antigen, such a PPD, are capable of killing syngeneic, allogencic, or xenogeneic target cells stands in marked contrast to the exquisite specificity in the membranebound antigen systems. (Also, see Section VI.) D. CORRELATION OF DIRECTLYhlPMOCYTE CYTOTOXICITY WITH CELL-MEDIATED IhlXfUXITY

Unquestionably many studies on cytotoxicity of cells bearing membrane-associated antigens show an excellent correlation between degree of cytotoxicity arid cnpacity of donor animals to rcjcct grafts bearing 1968a; the same transplantation antigens ( Brondz, 1964; Brunncr et d., Perlmann and Holm, 1969a). Yet lymphocyte donors also may have

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cytotoxic alloantibodies. For example, in studies of cytotoxicity across the H-2 histocompatibility barrier, there was a high degree of correlation between cytotoxic activity of spleen cells and formation of IgM antibodies, whereas no correlation was seen with 7 S antibodies (Brunner et al., 1968a,b). The observation (Denham et al., 1970) of two successive populations of cytotoxic cells, one arising in 6 to 12 days, the other appearing at 21 days, suggests that correlation with both types of antibody responses is possible. Two methods test whether this system correlates exclusively with cell-mediated immunity or also with antibody formation. The first determines whether nonsensitized lymphocytes can be cytotoxic in the presence of antibodies; the second utilizes a system in which antibodyforming cells can be totally excluded. Considerable literature ( Perlmann and Holm, 1969a) indicates that antibodies may, in fact, drastically influence the cytotoxic activity of lymphocytes in uitro. Target cells coated by antibodies in the absence of added complement could be lysed by the addition of normal unsensitized lymphocytes. Nucleated target cells, such as Chang cells, when coated by heteroantibodies, were lysed by normal lymphocytes from normal rats or rabbits ( MacLennan and Harding, 1970a). Antibodies from the same species directed against alloantigens could also be used (E. Moller, 1965a; Bubenik et al., 1970a). In the latter, cytotoxicity was effected by lymphocytes from donors tolerant to the alloantigens of the target cell which were precoated with antibodies. Both erythrocyte and fibroblast target cells were susceptible. Further, even with soluble antigens, if antibodies were added to antigen-coated erythrocytes, the erythrocytes were lysed by normal lymphocytes (Perlmann et al., 1968; Wasserman and Packalehi, 1965). In general, lymphocytes of spleen, lymph node, or peripheral blood are active, whereas thymus cells or leukemic cells are inactive. Notably, the quantity of antibody required for coating of target cells to sensitize them to lysis by normal lymphocytes is orders of magnitude lower than that required to lyse the cells in the presence of complement. In other words, dilutions at which the antibody would not be cytotoxic, even if complement were added, render lymphocytes cytotoxic. By chromatographic separation of immunoglobulin components on Sephadex G-150, MacLennan and Harding (1970b) found that the first peak, 19 S, was highly cytotoxic for Chang cells in the presence of guinea pig of complement; whereas, the second peak, containing 7 S antibodies were highly cytotoxic in the presence of normal spleen cells. The 7 s peak was effective at a 1000-fold lower concentration than the maximal dilution of the compleinent-dependent antibody activity. In studies in which PHA activated normal thymus cells, thymus and

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thoracic duct cells thus activated, as measured by thymidine incorporation, were totally unable to kill antibody-coated Chang cells (MacLennan and Harding, 1970a). Conversely, when spleens were depleted of thymus-derived cells by thoracic duct drainage, there was little mitogenic activity of PHA on spleen cells but a markedly enhanced cytotoxicity. It thus appeared that, under appropriate conditions, bone marrow-derived lymphocytes might be capable of cytotoxicity in the presence of antibody-coated target cells. This stands in marked contrast to experiments with mouse alloantigen where direct kill by sensitized lymphocytes required thymus-derived lymphocytes and was clearly independent of antibody-forming cells ( Cerottini et al., 1970a,b; Brunner et al., 1971). In terms of mechanism, this cytotoxicity was not inhibited by puromycin, did not require protein synthesis ( MacLennan and Harding, 1970a), but was inhibited by antimycin A (Perlmann and Holm, 1969b). The possibility that lymphocytes may supply components of complement, especially the lytic components, is suggested by experiments demonstrating that red cells, coated by antibodies to which have been added complement components 1-7, are lysed in the presence of normal lymphocytes ( Perlmann et al., 1969). These results suggest that lymphocytes may possess the components of complement required for cytolysis, namely C8 and C9. If they contain other components as well, a great deal of lymphocyte-mediated cytolysis might be complement-dependent, utilizing membrane-bound complement. Conversely, antibodies have been shown to inhibit the cytotoxic activity of sensitized lymphocytes analogously to immunological enhancement of tumors in vivo (Hellstrom and Hellstrom, 1970). In the mouse H-2 system, target cells exposed to heat-inactivated alloantibodies before exposure to lymphocytes from immunized mice, reduced or abolished cytotoxicity ( E . Moller, 1965b). In a variant of the cytotoxic systems, using the colony inhibition or growth inhibition assay, some patients have antibodies that are cytotoxic for their tumors, many have lymphocytes that are cytotoxic or growth-inhibitory for the tumors, and some have antibodies that block the cytotoxic activity of the patient’s lymphocytes for their tumors (Hellstrom et al., 1968a,b; reviewed, Hellstrom and Hellstrom, 1970).

E. CONCLUSIONS The studies on cytotoxicity of lymphocytes for target cells have provided a variety of model systems in vitm for studying allograft rejection, delayed hypersensitivity, and immunological enhancement. It is clear that in some systems, cytotoxicity is dependent upon sensitized

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thymus-derived cells which act directly on target cells in the absence of any detectable humoral antibodies or complement. When the antigens of target cells are membrane-associated, in general, cytotoxicity of lymphocytes is exquisitely specific for target cells carrying the sensitizing antigen. In other situations lymphocytes stimulated by mitogens or soluble antigens become activated, undergo blast cell transformation, and become nonspecifically cytotoxic for a variety of syngeneic, allogeneic, or xenogeneic target cells not known to carry any specificity related to that which induced the lymphocytes activation. In both types of systems, metabolic activity is required by the lymphocytes to exert cytotoxicity, but the cytotoxic state seems clearly indepcndent and dissociable from the processes of DNA synthesis and blast cell transformation. In most systems studied, contact seems essential for cytotoxicity, although the possible role of a cytotoxic factor released locally cannot be ruled out. The correlation of these systems with cell-mediated immunity is often excellent. Unfortunately, in many instances correlation also exists with development of circulating antibodies particularly the 19 S variety. In addition, in similar experiments target cells coated by specific antibodies could be lysed either by sensitized or normal lymphocytes in the absence, or presence, of exogenous complement components. In related circumstances, it appeared that bone marrow-derived, potentially antibody-producing lymphocytes, also engendered direct cytotoxicity of lymphocytes. It would be difficult to conclude at this time that there is one mechanism by which sensitized lymphocytes exert cytotoxicity on target cells. It appears that Nature, in her infinite diversity and wisdom, has provided a subtle variety of mechanisms for immune self-destruction. The relative contribution of each to thc rejection of allografts or tumors in uiuo is presently being pursued in a number of laboratories. And the possibility that some mechanisms found in uitro may have no corresponding reality in V ~ V Omust also be contemplated. Currently they provide probably the most important models for interpreting cases of tumor immunity, allograft rejection, autoimmunity, and enhancement. IV. Mediators-Qualitative

Basis of the Response

A. MIGRATION INHIBITORY FACTORAND FACTORS AFFECTING MACROPHAGES

The first in uitro model for studying delayed-type hypersensitivity, described by Rich and Lewis in 1932, was based on the observation that

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the migration of cells from spleen or lymph node explants obtained from tuberculous rabbits or guinea pigs was markedly inhibited when tuberculin was added to the tissue culturc media. There were subsequent interpretation problems, partially due to the toxicity and variability of diffcreiit batches of Old Tuberculin and partially due to the difficulty in quantitating migration i n the cxplant method. Nevertheless, the mechanism of cell-mediated immunity was studied by model systems in uitro in many laboratories (Heilnian, 1963; Carpenter, 1963). Although inodifications of the explaiit niethocl became highly reproducible ( Svejcar and Johanovsky, 1961) , the introduction of capillary tube migration by George and Vaughn (1962) provided a simple, reproducible, and quantitative in uitro system. In this test, peritoneal exudate cells migrate from capillary tubes in tissue culturc chambers in the presence or absence of specific antigen. When the cells derive from hypcrsensitive donors the migration of peritoneal cells is inhibited by the presence of specific antigcn. In addition to thc technicnl advantages, the use of peritoneal exudate cells offers two additional advantages. First, these cells are the most active sourcc for passive transfer of delayed-type hypcrsensitivity in uioo ( Bloom and Chasc, 1967), and, second, these cells includc the two major typcs invol\ v d in delnycd-type l ~ y p e r s ~ ~ ~ i s i tivity reactions in uiuo-the Iyiiiphocytc~and the macrophage. Inhibition of macrophage migration will be dealt with in some detail, not becausc it is the most important in uitro system, but because it has been the most extensively studied and illustrates some general principles governing the release of putative mcdiators by aiitigcii-stimulated lymphocytes. 1. Correlation with Cell-Mediated Immunity

a. Inhibition by Antigen of Migration of Cells from Animals Possessing Deluyecl-Type Hypersensitivity without Detectable Circulating Antibodies. Guinea pigs sensitized with living bacillus Calnictte-Gu6rin (BCG) had delayed hypersensitivity of PPD within 4 to 7 days, whereas anti-PPD antibodies were not found until 9 days or later. Peritoneal exudate cells from such animals were inhibited in their migration by PPD on the seventh day, before circulating antibodics could be detected (George and Vaughn, 1962; Bloom and Bennett, 1966; Ricci et al., 1969; Marcus, 1970). Similarly, i n guinea pigs sensitized to ovalbumin and diphtheria toxoid by injection of soluble antigen-antibody complexes in slight antibody excess, migration of peritoneal cells was inhibited by specific antigen. Such animals were previously shown to have delayed-type hypersensitivity without circulating antibodics ( Uhr et al., 1957).

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b. Failure to Inhibit Cells from Animals Possessing Only Circulating Antiboclies. In guinea pigs immunized with ovalbumin intravenously, boosted intramuscularly, and possessing considerable amounts of antibody without detectable delayed-type hypersensitivity, migration of peritoneal cells was not inhibited by the presence of antigen (J. R. David et al., 1964a). Similarly, peritoneal exudate cells obtained from guinea pigs immunized with a variety of antigens, including PPD on alumina, were not inhibited in their migration by specific antigens, although the animals had high titers of antibody (Bloom and Bennett, 1966). c. Failure of Cells from Tolerant Animals to Respond. Bore1 and David (1970) showed that guinea pigs rendered tolerant to haptenprotein conjugates with respect to delayed hypersensitivity had peritoneal populations uninhibited in migration by specific antigen. This was true even in partially tolerant animals which had circulating antibody but not delayed hypersensitivity. 2. Specificity The specificity of delayed-type hypersensitivity reactions in vivo to hapten-protein conjugates is largely conjugate- or carrier-specific ( see Section VII ). In general, immediate-type reactivity and reactions of antibody in vitro appear to be more hapten-specific. Accordingly, J. R. David et al. ( 1 9 6 4 ~ )demonstrated that the migration of peritoneal cells from guinea pigs sensitized to DNP-guinea pig albumin was inhibited by DNP-guinea pig albumin but not by DNP-BGG, although the sera had comparable titers of anti-DNP antibodies in passive cutaneous anaphylaxis reactions against both. (See Fig. 3 . ) Further, Schlossman et a2. (1966)) in studies on reactivity to DNPoligolysines, observed that the oligopeptide must contain seven or more lysyl residues in order to elicit a delayed hypersensitivity response in vivo. This was precisely the size required for peptides to be immunogenic. In this system, however, oligopeptides containing three to six lysines elicited Arthus-type reactions in immunized animals and were capable of binding antibodies to about 96% of the binding sites. J. R. David and Schlossman (1968) observed perfect correlation of in vitro cell migration with the findings in vivo. Migration inhibition was produced only by DNP-heptalysine or higher oligomeres and not by smaller peptides. The remarkable correlation of this system with the state of delayed-type hypersensitivity was supported by a study on response of guinea pigs to protein antigens of tobacco mosaic virus (Spitler et al., 1970); migration inhibition was obtained with a pentapeptide or an octanoylated tripeptide from the virus. These peptides were also active in eliciting skin reactions but were incapable of

FIG.3. Specificity of inhibition of migration of peritoneal exudate cells from guinea pigs sensitized to hapten-protein conjugates. The top row shows migration of cells from an animal exhibiting delayed hypersensitivity to dinitrophenyl (DNP)-guinea pig albumin (GPA). Cells were inhibited by the immunizing antigen, but not by the other DNP-conjugated carrier proteins, bovine y-globulin ( B G G ) or bovine serum albumin (BSA). The lower row shows migration of cells from an animal with delayed hypersensitivity t o DNP-BGG. The cells were only inhibited by the immunizing antigen and not by dinitrophenylated heterologous carrier proteins. (From J. R. David et al., 1964b.)

E+ n =]

0

5

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stimulating lymphocyte transformation and were not immunogenic. This argues against the need for an immunogenic antigen in delayed hypersensitivity reactions in vivo or in vitro but does substantiate the correlation between the migration inhibition assay and skin reactivity. Gerety et al. (1970) observed migration inhibition of peritoneal cells from guinea pigs immunized with a pneumococcal polysaccharide in complete Freund's adjuvant but not from animals immunized with polysaccharides in incomplete adjuvant. The former animals showed delayed skin reactions, the latter only Mote-Jones reactivity. Godfrey et al. (1969) made similar observations with a tuberculocarbohydrate. The earliest appearance of cutaneous hypersensitivity in vivo seems to have been 4 days. Marcus (1970) observed migration inhibition correlated with the onset of tuberculin skin conversion in vivo on the fourth day. In tuberculin reactivity, the duration of dermal hypersensitivity may last up to a year after inoculation of Freund's adjuvant, and migration inhibition has been observed even 120 days after sensitization (Salvin et al., 1970). However, it is clear that the magnitude of migration inhibition does not correlate with the diameter of skin reactions, possibly owing to the complexity and multiplicity of events required for skin reactivity. Although the migration assay correlates with hypersensitivity in vivo, it is possible that inhibition of macrophage migration may be produced by other causes than delayed hypersensitivity. For example, antigenantibody complexes (Bloom and Bennett, 1966; Spitler et al., 1969) and some cytophilic antibodies (Amos et al., 1967; Heise et al., 1968) inhibited migration of normal macrophages. Although cytophilic antibodies could cause inhibition, guinea pigs sensitized with BCG and lacking cytophilic antibodies gave migration inhibition reactions, clearly indicating that not all migration inhibition could be attributed to cytophilic antibodies. Finally, Stastny and Ziff (1970) reported a factor released from neutrophiles which could nonspecifically inhibit migration of guinea pig macrophages. 3. Production of M I F

Small numbers of peritoneal cells from sensitive guinea pigs (2.529, when admixed with normal peritoneal cells, were capable of inhibiting the migration of the mixture (J. R. David et al., 1964b). In order to ascertain which cell type was responsible for this inhibition of migration in uitro, peritoneal exudates from tuberculin hypersensitive guinea pigs were separated into their component types, macrophage populations of 99.5% or greater homogeneity and lymphocyte populations of greater than 94%purity (Bloom and Bennett, 1966). Purified macrophages ob-

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tained from sensitized animals were not inhibited by PPD. In contrast, purified peritoneal lymphocytes from the same exudate were able to inhibit the migration of normal macrophages. In these studies, as few as 1%sensitized lymphocytes were sufficient to inhibit the migration of a population of normal unsensitized macrophages. Thus, in this system the lymphocyte possesses the immunological information, the macrophage serving as an indicator cell that migrates. Macrophages did not seem to have specificity in this system; however, a cytophilic antibody might well have been lost during the purification of the macrophages. The fact that so few sensitized lymphocytes were able to inhibit the migration of normal macrophages suggested that this inhibition was not likely to be mediated by a direct lymphocyte-macrophage cytotoxicity but rather by a soluble material elaborated by the lymphocytes. To investigate this possibility, purified populations of sensitized peritoneal lymphocytes were incubated with or without PPD for various periods of time. After removal of the cells the supernatants were used as the chamber media to study the migration of normal peritoneal exudate cells. It became clear that sensitized lymphocytes, upon interaction with specific antigen, elaborated a soluble factor which inhibited the migration of normal macrophages (Bloom and Bennett, 1966). (See Fig. 4.) At the same time, David found that supernatants of sensitized lymph node lymphocytes cultured with specific hapten-protein conjugates were able to inhibit migration of normal macrophages (J. R. David, 1966). Similar results were obtained in the explant system by Svejcar et al. (1967) and by Dumonde (1967; Dumonde et al., 1968) in the capillary system. The factor responsible for mediating this reaction in vitro was termed “migration inhibitory factor” or MIF. This factor was detected as early as 6 hours after the lymphocytes were stimulated with PPD (Bennett and Bloom, 1967; Svejcar et al., 1969) and production continued for 4 days with daily changes of medium, suggesting continuous production rather than simply release of preformed material (Bloom and Bennett, 1968). Antigen-induced migration inhibition or production of MIF have been observed in guinea pigs, mice (Al-Askari et al., 1965; Feinstone et al., 1969; Taubler and Mudd, 1968; Halliday and Webb, 1969; W. A. F. Tompkins et al., 1970a), and rabbits (Svcjcar et al., 1967; Galindo and Myrvik, 1970). In man, supernatants of sensitized peripheral lymphocytes stimulated by specific antigen have been found to inhibit migration of human lymph node cells (Thor and Dray, 1968) or, after concentration, to inhibit migration of guinea pig exudate cells (Thor et al., 1968; Rocklin et al., 1970b). Human lung macrophages have also been inhibited (Bartfeld and Atoynatan, 1970). When sensitized human

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FIG. 4. Inhibition of normal peritoneal exudate cells by the migration inhibitory factor. Normal guinea pig exudate cells migrated in supernatants obtained from 20hour cultures of purified peritoneal lymphocytes obtained from tuberculin-hypersensitive guinea pigs. Chambers contained supernatants of cells cultured in ( A ) medium (15% normal guinea pig serum-Medium 199) alone; ( B ) medium containing purified protein derivative( PPD); ( C ) medium alone, PPD added after removal of the lymphocytes at 20 hours; ( D ) medium containing a heterologous antigen, coccidioidin. ( From Bloom and Bennett, 1966.)

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peripheral leukocytes were added to normal guinea pigs macrophages, in the presence of high doses of antigen (100 pg. PPD/ml.), inhibition of migration was detected ( Rajapakse and Glynn, 1970). Supernatants of stimulated human lymphocyte cultures have been shown to inhibit guinea pig macrophages, but they require concentration about tenfold (Thor et al., 1968; Rocklin et al., 1970b). Attempts to inhibit migration of human, mouse, or rabbit peritoneal macrophages with guinea pig MIF were not successful (Bloom et al., 1967). It therefore may be that MIF is species specific, but, like interferon, not absolutely so. With tenfold concentration, MIF may affect heterologous macrophages. Whether the problem in detecting human MIF is simply the low amount produced by very few sensitized cells, or a paucity of receptors on guinea pig macrophages is not clear. Cell sources used to prepare MIF have been peritoneal lymphocytes (Bloom and Bennett, 1966), lymph node lymphocytes (J. R. David, 1966), peripheral blood lymphocytes in the guinea pig (Bartfeld and Kelley, 1968) and in man (Thor et al., 1968), or spleen cells (Svejcar et al., 1967, 1969). Alveolar cells from sensitized individuals have been found to be inhibited in their migration (Galindo and Myrvik, 1970; Bartfeld et al., 1969), bdt production of MIF by them has not been reported. Curiously Salvin et al. (1970), using a migration technique on agar, found that bone marrow and thymus cells from sensitized animals, though unable to effect passive transfer of hypersensitivity in vivo, were inhibited in their migration. These findings contrast with those observed with the capillary tube technique. A variety of antigens was capable of inducing migration inhibition or MIF production: ( a ) bacterial products such as PPD (Bloom and Bennett, 1966; J. R. David, 1966; Svejcar et al., 1967; Ricci et al., 1969); ( b ) protein antigens (George and Vaughn, 1962; J, R. David et al., 1964a); ( c ) hapten-protein conjugates (J. R. David et al., 1964c; J. R. David, 1966); ( d ) synthetic polypeptides (J. R. David and Schlossman, 1968); ( e ) carbohydrates (Godfrey et al., 1969; Gerety et al., 1970; Chaparas et al., 1970); ( f ) viruses, such as influenza and mumps (Feinstone et al., 1969), vaccinia (Pilchard et al., 1970) and fibroma virus (W. A. F. Tompkins et al., 1970a); ( 6 ) allergenic antibiotics such as neomycin ( Nordqvist and Rorsman, 1967); ( 1 2 ) transplantation alloantigens in the mouse (Al-Askari et al., 1965) and in the guinea pig (Malmgren et al., 1969); ( i ) tumor antigens, in the guinea pig, using whole tumor cells as source of antigen (Kronman et al., 1969) or soluble tumor-specific antigen preparations in the guinea pig (BIoom et al., 1969) and in the mouse (Halliday and Webb, 1969).

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In addition, it has recently been found that MIF is produced by lymphocytes of inbred guinea pigs in mixed lymphocyte interaction but, interestingly, only after 4 days in culture (Bartfeld and Atoynatan, 1971).

4. Inhibition of M I F Production Studies on the passive transfer of delayed-type hypersensitivity in the guinea pig indicated that living lymphoid cells were required (Chase, 1945; Bloom and Chase, 1967) and that inhibition of RNA synthesis by mitomycin C abolished the capacity to transfer passively contact and tuberculin reactivity (Bloom et al., 1964). In the in vitro migration system, inhibition of protein synthesis by puromycin and its analogs prevented the inhibition of migration of sensitized peritoneal cells by antigen (J. W. David, 1965). Mitomycin C, under conditions that completely suppressed RNA synthesis (Bloom and Bennett, 1966) and puromycin (J. W. David, 1965), abolished the elaboration of MIF from antigen-stimulated peritoneal and lymph node lymphocytes. These results suggest but do not prove that MIF may be a protein. Quite conceivably protein synthesis is required to activate some other system, perhaps enzymes required for release, without the MIF necessarily being a protein. Together with the observation that MIF is elaborated continuously into the medium by PPD-stimulated lymphocytes over a 4-day period, these findings suggest that MIF may be produced d e novo after stimulation rather than merely being a release of preformed material, although this point is by no means proven. For example, if cells are not activated synchronously by antigen, the continuous production of MIF in the medium may only reflect the slow time course of activation of many cells by antigen, i.e., some on the first day, others on the second, etc. Treatment of sensitized exudates with trypsin was found by J. R. David et al. (1964) and by Pochyly (1967) to abolish the inhibition of migration produced by specific antigen. The effect of trypsin on sensitized cells was only temporary; when the trypsinized cells were cultured for 24 hours and then assayed, they were inhibited by antigen. Trypsin might have acted either on the sensitized lymphocytes, preventing their recognition of antigen or activation, or on the macrophages, blocking receptor sites for MIF attachment. When trypsinized, sensitized, exudate cells were added to normal peritoneal cells, inhibition of migration was not produced by PPD, suggesting that the sensitized lymphocyte population was affected by the enzyme. Bennett and Bloom (1967) observed that trypsinized lymph node lymphocytes failed to produce MIF upon stimulation with PPD. When cultured with

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lima bean trypsin inhibitor, they regained their ability to become activated by PPD to produce MIF. The interpretation made was that a specific receptor for antigen on the surface of the lymphocytes might have been removed by trypsin but since enzymatic treatment of cells also results in depressed metabolic activity, the mechanism of trypsin suppression was not established. Bartfeld and Atoynatan ( 1969), could not depress the ability of guinea pig peripheral blood lymphocytes to produce MIF by trypsin treatment, but destroyed receptors for MIF on macrophages.

5. Nature of M l F a. Specificity for Antigen. Shortly after obtaining active supernatants in the migration assay, a number of laboratories sought to determine whether this factor acted directly on macrophages in a pharmacological fashion, i.e., nonspecifically, or if the effect required the presence of specific antigen. When tuberculin-sensitive guinea pig lymph node cells were “pulsed with PPD for 1 to 2 hours, washed 5 times, and cultured overnight in antigen-free medium, the supernatnats were found to have a small degree of migration inhibitory activity. Restoration of PPD to those supernatants doubled the migration inhibitory activity ( Bennett and Bloom, 1967). Similarly, when PPD containing supernatants which were in migration inhibition were dialyzed under conditions where approximately 50%of the PPD was lost, these supernatants partially lost their inhibitory activity. Again restoration of PPD restored full migration inhibitory activity. In experiments with rabbits and guinea pigs using the explant system, lymph node cells were incubated in the presence of 1 or 0.1 pg./ml. PPD. Supernatants of these cultures made at low PPD concentrations were inactive in inhibiting migration of normal spleen cells. Restoration of PPD to these minimally stimulated supernatants returned complete migration inhibitory activity ( Svejcar et al., 1967, 1968a,b, 1969). These experiments suggested that MIF apparently did have some specificity, or more properly, antigen-dependence, and required specific antigen for biological activity. Yet all of these experiments were subject to the criticism that antigen was not completely removed. For example, in the pulsing experiments, 10% of labeled PPD added initially was found to adhere to cells, and, in the low-dose experiments, some PPD was present in all supernatants. The critical test of a specific MIF was made using an insolubilized antigen (Amos and Lachmaim, 1970). Purified protein derivative or p-lactoglobulin was conjugated to a polyaniinopolystyrene matrix. Although the insoluble agents were only 1/500 as effective as soluble antigen, they were each able to

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elicit MIF production by lymph node cells from guinea pigs immunized with BCG or P-lactoglobulin in Freund’s adjuvant, respectivcly. Supernatants from cells stimulated by insolubilized antigcns contained no free antigen and were totally inactive at inhibiting migration. When antigen was restored to aliquots of the same supernatants, complete migration inhibitory activity was found. These experiments strongly indicate that some species of MIF have specificity for antigen and require the presence of specific antigen for biological activity. J. R. David ( 1968), Remold et al. ( 1970), and J. R. David and Schlossman ( 1968) obtained strong and reproducible migration inhibition using purified Sephadex and acrylamide gel fractions which contained only infinitesimal amounts of the antigens used, either o-chlorobenzoyl ( CB )BGG or DNP-nonalysine. Further addition of antigens to these fractions was without effect. At this point we are left with an unhappy inconsistency of results. Either there are trace amounts of antigen remaining in David’s system sufficient to complex with MIF, or there are several MIFs produced to different antigens or after different immunization schedules, at least one of which is antigen-dependent and one is independent. Alternatively, it is possible that where there is apparent MIF specificity, antibodies or immunoglobulin fragments are actually being measured. However this is unlikely in view of fractionation studies in which the specific MIF activity was associated with the second fraction obtained from Sephadex G-100 columns, estimated to have a relative molecular mass ( molecular weight ) smaller than known immunoglobulins (Amos and Lachmann, 1970). A recent, very exciting observation on the possible antigen receptor may be pertinent here. W. C. Hill (1971), reacting a DNP affinity label with lymphocytes from guinea pigs sensitized to DNCB, was able to extract the reacted label from the cells by means of a nonionic detergent. The label was covalently bound to a molecule of about 25,000 molecular weight, which was presumed to be the cell receptor for antigen. By treating sensitized lymphocytes with detergent in the absence of any affinity label, W. C. Hill and Nissen (1971) were able to extract this putative receptor molecule free of antigen, which appeared to be capable of transferring dermal reactivity to the DNCB group or to PPD in normal guinea pigs. The preliminary chemical characterization of this receptor makes it at the present time indistinguishable from the low molecular weight MIF, i.e., mol. wt. of 25,000, behavior on diethylaminoethyl (DEAE) cellulose columns and electrophoresis similar to an CYglobulin or albumin, and failure to be removed or neutralized by antibodies to immunoglobulin light or heavy chains. If the sensitized

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lymphocyte, indeed, produces an antigen-specific MIF or effector molecule which is not antibody, it is unlikely that it would be a chemically different molecule from the antigen receptor. Therefore, in addition to the possible importance of a mediator with specificity, any antigenspecific molecule derived from sensitized lymphocytes, distinct from immunoglobulins, has basic interest in terms of being a possible receptor molecule. b. Chemistry. To most immunologists any soluble substance produced by lymphoid cells involved in an immunological reaction probably is an immunoglobulin. Consequently early work on MIF sought to ascertain whether or not it was an immunoglobulin or immunoglobulin fragment. In early fractionation studies, the presence of serum protein components in the tissue culture media (usually 15%guinea pig serum) was a source of difficulty because of toxicity of concentrated supers or fractions. This was especially true of albumin containing bound fatty acids, which can be quite toxic both in vitro and in vivo. Tissue culture conditions were devised for producing MIF in the absence of added serum proteins, which, in principle, should render characterization of MIF a great deal simpler (Bennett and Bloom, 1968). Fractionation on Sephadex G-100 columns indicated that the migration inhibitory activity induced by soluble hapten-protein-antigen conjugates (Remold et al., 1970) or tuberculin (Bloom and Bennett, 1969) eluted in the second peak along with a labeled serum albumin marker (molecular mass 67,000 daltons ) . However, upon sucrose gradient centrifugation (Remold et al., 1970) the molecular mass seemed closer to 3555,000 daltons. When tuberculin-induced MIF was fractionated on Sephadex G-100 (Yoshida and Reisfeld, 1970) or G-75 columns (Bloom and Jimenez, 1970), many fractions were tested to pinpoint the elution position of migration inhibitory activity. Two peaks of migration inhibitory activity were seen, approximately 50-67,000 daltons and 12-25,000 daltons. Thus, at least two molecular species appear capable of inhibiting inacrophage migration in the guinea pig, although a polymeric relationship cannot be excluded, It is unknown which, if either, of these has antigen dependence in the tuberculin system. However, in haptenprotein conjugates, the one species of MIF obtained does not require antigen for its activity (Remold et al., 1970). In any case, the elution behavior of all MIF on Sephadex and sucrose density gradients indicates that these molecules are smaller than any known class of immunoglobulin. The charge properties of MIF are similarly consistent with the view that MIF is not an immunoglobulin. When MIF containing supernatants or Sephadex G-100 second peak fractions were further fractionated on

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DEAE cellulose, fractions containing y- and p-globulins were entirely devoid of migration inhibitory activity, which eluted with the a-globulins and albumin ( Bloom and Bennett, 1970b). Unfortunately, the active fractions from DEAE cellulose contained at least five components. Acrylamide gel electrophoresis, which gives much greater resolution, was used to fractionate MIF induced by o-CB-BGG (Remold et al., 1970). The migration inhibitory activity eluted from pH 9.1 gels in the prealbumin fraction, i.e., just anodally to albumin. Interestingly, it could be separated from the macrophage chemotactic factor (see Section IV,D ) which eluted with the a-globulin fraction. Supernatants from unstimulated, control cultures gave the same number of bands as those containing MIF, indicating no significant increase in any chemically detectable protein species. Since as little as 40 ng. of highly purified MIF can inhibit migration of normal macrophages (Remold et al., 1970), this may be a substance, like interferon, which, in extremely small quantity, exhibits enormous biological activity. These experiments also illustrate the major problems in fractionation studies; namely, the paucity of starting material and the relatively low yields from fractionation methods, Two curious observations in the literature may be indirectly related to chemical studies on MIF. First, an antibody-like substance in the a2-globulin fraction of human tuberculous sera, which has the capacity to react with PPD, has been reported (Allerhand and Zitrin, 1962). It was suggested that this factor is specific for tuberculin is not an ordinary antibody. A similar antibody, with an a1 globulin electrophoretic mobility on pevikon, was found in the sera of mice immunized with SRBC in Freund's complete adjuvant by Nelson (1970). It is not beyond the realm of possibility that these might be analogs of the antigen-dependent MIF in guinea pigs. Second, a purified protein extrated from the invertebrate sea star, having a molecular mass of 32,000 daltons on Sephadex G-75 and electrophoretically homogeneous, can cause aggregation of coelomocytes of the sea star and also inhibit migration of guinea pig macrophages (Prendergast and Suzuki, 1970). This factor behaves as a y,-globulin, and is destroyed by heating at 56°C for 30 minutes. Migration inhibitory factor obtained from guinea pigs was resistant to heating at 56°C but was destroyed by heating at 80°C. Remold and David (1970) found that the nonspecific migration inhibitory activity is abolished by treatment with small quantities of neuraminidase. MIF is generally resistant to trypsin, pepsin, and chymotrypsin except when they are used in large quantities, in the case of water-insolubilized enzymes (Remold and David, 1970). MIF is not destroyed by reduction, alkylation, or treatment with 8 M urea, 6 A4 guanidine, or 0.1 M

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sodium dodecyl sulfate for up to 4 hours (Bloom, 1970). It is nevertheless most likely that MIFs are glycoprotcins. In summary, at least two MIFs in guinea pigs, chemically distinguishable by size and possibly by antigen-dependence, neither of which appears to be an immunoglobulin or fragment, seem to exist. They appear to be glycoprotcins and are biologically active in extraordinarily small quantities, but are difficult to characterize chemically. Antibodies against mammalian MIF have not yet been demonstrated. 6. Efects on Macrophages

Important studies on the activation of macrophages and phagocytosis (reviewcd by Cohn, 1968; Dannenbcrg, 1968) and the relation of activation to resistance to infection ( Mackaness and Blanden, 1967) led to the expectation that MIF would cause activation of macrophages, i.c., increased attachment to glass, hypcrnmobility of thc pseudopodial membranes, and increase in lysosomes and lysosomal hydrolases. In the earliest study of the morphological cffect of antigen on peritoneal niacrophages, the development of large mononuclear cells containing large cytoplasmic granules ( Waksman and Matoltsy, 1958), beginning at 24 hours and extending over several days, corresponds to what is now known to be blast cell transformation. One suspects that some of the large cells containing vacuoles may, in fact, have been blast cells. In support of a possible function of a lymphocyte-produced factor in activating macrophages is the observation that normal rabbit peritoneal macrophages, in the presence of supernatants of antigen-stimd a t e d lymphocyte cultures, show enhanced ability to phagocytize sheep erythrocytes ( Barnet et al., 1968). A preliminary report indicates that the acid phosphatase activity in lysosomes of guinea pig peritoneal macrophages may increase after stimulation in vitro with PPD (Adams et al., 1970). Supcrnatants of tuberculin-stimulated rabbit lymphocyte cultures were found to induce activation of monolayers of rabbit macrophages (Mooney and Waksman, 1970). In general, attempts to demonstrate activation of macrophages have been disappointing. For example, addition of antigen to sensitized mouse peritoneal cxudatcs, rather than leading to increased attachment and spreading expected from activated cells, resulted in marked diminution in spreading of the cells on a hemocytometer chamber (Fauve and Dekaris, 1968). Within 30 minutcs after exudatcs from guinea pigs scnsitized with BCG or from mice infcctcd with Listeria monocytogenes were exposcd in vitro to the respective antigens, there was marked inhibition of the number of macrophages which spread on the

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glass, Guinea pigs immunized against ovalbumin and producing circulating antibodies showed no such inhibition of spreading. It is clear that MIF does react with macrophages. Migration inhibitory activity could be removed from active supernatants by absorption with macrophages ( Dumonde, 1967; Svejcar et al., 1969). Macrophages taken from sensitized guinea pigs 1 hour after challenge with tuberculin intraperitoneally had markedly decreased negative surface charges and electrophoretic mobility ( Diengdoh and Turk, 1968) . Trypsin treatment of guinea pig alveolar macrophages removed a receptor for MIF (Bartfeld and Atoynatan, 1969). Several tuberculin preparations, which were not toxic for normal macrophages but which inhibited migration of macrophages from splenic explant of BCG-sensitized mice, produced morphological changes suggesting a cytotoxic activity on the macrophages (Shea and Morgan, 1957). The effect on macrophages of antigen and MIF was studied cinemicrographically by modified migration on agar with antigen or MIF incorporated into the agar (Salvin et al., 1970). Clearly macrophages were affected by MIF-they were rounded, their mobility as single cells was drastically slowed, and the ruffling of the pseudopodial membrane was markedly depressed. Later the cells developed granules, but it is not clear whether the granule formation represented increased lysosomal activity or merely ingestion of agar. Electron-microscopic studies of such macrophages indicated intertwined microvilli, but no cytoplasmic alteration was seen other than an increase in microtubules (Smyth and Weiss, 1970). The relation of MIF’s effect on macrophages in vitro to observed activation in vivo remains problematic, but clearly macrophages are not killed. An encouraging report indicates that supernatants of tuberculin-stimulated mouse lymphocytes had the capacity to confer resistance to infection to virulent tubercle bacilli onto normal macrophages ( Patterson and Youmans, 1970). Sensitized guinea pig exudate cells agglutinate in the presence of antigen over a 6-hour period, and the factor released into the medium which is responsible for this has been termed a “macrophage aggregation factor” (MAF) (Lolekha et al., 1970). By quite a different technique, macrophage aggregation was observed when normal human monocytes were cultured in supernatants of PPD-stimulated human peripheral blood leukocytes ( Nordqvist and Rorsman, 1970), Normal peripheral cells were allowed to migrate from capillary tubes for several days. After most of the neutrophiles had lysed, the chambers were inverted and the nonadherent lymphocytes removed, leaving a monolayer of monocytes. The chambers were then refilled with the supernatants of cultures of sensitized lymphocytes. Antigenic stimulation of human blood leukocytes from tuberculin-sensitive donors was found to induce intercel-

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lular cytoplasmic bridging of monocytes or macrophages, although to a smaller degree than PHA ( Maclaurin, 1969). It must be emphasized that factors other than MIF can affect macrophages. A toxic factor was obtained from neutrophiles which inhibited migration of guinea pig macrophages (Stastny and Ziff, 1970). This factor, however, was dialyzable and has a molecular mass of about 4000 daltons. Effects of antibodies, particularly cytophilic antibodies, will be discussed in Section IV,F.

B. LYMPHOTOXIN AND GROWTH INHIBITORY FACTORS In trying to understand the mechanism by which allografts or tumors are rejected by immune individuals, the possibility of release of soluble factors from lymphocytes which could amplify the effects of a small number of sensitized lymphocytes is attractive. Ruddle and Waksman ( 1967) reported that cell-free supernatants, obtained from cultures in which direct lymphocyte-target cell cytotoxicity had been induced by PPD stimulation of tuberculin-sensitive lymphocytes had a slight but discernible cytotoxic or growth inhibitory activity on monolayers of rat fibroblasts. On the premise that nonspecific mitogens such as PHA could engage many more lymphocytes than could specific antigens, Granger and Williams ( 1968) stimulated mouse spleen lymphocytes with PHA, recovered the cell-free culture supernatants and tested them for cytotoxic activity on mouse L cells. Their assay for cytotoxicity was the supernatant’s ability to diminish the incorporation of labeled amino acids into protein by the target cells. Amino acid incorporation by L cells was inhibited about 95%by the PHA-stimulated supernatants. Supernatants of mixed lymphocyte cultures exhibited a similar effect. They termed the factor responsible for this effect “lymphotoxin” ( LT) . A possible role for LT as a mediator of delayed hypersensitivity reactions was established when a similar cytotoxic activity was found in supernatants of tuberculin-sensitive guinea pig or human lymphocytes stimulated in vitro with PPD (Granger et al., 1969). 1 . Production of LT

The subject of cytotoxins produced by lymphocytes was reviewed by Granger ( 1970) and Pincus ( 1970). Lymphotoxin-like activity has been found in culture supernatants of PHA-stimulated lymphocytes obtained from mouse, rat, cat, rabbit, human (T. W. Williams and Granger, 1968), and guinea pig (Pincus et al., 1970) but was not produced by thymocytes or lymphocytes from patients with chronic lymphocytic leukemia or Hodgkin’s disease (Granger, 1970). Other agents found to induce production of L T were antilymphocytic

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sera, pokeweed mitogen, streptolysin A, PPD, and histoplasmin. Lyniphotoxin activity may be detected in the culture medium as early as 6 hours after stimulation of lymphocytes by PHA. In the guinea pig, production of LT by PPD-stimulated lymphocytes is approximately related in first-order fashion to the number of cells employed ( Pincus, 1970). Addition of nonsensitive lymphocytes does not lead to augmented levels of LT, suggesting that recruitment of normal cells into LT production is not significant in this case. Lymphoid cells from mice infected with or immunized against lymphocytic choriomeningitis virus, when incubated in vitro with living or killed virus, produce a lymphotoxin-like factor, which destroyed or inhibited growth of mouse or monkey fibroblasts (Oldstone and Dixon, 1970). Lyniphotoxin-like factor was produced by a large percentage of long-term lymphocyte lines tested (Granger et al., 1970). These lines were not stimulated by antigens or other mitogens, but behaved as activated lymphoid cells in that they divide continuously in culture. The use of these lines may be very helpful in producing many of the mediators in large quantity. In this regard it may be mentioned that Baumal et al. (1971) used antigen together with Epstein-Barr virus to produce long-term lyinphoblastoid lines from blood of tuberculin- or SKSD-positive human donors. Whether the lines retain specificity for antigen has not yet been established. It is not clear why LT is not found in the supernatants of cultures in which direct lymphocyte-target cell cytotoxicity is occurring (see Perlmann and Holm, 1969a). One possible reason is that the cell density of lymphocytes in that system is usually lower than that used for production of LT, which is commonly 10-20 x lo6lymphocytes per milliliter. Production of LT is deterred by a variety of metabolic inhibitors including DNP, puromycin, cycloheximide, and hydrocortisone (T. W. Williams and Granger, 1969). However, because of the speed of L T production after stimulation (4-8 hours) and the observation that Xirradiation does not depress its release, it may be concluded that production of LT is not related to DNA synthesis. 2. Chemistry of LT The nonspecific activity of the cytotoxic supernatants on a variety of target cells, discussed below, and the fact that its production is induced by nonspecific mitogens, suggests that LT is not an immunoglobulin. When LTs prepared by PHA activation of murine spleen cells and human adenoidal cells were compared chemically, they had distinct differences, yet they shared the common properties of many

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proteins, i.e., destruction by phenol extraction, stability to cthcr extraction, resistance to DNase and RNase, and buoyant density of 1.33 ( Grangcr, 1970). After gel filtration on Sephadcx G-100 columns, mouse LT eluted between the exclusion volume aiid alkaliiic pliosphatase, suggesting a niolccular mass of 90 to 150,000 daltons. Human LT seemed to have a slightly smaller molecular mass, estimated to bc approximately 80,000 daltoiis (Kolb and Granger, 1970). Guinea pig LT elutes from Sephadex approximately with albuiiiiiis ( Pincus, 1967; Heise and Weiser, 1969). Whereas human LT precipitated at 0 to 40% amnioniuiii sulfate saturation and was inactivatccl by heating at SO"C, niuriiie LT precipitated between 60 and SO%aninioniiini sulfate and was stable to 100°C for 15 minutes. Even by exposing large numbers of lymphocytes to PHA, the quantities of LT obtained, as with MIF, were exceedingly small aiid difficult to work with chemically. By acrylaiiiide gel electrophoresis, tlie best preparations were not homogeneous and activity was difficult to recover. Nevertheless, the niousc LT was reported to havc a relatively high charge, migrating aiiodally approximately with the albumins; in contrast, human LT migrated with the ,8- and ./-globulins (Granger, 1970). The LT produced by long-term human lyrnphoblastoid lines resembled, in precipitability at 40% ammonium sulfate aiid elution from DEAE-Sephadex coluiiiiis betwccn ,8- aiid 7-globulin, the L T produced by PHA-stimulated human lympliocytcs. Comparison of tlie chemical properties of mouse L T and guinea pig hlIF indicated that the chemical evidence was iiisufficieiit to draw significant distiiictions between them (Heise and Weiser, 1969). 3. Efects on Target Cells

In analyzing the cytotoxic effect of lymphocyte products on targct cells, the assay for cytotoxicity is critical. The assay used by Granger and his colleagues is the supernatant's ability to block incorporation of amino acids into protein which corr~~latcs \[.ith vital staining and morphological criteria (Granger and Kolb, 196s). However, there are circumstances in which protein synthesis is inhibited, but cell lysis does not occur. For detecting lysis of cells, other tests such as release of thymidine from prelabcled target cells (G. Klein and Perlmann, 1963) or chromium rcleasc ( Sanderson, 1964) have been used. In morphological studies on the effects of LT, it was found that rounding up, vacuolization, and detachment from glass occur in high concentrations of LT and arc markedly diminished with dilution of the active factor. In contrast to direct lymphocyte cytotoxicity, the effects of LT arc delayed, requiring 24-48 hours for maximal effect. Although

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LT has been reported to act on a great variety of target cells including fibroblasts, HeLa cells, L cells, and erythrocytes of several species, the niouse L cell is the most sensitive indicator of L T activity (T. W. Williams and Granger, 1969). (See Fig. 5 . ) Less than 1 pg. of purified LT can be detected by L cells (Granger, 1971). Although one batch of LT at a dilution of 1:20 was cytotoxic for 98% of L cells, it had no discernible effect on Chang cells, NBK cells, or KB cells; at higher concentration it had some cytotoxic effect on all these cells (Granger, 1971). One L T produced by PPD stimulation of sensitized guinea pig lymphocytes, caused a high degree of thymidine release from prelabeled L cells, but no release from guinea pig lung fibroblasts, 3T3 mouse tissue culture cells or KL human fibroblasts in culture (Bloom, 1971b). These experiments raise the question whether the amount of LT produced by sensitized lymphocytes after antigenic challenge is sufficient to permit a cytotoxic effect on target cells in vivo or whether its primary effect might be growth inhibition. In preliminary experiments,

Dilution of H L T -

FIG.5. Cytotoxic effect of lymphotoxin (HLT) on different target cells. The same batch of human lymphotoxin obtained from cultures of PHA-stimulated human adenoid cells were tested on HeLa (human), Chang (human), KB (human); MBK (bovine) WISH (human), and L Cells (mouse). Cytotoxicity was derived from the inhibition of amino acid incorporation by the labeled target cells. (From C . A. Granger, 1971.)

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Chang liver cells, prelabeled with W r and tested for ability to incorporate amino acids into protein showed marked reduction in amino acid incorporation, as found by Granger, but surprisingly, no concomitant release of “Cr, suggesting that lysis had not occurred (Perlmaim and Holm, 1970). Similarly, in comparative studies on the biological activities of partially purified fractions from antigen-stimulated culture supernatants, Dumonde et al. (1969) found that the same active Sephadex fraction caused a ten-fold increase in thymidine incorporation by normal lymphocytes, a 58%inhibition of macrophage migration, but only a 10%release of “Cr from target L cells. If one were to accept cytolytic activity as the main action of LT, another question arises; namely, how is it possible for a cytotoxic factor to be produced without destroying the lymphocytes that produce it? Granger (1970) pointed out that human lymphocytes are among the most resistant cells to the effects of LT, yet mouse lymphocytes are quite easily destroyed by it. In terms of potential usefulness of LT in an in vitro assay for delayed-type hypersensitivity, Granger et al. ( 1969) reported that significant LT activity could be found in supernatants of PPD-stimulated lymphocytes. However, using a less sensitive but more rigid criterion for cell lysis, i.e., release of thymidine from prelabeled L cells, supernatants of antigen-stimulated human lymphocytes, known to contain MIF, were cytotoxic for L cells in only 5 of 22 experiments (Rocklin et al., 1970). This again suggests that the quantity of LT produced by sensitized cells upon antigen stimulation is much lower than that produced by mitogen-stimulated cultures. More generally, do supernatants of antigen-stimulated cells, not invariably cytolytic, have a general inhibitory activity on cell growth? Lebowitz and Lawrence (1969) found that supernatants of PPD-stimulated tuberculin-sensitive human lymphocytes inhibited the cloning of individual HeLa cells in tissue culture over a 10-day period. From morphological observation of the flasks, although many of the single HeLa cells originally seeded undoubtedly died, it was possible to see small colonies of cells which had undergone one or two divisions but were restricted in their ability to divide continuously to form a large clone. They termed this activity “cloning inhibitory factor.” Similar findings were made with supernatants from PHA-stimulated cells which were tested on a variety of target cells (Green et al., 1970). They observed inhibition of thymidine incorporation into DNA, of thc number of mitotic figures, and of cloning. For these experiments the supernatants were diluted 1:10 or more, and little evidence of cellular destruction was found. The monolayers maintained their integrity and excluded trypan blue. The material responsible for

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this activity, which was nondialyzable, trypsin-sensitive, and inactivated by heating at lOO”C, has been termed “proliferation inhibitory factor.” In fact, in the original observation of Ruddle and Waksman (1967)) supernatants of PPD stimulated guinea pig lymphocyte cultures were reported cytotoxic for fibroblasts, but the method could not discriminate between cytolysis and growth inhibition. After incubation in the supernatants, the fibroblast monolayers were trypsinized, and the number of cells released from the glass were counted. If the control cultures were doubling, while those in the active supernatants merely failed to proliferate, a significantly smaller number of test cells would be observed. Another growth inhibitory factor, distinguishable chemically from the above, is produced by mouse cells stimulated by PHA, by antigen or allogeneic lymphocytes, or by human lymphoblastoid cell lines (R. T. Smith et al., 1970). This factor suppresses incorporation of thymidine into DNA by lymphocytes and has been designated “inhibitor of DNA synthesis” (IDS) (R. T. Smith et al., 1970). It was dialyzable, with molecular mass less than 10,000 daltons, and was inactivated by heating at 56°C. That it did not simply result from exhaustion of some nutrient in the medium was indicated by the fact that it exerted its inhibitory effect even in dilution of 1:500 with fresh medium. This factor apparently did not kill lymphocytes, because they could be seen to transform normally but, rather, seemed to have a specific inhibitory effect on DNA synthesis. A similar factor was found in leukocyte and lymphocyte extracts (Moorehead et al., 1969) and perhaps was released from dead cells in culture. It should also be noted that interferons may also have growth inhibitory activity (Gresser et al., 1970). It is important to note that the activity of LT is increased when the target cells are treated with the metabolic inhibitor, DNP (Granger, 1971), clearly indicating that the target cells need no metabolic activity in order to be killed. This rules out “burning out” or “suicide” mechanisms for target cell destruction. Detailed studies on the correlation of production of cytotoxic or growth inhibitory factors and cell-mediated immunity have not been made. It is clear that these activities can be released by sensitized lymphocytes stimulated with specific antigens. However, similar factors may be produced by long-term lymphocyte cultures and may be nonspecific products of activated lymphocytes. These activities point to the importance of ascertaining whether stimulation of any lymphocytes by any means results in identical processes of activation and elaboration of common products. In more practical terms, obviously the choice of assays is critical for study of cytotoxic factors.

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In addition, cytotoxic factors can be produced by cells other than lymphocytes, for example, by polymorphonuclear leukocytes ( G. Moller and Lundgren, 1969; Lundgren et al., 1968b; Falk et al., 1970a,b). A factor produced by human buffy coat and granulocytes, which inhibited migration of macrophages, has been partially characterized; it is dialyzable and has a molecular mass of about 4000 daltons (Stastny and Ziff, 1970). Dead or dying lymphoid cells as well as neutrophiles have been reported cytotoxic for human fibroblasts in a monolayer (Lundgren et al., 1968b). Lastly, macrophages from tuberculin-sensitive guinea pigs upon stimulation with PPD or, on occasion, normal macrophages, were found to release cytotoxins for L cells (Pincus, 1967; Heise and Weiser, 1969). It is perhaps worth considering whether cytotoxins are released only by activated lymphoid cells or whether they may, in fact, be a more general product of actively metabolizing cells. Growth inhibitory activity has been reported in supernatants of nondividing tissue culture cells ( Garcia-Giralt et al., 1970). Additionally, a factor with LT-like activity on L cells has recently been found in supernatants of HeLa cells and other tissue culture cells (Glade and Granger, 1971).

C. BLASTOCENIC FACTOR AND FACTORS AFFECTING LYMPHOCYTES 1. Mitogenesis The first report that lymphocytes produced a factor which could stimulate other normal lymphocytes to transform was based on studies in mixed leukocyte cultures (Kasakura and Lowenstein, 1965; Gordon and MacLean, 1965). Human lymphocytes, stimulated by allogeneic peripheral blood cells, released into the medium a substance capable of causing morphological transformation and incorporation of thymidine into DNA or uridine into RNA of other normal human lymphocytes (Kasakura and Lowenstein, 1967). Although there was some mitogenic activity released into the medium in cultures of single donor cells, considerably more material was released from the mixed leukocyte cultures. Deoxyribonucleic acid synthesis was maximal at about 120 hours and RNA synthesis at about 168 hours after culture in the blastogenic supernatant. Stimulation by transplantation antigens which may have been released from dead cells into the medium might have occurred, since after ultracentrifugation, mitogenic activity was found in the precipitate containing cell membranes and debris. Preliminary observations on the effects of MIF-containing supernatants obtained from antigen-stimulated guinea pig lymph node cell cultures suggested that there was mitogenic activity (Bennett and

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Bloom, 1968; Dumonde et al., 1967), which was later clearly established in guinea pigs by Wolstencroft and Dumonde (1970). In studies on peripheral lymphocytes from tuberculin-sensitive human donors, Valentine and Lawrence (1969) observed that sensitized human peripheral blood cells stimulated for 36 hours with low doses of PPD (0.6 pg./ml.) elaborated a blastogenic factor into the medium. When they pulsed cells for 1 hour with PPD, similar to the experiments on MIF for detection of the antigen-dependency of the factor, they found that the culture supernatants showed only minimal blastogenic activity on normal lymphocytes, However, the addition of increasing amounts of PPD to these supernatants resulted in further stimulation, suggesting that this blastogenic factor was antigen-dependent or had specificity for PPD. The active factor, termed “lyniphocyte-transforming factor,” was nondialyzable and nonsedimentable by ultracentrifugation. Independently, using relatively similar conditions, Maini et al. (1969) reported similar observations in man in the tuberculin system and termed the activity “mitogenic factor.” In both experiments, the leukocytes were cultured in the presence of autologous serum, which probably contained antiPPD antibodies, and it is known that transformation can be induced by antigen-antibody complexes ( Bloch-Shtacher et al., 1968). However, both experiments were controlled with unstimulated supernatants and, after removal of the cells, PPD was restored to the same concentration as used for stimulation. Neither control supernatant had activity, indicating that the mitogenic factor was not simply antigen-antibody complexes. A “blastogenic factor” which did not require added PPD for action was obtained from inbred guinea pig cells (Spitler and Fudenberg, 1970), although this contrasts with a previous report (Spitler and Lawrence, 1969) of some antigen dependency. The mitogenic effect was somewhat irregular, but the system of inbred animals demonstrated a blastogenic factor that was not simply a transplantation antigen. Study of the response of mouse cells to antigens has generally been hampered by poor survival of mouse lymphocytes in culture. By supplementing the medium with arginine and culturing in the presence of low levels of human serum, which apparently contained a natural antibody to mouse cells, Adler et al. (1970a,b) were able to study the response of sensitized mouse spleen cells to antigens and mitogens. In attempting to find a blastogenic factor in supernatants of human lymphoblastoid lines, they had difficulty in achieving normal lymphocyte survival in culture supernatants. The difficulty was traced to the presence of IDS which suppressed the activity or detection of the blastogenic factor (R. T. Smith et al., 1970). Interestingly, IDS appeared selectively

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to affect DNA synthesis and not morphological transformation. When the culture supernatants were heated to 56”C, IDS was destroyed or inactivated, and the supcrnatants exerted a strong mitogenic effect on normal mouse lymphocytes. The acronym given to this mitogenic activity, somewhat facetiously, was EGO ( enhanced gene operator) (Smith et al., 1970). A factor similar to IDS was found in extracts of lymphocytes by Moorhead et al. (1969). These studies illustrate two experimental problems. One is the establishment of a single activity using supernatants containing multiple factors, some of which may be cytotoxic or growth inhibitory; the other is that study of thymidine incorporation without concomitant observation of the number of surviving cells may give misleading results, especially if many cells die leaving only a few surviving to incorporate thymidine. A blastogenic factor arising from mixed leukocyte culture had the capacity to activate norinal lymphocytes to exert cytotoxicity on target cells (Falk et al., 1970a). When the cultures contained few neutrophiles, the supernatants wcre not toxic for target cells. A similar activity, i.e., inducing normal lymphocytes to become cytotoxic, was reported by Chernyakhovskaya et al. ( 1970) for interferon-containing cuIture supernatants. As mentioned previously, a factor capable of permitting purified lymphocytes to respond to antigens in the absence of niacrophages, was reported (Alter and Bach, 1970; F. H. Bach et al., 1970). This factor the “conditioned medium reconstituting factor” ( CMRF ) was produced by normal glass-adherent cells from human peripheral blood in the absence of antigenic stimulation. Presumably, it permitted lymphocytes to respond by providing some growth stimulant or nutrient required for lymphocyte survival, although analysis of viability was not made. Probably the most complicated of all the in vitro activities is a factor that does not stimulate normal or sensitized lymphocytes by itself, but, when added to sensitized lymphocytes stimulated by specific antigens, causes a marked increase in response above the level achieved by antigen alone. This activity, “potentiating factor” or PF (Janis and Bach, 1970), was released into mediuin by PPD-stimulated tuberculinsensitive lymphocytes. When added to lymphocytes of a second donor sensitive to PPD, in the absence of antigen, stimulation was minimal. Howevcr, when the second donor’s cells were stimulated with PPD in the prescnce of PF, thymidine incorporation was stimulated threefold over antigen alone. Apparently antigenic stimulation was not required for release of PF. These results suggest that both CMRF and PF acted nonspccifically by providing lymphocytes with some important nutricnt required for survival or activation. Mitoniycin treatment of

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leukocytes in mixed leukocyte cultures did pot affect their ability to serve as sources of antigen (F. H. Bach and Voynow, 1966) but blocked their ability to release PF. The question remains whether the blastogenic activity observed in these experiments is produced only from lymphocytes involved in cellmediated immunity. Recently, blastogenic activity was reported in supernatants of stimulated lymphocytes from patients with immediate reaginic hypersensitivity to pollens (Brostoff et al., 1969). Since lymphocytes of pollen-sensitive patients have been shown to transform (see Section I ) , it is not clear whether the blastogenic activity is released only from lymphocytes stimulated by antigens or whether it is produced by minute amounts of reaginic antibody known to be cytotropic in combination with antigen. It should be recalled that antigen-antibody complexes are capable of transforming normal lymphocytes ( Bloch-Shtacher et al., 1968). In summary, a factor is released into the medium of antigen-stimulated cultures capable of “recruiting” normal lymphocytes to transform and to incorporate thymidine. Sufficient chemical evidence is not available to discriminate this activity from the other factors, although in two instances evidence indicates that it may require specific antigen for activity. (Also see Section VII.) 2. Effect of Antigen on Migration of Lymphocytes Recent interest has focused on two systems for detecting human cellmediated immunity in vitro (Bloom and Glade, 1971). In the first buffy coat cells from Brucella-positive patients were allowed to migrate from capillary tubes in the presence or absence of killed organisms, Striking inhibition of sensitized buffy coat cells was found (S@borg,1967). This system was then applied to detect hypersensitivity to other antigens such as colon antigens in ulcerative colitis and tumor antigens (Bendixen and Sgborg, 1970). When applied to the tuberculin system, this method was successful in one laboratory (Mookerjee et al., 1969)) equivocal in the original laboratory (Bendixen and Sgborg, 1969), and not reproducible in two others (Kaltreider et al., 1969; Lockshin, 1969). (See Bendixen and Sgborg, 1970, for discussion of technical differences. ) Because of the availability of human cells for this test, it deserves critical evaluation, especially regarding the possible role of antibody. It has been impossible to coat normal lymphocytes with serum from immunized donors, wash them and demonstrate migration inhibition. On the other hand, migration of buffy coat cells of hypersensitive guinea pigs immunized to thyroglobulin can be inhibited by the protein under conditions where it is unlikely that the inhibition is related to delayed-type

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hypersensitivity (\Vnsserman and Pakal6n, 1965). Results of this test are somewhat ambiguous as it inay detcct either antibody or cellmediated immunity, since neutrophiles arc required for migration inhibition ( S@borg, 1969; Clausen, 1970). It is \vell known that certain classes of immunoglobulin, especially IgG, bind firmly to neutrophiles (Ishizaka et uZ., 1970). Further cxperimentatioii mill be required to clarify this point. Another system, designed to obviate the confusion arising from the presence of neutrophiles, employs periphcral blood lymphocytes, purified by gelatin sedinicntation and removal of phagocytic cells with carbonyl iron followed by ainnioniz~~n chloride lysis of erythrocytes. Most strikingly, migration of lymphocytes from tuberculin or Streptococcussensitive individuals is completely unaffected by soluble specific antigens, e.g., PPD or SKSD, although it is inhibited by the antigens in particulate form, e.g., tubercle bacilli or streptococcal membranes ( Falk et nl., 1969; Zabriskie and Falk, 1970). Previously migration of guinea pig lymph node cells was not inhibited by soluble antigens (Bloom and David, 1964). The inhibition was found to be mediated by a solublc factor ( Falk et nl., 1969), and ingenuity in using a rat histocompatibility antigen system periiiittecl exclusion of the possibility that the factor was antibody. I n this case, Fisher cclls were stimulated by Wistar antigenic cells, and the supcrnatant was tcstecl for inhibition of lymphocyte migration on normal Fisher cells. The principal objections to the procedure, using direct inhibition of sensitized lymphocytes by specific antigens, are the probability that significant numbers of neutrophiIes remain after purification with carbonyl iron and lack of proof that inhibition of leukocyte niigration is related exclusively to cell-mediated immunity. Lymphocytes from animals primed to produce circulating antibodies can bind antigens ( Sulitzcanu, 1968; see Section V ) , Yet critical tests of whether this binding mensiires cell-mediated immunity exclusively remain to be done. D. CHEMOTACTIC FACTOR The vast majority of cells at classic clelayed-type hypersensitivity reaction sites are nonsensitized host cclls, probably macrophages ( discussed in Section VI ) . In attempting to discovcr how they accumulate, an in vitro system used to study chemotactic effects on leukocytes was employed; namely, the ability of cells to pass through a micropore filter in the direction of a chemotactic stimulus. Supernatants of antigenstimulated guinea pig lymph node cells, known to contain M I F were tested. When normal guinea pig or rabbit macrophages were placed

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in ordinary medium on one side of the micropore filter, and the antigenstimulatcd supernatant on the other, a marked increase in the number of macrophages passing through the filtcr toward the active supernatants was observed (Ward et al., 1969). Supernatants of unstindated cultures did not induce chemotaxis, and when active supernatant was placed on both sides of the filter, there was similarly no chemotaxis. These results indicate a soluble factor produced by antigen-stimulated, delayed hypersensitive lymphocytes which causes macrophages to exhibit positive chemotaxis. This factor elutes from Sephadex G-100 columns, in fractions containing albumin or molecules slightly smaller, with an estimated molecular mass of about 50,000 daltons. Upon fractionation by acrylamide gel electrophoresis, under conditions where MIF migrates as a prealbuniin, the inacrophage chemotactic activity is associated with the a-globulin and albumin fractions (Ward et al., 1970). These results provide strong evidence that the chemotactic factor and MIF are chemically distinct. The active fraction seems to have specific chemotactic activity for inacrophages and not for neutrophiles. Interestingly, as mentioned earlier, supernatants of antigen-stimulated guinea pig lymphocytes do not regularly inhibit the migration of rabbit macrophages but do inhibit migration of guinea pig macrophages, whereas the chemotactic factor acts equally well on both.

E. INTERFERON The term interferon was first applied to a soluble substance produced by chick chorioallantoic membrane exposed to heat-inactivated influenza virus which had the capacity of diminishing virus replication in fresh pieces of membrane ( Isaacs and Lindenmann, 1957). The first clinical trial of interferon, and a highly successful one at that, was reported in the cartoon series, Flash Gordon (Gordon, 1961). As with so many clinical trials, however, the enthusiasm generated by the first report has been tempered as time and more critical evaluation have intervened. In any case, it appears that interferon is not a single substance; rather, there are several substances of different sizes able to protect a variety of cells from infection by many viruses. A brief review of interferon’s qualities will uncover parallels to present studies on the mediators of hypersensitivity. Interferon must rank among one of the most active biological molecules since it may protect against virus infection in dilution of 1: 1,000,000. Amounts of interferon produced are so infinitesimal that even with massive culture techniques and 13 years of skilled and intensive chemical study, it has not been isolated and characterized in pure form (Fantes, 1970). Finally, at least one species of interferon is known to be produced by lymphoid

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cells, including lymphocytes from delayed hypersensitive individuals stimulated by antigen, and must be considered as a possible mediator. In early studies, interferon was found to be produced from virtually all tissues or cells in the body or in zjitro, following infection by certain live or killed viruses. Many of the viruses that induce interferon also induce delayed-type hypersensitivity in vivo, such as mumps, influenza, vaccinia, and measles ( Raff el, 1961) . Interestingly, cells induced by viruses to produce interferon are themselves not protected against viral infection. In addition to viruses, there are a wide variety of nonviral inducers of interferon ( H o et al., 1967; Borecky and Lackovic, 1968; Merigan et al., 1970; Merigan, 1971). The earliest nonviral inducer to be recognized was endotoxin; as little as 8 ng. of Escherichia coli lipopolysaccharidcs injected intracerebrally into mice was able to block the neurotoxic effects of WS influenza virus (Hook and Wagner, 1959). Although these results in vivo have been confirmed and extended, no direct protection by endotoxin could be shown on cells in tissue culture, e.g., chick cells and kidney cells (Wagncr et al., 1959; Youngner and Stincbring, 1964; Ho, 1964). These results suggest that endotoxin may induce interferon by affecting lymphoid cells, perhaps as a consequence of a cell-mediated reaction. It should be noted that interferon can be produced by macrophages (T. J. Smith and Wagner, 1967), but macrophages are not stimulated by PHA to produce interferon (Merigan, 1971). Mitogens, such as PHA were shown (Wheelock, 1965; Friedman and Cooper, 1967) to induce interferon production by human leukocytes, detectable as early as 2 hours after stimulation and maximal at 24 hours. Other nonviral inducers of interferon (as reviewed by Borecky and Lackovic, 1968; Merigan, 1971) are ( 1) bacteria, including Listeria monocytogenes as well as Rickettsia and Mycoplasma; ( 2 ) microbial products, such as streptolysin 0; ( 3 ) mitogens, such as PHA and pokeweed mitogens; ( 4 ) multistranded polynucleotides; ( 5 ) plastics, such as pyran copolymer and polyvinyl sulfate; ( 6 ) polysaccharides, such as statolon and mannans; and ( 7 ) certain antibiotics such as kanamycin. Production of interferon by long-term cultures of human lymphoblastoid cell lines has been reported (Kasel et al., 1968; Haase et d., 1970). These cells may represent an ideal source for large-scale production of interferon, since the only limitations on the amount obtained would be the size of the vessel for culturing cells and the size of the budget for providing medium. In relation to the immune response, enhanced production of interferon took place in vitro to a specific viral inducer when the cells were

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taken from animals previously infected and, thus, immune to that virus ( Glasgow, 1966). However, the relationship between interferon production and cellmediated immunity was established by Green et al. (1969) who showed that culture supernatants of human peripheral lymphocytes from donors sensitized to tuberculin, when stimulated in vitro with PPD, possessed interferon activity. The production of interferon first appeared 4 days after stimulation and continued to the seventh. This contrasted to the production of interferon induced by viruses which was maximal at 24 hours, and from the kinetics of production of other mediators, such as MIF, which were detectable between 6 hours and 3 days after stimulation. Unique among these factors, all of which are found prior to blast cell transformation, interferon production appears to follow the morphological transformation of lymphocytes. Recently, it was reported that C3H mouse lymphocytes treated with chick interferon were capable of killing syngeneic target L cells in vitro (Chernyakhorskaya et al., 1970). A variety of molecular species has interferon activity (Fantes, 1967, 1970). Endotoxin and virus-induced rabbit interferons contained components of about 50,000 and 100,000 daltons by Sephadex G-100 (T. J. Smith and Wagner, 1967); however, the molecular mass of PHA-induced white blood cell interferon from human leukocytes, prepared by Wheelock and analyzed on Sephadex G-100 by Merigan, was approximately 18,000 daltons. This was precisely the same size as Newcastle disease virus ( NDV) -stimulated interferon from the same cells ( Merigan, 1971) . Chick interferon remained soluble after precipitation of the entire culture at pH 2 and could be precipiated by zinc ions Fantes, 1967). Rather interestingly, MIF produced by tuberculinsensitive guinea pig lymphocytes behaved similarly ( Bloom, 1970). Mouse and chick interferons apparently have an isoelectric point of about 6.5 to 7.0 and slight anodal mobility in electrophoresis. Chick interferon has been purified over 20,000-fold by ion-exchange chromatography, and activity was obtained with a fraction which neither absorbed ultraviolet light nor stained on acrylamide gels. Parenthetically, these experiments started with 22.5 liters of chorioallantoic fluid. Interferon is probably a glycoprotein and is destroyed by trypsin (Fantes, 1967). Another lesson to be learned from considerable experience with interferon concerns species specificity. Originally interferon was found effective in protecting only cells of the same species against virus infection. However, mouse interferon showed 5%of its activity on hamster cells and 1% on rat cells (Buckler and Baron, 1966). Generally interferon of

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one species used across a species barrier resulted in a loss of between one and three logs of activity. However, there are important exceptions; for example, human interferon made from NDV-infected fibroblast cultures protected rabbit embryo cells almost as well as rabbit interferon (Desinyter e t al., 1968). To confuse the problem of mediators even further, it must be mentioned that interferons, not of lymphoid origin, may have growth inhibitory activity. Interferon produced by mouse sarcoma cells after infection by NDV or treatment with poly I-poly C inhibited growth of leukemia 1210 cells ( Gresser e t al., 1970). In summary, interferon appears to be one of the substances released by delayed hypersensitive lymphocytes upon contact with antigen. In fact, lymphoid cells appear to be a major source of interferon in uiuo. In terms of difficulties in chemical purification and problems in species specificity and mechanism of action, many of the observations made on these highly active biological molecules relate to studies of mediators of hypersensitivity.

F. ANTIBODIES Because the specificity and variability inherent in the cell-mediated immune response so resemble the pattern known to exist in humoral antibodies, many immunologists assumed that delayed-type hypersensitivity must be mediated by antibody. There have been many published and unpublished attempts to transfer delayed hypersensitivity passively by means of serum from hypersensitivc donors, but, in general, these were unsuccessful or irreproducible ( Bloom and Chase, 1967). Possibly, then, only a special type or class of antibodies might be responsible. Boyden and Sorkin (1960) found that rabbits immunized against human serum had certain antibodies termed “cytophilic antibodies,” which more readily adsorbed onto spleen mononuclear cells than did Ig molecules as a whole. The assay consisted of incubating normal spleen cells at 0°C for 1 hour with the rabbit antiserum, and, after washing, testing for the adsorbed antibodies by uptake of 13’I-labeled serum albumin. Generally the amount of cytophilic antibody ranged between 0.1 and 1%of the total antibody to human serum albumin ( H S A ) . In a later study, guinea pigs were immunized with SRBC mixed in Freund’s complete adjuvant. In addition to developing circulating antibodies, these animals showed strong delayed-type reactions to a urea extract of SRBC ( Boyden, 1964). When tested for their cytophilic properties, the sera were quite active in adhering to peritoneal macrophages in uitro. In contrast, when animals were immunized with SRBC in incomplete Freunds adjuvant, henlagglutinin titers developed, but the animals did

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not show delayed-type hypersensitivity reactions upon skin test. When their sera were tested for ability to adsorb to macrophages, lower or undetectable amounts of a cytophilic antibody were found (Boyden, 1964). In repeated experiments the existence of cytophilic or cell-bound antibodies reactive against tuberculin in tuberculous guinea pigs could not be demonstrated. On the basis of these experiments, Boyden postulated that cytophilic antibodies adsorbed onto macrophages from the serum were involved in mediating delayed hypersensitivity. Cogent arguments in support of this hypothesis have been summarized by Nelson ( 1969). The nature of these cytophilic antibodies was studied in more detail by Berken and Benacerraf (1966). Their animals were similarly immunized with SRBC in complete and incomplete Frenud’s adjuvant. Although hemagglutinating antibody titers were similar in both courses of immunization, cytophilic antibody was only detectable in guinea pigs which had received SRBC mixed with complete Freund’s adjuvant. The majority of the cytophilic antibodies were found to be 7,-globulins, distinct from anaphylactic antibodies which are y,-globulins in the guinea pig. The site required for adsorption to macrophages resided on the Fc fragment, and the cytophilic activity was destroyed by pepsin digestion. Reversibility of the binding reaction and ease with which cytophilic antibody was eluted from macrophages was demonstrated by the fact that the majority of activity was eluted simply by culturing macrophages at 37°C for 30 minutes. In addition, the cytophilic antibodies exchanged with 7,-globulins from normal serum, showing competitive dissociation. Thus the attachment of the cytophilic antibody was weak, although the strength of binding was increased when the antibody was bound to antigen in complex form. Because of the ease of dissociation and ability to exchange with normal serum y:, components, cytophilic antibody seemed unlikely to mediate delayed hypersensitivity alone. Attempts to transfer delayed hypersensitivity locally by the intradermal method in guinea pigs using niarcophages to which were adsorbed cytophilic antibodies or which had been incubated with serum from delayed hypersensitive guinea pigs were unsuccessful, even using autologous or syngeneic peritoneal macrophages ( Blazkovec et al., 1965). Further, in guinea pigs sensitized with HSA-anti-HSA complexes in adjuvant, delayed hypersensitivity appeared 7 days after sensitization, yet no cytophilic antibody was present in the serum (Holtzer and Winkler, 1967). One interpretation of these findings is that immunization with Freund’s complete adjuvant elicits formation of y 3 antibodies,

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often concomitant with, but independent of induction of delayed-type hypersensitivity reactions. For example, human monocytes have been reported to have receptor sites for IgG molecules (Huber and Fudenberg, 1968). Cytophilic antibodies in sera of delayed hypersensitive guinea pigs, after attaching to macrophages, caused inhibition of macrophage of migration in vitro when specific antigen was added to the medium (Amos et al., 1967). However, of three different immunization procedures, all of which produced delayed hypersensitivity in vivo, only one elicited formation of cytophilic antibodies that inhibited macrophage migration. Cytophilic antibody was also found to inhibit macrophage migration when serum of BCG immunized guinea pigs was used. Alternatively, cytophilic antibodies could be eluted from peritoneal macrophages of such animals by heating the cells at 56°C for 1 hour (Heise et al., 1968). Sensitivity to migration inhibition was conferred upon normal macrophages or upon trypsinized macrophages from sensitized guinea pigs by incubation either with serum or heat eluates prepared from the BCG-sensitized peritoneal cells. Migration of passively sensitized cells was inhibited only by specific antigen. Cytophilic activity in serum or eluates was found in the second peak of G-200 Sephadex columns (containing 19 S and 17 S immunoglobulins ) which contained IgG, and probably y2, as detected with immunoelectrophorcsis. It was not found in eluates of lymph node cells from the same animals (Heise et al., 1968). Attempts to sensitize macrophages passively with known guinea pig cytophilic and noncytophilic anti-HSA antibodies, provided by Dr. E. Sorkin, were unsuccessful ( Bloom and Sorkin, 1967). It has been suggested that the principal function of cytophilic antibodies is opsonization. Macrophages coated with cytophilic antibodies against SRBC, for example, were highly phagocytic (Berken and Benacerraf, 1966). Macrophages from mice immunized against histocompatibility antigens of another inbred strain were capable both of opsonization and phagocytosis of tumor cells of that strain (Bennett et al., 1963) and of actual cytotoxicity and plaque formation in monolayers of target tumor cells (Granger and Weiser, 1966). In the latter case, an antibody could be eluted from immune cytotoxic macrophages by heating them to 56"C, which conferred on nonsensitized macrophages specific cytotoxicity for target cells carrying the proper histocompatibility antigens. In vivo, purified macrophages from immune mice could be specifically cytotoxic for tumor target cells in vitro (Bennett, 1965). Similarly, human blood monocytes coated with IgG antibodies

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have been found to bind erythrocytes and to cause their sphering, increased osmotic fragility, deformation, and fragmentation ( Lo Buglio et al., 1967). A test of the relationship between cytophilic antibodies and cellmediated immunity was made by sensitizing guinea pigs with sheep erythrocyte-anti-SRBC complexes in 5% antibody excess in complete Freunds adjuvant (Holtzer and Winkler, 1967). The animals developed strong delayed hypersensitive dermal reactivity but had no detectable cytophilic antibodies or hemolysins. Later, when hemolysins appeared, cytophilic antibodies appeared as well. These experiments and others in which cytophilic antibodies disappeared first after immunization while delayed-type hypersensitivity remained ( Gowland, 1968) indicate strongly that cytophilic antibodies are not required for, or invariably associated with, cell-mediated immune reactions, but do not exclude the possibility that they may participate if present. Sell and Gel1 (1965) showed that immunoglobulins were associated with the surface of lymphocytes by demonstrating lymphocyte transformation with anti-immunoglobulin sera. They postulated that this might be the cell receptor for antigen. Another concept of the involvement of antibodies in cell-mediated immunity was developed by Mitchison (1969). Because of the similarities of recognition by the receptor involved in delayed hypersensitivity and the specificity of circulating antibodies, he proposed that the receptor for cell-mediated immunity would be a special class of immunoglobulin which he termed IgX. An ingenious series of experiments was designed by Dupuy and Good and their collaborators to isolate such a cellbound immunoglobulin receptor and attempt passive transfer of hypersensitivity with it. Their premise was that IgX would be found normally only on living cells and not in the plasma of normal animals. However, when sensitized animals were lethally irradiated, lymphocytes would die and perhaps release IgX into the plasma (Dupuy et al., 1969). Accordingly, guinea pigs infected with BCG were lethally irradiated, heparinized plasma was obtained 3-5 days later, and injected into normal recipients. When these recipients were skin tested with tuberculin 6-9 days later, positive reactivity was found in a little over half the recipients. Further, the active principle had an affinity for cells and could be removed by absorption of the plasma with spleen cells, latex particles, or erythrocytes ( DUPLIY and Good, 1970). Fractionation of the plasma by agar block electrophoresis (Dupuy et at., 1970a) disclosed the activity to be solcly associated with the most anodal fractions, containing only albumin. Fractions with y - and ,8-globulin mobility were entirely inactive. The electrophoretic mobility of the IgX was

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remarkably similar to that reported by Allerhand and Zitrin (1962) for antibody to tuberculin in the serum of tuberculous patients and to a cytophilic antibody found in mice only after iminuiiization with SRBC (Nelson, 1970) and that of guinea pig MIF (Remold et al., 1970). Detailed histology of the reactions produced by IgX has not been reported at this time, although macroscopically they are 5-8 nim. diameter with weak or moderate induration. It has been argued that radiation of BCG-infected donors would supprcss cellular resistance and perhaps produce bactereniia resulting in transfer of living organisms with plasma. Thus one interpretation of the results could be active sensitization of recipients either by living BCG or immunogenic fragments. To the contrary, the same general procedure was used in mice to transfer accelerated allograft rejection with plasma. In this case, even though C3H and C57 mice do share some antigens in common, transfer of IgXcontaining plasma or normal spleen cells incubated in such plasma resulted in a 2-day acceleration in rejection of tail-to-back skin grafts. When guinea pigs were sensitized with killed mycobacteria, under conditions in which high levcls of delayed hypersensitivity developed, the transfer with IgX could not be confirnmed (Chase, 1970). Plasma taken from such donors after irradiation did, indeed, transfer dermal reactivity, but it was judged to be of the iinniediate type and contained passive cutaneous anaphylaxis antibodies producing maximal reactions about 10 hours after the test with marked permeability changes to Evans blue dye. The erythema seen later than 6-10 hours was thought to be the residue of the immediate reaction rather than a delayed hypersensitivity reaction. Plasina from nonirradiated donors was as effective as that from irradiated guinea pigs. Another serious published attempt to verify the IgX concept by plasma transfer from irradiated donors has failed to provide confirmation of the original reports. Collins et nl. (1970) repeated the protocol established for the tuberculin system in guinea pigs as precisely as possible, using living BCG- ( and ovalbumin ) sensitized animals and transferring post-irradiation plasma. By careful sequential observation of reactions to skin tests in recipients at various times, they found that there was a transfer by the plasma of Arthus-like inimediate-type reactivity, which in varying percentages of recipients, persisted to 24 hours as weak chronic type inflammatory reactions. These investigators failed to find any experimental support for an IgX in the plasma which could be related to delayed-type hypersensitivity, and have questioned the validity of previous interpretations. One critical test whether I g S is a specific mediator of delayed hypersensitivity reactions or is simply a cytotropic antibody could be made by

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a careful study of specificity of transfer after sensitization with haptenprotein conjugates. One would cxpcct that, if thc plasma transfer were niediatcd simply by an ordinary antibody, the reactions in recipients would be a hapten-specific. Conversely, if the specificity of IgX is the same as that of delayed hypersensitive cells, then the transfer by means of plasma should be carrier-specific. Until the specificity, histology and vascular permeability of the reactions transferred by IgX are better understood, it will be difficult to evaluate the role of IgX in cell-mediated immune reactions.

G. SKIN-REACTIVE FACTORS By definition, delayed-type hypersensitivity is a reaction of living organisms to antigenic challenge; therefore, the validity of assumptions that factors produced in vitro may be mediators can only be tested in vivo. Perhaps the earliest observation that lymphocytes from hypersensitive individuals might produce a factor in vitro which could engender skin reactivity in vivo was that of WesslCn (195213). Thoracic duct lymphocytes from tuberculous rabbits or guinea pigs were collected and incubated in the presence or absence of tuberculin. When the cellfree supernatants, adjusted to contain equal amounts of tuberculin, were inoculated into tuberculous animals, there was great increase in reactivity to tuberculin in the antigen-stimulated supernatant. Although detailed studies were not reported, some cvidence suggests that the factor might havc been active in nonsensitized animals as well. Attempts to determine whether supernatants containing MIF and the other mediators had biological reactivity in vivo were hampered when inoculation of concentrated fractions from cultures in the presence of serum frequently led to toxic reactions. The principal serum component rcsponsible for this toxicity was albumin or perhaps fatty acids associated with it. Consequently, conditions were developed for culturing guinea pig lymph node lymphocytes in serum-free medium which still enabled them to produce MIF (Bennett and Bloom, 1968). When these serum-free supernatants were concentrated ten- to twentyfold and inoculated into the skin of normal guinea pigs, supernatants only of antigen-stimulated lymphocytes produced reactions in the skin of normal guinea pigs; the rcactions arosc at 3 hours, reached a peak at 6 to 12 hours, and faded by 24 hours. Concentrated control supernatants, i.e., supernatants of equal numbers of sensitized cells cultured in the absence of antigen, to which an equivalent amount of PPD was added upon removal of the cells, produced no reactivity. The skin reactions produced by the stimulated supernatants were characterized primarily by palpable induration with little or no erythema. Histo-

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logically, at 4 hours these reactions were predominantly mononuclear with relatively few neutrophiles. (See Fig. 6.) By 16 hours the histological picture changed and infiltration of about equal numbers of neutrophiles and mononuclear cells occurred possibly as a consequence of late focal epidermal necrosis (Bloom and Bennett, 1969). Confirming that histocompatibility antigens in the supernatant had not caused these reactions, experiments with inbred strain 13 guinea pigs gave identical results. When recipient animals were injected intravenously with Evans blue dye, prior to intradermal challenge or 4 hours later, there was either no increased permeability at the skin sites or only a thin ring around the indurated area, just as in actively sensitized guinea pigs. Similar observations were made in the rabbit (Krejci et al., 1969). Supernatants of lymph node cells challenged in uitro with high doses of PPD produced indurated reactions in nomial rabbit skin. Supernatants of such cells challenged with low doses of PPD were much less active, even upon subsequent addition of increasing amounts of antigen. Histologically these reactions were primarily mononuclear and consistent with delayed-type hypersensitivity, especially in the deep dermis; rather than increased vascular permeability, the supernatants caused anemization and pallor of the site (Pekarek et al., 1969). Fractionation studies of the skin-reactive factor ( SRF ) indicated that activity eluted from Sephadex G-100 columns in the second peak (Bennett and Bloom, 1968) or from G-200 columns in the third peak (Pick et al., 1969) with albumin. This activity has not yet been distinguished from MIF, L T or blastogenic factors (Dumonde et al., 1969; Wolstencroft and Dunionde, 1970). Recently stimulation of nonsensitized lymphocytes by mitogens such as Con A has led to production of SRF (Pick et a]., 1970a,b; Schwartz ef al., 1970). Potentially great advantages result from using mitogens, since theoretically many more cells can be activated. But there are also pitfalls; by themselves mitogens produce inflammatory reactions in normal guinea pig skin, resembling delayed hypersensitivity reactions (Kind and Peterson, 1968). In these studies, Con A was removed from the supernatants before in uiuo testing by passing them through Sephadex which binds Con A. The best source of SRF appears to be lymph node cells. Although peritoneal lymphocytes are very active, contamination with macrophages and release of nonspecific toxic substances (Pincus, 1967; Heise and Weiser, 1969) commonly leads to high pcrcentages of neutrophiles in skin reactions. Another type of skin reactivity has been reported to be released from mixed lymphocyte cultures in uitro ( Ramseier, 1967, 1969). Super-

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natants of mixed lymphocyte cultures of inbred mice or rat cells produced strong inflammatory reactions when injected into the skin of irradiated hamsters. The active principle, “product of antigen recognition” (PAR), was found to be produced only when the lymphocytes were allogeneic and immunologically competent. For example, thymocytes or specifically tolerant lymphocytes were not competent to produce this factor, nor were lymphocytes of which the intermediary metabolism was inhibited by cyanide, DNP, puromycin, or iodoacetate (Raniseier, 1969). Skin-reactivity could be detected as early as 4 hours after cocultivation. It was also found to be produced by lymphocytes of mice immunized against SRBC upon cocultivation with SRBC. The histology of the reactions was virtually pure polymorphonuclear infiltration, and PAR apparently was not active in guinea pig skin. It is not clear from the histology of the response whether PAR has biological significance as a potential mediator of cell-mediated immune response or whether it serves only as a useful tool for detecting immunological reactions. A “lymph node permeability factor” (LNPF) has been found in aqueous extracts of lymph node cells injected into the skin of normal guinea pigs ( Willoughby et al., 1962). This factor could be distinguished from other permeabiIity factors, such as histamine and bradykinin, by pharmacological tests. Extraction of skin reaction sites in contact and tuberculin hypersensitivity also yiclded activity ( Willoughby et al., 1963,1964; Boughton and Spector, 1963). Although permeability changes occurred quickly after injection of LNPF, later histological examinations of the sites indicated a pattern of neutrophile and mononuclear cell infiltration not widely different from that of other delayed-type reactions ( Willoughby et al., 1964). In contrast to all the mediators so far discussed, yields of LNPF from sensitized and normal lymphocyte extracts were approximately the same. Chemical fractionation of lymphocytic extracts containing LNPF indicates that vascular activity is associated with many protein fractions and that LNPF may not be a macromolecule but rather a small substance that adsorbs noncovalently FIG. 6. Histology of reactions produced in normal guinea pig skin by Sephadex G-100 purified supernatants of purified protein derivative ( PPD )-stimulated lymphocyte culttires. ( A ) Site of injection of Sephades peak 2 material from PPDstimulated lymphocytes, 4 hours; ( B ) site of injection of Sephadex peak 2 material from control, unstiniulated lymphocytes, 4 hours; ( C ) higher magnification of the inflammatory cell exudate in A, showing infiltrate to be predominantly mononuclear; ( D ) site 18 hours after injection of Sephades peak 2 material from PPD-stimulated lymphocytes. The arrow indicates an area of focal epidermal necrosis. (From Bloom and Bennett, 1969.)

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to a variety of serum and tissue proteins (Meacock and Willoughby, 1968). Although permeability changes have been noted in careful studies of cell-mediated immune reactions (Voisin et aZ., 1964), they generally occur later in the course of reactivity, possibly secondary to the initial events. The facts that LNPF is found in normal as well as sensitized lymphocytes and that supernatants of antigen-stimulated lymphocytes do not increase permeability, rather if anything, cause anemization, raise serious questions whether LNPF can be considered a mediator in terms of initiating the events of the reaction. However, once the early events take place, LNPF may well contribute to the vascular changes and cell damage associated with later stages of delayed-type hypersensitivity reactions. In summary, sensitized lymphocytes have the capacity, upon stimulation in vitro with specific antigens or mitogens to release one or more factors which have biological activity, not only in vitro but in vivo as well. In normal animals, at least one of these factors is capable of eliciting reactions which resemble histologically active delayed-type hypersensitivity. In general, these factor-induced reactions arise considerably earlier than antigen-induced reactions in sensitive animals. One possible explanation is that the time ordinarily required for activation of the sensitized cells and production of the factor has already occurred in vitro over at least a 24-hour period. W. Hill (1969) has shown that the time course of active delayed-type skin reactions could be accelerated by injection of macrophages locally. Such observations are consistent with the hypothesis that some factors produced in vitro might be nicdiators of cell-mediated reactions in viuo. Indeed, Dumonde et al. (1969) coined the term “lymphokines” to describe collectively the various nonantibody mediators of cellular immunity. However, for proof that they are mediators, it would have to be demonstrated either that the same factors were present in viuo at the site of active cellmediated immune reactions or that agents, such as antibodies to them, which could specifically block or suppress one or more of these factors, also suppressed cell-mediated reactions. V. Enumeration of Specifically Sensitized Cells-Quantitative Basis of the Response

A. ANTIBODY FORMATION The antibody rcsponse can now bc analyzed owing to veiy sensitive techniques for detecting production of antibody by single cells ( Dutton, 1967; Makinodan and Albright, 1967). The most widely used approach is the Jerne plaque assay (Jerne et al., 1963) in which a cell

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is detected merely by the efiect of its product, the antibody, in lysing sheep erythrocytes. A second approach is antigen binding to lymphoid cells from normal and immune animals (Sulitzeanu, 1968), e.g., the ability of immune cells to adsorb and immobilize bacteria (Makela and Nossal, 1961), of erythrocytes to adhere leading to the formation of rosettes (Nota et al., 1964; Biozzi et al., 1968), or of radioactive soluble antigens to adhere and be visualized (Naor and Sulitzeanu, 1967, 1968; Byrt and Ada, 1969; Mandel et al., 1969). Several points are relevant for discussion of quantitative problems on delayed hypersensitive cells. First is the number of antibody-producing cells found in the peak of a hyperimmune or secondary response. In the Jerne assay, this figure is of the order of cells (Jerne and Nordin, 1963). In contrast, assays not directly measuring antibody but the ability of cells to bind antigen, invariably give a higher figure. With bacterial adherence the figure was 0.8%of lymphoid cells, although 40% of the adherent cells were plasma cells (Makela and Nossal, 1961). In the rosette assay, approximately 2% of lymphocytes in rabbits and approximately 4% in mice were maximally capable of rosette formation (Nota et al., 1964; Biozzi et al., 1968). Studies on the binding, of radioactive albumins indicated a figure of approximately 0.5% of lymphoid cells with greater than 10 grains of 1251-bovineserum albumin (BSA) (Naor and Sulitzeanu, 1967, 1968). Thus the numbcr of antigen-reactive cells is greater than the number of antibody-producing cells. The second consideration is the number of cells in a nonimmunized animal that either produces antibody or binds antigen specifically. An overall estimate of the frequency of stem cells or antigen-reactive cells in the normal animal was given by Jerne (1967) as In terms of actual antigen-binding studies, the figures vary considerably from experiment to experiment and tissue to tissue. For example, spleen showed 860/ lo6 hemocyanin-binding cells, yet bone marrow had 20,000/ 10' (Byrt and Ada, 1969) or essentially none ( Makela and Nossal, 1961). In humans immunized against flagellin, the average maximal number of antigen-binding cells in peripheral blood was 40/1000 at 14 days (Dwyer and Mackay, 1970). Interestingly, it has been rather difficult to show a significant increase in antigen-binding cells in animals that have been primed to make antibodies but have rested for considerable time (Sulitzeanu, 1968). Low doses of antigen have been shown to increase greatly the number of rosette-forming cells without significantly increasing the number of plaque-forming cells, and many of the rosette-forming cells are thymus-derived (Greaves et al., 1970; Greaves and Moller, 1970; Doenhoff et al., 1970).

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In some systems the thymocytes were totally unable to bind antigen ( Byrt and Ada, 1969), whereas in others, normal thymocytes showed high reactivity, e.g., approximately 0.1%for p-galactosidase ( Modabber et al., 1970). The last point concerns the kinetics by which this small number of stem cells generate antigen-reactive or antibody-producing cells. In almost all experiments, this increase is approximately exponential ( Perkins et al., 1969), and the increase in antibody-producing cells may cover a span of four logs in an individual animal. Therefore cell proliferation is required for the increase of antibody-forming and antigen-binding cells after immunization.

B. MIXEDLYMPHOCYTE INTERACTION The graft-versus-host reaction, i.e., the reaction of lymphocytes against an immunologically unresponsive foreign host, leading to splenoniegaly or runting, has been an extraordinarily useful tool in uiuo to study the early events in initiation of an apparent immune response. In uitro models for this response have consisted either of reaction of lymphocytes in allogeneic chick embryos or simply the reaction of allogeneic lymphocytes when mixed, leading to activation and proliferation (reviewed by Simonsen, 1962; Dutton, 1966; McBride, 1966). In the chick embryo chorioallantoic membrane (CAM) and in the mixed lymphocyte interaction (MLI) in the rat (Wilson, 1967; Wilson et al., 1967) the reaction is believed to be an immunological one for the following reasons: ( a ) the reacting cell is the small lymphocyte; ( b ) reactions occur only across major histocompatibility loci; ( c ) lymphocytes of thymectomized animals give negligible or diminished responses; and ( d ) lymphocytes of immunologically tolerant donors fail to respond against tissues containing the specific antigen. However; several peculiarities raise some question as to the immunological nature of MLI, especially as a primary immune response. First, reactions occur only across major histocompatibility loci and not across minor ones, although the latter are perfectly adequate to cause graft rejection in d u o . Second, response of preimmunized lymphocytes in mixed lymphocyte culture is not significantly greater than that of normal lymphocytes, whereas grafts in uiuo are rejected in accelerated fashion in the immunized hosts (Wilson et al., 1967). Third, although high reactivity is found in allogeneic combinations, xenogeneic lymphoid tissue, i.e., tissue from another species, does not react ( LafFerty and Jones, 1969; Wilson, 1971). In the CAM systems, as few as 36-50 lymphocytes were capable of initiating a response in an allogeneic embryo (Simonsen, 1967). In the MLI, a count of lymphocytes entering into the first mitosis showed that 1 3 % of nonsensitized lymphocytes reacted in a one-way situation

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(Wilson et al., 1968). By liniiting-dilution studies of responding cells in one-way mixed lymphocyte cultures, response was seen in about 50% of allogeneic mixtures when the number of responding cells ranged between 750 and 3000, again suggesting a frequency of responding cells of approximately O S - l % (F. H. Bach et al., 1969). In the rat MLI system, the number of cells incorporating thymidine or undergoing mitosis early was probably the initial reacting population, and secondary recruitment of bystander cells seemed very low (Wilson et al., 1968). Using the virus plaque assay to measure activated lymphocytes ( described in Section V,C), again the number of cells involved in a oneway MLI was about 1%(Jimenez and Bloom, 1971), and the number of lymphocytes initially activated by PHA, about 10% (Willems et al., 1969). The major problem raised by these results is that of the potentiality of the immunocompetent cells in cell-mediated immunity. Because there is little reactivity across minor histocompatibility barriers, it is most unlikely that all lymphocytes are omnipotential, i.e., capable of reacting against all antigens. However, the fact that 1-558 or more of lymphocytes from nonimniune donors may react against certain antigens strongly suggests that they may be multipotential. If there are at least 50 major histocompatibility loci, then all lymphocytes in peripheral blood could be engaged and activated simply by allogeneic transplantation antigens. Clearly the fact that immunization to other antigens can occur implies that if lymphocytcs were only unipotential, one would soon run out of available cells. Thus alternatives are that ( 1 ) lymphocytes are multipotential, ( 2 ) the mixed lymphocyte and graft-versus-host systems involve nonimniunological reactions, or ( 3 ) there is a high level of natural and continuous immunization to antigens in the environment, crossreacting with major transplantation antigens. A fourth possibility suggested by Jerne (1971) is that all the genes controlling specificity in the immune system derived from a smaller number of genes controlling reactivity only to alloantigens. These genes underwent mutation and variation such that specificities to all antigens derive from altered genes controlling reactivity to sclf- or nllogcneic antigens. Thc cells that did not mutate and had antisclf-specificity were eliminated, accounting for tolerance. This is an ingeneous model, selecting for variability and against initial specificities. Recent evidence, indicating that xenogeneic reactivity disappears in MLI among lymphocytes from germ-free animals but remains high in lymphocytes from allogeneic germ-free animals, argues against the third possibility, continuous immunization by crossreacting antigens ( \Vilson, 1971 ) . The fact that lymphocytcs from immunologically tolerant animals do not react in these systems does suggest, but does not prove, that these are truly immunological reactions.

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Before leaving the subject of the MLI, it is necessary to discuss briefly the possible implications of some recent experiments indicating sensitization in vitro. It had previously been reported that human lymphocytes, cultured on allogencic fibroblasts for 7 to 8 days in the absence of PHA or known antigen, when replated on fresh monolayers of the same origin, caused rapid destruction of the specific target cells but not of other fibroblasts (K. Hirschhorn et al., 1964). In more detailed experiments, rat lymphocytes could be sensitized in vitro by culture on mouse fibroblasts, and further, the resulting cytotoxicity was specific only for strong histocompatibility antigens, i.e., H-2 specificity was developed ( Ginsburg, 1968). Curiously the rat lymphocytes acquired H-2 specificity for mouse determinants, although rat antimouse antisera in general are species-specific and not H-2-specific. Bone marrow grafts in irradiated mice were rejected by irradiated mice in only 2 to 3 days, if the grafts crossed the H-2 locus (Cudkowicz, 1971). Morc reccntly, reports indicate that mouse and human lymphocytes can be sensitized by mixed lymphocyte culture (Hayry and Defendi, 1970a,b; Canty and Wunderlich, 1970; Wunderlich and Canty, 1970; Solliday and Bach, 1970). These cultures were set up for 4 to 5 days, either in the classic fashion or with rocking analogous to the in vitro immunization developed by Mishell and Dutton ( 1967) against SRBC ( Canty and Wunderlich, 1970; Wunderlich and Canty, 1970). The lymphocytes were then tested for their cytotoxic activity on either monolayers or suspensions of Wr-labeled target cells bearing the same alloantigens as the sensitizing lymphocytes in the MLI. A remarkably high degree of cytotoxicity was obtained, often as high as with cells immunized in vivo, which was apparently specific only for major transplantation antigens of the immunizing strain.3 As exciting as they are, these experiments pcse a serious conceptual problem. If the number of initially reacting cells in the MLI is 1-38, as discussed earlier, then why do not these cells, which certainly recognize major histocompatibility antigens and undergo transformation, also have the functional capacity to exert cytotoxicity? If 13%of the cells are already committed to a given histocompatibility antigen, why do they have to be sensitized by a 4-5 day incubation period before they acquire cytotoxicity? One wonders whether the cytotoxicity observed in these systems is produced ( a ) by the same cells which have undergone blastogenesis, ( b ) by their progeny, or ( c ) by other, perhaps nondividing cells. In any case, these fascinating systems raise important questions concerning the nature of sensitization for cell-mediated immunity and the relation of recognition to effector function. Similar results using subcellulnr materials to ‘sensitize’ lymphocytes in vitro have been observed (Simmons and Manson, 1971).

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C. DELAYED-TYPE HYPERSENSITIVITY The binding of labeled antigens to lymphoid cells from delayed hypersensitive animals has been studied by many investigators. By using 1311-labcledPPD, bovine serum albumin (BSA), and BGG, Turk (1960) found a relatively high background of binding to normal spleen cells, but a very slight increase in binding to cells from sensitized donors. He concluded, “perhaps the clearest finding from these experiments is that specific uptake, insofar as it occurs at all, is very small and hard to detect.” Using a modified antigen consumption technique, specific binding of antigen to ovalbuniin-hypersensitive lymph node cells, obtained from guinea pigs immunized with ovalbumin-antiovalbumin complexes, was inferred (Steffen and Rosak, 1962). The sensitized cells were allowed to react with ovalbumin, and, then, after washing, the cells were reacted with antiovalbumin globulin of known titer. That sensitized lymphocytes bound ovalbumin was deduced by finding a diminution in the standard antiovalbumin reagent. In a similar approach, in which sensitized guinea pig lymph node cells were reacted with HSA or ovalbumin, and the cells binding ovalbumin lysed with antiovalbumin antibodies plus complement, no difference in binding was detected between normal and sensitized cells (Peltier and Kourilsky, 1966). A slight increase in binding above control levels of Y - P P D to sensitized lymphocytes in vitro has been noted by Kay and Rieke (1963) and of fluoresceinated tuberculin (Gillissen, 1963). The PPD was attached to lymphocytes but also to granulocytes. In careful study of binding of tuberculoprotein to lymph node cells of tuberculin-hypersensitive guinea pigs, using fluoresceinated antituberculoprotein antibody to stain cells in living preparations which had bound the tuberculoprotein, it was estimated that between 5 and 20%of the cells were reactive (Martins et d., 1964). In applying the same system to allergic encephalomyelitis in guinea pigs, approximately 5% of the cells bound the basic protein antigen (Rauch and Raffel, 1964). Similar degrees of binding were found in studies using fluorescein-conjugated PPD in human tuberculin-hypersensitive donors ( Witten et al., 1963) and fluorescein-labeled iiisulin in insulin-hypersensitive patients (Federlin et al., 1968). By using blast cell transformation, Coulson and Chalmers (1967) found about 1%of the starting population of tuberculin-reactive cells transformed by PPD at 40 hours; at 90 hours, the range was 6 2 0 % . The number of immunized lymphocytes taking up ferritin was approximately 10%after immunization in Freund’s complete adjuvant and 1%after intramuscular injection (Pernis et al., 1970). In these experiments, thymocytes failed to bind antigen, and of those lymphocytes binding antigen, 90%had IgM detectable on their surface.

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The difficulties in interpreting antigen-binding studies, even the most careful experiments, revolve about the following questions: 1. Is it possible to distinguish a hypersensitive cell from an antibodyproducing cell by antigen binding? 2. Can one distinguish a cell with immunological competence from one which has merely adsorbed antibodies onto its surface? 3. Is it possible to distinguish specific binding to surface receptors from pinocytosis and ingestion of labeled antigens? 4. What is the level of nonspecific adsorption or surface attraction of the particular antigen in relation to the magnitude of binding? Are the differences significant? The dilemma is illustrated by two experiments, essentially the same in design, in which ferritin was added to lymph node cells from sensitized rabbits or guinea pigs and the binding studied by electron microscopy. In one case, a great number, in fact practically all, lymphocytes bound ferritin (Clarke and Periman, 1962), in the other there was no binding at all ( Goldberg et al., 1962). A number of attempts to avoid these difficulties and to detect specifically sensitized cells by their ability to produce mediators, such as MIF and LT, have been unsuccessful (Bloom, 1970). One difficulty with this approach, in contrast to measurement of antibody production in the Jerne assay, is the fact that the absolute amount of these factors produced is infinitesimally small, and activity may not be detected even at small distances from target cells. Another approach was designed to detect intrinsic changes in sensitized lymphocytes upon specific interaction with antigen. The ability of these cells to replicate certain RNA viruses was examined and used to develop a plaque assay for enumerating antigen-reactive cells in delayed hypersensitivity (Bloom et al., 1970; Bloom and Jimenez, 1970). It has long been known that resting lymphocytes were unable to replicate a variety of RNA and DNA viruses, or did so very poorly (see Edelman and Wheelock, 1966; Bloom et al., 1970). However, activation of lymphocytes by PHA was shown to enhance markedly the replication of several viruses, including measles, enteric cytopathogenic human orphan (ECHO), herpes simplex, NDV, mumps, polio, and vesicular stomatitis ( VSV) viruses. Lymphocytes were obtained from tubzrculinsensitive guinea pigs or humans and cultured in the presence or absence of PPD under conditions used for production of MIF. Virus was not present during the cultures but was simply used as an indicator to reveal the number of cells which had been activated by PPD and had now become capable of virus replication. Although most previous studies were concerned with the number of viruses produced per cell,

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a simple modification of the system, known as an infectious centers assay, permitted determination of the number of lymphocytes capable of producing viruses. In the guinea pig system, results indicated that after 24 hours of stimulation with PPD, the number of virus-forming cells above background was approximately 1:1000 and this increased over a 4-day period to approximately 20: 1000 (Bloom et nl., 1970; Bloom and Jimenez, 1970). Lymphocytes from guinea pigs, immunized with PPD on alumina to produce only circulating antibodies did not respond in this system, thereby establishing a correlation with cell-mediated immunity. The number of plaques observed was solely a function of the lymphocytes, and independent of the virus used to detect it, e.g., NDV or VSV. In healthy human donors hypersensitive to tuberculin the maximum number of cells detected at 4 days by this assay was approximately 5 : 1000 (Jimenez et al., 1971). Using a similar technique, Willems et al. (1969), found 10%of human lymphocytes to be activated by PHA. In this system, the increase in virus plaque-forming cells with time after antigen stimulation has been found to be remarkably linear (Bloom et al., 1970; Jimenez et al., 1971), in contrast to both antibody formation (Jerne and Nordin, 1963) and blast cell transformation (Marshall et al., 1969). That this linearity is not simply fortuitous was demonstrated by experiments in which inhibitors of mitosis, vinblastine, colchicine, and thymidine block, were found to have no effect on the linear kinetics of the system. In fact, in studies on human cells, under conditions where vinblastine inhibited mitosis (Palmer et al., 1960) and completely suppressed the incorporation of thymidine, the increase in virus plaques remained (Jimenez et al., 1971), indicating a dissociation between the mitogenic response and the virus plaque assay (see Fig. 7 ) . These experiments indicate that the number of cells specifically reactive to a single antigen in a hypersensitive individual is of the order of 5 to 50:lOOO. Although this is a rather small number from the point of view of biochemical studies, it is a large number to be committed to a single antigen. Yet, since there is little detectable reactivity in noninimunized individuals, this response reflects the end product of possible clonal proliferation as a result of immunization. Whether the effector cell in delayed hypersensitivity corresponds to the proliferating model as in blast cell transformation, or to thc nondividing cell, as in the virus plaque assay, will be discussed in Section VII,A,4. It is important here only to point out that under the same conditions both responses occur in the same test tube and can be distinguished. In quantitative kinetic studies on the development of plaques on the chorioallantoic membrane of chick embryos by allogeneic lymphocytes, Coppleson and Michie (1966) developed a relationship between the

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5 0 0 0 A~

-100004.

;

I

I

3

4

DAYS

FIG.7. Kinetics of lymphocyte stimulation in the virus plaque assay and mitogenic assay. ( A ) Effect of vinblastine on the increase in antigen-sensitive cells ( A P F U ) detected by the virus plaque assay of sensitized human lymphocytes following stimulation with purified protein derivative (PPD ). Closed circles (solid line) indicate the average for 5 donors cultured in the absence of mitotic inhibitor; open circles (dotted line) indicate the results of the same donor’s cells cultured in the presence of vinblastine ( 1 pg./nil. ), ( B ) Effect of vinblastine on the incorporation of “C-thymidine into DNA following PPD stimulation. Closed circles (solid line) indicate the average increase in incorporation for paired cultures from the same 5 donors as in A in the absence of mitotic inhibitor; open circles (dotted line) indicate the average results of the same donors’ cells cultured in the presence of vinblastine ( 1 pg./ml.). (From Jirnenez et al., 1971.)

log of the plaques observed and the log of the number of cells plated. The slope of this plot indicated the number of cell interactions required to give 1 plaque. When applied to the interaction of cells required to initiate a primary antibody response against SRBC in uitro, the slope was found to be 3, indicating a three-cell interaction was required (Mosier and Coppleson, 1968). It is now known that thymus, bone marrow, and a glass-adherent cell, probably a macrophage, are involved in the response. When data from the virus plaque assay of tuberculin-stimulated guinea pig lymphocytes were plotted on such a logarithmic kinetic

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plot, the average slope of sixteen experiments was found to be 2.008, indicating that interaction of two cells was required to produce 1 virus plaque (Jimenez, 1971). Removal of glass-adherent cells completely abolished the stimulation of lymphocytes by antigen to replicate VSV, suggesting that macrophages may be required for activation of lymphocytes in this system, just as they have been found to be required for blast cell transformation (Hersh and Harris, 1968; Levis and Robbins, 1970a,b ) . The level of reactivity in the MLI and to specific antigen after hypersensitization implies that lymphocytes involved in cell-mediated immunity are multipotential, or at least bipotential, and able to respond to a histocompatibility antigen and another antigen. With respect to soluble antigens, Zoschke and Bach ( 1970), studied transformation of lymphocytes from donors immunized against multiple antigens by sequential addition of antigen and elimination of previously responding cells using BUDR. The incorporation of thymidine for any given antigen was unaffected by previous stimulation with an irrelevant antigen, indicating eiitircly different populations of cells were being stimulated. Whether BUDR kills the cells, or simply blocks their ability to incorporate thymidine, is important to establish to clarify the functional capability of these cells. VI. Reality Testing-Relationships

between in Vifro Results a n d

Cell-Mediated Immunity in Vivo

A. MODELS From the foregoing survey of in vitro studies, two models emerge to explain effector function in cell-mediated immunity. In the first, based on direct lyniphocyte-target cell cytotoxicity, immunological information is carried by small, thymic-derived lymphocytes. These cells react with antigens on the target cell and are activated to become cytotoxic. The essential premises are that antigen recognition is specific and that effector function is specific. Only the specific target cell, containing antigens to which thc donor lymphocytes are immune is killed, and the entire reaction is mediated directly by the sensitized lymphocytes. In the second model, based on activities and factors produced by antigen-stimulated lymphocytcs, information is similarly carried by thymic-dcrived lymphocytes activated only by specific antigen; however, once lymphocytes are specifically activated, they can affect other cells in their environment nonspecifically by means of factors released, including MIF and chemotactic factor which affect macrophages, LT and interferon which affect target cells, blastogenic factor which stimulates other lymphocytes, etc.

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In this model a specific reaction at the level of the lymphocyte leads to nonspecific effector function. Before looking at these models in terms of their applicability to various observations in vivo, one should perhaps say a word about the immunologists concept of “reality.” Simply stated, it is the tradition that immunological competence is defined only by the result observed in vivo, which is the standard, i.e., the reality, by which in vitro experiments must be measured. It is amusing to consider a few systems in which in vitro experiments probably reveal greater “reality” than reactions in vivo. For example, scorbutic guinea pigs apparently cannot be sensitized well and give only very poor skin reactions. Yet, if one obtains lymphocytes from scorbutic animals after sensitization and transfers them to normal guinea pigs, equivalent reactivity is transferred as if sensitized cells from healthy guinea pigs were used (Zweiman et al., 1966). Similar findings were reported in the case of depression of tuberculin hypersensitivity by 7-mercaptopurine ( Zweiman and Phillips, 1970). Extrapolating to the case of tumor immunity, while many patients have tumors, it is possible that they also have immunological competence, tumors developing either because of their own aggressiveness or because enhancing antibodies prevent the sensitized lymphocytes from killing them ( Hellstrom and Hellstrom, 1970). AND ORIGINOF CELLSIN B. HISTOLOGY-NATURE DELAYED-TYPE HYPERSENSITIVITY REACTIONS

The histology of classic delayed-type reactions has been extensively reviewed (Dienes and Mallory, 1932; LaPorte, 1934; Boughton and Spector, 1963; Turk, 1967; Waksman, 1960). In general there is agreement that, after challenge, an early polymorphonuclear infiltrate occurs similarly in sensitized and nonsensitized animals and is probably unrelated to specific reactivity. Subsequently there is an increasing infiltration of mononuclear cells. The picture in homograft rejection is remarkably similar ( Gibson and Medawar, 1943). The percentage of mononuclear cells varies and the extent of polymorphonuclear leukocytes present probably reflects the contributions of toxicity of the antigen, degree of tissue damage, and the presence of antibody. Although it is difficult in ordinary histological sections to distinguish between lymphocytes and macrophages, by electron microscopy (Goldberg et al., 1962; Wiener et al., 1964) and by histochemical staining (Turk et al., 1966), it has been possible to identify the majority of cells as macrophages or monocytes. In passive transfer experiments, using isotopically labeled sensitized lymphocytes, it was observed that only a small number (0.15%)

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of the lymphoid cells at the test site were labeled (Najarian and Feldman, 1961, 1963). No correlation has been found between the number of labeled sensitized cells found at test sitcs aiid the intensity of the reaction (Hamilton and Chase, 1962; Turk, 1962; Kay aiid Rieke, 1963). Consequently, it appears that only a very sinall number of transferred lyiiiphocytcs from the specifically sensitized donors are found at reaction sites, although these cells may be sufficient to initiate the reaction. The vast majority of cells at reaction sites have been shown to accumulate iionspecifically at the cell-mediated reaction site. When cells of a normal recipient were labeled by a single injection of tritiated thyniidine and sensitized unlabeled lymph node cells were passively transferred, it was found that 80-90% of the cells at the positive reaction site were labeled. Thcrefore ~ionscnsitizcdhost cells, presumably derived from a rapidly dividing population, constituted the majority of infiltrating cells (McCloskey et al., 1963; S. Cohen et al., 1967). These cells, probably iiionocytes or macrophages, originate in the bone marrow from rapidly dividing precursors, arri\.c at thc tcst site by ivay of the blood as monocytes and differentiate therc into histiocytes ( Lubaroff and \Vaksman, 1968a,b; LiclCn, 1967; Volkman and Gonwis, 1965; Wiener et al., 1965; Feldn,an and Lee, 1967; Van Furth and Colin, 1968; reviewed by NeIson, 1969; Colin, 1968; Blooni and Bcnnett, 1970a; Bosinan and Feldman, 1970). Essentially thc samc conclusion was drawn in careful studies of the homograft reaction, namely that the infiltrate was predominantly nonspecific ( R. A. Prendergast, 1964 ) and primarily macrophages ( Gillette and Lancc, 1971) . When normal macrophages were injected into the skin of a sensitized animal along with antigen, a delayed-type reaction developed much earlier and much more intensely than when testing with antigen alone, suggesting that a major component of thc delayed time course of the classic reaction is simply thc tiiiic required for accuniulation of niacrophages at the site (W. Hill, 1969). Another phenomenon in uiuo which is best explained by the second model is h4acrophagc Disappearance. W h ~ n1iypersensitiL.e animals were challenged iiitraperitoneally with antigen, therc was a disappearance of inacrophages from the peritoneal cavity (Nclson and Boyden, 1963; Nelson aiid North, 1965 reviewcd by Nelson 1969). Similarly, when tubcrculin-sensitive guinea pigs were challcngcd \vith PPD intravenously therc was a marked depression of circulating monocytcs ( Yoshida et al., 1969). Recently, the macrophage disappearance reaction in viuo has been produced by supernatants of aiitigen-stimulated semitized lymphocyte culture in uitro (Sonazaki and Cohen, 1971). The possible release and action of MIF or hlAF in viuo are clearly suggested. This, although it is

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clear that the information and specificity for these reactions reside in small lymphocytes, the majority of cells actually at the reaction sitcs are nonspecific cells, primarily inacrophagcs. Regarding the classic histological picture of delayed hypersensitivity reactions, then, the second model would seem to provide a more satisfactory description of the events, although the precise function of thc nonspecific niononuclear cells at the sites is not established. C. REJECTIONOF ALLOGRAFTS AND TUMORS The specificity predicted by the first model was nowhere better demonstrated than by the Kleins aiid their colleagues studying rejection of F, tumors by parental strain congenic mice differing at a single H-2 locus (G. Klein and Klein, 1956, 1958). Chemically induced tumor cells with H-2 a/s aiitigcns were injected into parental A strain (H-2 a / a ) mice, aiid 5 x 10‘ cells were invariably rejected. As a test of specificity, a small number of parental strain cells (H-2 a / a ) were admixed with the 5 x 10‘ F, hybrid cells. After the 5 x 10’ F, cells were rejected, 200 syngeneic (parental) cells grew in 100%of the cases and 20 syngeneic cells in about 40%of the cases. The Kleins stated that these experiments “emphasized the extreme specificity exhibited by the homograft reaction and its ability to recognize a very small number of cells of compatible genotype within a large popul at‘ion of incompatible cells of similar morphological origin and diff cring from the former only with regard to a single gene locus.” These experiments were prcliniinary to asking the more general question whether, under the selective pressure of allograft rejection, mutations would occur permitting cells to lose incompatible transplantation antigens. In related experiments, it was found that if tumor cells with foreign histocompatibility antigcns were irradiated and admixed with syngeneic viable tumor cells, cvcn though a more intense, presumably delayedtype hypersensitivity reaction occurred, rather than nonspecific suppression of the tumor growth, there was, in fact, a slight enhancement (Revesz, 1956). In studies on local rejection of tumors in mice across the H-2 barrier, once again extraordinary specificity in the effcctor process was demonstrated (Bennett, 1965). Whcn C57B1/6 lymphocytes or macrophages immunized against Sarcoma 1 were admixed with Sarcoma 1 cells and EL 4 cells (the ratio of Sa 1 to EL4 was 200:1), and the mixture was injected iiitraderrnally into C57B1/6 mice, under conditions in which theoretically 200 specific tumor cells were killed, the one corresponding EL 4 cell was invariably able to survive. Ingenious experiments on rejection of pigmented cells in guinea

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pigs across a major transplantation barrier indicated that the above specificity is a general principle and not simply a function of the mouse or tumor systems. In these experiments, Family 2 pigmented skin cells were admixed with F, ( 2 x 13) nonpigmcnted cells and grafted onto a ( 2 13) F, guinea pig. After these cells had grown confluently to form a chimeric patch of skin, this skin was removed and grafted onto a Family 2 guinea pig sensitized against the F, strain 13antigens ( Billingham and Silvers, 1963, 1970). Again the F, cells were rejected, but melanocyte islands, which were of syngcncic Family 2 cells easily detected by their pigmentation, survived. These results were extended using allophenic skin grafts, i.e., skin from mouse embryos artificially created by dissociating and recombining cells from diffcrent embryos at the earliest stages (Mintz and Silvers, 1970). When thc mice develop, they are chimeric in the sense that cells of each genotype differentiate and cells coexist next to cells of other genotypes, producing a mosaic. Again studying the rejection of skin grafts by sensitized recipients, localized rejection occurred only against the genotype of cells that was foreign, even when the cells were randomly dispersed in a single hair bulb. These results corresponded closely to others (Section II1,B) in the in vitro rejection by sensitized lymphocytes of target cells containing strong histocompatibility antigens to which lymphocytes were sensitized ( Wunderlich and Canty, 1970; Hayry and Defendi, 1970b; Brunner et al., 1971; Cerottini, 1971). Notably cytotoxicity was more difficult to achieve in vitro against tumor-specific antigens in a syngeneic system ( Brunner et al., 1970) than against histocompatibility antigens in the tumors. It cannot be inferred from these results that the sole mechanism of rejection is direct lymphocyte-target cell cytotoxicity, although this is undoubtedly most important. From the experiments of Bennett ( 1965) in which highly purified macrophages from sensitized mice caused specific local rejection of tumors and from the in vitro experiments of Granger and Weiser (1964) in which target cells were destroyed specifically by mouse macrophages from immune donors, macrophages could be equipped for specific cytotoxicity, either by cytophilic antibodies, opsonization by free antibodies of target cells (Bennett et al., 1963), or possibly by the antigen-specific MIF (Bennett and Bloom, 1967; Amos and Lachmann, 1970). Apparently certain macrophages have an exceptional ability to concentrate antibodies, even passively transfused antibody, above serum concentrations and can transfer and release substantial levels of these antibodies in normal recipients (Hunt and Myrvik, 1964). Similarly, immune mouse macrophages, when mixed with tumor cells in vitro caused rejection following transfer to X-irradiated hosts (Tsoi and Weiser, 1968). However, if the tumor was inoculated subcutaneously

x

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and the macrophages intraperitoneally, no inhibition of tumor growth was seen, indicating that contact was required. Common to all these examples is the fact that rejection occurred across major histocompatibility barriers. Other experiments involve rejection of grafts or tumors with nonspecific effects. When kidney from a Lewis rat which contained Lewis spleen cells under its capsule was transplanted into a (Lewis x B N ) F, recipient, no graft rejection would have been expected to take place because the F, shares the parental Lewis antigens. In fact rejection occurred which looked very much like ordinary allograft rejection (Elkins and Guttman, 1968). The interpretation offered was that the lymphoid cells under the capsule of the parental strain kidney reacted with the F, incompatible antigen in a kind of MLI. Destruction of the kidney tissue was apparently a nonspecific consequence of an immunological, unidirectional interaction between donor and host lymphoid cells. Recently, a similar finding involved skin grafts in rabbits. After remaining as allografts long enough for vascularization and sensitization to occur (4 days), grafts were returned to their original rabbit donor and were promptly rejected (Lambert and Frank, 1970). Possibly mononuclear cells infiltrated the graft from the intermediate host and when the graft was returned to the original host served as a source of foreign antigens stimulating a MLI-type reaction, leading to the nonspecific rejection of the animal’s own skin. The most striking observation in this category is that of Edmund Klein and his colleagues (E. Klein, 1968; A. C. Williams and Klein, 1970) on the eradication of certain skin cancers by the application of chemical allergens. Patients with basal cell or squamous cell carcinomas, and premalignant keratoses were sensitized to the chemical allergen, 2,3, 5-triethyleneimino-( 1,4)-benzoquinone (TEIB), to which they commonly developed a contact-type hypersensitivity. After the period of sensitization, the allergen was painted over large areas of affected skin. In 1 or 2 days there was a great flare-up of most visible as well as previously invisible lesions. After the reaction subsided, desquamation and healing occurred, and the tumors, remarkably enough, disappeared. Still another example of nonspecific rejection of tumors following a specific cell-mediated immune reaction was reported in studies on diethylnitrosamine-induced tumors in inbred strain 2 guinea pigs. The experimental system was entirely syngeneic, the only antigenic difTerences being tumor-specific antigens. Line 1 tumors were used to sensitize normal animals so that when cells of line 1 were later transplanted, they were promptly rejected. When line 7 tumor cells, which did not cross-react immunologically, were injected into the same animal, they,

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of course, grew. However, when line 1 and line 7 cells were admixed and injected intradermally into a guinea pig sensitized only to line 1, in contrast to Klein’s experiment in which the nonspecific tumor invariably grew, in this case both tumors failed to grow (Zbar et al., 1970a). The implication was that a specific iniinune reaction against the tumorspecific line 1 antigen caused a nonspecific effector reaction which destroyed the irrelevant line 7 tumor as well. Additionally line 7 tumor cells were injected intradermally with PPD into strain 2 guinea pigs sensitized only to tuberculin. The tumors were not completely rejected, but there was a delay of considerable time before the outgrowth was visible, suggesting sonie destruction of tumor cells present at the site of an irrelevant tuberculin reaction. There is actually one example of this type of nonspecific rejection from the laboratory of G. Klein. Syngcneic tumors were rejected, in primarily or preimmunized hosts when lymphoma cells, admixed with the syiigeneic tumor cells, were the large, incompatible component ( Ringertz et al., 1959). A specific cell-mediated reaction may manifest nonspecific effector mechanisms, although there are often equally plausible alternative explanations ( see Section V1,D). In experiments of Holm and Perlmann (1967), Granger et a / . (1969), and Ruddle aiid Waksinan ( 1968a,b), lymphocytes activated in vitro either by tuberculin or MLI became nonspecifically cytotoxic for a variety of target cells. Elsewhere sensitized lymphocytes from DBA/2 mice were incapable of killing a syngciieic lymphoma, although they destroyed allogeneic tumor cells. However, when macrophages were added, destruction of the lymphoma could proceed. It is not clear whether bystander tumor cells unrelated to sensitization would also have been killed (Evans and Alexander, 1970). In experiments of Bennett (1965) and Tsoi and Weiser (1968) in vivo and of Granger and Weiser (1964) in vitro, macrophages were cytotoxic for tumor cells. Bloom (1971a) has pointed out that the nonspecific rejection in most of these cases involved syngencic tumor or skin cells and suggested thc possibility that mechanisms involved in recognizing allogeneic cells may be different from those for recognizing other antigens, including tumorspecific transplantation antigens, This point was originally raised in the context of the MLI aiid graft-vcrsus-host systems, in which the number of cells from normal, unininiunized donors capable of responding to allogeneic antigens was as high as 1-20% (Section V). This possibility remains to be tested, but there are alternative explanations of the data in vivo less drastic than dividing effector mechanisms on the basis of major histocompatibility antigens and weaker antigens.

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D. ADJWANTEFFEC~S One alternative to a nonspecific cytotoxic mechanism in graft rejection following stimulation with specific antigen would be a specific delayed-type reaction serving as a natural biological adjuvant for augmenting responsiveness to other weak antigens. For example, in experiments of Edmund Klein, the allergic reaction TEIB might have attracted macrophages that carried antigen back into the lymphoid tissue or to lymphocytes which could be actively sensitized to the tumor antigens. Similarly, in the experiments of Zbar and colleagues, since guinea pig tumors grow rather slowly, possibly the specific reaction to line 1cells enhanced scnsitization and rejection of line 7 tumor cells when they were admixed with line 1 cells. There is evidence that this may be a real role of cell-mediated immune reactions. When protein antigens such as human y-globulin were injected together with PPD intradermally into tuberculin-sensitive guinea pigs, antibody response to the unrelated protein antigen present in the injection site was enhanced (Humphrey and Turk, 1963). Similarly, histological studies of corneal reactions of guinea pigs sensitized with azobenzenearsonate ( ABA ) -tyrosine conjugates, showed delayed plasmacytosis. In this hapten-specific hypersensitivity (see Section VII,A,3 ), when the ABA group was attached to a heterologous protein, an accelerated primary response to the protein was seen (Flax et al., 1969). Bovine y-globulin injected intradermally into tuberculin-sensitive guinea pigs failed to sensitize, but if it was mixed with PPD and injected, sensitization occurred in 50%of the animals (Halpern and Bloom, in Bloom, 1970). Indeed, it seems most likely that the adjuvant effect of mycobacteria on immune responses, in general, depends critically on the establishment of cell-mediated immunity or delayed hypersensitivity. This can be especially important in the case of weakly immunogenic tumors (Old et al., 1959, 1961; Math6 et al., 1969; Morton et al., 1970; . the precise mechanism of the mycobacterial Zbar et al., 1 9 7 0 ~ )Although adjuvant effect on tumor rejection is not clear, it is enhanced when the adjuvant is mixed with or injected into the tumor. It is known that macrophages can transport and actively transfer antigen and, thus, more efficiently activate lymphocytes (Unanue et al., 1969; Clarke et al., 1970), but it is also possible that mediators that act nonspecifically to inhibit tumor growth may be released. It has recently been shown that macrophages, some time after phagocytizing SRBC can sensitize normal guinea pigs for delayed-type hypersensitivity more effectively than antigen alone ( Pearson and RafTel, 1971) .

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E. CELLULAR RESISTANCE Macrophage obtained from hypersensitve animals, under appropriate conditions, show enhanced resistance in vivo and in vitro to infection with microorganisms. This phenomenon has been termed cellular resistance or cellular immunity (reviewed by Mackaness and Blanden, 1967). In the mouse, resistance to Listeria monocytogenes closely parallels development of delayed-type hypersensitivity and appears to be independent of thc humoral responsc (Mackaness, 1962). In fact, there is evidence in the pertussis system that when antibody arises, cellular immunity is depressed or regulated (Gray and Cheers, 1969). For this resistance to become clearly manifest, challenge of the sensitized animal with the specific microorganism is required. However, once challenged, the animal is not only resistant to the specific microorganism but also to unrelated organisms such as brucellae and niycobacteria ( Mackaness, 1964). The same holds true for mice sensitized to BCG (Blanden et al., 1969). Perhaps this is the best example of the second model, in which a specific cell-mediated reaction leads to a nonspecific effect. Although it is the macrophages that demonstrate resistance to infection, the immunological specificity for initiating this protective effect is carried by lymphoid cells and can be passively transferred to normal animals only with lymphocytes ( Mackaness, 1969). The specific sensitization apparently need not even be to a microorganism, since cellular immunity to Listeria was found in mice sensitized and challenged with BGG (Dodd, 1970). Even a graft-versus-host reaction in mice can stimulate antibacterial cellular resistance ( Blanden, 1969). Cellular resistance of immune macrophages to viruses, e.g., vaccinia, has also been reported (W. A. F. Tompkins et al., 1970b). In the passive transfer cxperiinent, specifically sensitized lymphocytes, when challenged with specific antigen, have the ability to activate and cause resistance in nonscnsitized macrophages, suggesting that one or more of the mediators affecting macrophages described earlier may be involved. Sensitized spleen lymphocytes challenged with viable H,,Rv mycobacteria in vitro released into the supernatant a soluble factor that conferred resistance to challenge with H,;Rv upon normal macrophages (Patterson and Youmans, 1970). F. UNIFIEDMODEL Each in vivo model has some clear validity. In the case of allograft and tumor rejection across major histocompatibility barriers, most of the evidence suggest a high degrce of specificity in target cell destruction.

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In the case of weaker antigens, bacterial antigens and, possibly, tumorspecific transplantation antigens in syngeneic hosts, there is some possibility that nonspecific cytotoxic mechanisms may also occur. A syncretic model might unify thinking about the effector mechanisms. The reactions involving specific destruction occur both in vivo and in vitro when the antigen is cell-membrane associated. Conversely, many nonspecific reactions occur when lymphocytes are stimulated by soluble antigens. One could argue teleologically that the natural evolutionary function of cellmediated immunity deals with foreign antigens which are cell-associated, whereas the humoral response can control most extracellular infections, toxins, and parasites. The actual process by which lymphocytes destroy target cells upon direct contact is not known. However, perhaps stimulation of lymphocytes by soluble antigens provides insight into mechanisms by which activated lymphocytes exert their effects directly on target cells, that is, by elaboration of the various cell-free factors or mediators. Perhaps sensitized lymphocytes recognize and bind to cell-membraneassociated antigens, become activated, and release or inject some or all of the mediators, not into the medium, but directly through the plasma membrane into the target cell. One could, thus, explain why the specificity is so exquisite, why only the specific target cells are killed, and why no substantial amount of the factors are released into the medium. By time-lapse cinemicrography (Ax et al., 1968) on lymphocyte-target cell cytotoxicity, occasionally a lymphocyte is seen to attach to the target cell for a few minutes, detach, and move away, but the target cell does not lyse for 5 to 10 minutes, suggesting that something was released by the lymphocyte into thc target cell which caused its subsequent demise. This mechanism might also apply to intracellular bacterial and viral infections. Some of the mediators, such ns cytophilic antibodies and the antigen-specific MIF, could also be involved in amplifying the reactions in a specific fashion. Nonspecific aspects of these reactions, including infiltration of macrophages, may serve not only to amplify the reaction produced by a few sensitized lymphocytes, but also as biological adjuvants to increase specific sensitization to new or weak antigens. VII. Relationships between Cell-Mediated Immunity and Antibody Formation

Traditionally antibody formation and delayed-type hypersensitivity have been regarded as two quite separate immune responses, related but differing in basic mechanisms. In this section, an attempt will be made to characterize the effector cell in cell-mediated immune reactions and to suggest relationships with one of the cell types critically involved in antibody formation.

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A. EFFECTOR CFLLSIN DELAYED-TYPE HYPERSENSITIVITY 1. Role of Thymus The thymus or thymus-dependent lymyphocytes are absolutely required for cell-mediated immune reactions (J. F. A. P. Miller et d., 1962; Peterson et al., 1966; J. F. A. P. Miller and Osoba, 1967; Meuwissen et ul., 1969). In the earliest experiments, thymectomized mice retained allogeneic and sometimes xenogeneic skin grafts for extended periods of time rather than rejecting them within the first week or two. Immunological reactivity could be reestablished by grafting of thymuses or injecting adult spleen or lymph node cells from syngeiieic donors (J. F. A. P. Miller, 1961; Martinez et al., 1962). Lymphocytes from thymectomized animals failed to initiate graft-versus-host reactions (J. F. A. P. Miller et al., 1962). Later it was found that thymectomized rats failed to develop delayed-type hypersensitivity to tuberculin or BSA but could show Arthus reactivity to BSA. Homograft rejection was also considerably delayed in these rats (Arnason et ul., 1964). In the chicken, cellmediated immunity and production of humoral antibodies were shown to be controlled by different anatomical organs. Removal of the bursa of Fabricius combined with irradiation resulted in complete failure of immunoglobulin and antibody production, but the birds were capable of rejecting allografts normally (Warner and Szenberg, 1964; M. D. Cooper et al., 1966) or developing graft-versus-host reactivity. Conversely, thymectomy followed by irradiation completely suppressed the development of delayed hypersensitivity to diphtheria toxoid but allowed production of both 19 S and 7 S immunoglobulins. In graft-versus-host reactions, lymphocytes from chromosomally marked thymuses were found to proliferate (Davies et al., 1966). As mentioned earlier, thymusderived lymphocytes acquired an antigen, presumably in the thymus, known as 0 (Reif and Allen, 1964) and alloantibodies against 0 could be used, with complement, to destroy selectively thymus-derived lymphocytes (Raff, 1969). Treatment of mouse lymphocytes with anti4 in the presence of complement almost totally abolished specific lymphocyte-target cell cytotoxicity in vitro (Cerottini, 1971; Cerottini et al., 1970b; Brunner et al., 1971). However, ability to produce antibodies to the specific transplantation antigens was unaffected. In reconstitution experiments in thymectomized rats, using F, thymus-derived lymphocytes, detected by fluorescent antibodies to transplantation antigens, R. M. Williams and Waksman (1969) were able to demonstrate clearly that the lymphocytes which infiltrated tuberculin reactions 8-16 hours after antigen challenge were thymus-derived. From a large body of evidence

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in addition to the few experiments cited, the conclusion is inescapable that the effector cell in cell-mediated immune reactions is thymusderived or thynius-dependent. With the use of specific reagents such as anti-B antibodies and chromosomal markers, the precise distribution and effect of the thyniic-dependent lymphocytes in these reactions should be clearly defined in the near future. 2. Efector Cells As Nonantibody-Producing Cells Many attempts have been made to transfer delayed-type hypersensitivity passively by means of serum or serum fractions, and until now no reproducible procedure has been established (Bloom and Chase, 1967). There is considerable indirect evidence indicating that the cell-mediated inimune response is dissociable and independent from antibody formation. In human thymic deficiency states, such as Di George’s and Nezelof syndromes, a humoral antibody response can exist in the absence of cell-mediated immunity. Conversely, in the Brutontype and acquired types of hypogammaglobulineniia, normal cell-mediated immune reactivity is commonly found in the absence or vast reduction of circulating antibodies (Peterson et al., 1966; Meuwissen et al., 1969). Similarly, in the bursectomized, irradiated chicken, which has little or no detectable immunoglobulin in the circulation or ability to form antibodies, rejection of skin allografts proceeds relatively normally (Perey et al., 1967). Interestingly, their lymphocytes do not transform with anti-immunoglobulin antibodies but do with PHA (Alm and Peterson, 1969). That the effector cell does not produce antibodies was shown by Silverstein et al. (1963, 1964) with skin homografts made in utero in the fetal lamb during various stages of the 150-day gestation period. Fetal lambs acquired the capacity to reject allografts in the second half of the gestation period, and rejection took place in the same time course as in adults. At the time of grafting and rejection, no IgG or IgA could be found, and only faint trace amounts of IgM were present. After rejection of the grafts, there was no detectable increase in any of the immunoglobulins. Fetuses were injected with rabbit antibodies to sheep-immune globulins to complex fetal antibodies that might have been synthesized. Again, grafts were rejected in normal fashion. The experiments in which cytotoxic sensitized lymphocytes were completely destroyed by anti-B antibodies, leaving the anti-H, antibodyproducing cells completely intact and functional ( Cerottini et al., 1970b; Brunner et al., 1971), indicated dissociation. Information on the specificity of the response and on thymus-derived cells in antibody formation, makes it very unlikely that the thymus-derived effector cell is significantly involved in secretion of antibodies. However, the possibility that these

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cells may synthesize small amounts of immunoglobulins or that their receptor may be an immunoglobulin is not ruled out. 3. Specificity of Antigen Recognition

Stemming from the work of Landsteiner (1945), specificity has been the most critical parameter for recognition of antigen by antibodies and cells. It is a widely held view that haptens are primarily small aromatic groups attached to large backbones, such as proteins, yet the principles that govern antigenic determinants behaving as haptens or carriers are not well understood. Before considering the specificity shown in cellmediated immune reactions, perhaps a review of two recent experiments would shed some light on the meaning of hapten specificity and carrier specificity. Weakly immunogenic red cell isoantigens in the chick are known as A, D, and L, and a strong isoantigen is known as B. When red cells are injected into recipients which have the same B locus but differ at the A locus, no antibodies are found to the A determinant (Schierman and McBride, 1967). However, when the recipient differs in both A and B loci, a response is made to B but also invariably to A. The A and B loci must be on the same cells; mixtures of cells containing different antigens do not lead to response to the A antigen. The interpretation was made that in order to see antigen A, antigen B had to be on the cell and the chick had to bc capable of responding to it. In the same experiments, if chicks were tolerant to B, when cells containing B and A antigens were injected, there was no “adjuvant” effect of B permitting response to A. The system was expanded to include other isoantigens, and a generalization was established. If cells containing both antigens were injected into a normal chicken which was either preimmunized to B or received passive antibodies to B, the bird was capable of recognizing the A determinant. If it had antibodies to the A determinant, then it was capable of recognizing the L determinant (McBride and Schierman, 1970). When cells containing isoantigens A and B are injected into normal chickens differing at these loci, their principal response seems invariably against the immunodominant B antigen and not to the A antigen. When animals are preimmunized to the B antigen or passively immunized with antibodies to the B antigen, the B determinant behaves as a carrier and permits antibodies to be developed against the A antigen, which behaves as a hapten. Similarly, when there is a response against the A antigen, it behaves as a carrier, permitting formation of antibodies to the L antigen, which is then treated as a haptenic determinant. Interestingly, in this system the antigens, i.e., chicken erythrocytes, are the same in all the experiments. Thus whether a

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determinant behaves as a carrier or as a hapten does not depend primarily upon chemistry but, rather, upon biological response. Conversely, could the DNP group, then, serve as a carrier for a protein determinant? Indeed, this has been the case in one system. Iverson (1970), in attempting to produce high-titered anti-idiotypic antibodies against a mouse myeloma protein, presensitized mice to dinitrofluorobenzene by skin painting. After sensitization, the mice were immunized with the idiotypic myeloma protein coupled with DNP. The procedure was quite successful in producing anti-idiotypic antibodies. In this case, one must conclude that the DNP group against which the animals were presensitized served as a carrier and enabled the mice to recognize the idiotypic determinant in the globulin which behaved as a hapten. And lastly, possibly the same chemical group could serve on one molecule as a hapten and on another as a carrier, depending upon the immunological status of the cells with which it interacts (Bretscher and Cohn, 1968, 1970). In spite of these complexities, there is a vast amount of evidence indicating that when individuals are sensitized to hapten-protein conjugates, the antibody response is primarily hapten-specific, whereas the cell-mediated response is primarily carrier-specific or conjugate specific. For example, guinea pigs sensitized with picrylated BGG showed delayed-type reactivity both to picryl-BGG and to the unconjugated BGG carrier ( Benacerraf and Gell, 1959). Arthus reactivity or anaphylaxis was found to occur after challenge with picryl-BGG or picryl-guinea pig y-globulin but not after BGG alone. Similar results were obtained using conjugated albumins (Salvin and Smith, 1960). Much of the specificity to carrier may be the result of sensitization to denatured carrier, with altered antigenic determinants, as in the case of heatdenatured albumins which, although unable to stimulate antibody formation, stimulated delayed hypersensitivity to both heat-denatured and native albumin (Gell and Benacerraf, 1961) , The less immunogenic the carrier, the greater degree of delayed sensitivity to the hapten or conjugate (Gell and Benacerraf, 1961; Chase, 1954). When the same haptenic group was coupled to different sites on the carrier, either by diazotization to aromatic groups or by acylation of amino groups, guinea pigs sensitized to either showed no cross-reactivity to challenge with the same determinant on the same carrier but coupled through a different linkage. Two possible explanations are that recognition either must include the linkage position between the hapten and carrier, or coupling to the same carrier through two different linkages denatures or alters the carrier in different ways so as to not be recognized as cross-reactive ( Gell and Silverstein, 1962a; Silverstein and Gell, 1962).

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To generalize, cell-mediated immunity is primarily carrier-specific, yet there are circumstances in which at least some degree of haptenspecificity can be induced. When the carrier is weakly immunogenic or in fact is a host component, some hapten specificity of cell-mediated immunity develops (Gel1 and Benacerraf, 1961; Chase, 1954; Bloom and Chase, 1967). In some cases, challenge with very high concentrations of haptens coupled to unrelated carriers may also demonstrate some hapten specificity (Phair and Kantor, 1970). There is one system in which delayed-type hypersensitivity is apparently entirely haptenspecific. This involves sensitization with the ABA-tyrosine conjugate. Delayed hypersensitivity can be detected either with ABA-hexatyrosine or simply arsonyl tyrosine itself ( Leskowitz, 1963; Leskowitz and Zak, 1966; Borek et al., 1965; reviewed by Borek, 1968). Antibodies are produced and are specific for the polytyrosine carrier (Borek, 1968; Borek et al., 1967). The uniqueness of the arsonate group is surprising-sulfanilate, iodophenylaniline, or benzoate conjugates have no activity. One wonders whether the azobenzoate residue serves not as hapten but as a chemically reactive group which conjugates to a host protein, perhaps serum protein. If this occurs, then the interpretation could be the classical one, namely, that the ABA-polytyrosine couples through the arsenate group to a host protein. The true hapten would be the polytyrosine determinant, and the carrier would be the unknown host protein to which it was coupled. Thus the cell-mediated immunity which requires the arsonate group might actually be carrier-specific for a conjugated host protein. The size of the determinant also appears to be important. For example, in the DNP-oligolysine system, only polymers of seven or more lysine residues are immunogenic (Schlossman et al., 1965). Although antibodies are perfectly capable of binding to DNP-oligolysines smaller than (lys),, delayed hypersensitivity reactions in vivo and in vitro are found only to DNP-heptalysine or larger oligonieres (J. R. David and Schlossman, 1968). Even smaller determinants, including a tripeptide and a pentapeptide from tobacco mosaic virus (TMV) have been reported to produce delayed hypersensitivity skin reactions and to inhibit macrophage migration, although they are not immunogenic ( Spitler et al., 1970). In these CRSCS, it is difficult to see what the carrier determinant could be, and one might conclude that they demonstrate a haptenic specificity. However, polylysines are strongly basic and might bind to serum proteins, especially albumin which is acidic, and the tobacco mosaic peptides used in a 1000-fold higher concentration than was required for the wholc TMV protein may have to bind to a host carrier. The possibility exists that these small determinants might, in

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part, behave as haptens by binding to a host carrier protein, to which the cell-mediated immune response is directed. Thus whether or not all cell-mediated immune reactions are carrier-specific and require immunogenic antigens remains to be determined, but the specificity of cellmediated reactions is generally distinct from the hapten specificity found among humoral antibodies, although there can be great overlap. 4 . Ejeector Cells As Nondividing Cells

Whether the effector cell in delayed-type hypersensitivity poliferates has not received a great deal of study, but a number of lines of evidence suggest that it may not d i ~ i d e .In~ histological studies on tuberculintype or contact-type hypersensitivity reactions, there is little evidence for proliferation of lymphocyes at reaction sites ( Lid& 1967; Turk, 1970). Many cell-mediated reactions in vivo reach their maximum in 24 to 36 hours, declining thereafter. If the kinetics of proliferation in vivo follow those of blast cell transformation studies in uitro, which indicate that DNA synthesis does not begin until approximately 48 hours and is maximal often 6-7 days after stimulation, then delayed-type reactions in sensitized individuals would be over before significant proliferation of sensitized lymphocytes began. Indications are that the delayed-type response is much more radiation-resistant than is the humoral antibody response (Salvin and Smith, 1959; Uhr and Scharff, 1960). For example, 400500 r were capable of abolishing an antibody response, but without effect on delayed hypersensitivity in sensitized animals. In addition, sensitized lymphocytes, treated with niitomycin C under conditions in which DNA was cross-linked to the extent that a detectable diminution in RNA synthesis was seen and, therefore, most likely unable to divide (cf. F. H. Bach and Voynow, 1966), were capable of passively transferring high levels of reactivity to picryl chloride and tuberculin (Bloom et al., 1964). Similarly, sensitized lymphocytes, X-irradiated in vitro, were found to be capable of effecting passive transfer of delayed hypersensitivity reactions in guinea pigs (Asherson and Loewi, 1967) and rats ( Feldman, 1968). Although development of cellular immunity to Listeria in mice could be blocked by the mitotic inhibitor, vinblastinc, once cellular immunity was established it was unaffectcd by the mitotic inhibitor (North, 1970). These in vivo results appear to resemble more the model of the ' In this context, nondividing refers to the concept that at the time that the lymphocyte carries out its affector function, it does not divide. However, all evidence indicates that efl'ector cclls d o arise by division from precursors, and there is n o evidence to exclude the possibility that they may divide at times subsequent to their serving as effector cells.

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virus plaque assay (Section VI), in which the kinetics of lymphocyte activation in vitro were linear and unaffected by inhibitors of mitosis, than the model of blast cell transformation, with exponential kinetics and apparent clonal proliferation (Section 11). The suggestion was made (Bloom and Jimenez, 1970) that the nonproliferating reaction most resembles the effector arm of cell-mediated immune reactions. The proliferating response in vitro would then correspond to the “memory function,” that is, the generation of more effector cells from a small number of preexistent stem cells. This conclusion is supported by data from patients and chronic mucocutaneous candidiasis and other immunological deficiency states whose lymphocytes recognize antigen and proliferate but fail to produce MIF and delayed-type skin reaction (Rocklin et al., 1971) . In addition, cytotoxic lymphocytes are apparently not stimulated with PHA ( MacLennan and Harding, 1970a). The effector process would occur in various tissues and target sites in the body, whereas the generation of memory cells would primarily occur in the lymph nodes and spleen. Teleologically, it might be deleterious for all the mediators to be released in the nodes after antigenic stimulation. Clearly a proliferative phase is required during the process of sensitization, because X-irradiation and antimitotic drugs can suppress the process of sensitization. Although it is possible that the thymus-derived effector cell and the cell that proliferates presumably to generate more memory cells are separate types, it is also possible that one cell type might have either function depending upon the environment where it encounters antigen.

B. CELLSINVOLVED IN ANTIBODY FORMATION Many immunologists, struck by similarities between the specificity and diversity of the cell-mediated and circulating antibody responses, have attempted to bridge the gap and establish connections. Perhaps the first to suggest a relationship was Dienes (1936), based on the histology of lesions in animals immunized against bacterial infections, especially the mycobacteria, the time course of delayed hypersensitivity and antibody formation and differences between active and passive immunization. In studies on the kinetics of immune responses, Salvin and Smith (1960) observed that when small doses of antigen were used, delayed-type hypersensitivity developed first followed by antibody production and Arthus reactivity. In fact, when minute doses of antigen (0.5 pg. protein) were given as a primary stimulus, often delayed hypersensitivity alone developed; this, nevertheless, served as a primary stimulus for a secondary antibody response when a larger antigenic challenge followed. On the basis of this sequential relationship, it was suggested that delayed hypersensitivity was a necessary and preliminary

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stage in antibody formation (Salvin and Smith, 1959, 1960; Pappenheimer et al., 1959). An ingenious model for delayed hypersensitivity, postulating not a distinct immune reaction but mediation by a high affinity antibody, was put forward by Karush and Eisen (1962). HOWever, ( a ) failure to effect passive transfer of delayed hypersensitivity even with copious volumes of sera, ( b ) the development of homograft immunity in the fetal lamb before any detectable antibodies or immunoglobulins could be found, ( c ) failure to depress the cell-mediated immune response by injection of large doses of antigen intravenously, which would be expected to bind preferentially to high affinity antibodies (Silverstein and Borek, 1966), and ( d ) different specificities of the two reactions to the same hapten-protein conjugate have militated against this hypothesis. As more information on the mechanism of antibody production became available clearly some antibody systems required cooperation between two types of cells-a thymus-derived and a bone marrow-derived lymphocyte. Roitt et al. ( 1969), Gel1 ( 1970), and Richter ( 1970) have suggested that the thynius-derived cell might function in both systems.

1. Role of Thymus Although it is not clear in many of the commonly studied in vitro systems in antibody formation whether a primary or secondary immune response is occurring, discrimination between these two alternatives is not crucial for the present argument. It may be germane to recall earlier studies in which a genetic study was made of natural antibody formation to sheep and chicken erythrocytes in common inbred stains of mice (Stern and Davidsohn, 1954; Davidsohn and Stern, 1950). Essentially, some members of all strains examined produced natural hemolysins and agglutinins, but the percentage producing such natural antibodies, and the titers, varied according to strain. This would suggest that many of these studies probably measure secondary-type responses. Three cell types seem required for initiation of an antibody response to sheep erythrocytes and several other antigens in mice-a bone marrow-derived cell ( B cell), a thymus-dependent cell ( T cell), and a glass-adherent cell, possibly a macrophage (J. F. A. P. Miller and Mitchell, 1969; Davies, 1969; Clainan and Chaperon, 1969; Taylor, 1969; Talmage et al., 1970).5 The evidence in vivo is derived primarily from repopulation experiments, in which irradiated, thymectomized animals Until the origin of the T cells is more definitely established, it is preferable to refer to them as thynius-dependent rather than derived cells.

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had to be given both thymus and bone marrow cells in order to produce heinolysins to SRBC (Claman et al., 1966a,b; J. F. A. P. Miller and Mitchell, 1968; Mitchell and Miller, 1968). From kinetic studies of antibody induction, a three-cell interaction was predicted ( Mosier and Coppleson, 1968). Histocompatibility markers or chromosomal markers clearly demonstrated that the cell which produced the antibody was bone marrow-derived ( Alitchc~lland Miller, 1968; Nossal et al., 1968). Obviously, the synergism seen in the mouse to immunization with SRBC and certain other antigens inay not necessarily apply to formation of all classes of antibodies to all antigens. In the rat, for example, evidence suggested that the bone marrow-derived cell was entirely capable of being stimulated by SRBC directly although a few T cells in the bone marrow population could have bcen present ( Haskill, 1969). Any possible relationships bctwecn the cffcctor cell in cell-mediated ininiunity and the forination of antibodies must involve the T cell. The third cell, which is glass-adherent and possibly a macropage, may, in fact, not be required, since the supernatant of these cells can support initiation of an antibody response in vitro by nonadherent cells alone (Dutton et al., 1970). Thyniectomized mice or irradiated mice were found to be poor producers or unable to produce antibody-forming cells against SRBC. When they received normal thoracic duct or thymus cells, they became inimunologically competent and producccl antibody-forming cells (J. F. A. P. Miller and Mitchell, 1968; Claman et al., 1966a,b). Thymus cells from mice rendered immunologically tolerant to an antigen, i.e., BSA, were incapablc of cooperating in thc induction of immunization ( Taylor, 1968; Davies, 1969; Doenhoff et al., 1970; Chiller et al., 1970). Finally, cytotoxic antisera to B antigen markedly diminished the capacity of mouse spleen cells to generate direct and indirect plaque-forming cells to SRBC in vitro, but did not affcct plaque-forming cells or their precursors (Chan et al., 1970; Schinipl and TVecker, 1970). 2. Helper Cells As IVo~iut~til~otl~~-P,.otlrtcing Cells Tracing thymus cells and bone marrow cells after transfer to irradiated and thymectomized hosts by means of chromosomal markers or surface antigens has deinonstratcd that thymus-derived cells do not produce antibody and that bone marrow-derived cells do. The markers have included the T6T6 chromosoinnl markers ( Davies et a/., 1967; Nossal et al., 1968), transplantation antigens, ( Mitchell and Miller, 1968), and 6 antigens (Takahashi et al., 1970). Yet the thymic cell has specificity, and if rendered tolerant, fails to permit bone marrow cells to become antibody-forming (Taylor, 1968; Chiller et al., 1970).

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3. Specificity of Antigen Recognition

Studies on the specificity of the inimediate-type and delayed-type hypersensitivity responses to hapten-protein conjugates in animals have indicated, in general, that the Arthus reaction was hapten-specific, whereas thc delaycd-type sensitivity was conjugatc- or carrier-specific (Benacerraf and Gell, 1959; Salviii and Smith, 1960; Gell and Benacerraf, 1961). But from the work of Landsteiner (1945) and these same studies, a variable degree of carrier specificity or conjugate specificity was noted even in primary responses. However, secondary antibody responses showed a considerably greater degree of carrier specificity (Ovary and Benacerraf, 1963; Paul et d.,1967). In terms of cell cooperation, cnhaiiced or secondary antibody responses to the NIP-hapten were obtained when spleen cells of animals prcimmunized to the carricr albumin were transferred to irradiated syngeneic mice before challengc with the conjugate (Mitchison, 1969). These experiments, and observations that animals tolerant to the BSA carrier failed to show carrier function when given NIP-BSA (Taylor, 1969), suggested that the carrier cell might be a thymus-derived cell. In studies on the antibody response to isozynies of lactic dehydrogenase ( L D H ) , which consist of four subunits of two different types, a carrier effect was seen when the animals were challenged with LDH-3 containing both A and B subunits (Rajewsky et al., 1969). From these experiments, it was postulated that at least two different antigenic determinants wore rcquired for immunogenicity. Similarly, enhanced responses to the DNP group were obtained in animals primed with DNPovalbuniin, immunized to BGG 2 weeks later, and then challenged with DNP-BGG (Katz et al., 1970a,b). Attempted substitution of passive antibody for sensitization to the future carrier, BGG, was unsuccessful, suggesting that cellular cooperation was required, one cell being primed to the carrier. This was demonstrated by transferring lymph node cells, sensitized to the carrier, into syngeneic guinea pigs previously sensitized with DNP-ovalbumin. Upon challenge with DNP-BGG, an enhanced antihapten response was observed (Paul et al., 1970). By analogy with cooperation between thymus and bone marrow cells, since the antibody-producing cell is hapten-specific, thymus-derived cells were deduced to be carrier-specific. The recent development of an in vitro system for ininiunization to a hapten, permitted this to be tested directly (Kettman and Dutton, 1970). A primary response to the trinitrophenyl (TNP) hapten was induced by culturing spleen cells with TNP erythrocytes. When T cells were primed to the carrier, sheep erythocytes alone enhanced plaque-forming response to TNP. It remained

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only to show that the carrier-specific cells could be destroyed by e alloantibodies ( Dutton, 1971a,b ) . That antigen-binding cells, in particular rosette-forming cells with sheep erythrocytes, are thymus-derived (J. F. Bach et al., 1970), is most clearly shown by the ability of anti-6’ antibodies to suppress them (Greaves and Moller, 1970). These antigenbinding cells derived from thymus also appear to carry immunological memory (Paul et al., 1970; J. F. Bach et al., 1970; Greaves et al., 1970). The experiments suggest that many of the rosette-forming cells and antigen-binding cells are thymus-derived and serve as helper cells in the cooperative initiation of the antibody response.

4 . Helper Cells As Nondividing Cells As described above, the antibody response in vivo is known to be much more radiation-sensitive than is the cell-mediated immune response (Salvin and Smith, 1959; Uhr and Scharff, 1960). There is substantial evidence that the bone marrow-derived cell must undergo divisions, possibly as many as ten, before it matures into the antibody-forming cell ( Makinodan and Albright, 1967; Dutton and Mishell, 1967). Thymusderived, helper cells and presumably antigen-binding cells also appear capable of proliferation upon antigenic stimulation (Davies et al., 1966; Biozzi et al., 1968; Greaves et al., 1970). Yet a radiation-resistant cell is required for induction of antibody formation in vitro (Roseman, 1969; Goldie and Osoba, 1970; Osoba, 1970). The specificity of this cell has been established in a cell transfer system (Katz et al., 1970b). Lymph node cells from inbred guinea pigs immunized against BGG were transferred into syngeneic recipients which had been previously immunized with DNP-ovalbumin so that they would provide the carrier specificity required for enhanced anti-DNP response upon challenge with DNP-BGG. Irradiation of these cells with up to 5000 r completely abolished their ability to undergo blast cells transformation in vitro upon challenge with BGG but did not affect their capacity to transfer immunological memory. These results are consistent with the view that proliferation of both bone marrow and thymus-derived cells may occur during the process of immunization, but once sufficient memory cells have been generated, the “helper” function does not require division and is radiation-resistant.

5 . Mechanisms By pursuing the idea that the thymus-derived lymphocyte responsible for cell-mediated immunity and the helper cell in antibody formation may be the same, we find that at the present state of knowledge these cells cannot be distinguished. A model in which the same T cell functions

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in both immunoresponses is very attractive but biologically simplistic in implying only one type of T cell. The heterogeneity of lymphocyte populations (Gowans and McGregor, 1965) is such that there may be a number of classes of T cells which may have different functions, or some may engage in both functions. However, the concept does provide a framework for understanding ( I ) how it is possible to have separate memory and antibody specificity; (2) how it is possible that unresponsiveness can be induced separately to either specificity (Chiller et al., 1970; Bore1 and David, 1970); ( 3 ) why immunization with small doses of antigen initiates cell-mediated immunity ( Salvin and Smith, 1960) and increases rosette-forming cells (Greaves et al., 1970; Greaves and Moller, 1970), establishes immunological memory, but does not necessarily increase antibody-forming cells; and ( 4 ) why immunological memory and delayed-type hypersensitivity are radiation-resistant, whereas antibody formation is radiation-sensitive. If antigen-binding cells in antibody formation are thymus-derived and cells measured in the virus plaque assay or in mixed lymphocyte reaction are thymus-derived and equivalent, one has to explain how it is possible that they occur in the immunized animal in approximately 1%of the lymphoid populations studied. There is evidence that the bone marrow-derived cell in antibody formation can be made tolerant (Chiller et al., 1970), has hapten-specific receptors ( Wigzell and Anderson, 1969), and can be primed by immunization (Kennedy et al., 1970; H. C. Miller and Cudkowicz, 1970; Cudkowicz, 1971). If these cells, as the result of immunization, could be primed to the level of 195, and if cooperation with the 1%T cells is required for antibody production, then the probability of a successful interaction, assuming random interaction, should be the product of the separate probabilities, or le4. This is precisely the order of magnitude of antibody-forming cells observed in the Jerne plaque assay. Alternatively, if the B cell were limiting, in a concentration of then an excess of T cells would be needed to insure a high response. However, inside lymph nodes or spleens, interactions are undoubtedly anatomically restricted, so the correct prediction of the probability of an antibody-forming cell on the basis of 1%of T cells presumably being sensitized may well be fortuitous. At least four mechanisms to explain the role of the helper cell in antibody formation can be elaborated. a. The helper cell, by means of its carrier-specific receptors, serves to concentrate antigen and to present it in high density to the bone marrow-derived antibody precursor cell, a view presented by Mitchison ( 1969). b. The carrier-specific cell secretes a special class of anticarrier anti-

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body which must interact with antigen in order for the antigen to be recognized and to stimulate the bone marrow-derived cell. This view, in more sophisticated form, was taken by Bretscher and Cohn (1968, 1970), and is consistent with the results of McBride and Schierman (1970) and with observations that a soluble factor, possibly antibodies, can be eluted from peritoneal cells (Kennedy et al., 1970) or fractions of spleen cells (Dutton et al., 1970) which might perform the carrier function. It is inconsistent with experiments in vivo in which carrier antibodies were unable to cause an enhanced antihapten response (Katz et al., 1970a,b). c. The helper cell processes antigen, which then becomes able to stimulate the haptenspecific B cell or to induce the formation of an informational molecule which is transferred to the bone marrow-derived cell, permitting it to form antibodies (Fishman and Adler, 1963; Lawrence, 1969). The difficulty with this interpretation is to conceive of a cell, which generates its own carrier-specific receptor and yet transfers an informational molecule to the B cell which is of a different specificity than its own receptor. d. Another possibility is suggested by the production of soluble inediators by delayed hypersensitive lymphocytes upon contact with specific antigen. It would hold that bone-marrow derived lymphocytes, even when they have bound hapten-specific determinants of antigen to their receptors, are unable to proliferate and differentiate into antibody-forming cells. T cells reacting with carrier determinants on the antigen then would release the mediators described previously, one of which would be able to activate those B cells that have bound hapten-specific determinants. This model is schematically represented in Fig. 8. In support of this possibility is the interesting observation of Katz et al. (1971) that the transfer of allogeneic lymphoid cells into guinea pigs primed to DNP-ovalbumin stimulated the synthesis of anti-DNP antibodies in the absence of antigenic challenge and markedly heightened a secondary anti-DNP response when the recipients were challenged with DNP-BGG. The nonspecific stiniulatory effect seemed to be attributable to a graft-versus-host reaction of the allogeneic lymphocytes against the primed recipient. Against this notion stands the finding that when hapten and carrier are on different molecules rather than on a single molecule, apparcntly no stimulation of antihapten antibody production from carrier-primed spleens results in vivo or in vitro ( Dutton, 1971c; Paul, 1971). This hypothetical activating factor could well be one of the known mediators, such as MIF, blastogenic factor, or a new substance. A simple blastogenie activity on all B cells would be unlikely, otherwise antibody-forming cells to all antigens would in-

ANTIBODY FORMATION DELAYED HYPERSENSITIVITY

5cc m

Anti

- hapton

"Memory"Ce1Is

FIG. 8. Schematic representation of possible relationships between cell-mediated immunity and antibody formation. MIF-migration inhibitory factor; CLF--chemotactic factor; LT-lymphotoxin; ClIF-cloning inhibitory factor; PIF-proliferation inhibitory factor; SRF-skin-reactive factor. ( From Bloom, 1971b. )

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crease after any single antigenic challenge. The mediator may resemble the potentiating factor of Alter and Bach (1970) or the soluble factors described by Dutton et al. (1970) and Kennedy et al. (1970) in the SRBC system,factors which can replace one of the cells involved in the in uitro antibody response. The true nature of the interaction between thymus and bone marrow cells in the antibody response is of course unknown. Through the use of in uitro systems, cell fractionation and analysis of antigenic markers on different cell populations, the detailed mechanisms should be accessible. Clearly, the relationships discussed here, even if possible, have been treated in a most simplistic way. In particular, the known heterogeneity of lymphocytes suggests an almost inevitable existence of subpopulations of T and B cells, perhaps distinguishable on the basis of their origin, environment, and life history. Nevertheless, the unified model introduces two simplifications in thinking about the problem. First, only one set of information for diversity and variability need be carried for both types of immune responses, and, second, information derived from the study of either response should be used cooperatively and synergistically to attain a complete understanding of the immune response. REFERENCES Able, M. E., Lee, J. C., and Rosenau, W. (1970). Amer. J . Pathol. 60, 421. Achard, C., and BCrnard, H. (1909). C. R . SOC. Biol. 67, 502. Adams, D. O., Biesecker, J. L., and Koss, L. G. (1970). Fed. Proc., Fed. Amer. SOC. Exp. Biol. 29, 359 (abstr. ), Adler, W. H., Takiguchi, T., Marsh, B., and Smith, R. T. (1970a). J . Exp. Med. 131, 1049. Adler, W. H., Peary, D., and Smith, R. T. (1970b). Cell. Immunol. I, 78. Al-Askari, S., David, J., Lawrence, H. S., and Thomas, L. ( 1965). Nature (London) 205, 916. Allerhand, J., and Zitrin, C. M. (1962). 1. Immunol. 89, 252. Alm, G. V., and Peterson, R. D. A. (1969). J . Exp. Med. 129, 1247. Alter, B. J., and Bach, F. H. (1970). Cell. Immunol. 1, 207. Amos, H. E., and Lachmann, P. (1970). Immunology 18, 269. Amos, H. E., Gurner, B. W., Olds, R. S. J., and Coombs, R. R. A. (1967). Int. Arch. Allergy Appl. Immunol. 32, 496. Arnason, B. O., Jankovic, B. D., and Waksman, B. H. (1964). In “The Thymus in Immunology” ( R . A. Good and A. E. Gabrielson, eds.), p. 148. Harper (Hoeber), New York. Asherson, G. L., and Loewi, G. (1967). Immunology 13, 509. Ax, W., Malchow, H., Zeiss, I., and Fischer, H. (1968). Exp. Cell Res. 53, 108. Bach, F. H., and Voynow, M. K. (1966). Science 153, 545. Bach, F. H., Bock, H., Graupner, K., Day, E., and Klostermann, H. (1969). Proc. Nat. Acad. Sci. U.S . 62, 377.

194

BARRY R. BLOOM

Bach, F. H., Alter, B. J., Solliday, S., Zoschke, D. C., and Janis, M. (1970). Cell Immunol. 1, 219. Bach, J. F., Muller, J, Y., and Dardenne, M. (1970). Nature (London) 227, 1251. Balner, H. (1970). Rev. Eur. Etud. Clin. Biol. 15, 599. Barnet, K., Pekarek, J., and Johanovsky, J. (1968). Experentia 24, 948. Bartfeld, H., and Atoynatan, T. (1969). Proc. Soc. Exp. Biol. Med. 130, 497. Bartfeld, H., and Atoynatan, T. (1970). Int. Arch. Allergy Appl. Immunol. 38, 549. Bartfeld, H., and Atoynatan, T. (1971). Nature (London) ( i n press). Bartfeld, H., and Kelly, R. (1968). I . Immunol. 100, 1000. Bartfeld, H., Atoynatan, T., and Kelly, R. (1969). Transplantation 7, 242. Baumal, R., Bloom, B., and Scharff, M. D. (1971). Nature (London) 230, 20. Benacerraf, B., and Gell, P. G. H. (1959). Immunology 2, 53. Bendixen, G., and S$borg, M. (1969). Dan. Med. Bull. 15, 1. Bendixen, G., and SZborg, M. (1970). 1. Immunol. 104, 1551. Benezra, D., Gery, I., and Davies, A. M. (1970). C h . E x p . Immunol. 5, 155. Bennett, B. (1965). J . Immunol. 95, 656. Bennett, B., and Bloom, B. R. (1967). Transplantation 5, 996. Bennett, B., and Bloom, B. R. (1968). Proc. Nut. Acad. Sci. U. S. 59, 756. Bennett, B., Old, L. J., and Boyse, E. A. (1963). Nature (London) 188, 10. Berken, A,, and Benacerraf, B. (1966). J . Exp. Med. 123, 119. Bianco, C., Patrick, R., and Nussenzweig, V. (1970). I. Exp. Med. 132, 702. Billingham, R. E., and Silvers, W. K. (1963). Ann. N . Y. Acad. Sci. 100, 348. Billingham, R. E., and Silvers, W. K. (1970). 1. Exp. Med. 131, 161. Biozzi, G., Stiffel, C., Mouton, D., Bouthillier, Y., and Decreusefond, D. (1968). Immunology 14, 7. Blanden, R. V. ( 1969). Transplantation 7, 484. Blanden, R. V., Lefford, M. J., and Mackaness, G . B. (1969). J . E x p . Med. 129, 1079. Blazkovec, A. A,, Sorkin, E., and Turk, J. L. (1965). Int. Arch. Allergy Appl. Immunol. 28, 178. Bloch-Shtacher, N., Hirschhorn, K., and Uhr, J. W. (1968). Clin. Exp. Immunol. 3, 889. Bloom, B. R. (1964). Unpublished observation. Bloom, B. R. (1970). In “Mediators of Cellular Immunity” (H. S . Lawrence and M. Landy, eds. ), p. 249. Academic Press, New York. . Bloom, B. R. ( 1971a). In “Immunological Surveillance” (R. T. Smith and M. Landy, eds.), p. 231. Academic Press, New York. Bloom, B. R. (1971b). In “In Vitro Methods in Cell-Mediated Immunity” (B. R. Bloom and P. Glade, eds.), p. 353. Academic Press, New York. Bloom, B. R., and Bennett, B. (1966). Science 153, 80. Bloom, B. R., and Bennett, B. (1968). Fed. Proc., Fed. Amer. SOC. E x p . B i d . 27, 13. Bloom, B. R., and Bennett, B. ( 1969). In “Cellular Recognition” ( R . T. Smith and R. A. Good, eds.), p. 229. Appleton, New York. Bloom, B. R., and Bennett, B. (1970a). Sen. Hematol. 7, 215. Bloom, B. R., and Bennett, B. (1970b). Ann. N. Y. Acad. Sci. 169, 258. Bloom, B. R., and Chase, M. W. (1967). Progr. Allergy 10, 151. Bloom, B. R., and David, J. R. (1964). Unpublished observations. Bloom, B. R., and Glade, P., eds. (1971). “In Vitro Methods in Cell-Mediated Immunity.” Academic Press, New York. Bloom, B. R., and Jimenez, L. (1970). Amer. J . Pathol. 60, 125. Bloom, B. R., and Sorkin, E. ( 1967). Unpublished observation. Bloom, B. R., Hamilton, L. D., and Chase, M. W. (1964). Nature (London) 201,

MECHANIShZ OF CELL-MEDIATED IhZhZUNE REACTIONS

195

689-691. Bloom, B. R., Bennett, B., and Dattner, A. W. (1967). Unpublished observations. Bloom, B. R., Bennett, B., Oettgen, H. F., hlclean, E. P., and Old, L. J. (1969). Proc. Nut. Acad. Sci. U . S. 64, 1176. Bloom, B. R., Jimenez, L., and hiarcus, P. I. (1970). J. E x p . Med. 132, 16. Borecky, L., and Lackovic, V. (1968). I n “The Interferons” (G. Rita, ed.), p. 9. Academic Press, New York. Borek, F. (1968). Cirrr. Top. Alicrobiol. Itninunol. 43, 126. Borek, F., Stupp, Y., and Sela, hl. (1965). Science 150, 1177. Borek, F., Stupp, Y., and Sela, hl. (1967). J. Immunol. 98, 739. Borel, Y., and David, J. R. (1970). J. E x p . Aied. 131, 603. Bosnian, C., and Feldman, J. D. (1970). Amer. J. Patliol. 58, 201. Boughton, B., and Spector, W. G. (1963). J. Pathol. Bacterial 85, 371. Boyden, S. V. (1964). Immunology 7, 474. Boyden, S. V., and Sorkin, E. (1960). Immunology 3, 272. Bretscher, P. A,, and Colin, hl. (1968). Naturc ( LoticZon) 220, 444. Bretscher, P. A., and Colin, hl. (1970). Science 169, 1042. Brittinger, G., Hirschhorn, R., D O L I ~ ~S.~ ID., S , and Weissman, G. (1968). J. Cell Biol. 37, 394. Brondz, B. D. (1964). Folia B i d . (Prague) 10, 164. Brondz, B. D. (1968). Folia B i d . (Prague) 14, 115. Brondz, B. D., and Sidorova, E. V. (1969). Biokhimiya 34, 1168. Brostoff, J., and Roitt, I. hl. (1969). Lancet 2, 1269. Brostoff, J., Greaves, hl. F., and Roitt, I. h i . (19G9). Lancet 1, 803. Brunner, K. T., Slauel, J., and Schindler, R. ( 1967). Nattrrc (London) 213, 1246. Brunner, K. T., hlauel, J., Cerottini, J. C., Rudolf, H., and Chapuis, B. (1968a). It,it,ictno/,athol. I n t . S y n i p . , Sth, 1965 p. 342. Brunner, K. T., hlauel, J., Cerottini, J. C., and Chapuis, B. (1968b). Immunology 14, 181. Brunner, K. T., hlauel, J., Rudolf, H., and Chapuis, B. (1969). In “Cellular Recognition” ( R . T. Smith and R. A. Good, eds.). p. 243. Appleton, New York. Brunner, K. T., hlauel, J., Rudolf, H., and Chapuis, B. (1970). Immunology 18, 501. Brunner, K . T., Nordin, A. A., and Cerottini, J. C. (1971). Int. Conooc. Immunol. 2nd, 1970 (in press). Bubenik, J., Perlniann, P., and Hasek, hl. (1970a). Transplantation 10, 290. Bubenik, J., Perlmann, P., Helmstein, K., and hloberger, G. (1970b). Int. J. Cancer 5, 310. Buckler, C. E., and Baron, S. (1966). J. Bacterial 81, 231. Burnet, F. hl. (1959). “The Clonal Selection Theory of Acquired Immunity.” Cambridge Univ. Press, London and New York. Burnet, F. M. (1967). Lancet 1, 1171. Byrt, P., and Ada, G. L. (1969). Immunology 17, 503. Canty, T. C., and Wunderlich, J. R. (1970). 1. Nut. Cancer Inst. 45, 761. Caron, G. A. (1967a). Int. Arch. Allergy Appl. Immunol. 31, 521. Caron, G. A. (1967b). Int. Arch. Allergy Appl. Inimonol. 32, 98. Carpenter, R. R. (1963). J. Inimunol. 91, 803-818. Cergttini, J. C. (1971). I n “ I n Vitro hlethods in Cell-Mediated Immunity” (B. R. Bloom and P. Glade, eds.), p. 47. Academic Press, New York. Cerottini, J. C., Nordin, A. A., and Brunner, K. T. (1970a). Nature (London) 227, 72.

196

BARRY R. BLOOM

Cerottini, J. C., Nordin, A. A., and Brunner, K. T. (1970b). Nature (London) 228, 1308. Chan, E. L., Mishell, R. I., and Mitchell, G. F. (1970). Science 170, 1215. Chaparas, S. D., Thor, D. E., Godfrey, H. P., Baer, H., and Hedrick, S. R. (1970). Science 170, 637. Chase, M. W. (1945). Proc. SOC.E x p . B i d . Med. 59, 134. Chase, M. W. (1954). Int. Arch. Allergy Appl. Immunol. 5, 163. Chase, hl. W. (1970). Fed. Proc., Fed. Amer. SOC. E x p . B i d . 29, 701 (abstr.). Chernyakhovskaya, I. Y.,Slavina, E. G., and Svet-Moldovsky, G . J. (1970). Nature (London) 228, 70. Chilgren, R. A., Quie, P. G., Meuwissen, H. J., and Hong, R. (1967). Lancet 2, 688. Chiller, J. M., Habichi, G. S., and Weigle, 'IV. 0. (1970). Proc. Nut. Acad. Sci. U. S . 65, 551. Claman, H. N., and Chaperon, E. A. (1969). Transplant. Reu. 1, 92. Claman, H. N., Chaperon, E. A., and Triplett, R. F. (1966a). Proc. SOC. Exp. Biol. Med. 122, 1167. Claman, H. N., Chaperon, E. A., and Triplett, R. F. (1966b). 1. Immunol. 97, 828. Clarke, J. A., Salsbury, A. J., and Willoughby, D. A. (1970). Nature (London) 227, 69. Clarke, S. L., Jr., and Periman, P. 0. (1962). 2nd Annu. Meet. Amer. SOC.Cell Bio2. Abstr., p. 35. Clausen, J. E. (1970). Acfa Med. Scand. 188, 59. Cline, M. J., and Swett, V. C. (1968). J. E x p . Med. 128, 1309. Cohen, J. R., and Feldman, hl. (1971). Cell. Immuno~.1, 521. Colien, S., McCluskey, R. T., and Benacerraf, B. (1967). I. Immunol. 98, 269. Colin, Z. A. (1968). Aduan. Immunol. 9, 164. Collins, F. M., Volkman, A., and McGregor, D. D. (1970). Immunology 19, 501. Cooper, H. L. (1969). J. Biol. Chem. 244, 1946. Cooper, M. D., Peterson, R. D. A., South, M. A,, and Good, R. A. (1966). J. Exp. Med. 123, 75. Coppleson, L. W., and Michie, D. (1966). Proc. Roy. SOC., Ser. B 163, 555. Coulson, A. S., and Clialmers, D. G. (1967). Immunology 12, 417. Coulson, A. S., Turk, A., Glade, P. R., and Chessin, L. N. (1968). Lancet 1, 89. Cudkowicz, G. (1971). Int. Conuoc. Immunol., Znd, 1970 (in press), Dannenberg, A. M., Jr. (1968). Bacteriol Rev. 32, 85. David, J. R. (1965). 1. E x p . Med. 122, 1125. Dir did, J. R. (1966). Proc. Nut. Acad. Sci. U . S. 56, 72. D wid, J. R. ( 1968). Fed. Proc., Fed. Amer. SOC. E x p . Biol. 27, 6. David, J. R., and Schlossnian, S. F. (1968). J. E x p . Med. 128, 1451. David, J. R., Al-Askari, S., Lawrence, H. S., and Thomas, L. (1964a). J. Immunol. 93, 264. David, J. R., Lawrence, H. S., and Thomas, L. (1964b). J. Immunol. 93, 274-78. . Immunol. 93, 279. David, J. R., Lawrence, H. S., and Thomas, L. ( 1 9 6 4 ~ )J. David, J. R., Lawrence, €I. S., and Thomas, L. (1964d). J. Exp. Med. 120, 1189. Davidsohn, I., and Stern, K. (1950). Cancer Res. 10, 571. Davies, A. J. S. (1969). Trunsplant. Reu. 1, 43. Davies, A. J. S., Leuchars, E., Wallis, V., and Kaller, P. C. (1966). Transplantation 4, 438. Davies, A. J. S., Leucliars, E., Wallis, V., Marchant, R., and Elliot, E. V. (1967). Transplantation 5, 222.

MECHANISM OF CELL-MEDIATED IMMUNE REACTIONS

197

Davies, A. J . S., Festenstein, H., Leuchars, E., Wallis, V. J., and Doenhoff, M. J. (1968). Lancet 1, 183. Denham, S., Grant, C. K., Hall, J. G., and Alexander, P. (1970). Transplantation 9, 366. Desniyter, J., Rawls, W. E., and Melnick, J. L. (1968). Proc. Nut. Acod. Sci. U . S. 59, 69. Dienes, L. (1936). Arch. Pathd. 21, 357. Dienes, L., and Mallory, T. B. (1932). Amer. J. Pathol. 8, 689. Diengdoh, J. V., and Turk, J. L. (1968). Int. Arch. Allergy Appl. Immunol. 34, 297. Dodd, R. Y. (1970). Infec. Immunity 1, 511. Doenhoff, M. J., Davies, A. J. S., Leuchars, E., and Wallis, V. (1970). Proc. Roy. Soc., Ser. B 176, 69-85. Dumonde, D. C. (1967). Brit. Afed. Bull. 23, 9. Dunionde, D. C., Howson, W. T., and Wolstencroft, R. A. (1968). Immunopathol., lnt. Symp., 5th, I967 p. 263. Dumonde, D. C., Wolstencroft, R. A., Panayi, G. S., Matthew, M., Morely, J., and Howson, W. T. (1969). Nature (London) 224, 38. Dupuy, J. M., and Good, R. A. (1970). J. Immunol. 105, 1111. Dupuy, J. M., Perey, D. Y. E., and Good, R. A. (1969). Lancet 1, 551. Dupuy, J. hl., Kalpaktosogloa, P., and Good, R. A. (1970a). J . Immunol. 104, 1384. Dupuy, J. M., Perey, D. Y. E., and Good, R. A. (1970b). 1. Immunol. 104, 1523. Dutton, R. W. (1966). Bncteriol. Reu. 30, 397. Dutton, R. W. (1967). Aduan. Immunol. 6, 253. Dutton, R. W. (1971a). Int. Conooc. Immunol, 2nd, 1970 (in press). Dutton, R. W. (1971b). Proc. Nut. Acad. Sci. U . S. (in press). Dutton, R. W. ( 1 9 7 1 ~ )Personal . communication. Dutton, R. W., and Bulman, H. N. (1966). Immunology 7, 54. Dutton, R. W., and Mishell, R. I. (1967). J . Exp. Med. 126, 443. Dutton, R. W., and Pearce, J. D. (1962). Nature (London) 194, 93-94. Dutton, R. W., McCartliy, M. M., Mishell, R. I., and Raidt, D. J. (1970). Cell. lnimunol. 1, 196. Dwyer, J. M., and Mackay, I. R. (1970). Lancet 1, 164. Edelman, R., and Wlieelock, E. F. (1966). Science 154, 1053. Elkins, W. L., and Guttman, R. D. (1968). Science 159, 1250. Elves, M. W. (1970). Nature (London) 227, 725. Evans, R., and Alexander, P. ( 1970). Nature (London) 228, 620. Falk, R. E., Collste, L., and Moller, G. (1969). Nature (London) 224, 1206. Falk, R. E., Falk, J. A., Moller, E., and Moller, C . (1970a). Cell. Immunol. 1, 150. Falk, R. E., Collste, L., and Moller, G. (1970b). J. Immunol. 104, 1287. Fnntes, K. H. (1967). In “Interferons” ( N . B. Finter, ed.), p. 119. Wiley, New York. Fantes, K. H. (1970). J. Gen. Physiol. 56, 113. Fauve, R. M., and Dekaris, D. (1968). Science 160, 795. Federlin, K., Renold, A. E., and Pfeiffer, E. F. (1968). Immunol., Int. Symp., 5th, 1967 p. 107. Feinstone, S. M., Beachy, E. H., and Rytel, M. W. (1969). J . Immunol. 103, 844. Feldman, J. D. (1968). J. Immurwl. 101, 563. Feldman, J. D., and Lee, S. (1967). J. E x p . Med. 126, 783. Feldman, J. D., and Najarian, J. S. (1963). J. Immunol. 91, 306-312.

198

BARRY R. BLOOM

Fellner, M. J., Baer, R. L., Ripps, C., and Hirschhorn, K. (1967). Nature (London) 216, 803. Fisher, D. B., and Mueller, G. C. (1968). Proc. Nut. Acad. Sci. U. S. 60, 1396. Fishman, M., and Adler, F. L. (1963). J. Exp. Med. 117, 595. Flax, M. H., Elliot, J. H., Daly, J. J., Willms-Kretschmer, K., McCarthy, J. S., and Leskowitz, S. (1969). 3. lmmunol. 102, 1214. Freedman, S. O., Turcotte, R., Fish, A. J., and Sehon, A. H. (1963). J . lmmunol. 90, 52-59. Friedman, R. M., and Cooper, H. L. (1967). Proc. SOC.Exp. Biol. Med. 152, 901. Galindo, B., and Myrvik, Q. W. (1970). J . lmmunol. 105, 227. Garcia-Giralt, E., Beauman, L., and Macieira-Coelho, A. (1970). J . Nut. Cancer Inst. 45, 649. Gell, P. G. H. (1970). Ann. N. Y. Acad. Sci. 169, 245. Gell, P. G. H., and Benacerraf, B. ( 1961). J. Exp. Med. 113, 571. Gell, P. G. H., and Silverstein, A. M. (1962a). J. Exp. Med. 115, 1037-1051. Gell, P. G. H., and Silverstein, A. M. (196213). lmmunology 5, 278. George, M., and Vaughn, J. H. (1962). Proc. SOC.E x p . Biol. Med. 111, 514. Gerety, R. J., Ferraresi, R. W., and Raffel, S. (1970). 1. E x p . Med. 131, 189. Gibson, T., and Medawar, P. B. (1943). J. Anat. 77, 299. Gillette, R. W., and Lance, E. M. (1971). Transplantation 11, 94. Gillissen, G. (1963). Rev. lmmunol. 27, 43. Ginsburg, H. (1968). Immunology 14, 621. Girard, J. P., Rose, N. R., Kunz, M. L., Kobayashi, S., and Arbesman, C. E. (1967). J. Allergy 39, 65. Glade, P., and Granger, G. A. ( 1971). Personal communication. Glasgow, L. A. (1966). J. Bacteriol. 91, 2185. Godfrey, H. P., Baer, H., and Chaparas, S. D. ( 1969). J . Immunol. 102, 1466. Goldberg, B., Kantor, F. S., and Benacerraf, B. (1962). Brit. J. Exp. Pathol. 43, 621. Goldie, J. H., and Osoba, D. (1970). Proc. SOC. Exp. Bid. Med. 133, 1265. Golub, E. S., and Spitznagel, J. K. (1970). J. Immunol. 95, 1060. Gordon, F., quoted by Isaacs, A. ( 1961). Sci. Amer. 204, 57. Gordon, J. (1968). Proc. SOC.Exp. Biol. Med. 127, 30. Gordon, J., and MacLean, L. D. (1965). Nature (London) 208, 795. Govaerts, A. (1960). J . lmmunol. 85, 516. Gowans, J. L., and McGregor, D. D. (1965). Progr. Allergy 9, 1. Gowans, J. L., and Uhr, J. W. (1966). J. Exp. Med. 124, 1017. Gowans, J. L., McGregor, D. D., and Cowan, D. M., and Ford, C. E. (1962). Nature ( London) 196, 651-655. Gowland, E. (1968). Aust. J. Exp. Biol. Med. Sci. 46, 83. Granger, G. A. (1970) In “Mediators of Cellular Immunity” ( H . S. Lawrence and M. Landy, eds.), p. 321. Academic Press, New York. Granger, G. A. ( 1971 ). In “In Vitro Methods in Cell-Mediated Immunity” (B. R. Bloom and P. Glade, eds.), p. 38. Academic Press, New York. Granger, G. A,, and Kolb, W. P. (1968). J. Immunol. 101, 111. Granger, G. A., and Weiser, R. S. (1964). Science 145, 1427. Granger, G. A., and Weiser, R. S. (1966). Science 151, 97. Granger, G. A., and Williams, T. W. (1968). Nature (London) 218, 1253. Granger, G. A., Shacks, S. J., Williams, T. W., and Kolb, W. P., (1969). Nature (London) 221, 1155.

MECHANISM OF CELL-MEDIATED IMMUNE REACTIONS

199

Granger, G. A., Moore, G. E., White, J. G., Matzinger, P., Sundsmo, J. S., Shupe, S., Kolb, W. P., Kramer, J., and Glade, P. R. (1970). 1. Zmmunol. 104, 1476. Gray D. F., and Cheers, C. (1969). lmmunobgy 17, 889. Greaves, M. F., and Moller, E. (1970). Cell. Zmmunol. 1, 372. Greaves, M. F., Torrigiani, G., and Roitt, I. M. (1969). Nature (London) 222, 885. Greaves, M. F., Miiller, E., and Mdler, G. (1970). Cell. Irnniunol. 1, 386. Green, J. A., Cooperband, S. R., and Kibrick, S . (1969). Science 164, 1415. Green, J. A., Badger, A., and Cooperband, S. R. (1970). J. Immunol. 105, 48. Gresser, I., Brouty-BoyC, D., Thomas, M.-T., and Macieira-Coelho, A. ( 1970). Proc. Nut. Acad. Sci. U . S. 66, 1052. Haase, A. T., Johnson, J. S., Kasel, J. A., Margolis, S., and Levy, H. B. (1970). Proc. SOC. E x p . Biol. Med. 133, 1076. Hall, J. G. (1969). Lancet 1, 25. Halliday, W. J., and Webb, M. (1969). J. Nat. Cancer Inst. 43, 141. Halpern, B., Ky, N. T., Amache, N., Lagrue, G., and Hazard, J. (1967). Presse Med. 75, 461. Hamilton, L. D., and Chase, M. W. (1962). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 21, 40. Hardy, D. A., Ling, N. R., Wallin, J., and Aviet, T. (1970). Nature (London) 227, 723. Haskill, J. S . (1969). I. Exp. Med. 130, 877. Hayry, P., and Defendi, V. (1970a). Science 168, 133. Hayry, P., and Defendi, V. (1970b). Clin. E x p . Immunol. 6, 345. Heilman, D. H. (1963). Tex. Rep. Biol. Med. 21, 136. Heise, E. R., and Weiser, R. S. (1969). J. Immunol. 103, 570. Heise, E. R., and Weiser, R. S. (1970). J. Immunol. 104, 704. Heise, E. R., Han, S., and Weiser, R. S. (1968). J . Zmmunol. 101, 1004. Hellstrom, I. (1967). Int. J. Cancer 2, 65. Hellstriim, I., and Hellstriim, K. E. ( 1971). I n “In Vitro Methods in Cell-Mediated Immunity” ( B . R. Bloom and P. Glade, eds.), p. 409. Academic Press, N. Y. Hellstrom, I., Hellstriini, K. E., Pierce, G. E., and Bill, A. H. (1968a). Proc. Nut. Acad. Sci. U . S. 60, 1231. HellstrGm, I., Hellstriim, K. E., and Pierce, G. E. ( 196811). Int. J. Cancer 3, 467. Hellstrom, K. E., and Hellstrom, I. (1970). Atinu. Reo. Microbwl. 24, 373. Henney, C. S. (1970a). Nature (London) 226, 1167. Henney, C. S. (1970b). J. Zmniunol. 105, 919. Hersh, E. M., and Harris, J. E. (1968). J. Immunol. 100, 1184. Hill, W.C. (1969). J. E x p . Mcd. 129, 363. Hill, W. C. (1971). J. Immunol 106, 414. Hill, W. C., and Nissen, B. (1971). 1. Immtrno!. 106, 421. Hirschhorn, K., Firschein, I. L., and Bach, F. H. (1964). In “Histocompatibility Testing” (P. S. Russell and H. J. Winn, eds.), p. 131. Nat. Acad. Sci.-Nat. Res. Counc., Washington, D. C. Hirschhorn, R., Kaplan, J. M., Goldberg, A. F., Hirschhorn, K., and Weissman, G. ( 1965). Science 147, 55. Hirschhorn, R., Brittinger, G., Hirschhorn, K., and Weissman, G. (1968). J. CeU Biol. 37, 412. Ho, M . ( 1964). Scieiice 146, 1472.

200

BARRY R. BLOOM

Ho, M., Fantes, K. H., Burke, D. C., and Finter, W. B. (1967). In “Interferons” ( N . B. Finter, ed.), p. 181. Wiley, New York. Holm, G. (1967). E x p . Cell Res. 48, 327. Holm, G., and Perlniann, P. (1965). Nature (London) 207, 818. Holm, G.,and Perlniann, P. (1967). J. E x p . Med. 125, 721. Holm, G., Perlmann, P., and Werner, B. (1964). Nature (London) 203, 841-843. Holtzer, J. D., and Winkler, K. C. (1967). Immunology 12, 701. Hook, E. W., and Wagner, R. R. (1959). J . lmmunol. 83, 310. Huber, H.,and Fudenberg, H. H. (1968). Int. Arch. Allergy Appl. lmmunol. 34, 18. Humphrey, J. H., and Turk, J. L. (1963). lmmunology 6, 119. Hunt, W. B., and Myrvik, Q. W. (1964). J . lmmunol. 93, 677-681. Isaacs, A., and Lindenmann, J. (1957). Proc. Roy. SOC., Ser. B 147, 258. Ishizaka, K., Tomioka, H., and Ishizaka, T. (1970). J. lmmunol. 105, 1459. Iverson, G. M. (1970). Nature (London) 227, 273. Janis, M., and Bach, F. H. (1970). Nature (London) 225, 238. Jerne, N. K., (1967). Cold Spring Harbor Symp. Quont. Biol. 32, 591. Jerne, N. K. (1971). I n “Immunological Surveillance” (R. T. Smith and M. Landy, eds.), p. 345. Acadeniic Press, New York. Jerne, N . K., and Nordin, A. A. ( 1963). Science 140, 405. Jerne, N. K., Nordin, A. A., and Henry, C. (1963). In “Cell-Bound Antibodies” ( D . B. Amos and H. Koprowski, eds.). Wistar Inst. Press, Philadelphia, Pennsylvania. Jimenez, L., and Bloom, B. R. (1971). Cell Immunol. In press. Jimenez, L., and Bloom, B. R. (1971). Leucocyte Culture Conf., Sth, 1970 (in press). Jimenez, L., Bloom, B. R., and Marcus, P. (1970). Fed. Proc., Fed. Amer. SOC.E x p . Biol. 29, 501 (abstr. ). Jinienez, L., Bloom, B. R., Blume, M. R., and Oettgen, H. F. (1971). J. E x p . Med. 133, 740. Kaltreider, H. B., Soghor, D., Taylor, J. B., and Decker, J. L. (1969). J. Immunol. 103, 179. Karush, F., and Eisen, H. N. (1962). Science 136, 1032. Kasakura, S., and Lowenstein, L. (1965). Nature (London) 208, 795. Kasakura, S., and Lowenstein, L. (1967). Nature (London) 215, 80. Kasel, J. A,, Haase, A. T., Glade, P. R., and Chassin, L. N. W. (1968). Proc. SOC. E x p . Biol. Med. 128, 351. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B. (1970a). J. Erp. Med. 132, 261. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B. (1970b). Science 170, 462. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B. (1971). J . E x p . Med. 133, 169. Kay, W., and Rieke, W. 0. (1963). Science 139, 487. Kennedy, J. C., Treadwell, P. E., and Lennox, E. S. (1970). J . E x p . Med. 132, 353. Kettnian, J., and Dutton, R. W. (1970). J. lmmunol. 104, 1558. Killander, D., and Rigler, R. (1969). E x p . CeZZ. Res. 54, 163. Kind, L. S., and Peterson, W. A. (1968). Science 160, 312. Klein, E. (1968). N . Y. State J. Med. 68, 900. Klein, E.,Ringertz, N. W., and Revesz, L. (1959). Cancer 12, 697. Klein, G.,and Klein, E. (1956). Nature (London) 178, 1389. Klein, G., and Klein, E. (1958). J. Cell. Comp. Physiol. 52, Suppl. 1, 125.

MECHANISM OF CELL-MEDIATED IMMUNE REACTIONS

201

Klein, G., and Perlmann, P. (1963). Nature (London) 199, 451. Kolb, W. P., and Granger, G. A. (1970). Cell. lmmunol. 1, 122. Kolin, A., Johanovsky, J., and Pekarek, J. (1965). lnt. Arch. Allergy A p p l . lmmunol. 26, 167. Krejci, J., Pekarek, J., Johanovsky, J., and Svejcar, J. (1969). Immunology 16, 677. Kronman, B. S., Wepsic, H. T., Churchill, W. H., Jr., Zbar, B., Borsos, T., and Rapp, H. J. (1969). Science 165, 296. Lafferty, K. J., and Jones, M. A. S. (1969). Aust. J. E x p . Biol. Med. Sci. 47, 17. Lanibert, P. B., and Frank, H. A. (1970). J. E x p . Med. 132, 868. Landsteiner, K. ( 1945). Harvard Univ. Press, Cambridge, Massachusetts (rev. ed., Dover, New York, 1962). LaPorte, R. (1934). Ann. lnst. Pasteur, Paris 53, 598. Lawrence, H. S. (1969). Aduan. lmmunol. 11, 195. Lawrence, H. S., and Landy, M., eds. (1970). “Mediators of Cellular Immunity,” p. 469. Academic Press, New York. Lebowitz, A., and Lawrence, H. S. (1969). Fed. Proc., Fed. Amer. SOC. E x p . Biol. 28, 630 (abstr.). Leskowitz, S. (1963). Nature (London) 199, 291. Leskowitz, S., and Zak, S. J. (,1966). Nature (London) 211, 246. Levis, W. R., and Robbins, J. H. (1970a). J. lmmunol. 104, 1295. Levis, W. R., and Robbins, J. H. (1970b). E x p . Cell Res. 61, 153. Lid&, S. (1967). Acta Pathol. Microbiol. Scand. 70, 58. Ling, N. R. (1968). “Lymphocyte Activation.” Wiley, New York. Lo Buglio, A. F., Cotran, R. S., and Jandl, J. H. (1967). Science 158, 1582. Lockshin, M. D. (1969). Proc. SOC.E x p . Biol. Med. 132, 928. Loewi, G., Temple, A., and Vischer, T. L. (1968). lmmunol. 14, 257. Lolekha, S., Dray, S., and Gotoff, S. P. (1970). J. Immunol. 104, 296. Lubaroff, D. M., and Waksman, B. H. (1968a). J. E x p . Med. 128, 1425. Lubaroff, D. M., and Waksman, B. H. (1968b). J. E x p . Med. 128, 1437. Lundgren, G., Collste, L., and Moller, G. (1968a). Nature (London) 220, 289. Lundgren, G., Zukosk, C. F., and Moller, G. (1968b). Clin. E x p . lmmunol. 3, 817. McBride, R. A. (1966). Cancer Res. 26, 1135. McBride, R. A., and Schierman, L. W. (1970). J. E x p . Med. 131, 377. McCluskey, R. T., Benacerraf, B., and McCloskey, J. W. (1963). J. lmmunol. 90, 466. McFarland, W., and Schechter, G. P. (1970). Blood 35, 683. McFarland, W., Heilman, D. H., and Moorhead, J. F. (1966). I. E x p . Med. 124, 851. Mackaness, G. B. (1962). J. E x p . Med. 116, 381. Mackaness, G. B. (1964). J. E x p . Med. 120, 105. Mackaness, G. B. (1969). J. E x p . Med. 129, 973. blackmess, G . B., and Blanden, R. V. (1967). Progr. Allergy 11, 89. Maclaurin, B. P. (1969). Aust. J. E x p . Biol. Med. Sci. 47, 105. hlaclennan, I. C. M., and Harding, B. ( 1970a). Nature (London) 227, 1246. hlaclennan, I. C. M., and Harding, B. (1970b). lmmunol. 18, 405. Maini, R. N., Bryceson, A. D. M., Wolstencroft, R. A., and Dumonde, D. C. (1969). Nature (London) 224, 43. Xlakelii, O., and Nossal, G. J. V. (1961). J. lmmunol. 87, 447. hlakinodan, T., and Albright, J. F. (1967). Prog. Allergy 10, 1.

202

BARRY R. BLOOM

Malmgren, R. A., Holmes, E. C., Morton, D. L., Yee, C. L., Marrone, J., and Myers, M. W. (1969). Transplantation 8, 485. Mandel, T., Byrt, P., and Ada, G. L. (1969). E x p . Cell Res. 58, 179. Marcus, Z. ( 1970). Fed. Proc., Fed. Anwr. SOC. Exp. Biol. 29, 338 (abstr.). Marshall, W. H., and Roberts, K. B. (1963). Lancet 1, 773. Marshall, W. H., Valentine, F. T., and Lawrence, H. S. (1969). J. Exp. Med. 130, 327. Martinez, C., Kersey, J., Papermaster, B. W., and Good, R. A. (1962). Proc. SOC. E x p . Biol. Med. 109, 193. Martins, A. B., Moore, W. D., Dickenson, J. B., and Raffel, S . (1964). J. Immunol. 93, 953. Math&,G., Aniiel, J. L.,Schwartzenberg, L., Schneider, M., Catten, A., Schlumberger, J. R., Hayat, M., and de Vassal, F. (1969). Lancet 1, 697. Matisova, E., Butorova, E., Lackovic, V., and Borecky, L. (1969). Acta Virol. (Prague), Engl. Ed. 14, 1-7. Mauel, J., Rudolf, B., Chapuis, B., and Brunner, K. T. (1970). Immunology 18, 517. Meacock, S . C. R., and Willoughby, D. A. (1968). Immunology 15, 101. Medawar, P. B. (1959). In “Cellular and Humoral Aspects of the Hypersensitive States” (H. S. Lawrence, ed.), p. 504. Harper (Hoeber), New York. Merigan, T. C. (1971). In “In Vitro Methods in Cell-Mediated Immunity” ( B . R. Bloom and P. Glade, eds.), p, 81. Academic Press, New York. Merigan, T. C., De Clercq, E., and Bausek, G . H. (1970). J. Gen. Physiol. 56, 57. Metzger, H. (1970). Ann. Reu. Biochem. 39, 889. Meuwissen, H. J., Stutman, O., and Good, R. A. (1969). Sem. Hematol. 6, 28. Miller, H. C., and Cudkowicz, G. (1970). 3. E x p . Med. 132, 1122. Miller, J. F. A. P. ( 1961). Lancet 2, 748. Miller, J. F. A. P., and Mitchell, G. F. (1968). 3. E x p . Med. 128, 801. Miller, J. F. A. P., and Mitchell, G . F. (1969). Transplant. Reu. 1, 3. Miller, J. F. A. P., and Osoba, D. (1967). Physiol. Reu. 47, 437. Miller, J. F. A. P., Marshall, A. H. E., and White, R. G. (1962). Aduan. Immunol. 2, 111. Mills, J. A. (1966). J. Immunol. 97, 239. Mintz, B., and Silvers, W. K. (1970). Transplantation 9, 497. Mishell, R. I., and Dutton, R. W. (1967). 1. E x p . Med. 126, 423. Mitchell, G. F., and Miller, J. F. A. P. (1968). J. Exp. Med, 128, 821. Mitchison, N. A. ( 1969). In “Immunological Tolerance” ( M . Landy and W. Braun, eds.), p. 115. Academic Press, New York. Modabber, F., Morikawa, S., and Coons, A. H. (1970). Science 170, 1102. Moller, E. ( 1965a). Science 147, 873. Moller, E. (1965b). J. E x p . Med. 122, 11. Moller, G., and Lundgren, G . (1969). I n “Cellular Recognition” (R. T. Smith and R. A. Good, eds.), p. 177. Appleton, New York. Mookerjee, B., Ackman, C. F. D., and Dorreter, J. B. (1969). Transplantation 8, 745. Mooney, J. J., and Waksman, B. H. (1970). J. Immunol. 105, 1138. Moorhead, J. F., Paraskova-Tchernozenska, E., Pirrie, A,, and Hayes, C . (1969). Nature (London) 224, 1207. Morton, D., Eilber, F. R., Malnigren, R. A,, and Wood, W. (1970). Surgery 68, 158. Mosier, D. E., and Coppleson, L. W. (1968). Proc. Nut. Acad. Sci. U.S. 61, 542.

MECHANISM O F CELL-MEDIATED I h l M U X E REACTIONS

203

Najarian, J. S., and Feldnian, J. D. (1961). J. Ex)?. Med. 114, 779. Najarian, J. S., and Feldman, J. D. (1963). J. E x p . Med. 118, 341-352. Naor, D., and Sulitzeanu, D. ( 1967). Nattrrc ( L o n d o n ) 214, 687-688. Naor, D., and Sulitzeanu, D. (1968). Life Sci. 7, 377. Naspitz, C. K., and Richter, hl. (1968). Progr. Allergy 12, 1. Nelson, D. S. ( 1969 ). “hlacrophages and Immunity.” Wiley, Nen. York. Nelson, D. S. (1970). Austr. J. E x p . Biol. hled. Sci. 48, 329. Nelson, D. S., and Boyden, S. V. (1963). Immunology 6 , 264. Nelson, D. S., and North, R. J. (1965). Lab. Inoest. 14, 89. Nordqvist, B., and Rorsnian, H. (1967). Trans. S t . John’s Hosp. Dermotol. Soc. 53, 154. Nordqvist, B., and Rorsman, H. (1970). Znt. Arch. Allergy Appl. Immunol. 39, 172. North, R. J. (1970). J. E x p . hied. 132, 535. Nossal, G. J. V., Cunningham, A., hlitchell, G. F., and Miller, J. F. A. P. (1968). J. E x p . Mecl. 128, 839. Nota, N. R., Liacopoulos-Briot, hl., Stiffel, C., and Biozzi, G. (1964). C. R. Acad. Sci., Scr. D 259, 1277. Nowell, P. C. ( 1960). Cancer Res. 20, 462. Old, L. J., Clarke, D. A., and Benacerraf, B. (1959). Nature (London) 184, 291. Old, L. J., Benacerraf, B., Clarke, D. A., Carswell, E. A., and Stockert, B. (1961). Cancer Res. 21, 1281. Oldstone, hl. B., and Dixon, F. J. (1970). Virology 42, 805. Oort, J., i c . 1 Turk, J. L. (1965). Brit. J. E x p . Pathol. 46, 147. Oppenheim, J. J. (1968). Fed. Proc., Fed. Amer. SOC. E x p . Biol. 27, 21. Oppenheini, J. J., Wolstencroft, R. A., and Gell, P. G. H. (1967). Immunology 12, 89. Osoba, D. (1970). J. E x p . Med. 132, 368. Ovary, Z., and Benacerraf, B. (1963). Proc. Soc. E r p . Biol. Med. 114, 72. Palmer, C. G., Livergood, D., Warren, A. K., Sinipson, P. J., and Johnson, J. S. (1960). E x p . Cell Res. 20, 198. Pappenheimer, A. hf., Jr., Scharff, hl., and Uhr, J. W. (1959). I n “Mechanisms of Hypersensitivity” ( J . H. Schaffer, G. A. Lo Grippo and M. W. Chase, eds.), p. 417. Little, Brown, Boston, hlassachusetts. Patterson, R. J., and Younians, G. (1970). Znfec. Immirnity 1, GOO. Paul, W. E. ( 1971 ). Personal communication. Paul, W. E., Siskind, G. W., Benacerraf, B., and Ovary, Z. (1967). J. Immunol. 99, 760. Paul, W. E., Katz, D. H., Goidl, E. A., and Benacerraf, B. (1970). J. E x p . Med. 132, 283. Peamiain, G., Lycette, R. R., and Fitzgerald, P. H. (1963). Lancet 1, 637. Pearson, M. N., and Raffel, S. (1971). J. E x p . Med. 133, 494. Pederson, N. C., and Morris, B. (1970). J. Exp. Med. 131, 936. Pekarek, J., Svejcar, J., Krejci, J., and Johanovsky, J. ( 1969). Foliu Microbiol. (Prague) 14, 417. Peltier, A. P., and Kourilsky, R. (1966). Ann. Inst. Pasteur, Paris 110, 813. Perey, D. Y., Cooper, hl. D., and Good, R. A. (1967). Transplantation 5, 615. Perkins, E. H., Sato, T., and hlakinodan, T. (1969). J. Zmmunol. 103, 668. Perlmann, P. ( 1970). Personal communication. Perlmann, P., and Holm, G. (1969a). Adoan. Zmmunol. 11, 117.

204

BARRY R . BLOOM

Perlmann, P., and Holm, G . ( 1969h). I n “Cellular Recognition” ( R . T. Smith and R. A. Good, eds. ), p. 486. Appleton, New York. Perlmann, P., and Holm, G . ( 1970 ) . Personal coniniunication. Perlniann, P., Perlmann, H., and Holm, G. (1968). Science 160, 306. Perlniann, P., Perlniann, H. Miiller-Eberhard, H. J., and Manni, J. A. (1969). Science 163, 937. Perlmann, P., Perlmann, H., Wasserman, J., and Packalh, T. ( 1970a). Int. Arch. Allergy AppI. Inimiinol. 38, 204. Perlmann, P., Nilsson, H., and Leon, M. A. (1970b). Science 168, 1112. Pernis, B.,Forni, L., and Amante, L. (1970). J. E x p . M e d . 132, 1001. Peterson, R. D. A , , Cooper, M. D., and Good, R. A. (1966). Amer. J. Med. 41, 342. Phair, J. P., and Kantor, F. S. (1970). J. Immunol. 105, 471. Pick, E.,Krejci, J., Cech, K., and Turk, J. L. (1969). Immunology 17, 741. Pick, E.,Krejci, J., and Turk, J. L. ( 1970a). Nature (London) 225, 236. Pick, E.,Brostoff, J., Krejci, J., and Turk, J. L. (1970b). Cell. Immrrnol. 1, 92. Pilchard, E. I., Gold, E. E., and Gray, H. K. (1970). Int. Arch. Allergy Appl. Immunol. 37, 337. Pincus, W.B. (1967). J. Reticiiloe~idothel.SOC. 4, 122. Pincus, W. B. ( 1970). Ann. Allergr~28, 93. Pincus, W.B., Woods, W. W., and Pang, R. K. (1970). J. Reticuloendothel. Soc. 7, 220. Pochyly, D. F. ( 1967). Fed. Proc., Fed. Amer. Sac. E x p . B i d . 26, 478 (abstr.). Pogo, B. G. T., Allfrey, V. G., and hiirsky, A. E. (1966). Proc. Not. Acad. Sci. U. S. 55, 805. Polgar, P. R., and Kibrick, S. ( 1970). Nature (London) 225, 857-858. Prendergast, R., and Suzuki, M. (1970). Nature (London) 227, 277. Prendergast, R. A. (1964). J. Exp. Med. 119, 377. Raff, M.C. ( 1969). Nature (London) 224, 378. Raff, M. C. (1970). Nature (London) 226, 1257. Raffel, S. (1961). “Immunity,” p. 333. Appleton, New York. Rajapakse, D. A., and Glynn, L. E. (1970). Nature (London) 226, 857. Rajewsky, K. V., Schirrmacher, V. Nase, S., and Jerne, N. K. (1969). J. E x p . Med. 129, 1131. Ramseier, H. (1967). Science 157, 554. Ramseier, H.(1969). J. E x p . Med. 130, 1279. Rauch, H. C., and Raffel, S. (1964). J. Immunol. 93, 960. Reif, A. E., and Allen, J. M. V. (1964). 1. E x p . Med. 120, 413. Reisfeld, R. A., and Yoshida, T. ( 1970). Nature (London) 226, 856. Remold, H. G., and David, J. R. (1970). Fed. Proc., Fed. Amer. S O C . E x p . Biol. 29, 339 (abstr.). Remold, H. G., Katz, A. B., Haber, E., and David, J. R. (1970). Cell. Immunol. 1, 133. Revesz, L. (1956). Nature (London) 178, 1391. Ricci, M., Romagnani, S., Passaleva, A., and Biliotti, G. (1969). Clin. E x p . Immunol. 5, 659. Rich, A. R., and Lewis, M. R. (1932). Bull. ]ohns Hopkins Hosp. 50, 115. Richter, M. (1970). Proc. Nut. Acad. Sci. U . S. 66, 1127. Ringertz, N.,Klein, E., and Revesz, L. (1959). Cancer 12, 697. Rocklin, R. E., Churchill, W . H., Sheffer, A., and David, J. R. (1970a). I . Clin. Inoest. 49, 81 (abstr.).

MECHANISM OF CELL-MEDIATED IMMUNE REACTIONS

205

Rocklin, R. E., Meyers, 0. L., and David, J. R. (1970b). J. Immunol. 104, 95. Rocklin, R. E., Sheffer, A,, Churchill, W., and David, J. R. (1971). Leucocyte Culture Conf., 5th, 1970 (in press). Rocklin, R. E., Bloom, B. R., and David, J. R. (1970). Unpublished observation. Roitt, I. M., Torrigiani, G., Greaves, M. F., Brostoff, J., and Playfair, J. H. L. (1969).

Lancet 2, 367. Rosernan, J. M. (1969). Science 165, 1125. Rosenan, W. ( 1963). I n “Cell-Bound Antibodies” ( D . B. Amos and H. Koprowski, eds. ), p. 75. Wistar Inst. Press, Philadelphia, Pennsylvania. Rosenau, W., and Moon, H. D. (1961). J. Nut. Cancer Int. 27, 471-83. Rosenau, W., and Morton, D. L. (1966). J. Nut. Cancer Inst. 36, 825. Ruddle, N. H., and Waksman, B. H. (1967). Science 157, 1060. Ruddle, N. H., and Waksnian, B. H. (1968a). J. E x p. Med. 128, 1237. Ruddle, N. H., and Waksnian, B. H. (1968b). J. E x p. Med. 128, 1255. Ruddle, N . H., and Waksman, B. H. ( 1 9 6 8 ~ )J.. Exp. Med. 128, 1267. Salvin, S. B., and Smith, R. F. (1959). J. E x p . Med. 109, 325. Salvin, S. B., and Smith, R. F. (1960). J. E x p . Med. 111, 465. Salvin, S. B., Nishio, J., and Gribik, hl. (1970). Cell. Immunol. 1, 62. Sanderson, A. R. (1964). Nature (London) 204, 250. Schechter, G. P., and hlcFarland, W. (1970). J. Immunol. 105, 661. Schierman, L. W., and McBride, R. A. (1967). Science 156, 658. Schinipl, A., and Wecker, E. (1970). Nature (London) 226, 1258. Schlossman, S. F., Yaron, A., Ben-Ephraini, S., and Sober, H. A. (1965). Biochemistry 8, 1638. Schlossman, S. F., Ben-Ephraini, S., Yaron, A., and Sober, H. A. (1966). J. E x p . Med. 123, 1083. Schrek, R. (1963). Amer. Rev. Resp. Dis. 87, 734. Schwartz, H. J., Leon, M. A,, and Pelley, R. P. (1970). J . Zmmunol. 104, 265. Seeger, R. D., and Oppenheim, J. J. (1970). J. E x p . Med. 132, 44. Sehon, A. H. (1962). Protides Biol. Fluids, Proc. Collog. 11, 67-69. Sell, S., and Gell, P. G. H. (1965). J. Exp. Med. 122, 423. Shea, J. D., and Morgan, H. R. (1957). Proc. SOC.Exp. Biol. Med. 94, 436. Silverstein, A. M., and Borek, F. (1966). J. Immunol. 96, 953. Silverstein, A. M., and Gell, P. G. H. (1962). 3. Exp. Med. 115, 105S1064. Silverstein, A. M., Prendergast, R. A., and Kraner, K. L. (1963). Science 142, 1172. Silverstein, A. M., Prendergast, R. A,, and Kraner, K. L. (1964). J. Exp. Med. 119, 955. Simmons, T., and Manson, L. A. (1971). Transplant. Proc. 3, 253. Simonsen, M. (1962). Progr. Allergy 6, 349. Simonsen, M. (1967). Cold Spring Harbor Symp. Quant. Biol. 32, 517. Smith, R. T., Bauscher, J. A. C., and Adler, W. H. (1970). Amer. J. Pathol. 60, 495. Smith, T. J., and Wagner, R. R. (1967). J. E x p . Med. 125, 579. Smyth, A. C., and Weiss, L. (1970). J. Immunol. 105, 1360. S@borg,M. (1967). Acta Med. Scand. 102, 167. Seborg, M. (1969). Acta Med. Scand. 185, 221. Solliday, S., and Bach, F. H. (1970). Science 170, 1406. Sonozaki, H., and Cohen, S. (1971). Cell. Zmmunol. (in press). Spitler, L. E., and Fudenberg, H. H. (1970). J. Immunol. 104, 544. Spitler, L. E., and Lawrence, H. S. (1969). J. Immunol. 103, 1072. Spitler, L., Huber, H., and Fudenberg, H. H. (1969). J . Immunol. 102, 404.

206

BARRY R. BLOOM

Spitler, L., Benjamini, E., Young, J. D., Kaplan, H., and Fudenberg, H. H. (1970). J. E x p . Med. 131, 133. Stastny, P., and Ziff, M. (1970). J. Reticuloendothel. SOC. 7, 140. Steel, T., and Hardy, D. A. (1970). Lancet 1, 1322. Steffen, C., and Rosak, M. (1962). J. Immunol. 90, 337. Stern, K., and Davidsohn, I. (1954). J . Immunol. 72, 209. Strober, S., and Gowans, J. L. (1965). J. Exp. Med. 122, 347. Sulitzeanu, D. (1968). Bacteriol. Reu. 32, 404. Svejcar, J,, and Johanovsky, J. ( 1961 ). Z. Immunitaetsforsch 112, 420436. Svejcar, J., Johanovsky, J., and Pekarek, J. ( 1967). 2. Immunitaetsforsch., Allerg. Klin. Immunol. 133, 259. Svejcar, J,, Pekarek, J., and Johanovsky, J. (1968a). Immunology 15, 1. Svejcar, J., Pekarek, J., and Johanovsky, J. ( 196813). Folia Microbiol. (Prague) 13, 3. Svejcar, J., Pekarek, J., and Johanovsky, J. ( 1969). 2. Immunitaetsforsch., Allerg. Klin. Immunol. 138, 342. Takahashi, T., Carswell, E. A., and Thorbecke, G. J. (1970). J. E r p . Med. 132, 1181. Takasugi, M., and Klein, E. (1970). Transplantation 9, 219. Talmage, D. W., Radovich, J., and Hemmingsen, H. (1970). Advan. Immunol. 12, 184. Taubler, J. H., and Mudd, S. (1968). J. Immunol. 101, 550. Taylor, R. B. (1968). Nature (London) 220, 611. Taylor, R. B. (1969). Transplant. Reu. 1, 114. Thomas, L. (1959). In “Cellular and Humoral Aspects of the Hypersensitive States” ( H . S. Lawrence, ed.), p. 529. Harper (Hoeber), New York. Thor, D. E., and Dray, S. (1968). J. Immunol. 101, 459. Thor, D. E., Jureziz, R. S., Veach, S. R., Miller, E., and Dray, S. (1968). Nature (London) 219, 755. Tompkins, G . M., Gelehrter, T. D., Granner, D., Martin, D., Samuels, H. H., and Thompson, E. B. (1969). Science 166, 1474. Tompkins, W. A. F., Adams, C., and Rawls, W. E. (1970a). J. Imnirrnol. 104, 502. Tompkins, W. A. F., Zarling, J. M., and Rawls, W. E. (1970b). Infec. ImmunoZ. 2, 783. Tsoi, M., and Weiser, R. S. (1968). J . Nut. Cancer Inst. 40, 23. Turk, J. L. (1960). I n t . Arch. Allergy Appl. Immunol. 17, 338-351. Turk, J. L. (1962). Immunology 5, 478. Turk, J. L. (1967). Brit. Med. BUZZ. 23, 3. Turk, J. L. (1970). In “Mediators of Cellular Immunity” ( H . S. Lawrence and M. Landy, eds.), p. 430. Academic Press, New York. Turk, J. L., Heather, C. J., and Diengdoh, J. V. (1966). Int. Arch. Allergy A p p l . Immunol. 29, 278. Turner, K. J., and Forbes, I. J. (1966). J. Immunol. 96, 926. Uhr, J. W., and Scharff, M. (1960). J. E x p . Med. 112, 65. Uhr, J. W., Salvin, S. B., and Pappenheimer, A. M., Jr. (1957). J. EX),. Med. 105, 11.

Unanue, E. R., Cerottini, J. C., and Bedford, M. (1969). Nature (London) 222, 1193. Valdimarsson, H., Riches, H. R. C., Holt, L., and Ilobbs, J. R. (1970). Lancet 1, 1259.

MECHANISM OF CELL-MEDIATED IMMUNE REACTIONS

207

Valentine, F. T., and Lawrence, H. S. (1969). Science 165, 1014. Van Furth, R., and Colin, Z. A. (1968). J. Eqi. Aled. 128, 415. Voisin, G. A., Touillet, F., and Voisin, J. (1964). Ann. Ztist. Pasteur, Paris 106, 353. Volknian, A., and Goivans, J. L. (1965). Brit. J. Ex)). Pathol. 46, 62-70. and Puttrell, C. N. (1959). J. Immunol. \Vagner, R. R., Snyder, R. hl., IIook, E. W., 83. 87. R'aksman, B. €I. (1960). Cell. A.rpects Immtrnity, CiOa Found. Symp. 1959 p. 280. \?'~lkSlii~lll, B. II., illld >fatOltSy, h i . (1958). J. ~ t t l l 7 l I r ~ O81, ~ . 220. Ward, P. A., Remold, H. G., and David, J. R. ( 1969). Science 163, 1079. \I.'ard, P. A., Remold, H. G., a n d David, J. H. (1970). Cell. Zmmonol. 1, 162. M'arner, N. L., and Szenberg, A. (1984). I n "The Thymus in Immunology" ( R . A. Good and A. E. Gabrielson, eds.), p. 395. Harper ( Hoeber), New York. \\'assernian, J , , and Packalim, T. ( 1965). Zmrtitriiology 9, 1. Wesslkn, T. (1952a). Acta Trrbcrc. Scatid. 26, 38-53. Wessl~in,T. ( 1952b). Acta Dermato-Vcnereol. 32, 195. Wheelock, E. F. (1965). Science 149, 310. Wiener, J., Spiro, D., and Russell, P. S. (1964). Amcr. J. Pathol. 44, 319. Wiener, J., Spiro, D., and Zunker, H. 0. (1965). Anier. J. Pathol. 47, 723. \\'igzell, H., and Andersson, B. (1969). J. E x ) ) . Jled. 129, 23. \\'illems, F. T. C., hlelnick, J. L., and Rawls, W.E. (1969). 1. Viral. 3, 451. Williams, A. C., and Klein, E. (1970). Cancer 25, 450. Williams, R. hl., and Waksnian, B. H. (1969). J. Imitiutiol. 103, 1435. Williams, T. W.,and Granger, G. A. (1968). Natrrre (London) 219, 1076. \\Jillianis, T. W.,and Granger, G. A. (1969). J. Zmmrrnol. 102, 911. \\Jilloughby, D. A,, Boughton, B., Spector, \V. G., and Schild, H. 0. ( 1962). Life Sci. 7, :347. \Villoughl,y, D. A , , Roughton, B., and Schild, €1. 0. ( 1963). Imn~rrnology6, 484. \Yilloughl)y, 1). .I.,Spector, W. G., and Bougliton, B. ( 1964). J. Pathol. Bacteriol. 87, 353. \Vilson, D. B. (1965a). J. E s p . Aled. 122, 143. \Vilson, D. B. (1965b). J. Ex)). Mcd. 122, 167. \Vilson, D. B. (1967). J . Ex)). Alcd. 126, 625. \Vilson, 1). B. ( 1971 ). Pcrsonal communicntion. Wilson, D. B., and Nowell, P. C. (1971). J. E s p . Afcd. 133, 442. \Vilson, D. B., Silvtw, \\'. K., and So\vcll, P. C. ( 1967). J . E X ~ IAled. . 126, 655. \Vilsoii, D. B., BIyth, J. L., d Sowell, P. C. ( 1968). J. Esp. Mcd. 128, 1157. Witten, T. A., U'ong, \\'. L., aiid Killinn, 11. (196.3). Science 142, 596. l O ~ 599. ~ ~ ~ / \\'olhtencroft, R . .4., id Dlllilontlc, 11. c. ( 1970). ~ t t i l l l ~ ~ l 18, Wunderlich, J. R., and Cants, T. C. ( 1970). Natrrre ( L o n d o n ) 228, 62. Yoshida, T., and Rcisfeld, R. (1970). Natrrre ( L o n d o n ) 226, S5G. Yoshida, T., Rcw;icc~rraf.B.. J l c C l d i c ~ ! . , R. T., ant1 \'assalli, P. ( 1965 ) . J . I t t r ! t r r r t r o l . 102, 804. Youngner, J. S., and Stincl)ring, W. H. ( 19G4). Scieticc 144, 1022. Zabriskie, J. B., and Falk, R. E. ( 1970). A'atrrrc (Loridon) 226, 943. Zbur, B., \\'cpsic, 11. T., Borsns, T., am1 Rapp, H. J. ( 1970a). J. A'at. C~rr~ccr Ztist. 44, 473. Z b u , B., \\'epsic, 11. T., Rupp, H. J.. Stc\\.nrt, L. C., and Borsos, T. ( 1970b). J . A'at. Cancer Znst. 44, 701. Zbar, B., Bernstein, I., Taunkn, T., and Rapp. H. J. ( 1'3'iOc). Sciericc 170, 1217.

208

BARRY R. BLOOM

Zeitz, S. J., McClure, D., and Van Arsdel, P. P., Jr. (1965). 1. Allergy 36, 197 (abstr.). Zoschke, D. C., and Bach, F. H. (1970). Science 170, 1404. Zweiman, B., and Phillips, S. M. (1970). Science 169, 284. Zweiman, B., Schoenwetter, W. F., and Hildreth, E. A. (1966). 1. Zmmunol. 96, 296.

Immunological Phenomena in Leprosy and Related Diseases J. 1. TURK AND A. D. M. BRYCESON Department of Pathology, Royal College of Surgeons o f England, Lincoln’s Inn Fields, London, England and Department o f Medicine, Ahmadu Bello University, Zario, Nigeria

I. Introduction . . 11. Leprosy . . . A. Clinical Spectrum of B. Histological Features C. The Leproniin Test U. Histological Changes E. Allergic Reactions in

. .

. .

. . Leprosy .

. . .

. . .

. . .

. . . of the Leprosy Spectrum . . . . . . . .

. . . . . in Lymphoid Tissue in Lcprosy .

.

.

.

.

Leprosy

.

.

. . . . . .

.

F. Experimental Production of Leprosy in Laboratory Animals . G. Depression of Cell-Mediaiecl Immunity i n Human Leprosy ,

. . . . . . . . .

H. Immunoglobulins, Complement, and Humoral Antibody . . . . . . . . . . Production . . 111. Leishiiianiasis . . . . . . . . . . . . A. Introduction . . . . . . . . . . . B. Lcishmaniasis as a Disease Spectrum . . . . . . C. The Leishmnnin Test . . . . . . . . . . D. Immunodiagnosis . . . . . . . . . . E. Artificial Immunization . . . . . . . . . F. Experimental Leisliiiianiasis: Lcishniunici cnriettii Infection . . . . . . . . . . of the Guinea Pig IV. Concept of a Host-Determinccl Spectrum of Clinical Manifestations . . . . . . . in Other Chronic Infections in hlan References . . . . . . . . . . . . .

. . . . . . . . .

209 210 210 212 214 216 222 225 227

. .

.

234 237 237 238 247 248 249

.

250

.

. .

.

. .

259 261

I. Introduction

The clinical fonii of sonic infectious discases may be more related to the inirnunological responscs of the paticnt than to variations in the infectivity or metabolic activity of thc microorganism. Increased proliferation of the microorganism can result as much from a defect in the host’s inimunc mcchanisni as froin a chnngc in antigenic nature or toxin production. Considerable tissue damage has been found to be caused by hypersensitivity reactions directcd against antigens either resident in or derived from infecting microorganisms or diffused from them into surrounding tissues. Dcrived soluble antigens can also cliffuse into the bloodstream and form iniinimc coinplexes which can cause tissue daniagc at a considerablc distance from the sitc of proliferation of the infecting organism. Failure of host resistancc frequently results from a defect in “9

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J. L. TURK AND A. D. hi. BRYCESON

cell-mediated immunity ( CMI ) . However, hypersensitivity reactions resulting in tissue damage can occur as rcadily from ChlI as from the deposition of immune complexes involving hunioral antibody. Such an interaction between immune procedures and a given microorganism can display a wide spectrum of pathological processes, which, in turn, leads to markedly different clinical manifest a t‘1011s. Such a spectrum is particularly well demonstrated by the recent elaboration of the varied clinical patterns in leprosy. Postulations from time to time related these differences to variations in the host’s resistance, yet experimental and clinical evidence has been available only during the last few years. With elucidation of the immunological basis for the disease spectrum in leprosy, a parallel has been sought and found in other infectious discases. Thcse include especially certain protozoal diseases such as leishnianiasis and others caused by yeasts and fungi such as candidiasis and the sytemic mycoses. This review is, therefore, concerned with immunological concepts in leprosy leading to a discussion of analogous states now recognized in other infectious diseases. II. Leprosy

A. CLINICALSPECTRUMOF LEPROSY The typical nodular form of leprosy, associated with massive infiltration of the tissues with histiocytcs packed with colonies of mycobacteria and an apparent inability of the body to eliminate the organism, is one of the two main types of the disease. The other, the anesthetic or maculoanesthetic form, is associated with peripheral nerve damage with or without cutaneous lesions. Here the microorganisms may be scanty or difficult to demonstrate, and the skin lesions may come to resemble those in lupus vulgaris. Tuberculoid has, therefore, been used as a generic term to describe this second form of the disease; the nodular form of leprosy is now referred to a lepronzatous. It has long been accepted that tuberculoid leprosy is a “high-resistant” form, whereas leproniatous leprosy is a “low-resistant” form. A further development was recognition that sonic paticnts have a leprosy combining various features of both the tuberculoid and the lepromatous types. This manifestation of the infection has been called borderline (Wade, 1940) or dimorphous ( Khanolkar and Cochrane, 1956). Patients with this borderline form show varying patterns forming a spectrum between the tuberculoid and lepromatous poles. Some are more tuberculoid in character, whereas others are more leproniatous (see Table I ) . Manifestations of leprosy form a spectrum, depending on the host’s resistance, between high resistance and low resistance. Cochrane and Sniyly (1964)

211

LEPROSY AND RELATED DISEASES

,

TABLE I THE:SPECTRUM OF LEPROSY ,Indeterate

Tuberculoid+Borderlinee=Lepromatous

Characteristics

Alycobacterium leprae in tissues Lymphocytic infiltration Lepromin test Antimycobacterial antibodies (% patients with precipitins in serum) Plasma cells in lymphoid tissue Immune complex disease (erythema nodosum leprosum) Autoantibodies in serum (yo) Delayed hypersensitivity (o/c) to Dinitrochlorohenzene Hemocyanin

- or &

+++ +++ 11-28

+ or + + ++++ -

+-

-

82

95

+

+++ +++

*

30-50

3-1 1

90 100

75 100

50 100

added another point which they called “low-resistance tuberculoid lying between the tuberculoid pole and the central position on the spectrum formed by the borderline types of the disease. Ridley and Jopling (1962, 1966) introduced a scale which has been extremely useful in these immunological studies and was extended by Ridley and Waters (1969). The following classification was developed as a result of careful correlation of the clinical features of the disease and the histological pattern observed in skin biopsies. The leprosy spectrum TT-tuberculoid polar (high-resistance form) BT-borderline tuberculoid BB-borderline BL-borderline lepromatous LI-indefinite lepromatous LL-lepromatous polar (low-resistance form)

These are no more than arbitrary points extending from polar TT to polar LL, and patients may be at any point. In addition, in the early stages a patient may have not developed sufficiently definite characteristics to be placed accurately on the spectrum. Such patients are considered to have indeterminate leprosy and may later develop a more typical form of the disease or recover spontaneously. They may have no more than one to four areas of depigmentation of the skin with or without localized anesthesia. The classification of Ridley and Jopling (1966) with the modifications

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of Ridley and Waters (1969) is used throughout this review in order to provide a standard frame of reference. In order to place a patient in this spectrum it is important that simultaneous histological and immunological evaluations be made since the disease is not a stable one. The only stable points on the spectrum are the TT and LL extremes. Thus, a patient with BT may “downgrade” or lose resistance and move across the spectrum to BB or BL. Similarly a patient with LI may develop a “reversal reaction” and move to BB or BT as he develops immunity to the organism. Having swung across the spectrum in this way, he may regain or lose resistance again and swing back toward his original position on the spectrum. The most unstable point is BB, and a patient rarely stays at this point for long, moving rapidly to BT or BL. Interpretations of some investigative results are difficult because patients described as being tuberculoid may vary in their position from TT to near BB, and patients described as lepromatous may vary between LL and BL. Such investigations are not of homogeneous clinical groups, but on patients varying widely in their degree of resistance to Mycobacterium leprae or in the extent of their hypersensitivity reaction against the organism.

B. HISTOLOGICAL FEATURES OF THE LEPROSY SPECTRUM Much of our current knowledge about the immunological status of patients with leprosy is derived from histological examination of skin biopsies taken from individuals as the clinical picture changes and moves across the leprosy spectrum. There is a close relationship among the extent of the parasitism of the tissues with Mycobacterium leprae, the degree of lymphocytic infiltration, and the character of cells of the macrophage-histiocyte series which make up a large proportion of the infiltrate in the dermis. These skin biopsies show a striking inverse relationship between the degree of lymphocytic infiltration and the number of bacilli in the lesions of untreated patients. Patients with tuberculoid leprosy ( T T or BT) have scanty bacilli in their lesions and a high degree of lymphocytic infiltration. In lepromatous leprosy ( L I or LL) the tissues are infiltrated with sheets of macrophages containing masses of bacilli often in the form of “microglobi.” As well as the concentration of bacilli present in a lesion, the morphological appearance of the organisms is also important. The bacilli may appear solid, fragmented, or granular when stained by the Ziehl-Neelsen method. It is probable that those bacilli that are fragmented or granular ( nonsolid-staining ) are dead either as a result of chemotherapy or of the body’s own immune mechanisms ( Rees and Valentine, 1964; Shepard and McRae, 1965). Observations can be made of the different proportions of morphological

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forms seen in biopsics, and thc viability of thc organisms in any particular lcsions can be assessed and given numcrical values on an arbitrary scale called “the morphological index” ( \Vatcrs and Rces, 1962; Waters et al., 1967). Leprologists h n \ ~ long been a\varc~of morphological \.ariations of thc histiocyte-iiincrophagc. sc.ric,s i n lcsioiis biopsicd across the leprosy spectrum. The tcmiis epithelioid cell and lepru cell describe the cellular forms at the. tubc~rculoitl mtl lcpromatous cwds of the spectrum, respectively, and presumably reflect clificrences in the imniunological response of the host to the presensc of leprosy bacilli (Khanolkar, 1964). The lepra cell is a large uiidifl‘erentiatcd histiocyte, similar to that seen in experinicntal animals at thc site of the intradermal injcction of a nonantigenic colloidal material such as aluminum hydroxide or colloidal silica. These cells may be packed with microglobi of Jfycobncterium Zeprne in untreated patients They change gradually into cpithelioid cells, as the spectrum is crossed through borderlincx to tubcrculoid. The iinportance of this change in the histiocyte to the epithclioid form is associated with an increasing infiltration of the tissues with Iyniphocytcs and an incrc,asing ability of thc histiocytcs thcmsclves to eliminate Af . lepme. Thcre is cvidencc from cxperimcntal studies that thc cpithrlioitl a p p c ~ r a n c eof iiiacrophagcs may be rclatcd to their participation in a CAI1 reaction. Blanden ( 1968) showed that cpithelioid macrophages from Bacillus Calmette-Gut.rin ( BCG ) -infcctecl mice spread more rapidly and fully on glass and had much more extensive cytoplasm with morc numerous mitochondria than macrophages from normal animals. Godal et nl. (1971) fouiid that supernatants from mixed lyniphocytc cultures activated rabbit macrophages in vitro SO that they prolifcratecl, clongatcd, and foriiicd intracellular bridges and giant cells. They found similar activation of blood-derived macrophages from patients with tuberculoid leprosy in the presence of lymphocytes; no such activation of macrophages was obscrved in cultures from patients with leproniatous lcprosy. These histological observations coinbincd with the apparent lack of a relationship betwccn hunioral antibodies against niycobacterial antigens and resistance to infcction led to the suggestion that resistance to infcction with h f . leprae and other mycobactcria, such as Rdycobacterium tuberculosis, depends on CMI rather than any mechanism involving humoral (immunoglobulin) antiboclics (Turk, 1969). This interpretation would place lepromatous leprosy as an immunity deficiency disease in which the subject does not have the ability to develop a CMI response against the infecting organism. As the disease crosses the spectrum, CMI develops progressively. At the tuberculoid end of the spectrum, CMI is so great that in the process of eliminating the organism

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a hypersensitivity reaction is produced wherever mycobacterial antigen resides. Mycobacterium leprae resides preferentially in the skin and the peripheral nerves (Weddell et al., 1964); thus, tissue damage from delayed hypersensitivity reactions occurs especially in these tissues, accounting for the typical clinical and histological picture seen in tuberculoid leprosy. The histological picture of lesions of BT or low-resistance tuberculoid leprosy is particularly interesting. In this part of the spectrum the histological pattern in the tissues, especially the lymph nodes, may resemble that seen in sarcoidosis. The infiltrate is often multicentric consisting of discrete whorls of epithelioid cells with small numbers of typical Langhans-type giant cells surrounded by lymphocytes. The sarcoid appearance of the infiltrate in lymph nodes parallels the appearance of similar lesions in the skin. With the stronger CMI response seen in TT, histiocytes are far less prominent and do not, therefore, form such a typical pattern. In BB the histiocytes are less differentiated and do not have such a regular arrangement in the tissues. It would, therefore, appear that a sarcoid pattern of infiltrating cells is related to a particular position on an immunological spectrum. The relationship between the immunological capabilities of the sensitized cells and the load of the infecting organism would appear to be critical for the production of this particular pattern. It is well known that sarcoidlike patterns can be found in tissue infiltrates of other nonsarcoid conditions. An example of this is in lymph nodes draining certain carcinomas. It could well be that the sarcoid histological picture, far from being specific, merely reflects the tissue changes that occur following a chronic hypersensitivity reaction when the potential exists to eliminate most, but not all, of the antigen or infecting organism. Under these conditions as in tuberculoid leprosy it may be di5cult to demonstrate the infectious agent. C. THE LEPROMIN TFST Owing to the inability to culture Mycobacterium leprae, workers encountered considerable difficulty in providing a specific antigenic extract of this organism for immunological procedures. The only source of specific antigens of M . leprae was a crude preparation of autoclaved infected human or animal tissues (Cochrane, 1964; Rees, 1964). An attempt was made to purify this material by extraction with chloroform and ether (Dharmendra, 1942). Although it might be logical to use the purified preparation, many leprologists considered that the cruder preparation gave more consistent results and stronger, more interpretable reactions (Wade, 1961). Recently a cleaner preparation was made by

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enzyme digestion. In a comparison between lepromin produced in the latter way and the classic crude lepromin, no significant difference was found in 49 patients between reactions to the two preparations (Draper et al., 1968). Suspensions of lepromin are usually standardized to contain 1.6 x lo8 acid-fast bacilli per milliliter, and 0.1 ml. of the suspension is injected intradermally. The skin reaction to lepromin is biphasic. The first phase is a typical delayed-hypersensitivity reaction (the Fernandez reaction) read between 48 and 72 hours. The second (Mitsuda reaction) appears between 2 and 4 wecks and is generally read at 3 weeks. This reaction takes the form of an indurated skin nodule greater than 4 nim. in diameter, which usually ulcerates in strong reactions. It is generally considered that both the initial and late reactions are specific for antigens of M . leprae, and markedly parallel in most patients. However, most leprologists use the late reaction as an indication of lepromin positivity. With the chloroform- and ether-extracted preparation, the early reaction is stronger than the late one; whereas, with the more commonly used crude preparation, the late reaction is stronger and easier to interpret (Report of the Panel on Bacteriology and Immunology, 1963). A reaction to contaminating tissue components is thought to play a role in the late ( Mitsuda) reaction (Hart and Rees, 1967), yet it is also likely that the late release of antigen from intact bacilli may be important. Another factor to consider is the intradermal injection of this material which is also acting as an antigenic stimulus. The late reaction may result as the subject develops an immune response (either primary or secondary) to the material. A number of reports describe sensitization by lepromin and induction of lepromin positivity by BCCJ vaccination (J. A. K. Brown and Stone, 1961). Lepromin positivity does not necessarily indicate any previous contact with M . leprae or other mycobacteria ( Shepard and Saitz, 1967). Waters ( 1971) lepromin tested 65 normal Caucasians who had never been exposed to a leprosy endemic area nor received BCG and who were tuberculin negative. Only 1 gave a delayed hypersensitivity reaction at 72 hours; 38%gave positive Mitsuda-type reactions at 3 weeks. As the subjects gave negative delayed hypersensitivity reactions at 72 hours, the 3 week reactions strongly suggest primary sensitization by the lepromin itself. In a similar group who were tuberculin positive, one-third gave a positive delayed hypersensitivity reaction to lepromin at 72 hours, and all gave positive klitsucla-type reaction at 3 weeks. This would indicate that the antigens in lepromin, to which skin reactivity developed, were common to other bacterial species and not specific to M . leprae. The lepromin reaction therefore cannot be used to indicate whether or not a patient has been infected with M . leprae. It does, however, have

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a very important use in both clinical and experimental investigation. The ability of a patient to respond with the late or Mitsuda reaction appears to parallel his ability to eliminate M . leprae from the body. Thus lepromin reactions are negative in BL, LI, and LL leprosy and strongly positive in TT. In BT leprosy reactions are up to 8 nini. of induration, whereas in BB at the center of the spectrum reactions are up to 4 to 5 mm. The lepromin test also aids assessment of the direction in which a patient with indeterminate leprosy will pass. A patient with indeterminate leprosy who is lepromin positive may either recover spontaneously, as occurs in three-quarters of the patients, or develop leprosy on the tuberculoid side of the spectrum. A patient who is lepromin negative, however, does not have the capacity to eliminate the organism, and a strong probability exists that he will develop lepromatous type leprosy ( Khanolkar, 1964). The correlation between lepromin reactivity and host resistance to M . leprae is reflected in the histological appearance of the skin 4 weeks after the intradermal injection of lepromin. At the lepromatous end of the spectrum the host’s injection site consists of an accumulation of histiocytes unaccompanied by lymphocyte infiltration. At the tuberculoid end of the spectrum the nodule which develops consists of typical epithelioid cell follicles interspersed with a strong lymphocytic infiltration. In the middle of the spectrum the histiocytic infiltration may be similar to that seen in lepromatous leprosy, however. There may be considerable infiltration with lymphocytes, although less extensively than in tuberculoid leprosy (Kuper, 1964). Saul et aZ. ( 1969) looked at the histology of lepromin test sites 4 hours after intradermal injection in leprosy patients and those with other skin diseases. They found that no bacilli remained at the skin test site in patients with tuberculoid leprosy. However, in lepromatous leprosy patients, the bacilli were still present and had not been phagocytized. These authors felt that the inability of patients with lepromatous leprosy to phagocytize the bacilli, was related to the patients’ defect in CMI.

D. HISTOLOGICAL CHANGES IN LYMPHOID TISSUE IN LEPROSY The appearance of peripheral lymph nodes reflects fairly accurately the skin changes seen across the leprosy spectrum (Turk and Waters, 1971). In LI and LL, small lymphocytes in the paracortical area are strikingly depleted and are replaced by undifferentiated macrophages loaded with microglobi of Mycobacterium leprae (Turk and Waters, 1968) (Fig. 1 ) . However, those parts of the lymph node involved in humoral antibody production, far from being deficient, appear to be stimulated. The germinal centers are enlarged and surrounded by a normal cuff of

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FIG. 1. Lepromatous polar ( L L ) leprosy. Paracortical areas virtually depleted of small lymphocytes and replaced by histiocytes. Germinal centers, with marginal cuff of small lymphocytes and medullary plasma cells well developed. Stain: pyronin methyl green. Magnification: X160. (From Turk and Waters, 1971.)

small lymphocytes. There is also marked hyperplasia of plasma cells at the corticomedullary junction and in the medullary cords. This appearance is in keeping with drainage of parasitized macrophages from the peripheral lesions through the afferent lymphatics, Infiltration of similar undifferentiated macrophages into the paracortical areas via the afferent lymphatics has been observed in experimental animals following the induction of histiocytic infiltration in the skin of guinea pigs by the intradermal injection of nonantigenic substances, such as colloidal aluminum or silica (Gaafar and Turk, 1970) (Fig. 2 ) . Similar replacement of the paracortical areas with cells of the histiocyte macrophage series has been found in conditions where there was a depletion of the mobile

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FIG.2. Afferent lymphatic, draining site of intradermal injection of Al( OH)r, entering cortex of lymph node. The afferent lymphatic and adjoining lymph node cortex contain undifferentiated histiocytes similar to those seen at the skin injection site. Stain: hematoxylin and eosin. Magnification: X 1000.

pool of small lymphocytes involved in CMI, e.g., following treatment with antilymphocyte serum ( ALS ) or neonatal thymectomy (Turk and Oort, 1971). However, where infiltration of, these areas was in lymph nodes draining a peripheral site of histiocyte accumulation, these changes did not necessarily indicate a nonspecific failure of CMI. Such changes might be found in the lymph nodes draining the site of the intradermal injection of BCG vaccine into guinea pigs where a high level of CMI against Mycobacterium tuberculosis existed ( Gaafar and Turk, 1970) (Fig. 3 ) . The proliferation of plasma cells and marked germinal center formation in lepromatous lymph nodes is in keeping with the high incidence of precipitating antibodies against mycobacterial antigens ( Rees et al., 1965) and autoantibodies (Bonomo and Dammacco, 1968) in these patients. In TT, at the other extreme, there is no infiltration of the lymph nodes with cells of the histiocyte-macrophage series, but there may be changes suggestive of local stimulation of CMI. This consists of hyperplasia of lymphocytes in the paracortical areas (Fig. 4), with immunoblasts present, some of which may be seen in mitosis. Plasma cell proliferation and germinal center formation are not marked. In BT leprosy the lymph nodes show infiltration of the paracortical

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FIG. 3. Histiocytic replacement of paracortical area of draining lymph node 28 days after the intradermal injection of Bacillus Calmette-GuCrin vaccine. Note sparing of cells round the germinal centers and medullary sinuses. Stain: Methyl green pyronin. hlagnification: X 160. (From Gaafar and Turk, 1970.)

areas with epithelioid cells which form discrete whorls and tubercles. The small lymphocytes in these areas do not appear depleted, and the general imprcssion is of sarcoidlike changes similar to those seen in the skin (Fig. 5). As the disease moves across BB and BL, the histiocytic infiltrate in the paracortical areas increases, and the cells become less and less differentiated until at the lepromatous pole they are typical undifferentiated lepra cells (Fig. 6 ) . Concentrations of Mycobucterium Zeprne in these cells increase. In the TT forin, bacilli are not seen; in BT, they are scanty; by BB, they begin to be found more regularly. Similarly there is an increase in germinal centers and plasma cell proliferation toward the lepromatous poIe. In patients with LL who do not regain CMI when treated, despite elimination of mycobacteria from the tissues, the paracortical areas of the lymph nodes may still be infiltrated with histiocytcs after 10 years. In patients with LI leprosy who regain CMI on antibacterial therapy and move across the spectrum to BB or RT, there is a progressive increase in the number of small lymphocytcs populating the paracortical areas. These cells are often initially seen around the postcapillary venules (Fig. 7).

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FIG. 4. Tuberculoid polar ( T T ) leprosy. Hyperplasia of lymphocytes in paracortical area. Stain: hematoxylin. Magnification: X400. (From Turk and Waters, 1971).

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FIG. 5. Borderline tu1)erculoid ( BT) leprosy. Paracortical area replaced by multicentric discrete whorls of epithelioid cells. Stain: hematoxylin and eosin. Magnification: X300. (From Turk and Waters, 1971.)

FIG.6. Indefinite lepromatous ( LI ) leprosy. Undifferentiated histiocytes infiltrating paracortical area. Stain: hematoxylin and eosin. Magnification: X750. (From Turk and Waters, 1971.)

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BRYCESON

FIG. 7. Reversal reaction from indefinite lepromatous ( L I ) to borderline ( B B ) leprosy. Lymphocytes repopulating the paracortical area around a postcapillary venule. Histiocytes still have “lepromatous” appearance. Stain: heniatoxylin and eosin. Magnification: X300.

E. ALLERGICREACITONS IN LEPROSY Although leprosy is basically a chronic disease, the course of the condition is sometimes punctuated by acute exacerbations. TWOtypes of allergic reaction states can be distinguished. The state referred to as reversal reaction (Ridley, 1969) occurs in patients at the lepromatous end of the spectrum appears to be associated with a return of CMI and a rapid swing across the spectrum from LI or BL to BB or BT. It has many characteristics of an acute delayed hypersensitivity reaction. The other reaction also occurs in patients at the lepromatous pole and appears due to deposition of immune complexes in the tissues, especially the skin. This condition is referred to as erythema nodosum leprosum by some leprologists and the lepra reaction by others.

I. Reversal Reactions These reactions occur in LI and BL patients, when the bacterial load is diminished as a result of treatment. Clinically there is erythema and swelling of the skin lesions, which may ulcerate. In severe cases patients may be febrile, and there may be peripheral nerve involvement. Histologically skin lesions show increased infiltration by small lympho-

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cytes, the cells of the histiocyte-macrophage series become more epithelioid, and the number of bacilli in the lesions diminishes. Serial lymph nodes biopsies have recently been studied in four patients with reversal reactions (Turk and Waters, 1971). Little change was noted in two patients who had mild reversal reactions. However, in two patients who had more severe reactions shifting from LI to BB in one case and BT in the other, the histological appearance of the lymph nodes showed marked changes. Although the histiocytes infiltrating the paracortical areas remained vacuolated and contained mycobacteria, the infiltrate was broken up by broad bands of lymphocytes, which in some areas were scattered diffusely among the infiltrate cells. In other areas small or large accumulations of lymphocytes could be seen situated around the postcapillary venules. The mycobacteria were extremely granular and fragmented. Germinal center formation and plasma cell hyperplasia were still prominent and apparently unaffected. It would, therefore, appear that reversal reactions are caused by a rapid increase in CMI against Mycobacterium leprae in patients where CMI was originally low. Immunity returns in association with the decrease in antigenic load following the onset of therapy, and with it the patient regains the capacity to produce hypersensitivity reactions at the site of residual bacterial deposition. Thus the sites of reaction are those where M . leprae accumulate-the skin and peripheral nerves Clinically and histologically the reactions are those associated with CMI.

2. Erythema Nodosum Leprosum Erythema nodosum leprosum (ENL) occurs also in patients at the lepromatous side of the spectrum. As its name implies the condition is associated with the development of crops of red nodules in the skin. These nodules last for 24 to 48 hours, and the condition is frequently accompanied by fever. However, in more severe cases there may be other systemic manifestations such as arthritis, iridocyclitis, orchitis, and an acute painful neuritis. Fever is often associated with proteinuria. Although the skin lesions are generally erythematous and nodular they may become hemorrhagic and necrotic. Histologically the lesions in the skin differ from those seen in reversal reactions as they are the site of intense polymorphonuclear leukocyte infiltration, and the blood vessels in the dermis show acute fibrinoid necrosis (Waters and Ridley, 1963). Although ENL was seen occasionally in the past, it has become much more common since the use of chemotherapy in leprosy. Previously, ENL was recorded as being precipitated by many causes such as malaria, typhoid fever, appendicitis, and even emotional upsets. Nowadays as many as 50%of patients at the lepromatous side of the spectrum

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develop evidence of ENL by the end of their first year of treatment with sulfones. It, therefore, appears that ENL is related in some way to the destruction of mycobacteria and the possible release of mycobacterial antigens. The clinical and histological features of ENL in the skin are reminiscent of those found in the Arthus reaction. The systematic manifestations are among those of an immune complex disease caused by soluble circulating immune complexes formed with humoral antibody. Erythema nodosum leprosum occurs in that phase of the disease when there is high incidence of precipitating antibody in the circulation against mycobacterial antigens, and soluble mycobacterial antigens can sometimes be detected in the circulation (Rees et al., 1965). In biopsies of ENL lesions in 38 lepromatous patients, immunoglobulins and PIC/PIA globulin were detected in the areas of polymorph infiltration in 20 patients (Wemambu et al., 1969; Waters et al., 1971) (Fig. 8 ) . No such deposits were found in lesions from 13 patients with lepromatous leprosy who had not had ENL. The deposits were granular in form and did not correspond to the areas of bacillary infiltration. Deposits were sometimes demonstrated within the walls of blood vessels in the deep dermis. In 7 of the patients, evidenec suggestive of soluble mycobacterial antigen could be detected in the deposits. Erythema

FIG.8. Granular deposits of immunoglobulins in the dermis in erythema nodosum leprosum localized in region of maximal polymorphonuclear leukocyte infiltration. Fluorescence microscopy. Magnification: X 100. ( From Wemambu et al.,

1969.)

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nodosum leprosum always occurred in that part of the spectrum where CMI against Mycobacterium leprae antigens appeared to be deficient and was not associated with the development of CMI and movement across the spectrum from the lepromatous to the tuberculoid side. It, therefore, appears due to action between humoral antibody and mycobacterial antigen under conditions in which CMI is deficient.

F. EXPERIMENTAL PRODUCTION OF LEPROSY IN LABORATORY ANIMALS Until 1960 it was not possible to transmit Mycobacterium leprae to experimental animals. Even now it cannot be grown in vitro. Much information may be obtained as to immunological changes which occur in human leprosy from a study induction of a similar disease in mice inoculated with M . leprae. The first regularly reproducible means of transmitting M . leprae to experimental animals was described by Shepard in 1960. From lo2 to loGM . Zeprae bacilli, derived mainly from human nasal washings, were inoculated intradermally into the footpads of mice. With a dose of 10 to lo6 bacilli, macroscopic granulomas were detected at the inoculation site after 1 to 2 months. However with lo3 organisms, granulomata took up to 6 months to develop. After the development of the lesion the number of bacilli present never exceeded lo6 organisms regardless of the number injected. This indicated that host resistance kept the infection in check so that further proliferation could not occur. In an attempt to overcome this barrier, Rees and his colleagues (Rees, 1965; Rees et al., 1967) injected M. leprae into mice which had been thymectomized, given total body irradiation of 900 r, and transfused with syngeneic bone marrow at the age of 6 weeks. Thymectomy and irradiation considerably enhanced the infection so that lo9 bacilli could be obtained from the injection site. Moreover the skin covering the injected mouse foot developed nodular swellings similar to those seen in lepromatous leprosy. A disseminated infection was obtained in thymectomized irradiated mice which were injected intravenously with 3 x lo7 M. kprae bacilli. In this case nodular lesions were observed in the nose and ears and in both the fore and hind paws. The skin of these lesions was histologically similar to that seen in lepromatous leprosy. Apparently to obtain a disease similar to lepromatous leprosy in mice, the animal's immunological activity and especially CMI have to be depleted enough to accept and retain allogeneic skin grafts. Despite this it still took about 19 months after intravenous injection of M . leprae for the mouse to develop a disease similar to Iepromatous leprosy. In further experiments ( Rees and Weddell, 1968), thymectomized irradi-

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ated mice were injected intraperitoneally with lymphocytes equivalent to those present in the normal mouse 10 months after the injection of M . leprae, when macroscopic lepromatous lesions had already developed in the footpad. Six to 10 days later the areas of skin in which M . leprae were present became red and swollen. Bacteriological examination showed that the bacilli became degenerate, and the yield of organisms obtainable from the tissues dropped between ten- and a hundred-fold. Histologically there was evidence of increased lymphocytic infiltration of the infected tissues. Bacilli undergoing degeneration could also be seen in Schwann cells, and occasionally lymphocytes were seen invading the endoneurium. These changes were similar to those seen in the human with lepromatous leprosy undergoing a reversal reaction and were consistent with this condition, caused by return of CMI directed against the organism. In other experiments (Gaugas et al., 1971)) a massive reversal reaction was obtained 5 weeks after implantation of neonatal syngeneic thymus tissue. A further point on the leprosy spectrum reproduced in experimental animals was described by Rees et a2. (1969). In the original model, normal mice were infected with M . leprae (Shepard, 1960) and were followed for no longer than 1 year. Rees et al. (1969) fallowed mice infected in the same way for between 2 and 3 years and found that despite the low bacteriological counts pronounced histological changes indicated that the infection had not died out. The histological appearance was of an epithelioid cell granuloma containing very few bacilli and surrounded by loosely packed lymphocytes similar to BB to BT in man. The lesions were mainly localized to the injection site, yet they were occasionally disseminated. As in borderline leprosy in man, there was disorganization and cellular infiltration of nerve bundles in the deep dermis. Lepromatous leprosy seems to be a form of the disease in which CMI is deficient, and borderline leprosy a form in which CMI is normal with a balance struck between host resistance and bacterial growth. Any situation that depends on the maintenance of equilibrium tends to be unstable. This would, therefore, account for the known instability of patients with the borderline forms of leprosy who can move toward the lepromatous pole if CMI is reduced or to the tuberculoid pole if it is intensified. If enough infected, thymectomized, irradiated mice are followed for a long enough period they show a wide range of disease states corresponding to a number of points on the spectrum from LL to BT. About 10% develop the lepromatous form of the disease with enlarged footpads. Other mice, after passing through a highly bacilliferous phase, develop a form of the disease resembling BL or BT. This is probably because

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the syngeneic bone marrow, transfused to repopulate the hemopoietic system, contains a small number of lymphocytes which can themselves eventually reconstitute the lymphoreticular system. Although originally depleted, the paracortical areas of the lymph nodes became repopulated with lymphocytes 6 months after thymectomy and irradiation ( Shepard, 1971) . Attempts have been made to enhance leprosy infection in mice by the use of ALS (Gaugas, 1968). In these studies, ALS treatment was combined with thymectomy of 6-week-old animals. Nine and a half months after infection the yield of the bacilli from the footpads was 30 times higher than in mice which had been thymectomized and not treated with ALS. The mice could not, however, be followed longer as they all developed polyoma-type tumors (Gaugas et al., 1969). OF CELL-MEDIATED IMMUNITY IN G. DEPRESSION HUMANLEPROSY

As disease similar to lepromatous leprosy could be induced in mice following nonspecific suppression of CMI, interest was aroused in observations suggesting that a similar deficiency might underlie leprosy in man. This approach high-lighted recognition that possibly people developed lepromatous rather than tuberculoid leprosy related to constitutional predisposition to this form of the disease. Lepromatous leprosy seems not to occur in a fixed proportion of leprosy infections. Newell (1966) observed that a population in which the rate of leprosy infection was decreasing did not have a parallel fall in the incidence of lepromatous leprosy. He, therefore, suggested that the development of lepromatous leprosy was a host-determined characteristic present in a fixed proportion of all people everywhere. Moreover, evidence that leprosy in its various forms occurred more frequently in certain families in endemic areas has been cited by a number of workers since it was first described by Danielsen and Boeck in 1848. Thus a search was made for a genetically controlled defect in the immune response that might contribute to the failure of host resistance. Guinto and Mabalay (1962) observed that only 47.4%of patients with lepromatous leprosy were tuberculin positive, whereas the incidence of tuberculin positivity in the normal population from which the patients came was 81.3%.Jamison and Vollum (1968) recently cited evidence which suggested that leprosy occurred in families in which there was a natural weakness in ability to develop CMI. When tuberculin-negative children with a family history of leprosy were vaccinated with an avirulent strain of Mycobacterium containing antigens that cross-reacted with M . tuberculosis (vole tuberculosis vaccine) only 18%were con-

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verted to tuberculin positivity, whereas 90%of children who came from families where there was no history of leprosy were converted to tuberculin positivity by the vaccine. This could indicate that children from families in which leprosy occurred were less able to develop strong CMI than children from families in which leprosy did not occur. The generalized depression of the ability of leprosy patients to mount CMI has been investigated very fully over the past 5 years, in an attempt to detect a correlation between nonspecific depression and the development of the lepromatous state. Diminished ability of patients with leprosy to become sensitized to a chemical sensitizing agent has now been shown in a number of centers ( Waldorf et al., 1966; Bullock, 1968; Turk and Waters, 1969). Waldorf et al. ( 1966) found that 75%of patients with lepromatous leprosy without ENL failed to be sensitized to dinitrochlorobenzene (DNCB), whereas only 1 out of 5 patients with borderline leprosy failed to be sensitized in this way. Bullock (1968) failed to sensitize 70%of a group of lepromatous patients, who had been under treatment for less than 18 months, to picryl chloride, although he could sensitize 94%of normal people with this agent. In a second group of lepromatous patients, who had been under treatment for over 18 months, 47% could not be sensitized with picryl chloride. Of patients with tuberculoid leprosy, 55% could not be sensitized to picryl chloride. This study raised a number of interesting points. The first was that this author found that 15%of his lepromatous patients were lepromin positive. The second was that, although 70%of the patients in his lepromatous group treated for less than 18 months failed to be sensitized to DNCB, only 50%were tuberculin negative. The third point was that diminished CMI was not restricted to the lepromatous group but was also found in the tuberculoid group. Thus there was a lack of correlation between the deficiency in ability to develop contact sensitivity and tuberculin reactivity with the loss of host resistance to Mycobacterium leprae. The lack of correlation between tuberculin reactivity and ability to be sensitized to a chemical contact sensitizer indicated that failure of chemical contact sensitivity probably detected only a partial failure of CMI and that patients who could not be sensitized with a contact agent might be induced to develop delayed hypersensitivity to a more powerful antigen. With this in mind, Turk and Waters (1969) compared the ability of patients with lepromatous leprosy to develop contact sensitivity to DNCB and delayed hypersensitivity to keyhole limpet hemocyanin (KLH) (Swanson and Schwartz, 1967). All 10 patients who failed to be sensitized with DNCB could, however, be induced to develop delayed hypersensitivity to KLH. It was concluded that contact sensitization with DNCB represented a relatively weak stimulus of CMI and

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should not, therefore, be considered as an absolute indicator of ability to manifest CMI as several investigators have assumed. A further indication of some degree of nonspecific failure of CMI in leprosy was the observation of Job and Karat (quoted by Hart and Rees, 1967) that a delay i n rejection of skin homografts could be demonstrated in 4 patients with lepromatous leprosy using skin from donors with a similar type of leprosy. One graft was still healthy after 10 days, and the other three showed signs of rejection 19, 20, and 30 days after grafting. Han et nl. (1970b) compared the rejection of skin homografts in 10 patients with leprosy and 10 normal subjects. The mean graft survival times were for nornial subjects 11.22 days, lepromatous patients 15.2 days, and tuberculoid patients 13.44 days. In a further study, Han et al. (1970a) found suppression of the ability to manifest a normal lymphocyte transfer reaction ( Brent and Medawar, 1963) in patients with lepromatous leprosy. Patients with tuberculoid leprosy also showed a mild degree of reduction in response. Ptak et al. (1970) investigated the nonspecific depression of CMI in mice infected with A4ycobacteriuni leprae muriuni. Contact sensitivity to picryl chloride was suppressed as early as the thirteenth week after infection, and response to oxazolone, a stronger sensitizer, was diminished by 16 weeks. Infected mice also showed a prolongation of skin graft survival. These mice had a similar histological appearance in their lymph nodes to humans with lepromatous leprosy. It, therefore, appeared likely that nonspecific suppression of CMI in lepromatous leprosy might be related to replacement of the paracortical areas of lymphoid tissue by the histiocytic infiltrate and diminished ability of lymphocytes to proliferate in these areas (Turk and Waters, 1968). The situation would then be comparable to Hodgkin’s disease where the degree of CMI loss was related to lymphoid tissue replacement (R. S . Brown et al., 1967). However, in R further study (Turk and Waters, 1969), no difference could be found in the degree of replacement of the paracortical areas of lymph nodes from patients with lepromatous leprosy who were DNCB reactors or nonreactors. Moreover no correlation existed in individual patients betwcen DNCB reactivity and the degree of host resistance to M . leprae. Nonspecific suppression of CMI was found as readily in patients with LI leprosy who regained CMI when the bacterial load was reduced as in LL leprosy where host resistance to M . Zeprae was lost for good. Moreover, inability to be sensitized to picryl chloride has been described in tuberculoid leprosy ( Bullock, 1968). Further evidence for a loss in CMI has been sought in studies of the ability of lymphocytes from patients with leprosy to respond in vitro by transformation into blastlike cells when stimulated with antigens or

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mitogens. The first investigation of this type was by Dierks and Shepard (1968) in which lymphocyte transformation was determined only by morphological criteria. Lymphocytes from 8 active lepromatous patients had a low response to phytohemagglutinin (PHA) (only 10% of the cells being transformed by the mitogen). In another series, 3 patients with active lepromatous leprosy had markedly diminished response. In keeping with the finding of Bullock (1968) that patients with tuberculoid leprosy also had reduced ability to be sensitized with picryl chloride, Dierks and Shepard (1968) reported 3 patients with tuberculoid leprosy whose lymphocytes responded poorly to transformation in vitro with PHA. Since this initial study of reduced lymphocyte responsiveness in leprosy, the literature has been confused. Thus Sheagren et d. (1969) found no differences between the mean of the percent transformed cells of a control group and groups of patients with leprosy, using PHA as mitogen. However, they did find reduced responsiveness to the specific antigen streptolysin O( SLO). These authors also assessed lymphocyte transformation using morphological criteria, supplemented by radioautography for the uptake of 3H-thymidine by the transformed cells. Bullock and Fasal (1968) were able to detect depression of transformation both by PHA and by SLO. However depression of response to SLO only occurred when the cells from lepromatous patients were cultured in autologous plasma. Moreover certain lepromatous plasmas would depress the ability of the lymphocytes from normal people to respond to SLO. These plasmas had no effect on PHA transformation. The three initial reports were from the United States, and no attempt was made to correlate the phenomena with the racial groups involved. Talwar ( 1970) and his colleagues in India investigated PHA responsiveness of leprosy patients using precise quantitative techniques for estimating the uptake of 3H-thymidine by transformed lymphocytes stimulated with PHA. They emphasize that peak days of mitotic activity might vary from patient to patient and that comparison should be made between the rcsponses of lymphocytes on peak days of culture. They observe that so many factors including chemotherapeutic treatment might modify the responsiveness of lymphocytes in vitro that results are often impossible to interpret. The difficulties in interpreting results of this type of experiment were emphasized in two other recent investigations, those of Han and his colleagues in Taiwan and of Nelson and his colleagues in Malaysia. Han (1970) found depression of lymphocyte transformation to both PHA and tuberculin-purified protein derivative ( PPD ) in leprosy patients. Responsiveness was partially regained during treatment. However, he also observed that pooled plasma from leprosy patients appearcd to enhance PHA-induced deoxyribonucleic acid ( DNA)

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synthesis of normal, lepromatous, and tuberculoid leprosy lymphocytes. Nelson et al. (1970) found differences in responsiveness of lepromatous lymphocytes to PHA in different ethnic groups studied. Thus lymphocytes from lepromatous Chinese transformed more extensively than those from normal Chinese, whether cultured in autologous or a standard normal serum. Cells from lepromatous Malays and Indians transformed less well when cultured in autologous serum, although they transformed normally when cultured in normal serum. Serum from Malays or Indians having tuberculoid leprosy depressed the transformation of normal lymphocytes, compared with the serum of normal people of the same ethnic groups. Serum from lepromatous Chinese patients depressed the response of the lymphocytes of 1 normal subject but not those of another. Normal lymphocytes transformed significantly less well when cultured in the serum from lepromatous or tuberculoid Malays or from lepromatous Indians than when cultured in serum from normal controls. There was, thus, no evidence of an intrinsic defect in lymphocyte function in this study. The differences in the effect of serum on the response of cells from two normal donors wcre interesting and could well have been due to differences in the HL-A antigens of the cell donor. The overall impression from these studies is extremely confusing and difficult to interpret. It is most unlikely that they bear any relevance to the underlying defect in CMI since serum inhibitory factors were found in patients with tuberculoid as well as lepromatous leprosy. Further clarification may come from experiments along these lines. If these results are related to the pathogenesis of lepromatous leprosy, there may be two mechanisms by which they influence the defect in CMI. One could be direct disturbance of lymphocyte function either as a result of possible virus infection or a more fundamental genetically controlled enzyme dysfunction. The other could be related to the circulating antilymphocyte factor. This factor might be a by-product of the metabolism of microorganisms such as an enzyme akin to the immunosuppressive ribonuclease described by Mowbray et al. ( 1969). Another possibility is that the inhibitory factor may be an antilymphocyte autoantibody, as there is a high incidence of autoantibody formation by these patients. Patients with circulating antilymphocyte antibodies have severe depression of the response of their lymphocytes to PHA. However, as in the leprosy patients, this only occurs if the cells are cultured in autologous plasma, In the presence of normal homologous plasma the response of the cells is normal. Correspondingly the plasma of patients with antilymphocyte antibodies inhibits the ability of PHA to transform lymphocytes from normal people ( hqelli et al., 19SS). Finally in assessing the significance of failure of lymphocyte func-

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tion in the pathogenesis of lepromatous leprosy, it must be mentioned that this failure is far from universal. In early reports such defects were only found in a proportion of patients studied, and in one series from Mexico, no such defect could be detected ( Salazar-Mallen et al., 1970). There have been a number of studies aimed at detecting specific failure of CMI to Mycobacterium leprae in lepromatous patients. The specificity of immunological unresponsiveness underlying the loss of host resistance in lepromatous leprosy has been stressed previously (Turk, 1969). Also, Convit (1970) in Venezuela compared specificity of unresponsiveness in two similar diseases, lepromatous leprosy and diffuse cutaneous leishmaniasis ( see below). Patients with the lepromatouslike disease caused by Leishmania who were leishmanin negative responded with a positive Mitsuda reaction to the intradermal injection of lepromin. Patients with lepromatous leprosy were then injected intradennally with live Leishmania. They did not develop diffuse cutaneous leishmaniasis, only a delayed hypersensitivity reaction at the inoculation site. Salazar-Mallen et al. (1970) could find no diminution in skin reactivity to coccidioidin and histoplasmin in lepromatous patients. A further approach to study failure of host resistance in leprosy has been to observe the ability of blood monocytes to lyse Mycobacterium leprae in oitro. Beiguelman (1967) reported that blood macrophages from both lepromatous and tuberculoid patients actively phagocytized dead leprosy bacilli in tissue culture. The macrophages from the tuberculoid patients completely lysed the ingested bacilli freeing them of lipid. However, the macrophages from the lepromatous patients were unable to lyse the organisms and developed the appearance of typical lepra cells, containing numerous bacilli and drops of lipid material which stained with Sudan black. Similarly, Hanks ( 1947) cultured phagocytic cells, morphologically resembling fibroblasts, from patients with leprosy. Those from tuberculoid patients were able to lyse the organisms, whereas those from lepromatous patients became degenerate and released the organisms into the medium. Although unable to lyse M . leprae, cultured macrophages from patients with lepromatous leprosy were able to lyse M . leprae murium and Mycobacterium tuberculosis indicating that in this system, the failure of cellular immunity was specific to M . leprae. This system appears ideal for investigating the failure of CMI in leprosy, yet reproduction in two other laboratories was difficult ( Delville, 1970; Rees, 1970). However, this appears to be a most promising approach to the eventual elucidation of the fundamental defect in lepromatous leprosy. In attempting to analyze the underlying basis for development of

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lepromatous leprosy one must start by accepting Newell’s (1966) suggestion of genetic determination. However, although a nonspecific depression of CMI can be detected in some patients, it cannot be detected in all and must, therefore, be a secondary development, possibly related to the massive infiltration of the thynius-dependent areas of lymphoid tissue with lepromatous tissue. Despite this, there must be some inherent defect, as yet uncovered, which underlies the inability of the patient to eliminate the organism before it can take a hold in the tissues. In untreated borderline patients, factors ( e.g., intercurrent infection or pregnancy) which lower nonspecific resistance may allow the leprosy bacillus to multiply, possibly as early as the indeterminate stage of the disease, and induce a state of specific immunological tolerance. This would result in deterioration of the disease toward the lepromatous pole. In such patients the killing of the organism by esective treatment may allow tolerance to be broken, rcsulting in a reversal reaction and a shift in the disease toward the tuberculoid side of the spectrum. Such a shift occurs in 10%of patients with LI (i.e., lepromatous patients who passed through an earlier borderline phase) and in one-third of patients with BL leprosy by the end of the first year of effective antileprosy treatment (Ridley and Waters, 1969). It appears probable that patients with LL have a significant genetic impairment of response to antigens of Mycobacterium leprae. As a result, they develop tolerance more easily and, therefore, do not pass through a borderline phase. Moreover the degree of tolerance is so great that they cannot regain immunity when the antigenic load is reduced as a result of effective antileprosy therapy. Strikingly, tolerance to 31. leprae associated with failure of host resistance is restricted to CMI; there is no equivalent failure of humoral antibody production. This raises the question whether the CMI tolerance may be related to the high level of circulating antibody against the given antigen and result from immunological enhancement similar to that described against tumor antigens. Kaliss ( 1958) suggests that antibody reacts with antigen in the tumor graft in such a way that the antigen is no longer susceptible to the action of CMI directed against it. Such a mechanism could protect the RI. Zeprae antigens from the CMI involved in host resistance. An experimental model which parallels the impairment of CMI in lepromatous leprosy is referred to as “immune deviation” ( Asherson and Stone, 1965). Guinea pigs can be made incapable of CMI and y2 antibody production to a particular antigen. However, they still produce normal levels of y l antibody against this antigen when immunized. This form of unresponsiveness appears to occur as a direct result of changes in the cells involved in CMI and not to the inhibitory effect of a circulat-

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ing antibody, as the cells from such animals do not transfer delayed hypersensitivity reactions. Moreover transfer of serum from these animals does not affect the induction or peripheral manifestations of delayed hypersensitivity in animals sensitized subsequently or previously with the same antigen (Asherson, 1966). Specific CMI unresponsiveness can be produced inexperimental animals even after they have been shown to be sensitive by giving them as high an antigenic load as possible (Polak and Turk, 1968). In these experiments, although ability to develop CMI is suppressed, ability to develop a specific immunological reaction resembling the Arthus phenomenon is not modified.

H. IMMUNOGLOBULINS, COMPLEMENT, AND HUMORAL PRODUCTION ANTIBODY 1. Zmmunoglobulins The mean level of IgG reported in lepromatous patients varied from center to center (Table 11). Thus patients in the United States averaged 15.1 mg./ml. (normals-mean 10.1 mg./ml.) (Sheagren et al., 1969), in Formosa 25.7 mg./ml. (normals-mean 20.37 mg./ml.) Bullock et al., 1970). No rise in IgG was observed in lepromatous patients with ENL, although higher levels were found in patients with amyloid ( Sheagren et al., 1969). These slightly raised IgG levels probably reflected the high incidence of other infections in individuals with leprosy. Raised IgA levels (mean 4.0-5.0 mg./ml.) were found in all three series in lepromatous patients. Sheagren et al. (1969) and Waters et al. (1971) found no difference between patients with and without ENL. However, Bullock TABLE I1 MEANIMMUNOGLOBULIN LP:VE:I,SI N LEPROMATOUS LEPROSY mg./ml.

Authors

Country

Sheagren ct al. (1969) Bullock ct al. (1070)

Waters ct al. (1971) ENL-erythema

United States (with ENL)a (no ENL) Taiwan (with RNL) (no ENL) (Treated for longer than 24 months) (with ENL) (no ENL) (Treated for less than 12 months) Malaysia (with ENL) (no ENL)

nodosum leprosum.

IgG

IgA

IgM

13.5 15.1 28.6 25.4

3.9 3.6 7.1 3.5

1.7 1.3 1.1 1.1

23.7 25.2

4.6 4.7

1.3 1.3

23.5 23.8

5.0 5.0

2.2 2.8

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et a2. (1970) found higher levels of IgA in patients with reactive states who had been on antileprosy treatment for more than 24 months. All three studies described patients with lepromatous leprosy and ENL with IgA levels from 9 to 11 mg./ml. The significance of these findings was not clear but might be related to the heavy bacterial infiltration of the mucosa and the release of solublc antigen during therapy in the region where secretory IgA was produced. Moderately raised IgM levels were found in only one of the studies (Waters et al., 1971). 2. Complement Levels Sheagren et al. (1969) found that their leprosy patients lacking ENL had levels of hemolytic complement in the same range as controls, whereas those with ENL had a 30% increase. High levels of total hemolytic complement and especially C2 were also described in patients with ENL (Saitz et al., 1968). High levels of p,C/p,A globulin were found in patients with lepromatous leprosy with and without ENL ( Weniambu et al., 1969; Waters et al., 1971). High levels of complement in leprosy possibly associated with prolonged immune complex deposition could be related to similar high levels in acute pyogenic infection, rheumatic fever, Reiter's syndrome, acute nonspecific polyarthritis, acute gout, acute myocardial infarction, and rheumatoid arthritis ( Townes, 1967).

3. Antibody Production against Extrinsic Antigens Sera from all patients with lepromatous leprosy contained antibodies which reacted strongly causing precipitation in gel in a dilution of between 1:80 and 1:100 with culture filtrates of Mycobacten'um tuberculosis. Similar levels of precipitating antibodies were found in BL and fell steadily over a period of 24 months treatment. These antibodies were not found in patients with TT. However low levels could be detected in the serum of patients with BT (Rees et al., 1965). Antibodies were also detected in lepromatous leprosy patients against M . leprae which cross-reacted with antigens in other mycobacteria (e.g., MycoDacteritcm Oalnei, Mycobacterium niurium, and Mycobacterium p h k i ) . In a similar study (Norlin et al., 1966) precipitins against antigens from a number of common mycobacteria including Mycobacterium kansasii and Mycobacterium smegmatis were found in the sera of all of 19 lepromatous patients and in half of patients with tuberculoid lesions who were bacillary positive (probably BB or BT) . However precipitins were not found in the sera of 22 bacillary-negative tuberculoid patients (probably TT). Sheagren et al. (1969) immunized leprosy patients with Salmonella

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typhi antigen and found that patients with all types of leprosy were capable of producing the same amounts of hemagglutinating antibody against this antigen as normal subjects. Alnieida et al. (1964) found a slightly increased level of antibody production to S. typhi H antigen in patients with lepromatous leprosy when compared with normal subjects. There did not, therefore, appear to be any defect of the antibodyproducing mechanism to extrinsic antigens. The bacillary load in leprosy patients and the amount of antibody present at any time to mycobacterial antigens seemed directly related. Rees et al. (1965) and Norlin et al. (1966) found these antibodies in patients with bacilliferous forms of the disease only. Moreover a fall in the titer was observed over a period of 24 months of antileprosy treatment (Rees et al., 1965) while the bacillary load was falling. 4. Antibodies to Intrinsic Antigens

Numerous reports describe increased incidence of antibodies to intrinsic antigens in patients with lepromatous leprosy (Matthews and Trautman, 1965; Bonomo and Dammacco, 1968; Wager, 1969). The highest incidence of these autoantibodies was : antinuclear factor, 30%; lupus erythematosus (LE) cells, 8%; rheumatoid factors, 50%; thyroglobulin autoantibodies, 40%; cryoglobulins, 95%; biologically false positive tests for syphilis, 70%. These figures have tempted some authors to compare lepromatous leprosy with systemic lupus erythematosus (Matthews and Trautman, 1965). However, as Wager (1969) emphasized, circulating autoantibodies in general do not seem to be directly deleterious to the host, with the notable exception of autoantibodies causing red cell destruction in autoimmune hemolytic anemia. Since Matthews and Trautman (1965) failed to find cold agglutinins against human 0 erythrocytes in patients with leprosy, Wager (1969) concluded that circulating autoantibodies are not likely to play any major part in the pathogenesis of leprosy. The presence of a spectrum of autoantibodies in other infectious diseases is well documented. Antibodies against the nonspecies-specific antigen present in heart muscle occur not only in patients with syphilis, but in a wide range of other infectious diseases including malaria (Davis, 1944). Rheumatoid factor has been found in the sera of 10% of patients and cryoglobulins in sera of 15%of patients with syphilis (Mustakallio et al., 1967). A high incidence of rheumatoid factor was described in the sera of patients with kala azar (Kunkel et al., 1958) and also in subacute bacterial endocarditis (Williams and Kunkel, 1962). Autoantibodies against liver, lung, or both organs were present in 27% of patients infected with Sclaistosoma haematobium (Shamma et al., 1965).

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Autoantibodies against lung and other tissues were evident in serum of patients with chronic pulmonary tuberculosis ( Lehman-Fascius and Loeschkc, 1926; Fisclier, 1933) , Similar autoantibodies developed in rabbits within 12 weeks after infection with hlycobacterium tuberculosis or Pasteurelln psezirlotuberculosis ( Ali and Oakley, 1967). In leprosy tlie body probably harbors more infective organisms than in any other disease aiid for longer periods of tinic. itlycobacteriuin spp. are among the best adjuvants for antibody production in the human. The iiicidencc of antibodicxs produced to intrinsic antigens higher than in other infectious diseases is thus not surprising. How chronic stiniulation of bacteria and other microorganisins may encourage autoantibody production is outside tlie scope of this review; however, among the possibilities are ( I ) direct antigenic modification of host cells, e.g., by bacterial enzymes, ( 2 ) cross-reaction betwcen antigens carried by microorganisms and normal tissue coniponcnts, aiid ( 3 ) nonspecific stiniulation of those autoantibody-forming cells that arc present in small numbers in normal people and which increase with age (Rowley et aZ., 1968) . Ill. Leishmaniasis

A. INTRODUCTION In this section the concept is put forward that leishnianiasis is a spectral disease similar to leprosy, in which the clinical features of the disease depend on the CMI response of thc host toward the parasite. When this response is deficient diffuse cutaneous leishmaniasis ( DCL ) results. The possible reasons for such a deficiency in CMI will be discussed as well as the nature and specificity of the defect. There have been other general reviews of imniuiiity in leishnianiasis ( Evan-Paz and Sagher, 1961; Adler, 1963~1,1964, 1965; Stauber, 1963a; MansonBahr, 1961, 1964). This section will, therefore, be limited to those aspects of leishnianiasis that allow a coinparison between this disease and leprosy. As well as a discussion of thc clinical features of thc disease, information will be derived from the expcriniental animal niodel provided by the infection of guinea pigs with Leislzmnnia enriettii. Cutaneous infection of guinea pigs with L. enriettii produces a naturally healing lesion similar to that found in cutaneous leishmaniasis in man. This niodel has already providcd valuable information on the rclativc roles of cellular and humoral mcchanisms in the pathogenesis, healing, and rcsistance to infection in this diseasc. Human visceral leishmaniasis, kala azar, is a potentially fatal disease in which CMI, as indicated by cutaneous

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delayed hypersensitivity, does not develop or is suppressed. Such patients may not develop delayed hypersensitivity to leishmanin until the disease is cured chemotherapeutically. Active kala azar is characterized by a massive hyperglobulinemia, suggesting a high level of circulating antibody against the infecting organism. Characterization of these antibodies has begun but has been hampered by the lack of a complete antigenic analysis. Throughout Section I11 the terminology introduced by Hoare and Wallace (1966) is used, whereby amastigote refers to the LeishmanDonovan body, and promastigote to the flagellate or leptomonad form of the parasite. B. LEISHMANIASIS AS A DISEASE SPECRUM

The concept of leishmaniasis as a polar or spectral disease, like leprosy, was proposed by Destombes (1960). He pointed out that it was unnecessary to consider the condition of DCL (disseminated anergic cutaneous leishmaniasis ) , recently described in Venezuela ( Convit, 1958) and Ethiopia (Balzer et al., 1960), as a separate disease entity but rather as one polar form of cutaneous leishmaniasis the features of which depended upon the host's response to the parasite. The two polar forms of cutaneous leishmaniasis are DCL and lupoid or recidiva leishmaniasis (Table 111). The former is characterized by an absence of specific CMI and the latter by its exaggeration (Bryceson, 1970d). Kala azar also lies at a pole; it is the visceral equivalent of DCL but TABLE I11 FEATURES OF POLAR FORMS OF CUTANEOUS LEISHMANIASIS" Features

Diffuse cutaneous leishmaniasis

Clinical

Disseminated nodules

Histology

Parasitized macrophages (MM)b Negative

Delayed hypersensitivity leishmanin Antibodies Immunoglobulins Response to treatment From Brycesoii (1970d). MM-macrophage. TT-tuberculoid.

Variable Normal Poor, relapses

Lupoid or recidiva leishmaniasis Local scar with peripheral spread Tubercles (TT). Positive, occasional Arthus Not studied Not studied Variable, improved by steroids

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has special immunological characteristics which merit its consideration apart. 1. Clinical Features of Polar Forms of Cutaneous Leishmaniasis The characteristics of DCL are essentially the same in Venezuela (Convit, 1958; Convit et al., 1962; Convit and Kerdel-Vegas, 1965) and in Ethiopia (Balzer et al., 1960; Price and FitzHerbert, 1965). Bryceson (1969) has reviewed the literature and reported 33 cases. The primary nodule folIowing infection by the sand fly did not ulcerate; in most cases it remained unchanged for months or years, but in 11 cases it spread locally at once, and in 2 disappeared spontaneously. After intervals ranging from 4 months to 11years, metastatic lesions, presumably bloodborne, appeared on the body. Characteristically the metastatic lesions were nodules or macules; they did not ulcerate. Their distribution was, in order of frequency, on the face, extensor surfaces of legs and arms, buttocks, external genitalia, and trunk. The viscera were not involved. Lesions continued to grow indefinitely and caused no apparent harm other than disfigurement. Bryceson ( 1969) suggested that the distribution pattern of lesions was a feature of parasite vector adaptation, whereas the appearance of the lesions reflected the lack of the host’s immune response. Treatment with a parasiticidal drug was almost invariably followed by relapse ( Convit, 1958; Bryceson, 1970a). Lupoid leishmaniasis arises when a solitary oriental sore fails to heal completely or broke down again in situ ( Dostrovsky, 1936). Classic ulcerative nodules spread slowly from the edge of the scar; distant metastases do not develop. The patient is not immune as rechallenge produced a clinically and histologically “isophasic” response ( Dostrovsky et al., 1952). In the middle of the spectrum lie the vast majority of cases of cutaneous leishmaniasis, single lesions which ulcerated after 2 to 8 months and healed in 1 to 2 years leaving a scar (Rahim and Tartar, 1967; Lemma et al., 1969). 2. Histological Features of Polar Forms of Cutaneous Leishmaniasis The histological features of DCL have been described by Convit ( 1958) and Destombes et al. ( 1965). A histological classification analogous to that proposed by Ridley and Jopling (1962, 1966) for leprosy can be devised for the spectrum of leishmaniasis (Bryceson, 1969) extending that of Destombes ( 1960) (Table IV). In this classification the letter M is substituted for the letter L in the leprosy spectrum. The letter M is used to indicate the degree of macrophage as opposed to lymphocytic infiltration of the lesion. One extreme pole of the spectrum, equiv-

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L. TURK A N D A. D. M. BRYCESON

TABLE I V THEHISTOLOGICAL SPECTRUM I N LEISHMANIASIS’ Designation

Histological features

Thin intact epidermis, clear subepidermal zone. Dermal infiltration with macrophages, often vacuolated, full DCLb before treatof parasites; histiocytes, many vessels containing monocytes, absence of lymphocytes. MM together with scanty lymphocytes Macrophage/ ment scattered throughout or grouped intermediate deeply beneath the lesion. Epidermis thickened, intact early, may Intermediate ulcerate later. No clear zone. Lymphocytes intimately mixed with large fleshy histiocytes, moderate numbers of parasites or MM areas alongside IT or TT areas. Intermediate/ Epidermis ulcerated; where intact 3riental sore and shows reduplication of the layers, nutuberculoid DCL in relapse clear damage and hyperkeratosis. Lymphocytes predominate, early arrangement into tubercles around clumps of epithelioid cells, parasites scanty. Epidermis ulcerated. Tubercle formaTuberculoid tion, often with giant cells. Parasites Lupoid leishrare or invisible. maniasis Tubercles being destroyed by fibrosis. Tuberculoid All types healing fibrosis Re-epithelialization. Fibrosis Patchy perivascular infiltration with “Resolving” DCL healing lymphocytes and histiocytes. Abwithout imsence of tubercle formation and of munological parasites. shift

(MM) Macrophage

i

f

1

b

Adapted from Bryceson, 1969. Diffuse cutaneous leishmaniasis.

alent to LL in leprosy, is referred to as MM and in this group are found the more severe cases of DCL which are characterized by intense dermal infiltration with macrophages, often vacuolated and full of parasites, and an absence of small lymphocytes. In these cases the epidermis is thin and there may be a clear subepidermal zone. Less severe cases of DCL may show the macrophage intermediate ( M I ) or intermediate (11) patterns. The full spectrum which is a histological and not a clinical classification is as follows:

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MM-macrophage MI-macrophage intermediate II-intermediate IT-intermediate tuberculoid TT-tuberculoid TF-tuberculoid fibrosis F-fibrosis R-resolving

Diffuse cutaneous leishmaniasis cases before treatment range between

MM and 11. Oriental sore and DCL in relapse lie between MI and TF. Lupoid leishmaniasis lesions are found in ?T-TF range. All healing lesions pass through TF and F except for DCL healing without a shift across the immunological spectrum, which has been given a separate point R, which cannot as yet be placed accurately on the spectrum. The spectrum as such runs between MM and TT (analogous to T T leprosy). The points TF, F, and R have been added to include the histological appearance of lesions during healing occurring naturally or during therapy. The first histological sign of CMI across the leishmaniasis spectrum is found in the MI form and is associated with small numbers of lymphocytes scattered through the lesion or grouped at its base. This is followed by (11) partial differentiation of histiocytes into larger, paler cells, a dimunition in the number of parasites, and epidermal changes including hyperkeratosis, acanthosis, and intraepidermal necrosis leading to ulceration. In a detailed histological study of oriental sore undertaken to facilitate diagnoses of tuberculosis, sarcoidosis, and leprosy, Kurban et al. (1966) described dermal changes which, on this scale, ranged from MI to IT. There was a correlation between duration of infection and the development of a tuberculoid structure. They commented particularly upon the presence of basal cell degeneration and intraepidermal abscesses. Age of infection was also associated with decreasing numbers of parasites in oriental sore. Lemma et a2. (1969) were able to isolate Leishmania from 20 out of 43 cases of oriental sore less than 1 year old, and 2 out of 18 cases over 1 year old. Most cases of oriental sore healed within 1 to 2 years and did not go beyond IT. In recidiva leishmaninsis the TT pattern was fully developed ( Pettit, 1962; Kurban et al., 1966), and parasites wcre usually impossible to identify and difficult to isolate. Despite this gross manifestation of cellular hypersensitivity, the infection continued. In all forms of cutaneous leishmaniasis, plasma cells were found. Their number did not correlate with the position of the lesion in the spectrum.

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3. Delayed Hypersensitivity to Leishnianin Cases at the middle of the spectrum (oriental sore) developed delayed hypersensitivity after a variable period of time. In DCL, delayed hypersensitivity was usually absent ( Convit, 1958) ; some cases exhibited a weak response, and this correlated with histological evidence of partial CMI ( Bryceson, 1969). Cases of lupoid leishmaniasis exhibited “exquisite’’ delayed hypersensitivity to leishmanin. The leishmanin test was usually performed at only onc concentration, approximately loGparasites, corresponding to about 10 pg. purified leishmania1 protein antigen (Bryceson et al., 1970). 4. Movement along the Spectrum

Horizontal movement along the spectrum was suggested by a number of observations. Progression toward DCL could come in waves associated with systemic illness (Price and FitzHerbert, 1965) which might represent periods of diminished immunological activity. Reaction states have also been described, and these might be of two types. One, characterized by fever and local flare-up of lesions associated histologically with polymorphonuclear leukocyte infiltration ( Destombes et al., 1965), might be a form of Arthus reaction similar to ENL (Wemambu et al., 1969). The other, characterized by acute swelling and coalescence of early lesions associated with edema but not polymorphonuclear leukocyte infiltration (Bryccson, 1969), might be a form of downgrading reaction ( Ridley, 1969). Progression toward the tuberculoid end was shown by patients with DCL under the influence of prolonged treatment who might, in relapse, move to an intermediate or tuberculoid histology and a positive leishmanin test (Bryceson, 1970a). Without this immunological shift it was very difficult to cure the patient. Whether it was necessary for a patient with lupoid leishmaniasis to move away from the tuberculoid pole before his lesion could heal was unclear but supported by two observations. First, treatment with systemic steroid combined with antimonials was more effective than antimonials alone ( Evan-Paz and Sagher, 1961); second, intralesional steroid alone might cure the condition ( Bryceson, 1970e).

5. Nature and Specificity of Immune Deficiency in D C L Patients with DCL showed no evidence of diseases known to be caused by generalized depression of immune mechanisms ( Bryceson, 1970b). They handled intercurrent infections normally. Six patients tested with Kveim antigen were negative. Eight leishmanin-negative

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patients with DCL responded normally to lepromin and tuberculin when compared with 10 leishmanin-positive patients. Sensitization to DNCB by the standard technique (TValdorf et al., 1966) was achieved in 2 out of 8 patients which was less than might be expected (Turk and Waters, 1969), but normal controls were not tested. There was no correlation between state of disease and ability to be sensitized. Serum Ig levels in 9 cases of untrcated DCL did not differ from that of the population at large (Bryceson, 1969). Serum levels of IgG, IgM, and IgA were measured by single radial immune diffusion. There were no differences among 6 untreated cases and 3 treated cases of DCL, 5 cases of oriental sore, or 6 normals of comparable age. Four individual cases of DCL and one of oriental sore with niucosal involvement had IgA levels of 2.69 to 4.40 mg./ml. which were outside the range of the normal sera. Further studies on circulating and intralesional levels of IgA in cutaneous leishmaniasis are indicated. Studies of antibodies to the infecting organism in leishmaniasis have until recently been hindercd by lack of appropriate and sensitive techniques. Such work done on patients with DCL suggested normal antibody production. By using an indirect hemagglutination technique, Bray and Lainson (1967) found high titers of antibody in sera of 3 Ethiopian cases aftcr treatment. Examination of other sera showed moderate antibody titers (1:80) in 1 out of 4 untreated cases and in 1 out of 7 treated cases; comparable levels were found in 4 out of 12 cases of oriental sore (Bryceson, 1970b). From gel precipitation, low levels of precipitating antibody were detected in 2 out of 3 Venezuelan cases but none in 2 Ethiopian cases (Bray and Lainson, 1966). Torrealba (1967) also reported precipitating antibodies in the sera of 3 out of 5 Venezuelan cases. Indirect immunofluorescence has, in different hands, shown ( Bittencourt et al., 1968) and failed to show (Convit and Kerdel-Vegas, 1965) antibodies. Recently, however, Convit and Pinardi ( 1969) saw antibody titers up to 1:200 in the sera of 5 out of 5 patients using an indirect immunofluorescent technique with infected hamster spleen as substrate. Deane et al. (1966) were unable to demonstrate antibodies by complemcnt fixation in 1 case. These results were comparable with those found in oriental sore and South American leishmaniasis (see Section II1,D). These studies suggest that the immunological deficiency in DCL is a specific deficiency of CMI to Leishmania. The possibility of a more generalized deficiency being present in some patients needs further investigation. Attempts to reverse this deficiency in 4 patients by the intracutaneous transfer of 2.5 to 4 X 10' lymphocytes from leishmanin-sensitive donors

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were unsuccessful, although temporary hypersensitivity to 8 x lo5 intradermal promastigotes, lasting for 6 to 20 days, was achieved in 3 patients, Two out of 3 control recipients were likewise temporarily sensitized to leishmanin ( Bryceson, 1970b). This temporary transfer of cutaneous sensitivity was in contrast with previous reports. Adler and Nelken (1965) injected subcutaneously 5.4 x los leukocytes from an exquisitely sensitive donor into a normal recipient and skin tested at various intervals from 1 to 10 days later. They then attempted transfer with 3.1 x lo9 leukocytes by whole blood transfusion. It is possible that failure to elicit hypersensitivity was caused by inadequate doses of antigen ( l o 5 promastigotes 2 1 pg. protein). This form of therapy was theoretically unlikely to be successful if the recipient’s immune deficiency was specific. Moreover the therapeutic use of purified transfer factor would also be unsuccessful if the clone of lymphocytes potentially able to respond to the appropriate leishmanial antigen were deficient ( Bryceson, 1970b). 6. Pathogenesis of DCL

Specific deficiency of CMI to Leishmania in patients with DCL accounts for the unusual features of this form of leishmaniasis. MansonBahr (1964) has summarized the evidence that the features of lupoid leishmaniasis likewise reflect the host’s immune response and are not due to a different parasite (see also, Bryceson, 1 9 7 0 ~ ) The . genesis of the deficiency in DCL is unknown. Any attempted explanation must take into account several facts: ( a ) the geographical distribution of this form of the disease-it is absent in the Middle East and India; ( b ) in countries where it is found, it is a rare presentation of a common disease; ( c ) the immune deficiency, though parasitologically specific, is probably to several leishmanial antigens; ( d ) in Venezuela the causative agent shows certain differences from that causing simple cutaneous leishmaniasis which led Medina and Romero (1959, 1962) to designate it Leishmania pifanoi; ( e ) in Ethiopia the causative parasite is, on epidemiological (Lemma et al., 1969) and experimental (Bray and Bryceson, 1969) grounds, probably the same as that causing simple oriental sore. Bryceson ( 1 9 7 0 ~ )considered that these facts implied two things. First that certain less antigenic strains of Leishmania were more capable of causing DCL than other strains and, second, that they only did so in a host whose immune response was in some way compromised. Different species and strains of Leishmania are antigenically distinct ( Adler, 1965; Adler and Foner, 1966; Bray and Lainson, 1966, 1967) but also share many antigens in common. Bray and Rahim (1969) used an indirect hemagglutination technique and cross-absorbed rabbit antisera to com-

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pare several Iraqi strains of Leishmania tropica which did not cause DCL with an Ethiopian strain which did. They found that, whereas the Iraqi strains were antigenically identical, the Ethiopian strain was distinct. Antigens of the Iraqi strains absorbed all anti-Ethiopian serological activity, yet the reverse was not so. This impliecl a quantitativc difference in antigens, thc Ethiopian strain lacking an antigen or antigens possessed by the Iraqi strain. The possibility that certain strains of Leishmania possess antigens specifically capable of paralyzing the host’s response to the parent organism ( tolcrogens ) should also be considered ( Adler, 1965; Bryceson, 1 9 7 0 ~ )To . conform with accepted concepts of imniunological specificity this would be a secreted antigen which would leave the parasitized macrophage, and, by virtue of its molecular size, structure, or inode of prcsentation to lymphoid tissue, induce tolerance in low dosc. Tlic clone of lymphoid cells so rendered inactive would not recognize the macrophage bearing the secreted antigen. Destruction of this target cell would not take place and parasite antigens evoking a protective immune response would not be released. This hypothesis could be investigated in an experimental model when individual antigens have been isolated. A start has been made by Benex and Lamy (1968) also Bencx (1970). Possible mechanisms by which the host’s ininiune response might be rendered incapable of responding normally to a parasite potentially capable of causing DCL have been discussed (Bryceson, 1 9 7 0 ~ ) .In 30%of cases of DCL in Ethiopia the primary lesion was on the leg, whereas in oriental sore the lesion was on the face in 99% of the cases. Half of the former cases had evidence of gross lymphatic abnormality of the lower limbs. It might be argucd from analogies in other systems (Jancovic, 1962; Neveu, 1964) that the induction of the immune response in a lymph node which was damaged or immunologically preoccupied by competing antigens might be delayed or reduced and allow the parasite to multiply unusually rapidly, producing a state of high zone tolerance (Bryceson, 1970b). Although reduction of antigen load following treatment permitted expression or development of CMI in many cases, this hypothesis failed to account for the long steady state which preceded metastasis in many patients. It would, however, account for the rarity of thc condition and for many of its epidemiological features. Experimental studies of guinea pigs infected with Leishmania enriettii showed that intraderinal injcctioiis of heterologous antigens in adjuvant beforc infection with parasites altered the course of subsequent infection. One group of animals, pretreated with Corynebacterium paruuin in Freund’s incoinpletc adjuvant, developed infections showing some of the features of DCL (see Section 111,F).

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7 . Visceral Leishmaniasis Infection with Leishmania donovani usually results in kala azar, a disease characterized by gross parasitization of systemic macrophages, notably in spleen, liver, and blood, usually terminating fatally. Delayed hypersensitivity to leishmanin is absent. Immunologically the condition seems to lie at the diffuse or MM end of the spectrum, in which the race for CMI has been lost (Garnham and Humphrey, 1969). It is not known, however, whether CMI fails to develop (immune deviation) or whether it becomes suppressed as the infection progresses possibly by immunological enhancement. No studies of cutaneous hypersensitivity or of in vitro cellular hypersensitivity have been performed during the incubation period of the disease; the patients described by Cahill (1964, 1970) had solitary skin lesions, not kala azar, and the study did not resolve this point. One important immunological difference between kala azar and DCL lies in the enormous increase of serum globulins in patients with kala azar. Adler (1965)) and Garnham and Humphrey (1969) suggested that most of this globulin was not specific antibody, analogous to the situation in malaria (Neal et al., 1969), but no quantitative study has been published. The IgG and 7 S y-globulin levels were consistently high (Priolisi and GuiffrA, 1967; Martins et al., 1969; Hobbs, 1970); IgM levels might also have been raised (Chaves and Ferri, 1966), if only transiently, and fell rapidly following treatment ( Irunberry et al., 1968). The IgG pattern on imniunoelectrophoresis was characteristically skewed, and IgM sometimes precipitated spontaneously in the absence of antihuman serum (Irunberry et al., 1968). Antibodies were present in both 1 9 s and 7 s fractions (Ferri and Chaves, 1968). Newer techniques have replaced the traditional complement fixation test using a mycobacterial antigen. Bray and Lainson (1966) detected antibodies by precipitation in gel in 4 out of 4 kala azar sera. They also developed (Bray and Lainson, 1967) a passive hernagglutination technique, whereby naked or, better, tanned sheep erythrocytes were coated with 1:20,000 w/v solution of freeze-dried extract of cultured promastigotes. Titers up to 1: 10,240 were found in 4 out of 4 kala azar sera. Mukherjee et al. (1968) used this technique to detect antibodies in 6 out of 6 patients. Manson-Bahr (1967), using a similar technique found antibodies in sera of 8 out of 11 patients with active kala azar and none in 25 patients 1-3 years after cure. Ranque and Quilici (1970; see also Quilici & al., 1968) considered indirect immunofluorescence to be the most sensitive technique; they detected antibodies in 100%of kala azar cases (see Section 111,D). After treat-

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ment, antibody levels fell and CMI, as indicated by delayed hypersensitivity, emerged ( Manson-Bahr et al., 1959; Chaves and Torrealba, 1967). This shift along the spectrum was accompanied by the development of immunity to reinfection. As yet, it is not known whether the depression of CMI in active kala azar is maintained by high antigen levels ( split, high-zone tolerance), by antibody ( immunological enhancement), or by some other mechanism. 8. Post-kala azar Dermal Leishmaniasis

This condition, a purely cutaneous relapse following visceral leishmaniasis, remains unexplained. Adler ( 1964) suggested that it represented a state of premunition in that CMI was adequate to protect the viscera but not the skin. Sen Gupta (1966) reviewed the problem and considered that either there was local immunity, as in tuberculosis, or the organism became dermatotropic. In support of the latter idea were epidemiological observations correlating high incidence, early onset, and long duration of the condition when kala azar became endemic, as opposed to epidemic, in a given area. Bray and Lainson (1967) were unable to distinguish a strain of Indian post-kala azar leishmaniasis from that of Israeli Leishmania tropica. Later reversion to the viscerotropic strain was suggested by 5 unusual cases of chronic post-kala azar dermal leishmaniasis in patients who redeveloped visceral disease after 1 to 20 years (Sen Gupta and Mukherjee, 1968). Wide variations in skin test response and histological pattern (see review by Majundar, 1968) suggested that post-kala azar dermal leishmaniasis might lie at almost any point of the leishmaniasis spectrum. C. THELEISHMANIN TEST The intradermal leishmanin ( Montenegro) test bears a relationship to leishmaniasis as the tuberculin ( Mantoux) test does to tuberculosis. It is an adjunct to diagnosis. It indicates delayed hypersensitivity and, in cutaneous leishmaniasis, becomes positive before immunity to reinfection develops (see Manson-Bahr, 1961; Adler, 1963a). Differing observations as to how soon the test becomes positive after infection can be related to differences of infecting and challenge doses of organisms (see Section 111,F).The test is useful in assessing the degree of CMI in patients with DCL (Bryceson, 1969, 1970d). Leishmanin positivity correlates well with in vitro lymphocyte transformation in the presence of leishmania1 antigens (Trenlonti and Walton, 1970). Lymphocytes from leishmanin-sensitive donors have been shown to generate mitogenic factor when incubated with leishmania1 antigens ( Bryceson et al., 1971d). Hoogstraal and Heyneman (1969) reviewed the use of

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the leishmanin test in the field. In endemic areas of cutaneous leishmaniasis, leishmanin positively increased with age ( Imperato and Diakiti., 1969; Lemma et al., 1969). In endemic areas of visceral leishmaniasis, naturally acquired leishmanin positivity varied inversely with the incidence of kala azar (Southgate and Oviedo, 1967) and indicated population immunity ( Southgate and Manson-Bahr, 1967a). Leishmanin positivity is not species specific and does not indicate heterologous immunity ( Manson-Bahr, 1964) in the individual, nor does it indicate the species of immunizng parasite in populations immune to kala azar ( Southgate and Manson-Bahr, 1967a). The leishmanin test still suffers from certain technical defects-the antigen is never pure but is a suspension of organisms, and the washing and the suspending fluid of the organism, the number of organisms in concentrations, and antigenicity are not universally standardized. It is, therefore, difficult to compare the results of different workers as would be possible using any one standardized preparation.

D. IMMUNODIAGNOSIS In those forms of leishmaniasis commonly associated with circulating antibody the detection of antibody by indirect immunofluorescence is proving a useful technique. It can be performed with serum eluted from finger prick blood dried on filter paper (Duxbury and Sadun, 1964). Carmago and Rebonato (1969) found that washed fresh promastigotes from culture, formolized washed promastigotes, or freeze-dried washed promastigotes (which could be stored in ampoules) served equally well as substrate. Convit and Pinardi (1969) used splenic smears from hamsters infected with Leishmania donouani, as substrate, but Ranque and Quilici (1970) found promastigotes better than amastigotes. It was necessary to stick the parasites well to the slide using 6% dextran (Carmago and Rebonato, 1969), 2%bovine serum albumin (Radwanski et d., 1971), or cooled methanol (Convit and Pinardi, 1969). Ranque and Quilici ( 1970) considered that fluorescein labeling and counterstaining with Evans blue gave the best results. Ranque and Quilici (1970) in France used this test to examine 123 sera of patients with leishmaniasis. They found antibodies in 100%cases of kala azar, as compared with 95% when using gel precipitation and 654%using complement fixation techniques. They were unable to detect antibodies in sera of 22 patients with uncomplicated oriental sore, but could in those with clinical involvement of lymph nodes. It was not clear whether this distinction was related to time or sensitivity of technique or was absolute, as the leishmanin test was positive in both groups. In South America, Bittencourt et al. (1968) found antibodies by

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indirect immunofluorescence in 29 cases of leishmaniasis (cutaneous, mucosal, or mucocutaneous) but not in sera of 28 controls. Titers were highest in those patients with mucosal involvement ( Nery-Guimarez et al., 1969). The test became negative 2-6 months after cure (Bittencourt et al., 1968; Walton, 1970) and could be used as a criterion of success in drug trials. Walton (1970) considered that a persisting positive test indicated the persistence of parasites. Commonly indirect immunofluorescence detected antibodies in patients with uncomplicated South American leishmaniasis, whereas indirect hemagglutination did not ( Bray and Lainson, 1967). This might be a question of sensitivity, as Convit and Pinardi (1969) stated that titers were related to duration of disease; but experimentally (see Section II1,F) these two tests detected antibodies to different antigens. Indirect immunofluorescence could not differentiate among species of Leishmania, and there was complete cross-reaction with Trypanosoma cruzi (Aranjo and Mayrink, 1968; Ranque et al., 1969). Aranjo and Mayrink (1968) and Ranque and Quilici (1970) found it possible to dilute out the antileishmanial activity of sera from patients with Chagas disease. Carmago and Rebonato ( 1969) described in detail satisfactory cross-absorption techniques.

E. ARTIFICIALIMMUNIZATION The failure of the Kenya rodent strain of Leishmania to protect against naturally acquired kala azar was a disappointment after the strain was shown to be protective against artificially induced infection with Leishmania donovani ( Manson-Bahr, 1961). This failure might have been due to loss of virulence of the rodent strain during subculture ( Manson-Bahr and Southgate, 1964). Work with this strain and with Leishmania adleri of lizards was not followed up despite epidemiological and experiniental evidence of their immunizing potential ( Southgate and Manson-Bahr, 1967b). Artificial immunization against cutaneous leishmaniasis was carried out in Southern Russia, using a freeze-dried live vaccine of Leishmania tropica ( Neal et al., 1969). This strain was fully virulent ( Kellina, 1966), and immunity accompanied healing of the lesion, which lasted 3-5 months. There are no accounts of successful attempts to vaccinate against Leishmania brasiliensis in man. Lainson and Bray ( 1966) successfully immunized a monkey against L. brasiliensis by infecting it with Leishmania mericana, a nonmetastasing parasite. As virulence and immunogenicity go together in leishmaniasis, new approaches are necessary if n vaccine is to be produced. Artificial immunization using protein-

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M. BRYCESON

rich leishmania1 antigens of Leishmania enriettii in Freund's complete adjuvant has been achieved in the guinea pig (Bryceson et al., 1970). This suggests that an immunizing antigen can be isolated.

F. EXPERIMENTAL LEISHMANIASIS: Leislimunia enriettii INFECTION OF THE GUINEAPIG Cutaneous leishmaniasis of the guinea pig is caused by Leishmania enriettii. Inoculation of the parasite into the skin was followed by a single lesion which ulcerated and healed in 2 to 3 months (Paraense, 1953). It was accompanied by delayed hypersensitivity (Glazunova, 1965) and followed by long-lasting immunity to reinfection (von Kretschmar, 1965). Cutaneous metastases appeared in a varying proportion of infected animals. This infection has been used as a model for the study of the pathology and immunology of human cutaneous leishmaniasis by Bryceson et al. (1970). In these studies, a standardized protein-rich urea-extracted soluble antigen (PSA ) of cultured promastigotes of L. enriettii was used. 1. Cell-Mediated Immunity a. Evidence of Cellular Hypersensitivity and Cellular Immunity. Guinea pigs infected in the ear with lo6 amastigotes developed delayed hypersensitivity within 1 to 2 weeks (Bryceson et al., 1970). Delayed hypersensitivity could be transferred to normal guinea pigs with 1 x lo* viable peritoneal exudate cells or 5 x lo8 viable lymph node cells from infected animals or animals immunized with PSA in Freund's complete adjuvant. Lymph node cells from infected or immunized animals, cultured at a concentration of 106/ml.,transformed in the presence of PSA; this response was antigen dose-dependent in the range 0.1-100 pg./ml. The migration of macrophages from peritoneal exudates of infected or immunized animals was inhibited in the presence of PSA; this response was sensitized-cell dose-dependent, detecting a tenfold dilution of sensitized cells with normal cells. There was no cross-reaction with PPD in either system. Supernatant fluids of lymphocytes of infected or immunized animals cultured at a concentration of lo7 cells/ml. in the presence of 100 mg. PSA/ml. were tested for the presence of migration inhibition factor and mitogenic factor. Lymphocytes from half the infected animals examined generated both factors; control animals and control cultures were consistently negative. The suppression of CMI with daily injections of ALS raised against unpurified lymph node or thymus cells permitted the growth of larger primary lesions and widespread cutaneous metastasis in all animals

251

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enriettii in groups of 10 guinea pigs treated with antilymphocyte serum ( ALS) (0) and with normal rabbit serum Growth of lesion in 4 ALS-treated animals that were not killed at 10 weeks ( 0). (From Bryceson and Turk, 1971.)

(a).

(Bryceson and Turk, 1971) (Figs. 9 to 11). This prolonged effect of ALS was only seen in animals rendered tolerant neonatally to the heterologous globulin. Delayed hypersensitivity was suppressed. The clinical condition of these animals resembled that seen in human DCL. The early lag of lesion growth in ALS-treated animals (Fig. 9) recalled the reduced susceptibility of mice to Leishmania mexicanu after whole-body irradiation (Shaw and Voller, 1968). Both effects could be attributed to an antimacrophage effect. However, guinea pigs treated with purified antimacrophage serum healed sooner than did controls, and there was no early lag of growth of the lesions (Bryceson et al., 1971a). Pretreatment of guinea pigs with three intravenous injections of 10 mg. PSA rendered them more susceptible to infection with Leishmania enriettii. Lesions were twice the size of controls from 2 to 9 weeks, and healing was delayed (Bryceson et al., 1971a). In such animals, delayed hypersensitivity was depressed; lymphocytes from animals infected for 4 weeks failed to transform in vitro in the presence of antigen; and the inhibition of migration of peritoneal exudate cells was not detected until after 3 weeks (Bryceson et al., 1970). Sera from both groups were

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FIG. 10. Multiple cutaneous metastases in a guinea pig infected with Leishmania enriettii and treated with antilymphocyte serum. (From Bryceson and Turk, 1971.)

examined for antibody by indirect immunofluorescence. At 3 weeks, antibodies were not detected in either groups; at 9 weeks, antibody titers were comparable (1:160-1:1560) in both groups (Bryceson et al., 1971a). Further experiments, which might be interpreted as showing the effect of high antigen levels upon the course of infection and the development of immunity, were those in which different numbers of amastigotes of L. enriettii in the range 10'-1 )( los were used to infect groups of guinea pigs (Bryceson et al., 1971~).The incubation period varied inversely with infecting dose; animals infected with 2 x 108 parasites developed lesions within 5 days, animals infected with 10' parasites developed lesions after 4 weeks. The duration of overt infection was constant (7-9 weeks) in animals infected with 10' to 109 parasites but increased up to 18 weeks in animals infected with 2 )( 10s parasites. Maximum lesion size was independent of infecting dose in the range 101-105parasites (mean 700-1000 mrns3) but thereafter increased with infecting dose; animals infected with 1 )( 108 parasites

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FIG. 11. Testicular metastasis in guinea pig infected with Leishmania enriettii and treated for 10 weeks with antilymphocyte serum. Beside the one intact tubule are two degenerating tubules. The rest of the testis is replaced with heavily parasitized histiocytes and plasma cells. Stain: hematosylin and eosin. Magnification: X 1500. (From Bryceson and Turk, 1971.)

developed lesions of 4000 mm.3 mean volume. Similarly the percentage of animals developing cutaneous metastases was independent of infecting dose up to 10’ parasites (16-2m) but increased sharply thereafter; 40 and 92%of animals infected, respectively, with lo7 and 2 x lo8 parasites developed metastases. There seemed to be a threshold infecting dose of lo4 to lo5 parasites. Infection with up to 10‘ to lo5 parasites produced a similar degree of local infection which suggested that an antigenic dose beyond this was required for the development of immunity. Higher doses produced more severe infections. This effect was most marked in animals infected with 2 x lo8 parasites; 7 out of 20 died with multiple cutaneous metastases. In those animals that survived, metastases continued to grow, or even to appear, long after the primary lesions had healed, and the animals were immune to rechalIenge. This suggested that the parasites of the metastatic lesion were “hidden” from an effective immune response; the mechanism is unknown. Immunological investigations incIuded the measurement of delayed and immediate cutaneous hypersensitivity and of antibody by indirect

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immunofluorescence. The development of delayed hypersensitivity accompanied the appearance of the primary lesion. In animals infected with 1 0 parasites, the intensity of delayed hypersensitivity to 100 pg. PSA increased over the next 2 weeks, leveled off, and, as the lesion healed, finally rose to a new level which was then maintained indefinitely. In animals infected with 2 x los parasites, delayed hypersensitivity increased rapidly in intensity for the first 3 weeks and then fell sharply as the mean lesion volume exceeded 1000 n ~ m and . ~ metastases appeared. Delayed hypersensitivity to 100 pg. PSA could not be elicited in some severely affected animals. With clinical recovery, delayed hypersensitivity increased to the same final level of intensity as in the other groups. Immediate hypersensitivity developed after 8 to 10 weeks, regardless of infecting dose, at about the time lesions began to heal. The appearance of inimunofluorescent antibody also correlated with the appearance of the lesion. Maximum titers of 1:640 to 1:10,240 were reached after 2 to 4 weeks and maintained throughout the infection, Antibody titers did not correlate with infecting dose. These experiments suggested that severe infection selectively suppressed CMI independently of antibody production. b. Role of Macrophages in Resistance to Infection. Leishmania are obligatory intramacrophage parasites. Antimacrophage serum partially protected guinea pigs against infection with Leishmania enriettii ( Bryceson et al., 1971a). The early histology of the lesion which followed inoculation of L. enriettii into the ear of guinea pigs was characterized by a massive infiltration of mononuclear cells. Most of these, for the first 3-4 weeks, appeared to be monocytes/macrophages (Bryceson et al., 1970). Parasites multiplied in these macrophages. From 3 to 4 weeks onward, lymphocytes collected at the periphery of the macrophage mass, and from 4 to 6 weeks the lesion started to resolve. It is not known whether, as the infection proceeds, macrophages full of parasites rupture and fresh macrophages are invaded or whether parasitized macrophages divide. Also it is not known whether parasite multiplication is arrested as the infection heals or whether macrophage recruitment is inhibited. In vitro Bray and Bryceson (1968) showed that a monolayer of guinea pig niacrophages took up amastigotes of L. enriettii and allowed their multiplication over 48 to 72 hours. Uptake of parasites and their subsequent growth were unaffected by the presence or absence of complement or serum from recovered, immune animals. However, failure to detect antibody opsonizing for macrophages did not exclude the presence of antibody opsonizing for peritoneal polymorphs. Macrophages from immune guinea pigs were found to take up 1.8 to 5.7 (mean 2.7)

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times as many amastigotes as did macrophages from normal animals. Increased phagocytosis was species specific. Attempts to demonstrate antibody on the surface of macrophages or circulating antibody cytophilic for macrophages were unsuccessful (Bryceson et al., 1970). Peritoneal macrophages from convalescent or recovered guinea pigs did not inhibit or retard the growth of ingested amastigotes. In some, but not all, experiments in which immune guinea pigs were challenged intraperitoneally, as opposed to subcutaneously, with live parasites 3 days before peritoneal macrophages were harvested, the intracellular growth of parasites was significantly reduced ( Bryceson, 1970e). This suggested that enhanced macrophage activity might be found in the presence of a local immune response. Miller and Twohy (1969) found that macrophages from mice infected intraperitoneally showed some ability to inhibit growth of Leishmania donovani. However, Twohy (1970) was unable to repeat the experiment when he used a different strain of mice. c. Role of Lymphocytes in Resistance to Infection. The response of the draining lymph node to the cutaneous inoculation of parasites was found to be a dual one (Bryceson et al., 1970). Within a week, germinal centers developed in the cortex and paracortical areas hypertrophied with the appearance of imniunoblasts. These activities increased over the next 4 weeks during which time the medullary cords became thick with plasma cells, which contained immunoglobulin, and the afferent lymphatics filled with small round, nonpyroninophilic lymphocytes. Accompanying these changes in the node were changes in the skin lesion which consisted mainly of parasitized macrophages. Around the base of the lesion, small round cells began to appear in controls but not in ALS-treated animals, of which the lymph nodes showed depletion of paracortical areas and empty efferent lymphatics (Bryceson and Turk, 1971). Epidermal changes leading to ulceration developed at this time. From then onward the mass of infected macrophages diminished. The ulcer debris consisted of degenerating parasites and parasitized macrophages. The lesion became demarcated by a zone of new vessels. Complement and fibrinogen were shown by immunofluorescence to be deposited in this zone (Bryceson et al., 1971b). The distribution of complement did not correspond with that of immunoglobulin, and evidence of classic immune complex deposition was not found. The appearance recalled that of homograft rejection. It was, therefore, suggested that sensitized lymphocytes rejected the mass of parasitized macrophages ( Bryceson et al., 1970). In vitro lymphocyte-macrophage interaction was studied in a monolayer system (Bray and Bryceson, 1968; Bryceson et al., 1970). Monolayers of guinea pig macrophages in Carrel flasks were

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infected with Leishmania enriettii and overlaid with lymph node cells at a concentration of 1 to 5 x lo7 cells/ml. Lymphocytes from normal guinea pigs had an equal effect upon either infected or uninfected macrophage monolayers-about 50% reduction of macrophages after 24 hours. Lymphocytes from convalescent guinea pigs totally destroyed the monolayer within 24 hours. Examination of the monolayers after 2 hours revealed a 51%destruction of macrophages. Macrophages appeared to round up and lose their grip on the glass. Parasitized macrophages were preferentially destroyed. Similar preferential destruction was also seen when macrophages coated passively with antigen were used as target cells, uncoated macrophage monolayers being used as controls. Interestingly, lymph node cells of guinea pigs immunized with antigens with or without adjuvants also destroyed the target cells, even though such animals were partially or fully susceptible to infection and delayed hypersensitivity was not necessarily demonstrable. Target cell destruction was not specific in that lymph node cells from animals injected with Freund's complete adjuvant alone were equally effective. Whether this in vitro system is a true model of the process of rejection is uncertain, but a major role for the lymphocyte as the effector cell of CMI in this system is implied. Supernatants taken off lymphocyte-treated macrophage monolayers, or prepared for the production of migration inhibition factor and mitogenic factor, have not so far shown cytotoxicity for parasitized macrophage monolayers (Bryceson et al., 1970; Bray, 1970). This might suggest that lymphocyte macrophage contact was a necessary prerequisite for destruction. Further experiments to define the mechanism and antigenic specificity of cytotoxicity and to establish whether lymphocytes have a direct action on free Leishmania are, therefore, indicated.

2. Antibody a. Detection of Antibody. Antibody could be detected by indirect immunofluorescence in the serum of guinea pigs infected with Leishmania enriettii (Radwanski et al., 1971). The appearance of antibodies coincided with development of the lesion and correlated with infecting . use of conjugated antidose of organisms (Bryceson et al., 1 9 7 1 ~ )The globulin to partially purified guinea pig 7,-globulin increased the intensity of fluorescence and suggested that the antibody might be in the y. fraction ( Radwanski et al., 1971). Heterologous complement fixation was demonstrated by the application of fresh rat serum to frozen sections of infected guinea pig nose after layering with infected guinea pig serum. Fluorescein-conjugated antirat globulin produced bright fluorescence of intracellular parasites. Evidence of immune complex

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formation was, however, lacking; the distributions of y-globulin and of complement did not coincide, chunky deposits were not seen, and polymorphonuclear vasculitis and Arthus reactions were not elicited (Bryceson et al., 1970). Antibodies to PSA could not be demonstrated to the serum of infected animals by precipitation ( Bryceson, 1970e). Presence of antibodies was sought by an indirect hemagglutination technique (Bray and Lainson, 1967) using tanned sheep erythrocytes coated with a crude soluble extract of promastigotes which contained both polysaccharide and protein. Antibodies were not detected at any time in the serum of infected animals ( Bryceson et al., 1970). Antibodies were consistently demonstrated in the serum of animals immunized with this antigen, or with PSA, in Freund's complete or incomplete adjuvant; titers reached 1:5120 in some animals 4 weeks after immunization. The presence of this antibody did not correlate with protection. By contrast immunofluorescent antibody was never detected in the serum of artificially immunized animals (Radwanski et al., 1971), and this was interpreted that the two techniques detected antibodies to different antigens. Anaphylactic antibodies, demonstrable by immediate hypersensitivity on skin testing with PSA, developed in infected guinea pigs at about 5 to 9 weeks when the lesions began to heal (Bryceson et al., 1970). Reaginic antibodies, however, could not be demonstrated by passive cutaneous anaphylaxis in the serum of infected or recovered animals. Animals immunized with antigens in Freund's complete adjuvant developed anaphylactic antibody, reaginic antibody, and Arthus hypersensitivity. The serum of guinea pigs that had recovered from infection with Leishmania enriettii contained a factor that inhibited growth of promastigotes in culture (Rezai, 1970) and caused them to be agglutinated and lysed in uitro (Preston and Dumonde, 1970). D'Alesandro (1954) had previously demonstrated agglutinating antibody in the serum of hamsters infected with Leishmania donovani. Serum of rabbits sensitized hy repnated intravenous injections of promastigotes has long been known to alter the growth of cultured promastigotes in vitro (Adler, 1963b). Syncytia of a flagellar and immobile form develop. The specificity of this phenomenon has been used to confirm the identity of Sudanese strains of Leishmania isolated from man, sand fly, and rodents (Adler et al., 1966) and of Israeli strains isolated from man and from Psammomys obesus in the Jordan Valley (Gunders et al., 1968). Werthein et al. (1970) demonstrated that this rabbit antibody formed precipitates with antigens secreted by the organisms which then became emmeshed in flakes and hyaline masses of precipitate. Flagellar mobility

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was lost, the flagellar vacuolc increased in size, and a vesicle grew around the base of the flagellum. Organisms continued to divide, but the flagella remained stuck and prevented separation. Antibody seemed to increase the permeability of promastigotes but failed to kill them. Rezai et al. (1969) used a counting technique to show that this antibody reduced the multiplication of promastigotes. It would be important to know whether the properties of these two agglutinating antibodies, one from natural infection with amastigotes and the other from passive sensitization with promastigotes, were the same. Their relevance in vim is unknown. b. Role of Antibody. Natural immunity and susceptibility to leishmania1 infection were reviewed by Stauber (1963a). Ulrich et al. (1968) found two heat-labile factors in the sera of rabbits, guinea pigs, and rats which immobilized promastigotes and caused them to round up. One of these factors could be absorbed out by parasites. They suggested it could be natural antibody. It was not species specific. Natural immunity to leishmania1 infection did not correlate with the presence of these factors. These observations questioned the significance of Adler’s (1963a) finding that the sera of Mediterranean adults, as opposed to those from children, contained a factor that inhibited the growth of Leishmania infantum in vitro. The passive transfer of sera from guinea pigs that had recovered from infection with Leishmania enriettii failed to protect normal guinea pigs against infection with L. enriettii ( von Kretschniar, 1965). Studies using large quantities of convalescent immunoglobulin fractions should be performed to confirm the lack of protection by antibody. Protection would only be expected in the hours following inoculation of parasites and before their uptake by macrophages. Bryceson et al. (1971b), using an indirect immunofluorescent technique, failed to demonstrate immunoglobulin inside parasitized macrophages at a time when circulating antibody titers were high and immunoglobulin-producing cells were abundant in medullary cords of the draining lymph node. If, as has been suggested, sensitized lymphocytes destroyed parasitized macrophages from the fourth week of infection onward (Bryceson et al., 1970), liberated parasites would be liable to contact antibody. The appearance of anaphylactic antibody at this time was of interest. Whether it was produced in response to antigens liberated by dead parasites or whether it was protective was undetermined. Anaphylactic antibodies have also been demonstrated by passive cutaneous anaphylaxis in some immune human sera (Bray and Lainson, 1965). The possible phagocytic role of eosinophiles which characterize the immediate response to reinfection (unpublished) is under investigation.

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The possible enhancing effect of antibody has to be considered (Garnhani and Humphrey, 1969). Stauber ( 1963b) quoted an unpublished experiment of D’Alesandro in which hamsters’ susceptibility to infection with Leishinnilin donovani increased after passive receipt of immunc serum. Bryceson et al. (1971a) studied the effect of antigenic competition and of lymph node blockade upon the course of Leishmania enriettii infection in guinea pigs. Guinea pigs pretreated with Corynebacterium paruum or hlycobacterium tuberculosis in adjuvant were found to develop, respectively, more severc and more chronic metastasizing infections with L. enriettii. Delayed hypersensitivity was not impaired and titers of imiiiuiiofluorescent antibody were normal 6 weeks after infection ( 1:640-1 :2560). Heniagglutinating antibody was also detected in 3 out of 3 C. pnroun-treated animals (1:640-1:5120) and in 2 out of 3 A l . tu1,ercolosis-trcated animals ( 1: 10-1: 1280). Metastatic lesions in onc Al. tuberculosis-treated animal showed a poor lymphocyte response, despite the animal being immune to rechallenge. The possible enhancing role of antibody, prescnt in high titer as measured by immunofluorescence or indirect heinagglutiiiatioii in human leishnianiasis ( Bray and Lainson, 1967; Ranque and Quilici, 1970) needs investigation. IV. Concept of a Host-Determined Spectrum of Clinical Manifestations in Other Chronic Infections in M a n

The spectrum of clinical manifestations in a disease due to an individual organism is not limited to leprosy and leishmaniasis. Evidence exists for low resistance or “lepromatous” fornis of a number of diseases. Low-resistance fulminating tuberculosis without obvious tubercle fonnation is a recognized, although rare, clinical entity. This form of the disease is associated with widespread infiltration of the infected tissues with large numbers of organisms. Such patients are generally tuberculin negative, Clinical improvement following chemotherapy may be associated with a return of CMI indicated by patients regaining tuberculin sensitivity (Howard et al., 1970). Low-resistance forms of the chronic mycoses are also well documented. Chronic mucocutaneous candidiasis develops regularly under conditions where CMI becomes depressed nonspecifically. This may be due to a failure of thymus function in fetal life (Gitlin and Craig, 1963; Fulginiti et al., 1966; Nahmias et al., 1967) or to a failure of the production of soluble mediators of CMI (Chilgren et al., 1969; Valdimarsson et al., 1970). This condition is also found when CMI is suppressed nonspecifically by immunosuppressive drugs, following defects of endocrine function and in neoplastic disorders of lymphoid tissue such as Hodgkin’s disease. Low-resistance forms occur in the systemic mycoses, such as

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South American blastomycosis ( d a Silva Lacaz, 1967) and histoplasmosis. The lesions are generally systemic, but in South American blastomycosis there is also a diffuse cutaneous form of the disease. In the localized cutaneous form of South American blastomycosis, there is delayed hypersensitivity to paracoccidioniycin, a low level of complement-fixing antibodies to the polysaccharide antigens of Paracoccidiomyces brosiliensis, and a good response to treatment with sulfonamides. The disseminated cutaneous or systemic forms of the disease are associated with negative skin reactivity to paracoccidiomycin and a high level of circulating antibody, even detectable precipitins, against antigens derived from the infecting organism. These low-resistance forms respond poorly to treatment, and relapses occur even after treatment with amphotericin B. The role of a failure of CMI in the pathogenesis of other infectious diseases is even more speculative. In a recent survey of patients with suspected brucellosis, 60%with titers of antibrucella antibodies of 1:80 or more had negative brucellin tests and 50%with positive delayed hypersensitivity had low titers of circulating antibody (Bradstreet et al., 1970). This could indicate that this disease had a form in which there was low resistance associated with a poor host CMI response. In syphilis it has also been postulated that failure of CMI might play a role in the pathogenesis of the disease (Turk, 1970). Indications of this can be found in two sets of observations. First, delayed hypersensitivity to treponemal antigens, although present in latent and tertiary syphilis, is absent in the primary and secondary phases of the disease (Noguchi, 1911; Laymon, 1933; Marshak and Rothman, 1951; Temine et al., 1966; Ranque et al., 1967). Second, lymphocytic depletion of spleen follicles was found in 22 out of 37 human infants with congenital syphilis, 34 of whom had died within the first 6 months of life (Levene et al., 1971). A s’imilar state has been found in the spleen of neonatal rabbits, infected with Treponema pallidurn, which developed a runting syndrome resulting in thfir death within 3 months (Festenstein et al., 1967). Such depletion has been shown by Parrott et al. (1966) to develop when CMI is suppressed by neonatal thyrnectomy. It may be that failure of CMI develops in early acquired syphilis and congenital syphilis as a result of either the direct toxic effect of treponemal products or due to the high level of circulating antibody causing immunological enhancement. In this state secondary syphilis with its occasional associated arthritis, iridocyclitis, and nephritis could be an immune complex disease. The development of CMI in latent and tertiary syphilis produces a state of immunological balance and results in the development of a chronic state in these forms of the disease.

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REFERENCES Adler, S. (1963a). I n “Iinmunity to Protozoa” (P. C. C. Garnhani, A. E. Pierce, and I. hl. Hoitt, eds. ), pp. 235-245. Blackwell, Oxford. Adler, S. ( 196:3b). Reo. Inst. Salrrbridad Enfernicdades Trop. (Alex.) 23, 139. Adler, S. (1964). Adoan. Parasitol. 2, 35. Adler, S. (1965). Z.sr. J. Xfcd. Sci. 1, 9. Adler, S., and Foner, A. (1966). Proc. Irit. Congr. Parasitol. lst, 1964 Vol. 1, p. 343. Adler, S., and Nelken, D. (1965). Trans. Roy. SOC.Trop. Med. Ilyg. 59, 59. Adler, S., Foner, A,, and hlontilio, B. (1966). Trans. Roy. SOC. Trop. Med. Hyg. 60, 380. Ali, A. J. T., and Oakley, C. L. (1967). J. Pathol. Bacterial. 93, 413. .4lmeida, J. O., Brandaeo, H., and de Lima, E. G. ( 1964). Znt. J. Lepr. 32, 293. Aranjo, F. G., and hlayrink, W. (1968). Rev. Inst. Mcd. Trop. Sao Paul0 10, 41. Asherson, (3. L. (1966). It~inirrnology 10, 179. Asherson, G. L., and Stone, S. H. (1965). Zttimtrnology 9, 205. Balzer, R. J., Destombes, P., Schaller, K. F., and SCrik, C. (1960). Bull. Soc. Pathol. Exot. 53, 293. Beiguelinan, B. (1967). Bull. W . H . 0. 37, 461. Benes, J. (1970). J. Parasitol. 56, 396. Benex, J., and Lamy, L. (1968). Anti. Inst. Pastcur, Paris 115, 91. Bittencourt, A. L., Sodri., A., and AndradC, Z. A. (1968). Reu. Inst. Med. Trop. Sao Paulo 10, 247. Blanden, R. V. (1968). J. Reticulocndothcl. SOC.5, 179. Bonomo, L., and Dammncco, F. (1968). I n “Proceedings of the International Symposium on Ganimapathies, Infections, Cancer and Immunity” ( V. Chini, L. Bonomo, and C. Sirtori, eds.). Carlo Erba Found., hfilan. Borrel, A. (1920). Ann. Inst. Pastcur, Paris 34, 105. Bradstreet, C. hl. P., Tannahill, A. J., Pollock, T. hl., and hlogford, H. E. (1970). Lancet 2, 653. Bray, R. S. (1970). Unpublished data. Bray, R. S., and Bryceson, A. D. h4. (1968). Lancet 2, 898. Bray, R. S., and Bryceson, A. D. hl. (1969). Trans. Roy. Soc. Trop. Med. Hyg. 63, 524. Bray, R. S., and Lainson, R. (1965). Trans. Roy. SOC.Trop. Aled. Hyg. 59, 221. Bray, R. S., and Lainson, R. (1966). Trani,. Roy. Trop. bled. Hyg. 60, 605. Bray, R. S., and Lainson, R. (1967). Trans. Roy. SOC.Trop. Mcd. Hyg. 61, 490. Bray, R. S., and Rahim, G . A. F. (1969). Trans. Roy. SOC. Trop Med. Hyg. 63, 383. Brent, L., and hledawar, P. B. (1963). Brit Alcd. J. 2, 269. Brown, J. A. K., and Stone, hl. hl. (1961). Trans. Roy. Soc. Trop. Med. Hug. 55, 443. Brown, R. S., Haynes, I<. A,, Foley, H. T., Godwin, H. A., Bevard, C. W., and Carbone, P. P. (1967). Ann. Intern. Alcd. 67, 291. Bryceson, A. D. hl. (1969). Trans. Roy. SOC.Trop. Aled. Hyg. 63, 708. Bryceson, A. L). h i . (197Oa). Tmrir.. Roy. SOC.Trop. Med. Hyg. 64, 369. Bryceson, A,. D. hl. (197011). Trans. Roy. SOC. Trop. Med. Hyg. 64, 380. . Roy. SOC.T r o p bled. Hyg. 64, 387. Bryceson A. D. hf. ( 1 9 7 0 ~ )Trans. Bryceson, A. D. hl. (1970d). Proc. Roy. SOC.Aled. 63, 1056. . esperiments. Bryceson, A. D. hl. ( 1 9 7 0 ~ ) Unpublished

262

1. L.

TURK AND A. D. hl. BRYCESON

A. D. hl., and Turk, J. L. (1971). J. Pathol. (in press). A. D. M., Bray, R. S., Wolstencroft, R. A., and Dunionde, D. C. (1970). E x p . Zmmunol. 7, 301. A. 1). M . , Prcston, P. M., Bray, R. S., and Dulnonde, D. C. ( 1971a). Clin. Ex),. Z m t t i ~ t t i o / ,( i n press). Bryceson, A. 1). M., Radwanski, Z., Ryder, G . , and Dumonde, D. C. (19711~).In prepar:ition. Bryceson, A. D. M., Bray, R. S., and Dunionde, D. C. ( 1 9 7 1 ~ ) In . preparation. Bryccson, A. D. M., Maini, R. N., Bowers, E. J., and Dumonde, D. C . (1971d). In preparation. Bullock, W. E. (1968). N . Etigl. J. hled. 278, 298. Bullock, W. E., and Fmil, P. ( 1968). Abstr. Zrit. Letir. Congr., 9th, 1968 p. 46. Bullock, W. E., Ho, M-F., and Chen, M-J. (1970). J. Lab. Clin. Med. 75, 863. Cahill, K. (1964). Amcr. J. Trop. Med. Hyg. 13, 794. Caliill, K. (1970). Tram. Roy. Soc. Trop. Med. Hyg. 64, 107. Carmago, h l . E., antl Rehonato, C. (1969). Amer. J. Trop. Med. Hyg. 18, 500. Chaves, J., and Ferri, R. C. (1966). Rev. Inst. Med. Trop. Sao Paulo 8, 225. Chaves, J., and Torrealba, J. W. (1967). Gac. Med. Caracas 75, 19. Chilgrcn, R. A., Mcuwissen, H. J., Quie, P. G . , Good, R. A., and Hong, R. (1969). Lancet 1, 1286. Cochrane, R. G. (1964). 111 “Leprosy in Theory and Practice” ( R . G. Cochrane antl T. F. Davey, eds.), p. 623. Wright, Bristol. Cochrane, R. G., and Smyly, H. J. (1964). In “Leprosy in Theory and Practice” ( R . G. Cochrane and T. F. Davey, eds.), p. 299. Wright, Bristol. Convit, J. (1958). R e v . Sanid. Asistencia S O C . 23, 1. Convit, J. ( 1970). Alistr. Znt. Lepr. Colloq. 1970 p. 85. Convit, J., and Kertlel-Vegas, F. (1965). Arch. Derniatol. 91, 439. Convit, J., and Pinardi, M. E. (1969). Dermatol. Znt. 8, 17. Convit, J., Kerdel-Vegas, F., and Gordon, B. (1962). Brit. J. Dermatol. 74, 132. D’Alesandro, P. A. (1954). M.Sc. Thesis, pp. 10-37. Rutgers University, New Brunswick, New Jersey. Danielsen, D. C., and Boeck, C. W. (1848). “Trait6 de la Spedalskhed ou Elephantiasis des Grecs.” BailliBre, Paris. da Silva Lacaz, C. (1967). “Compendio de Micologia Medica.’’ Sarvier, Sao Paulo. Ilavis, B. D. ( 1944). hlctlicitie (Baltimore) 23, 359. Deane, hl. P., Chaves, J., Torrealha, J. \V,, and Torrealba, J. F. (1966). Gac. Med. Caracas 74, 367. Delville, J . ( 1970). A h t r . Z t i f . Lepr. Colloc/., 1970. Forschrt,ifi.Piri,P.titcrt,Bor.rtel., p. 31. Destombes, 1’. ( 1960). Btrll. Soc. Pathol. E x p t . 53, 299. Destombes, P., Poirier, A., and SkriB, C. (1965). Arch. Inst. Pmteur Alger. 43, 9. Dharmcndra ( 1912). Lcpros!/ i n Itidia 14, 122. Dierks, R. E., and Shepard, C. C . (1968). Proc. Soc. E x p Biol. Metl. 111, 514. Dostrovsky, A. (1936). A m . Trop. Mcd. Parasitol. 30, 267. Dostrovsky, A., Zuckcrnian, A,, and Sagher, F. ( 1952). Harefrtah 43, 29. Ilraper, P., Rees, R. J. W., and Waters, M. F. R. (1968). Clin. E x p . Zmmunol. 3, 809. I ~ n x b ~ ~11. r y E., , and Satlun, E. H. ( 1964). Amcr. J. Trop. Aled. Hyg. 13, 525. Evan-Paz, Z., and Sagher, F. (1961). S. Afr. Med. J . 35, 576. Ferri, 11. C., and Chaves, J. (1968). Arq. Castroenterol. 5, 169.

Bryceson, Bryceson, Clin. Bryceson,

LEPROSY AND RELATED DISEASES

263

Festenstein, H., Abraham, C., and Bokkenheuser, V. (1967). Clin. E x p . Zmmunol. 2, 311. Fischer, 0. (1933). Z. Trrberk. 68, 50. Fulginiti, V. A,, Hathaway, W. E., Pearlman, D. S., Blackburn, W. R., Reiquam, C. W., Githens, J. H., Claman, H. N., and Kenipe, C. H. (1966). Lancet 2, 5. Gaafar, S., and Turk, J. L. (1970). J. Pathol. 100, 9. Garnhani, P. C. C., and Humphrey, J. H. (1969). Curr. Top. Microbial. Immunol. 48, 29. Gaugas, J . (1968). Nature (London) 220, 1246. Gaugas, J., Chesterman, F. C., Hirscli, M. S., Rees, R. J. W., Harvey, J. J., and Gilchrist, C. ( 1969). Nature (London) 221, 1033. Gaugas, J., Rees, R. J. W., Weddell, A. G. M., and Palmer, E. (1971). Int. J. Lepr. (in press). Gitlin, D., and Craig, J. M. (1963). Pediatrics 32, 517. Glazunova, Z. I . (1965). A4ed. Pararitol. Parazit. Bolez. 34, 582. Godal, T., Rees, R. J. W., and Lanivik, J. 0. (1971). Clin. Exp. Irnmunol. 8, 625. Guinto, R. S., and Mabalay, M. C. (1962). Int. J. Lepr. 30, 278. Gunders, A. E., Foner, A,, and Montilio, B. (1968). Nature (London) 219, 85. Han, S. H. ( 1970). Personal coniniunication. Han, S. H., Tseng, J. J., and Kau, S. T. (1970a). Personal communication. Han, S. H., Weiser, R. S., and Kau, S. T. (1970b). Personal communication. Hanks, J. H. (1947). Int. J. Lepr. 15, 21. Hart, P. D., and Rees, R. J. W. (1967). Brit. Med. Bull. 23, 80. Hoare, C. A., and Wallace, F. G. ( 1966). Nature (London) 212, 1385. Hobbs, J. R. (1970). Brit. J. Hosp. Med. 3, 669. Hoogstraal, H., and Heyneman, D. (1969). Amer. J. Trop. Med. Hyg. 18, 1091. Howard, W. L., Klopfenstein, M. D., Steininger, W. J., and Woodruff, C. E. (1970). Chest 57, 530. Imperato, P. J., and DiakitC, S. (1969). Trans. Roy. SOC. Trop. Med. Hyg. 63, 236. Irunberry, J., Benalkgue, A., Grangaud, J. P., Mazonni, M., Khati, B., and Khedair, M. (1968). Arch. Inst. Pasteur Alger. 46, 102. Jamison, D. G., and Vollum, R. L. (1968). Lancet 2, 1271. Jancovic, B. D. (1962). Nature (London) 193, 789. Kaliss, N. (1958). Cancer Res. 18, 992. Kellina, 0. (1966). Med. Parazitol. 35, 679. Khanolkar, V. R. ( 1964). In “Leprosy in Theory and Practice” ( R . G. Cochrane and T. F. Davey, eds.), 13. 125. Wright, Bristol. Khanolkar, V. R., and Cochrane, R. G. (1956). Indian J . Med. Sci. 10, 499. Kunkel, H. G., Simon, N. J., and Fudenberg, H. (1958). Arthritis Rheum. 1, 289. Kuper, S. W. A . (1964). In “Leprosy in Theory and Practice” (R. G. Cochrane and T. F. Davey, eds.), p. 183. Wright, Bristol. Kurban, A. K., Malok, J. A., Farah, F. S., and Chaglassian, H. T. (1966). Arch. Derniatol. 93, 396. Lainson, R., and Bray, R. S. (1966). Trans. Roy. SOC. Trop. Med. Hyg. 60, 526. Laymon, C. W.(1933). Arch. Dermatol. Syph. 27, 518. Lehman-Fascius, H., and Loeschke, H. ( 1926). Muenchen. Med. Wochenschr. 73, 1578. Lemma, A,, Foster, W. A., Gemetchew, T., Preston, P. M., Bryceson, A. D. M., and Minter, D. M. (1969). Ann. Trop. Med. Parasitol. 63, 455.

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Levene, G. M., Wright, D. J, M., and Turk, J. L. (1971). Proc. Roy. SOC. Med. 64, 426. Majundar, T. D. (1968). Dermatol. Int. 6, 174. Manson-Bahr, P. E. C. (1961). Trans. Roy. SOC. Trop. Med. Hyg. 55, 550. Manson-Bahr, P. E. C. (1964). J. Trop. Med. Hyg. 67, 85. Manson-Bahr, P. E. C. (1967). East Afr. Med. J. 44, 177. Manson-Bahr, P. E. C., and Southgate, B. A. (1964). J. Trop. Med. Hug. 67, 79. Manson-Bahr, P. E. C., Heisch, R. B., and Garnham, P. C. C. (1959). Trans. Roy. SOC. Trop. Med. Hyg. 53, 380. Marshak, L. C., and Rothman, S. (1951). Amer. J. Syph. 35, 35. Martins, J. M., Cunha, R. N., and Pitombeira, M. da S. (1969). Reo. Brasil. Pesquisas Med. Biol. 2, 89. Matthews, L. J., and Trautman, J. R. (1965). Lancet 2, 915. Medina, R., and Romero, J. (1959). Arch. Venez. Med. Trop. Parasitol. Med. 3, 298. Medina, R., and Romero, J. (1962). Arch. Venez. Med. Trop. Parasitol. Med. 4, 349. Melli, C., Mazzei, D., Rugarli, C., Ortolani, C., and Bazzi, C. (1968). Abstr., Int. Congr. Microbiol. Stand., 9th, 1968 p. 47. Miller, H . C., and Twohy, D. W. (1969). J. Parasitol. 55, 200. Mowbray, J. F., Boylston, A. W., Milton, J. D., and Weksler, M. (1969). Antibiot. Chemother. (Basel) 15, 349. Mukherjee, A. C., Neogy, K. N., and Sen Gupta, P. C. (1968). Bull. Calcutta Sch. Trop. Med. 16, 38. Mustakallio, K . K., Lassus, A., and Wager, 0. (1967). Int. Arch. Allergy Appl. Immunol. 31, 417. Nahmias, A. J., Griffith, D., Salsbury, C., and Yoshida, K. (1967). J. Amer. Med. Ass. 201, 729. Neal, R. A., Garnham, P. C. C., and Cohen, S. (1969). Brit. Med. Bull 25, 194. Nelson, D. S., Nelson, M., Thurston, J. M., Waters, M. F. R., and Pearson, J, M. (1971). Clin. Exp. Immunol. (in press). Nery-Cuimarez, F., Lage, H. A,, Venancio, I. A,, and Grynberg, N. F. (1969). Hospital (Rio de Janeiro) 5, 1811. Neveu, T. (1964). Ann, Inst. Pasteur, Paris 107, 320. Newell, K. W. (1966). Bull. W. H . 0. 34, 827. Noguchi, H. (1911). Muenchen. Med. Wochenschr. 58, 2372. Norlin, M., Navalkar, R. G., Ouchterlony, O., and Lind, A. (1966). Acta Pathol. Microbiol. Scand. 67, 555. Paraense, L. W. (1953). Trans. Roy. SOC. Trop. Med. Hyg. 47, 556. Parrott, D. M. V., de Sousa, M. A. B., and East, J. (1966). J. Exp. Med. 123, 191. Pettit, J . H. S. (1962). Brit. J. Dermatol. 74, 127. Polak, L., and Turk, J. L. (1968). Clin. Erp. Immunol. 3, 245. Preston, P. M., and Dumonde, D. C. (1970). Personal communication. Price, E. W., and FitzHerbert, M. (1965). Ethiop. Med. J. 3, 57. Priolisi, A., and Cuiffri., L. (1967). Pathol. Microbiol. 30, 215. Ptak, W., Caugas, J. M., Rees, R. J. W., and Allison, A. C. (1970). Clin. Exp. Immunol. 6, 117. Quilici, M., Dunan, S., and Ranque, J. (1968). Med. Trop. (Marseilles) 28, 37. Radwanski, Z., Bryceson, A. D. M., Chaffee, N., and Dumonde, D. C. (1971). In preparation. Rahim, G. F., and Tartar, I. H. (1967). Bull. Endem. Dis. 8, 29.

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Ranque, J., and Quilici, M. (1970). J. Parasitol. 56, 277. Ranque, J., Quilici, M., and Assadourian, Y. (1967). Med. Trop. (Marseilles) 27, 519. Ranque, J., Quilici, M., Dunan, S., and Assadourian, Y. (1969). Med. Trop. (Marseilles) 29, 70. Rees, R. J. W. ( 1964). Progr. Allergy 8, 224. Rees, R. J. W. (1965). Nature (London) 211, 657. Rees, R. J. W. (1970). Personal communication. Rees, R. J. W., and Valentine, R. C. (1964). In “Leprosy in Theory and Practice” ( R . G. Cochrane and T. F. Davey, eds.), p. 26. Wright, Bristol. Rees, R. J. W., and Weddell, A. G. M. (1968). Ann. N. Y. Acad. Sci. 154, 214. Rees, R. J. W., Chaterjee, K. R., Pepys, J., and Tee, R. R. (1965). Amer. Rev. Resp. Dis. 92, 139. Rees, R. J. W., Waters, M. F. R., Weddell, A. G. M., and Palmer, E. (1967). Nature (London) 215, 599. Rees, R. J. W., Weddell, A. G . M., Palmer, E., and Pearson, J. (1969). Brit. Med. 1. 3, 216. Report of the Panel on Bacteriology and Immunology. (1963). Int. J. Lepr. 31, 495. Rezai, H. R. ( 1970). Personal communication. Rezai, H. R., Slier, R., and Cettner, S. (1969). Exp. Parasitol. 26, 257. Ridley, D. S. (1969). Lepr. Rev. 40, 77. Ridley, D. S., and Jopling, W. H. (1962). Lepr. Rev. 33, 119. Ridley, D. S., and Jopling, W. H. (1966). Int. J. Lepr. 34, 255. Ridley, D. S., and Waters, M. F. R. (1969). Lepr. Rev. 40, 143. Rowley, hl. J., Buchanan, H., and Mackay, I. R. (1968). Lancet 2, 24. Saitz, E. W., Dierks, R. E., and Shepard, C. C. (1968). Int. J. Lepr. 36, 400. Salazar-Mallen, M., Montes-Montes, J., Escobar-Gutienes, A., Amezcua-Chavarria, h4. G., and Chavez, A. (1970). Abstr., I n t . Congr. Microbial., loth, 1970 p. 81. Saul, A., Rodriguez, O., Novales, J., and Navarro, E. (1969). Dermutol. Rev. Mex. 13, 301. Sen Cupta, P. C. (1966). Proc. Int. Congr. Parasitol., lst, 1964 Vol. 1, p. 350. Sen Gupta, P. C., and Mukherjee, A. M. (1968). J. Indian Med. Ass. 50, 1. Shamma, A. H., Ali, A. J . T., and El-Shawi, N. N. (1965). J. Pathol. Bacteriol. 90, 659. Shaw, J. J., and Voller, A. (1968). Ann. Trop. Med. Parasitol. 62, 174. Sheagren, J. N., Block, J. B., Trautman, J. R., and Wolff, S. M. (1969). Ann. Intern. Med. 70, 295. Shepard, C. C. (1960). J. Exp. Med. 112, 445. Shepard, C. C. ( 1971). Personal communication. Shepard, C. C., and McRae, D. H. (1965). J. Bacteriol. 89, 365. Shepard, C. C., and Saitz, E. W. (1967). J. Immunol. 99, 637. Southgate, B. A., and Manson-Bahr, P. E. C. (1967a). J. Trop. Med. Hyg. 70, 29. Southgate, B. A., and Manson-Bahr, P. E. C. (196713). J. Trop. Med. Hyg. 70, 33. Southgate, B. A., and Oviedo, B. V. E. (1967). J. Trop. Med. Hyg. 70, 1. Stauber, L. (1963a). Ann. N. Y. Acad. Sci. 113, 409. Stauber, L. (1963b). J. ParasitoZ. 49, 3. Swanson, M. A., and Schwartz, R. S. (1967). N. EngZ. J. Aled. 277, 164. Tal\var, G. P. ( 1970 ). Personal communication. Temine, P., Tramier, G., and Privat, Y. (1966). Proph. Satiit. Morale 38, 154. Torrealba, J. W. ( 1967 ). Personal communication. Townes, A. S. (1967). Johns Hopkins hled. J . 120, 337.

266

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L. TURK AND A. D. M. BRYCESON

Tremonti, L., and Walton, B. C. (1970). Amer. J. Trop. Med. Hyg. 19, 49. Turk, J. L. (1969). Bull. W. H . 0. 41, 779. Turk, J. L. (1970). Brit. Med. J. 3, 363. Turk, J. L., and Oort, J. ( 1971 ). I n “Handbuch der allgemeinen Pathologie” ( H . Cottier and A. Studer, eds.), Vol. 7, Part 3, p. 392. Springer-Verlag, Berlin and New York. Turk, J. L., and Waters, M. F. R. (1968). Lancet 2, 436. Turk, J. L., and Waters, M. F. R. (1969). Lancet 2, 243. Turk, J. L., and Waters, hl. F. R. ( 1971). Clin. E x p . Imrnunol. 8, 363. Twohy, D. W. ( 1970). Personal communication. Ulrich, M., Trujillo, 0. D., and Convit, J. (1968). Trans. Roy. SOC. Trop. Med. Hug. 62, 825. Valdimarsson, H., Holt, L., Riches, H. R. C., and Hobbs, J. R. (1970). Lancet 1, 1259. von Kretschmar, W. (1965). 2. Tropenrned. Parasitol. 16, 277. Wade, H. W. (1940). I n t . J. Lepr. 8, 307. Wade, H. W. (1961). Int. J. Lepr. 29, 99. Wager, 0.(1969). Bull. W. H . 0. 41, 793. Waldorf, D. S., Sheagren, J. N., Trautman, J. R., and Block, J. B. (1966). Lancet 2, 773. Walton, B. C. (1970). J. Parasitol. 56, 480. Waters, M. F. R. (1971). Personal communication. Waters, M. F. R., and Recs, R. J. W. (1962). I n t . J. Lepr. 30, 266. Waters, hl. F. R., and Ridley, D. S. (1963). Int. J. Lepr. 31, 418. Waters, M. F. R., Rees, R. J. W., and Sutherland, I. (1967). Int. J. Lepr. 35, 311. Waters, hl. F. R., Turk, J. L., and Wemambu, S. N. C. (1971). Int. J. Lepr. (in press ) . Weddell, A. G. M.,Jamison, D. G., and Palmer, E. (1964). In “Leprosy in Theory and Practice” (R. G. Cochrane and T. F. Davey, eds.), p. 205. Wright, Bristol. Wenianibu, S. N. C., Turk, J. L., Waters, hl. F. R., and Rees, R. J. W. (1969). Lancet 2, 933. Werthein, G., Foner, A., and Montilio, B. (1970). Nature (London) 226, 267. Williams, R. C., Jr., and Kunkel, H. G . (1962). J. Clin. Inoest. 41, 666.

Nature and Classification of Immediate-Type Allergic Reactions ELMER 1. BECKER Deportment of Pofhology, University of Connecticut Schools o f Medicine and Dentistry, Farmington, Connecticuf

I. Introduction . . . . . . . . 11. Sensitization . . . . . . . . 111. Components of the Allergic Reaction . . . A. Antigens . . . . . . . . B. Antibodies . . . . . . . . C. Effector Enzyme Systems . . . . D. Mediators . . . . . . . . E. Cells . . . . . . . . . IV. Sites of the Antigen-Antibody Reaction . . V. Time Course of Allergic Reactions . . . VI. The Terrain . . . . . . . . VII. Basis and General Description of the Classification VIII. Direct Responses (Non-mediator Determined) . IX. Indirect Responses (Mediator Determined) . A. Anaphylactic-Type Reactions . . . . B. Macromolecular Mediator-Determined Reactions C. Unknown hlediator-Determined Reactions . X. Mixing of Categories in Natural Reactions . . XI. Pseudoallergic Reactions . . . . . References . . . . . . . .

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As ideas are preserved and communicated by means of words it necessarily follows that we can not improve the language of any science without at the same time improving the science itself; neither can we on the other hand, improve a science without improving the language or nomenclature which belongs to it. HOWever certain the facts of any science may be and however just the ideas we have formed of the facts we can only communicate false impressions to others while we want words by which these may be properly expressed. A. Lavoisier, “Elements of Chemistry”

I. Introduction

According to William James, infants find the world a booming, buzzing confusion, and until relatively recently this is how even mature workers found the field of immediate-type allergic reactions. However, work of the past 10 years or so has resulted in a large reduction in the confusion, if not in the booming and the buzzing. In certain instances, this increase in our understanding of the immediate-type allergic re267

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sponses has come about through a better definition of the antigens involved; more generally, it has been due both to a better appreciation of the nature and differing capacities of the various antibodies concerned in the reactions and a more detailed comprehension of the manifold biochemical pathways of tissue injury triggered by the reaction of antigen with these various antibodies. The body of knowledge which is emerging is still a skeleton, imperfectly articulated and only partially fleshed. Nevertheless, with the present and growing importance of allergy, a general survey of the nature of the immediate-type allergic reactions which focuses attention on our present understanding of the mechanisms involved should be of value. Any allergic manifestation is the visible result of a long series of steps, each step frequently comprising one or more sequences of reactions. In what follows, after some general definitions and comments, I shall attempt to identify these various steps in the mechanisms, the components that are involved, and then indicate how a tentative classification of the immediate-type allergic reactions can be erected based on our present understanding of these mechanisms. Von Pirquet originated the term allergy and defined it as follows: The vaccinated person behaves toward vaccine lymph, the syphilitic toward the virus of syphilis, the tuberculous patient toward tuberculin, the person injected with serum toward this serum, in a different manner from him who has not yet been in contact with such an agent. Yet, he is not insensitive to it. We can only say of him that his power to react has undergone a change. For this concept of a changed reactivity I propose the term “Allergy.”’

In the same paper, von Pirquet explicitly stated that allergy included not only hypersensitivity-the altered reactivity injurious to the hostbut also immunity, the alteration beneficial to the host in resisting infection ( I ) . Over the years, contrary to von Pirquet’s original intention, the term allergy in common medical, scientific, and lay usage has come to refer only to those immunological reactions that are damaging to the host; hypersensitivity and allergy have become synonymous. Recognizing the need for a term that would encompass damaging, beneficial, and neutral immunological reactivities, Coombs and Gel1 have suggested a return to von Pirquet’s original definition of allergy (2 ). This suggestion has every virtue except the possibility of general acceptance. Such a reactionary step is not really required, however. The term immunology or the adjectival form immunological in the majority, if Doerr, somewhat later made explicit the necessary restriction that the induced, specific, altered reactivity must arise through previous exposure to an antigenic substance.

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not all, of its present uses is fundamentally in accord with von Pirquet’s original definition of allergy. It is only necessary to restrict the term imniunological to those antigen-antibody-induced‘ reactions in which neither injury to the host nor rcsistancc to infection is necessarily iniplied as the final outcome of the reaction. Iniiiiiiiie reactioiis or imntunity are restricted to imniunological reactions beneficial or protective to the host organism3 Allergic reactions are, then, defined according to con~monusage as those immunological reactions that are damaging to the tissues or disruptive of the physiology of the vertebrate host. Although, as is indicated above, we carefully distinguish in words and in fact between allcrgy and immunity the sanic mechanisms operate in both; the fundamental difference in any individual reaction is who or what is the primary target of thc given immunological niechanisniwhether the invading organism and its products are affected in the case of immunity or the vertebrate host in the case of allergy. If microorganisms wrote textbooks there is no doubt which rcactions they would term allergic. Unlike Caesar’s Gaul, allergic reactions are grossly divided into only t\vo types-the immcrliatc-typc,’ thc subject of this discussion and the delayed-type. The imniediatc-type reactions include all reactions determined by one or another of the various kinds of antibodies found in the circulation and body fluids. They can be transferred from one animal to another by the appropriate antiserum. The delayed-type ‘Antiliody is used here in von Pirquet and Schick‘s original sense of “the sum of the specific products of the organism created by the introduced antigen” ( 3 , p. 119). Thus, its meaning includes not only the kinds of antibody found in the blood and tissne fluids but also the recognition factors arising on lymphoid cells as a result of inimunization which are presumed to be associated with delayed hypersensitivity. In what follows, however, I shall generally usc the term “antibody” in its more usual restricted sense as the antibody found in the circulation and body fluids. The term ccZZdar inimtinity is used in a t least two senses, I n the first it refers to those situations involving resistance to infection with intracellular microorganisms where the resistance is associated with activated macrophages. In the second, cellular immunity has been made synonymous \vith rlclaycd hypcrscnsiticity or used to refer to situations such as rejection of solid tissue grafts, \vhere it is believed delayed hypersensitivity operates. I can only make a n heartfelt plea that cellular immunity be restricted to the first sense. This is not only because ivhen it is employed as a synonym for delayed hypersensitivity it misuses ( o n thc basis of the preceding discussion) the term immunity but also that its use in this sense implicitly denies the importance of cells in immediate-t)~pe hypersensitivity. ‘ Immediate-type allergic reaction.; were originally termed “immediate allergic reactions”; they are now termed “immediate-type” ( 4 , p. 52G) to emphasize that they refer not only to reactions occurring within minutes but also to those that evolve over a matter of hours.

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reactions, in general, are not transferable by serum but are transferable by lymphoid cells. So far as present evidence goes, none of the presently recognized circulating intibodies is demonstrably responsible for delayed reactions, and uie nature of the specific recognition factor( s ) for the delayed responses is unknown.5 The delaycd reactions will not be considered any further here and, in what follows, when the terms allergic or allergy are used, only reactions of the immediate-type will be implied. II. Sensitization

The first step in an allergic reaction is sensitization. This is the preparation of the animal to give an allergic reaction when challenged by either antigen or antibody. The preparation of the animal so that challenge with antigen triggers the response is termed direct sensitization; reverse sensitization, involves the preparation of the animal so that challenge with antibody initiates the reaction. Induction of the allergic state by administration of antigen so that the organism makes its own antibody is spoken of as direct active sensitization or more commonly just as active sensitization. One could, if one wished, logically speak of reverse active sensitization in those situations where the antigen is an integral part of the subject’s own tissues, although, as far as I know, this is never done. Humans who undergo transfusion reactions due to the antibody contained in the transfused blood of a “dangerous universal donor” reacting with antigen present on their own erythrocytes are victims of reverse active sensitization. The guinea pig reacting to antibody against the Forssman antigen could be considered to be reverse actively sensitized by virtue of its possession of Forssman antigen as an integral part of its tissues. Direct passive sensitization or usually just passive sensitization occurs when an organism is prepared for an allergic reaction by administration of preformed antibody. Direct passive sensitization for the passive cutaneous anaphylactic (PCA) reaction can be induced by injecting antibody into guinea pig skin and then giving antigen and a blue dye intravenously 2 to 4 hours later to make manifest the consequent increase in vascular permeability. Direct passive sensi’ A number of workers have supposed that these recognition factors are the same as the kinds of antibody found in the circulation. Moreover, the assumption is also frequently made that such antibody is produced and/or is an integral part of the cell concerned. If these suppositions are borne out by future work w e shall then b e able to speak of “delayed hypersensitivity” as an “endogenous antibodydetermined allergic reaction” and immediate-type allergic reactions as “exogenous antibody-determined allergic reactions.’’ But such terminology must await upon future experimental proof of these assumptions.

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tization for an Arthus reaction consists of giving the antibody intravenously and then injecting the antigen into the skin. Reverse passive sensitization ensues when antigen is administered to an organism followed by antibody. Reversed passive cutaneous anaphylaxis( RPCA) occurs when a guinea pig is sensitized by administering an antigen such as rabbit 7-globulin into its skin; the reaction is triggered by giving antibody sometime later ( 5 ) . Similarly, a rabbit or guinea pig may undergo reverse passive sensitization for an Arthus reaction by giving antigen intravenously followed with antibody injected into the skin, In direct passive sensitization for certain kinds of anaphylactic reactions, an interval of time following the administration of antibody must elapse before challenge with antigen will elicit a maximum response or, in some circumstances, any response at all. This interval of time is called the latent ,period. The existence of a latent period was and generally still is taken as strong evidence of the necessity for the antibody to diffuse and fix to trigger cells or target mediator cells (see Section II1,C) in order to give an allergic reaction with antigen. This is probably true in many instances. That it is true in all instances, or when true that it is the complete explanation for the latent period is unlikely [see Binaghi ( S ) ] . Many reactions including a number of anaphylactic-type reactions, as well as others such as the Arthus reaction, have no latent period. I l l . Components of the Allergic Reaction

A. ANTIGENS Antigens considered from the viewpoint of their capacity to take part in either delayed or immediate-type allergic responses are spoken of as allergens. Allergens can be either complete antigens, in which case they can both induce and elicit an allergic reaction, or they can be haptens only capable of eliciting the response but not inducing it. Allergy to drugs is very largely allergy to haptens. Drug allergies arise mainly through the ability of the drug or its metabolic products to conjugate with body proteins to form a complete sensitizing antigen (or a complete “allergen”). In many cases the drug or its transformation product must also conjugate with body proteins before it can elicit the reactions. In a few instances, it is probable that prior conjugation is not necessary; for example, the ability of the drug when added in vitro to serum from cases of thrombocytopenic purpura due to Sedormid, quinidine sensitivity, etc., to give complement-dependent damage to platelets suggests that, in this instance, covalent conjugation of drug and protein is not required to elicit the given reaction (see below).

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An experimental animal or a human being can be sensitized to exogenous (extrinsic) or to endogenous (intrinsic) antigens. In the latter instance, reactions to the endogenous antigen are spoken of as autoallergic (the complete inappropriateness of the more commonly used term autoimmune should be readily apparent from the previous discussion; autoimmune in this context should be completely abandoned, [see also Vaughn et al. (7)]. Even though these are a large group of important reactions the pathogenetic mechanisms they involve are essentially the same as those involved in allergic reactions to exogenous antigens. Therefore, in what follows, no attempt will be made to consider autoallergic reactions as a special class.6 For a critical review of some of the problems of autoallergic disease the reader may consult Vaughn et al. ( 7 ) and Kunkel and Tan (8). In direct active sensitization, the sensitizing antigen is usually cleared from the organism before sufficient antibody is evoked to cause a reaction. In these circumstances, a second exposure to antigen is required to trigger the reaction. Under a number of other circumstances, all of the antigen is not cleared from the organism before antibody arises and that which remains can combine with the newly formed antibody and trigger an allergic reaction. When this occurs the various kinds of responses possible are all grouped under serum sickness, or when serum is not the inciting antigen, serum sickness-like reactions. Historically, the administration of foreign serum in the form of diphtheria or tetanus antitoxin prepared in horses was the most common cause of serum sickness; hence the name given the syndrome by von Pirquet and Schick in their great monograph of 1905 ( 3 ) . At present, serum sickness-like reactions in humans are most commonly seen following the administration of drugs, especially penicillin ( 9 ) . In addition, the delayed thrombocytopenic purpura occasionally seen after transfusion (10) and the very rare cases of delayed anti-Rh hemolytic disease following transfusion (11) are other examples of serum sicknesslike reactions in humans. The vasculitis and glomerulitis arising after large doses of antigens in rabbits ( 1 2 ) or the “autologous phase” of glomerulonephritis arising a week or more after the administration of heterologous, antiglomerular, basement membrane antiserum ( 1 3 ) are all examples of experimental serum sickness or serum sickness-like reactions in experimental animals even though the pathogenesis of the various reactions differ. Serum sickness-like reactions follow the administration of widely a There is evidence that delayed hypersensitivity may be that the basis of certain autoallergic reactions, but some of them, such as the autoallergic hemolytic anemias (see below), are clearly of the immediate type.

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different kinds of antigens and involve variegated mechanisms, as will become more evident later. Some have attempted to make the term serum sickness synonomous with allergy due to antigen-antibody aggregates; for example, citing the glomerulonephritis resulting from the administration of preformed antigen-antibody complexes as an example of passive serum sickness ( 1 3 ) .This implies that all reactions commonly termed serum sickness or serum sickness-like have a single pathogenesis due to the presence of antigen-antibody complexes. It is unlikely this is so; there is evidence, for example that the urticaria of human serum sickness or human serum sickness-like reactions may be due to the reaction of homocytotropic antibody with antigen ( 1 P 1 6 ) . It seems best, therefore, to keep the terms serum-sickness or serum sickness-like reactions, for those reactions, whatever their nature, where the same depot of antigens serves both to sensitize the organism and to trigger the allergic responses. This usage, unfortunately is not without its own difficulties. The reactions occurring within minutes after the administration of serum are, as von Pirquet and Schick originally postulated, due to antibody already present; this reaction would be termed serum anaphylaxis and not be considered an example of serum sickness despite the usual practice of classifying such reactions as part of the serum sickness syndrome. Similarly, the urticaria1 reactions occurring within a few hours after the administration of penicillin would bc differentiated from the serum-sickness-like reaction occurring 3 to 4 days or a week after the administration of penicillin. The time interval in the two kinds of responses to penicillin are not only different but arise through a somewhat different process. In the former, antibody is present at the time of administration of the penicillin, and the hour or so time interval between administration and visible manifestation is probably due to the time required for the penicillin to form the reactive penicilloyl conjugate in the body. The urticaria, etc., in this first instance would not be classed as a serum sickness-like reaction. In the latter instance, the injection of penicillin a few days or a week or more previously induces antipenicilloyl antibody which reacting with the penicilloyl conjugate formed from the penicillin so administered engenders the urticaria. This would then be an example of a serum sickness-like reaction.

B. ANTIBODIES Central to any discussion and description of immediate-type allergic reactions are the two general properties of antibodies, their specificity of combination and, even more, their nonspecific functional capacities. The relative importance of these two general properties of antibodies

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varies from one kind of allergic reaction to another. Certain, although not all of the allergic hemolytic anemias and thrombocytopenic purpuras, the reactions of anti-Forssman antibody in guinca pigs, and the early or heterologous phase (13) of the glonlerulonephritis produced by antiglomerular basement membrane antibodies, are examples of reactions in which the specificity of the antibody for a given antigen is a necessary condition for the particular kind of damage suffered. Nevertheless, even in thesc ancl similar examplcs, the ability of antibody to combine specifically with a givcn antigcn is not sufficient; the antibody must have the requisite nonspecific functional abilities in order for combination of the antibody with the antigen to result in damage, such as, in the examples given, the capacity to fix complement. The specificity of the antibody, however, is a matter of indifference in dctcrinining thc various forms of the anaphylactic-type reactions ( Section IX,A), the Arthus rcactioii ( Section IX,B ), and in many other kinds of allergic response. The crucial properties of the antibody are its nonspecific capabilities, such as the ability of antibody when aggregated with antigen to activate the Itinin-forming, the clotting or the complement systems, or to directly or indirectly prepare mast cells and other cells to release histamine and other mediators on the addition of antigen, or to attach to macrophages and thus direct the way in which the macrophages will react with other cells. Antibodies fall into different immunoglobulin classes with different chemical structures and with widely different nonspecific functional capacities (17). One such functional capacity is the ability to fix complement. In both the guinea pig and the mouse the electrophoretically slow moving y 2 class of yG antibodies are complement fixing, but the faster moving yl are not.s In humans, the subclasses (19) of the slow moving yG antibodies, yG,, yG,, and to a lesser extent, yG2 are all complement fixing; yG, is not. The Rh yG antibodies belong to both the noncompleinent and complement-fixing yG subgroups ( 2 0 ) . Nevertheless, they do not generally fix complement. It is possible that these latter antibodies are noncompleinent fixing because the paucity of Rh antigenetic sites on thc red cell does not permit two or more of the sites to occur together, as required for complement fixation by yG antibodies [ ( 2 1 ) ;see also Klun and Muschel (22) and Rosse ( 2 2 u ) l . Most of the y M human antibodies are complement fixing, although the guinea pig yM antibodies and some mouse and rabbit yM antibodies so far studied 'Recent work has shown that preformed coniplexes of guinea pig yl antibody with antigen are complement-fixing even though, on the addition of antigen and antibody to complement, no fixation occurs (18).

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are not (23, 24). Human yA and y E antibodics do not fix complement (25). Another kind of functional capacity of antibodies is their cytotropic properties ( 2 6 ) . This is the ability of certain antibodics to sensitize for anaphylactic-type reactions by fixing with greater or lesser persistence to certain cells in such a manner that specific combination with antigen leads to the release of pharmacological mediators (see Section II1,D) from these or other cells. Cytotropic antibodies are of two general kinds-homocytotropic and lieterocytotropic. Homocytotropic antibodies are cytotropic antibodies that can fix to cells of animals of the species in which they originate or of closely related species. Heterocytotropic antibodies do not sensitize by fixing to cells of their own species but do sensitize by fixing to cells of foreign specics.’ Human, rats, rabbits, mice, dogs, and guinea pigs (27) all produce a heat-labile homocytotropic antibody (28). The heat-labile homocytotropic antibody of man belongs to a separate immunoglobulin class, yE (29). There is direct evidence that the heat-labile homocytotropic antibody of the rabbit is homologous with the yE of humans and can be called y E of the rabbit ( 3 0 ) . It is quite likely that the heat-labile antibody of the other species, for example the rat and mouse ( 3 1 ) ,are also homologous with human YE; this has yet to be determined. In view of the great similarity in their properties to human yE, they may be called “YE-like.”The human YE antibody has been shown to sensitize one or several kinds of human leukocytes ( 3 2 ) , and the corresponding antibody in the rabbit probably sensitizes rabbit leukocytes; the antibodies of the rat and the mouse sensitize mast cells of their respective species ( 3 3 4 % ) . The heat-labile homocytotropic antibodies, YE-like or Class I antibodies as they have been called ( 2 8 ) are not the only homocytotropic antibodies. The noncomplement-fixing y l antibodies of the mouse and guinea pig also sensitize their own species but generally after a shorter latent period. They are heat stable and might provisionally be termed heat stable, homocytotropic or Class I1 antibodies (28). The mouse -yl antibody fixes to mouse mast cells, but much less firmly than the corresponding heat-labile homocytotropic antibody ( 3 6 ) . The reported

” In the definition of homocytotropic, the primary consideration is that the antibodies arc able to fiu to cells of the species in which they originate to give mediator release. The fact that in some instances they are capable of fixing to cells and tissues of unrelated species to give mediator release is not crucial so far as the definition is concerned. Similarly, in regard to the definition of heterocytotropic, the fact that such antibodies are not able to fix to cells of their species of origin to give pharmacological mediator release is as critical as their ability to fix to foreign cells.

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heterogeneity of sltin-sensitizing antibodies in humans ( 37) might possibly reflect the existence in this species of not only yE homocytotropic antibodies but antibodies analogous to the yl of the mouse and guinea pig and to yG of the rat (38, 39). There are species differences and even more striking similarities in the kinds of reactions the various homocytotropic antibodies can determine. The reaction with antigen of YE antibody in humans (38) and of yl antibody in guinea pigs (39) leads to the release of both histamine and SRS-A (see Section III,D for definition). In rats, the reaction of antigen with the heat-labile homocytotropic antibody ( YE-like ) or with the heat-stable yGa antibody causes release of histamine and slow-reacting substance of anaphylaxis (SRS-A) ( 4 0 ) . In this latter species, the release of the two mediators by the reaction of antigen with the two antibodies occurs under somewhat different circumstances of passive sensitization (38, 4 0 ~ ) . The y 2 antibodies of the mouse and guinea pig and the yG antibodies of the rabbit or dog are not homocytotropic, being incapable of sensitizing their own species of origin for anaphylaxis by fixing to mediator-target or trigger cells. However, they are heterocytotropic, that is, they are capable of sensitizing foreign species. The yG,, yG,, and yG4 but not the uG2 subclass of human yG antibodies are also heterocytotropic (41 ), sensitizing guinea pigs for PCA. Thus with human antisera, a Prausnitz-Kustner reaction in which human antibody is passively transferred to the skin of another human or a PCA reaction with the same antiserum in monkeys detects the homocytotropic YE antibody, whereas the heterocytotropic yG antibody is detected by PCA reaction in guinea pigs. Antibodies found in the yG fraction in the rabbit and in the y? fraction in guinea pigs are able to fix specifically to macrophages either before or after they have combined with antigen ( 4 2 ) . These antibodies have been termed “cytophilic antibodies” by Boyden and Sorkin ( 4 3 ) . In humans, the yG, incomplete Rh antibodies recently have been shown to be cytophilic ( 4 4 ) . Cytophilic antibodies differ from homocytotropic antibodies in the type of cell or cells with which they are capable of combining and, correspondingly, in the kinds of allergic reactions they determine. In addition, Uhr and Phillips (45) have shown that antigen-antibody complexes made with either rabbit or guinea pig 7s antibody are capable of fixing to homologous macrophages although the antibody alone does not. The pathogenetic significance of such antibody is unknown. Analogous to this is the possibility that subclasses of antibodies

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exist which can form complexes capable of specifically binding to cells other than macrophages. This is suggestcd by the fact that the human yG, antiquinidine antibody forms complexes with quinidine that specifically fix to thrombocytes, whereas the human y M, antistibophen antibody forms complexes with stibophen that specifically fix to red cells ( 4 6 ) . Thesc latter complexes activate the complement system and the red cells are destroyed. C. EFFECTOR EXZYMESYSTEMS In the long sequence of steps leading to any given allergic nianifestation, the activation of one or several enzyme systems of the blood or of certain kinds of cells is frequently the first event after the antigenantibody reaction. The enzyme systems which when activated by the antigen-antibody reaction are responsible for one or another of the subsequent events in the allergic reaction are termed “effector enzyme systems” (Table I ) . In a very real sense, differences in the ability of the effector enzyme systems to be activated by various types of antibody ( after aggregation with antigen) detcrmine, at the biochemical level, the roles of the antibody in mediating the different kinds of allergic reaction. In origin, the effector enzyme system may be purely humoral, purely cellular, or a combination of the two, humoral-cellular. The humoral effector enzymc systems consist of the complement system ( 4 7 ) , the kinin-forming system ( 48), the clotting system ( 4 9 ) , and possibly the plasniin system (49). Complement is the only one of the humoral enzyme systems unequivocally shown to be directly activated by the antigen-antibody reaction. As already mentioned, antibodies belonging to only certain immunoglobulin classes or subclasses are capable of activating the complement system. Different types of cells vary widely in their susceptibility to the cytotoxic or other actions TABLE I EFFECTOR ENZYME

SYSTEMS

I. Humoral A. Complement B. Permeability globulin (kinin-forming system) C. Clotting system 11. Cellular A. Antigen-antibody-activated esterases of mast cells 111. Cellular-humoral A. Complement-dependent release of histamine from rat peritoneal mast cells by rabbit antirat +globulin B. Chemotaxis of neutrophiles by complement-dependent rhemotactic factor

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of complement. Thus the role of complement in allergic reactions is distinctly limited since only certain antibodies can activate the system and only certain target or trigger cells are susceptible to its action. The cytotoxic activity of complement, inducing the irreversible loss of the selective membrane permeability of the target cell, usually requires the sequential activation of all nine components of the system ( 4 7 ) . However, the functions of complement in allergic reactions are not restricted to its cytotoxic effect nor to the completion of the entire sequence of component reactions. The cell-bound reaction of the first four components is sufficient to form a cell capable of immune adherence (47,51), to prepare the cell to be phagocytized ( 5 2 ) , to release a fragment C3a with anaphylatoxin and chemotactic activities (53,5 4 ) . Chemotactic and anaphylatoxin activity arise from a fragment (C5a) formed from the fifth component of complement (54, 55). Following the sequential action of the first four components, a factor chemotactic for polymorphonuclear leukocytes is formed as trimolecular complex of the fifth, sixth, and seventh components ( 5 4 ) . Recent work (55q 55Z,), has indicated that in the kinin-forming system, activated Hageman factor or Hageman factor fragments can activate prekallikrein to the proteolytic enzyme kallikrein. Activation and release of the proteolytic enzyme, kallikrein, has been demonstrated following an antigen-antibody reaction in guinea pig lung (56, 57). The properties of the knllikrein released in this manner are essentially the same as plasma kallikrein ( 5 7 ) . This suggests the probability that the kinin-forming system activated during certain anaphylactic responses may be purely humoral in nature. The immunoglobulin class ( es) capable of activating the kinin-forming system are not known. The activation of the clotting system is evident in certain forms of anaphylaxis in the mouse ( 5 8 ) , in the rabbit ( 5 9 ) , and in the heterologous or early autologous phase of nephrotoxic nephritis of rats and rabbits injected with large quantities of nephrotoxic serum (60). Rabbit -yG antibody can apparently activate the clotting system in the plasma of rabbits ( 4 9 ) ; the nature of other antibodies capable of doing this in other species is not known. Thc complcmcnt-dependent release of platelet Factor 3 from platelets altered by an antigen-antibody reaction is another general way in which allergic increase of the clotting function is accomplished; it is probable that there are others ( 4 9 ) . Plasminogen has been reported to be activated in vivo during allergic reactions in thc rabbit, guinea pig, dog, and human ( 6 1 ) . The nature of the antibody responsible and the mode of activation is not known since this has not been reproducibly accomplished in vitro ( 4 9 ) . There is no convincing evidence that the plasmin so released plays

IMMEDIATE-TYPE ALLERGIC REACTIONS Antigen - Antibody Complca

Antiqen-Antibody Complea t Hogcmon Factor (XU)

[

ACTIVATED HAGEMAN FACTOR

1

279

C'I

+ C'I

ESTERA I I

Plotelet

Pro PF/dil +PF/dil

ID)Prothrombin

ZThrombin

J

1 Klninogcn-Kinin

(I) Fibrinoqen+Fibrin

!

+ Domogcd cell, Anophylotoxin Chcmotoctic Factor

CLOTTING

PERMEABILITY GLOBULIN

COMPLEMENT

FIG. 1. The interrelationships of humoral effector enzyme systems ( 4 9 ) .

any role in any allergic reaction; it is, therefore, uncertain whether or not the plasminogen system should be classified as an effector enzyme system. Although the humoral enzyme systems have been discussed separately, it must always be kept in mind that they form an interrelated network, at least potentially capable of acting sequentially and in concert (49, 50). Figure 1 shows some of the actually demonstrated relationships of the complement, plasminogen, permeability globulin ( kininforming), and clotting systems (49). The complement, permeability globulin and plasminogen systems share a common inhibitor, Ci inhibitor, capable of inhibiting Ci,the activated first component of complement kallikrein and PF/dil of the kinin-forming system (49), and plasmin ( 62). The antigen-antibody reaction can activate the complement system, which in turn can act on platelets to release Factor 3 to activate the clotting system. The clotting and plasma kinin-forming systems share a common initial component, the Hageman factor (Factor XII).Becker and Jonasson and Movat have postulated that the antigenantibody reaction can also activate Hageman factor (49). Moreover, kallikrein can possibly activate the proenzyme form of the first component of complement to its active form (63). Thus, potentially at least, an antigen-antibody reaction can cause a massive and reverberating upheaval in the enzymatic makeup of the blood. So far as we know, cellular reactions dependent on homocytotropic antibody are due to the activation of cellular effector enzyme systems without the intervention of humoral effector enzymes. The antigen-induced release of histamine from rat peritoneal cells sensitized with rat heat-labile homocytotropic antibody ( 33) and the antigen-induced release of histamine from guinea pig lung slices sensitized with guinea

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pig yl antibody ( 6 4 ) are two examples of homocytotropic antibodymediated reactions releasing pharmacological mediators which involve the activation of a cellular effector enzyme system. The release of histamine from the leukocytes of ragweed-sensitive humans on the addition of ragweed antigen E ( 6 5 ) , the histamine release from human leukocytes by rabbit antibody against human YE immunoglobulin (66, 67) or from mouse peritoneal mast cells by complexes of antigen and mouse yl antibody (36, 68) or from rat peritoneal mast cells by high concentrations of antirat 7-globulin ( 6 9 ) , all occur in the absence of added humoral elements, Presumably, only cellular effector enzyme systems are required, although only in the last instance is there any direct evidence for such an assumption, and even here the evidence is quite incomplete. Phagocytosis of antigen-antibody complexes by neutrophilic polymorphonuclear leukocytes leads to the degranulation of the lysosomes of the leukocyte and extracellular release of their contents ( 7 0 ) . The possibility that this also involves a cellular effector enzyme system is suggested by the observation that phagocytosis of sensitized sheep erythrocytes by neutrophiles probably involves the activation of a cell-bound proesterase ( 7 1 ) . Not all reactions leading to the release of histamine or other pharmacological mediators are due to the activation of purely cellular effector enzyme systems. The release of histamine from rabbit platelets by small amounts of antigen and antibody is by a complement-dependent cytotoxic mechanism (72, 7 3 ) , as is the release of histamine from rabbit peritoneal mast cells by rabbit antimast cell antibody ( 7 4 ) . These reactions presumably require only the activation of the humoral effector enzyme system, complement, although this has not been rigorously proved in either case. Certain reactions require both cellular and humoral effector enzyme systems. The complement-dependent release of histamine by relatively low concentrations of antirat 7-globulins is by a noncytotoxic mechanism ( 7 5 ) and involves not only complement but also a cellular activatable esterase ( 7 6 ) .The chemotactic factor C 5 w formed from the fifth, sixth, and seventh components of complement as well as the protesterase of the rabbit neutrophilic leukocyte (heterophile) are involved in the chemotactic response of the latter cell ( 7 7 ) . Reference has already been made to the phagocytosis of sensitized erythrocytes containing the first four components of complement, EAC1423, by rabbit neutrophilic leukocytes probably requiring the activation of an endogenous esterase ( 7 1 ) . It is likely that this required coaction of cellular and humoral effector enzyme systems will be found to be a common mechanism of cellular alteration in many allergic reactions.

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D. MEDIATORS The term mediators, as used here, is restricted to substances of which the release or formationg is triggered directly or indirectly by the initiating antigen-antibody reaction and of which the action is responsible for one or more of the manifestations of a given allergic reaction, As is brought out below, not all types of allergic response are due to the action of mediators. Nevertheless, a large number of different kinds of allergic reactions are, and in these the differences among the reactions are due, in large pait, to the different kinds of mediators released (see below). Table I1 lists and classifies many of the presently known mediators. TABLE I1 hlEDIATORS

I. Low molecular weight mediators 1. Histamine 2. Serotonin 3. Kinins 4. Slow-reacting substance of anaphylaxis 11. Macromolecular mediators 1. Lysosomal enzymes 2. Cationic proteins of polymorphonuclear leukocytes 3. Heparin 4. Anaphylatoxins a. C3a b. C5a 5 . Complement-dependent chemotactic factors

The low molecular weight or pharmacological mediators, listed in Table 11 are substances of molecular weight not much greater than 1000, in the case of the kinins, and molecular weights substantially less than this for others. They all have marked, direct pharmacological activity. They consist of histamine, 5-hydroxytryptamine ( 5-HT, serotonin), SRS-A, and the kinins ( bradykinin, etc. ).lo Histamine and serotonin exist

' Some mediators, e.g., histamine, serotonin, cationic proteins, exist in cells preformed, others, such as SRS-A and kinins, are produced during the course of the allergic reaction from precursors either in cells or in body fluids. In most instances, the term release as used here will not differentiate between these two cases. I" Recent work has established that prostaglandins and a substance of unknown constitution capable of more-or-less specifically contracting the rabbit aorta are released during an anaphylactic-type reaction in perfused guinea pig lung ( 7 8 ) . These compounds shonld presumably be added to the list of pharmacological niediators, and this will be done as subsequent work better defines their role in anaphylactictype reactions. In addition the eosinophil chemotactic factor of anaphylaxis ( ECF-A) released when specific antigen is added to human lung slices should undoubtedly be included in the list of small molecular weight mediators ( 7 8 ~ ) .

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preformed in mediator cells, such as mast cells ( S C C Section 111,E). The SRS-A is formed as a consequence of the antigen-antibody reaction; kinins are formed from a y-globulin precursor or from precursors in the blood ( 4 8 ) . Histamine, serotonin, and bradykinin share the general properties of contracting smooth muscle, increasing vascular permeability, particularly of venules immediately distal to capillaries, and stimulating glands of exocrine secretion. Within a given species, there are great variations in the susceptibility of different organs and tissues to any of the actions of any one of the mediators. Histamine, for example, contracts the smooth muscle of the large arterioles of the guinea pig but relaxes the smooth muscle of the smallest ones. There are also great variations in the relative susceptibility of the same structure from species to species to a given activity of the same or different mediator (79). Contraction of smooth muscle ( 8 0 ) and increase in vascular permeability ( 4 0 4 are the two demonstrated pharmacological activities of SRS-A (81 ). The same variability of activity between and within species is also found for this mediator. I n addition to the low molccular weight mediators, a number of other kinds of intermediates, the high molecular weight mediators, or macromolecular mediators are released as a consequence of the antigen-antibody reaction. These latter mediators are generally of higher molecular weight than the pharmacological mediators, having molecular weight distinctly greater than 1OOO. They are otherwise remarkably diverse in source, structure, and function." Certain of them are released from cells, for example, the lysosomal enzymes and cationic proteins released from polymorphonuclear leukocytes ( 8 3 ) , or heparin, primarily released ( a t least in the dog) from the mast cells of the liver. Others are factors released from the humoral complement system as a consequence of its activation. Examples of the latter are the anaphylatoxins, C3a and C5a, physiologically active fragments produced from either C3, the third component, or from C5, the fifth component of complement ( 53, 5 5 ) , or complement-dependent chemotactic factors, such as C567, the activated trimolecular complex of the fifth, sixth, and seventh components of complement ( 5 4 ) .Thus, the complement system is not only an effector enzyme system but also a source of macromolecular mediators. The lysosomal enzymes, particularly the proteases can induce tissue T h e differentiation between pharmacological and macromolecular mediator does not imply that macromolecular mediators may not have direct pharmacological activity, some of them such as the anaphylatoxins d o [see Vogt et al. (SZ)]but in general this is not their major or primary activity.

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damage by a direct attack on cell products such as the glomerular or vascular basement membrane (83). In addition, there are a number of cationic proteins with various functions obtained from the polymorphonuclear leukocytes ( 8 3 ) . Some are chemotactic, and others release histamine from mast cells ( 8 4 ) or increase vascular permeability in other undefined ways ( 8 5 ) .The cationic proteins can be considered secondary mediators, when they act on cells such as mast cells to release pharmacological mediators or call forth cells which are a source of lysosomal enzymes. Whether any of the cationic proteins so far identified play a role in any allergic reaction is not known at present. The anaphylatoxins release histamine from mast cells (53) and also have a direct action on smooth muscle (82). In their role as histamine releasers they act as secondary mediators. Whether the anaphylatoxins play a role in any of the allergic reactions, and in fact, whether they are released in an active form in vivo has still to be ascertained.

E. CELLS Cells have three general roles in allergic reactions. First, damage to cells may be the direct cause of the allergic manifestations without the intervention of mediators; such cells will be termed end cells. Second, cells may serve as a source of macromolecular or low molecular weight mediators; these cells will be termed mediator cells. Mediator cells on which occurs the antigen-antibody reaction leading to mcdiator release will be called target mediator cells. Mediator cells that release their mediator without an antigen-antibody reaction occurring on their surface will be called secondary mediator cells. This secondary mediator release may arise either through the action of mediators such as one or another of the anaphylaxtoxins or cationic proteins of the polymorphonuclear leukocyte, or by an interaction with another cell (trigger or effector cell, see below). Thirdly, a cell called an efector or trigger cell may react with either an end cell or a mediator cell to induce allergic alteration in them. Reactions in which an end cell or a mediator cell is damaged through interaction with a trigger cell are spoken of as cooperative reactions. End cell, mediator cell, and trigger cell are functional designations and do not necessarily imply any fixed cell type. In fact, the same cell type under one set of circumstances can play one role and under a differing set of circumstances can play another, for example, the platelet, in allergic thrombocytopenic purpura serves as an end cell, whereas, in certain anaphylactic-type reactions, by releasing vasoactive amines, it serves as a mediator cell. End cells may be of various kinds. In allergic hemolytic anemia,

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allergic thrombocytopenic purpura, or allergic leukopenia, the red cell, platelet, and neutrophile, respectively act as end cells. If the suggestion is correct that certain forms of atrophic gastritis are due to the reaction of an antibody with the gastric parietal cell [(86); see, however Tai and McGuigan (87)], then this cell, under this circumstance, is an end cell. Mediator cells are also of various types as would be expected from the manifold kinds of mediators that exist. They are similar, however, in possessing granular or vesicular structures where the mediators are synthesized and/or stored. A number of the mediator cells are listed in Table 111. The mast cell, enterochromaffin cells, platelets, and basophiles are sources of histamine and serotonin. Whether one or both amines are released from any one of the above cells depends on the cell and the species of animal (79). The neutrophile is required for the allergic release of SRS-A in the rat when yGa is the antibody involved (88) and under these circumstances may be its source. The neutrophile is also a source of lysosomal enzymes and cationic proteins. The classification of the eosinophile as a mediator cell is highly tentative and based largely on the disputed report that it may contain histamine. In our present state of ignorance, the eosinophile is mainly a source of confusion and uncertainty. Recent work (78a, 87u-87~)makes a start on suggesting why the association exists but what, if anything, the eosinophile does in allergic reactions is essentially a mystery (89). Treadwell has reported that in aggregate anaphylaxis (see Section IX,A,5) of mice there is damage to macrophages of the reticuloendothelial system with release of a number of their lysosomal enzymes (90). Whether the substances released play any role in this type of anaphylaxis of the mouse has not been established; thus, whether the macrophages are mediator cells must await the results of further work. As already mentioned, both mediator and end cells can undergo cooperative reactions, and in such reactions there are various trigger cells. Red cells sensitized with yG, incomplete, noncomplement-fixing TABLE I11 MEDIATOR CELLS 1. Neutrophilic leukocytes 2. Mast cells 3. Enterochromaffin cells 4. Basophiles 5 . Platelets 6. Macrophages 7. Eosinophiles (truly a cell in search of a function, in allergy as elsewhere)

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Rh antibody are taken out of circulation, segmented, and heniolyzed by the reticuloendothelial cells of the spleen and liver ( 4 4 ) . Under such Circumstances the reticuloendothelial macrophages of these organs act as effector cells. Thc antibody is cytophilic, so that specific combination with thc erythrocyte results in a cell with a particular affinity for macrophages ( 4 4 ) . Red cells sensitized with too low a concentration of complement-fixing antibody to undergo heinolysis by coinplement in the circulation ( intravascular hemolysis ) may, nevertheless, activate the beginning steps of the complement sequence on the cells (91). Under these circumstances thc inconiplete reaction of complement powerfully enhances the extravascular lytic action of the littoral macrophages of the reticuloendothelial system, possibly through imniune adherence (51) and subsequent erythrophagocytosis (42) . Sheep erythrocytes containing the first four components of complement ( EAC1423) can scrvc' as trigger cc4l\ by inducing rabbit platelets to release histamine ( 7 2 ) . Sensitized rabbit inononuclear cells, possibly lymphocytes when reacted with antigen can also induce histamine release froin rabbit platelets and, thus, serve as trigger cells (72, 9 2 ) . At present, these cooperative reactions arc' only known for rabbit platelets; it is very likely they will prove to involve other mediator cells and other mediators. IV. Sites of the Antigen-Antibody

Reaction

Dcpending upon the site of the antigen-antibody reaction, inimediate-type allergic responses may be described as either antibody-adherent, antigen-adherent, or aggrcgate. Antibody-adherent reactions arc those reactions in which cell damage and/or mediator releasc is induced by the combination of antigen with an antibody which is adhercnt to either a target mediator cell or a trigger cell. So far as evidence is presently available, all antibodyadherent reactions involve cytotropic antibody (see above) and either trigger cells and niediator cells or target mediator cells but not end cells. Pharmacological mediators are the primary although not the only kinds of mediators released in antibody-adherent reactions; pari passu, as will be brought out later, all presently known antibody-adherent reactions are anaphlylactic type in nature; however, as will also be brought out later, the reverse is not true. Antigen-adherent reactions arc those that occur following the combination of antibody with antigen which is adherent to cells or to extracellular structures. The antigen may bc an integral part of or passively adsorbed to a cell surface or an extracelIuIar structure. The intravascular or extravascular lysis of human red cells sensitized with

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antibodies against the ABO, Rh, or other blood groups are examples of antigen-adherent reactions in which the antigen is an integral part of the cell. The kinds of nephrotoxic nephritis resulting when antibody combines with antigen on the basement membrane of the kidney glomerulus are examples of antigen-adherent reactions where the antigen is on an extracellular structurc. Thc complement-dependent release of histamine from rat peritoneal mast cells obtained when rabbit antirat yG-globulin reacts with rat y-globulin adsorbed to the surface of a rat peritoneal cell is an example of an antigen-adherent reaction in which the antigen is only loosely adsorbed to a cell. Responses triggered by antigen-antibody aggregates in which the aggregate is formed in the plasma, extravascular body fluids, or in interstitial spaces are called aggregate reactions. Abrupt responses (see Section V ) induced in guinea pigs by preformed antigen-antibody aggregates or possibly in the rabbit sensitized with hyperimmune rabbit yG antibody are examples of anaphylactic-type aggregate reactions. The allergic vasculitis seen in the Arthus reaction and the arteritis and glomerulonephritis of experimental serum sickness are also examples of aggregate reactions. In human disease, there is evidence (see below) that poststreptococcal glomerulonephritis, the glonierulonephritis of lupus erytheniatosus, and the thrombocytopenic purpura arising from quinidine and similar drug sensitivities are aggregate reactions. As is evident from just this short and incomplete list, aggregate reactions or “immunological complex diseases,” as they have been called, differ widely both in their manifestations and their mechanisms, V. Time Course of Allergic Reactions

The time course of immediate-type allergic reactions under certain but not all conditions is fundamentally related to the mechanism of the reaction and is, therefore, worth discussing from that standpoint, In this consideration, it is convenient to take as the starting point of the reaction the time at which antigen combines wih antibody (or the time of administration of preformed antigen-antibody complexes) even though the fornier may sometimes be difficult or impossible to define operationally. By this choice of starting point, in serum sicknesslike reactions, the interval from the introduction of the antigen to the formation of the antibody is not considered and attention is focused on how long it takes for one or another allergic reaction to occur once the antigen remaining in the organism has combined with the newly formed antibody. In the already sensitized subject given antigen or antibody, we also do not take into account the varying amount of time required for antigen to be adsorbed, disseminated, and brought to the antibody.

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Even when taking the time of antigen-antibody combination as the starting point, the time taken for the manifestations of the different kinds of allergic reactions to appear may vary from minutes to hours depending on the kind of reaction. Reactions of which the clinical manifestations occur within minutes after antigen-antibody combination are defined as abrupt reuctio12s.12 Those reactions of which the clinical manifestations occur hours or later after antigen-antibody combination are termed Although, both nonmediator-determined (direct) and mediator-determined (indirect) reactions (see Section VII for discussion of these terms) may be either abrupt or retarded, the significance of the difference in the time course is not the same in the two groups of reactions. The time course of mediator-determined ( indirect ) reactions bears a much more direct relationship to the underlying mechanism than is the case with nonmediator-determined (direct ) responses. So far as indirect reactions are concerned, the various kinds of anaphylactic-type reactions (pharmacological mediator-determined responses; see Section VII) are typically abrupt reactions. This is because the trigger and/or mediator cells are present or are readily available at the site of the antigen-antibody reaction, the release of the pharmacological mediators is rapid, as is the action on and response to them of the shock tissues and shock organs (see below for definition of these later terms). When the mediator cells are the mast cells in the tissue or the basophiles in the circulation, which either carry cytotropic antibody on their surface or are accompanied by their trigger cell, the availability of the necessary cells at the site of the antigen-antibody reaction is assured. However, in abrupt reactions mediated by the production of SRS-A, where under certain circumstances, there is presumably a requirement for the infiltration of polymorphonuclear leukocytes to the site of the antigen-antibody reaction (88) the situation is not as clear. In the reaction where SRS-A production is consequent to the injection of antibody into the peritoneum or skin of the rat, a major part of the latent period may be the time required for neutrophile leukocytes, called forth by the mild inflammatory stimulus of the injected serum, to infiltrate the site; however, there is also evidence that neutrophile leukocytes can infiltrate the site of a PCA reaction in appreciable although not massive numbers within minutes following the challenge dose of antigen (93). So far as we know now, the various kinds of indirect, macromolecular mediator-determined reactions (see Sections VII and IX,B for disI2By using the terms abrupt and retarded, it is hoped to avoid confusion with immediate and delayed reactions.

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cussion of these reactions) typically are retarded in their time course. The edema and hemorrhage of the Arthus reaction and proteinuria of the acute glomerulonephritis of experimental animals, occurring after the intravenous injection of heterologous antikidney, basement membrane antibodies, require an hour or more to develop after antigenantibody combination. In both instances, a major part of this time interval is taken up with the accumulation of polymorphonuclear leukocytes at the site of antigen-antibody union. An additional reason for the relative slowness of these reactions compared to the abrupt responses may be due to the relatively slow action of the lysosomal mediators they contain in producing damage to the venular basement membrane in the case of the Arthus reaction, and the glomerular basement membrane in the nephrotoxic reaction ( 8 3 ) . Thus, one of the major factors responsible for the difference in time course between the indirect, anaphylactic-type and the indirect, nonanaphylactic-type responses is the ready availability or lack of availability of sufficient numbers of trigger and /or mediator cells at the site of the antigen-antibody reactions. The pathogenetic mechanism of the induction of the arteritis of experimental serum sickness is very similar to that of the Arthus reaction and heterologous nephrotoxic nephritis. In the arteritis of experimental serum sickness, there is an infiltration of polymorphonuclear leukocytes and the consequent damage to the internal elastic membrane of the artery ( 8 3 ) . Whether, in fact, the arteritis of experimental serum sickness is also retarded, as one would expect, cannot be determined. As already mentioned, the time course of direct cellular reactions less clearly and fundamentally differentiates the mechanisms underlying the cell damage. Complement-dependent, lytic ( cytotoxic ) , end cell reactions are abrupt. On the other hand, effector cell-dependent, end cell reactions (see below for a further description of these terms) may vary from abrupt to retarded in their course depending basically on the rate of the reaction of the end cell with the effector cell. The rate of this interaction depends, in turn, upon the concentration and avidity of the antibody and, secondarily, upon whether the antibody is complementfixing (91). Thus the time course is not really characteristic of a given kind of direct effector cell reaction but of the attendant circumstances. The interval between antigen and antibody combination and clinical manifestations may be even longer than a few hours if the result of such union must be cumulative in order for the damage to be detectable. An example of this is the delayed renal injury produced in rats given small amounts of rabbit antibody to rat kidney and immunized to rabbit y-globulin ( 1 3 ) . However, it is doubtful if the mechanisms of this

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cumulative, subliminal damage are different from what has been or will be discussed. Immediate-type allergic reactions may be either self-limited in their duration or prolonged, that is, chronic. The chronicity may be due either to continuous recurrence of the initiating stimuli, as in chronic allergic rhinitis where the patient is continuously exposed to the offending allergen, or to the secondary changes induced by the reactions, or to both. Although chronicity or lack of it may be important clinically, it is not a fundamental differentiating characteristic among the various immediate-type responses. VI. The Terrain

Each of the primary components of the allergic reaction which we have just discussed-the antibodies, cells, mediators, the effector enzyme systems, as well as the tissues and organs in which they occur-can all be profitably studied in isolation. In the whole animal, however, they do not exist in this convenient but artificial privacy. In the whole animal, the antigen-antibody stimulus may and very often does initiate a more or less profound perturbation of the entire, integrated, internal environment of the organism. The organized structures which suffer the consequences of the primary extracellular and cellular alterations initiated by the union of antigen with antibody, and the various neural, pharmacological, and endocrine influences which modify these consequences, I subsume under the single word “terrain.” The degree and kind of involvement of the terrain depends to a large extent on whether the allergic reactions are direct or indirect (see above and Section VII). Direct reactions, as already stated, are those in which the clinical manifestations require no mediators for their expression. In direct reactions, due to damage to end cells, the effects on the terrain are mainly those resulting from an interference with the functions of the end cell, for example, the anoxia due to the hemolytic anemia or the bleeding as a consequence of allergic thronibocytopenia. This, however, is not invariably the case, The effect of allergic heniolysis in engendering hypersplenisni, and the effect of hypersplenism in enhancing some cases of allergic hemolytic disease is but one example of the interaction of allergic, end cell damage and the terrain in which it occurs (94). Indirect reactions have alreadly been defined as those reactions that result from the response of various tissues to the mediators released as a consequence of the anitgen-antibody reaction. The tissues that react to the mediators so released are termed s71ock tissues. The organs which bear the brunt of the allergic insult and in which the resulting changes define the clinical nature of the allergic reaction are termed shock

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organs. The content of mediator cells, the concentration and organization of the shock tissues, and the degree of susceptibility of the shock tissues to the mediator or mediators all play a role in determining the shock organ. An example of the influence of the nature of the anatomical organization of the shock organ in defining the allergic reaction is seen in the more-or-less characteristic pattern of anaphylactic response which a particular species gives and the variation of this pattern between species, The predominance of asphyxia1 signs in anaphylaxis in the guinea pig, of right heart failure in anaphylaxis of the rabbit, and of hepatic congestion in anaphylaxis of the dog are considered to be due, respectively, to the especially large concentration of smooth muscle (the shock tissue) in the bronchi of the guinea pig, in the pulmonary arterioles of the rabbit, and of its high concentration and particular arrangement in the hepatic venous tree of the dog. However, the availability of mediator cells in these organs, as exemplified by the high mast cell content of the liver of the dog, and the susceptibility of the smooth muscle to the mediators they release, also plays a role. Physiological, pharmacological, neurological, and in the case of humans, even psychic factors are all capable of modifying allergic reactions, and these modifiers are important parts of the terrain. An example of the importance of a purely physiological factor is the role of the hydrodynamic force of the blood in influencing the site of deposition of antigen-antibody complexes in the arteries and, thus, helping localize the lesions of expcrimental serum sickness ( 9 5 ) . The effect of corticosteroids in depressing antibody formation (96, 9 7 ) and supressing Arthus reactions, the effects of sex, thyroid, and growth hormones in sustaining antibody production (97, 98), the effect of adrenalectomy in increasing the intensity of anaphylactic-type reactions, Arthus reactions, and the arteritis of experimental serum sickness in various species of animals, and the role of thyroid function in various allergic responses (98-loo), are all examples of the importance of endocrine influences in modifying allergic reactions. Histamine and most of the other pharmacological mediators are known to cause the release of their antagonists, the catecholamines, epinephrine and norepinephrine (101 ). The injection of Bordetella pertussis increases the susceptibility of the mouse to anaphylactic shock and to histamine and serotonin (1.01, 102). Szentivanyi and Fishel have suggested that B . pertussis directly or indirectly blocks the antagonistic action of the catecholamines at the level of the ,8 adrenergic receptors, thus increasing the sensitivity of the mice to the pharmacological mediators (101 ). The evidence for this suggestion is as yet indirect. Szentivanyi has also postulated that human asthma is due primarily to a partial block

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of the p adrenergic receptors of thc smooth muscles, glands, and vessels of the bronchi. This hypothesis has been elaborated on by Szentivanyi (103) and by others ( 104). The evidence is still indirect and conflicting (105). Math&, on the other hand, considers that the lowering of the concentration of epinephrine in the vicinity of bronchial smooth muscle is the important pharmacological factor triggering human asthma (106). No matter what might prove to be the validity of these hypotheses, they are, nevertheless, significant as illustrations of the possible ways in which pharmacological aspects of the terrain may be of importance in certain types of allergic reactions. Interrelated to these postulated pharmacological aspects of the terrain in asthma are the various suggestions as to the relationships of the autonomic nervous system in this human disease ( 1 0 4 ) . Here again, the evidence for the role of the latter in asthma or any abrupt allergic reaction is only suggestive. The studies of the effects of ccntral nervous system function on allergic reactions carried out so far do not allow any definitive judgment to be made. There is, however, quite a good deal of work suggesting that the central nervous system may well play an indirect role in experimental anaphylaxis in lower animals (107). The effect of psychic influences in humans on allergic reactions has been the frequent subject of uncontrolled observation and ebullient speculation ( 108). However, carefully controlled studies leave no doubt as to the importance of such influences (109). That the role of the autonomic and central nervous system in various allergic reactions has not received more attention from experimental scientists of the United States and Western Europe is a distinct deficiency (cf. 110), but it is hoped, one which further work will remedy. VII. Basis and General Description of the Classification

Tables IV and V present a classification of the immediate-type allergic reactions based upon the mechanisms of allergic alteration and damage just discussed. Table IV outlines and defines, in summary fashion, the various categories in the classification, and Table V applies the classification to a number of experimental and clinical responses. The primary focus and basis of the classification are the chemical mediators; whether they are or are not involved in a given reaction, and if involved, their nature. On this basis, allergic reactions are first divided into direct reactions ( non-mediator-determined reactions ) and indirect reactions (mediator-determined reactions) ( I and 11, Tables IV and V ) . In the first, the manifestations arise directly from the allergic damage

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TABLE IV IMMEDIATE-TYPE ALLERGICRE.4CTIONS

S Y N O P S I S O F T H E CLASSIFICATION O F

I. Non-mediator-determined rcactions (direct reactions)-those reactions of which the manifestations arise directly from the antigen-antibody-initiated alterations of cells or noncellular substances. A. Noncellular reactions-those direct reactions in which the allergic damage or alteration is directed to noncellular structures. B. Cellular reactions-reactions involving end cells, that is, cells the damage of which directly gives rise to the allergic manifestations. 1. Antigen-adherent reactions-those reactions in which the end cell damage is initiated by antibody reacting with antigen which is either an integral part of or adsorbed to the cell. 2. Aggregate reactions-those reactions in which the end cell damage is initiated by antigen-antibody aggregates formed without the intervention of cells. 11. Mediator-determined reactions (indirect reactions)-those responses of which the major manifestations arise from the action of mediators released or produced as a result of antigen-antibody-initiated reactions. A. Anaphylactic-type reactions-those reactions of which the major manifestations are the result of the release of small molecular weight mediators. 1. Antibody-adherent reactions-those reactions initiated by antigen reacting with antibody on a mediator or trigger cell. 2. Antigen-adherent reactions-reactions in which small molecular weight mediator release is initiated by antibody reacting with antigen which is an integral part of or adsorbed to trigger or mediator cells. 3. Aggregate reactions-reactions in which small molecular weight mediator release is initiated by antigen-antibody aggregates formed without the intervention of cells or extracellular structure. B. Macromolecular mediator-determined reactions-those reactions of which the major manifestations are due to the direct action of macromolecular mediators. 1. neutrophile lysosomal reactions-those reactions of which the major manifestations are due to the action of the contents of the lysosomes of neutrophilic leukocytes. a. Antigen-adherent reactions-are those reactions in which the release of lysosomal products is initiated by antibody reacting with antigen which is an integral part of or adsorbed to cells or extracellular structures. b. Aggregate reactions-responses in which the release of lysosomal products is the result of reactions initiated by antigen-antibody aggregates formed in the fluid phase. C. Unknown mediator reactions-a catch-all category of indirect responses or presumed indirect responses where the mediator or mediators responsible for the manifestations are not known.

to extracellular or cellular structures without chemical mediators being involved in any major sense. The direct reactions are further subdivided into direct non-cellular and direct cellular reactions. (Tables IV and V, IA and B, respectively). The indirect reactions are those of which the major manifestations stem from the action of the mediators arising as a result of antigen-antibody-initiated changes. The further classification

293

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CL.\SSIFIC.\TION

OF

TABLE V V.\RIoUs IMMEDI.ITE-TTPI< ALLERGIC ItE.\CTIONS

I. Non-mrdiator-dctrrni incd (dircet ) A. Noncrllitlar reactions 1. Interferencc with clotting induccd by antibody to Factor VIII (AHG). 2. Cases of iiisrilin resistance due to antibody against insulin. B. Cellular reactions 1. Anligcn-adhrrcnt reactions a. Intravascular hemolysis of acute transfusion accidents. b. Acute crises of patients with paroxysmal cold hemoglobinuria. c. Abrupt complement-dependent rejection of kidney homografts. d. Systemic Forssman reaction in guinea pigs. e. Erythroblastosis fetalis due to Rh incompatibility. f. Autoallergic hemolytic anemia due to complement-fixing antibody, e.g., cold hemagglutinins with anti-I specificity, or to noncomplement-fixing antibodies with anti-Hh specific sp. 2 . Aggregate rraetions a. Bystander reactions of thrombocytopenic purpura or hemolytic anemia due to sensitivity to quinidine, stibophen, digoxin, Sedormid, etc. 11. Mediator-dctcrminrd (Indirect) A. Anaphylactic-type rraetions 1. Antibody-adhcrcnl reactions a. Release of histamine from sensitized human leukocytes by the specific antigen. b. Antigen-induced release of histamine from rat peritoneal cells sensitized with rat homocytotropic antibody. c. Antigen-induced release of histamine from guinea pig Iring slices sensitized with guinea pig 71 (homocytotropic) or rabbit yG (heterocytotropic) antibody. d. Praiisnitz-Kiistner ( P K) reaction induced in humans by human yE antibody. e. Passive cutaneous anaphylactic reaction of guinea pigs induced by human or rabbit heterocytotropic antibody. f. Passive cutaneous anaphylactic reactions of monkeys sensitized with human yE (homocytotropic) or human secretory yA (heterocytotropic) antibody. g. Human anaphylaxis, hay fever, some forms of human allergic asthma. h. Urticaria of human serum sickness and human serum sickness-like reactions (?). 2 . Antigen-adhcrcnl reactions a. Histamine release from humaii leukocytes by rabbit antibody against human yE immunoglobulin. b. Histamine release from rat mast cells on the addition of a high concentration of rabbit antirat 7-globulin. c. Wheal and flare induced in human skin by rabbit antibody against human 7E immunoglobulin. d. Complement-dependent release of histamine from rat mast cells by rabbit, antirat, mast cell antibody. e. Passive cutaneous and systemic anaphylaxis in rats induced by rabbit, antirat, mast cell antibody.

(continued)

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TABLE V (Continued) f. Complement-dependent release of histamine from rat mast cells by rela-

tively low concentrations of rabbit anti-yG globulin. g. Histamine release from rabbit platelets by EAC1423. 3. Aggregate Reactions a. Release of histamine from mouse mast cells by complexes of antigen with mouse y1 antibody. b. Wheal and flare reactions in human skin injected with preformed complexes of antigen and human yE antibody. c. Complement-dependent release of histamine from rabbit platelets induced by small amounts of antigen and antibody added in the presence of platelets. d. Complement-dependent release of histamine from rabbit platelets induced by large amounts of preformed antigen-antibody aggregates. e. Rabbit anaphylaxis induced by large amounts of hyperimmune rabbit antiserum (?). B. Macromolecular mediator-determined reactwns 1. ,Veutrophile lysosomal reactions a. Antigen-adherent i. Nephrotoxic nephritis induced within hours by relatively low concentrations of mammalian complement-fixing antibody against glomerular basement membrane. ii. Goodpasture’s syndrome (?). iii. Subacute and chronic cases of glomerulonephritis in humans with linear deposition of antigen-antibody complexes (?). b. Aggregate 1. Arthus reaction. 2. Arteritis of experimental serum sickness. 3. Certain cutaneous vasculitides. 4. Glomerulonephritis of systemic lupus erythematosus ( 1 ) . 5 . Posts trep tococcal nephritis (?) . C. Unknown mediator reactions 1. Arthus reaction in neutropenic rabbits. 2. Glomerulonephritis induced by injecting mammalian complement-fixing antibody into neutropenic rabbits. 3. Heterologous glomerulonephritis due to duck antibasement membrane antibody. 4. Serum sickness-like phase of riephrotoxic nephritis (autologous or secondary phase). 5. Anaphylactic-type reactions in guinea pigs induced by antigen-antibody complexes or by antigen and antibody given simultaneously. 6. Glomerulonephritis of experimental acute serum sickness.

of the indirect reactions is based on the nature of the mediators involved. As already described in Section III,D, these substances are of widely differing nature and correspondingly, of widely differing capabilities; even for the same mediator, the capabilities differ greatly from species to species and from tissue to tissue in the same animal. These

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mediators have been grouped into low molecular weight ( pharmacological) mediators and macromolecular mediators ( Section II1,D). Corresponding to this distinction, the first category of indirect reactions is the anuphylactic-type reactions (IIA, Tables IV and V), those allergic responses of which the major manifestations are due to the action of pharmacological mediators released as a direct or indirect consequence of the triggering antigen-antibody reaction. The second category is the macromolecular-mediator determined reactions, comprising those indirect responses of which the major manifestations are due to the activity of macromolecular mediators released as a consequence of the antigenantibody reaction. The macromolecular mediator-determined reactions essentially have only one subclass, the nsutrophile lysosomal reactions ( I I B l ) , Tables IV and V) where the lysosomes of the neutrophile leukocytes are the primary source of the mediators. Although it is generally considered poor classificatory practice to have a category with only one subclass, this is done here as an expression of faith that further study will reveal macromolecular-determined reactions other than those belonging to the neutrophile lysosomal group. In addition to the two more-or-less well-defined groups of indirect reactions, there is a third group of immediate-type allergic responses which are very similar to one or another of the indirect reactions, but the nature of the mediator or mediators concerned (if they, in fact exist) in each case is unknown. In order to have a place for our ignorance as well as our knowledge, these reactions have been placed under the rubric of unknown mediator-determined reactions (IIC, Tables IV and V). Although any allergic reactions of which the mechanism is sufficiently known can be grouped into one or another of the categories given above, nevertheless, further description and, thus, further subdivision of these reactions is possible. This further description and classification can be based either on where the reaction occurs or how it is carried out. Thus, it can be based on the site and mode of the initiating antibodyantigen reaction or on the nature of the effector enzyme systems involved, or on the nature of the primary cell damage, that is, whether or not the reaction is cytotoxic. To a large extent, like the use of the chemical mediators in the initial classification, the choice of the basis to be used for further classification is arbitrary. The choice is largely a matter of judgment as to what can serve as the most consistent and complete framework for our knowledge and what is likely to be most consistently useful without making the classification unbearably complex. My present feeling is that a further subdivision of the categories already given on

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the basis of the site and nature of the initiating antigen-antibody combination, as either antibody-adherent, antigen-adherent, or aggregate reactions should prove the most consistently informative. To recapitulate, antibody-adherent responses are those in which antigen reacts with antibody adherent to the mediator or effector cell to initiate the reaction; antigen-adherent reactions are those in which antibody reacts with antigen passively or integrally adherent to the cell; whereas, with aggregate reactions, antigen-antibody combination occurs without the intervention of cells. The classification given in Tables IV and V has used these categories where applicable to subdivide further the major groups of reactions. Thus, after segregating the relatively minor group of direct noncellular reactions (IA, Tables IV and V) from the remaining direct cellular reactions (IB, Tables IV and v ) , the latter are broken down into antigenadherent (IB1, Tables IV and V ) and aggregate reactions (IB2, Tables IV and V) . No antibody-adherent direct reactions are presently known. If allergic reactions are found where cytophilic antibody bound to macrophages combines with antigen on end cells to initiate damage to the latter, this third category will have to be created. So far only the possibility of this last kind of allergic reaction has been definitely established. Turning to the indirect reactions we see from Tables IV and V that anaphylactic-type reactions are divided into antibody-adherent ( IIAl }, antigen-adherent ( IIA2), and aggregate ( IIA3) reactions. This is due in large part to the diversity of the kinds of antibody which when combined with antigen are capable of liberating pharmacological mediators. However, the clinically important anaphylactic-type reactions are primarily antibody adherent in nature (IIA1, Table V), the antigen-adherent and aggregate responses arising mainly from experimental manipulation of man or lower animals. Neutrophile lysosomal reactions are broken down into antigen-adherent and aggregate reactions ( IIBla and b ). No antibody-adherent reactions of this kind are known, presumably reflecting the inability of cytotropic antibodies to play a major role in macromolecular mediator-determined responses. Although it is frequently possible to classify unknown mediator responses on the basis of the site of the initiating antigen-antibody reaction, in view of our confessed ignorance of these reactions I have refrained from doing so here. As already discussed, the great diversity of aggregate reactions further stresses the multiplicity of responses which immunological complexes are capable of eliciting. They also stress how ill-defined are the

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terms “immunological complex disease” or “aggregate disease.” Thus, immunological complexes can be responsible for several kinds of direct cellular reactions, for several kinds of anaphylactic-type reactions and for several different forms of neutrophile lysosomal responses, As pointed out earlier, the mechanisms of primary cell damage, and the effector enzymes involved in allergic reactions can also serve to classify the various kinds of allergic reactions. As already described, cells can be damaged by a cytotoxic mechanism. Here there is an irreversible loss of membrane integrity due primarily to the activation of the entire complement sequence. In addition, there are noncytotoxic mechanisms of cellular alteration which involve the reversible enhancement or derangement of the normal secretory processes of the cell. The dichotomy between these two processes is largely of use in distinguishing between various types of anaphylactic-type release mechanisms from mediator cells such as are featured in Table V, IIA. On the other hand, in discussing direct reactions, it is frequently useful to discuss damage to end cells in terms of two somewhat different mechanisms-a cytotoxic and an effector or trigger cell mechariism of damage. In direct cellular reactions the effector cells are usually the cells of the reticuloendothelial system, and the general distinction made here between cytotoxic and effector cell direct reactions is usually the same as the distinction the hematologists make between intravascular lysis and extravascular lysis in regard to the fate of allergically damaged erythrocytes (91). Thus, one may, if one wishes, superimpose two further categories on those given in Tables IV and V and speak of cytotoxic or effector cell aggregate direct reactions as the occasion calls for. Especially in discussing mediator release by various anaphylactictype reactions, it is frequently useful to define them in terms of the different kinds of effector systems involved. The antibody-adherent anaphylactic-type reactions require only cellular effector enzymes; they are all cellular antibody-adherent anaphylactic reactions. Antigen-adherent and aggregate reactions, on the other hand, may involve cellular, humoral, or humoral cellular mechanisms, that is, one may have cellular antigen-adherent, humoral antigen-adherent, and humoral-cellular antigen-adherent anaphylactic-type reactions. Similarly, one may have cellular aggregate, humoral aggregate, and hunioral-cellular aggregate reactions. No attempt has been made to exemplify these categories in Tables IV or V, but examples of each of these categories may be obtained by matching the anaphylactic-type release reactions given in Section II1,C with the same reactions given in Table V. Cytotoxic mechanisms are humoral by definition since in general they require either all or at least the late acting components of comple-

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nient. It is possible that. when we know more of the biochemical mechanisms of end cell destruction by cells of the reticuloendothelial system, we shall be able to talk of effector cell mechanisms as cellular or humoralcellular based upon positive knowledge of the effector enzymes concerned. This stage in our knowlcdge has not been reached. Similarly, it might be possible in the future to discuss macromolecular mediatordetermined reactions in terms of all of the effector enzymes concerned, not only conplement. It is evident from this, as well as the whole tenor of this contribution that I do not consider the field of immediate-type hypersensitivity overcropped and exhausted. VIII. Direct Responses (Non-mediator Determined)

The direct reactions, as already discussed, are subdivided on the basis of whether the allergic damage or alteration primarily affects noncellular structures (direct noncellular reactions, IA, Tables IV and V ) or cells (direct cellular reactions, IB, Tables IV and V). The first example of a direct noncellular reaction in Table V is the rare instances of the bleeding tendency resulting from the interference with blood coagulation caused by antibody spontaneously arising against Factor VIII (111, 1 1 2 ) . The second is the few cases of insulin fastness which have been related to the presence of high titers of anti-insulin antibody (113). Certain cases of pernicious anemia have been shown to possess blocking and/or binding antibodies against intrinsic factor (114). If the suspicion of some turns out to be correct that these antibodies interfere with the in v i m absorption of vitamin B,, and, thus, cause or contribute to the pernicious anemia, then these cases also would be classed among the direct, noncellular, immediate-type allergic reactions. The direct cellular reactions are essentially reactions involving end ~ e 1 l s . lThe ~ division of direct cellular reactions into antigen-adherent direct cellular reactions (IBI, Tables IV and V ) and aggregate direct cellular reactions (IB2, Tables IV and V ) has already been described. The intravascular heinolysis seen following acute transfusion reactions or in the acute crises of patients with paroxysmal cold hemoglobinuria are two examples of antigen-adherent, direct cellular reactions involving blood cells (Table V) . The complement-dependent, very abrupt, hyperacute rejection of kidney xenografts and the pulmonary hemorrhage and edema in guinea pigs given antiserum containing Forssman antibody are antigen-adherent, direct cellular reactions involving end cells other than blood cells. All four reactions are cytotoxic (see above). 'I Although all direct cellular reactions involve end cells, not all reactions involving end cells are necessarily direct in nature.

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Erythroblastosis fetalis due to Kh incompatibility or auto-allergic hemolytic anemias due to noncomplement-fixing antibodies are two further illustrations of antigen-adherent, direct cellular reactions ( Table V ) . In these two instances, the sensitized red cells arc taken out of the circulation and hemolysed by the cells of the reticuloendothelial system; the noncoiiiplenient-fixiiig antibody sensitizing the cells is apparently cytophilic ( 4 P 4 4 b ) . The extravascular lysis of red cells sensitized with coniplement-fixing antibodies, such as the hemolytic anemia in cases with high titer of complement-fixing, poorly hemolytic, cold hemagglutinins ( 115) is another example of an antigen-adherent, direct cellular reaction ( IB1, Table V ) . In these responses, the reaction of sensitized cells with the first four components of complement develops receptors for macrophages ( 116, 117). Presumably similar reactions are responsible for those few cases of posttransfusion thrombocytopenic purpura in which a complement-fixing antibody has been detected (10). Examples of aggregate direct cellular reactions (IB2, Table V ) are the cases of thrombocytopenic purpura due to sensitivity to drugs such as Sedormid, quinidine, and quinine or hemolytic anemia due to sensitivity to stibophen, etc. In these cases the antibody reacts with the drug, and then the drug-antibody complexes adsorb either to the platelet or the red cell, this, in turn, leading to platelet or red cell destruction ( 4 6 ) . The antibodies are complement-fixing; usually, although probably not invariably, they either are not present in sufficient concentration and/ or they are not efficient enough to be directly cytotoxic. Rather an effector cell-dependent mechanism generally operates, in which the extra-vascular clearing is presumably complement-dependent.

IX. Indirect Responses (Mediator Determined)

A. ANAPHYLACTIC-TYPE REACTIONS 1. Differentiation From Nonanupliylactic Reactions

As already discussed, anaphylactic-type responses are generally abrupt in their time course. They included anaphylaxis, the dramatic, abrupt, systemic reaction discovered by Richet and Portier in 1902, and a little later termed “anaphylactic shock by Besredka. Also included in this group are superficially different syndromes such as hay fever, some forms of asthma, some kinds of generalized urticaria, and angio-edema as well as the PCA reaction in lowcr animals and the Prausnitz-Kustner (PK) reaction, its analog in humans (14; see Section IX,A,M for further examples ). Thc mechanism( s ) of the urticaria, fever, and ar-

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thralgia of human serum sickness, or the serum sickness-like reactions developing within days after the injection of a foreign serum or drugs has not been established with certainty. These, nevertheless, must be considered anaphylactic-type responses if suggestions are correct that they are due to homocytotropic antibodies ( 1 4 1 6 , 1 8 ) . However, Idse et al. ( 9 ) have not been able to find the clear correlation between the presence of homocytotropic antibody against the penicilloyl group and the presence of serum sickness-like syndrome in cases of penicillin allergy which Levine and his coworkers have reported. There are a number of immediate-type alIergic reactions that are similar in one respect or another to anaphylactic-type reactions but differ from them in not being due primarily to the action of released pharmacological mediators. Among these are the systemic Forssman reaction resulting when specific antibody reacts with the Forssman antigen present in the endothelial and other cells of certain species such as the guinea pig (118).No evidence for a role for pharmacological mediators has been adduced in these reactions ( 2 6 ) . As already discussed, there is no direct evidence that pharmacological mediators play a role in the vasculitis of the Arthus reaction even though Arthus originally referred to this reaction as “local anaphylaxis”; a misnomer which is occasionally seen even now ( see, however, Section X). Sites on guinea pig skin injected with certain guinea pig y2 antibodies show increased vascular permeability 0.5-4 hours after challenge with antigen ( 119). As emphasized, anaphylactic-type reactions occur within minutes of antigen-antibody reaction. This consideration in itself suggests that the above reaction of guinea pig y2 antibodies is probably not an example of PCA and should not be classified as such without further study of the mechanisms involved. In addition to the abrupt, systemic reaction of anaphylaxis, various species (guinea pigs, rats, mice, dogs, etc.,) show a distinctly more slowly progressing reaction which has been termed delayed or protracted anaphylactic shock. This syndrome is characterized by a slowly developing hypotension and hypothermia, labored breathing, and collapse; the animals so affected may die 2-18 hours after antigenic challenge in irreversible shock or may completely recover [reviewed in Becker and Austen ( 2 6 )1. Although the signs, symptoms, and pathology resemble generalized tuberculin shock or the delayed phase of endotoxin shock, the reaction can be transferred by antibodies; it is clearly, therefore, an immediate-type allergic response. In no species has the mechanism or mechanisms of the delayed shock been distinctly and completely identified. In view of our ignorance of the mechanisms of such reaction, it would be better to substitute for the terms delayed or protracted an-

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aphylaxis, the more noncommittal term, immediate-type protracted shock. 2. Cfassification of Amphylactic-Type Reactions

The classification of the anaphylactic type clinical responses corresponds directly to the nature of the release reactions, that is, the reactions responsible for the release of the pharmacological mediator( s ) of which the subsequent activity results in the clinical response. As already brought out, the release reactions and, thus, the anaphylactic-type responses are primarily classified according to the nature and site formation of the triggering antigen-antibody complex. Thus, one has antibody-adherent ( IIA1, Table IV), antigen-adherent ( IIA2, Table IV) and aggregate ( IA3, Table IV ) anaphylactic-type responses. 3. Antibody-Adherent Reactions

A number of examples of antibody-adherent release reactions are given in Table V (IIA1). In general, antibody-adherent releasc reactions are due to antigen reacting with either homocytotropic or heterocytotropic antibody adhering to the appropriate cells. Nevertheless, although all antibody-adherent reactions are due to cytotropic antibody (so far as we know now), not all cytotropic antibody-determined reactions are antibody adherent (see below), In human medicine, the great majority of anaphylactic-type responses, such as anaphylaxis, hay fever, certain forms of allergic asthma, probably the urticaria of serum sickness and serum sickness-like reactions, are all examples of antibody-adherent, homocytotropic reactions (IIA1, Table V ) , Also included in this group of reactions are the PK reactions of humans and the PCA and Schulz-Dale reactions in monkeys sensitized with human YE antibody (120), and in guinea pigs sensitized with guinea pig rl antibody (IIA1, Table V ) . The antigen-induced release of histamine from slices of guinea pig lung sensitized with rabbit antibody is an example of an heterocytotropic antibody-adherent release reaction; the PCA reaction or systemic anaphylaxis of guinea pigs given rabbit or human rG antibody, or the PCA reaction of monkeys injected with human secretory yA antibody (121 ) are examples of anaphylactic-type heterocytotropic antibody-adherent responses. 4. Antigen-Adherent Reactions

Table V (IIAB) gives a number of examples of antigen-adherent release reactions. In many instances, antigen-adherent release reactions give rise to clinical manifestations and the latter are classified accord-

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ingly. The wheal aiid flare resulting from the injection of rabbit antibody against huinan YE immunoglobulin (122) and the PCA and systemic anaphylaxis in rats induced by the injection of rabbit, antirat, mast cell antibody ( 7 4 ) are antigen-adherent anaphylactic-type responses (Table V ) , the former being cellular and the latter humoral in nature. Whether the shocklike reaction resulting from the intravenous injection of rabbit antirat ./-globulin is an antigcn-adherent response is uncertain ( 123).

5. Aggregate Reactions The release of histamine from mouse peritoneal mast cells by complexes of antigen and mouse y l antibody (68) is an example of an aggregate, pharmacological mediator, release reaction ( Table V, IIA3 ) . It should be noted that the antibody in this instance is homocytotropic, that is, the release of histamine from mouse mast cells can also occur with mouse 7, lightly bound to mouse peritoneal cells. The wheal and flare response in normal human skin induced by preformed complexes of antigen aiid human YE antibody (124) is presumably an example of an aggregate anaphylactic-type reaction, duc in this case also to allergic complexes of antigen with homocytotropic antibody ( Table V ) . Tlic release of liistaminc from normal guinea pig lung by complexes of antigen with cither human or rabbit antibody (125, 126) is presumably an aggrcgatc releasc reaction due to allergic complexes fornied with heterocytotropic antibody. Systcmic anaphylaxis or the PCA reaction induced in guinea pigs by coiiiplescs of antigen and rabbit antibody (127) may be an aggregate anaphylactic-type reaction, but this or any othcr suggested mechanism rcniains to be rigidly proved (see Section X ) . Thc complement-dependent release of histamine from rabbit platelets by relatively small amounts of antigcn and antibody is cytotoxic (72, 7 3 ) and is presumably an exainplc of an aggregate release reaction. Whethcr the plasma-dependent releasc of histamine from rabbit platelets by relatively Iarge amounts of preformed antigen-antibody complcses ( 128) is a humoral ( 7 2 ) or possibly humoral-cellular (129) release reaction remains to bc proved. It is considered that anaphylaxis in rabbits induced in viva by combination of antigen with large amounts of hyperimmune rabbit antibody is an cxainple of aggregate anaphylaxis (26, 130). B. MACROMOLECULAR MEDIATOR-DETERMINED REACTIONS As alrcady discussed, thc only macromolecular mediator-determined reactions (IB aiid IIB, Tables IV and V) so far known are the neutrophile lysosomal reactions (IIB1, Tables IV and V). These reactions

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involve the fixation of complement by an in vivo antigen-antibody reaction with release of chcmotactic factors leading to an accumulation of neutrophiles at the site of specific combination. The neutrophiles phagocytize the antigen-antibody complex releasing lysosomal products including cathcptic enzymes which damage the tissue ( 8 3 ) . As already pointed out, the neutrophile lysosomal reactions are further subdivided on the basis of the nature of the initiating antigenantibody reactions into antigen-adherent neutrophile lysosomal reactions (IIBla, Tables IV and V ) and aggregate neutrophile lysosomal reactions ( IIBlb, Tables IV and V ) .

1. Antigen-Adherent React ions These neutrophile lysosomal reactions are macromolecular mediatordetermined responses where antibody reacting with antigen on cells or on defined extracellular structurcs initiates the infiltration of neutrophiles. The release of their lysosomal contents causes the major part of the tissue damage. As in other antigen-adherent responses the antigen may be an integral part of or otherwise bound to the structure concerned. There is a great deal of varied evidence that the nephrotoxic nephritis induced within hours in rats, rabbits, and guinea pigs by relatively low concentrations of mammalian coniplcment-fixing antibody against gloinerular basenient membrane depends largely upon complement and the lysosomal products of the neutrophile, particularly the catheptic enzymes (13, 8 3 ) . The antibody inducing the glonierulitis is bound in a linear fashion to the basement membrane as demonstrated by fluorescence or electron microscopy. It is thus an antigen-adherent, neutrophile lysosoma1 reaction (Table B, IIB1). In humans, Dixon and his colleagues have found antiglomerular basement membrane antibody in all cases of Goodpasture’s syndrome, in somewhat less than half of the cases of subacute and chronic cases of glomerulonephritis of adults, and in a smaller proportion of the membranous glomerulonephritides of children ( 131, 132). They have presented experimental evidence of the pathogenetic significance of this antibody in causing human glomcrulonephritis ( 131, 132). This evidence and the similarity in morphological features to experimental glomerulonephritis induced by antiglomerular bascmcnt membrane antibody provide powerful support for the idea that thrse human syndromes are antigenadherent reactions. There is no direct evidence in these various kinds of human syndromes that neutrophilic lysosomal products play the determining role as in the experimental examples given. Although there is indirect evidence ( 83) that they might be antigen-fixed neutrophilic lysosomal re-

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actions, these human syndromes frequently show a lack or paucity of neutrophils at the site of injury. This might very well indicate that they should be classified among the unknown mediator reactions. Thus, although I have provisionally classified them as antigen-fixed neutrophilic lysosomal reactions (see Table V, Sections IIBlaii and iii), the question marks indicate the uncertainty involved. 2. Aggregate Reactions Neutrophile lysosomal reactions initiated by antigen-antibody complexes formed with neither specific component being present at the time of formation on cells or extracellular structures are termed aggregate neutrophile lysosomal reactions ( IIBlb, Tables IV and V ) . The paradigm of these kinds of responses is the Arthus reaction (Table V ) even though, as will be brought out below, the Arthus reaction can apparently also be obtained by other than an aggregate neutrophile lysosomal mechanism. When, however, it is induced by moderate or relatively low concentrations of precipitation antibody, there is good evidence for complement and neutrophilic leukocytes being required for the reaction ( 8 3 ) . Moreover, there is demonstrable by fluorescence microscopy and/ or electron microscopy, the granular “lumpy bumpy” deposition of allergic complexes which is the morphological hallmark of aggregate reactions, as the linear deposition of allergic complexes is of antigen-fixed reactions. Similar evidence that complement and neutrophiles are required is available for the arteritis of experimental serum sickness ( 8 3 ) (Table V ) . Certain cases of allergic vasculitis, cutaneous arteriolitis and polyarteritis nodosa show neutrophil infiltration disruption of vessels and fibrinoid necrosis. They also have been shown to contain a patchy distribution of y-globulin and Complement in the lesions (132a), and in certain cases what is presumably the antigen has been found there. Thus, there is sound although still indirect evidence for classifying these cases as aggregate neutrophilic lysosomal reactions. There is immunofluorescent and ultramicroscopic evidence that the granular deposits along the glomeruli from patients with the nephritis of systemic lupus erythematosus and poststreptococcal nephritis are preformed antigen-antibody complexes containing bound complement (133). Anti-DNA and DNA have been isolatcd from the glomeruli of systemic ~ L I ~ L I Serythematosus with nephritis ( 1 3 4 ) . Thus, there is good evidence that these forms of human glomeruloiiephritis represent an aggregate reaction. There is no direct evidence, however, that the pathogenesis of these forms of glomcrulonephritis involves a neutrophil lysosomal mechanism. Thc uncertainty here is the same as already discussed with respect to the human antigen adherent

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reactions and my placing these diseases in the category of aggregate neutrophilic lysosomal reactions is an act of faith, and, as can be seen by reference to Table V (IIBlb; 3 4 ) , the act of faith is punctuated by question marks.

C. UNKNOWN MEDIATOR-DETERMINED REACTIONS David Ogg has pointed out that “To be worthy of the name, a profession must sometimes see that work is left for posterity. . . .” The subject of unknown mediator reactions indicates that in this regard experimental and clinical allergists have conscientiously, if unconsciously followed Ogg’s dictum. There is a number of rcactions where the nature of the mediators is largely unknown. In the anaphylactic-type aggregate reaction of guinea pigs given preformed antigen-antibody complexes ( 127) or antigen and antibody given simultaneously ( 135), there is apparently conflicting evidence as to the mediators involvcd (136, 137). Arthus reactions can be obtained in neutropenic rabbits, but higher concentrations of antibody have to be used than when the reaction is induced in normal animals ( 8 3 ) . Present cvidence indicates that the genesis of the glomerulonephritis of acute experimental seruni sickness involves neither complement nor neutrophiles ( 1 3 8 ) , even though there is prominent binding of complement in the glomerular lesions. Thc difference in pathogenesis of the arteritis of serum sickness is evident (83, 138). In mammals, given nonconiplement-fixing, duck, antiglomcrular, basement membrane antibody, an acutc gloinerulonephritis develops which depends neither on compleincnt nor on neutrophiles ( 8 3 ) . It is an antigenadherent unknown mediator reaction (Table V ) . In the same category is the glomerulonephritis induccd in ncutropenic rabbits by relatively large amounts of complement-fixing, antiglomerular, basement membrane antibody. Probably, although for technical reasons this is not certain, the glomerulonephritis which devclops after a delay of a week or more following the injection of heterologous basement menibranc antibody should probably also be placed in this category (83, 139). Hcrc the antibody concerned arises following thc injcction of the heterologous antiserum and then reacts with the heterologous antibody bound to the basemcnt membrane. The glomcruloncphritis has been termed “autologous” or “secondary nephrotoxic nephritis”; in accord with the prcceding discussion, it can be more precisely termed “serum sickness-like phase of antigen-fixed nephritis.” X. Mixing of Categories in Natural Reactions

In logic, the categories used in classification must be mutually exclusive; nature does not know this requirement. Thus, any given allergic syndrome, as it is observcd in the laboratory or the clinic may very

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well and often does have several components and these may fall into more than one of the categories given in Table IV. This is particularly true when, as in most clinical situations, the reaction occurs in actively sensitized subjects. H u e , there is a good chance to develop simultaneously a multiplicity of different antibodies with differing functional capacities and, thus, capable of giving risc to different kinds of reactions, This can also occur in passively sensitized subjects given unfractionated antiserum containing several different kinds of antibody. There are a number of examples of this multiplicity of components in the reactions we have already discussed. The acute hemolytic transfusion accident is a case in point. In Table V, this has been classified as a direct cellular antigen-fixed reaction (IB1). There is 110 doubt that the immediate intravascular hemolysis occurring in these reactions fits into this category. In addition to lysis of their red cells, these patients also frequently suffer from shock, urticaria, fever, flushing, etc. These latter signs and symptoms may very well be antigen-fixed, anaphylactic in nature according to the cvidence of Jandl (140) and others who found that small amounts of sensitized but not unsensitized red cell stroma can initiate the same signs and symptoms. If further work on the mechanisms involved should substantiate this, then one might well speak of an antigen-fixed direct cellular component and an antigen-fixed anaphylactic-type component of the acute hemolytic transfusion accident. Treadwell has presented clear-cut evidence that products of the macrophage lysosonies are released during the course of aggregate anaphylaxis in the mouse (90). If it should turn out that these macromolecular products directly induce some of the manifestations of this form of anaphylaxis in mice, one would have to speak of a macromolecular niediator-determined component of this typical anaphylactic-type reaction. This, however, remains to be proved. A more certain example of a macromolecular mediator response in what is essentially a typical anaphylactic-type reaction is the marked incoagulability of the blood in anaphylaxis of dogs. This is due primarily to the release of heparin, a typical macromolecular inhibitor (Table I1 and Section 111,D). Although a very evident manifestation of anaphylaxis, the incoagulability of the blood is hardly a major one. Nevertheless it is a minor, macromolecular, mediator-dependent component of this particular anaphylactic-type response. At the site of a cutaneous Arthus reaction, a typical macromolecular mediator-determined reaction, there is often an increase in vascular permeability occurring within minutes of antigen-antibody combination; this is a PCA reaction, a typical anaphylactic-type response. There is evidence that the PCA reaction enhances the later developing (re-

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tarded) Arthus reaction occurring some circumstances is apparently ( 1 4 2 ) . In these instances one can an anaphylactic-type component of

307

at the same site (141),and under required for the full-blown lesion and should consider the former as this neutrophile lysosomal reaction.

XI. Pseudoallergic Reactions

Allergic reactions in general are uniquely distinguished from other responses only by their initiating stimulus-the antigen-antibody complex. (Antibody is used here in the very general sense of von Pirquet, see footnote 2.) The clinical manifestations of responses to certain nonantigen-antibody stiniuli may be more or less similar to one or another allergic reaction. In some instances, this is due to their arising, in whole or in part, through the samc type of interactions on the same cells, effector enzymes and mediators described for allergic reactions. Thus, both hunians and experimental animals can and do give pseudoallergic reactions, that is, responses that are not initiated by an antigen-antibody complex but which are more or less similar to allergic reactions in their manifestations and underlying mechanisms.’” The clearest example of a pseudoallergic reaction presently known is the human disease, hereditary angioneurotic edema. Patients with this disease have a genetically determined lack of C i inhibitor (previously termed C’1 esterase inhibitor) ( 1 4 3 ) . This is associated with an in oioo activation of the complement system and probably the kininforming system (see Section II1,C and Fig. 1 and, more especially, Refs. 49 and 49a for a more detailed discussion). Abrupt systemic reactions which differ in one or several ways from anaphylaxis have frequently been termed “anaphylactoid reactions.” The term has been applied to systemic Forssman shock (see Section VIII) and with some reason, since, as already discussed in Section IX,A,l, despite similarities to anaphylaxis, the mechanism is not anaphylactic. Nevertheless, for purpose of clarity and precision of expression it might be well to limit the use of the term anaphylactoid to pseudoallergic reactions similar to anaphylaxis. As is well known, the injection of endotoxin can lead to an abrupt, systemic response very similar to anaphylaxis ( 1 4 4 ) or to localized or generalized Shwartzman reactions or to a delayed shock syndrome. These all might be thought of as pseudoallergic responses, although there are those who consider them as resulting from the reactions of endotoxin with cross-reacting natural antibodies ( 1 4 5 ) , that is, they are allergic. “It becomes a nice point whether the biological reactions of aggregated yglobulin should be classified as allergic or pseudoallergic. I incline to think of them as allergic.

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The inflammatory response of rats to carageenin or to dextran can also be considered as pseudoallergic reactions, although some believe the latter depends upon the reaction of dextran with natural antibody. It is obvious from some of the examples just given that to decide whether a reaction is allergic or pseudoallergic sometimes requires more knowledge than we presently possess. The necessity to make the distinction in any given instance, emphasizes the almost platitudinous truism that both experimentally and in human medicine, the nature of the clinical manifestations may give reason to suspect a reaction is allergic but does not prove it. ACKNOWLEDGMENTS This work was written at Walter Heed Army Institute of Research and I wish to thank Mrs. Jean Zufall for typing the various drafts of the manuscript. It was revised at the Sir William Dunn School of Pathology, Oxford University, during a John Simon Guggenheim Memorial Fellowship, I wish to thank Professor Henry Harris and his staff for the aid given me during my stay. I also wish to thank the Royal Society for permission to reproduce Fig. 1.

REFERENCES 1. von Pirquet, C. (1906). Munch. Med. Wochenschr. 30, 145; English trans., in “Clinical Aspects of Immunology” (P. G. H. Gell and R. R. A. Coombs, eds.), 2nd ed., p. 1295. Blackwell, Oxford, 1968. 2. Coombs, R. R. A., and Gell, P. G. H. (1968). In “Clinical Aspects of Immunology” (P. G. H. Gell and R. R . A. Coombs, eds. ), 2nd ed., p. 575. Blackwell, Oxford. 3. von Pirquet, C., and Schick, B. (1905). “Die Serum Krankheit”; English transl., “Serum Sickness,” William & Wilkins, Baltimore, Maryland, 1951. 4. Davis, B. D., Dulbecco, R., Eisen, H. N., Ginsberg, H. S., and Wood, W. B. Jr. ( 1967). In “Microbiology,” p. 526. Harper, New York. 5. Ovary, 2. (1965). Fed. Proc., Fed. Amer. SOC. Exp. Biol. 24, 94. 6. Binaghi, R. A. ( 1968). In “Biochemistry of the Acute Allergic Reactions” (K. F. Austen and E. L. Becker, eds.), p. 53, Blackwell, Oxford. 7. Vaughn, J. H., Barnett, E. V., and Leddy, J. P. (1966). N . Engl. 1. Med. 275, 1426 and 1486. 8 . Kunkel, H. G., and Tan, E. M. (1964). Aduon. Immunol. 4, 351. 9. Idse, O., Guthe, T., Wilcox, R. R., and De Weck, A. L. (1968). Bull. W . H . 0. 38, 159. 10. Shulman, N. R., Aster, R. H., Leitoner, A., and Hiller, M. C. (1961). J. Clin. Invast. 49, 1597. 11. Mollison, P. L. (1967). In “Blood Transfusion in Clinical Medicine,” 4th ed., p. 578. Blackwell, Oxford. 12. Dixon, F. J. (1963). Haruey L e d . 58, 21. 13. Unanue, E. R., and Dixon, F. J. (1967). Aduan. Immunol. 6, 1. 14. Levine, B. B. (1966). N . Engl. J . Med. 275, 1115. 15. Levine, B. B., and Zolov, D. M. (1969). J . Allergy 43, 231.

IhfMEDIATE-TYPE ALLERGIC REACTIONS

309

16. Reisman, R. E., Rose, N. R., Witelisky, E., and Arl~esman,C. E. (1961). J . Allergy 32, 531. 17. Franklin, E. C., and Frangionc, B. (1969). Antiti. Rec. Afed. 20, 155. 18. Sandberg, A. L., Osler, A. G., Shin, H. S., and Oliveira, B. (1970). J. Immr~tiol. 104, 329. 19. Fahey, J. L., Franklin, E. C., Kunkel, H. G., Osscrman, E. F., and Terry, W. D. ( 1967). J . Zmmrrtio~.99, 465. 20. Frame, XI., hlollison, P. L., antl Terry, \V. D. (1970). Nature (London) 225, 641. 21. Cohen, S. ( 1968). J. Zmniroiol. 100, 407. 22. Klun, hl. J., and hluschcl, L. H. ( 1966). Nature (Lotidon) 212, 159. 22a. Rosse, W.F. (1968). J. Cliri. Iticert. 47, 2430. 23. Hoyer, L. W., Borsos, T., Rapp, H. J., antl Vannier, W. E. (1968). 1. Exp. Med. 127, 589. 24. Plotz, P. H., Colten, H., and Talal, N. (1968). J. Imnirtriol. 100, 752. 25‘. Ishizaka, T., Ishizaka, K., Borsos, T., and Rapp, H. ( 1966). J. Zmmrtno2. 97, 716. 26. Becker, E. L., and Austen, K. F. (1968). Iii “Textliook of I m ~ i i u n o p a ~ ~ o l o g ” (P. A. hliescher and H. J. SIiiiler-Elierliard, eds.), Vol. 1, p. 76. Grune & Stratton, New York. 27. Catty, D. ( 1969 ). “The Iniinunology of Nematode Infections. Trichinosis in Guinea Pigs as a Model.” Karger, Basel. 28. Blocli, K. J. ( 1969). In “Cellular and Humoral Mechanisms in Anaphylaxis and Allergy” (H. Z. hlovat, e d . ) , p. 1. Karger, Basel. 29. Bennich, H., Ishizaka, K., Ishizaka, T., and Johansson, S. G . 0. (1969). 1. Zmttirinol. 102, 826. 30. Zvaifler, N. J., a n d Robinson, J. 0. ( 1969). J. E s p . Aled. 130, 907. 31. hlota, I. (1968). I n “Biochemistry of the Acute Allergic Reactions” ( K . F. Austen and E. L. Becker, eds.), p. 189. Black\vell, Oxford. 32. Levey, D., and Osler, A. G. (19GG). J. Ittimt~tiol.97, 203. 33. Becker, E. L., and Austen, K. F. (19G6). J. E x ] ) . Med. 124, 379. 34. Orange, R. P., and Austcn, K. F. ( 1 9 6 8 ) . Proc. SOC. Exp. Biol. Med. 129, 836. 35. Prouvost-Danon, A. ( 1968). I n “Biochemistry of the Acute Allergic Reactions” ( K . F. Austen and E. L. Becker, eds.), p. 175. Blackwell, Oxford. 35a. Prouvost-Danon, A,, and Binaghi, R. ( 1970). Nattrre (Lotidon) 228, 66. 36. Vaz, N. hl., and Ovary, Z. (1968). J. Immtrnol. 100, 1014. 37. Reid, R. T., Minden, P., and Farr, ‘R. S. (1966). J . E x p . Med. 123, 845. 38. Orange, R. P., Stechschulte, D. J., and Austen, K. F. (1969). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 28, 1710. 39. Stechshulte, D. J., Austen, K. F., and Bloch, K. J. (1967). J. E x p . Med. 125, 127. 40. Morse, H. C., Bloch, K. J., and Austen, K. F. (1968). J. Zmmtrnol. 101, 658. 40a. Orange, R. P., and Austen, K. F. (19G9). Adcan. Znimunol. 10, 105. 41. Terry, W. D. (1965). J. Zmmunol. 95, 1041. 42. Nelson, D. S. ( 1969 ). “hlacrophages and Imniunity.” North-Holland Publ. Amsterdam . 43. Boyden, S. V., and Sorkin, E. (1960). Immunology 3, 272. 44. LoBuglio, A. F., Cotran, R. S., and Jandl, J. H. (1967). Science 158, 1582. 44a. Aliramson, N., LoBuglio, A. F., Jandl, J. H., and Cotran, R. S. (1970). I. E x p . Mcd. 132, 1191.

310

ELMER L. BECKER

44b. Abramson, N., Gelfand, E. W., Jandl, J. H., and Rosen, F. S. (1970). J. E x p . hied. 132, 1207. 45. Uhr, J. W., and Phillips, M. (1966). Ann. N. Y. Acad. Sci. 129, 793. 46. Shulman, N. R. (1963). Trans. Asso. Amer. Physicians 76, 72. 47. Miiller-Eberhard, H. J. (1968). Adoan. Immunol. 8, 2. 48. Webster, M. (1969). I n “Cellular and Humoral Mechanisms in Anaphylaxis and Allergy” ( H . Z. Movat, ed.), p. 207. Karger, Basel. 49. Becker, E. L. (1969). Proc. Roy. Soc., Ser. B 173, 383. 50. Ratnoff, 0. (1969). Aduan. Immunol. 10, 145. 51. Nelson, D. S. (1963). Adoan. Itnmunol. 3, 131. 52. Gigli, I., and Nelson, R. A. Jr. (1968). Exp. Cell Res. 51, 45. 53. Lepow, I. H., Dias da Silva, W., and Patrick, R. A. (1969). In “Cellular and Humoral Mechanisms in Anaphylaxis and Allergy” ( H . Z. Movat, ed.), p. 237. Karger, Basel. 54. Ward, P. A. (1969). In “Cellular and Humoral Mechanisms in Anaphflaxis and Allergy” ( H . Z. hlovat, ed.), p. 279. Karger, Basel. 55. Cochrane, C. G., and Miiller-Eberliard, H. J. (1968). J. E x p . Med. 127, 374. 55a. Kaplan, A. P., and Austen, K. F. (1971). J. Exp. Med. 133, 696. 55b. Cochrane, C. G. and Wuepper, K. D. (1971). Fed. Proceed. 30, 451. 56. Brocklehurst, W. E., and Lahiri, S. C. (1962). J. Pliysiol. (London) 165, 39P. 57. Jonasson, O., and Becker, E. L. (1966). J. E x p . Med. 123, 509. 58. Einbinder, J. M., \Velzer, A., and Nelson, C. T. (1964). J. Immunol. 93, 165. 59. Bliimback, hl., Johansson, S. A., and Sjoberg, H. E. (1967). Acta Physiol. Scarid. 69, 313. GO. Vassali, P., and McCluskey, R. T. (1964). Ann. N. Y. Acad. Sci. 116, 1052. 61. Ungar, G., and Hayashi, H. (1958). Ann. Albrgy 16, 542. 62. Ratnoff, 0. D., Pensky, J., Ogston, D., and Naff, G. B. (1969). J. Exp. Med. 129, 315. 63. Donaldson, V. H. (1968). J. Exp. Med. 27, 411. 64. Becker, E. L., and Austen, K. F. (1964). J. Exp. Med. 120, 491. 65. Osler, A. G., Lichtenstein, L. M.,and Levy, D. A. (1968). Aduan. Immunol. 8, 183. 66. Ishizaka, T., Ishizaka, K., Johansson, G. O., and Bennich, H. (1969). J. Imtnitnol. 102, 884. 67. Lichtenstein, L. M. (1969). I n “Cellular and Hunioral Mechanisms in Anaphylaxis and Allergy” ( H . Z. Movat, ed.), p. 176. Karger, Basel. 68. Vaz, N. M., and Prouvost-Danon, A. (1969). Progr. Allergy 13, 111. 69. Humphrey, J. H., Austen, K. F., and Rapp, H. J. (1963). ImmtinoZogy 6, 226. 70. Daems, W.T., and Cort, J. (1962). Exp. Cell Res. 28, 11. 71. Pearlman, D. S., Ward, P. A., and Becker, E. L. (1969). J. Exp. Med. 130, 745. 72. Henson, P. M. (1969). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 28, 1720. 73. Osler, A. G. (1969). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 28, 1729. 74. Valentine, hf. D., Bloch, K. J., and Austen, K. F. (1967). J. Immunol. 99, 98. 75. Johnson, R. A., and hloran, W. C. (1969). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 28, 1716. 76. Austen, K. F., and Becker, E. L. (1966). 3. Exp. A4cd. 124, 397. 77. Becker, E. L., and Ward, P. A. (1969). J. Exp. Bled. 129, 569. 78. Piper, P. J., and Vane, J. R. (1969). Nature (London) 223, 29.

IMMEDIATE-TYPE ALLERGIC REACTIONS

311

78a. Kay, A. B., Steclischulte, D. J. and Austen, K. F. (1971). J. E x p . Med. 133, 602. 79. Austen, K. F. (1965). In “Immunological Diseases” ( M . Samter and H. L. Alexander, eds. ), p. 211. Little, Brown, Boston, Massachusetts. 80. Brockleliurst, W. E. ( 1962). Progr. Albrgy 6, 539. 81. Vogt, W. (1969). In “Cellular and Humoral Mechanisms in Anaphylaxis and Allergy” ( H . Z. Movat, ed.), p. 187. Karger, Basel. 82. Vogt, W.,Zemen, N., and Garbe, C. (1969). Naunyn-Schmiedebergs Arch. Pharmakol. E x p . Pathol. 263, 399. 83. Cochrane, C. (1968). Aduan. Immunol. 91, 97. 84. Janoff, A. (1968). lmmunopathol. lnt. Symp., 5th, 1967 p. 44. 85. Ranadive, N. S., and Cochrane, C. G. (1968). J. Exp. Med. 128, 605. 86. Glynn, L. E., and Holborrow, E. J. (1965). “Autoimmunity and Disease,” p. 239. Blackwell, Oxford. 87. Tai, C., and McGuigan, J. E. (1969). Blood 34, 63. 87a. Basten, A., Boyer, M. H., and Beeson, P. B. (1970). J. Exp. Med. 131, 1271. 87b. Basten, A,, and Beeson, P. B. (1970). J. E x p . Med. 131, 1288. 87c. Cohen, S., and Ward, P. A. (1971). J. E x p . Med. 133, 133. 88. Orange, R. P., Valentine, M. D., and Austen, K. F. (1968). J. E x p . Med. 127, 767. 89. Litt, M. (1969). In “Allergology” (B. Rose et al., eds.), p. 38. Excerpta Med. Found., Amsterdam. 90. Treadwell, P. E. (1969). J. Reticuloendothel. Sac. 6, 354. 91. Mollison, P. L. (1965). Complement, Ciba Found. Symp., 1964 p. 323. 92. Sclioenbechler, M. J., and Barbaro, J. F. (1968). PTOC.Nut. Acad. Sci. U. S. 60, 1247. 93. Taicliman, N. S., and Movat, H. Z. (1966). lnt. Arch. Allergy Appl. Immunol. 30, 97. 94. Jandl. J. H. (1965). Ser. Haematol. 1, 35. 95. Kniker, W. T., and Cochrane, C. G. (1968). J. Exp. Med. 127, 119. 96. Darrack, M. (1959). 111 “Mechanisms of Hypersensitivity” (J. H. Shaffer, G. A. Lo Grippo, and M. W. Chase, eds.), p. 613. Little, Brown, Boston, Massachusetts. 97. Batchelor, J. R. ( 1968). In “Regulation of the Antibody Response” ( B . Cinader, ed. ), p. 276. Thomas, Springfield, Illinois. 98. Pierpaoli, W., Baroni, C., Fabris, N., and Sorkin, E. (1969). Immunology 16, 217. 99. Rose, B. (1959). In “Mechanisms of Hypersensitivity” (T. H. Shaffer, G. A. Lo Grippo, and M . W. Chase, eds.), p. 599. Little, Brown, Boston, Massachusetts. 100. Treadwell, P. E., and Rassmussen, A. F., Jr. (1961). J. Immunol. 87, 492. 101. Szentivanyi, A., and Fishel, C. i V . (1965). In “Immunological Diseases” (M. Saniter and H. L. Alexander, eds.), p. 226. Little, Brown, Boston, Massachusetts. 102. Munoz, J. (1964). Aduan. lmnmnol. 4, 397. 103. Szentivanyi, A. (1968). J. Allergy 42, 203. 104. Reed, C. E. (1969). In “Allergology” (B. Rose et al., eds.), p. 402. Excerpta Med. Found., Amsterdam. 105. Lecks, H. I., Wood, D. W., and Donsky, G. (1969). J. Allergy 44, 261. 106. Math&, A. A., and Knapp, P. H. (1969). N . Engl. J. Med. 281, 234.

312

ELMER L. BECKER

107. Rasmussen, A. F., Jr., Spencer, E. S., and March, J. T. (1965). Proc. SOC. Exp. Biol. Med. 100, 878. 108. Solomon, G. F., and Moss, R. H. (1964). Arch. Gen. Psychiat. 11, 657. 109. Luparello, T., Lyons, H. A., Bleecker, E. R., and McFadden, E. R., Jr. (1968). Psychosom. Med. 30, 819. 110. Kesztyus, L. ( 1967). “Immunitat und Nervensystem.” Akadbmiai Kiadb, Budapest. 111. Anderson, B. R., and Terry, W. D. (1968). Nature (London) 217, 174. 112. Feinstein, D. I., Rapoport, S. I., and Chong, M. N. Y. (1969). Blood 34, 85. 113. Berson, S. A., and Yalow, R. S. (1962). In “Clinical Diabetes Mellitus” (M. Ellenberg and H. Rifkin, eds.), p. 194. McCraw-Hill (Blakiston), New York. 114. Garrido-Pinson, G. C., Turner, M. D., Crookston, J. H., Samloff, I. M. Miller, L. L., and Segal, H. L. (1966). J. Immunol. 97, 897. 115. Leddy, J. P. (1966). Semin. Hematol. 3, 48. 116. Waltraut, H. L., and Nussenzweig, V. (1968). J. E z p . Med. 128, 991. 117. Huber, H., Polley, M. J., Linscott, W. D., Fudenberg, H. H., and MiillerEberhard, H. J. (1968). Science 162, 1281. 118. Tanaka, N., and Leduc, E. H. (1956). J. Immunoz. 77, 198. 119. Strejan, G., and Campbell, D. A. (1968). J. Immunol. 100, 1245. 120. Ishizaka, K., Ishizaka, T., and Arbesman, C. E. (1967). J. Allergy 39, 254. 121. Arbesmann, C. E., Dolovich, J,, Wicher, K., Duskenski, L. A,, Reisman, R. E., and Tomasi, T. B., Jr., quoted by Tomasi, T. B., Jr., and Bienenstock, J. (1968). Aduan. Immunol. 9, 78. 122. Ishizaka, K., and Ishizaka, T. (1968). J. Immunol. 100, 554. 123. Keller, R. (1965). Int. Arch. Allergy Appl. Immunol. 28, 288. 124. Ishizaka, K., and Ishizaka, T. (1968). J. Immunol. 101, 68. 125. Broder, I., Baumal, R., and Keystone, E. (1968). Clin. Exp. Immunol. 3, 537. 126. Baumal, R., and Broder, I. (1968). Clin. E x p . Immunol. 3, 555. 127. Ishizaka, K. ( 1963). Progr. Allergy 7, 32. 128. Barbaro, J. F. (1961). J. Immunol. 86, 377. 129. Des Pres, R. M., and Bryant, R. E. (1969). J. Immunol. 102, 241. 130. Movat, H. Z., Urihara, T., Taichman, N. S., Rowsell, H. C . , and Mustard, J. F. (1968). Immunology 14, 637. 131. Lerner, R. A., Classcock, R. J., and Dixon, F. J. (1967). J. Exp. Med. 126, 989. 132. Dixon, F. J. (1968). Amer. J. Med 44, 493. 132a. Parish, W. E. (1970). Proc. Int. Symp. Immune Complex Diseases p. 98. Erba, Milan. 133. Burkholder, P. M. ( 1968). In “Structural Basis of Renal Disease” (E. L. Becker, ed.), p. 197. Harper, New York. 134. Koffler, D., Schur, P. H., Kunkel, H. G., and Graf, M. (1968). Zmmunopathol., Int. Symp., 5th, 1967 p. 70. 135. Feinman, L., Cohen, S., and Becker, E. L. (1969). J. Zmmunol. 103, 395. 136. Mota, I. (1961). Nature (London) 191, 572. 137. Weigle, W. O., Cochrane, C. G., and Dixon, F. J. (1960). J. Immunol. 85, 469. 138. Henson, P. M., and Cochrane, C. G. (1971). J. E x p . Med. 133, 554. 139. Rother, K., Rother, U., Vassali, P., and McCluskey, R. T. (1967). J. Immunol. 98, 965. 140. Jandl, J. H., and Tomlinson, A. S. (1958). J. Clin. Inuest. 37, 1202. 141. Voisin, G. A., and Maillard, J. (1968). Ann. Inst. Pasteur, Paris 114, 173.

IMMEDIATE-TYPE ALLERGIC REACTIONS

142. 143. 144. 145.

313

Maillard, J. L., and Voisin, G. A. (1970). Proc. SOC.E x p . B i d . Med. 133, 1188. Donaldson, V. H., and Evans, R. R. (1963). Arner J . Med. 35, 37. Weil, M. H., and Spink, W. W. (1957). J. Lab. Clin. Med. 50, 501. Lee, L., and Stetson, C. A., Jr. (1965). I n “Inflammatory Process” ( B . W. Zweifach, L. Grant, and R. T. McCluskey, eds.), p. 791. Academic Press, New York.

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AUTHOR INDEX Numbers in parentheses are reference numbers and indicate that an author's work is referred to, although his name is not cited in the text. Nunibers in italics show the page on which the complete reference is listed.

A

Andradk, Z. A., 243, 248, 249, 261 Appella, E., 59, 73, 97 Aranjo, F. G., 249, 261 Arbesman, C. E., 10, 53, 107, 198, 273 (16), 300( 16), 301( 120, 121), 309, 312 Arnason, B. O., 179, 193 Arner, B., 45, 51 Arthris, M., 39, 55 Asherson, G. L., 184, 193, 233, 234, 261 Assadourinn, Y., 249, 260, 265 Aster, R. H., 272(10), 299(10), 308 Atoynatan, T., 127, 129, 130, 131, 136, 194 Augustin, R., 4, 51, 51 Austen, K. F., 39, 55, 275( 26, 33, 34), 276( 38, 39, 40, 40a), 278( 55a), 279 ( 3 3 ) , 280(64, 69, 74, 76), 281 (78a), 282( 40a, 79), 284( 78a, 79, 8 8 ) , 287( 8 8 ) , 300( 26), 302( 26, 74), 305(34), 309, 310, 311 Austin, C. M., 82, 98 Averbeck, A. K., 29, 53 Aviet, T., 113, 199 Avrameas, S., 10, 12, 51 Ax, W., 114, 119, 178, 193 A x h , R., 10, 51, 54

Aas, K., 31, 39, 51 Able, M. E., 115, 193 Abrahams, C., 260, 263 Abranison, N., 299(44a, 44b), 309, 310 Achard, C., 105, 193 Ackman, C . F . D., 146, 202 Ada, G. L., 82, 98, 161, 162, 195, 202 Adanis, C., 127, 129, 206 Adanis, D. O., 135, 193 Adler, F. L., 191, 198 Adler, S., 237, 244, 245, 246, 247, 257, 258, 261 Adler, W. H., 111, 142, 144, 145, 193, 205 Ahlinder, S., 50, 51 Aicardi, J., 39, 55 Al-Askari, S., 124, 127, 129, 193, 196 Albright, J. F., 76, 98, 160, 189, 201 Aldrich, R. A., 37, 51 Alescio, L., 76, 98 Alexander, P., 114, 120, 175, 197 Ali, A. J. T., 236, 237, 261, 265 Allen, J . M. V., 114, 179, 204 Allerhand, J., 134, 155, 193 Allfrey, V. G., 105, 204 Allison, A. C., 229, 264 Alm, G. V., 180,193 Almeidn, J. O., 236, 261 B Alter, B. J., 110, 145, 193, 193, I94 Altounyan, R. E. C., 45, 51 Bach, F. H., 106, 110, 145, 146, 163, Amache, N., 107, 199 164, 169, 184, 193, 193, 194, 199, Amante, L., 165, 204 200, 205, 208 Amezcua-Chavarria, M. G., 232, 265 Bach, J. F., 110, 189, 194 Amiel, J, L., 176, 202 Badger, A., 141, 199 Ammann, A. J., 39, 40, 51, 52 Baer, H., 108, 126, 129, 196, 198 Amos, H . E., 126, 131, 132, 153, 173, Baer, R. L., 107, 198 193 Baglioni, C., 91, 97 Anderson, B. R., 16, 51, 298(111), 312 Ball, P. A. J., 43, 49, 52, 53, 55 Anderson, B., 190, 207 Balner, H., 102, 194 315

316

AUTHOR INDEX

Balzer, R. J., 238, 239, 261 Barbaro, J. F., 285(92), 302( 128), 311, 312 Barlett, A., 43, 52 Barnet, K., 135, 194 Barnett, E. V., 272( 7 ) , 308 Baron, S., 150, 195 Baroni, C., 290(98), 311 Barrat, T. M., 34, 55 Bartfeld, H., 127, 129, 130, 131, 136, 194 Basten, A., 284(87a, 87b), 311 Batclielor, J. R., 290( 97), 311 Baumal, R., 138, 194, 302(125, 126), 312 Bauscher, J. A. C., 142, 144, 145, 205 Bausek, G. H., 149, 202 Bazzi, C., 231, 264 Beachy, E. H., 127, 129, 197 Beauman, L., 143, 198 Becker, E. L., 275(26, 33), 277(49), 278(49, 57), 279(33, 49), 280(84, 71, 76, 77), 300(26), 302(26), 305 (135), 309, 310, 312 Bedford, M., 176, 206 Beeson, P. B., 284(87a, 87b), 311 Beiguelman, B., 232, 261 Benacerraf, B., 107, 152, 153, 166, 170, 171, 176, 182, 183, 188, 189, 191, 194, 196, 198, 200, 201, 203, 207 Benallhgue, A., 246, 263 Bendken, C., 146, 194 Ben-Ephraim, S . , 124, 183, 205 Benex, J., 245, 261 Benezra, D., 107, 194 Benjamini, E., 108, 124, 183, 206 Bennett, B., 123, 124, 126, 127, 128, 129, 130, 131, 133, 134, 143, 153, 156, 157, 159, 171, 172, 173, 175, 194, 195 Bennett, J. C., 84, 97 Bennich, H., 2, 4, 5, 7, 8, 9, 10, 15, 16, 17, 19, 20, 22, 23, 25, 27, 28, 29, 31, 32, 33, 34, 35, 37, 39, 40, 42, 43, 44, 45, 46, 48, 49, 50, 52, 53, 54, 55, 275( 29), 280( 66), 309, 31 0 Berdal, P., 33, 44, 52 Berg, T., 29, 30, 31, 36, 37, 38, 43, 44, 48, 49, 52, 53 Berggird, I., 23, 52

Berglund, G., 37, 52 Berken, A,, 152, 153, 194 BBrnard, H., 105, 193 Bernstein, I., 176, 207 Berson, S . A., 298( 113), 312 Bertler, A., 45, 51 Bettane, M., 94, 98 Bevard, C. W., 229, 261 Bianco, C., 110, 194 Biesecker, J. L., 135, 193 Biliotti, C . , 123, 129, 204 Bill, A. H., 113, 116, 121, 199 Billingham, R. E., 173, 194 Binaghi, R. A,, 271( 6 ) , 275( 35a), 308. 309 Biozzi, G., 161, 189, 194, 203 Birke, G., 50, 51 Bittencourt, A. L., 243, 248, 249, 261 Bjernulf, A., 39, 52 Blackburn, W. R., 259, 263 Blanden, R. V., 135, 177, 194, 201, 213, 261 Blazkovec, A. A., 152, 194 Bleecker, E. R., 291( log), 312 Bloch, K. J., 79, 81, 98, 275(28), 276 (39, 40), 280(74), 302(74), 309, 310 Bloch-Shtacher, N., 107, 144, 146, 194 Block, J. B., 228, 230, 234, 235, 243, 265, 266 Blomback, M., 278(59), 310 Bloom, B. R., 103, 123, 124, 126, 127, 128, 129, 130, 131, 133, 134, 135, 138, 140, 141, 144, 146, 147, 150, 151, 153, 156, 157, 159, 163, 166, 167, 168, 171, 173, 175, 176, 180, 184, 185, 192, 194, 195, 200, 205 Blume, M. R., 167, 168, 200 Blyth, J. L., 163, 207 Bock, H., 163, 193 Boeck, C. W., 227, 262 Boerma, F. W., 62, 97 Bokkenheuser, V., 260, 263 Bonomo, L., 218, 236, 261 Bordenave, G., 69, 90, 97, 9s Borecky, L., 149, 195, 202 Borek, F., 183, 186, 195, 205 Borel, Y., 124, 190, 195 Borrel, A., 261

317

AUTHOR INDEX

Borsos, T., 129, 175, 201, 207, 275(23, 25), 309 Bosnian, C., 171, 195 Boughton, B., 159, 170, 195, 207 Bouthillier, Y., 161, 189, 194 Bowers, E. J., 247, 262 Boyden, S. V., 151, 152, 171, 195, 203, 276(43), 309 Boyer, &I. H., 284(87a), 311 Boyle, J. A., 32, 54 Boylston, A. W., 231, 264 Boyse, E. A., 153, 173,194 Bradstreet, C. M. P., 260, 261 Brandaeo, H., 236, 261 Brandtzaeg, P., 32, 52 Braun, D. G., 70, 71, 79, 80, 97 Bray, R. S., 242, 243, 244, 246, 247, 249, 250, 2-51, 252, 254, 255, 256, 257, 258, 259, 261, 262, 263 Brent, L., 229, 261 Bretscher, P. A., 182, 191, 195 Brient, B., 85, 86, 88, 97 Brittinger, G., 105, 195, 199 Brocklehurst, iV. E., 278(56), 282( 8 0 ) , 310, 311 Broder, I., 302( 125, l 2 6 ) , 312 Brondz, B. D., 113, 115, 116, 195 Brostoff, J., 107, 146, 157, 186, 195, 204, 205 Brouty-Boyi, D., 142, 151, 199 Brown, J. A. K., 215, 261 Brown, R. S., 229, 261 Brrrnner, K. T., 113, 114, 115, 116, 117, 119, 120, 121, 173, 179, 180, 195, 196, 202 Bryant, R. E., 302( 129), 312 Bryceson, A . D. hl., 144, 201, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 250, 251, 2-52, 253, 2.54, 255, 2.56, 257, 258, 259, 261, 262, 263, 264 Bnbenik, J., 113, 120, 195 Buchanan, H., 237, 265 Buchanan, W. W., 32, 54 Buckler, C. E., 150, 195 Bullock, W. E., 228, 229, 2330, 234, 262 Bulnian, H. N., 107, 197 Burke, D. C., 149, 200 Burkholder, P. M., 304( 133), 312 Burnet, F. M., 102, 103, 195

Biitorova, E., 20," Byrt, P., 161, 162, 195, 202

C Cahill, K,,246, 262 Cuin, W.A,, 39, 40, 51, 52 Campbell, D. A., 300( 119), 312 Campbell, D. C., 37, 51 Canty, T. C., 117, 164, 173, 195, 207 Capra, J. D., 62, 63, 74, 97, 99 Carbonara, A. O., 4, 5, 14, 19, 29, 54 Carbone, P. P., 229, 261 Carmago, hl. E., 248, 249, 262 Caron, C . A,, 106, 195 Carpenter, R. R., 123, 195 Cnrs~vcll,E. A., 176, 187, 203, 206 Catt, K., 29, 52 Catten, A , , 176, 202 Catty, D., 275( 27), 309 Cech, K., 157, 204 Cerottini, J. C., 114, 115, 116, 117, 119, 120, 121, 173, 176, 179, 180, 195, 136, 206 Ccsari, I. A!., 59, 73, 99 Chaffee, N., 248, 256, 257, 264 Choglassinn, H. T., 241, 263 Clialmers, D. C., 111, 163, 196 Chan, E. L., 187, 196 Chaparas, S. D., 108, 126, 129, 196, 198 Chaperon, E. A,, 186, 187, 196 Chapuis, B., 113, 115, 119, 120, 173, 195, 202 Chase, hl. W.,102, 104, 123, 130, 151, 155, 171, 180, 182, 183, 184, 194, 196, 199 Clmssin, L. N. W.,149, 200 Chaterjee, K. R., 218, 224, 235, 236, 265 Chaves, J., 243, 246, 247, 262 Chavez, A., 232, 265 Cheers, C., 177, 199 Chen, hl-J., 234, 235, 262 Chernyakhovskaya, I. Y., 145, 150, 196 Chessin, I,. N., 111, 196 Chesterman, F. C., 227, 263 Chilgren, R. A,, 108, 196, 259, 262 Chiller, J. hl., 187, 190, 196 Chong, M. N. Y., 298( 112), 312 Christensen, T.. 63, 9 8 Churchill, W. H., 108, 185, 204, 205

318

AUTHOR INDEX

Churchill, W. H., Jr., 129, 201 Cioli, D., 91, 97 Claman, H. N., 29, 52, 186, 187, 196, 259, 263 Clamp, J. R., 17, 27, 52 Clarke, D. A., 176, 203 Clarke, J. A., 176, 196 Clarke, S. L., Jr., 166, 196 Clausen, J. E., 147, 196 Cline, M. J., 110, 196 Cochrane, C. G., 278(55, 55b), 282(55, 83), 283( 83, 85), 288( 8 3 ) , 290 ( 9 5 ) , 303( 8 3 ) , 304(83), 305( 83, 137, 138), 310, 311, 312 Cochrane, R. G., 210, 214, 262, 263 Cohen, J. R., 117, 196 Cohen, S., 171, 196, 205, 246, 249, 264, 274(21), %4( 87c), 305( 135), 309, 311, 312 Colin, M., 59, 72, 73, 91, 94, 97, 99, 182, 191, 195 Colin, Z. A., 135, 171, 196, 207 Collins, F. M., 155, 196 Collins-Williams, C., 29, 52 Collste, L., 118, 143, 147, 197, 201 Colten, H., 275( 24), 309 Convit, J., 232, 238, 239, 242, 243, 248, 249, 258, 262, 266 Coonibs, R. R. A., 45, 46, 52, 126, 153, 193, 268(2), 308 Coons, A. H., 162, 202 Cooper, H. L., 105, 149, 196, 198 Cooper, M. D., 102, 103, 179, 180, 196, 203, 204 Cooperband, S. R., 141, 150, 199 Coppleson, L. W., 167, 168, 187, 296, 202 Cort, J., 280( 70), 310 Cotran, R. S., 154, 201, 276(44), 285 ( 4 4 ) , 299(44, 44a), 309 Coulson, A. S., 111, 165, 196 Cowan, D. M., 104, 105, 198 Crabbk, P. A., 39, 53 Craig, J. M., 259, 263 Craig, L. C., 84, 98 Crookston, J. H., 298(114), 312 Cross, J. H., 43, 55 Cudkowicz, G., 164, 190, 196, 202 Cunha, R. N., 246, 264 Cunningham, A,, 187, 203

D ~

~

~

~

;

~

~

;

o

~

Daly, J. J., 176, 198 Damniacco, F., 218, 236, 261 Dandliker, W. B., 13, 52 Danielsen, D. C., 227, 262 Dannenberg, A. M., Jr., 135, 196 Danon, F., 38, 54 Dardenne, M., 110, 189,194 Darracky M’, 290(96), 311 ~ ~ ; , ~ $ , ~ g ~ 6 2 Daugharty, H., 66, 71, 75, 76, 97, 98 David, J. R., 108, 124, 125, 126, 127, 129, 130, 132, 133, 134, 141, 147, 148, 155, 183, 185, 190, 193, 194, 195, 196, 204, 205, 207 Davidsohn, I., 186, 196, 206 Davies, A. J. S., 105, 118, 161, 179, 186, 187, 189, 196,197 Davies, A. M., 107, 194 Davis, B. D., 236, 262, 269( 4), 308 Day, E., 163,193 Deane, M. P., 243,262 Decker, J. L., 146, 200 De Clercq, E., 149, 202 Decreusefond’ D ’ 3 1897 Defendi, V., 117, 164, 173, 199 Dekaris, D., 135, 197 de Lima, E. G., 236, 261 Delville, J’>232y 262 Denham, S., 114, 120, 197 de Saussure, V. A., 13, 52 Desmyter, J.> 151*Ig7 de Sousa, M. A. B., 260, 264 Des Pres, R. M., 302(129), 312 Destombes, P., 238, 239, 242, 261, 262 Deuschl, H., 32, 52 de Vassal, F., 176, 202 Deverill, J., 62, 63, 68, 98, 99 De Weck, A. L., 272(9), 300(9), 308 Dharmendra, 214, 262 Diakitk, S., 248, 263 Dias da Silva, W., 278(53), 282(53), 283(53), 310 Dickenson, J. B., 165, 202 Dienes, L., 170, 185, 197

;

~

~

319

AUTHOR INDEX

Diengdoh, J. V., 136, 170, 197, 206 Dierks, R. E., 230, 235, 262, 265 Dixon, F. J., 138, 203, 272(12, 13), 273 (13), 274( 13), 288( 13), 303( 13, 131, 132), 305( 137), 308, 312 Dodd, R. Y.,177, 197 Doenhoff, M. J., 105, 118, 161, 187, 197 Dolovich, J., 301( 121), 312 Donaldson, V. H., 279( 63), 307( 143), 310, 313 Donovan, R., 33, 44, 52 Donsky, G., 291(105), 311 Dorreter, J. B., 146, 202 Dorrington, K. J., 16, 19, 25, 53 Dostrovsky, A., 239, 262 Douglas, A. P., 39, 52 Douglas, S. D., 105, 195 Draper, P., 215, 262 Dray, S., 68, 97, 127, 129, 136, 201, 206 Dreyer, W. J., 84, 97 Dubert, J. M., 94, 98 Dulbecco, R., 269( 4 ) , 308 Dunionde, D. C., 127, 136, 141, 144, 157, 160, 197, 201, 207, 242, 2.17, 248, 250, 251, 252, 254, 255, 256, 257, 258, 259, 262, 264 Dunan, S., 246, 249, 264, 265 Duplan, J. F., 94, 98 Dupuy, J. M., 154,197,203 Duskenski, L. A,, 301(121), 312 Dutton, R. W., 107, 160, 162, 164, 187, 188, 189, 191, 193, 197, 200, 202 Duveen, G. W., 44, 54 Duxbury, R. E., 248, 262 Dwyer, J. M., 161, 197 E

East, J., 260, 264 Edelhoch, H., 16, 17, 53 Edelnian, R., 166, 197 Ehrcnberg, A., 15, 53 Eichmann, K., 71, 79, 80, 97 Eilber, F. R., 176, 202 Ein, D., 84, 98 Einbinder, J. M., 278(58), 310 Eisen, H. N., 186, 200, 269( 4 ) , 308 Elkins, W.L., 174, 197 Elliot, E. V., 187, 196 Elliot, J. H., 176, 198

El-Shawi, N. N., 236, 265 Elves, M. W., 104,197 Engle, R. L., Jr., 62, 91, 98 Epstein, W., 91, 99 Eriksen, J., 63, 98 Ernback, S., 10, 51, 54 Escobar-Gutienes, A,, 232, 265 Evans, R., 175, 197 Evans, R. R., 307( 143), 313 Evans-Paz, Z., 237, 242, 262

F Fabris, N., 290(98), 311 Fahey, J. L., 39, 54, 274( 19), 309 Falk, R. E., 143, 145, 147, 197, 207 Fantes, K. H., 148, 149, 150, 197, Farah, F. S., 241, 263 Farr, R. S., 35, 54, 276(37), 309 Fasal, P., 230, 262 Fauve, R. M., 135, 197 Federlin, K., 165, 197 Feinman, L., 305( 135),312 Feinstein, D. I., 298( 112), 312 Feinstone, S. M., 127, 129, 197 Feizi, T., 71, 79, 80, 97 Feldman, J. D., 171, 184, 195, 197, Feldman, M., 117, 196 Fellner, M. J., 107, 198 Fermresi, R. W., 126, 129, 198 Ferri, R. G., 246, 262 Festenstein, H., 105, 118, 197, 260, Finnstrom, O., 37, 52 Finter, W. B., 149, 200 Firschein, I. L., 164, 199 Fischer, H., 114, 119, 178, 193 Fischer, O., 2.37, 263 Fish, A. J., 107, 198 Fishel, C. W., 290( 101), 311 Fisher, D. B., 105, 119, 198 Fishkin, B. G., 50, 54 Fishman, M., 191, 198 Fikgerald, P. H., 106, 203 Fitz-Herbert, M., 239, 242, 264 Flas, M. H., 176, 198 Foley, H. T., 229,261 Foner, A,, 244, 257, 261, 263, 266 Forbes, I. J., 105, 206 Ford, C. E., 104, 105,198 Forni, L., 165, 204 Foster, W. A., 239, 241, 244, 248,

200

203

263

263

320

AUTHOR INDEX

Foucard, T., 32, 34, 44, 48, 52, 53 Frame, M., 274( ZO), 309 Frangione, B., 274( 17), 309 Frank, H. A., 174, 201 Franklin, E. C . , 29, 53, 274( 17, 19), 309 Freedman, S. O., 107, 198 Friedman, R. M., 149,198 Fudenberg, H. H., 28, 29, 39, 53, 54, 55, 63, 83, 84, 85, 90, 93, 98, 99, 108, 124, 126, 144, 153, 183, 200, 205, 206, 236, 263, 299( 117), 312 Fulginiti, V. A., 259, 263

Gleich, G. J., 29, 53 Glynn, L. E., 129, 204, 284(86), 311 Godal, T., 213, 263 Godfrey, H. P., 108, 126, 129, 196, 198 Godwin, H. A., 229, 261 Goidl, E. A., 188, 189, 191, 200, 203 Gold, E. E., 129, 204 Goldberg, A. F., 199 Goldberg, B., 166, 170, 198 Goldie, J. H., 189, 198 Golub, E. S., 198 Good, R. A., 39, 40, 51, 52, 102, 103, 154, 179, 180, 196, 197, 202, 203, 204, 259, 262 Gordon, B., 239, 262 Gordon, F., 148, 198 Gordon, J., 110, 143, 198 Gotoff, S. P., 136, 201 Govaerts, A., 112, 198 Gowans, J. L., 104, 105, 171, 190, 198, 206, 207 Gowland, E., 154, 198 Graf, M., 304( 134), 312 Grangaud, J. P., 246, 263 Granger, G. A., 119, 137, 138, 139, 140, 141, 142, 143, 153, 173, 175, 198, 199, 201, 207 Granner, D., 104, 206 Grant, C . K.,’114, 120, 197 Graupner, K., 163, 193 Gray, D. F., 177, 199 Gray, H. K., 129,204 Greaves, M. F., 107, 119, 146, 161, 186, 189, 190, 195, 199,205 Green, J. A., 141, 150,199 Green, N. M., 67, 91 Gresser, I., 142, 151, 199 Grey, H. M., 50, 54, 63, 84, 88, 91, 93,

G Gaafar, S., 217, 218, 219, 263 Galindo, B., 127, 129, 198 Garbe, G., 282(82), 283(82), 311 Garcia-Giralt, E., 143, 198 Garnham, P. C . C., 246, 247, 249, 259, 263, 264 Garrido-Pinson, G. C., 298( 114), 312 Gaugas, J. M., 226, 227, 229, 263, 264 Gelehrter, T. D., 104, 206 Gelfand, E. W., 299(44b), 310 Cell, P. G. H., 58, 59, 63, 65, 71, 75, 87, 94, 97, 98, 106, 154, 182, 183, 186, 188, 194, 198, 203, 205, 268 ( 2 ) , 308 Gemetchew, T., 239, 241, 244, 248, 263 George, M., 123, 129, 198 Gerety, R. J., 126, 129, 198 Gery, I., 107, 194 Gettner, S., 258, 265 Gibson, T., 170, 198 Gigli, I., 278( 52), 310 Gilchrist, C., 227, 263 Gillette, R. W., 171, 198 Gillissen, G., 165, 198 97, 99 Gilman, A., 68,97 Gribik, M., 126, 129, 136, 205 Ginsberg, H. S., 269(4), 308 Griffith, D., 259, 264 Ginsburg, H., 117, 164, 198 Grynberg, N. F., 264 Girard, J.-P., 8, 53, 107, 198 CuiffrB, L., 246, 264 Githens, J. H., 259, 263 Guinto, R. S . , 227, 263 Gitlin, D., 259, 263 Gunders, A. E., 257, 263 Glade, P. R., 103, 111, 138, 143, 146, Gurner, B. W., 126, 153, 193 149, 194, 196, 198, 199, 200 Guthe, T., 272(9), 300(9), 308 Glasgow, L. A., 150, 198 Gutman, R, A., 43,55 Glasscock, R. J., 303( 131), 312 Guttman, R. D., 174, 197 Glazunova, Z. I., 250, 263 Gyenes, L., 2, 5, 9, 53, 54

AUTHOR INDEX

H Haase, A. T., 149, 199, 200 Haber, E., 79, 81, 98, 132, 133, 134, 155, 204 Habich, von H., 98 Habichi, G. S., 187, 190, 196 Hall, J. G., 104, 114, 120, 197, 199 Hall, P., 42, 54 Halliday, W. J., 127, 129, 199 Halpern, B., 107, 199 Hamilton, L. D., 130, 171, 184, 194, 199 Han, S., 126, 153, 199, 229, 230, 263 Hanks, J. H., 232, 263 Hannestad, K., 63, 98 Hansson, L. A., 39, 54 Harboe, M., 62, 63, 68, 98, 99 Harding, B., 120, 121, 185, 201 Hardy, D. A., 113,199,206 Harris, J. E., 110, 169, 199 Hart, P. D., 215, 229, 263 Harvey, 3. J., 227, 263 Hasek, M., 120, 195 Haskill, J. S., 187, 199 Hassig, A., 99 Hathaway, W. E., 259, 263 Hayashi, H., 278( 61), 310 Hayat, M., 176, 202 Hayes, C., 142, 145, 202 Haynes, H. A., 229,261 Hayry, P., 117, 164, 173, 199 Hayward, B. J., 4, 51 Hazard, J., 107, 199 Heather, C. J., 170, 206 Hedrick, S. R., 108, 129, 196 Heilman, D. H., 102, 115, 123, 199, 201 Heisch, R. B., 247, 264 Heise, E. R., 126, 139, 143, 153, 157, 199 Hellstrom, I., 113, 116, 121, 170, 199 Hellstrom, K. E., 113, 116, 121, 170, 199 Helmstein, K., 113, 195 Hemmingsen, H., 186, 206 Henney, C. S., 113, 199 Henry, C., 160, 200 Henson, P. hl., 280(72), 285(72), 302 (72), 305( 138), 310, 312 Hepner, G. W., 39, 53 Heremans, J. F., 4, 5, 14, 19, 29, 39, 54

321

Hersh, E. M., 110, 169, 199 Heyneman, D., 247, 263 Hildreth, E. A., 170, 208 Hill, W. C., 132, 160, 171, 199 Hiller, M. C., 272( lo), 299( l o ) , 308 Hilschmann, N., 84, 98 Hirsch, M. S., 227, 263 Hirschhorn, K., 165, 107, 144, 146, 164, 194. 198. 199 Hirschhorn, R., 105, 195, 199 Ho, M., 149, 199, 200 Ho, M-F., 234, 235, 262 Hoare, C. A., 238,263 Hobbs, J. R., 39, 53, 108, 206, 246, 259, 263, 265 Hogarth-Scott, R. S., 42, 43, 49, 53 Hogman, C . F., 31, 37, 39, 40, 53, 54 Holborrow, E. J., 284(86), 311 Holm, G., 112, 113, 115, 117, 118, 119, 120, 121, 138, 141, 175, 200, 203, 204 Holmberg, K., 32, 54 Holmes, E. C., 129, 202 Holt, L., 108, 206, 259, 266 Holtzer, J. D., 152, 154, 200 Hong, R., 39, 40, 51, 52, 108, 196, 259, 262 Hood, L. E., 59, 74,84,98 Hoogstraal, H., 247, 263 Hook, E. W., 149, 200, 207 Hopper, J. E., 63, 66, 67, 68, 69, 71, 75, 76, 83, 84, 85, 90, 93, 97, 98, 99 Hornbrook, hl. M., 2, 3, 4, 16, 53 Housley, J., 20, 22, 23, 55 Howard, A. W., 45,52 Ho\vard, W. L., 259, 263 Howson, W. T., 127, 141, 144, 157, 160, 197 Hoyer, L. W., 275( 2 3 ) , 309 Hnber, H., 126, 153, 200, 205, 299 (117), 312 Huldt, G., 49, 53 Humphrey, J. H., 4, 22, 23, 54, 55, 176, 200, 246, 259, 263, 280( 69), 310 Hunt, W. B., 173, 200 Hunter, A., 45, 52 Hurez, D., 38, 54, 63, 89, 93, 98, 99

322

AUTHOR INDEX

I

K

ldse, O., 272(9), 300(9), 308 linperato, P. J., 248, 263 Irunberry, J., 246, 263 Isaacs, A., 148, 200 Ishizaka, K., 2, 3, 4, 8, 16, 35, 39, 40, 45, 51, 52, 53, 54, 55, 147, 200, 275(25, 29), 280(66), 301(120), 308( 122, 124, 127), 305( 127), 309,

Kaliss, N., 233, 263 Kalpaktosogloa, P., 154, 197 Kaltreider, H. B., 146, 200 Kantor, F. S., 166, 170, 183, 198, 204 Kaplan, A. P., 278(55a), 310 Kaplan, H., 108, 124, 183, 206 Kaplan, J. M., 199 Karush, F., 186, 200 Kasakura, S., 143, 200 Kasel, J. A., 149, 199, 200 Katz, A. B., 132, 133, 134, 155, 204 Katz, D. H., 188, 189, 191, 200, 203 Kau, S. T., 229, 263 Kay, A. B., 281(78a), 284(78a), 311 Kay, W., 165, 171, 200 Keller, R., 302( 123), 312 Kellina, O., 249, 263 Kelly, R., 129, 194 Kelus, A. S., 58, 59, 63, 65, 71, 75, 87. 94, 97, 98 Kempe, C. H., 259, 263 Kennedy, J. C., 190, 191, 193, 200 Kerdel-Vegas, F., 239, 243, 262 Kersey, J., 179, 202 Kesztyus, L., 291( 110), 312 Kettman, J., 188, 200 Keystone, E., 302( 125), 312 Khanolkar, V. R., 210, 213, 216, 263 Khati, B., 246, 263 Khedair, M., 246, 263 Kibrick, S., 105, 150, 199, 204 Killander, D., 105, 200 Killander, J., 31, 32, 39, 53, 54, 65, 91,

310, 312

Ishizaka, T., 2, 3, 4, 8, 16, 39, 45, 52, 53, 147, ZOO, 275( 25, 29), 280( 68), 301( E O ) , 302( 122, 124), 309, 310, 312

Ito, K., 10, 53 Iverson, G. M., 182, 200

J Jamison, D. G., 214, 227, 263, 266 Jancovic, B. D., 245, 263 Jandl, J. H., 154, 201, 276(44), 285 ( 4 4 ) , 289(94), 299(44, 44a, 44b), 306, 309, 310, 311, 312 Janis, M., 145, 194, 200 Jankovic, B. D., 179, 193 Janoff, A., 283(82), 311 Jaton, J. C., 79, 81, 98 Jerne, N. K., 103, 160, 161, 163, 167, 188, 200, 204 Jimenez, L., 133, 163, 166, 167, 168, 169, 185, 194, 195, 200 Johanovsky, J., 123, 127, 129, 131, 135, 136, 157, 194, 201, 203, 206 Johansson, S. A., 278(59), 310 Johansson, S. G. O., 2, 4, 5, 8, 9, 10, 15, 16, 20, 22, 23, 25, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 275( 29), 280( 66), 309, 310 Johnson, J. S., 149, 167, 199, 203 Johnson, R. A,, 280(75), 310 Jonas, W. E., 45, 52 Jonasson, O., 278( 57), 310 Jones, M. A. S., 162, 201 Jopling, W. H., 211, 239, 265 Juhlin, L., 37, 45, 49, 53, 54 Jureziz, R. S., 127, 129, 206

98, 99

Killian, M., 165, 207 Kind, L. S., 167, 200 Klein, E., 113, 172, 174, 175, 200, 204, 206, 207

Klein, G., 139, 172, 200, 201 Klopfenstein, M. D., 259, 263 Klostermann, H., 163, 193 Klun, M. J., 274, 309 Knapp, P. H., 291( 106), 311 Kniker, W. T., 290( 95), 311 Kobayashi, S., 107, 198 Kochwa, S., 40, 54 Koffler, D., 304( l a ) , 312 Kohler, H., 84, 98

AUTHOR INDEX

Kohler, P. F., 35, 54 Kolb, W. P., 137, 139, 141, 175, 198, 201 Kolin, A , , 201 Koller, P. C., 105, 179, 189, 196 Korngold, L., 61, 62, 98 Koss, L. G., 135, 193 Kourilsky, R., 165, 203 Kranier, J., 138, 199 Kraner, K. L., 180, 205 Krause, R. M., 70, 71, 79, 80, 97, 98 Krejci, J., 157, 201, 203, 204 Kronnian, B. S., 129, 201 Kuhns, W. J., 51, 54 Kunkel, H. G., 16, 29, 39, 53, 54, 58, 61, 62, 63, 64, 65, 70, 74, 84, 88, 91, 92, 93, 97, 98, 99, 236, 263, 266, 272, 274( 19), 304( 134), 308, 309, 312

Kunz, M. L., 107, 198 Kuper, S. W. A., 216, 263 Kurban, A. K., 241, 263 Ky, N. T., 107, 199

249,

108, 141, 202,

205, 207

Laymon, C. W., 260,263 Lebowitz, A., 141, 201 Lecks, H. I., 291(105), 311 Leddy, J. P., 272(7), 299(115), 308, 312

Leduc, E. H., 300( 118), 312 Lee, J. C., 115, 193 Lee, L., 307( 145), 313 Lee, S., 171, 197 Lefford, M. J., 177, 194 Lehnian-Fascius, H., 237, 263 Leitoner, A., 272( lo), 299( l o ) , 308 Lelievre, L., 94, 98 Lemma, A,, 239, 241, 244, 248, 263 Lennox, E. S., 190, 191, 193, 200 Leon, M. A., 73, 99, 118, 157, 204, 205 Lepow, I. H., 278(53), 282(53), 283 ( 5 3 ) , 310 Lerner, R. A., 303( 1311, 312 Leskowitz, S., 176, 183, 198, 201 Leuchars, E., 105, 118, 161, 179, 187, 189, 196, 197 Levene, G. M., 260, 264 Levey, D., 275(32), 309 Levin, A. S . , 85, 98 Levine, B. B., 273(14, 15), 300(14, 15), 308

I Lachmann, P., 131, 132, 173, 193 Lackovic, V., 149, 195, 202 Lafferty, K. J., 162, 201 Lage, H. A., 264 Lagrue, G., 107, 199 Lahiri, S. C., 278(56), 310 Lainson, R., 243, 244, 246, 247, 257, 258, 259, 261, 263 Lambert, P. B., 174, 201 Lamvik, J. O., 213, 263 Lamy, L., 245, 261 Lance, E. M., 171, 198 Landsteiner, K., 181, 188, 201 Landy, M., 102, 201 LaPorte, R., 170, 201 Lassus, A., 236, 264 Lawrence, H. S., 102, 103, 106, 124, 125, 126, 127, 129, 130, 144, 167, 191, 193, 196, 201,

323

Levis, W. R., 110, 169, 201 Levy, D. A., 280( 65), 310 Levy, H. B., 149, 199 Lewis, M. R., 102, 122, 204 Liacopoulos-Briot, M., 161, 203 Lichtenstein, L. M., 280(65, 67), 310 Lid&, S., 171, 184, 201 Lieberman, R., 59, 67, 72, 94, 99 Lind, A., 235, 236, 264 Lindeberg, L., 38, 55 Lindenmann, J., 148, 200 Ling, N. R., 104, 105, 113, 199, 201 Linscott, W. D., 299(117), 312 Lipari, R., 61, 98 Litt, M., 284( 89), 311 Livergood, D., 167, 203 Lockshin, M. D., 146, 201 Lo Buglio, A. F., 154, 201, 276(44), 285 ( 4 4 ) , 299(44, 44a), 309 Loeschke, H., 237, 263 Loewi, C., 107, 184, 193, 201 Lolekha, S., 136, 201 Lowenstein, L., 143, 200 Lubaroff, D. M., 171, 201 Lundgren, G., 118, 143, 201, 202 Lundkvist, U., 48, 53 Luparello, T., 291( log), 312 Lycette, €3. R., 106, 203

324

AUTHOR INDEX

Lyon, G., 39, 55 Lyons, H. A,, 291(109), 312

M Mabalay, M. C., 227, 263 McBride, R. A., 104, 162, 181, 191, 201, 205

McCarthy, M. M., 187, 191, 193, 197 McCarthy, J. S., 176, 198 McCloskey, J. W., 171, 201 McClure, D., 107, 208 McCluskey, R. T., 171, 196, 201, 207, 278( 6U),305( 139), 310, 312 McCormick, J. N., 83, 85, 99 MacDonald, A. B., 66, 67, 68, 69, 71, 75, 76, 77, 78, 97, 98 McFadden, E. R., Jr., 291( log), 312 McFarland, W., 110, 114, 115, 201, 205 McGregor, D. D., 104, 105, 155, 190, 196

McGregor, I. A., 42, 54 McGuigan, J. E., 2.84, 311 Macieira-Coelho, A., 142, 143, 151, 198, 199

McIntyre, 0. R., 40, 54 Mackaness, G. B., 135, 177, 194, 201 Mackay, I. R., 161, 197, 237, 265 McKean, D. J., 59, 74, 98 Mackey, G., 28, 29, 54 McLaughlin, C. L., 91, 99 Maclaurin, B. P., 137, 201 McLean, E. P., 129, 195 MacLean, L. D., 110, 143, 198 MacLennan, I. C. M., 120, 121, 185, 201 McRae, D. H., 212,265 Maillard, J. L., 307( 141, 142), 312, 313 Ma'ni, R. N., 144, 201, 247, 262 M..jundar, T. D., 247, 264 Makela, O., 161, 201 Makela, V., 38, 55 Makinodan, T., 76, 98, 160, 162, 189, 201, 203

Malak, J. A., 241, 263 Malchow, H., 114, 119, 178, 293 Mallory, T. B., 170, 197 Malmgren, R. A., 129, 176, 202 Mancini, G., 4, 5, 14, 19, 29, 54 Mandel, T., 161, 202 Mandema, E., 62, 97 Manni, J. A., 121, 204

Mannik, M., 58, 63, 64, 65, 70, 88, 93, 97, 98

Manson, L. A., 164, 205 Manson-Bahr, P. E. C., 237, 244, 246, 247, 248, 249, 264, 265 March, J. T., 291( 107), 312 Marchant, R., 187, 196 Marcus, P. I., 166, 167, 195, 200 Marcus, Z., 123, 126, 202 Margolies, M. N., 79, 81, 98 Margolis, S., 149, 199 Marrone, J., 129, 202 Marsh, B., 111, 144, 193 Marshak, L. C., 260, 264 Marshall, A. H. E., 103, 179, 202 Marshall, W. H., 106, 108, 167, 202 Martin, D., 104, 206 Martinez, C., 179, 202 Martins, A. B., 165, 202 Martins, J. M., 246, 264 Math&, A. A,, 291( 106), 311 MatliC, G., 176, 202 Matisova, E., 202 Matoltsy, M., 135, 207 Matsuoka, Y., 94, 98 Matthew, M., 141, 157, 160, 197 Matthews, L. J., 236, 264 hlatzinger, P., 138, 199 Mauel, J., 113, 115, 119, 120, 173, 195, 202

Mayrink, W., 249, 261 Mazonni, M., 246, 263 Mazzei, D., 231, 264 Meacock, S. C. R., 160, 202 Medawar, P. B., 111, 170, 198, 202, 229, 261

Medina, R., 244, 264 Mehrotra, T. N., 62, 98 Mellbin, T., 42, 53 Melli, G., 231, 264 Melnick, J. L., 151, 163, 167, 197, 207 Merigan, T. C., 149, 150, 202 Merrill, D., 29, 52 Meshaka, G., 63, 89, 93, 98, 99 Metzger, H., 19, 54, 103, 202 Meuwissen, H. J., 108, 179, 180, 196, 202, 259, 262 Meyers, 0. L., 127, 129, 205 Michel, M., 58, 59, 63, 64, 69, 70, 71, 75, 78, 81, 82, 83, 84, 98

325

AUTHOR INDEX

hlichie, D., 167, 196 hliliaesco, C., 63, 89, 93, 98, 99 hliliaesco, E., 38, 54 Miller, E., 127, 129, 206 Miller, F., 19, 54 hliller, H. C., 190, 202, 255, 264 hliller, J. F. A. P., 103, 104, 179, 186, 187, 202, 203 Miller, L. L., 298( 114), 312 Mills, J. A., 106, 202 Milstein, C., 84, 98 Milton, J. D., 231, 264 hlinden, P., 276( 3 7 ) , 309 hlintz, B., 173, 202 hlirsky, A. E., 105, 204 Mishell, R. I., 164, 187, 189. 191, 193, 196, 197, 202 Mitchell, C. F., 186, 187, 196, 202, 203 Mitchison, N. A,, 154, 188, 190, 202 Moberger, G., 113, 195 hloclnbber, F., 162, 202 hlogford, H. E., 260, 261 hloller, E., 115, 120, 121, 143, 145, 161, 189, 190, 197, 199, 202 Moller, G . , 118, 143, 145, 147, 161, 189, 190, 197, 199, 201, 202 hfoller, K. L., 37, 52 Mollison, P. L., 272( I l ) , 274( 2 0 ) , 2885 ( 9 1 ) , 288(91), 297(91), 308, 309, 311 hlontes-Montes, J., 2332, 265 hlontilio, B., 257, 261, 263, 266 hlookerjee, B., 146, 202 Moon, H. D., 112,205 hlooney, J. J., 135, 202 hloore, G. E., 94, 98, 138, 199 hloore, V., 84, 98 Moore, W. D., 165, 202 Moorhead, J. F., 115, 142, 145, 201, 202 hloran, 1%'. C., 280(75), 310 Morgan, H. R., 136, 205 hlorikawa, S., lG2, 202 hforley, J., 141, 157, 160, 197 hlorrell, A., 50, 54 hlorric, B., 104, 203 Morse, H. C., 276(40), 309 Morton, D. L., 113, 116, 129, 176, 202, 205 hfoscarello, hl., 29, 52 Moser, H. W., 39, 55

Mosier, D. E., 168, 187, 202 hloss, R. H., 291( 108), 312 Alota, I., 275(31), 305( 136), 309, 312 hlouton, D., 161, 189, 194 hlovat, 11. Z., 287(93), 302( 130), 311, 312 Slonbray, J. F., 231, 264 hloyne, hf. A., 94, 98 hludd, S., 127, 206 hlueller, G. C., 105, 119, 198 Xfukherjee, A. C., 246, 264 hlukherjce, A. hl., 247, 265 hluller, J. Y., 110, 189, 194 hfiiller-Eberhard, H. J., 121, 204, 277 ( 4 7 ) , 278(47, 5 5 ), 282(55), 299 ( 1 1 7 ) , 310, 312 hlunoz, J., 290( 102), 311 Sluschel, L. H., 274, 309 Alustard, J. F., 302( 130), 312 hlustakallio, K. K., 236, 264 Slyers, hl. LV., 129, 202 hlynors, L. S., 45, 52 hlyrvik, Q. W., 127, 129, 173, 198, 200

N Nachnlan, R. L., 62, 91, 99 Naff, C.. B., 279( G2),310 Nahmias, A. J., 259, 264 Najarian, J. S., 171, 197, 203 Saor, D., 161, 203 "ase, S., 188, 204 Naspitz, C. K., 104, 105, 203 Navalkar, R. G., 235, 236, 264 Navarro, E., 216, 265 Neal, R. A., 246, 249, 264 Nelken, D., 244, 261 Nelson, C. T., 278(58), 310 Selson, D. S., 134, 152, 155, 171, 203, 231, 264, 276(42), 278(51), 285 (42, 51), 309, 310 Nelson, hl., 231, 264 Nelson, R. A., Jr., 278(52), 310 Seogy, K. N., 246,264 Ncry-Guimnrez, F., 249, 264 Neveu, T., 245, 264 Newell, K. W., 227, 233, 264 Niall, H. D., 29, 52 Nilsson, H., 118, 204 Nilsson, K., 42, 54 Nishio, J., 126, 129, 136, 205

326

AUTHOR INDEX

Nisonofl, A., 66, G7, 68, 69, 71, 75, 76, 77, 78, 85, 86, 88, 90, 93, 97, 98, 99 Nissen, B., 132, 199 Noguchi, H., 260, 264 Norberg, R., 50, 51, 54 Nordbring, F., 40, 54 Nordin, A. A,, 114, 115, 116, 117, 121, 160, 173, 179, 180, 195, 196, 200 Nordqvist, B., 129, 136, 203 Norlin, M., 235, 236, 264 Notth, R. J., 171, 184, 203 Nossal, G . J. V., 82, 98, 161, 187, 201 203 Nota, N. R., 161, 203 Notani, G., 72, 94, 97 Novnles, J., 216, 265 Nowell, P. C., 104, 162, 163, 203, 207 Nussenzweig, V., 110, 194, 299( 116), 312

0 Oakley, C. L., 237, 261 Oettgen, H. F., 129, 167, 168, 195, 200 Ogawa, hl., 40, 54 Ogston, D., 279( 6 2 ) , 310 bhnian, S., 37, 54 Ohms, J., 84, 99 Old, L. J., 129, 153, 173, 176, 194, 195, 203 Olds, R. S. J., 123, 153, 193 Oldstone, hl. B., 138, 203 Olhngen, B., 50, 51 Oliveirn, B., 274( 18), 300( 18), 309 Oort, J., 104, 203, 218, 266 Oppcnheim, J. J., 106, 110, 203, 205 Orange, R. P., 275( 34), 276( 38, 40a), 282( 40a), 284( 8 8 ) , 287(88), 305 ( 3 4 ) , 309, 311 0 1 tolani, C., 231, 264 Osler, A. G., 274( 1 8 ) , 275( 32), 280( 65, 73), 300( 18), 302(73), 309, 310 Osobn, D., 103, 104, 179, 189, 198, 202, 203 Osserman, E. F., 274( 1 9 ) , 309 Oochterlony, O., 235, 236, 264 Oudin, J., 58, 59, 63, 64, 69, 70, 71, 75, 78, 81, 82, 83, 84, 97, 98 Ovary, Z., 107, 188, 203, 271(5), 275 ( 3 6 ) , 280(36), 308, 309

Oviedo, B. V. E., 248, 265

P PackalBn, T., 113, 120, 147, 179, 204, 207 Palmer, C. G., 167, 203 Palmer, E., 214, 215, 226, 263, 265, 266 Panayi, G. S., 141, 157, 160, 197 Pang, 11. K., 137, 204 Papermaster, B. W., 179, 202 Pappenheinier, A. M., Jr., 123, 186, 203, 206 Parnense, L. W., 250, 264 Paraf, A., 94, 98 Parnskova-Tchernozet~ska, E., 142, 145, 202 Parish, W. E., 304(132a), 312 Parrott, D. M. V., 260, 264 Parrow, A., 39, 52 Passaleva, A., 123, 129, 204 Patrick, R. A., 110, 194, 278(53), 282 ( 5 3 ) , 283( 53), 310 Patterson, R. J., 136, 177, 203 Paul, C., 84, 98 Paul, W. E., 188, 189, 191, 200, 203 Penrce, J. D., 107, 197 Pearlman, D. S., 259, 263, 280, 310 Peartiinin, G., 106, 203 Pearson, J. M., 226, 231, 264, 265 Pearson, hl. N., 176, 203 Peary, D., 111, 144, 193 Pederson, N. C., 104, 203 Pekarek, J., 127, 129, 131, 135, 136, 157, 194, 201, 203, 206 Pelley, R. P., 157, 205 Peltier, A. P., 165, 203 Penn, G. M., 84, 99 Pensky, J., 279(62), 310 Pepys, J., 44, 54, 218, 224, 235, 236, 265 Perey, D. Y. E., 154, 180, 197, 203 Perhnm, R. N.,59, 73, 97 Perkins, E. H., 162, 203 Periman, P. O., 166, 196 Perlniann, H., 113, 120, 121, 204 Perlmnnn, P., 37, 53, 112, 113, 115, 117, 118, 119, 120, 121, 138, 139, 141, 175, 195, 200, 201, 203, 204 Pernis, B., 165, 204 Peterson, R. D. A,, 102, 103, 179, 180, 193, 196, 204

327

AUTHOR INDEX

Peterson, W. A., 157, 200 Pettit, J. H. S., 241, 264 Pfeiffer, E. F., 165, 197 Phair, J. P., 183, 204 Phillips, hl., 276, 310 Phillips, S. hl., 170, 208 Pick, E., 157, 204 Pierpaoh, W., 290(98), 311 Pierce, G. E., 113, 116, 121, 199 Pilchard, E. I., 129, 204 Pinardi, h l . E., 243, 248, 249, 262 Pincus, W. B., 137, 138, 139, 143, 157, 204 Pink, J. R. L., 84, 99 Piper, P. J., 281( 78), 310 Pirrie, A., 142, 145, 202 Pitombeira, M. da S., 246, 264 Plantin, L. O., 50, 51 Playfair, J. H. L., 186, 205 Plotz, P. H., 275(24), 309 Pochyly, D. F., 130, 204 Pogo, B. G . T., 105, 204 Polak, L., 234, 264 Polgar, P. R., 105, 204 Polley, M. J., 299( 117), 312 Pollock, T. M., 260, 261 PontCn, J., 42, 54 Porath, J., 10, 29, 51, 54, 55 Porter, R. R., 20, 54 Potter, M . , 59, G7, 72, 73, 74, 94, 98, 99 Prendergast, R. A., 134, 171, 180, 204, 205 Pressman, D., 76, 94, 98, 99 Preston, P. M., 239, 241, 244, 248, 251, 252, 254, 2.57, 259, 262, 263, 264 Preudhomnie, J.-L., 38, 54 Price, E. W., 239, 242, 264 Priolisi, A., 246, 264 Privat, Y., 260, 265 Prouvost-Danon, A., 275( 35, 35a), 280 ( 6 8 ) , 302(68), 309, 310 229, 264 Ptak, W., Putnam, F., 84, 98 Puttrell, C. N., 149, 207

Q Quie, P. G., 108, 196, 2.59, 262 Quilici, M., 246, 248, 249, 259, 260, 264, 265

R Radovich, J., 186, 206 Radwanski, Z., 248, 255, 256, 257, 258, 262, 264 Radzimski, G., 76, 99 Raff, M. C., 114, 179, 204 Raffel, S., 126, 129, 149, 165, 176, 198, 202, 203, 204 Rahim, G. A. F., 239, 244, 261, 264 Raidt, D. J., 187, 191, 193, 197 Rajapakse, D. A., 129, 204 Rajewsky, K . V., 188, 204 Ramseier, H., 157, 159, 204 Ranadive, N. S., 283(85), 311 Rahim, G. A. F., 239, 244, 261, 264 265 Rapoport, S. I., 298(112), 312 Rapp, H. J., 129, 175, 176, 201, 207, 275(23, 25), 280(69), 309, 310 Rasanen, ." J. A., 38, 55 Rassmussen, A. F., Jr., 290( loo), 291 (107), 311, 312 Ratnoff, 0. D., 279(50, 62), 310 Rauch, H . C., 165, 204 Rawls, W. E., 127, 129, 151, 163, 167, 177, 197, 206, 207 Rebonato, C., 248, 249, 262 Reed, C. E., 291( 104), 311 Reerink-Broncers, E. E., 43, 54 Rees, R. J. W.,212, 213, 214, 215, 218, 224, 225, 226, 227, 229, 232, 235, 236, 242, 262, 263, 264, 265, 266 Reid, R. T., 276(37), 309 Reif, A. E., 114, 179, 204 Reiqnam, C. W., 259, 263 Reisfeld, R. A., 133, 204, 207 Reizenstein, P., 50, 51 Reisman, R. E., 273(16), 300(16), 301 ( E l ) , 309, 312 Remold, H . G., 132, 133, 134, 148, 155, 204, 207 Renolcl, A. E., 165, 197 Report of the Panel on Bacteriology and Immunology, 215, 265 Revesz, L., 172, 175, 200, 204 Rezai, H . R., 257,258,265 Ricci, M., 123, 129, 204 Rice, S. A., 72, 94, 97 Rich, A. R., 102, 122, 204 '

328

AUTHOR INDEX

Riches, H. R. C., 108, 206, 259, 266 Richter, M., 104, 105, 186, 203, 204 Ridley, D. S., 211, 212, 222, 223, 233, 239, 242, 265, 266 Rieke, W. O., 165, 171, 200 Rigler, R., 105, 200 Ringertz, N. W., 175, 200, 204 Ripps, C.,107, 198 Robbins, J. H., 110, 169, 201 Roberts, K. B., 106, 202 Robertson, C. L., 72, 99 Robinson, J. O., 275(30), 309 Rockey, J. H., 16, 39, 54 Rocklin, R. E., 108, 127, 129, 141, 185, 204, 205 Rodriguez, O., 216, 265 Roholt, 0.A., 76, 99 Rosenau, W., 112, 113, 115, 116, 193, 199, 205 Romagnani, S., 123, 129, 204 Romero, J., 244, 264 Rorsman, H., 129, 136, 203 Rosak, M., 185, 206 Rose, B., 290( 99), 311 Rose, N. R., 107, 198, 273(16), 300 (16), 309 Roseman, J. M., 189, 205 Rosen, F. S., 299(44b), 310 Rosenau, W., 112, 113, 115, 116, 193 205 Rosenberg, E. B., 43, 54, 55 Rosse, W. F., 274, 309 Rother, K., 305( 139), 312 Rother, U., 305( 139), 312 Rothman, S., 260, 264 Rouze, P., 94, 98 Rowe, D. S., 2, 28, 29, 31, 32, 39, 42, 52, 54 Rowley, M. J., 237, 265 Rowsell, H. C., 302( 130), 312 Ruddle, N. H., 118, 119, 137, 142, 175, 205 Rudolf, B., 115, 202 Rudolf, H., 113, 115, 119, 120, 173, 195 Rugarli, C., 231, 264 Russell, P. S., 170, 207 Ryder, G., 255, 258, 262 Rytel, M. W., 127, 129, 197

s Sadun, E. H., 248, 262 Sagher, F., 237, 239, 242, 262 Saitz, E. W., 215, 235, 265 Salazar-Mallen, M., 232, 265 Salmon, S. E., 28, 29, 54 Salsbury, A. J., 176, 196, 259, 264 Salvin, S. B., 123, 126, 129, 136, 182, 184, 185, 186, 188, 189, 190, 205, 206 Samloff, I. M., 298( 114), 312 Samuels, H. H., 104, 206 Sandberg, A. L., 274( 18), 300( 18), 309 Sanderson, A. R., 139,205 Sato, T., 162, 203 Saul, A,, 216, 265 Schaller, K. F., 238, 239, 261 ScharE, M. D., 138, 184, 186, 189, 194, 203, 206 Schechter, G. P., 110, 114, 201, 205 Scherrer, R., 94, 98 Schick, B., 269, 272, 308 Schierman, L. W . , 181, 191, 201, 205 Schild, H . O., 159, 207 Schimpl, A,, 187, 205 Schindler, R., 115, 195 Schirrmacher, V., 188, 204 Schlossman, S. F., 124, 129, 132, 183, 196, 205 Schumberger, J. R., 176, 202 Schneider, M., 176, 202 Schoenbechler, M. J., 285( 92), 311 Schoenwetter, W. F., 170, 208 Schooley, J. C., 76, 99 Schrek, R., 106, 205 Schubert, D., 91, 99 Schur, P. H., 304( 134), 312 Schwartz, H. J., 157, 205 Schwartz, R. S., 228,265 Schwartzenberg, L., 176, 202 Seeger, R. D., 110, 205 Segal, H. L., 298( 114), 312 Sehon, A. H., 2, 4, 5, 9, 53, 54, 107, 198, 205

Sela, M., 183, 195 Seligmann, M., 38, 54, 63, 89, 93, 98, 99 Sell, S., 154, 205 Sen Gupta, P. C., 246, 247, 264, 265 SBriB, C., 238, 239, 242, 261, 262

AUTHOR INDEX

Shacks, S. J., 137, 141, 175, 198 Shaninia, A. H., 236, 265 Shaw, J. J., 251, 265 Shea, J. D., 136, 205 Sheagren, J. N., 228, 230, 234, 235, 243, 265, 266 Sheffer, A., 108, 185, 204, 205 Shepard, C. C., 212, 215, 225, 226, 227, 230, 2.35, 262, 265 Sher, R., 258, 265 Shimizu, A., 84, 98 Shin, H. S., 2~74(18), 300( 18), 309 Shulnian, N. R., 272( lo), 277(46), 299 (10, 46), 308, 310 Shupe, S., 138, 199 Sidorova, E. V., 115, 195 Silvers, W. K.. 162. 173, 194, 202, 207 Silverstein, A. M., 180, 182, 186, 198, 205 Simmons, T., 164, 205 Simon, N. J., 236, 263 Simonsen, hl., 162, 205 Simpson, P. J., 167, 203 S Iskind, '. ' G. W., 188, 203 Sjoberg, H. E., 278(59), 310 Slater, R. J., 58, 61, 99 Slavina, E. G., 150, 196 Smith, C., 40, 54 Smith, R. F., 182, 184, 185, 186, 188, 189, 190, 205 Smith, R. T., 111, 142, 144, 145, 193, 205 Smith, S. J., 42, 54 Smith, T. J., 149, 150, 205 Smyly, H. J., 210, 262 Smyth, A. C., 136, 205 Snyder, R. hl., 149, 207 Sober, H. A., 124, 183, 205 S@borg, hl., 146, 147, 194, 205 Sodrt., A,, 243, 248, 249, 261 Soghor, D., 146, 200 Solheim, B. G., 63, 68,98, 99 Solliday, S., 145, 164, 194, 205 Solomon, A., 91, 99 Solomon, G. F., 2 9 l ( 108), 312 Sonozaki, H., 171, 205 Soothill, J, F., 33, 44, 52 Sorkin, E., 151, 152, 153, 194, 195, 276 ( 4 3 ) , 290( 98), 309, 311 South, M.A,, 179, 196

329

Southgate, B. A,, 248, 249, 264, 265 Spector, W.G., 159, 170, 195, 207 Spencer, E. S., 291(107), 312 Spiegelberg, H. L., 50, 54 Spink, W. W., 307( 144), 313 Spiro, D., 170, 171, 207 Spitler, L. E., 108, 124, 126, 144, 183, 205, 206 Spitznagel, J. K., 198 Spring, S. B., 78, 99 Stainislawski, M., 94, 98 Stanworth, D. R., 2, 4, 8, 20, 22, 23, 51, 52, 54, 55 Stastny, P., 126, 137, 143, 206 Stauber, L., 237, 258, 259, 265 Stechschulte, D. J., 276( 38, 39), 281 (78a), 284(78a), 309, 311 Steininger, W.J., 259, 263 Steel, T., 113, 206 Steffen, C., 165, 206 Stein, S., 62, 91, 99 Steinberg, A. G., 37, 51 Stenius, B., 48, 49, 55 Stern, K., 186, 196, 206 Stetson, C. A., Jr., 307( 145), 313 Stewart, L. C., 207 Stiehm, E. R., 29, 55 Stiffel, C., 161, 189, 194, 203 Stinclxing, \V. R., 149, 207 Stockert, B., 176, 203 Stone, hi. hl., 215, 261 Stone, S. II., 233, 261 Strejan, C., 300( 119), 312 Strickland, G. T., 43, 55 Strober, S., 104, 206 Stupp, Y., 183, 195 Stutnian, O., 179, 180, 202 Sulitzeanu, D., 147, 161, 203, 206 Sundvno, J. S., 138, 199 Sutherland, I., 213, 266 Suzuki, hl., 134, 204 Svejcar, J., 123, 127, 129, 131, 136, 157, 201, 203, 206 Svet-Moldovsky, G. J., 150, 196 Swanson, M. A., 228, 265 Swedluncl, H. A., 29, 53 Sn.ett, V. C., 110, I96 Szenlierg, A., 82, 98, 207 Szentivanyi, A,, 290( 101), 291, 311

330

AUTHOR INDEX

T Tada, T., 35, 55 Tai, C., 284, 311 Taichmnn, N. S., 287(93), 302( 130), 311, 312

Takahashi, T., 187, 206 Takasugi, M., 113, 206 Takiguchi, T., 111, 144, 193 Talal, N., 275( 24), 309 Taliaferro, W. H., 76, 99 Talniage, D. W., 76, 99, 186, 206 Talwar, G. P., 230, 265 Tan, E. M., 272, 308 Tan, hi., 91, 99 Tanaka, N., 300( 118), 312 Tannahill, A. J., 260, 261 Tartar, I. H., 239, 264 Tauaka, T., 176,207 Taubler, J. H., 127, 206 Taylor, J. B., 146, 200 Taylor, R. B., 186, 187, 188, 206 Tee, R. R., 218, 224, 235, 236, 265 Temine, P., 260, 265 Temple, A., 107, 201 Ternynck, T., 10, 12, 51 Terry, W. D., 2, 50, 52, 54, 274( 19, 2 0 ) , 276(41), 283(111), 309, 312 Tliieffry, S., 39, 55 Thomas, L., 102, 124, 125, 126, 127, 129, 130, 193, 196, 206 Thomas, M.-T., 142, 151, 199 Thompson, E. B., 104, 206 Thor, D. E., 108, 127, 129, 196, 206 Thorbecke, G. J., 187, 206 Thurston, J. M., 231, 264 Thyresson, N., 37, 54 Tkachyk, S. J., 29, 52 Toft, B., 29, 52 Tomasi, T. B., Jr., 301( 121), 312 Tomioka, H., 147, 200 Tomlinson, A. S., 306( 140), 312 Tompkins, G. M., 104, 206 Tompkins, W. A. F., 104, 127, 129, 177, 206

Torrealba, J. F., 243, 262 Torrealba, J. W., 243, 247, 262, 265 Torrigiani, G., 119, 199, 186, 205 Touillet, F., 160, 207 Townes, A. S., 235, 265 Tramier, G., 260, 265

Trautman, J. R., 228, 230, 234, 235, 236, 243, 264, 265, 266 Treadwell, P. E., 190, 191, 193, ZOO, 284( go), 290( l o o ) , 306( go), 311 Tregear, G. W., 29, 52 Trernonti, L., 247, 266 Triplett, R. F., 187, 196 Trujillo, 0. D., 258, 266 Tseng, J. J., 239, 263 Tsoi, hl., 173, 175, 206 Turcotte, R., 107, 198 Turk, J. L., 104, 111, 136, 152, 157, 165, 170, 171, 176, 184, 194, 196, 197, ZOO, 203, 204, 206, 216, 217, 218, 219, 220, 221, 223, 224, 228, 229, 231, 232, 234, 235, 242, 243, 251, 252, 253, 255, 260, 262, 263, 264, 266

Turner, Turner, Turner, Tnrohy,

K. J., 105, 206 M. D., 298( 114), 312 M. W., 32, 34, 42, 55 D. \\’., 255, 264, 266

U Uhr, J. W., 104, 107, 123, 144, 146, 184, 186, 189, 194, 198, 203, 206, 276, 310

Ulrich, M., 258, 266 Unanue, E. R., 176, 206, 272(13), 273 ( 1 3 ) , 274( 13), 288( 13), 303(13), 308

Ungar, G., 278(61), 309 Urihara, T., 302( 130), 312

V Vahlqvist, B., 42, 53 Valdiniarsson, H., 108, 206, 259, 266 Valentine, F. T., 106, 108, 144, 167, 202, 207

Valentine, M. D., 280(74), 284( 8 8 ) , 287( 881, 302( 74), 310, 311 Valentine, R. C., 67, 99, 212, 265 Van Arsdel, P. P., Jr., 107, 208 Vane, J. R., 281(78), 310 Van Furth, R., 171, 207 Van Leeuwen, G., 61, 62, 98 Vannier, W. E., 16, 51, 275( 23), 309 Vassali, P., 171, 207, 278(60), 305 (139), 310, 312 Vaughn, J. H., 123, 129, 198, 272, 308

AUTHOR INDEX

Vaz, N. M., 275(36), BO(36, 68), 302 (68), 309, 310 Veach, S. R., 127, 129, 206 Venancio, I. A., 264 Vischer, T. L., 107, 201 Vogt, W., 282(81), 283(82), 311 Voisin, G. A., 160, 207, 307( 141, 142), 312, 313

Voisin, J., 160, 207 Volknian, A., 155, 171, 196, 207 Voller, A,, 42, 43, 49, 53, 55, 251, 265 Volluni, R. L., 227, 263 van Kretschmar, W., 250, 258, 266 von Piquet, C., 268, 269, 308 Voynow, hl. K., 146, 184, 193

W Wade, H. W., 210, 214, 266 Wager, O., 38, 55, 236, 264, 266 Wagner, R. R., 149, 150, 200, 205, 207 Wahren, B., 40, 54 Waksnian, B. H., 118, 119, 135, 137, 142, 170, 171, 175, 179, 193, 201, 202, 205, 207

Waldman, T., 50, 54 Waldmann, T. A., 50, 55 Waldorf, D. S., 228, 243, 266 Wallace, F. G., 238, 263 Wallin, J., 113, 199 Wallis, V. J., 105, 118, 161, 179, 187, 189, 196, 197 Walton, B. C., 247, 249, 266 Waltraut, H. L., 299(116), 312 Wang, A. C., 63, 83, 84, 85, 90, 93, 99 Wang, I. Y. F., 83, 85, 99 Ward, P. A., 148, 207, 278(54), 280(71, 7 7 ) , 282(54), 284(87c), 310, 311 Ward, S. M., 58, 61, 99 Warner, N. L., 207 Warren, A. K., 167, 203 Wasserman, J., 113, 120, 147, 179, 204, 207

Waterfield, M. D., 79, 81, 98 Waters, M. F. R., 211, 212, 213, 215, 216, 217, 220, 221, 223, 224, 225, 228, 229, 231, 233, 234, 235, 242, 243, 262, 264, 265, 266 Watten, R. H., 43, 55 Webb, M., 127, 129, 199 Webster, M., 277( 48), 282(48), 310

331

Wecker, E., 187, 205 Weddell, A. C. M., 214, 225, 226, 263, 265, 266

Weigert, hl. C., 59, 73, 99 Weigle, W. O., 76, 99, 187, 190, 196, 305( 137), 312 Weil, hf. H., 307( 144), 313 Weiser, R. S., 126, 139, 143, 153, 157, 173, 175, 198, 199, 206, 229, 263 Weiss, L., 136, 205 Weissman, C., 105, 195, 199 Weksler, M., 231, 264 Welzer, A,, 278( 58), 310 \Vemambu, S. N. C., 224, 234, 235, 242, 266

Wepsic, H. T., 129, 175, 201, 207 Werner, B., 117, 200 Wertheim, G., 257, 266 \Vessl&i, T., 104, 156, 207 \Vhalen, C. E., 43, 54, 55 Wheelock, E. F., 149, 166, 197, 207 White, J. G., 138, 199 \Vhite, R. G., 105, 179, 202 Wicher, K., 10, 53, 301(121), 312 Wide, L., 29, 39, 45, 48, 49, 53, 54, 55 Wiener, J., 170, 171, 207 Wigzell, H., 190, 207 Wilcox, R. R., 272(9), 300(9), 308 Willenis, F. T. C., 163, 167, 207 Williams, A. C., 174, 207 Williams, K., 42, 54 Williams, R. C., 58, 62, 63, 64, 65, 70, 98, 99

Williams, R. C., Jr., 236, 266 Williams, R. M., 179, 207 Williams, T. W., 137, 138, 140, 141, 175, 198, 207

Willms-Kretschmer, K., 176, 198 Willoughby, D. A., 159, 160, 176, 196, 202, 207

\Vilson, D. B., 112, 115, 162, 163, 207 Wilson, S. K., 63, 83, 84, 85, 90, 93, 98, 99

Winkler, K. C., 152, 154, 200 Wiskott, A., 37, 55 Witebsky, E., 273( 16), 300( 16), 309 Witten, T. A., 165, 207 \Volff, S. M., 230, 234, 235, 265 Wollheim, F. A., 40, 55

332

AUTHOR INDEX

Wolstencroft, R. A., 106, 127, 141, 157, 160, 197, 201, 203, 207, 250, 251, 254, 255, 256, 257, 262 Wong, W. L., 165,207 Wood, D. W., 291( 105), 311 Wood, W. B., Jr., 269(4), 308 Woods, W. W., 137,204 Woodruff, C. E., 259, 263 Wright, D. J. M., 260, 264 Wuepper, K. D., 278( 55b), 310 Wuhrmann, F. H., 60,99 Wunderlich, J. R., 117, 164, 173, 207 Wunderly, C. H., 99

Y Yagi, Y., 94, 98 Yalow, R. S., 298(113), 312 Yaron, A., 124, 183, 205 Yee, C. L., 129, 202 Yonkovich, S. J., 59, 73, 99 Yoshida, K., 259, 264 Yoshida, T., 133, 171, 204, 207

144, 242, 258,

Youmans, G., 13'6, 177, 203 Young, J. D., 108, 124, 183, 206 Young, R. R., 39,55 Youngner, J. S., 149, 207 Yphantis, D. A., 8, 16, 19, 55

Z

195,

Zabriskie, J. B., 147, 207 Zak, S. J., 183, 201 Zarling, J. M., 177, 206 Zbar, B., 129, 175, 176, 201, 207 Zeiss, I., 114, 119, 178, 193 Zeitz, S. J., 107, 208 Zemen, N., 282(82), 283(82), 311 Ziff, M., 126, 137, 143, 206 Zitrin, C. M., 134, 155, 193 Zolov, D. M., 273( 15), 300( 15), 308 Zoschke, D. C., 106, 145, 169, 194, 208 Zuckerman, A., 239, 262 Zukosk, C. F., 143, 201 Zunker, H. O., 171,207 Zvaifler, N. J., 275(30), 309 Zweiman, B., 170, 208

SUBJECT INDEX A

minants

Adjuvants effects, cell-mediated immune reactions and, 176 Allergic reactions, see also Hypersensitivity, and Immediate-type leprosy and, 222-225 Allografts rejection, cell-mediated itiiniune reactions and, 172-175 Amino acid ( s ) sequence antibody persistance and change during prolonged immunization,

Antigen, immediate-type allergic reactions and, 271-273 Antigen-antiglobulin reaction, red celllinked, 4 5 1 6 Antiidiotypic sera cross-reactions different donors, 69-70 identical immunoglobulin molecules in different individuals, 73-75 myeloma proteins, 72-73 same individuals, 70-72

6

79-81 identical immunoglobulins in different individuals, 73-75 Antibody anti-ind, cross-reactions with nonspecific immunoglobulins, 92-93 anti-Salmonello, shared determinants, 81-83 cell-mediated immune reactions and,

151-156 combining site, individually specific antigenic determinants and, 86-87 formation cells involved, 185-193 enumeration of specifically sensitized cells and, 160-162 humoral, production in leprosy, 234-

from same individual,

70-72

Blastogenic

factors, lymphocytes

and,

143-147

C Cell-mediated immune reactions, 102-105 antibody formation, 178 cells involved, 185-193 effector cells in delayed-type hypersensitivity, 179-185 depression in leprosy, 227-234 direct cytotosicity of target cells by lymphocytes, 111-112, 121-122 correlation of, 119-121 niernbrane-associated antigens, 113-

237 idiotypic cross-reaction in different donors, 69-70 immediate-type allergic reactions and,

273-277

117 soluble antigens, 117-119 enumeration of specifically sensitized cells, antibody formation, 160-162 delayed-type hypersensitivity, 165-

169

normal populations, idiotypic specificity for limited heterogeneity, 75 persistence and change during prolonged immunization amino acid sequence evidence, 79-81 idiotypic specificities and, 7G-79 populations, individual antigenic specificities in, 6.3-69 idiotypic deterunrelatedness of

333

mixed Iyniphocyte interaction, 162-

164 lymphocyte transformation, 105-110 cell cooperation and lymphocyte activation, 110-111 mediators antibodies, 151-156 blastogenic factors, 143-147 chemotactic factor, 147-148

334

SUBJECT INDEX

interferon, 148-151 lymphotoxin, 137-143 migration inhibitory factor, 122-137 skin-reactive factor, 156-160 relationships between in vitro and in vivo results adjuvant effects, 176 cellular resistance, 177 histology, 170-172 models, 169-170 rejection of allografts and tumors, 172-175 unified model, 177-178 Chemotaxis, cell-mediated immune reactions and, 147-148 Complement, leprosy and, 234-237

E Enzymes, immediate-type actions and, 277-280

allergic re-

G Guinea pig, experimental leishmaniasis, in, 250-259

H Hypersensitivity delayed-type effector cells, 179-185 enumeration of specifically sensitized cells and, 165-169 histology and, 170-172

I Immediate-type allergic reactions, classification, basis and general description, 291-298 components antibodies, 273-277 antigens, 271-273 cells, 28S285 effector enzyme systems, 277-280 mediators, 281-283 direct responses, 298-299 general, 267-270 indirect responses anaphylactic-type, 299-302 macromolecular mediator-determined, 302-305 unknown mediator-determined, 305

mixing of categories in natural reactions, 305-307 pseudoallergic reactions and, 307-308 sensitization and, 270-271 sites of antigen-antibody reaction, 285286 the terrain, 289-291 time course, 286-289 Immunoglobulin( s ) amino acid sequence, identical molecules in different individuals, 7375 leprosy and, 234-237 nonspecific, cross-reaction with antiincl antibodies, 94 Immunoglobulin E chemical and phy.sica1 characteristics, 15-19 detection of antibody activity allergen antibodies in serum, 48-49 radioallergosorbent test, 46-48 red-cell-linked antigen-antiglobulin reaction, 4 5 4 6 general, 1-2 identification of myeloma protein ND,

2-4 isolation of, 4-15 levels in disease factors influencing, 44-45 secretions, 43-44 serum, 35-43 levels in health secretions, 32-35 serum, 29-32 metabolism, 49-51 methods for determination, 28-29 properties of active regions e chain, 19-20 enzymatic fragments, 20-28 Immunoglobulin( s ) G and M shared idiopathic determinants anti-Salmonella antibodies, 81-83 structural relationships of clonally produced, 83-85 Individual antigenic specificity antibody populations qualitative analysis, 63-66 quantitation of idiotypic specificities, 66-69 general, 58-60

335

SUBJECT INDEX

localization of determinants, 85-86 combining site and, 86-87 isolated heavy and light chains, 87-

91 light

chain variable

region and,

91-92 monoclonal origin, 94 monotypic proteins and, 60-63 Infections chronic, host-determined spectrum of clinical manifestations, 209-210,

259-260 Interferon, cellmediated actions and, 148-151

iminune

re-

L Leishmania enriettii, guinea pig infection,

250-259 Leishmaniasis artificial immunization, 249-250 disease spectrum, 238-247 experimental in guinea pig, 250-259 general, 237-238 imniunodiagnosis, 248-249 leishmanin test, 247-248 Leprosy allergic reactions in, 222-225 clinical spectrum, 210-212 experimental production in animals,

225-227 histological features, 212-214 lymphoid tissue, 216-222 human depression of cellmediated immunity and, 227-234 immunoglobulins, complement and humoral antibody production,

234-237 lepromin test, 214-21 6 Lymphocytes blastogenic factors and, 143-147 direct cytotoxicity of target cells correlation with cell-mediated immunity, 119-121 membrane-associated antigens, 113-

117 soluble antigens, 117-119

mixed interaction, enumeration of specifically sensitized cells and,

162, 164 transformation, cell-mediated immune reactions and, 105-1 11 Lymphoid tissue, histological changes in leprosy, 216-222 Lymphotosin, growth inhibitory factors and, 137-143

M Macrophages, factors affecting, 122-137 Models cell-mediated immune reactions, 169-

170 unified, 177-178 Myeloma proteins, cross-reactivity in different individuals, 72-73 Myeloma protein ND, identification of,

2 4

P Protein( s ) monotypic, individual antigenic specificity and, 60-63 Pseudoallergic reactions, immediate-type allergic reactions and, 307-308

R Radioallergosorbent test, immunoglobulin E class and, 4 6 4 8

S Serum allergen antibodies in, 4-9 immunoglobulin E levels disease, 35-43 health, 29-32 Skin-reactivity, cell-mediated reactions and, 15&160

immune

T Tumors, cell-mediated immune reactions and, 172-175

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